80-6abstracts 425..498 - J-Stage

S-13-5

SUGAWARA, Hideaki1 (1Center for Inform. Biol. &DNA Data Bank of Japan, Natl. Inst. Genet.)

Are Barcodes of Life retrievable from the InternationalNucleotide Sequence Databases (INSD) of DDBJ, EMBLand GenBank

DNA Data Bank of Japan (DDBJ) at the National Institute ofGenetics develops and diffuses the International NucleotideSequence Database (INSD) by collaborating with EMBL ofEuropean Bioinformatics Institute and GenBank of NationalCenter for Biotechnology Information. INSD has accumulatednucleotide sequences and their annotation obtained by diverseviewpoints such as analysis of genes and genomes. The size of thedatabase was over 400 million entries and 40 giga nucleotides.It is a central value of INSD that users are able to carry outcomprehensive retrieval and analysis of sequence data andannotation independent of the intention of the data submitters,biological species and types of sequences in INSD. Therefore, theConsortium of Barcode of Life (CBOL) and INSD and also thethree centers of INSD have discussed about the inclusion of theCBOL data into INSD. For example. DDBJ, EMBL and GenBankactually discussed about the standard data format to accommo-date the CBOL data at the INSD International CollaboratorsMeeting at Mishima in May, 2005. Therefore, the COBL data willbe retrievable from INSD in the same way as other entries inINSD in the near future.(The presentation will be in Japanese)

1A-01

WATANABE, Yutaka1, TATSUTA, Haruki1,2,TAKANO-SHIMIZU, Toshiyuki1,3 (1Div. Popul. Genet.,Natl. Inst. Genet., Research Org. Inform. and Systems,2Ecological Risk Assessment Section, Res. Cent. Env.Risk, Natl. Inst. Environmental Studies, 3Dept.Biosystems Science, Sch. Adv. Sci., The Grad. Univ.Adv. Stud. (Sokendai))

Molecular variation in 150-kb region around the DrosophilaScr gene in D. melanogaster and D. simulans

The HOX genes are well known to determine segmentalidentities, but may also be involved more directly in formationof morphological structures. Through QTL analyses for sex-combtooth-number variation within D. simulans, we identified severalsignificant regions in the genome. A candidate region is theAntennapedia complex (ANTC), where the Scr gene is located. Inspite of many functional and comparative studies, relatively littleis known about the regulatory mechanism and sequences of theHOX genes. Toward identifying QTN for the sex-comb tooth-number variation, we have taken the first step, surveyingmolecular variation in about 150-kb region surrounding the Scrgene in both D. melanogaster and D. simulans. The ANTP clusteris included in the large inversion between D. melanogaster and D.simulans sequences. We will report the data, particularly on themagnitude and pattern of linkage disequilibrium, which isimportant for determining the resolution of association study.

1A-02

TERANISHI, Chika1, YOSHIDA, Kentarou1,MIYASHITA, Naohiko1 (1Lab. Plant Genet., Grad.Sch. Agr., Kyoto Univ.)

Analysis of DNA polymorphism at the MADS-box gene(SUPERWOMAN1) locus of the wild rice Oryza rufipogon

To clarify the levels of polymorphism in SUPERWOMAN1(SPW1) gene of the wild rice (O. rufipogon), nucleotide variationin the SPW1 region was analyzed. SPW1 is one of the B-class-MADS-box genes controlling development of petals and stamens,and homologous to APETALA3 (AP3) in Arabidopsis thaliana.The estimated nucleotide diversity was 0.0031, which is similarto the values of other genes of O. rufipogon. However, novariation was detected in the MADS-box domain. This resultsuggests that the low level of variation in the MADS-box domainis related to the functional importance as a DNA-binding domain.The deviation from the neutrality was not detected. We willreport the results of MK test and HKA test comparing divergencebetween O. rufipogon and O. barthii, O. meridionalis and O.australiensis.

Genes Genet. Syst. (2005) 80444

1A-03

KADO, Tomoyuki1, MATSUMOTO, Asako1, UJINO-IHARA, Tokuko1, TSUMURA, Yoshihiko1 (1ForestryForest Products Res. Inst.)

Nucleotide variation at ten loci in two Japanese conifers, sugi(Cryptomeria japonica) and hinoki (Chamaecyparis obtusa)in Cupressaceae sensu lato.

Sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa)are the most important timber species in Japan. We surveyednucleotide variation at ten EST based nuclear loci, to understandthe mechanisms of molecular evolution in these conifers and toevaluate the details of genetic variation within and between thesespecies for the practical applications in the field of forestry.Lengths of genes analyzed in this study are 410 to 948bp. Wecollected seed samples from wide ranges of their distributions andextracted the haploid DNA from megagametophyte of 18 seeds.Average values of nucleotide diversity at silent site in sugi andhinoki are 0.004 and 0.007, respectively. No recombination eventwas detected across loci in either species. Levels of divergencebetween two species at synonymous sites of ten genes range from0.15 to 0.38 (0.28 on average). These values positively correlatewith G+C contents at third codon positions synonymous sites(GC3s). Deviation from standard neutral model was found atchitinase locus in hinoki, by tests of neutralities, such as Tajima’sD and Fay and Wu’s H.

1A-04

YUKUHIRO, Kennji1, KOMOTO, Natuo1, KOSEGAWA,Eiichi2, HIROKAWA, Masahiko2, TATEMATSU,Kenichiro2 (1Insect Molec. Evolution Lab., InsectEvolution and Genet. Dept., Natl. Inst. AgroBiol. Sci.,2Insect Genet. Lboratory, Insect Evolution and Genet.Dept., Natl. Inst. AgroBiol. Sci.)

Genetic diversity of molybdenum cofactor sulfurase gene (og)and mitochondrial cox 1 gene within the domesticatedsilkmoth, Bombyx mori

Recent advance of genomic technology has given us large amountof information that was unexpected. For example, degrees ofgenetic diversity within domesticated species tend to be largerthan those expected and as much as those of wild relatives. Thedomesticated silkmoth Bombyx mori is an insect that was highlydomesticated to be impossible to survive without human help.The domestication of B. mori is believed to have occurred inChina and the progenitor of B. mori is likely B. mandarina livedin China. Despite nearly completed of B. mori genome sequenc-ing, we know limited amount of information for molecular aspectof the domestication of B. mori. We studied molecular level ofpolymorphism in B. morimolybdenum cofactor sulfurase gene (og)whose mutants cause translucent skin because of lack ordeficiency of accumulation of uric acid under the skin. Weanalyzed regional races of B. mori and found five highlydiversified haplotypes. This result is highly contrasted to thatof mitochondrial cox 1 gene wherein low level of sequencediversity was confirmed.

1A-05

ISHIBASHI, Minaka1, TAKAHASHI, Aya1,2,TAKANO, Toshiyuki1,2, YOSHIURA, Koh-ichiro3,NIIKAWA, Norio3, SAITOU, Naruya1,2 (1Dept. Genet.,Sch. Life Sci., Grad. Univ. Adv. Stud., 2Div. Popul.Genet., Natl. Inst. Genet., 3Dept. Human Genet.,Grad. Sch. Biomedical Sciencs, Nagasaki Univ.)

Haplotype analysis of cerumen gene neighboring region

There are two types of cerumen (ear wax), wet and dry types. Thecerumen phenotype can be easily identified. Dry type allelefrequencies are higher in East Asia (e.g., about 90% of Japaneseare dry type) than other regions. Cerumen phenotypes arecontrolled by single locus at human chromosome 16p11.2-q12.1,and wet type is dominant. We obtained 30 SNPs distributing onabout 700kb region neighboring cerumen locus for 100 Japaneseindividuals. To reconstruct the evolutionary history of thecerumen locus region, we estimated haplotypes of these individ-uals using PHASE, and constructed haplotype networks. We alsoselected some sets of SNPs and compared some networks.Phylogenetic networks are useful to recognize recombinationevents. We also estimated ancestral haplotypes of this region bydetermining chimpanzee sequences.

1A-06

KIM, Hielim1, SATTA, Yoko1, TAKAHATA, Naoyuki1

(1Dept. Biosyst. Sci., The Grad. Univ. Adv. Stud.(Sokendai))

Evolutionary Genet. of human mental activity: evolution ofgenes associated to lipid storage diseases that cause mentalretardation

We are interested in genetic bases of human specific mentalactivity. To understand the bases from the evolutionary view-point, we carried out molecular population genetic analysis ofhuman mental retardation related genes. Because many of lipidstorage diseases (LSD) often accompany mental retardation, wefocused on nine human LSD associated genes and compared themwith chimpanzee orthologs. Among the nine, particularly inASAH1 that encodes N-acylsphingosine amidohydrolase, weobserved a strongly conserved pattern of nonsynonymousnucleotide substitutions. In contrast, an excess of nonsynon-ymous substitutions over synonymous ones in human populationSNP data is observed. Furthermore, a single major (~70% in thefrequency) ASAH1 haplotype of ~8kb length and a strong linkagedisequilibrium block encompassing the region are found. Thisobservation suggests recent operation of positive selection atASAH1 in the human population. To elucidate the history ofnatural selection at the gene in more detail, we are sequencingthe ASAH1 region for a worldwide human sample. We report theage and divergence of haplotypes and argue for natural selectionat ASAH1.

Genes Genet. Syst. (2005) 80 445

1A-07

YAMAGUCHI-KABATA, Yumi1, HAYAKAWA,Yousuke2,3, MINOSHIMA, Shinsei4, CHAKRABORTY,Ranajit5, IMANISHI, Tadashi1, GOJOBORI,Takashi1,6, CONSORTIUM, H-invitational1 (1Biol.Inform. Res. Cent., Natl. Inst. Adv. Industr. Sci. Tech.,2Hitachi Software Eng. Co. Ltd, 3Japan Biol. Inform.Consortium, Japan Biol. Inform. Research Center,4Photon Med. Research Center, Hamamatsu Univ.Sch. Med., 5Center for Genome Inform., Dept. Env.Health, Univ. Cincinnati, 6CIB/DDBJ, Natl. Inst.Genet.)

Large-scale analysis of human SNPs with possible effects ongene function

Using dbSNP and the gene structure of more than 21,000 humangenes that were revealed in the annotation project of human full-length cDNAs (H-Invitational), we examined the possible effectsof genetic polymorphisms on gene function. We classified SNPson human genes according to their effects on ORF, and identifiedsynonymous SNP, nonsynonymous SNP, and nonsense SNPscausing premature termination codons. As a result, we detected25,422 nonsynonymous SNPs and 682 nonsense SNPs. Theeffects of nonsense SNPs would be serious if there are no othergenes that can compensate the gene function. We thus examinedthe frequency of nonsynonymous SNPs and nonsense SNPs ongenes with or without duplicate genes. The numbers of SNPsaffecting ORF were about two-fold larger in genes with duplicatescompared with those without duplicate genes (singleton genes).This supports the idea that duplicate genes contribute to thegenetic robustness against mutations. The effects of ORF changefor singleton genes may be more serious, as far as any other genethat can compensate its function does not exist.

1A-08

INOMATA, Nobuyuki1, ITOH, Masanobu2, KONDO,Rumi3, OHSHIMA, Miki3, INOUE, Yutaka4, TAKANO-SHIMIZU, Toshiyuki5 (1Dept. Biol., Grad. Sch. Sci.s,Kyushu Univ., 2Dept. Appl. Biol., Faclt. Textile Sci.s,Kyoto Inst. Tech., 3Dept. Biol., Ochanomizu Univ.,4Dept. Internatl. Stud., Faclt. Foreign Stud., OsakaUniv. Foreign Stud., 5Dept. Popul. Genet., Natl. Inst.Genet.)

Detecting single-generation selection acting on inversionpolymorphism in Drosophila

Much effort has been made to search signatures of past naturalselection in DNA sequences, but currently acting selection israrely detected in extant populations because of uncommonnessor low detection power of available methods or both. Inversionpolymorphisms in Drosophila have long been thought to be underselection because of latitudinal clines in frequency. Here, wedeveloped a new method and applyed the method to In(2L)tinversion polymorphism in a natural population of Drosophilamelanogaster. Our results suggest that strong selection iscurrently acting on In(2L)t polymorphism in the investigatedpopulation.

1A-09

ITOH, Masanobu1,2, NANBA, Noriko1, HASEGAWA,Masako1, INOMATA, Nobuyuki3, KONDO, Rumi4,OSHIMA, Miki4, TAKANO-SHIMIZU, Toshiyuki5,6

(1Dept. Appl. Biol., Faclt. Textile Sci.s, Kyoto Inst.Tech., 2Insect Biomedical Research Center, Kyoto Inst.Tech., 3Dept. Biol., Grad. Sch. Sci.s, Kyushu Univ.,4Dept. Biol., Ochanomizu Univ., 5Dept. Popul. Genet.,Natl. Inst. Genet., 6Dept. Biosyst. Sci., Grad. Univ.Adv. Stud. (SOKENDAI))

Seasonal changes in the scale of long-distance linkagedisequilibrium and population structure in Drosophilamelanogaster

Linkage disequilibrium (LD) is a good indicator of demographichistory of a population and possible natural selection. We studiedthe amount and pattern of long-distance LD in the Drosophilamelanogaster genome in two seasonal fly samples (autumn andspring) from Kyoto, and a fly sample collected in 108 localities inand around Japan (entire Japan). LDs were measured between51 polymorphisms in 50 X-linked genes distributed throughoutthe chromosome, which included 11 chemoreceptor (Cr) genes.There was no difference in the amount of LD (average r2) betweenthe fly sample from the entire Japan and samples from the localpopulations. On the other hand, the average r2 for all gene pairswas slightly larger in the spring sample than in the autumnsample, but the difference was not statistically significant afterexcluding Cr gene pairs. Indeed, we found a significant increaseof the amount of LDs between the Cr genes in the spring samplewhen compared with the autumn and other samples. Environ-mental selective pressure may play an important role in shapingLD between this class of genes.

1A-10

ISHII, Kazushige1, MATSUDA, Hirotsugu2 (1Div. Biol.Sci., Grad. Sch. Sci., Nagoya Univ., 2Kyushu Univ.(Emeritus Professor))

Size effect and fluctuation effect on the genetic load – ParityModel –

When Kimura proposed the neutral theory of molecular evolutionin 1968,his main theoretical ground was that in order to explainthe then estimated large molecular evolution rate by a selectionmodel, a too large substitution load seemed to have been imposedon the population. The genetic load is a measure to study theevolutionary condition dependence of the population fitness, andit is judged that if its stationary value is too large a long termpersistence is difficult for the population, which makes thestudied condition inappropriate to explain the real evolution. Inthis presentation we adopt the Parity model as a simple modelthat incorporates selection, mutation, finite size and environmentfluctuation, and make a comprehensive study of its stationarygenetic load based on a power series solution of its gene frequencydistribution density. As generally useful concepts we propose thesize load and the fluctuation load, and we also clarify theusefulness and the limitation of classical concepts of the mutationload and the substitution load.


1A-11

MANO, Shuhei1 (1Grad. Sch. Natural Sci., NagoyaCity Univ.)

Evolution of linkage disequilibrium of founders in growingpopulations

Evolution of linkage disequilibrium which has already existed inthe founder population of exponentially growing populations wasconsidered by using a diffusion model. The ordinary differentialequation that the moments obey was obtained. It can be solvednumerically. In addition, a perturbative solution was obtainedwhen the growing rate is small enough. By using the perturba-tion, an asymptotic formula for the squared standard linkagedeviation after large number of generations was obtained; thesquared standard linkage deviation tends to 1/(4Nc) asymptoti-cally, where N is the current size of the population and c is therecombination fraction. According to the asymptotic formula,strength of linkage disequilibrium depends on neither of theeffective size of the founder population and the growing rate.

