Molecular analysis of UASE, a cis element containing stress responseelements responsible for ethanol induction of the KlADH4 gene

of Kluyveromyces lactis

Cristina Mazzoni*, Francesca Santori, Michele Saliola, Claudio Falcone

Pasteur Institute-Cenci Bolognetti Foundation, Department of Cell and Developmental Biology, University of Rome ‘La Sapienza’,Piazzale A. Moro, 00185 Rome, Italy

(Submitted 24 May 1999; accepted 14 September 1999)

Abstract — KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenaseactivity, which is specifically induced by ethanol and insensitive to glucose repression. In this work, we reportthe molecular analysis of UASE, an element of the KlADH4 promoter which is essential for the induction ofKlADH4 in the presence of ethanol. UASE contains five stress response elements (STREs), which have beenfound in many genes of Saccharomyces cerevisiae involved in the response of cells to conditions of stress.Whereas KlADH4 is not responsive to stress conditions, the STREs present in UASE seem to play a key rolein the induction of the gene by ethanol, a situation that has not been observed in the related yeast S. cerevisiae.Gel retardation experiments showed that STREs in the KlADH4 promoter can bind factor(s) undernon-inducing conditions. Moreover, we observed that the RAP1 binding site present in UASE binds KlRap1p.© 2000 Éditions scientifiques et médicales Elsevier SAS

Kluyveromyces lactis / gene expression / alcohol dehydrogenase / stress response element

1. Introduction

Kluyveromyces lactis is a lactose-metabolizingyeast with a predominantly aerobic metabolism.Despite its low fermentative capability, fouralcohol dehydrogenase (ADH) genes, termedKlADH1 to KlADH4, are present in this yeast.All four genes have been cloned and theirnucleotide sequences have been deter-mined [26, 28, 31]. KlADH1 and KlADH2 encodetwo cytoplasmic activities (KlADHI and KlA-DHII), preferentially expressed in cells grown inglucose [25], and responsible for ethanol pro-duction. KlADH3 and KlADH4, instead, encodetwo activities (KlADHIII and KlADHIV), local-

ized in mitochondria [26]. The only mitochon-drial activity present in S. cerevisiae, ADHIII, ispoorly expressed and does not appear to berelevant to cellular metabolism [35]. KlADH3and KlADH4, on the other hand, are highlyexpressed, but their precise role has not beendefined because their disruption does not con-fer a clear phenotype upon the cell [20]. Studiesof the regulation of KlADH4 have shown thatthis gene is induced at transcriptional level byethanol and not by other respiratory carbonsources, and is insensitive to glucose repres-sion [20, 25].

On the contrary, the transcription of KlADH3is repressed in the presence of ethanol added tothe medium or produced during fermentationand is depressed under respiratory condi-tions [25]. These observations indicate the exist-ence of a peculiar regulatory mechanism whichresults in the mutual exclusion of the KlADHIIIand KlADHIV activities, depending on the res-piratory or fermentative metabolism.

* Correspondence and reprintsTel.: +39 06 49 91 22 57; fax: +39 06 49 91 22 56;Mazzonic@axcasp.caspur.itAbbreviations: ADH, alcohol dehydrogenase; HSE, heatshock element; STRE, stress response element.

Res. Microbiol. 151 (2000) 19−28© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reservedS092325080000131-5

In a previous work [27], we identified bydeletion analysis a small region of the KlADH4promoter (UASE), which is responsible for theinduction of the gene in the presence of ethanol.Sequence analysis of UASE using the Transfac3.5 matrices [10], showed that this region con-tains five stress response elements (STREs),which have been found in many S. cerevisiaegenes, where they play an important role in theresponse and tolerance of yeast cells to severestress [24].

This is the first report on the presence ofSTREs in a K. lactis promoter and we demon-strate that although KlADH4 is not responsiveto stress conditions, these elements are involvedin ethanol induction of the gene.

We also provide evidence that the Rap1 bind-ing site present in the KlADH4 promoter isfunctional and can bind KlRap1p present incellular extracts from K. lactis.

2. Materials and methods

2.1. Strains and culture conditions

MW179–1D (α, lac4–8, ura3, leu2, trp1, ade2–1,metA-1) (kindly provided by Dr M. Wesolowski-Louvel), which lacks endogenous â-galacto-sidase activity, was used in the â-galactosidasefusion experiments.

