Comparative genomics and systems biology of ... - CORE

Comparative genomics and systems biology of environmental stress responses relevant to fungal

virulence

A thesis presented for the degree of

Doctor of Philosophy of the University of London

and the

Diploma of Imperial College

by

Andrew N. McDonagh

Department of Microbiology

Imperial College

Armstrong Road, London SW7 2AZ

OCTOBER 2010

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I certify that this thesis, and the research to which it refers, are the product of my own work, and that any ideas or quotations from the work of other people, published or otherwise, are fully acknowledged in accordance with the standard referencing practices of the discipline.

Signed: r

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For Dad (1945 - 2007).

For my son Fin. Stick to the arts subjects kid.

And for Mrs Chippy, who pioneered the way.

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Abstract

The ability to sense and adapt to environmental stress is a major factor for fungi such as Candida glabrata and Aspergillus fumigatus to colonize a host niche and therefore for patho-genesis. Herein, I assess the role of the putative C. glabrata stress activated protein kinase signalling architecture in hyperosmotic stress adaptation using gene disruption, and gene ex-pression microarrays. I report that, apart from the induction of glycerol biosynthetic genes, the transcriptional response of wild-type C. glabrata subjected to hyperosmotic stress is dissimilar to S. cerevisiae (< 20% concordance) despite sharing approximately 90% of their genetic repertoire. Transcription profiling of mutants disrupted in signalling genes (HOG 1 and STE11), revealed a significant component (approximately 30%) of transcription that was independent of the extant Shol signalling. In parallel, a library of null mutant strains disrupted in 186 C. glabrata putative transcription factor ORFs was developed and screened for sensitivity to the anti-fungal compound fluconazole, identifying novel determinants of fluconazole sensitivity (including a regulator of aromatic acid biosynthesis and several me-diator complex subunits). In a separate study, to avoid using in vitro approximations of host niche stressors, the transcriptional response of A. fumigatus cells during early stage infection in a murine model in invasive aspergillosis was investigated. This revealed an A. fumigatus gene expression programme governing metabolic and physiological adaptation, which may allow the organism to prosper within the mammalian niche. The transcrip-tional response to nutritional cues was also studied in C. glabrata and mapped to molecular interaction networks inferred by homology with S. cerevisiae. Transcription profiling of C. glabrata exposed to serum (10% v/v) revealed upregulation of amino acid uptake and catabolism and repression of carbon catabolic pathways in favour of high mannose glycan biosynthesis.

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Acknowledgments

There are many people to thank for their scientific and emotional support during this chal-lenging endeavour. Accordingly, I would like to thank my excellent mentor, and friend, Ken Haynes for his unfaltering support. I would also like to thank Elaine Bignell and Janet Quinn for giving me the opportunity to analyze some interesting transcription data and to Michael Stumpf for informative and entertaining discussions in bioinformatics and computa-tional biology. I am indebted to members of the Butler group at University College Dublin for schooling me in the practical aspects of microarray technology, notably Anne-Marie Jennings. I thank Geraldine Butler for her generous gift of Candida glabrata microarrays. I would also like to thank Darius Armstrong-James, Helen Findon, Hsueh-Lui Ho and Lucy Holcombe for their guidance in the early stages of my PhD, and Piers Ingram and fellow CisBIC bunker detainees for technical support in the latter stages. I extend special thanks to Tatiana Munera and Emily Cook at Imperial College for expert technical assistance and patience. I am thankful for the Medical Research Council doctoral training award that supported this research. Finally: thanks to Ernest, Frank, Thomas, Sir James Caird and Dudley Docker for inspiration.

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Table of contents

Abstract 4

1 Introduction 23 1.1 The increasing prevalence of opportunistic mycoses 23 1.2 The medical burden of invasive candidiasis and invasive pulmonary aspergillosis 24

1.2.1 Candida spp. and candidiasis 24 1.2.2 Aspergillus fumigatus and aspergillosis 28

1.3 Current treatment options for systemic mycoses: the need for better antifungal drugs 28

1.4 Identifying therapeutic targets: strategies for identifying virulence determinants in Candida spp. and A. fumigatus 31

1.5 A brief review of virulence factors important for Candida and Aspergillus mediated pathogenesis 34

1.6 Sensing and adapting to environmental stress responses 43 1.6.1 Oxidative stress response 43 1.6.2 Ambient pH adaptation 44 1.6.3 Hyperosmotic stress adaptation 44 1.6.4 Thermotolerance 45 1.6.5 The general stress response of S. cerevisiae 45

1.7 Identifying therapeutic targets: potential for a systems approach to fungal virulence studies 47

1.8 Specific aims addressed in this thesis 53

2 Materials and Methods 55 2.1 Experimental Methods 55

2.1.1 Fungal strains and culture media 55 2.1.2 Bacterial strains and culture media 56 2.1.3 Assays to identify altered growth phenotypes of mutant C. glabrata

and S.cerevisiae strains 56 2.1.3.1 Solid agar assays of C. glabrata and S.cerevisiae strain growth 56 2.1.3.2 Miniaturized optical density assays of mutant Candida glabrata

strain growth 57 2.1.4 Oligonucleotide primer synthesis 57 2.1.5 Polymerase Chain Reaction (PCR) 58

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2.1.6 Ribonucleic acid (RNA) extraction and quantification 2.1.7 Linear DNA and PCR extraction and purification 2.1.8 Agarose gel electrophoresis 2.1.9 Yeast DNA extraction 2.1.10 Transformation of yeast cells with electroporation 2.1.11 Western blots

2.1.11.1 Design of probes 2.1.11.2 Microarray slides

2.1.12 Reverse transcription and fluorescent labelling 2.1.12.1 Hybridization, washing and scanning 2.1.12.2 Image acquisition and data analysis

2.1.13 Statistical analysis of microarray data 2.2 Computational Methods

2.2.1 Computation of network statistics 2.2.2 k-clique percolation and community finding

3 The Hogip and Stellp dependent transcriptional effects of hyperosmotic stress in Candida glabrata 63 3.1 Chapter Overview 63 3.2 Specific Methods 70

3.2.1 Construction of a C. glabrata hogl null mutant 70 3.2.1.1 Western blot analysis 70 3.2.1.2 Genome-wide transcript profiling 71 3.2.1.3 Downstream bioinformatic analyses 71

3.3 Results 72

3.3.1 C. glabrata, hyperosmotic stress dependent, Hog1p phosphorylation is abolished in a stell background

72

3.3.2 The transcriptional response of wild-type C. glabrata cells exposed to transient hyperosmotic stress

74

3.3.3 Comparison of S. cerevisiae, C. albicans and C. glabrata transcriptional responses to hyperosmotic stress

85

3.3.4 The effect of 1-10G1 or STE11 deletion on hyperosmotic stress induced transcriptional responses in C. glabrata

88

3.3.5 The hogl and stel 1 specific transcriptional responses to hyperosmotic shock: evidence for transcriptional cross-talk between putative C glabrata MAPK cascades 92

3.3.6 Identification of a hypothetical hyperosmotic stress regulon in C glabrata

94 3.3.7 Identification of hyperosmotic stress sensitive C. glabrata transcrip-

tion factor null mutant strains 101 3.4 Discussion 120 3.5 Chapter Summary 133

of cDNA of cDNA microarrays

58 58 59 59 59 59 60 60 60 61 61 61 62 62 62

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4 Sub-telomere directed gene expression during initiation of invasive aspergillosis 134 4.1 Chapter Overview 134 4.2 Specific Methods 137

4.2.1 A. fumigatus strains and growth conditions 137 4.2.2 Murine Infections 138 4.2.3 RNA extraction and amplification 139 4.2.4 Estimation of fidelity of transcript abundance following mRNA am-

plification 139 4.2.5 Microarray hybridisations 140

4.3 Results 140 4.3.1 A. fumigatus RNA extraction and mRNA amplification 140 4.3.2 Impact of mRNA amplification on preservation of transcript ratios 141 4.3.3 A. fumigatus transcript profile during initiation of mammalian infection144 4.3.4 Lineage specificity and locational analyses of differentially expressed

genes 150 4.3.5 Clustering of induced genes 157 4.3.6 Comparative analyses of murine adaptation- and in vitro gene expres-

sion datasets 158 4.3.7 Nutrient limitation is a relevant physiological cue during A. fumigatus

initiation of murine infection 160 4.3.8 The LaeA regulon is represented among genes preferentially expressed

during intitation of murine infection 162 4.4 Discussion 163 4.5 Chapter Summary 171

5 Functional genomics of fluconazole sensitivity in Candida glabrata using a library of transcription factor knock-outs: a pilot study 172 5.1 Chapter Overview 172 5.2 Specific Methods 175

5.2.1 Construction of the CGTFKOLib library of transcription factor null mutants 175

5.2.2 Liquid culture assay to determine strain growth dynamics 177 5.2.3 Fitting growth dynamics data to a logistic model of population growth178 5.2.4 Fitting truncated growth dynamics data to an exponential model of

population growth 178 5.2.5 Fitting the inhibitory effect of fluconazole at a single time point in a

linear model 179 5.3 Unsupervised learning of strain clusters based on growth parameter estimates 179 5.4 Results 179 5.5 Discussion 182 5.6 Chapter Summary 192

6 Conclusions and future work 193 6.1 Future work 193

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A Chapter 1 Supplementary Data 199

B Media Recipes 205

C Strain Genotypes 206

D Chapter 3 Supplementary Data 207

E Chapter 4 Supplementary Data 274 E.1 Dataset S1 reference 306 E.2 Dataset S2 reference 306 E.3 Dataset S3 reference 306 E.4 Dataset S4 reference 306

F Chapter 5 Supplementary Data 307

G Transcription profiling of C. glabrata serum stress responses 330 G.1 Chapter overview 330 G.2 Specific methods 331 G.3 Transcriptional response of C. glabrata cells exposed to 10% Foetal Calf

Serum (FCS) 332

References 469

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List of Figures

1.1 Maximum likelihood phylogeny of 153 universally distributed fungal genes . 26

2.1 Overview of k-clique percolation algorithm 62

3.1 Architecture of the HOG pathway in S. cerevisiae 64 3.2 Architecture of MAPK cascades of the model organism S. cerevisiae, incuding

the HOG osmoadaptation cascade 65 3.3 The C. glabrata HOG pathway is activated by a single MAPKKK, Ste11p 73 3.4 Schematic of experimental design for transcriptional profiling of hyperosmotic

stress responses 76 3.5 Single channel normalization methods preserve slide intensity distributions

for cross-slide comparison and construction of synthetic log ratios of transcript abundance 77

3.6 Heatmap featuring column and row based cluster dendrograms of genome- wide transcriptional response to hyperosmotic stress in wild type, hogl and ste / / cells 78

3.7 Heatmap subset of genome featuring column and row based cluster dendro-grams of loge (Cy5T') - log2 (Cy3TM ) for transcripts induced by hyperosmotic stress in wild-type cells, compared with the equivalent values in hyperosmotic stressed hogl and sten cells 79

3.8 A transcript encoding glycerol dehydrogenase, an enzyme involved in glyc-erophospholipid metabolism is induced in wild-type C. glabrata cells exposed to 1 M sorbitol for 15 minutes 80

3.9 Three transcripts involved in glycerolipid metabolism are induced in wild- type C. glabrata cells exposed to 1 M sorbitol for 15 minutes 81

3.10 Three transcripts encoding enzymes involved in the ergosterol biosynthesis pathway, are repressed in wild-type C. glabrata cells exposed to 1 M sorbitol for 15 minutes 84

3.11 Venn diagram of transient hyperosmotic stress and basally regulated genes in wild-type, hogl and sten cells. 90

3.12 Architecture of MAPK cascades of the model organism S. cerevisiae, incuding the HOG osmoadaptation cascade annotated with evidence of transcriptional cross-talk in conserved C. glabrata transcripts 103

3.13 Overview of the hypothetical hyperosmotic stress regulon 104

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3.14 Distributions of betweenness centrality and degree in the hypothetical osmostress regulon network (HORN) 105

3.15 HORN transcription factor activity versus node connectivity 106 3.16 HORN subnetwork comprising putative hyperosmotic stress regulators Skolp,

Skn7p, Msn2p, Msn4p and their immediate k = 1 neighbours 107 3.17 HORN subnetwork comprising highly active regulators and their immediate

k = 1 neighbours 108 3.18 HORN subnetwork comprising hub regulators and their immediate k = 1

neighbours 109 3.19 HORN subnetwork comprising low activity and low connectivity regulators

and their immediate k = 1 neighbours 110 3.20 HORN subnetwork comprising the pair of transcriptional regulators account-

ing for the largest proportion of the regulated set and their k = 1 neighbours 111 3.21 HORN subnetwork comprising candidate GPD2 regulators and their k = 1

neighbours 112 3.22 HORN subnetwork comprising candidate GCY1 regulators and their k = 1

neighbours 113 3.23 HORN subnetwork comprising candidate ALD6 regulators and their k = 1

neighbours 114 3.24 HORN subnetwork comprising direct targets of Cin5p 115 3.25 HORN subnetwork comprising direct targets of Stel2p 116 3.26 HORN subnetwork comprising the minimal connections between 12 ortholo-

gous S. cerevisiae genes required for growth in hyperosmotic stress 117 3.27 Four C. glabrata mutants, each lacking a single putative transcription factor

are hypersensitive to 0.5 M KC1 in a liquid growth medium assay 118 3.28 Three C. glabrata mutants, each lacking a single putative transcription factor

are hypersensitive to 0.5 M KC1 in a liquid growth medium assay 119 3.29 A working model of C. glabrata ATCC 2001 hyperosmotic stress adaptation 122 3.30 Alternate hypotheses for hyperosmotic stress signal transduction through the

Shol branch of C. glabrata ATCC 2001 130 3.31 Four C. glabrata transcripts, orthologous to S. cerevisiae genes encoding core

and regulatory subunits of the 26S proteasome are induced in cells exposed to 1 M sorbitol for 15 minutes 131

3.32 Two C. glabrata transcripts, orthologous to S. cerevisiae genes encoding members of the adjacent cell integrity pathway are induced in cells exposed to 1 IVI sorbitol for 15 minutes 132

4.1 Comparative time-course of A. fumigatus Af293 germination and hyphal development in the murine lung, and laboratory culture 142

4.2 Correlation of log2 ratios resulting from comparative transcriptional analysis of the laboratory cultured A. fumigatus cell populations To and T60 under varying mRNA amplification protocols. 143

4.3 A genome-wide transcriptional snapshot of A. fumigatus Af293 during intiation of murine infection

151

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4.4 Distribution of lineage specific and telomere-proximal genes among differentially expressed host adaptation dataset 152

4.5 Overlap between murine adaptation and in vitro stress datasets 153 4.6 Comparative analysis of A. fumigatus gene expression datsets. 154 4.7 Characterisation of A. fumigatus growth on YPD, murine lung tissue and

minimal medium supplemented with ammonium or hydroxyproline 155 4.8 Expression of LaeA-regulated genes during initiation of murine infection 156

5.1 Molecular approach adopted for targeted disruption of C. glabrata transcription factor ORFS (schematic). 176

5.2 Logistic growth curves of a) wild-type strain ATCC 2001and b) the library parental strain his3 in YEPD and YEPD + fluconazole (5-10 pg-m1-1) . . . 183

5.3 Fluconazole dependent distribution of midpoint standard errors and number of inestimable growth midpoints 184

5.4 A liquid growth phenotype assay identifies two categories of fluconazole- sensitive null mutant strains 185

5.5 Growth curves of strains belonging to a cluster (C1) characterized by wild-type like growth in YEPD but sensitivity to YEPD + fluconazole (5-100 ug•ml-1) 186

5.6 Growth curves of strains belonging to a cluster (C5) characterized by slow growth in YEPD and YEPD + fluconazole (5-100 pg•ml-1) 187

5.7 Growth curves of strains belonging to a cluster (C3) characterized by wild- type like growth in YEPD and YEPD + fluconazole (5-100 mg•ml-1) . 188

A.1 Licensing timeline for antifungal drugs with systemic bioavailability 200 A.2 Species specific incidence rates of invasive candidiasis 201 A.3 Common antifungal drug molecules and their mechanism of action 202 A.4 Schematic summary of Candida albicans virulence factors 203 A.5 Proportion of conserved virulence factors in the Ascomycota 204

D.1 Solid agar phenotype assay screen of CGTFKOlib plate 1 on hypertonic agar (1 M Sorbitol) 262

D.2 Solid agar phenotype assay screen of CGTFKOlib plate 2 on hypertonic agar (1 M Sorbitol) 263

D.3 Calculated dN dS rates for Saccharomyces paradoxes, Saccharomyces bayanus, Saccharomyces mikatae and C. glabrata 264

E.1 Schematic representation of hybridisations performed in the amplification control study 275

E.2 Comparative analysis of A. fumigatus gene expression datasets 276 E.3 Concordance between estimates of relative abundance from triplicate qPCR

measurements and microarray experiments for selected A. fumigatus ORFs. 277 E.4 Box and whisker plots of Cy5TM and Cyr intensities for slides hybridized with

amplified and non-amplified RNA 278 E.5 Genes rejected in the amplification control experiment transposed onto cor-

relation plots 279

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F.1 Assessment of pipette channel cross contamination on a robotic platform . . 328 F.2 Example growth curves for four strains following incubation and sampling on

a robotic platform 329

G.1 Exploratory data analysis plots for C. glabrata environmental stress microarray hybridizations 337

G.2 MA plots for each hybridization performed in the C. glabrata environmental stress study 338

G.3 Volcano plots of differentially expressed transcripts in the C. glabrata environmental stress study 338

G.4 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous global metabolic map of S. cerevisiae 339

G.5 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous phenylalanine, tyrosine and trytophan biosynthetic pathway of S. cerevisiae 340

G.6 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous valine, leucine and isoleucine biosynthetic pathway of S. cerevisiae 341

G.7 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous valine, leucine and isoleucine degradation pathway of S. cerevisiae 342

G.8 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous arginine and proline metabolic pathway of S cerevisiae 343

G.9 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous alanine, aspartate and glutamate metabolic pathway of S. cerevisiae 344

G.10 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous lysine degradation pathway of S. cerevisiae . . . 345

G.11 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous glycolysis pathway of S. cerevisiae 346

G.12 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous pyruvate metabolism pathway of S. cerevisiae . 347

G.13 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous oxidative phosphorylation pathway of S. cerevisiae 348

G.14 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous biotin biosynthesis pathway of S. cerevisiae . . 349

G.15 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous pantothenoate and CoA biosynthesis pathway of S. cerevisiae 350

G.16 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous unsaturated fatty acid biosynthesis pathway of S. cerevisiae 351

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G.17 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous starch and sucrose metabolism pathway of S cerevisiae 352

G.18 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous high mannose type N-glycan biosynthesis pathway of S. cerevisiae 353

G.19 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous o-mannosyl glycan biosynthesis pathway of S cerevisiae 354

G.20 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous v-snare vesicular trafficking pathway of S. cerevisiae355

G.21 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous meiosis pathway of S. cerevisiae 356

G.22 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous cell cycle pathway of S. cerevisiae 357

G.23 Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous MAPK signalling pathways of S. cerevisiae . . . 358

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List of Tables

3.1 Conservation of induced transcripts between publicly available, hyperosmotic stress affected, transcript profiles in S. cerevisiae, C. albicans and this study 88

4.1 Over-represented Gene Ontology terms among differentially expressed genes 146 4.2 Functional classification of selected genes with altered transcript abundance,

relative to laboratory culture, in the murine lung 147 4.3 Distribution of induced and repressed genes among lineage specific cohorts

and chromosomal locations 150 4.4 Comparative analysis of genetic distribution among genes having differen-

tial transcript abundances in microarray analyses using A. fumigates RNA following exposure to in vitro or murine-adaptive stress, or laeA gene deletion.159

A.1 Factors involved in the development of opportunistic mycoses 199

B.1 Recipe for Synthetic Complete culture medium (1 L) 205 B.2 Recipe for Yeast Extract Peptone Dextrose culture medium (1 L) 205

C.1 Fungal strains used in this study 206

D.1 Oligonucleotides used in the three step PCR strategy for HOG1 disruption in C. glabrata 207

D.2 Biological effects encoded by linear model contrasts 208 D.3 Candida glabrata transcripts induced in wild-type cells following exposure to

YEPD + 1M sorbitol for 15 minutes 209 D.4 Candida glabrata transcripts repressed in wild-type cells following transient

exposure to 1M sorbitol for 15 minutes 224 D.5 Profile of transcripts involved in metabolism in wild-type, hog1 and stell cells228 D.6 Profile of transcripts involved in protein degradation in wild-type, hog1 and

ste11 cells 230 D.7 Profile of transcripts involved in protein trafficking in wild-type, hog1 and

stell cells 231 D.8 Profile of transcripts involved in transcriptional regulation in wild-type, hog1

and ste11 cells 233 D.9 Profile of induced transcripts conserved between S. cerevisiae (Gasch et al.,

and C. glabrata after exposure to hyperosmotic stress 234

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D.10 Profile of induced transcripts conserved between S. cerevisiae (O'Rourke & Herskowitz 1M sorbitol) and C. glabrata after exposure to hyperosmotic stress236

D.11 Profile of induced transcripts conserved between S. cerevisiae (Rep et al.,) and C. glabrata after exposure to hyperosmotic stress 237

D.12 Profile of induced transcripts conserved between S. cerevisiae (O'Rourke & Herskowitz 0.5M KC1) and C. glabrata after exposure to hyperosmotic stress 239

D.13 C. glabrata hyperosmotic stress repressed transcripts that depend on a functional putative SHOI signalling branch 241

D.14 C. glabrata hyperosmotic stress induced transcripts that are independent of HOG1 and STE11 245

D.15 HOG1-dependent hyperosmotic stress induced C. glabrata transcripts . . 250 D.16 STE/ / -dependent hyperosmotic stress induced C. glabrata transcripts . . 252 D.17 C. glabrata hyperosmotic stress repressed transcripts that depend on a func-

tional putative SHOI signalling branch 256 D.18 HOG1-dependent hyperosmotic stress repressed C. glabrata transcripts . 258 D.19 STE/ / -dependent hyperosmotic stress repressed C. glabrata transcripts . 259 D.20 S. cerevisiae transcripts whose hyperosmotic stress dependent induction is

affected by HOG1 deletion. Data from Rep et al., 260 D.21 Betweenness centrality measures and degree of tube stations in the London

Underground network 261 D.22 C. glabrata ORFs with dNIdS > 1 265 D.23 Profile of induced transcripts conserved between S. cerevisiae stell (O'Rourke

& Herskowitz 0.5M KC1 sorbitol) and C. glabrata after exposure to hyperosmotic stress 272

E.1 RNA Samples and associated amplification factors 280 E.2 Filtering per array in the amplification study 281 E.3 Over-represented Gene Ontology (GO) terms amongst genes having increased

transcript abundance. 282 E.4 Over-represented Gene Ontology (GO) terms amongst genes having repressed

transcript abundance. 287 E.5 A. fumigatus genes having increased transcript abundance, relative to labo-

ratory culture, in the murine lung and S. cerevisiae orthologs regulated by rapamycin-mediated TOR kinase inhibition 292

E.6 A. fumigatus genes having decreased transcript abundance, relative to labo-ratory culture, in the murine lung and S. cerevisiae orthologs regulated by rapamycin-mediated TOR kinase inhibition 294

E.7 A. fumigatus specific genes having elevated transcript abundance in the murine lung, relative to laboratory culture. 298

E.8 A. fumigatus specific genes having repressed transcript abundance in the murine lung, relative to laboratory culture. 299

E.9 Affc-specific specific genes having elevated transcript abundance in the murine lung, relative to laboratory culture 300

E.10 Affc-specific specific genes having repressed transcript abundance in the murine lung, relative to laboratory culture 301

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E.11 Nitrogen starvation gene clusters 303 E.12 Oligonucleotides used in qPCR 305

F.1 Summary of null mutant CGTFKOLib strain growth relative to his3 parental strain in YEPD and YEPD + 5 - 10 pg m1-1 fluconazole.

F.2 Summary of null mutant CGTFKOLib strains showing insufficent growth in YEPD (0-11.5 hours) for nonlinear regression based estimation of midpoint and upper asymptote

F.3 Summary of null mutant CGTFKOLib strains showing insufficent growth in YEPD + 5pgm1-1 (0-11.5 hours) for nonlinear regression based estimation of midpoint and upper asymptote

F.4 Summary of null mutant CGTFKOLib strains showing insufficent growth in YEPD + 10,u,gm1-1 (0-11.5 hours) for nonlinear regression based estimation of midpoint and upper asymptote

F.5 Summary of hierarchical clustering membership with associated median growth rates in YEPD ± fluconazole (5 - 10pg m1-1)

F.6 Effect of timepoint and growth condition on Z factor F.7 Fitted parameter values for a linear model describing optical density at 11.5

hours as a function of strain genotype, growth condition and the interaction between growth condition and genotype. Plate 1 of 2

F.8 Fitted parameter values for a linear model describing optical density at 11.5 hours as a function of strain genotype, growth condition and the interaction between growth condition and genotype. Plate 2 of 2

G.1 Candida glabrata transcripts induced in ATCC 2001 following exposure to Synthetic Complete + 10% (v/v) foetal calf serum (FCS)

G.2 Candida glabrata transcripts repressed in ATCC 2001 following exposure to Synthetic Complete + 10% (v/v) foetal calf serum (FCS)

G.3 Over-represented GO component terms for C. glabrata transcripts induced by 10% FCS (w/v) 432

G.4 Over-represented GO process terms for C. glabrata transcripts induced by 10% FCS (w/v) 432

G.5 Over-represented GO function terms for C. glabrata transcripts induced by 10% FCS 433

G.6 Over-represented GO component terms for C. glabrata transcripts repressed by 10% FCS (w/v) 433

G.7 Over-represented GO process terms for C. glabrata transcripts repressed by 10% FCS (w/v) 433

G.8 Over-represented GO function terms for C. glabrata transcripts repressed by 10% FCS 434

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Nomenclature

lc Micro - 10-6

Microgram

Ohm

°C Degree celsius

AA Absorbance at A

aH Activity of hydrogen ions

L. Chirality identifier: laevorotatory

as amino acid

ABC ATP binding cassette

ACM Aspergillus complete medium

AMM Aspergillus minimal medium

ATCC American type culture collection

ATP Adenosine triphosphate

BLAST Basic local alignment search tool

bp Base pairs

cDNA Complementary DNA

CFU Colony forming units

CHiP Chromatin Immunoprecipitation

COGEME Consortium for the Functional Genomics of Microbial Eukaryotes

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CWPs Cell wall proteins

Cy3 Cyanine 3. Fluorescent label incorporated in deoxyribonucleosides.

Cy5 Cyanine 5. Fluorescent label incorporated in deoxyribonucleosides.

dATP Deoxyadenosine triphosphate

dCTP Deoxycytidine triphosphate

dGTP Deoxyguanosine triphosphate

DMSO Dimethyl sulphoxide

DNA Deoxyribonucleic acid

dTT Dithiothreitol

dTTP Deoxythymidin triphosphate

EASE Expression Analysis Systematic Explorer

EDTA Ethylenediaminetetraacetic acid

ER Endoplasmic reticulum

ESR Environmental stress response

F Farad

FCS Foetal calf serum

g Gram

GDP Guanosine diphosphate

GO Gene ontology

GPR Genepix results

h Hour

H202 Hydrogen peroxide

JCVI The J Craig Venter Institute of Genomic Research

KAN Kanomycin

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kb Kilobase

KEGG Kyoto Encyclopaedia of Genes and Genomes

kg Kilogram

kV Kilo volt

L litre

LB Lurian Bertinin

log2 Binary logarithm. For 16 bit images, the theoretical range of microarray intensities

is 216. Binary logarithm transformation puts these values in the range r < 16 > 0

Lowess Locally weighted scatterplot smoothing

M Molar

m Milli - 10-3

M13 Bacteriophage M13.

MAPK Mitogen activated protein kinase. See MAPKKK

MAPKK Mitogen activated protein kinase kinase. See MAPKKK

MAPKKK Mitogen activated protein kinase kinase kinase. Hierarchical nature of this class of protein's activity is reflected in the nomenclature. MAPKKK phosphorylate

MAPKK which in turn phosphorylate MAPK

mg Milligram

min Minutes

MLT Murine lung tissue

mM Millimolar

mRNA Messenger RNA

Mw Molecular weight

n Nano - 10-9

NADPH Nicotinamidr adenine dinucleotide phosphate

NO Nitric oxide or nitrogen monoxide

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Null mutation A mutation that results in the complete loss of function of the encoded gene product

ODA Optical density at wavelength A

ORF Open reading frame. Stretch of DNA that could potentially be translated into a polypeptide or RNA

PAMP Pathogen associated molecular pattern

PCR Polymerase chain reaction

pH The decimal logarithm of the hydrogen ion activity in aqueous solution. A measure of acidity and alkalinity of a solution -logioax

PLOS Public library of science

R The R statistical computing framework

Regulon Collection of genes under regulation by the same regulatory protein

RIA Reductive iron assimilation

RNA Ribonucleic acid

ROS Reactive oxygen species

RPM Revolutions per minute

RT Reverse transcription

s Second

SAP Secreted aspartic proteases or secreted aspartyl proteases

SAPK Stress activated protein kinase

SDS Sodium dodecyl sulphate

SGD Saccharomyces genome database

SH3 SRC homology domain 3

STM Signature tagged mutagenesis

t-BOOH tertiary butyl hydroperoxide or ,1-1, dimethylethyl hydroperoxide

T-DNA Transferred DNA of tumour inducing plasmid

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T7 Bacteriophage T7

Tet Tetracycline

TF Transcription factor

TIGR The Institute of Genomic Research

TLR Toll-like receptor

TOR Target of rapamycin

UTR Untranslated region

UV Ultraviolet

UV Volts

v/v Volume / volume. Percentage solution expressing volume of the concentrated solution as a proportion of dissolving solvent (in excess)

v/v Weight / volume. Percentage solution expressing mass of the solute as a proportion of dissolving solvent

WT Wild-type

YEPD Yeast extract peptone dextrose

YKO Yeast knock-out

YPD YEPD

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Chapter 1

Introduction

1.1 The increasing prevalence of opportunistic mycoses

The increasing incidence of life-threatening mycoses emerged as a public health concern in the last three decades of the twentieth century (456; 156; 259; 479; 411). It steadfastly remains so, with the rapidly widening spectrum of opportunistic agents outpacing the emer-gence of effective, novel therapies (Supplementary Figure A.1 and A.2). This increase has been attributed, in part, to a shift in western demography and medical practise. The use of broad-spectrum antibiotics, cytotoxic chemotherapies and transplantation in combination with an increasingly aging, immunocompromised population elevates the risk for both com-mon and uncommon opportunistic mycoses (507; 541; 724). The majority of these mycoses come from the Candida (72.8 million cases annually), Cryptococcus (65.5 cases annually) and Aspergillus (12.4 million cases annually) species (542), ubiquitous organisms (107; 394) that can colonize a range of mammalian anatomical sites. Unlike primary pathogens, where disease incidence is influenced by the communicability of the organism, Candida and As-

pergillus - the pathogenic fungi studied in this thesis - are ubiquitous, and transmission of the organism to a susceptible individual is not a major factor in disease propagation. More important, is the degree and heterogeneity of immune dysfunction required for these otherwise commensal organisms (202; 704) to evade the immune system, proliferate and affect human health (Supplementary Table A.1).

1.2 The medical burden of invasive candidiasis and invasive pulmonary aspergillosis 24

1.2 The medical burden of invasive candidiasis and invasive pulmonary asp ergillosis

1.2.1 Candida spp. and candidiasis

Candida is a heterogeneous and taxonomically challenging genus of 154 species belonging to the Ascomycetes class, which accounts for, approximately, 50% of all known fungal species and 80% of primary and opportunistic-pathogen species (258). The majority of Candida

spp. are not associated with human morbidity or mortality and exist as commensals of a healthy oral, gut, skin, and vaginal flora as well as saprophytes in other non-mammalian niches'. Increasingly, in some at-risk groups, emerging Candida spp. can switch from be-nign commensals to well adapted and prolific pathogens. Systems for fungal taxonomy2 are

vital for accurate diagnosis of aetiological agents of candidiasis and aspergillosis, early and accurate diagnosis of which can improve clinical outcomes (720; 7). Candida are morpho-logically and sexually diverse, ranging from meiospores (714; 107) to obligate mitospores, with some organisms able to produce colonies containing budding yeasts, pseudohyphae and true hyphae (reviewed in (253; 92; 348)) - features that have been used as distinguishing characteristics in fungal systematics. Fungal taxonomists have traditionally used multiple classifying dimensions3, often without consensus, to classify species. Microscopically iden-tified morphological differences have perhaps been the most commonly adopted, but these techniques do do not accurately depict evolutionary relationships with a common ancestor. These traditional systems of Ascoinycete classification have been criticized for their reliance on an insufficient number of distinguishing morphological characteristics to resolve higher taxonomic relationships and have recently been augmented with methods based on molec-ular sequence evolution, which is becoming increasingly available in the post-genomic era of fungal biology.

In the post-genomic era (reviewed in (457)), fungal research is benefiting from the ac-crual of molecular sequence data (reviewed in (455)) and phylogenetic inference methods (198; 148; 763). Between 1990 and 2003, 560 fungal research papers reporting phylogenies were published, of which approximately 84% were derived using the ribosomal DNA (rDNA) (119). The 18S rDNA gene is frequently chosen for phylogenetic inference because it is ubiq-uitous, vertically transferred and has slowly evolving sites. Nevertheless, phylogenies based

'Such as on the surface of fruits in the soil and in leaf litter and the guts of insects that feed on it (497). C. glabrata can be a major cause of food spoilage. Paradoxically, it has also been used as a competitive agent in the prevention of Botrytis rot in strawberries (186)

2comprehensively reviewed in (258).

3These include morphological, physiological and biochemical techniques (194), diagnostic use of secondary metabolite profiles (527; 215), cell wall composition and protein composition (258)

Chapter 1. Introduction 25

on a single-gene do not necessarily reflect the ancestry of the organisms possessing them and can be affected by lateral gene transfer4. This is particularly applicable to protein coding genes - phylogenies based on genes involved with vital physiological or adaptive processes do not always correlate with rDNA derived trees, but they do have the capacity to resolve deep taxonomic relationships. In an attempt to combine the beneficial attributes of both phylogenetic approaches, researchers have sought to incorporate all available phylogenetic data to form consensus trees.

Recently, Fitzpatrick et al., constructed a fungal phylogeny based on 42 complete genomes derived from a supertree and combined gene analysis (205). This method used 4,805 orthologous genes families and the resulting gene-family phylogenies as input (Figure 1.1) for two different consensus tree inference methods: matrix representation with parsi-mony (MRP) and the average consensus method (AV). The consensus phylogenies provide supporting evidence for the classification of phyla, sub-phyla and orders and also shows ev-idence that the Saccharomycotina is a monophyletic Glade containing organisms that either translate CTG either as serine or as leucine. This Glade contains two sub-groups, one con-taining the fully sexual species Candida lusitaniae, Candida guilliermondii, Debaryomyces hansenii and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis and Lodderomyces elongisporus. This CTG Glade is distinct from species that have undergone a genome duplication event - which notably contains Candida glabrata, in phylogenetic terms a closer relative of Saccharomyces spp. than Candida spp.

Genome duplication events are believed to be important evolutionary processes in a variety of eukaryotes, where they play a role in the evolution of new molecular functions (515; 6; 654). The sequencing of Saccharomyces cerevisiae genome (246) has provided new insight into the evolutionary processes that have shaped its genome size and content. Shortly after the S. cerevisiae sequence was published, Wolfe and Shields (752) proposed that the species is a degenerate tetraploid resulting from whole genome duplication after its divergence from Kluyveromyces lactis based on the number and orientation of these conserved blocks, further evidence being provided by analyzing paired regions between the Kluyveromyces waltii and S. cerevisiae genomes (352). With the rapid accumulation of other hemiascomycete genome sequences (246)5 Dujon and co-workers (182) investigated the distribution of tandem gene duplications and blocks of ancestral duplications between S. cerevisiae, C. glabrata, K. lactis, D. hansenii and Y. lipolytica. They demonstrated that duplicate blocks exist in each of the four investigated species, but their number decreases with the evolutionary distance from S. cerevisiae and are highly conserved between S.

4this is common in prokaryotes

5For details of sequencing efforts consult references in (182)

1.2 The medical burden of invasive candidiasis and invasive pulmonary aspergillosis 26

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Figure 1.1: Maximum likelihood phylogeny of 153 universally distributed fungal genes. The concatenated alignment contains 42 taxa and exactly 38,000 amino acid posi-tions. Taken from Fitpatrick et al (205). At the bottom of the tree, the Saccharomycotina family consist of two clades encompassing Candida species that translate CTG as serine in-stead of leucine. This Glade can be further divided into species that are meiosporic. The second major Glade within the Saccharomycotina contains species who have undergone a whole genome duplication (WGD).


cerevisiae and C. glabrata but not between other species. Although the number and degree of conservation of these blocks is higher between S. cerevisiae and C. glabrata, they are structurally different, the latter, on average, being smaller (27 - 89 kb versus 42 - 243 kb in S. cerevisiae), a feature of reductive evolution in C. glabrata. These inferences from molecular sequence data imply that C. glabrata is phylogenetically distant from its other pathogenic relations, and is more closely related to organisms that have undergone a whole genome duplication and are not normally associated with disease (189). This evolutionary proximity of C. glabrata and Saccharomyces sensu strict() species presents the model organism S. cerevisiae, with its comprehensive molecular tools (55; 634; 595; 559; 749; 239; 173), as useful resource for the study of conserved biological processes of relevance to pathogenesis. Candida species can colonize a range of anatomical sites in immunocompetent hosts (causing genitourinary, oropharyngeal , vulvovaginal and cutaneous infections (107)), where evasion of the immune system and proliferation of the organism can lead to medical complaints with varying degrees of morbidity and mortality. Genitourinary (40) and superficial infections such as vulvovaginal and oropharyngeal candidiasis (28; 29) are common, a leading cause of GP visits and prescription expense, but not life-threatening (645). Although superficial Candida infections are trivial in terms of mortality, impairment of epithelial barriers at these anatomical sites can facilitate hematogeneous dissemination of the organism, a condition called invasive candidiasis (IC).

The medical burden of such invasive Candida infections has been evaluated using analy-ses of hospital discharge information (745; 542; 736) and population surveillance initiatives (751). Estimates vary substantially for the number of invasive candidiasis infections in the US (ranging from 7,000 (736) to 63,000 per annum (542)), but crude mortality is more stably estimated around 40%. Taken together, these results have been used to estimate the annual deaths associated with nosocomial candidiasis in the US to be in the range of 2,800 to 25,300. A long-term study of nosocomial fungal infections in the U.S.A6 reported that the Candida species were the most prevalent, accounting for 78.3% of cases (214). Although C. albicans remains the principal infective species, recent epidemiological evidence indicates an increased prevalence of the non-albicans species (496; 64)7. In a seven year study of 24,179 nosocomial bloodstream infections (BSI), conducted in the USA, Wisplinghoff et al., ranked Candida species as having the fourth highest BSI incidence rate, behind Staphylococcus au-reus and Enterococcus species (751). To date, more than 17 aetiological agents of human IC have been identified, but 90% of invasive infections are due to only five species: C. albicans,

6This study, of fungal and non-fungal infections included surveillance cultures from 115 hospitals between 1980 - 1990. The data are collected by the National Nosocomial Infections Surveillance system

7Concomitant advances in classification may have also increased detection rates, suggesting that the overall epidemiology might not have changed so significantly. See Haynes (278) and references therein

1.3 Current treatment options for systemic mycoses: the need for better anti-fungal drugs 28

C. glabrata, C. tropicalis and C. krusei. C. albicans is the most common of clinical Candida isolates in BSIs (66% of all Candida species). Surveillance studies have shown that the rates are decreasing with simultaneous increases in incidence rates of other Candida spp (Supplementary Figure A.2). Of these emergent species, C. glabrata has particular medical significance because it is associated with high mortality (748; 212) and increased anti-fungal resistance (543).

1.2.2 Aspergillus fumigatus and aspergillosis

Invasive aspergillosis describes several diseases (aspergilloma, allergic bronchopulmonary aspergillosis and invasive aspergillosis) that can affect several anatomical sites (lungs, liver, brain and kidney) whose aetiological agents belong to the genus Aspergillus. Of the ap-proximately 185 Aspergillus species identified to date, approximately 11% (20 species) are recognised as human pathogens. Prevalent species include Aspergillus fumigatus, Aspergillus nidulans, Aspergillus flavus, Aspergillus clavatus, and Aspergillus niger (394). Invasive pul-monary aspergillosis (IPA) is the most life-threatening of these diseases, with an associated mortality of 70-90 % (168) and is principally caused by A. fumigatus (394). Although the spores of this organism are among the least prevalent found in air samples (616), they are disproportionately linked to the majority of air-borne fungal infections (616). The size of A. fumigatus spores (2-3 ii1VI diameter) not only facilitates their sustained dispersion (394), but also permits entry into the pulmonary alveoli of the mammalian lung (576), whereupon the level of host immunocompetence (95; 169; 168; 583; 371), and the organism's ability to adapt to environmental stress, determines its fate, and conversely, the morbidity of the host (394; 773). Invasive pulomonary aspergillosis (IPA) has become a leading cause of death among haematology patients. The average incidence of IPA is estimated to be 5 to 25% in patients with acute leukemia, 5 to 10% after allogeneic bone marrow transplant, and 0.5 to 5.0% after cytotoxic treatment of blood diseases or autologous bone marrow transplant and solid-organ transplantation. IPA which follows solid-organ transplantation is most common in heart-lung transplant patients (19 to 26%) (reviewed in (394)). The emergence of such pernicious, life-threatening mycoses necessitates effective therapies. In the following section, treatment options and their limitations are briefly reviewed.

1.3 Current treatment options for systemic mycoses: the need for better anti-fungal drugs

In the absence of clinically available vaccines (311), the primary therapeutic option for the treatment of mycoses is the use of small molecule anti-fungals such as the polyenes, allylamines and azoles, the majority of which owe their activity to the physical disruption


of ergosterol or its biosynthesis (mechanisms of action are reviewed in (238)). Ergosterol is required for optimal functioning of membrane-bound enzymes and transporters (706; 155), pheromone signalling (332) and is a key determinant of plasma membrane structure (2). The most established drugs used in the treatment of invasive mycoses belong to the polyene class of anti-fungals. Members of this class, such as nystatin and amphotericin B, have been available for several decades, and amphotericin B was the standard therapy for candidemia for many years (668). By binding directly to cell membrane sterols the larger polyenes, such as amphotericin B are thought to form pores lined by the inward facing polyene hydroxyl residues (160; 294). These pores alter membrane permeablility and lead to leakage of important cytoplasmic components including intracellular potassium and ultimately cell death (354; 355). Although amphotericin B has been the gold standard therapy for invasive fungal infections (660), a narrow therapeutic index (238) and poor efficacy (53) necessitate other clinical options.

Despite the existence of many licensed anti-fungal molecules, less than half of them have a route of administration that is suitable for treatment of invasive mycoses8. The azole class of anti-fungals (273; 613; 599; 101; 503) was developed in order to improve upon the poor bioavailability of contemporary anti-fungals and until recently had been used ex-tensively in the treatment and prophylaxis of invasive candidiasis (124) and aspergillosis (187). The azoles (e.g. fluconazole, itraconazole and posaconazole) disrupt the biosyn-thesis of ergosterol by inhibiting catalytic enzymes involved in the step-wise conversion of lanosterol to ergosterol (Supplementary Figure A.3). The resulting altered flux in this path-way results in the depletion of ergosterol and the concomitant accumulation of 14a-methyl sterols (lanosterol, 4,14-dimethylzymosterol and 24-methylenedihydrolanosterol) whose C-4 methyl groups contribute to the altered structural properties of the azole effected fungal cell membrane. The primary target of the first azoles is encoded by ERG11 (also known as Cyp51 in Aspergillus), whose protein product lanosterol 14-a-demethylase co-catalyzes cytochrome P-450-dependent 14a-demethylation of lanosterol (288), although more recent triazole derivatives9 partially owe their activity to inhibition of cytochrome P-450 depen-dent 14a-sterol demethylase (encoded by ERG24 ) (604). The widespread use of the effica-ceous fluconazole has been tempered by the emergence of drug resistance in Aspergillus spp (298; 120; 250) and Candida spp. (543).

Although compensatory mechanisms exist to scavenge exogenous sterols for example in C. glabrata (492), azole resistance is generally caused by diminishing the concentration of the active compound at the active site of the target enzyme. This can be achieved in several

8Data obtained from http://www.accessdata.fda.gov/scripts/cder/drugsatfda/

8such as fluconazole, itraconazole and voriconazole

1.3 Current treatment options for systemic mycoses: the need for better anti-fungal drugs 30

ways, including mutations in the target enzyme effecting drug but not natural substrate binding (712; 741), overproduction of the target enzyme (711), biophysical changes in the membrane that impede intracellular azole accumulation (437), and active efflux mechanisms (reviewed in (110)). The molecular basis of such azole efflux mechanisms has been well characterized in S. cerevisiae, C. albicans and C. glabrata. There are two main classes of efflux pumps that act to translocate anti-fungal compounds across the cell membrane: i) the ABC (ATP-binding cassette (335)) transporters and ii) the MFS (major facilitator superfamily (600)) pumps. ABC transporters are primary transporters, using the energy derived from ATP hydrolysis to pump azoles out of the cell and include the pleiotropic drug resistance (PDR) group of transporters such as Pdr5p identified in S. cerevisiae (46; 69; 306) and orthologues in C. albicans, C. glabrata (697; 719; 606) and A. fumigatus. An elegant study by Thakur et al probed the mechanism of Pdrlp-mediated transcriptional control of efflux pumps and demonstrated that direct binding of anti-fungal molecules to Pdrlp causes this transcription factor to activate transcription of efflux pumps via an interaction with the mediator complex subunit Ca111p (687) in S. cerevisiae and C. glabrata. MFS transporters are secondary transporters that utilize proton-motive force across the plasma membrane to actively translocate azoles out of the cell and include the C. albicans Mdrlp, Flu1p and A. fumigatus Mdr3p proteins (110). The importance of efflux mechanisms in Aspergillus spp. is less clear. Much of the research performed in A. nidulans has identified candidate efflux transporters (21; 165; 23), however few clinical isolates of A. fumigatus overexpress efflux pumps, and there is evidence to suggest that target enzyme alteration is a major mechanism in addition to other uncharacterized mechanisms (154; 442; 493).

Clinicians have sought to quickly address the problem of emerging drug resistance by investigating the efficacy of combination therapy with existing drugs. Sanati at al (605) investigated the effects of amphotericin B and fluconazole monotherapy and combination therapy on mortality and tissue fungal density in neutropenic mice. They demonstrated that the survival of neutropenic mice with disseminated candidiasis was prolonged when animals were treated with a combination of amphotericin B and fluconazole. This seemingly strong in vitro evidence in favour of combination therapy belies the clinical, pharmacoeco-nomic and regulatory drawbacks. Clinical trials of combination therapy are limited and often empirical, and concerns remain over the putative mechanism and the increased po-tential for adverse pharmacokinetic interactions (370; 406; 713; 256). In their attempts to exploit the unmet medical need for efficacious, cost-effective anti-fungal therapies, pharma-ceutical companies have had some success with a new generation of molecules such as the triazoles (posaconazole, isavuconazole, ravuconazole and albaconazole) and echinocandins (caspofungin, micafungin and anidulafungin) (199; 244). Voriconazole is now the primary treatment of most forms of invasive aspergillosis. A clinical trial comprising 277 patients


with invasive pulmonary aspergillosis demonstrated that a successful outcome was observed in 52% of patients treated with voriconazole in comparison to 32% of patients who received amphotericin B (283). Caspofungin is better tolerated and as effective as amphotericin B (651) and voriconazole was as effective as amphotericin B followed by fluconazole in a large clinical trial that included 370 patients with systemic candidiasis (378). Mortality rates were comparable in the groups that received voriconazole or fluconazole with amphotericin B (36% versus 42% respectively), however voriconazole led to much better treatment out-comes for patients infected with C. tropicalis. These are small advances in clinical outcomes, however, significant cross-resistance to next generation triazoles has also been reported af-ter prolonged exposure to fluconazole and itriconazole (523). Although triazoles are an effective option for new cases, patients with persistent candidemia and prior second or first generation azole exposure may still have few effective treatment options. Clearly, this poses a major challenge for clinicians and drug discovery companies alike: how can such increas-ingly prevalent, drug-resistant, pernicious mycoses be managed? Focusing on such narrow range of targets restricts the chemical space that can be explored in designing potent, selec-tive and safe drug molecules. Indeed, a recent chemical genomics (287) study in which 316 unique compound treatment conditions spanning established anti-fugal molecules, natural products and chemical probes and showed that 97% of the knockout strains were suscepti-ble to at least one drug condition. This systematic investigation indicates that there is a largely unexplored chemical space available for exploitation. In this clinical context, biolo-gists aim to understand the molecular basis of virulence. In doing so they hope to identify and exploit new anti-fungal targets - aspects of fungal genetics that are important for fungal pathogenesis.

1.4 Identifying therapeutic targets: strategies for identifying virulence determinants in Candida spp. and A. fumigatus

Molecular mycologists have employed a variety of molecular and genetic techniques to iden-tify criticial determinants of virulence, including gene deletion strategies, gene expression strategies, and the use of systematic disruption libraries in the model organism S. cerevisiae to characterize processes required for virulence. Before the advent of genomics, classical for-ward genetic approaches' were the cornerstone of research in sexually reproductive species such as A. nidulans and S. cerevisiae and have been used extensively to formulate ge-netic maps of such diverse cellular processes as nitrogen-metabolite and carbon-catabolite

10These involve identifying a chemically or physically induced mutation that confers a phenotype of inter-est, followed by characterization of a wild-type copy of the mutated gene, e.g using functional complemen-tation.

1.4 Identifying therapeutic targets: strategies for identifying virulence determinants in Candida spp. and A. fumigatus 32

repression (34), conidial development and mitosis (481) Unfortunately, these classical techniques are not yet accessible to map the virulence phe-

notype or related phenotypes in Candida spp and A. fumigatus. Most Candida species are

diploid and devoid of a sexual cycle. This limits the frequency at which storable recessive mutations are obtained and prevents segregation of relevant genotypes of interest. Even with these limitations, chemical mutagenesis techniques were adopted in early A. fumiga-

tus studies (365; 372) but were largely abandoned in ensuing years. However, the recent exciting discovery of a sexual cycle in A. fumigatus (513) may yet re-open this avenue of

virulence research in A. fumigatus. In the absence of suitable forward genetics approaches, functional complementation studies (5) have been used in C. albicans, C. glabrata and A.

fumigatus. This technique works by cloning a gene of interest, or a library of genes into a heterologous host to complement a defined phenotype. It has been used to identify molec-ular determinants of epithelial adhesion (231; 220), multi-drug resistance (207; 8; 677) and morphogenesis (663), in C. albicans; cell wall maintenance (734; 490; 500) in C. glabrata

and calcium homeostasis (650) in A. fumigatus. Functional complementation experiments make an assumption that the transforming gene is expressed in the heterologous host in the same manner to its native locus in the donor strain, and that it has the same function in the host genetic context. However, fundamental differences relating to codon usage (608; 91), and to background-specific functions have been reported (677).

Reverse genetics approaches approaches have also been adopted to associate loci with phenotypes. Unlike classical forward genetics studies, reverse genetics methods expose a strain, typically deficient in a known protein-coding chromosomal locus, to a selection of in vitro assays designed to evaluate the contribution of that locus to a specific biological process. Typically, this leads to a loss of function in in vitro assays or in in vivo models of invasive mycosis, however some studies have revealed enhanced function in the null mutant strain. For example deletion of the C. glabrata ACE2 locus results in hyper-virulence

in a murine model of candidiasis (341), and deletion of CAS1, a C. neoformans putative

membrane protein rendered cells more virulent (324). The development of high copy number plasmids (545), robust transformation protocols (247; 582; 171) and multiple selectable markers (582; 628), facilitated gene disruption techniques (208; 746; 483; 750) in C. albicans, elucidating the function of genes involved in e.g. invasive growth and virulence (728), salt tolerance (647) and gluconase function (251). Similarly, transformation procedures (519) and single gene disruption techniques using antibiotic resistance markers (447; 561) or auxotrophic markers (166) have been developed in A. fumigatus, elucidating the function of genes involved in virulence related processes such as calcium tolerance (649), multi-drug resistance (460), cation transport (423), iron assimilation (618) and alkaline adaptation

(66).


Although some progress has been made in functional genomics of pathogenic fungi, the laborious nature of targeted gene disruption methods has limited its scope and impact in C. albicans, A. fumigatus and C. glabrata. To date there are no genome-wide knockout collections for these organisms and this still represents a significant technical challenge. A genome-wide knockout collection is of particular value because it enables unbiased pheno-typic screens, in contrast to small scale targeted single gene deletions which are usually chosen on the basis of homologues in a related species. In their excellent review, Scherens and Goffeau (614) summarized the value of this unbiased approach by focusing on the S. cerevisiae knockout library (749). At the time of their publication, they identified 33 pub-lished studies which used this collection to screen approximately 100 conditions, identifying 5000 new phenotypes including over 100 new genes resulting in a defective bipolar budding pattern (498) and 71 essential genes that were previously thought to be dispensable (257). Massively parallel random mutagenesis techniques were developed to maximize genome cov-erage with limited resources (595; 282; 98; 116; 135; 356). Variations on this approach have been adopted to successfully identify virulence determinants in A. fumigatus (94) and C. glabrata (342; 341; 139; 141). Despite these positive findings, the propensity for mobile elements to integrate in non-coding regions and limited transformation efficiency means that exhaustive coding region coverage has not yet been achieved and systematic unbiased screens consequently remain elusive.

In contrast, collaborative targeted disruption programs, although labour intensive, pro-vide much greater coverage of the target genome. This has been performed on an industrial scale in S. cerevisiae (749; 239), and there are near comprehensive libraries of null mu-tants in this organism. The collection has also been informative in dissecting the molecular basis of fundamental biological processes such as meiosis and sporulation (498), chromoso-mal segregation (445), cytoskeletal organization, cell-wall biosynthesis, microtubule-based chromosome segregation and DNA metabolism (692; 693), glycogen accumulation (747), accumulation of mutations (307), response to abiotic chemical stresses induced by a va-riety of drug molecules including anti-fungal agents and anti-cancer drugs (25; 26; 240) and has also made a large contribution to our understanding of conserved signal transduc-tion pathways pertinent to Candida spp. (484; 63; 739; 310) and A. fumigatus virulence, although a more complete insight into the function of pathogen specific genes necessitates gene disruption in the appropriate species (61), moreover conserved genes can have opposite functions in related species, for example ace2 strains of S. cerevisiae and C. albicans are avirulent, whereas the C. glabrata ace2 strain is hyper-virulent in an immunocompromised mouse model (430).Gene disruption studies and heterologous expression studies mentioned above have also been allied with techniques that monitor the expression of genes within the host or in conditions that mimic environmental cues experienced within the host niche. In


non-fungal pathogens, where virulence may be associated with a single expressed genetic element, transcriptional profiling techniques compare the mRNA abundance of each gene in one or more conditions pertinent to pathogenesis.

This technique allows researchers to identify genes whose altered expression may be part of an adaptive response to environmental stress signals. Before gene expression microarrays were widely adopted, this was monitored in pathogenic organism with reporter gene assays, differential subtraction (68), RNA fingerprinting (133) or in vitro expression technology (IVET)

1.5 A brief review of virulence factors important for Candida and As-pergillus mediated pathogenesis

The techniques described in the previous section have elucidated important processes and molecules involved in pathogenesis caused by Candida spp and Aspergillus spp.; the follow-ing section is a non-exhaustive overview of some of those critical determinants of patho-genesis. Unlike some primary enteric pathogens, whose pathogenic properties are tightly linked to a very small set of molecules (e.g. type 3 secretion proteins of Salmonella spp. (671)), virulence determinants (VDs) of opportunistic fungal pathogens are more diffuse, and pathogenesis is determined by the overall fitness of the fungus within the host. Although some researchers favour the strict definition of a virulence determinant as a microbial en-coded entity that damages the host, a more subtle definition which presents virulence as a continuum in which host immunocompetence and pathogen fitness has been suggested by Casadevall and Pirofski (112). Much research has focused on the molecular and genetic nature of processes that enable fungal pathogens to adapt to the host niche. Although Can-dida spp and A. fumigatus primarily colonize different anatomical sites, the host imposes some common constraints on these opportunistic pathogens: they must adhere to mucosal layers of the host and sense and adapt to the environmental and nutritional stresses of their mammalian niche. Colonization of the host niche requires adherence to a substrate; a process that has been studied in depth in C. albicans and to a lesser extent in C. glabrata and A. fumigatus. Radioligand binding assays were used in conjunction with human buccal (HBECs) or vaginal cells (HVECs) to identify spontaneous, cerulenin-resistant strains of C. albicans with impaired adherence (108; 289) and to demonstrate a correlation between the degree of innate adherence of Candida species and their virulence (358). Techniques were later developed to identify the molecular determinants of adhesion, initially in S. cerevisiae, where heterologous gain of function in non adherent strains (231; 220) demonstrated the genetic basis of adherence, and subsequently in C. albicans (208). The molecular basis of this process has largely been dissected by Lois Hoyer (304) and colleagues.


They initially described the similarity of ALS1 gene product to the S. cerevisiae a—agglutinin, a protein that is involved in adhesion of a mating types to a ligand on the complementary a type cells during mating (413). The agglutinin-like sequence ALS gene family consists of at least nine genes (302; 231; 340; 303; 300) that share a conserved (55% - 90% sequence iden-tity) 5' domain, one or more putatively glycosylated central motifs of 108bp and a variable 3' domain. Immunohistochemical studies have shown that the gene products of ALS genes are localized to the cell surface in vitro and in vivo due to the amino and carboxy-terminal hydrophobic sequences that act as a secretory signal sequence and glycosylphosphatidyli-nositol (GPI) anchor addition site respectively (303; 301; 346). The amino terminal domain has been the focus of other structural (76; 301), although not crystallographic, and func-tional studies, with some groups predicting that it assumes an immunoglobin fold motif (76; 129). The abundant glycosylation observed in the carboxy-terminal and central motifs is likely to confer an extended conformation of this domain, whose exposed hydrophobicity may explain the adhesive nature of the molecule to epithelial cells.

Possibly as a consequence of its epidemiological importance, and the earlier availability of genetic tools for systematic investigations of virulence, the Candida literature offers many more molecular studies of virulence attributes in C. albicans than other species. Although commonly used as point of reference for discussion of the molecular basis of virulence in C. glabrata and other non-albicans species, this is not always useful. For example, although relatively well defined in C. albicans, adhesins required for virulence in models of candidiasis are not conserved in most other aetiological agents of invasive candidiasis. Inspection of the Candida genome database" (CGD) data shows that two genes of the ALS family that are required for virulence in mouse models of candidiasis (ALS1 (340) and ALS/ (770)) are not conserved in any of the major aetiological agents of IC apart from C. tropicalis (Sup-plementary Figure A.5). Indeed adhesion is mediated by a separate family in C. glabrata (140; 117; 1; 141), the EPA adhesins. Most of the genes belonging to this family are located in subtelomeric regions (1), which is an important determinant of their regulation. The importance of the chromosomal location was demonstrated by inserting DNA cassettes be-tween the telomere and EPA6 and EPA7 in order to vary their telomeric proximity (117). Large insertions, which increase the distance between the telomere and the EPA genes by approximately 18 kb resulted in hyperadherance, mediated by the loss of transcriptional silencing associated with their close proximity to telomeric regions. Transcriptional silenc-ing is a function of the SIR-family (silent information regulator SIR2p, SIR3p, SIR4p) modulated heterochromatic state of these chromosomal regions, which serves to restrict transcription in those areas. Studies of subtelomeric silencing in S. cerevisiae showed that

lhttp://www.candidagenome.org


a complex of Sir2p/Sir3p/Sir4p is recruited to the telomere by interaction with two telom-ere associated proteins Rap1p and Hfdlp (465; 700). Sir2p is an NAD+ dependent histone deacetylase that has been implicated as a major regulator of urinary tract infection, where nicotinamide limiting conditions in the bladder is associated with derepression of EPA1, EPA6, EPA 7 and increased adherence in wild-type cells (180; 390; 682).

The cell wall of C. albicans harbours other important adhesins that are not conserved in C. glabrata such as the /3-glucan linked hypha cell wall specific protein Hwp1p (656) which encodes an outer surface mannoprotein. The proline and glutamine rich amino ter-minus of this protein has structural similarity to substrates for mammalian transglutaminase (TGases) involved in crosslinking epithelial cell wall proteins, and it was postulated that this protein contributes to the proliferation of C. albicans in keratinized epithelial cells by interacting with these TGases responsible for maintaining an essential host epithelial bar-rier. To test this hypothesis, Staab et al generated a recombinant, truncated Hwp1p and showed a fourfold elevation in covalent incorporation of a radiolabelled TGase2 substrate relative to a negative control. Furthermore, homozygous null mutants of HWP1 showed significantly decreased (23% relative to wild type) stable adhesion to BECs and less viru-lence than parental or single-gene deleted strains in a hematogenously disseminated murine model (655), although this was later re-evaluated by the authors themselves (673) and an-other prominent group in the field (626). They have questioned the veracity of the survival phenotype data and have demonstrated that the expression of the reconstituted URA3 gene, used as the selection marker in the disruption strategy may actually account for the filamentation and survival phenotypes observed. Although not identified as virulence factor in the CGD repository, further literature review of adhesins reveals that the C. albicans cell wall has integrin-like molecules that bind several extracellular matrix ligands (fibronectin, collagen I and IV and laminin) as well as specific monoclonal antibodies raised against the a-chain of integrin proteins.

A putative integrin-like molecule Int 1p, which has low sequence identity to the human integrin protein at the level of the ligand binding domain, has been described in C. albi-cans. This transmembrane protein has an external EF-hand domain that is consistent with a cation binding function, and a cytoplasmic talin interacting domain that may interact with the actin cytoskeleton. Molecular studies of INT1 gene disruptions reveal impaired adhesion to epithelia, attenuated virulence in mice and inhibition of hypha formation (224) as wells as abrogation of super-antigen like effects on human T-lymphocytes (174). Het-erologous gene transformation studies of non adherent S. cerevisiae strains showed that INT1 confers increased adherence and induced filamentous growth. The adhesins of A. fumigatus are poorly understood. A. fumigatus conidia also express proteins that permit specific binding to proteins expressed on host mucosal layers (24)(78; 79; 90; 241). To date,


most studies of A. fumigates adhesion have mainly focused on the conidial outer cell wall, and its largely proteinaceous rodlet layer in particular (59). This thin layer of regularly arranged rodlets is thought to confer buoyancy and aid dispersion in air currents, and is also where hydrophobins, a family of hydrophobic proteins (528), are located. Another important process determining the virulence of C. albicans is its ability to grow in differ-

ent morphological states (e.g. blastospores, pseudohyphae and hyphae) (86). C. glabrata can form pseudohyphae, but not true hyphae in response to nitrogen starvation (150) and germ-like tubes in certain colony forms. Pseudohyphae are not true septate hyphae and are distinguished from "true hyphae" by their method of growth, relative frailty and lack of cytoplasmic connection between the cells. C. albicans forms true hyphae in response to environmental cues associated with host colonization (92; 667; 760).

For example, simultaneous changes in ambient temperature and pH, both of which are departures from the optimal growth conditions (471; 438), induce filamentation. Further-more, many of the culture media used to induce hyphal growth in vitro impose some degree of nitrogen starvation. For example, serum, a powerful inducer of germ-tube formation is composed mainly of proteins that are an inaccessible nutrient source until they are hy-drolysed. N-acetylglucosamine, a poor source of carbon and nitrogen will induce hyphae alone, which can be reversed by supplementing the media with amino acids. These in vitro environmental cues reflect starvation or depletion of easily accessible nutrients. Moreover, this polymorphic behaviour is shared with more distant fungal relatives whose mycelial networks penetrate woodland floors where they can influence the control of nitrogen and phosphorus supply, scavenging and sequestering nutrients from the soil and leaf litter that they decompose (213). It has been suggested that in the mammalian host, different mor-phological states confer advantages at specific stages of pathogenesis. For example hyphae can penetrate endothelial and epithelial cell layers during invasion whereas the unicellular yeast form can disseminate more easily through the bloodstream (253; 593). Studies in S.

cerevisiae and C. albicans have identified several genes in distinct pathways that influence filamentous growth. Like in the studies of adhesion, S. cerevisiae was used extensively in

the early characterization of C. albicans morphogenetic regulation and focused on the re-lationship between filamentation and mating, the coherence of which is dependent on the transcription activator Ste12p and the mitogen activated protein kinases that regulate its activation (136; 414; 397; 469; 416). C. glabrata orthologues belonging to this putative pathway have also been investigated (105; 103; 104). Strains disrupted in the orthologous C. glabrata STEW and STE1 1 loci were shown to be attenuated for virulence in an im-munocompromised murine model of candidiasis. Ste2Op and Stellp are both required for adaptation to hyperosmotic stress, and Ste2Op is required for a fully functional cell integrity pathway whereas Ste11p is largely dispensable (103; 104).


The C. albicans homologues of STE12, CPH1 (415; 87) complements both the mating and pseudohyphal defects of ste12 S. cerevisiae strains. Functional homologues of S. cere-visiae STE genes have now been identified in C. albicans, and null mutations at each of these loci cause analagous hyphal defects in the cphl null mutant, providing evidence that these genes lie on the same signalling pathway. Further epistasis analyses have placed CST20, HST7 and CPH1 genes in positions equivalent to their homologues in S. cerevisiae and sug-gest a conserved architecture between the two species. Deletion of a different transcriptional activator in the Ras-protein kinase A pathway, EFGJ, which belongs to a family of basic helix-loop-helix transcription factors, results in decreased filamentous growth and attentu-ated virulence (529). Epistasis analysis has suggested that EFG1 and CPH1 lie on distinct, parallel pathways that activate hyphal development. Culture conditions that induce hypha formation also induce the expression of SAP4 and SAP6 genes encoding secreted aspartyl proteases. There is evidence to suggest that co-ordinated regulatory pathways exist for hy-pha production and proteinase secretion, since deletion of a transcriptional activator of Sap expression, TEC1 abrogates SAP4 and SAP6 expression and prevents hypha formation (620). Moreover, deletion of EFG1 results in impaired hypha formation and a reduction in SAP4 and SAP6 expression. Strains lacking regulators of morphogenetic shift have been used in studies of transcription during the yeast-hyphal transition (491) and provide fur-ther evidence for a co-ordinated regulation between hypha formation and SAP expression. C. albicans can undergo phenotypic switching in response to environmental stress and is implicated in the control of the sexual cycle and in virulence traits such as Sap secretion and hyphal formation. This phenomenon was also reported in C. glabrata by Lachke and colleagues, who showed rapid cell morphology changes, associated with altered expression of the metallothionein gene MTII and the haemolytic HLP in response to copper stress (384; 385). Increased high frequency phenotypic switching, identified in invasive, infectious C. albicans strains (336) suggests a role for this mechanism in virulence. Dimorphism, was also detected amongst the different copper-stress induced phenotypes after 2-3 days of growth. The proportions of budding and pseudohyphal cells was similar amongst four core phenotypes, but one phenotype displayed a far greater proportion of pseudohyphae. Previous work indicated that C. glabrata did not form pseudohyphae, and this assumption was used as a discriminating characteristic for its identification (45), but there are later reports of pseudohyphal-like projections under nitrogen starvation (150).

Hydrolytic enzyme secretion is a feature of many pathogenic organisms including bac-teria (203; 204), protozoa (454) and pathogenic yeasts (514). Although extracellular pro-teinases of saprophytic fungi such as A. niger or Neurospora crassa are secreted primarily to provide nutrients for the cell, pathogenic fungi appear to have adapted this biochem-ical property to fulfill a number of specialized functions during the infective process in


addition to the simple role of digesting molecules for nutrient acquisition, for example in adhesion (237). The 10 Secreted aspartyl proteases (Saps) of C. albicans are preproenzymes processed in the secretory pathway (211), and play an important and complicated role in virulence (597)12 . Between them, the Sap enzymes have a broad substrate specificity where hydrolysis of peptide linkages can provide access to vital nitrogen sources, but can also deactivate important host molecules. These include mucosal layer protective proteins such as mucin and IgA and host defence molecules such as lactoferrin, lactoperoxidase, cathep-sin D complement (115; 4; 158). Saps are expressed in vivo during leukocyte engulfment and in murine models of disseminated candidiasis and gastrointestinal persistance (491). In a model of vaginal candidiasis, Schaller et al showed that Saps may contribute to the induction of an inflammatory host immune response; sap mutants, lacking either SAP1 or SAPS caused reduced tissue damage and had a significantly reduced potential to stimulate cytokine expression. In contrast, the vaginopathic and cytokine-inducing potential of sap4 and sap6 mutants was similar to that of the wild-type strain (612).

Saps are also importantly associated with other virulence processes. Sap production is correlated with adherence - strongly proteolytic C. albicans strains adhere more strongly to HBECs (237) and show a higher degree of tissue colonization. Inhibition of Saps with proteinase inhibitors such as pepstatin A inhibit germ tube dependent invagination of the vascular epithelium (362). The mechanism by which Saps confer adherence is not fully understood, but two different hypotheses exist; the first hypothesis is that Saps have ad-hesive properties themselves by acting as ligands for surface moieties of host cells. The second hypothesis, perhaps more consistent with their known functions is that Sap depen-dent hydrolysis could the modify the structure and increase adherence of host molecules (474). A correlation between virulence and proteolytic activity has been demonstrated for A. fumgatus clinical isolates and in experimental models of infection (365; 373; 585), however a specific role for proteases in pathogenesis is unclear. The major secreted pro-tease of A. fumigates is a serine alkaline protease (Alpp), belonging to the subtilisin family ((217; 326; 365; 475; 573)), however a metalloprotease (Mepp) and a pepsin family aspartic protease (Pepp) have also been identified in this species ((398; 574; 574)). A mutant strain lacking the Alpp gene showed abrogated elastin but not collagen hydrolysis, however this mutant strain was no less virulent than an isogenic Alpp secreting strain in an experimental model of aspergillosis ((475; 640; 680; 681)). Similarly, cloning and disruption of Mepp (325; 636) in a A* background results in total abrogation of extracellular proteolytic ac-tivity at neutral pH in vitro is still virulent in immunosuppressed mice in a manner similar to the wild-type cells (325). Although proteolytic activity is correlated with virulence it

12comprehensively reviewed in (491)


may not be causative relationship. Indeed, histopathological studies have shown that in the lung tissue of humans or experimental animals, the collagen and elastin matrix is tra-versed without any apparent proteolytic degradation associated with the invading fungus (170; 325)). Furthermore, a monocolonal antibody that inhibits A. fumigatus elastase did not reduce the virulence of the fungus in immunocompromised mice (219; 218). Secreted proteases are important for nutrient acquistion in Candida spp. and A. fumigatus, however acquistion of nutrients other than carbon and nitrogen is also of importance in the host, for example siderophore biosynthesis is required for A. fumigatus iron acquisition and virulence in a murine model of aspergillosis (618). Recently, the zafA zinc-responsive transcriptional activator was shown to regulate the expression of genes required for growth in zinc depleted media in vitro (478). Moreno et al., demonstrated that the A. fumigatus zafA gene com-plements the ZAP1 transcription factor gene of S. cerevisiae, is induced in zinc deficient medium and is repressed by the addition of zinc. Deletion of zafA impairs the germination and maximal growth of A. fumgatus in zinc-limiting media and attentuates the virulence phenotype (478).

Fungi that produce fruiting bodies such as A. fumigatus, demonstrate co-ordinate expres-sion of biologically active, toxic and immunosuppresive secondary metabolites and spore-related products during development (109). Indeed, A. fumigatus is equipped with a di-verse arsenal of toxins ranging from specific inhibitors of the 28S ribosome (344; 345; 389) through natural steroid antibiotics (632; 470; 99), haemolytic proteins (184; 221; 377), afla-toxins (537; 510) and small molecular weight toxins such as gliotoxin (GT). Gliotoxin is a mycotoxin that belongs to the group of epipolythiodioxopiperazines, which damages ep-ithelial cells (67), is cytotoxic to mouse macrophages (731) impedes the function of ciliary cells (18), and inhibits the activiation of NADPH oxidase of polymorphonuclear leucocytes (701). This inhibitory action on a major killing mechanism employed by major effectors of cell-mediated defence, macrophages and neutrophils (611; 236), means that gliotoxin has been implicated in virulence (486; 526). Although the role of gliotoxin in virulence remains unclear (74; 145; 379; 652; 670), there has been renewed interest in the role of secondary metabolites in Aspergillus infection since the discovery of a global transcriptional regulator of secondary metabolite biosynthesis in this organism, laeA (73). laeA deletion does not affect growth or sporulation in media in vitro, but it does result in reduced virulence in mice. This impairment in the ability to cause infection can be correlated with a loss of detectable gliotoxin as well as mis-regulation of gene expression within 13 gene clusters (539). Siderophore-mediated iron uptake and storage, also partially under laeA control (539) is indispensable for A. fumigatus virulence (618; 619) and adequate nutrient acquisi-tion within the host niche is linked to pathogenesis of invasive disease being necessary to support sufficient growth to promote infection (281; 375; 374).


Biofilm formation is one of the major factors contributing to the virulence of Candida species (621). Candida biofilms are surface-attached microbial communities surrounded my a matrix of extracellular polymers and are widespread amongst medical devices involved with the systemic circulation (567)13 , where a breached epithelial layer poses a major risk for tissue invasion and invasive candidiasis. Biofilms often display phenotypic features that are inconsistent with those of their planktonic constituents. Candida biofilms were 30 - 2000 times as resistant as planktonic cells to various anti-fungal agents, including amphotereicin B, fluconazole, itraconazole and ketoconazole (276). The first, critical, step of adhesion14 is initially mediated by hydrophobic and electrostatic interactions but is subsequently stabi-lized by the expression of specific adhesion molecules. Iraqui et al (316) used an insertional mutant library of C. glabrata to isolate mutant strains with an increased ability to form biofilms. They showed that silencing mutations of C. glabrata, which displayed Yak1p de-pendent transcriptional induction of EPA6 and EPA 7 also showed dramatically induced in vitro biofilm formation. Candida species differ in their ability to form biofilms, with C. glabrata generally showing poor growth and scant blastosphore distribution in comparison to the more proilific biofilm forming species C. albicans, C. krusei and C. dublininensis. The YWP1 gene of Candida albicans is predicted to be a 533 amino acid polypeptide with an N-terminal. A homozygous ywpl/ywpl null mutant, created by recombination of both YWP1 alleles in C. albicans exhibited increased adhesiveness and biofilm formation sug-gesting that it may promote dispersal of yeast forms of C. albicans (254). This protein is absent from C. gabrata. A. fumigatus also forms biofilms

The discovery of melanin production in C. neoformans was a major breakthrough in un-derstanding the basis of virulence in that species and in other pathogens such as Wangiella dermatitidis (177) and Mycobacterium leprae (738). Fungal melanins are produced by at least two different pathways (738; 234). In C. neoformans, melanin synthesis is catalyzed by lactase, a reaction that requires exogeous substrate in comparison to other fungi that pro-duce melanin from dihydroxynapthalene by the pentaketide pathway (738). When grown in the presence of certain o-diphenolic compounds, such as 3,4-dihydroxyphenylalanine (L-dopa), C. neoformans cells turn a dark colour, due to the presence of melanin (113). Trans-mission electron micrographs and electron spin resonance show that this pigment has a high electron density and is located in the cell wall. Classic genetic and gene disruption studies have demonstrated that melanin producing C. neoformans strains are more virulent than their respective albino mutants (383; 382; 586; 602). Further evidence of the importance of

13such as shunts, stents, implants, endotracheal tubes, pacemakers and venous catheters

14A biofilm typically develops over four sequential steps: i) adhesion to a usually flat and hydrophobic sub-strate, ii) discrete colony formation, iii) secretion of extracellular polymeric substances and iv) dissemination of progeny biofilms


melanization in vivo came from studies were a hyperpigmented C. neoformans strain was

associated with reduced production of the pro-inflammatory tumor necrosis factor a and also lymphoproliferation in infected mice (308); furthermore, binding peptides which react with melanized C. neoformans cell walls were used in immunohistochemical studies to show

that infected tissues contained melanized C. neoformans cell walls (506). Moreover, the iso-lation of anti-melanin antibodies following infection of mice with C. neoformans indicated an immune response to this pigment during infection (506). Direct biochemical evidence for in vivo biosynthesis of melanin was provided by the isolation of melanin particles from the tissues of infected rodents (594). Melanin confers several physiological advantages that partially contribute to C. neoformans virulence, most notably a resistance to oxidative stress (725; 546; 321; 322; 188)), amphotericin B (726) and microbiocidal peptides (179). Melanized cells are are also less susceptible to ingestion by macrophages and killing by alveolar macrophages (418).

A. fumigatus strains lacking polyketide synthase and scyatolone dehydratase (encoded

by ALB1 and ARP1 respectively (696; 698), and pigmentless wild-type strains (394) are less pathogenic than pigmented wild-type strain. Pigmented, or "white" conidia bind to complement more effectively than do wild-type cells and are also more permeable to anti-fungal drugs. A recent study in which 0 ace2 mutants were shown to produce an orange pigment (185). The relevance of these findings in virulence is limited however, since the wild-type strains did not produce the pigment and the increased null strain virulence phenotype could be a result of the morphoogical changes associated with the disruption of a pleiotropic gene such as A CE2; furthermore, although "white" conidia are more virulent, all observed wild-type strains have green conidia with hyaline hyphae (394). C. albicans also produced

melanin in vitro and during infection of immunocompetent mice (482) although there are no studies to date on its mechanistic role in stress protection or virulence. There are also no reports of melanin production by C. glabrata, although studies of phenotypic switching

in C. glabrata (385; 384) identify core phenotypes that are light brown, dark brown or very dark brown this is assumed to be due to the graded levels of cupric and cuprous copper

ions in the cells. C. glabrata does however produce indole alkaloid pigments (pityriacitrin, malasseziaindole, pityrianhydride and) when tryptophan is the sole source of nitrogen in the culture medium (450). Pityriacitrin, first isolated in Paracoccis spp. are protective

against UV radiation, and pityarubins A, B and C are all capable of inhibiting the human neutrophil respiratory burst. Although the role of these pigments in C. glabrata remains

undetermined, Mayser et al., speculate that these pigments may be protective in vivo.


1.6 Sensing and adapting to environmental stress responses

Sessile microorganisms must be able to adapt to a wide range of physicochemical conditions. Such innate robustness requires homeostatic mechanisms (538; 368; 554; 50; 190; 315; 480; 261; 89; 292) that sense these fluctuations and direct adaptive processes which either mod-ulate the signal or obviate the effects of the perturbation. Understanding the molecular and genetic basis of these homeostatic mechanisms is of fundamental importance in biology. With a mechanistic insight, we can try to fix them when they are broken, or conversely try to break them when we want to sabotage this cellular robustness. Accordingly environmen-tal stress research transcends the field of mycology (627; 555; 260). However, the model fungal research communities have powerful molecular toolboxes at their disposal, and sig-nificant advances have been made in our understanding of environmental stress adaptation in S. cerevisiae and Schizosaccharomyces porn be. In particular, comprehensive genome-wide transcription surveys (230; 118) have significantly advanced our understanding of the core environmental stress response in S. cerevisiae, and specific stress responses pertinent to the host niche of a fungal pathogen. In the context of fungal virulence studies we are primarily interested in the specific environmental stresses of the host niche, and how fungi adapt to them. Although there is some debate over the definition of an opportunistic fungal viru-lence factor (511; 278), one of the uncontentious molecular Koch's postulates (195) is the requirement for virulence determinant expression within the host. So far, genome-wide transcription profiling in the host has been inaccessible due to technical limitations in re-covering sufficient fungal material for microarray experiments, however gene expression has been monitored within the host on a gene-family basis (22; 376). While these technical limitations remain, host environmental stress has been approximated in vitro (e.g. oxida-tive stress, thermotolerance, exposure to human polymorphonuclear leukocytes and human whole blood (191; 192; 210)), with the majority of genome-wide transcription profiling pe-formed in C. albicans and A. fumigatus. At the start of this thesis, the environmental stress

response of C. glabrata had not been characterized either by transcriptome surveys or in-vestigations of signal transduction pathways. A non-exhaustive discussion of environmental important environmental stress response follows.

1.6.1 Oxidative stress response

Several C. albicans studies have focused on important effector (611; 236; 366) cells of host immunity (210; 596) and the fungicidal molecules they release (192; 191; 305; 718). Can-

dida spp. and A. fumigatus are predominantly engulfed by macrophages and neutrophils, whose reactive oxygen species (ROS) burst is a fungicidal mechanism (421; 466). Envi-ronmental stress response studies in S. cerevisiae using hydrogen peroxide, menadione and

1.6 Sensing and adapting to environmental stress responses 44

diamide as sources of exogenous reactive oxygen species (230; 118) revealed co-ordinated expression of genes encoding oxidative stress protection proteins such as the thioredoxin TRX1 and glutaredoxin TTRI and the glutathione biosynthetic enzyme. These are largely conserved in C. albicans cells exposed to hydrogen peroxide (191) and after exposure to ploymorphonuclear leukocytes. Comparison of transcript profiles generated after exposure of C. albicans cells to 11202 and polymorponuclear leukocytes reveals conserved upregu-lation of transcripts encoding oxidative stress protection proteins: CTA1, TTRI, GLR1, TRX1, GTT, BMR, YHB2. Oxidative stress protection genes such as glutaredoxin GRX1 (125), catalase CTA1 (758), and cell surface superoxide dismutases SOD! and SOD5 (216) are also required for virulence in animal models of candidiasis.

1.6.2 Ambient pH adaptation

In addition to the specific stress imposed by the host immune system, the fungus must adapt to physicochemical constraints of the host such as the ambient pH, temperature and energy source availability. Candida spp. and A. fumigatus can adapt to a wide ambient pH range, an attribute that in part enables these fungi to survive in a diversity of habitats, ranging from compost and fruit surfaces where they are saprophytes to a wide range of anatomical sites in immunocompetent host where they are commensals. Significant insight into the molecular basis of ambient pH adaptation has come from studies of the signal transduction components and PacC transcription factor of A. nidulans (reviewed in (536; 33)), and orthologous Rim101 pathway counterparts in C. albicans (reviewed in (157)) and S. cerevisiae. Ambient pH adaptation is required for virulence in A. nidulans (66) and C. albicans but the role of Rim101/pacC in C. glabrata or A. fumigatus remains undetermined.

1.6.3 Hyperosmotic stress adaptation

Hyperosmotic stress adaptation pathways (89; 292) are particularly well characterized in S. cerevisiae and S. pombe. Although the assertion that hyperosmotic stress is imposed on pathogenic fungi in the host environment is not entirely convincing, a recent observation that Hog1p MAPK function has diverged from related model organisms and is required for virulence in C. albicans (14; 126; 192) and C. neoformans (42) makes this pathway especially interesting in the broader context of fungal virulence. The pathway architecture was deduced in S. cerevisiae with a series of genetic screens and consists of at least two signalling branches, defined by the putative osmo-sensors at the top of each cascade: the two component like Slnl phosphorelay branch (553; 575), and the mucin-like transmembrane receptor Shol branch (551; 152). A MAPKKK architecture lying at the core of each (551; 552), relays the upstream signal to the MAPKK Pbs2p (89) and ultimately the MAPK,


Hog1p (89). Osmoadaptation proceeds in part (737) by the Nmd5p (201) mediated nuclear translocation of phosphorylated Hog1p mediating the induction of glycerol biosynthetic enzymes (11; 505) and the closure of aquaglyceroporins (678; 428), resulting in the net accumulation of glycerol and decreased water potential of the cell. The role of orthlogous A. fumigatus osmosensing proteins in virulence is largely undetermined, however two genes encoding putative osmoregulatory proteins (mpkA and SHOT ) were shown to have severe growth defects in osmotic stress conditions but were not required for virulence (709; 710; 429).

1.6.4 Thermotolerance

Thermotolerance plays an important role in virulence gene expression in pathogenic bacte-ria, including Escherichia coli, Salmonella spp., Shigella spp., and Yersinia spp., where the heat shock response is an important determinant of pathogenesis, and this has long been suggested to be a key determinant of virulence in A. fumigatus, which unlike its mesophilic relatives can grow in temperatures ranging from 20°C - 60°C.To investigate the metabolic adaptation of A. fumigatus at such elevated temperatures, Nierman et al., (499) performed transcript profiling of A. fumigatus cultures at 30°C (tropical soil temperature), 37°C (mam-malian body temperature) and 48°C (compost temperature) and identified a cluster of 135 genes that were specifically induced at mammalian body temperature, including genes en-coding putative heat shock proteins and oxidative stress protection proteins. Heat shock proteins are thought to act as chaperones to maintain correct protein folding, however their role in virulence may be more complicated (521; 520), particularly in C. albicans where heat shock proteins are induced during the hyphal transition

1.6.5 The general stress response of S. cerevisiae

The concept of a co-ordinated, general stress response or environmental stress response (ESR) emerged after the effects of normally lethal doses of one stress were sublethal after pre-treatment with a sub-lethal dose of a different stress (467; 468). Furthermore, genes that were specifically thought to protect the cell against heat shock were shown to be induced under a variety of independent stressors. Analysis of the upstream promoter region of these genes identified a conserved sequence element that became known as the stress response element (STRE), which was later shown to be the consensus sequence binding site for the zinc-finger transcription factors, Msn2p and Msn4p (615; 615; 81). The ESR was originally described using microarray expression analysis in S. cerevisiae by the independent groups Gasch et al(230) and Causton et a/(118). In combination these groups investigated the transcriptional responses to a range of environmental cues including heat shock (230), ex-

1.6 Sensing and adapting to environmental stress responses 46

tremes of pH (118), oxidative stress (hydrogen peroxide (118) or menadione induced (230)), hyper-osmotic stress (NaC1 (118) or sorbitol (230; 118) induced), hypo-osmotic shock (230), amino acid starvation (230), nitrogen depletion (230), and sub-optimal carbon sources (230). Gasch et al identified a set of approximately 900 genes, whose transcript abundance was elevated or decreased across almost all of the environmental stress conditions. The set of stress decreased transcripts (600) and the set of stress-increased transcripts ('300), were remarkably similar in their average fold-change and kinetics, being almost equal and opposite across the time-course; transcripts that are co-expressed often behave with sim-ilar kinetics and this was taken as evidence that these genes were in part controlled by a common transcriptional regulation process. A remarkable feature of the ESR data is its large proportion of un-annotated transcripts (approximately 50% in the repressed set and approximately 45% in the induced set). Although common transcriptional patterns may be functionally related (716), a significant portion of the ESR remains poorly characterized. Induced ESR transcript functions spanned diverse processes, such as oxidative stress protec-tion, autophagy, protein degradation, carbohydrate metabolism and respiration; repressed ESR transcripts were predominantly involved in protein synthesis.

Since the identification of the general stress response, the model in which two tran-scription factors Msn2p and Msn2p are global regulators (615; 446) of the co-ordinated response has been refined. Although the majority (251, 88%) (230) of induced ESR genes contain the consensus binding site (STRE element CCCCT) for the "general" transcription factors Msn2p and Msn4p, they are not necessary for expression of all genes in the ESR. Indeed, several transcription factors regulate specific stresses within the broader context of the ESR. For example, Hot1p and Msnlp are required for the expression of hyperos-motic stress genes of the ESR (581). Further regulatory complexity was discovered by condition specific behaviour of transcription factor null strains in S. cerevesiae. A study (581) of msnl, msn2, msn4 and hotl transcription factor knockout strains subjected to osmotic stress and heat shock revealed that the gene encoding glycerol-3-phosphate dehy-drogenase GPD1 is predominantly dependent on Hot1p in response to osmotic shock, but was dependent on Msn2p and Msn4p in response to heat shock (581). Similar observa-tions supporting the notion of stress specific transcription factor behaviour come from heat shock studies. The heat shock proteins Hsp26p and Hsp104p are induced after a heatshock in an Hsflp dependent manner, however their induction in response to carbon starvation, osmotic shock and oxidative stress is dependent on Msn2p and Msn4p (19; 695). To fully understand the complex nature of these regulons, comprehensive time-course transcription profiling and phenotypic analysis of transcription factor null mutants and double mutants will be necessary. There is also evidence for epigenetic control of gene expression in re-sponse to stress ((641; 732; 756; 226; 579; 337)), however the relationship between these


regulatory factors, stress specific transcription factors and other pathways that regulate the ESR (e.g. the TOR (58) and PKA (227; 252; 638) pathways) is unclear. It has been proposed that this co-ordinated transcription programme is part of a conserved protective response to protect critical features of cellular physiology in budding yeast (229). How-ever, initial transcriptional studies in C. albicans did not reveal a general stress response (191) (in oxidative, hyperosmotic stress and a 23 - 27°C temperature shift) nor was any cross protection observed when cells pre-treated with a mild heat shock were subsequently exposed to oxidative stress. In a contradictory study, Smith et al., (639) did demonstrate HOG/ -dependent cross-protection to 0.3M NaC1 after pre-treatment with a sublethal dose of H202. A subsequent systematic comparison of general stress response data sets of C. albicans, S. pombe and S. cerevisiae (192) did reveal a general stress response with almost 50-fold fewer genes than in S. cerevisiae. While there is evidence for a conserved ESR in the model fungi S. cervisiae and S.pombe (229), the evidence for such a comprehensive ESR in C. albicans is unconvincing. At the start of this study, the general stress response of C. glabrata had not been investigated, and the relevance of the ESR in opportunistic fungal pathogens is still unclear.

In summary, gene deletion and transcription profiling methods have been used to iden-tify the critical components for growth, and to identify a set of genes that are adaptively regulated in response to stress conditions pertinent to the host. Eukaryotic pathogens such as the opportunistic fungi co-ordinate many processes in order to survive in the host. Fo-cusing on single gene deletion strategies alone may not uncover the complexity of these processes. For example, the virulence of A. fumigatus is increasingly regarded as a com-plex multi-factorial attribute (36), underpinned by different networks of genes that have evolved to support the organism in a variety of environments. A critical question remains: why is C. glabrata a pathogen when its close phylogenetic neighbour S. cerevisiae is rarely so? Similarly, what makes A. fumigatus such a prolific pathogen in relation to its close relatives? By understanding the co-ordination of these complex processes, rather than the role of individual genes, we may be able to better understand the molecular basis for their survival in an immunocompromised host. In order to do so, genomic data may need to be interpreted in the context of pathways and regulatory processes, a procedure that requires methods for system-wide data integration and analysis.

1.7 Identifying therapeutic targets: potential for a systems approach to fungal virulence studies

A systems approach to studying fungal virulence may overcome these limitations in the future. Although it is not a novel concept (635; 735), the confluence of post-genomic data


(424), advances in the understanding of complex networks (47; 48; 733) and a burgeoning research community (293) make it a tractable proposition in biology. The motivation of systems biology is to develop mathematical and computational techniques to predict the behaviour of a complex system (359; 648; 313; 272) which would otherwise be limited by a reductionist approach and has been the focus of much speculation and skepticism with regard to the potential value in understanding multigenic human diseases. Although the field is immature, there are signs that this approach might be of real value in the fight against complex multigenic diseases (617; 130; 610). Despite being regarded as the antithe-sis of the reductionist approach, systems biology is necessarily reductionist at this time, currently focusing on either subsets of complex pathways within a cell (755; 151; 128), or a specific organ (501) within an organ system. The distinction is not that systems biology is anti-reductionist, rather that it is integrative and simply less reductionist. By integrating information about the parts of the system, how they interact, and the kinetics of these interactions it is possible to identify emergent system-level properties - behaviours that are not explicable in terms of the components of the system alone. For example, swarming intelligence (530) is an emergent property of systems of unintelligent agents that collec-tively demonstrate intelligence (740). Individual ants are behaviorally simple insects with limited memory; collectively, however, they manage to solve complicated problems (114) such as searching for and retrieving food and the feeding of larvae by worker ants. When mycelial fungi sense carbon and nitrogen depletion, they develop hyphal projections which radiate from their established substrate towards richer nutrient sinks, eventually forming an aggregated mycelial network composed of other individual fungal cells. These mycelia aggregate behind the growing margin and fuse to form high capacity nutrient transport channels (570; 102). Although each cell within the aggregate each has individual mech-anisms for nutrient acquisition, recent studies in Phanerochaete velutina revealed highly specialized components of the mycelial aggregate that preferentially transport nitrogen to carbon rich areas of the network in an oscillatory fashion (213).

Emergent properties have also been discovered in cell signalling pathways of the cell. Bhalla and Iyenger (65) reconstructed in silico biochemical pathways using experimentally determined parameters and connected these pathways to create more complex networks composed of either two (epidermal growth factor - EGF - pathways) or four (glutamate synaptic transmission in hippocampal CAl neurons) of these discrete pathways, and demon-strated that the interconnected network possessed several properties distinct from the indi-vidual discrete pathways. For example, when two epidermal growth factor pathways (EGF mediated stimulation of MAPK 1 and MAPK2, EGF mediated activation of phospholipase C-'y) were connected with a feedback loop, the resulting system was bi-stable. Such a bi-stable system showed thresholding behaviour whereby only certain doses and exposure times


elicited sustained activation of MAPK or phospholipase C--y. In addition to this switching behaviour, bi-stable systems also demonstrated a memory property (759), whereby path-way activation can occur after the stimulus is removed. This is of particular importance to the EGF pathway, sustained activation of which is a hypothetical therapeutic strategy for cholangiocarcinoma (699; 765). bi-stable switches also direct the population level synthesis of antibiotics in Streptomyces coelicolor (the bytryolcatone regulon (458)), haematopoietic stem cell fate (393) and have a regulatory role in the cell cycle (547). Although Bhalla and Iyenger connected pathways into large networks composed of up to approximately 15 reaction members, it should be noted that the bistability can also occur in a simple reaction scheme composed of two stochastically expressed genes (30; 533).

The two best examples of integrated dynamic models in yeast to date have focused on hyperosmotic stress adaptation (361; 569) and cell cycle control (128). The Klipp-Hohmann integrative model of the response of yeast to osmotic shock (361) was constructed and pa-rameterized to fit experimental measurements of Hog1p activation, glycerol accumulation and glycerol biosynthetic enzyme activation in S. cerevisiae. Simulations from their pa-rameterized model closely matched experimental data and also provided new insight into the regulatory mechanisms of pathway activation. For example, although the Slnlp-Ypdlp phosphorelay is similar to the two-component signalling cascades of bacteria, the incorpo-ration of a second phosphorelay step provides a switch like behaviour that requires supra-threshold input to cause an effect, and a delayed activity analagous to the findings of Balla and Iyenger in their EGF pathway simulation (65). Another important prediction of the model was that sufficient glycerol accumulation can be achieved with basal glycerol pro-duction and rapid Fps1p closure (the aquaglyceroporin through which glycerol leaves the cell), which was supported by later evidence from Westfall et al., who demonstrated S. cere-visiae cells could still proliferate if Hog1p was prevented from entering the nucleus where it directs the transcription of glycerol biosynthetic enzymes (737). They also demonstrated that phosphatases do not need to be induced above a threshold in order to elicit feedback, moreover subsequent pathway activation would not be possible if phosphatase levels are induced greater than two-fold in their model. Chen et al., (128) used literature searches to create a wiring diagram of components involved in the cell cycle and represented the mech-anism as a series of algebraic and nonlinear differential equations. Using experimentally determined and fitted parameters, their model accurately predicts the phenotypic conse-quences of a panel of approximately 100 existing null mutant strains, and more importantly predicted the phenotypic effects of novel mutants. Another key feature of using integrated dynamical models is to identify inconsistencies with simulations and the consensus opinion of mechanisms. This interplay between simulation and experimentation serves to either improve parameterization or description of the model, or more fundamentally to formulate


hypotheses that form the basis of future experimentation. Although dynamic models are most desirable, the required physiological parameters are often immeasurable or unmea-sured (262). Consequently, contrary to the strict definitions of systems biology posited by some eminent researchers, other modelling techniques that may ordinarily be described as integrative genomics have been incorporated into the systems biology lexicon.

One such research field is that of network theory and its specific application to molec-ular interaction networks of model organisms (reviewed in (665). Network theory is a relatively new branch of statistical physics that extended the pioneering work of social scientists (463) and ecologists, and has advanced our understanding of how complex sys-tems are organized, how they may evolve, and how their topology is linked to system behaviour. The recent interest in network theory was sparked by a seminal paper published by Barabasi and Albert (47; 48) who overturned the tacit assumption that nodes in real networks were randomly wired together and best described by the random graphs of Eras and Renyi (193). By studying diverse "real" network data obtained for power grids, movie-collaborations and the world wide web they observed that the degree distribution of these graphs followed a power law and retained the small world property (733). These so-called scale-free networks characteristically have a majority of poorly connected nodes (i.e much lower than the average number of connections in the network), and a minority of highly connected (i.e. much greater than average number of connections in the network) nodes that are often termed hubs. Ensuing technological advances furnished the S. cerevisiae (705; 318; 232; 233; 693; 692), Caenorhabditis elegans (407) and Drosophila melanogaster (243) research communities with large-scale interaction data, whose topological character-istics have also been investigated and shown to be scale-free networks (328). In addition, metabolic networks whose nodes may represent metabolites, reactions or enzymes and whose edges connecting them represent mass flow or catalytic regulation are also scale-free (329). Scale free networks have particular topological properties that determine aspects of system function. For example, topology dependent robustness of scale-free networks has been stud-ied using vertex attack methods. In the context of scale-free networks, robustness refers to the persistence of network structural properties after sequential, random removal of nodes. Albert et al., (10) demonstrated that networks display an unexpected degree of robustness to random vertex removal, however, scale-free networks are not robust if high degree nodes are removed. Although it is clear that dynamical behaviour also governs system robustness, these in silico attack tolerance simulations have also been interpreted in the light of gene knockout data in S. cerevisiae. Genome-wide surveys of essentiality in S. cerevisiae indicate that approximately 10% of known ORFs with less than 5 connections are essential, whereas over 60% of proteins having 15 or more links are essential, i.e. targeted attack of hubs has a catastrophic effect on network robustness (328).


In addition to power law degree distributions, scale free networks have high average clus-tering coefficients (9), a measure of local connectedness in the network. The high average clustering coefficients of metabolic and protein-protein interactions reveal that they consist of subgraphs more highly connected to each other than the network as a whole, and that these subgraphs are connected to each other by hubs of high degree (764; 49). These bio-logical "modules" consist of highly interconnected and functionally related proteins (522), a property that has led some researchers to propose the function of uncharacterized pro-teins by their co-location in such modules (405; 625). Interaction networks have also been used as a scaffold for the integration of genome-wide data sets using visualization tools such as cytoscape (624; 137) and statistical methods to identify subgraphs of the network enriched with differentially expressed genes (314) and network motifs with putative regu-latory function (629). Transcriptome and deletome data have been integrated to identify cellular pathways activated in response to alkaline (31) and transition metal stresses in S. cerevisiae (333; 274). Haugen et al., (274) mapped arsenic III specific transcription profil-ing and gene deletion data to metabolic and protein interaction networks. This approach identified significant subgraphs of the protein network that were enriched with differentially expressed genes, and proteins central to that network with known regulatory function were proposed to be regulators of the arsenic III stress response (e.g. Msn2p, Msn4p, Yap1p, Hsflp). Furthermore, significant metabolic subnetworks were also identified by integrating gene deletion data with the global yeast metabolic network. In doing so they showed that genes conferring sensitivity to arsenic III belong to the shikimate and serine/threonine/ glutamate pathways lie upstream of the genes that are transcriptionally regulated. The development of the synthetic genetic array technology in the Boone and Andrews laborato-ries (693; 692) enabled a systematic global screen for synthetic global genetic interactions, which has recently been extended to approximately 75% of the genome in the DryGIN project (142). Using this technology they identified complex regulatory connections that only emerge in this unbiased genome-scale network approach. For example they identified a select subset of genes involved in cell polarity functions that appear to be regulated by the elongator and tRNA modification pathway; also they predict that the similar regula-tion of microtubule and cytoskeletal organization genes underlies a human disorder, familial dysautomia. The positive correlation between genetic interaction degree and the chemical genetic degree published by Giaever et al., (287) also revealed that hubs in the chemical genetic network are predictive of the hubs of the genetic interaction network. These data sets were integrated to propose a mechanism of action for an uncharacterized compound called erodoxin. Network analysis showed that the drug sensitivity profile of this drug most resembled the genetic interaction profile of ERO1 which encodes an enzyme participating in disulfide bond formation into its oxidized state.


The use of S. cerevisiae as a model organism has been described above, and has princi-pally focused on orthologous expression profiles and deletion phenotypes to elucidate diverse cellular processes. As S. cerevisiae enters an integrative systems based era, it is important to assess the degree to which a model organism can be used as a framework for a systems approach in a related organism that lacks fully described interaction data and functional genomics screens. In this thesis, parallel gene deletion assays and transcriptional profil-ing were conducted and integrated with a protein-protein interaction network inferred by othology to S. cerevisiae. This was performed in an attempt to identify significant subnet-works and to formulate hypotheses on the nature of environmental stress adaptation in C. glabrata. Specific aims are listed below.


1.8 Specific aims addressed in this thesis

1. Recent studies in the non-pathogenic models yeasts and C. albicans have provided evidence that signalling pathways, transcription factors and transcriptional responses to virulence related environment cues have significantly diverged in C. albicans. The environmental stress response of a yeast with higher innate drug resistance and an increasing cause of invasive candidiasis, C. glabrata is largely unexplored at the tran-scriptional level. Furthermore, to date, no studies have been conducted to investigate the contribution of stress signalling pathway components to these transcriptional re-sponses. In Chapter 3 I aimed to:

(a) Identify whether HOG1 is required for growth in hyperosmotic stress conditions by constructing a hogl strain and assessing its growth in hyperosmotic stress medium relative to rich culture medium.

(b) Assess how Hog1p functions under hyperosmotic stress conditions with respect to its phosphorylation kinetics and determine if any observed phosphorylation depends on upstream MAPKKK architecture conserved with related fungi.

(c) Further assess any divergence in fungal hyperosmotic stress adaptation by assay-ing the transcript profile of wild-type C. glabrata cells subjected to hyperosmotic stress and comparing it with published data for S. cerevisiae and C. albicans.

(d) Assess how divergent HOG1-dependent transcriptional responses are by further comparing the transcript profile of hogl cells subjected to hyperosmotic stress, with equivalent data in S. cerevisiae and C. albicans.

(e) Identify a candidate regulatory network by mapping regulated genes to a tran-scriptional regulatory network inferred from S. cerevisiae and use this to identify candidate regulators of hyperosmotic stress induced transcription.

2. Investigating the transcriptional responses to environmental cues in vitro is an ap-proximation of the host-niche, and is useful in Candida spp. where fungal material is virtually impossible to recover post-infection. In contrast, an immunocompromised mouse model of invasive aspergillosis does permit the re-capture of germlings in the early stages of infection. In Chapter 4, I report on joint work with Natalie Federova and Elaine Bignell. In this section we aimed to:

(a) Optimize the time-course for infection of mice immunocompetent mice with A. fumigates Af293 such that fungal germlings are simultaneously easy to recover and also in growth phase of relevance to infection.

1.8 Specific aims addressed in this thesis 54

(b) Employ bioinformatic and molecular strategies for global profiling of A. fumigatus gene expression in germlings rescued directly from the murine lung, a tool which will empower the analysis of virulence in this pathogen

(c) Identify fungal attributes preferentially employed during adaptation to the host niche, and thus contributing to the virulence of A. fumigatus

(d) Compare the transcriptomes of developmentally matched A. fumigatus isolates following laboratory culture or initiation of infection in the neutropenic murine lung.

(e) Compare host-niche regulated genes with data generated in vitro to identify environmental cues relevant to the host niche

(f) Assess the lineage specificity of genes preferentially expressed in the host-niche, which might provide evolutionary clues to the unique success of A. fumigatus as a pathogen in comparison to closely related Aspergilli.

3. The groundbreaking sequencing and annotation of the S. cerevisiae genome some thir-teen years ago (246) facilitated a new era of yeast research in which reverse genetics approaches became the catalyst for a burgeoning supply of genome-wide phenotypic data. Although the S. cerevisiae knockout libraries have been informative in elucidat-ing the molecular nature of conserved processes, a remaining challenge is to establish a comprehensive, genome-wide C. glabrata deletion mutants akin to that developed by the Saccharomyces genome deletion project. In chapter 5 I aimed to:

(a) Report on initial progress in creating a prototypic targeted disruption library in C. glabrata

(b) Provide a quantitative measure of C. glabrata fitness when exposed to fluconazole with a view to comparing equivalent null strains (lacking a single orthologous gene) in S. cerevisiae

55

Chapter 2

Materials and Methods

2.1 Experimental Methods

All chemicals used in this study were purchased from Sigma (UK) or Fisher Scientific (UK), unless otherwise stated. All culture media constituents were purchased from Oxoid Ltd (Hampshire, UK), BDH (Poole, UK) or Formedium (Norwich, UK). Unless otherwise

stated, all buffers and solutions were prepared according to standard recipes (603).

2.1.1 Fungal strains and culture media

Saccharomyces cerevisiae and Candida glabrata were routinely cultured in synthetic com-

plete (SC, Supplementary Table B.1) or yeast extract peptone dextrose (YEPD, Supple-mentary Table B.2) broths and equivalent solid media. Solid or liquid S. cerevisiae and C. glabrata cultures were incubated in either a static or an orbital shaking (180 rpm) incubator

(New Brunswick Scientific, USA) at 30°C or 37°C respectively.

All S. cerevisiae strains used in this study come from the Yeast Knockout (YKO) Mata

collection (749) (Open Bio Systems, USA). These strains, derived from BY4741, were cre-ated by homologous recombination with a kanamycin (KanMX) antibiotic resistance cas-sette. Accordingly, cultures of these strains were supplemented with geneticin G418 (200

µg/mL) to maintain selection of the correct genetic background. A. fumigates were routinely cultured at 37°C in supplemented Aspergillus Minimal

Media (AMM) (NaNO3, 6.0 g/L; KC1, 0.52g/L; KH2PO4, 1.52 g/L; MgSO4 . 7H20, 0.52

g/L; and 1 mL of trace element solution per litre) or Aspergillus Complete Media (ACM) (2.5% glucose (w/v), 0.5% yeast extract (w/v), 1.5% agar (w/v)) according to Pontecorvo

et al. (548) containing 1% (w/v) glucose as carbon source and 5 mM ammonium ± tartrate as nitrogen source. Non-standard Aspergillus culture media are explained in the relevant

results section (Chapter 4).

2.1 Experimental Methods 56

2.1.2 Bacterial strains and culture media

XL-10 Escherichia coli competent cells were cultured in Luria Bertani (LB) medium, 2% (w/v) GibcoBRL LB broth base (Life Technologies, UK). Liquid cultures were routinely incubated at 37°C, 150 rpm. Solid medium was produced by adding 2% (w/v) agar prior to autoclaving. When required, ampicillin was added to a final concentration of 100 pg/mL) after autoclaving and once the media had cooled to below 55°C.

2.1.3 Assays to identify altered growth phenotypes of mutant C. glabrata and S. cerevisiae strains

2.1.3.1 Solid agar assays of C. glabrata and S.cerevisiae strain growth

YKO library strains, individually stored as glycerol stocks in 96 well microtitre plates, were thawed close to a Bunsen flame and agitated in situ on a bench-top orbital plate-shaker (10 min, 10 rpm).

Stocks were aspirated from library plates using a multi-channel pipette and dispensed into new, sterile 96 well microtitre plates containing YEPD (final density 1 x 106 cells / ml) and incubated until the cells reached stationary phase (16 hours, 30°C, 180 rpm). In parallel, an equivalent cell density 5 mL culture of BY4741, the library parental strain, was created in a separate Sterilin universal tube.

Cells were harvested from sub-cultured microtitre plates with a centrifuge equipped with plate-adpators (Wolf Labs, UK, 3000 rpm, 5 min). The supernatant was discarded by aspiration with a multi-channel pipette, leaving cell pellets of YKO library strains, which were then washed twice with 100 pl sterile ddH2O using the harvesting steps described above.

Cell density was estimated for 5-7 wells using a microscope and a haemocytometer (Neubauer, USA). The average cell density was taken for these wells and used to calculate a global dilution factor for daughter plates containing wells seeded at a density of 106 cells / mL. The parental strain, at the same cell density, was added to an empty well on this plate.

Cells were then transferred from these working stock plates to pre-poured YEPD or SC solid agar (+ environmental stress) in 50 mL Petri dishes using a 96-pronged multi-replicator tool.

Inoculated plates were covered with lids and allowed to dry for one hour on the bench before inversion and incubation in a static incubator (30°C, 48-72 hours).

Colony growth of null mutant strains was qualitatively scored (either no growth or sen-sitive) in relation to the co-inoculated parental strain in YEPD and YEPD ± environmental stress.

Chapter 2. Materials and Methods 57

A conditional growth phenotype was defined for strains showing growth equivalent to the parental strain in YEPD but impaired growth in agar supplemented with an environmental stress on which the parental strain growth was unaffected.

2.1.3.2 Miniaturized optical density assays of mutant Candida glabrata strain growth

Glycerol stocks of each library strain (either the C. glabrata transcription factor knockout library CGTFKOLib or the equivalent null strains in the YKO library), collectively main-tained in two 96-well microtitre plates at -80°C, were sampled (5 itl) using a multi-channel pipette and dispensed into corresponding sterile, round-bottomed 96-well plate containing 150 it of YEPD broth. Library strains were sub-cultured to stationary phase for 18 hours in an orbital shaker (30°C, 180 RPM) before commencing the growth curve assay.

Sub-cultured cells were harvested (3000rpm, 5 mins) in a centrifuge rotor equipped with plate adaptors (Wolf Laboratories, UK) and washed three times with sterile, distilled water. Washed cells from each library plate were transferred (final OD600=0.1) into microtitre plates containing YEPD ± environmental stress. Plates were sealed with a breathable membrane and incubated for 12 hours. Optical density (0D600) measurements were bi-hourly recorded on a Multiskan Ascent absorbance plate-reader (Thermo Scientific, U.S.A).

Time course optical density (0D600) data, used as a measure of cell population dynamics, were fitted to a three parameter (slope, midpoint and upper asymptote) logistic function of growth using non-linear regression (SSLogis function) in the R statistical computing framework (562). The logistic function of growth adopted in the analysis is shown in equation 2.1:

f (x, (b, d, e)) = (2.1) 1 + exp{ b (log(x) — log(e))}

In this model, logistic growth is a function of time x, the maximum cell density d (upper asymtote), the midpoint of the growth e and the slope around this midpoint b. Parameters d, e and b are quantitative phenotypic traits of each strain and are estimated from samples of cell density at x = 0 — 11.5 hours.

Strains showing significant, environmental stress specific growth defects were identified in null strains either by a) a complete lack of logistic growth in the presence of environmen-tal stress but not YEPD (hypersensitive) or b) significantly greater midpoint (p < 0.05) estimates than wild-type strains in fluconazole but not YEPD (sensitive).

2.1.4 Oligonucleotide primer synthesis

All primers used in this study were purchased from Sigma-Genosys (Dorset, UK) and are listed in the relevant Appendices. See relevant chapters featuring molecular work.


2.1.5 Polymerase Chain Reaction (PCR)

Colony PCR cycling and reaction parameters, used for verifying the identity of yeast knock-out (YKO) strains, were adapted from published protocols (601). Genomic DNA template was obtained by adding a prior high-temperature lysis step (100°C, 15 minutes) to single colony picks of the knockout strain in the presence of oligonucleotide primers and reaction buffer; the reaction was initialized by addition of 0.5U of BioTaq polymerase (BioLine, UK). All reactions were performed in a thermocycler (Thermofisher, UK), in a total volume of 50 1d. YKO strain genotypes were validated using previously described primers, designed specifically for each null mutant (consult the original paper, (749) and the accompanying websitel). PCR products were inspected using agarose gel electrophoresis and a 1kb ladder (New England Biolabs, USA).

2.1.6 Ribonucleic acid (RNA) extraction and quantification

S. cerevisiae and C. glabrata cells (0D600 = 0.4 — 0.8) were harvested with a benchtop centrifuge (3000 rpm, 5 min) and washed twice in sterile distilled water. Fungal total RNA was extracted by mechanical disruption of cells (FastPrep and acid washed glass beads, BI0101, US) followed by purification with RNEasy mini-kit columns and reagents, incorporating an in situ DNase digestion step (Qiagen, UK). Quantification of samples (0 —3.7 pg/mL) was performed by measuring A260 on a nanodrop ND-1000 spectrophotometer (Labtech, US). A2601280 and A260/230 ratios were used as indicators of protein and salt contamination respectively. Samples were retained if these ratios where in the 1.8 — 2.5 and 2.0 — 2.5 ranges respectively.

RNA sample quality was assessed with the bioanalyzer nano series microfluidics platform (Agilent, UK); quanitification of 26S and 18S peaks was performed using the Eukaryotic RNA assay protocol within the Agilent Expert software (version B.02.02). Samples showing distinguishable 26S and 18S bands on the synthetic electropherogram, and 26S:18S peak height ratios > 1.7 were retained for use as templates in reverse transcription and fluorophore labelling reactions.

2.1.7 Linear DNA and PCR extraction and purification

Linear DNA and PCR fragments were routinely purified from 1% (w/v) agarose gels or from solution using a QlAquickTM (QIAGen LTD, UK) column according to the manufacturer's protocol.

Ihttp://www-sequence.stanford.edu/group/yeast_deletion_project/protocols.html


2.1.8 Agarose gel electrophoresis

Samples of DNA were routinely separated on 1% (w/v) agarose made with TBE (Tris-Borate: 4 mM EDTA, 8.9 mM TriZMATm base), containing a 1:10000 dilution of SYBR Safe DNA gel stain (Invitrogen, USA). Gels were inspected under a Gel Dock transilluminator (Alpha Innotech, USA).