1A-12

NAWA, Nobukazu1, TAJIMA, Fumio2 (1OsakaShijonawate High School, 2Dept. Biol. Sci.s, Grad. Sch.Sci., The Univ. Tokyo)

Pattern of DNA polymorphism after population bottleneck

It is known that a population bottleneck can reduce the amount ofDNA polymorphism. In this study, we have simulated thecoalescent process of sampled DNA sequences under severalpopulation bottleneck models, and investigated DNA polymor-phism patterns on them. In some cases an excess of raresegregating sites compared with a constant size model wasobserved, and the frequency spectrum of qi (=4Nm) which isestimated from the number of segregating sites(Si) with eachfrequency(i) became U-shape. In these cases Tajima’s D wasclearly negative. On the other hand, in some cases an interestingW-shaped frequency spectrum was observed, and the D value wasnearly 0. W-shaped distribution might reflect population sizesbefore and on the way and after the bottleneck.

1A-13

NISHINO, Jo1, TAJIMA, Fumio1 (1Dept. Biol. Sci.s,Grad. Sch. Sci., Univ. Tokyo)

On statistic for symmetry of genealogical shape under theneutral model

The shape of gene genealogy in a population is affected by thevarious factors (e.g., selection, migration, population structure).However, even the shape of gene genealogy in the standardneutral model is not sufficiently investigated. Here, assuming thestandard neutral model and n samples, the theoretical expressionfor the variance of the statistic (Sackin’s statistic, g), whichsummarize the shape of tree and measures tree symmetry, isobtained using coalescent theory. Then, the normalized g, G, byusing the expectation and variance of g is investigated. For n ≥

30, the distributions of G are almost identical. In addition, thecorrelation between G and Tajima’s D, and the values of Gestimated from the hypothetical molecular data by some methodsof phylogenetic reconstruction are investigated.

1A-14

NOTOHARA, Morihiro1, UMEDA, Takayoshi1 (1Grad.Sch. Natural Sci., Nagoya City Univ., 2Grad. Sch.Natural Sci., Nagoya City Univ.)

Post-data Distribution of Coalescence Time

There are many theoretical studies on the inference ofthecoalescence time of sampled DNA sequences from thenumber ofsegregating sites detected in these sequences.In a panmicticpopulation, We report the exact solution of the post datadistribution of the coalescence time(T) given the number ofsegregating sites(S) among sampled DNA sequences can berepresented by the weighted mean of the convolution of gammadistributions. And we also report the analytical results andtheresults by computer simulation on the post-data distributionof Tin the geographically structured population such as islandmodel and stepping stone model.


2A-01

TAKAHASHI, Aya1, TAKANO, Toshiyuki1 (1Div.Popul. Genet., Natl. Inst. Genet.)

A high-frequency null mutant of an odorant-binding proteingene, Obp57e, in Drosophila melanogaster

We have found a null mutant of an odorant-binding protein,Obp57e, in Drosophila melanogaster. This frameshift mutation,which is a 10-bp deletion in the coding region, is at a highfrequency in the Kyoto population and is also present in Taiwanand Africa. The analyses of the surrounding DNA region wereconducted in order to investigate if there was any pattern thatdeviated from neutrality. We sequenced a 1.5-kb region includingthe tandemly duplicated gene, Obp57d, from 16 inbred linessampled in Kyoto, Japan. The analyses showed a peak ofnucleotide diversity and strong linkage disequilibrium aroundthis mutation. This pattern suggests an elevated mutation rate oran influence of balancing selection in this region. The level ofnucleotide divergence between D. melanogaster and D. simulansdoes not support the former possibility. Thus, this presence/absence polymorphism may be due to balancing selection betweenwild-type and null alleles. In addition, the Obp57d gene regionshowed an excess of high-frequency derived mutants that isconsistent with a pattern predicted under positive naturalselection.

2A-02

MIYO, Takahiro1, CHARLESWORTH, Brian1,OGUMA, Yuzuru2 (1Inst. Evolutionary Biol., Schoolof Biol. Sci.s, Univ. Edinburgh, 2Inst. Biol. Sci.s, Univ.Tsukuba)

Genetic variation in susceptibility to organophosphate in-secticides within a natural population of Drosophila mela-nogaster

Although molecular genetic techniques have elucidated thedetails of several resistance factors, we have little informationon how these resistance factors contribute to genetic variation insusceptibility to insecticides within natural populations. Aftermany isofemale lines collected from the Katsunuma population ofDrosophila melanogaster were established, the susceptibility ofeach isofemale line to three organophosphate insecticides wasmeasured. Susceptibility of isofemale lines to the three organo-phosphate insecticides was continuously distributed within theKatsunuma population, ranging from susceptible to resistant.One-way analysis of variance showed highly significant variationamong isofemale lines in susceptibility to each insecticide. Highlysignificant variation in susceptibility to the three organophos-phates among isofemale lines provides evidence for a geneticbasis for the variation in susceptibility to the three chemicalswithin the Katsunuma population. Two resistance factorsgenetically mapped in the previous study (Miyo et al. 2002)may explain at least a part of the variation in susceptibilitywithin the Katsunuma population of D. melanogaster.

2A-03

OSAKABE, Masahiro1, GOKA, Kouichi2, TODA,Satoshi3, AMANO, Hiroshi4 (1Lab. Ecological Inform.,Grad. Sch. Agr., Kyoto Univ., 2Natl. Inst. Env. Studies,3Dept. Grape and Persimmon Research, Natl. Inst.Fruit Tree Sci., 4Facult. Horticulture, Chiba Univ.)

Meta-population structure and genetic variation relevant todevelopment of pesticide resistance in spider mites

Development of pesticide resistance is an important issueconnected with agriculture and medical care. Of spider mitesthat included many agricultural pests, a polyphagous species,Tetranychus urticae, was panmictic within a narrow area,whereas its populations separated by some distance were not.For a tiny organism such as spider mites, difference in host rangeintends the difference of habitats in spatial distribution anddraws variation in genetic structures, probably influencing on thedevelopment of pesticide resistance. Using three molecularmarkers: an esterase locus, one microsatellite and a pointmutation in the mitochondrial cytochrome oxidase subunit I,and susceptibility tests to two acaricides: fenpyroximate andetoxazole, the authors investigated the divergence of an oligoph-agous spider mite, Panonychus citri, according to a hierarchicalarrangement of geographical distance. We found that P. citripopulations maintain a higher level of variation between trees (orpatches within individual trees) within groves than betweengroves at the local level. Results are discussed in relation to thedispersal behaviors of spider mites.

2A-04

UESUGI, Ryuji1, OSAKABE, Masahiro1, GOKA,Koichi2 (1Grad. Sch. Agr., Kyoto Univ., 2Natl. Inst.Env. Stud.)

Associative cross-resistance by linkage of resistance genes

We investigated the effect of the “associative cross-resistance”,which is caused by the linkage of resistance genes and resemblescross-resistance by a simulation of the Hardy-Weinberg model oftwo loci. The simulation showed that migration of individualhomozygous for resistance alleles brought the increase of non-selected resistance, that is, the associative cross-resistance.Although the extent of the effect of the associative cross-resistance depended on genetic mode of the resistances, 1% ofrecombination rate between the resistance genes brought thestrong effect in any cases.


2A-05

YOSHIDA, Kentaro1, MIYASHITA, Naohiko1 (1Grad.Sch. Agr., Kyoto Univ.)

DNA polymorphism in highly expressed genes during theinteraction between the wild rice Oryza rufipogon and blastfungus Magnaporthe oryzae

To clarify maintenance mechanisms of DNA polymorphism in thegenes involved in the interaction between the wild rice Oryzarufipogon and rice blast fungus Magnaporthe oryzae, DNApolymorphism in the Pbz-1, Cht-1 (Class I chitinase), andOschib3H-b (Class III chitinase) of O. rufipogon was analyzed.These genes are highly expressed in O. sativa treated with theelicitor of M. oryzae. The level of polymorphism (q) at silent sitesof the Pbz-1, Cht-1 and Oschib3H-b of O. rufipogon was 0.007,0.004 and 0.043, respectively. Particularly, Oschib3H-b had threedivergent sequence types, which caused the high degree of silentpolymorphism. We examined whether the silent q in these geneswas expected from average silent q of the genes unrelated to theplant-pathogen interaction under the neutral coalescent process.The probability of obtaining q higher than the observed value inthe Oschi3H-b was less than 0.1%, suggesting that the q in theOschi3H-b was significantly higher than that expected under theneutral mutation model. Considering the three divergent se-quence types in the Oschi3H-b, balancing selection might be acandidate of the maintenance mechanisms of polymorphism inthis gene.

2A-06

MAEDA, Kaoru1, TAKESHIMA, Hirohiko2,TAKAHASHI, Kazuhiko3, SATO, Tetsu4, OKADA,Norihiro4, NISHIDA, Mutsumi2, TACHIDA, Hidenori5

(1Grad. Sch. Sci.s, Kyushu Univ., 2Ocean Res. Inst.,The Univ. Tokyo, 3Natl. Inst. Basic Biol., 4Grad. Sch.BioSci. and BioTech.,Tokyo Inst. Tech., 5Faclt. Sci.s,Kyushu Univ.)

Isolation of HapSTR markers from cichlid in Lake Victoriaand characterization of the population structure

Population sizes of Zooplanktivore cichlids living offshore(Ziko) inLake Victoria of East Africa had been drastically reduced in thelast century. But recently populations of some species of Ziko arerecovering. In order to investigate genetic structures of theirpopulations, we have developed twelve HapSTR makers forHaplochromine cichlids in Lake Victoria using DNA fromHaplochromis chilotes. A HapSTR maker consists of a micro-satellite part (STR) and its flanking region. We analyzed sevenpopulations of Ziko in Lake Victoria using only microsatelliteparts(STR) of the markers. The populations contain H.laparo-gramma,H.paropius,H.piceatus,H.plumbus,H.pyrrhhocephalus.Eleven out of 12 STR loci showed high levels of polymorphism.The average estimate of FIS was -0.0248. Estimates fo FST

between populations range from 0.0095 to 0.0916 and its overallaverage was 0.0653. Mean heterozygosities were in the rangefrom 0.6181 to 0.7628. All populations are almost random mating,and FST estimates between populations suggested that popula-tion structure across these seven populations is weak. Thus,these populations seem to exchange genes frequently, evenbetween different species.

2A-07

ISHIYAMA, Hiroko1, INOMATA, Nobuyuki1,YAMAZAKI, Tsuneyuki2, SZMIDT, Alfred E.1 (1Dept.Biol., Grad. Sch. Sci., Kyushu univ., 2The Res. Inst. ofEvolutionary Biol.)

Demographic history of Southeast Asian tropical tree speciesShorea parvifolia (Dipterocarpaceae)

The tropical trees which are currently widely distributed throughSoutheast Asia are considered to have been confined to parts ofSumatra and Borneo islands during Last Glacial Maximum(approximately 20,000 years ago). The objective of this study is toinfer the demographic history and population structure of atropical tree Shorea parvifolia (Dipterocarpaceae), which is one ofdominant canopy trees in the tropical forest in Southeast Asia. Toachieve our objective we studied the level and pattern of DNApolymorphism in the SBE2 (starch branching enzyme class II)gene region in two populations from Peninsular Malaysia, twopopulations from Sumatra island and three populations fromBorneo island.

2A-08

FUKUDA, Ichiro1, HORII, Yujiro1 (1The AsianEcology-Evolution Botanical Inst.)

Hybrid speciation of Trillium in the Tohoku region of Japan

Trillium camschatcense, T. apetalon & T. tschonoskii aredistributed in the Tohoku region. Several examples of hybridspeciation were found in our recent research. Of special mentionis the hybrid species T. miyabeanum. We found that this speciescan reproduce by seed when the plants have an even chromosomenumber,and makes a complete population. We also foundthat Thehybrid species T. yezoense & T. hagae in this region. We willdiscuss thespecial situation of hybrid speciation in Trilliumevolution in Japan compared with that of the North AmericanTrillium.


2A-09

SUMIDA, Masayuki1, ISLAM, Mohammad Mafizul1,KURABAYASHI, Atsushi1, MACHIYAMA, Fumiaki1,KUROSE, Naoko1, NISHIOKA, Midori1, KHAN,Mohammad Mukhlesur rahman2, ALAM, MohammadShafiqul2, MATSUI, Masafumi3, OTA, Hidetoshi4,KURAMOTO, Mitsuru5 (1Inst. Amphibian Biol., Grad.Sch. Sci., Hiroshima Univ., 2Faclt. Fisheries,Bangladesh Agricultural Univ., 3Grad. Sch. HumanEnv. Stud., Kyoto Univ., 4Tropical Biosphere ResearchCenter, Univ. the Ryukyus, 5Hikarigaoka)

Reproductive isolating mechanisms and genetic divergence inthe rice frog Fejervarya limnocharis complex from Asiaelucidated by crossing experiments and mitochondrial genesequence analyses

The rice frog Fejervarya limnocharis is one of the most widelydistributed species among Asian frogs. Conspicuous variations of thisspecies have been reported based on morphology and mating callsover the distribution range. In the present study, we conductedcrossing experiments among nine populations of the F. limnochariscomplex from five Asian countries in order to elucidate thereproductively isolating mechanisms among the Asian populationsof this species complex. We also carried out mtDNA gene sequenceanalyses to genetically estimate the intra- and interspecific diver-gences of this species complex using 27 populations from eight Asiancountries. The crossing experiments revealed that the Sri Lanka andBangladesh (small-type) populations are reproductively isolated fromthe other populations by complete hybrid inviability at the embryonicstage. The phylogenetic analyses based on the mitochondrial 16SrRNA gene sequences showed that the F. limnocharis complex fromSoutheast Asia diverged into two groups, and that the South Asianpopulations from Sri Lanka and India formed a cluster remotelyrelated to the above two groups, then diverged into severalpresumptive species.

2A-10

KAMIYA, Koichi1, TACHIDA, Hidenori1 (1Faclt. Sci.s,Kyushu Univ.)

Testing the isolation with migration model for divergence inthe closely related species of Shorea, a tropical tree genus inSoutheast Asian tropical forests

Both ancestral polymorphism and gene introgression often causeproblems in phylogenetic inference for recently diverged speciesgroups. Here we use “Isolation with Migration” (IM) model to testfor introgression between four species in a genus Shorea, thetrees having diverged into about 200 species in Southeast Asiantropical forests. Genealogies of three nuclear and a chloroplastgenes showed that haplotypes from each species were notexclusively monophyletic. However, because we could not findany shared haplotype between species, introgression seems not tohave occurred recently. Under the IM model, estimates ofpopulation mutation rates were smaller in the ancestral speciesthan in the descendant species. Certain extents of introgressionin the comparisons involving S. fallax were observed whereas noevidence of introgression was found in the other speciescomparisons. The results suggest that historical gene introgres-sion, probably having occurred at the earlier stage of thespeciation, rather than ancestral polymorphisms can explaindiscordant phylogenetic relationships in the Shorea species.

2A-11

JIN, Lihua1, IKEO, Kazuho1,2, SUZUKI, Yoshiyuki1,2,GOJOBORI, Takashi1,2 (1CIB/DDBJ research center,Natl. Inst. Genet., 2Grad. Univ. Adv. Stud.)

The analysis to the evolutionary relationship between geneduplication and alternative splicing

Living things increase their functional diversification by variableevolutionary mechanisms. Among these mechanisms, geneduplication and alternative splicing (AS) are known as the twoof major evolutionary ways that can bring the functionaldiversification by increasing gene variations. Our researchinterest is to clarify the evolutionary relationship between thesetwo different phenomena by utilizing all available data resources.The results of this study showed that the ratio of AS locus in thesingleton gene group was less than that in the duplicated genegroup and the duplicate genes show statistically more number ofAS isoforms than singleton genes. In spite of these results, whenthe duplicated genes grouped into different homologous families,there was no significant tendency of AS isoform number increasewhen the family size increases. Our results suggest that geneduplication would induce more alternative splicing events on theduplicated genes than on singletons by reducing the functionalconstraint on a gene, and the degree of reduction of functionalconstraint was significantly large at the step of singletonduplication into the homologous genes.

2A-12

SUGINO, Ryuichi1,2, INNAN, Hideki1 (1HumanGenet. Center, School of Public Health, Univ. TexasHealth Science Center at Houston, 2Lab. CropEvolution, Grad. Sch. Agr., Kyoto Univ.)