Cells were grown on synthetic medium (YNB0.67%, 0.2% glucose) to the exponential phase;2% ethanol was added to the cultures and theâ-galactosidase activity was measured after 1.5and 3 h.

Yeast transformants were selected for leucineprototrophy on minimal medium (SD).

CMK5 (α, thr, lys, pgi1, adh3, adh1::URA3,adh2::URA3) was the strain used in stressexperiments. This strain, which expresses onlyKlADH4, was obtained by crossing strains lack-ing single ADH activities [20].

2.2. Stress conditions

Cells were grown to the early exponentialphase on complete or selective media (YPD orSD-Leu) and exposed to different alcohols (at

the concentration of 0.5%), osmoshock (0.5 MNaCl or 2 M sorbitol) and oxidative stress(0.4 mM H2O2). In the nitrogen starvationexperiment, yeast cells were grown on YPD andthen transferred to STMD medium (0.17% yeastnitrogen base without amino acid and ammo-nium sulfate, 2% glucose and 5 mg/L of aux-otrophic requirements) [2]. In heat- and cryo-shock experiments, cultures were incubated at42 and 4 °C, respectively.

The â-galactosidase and the KlADHIV activi-ties were measured after 1 and 2 h, or 4 and 6 hof treatment, respectively.

2.3. Enzyme activity

The presence of KlADHIV in crude extractswas revealed by native gel electrophoresis [20]followed by staining the gels for ADH activ-ity [16]. Catalase activity was measured spectro-photometrically at 240 nm [1] in crude extractsfrom yeast cells broken with glass beads in abuffer containing 100 mM TrisHCl pH 8.0,1 mM dithiothreitol, 20% glycerol.

The â-galactosidase activity was measured incrude extracts prepared by breakage of yeastcells with glass beads [12].

Protein concentration was determined accord-ing to Bradford [5].

2.4. General methods

Restriction enzyme digestions, plasmid engi-neering, and other standard techniques wereperformed according to Sambrook et al. [29]. E.coli and yeast transformation was performed byelectroporation with a Biorad gene-pulser appa-ratus following the protocols supplied.

2.5. PCR amplification and cloningof KlADH4 promoter fragments

The oligonucleotides used in PCR amplifica-tions are listed in table I. PCR reactions wereperformed with the GeneAmp kit (Perkin ElmerCetus) using as template a 4.0-kbp genomicDNA region of K. lactis, which contained theKlADH4 gene. The reaction mixture (100 µL)contained 0.2 mM of each primer, 0.2 mM of the

20 Mazzoni et al.

four dNTPs, PCR buffer 1X, 25 ng of DNA,2 mM MgCl2, and 2.5 units of Ampli-Taq DNAPolymerase. PCR was carried out for 30 cyclesaccording to the following schedule: 94 °C for1’, 52 °C for 2’, 72 °C for 3’.

2.6. Construction of KlADH4 promoter-lacZfusion vectors

The region -931/-13 containing wild type ormutated STREs 5 and 4 was amplified by PCRusing oligos S1/9 and SIM/9, respectively(table I).

The region -791/-13 containing wild type ormutated STREs 3, 2 and 1 was amplified by PCRusing oligos S2/9 and S2M/9, respectively.

These EcoRI-HindIII fragments were clonedinto the polylinker of pKSII plasmid to yieldplasmids pKSII/S1, pKSII/SIM, pKSII/S2, andpKSII/S2M. The SpeI site, located at -196 in theKlADH4 promoter, was used to generate EcoRI-SpeI promoter fragments. These fragments werethen cloned into pCM19, a centromeric plasmidderivative of plasmid Kcplac13 (kindly pro-vided by Dr M. Wesolowski-Louvel) carryingthe KlADH4 promoter fused to lacZ gene, toyield plasmids pNS1, pNS2, pNS3, and pNS4,respectively.

2.7. Protein extract and bandshift experiments

Cellular extracts from MW179–1D grown ondifferent carbon sources (see text) were pre-pared by vortexing cells with glass beads in thefollowing buffer: 200 mM Tris-HCl pH 8.4,1 mM EDTA, 10% glycerol, 10 mMâ-mercaptoethanol, 10 mM MgCl2, 300 mM(NH4)2SO4. After removal of cell debris, thesupernatant was centrifuged at 100 000 g (S100)and used in binding assays.