2.1.9 Yeast DNA extraction

DNA extractions were performed using the MasterPure Yeast DNA Purification Kit (Epi-centre Biotechnologies, Madison, USA) according to manufacturer's instructions.

2.1.10 Transformation of yeast cells with electroporation

Yeast cells were transformed using a modification of the electroporation protocol (39). Briefly, 2.5 mL of a saturated overnight culture was transferred into 50 mL pre-warmed YEPD and incubated for a further 4 h at 37°C (C. glabrata) or 30°C (S. cerevisiae), 180 rpm. Cells were harvested by centrifugation, washed in sterile water and resuspended in 8 mL dd H20, 1 mL 10 x TE (10 mM Tris-HC1 (pH 8), 1 EDTA (pH 8)) and 1 mL LiAc (1 M) and incubated at 30°C for 30 mM at 130 rpm. Then 250 µL dithiotheitol DTT (1 M) was added and incubated for a further 60 min at 30°C at 130 rpm, 40 mL of dd H2O added and the cells harvested by centrifugation.

All subsequent procedures were performed at 4°C. The cells were resuspended in 25 mL ice-cold dd H20, harvested by centrifugation, washed in 5 mL sorbitol (1 M) and resuspended in 550 ,uL sorbitol (1 M). The competent cells (45 ,u,L per reaction) were transferred to pre-chilled electroporation cuvettes and 100 ng DNA added, mixed and left at 4°C for 10 mM. The cuvettes were pulsed at 1.5 kV, 25 pF, 200 ft The cells were recovered by gentle mixing with 950 it L YEPD and incubated for 4 h at 30°C and plated onto YEPD plates containing 200 pg/mL nourseothicin and incubated for 2-4 days at 30°C or 37°C

2.1.11 Western blots

To prepare samples for the phosphorylation assay, C. glabrata ATCC 2001 cells were cul-tured in 100 mL of YEPD (37°C, 180 rpm) to OD600= 0.55, at which time 25 mL samples were collected and diluted either in 25 mL YEPD or YEPD supplemented with sorbitol (1 M final concentration) to provide the hyperosmotic stress. Cultures were incubated for a further 15 mM prior to harvesting by centrifugation (2000 rpm, 2 min, 4°C). The super-natant was discarded, pellets were retained and snap-frozen in liquid nitrogen before storage at -80°C. Phosphorylation assays were performed as described previously in Candida albi-


cans using an anti-phospho-p38 MAPK (Thr180/Tyr182) antibody (9211S, New England Biolabs, USA).

2.1.11.1 Design of probes

S. cerevisiae microarray oligonucleotides were developed by Agilent using oligonucleotides designed in (134).

C. glabratra microarray oligonucleotides used in the Agilent platform were designed by Butler laboratory at University College Dublin, Eire and produced by custom production spotting 5908 69- or 70-mer oligonucleotides synthesized replicated in quadruplicate for each of the 4x 44k arrays on a slide. A. fumigatus microarray oligonucleotides were developed by TIGR, now the JCVI (499).

2.1.11.2 Microarray slides

The Agilent 4x 44k microarray platform was adopted for environmental stress studies in C. glabrata (AMADID 015761) and S. cerevisiae .

COGEME consortium microarrays were used for the hyperosmotic stress transcript profiling of ATCC 2001 and stell and hogl strains (Chapter 3)

All A. fumigatus transcription profiling experiments used Af293 DNA amplicon mi-croarrays (499).

2.1.12 Reverse transcription and fluorescent labelling of cDNA

For environmental stress experiments in S. cerevisiae and C. glabrata reverse transcription and fluorophore labelling was performed with the ChipShotTM direct labelling and clean-up system (Promega, UK) using a thermocycler for all incubation steps. Priming of mRNA within the total RNA population was achieved by incubating RNA template with oligo dT and random primers (65°C, 10 minutes) before rapid chilling to allow primer annealing (ice, 5 minutes) and subsequent addition of reverse transcription reaction components to the primed mix (final volume 40 pl).

Final reaction concentrations were as follows: total RNA template (1.25 pg p1-1), ran-dom primers (75 pg p1-1), oligo dT primer (50 pg p1-1), ChipShotTM reaction buffer (1 x), MgC12 (12.5 mM), dNTP mix (12.5 mM), Cy3Thor Cy5TM-dCTP (25 pg p1-1) and ChipShot'M reverse transcriptase. Reaction mixtures were briefly mixed with a vortex, spun in a micro-centrifuge (13000 rpm, 1 minute) and pre-incubated in a dark room (20-25°C, 30 minutes) before transfer to the thermocycler (42°C, 2 hours). Thereafter, cDNA synthesis reactions were terminated by incubation of samples with RNaseH (1 pl, 37°C, 15 minutes). The re-action was neutralized by adding 4 pl of sodium acetate (5 M). Fluorophore labelled cDNA


samples were added to ChipShotTM binding buffer and transferred to ChipShotTM columns for a cycle of three washes (500 pi, 80% (w/v) ethanol, 13000 rpm, 1 minute). Samples were eluted in 600 of elution buffer and quantified using the microarray procedure on the nanodrop ND-1000. Acceptable sample yield ranges for Cy3TM and Cy5TM labelled cDNA were 1.2 - 2.4 pg and 1.0 - 2.4 pg respectively; associated acceptable yields of incorporated dye were 100 - 225 and 50 - 160 pmol respectively.

2.1.12.1 Hybridization, washing and scanning of cDNA microarrays

2.1.12.2 Image acquisition and data analysis

For C. glabrata and S. cerevisiae (both agilent and COGEME arrays), intensities of hy-bridized probes were oriented and measured with a GenePix 4000b semiconfocal microarray scanner and GenePix version 6.0 software (Axon Instruments, USA). Scanning parameters were adjusted for the high density spotting format of the agilent slide. Pixel size was set to 5,um with 3 lines used to average the spot intensity. The photomultiplier tube (PMT) voltage settings for each channel (532 nm and 635 nm) were adjusted for each scan to give an overall intensity ratio across each slide of 1, a background intensity at 532 nm of approximately 100, and a maximal intensity close to saturation (2 x 1016) for a 16-bit TIFF image. The GenePix proprietary spotfinding algorithm was used in combination with the "gal" meta-data file to map intensity values to oligonucleotides on the array that represent ORF in either the S. cerevisiae or C. glabrata genome. Image files were ex-ported as GenePix results files (GPR) which were used in subsequent statistical analyses. Hybridised A. fumigatus slides were scanned using the Axon GenePix 4000B microarray scanner and the TIFF images generated were oriented and analyzed using TIGR Spotfinder (http : //www t igr org/ sof twar e /) to identify poor quality spots.

2.1.13 Statistical analysis of microarray data

All data processing and statistical analyses were performed using the R statistical frame-work (562) for scientific computing, bioconductor (643) and the limma package (644) for linear modelling of microarray data. Detailed descriptions of exploratory and design specific analysis can be found in the relevant chapters. Briefly, poor quality spots were filtered as previously described and systematic spatial effects were normalized within arrays using the print-tip lowess method and quantile between array normalization encoded in (644).

Least squares regression was used to fit the average log2 intensity ratios, residuals and the p values, corrected for multiple testing using empirical Bayes methods (644) or Benjamini-Hochberg correction. Non standard pre-processing steps, such as single channel normal-ization or estimation of amplification effects are detailed in the relevant chapters (Chapter

k-Clique Community Discovery Algorithm

5 1

°

1 1 0 0

1 1 0 0

O 0 0 0

O 0 0 0

0 0 0

5

1 0

O 0

O 0

O 0

1 0

a 0 0

5 3 2 1 3 1

4 2 1 1 1

2 2 3 2 1 2

Z 3 0 1

I 1, 0 1 2

1 1 2 1 2 4

clique clique overlap matrix •

(b) Outcome of 4 -clique percolation (a) Input network

2.2 Computational Methods 62

3 and 4). Least squares regression and linear modelling for microarrays are described in detail (642; 643; 644)

2.2 Computational Methods

2.2.1 Computation of network statistics

Graphical representation of networks and their statistical computation was performed using the nodebox and networkx toolboxes developed for python (263; 717). Cytoscape was also used for graphical representation of networks (624; 137).

2.2.2 k-clique percolation and community finding

The majority of community finding algorithms detect standard partitions of a network. The problem of graph partitioning concerns dividing the nodes into discrete groups of predefined size, such that the number of edges lying between them is minimal. Most of these methods prohibit multiple community membership for a single node, however in real graphs, single nodes are often shared between communities, and the problem of detecting overlapping communities has recently been addressed by Palla and colleagues (522) (Figure 2.1).

Figure 2.1: Overview of k-clique percolation algorithm adapted from (522)

63

Chapter 3

The Hogip and Stellp dependent transcriptional effects of hyperosmotic stress in Candida glabrata

3.1 Chapter Overview

Yeasts, devoid of any behavioural homeostatic facility, have evolved co-ordinated signalling pathways to adapt to the fluctuating physicochemical nature of their habitat. Several sens-ing and adaptive mechanisms have been discovered to date ((538; 368; 554; 50; 190; 315; 480; 261), however, the high osmolarity glycerol response (HOG) pathway (89; 292), and its role in the adaptation of S. cerevisiae to hyperosmotic stress in particular, is one of the best characterized. This model pathway forms the basis for studying hyperosmotic stress adaptation in related pathogenic fungi where homologous mitogen activated protein kinases coupled to stress adaptation are referred to as stress activated protein kinase (SAPK) cas-cades (192). A hyperosmotic stress is imparted on a cell when the external water potential of the culture medium is less than the sum of the water potential of the soluble component of the cell and the turgor pressure imparted by the mechanical structures bounding the cell, leading to a net outflow of water from the cell until equilibrium is reached (443).

The HOG pathway modulates the rate at which equilibrium is reached by inducing cellular changes leading to the accumulation of intracellular glycerol (12; 392), a compat-ible solute that serves to decrease intracellular water potential and therefore the rate of water outflow. This is achieved by increasing the metabolic flux through glycerol biosyn-thetic pathways, and also by closing aquaglyceroporins (678; 428; 473) located in the cell

Hyperobrpotic Signal

A #= no C. glabrata ortholog

= truncated in C.giabrata

PTP2 PTP3

Glycerate-3-phosphate D-glycerate

10

ALD4. ALD5, ALD6

04

B

3.1 Chapter Overview 64

membrane that permit glycerol efflux. This exemplar hyperosmotic adaptation pathway

architecture was deduced with a series of genetic screens and consists of at least two func-tionally redundant signalling branches, defined by the putative osmo-sensors at the top of

each cascade: the two component like Slnl phosphorelay branch (553; 575), and the mucin-like transmembrane receptor Shol branch (551; 152). A MAPKKK architecture lying at the core of each (551; 552), relays the upstream signal to the MAPKK Pbs2p (89) and ultimately the MAPK, Hog1p (89) - a major effector of hyperosmotic stress adaptation in

S. cerevisiae (Figure 3.1).

DAK1, DAK2

GRP

l'OD-glyceraldehyde

•,.0 Glycerol 0.4 04

Glycerone phosphate Glycerine

HOF72, RriN ' II &Hi

GPD1, Gun' 0

II.Nn-glycerot-3-phosphate

Adapted from Kegg pathway. Kaneisha laboratories (http://www.genome.jp/kegg/).

Figure 3.1: Architecture of the HOG pathway in S. cerevisiae. See text for descrip-tions.

• . • Several Factors

(71'

?

0 O!00

0 0 MAPKKK

0 O MAPKK O MAPKK

Chapter 3. The Hog1p and Stellp dependent transcriptional effects of hyperosmotic stress in Candida glabrata 65

Mating Morphological Cell Wall Osmoregulation Cell Wall Sporulation Response Switch Integrity Construction Response

Figure 3.2: Architecture of MAPK cascades of S. cerevisiae, incuding the HOG osmoadaptation cascade. Adapted from (292). Orange circles denote putative pro-teins embedded in the plasma membrane, connected to the upstream G-protein (Stel8p, Ste4p, Ras2p, Rholp, Cdc24p, Cdc42p, Gpalp, Rom2p) or phosphorelay (Ypdlp, Ssklp) control components (black circles). Kinases (either p21 activated kinases Ste2Op or serine/ threonine kinases Pkclp, Spslp), depicted by grey circles, lie upstream of the core MAP kinase architecure (IVIAPKKK (Ste11p, Ssk2p, Ssk22p, Bcklp) MAPKK (Ste7p, Pbs2p, Mkklp, Mkk2p) —p MAPK (Fus3p, Ksslp, Hoglp, Slt2p, Smklp)) whose activation re-sults in recruitment of transcription factors (Stel2p, Rlmlp) which mediate transcriptional changes in response to the detected signals. MAPKKK —÷ MAPK components are depicted as blue circles colour-graded in relation to their hierarchy; the darkest hue representing a MAPKKK and the lightest blue representing a transcription factor activated by the ap-propriate signal cascade. Coherence of osmoadaptation signalling is maintained by the Hoglp MAPK, which prevents cross-talk with the mating response pathway (516). Genes disrupted in this study are depicted by shadowed circles with red boundaries. Unknown inputs, regulators or transcriptional events are denoted by a question mark.


The HOG pathway comprises at least two signalling branches that transduce a signal detected in the plasma membrane by putative osmosensors (Figure 3.1 A orange circles) to the upstream G-protein related proteins (Cdc24p, Cdc42p) or phosphorelay (Ypdlp, Ssklp) control components (Figure 3.1 A black circles). These upstream regulatory components transduce the signal to a MAPKKK (Figure 3.1 A dark blue circles , Ssk2, Ste11) —> MAPKK (sky blue circles, Pbs2p) —> MAPK (Figure 3.1 A cyan circles, Hog1p) signalling cascade, in the case of the Slnl branch this is via p21-activated serine-threonine kinase (Ste20p, Figure 3.1 A grey circle). The Slnl branch (435; 531), similar to the canonical two-component-like signalling cascades found in prokaryotes and other fungi (reviewed in (662)), functions as a negative regulator of the HOG pathway and comprises the Sln1p histidine sensor-kinase, the Ypdlp phosphorelay protein (553) and the Ssklp response regulator (553). The autophosphorylation of Sln1p is modulated by external water potential, with the greatest level of phosphorylation occurring in hypotonic culture media (553; 552). Switching cells to a hypertonic environment (denoted by the flat arrow head and negative symbol in Figure 3.1 A) results in a relative dephosphorylation of Sln1p and Ypdlp, and ultimately the Ssklp response regulator. In its dephosphorylated form, Ssklp acts as a positive regulator of Ssk2p/Ssk22p activity, and also the downstream MAPKKs (Pbs2p) and MAPK (Hog1p) (435).

In the second branch, the Sholp transmembrane protein contains an intracellular Src homology 3 (SH3) domain, which binds to a proline-rich region of the Pbs2p MAPKK (435) which is activated in response to hypertonicity (denoted by the arrow head and positive symbol in Figure 3.1 A). Sho1p recruits Pbs2p to the cell membrane following osmotic stress, and both Sholp and Pbs2p bind to the MAPKKK Sten') (684). Sten') is acti-vated by phosphorylation which requires the Rho-like GTPase Cdc42p, the p21-activated serine/threonine protein kinase Ste20p, and the adaptor protein Ste5Op (715; 564) (scaf-fold denoted by grey oval shape encompassing these proteins in Figure 3.1 A). A third osmosensing branch requires the Msb2p protein, which has a single membrane-spanning do-main and a large predicted extracellular domain; this sensing branch is partially redundant with Sholp (517). Once activated, the phosphorylated Hog1p translocates to the nucleus in an Nmd5p mediated fashion (201), where it promotes adaptive transcriptional changes. Hog1p controls the transcriptional response to hyperosmotic stress by activating members of distinct transcription factor families that in turn drive transcriptional responses to hyper-osmotic stress in S. cerevisiae: Skolp (557; 558), Hot1p (13; 578), Msnlp, Msn2p/Msn4p (581), Sgdlp (410) and Gen4p (532) (not shown). Nuclear Hog1p mediates the transcrip-tion of key transcripts highlighted in this figure including glycerol-3-phosphate dehydro-genase (GPD1,GPP2) (reaction catalyzed shown in Figure 3.1 B), and phosphotyrosine phosphatases (Figure 3.1 green circles, PTP2, PTP3) which provide negative feedback by


dephosphorylating Hog1p (449; 757; 448). Type two phosphotyrosine proteases (Figure 3.1 green circles PTC1, PTC2) also dephosphorylate Hog1p (729; 767). During the course of this work, Gregori et al., showed that the C. glabrata ATCC 2001 strain contains a prema-ture stop codon in the SSK2 gene that leads to a dysfunctional Slnl signalling branch (255) - denoted by the * (Figure 3.1 A) adjacent to the Ssk2p component of the Shol branch. C. glabrata also lacks an Ssk22 homolog (denoted by the # in Figure 3.1 A) - a functionally redundant paralogue of the Ssk2p MAPKKK.

The HOG pathway and adjacent MAPK signalling cascades contain common functional elements both in the upper parts of the regulatory hierarchy, and in the downstream mod-ules that regulate the phosphorylation signal on MAPK proteins (Figure 3.2). Hoglp's position within the signalling cascades means that it not only plays an important role in signal transduction for hyperosmotic stress adaptation, but also maintains the coherence of this signal, namely the correct relay to the downstream effectors of osmotic stress adap-tation and not to the morphogenesis, cell integrity or mating pathways (516). O'Rourke and Herskowitz demonstrated that a hogl mutant subjected to an osmotic upshift aber-rantly activates the pheromone response pathway as evidenced by activation of a FUS1::lacZ reporter, morphological changes, and mating in ste4 and ste5 mutants. This cross-talk re-quired the osmosensor Sho1p, as well as Ste20p, Ste50p, the pheromone response MAPK cascade (Stellp, Ste7p, and Fus3p or Ksslp), and Stel2p but not Ste4p or the MAPK scaf-fold protein, Ste5p(516). More recently, Shock et al., demonstrated that the Hog1p MAPK interrupts signal transduction between the Ksslp MAPK and the Tecip transcription fac-tor to maintain pathway specificity between the osmoadaptation, mating and filamentous growth pathways (633). The transcriptional responses to hyperosmotic stress have been studied extensively in S. cerevisiae, both in broader investigations of the environmental stress response (ESR) (230; 118), and also in more focused studies of hyperosmotic stress imparted by high salinity (550) or high exogenous concentrations of an impermeable solute such as sorbitol (581; 230).

These pathways have taken on new importance in the context of related, pathogenic fungi; deletion of MAPKs in some human pathogens significantly attenuates virulence (14; 42), whilst such pathways are dispensable for virulence in a number of plant pathogens. For example, in C. albicans it has been demonstrated that the Hog1p MAPK is activated in response to diverse stimuli, many of which are likely to be encountered in the host, such as: oxidative stress (15), osmotic stress, the morphogenic agent serum, and the quorum-sensing molecule farnesol (639). The SAPK network in this pathogen has diverged from corresponding networks in the non-pathogenic model yeasts S. cerevisiae and Schizosac-charomyces pombe, which suggests that specialised, niche-specific, Hoglp-mediated stress responses have evolved in this pathogenic fungus (639). A mathematical modelling approach


has been used in conjunction with single-cell measures of GFP-promoter fusion strains (452) to investigate how signal specificity is maintained in MAPK/SAPK signalling cascades. Mc-Clean et al., showed that the hyperosmotic stress and pheromone response pathways which share a common MAPKKK (Ste11p) achieve specificity by mutual inhibition rather than insulation and that this mutual inhibition must occur downstream of the components com-mon to both pathways; Zou et al., later used a modelling approach to provide evidence that Pbs2p and Hog1p are essential for maintenance of this specificity (774). SAPKs are critical for survival of some opportunistic fungi in the host, as C. albicans cells lacking HOG1 display significantly attenuated virulence in a mouse model of systemic candidiasis (14). Similarly, a recent study in C. neoformans revealed that the Hog1p SAPK is essential for virulence in the highly pathogenic serotype A Glade, and, significantly, uncovered clear differences in the mechanisms underlying Hog1p regulation in highly virulent and less viru-lent serotypes (42). Collectively, these studies demonstrate: (i) that stress responses are an essential virulence attribute of human pathogenic fungi and (ii) the importance of SAPK pathways in these stress responses.

The role of C. glabrata SAPK pathways has been partially investigated in our labora-tory by Ana-Maria Calcagno-Pizarelli, who identified C. glabrata homologues encoding the S. cerevisiae upstream p21- activated serine/threonine kinase Ste2Op (103), the MAPKKK Stel1p (104) and the transcription factor Stel2p (105). Cloning, disruption and functional complementation studies were used to investigate the role of Ste2Op in hyperosmotic stress adaptation, filamentation and mating, cell wall integrity and in a mouse model of candidia-sis. S. cerevisiae ura3/ura3 ste20/ste20 cells were transformed with the pAMC86 plasmid bearing the C. glabrata STE20 gene and a URA3 marker, with subsequent selection of uracil prototrophs. These colonies were selected and tested in mating and filamentation defect assays which revealed that C. glabrata STE20 complements the mating and filamen-tation defects of S. cerevisiae ste20 strains. Although C. glabrata Ste2Op is required for growth on solid hypertonic growth medium (containing either 1M NaC1 or 1.5 M sorbitol) and agar supplemented with calcofluor white (lmg/ml), it is not required for nitrogen star-vation induced filamentation. Moreover, in a mouse model of candidiasis, ste20 strains are partially attenuated for virulence in comparison to reconstituted ste20:STE20 strains (103). Similar cloning, complementation and disruption studies were also performed for the C. glabrata STE11 gene encoding a MAPKKK: the mating and filamentation defects of a S. cerevisiae strain are complemented following transformation of a plasmid bearing the C. glabrata STEN gene. Cell wall integrity and hyperosmotic stress adaptation as-says were also carried out for the C. glabrata stel 1 strain and revealed that this gene is required for adaptation to hyperosmotic stress but is largely dispensable for maintenance of cell wall integrity (104). Survival analysis conducted in the murine model of candidiasis

Chapter 3. The Hogip and Stellp dependent transcriptional effects of hyperosmotic stress in Candida glabrata 69

demonstrated that sta./ mutants are also partially attenuated for virulence in comparison to the re-constituted stell:STE11 strain (104). These results suggested that although the components of SAPK pathways are conserved, they play different roles in the biology of each species. In this section we aim to expand our investigation into the divergence of this pathway by studying the role of an homologous MAPK (putative SAPK) in the re-lated pathogenic fungus C. glabrata. I present an investigation into the putative role of the homologous C. glabrata Hog1p as a regulator of hyperosmotic stress adaptation, although there are plans to assess the role of Hog1p in a variety of environmental stress conditions. In collaboration with Janet Quinn (University of Newcastle), we had the following aims:

1 Identify whether HOG1 is required for growth in hyperosmotic stress conditions by constructing a hog1 strain and assessing its growth in hyperosmotic stress medium relative to rich culture medium.

2. Assess how Hog1p functions under hyperosmotic stress conditions with respect to its phosphorylation kinetics and determine if any observed phosphorylation depends on upstream MAPKKK architecture conserved with related fungi.

3. Further assess any divergence in fungal hyperosmotic stress adaptation by assaying the transcript profile of wild-type C. glabrata cells subjected to hyperosmotic stress and comparing it with published data for S. cerevisiae and C. albicans.

4. Assess how divergent HOG1-dependent transcriptional responses are by further com-paring the transcript profile of hog1 cells subjected to hyperosmotic stress, with equiv-alent data in S. cerevisiae and C. albicans.

5. Identify a hypothetical hyperosmotic stress regulation network by mapping regulated transcripts to a hypothetical osmostress regulation network inferred from S. cerevisiae consensus transcription factor binding sites. This hypothetical network can be queried to identify potential regulators of the hyperosmotic stress response in C. glabrata.

It is important to stress that my involvement in this work has only been in the technical analysis and biological discussion of the gene expression microarray data in context of the initial phenotype discoveries. I am very grateful to Janet Quinn for involving me in this project. Unless otherwise stated, all figures in this section have been generated from my analysis.

3.2 Specific Methods 70

3.2 Specific Methods

3.2.1 Construction of a C. glabrata hogl null mutant

BLAST (16) searches of the C. glabrata genome (630) identied an open reading frame, CAGLOM11748g, encoding a protein with 85% amino acid identity to S. cerevisiae Hog1p. A three-step overlapping PCR strategy was devised by our collaborator, Janet Quinn (Uni-versity of Newcastle), to generate a HOG1 disruption cassette based on this nucleotide sequence, for subsequent lithium acetate based transformation of a C. glabrata histidine auxotrophic strain. A brief description of the construction method follows:

In the first step, two fragments, an 870 bp 5' fragment (-942 to -73) and a 512 bp 3' fragment spanning coding sequence, stop codon and 3' untranslated region (+1224 to +1735) were amplified with M13 overhangs from C. glabrata ATCC 2001 genomic DNA template by high fidelity PCR using primer pairs HOG15-5 + HOG15-3 and HOG13-5 + HOG13-3 respectively (Supplementary Table D.1). Secondly, primer pair HOG1-HIS5 + HOG1-HIS3 was used to amplify a HISS marker gene template that had been previously cloned into the M13 site of vector pTW25 (734), yielding the HISS gene with M13 overhangs and HOG1 5' (-73 to -94) and 3' (+1224 -+1243) sequences. Finally, the HOG1 disruption cassette was constructed using the products from the above two reactions. The disruption cassette was amplified by PCR using the 5' and 3' HOGI fragments from the first step which overlap the amplified HISS template from the second step at the 5' and 3' ends. The resulting fragment was used to transform C. glabrata AHT6 (734), and restoration of histidine prototrophy was used to select putative hog1 colonies. Putative hog1 mutants were tested for homologous integration and gene replacement by PCR, using the primers HOG13ex (external to the disruption cassette) and HIS3INTF (internal HIS3). The selected hog1 strain used in this study was given the laboratory name JG7.Two strains disrupted in the STE11 and STENO loci respectively were created by Ana Maria-Calcagno Pizarelli (106; 103; 104). Full details of these strains and JG7 can be found in the supplementary data and references therein (Supplementary Table C.1).

3.2.1.1 Western blot analysis

Western blot analysis was performed on whole cell extracts from wild-type, ste11 and ste20 C. glabrata cells following treatment with 1 M sorbitol. Blots were probed with an anti-p38 MAPK antibody (New England Biolabs 9211S), which recognises the phosphorylated, active form of C. glabrata Hoglp (phospho-Hoglp). Total levels of Hog1p were determined by stripping and reprobing the blot with an anti-Hoglp antibody (Y-215 Santa Cruz). Full details are described in the materials and methods section.

Chapter 3. The Hog1p and Ste11p dependent transcriptional effects of hyperosmotic stress in Candida glabrata

3.2.1.2 Genome-wide transcript profiling

Transcript profiling was performed in biological triplicate using C. glabrata strains and CO-GEME consortium arrays: ATCC 2001 (wild type), stell (AI\43, (104)) and hog' (JG7, this study). Hybridizations were also performed using a dye-swap to account for potential dye-bias in the experiments. Strains were cultured in YEPD at 37°C, 180 rpm to OD600 = 0.55 and harvested after 15 minutes of mock stress treatment with YEPD or hyperos-motic stress treatment with 1 1VI sorbitol. Hyperosmotic stress was induced by adding 60 ml of pre-warmed YEPD + 2.4 M sorbitol to 50 ml of OD600 = 0.55 cell suspension to give a final concentration of 1 M sorbitol and an OD600 = 0.23. Cells were collected by centrifugation (3000 rpm, 5 minutes), snap-frozen in liquid nitrogen and stored at -80°C before isolating total RNA. Total RNA was prepared by shearing frozen cell pellets with glass beads and a Mini-Beadbeater-8 (Biospec Products, Bartlesville, USA) and extraction with Trizol Reagent (GibcoBRL), as described previously (275). Hybridization and scan-ning was performed as described in the materials and methods section. Relative abundance of transcripts is quantified by log2 intensity ratios (also known as M values) of intensity measured in the CySTM channel (red) and the Cy3T' channel (green), values for which are dependent on each hybridization comparison. Average intensity A values are quantified as (0.5 x log2 (Cy5Tm .) + log2(Cy3m.)) and are used in conjunction with M values in quality control procedures to assess how log2 ratios vary as a function of spot brightness. All sta-tistical analysis was performed in the R statistical computing framework (562) using the limma package (643) for linear modelling of microarray data and single channel normaliza-tion procedures. Hierarchical clustering was performed using the hclust function, with the default settings of complete agglomeration and Euclidean measures of similarity:

For two n—dimensional points, P = (pi , p2, • • • ,pri) and Q = (gi,g2 ,• • • , g„) then Eu-clidean distance is

D _ Om. qi ) 2 + (192 (22 ) 2 ± (prt qn ) 2 (3.1)

Heatmaps of cluster dendrograms were created using the heatmap.2 function provided in the gplots package.

3.2.1.3 Downstream b ioinform at ic analyses

Over-representation of gene ontology terms (35) was investigated using the Saccharomyces Genome Database' hosted GO term finder (83), using a C. glabrata gene ontology inferred by homology to S. cerevisiae. Over-represented GO Process, GO Function and GO Corn-

lhttp://www.yeastgenome.org/

71

3.3 Results 72

ponent terms were identified in relation to this inferred whole-genome background set. The null distribution was approximated by a hypergeometric distribution and statistically sig-nificant over-represented terms were defined as having a Fisher's exact p value of < 0.05. Annotated metabolic pathways were created by permission of the Kyoto Encyclopaedia of Genes and Genomes (KEGG) (343). The colour pathway tool was used for the C. glabrata reference species. At this point, it is appropriate to note the following analysis of transcript regulation is encumbered with two caveats. First, transcription profiles are often discussed in lieu of protein levels, but gene expression profiles themselves describe only a part of the adaptive responses to stress. Turnover of mRNA, post-translational modification of pro-teins and patterns of localization can all significantly affect the exact nature of a cellular response. Nevertheless, these profiles provide useful clues to how the cell may orchestrate a response to an environmental stress. Second, unlike the collegial and prolific S. cerevisiae research community, the poorer C. glabrata cousins have very few giants' shoulders upon which to stand - much of our knowledge of C. glabrata is inferred by evolutionary com-putational annotation. With this in mind, homologous S. cerevisiae gene names are used, and unless otherwise stated, it should be assumed that appropriate S. cerevisiae comple-mentation studies have not been performed. Furthermore, functional assignment is based on descriptions cited in the SGD (131) database for each transcript discussed in these re-sults and is used throughout the discussion and results section. Supplementary data tables comprise C. glabrata ORF names alongside their S. cerevisiae homologous standard and canonical names.

3.3 Results

3.3.1 C. glabrata, hyperosmotic stress dependent, Hogip phosphorylation is abol-ished in a ste// background

Colony growth assays and western-blotting of Hog1p phosphorylation indicate that Hog1p is phosphorylated upon exposure to hyperosmotic stress, and that is required for growth in high osmolarity medium (Figure 3.3). All three mutant strains (hogl, stel 1 and stab') showed equivalent growth to the wild-type on YEPD agar, but impaired growth in solid medium supplemented with 1 M sorbitol. The hogl and stel 1 stains showed equivalent growth defects in 1 M sorbitol medium over the range of cell titres used in this study, whereas the ste20 was not as sensitive (some growth at highest cell titre - 104 cells).

A two-stage Western blotting analysis of the Hog1p putative SAPK, using an anti-p38 antibody to detect phosphorylation specific epitopes, and an S. cerevisiae anti-Hoglp revealed a low level of Hog1p phosphorylation in rich culture medium, that is rapidly up-regulated and sustained for twenty minutes upon application of exogenous sorbitol (1 M)


WI

hogld

ste118

ste20d

0 ze

*COOP • • ►t•

A

YPD

YPD + 1M Sorbitol

WT stelld ste2O4 0 10 20 0 10 20 0 10 20 mins

B Ism

Figure 3.3: The C. glabrata HOG pathway is activated by a single MAPKKK, Stellp. C. glabrata hogl and stell mutants display equivalent hyperosmotic stress growth phenotypes. (A) Exponentially growing wild-type and mutant C.glabrata strains (104 cells and 10-fold dilutions thereof) were spotted onto YEPD agar plates ± 1 M sorbitol, and incubated at 37°C for 24 hours. (B) Hyperosmotic stress activation of Hoglp in C. glabrata requires STEll and STE20. Western blot analysis of whole cell extracts from wild-type, step and ste20 C. glabrata cells following treatment with 1 M sorbitol. Blots were probed with an anti-p38 MAPK antibody, which recognises the phosphorylated, active form of C. glabrata HOG1 (Hog1p-P top panel B). Total levels of Hoglp were determined by stripping and reprobing the blot with an anti-Hoglp antibody (bottom panel B). Courtesy of Janet Quinn Personal Communication.

(Figure 3.3 B). Total Hoglp levels changed little over the same time course. These ober-

vations indicate that, as is the case for S. cerevisiae. C.glabrata Hoglp is required for pro-

liferation in high external osmolarity medium and that Hoglp phosphorylation coincides

with this environmental stress, implicating Hoglp phosphorylation in hyperosmotic stress

sensing and adaptation. It should be noted that we have not yet performed the experiments

to directly link Hoglp phosphorylation to this phenotype. However, unlike S. cerevisiae,

3.3 Results 74

where input signals from both osmosensing branches must be disrupted to block hyperos-motic stress induced Hogip phosphorylation (435), this can be achieved by deletion of a single MAPKKK belonging to the predicted Shol branch upstream of Cg-Hoglp suggesting that C. glabrata signals only through the Shol signalling branch. These results were later demonstrated independently by the Kuchler laboratory (255). We next investigated the transcriptional effects of hyperosmotic stress on C. glabrata, and the roles of Hog1p and Ste1 1p in regulating these transcriptional responses.

3.3.2 The transcriptional response of wild-type C. glabrata cells exposed to transient hyperosmotic stress

Hybridizations were designed (Figure 3.4) specifically to compare the wild-type against hogl and stell strains in rich culture medium and in rich culture medium supplemented with a source of exogenous hyperosmotic stress (1 M sorbitol).

To increase the value of this design, I also wanted to investigate the wild-type hyperos-motic stress affected transcriptome of C. glabrata so that it could be compared with pub-lished data in S. cerevisiae, even though conventional analysis of the initial design precluded this. To overcome this limitation, I treated the two-colour gene expression microarray data as a single-colour experiment where intensity values are measured for one channel only and relative abundances (log ratios) are constructed across channels. This is unusual for the analysis of two-colour chips but is common place in other platforms such as on Affymetrix chips. Since the specific hybridizations compare wild-type in one channel (Cy3TM) and a null strain in the other channel (Cy5TM) with and without hyperosmotic stress, it is theoreti-cally possible to obtain the wild-type hyperosmotic stress response by comparing the single channels of wild-type cells before and after stress (i.e. Cy3TM versus Cyr), and therefore construct a log ratio of intensities spanning hybridizations. This poses particular problems in removing systematic sources of noise to reveal the biological effects of interest. To this end, a standard lowess normalization technique (762), accounting for within-slide bias, was complemented with specific techniques that ensure comparable distributions of Cy5TM and Cy3TM intensities between slides, thus facilitating meaningful construction of log ratios across hybridizations. This was performed with an adapted quantile normalization by Yang and Thorne (690) (Figure 3.5). This method extends the idea of normalizing for equivalent medians or quartiles of the single-channels by requiring every quantile across channels be equivalent, and thus forcing each channel to share a common distribution.

The distribution is estimated by averaging across channels for each quantile. After normalizing within arrays using quantile normalization (Figure 3.5 a) and M values between slides using quantile normalization, intensity values were normalized with "A"-quantile


normalization to make intensity values comparable across slides and suitable for downstream comparison (Figure 3.5 c and d) in both the red and green channels (Figure 3.5 e and f). In this study, the genome-wide transcriptional response (Figure 3.6 and 3.7) of wild-type C. glabrata was constrained to a small proportion (< 5%) of the total genetic repertoire, in a similar manner to published data in S. cerevisiae (Supplementary Data Tables D.3 and D.4) .

This analysis reveals 169 induced and 39 repressed transcripts in the wild-type C. glabrata response to transient hyperosmotic stress. Functional classification of these tran-scripts was performed by inspection of the SGD annotation descriptions and by mapping differentially transcribed genes to annotated KEGG pathways (343) and a gene ontology (GO) (35) inferred by S. cerevisiae sequence homology. Collectively, these downstream analyses reveal the transcriptional regulation of several important biological processes, that are perhaps unsurprisingly categorized under the GO process terms: "Response to chemical stimulus" and "Response to abiotic stimulus". More specifically, induced transcripts are involved in: metabolic processes (14%), vesicular trafficking (9%), transcriptional regula-tion (8%), cell wall remodelling (7%), protein degradation and autophagy (7%), phosphate metabolism (5%), transport of inorganic ions and organic molecules (3%), the protein un-folding response (3%), oxidative stress and DNA damage repair (3%) in addition to a number of uncharacterized proteins (10%). Importantly, key glycerol biosynthetic enzymes are induced (Figure 3.8 and 3.9). The NAD-dependent glycerol 3-phosphate dehydrogenase GPD2 transcript levels are induced, however the array design does not include a specific probe for the isoforms GPD1 or GPD3 and so the transcriptional activity of these paralo-gous genes is indeterminable in this study. The NADP(+) coupled glycerol dehydrogenase GCY1, is proposed to be involved in an alternative pathway for glycerol production is also induced after hyperosmotic stress (122; 121; 504; 143) (Supplementary Data Table D.5).

3.3 Results 76

1

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Figure 3.4: Gene expression microarray hybridization scheme adopted to inves-tigate the HOG1 and STE11 dependency of hyperosmotic stress induced tran-criptional changes in C. glabrata. Cultures of stell, hogl and their parental strain - ATCC 2001 were incubated (YEPD, 37°C, 3.5 hours, 180 rpm) to mid-logarithmic phase (0D600=0.4) and either transiently exposed to hyperosmotic stress (1 M Sorbitol, 15 min) or left untreated. Bi-directional arrows denote dye-swapping of cDNA samples labelled with Cy5

TM or Cy3TM dCTP (black and white rectangles respectively). Biological effects are

numbered 1 through 9. Effects 1 - 4 are measured directly in the hybridization, effects 5 - 9 are estimated indirectly by fitting loge intensity values in a linear model followed by extraction of the relevant effect by application of a contrast matrix.

111111 I I I I I 1


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Figure 3.5: Single channel normalization methods preserve slide intensity distri-butions for cross-slide comparison and construction of synthetic log ratios of transcript abundance. The effects of normalization procedures on background corrected intensity distributions prior to single-channel linear modelling analysis. Box and whisker plots of M values (log2 (Cy5T9 ) - log2(Cy3TN )) are shown for each slide after lowess normal-ization (a) and lowess normalization + quantile normalization (b). Similarly, equivalent plots of A values (0.5 x log2 (CyC") log2 (Cy3-m )) are shown for each slide after lowess normalization (c) and lowess normalization + "A"-quantile normalization (d). Coloured boxes denote the biological effect (see Supplementary Data Table D.2) measured in the hybridization. Black boxes = STE.0 - WT.U, red boxes = HOG.0 - WT.U, blue boxes = STE.S - WT.S and cyan boxes = HOG.S - WT.S. The pattern of boxes repeats six times, accounting for three dye-swapped biological replicates of each effect. Density plots of red

3.3 Results 78

C o

0 (0

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0 0

Figure 3.6: Heatmap featuring column and row based cluster dendrograms of genome-wide log ratios (log2 (Cy5TM ) - log2 (Cy3TM )) for hyperosmotic stressed wild-type (WT.S - WT) cells compared with stell (STE.S - STE.U) and hogl cells (HOG.S HOG.U) (defined in Supplementary Data Table D.2). The heat-map depicts the magnitude of intensity ratios for each gene on a hue scale ranging from dark blue (repressed) to bright yellow (induced), as indicated by the colour key histogram of log ratio in the top left inset box. Cluster dendrograms, based on the hierarchical clustering of Euclidean distances, indicate the degree of similarity in expression profiles between genes (rows) and between experimental conditions (columns). Vertical dashed lines represent the median loge (Cy5TM ) - log2 (Cy3TM ). Transcripts induced by hyperosmotic stress in wild-type cells form the basis for a "zoomed" view in the related Figure 3.7

0 2 4 6 Value

STE. S

-STE

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Figure 3.7: Heatmap featuring column and row based cluster dendrograms of log2 (Cy5TM ) - log9 (Cy3TM ) for transcripts induced by hyperosmotic stress in wild-type cells, compared with the equivalent values in hyperosmotic stressed hogl and stel1 cells. The heat-map depicts the magnitude of intensity ratios for each gene in the subset on a hue scale ranging from dark blue (repressed) to bright yellow (induced) as indicated by the colour key histogram of log ratio in the top left inset box. Cluster den-drograrns, based on the hierarchical clustering of Euclidean distances, indicate the degree of similarity in expression profiles between genes (rows) and between experimental condi-tions (columns). Vertical dashed lines represent the median log2 (Cy5 - log2 (Cy3TM ) This heatniap is a "zoomed view" of the entire genome and is restricted to the subset of the genome that was significantly induced in the wild-type strain after hyperosmotic stress and compares the expression profile with the equivalent conditions in the hogl and ste11 strains. In this subset, the colour key histogram median is much higher than the genome-wide median of 0, because it is skewed by focusing on the comparison of transcripts induced by hyperosmotic stress in wild-type cells. Panel A denotes HOG1-dependent transcripts with lower log ratios in hyperosmotic stress treated hogl cells than wild type or stell cells. Panel B denotes STE/ / -dependent transcripts with lower log ratios in ste11 cells than wild-type or hogl cells. Panel D denotes transcripts with higher log ratio in hogl cells than stell or wild-type cells subjected to transient hyperosmotic stress. Contrasts are defined in Supplementary Data Table D.2

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3.3 Results 80

Figure 3.8: A transcript encoding the enzyme glycerol dehydrogenase (IUBMB Enzyme code EC 1.1.1.6), involved in glycerophospholipid metabolism is in-duced in wild-type C. glabrata cells exposed to 1 M sorbitol for 15 minutes. Differentially transcribed transcripts were identified by gene expression microarray analysis and mapped to C. glabrata pathways of metabolism in the KEGG database. Red boxes de-note enzymes whose transcripts were more abundant after exposure to hyperosmotic stress imposed by addition of 1 M sorbitol to rich culture medium for 15 minutes.


Figure 3.9: Three transcripts encoding the enzymes aldehyde dehydroge-nase, alcohol dehydrogenase and glycerol-1-phosphatase (IUBMB Enzyme code EC 1.2.1.3, EC 1.1.1.2 and 3.1.3.21 respectively), involved in glycerolipid metabolism are induced in wild-type C. glabrata cells exposed to 1 M sorbitol for 15 minutes. Differentially transcribed transcripts were identified by gene expression microarray analysis and mapped to C. glabrata pathways of metabolism in the KEGG database. Red boxes denote enzymes whose transcripts were more abundant after exposure to hyperosmotic stress imposed by addition of 1 M sorbitol to rich culture medium for 15 minutes.

3.3 Results 82

Transcriptional changes that may lead to the accumulation of alternative compatible solutes (e.g. induction of biosynthetic enzymes or transporters) adopted in other organisms such as inositol (664), arabitol, mannitol (319) and trehalose (459; 297; 296; 432) are not evident. Moreover, inositol monophosphatase INM2, which is involved in the biosynthesis of inositol, is repressed under hyperosmotic stress conditions. Taken together, these tran-script profiles provide indirect evidence for an increased metabolic flux through the glycerol biosynthetic pathway after hyperosmotic stress, similar to that reported previously S. cere-visiae (12; 392). Another key feature of the induced transcripts is the involvement of their gene products in energy production pathways such as glycolysis (pyruvate kinase CDC/9), the TCA cycle (pyruvate carboxylase PYC1, succinyl Co-A ligase LSC2, mitochondrial di-carboxylate carrier DIC1, pantothenate synthase PANG, riboflavin kinase FMN1), pentose phosphate pathway regulation (fatty acid dehydrogenase HFD1) and oxidative phospho-rylation (cytochrome b5 reductase MCR1). Transcript levels are also elevated for genes encoding proteins involved in the oxidative stress response (methionine-S-sulphoxide reduc-tase MXR1, monothiol glutaredoxin GRX7, NADPH-dependent aldo-keto reductase YPR1, and the cytosolic alcohol dehydrogenase ALD6). Transcripts annotated to protein degra-dation processes are induced in the wild-type strain after exposure to 1 M sorbitol for 15 minutes (Supplementary Data Table D.6). Within this set of transcripts there are those en-coding key proteins in ubiquitin conjugation (ubiquitin UBI4, the genes encoding ubiquitin protein ligase HRD1 and UFD2 a ubiquitin chain assembly factor and RODI a membrane protein that binds the ubiquitin ligase). These genes are co-induced with regulatory and catalytic subunits of the 26S proteasome, which degrades ubiquitinated substrates (RPN11, a metalloprotease subunit of the 19S regulatory particle; RPT3 an ATPase of the 19S reg-ulatory particle; RPN6, an essential, non-ATPase regulatory subunit of the 26S proteasome lid; PRES a2 subunit of the 20S proteasome, RPN3, an essential, non-ATPase regulatory subunit of the 26S proteasome lid), suggesting that hyperosmotic stress leads to an enhanced (relative to rich culture medium) turnover of proteins in the stressed wild-type.

Some transcripts related to protein trafficking are also induced by hyperosmotic stress (Supplementary Data Table D.7); gene products of which are involved in sorting proteins in the ER and Golgi (SEC8, SNX4) apparatus or in endocytosis and uptake of arginine (BTN2). Furthermore, gene products of several transcripts induced by hyperosmotic stress are located in the vesicles (VMA5, DAP2, PRB1, VPS31 and MVP/ ). Ubiquitination mediated by the induced ubiquitin-protein ligase HRD1 is also required for endoplasmic reticulum-associated degradation of misfolded proteins. Several other transcripts belonging to the heat-shock protein family, which are also involved in protecting misfolded proteins, are also induced in response to hyperosmotic stress (HSP78, HSP104, HRD1, KAR2 and APJ1)


Several transcriptional regulators are also induced, but none of the known downstream targets of S. cerevisiae Hog1p (Supplementary Data Table D.8) were shown to be induced in this study. However, Hog1p and Stellp dependent induction of the epigenetic modulator of transcription SIR2 - was demonstrated, as was a component of the Rpd3 histone deacetylase complex: PH023. Three basic leucine zipper transcription factor genes - FZF1, CIN5 and YAP1, and two zinc finger containing transcription factor genes SUT1 and SET5 were also induced. Hyperosmotic stress causes a largely Hog1p and Stel 1p independent induction of transcripts encoding polyamine (PUT4 ), glucose (HXT3) and multidrug transporter genes (YOR1) as well as the P-type ATPase sodium pump gene (ENA1), involved in Na+ and Li+ efflux to allow salt tolerance. Although no transcriptional changes were observed for the ac-tual signalling components of the HOG pathway, hyperosmotic stress did induce transcripts involved in modulation and feedback control of Hoglp phosphorylation state (PTC1). Other significantly induced phosphatase transcripts included the genes encoding both calcineurin subunits ( CMP2 and CNB1) which are required for activation of another stress response pathway mediated by the transcription factor Crzlp. Repressed transcripts largely belong to different biological processes (ribosome biogenesis, mRNA processing, inositol biosyn-thesis, GPI anchor biosynthesis, sterol synthesis, DNA repair/replication and other as yet uncharacterized functions), although some of those biological processes mentioned above are also represented in the repressed transcripts (cell wall maintenance and transcriptional regulation, endocytosis, ion transport). Transcripts of three enzymes involved in ergosterol biosynthesis are repressed upon exposure to hyperosmotic stress: squalene mono-oxygenase ERG1, C-5 sterol desaturase ERG3 and C-4 methyl sterol oxidase ERG25 (Figure 3.10). The hyperosmotic stress induced repression of both ERG1 and ERG2 is abolished in hog1 and sten cells, suggesting that the sorbitol dependent repression relies on a functional putative Shol signalling branch. In summary, transcription profiling of C. glabrata, and the summary of biological themes by inference from S. cerevisiae identified a largely divergent hyperosmotic stress response to that observed in S. cerevisiae. Although the induction of glycerol biosynthesis genes was observed, these analyses suggest that enhanced cellular traf-ficking, probably in delivery of remodelled cell wall components to the cell wall and targeted turnover of plasma membrane occurs in parallel with proteasomal degradation.

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3.3 Results 84

Figure 3.10: Three transcripts encoding enzymes involved in the ergosterol biosynthesis pathway, are repressed in wild-type C. glabrata cells exposed to 1 M sorbitol for 15 minutes. Repressed transcripts were identified by gene expres-sion microarray analysis and mapped to C. glabrata pathways of metabolism in the KEGG database. Red boxes denote enzymes whose transcripts where less abundant after exposure to hyperosmotic stress imposed by addition of 1 M sorbitol to rich culture medium for 15 minutes.


3.3.3 Comparison of S. cerevisiae, C. albicans and C. glabrata transcriptional re-sponses to hyperosmotic stress

Having found phenotypic evidence of a different hyperosmotic stress sensing architecture, we hypothesized that the core transcriptional responses of hyperosmotic stress would be conserved, assuming that a) phosphorylation of Hog1p was necessary and sufficient for osmoadaptation and b) the regulatory components downstream of Hog1p are functionally conserved in C. glabrata. To assess the conservation of transcriptional changes induced by hyperosmotic stress between related species, I performed a simple comparison of induced and repressed transcription profiles between C. glabrata, S. cerevisiae and C. albicans. Several studies have investigated the affect of an osmotic upshift on the transcriptional behaviour of S. cerevisiae, using a range of solutes and exposure times. In an early study, Rep et al., (581) applied hyperosmotic stress by exposing S. cerevisiae cells to 0.7M NaC1, while more recently O'Rourke and Herskowitz (518) investigated the effects of both 0.5 M KC1 and 1 M sorbitol in wild-type and hogl strains (Supplementary Data Table 3.1). In a broader investigation into the environmental stress response of S. cerevisiae, Gasch et al., (230) applied hyperosmotic stress with 1 M sorbitol and recorded the transcriptional changes over a time-course.

To assess the degree of functional conservation between each available data set, I iden-tified homologous genes of C. glabrata and S. cerevisiae that showed conserved2 transcrip-tional responses between the two species after exposure to hyperosmotic stress. Comparison of induced C. glabrata transcripts identified in this study with the S. cerevisiae equivalents identified by Gasch et al., revealed little functional conservation between the two species. Curiously, even though the vast majority (93.5%) of hyperosmotic stress affected transcripts have a S. cerevisiae homologue, less than ten percent of the induced (Supplementary Data Table D.9) (7.10%) and repressed (5.12%) transcripts are similarly regulated in S. cerevisiae cells exposed to virtually identical hyperosmotic stress treatment (1 M sorbitol, 15 minutes). This discordance was also shown when data from this study were compared finding to a separate study, by O'Rourke and Herskowitz (518), who also used 1 M sorbitol as the source of hyperosmotic stress (Supplementary Data Table D.10), providing greater support for a different transcriptional response of C. glabrata to hyperosmotic stress. 3

The dissimilarity of transcriptional responses between C. glabrata and S. cerevisiae also

tin terms of the relative abundance. Conserved induction requires homologues to be more abundant after hyperosmotic stress treatment, whereas conserved repression requires homologues to be less abundant after hyperosmotic stress treatment

3The set induced of C. glabrata transcripts was also compared to Gasch and colleagues' 1 M sorbitol affected transcriptome at all available time-points, to see if a similar profile existed but with a delayed onset, however there was no concordance at all with the later time points.

3.3 Results 86

extends to studies that have used permeable solutes such as NaC1 and KC1. In the study by Rep et al., where 0.7M NaC1 was used as the hyperosmotic stress treatment, only 11.32% of hyperosmotic stress induced transcripts are functionally conserved with the equivalent tran-scripts identified in this study (Supplementary Data Table D.11). The sets of functionally conserved, repressed transcripts were similarly discordant: only three repressed transcripts are conserved, including: FUR1, a gene encoding a uracil phosphoribosyltransferase in-volved in the pyrimidine salvage pathway, and two genes encoding enzymes involved in the ergosterol biosynthesis pathway (ERGS encoding a putative C-5 sterol desaturase and ERG25 encoding C-4 methyl sterol oxidase). Furthermore, O'Rourke and Herskowitz (518) extended their microarray experiments to incorporate a treatment with 0.5 M KC1 as well as sorbitol discussed above. Again, transcriptional changes caused by 0.5 M KC1 treatment showed little similarity with those caused by 1 M sorbitol in C. glabrata (Supplementary Data Table D.12). Hyperosmotic stress induced transcriptional changes were also compared between a wild-type C. albicans data set (192) and this study. Enjalbert et al., identified 95 induced transcripts in a wild-type C. albicans strain subjected to an osmotic shift imposed by 0.3M NaC1 for 10 minutes. As is the case for S. cerevisae, there is very little similarity between the transcriptional response of C. albicans reported by Enjalbert et al., and the findings in this study.

Following the same protocol adopted for the comparison with S. cerevisiae data above, only 8 induced transcripts were shared in the C. albicans and C. glabrata hyperosmotic stress data sets. Functionally conserved transcripts were annotated as (S. cerevisiae/ C. albicans): NADPH-dependent medium chain alcohol dehydrogenase ADH6/113F20/04 ; pheromone-regulated protein PRM1011PF13607; mitochondrial NADH-cytochrome b5 reductase MCR1 and DNA damage-responsive protein DDR48. Search criteria were also relaxed to identify induced genes that belong to similar gene families or where annotated to different paralo-gous genes. For example, both data sets show elevated transcript levels for different isoforms of glycerol 3-phosphate dehydrogenase. The annotations of isoforms are slightly different between studies: GPD2 was found to be homologous to YDL022w in the C. albicans study but is annotated as YOL059W on microarrays used in this study. Similarly, hallmarks of HOG pathway activation such a putative C. albicans hexose transporter, HXT3 and a sodium transporter ENA22, each having different S. cerevisiae homologues to those in C. glabrata are also induced.

Nevertheless, even under these relaxed criteria, there are few similarities between the hyperosmotic stress affected transcriptome shown in this study and that of Enjalbert et al. A meta-analysis of microarray data from several independent studies, spanning different laboratories and sources of transcriptional stress, is problematic. Uncontrolled systematic differences between studies may equally well explain the observed discordance, casting some


doubt over whether the proposed divergence is really a biological phenomenon. Indeed, the highest degree of similarity between transcript profiles is observed in experiments performed in the same study with different sources of hyperosmotic stress (1 M sorbitol and 0.5 M KC1 in O'Rourke and Herskowitz), where perhaps systematic bias is best controlled be-tween data sets. Notwithstanding these technical differences, the proportion of functionally conserved transcripts is greater within S. cerevisiae data sets utilizing different sources of hyperosmotic stress than between S. cerevisiae and C. glabrata data sets using the same source of hyperosmotic stress (Table 3.1), which suggests that the transcriptional response to hyperosmotic stress may be due to fundamental differences in the biology of the species.

3.3 Results 88

Table 3.1: Conservation of induced transcripts between publicly available, hy-perosmotic stress affected, transcript profiles in S. cerevisiae, C. albicans and this study. Figures shown are percentage of induced transcripts functionally conserved between each study.

1 M Sorbitol* 0.7M NaC1 0.5 M KC1I 1 M Sorbitol' 0.3M NaCF 1 NI Sorbitol 1 M Sorbitoll 61.46 74.14 53.44 12.01 12.50 0.7M NaClt 22.00 - 39.60 21.90 4.20 9.40 0.5 M KC1I 33.52 48.35 - 40.11 1.10 10.44 1 M Sorbitol* 51.90 63.29 92.41 - 6.33 12.66 0.3M NaCl° 17.02 21.28 21.28 10.64 - 4.26 1 M Sorbitol 7.55 11.32 11.95 6.29 1.26 -

* 1 M Sorbitol, S. cerevisiae, Gasch et al., (230); t 0.7M NaC1, S. cerevisiae, Rep et al., (581); t 0.5 M KC1, S. cerevisiae, O'Rourke & Herskowitz (518); 1 M Sorbitol KC1, S. cerevisiae, O'Rourke

& Herskowitz (518); ° 0.3M NaC1, C. albicans, Enjalbert et al., (192); m 1 1\4 Sorbitol, C. glabrata,

This study.

3.3.4 The effect of HOG1 or STEll deletion on hyperosmotic stress induced tran-scriptional responses in C. glabrata

Transcript profiling of hog1 and stell under hyperosmotic stress conditions serves three purposes. First, Hog1p dependent hyperosmotic stress responses can be determined by identifying transcripts whose level is altered as a consequence of HOG1 deletion. Second, the analagous consequences of STE11 deletion can be determined, and compared with the above to identify which transcripts are affected by an individual gene deletion, either deletion, or both deletions. The dependency of transcript profiles can be interpreted as gene specific (i.e. wild-type response altered as a consequence of HOG1 deletion only (Figure 3.7 panel A), or STET1 deletion only (Figure 3.7 panel B)), branch specific (i.e. wild-type response altered by either HOG1 or STE11 deletion) or branch independent (wild-type response unaffected by HOG1 or STE11 deletion). I define a signalling branch as a hierarchical input of phosphorylation signal to Hog1p, in this sense Ste11p phosphorylates Pbs2p which in turn phosphorylates Hog1p; for a working hypothesis I assume that these proteins act only as a relay and that phosphorylation of Hog1p is sufficient to cause the downstream transcriptional events. Third, by observing transcriptional changes specific to stressed hogl

or stell strains, it is possible to identify points of hyperosmotic stress induced cross-talk between signalling pathways. In this section, I report the effects of HOGI or STE11 deletion on wild-type transcriptional response to hyperosmotic stress by comparing overlapping and disjoint transcript sets (Figure 3.11). Dealing with hyperosmotic stress induced transcripts


first (Figure 3.11 b): Sixty two transcripts (36.70% of the induced set) show hyperosmotic stress dependent

induction that is abolished in either ste11 or hogl backgrounds. These transcripts (Supple-mentary Data Table D.13) are dependent on a functional putative signalling branch rather than on either of the two specific components investigated in this study (HOG1 or STE11). Notably, Shol branch dependent transcripts include those encoding key glycerol biosyn-thetic enzymes GPD2 and GCY2 and other metabolic process (alkaline ceramidase YPC1, /3 subunit of succinyl-CoA ligase LSC2, carbamoyl phosphate synthetase CPA1, serine hy-drolase FSH1, a pyruvate carboxylase isoenzyme PYC1, MCR1, malonyl-CoA:ACP trans-ferase MCT1 and mitochondrial dicarboxylate carrier involved in the TCA cycle D/C/ ). Some transcripts involved in the oxidative stress response are also dependent on the putative Shol branch (aldehyde dehydrogenase ALD6, glutaredoxin GRX7, methionine-S-sulfoxide reductase MXR1 and MBR1), although a key regulator of this response YAP1 depends specifically on STE11.

A small proportion of induced transcripts involved in protein degradation are Shol branch dependent (transcripts encode a subunit of a proteasomal metalloprotease RPN11, ubiquitin U1/3/4, mitochondrial inner membrane m-AAA protease YTA12), however these transcripts are largely uninvolved with the proteasome. Transcripts involved in vesicular fusion, transport and sorting (SEC18, VMA5, SWF1, GRH1, PKH2 and MVP/ ) are also induced by hyperosmotic stress. Similarly, there is small proportion of transcripts involved in cell wall maintenance that are generally dependent on the Shol branch (chitin synthase CHS1, a1,3-mannosyltransferase ALG3 and a cell wall beta-glucan assembly glycoprotein KRE9)

A functional Shol branch is also required for hyperosmotic stress dependent induction of transcripts involved with phosphate metabolism, whose protein products act either as direct regulators of MAPK phosphorylation status (e.g. transcripts encoding phosphotyrosine-specific protein phosphatase PTP3 and type 2C protein phosphatase PTC/ ) or in other metabolic processes such as glycogen metabolism (e.g. the regulatory subunit of protein phosphatase Glc7p GIP2) and sphingolipid metabolism, e.g. putative regulatory subunit of Nemlp-Spo7p phosphatase holoenzyme SPOT). A general disruption of Sho1 branch also affects the hyperosmotic stress dependent induction of several transcripts that share little or no sequence identity with S. cerevisiae (CAGLOE04554g) or are homologous to uncharacterized ORFS (YPR127W, YJL017W and YDR379W).

A surprisingly large proportion of transcripts are unaffected by deletion of HOG1 or STE11 (50, 29.58%) (Supplementary Data Table D.14), suggesting that a significant com-ponent of the transcriptional regulation is independent of the SHO1 signalling branch in C. glabrata. Protein products of these transcripts are involved in several biological pro-

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(a) hyperosmotic repressed (b) hyperosmotic induced

HOG .0-WT.0 STE.0-WT.0

2 0 0

5051

(c) Basal repressed (d) Basal induced

Figure 3.11: Venn diagrams of transient hyperosmotic stress repressed (a) and induced (b) genes in wild-type (WT.S - WT.U), hogl (HOG.S - HOG.U) and stell (STE.S - STE.U) cells (defined in Supplementary Data Table D.2). Induced and repressed genes were defined as having log2(Cy5TM) - log2(Cy3TM) >= 2.0 and <= -2.0 respectively and Benjamini-Hochberg corrected p values < 0.05. Basally repressed (c) and induced (d) genes are compared in hogl and stell cells grown in rich culture medium (HOG.0 - WT.U) and (STE.0 - WT.U) respectively. For description of contrasts: STE.0 - WT.0 etc see Supplementary Data Table D.2.


cess categories including stress protection such as the heat-shock proteins (HSP78, HSP42, HSP104 and APJ1), as well as transporters of inorganic ions (ENA1) and organic molecules (HXTS, YOR1, TPO4) and regulators of glucose availability (SKS1, NRG2 and RGS2). The most induced transcript, BTN2 is also induced in a hogl or ste11 background along with other transcripts encoding proteins involved with vesicular trafficking ( VPS/2 and

SBE22). Transcripts belonging to the cell integrity pathway (the putative cell integrity receptor MID2, and the calcineurin subunit encoding CMP2 and CPA2) are also indepen-

dent of HOG1 and STE11 deletion. Twenty one transcripts (12.42% of the induced set) are specifically HOG1-dependent. These transcripts (Supplementary Data Table D.15) are induced in both wild-type and ste11, but not hogl cells and largely encode proteins in-volved with vesicular sorting and transport (HSE1, YPT52) or protein degradation either in the vesicle (PRB/ ) or in the 26S proteasome (RPT3, PRE8, HRD1). However, there are several poorly characterized induced transcripts that are specifically HOG1-dependent (AIM7, AIMS, YMR253C, YLR392C).

Thirty six transcripts (21.30% of the induced set) are specifically STE11-dependent. These transcripts (Supplementary Data Table D.16) are induced in both wild-type and hogl cells, but not stell cells and are largely uncharacterized (YIL055C, YLR414C, YLR194C, YOL057W, YN:305C, YGR205W, YDL190C, RCN2 and YJL171C), although several tran-scripts are involved in cell wall maintenance (KTR6, GSC2, SLT2, MTL1 and MN/V4), transcription regulation (SUT1, TFG1, YAP1, BDF1, SRB6) and vesicular sorting (APM1, SNX4 and KAR2).The repressed transcripts show less dependency on a specific component of the SHO1 signalling branch than induced transcripts (Figure 3.11 a). Twenty nine tran-scripts (74.30% of the repressed set) show hyperosmotic stress dependent repression that is abolished in both ste11 and hogl backgrounds (Table D.17). HOG1 and STE11 inde-pendent repressed transcripts include two that encode key ergosterol biosynthesis enzymes (squalene monooxygnease ERG1 and ERG25 C4 methyl sterol oxidase). Transcripts are also involved in mRNA processing (HSH49, ALB1 and UTPIS) transcriptional regulation (POG1, MOT1 and TRA1).

Seven transcripts (18.00% of the repressed set) are specifically HOG1-dependent. These transcripts (Supplementary Data Table D.18) are repressed in both wild-type and ste11, but not hogl cells and encode proteins involved in axial bud-site selection Bud4p and iron acquisition Ftrlp respectively.Two transcripts (5.20% of the repressed set) are specifically Stel1p-dependent. These transcripts (Supplementary Data Table D.19) are repressed in both wild-type and hogl cells, but not stell cells and includes the gene encoding the C-5 sterol desaturase ERGS of the ergosterol biosynthetic pathway. In contrast to the hyperosmotic stress induced transcripts, only a single transcript (CAGLOI04026g/PBP4 ) (2.50% of the repressed set) is unaffected by deletion of HOG1 or STE11. Transcript

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repression therefore relies almost entirely on the Shol branch, in contrast to the induced genes which has a larger Sho1 branch independent component.

A small number of transcripts are basally regulated by HOG1 or STE11. These tran-scripts were identified by comparing wild-type and hogl or ste11 cells grown in YEPD with-out sorbitol. Two transcripts, encoding a mitochondrial protein CAGLOG07931g/MRPS/2 and a putative glucokinase, CAGLOF00605g/ GLK1, are more abundant in hogl cells than wild-type cells which indicates that HOG1 is required for basal repression of these genes. Three transcripts are less abundant in hogl cells than wild-type cells and therefore require HOG1 for basal expression in rich culture medium. These transcripts, encode a putative hexose transporter CAGLOA01804g/HXT3, a lectin-like protein CAGL0005665g/FL 0/0 and an uncharacterized ORF CAGLOA02277g/YCL049c.

The effects of HOG1 deletion on hyperosmotic stress induced transcriptional changes has also been investigated in S. cerevisiae (581). Publicly available data from the study by Rep et al., were mapped to the C. glabrata annotation (Supplementary Data Table D.20) and compared with the HOG/ -dependent, STE11-dependent and SHO/ -branch dependent transcripts identified in this study. No homologous, HOG1-dependent transcripts were functionally conserved between this study and that of Rep et al.,. This is largely due to differences in the wild-type transcriptional responses; twenty one (75%) HOG1-dependent transcripts identifed by Rep et al., are not induced in C. glabrata cells exposed to hyperos-motic stress. In this case, a relaxation of search criteria to include HOG/ -dependent mem-bers of conserved gene families revealed some degree of conservation. The HOG1-dependent induction of mitochondrial aldehyde dehydrogenase ALD4 in stressed S. cerevisiae cells is not observed in stressed C. glabrata cells, however a Shot branch-dependent, related cytoso-lic aldehyde dehydrogenease homologue ALD6 is evident. Similarly, uncharacterized genes belonging to the AIM (Altered Inheritance rate of Mitochondria) family are Shol branch dependent (AIM9) or STE11 dependent in this study, and HOG1 dependent in S. cere-visiae. Surprisingly, four Hoglp-dependent, stress induced S. cerevisiae transcripts (ENA1, HSP104, PRM10, PHM8) were found to be induced by hyperosmotic stress in C. glabrata, but independent of HOG1 and STE11 deletion, suggesting altered regulatory control over two genes encoding genes expressed in the response to pheromone (PR/1410 and PHM8).

3.3.5 The hogl and stel 1 specific transcriptional responses to hyperosmotic shock: evidence for transcriptional cross-talk between putative C. glabrata MAPK cascades

Null mutant strains, disrupted in either the HOG1 or STE11 loci, provide an insight into how C. glabrata maintains coherence in hyperosmotic stress signalling. Hyperosmotic stress


induced transcriptional responses, specific to each null mutant strain were investigated in the context of the orthologous MAPKKK MAPK architecture of S. cerevisiae. Remarkably, several components of the adjacent signalling cascades, spanning four levels of the hierarchy are only induced in the hogl strain under hyperosmotic stress (Figure 3.12).

Remarkably, several components of these signalling cascades, spanning four levels of the hierarchy, are aberrantly induced or repressed as a consequence of HOG1 deletion (Figure 3.12). The hogl specific osmostress-dependent induction of the gene encoding the tran-scription factor STE12 is intriguing because it regulates several downstream processes in S. cerevisiae, including the cell wall integrity pathway, the morphological switching pathway and the mating response pathway, and it's many functional guises, two of which - mor-phological switching and mating - are observed rarely or have never been observed in C. glabrata, make the interpretation of these findings problematic. Given that C. glabrata is currently thought to be an obligate haploid (754; 487), devoid of any observed sexual cycle, it is perplexing to not only see aberrant induction of a downstream transcriptional acti-vator of (amongst others) the mating response pathway, but to see an apparent feedback to a pheromone signalling sensor gene (STE2 (100)) in the plasma membrane, and regula-tory components upstream of the MAPKKK MAPK architecture. Furthermore, Far7p a protein involved in G1 cell cycle arrest in response to pheromone (353) is also aberrantly induced in hogl cells exposed to hyperosmotic stress in addition to other genes involved with the cell cycle progression (NISI encodes a protein localized to the bud neck at G2/M phase (320), WTM1 encodes a transcriptional modulator involved in regulation of meiosis (535), CLN3 a G1 cyclin involved in cell cycle progression and SPS1 a putative protein serine/threonine kinase expressed at the end of meiosis and localized to the prospore mem-brane of S. cerevisiae). S. cerevisiae Stel2p also activates genes involved in pseudohyphal/ invasive growth pathways of S. cerevisiae. Although, described as a third osmosensor in S. cerevisiae, Msb2p was also identified as an important regulator of filamentous growth by promoting differential activation of Ksslp - the MAPK for the filamentous growth pathway. Domain deletion studies show that Msb2 (153)plays an inhibitory role in mucin dependent MAPK pathway activation. If transcriptional induction results in increased amount of func-tional Msb2p in the plasma membrane and a greater inhibitory signal, then hogl cells will suppress the filamentous growth pathway when exposed to hyperosmotic stress.

In addition to incoherent signalling through, cross-talk is observed in another cascade in which Stel2p is not a downstream effector - the cell wall construction pathway. Although two genes, encoding important components of this pathway: the transmembrane spanning MUM and the MAPK, SLT2 are induced in stressed wild-type cells, the downstream tran-scriptional regulator RLM1 is only induced in stressed hogl cells, suggesting that Hog1p is required for suppression of this transcription factor in stressed wild-type cells. Other down-

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stream transcripts, relating to activation of this pathway are also only induced in stressed hog1 cells including cell-wall mannoprotein CWP1, chitin trans-glycosidase CRH1, DIE2 and CAX4. In addition to STE12, several other transcriptional regulators are specifically in-duced in hog1 cells exposed to hyperosmotic stress, including TAF3, VHR1, YAP7, SOK2, WTM1 and GCR1. These findings suggest that, like in S. cerevisiae MAPK pathways, Hogip is required to maintain coherent signalling.

3.3.6 Identification of a hypothetical hyperosmotic stress regulon in C. glabrata

In order to estimate the regulatory processes controlling the gene expression patterns ob-served after exposing C. glabrata to hyperosmotic stress, I used the YEASTRACT plat-form (686; 477) to construct a hypothetical osmostress regulon network (HORN) by infer-ring regulatory interactions from the model organism S. cerevisiae. Specifically, induced and repressed transcripts (identified using methods described above) were pooled to cre-ate a "regulated" set whose transcriptional control we sought to understand. For each C. glabrata gene in the regulated set, an S. cerevisiae orthologue was identified (using the IN-PARANOID determined orthologues available at Genolevures (630)); YEASTRACT was then used to identify all known conserved transcriptional regulators of this osmotic stress regulated gene. Assuming that these interactions are conserved between the two species, the regulatory interactions are represented by a directed edge from the transcription factor to the regulatory target. When performed for all genes, this process creates an orthologous network of transcriptional regulators and their interactions, with target genes having "in" edges from one or more transcription factors and transcription factors having "out" edges to one or more targets. Although the assumption of conserved regulatory interactions is quite strong, the analysis was performed only to generate hypotheses about regulation that can be experimentally verified or refuted in later studies.

I introduce some pertinent definitions prior to presenting the network analyses: a simple graph is a collection of items called nodes or vertices and their relations, called links or edges4 . I represent a simple graph as g (V, 5) where V = {vi, v2, , vM} is the set of M nodes and E = {ei, e2, , eEl is the set of E edges. Two nodes of a graph are adjacent if there is an edge between them. The entire graph can be represented by an adjacency matrix G E RM"71, containing entries of the form G (i, j), representing an edge between two nodes vi and 'Undirected graphs exclusively contain edges with no orientation and therefore have symmetric adjacency matrices. In contrast, directed graphs - also known as digraphs - have directed edges or arcs, a = (x, y) from the tail x to the head y and can have asymmetric adjacency matrices. These graphs can be used to depict influence

4for clarity in this section, we will consistently define graphs with nodes connected by edges


between variables, for example in a Bayesian network or to indicate physical constraints in real paths (over Konigsburg bridges for example). The degree of a node in a network is simply the number of edges it has to other nodes; betweenness centrality is a measure of a node's importance in a network's structure. Let o-st = ats denote the number of shortest paths from s E V to t E V where mss = 1. Let crst]( ,) denote the number of shortest paths from s to t that some v E V lies on, then a commonly used betweenness centrality CB (v) measure is:

C B (V) E sty (3.2) Est V

and and is computed using an implementation of the algorithm developed by Brandes in networkx and python (263; 717). In essence, high centrality scores indicate that a node can reach others on relatively short paths, or that that the node lies on a high proportion of shortest paths connecting other nodes - i.e. a node of high traffic. Drawing comparisons with more familiar networks, such as the London Underground rail system, Baker Street is an example of a station that has high betweenness centrality (Supplementary Data Table D.21); if a passenger were to travel between each pair of stations in the network - taking the shortest route possible then Baker Street lies on a higher proportion of those shortest routes than any other stations.

The resulting HORN consists of 282 nodes and 863 edges in two connected components (overview in Figure 3.13), the largest of which comprises > 99% of the nodes and edges (280 nodes and 862 edges), the smallest of which is simply a directed edge from the origin replication complex Sumlp to the induced PFS2 gene, an integral subunit of the pre-mRNA cleavage and polyadenylation factor (CPF) complex (an overview of this regulon is shown in Figure 3.13). The largest connected component comprises transcription factors (large nodes in Figure 3.13 that are both transcriptionally repressed (coloured blue in Figure 3.13), induced (coloured yellow in Figure 3.13) and unchanged (coloured grey in Figure 3.13) by hyperosmotic stress; target genes are represented by smaller rectangles. The majority of nodes in the HORN are targets (167 nodes, 59%) in comparison to transcription factors (115 nodes, 41%), however the HORN does capture the majority of nodes that are annotated as transcription regulators in YEASTRACT (115 out of 183, 63%), which suggests that the structure might not be specific to the hyperosmotic stress regulated genes. To address this question of specificity, although not performed here, future work will require a resampling analysis to assess if the appearance of these transcription factors is specifically dependent

5Although perhaps oxymoronic, a rational terrorist might select this tube station as a potential target to maximize disruption with a single terrorist strike

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on the choice of input data (i.e. the hyperosmotic stress signal) rather than the size of the regulated gene set used to construct the HORN. Although the specificity of the presented HORN may be in question, I investigated the phenotypic enrichment of the HORN by taking 10,000 random 282 node samples from the genome and calculating the number of nodes that were annotated with hyperosmotic stress growth decreased, abnormal or absent in SGD. The permuted null distribution had an average of < 5 sensitive phenotypes, whereas the observed number of hyperosmotic stress sensitive nodes in the HORN (12 denoted by red vertical line in INSET B of Figure 3.13) was significantly greater than expected in a random sample. This evidence suggests that the HORN controls the expression of genes of significant number of genes that are required for the adaptation to hyperosmotic stress than would be expected by chance selection of genes from the genome. On a cautionary note, this assertion is based on a rather weak inductive argument (because phenotypic enrichment is also calculated on the basis of homology with S. cerevisiae).

While the HORN approach described above makes strong assumptions about the con-servation of transcription factor binding sites several approaches have been developed to identify statistically significant motifs within nucleotide sequences. I adopted one such approach, a Gibbs motif recursive sampling strategy, to identify candidate transcription factor binding motifs in the upstream promoter regions (-1000 to -1 bp before start codon, downloaded from Genolevures database (630)) of the regulated set described above. This sampling strategy used the Eukaryotic default settings and took 72 hours to complete on a Macbook Pro laptop computer. The resulting position weight matrices were then used to create a sequence logo with the graphical seqLogo toolkit in the R statistical framework (Inset of Figure 3.13). This process identifies in a 16 nucleotide motif (GAGATAAACCCGCTCT),

containing two sub-motifs (GAGAT and GCTCT) with higher information content, separated by a ] portion (AAACCC). The entire motif was used to search YEASTRACT transcription factor binding site motifs, allowing 2 substitutions and aligned with S. cerevisiae consensus sequences of Gsmlp, Yrrlp, Yaplp, Xbplp, Uga3p, Tec1p, Stp2p, Stplp, Stb5p, Rtglp, Rtg3p, Rgtlp, Raplp, Pho4p, Nrg1p, Msn2p, Msn4p, Mot3p, Mcmlp, Haclp, Gln3p, Ace2p, Swi5p, Aft1p, Aship, Bas1p, Cat8p and Sip4p.

There is also some information in the set of transcription factors that are not incorpo-rated into the HORN. For example, GO term searches of the unrepresented transcriptional regulators factors reveals processes that may not be regulated during hyperosmotic stress in-cluding (not exhaustive list): protein deubiquitination and urmylation (OTU1, ELP6), neg-ative regulation of transcription by glucose (M/G2), nitrogen utilization ( UGA3), positive regulation of RNA polymerase II by pheromones (KAN), positive regulation of gluconeo-genesis (S1134 and CATS), purine base biosynthesis (BAS1), cellular zinc ion homeostasis (ZAP1) and reciprocal meiotic recombination (BAS/ ). One obvious limitation of this ap-

Chapter 3. The Hog1p and Stellp dependent transcriptional effects of hyperosmotic stress in Candida glabrata 97'

proach is the omission of transcripts lacking S. cerevisiae orthologues, i.e the C. glabrata specific genes that may confer a different hyperosmotic stress phenotype to the model or-ganism. Although using a model organism can be useful in studying conserved regulatory processes, there is a stark requirement for experimenal tools and statistical inference meth-ods to elucidate the nature of these regulatory process directly in the pathogenic fungi. Such a top-down approach to modelling is still in it infancy, but might yet be truly informative in understanding the molecular basis of environmental stress adaptation in non-model or-ganisms. Moreover, this approach is severely limited by the lack of dynamic transcription measurements.

In order to assess how the HORN structure is associated with hyperosmotic stress de-pendent transcription, I adopted two separate approaches to investigate the hypothetical network organization. The first method involved computing simple summary statistics of network structure (node degree and node betweenness centrality - Figure 3.14) and assess-ing the relationship between these measures and gene expression. The HORN contains very many nodes that are sparsely connected (i.e. with degree below the mean degree depicted by the red line in the degree histogram), and very few nodes that have many connections (bars to the far right of the distribution). For the sake of discussion, I defined a hub as a node with degree k > 20, and subsequently identified 13 hubs in the HORN: Sok2p (k = 45), Rap1p (k = 45), Fhllp (k = 45), Teel') (k = 34), Stel2p (k = 32), Uthlp (k = 27), Cin5p (k = 27), Roxlp, Phdlp (k = 25), ENA1 (k = 21) and Ino4p (k = 20). Interestingly, all of these hubs apart from ENA1 are transcription regulators; ENA1 is annotated as a P-type ATPase sodium pump, involved in sodium and lithium efflux, which is apparently under the regulatory influence of many transcription factors.

The connectivity of HORN nodes was then investigated in the context of the observed gene expression. I defined a simple expression of regulator activity, Ant and Ar,.. This mea-sure is simply the number of target genes that the regulator induces or represses respectively, expressed as a proportion of the total number of target genes k. These expressions pro-vide a measure of activity for each of the HORN transcriptional regulators and can be used to rank transcription factors implicated in hyperosmotic stress adaptation, with the top ranking regulators having the highest proportion of their targets regulated. Notably, transcription factors ranked in this manner do not necessarily regulate the largest number of targets in the regulated set. The HORN can also be classified in terms of An, and k, in order to identify groups of functionally related transcriptional regulators. A k-means clustering method with 4 centroids was adopted to do so (Figure 3.15), and revealed four distinct groups. The first group (red points and red shaded text box Figure 3.15) consists of non-hubs (k < 20) with the highest target activity Art (> 80%). Remarkably this highly active group contains four transcription factors that have been implicated in S. cerevisiae

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hyperosmotic stress regulation: 1VIsn2p, Msn4p, Skn7p, Sko1p in addition to Stel2p which has a role in the mating, cell wall maintenance and morphological switch pathways, which suggests that they may also be active in hyperosmotic stress regulation in C. glabrata. The subnetwork of these transcription factors and their immediate neighbour nodes is shown in Figure 3.16. The second group (green points and green shaded text box Figure 3.15) consists of HORN hubs (k > 20) with moderate target activity Ari (> 80%); these tran-scriptional regulators have the highest connectivity (Roxlp, Rap1p, Mcm1p, Ino4p, Cin5p, Tec1p, Sockp, Fhllp, Phdlp and Flo8p) within the HORN but activate or repress a lower proportion of their putative targets (Figure 3.18). Although these hubs do not activate or repress every target, they do regulate larger and distinct proportions of the overall reg-ulated set than those identified in the red group e.g. Sok2p (20%), Rap1p (18%), Fhllp (16%), Teclp (16%), Stel2p (15%); moreover when all pairings of transcription factors were considered, the combination of two transcription factors regulating the largest proportion (51 genes, 30%) of the regulated set was found to be Sok2p-Rap1p (Figure 3.20).

Although present in the HORN, IVIsn2p and Msn4p are not themselves transcription-ally induced and have only a few direct targets which include the induced genes HSP78, ROD1, BTN2, HSP104, LST8, CMP2, IPT1, APJ1, GSC2, PIH1 and HSP42, GO term analysis of which reveals that this set is enriched in the protein unfolding response genes (corrected p value < 0.05) and also in the membrane fraction (corrected p value < 0.05) of the GO component ontology terms(IPT1, LST8 and GSC2). Other direct targets include the putative transcriptional regulators Roxlp and Yap1p. Motif searching (implemented in cytoscape (624; 137) NetMatch based on the Alon motif finding algorithm (629; 464)) reveals that Msn4p and Msn2p both have feedforward loop structures (a potential regula-tory motif discussed in (629; 464)) with YAP1 and DDR48. By identifying the subgraph of Msn2p, Msn4p, Skn7p and Sko1p with the k = 1 neighbours it was also possible to identify other transcription factors that are local to this subnetwork and may co-operate with these putative hyperosmotic stress regulators to drive the observed transcriptional changes. For example, Skn7p appears to direct the transcription of NRG2 which encodes a transcription factor that mediates glucose repression and negatively regulates filamentous growth, and also appears to direct the transcription of ROX1 and YAP1 in co-operation with Msn2p and Msn4p. The induced direct targets of Skn7p that are not transcription factors in-clude genes encoding a polyamine transporter ( TP04), chitin synthase I ( CHS/ ) and a type 2C protein phosphatase (PTC/ ). Surprisingly, SUR7, a gene required for growth in hyperosmotic stress conditions in S. cerevisiae is repressed in this study, apparently due to the influence of Skn7p. Transcriptional regulators recruited by Skolp, Skn7p, Msn2p and Msn4p are themselves regulators of the oxidative stress response (Yaplp and Roxlp).

In order to predict hypothetical regulators of glycerol biosynthesis, I searched the HORN


for all transcriptional regulators with an edge incident upon induced transcripts having a putative role in glycerol biosynthesis (GPD2 - Figure 3.21, GCY1 - Figure 3.22 and ALD6 - Figure 3.23), all of which are Shol branch-dependent (as depicted by the hexagonal node shape in each figure). Although several transcription factors had edges to GPD2 (Tec1p, Hcm1p, Ino2p, Rap1p, Ino4p, Cin5p), only one (Cin5p) was induced after exposure to hy-perosmotic stress, and was induced independently of the extant signalling branch. This transcriptional regulator also has an edge to ALD6 (Figure 3.24) and several other tran-scripts, the majority of which are Shol branch dependent. Cin5p seems to be an obvious candidate regulator of glycerol biosynthesis, although by an inductive argument one might expect Cin5p transcription to also be Shol branch dependent if there is a direct regulatory role. However, this interpretation is confounded by the "hidden" activation profile of this protein. For example, it is conceivable that the transcriptional induction is not equivalent to activation, and some other Shol branch dependent activation event occurs that may account for the Shol branch dependent profile of its targets. Perhaps more important, in acknowledgement of the nature of complex networks, is the potential co-operative role with other transcriptional regulators and their targets (e.g. ALD6 has several candidate regulators: Pdr3p, Tec1p, Gcn4p, Flo8p, Pdrlp, Stel2p, Stb5p) in the network that are not tractable with the simple analyses adopted here, but may warrant investigation with approaches such as boolean networks. However, the observed Sho1 branch dependent in-duction of these glycerol biosynthetic enzyme does suggest that glycerol production under hyperosmotic stress conditions is regulated by the extant SAPK architecture in C. glabrata. Recent work conducted in our laboratory by Emily Cook (Personal Communication) shows that glycerol accumulation is independent of Hog1p in C. glabrata but Hog1p is required for growth in these conditions. Taken together with our findings of Shol branch dependent induction of glyerol biosynthetic enzymes, it appears that induction of these enzymes is not required for proliferation under hyperosmotic stress conditions, and there may exist other Hog1p dependent mechanisms that are required for proliferation in hyperosmotic stress. Glycerol accumulation is also the net effect of glycerol turnover and export, and the regu-lation of C. glabrata Fps1p may be an important determinant of glycerol accumulation in hyperosmotic stress conditions. One speculation is that Hog1p is involved in the regulation of Fps1p closure or endocytosis to reduce glycerol efflux in C. glabrata which is under pos-itive selection in comparison to sensu stricto species (Supplementary Data Table D.22 and Supplementary Figure D.3).

I used the same HORN approach to make predictions regarding the nature of C. glabrata stel2 null strain phenotypes observed in previous work conducted in our laboratory (Figure 3.25). A total of five (SKS1, UTH1, DDR48, BDF1 and SUT1) induced Stel2p targets are required for growth in S. cerevisiae cells exposed to hyperosmotic stress (depicted by red

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borders of nodes in Figure 3.25). The sensitivity of C. glabrata ste12 strains to sorbitol (1.5 M) may possibly be due to the regulatory effects of Stel2p on these transcripts however, these transcripts are also apparently regulated by other transcription factors in the HORN so future experimentation will be required to assess the regulatory influence on these genes whose S. cerevisiae required for growth. Moreover analysis methods that may take into account the complexity of the interactions (such as boolean networks (753; 743; 37; 708; 167) or hidden variable dynamical modelling (51; 52)) may be more complex approaches that are still easy to implement, providing time series experiments are conducted.

In general, there is a strong positive correlation (r = 0.89 between k and CB(v) such that hubs are also highly central to the HORN. However, there are some hubs that deviate from this correlation with some having higher centrality (above the dotted red line in Figure 3.14 C: Ino4p, ENA1, UTH1, Rap1p and Sok2p) and some that have lower than average centrality (below the dotted red line in Figure 3.14 C: Cin5p, Flo8p, Roxip, Fhllp, Tec1p, Stel2p and Phdlp). The more highly connected set consists exclusively of transcriptional regulators, whereas the more highly central set consists of transcriptional regulators and two target genes ENA1 and UTR1 that are both required for wild-type growth in S. cerevisiae cells subjected to hyperosmotic stress. In this study, the transcription of these highly central transcripts was independent of Hog1p and Ste11p and potentially controlled by several regulators.

In a second approach, I used a k-clique percolation search strategy to identify communi-ties in the HORN based purely on network topology, the aim being to find how transcription factors are organized in terms of their topological relationship to each other. In this sense, while the investigation described above deals with local network structure, the following approach addresses the topological structure at a higher level of organization. While there is no accepted mathematical definition of a "community", one interpretation comes from an arguably intuitive understanding of social community structure. A community therefore consists of entities that are more closely related to each other than to other members of the entire society (encyclopaedically reviewed by Porter (549), Newman (495) and also by Fortunato (209)). In terms of graph structure, a community finding algorithm seeks sub-networks that contain a greater proportion of edges between each other than to the wider network, and provides an unbiased strategy to identify modularity in the network based on topology alone. Most algorithms find partitions of the graph and therefore do not allow overlapping communities, which confines nodes to a single context. By analogy, standard partition methods may identify a community of British National Party (BNP) voters, but would not also permit simultaneous membership with a West Ham United Football Club (WHUFC) supporters community, whereas k-clique percolation may uncover membership of the BNP, WHUFC and Fathers 4 Justice. This strategy identified three overlapping com-

Chapter 3. The Hog1p and Ste11p dependent transcriptional effects of hyperosmotic stress in Candida glabrata 101

munities (Figure 3.13) that regulate 50% of the targets in the HORN. The largest identified community (community 2) has 15 members including several putative transcription factors (Phdlp, Teclp, Yap6p, Rim101p, Skolp, Pdrlp, Cin5p and Yap7p) and overlaps with a smaller community (community 3) that contains the putative transcription factors Roxlp, Msn2p and Skn7p. The points of overlap are not generally transcriptional regulators (apart form Skn7p): overlap b (Figure 3.13) for example consists of DDR48 and YPRO15C which appear to be under the complex regulation of two communities of transcriptional regulators.

In summary these approaches have assessed the relationship between local network struc-ture (degree and centrality measures) and hyperosmotic stress dependent transcription and the hierarchical organisation of these nodes (k-clique percolation). The local network statis-tics have been used to classify transcriptional regulators in terms of their activity and connectivity. I identified one class of transcription factors with high activity and low con-nectivity that contained several putative regulators of hyperosmotic stress adaptation in S. cerevisiae. In comparison to the HORN hubs, these regulators are incident on a smaller proportion of the whole regulated set. Hubs with moderate activity (e.g Mcmlp, Rap1p, Roxlp, Ino4p, Cin5p, Tec1p. Sok2p, Fhllp, PhDlp and Flo8p) are likely to be important regulators of hyperosmotic stress response in C. glabrata, Moreover, k-clique percolation identified communities of transcription regulators that show similarity in function. For example community 3 consists of transcriptional regulators (Roxlp, Hap1p, Msn2p and Skn7p) that regulate gene expression in response to hypoxia and oxidative stress whereas community 2 consists of transcriptional regulators involved in filamentation or morpholog-ical changes (Phdlp,Stb6p, Teclp and Sok2p). Although the observations are based on hypothetical network structure, the value of this approach is in the contextualization of gene expression in the network structure, and as such is merely a visualization tool rather than a true systems biology approach. Moreover, by only focusing on transcription data, to say this work is an integrative genomics approach might also be stretching the point. How-ever, such contextualization does permit hypothesis development for future experimental work that might not be immediately obvious from looking at gene lists alone.

3.3.7 Identification of hyperosmotic stress sensitive C. glabrata transcription factor null mutant strains

We screened 186 null mutants, with disrupted putative transcription factor ORFs to identify transcription factors that are required for growth in hyperosmotic stress (1 M sorbitol) using liquid growth and solid agar assays (Figures D.1 and D.2, performed by Lauren Ames). We reasoned that, if the downstream targets of Hog1p are conserved and required for growth in hyperosmotic stress then strains deficient in these orthologous genes would show impaired

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growth in 1 M sorbitol and 0.5 M KC1 which are similarly hypertonic (Emily Cook, Per-sonal Communication). The so-called C. glabrata Transcription Factor Knockout Library (CGTFKOLib) employed here comprises 186 strains (created by Biao Ma), disrupted in ORFs encode transcription regulators in S. cerevisiae. Growth was measured using solid agar screens (performed by Lauren Ames, Figures D.1 and D.2) assays (see materials and methods section).

The strains showing the highest degree of growth impairment were: pho4 (CAGLOD05170g) a bHLH transcription factor involved in the transcriptional control of the phosphatase reg-ulon (512); not3 (CAGLOL01001g) a subunit of the CCR4-NOT complex involved in tran-scription initiation, elongation and in mRNA degradation (417); srb8 (CAGLOE00627g) a subunit of the RNA polymerase II mediator involved in glucose repression (44); msn4 (CAGLOM13189g) transcriptional activator related to Msn2p; activated in stress conditions in both S. cerevisiae and C. glabrata (589; 615). The gain (CAGLOF00803g) subunit of the RNA polymerase II mediator complex; associates with core polymerase subunits to form the RNA polymerase II holoenzyme; affects transcription by acting as target of acti-vators and repressors(472); hstl (CAGL0005357g) a NAD(+)-dependent histone deacety-lase required for ORC-dependent silencing and mitotic repression; yox1 (CAGLOG07249g),

taf.14 (CAGLOIVI02739g) a major component in transcriptional regulation complexes TFIID, TFIIF, IN080, SWI/SNF, and NuA3 (694; 269; 400; 268; 489; 540; 399); gcr2 (CAGLOG05467g) a transcriptional activator of genes involved in glycolysis; and arg81 (CAGLOH06875g) a zinc-finger transcription factor of the Zn(2)-Cys(6) binuclear cluster domain type, involved in the regulation of arginine-responsive genes.


• • • Ste -12 Stel2 Several Factors Rim, ?

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Mating Morphological Cell Wall Osmoregulation Cell Wall Sporulation Response Switch Integrity Construction Response

Figure 3.12: Architecture of MAPK cascades of S. cerevisiae, incuding the HOG osmoadaptation cascade annotated with evidence of transcriptional cross-talk in conserved C. glabrata transcripts. Adapted from (292). Orange circles denote putative osmosensors embedded in the plasma membrane, connected to the upstream G-protein (Stel8p, Ste4p, Ras2p, Rho1p, Cdc24p, Cdc42p, Gpalp, Rom2p) or phosphorelay (Ypdlp, Ssklp) control components (black circles). Kinases (either p21 activated kinases Ste2Op or serine/threonine kinases Pkclp, Spslp), depicted by grey circles, lie upstream of the core MAP kinase architecure (MAPKKK (Stel 1p, Ssk2p, Ssk22p, Bcklp) —> MAPKK (Ste7p, Pbs2p, Mkklp, 1V1kk2p) —> MAPK (Fus3p, Ksslp, Hoglp, Slt2p, Smklp)) whose activation results in recruitment of transcription factors (Stel2p, R1m1p) which mediate transcriptional changes in response to the detected signals. Coherence of osmoadaptation signalling is maintained by the Hoglp MAPK, which prevents cross-talk with the mating response pathway (516). Genes disrupted in this study are depicted by shadowed circles with red boundaries. Unknown inputs, regulators or transcriptional events are denoted by a question mark. An aberrant transcriptional response, observed only in hog 1 cells, manifests itself as a hyperosmotic stress dependent transcriptional induction (yellow circle boundaries) or repression (blue circle boundary) in connected signalling branches that respond to a specific, non-hyperosmotic signalling input in wild-type cells.

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community 2: PH01, STR6, COC19, TEC], YAPR, MGA1, SOKZ YHM1, R1M101, FSH1, AGOZ SK01, PORI, OHS, YAP?, PITS, Aux. HSH49, HX71 PH023, KRA 71C

overlap b: DOR48,YPRO15C

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Figure 3.13: Overview of the hypothetical hyperosmotic stress regulon. See text for full descriptions (Subsection 3.3.6). A sequence logo of a statistically significant mo-tif (GAGATAAACCCGCTCT), discovered by Gibbs sampling of the upstream (-1000 to -1 bp) regions of C. glabrata genes, is displayed in the bottom left corner of the image. The mo-tif was discovered using the default Eukaryotic settings. This results in a 16 nucleotide motif (INSET A), containing two sub-motifs (GAGATAAACCCGCTCT and GCTCT) with higher information content, separated by a low scoring portion. This motif was used to search YEASTRACT transcription factor binding site motifs, allowing 2 substitutions and aligned S. cerevisiae consensus sequences of Gsmlp, Yrrlp, Yaplp, Xbplp, Uga3p, Tecip, Stp2p, Stplp, Stb5p, Rtglp, Rtg3p, Rgtlp, Raplp, Pho4p, Nrglp,Msn2p, Msn4p, Mot3p, Mcmlp, Haclp, Gln3p, Ace2p, Swi5p, Aft1p, Ashlp, Bas1p, Cat8p and Sip4p. Phenotypic enrich-ment of the HORN was investigated by taking 10,000 random 282 node samples from the genome and calculating the number of nodes that were annotated with hyperosmotic stress growth decreased, abnormal or absent in SGD. The permuted null distribution had an average of 1.38 (range = 0 - 10) sensitive phenotypes, whereas the observed number of hyperosmotic stress sensitive nodes in the HORN (12) was significantly greater than ex-pected in a random sample (INSET B). This evidence suggests that the HORN controls the expression of genes of significant number of genes that are required for the adaptation to hyperosmotic stress than would be expected by chance selection of genes from the genome.

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Figure 3.14: Distributions of degree (A) and betweenness centrality (B) and their correlation (C) in the hypothetical osmostress regulon network (HORN). The degree k of a node in a network is simply the number of edges it has to other nodes; betweenness centrality C B (V) (abbreviated as C in figure (B)) is a measure of a node's traffic in the network's structure (Equation 3.2). Vertical red lines depict the mean k (6.12, A) and CB(v) (0.008, B) observed in the HORN. In general there is a strong positive correlation (r = 0.89) between k and CB(v) (C) such that hubs are also highly central to the HORN. However, there are some hubs that have higher than average centrality (red dots, Ino4p, ENA1, UTH1, Rap1p and Sok2p) and some that have lower than average centrality (blue dots, Cin5p, Flo8p, Roxlp, Fhllp, Teclp, Stel2p and Phdlp). The hub threshold in C is depited by the horizontal dashed line at k = 20

MCM1, RAPS. ROX2, 1N04, CANS, TEC1, 5OK2, FHLI, PNDI, FLOG Proportion Upregulated v Degree

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OAF1,, MET32. SUMS, STP1, MIG1, NDT80, PDR8, ARG80, 0171, GCRI UPC2, Ph102, DOTE, STP2, GIF3 1XR1, RGT1, MET31, MAL33 AZF1, YRM1, TYE1, GAT1, TOSS, DAL81, MOTS, HSF1, mum M641, tION2 PDRI, ACE2, NISPE YOX1 XBP1, Ent GLA13, oz. STBS, HOME YAPT, GCR2,

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DPC2, DALSO, GA73, RGM1, META, ME CUPS, RFX1, HAP4, F2F1, NRG2 51J11 0 10 20 30 40

k

Figure 3.15: HORN transcription factor activity versus node connectivity. A simple expression of regulator activity Ant was defined as the number of target genes that the regulator induces expressed as a proportion of the total degree k for each node. Regulator activity (y axis, Ant ) is plotted versus node degree (x axis, k) and classified into four groups (red, blue, green and grey) on the basis of this profile using k-means clustering (4 centroids using the Hartigan-Wong method in the R statisical computing framework). Transcriptional regulators in the red class have the highest Ant (> 80%) and an associated low k (less than the mean value of 6). Transcriptional regulators in the green class are hubs (k > 20, threshold depicted by vertical red line) with the highest degree k > 20 and moderate Art , while those in the grey class are non-hubs with low Art . Blue class transcriptional regulators have moderate Art and k. Remarkably, this method co-classifies several transcription factors that are implicated as regulators of hyperosmotic stress adaptation with low k and high Art (Msn2p, Msn4p, Skolp, Skn7p)

U


Figure 3.16: HORN subnetwork comprising putative hyperosmotic stress reg-ulators Sko1p, Skn7p, Msn2p, Msn4p and their immediate k = 1 neighbours. Large rectangular nodes depict transcriptional regulators, whose directed arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same conditions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hog1p, Stel 1p or Sho1-branch dependence of expression. Ellipses represent Hoglp-specific regulation, triangles represent Stel 1p-specific regulation and octagons represent Shol-branch dependent expression. Rect-angular nodes whose expression is altered independently of Hog1p or Ste11p after exposure to hyperosmotic stress are represented as rounded rectangles.

3.3 Results 108

Figure 3.17: HORN subnetwork (red group in Figure 3.15) comprising highly active regulators and their immediate k = 1 neighbours. Large rectangular nodes depict transcriptional regulators, whose directed arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcrip-tion is repressed under the same conditions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hog1p, Stellp or Shol-branch dependence of expression. Ellipses represent Hog1p-specific regulation, triangles represent Stellp-specific regulation and octagons represent Shol-branch dependent expression. Rectangular nodes whose ex-pression is altered independently of Hog1p or Stellp after exposure to hyperosmotic stress are represented as rounded rectangles.


Figure 3.18: HORN subnetwork (green group in Figure 3.15) comprising hub regulators and their immediate k = 1 neighbours. Large rectangular nodes depict transcriptional regulators, whose directed arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same conditions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hog1p, Ste11p or Shol-branch dependence of expression. Ellipses represent Hog1p-specific regulation, triangles represent Ste11p-specific regulation and octagons represent Shol-branch dependent expression. Rectangular nodes whose ex-pression is altered independently of Hog1p or Stel1p after exposure to hyperosmotic stress are represented as rounded rectangles.

GAT3

3.3 Results 110

Figure 3.19: HORN subnetwork (grey group in Figure 3.15) comprising low ac-tivity and low connectivity regulators and their immediate k = 1 neighbours. Large rectangular nodes depict transcriptional regulators, whose directed arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same conditions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hog1p, Ste1 1p or Shol-branch dependence of expression. Ellipses represent Hog1p-specific regulation, triangles represent Stellp-specific regulation and octagons represent Sho1-branch dependent expression. Rect-angular nodes whose expression is altered independently of Hog1p or Stellp after exposure to hyperosmotic stress are represented as rounded rectangles.

...s161./


Figure 3.20: HORN subnetwork comprising the pair of transcriptional regulators accounting for the largest proportion of the regulated set and their k = 1 neigh-bours. Large rectangular nodes depict transcriptional regulators, whose directed arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same conditions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hoglp, Stellp or Shol-branch dependence of expression. Ellipses represent Hoglp-specific regulation, tri-angles represent Stellp-specific regulation and octagons represent Shol-branch dependent expression. Rectangular nodes whose expression is altered independently of Hoglp or Stellp after exposure to hyperosmotic stress are represented as rounded rectangles.

I

3.3 Results 112

Figure 3.21: HORN subnetwork comprising candidate GPD2 regulators and their k = 1 neighbours. Large rectangular nodes depict transcriptional regulators, whose di-rected arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same condi-tions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hoglp, Stellp or Shol-branch dependence of expression. Ellipses represent Hoglp-specific regula-tion, triangles represent Stellp-specific regulation and octagons represent Shol-branch de-pendent expression. Rectangular nodes whose expression is altered independently of Hoglp or Stellp after exposure to hyperosmotic stress are represented as rounded rectangles.


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Figure 3.22: HORN subnetwork comprising candidate G CY 1 regulators and their k = 1 neighbours. Large rectangular nodes depict transcriptional regulators, whose di-rected arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same condi-tions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hoglp, Stellp or Shol-branch dependence of expression. Ellipses represent Hoglp-specific regula-tion, triangles represent Stellp-specific regulation and octagons represent Shol-branch de-pendent expression. Rectangular nodes whose expression is altered independently of Hoglp or Stellp after exposure to hyperosmotic stress are represented as rounded rectangles.

3.3 Results 114

Figure 3.23: HORN subnetwork comprising candidate ALD6 regulators and their k = 1 neighbours. Large rectangular nodes depict transcriptional regulators, whose di-rected arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same condi-tions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hog1p, Stelip or Shol-branch dependence of expression. Ellipses represent Hog1p-specific regula-tion, triangles represent Ste11p-specific regulation and octagons represent Shot-branch de-pendent expression. Rectangular nodes whose expression is altered independently of Hogip or Steilp after exposure to hyperosmotic stress are represented as rounded rectangles.

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Figure 3.24: HORN subnetwork comprising direct targets of Cin5p. Large rectan-gular nodes depict transcriptional regulators, whose directed arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcrip-tion is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same conditions. Nodes with red borders rep-resent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hog1p, Stellp or Shot-branch dependence of expression. Ellipses represent Hog1p-specific regulation, triangles represent Ste11p-specific regulation and octagons represent Shol-branch dependent expression. Rect-angular nodes whose expression is altered independently of Hog1p or Stellp after exposure to hyperosmotic stress are represented as rounded rectangles.

3.3 Results 116

Figure 3.25: HORN subnetwork comprising direct targets of Stel2p. Large rectan-gular nodes depict transcriptional regulators, whose directed arrows represent a regulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcrip-tion is induced after exposure to hyperosmotic stress, whereas blue nodes represent genes whose transcription is repressed under the same conditions. Nodes with red borders rep-resent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hog1p, Stelip or Shol-branch dependence of expression. Ellipses represent Hoglp-specific regulation, triangles represent Ste11p-specific regulation and octagons represent Shol-branch dependent expression. Rect-angular nodes whose expression is altered independently of Hog1p or Stel.l.p after exposure to hyperosmotic stress are represented as rounded rectangles.

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Figure 3.26: HORN subnetwork comprising the minimal connections between or-thologous S. cerevisiae genes required for growth in hyperosmotic stress. Large rectangular nodes depict transcriptional regulators, whose directed arrows represent a reg-ulatory influence to smaller rectangle target nodes. Yellow nodes represent genes whose transcription is induced after exposure to hyperosmotic stress, whereas blue nodes repre-sent genes whose transcription is repressed under the same conditions. Nodes with red borders represent genes that are required for wild-type like growth in S. cerevisiae cells exposed to hyperosmotic stress. The shape of the node depicts the Hoglp, Stellp or Shol-branch dependence of expression. Ellipses represent Hoglp-specific regulation, triangles represent Stellp-specific regulation and octagons represent Shol-branch dependent expres-sion. Rectangular nodes whose expression is altered independently of Hogip or Stellp after exposure to hyperosmotic stress are represented as rounded rectangles.

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Gene name inferred by SC homology


Figure 3.27: Four C. glabrata mutants, each lacking a single, different putative transcription factor are hypersensitive to 0.5 M KC1 in a liquid growth medium assay. A library of 186 C. glabrata null mutants (plate one shown here), each disrupted in one putative transcription factor ORF (based on sequence homology with S. cerevisiae) was incubated in an orbital shaker to stationary phase (18 hours, 37°C, 180 RPM) in YEPD and then sub-cultured in clear well micro-titre plates containing YEPD with (A) or without (B) 0.5 M KC1. Bi-hourly recorded optical density (0D600) measurements, used in place of cell counts, were fitted to a three parameter logistic function of growth using non-linear regression function SSLogistic from the R statistical computing framework. The slowest growing strains in 0.5 M KC1 were identified as hyperosmotic stress sensitive strains: (hstl, srb8, msnl, ga11.1, gisl)


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Figure 3.28: Three C. glabrata mutants, each lacking a single, different putative transcription factor are hypersensitive to 0.5 M KC1 in a liquid growth medium assay. A library of 186 C. glabrata null mutants (plate two shown here), each disrupted in one putative transcription factor ORF (based on sequence homology with S. cerevisiae) was incubated in an orbital shaker to stationary phase (18 hours, 37°C, 180 RPM) in YEPD and then sub-cultured in clear well micro-titre plates containing YEPD with (A) or without (B) 0.5 M KC1. Bi-hourly recorded optical density (0D600) measurements, used in place of cell counts, were fitted to a three parameter logistic function of growth using non-linear regression function SSLogistic from the R statistical computing framework. The slowest growing strains in 0.5 M KC1 were identified as hyperosmotic stress sensitive strains (rgtl, tafl4, rnsn4).

3.4 Discussion 120

3.4 Discussion

Stress activated protein kinases are important molecular signalling molecules involved in environmental sensing and adaptation, a critical requirement for host colonization. In this study, we used the consensus HOG pathway architecture, taken from an array of genetic interaction and comparative genomics studies in S. cerevisiae (comprehensively reviewed in (292)), as a conserved signalling pathway for understanding Hog1p function in C. glabrata. Using hogl and stel 1 null mutants in tandem with transcription profiling, we report a difference between the genetic architecture of the HOG pathway in C. glabrata ATCC 2001 and that of the model organism S. cerevisiae (Figure 3.3). The latter has two signalling branches that must both be disrupted to prevent phosphorylation of Hog1p. In contrast, like C. albicans, C. glabrata ATCC 2001 relies on only one (Ste11p) MAPKKK for Hog1p phosphorylation and osmoadaptation after an osmotic upshift (126), but unlike C. albicans this MAPKKK (Ste1 1p belongs to the putative Shol signalling branch and not the SLN1 signalling branch. Initially, it was appealing to speculate on these differences in an evo-lutionary context; however during the course of this work, a study of C. glabrata ATCC 2001 by Gregori et al., (255) revealed the same sole dependency on the Shol branch for osmoadaptation, which was not evident in two other C. glabrata strains (BG2 and Cg2633) and therefore is not generalizable to the species.

In initial gene deletion studies, adopting a non-prototrophic selection method suitable for all three strains, Gregori and colleagues demonstrated that disruption of PBS2 led to an osmosensitive phenotype in all three strains, whereas deletion of SHO1 led to an osmosen-sitive phenotype only in the ATCC 2001 background. Crucially, this observation arises from strain-dependent differences in a single Slnl branch component: SSK2. Of the three strains, the ATCC 2001 strain uniquely possesses a SSK2 allele (ssk2-1 ) with a premature stop-codon upstream of the phosphotransfer domain, which blocks Sln1p branch dependent phosphorylation of Pbs2p. As such, Gregori and colleagues relay a cautionary message in in-terpreting phenotypic results of gene deletion studies: "all mutations or deletions generated in the ATCC 2001-derived genetic backgrounds are in fact double mutants also carrying a Cgssk2-1 allele. Thus, any stress-related phenotypes and molecular cross talk between different signaling pathways may be caused by synthetic genetic interactions due to the presence of the mutated Cgssk2-1 allele and the lack of the second gene". However, this can be said of many C. glabrata null mutants that have been created with mutagenic trans-formation protocols, since there exists no facility to back cross C. glabrata strains to obviate the mutagenic nature of direct gene disruption. Moreover, this genetic quirk provides an opportunity to investigate the Shol branch contribution to hyperosmotic stress adaptation without deletion of relay components upstream of Pbs2p in the Slnl branch.


Reconstituting ATCC 2001 with a full-length SSK2 ORF from another C. glabrata strain such as BG2 would ensure that the upstream architecture of S. cerevisiae and this C. glabrata strain are collectively orthologous (however this has been particularly difficult to achieve at the chromosomal locus, Christa Gregori Personal Communication). Transcrip-tional differences could then be explained in terms of how each component relays osmostress signals, or in terms of mechanisms downstream of Hog1p rather than differences in SAPK architecture per se. In the absence of such a reconstituted strain, it was possible to compare the transcriptional response of a S. cerevisiae sskl strain treated with 0.5 M KC1 to our "wild-type" (ssk2-1) ATCC 2001 strain (518). Although this comparison is not ideal, it per-mits comparison of C. glabrata and S. cerevisiae strains lacking a functional Slnl branch. Although the proportion of functionally conserved transcripts is slightly higher between these strains (20.10%) (Supplementary Data Table D.23) than in the comparison of ATCC 2001 strain to the wild-type S. cerevisiae strain, there are still prominent hyperosmotic transcription signatures in C. glabrata ATCC 2001 that are absent in S. cerevisiae ssk1. Therefore, even with a more similar SAPK architecture, we could still identify hyperosmotic stress induced transcriptional differences between two species, spanning several biological processes.

Although the comparisons with S. cerevisiae data are likely to be confounded by the lack of direct experimental comparison, my analyses recovered only a small conserved re-sponse (< 15%) between C. glabrata and S. cerevisiae (Supplementary Data Tables D.9, D.10, D.11 and D.12), encompassing genes encoding glycerol biosynthetic enzymes ( GPD1/ 2, GCY1, ALD6 (230)), DNA damage response proteins (DDR48 and RCN2 (230; 518)), a heat shock protein (HSP42 (230; 518)), a v-snare binding protein that facilitates spe-cific protein retrieval from the late endosome (BTN2 (230; 518)) and a proline permease (PUT4 (518)). The regulation of some (37%) transcripts is consistent with the S. cerevisiae consensus HOG pathway model (GPD1/2, GCY1, ALD6, PTC1, PTP3 are Shol branch dependent), moreover this analysis identifies a novel Sho1 branch dependency for some transcripts induced under hyperosmotic stress (ALG3,UBI4, RPN11 and CPA1). Impor-tantly, a large component (30%) of the hyperosmotic stress induced transcriptional changes occurs independently of the extant Shol branch (summarized in Figure 3.29), and includes transcripts whose hyperosmotic stress induction is also conserved with S. cerevisiae (BTN2, P HM8 and PRM10 and DDR48), a proteasomal lid subunit (RP N6) and a transcriptional regulator of the oxidative stress response (CIN5). Indeed, the BTN2 transcript showed the highest fold-induction in response to hyperosmotic shock in C. glabrata ATCC 2001 cells. This gene encodes a v-snare that mediates late endosome-Golgi sorting (339), and modulates L-arginine uptake through localization of Canlp. It is not clear if modulation of L-arginine levels is part of an adaptive response to hyperosmotic stress in S. cerevisiae or

Hyperosmotic Signal

'PWAISt4tmeRA

1

Independent

130%1 BTN2, PHM8, PRM1 HSP42, CIN5, ENA1, ROD1, VP513, HXT3, RPN6, CPA2, UTH1

Hog1p-specific (12 %)

NGL2, HSE1, HRD1, PRB1 RPT3,CSG2, PRES, SSN8

Stellp-specific (22%1 SLT2, KTR6,YAP1, MTL1, ROX1, RPN3, MNN4, KAR2, SNX4 GSC2

Shol branch dependent 07%1

GPV2, ALD6, GCY1, PTC1, PTP3, S1R2, KRE9, ALG3, CPA1 ...