The effece of concerted evolution against the genes derivedfrom whole genome duplication

A maximum-likelihood (ML) method is developed to estimate theduration of concerted evolution and the time to the whole genomeduplication (WGD) event in baker’s yeast Saccharomyces cer-evisiae. The models with concerted evolution fit the datasignificantly better than the molecular clock model, indicating acrucial role of concerted evolution via gene conversion after geneduplication in yeast. Our ML estimate of the time to the WGD isnearly identical to the time to the speciation event between S.cerevisiae and Kluyveromyces waltii, suggesting that the WGDoccurred in very early stages after speciation or the WGD mighthave been involved in the speciation event.


2A-13

NAKASHIMA, Yuko1,2, HIGASHIYAMA, Asako1,3,USHIMARU, Ayana1, NAGODA, Nozomi1,3, MATSUO,Yoshinori1 (1Faclt. Integr. Arts Sci.s., The Univ.Tokushima, 2Dept. Applied Biology, Kyoto Inst. Tech.,3Master’s Program in Biosystem Studies, Univ.Tsukuba)

Evolutionary analysis of the histone gene repeating unitsfrom Drosophila: D.lutescens, D.takahashii, D.pseudoobs-cura.

Analysis of the H3 genes from seven Drosophila species showedthat the GC contents at the third codon position from D.lutescensand D.takahashii were higher. While that from D.pseudoobscurawas about the same level as D.melanogaster. In this study the GClevel was checked for other histone genes from these species bycloning and analyzing the repeating units of histone genes. TheGC contents at the third codon position for four histone genes,H1, H2A, H2B, and H4, from D.lutescens and D.takahashii werehigher, indicating the same tendency to the H3 gene region.Those from D.pseudoobscura showed about the same level as D.melanogaster. The species differences of the GC content in theregions for spacer were smaller when compared with those in theregions for coding gene. The species difference of the GC contentas found for the H3 gene was observed for other histone genesindicated that the difference was not specific for the H3 gene butwas confirmed at least for whole repeating unit. From theseresults it was speculated that the high GC contents in D.lutescensand D.takahashii could be explained by the efficient selectionagainst A and T through the large size of population.

2A-14

HAYAKAWA, Toshiyuki1,2, ANGATA, Takashi1,LEWIS, Amanda L.1, MIKKELSEN, Tarjei S.3,VARKI, Nissi1, VARKI, Ajit1 (1Dept. Med., Path., Biol.Sci.s and Cellular & Molecular Medicine, Univ.California at San Diego, 2Sch. Adv. Sci., Dept. Biosyst.Sci., The Grad. Univ. Adv. Stud., 3Broad Institute ofMIT and Harvard)

Function and expression of human Siglec-11 were changedvia gene conversion by an adjacent pseudogene

Siglecs (sialic acid-binding immunoglobulin superfamily lectins)are a family of cell surface receptors involved in regulating theimmune response. Of these, Siglec-11 is expressed in brainmicroglial cells, which play important roles in brain immunity aswell as other brain functions. Gene conversion is a mechanism forcopying part of a genomic sequence into another, contributing togenetic diversity. We analyzed genomic sequences of SIGLEC11and adjacent pseudogene SIGLECP16 in several primates andfound the gene conversion of SIGLEC11 by SIGLECP16 only inthe human lineage. The converted region encompasses 5untranslated sequences and exons encoding the N-terminalregion, including the domain mediating sialic acid recognition.Therefore, the changes in function and expression of humanSiglec-11 were expected. The immunohistochemical analysesusing anti-Siglec-11 antibody and ELISA assays using recombi-nant proteins represent the difference of both expression andfunction between human and chimpanzee Siglec-11. It is likelythat the gene conversion by pseudogene caused changes infunction and expression in the human lineage.

2A-15

ZHENG, Songlin1, TAGA, Masatoki1 (1Grad. Sch.Natural Sci. Tech., Okayama Univ.)

Karyotype evolutoion in filamentous fungi: a case study of thegenus Fusarium

In contrast to the ample information of the karyotype evolutionfor higher eukaryotes, our knowledge of this subject is limited forfilamentous fungi. Here, we analyzed karyotype evolution infilamentous fungi using the genus Fusarium as a case study. Thisgenus was chosen because the previous data had shown that it iscomprised of species with considerably different chromosomenumbers (CNs). Fluorescence microscopy and pulsed field gelelectrophoresis (PFGE) were used for karyotyping, and molecularphylogeny was constructed based on the sequence data of thepartial TEF-1alpha gene. CNs of 19 Fusarium spp. fell into threegroups, namely, the group of n = 4, n = 5, n = 8, and n = 12,respectively. In the molecular phylogenetic tree, species with thesame CN clustered in a single branch, indicating that CN hasbeen conserved in the process of speciation. In addition, the orderof branching suggested the occurrence of chromosome fusionevents in the genome of n = 12 to yield independently n = 4 and n= 8. Interestingly, PFGE analysis showed that chromosomerearrangement is prevailing irrespective of the conservation ofCN.


3A-01

ASAI, Shin-ichi1, ITOH, Tateo1 (1Dept. Biol., Faclt.Sci., Shinshu Univ.)

Mechanism of primer synthesis by the plasmid ColE2replication initiator protein (Rep)

The plasmid ColE2 replication initiator protein (Rep) is a primasewhich synthesizes a 3-mer primer (ppApGpA) at a fixed region inthe replication origin (ori). The enzyme requires ADP as the firstnucleotide of the primer. The DXD sequences are highlyconserved among the Rep proteins of the ColE2-related plasmids.We speculate that DXD motif is a part of the catalytic domain forprimer synthesis. We found that Rep fused with His-tag bindsATP tightly. We report functional analysis of the DVD motif ofColE2 Rep by using point mutations and characterization of theATP free Rep.

3A-02

FUJIKANE, Ryosuke1, SHINAGAWA, Hideo2,ISHINO, Yoshizumi1 (1Dept. Genet. Resources Tech.,Faclt. Agr., Kyushu Univ., 2Dept. Molec. MicroBiol.,Res. Inst. for Microbial Diseases, Osaka Univ.)

A novel helicase related to replication fork repair in thehyperthermophilic archaeon, Pyrococcus furiosus

Impairment of replication fork progression causes genomeinstability, and is a serious threat to living organisms. Manyefforts of the researchers have established that recombinationevent and proteins involved in recombination play importantroles in restoration of the stalled replication fork. The archaealHjm (Holliday junction migration) is a structure-specific DNAhelicase, which was originally identified in the hyperthermophilicarchaeaon, Pyrococcus furiosus, by in vitro screening for Hollidayjunction migration activity. Further biochemical analyses of theHjm protein from P. furiosus showed that this protein specificallybinds to fork-related Y-structured DNAs and unwinds theirdouble-stranded regions in vitro, just like the Escherichia coliRecQ protein. Furthermore, genetic analyses showed that Hjmproduced in E. coli cells partially complemented the functions ofRecQ in a recQ mutant E. coli strain. These results suggest thatHjm may be a functional counterpart of RecQ in Archaea, inwhich it is necessary for the maintenance of genome integrity,although the amino acid sequences are not conserved.

3A-03

OHYA, Tomoko1, ARAI, Hirokazu1, SHINAGAWA,Hideo1, HISHIDA, Takashi1 (1Res. Inst. Microb. Dis.,Osaka Univ.)

Roles of chromatin silencing protein Esc2 required for DNAreplication and repair in Saccharomyces cerevisiae

Esc2 (Establishment silent chromatin 2) belongs to conservedRENi family of SUMO-like domain protein and is related tofission yeast Rad60 and human NIP45. Previous studies suggestthat Esc2 protein interacts with Sir2 protein and is required forestablishment of chromatin silencing at mating type locus. Theseresults raise the possibility that Esc2 recruits or stabilizes Sir2 tosilencing locus. However, Desc2 mutants have a slow growthphenotype, which is absent in Dsir2 cells. Fission yeast Rad60 isessential for growth. These results argue that Esc2 has anotherrole beside its role in establishment chromatin silencing, possiblyduring S-phase. In this study, we explored the possibility thatEsc2 plays a more direct role in DNA replication and repair. Wefound that esc2 null mutant was sensitive to MMS. Moreover, weprovide evidences suggesting that Esc2 plays an important role inS-phase checkpoint control and restoration of DNA replicationarrest. Taken together, this study may provide novel evidencesthat Esc2 is involved in protein complex that plays between DNAreplication, recombination and repair functions.

3A-04

OSHIUMI, Hiroyuki1, SHINOHARA, Miki1,SHINOHARA, Akira1 (1Inst. Prot. Res.)

DNA repair activities of Mre11 inhibit Ty1 retrotransposi-tion, and promote recombination.

Mre11 is important for DNA recombination of host genome andconserved among eukaryote. Recently, it was reported thatMre11 is involved in ligation of adenovirus or HIV genome ends,which leads to viurs suppression. Budding yeast has endogenousretrovirus Ty1 in its genome. Interestingly, Mre11 is reported tobe required for suppression of Ty1 transposition. These factssuggest a novel hypothesis that Mre11 interacts with non-hostgenome, such as virus and transposon. We tried to elucidate themolecular mechanism by using Ty1 as model system. When Ty1was expressed from LTR or galactose inducible promoter, Mre11suppressed Ty1 transposition, suggesting that suppression of Ty1by Mre11 is mediated at post-transcriptional steps. Ty1 homol-ogous recombination between cDNA and genome encoded Ty1was reduced in mre11 mutants suggesting that Mre11 promotesTy1 recombination between cDNA and genome encoded Ty1.Exo1 is a DNA exonuclease, and overexpression of Exo1suppresses repair defects of mre11 null mutants. We found thatoverexpression of Exo1 in mre11 mutants decreased Ty1trasnposition, suggesting that DNA end resection is involved inTy1 suppression by Mre11.


3A-05

IDE, Satoru1, WATANABE, Keiiti1, WATANABE,Hiromitu1, KOBAYASHI, Takehiko2, MAKI, Hisaji1

(1Dept. of Mol. Biol., Grad. School of Biol. Sci., NaraInst. of Sci. and Tech., 2Natl. Inst. Basic Biol.)

Involvement of rDNA locus in monitoring abnormal initiationprogram of chromosomal replication by DNA damage check-point response

Perturbation of origin firing in chromosome replication is apossible cause of spontaneous chromosome instability in multi-replicon organisms. We have shown that chromosomal abnormal-ities were significantly increased in yeast diploid cells withdefects in the origin recognition complex and that the DNAdamage checkpoint response was induced to maintain thegenome stability in these mutants. We recently found that upontemperature shift, DNA lesions were induced much morefrequently within rDNA locus than other chromosomal loci inorc2-1 mutants. When the copy number of rDNA units in thechromosome was decreased, the temperature-sensitive pheno-types of orc2-1 mutant strains were significantly suppressed andDNA damage checkpoint response was activated much lessefficiently in such cells at the restrictive temperatures, suggest-ing that the rDNA locus plays an important role for monitoringabnormal initiation program of chromosomal replication by DNA-damage checkpoint response.

3A-06

DAIGAKU, Yasukazu1, ENDO, Kingo1, YAMAMOTO,Kazuo1 (1Grad. Sch. life Sci., Tohoku Univ.)

Positive and negative effects of DNA damage for mitoticrecombination in Saccharomyces cerevisiae

Mitotic recombination functions as DNA damage tolerantmechanism and double-strand break repair. One of the con-sequences of mitotic recombination is gene conversion that isnonreciprocal transfer of genetic information from one strand toits homologs. During gene conversion, strand with duplicatedsequence and the strand with lost sequence are referred as “donorsequence” and “recipient sequence” respectively. The purpose ofthis study is to investigate the effect of damage on donor sequenceand recipient sequence during mitotic recombination. UV-induced DNA damage on recipient sequence induced geneconversion effectively, but damage on donor sequence did not.Furthermore, we observed that injured sequence functioned asdonor sequence less efficiently than intact sequence. Theseresults indicated that DNA damage on the recipient sequencehad a positive effect on induction of gene conversion, but damageon the donor sequence has negative effect. It can be interpreted tomean that damage on the recipient sequence induces substratefor recombination, that is double-strand breaks, and the damageon the donor strand inhibits the strand invasion or replication ofconverted sequence.

3A-07

KATOH, Kazutaka1, KUMA, Kei-ichi1, IWABE,Naoyuki2, MIYATA, Takashi3,4,5 (1Bioinform. Cent.,Inst. Chem. Res., Kyoto Univ., 2Grad. Sch. Sci., KyotoUniv., 3JT Biohistory Res. Hall, 4Sci. Eng., WasedaUniv., 5Grad. Sch. Sci., Osaka Univ.)

Evolutionary analysis of the catalytic subunit of DNApolymerase dd from various vertebrates

A eukaryotic genome is replicated mainly by DNA polymerases e,d and a. Since these polymerases causes mutations occurring inthe germ line, they might be unique and interesting proteins fromthe point of view of molecular evolution. We have cloned andsequenced the genes encoding the catalytic subunit of DNApolymerase dw from various vertebrate species including cartila-ginous fishes, lobe-finned fishes, holosteans, sturgeon, bichir andreptiles. The phylogenetic analyses using these data revealed acharacteristic evolutionary pattern in several independentlineages. We will discuss evolutionary implications of ourobservation.

3A-08

FUJIMITSU, Kazuyuki1, KATAYAMA, Tsutomu1

(1Dept. Molec. Biol., Grad. Sch. PharmaceuticalScience, Univ. Kyushu)

A specific chromosomal sequence reactivates DnaA protein,the initiator of chromosomal replication in E. coli.

In E. coli, DnaA protein, the initiator of chromosomal replicationtightly binds to ATP and ADP, and only the ATP-bound form isactive in the initiation of chromosomal replication. Whereas thecellular content of total DnaA protein molecules is kept constantduring cell cycle, the composition of the ATP form fluctuates in amanner coordinated with the replication cycle, temporarilyincreasing at the time of initiation. These observations suggestthat the regeneration of the ATP-DnaA from the ADP-DnaAwould play an important role in determining the initiationswitch. Although we have already indicated the presence of aregeneration pathway in vivo, a crucial factor that reactivatesDnaA by nucleotide exchange in vivo has not been identified yet.In this study, we searched for a novel factor that can reactivateDnaA. As the result, we found that a specific DNA sequence thatis carried on the E. coli genome can reactivate DnaA and canstimulate the initiation of chromosomal replication.


3A-09

TANAKA, Seiji1, ARAKI, Hiroyuki1 (1Div. MicrobialGenetics, Natl. Inst. Genet.)

ISOLATION OF THE MUTANT THAT BYPASSES THECDK REQUIREMENT IN THE INITIATION OF DNAREPLICATION

In eukaryotic cells, cyclin dependent kinases (CDKs) are requireffor the initiation of DNA replication.Although target(s) of CDK had been elusive for a long time, Sld2was reported as an essential target of CDK in initiation. Ourrecent analyses showed that Sld2 is essential but not sufficienttarget. To identify unknown target(s) of CDK, we screenedmutants that replicate their chromosome DNA untimely. Sur-prisingly, one mutant initiated DNA replication even when cellswere arrested in G1 phase. Our data suggest that CDK regulatesat least two essential processes in initiation.

3A-10

MORISHITA, Takashi1, SAKAGUCHI, Chikako1,FURUKAWA, Fumiko1, SHINAGAWA, Hideo1 (1Res.Inst. for Microbial Diseases, Osaka Univ.)

Role of the fission yeast F-box DNA helicase in recombina-tiopn repair

To identify novel genes involved in recombination repair, weisolated fission yeast Schizosaccharomyces pombe mutantssensitive to methyl methanesulfonate and a synthetic lethal withrad2. A gene which complements such mutations was isolatedfrom an S. pombe genomic library, and subsequent analysisidentified it as the fbh1 gene encoding the F-box DNA helicase,which is conserved in mammals but not in Saccharomycescerevisiae. An fbh1 deletion mutant is sensitive to UV, MMSand g-rays. rhp51 mutation is epistatic to fbh1. fbh1 is essentialfor viability in stationary phase cells, and in the absence of eitherSrs2 or Rqh1 DNA helicase. In each case lethality is suppressedby deletion of rhp57. These results suggests that fbh1 actsdownstream of rhp51 and rhp57. Following UV irradiation orentry into the stationary phase, nuclear chromosomal domains ofthe fbh1D mutant shrank and accumulation of some recombina-tion intermediates was suggested by pulsed-field gel electro-phoresis. Foci formation of Fbh1 protein was induced bytreatment that damages DNA. Thus the F-box DNA helicaseappears to process recombination intermediates, formation ofwhich is dependent on the function of Rhp51.