DNA probes (table I) were end-labelled withthe Kleenow enzyme using [α-32P]-dATP andthen purified on a Sephadex G50 column.Labelled oligonucleotides (5fM) were incubatedwith 20 mg of protein extract for 20’ at 25 °C inbuffer containing 10 mM Tris-HCl pH 7.5,50 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA, 1 mgpoly(d I-dC)poly(dI-dC), 100 mM NaCl, 10%glycerol, 15 mM ZnCl. Samples were thenloaded onto a 5% polyacrylamide gel and elec-trophoresis was performed in TAE 1X buffer at4 °C for 3 h at 35 mA.

3. Results3.1. Sequence analysis of UASE

UASE is a cis element of the KlADH4 pro-moter spanning from -953 to -741, which confers

Table I. Nucleotide sequence of the oligonucleotides used in PCR amplification of different regions of the KlADH4 promoter.Chromosomal sequences of K. lactis are in capital letters. S1 and S2 carry the wild-type STREs (boxed). Nucleotides mutagenized inSTREs are indicated in lower case (S1M and S2M). In bandshift experiments, double-strand oligonucleotides S1, S1M, S2 and S2M wereused. The sequence of oligonucleotides used in competition experiments for RAP1 and YAP1 binding sites are also shown.

Stress response elements and ethanol induction in K. lactis 21

ethanol-dependent activation of transcriptionwhen inserted into the promoters of heterolo-gous genes [27].

The sequence analysis of UASE (figure 1)revealed the presence of five repeats, three withthe same and two with the opposite orienta-tions, which perfectly matched the core consen-sus sequences (AGGGG or CCCCT) of theSTREs.

These elements have been found in the pro-moters of many S. cerevisiae genes, where theyare present in two or more copies with bothorientations. STREs have been shown to have apositive effect on transcription during theresponse to different stresses such as nitrogenstarvation, osmotic and oxidative shock, lowexternal pH, and high ethanol concentrations [2,13, 30, 33].

On the other hand, STREs are negativelyregulated by protein kinase A [2, 30].

Between positions -906/-900 of UASE, a puta-tive binding site for Yap1p is present, a tran-scription factor involved in stress response in S.cerevisiae and K. lactis [4, 14].

As shown in figure 1, UASE also containedtwo putative heat shock elements (HSEs). Theseelements, which have been found in combina-tion with STREs in many stress-responsivegenes of S. cerevisiae, are involved in heat shockresponse [17, 24].

Between positions -917 and -905 of the pro-moter, we also found a sequence which per-fectly matched the consensus sequence (RAAY-CCRYNCAYY) of the binding site of the Rap1protein [3]. RAP1 (repressor activator protein 1)is an essential gene, present in both S. cerevisiaeand K. lactis, which acts positively or negativelyon the expression of many genes [15, 32]. RAP1binding sites (UASRPG) have been found inmany promoters of glycolytic genes where theyplay a positive role in transcription [6, 7, 21, 23].

3.2. KlADH4 is not a stress-responsive gene

We sought to verify whether the KlADH4gene, as a result of the presence of five STREs inits promoter, could be expressed under stressconditions.

Therefore, cells were grown to the early expo-nential phase on rich medium (YPD) and thenexposed to osmotic, oxidative and temperatureshocks (for details see Materials and methods).

After 4 and 6 h of treatment, the presence ofthe KlADHIV activity in crude cell extracts wasanalysed by electrophoresis on native gels. Wealso checked for the presence of KlADHIV after4, 6, and 16 h in cells subjected to nitrogenstarvation. As reported in table II, the activitycould not be detected under all the stress con-ditions tested. To verify whether the lack ofactivity was due to the absence of transcription

Figure 1. Nucleotide sequences of the KlADH4 promoter region responsive to ethanol induction (UASE). The five STREs (shadowedboxes), the putative HSEs, the Rap1, and the Yap1 binding sites are also indicated. Nucleotides matching with the HSE consensussequence are underlined.