3,4 Discussion 122

Figure 3.29: A working model of C. glabrata ATCC 2001 hyperosmotic stress (1 M sorbitol, 15 min) adaptation. Transcription profiling was performed in wild-type, ste11 and hog1 strains exposed to sorbitol (1 M, 15 min) in order to assess the effects of stress dependent transcriptional changes. Our findings suggest that the wild-type strain, whose Slnl branch is dysfunctional due to a premature stop codon in SSK2 (255), transduces signal via the extant Shol signalling branch, which is required for the induction of glycerol biosynthetic enzymes ( GPD2, ALD6, GCY1) and feedback modulating phosphatases (PTC1 and PTP3). Our initial hypothesis that Stellp and Hog1p acted in a linear relay to direct differential transcription were rejected because STE11 and HOG1 deletion do not have an equivalent effect on hyperosmotic stress dependent (22% of the transcriptional changes are abrogated only in a sten strain; 12% of transcriptional changes are abrogated only in a hog1 strain, and 30% are unaffected by HOG1 or STE11 deletion). See Figure 3.30.

C. glabrata, or indeed if the primary role of Btn2p is in an endocytotic capacity. However,

the co-induction of carabomyl phosphate subunits (CPA1 and CPA2), which catalyzes the

production of L-citrulline, an L-arginine precursor and the sensitivity of an arg81 strain to

0.5 M KC1 suggests that an increase in L-arginine levels may occur during hyperosmotic

Chapter 3. The Hogip and Ste11p dependent transcriptional effects of hyperosmotic stress in Candida glabrata 123

stress adaptation. The inequivalent effects of HOG1 and STE11 deletion on hyperosmotic stress induced

transcription suggest that Stellp does not act as a simple relay to Hogip (via Pbs2p) to elicit transcriptional changes (Figures 3.29 and 3.30). The identification of a Stellp specific hyperosmotic transcriptional response (Figure 3.30 a) implies that 22% of the tran-scriptional changes are mediated by Stellp, independently of Hog1p, possibly by recruiting additional downstream signalling proteins that regulate transcriptional events. An alter-nate hypothesis is that Stellp is required to phosphorylate Pbs2p, which in turn has other non-Hoglp substrates that regulate the transcriptional hyperosmotic stress response. The striking, hyperosmotic stress dependent, transcriptional activation of components of the ad-jacent cell wall remodelling pathway (Figure 3.32) which was previously undescribed in C. glabrata and S. cerevisiae may be caused by cross-talk at the level of Stellp (as evidenced by Stellp specific transcription of SLT2, KTR6, GSC2 and MNN4 ). A key difference between this study and transcript profiling studies of hyperosmotic stress in S. cerevisiae, is this induction of genes whose products affect cell wall structural integrity, both at the regulatory and effector level. For example, the rapid induction of genes encoding enzymes involved in the biosynthesis of the carbohydrate components of the cell wall (chitin syn-thase I CHS1, KRE9 - required for /3-1,6 glucan assembly, GSC2 - the catalytic subunit of 0-1,6 glucan synthase) indicate that C. glabrata mounts a significant re-modelling of the cell wall carbohydrate content in response to sorbitol treatment. Moreover, genes involved in glycoprotein mannosylation (KRE2, KTR6, MNN4 and GUK/ ) and N-linked glycosy-lation (ALG3) are also induced in the same time-frame. The simultaneous repression of ergosterol biosynthetic enzymes (ERG1, ERGS and ERG25) and induction of chitin syn-thase I ( CHS1) are likely to have effects on the physical properties of the plasma membrane (decreased fluidity) and cell wall (increased tensile strength), which I hypothesize may in-crease the turgor pressure of the cell. However, the cell wall remodelling is likely to be more complicated. An alternate speculation is that pre-existing cross-linked glucan and chitin moieties are replaced with new components that do not become cross-linked, as suggested by the concomitant repression of UTR2, a cell wall protein that functions as a chitin trans-glycosidase in the cross-linking of chitin to 13(1-6)-glucan. Indeed, reduced cross-linking of chitin, as artificially imposed by calcofluor white staining in growing cells, may results in a (counter-intuitive) reduction in cell wall strength even when chitin levels are elevated (565). Furthermore, it is also important to consider other physical changes that may occur as a result of this proposed cell wall remodelling. For example how does cell wall permeability change with respect to its cell wall composition and does this instead confer some osmoad-aptive advantage? De Nobel and colleagues showed that N-linked mannoproteins (whose gene expression is induced in this study) play a major role in limiting cell wall porosity in

3.4 Discussion 124

S. cerevisiae (161). The upregulated N-linked mannosylation may alter the permeability of the cell to limit water efflux during osmotic stress.

In addition to an Stellp specific component, a Hog1 p specific hyperosmotic transcrip-tional response was identified (Figure 3.30 b), which implies that the hyperosmotic stress signal is partially transduced independently of Stellp. The majority of these Hoglp-specific, hyperosmotic stress induced transcripts are involved in mediating proteolysis, either in the vacuole or in the proteasome, although the regulation of transcripts involved in proteolysis was not exclusively Hog1p dependent. We identified C. glabrata-specific, Hogip and Stellp dependent induction of transcripts involved in the ubiquitination of proteins ( UB/-4, HRD1, UFD2). The covalent attachment of ubiquitin (encoded by UB/4 ) to a lysine residue of a substrate protein is a versatile regulatory post-translational modification that can de-termine the fate of a protein depending on the degree of conjugation. This process may involve conjugation of a single ubiquitin (monoubiquitination), several ubiquitin molecules (multiubiquitination) or the attachment of ubiquitin chains (polyubiquitination) to a sub-strate protein via a three step process involving the sequential action of activating (El), conjugating and ligase enzymes (E3) (HRD1 ), which can recruit proteins in a variety of cellular processes such as proteasomal and endoplasmic reticulum associated degradation, signal transduction, membrane trafficking, DNA repair, chromatin remodelling and peroxi-some biogenesis (175; 264; 285) that are important in environmental stress adaptation. The consequences of ubiquitination are diverse, and it is possible that one or more of the above cellular processes is recruited in response to hyperosmotic stress in C. glabrata. However, the co-induction of genes encoding subunits of the proteasomal (Figure 3.31) degradation ma-chinery (RPN11, RPT3, PRES and RPN3) provides supporting evidence for an osmostress induced, HOG1 and STE// -dependent induction of genes having protein degradation func-tion. The functional roles of 19S proteasome subunits remain poorly understood, however Rpn3p is required for controlling cyclin levels in Gi /S transition (43; 338). Recent stud-ies by Posas et al., showed that Hoglp mediates cell cycle S-phase delay in response to hyperosmotic stress in S. cerevisiae (761); in the light of these findings and this study, future work may involve studying the role of the proteasome in controlling cyclin levels and cell cycle progression under hyperosmotic stress in C. glabrata. The subunit (Rpn3, RpnS, Rpn6, Rpn7, Rpn8, Rpn9, Rpnl 1 and Rpn12) stoichiometry of the 19S proteasome is typically invariant, however mature proteasomes are also directly modified in response to environmental stress (reviewed in (245)). The differential expression of genes encoding these subunits observed in this study may be an artifact of transcript profiling noise, however it is possible that this leads to an altered stochiometry of these regulatory subunits and an altered regulatory function. Indeed, RPN6 - induced by hyperosmotic stress in a Hoglp and Stellp independent manner, encodes a non-ATPase regulatory subunit that binds to im-

Chapter 3. The Hog1p and Stel1p dependent transcriptional effects of hyperosmotic stress in Candida glabrata 125

portant components of adjacent MAPK signalling cascades of S. cerevisiae (Ksslp, Tec1p, Ste12p, Slt2p, Bcklp and Ste11p) and to other proteins induced in hyperosmotic stress conditions (Hxt6p, Grx3p, Pho84p - this study) (291). An intriguing possibility that Shol branch independent expression of Rpn6p may regulate core proteasomal degradation of MAPK components to maintain coherent hyperosmotic stress signalling warrants further investigation.

Why might the HOG pathway regulate protein degradation in hyperosmotic stress con-ditions? One explanation is that Stelip and Hogip control the proteasome in its function as a housekeeper - preventing the intracellular accumulation of aberrantly folded, functionally impaired and potentially toxic proteins that arise as a result of hyperosmotic stress (248). Evidence supporting this role includes the co-induction of heat-shock proteins encoded by HSP78, HSP104 and APJ1. Although the expression of heat-shock protein folding chaper-ones is known to be induced in response to heat-denatured proteins (674), they can also be expressed in a variety of other environmental stress conditions (230; 118) including osmotic stress. The proteasome has also been shown to act as a regulatory feedback mechanism in the preferential degradation of unphosphorylated Ssklp (609). Assuming C. glabrata ATCC 2001 cells maintain an active signalling branch (Ypdlp, Sln1p) upstream of the truncated Ssklp lacking a phosphoreceiver domain, then hyperosmotic stress would lead to an ac-cumulation of unphosphorylated Ssklp relative to cells with a complete and active Slnl branch exposed to hyperosmotic stress, which may require modulation by this degradative mechanism. Placed in the context of the physical changes that must occur in the plasma membrane and in the cell wall during hyperosmotic stress adaptation, this likely involves targeted endocytosis of proteins that would normally increase either the permeability of the cell to water or glycerol, or alter the composition of the cell wall in order to modulate tur-gor pressure. A second, explanation of Hog1p dependent control of the proteasome is that it may provide a more subtle, stress-dependent mechanism for targeted turnover of active proteins, which augments the transcriptional repression and induction employed to alter the protein content of the cell in response to hyperosmotic stress. Such a targeted turnover of active proteins, may also be achieved by altering the cellular location of proteins rather than by their degradation. For example, the co-induction of ROD1 (also known as ART4), an arrestin-related protein that targets specific plasma membrane proteins for endocytic downregulation by serving as adaptors for Rsp5 (409), suggests that one consequence of the induced ubiquitin machinery genes may involve targeting membrane protein cargoes for endocytosis (409). Such cargo specific endocytosis, mediated by arrestin-related proteins is believed to be important in stress induced plasma membrane remodelling of S. cerevisiae, particularly in alkaline stress adaptation (284; 631).

I also identified an hyperosmotic stress dependent induction of transcripts that are

3.4 Discussion 126

normally associated with oxidative stress protection in S. cerevisiae. Monothiol glutaredoxin (encoded by GRX7) and peptide methionine sulphoxide reductase (encoded by MXR1) are induced in response to hyperosmotic stress by a mechanism that requires Hog1p or Ste11p, as is the putative transcriptional regulator of the oxidative stress response ( YAPI). Maintaining the redox state of cysteine thiol groups - which govern protein tertiary structure and co-factor binding - is critically important for correct maintenance of protein function. Without intervention, reactive oxygen species can disrupt this optimal redox state leading to misfolded and functionally impaired proteins. Glutaredoxins typically act as glutathione-dependent disulphide oxidoreductases, reducing superoxide anion or hydroperoxide in place of proteins' reactive sulphydryl groups to prevent the formation of mixed disulphides in proteins (425), however GRX7 is not involved in the the general maintenance of disulphide bonds, but is required for reduction of cysteine residues in certain substrate proteins of the secretory pathway that require reduced thiol residues for their functionality (462).

Methionine residues and iron-sulphur clusters are similarly vulnerable to oxidation by cellular reactive oxygen species, and the methionine sulphoxide reductases, such as that encoded by MXR1 reduce oxidized methionines and protect iron-sulphur clusters from re-active oxygen species in an analagous fashion to the glutaredoxin mediated protection of cysteine-sulphydryl groups. The induction of oxidative stress response genes by (ostensi-bly) independent stressors is well documented in the S. cerevisiae (but not in C. albicans (191)) literature (230; 118), in what has become known as the general stress response, and latterly the environmental stress response (ESR). The cross-protection against difference stresses, evident from the fact that cells exposed to a mild dose of one stress become resis-tant to a normally lethal dose of another stress (467; 742), has been interpreted as a general mechanism of cellular protection that is induced when cells are exposed to stressful stimuli. However, a fundamental assumption underpinning this interpretation is that the "indepen-dent" stress conditions used have no common effect, or that during the adaptive response the cell itself does not produce stressful by-products in the course of finding an optimal solution to the immediate cellular threat. In this study, the induction of oxidative stress response genes by hyperosmotic stress treatment, is a likely consequence of the metabolic demands of ATP-dependent proteasomal degradation, glycerol production and remodelling of the cell wall. Indeed, induction of transcripts involved in glycolysis, the TCA cycle and oxidative phosphorylation would support this notion - oxidative phosphorylation, in partic-ular, is known to produce reactive oxygen species (235; 676; 525; 403; 80). Furthermore, the production of reactive oxygen species is an inevitable consequence of protein modification in the endoplasmic reticulum (440).


In his entertaining review paper, entitled "can a biologist fix a radio?" (396)6 Lezebnik asks how would molecular biologists and electrical engineers go about fixing a complex system that they know nothing about? Which methodologies typify the separate disciplines and what are the relative merits of each approach? More importantly: who would you bet on to fix the radio? In short, he advocates the approach of the engineer, based on a quantitative approach to reverse engineering the radio's wiring diagram. In systems biology, we have a considerably harder problem: our systems are noisy, model parts are not yet fully characterized7 and most model parameters are immeasurable with current technologies. Our knowledge of how model components interact is also still embryonic, but technological advances have furnished the community with some interaction data: methods exist to identify physical interactions between proteins (705; 318) and genetic interactions in single and double null mutant strains of S. cerevisiae (404; 693; 692). However, these data also have well documented caveats (568; 41; 661; 162; 363; 265). For example, the protein-protein interactome of S. cerevisiae is incomplete (271) both in terms of the number of known nodes that can participate in the network, and also the incompleteness of edges that exist in the nodes that are present. The ensuing subnetworks are also biased towards proteins from certain cellular environments, towards more ancient, conserved proteins and towards highly expressed proteins (721). This has important implications in assertions made about the properties of the unobserved "complete" network. Most studies of networks have only looked at the small subsets of the true network. Only if the degree distributions of the network randomly sampled subnets belong to the same family of probability distributions is it possible to extrapolate from sub-network properties to the full network from which the subgraph is taken. Although some theoretical graphs indeed satisfy these criteria, scale-free degree distributions, which caused such a stir a decade ago, do not satisfy this condition (666). Clearly problems are abound, even in a model organism.

The picture is even bleaker in non-model organisms, where the parts-list is largely in-ferred by computational annotation, and interaction data are virtually non-existent. In a progressive step, a project has recently commenced in our laboratory (Hsueh-Lui Ho, Per-sonal Communication) to obtain large-scale protein protein interaction data in C. glabrata. However, in the absence of these richer data sets, I have adopted a rather crude method to propose a candidate regulatory circuit for hyperosmotic stress in C. glabrata. The HORN was created by searching for transcription factor associations with the differentially tran-

6This biologist has never fixed a radio. However with an aging mother in-law, I have become proficient in explaining the intricacies of a VCR

7genome snapshot http://www.yeastgenome.org/cache/genomeSnapshot.html reveals 968 uncharacter-ized ORFs in september 2009, representing approximately 15% of predicted gene sequences. Moreover, a peptidocentric view underestimates the complexity of the parts list

3.4 Discussion 128

scribed orthologous C. glabrata genes which relies on a strong assumption that transcrip-

tional regulators share conserved regulatory behaviour in C. glabrata and S. cerevisiae.

Borneman et al., (77) used chromatin immunoprecipitation and gene expression microar-ray (CHIP-chip) analysis to demonstrate the divergence of Tec1p and Stel2p binding sites between members of the Saccharomyces sensu stricto species, with species specific Stel2p and Tec1p binding and activation profiles varying greater than gene sequences between these species. Such differences in gene regulation are likely to be a major cause in species divergence and should be investigated in C. glabrata which is even more distantly related, and potentially harbours greater TF binding site divergence. CHIP-chip technologies are likely to be superceded by higher resolution techniques in the near future, and any such in-vestigations of transcription factor binding site divergence should be developed with higher throughput, more cost efficient methods such as the multiplexed CHIP-seq protocol recently adopted in S. cerevisiae (401).

Mapping transcriptional status to the HORN does not fully describe the activation status of the incorporated transcriptional regulators, which may be affected by e.g. phos-phorylation (228), redox state (646) and cellular localization (658). A more detailed un-derstanding of hyperosmotic stress regulation may therefore require further experimental work to determine the phosphorylation status of potential targets using high throughput phosphoproteomic methods (71; 72; 683) or mathematical methods for their in silico pre-diction. Hidden variable dynamical modelling (HVDM) has been used with some success to predict downstream targets of p53 (52), and has since been improved upon by the same group (51) to remove the requirement of prior knowledge of some targets to train the model. In their revamped genome-wide transcriptional modelling (GWTM) approach, Barenco et al., directly explained 70% of the 200 most significantly upregulated genes in the DNA damage response of a mammalian cell line, but required time course measurements of gene expression and estimates of transcript degradation rate that have not yet been performed for hyperosmotic stress adaptation in C. glabrata. Despite these major caveats, some hy-potheses arise from the analysis of local network topology and community structure. Simple analysis of target activity and node degree suggest that there are transcription regulators other than the putative S. cerevisiae Hog1p targets (1\ilsn2p/Msn4p, Sko1p and Skn7p) in-volved in hyperosmotic stress regulation in C. glabrata. HORN hubs with moderate activity (i.e. approximately 80% of direct targets showing differential expression) such as Mcm1p, Rap1p, Roxlp, Ino4p, Cin5p, Sok2p, Fhllp, Phdlp and Flo8p may also play a role in governing the transcriptional response to hyperosmotic stress in C. glabrata. Furthermore, transcription factors are likely to be co-ordinated and redundant in their function as solid agar and liquid culture assays of single gene knockout CGTFKOlib strains did not identify any hypersensitive (i.e. completely abrogated growth) strains.


In the community structure analysis, three HORN communities were discovered by k—clique percolation (522), however the majority of nodes (85%, 242 nodes) were not at-tributed to any particular community. Accordingly, an incomplete and inaccurate picture of HORN organization may have been presented. The method of k—clique percolation is based on the concept of a k—clique, which is a complete subgraph of k connected with all k(k — 1)/2 possible links and relies on the observation that communities seem to consist of several smaller cliques that share many nodes with other cliques of the same commu-nity. A k—clique community is then defined by the union of adjacent k—cliques, which share k — 1 nodes and is particularly sensitive to the choice of k, with k = 2 effectively reducing to link percolation (k = 2 signifies an edge rather than any complicated subgraph) and larger values of k imposing more stringent requirement for communities (e.g cliques with large k values are likely to be multi-subunit complexes). The use of such cliques as a basis for community detection does enable the detection of overlapping communities, with nodes existing in multiple contexts, however this comes at the cost of detection sensitivity. Is this a worthwhile cost? Overlapping community detection appears to be of significant interest in the social sciences, where community membership might be used to infer po-litical (412) and, more controversially (and as yet unpublished), sexual preferences (331), in addition to consumer appetites (590). We would also like to make analagous inferences in computational biology; for example can a protein community be used to infer biological function? In this study, there seems to be little scope for such an approach, simply because of the small number of detected communities. However, DDR48 and the uncharacterized YPRO15C genes are the point of overlap for two functionally diverse HORN communities. Community 2 (Figure 3.13) contains transcriptional regulators involved in filamentation (Phdlp, Tec1p, Sok2 (242; 727; 387)), ambient pH adaptation (RimlOip (388)), hyperos-motic stress adaptation (Skolp (580)), multi-drug resistance (Pdrlp (441)) and oxidative stress adaptation (Cin5p (461; 222; 646) and Yap7p (200)). Community 3 is more homo-geneous and contains transcriptional regulators involved in hypoxic gene reguation and the oxidative stress response (Skn7p Roxlp, Hap1p and Msn2p (563; 771; 118; 380; 381)). The combined regulatory context of DDR48 and the uncharacterized YPRO15C indicates that they might be regulated during filamentation, alkaline adaptation, hypoxia and azole efflux. There is some literature to support these predictions: DDR48 is regulated in C. albicans during exposure to ketoconazole (419), hypoxia (622), hyperosmotic stress (191), ambient pH adaptation (62) and is also essential for filamentation (176). Although YPRO15C is less well characterized, the C. albicans orthologue of YPRO15C NRG1 lorf19.7150 is a negative regulator of filamentation (88). The community context identified here suggests that the gene product may also play a role in stress responses in a similar manner to Ddr48p.

(a)

Specific Hogip Requirement Hyperosmotic Signal

PLASMA M[~A9AAYL

Hogip-specific 112 961

NGL2, HSE1, NRD1, PRS1 RPT3,CSG2, PRES, SSN8

(b)

Specific Stellp Requirement

•

Stel le-specific 122%1

KT116,YAP1, 4."*.

• • ***** * MTLI, ROX1, RPN3, 2

MNN4, KAR2, SNX4 GSC2 ..

Hyperosmotic Signal

'ftW

3.4 Discussion 130

Figure 3.30: Alternate hypotheses for hyperosmotic stress signal transduction through the Sho1 branch of C. glabrata ATCC 2001. The identification of a Stellp specific hyperosmotic transcriptional response (a) implies that 22% of the transcriptional changes are mediated by Stellp independently from Hogip, possibly by recruiting addi-tional downstream signalling proteins (*1) that regulate the induction of genes such as (incomplete list) SLT2, KTR6, ROX1, RPN3 and MNN4. Another alternate explanation is that Stellp is required to phosphorylate Pbs2p (*2), which in turn has other non-Hogip substrates that are involved in the hyperosmotic stress response. The identification of a Hog1p specific hyperosmotic transcriptional response (b) implies that the hyperosmotic stress signal is partially transduced independently of Ste11p, leading to the transcriptional regulation of (incomplete list) NGL2, HSEJ, HRDI, RPT3, PRES. This suggests that sig-nal may be transduced via upstream components that are not yet characterized (?1), and /or directly to Pbs2p (?2)

Chapter 3. The Hog1p and Stel1p dependent transcriptional effects of hyperosmotic stress in Candida glabrata 131

Figure 3.31: Four C. glabrata transcripts, orthologous to S. cerevisiae genes en-coding core and regulatory subunits of the 26S proteasome are induced in cells exposed to 1 M sorbitol for 15 minutes. Differentially transcribed transcripts were identified by gene expression microarray analysis and mapped to C. glabrata pathways of metabolism and cell signalling in the KEGG database. Red boxes denote subunits whose transcripts were more abundant after exposure to hyperosmotic stress imposed by addition of 1 M sorbitol to rich culture medium for 15 minutes. Four transcripts encode regula-tory subunits of the 19S proteasome lid (RPNS, RPN6, RPN11) or base (RPT3) and one transcript encodes a subunit of the proteasome core a subunit (PRE8)

MAPIC

MAPK SIGNALING PATHWAY

MAPIOCK INAPECK

04011 1 1.9709 Kenebsa Laboratories

3.4 Discussion 132

Figure 3.32: Two C. glabrata transcripts, orthologous to S. cerevisiae genes encoding members of the adjacent cell integrity pathway are induced in cells exposed to 1 M sorbitol for 15 minutes. Differentially transcribed transcripts were identified by gene expression microarray analysis and mapped to C. glabrata pathways of metabolism and cell signalling in the KEGG database. Red boxes denote subunits whose transcripts were more abundant after exposure to hyperosmotic stress imposed by addition of 1 M sorbitol to rich culture medium for 15 minutes.


3.5 Chapter Summary

1. Disruption of a MAPKKK (STE11 ) or MAPK (HOG/ ) in the putative SHO1 sig-nalling branch is sufficient to prevent sorbitol stress (1 M) induced Hog1p phospho-rylation and proliferation in a strain derived from C. glabrata ATCC 2001. This observation was independently reported during the course of the study (255), and is not a universal feature of all C. glabrata strains.

2. Transcription profiling of ATCC 2001 exposed to sorbitol (1 M) for 15 minutes iden-tified a transcriptome that is dissimilar (< 15%) to relevant studies in S. cerevisiae and C. albicans. The transcript profile of S. cerevisiae sskl strain, a background more closely related to ATCC 2001, subjected to hyperosmotic stress is also poorly conserved (< 21%). Our hypothesis that Hoglp phosphorylation would be sufficient to cause a highly conserved transcriptional response to hyperosmotic stress between S. cerevisiae and C. glabrata was rejected.

3. Using an annotation inferred from S. cerevisiae, several biological themes emerged in the set of induced and repressed transcripts. In keeping with the current model of osmoadaptation, key glycerol biosynthetic enzymes were induced, although not all were Hoglp dependent. Other biological processes represented in the set of induced transcripts include, ubiquitin mediated proteolysis, cell wall re-modelling and response to oxidative stress.

4. Transcription profiling of putative C. glabrata HOG pathway mutants allowed us to investigate the architectural dependency of the hyperosmotic stress transcript pro-files. A total of 30% of transcripts induced on exposure to hyperosmotic stress were independent of Hoglp or Stellp; 37% of induced transcripts depended on Stellp or Hoglp; 13% of induced transcripts were specifically dependent on Hoglp and 20% were specifically dependent on Stellp.

5. The observation that STE11 deletion and HOG1 deletion do not have the same effects on hyperosmotic stress induced gene expression is inconsistent with a model in which Stellp and Hoglp function together only as a simple relay of signal status.

134

Chapter 4

Sub-telomere directed gene expression during initiation of invasive aspergillosis


Aspergillus fumigatus is a common mould whose small asexual spores (2-3 tiM diameter) enable prolonged suspension in the normal airborne flora (394). The size of these conidia not only facilitates their sustained dispersion, but also permits entry into the pulmonary alveoli of the mammalian lung (576), whereupon the level of host immunocompetence determines their fate, and conversely, the morbidity of the host (394; 773).The host niche imposes common constraints on opportunistic pathogens such as A. fumigatus and some Candida spp; to successfully exploit an immunocompromised host, these organisms must be able to withstand purgative mechanical forces in order to establish themselves on a substrate (164; 1), sense the local environment, exploit the available nutrients (584) and evade host defence mechanisms (657) that function to expunge the foreign organism. Such commonality is reflected in the study of A. fumigatus virulence, with much research focused on the study of host-pathogen adhesion (e.g. the effect of hydrophobins), the role of secreted proteolytic enzymes such as aspartic proteases, (572; 573) serine alkaline proteases (e.g. Alpp (365; 444; 485; 571) the metalloprotease (Mepp) (217; 327; 365; 476; 653)), dipeptidylpeptidases (56; 57) and enzymes involved in the dissipation of reactive oxygen species (266; 279). However, in keeping with their phylogenetic differences, virulence studies in A. fumigatus have also diverged, notably in an effort to understand the role of pigments (391; 323), allergens (364; 386; 566) and toxins in virulence. Fungi that produce fruiting bodies such as A. fumigatus, demonstrate co-ordinated expression of biologically active, toxic and immunosuppresive

Chapter 4. Sub-telomere directed gene expression during initiation of invasive aspergillosis 135

secondary metabolites and spore-related products during development (109). Indeed, A.

fumigatus is equipped with a diverse arsenal of toxins ranging from specific inhibitors of the 28S ribosome (344; 345; 389) through natural steroid antibiotics (632; 470; 99), haemolytic proteins (184; 221; 377), aflatoxins (537; 510) and small molecular weight toxins such as gliotoxin (GT).

Gliotoxin is a mycotoxin that belongs to the group of epipolythiodioxopiperazines, which damages epithelial cells (67), is cytotoxic to mouse macrophages (731) impedes the function of ciliary cells (18), and inhibits the activiation of NADPH oxidase of polymorphonuclear leucocytes (701). This inhibitory action on a major killing mechanism employed by major effectors of cell-mediated defence, macrophages and neutrophils (611; 236), means that gliotoxin has been implicated in virulence (486; 526). Although the role of gliotoxin in virulence remains unclear (74; 145; 379; 652; 670), there has been renewed interest in the role of secondary metabolites in Aspergillus infection since the discovery of a global transcriptional regulator of secondary metabolite biosynthesis in this organism, LaeA (73). LaeA deletion does not affect growth or sporulation in media in vitro, but it does result in reduced virulence in mice. This impairment in the ability to cause infection can be correlated with a loss of detectable gliotoxin as well as mis-regulation of gene expression within 13 gene clusters (539). Siderophore-mediated iron uptake and storage, also partially under LaeA control (539) is indispensable for A. fumigatus virulence (618; 619) and adequate nutrient acquisition within the host niche is linked to pathogenesis of invasive disease being necessary to support sufficient growth to promote infection (281; 375; 374).While single gene deletion strategies have been informative in the study of Saccharomyces cerevisiae biology (749; 239), and to some extent in the elucidation of virulence traits in C. albicans and C. glabrata (341), there is little evidence that single gene knockout methods alone are informative in study of A. fumigatus virulence (reviewed in (394)) because virulence is multifactorial (36). In the absence of practical methods for disrupting multiple loci, transcriptional profiling studies can be informative. In this scenario, it is possible to identify genes that are adaptively transcribed or repressed in response to a perturbation in the culture environment. Indeed this has been adopted in a variety of in vitro environmental cues (alkaline stress, calcium stress, Omar Loss PhD thesis (423)). Moreover, even before microarray technologies were widely adopted in post-genomic fungal research, groups studying bacterial infection foresaw the potential of profiling the transcriptional changes within the host environment (637; 22). In vivo expression technology (IVET) was originally conceived upon the premise that most virulence genes are transcriptionally induced at one or more times during infection, and although certain host environmental parameters can be mimicked in vitro to induce a subset

of virulence genes, the full repertoire is only expressed in vivo. The renewed focus of attention on an important transcriptional regulator of secondary


metabolism, and the paucity of data obtained from single gene deletion methods turned our attention to profiling the transcriptional changes that occur during the early stages of A. fumigatus infection in an immunocompromised murine lung, and to compare the findings with existing in vitro data that can be viewed as components of the overall host environment. The feasibility of performing microarray analyses on limited sample material has been tested by a number of researchers employing linear amplification of mRNA, an approach which has recently proven successful in murine candidiasis (688), though a truly global transcriptional signature has yet to be reported for any fungal pathogen initiating mammalian invasive disease. The Eberwine method of mRNA amplification (183) involves reverse transcription of mRNA with an oligo dT primer bearing a T7 RNA polymerase promoter site, to direct in vitro transcription of antisense RNA after double stranded cDNA synthesis and is favoured for linear mRNA amplification from limited quantities of starting material. It provides the basis for the methodology employed in our study, and in the majority of reported instances where mRNA amplification has been applied to samples destined for microarray analysis (509). To identify fungal attributes preferentially employed during adaptation to the host niche, and thus contributing to the virulence of the saprophytic fungus A. fumigatus, we compared the transcriptomes of developmentally matched A. fumigatus isolates following laboratory culture or initiation of infection in the neutropenic murine lung. In this section, I report the employment of a methodology for global profiling of A. fumigatus gene expression in germlings rescued directly from the murine lung, a tool which will empower the analysis of virulence in this pathogen.

Our methodology employs simple molecular manipulations which, combined with cus-tom bioinformatic scripts based on the latest annotations of the A. fumigatus Af293 genome, mark a significant advance in understanding orchestration of fungal gene expression in vivo. Our analyses identify iron limitation, alkaline stress and nutrient deprivation as relevant host-imposed stresses during early-stage A. fumigatus infection and reveal a biased distri-bution of host-adaptation genes (relative to laboratory culture) in subtelomeric regions of chromosomes. Finally we assess lineage specificity of functions favoured during initiation of infection with other members of the genus Aspergillus.

This work was a collaborative effort, across several departments and universities. To place my work in context, and to maintain a narrative flow, findings from other co-authors are included, and appropriately cited. My contribution, as a joint first author to the manuscript - apart from a major contribution to the writing, was in the analysis of gene expression microarray data and in assessing the fidelity of log ratios following linear ampli-fication. This work was published in PLoS Pathogens (453).



4.2.1 A. fumigatus strains and growth conditions

In vitro isolates of A. fumigatus Af293 used in murine infection experiments were cultured in liquid YEPD medium, in an orbital shaking incubator (37°C, 150 rpm). For oxidative stress treatment, Aspergillus complete medium (CM) (144) was inoculated with 107 Af293 conidia and cultured in an orbital shaking incubator (37°C, 16 hours, 150 rpm). The resulting hyphal culture was transferred to CM containing 17 mM H202 and aliquots were harvested for RNA isolation either instantaneously (To) or after 1 hour of H202 exposure (T60). To impose acid stress, the same fungal burden (107 conidia) was incubated in liquid CM (37°C, 6 hours, 150 rpm) prior to sampling of two batches: a) a batch required for pH adjustment (pH 3.0) using HC1 and b) an unadjusted batch used as a reference. Aliquots of each were taken at the initiation of the subculture (To), and after 1 hour (T60), and culture material was used for RNA isolation and microarray expression analysis. Culture pH was verfied at the initiation and termination of the sub-culturing period. To impose alkaline stress, A. fumigatus germlings were cultured in an orbital shaking incubator for 16 hours in liquid AMM containing 100 mM glycolic acid pH 5.0, 10 ml / 1 vitamin solution (526), 5 mM ammonium tartrate and 1% w/v glucose. Germlings were filtered using Miracloth and shifted to similar, pre-warmed medium containing 100 mM Tris-HC1 pH 8.0. After 1 hour of incubation at 37°C, mycelia were washed with cold AMM (pH 5.0) and RNA extraction was performed immediately. For anaerobic stress treatment, hyphae (107 conidia) were statically cultured in 100 mm Petri dishes containing 20 ml of CM (16 hours at 37°C). Three plates were placed in anaerobic chambers and anaerobic conditions were established using 2 palladium catalysts (GasPak Plus, Becton Dickinson). A control plate was placed in a chamber without palladium catalysts and all plates were returned to a 37°C incubator for the duration of the experiment. Plates placed in anaerobic chambers were removed after 1 hour, the mycelium rapidly harvested on Miracloth, rinsed with ice-cold water and rapidly frozen in liquid nitrogen. The RNA was extracted and labelled cDNA was used in competitive hybridization with the equivalent material derived from the 16 hour control RNA sample. For neutrophil exposure assays, 107 germlings were initially cultured in 20 ml of RPMI 1640 (Sigma) with L-glutamine, 25 mM HEPES, and 5% (v/v) fetal bovine serum for 7 hours at 37°C in 100 mm Petri dishes, prior to addition of 107 human neutrophils for 1 hour, after which time cells were harvested for RNA isolation.For iron limitation treatment, A. fumigatus isolate ATCC46645 was grown for 15 hours at 37°C in Aspergillus minimal medium with depleted iron (AMM -Fe, iron depleted conditions) according to Pontecorvo (548) containing 1% (w/v) glucose as carbon source, 20 mM L-glutamine as a nitrogen source. After this time, 10 itM FeSO4 was added to the medium and germlings


were harvested after a further 1 hour. Growth curve analyses were performed in triplicate using liquid YEPD, or AMM containing 1% glucose and either 5 mM ammonium tartrate or 5 mM hyroxyproline as nitrogen source. Cultures were inoculated to a final concentration of 5x 106 spores/ml and incubated under aerobic conditions at 37°C with shaking at 150 rpm. At selected timepoints mycelia were harvested on Miracloth, encased in Whatmann paper and dried at 37°C for 48 hours before weighing. Fungal growth is presented as the accumulation of biomass as a function of time. To prepare MLT medium we harvested uninfected murine lung tissue and homogenised the tissue in 100 ul of sterile distilled water per lung. The homogenate was added 70/30 v/v to a 2% water/oxoid No.1 agar mix and allowed to set prior to inoculation with A. fumigatus spores.

4.2.2 Murine Infections

Groups of 24 outbred male mice (strain CD1, 18-22 g, Harlan Ortech) were housed in indi-vidually vented cages and allowed free access to food and water. Infection was performed on day Do of the experiment and preceding steps are named in relation to this step e.g D_1 is one day prior to infection etc. Mice received cyclophosphamide (150 mg•kg-1), ENDOX-ANA, Asta Medica) by intraperitoneal injection on days D_3 and D_1 of the experiment. A single dose of hydrocortisone acetate (112.5 mg / kg), HYDROCORTISTAB, Sovereign Medical) was administered subcutaneously on day D_1. All mice received tetracycline hy-drochloride 1 mg / L and ciprofloxacin 64 mg / L in drinking water as prophylaxis against bacterial infection. Aspergillus spores for inoculations were grown on solid ACM, contain-ing 5 mM ammonium (-0-tartrate and 1% (w/v) Oxoid Agar number 3 for 5 days prior to infection. Conidia were freshly harvested using sterile saline (Baxter Healthcare Ltd. Eng-land) and filtered through MIRACLOTH (Calbiochem). Conidial suspensions were spun for 5 minutes at 3000g, washed twice with sterile saline, counted using a haemocytometer and re-suspended to a concentration of 2.5 x101° CFU / ml. Viable counts from administered inoculations were determined following serial dilution by plating on Aspergillus complete medium and growth at 37°C. Mice were anaesthetized by halothane inhalation and infected by intranasal instillation of 108 conidia in 40 ,u1 of saline solution. Groups of infected mice were culled and processed collectively during a 2 hour window corresponding to a time point 12-14 hours post-infection. Bronchoalveolar lavage was performed immediately after culling using three 0.5 ml aliquots of pre-warmed sterile saline. BALFs were snap frozen immediately following harvest using liquid nitrogen.


4.2.3 RNA extraction and amplification

Prior to RNA extraction snap-frozen BALF samples were harvested by centrifugation, washed with 1 ml of ice-cold sterile water to lyse contaminating host cells and pooled. Following a further cycle of snap freezing pelleted pooled product was homogenized using a pestle and mortar and liquid nitrogen. RNA extraction was performed immediately using the filamentous fungi protocol of the RNeasy mini kit (Qiagen). RNA concentration was quantified on a Nanodrop spectrometer. RNA from reference samples was prepared from snap-frozen homogenized germlings or young mycelium.

4.2.4 Estimation of fidelity of transcript abundance following mRNA amplification

The corrected, multiple t—testing methodology of Nygaard (509) formed the basis of our investigation. However, for computational ease it was modified to use the limma linear modelling framework for the R statistical environment (562) (Supplementary Data Figure E.1).Spots were filtered as previously described (509) and systematic spatial effects were normalized within arrays using the print-tip lowess method (limma package for R (643)); no between-array normalization was performed in order to preserve the amplification-protocol variation existent between arrays. Least squares regression was used to fit the average log ratio, and error term of each transcript in each of the amplification protocols. The resulting coefficient vectors contained the mean relative abundance of each transcript (To v T60) in one of the three amplification protocols: aRNAri (amplified one round), aRNAr2 (ampli-fied two rounds) or totRNA (total RNA, unamplified). The amplification protocol effects and their associated Benjamini-Hochberg (290) corrected p-values (contrasts, contrast2 and contrast3 in Supplementarty Figure E.1) were extracted by fitting a contrast matrix to the original linear model. We defined the null hypothesis Ho as no difference in log ratios due to the amplification protocol, and an alternative hypothesis H1 as loss of log ratio fidelity due to the amplification protocol. Spots were partitioned into the three sets based on the Benjamini-Hochberg (290) adjusted p-values associated with each of the fitted contrasts. The rejected, undetermined and conservative sets were populated by genes whose adjusted p-values were 0<p<0.01, 0.01<p<0.1 and 0.1<p<1 respectively. This approach rejected 2325 (8.49%) of the spots in the aRNAi - totRNA comparison, i.e. the genes whose log ratios are significantly altered as a result of one round of mRNA amplification. The anal-ogous rejected sets for the aRNA2 - totRNA and aRNAi - aRNA2 comparisons contained 2604 (9.52%) and 73 (0.27%) rejected spots respectively. Comparison of the rejection sets showed that 80.69% of the spots in the aRNAi- totRNA rejection set were also present in the aRNA2- totRNA rejection set. This finding, in addition to the observed small number of genes in the aRNAi - aRNA2 rejection set, suggests that most of the amplification specific

4.3 Results 140

noise is introduced in the first round.

4.2.5 Microarray hybridisations

All experiments used Af293 DNA amplicon microarrays (499). Labelling reactions with RNA and hybridisations were conducted as described in the TIGR standard operating proceduresl. Independent verification of selected log2 ratios was achieved using quantita-tive RT-PCR (See Supplementary Figure E.3 and Supplementary Table E.12). Hybridised slides were scanned using the Axon GenePix 4000B microarray scanner and the TIFF images generated were analyzed using TIGR Spotfinder2 to identify poor quality spots. mRNA underwent two rounds of linear amplification by T7 promoter-directed transcrip-tion. Data processing and analysis was performed in the R statistical framework, using the limma, multcomp and fdrtool packages3. The intensity data retained after filtering (91.5% of the total spots) were normalized within (print-tip lowess) and between (scale) arrays. Least squares regression (1mFit) was then used to estimate a vector of regression coefficients (representing the mean loge (Cy5/Cy3) across the 5 biological replicates for the in vivo effect), the associated residuals and unadjusted p-values. Differentially regulated transcripts (Benjamini-Hochberg (290) corrected p-value < 0.05) were further categorized by the magnitude and direction of the fitted log ratios. Up-regulated and down-regulated transcripts were defined as having log2 (Cy5/Cy3) greater than an arbitrary threshold of plus, or minus, two respectively. Accuracy of microarray data was independently verified by quantitative RT-PCR on selected transcripts (See Figure E.3). Oligonucleotides used for this analysis are detailed in Table E.12). In vitro stress experiments were performed as previously described (499).

4.3 Results

4.3.1 A. fumigates RNA extraction and mRNA amplification

Microarray analyses are constrained by the availability of sufficient RNA for fluorophore labelling and hybridisation. One strategy to overcome the requirement for large quantities of material is global amplification of the sample. This approach has been employed for a number of reported microarray expression analyses (509) with favourable results. We chose to analyse an early time point of infection to facilitate separation of fungal cells from

ihttp://atarrays.tignorg

2http://www.tignorg/software/

3http://www.bioconductor.org


those of the host with the advantage of studying a stage at which germinating hyphae have sensed, and are adapting to, their host. Early time points of A. fumigatus infection rep-resent a vulnerable phase of morphogenesis in vivo since epithelial invasion and formation of mycelial mass have yet to occur. They also mark a point at which diagnoses capable of distinguishing infection from carriage of fungal spores would be most desirable, and antifun-gal therapy most effective. We firstly characterised the time course of hyphal development in the sequenced clinical isolate Af293 by histopathological examination of infected neu-tropenic murine lung tissues (Figure 4.1). Lung sections collected and formalin-fixed at 4, 6, 8 and 12 hours post-infection contained numerous A. fumigatus spores in close associa-tion with murine epithelium in the bronchioles and alveoli (Figure 4.1 a). At 12 - 14 hours post-infection > 80% of A. fumigatus conidia had undergone germination and primary hy-phal production. We therefore performed all BAL extractions for downstream analyses on concurrently infected neutropenic mice within a two hour window of infectious growth cor-responding to 12 - 14 hours post-infection. At this time point recovery of germlings in BAL fluid was routinely achievable in the order of 103 germlings per lavaged lung (Figure 4.1 b).

4.3.2 Impact of mRNA amplification on preservation of transcript ratios

A suitable global mRNA amplification protocol should provide sufficient material for fluo-rophore cDNA labelling reactions whilst preserving the samples' original relative transcript abundance. To estimate amplification-related error (and distinguish such error from system-atic error inherent to microarray methodologies) a mock experiment was devised by Elaine Bignell to quantify the proportion of transcripts having significantly aberrant log2 ratios as a result of RNA amplification. This was achieved by indirectly comparing cDNA sam-ples from different amplification protocols in a statistical linear model and fitting relevant contrast matrices (see Materials and Methods and Figure E.1). For the mock experiment total RNA (totRNA) was isolated from two A. fumigatus cell populations To and T60, and subjected to either one (aRNAri) or two (aRNA,2) rounds of mRNA amplification prior to cDNA fluorophore labelling and microarray hybridisation (Figure 4.2). To evaluate whether ratios are preserved between amplification protocols, we adopted the approach of Nygaard et. at., (509) who used corrected gene-wise t-tests to identify differences in the mean log intensity ratios between aRNA2 and total RNA populations. In preparing the data for mul-tiple t—testing, we excluded spots that were flagged by the the TIGR spotfinder software, or where the intensity was lower than twice the background intensity in either the Cy5 or Cy3 channel. The proportion of excluded spots was used as an indication of the hybridis-ation quality of the slide as a whole. Slides hybridised with aRNA had fewer spots (13.49 - 18.22%) removed by the filtering process than did slides hybridised with cDNA (44.56 -48.95%), see Table E.2. This apparent amplification-related improvement in hybridisation

12 - 14 hours 12 hours

4 hours

8 hours

6 hours

12 hours

A

B C

4.3 Results 142

quality has been previously reported (508). In our study, this can be attributed to a greater

signal to noise ratio, and more specifically to increased foreground intensity on these slides

(Supplementary Figure E.4). We identified 8.49% of retained spots showing evidence of am-plification protocol dependent differences in the log2 ratios. Thus our estimated measures of

confidence from the above quality-control (QC) exercises fall within a range conducive for deriving biologically useful information, based upon reports of analytical studies published

to date (508) where Pearson correlation coefficients range from 0.75 to 0.99 (Figure 4.2)

and rejected gene sets approximate 10% of spots included in QC analysis.

Figure 4.1: Comparative time-course of A. fumigatus Af293 germination and hy-phal development in the murine lung, and laboratory culture. (A) Time-course of Af293 germination and hyphal development in the neutropenic murine lung. (B) Mi-croscopic appearance of Af293 germlings recovered from a typical single murine BALF, (harvested at 12-14 hours post-infection). (C) Microscopy of developmentally matched lab-oratory cultured Af293 germlings, following liquid culture for 12 hours in YEPD at 37°C. Infection/harvesting procedures and imaging performed by Elaine Bignell.

•i0

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Figure 4.2: Correlation of log2 ratios resulting from comparative transcriptional analysis of the laboratory cultured A. fumigatus cell populations To and To under varying mRNA amplification protocols. Correlation of technically replicated log2 ratios between competitive hybridizations using single (aRNA1), double (aRNA2) and non-amplified (totRNA) RNA samples. (A and B) Correlation between log2 ratios obtained using highest correlations between slides using cDNA derived from amplified RNA (aRNA1 v aRNA2 r=0.74-0.8). Cross-protocol pairings (C) revealed highest correlations between slides using cDNA derived from amplified RNA (aRNA1 v aRNA2 r = 0.88-0.01). Surprisingly technical replicates of slides derived using cDNA derived from total RNA (totRNA r = 0.8) were comparable to cross-protocol pairings. Rejected sets are transposed onto correlation plots in Figure E.5

4.3 Results 144

4.3.3 A. fumigatus transcript profile during initiation of mammalian infection

A common reference design was adopted for the microarray experiment from which data were analysed and processed as described in the Materials and Methods section. We per-formed the infection experiment 5 times in total generating pooled (n = 24) BALFs from five independent Af293 infections (samples A to E, Table E.1). These samples were co-hybridised with cDNA derived from similarly amplified mRNA prepared from developmentally matched laboratory cultured Af293 germlings (Materials and Methods and Table E.1). Fluorescent signals from 9075 out of a possible 9516 represented ORFs were detectable from these hybridisation analyses (Supplementary Dataset E.1 and Array Expresso). Of 2180 genes (22.6% of the whole genome) having a fold-change in log intensity ratio of 2 or greater, 1281 were up-regulated and 897 were down-regulated. The entire expression dataset is graphi-cally represented in Figure 4.3 which plots log2 ratios against chromosomal locus. Initial interpretations of the dataset were performed by the Expression Analysis Systematic Ex-plorer (295) (EASE) to infer function by homology to Saccharomyces cerevisiae and identify over-represented Gene Ontology (GO) terms (35) among differentially expressed genes. The results of these analyses are partially listed in Table 4.1 (full listings in Supplementary Ta-ble E.3 and E.4). Distinct trends among favoured cellular processes are evident from these analyses which reveal a marked utilization by the host-adapting A. fumigatus cell, of genes encoding proteins involved in transport of metal ion, cation, carbohydrate and siderophore iron. Given the importance of iron acquisition for microbial pathogenesis in general, and the absolute requirement for siderophore biosynthesis during murine A. fumigatus patho-genesis in this model of infection (618; 619) we expected transcripts from genes involved in iron mobilization and transport to be differentially abundant in this analysis, relative to iron-rich laboratory culture media. Accordingly we could identify a minimum of eleven siderophore biosynthesis/transport genes as important during growth in the murine lung (Table 4.2) including two ferric-chelate reductases (Afu1g17270 and Afu6g13750).

Thirteen amino acid permease genes were more abundantly represented during host-adaptation than growth in YEPD (Table 4.2) including 4 4-aminobutyrate GABA (Afu8g01450, Afu7g0040, Afu5g14660 and Afu5g00710), and three proline permeases (Afu2g11220, Afu8g02200 and Afu7g01090) as well as the general amino acid permease, Gapl (Afu7g04290). Nine genes annotated as maltose permeases or transporters in the current Af293 annotation were also more abundantly represented during initiation of murine infection (Table 4.2). Extracellular proteases have been implicated as virulence factors in invasive aspergillosis, as well as antigens causing inflammatory irregularity during allergic A. fumigatus disease (394). Our analysis identified increased abundance of transcripts from the elastinolytic

4http://www.ebi.ac.uk/microarray-as/aer/#?ae-main0AccessionnumberE-TABM-327


metalloprotease (Mep) (Afu8g07080), an aorsin-like serine protease (Afu6g6g10250) and three dipeptidylpeptidases (Afu4g09320, Afu2g09030 and Afu3g07850). Thus transcription of this subset of A fumigatus proteases is significantly higher in the murine lung relative to rich laboratory culture. Functional categories of ergosterol biosynthesis, haem biosynthe-sis and aerobic respiration were significant among genes underrepresented during infection, relative to laboratory culture (Table 4.1) as well as multiple functional categories represent-ing ribosome biogenesis and assembly, and protein biosynthesis and processing. This may reflect the poor nutritional value of murine lung relative to YEPD and/or reduced growth (due to any number of stresses) during host-adaptation compared to broth culture. This trend is evidenced on multiple levels within our dataset, comprising repression of genes directing ribosomal protein synthesis, rRNA synthesis, RNA polymerase I and II activity, translation initiation and elongation, tRNA processing and synthesis, intracellular traffick-ing, secretion and vesicular trafficking (Table 4.1 and Supplementary Table E.3 and E.4). While such metabolic dampening is often observed in microbial systems under stress, the observed A. fumigatus regulatory signature mimics that of rapamycin-mediated TOR ki-nase (111) inhibition and typifies fungal starvation. The S. cerevisiae TOR proteins TOR1p and TOR2p are phosphatidylinositol kinase homologues, first identified as the targets of the immunophilin-immunosuppressant complex FKBP-rapamycin (280), combined deletion of which causes yeast cells to arrest growth, undergo a reduction in protein synthesis, accu-mulate the storage carbohydrate glycogen and acquire thermotolerance. Comparison of our dataset to that obtained following rapamycin-induced TOR inhibition in S. cerevisiae (111) reveals extensive overlap in induced (n = 35) and repressed (n = 90) homologous genes be-tween the two datasets (Tables E.5 and E.6). Thus a clear TOR repression-like starvation signature, relative to laboratory culture, is observable during early-stage infection, which may derive from the relative nutritional status of the tested conditions and/or slower growth within the context of our experiment. The indicated cellular down-turn in metabolism ob-served is strongly countered by up-regulation of genes encoding functions associated with amino acid and carbohydrate catabolism (Tables 4.1 and E.3 and E.4). These analyses were performed by myself and Elaine Bignell.

4.3 Results 146

Table 4.1: Over-represented Gene Ontology terms among differentially expressed genes. Table lists selected biological processes significantly over-represented among dif-ferentially expressed genes, with respect to their occurrence in the A. fumigatus Af293

genome. To identify over-represented Gene Ontology terms, loci having significantly dif-ferent expression were analyzed by the Expression Analysis Systematic Explorer (EASE)

(PMID:14519205), which is implemented in MEV within the TIGR TM4 microarray data

analysis suite. Numbers of genes in the indicated Gene Ontology categories were subjected to statistical analysis by EASE to identify categories overrepresented compared with the whole genome data set. Only categories with Fisher's exact (FE) test probabilities < 0.05

were included. Induced genes are represented above the double horizontal line. Repressed genes are represented are represented below the double horizontal line. Full results of the

analysis can be found in Table E.3 and E.4

GO ID GO Term List hits List size Pop hits Pop size FE

GO:0006810 transport 133 448 761 4219 1.08 x 10-10

GO:0008643 carbohydrate transport 19 448 40 4219 1.08 x 10-19

GO:0009063 amino acid catabolism 14 448 35 4219 3.41X10-9

GO:0044242 cellular lipid catabolism 10 448 19 4219 5.37x10-6

GO:0044270 nitrogen compound catabolism 15 448 42 4219 6.30x10-6

GO:0030001 metal ion transport 19 448 74 4219 1.28X10-5

GO:0015892 siderophore-iron transport 5 448 8 4219 1.76x10-4

GO:0006812 cation transport 21 448 94 4219 5.63x10-4

GO:0019541 propionate metabolism 3 448 4 4219 6.45x10-4

GO:0006830 high-affinity zinc ion transport 2 448 2 4219 4.38x10-2

GO:0006631 fatty acid metabolism 13 448 62 4219 1.17 x 10-2

GO:0019538 protein metabolism 174 561 781 4219 7.35x10-15

GO:0042254 ribosome biogenesis and assembly 51 561 161 4219 5.56 X 10-19

GO:0044237 cellular metabolism 366 561 2251 4219 6.51 x 10-19

GO:0007046 ribosome biogenesis 46 561 140 4219 1.08 X 10-9

GO:0016072 rRNA metabolism 41 561 118 4219 1.37X10-9

GO:0006364 rRNA processing 40 561 115 421.9 2.12x10-9

GO:0044238 primary metabolism 334 561 2028 4219 3.30 x 10-9

GO:0006457 protein folding 23 561 50 4219 1.48x10-8

GO:0006412 protein biosynthesis 72 561 302 4219 1.63x10-7

GO:0006783 heme biosynthesis 6 561 13 4219 4.03x10-3

GO:0009060 aerobic respiration 13 561 48 4219 8.02 x 10-3

GO:0006696 ergosterol biosynthesis 11 561 38 4219 8.45X10-3


Table 4.2: Functional classification of selected genes with altered transcript abun-dance, relative to laboratory culture, in the murine lung.

GO ID GO Term List hits FE Ergosterol and Heme Biosynthesis

-2.07 Afu1g05720 c-14 sterol reductase -2.14 Afu1g03950 cytochrome P450 sterol C-22 desaturase -2.18 Afu8g07210 hydroxymethylglutaryl-CoA synthase -2.30 Afu5g02450 farnesyl-pyrophosphate synthetase -2.35 Afu7g03740 14-alpha sterol demethylase Cyp51I3 -2.90 Afulg07140 c-24(28) sterol reductase -2.92 Afulg03150 c-14 sterol reductase -2.97 Afu2g00320 sterol delta 5 -3.38 Afu6g05140 sterol delta 5 -3.42 Afu5g14350 c-24(28) sterol reductase -3.93 Afu4g06890 14-alpha sterol demethylase Cyp51A -4.40 Afu4g09190 S-adenosyl-methionine-sterol-C- methyltransferase -4.30 Afulg07480 coproporphyrinogen III oxidase -2.44 Afu5g06270 5-aminolevulinic acid synthase -2.13 Afu5g07750 ferrochelatase precursor -3.02 Afu6g07670 cytochrome c oxidase assembly protein cox15

Iron acquisition 2.39 Afu7g06060 siderochrome-iron transporter (Sitl) 2.55 Afu7g04730 siderochrome-iron transporter 3.97 Afu6g13750 ferric-chelate reductase 6.11 Afu3g03440 MFS family siderophore transporter 3.40 Afu4g14640 low affinity iron transporter 2.64 Afu3g03650 sidG 6.45 Afu3g03640 siderochrome-iron transporter (MirB) 6.14 Afu3g03420 sidD 6.45 Afu3g03400 siderophore biosynthesis acetylase Acel (sidF) 3.41 Afu3g03390 siderophore biosynthesis lipase/esterase 4.85 Afu3g03350 nonribosomal peptide synthase (sidE) 4.01 Afu3g01360 siderochrome-iron transporter 5.29 Afulg17270 ferric-chelate reductase (Fret) 2.66 Afulg17200 nonribosomal peptide synthase (sidC) 2.61 Afu8g01310 metalloreductase (FRE1)

Nitrate assimilation -4.05 Afu1g12840 nitrite reductase -0.30 Afu1g12850 nitrate transporter (nitrate. permease) -2.33 Afu1g12830 nitrate reductase NiaD 0.47 Afu5g10420 nitrate reductase 7.06 Afu1g17470 high affinity nitrate transporter NrtB 0.56 Afu6g13230 Nit protein 2

Carbohydrate transport 5.70 Afu7g06390 maltose permease 5.88 Afu7g05190 maltose permeate 4.69 Afu6g11920 maltose permease 2.72 Afu5g00500 maltose permeate 4.75 Afu3g01700 maltose permeate 2.92 Afu2g10910 maltose permease 2.61 Afu1g03280 maltose permease 3.20 Afu3g03380 maltose 0-acetyltransferase 2.56 Afu8g07070 maltase 4.68 Afu7g06380 maltase 3.48 Afu4g00150 MFS maltose transporter 2.24 Afu8g07240 MFS maltose permease 3.08 Afu6g01860 MFS lactose permeate 2.08 Afu1g17310 MFS lactose permease 4.35 Afu3g01670 MFS hexose transporter 4.69 Afu2g08120 MFS monosaccharide transporter (IIxt8) 3.31 Afu5g14540 MFS monosaccharide transporter

continued on next page

4.3 Results 148

continued from previous page GO ID GO Term List hits FE

4.86 Afu4g00800 MFS monosaccharide transporter 5.06 Afu7g00780 MFS monocarboxylate transporter 2.77 Afu3g03320 MFS monocarboxylate transporter 3.04 Afu3g03240 MFS monocarboxylate transporter 2.81 Afu6g03060 monosaccharide transporter 3.47 Afu5g01160 monosaccharide transporter 3.77 Afu4g13080 monosaccharide transporter 2.81 Afu7g05830 MFS sugar transporter 6.97 Afu6g14500 MFS sugar transporter 2.05 Afu5g06720 MFS sugar transporter 4.47 Afulg11050 MFS sugar transporter 2.42 Afu3g12010 high-affinity hexose transporter 2.89 Afu3g00430 high-affinity glucose transporter

Nitrate assimilation -4.05 Afu1g12840 nitrite reductase -0.30 Afulg12850 nitrate transporter (nitrate permease) -2.33 Afu1g12830 nitrate reductase NiaD 0.47 Afu5g10420 nitrate reductase 7.06 Afu1g17470 high affinity nitrate transporter NrtB 0.56 Afu6g13230 Nit protein 2

Secreted Proteins 7.40 Afu5g14190 beta-glucanase 5.90 Afulg17510 lipase/esterase 5.64 Afu2g09380 cutinase 5.50 Afu8g07090 extracellular proline-serine rich protein 5.07 Afu2g05150 cell wall galactomannoprotein Mpg 5.01 Afu5g00540 extracellular signaling protein FacC 4.97 Afu7g01180 extracellular lipase 4.93 Afulg16250 alpha-glucosidase B 4.70 Afu3g14030 alkaline phosphatase 4.68 Afu2g00490 glycosyl hydrolase 4.68 Afu7g06380 maltase 4.66 Afu8g01050 lipase/esterase 4.63 Afu3g14910 extracellular signalling protein (factor C) 4.63 Afu8g01130 alpha-galactosidase C 4.58 Afu4g01070 acid phosphatase 4.54 Afu7g05610 glucanase 4.32 Afu4g00870 antigenic cell wall galactomannoprotein 4.07 Afu6g02980 extracellular exo-polygalacturonase 3.88 Afu8g04710 xylosidase 2.18 Afu3g07850 dipeptidyl aminopeptidase Stel3 3.50 Afu2g09030 secreted dipeptidyl peptidase 3.82 Afu6g11500 dipeptidase

Antigens 5.64 Afu4g09320 antigenic dipeptidyl-peptidase Dpp4 4.32 Afu4g00870 antigenic cell wall galactomannoprotein 7.13 Afu4g09580 major allergen Asp F2 6.44 Afu8g07080 elastinolytic metalloproteinase Mep 3.22 Afu6g10250 alkaline serine protease AorO

Carbohydrate and/or protein glycosylation 0 2.54 Afu8g02020 glycosyl transferase 6.10 Afu4g14070 glycosyl transferase 2.23 Afu5g00670 glycosyl hydrolase family 35 2.27 Afu6g11910 glycosyl hydrolase family 3 3.18 Afu4g00390 glycosyl hydrolase 2.07 Afu2g03270 glycosyl hydrolase 4.68 Afu2g00490 glycosyl hydrolase 2.14 Afu2g03120 cell wall glucanase (Utr2) 3.08 Afu8g05610 cell wall glucanase (Scw11) 5.07 Afu2g05150 cell wall galactomannoprotein Mp2

Carbohydrate catabolism 2.83 Afu6g14490 beta-glucosidase



continued from previous page GO ID GO Term List hits FE

2.11 Afu3g00230 beta-glucosidase 7.40 Afu5g14190 beta-glucanase 3.17 Afu5g14550 beta-galactosidase 2.18 Afulg14170 beta-galactosidase 7.16 Afu7g06140 beta-D-g]ucoside glucohydro]ase 3.59 Afu6g08700 beta glucosidase 3.25 Afulg16700 beta galactosidase 4.93 Afu1g16250 alpha-glucosidase B 3.83 Afu4g10150 alpha-glucosidase 4.63 Afu8g01130 alpha-galactosidase C 6.51 Afulg01200 alpha-galactosidase 2.87 Afu8g07300 alpha/beta hydrolase 4.46 Afu8g00570 alpha/beta hydrolase 2.44 Afu8g00530 alpha/beta hydrolase 2.04 Afu7g00830 alpha/beta hydrolase 5.21 Afu3g01280 alpha/beta hydrolase

Amino acid transport 4.72 Afu7g04290 amino acid permease (Gapl) 4.05 Afu2g08800 amino acid permease (Dips)

amino acid permease 3.73 Afu8g06090 amino acid permease

2.96 Afu2g10560 amino acid permease 3.27 Afulg09120 amino acid permease 2.96 Afu8g02200 proline permease 5.71 Afu7g01090 proline permease 3.38 Afu2g11220 proline permease 4.19 Afu8g01450 GABA permease 2.39 Afu7g00440 GABA permease 2.91 Afu5g14660 GABA permease 6.28 Afu5g00710 GABA permease 6.73 Affilg14700 allantoate transporter

Metal ion transport/homeostasis 4.97 Afu5g09360 calcineurin A 5.71 Afu7g01030 Calcium-transporting ATPase 1 (PMC1) 3.30 Afu3g10690 calcium-translocating P-type ATPase(PMCA-type) 2.25 Afu3g08540 Ca2+ binding modulator protein (A1g2) 2.77 Afu6g00470 plasma membrane zinc ion transporter 4.26 Afu5g03550 plasma membrane H(+)ATPase 3.26 Afu1g02480 plasma membrane ATPase 4.13 Afulg01550 high affinity zinc ion transporter 3.10 Afu8g01890 Na+/H-I- exchanger family protein 3.32 Afu7g04570 Na/K ATPase alpha 1 subunit

Oxidative stress resistance 4.27 Afu1g14550 Mn superoxide dismutase MnSOD -0.71 Afu4g11580 Mn superoxide dismutase (SodB) 2.34 Afu8g01670 bifunctional catalase-peroxidase Cat2

4.3 Results 150

Table 4.3: Distribution of induced and repressed genes among lineage specific cohorts and chromosomal locations. x2 analysis (with one degree of freedom) was used to test the distribution of induced and repressed genes with respect to lineage specificity and sub-genomic locations, based upon the null hypothesis that equal numbers of induced and repressed genes occur in each cohort. Asterisked gene populations are listed in Tables E.7, E.8, E.9, E.10. Table entries above the horizontal line denote lineage statistics; table entries below the horizontal line denote chromosome location statistics. Key to abbreviations: n = number of genes; n de = number differentially expressed; Induced obs = observed number of genes induced; Repressed obs = observed number of genes repressed; Induced E = expected number of induced genes; Repressed E = expected number of repressed genes.

rt. nd, Induced obs Down obs Induced E Repressed E X2 Aspergillus core 5095 965 414 551 483 483 19.45 < 0.0001 Affc-core 6907 1468 791 677 734 734 8.86 0.0029

Affc-specific 180 64* 54 10 32 32 30.25 < 0.0001 Mu-specific 428 135* 77 58 68 68 2.68 0.102

0-300 kb 1326 470 367 103 235 235 148.289 < 0.0001

301-600 kb 1504 365 229 136 182.5 182.5 23.696 < 0.0001

>600 5779 1345 686 659 672.5 672.5 0.5 0.4795

4.3.4 Lineage specificity and locational analyses of differentially expressed genes

From an evolutionary perspective, relative proportions of genes being over- and underrepre-sented in the analysis differed significantly within lineage-specific gene cohorts (Figure 4.4 A and Tables E.7, E.8, E.9, E.10). Genes having increased transcript abundance during infection are significantly enriched (p<0.0001 by chi-square analysis, see Tables E.7, E.8, E.9, E.10) among differentially expressed genes (n = 64) unique to the A. fumigatus lin-eage. Thus 93.6% of A. fumigatus genes having orthologues restricted to two very closely related, but differentially virulent, species Neosartorya fischeri (anamorph of Aspergillus fis-cherianus) (Genbank sequenced genome identifier AAKE00000000) and Aspergillus clavatus (Genbank sequenced genome identifier AAKD00000000) are more abundantly represented during the initiation of infection (Supplementary Data Tables E.7, E.8, E.9, E.10). In contrast only 8% of genes having orthologues in all six Aspergillus species sequenced to

date (i.e. the Aspergillus core genome) are more abundantly represented under these con-ditions (n = 5095). This invariable core genome encodes many functions associated with information processing, central metabolism and cell growth, retention of which is most likely to be essential for cellular survival (197). Narrowing the phylogenetic sampling to include only A. fumigatus and its relatives N. fischeri and A. clavatus distinguishes several subtelomeric genomic islands upon which phenotypic variation between species, including differing pathogenicity, might depend (197). Accordingly we found differentially expressed genes to be unevenly distributed amongst A. fumigatus chromosomes (Figure 4.4 B and

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Chapter 4. Sub-telornere directed gene expression during initiation of invasive aspergillosis 151

Figure 4.3: A genome-wide transcriptional snapshot of A. fumigates A1293 dur-ing intiation of murine infection. Red and green horizontal lines correspond to individ-ual up- and down-regulated genes, respectively. Thin light gray horizontal lines indicate the positions of all other genes. (SM) and (aspcore) are density graphs of secondary metabo-lite and Aspergillus-core genes, respectively, expressed as a percentage of the total bases contained per gene type, per non-overlapping 2 kb of chromosomal sequence. Induced and repressed gene clusters, are depicted by red and green rectangles, respectively, below each chromosome. A complete listing of genes housed in these co-regulated clusters can be found in Table S6. Light blue/gray horizontal bars represent putative centromeres and the pink horizontal bar in chromosome 4 represents a region of ribosomal DNA. Figure produced by Jonathon Crabtree. Higher resolution figure available at http: //ukpmc .ac. uk/articlerender.cgi?artid=16630688mendertype=figure&id=ppat-1000154-g003

4.3 Results 152

Aspergillos core Affc-core Affc-specific

Afu-specific A n=5095 n - 6907 n = 180 n = 428 •

> 600 kb

301 - 600 kb

0 — 300 kb

(Distal)

(Intermediate)

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B n = 6494 n = 1634 n = 1509

Increased transcript abundance

II Decreased transcript abundance

Figure 4.4: Distribution of lineage specific and telomere-proximal genes among differentially expressed host adaptation dataset. (A) Lineage specificity of A. fu-migatus genes having altered transcript abundances, relative to laboratory culture, in the murine lung. The Aspergillus-core (Asp-core) set contains A. fumigatus Af293 proteins that have orthologues in A. clavatus (AAKD00000000), N. fischeri (AAKE00000000), As-pergillus terreus NIH2624 (AAJNO1000000), Aspergillus oryzae RIB40 (431), A. nidulans FGSC A4 (223) and Aspergillus niger CBS 513.55 (534). The Affc-core set were defined as A. fumigatus Af293 proteins that have orthologues in N. fischeri and A. clavatus. The Affc-unique set is a sub-set of Affc-core proteins that do not have orthologues in A. terreus, A. oryzae, A. nidulans or A. niger. Asterisks indicate gene sets which are listed in Table E.7, Table E.8, Table E.9 and Table E.8. Underlined values significantly deviate from the null hypothesis that an equal number of induced and repressed genes will occur in each cohort, as estimated by Chi-square analysis (Tables E.7, E.8, E.9, E.10). (B) Chromoso-mal distribution of A. fumigatus genes having altered transcript abundances, relative to laboratory culture, in the murine lung. Distances from telomeres (kb) are noted above pie charts. Asterisked gene sets are listed in Supplementary Table Table E.7, Table E.8, Table E.9 and Table E.8. Underlined values significantly deviate from the null hypothesis that an equal number of induced and repressed genes will occur in each cohort, as estimated by Chi-square analysis (Tables E.7, E.8, E.9, E.10).

7 0 0 11

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Alkaline (211)

Figure 4.5: Overlap between murine adaptation and in vitro stress datasets. Venn diagrammatic representation of overlap between murine adaptation dataset and those of nitrogen starvation, iron starvation and alkaline shift. Genes are listed in Dataset E.3. Figure produced by Elaine Bignell.

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4.3 Results 154

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Figure 4.6: Comparative analysis of A. fumigatus gene expression datsets.. A Cross experimental comparison of A. fumigatus gene expression aligning log2 ratios ob-tained during host adaptation (mice); exposure to neutrophils (neut), increased expression in parental strain versus AlaeA mutant, acid shift (acid), iron starvation (iron), oxygen depletion (anaer) and oxidative stress (H202) for various genes. The colour bar indicates the range of log2 expression ratios; grey bars indicate genes from which signals were un-detectable for technical reasons. Experimental conditions are described in Materials and Methods. LaeA dataset is taken from Perrin et. al. (539). Comparative analyses were implemented in TM46. Figure produced by Natalie Federova.

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Figure 4.7: Characterisation of A. fumigates growth, relative to YEPD. (A) Com-parative analysis of Af293 radial growth on YEPD and synthetic murine lung tissue medium (114LT). Triplicated, spot-inoculated plates containing single 100 spore inocula were incu-bated at 37°C. (B) Growth curve analysis of Af293, performed in triplicate using liquid YEPD, or AMM containing 1% glucose and either 5 mM ammonium tartrate or 5 mM hy-roxyproline as nitrogen source. Cultures were inoculated to a final concentration of 5 x 106 spores/ml and incubated under aerobic conditions at 37°C with shaking at 150 rpm. At selected timepoints mycelia were harvested on Miracloth, encased in Whatmann paper and dried at 37°C for 48 hours before weighing. Experiments and figures produced by Timothy Cairns

23 genes in secondary metabolism clusters

Subtelomeric (1509)

23 subtelomeric

4.3 Results 156

Up in vivo Up in wt versus A/aeA

(1266)

(415)

Up in vivo Up in WT versus laeA (1282) (415)

Secondary metabolism clusters (209 [133 subtelomeric])

Figure 4.8: Expression of LaeA-regulated genes during initiation of murine infec-tion. Venn diagram representation of overlap between genes repressed in AlaeA (539) and those having increased transcript abundance during murine infection, according to propor-tions having subtelomeric locations, and secondary metabolism functionality (on the basis of annotation).


Supplementarty Data Tables E.7, E.8, E.9, E.10). Induced genes form a significantly in-creased proportion of differentially regulated functions in intermediate (p < 0.001) and subtelomeric (p < 0.001) regions of the chromosomes (Figure 4.4 B and Supplementary Data Tables E.7, E.8, E.9, E.10). While only 16% of the predicted A. fumigatus gene reper-toire is housed within 300 kb of telomeres (classed as the subtelomeric gene repertoire in our analyses), 29% of transcripts having increased abundance, relative to laboratory cul-ture, in the murine lung are located to such subtelomeric areas, compared to just 11% of down-regulated transcripts. Moreover, 28% of the entire subtelomeric gene repertoire is represented in the induced dataset compared to only 8% of subtelomeric genes represented among down-regulated functions (Supplementary Data Tables E.7, E.8, E.9, E.10). These analayses were performed by Natalie Federova.

4.3.5 Clustering of induced genes

Regarding our A. fumigatus gene expression dataset as a function of chromosomal locus (Figure 4.3) we discovered that many induced A. fumigatus genes are found in contigu-ous clusters. To investigate this further a custom script, developed by Jonathon Crabtree, was used to automate cluster identification. This identified numerous genomic loci within which co-ordinate regulation of a minimum of 5 closely neighbouring genes can be observed (Figure 4.3 and Table S6). Co-ordinated expression of physically clustered genes is a promi-nent feature of the induced, but not repressed, gene set and we observe a large proportion (40%) of up-regulated physically clustered genes to reside within 300 kb of chromosome ends (Figure 4.3 and Table 4.4). The clusters are comprised of up to 34 co-ordinately expressed genes and include loci directing biosynthesis of siderophores (cluster 33) and two known sec-ondary metabolites, pseurotin and gliotoxin (clusters 72 and 60, respectively). The gliotoxin biosynthetic cluster is not subtelomerically located, being 700 kb from the telomere (as annotated by Perrin et. al. (539)). The pseurotin biosynthetic cluster, however, (as anno-tated by Maiya et. al. (439)) is contained within the fumitremorgen cluster (Afu8g00100-Afu8g00720) at 100 kb from the telomere (539). Pseurotin (439; 367; 277) is a neuritogenic, nematicidal quinone and gliotoxin an immunotoxin which supports A. fumigatus virulence in some murine models of invasive pulmonary aspergillosis (74; 145; 379; 652; 670). We observed four other postulated, but uncharacterised, secondary metabolite gene clusters in-duced during early stage A. fumigatus infection, including a large proportion of genes on the left arm of chromosome 8, predicted to encode a fumitremorgen biosynthesis supercluster (75). Thus, it would seem that selective expression of a subset of secondary metabolite loci facilitates initiation of mammalian infection. While gliotoxin biosynthesis is dispensable for virulence in some murine models, our analysis demonstrates that this host environment is nonetheless conducive to immunotoxin production, and further insights on virulence mech-

4.3 Results 158

anisms relevant to neutropenia and/or corticosteroid therapy await comparative analyses of fungal gene expression in each of these strikingly different host settings, an analysis which is currently underway in our laboratory. A fully annotated table of clusters, indicating between-species synteny within cluster loci, and all accession numbers, can be viewed in Supplementary Dataset E.2.

4.3.6 Comparative analyses of murine adaptation- and in vitro gene expression datasets

The complexity of the transcriptional signature derived from host adaptation analyses is likely to originate from convergence of multiple environmental cues, coupled with metabolic and morphologic effects. Although functional categorisation of differentially expressed func-tions (Tables 4.1, E.3 and E.4) could identify metabolic and physiological trends during initiation of infection, environment-related signatures were less easy to discern. To ascer-tain physiologically relevant features of the host environment we compared the transcrip-tome of host-adapting germlings to those of in vitro A. fumigatus cultures exposed to iron limitation, nutrient limitation, alkaline stress, acid stress, neutrophils, oxidative stress or anaerobic stress. The resulting transcriptomic responses varied in magnitude (Table 4.4) but assignment of cut-off log2 ratio values of +2 and -2 across all of the analyses enabled us to distinguish several important aspects of the A. fumigatus host-adaptive response (Fig-ure 4.5). The transcriptional signatures of paramount importance among those examined were alkaline adaptation, iron deprivation and nutrient starvation, which are remarkably prominent in the infection dataset. Using the aforementioned log2 cut-offs, 24 of 43 iron-regulated genes were identified as differentially expressed during host adaptation, of these 18 were more abundantly represented (Figure 4.5 and Supplementary Dataset E.3), and 6 less abundantly represented.


Table 4.4: Comparative analysis of genetic distribution among genes having dif-ferential transcript abundances in microarray analyses using A. fumigatus RNA following exposure to in vitro or murine-adaptive stress, or laeA gene deletion. Gene density in telomere-proximal (0 - 300 kb from telomeres), intermediate (300 - 600 kb from telomeres) and telomere-distal (>600 kb from telomeres) regions of the A. fumiga-tus genome are indicated (Genome stats); (no sec mets) indicates omission of secondary

metabolite biosynthetic genes (identified as detailed in the Materials and Methods section) from the tested dataset.

Experimental condition Total genes in dataset Proximal % Intermediate % Distal % A. fumigates-specific % Genome stats 9632 1509 16 1634 17 6494 67 2201 23

Clustered in vivo up 658 227 34 129 20 302 46 218 33 AlaeA up 415 102 32 59 14 254 61 122 29 in vivo up 1282 367 29 229 18 686 54 374 30

Nitrogen starvation up 626 180 29 115 18 331 53 33 33 In vivo up (no sec mets) 1196 327 27 223 19 646 54 355 30

Neutrophils 60 min up 312 83 27 81 26 148 47 80 26 Alkaline adaptation up 211 53 25 39 18 113 54 6 6

AlaeA up (no sec mets) 318 70 22 58 18 190 60 50 16 Acid stress up 83 18 22 21 25 44 53 43 52

Iron limitation up 28 4 14 4 15 20 71 8 30 AlaeA down 528 93 18 99 19 336 64 108 20

Oxidative stress up 54 9 17 9 17 36 67 22 54 In vivo down 898 103 11 136 15 659 73 126 14

Relaxing the cut-off criterion to encompass all differentially regulated genes in the host

adaptation dataset allowed complete capture of the iron regulon (Figure 4.6). Among these genes are the siderophore biosynthetic genes sidA (Afu2g07680), sidD (Afu3g03420) and sidC (Afu1g17200), the latter two discerning biosynthesis of both intra- and extracel-

lular siderophores during infection as substantiated by previous findings (619), and four siderochrome/siderophore transporter proteins Afu7g06060, Afu7g04730, Afu3g03440 and Afu3g03640. Preferential gene expression following a 60 minute shift from acid to alka-line medium could also be strongly correlated with that observed during infection (Figure

4.5 and Supplementary Dataset E.3). Alkaline adaptive capability, previously found to be essential for A. nidulans virulence in neutropenic mice (66), is likely to be important for growth of A. fumigatus spores at physiological pH. Accordingly we identified 102 genes pref-erentially expressed during both murine infection and in vitro alkaline adaptation (Figure 4.5 and Supplementary Dataset E.3). Among them are 36 genes having unknown function, two sodium ATPases (Afu6g03690 and Afu4g09440), the plasma membrane zinc ion trans-

porter (Afu6g00470) and an alkaline phosphatase (Afu3g14030). Interestingly, we found no concordance between iron starvation and alkaline adaptation (Figure 4.5). Since acidifica-tion of the macrophage phagolysosome is an essential step in ROS-mediated A. fumigatus killing we also assessed the transcriptome of Af293 germlings upon shift from pH 7 to pH 3, using a rich medium and hydrochloric acid. No concordance between the resulting dataset and that of host adaptation was evident, indeed (despite the magnitude of the

4.3 Results 160

murine infection dataset) most functions upregulated in response to an in vitro acid shift were less abundantly represented in our infection analyses (n = 18, Supplementary Figure E.2) thus we conclude that acid stress, at least within the context tested in this analysis (which can only approximate conditions encountered in the host) is not relevant during host adaptation. This agrees with our observation that alkaline adaptation is a physiologi-cal cue of primary importance in this murine model of infection (Figure 4.5). An essential component of phagocyte defence against A. fumigatus conidia and hyphae is the NADPH oxidase-mediated respiratory burst which generates reactive oxygen species (ROS) required for fungal killing. To assess the A. fumigatus transcriptional response to oxidative stress, conidia were grown in rich medium at 37°C prior to shift into similar medium containing 17 mM hydrogen peroxide. Comparison of the resulting dataset to that obtained from host-adapting germlings revealed some concordance, this time revealing a subset of genes having decreased transcript abundance both in vitro and during murine infection (Figure E.2).

A common theme among this group of genes is ergosterol and haem biosynthesis, evidenced by common behaviour of both 14-alpha sterol demethylase Cyp51A-encoding genes (Afu4g06890 and Afu7g03740) and a coproporphyrinogen III oxidase homologue (Afu1g07480). Oxygen depletion was achieved by transfer of A. fumigatus hyphae, fol-lowing 16 hour growth in rich medium, to anaerobic chambers containing two palladium catalysts. We were unable to correlate gene expression under these anaerobic stress con-ditions with gene expression during infection (Figure 4.6). Finally, we assessed germlings grown in 20 ml of RPMI1640 with L-glutamine, 25 mM HEPES and 5% fetal bovine serum for 7 hours at 37°C following exposure to human neutrophils at a multiplicity of infection of 1:1 for 60 minutes, the reference sample for this analysis being germlings incubated in the absence of neutrophils. Of 57 A. fumigatus genes upregulated in response to neutrophil ex-posure in vitro, 18 (60%) were also more abundantly represented during initiation of murine infection (Figures 4.6 and E.2). Interestingly these included the two major known A. fu-migatus antioxidant enzymes, Mn superoxide dismutase (Afu1g14550) and the bifunctional catalase-peroxidase Cat2 (Afu8g01670). Whether representation of these transcipts among those differentially expressed in both murine and laboratory culture is indeed neutrophil-specific remains to be determined.

4.3.7 Nutrient limitation is a relevant physiological cue during A. fumigatus initia-tion of murine infection

To investigate the relevance of nutrient starvation during host adaptation we made sev-eral growth and gene expression analyses. Hypothesising that YEPD is nutritionally more robust than murine lung tissue we compared radial growth of A. fumigatus, in triplicate

Chapter 4. Sub-telomere directed gene expression during initiation of invasive asp ergillosis 161

from an inoculum of 100 spores grown at an agar/air interface on Petri dishes, on YEPD and on a synthesized murine lung tissue medium (MLT) composed of homogenised murine lung tissue (80%) and water/agar (20%), overlaid upon a water/agar baseplate. Growth of A. fumigatus was completely unsupported by water/agar base with no evidence of conidial germination after 7 days. Growth on YEPD produced conidiating colonies averaging 66 mm (n = 3) in diameter after 7 days at 37°C, whereas MLT as growth medium supported significantly less growth, reaching a maximum colony diameter of 44 mm (Figure 4.7A). Any concern that reduced radial growth observed on MLT medium originates from iron deprivation can be allayed by reference to radial growth analysis of two independent wild type A. fumigatus isolates, CEA10 and ATTC46645 (618), where equivalent growth is ob-served in the presence and absence of iron, and on blood agar medium. Thus, from this solid growth analysis, we conclude that MLT supports slower A. fumigatus colony growth than YEPD under laboratory conditions. Our MLT analysis could not support the volume of liquid culture required to perform growth curve analyses, moreover, the viscosity of the medium would have hindered dry weight measurements. To support our conclusions on nutritive status of the host environment, within the context of the experimentation per-formed during our infection analyses, we assessed log2 ratios obtained from competitive microarray hybridisation, using doubly amplified A. fumigatus mRNA extracted from ni-trogen starved germlings, and the same YEPD reference sample used for the initial host adaptation analysis (samples F and G, respectively, Supplementary Table E.1). Nitrogen starvation was exerted in shaken liquid culture using minimal medium, with hydroxyproline as nitrogen source, for a period of five hours. Hydroxyproline is a rational candidate nitrogen source during initiation of mammalian pulmonary infection, being a widely used surrogate marker of lung injury whose concentration in bronchoalveolar lavage fluid permits quantita-tive assessment of collagen breakdown (3; 451). Transcript levels of the proline permeases Afu2g11220, Afu8g02200 and Afu7g01090 suggest induction of proline uptake during initia-tion of infection, and laboratory culture on solid medium confirms that hydroxyproline can support aconidial filamentous growth of A. fumigatus on minimal medium in the presence of a repressing carbon source such as glucose (data not shown). To confirm the inferiority of hydroxyproline as a nitrogen source (relative to YEPD) we performed dry weight growth curve analyses (Figure 4.7B) including a widely used Aspergillus minimal medium (MM) for comparison. Nitrogen starvation rendered 1047 genes subject to differential expression, relative to the doubly amplified YEPD reference. Several notable features of the resultant dataset (Dataset E.4) support our conclusion that, relative to YEPD laboratory culture, initiation of murine infection occurs under nutrient stress. As with our analyses of host adaptation, over-represented Gene Ontology (GO) terms among differentially expressed genes identified ribosome biogenesis and assembly, and protein biosynthesis and processing

4.3 Results 162

as the most significantly down-regulated functional categories (Table E). We also identified significant over-representation of cell cycle-related functions among

genes preferentially expressed during growth in hydroxyproline, including cell cycle regula-tion, mitosis, nuclear migration, chromosome segregation and karyogamy (Table E). This is a particularly satisfying finding since, supported by our growth curve analyses (Figure 4.7 B), a clear distinction between A. fumigatus growth phase in the compared media is discern-able. This was not a feature of the murine versus YEPD comparison which, importantly, lessens the probability that differences associated with cell cycle stage or growth rate pre-side over environmental cues within the host adaptation dataset. Direct comparisons of the murine and nitrogen starvation datasets identified an overlap of 280 differentially expressed genes common to both, of these 24 genes are also common to the TOR kinase nutrient limitation geneset, and are indicated in Supplementary Data Tables E.5 and E.6. 156 genes preferentially expressed under nitrogen starvation conditions (relative to YEPD laboratory culture) were similarly favoured during host adaptation (Supplementary Dataset E.4).

Assessing locational bias among the hydroxyproline dataset, 29% of the geneset was found to reside subtelomerically (Table 4.4) with 180 out of 634 preferentially expressed genes housed within 300 kb of telomeres. This is comparable to the level of subtelomeric gene expression identified during initiation of murine infection (Table 4.4) and indicates that, relative to growth in a nutrient-rich laboratory culture, adaptation to growth in a nu-tritionally challenging environment prompts expression of a subtelomeric gene repertoire. A number of physically linked coregulated genes came to prominence in the hydroxyproline starvation dataset (Supplementary Data Table E.11). 16 clusters conforming to the previ-ously applied cluster algorithm were identified, 8 of which were subtelomeric and 3 of which encompass genes in secondary metabolite loci, as defined by bioinformatic analyses. Of the three latter loci one cluster (Afu6g03390 - Afu6g03490) subject to regulation by the LaeA methyltransferase (539), the product of which is currently unknown, is also expressed dur-ing murine infection. Beyond this, correlation between clustered gene regulation in murine and nitrogen starved growth was modest.

4.3.8 The LaeA regulon is represented among genes preferentially expressed during intitation of murine infection

Given the predominance of clustered and subtelomeric gene loci among host adaptation genes, we compared our dataset to that produced by Perrin et. al. (539) during study of laeA gene deletion. An A. fumigatus AlaeA (Afu1g14660) mutant, which has decreased virulence in both neutropenic and hydrocortisone treated mice (350; 669), demonstrates significantly lower expression of genes in 13 secondary metabolite biosynthetic clusters in-


eluding that of gliotoxin. LaeA was found to influence expression of a subset of lineage and species-specific genes therefore we tested the overlap between functions down-regulated in the absence of LaeA and functions having greater transcript abundance during murine infec-tion, hoping to decipher a link between genes under LaeA control, and those active during initiation of murine infection. Out of 415 genes down-regulated in the absence of LaeA, we identified 99 genes having increased abundance during initiation of murine infection (Figure 4.8). Functional categorisation of shared genes revealed that 40% (n = 40) were involved in secondary metabolite biosynthesis, among these we could identify three complete secondary metabolite clusters, those directing gliotoxin and pseurotin biosynthesis as well as the ge-netic locus mentioned above (Afu6g03390) whose biosynthetic product is unknown. Perrin et. al. also found 54% of the LaeA-regulated gene clusters showing differential expression under laboratory conditions were located within 300 kb of the telomeres. We therefore ex-tended our analysis still further, determining the proportions of subtelomeric and secondary metabolism cluster genes shared between the two datasets. This identified 49 and 40 genes, having subtelomeric locations and secondary metabolite biosynthetic functions respectively (Figure 4.8).

Finally we analysed all of the in vitro and in vivo datasets comparatively to assess the occurrence of subtelomeric bias among differentially expressed genes, aiming to deter-mine whether the induction of genes at the telomeric extremities of chromosomes was a standard feature of adaptation to environmental alterations rather than a host adaptation phenomenon (Table 4.4). The analysis identified murine adaptation, LaeA deletion, neu-trophil exposure and nitrogen starvation as the conditions most enriching for transcript abundance among subtelomeric genes, where 29%, 32%, 27% and 29% of the respective cohorts were located. Interestingly, removal of secondary metabolism genes from the anal-ysis dramatically reduced the LaeA regulated subtelomeric gene cohort to 22% while only minimally impacting representation of murine adaptation genes (Table 4.4). Taken together these analyses indicate that a significant component of the LaeA regulon, comprised mainly of secondary metabolism genes, is represented among transcripts more abundant during in-fection. Furthermore the subtelomeric bias observed among differentially expressed murine adaptation genes extends beyond secondary metabolite biosynthesis and does not appear to be a general feature of adaptation to environmental change.

4.4 Discussion

We present a methodology for A. fumigates transcript profiling during initiation of murine infection and a comparative analysis of global transcriptional programming, in laboratory culture and the mammalian lung. We were able to optimize a technical and statistical

4.4 Discussion 164

framework sufficiently robust to reproducibly quantify relative transcript abundances using minute samplings of A. fumigatus germlings. We chose to co-hybridize mock samples from the same amplification protocol (thereby determining the true biological effect in a bench-mark sample, in this case total RNA) and then, by comparison to cohybridisations using unamplified samples, indirectly estimate the systematic effect of the amplification in our analysis. The t—testing framework (509; 508) then allowed us to identify genes where the log ratios are significantly different due to one factor i.e. the amplification protocol. This en-abled an estimation of the proportion of genes showing amplification-dependent bias which we carried into the murine experiment, where comparison of unamplified versus amplified material is impossible. Clearly, we assume that this estimate can be reliably applied to sim-ilar experiments, in doing so we also assume that the amplification process depends only on the protocol adopted, and not the underlying gene expression dynamics. A further technical consideration in planning experiments comparing murine and laboratory samples is differ-ential treatments. Given the nature of our analysis, differential treatment of the germling samples was necessary to avoid osmotic shock in either instance. Water lavage would impose an osmotic shift on germlings rescued from the lung and saline treatment would osmotically shock laboratory cultured germlings. Thus the technical limitations imposed by such com-parative analysis must be accepted, however, in terms of maximally preserving transcript abundances within the context of the experiment, we believe our treatments of the samples to be appropriate.

To identify factors governing adaptation to the host niche we compared laboratory and murine lung samples aiming to find fungal attributes preferentially employed during in-fection. Therefore our findings document genes having increased transcript abundance in the A. fumigatus sample of murine origin, relative to that derived from laboratory cul-tured fungus. We performed our comparative analysis using doubly amplified mRNA from developmentally matched A. fumigatus germlings following laboratory culture, or growth in neutropenic murine lungs. In total we identified 2164 genes having altered transcript abundance. Functional analysis of the dataset flagged certain putatively relevant physio-logical cues which we then pursued by additional in vitro analyses. Careful interpretation of the dataset with respect to specific nutrient acquisition mechanisms, and within the con-text of the comparison performed here, can lend powerful insight into accessible nutrients in the mammalian niche. Nitrate assimilation (which is strongly inducible by nitrate in the absence of preferred nitrogen sources ammonium and glutamine) (17) is not preferen-tially employed in the murine host as evidenced by co-ordinate down-regulation of crnA (Afu1g12850), niaD (Afu12830) and niiA (Afu1g12840). This may be due to a) equivalent nutritional status of YEPD and murine lung or b) to the absence of nitrate as a nitrogen source during initiation of infection, or both. We were able to confirm, by radial growth


analyses in vitro using YEPD and a synthetic lung tissue medium IVILT, that YEPD is nu-tritionally superior to MLT based upon the rate of radial growth supported (Figure 4.7A). Coupled with the observation that the nitrogen metabolite repression gene product AreA is required for full virulence (281) a likely explanation for slowed growth and repression of nitrogen assimilation, is the utilisation of alternative non-preferred nitrogen sources during establishment of disease, such as amino acids.

Strong support for this conclusion is provided by high level induction of the areA-dependent nitrogen-scavenging enzyme L-amino acid oxidase LaoA (Afu7g06810) which enables Aspergilli to catabolise a broad spectrum of amino acids in nitrogen starvation conditions (436). Notably a by-product of such catabolism is ammonium. However the likelihood that sufficient ammonium is produced by these reactions to prevent starvation is diminished by the observed general starvation response in our dataset. Catabolism of amino acids during initiation of infection is also evidenced by induction of the methyl citrate synthase enzyme (Afu6g03590) which acts to detoxify the intermediates of propionyl-coA generating carbon sources (436), such as cysteine, isoleucine and methionine. An essen-tial role for methylcitrate synthase in murine aspergillosis has recently been demonstrated (312) thereby demonstrating the value of our approach in generating physiological infor-mation on virulence mechanisms within the context of murine infection. Strong themes among genes having lowered transcript abundance during murine infection are ribosome biogenesis and assembly, and protein bisoynthesis and folding (Table 4.1). Such signatures are commonly observed among microbes under stress, and in this instance might indicate a slowing of growth in the murine lung, relative to laboratory culture. We reasoned that a mechanism possibly linking such transcriptional profiles to nutrient starvation is TOR mediated ribosomal gene regulation, which tightly couples protein synthesis and cellular growth to availability of nutrients and physiological status to balance the opposing forces of protein synthesis and degradation. This is pivotal for cellular fate determination in many organisms (270; 172) directing cells towards either proliferation (through the cell cycle) or vegetative growth (increase in size). Comparison of our dataset to that generated following rapamycin-mediated TOR kinase inhibition in S. cerevisiae revealed a very marked overlap (n = 125) in differentially regulated homologous genes (Supplementary Data Tables E.5 and E.6). This is in keeping with defined roles for TOR kinase function in S. cerevisiae, which includes the regulation of transcription in response to nutrients (111). A strong correlation between developmental programming and microbial secondary metabolite biosynthesis has been well-documented (351) and while plasticity of nitrogen metabolism demonstrably sup-ports A. fumigatus virulence (375) the role of the single, and likely essential, A. fumigatus TOR kinase homologue, TorA, (Afu2g10270) in this process remains untested. Intriguingly, however, a link between TOR kinase function and secondary metabolite production, par-

4.4 Discussion 166

tially through AreA, has recently been established in the rice pathogen Fusarium fujikuroi (685) where, in addition to the target genes in common with yeast and other eukaryotes, the AreA-regulated giberellin and bikaverin biosyntheis genes are also under the control of TOR. This raises the possibility of a relatively limited investment in secondary metabolism, within the context of our analysis, at the tested timepoint of murine infection.

Genes expressed during nitrogen starvation are also expressed during virulence in Mag-neporthe grisea (181), where two wide-range regulators of nitrogen catabolism genes, NPR1 and NPR2 (395) are required for virulence. Contrary to our analysis nitrate and nitrite reductase activities were found to be relevant for M. grisea during rice infection (181), how-ever, a number of amino acid transporting proteins predominated among induced functions during both in vitro nitrogen starvation and infection.

Common to that analysis, and ours, was increased abundance of proline oxidase (Afu3g02300) and proline permease (Afu2g11220, Afu7g10190, Afu8g02200) encoding genes, which, in the context of our experiment might have special relevance given the high hydroxyproline of collagen tissue, and the notable proline requirement of an attenuated A. fumigatus deletion mutant lacking the Ras-related protein RhbA (524). To test the effect of nitrogen limitation on gene expression, in the context of the host adaptation study performed, we returned to the YEPD reference sample employed for the initial analysis (sample F, Supplementary DataTable E.1), this time performing competitive hybridisations with doubly-amplified RNA obtained from a nitrogen-starved laboratory culture (sample G, Supplementary Data Table E.1). Given the predominance of hydroxyproline among collagen amino acids content, and its release into bronchoalveolar lavage fluid upon tissue injury (3) we used hydroxypro-line as sole source of nitrogen for the starvation experiment. Hydroxyproline supported markedly slowed growth of A. fumigatus in liquid culture (Figure 4.7 B) relative to YEPD. We identified a significant overlap between the two datasets amounting to 280 genes.

Functional anlaysis of the resulting datasets revealed that, in keeping with the murine adaptation dataset, ribosome biogenesis and protein biosynthesis were markedly down reg-ulated, moreover, functions associated with mitosis and cell cycle were more abundantly represented among the categories favoured under nitrogen starvation, relative to rich labo-ratory growth. Given the differences between the tested media in terms of ability to support A. fumigatus growth (Figure 4.7 B) it is pleasing to see that such a theme was not apparent from the murine adaptation dataset where the degree of nutrient limitation is unlikely to be as severe as that imposed in the nitrogen starvation conditions we used here. Interestingly 29% of the genes preferentially expressed during nitrogen starvation were subtelomerically located, and a degree of clustered gene expression was observable. Thus induction of the subtelomeric gene repertoire becomes important during nutrient deprivation, a trend which was not observable in response to any of the other in vitro stresses, other than neutrophil


exposure the physiological relevance of which requires further investigation. The uptake and catabolism of other amino acids released by proteolytic digestion of

murine lung parenchyma might provide a source of nitrogen during A. fumigatus infection, which is supported in our analyses by increased transcript abundance of various secreted proteases (Table 4.2). Extracellular proteases are implicated as virulence factors in invasive aspergillosis (394). An elastolytic protease, produced when A. fumigatus is cultured on the insoluble matrix from bovine lung is also produced during spore germination in infected lungs of neutropenic mice, as judged by immunogold cytochemical localization (365), and mutants unable to produce this protease were deficient for virulence in the murine model. An elastinolytic metalloprotease, characterized by the same group was similarly visualized during murine lung infection (444). Many species of human pathogenic fungi secrete pro-teases in vitro or during infection. Full virulence associated with A. fumigatus protease mutants (394; 325) is presumed to be due to redundancy among the many enzymes pro-duced by this organism which may degrade the lung parenchyma to release utilisable carbon and nitrogen during infection. A directed analysis of the role of such proteins in virulence might now be possible on the strength of our data. Time course analyses of A. fumigatus growth during murine infection will reveal stage-specific gene expression in the absence of confounding sample treatments. These analyses are underway in our laboratories.

Fungal oxygen-sensing mechanisms have been linked to cell membrane sterol levels in S. pombe and C. neoformans where homologues of the mammalian Sterol Regulatory Element Binding Protein (SREBP) transcription factors, in complex with SREBP cleavage-activating protein (SCAP) partners, undergo cellular translocation (from the endoplasmic reticulum to the golgi) prior to proteolytic SREBP activation (309), (123). The C. neoformans SREBP homologue, Sre1p, plays an important role in low oxygen adaptation and infection (123). Biosynthesis of sterols and unsaturated fatty acids is an aerobic process in S. cerevisiae and in C. neoformans, the hypoxia-mimicking agent cobalt chloride and oxygen limitation target sterol biosynthetic gene expression. This was intriguing to us given the broadly observed reduction in transcript abundance among ergosterol biosynthetic genes, relative to laboratory culture (Table 4.2).

Our initial hypothesis attributed this effect to oxygen limitation in the murine lung environment since airway obstruction, intraalveolar exudates and inflammation, or damage to alveolar capillaries (all observed in our murine modelling of pulmonary aspergillosis) pose a significant barrier to proper oxygenation in human lungs (703). Oxygen depriva-tion in mammals leads to a transcriptional induction of genes for adaptation to hypoxia (84) and efforts to characterise the transcription profile of murine immune responses to A. fumigatus infection indicate that hypoxia is relevant in the neutropenic murine lung at 24 hours post-infection (32). Co-hybridisation of murine lung cDNAs, derived from in-

4.4 Discussion 168

fected immunocompetent and immunocompromised mice with a murine immunology array set identified upregulation of murine ARNT (log2 ratio = 2.09) the obligate heterodimeric binding partner for the hypoxia induced factor HIF-1a (84). However, on comparing the A. fumigatus murine adaptation gene expression signature to that observed following exposure to anaerobic stress in vitro (Figure 4.5A) no support for this hypothesis could be gleaned. Rather, repression of the two A. fumigatus 14-alpha sterol demethylase genes (Afu4g06890 and Afu03740), representing a critical step in ergosterol biosynthesis, was observed following hydrogen peroxide-mediated oxygen stress. Therefore, it would seem that this component of the H202-mediated oxidative stress response is relevant in vivo.

This was found to be distinct from antioxidative action of the A. fumigatus Mn super-oxide dismutase (Afu1g14550) and the bifunctional catalase-peroxidase Cat2 (Afu8g01670), both of which were more abundant following murine lung (Table 4.2) or neutrophil expo-sure (Figures 4.6 and E.2), suggesting multiple modes of oxidative stress encountered dur-ing murine infection. Interestingly H202 gradients are detectable across the S. cerevisiae plasma membrane upon H202 exposure, suggesting a mechanism other than diffusion for H202 entry into cells. This, coupled with the observation that S. cerevisiae mutants erg30 and erg60, having increased ergosterol biosynthesis, show increased permeability to H202 might suggest down-regulation of ergosterol biosynthesis as a protective response against oxidative stress (85). All of the previously characterised components of the A. fumigatus siderophore biosynthetic pathway (618; 619) were more abundantly represented at tran-script level during murine infection. Comparison of the murine dataset with that generated during in vitro iron limitation confirmed the importance of this environmental deficit dur-ing murine adaptation (Figure 4.6). With respect to extracellular iron mobilization fungal siderophores bind ferric iron with a high affinity, delivering the ferric chelate to specific receptors at the cell surface for translocation into the cytoplasm. Existing evidence sup-ports an essential role for this uptake mechanism during infection (618; 619). Reductive iron assimilation (RIA) is dispensable for murine virulence (618) but may assist in iron acquisition during infection since RIA inhibition in the absence of extracellular siderophore biosynthesis prevents growth in vitro (619). This hypothesis is supported by our finding that numerous RIA components are induced during initiation of infection (Table 4.2) in addition to siderophore biosynthetic genes. We were also able to correlate differential gene expression following in vitro alkaline shift to the host adaptation transcriptome (Figure 4.5). This was to be expected given the broad requirement for fungal pH adaptation dur-ing mammalian pathogenesis (536) but is nonetheless pleasing to observe particularly given that the in vitro acid stress transcriptome showed an opposite trend (Supplementary Fig-ure E.2) and since pleiotropic activity of the virulence-directing family of PacC/Rim101 pH sensing transcription factors (536) does not permit virulence defects to be solely, or thus


far absolutely, correlated with pH growth phenotypes. Fungal secondary metabolite biosynthesis is mediated at the level of transcription, from

clusters of physically linked, co-ordinately regulated genes and is profoundly affected by en-vironmental factors such as pH and nutrient availability (351). For the most part, defined cues prompting gene expression from such clusters remain unknown, as do the functions of the molecules produced by them in the natural environment, but popular theory regards mi-crobial secondary metabolites as chemical determinants of selective advantage (591). Noting that clusters of physically linked, co-ordinately regulated genes were a prominent feature of our dataset we applied a custom script to systematically identify them. To allow for noise in the microarray analysis, which can lead to exclusion of some spots in the final analysis, we allowed for gaps of up to a maximum of 4 non-up- or down- regulated genes permissable per cluster. Gap size variation influenced the architecture of clusters detectable in the dataset, further widening the extremities of pre-existing clusters rather than creat-ing new ones (a complete breakdown of the cluster anlaysis is available as Supplementary Dataset E.2). Thus a gap size of zero identified 14 upregulated gene clusters and a gap size of one identified 38. The maximum gap size applied (gap size = 4) identified 65 groups of physically linked genes having increased transcript abundance during murine infection (Figure 4.3). A large proportion (34%) of up-regulated, physically clustered, genes was found to reside within 300 kb of chromosome ends (Figure 4.3 and Table 4.4). The clusters are comprised of up to 34 co-ordinately expressed genes and include loci directing biosyn-thesis of siderophores (cluster 33) and two known secondary metabolites, pseurotin and gliotoxin (clusters 70 and 59, respectively). The importance of fungal gene clusters in viru-lence has recently been characterised in the fungal plant pathogen, Ustilago maydis where co-induction of physically linked, secreted protein-encoding genes is seen during infection (299).

We were unable to find evidence of clustered secreted protein genes from our analyses of A. fumigatus (data not shown) however, from an evolutionary perspective, clusters 8, 40, 53, 68 (down-regulated), 69 and 70 (Table S6) deviate significantly from the least viru-lent sequenced species considered here, N. fischeri, and therefore merit further analysis as they are linked to a specific, virulent Glade. N. fischeri is extremely rarely identified as a human pathogen (132; 420; 672), while prolonged exposure to A. clavatus spores can cause extrinsic allergic alveolitis known as malt worker's lung (70). From a functional perspective some relevant deductions regarding the clusters identified by our analyses might be possible using present genome annotations. However, closer scrutinization of clusters is required to address the likelihood that A. fumigatus genes located in them encode functions required for virulence. We anticipate that the inference of shared functionality among neighbouring co-regulated genes in our dataset will empower the functional annotation of the A. fumiga-

4.4 Discussion 170

tus genome; moreover, it significantly raises the profile of such gene-regulatory paradigms within the context of fungal pathogenicity. Our observations of biased localization among genes overrepresented during growth in the murine lung (Figures 4.3 and 4.4, Tables E.7, E.8, E.9, E.10) prompted comparison of our dataset to that obtained by Perrin et. al. (539) who identified genes under control of the global seconday metabolism regulator, LaeA. Sig-nificant overlap is observable, notably among secondary metabolism genes, most specifically between genes in the gliotoxin and pseurotin biosynthetic gene clusters.

Hypovirulence of a AlaeA deletion mutant cannot be explained solely on the basis of a lack of gliotoxin biosynthesis, and no investigation of the role of other secondary metabolite clusters, with respect to virulence, has been undertaken in A. fumigatus, therefore at this time it is not possible to reach any conclusions on the contribution made to virulence by the molecules whose synthesis is directed by these loci. With regard to our comparison of functions under LaeA control and those important during murine infection, differing ex-perimental conditions (Perrin study (539) performed at 25°C in liquid shaking culture and glucose minimal medium (548) for 60 hours) and methodologies pose a contentious issue. A truly illuminating analysis of LaeA activity during murine pathogenesis might be forth-coming from time course analyses in neutropenic mice, which is currently being attempted (Timothy Cairns, Bignell Lab). DNA sequences in subtelomeric regions undergo ectopic recombination at a much higher rate than expected for homologous recombination (196) allowing the expansion and diversification of gene families located at chromosome ends. For some organisms gene expression is governed by subtelomeric localisation, for example two A. nidulans secondary metabolism gene clusters, directing penicillin and sterigmatocystin biosynthesis, are activated following deletion of the hdaA histone deacetylase gene [65]. In other organisms subtelomeres provide an ideal setting for genes involved in antigenic varia-tion, such as in the parasites Plasmodium falciparum, and Trypanosoma brucei (54) and in cytoadhesion, such as the EPA family of C. glabrata adhesins (1). Thus the importance of sub-telomeric chromosomal regions within the context of eukaryotic pathogenesis is gaining significance in this post-genomic era of microbial studies.

These analyses of transcript abundance, drawn from minute quantities of fungal material convey a programme of A. fumigatus cellular regulation directly from the site of pulmonary infection. From a fungal physiological perspective the mammalian host restricts iron, and likely various nutrients, as well as exerting multiple degrees of oxidative stress. Regarded as a function of the genomic landscape the observed transcriptional changes reveal genome organisation and subtelomeric diversity as effectors of the remarkable versatility of A. fu-migatus with respect to the niches it successfully inhabits, one of which is the neutropenic human lung.


4.5 Chapter Summary

1. Recent genome sequencing of several Aspergillus species provides an exceptional op-portunity to analyse the transcription of potential fungal virulence attributes within a genomic and evolutionary context.

2. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection

3. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature

4. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative anal-yses of richly and poorly nourished laboratory cultured germlings.

5. We found remarkable concordance between the A. fumigatus host-adaptation tran-scriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA

6. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

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Chapter 5

Functional genomics of fluconazole sensitivity in Candida glabrata using a library of transcription factor knock-outs: a pilot study


The groundbreaking sequencing and annotation of the S. cerevisiae genome some thirteen years ago (246) ushered in a new era of yeast research in which reverse genetics approaches became the catalyst for a burgeoning supply of genome-wide phenotypic data. Unlike for-ward genetics, reverse genetics exposes a strain, typically deficient in a known protein-coding chromosomal locus, to a selection of in vitro assays designed to evaluate the contribution of that locus to a specific biological process. With an abstract notion of systematically removing each functional gene, and a research community newly furnished with sequence data, various implementations emerged. These were reliant either on the promiscuity of mobile genetic elements in target genomes, or the collaborative spirit of yeast researchers and their collective purpose to disrupt open reading frames in a targeted fashion.

The earliest comprehensive deployment of mobile genetic elements, was undertaken by Ross-Macdonald et al. (595) who used a mTn3xHA/lacZ mini transposon to determine dis-ruption phenotypes for nearly 8,000 strains in 20 different growth conditions. This technique provided supporting evidence for some established gene functions, but more importantly elu-cidated the function of previously uncharacterized ORFs involved in sporulation and vege-tative growth. The collection of mutants was also used in separate studies to identify genes required for growth in high-salt medium (159), survival of hypo-osmotic shock and growth

Chapter 5. Functional genomics of fluconazole sensitivity in Candida glabrata using a library of transcription factor knock-outs: a pilot study 173

at 15°C in addition to nitrogen starvation, invasive or filamentous growth and sporulation (675).Although transposons are widely adopted for random mutagenesis in yeast, other approaches have also been used, including mutagenesis by T-DNA from Agrobacterium tumefaciens (98), linearized plasmid DNA (135) and the use of non-homologous insertion of DNA cassettes (356). There are some benefits to using a random mutagenesis approach (e.g. promoter regions can be disrupted, gain-of-function phenotypes can be observed), however the propensity for mobile elements to integrate in non-coding regions means that exhaustive coding region coverage has not yet been achieved; on the other hand, targeted disruption techniques, although labour intensive, provide much greater coverage of the tar-get genome. The best example of this co-ordinated disruption effort is the library of deletion mutants provided by Winzeler et al (749) and Giaever et a/.,(239).

This library comprises over 20,000 haploid and diploid knockout strains corresponding to deletions of each of 5,916 genes (including 1,159 essential genes) and one haploid strain of each mating type for every non-essential gene (4,757 genes). It has been used extensively to uncover the role of genes in a range of environmental stresses including: extremes of salinity (730; 239), hyperosmotic stress (239), alternative carbon sources (659; 239), alkaline stress (239), temperature shock (239), oxidative stress (239) and DNA damaging agents (60; 225). The collection has also been informative in dissecting the molecular basis of fundamental biological processes such as meiosis and sporulation (498), chromosomal segregation (445), cytoskeletal organization, cell-wall biosynthesis, microtubule-based chromosome segregation and DNA metabolism (692; 693), glycogen accumulation (747), accumulation of mutations (307), response to abiotic chemical stresses induced by a variety of drug molecules including anti-fungal agents and anti-cancer drugs (25; 26; 240). A valuable feature of this library is the incorporation of unique identifiers within the disruption cassette sequence. This enables parallel screening of pooled mutants in a range of environmental stress conditions, in an analogous manner to the signature tagged mutagenesis (STM) approach developed by the Holden laboratory (282). This technology relies on hybridization to microarrays, rather than filter membranes, spotted with complementary barcode sequences and allows data to be analyzed with statistical measures and unsupervised learning techniques for classification and functional prediction of genes (93).

As S. cerevisiae research enters a new integrative phase (reviewed in (293)), it does so from a solid platform of reductionist biology based, in part, on the use of disruption libraries described above. Meanwhile, Candida researchers lag somewhat behind, lacking the equiva-lent comprehensive disruption libraries. Existing S. cerevisiae disruption libraries have been useful in characterizing orthologous C. abicans pathways (63; 739), but do not provide any insight into the function of genes specific to Candida species that may explain differences in virulence phenotype between species. Candida researchers have tried to follow suit. In-


deed, mutagenesis protocols have been successfully adopted in C. albicans and C. glabrata. The Haynes laboratory adopted a signature tagged mutagenesis approach to identify a C. glabrata mutant, aces, with a cell separation defect, that is hypervirulent in a murine model of candidiasis aces (341); Cormack and Falkow (140; 139) developed a random mutagen-esis approach based on transformation of non homologous linearized DNA to identify a novel family of genes (EPA) mediating adhesion of C. glabrata to human epithelial cells. A Tn7 transposon based mutatgenesis protocol developed by (116) has been used to screen for altered sensitivity to fluconazole, (347) however coverage was limited to approximately 25% of the genome, with 75% of disruptions occuring in coding sequences. Therefore, a remaining challenge is the establishment of a comprehensive, genome-wide collection of C. alb jeans and C. glabrata deletion mutants akin to that developed by the Saccharomyces genome deletion project. In this section I report on initial progress in creating a prototypic targeted disruption library in C. glabrata. The overall aim is to provide genome-wide cov-erage, however for the initial pilot study, a collection of putative functionally related ORFs was chosen. In the first instance, we chose to disrupt ORFs encoding putative regulators of transcription, because the laboratory's earlier work with ACES suggested that transcrip-tion factors play an important role in modulating virulence. Moreover, the altered function of transcriptional circuitry in evolutionary timescales has emerged as an important source of diversity between organisms that share similar transcriptional components (reviewed in (702) and (592)). With an orthologous S. cerevisiae transcription factor knockout library already available, we hope that a quantitative growth assay will form the first part of a study of transcriptional rewiring between non pathogenic fungus and related opportunis-tic pathogen. Creation of this library will facilitate comparison of S. cerevisiae and C. glabrata transcription factor regulons in gene expression microarray experiments, and also phenotypic traits of null mutants exposed to a variety of environmental stress conditions.

The aims of this pilot study were to provide a quantitative measure of fitness in envi-ronmental stress conditions so that S. cerevisiae and C. glabrata phenotypic traits can be compared.