3A-11

ISHIKAWA, Ken1,2, WATANABE, Miki2, KUROITA,Toshihiro3, UCHIYAMA, Ikuo4, BUJNICKI, Janusz5,KAWAKAMI, Bunsei3, TANOKURA, Masaru6,KOBAYASHI, Ichizo1,2 (1Graduate Program inBiophysics and Biochem., Grad. Sch. Sci., Univ. Tokyo,2Dept. Medical Genome Sci., Grad. Sch. Front. Sci.,Univ. Tokyo, 3Tsuruga Inst. of Biotec., Toyobo CO.,LTD., 4Natl. Inst. Basic Biol., 5Lab. Bioinform. andProtein Eng., InterNatl. Inst. Molecular and CellBiology, Warsaw, Poland, 6Dept. Applied BiologicalChemistry, Grad. Sch. Agricultural and Life Sci., Univ.Tokyo)

Discovery of a novel restriction endonuclease by genomecomparison and application of a wheat-germ-based cell-freetranslation assay: PabI (5’GTA/C) from the hyperthermo-philic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-basedcell-free translation procedure that bypasses the toxicity of theseenzymes. To identify candidate genes, the related genomes of thehyperthermophilic archaea Pyrococcus abyssi and Pyrococcus hori-koshii were compared. In line with the selfish mobile gene hypothesisfor restriction-modification systems, apparent genome rearrange-ment around putative restriction genes served as a selectingcriterion. Several candidate restriction genes were identified andexpressed with a wheat germ-based cell-free protein synthesissystem. The resulting solution could be directly assayed forrestriction activity. We identified two deoxyribonucleases. The novelenzyme was denoted as PabI, purified and found to recognize 5’GTACand leave a 3’TA overhang (5’GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90˚C and optimally active ata pH of around 6 and in NaCl concentrations ranging from 100 to 200mM. We predict that it has a novel three-dimensional structure.

3A-12

ICHIGE, Asao1, KOBAYASHI, Ichizo1,2 (1Dept. Medi-cal Genome Sci., Univ. Tokyo, 2Inst. Med. Sci., Univ.Tokyo)

Stability of EcoRI restriction modification enzymes in vivodifferentiates EcoRI restriction-modification system fromother postsegregational cell killing systems

Certain Type II restriction modification gene systems can killhost cells when these gene systems are eliminated from the hostcells. Such ability to cause postsegregational killing of host cellsis the feature of bacterial addiction modules, each of whichconsists of toxin and antitoxin genes. With these addictionmodules, differential stability of toxin and antitoxin moleculesin cells plays an essential role in execution of postsegregationalkilling. We here examined in vivo stability of the EcoRIrestriction enzyme (toxin) and modification enzyme (antitoxin)in Escherichia coli. Using Western blot analysis and pulse-chaseimmunoprecipitation analysis, we demonstrated that both theEcoRI restriction enzyme and modification enzyme are as stableas bulk cellular proteins and that there is no marked difference intheir stability. We also monitored changes in cellular levels of theEcoRI restriction and modification enzymes during postsegrega-tional killing. Results from these analyses together suggest thatthe EcoRI gene system does not rely on differential stabilitybetween the toxin and the antitoxin molecules for execution ofpostsegregational cell killing.


3A-13

FURUYA, Nobuhisa1, KOMANO, Teruya1 (1Dept.Biol. Sci.s, Tokyo Metropolitan Univ.)

Specific recognition of NikA protein to the origin of transfer ofconjugative plasmid R64

Conjugative DNA transfer of bacterial plasmids are initiated byintroduction of a specific nick within the origin of transfer, oriT.During conjugation, the nicked strand is transferred, beingaccompanied with rolling-circle DNA replication. In the case ofplasmid R64, that moves among various enterobacteria such asEscherichia, Shigella and Salmonella, the specific nick isintroduced by NikB nickase (relaxase). However, NikB is notable to recognize double-stranded oriT DNA without anothercomponent, NikA. NikA, containing ribbon-helix-helix (RHH)DNA-binding motif, binds to the 17-bp specific DNA sequence(repeat A) 8-bp apart from the nick site. However, repeat Asequence does not have distinct symmetric features that would beexpected for DNA-binding protein with RHH motif. To inves-tigate the mechanism of NikA recognition of repeat A, NikA-bound DNA fragment was selected from synthetic, short DNAfragments with random sequences. The selected sequences boundto NikA were found to have the 5-nt successive sequence incommon, that is identical to the nick site-distal portion of RepeatA sequence. However, only the 5-bp sequence was not sufficientfor NikA-binding.

3A-14

SATO, Hajime1, TOMIMURA, Yoshihiko2, OGAWA,Ryouko1, MATSUDA, Muneo1 (1Sch. Med., KyorinUniv., 2Shiba Gakuen)

The Gene Arrangement in the Proximal Region of the 2ndChromosome of Drosophila ananassae

In Drosophila ananassae, the enhancer of male recombinationand the hotspot of male recombination have been mapped within0.4 cM of the proximal region of 2nd chromosome. However, thedetailed gene arrangement of this region has not been previouslyclarified. For fine mapping of the hotspot and the enhancer ofmale recombination, we have performed experiments to deter-mine the cytological sites of molecular markers selected from theFlybase sequence data (Institute for Genomic Research; TIGR) ofD. ananassae by in situ hybridization and to detect polymorphicsites of these markers among the strains used for the mappingexperiment. By comparing the gene order in D. ananassae to thatin D. melanogaster, the role of gene rearrangement in theevolutionary history between these species is discussed.

1B-01

YUGE, Kazuya1,2, IKEO, Kazuho1,2, GOJOBORI,Takashi1,2 (1Cent. Inform. Biol. DNA Data BankJapan, Natl. Inst. Genet., 2Grad. Univ. Adv. Stud.)

Comparison of gene expression of mouse adult brain betweenmale and female

It has been noted that male and female animals show variousphenotypic differences between male and female, includingreproductive organs. It is suspected that differences in the brainbetween male and female are responsible for differences inbehavioral pattern between sexes. However, little is known aboutsex differences in the brain at the molecular level. Therefore weexamined whether there are significant differences in geneexpression between the sexes in the mouse brain. We comparedmRNA expression levels between sexes in preoptic area,hypothalamus, olfactory bulb and pituitary using Microarrayand RT-PCR. We found that there are significant differencesbetween the sexes in these regions of the brain at the level of geneexpression and that genes which are expressed differentiallybetween sexes include many receptor and receptor binding genes.

1B-02

OSATO, Naoki1,2, IKEO, Kazuho1,2, GOJOBORI,Takashi1,2 (1Lab. for DNA Data Analysis, Cent.Inform. Biol. DNA Data Bank Japan, Natl. Inst.Genet., 2Inst. for Bioinform. Research and Dev., JapanSci. and Tech. Agency, Japan)

POSSIBLE REGULATORY ROLES OF HUMAN ANDMOUSE OVERLAPPING TRANSCRIPTS IN GENE EX-PRESSION

Overlapping transcripts have been found from various speciesthrough the analyses of complete genome and cDNA sequences.Some overlapping transcripts are known to have a function ofSense-antisense mRNAs suppressing their expression. However,the properties of overlapping transcripts that affect theirexpression have not been studied well. To remedy this situation,we examined the relationship between the expression level andgenomic arrangement of human and mouse overlapping tran-scripts by using SAGE (Serial Analysis of Gene Expression) datain NCBI SAGEmap database. As a result, the expression level ofoverlapping transcripts was changed according to their over-lapping pattern, compared with that of non-overlapping tran-scripts. It is suggested that the genomic arrangement ofoverlapping transcripts is an important factor in their regulation.


1B-03

KUROKI, Yoko1, TOYODA, Atsushi1, NOGUCHI,Hideki1,2, TAYLOR, Todd1, ITOH, Takehiko3, KIM,Dae-soo4, KIM, Dae-won4, CHOI, Sang-haeng4, KIM,Il-chul4, CHOI, Han-ho4, KIM, Yong-sung4, SATTA,Yoko5, SAITOU, Naruya6, YAMADA, Tomoyuki2,MORISHITA, Shinichi2, HATTORI, Masahira1,7,SAKAKI, Yoshiyuki1, PARK, Hong-seog4, FUJIYAMA,Asao1,8 (1Sequence Tech. Team, Genome Core Tech.Facilities, RIKEN Genomic Sci.s Cent., Kanagawa,Japan, 2Grad. Sch. Front. Sci.s, The Univ. Tokyo,3Mitsubishi Res. Inst. Inc., 4Genome Research Center,Korea Reaearch Inst. of Biosci. and Biotec., 5Grad.Univ. Adv. Stud., 6Natl. Inst. Genet., 7Kitasato Univ.,8Natl. Inst. Inform.)

Comparative analysis of human and chimpanzee Y chromo-somes to understand their evolutional and biological charac-teristics

Human chromosome Y is known to have male-specific characteristicsand a peculiar genomic structure including abundant chromosomal-specific repeats, large-scale duplications, and complicated chromoso-mal rearrangements. To understand the evolutional and biologicalcharacteristics of the Y chromosome, we have started a comparativesequence analysis between the human and chimpanzee Y chromo-somes. We first constructed a physical map of chimpanzee Ychromosome, and then started sequencing clone by clone. We havecompletely finished the sequencing of 100 BAC and fosmid clones; thetotal non-redundant length of the sequenced region is about 11 Mb.This region contains 32 genes which are derived from human Yp andYq. Many structural differences such as rearrangements, insertion-deletions, and duplications have been found so far in this study. Wewill report the dynamic changes which have occurred on the non-recombining region of the Y chromosome in the past 5-6 million yearsafter the separation of the two species.

1B-04

TAGUCHI, Takahiro1, HIRAI, Hirohisa2, HIRAI,Yuriko2, TOMINAGA, Akira1 (1Dept. Human Healthand Med. Sci., Grad. Sch. Kuroshio Sci., Kochi Univ.,2Primate research Inst., Kyoto Univ.)

Regeneration of Y chromosome probes from monkeys with asmall Y by chromosome microdissection

We generated FISH (fluorescence in situ hybridization) probesfrom Y chromosomes of common marmoset (Callithrix jacchus),Rhesus monkey (Macaca mulatta), and agile gibbon (Hylobatesagilis) by chromosome microdissection. Probes obtained from theY chromosomes show higher specificity for the Y chromosome ofeach species, confirmed by FISH. The probes from Rhesusmonkey and common marmoset hybridized constitutive hetero-chromatin, and one from agile gibbon showed whole long arm ofthe Y chromosome. By microcloning of Y probes of Rhesusmonkey, a unique sequence was isolated and sequenced.Sequence and alignment analyses of the probe revealed that thesize was 870bp and an alphoid DNA had the highest affinity withthe probe. Comparative FISH-mapping disclosed that this DNA-sequence cluster was located at different sites on the Ychromosome in several species of the Old World monkey.Microclonig of probes from two other species is in progress.

1B-05

KURAKU, Shigehiro1, ISHIJIMA, Junko2, NISHIDA-UMEHARA, Chizuko2,3, AGATA, Kiyokazu4, KURATANI,Shigeru1, MATSUDA, Yoichi2,3 (1Laboratory forEvolutionary Morphology, RIKEN Center for Dev.Biol., 2Lab. Animal Cytogenetics, Div. GenomeDynamics, Creative Res. Initiative "Sousei", HokkaidoUniv., 3Div. Biol. Sci.s, Grad. Sch. Sci., HokkaidoUniv., 4Lab. Molec. Dev. Biol., Dept. Biophysics, Grad.Sch. Sci., Kyoto Univ.)

Isochore in reptile genomes: cDNA-based studies on intra-genome GC-heterogeneity in Chinese soft-shelled turtle

Mammalian and avian genomes consist of several classes ofchromosomal segments that dramatically vary in GC-content, namely“isochores”. To understand evolutionary history of this intra-genomeGC-heterogeneity in amniotes, we cloned cDNAs in Chinese soft-shelled turtle Pelodiscus sinensis, and localized them to chromosomesby FISH. Our analysis using P. sinensis cDNA sequences detectedmarked heterogeneity in GC-content of exonic third positions (GC3)as previously shown for human and chicken, and each turtle geneexhibited similar levels of GC3 to its orthologs of human and chicken.In addition, our gene mapping indicated that turtle GC-rich geneswere localized more preferentially to microchromosomes, as observedin chicken. These results imply that the GC-heterogeneity is derivedfrom an event that occurred in the common ancestor of extantamniotes. Moreover, it has been suggested that this GC-heteroge-neity has been maintained by coexistence of GC-rich and GC-poorregions in individual chromosomes in a mosaic manner in themammalian lineage, while GC-rich fractions of genome tend to beconfined to microchromosomes in the sauropsidan lineage.

1B-06

MOCHIDA, Keiichi1, KAWAURA, Kanako2,OGIHARA, Yasunari2 (1Faclt. BioSci., NagahamaInst. Bio-Sci. Tech., 2Lab. Genetic Eng., KyotoPrefectural Univ.)

Discovery of miRNAs by large-scale EST analysis of hex-aploid wheat

MicroRNAs(miRNAs) are non-coding RNA molecules that post-transcriptionally regulate the expression of the target genes ineukaryote transcriptome. So far, over 300 miRNA genes of modelplant species have been discovered by computational predictionfrom genome sequences as well as cloning method of small RNAmolecules. Large-scale EST data is a possible genome wideresource to enable the discovery of miRNAs as well as tocharacterize their expression patterns. To discover the candi-dates of wheat miRNA, we grouped 335,014 wheat ESTs derivedfrom 32 various cDNA libraries into 84,700 individual contigswhich can distinguish each homoeo-loci using CAP3 program. Weperformed homology search using blastn program with previouslyknown plant miRNAs precursor sequences obtained from Rfamdatabase as the queries. In total, 135 candidates of wheat miRNAgene were identified. Then, we validated secondary structure ofthese miRNA precursor candidates using RNAfold, and identified16 contigs as wheat miRNA gene. Furthermore, we monitored theexpression patterns of these wheat miRNA genes using a digitalgene expression map, Virtual Display.


1B-07

KAWAURA, Kanako1, OGIHARA, Yasunari1 (1Lab.Genetic Eng., Kyoto Prefectural Univ.)

Structure and expression analyses of three homoeologousAP2-like genes in common wheat

APETALA2 (AP2) is a floral homeotic gene and a candidate forthe Q gene in wheat. The Q gene played important role indomestication of wheat and locates at the long arm of 5Achromosome. Here, we cloned cDNAs corresponding to threehomoeologous AP2-like genes from Chinese Spring wheat.According to the RT-PCR, three AP2-like genes were expressedin the young spikelets. The real-time RT-PCR analysis using thelocus-specific primer set showed that the gene from the A genomewas preferentially expressed among the three homoeologs. TheTAC library screening and the inverse-PCR were carried out, sothat genomic sequences around the AP2-like gene region wereobtained. Comparing among the three homoeologs, some struc-ture alternations of wheat AP2-like genes were disclosed: thegenes from the B genome contained deletion in its exons. Twodeletions and two independent deletions were found in the 9thintrons of the genes from the A, and both of B and D genomes,respectively. Furthermore, certain cis-elements specific to the Agenome were found in the up-stream region of the gene,suggesting an implication for the differences of the expressionlevels among the three homoeologous genes.