22 Mazzoni et al.

of the KlADH4 gene, we fused the promoterregion to the lacZ gene (plasmid pNS1; seeMaterials and methods) and followed the pro-duction of â-galactosidase in cells exposed toethanol, and osmotic and oxidative stress. Ascan be seen in figure 2, the â-galactosidase wasproduced only after the addition of ethanol, andits level increased during the 2-h period. On thecontrary, no activity could be detected in thesame period in cells exposed to stress, indicat-ing that the KlADH4 promoter was not acti-vated under these conditions.

We also verified whether other alcoholsbesides ethanol could activate KlADH4.

Cells exposed for 4 h to different alcohols(table II) showed amounts of KlADHIV similarto those observed after ethanol treatment, withthe exception of methanol which induced theactivity at a lower level.

Almost nothing is known about the stressresponse in K. lactis. For this reason, we per-formed preliminary experiments concerning theinduction of catalase and found that this activ-ity, which in S. cerevisiae is considered to be amarker of the response to different stress condi-tions [30], can be induced in K. lactis cells duringosmostress, stationary phase and, to a lesserextent, by high ethanol concentrations (figure 3).

3.3. STREs are involved in the ethanol response

To verify whether the STREs present in theKlADH4 promoter were involved in ethanolinduction, we constructed a series of centro-

meric plasmids carrying the lacZ gene under thecontrol of regions of the KlADH4 promotercontaining wild type and mutagenized STREs(figure 4).

Table II. Effects of different stress conditions and different alco-hols on the induction of KlADHIV activity in K. lactis strain CMK5.Plus sign indicates levels of KlADHIV similar to those observedafter ethanol induction. Minus sign indicates undetectable levels.

Induction of KIADH4

Stress conditions Alcohols

0.5 M NaCl – 0.5% methanol +/–2 M Sorbitol – 0.5% ethanol +Heat shock (42 °C) – 0.5% propanol +Cro shock (4 °C) – 0.5% isopropanol +0.4 mM H2O2 – 0.5% butanol +Nitrogenstarvation

–

Stationary phase –

Figure 2. Kinetics of â-galactosidase production in cells exposedto ethanol, osmotic and oxidative stress; 2% ethanol, 0.5 M NaCl,and 0.4 mM H2O2 were added to cultures pregrown on syntheticmedium containing glycerol as the carbon source. Standard devia-tion is also shown.

Figure 3. Induction of catalase under stress conditions. CMK5cells were grown in YPD to the exponential phase. Ethanol andNaCl were added at the indicated concentrations and catalaseactivity was measured in crude extracts after 1 h (see Materialsand methods). Catalase was also measured in stationary phasegrown cells.

Stress response elements and ethanol induction in K. lactis 23

All constructs were transformed intoMW179–1D, a â-galactosidase-deficient strain,and transformant clones were grown in thepresence of low glucose concentration (0.2%) toavoid ethanol production by fermentation.

Since KlADHIV activity is induced by ethanolwithin a few hours [20], we measured theâ-galactosidase activity 90 and 180 min after theaddition of ethanol to the medium.

In these experiments, the control plasmid waspNS1 which contained the first 931 nucleotidesof the promoter region and allowed full induc-tion of the â-galactosidase gene in the presenceof ethanol. As can be seen in figure 4, the addi-tion of ethanol clearly induced the productionof â-galactosidase within 3 h in cells carryingpNS1.

Next, we mutagenized the two Gs present inSTREs (see oligonucleotides in table I), whichhave been shown to be important for function inS. cerevisiae [18].

After mutagenesis of STREs 4 and 5 (plasmidpNS2), we still observed induction ofâ-galactosidase by ethanol but the activity levelafter 3 h was 70% of the control plasmid. The

results obtained with plasmid pNS3, in whichSTREs 4 and 5 were completely deleted andonly STREs 1, 2, and 3 were present, showedthat induction of â-galactosidase still occurred,but the level of the activity was further reducedto 35% of the control after 3 h. Moreover,mutagenesis and deletion of STREs 4 and 5resulted in a delay in the induction process, asindicated by the low levels of â-galactosidasemeasured after 90 min.

Finally, we mutagenized STREs 1, 2, and 3present in pNS3 and obtained plasmid pNS4.When ethanol was added to cells carrying thisplasmid, the level of â-galactosidase activity attime zero was very similar to that observed withpNS3 after ethanol induction and remainedconstant for 3 h, indicating that inactivation ofSTREs 1, 2, and 3 resulted in constitutive activ-ity of the promoter.