This work was presented at the following conference: Third FEBS Advanced Lecture CourseHuman Fungal Pathogens:

Molecular Mechanisms of Host-Pathogen Interactions and Virulence May 2 - 8, 2009

Biao Ma, Andrew McDonagh, Emily Cook, Melanie Puttnam and Ken Haynes Functional genomics of fluconazole sensitivity in Candida glabrata using a library of

transcription factor knock-outs.


5.2.1 Construction of the CGTFKOLib library of transcription factor null mutants

Through the study of sequence data on the Saccharomyces Genome Database 1 and liter-ature searches, Biao Ma - a post-doctoral colleague in the Haynes laboratory - identified 208 non-essential S. cerevisiae genes encoding known or putative regulators of transcrip-tion, encompassing the many categories of genes that can regulate transcription, including: DNA-binding TFs, subunits of large protein complexes that regulate gene transcription and also chromatin re-modelling factors that play important accessory roles in transcrip-tion regulation. The C. glabrata homologues of these 208 genes were identified by BLAST (16) search on the Genolevures website2 (630). For ten S. cerevisiae genes, two C. glabrata paralogues can be identified. Hence, in total, 218 C. glabrata genes were identified as dis-ruption candidates for inclusion in a gene knock-out collection known as the CGTFKOLib (Candida glabrata Transcription Factor Knockout Library).

Based on published methods for constructing gene knock-outs on a large scale in C. albicans (502), and methods previously developed in the Haynes and Cormack laboratories, a highly efficient knockout protocol was developed by Biao Ma over the course of a year. The method (Figure 5.1) is summarized below. Approximately 500 bp of the 5' and the 3' un-translated regions (UTRs) of each target gene in addition to a 1.4 kb selectable NATR cassette, which confers resistance to nourseothricin, are individually amplified by PCR and purified by gel extraction. Several features are also integrated in the amplified 5' and 3' UTRs through the primers `-1-NATR' and `4-1-NAT-F'. For the amplified 5' UTR, two 18 bp universal priming sequences, Ul and U2, flanking a unique 20 bp sequence tag (Up tag) are incorporated at its 3' end. For the amplified 3' UTR, two 18 bp universal priming sequences, D2 and D1, flanking another unique 20 bp sequence tag (Down tag) are incorporated at its 5 end. The 'Up tag' and the 'Down tag' enable the future identification

lhttp://www.yeastgenome.org

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Figure 5.1: Molecular approach adopted for targeted disruption of C. glabrata transcription factor ORFS (schematic). The procedure consists of three stages: 1) The creation of three fusion components. This involves the amplification of approximately 500 bp of the 5' (-500-F, -1-NATR primer pair) and the 3' (1-NAT-F, +500-R primer pair) un-translated regions (UTRs) of each target gene in addition to a 1.4 kb nourseothricin (628) resistance cassette (NATR) (Top panel). Amplified upstream and downstream UTRs comprise 151-U2 and D2-D1 tags respectively - universal amplification sites flanking molec-ular barcodes that uniquely identify the disrupted strain. 2) Fusion. U2 and D2 sequences incorporated in the amplified 5' and 3' UTRs, respectively, overlap with the 5' and 3' end of the amplified NATR cassette. Thus, the 5' UTR, the NATR cassette, and the 3' UTR of the target gene can be joined together through both overlaps by a fusion PCR to yield a deletion construct. 3) Transformation and screening. Each deletion construct is individu-ally transformed into a C. glabrata his3 strain by electroporation and disrupted strains are identified by screening 20 to 60 transformants by PCR (-600-F, -int-F and -int-R. + 600-R primer pairs for amplification of targeted ORF - present if not disrupted; -600-F, NAT-5'-R. NAT-3'-F, +600-R for amplification of disruption cassette at the chromosomal locus).

400-F 5' UTR


of each gene knock-out through a tag-array-based method, and the Ul, U2 sites and the D2, D1 sites can be used to amplify all the Up tags and Down tags, respectively. The `Up tag' and 'Down tag sequences were chosen to match the homologous identifiers used in generating the complete S. cerevisiae gene knock-out strain collection, allowing existing parallel knockout screening tools to be re-deployed in C. glabrata. Moreover, the U2 and D2 sequences incorporated in the amplified 5' and 3' UTRs, respectively, overlap with the 5' and 3' end of the amplified NATR cassette. Thus, the 5' UTR, the NATR cassette, and the 3` UTR of the target gene can be joined together through both overlaps by a fusion PCR to yield a deletion construct. Each deletion construct is individually transformed into a C. glabrata his3 strain (Schwarzmiiller unpublished data (691)) (a derivative of C. glabrata ATCC 2001) by electroporation. Subsequently, 20 to 60 transformants are screened by PCR for the desired gene knock-out through PCR. The PCR screening confirms the correct integration of the deletion construct in the target gene locus and the loss of the open reading frame of the target gene. Finally, individual frozen stocks are made of single colonies derived from two independent transformants that contain the correct knock-out of each target gene. For the 218 candidate genes, 1,960 DNA primers were designed for generating deletion constructs as well as for confirming correct gene knock-outs. Using the protocol described above, gene knock-out strains for 186 genes have been generated, with a success rate of 85%.

5.2.2 Liquid culture assay to determine strain growth dynamics

Glycerol stocks of each library strain, collectively maintained in two 96-well microtitre plates at -80°C, were sampled (5 pl) using a multi-channel pipette and dispensed into correspond-ing sterile, round-bottomed 96-well plate containing 150 µl of YEPD broth. Library strains were sub-cultured to stationary phase for 18 hours in an orbital shaker (30°C, 180 RPM) before commencing the growth curve assay. Sub-cultured cells were harvested (3000rpm, 5 mins) in a centrifuge rotor equipped with plate adaptors (Wolf Laboratories, UK) and washed three times with sterile, distilled water. Washed cells from each library plate were transferred (final OD600=0.1) into microtitre plates containing YEPD, YEPD + 5 µg-m1-1 fluconazole, YEPD + 10 iug.m1-1, YEPD + 30 µg•ml-1 and YEPD + 100 µg•ml-1 (fi-nal concentration). Plates were sealed with a breathable membrane and incubated for 12 hours. Optical density (0D600) measurements were bi-hourly recorded on a Multiskan Ascent absorbance plate-reader (Thermo Scientific, U.S.A). Assays were performed in bio-logical quadruplicate unless otherwise stated.


5.2.3 Fitting growth dynamics data to a logistic model of population growth

Time course optical density (0D600) data, used as a measure of cell population dynamics, were fitted to a three parameter (slope, midpoint and upper asymptote) logistic function of growth using non-linear regression (SSLogis function) in the R statisitical computing framework. The logistic function of growth adopted in the analysis is shown in equation 5.1:

f (x, (b, d, e)) = (5.1) 1 + exp{ b (log(x) — log(e))}

In this model, logistic growth is a function of time x, the maximum cell density d (upper asymtote), the midpoint of the growth e and the slope around this midpoint b. Parameters d, e and b are quantitative phenotypic traits of each strain and are estimated from samples of cell density at x = 0 —11.5 hours. Strains showing significant, fluconazole specific growth defects were identified in null strains either by a) a complete lack of logistic growth in the presence of fluconazole but not YEPD (hypersensitive) or b) significantly greater midpoint (p < 0.05) estimates than wild-type strains in fluconazole but not YEPD (sensitive). Missing midpoint estimates, resulting from failed non-linear regression were imputed and set to a value outside the measured range (15 hours).

5.2.4 Fitting truncated growth dynamics data to an exponential model of population growth

Optical density data (0 - 8 hours), were considered for estimating the growth rate of strains in an exponential model of growth, in an attempt to disregard the systematic problems of estimating a logistic fit to data that poorly defined the stationary phase of growth. In this approach, optical density y is described by a simple model of exponential growth:

y = a + bert (5.2)

Where a is the initial population at time t = 0 and r is the growth rate of the strain. Model parameters were estimated using the non-linear least squares regression function in the R statistical framework. Starting values for estimated parameters were fixed for each strain: a = 0.04, r = 0.2, and 1000 iterations were allowed for convergence. 95% Confidence intervals were simulated from the standard errors of the estimated parameters using the confint function provided by the multcomp package in the R statistical framework. Sensitive strains were identified if the upper confidence interval of r for the mutant strain was less than the lower confidence of interval of r for the parental strain in media supplemented with fluconazole. Furthermore, these strains must show no difference in growth in YEPD


medium lacking fluconazole.

5.2.5 Fitting the inhibitory effect of fluconazole at a single time point in a linear model

In a departure from the above non-linear regression modelling approaches, the optical den-sity y at a single time point is modelled as a linear relationship between the strain genotype 131, the environmental condition 02 and an interaction term between the two, which repre-sents the fluconazole-specific sensitivity of each strain.

y =131X+02X+ /302X +E (5.3)

Least squares regression estimates of these terms and the residuals E was performed using the lm function of the R framework for statistical computing.

The Z factor statistic, commonly used in high throughput screening assays as a quality metric (769) was used as a rational basis for choosing the single time point that maximized the signal to noise ratio. The Z factor is defined as:

=7 1 3 x (6p + &n)

I uip —fin l

(5.4)

Where i7p is the estimated sample standard deviation for the positive control samples (i.e. the parental strain growth), ern is the estimated sample standard deviation for the negative control samples (i.e. blank wells), A p is the mean of the positive control samples and ri p is the mean of the negative control samples.

5.3 Unsupervised learning of strain clusters based on growth parameter estimates

Hierarchical clustering was performed with the hclust function in the R statistical framework using a Euclidean distance metric between midpoint estimates and average linking. Where insufficient growth occurred for logistic regression based estimation of parameters, growth of each null strain was expressed as a percentage of the parental strain growth, his3 in each growth condition.

5.4 Results

My aim was to develop an assay that could identify null mutant strains showing either hypersensitivity or sensitivity to fluconazole, and where possible, to provide estimates of growth rate, so that strains could be classified by unsupervised learning techniques such

5.4 Results 180

as hierarchical clustering. This would form the basis for quantitative comparison of S. cerevisiac and C. glabrata environmental stress regulation by putative transcription factors using a functional genomics approach, instead of gene expression microarrays. The standard phenotypic growth assay adopted in the Haynes lab relies on a discrete scoring (growth, no growth or sensitive) of colonies spotted onto solid agar plates, and thus provides only a very coarse classification of strains that is uniform across each environmental stress condition. For this newer assay to be valuable to the overall aim, it must not only be possible to easily identify each of the categories described above, but also to provide a measure of sensitivity to provide finer grained categorization. This relies on reproducible estimates of the growth midpoints for each strain, but most importantly the control strain, with which the null mutant strains are compared.

Hypersensitive strains, classified as "no growth" strains in the existing method, were easily identified even in the untreated YEPD culture medium. Ten null mutant strains (rpd3, gln3, pop2, ssn6, nggl, snf6, gcn5, rtfl, asfl and spt3) grew so slowly in relation to the parental his3 strain (range = 11.64% - 74.42% of his3 growth at 11.5 hours) that logistic regression estimates of growth parameters were not achieved with the SSlogis function (Table F.2). For the sake of discussion, these strains were defined as "growth defect" strains. Were logistical fits were impossible, curves are omitted from all plots.

In contrast, both the library parental strain, his3, and the wild-type ATCC 2001 strain showed logistic growth in YEPD that was sensitive to fluconazole in a concentration-dependent manner (Figure 5.2). Disappointingly, variable midpoint estimates of these reference strains prevented the identification of "sensitive" null mutant strains with the hypothesis testing methods adopted. The estimate of ATCC 2001 growth midpoint in YEPD, showed the least error of the two reference strains (8.44 hours, SEM = 0.37 hours), corresponding to a coefficient of variance of 7.58%.

Estimation of 95% confidence intervals, based on assumption of normally distributed midpoints, places the midpoint in the range of 7.72 - 9.16 hours; addition of the lowest concentration of fluconazole (5pg-m1-1) to the culture medium shifts the upper confidence interval of the midpoint beyond the detectable range (8.134 - 14.46 hours). Thus, although 81 strains have midpoint estimates greater than that of the ATCC 2001 strain, error in the estimates of reference and null strains prevents the rejection of the null hypothesis i.e. no difference in growth rates as a consequence of gene deletion.

The same is true if the his3 strain is used as the reference strain - a more suitable control because all null mutant strains in the CGTFKOLib were derived from this background. The estimate of the his3 growth midpoint in YEPD, was greater than ATCC 2001 (9.39 hours, SEM = 1.33 hours), corresponding to a coefficient of variance of 24.49%,and shows a more poorly defined upper asymptote than that of the ATCC 2001 strain (Figure 5.2), indicating


that its growth is further away from the stationary phase at the maximum sampling time-point. Similar estimates of 95% confidence intervals, place the midpoint in the range of 8.13 -14.46 hours; addition of the lowest concentration of fluconazole (5jzg•m1-1) to the culture medium also shifts the midpoint beyond the detectable range (3.33 - 19.39 hours). Errors associated with midpoint estimates increased with fluconazole concentration (Figure 5.3), such that only data from the 5µg•ml-1 and 10pg•m1-1 fluconazole treatment were retained in the final analysis.

Twenty four strains were identified as hypersensitive in YEPD supplemented with 5teg•ml-1 fluconazole. These stains showed no obvious increased in optical density be-tween 0 - 8 hours. This includes 14 strains that showed no difference in growth to reference strains in YEPD (), in addition to those 10 strains previously defined as having growth defects in YEPD (Table F.4). Of the 14 strains that showed fluconazole-specific growth inhibition, two strains carried disruption cassettes in ORFs encoding known multi-drug resistance effectors, PDR1 and MIG1.

Other strains that were hypersensitive to fluconazole (5,ug-m1-1) are disrupted in ORFs whose S. cervisiae orthologues are involved in histone modification (hat2, ada2, rphl and hosl), chromatin modification (taf14), whereas others are transcriptional activators of genes modulated by levels of purines (basl), aromatic amino-acids (aro80), or the levels of haem and oxygen (hap1). Some fluconazole sensitive strains are disrupted in ORFs that encode proteins belonging to multi-subunit regulators of RNA polymerase II (ssn6) such as the SAGA complex (spt8) and the mediator complex (ssn2)

Hierarchical clustering of midpoint estimates was used to classify strains based on their growth in untreated YEPD, and YEPD supplemented with fluconazole (5-10pg•ml-1). Where midpoint estimates were missing, due to insufficient growth, data were imputed and set to an arbitrary value beyond the detectable range (15 hours), thus artificially plac-ing hypersensitive strains more closely together than may actually be the case if the range of timepoints was increased. Nevertheless, hierarchical clustering of midpoints identified seven categories of null mutant strains, two of such categories are shown in Figure 5.4.

Although the initial aim was to fully characterize the logistic growth of C. glabrata strains, growth curves (Figure 5.2, 5.5 and 5.7) show that stationary phase is not reached in every strain with the experimental method adopted. To achieve improved logistic fits, the experimental system can be adjusted to increase the range of sampling times or to increase the incubation temperature so that growth midpoints are decreased. However, comparison of phenotypic traits at this elevated temperature may need to be complemented with comparison at 30°C in order to identify temperature dependent growth sensitivities in S. cerevisiae.

To be practicable, a longer time-course assay requires automation, the initial results of

5.5 Discussion 182

which have shown promise, in terms of the reproducibility of data, the lack of contamination (Figure F.1) and the definition of the stationary phase of growth (Figure F.2). With a robotic platform, the assay can run overnight without supervision and be extended to 20 hours. In initial experiments, only three strains out of 96 failed to reach stationary phase after incubation in YEPD, for 20 hours (37°C, 300 RPM).

Moreover, it was possible to identify 32 strains that saturated at significantly lower optical density (< 0D600 9.04 - 10.05, p < 0.05) in comparison to the parental strain, and to identify 16 sensitive strains that showed logistic growth but with a greater midpoint than the parental strain (> 4.12 — 5.22 hours, p < 0.05). Unfortunately, this system has not yet been deployed in a screen of fluconazole sensitivity because it is awaiting repair.

Two alternate approaches, which disregard the requirement for a logistic model and make use of the existing data, are a) to estimate the fluconazole dependent inhibition of growth at a single time point, or b) to use the range of time points that determine the exponential phase of growth and therefore do not require a defined stationary phase. In a), the inhibitory concentration of fluconazole and the choice of time point must be considered carefully. In this method, only the inhibitory effects of YEPD supplemented with 5 pg•ml-1 fluconazole were considered as the assay showed the least systematic variability at this concentration (Figure 5.3). Furthermore, a time-point that maximizes the signal to noise ratio, was chosen.

In this instance, the Z factor assay quality score (see methods above) was maximized at 11.5 hours (Table F.6). This method identified eight strains with significant (p < 0.05), flu-conazole (5 pg-m1-1) specific growth sensitivity. Four strains were identified as fluconazole sensitive in both the linear regression and logistic fitting method (pdrl, srb8, hap/ -like and rph1). The linear regression method also classified strains differently: two were classified as having growth defects in the logistic method, but fluconazole sensitivity in the linear regression method (nggl, gcn5) (Table F.7 and Table F.8).

5.5 Discussion

In this pilot study, I aimed to develop a quantitative assay that could identify null mu-tant strains whose growth is significantly more susceptible to environmental stress than the parental strain. In this instance we used fluconazole sensitivity as an exemplar con-dition. Although well established methods already exist for assessing microbial sensitivity to antimicrobial compounds (544; 494), we intend this screen to be applicable to a wider number of environmental stress conditions, and aim to quantify differences in growth rate, and population size at stationary phase.

In recent years, quantitative phenotype scores have been adopted proved advantageous.

CG2001

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Figure 5.2: Logistic growth curves of a) wild-type strain ATCC 2001 and b) the library parental strain his3 in YEPD and YEPD fluconazole (5-10 µg-m1-1). Panels show optical density measurement (0D600= 0 - 1.5, as a function of time = 0 - 11.5 hours) for both strains, each depicting growth in YEPD (closed blue circles), YEPD + 5 pg-m1-1 fluconazole (open red triangles) and YEPD + 10 µg-m1-1 fluconazole (open black squares). Data points derived from growth assays in higher concentrations of fluconazole are omitted for clarity. ATCC 2001 showed logistic growth in YEPD (midpoint estimate =-8.44 hours, SEM = 0.37 hours) that was inhibited by the addition of 5 µg•ml-1 fluconazole (midpoint estimate = 11.29, SEM = 1.61 hours). Growth was sufficiently impaired in YEPD + fluconazole (10-100 µg•ml-1) to prevent logistic regression estimates of growth parameters. At these higher concentrations of fluconazole (YEPD + 10 iug•m1-1, YEPD + 50 µg-ml-1 and YEPD + 100 pg-m1-1), growth was 41.03%; SEM = 5.00%, 34.02%; SEM =-6.58%, 30.80%; SEM = 13.21% of that observed in YEPD without fluconazole respectively. The his3 parental strain also showed logistic growth in YEPD (midpoint estimate = 9.39 hours, SEM = 1.33 hours) and YEPD + 5 pg-m1-1 (midpoint estimate = 11.32, SEM = 4.62 hours), and a pattern or failed logistic regression fits in YEPD + fluconazole (10-100 ,ug•m1-1). Growth of his3 at these higher concentations of fluconazole (YEPD + 10 tig-m1-1,YEPD + 50 pg•ml-1 and YEPD + 100 pg-m1-1) was 62.46%; SEM = 14.65%, 35.21%; SEM = 9.38% and 34.68%; SEM = 10.13 respectively.

0 0

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5.5 Discussion 184

0 5 10 50 1000 Fluconazole concentration (µg/m1)

(a)

Number of strains

0 5 10 50 100 Fluconazole concentration (µ,g/m1)

(b)

Figure 5.3: Fluconazole dependent distribution of midpoint standard errors and number of inestimable growth midpoints. (a) Box and whisker plots depict increasing median standard error as a function of increasing fluconazole concentration. Median stan-dard errors of midpoint estimates ranged from 4.68 (5pg•ml-lml fluconazole) - 76 hours (100pg•ml-lml fluconazole). (b) The number of strains showing insufficient growth for estimation of midpoint also increased with fluconazole concentration, reaching a peak at 10µg•m1-1 fluconazole.

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Chapter 5. Functional genotnics of fluconazole sensitivity in Candida glabrata using a library of transcription factor knock-outs: a pilot study 185

Figure 5.4: A liquid growth phenotype assay identifies two clusters of fluconazole-sensitive null mutant strains. A library comprising wild-type and parental auxotrophic strains and 186 C. glabrata null mutants, each disrupted in one putative transcription factor ORF (based on sequence homology with S. cerevisiae) was incubated overnight in an orbital shaker to stationary phase (18 hours, 30°C, 180 RPM, in YEPD broth) and then sub-cultured (18 hours, 30°C, 180 RPM, in YEPD broth) in micro-titre plates containing YEPD broth ± fluconazole (5 - lOug /ml). Optical density (0D600) measurements were bi-hourly recorded on a Multiskan Ascent absorbance plate-reader (Thermo Scientific, U.S.A), and data were fitted to a three parameter (slope, midpoint and upper asymptote) logistic function of growth using non-linear regression in the R statisitical computing framework. Hierarchical clustering of midpoint estimates was used (average linkage, Euclidean distance) to classify strains based on their growth in untreated YEPD, and YEPD supplemented with fluconazole (5-10 pg•ml-1). Where midpoint estimates were missing, due to insufficient growth, data were imputed and set to an arbitrary value beyond the detectable range (15 hours). Two clusters, Cl and C5, showing fluconazole sensitivity and general growth defects respectively are depicted. Midpoint estimates are depicted in a hue scale rangeing from red (low) to white (highh)

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5.5 Discussion 186

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Figure 5.6: Growth curves of strains belonging to a cluster (C5) characterized by slow growth in YEPD and YEPD fluconazole (5-10 pg-m1-1). Panels show optical density measurement (0D600= 0 - 1.5, as a function of time = 0 - 11.5 hours) for twelve null mutant strains, each depicting growth in YEPD (closed blue circles), YEPD 5 pg-m1-1 (open red triangles) and YEPD 1- 10 itg-m1-1 (open black squares). Data points derived from growth assays in higher concentrations of fluconazole are omitted for clarity.

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5.5 Discussion 188

Figure 5.7: Growth curves of strains belonging to a cluster (C3) characterized by wild-type like growth in YEPD and YEPD fluconazole (5-10 pg•ml -1 ). Panels show optical density measurement (0D600= 0 - 1.5, as a function of time = 0 - 11.5 hours) for twelve null mutant strains, each depicting growth in YEPD (closed blue circles), YEPD + 5 µg•m1-1 (open red triangles) and YEPD + 10 pg•ml-1 (open black squares). Data points derived from growth assays in higher concentrations of fluconazole are omitted for clarity.


Synthetic genetic array scoring has facilitated correlation maps between proteins that al-low cross species comparison (178) as well as guilt by association functional predictions of uncharacterized S. cerevisiae proteins (178). The methods developed in the Andrews lab have relied on image analysis of colony size, although the SGA scoring algorithm and software analysis has yet to be published, this may be something that we re-visit in the future. Furthermore, although the strains have been barcoded, and are amenable to fitness profiling using the microarray method described previously (239); we proceeded with the growth curve assay on economic grounds and, more importantly, because strains do not need to be pooled before the assay.

Optical density measurements are used in lieu of cell counts, although currently there exists no calibration curve relating cell number to the optical density on our absorbance measurement instrument, this is an important technical requirement for future screening. Furthermore, by using optical density in place of cell counts I have made an assumption that differences in optical density between strains exposed to stress over a timecourse are due only to population size. However, it is conceivable that null mutations can confer differences in size and shape, which can affect optical density measurements. Although I expect cell morphological differences to be a small contributing factor to differences in optical density measurements, future work should include a comparison of the morphological characteristics of the null mutant strains in normal culture medium and culture medium exposed to fluconazole.

The manual methods initially adopted here suffered from variability and a poorly defined stationary phase in most strains. The initial development of a robotic screening platform has addressed these two problems, and allows us to perform high throughput screening. The reliability of midpoint estimates of 96 parental strain growth curves conducted on this platform is good (with a midpoint coefficient of variance of 5.23%). We anticipate this platform to be the basis for a large scale test of null mutant strains of C. glabrata and strains deleted in orthologous S. cerevisiae genes in a large number of environmental stress conditions.

Extending the screen to incorporate different environmental stress conditions is a vital step in dissecting the functional role of transcription factor ORFs in environmental stress adaptation, at least two of which are likely to be pleiotropic (e.g. Msn2p and Msn4p regulate the expression of approximately 200 genes in response to several stresses, including heat shock, osmotic shock, oxidative stress, low pH, glucose starvation, sorbic acid and high ethanol concentrations (446; 230; 118).

In this pilot study, fluconazole, a specific inhibitor of lanosterol C14-demethylase (766), acts to knock down the activity of a single gene; as a result, sensitive or hypersensitive null mutant strains effectively reveal synthetic sick or synthetic lethal interactions with

5.5 Discussion 190

disrupted ergosterol biosynthesis. Such synthetic interactions may occur in the ergosterol biosynthetic pathway itself ((20; 623)), in a dependent process (e.g. calcium signalling (347; 707), respiration (369), iron uptake (556)), or in downstream processes that are affected by ergosterol levels in the plasma membrane (e.g tryptophan permease targeting to the plasma membrane (706)). The dissection of this phenotype is further complicated by the type of genes profiled in this analysis. Null mutants like those profiled in this pilot study -transcription factors - are likely to be regulators of one or more of those pathways, rather than effectors in the pathway.

Further work should assess the growth of these null mutants in a range of antibiotic agents, including related azole compounds (e.g. itraconazole, posaconazole, ketoconazole and voriconazole) as well as antibiotics that do not target the ergosterol biosynthetic path-way (e.g. cyclohexamide, dexamethosone, hygromyin, amphotericin B). This will help to identify which transcription factors are specifically involved in azole sensitivity. Further-more, fitness profiling in conditions that identify respiratory deficient mutants will be nec-essary. Typically, respiratory mutants have the petite phenotype (96; 163), and therefore spotted colony phenotypic screens on 3% yeast peptone glycerol agar (in addition to the liquid screens described) should be performed; other sole carbon sources such as ethanol, acetate and 2-deoxyglucose should be considered (357; 127; 408). Null mutants with an iron auxotrophy can be identified either with dropout synthetic medium or by adding chelators bathophenanthrolene disulfonic acid and ferrozine to the YEPD medium (556).

Apart from adding the suggested environmental stress conditions to the phenotypic screen, the function of each of these transcription factors may be further elucidated by whole genome experimental approaches. It will be beneficial to compare the transcriptional changes that occur after exposure to fluconazole between parental and null mutant strains using gene expression microarrays. These experiments will help to define the sets of genes that are induced and repressed in response to fluconazole exposure, and how they depend on each transcription factor (e.g. (719)). Chromatin immunoprecipitation in tandem with gene expression microarray profiling (CHiP-chip analysis) may also complement the gene expres-sion microarray approaches described above (577; 27). Although null mutant libraries are informative tools in functional genomics, they only identify a subset of regulatory influences, that can be complemented by overexpression libraries (334; 82).

In this screen, I identified two clusters of strains that were defined by either an impaired growth profile in YEPD (C5) or YEPD supplemented with fluconazole 5 - 10 iug•ml-1 flu-conazole (C1). Several fluconazole sensitive C. glabrata strains identified in this study were disrupted in ORFs whose S. cerevisiae orthologues encode components of the C. glabrata RNA polymerase II mediator complex (CAGLOE00627g/SRB8, CAGLOJ10472g/SSN2 and CAGLOM06875g/SSN8), a requirement for activator-dependent stimulation of RNA poly-


merase II (349; 206) and Tupl-Ssn6 co-mediated transcriptional repression (641; 768). A specific co-operative role for a mediator complex subunit in directing xenobiotic resis-

tance gene expression was recently discovered in S. cerevisiae and C. glabrata (687). After studying orthologous multi-drug resistance pathways in mammalian cells, where the preg-nane X receptor upregulates ABC transporters after binding structurally diverse xenobiotics (360; 744), Thakur et al., demonstrated that the S. cerevisiae and C. glabrata multidrug resistance transcription factor Pdrlp binds to a related antifungal - ketoconazole - and that deletion of the mediator subunit Gall 1p results in hypersensitivity to this compound.

In their screen of S.cerevisiae mutant strains, Thakur et al., also profiled other medi-ator subunit mutants for ketoconazole hypersensitivity (ssn2, srb8, sr1)2, cse2, ssn8, pgdl, medl, sohl, sin4 and srb5) but did not identify any additional hypersensitive strains. In this study, a C. glabrata srb8 strain was identified as fluconazole sensitive. Although C.

glabrata srb8 mutants were not screened for fluconazole sensitivity in (687), a separate study identified three other hypersensitive strains disrupted in ORFs encoding mediator subunits, including SRB8 (347). In addition, two other mediator subunit mutants from the CGTFKOlib profiled here(ssn2 and ssn8), were also fluconazole sensitive, suggesting a different role for these mediator subunits in fluconazole versus ketoconazole susceptibility, or intriguingly between C. glabrata and S. cerevisiae.

Zinc cluster proteins (434) and basic leucine zipper proteins have a more established mechanistic role in the regulation of multi-drug resistance gene expression (? ). Therefore, it was not surprising to identify several fluconazole sensitive strains that were disrupted in ORFs encoding zinc finger domain transcription factors. The role of Pdrlp in regulat-ing the expression of multi-drug transporters, which confer fluconazole resistance, is well known in both S. cerevisiae (433) and C. glabrata (719). Other well established fluconazole dependent processes were fluconazole sensitive. Ergosterol synthesis in S. cerevisiae is an energy expensive, aerobic process (772) requiring haem (249; 38; 422) and molecular oxy-gen (422; 598). Perhaps unsurprisingly, this study identified fluconazole sensitive mutant strains lacking ORFs whose S. cerevisiae orthologs have regulatory roles in these processes (CAGLOK05841g/hap/ (286; 679), CAGL0001485g/cti6 (560; 149) and CAGLOA01628g I mig.1 (427), reviewed in (607)),In contrast, there are no previous reports of fluconazole sensitivity in an aro80 strain in C. glabrata (CAGLOF03025g), S. cerevisiae or C. albicans. Although a role for a transcriptional regulator of aromatic amino acid catabolism in flucona-zole sensitivity is intriguing, it remains unclear why aro80 strains are more susceptible to fluconazole than the parental his3 strain. In S. cerevisiae, Aro8Op positively regulates the transcription of the aromatic transferase II Aro9p which catalyzes the first step of trypto-phan, phenylalanine and tyrosine (317) catabolism. Previous work in S. cerevisiae reveals a link between depleted ergosterol levels and the improper targeting of the high affinity

5.6 Chapter Summary 192

tryptophan permease in the plasma membrane (706). Thus, a plausible explanation for the fluconazole sensitivity observed in the aro80 strain might be that reduced ergosterol levels in the cell membrane lead to impaired tryptophan uptake, which might otherwise be rescued by an intact aromatic acid catabolic pathway, governed by Aro80p. This can be investigated by supplementing the YEPD + fluconazole test condition with a tenfold excess of tryptophan, or by over expressing the genes encoding effectors downstream of Aro80p, ARO9 and AR010, in a aro80 background.

5.6 Chapter Summary

This preliminary work has provided the basis for the development of an automated plat-form for functional genomics studies in C. glabrata although significant optimization is still required. A medium throughput knockout methodology has been adopted to differentiate between wild-type and null mutant growth rates in medium supplemented with fluconazole, although this can be extended to other environmental cues. This study has identified some candidate genes that may play a role in fluconazole sensitivity that have not previously been reported in the S. cerevisiae literature.

193

Chapter 6

Conclusions and future work

6.1 Future work

The following points for further research are applicable to chapter 3:

1. In chapter 3 we constructed a Candida glabrata hog1 null mutant and performed parallel phosphorylation, growth and transcription profiling assays in cells that were either mock treated or exposed to hyperosmotic stress (sorbitol, 15 minutes). The hogl mutant showed simultaneous growth and phosphorylation defects in relation to the parental strain, and wild-type equivalent growth and Hog1p phosphorylation in rich culture medium (YEPD).

2. Although this implicated Hog1p phosphorylation in hyperosmotic stress adaptation, no direct link was demonstrated. The evidence in support of Hog1p phosphorylation mediated hyperosmotic stress adaptation would be greatly enhanced by preventing phosphorylation of conserved target residues (Thr174 and Tyr176) using site directed mutagenesis, or by overexpression of the specific Hog1p phosphatase PTC1. To be consistent with our hypothesis, we would expect such an expressed and mutated Hog1p allele to confer normal growth in rich culture medium, and conversely, abrogated phosphorylation and growth in culture medium supplemented with sorbitol (1 M).

3. Transformation protocols adopted in gene disruption can be mutagenic and introduce secondary site mutations that confound the interpretation of phenotypic results. In order to confirm that the hyperosmotic stress induced growth defect is indeed spe-cific to HOG1 deletion, phosphorylation, growth and transcription profiling assays should be performed with the reconstituted strain. If the observed phenotypic and transcription traits are specific to HOG1 deletion, a reconstituted strain would res-cue wild-type behaviour in the above assays. Heterologous complentation assays of

6.1 Future work 194

S. cerevisiae hogl strain would also provide evidence that the gene has a similar biochemical profile when expressed in a heterologous host.

4. Apart from the secondary mutations arising from mutagenic transformation proce-dures, gene deletion methods can lead to multiple (non-targeted) sites of cassette integration. It will be prudent to a) confirm the genotype of the HOG1 locus null by using a PCR based method (upstream HOG1 and internal HOG1 primer pair versus upstream HOG1 and internal HISS pair and b) perform a Southern analysis using a HISS probe directed to genomic DNA extracted from the ATCC 2001, AHT6 and JG7 to confirm a single site of integration in the JG7 strain.

5. Gene expression microarray profiling identified a radically different osmostress tran-scriptome to published data for Candida albicans and S. cerevisiae (references in chapter 3), however comparing gene expression data sets across laboratories and mi-croarray platforms does not adequately control for different sources of systematic noise. In order to make more convincing comparisons with related species, the tran-scription profiling should be performed in parallel with equivalent S. cerevisiae and C. albicans strains. A particularly good example of this approach was recently published by Lelandais et al., (402) who simultaneously performed transription profiling of S. cerevisiae and C. glabrata after benomyl exposure.

6. During the course of the study, a truncated SSK2 allele was identifed as the cause of the functionally impaired Slnl branch of C. glabrata (references in chapter 3), and this was not evident in two other common C. glabrata strains. Prior to our transcription profiling we hypothesized that Hog1p phosphorylation would be sufficient to recover a S. cerevisiae-like transcription profile if the downstream transcriptional modules are dependent on Hog1p and conserved in C. glabrata, We rejected this hypothesis on the evidence of the observed transcript profiles, however the fact that the specific architecture of the ATCC 2001 strain is not generalizable to the whole species, raises the question of whether these transcript profiles are an aberration, peculiar to the ATCC 2001 strain background. The study would be strengthened by profiling other mutants derived from other C. glabrata strain backgrounds such as BG2, that have both signalling branches in tact or by reconstituion of a functional SSK2 gene either episomally or at the chromosomal locus. HOG1-dependent transcripts should be the same in the ATCC 2001 and BG2 backgrounds if this depends only on Hoglp phosphorylation rather than a specific background.

7. Assertions regarding the transcriptional activity of important transcripts and their STEII and HOG1 dependency should be independently verified with non-microarray

Chapter 6. Conclusions and future work 195

based methods that ideally use the same RNA samples, e.g qFCR or Northern blot analysis.

8. Apart from the technical verification steps above, there are some potentially interest-ing biological phenomena that should be investigated. Several transcriptional regula-tors are induced wild-type ATCC 2001 cells exposed to 1 M sorbitol. CIN5 is expressed in hogl and sten cells exposed to 1 M sorbitol. Inspection of CHiP-chip data for S. cerevisiae reveals that Cin5p binds upstream of GPD1, a key glycerol biosynthetic enzyme. Assuming that C. glabrata Cin5p binds to the orthologous GPD2 promoter region, there may exist a HOG1-independent regulator of GPD2 transcription and glycerol biosynthesis. Electrophoretic mobility shift assays should be designed to as-sess if C. glabrata Cin5p binds to the upstream GPD2 promoter region. A cin5 null mutant should also be constructed. GPD2 transcript levels should be measured anal-ysis in ATCC 2001 and cin5 cell extracts grown in rich culture medium ± sorbitol (1 M)

9. To further investigate the role of CIN5 in glycerol production, internal glycerol ac-cumulation assays should be performed in cin5 and gpd2 cells grown in rich culture medium ± sorbitol (1 M). Since internal glycerol accumulation is a net effect of glyc-erol biosynthesis and efflux, the study may have higher resolution if the efflux system is impaired. Accordingly these experiments may need to be performed in an fpsl background or in strains that have a constitutively closed Fps1p aquaglyceroporin.

10. The analysis of the STE11 and HOG1 dependency of the transcriptional response highlighted four major sets of genes: a) a set of genes that were dependent on the extant Shot signalling branch, b) a set of genes that were not dependent on HOG1 or STE11, c) a set of genes that were specifically dependent on STE11 and d) a set of genes that were specifically dependent on HOG1. Analysis of over-represented GO terms showed that the Shol branch dependent genes predominantly encode genes in-volved in metabolism, including glycerol biosynthetic genes. Those genes that were independent of HOG1 and STE11 contained calcineurin signalling components sug-gesting that the calcineurin pathway is not under the control of Hog1p or Ste11p. The STE11 dependent genes were predominantly associated with cell wall remodelling and oxidative stress protection whereas the HOG1 dependent genes predominantly encoded genes involved in proteolysis.

11. The induction of genes involved in cell wall remodelling, both at the level of signalling and catalysis suggests that changes in the cell wall may be part of the adaptive response to hyperosmotic stress in C. glabrata. Notably, genes encoding signalling

6.1 Future work 196

components of the calcineurin and protein kinase C cell integrity pathways are co-induced with their downstream targets (see data in Chapter 3). To individually test whether transcriptional induction of the PKC cell integrity signalling genes SLT2, MID2 and BCK1 is required for proliferation in hyperosmotic stress null mutants of these strains should be constructed and tested for growth on agar supplemented with 1 M sorbitol or 1 M Na Cl. Assuming that deletion of a single component of the PKC cell integrity pathway is sufficient to disrupt its function, one would expect all of these strains to be sensitive to the above supplementations if this pathway is required for the hyperosmotic stress adapation in C. glabrata. A similar experiment can be conducted by chemical epistasis if such knockout strains prove hard to obtain. The protein kinase C pathway can be inhibited by supplementation of the culture medium with caffeine (1- 10 mM) (267). If the protein kinase C pathway is required for osmoadaption then wild-type C. glabrata cells grown in 1 M sorbitol and caffeine (1- 10 mM) will have more impaired growth than strains cultured in sorbitol alone or caffeine alone. Physical evidence of cell wall remodelling can also be investigated by assaying chitin levels qualitatively and quantitatively using calcofluor white staining and (722; 488)


1. In chapter 4 we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection in order to identify genes preferentially expressed during adaptation to the mammalian host niche. This analy-sis revealed a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a promi-nent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, sub-telomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation tran-scriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase laeA, a putative global regulator of sec-ondary metabolite production.

Chapter 6. Conclusions and future work 197

2. The regulation of such secondary metabolite production is unclear, however envi-ronmental signals such as pH changes and carbon availability may alter production of metabolites through the actions of proteins such as PacC (146). Although tran-scriptional regulation of secondary metabolites by cis-acting transcriptional regulators which simultaneously activate or repressall the genes of the cluster has been described (74), chromatin regulation is also thought to play a significant role. Deletion of the laeA gene has been shown to result in the loss of numerous secondary metabolites (75; 539)), and transcriptional comparisons between in vitro cultures of wild-type and

laeA strains using microarrays revealed 13 out of 22 secondary metabolite clusters were down regulated in the AlaeA mutant. In these clusters, regulation by laeA is limited to genes within the cluster and not to surrounding genes (539). Additionally, the laeA protein contains motifs which are similar to histone methlytransferases, sug-gesting laeA regulation via chromatin remodelling. Interestingly, the AlaeA strain shows attenuation in murine models of virulence, with no significant difference in in vitro growth rates (73). It is presumed that the attenuation of virulence is in some way related to the decrease in multiple toxin production.

3. A clearer mechanistic understanding of the putative epigenetic control of transcription and secondary metabolite production may elucidate a major mechanism of A. fumi-gates adaptation within the host and also identify future candidates for therapeutic intervention. Future work should focus on the functional role of LaeA in histone mod-ification and its mediatory role in transcription within the host. The first step will be to evaluate those LaeA dependent secondary metabolism clusters by comparing the virulence and transcription profiles of AlaeA strain and wild-type strains in vivo. In addition, the role of secondary metabolite production in virulence can be more specifically addressed by creating mutants with deficiencies in secondary metabolite production. For example ApsoA and AftmA lack a hybrid NRPS/PKS gene and a NRPS gene respectively. The virulence of these strains should be investigated in both cyclophosphamide and hydrocortisone treated models of murine infection.

4. Whilst our initial investigations are a snapshot of the fungal response to the host, relative to a nutrient rich in vitro culture, a more detailed time-series investigation of transcription in wild-type and A.laeA strains, coupled with NMR and LCMS analy-sis of secondary metabolite production will determine whether secondary metabolite synthesis is stage-specific and laeA-dependent.


1. In chapter 5, we showed that targeted disruptions of C. glabrata open reading frames

6.1 Future work 198

can be achieved systematically. We adopted these methods to disrupt orthologues of S. cerevisiae transcription factors in C. glabrata. We used these genetic tools for a functional genomics study of fluconazole susceptibilty in order to identify novel mediators of resistance mechanisms.

2. Sensitive strains should now be profiled in clinically acceptable sensitivity assays such as the NCLSS (138) or EUCAST (588) microbroth methods. Furthermore, several mechanisms involved in fluconazole sensitivity have been previously described (see (97)). Sensitive strains should therefore be profiled for respiratory deficiency by growth on yeast peptone glycerol agar or by cytometric analysis of rhoadmine 123 accumulation in the mitochondria; in addition the ergosterol content of strains should also be measured to rule out any defects in ergosterol biosynthesis that may be com-pounded by fluconazole treatment.

3. Gene expression microarray experiments of sensitive transcription factor null strains can be performed to define the regulon of these genes with and without exposure to fluconazole to further define the processes that are controlled by these genes, which may identify novel pathways involved in fluconazole resistance.

Factor Fungal Pathogens Mucosal and cutaneous barrier disruption

Neutrophil dysfunction

Candida spp. Aspergillus spp

Candida spp. Aspergillus spp.

Trichosporon

Defects in cell-mediated immunity Cryptococcus endemic mycoses

Metabolic disorders Candida spp. Zygomycetes

Exposures Aspergilllus spp

endemic mycoses

Extremes of age (< 1 > 70 years) Candida spp

199

Appendix A

Chapter 1 Supplementary Data

Table A.1: Factors involved in the development of opportunistic mycoses. Par-tial list of pre-disposition categories involved in the development of opportunistic mycoses. Taken from (542)

Ketoc663zoie

Itracanazole

Posacannale

Yori tale

Flucoataanie

1450 1972 1976 1983 15146 1990 15051 6

Appendix A. Chapter 1 Supplementary Data 200

KEY: Anti-metabolite Atoles

2006

Figure A.1: Licensing timeline for antifungal drugs with systemic bioavailability. Data taken from Drugs@FDA http://www.accessdata.fda.gov/scripts/cder/drugsatfda/

A B

2003 1998 1999 2000 2001 2002 2003

year

1998 1999 2000 2001 2002

year

C

ro

.0 a

2000 2001 2002 2003


year

Figure A.2: Species specific incidence rates of invasive candidiasis. Aetiological agents of invasive candidiasis (IC) and their associated incidence rates. Incidence data taken from (542) indicate Candida albicans is the primary aetiologial agent of invasive candidiasis (IC) (A), its prevalence decreased between 1998 and 2003, with concomitant increases in non-albicans species Candida glabrata, Candida tropicalis, Candida parapsilop-sis and Candida krusei (B). The shaded panel in (B) denotes the period in which new aetiological agents rapidly emerged as causes of IC (C).

5 f,

0 0 - •9

ro a

.,,

2

o

— C. albicans - - C.glabrata — C.tropiCalis - - C.parepsilopsis — C.krusei

— C. inconspicua - C.norvengsis

— C.dubliniensis Clipolytica

— C.zelanoides - - C.pelliculose

Amphotericin B VON, 0

N CI

0 OH rTh,

CI 0

Itraconazole

CI


off

Ketoconazole

N

F

Fluconazole

---1 Hydroxy-methyl-glutaryl (HMG)-CoA)

1,1vIG-CoA Reductase

Al Iylamines

Mevalonic acid

Squalene

I Squalene epoxidase IERG/)

Lanosterol

A"— saturase (ERG3) Azoles ---

eplethyl-lanosterol cp1,-,d

L1-1-t eductase (ERG24)

Fecosterol

- .sorne rase (ERG21

Polyenes_

Episterol

Ergosterol

Figure A.3: Common antifungal drug molecules and their mechanism of action. Structure of commonly used antifungals (A) and schematic represenation of the mechanism of action of selected classes of antifungal drugs affecting the ergosterol biosynthesis pathway (B). Biochemical precursors are depicted as blue rectangles; the enzymes catalyzing reaction steps are shown to their right with encoding genes in parentheses. Antifungal drug classes are reported in grey outlined rectangles to the left of the relevant biosynthetic step, or in the case of polyenes directly toward the molecular target. Adapted from Lupetti et al (426)

C14a demethylase (ERG11)

14a-methyl-3,6-diol Amo ro Hine

Blofilm Formation

Fit. rrent ati on

51, PEP1-7, /PC CST20

T Issue Invasion

R.4052,1E11

1,

Endosomal Transport

Cell Wall integrity

CHS2,CHS.i.1114. G5C1,440041

COMPit475 JradEMVPi

CCCI. CTIY3 FRE10,F1,112 GC/44,6,01 I801,11ArG1, PEP7A4P1-8 Str1.114,1,

SAP1-8

Adhesiori

71,5t 11 PAS1

Al A. VPSJ

CiAL I


Figure A.4: Schematic summary of Candida albicans virulence factors stored in the CGD database. Gene names bounded by a white box are required for the associated function, which is denoted by the caption in the grey box, but are not necessary or virulence. Gene names bounded by the red boxes are required for virulence. Data sourced from CGD.


N

Y.lipolytica 111.111.1111111111111.1.1.111111.1111111.11

C. g I ab rata 1111111111.111111111111.111.11.111111.111

S.cereyistae

S.kluyveri

A.gossypii

K.lactis 1111111111111111111111111111111•111111111111111111111111111111111111•111

C.lusitanae

C.guilliermondii

D.hansenii

C. parapsilopsis

C.tropicalis 1111111111111111111111111111111111111111 -11111111111.1111111111111111111.11111111111111111111111111111

0 10 20 30 40 50 60 70 80 90

Proportion of conserved virulence genes (%)

en Ascomycota l'arrmi.ra lrlrnlmiia

Candido pirotmitta

Sacchairomycohtna

W OD S Chan' Air '4•1 CM lent!

Sur Y: baron iy4 CA AlikarCle

100 SYa OYA111141.1

ril-ht4r7Nrity.reN rroinvir3 141. 1.11 1117.1T 4' tAtlyitill,11A

Aro (741r. ce.y iiiinur LI: rill irsq.preJni s Iva {

Ita.5-1'ffir(.1711Y'et kiatyvero

‘Way,;.1 gym rai

,5. rit,,vemovy4-ew ?at' A

CC4Flifi4*/ iitraraniar C'artdiriro ,gr4diarrniornlii

100

GTG 00

Dri'xu-viArr5rrr hiamer4 Candider itklritiMike. AA

uruliilaa trrviralit

7C:10 etlablatir bbirli rri 100 Ca 'Oak OHM lAITA

Figure A.5: Proportion of virulence factor (VF) reciprocal best BLAST (587; 16) hits in the Saccharomycotina as a function of evolutionary distance from Candida albicans. Species located in the same subgroup as C. albicans (orange bars), that translate the colon CTG as serine, show a higher proportion of conserved, published VFs than whole genome duplication (WGD) species (green bars).

205

Appendix B

Media Recipes

Table B.1: Recipe for Synthetic Complete culture medium (1 L). Components above the double solid line are added to 800 ml of distilled water and dissolved with the aid of a magnetic stirrer. Solutions are made up to 1 L and agar is added if solid medium is required. Culture media are sterilized by heating at 121°C for 15 minutes.

Constituent Mass (g) Final Concentration YNB - amino acids, - ammonium sulphate

6.78 0.68 % (w/v)

Complete Supplement Medium 0.798 0.0798 % (w/v) d-Glucose 20 2 % (w/v) Agar 20 2 % (w/v)

Table B.2: Recipe for Yeast Extract Peptone Dextrose culture medium (1 L). Components above the double solid line are added to 800 ml of distilled water and dissolved with the aid of a magnetic stirrer. Solutions are made up to 1 L and agar is added if solid medium is required. Culture media are sterilized by heating at 121°C for 15 minutes.

Constituent Mass (g) Final Concentration Bacteriological peptone 20 2 % (w/v) Yeast extract 10 1 % (w/v) d-Glucose 20 2 % (w/v) Agar 20 2 % (w/v)

206

Appendix C

Strain Genotypes

Table C.1: Fungal strains used in this study. The genotype describes the strain back-

ground used and the gene disrupted separated by a ",". Reconstitution is represented by

genes between square braces. Plasmid details are in the accompanying plasmid table.

Species Strain Name Genotype Origin

S. CCT,I4Siae BY4741 MATa Ahis3Aleu2Amet15Aura3 EuroScarf C. glabrata C. glabrata C. glabrata C. glabrata C. glabrata C. glabrata

ATCC 2001 2001AHT6

AM3 AM22 AM25 JG7

Type strain Aura3::pUC18, Atrp/ Aura3,his3::URA3

2001AHT6, ste11,HIS3 2001AHT6, .ste20,HIS3

AM22, pAMC85 [STE20, TRP1] 2001AHT6, hog1,111S3

American Type Culture Collection (734)

(106; 104) (106; 103) (106; 103) This study

207

Appendix D


Table D.1: Oligonucleotides used in the three step PCR strategy for HOG1 disruption in C. glabrata

Oligonucleotide Sequence 5'-3' HOG15-5 GTATCCATACTTGCGTTTTGG HOG15-3 gtcatagctgttt cctgTGTATAGTGTAGTATATCCAAG HOG13-5 tacaacgt cgtgactgggCAGCTCGAATTCTGCAACAA HOG13-3 TTTTGGAGGACTATTTCCGATT

HOG1-HIS5 CTTGGATATACTACACTATACAcaggaaacagctatgac HOG1-HIS3 TTGTTGCAGAATTCGAGCTGcccagt cacgacgttgt a HOG13-ex CGAAGGAGCAACAAAAGAGG HIS3INTF TGGGCAAGGAATATGCTAGTGG CgHOG F1 GCGCGGATCCGAACTGATTAAGATGTTCAGAGACGAGG CgHOG R1 GCGCGGATCCTGAAATATTCGCTAAGGGTATGTAG

Appendix D. Chapter 3 Supplementary Data 208

Table D.2: Biological effects encoded by linear model contrasts

Figure 3.4 ID

Effect Name Biological Effect

1 STE.0 - WT.0 Untreated stell cells versus untreated ATCC 2001 cells

2 HOG.0 - WT.0 Untreated hog1 cells versus untreated ATCC 2001 cells

3 STE.S - WT.S Hyper-osmotic stress treated stell cells versus untreated ATCC 2001 cells

4 HOG.S - WT.S Hyper-osmotic stress treated stell cells versus untreated ATCC 2001 cells

5 WT.S - WT.0 Hyper-osmotic stress treated ATCC 2001 cells versus untreated ATCC 2001 cells

6 HOG.S - HOG.0 Hyper-osmotic stress treated hog1 cells versus untreated hog1 cells

7 STE.S - STE.0 Hyper-osmotic stress treated ste11 cells versus untreated stell cells

8 HOG.0 - STE.0 Untreated hog1 cells versus untreated stell cells

9 HOG.S - STE.S Hyper-osmotic stress treated hog1 cells versus Hyper-osmotic stress treated stell cells


Table D.3: Candida glabrata transcripts induced in wild-type cells following exposure to YEPD 1M sorbitol for 15 minutes. Induced transcripts are defined as having log2(Cy5) - log2(Cy3) > 2.0 and Benjamini-Hochberg corrected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 effect described in Table D.2

CG ORF SC ORF gene name Description WT.S - WT.0 CAGLOI10010g YGR142W BTNS v-SNARE binding protein that fa-

cilitates specific protein retrieval from a late endosome to the Golgi; modulates arginine uptake, possible role in mediating pH homeosta-sis between the vacuole and plasma membrane H(+)-ATPase

5.55

CAGL0CO2321g YER037W PHM8 Protein of unknown function, expression is induced by low phosphate levels and by inactivation of

5.41

Pho85p CAGL0G04433g YJL108C PRM10 Pheromone-regulated protein, pre-

to have 5tpreadnsmembrane dictedt segments; induced by treatment with 8-methoxypsoralen and UVA irradiation Description not

map 5.14

CAGLOE04554g - - 4.96 CAGLOM09713g YMR152W YIMI Protein of unknown function; null

mutant displays sensitivity to DNA damaging agents; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

4.9

CAGLOK08338g YOR385W - Description not mapped 4.57 CAGLOA02816g YDR368W YPRI NADPH-dependent aldo-keto

reductase, reduces multiple substrates including 2- methylbutyraldehyde and D,L- glyceraldehyde, expression is induced by osmotic and oxidative stress; functionally redundant with other aldo-keto reductases

4.43

CAGLOM06963g YNR034W SOLI Protein with a possible role in tRNA export; shows similarity to 6-phosphogluconolactonase non-catalytic domains but does not exhibit this enzymatic activity; homologous to Sol2p, Sol3p, and

4.31

Sol4p CAGLOJ10846g YHR071W PCL5 Cyclin, interacts with and phos-

phorylated by Pho85p cyclin- dependent kinase (Cdk), induced by Gcn4p at level of transcription, specifically required for

4.09

Gcn4p degradation, may be sensor of cellular protein biosynthetic capacity

CAGL0105390g YPL026C SKSI Putative serine/threonine protein kinase; involved in the adaptation to low concentrations of glucose in-dependent of the SNF3 regulated pathway

3.97



continued from previous page CG ORF SC ORF gene name Description WT.S - WT.0

CAGLOM08822g YDR258C HSP78 Oligomeric mitochondria] matrix chaperone that cooperates with

3.93

Ssclp in mitochondrial thermotol-erance after heat shock; able to pre-vent the aggregation of misfolded proteins as well as resolubilize pro-tein aggregates

CAGLOH08151g - - Description not mapped 3.86 CAGLOE00803g YDR171W HSP42 Small heat shock protein (sHSP)

with chaperone activity; forms barrel-shaped oligomers that sup-press unfolded protein aggregation; involved in cytoskeleton reorganiza-tion after heat shock

3.82

CAGLOL05016g YKL072W STB6 Protein that binds Sin3p in a two- hybrid assay

3.64

CAGLOG03883g YLL026W HSP/04 Heat shock protein that cooperates with Ydjlp (Hsp40) and Ssalp

3.61

(Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses in-cluding: heat, ethanol, and sodium arsenite; involved in [PSI-I-] propa-gation

CAGLOH06523g YIL145C PANG Pantothenate synthase, also known as pantoate-beta-alanine ligase, re-quired for pantothenic acid biosyn-thesis, deletion causes pantothenic acid auxotrophy, homologous to E. coli panC

3.6

CAGLOF08745g YLR327C TAIA10 Protein of unknown function that associates with ribosomes

3.53

CAGLOH08173g YOR028C CIN5 Basic leucine zipper (bZIP) transcription factor of the yAP-1 family, mediates pleiotropic drug resistance and salt tolerance; nude-arly localized under oxidative stress and sequestered in the cytoplasm by

3.49

Lot6p under reducing conditions CAGLOG01540g YNL036W NCE103 Carbonic anhydrase; poorly tran-

scribed under aerobic conditions and at an undetectable level under anaerobic conditions; involved in non-classical protein export path-way

3.47

CAGLOM12474g YIL055C - Description not mapped 3.46 CAGLOM07381g YPL053C KTR6 Probable mannosylphosphate

transferase involved in the synthesis of core oligosaccharides in protein glycosylation pathway; member of the KRE2/MNT1 mannosyltransferase family

3.45

CAGLOF09119g YHR207C SET5 Zinc-finger protein of unknown function, contains one canonical and two unusual fingers in unusual arrangements; deletion enhances replication of positive-strand RNA virus

3.38

CAGLOK00715g YGR213C RTA1 Protein involved in 7- aminocholesterol resistance; has seven potential membrane-spanning regions; expression is induced un-der both low-heme and low-oxygen conditions

3.3




CAGLOM12320g

CAGLOG00858g

YAL053W

YLR332W

FLC2

MID2

Putative FAD transporter; required for uptake of FAD into endoplas-mic reticulum; involved in cell wall maintenance 0-glycosylated plasma membrane protein that acts as a sensor for cell wall integrity signaling and activates the pathway; interacts with Rom2p, a guanine nucleotide exchange factor for Rho1p, and with cell integrity pathway protein

3.28

3.23

Zeolp CAGL0G00242g YGR281W YORI Plasma membrane ATP-binding

cassette (ABC) transporter, rnul- tidrug transporter mediates export of many different organic anions including oligomycin; similar to hu-man cystic fibrosis transmembrane receptor (CFTR)

3.21

CAGL0102684g YMR285C NGL2 Protein involved in 5.85 rRNA processing; Ccr4p-like RNase required for correct 3'-end formation of 5.8S rRNA at site E; similar to Ngllp and Ngl3p

3.2

CAGLOK02761g YHLOO2W HSE1 Subunit of the endosomal Vps27p- 3.2 H se cnInert requiredm for sorting of ubiquitinated membrane proteins into intralumenal vesicles prior to vacuolar degradation, as well as for

Description

1p app ed

recycling of Golgi proteins and for-mation of lumens] membranes

CAGLOL09251g - - 3.17 CAGL00O2233g YER042W AfXR / Peptide methionine sulfoxide reduc-

tase, reverses the oxidation of me-thionine residues; involved in oxida-tive damage repair, providing resis-tance to oxidative stress and regu-lation of lifespan

3.14

CAGLOJ00539g YHR030C SLT2 Serine/threonine MAP kinase involved in regulating the maintenance of cell wall integrity and pro-gression through the cell cycle; reg-ulated by the PKC1-mediated sig-naling pathway

3.13

CAGLOK00539g YPL259C APM1 Mul-like medium subunit of the clathrin-associated protein complex

3.1

(AP-1); binds clathrin; involved in clathrin-dependent Golgi protein sorting

CAGLOD03916g YHR034C P1111 Protein of unresolved function; may function in protein folding and/or rRNA processing, interacts with a chaperone (Hsp82p), two chromatin remodeling factors (Rvblp, Rvb2p) and two rRNA processing factors

3.1

(Rrp43p, Nop58p) CAGLOM12034g YAL038W CDC19 Pyruvate kinase, functions as a ho-

motetramer in glycolysis to convert phosphoenolpyruvate to pyruvate, the input for aerobic (TCA cycle) or anaerobic (glucose fermentation) respiration

3.09




CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein, expression is increased in response to heat-shock stress or treatments that produce DNA lesions; contains multiple repeats of the amino acid sequence NNNDSYGS

3.06

CAGL0104246g YGL162W S UT/ Transcription factor of the 3.04 Zn[II]2Cys6 family involved in sterol uptake; involved in induction of hypoxic gene expression

CAGLOD05082g YLL039C U/34 Ubiquitin, becomes conjugated to proteins, marking them for selective degradation via the ubiquitin-

2.99

26S proteasome system; essential for the cellular stress response; en-coded as a polyubiquitin precursor comprised of 5 head-to-tail repeats

CAGLOH04631g YML007W YAPI Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance; activated by

2.98

11202 through the multistep forma-tion of disulfide bonds and transit from the cytoplasm to the nucleus; mediates resistance to cadmium

CAGLOD05368g YBR253W SRB6 Subunit of the RNA polymerase II mediator complex; associates with core polymerase subunits to form the RNA polymerase II holoenzyme; essential for transcriptional regulation

2.97

CAGLOF08107g YGR244C LSC2 Beta subunit of succinyl-CoA ligase, which is a mitochondrial enzyme of the TCA cycle that catalyzes the nucleotide-dependent conversion of succinyl-CoA to suc-cinate

2.96

CAGLOB04257g YBRO31W RPL4A N-terminally acetylated protein component (60S) ribosomal subunit, nearly identical to Rpl4Bp and has similarity to

2.96

E. coli L4 and rat L4 ribosomal proteins Description not

o mtahpepedlarge

CAGLOE03025g - - 2.94 CAGLOM02255g YPL 152W RRD2 Activator of the phosphoty-

rosyl phosphatase activity of 2.92

PP2A,peptidyl-prolyl cis/trans- isomerase; regulates G1 phase progression, the osmoresponse, microtubule dynamics; subunit of the Tap42p-Pph2lp-Rrd2p complex

CAGLOJ01595g YPRO15C - Description not mapped 2.91 CAGLOJ00385g YHR049W FSHI Putative serine hydrolase that lo-

calizes to both the nucleus and cytoplasm; sequence is similar to S. cerevisiae Fsh2p and Fsh3p and the human candidate tumor suppressor

2.89

OVCA2 CAGLOE05632g YOR348C PUT4 Proline permease, required for high-

affinity transport of proline; also transports the toxic proline ana-log azetidine-2-carboxylate (AzC);

2.85

PUT4 transcription is repressed in ammonia-grown cells




CAGLOG04763g YOR107W RGS2 Negative regulator of glucose- induced cAMP signaling; directly activates the GTPase activity of the heterotrimeric G protein alpha subunit Gpa2p

2.84

CAGLOJ00253g YGRO23W MTL1 Protein with both structural and functional similarity to Mid2p, which is a plasma membrane sensor required for cell integrity signaling during pheromone-induced morpho-genesis; suppresses rgdl null muta-tions

2.83

CAGLOJ03256g YER075C PTP3 Phosphotyrosine-specific protein phosphatase involved in the inactivation of mitogen-activated protein kinase (MAPK) during osmolarity sensing; dephosporylates Hog1p MAPK and regulates its localization; localized to the cytoplasm

2.83

CAGLOL05434g YKR042W UTH/ Mitochondrial outer membrane and cell wall localized SUN family mem-ber required for mitochondrial au-tophagy; involved in the oxidative stress response, life span during starvation, mitochondrial biogene-sis, and cell death

2.83

CAGL0005137g YOL059W GPD2 NAD-dependent glycerol 3- phosphate dehydrogenase, homolog of Gpdlp, expression is controlled by an oxygen-independent signal-ing pathway required to regulate metabolism under anoxic conditions; located in cytosol and mitochondria

2.81

CAGLOG07601g YBR287W - Description not mapped 2.79 CAGLOA02046g YOR018W ROD1 Membrane protein that binds the

ubiquitin ligase Rsp5p via its 2 PY motifs; overexpression confers resistance to the GST substrate o- dinitrobenzene,zinc, and calcium; proposed to regulate the endocyto-sis of plasma membrane proteins

2.79

CAGLOM01628g YDR389W SAC7 GTPase activating protein (GAP) for Rho1p, involved in signaling to the actin cytoskeleton, null muta-tions suppress tor2 mutations and temperature sensitive mutations in actin; potential Cdc28p substrate

2.77

CAGLOH09812g YDROO6C SOK1 Protein whose overexpression suppresses the growth defect of mu-tants lacking protein kinase A ac-tivity; involved in cAMP-mediated signaling; localized to the nucleus; similar to the mouse testis-specific protein PBS13

2.77

CAGLOF09163g YER182W FMP10 Putative protein of unknown function; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

2.74




CAGLOG01166g YLR348C Did Mitochondria] dicarboxylate carrier, integral membrane protein, catalyzes a dicarboxylate- phosphate exchange across the inner mitochondrial membrane, transports cytoplasmic dicar- boxylates into the mitochondria] matrix

2.7

CAGLOH07557g YGL254W FZF1 Transcription factor involved in sulfite metabolism, sole identified reg-ulatory target is SSU1, overexpres-sion suppresses sulfite-sensitivity of many unrelated mutants due to hy-peractivation of SSU1, contains five zinc fingers

2.68

CAGLOF06941g YGL062W PYC1 Pyruvate carboxylase isoform, cytoplasmic enzyme that converts pyru-vate to oxaloacetate; highly similar to isoform Pyc2p but differentially regulated; mutations in the human homolog are associated with lactic acidosis

2.66

CAGLOH02519g YMR253C - Description not mapped 2.65 CAGLOL07480g YBRO66C NRG2 Transcriptional repressor that me-

diates glucose repression and nega-tively regulates filamentous growth; has similarity to Nrglp

2.64

CAGLOM11066g YGR109C CLB6 B-type cyclin involved in DNA replication during S phase; activates Cdc28p to promote initiation of DNA synthesis; functions in formation of mitotic spindles along with C1b3p and Clb4p; most abun-dant during late GI

2.64

CAGL0003201g YLL040C VPS13 Protein of unknown function; heterooligomeric or homooligomeric complex; peripherally associated with membranes; homologous to human COH1; involved in sporula-tion, vacuolar protein sorting and protein-Golgi retention

2.63

CAGLOL00803g YER054C CIP2 Putative regulatory subunit of the protein phosphatase Glc7p, involved in glycogen metabolism; contains a conserved motif (GVNK motif) that is also found in Gaclp, Pig1p, and Pig2p

2.62

CAGL0,11.0296g YNL077W APJ1 Putative chaperone of the HSP40 2.62 (DNAJ) family; overexpression interferes with propagation of the [Psi+) prion; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

CAGLOK12034g YDR040C ENA 1 P-type ATPase sodium pump, involved in Nab- and Li+ efflux to al-low salt tolerance

2.61

CAGLOM01782g YBRO80C SEC18 ATPase required for the release of 2.59 Secl7p during the 'priming' step in homotypic vacuole fusion and for ER to Golgi transport; homolog of the mammalian NSF




CAGLOJ01353g YMR089C YTA.12 Component, with Afg3p, of the mitochondrial inner membrane m-

2.57

AAA protease that mediates degra-dation of misfolded or unassembled proteins and is also required for cor-rect assembly of mitochondrial en-zyme complexes

CAGLOI01100g YOR120W GCY1 Putative NADP(-1-) coupled glycerol dehydrogenase, proposed to be involved in an alternative pathway for glycerol catabolism; member of the aldo-keto reductase (AKR) fam-ily

2.56

CAGLOI07007g YGL219C MDM3.4 Mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance; localizes to dots on mitochondrial surface near mtDNA nucleoids; ubiquitination target of

2.55

Mdm30p; interacts genetically with MDM31 and MDM32

CAGLOB01991g YDR126W S WF/ Palmitoyltransferase that acts on the SNAREs Snclp, Syn8p, Tlglp and likely on all SNAREs; mem-ber of a family of putative palmi-toyltransferases containing an Asp-

2.55

His-His-Cys-cysteine rich (DHHC- CRD) domain; may have a role in vacuole fusion

CAGLOF07095g YGL122C NAB2 Nuclear polyadenylated RNA- binding protein required for nuclear mRNA export and poly(A) tail length control; binds nuclear pore protein MIplp; autoregulates mRNA levels; related to human hnRNPs; nuclear localization sequence binds Kap104p

2.54

CAGLOH05137g YPL061W ALD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes

2.53

NADP4- as the preferred coenzyme; required for conversion of acetaldehyde to acetate; constitutively expressed; locates to the mitochondrial outer surface upon oxidative stress

CAGLOE00649g YCR079W PTC6 Mitochondrial protein phosphatase of type 2C with similarity to mammalian PP1Ks; involved in mi- tophagy; null mutant is sensitive to rapamycin and has decreased phos-phorylation of the Pdal subunit of pyruvate dehydrogenase

2.53

CAGLOM06347g YBR183W YPC1 Alkaline ceramidase that also has reverse (CoA-independent) ceramide synthase activity, catalyzes both breakdown and synthesis of phytoceramide; overexpression confers fumonisin B1 resistance

2.52

CAGLOL09152g YFROO4W BPN11 Metalloprotease subunit of the 19S regulatory particle of the 26S pro-teasome lid; couples the deubiqui-tination and degradation of protea-some substrates

2.52




CAGLOA02024g

CAGLOJ11176g CAGLOJ04466g CAGLOH03729g

YOL025W

YNL176C YLR4I4C YNLOO6W

LAG2

- -

LST8

Protein involved in determination of longevity; LAG2 gene is preferen-tially expressed in young cells; over-expression extends the mean and maximum life span of cells Description not mapped Description not mapped Protein required for the transport of amino acid permease Gap1p from the Golgi to the cell surface; com-ponent of the TOR signaling pathway; associates with both Tor1p and Tor2p; contains a WD-repeat

2.52

2.51 2.5

2.48

CAGLOG07689g YKR014C YPT52 GTPase, similar to Ypt5lp and 2.48 Ypt53p and to mammalian rab5; re-quired for vacuolar protein sorting and endocytosis

CAGLOL04708g YGR111W - Description not mapped 2.48

CAGLOA04081g YLR194C - Description not mapped 2.48

CAGLOL00605g YKL190W CNB1 Calcineurin B; the regulatory subunit of calcineurin, a

2.46

Cal-l-/calmodulin-regulated type 2B protein phosphatase which regulates Crzlp (a stress-response transcription factor), the other calcineurin subunit is encoded by CNA1 and/or CMP1

CAGLOL10186g YOR052C - Description not mapped 2.45

CAGLOL11572g YHR103W SBE'22 Protein involved in the transport of cell wall components from the

2.43

Golgi to the cell surface; similar in structure and functionally redun-dant with Sbe2p; involved in bud growth

CAGLOE06424g YKL150W MCR1 Mitochondrial NADH-cytochrome b5 reductase, involved in ergosterol biosynthesis

2.42

CAGLOK00891g YGR205W - Description not mapped 2.42

CAGLOJ03322g YER080W AIMS Putative protein of unknown function; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

2.4

CAGLOM10263g YCLOO2C - Description not mapped 2.39

CAGLOJ09108g YDL190C UFD2 Ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme

2.39

(E2), and a ubiquitin protein ligase (E3) to conjugate ubiquitin to substrates; also functions as an E3

CAGLOD05434g YPRO65W ROX1 Heme-dependent repressor of hypoxic genes; contains an HMG do-main that is responsible for DNA bending activity

2.38

CAGLOL06028g YGL181W GTS1 Protein that localizes to the nucleus and is involved in transcription regulation; also localizes to actin patches and plays a role in en-docytosis; N-terminus contains an

2.38

ARF-GAP domain and C-terminus contains a Gln-rich domain




CAGLOM14047g

CAGL0004499g

YMR318C

YDLOO6W

ADM;

PTC1

NADPH-dependent medium chain alcohol dehydrogenase with broad substrate specificity; member of the cinnamyl family of alcohol dehydro-genases; may be involved in fusel al-cohol synthesis or in aldehyde toler-ance Type 2C protein phosphatase (PP2C); inactivates the osmosens-ing MAPK cascade by dephosphorylating Hog1p; mutation delays mitochondria] inheritance; deletion reveals defects in precursor tRNA splicing, sporulation and cell separation

2.36

2.35

CAGLOJ04158g YOR220W RCN2 Protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm and is induced in response to the

2.35

DNA-damaging agent MMS; phos-phorylated in response to alpha fac-tor

CAGL0104554g YBRO14C GRX7 Cis-golgi localized monothiol glutaredoxin; more similar in activ-ity to dithiol than other monothiol glutaredoxins; involved in the oxidative stress response; does not bind metal ions; functional overlap with GRX6

2.34

CAGLOGD5313g YDR072C IPTI Inositolphosphotransferase, involved in synthesis of mannose-

2.34

(inositol-P)2-ceramide (M(IP)2C), the most abundant sphingolipid;, can mutate to resistance to the antifungals syringomycin E and DmAMP1 and to K. lactis zymocin

CAGLOD04752g YPR127W - Description not mapped 2.33 CAGLOB00836g YCL033C - Description not mapped 2.33

CAGLOE02299g YOL013C HRD1 Ubiquitin-protein ligase required for endoplasmic reticulum- associated degradation (ERAD) of misfolded proteins; genetically linked to the unfolded protein response (IJPR); regulated through association with Hrd3p; contains an H2 ring finger

2.31

CAGLOG06886g Y.11.017W - Description not mapped 2.3 CAGLOK03509g YMR110C 11FD1 Putative fatty aldehyde dehydro-

genase, located in the mitochondria) outer membrane and also in lipid particles; has similarity to hu-man fatty aldehyde dehydrogenase

2.3

(FALDH) which is implicated in Sjogren-Larsson syndrome

CAGL0G05049g YD11.063W AIM7 Putative protein of unknown function; green fluorescent protein

2.28

(GFP)-fusion protein localizes to the cytoplasm and nucleus; YDR063W is not an essential gene

CAGLOA02321g YDR345C HXT3 Low affinity glucose transporter of the major facilitator superfamily, expression is induced in low or high glucose conditions

2.28



continued from previous page CG ORF SC ORF gene name Description WT.S - WT.IJ

CAGLOA03058g YDR379W RGA2 GTPase-act ivating protein for the polarity-establishment protein

2.27

Cdc42p; implicated in control of septin organization, pheromone response, and haploid invasive growth; regulated by Pho85p and Cdc28p

CAGL0000363g YJL174W KRE9 Glycoprotein involved in cell wall beta-glucan assembly; null mutation leads to severe growth defects, aberrant multibudded morphology, and mating defects

2.27

CAGLOL07238g YDL097C RPN6 Essential, non-ATPase regulatory subunit of the 26S proteasome lid required for the assembly and activ-ity of the 26S proteasome; the hu-man homolog (S9 protein) partially rescues Rpn6p depletion

2.25

CAGLOI04774g YBRO36C CSG2 Endoplasmic reticulum membrane protein, required for mannosylation of inositolphosphorylceramide and for growth at high calcium concen-trations

2.25

CAGLOJ11506g YNL192W CHSI Chitin synthase I, requires activation from zymogenic form in order to catalyze the transfer of

2.25

N-acetylglucosamine (G1cNAc) to chitin; required for repairing the chitin septum during cytokinesis; transcription activated by mating factor

CAGLOG04873g YLR371W ROM2 GDP/GTP exchange protein (GEP) for Rho1p and Rho2p; mutations are synthetically lethal with muta-tions in roml, which also encodes a

2.25

GEP CAGLOM06545g YKL124W SSH4 Specificity factor required for 2.24

Rsp5p-dependent ubiquitination and sorting of cargo proteins at the multivesicular body; identified as a high-copy suppressor of a SHR3 deletion, increasing steady-state levels of amino acid permeases

CAGLOH07007g YML092C PRE8 Alpha 2 subunit of the 20S proteasome

2.23

CAGLOA04587g YBL082C ALG3 Dolichol-P-Man dependent alpha(1- 2.23 3) mannosyltransferase, involved in the synthesis of dolichol-linked oligosaccharide donor for N-linked glycosylation of proteins

CAGL0004433g YDL003W MCDI Essential subunit of the cohesin complex required for sister chro-matid cohesion in mitosis and meio-sis; apoptosis induces cleavage and translocation of a C-terminal fragment to mitochondria; expression peaks in S phase

2.22

CAGLOM11242g YMR226C TMA29 NADP(+)-dependent dehydrogenase; acts on serine, L-allo- threonine, and other 3-hydroxy acids; green fluorescent protein fusion protein localizes to the cytoplasm and nucleus; may interact with ribosomes, based on co-purification experiments

2.21




CAGLOM09108g YJR091C JSNI Member of the Puf family of 2.21 RNA-binding proteins, interacts with mRNAs encoding membrane-associated proteins; overexpression suppresses a tub2-150 mutation and causes increased sensitivity to beno-myl in wild-type cells

CAGL0005093g YOL057W - Description not mapped 2.21 CAGLOM03839g YNL305C - Description not mapped 2.2 CAGL0108239g YER021W RPN3 Essential, non-ATPase regulatory

subunit of the 26S proteasome lid, similar to the p58 subunit of the hu-man 26S proteasome; temperature-sensitive alleles cause metaphase ar-rest, suggesting a role for the pro-teasome in cell cycle control

2.2

CAGLOK12892g YFLO47W RGD2 GTPase-activating protein 2.2 (RhoGAP) for Cdc42p and Rho5p

CAGLOM04081g YNL317W PFS2 Integral subunit of the pre-mRNA cleavage and polyadenylation factor

2.2

(CPF) complex; plays an essential role in mRNA 3'-end formation by bridging different processing factors and thereby promoting the assem-bly of the processing complex

CAGLOJ04136g YOR221C MC T/ Predicted malonyl-CoA:ACF transferase, putative component of a type-II mitochondrial fatty acid synthase that produces intermedi-ates for phospholipid remodeling

2.19

CAGLOK11022g YDR236C FMNI Riboflavin kinase, phosphorylates riboflavin to form riboflavin monophosphate (FMN), which is a necessary cofactor for many enzymes; localizes to microsomes and to the mitochondrial inner membrane

2.18

CAGLOD01474g YBR108W AIMS Protein interacting with Rvs167p; 2.17 YBR108W is not an essential gene

CAGLOG08382g YDR195W REF2 RNA-binding protein involved in the cleavage step of mRNA 3'-end formation prior to polyadenylation, and in snoRNA maturation; part of holo-CPF subcomplex APT, which associates with 3'-ends of snoRNA-and mRNA-encoding genes

2.17

CAGLOF07513g YKL093W MBRJ Protein involved in mitochondrial functions and stress response; overexpression suppresses growth defects of hap2, hap3, and hap4 mu-tants

2.16

CAGLOL05126g YKL080W VMA5 Subunit C of the eight-subunit 2.15 V1 peripheral membrane domain of vacuolar H-k-ATPase (V-ATPase), an electrogenic proton pump found throughout the endomembrane system; required for the V1 domain to assemble onto the vacuolar mem-brane




CAGLOM06875g YNL025C SSN8 Cyclin-like component of the RNA polymerase II holoenzyme, involved in phosphorylation of the RNA polymerase II C-terminal domain; involved in glucose repression and telomere maintenance

2.15

CAGLOI07513g YOL 100W PKH2 Serine/threonine protein kinase involved in sphingolipid-mediated sig-naling pathway that controls endocytosis; activates Ypklp and

2.15

Ykr2p, components of signaling cascade required for maintenance of cell wall integrity; redundant with Pkhlp

CAGLOH09130g YKL201C MNN4 Putative positive regulator of mannosylphosphate transferase

2.14

(Mnn6p), involved in manno- sylphosphorylation of N-linked oligosaccharides; expression increases in late-logarithmic and stationary growth phases

CAGLOM08206g YJL171C - Description not mapped 2.14 CAGLOI00418g YGL055W OLEI Delta(9) fatty acid desaturase, re-

quired for monounsaturated fatty acid synthesis and for normal dis-tribution of mitochondria

2.13

CAGLOD03322g YLR023C IZH3 Membrane protein involved in zinc metabolism, member of the four-protein IZH family, expression induced by zinc deficiency; deletion reduces sensitivity to elevated zinc and shortens lag phase, overezpres-sion reduces Zap1p activity

2.12

CAGLOJ00583g YHR028C DAPS Dipeptidyl aminopeptidase, synthesized as a glycosylated precursor; localizes to the vacuolar membrane; similar to Stel3p

2.12

CAGLOL10912g YOR273C TPO4 Polyamine transport protein, recognizes spermine, putrescine, and spermidine; localizes to the plasma membrane; member of the major fa-cilitator superfamily

2.12

CAGLOF00583g YDR517W GRH1 Acetylated, cis-golgi localized protein involved in ER to Golgi trans-port; homolog of human GRASP65; forms a complex with the coiled- coil protein Bug1p; mutants are compromised for the fusion of ER-derived vesicles with Golgi mem-branes

2.12

CAGLOD02948g YJL034W KAR2 ATPase involved in protein import into the ER, also acts as a chap-erone to mediate protein folding in the ER and may play a role in ER export of soluble proteins; regulates the unfolded protein response via interaction with Ire1p

2.12

CAGLOK05995g YLR26OW LCB5 Minor sphingoid long-chain base ki- ease, paralog of Lcb4p responsible for few percent of the total activ-ity, possibly involved in synthesis of long-chain base phosphates, which function as signaling molecules

2.12




CAGLOK01287g YGRO74W SMD I Core Sm protein Sm Dl; part of heteroheptameric complex (with

2.11

Smblp, Smd2p, Smd3p, Smeip, Smx3p, and Smx2p) that is part of the spliceosomal Ul, U2, U4, and U5 snRNPs; homolog of human Sm D1

CAGLOA03806g YOR298W MUMS Protein of unknown function involved in the organization of the outer spore wall layers; has similar-ity to the tafazzins superfamily of acyltransferases

2.11

CAGL00O2673g YLR392C - Description not mapped 2.11 CAGL00O2717g YAL009W SPO T Putative regulatory subunit of 2.1

Nemlp-Spo7p phosphatase holoen-zyme, regulates nuclear growth by controlling phospholipid biosynthesis, required for normal nuclear envelope morphology, premeiotic replication, and sporulation

CAGLOJ01001g YJL036W SNX4 Sorting nexin, involved in retrieval of late-Golgi SNAREs from post-

2.1

Golgi endosomes to the trans- Golgi network and in cytoplasm to vacuole transport; contains a PX phosphoinositide-binding domain; forms complexes with Snx4lp and with Atg2Op

CAGLOL09889g YKL025C PA N3 Essential subunit of the Pan2p- 2.09 Pan3p poly(A)-ribonuclease complex, which acts to control poly(A) tail length and regulate the stoi-chiometry and activity of postrepli-cation repair complexes

CAGLOK09526g

.

YDR394W riPT3 One of six ATPases of the 19S regulatory particle of the 26S protea-some involved in the degradation of ubiquitinated substrates; substrate of N-acetyltransferase B

2.09

CAGLOB03619g YEL060C P 1-03 1 Vacuolar proteinase B (yscB), a serine protease of the subtilisin fam-ily; involved in protein degradation in the vacuole and required for full protein degradation during sporula-tion

2.09

CAGLOJ09328g YDL127W PCL2 G1 cyclin, associates with Pho85p cyclin-dependent kinase (Cdk) to contribute to entry into the mitotic cell cycle, essential for cell morpho-genesis; localizes to sites of polar-ized cell growth

2.09

CAGLOG01034g YGRO32W GSC2 Catalytic subunit of 1,3-beta- glucan synthase, involved in formation of the inner layer of the spore wall; activity positively regulated by Rholp and negatively by Smklp; has similarity to an alternate catalytic subunit, Fkslp

2.08

(Gsclp) continued on next page



CAGLOD05478g YGR186W TFG1 TFIIF (Transcription Factor II) largest subunit; involved in both transcription initiation and elonga-tion of RNA polymerase II; homol-ogous to human RAP74

2.08

CA GLOJ11704g YMR004W MVPI Protein required for sorting proteins to the vacuole; overproduction of Mvplp suppresses several domi-nant VPS1 mutations; Mvplp and

2.08

Vpslp act in concert to promote membrane traffic to the vacuole

CAGL0G06556g YNL097C PH023 Probable component of the Rpd3 histone deacetylase complex, involved in transcriptional regulation of PHO5; C-terminus has similarity to human candidate tumor suppres-sor p33(ING1) and its isoform ING3

2.07

CAGLOF04829g YPR154W PINS Protein that induces appearance of 2.05 [PIN-1-] prion when overproduced

CAGLOL11110g YML057W CMP2 Calcineurin A; one isoform (the other is CNA1) of the catalytic subunit of calcineurin, a

2.05

Ca4--Ficalmodulin-regulated protein phosphatase which regulates Cr. 1p (a stress-response transcrip-tion factor), the other calcineurin subunit is CNB1

CAGLOE04070g YHL039W - Description not mapped 2.05 CAGL00O2541g YLR399C BDF1 Protein involved in transcription

initiation at TATA-containing promoters; associates with the basal transcription factor TFIID; contains two bromodomains; corresponds to the C-terminal region of mammalian TAF1; redundant with

2.04

Bdf2p CAGLOK11946g - - Description not mapped 2.04 CAGLOF01111g YOL032W OP110 Protein with a possible role in

phospholipid biosynthesis, based on inositol-excreting phenotype of the null mutant and its suppression by exogenous choline

2.04

CAGL0004917g YJR109C CPA 2 Large subunit of carbamoyl phosphate synthetase, which catalyzes a step in the synthesis of citrulline, an arginine precursor

2.03

CAGL0005357g YDL042C SIRS Conserved NAD4- dependent histone deacetylase of the Sirtuin family involved in regulation of lifespan; plays roles in silencing at

2.02

HML, HMR, telomeres, and the rDNA locus; negatively regulates initiation of DNA replication

CAGL0C04785g YJR115W - Description not mapped 2.02 CAGLOK02013g -- Description not mapped 2.02 CAGLOE04510g YOR022C - Description not mapped 2.01 CAGL0109592g YOR303W CPAI Small subunit of carbamoyl phos-

phate synthetase, which catalyzes a step in the synthesis of citrulline, an arginine precursor; translation-ally regulated by an attenuator pep-tide encoded by YOR302W within the CPAI mRNA 5'-leader

2



continued from previous page CC ORF SC ORF gene name Description r WT.S - WT.0


Table D.4: Candida glabrata transcripts repressed in wild-type cells following transient exposure to 1M sorbitol for 15 minutes. Repressed transcripts are defined

as having log2(Cy5) - log2(Cy3) < -2.0 and Benjamini-Hochberg corrected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 effect described in Table D.2

CG ORF SC ORF gene name Description WT.S - WT.0

CAGLOH01089g YDR287W INM2 Inositol monophosphatase, involved in biosynthesis of inositol; enzymatic activity requires magnesium ions and is inhibited by lithium and sodium ions; inml inm2 double mu-tant lacks inositol auxotrophy

-3.41

CAGL0L10384g YOR064C YNGI Subunit of the NuA3 histone acetyltransferase complex that acetylates histone H3; contains PHD finger domain that interacts with methy-lated histone H3, has similarity to the human tumor suppressor ING1

-3.38

CAC LOH09064g YHR128W FUR/ Uracil phosphoribosyltransferase, synthesizes UMP from uracil; involved in the pyrimidine salvage pathway

-2.97

CAGLOL05500g YJL 122W ALB1 Shuttling pre-60S factor; involved in the biogenesis of ribosomal large subunit; interacts directly with

-2.91

Arxlp; responsible for Tif6p recy-cling defects in absence of Reilp

CAGL0000737g YLR222C UTP13 Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

-2.91

CAGLOK09416g YGL211W NCS6 Protein required for thiolation of the uridine at the wobble position of Gln, Lys, and Glu tRNAs; has a role in urmylation and in invasive and pseudohyphal growth; inhibits replication of Brome mosaic virus in

-2.81

S. cerevisiae CAGL0000352g YGR277C - Description not mapped -2.81 CAGL0106743g YER145C FTR1 High affinity iron permease involved

in the transport of iron across the plasma membrane; forms complex with Fet3p; expression is regulated by iron

-2.74

CAGL0106809g YER142C MAGI 3-methyl-adenine DNA glycosylase involved in protecting DNA against alkylating agents; initiates base ex-cision repair by removing damaged bases to create abasic sites that are subsequently repaired

-2.71

CAGLOG04411g YJL109C UTPIO Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

-2.7

CAGLOK03795g YMR128W ECM16 Essential DEAR-box ATP- dependent RNA helicase specific to the U3 snoRNP, predominantly nucleolar in distribution, required for 18S rRNA synthesis

-2.63




CAGLOG00484g CAGLOA01914g

YGR272C YHR099W

- TRA I

Description not mapped Subunit of SAGA and NuA4 histone acetyltransferase complexes; interacts with acidic activators (e.g., Gal4p) which leads to transcription activation; similar to human TR-

-2.59 -2.58

RAP, which is a cofactor for c-Myc mediated oncogenic transformation

CAGLOG07953g YNR037C RSMI;) Mitochondrial ribosomal protein of the small subunit, has similarity to

-2.53

E. coli S19 ribosomal protein CAGLOI09900g YOR319W HSH49 U2-snRNP associated splicing fac-

tor with similarity to the mam-malian splicing factor SAP49; pro-posed to function as a U2-snRNP assembly factor along with Hsh155p and binding partner Cuslp; contains two RNA recognition motifs

-2.51

(RRM) CAGLOD05940g YGR175C ERG1 Squalene epoxidase, catalyzes the

epoxidation of squalene to 2,3- oxidosqualene; plays an essential role in the ergosterol-biosynthesis pathway and is the specific target of the antifungal drug terbinafine

-2.48

CAGLOKO6281g YDR454C GUK1 Guanylate kinase, converts GMP to -2.48 GDP; required for growth and man-nose outer chain elongation of cell wall N-linked glycoproteins

CAGL0104026g YDL053C PBP4 Pbplp binding protein, interacts strongly with Pablp-binding pro-tein 1 (Pbplp) in the yeast two-hybrid system; also interacts with

-2.47

Lsml2p in a copurification assay CAGLOF01793g YLR056W ERGS C-5 sterol desaturase, catalyzes the

introduction of a C-5(6) double bond into episterol, a precursor in ergosterol biosynthesis; mutants are viable, but cannot grow on non- fermentable carbon sources

-2.43

CAGL0101826g YHR148W IMP3 Component of the 55LT processome, which is required for pre-18S rRNA processing, essential protein that interacts with MpplOp and mediates interactions of Imp4p and

-2.42

MpplOp with U3 snoRNA CAGLOL03025g YKR025W RPC37 RNA polymerase III subunit C37 -2.4 CAGLOH04301g YML078W CPR3 Mitochondrial peptidyl-prolyl cis-

trans isomerase (cyclophilin), cat-alyzes the cis-trans isomerization of peptide bonds N-terminal to proline residues; involved in protein refold-ing after import into mitochondria

-2.4

CAGLOK02233g YER126C NSA2 Protein constituent of 66S pre- ribosomal particles, contributes to processing of the 27S pre-rRNA

-2.38

CAGLOM08448g YKL165C MCD4 Protein involved in glycosylphosphatidylinositol (GPI) anchor synthesis; multimembrane-spanning protein that localizes to the endo-plasmic reticulum; highly conserved among eukaryotes

-2.32




CAGLOH05533g YPL082C MOT] Essential abundant protein involved in regulation of transcription, removes Sptl5p (TBP) from DNA via its C-terminal ATPase activity, forms a complex with TBP that binds TATA DNA with high affin-ity but with altered specificity

-2.31

CAGL00O2211g YELO4OW UTR2 Cell wall protein that functions in the transfer of chitin to beta(1-

-2.27

6)glucan; putative chitin transgly-cosidase; glycosylphosphatidylinos-itol (GPI)-anchored protein local-ized to the bud neck; has a role in cell wall maintenance

CAGLOE05324g YOR330C MIP1 Catalytic subunit of the mitochondrial DNA polymerase; conserved

-2.24

C-terminal segment is required for the maintenance of mitochondria] genome.

CAGLOD00748g YDL067C COX9 Subunit VIIa of cytochrome c oxi- dose, which is the terminal member of the mitochondrial inner mem-brane electron transport chain

-2.18

CAGLOB00638g YCL047C - Description not mapped -2.16 CAGLOM09086g YJR0924V BUD4 Protein involved in bud-site selec-

tion and required for axial budding pattern; localizes with septins to bud neck in mitosis and may con-stitute an axial landmark for next round of budding; potential Cdc28p substrate

-2.16

CAGLOK04477g YGRO6OW ERG25 C-4 methyl sterol oxidase, catalyzes the first of three steps re-quired to remove two C-4 methyl groups from an intermediate in ergosterol biosynthesis; mutants accumulate the sterol intermediate

-2.15

4,4-dimethylzymosterol CAGLOG08646g YIL122W POG1 Putative transcriptional activa-

tor that promotes recovery from pheromone induced arrest; inhibits both alpha-factor induced G1 arrest and repression of CLN1 and CLN2 via SCB/MCB promoter elements; potential Cdc28p substrate; SBF regulated

-2.12

CAGLOL13156g YLR073C - Description not mapped -2.1 CAGLOJ02442g YIL022W TL)144 Peripheral mitochondrial mem-

brane protein involved in mitochondria] protein import, tethers essential chaperone Ssclp to the translocon channel at the matrix side of the inner membrane

-2.09

CAGLOL01991g YKL033W TTI1 Putative protein of unknown function; subunit of the ASTRA complex which is part of the chromatin remodeling machinery; similar to S. pombe Ttilp; detected in highly purified mitochondria in high-throughput studies

-2.09




CAGL00O2739g YAL008W FUN.14 Mitochondria) protein of unknown function

-2.05

CAGLOL01551g YML052W SUR7 Putative integral membrane protein; component of eisosomes; associated with endocytosis, along with

-2.04

PiIlp and Lsplp; sporulation and plasma membrane sphingolipid con-tent are altered in mutants

CAGLOL08470g - - Description not mapped -2.03


Table D.5: Profile of transcripts involved in metabolism in wild-type, hog)

and stel 1 cells. Key to abbreviations: CG= Candida glabrata open reading frame ID;

SC=Saccharomyces cerevisiae open reading frame ID; WT=wild-type C. glabrata cells;

hog 1 =hog 1 C. glabrata strain; stel 1 =stel 1 C. glabrata strain. The digits in each column

indicate whether the transcript is induced (1), repressed (-1) or not differentially expressed (0) after 15 minutes exposure to 1M sorbitol. All descriptions are inferred by sequence

homology between C. glabrata and S. cerevisiae open reading frames.

CG ORF SC ORF Gene name Description WT hog) stet 1

CAGLOM07381g YPL053C KTR6 Probable mannosylphosphate transferase involved in the synthesis of core oligosaccharides in protein glycosylation path-way; member of the KRE2/MNT1 mannosyltransferase family

1 1 0

CAGL0104554g YBRO14C GRX7 Cis-golgi localized monothiol glutaredoxin; more similar in activity to dithiol than other monothiol glutaredoxins; involved in the oxidative stress response; does not bind metal ions; func-tional overlap with GRX6

1 0 0

CAGLOG01166g YLR348C DIC1 Mitochondrial dicarboxylate carrier, integral membrane protein, catalyzes a dicarboxylate-phosphate exchange across the inner mitochondrial membrane, transports cytoplasmic dicar-boxylates into the mitochondrial matrix

1 0 0

CAGLOK11022g YDR236C FMN1 Riboflavin kinase, phosphorylates riboflavin to form riboflavin monophosphate (FMN), which is a necessary cofactor for many enzymes; localizes to microsomes and to the mitochondrial. in-ner membrane

1 0 1

CAGL0004917g YJR1O9C CPA 2 Large subunit of carbamoyl phosphate synthetase, which catalyzes a step in the synthesis of citrulline, an arginine precursor

1 1 1

CAGLOH05137g YPL061W A LD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes NADP+ as the preferred coenzyme; required for conver-sion of acetaldehyde to acetate; constitutively expressed; lo-cates to the mitochondrial outer surface upon oxidative stress

1 0 0

CAGL00O2233g YER042W If XR 1 Peptide methionine sulfoxide reductase, reverses the oxidation of methionine residues; involved in oxidative damage repair, providing resistance to oxidative stress and regulation of lifes-pan

1 0 0

CAGLOI09592g YOR303W CPA I Small subunit of carbamoyl phosphate synthetase, which catalyzes a step in the synthesis of citrulline, an arginine pre-cursor; translationally regulated by an attenuator peptide en-coded by YOR302W within the CPA1 mRNA 5'-leader

1 0 0

CAGLOG04763g YOR107W RGS2 Negative regulator of glucose-induced cAMP signaling; directly activates the GTPase activity of the heterotrimeric G protein alpha subunit Gpa2p

1 1 1

CAGL0005137g YOL059W GPD2 NAD-dependent glycerol 3-phosphate dehydrogenase, homolog of Gpdlp, expression is controlled by an oxygen-independent signaling pathway required to regulate metabolism under anoxic conditions; located in cytosol and mitochondria

1 0 0

CAGLOF08107g YGR244C LSC2 Beta subunit of succinyl-CoA ligase, which is a mitochondrial enzyme of the TCA cycle that catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate

1 0 0

CAGLOM12034g YALO3SW CDCI9 Pyruvate kinase, functions as a homotetramer in glycolysis to convert phosphoenolpyruvate to pyruvate, the input for aero-bic (TCA cycle) or anaerobic (glucose fermentation) respira-tion

1 1 0

CAGLOF06941g YGL062W PYCI Pyruvate carboxylase isoform, cytoplasmic enzyme that converts pyruvate to oxaloacetate; highly similar to isoform Pyc2p but differentially regulated; mutations in the human homolog are associated with lactic acidosis

1 0 0

CAGLOG01540g YNL036W NCE103 Carbonic anhydrase; poorly transcribed under aerobic conditions and at an undetectable level under anaerobic conditions; involved in non-classical protein export pathway

1 1 1



continued from previous page CC ORF SC ORF Gene Name Description WT hogI ste11

CAGLOM14047g YMR318C ADH6 NADPH-dependent medium chain alcohol dehydrogenase with broad substrate specificity; member of the cinnamyl family of alcohol dehydrogenases; may be involved in fusel alcohol syn-thesis or in aldehyde tolerance

1 0 0

CAGLOH06523g YIL145C PANG Pantothenate synthase, also known as pantoate-beta-alanine ligase, required for pantothenic acid biosynthesis, deletion causes pantothenic acid auxotrophy, homologous to E. coli panC

1 1 1

CAGLOK03509g YMR110C HFDI Putative fatty aldehyde dehydrogenase, located in the mitochondrial outer membrane and also in lipid particles; has simi-larity to human fatty aldehyde dehydrogenase (FALDH) which is implicated in Sjogren-Larsson syndrome

1 1

CAGL0F01111g YOL032W OPII0 Protein with a possible role in phospholipid biosynthesis, based on inositol-excreting phenotype of the null mutant and its sup-pression by exogenous choline

1 0 0

CAGLOG05313g YDR072C IPT1 Inositolphosphotransferase, involved in synthesis of mannose- 1 1 1 (inositol-P)2-ceramide (M(IP)2C), the most abundant sphingolipid;, can mutate to resistance to the antifungals syringomycin E and DmAMP1 and to K. lactis zymocin

CAGLOI01100g YOR120W GCY1 Putative NADP(+) coupled glycerol dehydrogenase, proposed to be involved in an alternative pathway for glycerol catabolism; member of the aldo-keto reductase (AKR) family

1 0 0


Table D.6: Profile of transcripts involved in protein degradation in wild-type, hogl and sten cells. Key to abbreviations: CG=Candida glabrata open reading frame

ID; SC=Saccharomyces cerevisiae open reading frame ID; WT=wild-type C. glabrata cells;

hogl=hogl C. glabrata strain; stell=stel 1 C. glabrata strain. The digits in each column

indicate whether the transcript is induced (1), repressed (-1) or not differentially expressed

(0) after 15 minutes exposure to 1M sorbitol. All descriptions are inferred by sequence


CG ORF SC ORF Gene name Description WT hog 1 stell

CAGLOD05082g YLL039C UBLI Ubiquitin, becomes conjugated to proteins, marking them for selective degradation via the ubiquitin-26S proteasome system; essential for the cellular stress response; encoded as a polyu-biquitin precursor comprised of 5 head-to-tail repeats

1 0 0

CAGLOL09152g YFROO4W RPN11 Metalloprotease subunit of the 19S regulatory particle of the 1 0 0 265 proteasome lid; couples the deubiquitination and degrada-tion of proteasome substrates

CAGLOK09526g YDR394W RPT3 One of six ATPases of the 19S regulatory particle of the 26S proteasome involved in the degradation of ubiquitinated sub-strates; substrate of N-acetyltransferase B

1 0 1

CAGLOL07238g YDL097C RPN6 Essential, non-ATPase regulatory subunit of the 265 proteasome lid required for the assembly and activity of the 26S proteasome; the human homolog (S9 protein) partially rescues

1 1 1

Rpn6p depletion CAGLOH07007g YML092C PRES Alpha 2 subunit of the 20S proteasome 1 0 1 CAGLOM06545g YKL124W SSH4 Specificity factor required for Rsp5p-dependent ubiquitination

and sorting of cargo proteins at the multivesicular body; iden-tified as a high-copy suppressor of a SHR3 deletion, increasing steady-state levels of amino acid permeases

1 1 1

CAGLOE02299g YOL013C HRD1 Ubiquitin-protein ligase required for endoplasmic reticulum- associated degradation (ERAD) of misfolded proteins; geneti-cally linked to the unfolded protein response (UPR); regulated through association with Hrd3p; contains an 112 ring finger

1 0 1

CAGL0108239g YER021W RPN3 Essential, non-ATPase regulatory subunit of the 26S proteasome lid, similar to the p58 subunit of the human 26S pro-teasome; temperature-sensitive alleles cause metaphase arrest, suggesting a role for the proteasome in cell cycle control

1 1 0

CAGLOJ09108g YDL19OC UFD2 Ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (El), a ubiquitin-conjugating en-zyme (E2), and a ubiquitin protein ligase (E3) to conjugate ubiquitin to substrates; also functions as an E3

1 1 0

CAGLOL05434g YKR042W UTH1 Mitochondria] outer membrane and cell wall localized SUN family member required for mitochondria] autophagy; involved in the oxidative stress response, life span during starvation, mitochondrial biogenesis, and cell death

1 1 1

CAGLOA02046g YOR018W ROD] Membrane protein that binds the ubiquitin ligase Rsp5p via its 2 PY motifs; overexpression confers resistance to the GST substrate o-dinitrobenzene,zinc, and calcium; proposed to reg-ulate the endocytosis of plasma membrane proteins

1 1 1


Table D.7: Profile of transcripts involved in protein trafficking in wild-type, hogl

and stel 1 cells. Key to abbreviations: CG=Candida glabrata open reading frame ID;

SC=Saccharomyces cerevisiae open reading frame ID; WT=wild-type C. glabrata cells;

hogl =hogl C. glabrata strain; stel 1 =stel 1 C. glabrata strain. The digits in each column

indicate whether the transcript is induced (1), repressed (-1) or not differentially expressed

(0) after 15 minutes exposure to 1M sorbitol. All descriptions are inferred by sequence


CG ORF SC ORF Gene name Description WT hog1 stel 1

CAGLOL11572g YHR103W SBE22 Protein involved in the transport of cell wall components from the Golgi to the cell surface; similar in structure and function-ally redundant with Sbe2p; involved in bud growth

1 1 1

CAGLOL05126g YKL080W VMA 5 Subunit C of the eight-subunit VI peripheral membrane domain of vacuolar HA--ATPase (V-ATPase), an electrogenic pro-ton pump found throughout the endomembrane system; re-quired for the VI domain to assemble onto the vacuolar mem-brane

1 0 0

CAGLOI10010g YGR142W BTN2 v-SNARE binding protein that facilitates specific protein retrieval from a late endosome to the Golgi; modulates arginine uptake, possible role in mediating pH homeostasis between the vacuole and plasma membrane H(-1-)-ATPase

1 1 1

CAGLOH03729g YNLOO6W LST8 Protein required for the transport of amino acid permease 1 0 0 Gap1p from the Golgi to the cell surface; component of the TOR signaling pathway; associates with both Tor1p and Tor2p; contains a WD-repeat

CAGLOJ00583g YHR028C DA P2 Dipeptidyl aminopeptidase, synthesized as a glycosylated precursor; localizes to the vacuolar membrane; similar to Stel3p

1 0 0

CAGLOM01782g YBRO80C SEC18 ATPase required for the release of Sec17p during the 'priming' step in homotypic vacuole fusion and for ER to Golgi trans-port; homolog of the mammalian NSF

1 0 0

CAGL0B03619g YEL060C PRB1 Vacuolar proteinase B (yscB), a serine protease of the subtilisin family; involved in protein degradation in the vacuole and required for full protein degradation during sporulation

1 0 1

CAGLOB01991g YDR126W S WEI Palmitoyltransferase that acts on the SNAREs Snclp, Syn8p, Tlglp and likely on all SNAREs; member of a family of pu-tative palmitoyltransferases containing an Asp-His-His-Cys-cysteine rich (DHIJC-CRD) domain; may have a role in vacuole fusion

1 0 0

CAGLOL06028g YGL181W GTS1 Protein that localizes to the nucleus and is involved in transcription regulation; also localizes to actin patches and plays a role in endocytosis; N-terminus contains an ARF-GAP domain and C-terminus contains a Gln-rich domain

1 0 0

CAGL000320Ig YLL040C VPS13 Protein of unknown function; heterooligomeric or homooligomeric complex; peripherally associated with membranes; homologous to human COH1; involved in sporulation, vacuolar protein sorting and protein-Golgi retention

1 1 1

CAGLOK00539g YPL259C APMI Mul-like medium subunit of the clathrin-associated protein complex (AP-1); binds clathrin; involved in clathrin-dependent

1 1 0

Golgi protein sorting CAGLOJ01001g YJL036W SNX4 Sorting nexin, involved in retrieval of late-Golgi SNAREs from

post-Golgi endosomes to the trans-Golgi network and in cyto-plasm to vacuole transport; contains a PX phosphoinositide- binding domain; forms complexes with Snx41p and with

1 1 0

Atg2Op CAGLOK02761g YHLOO2W HSEI Subunit of the endosomal Vps27p-Hselp complex required for

sorting of ubiquitinated membrane proteins into intralumenal vesicles prior to vacuolar degradation, as well as for recycling of Golgi proteins and formation of lumenal membranes

1 0 1



continued from previous page CG ORF SC ORF Gene Name Description WT hogl step

CAGLOJ11704g YMR004W MVP1 Protein required for sorting proteins to the vacuole; overproduction of Mvplp suppresses several dominant VPS1 muta-tions; Mvplp and Vpslp act in concert to promote membrane traffic to the vacuole

1 0 0


Table D.8: Profile of transcripts involved in the transcriptional regulation in wild-type, hogl and stell cells. Key to abbreviations: CG=Candida glabrata open reading

frame ID; SC=Saccharomyces ccrevisiae open reading frame ID; WT=wild-type C. glabrata

cells; hagl=hogl C. glabrata strain; stell=stell C. glabrata strain. The digits in each

column indicate whether the transcript is induced (1), repressed (-1) or not differentially

expressed (0) after 15 minutes exposure to 1M sorbitol. All descriptions are inferred by

sequence homology between C. glabrata and S. cerevisiae open reading frames.

CG ORF SC ORF Gene name Description WT hog! ate!!

CAGLOI04246g YGL162W S UT/ Transcription factor of the Zn[III2Cys6 family involved in sterol uptake; involved in induction of hypoxic gene expres-sion

1 1 0

CAGLOM06875g YNL025C SSN8 Cyclin-like component of the RNA polymerase II holoenzyme, involved in phosphorylation of the RNA polymerase II C-terminal domain; involved in glucose repression and telomere maintenance

1 0 1

CAGL0005357g YDL042C SIR2 Conserved NAD+ dependent histone deacetylase of the Sirtuin family involved in regulation of lifespan; plays roles in silencing at HML, HMR, telomeres, and the rDNA locus; negatively regulates initiation of DNA replication

1 0 0

CAGLOD05368g YBR253W SR136 Subunit of the RNA polymerase II mediator complex; associates with core polymerase subunits to form the RNA poly-merase II holoenzyme; essential for transcriptional regulation

11 0

CAGL0CO2541g YLR399C DDF1 Protein involved in transcription initiation at TATA- containing promoters; associates with the basal transcription factor TFIID; contains two bromodomains; corresponds to the

1 1 0

C-terminal region of mammalian TAF1; redundant with Bdf2p CAGLOD05434g YFRO65W ROX I Heme-dependent repressor of hypoxic genes; contains an HMG

domain that is responsible for DNA bending activity 1 1 0

CAGLOH07557g YGL254W FZF1 Transcription factor involved in sulfite metabolism, sole identified regulatory target is SSU1, overexpression suppresses sulfite-sensitivity of many unrelated mutants due to hyperac-tivation of SSU1, contains five zinc fingers

1 1 0

CAGLOL06028g YGL181W GTSI Protein that localizes to the nucleus and is involved in transcription regulation; also localizes to actin patches and plays a role in endocytosis; N-terminus contains an ARF-GAP domain and C-terminus contains a Gln-rich domain

1 0 0

CAGLOD05478g YGR186W TFG1 TFIIF (Transcription Factor II) largest subunit; involved in both transcription initiation and elongation of RNA poly-merase II; homologous to human RAP74

1 1 0

CAGL0G06556g YNL097C PH023 Probable component of the Rpd3 histone deacetylase complex, involved in transcriptional regulation of PHO5; C-terminus has similarity to human candidate tumor suppressor p33(ING1) and its isoform ING3

1 0 0

CAGLOH08173g YOR028C CIN5 Basic leucine zipper (bZIP) transcription factor of the yAP-1 family, mediates pleiotropic drug resistance and salt tolerance; nuclearly localized under oxidative stress and sequestered in the cytoplasm by Lot6p under reducing conditions

1 1 1

CAGLOF09119g YHR207C SETS Zinc-finger protein of unknown function, contains one canonical and two unusual fingers in unusual arrangements; deletion enhances replication of positive-strand RNA virus

1 0 0

CAGLOH04631g YML007W YAPI Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance; activated by H202 through the mul-tistep formation of disulfide bonds and transit from the cyto-plasm to the nucleus; mediates resistance to cadmium

1 1 0


Table D.9: Profile of induced transcripts conserved between S. cerevisiae (Gasch

et al., and C. glabrata after exposure to hyperosmotic stress. Key to abbreviations:

CG=Candida glabrata open reading frame ID; SC=Saccharornyces cerevisiae open reading

frame ID. All descriptions are inferred by sequence homology between C. glabrata and S.

cerevisiae open reading frames. S. cerevisiae data were obtained from a study by Gasch et

a/.(230) in which S. cerevisiae cells were treated with 1M sorbitol.

CG ORF SC ORF Gene name Description WT

CAGLOI10010g YGR142W BTN2 v-SNARE binding protein that facilitates specific protein retrieval from a late endosome to the Golgi; modulates arginine uptake, possible role in mediating pH homeostasis between the vacuole and plasma membrane H(+)-ATPase

5.55

CAGLOG04433g YJL108C PRM10 Pheromone-regulated protein, predicted to have 5 transmembrane segments; induced by treatment with 8-methoxypsoralen and UVA irradiation

5.14

CAGLOE00803g YDR171W HSP42 Small heat shock protein (sHSP) with chaperone activity; forms barrel-shaped oligomers that suppress unfolded protein aggregation; involved in cytoskeleton reorganization after heat shock

3.82

CAGLOG03883g YLL026W HSP/0.4 Heat shock protein that cooperates with Ydjlp (Hsp40) and 3.61 Ssalp (Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in [PSI-I-] propagation

CAGLOH08173g YOR028C CIN5 Basic leucine zipper (bZIP) transcription factor of the yAP-1 family, mediates pleiotropic drug resistance and salt tolerance; nuclearly localized under oxidative stress and sequestered in the cytoplasm by Lot6p under reducing conditions

3.49


3.47

CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein, expression is increased in response to heat-shock stress or treatments that produce DNA lesions; contains multiple repeats of the amino acid sequence

3.06

NNNDSYGS CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein, expression is increased in re-

sponse to heat-shock stress or treatments that produce DNA lesions; contains multiple repeats of the amino acid sequence

3.06

NNNDSYGS CAGLOH08844g YlvIR173W DDR48 DNA damage-responsive protein, expression is increased in re-


3.06

NNNDSYGS CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein, expression is increased in re-


3.06

NNNDSYGS CAGLOI01100g YOR120W GCY1 Putative NADP(+) coupled glycerol dehydrogenase, pro-

posed to be involved in an alternative pathway for glycerol catabolism; member of the aldo-keto reductase (AKR) family

2.56

CAGLOH05137g YPL061W ALD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes NADP+ as the preferred coenzyme; required for conver-sion of acetaldehyde to acetate; constitutively expressed; lo-cates to the mitochondrial outer surface upon oxidative stress

2.53

CAGLOM06347g YBR183W YPC1 Alkaline ceramidase that also has reverse (CoA-independent) ceramide synthase activity, catalyzes both breakdown and syn-thesis of phytoceramide; overexpression confers fumonisin B1 resistance

2.52

CAGLOJ04158g YOR220W RCN2 Protein of unknown function; green fluorescent protein (GFP)- fusion protein localizes to the cytoplasm and is induced in re-sponse to the DNA-damaging agent MMS; phosphorylated in response to alpha factor

2.35



continued from previous page CC ORF SC ORF Gene Name Description WT.S - WT.0

CAGLOF07513g YKL093W MBR1 Protein involved in mitochondrial functions and stress response; overexpression suppresses growth defects of hap2, hap3, and hap4 mutants

2.16


Table D.10: Profile of induced transcripts conserved between S. cerevisiae

(O'Rourke & Herskowitz 1M sorbitol) and C. glabrata after exposure to hy-

perosmotic stress. Key to abbreviations: CG=Candida glabrata open reading frame ID;

SC=Saccharomyces cerevisiae open reading frame ID. All descriptions are inferred by se-

quence homology between C. glabrata and S. cerevisiae open reading frames. S. cerevisiae

data were obtained from a study by O'Rourke & Herskowitz (518) in which S. cerevisiae

cells were treated with 1M sorbitol

CG ORF SC ORF Gene name Description WT


5.55

CAGLOG04433g YJLI08C PRM1O Pheromone-regulated protein, predicted to have 5 transmembrane segments; induced by treatment with 8-methoxypsoralen and UVA irradiation

5.14


3.82

CAGLOG03883g YLL026W HSPI 04 Heat shock protein that cooperates with Ydjlp (Hsp40) and 3.61 Ssalp (Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in [PSI-] propagation

CAGLOF08745g YLR327C TMA I 0 Protein of unknown function that associates with ribosomes 3.53 CAGLOG01540g YNL036W NCE103 Carbonic anhydrase; poorly transcribed under aerobic condi-

tions and at an undetectable level under anaerobic conditions; involved in non-classical protein export pathway

3.47

CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein, expression is increased in response to heat-shock stress or treatments that produce DNA lesions; contains multiple repeats of the amino acid sequence

3.06

NNNDSYGS CAGLOE05632g YOR348C PUT4 Proline permease, required for high-affinity transport of pro-

line; also transports the toxic proline analog azetidine- 2.85

2-carboxylate (AzC); PUT4 transcription is repressed in ammonia-grown cells


2.35

CAGLOF01111g YOL032W OPIIO Protein with a possible role in phospholipid biosynthesis, based on inositol-excreting phenotype of the null mutant and its sup-pression by exogenous choline

2.04


Table D.11: Profile of induced transcripts conserved between S. cerevisiae (Rep

et al.,) and C. glabrata after exposure to hyperosmotic stress. Key to abbrevia-

tions: CG=Candida glabrata open reading frame ID; SC=Saccharomyces cerevisiae open

reading frame ID. All descriptions are inferred by sequence homology between C. glabrata

and S. cerevisiae open reading frames. S. cerevisiae data were obtained from a study by

Rep et al. (581) in which S. cerevisiae cells were treated with 0.71\4 NaCl.