1B-08

OGIHARA, Yasunari1, KAWAURA, Kanako1,MOCHIDA, Keiichi2, ENJU, Akiko3, SAKURAI,Tetsuya4, SEKI, Motoaki3, SHINOZAKI, Kazuo4,KAWAI, Jun3, KAI, Chikatoshi3, HAYASHIZAKI,Yoshihide3, TOYODA, Atsushi3, TOTOKI, Yasushi3,SAKAKI, Yoshiyuki3 (1Lab. Genetic Eng., Grad. Sch.Agr., Kyoto Pref. Univ., 2Nagahama Institute ofBioSci. and Tech., 3RIKEN Genomic Sciences Center,4RIKEN Plant Science Center)

Accelerated evolution of generative organ genes duringdifferentiation course between wheat and rice as revealedby the full length cDNA sequences

The polyploid nature of wheat is a key characteristic of the plant.We have constructed a new full length cDNA library derived fromthe pooled RNAs extracted from the 17 different tissues ofChinese Spring (6x) wheat. The 19,968 clones were sequencedfrom both ends. These sequences were classified into 25,502contigs with the phrap method, and 15,197 gene clusters with theblastn. Subsequently, these gene clusters were assembled intothe 7,149 unigene clusters. Out of the 7,149 genes, 6,162 cDNAswere entirely sequenced. 10 % of wheat cDNAs were unique afterthe homology search against rice pseudomolecules in TIGR.These results strongly suggest that full length cDNA library ofcommon wheat might provide efficient source to study compara-tive functional genomics of cereals. 3061 genes having their ricecounterparts were examined their expression profiles duringwheat life cycle. Using amino acid sequences of 3061 genesbetween wheat and rice, Ka and Ks were calculated. Judged fromKa/Ks values, genes expressed in generative organs revealedaccelerated evolution during differentiation course betweenwheat and rice.

1B-09

SANO, Yukie1,4, KANAMORI, Hiroyuki2, NAMIKI,Nobukazu2, YAMAZAKI, Yukiko3, MIYABAYASHI,Toshie4, KURATA, Nori1,4 (1Dept. Life Sci., Grad.Univ. Adv. Stud., 2Sequencing group, Rice GenomeResearch Program, SOCIETY for TECHNO-INNOVATION of AGRICULTUER, FORESTRY andFISHERIS-institute, 3Genetic Inform. Lab., Natl. Inst.Genet., 4Plant Genet. Lab./Experimental Farm, Natl.Inst. Genet.)

Analysis of gene variation in rice based on the comparativegenomics

Almost wild rice have rarely been analyzed molecularly andgenetically, and the molecular details of various genome specieshave not been solved yet. We plan to examine genome differencesby comparing expressed sequences of BB and CC genome specieswith AA genome.The cDNA libraries were constructed using mRNA isolated fromO. punctata (BB) shoot and O. officinalis(CC) panicle and 2,000each randomly picked up clones were sequenced. After theremoval of ambiguous sequences, 973 clones of O. punctata and1,425 clones of O. officinalis were used for BLAST searchanalysis.At the nucleotide level, comparison against rice cDNA and ESTsequence databases revealed that 88-96% clones showed highhomology (90-100%) to O. sativa sequence. Candidate cloneswhich showed low homology to O. sativa were hybridized to allgenome DNAs of AA, BB, CC, CCDD, EE, FF genome species andit was revealed that 13 clones could be genome specific or wildrice specific genes.

1B-10

MATSUOKA, Satoshi1, ASAI, Kei1, HABU, Chiemi2,NANAMIYA, Hideaki2, KAWAMURA, Fujio2, SADAIE,Yoshito1 (1Dept. Biochem. and Molec. Biol., Facaluty ofscience, Saitama Univ., 2Dept. Life Sci., College of Sci.,Rikkyo Univ.)

Analysis of the mechanism of protection against infection ofSP10 phage in Bacillus subtilis Marburg

Inactivation of both nonA and nonB genes render B. subtilis cellpermissive to infection by SP10. In a previous study, wedetermined the mutation sites of nonA and nonB. The nonAmutation is the cured state of endogenous prophage SPb and thenonB mutation is a nonsense mutation in ydiR which is acomponent of BsuM system. In this study, we investigated themechanism of protection against infection of SP10. First, weanalyzed effect of the BsuM system to SP10. SP10 grown in the R/M-deficient strain could not make plaques on the restrictionproficient indicator strain. An enforced expression of the wildtype BsuMM operon in the BsuMR+ indicator strain, however,allowed more than thousand times plaques. Thus, the SP10genome DNA is able to be nealy full-methylated but some BsuMsites are considered to be unmethylated. Secondly, to investigatewhich genes on SPb are responsible for the interference of SP10growth, we constructed partial deletion mutants of SPb. Tworegions on SPb were found to be concerned with the interferenceagainst infection of SP10. We are investigating above regions toidentify genes which are involved in the interference againstinfection of SP10.


1B-11

FUJITA, Kiyohito1, OHTA, Niji1 (1Grad. Sch. Sci.,Univ. Saitama)

Analysis of cfxQ function that is encoded in the cell nucleusand plastid genome of unicelluar red alga Cyanidioschyzonmerolae 10D

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isthe key enzyme of the calvin Benson cycle. RuBisCO contains oftwo kinds of subunits, and their genes are rbcL (encodingRuBisCO large subunit) and rbcS (encoding RuBisCO smallsubunit). Red and cryptomonad algae possess red-type RuBisCOgenes that constitute a rbcL-rbcS-cfxQ operon. The cfxQ ispredicted to be necessary for the expression of RuBisCO genes(rbcL and rbcS), but the function of this gene has not beeninvestigated.Recently, complete genome sequence of primitivered alga Cyanidioschyzon merolae 10D was determined. cDNAanalysis indicated that cfxQ is encoded both in the plastid genomeand in the nuclear genome. There are few genes that areduplicated both in plastid genome and nucleus genome. In gelmobility sift assays, we found the affinity of both CfxQs for rbcLpromoter. These results suggest that both CfxQs bind to rbcLpromoter, and may regulate the transcription of the rbcL operon.

1B-12

NAGAI, Takeshi1, FURUTA, Taku1, OHTA, Niji1

(1Dept. Molec. Biol., Faclt. Sci., Saitama Univ.)

Genome Analyses of Cyanidium caldarium strain RK-1

Cyanidium caldarium, Cyanidioschyzon merolae and Galdieriasulphuraria belong to Cyanidiales, which are thought to beamong the most primitive plants. They are unicellular red algaeand live in acidic hot springs. The three species differ in the sizeand the shape of the cell, content of DNA, their mode of the celldivision, and so forth. The genome sequence of C. merolae wascompleted, though, only part of the sequence of plastid genomewas analyzed in C. caldarium RK-1 that are produced in Japan.More than 70% of the plastid genome was analyzed. So far,nucleotide sequence and the gene order resemble to those of C.merolae rather than European C. caldarium. We began thegenome sequence of C. caldarium by using the shot-gun cloningmethod. Many genes that have high similarity with those of C.merolae are found.

1B-13

TAKASHIMA, Yasuo1, BANDO, Tetsuya1, KAGAWA,Hiroaki1 (1Grad. Sch. Natural Sci. Tech., OkayamaUniv.)

The CE1 elements enhance the adjacent gene expressionsand bind transcription factors and chromatin recognitionproteins

Repetitive elements are dispersed into eukaryotic genomes andconsists high proportion of the genomes. Here we report a novelrepetitive family CE1, with 293 copies, in the nematode C.elegans genome. The CE1 elements were constituted a largepalindrome and associated with or within protein-coding genes.The CE1 elements formed a hairpin structure and containedmany target sites of transcription factors. CE1(bs258), a memberof the CE1 family, was located in the first intron of C46H11.6.Transcription factors and chromatin recognition proteins wereisolated by yeast one-hybrid screening using CE1(bs258) as bait.The gfp reporter gene with CE1(bs258) was expressed in skeletalmuscles, whereas the gfp reporter expression without CE1(bs258)was hardly detectable, suggesting that CE1(bs258) functions tothe expression of C46H11.6 gene. The CE1(bs258) transcript wasdetected by RT-PCR, suggesting that CE1(bs258) is transcribedand remains stable in the cytoplasm. We conclude that the CE1elements are involved in the regulation of the associated geneexpressions as an enhancer property and a chromatin remodel-ing, and thus are retained in the C. elegans genome throughouttheir evolution.

1B-14

SEZUTSU, Hideki1, UCHINO, Keiro1, KOJIMA,Katsura1, KOBAYASHI, Isao1, KANDA, Toshio1,TAMURA, Toshiki1 (1Natl. Inst. AgroBiol. Sci.)

Development of enhancer trap mutagenesis in silkworm

The genome draft sequence of silkworm (Bombyx mori) werepublished last year. In the functional analysis of the Bombyxgenome, mutagenesis and enhancer trap are important foridentifying novel genes and regulatory elements. We have beendeveloping GAL4 enhancer trap system in Bombyx to study genefunction and control the stage and tissue specific expression oftransgenes. GAL4/UAS elements were adopted with GFP as thereporter using piggyBac transposon. We have also establishedjumpstarter strain which provide piggyBac transposase usingminos transposon. For feasibility study, we analyzed theefficiency of transposition of mutator elements and the positioneffect. Cytoplasmic actin A3 promoter of Bombyx was used forGAL4 mutator (A3-GAL4) in the test. Some other promoters fromDrosophila melanogaster seem to do not work well. We mightneed to utilize useful basal promoter of Bombyx for mutators.Although in A3-GAL4 lines, the expression patterns of UAS-GFPreporter varied greatly due to the insertion positions of themutator element. GAL4/UAS elements should provide a usefulsystem for enhancer trap in silkworm.


1B-15

YOSHIDO, Atsuo1, IHARA, Jyunichiro1, YASUKOCHI,Yuji2, SAHARA, Ken1 (1Div. Appl. BioSci., Grad. Sch.Agr., Hokkaido Univ., 2Natl. Inst. Agribiological Sci.)

BAC-FISH karyotyping and the application for chromosomeanalysis in Bombyx mori

Lepidopteran species have relatively large number of holocentricchromosome and the size is similar each other. These have longhampered chromosome identification in the silkworm. In thisstudy, we have developed the chromosome identification methodfor the silkworm by FISH (Fluorescence in situ hybridization)using BAC (bacterial artificial chromosome) as probes. Thecombination of one to four either fluorescein- or Cy3-labeledprobes per chromosome allowed us to recognize unequivocallyeach of 28 pachytene bivalents of Bombyx mori karyotype by itslabeling pattern. The pattern on the most bivalents has goodcorrespondence with the determined position on the respectivelinkage map. We expect this method is applicable for genemapping and the analysis in chromosome aberration in B. mori.We will show the empirical data in chromosome analysis usingthe B. mori BAC-FISH.

2B-01

MURAGUCHI, Hajime1, MURAYAMA, Tomomi1,KAMADA, Takashi2, YANAGI, Sonoe1 (1Dept. BioTech.,Akita Prefectural Univ., 2Dept. Biol., Okayama Univ.)

Positional cloning of the Coprinus cinereus eln6 gene usingtransformation-competent BAC clones

We constructed a BAC library of Coprinus cinereus strainOkayama-7, which was used for the genome project, using avector carrying the C. cinereus trp1 gene as a selectable marker.We then assigned BAC clones to specific regions of the publishedgenome sequences by fingerprinting, the BACFinder programand BAC end-sequencing. We used this library to clone the eln6gene, which is involved in stipe elongation in the maturationstage of fruiting, and has been mapped onto chromosome XIII. Wetransformed a trp1 auxotrophic strain carrying the eln6-1mutation with BACs mapped onto chromosome XIII, resultingin identification of a BAC clone that complements the eln6-1mutation. Using a genome browser of C. cinereus, we arenarrowing the region with the complementing activity.

2B-02

TORIBA, Taiyo1, HARADA, Kohsuke1, TAKAMURA,Atsushi1, HIRANO, Hiro-yuki1,2 (1Grad. Sch. Agr. andLife Sci., Univ. Tokyo, 2Grad. Sch. Sci., Univ. of Tokyo)

Functional analysis of the rice YABBY genes by constitutiveexpression and suppression of gene function

Members of the YABBY gene family, which encode plant specifictranscriptional factors, are required for the regulation of abaxialidentity in lateral organs of Arabidopsis. On the other hand, wefound that expression patterns of rice YABBY genes differed fromthose of Arabidopsis YABBY genes, suggesting that the rice geneshave different functions. We have identified eight members of theYABBY gene family in rice. Phylogenetic analysis suggests thatOsYABBY1, OsYABBY2 and OsYABBY6 are specific to monocots.To reveal the functions of the YABBY genes specific to monocots,we produced transgenic plants in which either OsYABBY1 orOsYABBY2 mRNA expression was suppressed by the gene-specific RNAi construct. Although transcript levels of the targetmRNA decreased in the transgenic plants, no significantalterations were observed. For further investigation, we gener-ated transgenic plants in which each of the three YABBY geneswas constitutively expressed under the control of the rice actin1gene promoter. The observations of these transgenic plantssuggest that the three YABBY genes have both distinct andoverlapping functions.


2B-03

IWASAKI, Mayumi1, NITASAKA, Eiji1 (1Dept. Biol.Sci., Grad. Sch. Sci., Kyushu Univ.)

Genes controlling abaxial organ identity in the Japanesemorning glory

In the Japanese morning glory (Ipomoea nil), many mutants forleaf and flower morphology were isolated in late Edo period, andare still maintained in our laboratory. Some classes showmonstrous morphologies, e. g. a classic mutation, feathered (fe)causes loss of abaxial organ identities in leaves and flowers. FEencodes a GARP transcription factor closely related to Arabidop-sis KANADI1 (KAN1) that determines abaxial cell fate. fe strainswith stronger phenotypes are shown to have additional mutationsenhancing fe phenotype by genetic and molecular analyses. Thesemodifier genes are thought to specify the abaxial identities. Othermutations than fe modifier genes such as crepe (cp), delicate (dl)and crumpled (cm) are also known to enhance fe phenotype, andthey might be involved in adaxial-abaxial organ identity. Tounderstand the mechanism of plant development via axisdetermination, we are seeking responsible genes for thesemutations, and InCRC (Ipomoea nil’s club’s claw) was identifiedas one of modifier mutation of fe.

2B-04

MAEDA, Reo1, HOZUMI, Shunya1, TANIGUCHI,Kiichiro1, SHIRAKABE, Syuuichi1, FUJIWARA,Hiroo1, SASAMURA, Takeshi1, AIGAKI, Toshiro2,MURAKAMI, Ryutaro3, MATSUNO, Kenji1 (1Dept.Biol. Sci. and Tech., Tokyo Univ. Sci., 2Dept. Biol. Sci.s,Tokyo Metro. Univ., 3Dept. Physics, Biol. and Inform.,Yamaguchi Univ.)

Actin cytoskeleton is involved in left-right asymmetry of theembryonic gut in Drosophila melanogaster.

Bilateral animals often show left-right (L-R) asymmetry in theirinternal organs. In vertebrate, mechanisms of L-R axis formationare well understood. In mouse, directional extra-cellular flowinduced by monocilia in the node is a primary cue for L-R axisformation, and this mechanism seems to be conserved invertebrates. In contrast, it is suggested that different mecha-nisms are employed for the L-R axis formation in invertebrates.To elucidate the mechanisms of L-R axis formation in inverte-brates, we studied genetic mechanisms responsible for L-Rasymmetry in Drosophila. Here, we attempted to identify thegenes affecting handedness of the embryonic gut. We generatedhomozygous embryos for P-element insertion mutants of a largecollection of P-element insertion lines, the gene search (GS) lines.Approximately 4100 GS lines have been screened, and we isolated11 mutants, which affected L-R asymmetry of the embryonic gut.These mutants can be classified into four groups phenotypically.Analyses of these mutants and identified genes suggest that theactin cytoskeleton has an important role in L-R axis formation ofDrosophila melanogaster.

2B-05

TANIGUCHI, Kiichiro1, HOZUMI, Shunya1, MAEDA,Reo1, SASAMURA, Takeshi1, AIGAKI, Toshiro2,MATSUNO, Kenji1 (1Dept. Biol. Sci. and Tech., TokyoUniv. Sci., 2Dept. Biol. Sci., Tokyo Metropolitan Univ.)