3.4. Gel shift assay with protein extractsfrom K. lactis

Protein extracts from strain MW179–1D wereused in bandshift experiments with differentDNA probes from the UASE region (table I).

Figure 4. Effect of STRE inactivation on expression of the lacZ gene. Cells were grown overnight on synthetic medium containing 0.2%glucose and the â-galactosidase activity was measured 90 and 180 min after the addition of 2% ethanol to the cultures. Wild-type andmutagenized STREs in the KlADH4 promoter are indicated by circles and stars, respectively. Standard deviation is also indicated. aMeanfor three independent transformants.

24 Mazzoni et al.

As can be seen in figure 5, using probe S2containing STREs 1, 2, and 3, we could notobserve any complex with the protein extractfrom ethanol-grown cells (lane 2), while twobands were detected with the extracts fromglycerol-grown cells (lanes 6).

These two bands were specifically competedfor by the addition of the unlabelled S2 oligo-nucleotide (lanes 7 and 8), while oligonucle-otide S2M, which carried the mutagenizedSTREs, had no effect (lanes 9), indicating thatthese elements are directly involved in theobserved binding.

We also performed gel shift experimentsusing oligonucleotide S1 with protein extractsfrom cells grown on glucose (figure 6). In thiscase, we observed a single band (lane 2), whichwas competed for by an excess of either theunlabelled oligonucleotide S1 or S1M, contain-ing the wild-type or mutated STREs 4 and 5,respectively (lanes 3 and 4), and demonstratedthat the observed complex was not due toSTREs. Similar results were obtained with pro-tein extracts from cells grown on glycerol andethanol (not shown).

As shown in table I, in both oligonucleotidesS1 and S1M, the consensus sequences for thetranscription factors Rap1p and Yap1p werepresent. For this reason, in competition experi-ments, we used an oligonucleotide containingthe consensus sequence for RAP1 derived fromthe yeast Kluyveromyces marxianus, which canalso bind KlRap1p [3, 11], and an oligonucle-otide containing the putative Yap1p bindingsite.

As can be seen in figure 6, the RAP1 oligo-nucleotide efficiently competed for the forma-tion of the complex (lane 5), while the YAP1oligonucleotide had no effect (lane 6).

In addition, when the Rap1 sequence wasused as a probe in binding assays, we observedthe formation of a complex which could becompeted for by an excess of the S1 oligonucle-otide (not shown).

These results indicated that KlRap1p can bindto its recognition site present in UASE.

Figure 5. STRE binding with extracts from uninduced andethanol-induced K. lactis cells. The labelled double-strand oligo-nucleotide S2 was used in electrophoretic mobility shift assaywith protein extracts from the MW179–1D strain of K. lactisgrown on rich medium containing 2% ethanol (lanes 2–5) or 3%glycerol (lanes 6–9). An excess of unlabelled double-strand oligo-nucleotides at increasing concentrations, as indicated in thefigure, were used in competition experiments. Lane 1: free probe. Figure 6. KlRap1p binds to the KlADH4 promoter. The labelled

double-strand oligonucleotide S1 was used in electrophoreticmobility shift assay with protein extracts from the MW 179–1Dstrain of K. lactis grown on YPD. A 50x molar excess of theindicated unlabelled double-strand oligonucleotide was used ascompetitor (lanes 3–6). Lane 1: free probe.

Stress response elements and ethanol induction in K. lactis 25

4. Discussion

KlADH4 is an alcohol dehydrogenase genepeculiar to K. lactis in that it is specificallyinduced by ethanol and insensitive to glucoserepression.

We previously identified in the promoter anethanol-responsive element (UASE), which con-tains five STREs [13, 18].

Despite the presence of the STREs, KlADH4 isnot a general stress-response gene. In fact, wedemonstrated that the gene is not induced aftercell exposure to conditions that activate com-mon sets of stress genes in S. cerevisiae. More-over, in the latter yeast, 7.5% ethanol is neededto induce stress response [30], while the concen-tration of ethanol sufficient to induce KlADH4 isas low as 0.1% [25]. We also demonstrated thatother alcohols such as methanol, butanol, pro-panol, and isopropanol in low concentrationscan act as inducers of KlADH4, and it is notknown whether they might also be physiologi-cal substrates for KlADHIV activity.