CG 0 RF SC ORE Gene name Description WT.S - WT.0 CAGL00O2321g YER037W PHM8 Protein of unknown function, expression is induced by low

phosphate levels and by inactivation of Pho85p 5.41

CAGLOG04433g YJL108C PR 114.10 Pheromone-regulated protein, predicted to have 5 transmembrane segments; induced by treatment with 8-methoxypsoralen and UVA irradiation

5.14


3.82

CAGLOG03883g YLL026W HSP/04 Heat shock protein that cooperates with Ydjlp (Hsp40) and 3.61 Ssalp (Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in [PSI-I-1 propagation

CAGLOF08745g YLR327C TMA 10 Protein of unknown function that associates with ribosomes 3.53 CAGLOH08173g YOR028C CINS Basic leucine zipper (bZIP) transcription factor of the yAP-1

family, mediates pleiotropic drug resistance and salt tolerance; nuclearly localized under oxidative stress and sequestered in the cytoplasm by Lot6p under reducing conditions

3.49

CAGLOG01540g YNL036W NCE 103 Carbonic anhydrase; poorly transcribed under aerobic conditions and at an undetectable level under anaerobic conditions; involved in non-classical protein export pathway

3.47

CAGLOM12474g YIL055C - - 3.46 CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein, expression is increased in re-


3.06




CAGLOL00803g YER054C GIP2 Putative regulatory subunit of the protein phosphatase Glc7p, involved in glycogen metabolism; contains a conserved motif

2.62

(GVNK motif) that is also found in Gaclp, Pig1p, and Pig2p CAGLOK12034g YDR040C ENA 1 P-type ATPase sodium pump, involved in Na+ and Li+ efflux

to allow salt tolerance 2.61

CAGLOH05137g YPL061W ALD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes NADP+ as the preferred coenzyme; required for conver-sion of acetaldehyde to acetate; constitutively expressed; lo-cates to the mitochondrial outer surface upon oxidative stress

2.53

CAGLOJ04466g YLR414C - - 2.5 CAGLOE06424g YKL150W MCR/ Mitochondrial NADH-cytochrome b5 reductase, involved in er-

gosterol biosynthesis 2.42


2.35

CAGLOI04554g YBRO14C GRX7 Cis-golgi localized monothiol glutaredoxin; more similar in activity to dithiol than other monothiol glutaredoxins; involved in the oxidative stress response; does not bind metal ions; func-tional overlap with GRX6

2.34



continued from previous page CG ORF SC ORF Gene Name Description WT.S - WT.0

CAGLOB03619g YEL060C PRB1 Vacuolar proteinase B (yscB), a serine protease of the subtilisin family; involved in protein degradation in the vacuole and required for full protein degradation during sporulation

2.09


Table D.12: Profile of induced transcripts conserved between S. cerevisiae

(O'Rourke & Herskowitz 0.5M KC1) and C. glabrata after exposure to hy-

perosmotic stress. Key to abbreviations: CG.Candida glabrata open reading frame ID;

SC=Saccharomyces cerevisiae open reading frame ID. All descriptions are inferred by se-

quence homology between C. glabrata and S. cerevisiae open reading frames. S. cerevisiae

data were obtained from a study by O'Rourke & Herskowitz (518) in which S. cerevisiae

cells were treated with 0.5M KC1.

CG ORF SC ORF Gene name Description WT CAGLOI10010g YGR142W HTN2 v-SNARE binding protein that facilitates specific protein re-

trieval from a late endosome to the Golgi; modulates arginine uptake, possible role in mediating pH homeostasis between the vacuole and plasma membrane 1-1(+)-ATPase

5.55

CAGLOG04433g YIL108C PRI V I 10 Pheromone-regulated protein, predicted to have 5 transmembrane segments; induced by treatment with 8-methoxypsoralen and UVA irradiation

5.14

CAGLOM06963g YNR034W SOL 1 Protein with a possible role in tRNA export; shows similarity to 6-phosphogluconolactonase non-catalytic domains but does not exhibit this enzymatic activity; homologous to Sol2p, Sol3p, and Sol4p

4.31

CAGLOE00803g YDR171W LISP42 Small heat shock protein (sHSP) with chaperone activity; forms barrel-shaped oligomers that suppress unfolded protein aggregation; involved in cytoskeleton reorganization after heat shock

3.82

CAGLOG03883g YLL026W HSP104 Heat shock protein that cooperates with Ydjlp (Hsp40) and 3.61 Ssalp (Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in EPSI4-1 propagation

CAGLOF08745g YLR327C TMAIO Protein of unknown function that associates with ribosomes 3.53 CAGLOG01540g YNL036W NCE103 Carbonic anhydrase; poorly transcribed under aerobic condi-

tions and at an undetectable level under anaerobic conditions; involved in non-classical protein export pathway

3.47

CAGLOJ00539g YHR030C SLT2 Serine/threonine MAP kinase involved in regulating the maintenance of cell wall integrity and progression through the cell cycle; regulated by the PKC1-mediated signaling pathway

3.13

CAGLOE05632g YOR348C PUT4 Proline permease, required for high-affinity transport of proline; also transports the toxic proline analog azetidine-

2.85

2-carboxylate (AZC); PUT4 transcription is repressed in ammonia-grown cells

CAGLOK12034g YDR040C ENA I P-type ATPase sodium pump, involved in Na+ and Li-{- efflux to allow salt tolerance

2.61

CAGLOI01100g YOR120W GCYI Putative NADP(+) coupled glycerol dehydrogenase, proposed to be involved in an alternative pathway for glycerol catabolism; member of the aldo-keto reductase (AKR) family

2.56

CAGLOHD5137g YPL061W ALD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes NADP+ as the preferred coenzyme; required for conver-sion of acetaldehyde to acetate; constitutively expressed; lo-cates to the mitochondrial outer surface upon oxidative stress

2.53

CAGLOM06347g YBR183W YPCI Alkaline ceramidase that also has reverse (CoA-independent) ceramide synthase activity, catalyzes both breakdown and syn-thesis of phytoceramide; overexpression confers fumonisin B1 resistance -

2.52

CAGLOL10186g YOR052C -2.45 CAGLOE06424g YKL150W MCR / Mitochondrial NADH-cytochrome b5 reductase, involved in er-



2.35




CAGL0104554g

CAGLOF01111g

Yl3R014C

YOL032W

GRX7

OPI10

Cis-golgi localized monothiol glutaredoxin; more similar in activity to dithiol than other monothiol glutaredoxins; involved in the oxidative stress response; does not bind metal ions; func-tional overlap with GRX6 Protein with a possible role in phospholipid biosynthesis, based on inositol-excreting phenotype of the null mutant and its sup-pression by exogenous choline

2.34

2.04


Table D.13: C. glabrata hyperosmotic stress repressed transcripts that depend on a functional putative SHO1 signalling branch. C. glabrata transcripts induced in

wild-type, but not hogl or stell cells following exposure to transient hyper-osmotic shock (1M sorbitol, 15 minutes). repressed transcripts are defined as having log2(Cy5) - log2(Cy3) > 2.0 and Benjamini-Hochberg corrected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 in Table D.2

CG ORF SC ORF Gene name Description WT.S - WT.0

CAGLOE04554g NORBH - Description not mapped 4.96 CAGLOM06963g YNR034W SOLI Protein with a possible role in tRNA export; shows similar-

ity to 6-phosphogluconolactonase non-catalytic domains but does not exhibit this enzymatic activity; homologous to Sol2p, Sol3p, and Sol4p

4.31

CAGLOL05016g YKL072W STB6 Protein that binds Sin3p in a two-hybrid assay 3.64 CAGLOF09119g YHR207C SET5 Zinc-finger protein of unknown function, contains one canoni-

cal and two unusual fingers in unusual arrangements; deletion enhances replication of positive-strand RNA virus

3.38

CAGLOM12320g YAL053W FLC2 Putative FAD transporter; required for uptake of FAD into endoplasmic reticulum; involved in cell wall maintenance

3.28

CAGLOL09251g NORBH - Description not mapped 3.17 CAGL00O2233g YER042W MXR1 Peptide methionine sulfoxide reductase, reverses the oxidation

of methionine residues; involved in oxidative damage repair, providing resistance to oxidative stress and regulation of lifes-pan

3.14

CAGLOD05082g YLL039C UBI4 Ubiquitin, becomes conjugated to proteins, marking them for selective degradation via the ubiquitin-26S proteasome system; essential for the cellular stress response; encoded as a polyu-biquitin precursor comprised of 5 head-to-tail repeats

2.99

CAGLOF08107g YGR244C LSC2 Beta subunit of succinyl-CoA ligase, which is a mitochondrial enzyme of the TCA cycle that catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate

2.96

CAGLOB04257g YBRO31W RPL4A N-terminally acetylated protein component of the large (60S) ribosomal subunit, nearly identical to Rpl4Bp and has similar-ity to E. coli L4 and rat L4 ribosomal proteins

2.96

CAGLOJ01595g YPRO15C - Description not mapped 2.91 CAGLOJ00385g YHR049W FSH1 Putative serine hydrolase that localizes to both the nucleus

and cytoplasm; sequence is similar to S. cerevisiae Fsh2p and 2.89

Fsh3p and the human candidate tumor suppressor OVCA2 CAGLOJ03256g YER075C PTPS Phosphotyrosine-specific protein phosphatase involved in the

inactivation of mitogen-activated protein kinase (MAPK) dur-ing osmolarity sensing; dephosporylates Hog1p MAPK and regulates its localization; localized to the cytoplasm

2.83

CAGL0005137g YOL059W GPD2 NAD-dependent glycerol 3-phosphate dehydrogenase, homolog of Gpdlp, expression is controlled by an oxygen-independent signaling pathway required to regulate metabolism under anoxic conditions; located in cytosol and mitochondria

2.81

CAGLOF09163g YER182W FMP10 Putative protein of unknown function; the authentic, non- tagged protein is detected in highly purified mitochondria in high-throughput studies

2.74

CAGLOG01166g YLR34SC DICI Mitochondrial dicarboxylate carrier, integral membrane protein, catalyzes a dicarboxylate-phosphate exchange across the inner mitochondria] membrane, transports cytoplasmic dicar-boxylates into the mitochondria] matrix

2.7

CAGLOF06941g YGL062W PYCI Pyruvate carboxylase isoform, cytoplasmic enzyme that converts pyruvate to oxaloacetate; highly similar to isoform Pyc2p but differentially regulated; mutations in the human homolog are associated with lactic acidosis

2.66

CAGLOL00803g YER054C GIP2 Putative regulatory subunit of the protein phosphatase Glc7p, involved in glycogen metabolism; contains a conserved motif

2.62

(GVNK motif) that is also found in Gaclp, Pig1p, and Pig2p continued on next page


continued from previous page CG ORF SC ORF Gene name Description WT.S - WT.0

CAGLOM01782g YBRO80C SEC18 ATPase required for the release of Secl7p during the 'priming' step in homotypic vacuole fusion and for ER to Golgi trans-port; homolog of the mammalian NSF

2.59

CAGLOS01353g YMR089C YTA /2 Component, with Afg3p, of the mitochondrial inner membrane m-AAA protease that mediates degradation of misfolded or unassembled proteins and is also required for correct assembly of mitochondrial enzyme complexes

2.57

CAGLOIO1100g YOR120W GCY1 Putative NADP(+) coupled glycerol dehydrogenase, proposed to be involved in an alternative pathway for glycerol catabolism; member of the aldo-keto reductase (AKR) family

2.56

CAGLOB01991g YDR126W S WF/ Palmitoyltransferase that acts on the SNAREs Snclp, Syn8p, Tlglp and likely on all SNAREs; member of a family of pu-tative palmitoyltransferases containing an Asp-His-His-Cys-cysteine rich (DHHC-CRD) domain; may have a role in vacuole fusion

2.55

CAGLOF07095g YGL122C NA B2 Nuclear polyadenylated RNA-binding protein required for nuclear mRNA export and poly(A) tail length control; binds nuclear pore protein MIplp; autoregulates mRNA levels; re-lated to human hnRNPs; nuclear localization sequence binds

2.54

Kap104p CAGLOH05137g YPL061.W ALD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and uti-

lizes NADP+ as the preferred coenzyme; required for conver-sion of acetaldehyde to acetate; constitutively expressed; lo-cates to the mitochondria] outer surface upon oxidative stress

2.53

CAGLOM06347g YBR183W YPCI Alkaline ceramidase that also has reverse (CoA-independent) ceramide synthase activity, catalyzes both breakdown and syn-thesis of phytoceramide; overexpression confers fumonisin B1 resistance

2.52

CAGLOL09152g YFR0O4W RPN11 Metalloprotease subunit of the 19S regulatory particle of the 2.52 26S proteasome lid; couples the deubiquitination and degrada-tion of proteasome substrates

CAGLOH03729g YNLOO6W LST8 Protein required for the transport of amino acid permease 2.48 Gaplp from the Golgi to the cell surface; component of the TOR signaling pathway; associates with both Torlp and Tor2p; contains a WD-repeat

CAGLOL00605g YKL 190W CNB1 Calcineurin B; the regulatory subunit of calcineurin, a 2.46 Ca++/calmodulin-regulated type 2B protein phosphatase which regulates Crzlp (a stress-response transcription factor), the other calcineurin subunit is encoded by CNA1 and/or CMP1

CAGLOE06424g YKL150W 11,1 CR 1 Mitochondrial NADII-cytochrome b5 reductase, involved in ergosterol biosynthesis

2.42

CAGLOJ03322g YER080W A IA12 Putative protein of unknown function; the authentic, non- tagged protein is detected in highly purified mitochondria in high-throughput studies

2.4

CAGLOM10263g YCLOO2C - Description not mapped 2.39 CAGLOL06028g YGL181W GTS1 Protein that localizes to the nucleus and is involved in tran-

scription regulation; also localizes to actin patches and plays a role in endocytosis; N-terminus contains an ARF-GAP domain and C-terminus contains a Gln-rich domain

2.38

CAGLOM14047g YMR318C ADH6 NADPH-dependent medium chain alcohol dehydrogenase with broad substrate specificity; member of the cinnamyl family of alcohol dehydrogenases; may be involved in fusel alcohol syn-thesis or in aldehyde tolerance

2.36

CAGL0004499g YDLOO6W PTC1 Type 2C protein phosphatase (PP2C); inactivates the osmosensing MAPK cascade by dephosphorylating Hoglp; muta-tion delays mitochondria] inheritance; deletion reveals defects in precursor tRNA splicing, sporulation and cell separation

2.35


2.34




CAGLOD04752g YPR127W Description not mapped 2.33 CAGLOB00836g YCL033C - Description not mapped 2.33 CAGLOGO6886g YJL017W - Description not mapped 2.3 CAGLOA03058g YDR379W RGA2 GTPase-activating protein for the polarity-establishment pro-

tein Cdc42p; implicated in control of septin organization, pheromone response, and haploid invasive growth; regulated by Pho85p and Cdc28p

2.27

CAGL0000363g YJL174W KRE9 Glycoprotein involved in cell wall beta-glucan assembly; null mutation leads to severe growth defects, aberrant multibudded morphology, and mating defects

2.27

CAGLOJ11506g YNL192W CHS1 Chitin synthase I, requires activation from zymogenic form in order to catalyze the transfer of N-acetylglucosamine (GIcNAc) to chitin; required for repairing the chitin septum during cy-tokinesis; transcription activated by mating factor

2.25

CAGLOG04873g YLR371W ROM2 GDP/GTP exchange protein (GEP) for Rho1p and Rho2p; mutations are synthetically lethal with mutations in roml, which also encodes a GEP

2.25

CAGLOA04587g YBL082C ALG3 Dolichol-P-Man dependent alpha(1-3) mannosyltransferase, involved in the synthesis of dolichol-linked oligosaccharide donor for N-linked glycosylation of proteins

2.23

CAGLOM11242g YMR226C TMA29 NADP(+)-dependent dehydrogenase; acts on serine, L-allo- threonine, and other 3-hydroxy acids; green fluorescent protein fusion protein localizes to the cytoplasm and nucleus; may in-teract with ribosomes, based on co-purification experiments

2.21

CAGLOJ04136g YOR221C MGT/ Predicted malonyl-CoA:ACP transferase, putative component of a type-II mitochondrial fatty acid synthase that produces intermediates for phospholipid remodeling

2.19

CAGLOG08382g YDR195W REFS RNA-binding protein involved in the cleavage step of mRNA 2.17 3'-end formation prior to polyadenylation, and in snoRNA maturation; part of holo-CPF subcomplex APT, which asso-ciates with 3'-ends of snoRNA- and mRNA-encoding genes

CAGLOF07513g YKL093W MBR1 Protein involved in mitochondrial functions and stress response; overexpression suppresses growth defects of hap2, hap3, and hap4 mutants

2.16

CAGLOL05126g YKLOSOW VMA5 Subunit C of the eight-subunit V1 peripheral membrane domain of vacuolar H-I--ATPase (V-ATPase), an electrogenic pro-ton pump found throughout the endomembrane system; re-quired for the V1 domain to assemble onto the vacuolar mem-brane

2.15

CAGL0107513g YOL100W PKH2 Serine/threonine protein kinase involved in sphingolipid- mediated signaling pathway that controls endocytosis; acti-vates Ypklp and Ykr2p, components of signaling cascade re-quired for maintenance of cell wall integrity; redundant with

2.15

Pkhlp CAGLOI00418g YGL055W OLEI Delta(9) fatty acid desaturase, required for monounsaturated

fatty acid synthesis and for normal distribution of mitochon-dria

2.13

CAGLOJ00583g YHR028C DAP2 Dipeptidyl aminopeptidase, synthesized as a glycosylated precursor; localizes to the vacuolar membrane; similar to Ste13p

2.12

CAGLOF00583g YDR517W GRH1 Acetylated, cis-golgi localized protein involved in ER to Golgi transport; homolog of human GRASP65; forms a complex with the coiled-coil protein Bug1p; mutants are compromised for the fusion of ER-derived vesicles with Golgi membranes

2.12

CAGLOK01287g YGRO74W SMD1 Core Sm protein Sm DI; part of heteroheptameric complex 2.11 (with Smblp, Smd2p, Smd3p, Smelp, Smx3p, and Smx2p) that is part of the spliceosomal Ul, U2, U4, and US snRNPs; homolog of human Sm D1

CAGL00O2717g YAL009W SPOT Putative regulatory subunit of Nemlp-Spo7p phosphatase holoenzyme, regulates nuclear growth by controlling phospho-lipid biosynthesis, required for normal nuclear envelope mor-phology, premeiotic replication, and sporulation

2.1

C AG L0J11704g YMR004W MVP1 Protein required for sorting proteins to the vacuole; overproduction of Mvplp suppresses several dominant VPS1 muta-tions; Mvplp and Vpslp act in concert to promote membrane traffic to the vacuole

2.08



continued from previous page CC ORF SC ORF Gene name Description WT .S - WT. U

CAGLOG06556g YNL097C PH02.9 Probable component of the Rpd3 histone deacetylase complex, involved in transcriptional regulation of PHO5; C-terminus has similarity to human candidate tumor suppressor p33(ING1) and its isoform ING3

2.07

CAGLOE04070g YHL039W - Description not mapped 2.05 CAGLOFO1111g YOL032W OP/I0 Protein with a possible role in phospholipid biosynthesis, based

on inositol-excreting phenotype of the null mutant and its sup-pression by exogenous choline

2.04

CAGL0005357g YDL042C SIRS Conserved NAD+ dependent histone deacetylase of the Sirtuin family involved in regulation of lifespan; plays roles in silencing at HML, HMR, telomeres, and the rDNA locus; negatively regulates initiation of DNA replication

2.02

CAGLOK02013g NORBH - Description not mapped 2.02 CAGL0109592g YOR303W CPA1 Small subunit of carbamoyl phosphate synthetase, which cat-

alyzes a step in the synthesis of citrulline, an arginine pre-cursor; translationally regulated by an attenuator peptide en-coded by YOR302W within the CPA1 mRNA 5'-leader

2


Table D.14: C. glabrata hyperosmotic stress induced transcripts that are inde-

pendent of HOG1 and STE11. C. glabrata transcripts induced in wild-type, hogl and

stell cells following exposure to transient hyper-osmotic shock. Induced transcripts are defined as having log2(Cy5) - log2(Cy3) > 2.0 and Benjamini-Hochberg corrected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.U, HOG.S - HOG.0 and STE.S - STE.0 effects described in Table D.2

CG ORF SC ORF Gene name Description WT.S - WT.0 HOGS - HOG.0 STE.S - STE.0

CAGLOI10010g YGR142W BTN2 v-SNARE binding protein that facilitates specific protein retrieval from a late endosome to the Golgi; modulates arginine uptake, possible role in mediating pH homeosta-sis between the vacuole and plasma membrane H(-1-)-ATPase

5.55 5.26 6.39

CAGL00O2321g YER037W PHM8 Protein of unknown function, expression is induced by low phosphate levels and by inactivation of

5.41 2.93 2.86

Pho85p CAGLOG04433g YJL108C PRMIO Pheromone-regulated protein, pre-

dicted to have 5 transmembrane segments; induced by treatment with 8-methoxypsoralen and UVA irradiation

5.14 4.41 4.12

CAGLOM09713g YMR152W YIM1 Protein of unknown function; null mutant displays sensitivity to DNA damaging agents; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies -

4.9 4.7 4.16

CAGLOK08338g YOR385W -4.57 5.14 3.71 CAGLOA02816g YDR368W YPRI NADPH-dependent aldo-keto

reductase, reduces multiple substrates including 2- methylbutyraldehyde and D,L- glyceraldehyde, expression is induced by osmotic and oxidative stress; functionally redundant with other aldo-keto reductases

4.43 2.1 2.19

CAGLOJ10846g YHR071W PCL5 Cyclin, interacts with and phosphorylated by Pho85p cyclin- dependent kinase (Cdk), induced by Gcn4p at level of transcription, specifically required for

4.09 4.3 3.39


CAGL0105390g YPL026C SKS1 Putative serine/threonine protein kinase; involved in the adaptation to low concentrations of glucose in-dependent of the SNF3 regulated pathway

3.97 3.69 3.22

CAGLOM08822g YDR258C HSP7R Oligomeric mitochondrial matrix chaperone that cooperates with

3.93 3.91 4.94

Ssclp in mitochondrial thermotol-erance after heat shock; able to pre-vent the aggregation of misfolded proteins as well as resolubilize pro-tein aggregates



continued from previous page CG ORF SC ORF Gene name Description WT.S - WT.0 HOG.S - HOG.0 STE.S - STE.0

CAGLOH08151g NORBH - - 3.86 3.67 3.1 CAGLOE00803g YDR171W HSP42 Small heat shock protein (sIISP)

with chaperone activity; forms barrel-shaped oligomers that sup-press unfolded protein aggregation; involved in cytoskeleton reorganiza-tion after heat shock

3.82 4.38 4.02

CAGLOG03883g YLL026W HSP/04 Heat shock protein that cooper- ales with Ydjlp (Hsp40) and Ssalp

3.61 3.51 3.38

(Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses in-cluding: heat, ethanol, and sodium arsenite; involved in [PSI-I-] propa-gation

CAGLOH06523g YIL145C PANG Pantothenate synthase, also known as pantoate-beta-alanine ligase, re-quired for pantothenic acid biosyn-thesis, deletion causes pantothenic acid auxotrophy, homologous to E. coli panC

3.6 3.23 2.82

CAGLOF08745g YLR327C TMAIO Protein of unknown function that associates with ribosomes

3.53 4.7 2.16

CAGLOH08173g YOR028C CIN5 Basic leucine zipper (bZIP) transcription factor of the yAP-1 family, mediates pleiotropic drug resistance and salt tolerance; nuclearly localized under oxidative stress and sequestered in the cytoplasm by

3.49 2.14 2.61

Lot6p under reducing conditions CAGLOG01540g YNL036W NCE103 Carbonic anhydrase; poorly tran-

scribed under aerobic conditions and at an undetectable level under anaerobic conditions; involved in non-classical protein export path-way

3.47 2.7 2.44

CAGLOK00715g YGR213C RTA1 Protein involved in 7- aminocholesterol resistance; has seven potential membrane-spanning regions; expression is induced un-der both low-heme and low-oxygen conditions

3.3 5.14 3.11

CAGLOG00858g YLR332W MID2 0-glycosylated plasma membrane protein that acts as a sensor for cell wall integrity signaling and activates the pathway; interacts with Rom2p, a guanine nucleotide exchange factor for Rholp, and with cell integrity pathway protein

3.23 4.33 3.14

Zeolp CAGLOG00242g YGR281W YOR1 Plasma membrane ATP-binding

cassette (ABC) transporter, multidrug transporter mediates export of many different organic anions including oligomycin; similar to hu-man cystic fibrosis transmembrane receptor (CFTR)

3.21 3.25 3.28

CAGLOD03916g YHR034C PIHI Protein of unresolved function; may function in protein folding and/or rRNA processing, interacts with a chaperone (Ilsp82p), two chromatin remodeling factors (Rvblp, Rvb2p) and two rRNA processing factors

3.1 2.88 3.05

(Rrp43p, Nop58p) continued on next page


continued from previous page CG ORF SC ORF Gene name Description WT.S - WT.1.1 HOG.S - HOG.0 STE.S - STE.0

CAGLOH08844g

CAGLOE03025g CAGLOM02255g

YMR173W

NORBH YPL152W

DDR48

- RRD2

DNA damage-responsive protein, expression is increased in response to heat-shock stress or treatments that produce DNA lesions; contains multiple repeats of the amino acid sequence NNNDSYGS - Activator of the phosphoty- rosyl phosphatase activity of PP2A,peptidyl-prolyl cis/trans- isomerase; regulates G1 phase progression, the osmoresponse, microtubule dynamics; subunit of the Tap42p-Pph2lp-Rrd2p complex

3.06

2.94 2.92

2.32

2.92 3.37

2.39

3.06 3.12

CAGLOG04763g YOR1071V RGS2 Negative regulator of glucose- induced cAMP signaling; directly activates the GTPase activity of the heterotrimeric G protein alpha subunit Gpa2p

2.84 3.3 2.39

CAGLOL05434g YKR042W UTHI Mitochondria' outer membrane and cell wall localized SUN family mem-ber required for mitochondrial au-tophagy; involved in the oxidative stress response, life span during starvation, mitochondrial biogene-sis, and cell death

2.83 4.39 3.62

CAGLOG07601g YBR287W - - 2.79 2.43 3.46 CAGLOA02046g YOR018W ROD] Membrane protein that binds the


2.79 2.92 3.7

CAGLOL07480g YBRO66C NRG2 Transcriptional repressor that mediates glucose repression and nega-tively regulates filamentous growth; has similarity to Nrglp

2.64 2.49 3.15

CAGL0003201g YLL040C VPS13 Protein of unknown function; heterooligomeric or homooligomeric complex; peripherally associated with membranes; homologous to human COH1; involved in sporula-tion, vacuolar protein sorting and protein-Golgi retention

2.63 2.76 3.01

CAGLOJ10296g YNL077W A PJ/ Putative chaperone of the HSP40 2.62 4.21 2.7 (DNAJ) family; overexpression interferes with propagation of the 1Psi-1-] prion; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

CAGLOK12034g YDR040C ENA1 P-type ATPase sodium pump, involved in Nal- and Li-t- efflux to al-low salt tolerance

2.61 3.29 2.39

CAGLOI07007g YGL219C Ill DM34 Mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance; localizes to dots on mitochondrial surface near mtDNA nucleoids; ubiquitination target of

2.55 3.07 2.16

Mdm30p; interacts genetically with MDM31 and MDM32



continued from previous page CG ORF SC ORF Gene name Description WT.S - WT. U HOGS - HOG.0 STE.S - STE. U

CAGLOA02024g YOL025W LAG2 Protein involved in determination of longevity; LAG2 gene is preferen-tially expressed in young cells; over-expression extends the mean and maximum life span of cells -

2.52 2.84 2.64

CAGLOJ11176g YNL176C -2.51 3.31 2.64 CAGLOL04708g YGR111W - - 2.48 2.28 2.3 CAGLOL11572g YHR103W SDE22 Protein involved in the transport

of cell wall components from the 2.43 2.56 2.29

Golgi to the cell surface; similar in structure and functionally redun-dant with Sbe2p; involved in bud growth

CAGLOG05313g YDR072C IP TI Inositolphosphotransferase, involved in synthesis of mannose-

2.34 2.65 2.15

(inositol-P)2-ceramide (M(IP)2C), the most abundant sphingolipid;, can mutate to resistance to the antifungals syringomycin E and DmAMP1 and to K. lactis zymocin

CAGLOK03509g YMR110C HFD 1 Putative fatty aldehyde dehydrogenase, located in the mitochondria) outer membrane and also in lipid particles; has similarity to hu-man fatty aldehyde dehydrogenase

2.3 3.32 2.26


CAGLOA02321g YDR345C HXT3 Low affinity glucose transporter of the major facilitator superfamily, expression is induced in low or high glucose conditions

2.28 2.36 2.11

CAGLOL07238g YDL097C RP N6 Essential, non-ATPase regulatory subunit of the 26S proteasome lid required for the assembly and activ-ity of the 265 proteasome; the hu-man homolog (S9 protein) partially rescues Rpn6p depletion

2.25 2.01 2.76

CAGLOM06545g YKL124W SSH4 Specificity factor required for 2.24 2.69 2.21 Rsp5p-dependent ubiquitination and sorting of cargo proteins at the multivesicular body; identified as a high-copy suppressor of a SHR3 deletion, increasing steady-state levels of amino acid permeases

CAGLOK12892g YFLO47W RC D2 GTPase-activating protein 2.2 3.62 2.14 (RhoGAP) for Cdc42p and Rho5p

CAGLOL10912g YOR273C TPO4 Polyamine transport protein, recognizes spermine, putrescine, and spermidine; localizes to the plasma membrane; member of the major fa-cilitator superfamily

2.12 2.27 3.14

CAGLOA03806g YOR298W MUMS Protein of unknown function involved in the organization of the outer spore wall layers; has similar-ity to the tafazzins superfamily of acyltransferases

2.11 2.2 2.97

CAGLOJ09328g YDL127W PCL2 G1 cyclin, associates with Pho85p cyclin-dependent kinase (Cdk) to contribute to entry into the mitotic cell cycle, essential for cell morpho-genesis; localizes to sites of polar-ized cell growth

2.09 3.68 3.02



continued from previous page CG ORF SC ORF Gene name Description WT.S - WT.0 HOG.S - HOG.0 STE.S - STE.0

CAGLOL11110g YML057W CAIP2 Calcineurin A; one isoform (the other is CNA1) of the catalytic subunit of calcineurin, a

2.05 2.68 2.74

Ca+-1-/calmodulin-regulated protein phosphatase which regulates Crzlp (a stress-response transcrip-tion factor), the other calcineurin subunit is CNB1

CAGL0004917g YJR109C CPA2 Large subunit of carbamoyl phosphate synthetase, which catalyzes a step in the synthesis of citrulline, an arginine precursor

2.03 2.93 3.31

CAGL0004785g YJR115W - - 2.02 2.81 2.63


Table D.15: HOG/-dependent hyperosmotic stress induced C. glabrata tran-scripts. C. glabrata transcripts induced in wild-type and stel 1 cells, but not hog] cells

following exposure to transient hyper-osmotic shock (1M sorbitol, 15 minutes). Induced transcripts are defined as having log2(Cy5) - log2(Cy3) > 2.0 and Benjamini-Hochberg cor-rected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 in Table D.2

CG ORF SC ORF Gene Name Description WT.S - WT.0 STE.S - STE.0

CAGL0I02684g YMR2S5C NGL2 Protein involved in 5.8S rRNA processing; Ccr4p-like RNase required for correct 3'-end formation of 5.8S rRNA at site E; similar to Ngllp and Ngl3p

3.2 2.19

CAGLOK02761g YHLOO2W FISE1 Subunit of the endosomal Vps27p- 3.2 3.01 Hselp complex required for sorting of ubiquitinated membrane proteins into intralumenal vesicles prior to vacuolar degradation, as well as for recycling of Golgi proteins and for-mation of lumenal membranes - CAGLOH02519g YMR253C -2.65 3.46

CAGL0G07689g YKR014C YPT52 GTPase, similar to Ypt5lp and 2.48 2.47 Ypt53p and to mammalian rab5; re-quired for vacuolar protein sorting and endocytosis

CAGLOL10186g YOR052C - - 2.45 2.92

CAGLOE02299g YOL013C HRD1 Ubiquitin-protein ligase required for endoplasmic reticulum- associated degradation (ERAD) of misfolded proteins; genetically linked to the unfolded protein response (UPR); regulated through association with Hrd3p; contains an H2 ring finger

2.31 2.18

CAGLOG05049g YDR063W AIM7 Putative protein of unknown function; green fluorescent protein

2.28 2.75


CAGL0104774g YB R036C CSG2 Endoplasmic reticulum membrane protein, required for mannosylation of inositolphosphorylceramide and for growth at high calcium concen-trations

2.25 3.48

CAGLOH07007g YML092C PRE8 Alpha 2 subunit of the 20S proteasome

2.23 2.03

CAGLOM09108g YJR091C JSNI Member of the Puf family of 2.21 2.07 RNA-binding proteins, interacts with mRNAs encoding membrane-associated proteins; overexpression suppresses a tub2-150 mutation and causes increased sensitivity to beno-myl in wild-type cells

CAGLOK11022g YDR236C FAIN1 Riboflavin kinase, phosphorylates riboflavin to form riboflavin monophosphate (FMN), which is a necessary cofactor for many enzymes; localizes to microsomes and to the mitochondria] inner membrane

2.18 2.74



continued from previous page CC ORF SC ORF Gene name Description WT.S - WT.0 STE.S - STE.0

CAGLOD01474g

CAGL0M06875g

CAGL0D03322g

YBR108W

YNL025C

YLR023C

AIMS

SSN8

IZH3

Protein interacting with Rys167p; YBR108W is not an essential gene Cyclin-like component of the RNA polymerase II holoenzyme, involved in phosphorylation of the RNA polymerase II C-terminal domain; involved in glucose repression and telomere maintenance Membrane protein involved in zinc metabolism, member of the four-protein IZII family, expression induced by zinc deficiency; deletion reduces sensitivity to elevated zinc and shortens lag phase, overexpres-sion reduces Zap1p activity

2.17

2.15

2.12

2.39

2.38

2.01

CAGL00O2673g YLR392C - - 2.11 2.03

CAGLOL09889g YKL025C PAN3 Essential subunit of the Pan2p- 2.09 2.68

Pan3p poly(A)-ribonuclease complex, which acts to control poly(A) tail length and regulate the stoi-chiometry and activity of postrepli-cation repair complexes

CAGL0K09526g YDR394W RPTS One of six ATPases of the 19S regulatory particle of the 26S protea-some involved in the degradation of ubiquitinated substrates; substrate of N-acetyltransferase B

2.09 2.11

CAGLOB03619g YEL060C PRI31 Vacuolar proteinase B (yscB), a serine protease of the subtilisin fam-ily; involved in protein degradation in the vacuole and required for full protein degradation during sporula-tion

2.09 2.17

CAGLOF04829g YPR154W PINS Protein that induces appearance of 2.05 3.37 (PIN-k] prion when overproduced

CAGLOE04510g YOR022C - 2.01 2.29


Table D.16: STE11-dependent hyperosmotic stress induced C. glabrata tran-

scripts. C. glabrata transcripts induced in wild-type and hog] cells, but not stell cells

following exposure to transient hyper-osmotic shock (1M sorbitol, 15 minutes). Induced

transcripts are defined as having log2(Cy5) - log2(Cy3) > 2.0 and Benjamini-Hochberg cor-

rected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 in Table D.2

CG ORF SC ORF Gene Name Description WT.S - WT.0 HOGS - HOG . U

CAGLOM12474g YIL055C - - 3.46 3.07

CAGLOM07381g YPL053C KTR6 Probable mannosylphosphate transferase involved in the synthesis of core oligosaccharides in protein glycosylation pathway; member of the KRE2/MNT1 mannosyltransferase family

3.45 3.54

CAGLOJ00539g YHR030C SLT2 Serine/threonine MAP kinase involved in regulating the maintenance of cell wall integrity and pro-gression through the cell cycle; reg-ulated by the PKC1-mediated sig-naling pathway

3.13 4.02

CAGLOK00539g YPL259C APMI Mul-like medium subunit of the clathrin-associated protein complex

3.1 3.96

(AP-1); binds clathrin; involved in clathrin-dependent Golgi protein sorting

CAGLOM12034g YAL038W CDC19 Pyruvate kinase, functions as a homotetramer in glycolysis to convert phosphoenolpyruvate to pyruvate, the input for aerobic (TCA cycle) or anaerobic (glucose fermentation) respiration

3.09 4.32

CAGL0104246g YGL162W S UT/ Transcription factor of the 3.04 4.21 Zn[II]2Cys6 family involved in sterol uptake; involved in induction of hypoxic gene expression

CAGLOH04631g YMLOO7W YAP1 Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance; activated by

2.98 3.6

H202 through the multistep forma-tion of disulfide bonds and transit from the cytoplasm to the nucleus; mediates resistance to cadmium

CAGLOD05368g YBR253W SRB6 Subunit of the RNA polymerase II mediator complex; associates with core polymerase subunits to form the RNA polymerase II holoenzyme; essential for transcriptional regulation

2.97 2.55

CAGLOE05632g YOR348C PUT4 Proline permease, required for high- affinity transport of proline; also transports the toxic proline ana-log azetidine-2-carboxylate (AzC);

2.85 3.55

PUT4 transcription is repressed in ammonia-grown cells

CAGLOJ00253g YGRO23W MTL1 Protein with both structural and functional similarity to Mid2p, which is a plasma membrane sensor required for cell integrity signaling during pheromone-induced morpho-genesis; suppresses rgdl null muta-tions

2.83 2.86

continues on next page


continued from previous page CG ORF SC ORF Gene name Description WT.S - WT.0 HOG.S - HOG.0

CAGLOM01628g

CAGLOH09812g

YDR389W

YDROO6C

S AC7

SOK1

GTPase activating protein (GAP) for Rho1p, involved in signaling to the actin cytoskeleton, null muta-tions suppress tort mutations and temperature sensitive mutations in actin; potential Cdc28p substrate Protein whose overexpression suppresses the growth defect of mu-tants lacking protein kinase A ac-tivity; involved in cAMP-mediated signaling; localized to the nucleus; similar to the mouse testis-specific protein PBS13

2.77

2.77

4.08

3.42

CAGLOH07557g YGL254W FZF/ Transcription factor involved in sulfite metabolism, sole identified reg-ulatory target is SSU1, overexpres-sion suppresses sulfite-sensitivity of many unrelated mutants due to hy-peractivation of SSU1, contains five zinc fingers

2.68 2.24

CAGLOM11066g YGR109C CLB6 B-type cyclin involved in DNA replication during S phase; activates Cdc28p to promote initiation of DNA synthesis; functions in formation of mitotic spindles along with Clb3p and C1b4p; most abun-dant during late G1

2.64 2.23

CAGLOE00649g YCR079W PTC6 Mitochondrial protein phosphatase of type 2C with similarity to mammalian PP1Ks; involved in mi- tophagy; null mutant is sensitive to rapamycin and has decreased phos-phorylation of the Pdal subunit of pyruvate dehydrogenase

2.53 4.06

CAGL0,104466g YLR414C - - 2.5 2.92 CAGLOA04081g YLR194C - 2.48 3.88 CAGLOK00891g YGR205W - - 2.42 2.53 CAGLOJ09108g YDL190C UFD2 Ubiquitin chain assembly fac-

tor (E4) that cooperates with a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme

2.39 2.07

(E2), and a ubiquitin protein ligase (E3) to conjugate ubiquitin to substrates; also functions as an E3

CAGLOD05434g YPRO65W ROX1 Heme-dependent repressor of hypoxic genes; contains an HMG do-main that is responsible for DNA bending activity

2.38 2.58


2.35 2.1


CAGL0004433g YDL003W MOD 1 Essential subunit of the cohesin complex required for sister chromatid cohesion in mitosis and meio-sis; apoptosis induces cleavage and translocation of a C-terminal fragment to mitochondria; expression peaks in S phase

2.22 2.41

continues on next page



CAGL0005093g CAGLOM03839g CAGL0108239g

YOL057W YNL305C YER021W

- -

RPN3

- - Essential, non-ATPase regulatory subunit of the 26S proteasome lid, similar to the p58 subunit of the hu-man 26S proteasome; temperature-sensitive alleles cause metaphase ar-rest, suggesting a role for the pro-teasome in cell cycle control

2.21 2.2 2.2

3.05 2.94 2.55

CAGLOM04081g YNL317W PFS2 Integral subunit of the pre-mRNA cleavage and polyadenylation factor

2.2 3.07

(CPF) complex; plays an essential role in mRNA 3'-end formation by bridging different processing factors and thereby promoting the assem-bly of the processing complex

CAGLOH09130g YKL201C MNN4 Putative positive regulator of mannosylphosphate transferase

2.14 2.63

(Mnn6p), involved in manno- sylphosphorylation of N-linked oligosaccharides; expression increases in late-logarithmic and stationary growth phases

CAGLOM08206g YJL171C - 2.14 3.13

CAGLOD02948g YJL034W KAR2 ATPase involved in protein import into the ER, also acts as a chap-erone to mediate protein folding in the ER and may play a role in ER export of soluble proteins; regulates the unfolded protein response via interaction with Ire1p

2.12 2.29

CAGLOK05995g YLR260W LCB5 Minor sphingoid long-chain base kinase, paralog of Lcb4p responsible for few percent of the total activ-ity, possibly involved in synthesis of long-chain base phosphates, which function as signaling molecules

2.12 2.35

CAGLOJ01001g YJL036W SNX4 Sorting nexin, involved in retrieval of late-Golgi SNAREs from post-

2.1 2.84

Golgi endosomes to the trans- Golgi network and in cytoplasm to vacuole transport; contains a PX phosphoinositide-binding domain; forms complexes with Snx4lp and with Atg2Op

CAGLOG01034g YGR032W GSC2 Catalytic subunit of 1,3-beta- glucan synthase, involved in formation of the inner layer of the spore wall; activity positively regulated by Rho1p and negatively by Smklp; has similarity to an alternate catalytic subunit, Fkslp

2.08 2.73

(Gsclp) CAGLOD05478g YGR186W TFG1 TFIIF (Transcription Factor II)

largest subunit; involved in both transcription initiation and elonga-tion of RNA polymerase II; homol-ogous to human RAP74

2.08 3.07

CAGL00O2541g YLR399C BDF1 Protein involved in transcription initiation at TATA-containing promoters; associates with the basal transcription factor TFIID; contains two bromodomains; corresponds to the C-terminal region of mammalian TAF1; redundant with

2.04 2.25

Bdf2p continues on next page



CAGLOK11946g I NORBH I - I - 2.04 3.9


Table D.17: C. glabrata hyperosmotic stress repressed transcripts that depend on a functional putative SHO1 signalling branch. C. glabrata transcripts repressed in

wild-type, but not hogl or sten cells following exposure to transient hyper-osmotic shock (1M sorbitol, 15 minutes). repressed transcripts are defined as having log2(Cy5) - log2(Cy3) < -2.0 and Benjamini-Hochberg corrected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 in Table D.2

CG ORF SC ORF Gene Name Description WT.S - WT.0

CAGLOL08470g NORBH - Description not mapped -2.03 CAGLOL01551g YML052W SUR7 Putative integral membrane protein; component of eisosomes;

associated with endocytosis, along with Pillp and Lsplp; sporulation and plasma membrane sphingolipid content are al-tered in mutants

-2.04

CAGL00O2739g YAL008W FUNI4 Mitochondria] protein of unknown function -2.05

CAGLO/02442g YIL022W TIM44 Peripheral mitochondria] membrane protein involved in mitochondrial protein import, tethers essential chaperone Ssclp to the translocon channel at the matrix side of the inner mem-brane

-2.09

CAGLOL01991g YKL033W TTI1 Putative protein of unknown function; subunit of the ASTRA complex which is part of the chromatin remodeling machin-ery; similar to S. pombe Ttilp; detected in highly purified mitochondria in high-throughput studies

-2.09

CAGLOL13156g YLR073C - Description not mapped -2.1

CAGL0G08646g YIL122W POC/ Putative transcriptional activator that promotes recovery from pheromone induced arrest; inhibits both alpha-factor induced

-2.12

G1 arrest and repression of CLN1 and CLN2 via SCB/MCB promoter elements; potential Cdc28p substrate; SBF regulated

CAGLOK04477g YGR060W ERG25 C-4 methyl sterol oxidase, catalyzes the first of three steps required to remove two C-4 methyl groups from an intermedi-ate in ergosterol biosynthesis; mutants accumulate the sterol intermediate 4,4-dimethylzymosterol

-2.15

CAGLOB00638g YCL047C - Description not mapped -2.16

CAGLOD00748g YDL067C COX9 Subunit VIIa of cytochrome c oxidase, which is the terminal member of the mitochondria] inner membrane electron trans-port chain

-2.18

CAGL00O2211g YELO4OW UTR2 Cell wall protein that functions in the transfer of chitin to beta(1-6)glucan; putative chitin transglycosidase; glycosylphosphatidylinositol (GPI)-anchored protein localized to the bud neck; has a role in cell wall maintenance

-2.27

CAGLOH05533g YPL082C MO T/ Essential abundant protein involved in regulation of transcription, removes Sptl5p (TBP) from DNA via its C-terminal AT-

-2.31

Pase activity, forms a complex with TBP that binds TATA DNA with high affinity but with altered specificity

CAGLOM08448g YKL165C MCD4 Protein involved in glycosylphosphatidylinositol (GPI) anchor synthesis; multimembrane-spanning protein that localizes to the endoplasmic reticulum; highly conserved among eukaryotes

-2.32

CAGLOK02233g YER126C NSA2 Protein constituent of 66S pre-ribosomal particles, contributes to processing of the 27S pre-rRNA

-2.38

CAGLOL03025g YKR025W RPC37 RNA polymerase III subunit C37 -2.4

CAGLOI01826g YHR148W IMPS Component of the SSU processome, which is required for pre- -2.42 18S rRNA processing, essential protein that interacts with MpplOp and mediates interactions of Imp4p and MpplOp with U3 snoRNA

CAGLOD05940g YGR175C ERG1 Squalene epoxidase, catalyzes the epoxidation of squalene to -2.48 2,3-oxidosqualene; plays an essential role in the ergosterol- biosynthesis pathway and is the specific target of the antifun-gal drug terbinafine

CAGLOK06281g YDR454C GUK1 Guanylate kinase, converts GMP to GDP; required for growth and mannose outer chain elongation of cell wall N-linked gly-coproteins

-2.48




CAGL0109900g YOR3I9W HSH49 U2-snRNP associated splicing factor with similarity to the mammalian splicing factor SAP49; proposed to function as a

-2.51

U2-snRNP assembly factor along with Hsh155p and binding partner Cus1p; contains two RNA recognition motifs (RRM)

CAGLOA01914g YHR099W TRAI Subunit of SAGA and NuA4 histone acetyltransferase corn- plexes; interacts with acidic activators (e.g., Gal4p) which leads to transcription activation; similar to human TRRAP, which is a cofactor for c-Myc mediated oncogenic transforma-tion

-2.58

CAGLOG00484g YGR272C - Description not mapped -2.59 CAGL0106809g YER142C MAGI 3-methyl-adenine DNA glycosylase involved in protecting DNA

against alkylating agents; initiates base excision repair by re-moving damaged bases to create abasic sites that are subse-quently repaired

-2.71

CAGLOK09416g YGL211W NCS6 Protein required for thiolation of the uridine at the wobble position of Gln, Lys, and Glu tRNAs; has a role in urmylation and in invasive and pseudohyphal growth; inhibits replication of Brome mosaic virus in S. cerevisiae

-2.81

CAGLOLO5500g YJL122W ALBI Shuttling pre-60S factor; involved in the biogenesis of ribosomal large subunit; interacts directly with Arxlp; responsible for Tif6p recycling defects in absence of Reilp

-2.91

CAGL0000737g YLR222C UTP13 Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in process-ing of pre-18S rRNA

-2.91

CAGLOH09064g YHR128W PURI Uracil phosphoribosyltransferase, synthesizes UMP from uracil; involved in the pyrimidine salvage pathway

-2.97

CAGLOL10384g YOR064C YNG1 Subunit of the NuA3 histone acetyltransferase complex that acetylates histone H3; contains PHD finger domain that inter-acts with methylated histone I-13, has similarity to the human tumor suppressor ING1

-3.38

CAGLOH01089g YDR287W INM2 Inositol monophosphatase, involved in biosynthesis of inositol; enzymatic activity requires magnesium ions and is inhibited by lithium and sodium ions; inml inm2 double mutant lacks inositol auxotrophy

-3.41


Table D.18: HOG1-dependent hyperosmotic stress repressed C. glabrata tran-

scripts. C. glabrata transcripts repressed in wild-type and stell cells, but not hogl cells following exposure to transient hyper-osmotic shock (1M sorbitol, 15 minutes). repressed transcripts are defined as having log2(Cy5) - log2(Cy3) < -2.0 and Benjamini-Hochberg corrected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 in Table D.2

CG ORF SC ORF Gene Name Description WT.S - WT.0 STE.S - STE.0

CAGLOM09086g YJR092W BUD4 Protein involved in bud-site selection and required for axial budding pattern; localizes with septins to bud neck in mitosis and may con-stitute an axial landmark for next round of budding; potential Cdc28p substrate

-2.16 -3.74

CAGLOE05324g YOR330C MIP1 Catalytic subunit of the mitochondrial DNA polymerase; conserved

-2.24 -3.57

C-terminal segment is required for the maintenance of mitochondrial genome.

CAGLOH04301g YML078W CPB3 Mitochondrial peptidyl-prolyl cis- trans isomerase (cyclophilin), cat-alyzes the cis-trans isomerization of peptide bonds N-terminal to proline residues; involved in protein refold-ing after import into mitochondria

-2.4 -3.31

CAGLOG07953g YNR037C RSM19 Mitochondrial ribosomal protein of the small subunit, has similarity to

-2.53 -3.36

E. coli S19 ribosomal protein CAGLOK03795g YMR128W ECM/6 Essential DEAD-box ATP-

dependent RNA helicase specific to the 1J3 snoRNP, predominantly nucleolar in distribution, required for 18S rRNA synthesis

-2.63 -3.71

CAGLOG04411g YJL109C UTP/ 0 Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

-2.7 -3.39

CAGL0106743g YER145C FTR1 High affinity iron permease involved in the transport of iron across the plasma membrane; forms complex with Fet3p; expression is regulated by iron

-2.74 -2.54


Table D.19: STE11-dependent hyperosmotic stress repressed C. glabrata tran-

scripts. C. glabrata transcripts repressed in wild-type and hogl cells, but not stel 1 cells following exposure to transient hyper-osmotic shock (1M sorbitol, 15 minutes). repressed transcripts are defined as having log2(Cy5) - log2(Cy3) > 2.0 and Benjamini-Hochberg cor-rected p-values < 0.05. Estimated log-ratios correspond to the WT.S - WT.0 in Table D.2

CG ORF SC ORF Gene Name Description WT.S - WT.0 HOGS - HOG.0 CAGLOF01793g YLR056W ERG3 C-5 sterol desaturase, catalyzes the

introduction of a C-5(6) double bond into episterol, a precursor in ergosterol biosynthesis; mutants are viable, but cannot grow on non- fermentable carbon sources

-2.43 -2.73

CAGLOG00352g YGR277C - - -2.81 -3.46


Table D.20: S. cerevisiae transcripts whose hyperosmotic stress dependent in-duction is affected by HOG1 deletion. Data obtained from Rep et al., (581)

CG ORF SC ORF Gene name Description CAGLOI09878g YOR317W FAA 1 Long chain fatty acyl-CoA synthetase with a preference for C12:0-C16,0

fatty acids; involved in the activation of imported fatty acids; localized to both lipid particles and mitochondrial outer membrane; essential for stationary phase

CAGLOG05984g YHR139C SPS100 Protein required for spore wall maturation; expressed during sporula-tion; may be a component of the spore wall

CAGLOL11374g YML070W DAK I Dihydroxyacetone kinase, required for detoxification of dihydroxyace-tone (DHA); involved in stress adaptation

CAGLOD06688g YOR374W ALD4 Mitochondrial aldehyde dehydrogenase, required for growth on ethanol and conversion of acetaldehyde to acetate; phosphorylated; activity is K+ dependent; utilizes NADP+ or NAD+ equally as coenzymes; ex-pression is glucose repressed

CAGLOG06028g YHR137W AR09 Aromatic aminotransferase II, catalyzes the first step of tryptophan, phenylalanine, and tyrosine catabolism

CAGLOB02563g YML128C MSC1 Protein of unknown function; mutant is defective in directing meiotic recombination events to homologous chromatids; the authentic, non-tagged protein is detected in highly purified mitochondria and is phos-phorylated

CAGLOE05280g YOL151W GRES 3-methylbutanal reductase and NADPH-dependent methylglyoxal re-ductase (D-lactaldehyde dehydrogenase); stress induced (osmotic, ionic, oxidative, heat shock and heavy metals); regulated by the HOG path-way

CAGLOK08844g YHL021C AIM17 Putative protein of unknown function; the authentic, non-tagged pro-tein is detected in highly purified mitochondria in high-throughput studies

CAGLOB00286g YCL064C CHA1 Catabolic L-serine (L-threonine) deaminase, catalyzes the degradation of both L-serine and L-threonine; required to use serine or threonine as the sole nitrogen source, transcriptionally induced by serine and threo-nine

CAGLOK12034g YDR040C ENA I P-type ATPase sodium pump, involved in Na-F and Li+ efflux to allow tolerance _salt

CAGLOJ01331g YMR090W - CAGLOG03883g YLL026W HSP/04 Heat shock protein that cooperates with Ydjlp (Hsp40) and Ssalp

(Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in [PSI+, propagation

CAGLOE06380g YKL151C - - CAGLOI10516g YGR130C - - CAGLOG03773g YLL023C - - CAGLOJ04202g YFLO14W HSPI2 Plasma membrane localized protein that protects membranes from des-

iccation; induced by heat shock, oxidative stress, osmostress, station-ary phase entry, glucose depletion, oleate and alcohol; regulated by the HOG and Ras-Pka pathways

CAGLOG04433g YJL108C PRIV110 Pheromone-regulated protein, predicted to have 5 transmembrane seg-ments; induced by treatment with 8-methoxypsoralen and UVA irradi-ation

CAGLOH02101g YHR087W RTC3 Protein of unknown function involved in RNA metabolism; has struc-tural similarity to SBDS, the human protein mutated in Shwachman- Diamond Syndrome (the yeast SBDS ortholog = SDO1); null mutation suppresses cdc13-1 temperature sensitivity

CAGLOF04807g YIL136W 0M45 Protein of unknown function, major constituent of the mitochondrial outer membrane; located on the outer (cytosolic) face of the outer mem-brane

CAGLOM11660g YIL053W 1311132 Constitutively expressed isoform of DL-glycerol-3-phosphatase; involved in glycerol biosynthesis, induced in response to both anaerobic and, along with the Hor2p/Gpp2p isoform, osmotic stress

C AG LOI07865g YOL084W PHM7 Protein of unknown function, expression is regulated by phosphate lev-els; green fluorescent protein (GFP)-fusion protein localizes to the cell periphery and vacuole



continued from previous page CG ORF SC ORF Gene Name Description

CAGLOK12958g YML131W - - CAGLOG05335g YDR074W TPS2 Phosphatase subunit of the trehalose-6-phosphate syn-

thase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; expression is induced by stress conditions and repressed by the Ras-cAMP pathway

CAGLOF08085g YGR243W FMP43 Putative protein of unknown function; expression regulated by osmotic and alkaline stresses; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

CAGLOG02915g YGR086C DILI Primary component of eisosomes, which are large immobile cell cortex structures associated with endocytosis; null mutants show activation of Pkclp/Ypklp stress resistance pathways; detected in phosphorylated state in mitochondria

CAGLOF07579g YKL096W CWP1 Cell wall mannoprotein, linked to a beta-1,3- and beta-1,6-glucan het-eropolymer through a phosphodiester bond; involved in cell wall orga-nization

CAGLOL07656g YMLOO4C GL 0 1 Monomeric glyoxalase I, catalyzes the detoxification of methylglyoxal (a by-product of glycolysis) via condensation with glutathione to pro-duce S-D-lactoylglutathione; expression regulated by methylglyoxal lev-els and osmotic stress

CAGLOF07777g YMR170C AL D2 Cytoplasmic aldehyde dehydrogenase, involved in ethanol oxidation and beta-alanine biosynthesis; uses NAD+ as the preferred coenzyme; ex-pression is stress induced and glucose repressed; very similar to Ald3p

CAGLOI01122g YHR1O4W GRE3 Aldose reductase involved in methylglyoxal, d-xylose and arabinose metabolism; stress induced (osmotic, ionic, oxidative, heat shock, star-vation and heavy metals); regulated by the HOG pathway

Table D.21: Betweenness centrality measures and degree of tube stations in the London Underground network London underground tube- map data sourced from http: //research. cs . queensu . ca/-daver/235/C1352963146/ E20070302133910/index.html were used to build a network of stations and edges be-tween stations. The degree of each station is the number of adjacent stations i.e. those that are one stop away. The betweenness centrality CB(v) is defined in section (3.3.6). High centrality scores indicate that a node can reach others on a relatively short path or that a node lies on a large proportion of shortest paths connecting other nodes. In the case of the London Underground tube-map, Baker Street is the most central node and King's Cross, although it has a high degree is ranked five places below Baker Street in terms of betweenness centrality.

Station Degree CB(v) Baker Street 7 0.34 Green Park 6 0.33

Bank 7 0.3 Waterloo 5 0.29 Stratford 5 0.22

Liverpool Street 6 0.19 King's Cross St Pancras 7 0.19

Turnham Green 5 0.11 Oxford Circus 6 0.1

Shadwell 5 0.06

(b)

(c)

YEPD + 1 M SORBITOL CGTFKOLIB Plate 1


062001 C „2,03B1 PO81 14161 H052 GAL83 SASS 5EFI RTG2 KARA TO54

81103 11102 MEW NAP1 Han RFXI EIL I LTA'S CUP. GLIY3 MI53 POP2

TOPS- like (Rot RTG1 HST1 45111

ATO)

SFP1-

(not A1.01 SSAAS 11051 11051 PP131 BRE2 31104 46 00?

TUPI 5000 HGGI SWIS SBA HIRI SHF6 8I14101 STP2 AHC1 ADRI

TYE7 6AL11 YAP] SITS ARCOT 5131 CADJ 11574 TSPI MCI 1

0.4081 51134 hISSI 1 0021 GCHS SWDl X501 45610 FLOE G273 5362

IIII• P11023 YOX1 GIS 1 POGI A5G1 FX211 AFT2 TOSS SP01 WARS #101

%V- 1032 . ARGR1 FIFE RTF1 HAP2 0051 0533 11102 STBS COPY

(a)

YEPD CGTFKOLib Plate 1

• 4111•• 4116••••• •••••••• • 6 II 41 41 41 • 41 41 44 41 41 40 41 ••

4114

• 40 ED • 0 • • ',10 • • • • • • IA •••••••• • •••• • 111 411111•• 4Il • • 11 • • fir 41 • • • 410 11104141•64.0•41 0.41

Figure D.1: Solid agar phenotype assay screen of CGTFKO1ib plate 1 on hyper-tonic agar (1 M Sorbitol). Frozen glycerol stocks of plate 1 of the CGTFKOLib arrayed in 96 well format (a) were defrosted by a Bunsen flame, subcultured (18 hours, 37°C, 180 rpm), washed twice in distilled water (3000 rpm, 5 minutes) and replicated (104 cells) onto solid YEPD agar or solid YEPD agar + 1 M sorbitol and incubated for 24 hours. Feint red squares highlight mutant strains relevant to hyperosmotic stress and/or SAPk pathways. Experiments performed by Lauren Ames

•••••••••fie ••••••••••••

•••••••••111 ••••••••••• +60 • • • • • ••• • •

• • • • • * 411 zi • • • • 11111 • • • iik • 81 41 • 1 0 41 41 0 • ••

(b)

YEPD + 1 M SORBITOL CGTFKOLIB Plate 2

* II • 0 • 114\ 041000111/00 411•••••• • • • *GOO* • • • ••••• o• • • • ••• ►•••••••• 410•••

(c)


(111001 C20C" SUT1 CST6 5111 MO 15115 MCMI 1-041 Han 1 1013

ZAP1 HOU III PIP2 GISI San 11142 MOSS

10,

5502 WTI SAU 1 RINI1

5614 EMl1 4.61- like

ADA2 STP1 5514-11e GAT1 1464 METH YAPS MI2.2 NAPS

SUM LY514 HOU 0013 WTI SAPS OW StS3 auto SiP0 2211' YHPI LIVE

YAM AS11 GT51 TIM MCI 44312 14.401 .61 11111 PUTS sprz CHOI

*PHI SSIO 0501 IOW IIIASET31 if AIM SPT3 UNIZI SPP1

3052 TA114 CAR SRL DAM ACF2 SPAM 5053 CALI SSW FAPI SON/

0464 MEI Rira VAPilikr 0444

(a)

YEPD CGTFKOLib Plate 2

Figure D.2: Solid agar phenotype assay screen of CGTFKOlib plate 2 on hyper-tonic agar (1 M Sorbitol). Frozen glycerol stocks of plate 1 of the CGTFKOLib arrayed in 96 well format (a) were defrosted by a Bunsen flame, subcultured (18 hours, 37°C, 180 rpm), washed twice in distilled water (3000 rpm, 5 minutes) and replicated (104 cells) onto solid YEPD agar or solid YEPD agar + 1 M sorbitol and incubated for 24 hours. Feint red squares highlight mutant strains relevant to hyperosmotic stress and/or SAPk pathways. Experiments performed by Lauren Ames

ver.812C Yogi SW

YGFI133W Y13191,03W

YEN75C o 0 8 c o

0 o 0 o

° SO 43-V-43

YLL09W Y011906C

YPRgI36W

__1'0.55.11.R22 S ..................... _

i i i 1000 2000 3000

arbitrary gene Index

4000 500 1 000 1500 0000 2500 3000

arbitrary gene index

012


z

S. bayanus dblidS

YILV4W

YILI,F3C 0

;PAW/

vp111,55c ° o

° o

-7-

S. mikatae dblidS

YP147W

VIL1.;4W

V.1,4,70W

Velr_ig51W rr

1000 2000 3000 40011 1000

2000 3000 4000

arbitrary gene Index arbitrary gene Index

(a)

(b)

S. paradoxus dNIdS C. glabrata dNIdS

(c)

(d)

Figure D.3: Calculated dN/dS > rates for Saccharomyces bayanus (A), Sac-charomyces mikatae (B), Saccharomyces paradoxus (C) and C. glabrata (D). Genome sequences of Saccharomyces paradoxus, Saccharomyces bayanus, Saccharomyces mikatae and C. glabrata were obtained along with pre-computed orthologue lists and used to construct a table of ORFs that are conserved between S. cerevisiae and the other Sac-charomycotina. In order to calculate the ratio of non-synonymous substitution rate to synonymous substitution rate (dN/dS), nucleic acid sequences were first translated to their amino acid sequences using translfas (147; 148) and the default universal codon tables. This step is performed to preserve the alignment of codons and permits the use of substitution matrices, such as those described in, for the estimation of evolutionary rates. Multiple align-ment of conserved Saccharomycotina proteins was performed using clustalw (723; 689) and putgaps (147; 148) was used to create a gapped, in-frame codon alignment of nucleic acids for subsequent estimation of dN/dS using the Creevey-McInerney method. The average C. glabrata dN/dS (0.32) is significantly greater than dN/dS for S. paradoxus (mean = 0.15, p < 0.05, t = 31.54, df = 4355), S. bayanus (mean = 0.15, p < 0.05, t = 32.00, df = 4105) and S. mikatae (mean = 0.16, p < 0.05, t = 30.00, df = 4581). A larger proportion of C. glabrata orthologue have dN/dS greater than 1. The dN/dS > 1 threshold is depicted by a horizontal red, dashed line at dN/dS = 1


Table D.22: C. glabrata ORFs with dN/dS > 1. Genome sequences of Saccharomyces paradoxus, Saccharomyces bayanus, Saccharomyces mikatae and C. glabrata were obtained along with pre-computed orthologue lists and used to construct a table of ORFs that are conserved between S. cerevisiae and the other Saccharomycotina. In order to calculate the ratio of non-synonymous substitution rate to synonymous substitution rate (dN dS), nucleic acid sequences were first translated to their amino acid sequences using translfas (147; 148) and the default universal codon tables. This step is performed to preserve the alignment of codons and permits the use of substitution matrices, such as those described in, for the estimation of evolutionary rates. Multiple alignment of conserved Saccharomycotina proteins was performed using clustalw (723; 689) and putgaps (147; 148) was used to create a gapped, in-frame codon alignment of nucleic acids for subsequent estimation of dN/dS using the Creevey--McInerney method. The average C. glabrata dNI dS (0.32) is significantly greater than dN/dS for S. paradoxus (mean = 0.15, p < 0.05, t = 31.54, df = 4355), S. bayanus (mean = 0.15, p < 0.05, t = 32.00, df = 4105) and S. mikatae (mean = 0.16, p < 0.05, t = 30.00, df = 4581). A larger proportion of C. glabrata orthologue have dN dS greater than 1.

homologous SC ORF Gene Name Description YALOO5C SSA1 ATPase involved in protein fold-

ing and nuclear localization signal (NLS)-directed nuclear transport; member of heat shock protein 70 (HSP70) family; forms a chaperone complex with Ydjlp; localized to the nucleus, cytoplasm, and cell wall

YAL047C SPC7.2 Component of the cytoplasmic Tub4p (gamma-tubulin) complex, binds spindle pole bodies and links them to microtubules; has roles in astral microtubule formation and stabilization

YBR127C VMA2 Subunit B of the eight-subunit Y1 peripheral membrane domain of the vacuolar H-1-ATPase (V-ATPase), an electrogenic proton pump found throughout the endomembrane system; contains nucleotide binding sites; also detected in the cytoplasm

YCL036W GFD2 Protein of unknown function, iden-tified as a high-copy suppressor of a dbp5 mutation

YCR024C SLM5 Mitochondrial asparaginyl-tRNA synthetase

YCR087C-A Putative protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the nucleolus; YCR087C-A is not an essential gene



continued from previous page homologous SC ORF Gene Name Description

YDL155W

YDL237W

YDR071C

YDR075W

YDR103W

YDR137W

YDR168W

YDR350C

YDR452W

YDR463W

CLB3

AIMS

PA A 1

PPH3

STE5

RGP1

CD C37

ATP22

PPN1

STPI

B-type cyclin involved in cell cy-cle progression; activates Cdc28p to promote the G2/M transition; may be involved in DNA replication and spindle assembly; accumulates dur-ing S phase and G2, then targeted for ubiquitin-mediated degradation Putative protein of unknown function, required for respiratory growth; YDL237W is not an essential gene Polyamine acetyltransferase; acetylates polyamines (e.g. putrescine, spermidine, spermine) and also aralkylamines (e.g. tryptamine, phenylethylamine); may be involved in transcription and/or DNA replication Catalytic subunit of an evolution-arily conserved protein phosphatase complex containing Psy2p and the regulatory subunit Psy4p; required for cisplatin resistance; involved in activation of GIn3p Pheromone-response scaffold protein; binds Ste11p, Ste7p, and Fus3p kinases, forming a MAPK cascade complex that interacts with the plasma membrane and Ste4p-Stel8p; allosteric activator of Fus3p that facilitates Ste7p-mediated ac-tivation Subunit of a Golgi membrane ex-change factor (Riclp-Rgplp) that catalyzes nucleotide exchange on Ypt6p Essential HspOOp co-chaperone; necessary for passage through the START phase of the cell cycle; stabilizes protein kinase nascent chains and participates along with IfspOOp in their folding Mitochondrial inner membrane pro-tein required for assembly of the FO sector of mitochondria' FIFO ATP synthase, which is a large, evolu-tionarily conserved enzyme complex required for ATP synthesis Vacuolar endopolyphosphatase with a role in phosphate metabolism; functions as a ho- modimer Transcription factor, undergoes proteolytic processing by SPS (Ssylp-Ptr3p-Ssy5p)-sensor component Ssy5p in response to extracellular amino acids; activates transcription of amino acid perme-ase genes and may have a role in tRNA processing




YER058W

YER168C

YER175C

YFRO12W

YFRO36W

YGL110C

YGL112C

YGL153W

YGRO13W

YGRO78C

YGR133W

YGR199W

PETI17

CCAI

TMT1

CDC26

CUES

TAPS

PEXI4

SNU7I

PACIO

PEX4

PMTG

Protein required for assembly of cy-tochrome c oxidase ATP (CTP):tRNA-specific tRNA nucleotidyltransferase; different forms targeted to the nucleus, cytosol, and mitochondrion are generated via the use of multiple transcriptional and translational start sites Trans-aconitate methyltransferase, cytosolic enzyme that catalyzes the methyl esterification of 3- isopropylmalate, an intermediate of the leucine biosynthetic pathway, and trans-aconitate, which inhibits the citric acid cycle Putative protein of unknown func-tion Subunit of the Anaphase- Promoting Complex/Cyclosome (APC/C), which is a ubiquitin- protein ligase required for degradation of anaphase inhibitors, including mitotic cyclins, during the metaphase/anaphase transition Protein of unknown function; has a CUE domain that binds ubiquitin, which may facilitate intramolecular monoubiquitination Subunit (60 kDa) of TFIID and SAGA complexes, involved in tran-scription initiation of RNA poly-merase II and in chromatin modi-fication, similar to histone H4 Peroxisomal membrane peroxin that is a central component of the peroxisomal protein import machinery; interacts with both PTS1 (Pex5p) and PTS2 (Pex7p), peroxisomal matrix protein signal recognition factors and membrane receptor Pexl3p Component of Ul snRNP required for mRNA splicing via spliceosome; yeast specific, no metazoan counter-part Part of the heteromeric co- chaperone GimC/prefoldin complex, which promotes efficient protein folding Peroxisomal ubiquitin conjugating enzyme required for peroxisomal matrix protein import and peroxi-some biogenesis Protein 0-mannosyltransferase, transfers mannose from dolichyl phosphate-D-mannose to protein serine/threonine residues of secretory proteins; reaction is essential for cell wall rigidity; member of a family of mannosyltransferases




YHROO5C

YHR032W

YIL018W

YIL044C

YIL066C

YIL079C

YIR031C

YJL110C

YJL176C

GPA 1

RPL2B

AGE2

RNR3

A IR I

DA L7

GZ.F3

SW13

GTP-binding alpha subunit of the heterotrimeric G protein that cou-ples to pheromone receptors; nega-tively regulates the mating pathway by sequestering G(beta)gamma and by triggering an adaptive response; activates Vps34p at the endosome Putative protein of unknown function; putative substrate of the cAMP-dependent protein kinase (PKA) Protein component of the large (60S) ribosomal subunit, identical to Rpl2Ap and has similarity to E. coli L2 and rat L8 ribosomal pro-teins; expression is upregulated at low temperatures ADP-ribosylation factor (ARF) GTPase activating protein (GAP) effector, involved in Trans-Golgi- Network (TGN) transport; contains C2C2142 cysteine/histidine motif One of two large regulatory sub-units of ribonucleotide-diphosphate reductase; the RNR complex cat-alyzes rate-limiting step in dNTP synthesis, regulated by DNA repli-cation and DNA damage checkpoint pathways via localization of small subunits Zinc knuckle protein, involved in nuclear RNA processing and degradation as a component of the TRAMP complex; stimulates the poly(A) polymerase activity of Pap2p in vitro; functionally redun-dant with Air2p Malate synthase, role in allantoin degradation unknown; expres-sion sensitive to nitrogen catabo-lite repression and induced by allo-phanate, an intermediate in allan-toin degradation GATA zinc finger protein and Da180p homolog that negatively regulates nitrogen catabolic gene expression by competing with Gat1p for GATA site binding; function requires a repressive carbon source; dimerizes with Da180p and binds to Tor1p Subunit of the SWI/SNF chromatin remodeling complex, which regulates transcription by remodeling chromosomes; required for transcription of many genes, including ADH1, ADH2, GALI, HO, INC)]. and SUC2




YJR006W

YJR136C

YKL201C

YKR039W

YKR055W

YLL029W

YLL036C

YLL043W

YLRO09W

YLR369W

YLR438C-A

POL31

TTI2

MNN4

GAP1

RHO4

FRA I

PRP19

FPS1

RLP24

SSQI

LSM7

DNA polymerase III (delta) sub-unit, essential for cell viability; in-volved in DNA replication and DNA repair Putative protein of unknown func-tion; subunit of the ASTRA com-plex which is part of the chromatin remodeling machinery; similar to S. pombe Tti2p; may interact with Rsm23p; GFP-fusion protein local-izes to the cytoplasm Putative positive regulator of mannosylphosphate transferase (Mnn6p), involved in manno- sylphosphorylation of N-linked oligosaccharides; expression increases in late-logarithmic and stationary growth phases General amino acid permease; lo-calization to the plasma membrane is regulated by nitrogen source Non-essential small GTPase of the Rho/Rac subfamily of Ras-like pro-teins, likely to be involved in the es-tablishment of cell polarity Protein involved in negative regula-tion of transcription of iron regulon; forms an iron independent complex with Fra2p, Grx3p, and Grx4p; cy-tosolic; mutant fails to repress tran-scription of iron regulon and is de-fective in spore formation Splicing factor associated with the spliceosome; contains a U-box, a motif found in a class of ubiquitin ligases Plasma membrane channel, mem-ber of major intrinsic protein (MIP) family; involved in efflux of glycerol and in uptake of acetic acid and the trivalent metalloids arsen-ite and antimonite; phosphorylated by Hoglp MAPK under acetate stress Essential protein with similarity to Rp124Ap and Rp12413p, associated with pre-60S ribosomal subunits and required for ribosomal large subunit biogenesis Mitochondrial hsp70-type molecu-lar chaperone, required for assem-bly of iron/sulfur clusters into pro-teins at a step after cluster synthe-sis, and for maturation of Yfhlp, which is a homolog of human frataxin implicated in Friedreich's ataxia Lsm (Like Sm) protein; part of het-eroheptameric complexes (Lsm2p-7p and either Lsmlp or Sp): cyto-plasmic Lsmlp complex involved in mRNA decay; nuclear Lsm8p com-plex part of U6 snRNP and pos-sibly involved in processing tRNA, snoRNA, and rRNA




YLR449W

YML041C

YML120C

YMR291W

YNL099C

YNR003C

YNR004W

YOL022C

YOR006C

YOR104W

YORHOW

YOR160W

FP./1.4

VPS71

ND//

OCA I

RPC34

PIN2

TFC7

A I TR 10

Peptidyl-prolyl cis-trans isomerase (PPIase) (proline isomerase) local-ized to the nucleus; catalyzes iso-merization of proline residues in hi-stones H3 and H4, which affects ly-sine methylation of those histones Nucleosome-binding component of the SWR1 complex, which exchanges histone variant H2AZ (Htzlp) for chromatin-bound histone H2A; required for vacuolar protein sorting NADHaMiquinone oxidoreductase, transfers electrons from NADH to ubiquinone in the respiratory chain but does not pump protons, in contrast to the higher eukaryotic multisubunit respiratory complex I; phosphorylated; homolog of human AMID Putative kinase of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm and nucleus; YMR291W is not an essential gene Putative protein tyrosine phosphatase, required for cell cycle ar-rest in response to oxidative dam-age of DNA RNA polymerase III subunit C34; interacts with TFIIIB70 and is a key determinant in pol III recruit-ment by the preinitiation complex Putative protein of unknown function; haploid disruptant exhibits cold-sensitive growth and elongated buds Cytoplasmic protein of unknown function; essential gene in S288C background, while tsr4 null muta-tions in a CEN.PK2 background confer a reduced growth rate Putative protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to both the cytoplasm and the nucleus Protein that induces appearance of [PIN-I-] prion when overproduced; predicted to be palmitoylated One of six subunits of the RNA polymerase III transcription initia-tion factor complex (TFIIIC); part of the TauA globular domain of TFIIIC that binds DNA at the BoxA promoter sites of tRNA and similar genes Nuclear import receptor, mediates the nuclear localization of proteins involved in mRNA-nucleus export; promotes dissociation of mRNAs from the nucleus-cytoplasm mRNA shuttling protein Npl3p




YOR247W SRL1 Mannoprotein that exhibits a tight association with the cell wall, required for cell wall stability in the absence of GPI-anchored mannoproteins; has a high serine-threonine content; expression is in-duced in cell wall mutants


Table D.23: Profile of induced transcripts conserved between S. cerevisiae sten (O'Rourke & Herskowitz 0.5M KC1, 10 minutes) and C. glabrata after exposure to hyperosmotic stress. Key to abbreviations: CG= Candida glabrata open reading frame

ID; SC=Saccharomyces cerevisiae open reading frame ID. All descriptions are inferred by

sequence homology between C. glabrata and S. cerevisiae open reading frames. S. cerevisiae data were obtained from a study by O'Rourke & Herskowitz (518) in which S. cerevisiae cells were treated with 0.5M KC1.

CC ORF SC ORF Gene name Description WT V1 V2 V3 V4 V5


5.55

CAGL0CO2321g YER037W PHM8 Protein of unknown function, expression is induced by low phosphate levels and by inactivation of Pho85p

5.41

CAGLOG04433g YJL108C PRM10 Pheromone-regulated protein, predicted to have 5 transmem- brave segments; induced by treatment with 8-methoxypsoralen and UVA irradiation

5.14

CAGLOM06963g YNR034W SOLI Protein with a possible role in tRNA export; shows similarity to 6-phosphogluconolactonase non-catalytic domains but does not exhibit this enzymatic activity; homologous to Sol2p, Sol3p, and Sol4p

4.31

CAGLOM08822g YDR258C HSP78 Oligomeric mitochondrial matrix chaperone that cooperates with Ssclp in mitochondrial thermotolerance after heat shock; able to prevent the aggregation of misfolded proteins as well as resolubilize protein aggregates

3.93


3.82

CAGLOG03883g YLL026W HSP/04 Heat shock protein that cooperates with Ydjlp (Hsp40) and 3.61 Ssalp (Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses including: heat, ethanol, and sodium arsenite; involved in [PSI-H propagation

CAGLOF08745g YLR327C TMAIO Protein of unknown function that associates with ribosomes 3.53 CAGLOH08173g YOR028C CIN5 Basic leucine zipper (bZIP) transcription factor of the yAP-1

family, mediates pleiotropic drug resistance and salt tolerance; nuclearly localized under oxidative stress and sequestered in the cytoplasm by Lot6p under reducing conditions

3.49


3.47

CAGLOM12474g YIL055C - Description not mapped 3.46 CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein, expression is increased in re-


3.06




CAGLOF09163g YER182W FM -PIO Putative protein of unknown function; the authentic, non- tagged protein is detected in highly purified mitochondria in high-throughput studies

2.74

CAGLOL07480g YBRO66C NRG2 Transcriptional repressor that mediates glucose repression and negatively regulates filamentous growth; has similarity to

2.64

Nrglp CAGLOL00803g YER054C GIP2 Putative regulatory subunit of the protein phosphatase Glc7p,

involved in glycogen metabolism; contains a conserved motif 2.62

(GVNK motif) that is also found in Gac1p, Pig1p, and Pig2p continued on next page



CAGLOK12034g YDR040C ENA1 P-type ATPase sodium pump, involved in Na+ and Li+ efflux to allow salt tolerance

2.61

CAGLOI01100g YOR12OW GCYI Putative NADP(+) coupled glycerol dehydrogenase, proposed to be involved in an alternative pathway for glycerol catabolism; member of the aldo-keto reductase (AKR) family

2.56

CAGLOH05137g YPLO61W ALD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes NADP+ as the preferred coenzyme; required for conver-sion of acetaldehyde to acetate; constitutively expressed; lo-cates to the mitochondrial outer surface upon oxidative stress

2.53

CAGLOM06347g YBR183W YPC/ Alkaline ceramidase that also has reverse (CoA-independent) ceramide synthase activity, catalyzes both breakdown and syn-thesis of phytoceramide; overexpression confers fumonisin B1 resistance

2.52

CAGLOJ04466g YLR414C - - 2.5 CAGLOL10186g YOR052C - Description not mapped 2.45 CAGLOB06424g YKL150W - Mitochondrial NADH-cytochrome b5 reductase, involved in er-


CAGLOJ04158g YOR22OW RCN2 Protein of unknown function; green fluorescent protein (GFP)- fusion protein localizes to the cytoplasm and is induced in re-sponse to the DNA-damaging agent MMS; phosphorylated in response to alpha factor

2.35


2.34

CAGLOM06545g YKL124W SSH4 Specificity factor required for Rsp5p-dependent ubiquitination and sorting of cargo proteins at the multivesicular body; iden-tified as a high-copy suppressor of a SHR3 deletion, increasing steady-state levels of amino acid permeases

2.24

CAGLOL10912g YOR273C TPO4 Polyamine transport protein, recognizes spermine, putrescine, and spermidine; localizes to the plasma membrane; member of the major facilitator superfamily

2.12

CAGLOFO1111g YOL032W 01'110 Protein with a possible role in phospholipid biosynthesis, based on inositol-excreting phenotype of the null mutant and its sup-pression by exogenous choline

2.04

274

Appendix E


411101•1•11100., Atu203 t130 aRNA2 CCrIl A1u293 IC a A

03- (Afu29330 - Afu293.t60) + totRNA

Appendix E. Chapter 4 Supplementary Data 275

31- (Aftt293.t0 - Afu293.t60) + ARNA1

c..ortrast, 32 - (Afu293.t0 - Afu293.t60) aRNA2

co,trast2

contrast,- aRNA2 aRNA1

contrast2- totRNA - aRNA2

contrast3- totRNA - aRNA1

Figure E.1: Schematic representation of hybridisations performed in the ampli-fication control study. Boxes represent the tissue from which the RNA was isolated and used as a template for reverse transcription and concomitant fluorophore labelling. The open and shaded arrows demonstrate that samples were dye-swapped to account for preferential fluorophore labelling in some transcripts. In all cases, labelled cDNA from A. fumigatus strain 237 at time = 0 minutes (Afu237.To ) was co-hybridised with equivalent material derived from the RNA isolated from A. fumigatus strain 237 at time = 60 minutes (Afu237.t60). The top row of boxes shows co-hybridisation of cDNA derived from RNA sam-ples that had been diluted and subjected to 1 round of mRNA amplification prior to reverse transcription and fluorophore labelling. The second row of boxes shows co-hybridisation of samples that had been diluted and subjected to 2 rounds of mRNA amplification, and the third row of boxes shows co-hybridisation of samples that did not undergo mRNA amplifi-cation prior to reverse transcription. We investigated the relative abundance of transcripts at To minutes and T60 minutes, and used a linear regression framework to identify am-plification protocols that provided evidence for differences in those relative abundances, flagging those genes that showed significant difference in relative abundance after linear amplification. The treatment effects (i3i , /32 and 33) and contrasts (contrasts , contrast2 and contrast3 ) estimated in the linear model are also depicted.


-3.0

0.0

3.0

4 t1 C

U 0

4 4 m htu2/13110: cytochresee c kfu4/01110: 14-alpha sterol deeethylthe C1)151 "hfu4r0/4110, coproporphythnopes III oridase, putative" 'hfu4q15140: nitochondrial phosphate carrier protein (Kiel), MO/0740 li•olpha sterol &Aoki'''. Cip5111 Ifu11105600: conserved hypothetical protein kfuSgOt230: citrate synthase CitA "Aft101120: fatty acid hydroxylue, putative" 1E0603400' eor.served hnethetieal protein "Afulr110/0 pyruvite decarboxylase Pdck, putative" 'Mu400100 :hoe! chain dehydrogenase Oast), putative" "AN201100: wane acid manse (Dip , putative" Afudg10720: alpha-ketollutazate-dependent twine &ionic's.: ifuSg13060: 001275 dada protein "Afu1g02310: actin cortical patch, protein. Sue, putative hfuSg01220, conserved hypothetical protein Aing142$0 flavia-hnding aoloorypenne-like protein "Afu3g0940: 2,3-diketo-S•aethylthio-1-phosphopentone phosphatase, putat: IftelgOOda cell surface pnteir., putative" hfulq16440 hypothetical protein Afulg01690 conserved hypothetical protein "If:409110. suralefferredosin•like (oily protein Apdl, putative" kfu1g00350• conserved hypothetical protein "Afu301010: pyravate decirboxylase Pdch, ?native "Afulg064/0 . hest :hock protein !I:pH-like, putative "Aft:40116D: tartrate dehydrogenate, putative' Afu3g10310 conserved hypothetical protein "AfulgISPSO Elwin-binding aotooxygerase, putative "Aftag09330 tipe-like antibiotic response protein, putative AftrtgOPIPO S-netosyliaetbiorine-sterol-C- sethyltreateferae 'Aft:401140. VS Raltidrag tresporter, putative' AfuSg01820: estratellulas ?reline-rich protein 'Aft13,09890: extractllalu thmattin Wain protein, putative' 1(001960: VI anchored cell rill protein, putative "1:n902700: KV antidrug transporter, putative' "X614900240 . eine-conturing alcohol &hydrogens, putative' Afir4q13510 isocitrate irate AtuD hfu4g00150 conserved hypothetical protein 'A6502100 VS aultietral transporter, putative' Afu1g13310 isocitrite lyase kcu "Afseg01000. potusiwattivated aldehyde dehydrogenase 1114 putative" hfaSg011130 conserved hypothetical protein `Afu3g09110 sucruelierredorin-like fussily protein Apt11, putative' "UuSg01920 GPI anchored protein, putative' Afulg05100. "AfuSg00710 ZIA pessease, putative "kfulg0100 isomayl alcohol oxidant, putative' 'Aft:204200 4.hydeorypher.ylpyrovate dioxygenne, putative "Ain/01240 tint-containing alcohol dedydrogenue, putative' "Aft151012$0 ondontactase, putative' Afu410190 arkyria repeat protein "kfu3I00110 short eltair, dekdraprese rktsel , putative' Afu3g11680 lysophospholipue Pill kfu6111190. conserved hypothetical peoteta A10;0161 bifunctional eatalase-perexidue Catl 'AfuSg03330 ktyph-like protein, putative' 11003010 phosphite-ripreszille ho/phosphate cotransporter Phe$9, 'hfulq46020 skra folly transcriptional regulator, putative 'At p21313 ferric-chelate eeductase Int), putative 1E15100120 ZAI folly acetyltronsferase, putative Afull14550 Hz superoxide dismutose

Figure E.2: Comparative analysis of A. fumigates gene expression datasets. A pan-experimental comparison of A. furnigatus gene expression comparing log2 ratios ob-tained during host adaptation (mice); exposure to neutrophils(neut), increased expression in parental strain versus AlaeA mutant, acid shift (acid), iron starvation (iron), oxygen depletion (anaer) and oxidative stress (H202) for various genes. The colour bar indi-cates the range of loge expression ratios, grey bars indicate genes from which signals were undetectable. Experimental conditions are described in the materials and methods sec-tion. LaeA dataset take from (539). Comparative analysis performed with TM4 software http://www.jcvi.org/cms/research/software/.


10.00

5.00

0.00

-5.00

-10.00

-15.00

..-Liti Lij 1 I

Figure E.3: Concordance between estimates of relative abundance from triplicate qPCR measurements (yellow) and microarray experiments (grey) for selected A. fumigates ORFs. Oligo sequences are given in Table E.12

6 ' 8

ci : 2 I0o C

l',_

`4'

Iiiiiiiiiiillill

O

a -

1—


Foreground Intensity Boxplots of Cy5 Channel Foreground Intensity Boxplots of Cy3 Channel

1 2

3 4 5 8 8 4 5 7 8

Slide tilde

(a)

(b)

Background Intensity Boxplols of Cy5 Channel Background Intensity Boxplots of Cy3 Channel

8 _ -

8

a a a

a 82 3 a I I

4441-1-11.1: 8

O -

1 2 3 4 5 6 7 8

Side

(C)

I III I

1 2 3 4 5 5 7

Sbde

(d)

Figure E.4: Box and whisker plots of Cy5TM and Cyr intensities for slides hy-bridized with amplified and non-amplified RNA. Foreground intensities are shown for Cy5rm and Cye in a) and b). Background intensities are shown for Cy5TM and Cy3

TM

in c) and d). Slides 1-6 (blue) represent slides that were hybridized with cDNA derived from amplified RNA. Slides 7-8 (orange) represent slides that were hybridized with cDNA derived from unamplified RNA.


a) To

a

difference

(a)

aRNA 2 v totRNA

R. 0.75

log2

ratio

totR

NA

a?

-5

5

10

log2 ratio aRNA 2

(b)

Figure E.5: Genes rejected in the amplification control experiment transposed onto correlation plots. Plot of the difference in log2 ratio between the amplified (aRNA2) and the non-amplified material (totRNA) for the loge ratio for the two samples vs. the p-value for each of the retained genes a). Correlation of log2 ratios between non-amplified and amplified material. The genes in the "conservative set" are coloured gold, the genes in the "rejected set" are coloured black and the genes in the "undetermined set" are coloured grey b). Sets are defined in the materials and methods and follow criteria originally described in (509).


Table E.1: RNA Samples and associated amplification factors. Key to abbrevia-

tions: ID = sample identifier; tRNA = total RNA, mRNA = messenger RNA; aRNAi = RNA after 1 round of amplification; aRNA2 = RNA after 2 rounds of amplification; AF = amplification factor.

ID Strain tRNA ng mRNA ng aRNA1 mg aRNA2 tig AF To Overnight broth

culture 1000 20 25.95 184.76 9.2 X103

T60 Overnight broth plus media shift for 60 minutes

1000 20 87.33 174.60 8.7 X 103

A (in vivo)1 323 13 11.08 251.14 2.0 x 104 B (in vivo)2 130 3 6.25 164.02 6.3 X 104 C (in vivo)3 240 10 9.02 172.63 1.8 x 104 D (in vivo)4 800 64 19.27 177.11 2.8 x 103 E (in vivo)5 110 2 12.27 258.39 1.2 X 105 F (in vitro) 1136 23 3.72 186.96 8.2 X 103 G (in vitro) 1000 20 56.00 566.00 2.8 X 104


Table E.2: Filtering per array in the amplification study. Filtering criterion A refers

to spots flagged by the TIGR spotfinder software. Filtering criterion B excludes spots whose

foreground intensity is less than twice its background intensity in either the Cy5Th or Cy3TM

channel

Array Number 1 2 3 4 5 6 Criterion A 1895 2080 1867 3630 8079 7151

6.33% 6.94% 6.23% 12.11% 26.97% 23.90% Criterion B 2147 2008 1687 1827 6584 6197

7.17% 6.70% 5.63% 6.10% 21.98% 20.69% Sum 4042 4088 3554 5457 14663 13348

13.49% 13.65% 11.87% 18.22% 48.95% 44.56%

Table Found at http : //ukpmc . ac .uk/picrender . cgi?artid=16630688thlobnarne=ppat

1000154.s006.doc


Table E.3: Over-represented Gene Ontology (GO) terms amongst genes having increased transcript abundance.

GO ID GO Term List hits List size Pop hits Pop

GO:0019538 protein metabolism 174 561 781 4

GO:0044267 cellular protein metabolism 159 561 695 4

G0:0044260 cellular macromolecule metabolism 168 561 775 4

G0:0043170 macromolecule metabolism 254 561 1383 4

GO:0042254 ribosome biogenesis and assembly 51 561 161 4

GO:0007028 cytoplasm organization and biogenesis 51 561 161 4

GO:0044237 cellular metabolism 366 561 2251 4

GO:0007046 ribosome biogenesis 46 561 140 4

GO:0016072 rRNA metabolism 41 561 118 4

GO:0006364 rRNA processing 40 561 115 4

GO:0044238 primary metabolism 334 561 2028 4

GO:0006457 protein folding 23 561 50 4

00:0006412 protein biosynthesis 72 561 302 4

GO:0008152 metabolism 394 561 2570 4

GO:0043037 translation 30 561 96 4

00:0016070 RNA metabolism 58 561 247 4

G0:0009059 macromolecule biosynthesis 74 561 346 4

GO:0006365 35S primary transcript processing 18 561 46 4

GO:0006413 translational initiation 17 561 42 4

166 RNA processing 43 561 175 4

GO:0030490 processing of 20S pre-rRNA 15 561 37 4

G0:0006996 organelle organization and biogenesis 100 561 522 4

GO:0009058 biosynthesis 123 561 696 4 mitochondria] electron transport, ubiquinol to cytochrome c 5 561 6 4

GO:0001302 replicative cell aging 6 561 9 4

GO:0000027 ribosomal large subunit assembly and maintenance 11 561 28 4

00:0007569 cell aging 6 561 11 4

GO:0006359 regulation of transcription from RNA polymerase III promoter 4 561 5 4

GO:0006360 transcription from RNA polymerase I promoter 7 561 15 4

G0:0009987 cellular process 461 561 3265 4

G0:0043283 biopolymer metabolism 151 561 930 4

00:0042257 ribosomal subunit assembly 12 561 37 4

GO:0000128 flocculation 3 561 3 4

GO:0006356 regulation of transcription from RNA polymerase I promoter 3 561 3 4

GO:0044249 cellular biosynthesis 105 561 616 4

GO:0042255 ribosome assembly 13 561 43 4

G0:0043412 biopolymer modification 73 561 406 4

00:0050875 cellular physiological process 454 561 3222 4

GO:0007568 aging 6 561 13 4

G0:0006783 heme biosynthesis 6 561 13 4

00:0042168 hems metabolism 6 561 13 4

GO:0000154 rRNA modification 6 561 13 4

GO:0006628 mitochondrial translocation 7 561 17 4

GO:0006508 proteolysis 27 561 125 4

00:0006778 porphyrin metabolism 6 561 14 4

GO:0006779 porphyrin biosynthesis 6 561 14 4

GO:0006626 protein targeting to mitochondrion 6 561 14 4

GO:0006461 protein complex assembly 19 561 79 4

G0:0016337 cell-cell adhesion 4 561 7 4 00:0009060 aerobic respiration 13 561 48 4 GO:0045333 cellular respiration 13 561 48 4 GO:0016043 cell organization and biogenesis 135 561 849 4

GO:0006696 ergosterol biosynthesis 11 561 38 9

GO:0008204 ergosterol metabolism 11 561 38 4

GO:0007155 cell adhesion 5 561 11 4

G0:0042273 ribosomal large subunit biogenesis 5 561 11 4

G0:0018193 peptidyl-amino acid modification 5 561 11 4

G0:0006091 generation of precursor metabolites and energy 32 561 161 4

conti


continued from previous page GO ID GO Term List hits List size Pop hit:

GO:0006464 protein modification 64 561 372 mitochondrial electron transport, succinate to ubiquinone 2 561 2

GO:0042126 nitrate metabolism 2 561 2 GO:0042128 nitrate assimilation 2 561 2 GO:0044257 cellular protein catabolism 20 561 92 GO:0046148 pigment biosynthesis 7 561 22 GO:0042440 pigment metabolism 7 561 22 GO:0009451 RNA modification 9 561 32 GO:0051603 proteolysis during cellular protein catabolism 19 561 SE GO:0016126 sterol biosynthesis 11 561 42 GO:0015980 energy derivation by oxidation of organic compounds 24 561 12C GO:0006383 transcription from RNA polymerase III promoter 7 561 24 GO:0019941 modification-dependent protein catabolism 18 561 87 GO:0043632 modification-dependent macromolecule catabolism 18 561 87 GO:0000002 mitochondria] genome maintenance 5 561 15 GO:0000283 establishment of cell polarity (sensu Saccharomyces) 5 561 15 GO:0016125 sterol metabolism 11 561 47 GO:0006139 nucleobase, nucleoside, nucleotide and nucleic acid metabolism 127 561 835 GO:0006694 steroid biosynthesis 11 561 45 GO:0030163 protein catabolism 21 561 105 GO:0042798 protein neddylation during NEDD8 class-dependent protein catabolism 2 561 2 GO:0045041 protein import into mitochondrial intermembrane space 2 561 G0:0018195 peptidyl-arginine modification 2 561 00:0019942 NEDD8 class-dependent protein catabolism 2 561 GO:0006534 cysteine metabolism 2 561 GO:0016574 histone ubiquitination 2 561 GO:0006549 isoleucine metabolism 2 561 2 GO:0006839 mitochondrial transport 6 561 21 GO:0006512 ubiquitin cycle 22 561 119 GO:0006511 ubiquitin-dependent protein catabolism 17 561 85 GO:0043285 biopolymer catabolism 31 561 177 137 tRNA aminoacylation 6 561 22 G0:0006418 tRNA aminoacylation for protein translation 6 561 22 GO:0006403 RNA localization 8 561 32 G0:0046112 nucleobase biosynthesis 4 561 12 00:0006399 tRNA metabolism 11 561 52 GO:0008202 steroid metabolism 11 561 52 GO:0044265 cellular macromolecule catabolism 31 561 181 00:0019856 pyrimidine base biosynthesis 3 561 8 GO:0006743 ubiquinone metabolism 3 561 E GO:0016571 historic methylation 3 561 5 GO:0043414 biopolymer methylation 5 561 15 00:0032259 methylation 5 561 15 GO:0006090 pyruvate metabolism 7 561 25 00:0006730 one-carbon compound metabolism 7 561 25 G0:0008151 cell growth and/or maintenance 4 561 13 G0:0015931 nucleobase, nucleoside, nucleotide and nucleic acid transport 8 561 35 00:0007005 mitochondrion organization and biogenesis 9 561 41 GO:0006080 mamma metabolism 2 561 4 G0:0006097 glyoxylate cycle 2 561 4 G0:0016074 snoRNA metabolism 2 561 4 GO:0006835 dicarboxylic acid transport 2 561 4 G0:0096487 glyoxylate metabolism 2 561 9

deadenylation-dependent decapping 2 561 4 Molecular Function 0 0 0 C G0:0003754 chaperone activity 17 568 35 GO:0003743 translation initiation factor activity 16 568 34 G0:0008135 translation factor activity, nucleic acid binding 20 568 5C GO:0045182 translation regulator activity 21 568 55 G0:0016627 oxidoreductase activity, acting on the CH-CH group of donors 11 568 19 00:0030515 snoRNA binding 11 568 21 GO:0003723 RNA binding 27 568 10C


continued from previous page GO ID

GO Term

GO:0003735 GO:0003724 GO:0016628 oxidoreductase activity,

GO:0004680 GO:0004004 GO:0008186 GO:0004682 GO:0016274 GO:0016273 GO:0004176 GO:0008026 GO:0016876 GO:0016875 GO:0004812 GO:0003773 GO:0008369 GO:0004175 GO:0004386 GO:0008565 GO:0004691 GO:0004690 GO:0008398 GO:0008177 GO:0008097 GO:0004739 GO:0016635 oxidoreductase activity, acting on the

GO:0000246 GO:0004827 GO:0000339 GO:0004738 GO:0008121 GO:0016681 oxidoreductase activity, acting on c

GO :0003925 GO:0016679 oxidoreduc

GO:0015078 G 0:0004722 GO :0016705 oxidoreductase activity, acting on I

GO :0008170 GO :0003676 GO :0005198 GO:0016709 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAI

GO:0008233 GO :0008757 GO :0016251 GO:0019202 GO:0000248 GO:0016661 oxidore

GO:0000171 GO:0047956 GO:0005294 GO:0000102 GO:0019206 GO:0004526 GO:0000049 GO:0016624 oxidoreductase activity, a

GO:0016741 GO:0004674 GO:0016455 GO:0015077 GO:0003674 GO:0008168 GO:0042054


continued from previous page GO ID GO Term List hits List size Pop hits F

GO:0019205 nucleobase, nucleoside, nucleotide kinase activity 3 568 8 GO:0016491 oxidoreductase activity 68 568 441 G0:0016215 CoA desaturase activity 2 568 4 GO:0015191 L-methionine transporter activity 2 568 4 00:0004768 stearoyl-CoA 9-desaturase activity 2 568 4 G0:0030276 clathrin binding 2 568 4 GO:0005310 dicarboxylic acid transporter activity 2 568 4 GO:0004128 cytochrome-b5 reductase activity 2 568 4 60:0004721 phosphoprotein phosphatase activity 7 568 30 GO:0016830 carbon-carbon lyase activity 8 568 36 GO:0004497 monooxygenase activity 6 568 25 Cellular component 0 0 0 0 GO:0044429 mitochondria! part 65 526 222 GO:0031974 membrane-enclosed lumen 96 526 395 G0:0043233 organelle lumen 96 526 395 G0:0043234 protein complex 149 526 711 GO:0005730 nucleolus 40 526 130 GO:0005739 mitochondrion 95 526 426 GO:0030529 ribonucleoprotein complex 62 526 243 GO:0043228 non-membrane-bound organelle 96 526 442 GO:0043232 intracellular non-membrane-bound organelle 96 526 442 GO:0005759 mitochondrial matrix 31 526 93 GO:0031980 mitochondrial lumen 31 526 93 G0:0005852 eukaryotic translation initiation factor 3 complex 9 526 12 GO:0031966 mitochondria! membrane 31 526 97 GO:0044455 mitochondrial membrane part 13 526 24 GO:0005622 intracellular 441 526 2891 GO:0044424 intracellular part 438 526 2868 GO:0044452 nucleolar part 17 526 39 GO:0005761 mitochondria! ribosome 17 526 41 G0:0005840 ribosome 40 526 151 00:0000313 organellar ribosome 17 526 43 GO:0005746 mitochondrial electron transport chain 8 526 12 GO:0005743 mitochondrial inner membrane 25 526 80 GO:0019866 organelle inner membrane 25 526 82 GO:0031981 nuclear lumen 60 526 270 GO:0005740 mitochondrial envelope 34 526 129 GO:0005732 small nucleolar ribonucleoprotein complex 10 526 23 GO:0016282 eukaryotic 43S preinitiation complex 15 526 47 GO:0005844 polysome 4 526 5 GO:0045285 ubiquinol-cytochrome-c reductase complex 4 526 5 00:0005750 respiratory chain complex III (sensu Eukaryota) 4 526 5 G0:0045275 respiratory chain complex III 4 526 5

proteasome regulatory particle, lid subcomplex (sensu Eukaryota) 5 526 8 G0:0031967 organelle envelope 37 526 167 GO:0031975 envelope 37 526 167 00:0005956 protein kinase CK2 complex 3 526 3 GO:0005762 mitochondrial large ribosomal subunit 9 526 24 GO:0044422 organelle part 177 526 1086 GO:0044446 intracellular organelle part 177 526 1086 GO:0000315 organellar large ribosomal subunit 9 526 25 GO:0005737 cytoplasm 306 526 2010 GO:0005763 mitochondrial small ribosomal subunit 6 526 14 G0:0043229 intracellular organelle 335 526 2228 GO:0043226 organelle 335 526 2228 G0:0045254 pyruvate dehydrogenase complex 3 526 4 GO:0000314 organellar small ribosomal subunit 6 526 15 GO:0015934 large ribosomal subunit 16 526 65 GO:0005845 mRNA cap complex 2 526 2 GO:0005749 respiratory chain complex II (sensu Eukaryota) 2 526 2 GO:0045283 fumarate reductase complex 2 526 2 GO:0045273 respiratory chain complex II 2 526 2

Co


continued from previous pare GO ID GO Term List hits List size Pop hits

GO:0045257 succinate dehydrogenase complex (ubiquinone) 2 526 2

GO:0045281 succinate dehydrogenase complex 2 526 2 GO:0044464 cell part 490 526 3436

GO:0005742 mitochondrial outer membrane translocase complex 3 526 5

GO:0005741 mitochondrial outer membrane 6 526 17 GO:0044444 cytoplasmic part 194 526 1254 GO:0031968 organelle outer membrane 6 526 18

00:0019867 outer membrane 6 526 18

GO:0005623 cell 490 526 3444 GO:0015935 small ribosomal subunit 12 526 50

proteasome core complex, beta-subunit complex (sensu Eukaryota) 3 526 6

GO:0030663 COPI coated vesicle membrane 3 526 6 GO:0030126 COPI vesicle coat 3 526 6 00:0005736 DNA-directed RNA polymerase I complex 4 526 10

00:0000502 proteasome complex (sensu Eukaryota) 8 526 29 G0:0030677 ribonuclease P complex 2 526 3

GO:0000172 ribonuclease MRP complex 2 526 3 proteasome regulatory particle, base subcomplex (sensu Eukaryota) 2 526 3

GO:0005655 nucleolar ribonuclease P complex 2 526 3 GO:0012506 vesicle membrane 6 526 22

GO:0030120 vesicle coat 6 526 22 00:0030659 cytoplasmic vesicle membrane 6 526 22 GO:0030662 coated vesicle membrane 6 526 22 GO:0043231 intracellular membrane-bound organelle 301 526 2061 GO:0043227 membrane-bound organelle 301 526 2061 GO:0031090 organelle membrane 51 526 307

G0:0005839 proteasome core complex (sensu Eukaryota) 4 526 13 GO:0019897 extrinsic to plasma membrane 4 526 13 G0:0012510 trans-Golgi network transport vesicle membrane 2 526 4

GO:0030130 clathrin coat of trans-Golgi network vesicle 2 526 4 GO:0030121 AP-1 adaptor complex 2 526 4


Table E.4: Over-represented Gene Ontology (GO) terms amongst genes having repressed transcript abundance.