The arrangement of visceral musculature cells controlled byJNK signaling is involved in the left-right asymmetriclooping of the anterior-midgut in Drosophila

In Drosophila, the embryonic alimentary canal mainly consists ofthe foregut, midgut and hindgut, and each of which showsstereotypic left-right asymmetry. We performed a genetic screento identify mutations affecting the left-right (LR) asymmetry inthe embryonic gut and identified puckered (puc). Homozygousembryos of puc showed inversion of the anterior-midgut (AMG)handedness.puc encodes a Drosophila JNK phosphatase, which mediatesnegative feedback regulation in the signal cascade of JNK. Wefound puc was required in the visceral mesoderm at stage11-14for the proper LR patterning of the AMG. The disruption of theLR asymmetry in puc was caused by the hyper activation of JNKsignaling in the visceral mesoderm. In wild type embryos, thearrangement of circular visceral musculature cells surroundingthe AMG was organized LR asymmetrically before the AMGlooping. In contrast, puc mutant failed to arrange these cells LRasymmetrically. Our results suggest that the down regulation ofJNK signaling by Puc in the visceral mesoderm is requited for theLR asymmetric arrangement of circular visceral musculaturecells to elicit handedness of the AMG.

2B-06

FUSE, Naoyuki1, KANESAKI, Takuma1, HIROSE,Susumu1 (1Dept. Dev. Genet., Natl. Inst. Genet.)

The heterotrimeric G proteins differentially regulate cellmovements during Drosophila gastrulation.

Gastrulation is a common developmental process and createsgerm layers by dynamic morphogenetic movements. In gastrula-tion of a Drosophila embryo, the ventral cells acquire themesodermal cell fate and undergo sequential cell movements. Ithas been shown that the concertina (cta), encoding a Ga subunitof the heterotrimeric G proteins (G proteins), is required for aninitial event of cell movements. However, mechanisms under-lying other cell movements and their coordination are largelyunknown. To address the issues, we investigate roles of the Gproteins in the gastrulation process.Since the Cta and other Ga subunits are expressed ubiquitouslyat the gastrulation stage, we examined mutants for these Gasubunits. Among them, the Gai mutant embryos showedgastrulation defects, which are distinct from the phenotypes ofthe cta mutant, suggesting that two Ga subunits regulate distinctcell movements. We also found that a novel activator of the Gprotein, DRic-8, is involved in both Cta- and Gai- dependentprocesses. We will discuss the G protein signaling for organizingvarious cell movements during gastrulation.


2B-07

KAGAWA, Hiroaki1, NAKAYAMA, Noriko1,NAKAGAWA, Kimiko1, OUCHI, Masaaki1 (1Grad.Sch. Sci. and Tech., Okayama Univ.)

Control mechanisms of LIM-8 and CeTis11 for body wall geneexpressions in Caenorhabditis elegans

The nematode Caenorhabditis elegans has pharyngeal and bodywall muscle-cells. Expression of genes in body-wall muscles ismainly controlled by CeMyoD gene, hlh-1. We isolated LIM-8/ZYX-1 that bound to CeMyoD and CeTis11, which regulatemuscle genes and hlh-1 expressions in body-wall muscles. Knockdown of CeTis11 with RNAi dramatically decreased motility ofwild type worm, on the other hand the LIM-8 (RNAi) decreased20 % of the wild type. This was consistent to the result thatCeTis11 bound to the enhancer of hlh-1 but LIM-8 did not. Theseresults suggest that LIM-8 function to modulate enhancerbinding transcription factors. We also present tissue localizationsof LIM-8 and CeTis11.

2B-08

KITAOKA, Shun1, BANDO, Tetsuya1, KAGAWA,Hiroaki1 (1Grad. Sch. Natural Sci. Tech., OkayamaUniv.)

Identification of tissue-expression control factors of thetroponin I gene, tni-3 in C. elegans

In C. elegans, three TnI genes; tni-1, -2 and -3 are expressed inbody-wall muscle and tni-4 is expressed in pharyngeal muscle.Expression patterns of three TnI genes in body-wall wereobserved by using gfp or rfp fusion construct. Three TnI geneswere co-expressed in body-wall muscle. tni-3 was stronglyexpressed in the head region of body-wall muscle. To figure outtranscription factors which regulate expression in the headregion of body-wall muscle, we performed a promoter deletionanalysis of tni-3::rfp reporters. Sequence comparisons of tni-3genes between C. elegans and C. briggsae suggested two highlyconserved sequence motifs, motif 1 and motif 2. The results ofpromoter deletion analysis showed the 168 bp sequence, whichcontains motif 1, in the 5’ upstream of tni-3 regulated expressionin the head region of body-wall muscle. We designated this regionas HE (Head Enhancer) region, and employed this region as abait for yeast one-hybrid screening. We are searching tran-scription factors regulate expression in the head region of body-wall muscle.

2B-09

OHHATA, Tatsuya1,2, HOKI, Yuko1,2, SASAKI,Hiroyuki2,3, SADO, Takashi1,2,3 (1PRESTO, Japan Sci.Tech. Agency, 2Div. Human Genet., Natl. Inst. Genet.,3Dept. Genet., The Grad. Sch. for Adv. Stud.)

Significance of the antisense transcription across the Xistpromoter in the transcriptional control of Xist

To equalize the dosage differences of X-linked genes betweenmales and females, one of the two X chromosomes is inactivatedduring the early development in female mammals. This process,termed X-inactivation, is regulated by non-coding RNAs, Xist andTsix. Recently, we reported that Tsix repressed Xist in cisthrough modification of the chromatin structure in the Xistpromoter. In this study, we attempted to terminate Tsixtranscription prematurely by introducing multiple polyadenyla-tion signals before it runs across the Xist promoter. This new Tsixmutant allele (TsixpA) allowed us to examine whether theantisense transcription of the promoter region is important forthe function of Tsix during mouse development. We will discussthe effect of the premature termination on the establishment ofthe chromatin structure in the Xist promoter.

2B-10

SADO, Takashi1,2,3, HOKI, Yuko1,3, SASAKI,Hiroyuki1,2 (1Div. Human Genet., Natl. Inst. Genet.,2Dept. Genet., The Grad. Univ. Advanced Studeis,3PRESTO, Japan Sci. Tech. Agency)

Functional analysis of antisense RNA in the regulation ofXist

X-inactivation is the one of the most critical events during thedevelopment of female mammals and the non-coding Xist geneplays a crucial role in this process. We and others previouslyshowed that expression of Xist is regulated in cis by its non-coding antisense gene Tsix. More recently, we have shown thatTsix silences Xist through modification of the chromatinstructure. It is still unknown, however, whether the RNA productis important for the function of Tsix or whether the antisensetranscription is sufficient. It is known that Tsix RNA is present inboth spliced and unspliced forms. We are currently studying thefunctional significance of the spliced Tsix RNA in its proposedrole as a chromatin modulator. We have created mice harboring anew Tsix mutation (TsixDSA), which prevents the production ofthe spliced Tsix RNA and analyzed the effect of the mutation inthe mouse embryos. We will discuss the role for the spliced RNAin the antisense regulation at the Xist locus.


2B-11

YAMAGUCHI, Masahiro1, TONOU-FUJIMORI,Noriko1, KOMORI, Atsuko1, MAEDA, Ryu2, NOJIMA,Yasuhiro3, LI, Haichang3, OKAMOTO, Hitoshi2,3,MASAI, Ichiro1 (1Masai Initiative Res. Unit, RIKEN(The Inst. Physical and Chem. Res.), 2Lab. Dev. GeneRegulation, Brain Res. Inst., RIKEN (The Inst.Physical and Chem. Res.), 3CREST, JST)

Histone deacetylase 1 regulates the timing of neurogenesis inthe zebrafish retina

In the zebrafish retina, progenitor cells initially proliferate butbegin to produce postmitotic neurons when neuronal differ-entiation occurs. However, the mechanism underlying the timingof retinal neurogenesis is largely unknown. In our large-scalemutagenesis, we identified a zebrafish mutant, ascending anddescending (add), in which retinal cells fail to differentiate butinstead overproliferate. The cloning of the add gene revealed thatit encodes Histone deacetylase 1 (HDAC1). HDAC1 is associatedwith the transcription repressors and represses the transcriptionof target genes by altering the acetylation status of histone. Wntsignaling promotes cell proliferation and Notch signaling inhibitsneurogenesis in the zebrafish retina. We found that both the Wntand Notch pathways are de-suppressed in the add mutant retina,and that the blockade of these pathways inhibits the over-proliferation in add mutant retina, suggesting that HDAC1antagonizes these pathways to promote retinal neurogenesis.From these data, we propose that the balance between HDAC1and Wnt/Notch activity is important for the timing of retinalneurogenesis in zebrafish.

2B-12

ISHIDA, Ryutaro1, INADA, Eriko1, TANAKA,Shigekazu2, TAMURA, Masaru2, MASUYA, Hiroshi3,WAKANA, Shigeharu3, SHIROISHI, Toshihiko2,3,YAMAMOTO, Hiroaki1 (1Dept. Dev. Biol. and NeuroSci.,Grad. Sch. Life Sci., Tohoku Univ., 2MammalianGenet. Lab., Natl. Inst. Genet., 3Mouse FunctionalGenomics Res. Group, RIKEN GSC)

Characterization of a new mouse coat color mutant strainwith age- and hair cycle- dependent coat color changedifferent from graying

In the previous meeting, we reported the mutant mouse strainM100288, generated by ENU-mutagenesis, changes its coat colorfrom black to brown with aging and exhibits aberrant skinstructures associated also with aberrant pigmentation-geneexpression. Here we report another coat color mutant strainM100212 also generated by the same mutagenesis. This mutantresembles the M100288 mutant strain but has much intensivephenotypes both in the coat color and the skin structures.Analyses of pigmentation-gene expression by both RT-PCR andhistochemical methods throughout the hair cycle showeddecreased activity of these genes in the mutant in anagen.Normally, genes involved in the melanization are highly activeand the melanin pigment is produced during this phase. Ourresults suggest that mechanisms underlying maintenance of thepigment production during anagen in the wild type function andthese pigmentation genes are affected all together directly orindirectly by the causative gene of the M100212 strain in thephase.

2B-13

TAMURA, Masaru1,2, FUJII, Tomoaki1,2, SHIROISHI,Toshihiko1,2 (1Mamamlian Genet. Lab., Natl. Inst.Genet., 2Sch. Life Sci., The Grad. Univ. Adv. Stud.)

GsdmC4, a novel member of the gene family Gsdm, ispreferentially expressed in lower gastrointestinal tract

Previously, we isolated a novel gene family, named Gsdm. One ofthe family members was originally identified as causative gene ofa mouse mutant Ricombinant-induced mutation 3 (Rim3), whichexhibits epidermal hyperplasia, hyperkeratosis and abnormalhair development. Here we report a novel member of the Gsdmfamily, GsdmC4, which is one of structurally related four genesclustering in the mouse chromosome 15. Northern blot andquantitative RT-PCR analyses showed that GsdmC4 is expressedspecifically in lower gastrointestinal tract. In situ and immuno-histochemical analyses revealed that GsdmC4 mRNA is localizedin crypt region of the intestinal epithelium, and that the GsdmC4expressing cells do not overlapped proliferating cell (PCNA-positive cell). The results of this study together with the previousanalysis of the mutant mice, Rim3, suggested that GsdmC4 playa key role in regulation of cell proliferation and/or differentiationof epithelium in lower gastrointestinal tract.

2B-14

FUJII, Tomoaki1,2, TAMURA, Masaru1,2, SHIROISHI,Toshihiko1,2 (1Shcool of Life Sci., The Grad. Univ. Adv.Stud., 2Mammalian Genet. Labolatory, Genetic StrainsRes. Cent., Natl. Inst. Genet.)

Expression and functional analyses of GasderminD (GsdmD)

In the previous meeting, we reported a novel gene family, namedGasdermin (Gsdm), one of the family members was identified ascausative gene for a mouse skin mutant Rim3. Here we reportresults of expression and functional analyses of GsdmD, amember of Gsdm family gene. The expression analysis demon-strated that GsdmD is predominantly expressed in lowerintestinal tract and in villi of intestine in which differentiatedcells exist. In order to elucidate the function of GsdmD in vivo, wegenerated transgenic mice that contain GsdmD harboring Rim3-type mutation. The mice exhibit hyper-proliferation of intestinalepithelial cells. These results suggest that GsdmD is involved incontrolling proliferation of intestinal epithelial cells, although itis expressed in differentiated cells.


2B-15

SUGIMOTO, Michihiko1, MEKADA, Kazuyuki2,KARASHIMA, Yuko3, YUZURIHA, Misako1, KO,Minoru Sh4, NAGARAJA, Ramaiah4, TAN, Seong-seng5, TAKAGI, Nobuo3, ABE, Kuniya1 (1Technologyand Development Team for Mammalian CellularDynamics, BioResource Center, RIKEN, 2ExperimentalAnimal Division, 3Div. Bioscience, Grad. Sch.Environmental Earth Science, Hokkaido Univ., 4NIA/NIH, USA, 5The Univ. Melbourne, Australia)

Rescue of the t-complex recessive lethal mutation tclw5 by a180kb BAC clone

The mouse t-complex is a variant of the proximal region ofchromosome 17. The t-complex contains four tandem inversionsrelative to the wildtype, and therefore recombination in t-heterozygotes is severely suppressed along the t-complex. Thevariant form of the t-complex found in wild mice is known as t-haplotypes, which usually contain a recessive lethal mutation.tclw5 is a t-complex recessive mutation in the tw5-haplotype,which causes embryonic lethality at the gastrulation stage. In thehomozygotes, extensive death of the embryonic ectoderm wasobserved, whereas the extraembryonic tissues were less affected.However, our tetraploid rescue experiments have shown that theproduct of tclw5 likely functions in extraembryonic tissues andsupports proliferation/differentiation of embryonic ectodermthrough cell-cell interactions. These results point to the tclw5product as an important regulator of pluripotent cell developmentin mammalian embryos. In this study, we carried out transgenicrescue using five BACs, and successfully rescued the lethality oftw5/tw5 embryos by one BAC, indicating that tclw5 is locatedwithin a 180kb BAC, harboring 15 transcription units.

3B-01

FUJITA, Mika1, KATO, Hiroaki1, SAITO, Tamao1,KATO, Atsushi1 (1Div. Biol. Sci.s, Grad. Sch. Sci.,Hokkaido Univ.)

Analysis of non-coding RNAs found in Arabidopsis thariana

Many non-coding RNAs (ncRNAs) exept for rRNAs and tRNAshave been found in various organisms. Also in Arabidopsisthariana, a lot of ncRNAs which functions are unknown werereported. In order to esstimate their function, we analyzed someArabidopsis ncRNAs. Sequencing analysis of RT-PCR productsrevealed that different transcripts were produced from one geneby alternative splicing and using different polyadenylation sits.Expression analysis showed that the quantities of some ncRNAswere different among several organs and the transcriptions ofsome ncRNAs were regulated by some plant hormones. Theseresults suggest that these ncRNAs play an active role in vivo.

3B-02

KATO, Hiroaki1, FUJITA, Mika1, KOMEDA,Yoshibumi2, SAITO, Tamao1, KATO, Atsushi1 (1CellStructure and Function, Div. Biol. Sci.s, Grad. Sch.Sci., Hokkaido Univ., 2Plant Sci., Dept. Biol. Sci.s,Grad. Sch. Sci., The Univ. Tokyo)

Analysis of an acl2 mutant defective in flower stalkelongation in Arabidopsis thaliana

An acaulis2 (acl2) mutant has a defect in flower stalk elongation.No ORFs were detected around a changed base that is assumed tobe the origin of this mutant. However, when a wild-type DNAfragment containing the mutation point was introduced into theacl2, the mutant phenotype was complemented. Since theseresults suggested that non-coding RNAs were transcribed nearthe mutation point, we performed the isolation of them usingRACE procedure. In consequence, several kinds of transcriptscontaining the mutation point, probably non-coding RNAs, wereidentified. We considered the possibility that these non-codingRNAs regulated the expression of other genes to affect flowerstalk elongation. Therefore we conducted microarray analysis. Asa result, multiple genes the expression of which changed in themutant were identified.


3B-03

TAKUMI, Shigeo1, SUGIE, Atsushi1, MATSUOKA,Yoshihiro2, MURAI, Koji2, NAKAMURA, Chiharu1

(1Faclt. Agr., Kobe Univ., 2Dept. BioSci., Fukui Pref.Univ.)