The mutagenesis of STREs 4 and 5 resulted inthe loss of 30% of the â-galactosidase activitywhile the deletion of the same STREs resulted ina further reduction of up to 65%. This differencecould be due to the deletion of other sequencespresent in the promoter besides STREs. Forinstance, between STREs 4 and 5 is located aUASRPG-box, a binding site for the Rap1 pro-tein, which has been found to have a positiverole toward the promoters of many glycolyticgenes [6, 7, 21, 23]. This also seems to be the casein K. lactis, since the RAP1 site present in theKlADH4 promoter can bind a factor that isspecifically competed for by oligonucleotidescarrying the RAP1 consensus site.

Surprisingly, the absence of functional STREsresulted in constitutive activity of the KlADH4promoter. One possible explanation is that theinactivation of STREs eliminates a negative con-trol transforming the KlADH4 regulated pro-moter into a constitutive one. Although addi-tional experiments are needed to clarify the roleof each of the STREs present in UASE, our

results indicate that these elements are clearlyinvolved in the induction of KlADH4 by etha-nol.

We could not detect any protein bound toSTREs in gel retardation experiments with pro-tein extracts from K. lactis cells grown on etha-nol, i.e. under conditions inducing KlADH4,while two bands were observed with extractsfrom cells nonexpressing KlADH4.

These results, together with those obtainedwith the mutagenesis of STREs, support thehypothesis that negative factors bind STREsunder noninducing conditions and that theaddition of ethanol removes these factors andfacilitates the access of the transcriptionalmachinery to the promoter. In S. cerevisiae, it hasbeen reported that two transcriptional activa-tors, Msn2p and Msn4p, are required for highexpression levels of most of the genes activatedvia STREs [8, 9, 19, 24].

In this yeast, STREs, in combination withother known (i.e. HSE) and unknown cis ele-ments, modulate the activation of the STRE-containing promoter, and some of them do notrequire Msn2p or Msn4p for their function-ing [22, 34].

In the case of KlADH4, the regulatory factorscould be different from Msn2p and Msn4p,since the role of STREs in the context of theKlADH4 promoter seems limited to theresponse to ethanol.

At present, due to the limited number ofgenomic sequences available, nothing is knownabout the presence of STREs in K. lactis promot-ers.

More information on this subject will benecessary to determine whether ethanol regu-lates the expression of other genes throughSTREs.

Résumé — Analyse moléculaire de l’élémentUASE, un élément cis contenant les élémentsSTREs responsables de l’induction par l’éthanol dugène KlADH4 de Kluyveromyces lactis. Le gèneKlADH4 de K. lactis code une activité alcool-déshydrogénase mitochondriale spécifiquementinduite par l’éthanol et insensible à la répression duglucose. Nous rapportons ici l’analyse moléculaire

26 Mazzoni et al.

de l’élément UASE du promoteur KlADH4, qui estessentiel dans l’induction de KlADH4 en présenced’éthanol. Cet élément UASE contient cinq élémentsde réponse aux stress (STREs) trouvés chez de nom-breux gènes de Saccharomyces cerevisiae impliquésdans la réponse cellulaire en situation de stress.Alors que KlADH4 reste insensible aux stress, leséléments STREs présent dans l’UASE semblent jouerun rôle clé dans l’induction du gène par l’éthanol,situation non observée chez la levure apparentée, S.cerevisiae. Des expériences de retardation en gel ontmontré que les STREs du promoteur KlADH4 peu-vent lier des facteurs dans des conditions de non-induction. De plus, on observe que le site de liaisondu RAP1 présent dans l’élément UASE lie KlRap1p.© 2000 Éditions scientifiques et médicales ElsevierSAS

Kluyveromyces lactis / expression du gène /alcool-déshydrogénase / élément STRE

Acknowledgments

We would like to thank Dr M. Wesolowski-Louvel for providing strain MW179–1D and F.Castelli for technical assistance. Dr CristinaMazzoni was supported by a fellowship fromthe MURST (Ministero della Ricerca Scientifica eTecnologica – Progetti di Ateneo). This workwas supported by EC contract BIO4-CT96–0003,partially by MURST (Ministero della RicercaScientifica e Tecnologica – Progetti di Ateneo)and the CNR Target Project on Biotechnologycontract n° 97.01162.49. This work has beencarried out in compliance with current Italianlaws governing genetics experimentation.

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