GO ID GO Term List hits List size Pop hits Pop size FE GO:0019538 protein metabolism 174 561 781 4219 < 0.05 GO:0044267 cellular protein metabolism 159 561 695 4219 < 0.05 GO:0044260 cellular macromolecule metabolism 168 561 775 4219 < 0.05 GO:0043170 macromolecule metabolism 254 561 1383 4219 < 0.05 GO:0042254 ribosome biogenesis and assembly 51 561 161 4219 < 0.05 GO:0007028 cytoplasm organization and biogen-

esis 51 561 161 4219 < 0.05

GO:0044237 cellular metabolism 366 561 2251 4219 < 0.05 00:0007046 ribosome biogenesis 46 561 140 4219 < 0.05 GO:0016072 rRNA metabolism 41 561 118 4219 < 0.05 GO:0006364 rRNA processing 40 561 115 4219 < 0.05 GO:0044238 primary metabolism 334 561 2028 4219 < 0.05 GO:0006457 protein folding 23 561 50 4219 < 0.05 GO:0006412 protein biosynthesis 72 561 302 4219 < 0.05 00:0008152 metabolism 394 561 2570 4219 < 0.05 GO:0043037 translation 30 561 96 4219 < 0.05 GO:0016070 RNA metabolism 58 561 247 4219 < 0.05 00:0009059 macromolecule biosynthesis 74 561 346 4219 < 0.05 GO:0006365 35S primary transcript processing 18 561 46 4219 < 0.05 GO:0006413 translational initiation 17 561 42 4219 < 0.05

166 RNA processing 43 561 175 4219 < 0.05 60:0030490 processing of 20S pre-rRNA 15 561 37 4219 < 0.05 G0:0006996 organelle organization and biogene-

sis 100 561 522 4219 < 0.05

GO:0009058 biosynthesis 123 561 696 4219 < 0.05 mitochondrial electron transport, ubiquinol to cytochrome c

5 561 6 4219 < 0.05

00:0001302 replicative cell aging 6 561 9 4219 < 0.05 00:0000027 ribosomal large subunit assembly

and maintenance 11 561 28 4219 < 0.05

GO:0007569 cell aging 6 561 11 4219 < 0.05 GO:0006359 regulation of transcription from 4 561 5 4219 < 0.05

RNA polymerase III promoter G0:0006360 transcription from RNA polymerase 7 561 15 4219 < 0.05

I promoter GO:0009987 cellular process 461 561 3265 4219 < 0.05 GO:0043283 biopolymer metabolism 151 561 930 4219 < 0.05 GO:0042257 ribosomal subunit assembly 12 561 37 4219 < 0.05 GO:0000128 flocculation 3 561 3 4219 < 0.05 GO:0006356 regulation of transcription from 3 561 3 4219 < 0.05

RNA polymerase I promoter GO:0044249 cellular biosynthesis 105 561 616 4219 < 0.05 GO:0042255 ribosome assembly 13 561 43 4219 < 0.05 GO:0043412 biopolymer modification 73 561 406 4219 < 0.05 GO:0050875 cellular physiological process 454 561 3222 4219 < 0.05 GO:0007568 aging 6 561 13 4219 < 0.05 GO:0006783 heme biosynthesis 6 561 13 4219 < 0.05 GO:0042168 heme metabolism 6 561 13 4219 < 0.05 GO:0000154 rRNA modification 6 561 13 4219 < 0.05 60:0006628 mitochondrial translocation 7 561 17 4219 < 0.05 GO:0006508 proteolysis 27 561 125 4219 < 0.05 GO:0006778 porphyrin metabolism 6 561 14 4219 < 0.05 GO:0006779 porphyrin biosynthesis 6 561 14 4219 < 0.05 GO:0006626 protein targeting to mitochondrion 6 561 14 4219 < 0.05 GO:0006461 protein complex assembly 19 561 79 4219 < 0.05 GO:0016337 cell-cell adhesion 4 561 7 4219 < 0.05 GO:0009060 aerobic respiration 13 561 48 4219 < 0.05 00:0045333 cellular respiration 13 561 48 4219 < 0.05 00:0016043 cell organization and biogenesis 135 561 849 4219 < 0.05



continued from previous page GO ID GO Term List hits List size Pop hits Pop size FE

G0:0006696 ergosterol biosynthesis 11 561 38 4219 < 0.05 GO:0008204 ergosterol metabolism 11 561 38 4219 < 0.05 00:0007155 cell adhesion 5 561 11 4219 < 0.05 GO:0042273 ribosomal large subunit biogenesis 5 561 11 4219 < 0.05 00:0018193 peptidyl-amino acid modification 5 561 11 4219 < 0.05 GO:0006091 generation of precursor metabolites

and energy 32 561 161 4219 < 0.05

GO:0006464 protein modification 64 561 373 4219 < 0.05 mitochondrial electron transport, succinate to ubiquinone

2 561 2 4219 < 0.05

G0:0042126 nitrate metabolism 2 561 2 4219 < 0.05 00:0042128 nitrate assimilation 2 561 2 4219 < 0.05 00:0044257 cellular protein catabolism 20 561 93 4219 < 0.05 GO:0046148 pigment biosynthesis 7 561 22 4219 < 0.05 GO:0042440 pigment metabolism 7 561 22 4219 < 0.05 GO:0009451 RNA modification 9 561 32 4219 < 0.05 GO:0051603 proteolysis during cellular protein

catabolism 19 561 88 4219 < 0.05

GO:0016126 sterol biosynthesis 11 561 43 4219 < 0.05 G0:0015980 energy derivation by oxidation of

organic compounds 24 561 120 4219 < 0.05

GO:0006383 transcription from RNA polymerase 7 561 24 4219 < 0.05 III promoter

GO:0019941 modification-dependent protein catabolism

18 561 87 4219 < 0.05

GO:0043632 modification-dependent macromolecule catabolism

18 561 87 4219 < 0.05

GO:0000002 mitochondrial genome maintenance 5 561 15 4219 < 0.05 GO:0000283 establishment of cell polarity (sensu 5 561 15 4219 < 0.05

Saccharomyces) G0:0016125 sterol metabolism 11 561 47 4219 < 0.05 GO:0006139 nucleobase, nucleoside, nucleotide

and nucleic acid metabolism 127 561 839 4219 < 0.05

G0:0006694 steroid biosynthesis 11 561 48 4219 < 0.05 G0:0030163 protein catabolism 21 561 109 4219 < 0.05 GO:0042798 protein neddylation during NEDD8

class-dependent protein catabolism 2 561 3 4219 < 0.05

GO:0045041 protein import into mitochondrial intermembrane space

2 561 3 4219 < 0.05

G0:0018195 peptidyl-arginine modification 2 561 3 4219 < 0.05 GO:0019942 NEDD8 class-dependent protein

catabolism 2 561 3 4219 < 0.05

GO:0006534 cysteine metabolism 2 561 3 4219 < 0.05 00:0016574 histone ubiquitination 2 561 3 4219 < 0.05 GO:0006549 isoleucine metabolism 2 561 3 4219 < 0.05 G0:0006839 mitochondrial transport 6 561 21 4219 0.0504 GO:0006512 ubiquitin cycle 22 561 116 4219 0.0509 GO:0006511 ubiquitin-dependent protein

catabolism 17 561 85 4219 0.0523

00:0043285 biopolymer catabolism 31 561 177 4219 0.0614 137 tRNA aminoacylation 6 561 22 4219 0.0618

GO:0006418 tRNA aminoacylation for protein translation

6 561 22 4219 0.0618

GO:0006403 RNA localization 8 561 33 4219 0.0625 60:0046112 nucleobase biosynthesis 4 561 12 4219 0.0638 GO:0006399 tRNA metabolism 11 561 52 4219 0.0762 GO:0008202 steroid metabolism 11 561 52 4219 0.0762 GO:0044265 cellular macromolecule catabolism 31 561 181 4219 0.0783 00:0019856 pyrimidine base biosynthesis 3 561 8 4219 0.0783 GO:0006743 ubiquinone metabolism 3 561 8 4219 0.0783 G0:0016571 histone methylation 3 561 8 4219 0.0783 GO:0043414 biopolymer methylation 5 561 18 4219 0.0799 GO:0032259 methylation 5 561 18 4219 0.0799 GO:0006090 pyruvate metabolism 7 561 29 4219 0.0805




GO:0006730 one-carbon compound metabolism 7 561 29 4219 0.0805 GO:0008151 cell growth and/or maintenance 4 561 13 4219 0.0828 00:0015931 nucleobase, nucleoside, nucleotide

and nucleic acid transport 8 561 35 4219 0.0838

00:0007005 mitochondrion organization and biogenesis

9 561 41 4219 0.0853

00:0006080 mannan metabolism 2 561 4 4219 0.0881 GO:0006097 glyoxylate cycle 2 561 4 4219 0.0881 GO:0016074 snoRNA metabolism 2 561 4 4219 0.0881 00:0006835 dicarboxylic acid transport 2 561 4 4219 0.0881 GO:0046487 glyoxylate metabolism 2 561 4 4219 0.0881

deadenylation-dependent decapping

2 561 4 4219 0.0881

Molecular Function 0 0 0 0 0 < 0.05 GO:0003754 chaperone activity 17 568 35 4324 < 0.05 GO:0003743 translation initiation factor activity 16 568 34 4324 < 0.05 G0:0008135 translation factor activity, nucleic

acid binding 20 568 50 4324 < 0.05

GO:0045182 translation regulator activity 21 568 55 4324 < 0.05 00:0016627 oxidoreductase activity, acting on

the CH-CH group of donors 11 568 19 4324 < 0.05

00:0030515 snoRNA binding 11 568 21 4324 < 0.05 GO:0003723 RNA binding 27 568 100 4324 < 0.05 00:0003735 structural constituent of ribosome 28 568 112 4324 < 0.05 G0:0003724 RNA helicase activity 13 568 39 4324 < 0.05 GO:0016628 oxidoreductase activity, acting on

the CH-CH group of donors, NAD or NADP as acceptor

4 568 5 4324 < 0.05

GO:0004680 casein kinase activity 4 568 5 4324 < 0.05 00:0004004 ATP-dependent RNA helicase ac-

tivity 10 568 27 4324 < 0.05

GO:0008186 RNA-dependent ATPase activity 10 568 27 4324 < 0.05 GO:0004682 protein kinase CIC2 activity 3 568 3 4324 < 0.05 GO:0016274 protein-arginine N-

methyltransferase activity 3 568 4 4324 < 0.05

GO:0016273 arginine N-methyltransferase activity

3 568 4 4324 < 0.05

GO:0004176 ATP-dependent peptidase activity 3 568 4 4324 < 0.05 GO:0008026 ATP-dependent helicase activity 11 568 40 4324 < 0.05 GO:0016876 ligase activity, forming aminoacyl-

tRNA and related compounds 10 568 35 4324 < 0.05

00:0016875 ligase activity, forming carbon- oxygen bonds

10 568 35 4324 < 0.05

G0:0004812 aminoacyl-tRNA ligase activity 10 568 35 4324 < 0.05 GO:0003773 heat shock protein activity 4 568 8 4324 < 0.05 GO:0008369 obsolete molecular function 38 568 204 4324 < 0.05 GO:0004175 endopeptidase activity 13 568 52 4324 < 0.05 GO:0004386 helicase activity 16 568 69 4324 < 0.05 60:0008565 protein transporter activity 9 568 31 4324 < 0.05 G0:0004691 cAMP-dependent protein kinase ac-

tivity 2 568 2 4324 < 0.05

G0:0004690 cyclic nucleotide-dependent protein kinase activity

2 568 2 4324 < 0.05

GO:0008398 sterol 14-demethylase activity 2 568 2 4324 < 0.05 GO:0008177 succinate dehydrogenase 2 568 2 4324 < 0.05

(ubiquinone) activity G0:0008097 5S rRNA binding 2 568 2 4324 < 0.05 GO:0004739 pyruvate dehydrogenase (acetyl-

transferring) activity 2 568 2 4324 < 0.05

GO:0016635 oxidoreductase activity, acting on the CH-CH group of donors, quinone or related compound as acceptor

2 568 2 4324 < 0.05




00:0000246 delta24(24-1) sterol reductase activity

2 568 2 4324 < 0.05

GO:0004827 proline-tRNA ligase activity 2 568 2 4324 < 0.05 GO:0000339 RNA cap binding 2 568 2 4324 < 0.05 GO:0004738 pyruvate dehydrogenase activity 2 568 2 4324 < 0.05 GO:0008121 ubiquinol-cytochrome-c reductase

activity 3 568 5 4324 < 0.05

GO:0016681 oxidoreductase activity, acting on diphenols and related substances as donors, cytochrome as acceptor

3 568 5 4324 < 0.05

GO:0003925 small monomeric GTPase activity 3 568 5 4324 < 0.05 GO:0016679 oxidoreductase activity, acting on

diphenols and related substances as donors

3 568 5 4324 < 0.05

GO:0015078 hydrogen ion transporter activity 5 568 13 4324 < 0.05 00:0004722 protein serine/threonine phos-

phatase activity 6 568 18 4324 < 0.05

GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen

6 568 18 4324 < 0.05

GO:0008170 N-rnethyltransferase activity 6 568 18 4324 < 0.05 GO:0003676 nucleic acid binding 73 568 448 4324 < 0.05 00:0005198 structural molecule activity 31 568 168 4324 < 0.05 00:0016709 oxidoreductase activity, acting on

paired donors, with incorporation or reduction of molecular oxygen, NAD or NADH as one donor, and incorporation of one atom of oxy-gen

3 568 6 4324 < 0.05

GO:0008233 peptidase activity 23 568 119 4324 < 0.05 GO:0008757 S-adenosylmethionine-dependent

methyltransferase activity 12 568 53 4324 < 0.05

00:0016251 general RNA polymerase II transcription factor activity

9 568 36 4324 < 0.05

GO:0019202 amino acid kinase activity 2 568 3 4324 < 0.05 GO:0000248 C-5 sterol desaturase activity 2 568 3 4324 < 0.05 GO:0016661 oxidoreductase activity, acting on

other nitrogenous compounds as donors

2 568 3 4324 < 0.05

GO:0000171 ribonuclease MRP activity 2 568 3 4324 < 0.05 GO:0047956 glycerol dehydrogenase (NADP+)

activity 2 568 3 4324 < 0.05

GO:0005294 neutral L-amino acid porter activity 2 568 3 4324 < 0.05 G0:0000102 L-methionine porter activity 2 568 3 4324 < 0.05 GO:0019206 nucleoside kinase activity 2 568 3 4324 < 0.05 GO:0004526 ribonuclease P activity 2 568 3 4324 < 0.05 GO:0000049 tRNA binding 2 568 3 4324 < 0.05 GO:0016624 oxidoreductase activity, acting on

the aldehyde or oxo group of donors, disulfide as acceptor

2 568 3 4324 < 0.05

GO:0016741 transferase activity, transferring one-carbon groups

15 568 73 4324 < 0.05

00:0004674 protein serine/threonine kinase activity

11 568 49 4324 < 0.05

00:0016455 RNA polymerase II transcription mediator activity

3 568 7 4324 0.0525

GO:0015077 monovalent inorganic cation transporter activity

5 568 17 4324 0.0617

GO:0003674 molecular! unction 549 568 4124 4324 0.0691 GO:0008168 methyltransferase activity 14 568 71 4324 0.075 GO:0042054 histone methyltransferase activity 3 568 8 4324 0.076 G0:0019205 nucleobase, nucleoside, nucleotide

kinase activity 3 568 8 4324 0.076




GO:0016491 oxidoreductase activity 68 568 441 4324 0.0792 GO:0016215 CoA desaturase activity 2 568 4 4324 0.0862 GO:0015191 L-methionine transporter activity 2 568 4 4324 0.0862 GO:0004768 stearoyl-CoA 9-desaturase activity 2 568 4 4324 0.0862 GO:0030276 clathrin binding 2 568 4 4324 0.0862 GO:0005310 dicarboxylic acid transporter activ-

ity 2 568 4 4324 0.0862

GO:0004128 cytochrome-b5 reductase activity 2 568 4 4324 0.0862 GO:0004721 phosphoprotein phosphatase activ-

ity 7 568 30 4324 0.0889

GO:0016830 carbon-carbon lyase activity 8 568 36 4324 0.0907 GO:0004497 monooxygenase activity 6 568 25 4324 0.0998

Cellular component 0 0 0 0 0 < 0.05 GO:0044429 mitochondrial part 65 526 222 3787 < 0.05 GO:0031974 membrane-enclosed lumen 96 526 395 3787 < 0.05 GO:0043233 organelle lumen 96 526 395 3787 < 0.05 GO:0043234 protein complex 149 526 711 3787 < 0.05 GO:0005730 nucleolus 40 526 130 3787 < 0.05 GO:0005739 mitochondrion 95 526 426 3787 < 0.05 GO:0030529 ribonucleoprotein complex 62 526 243 3787 < 0.05 GO:0043228 non-membrane-bound organelle 96 526 442 3787 < 0.05 GO:0043232 intracellular non-membrane-bound

organelle 96 526 442 3787 < 0.05

GO:0005759 mitochondrial matrix 31 526 93 3787 < 0.05 GO:0031980 mitochondrial lumen 31 526 93 3787 < 0.05 GO:0005852 eukaryotic translation initiation

factor 3 complex 9 526 12 3787 < 0.05

GO:0031966 mitochondrial membrane 31 526 97 3787 < 0.05 GO:0044455 mitochondria] membrane part 13 526 24 3787 < 0.05 GO:0005622 intracellular 441 526 2891 3787 < 0.05 00:0044424 intracellular part 438 526 2868 3787 < 0.05 GO:0044452 nucleolar part 17 526 39 3787 < 0.05 00:0005761 mitochondrial ribosome 17 526 41 3787 < 0.05 00:0005840 ribosome 40 526 151 3787 < 0.05 GO:0000313 organellar ribosome 17 526 43 3787 < 0.05 GO:0005746 mitochondrial electron transport

chain 8 526 12 3787 < 0.05

GO:0005743 mitochondrial inner membrane 25 526 80 3787 < 0.05 GO:0019866 organelle inner membrane 25 526 82 3787 < 0.05 GO:0031981 nuclear lumen 60 526 270 3787 < 0.05 GO:0005740 mitochondrial envelope 34 526 129 3787 < 0.05 GO:0005732 small nucleolar ribonucleoprotein

complex 10 526 23 3787 < 0.05

GO:0016282 eukaryotic 43S preinitiation complex

15 526 47 3787 < 0.05

GO:0005844 polysome 4 526 5 3787 < 0.05 GO:0045285 ubiquinol-cytochrome-c reductase

complex 4 526 5 3787 < 0.05

GO:0005750 respiratory chain complex III (sensu 4 526 5 3787 < 0.05 Eukaryota)

GO:0045275 respiratory chain complex III 4 526 5 3787 < 0.05 proteasome regulatory particle, lid subcomplex (sensu Eukaryota)

5 526 8 3787 < 0.05

GO:0031967 organelle envelope 37 526 167 3787 < 0.05 GO:0031975 envelope 37 526 167 3787 < 0.05 GO:0005956 protein kinase CK2 complex 3 526 3 3787 < 0.05 GO:0005762 mitochondria] large ribosomal sub-

unit 9 526 24 3787 < 0.05

GO:0044422 organelle part 177 526 1086 3787 < 0.05 GO:0044446 intracellular organelle part 177 526 1086 3787 < 0.05 GO:0000315 organellar large ribosomal subunit 9 526 25 3787 < 0.05 GO:0005737 cytoplasm 306 526 2010 3787 < 0.05 00:0005763 mitochondrial small ribosomal sub-

unit 6 526 14 3787 < 0.05




00:0043229 intracellular organelle 335 526 2228 3787 < 0.05 GO:0043226 organelle 335 526 2228 3787 < 0.05 GO:0045254 pyruvate dehydrogenase complex 3 526 4 3787 < 0.05 GO:0000314 organellar small ribosomal subunit 6 526 15 3787 < 0.05 GO:0015934 large ribosomal subunit 16 526 65 3787 < 0.05 GO:0005845 mRNA cap complex 2 526 2 3787 < 0.05 00:0005749 respiratory chain complex II (sensu 2 526 2 3787 < 0.05

Eukaryota) GO:0045283 fumarate reductase complex 2 526 2 3787 < 0.05 G0:0045273 respiratory chain complex II 2 526 2 3787 < 0.05 00:0045257 succinate dehydrogenase complex 2 526 2 3787 < 0.05

(ubiquinone) GO:0045281 succinate dehydrogenase complex 2 526 2 3787 < 0.05 GO:0044464 cell part 490 526 3436 3787 < 0.05 GO:0005742 mitochondria] outer membrane

translocase complex 3 526 5 3787 < 0.05

00:0005741 mitochondria] outer membrane 6 526 17 3787 < 0.05 G0:0044444 cytoplasmic part 194 526 1254 3787 < 0.05 GO:0031968 organelle outer membrane 6 526 18 3787 < 0.05 GO:0019867 outer membrane 6 526 18 3787 < 0.05 GO:0005623 cell 490 526 3444 3787 < 0.05 GO:0015935 small ribosomal subunit 12 526 50 3787 < 0.05

proteasome core complex, beta- subunit complex (sensu Eukaryota)

3 526 6 3787 < 0.05

GO:0030663 COPI coated vesicle membrane 3 526 6 3787 < 0.05 GO:0030126 COPI vesicle coat 3 526 6 3787 < 0.05 GO:0005736 DNA-directed RNA polymerase I

complex 4 526 10 3787 < 0.05

GO:0000502 proteasome complex (sensu Eukary- ota)

8 526 29 3787 < 0.05

GO:0030677 ribonuclease P complex 2 526 3 3787 0.0525 GO:0000172 ribonuclease MRP complex 2 526 3 3787 0.0525

proteasome regulatory particle, base subcomplex (sensu Eukaryota)

2 526 3 3787 0.0525

GO:0005655 nucleolar ribonuclease P complex 2 526 3 3787 0.0525 GO:0012506 vesicle membrane 6 526 22 3787 0.0736 GO:0030120 vesicle coat 6 526 22 3787 0.0736 GO:0030659 cytoplasmic vesicle membrane 6 526 22 3787 0.0736 GO:0030662 coated vesicle membrane 6 526 22 3787 0.0736 G0:0043231 intracellular membrane-bound or-

ganelle 301 526 2061 3787 0.0895

Table E.5: A. fumigatus genes having increased transcript abundance, relative to laboratory culture, in the murine lung and S. cerevisiae orthologs regulated by rapamycin-mediated TOR kinase inhibition

Accesion SC ORF Description Afu4g13660 YBRO43C Multidrug transporter required for resistance to quinidine, barban, cis-

platin, and bleomycin Afu1g15520 YBR208C Urea amidolyase, contains both urea carboxylase and allophanate hy-

drolase activities, degrades urea to CO2 and NH3; expression sensitive to nitrogen catabolite repression.

Afu7g06380 YBR299W Maltase (alpha-D-glucosidase), inducible protein involved in maltose catabolism; encoded in the MAL3 complex locus; functional in genomic reference strain S288C

Afulg06150 YCL064C Catabolic L-serine (L-threonine) deaminase, catalyzes the degradation of both L-serine and L-threonine; required to use serine or threonine as the sole nitrogen source.

Afulg10780 YDR019C T subunit of the mitochondria] glycine decarboxylase complex, required for the catabolism of glycine to 5,10-methylene-THF.

Afu2g04260 YDR421W Zinc finger transcriptional activator of the Zn2Cys6 family; activates transcription of aromatic amino acid catabolic genes in the presence of aromatic amino acids



continued from previous page Accesion SC ORF Description

Afu7g06060

Afu1g17370

Afu5g08090

Afulg17150

Afu3g07850

Afu5g13810

Afu5g01520

Afu6g03540

Afu8g04760

Afu2g17300

Afu7g00850 Afu1g09690

Afu7g04290

Afu6g07720

Afu1g12620 Afu3g02300

Afu7g04970

Afu4g14640 Afu7g04710

Afu7g00830

Afu2g02480 Afu6g08570

Afu7g01090

Afu7g01000

Afulg01600

Afu1g04780

YEL065W

YFLO14W

YFLO59W

YGR288W

YHR028C

YHR112C

YIL121W

YIR031C

YIR032C

YIR038C

YIR042C Y.IL087C

YKR039W

YKR097W

YKR105C YLR142W

YLR214W

YMR319C YNL023C

YNR064C

YOL048C YOR346W

YOR348C

YOR374W

YOR386W

YPL147W

Ferrioxamine B transporter, member of the ARN family of transporters that specifically recognize siderophore-iron chelates; transcription is in-duced during iron deprivation. Plasma membrane localized protein that protects membranes from des-iccation; induced by heat shock, oxidative stress, osmostress, stationary phase entry, glucose depletion. Member of a stationary phase-induced gene family; transcription of SNZ2 is induced prior to diauxic shift, and also in the absence of thi-amin in a Thi2p-dependent manner;. MAL-activator protein, part of complex locus MALI.; nonfunctional in genomic reference strain S288C Dipeptidyl aminopeptidase, synthesized as a glycosylated precursor; lo-calizes to the vacuolar membrane; similar to Stel3p Putative protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm Multidrug transporter required for resistance to quinidine, barban, cis-platin, and bleomycin; may have a role in potassium uptake; member of the major facilitator superfamily. Malate synthase, role in allantoin degradation unknown; expression sen-sitive to nitrogen catabolite repression and induced by allophanate, an intermediate in allantoin degradation Ureidoglycolate hydrolase, converts ureidoglycolate to glyoxylate and urea in the third step of allantoin degradation; expression sensitive to nitrogen catabolite repression ER associated glutathione S-transferase capable of homodimerization; expression induced during the diauxic shift and throughout stationary phase. Putative protein of unknown function; YIR042C is a non-essential gene tRNA ligase, required for tRNA splicing; composed of three essential domains containing the phosphodiesterase, polynucleotide kinase, and ligase activities. General amino acid permease; localization to the plasma membrane is regulated by nitrogen source Phosphoenolpyruvate carboxykinase, key enzyme in gluconeogenesis, catalyzes early reaction in carbohydrate biosynthesis, glucose represses transcription. Putative transporter of the Major Facilitator Superfamily (MFS) Proline oxidase, nuclear-encoded mitochondrial protein involved in uti-lization of proline as sole nitrogen source; PUT1 transcription is induced by Put3p in the presence of proline Ferric reductase and cupric reductase, reduces siderophore-bound iron and oxidized copper prior to uptake by transporters; expression induced by low copper and iron levels Low-affinity Fe(II) transporter of the plasma membrane Protein that binds to Fprlp (FKBP12), conferring rapamycin resistance by competing with rapamycin for Fprlp binding; has similarity to pu-tative transcription factors. Epoxide hydrolase, member of the alpha/beta hydrolase fold family; may have a role in detoxification of epoxides Putative protein of unknown function Deoxycytidyl transferase, forms a complex with the subunits of DNA polymerase zeta, Rev3p and Rev7p; involved in repair of abasic sites in damaged DNA Proline permease, required for high-affinity transport of proline; also transports the toxic proline analog azetidine-2-carboxylate (AzC);. Mitochondrial aldehyde dehydrogenase, required for growth on ethanol and conversion of acetaldehyde to acetate; activity is K+ dependent. DNA photolyase involved in photoreactivation, repairs pyrimidine dimers in the presence of visible light; induced by DNA damage; regu-lated by transcriptional repressor Rphlp Subunit of a heterodimeric peroxisomal ATP-binding cassette trans-porter complex (Pxalp-Pxa2p), required for import of long-chain fatty acids into peroxisomes.




Afu2g08800

Afufig03730

Afu3g02280

YPL265W

YPROO2W

YPRO26W

Dicarboxylic amino acid permease, mediates high-affinity and high-capacity transport of L-glutamate and L-aspartate; also a transporter for Gln, Asn, Ser, Ala, and Gly Mitochondria] protein that participates in respiration, induced by di-auxic shift; homologous to E. coli PrpD, may take part in the conversion of 2-methylcitrate to 2-methylisocitrate Acid trehalase required for utilization of extracellular trehalose

Table E.6: A. fumigatus genes having decreased transcript abundance, relative to laboratory culture, in the murine lung and S. cerevisiae orthologs regulated by rapamycin-mediated TOR kinase inhibition

Accesion SC ORF Description Afu1g05560 YAL036C Member of the DRG family of GTP-binding proteins; interacts with

translating ribosomes and with Tma46p Afu8g05580 YBL015W Acetyl-coA hydrolase, primarily localized to mitochondria; required for

acetate utilization and for diploid pseudohyphal growth Afu1g07470 YBL078C Protein required for autophagy; modified by the serial action of Atg4p,

Atg7p, and Atg3p, and conjugated at the C terminus with phos-phatidylethanolamine,.

Afulg06190 YBRO34C Nuclear SAM-dependent mono- and asymmetric arginine dimethylating methyltransferase that modifies hnRNPs, including Npl3p and Hrplp.

Afulg14110 YBR154C RNA polymerase subunit ABC27, common to RNA polymerases I, II, and III; contacts DNA and affects transactivation

Afu6g13600 YBR155W TPR-containing co-chaperone; binds both HspS2p (Hsp90) and Ssalp (Hsp70) and stimulates the ATPase activity of SSA1, is mutants reduce Hsp82p function.

Afu1g09770 YCL037C Cytoplasmic RNA-binding protein that associates with translating ri-bosomes; involved in heme regulation of Hap1p as a component of the HMC complex.

Afu5g02760 YCR034W Fatty acid elongase, involved in sphingolipid biosynthesis; acts on fatty acids of up to 24 carbons in length; mutations have regulatory effects on 1,3-beta-glucan synthase.

Afu5g05610 YCR063W Protein involved in bud-site selection; analysis of integrated high- throughput datasets predicts an involvement in RNA splicing.

Afu1g14220 YDL014W Nucleolar protein, component of the small subunit processome com-plex, which is required for processing of pre-18S rRNA; has similarity to mammalia fibrillarin

Afulg11170 YDL154W Protein of the MutS family, forms a dieter with Msh4p that facilitates crossovers between homologs during meiosis; Insh5-Y823H mutation confers tolerance to DNA alkylating agents.

Afu5g02160 YDL167C Protein of unknown function, rich in asparagine residues Afu5g02410 YDR021W Nucleolar protein required for maturation of 18S rRNA, member of the

eIF4A subfamily of DEAD-box ATP-dependent RNA helicases Afulg10310 YDR091C Essential iron-sulfur protein required for ribosome biogenesis and trans-

lation initiation;. Afu2g16820 YDR101C Shuttling pre-60S factor; involved in the biogenesis of ribosomal large

subunit biogenesis; interacts directly with Albl; responsible for Tif6 recycling defects in absence of Reil.

Afu6g13630 YDR124W Putative protein of unknown function; non-essential gene; expression is strongly induced by alpha factor

Afulg05070 YDR165W Subunit of a tRNA methyltransferase complex composed of Trm8p and Trm82p that catalyzes 7-methylguanosine modification of tRNA

Afu2g03930 YDR324C Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

Afu5g10540 YELO11W Glycogen branching enzyme, involved in glycogen accumulation; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm in a punctate pattern

Afu6g09160 YEL012W Ubiquitin-conjugating enzyme that negatively regulates gluconeogen-esis by mediating the glucose-induced ubiquitination of fructose-1,6-bisphosphatase (FBPase).




Afu2g08360

Afu4g07580

Afu5g07020

Afu3g13280

Afu7g05920

Afu2g06310

Afu5g06700

Afu2g04780

Afu6g11800

Afu3g13320

Afu6g07880

Afu2g16010

Afu8g04820

Afu5g13480

Afu2g12440

Afu4g07080

Afu6g11070

Afu6g13370

Afu6g11310

Afulg06810

Afu3g13480

Afulg04070

Afu8g01970

Afu5g13470

Afu7g02420

YEL021W

YER025W

YER036C

YFLOO2C

YGL055W

YGL078C

YGR123C

YGR145W

YGR213C

YGR214W

YHR016C

YHRO2OW

YHR062C

YIL061C

YIL103W

YIR026C

YJL033W

YJL109C

YJL130C

YJL200C

YJR007W

YJR047C

YJR153W

YKL009W

YKL029C

Orotidine-5'-phosphate (OMP) decarboxylase, catalyzes the sixth enzy-matic step in the de novo biosynthesis of pyrimidines, converting OMP into uridine monophosphate (UMP) Gamma subunit of the translation initiation factor eIF2, involved in the identification of the start codon; binds GTP when forming the ternary complex with GTP and tRNAi-Met ATPase of the ATP-binding cassette (ABC) family involved in 40S and 60S ribosome biogenesis, has similarity to Gcn20p. Putative ATP-dependent RNA helicase, nucleolar protein required for synthesis of 60S ribosomal subunits at a late step in the pathway; sed-iments with 66S pre-ribosomes in sucrose gradients Fatty acid desaturase, required for monounsaturated fatty acid synthe-sis and for normal distribution of mitochondria Putative ATP-dependent RNA helicase of the DEAD-box family in-volved in ribosomal biogenesis Protein serine/threonine phosphatase with similarity to human phos-phatase PPS; present in both the nucleus and cytoplasm; expressed during logarithmic growth Essential nucleolar protein of unknown function; contains WD repeats, interacts with MpplOp and Bfr2p, and has homology to Spblp Protein involved in 7-aminocholesterol resistance; has seven potential membrane-spanning regions Protein component of the small (40S) ribosomal subunit, nearly iden-tical to RpsOBp; required for maturation of 18S rRNA along with RpsOBp. Protein involved in the organization of the actin cytoskeleton; contains SH3 domain similar to Rys167p Protein of unknown function that may interact with ribosomes, based on co-purification experiments; has similarity to proline-tRNA ligase; YHRO2OW is an essential gene Subunit of both RNase MRP, which cleaves pre-rRNA, and nuclear RNase P, which cleaves tRNA precursors to generate mature 5' ends Component of Ul snRNP required for mRNA splicing via spliceosome; may interact with poly(A) polymerase to regulate polyadenylation; ho-molog of human U1 70K protein Protein required, along with Dph2p, Ktillp, Jjj3p, and Dphip, for syn-thesis of diphthamide, which is a modified histidine residue of transla-tion elongation factor 2 (Eft 1p or Eft2p). Protein phosphatase involved in vegetative growth at low temperatures, sporulation, and glycogen accumulation; transcription induced by low temperature and nitrogen starvation;. Putative nucleolar DEAD box RNA helicase; high-copy number sup-pression of a U14 snoRNA processing mutant suggests an involvement in 18S rRNA synthesis Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA Bifunctional carbamoylphosphate synthetase (CPSase)-aspartate tran-scarbamylase (ATCase), catalyzes the first two enzymatic steps in the de novo biosynthesis of pyrimidines Putative mitochondria] aconitase isozyme; similarity to Acolp, an aconitase required for the TCA cycle; expression induced during growth on glucose, by amino acid starvation via Gcn4p Alpha subunit of the translation initiation factor eIF2, involved in the identification of the start codon; phosphorylation of Ser51 is required for regulation of translation Translation initiation factor eIF-5A, promotes formation of the first peptide bond; similar to and functionally redundant with Hyp2. Endo-polygalacturonase, pectolytic enzyme that hydrolyzes the alpha-1,4-glycosidic bonds in the rhamnogalacturonan chains in pectins Protein involved in mRNA turnover and ribosome assembly, localizes to the nucleolus Mitochondria' malic enzyme, catalyzes the oxidative decarboxylation of malate to pyruvate, which is a key intermediate in sugar metabolism and a precursor for synthesis of several amino acids




Afu6g04570 Afu6g02150

Afu5g09850 Afu2g02190

Afu3g12300

Afu1g03930

Afu2g12150

Afu3g06010

Afu2g13980

Afu6g12930

Afu2g13020

Afu3g04310

Afu6g02060

Afu4g09140

Afu5g05450

Afu4g12480

Afu3g08640

Afu2g16040

Afu6g13490

Afu6g02520

Afu2g02120

Afu3g05840

Afu3g06580

Afu8g03930

Afu3g05320

YKL081W YKL100C

YKL205W YLL035W

YLR061W

YLR067C

YLR106C

YLR186W

YLR276C

YLR304C

YLR348C

YLR409C

YLR43OW

YLR438W

YML063W

YML096W

YMR146C

YMR229C

YMR250W

YMR260C

YMR278W

YNL175C

YNL182C

YNL209W

YNL227C

Translation elongation factor EF-1 gamma Putative protein of unknown function with similarity to a human minor histocompatibility antigen; YKL100C is not an essential gene Nuclear pore protein involved in nuclear export of pre-tRNA Protein of unknown function, required for cell growth and possibly in-volved in rRNA processing; mRNA is cell cycle regulated Protein component of the large (60S) ribosomal subunit, has similarity to Rp122Bp and to rat L22 ribosomal protein Specific translational activator for the COX1 mRNA, also influences stability of intron-containing COX1 primary transcripts; located in the mitochondrial inner membrane Huge dynein-related AAA-type ATPase (midasin), forms extended pre-60S particle with the Rixl complex (Rix1p-Ipilp-Ipi3p), may mediate ATP-dependent remodeling of 60S subunits Protein required for the maturation of the 18S rRNA and for 40S ribo-some production; associated with spindle/microtubules; nuclear local-ization depends on physical interaction with Nopl4p ATP-dependent RNA helicase of the DEAD-box family involved in bio-genesis of the 60S ribosomal subunit Aconitase, required for the tricarboxylic acid (TCA) cycle and also inde-pendently required for mitochondrial genome maintenance; component of the mitochondria] nucleoid. Mitochondria] dicarboxylate carrier, integral membrane protein, cat-alyzes a dicarboxylate-phosphate exchange across the inner mitochon-drial membrane. Possible U3 snoRNP protein involved in maturation of pre-18S rRNA, based on computational analysis of large-scale protein-protein interac-tion data Putative helicase required for RNA polymerase II transcription termi-nation and processing of RNAs; homolog of Senataxin which causes Ataxia-Oculomotor Apraxia 2. L-ornithine transaminase (OTAse), catalyzes the second step of arginine degradation, expression is dually-regulated by allophanate induction and a specific arginine induction process. Ribosomal protein 10 (rp10) of the small (40S) subunit; nearly identical to RpslAp and has similarity to rat S3a ribosomal protein Putative protein of unknown function with similarity to asparagine syn-thetases; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm;. Subunit of the core complex of translation initiation factor 3(eIF3), which is essential for translation Protein required for the synthesis of both 18S and 5.8S rRNA; C-terminal region is crucial for the formation of 18S rRNA and N-terminal region is required for the 5.85 rRNA. Glutamate decarboxylase, converts glutamate into gamma- aminobutyric acid (GABA) during glutamate catabolism; involved in response to oxidative stress Translation initiation factor eIF1A, essential protein that forms a com-plex with Suilp (eIF1) and the 40S ribosomal subunit and scans for the start codon. Putative protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm and nucleus; YMR278W is not an essential gene Protein of unknown function, localizes to the nucleolus and nucleo-plasm; contains an RNA recognition motif (RR/t4) and has similarity to Nopl2p. Essential component of the Rixl complex (Rix1p, Ipilp, Ipi3p) that is required for processing of ITS2 sequences from 35S pre-rRNA; highly conserved and contains WD40 motifs. Cytoplasmic ATPase that is a ribosome-associated molecular chaper-one, functions with J-protein partner Zuolp; may be involved in the folding of newly-synthesized polypeptide chains. Co-chaperone that stimulates the ATPase activity of Scalp, required for a late step of ribosome biogenesis; associated with the cytosolic large ribosomal subunit.




Afu5g04240 YNL251C RNA-binding protein that interacts with the C-terminal domain of the RNA polymerase II large subunit (Rpo21p), required for transcription termination.

Afu1g07630 YNL255C Protein with seven cysteine-rich CCHC zinc-finger motifs, similar to human CNBP, proposed to be involved in the RAS/cAMP signaling pathway

Afu5g12310 YNL292W Pseudouridine synthase, catalyzes only the formation of pseudouridine- 55 (Psi55), a highly conserved tRNA modification, in mitochondria] and cytoplasmic tRNAs;.

Afu7g04130 YNL299W Poly (A) polymerase involved in nuclear RNA quality control based on, homology with Trf4p, genetic interactions with TRF4 mutants, physical interaction with Mtr4p (TRAMP subunit).

Afu2g05430 YNR012W Uridine/cytidine kinase, component of the pyrimidine ribonucleotide salvage pathway that converts uridine into UMP and cytidine into CMP.

Afu4g08930 YNR053C Putative GTPase that associates with pre-60S ribosomal subunits in the nucleolus and is required for their nuclear export and maturation

Afulg06690 YOR006C Putative protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to both the cytoplasm and the nucleus

Afulg16730 YOR243C Pseudouridine synthase, catalyzes pseudouridylation at position 35 in U2 snRNA, position 13 in cytoplasmic tRNAs, and position 35 in pre-tRNA(Tyr).

Afu4g07630 YOR272W Constituent of 66S pre-ribosomal particles, required for maturation of the large ribosomal subunit

Afulg02030 YOR361C Subunit of the core complex of translation initiation factor 3(eIF3), essential for translation.

Afu2g11810 YPL012W Protein required for export of the ribosomal subunits; associates with the RNA components of the pre-ribosomes; contains HEAT-repeats

Afu3g05570 YPL054W Zinc-finger protein of unknown function Afu7g02610 YPL126W LT3 snoRNP protein, component of the small (ribosomal) subunit (SSU)

processosome containing U3 snoRNA; required for the biogenesis of18S rRNA

Afu6g07490 YPL183C Cytoplasmic protein of unknown function Afu6g11510 YPL207W Protein required for the synthesis of wybutosine, a modified guano-

sine found at the 3'-position adjacent to the anticodon of phenylalanine tRNA.

Afu2g08780 YPRO95C Guanine nucleotide exchange factor (GEF) for Arf proteins; involved in vesicular transport; suppressor of ypt3 mutations; member of the Sec7-domain family

Afu4g07500 YPR137W Protein involved in pre-rRNA processing, associated with U3 snRNP; component of small ribosomal subunit (SSU) processosome; ortholog of the human U3-55k protein

Afu8g05430 YPR144C Nucleolar protein, forms a complex with Nopl4p that mediates matu-ration and nuclear export of 40S ribosomal subunits

Afu3g06540 YPR167C 3'-phosphoadenylsulfate reductase, reduces 3'-phosphoadenylyl sulfate to adenosine-3',5'-bisphosphate and free sulfite using reduced thiore-doxin as cosubstrate.


Table E.7: A. fumigatus specific genes having elevated transcript abundance in the murine lung, relative to laboratory culture. The Affc-core set were defined as

A. fumigatus Af293 proteins that have orthologs in N. fischeri and A. clavatus. The Affc-

unique set is a sub-set of Affc-core proteins that do not have orthologs in A. terreus, A.

oryzae, A. nidulans or A. niger

Accesion Old Accession Protein name Location SignalP Process AFUA_IG00100 Afulg00100 MFS monocarboxylate transporter,

putative subtelomeric SignalP transport

AFUA_1G00450 Afulg00450 N-acetylglucosamine-6-phosphate deacetylase (NagA), putative

subtelomeric metabolism

AFUA_1G01580 Afulg01580 hypothetical protein unknown AFUA_1G05400 Afulg05400 hypothetical protein unknown AFUA_1G11030 Afulg11030 xylitol dehydrogenase metabolism AFUA_1G11240 Afulg11240 hypothetical protein unknown AFUA_1G15190 Afulg15190 hypothetical protein unknown AFUA_2G00490 Afu2g00490 glycosyl hydrolase, family 31 subtelomeric metabolism AFUA_2G00550 Afu2g00550 conserved hypothetical protein subtelomeric unknown AFUA_2G00700 Afu2g00700 hypothetical protein subtelomeric unknown AFUA_2G02330 Afu2g02330 hypothetical protein unknown AFUA_2G04530 Afu2g04530 hypothetical protein unknown AFUA_2G05300 Afu2g05300 hypothetical protein unknown AFUA_2G11530 Afu2g11530 hypothetical protein unknown AFUA_2G12740 Afu2g12740 methyltransferase, putative metabolism AFIJA_2017640 Afu2g17640 conserved hypothetical protein subtelomeric unknown AFUA_2G17910 Afu2g17910 conserved hypothetical protein subtelomeric SignalP unknown AFUA_2G18050 Afu2g18050 FAD binding oxidoreductase CpoX1 subtelomeric SignalP metabolism AFUA_2G18060 Afu2g18060 AdoMet:dimethylallyltryptophan subtelomeric metabolism

N-methyltransferase EasF AFUA_3G01060 Afu3g01060 hypothetical protein subtelomeric unknown AFIJA_3G01140 Afu3g01140 hypothetical protein subtelomeric unknown AFIJA_3001230 Afu3g01230 MFS sugar transporte, putative transport AFUA_3G01730 Afu3g01730 hypothetical protein unknown AFIJA_3G02110 Afu3g02110 MFS multidrug transporter, puta-

tive SignalP transport

AFIJA_3G02130 Afu3g02130 oxidoreductase, zinc-binding, putative

metabolism

AFUA_3G02420 Afu3g02420 ThiJ/PfpI family transcriptional regulator, putative

transcriptional regulation

AFUA_3 G 03600 Afu3g03600 carboxylesterase, putative metabolism AFUA_3G03750 Afu3g03750 hypothetical protein unknown AFUA_3007090 Afu3g07090 hypothetical protein unknown AFUA_3G11880 Afu3g11880 hypothetical protein unknown AFUA_3G14740 Afu3g14740 conserved hypothetical protein subtelomeric unknown AFUA_4G00860 Afu4g00860 cell surface protein, putative subtelomeric cell wall biogenesis AFIJA_4006580 Afu4g06580 hypothetical protein unknown AFUA_4G06660 Afu4g06660 hypothetical protein unknown AFUA_4G08840 Afu4g08840 RING finger protein, putative SignalP unknown AFUA_4G13520 Afu4g13520 oxidoreductase, short-chain dehy-

drogenase/reductase family metabolism

AFIJA_5G00190 Afu5g00190 hypothetical protein subtelomeric unknown AFUA_5G00390 Afu5g00390 hypothetical protein subtelomeric unknown AFUA_5G00490 Afu5g00490 hypothetical protein subtelomeric unknown AFUA-5001410 Afu5g01410 hypothetical protein unknown AFLJA_5G01610 Afu5g01610 hypothetical protein unknown AFUA_5G02960 Afu5g02960 hypothetical protein unknown AFUA_5010350 Afu5g10350 conserved hypothetical protein SignalP unknown AFUA_5G12220 Afu5g12220 conserved hypothetical protein unknown AFLTA_5G13770 Afu5g13770 hypothetical protein unknown AFUA_5G14280 Afu5g14280 integral membrane protein subtelomeric unknown



continued from previous page Accesion Old Accession Protein name Location SignalP Process

AFUA_5G14900 Afu5g14900 hypothetical protein subtelomeric unknown AFUA_6G00140 Afu6g00140 hypothetical protein subtelomeric SignalP unknown AFUA_6G00690 Afu6g00690 conserved hypothetical protein subtelomeric SignalP unknown AFUA_6G02290 Afu6g02290 hypothetical protein unknown AFUA_6003220 Afu6g03220 hypothetical protein unknown AFUA-6G07790 Afu6g07790 hypothetical protein unknown AFUA_6G10180 Afu6g10180 spherulin 4 family protein, putative unknown AFUA_6G10190 Afu6g10190 hypothetical protein unknown AFUA_6G11500 Afu6g11500 dipeptidase, putative metabolism AFUA_6G11950 Afu6g11950 hypothetical protein unknown AFUA-6G11990 Afu6g11990 hypothetical protein unknown AFUA_6G12100 Afu6g12100 nitrilase family protein metabolism AFUA-7G00790 Afu7g00790 hypothetical protein subtelomeric unknown AFUA_7G01020 Afu7g01020 hypothetical protein subtelomeric unknown AFUA_7G04560 Afu7g04560 hypothetical protein unknown AFUA_7G07130 Afu7g07130 hypothetical protein unknown AFUA_7G08450 Afu7g08450 ornithine decarboxylase, putative subtelomeric metabolism AFUA_8G00100 Afu8g00100 aspartate-tRNA ligase, putative subtelomeric metabolism AFUA_8G00490 Afu8g00490 PKS-like enzyme, putative subtelomeric metabolism AFUA_8000500 Afu8g00500 acetate-CoA ligase, putative subtelomeric metabolism AFUA_8G00810 Afu8g00810 hypothetical protein subtelomeric unknown AFUA_8G01460 Afu8g01460 hypothetical protein unknown AFUA_8G01650 Afu8g01650 hypothetical protein unknown AFUA_8G01950 Afu8g01950 hypothetical protein SignalP unknown AFUA_8G02260 Afu8g02260 neutral amino acid permease transport AFUA_8G05050 Afu8g05050 hypothetical protein unknown AFUA_8G05080 Afu8g05080 hypothetical protein unknown AFUA_8G05470 Afu8g05470 hypothetical protein unknown AFUA_8G05770 Afu8g05770 hypothetical protein unknown AFUA_8G05780 Afu8g05780 NACHT and Ankyrin domain pro-

tein signaling

AFUA_8G06640 Afu8g06640 ubiE/COQS methyltransferase, putative

subtelomeric metabolism

Table E.8: A. fumigatus specific genes having repressed transcript abundance in the murine lung, relative to laboratory culture. The Affc-core set were defined

as A. fumigatus Af293 proteins that have orthologs in N. fischeri and A. clavatus. The

Affc-unique set is a sub-set of Affc-core proteins that do not have orthologs in A. terreus,

A. oryzae, A. nidulans or A. niger

Accesion Old Accession Protein name Location SignalP Process AFUAAG00270 Afulg00270 hypothetical protein subtelomeric unknown AFUA_1G00310 Afulg00310 class V chitinase, putative subtelomeric SignalP cell wall biogenesis AFUAAG01010 Afulg01010 polyketide synthase, putative metabolism AFUA_1G02220 Afu1g02220 hypothetical protein unknown AFUA_1G02960 Afu1g02960 conserved hypothetical protein unknown AFUA_1G03230 Afu1g03230 ABC multidrug transporter, puta-

tive transport

AFUA_1G03270 Afu1g03270 hypothetical protein unknown AFUA_1G17420 Afu1g17420 hypothetical protein subtelomeric unknown AFUA_2G01470 Afu2g01470 hypothetical protein unknown AFUA_2G05280 Afu2g05280 F-box domain protein unknown AFUA_2G06160 Afu2g06160 alpha-1,3-glucanase/mutanase, pu-

tative cell wall biogenesis

AFUA_2G09460 Afu2g09460 potassium transporter transport AFUA_2G16440 Afu2g16440 hypothetical protein unknown AFUA_3G02180 Afu3g02180 NRPS-like enzyme, putative metabolism AFUA_3007010 Afu3g07010 hypothetical protein unknown AFUA_3009120 Afu3g09120 hypothetical protein unknown AFUA_3G09510 Afu3g09510 3-oxoacyl-(acyl-carrier-protein) re-

duct ase metabolism




AFUA_3009660 Afu3g09660 hypothetical protein unknown AFUA_3013710 Afu3g13710 GTP cyclohydrolase I, putative metabolism AFUA_3G13720 Afu3g13720 oxidoreductase, 20G-Fe(II) oxyge-

nose family, putative metabolism

AFUA_4G00510 Afu4g00510 hypothetical protein subtelomeric unknown AFUA_4G00850 Afu4g00850 hypothetical protein subtelomeric unknown AFUA_4G00880 Afu4g00880 hypothetical protein subtelomeric unknown AFUA_4G01430 Afu4g01430 hypothetical protein SignalP unknown AFUA_4G06810 Afu4g06810 hypothetical protein unknown AFUA_4G07920 Afu4g07920 hypothetical protein unknown AFUA_4G08860 Afu4g08860 hypothetical protein unknown AFUA_5G00170 Afu5g00170 extracellular serine threonine rich

protein, putative subtelomeric SignalP cell wall biogenesis

AFUA_5G00220 Afu5g00220 hypothetical protein subtelomeric unknown AFUA_5G00230 Afu5g00230 hypothetical protein subtelomeric unknown AFUA_5G00860 Afu5g00860 Ankyrin repeat protein subtelomeric unknown AFUA_5G01070 Afu5g01070 C6 transcription factor, putative subtelomeric transcriptional regulation AFUA_5G02710 Afu5g02710 hypothetical protein unknown AFUA_5G13870 Afu5g13870 mRNA export factor Mlo3, putative metabolism AFUA_5G15140 Afu5g15140 hypothetical protein subtelomeric unknown AFUA_6G00180 Afu6g00180 hypothetical protein subtelomeric unknown AFUA_6G00350 Afu6g00350 hypothetical protein subtelomeric unknown AFUA_6G03120 Afu6g03120 hypothetical protein unknown AFUA_6G09310 Afu6g09310 class V chitinase, putative SignalP cell wall biogenesis AFUA_6009340 Afu6g09340 hypothetical protein unknown AFUA_6G10150 Afu6g10150 hypothetical protein unknown AFUA_6G10350 Afu6g10350 hypothetical protein unknown AFUA_6G11630 Afu6g11630 FAD-dependent isoamyl alcohol ox-

idase, putative SignalP metabolism

AFUA_6G11700 Afu6g11700 hypothetical protein unknown AFUA_7G00400 Afu7g00400 hypothetical protein subtelomeric unknown AFUA_7G04750 Afu7g04750 hypothetical protein unknown AFUA_7G05710 Afu7g05710 hypothetical protein unknown AFUA_7G06000 Afu7g06000 hypothetical protein unknown AFUA_7G07040 Afu7g07040 hypothetical protein unknown AFIJA_7G07060 Afu7g07060 hypothetical protein unknown AFUA_7G07140 Afu7g07140 hypothetical protein subtelomeric unknown AFUA_7G08250 Afu7g08250 C6 finger domain protein, putative subtelomeric transcriptional regulation AFUA_7G08380 Afu7g08380 hypothetical protein subtelomeric unknown AFUA_7G08470 Afu7g08470 peroxisomal copper amine oxidase,

putative subtelomeric metabolism

AFUA_7G08520 Afu7g08520 hypothetical protein subtelomeric unknown AFIJA_SG02110 Afu8g02110 hypothetical protein unknown AFUA_8G06150 Afu8g06150 serine-threonine rich protein, puta-

tive unknown

AFIJA_8G06370 Afu8g06370 monooxygenase, putative metabolism

Table E.9: Affc-specific genes having elevated transcript abundance in the murine lung, relative to laboratory culture. The Affc-core set were defined as A. fumigatus Af293 proteins that have orthologs in N. fischeri and A. clavatus. The Affc-unique set is a sub-set of Affc-core proteins that do not have orthologs in A. terreus, A. oryzae, A. nidulans

or A. niger

Accesion Old Accession Protein name Location SignalP Process AFUA_1G04160

AFUAAG05910 AFUA_1G11370 AFUA_IG13410 AFUA_1G16740 AFUA_1G17370 AFUAAG17600

Afulg04160

Afulg05910 Afulg11370 Afulg13410 Afu1g16740 Afu1g17370 Afulg17600

aspartate aminotransferase, putative conserved hypothetical protein GMC oxidoreductase, putative conserved hypothetical protein conserved hypothetical protein HSP9/12 family heat shock protein conserved hypothetical protein

subtelomeric subtelomeric

signalP

metabolism

unknown metabolism

unknown unknown unknown unknown




AFUA_2G00580 Afu2g00580 conserved hypothetical protein subtelomeric unknown AFUA_2G00990 Afu2g00990 glycerophosphoryl diester phospho-

diesterase family protein subtelomeric metabolism

AFUA_2G02500 AFUA_2C12780

Afu2g02500 Afu2g12780

conserved hypothetical protein von Willebrand domain protein

unknown

AFUA_2G12850 Afu2g12850 1,3-beta-glucanosyltransferase Ge13 signalP cell wall biogenesis AFUA_2G17420 Afu2g17420 Pfs and NB-ARC domain protein subtelomeric signaling AFUA_2G17650 Afu2g17650 DUF907 domain protein subtelomeric signalP unknown AFUA_3G01130 Afu3g01130 cell wall protein, putative subtelomeric signalP cell wall biogenesis AFUA_3G01320 Afu3g01320 homocysteine S-methyltransferase,

putative metabolism

AFUA_3G02800 Afu3g02800 lipase/esterase, putative metabolism AFUA_3G12220 Afu3g12220 ABC transporter, putative transport AFUA_3G12610 Afu3g12610 conserved hypothetical protein unknown AFUA_3G13130 Afu3g13130 UBE domain protein metabolism AFUA_3G13290 Afu3g13290 conserved hypothetical protein unknown AFUA_3G14770 Afu3g14770 NAD dependent

epimerase/dehydratase family protein

subtelomeric signalP metabolism

AFUA_3G15080 Afu3g15080 conserved hypothetical protein subtelomeric unknown AFUA_4G01360 Afu4g01360 MFS transporter of unkown speci-

ficity transport

AFUA_4G01370 Afu4g01370 pyoverdine/dityrosine biosynthesis family protein

signaling

AFUA_4C01392 Afu4g01390 C6 transcription factor, putative transcriptional regulation AFUA_4G01470 Afu4g01470 C6 finger domain protein, putative transcriptional regulation AFUA_4G08390 Afu4g08390 conserved hypothetical protein unknown AFUA_4G13950 Afu4gI3950 GNAT family acetyltransferase, pu-

tative subtelomeric metabolism

AFUA_4014170 Afu4g14170 conserved hypothetical protein subtelomeric signalP unknown AFUA_4G14180 Afu4g14180 conserved hypothetical protein subtelomeric signalP unknown AFUA_4G14200 Afu4g14200 conserved hypothetical protein subtelomeric signalP unknown AFUA_5G00810 Ant500810 conserved hypothetical protein subtelomeric unknown AFUA_5G01130 Afu5g01130 conserved hypothetical protein subtelomeric unknown. AFUA.5G01300 Afu5g01300 integral membrane protein signalP unknown AFUA_5G08180 Afu5g08180 cell wall protein, putative signalP cell wall biogenesis AFUA.5G09130 Afu5g09130 polysaccharide deacetylase family

protein metabolism

AFUA-5G09440 Afu5g09440 amino acid permeate, putative transport. AFUA_6G00620 Afu6g00620 GPI anchored hypothetical protein subtelomeric cell wall biogenesis AFIJA-6003550 Afu6g03550 conserved hypothetical protein unknown AFUA_6004280 Afu6g04280 integral membrane protein signalP unknown AFUA-6G07260 Afu6g07260 purine-cytosine permease, putative transport AFUA_6G07920 Afu6g07920 acetyltransferase, GNAT family

family metabolism

AFUA_6G08500 Afu6g08500 phosphoglycerate mutate family protein

metabolism

AFUA_6008650 Afu6g08650 conserved hypothetical protein unknown AFUA_6G12150 Afu6g12150 bZIP transcription factor, putative transcriptional regulation AFUA_7G00690 Afu7g00690 aminotransferase, putative subtelomeric metabolism AFUA_7001000 Afu7g01000 aldehyde dehydrogenate, putative subtelomeric metabolism AFUA_7G04990 Afu7g04990 dUTP diphosphatase, putative uracil metabolism AFUA_7G05650 Afu7g05650 XYPPX repeat family protein unknown AFUA_8G00580 Afu8g00580 glutathione S-transferase, putative subtelomeric metabolism. AFUA_8000710 Afu8g00710 secreted antimicrobial peptide, pu-

tative subtelomeric signalP unknown

AFUA_8G01770 Afu8g01770 conserved hypothetical protein signalP unknown AFUA_8G05930 Afu8g05930 conserved hypothetical protein unknown

Table E.10: Affc-specific genes having repressed transcript abundance in the murine lung, relative to laboratory culture. The Affc-core set were defined as A.

fumigates Af293 proteins that have orthologs in N. fischeri and A. clavatus. The Affc-

unique set is a sub-set of Affc-core proteins that do not have orthologs in A. terreus, A.

oryzae, A. nidulans or A. niger


Accesion Old Accession Protein name Location SignalP Process

AFUA_1G12220 Afulg12220 conserved hypothetical protein unknown

AFITA_1G13120 Afulg13120 MIT/CorA family metal ion transporter

unknown

AFUA_2G02650 Afu2g02650 conserved hypothetical protein unknown

AFUA_3G09260 Afu3g09260 conserved hypothetical protein SignalP unknown

AFUA_3G12680 Afu3g12680 conserved hypothetical protein unknown AFUA_4G08890 Afu4g08890 aldo-keto reductase family protein metabolism

AFUA_5G10940 Afu5g10940 conserved hypothetical protein unknown

AFUA_6G00500 Afu6g00500 chitosanase, putative subtelomeric SignalP cell wall biogenesis

AFUA_7G06440 Afu7g06440 F-box domain protein unknown

AFUA_8G01820 Afu8g01820 hypothetical protein unknown

Table S6 Found at http : //ukpmc . ac .uk/picrender . cgi?artid=1663068&blobname=

ppat . 1000154 . s006 . doc Table S7 Found at http : //ukpmc . ac .uk/picrender . cgi?artid=1663068&blobnarne=

ppat 1000154 . s010.xls

Table E.11: Nitrogen starvation gene clusters

Cluster Number Accession Description log2 ratio Sub-telomeric Murine gene cluster

1 Afulg00500 FMN dependent dehydrogenase, putative

6.56

ZZ

z ZZ

z Z

ZZ

ZZ

ZZ

z ZZX

ZZ

ZZ

ZZ

ZZ

Z. <

Afulg00530 thermoresistant gluconokinase family protein

5.50

Afulg00550 conserved hypothetical protein 4.13 Afulg00560 Pfs domain protein 4.24 Afulg00570 Tel-mariner transposase, putative 2.25

2 Afu1g03280 maltose permease, putative 2.05 4 Afulg03300 carboxylesterase, putative 2.27 4

Afu1g03330 hypothetical protein 4.98 Afulg03340 hypothetical protein 3.75 4 Afu1g03350 alpha-1,3-glucanase, putative 2.41 4 Afu1g03360 hypothetical protein 3.00

3 Afulg16020 hypothetical protein 3.82 Afulg16030 conserved hypothetical protein 4.82 Afulg16050 conserved serine-proline rich pro-

tein 5.07

Afulg16060 conserved hypothetical protein 6.13 Afulg16080 hypothetical protein 5.04 Afulg16110 arsenic methyltransferase Cyt19,

putative 3.16

Afu1g16130 copper-transporting ATPase, putative

3.71

Afulg16170 hypothetical protein 2.71 4 Afu3g02520 MSF multidrug transporter, puta-

tive 4.76

Afu3g02550 conserved hypothetical protein 4.91 Afu3g02570 polyketide synthase, putative 2.48 Afu3g02600 hypothetical protein 4.54 Afu3g02610 MFS transporter, putative 3.85 Afu3g02640 nucleoside-diphosphate-sugar

epimerase family protein 2.24

5 Afu3g03280 FAD binding monooxygenase, putative

2.50 33

Afu3g03290 hypothetical protein 2.89 Afu3g03330 mitochondria] enoyl reductase, pu-

t at ive 6.03

Afu3g03350 nonribosomal peptide synthase, putative

5.84 33

Afu3g03360 hypothetical protein 5.29 6 Afu3g13620 conserved hypothetical protein 2.38



continued from previous page Cluster Number Accession Description log2 ratio Sub-telomeric Murine gene cluster

Afu3g13640 extracellular serine-rich protein 2.12 N Afu3g13650 integral membrane protein 2.17 N Afu3g13660 Ctr copper transporter family pro-

tein 3.61 N

Afu3g13670 siderochrome-iron transporter, putative

5.30 N

Afu3g13680 conserved hypothetical protein 5.04 N Afu3g13700 transferase family protein 3.47 N

7 Afu4g00490 conserved hypothetical protein 4.21 Y Afu4g00510 hypothetical protein 3.72 Y Afu4g00530 conserved hypothetical protein 3.40 Y Afu4g00550 membrane transport protein, puta-

tive 4.09 Y

Afu4g00580 hypothetical protein 2.78 Y Afu4g00590 monooxygenase, putative 4.31 Y Afu4g00600 integral membrane protein 4.32 Y Afu4g00610 aryl-alcohol dehydrogenase, puta-

tive 4.67 Y

8 Afu4g14000 tripeptidyl peptidase A 3.51 Y Afu4g14040 Hsp70 family protein 4.50 Y 44 Afu4g14060 conserved hypothetical protein 2.52 Y Afu4g14070 glycosyl transferase, putative 5.61 Y 44

Afu4g14080 cell surface antigen spherulin 4, putative

2.37 Y 44

Afu4g14090 UDP-glucose 4-epimerase 3.36 Y 44

9 Afu4g14160 hypothetical protein 3.97 Y Afu4g14170 hypothetical protein 3.39 Y 44

Afu4g14180 hypothetical protein 2.58 Y 44

Afu4g14190 hypothetical protein 2.47 Y Afu4g14230 MFS transporter, putative 3.17 Y Afu4g14240 0-methyltransferase 2.95 Y Afu4g14250 hypothetical protein 2.62 Y Afu4g14290 conserved hypothetical protein 2.40 Y

10 Afu5g03690 CRAL/TRIO domain protein 2.33 N Afu5g03710 conserved hypothetical protein 2.93 N Afu5g03750 WW domain protein 3.78 N Afu5g03760 class III chitinase ChiAl 2.92 N Afu5g03800 high-affinity iron permease 2.19 N

CaFTR2 11 Afu5g14860 cytochrome P450 4.64 Y

Afu5g14870 protein kinase, putative 5.27 Y Afu5g14880 hypothetical protein 5.67 Y Afu5g14900 hypothetical protein 5.56 Y Afu5g14920 hypothetical protein 3.39 Y. Afu5g14930 conserved hypothetical protein 4.72 Y Afu5g14940 cell surface metalloreductase 5.13 Y

(FreA), putative Afu5g14960 hypothetical protein 5.83 Y Afu5g14970 conserved hypothetical protein 4.82 Y Afu5g14990 hypothetical protein 4.36 Y Afu5g15010 arsenite permease (ArsB), putative 3.42 Y Afu5g15040 hypothetical protein 3.06 Y Afu5g15070 AhpC/TSA family thioredoxin per-

oxidase, putative 2.41 Y

Afu5g15080 Rho-associated protein kinase, putative

2.71 Y

12 Afu6g00230 isoflavone reductase family protein 4.53 Y Afu6g00260 phosphatidylserine decarboxylase

family protein 3.98 Y

Afu6g00280 isoflavone reductase family protein 2.02 Y Afu6g00290 aminotransferase, putative 2.27 Y Afu6g00310 serine carboxypeptidase (CpdS),

putative 2.48 Y



continued from previous page Cluster Number Accession Description log2 ratio Sub-telomeric Murine gene cluster

13 Afu6g00550 glycosyl transferase family 8 family, putative

3.19

zzzzzzz z Z

>.>

.?.

>..)-.}.Z

ZZ

ZZ

Afu6g00560 hypothetical protein 2.76

Afu6g00600 hypothetical protein 2.22 55


Afu6g00620 GPI anchored hypothetical protein 2.71

Afu6g00630 MFS transporter, putative 2.19 55

Afufig00640 integral membrane protein 3.14 55

14 Afu6g03390 glutathione S-transferase family protein

2.86

Afu6g03410 2.15

Afu6g03420 trehalose synthase, putative 2.21

Afu6g03430 C6 finger domain protein, putative 3.62 58

Afu6g03440 fructosyl amine 5.72 58

Afu6g03450 conserved hypothetical protein 4.33 58

Afu6g03460 hypothetical protein 4.00 58

Afu6g03470 ABC multidrug transporter, putative

3.49 58

Afu6g03480 nonribosomal peptide synthetase, putative

3.19 58

Afu6g03490 phenol 2-monooxygenase, putative 4.08 58

15 Afu7g08520 hypothetical protein 6.35

Afu7g08530 hypothetical protein 4.07 Afu7g08540 ankyrin repeat protein 4.41


Afu7g08590 hypothetical protein 5.07 Afu7g08600 hypothetical protein 3.96

16 Afu8g06050 salicylate hydroxylase, putative 3.49

Afu8g06060 conserved hypothetical protein 4.18


Afu8g06090 amino acid permease, putative 3.32

Afu8g06100 integral membrane protein 3.19

Table E.12: Oligonucleotides used in qPCR

Oligo Name Oligo Sequence

Afu3g03080F ATACTIGGCGCATCGICTGG Afu3g0308OR ACGGCCACGTTGAGAATGAA Afu2g07770F TACGATGTCGTTCGCATGCT Afu2g0777OR TCACAGGATGACGCACTTGAT Afu3g12060F GTGTIGAAGAGGCCCTCAAG Afu3g1206OR GGGCCAGATATCGTTAGCAAT Afulg12310F CTATGCCCGCAAGCATTTCA Afu lgl 231 0 R TCTTGGAGGACATCGATCCAT Aful g16890F CCAGATGATGTGCCCGTGAA Afulg16890R CGGCTGGAATAGATTCCTTC Afu2g07910F TTIGGGTTCTATGCTGGGAT Afu2g0791OR GCTTCTGCCTCTCCTGAAGTA Afu2g06210F AGTTCGGCCGGCTTTGGTA Afu2g0621OR TGAATACAAAGGGGACGCAA Afulg15970F GGATTGATTGCAATCCCAGG Afulg1597OR TAATCACCGACCATCGATTG Afu2g05360F CGGATTTCGATTCACGAAGC Afu2g05360R TGGTATACATGCTCTCCGTGT Afu2g1499 OF TCGACCTCATGTACTCGAAG Afu2g 1499 OR CCTTCCTCCTCTGGTTCCATA Afu6g09610F ATGCGCCAT9CTCGAGATCA Afu6g096 lOR GCGAGACCTTCAAGGCATT Afu6g09620F CGACGATAAGTTCGCGCCGT Afu6g0962OR AAAATCTOCCGGICCGTGAAA Afu6g09630F TCTITGAACCCTATGATCCG Afu6g0963OR AGTAGGTTGCACAATCGAGCC

E.1 Dataset Si reference 306

E.1 Dataset S1 reference

Found at http : //ukpmc . ac . uk/picrender . cgi?artid=1663068&blobname=ppat .1000154 .

s013 .xls


Found at http : //ukpmc . ac .uk/picrender. cgiTartid=1663068&blobname=ppat . 1000154 .

s014.xis


Found at http://ukpmc.ac.uk/picrender . cgilart id=1663068&blobname=ppat .1000154.

s015 . xls


Found at http: //ukpmc . ac .uk/picrender . cgi?artid=1663068&blobname=ppat . 1000154 .

s016.xls

307

Appendix F


Table F.1: Summary of null mutant CGTFKOLib strain growth relative to his3

parental strain in YEPD and YEPD + 5 - 10 pg m1-1 fluconazole..

Gene Name YEPD % Control

YEPD % SEM

5 pg ml-' fluconazole % Control

5 pg ml-' fluconazole SEM

10 pg ml-1 fluconazole % Control

10 pg ml-1 fluconazole SEM

ASF1 13.91 2.67 16.42 4.19 34.03 1.53

POPS 17.95 5.84 9.42 5.18 9.02 13.94 SNF6 21.13 4.26 8.33 2.25 9.02 5.07

SPT3 21.64 5.73 17.88 4.33 26.05 5.48 GCN5 38.13 5.54 10.93 4.83 14.49 11.15

SNF5 42.15 10.73 31.20 8.04 56.51 15.64 NGGI 44.48 5.96 12.70 4.02 17.53 7.64 SSN6 53.85 2.87 75.27 14.12 81.45 66.53 RTF1 55.12 6.41 46.09 14.75 45.11 43.83

TAFf4 61.76 2.98 42.47 9.86 49.16 1.77 ADA2 67.45 12.59 21.28 3.80 36.13 6.67

HAT2 68.63 8.82 49.45 18.46 41.68 24.08 SFLI 69.73 7.30 93.88 10.50 106.30 5.98 EMIT 70.95 11.04 84.84 12.87 176.05 2.29 SSN8 72.50 22.60 75.80 18.85 182.77 25.54 RPD3 72.84 4.79 72.83 6.08 87.55 45.68 GLN3 74.42 17.92 76.11 18.27 108.77 69.38 AR080 76.49 11.71 61.14 27.55 50.70 37.99 CTI6 78.00 11.80 48.28 21.00 36.85 35.50 SPT8 78.40 15.92 55.93 16.21 67.47 24.24 SSN2 79.17 7.42 90.86 23.93 91.60 17.97 MSNI 79.98 8.71 52.40 21.85 48.92 31.92 MKS1 80.07 15.21 96.31 20.83 105.46 23.74 CST6 80.15 6.30 97.86 8.22 204.20 25.54

SRB8 81.02 6.24 53.91 8.00 34.94 12.80

HOST 81.89 13.64 57.70 26.35 49.30 66.02 M1G/ 83.24 4.60 63.84 10.51 54.38 8.15

FKH2 83.97 18.76 92.61 20.59 210.50 39.12

BA S1 84.05 15.83 89.70 15.13 203.57 40.92 TECI 85.92 21.25 96.60 15.45 170.59 10.97 SFP1-like (not OT- tholoque)

86.18 14.50 62.07 21.56 53.88 40.11

SUT2 87.55 5.77 108.84 19.16 224.79 53.70

SINS 88.09 5.47 97.39 5.52 121.98 6.17

STP2 88.40 9.86 100.84 18.05 128.97 13.72


Appendix F. Chapter 5 Supplementary Data 308

continued from previous page Gene Name YEPD %

Control YEPD % SEM

5 pg m1-1 fluconazole % Control



10 ttg ml-1 fluconazole SEM

GAT1 89.91 1.60 101.36 14.45 171.43 36.37 PHO2 90.15 12.09 105.83 22.16 241.39 54.89 ASG1 90.55 10.15 86.46 13.45 84.50 29.22 HS T3 91.02 8.38 80.32 15.73 71.66 35.25

ACE2 91.13 17.71 105.93 14.74 212.82 0.39 SPO1 91.74 7.65 95.46 18.26 130.88 20.23

MSN4 91.94 4.25 111.76 17.46 211.97 48.97 CG2001 92.03 15.96 103.50 9.62 100.00 30.59 CRZI 92.03 19.90 109.91 19.12 223.74 0.76 SNF4-like 92.43 4.41 107.48 21.11 133.19 24.31

FAP1 93.00 14.58 113.61 16.69 226.89 1.66

PHO23 93.17 11.06 93.61 21.41 131.77 57.17 STP/ 93.25 7.37 110.20 8.56 187.61 27.15

PUT3 93.49 13.39 112.73 16.80 207.14 41.19

YAP6 93.82 26.64 117.30 26.47 224.79 14.34

RFX1 93.88 13.84 82.00 13.72 72.30 59.19

MSN2 94.12 5.12 96.47 11.26 127.06 21.77

SWD/ 94.36 4.92 97.56 6.60 100.76 22.64

FKH1 94.44 8.72 94.79 17.03 126.30 42.09 SPP I 94.79 6.56 107.19 10.59 78.15 13.22

SIPI 95.00 4.38 92.68 18.02 122.74 20.97 CATS 95.04 16.27 112.93 18.80 151.68 10.40

/ME/ 95.36 16.39 107.87 13.75 139.92 17.06

RTG2 95.63 10.07 100.76 19.43 80.94 7.04 RIM101 95.71 6.74 98.91 11.65 134.43 26.67

SSN3 95.85 10.37 83.09 12.67 151.47 30.63

.51P4 96.01 16.59 117.69 18.64 197.06 31.21

GAL83 96.27 4.71 102.61 2.82 85.13 4.05 SUT/ 96.58 26.40 117.01 28.90 209.87 52.87

OAFI 96.58 10.07 120.80 10.55 237.82 35.44 RGT1 96.66 3.93 117.20 18.34 163.24 32.49

SMP1 96.66 14.75 117.40 15.50 223.95 33.32

TUP1 96.82 6.45 108.33 17.11 124.65 57.94 HA P1-like 96.83 14.13 77.16 10.94 42.86 0.59

LYS 14 96.83 3.68 100.29 8.53 110.92 3.87

GIS2 96.83 6.90 119.24 16.25 206.72 39.23

EMI5 96.91 14.06 116.72 28.56 159.03 41.60

YRR1 96.91 6.57 119.14 6.45 102.10 5.46

MBP1 97.06 6.59 104.79 12.59 144.09 47.80 DAL80 97.15 8.41 120.89 24.60 202.10 44.99 GAL 1 1 97.22 8.03 84.95 17.56 74.97 36.94

DAL82 97.40 17.10 115.16 14.72 211.34 22.27

HCM1 97.40 8.06 117.20 10.66 221.22 28.76

HOT I 97.46 2.03 96.72 12.01 136.72 14.53

BRE2 97.46 14.34 100.76 16.54 107.12 47.91

KA R4 97.54 11.24 94.45 18.58 118.81 33.08

ASK10 97.54 13.73 100.17 16.73 139.90 27.52

RPH1 97.56 21.82 76.97 17.76 102.73 12.10

RTG1 97.62 7.12 87.80 19.52 75.22 26.88

SDS3 97.64 15.35 106.12 17.23 184.03 42.39

HOS2 97.70 4.64 101.35 10.54 128.08 20.26 GZF3 97.86 16.61 100.42 10.69 108.26 5.76

HDA 1 97.97 11.54 118.17 20.74 176.47 43.53 MCM1-like. 98.05 8.07 117.40 23.93 182.98 45.01

FZF1 98.09 8.57 102.02 15.69 108.39 34.41

YR A/ / 98.21 26.55 109.91 20.85 155.46 4.80 HAP2 98.25 13.54 99.58 31.33 146.12 45.56

HMS1 98.33 10.05 99.07 22.18 141.68 36.12

SKN7 98.33 14.08 103.20 19.90 132.53 44.06

HIR1 98.33 4.56 104.21 9.94 106.23 28.94




Control YEPD % SEM

5 og ml-' fluconazole % Control

5 og ml-1 fluconazole SEM

10 og m1-1 fluconazole % Control

10 og ml-' fluconazole SEM

YAPS 98.37 12.15 104.96 15.52 151.05 22.04

ND T80 98.45 12.66 117.49 12.78 222.06 37.49

HIR2 98.49 4.43 106.56 12.98 151.72 26.05 CHA4 98.62 15.32 117.40 13.41 183.82 11.50

CG2001 98.65 8.45 74.10 25.38 65.69 46.33

PPR I 98.65 9.28 102.69 20.07 144.09 62.49

UMEI 99.02 20.02 110.50 15.14 187.18 4.17 AZF I-LIKE 99.10 9.30 119.24 12.62 189.29 29.53 TEC1-L IKE 99.13 3.37 94.28 8.99 70.78 27.82

TOSS 99.13 11.97 98.74 20.76 139.26 30.66 SIPS 99.19 12.87 117.49 17.15 168.07 29.77

STE 12-LIKE 99.19 9.19 119.24 22.42 220.38 44.92

PIPS 99.35 35.39 120.41 38.24 216.39 85.41 XBP1 99.36 10.98 94.20 15.70 93.14 23.34 SASS 99.43 13.35 116.62 19.78 234.24 44.06

ASH1 99.44 4.41 80.24 11.33 82.59 15.09 AFT2 99.44 11.53 100.25 22.55 112.07 108.38 IMPS' 99.52 17.18 86.29 28.99 ' 75.73 82.36

MO T3 99.59 5.69 114.87 19.10 196.22 44.17 Z MS 1 99.67 7.61 120.21 18.53 210.71 46.72

CUPS 99.92 5.23 101.18 6.09 99.11 22.90 HST4 99.92 5.18 103.20 12.68 141.55 22.78

SOK 2 99.92 11.33 114.38 9.78 166.39 1.13 his3 100.00 ' 0.00 100.00 0.00 100.00 0.00

his3 100.00 0.00 100.00 0.00 100.00 0.00 TUP1 -lac (not ortholog)

100.08 9.02 103.62 24.95 160.99 197.00

PDR1 100.16 5.13 28.34 5.07 35.96 12.52 POG1 100.16 10.78 101.77 20.18 141.55 30.84 GCRS 100.24 19.19 98.07 21.96 129.35 43.64

CAD I 100.24 4.24 105.97 14.86 106.23 30.63

NOT3 100.24 26.37 112.54 25.40 236.34 35.83 SPT2 100.33 6.99 114.77 12.12 224.37 46.25

SASS 100.40 13.54 82.84 26.96 63.91 62.41 SKOI 100.41 7.15 100.00 15.10 91.39 13.79 HAA I 100.41 9.94 118.46 25.71 224.58 54.54 SEE/ 100.48 1.33 98.32 0.46 129.73 24.14 /MG/ 100.49 23.58 115.35 25.69 219.75 5.29

HAP5 100.65 9.04 119.34 9.00 96.01 1.18 GTS1 100.73 14,54 108.45 7.29 142.44 3.89 THIS 100.73 14.88 114.38 14.75 200.00 8.22 SW15 100.87 14.28 102.02 29.53 131.51 42.54

RME1 100.90 5.38 116.81 10.75 125.84 4.50 YA P7 100.95 5.73 99.33 15.08 125.67 11.29 INOS 101.19 8.51 88.14 18.43 76.37 42.23 CUPS 101.19 13.38 103.03 20.24 120.46 35.80 METS8 101.22 15.77 114.77 10.62 169.12 12.08 MET32 101.27 4.80 99.07 3.91 95.04 2.02 TSPI 101.27 10.85 105.55 12.55 129.73 20.71 SNF4 101.43 9.98 106.64 20.14 128.46 37.26

HA P4 101.46 10.43 116.13 21.82 180.46 40.52 RLM I 101.67 7.19 99.92 16.79 124.02 40.45 SASS 102.12 21.25 118.46 28.01 212.61 59.91

CHD1 102.12 5.20 120.21 5.35 176.05 25.11 HA PI 102.22 10.47 96.55 15.54 85.01 32.51

ZAP I 102.28 23.67 101.36 19.54 104.62 39.10

A HC1 102.46 0.99 105.30 3.96 117.15 2.25

MCM/ 102.62 21.13 100.76 40.07 101.78 57.48

GIS I 102.70 10.20 103.11 19.60 132.53 32.28

WARI 102.70 5.87 104.63 8.55 127.45 17.89 continued on next page



Control YEPD % SEM

5 pg ml - I fluconazole % Control

5 pg ml -1 fluconazole SEM



MIG2 102.77 '5.16 115.16 16.30 175.84 26.65 EZL I 102.94 5.05 109.42 18.64 144.98 60.25 HIR3 103.09 12.66 108.94 19.31 179.62 42.30 STE12 103.10 4.53 100.34 13.67 86.66 27.35 UPC2 103.10 7.21 104.29 12.38 146.63 21.96 YOX1 103.18 7.80 92.85 8.96 95.55 45.31 HAC1 103.25 5.22 110.50 10.73 167.65 20.26 HATI 103.25 12.07 119.53 18.33 226.47 50.15 CAL//-like 103.34 5.94 54.33 10.66 62.90 5.96 ST135 103.42 9.04 104.54 17.37 124.02 24.91 HALO 103.66 7.32 123.32 11.58 210.71 42.16 MET3I 103.82 4.87 117.78 6.31 219.75 38.32 RDS1 103.89 7.75 97.22 11.53 133.04 17.74 DA L81 103.97 10.18 110.34 23.61 155.91 49.06 H033 103.99 6.97 93.97 15.58 118.70 16.28 ARC81 104.37 5.84 103.62 16.46 133.42 39.36 ADR1 104.45 12.77 99.50 21.48 107.37 21.67 MILS 104.45 3.85 101.68 6.38 80.05 21.09 HS TI 104.53 16.79 105.30 20.66 133.42 33.95 RDS2 104.64 30.59 120.70 21.05 245.38 20.72

FL O8 104.69 12.98 112.45 26.33 154.89 52.86 YAP3-like 104.80 10.88 115.94 7.77 216.39 29.89 HA P3 104.80 4.64 121.28 15.18 80.46 1.93 HS T2 105.04 12.67 117.78 26.33 210.08 54.73

PHO4 105.16 9.54 104.21 15.07 120.84 12.90 SFPI 105.29 22.08 95.92 19.42 173.95 4.35 STB1 105.29 32.50 113.80 35.65 219.12 18.97 TYE7 105.40 5.96 108.49 15.90 143.33 26.19

S/H4 106.02 26.35 114.87 24.86 188.24 55.97

TOS4 106.04 5.00 103.11 13.15 97.84 11.97

SPT4 106.92 21.30 115.65 18.12 158.40 28.25 MSS1 / 107.23 14.76 116.32 27.27 155.27 72.20

RTG3 107.97 12.96 114.48 18.12 159.45 4.30


Table F.2: Summary of null mutant strains showing insufficent growth in YEPD (0-11.5 hours) for nonlinear regression based estimation of midpoint and upper asymptote

CG ORF SC ORF Gene Name Description CAGLOB01441g YNL330C RPD3 Histone deacetylase; regulates tran-

scription and silencing CAGL00O2277g YER040W GLN3 transcriptional activator of

nitrogen-regulated genes CAGL0003399g YNR052C POP2 transcription factor (putative) CAGLOD01364g YBR112C SSN6 General repressor of transcription CAGLOE00693g YDR176W NGG1 Involved in glucose repression of

GAL4p-regulated transcription; transcription factor; genetic and mutant analyses suggest that Ngglp (Ada3p) is part of two transcriptional adaptor/HAT (histone acetyltransferase complexes, the 0.8 MD ADA complex and the 1.8 MD SAGA complex

CAGLOE03718g YHL025W SNF6 RNA polymerase II transcription factor activity

CAGLOF08283g YGR252W GCN5 Ada2/Gcn5/Ada3 transcription ac-tivator complex

CAGLOH07711g YGL244W RTF1 Subunit of the RNA polymerase II-associated Pafl complex; directly or indirectly regulates DNA-binding properties of Sptl5p and relative activities of different TATA elements

CAGLOL02123g YBR289W SNF5 RNA polymerase II transcription factor activity

CAGLOL05412g YJL115W ASF1 Nucleosome assembly factor, involved in chromatin assembly and disassembly, anti-silencing protein that causes derepression of silent loci when overexpressed

CAGLOM01540g YDR392W SPTS transcription factor

Table F.3: Summary of null mutant CGTFKOLib strains showing insufficent growth in YEPD 5//gm1-1 (0-11.5 hours) for nonlinear regression based estimation of midpoint and upper asymptote. Strains above the line show a general

growth defect in YEPD and can also be found in Table F.2

CG ORF SC ORF Gene Name Description CAGLOE03718g YHL025W SNF6 RNA polymerase II transcription

factor activity CAGL0C03399g YNR052C POPS transcription factor (putative) CAGLOF08283g YGR252W GCN5 Ada2/Gcn5/Ada3 transcription ac-

tivator complex CAGLOE00693g YDR176W NGG1 Involved in glucose repression of





CAGLOL05412g

CAGLOM01540g CAGL0L02123g

CAGLOH07711g

CAGLOE00627g CAGLOB01441g

CAGLOD01364g CAGL00O2277g

YJL115W

YDR392W YBR289W

YGL244W

YCRO81W YNL330C

YBR112C YER040W

ASFI

SPT3 SNF5

RTF1

SRB8 RPD3

SSNG GLN3

Nucleosome assembly factor, involved in chromatin assembly and disassembly, anti-silencing protein that causes derepression of silent loci when overexpressed transcription factor RNA polymerase II transcription factor activity Subunit of the RNA polymerase II-associated Pafl complex; directly or indirectly regulates DNA-binding properties of Spt15p and relative activities of different TATA elements transcription factor complex Histone deacetylase; regulates tran-scription and silencing General repressor of transcription transcriptional activator of nitrogen-regulated genes

CAGLOK06193g

CAGLOA00451g

CAGLOM02739g CAGL0001485g CAGLOB03575g CAGLOE02079g CAGLOF01837g CAGLOD01430g

CAGLOF03025g

CAGLOA01628g

CAGLOM06875g CAGLOL11880g

CAGLOL12650g CAGLOL02585g

CAGLOL10472g

YDR448W

YGL013C

YPL129W YPL181W YEL056W YOL116W YLR055C YPRO68C

YDR421W

YGL035C

YNL025C YER169W

YPL042C YKR099W

YDR443C

ADA2

PDR1

TAF14 OT/6 HAT2 MSNI SPT8 HOST

ARO8O

MIGI

SSN8 RPH1

SSN3 BAS/

SSN2

transcription factor, member of ADA and SAGA, two transcriptional adaptor/HAT (histone acetyltransferase)complexes zinc finger transcription factor of the Zn(2)-Cys(6) binuclear cluster domain type transcription factor TFIID complex transcription factor binding Histone acetyltransferase subunit 2, transcriptional activator transcription factor Putative class I histone deacetylase (HDAC) with sequence similarity to Hdalp, Rpd3p, Hos2p, and Hos3p; deletion results in increased histone acetylation at rDNA repeats; specific RNA polymerase II tran-scription factor activity transcription factor involved in glucose repression; C2H2 zinc finger protein which resembles the mam-malian Egr and Wilms tumour pro-teins transcription factor complex JmjC domain-containing histone demethylase which can specifically demethylate H3K36 tri- and dimethyl modification states; tran-scriptional repressor of PHR1 transcription factor complex Myb-related transcription factor in-volved in regulating basal and in-duced expression of genes of the purine and histidine biosynthesis pathways Required for stable association of SrblOp-Srbllp kinase with RNA polymerase holoenzyme; regulates YGP 1 epxression; component of RNA polymerase II holoenzyme and Kornberg's mediator (SRB) subcomplex; transcription factor




CAGLOK05841g YLR256W-like HARI-like Zinc finger transcription factor in-volved in the complex regulation of gene expression in response to levels of heme and oxygen

Table F.4: Summary of null mutant CGTFKOLib strains showing insufficent growth in YEPD 10pgm1-1 (0-11.5 hours) for nonlinear regression based estimation of midpoint and upper asymptote. Strains above the line show a general

growth defect in YEPD and can also be found in Table F.2

CC ORF SC ORF Gene Name Description CAGL0003399g YNR052C POP2 transcription factor (putative) CAGLOF08283g YGR252W GCN5 Ada2/Gcn5/Ada3 transcription ac-

tivator complex CAGLOE00693g YDR176W NGGI Involved in glucose repression of


CAGLOK06193g YDR448W ADA2 transcription factor, member of ADA and SAGA, two transcriptional adaptor/HAT (histone acetyltransferase)complexes

CAGLOL02123g YBR289W SNF5 RNA polymerase II transcription factor activity

CAGLOA00451g YGL013C PDRI zinc finger transcription factor of the Zn(2)-Cys(6) binuclear cluster domain type

CAGL0001485g YPL181W CTI6 transcription factor binding CAGLOB03575g YEL056W HAT2 Histone acetyltransferase subunit 2 CAGLOE03718g YHL025W SNF6 RNA polymerase II transcription

factor activity—Function— CAGL0D01430g YPRO68C HOS1 Putative class I histone deacetylase

(HDAC) with sequence similarity to Hdalp, Rpd3p, Hos2p, and Hos3p; deletion results in increased histone acetylation at rDNA repeats;

CAGLOF03025g YDR421W AR080 specific RNA polymerase II tran-scription factor activity

CAGLOA01628g YGL035C MIG1 transcription factor involved in glucose repression; C2H2 zinc finger protein which resembles the mam-malian Egr and Wilms tumour pro-teins

CAGLOF01837g YLR055C SPT8 transcription factor CAGLOD01364g YBR112C SSN6 General repressor of transcription CAGLOB03421g YLR256W HAPI Heme-responsive zinc finger tran-

scription factor of the Zn(2)-Cys(6) binuclear cluster domain type; redox sensing regulator of gene expression (activates CYC1, CYC7, CYP3, CYB2, CTT1, COR2, ROX1, ERGS, ERG11, SOD2 and YHB1; represses HEM13)



continued from previous page CG ORF SC ORF Gene Name Description CAGLOB01441g

CAGL00O2277g

CAGLOE02079g

YNL330C

YER040W

YOL116W

RPD3

GLN3

MSNI

Histone deacetylase; regulates tran-scription and silencing transcriptional activator of nitrogen-regulated genes transcriptional activator

CAGLOM01540g CAGLOL05412g

CAGLOK05841g

CAGLOM02739g

CAGLOE00627g CAGLOH07711g

CAGLOM02585g CAGLOJ04400g

CAGLOD00682g

CAGLOJ06182g

CAGLOJ10472g

CAGLOK09900g

CAGLOL04400g

CAGLOL11880g

CAGLOA04235g

CAGLOL03916g

YDR392W YJL115W

YLR256W-like

YPL129W

YCR081W YGL244W

YPL138C YI3L021C

YLR403W

YNL167C

YDR443C

YOR358W

YOR162C

YER169W

YBL052C

YOR113W

SPT3 ASF1

HAP1-like

TAF14

SR138 RTF1

SPP1 HAP3

SFP1-like (not orthologuc)

SKO1

SSNS

HAP5

YRR1

RPH1

SASS

AZFI-LIKE

transcription factor Nucleosome assembly factor, involved in chromatin assembly and disassembly, anti-silencing protein that causes derepression of silent loci when overexpressed Zinc finger transcription factor in-volved in the complex regulation of gene expression in response to levels of heme and oxygen transcription factor TFIID complex—Component— transcription factor complex Subunit of the RNA polymerase II-associated Pafl complex; directly or indirectly regulates DNA-binding properties of Sptl5p and relative activities of different TATA elements transcriptional activator activity transcriptional activator protein of CYCI (component of HAP2/HAP3 heteromer) transcription factor activity Func-tion Basic leucine zipper (bZIP) tran-scription factor of the ATF/CREB family that forms a complex with Tuplp and Ssn6p to both activate and repress transcription; cytoso- lie and nuclear protein involved in the osmotic and oxidative stress re-sponses Required for stable association of SrblOp-Srbllp kinase with RNA polymerase holoenzyme; regulates YGP1 epxression; component of RNA polymerase II holoenzyme and Kornberg's mediator (SRB) subcomplex; transcription factor CCAAT-binding transcription fac-tor component (along with Hap2p and Hap3p) Zn2-Cys6 zinc-finger transcription factor that activates genes involved in multidrug resistance; paralog of Yrrlp, acting on an overlapping set of target genes JmjC domain-containing histone demethylase which can specifically demethylate H3K36 tri- and dimethyl modification states; tran-scriptional repressor of PHR1 Protein with histone acetyltransferase activity Zinc-finger transcription factor, in-volved in induction of CLN3 tran-scription in response to glucose; ge-netic and physical interactions in-dicate a possible role in mitochon-dria] transcription or genome main-tenance



continued from previous page CG ORF SC ORF Gene Name Description CAGLOJ05060g

CAGL0.110274g

CAGL0107183g

CAGLOK11902g CAGLOF04081g

CAGLOH08239g CAGLOJ06974g

CAGLOF00803g

CAGL0005335g

CAGLOH06677g CAGLOB01947g

CAGLOK04543g

CAGL00O2519g

CAGLOD00462g CAGLOK07161g

CAGLOG08844g

CAGLOH02145g

YJL056C

YNL076W

YOR140W

YDR034C YBRO83W

YOR025W YPL116W

YOLO51W

YOL067C

YIL154C YDR123C

YGRO63C

YER028C

YKL185W YGL115w

YIL13OW

YHR084W

ZAPI

MKS1

SFL1

LYSI4 TEC1-LIKE

HST3 HOS3

GALII

RTG1

IMP2' INO2

SPT4

MIG3

ASH1 SNR4-like

ASG/

STEI2

Zinc-regulated transcription factor, binds to zinc-responsive promoter elements to induce transcription of certain genes in the presence of zinc; regulates its own transcription; contains seven zinc-finger do-mains Negative regulator of nitrogen metabolism transcription factor with domains homologous to Hsflp and to myc oncoprotein; required for normal cell surface assembly and floccu- lence transcriptional activator activity transcription factor of the TEA/ATTS DNA-binding domain family, regulator of Tyl expression Protein similar to Sir2p Trichostatin A-insensitive homod-imeric histone deacetylase (HDAC) with specificity in vitro for histones H3, H4, H2A, and H2B; similar to Hdalp, Rpd3p, Hos1p, and Hos2p; Component of the Mediator complex; interacts with RNA polymerase II and the general transcrip-tion factors to form the RNA poly-merase II holoenzyme; affects tran-scription by acting as target of ac-tivators and repressors transcription factor (bHLH) involved in interorganelle communi-cation between mitochondria, per-oxisomes, and nucleus transcription factor transcription factor required for derepression of inositol-choline- regulated genes involved in phospholipid synthesis; potential Cdc28p substrate; helix-loop-helix protein Protein involved in chromatin structure that influences expression of many genes transcription factor activity Func-tion GATA-type transcription factor Protein involved in derepression of glucose-repressed genes, acts with Snflp Proposed transcriptional activator, member of the Gal4p family of zinc cluster proteins transcription factor that is activated by a MAP kinase signaling cascade, activates genes involved in mating or pseudohyphal/invasive growth pathways; cooperates with Teclp transcription factor to regulate genes specific for invasive growth



continued from previous page CG ORF SC ORF Gene Name Description CAGLOM09405g

CAGLOL06028g CAGLOK08756g CAGLOL12650g

CAGLOM03025g

CAGLOB02651g

CAGLOG07249g

CAGLOL04576g

CAGL0108085g

CAGLOM07634g

CAGLOK12540g

CAGLOJ07942g

CAGLOK08668g

CAGLOM01716g

CAGLOE02805g CAGLOK07634g

CAGLOE04884g

CAGLOM09955g

CAGLOG04389g

CAGLOK09372g

YBL103C

YGL181W YIR018W YPL042C

YMR280C

YDR253C

YML027W

YOR172W

YOL071W

YMRO16C

YEL031W

YNL257C

YIR017C

YBRO83W

YBLOO8W YFLO21W

YDR216W

YLR403W

YJL110C

YGL209W

RTG3

GTSI YAPS SSN3

CATS

MET32

YOX1

YRMI

EMI5

SOK2

HAC1

SIPS

MET28

TEC1

HIRI GAT1

ADRI

SFP1

GZF3

MIG2

Basic helix-loop-helix-leucine zipper (bHLH/Zip) transcription fac-tor that forms a complex with an-other bHLH/Zip protein, Rtglp, to activate the retrograde (RTG) and TOR pathways transcriptional activator activity bZIP transcription factor transcription factor complex—Component— specific RNA polymerase II tran-scription factor activity specific RNA polymerase II tran-scription factor activity Homeodomain-containing transcriptional repressor, binds to Mcmlp and to early cell cycle boxes (ECBs) in the promoters of cell cycle-regulated genes expressed in M/G1 phase; expression is cell cycle-regulated; potential Cdc28p substrate Zn2-Cys6 zinc-finger transcription factor that activates genes involved in multidrug resistance; paralog of Yrrlp, Non-essential protein of unknown function required for transcriptional induction of the early meiotic-specific transcription factor IME1, also required for sporulation Protein that can when overexpressed suppress mutants of cAMP-dependent protein kinase; displays homologies to several transcription factors bZIP transcription factor (ATF/CREB1 homolog) that regulates the unfolded-protein response, via UPRE binding, and membrane biogenesis; ER stress- induced splicing pathway utilizing Ire1p, Trllp and Ada5p facilitates efficient Haclp synthesis transcriptional activator, interacting with Snflp transcriptional activator in the Cbflp-Met4p-Met28p complex Transcription factor required for full Tyl epxression, Tyl-mediated gene activation, and haploid invasive and diploid pseudohyphal growth Histone transcription inhibitor transcriptional activator with GATA-1-type Zn finger DNA- binding motif transcription factor activity Func-tion transcription factor activity Func-tion specific RNA polymerase II tran-scription factor activity specific RNA polymerase II tran-scription factor activity



continued from previous page CG ORF SC ORF Gene Name Description CAGLOK05797g

CAGLOL11770g

CAGLOJ03454g

CAGLOM00286g CAGLOK08624g

CAGLOM06875g

CAGLOI10769g

CAGLOM 11440g

CAGLOM06699g CAGLOM02563g CAGLOD02926g

CAGLOK03003g

CAGLOL03377g

CAGLOH05621g CAGLOL06842g CAGLOL03157g

CAGLOL02585g

CAGLOG05379g CAGLOI04246g

CAGLOM06831g

YDR512C

YER164W

YNL021W

YJR140C YKL109W

YNL025C

YMR043w

YLR098C

YIL084C YPL139C YLR015W

YMR070W

YJL089W

YPL089C YDL080C YKR034W

YKR099W

YNL199C YGL162W

YNL027W

EMII

CHDI

HDAI

HIR3 HAP4

SSN8

MCM/-like

CHA4

SDS3 UME1 BRE2

MOTS

SIP4

RLMI THIS

DAL80

BASI

GDR2 SUT1

CRZ1

Non-essential protein of unknown function required for transcriptional induction of the early meiotic-specific transcription factor IME1, also required for sporulation Nucleosome remodeling factor that functions in regulation of transcrip-tion elongation; component of both the SAGA and SILK complexes Component of histone deacetylase A, 75 kDa subunit Histone transcription regulator 3 transcriptional activator protein of CYC1 (component of HAP2/HAP3 heteromer) transcription factor corn- plex—Component— Transcription factor involved in cell-type-specific transcription and pheromone response DNA binding transcriptional acti-vator of CHA1 Transcriptional regulator Negative regulator of meiosis Subunit of the COMPASS (Set1C) complex, which methylates histone H3 on lysine 4 and is required in transcriptional silencing near telomeres; involved in telom-ere maintenance; Nuclear transcription factor with two Cys2-His2 zinc fingers; involved in repression of a subset of hypoxic genes by Roxlp, repression of sev-eral DAN/TIR genes during aerobic growth, and repression of ergosterol biosynthetic genes specific RNA polymerase II tran-scription factor activity transcriptional activator activity transcriptional activator activity Negative regulator of genes in multiple nitrogen degradation pathways; member of the GATA-binding family, forms homodimers and het-erodimers with Dehlp Myb-related transcription factor in-volved in regulating basal and in-duced expression of genes of the purine and histidine biosynthesis pathways transcription factor specific RNA polymerase II tran-scription factor activity transcription factor that activates transcription of genes involved in stress response; nuclear localization is positively regulated by calcineurin-mediated dephosphory-lation



continued from previous page CG ORF SC ORF Gene Name Description CAGLOM06919g YNL023C FAP1 transcription factor homolog; sim-

ilarity to Drosophila melanogaster shuttle craft protein; similarity to human NEX1 protein; similar-ity to human DNA-binding protein tenascin

CAGLOK04257g YGRO44C RME1 Transcriptional repressor of meiosis in non-a/alpha cells

CAGLOM09042g YJR094C /ME/ Transcription factor required for sporulation, positive regulator of IME2 and many sporulation genes


Table F.5: Summary of hierarchical clustering membership with associated me-dian growth rates in YEPD f fluconazole (5 10/./g m1-1). Cluster Cl and C5

containing fluconazole sensitive and growth defect strains respectively are discussed in the

main text and are depicted in 5.4

Cluster Members YEPD 5pgm1-1 Fluc (hrs) lOugml-1 Flue (hrs)

1 pdrl, migl, hos2, hat2, cti6, ashl, sfpl, sfpl, hosl, srb8, ,nsnl, aro80, sste2, hapl-like, hap4, rph1, .sar13, sfpl -like

10.11 15.00 15.00

2 ga183, hst1, pho4, tupl, swi5, heel, shcl, adr1, yap7, cad1, tspl, mcml, snf4, rbp1, pho23, OM rlml, fzfl, sut1, cst6, hcm1, pip2, gist, sir4, rgtl, haal, spt2, mct31, dig1, redt80, smp1, crzl, fapl, msn4

9.20 10.01 9.00

3 sass, ino2, met32, hapl, mig3, rtg1, bra, ga111, tecl-likea, 8711(11, yor1, asgl, ste12, imp2', hst3, ATCC

8.90 10.79 15.0

2001, 41,13, cha4, cmi5, mcml-like, hdal, hap3, zap/, .skol, hos3, sip3, mots, renel, spt4, 8144-like, gat1, met28, yaps, mia, hap5, lys14, hac1, da180, sip4, azfl-like, yrrl, yrrn/, gtsl, this, chill, tec1, umel, sppl, cats, sok2, imc1, rtg3, sf11

4 sell, kar4, rfr1, cup9, idol, rint101, hst4, msn2, ask10, gist, tos8, wail, hot1, stb5, hat1, znes1, stc12-like, da182

9.20 12.00 7.26

5 Itg2, rpd3, 91713, pop2, SSTE6, nggl, 8710, spt8, gen5, rt11, taf14, treks1, ada2, snf5, asf1, .spt3

15.00 14.88 14.93

6 tos4, sin3, gzf3, gcr2, fkh2, sas2, emil, basl, sass, pho2, put3, hir3, odes

11.26 12.54 9.76

7 ez1/, °aft, mbpl, pprl, stp2, tye7, stip1, da181, mss11, upc2, skn7, flo8, pogl, fkh1, spot, guilt-like, arg81, hap.2, hinst, hir2, cup2, hal9, stpl, not3, hst2, sut2, rds2, stbl, ace2, yap6, yaps-like, tupl-like

8.25 9.45 7.23

Table F.6: Effect of timepoint and growth condition on Z factor. The Z factor

statistic, is commonly used in high throughput screening assays as a quality metric (769)

and is defined in equation 5.4. Values for Z were calculated for cells grown in YEPD (YEPD

Z) or YEPD supplemented with fluconazole (5pg iul-1) (Fluconazole Z).

Timepoint (hrs) YEPD Z Fluconazole Z

1.5 -1.22 -1.85

4 -1.75 -2.07 6 -1.16 -1.61 8 -0.54 -1.12

10 0.13 -0.43

11.5 0.84 0.29


Table F.7: Fitted parameter values for a linear model describing optical density at 11.5 hours as a function of strain genotype, growth condition (YEPD or YEPD supplemented with fluconazole (5,ag ii1-1)) and the interaction between growth condition and genotype (equation 5.3). Plate 1 of 2. Results are for strains

contained on the first of two library plates of the CGTFKOlib. The residual standard

error of the fit was 0.159 on 576 degrees of freedom, with a multiple R2= 0.9864, adjusted

R2=0.9819 , F-statistic= 218.2 on 192 and 576 degrees of freedom, p < 2.2e - 16. The

genotype effect is of the form straincoden where n is the number of the strain in the plate.

The interaction term is of the form straincodesn:fluconazole5ugml-1. Note that significant

interactions (p < 0.05) are reported for strain 4 (pdrl), 38 (srb8), 39 (nggl), 65 (gcn5) and

85 (ga111-like) on this plate. Strain 97 is a dummy code representing the only blank well

on this plate

Effect Average 0D600 S.E.M t value p straincodes97 0.101 0.079 1.28 0.201 straincodes2 1.272 0.079 16.04 < 2e-16 straincodesl 1.274 0.079 16.074 < 2e-16 straincodes4 1.246 0.079 15.718 < 2e-16 straincodes5 1.193 0.079 15.043 < 2e-16 straincodes6 1.277 0.079 16.103 < 2e-16 straincodes7 1.222 0.079 15.412 < 2e-16 straincodes8 1.301 0.079 16.402 < 2e-16 straincodes9 1.274 0.079 16.062 < 2e-16 straincodes10 1.144 0.079 14.425 < 2e-16 straincodesll 1.265 0.079 15.958 < 2e-16 straincodes12 1.342 0.079 16.929 < 2e-16

straincodesl3 1.117 0.079 14.091 < 2e-16 straincodesl4 1.325 0.079 16.711 < 2e-16

straincodesl5 1.259 0.079 15.876 < 2e-16

straincodesl6 1.245 0.079 15.702 < 2e-16 straincodes17 1.29 0.079 16.273 < 2e-16

straincodesl8 1.227 0.079 15.475 < 2e-16 straincodesl9 1.324 0.079 16.702 < 2e-16

straincodes20 1.314 0.079 16.576 < 2e-16 straincodes21 1.242 0.079 15.667 < 2e-16


straincodes24 0.411 0.079 5.184 < 2e-16


straincodes27 1.246 0.079 15.712 < 2e-16


straincodes30 1.308 0.079 16.5 < 2e-16 straincodes3l 0.917 0.079 11.565 < 2e-16


straincodes34 1.242 0.079 15.667 < 2e-16

straincodes35 1.176 0.079 14.838 < 2e-16


straincodes38 1.209 0.079 15.245 < 2e-16

straincodes39 0.796 0.079 10.046 < 2e-16

straincodes40 1.274 0.079 16.074 < 2e-16



continued from previous page Effect Average ()Dom S.E.M t value p straincodes4l 1.29 0.079 16.267 < 2e-16 straincodes42 1.129 0.079 14.242 < 2e-16 straincodes43 1.229 0.079 15.497 < 2e-16 straincodes44 0.411 0.079 5.184 < 2e-16 straincodes45 1.193 0.079 15.049 < 2e-16 straincodes46 1.172 0.079 14.778 < 2e-16 straincodes47 1.29 0.079 16.267 < 2e-16 straincodes48 1.257 0.079 15.85 < 2e-16 straincodes49 1.289 0.079 16.263 < 2e-16 straincodes50 1.289 0.079 16.263 < 2e-16 straincodes5l 1.241 0.079 15.652 < 2e-16 straincodes52 1.038 0.079 13.091 < 2e-16 straincodes53 1.302 0.079 16.418 < 2e-16 straincodes54 1.156 0.079 14.586 < 2e-16 straincodes55 1.235 0.079 15.573 < 2e-16 straincodes56 1.263 0.079 15.929 < 2e-16 straincodes57 1.271 0.079 16.036 < 2e-16 straincodes58 1.215 0.079 15.321 < 2e-16 straincodes59 1.283 0.079 16.178 < 2e-16 straincodes60 1.183 0.079 14.927 < 2e-16 straincodes61 1.294 0.079 16.32 < 2e-16 straincodes62 1.272 0.079 16.049 < 2e-16 straincodes63 1.39 0.079 17.531 < 2e-16 straincodes64 1.314 0.079 16.576 < 2e-16 straincodes65 0.888 0.079 11.2 < 2e-16 straincodes66 1.315 0.079 16.582 < 2e-16 straincodes67 1.221 0.079 15.399 < 2e-16 straincodes68 1.255 0.079 15.831 < 2e-16 straincodes69 1.2 0.079 15.138 < 2e-16 straincodes70 1.386 0.079 17.48 < 2e-16 straincodes7l 1.069 0.079 13.489 < 2e-16 straincodes72 1.227 0.079 15.478 < 2e-16 straincodes73 1.171 0.079 14.769 < 2e-16 straincodes74 1.306 0.079 16.465 < 2e-16 straincodes75 1.286 0.079 16.222 < 2e-16 straincodes76 1.241 0.079 15.649 < 2e-16

straincodes77 1.222 0.079 15.418 < 2e-16 straincodes78 1.261 0.079 15.907 < 2e-16 straincodes79 1.259 0.079 15.882 < 2e-16 straincodes80 1.318 0.079 16.629 < 2e-16 straincodes81 1.192 0.079 15.037 < 2e-16 straincodes82 1.236 0.079 15.589 < 2e-16 straincodes83 1.314 0.079 16.569 < 2e-16 straincodes84 1.284 0.079 16.197 < 2e-16 straincodes85 1.272 0.079 16.046 < 2e-16 straincodes86 1.217 0.079 15.346 < 2e-16

straincodes87 1.274 0.079 16.074 < 2e-16 straincodes88 1.257 0.079 15.853 < 2e-16 straincodes89 1.082 0.079 13.646 < 2e-16 straincodes90 1.224 0.079 15.444 < 2e-16 straincodes9l 1.243 0.079 15.68 < 2e-16 straincodes92 1.096 0.079 13.823 < 2e-16 straincodes93 1.213 0.079 15.299 < 2e-16 straincodes94 1.284 0.079 16.194 < 2e-16

straincodes95 1.326 0.079 16.721 < 2e-16 straincodes96 1.227 0.079 15.472 < 2e-16 fluconazole5ugml-1 0.012 0.112 0.103 0.918 straincodes2:fluconazole5ugml-1 -0.045 0.159 -0.284 0.777 straincodesl:fluconazole5ugml-1 -0.157 0.159 -0.987 0.324 straincodes4:fluconazole5ugml-1 -0.846 0.159 -5.332 0 straincodes5ffluconazole5ugm1-1 -0.183 0.159 -1.151 0.25 straincodes6:fluconazole5ugnal-1 -0.117 0.159 -0.739 0.46



continued from previous page Effect Average OD600 S.E.M t value p straincodes7:fluconazole5ugm1-1 -0.143 0.159 -0.899 0.369 straincodes8:fluconazole5ugml-1 -0.115 0.159 -0.722 0.471 straincodes9:fluconazole5ugm1-1 -0.159 0.159 -1 0.318 straincodes10:fluconazole5ugm1-1 -0.171 0.159 -1.08 0.281 straincodes11:fluconazole5ugm1-1 -0.124 0.159 -0.784 0.434 straincodes12:fluconazole5ugml-1 -0.224 0.159 -1.414 0.158 straincodesl3:fluconazole5ugml-1 -0.043 0.159 -0.273 0.785 straincodesl4:fluconazole5ugml-1 -0.092 0.159 -0.58 0.562 straincodes15:fluconazole5ugm1-1 -0.157 0.159 -0.988 0.323 straincodes16:fluconazole5ugm1-1 -0.154 0.159 -0.97 0.333 straincodesl7:fluconazole5ugml-1 -0.217 0.159 -1.368 0.172 straincodes18:fluconazole5ugm1-1 -0.184 0.159 -1.162 0.246 straincodes19:fluconazole5ugm1-1 -0.039 0.159 -0.248 0.805 straincodes20:fluconazole5ugm1-1 -0.288 0.159 -1.818 0.07 straincodes21:fluconazole5ugm1-1 -0.138 0.159 -0.869 0.385 straincodes22:fluconazole5ugm1-1 -0.02 0.159 -0.126 0.9 straincodes23:fluconazole5ugm1-1 -0.156 0.159 -0.984 0.326 straincodes24:fluconazole5ugm1-1 -0.183 0.159 -1.154 0.249 straincodes25:fluconazole5ugml-1 -0.056 0.159 -0.355 0.723 straincodes26:fluconazole5ugml-1 -0.064 0.159 -0.404 0.687 straincodes27:fluconazole5ugm1-1 -0.057 0.159 -0.359 0.719 straincodes28:fluconazole5ugm1-1 -0.161 0.159 -1.012 0.312 straincodes29:fluconazole5ugml-1 -0.207 0.159 -1.304 0.193 straincodes30:fluconazole5ugml-1 -0.088 0.159 -0.552 0.581 straincodes31:fluconazole5ugm1-1 0.064 0.159 0.405 0.686 straincodes32:fluconazole5ugm1-1 -0.151 0.159 -0.952 0.341 straincodes33:fluconazole5ugm1-1 -0.125 0.159 -0.785 0.433 straincodes34:fluconazole5ugm1-1 -0.022 0.159 -0.139 0.89 straincodes35:fluconazole5ugml-1 -0.09 0.159 -0.566 0.572 straincodes36:fluconazole5ugm1-1 -0.077 0.159 -0.487 0.626 straincodes37:fluconazole5ugm1-1 -0.011 0.159 -0.066 0.947 straincodes38:fluconazole5ugml-1 -0.407 0.159 -2.567 0.011 straincodes39:fluconazole5ugml-1 -0.556 0.159 -3.503 0 straincodes40:fluconazole5ugm1-1 -0.083 0.159 -0.52 0.603 straincodes4l:fluconazole5ugml-1 -0.2 0.159 -1.258 0.209 straincodes42:fluconazole5ugm1-1 -0.097 0.159 -0.612 0.541 straincodes43:fluconazole5ugm1-1 -0.045 0.159 -0.285 0.775 straincodes44:fluconazole5ugm1-1 -0.224 0.159 -1.411 0.159 straincodes45:fluconazole5ugm1-1 -0.108 0.159 -0.678 0.498 straincodes46:fluconazole5ugml-1 -0.048 0.159 -0.303 0.762 straincodes47:fluconazole5ugm1-1 -0.105 0.159 -0.661 0.509 straincodes48:fluconazole5ugml-1 -0.104 0.159 -0.657 0.511 straincodes49:fluconazole5ugm1-1 -0.022 0.159 -0.137 0.891 straincodes50:fluconazole5ugm1-1 -0.041 0.159 -0.255 0.799 straincodes51:fluconazole5ugm1-1 -0.111 0.159 -0.698 0.485 straincodes52:fluconazole5ugm1-1 -0.287 0.159 -1.811 0.071 straincodes53:fluconazole5ugm1-1 -0.189 0.159 -1.189 0.235 straincodes54:fluconazole5ugm1-1 -0.015 0.159 -0.091 0.927 straincodes55:fluconazole5ugml-1 -0.024 0.159 -0.151 0.88 straincodes56:fluconazole5ugm1-1 -0.1 0.159 -0.631 0.529 straincodes57:fluconazole5ugm1-1 -0.148 0.159 -0.935 0.35 straincodes58:fluconazole5ugm1-1 -0.108 0.159 -0.681 0.496 straincodes59:fluconazole5ugm1-1 -0.053 0.159 -0.336 0.737 straincodes60:fluconazole5ugm1-1 -0.082 0.159 -0.514 0.607 straincodes61:fluconazole5ugml-1 -0.002 0.159 -0.014 0.989 straincodes62:fluconazole5ugm1-1 -0.053 0.159 -0.334 0.738 straincodes63:fluconazole5ugm1-1 -0.067 0.159 -0.421 0.674 straincodes64:fluconazole5ugml-1 -0.127 0.159 -0.798 0.425 straincodes65:fluconazole5ugml-1 -0.603 0.159 -3.801 0 straincodes66:fluconazole5ugm1-1 -0.067 0.159 -0.424 0.672 straincodes67:fluconazole5ugm1-1 -0.136 0.159 -0.858 0.391 straincodes68:fluconazole5ugm1-1 -0.093 0.159 -0.586 0.558

continued- on next page


continued from previous page Effect Average OD900 S.E.M t value p straincodes69:fluconazole5ugm1-1 -0.063 0.159 -0.396 0.692 straincodes70:fluconazole5ugm1-1 -0.041 0.159 -0.259 0.796 straincodes71:fluconazole5ugm1-1 -0.044 0.159 -0.279 0.78 straincodes72:fluconazole5ugm1-1 -0.073 0.159 -0.462 0.644 straincodes73:fluconazole5ugml-1 -0.035 0.159 -0.218 0.828 straincodes74:fluconazole5ugml-1 -0.127 0.159 -0.799 0.424 straincodes75:fluconazole5ugm1-1 -0.145 0.159 -0.916 0.36 straincodes76:fluconazole5ugm1-1 -0.132 0.159 -0.834 0.405 straincodes77:fluconazole5ugml-1 -0.111 0.159 -0.702 0.483 straincodes78:fluconazole5ugm1-1 -0.063 0.159 -0.396 0.692 straincodes79:fluconazole5ugm1-1 -0.061 0.159 -0.385 0.701 straincodes80:fluconazole5ugm1-1 -0.078 0.159 -0.493 0.622 straincodes81:fluconazole5ugm1-1 -0.108 0.159 -0.679 0.497 straincodes82:fluconazole5ugm1-1 -0.076 0.159 -0.476 0.634 straincodes83:fluconazole5ugm1-1 -0.094 0.159 -0.59 0.556 straincodes84:fluconazole5ugm1-1 -0.059 0.159 -0.37 0.711 straincodes85:fluconazole5ugm1-1 -0.732 0.159 -4.614 0 straincodes86:fluconazole5ugm1-1 -0.012 0.159 -0.073 0.942 straincodes87:fluconazole5ugml-1 -0.063 0.159 -0.396 0.692 straincodes88:fluconazole5ugm1-1 -0.065 0.159 -0.407 0.684 straincodes89:fluconazole5ugm1-1 -0.111 0.159 -0.702 0.483 straincodes90:fluconazole5ugm1-1 0.018 0.159 0.114 0.91 straincodes91:fluconazole5ugm1-1 0.002 0.159 0.016 0.987 straincodes92:fluconazole5ugm1-1 -0.018 0.159 -0.115 0.908 straincodes93:fluconazole5ugm1-1 -0.063 0.159 -0.394 0.694 straincodes94:fluconazole5ugml-1 0 0.159 0 1 straincodes95:fluconazole5ugm1-1 -0.039 0.159 -0.246 0.806 straincodes96:fluconazole5ugm1-1 -0.054 0.159 -0.337 0.736


Table F.8: Fitted parameter values for a linear model describing optical density at 11.5 hours as a function of strain genotype, growth condition (YEPD or YEPD supplemented with fluconazole (5/.ig p1-1) and the interaction between growth condition and genotype (equation 5.3). Plate two of two. Results are for strains contained on the second of two library plates of the CGTFKO1ib. The residual standard error of the fit was 0.183 on 576 degrees of freedom, with a multiple R2= 0.9802,

adjusted R2=0.9735 , F-statistic= 148.1 on 192 and 576 degrees of freedom, p < 2.2e - 16. The genotype effect is of the form straincoden where n is the number of the strain in the plate. The interaction term is of the form straincodesn:fluconazole5ugm1-1. Note that significant interactions (p < 0.05) are reported for strain 27 (hap)-like), 28 (ada2) and 61

(rphl) on this plate. Strain codes 93-97 inclusive are blank wells on this plate

Effect Average ()Dm) S.E.M t value p

straincodes97 0.063 0.092 0.69 0.49 straincodes2 1.264 0.092 13.796 0

straincodesl 1.259 0.092 13.747 0 straincodes4 1.254 0.092 13.687 0

straincodes5 1.071 0.092 11.695 0 straincodes6 1.026 0.092 11.193 0

straincodes7 1.274 0.092 13.905 0 straincodes8 1.097 0.092 11.979 0 straincodes9 1.192 0.092 13.013 0

straincodes10 1.119 0.092 12.208 0

straincodesll 1.25 0.092 13.643 0 straincodesl2 1.28 0.092 13.97 0

straincodesl3 1.217 0.092 13.288 0 straincodesl4 1.289 0.092 14.071 0

straincodesl5 1.238 0.092 13.518 0

straincodes16 1.257 0.092 13.719 0

straincodes17 1.205 0.092 13.157 0 straincodes18 1.143 0.092 12.478 0 straincodesl9 0.863 0.092 9.414 0 straincodes20 0.851 0.092 9.294 0 straincodes2) 1.051 0.092 11.474 0 straincodes22 1.246 0.092 13.602 0

straincodes23 1.138 0.092 12.418 0

straincodes24 1.282 0.092 13.987 0


straincodes27 1.266 0.092 13.823 0 straincodes28 0.92 0.092 10.039 0 straincodes29 1.208 0.092 13.182 0 straincodes30 1.192 0.092 13.015 0

straincodes3l 1.195 0.092 13.04 0

straincodes32 1.249 0.092 13.635 0


straincodes35 1.246 0.092 13.605 0


straincodes38 1.291 0.092 14.088 0 straincodes39 1.271 0.092 13.872 0 straincodes40 1.274 0.092 13.905 0

straincodes4) 1.239 0.092 13.52 0 straincodes42 0.492 0.092 5.37 0



continued from previous page Effect Average OD600 S.E.M t value p straincodes43 0.891 0.092 9.719 0 straincodes44 1.217 0.092 13.286 0 straincodes45 1.195 0.092 13.043 0 straincodes46 1.144 0.092 12.481 0 straincodes47 1.274 0.092 13.908 0 straincodes48 1.291 0.092 14.091 0 straincodes49 1.244 0.092 13.578 0 straincodes50 0.32 0.092 3.495 0.001 straincodes5l 1.271 0.092 13.875 0 straincodes52 1.285 0.092 14.022 0 straincodes53 1.043 0.092 11.378 0 straincodes54 1.205 0.092 13.152 0 straincodes55 1.285 0.092 14.02 0 straincodes56 1.211 0.092 13.215 0 straincodes57 1.155 0.092 12.612 0 straincodes58 1.251 0.092 13.659 0 straincodes59 1.248 0.092 13.618 0 straincodes60 1.29 0.092 14.082 0 straincodes6l 1.22 0.092 13.318 0 straincodes62 1.256 0.092 13.709 0 straincodes63 1.194 0.092 13.032 0 straincodes64 1.28 0.092 13.97 0 straincodes65 1.265 0.092 13.807 0 straincodes66 1.185 0.092 12.934 0 straincodes67 1.244 0.092 13.578 0 straincodes68 1.265 0.092 13.807 0 straincodes69 0.272 0.092 2.969 0.003 straincodes70 1.173 0.092 12.8 0 straincodes7l 1.259 0.092 13.741 0 straincodes72 1.169 0.092 12.759 0 straincodes73 1.277 0.092 13.935 0 straincodes74 1.076 0.092 11.744 0 straincodes75 1.263 0.092 13.782 0 straincodes76 1.22 0.092 13.321 0 straincodes77 1.279 0.092 13.957 0

straincodes78 1.122 0.092 12.243 0 straincodes79 1.276 0.092 13.93 0 straincodes80 1.071 0.092 11.687 0 straincodes81 1.204 0.092 13.146 0 straincodes82 0.929 0.092 10.14 0

straincodes83 1.218 0.092 13.299 0 straincodes84 1.296 0.092 14.145 0 straincodes85 1.179 0.092 12.874 0 straincodes86 1.241 .0.092 13.548 0 straincodes87 1.278 0.092 13.951 0 straincodes88 1.143 0.092 12.478 0 straincodes89 1.303 0.092 14.222 0 straincodes90 1.285 0.092 14.025 0 straincodes9l 1.272 0.092 13.883 0 straincodes92 1.187 0.092 12.958 0 straincodes93 0.07 0.092 0.761 0.447 straincodes94 0.066 0.092 0.718 0.473

straincodes95 0.067 0.092 0.734 0.463 straincodes96 0.068 0.092 0.748 0.455 conditionsB 0.001 0.13 0.004 0.997 straincodes2:conclitionsB -0.059 0.183 -0.323 0.747 straincodesl:conditionsB -0.08 0.183 -0.437 0.663 straincodes4:conditionsB -0.054 0.183 -0.296 0.767 straincodes5:conditionsB 0.027 0.183 0.147 0.883 straincodes6:conditionsB -0.028 0.183 -0.154 0.878 straincodes7:conditionsB -0.064 0.183 -0.349 0.727 straincodes&conditionsB -0.085 0.183 -0.464 0.643

continue. on next page


continued from previous page Effect Average 00600 S.E.M t value p

str aincodes9:conditionsB -0.11 0.183 -0.599 0.549 straincodes10:conditionsB -0.035 0.183 -0.191 0.849 straincodes11:conditionsB -0.051 0.183 -0.277 0.782 straincodes12:conditionsB -0.083 0.183 -0.453 0.651 straincodesl3:conditionsB 0.006 0.183 0.03 0.976 straincodesl4:conditionsB -0.202 0.183 -1.104 0.27 straincodes15:conditionsB -0.134 0.183 -0.734 0.463 straincodes16:conditionsB -0.022 0.183 -0.117 0.907 straincodesl7:conditionsB -0.067 0.183 -0.367 0.714 straincodesl8:conditionsB -0.034 0.183 -0.186 0.853 straincodes19:conditionsB -0.046 0.183 -0.25 0.803 straincodes20:conditionsB -0.02 0.183 -0.109 0.913 straincodes21:conditionsB -0.286 0.183 -1.559 0.119 straincodes22:conditionsB -0.151 0.183 -0.825 0.409 straincodes23:conditionsB -0.113 0.183 -0.618 0.537 straincodes24:conditionsB -0.084 0.183 -0.458 0.647 straincodes25:conditionsB -0.024 0.183 -0.128 0.898 straincodes26:conditionsE -0.067 0.183 -0.366 0.715 straincodes27:conditionsB -0.42 0.183 -2.293 0.022 straincodes28:conditionsB -0.599 0.183 -3.27 0.001 straincodes29:conditionsB -0.062 0.183 -0.34 0.734 straincodes30:conditionsB -0.167 0.183 -0.911 0.362 straincodes31:conditionsB -0.224 0.183 -1.222 0.222 straincodes32:conditionsB -0.187 0.183 -1.023 0.307 straincodes33:conditionsB -0.116 0.183 -0.636 0.525 straincodes34:conditionsB -0.246 0.183 -1.342 0.18 straincodes35:conditionsB -0.105 0.183 -0.573 0.567 straincodes36:conditionsB -0.031 0.183 -0.171 0.865 straincodes37:conditionsB -0.067 0.183 -0.363 0.717 straincodes38:conditionsB -0.109 0.183 -0.592 0.554 straincodes39:conditionsB -0.101 0.183 -0.554 0.58 straincodes40:conditionsS -0.116 0.183 -0.63 0.529 straincodes41:conditionsB -0.2 0.183 -1.09 0.276 straincodes42:conditionsB -0.182 0.183 -0.995 0.32 straincodes43:conditionsB -0.057 0.183 -0.308 0.758 straincodes44:conditionsB -0.15 0.183 -0.817 0.414

straincodes45:conditionsB -0.109 0.183 -0.596 0.551 straincodes46:conditions8 -0.131 0.183 -0.715 0.475

straincodes47:conditionsB -0.075 0.183 -0.409 0.682 straincodes48:conditionsB -0.028 0.183 -0.15 0.881 straincodes49:conditionsB -0.087 0.183 -0.473 0.636 straincodes50:conditionsB 0.04 0.183 0.218 0.827 straincodes51:conditionsB -0.042 0.183 -0.226 0.821 straincodes52:conditionsB -0.134 0.183 -0.734 0.463 straincodes53:conditionsB -0.147 0.183 -0.804 0.422 straincodes54:conditionsB -0.115 0.183 -0.63 0.529 straincodes55:conditionsB -0.181 0.183 -0.989 0.323 straincodes56:conditionsB -0.17 0.183 -0.925 0.355

straincodes57:conditionsB -0.014 0.183 -0.075 0.94 straincodes58:conditionsB -0.136 0.183 -0.742 0.458

straincodes59:conditionsB -0.151 0.183 -0.821 0.412 straincodes60:conditionsB 0.017 0.183 0.09 0.928 straincodes61:conditionsB -0.366 0.183 -1.999 0.046 straincodes62:conditionsB -0.027 0.183 -0.146 0.884 straincodes63:conditionsB -0.257 0.183 -1.405 0.16 straincodes64:conditionsB -0.103 0.183 -0.563 0.573 straincodes65:conditionsB -0.092 0.183 -0.502 0.616 straincodes66:conditionsB -0.15 0.183 -0.817 0.414 straincodes67:conditionsB -0.122 0.183 -0.663 0.508 straincodes68:conditionsB -0.21 0.183 -1.147 0.252 straincodes69:conditionsB -0.051 0.183 -0.277 0.782 straincodes70:conditionsB -0.072 0.183 -0.392 0.696



continued from previous page Effect Average 0D600 S.E.M t value p straincodes71:conditionsB -0.076 0.183 -0.416 0.677 straincodes72:conditionsB -0.03 0.183 -0.162 0.871 straincodes73:conditionsB 0.03 0.183 0.161 0.872 straincodes74:conditionsB -0.272 0.183 -1.486 0.138 straincodes75:conditionsB -0.031 0.183 -0.168 0.867 straincodes76:conditionsB -0.098 0.183 -0.533 0.594 straincodes77:conditionsB -0.033 0.183 -0.18 0.857 straincodes78:conditionsB 0.002 0.183 0.012 0.99 straincodes79:conditionsB -0.061 0.183 -0.333 0.739 straincodes80:conditionsB -0.038 0.183 -0.206 0.837 straincodes81:conditionsB -0.027 0.183 -0.149 0.882 straincodes82:conditionsB -0.159 0.183 -0.87 0.384 straincodes83:conditionsB -0.04 0.183 -0.218 0.827 straincodes84:conditionsB -0.039 0.183 -0.213 0.832 straincodes85:conditionsB 0.066 0.183 0.362 0.718 straincodes86:conditionsB -0.011 0.183 -0.059 0.953 straincodes87:conditionsB -0.022 0.183 -0.119 0.906 straincodes88:conditionsB -0.07 0.183 -0.379 0.705 straincodes89:conditionsB -0.065 0.183 -0.353 0.724 straincodes90:conditionsB -0.039 0.183 -0.21 0.834 straincodes9I:conditionsB 0.002 0.183 0.014 0.989 straincodes92:conditionsB -0.103 0.183 -0.563 0.573 straincodes93:conditionsB -0.003 0.183 -0.016 0.987 straincodes94:conditionsB -0.001 0.183 -0.007 0.995 straincodes95:conditionsB -0.001 0.183 -0.004 0.997 straincodes96:conditionsB -0.003 0.183 -0.018 0.986

(a) tO (b) t2

(e) t8 (f) t10

1

(g) t12 (h) t14

3 • 6 .6 7 • 10 11 12 7 1 10 11 12

1 3 0

(c) t4 (d) t6

5 8 7 • 1 18 11 12 1 2 1 • .1 8 7 8 9 10 11 12


Figure F.1: Assessment of pipette channel cross contamination on a robotic plat-form. Two fold serial dilutions of a C. glabrata culture (0D600=0.5) were dispensed into column 1 (rows A-H inclusive) and 7 (rows A-E inclusive) of a 96 well deep well plate. Columns 2-6 and 8-12 contained growth medium (YEPD) without cells. Samples (10p/1) were taken from the deep-well plate every 2 hours for 14 hours and transferred to a mi-crotitre plate containing 90 pd of sodium azide (final concentration 100mM, final OD600 = 0.1 x initial inoculation OD600) to stop cell growth. Wells lacking inoculum were inspected


CG2001

CG2001 his3

0

0 10 15 20

5 10

1(hr.) t (hrs)

(a) ATCC CG2001

(b) his.