Increased expression of the alternative oxidase gene Waox1ain wheat accessions showing hybrid necrosis

In Triticum and Aegilops, F1 progeny derived from somecombinations of inter- and intraspecific crosses are bound tonecrotic death. This phenomenon called hybrid necrosis isconsidered to be one of genetic mechanisms for reproductiveisolation. Ne1 and Ne2 loci are well known to control hybridnecrosis in wheat. We compared expression patterns of severalgenes encoding mitochondrial proteins between wild-type andnecrosis-induced plants. Transcripts of alternative oxidase (AOX)gene Waox1a were more abundant in the necrotic F1 plants thanin the wild-type plants. The increased expression of Waox1a wasalso observed in a novel lesion-mimic mutant of common wheat.Paralogous AOX gene Waox1c, however, showed no significantalterations in its expression level. Moreover, other types of hybridnecrosis independent of the Ne1/Ne2 system were found incrossed progeny between Aegilops tauchii accessions and anemmer wheat cultivar Langdon. Study of the geographicaldistribution of hybrid necrosis-related genes showed that mostAfghanistan and Pakistan accessions of Aegilops tauschiipossessed the type-II hybrid necrosis-related gene.

3B-04

YOSHIMOCHI, Takeshi1, ADACHI, Masanori1, KIBA,Akinori1, HIKICHI, Yasufumi1, OHNISHI, Kouhei2

(1Dept. Biores. Sci., Kochi Univ., 2Res. Inst. ofMolecular Genetics)

Global regulation of pathogenicity genes at early stages of theinfection process of Ralstonia solanacearum

Plant pathogen Ralstonia solanacearum causes wilt disease inmany plants. R. solanacearum attaches to host plant rootsurfaces and invades the intercellular spaces of the root cortex,within which it rapidly multiplies and colonizes. Deliveringeffector proteins into the host cell through a type III secretionsystem is necessary for R. solanacearum cells to multiply.Structural genes for the type III secretion apparatus and theeffectors form a hrp regulon. The expression of the hrp regulon isactivated by plant cell signals, which are recognized by PrhA onthe bacterial outer membrane and transferred to hrpB expressionvia a signal cascade; PrhA-PrhR/PrhI-PrhJ-HrpG. By cocultivat-ing R. solanacearum with Arabidopsis thaliana, we haveinvestigated the regulation of signal cascade gene expression.While the expression of prhA was constitutive, other genesinvolved in plant signal cascade were expressed depending on celldensities. A negative regulator PhcA in the hrp regulon repressedthe expression of the prhR/prhI operon. Taken together, at highcell densities the activated PhcA represses the prhR/prhI operonand resulted in the repression of hrpB and the entire hrp regulon.

3B-05

HIRONO, Satomi1, TANIKAWA, Kaori1, HARA,Hiroshi1, MATSUMOTO, Kouji1 (1Dept. Biochem. Mol.Biol., Faclt. Sci., Saitama Univ.)

Analysis of the function of the acidic phospholipid in Bacillussubtilis by using multicopy suppressors

In Bacillus subtilis, phosphatidylglycerol (PG), the acidic phos-pholipid, has been considered to be essential for cell viability. ThepgsA gene is the structural gene of the enzyme that catalyzes thecommitted step in the biosynthesis of the major acidic phospho-lipid. The reduction of the expression of pgsA results in a reducedcontent of PG in the cell membrane and causes cell lysis. Tounderstand the function of the acidic phospholipid, we identifiedyqgB, yceC, yufM, ywnF genes as multicopy suppressors whichsuppress the lethal phenotype caused by reduced expression ofpgsA. We examined the conditions required for the efficientsuppression to elucidate how these suppressors suppress thelethal phenotype. Our goal is to understand the function of theacidic phospholipid in Bacillus subtilis.

3B-06

SHIBA, Yasuhiro1, MATSUMOTO, Kouji1, HARA,Hiroshi1 (1Dept. Biochem. and Molec. Biol., Faclt. Sci.,Saitama Univ.)

Stimulation of Escherichia coli Rcs phosphorelay regulatorysystem by stagnation of outer membrane lipoprotein RcsF inthe inner membrane

It is not clear by what mechanism RcsC, the inner membrane-associated sensor kinase of the Rcs phosphorelay regulatorysystem of Escherichia coli, senses external stimuli. Among outermembrane and periplasmic proteins, only RcsF, an outermembrane lipoprotein, is known as an essential compornent ofthe Rcs system. The precursor of RcsF undergoes multistepmodification and processing on the periplasmic side of the innermembrane, and the matured RcsF is then transported to theouter membrane.In this study, we showed that stagnation of RcsF in the innermembrane activated the Rcs system. Inhibition of lipoproteinsignal peptidase by globomycin caused the activation of thesystem. Alternation of lipoprotein-sorting signals at positions 2and 3 of the mature form indicated that mislocalization of RcsF tothe inner membrane led to higher Rcs signaling, depending onthe amount of RcsF stagnated in the inner membrane. Wepropose a model that RcsC senses direct interaction of thestagnated RcsF with it as a stimulus. Changes in the externalenvironment that somehow affect the maturation and/or trans-port of newly synthesized RcsF would activate the Rcs system.


3B-08

KUTSUKAKE, Kazuhiro1,2, WADA, Takeo2,3,OOZAWA, Mizuki2 (1Grad. Sch. Natural Sci. Tech.,Okayama Univ., 2Dept. Biol., Faclt. Sci., OkayamaUniv., 3Grad. Sch. Eng., The Univ. Tokyo)

A novel addiction module in Salmonella enterica serovarTyphimurium

RpoS is a sigma factor specific for stationary phase and stressfulconditions and degraded by ClpXP protease during exponentialgrowth. LT2, a standard laboratory strain of Salmonella entericaserovar Typhimurium, has an rpoS gene whose initiation codon isTTG and does not show a detectable RpoS activity. An LT2derivative in which this initiation codon has been replaced withATG shows a high RpoS activity. In this study, we found that theclpP gene could not be replaced with its disrupted version in thisRpoS+ strain. This suggests that overproduction of RpoS causesgrowth inhibition in Salmonella. We isolated a mutant in whichthe RpoS-induced growth inhibition was suppressed. This mutantwas found to have a defect in a not-yet-characterized ORF. Wenamed this gene rsaB. We showed that transcription of the rsaBgene was impaired by an rpoS mutation and overexpression ofthis gene caused growth inhibition. We showed further thatoverexpression of another gene rsaA locating just upstream of thersaB gene could suppress the rsaB-induced growth inhibition. Weconclude that the rsaB and rsaA genes constitute together anaddiction module encoding a toxin and an antitoxin, respectively.

3B-09

YAMAMOTO, Shinji1, SUZUKI, Katsunori1 (1Dept.Biol. Sci., Grad. Sch. Sci., Hiroshima Univ.)

Characterization of stability genes in the plant-tumorinducing plasmid pTi-SAKURA

Stability of Ti plasmids differs depending on strains in Agro-bacterium tumefaciens. High stability sometimes causes troublein deletion. In this study, we localized what determines stabilitydifference between two nopaline type Ti plasmids, pTi-SAKURAand pTiC58 in 2.6-kbp NheI fragment of pTi-SAKURA. Two openreading frames tiorf24 and tiorf25 are situated on the fragment.Deletion of either one of the two ORFs abolished the plasmidstabilization effect. RT-PCR experiments indicated that the twoORFs form an operon. The two ORFs conferred similar effect onan incP-type replicon in cis-acting manner.

3B-10

KOMANO, Teruya1, AKAHANE, Kenji1, SAKAI,Daisuke1, FURUYA, Nobuhisa1 (1Dept. Biol., TokyoMetro. Univ.)

Genetic analysis of the pilU gene encoding the prepilinpeptidase for the biogenesis of type IV pili in plasmid R64

The PilS prepilin of plasmid R64 type IV pili is processed by pilUprepilin peptidase to produce the mature pilin. To study therelationship between the structure and function of the PilUprepilin peptidase of plasmid R64, 43 missense pilU mutationswere constructed by PCR and site-directed mutagenesis, and theability of these pilU mutants to complement a pilU-null mutantin liquid matings was analyzed.Although practically no conjugation was noted for 16 of themutants, the remaining 27 exhibited varying levels of residualtransfer activity. Two mutants with aspartic acid replacements inconserved motifs exhibited no pilU activity, suggesting that theR64 pilU gene product is an aspartic acid peptidase, as was thecase for Vibrio cholerae Tcp protein. No PilS processing wasdetected for 16 of the mutants, but the remaining 27 mutantsexhibited varying levels of residual PilS processing.From the assay of pilU-phoA and pilU-lacZ fusion genes carryingdifferent lengths of pilU, a PilU membrane topology waspredicted. Changes in the PilU membrane topology wereobserved from some amino acid substitutions in the pilU portionof the pilU-phoA and pilU-lacZ fusion genes.

3B-11

TANAKA, Yoshiharu1, KONDO, Ryouta1 (1Dept. LifeSci.s, Faculty of Liberal Arts Sci.s, Osaka PrefectureUniv.)

Relationship between the expression patterns of Drosophilasodium channel alternative exons and the phenotype.

Mutants of the DROSOPHILA sodium channel gene show heat-induced paralysis and hypersensitivity to diethylether anesthe-sia. Two alleles, parahd838 and parats1 show alternative sensitiv-ities to ether anesthesia and heat-induced paralysis relative withthe wild type (Canton-S). The mutations would cause aberrationsof RNA splicing that result in alternatively spliced mRNAs thatmay produce proteins with abnormal or reduced functions. Tocompare the usage of alternative exons of the sodium channelgene between the wild type, the parahd838 and the parats1, andinvestigate the correlation between the mRNA types and thephenotypes of the para alleles, RT-PCR was performed with theRNAs from the adults and larvae of the three strains, using theprimers of several constitutive exons and alternative exons.


3B-12

IBARAGI, Yoko1, KANKI, Tomotake2, TANIMURA,Teiichi3, KANG, Dongchon2, MATSUURA, Etsuko4

(1Dept. Molec. Biol. and Biochem., Ochanomizu Univ.,2Dept. Clinical Chemistry and Lab. Med., KyushuUniv. Grad. Sch. of Med. Sci., 3Dept. Biol., Grad. Sch.Sci.s, Kyushu Univ., 4Dept. Biol., Ochanomizu Univ.)

Effects of mitochondrial transcription factor A in ageing ofDrosophila

Mitochondrial transcription factor A (TFAM) is the primarycomponent necessary for transcription initiation of mitochondrialDNA (mtDNA). TFAM has been recently shown to packagemtDNA without sequence specificity and to be essential formtDNA maintenance. To further understand TFAM functions indevelopment and ageing, human and Drosophila TFAM-DCwhich lack C-terminal 25 amino acids necessary for the tran-scription initiation were expressed in D.melanogaster by GAL4/UAS system. When the two kinds of TFAM-DC were expressed bythe Actin-Gal4 driver at 25˚C and 28˚C, only flies possessinghuman TFAM-DC were obtained at 25˚C. When their lifespan wasexamined at 28˚C, it significantly reduced. These suggest that anexcess of TFAM-DC extremely lower viability during developmentand that TFAM may be involved in ageing. In contrast, whenexpression of TFAM-DC was induced by the hs-Gal4 driver, flieswere viable at 25˚C for both human and Drosophila TFAM-DC.The amount of mtDNA surprisingly increased to about 8-fold ofcontrols when they were maintained at 25˚C or 30˚C for 5 daysafter eclosion. Examination of their lifespan is now in progress.

3B-13

MASUYA, Hiroshi1, SHIMIZU, Kunihiko2, MIURA,Ikouo1, SEZUTSU, Hideki3, SAKURABA, Yoshiyuki1,MAEDA, Takahide2, GONDO, Yoichi1, NODA, Tetsuo1,WAKANA, Shigeharu1, SHIROISHI, Toshihiko1

(1Functional Genomics Res. Group, RIKEN GSC,2Dept. Pediatric Dentistry, Nihon Univ. School ofDentistry at Matsudo, 3Insect Gene Eng. Lab., InsectBioTech. and Sericology Dept.)

Amelogenesis imperfecta mouse mutants induced by ENUmutagenesis

ENU is a powerful chemical mutagen that randomly inducespoint mutations in genomic DNA at a high frequency. Phenotype-based screening allows comprehensive collection of mutations ingenes involved in specific biological pathways.We isolated multi-ple ENU-induced dominant mouse mutants which caused AI-likephenotypes. Three of these mutants were mapped to a region ofchromosome 5 and it was revealed that they are mutations ofgenes encoding enamelin (Enam). Another mutant was mappedon chromosome X. Sequence analysis revealed there is single-base substitution in amelogenin (Amelx).

3B-14

WADA, Adumi1, NISHIMURA, Masahiko2, OHKAWA,Kiyoshi1, TSUDZUKI, Masaoki3 (1Lab. AnimalFacility, Res. Cent. Med. Sci., Jikei Univ. Sch. Med.,2The Inst. Lab. Animal Res., Grad. Sch. Med., NagoyaUniv., 3Lab. Animal Breeding and Genet., Grad. Sch.Biosphere Sci., Hiroshima Univ.)

Dominant Yellow Coat Color Mutation Found in PhodopusHamster-Establishment of Congenic Strain and Research for Nucleo-tide Substitution Responsible for the Coat Color Mutation-

Phodopus hamsters are the small rodents having hairy feet, andthis genus has three species, P. campbelli, P. sungorus, and P.roborovskii.In December, 2001, we established an inbred strain of P.campbelli (PMI) by full-sib matings. In July, 2005, we got thehamsters at the 32nd generation. In November, 1999, we found amutant sungorus hamster having yellow coat and black eyes. Thephenotype was controlled by an autosomal dominant gene, andthe mutant gene has been introduced into the Pmi line (afterDecember, 2001, PMI strain) of the campbelli hamster bysuccessive backcrosses. At present, the congenic strain hasreached the 15th generation. Mating experiments suggested thathomozygotes for the mutant gene show prenatal lethality.Furthermore, heterozygotes (yellow hamsters) exhibited obeseand hyperglycemia. These facts suggested that this hamstermutation is similar to the mouse yellow (Ay) mutant. Thus, wedecided the sequence of the nucleotide (394 bp) including partlythe coding region of the agouti gene of the hamster. Moreover,RT-PCR revealed that agouti gene expression is enhanced in theskin of the yellow mutant hamster.


1C-01

YOKOYAMA, Katsushi1,2, OYAMADA, Tomoya3,SHINAGAWA, Hideo4, SUZUKI, Masashi1, MAKINO,Kozo3 (1Natl. Inst. Adv. Industr. Sci. and Tech. (AIST),AIST Tsukuba Center 6-10, 2Japan Sci. Tech. Agency(JST), 3Dept. Applied Chemistry, Natl. Defense Acad.,4Res. Inst. Microb. Dis., Osaka Univ.)

Molecular analysis of the hdiI gene in EHEC O157 which issimilar to the dinI gene in E. coli K-12.

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strainRIMD 0509952 derived from the Sakai outbreak in 1996 has theShiga toxin 1 genes that are carried by the prophage, VT1-Sakai.VT1-Sakai carries a unique gene named as hdiI (homolog of dinI)and the HdiI protein is homologous to DinI of E. coli K12. Wehave shown that hdiI gene belongs to the SOS regulon and theoverproduction of HdiI protein inhibits not only the transcriptioninitiation of hdiI gene but also those of other SOS genes such asrecA, lexA and umuD. The overproduction of HdiI protein alsodecreases lambda phage production from the lysogenic E. coliK12, suggesting that HdiI protein inhibits cleavage of CI, therepressor of lambda. We also indicated that the overproduction ofHdiI protein decreased the transcription initiation of N genewhich is negatively regulated by CI repressor. These data suggestthat HdiI protein inhibits the RecA activity in response to SOS.