PDR1

MIG1

_

-

0 > _ 8

/ 1

•

0 5 10 15 20

5 10 20

t(hrs) t (hrs)

(c) pdrl

(d) migl

Figure F.2: Example growth curves for four strains following incubation and sampling on a robotic platform. Glycerol stocks of each library strain, collectively maintained in two 96-well microtitre plates at -80°C, were sampled (5p,1) using a multi-channel pipette and dispensed into corresponding sterile, round-bottomed 96-well plate containing 150/21 of YEPD broth. Library strains were sub-cultured to stationary phase for 18 hours in an orbital shaker (30°C, 180 RPM) before commencing the growth curve assay. Cells from each library plate were transferred (final OD600=0.2) into square bottom, deep well plates (Anachem) containing a final volume of 500 µl of YEPD and incubated at (37°C, 180 RPM), rather than 30°C (the temperature used in the manual assay). The robot was programmed to take 100 samples of cells every two hours and dispense them into microtitre plates containing 900 of sodium azide (final concentration of 100mM). The 8-tip pipette head was washed five times with a mixture of isopropanol and copper sulphate (100mM) and rinsed with distilled water before each move to a new column on the deep well plate. Plates were stored at room temperature and 0D600 measurements where made using a Multiskan Ascent absorbance plate-reader (Thermo Scientific, U.S.A).

330

Appendix G

Transcription profiling of C. glabrata serum stress responses

G.1 Chapter overview

This truncated chapter is a record of the current status of some of the environmental stress transcription profiling I have performed. The scope of this work was large and has not yet been fully summarized (including oxidative - 1mM tBOOH, osmotic stress - 0.5 M KC1 and carbon starvation 0.01% glucose w/v), partially due to time constraints and also because of uncertainties surrounding the use of orthologous integrated molecular interaction networks, which we intended to use to identify conserved orthologous active modules. For example, to date we do not have a C. glabrata protein interaction network, so the well documented health warnings that come with protein interaction data (excellently reviewed in (265)) are further compounded by assuming that orthologous proteins have the same connections in related species. Furthermore, current protein interaction data take no account of how compartmentalization, and dynamical expression and transcription may affect the structure of these ostensibly scale-free networks (47; 49; 48; 328; 329; 330).

The concept of a co-ordinated, general stress response or environmental stress response (subsequently called the environmental stress response - ESR) emerged after the effects of normally lethal doses of one stress were sublethal after pre-treatment with a sub-lethal dose of a different stress (467; 468). Furthermore, genes that were specifically thought to protect the cell against heat shock were shown to be induced under a variety of independent stressors. Analysis of the upstream promoter region of these genes identified a conserved sequence element that became known as the stress response element (STRE), which was later shown to be the consensus sequence binding site for the zinc-finger transcription fac-tors, Msn2p and Msn4p (615; 615; 81). The ESR was originally described using microarray

Appendix G. Transcription profiling of C. glabrata serum stress responses 331

expression analysis in S. cerevisiae by the independent groups Gasch et al and Causton et al. In combination these groups investigated the transcriptional responses to a range of environmental cues including heat shock (230), extremes of pH (118), oxidative stress (hydrogen peroxide (118) or menadione induced (230)), hyper-osmotic stress (NaC1 (118) or sorbitol (230; 118) induced), hypo-osmotic shock (230), amino acid starvation (230), nitrogen depletion (230), and sub-optimal carbon sources (230).

It had been proposed that this co-ordinated transcription programme is part of a con-served protective response to protect critical features of cellular physiology in budding yeast (229). However, initial transcriptional studies in C. albicans did not reveal a general stress response (191) (in oxidative, hyperosmotic stress and a 23 - 27°C temperature shift) nor was any cross protection observed when cells pre-treated with a mild heat shock were sub-sequently exposed to oxidative stress. In a contradictory study, Smith et al., (639) did demonstrate HOG/ -dependent cross-protection to 0.3M NaC1 after pre-treatment with a sublethal dose of H202. A subsequent systematic comparison of general stress response data sets of C. albicans, S. pombe and S. cerevisiae (192) did reveal a general stress re-sponse with almost 50-fold fewer genes than in S. cerevisiae. While there is evidence for a conserved ESR in the model fungi S. cervisiae and S.pombe (229), the evidence for such a comprehensive ESR in C. albicans is unconvincing. At the start of this study, the general stress response of C. glabrata had not been investigated, and the relevance of the ESR in opportunistic fungal pathogens is still unclear. This work was also put on hold after a similar publication addressed the specific question of whether or not an ESR is detectable in C. glabrata (589).

The following section is a summary of transcription profiling performed when C. glabrata cells were exposed to culture medium containing foetal calf serum (10% w/v), an environ-mental cue that is often used as a morphogenetic inducer in C. albicans. In this instance, we used synthetic medium supplemented with foetal calf serum to approximate the nitrogen availability that may be found in the blood. Blood products have been used in transcrip-tional profiling studies in C. albicans (210), and would have been a much better option to address this specific question. This is another reason why the following analysis has been relegated to the supplementary data section.

G.2 Specific methods

C. glabrata ATCC 2001 cells were re-streaked onto YEPD agar from frozen glycerol stocks 3 days prior to the microarray experiment. On the day prior to the experiment one colony was picked from the plate and used to inoculate 50 ml of synthetic complete medium (supplemented with 100mM succinic acid and buffered to pH 4.0) which was incubated

G.3 Transcriptional response of C. glabrata cells exposed to 10% Foetal Calf Serum (FCS) 332

in an orbitol shaking incubator for 18 hours (37° C, 180 rpm). Cells were harvested by centrifugation (3000 rpm, 5 minutes), resuspended to a volume of 50 ml and washed twice with sterile distilled water before estimating the cell density using a multiskan ascent at absorbance plate reader at OD600. The experiment was initialized by inoculating 1 L of synthetic complete medium in a 2 L flask with cells to a final OD600 = 0.2. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented with 10% foetal calf serum (FCS). The stress treated sub-culture and one control culture (untreated) were both incubated for a further sixty minutes before harvesting by centrifugation (3000 rpm, 5 minutes), snap-freezing in liquid nitrogen and storage at -80°C. RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2).

G.3 Transcriptional response of C. glabrata cells exposed to 10% Foetal Calf Serum (FCS)

Transcription profiling of cells exposed to 10% (v/v) FCS for one hour identified 501 in-duced transcripts (Supplementary Table G.1) and 448 repressed (Supplementary Table G.2) transcripts (approximately 10% of the genome in each case). These data were summarized by searching for over-represented GO component, process and function terms. Furthermore, repressed and induced transcript sets were combined and filtered to retain genes with S. cervisiae orthologues so that the differentially transcribed set could be mapped to KEGG pathways, which summarize gene expression in the context of the metabolic and signalling pathways curated for the closely related model organism. These tools provided insight into the effects of serum exposure (10% (v/v) FCS, 1 hour) on orthologous metabolic pathways, RNA metabolism and ribosome biogenesis, in addition to the activation of important sig-nalling pathways involved in nutrient and stress sensing. For brevity, all gene descriptions are taken from the functional annotation in SGD (131) and refer to references on the re-spective summary page of each gene. If it is deemed appropriate to write this section fully, proper citations will be included.

In particular, these analyses reveal an over-representation of induced transcripts local-ized in the nucleolus (Supplementary Table G.3) involved in rRNA and mRNA process-ing, ribosome biogenesis and assembly and also in the subunits of DNA directed RNA polymerases I and III (Supplementary Table G.4). The over-representation of transcripts involved in tRNA modification in combination with the above points to a cell that is up-regulating transcription and translation processes relative to the synthetic complete medium


reference at the same time-point. Although C. glabrata and S. cerevisiae have different metabolic capabilities (e.g. the C. glabrata genome has lost genes involved in galactose and sucrose assimilation, phosphate, nitrogen and sulfur metabolism, as well as thiamine, pyridoxine and nicotinic acid biosynthesis (348)), mapping of transcript fold-changes to the orthologous global S. cerevisiae metabolic map can be useful to generate hypotheses regard-ing the metabolic adaptation that occurs following exposure to serum, an environmental cue that may approximate the nutrient availability during a bloodstream infection. Doing so reveals a co-ordinated change in gene expression, where repressed and induced transcripts are located to specific areas of the global metabolic map (Supplementary Figure G.4). In general this global view indicates that enzymes catalyzing reactions in fatty acid biosynthe-sis, starch and sucrose metabolism, glycerophospholipid metabolism, pyruvate metabolism, citrate cycle, glyoxylate metabolism and oxidative phosphorylation are repressed on expo-sure to serum. In contrast, induced genes are predominantly involved in other pathways including nucleotide metabolism, nitrogen metabolism, amino acid metabolism, the pentose phosphate shunt, the urea cycle, C5-branched dibasic acid metabolism, high mannose N-glycan biosynthesis and cyanoaminoacid metabolism. This high-level view of metabolism at a single time-point is of course an over-simplification of the dynamic nature of metabolism and gives no quantitative summary of reactant fluxes, however the hypothetical repression of C. glabrata energy production pathways which utilize glucose, and the concomitant hy-pothetical induction of C. glabrata pathways involved in amino acid metabolism indicate that glucose is not primarily used in the production of energy in serum treated cells.

The induction of genes whose products catalyze amino acid biosynthetic reactions, in conjunction with the up-regulation of permeases that transport such nutrients into the cell (e.g. the high-affinity glutamine permease GNP1 - also a transporter of leucine, threonine, cysteine and methionine, the dicarboxylic amino acid permease DIP5, the high-affinity histidine permease HIP1, a polyamine and urea transporter DUR3, an ammonium permease involved in regulation of pseudohyphal growth MEP2) and a cytosolic aspartate aminotransferase, involved in nitrogen metabolism AAT2 and CHA1 a catabolic L-serine and L-threonine deaminase which is required to use serine or threonine as the sole nitrogen source, indicates that a programme of nitrogen compound assimilation and metabolism is orchestrated by C. glabrata in response to serum exposure. Indeed, transcripts encoding catalytic enzymes in several amino acid metabolic pathways were identified. For example eight genes encoding enzymes of the phenylalanine, tyrosine and tryptophan biosynthesis pathways are induced (Supplementary Figure G.5). Such aromatic acid biosynthesis requires conversion of phosphoenolpyruvate or erythrose-4-phosphate precursors for the production of chorismate, the branch point of aromatic acid biosynthesis which is either converted to an-thranilate for tryptophan biosynthesis or to prephenate for tyrosine or phenylalanine biosyn-


thesis. The induction of enzymes catalyzing precursor condensation (EC 2.5.1.54 ARO3), chorismate synthesis (EC 4.2.3.5 ARO2), anthranilate synthesis (EC 4.1.3.27 TRPE) and transamination (EC 2.6.1.1 AAT1 and EC 2.6.1.57 ARO8) suggest that phenylalanine, ty-rosine and tryptophan biosynthesis are all enhanced on serum exposure. The net effect of en-zyme differential transcription on valine, leucine and isoleucine is more difficult to interpret because both biosynthetic and catabolic enzymes are co-induced (Supplementary Figures G.6 and G.7). However, co-induction of enzymes catalyzing the biosynthesis (EC 4.3.1.19 CHAl, EC 2.2.1.6 ILV6,EC 4.2.1.9 , EC 2.3.3.13 ILV3) and subsequent degradation (EC 2.6.1.42 BAT1) of these amino acids may suggest that isoleucine, leucine and valine catabo-lites are preferred. Although this interpretation is confounded by the dual role of BAT1 in both synthesis and degradation of these amino acids, the concomitant repression of NAME (EC 6.1.1.4) which catalyzes leucine aminoacyl-tRNA biosynthesis further supports the no-tion that leucine biosynthesis is channeled to downstream metabolic reactions rather than into biopolymer production at the ribosome. Inspection of the arginine/proline metabolic map (Supplementary Figure G.8) and alanine/aspartate/glutamate metabolic map (Supple-mentary Figure G.9) reveals induction of enzymes that may increase flux through the citrate cycle, including arginosuccinate synthase (EC 6.3.4.5 ARG1) which catalyzes the formation of L-argininosuccinate from citrulline and L-aspartate leading to the production of fumarate a substrate in the citrate cycle. The induction of aspartate aminotransferase (EC 2.6.1.1 AAT1), L-asparaginase (EC 3.5.1.1 ASP1) and glutamine hydrolysing asparagine synthase (EC 6.3.4.5 ASNE) may lead to an increased L-arginosuccinate production and, following by, increased fumarate production. Induction of bifunctional carbamoylphosphate synthetase (EC 6.3.5.5 URA2) may also lead to an increased production of carabomyl phosphate, a precursor for the urea cycle after conversion to L-citrulline by ornithine carbamoyltrans-ferase (EC 2.1.3.3 ARG3). The concomitant repression of other enzymes that catalyze the catabolism of L-Glutamate (EC 1.2.1.16 UGAE, EC 2.6.1.19 UGA1, EC 4.1.1.15 GAD1, EC 1.4.1.2 GDH2 and EC 1.5.1.12 PUTS) suggests that the production of carabomyl phos-phate and subsequent increased flux though the urea cycle to produce fumarate rather than succinate is favoured.

Analysis of carbon catabolite pathways shows that the majority of glycolytic and elec-tron transport enzymes are repressed after exposure to serum (Supplementary Figures ??, G.12 and G.13). Notably, pyruvate kinase (EC 2.1.7.40 CDC19), acetyl CoA synthetase (EC6.2.1.1 A CS/ ), aldehyde dehydrogenase (EC 1.2.1.5 ALD3) and hexokinase (EC 2.7.1.1 GLK1) are all repressed whilst fructose-1,6-bisphosphatase I (EC 3.1.3.11 FBP1) Biotin, Co A and panthenoic acid biosynthetic genes are also induced (Supplementary Figure G.14 and Supplementary Figure G.15), suggesting a reduced production of pyruvate and an increased production of acetyl Co A. Induced carnitine transporter transcription and carnitine syn-


thesis (EC 1.2.1.3 ALD5) with concomitant reduced fatty acid biosynthesis (Supplementary Figure G.16) suggest that unsaturated fatty acid oxidation is a preferred energy produc-tion pathway (this process requires acyl Co A and carnitine for uptake of fatty acids into the mitochondrion). This hypothetical decrease in carbon catabolism and carbon storage (Supplementary Figure G.17) indicates that carbon sources are diverted away from energy production pathways, which is perplexing as a glucose shortage is unlikely with the chosen experimental conditions (culture medium contains 2% (w/v) glucose and optical density of harvested cells was below saturation (0D600 = 0.45)).

Why are carbon catabolic energy production pathways down-regulated in favour of the hypothetical amino acid and fatty acid degradation proposed above? One explanation might be that carbon compounds are diverted to high mannose N-glycan biopolymer pro-duction in the cell wall. Exposure to serum leads to the differential transcription of genes whose products are either located in the cell wall or are involved in the modification of cell wall carbohydrates. For example, mannan polymerase II complex 1VINN10 subunit (EC 2.4.1 MNN10), the alpha-1,6-mannosyltransferase involved in cell wall mannan biosynthe-sis (HOC1) and the alpha-1,2-mannosyltransferase responsible for addition of the second alpha-1,2-linked mannose of the branches on the mannan backbone of oligosaccharides (EC 2.4.1 MNN5) are induced whilst mannosylphosphate transferase (EC 2.7.8 MNN6) is re-pressed (Supplementary Figure G.18). Furthermore, alpha-1,3-mannosyltransferase, which adds the fourth and fifth alpha-1,3-linked mannose residues to 0-linked glycans during pro-tein 0-glycosylation (EC 2.4.1 MNT3) is also induced (Supplementary Figure G.19) as is the v-snare SEC20 required for N- and 0-glycosylation in the Golgi (Supplementary Figure G.20). The induction of EXG1 EXG2 genes encoding exo-1,3-beta-glucanase and CWP1 a cell wall mannoprotein, linked to a beta-1,3- and beta-1,6-glucan provides further evidence in support of the hypothesis that serum treated C. glabrata cells undergo a programme of high mannose N-glycan biosynthesis and assembly in the cell wall. In addition, the induc-tion of UTR2 a chitin transglycosylase that functions in the transfer of chitin to beta(1-6) and beta(1-3) glucans in the cell wall suggest that newly synthesized and assembled beta glucans are also cross-linked to chitin. Further to the hypothetical reduction in carbon catabolism and increased amino acid catabolism, the induction of many transporters in the plasma membrane also provides clues regarding the nutritional demands of the serum treated cells. The co-induction of the genes encoding a putative divalent metal ion trans-porter involved in iron homeostasis (SMF3), a low affinity iron transporter (FET4) and a siderophore-iron chelate uptake transporter (ARN1) suggests that iron uptake is increased in this environmental perturbation. Furthermore, the co-induction of the biotin transporter gene VTH1 and biotin synthesis pathways, strongly suggest a nutritional requirement for this vitamin at this stage of growth in cells exposed to serum. Other vitamin transporter


genes, whose protein products are located in the plasma membrane are also induced (the vitamin B2 transporter MGRS and the vitamin B6 transporter TPN6, which is a cofactor required for amino acid biosynthesis, whose upregulation is described above).

Cells exposed to foetal calf serum (10% v/v) also demonstrate differential transcrip-tion in non-metabolic functions relative to untreated cells, notably in the cell cycle (Figure G.22), a meiosis-like (Figure G.21) pathway and a MAPK signalling pathway (Figure G.23) inferred by orthology to S. cerevisiae. Inspection of the cell cycle pathway (Figure G.22) reveals serum-specific regulation of G1 phase (YOX1, PCL1 and CLN1), S phase (APC4 and CDC6) and 02 /M phase transcripts (RAD24, CHK1, CDC55 and PF2A). Although it is difficult to assess the dynamic nature of the cell cycle from one time point and also from dealing with transcripts in isolation rather than in an integrated mathematical model, speculatively one might suggest that the induction of YOX1 and CLN1 indicates the cells can progress from G1 phase to G2 phase. Intriguingly, genes involved in orthologous mating, filamentation and sporulation pathways of S. cerevisiae are induced after exposure to serum. Two genes encoding pheromone regulated S. cerevisiae orthologues (a transcription factor required for gene regulation in response to pheromones, Kar4p and Prm3p, a pheromone-regulated protein required for nuclear envelope fusion during karyogamy) in addition to a cAMP dependent protein kinase involved in regulating vegetative growth (Tpk2p), and two genes encoding Mat al orthologues (CAGLOB00242g/YCRO4OW and CAGLOB01243g/ YCR040W) indicate an activation of pathways whose orthologous functions in S. cerevisiae are to regulate the mating response pathway. Mapping of regulated transcripts to ortholo-gous MAPK pathways (Figure G.23) is consistent with this. For example, the concomitant induction of SHO1 and TEC1 and repression of MSN2 suggests an activation of the fil-amentation response signalling cascade rather than the hyperosmotic stress pathway that also lies downstream of Sholp. The induction of BEM1, a gene encoding a protein involved in establishing cell polarity and morphogenesis is also induced and two genes encoding sporulation specific genes (DIT1 and TPK2) was also observed in serum treated cells.


Ad

6

no normalization

1 2 3 4 5 8 7 8 9 11 13 15

log2(Cy6 / Cy3)

(a)

RG densities

10

15

Intensity

(c)

loess + quantile normalization

2

1 2 3 4 5 6 7 8 9 11 13 15

log2(Cy5 / Cy3)

(b)

RG densities

o

5 10

15

Intensity

(d)

0 o

Figure G.1: Exploratory data analysis plots for C. glabrata environmental stress microarray hybridizations. The effects of normalization procedures on background cor-rected intensity distributions prior to linear modelling analysis. Box and whisker plots of M values (log2(Cy5TM) - log2(Cy3Tm )) are shown for each slide before normalization (a) and after lowess normalization and background correction (b). Coloured boxes depict the en-vironmental stress added in each experiment. Black boxes = synthetic complete + 10% (v/v) foetal calf serum (FCS), red boxes = synthetic medium + 0.01% dextrose (w/v), blue boxes = synthetic complete + 0.5M KC1 and blue boxes = synthetic complete + 1mM tertiary butyl hydroperoxide (t-BOOH). The pattern of boxes repeats four times, account-ing for each of the four biological replicates. The Cy5

TM (red) and Cy3TM (green) intensity

density distributions are plotted before (c) and after normalization (d). Both channels are comparable after normalization.

6 8 10 12 14 2 4 8 8 10 12 14

0

2 4 6 8 10 12 14

• •

•

SC + 10% FCS

0

01 -0

0 C0 0 0 _J

O

-6 -4 -2

0

2

Log Fold Change


Slide 1

Slide 5

Slide 9

Slide 13

2 4 6 8 10 1.2 14

(a) Slide 1, 10% FCS (b) Slide 5, 10% FCS (C) Slide 9, 10% FCS (d) Slide 13, 10% FCS

Figure G.2: MA plots for each hybridization performed in the C. glabrata en-vironmental stress study. The log ratio of intensities M (log2 (Cy5TM ) - log2(Cy3T )) is plotted on the y axis as a function of average spot brightness A (0.5 x (log2(Cy5''') log2(Cy3TM))). The dotted red line (M = 0) depicts the line of no differential expression. These plots show little brightness dependent bias in M values.

Figure G.3: Volcano plots of differentially expressed transcripts in the C. glabrata environmental stress study. The volcano plots show the log odds of differential expres-sion (y axis) versus the fold change in expression (x axis) for each gene. The vertical dashed blue line depicts the threshold for a two-fold absolute repression; similarly the ver-tical dashed yellow line depicts the threshold for two-fold absolute induction. The dashed, horizontal red line marks the threshold for the log odds of differential expression = 5.


TNT

11

1

Figure G.4: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous global metabolic map of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultra-centrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic com-plete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Pathways containing induced and repressed genes are coloured black and dark blue respectively.

1 2 6.1571 1 2,6.19

Tymaine yieC Isoquinoline alkaloid biosynthesis)

1 '5 1 (- Puronnycin biosynthesis

( Tyrosine metabolism ) TIVIICStanDia

PHENYLALANINE, TYROSINE AND TRYPTOPHAN BIOSYNTHESIS

Panama phosphate pethoray

L-Aspirtale 4-semiakiehyle

6-Deoxy-5-ketofructose phosOiale

7P-2-Dehydra-3-cleory-D arabwo-heptatate

04-1 QuiC 1-10.0stale rho- mate Protocatechua*

Olycolysis}-- i> Phosphoenol-pyrwate

• CEIZI A• T 3-De®te 142.1.101

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E 1445-Phosiho 8-D-nloos anthianila

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4 [Biosylithesisofhopine,

pyrinkadine, rec and pipeiiiline allealciels

onne 1

1 4 !

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00400 8128109 (c) Kanehise Laboratories

5-0-(1-Carboxyriny1)- posynthesis of ilinlielrnt'n117471ides

1

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EMS (Ubiquinone and other tert:enced-cpanone biosynthesis

4-H n private

• 3-phosphoshikarriabe

11.3.1.121 26.1.57

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114 201

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Slukimate 3-phosphate


Figure G.5: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous phenylalanine, tyrosine and trytophan biosyn-thetic pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and har-vested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (ref-erence sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully de-scribed in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

PHENYLALANINE, TYROSINE AND TRYPTORIAN BIOSYNTHESIS

4.1.1.51

( Phynyialanine metabolism ) 0 Piety/way 0

Ithigainorie and other 4-H terpenoid-gasonebiosyntlems ) ydioxy- phenylpyruvate

( Pentonphosphide) pathway

Photosynthesis)- 1

Quints

111.1.241 2-Athino-3,7-cluteoxy- 1119925 111 2s2 I D-6areo-1M,1- 6-ulosomc acid

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6-Deoxy-5-ketufructose Phosphoenol- D-arabuth- pynivate

, _ 111 1 n,,o„dm_, 1-phospmle heptouate

41-1 1 QuiC -m0 stikima --,t-i,"- Proton:decimate

L-Aspartata 4-sereialdelmnle •

lird:1:6-142.1 20 f 4,C Ptensostainoue 1 1

biosynthesis LEEE3

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(LYttbteoft}F—j1 2-C 14.1.1.431

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125.1.191

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c Biosynthesis of ) no$iderueosormarrquolos

4

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(gitegCtin'90 .0 rhecLk.nyi 1.1416.1 1 1

1

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Figure G.6: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous valine, leucine and isoleucine biosynthetic pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

0(S)-3-Hydroxy isobutyrate

2-Methyl-acetorreetyl-CoA

123.1161

•

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MEE IBM

Thpp 2-Methyl- 1-11 xyb DPP

Lipoarnide-E

•

VALINE, LEUCINE AND ISOLEUCINE DEGRADATION

V aline, leucine and ) isolerxine biosynthesis

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2.6.1.42

•

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c Biosynthesis of 12-, 14- and ,- —Lt

) ) 16-membered raernolides

.

00280 8.128.09 (c) Ranehisa Laboratories

•

11.3 99.21 1399.12 1 3 99 3 1.3.99.3

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Figure G.7: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous valine, leucine and isoleucine degradation pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

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Figure G.8: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous arginine and proline metabolic pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

ALANINE. ASP ARTATE AND GLUTAMATE METABOLISM

1 26.11 14

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Figure G.9: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous alanine, aspartate and glutamate metabolic pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

LYSINE DEGRADATION

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Figure G.10: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous lysine degradation pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA la-belling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

1.2.1.3

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4

I GLYCOLYSIS 1 GLUCONEOGENESIS

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Figure G.11: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous glycolysis pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

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41-11.3.1.491-Ct.

D-Leclakieh3de


Figure G.12: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous pyruvate metabolism pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA la-belling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.


Figure G.13: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous oxidative phosphorylation pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

BIOTIN METABOLISM

Panelate 0 , Pimelovl-CoA rnino- 6.2.1.141-0-0 2.3.1.471---10.? toAxononanoate

Biotint- Biotinyl-CoA 5'-Ale

Detinobiotin ?4H 6.3.3.3

2.8.1.6

6 2 1 11

2 6 1 62

_011r 7,8-Diainino-noranoate

0 4 6 2.1 11 •

degradation

► 0— 3.4 - -

Holo- [cathoxylase]

• 0

Biotin J. .1.12 •

L-Lysine

Trope, .pericline anc0 id

iosynthesis

6.3.4.10 6.3.4.9

6.3.4.11 6.3.4.15

6 .3.4.10 6.3.4.9

6 3 4 15 6 3 4 11

00780 911/09 (c) Kanehisa Laboratories


Figure G.14: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous biotin biosynthesis pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA la-belling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

PANTOTHENATE AND CoA BIOSYNTHESIS Olscolres )

•r7 0__H 2.2.1.6 ,i krif ietBIE

Eynerate

16.1.6 4 10.0 3-Methyl-7- 3.6.1.41 L. V aline obobutarbab

2-Debycleopentoete

(F1)-3,3-Dimethyhnalax

(R)-P oaken* 13-4-Phos bthenate Cysteme and metloonme

L-Cystaine l metaboborn (R).Panbata

(R)-4-Phospbc pantotkamo}4-1.-crieMe

Pareathatiot

Alm" ( Propenotee Uresil N-Cesbarno

metabolism

P-ATM

(- Mamma, aspadale and )_ — glutamate membolisna

kuspho-ranlethem

• 0

Adenosine 3',5'-berpltosplele

Alb-Wp] Dephospho-CoA 0-1111241 0

Acyl-canter Protein Peatetheme

A • L-Aepartate

00770 9,1J99 (c) )(audio(' Laboratone


Figure G.15: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous pantothenoate and CoA biosynthesis pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

'FAIT? ACID BIOSYNTHESIS-I

—(Caalf orb Acell.Caq,

ACP 0—, ASV

rpm IimmOL_Ctwil (F) Fr

Wear aid

COI ErIrcuboryl.

fonS1398109 (c)Kalehin Lakesieres


Figure G.16: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous unsaturated fatty acid biosynthesis biosynthe-sis pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and har-vested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (ref-erence sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully de-scribed in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

• MEI • ME • UDP-0 UDP

2.4.1.14, qd-Defielose.SP

UDP glazer IIIIII 2419

1.43111.43.6P Pnicbse

241.3 HPoSu. C7clen.4.4.44. ? Mabee Nxtrawkalma)

•1431k4Inlxie 5E40. 04

Colulose 111111111111,13.2.1.201 w Amy

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( Nob* athglacutotele °444c" vetronverafors 2 1 31 J-4;81. 0-0-21owewtsich

)4L l'-1=2"'; PD

041 5 4 2 61 lk 4 pl-D-Ohicose-IP

1 311....LH41 3.2.1.93 1.0

3.Ketormase04--i I 159133 324 32 1 20 1 10,,,,,,,, 4--74-1.1 13

32 1 4 r 1 2.4.11 2413 1 241.131

0 D.Frodnee D-Oh.... 3.2 139 4-124.1341--

32138 1.3-13-Glocen ADP-glucaa

,41- 2.4.135 ...--1.0--1.1.1.21 (-12.41.121--' LI 6 1211 2.7.1.4 1 Cellthiose 37779 4 eligkeavose

al-P.71341,_,..— 4---) ci-D-0lums-1P

Dexem `-1 32.1.1

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A.Dreennse.6P

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4-0-011rose.64

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Figure G.17: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous orthologous starch and sucrose metabolism pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

HIGH-MANNOSE TYPE N-GLYCAN BIOSYNTHESIS

AIN1410 ANP1

MNNI I HOCI MNN9

MNN2 MUN5 1-11.0-111111F0•0-114NN1 Ho Maud 6Manal , MNN9

14N59 I. 6 2Manal MNN9 Manal 3Manal MNN2

LiN51 MNN5Manal MN 5 Manal Manal 3Manal 2Mana1 \

WW1 !Manal Man al 3Mana1 2Mana1 MNP2 149 Manal

mand / \

MNN1 WINN5 MNN9 Man al 3Manal 2Manal MNN2 6 1 Mani11-4GINAc 31— 4G1cNAc PI—Pun MNN1 MN145 MIII46

2 Mahal ] 6Manal / 3

Man al 3Man I —P., MNN9 OCHI 01111'51 5Man al MNN2

Mimi

Manal 3Manal / MNI41 MNI45 Man al 3Manal — 2Manal

MNN1

00513 8128109 (c)Kanehisa Laboralosien

• EMI 2.41232 OCH1

I1INN9 VANI1

C N-Olyranbionyallesis — —1>V-1321113 0-1 mow 1—ro


Figure G.18: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous high mannose type N-glycan biosynthesis pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

I 0-I'.NNOSYL GLYCAN BIOSYNTHESIS

Mania 3Mano(

Mani) I 2 hula n 15 .1

Man a t---- 2 Man a I/

Galpo:

3 3 2rulan a 2Mano( 1 2 2

/

Galp0:

2 Man Ser/Thr 2

GloNAo15

00514 412/09 (c) Kanads& Laboratories


Figure G.19: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous orthologous o-mannosyl glycan biosynthesis pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, eDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

PM !.plasma membrane)

I SNARE INTERACTIONS IN VESICULAR TRANSPORT

I Ykt6 LE date endosome)

Golgi body Sts

Sb:17 S Cc] 8 Use1

ER (ondoplasmic Miniboom) Sy-p7

S tc11 TGN

(trans Golgi network) STX6

Bost See22

I Batt

S Gast Ykt6

SNAP29

S ec20

Nucleus

04130 3/31/09 (c) Kanehisa Laboratories

apical PM (apical plasma membrane)

11 IVAMP4

RE (recycling endoseme)

VAMP?

I vAivrps (muscle)

IVAMP4

EE putty endeseme)

lysosome


Figure G.20: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous orthologous v-snare vesicular trafficking pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

MEICOSIS - yeast

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Figure G.21: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous meiosis pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

r I

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Figure G.22: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous cell cycle pathway of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA labelling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.

MAPI MANGE MAPK

MAR{ SIGNALING PATHWAY

04011 3,31,99 (c) Kam hien Labotatoties

S te4 Stel$


Figure G.23: Genes differentially transcribed following exposure to 10% FCS (1 hour) mapped to the orthologous MAPK signalling pathways of of S. cerevisiae. Cells were incubated for 4 hours (0D600 = 0.8) and harvested using a Sorvall SLA-3000 ultracentrifuge rotor (suitable for large volume harvesting), washed with sterile distilled water and then subcultured in synthetic culture medium (reference sample) or synthetic complete medium supplemented 10% foetal calf serum (FCS). RNA Isolation, cDNA la-belling, microarray hybridizations and data analyses are fully described in the the materials and methods section (Chapter 2). Induced and repressed genes are coloured yellow and dark blue respectively.


Table G.1: Candida glabrata transcripts induced in ATCC 2001 following ex-posure to Synthetic Complete + 10% (v/v) foetal calf serum (FCS) Induced transcripts are defined as having log2(Cy5) - log2(Cy3) > 2.0 and Benjamini-Hochberg corrected p-values < 0.05.

CG ORF SC ORF gene name Description Log Ratio CAGLOI09108g YNL125C ESBP6 Protein with similarity to monocar-

boxylate permeases, appears not to be involved in transport of mono-carboxylates such as lactate, pyru-vate or acetate across the plasma membrane

8.15

CAGLOF00407g YJR070C LIA1 Deoxyhypusine hydroxylase, a 6.6 HEAT-repeat containing metal- loenzyme that catalyses hypusine formation; binds to and is required for the modification of Hyp2p (eIF5A); complements S. pombe mmdl mutants defective in mitochondrial positioning

CAGLOG09086g - - Description not mapped 6.39 2cox3 - Description not mapped 6.21 rner - Description not mapped 6.06

CAGLOL03828g YNL111C CYB5 Cytochrome b5, involved in the sterol and lipid biosynthesis path-ways; acts as an electron donor to support sterol C5-6 desaturation

5.98

CAGL0003289g YLL048C YBT1 Transporter of the ATP-binding cassette (ABC) family involved in bile acid transport; similar to mam-malian bile transporters

5.77

CAGLOI00484g YLR300W EXG1 Major exo-1,3-beta-glucanase of the cell wall, involved in cell wall beta-glucan assembly; exists as three dif-ferentially glycosylated isoenzymes

5.69

CAGLOL10868g YOR271C FSF1 Putative protein, predicted to be an alpha-isopropylmalate carrier; belongs to the sideroblastic-associated protein family; non-tagged protein is detected in purified mitochondria; likely to play a role in iron homeostasis

5.68

CAGLOK00605g YJL194W CDC6 Essential ATP-binding protein required for DNA replication, component of the pre-replicative complex

5.61

(pre-RC) which requires ORC to as-sociate with chromatin and is in turn required for Mcm2-7p DNA as-sociation; homologous to S. pombe Cdcl8p

- - Description not mapped 5.54 CAGLOA00511g - - Description not mapped 5.54

- - Description not mapped 5.5 CAGLOE00792g YCL037C SR09 Cytoplasmic RNA-binding protein

that associates with translating ri-bosomes; involved in heme regula-tion of Hap1p as a component of the

5.45

HMC complex, also involved in the organization of actin filaments; con-tains a La motif



continued from previous page CG ORF SC ORF gene name Description Log Ratio

CAGL0G01848g CAGLOG04741g

- YNL104C

- LEU4

Description not mapped Alpha-isopropylmalate synthase

5.38 5.32

(2-isopropylmalate synthase); the main isozyme responsible for the first step in the leucine biosynthesis pathway

CAGL0003531g - - Description not mapped 5.29 CAGLOG08668g YNL066W SUN4 Cell wall protein related to glu-

canases, possibly involved in cell wall septation; member of the SUN family

5.03

CAGLOF01485g YER011W TIR 1 Cell wall mannoprotein of the 5 Srplp/Tiplp family of serine- alanine-rich proteins; expression is downregulated at acidic pH and induced by cold shock and anaer-obiosis; abundance is increased in cells cultured without shaking

CAGLOM03487g YNR003C RPC34 RNA polymerase III subunit C34; 4.94 Be70 and is a

key determinant in pol III recruit-ment by the preinitiation complex

siown

Description

otinthTmFaIpHpd

CAGLOE06094g - - 4.93 CAGLOJ01848g YPRO10C RPA 135 RNA polymerase I subunit A135 4.79 CAGLOK01155g YGR079W - Description not mapped 4.69 CAGLOJ10252g YNL075W IMP4 Component of the SSU processome,

which is required for pre-18S rRNA processing; interacts with MpplOp; member of a superfamily of proteins that contain a sigma(70)-like motif and associate with RNAs

4.59

CAGLOJ01265g YMR093W UTP 15 Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

4.5

CAGLOI11011g YARO5OW F LO 1 Lectin-like protein involved in flocculation, cell wall protein that binds to mannose chains on the surface of other cells, confers floc-forming ability that is chy- motrypsin sensitive and heat resis-tant; similar to FloSp

4.48

CAGLOK00781g YGR210C - Description not mapped 4.47 CAGLOL03872g YNL113W RPC 19 RNA polymerase subunit, common

to RNA polymerases I and III 4.46

CAGLOJ06666g YML108W - Description not mapped 4.37 CAGLOLO8294g YIL14OW A XL 2 Integral plasma membrane protein

required for axial budding in hap-loid cells, localizes to the incipient bud site and bud neck; glycosylated by Pmt4p; potential Cdc28p sub-strate

4.34

CAGLOH01589g - - Description not mapped 4.29 CAGL0G08624g YIL121W QDR2 Multidrug transporter of the major

facilitator superfamily, required for resistance to quinidine, barban, cis-platin, and bleomycin; may have a role in potassium uptake

4.24

CAGLOG04499g YJL 105W SET4 Protein of unknown function, contains a SET domain

4.2

CAGLOG02409g YKR092C SRN° Nucleolar, serine-rich protein with a role in preribosome assembly or transport; may function as a chap-erone of small nucleolar ribonucleoprotein particles (snoRNPs); im- munologically and structurally to rat Nopp140

4.15




CAGLOL02937g CAGLOA04037g

CAGLOE02343g

-- YLR196W

YOLO1OW

PWPI

RCL I

Description not mapped Protein with WD-40 repeats involved in 'RNA processing; associates with trans-acting ribosome biogenesis factors; similar to beta-transducin superfamily Subunit of U3-containing 90S preribosome processome complex in-volved in production of 18S rRNA and assembly of small ribosomal subunit; similar to RNA terminal phosphate cyclase-like proteins but RNA cyclase activity not detected

4.13 4.12

4.12

CAGLOL06072g YER130C COM2 Protein of unknown function 4.11 CAGL0G05874g - - Description not mapped 4.1 CAGLOE04092g YHL040C ARN1 Transporter, member of the ARN

family of transporters that specifically recognize siderophore-iron chelates; responsible for uptake of iron bound to ferrirubin, fer- rirhodin, and related siderophores

4.09

CAGLOL00671g YER056C FCY2 Purine-cytosine permease, mediates purine (adenine, guanine, and hy-poxanthine) and cytosine accumu-lation

4.08

CAGLOD05918g YGR177C ATF2 Alcohol acetyltransferase, may play a role in steroid detoxification; forms volatile esters during fermen-tation, which is important in brew-ing

4.06

CAGLOK02233g YER126C NSA2 Protein constituent of 665 pre- ribosomal particles, contributes to processing of the 27S pre-rRNA

4.05

CAGLOM11660g YIL053W RHR2 Constitutively expressed isoform of DL-glycerol-3-phosphatase; involved in glycerol biosynthesis, in-duced in response to both anaerobic and, along with the Hor2p/Gpp2p isoform, osmotic stress

4.04

CAGLOI08613g YHLO16C DUR3 Plasma membrane transporter for both urea and polyamines, expres-sion is highly sensitive to nitrogen catabolite repression and induced by allophanate, the last intermediate of the allantoin degradative pathway

4

CAGLOD05060g YGR123C PPTI Protein serine/threonine phosphatase with similarity to human phosphatase PP5; present in both the nucleus and cytoplasm; expressed during logarithmic growth; computational analyses suggest roles in phosphate metabolism and rRNA processing

3.96

CAGLOF00187g YMR319C FEZ{ Low-affinity Fe(II) transporter of the plasma membrane

3.94

CAGLOF02013g YFLO10C WWM1 WW domain containing protein of unknown function; binds to

3.94

Mcalp, a caspase-related protease that regulates H202-induced apoptosis; overexpression causes Gi phase growth arrest and clonal death that is suppressed by overex-pression of MCA1




CAGL01102937g YJL069C UTPI8 Possible U3 snoRNP protein involved in maturation of pre-18S rRNA, based on computational analysis of large-scale protein- protein interaction data

3.93

CAGL0107689g YOL092W - Description not mapped 3.91 CAGL0003369g - - Description not mapped 3.9 CAGL0109746g YOR307C SLY4/ Protein involved in ER-to-Golgi

transport 3.9

CAGLOD04180g YIL091C - Description not mapped 3.88 CAGLOM07227g YHR196W UTP9 Nucleolar protein, component of

the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

3.88

CAGLOE02035g YOL119C MCH4 Protein with similarity to mammalian monocarboxylate permeases, which are involved in trans-port of monocarboxylic acids across the plasma membrane; mutant is not deficient in monocarboxylate transport

3.86

CAGLOL07678g YPL266W DIM1 Essential 18S rRNA dimethy- lase (dimethyladenosine transferase), responsible for conserved m6(2)Am6(2)A dimethylation in

3.86

3'-terminal loop of 18S rRNA, part of 90S and 40S pre-particles in nucleolus, involved in pre-ribosomal RNA processing

CAGLOJ11286g YNL182C 1P13 Essential component of the Rixl complex (Rix1p, Ipilp, Ipi3p) that is required for processing of ITS2 sequences from 35S pre-rRNA; highly conserved and contains

3.85

WD40 motifs; Rixl complex associates with Mdnlp in pre-60S ribosomal particles

CAGLOB02343g YML116W ATRI Multidrug efflux pump of the major facilitator superfamily, required for resistance to aminotriazole and 4-nitroquinoline-N-oxide

3.84

CAGLOL04950g YMR049C ERD1 Protein required for maturation of the 25S and 5.8S ribosomal RNAs; constituent of 66S pre-ribosomal particles; homologous to mammalian Bopl

3.79

CAGL0003938g YIL014W MNT3 Alpha-1,3-mannosyltransferase, adds the fourth and fifth alpha-

3.77

1,3-linked mannose residues to 0-linked glycans during protein 0-glycosylation

5i — Ugh - Description not mapped 3.75 CAGLOE01155g -- Description not mapped 3.75 CAGLOM01056g YDR339C FCF1 Putative PINc domain nuclease re-

quired for early cleavages of 35S pre-rRNA and maturation of 18S rRNA; component of the SSU

3.75

(small subunit) processome involved in 40S ribosomal subunit biogenesis; copurifies with Faflp




CAGLOG04763g YORIO7W RGS2 Negative regulator of glucose- induced cAMP signaling; directly activates the GTPase activity of the heterotrimeric G protein alpha subunit Gpa2p

3.74

CAGLOL09581g YBR252W DUT1 dUTPase, catalyzes hydrolysis of dUTP to dUMP and PPi, thereby preventing incorporation of uracil into DNA during replication; crit-ical for the maintenance of genetic stability

3.73

CAGLOI06006g YJL148W RPA 34 RNA polymerase I subunit A34.5 3.71 CAGLOD03124g YLLOO8W DRS1 Nucleolar DEAD-box protein re-

quired for ribosome assembly and function, including synthesis of 60S ribosomal subunits; constituent of

3.69

66S pre-ribosomal particles CAGLOE02233g YNL289W PCL1 Pho85 cyclin of the Pc11,2-like sub-

family, involved in entry into the mitotic cell cycle and regulation of morphogenesis, localizes to sites of polarized cell growth

3.65

CAGLOK09416g YGL211W NCS6 Protein required for thiolation of the uridine at the wobble position of GIn, Lys, and Glu tRNAs; has a role in urmylation and in invasive and pseudohyphal growth; inhibits replication of Brome mosaic virus in

3.58

S. cerevisiae CAGLOL10890g YOR272W YTM1 Constituent of 66S pre-ribosomal

particles, required for maturation of the large ribosomal subunit

3.58

CAGLOJ00869g YKL144C RPC25 RNA polymerase III subunit C25, required for transcription initiation; forms a heterodimer with Rpc17p; paralog of Rpb7p

3.57

CAGLOJ09592g YDL2O9C CWG2 Protein involved in pre-mRNA splicing, component of a complex containing Ceflp; interacts with

3.51

Prpl9p; contains an RNA recognition motif; has similarity to S. pombe Cwf2p

CAGLOK09262g YCR059C YIH1 Protein that inhibits activation of 3.46 Gcn2p, an eIF2 alpha subunit pro-tein kinase, by competing for Gcnlp binding, thus impacting gene ex-pression in response to starvation; has sequence and functional simi-larity to the mouse IMPACT gene

CAGLOK06391g YDR462W MRPL28 Mitochondrial ribosomal protein of the large subunit

3.45

CAGLOG05093g YDR061W - Description not mapped 3.43 CAGLOM12430g YIL053W RHR2 Constitutively expressed isoform

of DL-glycerol-3-phosphatase; involved in glycerol biosynthesis, in-duced in response to both anaerobic and, along with the Hor2p/Gpp2p isoform, osmotic stress

3.43

9„, tp6 - - Description not mapped 3.41 CAGLOB01232g YLR186W EMG 1 Member of the alpha/beta knot

fold methyltransferase superfamily; required for maturation of 18S rRNA and for 40S ribosome production; interacts with RNA and with S-adenosylmethionine; associates with spindle/microtubules; forms homodimers

3.39




CAGLOJ09746g YDL201W TRM8 Subunit of a tRNA methyltransferase complex composed of Trm8p and Trm132p that catalyzes 7- methylguanosine modification of tRNA

3.38

CAGLOE00363g - - Description not mapped 3.37 CAGLOJ07766g YNL248C RPA4O RNA polymerase I subunit A49 3.37 CAGLOI09592g YOR303W CPAI Small subunit of carbamoyl phos-

phate synthetase, which catalyzes a step in the synthesis of citrulline, an arginine precursor; translation-ally regulated by an attenuator pep-tide encoded by YOR302W within the CPA1 mRNA 5'-leader

3.36

CAGLOF00253g YKL211C TRP3 Bifunctional enzyme exhibiting both indole-3-glycerol-phosphate synthase and anthranilate synthase activities, forms multifunctional hetero-oligomeric anthranilate synthase:indole-3-glycerol phosphate synthase enzyme complex with Trp2p

3.35

6i — CglII - - Description not mapped 3.32 CAGLOF02431g YJL200C ACO2 Putative mitochondrial aconitase

isozyme; similarity to Acolp, an aconitase required for the TCA cycle; expression induced during growth on glucose, by amino acid starvation via Gcn4p, and repressed on ethanol

3.31

CAGLOH07975g YDL051W LHP1 RNA binding protein required for maturation of tRNA and U6 snRNA precursors; acts as a molecular chaperone for RNAs transcribed by polymerase III; homologous to hu-man La (SS-B) autoantigen

3.31

CAGL0105764g YNR009W NRMI Transcriptional co-repressor of 3.3 MBF (MCB binding factor)- regulated gene expression; Nrmlp associates stably with promoters via MBF to repress transcription upon exit from 01 phase

2 - Description not mapped 3.29 CAGLOB04543g YOR100C CRC.1 Mitochondrial inner membrane car-

nitine transporter, required for carnitine-dependent transport of acetyl-CoA from peroxisomes to mi-tochondria during fatty acid beta-oxidation

3.29

CAGL0.102398g YIL020C HIS6 Phosphoribosyl-5-amino- 3.29 1-phosphoribosy1-4- imidazolecarboxiamide isomerase, catalyzes the fourth step in histidine biosynthesis; mutations cause histidine auxotrophy and sensitivity to Cu, Co, and Ni salts

CAGLOJ00473g YHR052W C/C/ Essential protein that interacts with proteasome components and has a potential role in proteasome substrate specificity; also copurifies with 66S pre-ribosomal particles

3.28



continued from previous page CG O RF SC ORF gene name Description Log Ratio

CAGLOI03608g YDL153C SASIO Essential subunit of U3-containing 3.27 Small Subunit (SSU) processome complex involved in production of 18S rRNA and assembly of small ri-bosomal subunit; disrupts silencing when overproduced

CAGLOK06215g YDR449C UTP6 Nucleolar protein, component of tpliescsmripatlliosnubnintitm(aSpSpUe)dprocessome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

3.26

I,ox2 - - 3.2 CAGLOB00220g YLR186W EMG1 Member of the alpha/beta knot

fold methyltransferase superfamily; required for maturation of 18S rRNA and for 40S ribosome pro-duction; interacts with RNA and with S-adenosylmethionine, associates with spindle/microtubules; forms homodimers

3.24

CAGLOF04983g YLR276C DBP9 ATP-dependent RNA helicase of the DEAD-box family involved in biogenesis of the 60S ribosomal sub-unit

3.24

CAGLOK09284g YCR057C PWP2 Conserved 90S pre-ribosomal corn- ponent essential for proper endonu-cleolytic cleavage of the 35 S rRNA precursor at AO, Al, and A2 sites; contains eight WD-repeats; PWP2 deletion leads to defects in cell cy-cle and bud morphogenesis

3.24

CAGL0004279g YBROO2C RER2 Cis-prenyltransferase involved in dolichol synthesis; participates in endoplasmic reticulum (ER) protein sorting

3.22

CAGLOL10648g YMR199W CLN1 G1 cyclin involved in regulation of the cell cycle; activates Cdc28p ki-nase to promote the GI to S phase transition; late G1 specific expres-sion depends on transcription factor complexes, MBF (Swi6p-Mbp1p) and SBF (Swi6p-Swi4p)

3.22

CAGLOM05621g YPL057C SUR/ Probable catalytic subunit of a mannosylinositol phosphorylce- ramide (MIPC) synthase, forms a complex with probable regulatory subunit Csg2p; function in sphin-golipid biosynthesis is overlapping with that of Cshlp

3.21

CAGLOM05775g YPL211W NIP7 Nucleolar protein required for 60S ribosome subunit biogenesis, constituent of 66S pre-ribosomal particles; physically interacts with

3.21

Nop8p and the exosome subunit Rrp43p

CAGLO.J07590g - - Description not mapped 3.2 CAGLOK09460g YOR145C PNO I Essential nucleolar protein required

for pre-18S rRNA processing, in-teracts with Dim1p, an 18S rRNA dimethyltransferase, and also with

3.2

Nob1p, which is involved in protea-some biogenesis; contains a KH do-main




CAGL0004213g CAGLOB02387g

YBROO5WP YMR285C

- NGL2

Description not mapped Protein involved in 5.8S rRNA processing; Ccr4p-like RNase required for correct 3'-end formation of 5.8S rRNA at site E; similar to Ngllp and Ngllp

3.19 3.18

CAGLOE00341g -- Description not mapped 3.18 CAGLOG06248g YAL025C MAK16 Essential nuclear protein, con-

stituent of 665 pre-ribosomal particles; required for maturation of

3.17

25S and 5.8S rRNAs; required for maintenance of M1 satellite double-stranded RNA of the L-A virus

3,yb -- Description not mapped 3.16 CAGLOH02057g YHR089C GAR1 Protein component of the H/ACA

snoRNP pseudouridylase complex, involved in the modification and cleavage of the 18S pre-rRNA

3.15

CAGL0.110912g YHR065C RRP3 Protein involved in rRNA processing; required for maturation of the

3.15

35S primary transcript of pre-rRNA and for cleavage leading to mature 18S rRNA; homologous to eIF- 4a, which is a DEAD box RNA-dependent ATPase with helicase ac-tivity

CAGLOE05522g YOR342C - Description not mapped 3.14 CAGLOF04499g YBL042C FUJI High affinity uridine permease, lo-

calized to the plasma membrane; not involved in uracil transport

3.14

CAGLOB04125g YPR110C RPC40 RNA polymerase subunit, common to RNA polymerase I and III

3.12

CAGLOG09108g YPL199C - Description not mapped 3.12 CAGLOH00935g -- Description not mapped 3.12 CAGLOH08019g YNL207W R102 Essential serine kinase involved in

the processing of the 20S pre-rRNA into mature 18S rRNA; has similar-ity to Rio1p

3.12

8,,, tp8 -- Description not mapped 3.1 CAGLOB00242g YCRO4OW MATALPHAI Transcriptional co-activator in-

volved in regulation of mating- type-specific gene expression; targets the transcription factor

3.1

Mcmlp to the promoters of alpha- specific genes; one of two genes encoded by the MATalpha mating type cassette

CAGLOE00979g YDR165W TRM82 Subunit of a tRNA methyltransferase complex composed of Trm8p and TrmS2p that catalyzes 7- methylguanosine modification of tRNA

3.1

CAGL0107975g YOL077C BRXI Nucleolar protein, constituent of 3.1 66S pre-ribosomal particles; depletion leads to defects in rRNA processing and a block in the assembly of large ribosomal subunits; possesses a sigma(70)-like RNA- binding motif




CAGLOB02057g YDR130C FINI Spindle pole body-related intermediate filament protein, forms cell cycle-specific filaments between spindle pole bodies in mother and daughter cells, able to self- assemble, expression induced dur-ing S/G2, localization cell-cycle de-pendent

3.09

CAGLOK09570g YNL130C CPT] Cholinephosphotransferase, required for phosphatidylcholine biosynthesis and for inositol- dependent regulation of EPT1 transcription

3.08

CAGLOJ00715g YOR101W RASI GTPase involved in G-protein signaling in the adenylate cyclase activating pathway, plays a role in cell proliferation; localized to the plasma membrane; homolog of mammalian RAS proto-oncogenes

3.07

CAGLOL06138g YGL186C TPN1 Plasma membrane pyridoxine (vitamin B6) transporter; member of the purine-cytosine permease sub-family within the major facilitator superfamily; proton symporter with similarity to Fcy21p, Fcy2p, and

3.07

Fcy22p CAGLOH04939g YLR377C FBPI Fructose-1,6-bisphosphatase, key

regulatory enzyme in the gluconeogenesis pathway, required for glucose metabolism; undergoes either proteasome-mediated or autophagy-mediated degradation depending on growth conditions; interacts with Vid3Op

3.06

CAGLOK12210g YBR104W YMC2 Mitochondrial protein, putative inner membrane transporter with a role in oleate metabolism and glu-tamate biosynthesis; member of the mitochondria] carrier (MCF) fam-ily; has similarity with Ymclp

3.05

CAGLOP00561g YJR063W RPA12 RNA polymerase I subunit Al2.2; contains two zinc binding domains, and the N terminal domain is re-sponsible for anchoring to the RNA pol I complex

3.04

CAGLOM02871g YJL186W MNN5 Alpha-1,2-mannosyltransferase, responsible for addition of the sec-ond alpha-1,2-linked mannose of the branches on the mannan backbone of oligosaccharides, localizes to an early Golgi compartment

3.04

CAGLOM14113g YLR237W TH17 Plasma membrane transporter responsible for the uptake of thiamine, member of the major facil-itator superfamily of transporters; mutation of human ortholog causes thiamine-responsive megaloblastic anemia

3.04




CAGLOB03773g

CAGLOF09207g

YGRI91W

YHR208W

HIP/

BAT1

High-affinity histidine permease, also involved in the transport of manganese ions Mitochondria] branched-chain amino acid aminotransferase, homolog of murine ECA39; highly expressed during logarithmic phase and repressed during stationary phase

3.03

3.03

CAGLOF08371g YGR26OW TNA1 High affinity nicotinic acid plasma membrane permease, responsible for uptake of low levels of nicotinic acid; expression of the gene increases in the absence of extracellular nicotinic acid or para-aminobenzoate (PABA)

3.02

CAGLOL10516g YKR081C RPF2 Essential protein involved in the processing of pre-rRNA and the as-sembly of the 60S ribosomal sub-unit; interacts with ribosomal protein L11; localizes predominantly to the nucleolus; constituent of 66S pre-ribosomal particles

3.02

CAGLOH10010g - - Description not mapped 3.01 CAGLOJ03476g YCR072C RSA4 WD-repeat protein involved in ribo-

some biogenesis; may interact with ribosomes; required for maturation and efficient intra-nuclear transport or pre-60S ribosomal subunits, lo-calizes to the nucleolus

3.01

CAGLOE02893g YBL014C RRN6 Protein involved in the transcription of 35S rRNA genes by RNA polymerase I; component of the core factor (CF) complex also com-posed of Rrnllp, Rrn7p and TATA-binding protein

2.99

CAGLOK01771g YAR035W YAT1 Outer mitochondrial carnitine acetyltransferase, minor ethanol- inducible enzyme involved in transport of activated acyl groups from the cytoplasm into the mito-chondrial matrix; phosphorylated

2.99

CAGLOL03806g YNL11.0C NOP15 Constituent of 66S pre-ribosomal particles, involved in 60S ribosomal subunit biogenesis; localizes to both nucleolus and cytoplasm

2.99

2,, ar 1 - - Description not mapped 2.96 CAGLOF01023g YOL041C NOP12 Nucleolar protein, required for pre- 2.96

25S rRNA processing; contains an RNA recognition motif (RRM) and has similarity to Nopl3p, Nsrlp, and putative orthologs in Drosophila and S. pombe

CAGLOF04103g YBL028C - Description not mapped 2.96 CAGLOI00770g YMR144W - Description not mapped 2.96 CAGLOF05709g YDR184C ATC1 Nuclear protein, possibly involved

in regulation of cation stress responses and/or in the establishment of bipolar budding pattern

2.95

CAGLOJ03916g YPL145C KES1 Member of the oxysterol binding protein family, which includes seven yeast homologs; involved in nega-tive regulation of Secl4p-dependent

2.95

Golgi complex secretory functions, peripheral membrane protein that localizes to the Golgi complex




CAGLOL03025g CAGLOD04884g

YKR025W YPR137W

RPC37 RRP9

RNA polymerase III subunit C37 Protein involved in pre-rRNA processing, associated with U3 snRNP; component of small ribosomal sub-unit (SSU) processosome; ortholog of the human U3-55k protein

2.95 2.94

CAGLOH01309g YDR294C DPL1 Dihydrosphingosine phosphate lyase, regulates intracellular levels of sphingolipid long-chain base phosphates (LCBPs), degrades phosphorylated long chain bases, prefers C16 dihydrosphingosine-l- phosphate as a substrate

2.94

CAGLOL02849g YOR207C RET1 Second-largest subunit of RNA polymerase III, which is responsi-ble for the transcription of tRNA and 5S RNA genes, and other low molecular weight RNAs

2.91

CAGLOJ02222g YER002W NOP16 Constituent of 665 pre-ribosomal particles, involved in 60S ribosomal subunit biogenesis

2.9

CAGLOB01243g YCRO4OW MATALPHA1 Transcriptional co-activator involved in regulation of mating- type-specific gene expression; targets the transcription factor

2.89

Mcm1p to the promoters of alpha- specific genes; one of two genes encoded by the MATalpha mating type cassette

CAGLOL03846g - - Description not mapped 2.89 CAGLOD01606g YPRO58W YMC1 Mitochondrial protein, putative in-

ner membrane transporter with a role in oleate metabolism and glu-tamate biosynthesis; member of the mitochondrial carrier (MCF) fam-ily; has similarity with Ymc2p

2.88

CAGLOL13354g YLR237W THI7 Plasma membrane transporter responsible for the uptake of thiamine, member of the major facil-itator superfamily of transporters; mutation of human ortholog causes thiamine-responsive megaloblastic anemia

2.88

CAGLOM08074g YJR030C - Description not mapped 2.88 CAGLOM13563g YMR292W GOT1 Evolutionarily conserved non-

essential protein present in early 2.86

Golgi cisternae that may be involved in ER-Golgi transport at a step after vesicle tethering to Golgi membranes, exhibits membrane topology similar to that of Sft2p

CAGLOF08019g YGR239C PEX21 Peroxin required for targeting of peroxisomal matrix proteins con-taining PTS2; interacts with Pex7p; partially redundant with PexlSp

2.84

CAGLOM04631g YLR147C SMD3 Core Sm protein Sm D3; part of heteroheptameric complex (with

2.84

Smblp, Smdlp, Smd2p, Smelp, Smx3p, and Smx2p) that is part of the spliceosomal Ul, U2, U4, and U5 snRNPs; homolog of human Sm D3




CAGLOM05599g YBR162C TOS1 Covalently-bound cell wall protein of unknown function; identified as a cell cycle regulated SBF target gene; deletion mutants are highly resistant to treatment with beta-

2.84

1,3-glucanase; has sequence similar-ity to YJL171C

CAGL0001441g YPL183C RTTIO Cytoplasmic protein with a role in regulation of Tyl transposition

2.83

CAGL00O2211g YELO40W UTR2 Cell wall protein that functions in the transfer of chitin to beta(1-

2.83

6)glucan; putative chitin transgly-cosidase; glycosylphosphatidylinos-itol (GPI)-anchored protein local-ized to the bud neck; has a role in cell wall maintenance

CAGLOH08712g YBR200W BEM1 Protein containing SI13-domains, involved in establishing cell polarity and morphogenesis; functions as a scaffold protein for complexes that include Cdc24p, Ste5p, Ste20p, and

2.83

Rsr 1p CAGLOJ00385g YHR049W FSH1 Putative serine hydrolase that lo-

calizes to both the nucleus and cy-toplasm; sequence is similar to S. cerevisiae Fsh2p and Fsh3p and the human candidate tumor suppressor

2.83

OVCA2 CAGLOJ00649g YHR025W THR1 Homoserine kinase, conserved pro-

tein required for threonine biosynthesis; expression is regulated by the GCN4-mediated general amino acid control pathway

2.81

CAGLOL02079g YBR291C GTP1 Mitochondria] inner membrane citrate transporter, member of the mi-tochondrial carrier family

2.81

CAGLOB00352g YCL059C KRR1 Essential nucleolar protein required for the synthesis of 18S rRNA and for the assembly of 40S ribosomal subunit

2.8

CAGLOF02849g YDR412W RRPI7 Component of the pre-60S pre- ribosomal particle; required for cell viability under standard (aerobic) conditions but not under anaerobic conditions

2.8

CAGL0106743g YER145C FTR1 High affinity iron permease involved in the transport of iron across the plasma membrane; forms complex with Fet3p; expression is regulated by iron

2.8

CAGLOA03014g YDR376W ARH1 Oxidoreductase of the mitochondrial inner membrane, involved in cytoplasmic and mitochondrial iron homeostasis and required for activ-ity of Fe-S cluster-containing en-zymes; one of the few mitochondrial proteins essential for viability

2.79

CAGLOD01694g YPRO54W SMK1 Middle sporulation-specific mitogen-activated protein kinase

2.79

(MAPK) required for production of the outer spore wall layers; negatively regulates activity of the glucan synthase subunit Gsc2p



continued from previous page CC ORF SC ORF gene name Description Log Ratio

CAGL0G01210g

CAGLOK10186g

YLR351C

YDR076W

NITS

RAD55

Nit protein, one of two proteins in S. cerevisiae with similarity to the Nit domain of NitFhit from fly and worm and to the mouse and human Nit protein which interacts with the Fhit tumor suppressor; nitrilase su-perfamily member Protein that stimulates strand exchange by stabilizing the binding of Rad5lp to single-stranded DNA; involved in the recombinational repair of double-strand breaks in DNA during vegetative growth and meiosis; forms heterodimer with

2.79

2.79

Rad57p CAGLOM06435g YBR186W PCH2 Nucleolar component of the

pachytene checkpoint, which prevents chromosome segregation when recombination and chromosome synapsis are defective; also represses meiotic interhomolog recombination in the rDNA

2.79

CAGLOM12122g YAL036C RBGI Member of the DRG family of GTP- binding proteins; interacts with translating ribosomes and with

2.79

Tma46p CAGLOF02695g YDR405W MRP20 Mitochondrial ribosomal protein of

the large subunit 2.78

CAGLOK01485g YOL066C R1132 DRAP deaminase, catalyzes the third step of the riboflavin biosyn-thesis pathway; cytoplasmic tRNA pseudouridine synthase involved in pseudouridylation of cytoplasmic tRNAs at position 32

2.78

CAGLOK08360g YKR056W TRM2 tRNA methyltransferase, 5- methylates the uridine residue at position 54 of tRNAs and may also have a role in tRNA stabilization or maturation; endo- exonuclease with a role in DNA repair

2.77

0,,. tp9 - - Description not mapped 2.76 CAGL00O2519g YER028C MIG3 Probable transcriptional repressor

involved in response to toxic agents such as hydroxyurea that inhibit ri-bonucleotide reductase; phosphory-lation by Snflp or the Meclp path-way inactivates Mig3p, allowing in-duction of damage response genes

2.76

CAGLOM10219g YKLOO8C LACI Ceramide synthase component, involved in synthesis of ceramide from C26(acyl)-coenzyme A and dihydrosphingosine or phytosphingosine, functionally equivalent to

2.76

Laglp CAGLOA04323g YEL057C PTH2 One of two (see also PTill)

mitochondrially-localized peptidyl- tRNA hydrolases; negatively regulates the ubiquitin-proteasome pathway via interactions with ubiquitin-like ubiquitin-associated proteins; dispensable for cell growth

2.75




CAGLOB03883g YPRO58W YMC1 Mitochondrial protein, putative inner membrane transporter with a role in oleate metabolism and glu-tamate biosynthesis; member of the mitochondrial carrier (MCF) fam-ily; has similarity with Yme2p

2.75

CAGL0003575g YJR151C DAN4 Cell wall mannoprotein with similarity to Tirlp, Tir2p, Tir3p, and

2.75

Tir4p; expressed under anaerobic conditions, completely repressed during aerobic growth

CAGLOL00341g YKL172W EBP2 Essential protein required for the maturation of 25S rRNA and 60S ri-bosomal subunit assembly, localizes to the nucleolus; constituent of 66S pre-ribosomal particles

2.75

CAGLOH02079g YHR088W RPFI Nucleolar protein involved in the assembly of the large ribosomal subunit; constituent of 66S pre- ribosomal particles; contains a sigma(70)-like motif, which is thought to bind RNA

2.74

CAGLOII10142g YDR035W ARO.? 3-deoxy-D-arabino-heptulosonate- 2.74 7-phosphate (DAHP) synthase, catalyzes the first step in aromatic amino acid biosynthesis and is feedback-inhibited by phenylalanine or high concentration of tyrosine or tryptophan

CAGLOI05500g YER099C PRS2 5-phospho-ribosyl-1(alpha)- pyrophosphate synthetase, synthesizes PRPP, which is required for nucleotide, histidine, and tryptophan biosynthesis; one of five related enzymes, which are active as heteromultimeric complexes

2.74

CAGLOK00627g YJL193W Description not mapped 2.74 CAGL0.103278g YER077C Description not mapped 2.73 CAGLOB02926g - - Description not mapped 2.72 CAGLOI02354g YHR169W DBP8 ATPase, putative RNA helicase of

the DEAD-box family; component of 90S preribosome complex involved in production of 18S rRNA and assembly of 40S small riboso-mal subunit; ATPase activity stim-ulated by association with Esp2p

2.72

CAGLOK04477g YGRO6OW ERG25 C-4 methyl sterol oxidase, catalyzes the first of three steps re-quired to remove two C-4 methyl groups from an intermediate in ergosterol biosynthesis; mutants accumulate the sterol intermediate

2.72

4,4-dimethylzymosterol CAGLOM06941g YNL022C Description not mapped 2.72

- Description not mapped 2.71 CAGL0001617g - - Description not mapped 2.71 CAGLOE01903g YOL125W TRAD 3 2'-O-methyltransferase responsible

for modification of tRNA at position 4; exhibits no obvious similarity to other known methyltrans-ferases

2.71




CAGLOF05753g YDR182W CDC1 Putative lipid phosphatase of the endoplasmic reticulum; shows

2.71

Mn2+ dependence and may affect Ca2+ signaling; mutants display actin and general growth defects and pleiotropic defects in cell cycle progression and organelle distribution

CAGLOJ01045g YJL033W fICA.4 Putative nucleolar DEAD box RNA helicase; high-copy number sup- pression of a U14 snoRNA process-ing mutant suggests an involvement in 18S rRNA synthesis

2.71

CAGLOJ02816g YER049W TPA1 Protein of unknown function; interacts with Sup45p (eRF1), Sup35p

2.7

(eRF3) and Pablp; has a role in translation termination efficiency, mRNA poly(A) tail length and mRNA stability

CAGLOJ11066g YLROO2C NOC3 Protein that forms a nuclear corn- plex with Noc2p that binds to

2.7

665 ribosomal precursors to mediate their intranuclear transport; also binds to chromatin to promote the association of DNA replication factors and replication initiation

CAGLOG05610g YDL219W DTD1 D-Tyr-tRNA(Tyr) deacylase, functions in protein translation, may af-fect nonsense suppression via alter-ation of the protein synthesis ma-chinery; ubiquitous among eukary-otes

2.69

CAGLOG07106g YML022W APTI Adenine phosphoribosyltransferase, catalyzes the formation of AMP from adenine and 5- phosphoribosylpyrophosph ate; involved in the salvage pathway of purine nucleotide biosynthesis

2.69

CAGLOI10670g YPR144C NOC4 Nucleolar protein, forms a complex with Nopl4p that mediates matura-tion and nuclear export of 40S ribo-somal subunits

2.69

CAGLOG01991g YOR056C NO131 Essential nuclear protein involved in proteasome maturation and syn-thesis of 40S ribosomal subunits; re-quired for cleavage of the 205 pre-rRNA to generate the mature 18S rRNA

2.68

CAGLOE04224g YBRO71W - Description not mapped 2.67 CAGLOI05016g YER090W TR P2 Anthranilate synthase, catalyzes

the initial step of tryptophan biosynthesis, forms multifunctional hetero-oligomeric anthranilate synthase.indole-3-glycerol phosphate synthase enzyme complex with Trp3p

2.67

CAGLOI09438g YBR271W - Description not mapped 2.67 CAGLOE02409g YOLOO7C CS/2 Protein of unknown function; green

fluorescent protein (GFP)- fusion protein localizes to the mother side of the bud neck and the vacuole;

2.66

YOLOO7C is not an essential gene continued on next page



CAGLOF01177g CAGLOG10043g

CAGLOJ10010g

YOL036W YPR187W

YNL062C

- RP026

GCD10

Description not mapped RNA polymerase subunit ABC23, common to RNA polymerases I, II, and III; part of central core; similar to bacterial omega subunit Subunit of tRNA (1- methyladenosine) methyltransferase with Gcd14p, required for the modification of the adenine at position 58 in tRNAs, especially tRNAi-Met; first identified as a negative regulator of GCN4 expression

2.66 2.66

2.66

CAGLOJ05412g YGL099W LSG1 Putative GTPase involved in 60S ribosomal subunit biogenesis; required for the release of Nmd3p from 60S subunits in the cytoplasm

2.65

CAGLOJ10802g - - Description not mapped 2.64 CAGLOL13024g YHR121W LSM12 Protein of unknown function that

may function in RNA processing; interacts with Pbplp and Pbp4p and associates with ribosomes; con-tains an RNA-binding LSM domain and an AD domain; GFP-fusion protein is induced by the DNA-damaging agent MMS

2.63

CAGLOK08030g YPRO91C - Description not mapped 2.62

CAGLOD03344g YLR024C UDR2 Cytoplasmic ubiquitin-protein ligase (E3); required for ubiquitylation of Rpn4p; mediates formation of a Mublp-Ubr2p-Rad6p complex

2.61

CAGL0106490g - - Description not mapped 2.61

CAGLOE01925g YOL124C TRMI1 Catalytic subunit of an adoMet- dependent tRNA methyltransferase complex (Trmllp-Trm112p), required for the methylation of the guanosine nucleotide at position

2.6

10 (m2G10) in tRNAs; contains a THUMP domain and a methyl-transferase domain

CAGLOE05126g YGL155W CDC43 Beta subunit of geranylgeranyl- transferase type I, catalyzes ger- anylgeranylation to the cysteine residue in proteins containing a C-terminal CaaX sequence ending in

2.6

Leu or Phe; has substrates impor-tant for morphogenesis

CAGL0107315g YOR131C - Description not mapped 2.6 CAGLOJ10604g YDR437W GPI19 Subunit of GPI-G1cNAc trans-

ferase involved in synthesis of N-acetylglucosaminyl phosphatidylinositol (GIcNAc-PI), which is the first intermediate in glycosylphosphatidylinositol (GPI) anchor synthesis, shares similarity with mammalian PIG-P

2.6

CAGLOL02799g - - Description not mapped 2.6 CAGLOM05797g YGL063W PUS2 Mitochondrial tRNA:pseudouridine

synthase; acts at positions 27 and 2.6

28, but not at position 72; effi- ciently and rapidly targeted to mitochondria, specifically dedicated to mitochondria) tRNA modifica-tion




CAGLOM10197g

CAGLOM13519g

YKL009W

YMR290C

MRT4

HAS1

Protein involved in mRNA turnover and ribosome assembly, localizes to the nucleolus ATP-dependent RNA helicase; localizes to both the nuclear periph-ery and nucleolus; highly enriched in nuclear pore complex fractions; constituent of 66S pre-ribosomal particles

2.6

2.6

CAGLOD05610g YLL010C PSR1 Plasma membrane associated protein phosphatase involved in the general stress response; required along with binding partner

2.59

Whi2p for full activation of STRE-mediated gene expression, possibly through dephosphorylation of Msn2p

CAGLOE03245g YGR159C NSRI Nucleolar protein that binds nuclear localization sequences, required for pre-rRNA processing and ribosome biogenesis

2.59

CAGL0001639g YBR247C ENP1 Protein associated with U3 and U14 snoRNAs, required for pre-rRNA processing and 40S ribosomal sub-unit synthesis; localized in the nu-cleus and concentrated in the nucle-olus

2.58

CAGL0107799g YBR154C RPB5 RNA polymerase subunit ABC27, common to RNA polymerases I, II, and III; contacts DNA and affects transactivation

2.58

CAGLOJ02948g YER060W FC1,11./ Putative purine-cytosine permease, very similar to Fcy2p but cannot substitute for its function

2.57

CAGLOD03410g YJL025W RRN7 Protein involved in the transcription of 35S rRNA genes by RNA polymerase I; component of the core factor (CF) complex also com-posed of Rrnllp, Rrn6p and TATA-binding protein

2.54

CAGL0,110890g YHRO66W SSF1 Constituent of 66S pre-ribosomal particles, required for ribosomal large subunit maturation; function-ally redundant with Ssf2p; member of the Brix family

2.54

CAGLOK02211g YER127W LCP5 Essential protein involved in maturation of 18S rRNA; depletion leads to inhibited pre-rRNA processing and reduced polysome levels; local-izes primarily to the nucleolus

2.54

- - Description not mapped 2.53 CAGL00O2497g YER029C SMR/ Core Sm protein Sm B; part

of heteroheptameric complex (with 2.53

Smdlp, Smd2p, Smd3p, Smelp, Smx3p, and Smx2p) that is part of the spliceosomal Ul, 112, U4, and 1.15 snRNPs; homolog of human Sm B and Sm B'

CAGLOJ09614g YDL208W NHP2 Nuclear protein related to mammalian high mobility group (HMG) proteins, essential for function of

2.53

H/ACA-type snoRNPs, which are involved in 18S rRNA processing




CAGL0G07535g YOR294W RRS1 Essential protein that binds ribosomal protein L11 and is required for nuclear export of the 60S pre-ribosomal subunit during ribosome biogenesis; mouse homolog shows altered expression in Huntington's disease model mice

2.52

CAGL0003355g YNR054C ESF2 Essential nucleolar protein involved in pre-18S rRNA processing; binds to RNA and stimulates ATPase ac-tivity of Dbp8; involved in assembly of the small subunit (SSU) proces-some

2.51

CAGL0004961g YJR106W ECM27 Non-essential protein of unknown function

2.51

CAGLOD02018g - - Description not mapped 2.51 CAGLOHOO11Og YIR019C MUC/ GPI-anchored cell surface glyco-

protein (flocculin) required for pseudohyphal formation, invasive growth, flocculation, and biofllms; transcriptionally regulated by the

2.51

MAPK pathway (via Stel2p and Teclp) and the cAMP pathway (via Flo8p)

CAGLOH10252g YBRO61C TRM7 2'-O-ribose methyltransferase, methylates the 2'-O-ribose of nu-cleotides at positions 32 and 34 of the tRNA anticodon loop

2.51

CAGLOJ03388g YDL175C AIR2 RING finger protein that interacts with the arginine methyltransferase

2.51

Hmtlp; may regulate methylation of Npl3p, which modulates Npl3p function in mRNA processing and export; has similarity to Air1p

CAGLOJ11110g YJR003C - Description not mapped 2.51 CAGLOK09042g YOR252W TMA15 Protein of unknown function that

associates with ribosomes 2.51

CAGLOE03069g - - Description not mapped 2.5 CAGLOI10747g YPR138C MEPS Ammonium permease of high ca-

pacity and low affinity; belongs to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium (NII4-1-); expression is under the nitrogen catabo-lite repression regulation ammonia permease

2.5

CAGLOJ11242g YNL180C RHOS Non-essential small GTPase of the 2.5 Rho/Rac subfamily of Ras-like pro-teins, likely involved in protein kinase C (Pkclp)-dependent signal transduction pathway that controls cell integrity

CAGLOM05687g YGL067W NPY1 NADH diphosphatase (pyrophos- phatase), hydrolyzes the pyrophos-phate linkage in NADH and related nucleotides; localizes to peroxisomes

2.5

CAGLOF07359g YGL117W - Description not mapped 2.49 CAGLOF07557g YKL095W YJU2 Essential protein required for pre-

mRNA splicing; associates transiently with the spliceosomal NTC

2.49

(nineteen complex) and acts after Prp2p to promote the first catalytic reaction of splicing



continued from previous page CG ORF SC ORE gene name Description Log Ratio

CAGLOG10219g YHR211W FLO5 Lectin-like cell wall protein (flocculin) involved in flocculation, binds to mannose chains on the surface of other cells, confers floc-forming ability that is chy- motrypsin resistant but heat labile; similar to Flo1p

2.49

CAGLOJ00297g YHR045W - Description not mapped 2.49 CAGL0D05588g YLL011W SOFT Essential protein required for bio-

genesis of 40S (small) ribosomal subunit; has similarity to the beta subunit of trimeric G-proteins and the splicing factor Prp4p

2.48

CAGLOI02508g YIIR175W CTR2 Putative low-affinity copper transporter of the vacuolar membrane; mutation confers resistance to toxic copper concentrations, while over-expression confers resistance to cop-per starvation

2.48

CAGLOM01716g YBRO83W TECI Transcription factor required for fpuellscTriypltioenpx.rissmioanp,pTedyl-mediated gene activation, and haploid invasive and diploid pseudohyphal growth; TEA/ATTS DNA-binding domain family member

2.48

CAGLOA03905g - - 2.47 CAGLOG01254g YGL202W ARO8 Aromatic aminotransferase I, ex-

pression is regulated by general con-trol of amino acid biosynthesis

2.47

CAGLONI11462g YLR099C ICT1 Lysophosphatidic acid acyltransferase, responsible for enhanced phospholipid synthesis during organic solvent stress; null displays increased sensitivity to Calcofluor white; highly expressed during or-ganic solvent stress

2.47

CAGLOA00385g YGL01.6W KAP1.22 Karyopherin beta, responsible for import of the Toalp-Toa2p complex into the nucleus; binds to nucleo-porins Nuplp and Nup2p; may play a role in regulation of pleiotropic drug resistance

2.45

CAGL0I00724g YOR306C MCH5 Plasma membrane riboflavin transporter; facilitates the uptake of vitamin B2; required for FAD- dependent processes; sequence simi-larity to mammalian monocarboxylate permeases, however mutants are not deficient in monocarboxy-late transport

2.45

CAGLOM03949g YNL310C Z/M/ 7 Heat shock protein with a zinc finger motif; essential for protein import into mitochondria; may act with Paml8p to facilitate recogni-tion and folding of imported pro-teins by Ssc1p (mtHSP70) in the mitochondrial matrix

2.45

CAGLOM04477g YLR141W RRN5 Protein involved in transcription of rDNA by RNA polymerase I; tran-scription factor, member of UAF

2.45

(upstream activation factor) family along with Rrn9p and RrnlOp




CAGLOA00627g CAGLOA03212g

YAR002CA YDR384C

- ATO3

Description not mapped Plasma membrane protein, regulation pattern suggests a possible role in export of ammonia from the cell; phosphorylated in mitochondria; member of the TC 9.B.33

2.44 2.44

YaaH family of putative transporters

CAGLOM08096g YOR208W PTP2 Phosphotyrosine-specific protein phosphatase involved in the inactivation of mitogen-activated protein kinase (MAPK) during osmolarity sensing; dephosporylates Hog1p MAPK and regulates its localization; localized to the nucleus

2.44

CAGLOA04257g YER088C DOT6 Protein involved in rRNA and ribosome biogenesis; binds polymerase

2.43

A and C motif; subunit of the RPD3L histone deacetylase complex; similar to Tod6p; has chromatin specific SANT domain; in-volved in telomeric gene silencing and filamentation

CAGLOF06853g YKL021C MA KI 1 Protein involved in an early, nude- o] ribosomal subunit biogenesis; essential for cell growth and replication of killer M1 dsRNA

2.43

CAGLOD00396g - -

60S.tmd ofonn

virus; contains four beta-transducin repeats

r) easr crs itpetpi appe

2.42 CAGLOD00880g YDLO6OW TSR1 Protein required for processing of 2.42

20S pre-rRNA in the cytoplasm, associates with pre-40S ribosomal particles

CAGLOF06809g YIR026C YVH1 Protein phosphatase involved in vegetative growth at low tempera-tures, sporulation, and glycogen accumulation; transcription induced by low temperature and nitrogen starvation; member of the dual- specificity family of protein phos-phatases

2.42

CAGLOJ11154g YNL175C HOPIS Protein of unknown function, localizes to the nucleolus and nucleo-plasm; contains an RNA recogni-tion motif (RRM) and has similar-ity to Nopl2p, which is required for processing of pre-18S rRNA

2.42

CAGLOA02431g YDR349C YPS7 Putative GPI-anchored aspartic protease, located in the cytoplasm and endoplasmic reticulum

2.41

CAGLOB02970g - - Description not mapped 2.41 CAGL0102398g YHR170W NMD3 Protein involved in nuclear export

of the large ribosomal subunit; acts as a Crmlp-dependent adapter pro-tein for export of nascent ribosomal subunits through the nuclear pore complex

2.41




CAGLOJ00517g

CAGLOJ09702g CAGLOM00308g CAGLOM00572g CAGLOG07018g CAGLOL04928g

YHR031C

-- --

YJR129C YML018C YMR047C

RRM3

- NUP116

DNA helicase involved in rDNA replication and Tyl transposition; relieves replication fork pauses at telomeric regions; structurally and functionally related to Pif1p Description not mapped Description not mapped Description not mapped Description not mapped Subunit of the nuclear pore corn- plex (NPC) that is localized to both sides of the pore; contains a repeti-tive GLFG motif that interacts with mRNA export factor Mex67p and with karyopherin Kap95p; homolo-gous to Nup100p

2.41

2.41 2.41 2.41 2.4 2.4

CAGLOM01430g YDR324C UTP4 Subunit of U3-containing 90S preribosome and Small Subunit (SSU) processome complexes involved in

2.4

Production of 18S rRNA and assembly of small ribosomal subunit; member of t-Utp subcomplex involved with transcription of 35S rRNA transcript

CAGL00O2255g YER041W YEN1 Holliday junction resolvase; homolog of human GEN1 and has sim-ilarity to S. cerevisiae endonuclease

2.39

Rthlp; potentially phosphorylated by Cdc28p

CAGLOG02761g YIL103W DPH1 Protein required, along with 2.39 Dph2p, Ktillp, Jjj3p, and Dph5p, for synthesis of diphthamide, which is a modified histidine residue of translation elongation factor 2 (Eft1p or Eft2p); may act in a complex with Dph2p and Ktillp

CAGLOI05544g YER095W RAD51 Strand exchange protein, forms a helical filament with DNA that searches for homology; involved in the recombinational repair of double-strand breaks in DNA dur-ing vegetative growth and meiosis; homolog of Dmclp and bacterial

2.39

RecA protein CAGLOJ10208g YNL072W RNH201 Ribonuclease H2 catalytic sub-

unit, removes RNA primers during Okazaki fragment synthesis; homolog of RNAse HI (the S. cerevisiae homolog of mammalian

2.39

RNAse HII is RNH1); related to human AGS4 that causes Aicardi- Goutieres syndrome

CAGLOM11110g YDR087C RRP1 Essential evolutionarily conserved nucleolar protein necessary for bio-genesis of 60S ribosomal subunits and processing of pre-rRNAs to ma-ture rRNAs, associated with several distinct 66S pre-ribosomal particles

2.39

CAGLOD05874g YJR002W MPPIO Component of the SSU processome and 90S preribosome, required for pre-18S rRNA processing, interacts with and controls the stability of

2.38

Imp3p and Imp4p, essential for vi-ability; similar to human Mpp lOp




CAGLOG02585g YIL094C LYSI2 Homo-isocitrate dehydrogenase, an NAD-linked mitochondria] enzyme required for the fourth step in the biosynthesis of lysine, in which homo-isocitrate is oxidatively de- carboxylated to alpha-ketoadipate

2.38

CAGLOL02035g YKL029C MAE! Mitochondrial malic enzyme, catalyzes the oxidative decarboxylation of malate to pyruvate, which is a key intermediate in sugar metabolism and a precursor for syn-thesis of several amino acids

2.38

CAGLOH02189g YIVIR269W TMA23 Nucleolar protein of unknown function implicated in ribosome biogenesis; TMA23 may be a fungal-specific gene as no homologs have been yet identified in higher eukary-otes

2.37

CAGLOH07051g YDR321W ASP1 Cytosolic L-asparaginase, involved in asparagine catabolism

2.37

CAGLOH07469g YFRO19W FABI 1-phosphatidylinositol-3-phosphate 2.36 5-kinase; vacuolar membrane kinase that generates phosphatidylinositol (3,5)P2, which is involved in vacuolar sorting and homeostasis

CAGLOK06171g - - Description not mapped 2.36 CAGLOK11506g YKL113C RAD27 5' to 3' exonuclease, 5' flap endonu-

ment processing and maturation as well as for long-patch base-excision

cpleeascsrei,ptrieociunirstd rppOedkazaki frag-

repair; member of the S. pombe

2.36

RAD2/FEN1 family CAGLOL07590g - - mapped 2.36 CAGLOM09801g YLR285W NNT1 Putative nicotinamide N-

methyltransferase, has a role in rDNA silencing and in lifespan determination

2.36

CAGLOA03476g YLR034C SMF3 Putative divalent metal ion transporter involved in iron homeostasis; transcriptionally regulated by metal ions; member of the Nramp family of meta/ transport proteins

2.35

CAGL00O2981g YCR048W ARE1 Acyl-CoA:sterol acyltransferase, isozyme of Are2p; endoplasmic reticulum enzyme that contributes the major sterol esterification activity in the absence of oxygen

2.35

CAGLOG03003g YGRO9OW UTP22 Possible U3 snoRNP protein involved in maturation of pre-18S rRNA, based on computational analysis of large-scale protein- protein interaction data

2.35

CAGLOH06017g YBROO8C FLR1 Plasma membrane multidrug transporter of the major facilitator superfamily, involved in efflux of fluconazole, diazaborine, benomyl, methotrexate, and other drugs

2.35

CAGLOK06919g YBR227C MCX1 Mitochondrial matrix protein; putative ATP-binding chaperone with non-proteolytic function; similar to bacterial CIpX proteins

2.35

CAGL0003003g YCR051W - Description not mapped 2.34 CAGLOK01287g YGRO74W SMD1 Core Sm protein Sm Dl; part

of heteroheptameric complex (with 2.34

Smblp, Smd2p, Smd3p, Smelp, Smx3p, and Smx2p) that is part of the spliceosomal Ul, U2, U4, and U5 snRNPs; homolog of human Sm D1




CAGLOF03091g CAGLOG03443g

-- YER110C KAP123

Description not mapped Karyopherin beta, mediates nuclear import of ribosomal proteins prior to assembly into ribosomes and im-port of histones H3 and H4; localizes to the nuclear pore, nucleus, and cytoplasm; exhibits genetic in-teractions with RAIL

2.33 2.33

CAGLOG07249g YML027W YOX1 Homeodomain-containing transcriptional repressor, binds to

2.33

Mcm1p and to early cell cycle boxes (ECBs) in the promoters of cell cycle-regulated genes expressed in M/G1 phase; expression is cell cycle-regulated; potential Cdc28p substrate

CAGLOH05731g YPL094C SEC62 Essential subunit of Sec63 corn- plex (Sec63p, Sec62p, Sec66p and Sec72p); with Sec61 complex, Kar2p/BiP and Lhslp forms a channel competent for SRP- dependent and post-translational

2.33

SRP-independent protein targeting and import into the ER

CAGLOL10714g YMR202W ERG2 C-8 sterol isomerase, catalyzes the isomerization of the delta-8 double bond to the delta-7 position at an intermediate step in ergosterol biosynthesis

2.33

CAGLOM08756g YDR261C EXG2 Exo-1,3-beta-glucanase, involved in cell wall beta-glucan assembly; may be anchored to the plasma mem-brane via a glycosylphosphatidyli-nositol (GPI) anchor

2.33

CAGL01103465g - - Description not mapped 2.32 CAGLOH10626g YARO5OW FLO1 Lectin-like protein involved in floc-

culation, cell wall protein that binds to mannose chains on the surface of other cells, confers floc-forming ability that is chy- motrypsin sensitive and heat resis-tant; similar to Flo5p

2.32

CAGL0105676g YNFt011C PRP2 RNA-dependent ATPase in the 2.32 DEAR-box family, required for acti-vation of the spliceosome before the first transesterification step in RNA splicing

CAGLOK09922g YOR359W VTSI Post-transcriptional gene regulator, RNA-binding protein containing a

2.32

SAM domain; shows genetic inter-actions with Vtilp, which is a v- SNARE involved in cis-Golgi mem-brane traffic

CAGLOL06314g YDR101C ARX1 Shuttling pre-60S factor; involved in the biogenesis of ribosomal large subunit biogenesis; interacts directly with Albl; responsible for

2.32

Tif6 recycling defects in absence of Reil; associated with the ribosomal export complex




CAGLOL13332g

CAGLOA01199g

YAL063C

YPL265W

FLOP

DIPS

Lectin-like protein with similarity to Flo1p, thought to be expressed and involved in flocculation Dicarboxylic amino acid permease, mediates high-affinity and high- capacity transport of L-glutamate and L-aspartate; also a transporter for Gln, Asn, Ser, Ala, and Gly

2.32

2.31

CAGLOE02673g YOR004W UTP23 Essential nucleolar protein that is a component of the SSU (small sub-unit) processome involved in 40S ribosomal subunit biogenesis; has homology to PING domain protein

2.31

Fcflp, although the PING domain of Utp23p is not required for function

CAGLOF06963g YGLO61C DUOI Essential subunit of the Daml corn- plex (aka DASH complex), couples kinetochores to the force produced by MT depolymerization thereby aiding in chromosome segregation; is transferred to the kinetochore prior to mitosis

2.31

CAGLOF08129g YGR245C SDAI Highly conserved nuclear protein required for actin cytoskeleton organization and passage through

2.31

Start, plays a critical role in G1 events, binds Nap1p, also involved in 60S ribosome biogenesis

CAGL0G03597g YER118C SHOT Transmembrane osmosensor, participates in activation of both the Cdc42p- and MAP kinase- dependent filamentous growth pathway and the high-osmolarity glycerol response pathway

2.31

CAGLOJ02376g YIL019W FAFI Protein required for pre-rRNA processing and 40S ribosomal subunit assembly

2.31

CAGLOL05566g YJL125C GCDI4 Subunit of tRNA (1- methyladenosine) methyltransferase, with GcdlOp, required for the modification of the adenine at position 58 in tRNAs, especially tRNAi-Met; first identified as a negative regulator of GCN4 expression

2.31

CAGLOM03751g YNL299W TRFS Poly (A) polymerase involved in nuclear RNA quality control based on: homology with Trf4p, genetic interactions with TRF4 mutants, physical interaction with Mtr4p

2.31

(TRAMP subunit), and by direct assay; disputed role as a sigma DNA polymerase

CAGLOM06457g YBR187W GDTI Putative protein of unknown function; expression is reduced in a gcrl null mutant; GFP-fusion protein localizes to the vacuole; expression pattern and physical interactions suggest a possible role in ribosome biogenesis

2.31




CAGLOG05544g CAGLOH01595g

- YDR527W

- RBA50

Description not mapped Protein involved in transcription; interacts with RNA polymerase II subunits Rpb2p, Rpb3, and

2.3 2.3

Rpbllp; has similarity to human RPAP 1

CAGLOI07777g YOL088C IlIPD2 Member of the protein disulfide isomerase (PDI) family, exhibits chap-erone activity; overexpression sup-presses the lethality of a pdil dele-tion but does not complement all

2.3

Pdilp functions; undergoes oxida-tion by Erolp

CAGLOL08998g YFLOO1W DEG1 Non-essential tRNA:pseudouridine synthase, introduces pseu- douridines at position 38 or 39 in tRNA, important for maintenance of translation efficiency and normal cell growth, localizes to both the nucleus and cytoplasm

2.3

CAGLOB00286g YCL064C CHA I Catabolic L-serine (L-threonine) deaminase, catalyzes the degradation of both L-serine and L- threonine; required to use serine or threonine as the sole nitrogen source, transcriptionally induced by serine and threonine

2.29

CAGLOB03663g - - Description not mapped 2.29 CAGLOD04796g YPR131C NAT3 Catalytic subunit of the NatB N-

terminal acetyltransferase, which catalyzes acetylation of the amino-terminal methionine residues of all proteins beginning with Met-Asp or

2.29

Met-Glu and of some proteins be-ginning with Met-Asn or Met-Met

CAGLOH01441g YDR300C PRO1 Gamma-glutamyl kinase, catalyzes the first step in proline biosynthesis

2.29

CAGLOJ04576g YKL191W DPH2 Protein required, along with 2.29 Dphlp, Ktillp, Jjj3p, and Dph5p, for synthesis of diphthamide, which is a modified histidine residue of translation elongation factor 2 (Eftlp or Eft2p); may act in a complex with Dphlp and Ktillp

CAGLOL06710g YOL092W - Description not mapped 2.29 CAGLOD01892g YLR027C AAT2 Cytosolic aspartate aminotrans-

ferase, involved in nitrogen metabolism; localizes to peroxisomes in oleate-grown cells

2.28

CAGLOH06545g YIL146C ECM37 Non-essential protein of unknown function

2.28

CAGLOI02926g YDR496C PUF6 Pumilio-homology domain protein that binds ASH1 mRNA at PUF consensus sequences in the 3' UTR and represses its translation, result-ing in proper asymmetric localiza-tion of ASH1 mRNA

2.28

CAGLOI02860g YOR112W CEX1 Cytoplasmic component of the nuclear aminoacylation-dependent tRNA export pathway; interacts with nuclear pore component

2.27

Nup116p; copurifies with tRNA export receptors Loslp and Msn5p, as well as eIF-la and the RAN GTPase Gsplp




CAGL0103806g YDL141W BPL1 Biotin:apoprotein ligase, covalently modifies proteins with the addition of biotin, required for acetyl-

2.27

CoA carboxylase (Acclp) holoen-zyme formation

CAGLOJ03014g YIL056W VHR1 Transcriptional activator, required for the vitamin H-responsive ele-ment (VHRE) mediated induction of VHT1 (Vitamin H transporter) and BI05 (biotin biosynthesis inter-mediate transporter) in response to low biotin concentrations

2.27

CAGLOJ05390g - - Description not mapped 2.27 CAGLOL02871g YOR206W NOC2 Protein that forms a nucleolar corn-

plex with Mak21p that binds to 2.27

90S and 66S pre-ribosomes, as well as a nuclear complex with Noc3p that binds to 66S pre-ribosomes; both complexes mediate intranuclear transport of ribosomal precur-sors

CAGLOL11308g YML067C ERV4/ Protein localized to COPII-coated vesicles, forms a complex with

2.27

Erv46p; involved in the membrane fusion stage of transport; has ho-mology to human ERGIC2 (PTX1) protein

CAGLOM02805g YPL126W NANI U3 snoRNP protein, component of the small (ribosomal) subunit

2.27

(SSU) processosome containing U3 snoRNA; required for the biogene-sis ofl8S rRNA

CAGLOM13915g YMR310C - Description not mapped 2.27 CAGLOA02673g YDR361C BCP1 Essential protein involved in nu-

clear export of Mss4p, which is a lipid kinase that generates phosphatidylinositol 4,5-biphosphate and plays a role in actin cytoskeleton organization and vesicular transport

2.26

CAGL0103542g YDL156W CMRI Putative protein of unknown function; protein sequence contains three WD domains (WD-40 repeat); green fluorescent protein (GFP)- fusion protein localizes to the cyto-plasm and nucleus

2.26

CAGLOJ05984g YNL141W AAH1 Adenine deaminase (adenine amino- hydrolase), converts adenine to hy-poxanthine; involved in purine sal-vage; transcriptionally regulated by nutrient levels and growth phase;

2.26

Aahlp degraded upon entry into quiescence via SCF and the protea-some

CAGLOL13156g YLR073C - Description not mapped 2.26 CAGLOA01177g YPL268W PLC/ Phospholipase C, hydrolyzes phos-

phatidylinositol 4,5-biphosphate 2.2

(PIP2) to generate the signaling molecules inositol 1,4,5- triphosphate (IP3) and 1,2- diacylglycerol (DAG); involved in regulating many cellular processes



continued from previous page CG O RF SC ORF gene name Description Log Ratio

CAGLOI00198g CAGLOL05610g

-- YJL127C SPTIO

Description not mapped Putative histone acetylase, sequence-specific activator of histone genes, binds specifically and highly cooperatively to pairs of UAS elements in core histone promoters, functions at or near the

2.2 2.2

TATA box CAGLOL10846g YCR047C B UD23 Protein involved in bud-site selec-

tion; diploid mutants display a ran-dom budding pattern instead of the wild-type bipolar pattern

2.2

CAGLOM06589g YDR020C DAS2 Putative protein of unknown function; not an essential gene; weak similarity with uridine kinases and with phosphoribokinases

2.2

- - Description not mapped 2.24 CAGL0E04334g YHROO7C ERG11 Lanosterol 14-alpha-demethylase,

catalyzes the C-14 demethylation of lanosterol to form 4,4"-dimethyl cholesta-8,14,24-triene-3-beta-ol in the ergosterol biosynthesis pathway; member of the cytochrome

2.24

P450 family CAGLOF03261g - - Description not mapped 2.24 CAGLOH09724g YER006W NUG1 GTPase that associates with nu-

clear 60S pre-ribosomes, required for export of SOS ribosomal sub-units from the nucleus

2.24

CAGL0105588g YNR015W SMMI Dihydrouridine synthase, member of a family of dihydrouridine syn- thases including Duslp, Smm1p, Dus3p, and Dus4p; modifies uridine residues at position 20 of cytoplas-mic tRNAs

2.24

CAGLOM03905g YNL308C KRII Essential nucleolar protein required for 40S ribosome biogenesis; physically and functionally interacts with Krrlp

2.24

CAGLOH04235g YML080W DUSI Dihydrouridine synthase, member of a widespread family of conserved proteins including Smmlp, Dus3p, and Dus4p; modifies pre- tRNA(Phe) at U17

2.23

CAGL0109834g - Description not mapped 2.23 CAGLOJ07986g YNL260C Description not mapped 2.23 CAGLOL06116g YGL185C - Description not mapped 2.23 CAGLOF03443g YMR274C RCE1 Type II CAAX prenyl protease in-

volved in the proteolysis and matu-ration of Ras and the a-factor mat-ing pheromone

2.22

CAGLOF08525g YGROO1C AML 1 Putative protein of unknown function with similarity to methyltrans-ferase family members; green flu-orescent protein (GFP)-fusion pro-tein localizes to the cytoplasm; re-quired for replication of Brome mo-saic virus in S. cerevisiae

2.22

CAGLOI03080g YEL021W URA3 Orotidine-5'-phosphate (OMP) decarboxylase, catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, converting

2.22

OMP into uridine monophosphate (UMP); converts 5-FOA into 5- fluorouracil, a toxic compound




CAGLOJ00957g YLROO9W RLP24 Essential protein with similarity to 2.22 Rp124Ap and Rp124Bp, associated with pre-60S ribosomal subunits and required for ribosomal large subunit biogenesis

CAGLOJ10846g YHR071W PCL.5 Cyclin, interacts with and phosphorylated by Pho85p cyclin- dependent kinase (Cdk), induced by Gcn4p at level of transcription, specifically required for

2.22


CAGLOF04433g YBL039C URA 7 Major CTP synthase isozyme (see also URA8), catalyzes the ATP- dependent transfer of the amide nitrogen from glutamine to UTP, forming CTP, the final step in de novo biosynthesis of pyrimidines; involved in phospholipid biosynthe-sis

2.21

CAGLOJ05698g YNL151C RPC31 RNA polymerase III subunit C31; contains HMG-like C-terminal do-main

2.21

CAGLOK128O4g YLR074C BUD20 Protein involved in bud-site selection; diploid mutants display a ran-dom budding pattern instead of the wild-type bipolar pattern

2.21

CAGLOM02409g YPL146C NOP53 Nucleolar protein; involved in biogenesis of the 60S subunit of the ri-bosome; interacts with rRNA pro-cessing factors Cbf5p and Nop2p; null mutant is viable but growth is severely impaired

2.21

CAGLOD04202g YIL090W ICE2 Integral ER membrane protein with type-III transmembrane domains; mutations cause defects in cortical

2.2

ER morphology in both the mother and daughter cells

CAGLOD05104g -- Description not mapped 2.2 CAGL0110879g YPL112C PEX25 Peripheral peroxisomal membrane

peroxin required for the regulation of peroxisome size and maintenance, recruits GTPase Rho1p to peroxisomes, induced by oleate, interacts with homologous protein

2.2

Pex27p CAGLOJ01463g YKL096W CWPI Cell wall mannoprotein, linked to a

beta-1,3- and beta-1,6-glucan het-eropolymer through a phosphodi-ester bond; involved in cell wall or-ganization

2.2

CAG LOB 00462g YC L055W KAR4 Transcription factor required for gene regulation in repsonse to pheromones; also required during meiosis; exists in two forms, a slower-migrating form more abun-dant during vegetative growth and a faster-migrating form induced by pheromone

2.19




CAGL0000737g YLR222C UTP13 Nucleolar protein, component of the small subunit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

2.19

CAGLOH08415g YDR045C RPC// RNA polymerase III subunit C11; mediates pol III RNA cleavage ac-tivity and is important for termina-tion of transcription; homologous to

2.19

TFIIS CAGLOL03333g YKR044W UIP5 Protein of unknown function

that interacts with IJIplp, a Ubl 2.19

(ubiquitin-like protein)-specific protease for Smt3p protein conju-gates

CAGLOD02838g YLL034C RIX7 Putative ATPase of the AAA family, required for export of pre- ribosomal large subunits from the nucleus; distributed between the nucleolus, nucleoplasm, and nuclear periphery depending on growth con-ditions

2.18

CAGLOM07425g YMR0281V TAP42 Essential protein involved in the 2.18 TOR signaling pathway; physically associates with the protein phos-phatase 2A and the SIT4 protein phosphatase catalytic subunits

CAGLOM09493g YMR160W - Description not mapped 2.18 CAGLOA00913g YLR320W MMS22 Protein that acts with Mmslp in

a repair pathway that may be in-volved in resolving replication inter-mediates or preventing the damage caused by blocked replication forks; required for accurate meiotic chro-mosome segregation

2.17

CAGLOH01023g YDR279W RNH202 Ribonuclease H2 subunit, required for RNase H2 activity; related to human AGS2 that causes Aicardi-

2.17

Goutieres syndrome CAGLOJ05896g YNL158W PGAI Essential component of GPI-

mannosyltransferase II, responsible for second mannose addition to

2.17

GPI precursors as a partner of Gpil8p; required for maturation of Gas1p and Pho8p; has synthetic genetic interations with secretory pathway genes

CAGLOK11231g YDR245W MNNIO Subunit of a Golgi mannosyltransferase complex also containing Anplp, Mnn9p, Mnnllp, and

2.17

Hoc1p that mediates elongation of the polysaccharide mannan backbone; membrane protein of the mannosyltransferase family

CAGLOG00264g YGR280C PXR1 Essential protein involved in rRNA and snoRNA maturation; competes with TLC1 RNA for binding to

2.16

Est2p, suggesting a role in negative regulation of telomerase; human ho-molog inhibits telomerase; contains a G-patch RNA interacting domain




CAGLOJ03146g

CAGLOK03927g

CAGLOL00759g

CAGLOM09691g

CAGL0001111g CAGLOK01463g

CAGLOM09911g

CAGLOB04169g

CAGLOD06050g

CAGLOF02563g

CAGLOF02871g

YER070W

YMR134W

YER055C

YMR153W

- YDL042C

YLR405W

YPR112C

YJR054W

YDR399W

YDR414C

RNR1

ERG29

11151

NUP53

- SIR2

DUS4

MRD/

ERAIG

HPT1

ERD1

One of two large regulatory subunits of ribonucleotide-diphosphate reductase; the RNR complex cat-alyzes rate-limiting step in dNTP synthesis, regulated by DNA repli-cation and DNA damage checkpoint pathways via localization of small subunits Protein of unknown function that may be involved in iron metabolism; mutant bm-8 has a growth defect on iron-limited medium that is comple-mented by overexpression of Yfhlp; shows localization to the ER; highly conserved in ascomycetes ATP phosphoribosyltransferase, a hexameric enzyme, catalyzes the first step in histidine biosynthesis; mutations cause histidine auxotro-phy and sensitivity to Cu, Co, and Ni salts; transcription is regulated by general amino acid control Subunit of the nuclear pore corn-

pherin Kap121p or with Nup170p via overlapping regions of Nup53p,

ape with karyo-interacts), tmpd

involved in activation of the spindle

ppleesxcr(iNPi.C. no

checkpoint mediated by the Mad1p-Mad2p complex

pt

Conserved NAD+ dependent histone deacetylase of the Sirtuin fam-ily involved in regulation of lifespan; plays roles in silencing at HML, HMR, telomeres, and the rDNA locus; negatively regulates initiation of DNA replication Dihydrouridine synthase, member of a widespread family of conserved proteins including Smmlp, Duslp, and Dus3p Essential conserved protein that is part of the 90S preribosome; required for production of 18S rRNA and small ribosomal subunit; con-tains five consensus RNA-binding domains Vacuolar protein of unknown func- Lion; potential Cdc28p substrate Dimeric hypoxanthine-guanine phosphoribosyltransferase, catalyzes the formation of both inosine monophosphate and guano-sine monophosphate; mutations in the human homolog HPRT1 can cause Lesch-Nyhan syndrome and Kelley-Seegmiller syndrome Predicted membrane protein required for the retention of lume-nal endoplasmic reticulum proteins; mutants secrete the endogenous ER protein, BiP (Kar2p)

2.16

2.16

2.16

2.16

2.15 2.15

2.15

2.14

2.14

2.14

2.14




CAGLOF07535g

CAGLOI02948g

YKL094W

YDR498C

YJU3

SEC20

Serine hydrolase with sequence similarity to monoglyceride lipase (MGL), localizes to lipid particles Membrane glycoprotein v-SNARE involved in retrograde transport from the Golgi to the ER; required for N- and 0-glycosylation in the

2.14

2.14

Golgi but not in the ER; forms a complex with the cytosolic Tip2Op

CAGLOK00957g YOR200C ELP2 Subunit of Elongator complex, which is required for modification of wobble nucleosides in tRNA; target of Kluyveromyces lactis zymocin

2.14

CAGLOB04961g YLR172C DPH5 Methyltransferase required for synthesis of diphthamide, which is a modified histidine residue of trans-lation elongation factor 2 (Eft1p or

2.13

Eft2p); not essential for viability; GFP-Dph5p fusion protein localizes to the cytoplasm

CAGLOD00220g YAL059W ECM1 Protein of unknown function, localized in the nucleoplasm and the nucleolus, genetically interacts with

2.13

MTR2 in 60S ribosomal protein subunit export

CAGLOE05016g YGL148W AR02 Bifunctional chorismate synthase and flavin reductase, catalyzes the conversion of 5- enolpyruvylshikimate 3-phosphate

2.13

(EPSP) to form chorismate, which is a precursor to aromatic amino acids

CAGLOG07843g YGR103W NOP7 Nucleolar protein involved in rRNA processing and 60S ribosomal subunit biogenesis; constituent of several different pre-ribosomal particles; required for exit from

2.13

Gisub2,0j/subi, and the initiation of cell proliferation

CAGLOI02596g -- Description not mapped 2.13 CAGLOK04917g - - Description not mapped 2.13 CAGLOL04114g YNL124W NAF1 RNA-binding protein required

for the assembly of box H/ACA snoRNPs and thus for pre-rRNA processing, forms a complex with

2.13

Shqlp and interacts with II/ACA snoRNP components Nhp2p and Cbf5p; similar to Gar1p

CAGL0001595g YER248C HIS7 Imidazole glycerol phosphate synthase (glutamine amidotrans- ferase:cyclase), catalyzes the fifth and sixth steps of histidine biosynthesis and also produces

2.12

5-aminoimidazole-4-carboxamide ribotide (AICAR), a purine precursor

CAGLOG05445g YDL226C GCS1 ADP-ribosylation factor GTPase activating protein (ARF GAP), involved in ER-Golgi transport; shares functional similarity with

2.12

Glo3p continued on next page



CAGLOK06787g YBR218C PYC2 Pyruvate carboxylase isoform, cytoplasmic enzyme that converts pyru-vate to oxaloacetate; highly similar to isoform Pyclp but differentially regulated; mutations in the human homolog are associated with lactic acidosis

2.12

CAGLOK10780g YML056C titfD4 Inosine monophosphate dehydrogenase, catalyzes the first step of

2.12

GMP biosynthesis, member of a four-gene family in S. cerevisiae, constitutively expressed

CAGLOM06699g YIL084C SDS3 Component of the Rpd3p/Sin3p deacetylase complex required for its structural integrity and catalytic activity, involved in transcriptional silencing and required for sporula-tion; cells defective in SDS3 display pleiotropic phenotypes

2.12

CAGLOH03905g YCLOO9C MVO Regulatory subunit of acetolactate synthase, which catalyzes the first step of branched-chain amino acid biosynthesis; enhances activity of the Ily2p catalytic subunit, localizes to mitochondria

2.11

CAGLOH05115g YLR385C SWC7 Protein of unknown function, corn- ponent of the Swrlp complex that incorporates Htzlp into chromatin

2.11

CAGL0101254g YHR111W UBA4 Protein that activates Urmlp before its conjugation to proteins

2.11

(urmylation); one target is the thioredoxin peroxidase Ahplp, sug-gesting a role of urmylation in the oxidative stress response

CAGLOK03465g YMR108W ILV2 Acetolactate synthase, catalyses the first common step in isoleucine and valine biosynthesis and is the target of several classes of inhibitors, localizes to the mitochon-dria; expression of the gene is under general amino acid control

2.11

CACLOL08976g YFLOO2C SPB4 Putative ATP-dependent RNA helicase, nucleolar protein required for synthesis of 60S ribosomal subunits at a late step in the pathway; sedi-ments with 66S pre-ribosomes in su-crose gradients

2.11

CAGLOL10678g YOR276W CAF20 Phosphoprotein of the mRNA cap- binding complex involved in translational control, repressor of cap- dependent translation initiation, competes with eIF4G for binding to eIF4E

2.11

CAGL0003960g YIL014W MNT3 Alpha-1,3-mannosyltransferase, adds the fourth and fifth alpha-

2.1

1,3-linked mannose residues to 0-linked glycans during protein 0-glycosylation

CAGLOG05742g YDL213C NOP6 Putative RNA-binding protein implicated in ribosome biogenesis; contains an RNA recognition mo-tif (RRM) and has similarity to hy-drophilins; NOP6 may be a fungal-specific gene as no homologs have been yet identified in higher eukary-otes

2.1




CAGLOH02607g YOL052C SPE2 S-adenosylmethionine decarboxy-

of spermidine and spermine; cells lacking Spe2p require spermine or spermidine for growth in the

liesesc,rirpetqlenclotfomr atphpeedbiosynthesis 2.1

presence of oxygen but not when grown anaerobically

CAGLOK07183g - - 2.1 7i — C91111 - - Description not mapped 2.09

CAGLOA03674g YBR141C - Description not mapped 2.09 CAGLOB03993g YJR016C ILVS Dihydroxyacid dehydratase, cat-

alyzes third step in the common pathway leading to biosynthesis of branched-chain amino acids

2.09

CAGLOE01573g YNL313C - Description not mapped 2.09 CAGLOF00297g YJR075W HOC1 Alpha-1,6-mannosyltransferase in-

volved in cell wall mannan biosyn-thesis; subunit of a Golgi-localized complex that also contains Anplp, Mnn9p, Mnnllp, and MnnlOp; identified as a suppressor of a cell lysis sensitive pkcl-371 allele

2.09

CAGLOH05995g YAL049C AIMS Cytoplasmic protein of unknown function, potential Hsp82p interac-tor; YAL049C is not an essential gene

2.09

CAGLOH08910g YGL138C - Description not mapped 2.09 CAGL0109350g YBR267W REI1 Cytoplasmic pre-60S factor; re-

quired for the correct recycling of shuttling factors Albl, Arxl and

2.09

Tif6 at the end of the ribosomal large subunit biogenesis; involved in bud growth in the mitotic signaling network

CAGLOJ01375g YMRO88C VBAI Permease of basic amino acids in the vacuolar membrane

2.09

CAGLOJ03344g YER082C UTP7 Nucleolar protein, component of the small subunit (SSU) processome containing the XJ3 snoRNA that is involved in processing of pre-18S rRNA

2.09

CAGLDL05500g YJL 122W ALB1 Shuttling pre-60S factor; involved in the biogenesis of ribosomal large subunit; interacts directly with

2.09

Arxlp; responsible for Tif6p recy- ,..

cling defects in absence of Reilp CAGLOM00946g YLR435W TSR2 Protein with a potential role in pre-

rRNA processing 2.09

- - Description not mapped 2.08 CAGLOD03652g YMR179W SPT21 Protein required for normal tran-

scription at several loci including 2.08

HTA2-HTB2 and HHF2-HHT2, but not required at the other histone loci; functionally related to SptlOp; involved in telomere maintenance

CAGLOD03960g YHR036W BRL1 Essential nuclear envelope integral membrane protein identified as a suppressor of a conditional mutation in the major karyopherin, CRM1; homologous to and interacts with Brr6p, a nuclear envelope pro-tein involved in nuclear export

2.08




CAGLOE01067g YDR161W - Description not mapped 2.08 CAGLOG04983g YLR363WA - Description not mapped 2.08 CAGLOH01375g YDR297W SUR2 Sphinganine C4-hydroxylase, catal-

yses the conversion of sphinganine to phytosphingosine in sphingolipid biosyntheis

2.08

CAGLOI10648g YGR124W ASN2 Asparagine synthetase, isozyme of 2.08 Asnlp; catalyzes the synthesis of L-asparagine from L-aspartate in the asparagine biosynthetic pathway

CAGLOJ01727g YIR019C MUC1 GPI-anchored cell surface glycoprotein (flocculin) required for pseudohyphal formation, invasive growth, flocculation, and biofilms; transcriptionally regulated by the

2.08

MAPK pathway (via Stel2p and Tee1p) and the cAMP pathway (via Flo8p)

CAGLOK02497g YPRO16C TIF6 Constituent of 66S pre-ribosomal particles, has similarity to human translation initiation factor 6

2.08

(eIF6); may be involved in the bio-genesis and or stability of 60S ribo-somal subunits

CAGLOL08756g YPL012W RRP12 Protein required for export of the ribosomal subunits; associates with the RNA components of the pre-ribosomes; contains HEAT-repeats

2.08

CAGLOM12815g YIL073C SP022 Meiosis-specific protein essential for chromosome synapsis, involved in completion of nuclear divisions dur-ing meiosis; induced early in meiosis

2.08

CAGLOG08019g YDR090C - Description not mapped 2.07 CAGLOG08195g YLR168C UPS2 Putative protein of unknown func-

tion; similar to Ups1p, Ydr185Cp and to human PRELI; GFP-tagged protein localizes to mitochondria; required for wild-type respiratory growth

2.07

CAGLOH07183g YHRO7OW TRM5 tRNA(m(1)G37)methyltransferase, methylates a tRNA base adjacent to the anticodon that has a role in prevention of frameshifting; highly conserved across Archaea, Bacteria, and Eukarya

2.07

CAGLOI08195g YER020W GPA2 Nucleotide binding alpha subunit of the heterotrimeric G protein that interacts with the receptor Gprlp, has signaling role in response to nutrients; green fluorescent protein

2.07

(GFP)-fusion protein localizes to the cell periphery

CAGLOK07436g YMR241W YHM2 Mitochondria] DNA-binding protein, component of the mitochon-drial nucleoid structure, involved in mtDNA replication and segregation of mitochondria] genomes; member of the mitochondrial carrier protein family

2.07




CAGLOL02915g YOR204W DEDI ATP-dependent DEAD (Asp-Glu- 2.07 Ala-Asp)-box RNA helicase, required for translation initiation of all yeast mRNAs; mutations in hu-man DEAD-box DBY are a frequent cause of male infertility

CAGLOB02145g YHR149C SKG6 Integral membrane protein that localizes primarily to growing sites such as the bud tip or the cell periphery; potential Cdc28p substrate; Skg6p interacts with Zdslp and Zds2p

2.06

CAGLOD06534g -- Description not mapped 2.06 CAGLOF07645g YKL099C UTPII Subunit of U3-containing Small 2.06

Subunit (SSU) processome complex involved in production of 18S rRNA and assembly of small ribosomal subunit

CAGL0G01947g YKR071C DRE2 Protein of unknown function required for iron-sulfur cluster assem-bly and sister chromatid cohesion; mutation displays synthetic lethal interaction with the po13-13 allele of CDC2

2.06

CAGLOH09152g YJR059W PTK2 Putative serine/threonine protein kinase involved in regulation of ion transport across plasma membrane; enhances spermine uptake

2.06

CAGLOJ03212g YER073W ALD5 Mitochondrial aldehyde dehydrogenase, involved in regulation or biosynthesis of electron transport chain components and acetate for-mation; activated by K+; utilizes

2.06

NADP+ as the preferred coenzyme; constitutively expressed

CAGLOL11660g YOR116C RP031 RNA polymerase III subunit C160, part of core enzyme; similar to bac-terial beta-prime subunit

2.06

CAGLOA03608g YOL155C HPF1 Haze-protective mannoprotein that reduces the particle size of aggre-gated proteins in white wines

2.05

C AG LOE100572g - - Description not mapped 2.05 CAGLOE02937g YGL029W CGRI Protein involved in nucleolar in-

tegrity and processing of the pre-rRNA for the 60S ribosome subunit; transcript is induced in response to cytotoxic stress but not genotoxic stress

2.05

CAGLOI03014g YNL282W POPS Subunit of both RNase MRP, which cleaves pre-rRNA, and nu-clear RNase P, which cleaves tRNA precursors to generate mature 5' ends

2.05

CAGL0107997g YOL076W MDM20 Non-catalytic subunit of the NatB 2.05 N-terminal acetyltransferase, which catalyzes N-acetylation of proteins with specific N-terminal sequences; involved in mitochondrial inheritance and actin assembly

CAGLOJ06028g YNL142W MEP2 Ammonium permease involved in regulation of pseudohyphal growth; belongs to a ubiquitous family of cytoplasmic membrane proteins that transport only ammonium

2.05

(NH4+); expression is under the ni-trogen catabolite repression regula-tion




CAGLOL04290g

CAGLOB04191g CAGL0001419g

YOR156C

YPR114W YPL184C

TIFI1

- MRN1

SUMO ligase, catalyzes the covalent attachment of SUMO (Smt3p) to proteins; involved in maintenance of proper telomere length Description not mapped RNA-binding protein proposed to be involved in translational regulation; binds specific categories of mRNAs, including those that con-tain upstream open reading frames (uORFs) and internal ribosome en-try sites (IRES)

2.05

2.04 2.04

CAGLOD02596g YOL146W PSF3 Subunit of the GINS complex 2,04 (Sld5p, Psflp, Psf2p, Psf3p), which is localized to DNA replication ori-gins and implicated in assembly of the DNA replication machinery

CAGLOH04917g YDR088C SLUT RNA splicing factor, required for 2.04 ATP-independent portion of 2nd catalytic step of spliceosomal RNA splicing; interacts with Prpl8p; contains zinc knuckle domain

CAGLOH10384g YDL111C RRP4 2 Exosome non-catalytic core component; involved in 3'-5' RNA process-ing and degradation in both the nu-cleus and the cytoplasm; has simi-larity to E. coli RNase PH and to human hRrp42p (EXOSC7)

2.04

CAGL0105148g YDL174C DLD1 D-lactate dehydrogenase, oxidizes 2.04 D-lactate to pyruvate, transcription is heme-dependent, repressed by glucose, and derepressed in ethanol or lactate; located in the mitochon-dria' inner membrane

CAGLOK02937g YKL027W - Description not mapped 2.04 CAGLOK11319g YDR249C - Description not mapped 2.04 CAGL0005115g YOL058W ARG1 Arginosuccinate synthetase,

catalyzes the formation of L- argininosuccinate from citrulline and L-aspartate in the arginine biosynthesis pathway; potential

2.03

Cdc28p substrate CAGLOF02651g YDR403W DIT1 Sporulation-specific enzyme re-

quired for spore wall maturation, involved in the production of a soluble LL-dityrosine-containing precursor of the spore wall; transcripts accumulate at the time of prospore enclosure

2.03

CAGLOG09229g YPL192C PRM3 Pheromone-regulated protein required for karyogamy; localizes to the inner membrane of the nuclear envelope

2.03

CAGLOH05973g YPL108W - Description not mapped 2.03 CAGLOK03861g YMR131C RRB1 Essential nuclear protein involved

in early steps of ribosome biogene-sis; physically interacts with the ri-bosomal protein Rpl3p

2.03

CAGLOL08206g YCR035C RI1P43 Exosome non-catalytic core component; involved in 3'-5' RNA process-ing and degradation in both the nu-cleus and the cytoplasm; has simi-larity to E. coli RNase PH and to human hRrp43p (01E2, EXOSCS)

2.03


Appendix G. Transcription profiling of C. glabrata serum stress responses395


CAGLOM01892g

CAGLOM09845g

YBR076W

YLR409C

ECM8

UTP21

Non-essential protein of unknown function Subunit of U3-containing 90S preribosome and Small Subunit (SSU) processome complexes involved in production of 18S rRNA and assembly of small ribosomal subunit; synthetic defect with STI1

2.03

2.03

Hsp90 cochaperone; human homolog linked to glaucoma

CAGL0B04785g YCL010C SGF29 Probable subunit of SAGA histone acetyltransferase complex

2.02

CAGLOF03553g YPL170W DAP1 Heme-binding protein involved in regulation of cytochrome P450 pro-tein Ergl 1p; damage response pro-tein, related to mammalian mem-brane progesterone receptors; mu-tations lead to defects in telomeres, mitochondria, and sterol synthesis

2.02

CAGLOG04477g - - Description not mapped 2.02 CAGLOG06578g YNL099C OCA1 Putative protein tyrosine phos-

phatase, required for cell cycle ar-rest in response to oxidative dam-age of DNA

2.02

CAGLOH03773g YNLOO2C RLP7 Nucleolar protein with similarity to large ribosomal subunit L7 proteins; constituent of 66S pre- ribosomal particles; plays an essential role in processing of precursors to the large ribosomal subunit

2.02

RNAs CAGLOJ04070g YOR224C RPB8 RNA polymerase subunit ABC14.5,

common to RNA polymerases I, II, and III

2.02

CAGL0001287g YIL114C POR2 Putative mitochondrial porin 2.01 (voltage-dependent anion channel), related to Porlp but not required for mitochondrial membrane per-meability or mitochondrial osmotic stability

CAGLOF01793g YLR056W ERGS C-5 sterol desaturase, catalyzes the introduction of a C-5(6) double bond into episterol, a precursor in ergosterol biosynthesis; mutants are viable, but cannot grow on non- fermentable carbon sources

2.01

CAGLOG08294g YLR243W Description not mapped 2.01 CAGLOH00440g YJR124C - Description not mapped 2.01 CAGLOJ09460g YIL079C AIR/ RING finger protein that inter-

acts with the arginine methyltrans-ferase Hmtlp to regulate methy-lation of Npl3p, which modulates

2.01

Npl3p function in mRNA processing and export; has similarity to Air2p

CAGLOK05753g YDR508C GNP1 High-affinity glutamine permease, also transports Leu, Ser, Thr, Cys, Met and Asn; expression is fully de-pendent on Grrlp and modulated by the Ssylp-Ptr3p-Ssy5p (SPS) sensor of extracellular amino acids

2.01



Description not mapped Description not mapped Protein required for mismatch re-pair in mitosis and meiosis, forms a complex with Msh2p to repair both single-base

Nucleolar protein required for ex-port of tRNAs from the nucleus; also copurifies with the small sub-unit (SSU) processome containing the U3 snoRNA that is involved in processing of pre-18S rRNA

MSH6

UTP8

YML053C

YDR097C

YGR128C

2.01 2.01

insertion-deletion mispairs; potentially phosphorylated by I

2

CAGLOL01573g GAGLOL09361g CAGLOG08129g

2 CAGL0110560g



Table G.2: Candida glabrata transcripts repressed in ATCC 2001 following ex-posure to Synthetic Complete + 10% (v/v) foetal calf serum (FCS) repressed transcripts are defined as having log2(Cy5) - log2(Cy3) < -2.0 and Benjamini-Hochberg corrected p-values < 0.05.

CG ORF SC ORF gene name Description Log Ratio CAGLOE04884g YDR216W ADR1 Carbon source-responsive zinc-

finger transcription factor, required for transcription of the glucose- repressed gene ADH2, of peroxi-somal protein genes, and of genes required for ethanol, glycerol, and fatty acid utilization

-9.77

CAGL0107249g YDR389W SAC7 GTPase activating protein (GAP) for Rho1p, involved in signaling to the actin cytoskeleton, null muta-tions suppress tort mutations and temperature sensitive mutations in actin; potential Cdc28p substrate

-9.63

CAGLOB01875g NORBH - Description not mapped -9.48 CAGLOJ03080g NORBH - Description not mapped -8.95 CAGLOG-09977g YPR184W GDB1 Glycogen debranching enzyme

containing glucanotranferase and alpha-1,6-amyloglucosidase activities, required for glycogen degradation; phosphorylated in mitochondria

-8.27

CAGLOK03421g YMR105C PGM2 Phosphoglucomutase, catalyzes the conversion from glucose-1- phosphate to glucose-6-phosphate, which is a key step in hexose metabolism; functions as the acceptor for a Glc-phosphotransferase

-8.04

CAGLOE06380g YKL151C Description not mapped -8 CAGLOA02002g YFRO17C - Description not mapped -7.75 CAGLOH02387g YMR261C TPS8 Regulatory subunit of trehalose- -7.61

6-phosphate synthase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; ex-pression is induced by stress con-ditions and repressed by the Ras-cAMP pathway

CAGLOI06050g YJL153C INOI Inositol 1-phosphate synthase, involved in synthesis of inositol phosphates and inositol-containing phospholipids; transcription is coregulated with other phospholipid biosynthetic genes by Ino2p and Ino4p, which bind the CASINO

-69.05

DNA element CAGLOF04719g YLR258W GSY2 Glycogen synthase, similar to -6.89

Gsylp; expression induced by glucose limitation, nitrogen starvation, heat shock, and stationary phase; activity regulated by cAMP- dependent, Snflp and Pho85p kinases as well as by the Gaclp- Glc7p phosphatase




CACLOJ04202g YFLO14W HSP12 Plasma membrane localized protein that protects membranes from des-iccation; induced by heat shock, ox-idative stress, osmostress, station-ary phase entry, glucose depletion, oleate and alcohol; regulated by the HOG and Ras-Pka pathways

-6.76

CAGLOF00605g YCLO4OW GLK1 Glucokinase, catalyzes the phosphorylation of glucose at C6 in the first irreversible step of glucose metabolism; one of three glucose phosphorylating enzymes; expression regulated by non-fermentable carbon sources

-6.73

CAGLOE05610g YAL038W CDC19 Pyruvate kinase, functions as a homotetramer in glycolysis to convert phosphoenolpyruvate to pyruvate, the input for aerobic (TCA cycle) or anaerobic (glucose fermentation) respiration

-6.67

CAGLOL06094g YGL184C STR3 Cystathionine beta-lyase, converts cystathionine into homocysteine

-6.66

CAGLOF02717g NORBH - Description not mapped -6.54 CAGLOF08107g YGR244C LSC2 Beta subunit of succinyl-CoA lig-

ase, which is a mitochondrial enzyme of the TCA cycle that catalyzes the nucleotide-dependent conversion of succinyl-CoA to suc-cinate

-6.5

CAGLOJ01699g NORBH - Description not mapped -6.27 CAGLOK05687g YHR179W OYEZ Widely conserved NADPH oxidore-

ductase containing flavin mononu- cleotide (F MN), homologous to

-5.89

Oye3p with slight differences in lig- and binding and catalytic properties; may be involved in sterol metabolism

CAGLOHO1111g YDR286C - Description not mapped -5.76 CAGLOK05813g NORBH - Description not mapped -5.67 CAGLOF04807g YIL136W OM45 Protein of unknown function, ma-

jor constituent of the mitochondrial outer membrane; located on the outer (cytosolic) face of the outer membrane

-5.63

CAGLOK03459g NORBH _ Description not mapped -5.54 CAGLOK07480g YMR105C PGM2 Phosphoglucomutase, catalyzes

the conversion from glucose-1- phosphate to glucose-6-phosphate, which is a key step in hexose metabolism; functions as the acceptor for a Glc-phosphotransferase

-5.46

CAGLOL01925g YKL035W UGP1 UDP-glucose pyrophosphorylase -5.43 (UGPase), catalyses the reversible formation of UDP-Glc from glucose 1-phosphate and UTP, involved in a wide variety of metabolic pathways, expression modulated by Pho85p through Pho4p




CAGLOB01078g CAGLOL00957g

YLR177W YER048C

- CAJ1

Description not mapped Nuclear type II J heat shock protein of the E. coli dnaJ family, contains a leucine zipper-like motif, binds to non-native substrates for presenta-tion to Ssa3p, may function during protein translocation, assembly and disassembly

-5.42 -5.31

CAGLOB01925g YDR122W KIN1 Serine/threonine protein kinase involved in regulation of exocytosis; localizes to the cytoplasmic face of the plasma membrane; closely re-lated to Kin2p

-5.29

CAGLOG05698g YDL215C GDH2 NAD(+)-dependent glutamate dehydrogenase, degrades glutamate to ammonia and alpha-ketoglutarate; expression sensitive to nitrogen catabolite repression and intracellular ammonia levels

-5.15

CAGLOD06512g YLR310C CDC25 Membrane bound guanine nucleotide exchange factor (GEF or

-5.08

GDP-release factor); indirectly regulates adenylate cyclase through activation of Ras1p and Ras2p by stimulating the exchange of GDP for GTP; required for progression through G1

CAGL0004587g YJR098C Description not mapped -5.03 CAGLOH0826Ig YOR019W - Description not mapped -5 CAGLOL04598g YLR270W DCS1 Non-essential hydrolase involved in

mRNA decapping, may function in a feedback mechanism to regulate deadenylation, contains pyrophos- phatase activity and a HIT (histidine triad) motif; interacts with neutral trehalase Nth1p

-4.92

CAGLOK04301g YGRO52W FMP48 Putative protein of unknown function; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies; induced by treatment with

-4.9

8-methoxypsoralen and UVA irradi-ation

CAGLOE05148g YGL156W AMS1 Vacuolar alpha mannosidase, involved in free oligosaccharide (fOS) degradation; delivered to the vac-uole in a novel pathway separate from the secretory pathway

-4.82

CAGLOF07029g YGL125W MET13 Major isozyme of methylenetetrahydrofolate reductase, catalyzes the reduction of 5,10- methylenetetrahydrofolate to

-4.8

5-methyltetrahydrofolate in the methionine biosynthesis pathway

CAGLOI01980g YNL257C SIPS Protein that activates transcription through interaction with DNA-bound Snflp, C-terminal region has a putative leucine zipper motif; po-tential Cdc28p substrate

-4.79

CAGLOM03377g YEL011W GLC3 Glycogen branching enzyme, involved in glycogen accumulation; green fluorescent protein (GFP)- fusion protein localizes to the cyto-plasm in a punctate pattern

-4.79




CAGLOE00803g YDR171W HSP42 Small heat shock protein (sHSP) with chaperone activity; forms barrel-shaped oligomers that sup-press unfolded protein aggregation; involved in cytoskeleton reorganiza-tion after heat shock

-4.72

CAGLOL11902g YER170W ADK2 Mitochondrial adenylate kinase, catalyzes the reversible synthesis of GTP and AMP from GDP and

-4.71

ADP; may serve as a back-up for synthesizing GTP or ADP depending on metabolic conditions; 3' sequence of ADK2 varies with strain background

CAGLOL00473g YMR187C - Description not mapped -4.66 CAGL0107359g YOL108C INN Transcription factor required for

derepression of inositol-choline- regulated genes involved in phospholipid synthesis; forms a complex, with Ino2p, that binds the inositol-choline-responsive element through a basic helix-loop-helix domain

-4.62

CAGLOH08778g YPRO42C PUF2 Member of the PUF protein family, which is defined by the pres-ence of Pumilio homology domains that confer RNA binding activity; preferentially binds mRNAs encod-ing membrane-associated proteins

-4.49

CAGLOB02860g YLR356W - Description not mapped -4.47 CAGLOE01837g YDR144C AI KC7 GPI-anchored aspartyl protease -4.45

(yapsin) involved in protein processing; shares functions with Yap3p and Kex2p

CAGLOF03267g YDR326C YSP2 Protein involved in programmed cell death; mutant shows resistance to cell death induced by amiodarone or intracellular acidification

-4.41

CAGLOM04763g YOR289W - Description not mapped -4.35 CAGLOM07403g YPL052W OAZ I Regulator of ornithine decarboxy-

lase (Spelp), antizyme that binds to Spelp to regulate ubiquitin- independent degradation; ribosomal frameshifting during synthesis of Oazlp and its ubiquitin- mediated degradation are both polyamine-regulated

-4.34

CAGLOH02585g YMR250W GAD1 Glutamate decarboxylase, converts glutamate into gamma- aminobutyric acid (GABA) during glutamate catabolism; involved in response to oxidative stress

-4.31

CAGLOK00583g YCR034W FEN1 Fatty acid elongase, involved in sphingolipid biosynthesis; acts on fatty acids of up to 24 carbons in length; mutations have regulatory effects on 1,3-beta-glucan synthase, vacuolar ATPase, and the secretory pathway

-4.3




CAGLOJ09812g YBR126C TPS1 Synthase subunit of trehalose-6- phosphate synthase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; also found in a monomeric form; expression is induced by the stress response and repressed by the

-4.2

Ras-cAMP pathway CAGLOE01881g YLR120C YPS1 Aspartic protease, attached to the

plasma membrane via a glycosylphosphatidylinositol (GPI) anchor

-4.15

CAGLOM04367g YLR133W CKII Choline kinase, catalyzing the first step in phosphatidylcholine synthesis via the CDP-choline

-4.15

(Kennedy pathway); exhibits some ethanolamine kinase activity contributing to phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway

CAGLOJ06050g YNL160W YGP1 Cell wall-related secretory glycoprotein; induced by nutrient deprivation-associated growth arrest and upon entry into stationary phase; may be involved in adapta-tion prior to stationary phase entry; has similarity to Sps100p

-4.13

CAGLOB01947g YDR123C ING2 Component of the heteromeric -4.12 Ino2p/Ino4p basic helix-loop- helix transcription activator that binds inositol/choline-responsive elements (ICREs), required for derepression of phospholipid biosynthetic genes in response to inositol depletion

CAGLOD00198g YAL060W BDH1 NAD-dependent (R,R)-butanediol dehydrogenase, catalyzes oxidation of (R,R)-2,3-butanediol to

-4.12

(3R)-acetoin, oxidation of meso- butanediol to (3S)-acetoin, and reduction of acetoin; enhances use of 2,3-butanediol as an aerobic carbon source

CAGLOI05874g YIL053W RHR2 Constitutively expressed isoform of DL-glycerol-3-phosphatase; involved in glycerol biosynthesis, in-duced in response to both anaerobic and, along with the Hor2p/Gpp2p isoform, osmotic stress

-4.11

CAGLOH06699g YIL155C GUT2 Mitochondrial glycerol-3-phosphate dehydrogenase; expression is repressed by both glucose and cAMP and derepressed by non-fermentable carbon sources in a Snflp, Rsflp, Hap2/3/4/5 complex dependent manner

-4.1

CAGLOH06281g YAL016W TPD3 Regulatory subunit A of the heterotrimeric protein phosphatase 2A, which also contains regulatory subunit Cdc55p and either catalytic subunit Pph2lp or Pph22p; required for cell morphogenesis and for transcription by RNA polymerase III

-4.09




CAGLOJ01870g YGL167C PMR1 High affinity Ca2+/Mn2+ P-type -4.05 ATPase required for Ca2+ and Mn2+ transport into Golgi; involved in Ca2-1- dependent protein sorting and processing; mutations in human homolog ATP2C1 cause acantholytic skin condition Hailey- Hailey disease

CAGLOG05335g YDR074W TPS2 Phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; expression is induced by stress conditions and repressed by the Ras-cAMP pathway

-4.04

CAGL0000275g YDR533C HSP31 Possible chaperone and cysteine protease with similarity to E. coli Hsp31; member of the DJ-

-4.01

1/ThiJ/PfpI superfamily, which includes human DJ-1 involved in Parkinson's disease; exists as a (timer and contains a putative metal-binding site

CAGLOL10186g YOR052C Description not mapped -3.96 CAGLOH02541g YMR252C - Description not mapped -3.95 CAGLOE05654g YPL206C PGC1 Phosphatidyl Glycerol phospho-

lipase C; regulates the phos- phatidylglycerol (PG) content via a phospholipase C-type degradation mechanism; contains glycerophos- phodiester phosphodiesterase motifs

-3.81

CAGLOL02827g YOR208W PTPS Phosphotyrosine-specific protein phosphatase involved in the inactivation of mitogen-activated protein kinase (MAPK) during osmolarity sensing; dephosporylates Hog1p MAPK and regulates its localization; localized to the nucleus

-3.75

CAGLOI10516g YGR130C Description not mapped -3.72 CAGL01102893g YJL070C - Description not mapped -3.65 CAGLOH08558g YPRO49C ATG11 Adapter protein for pexophagy and

the cytoplasm-to-vacuole targeting -3.65

(Cvt) pathway; directs receptor- bound cargo to the phagophore assembly site (PAS) for packaging into vesicles; required for recruiting other proteins to the (PAS)

CAGL0003223g YLL041C SDH2 Iron-sulfur protein subunit of succinate dehydrogenase (Sdhlp, Sdh2p, Sdh3p, Sdh4p), which couples the oxidation of succinate to the trans-fer of electrons to ubiquinone

-3.63

CAGLOF02519g YJL206C Description not mapped -3.63 CAGLOG03245g YKR018C - Description not mapped -3.63 CAGLOH05775g YPL097W MSYI Mitochondrial tyrosyl-tRHA syn-

thetase -3.61

CAGLOL12012g YER175C TMTI Trans-aconitate methyltransferase, cytosolic enzyme that catalyzes the methyl esterification of 3- isopropylmalate, an intermediate of the leucine biosynthetic pathway, and trans-aconitate, which inhibits the citric acid cycle

-3.58




CAGLOF05071g YLR284C EC11 Peroxisomal delta3,delta2-enoyl- -3.53 CoA isomerase, hexameric protein that converts 3-hexenoyl-CoA to trans-2-hexenoyl-CoA, essential for the beta-oxidation of unsaturated fatty acids, oleate-induced

CAGLOI09482g YBR273C UBX7 UBX (ubiquitin regulatory X) domain-containing protein that interacts with Cdc48p

-3.53

CAGLOJ08976g NORBH - Description not mapped -3.53 CAGLOL06820g YMR139W RIM11 Protein kinase required for sig-

nal transduction during entry into meiosis; promotes the formation of the Imelp-Ume6p complex by phosphorylating Imelp and Ume6p; shares similarity with mammalian glycogen synthase kinase 3-beta

-3.51

CAGLOH10362g YDL110C TMA17 Protein of unknown function that associates with ribosomes; het- erozygous deletion demonstrated increases in chromosome instability in a rad9 deletion background; pro-tein abundance is decreased upon intracellular iron depletion

-3.5

CAGLOL10582g YMR196W Description not mapped -3.5 CAGLOM08552g NORBH - Description not mapped -3.47 CAGLOI04246g YGL162W SUT1 Transcription factor of the -3.44

Zn[II]2Cys6 family involved in sterol uptake; involved in induction of hypoxic gene expression

CAGLOM02981g YMR278W PCMS Phosphoglucomutase, catalyzes interconversion of glucose-1- phosphate and glucose-6-phospate; transcription induced in response to stress; green fluorescent protein

-3.43

(GFP)-fusion protein localizes to the cytoplasm and nucleus; non-essential

CAGLOH04037g YOR178C CACI Regulatory subunit for Glc7p type- -3.42 1 protein phosphatase (PP1), teth-ers Glc7p to Gsy2p glycogen syn-thase, binds Hsflp heat shock tran-scription factor, required for induc-tion of some HSF-regulated genes under heat shock

CAGLOF03817g YMR210W - Description not mapped -3.41 CAGLOB02431g YML120C MDI1 NADFLubiquinone oxidoreductase,

transfers electrons from NADH to ubiquinone in the respiratory chain but does not pump protons, in contrast to the higher eukaryotic multisubunit respiratory complex I; phosphorylated; homolog of human

-3.4

AMID CAGLOK02629g YNL134C - Description not mapped -3.4 CAGLOK04367g YGRO55W MUPI High affinity methionine perme-

ase, integral membrane protein with -3.4

13 putative membrane-spanning re-gions; also involved in cysteine up-take




CAGLOH03289g CAGLOH06259g

YGL082W YAL017W

- PSK1

Description not mapped One of two (see also PSK2) PAS domain containing S/T protein kinases; coordinately regulates protein synthesis and carbohydrate metabolism and storage in response to a unknown metabolite that re-flects nutritional status

-3.39 -3.39

CAGLOF02101g YFLOO7W BLMIO Proteasome activator subunit; found in association with core particles, with and without the

-3.38

19S regulatory particle; required for resistance to bleomycin, may be involved in protecting against oxidative damage; similar to mammalian PA200

C AG LOM12474g YIL055C - Description not mapped -3.37 CAGLOA01221g YPR192W AQYI Spore-specific water channel that

mediates the transport of water across cell membranes, developmen-tally controlled; may play a role in spore maturation, probably by al-lowing water outflow, may be in-volved in freeze tolerance

-3.36

CAGLOE06138g YPL231W FAS2 Alpha subunit of fatty acid synthetase, which catalyzes the syn-thesis of long-chain saturated fatty acids; contains beta-ketoacyl reduc-tase and beta-ketoacyl synthase ac-tivities; phosphorylated

-3.35

CAGLOH02695g YJL137C GLG2 Self-glucosylating initiator of glycogen synthesis, also glucosylates n- dodecyl-beta-D-maltoside; similar to mammalian glycogenin

-3.34

CAGLOD01782g YGL197W MDS3 Protein with an N-terminal kelch- like domain, putative negative regulator of early meiotic gene expression; required, with Pmdlp, for growth under alkaline conditions

-3.32

CAGLOF04917g YOR178C GACI Regulatory subunit for Glc7p type- -3.29 1 protein phosphatase (PP1), teth-ers Glc7p to Gsy2p glycogen syn-thase, binds Hsflp heat shock tran-scription factor, required for induc-tion of some HSF-regulated genes under heat shock

CAGL0005027g YAR035W YAT1 Outer mitochondrial carnitine acetyltransferase, minor ethanol- inducible enzyme involved in transport of activated acyl groups from the cytoplasm into the mito-chondrial matrix; phosphorylated

-3.27

CAGLOL00649g YAL054C ACSI Acetyl-coA synthetase isoform which, along with Acs2p, is the nuclear source of acetyl-coA for histone acetlyation; expressed during growth on nonfermentable carbon sources and under aerobic conditions

-3.27




CAGLOG05830g

CAGLOM11792g

YHR146W

YAL031C

CRP]

GIP4

Protein that binds to cruciform DNA structures Cytoplasmic Glc7-interacting protein whose overexpression relocal-izes Glc7p from the nucleus and prevents chromosome segregation; potential Cdc28p substrate

-3.26

-3.24

CAGLOM13761g YMR302C YME2 Integral inner mitochondria] membrane protein with a role in maintaining mitochondrial nucleoid structure and number; mutants ex-hibit an increased rate of mitochon-drial DNA escape; shows some se-quence similarity to exonucleases

-3.24

CAGLOM08822g YDR258C HSP78 Oligomeric mitochondrial matrix chaperone that cooperates with

-3.22

Ssclp in mitochondria] thermotol-erance after heat shock; able to pre-vent the aggregation of misfolded proteins as well as resolubilize pro-tein aggregates

CAGLOK00231g YKL215C - Description not mapped -3.21 CAGLOA04455g YBL066C SEFI Putative transcription factor, has

homolog in Kluyveromyces lactis -3.2

CAGLOA00957g YLR324W PEX30 Peroxisomal integral membrane protein, involved in negative regulation of peroxisome number; partially functionally redundant with Pex31p; genetic interactions suggest action at a step downstream of steps mediated by

-3.19

Pex28p and Pex29p CAGLOKOOS91g YGR205W Description not mapped -3.19 CAGLOG07601g YBR287W - Description not mapped -3.18 CAGLOF07975g YGR237C - Description not mapped -3.17 CAGLOG02717g YIL099W SGA1 Intracellular sporulation-specific

glucoamylase involved in glycogen degradation; induced during starvation of a/a diploids late in sporulation, but dispensable for sporulation

-3.17

CAGLOJ05830g YNL144C Description not mapped -3.14 CAGLOK03663g NORBH - Description not mapped -3.14 CAGLOK08624g YKL109W HAP4 Subunit of the heme-activated,

glucose-repressed Hap2p/3p/4p/5p -3.14

CCAAT-binding complex, a tran-scriptional activator and global reg-ulator of respiratory gene expres-sion; provides the principal activa-tion function of the complex

CAGLOF00363g YJR073C OPI3 Phospholi pi d methyltransferase -3.1 (methylene-fatty-acyl-phospholipid synthase), catalyzes the last two steps in phosphatidylcholine biosynthesis

CAGLOG07062g YML020W - Description not mapped -3.1 CAGLOH01287g YDR293C SSD1 Protein with a role in maintenance

of cellular integrity, interacts with components of the TOR pathway; ssdl mutant of a clinical S. cere-visiae strain displays elevated vir-ulence

-3.09




CAGLOF03839g CAGLOD01270g CAGLOF07513g

YMR209C NORBH

YKL093W -

MBR1

Description not mapped Description not mapped Protein involved in mitochondrial functions and stress response; over-expression suppresses growth de-fects of hap2, hap3, and hap4 mu-tants

-3.08 -3.07 -3.07

CAGLOK10824g YLR149C - Description not mapped -3.07 CAGLOG03289g YBL075C SSA3 ATPase involved in protein folding

and the response to stress; plays a role in SRP-dependent cotransla-tional protein-membrane targeting and translocation; member of the heat shock protein 70 (HSP70) fam-ily; localized to the cytoplasm

-3.06

CAGLOM13255g YKL065C YETI Endoplasmic reticulum transmembrane protein; may interact with ribosomes, based on co-purification experiments; homolog of human

-3.06

BAP31 protein CAGLOG04829g YML017W PSP2 Asn rich cytoplasmic protein that

contains RGG motifs; high-copy suppressor of group II intron- splicing defects of a mutation in

-3.02

MRS2 and of a conditional muta-tion in POLL (DNA polymerase al-pha); possible role in mitochondrial mRNA splicing

CAGLOI01012g YOR036W PEP12 Target membrane receptor (t- -3.02 SNARE) for vesicular intermediates traveling between the Golgi apparatus and the vacuole; controls entry of biosynthetic, endocytic, and retrograde traffic into the prevacuolar compartment; syntaxin

CAGLOG09020g YPL203W TPK2 cAMP-dependent protein kinase catalytic subunit; promotes vegeta-tive growth in response to nutrients via the Ras-cAMP signaling pathway; inhibited by regulatory subunit Bcy1p in the absence of cAMP; partially redundant with

-3.01

Tpklp and Tpklp CAGLOJ06468g YMR261C TPS3 Regulatory subunit of trehalose- -3.01

6-phosphate synthase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; ex-pression is induced by stress con-ditions and repressed by the Ras-cAMP pathway

CAGLOH08888g YPL221W FLC1 Putative FAD transporter; required for uptake of FAD into endoplas-mic reticulum; involved in cell wall maintenance

-3

CAGLOK03575g YMR114C - Description not mapped -3 CAGLOI06644g YDR077W SEDI Major stress-induced structural -24.11

GPI-cell wall glycoprotein in stationary-phase cells, associates with translating ribosomes, possi-ble role in mitochondrial genome maintenance; ORF contains two distinct variable minisatellites




CAGLOM11000g CAGLOJ04004g CAGLOL10912g

NORBH YOR228C YOR273C

- -

TPO4

Description not mapped Description not mapped Polyamine transport protein, recognizes spermine, putrescine, and spermidine; localizes to the plasma membrane; member of the major fa-cilitator superfamily

-21.21 -2.99 -2.99

CAGLOI01408g YJR048W CYC1 Cytochrome c, isoform 1; electron carrier of the mitochondrial inter-membrane space that transfers elec-trons from ubiquinone-cytochrome c oxidoreductase to cytochrome c oxidase during cellular respiration

-2.96

CAGLOL11880g YER169W RPH1 JmjC domain-containing histone demethylase which can specifically demethylate H3K36 tri- and dimethyl modification states; transcriptional repressor of PHR1;

-2.95

Rphlp phosphorylation during DNA damage is under control of the MEC1-RAD53 pathway

CAGLOD06600g YLL019C KNSI Nonessential putative protein kinase of unknown cellular role; member of the LAMMER family of protein kinases, which are serine/threonine kinases also capable of phosphorylating tyrosine residues

-2.94

CAGLOM02211g YPL154C PEP4 Vacuolar aspartyl protease (proteinase A), required for the post-translational precursor maturation of vacuolar proteinases; important for protein turnover after oxidative damage; synthesized as a zymogen, self-activates

-2.94

CAGLOB03619g YEL060C PR131 Vacuolar proteinase B (yscB), a serine protease of the subtilisin fam-ily; involved in protein degradation in the vacuole and required for full protein degradation during sporula-tion

-2.93

CAGLOF04213g YBL030C PETS Major ADP/ATP carrier of the mitochondrial inner membrane, exchanges cytosolic ADP for mito-chondrially synthesized ATP; phosphorylated; required for viability in many common lab strains carry-ing a mutation in the polymorphic

-2.93

SAL1 gene CAGLOH03971g YCROO4C YCP4 Protein of unknown function, has

sequence and structural similarity to flavodoxins; predicted to be pahnitoylated; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

-2.93

CAGLOB02838g YGRO55W MUPI High affinity methionine permease, integral membrane protein with

-2.92

13 putative membrane-spanning re-gions; also involved in cysteine up-take




CAGL0C05357g CAGLOK03245g CAGLOL12914g CAGLOD04026g

YNL200C NORBH

YMR031C YGRO19W

-

- UGA1

Description not mapped Description not mapped Description not mapped Gamma-aminobutyrate (GABA) transaminase (4-aminobutyrate aminotransferase) involved in the

-2.92 -2.92 -2.92 -2.91

4-aminobutyrate and glutamate degradation pathways; required for normal oxidative stress tolerance and nitrogen utilization

CAGLOG02915g YGRO86C PILL Primary component of eisosomes, which are large immobile cell cor-tex structures associated with en-docytosis; null mutants show acti-vation of Pkclp/Ypklp stress resis-tance pathways; detected in phos-phorylated state in mitochondria

-2.91

CAGLOI03190g YEL024W RIP/ Ubiquinol-cytochrome-c reductase, a Rieske iron-sulfur protein of the mitochondria] cytochrome bc1 complex; transfers electrons from ubiquinol to cytochrome cl during respiration

-2.91

CAGLOL10494g YOR070C GYPI Cis-golgi GTPase-activating protein (GAP) for the Rab family members Yptlp (in vivo) and for

-2.91

Yptlp, Sec4p, Ypt7p, and Ypt5lp (in vitro); involved in vesicle dock-ing and fusion

CAGLOI01100g YOR120W GCYI Putative NADP(+) coupled glycerol dehydrogenase, proposed to be involved in an alternative pathway for glycerol catabolism; member of the aldo-keto reductase (AKR) fam-ily

-2.9

CAGL0101496g NORBH - Description not mapped -2.9 CAGL0107447g YOL103W ITR2 Myo-inositol transporter with

strong similarity to the major myo-inositol transporter Itrlp, member of the sugar transporter superfam-ily; expressed constitutively

-2.88

CAGLOK05137g YPRO26W ATH1 Acid trehalase required for utilization of extracellular trehalose

-2.87

CAGLOL04268g YOR155C ISN1 Inosine 5'-monophosphate (IMP)- specific 5'-nucleotidase, catalyzes the breakdown of IMP to inosine, does not show similarity to known

-2.87

5'-nucleotidases from other organ-isms

CAGLOE04548g NORBH Description not mapped -2.86 CAGLOK04037g NORBH - Description not mapped -2.86 CAGLOL00825g YIL044C AGE2 ADP-ribosylation factor (ARF) -2.86

GTPase activating protein (GAP) effector, involved in Trans-Golgi- Network (TGN) transport; contains C2C2H2 cysteine/histidine motif

CAGLOA04829g YGL253W HXK2 Hexokinase isoenzyme 2 that catalyzes phosphorylation of glucose in the cytosol; predominant hexokinase during growth on glucose; functions in the nucleus to repress expression of HXK1 and GLK1 and to induce expression of its own gene

-2.85




CAGLOK12892g

CAGLOL06006g

YFLO47W

YGL180W

RGD2

ATGI

GTPase-activating protein (RhoGAP) for Cdc42p and Rho5p Protein ser/thr kinase required for vesicle formation in autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway; structurally required for phagophore assembly site formation; during autophagy forms a complex with Atgl3p and Atgl7p

-2.85

-2.85

CAGLOI06094g YJL155C FBP26 Fructose-2,6-bisphosphatase, required for glucose metabolism

-2.84

CAGLOM06413g YPL084W BROI Cytoplasmic class E vacuolar protein sorting (VPS) factor that coor-dinates deubiquitination in the mul-tivesicular body (MVB) pathway by recruiting Doa4p to endosomes

-2.84

CAGLOMI2793g YER079W - Description not mapped -2.84 CAGLOH01089g YDR287W INM2 Inositol monophosphatase, involved

in biosynthesis of inositol; enzymatic activity requires magnesium ions and is inhibited by lithium and sodium ions; inml inm2 double mu-tant lacks inositol auxotrophy

-2.83

CAGLOL03982g YNL117W MLSI Malate synthase, enzyme of the glyoxylate cycle, involved in utilization of non-fermentable carbon sources; expression is subject to carbon catabolite repression; local-izes in peroxisomes during growth in oleic acid medium

-2.83

CAGLOK04719g YNL208W - Description not mapped -2.82 CAGLOM07381g YPL053C KTR6 Probable mannosylphosphate

transferase involved in the synthesis of core oligosaccharides in protein glycosylation pathway; member of the KRE2/MNT1 mannosyltransferase family

-2.82

CAGLOD00528g YKL182W FASI Beta subunit of fatty acid synthetase, which catalyzes the syn-thesis of long-chain saturated fatty acids; contains acetyltransacylase, dehydratase, enoyl reductase, malonyl transacylase, and palmitoyl transacylase activities

-2.81

CAGLOK10868g YDR256C CTA1 Catalase A, breaks down hydrogen peroxide in the peroxisomal matrix formed by acyl-CoA oxidase

-2.8

(Pox1p) during fatty acid beta- oxidation

CAGL0000110g YKR102W FLO10 Lectin-like protein with similarity to Flo1p, thought to be involved in flocculation

-2.78

CAGLOH03619g YNL011C - Description not mapped -2.78 CAGLOH08844g YMR173W DDR48 DNA damage-responsive protein,

expression is increased in response to heat-shock stress or treatments that produce DNA lesions; contains multiple repeats of the amino acid sequence NNNDSYGS

-2.77

CAGLOI05610g YNR014W - Description not mapped -2.77 CAGLOA04477g YBL067C UBP13 Putative ubiquitin carboxyl-

terminal hydrolase, ubiquitin- specific protease that cleaves ubiquitin-protein fusions

-2.76




CAGLOE03388g YJL024C APS3 Small subunit of the clathrin- associated adaptor complex AP-3, which is involved in vacuolar protein sorting; related to the sigma subunit of the mammalian clathrin

-2.76

AP-3 complex; suppressor of loss of casein kinase 1 function

CAGLOJ06270g YEL176W - Description not mapped -2.76 CAGLOL10780g YNR016C ACCI Acetyl-CoA carboxylase, biotin

containing enzyme that catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA; required for de novo biosynthesis of long-chain fatty acids

-2.76

CAGLOF06325g YMR053C STBd Protein that interacts with Sin3p in a two-hybrid assay and is part of a large protein complex with Sin3p and Stblp

-2.75

CAGLOA02211g YDR343C HXT6 High-affinity glucose transporter of the major facilitator superfamily, nearly identical to Hxt7p, expressed at high basal levels relative to other

-2.74

HXTs, repression of expression by high glucose requires SNF3

CAGLOE04554g NORBH - Description not mapped -2.74 CAGLOF02255g YFR003C YP11 Inhibitor of the type I protein

phosphatase Glc7p, which is involved in regulation of a variety of metabolic processes; overproduc-tion causes decreased cellular con-tent of glycogen

-2.74

CAGLOH08063g YNL202W SPS19 Peroxisomal 2,4-dienoyl-CoA reductase, auxiliary enzyme of fatty acid beta-oxidation; homodimeric enzyme required for growth and sporulation on petroselineate medium; expression induced during late sporulation and in the presence of oleate

-2.74

CAGLOK08668g YIR017C MET28 Basic leucine zipper (bZIP) transcriptional activator in the Cbflp-

-2.74

Met4p-Met28p complex, participates in the regulation of sulfur metabolism

CAGLOG10153g YDR529C QCR7 Subunit 7 of the ubiquinol cytochrome-c reductase complex, which is a component of the mitochondrial inner membrane electron transport chain; oriented facing the mitochondria] matrix;

-2.73

N-terminus appears to play a role in complex assembly

CAGLOK00803g YGR209C TRX2 Cytoplasmic thioredoxin isoenzyme of the thioredoxin system which protects cells against oxidative and reductive stress, forms LMA1 com-plex with Pbi2p, acts as a cofactor for Tsalp. required for ER-Golgi transport and vacuole inheritance

-2.73


Appendix G. Transcription profiling of C. gtabrata serum stress responses 411


CAGLOH06721g

CAGLOH04851g

YIL156W

YML016C

UBP7

PPZI

Ubiquitin-specific protease that cleaves ubiquitin-protein fusions Serine/threonine protein phosphatase Z, isoform of Ppz2p; involved in regulation of potassium transport, which affects osmotic stability, cell cycle progression, and halotolerance

-2.72

-2.71

CAGLOB03685g YCROO4C YCP4 Protein of unknown function, has sequence and structural similarity to flavodoxins; predicted to be palmitoylated; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

-2.69

CAGLOD00704g YDL072C YET3 Protein of unknown function: YET3 null mutant decreases the level of secreted invertase; homolog of hu-man BAP31 protein

-2.69

CAGL0,107150g YALO51W OAF1 Oleate-activated transcription factor, acts alone and as a heterodimer with Pip2p; activates genes involved in beta-oxidation of fatty acids and peroxisome organization and biogenesis

-2.69

CAGLOL06204g YGL191W COX13 Subunit Via of cytochrome c oxidase, which is the terminal member of the mitochondrial inner mem-brane electron transport chain; not essential for cytochrome c oxidase activity but may modulate activity in response to ATP

-2.69

CAGLOE06116g YPL230W US lil Putative transcription factor containing a C2H2 zinc finger; muta-tion affects transcriptional regula-tion of genes involved in growth on non-fermentable carbon sources, re-sponse to salt stress and cell wall biosynthesis

-2.68

CAGL0106765g YDR069C D0.4.4 Ubiquitin isopeptidase, required for recycling ubiquitin from proteasome-bound ubiquitinated intermediates, acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole

-2.68

CAGLOE04004g YHL036W MUP3 Low affinity methionine permease, similar to Muplp

-2.67

CAGL0100748g YMR145C NDEI Mitochondrial external NADH dehydrogenase, a type II

-2.67

NAD(P)H:quinone oxidoreductase that catalyzes the oxidation of cytosolic NADH; Ndelp and Nde2p provide cytosolic NADH to the mitochondrial respiratory chain

CAGLOI08591g YER158C - Description not mapped -2.67 CAGLOK03509g YMR110C HFDI Putative fatty aldehyde dehydro-

genase, located in the mitochondria] outer membrane and also in lipid particles; has similarity to hu-man fatty aldehyde dehydrogenase

-2.67





CAGLOM13189g

CAGLOB03069g

YKL062W

YLR354C

MSN4

TALI

Transcriptional activator related to Msn2p; activated in stress conditions, which results in translocation from the cytoplasm to the nucleus; binds DNA at stress response ele-ments of responsive genes, inducing gene expression Transaldolase, enzyme in the non- oxidative pentose phosphate pathway; converts sedoheptulose 7- phosphate and glyceraldehyde 3- phosphate to erythrose 4-phosphate and fructose 6-phosphate

-2.67

-2.66

CAGLOE00869g YOL048C - Description not mapped -2.65 CAGL0G09449g YGRI89C CRH1 Putative chitin transglycosidase,

cell wall protein that functions in the transfer of chitin to beta(1-

-2.65

6)glucan; localizes to sites of polar-ized growth; expression is induced under cell wall stress conditions

CAGLOE02981g YGR149W - Description not mapped -2.64 CAGLOJ07304g YNL223W ATG4 Conserved cysteine protease re-

quired for autophagy; cleaves Atg8p to a form required for autophago-some and Cvt vesicle generation

-2.64

CAGLOJOS316g YNL277W MET2 L-homoserine-O-acetyltransferase, catalyzes the conversion of homoserine to 0-acetyl homoserine which is the first step of the methionine biosynthetic pathway

-2.64

CAGLOL08932g YPLOO4C LSP1 Primary component of eisosomes, which are large immobile patch structures at the cell cortex asso-ciated with endocytosis, along with

-2.64

PiIlp and Sur7p; null mutants show activation of Pkclp/Ypklp stress resistance pathways

CAGLOM05467g YBR204C - Description not mapped -2.64 CAGLOM13783g YMR304W UBP15 Ubiquitin-specific protease that

may play a role in ubiquitin precursor processing

-2.64

CAGLOCO3113g YLR270W DCSI Non-essential hydrolase involved in mRNA decapping, may function in a feedback mechanism to regulate deadenylation, contains pyrophos- phatase activity and a HIT (histidine triad) motif; interacts with neutral trehalase Nth1p

-2.62

CAGLOG01738g YGRO86C PIL1 Primary component of eisosomes, which are large immobile cell cor-tex structures associated with en-docytosis; null mutants show acti-vation of Pkclp/Ypklp stress resis-tance pathways; detected in phos-phorylated state in mitochondria

-2.61

CAGL0G05049g YDR063W AIM7 Putative protein of unknown function; green fluorescent protein

-2.61





CAGLOK06083g NORBH - Description not mapped -2.61 CAGLOL02717g YOR215C A111441 Putative protein of unknown func- -2.61

Lion; the authentic protein is de-tected in highly purified mitochondria in high-throughput studies; YOR215C is not an essential gene

CAGLOM12551g YIL057C RGI2 Putative protein of unknown func- -2.61 Lion; expression induced under car-bon limitation and repressed under high glucose

CAGLOB01595g NORBH - Description not mapped -2.6 CAGLOL07744g YCR011C ADPI Putative ATP-dependent permease

of the ABC transporter family of proteins

-2.6

CAGLOL08426g YPR151C SUE/ Mitochondrial protein required for degradation of unstable forms of cy-tochrome c

-2.6

CAGLOL10406g YOR065W CYTI Cytochrome cl, component of the mitochondria] respiratory chain; expression is regulated by the heme-activated, glucose-repressed

-2.6

Hap2p/3p/4p/5p CCAAT-binding complex

CAGLOF04521g YBL043W ECM13 Non-essential protein of unknown function; induced by treatment with 8-methoxypsoralen and UVA irradiation

-2.59

CAGLOH05621g YPL089C RLM/ MADS-box transcription factor, component of the protein kinase

-2.59

C-mediated MAP kinase pathway involved in the maintenance of cell integrity; phosphorylated and activated by the MAP-kinase Sit2p

CAGLOJ04488g YLR417W VrS36 Component of the ESCRT-II corn- plea; contains the GLUE (GRAM

-2.59

Like Ubiquitin binding in EAP45) domain which is involved in interac-tions with ESCRT-I and ubiquitin-dependent sorting of proteins into the endosome

CAGL0005533g YDL237W LRC1 Putative protein of unknown function; YDL237W is not an essential gene

-2.58

CAGLOM06325g YPL089C RLMI MADS-box transcription factor, component of the protein kinase

-2.58

C-mediated MAP kinase pathway involved in the maintenance of cell integrity; phosphorylated and activated by the MAP-kinase Slt2p


-2.57


CAGLOF04565g YBL045C COR/ Core subunit of the ubiquinol- cytochrome c reductase complex

-2.57

(bc1 complex), which is a component of the mitochondrial inner membrane electron transport chain

CAGLOJ04268g YBL015W ACHI Acetyl-coA hydrolase, primarily localized to mitochondria; phospho-rylated; required for acetate utiliza-tion and for diploid pseudohyphal growth

-2.57




CAGLOK07678g CAGLOK12760g CAGLOF07777g

NORBH YEL042C YMR17OC

- -

ALD2

Description not mapped Description not mapped Cytoplasmic aldehyde dehydrogenase, involved in ethanol oxidation and beta-alanine biosynthesis; uses NAD+ as the preferred coen-zyme; expression is stress induced and glucose repressed; very similar to Ald3p

-2.57 -2.56 -2.55

CAGLOL01265g YJR049C UTRI ATP-NADH kinase; phosphorylates both NAD and NADH; active as a hexamer; enhances the activity of ferric reductase (Fre1p)

-2.55

CAGLOL03498g YJL094C KHA 1 Putative K+/I-1-1- antiporter with a probable role in intracellular cation homeostasis, localized to Golgi vesi-cles and detected in highly purified mitochondria in high-throughput studies

-2.55

CAGLOB03201g YKR011C - Description not mapped -2.54 CAGLOF04631g YBL049W MOH/ Protein of unknown function, has

homology to kinase Snf7p; not required for growth on nonfermentable carbon sources; essential for viability in stationary phase

-2.54

CAGLOG01100g YLR345W - Description not mapped -2.53 CAGLOM03839g YNL305C Description not mapped -2.53 CAGLOF04697g YLR257W - Description not mapped -2.52 CAGL0104994g YER091C METS Cobalamin-independent methionine

synthase, involved in amino acid biosynthesis; requires a minimum of two glutamates on the methylte-trahydrofolate substrate, similar to bacterial metE homologs

-2.52

CAGLOJ00803g YJL042W MHP1 Microtubule-associated protein involved in assembly and stabilization of microtubules; overproduction results in cell cycle arrest at

-2.52

G2 phase; similar to Drosophila protein MAP and to mammalian MAP4 proteins

CAGL0C04763g YCLO01W liERI Protein involved in retention of membrane proteins, including

-2.51

Secl2p, in the ER; localized to Golgi; functions as a retrieval receptor in returning membrane proteins to the ER

CAGL0,102200g YIROO1C SGNI Cytoplasmic RNA-binding protein, contains an RNA recognition motif (RRM); may have a role in mRNA translation, as suggested by genetic interactions with genes en-coding proteins involved in transla-tional initiation

-2.51

CAGLOK06237g YDR452W PPN1 Vacuolar endopolyphosphatase with a role in phosphate metabolism; functions as a ho- modimer

-2.51

CAGLOL06182g YGL190C CDC55 Non-essential regulatory subunit B of protein phosphatase 2A, which has multiple roles in mitosis and protein biosynthesis; involved in regulation of mitotic exit; found in the nucleus of most cells, also at the bud neck and at the bud tip

-2.51




CAGLOH10472g

CAGL0004191g

YDL115C

YBROO6W

IWR1

UGA2

Protein of unknown function, deletion causes hypersensitivity to the K1 killer toxin Succinate semialdehyde dehydrogenase involved in the utilization of gamma-aminobutyrate (GABA) as a nitrogen source; part of the 4-aminobutyrate and glutamate degradation pathways; localized to the cytoplasm

-2.5

-2.49

CAGLOH10428g YDL113C A TG20 Sorting nexin family member required for the cytoplasm-to- vacuole targeting (Cvt) pathway and for endosomal sorting; has a

-2.49

Phox homology domain that binds phosphatidylinositol-3-phosphate; interacts with Snx4p; potential Cdc28p substrate

CAGL0105236g YIL033C BCY1 Regulatory subunit of the cyclic -2.49 AMP-dependent protein kinase (PICA), a component of a signaling pathway that controls a variety of cellular processes, including metabolism, cell cycle, stress response, stationary phase, and sporulation

CAGLOA03564g YLR039C RIC1 Protein involved in retrograde transport to the cis-Golgi network; forms heterodimer with Rgplp that acts as a GTP exchange factor for

-2.48

Ypt6p; involved in transcription of rRNA and ribosomal protein genes


-2.48


CAGLOK03003g YMR070W MOTS Nuclear transcription factor with two Cys2-His2 zinc fingers; involved in repression of a subset of hypoxic genes by Roxlp, repression of sev-eral DAN/TIR genes during aerobic growth, and repression of ergosterol biosynthetic genes

-2.48

CAGLOA03542g NORBH - Description not mapped -2.47 CAGL00O2321g YER037W PHMS Protein of unknown function, ex-

pression is induced by low phosphate levels and by inactivation of

-2.47

Pho85p CAGL0005137g YOL059W GPD2 NAD-dependent glycerol 3-

phosphate dehydrogenase, homolog of Gpdlp, expression is controlled by an oxygen-independent signal-ing pathway required to regulate metabolism under anoxic conditions; located in cytosol and mitochondria

-2.47




CAGLOF04741g YOL016C CMK2 Calmodulin-dependent protein kinase; may play a role in stress response, many CA++/calmodulan dependent phosphorylation substrates demonstrated in vitro, amino acid sequence similar to

-2.47

Cmklp and mammalian Cam Kinase II

CAGLOI00880g YMR137C P502 Required for a post-incision step in the repair of DNA single and double-strand breaks that result from interstrand crosslinks produced by a variety of mono- and bi-functional psoralen derivatives; in-duced by UV-irradiation

-2.47

CAGLOK09350g YGL208W SIP2 One of three beta subunits of the -2.47 Snfl serine/threonine protein kinase complex involved in the response to glucose starvation; null mutants exhibit accelerated aging; N-myristoylprotein localized to the cytoplasm and the plasma mem-brane

CAGLOB03817g Y.111008W Description not mapped -2.46 CAGLOD02266g YOR352W - Description not mapped -2.45 CAGLOK02783g YKL007W CAPI Alpha subunit of the capping pro-

rein (CP) heterodimer (Cap1p and -2.45

Cap2p) which binds to the barbed ends of actin filaments preventing further polymerization; localized predominantly to cortical actin patches

CAGLOL00803g YER054C GIP2 Putative regulatory subunit of the protein phosphatase Glc7p, involved in glycogen metabolism; contains a conserved motif (GVNK motif) that is also found in Gaclp, Pig1p, and Pig2p

-2.45

CAGLOF03707g YOR267C HRK1 Protein kinase implicated in activation of the plasma membrane H(4-)-

-2.44

ATPase Pmalp in response to glu-cose metabolism; plays a role in ion homeostasis

CAGLOI07821g YOL087C DUF1 Putative protein of unknown function; green fluorescent protein

-2.44

(GFP)-fusion protein localizes to the cytoplasm; deletion mutant is sensitive to various chemicals in-cluding phenanthroline, sanguinar-ine, and nordihydroguaiaretic acid

CAGLOJ02134g YILOO2C INP51 Phosphatidylinositol 4,5- bisphosphate 5-phosphatase, synaptojanin-like protein with an

-2.44

N-terminal Sacl domain, plays a role in phosphatidylinositol 4,5- bisphosphate homeostasis and in endocytosis; null mutation confers cold-tolerant growth



continued from previous page CO ORF SC ORF gene name Description Log Ratio

CAGLOJ05962g CAGLOM13937g

YNL155W YMR311C

- GLC8

Description not mapped Regulatory subunit of protein phosphatase 1 (Glc7p), involved in glycogen metabolism and chromo-some segregation; proposed to reg-ulate Glc7p activity via conforma- tional alteration; ortholog of the mammalian protein phosphatase in-hibitor 2

-2.44 -2.44

CAGL0CO2189g YER043C SAH1 S-adenosyl-L-homocysteine hydrolase, catabolizes S-adenosyl-L- homocysteine which is formed after donation of the activated methyl group of S-adenosyl-L-methionine

-2.43

(AdoMet) to an acceptor CAGLOI10054g YGR143W SKN1 Protein involved in sphingolipid

biosynthesis; type II membrane pro-tein with similarity to Kre6p

-2.43

C AG LOK09900g YOR358W HAP5 Subunit of the heme-activated, glucose-repressed Hap2/3/4/5

-2.43

CCAAT-binding complex, a transcriptional activator and global regulator of respiratory gene expression; required for assembly and DNA binding activity of the complex

CAGLOE05588g YOR346W REV1 Deoxycytidyl transferase, forms a complex with the subunits of DNA polymerase zeta, Rev3p and Rev7p; involved in repair of abasic sites in damaged DNA

-2.42

CAGLOM03025g YMR280C CATS Zinc cluster transcriptional activator necessary for derepression of a variety of genes under non-fermentative growth conditions, ac-tive after diauxic shift, binds car-bon source responsive elements

-2.42

CAGL0G08712g YIL125W KGD1 Component of the mitochondria' alpha-ketoglutarate dehydrogenase complex, which catalyzes a key step in the tricarboxylic acid (TCA) cycle, the oxidative decarboxylation of alpha-ketoglutarate to form succinyl-CoA

-2.41

CAGLOL06600g YGR232W NAS6 Regulatory, non-ATPase subunit of the 26S proteasome; homolog of the human oncoprotein gankyrin, which interacts with the retinoblastoma tumor suppressor (Rb) and cyclin-dependent kinase 4/6

-2.41


-2.4


CAGLOK08338g YOR385W - Description not mapped -2.4 CAGLOB00814g YCL034W LSB5 Protein of unknown function; binds -2.39

Las17p, which is a homolog of human Wiskott-Aldrich Syndrome protein involved in actin patch as-sembly and actin polymerization




CAGLOJ06380g CAGLOK08734g CAGLOJ10428g

YDL130WA YIR014W YKL092C

- -

BUD2

Description not mapped Description not mapped GTPase activating factor for Rsrlp/Budlp required for both axial and bipolar budding patterns; mutants exhibit random budding in all cell types

-2.39 -2.39 -2.38

CAGLOM03179g YJL082W IML2 Protein of unknown function; the authentic, non-tagged protein is de-tected in highly purified mitochon-dria in high-throughput studies

-2.38

CAGLOM04125g YNL320W - Description not mapped -2.38 CAGL0004785g YJR115W - Description not mapped -2.37 CAGL0105830g YNROO6W VPS27 Endosomal protein that forms a

complex with Hselp; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined for degradation; has Ubiquitin Inter- action Motifs which bind ubiquitin

-2.37

(Ubi4p) CAGLOK09878g YNL094W APPI Protein of unknown function, in-

teracts with Rvs161p and Rvs167p; computational analysis of protein- protein interactions in large-scale studies suggests a possible role in actin filament organization

-2.37

CAGLOD03982g YHR037W PUT2 Delta-1-pyrroline-5-carboxylate dehydrogenase, nuclear-encoded mito-chondrial protein involved in utilization of proline as sole nitrogen source; deficiency of the hu-man homolog causes HPII, an au-tosomal recessive inborn error of metabolism

-2.36

CAGLOI01122g YHRIO4W GRE.? Aldose reductase involved in methylglyoxal, d-xylose and arabinose metabolism; stress induced

-2.36

(osmotic, ionic, oxidative, heat shock, starvation and heavy metals); regulated by the HOG pathway

CAGLOL04818g YG111.17C - Description not mapped -2.36 CAGL0G02849g YPL186C UIP4 Protein that interacts with Ulplp, a -2.35

Ubl (ubiquitin-like protein)-specific protease for Smt3p protein conju-gates; detected in a phosphorylated state in the mitochondrial outer membrane; also detected in ER and nuclear envelope

CAGLOH05291g YPLO7OW MUKI Cytoplasmic protein of unknown function; computational analysis of large-scale protein-protein interac-tion data suggests a possible role in transcriptional regulation

-2.35

CAGLOK10626g YFRO15C GSY1 Glycogen synthase with similarity to Gsy2p, the more highly expressed yeast homolog; expression induced by glucose limitation, nitrogen starvation, environmental stress, and entry into stationary phase

-2.35




CAGLOL05258g CAGLOH02959g

NORBH YGL096W

- TOSS

Description not mapped Homeodomain-containing protein and putative transcription factor found associated with chromatin; target of SBF transcription factor; induced during meiosis and under cell-damaging conditions; similar to Cup9p transcription factor

-2.34 -2.33

CAGL0102420g YHR171W ATG7 Autophagy-related protein and dual specificity member of the

-2.33

El family of ubiquitin-activating enzymes; mediates the conjugation of Atgl2p with Atg5p and Atg8p with phosphatidylethanolamine, required steps in autophagosome formation

CAGLOJ04906g YJL049W - Description not mapped -2.33 CAGLOM05951g YKR049C FMP46 Putative redox protein containing

a thioredoxin fold; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

-2.33

CAGLOF07051g YGL124C MON1 Protein required for fusion of cvt- vesicles and autophagosomes with the vacuole; associates, as a com-plex with Cczlp, with a perivacuo-lar compartment; potential Cdc28p substrate

-2.32

CAGL0102662g YMR284W YKU70 Subunit of the telomeric Ku corn- plex (Yku70p-Yku80p), involved in telomere length maintenance, struc-ture and telomere position effect; relocates to sites of double-strand cleavage to promote nonhomologous end joining during DSB repair

-2.32

CAGL0104972g NORBH - Description not mapped -2.32 CAGL0106182g YKL164C PIR1 0-glycosylated protein required for

cell wall stability; attached to the cell wall via beta-1,3-glucan; medi-ates mitochondrial translocation of

-2.32

Apnlp; expression regulated by the cell integrity pathway and by Swi5p during the cell cycle

CAGLOE01353g YLR130C ZRT2 Low-affinity zinc transporter of the plasma membrane; transcription is induced under low-zinc conditions by the Zap1p transcription factor

-2.31

CAGLOF06061g YMR041C ARA2 NAD-dependent arabinose dehydrogenase, involved in biosynthesis of dehydro-D-arabinono-1,4-lactone; similar to plant L-galactose dehy-drogenate

-2.31

CAGL0110582g YGR127W - Description not mapped -2.31 CAGLOK03113g YIIL014C YLF2 Protein of unknown function, has

weak similarity to E. coli GTP- binding protein gtpl; the authen-tic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

-2.31




CAGLOH02491g CAGLOH03025g

NORBH YGL094C

- PANS

Description not mapped Essential subunit of the Pan2p- Pan3p poly(A)-ribonuclease complex, which acts to control poly(A) tail length and regulate the stoi-chiometry and activity of postrepli-cation repair complexes

-2.3 -2.3

CAGLOI00836g YMR140W SIPS Protein of unknown function; interacts with both the Reglp/Glc7p phosphatase and the Snflp kinase

-2.3

CAGLOK08800g YMR009W ADI1 Acireductone dioxygenease involved in the methionine salvage pathway; ortholog of human MTCBP-

-2.3

1; transcribed with YMR010W and regulated post-transcriptionally by RNase III (Rntlp) cleavage; ADI1 mRNA is induced in heat shock con-ditions

CAGL0001771g YBR241C - Description not mapped -2.29 CAGLOK10714g YOR018W ROD1 Membrane protein that binds the


-2.29

CAGLOG06952g YJL012C VTC4 Vacuolar membrane protein involved in vacuolar polyphosphate accumulation; functions as a regu-lator of vacuolar H-F-ATPase activ-ity and vacuolar transporter chap-erones; involved in non-autophagic vacuolar fusion

-2.27

CAGLOL05236g YKL085W AIDH1 Mitochondria] malate dehydrogenase, catalyzes interconversion of malate and oxaloacetate; involved in the tricarboxylic acid (TCA) cy-cle; phosphorylated

-2.27

CAGLOG01342g YNL054W VAC7 Integral vacuolar membrane protein involved in vacuole inheritance and morphology; activates Fab1p kinase activity under basal conditions and also after hyperosmotic shock

-2.26

CAGLOG04081g YLROO4C THI73 Putative plasma membrane permease proposed to be involved in carboxylic acid uptake and repressed by thiamine; substrate of

-2.26

Dbf2p/Moblp kinase; transcription is altered if mitochondrial dysfunc-tion occurs

CAGLOJ08294g YNL275W DORI Boron efflux transporter of the plasma membrane; binds HCO3-, I- , Br-, NO3- and Cl-; has similarity to the characterized boron efflux transporter A. thaliana BOR1

-2.26

CAGLOI00660g YLR289W GUF1 Mitochondria] matrix GTPase that associates with mitochondrial ribosomes; important for translation under temperature and nutri-ent stress; may have a role in trans-lational fidelity; similar to bacterial

-2.25

LepA elongation factor continued on next page



CAGLOI00902g

CAGLOI07139g

YMR136W

YOR142W

GAT2

LSC1

Protein containing GATA family zinc finger motifs; similar to Gln3p and Dal80p; expression repressed by leucine Alpha subunit of succinyl-CoA ligase, which is a mitochondrial enzyme of the TCA cycle that catalyzes the nucleotide-dependent conversion of succinyl-CoA to suc-cinate; phosphorylated

-2.25

-2.25

CAGLOJ04180g YFLO13C IESI Subunit of the IN080 chromatin remodeling complex

-2.25

CAGLOM13541g YMR291W - Description not mapped -2.25 CAGLOB01639g YDR137W RGP1 Subunit of a Golgi membrane ex-

change factor (Riclp-Rgplp) that catalyzes nucleotide exchange on

-2.24

Ypt6p CAGLOH06765g YIL159W BNRI Formin, nucleates the formation of

linear actin filaments, involved in cell processes such as budding and mitotic spindle orientation which require the formation of polarized actin cables, functionally redundant with BNI1

-2.24

CAGLOI08305g YER024W YAT2 Carnitine acetyltransferase; has similarity to Yatlp, which is a car-nitine acetyltransferase associated with the mitochondrial outer mem-brane

-2.24

CAGLOJ08613g YOR088W - Description not mapped -2.24 CAGLOM08206g YJL171C - Description not mapped -2.24 CAGLOD00660g YDL073W - Description not mapped -2.23 CAGLOE00583g YCR083W TRX3 Mitochondria! thioredoxin, highly

conserved oxidoreductase required to maintain the redox homeostasis of the cell, forms the mitochondrial thioredoxin system with Trr2p, re-dox state is maintained by both

-2.23

Trr2p and Glrlp CAGLOF01397g YOR014W RTS1 B-type regulatory subunit of pro-

tein phosphatase 2A (PP2A); ho-molog of the mammalian B' subunit of PP2A

-2.23

CAGLOG09603g YOR186W - Description not mapped -2.23 CAGL0104620g YBRO42C CST26 Protein of unknown function, af-

fects chromosome stability when overexpressed

-2.23

CAGLOM02255g YPL152W REDS Activator of the phosphoty- rosy) phosphatase activity of

-2.23

PP2A,peptidyl-prolyl cis/trans- isomerase; regulates GI phase progression, the osmoresponse, microtubule dynamics; subunit of the Tap42p-Pph2lp-Rrd2p complex

CAGLOI10494g YPR149W NCE102 Protein of unknown function; contains transmembrane domains; involved in secretion of proteins that lack classical secretory signal sequences; component of the detergent-insoluble glycolipid- enriched complexes (DIGs)

-2.22




CAGLOL07128g YDL091C UBX3 UBX (ubiquitin regulatory X) domain-containing protein that interacts with Cdc48p, green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm in a punctate pattern

-2.22

CAGLOB01727g YDR109C - Description not mapped -2.21 CAGLOL08668g YPL015C HST2 Cytoplasmic member of the silent-

ing information regulator 2 (Sir2) family of NAD(+)-dependent pro-tein deacetylases; modulates nude-olar (rDNA) and telomeric silencing; possesses NAD(+)-dependent histone deacetylase activity in vitro

-2.2

CAGLOB02035g YDR129C SAC6 Fimbrin, actin-bundling protein; cooperates with Scplp

-2.19

(calponin/transgelin) in the organization and maintenance of the actin cytoskeleton

CAGLOF02513g NORBH - Description not mapped -2.19 CAGLOH03113g YGLO9OW LIF1 Component of the DNA ligase IV

complex that mediates nonhomol-ogous end joining in DNA double-strand break repair; physically in-teracts with Dn14p and Nejlp; ho-mologous to mammalian XRCC4 protein

-2.19

CAGL0109812g YOR311C DGK1 Diacylglycerol kinase, localized to the endoplasmic reticulum

-2.19

(ER); overproduction induces enlargement of ER-like membrane structures and suppresses a temperature-sensitive slyl mutation; contains a CTP transferase domain

CAGLOK07315g NORBH - Description not mapped -2.19 CAGLOL04378g YOR161C PNS 1 Protein of unknown function; has

similarity to Torpedo californica tCTL1p, which is postulated to be a choline transporter, neither null mutation nor overexpression affects choline transport

-2.19

CAGLOB00748g YCL039W GID7 Protein of unknown function, involved in proteasome-dependent catabolite inactivation of fructose-

-2.18

1,6-bisphosphatase; contains six WD40 repeats; computational analysis suggests that Gid7p and Mohlp have similar functions

CAGLOG05247g YDR069C DOA4 Ubiquitin isopeptidase, required for recycling ubiquitin from proteasome-bound ubiquitinated intermediates, acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole

-2.18




CAGLOH05137g YPL061W ALD6 Cytosolic aldehyde dehydrogenase, activated by Mg2+ and utilizes NADP+ as the preferred coenzyme; required for conversion of acetalde-hyde to acetate; constitutively expressed; locates to the mitochondria] outer surface upon oxidative stress

-2.18

CAGLOH08437g YBRO97W VPS15 Myristoylated serine/threonine protein kinase involved in vacuolar protein sorting; functions as a membrane-associated complex with Vps34p; active form recruits

-2.18

Vps34p to the Golgi membrane; interacts with the GDP-bound form of Gpalp

CAGLOG07689g YKR014C YPT52 GTPase, similar to Ypt5lp and -2.17 Ypt53p and to mammalian rab5; re-quired for vacuolar protein sorting and endocytosis

CAGLOJ05940g YHRI35C YCK1 Palmitoylated plasma membrane- bound casein kinase I isoform; shares redundant functions with

-2.17

Yck2p in morphogenesis, proper septin assembly, endocytic traffick-ing; provides an essential function overlapping with that of Yck2p

CAGLOJ08118g YNL265C ISTI Protein with a positive role in the multivesicular body sorting path-way; functions and forms a com-plex with Did2p; recruitment to en-dosomes is mediated by the Vps2p-

-2.17

Vps24p subcomplex of ESCRT-III; also interacts with Vps4p

CAGLOL03135g YKR031C SPO/4 Phospholipase D, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phospha- tidic acid; involved in Secl4p- independent secretion; required for meiosis and spore formation; differently regulated in secretion and meiosis

-2.17

CAGLOB01100g NORBH Description not mapped -2.16 CAGLOB02563g YML128C MSC/ Protein of unknown function; mu-

taut is defective in directing meiotic recombination events to homologous chromatids; the authen-tic, non-tagged protein is detected in highly purified mitochondria and is phosphorylated

-2.16

CAGLOG08932g YOL018C TLG2 Syntaxin-like t-SNARE that forms a complex with Tlglp and Vtilp and mediates fusion of endosome-derived vesicles with the late Golgi; binds Vps45p, which prevents

-2.16

T1g2p degradation and also facili-tates t-SNARE complex formation

CAGL0102046g YPR127W - Description not mapped -2.16 CAGLOL1111Og YML057W CMP2 Calcineurin A; one isoform (the

other is CNA1) of the catalytic subunit of calcineurin, a

-2.16

Ca+-1-/calmodulin-regulated protein phosphatase which regulates Crzlp (a stress-response transcription factor), the other calcineurin subunit is CNB1




CAGLOM10956g CAGLOG01474g

CAGLOH04983g

YCR073WA YNL042W

YDL055C

- BOPS

PSAI

Description not mapped Protein of unknown function, potential Cdc28p substrate; overproduction confers resistance to methylmercury GDP-mannose pyrophosphorylase (mannose-1-phosphate guanyltransferase), synthesizes

-2.16 -2.15

-2.15

GDP-mannose from GTP and mannose-l-phosphate in cell wall biosynthesis; required for normal cell wall structure

CAGL0106072g Y.11.154C VPS35 Endosomal subunit of membrane- associated retromer complex required for retrograde transport; re-ceptor that recognizes retrieval sig-nals on cargo proteins, forms sub-complex with Vps26p and Vps29p that selects cargo proteins for re-trieval

-2.15

CAGLOL00583g YPL230W USVI Putative transcription factor containing a C2112 zinc finger; muta-tion affects transcriptional regula-tion of genes involved in growth on non-fermentable carbon sources, re-sponse to salt stress and cell wall biosynthesis

-2.15

CAGLOL09933g YOR042W CUES Protein containing a CUE domain that binds ubiquitin, which may fa-cilitate intramolecular monoubiqui-tination; green fluorescent protein

-2.15

(GFP)-fusion protein localizes to the cytoplasm in a punctate pattern

CAGLOE02695g YOR005C DNL4 DNA ligase required for nonhomologous end-joining (NHEJ), forms stable heterodimer with re-quired cofactor Liflp, interacts with

-2.14

Nejlp; involved in meiosis, not es-sential for vegetative growth

CAGLOG07711g YKR016W A111128 Mitochondrial protein of unknown function; null mutation displays a synthetic slow growth defect in combination with a null mutation in

-2.14

GEM1, which encodes a mitochon-drial GTPase

CAGLOM02013g YPL166W ATG29 Autophagy-specific protein that is required for recruitment of other ATG proteins to the pre- autophagosomal structure (PAS); interacts with Atgl7p and localizas to the PAS in a manner interde-pendent with Atgl7p and Cis1p; not conserved

-2.14

CAGL0001397g YIL107C PFK26 6-phosphofructo-2-kinase, inhibited by phosphoenolpyruvate and sn- glycerol 3-phosphate, has negligi- ble fructose-2,6-bisphosphatase ac-tivity, transcriptional regulation in-volves protein kinase A

-2.13




CAGLOD02464g YNL325C FIG4 Phosphatidylinositol 3,5- bisphosphate (PtdIns[3,51P) phosphatase; required for efficient mating and response to osmotic shock; physically associates with and regulated by Vacl4p; contains a SAC1-like domain

-2.13

CAGLOE06006g YPL224C MMT2 Putative metal transporter involved in mitochondrial iron accumulation; closely related to Mmtlp

-2.13

CAGLOH10120g YBRO56W - Description not mapped -2.13 CAGL0101034g YOR035C SH.E.4 Protein containing a UCS (UNC- -2.13

45/CRO1/SHE4) domain, binds to myosin motor domains to regulate myosin function; involved in endo-cytosis, polarization of the actin cy-toskeleton, and asymmetric mRNA localization

CAGL0105962g YJLI46W IDS2 Protein involved in modulation of -2.13 Ime2p activity during meiosis, ap-pears to act indirectly to promote Ime2p-mediated late meiotic func-tions; found in growing cells and de-graded during sporulation

CAGL0110384g YGR138C TPO2 Polyamine transport protein specific for spermine; localizes to the plasma membrane; transcription of

-2.13

TPO2 is regulated by Haalp; mem-ber of the major facilitator super-family

CAGLOL13178g YLR077W FMP25 Putative protein of unknown function; the authentic, non-tagged protein is detected in highly purified mitochondria in high-throughput studies

-2.13

CAGLOM02387g YPL147W PXA1 Subunit of a heterodimeric peroxisomal ATP-binding cassette trans-porter complex (Pxalp-Pxa2p), required for import of long-chain fatty acids into peroxisomes; simi-larity to human adrenoleukodystro-phy transporter and ALD-related proteins

-2.13

CAGLOM11902g YAL034C FUNI9 Non-essential protein of unknown function; expression induced in re-sponse to heat stress

-2.13

CAGLOD04070g YGRO21W - Description not mapped -2.12 CAGLOD06204g YGL045W RIMS Protein of unknown function, in-

volved in the proteolytic activation of RimlOip in response to alkaline pH; has similarity to A. nidulans

-2.12

Pall"; essential for anaerobic growth CAGLOG10131g YPR191W QCR2 Subunit 2 of the ubiquinol

cytochrome-c reductase complex, which is a component of the mitochondrial inner membrane electron transport chain; phosphorylated; transcription is regulated by Hap1p, Hap2p/Hap3p, and heme

-2.12




CAGL01104631g YML007W YAP1 Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance; activated by H202 through the multistep forma-tion of disulfide bonds and transit from the cytoplasm to the nucleus; mediates resistance to cadmium

-2.12

CAGLOH05049g YLR382C NAM2 Mitochondria' leucyl-tRNA synthetase, also has a direct role in splicing of several mitochondria] group I introns; indirectly required for mitochondrial genome maintenance

-2.12

CAGLOI02904g YOR110W TFC7 One of six subunits of the RNA polymerase III transcription initia-tion factor complex (TFIIIC); part of the TauA globular domain of

-2.12

TFIIIC that binds DNA at the BoxA promoter sites of tRNA and similar genes

CAC LOL04686g YGRHOW - Description not mapped -2.12 CAGLOK01903g NORBH - Description not mapped -2.11 CAGLOK07590g YKL129C MYO3 One of two type I myosins; lo-

calizes to actin cortical patches; deletion of MY03 has little effect on growth, but myo3 myo5 dou-ble deletion causes severe defects in growth and actin cytoskeleton orga-nization

-2.11

CAGLOK10252g YOR124C UBP2 Ubiquitin-specific protease that removes ubiquitin from ubiquitinated proteins, cleaves at the C terminus of ubiquitin fusions; capable of cleaving polyubiquitin and pos-sesses isopeptidase activity

-2.11

CAGLOL03674g YJL103C GSMI Putative zinc cluster protein of unknown function; proposed to be in-volved in the regulation of energy metabolism, based on patterns of expression and sequence analysis

-2.11

CAGLOL03938g YNL115C - Description not mapped -2.11 CAGLOM01760g YOR153W PDR5 Plasma membrane ATP-binding

cassette (ABC) transporter, multidrug transporter actively reg-ulated by Pdrlp; also involved in steroid transport, cation resistance, and cellular detoxification during exponential growth

-2.11

CAGLOM09537g YBL095W - Description not mapped -2.11 CAGLOA03718g YGL206C CHC1 Clathrin heavy chain, subunit of

the major coat protein involved in intracellular protein transport and endocytosis; two heavy chains form the clathrin triskelion structural component; the light chain (CLC1) is thought to regulate function

-2.1

CAGL0004631g YJR100C AIM25 Putative protein of unknown function; non-tagged protein is detected in purified mitochondria in high-throughput studies; similar to murine NORA; YJR100C is not an essential gene

-2.1




CAGLOD02134g YMR115W MGRS Subunit of the mitochondrial (mt) i- AAA protease supercomplex, which degrades misfolded mitochondrial proteins; forms a subcomplex with

-2.1

Mgrlp that binds to substrates to facilitate proteolysis; required for growth of cells lacking mtDNA

CAGLOG04873g YLR371W ROM2 GDP/CTP exchange protein (GEP) for Rholp and Rho2p; mutations are synthetically lethal with muta-tions in roml, which also encodes a

-2.1

GEP CAGL0103652g YDL149W ATG9 Transmembrane protein involved in

forming Cvt and autophagic vesi-cles; cycles between the phagophore assembly site (PAS) and other cytosolic punctate structures, not found in autophagosomes; may be involved in membrane delivery to the PAS

-2.1

CAGLOJ07612g YNL241C ZWF1 Glucose-6-phosphate dehydrogenase (G6PD), catalyzes the first step of the pentose phosphate pathway; involved in adapting to oxidatve stress; homolog of the human G6PD which is deficient in patients with hemolytic anemia

-2.1

CAGLOK11275g YDR247W VHS1 Cytoplasmic serine/threonine protein kinase; identified as a high- copy suppressor of the synthetic lethality of a sis2 sit4 double mutant, suggesting a role in G1/S phase progression; homolog of

-2.1

Skslp CAGLOE01287g YDR148C KGD2 D ihydrolipoyl transsuccinylase,

component of the mitochondrial alpha-ketoglutarate dehydrogenase complex, which catalyzes the oxidative decarboxylation of alpha- ketoglutarate to succinyl-CoA in the TCA cycle; phosphorylated

-2.09

CAGLOE04400g YFIR011W DMA Probable mitochondrial seryl-tRNA synthetase, mutant displays increased invasive and pseudohyphal growth

-2.09

CAGLOH09812g YDROO6C SOK1 Protein whose overexpression suppresses the growth defect of mu-tants lacking protein kinase A ac-tivity; involved in cAMP-mediated signaling; localized to the nucleus; similar to the mouse testis-specific protein PBS13

-2.09

CAGLOJ00429g YHR051W COX6 Subunit VI of cytochrome c oxidase, which is the terminal member of the mitochondrial inner membrane elec-tron transport chain; expression is regulated by oxygen levels

-2.09

CAGLOL02167g YKROO9C FOX2 Multifunctional enzyme of the peroxisomal fatty acid beta-oxidation pathway; has 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hy-dratase activities

-2.09




CAGLOA01672g YGR009C SEC9 t-SNARE protein important for fusion of secretory vesicles with the plasma membrane; similar to but not functionally redundant with

-2.08

Spo20p; SNAP-25 homolog CAGL0B00946g YCL028W RNQI [PIN(+)] prion, an infectious pro-

tein conformation that is generally an ordered protein aggregate

-2.08

CAGL0104796g YB R037C SCO/ Copper-binding protein of the mitochondrial inner membrane, required for cytochrome c oxidase activity and respiration; may function to de-liver copper to cytochrome c oxi-dase; has similarity to thioredoxins

-2.08

CAGLOLI0604g YMR197C VTII Protein involved in cis-Golgi membrane traffic; v-SNARE that inter-acts with two t-SNARES, Sed5p and Pepl2p; required for multiple vacuolar sorting pathways

-2.08

CAGL01-100979g YPL236C ENV7 -2.07 CAGL0100682g YMR150C IMPI Catalytic subunit of the mitochon-

drial inner membrane peptidase complex, required for maturation of mitochondrial proteins of the inter-membrane space; complex contains

-2.07

Imp1p and Imp2p (both catalytic subunits), and Som1p

CAGLOJ06820g YPL123C RNYI RNAse; member of the T(2) family of endoribonucleases

-2.07

CAGLOL06798g YDL078C MDH3 Peroxisomal malate dehydrogenase, catalyzes interconversion of malate and oxaloacetate; involved in the glyoxylate cycle

-2.07

CAGLOM04565g YLR144C ACF2

-

Intracellular beta-1,3- endoglucanase, expression is induced during sporulation; may have a role in cortical actin cytoskeleton assembly

-2.07

CAGLOH00682g YMR196W - Description not mapped -2.06 CAGL0109.504g YBR274W CHK I Serine/threonine kinase and DNA

damage checkpoint effector, mediates cell cycle arrest via phosphorylation of Pdslp; phosphorylated by checkpoint signal trans- ducer Meclp; homolog of S. pombe and mammalian Chkl checkpoint kinase

-2.06

CAGL0,104796g YLR427W MAG2 Cytoplasmic protein of unknown function, predicted by computational analysis of interaction data to encode a DNA-3-methyladenine glycosidase II that catalyzes the hy-drolysis of alkylated DNA

-2.06

CAGLOJ11704g YMR004W MVPI Protein required for sorting proteins to the vacuole; overproduction of Mvplp suppresses several domi-nant VPS1 mutations; Mvplp and

-2.06

Vpslp act in concert to promote membrane traffic to the vacuole




CAGLOMOS492g YKL164C PH11 O-glycosylated protein required for cell wall stability; attached to the cell wall via beta-1,3-glucan; medi-ates mitochondrial translocation of

-2.06

Aprilp; expression regulated by the cell integrity pathway and by Swi5p during the cell cycle

CAGL0G06182g YHR131C - Description not mapped -2.05 CAGLOI01672g YJR036C HUL4 Protein with similarity to hect do-

main E3 ubiquitin-protein ligases, not essential for viability

-2.05

CAGLOJ09768g YDL200C MGT/ DNA repair methyltransferase (6- -2.05 O-methylguanine-DNA methylase) involved in protection against DNA alkylation damage

CAGLOM08360g YJL165C HAL5 Putative protein kinase; overexpression increases sodium and lithium tolerance, whereas gene disruption increases cation and low pH sensitivity and impairs potassium uptake, suggesting a role in regulation of Trklp and/or

-2.05

Trk2p transporters CAGLOB01859g YDR118W APC4 Subunit of the Anaphase- -2.04

Promoting Complex/Cyclosome (APC/C), which is a ubiquitin- protein ligase required for degradation of anaphase inhibitors, including mitotic cyclins, during the metaphase/anaphase transition

CAGLOD01672g YPRO55W SECS Essential 121kDa subunit of the exocyst complex (Sec3p, Sec5p, Sec6p, Sec8p, SeclOp, Secl5p, Exo70p, and Exo84p), which has the essential function of mediating polarized targeting of secretory vesicles to active sites of exocytosis

-2.04

CAGLOG03883g YLL026W HSP/04 Heat shock protein that cooperates with Ydjlp (Hsp40) and Ssalp

-2.04

(Hsp70) to refold and reactivate previously denatured, aggregated proteins; responsive to stresses in-cluding: heat, ethanol, and sodium arsenite; involved in [PSI+] propa-gation

CAGLOI08745g YGR170W PSD2 Phosphatidylserine decarboxylase of the Golgi and vacuolar mem-branes, converts phosphatidylserine to phosphatidylethanolamine

-2.04

CAGLOK12914g YFLO48C EMP47 Integral membrane component of endoplasmic reticulum-derived

-2.04

COPII-coated vesicles, which function in ER to Golgi transport

CAGL0M13343g YHR183W GND1 6-phosphogluconate dehydrogenase -2.04 (decarboxylating), catalyzes an NADPH regenerating reaction in the pentose phosphate pathway; required for growth on D-glucono- delta-lactone and adaptation to oxidative stress




CAGLOD05632g CAGLOK09482g

NORBH YOR147W

- MDM32

Description not mapped Mitochondrial inner membrane protein with similarity to Mdm31p, required for normal mitochondria' morphology and inheritance; interacts genetically with MMM1,

-2.03 -2.03

MDM10, MDM12, and MDM34 CAGLOL06512g NORI3H - Description not mapped -2.03 CAGLOF06567g YMR064W AEP1 Protein required for expression of

the mitochondrial OLD gene encod-ing subunit 9 of Fl-FO ATP syn-thase

-2.02

CAGLOG05467g YNL199C GCR2 Transcriptional activator of genes involved in glycolysis; interacts and functions with the DNA-binding protein Gcrlp

-2.02

CAGL0I00638g YLR290C - Description not mapped -2.02 CAGLOI01210g YIIR109W CTI111 Cytochrome c lysine methyltrans-

ferase, trimethylates residue 72 of apo-cytochrome c (Cyc1p) in the cytosol; not required for normal res-piratory growth

-2.02

CAGLOK02145g YER13OC COM2 Protein of unknown function -2.02 CAGLOL06160g YGL187C COX4 Subunit IV of cytochrome c oxidase,

which is the terminal member of the mitochondrial inner membrane elec-tron transport chain; N-terminal 25 residues of precursor are cleaved during mitochondria' import; phos-phorylated

-2.02

CAGLOL06292g YDR100W TVP1.5 Integral membrane protein localized to late Golgi vesicles along with the v-SNARE TIg2p

-2.02

CAGLOL08712g YPL014W - Description not mapped -2.02 CAGLOM03135g YJL084C A LY2 Protein proposed to regulate the

endocytosis of plasma membrane proteins; interacts with the cyclin

-2.02

Pcl7p and ubiquitin ligase Rsptp; phosphorylated by the cyclin-CDK complex, Pc17p-Pho85p; mRNA is cell cycle regulated, peaking in M phase

CAGLOM11682g YLI1108C - Description not mapped -2.02 CAGLOB04367g YBRO24W SCO2 Protein anchored to the mitochon-

drial inner membrane, similar to -2.01

Scolp and may have a redundant function with Scolp in delivery of copper to cytochrome c oxidase; in-teracts with Cox2p

CAGLOE05918g YPL219W PCL8 Cyclin, interacts with Pho85p cyclin-dependent kinase (Cdk) to phosphorylate and regulate glycogen synthase, also activates

-2.01

PhoS5p for Glc8p phosphorylation CAGLOG03179g YGRO97W ASK 10 Component of the RNA polymerase -2.01

II holoenzyme, phosphorylated in response to oxidative stress; has a role in destruction of Ssntp, which relieves repression of stress-response genes




CAGL0105522g

CAGLOK03377g CAGLOK11990g

YER098W

YMR102C YBRO59C

UBP9

- AKLI

Ubiquitin carboxyl-terminal hydrolase, ubiquitin-specific protease that cleaves ubiquitin-protein fusions Description not mapped Ser-Thr protein kinase, member (with Ark1p and Prklp) of the Ark kinase family; involved in endocyto-sis and actin cytoskeleton organiza-tion

-2.01

-2.01 -2.01

CAGLOL02431g YIL005W EPS1 ER protein with chaperone and co- chaperone activity, involved in re-tention of resident ER. proteins; has a role in recognizing proteins tar-geted for ER-associated degrada-tion (ERAD), member of the pro-tein disulfide isomerase family

-2.01

CAGLOL07634g YML002W - Description not mapped -2.01 CAGLOL12276g YLR090W XDJI Putative chaperone, homolog of E.

coli DnaJ, closely related to Ydjlp; the authentic, non-tagged protein is detected in highly purified mito-chondria in high-throughput stud-ies

-2.01

CAGL0104026g YOR227W HERI Protein of unknown function required for proliferation or remod-eling of the ER that is caused by overexpression of Hmg2p; may in-teract with ribosomes, based on co-purification experiments

-2

CAGLOL03267g YKR039W GAPI General amino acid permease; localization to the plasma membrane is regulated by nitrogen source

-2

CAGLOL03960g YNL116W DMA2 Protein involved in ubiquitin liga- tion; plays a role in regulating spin-dle position and orientation; func-tionally redundant with Dmalp; or-thologous to human RNF8 protein, also has sequence similarity to hu-man Chfr.

-2

CAGLOL11968g YER173W RAD24 Checkpoint protein, involved in the activation of the DNA damage and meiotic pachytene checkpoints; sub-unit of a clamp loader that loads

-2

Radl7p-Mec3p-Ddclp onto DNA; homolog of human and S. pombe Rad17 protein

CAGLOF08745g YLR327C TMAIO Protein of unknown function that associates with ribosomes

-14.24

CAGLOH04279g NORBH - Description not mapped -13.58 CAGLOH02101g YHROS7W RTC3 Protein of unknown function in-

volved in RNA metabolism; has structural similarity to SBDS, the human protein mutated in

-11.37

Shwachman-Diamond Syndrome (the yeast SBDS ortholog = SDO1); null mutation suppresses cdc13-1 temperature sensitivity




CAGLOF04895g YPR160W GPH1 Non-essential glycogen phosphory- lase required for the mobilization of glycogen, activity is regulated by cyclic AMP-mediated phospho-rylation, expression is regulated by stress-response elements and by the HOG MAP kinase pathway

-10.49

Table G.3: Over-represented GO component terms for C. glabrata transcripts induced by 10% FCS (w/v).

Process Term p , nucleolus < 1 X 10—' nuclear lumen < 1 x 10-5 cell part < 1 x 10-5 cell < 1 x 10-5 membrane-enclosed lumen < 1 x 10-5 organelle lumen < 1 X 10-5 nucleus < 1 x 10-5 nuclear part < 1 x 10-5 membrane-bound organelle < 1 x 10-5 intracellular membrane-bound organelle < 1 x 10-5 nucleolar part < 1 x 10-5 intracellular < 1 x 10-5 non-membrane-bound organelle < 1 X 10-5 intracellular non-membrane-bound organelle < 1 x 10-5 intracellular part < 1 x 10-5 intracellular organelle < 1 x 10-5 organelle < 1 x 10-5 RNA polymerase complex < 1 x 10-5 DNA-directed RNA polymerase III complex < 1 x 10-5 DNA-directed RNA polymerase I complex < 1 x 10-5 organelle part < 1 x 10-5 intracellular organelle part < 1 x 10-5 small nucleolar ribonucleoprotein complex < 1 x 10-5 preribosome < 1 x 10-5 small subunit processome < 1 X 10-5 nucleolar preribosome < 1 x 10-5

Table GA: Over-represented GO process terms for C. glabrata transcripts in-duced by 10% FCS (w/v).

Process Term p ribosome biogenesis < 1 x 10—' cytoplasm organization and biogenesis < 1 X 10-5 ribosome biogenesis and assembly < 1 x 10-5 rRNA processing < 1 x 10-5 rRNA metabolism < 1 x 10-5 RNA processing < 1 x 10-5 RNA metabolism < 1 X 10-5 35S primary transcript processing < 1 x 10-5 nucleobase, nucleoside, nucleotide and nucleic acid metabolism < 1 x 10-5 organelle organization and biogenesis < 1 x 10-5 ribosomal large subunit biogenesis < 1 x 10-5 processing of 20S pre-rRNA < 1 x 10-5 cell organization and biogenesis < 1 x 10-5 biopolymer metabolism < 1 X 10-5 cellular process < 1 x 10-5 cellular physiological process < 1 x 10-5 primary metabolism < 1 x 10-5 physiological process < 1 x 10-5 transcription from RNA polymerase I promoter < 1 X 10-5



continued from previous page Process Term p

metabolism < 1 x 10-5 tRNA modification < 1 x 10-5 cellular metabolism < 1 x 10-5 transcription from RNA polymerase III promoter < 1 x 10-5 ribosome assembly < 1 x 10-5 amino acid and derivative metabolism < 1 x 10-5 tRNA methylation < 1 X 10-5 amine biosynthesis < 1 x 10-5 nitrogen compound biosynthesis < 1 x 10-5 amino acid biosynthesis < 1 X 10-5 ribosomal large subunit assembly and maintenance < 1 X 10-5 amino acid metabolism < 1 X 10-5 tRNA metabolism < 1 x 10-5 nitrogen compound metabolism < 1 x 10-5 ribosomal subunit assembly < 1 X 10-5 processing of 27S pre-rRNA < 1 X 10-5 ribosomal large subunit export from nucleus < 1 x 10-5 macromolecule metabolism < 1 x 10-5 amine metabolism < 1 X 10-5

Table G.5: Over-represented GO function terms for C. glabrata transcripts in-duced by 10% FCS.

Function Term p snoRNA binding < 1 x 10-5 DNA-directed RNA polymerase activity < 1 X 10-5 catalytic activity < 1 X 10-5 tRNA methyltransferase activity < 1 X 10-5 RNA methyltransferase activity < 1 x 10-5 RNA binding < 1 X 10-5 transferase activity < 1 x 10-5 methyltransferase activity < 1 X 10-5 nucleotidyltransferase activity < 1 x 10-5 transferase activity, transferring one-carbon groups < 1 X 10-5 ATP-dependent RNA helicase activity < 1 X 10-5 RNA helicase activity < 1 x 10-5 RNA-dependent ATPase activity < 1 X 10-5 rRNA primary transcript binding < 1 X 10-5 S-adenosylmethionine-dependent methyltransferase activity < 1 X 10-5

Table G.6: Over-represented GO component terms for C. glabrata transcripts repressed by 10% FCS (w/v).

Process Term p cytoplasm < 1 X 10-5 cell < 1 X 10-5 cell part < 1 X 10-5 intracellular < 1 X 10-5 intracellular part < 1 X 10-5 mitochondrion < 1 X 10-5 cytoplasmic part < 1 X 10-5 mitochondrial electron transport chain < 1 x 10-5 respiratory chain complex III (sensu Eukaryota) < 1 X 10-5 respiratory chain complex III < 1 x 10-5 ubiquinol-cytochrome-c reductase complex < 1 x 10-5 protein serine/threonine phosphatase complex < 1 x 10-5

Table G.7: Over-represented GO process terms for C. glabrata transcripts re-pressed by 10% FCS (w/v).

Process Term p generation of precursor metabolites and energy < 1 X 10-5



continued from previous page Process Term P

energy derivation by oxidation of organic compounds < 1 x 10-5

carbohydrate metabolism < 1 x 10-5

cellular carbohydrate metabolism < 1 x 10-5

energy reserve metabolism < 1 x 10-5 main pathways of carbohydrate metabolism < 1 X 10-5 glycogen metabolism < 1 x 10-5

monosaccharide metabolism < 1 x 10-5 glucan metabolism < 1 X 10-5 response to stress < 1 X 10-5 hexose metabolism < 1 x 10-5

alcohol metabolism < 1 X 10-5 carbohydrate catabolism < 1 x 10-5 cellular carbohydrate catabolism < 1 X 10-5 phosphorus metabolism < 1 X 10-5 phosphate metabolism < 1 x 10-5 acetyl-CoA metabolism < 1 x 10-5 response to stimulus < 1 x 10-5 organic acid metabolism < 1 x 10-5

carboxylic acid metabolism < 1 x 10-5

glucose metabolism < 1 x 10-5 monosaccharide catabolism < 1 X 10-5 polysaccharide metabolism < 1 x 10-5

cellular polysaccharide metabolism < 1 x 10-5 glycogen biosynthesis < 1 X 10-5 aerobic respiration < 1 X 10-5 alcohol catabolism < 1 x 10-5 response to external stimulus < 1 X 10-5 response to extracellular stimulus < 1 X 10-5 response to nutrient levels < 1 X 10-5 ATP synthesis coupled electron transport < 1 X 10-5 ATP synthesis coupled electron transport (sensu Eukaryota) < 1 X 10-5 cellular respiration < 1 x 10-5

electron transport < 1 x 10-5 glucan biosynthesis < 1 x 10-5

glucose catabolism < 1 x 10-5 carbohydrate biosynthesis < 1 x 10-5 tricarboxylic acid cycle < 1 x 10-5 acetyl-CoA catabolism < 1 x 10-5

Table G.8: Over-represented GO function terms for C. glabrata transcripts re-pressed by 10% FCS.

Function Term p catalytic activity < 1 x 10-5

oxidoreductase activity < 1 x 10-5 kinase activity < 1 x 10-5 phosphotransferase activity, alcohol group as acceptor < 1 X 10-5 enzyme regulator activity < 1 x 10-5

phosphoric monoester hydrolase activity < 1 X 10-5 phosphoric ester hydrolase activity < 1 x 10-5 cysteine-type peptidase activity < 1 X 10-5

435

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