1C-02

OYAMADA, Tomoya1, YOKOYAMA, Katsushi2,3,SUZUKI, Masashi2, MAKINO, Kozo1 (1Dept. AppliedChemistry, Natl. Defense Acad., 2Natl. Inst. Adv.Industr. Sci. and Tech. (AIST), AIST, 3Japan Sci. Tech.Agency (JST))

Identification of CRP-cAMP dependent promoters fromEnterohemorrhagic E. coli O157

We have cloned CRP-cAMP dependent promoters from Enter-ohemorrhagic E. coli O157:H7 genome by the whole genomeshotgun method. The 51 identified CRP-cAMP dependentpromoters were scattered in both regions common to the non-pathogenic strain, K12 and specific to the O157:H7. Thepromoters conserved in K12 regulated many genes includingtwo-component regulatory genes and Y genes whose functionswere unclear. The promoters specific to O157:H7 regulated phagegenes, ATP dependent DNA helicase gene, etc.

1C-03

YOKOYAMA, Katsushi1,2, OYAMADA, Tomoya3,SHINAGAWA, Hideo4, SUZUKI, Masashi1, MAKINO,Kozo3 (1Natl. Inst. Adv. Industr. Sci. and Tech. (AIST),2Japan Sci. Tech. Agency (JST), 3Dept. Appl. Chem.,Natl. Defense Acad., 4Res. Inst. Microb. Dis., OsakaUniv.)

Molecular analysis of the specific two component regulatorysystems in the EHEC O157:H7.

We searched new response regulator and sensor kinase genes bycomparison of two Escherichia coli genomes, enterohemorrhagicE. coli (EHEC) O157:H7 strain RIMD 0509952 derived from theSakai outbreak in 1996 and E. coli K12. We found new two pairsof response regulator and sensor kinase genes in the O157genome. To search promoter regions regulated by these responseregulators, the O157 DNA fragments were randomly cloned intothe plasmid containing the promoterless lacZ gene as a reportergene. Overproduction of these respose regulators positivelyregulated the transcription initiation of the operons present inthe O157 specific regions. We also indicated that the expression ofone of these response regulator genes was regulated by CRP-cAMP.

1C-04

YOSHIDA, Yusuke1, IIGUSA, Hideo1, HASUNUMA,Kohji1 (1Kihara Inst. Biol. Res., Yokohama City Univ.)

Roles of reactive oxygen species in the light-dependentcarotenogenesis in Neurospora crassa

In Neurospora crassa, carotenogenesis in the mycelia is inducedby light. A sod-1 mutant, with a defective superoxide dismutasecatalyzing conversion of superoxide to hydrogen peroxide, showedhyper-accumulation of light induced carotenoid. These suggestedthat intracellular reactive oxygen species enhanced light-depend-ent carotenogenesis. Moreover, we found that the light-inducedcarotenogesis was promoted by the exposure to air. Myceliatreated with oxygen gas and hydrogen peroxide were moreaccumulated than that of the non-treatments. These resultssuggested that stimuli caused by the exposure of the mycelia toair containing oxygen gas triggered the light-induced caroteno-genesis.


1C-05

WANG, Ni-yan1, YOSHIDA, Yusuke1, LEE, Bumkyu1,HASUNUMA, Kohji1 (1Kihara Inst. Biol. Res., YokohamaCity Univ.)

Conidial germination rate is reduced in a Catalase-1 loss-of-function mutant in Neurospora crassa

Catalase-1(CAT-1) in Neurospora crassa is one of the threecatalases which are known to detoxify hydrogen peroxide (H2O2),and is largely synthesized during conidial formation andmaturation. Conidia have 60 times more catalase activity thanhyphae growing in liquid medium. Here we investigated theeffect of CAT-1 inactivation on conidia by using CAT-1 loss-of-function mutant (cat-1RIP) obtained by RIPing method. No CAT-1activity was detected in cat-1RIP, while the mRNA abundancy ofcat-1 was normal. DNA sequencing analysis of cat-1RIP showedthat 40 guanines were replaced with adenines which caused 30amino acid substitutions. The mutant strain grew normally, butmycelia of mutant were more sensitive to high concentration ofH2O2 (20mM) than those of wild type. Moreover the lack of CAT-1activity reduced the conidial germination rate significantly.

1C-06

OBATA, Kazue1, OTSUJI, Takafumi1, MATSUMOTO,Takashi1, ASAI, Kei1, SADAIE, Yoshito1 (1Dept.Biochem. and Molec. Biol., Faclt. Sci., Saitama Univ.)

Analysis of the SigI function in Bacillus subtilis

SigI of Bacillus subtilis is a sigma factor of sigma 70 family.Transcription of the sigI operon was measured as activity of heatstable BgaB from the bgaB gene fused to the sigI promoter. Heatinduces the transcription of the sigI operon and this inductionwas stimulated greatly by disrupting ykrI gene that is locatedimmediate downstream of the sigI. Furthermore yeast two-hybridanalysis showed that N-terminal portion of the YkrI proteininteracted directly with SigI, and disruption of dnaK abolishedabove heat stimulation of the sigI operon. These results suggestthat SigI is a sigma factor of stress response but is not a sigmafactor of ECF sigma type, and YkrI plays a role of anti-sigmafactor protein like and DnaK is necessary for SigI function.

1C-07

UEHARA, Takuya1, MIURA, Daisuke1, ASAI, Kei1

(1The Dept. Biochem. and Molec. Biol., Saitama Univ.)

Expression control of ECF ssM and ssW in Bacillus subtilis

In Bacillus subtilis, ECF sigma factors are activated to transcribethe genes responding to various stresses from the outside. It is acharacteristic that an activity of ECF sigma factor is negativelycontrolled by anti-sigma protein. In this study, the purpose isunderstanding of activation mechanism of ECF sigma factor, sM

and sW.We found that the degradation of anti-sigma led to the activationof sM and sW. In addition, we identified some mutations whichaffected activity of sM or sW by the method of transposonmutagenesis (20000 colonies in sM and 40000 colonies in sW weretested).

1C-08

SHIMURA, Daisuke1, ASAI, Kei1, SADAIE, Yoshito1,SATOMURA, Takenori2, HIROOKA, Kazutake2,FUJITA, Yasutaro2 (1Dept. Biochem. and MorecularBiology, Faclt. Sci., Saitama Univ., 2Dept. BioTech.,Faclt. Life Sci. and BioTech., Fukuyama Univ.)

Analysis of the response regulators of unknown function inBacillus subtilis

There are many two-component systems of unknown function inBacillus subtilis. DNA microarray analysis revealed the geneswhose transcription was activated when response regulatorswere overproduced. In this study, above transcriptional activa-tion was confirmed by lacZ assay in vivo and regulon genes werefurther analyzed. In addition to the above transcriptionalanalysis, gel mobility shift analysis was performed to show directbinding of response regulators (YcbB,YbdJ,YesN,YvfU) to reg-ulon genes in vitro. The genes of response regulators wereadjacent to regulon genes and response regulators showed directbinding ability to the promoter DNA of the regulon. In the future,we will reveal the function of two-component systems by studyingthe function of regulon genes.


1C-09

KISHIMOTO, Naoki1, FUJII, Fumiko1, YAZAKI,Junshi1, TAKEUCHI, Keiko1, TOYOSHIMA, Kazuko1,SHIMBO, Kanako2, SHIMATANI, Zenpei2, NAGATA,Yuko2, HASHIMOTO, Akiko2, SUZUKI, Koji3,KOJIMA, Keiichi3, ISHIKAWA, Masahiro1, SASAKI,Takuji1, KIKUCHI, Shoshi1 (1Natl. Inst. AgroBiol.Sci., 2STAFF-Inst., 3Hitachi Software Eng.)

Microarray analysis of plant cultured cells treated withhistone deacetylase inhibitors

KISHIMOTO, Naoki1, FUJII, Fumiko1, YAZAKI, Junshi1, TAKE-UCHI, Keiko1, TOYOSHIMA, Kazuko1, SHIMBO, Kanako2,SHIMATANI, Zenpei2, NAGATA, Yuko2, HASHIMOTO, Akiko2,SUZUKI, Koji3, KOJIMA, Keiichi3, ISHIKAWA, Masahiro1,SASAKI, Takuji1 and KIKUCHI, Shoshi1 (1Natl. Inst. Agrobiol.Sci., 2STAFF-Institute, 3Hitachi Software Engineering)Microarray analysis of plant cultured cells treated with histonedeacetylase inhibitorsHistone deacetylase inhibitors (HDAI) are pivotal research toolsfor epigenetic regulation of gene expression. We carried outexpression profiling of cultured rice cells treated with one of 3HDAIs (butyrate, HC toxin and trichostatin A(TSA)) using cDNAmicroarray system and clustering analysis of the microarray datausing the Rice Expression Database. Rice cells treated withbutyrate and HC toxin, showing similar expression profiles, weregrouped in a cluster including rice plants infected by blast andrice cells treated with aphidicolin and metal ions (Al and Ni). Ricecells treated with TSA was grouped in another cluster including asilencing transgenic plant, an epigenetic virescent mutant andrice cells treated with 5-azacytidine (DNA methylation inhibitor).

1C-10

ITO, Yukihiro1,2, KURATA, Nori1,2 (1Plant Genet.Lab., Genetic Strains Res. Cent., Natl. Inst. Genet.,2Life Sci.s, SOKENDAI)

Cytokinin-induced expression of a KNOX homeobox gene inrice

KNOX homeobox genes play a pivotal role for shoot apicalmeristem (SAM) formation and maintenance, and their specificexpression in the SAM is essential for normal development ofplants. We found that cytokinin induces expression of a riceKNOX homeobox gene OSH1 upon regeneration of shoot fromcallus, and examined whether His-Asp phosphorelay genesmediate the OSH1 expression. Five histidine kinase (HK) genes,five phosphotransmitter (HPt) genes and six B-type responseregulator (B-RR) genes relating to the His-Asp phosphorelaywere identified in the rice genome. Overexpression of an HK gene(COS3), an HPt gene (OHP2) or a B-RR gene (ORR1) in callusenhanced cytokinin-induced expression of OSH1, and converselyexpression of a dominant negative form of COS3 reduced it. Thesethree genes are expressed in overlapped regions of callus. In theyeast two-hybrid analysis OHP2 interacted with COS3 andORR1. These results show that these cytokinin-signaling genesmediate cytokinin-induced expression of OSH1 and that they arepositive regulators of the OSH1 expression in rice.

1C-11

AOKI, Setsuyuki1, TSUKUDA, Masashi1, OKADA,Ryo1, KONDO, Sayo1, TEZUKA, Yuki2 (1Grad. Sch.Inform. Sci., Nagoya Univ., 2Sch. Inform. and Sci.s)

Circadian clock-related genes in the moss Physcomitrellapatens

We started to study the circadian clock using the mossPhyscomitrella patens from the viewpoint of the evolution ofcircadian clocks in plants. Gene targeting techniques based onhomologous recombination are applicable to this moss. Wereported that the Lhcb genes encoding the light-harvestingproteins of the photosystem II are controlled by the clock inconstant darkness in P. patens (Aoki et al., PCP, 2004). Here wereport the isolation and structural and expression analyses ofPpCCA1a and PpCCA1b encoding the P. patens homologs for theArabidopsis thaliana clock genes CCA1 (Circadian Clock Asso-ciated 1) and LHY (Long elongated HYpocotyl).

1C-12

SASAMURA, Takeshi1,2, ISHIKAWA, Hiroyuki3,SASAKI, Nobuo1, HIGASHI, Syunsuke1, KANAI,Maiko1,2, NAKAO, Shiho1, AYUKAWA, Tomonori1,AIGAKI, Toshiro4, NODA, Katsuhisa5, MIYOSHI,Eiji5, TANIGUCHI, Naoyuki5, MATSUNO, Kenji1,2,3

(1Dept. Biol. Sci. Tech., Tokyo Univ. Sci., 2PRESTO,JST, 3Genome Drug Res. Cent., Tokyo Univ. Sci.,4Dept. Biol. Sci.s, Tokyo Metropolitan Univ., 5Dept.Biochem., Osaka Univ., Grad. Sch. Med.)

Drosophila O-fucosyltransferase, O-fut1 regulates Notchturnover and intracellular sorting

Notch signaling is an evolutionarily conserved mechanism thatregulates a broad spectrum of cell-specification events throughlocal cell-cell communication. Drosophila O-fut1 catalyzes the O-fucosylation of Notch, a modification essential for Notch signalingand ligand binding. Here, we show that O-fut1 has additionalfunctions in the regulation of Notch signaling: it regulates theturnover and trafficking of the receptor. First, we found O-fucosylation is essential for the localization of Notch to adherensjunctions.Recently, it was reported that O-fut1 has a Notch chaperoneactivity, and in O-fut1 mutant cells, Notch is retained in ER byquality control mechanism. However, our results indicate thatNotch is not accumulated in ER, but in early endocytic vesicles inO-fut1 mutant cells. Moreover, O-fut1 is indispensable fortranslocation of Notch to early endosomes. This function,however, is independent of O-fut1’s O-fucosyltransferase activity.We propose that extracellular O-fut1 regulates Notch intra-cellular sorting in O-fucosylation independent manner.


1C-13

OHTA, Hiromitsu1, FUJIWARA, Manabi1, OHSHIMA,Yasumi2, ISHIHARA, Takeshi1 (1Dept. Biol., Faclt.Sci.s, Kyushu Univ. Grad. Sch., 2Dept. Applied LifeSci., Faclt. BioTech. and Life Sci., Sojo Univ.)

Identification of new genes involved in body size regulation inC. elegans

Animals have a specific body size for species. There are noelephant-size mouse and mouse-size elephant. How body sizes ofanimals are determined so exactly? To reveal molecular mecha-nisms of body size regulation, we utilized C. elegans. In C. elegansas a model organism, body size is mainly controlled by TGFbpathway. To identify new genes involved in body size regulation,we screened for the mutants with changed activity of the TGFbpathway, by monitoring the expression levels of genes, which areregulated by TGFb pathway. In this screening, we isolated 10lines of mutants and found that among them, 2 mutants exhibitlarge body size. Positional cloning of one of them, qj1, revealedthat the gene encodes a novel protein with no homologue in otherspecies. To identify the function in body size regulation, weanalyzed body size of double mutants of qj1 with other mutationsof TGFb pathway, and found that qj1 seems to negativelyregulate Smad transcription factors independent of TGFb. qj1 isexpressed in hypodermis, where Smad functions, and localized innuclei. These results indicate that qj1 controls body size byinhibiting Smad activity.

1C-14

ANOKYE-DANSO, Frederick1, ANYANFUL, Akwasi2,SAKUBE, Yasuji1, KAGAWA, Hiroaki1 (1Grad. Sch.Natural Sci. Tech., Okayama Univ., 2Emory Univ.,Atlanta, GA)

EGL-18, a GATA transcription factor, regulates tropomyosinisoforms III and IV expressions in pharyngeal and intestinalmuscles of Caenorhabditis elegans

The C. elegans single tropomyosin gene, tmy-1 generates fourisoforms -CeTMI/II/III and IV. CeTMI/II are expressed in bodywall, anal and sex muscles and, CeTMIII/IV expressed inpharynx and intestines. To identify factors involved in expressionregulation of isoforms CeTMIII/IV, the internal promoter of tmy-1was analyzed. Promoter deletion assay showed that the regu-latory element for CeTMIV expression was 405 to 505 bpupstream of the transcription start codon. The GATA-like sitecontributed to intestinal expression but not pharyngeal expres-sion as shown by mutation of the GATA-like site. In a yeast one-hybrid screen, EGL-18 bound to a bait taken from the internalpromoter. Knockout of EGL-18 by RNA-mediated interferenceresulted in embryonic lethality and L1 larva that survived died atthe L1 stage. CeTMIV reporter genes were not expressed in theL1 larva that survived the egl-18 RNA-mediated interferencetreatment. Consistent with RNAi knockout, egl-18(mt162) andegl-18(ew34) mutant worms did not express CeTMIV reporters.These results suggest that EGL-18 controls transcription of tmy-1from the internal promoter.

2C-01

ITO, Hidetaka1, MIURA, Asuka1, TAKASHIMA,Kazuya1, KAKUTANI, Tetsuji1 (1Dept. IntegratedGenetics, Natl. Inst. Genet.)

Ecotype-specific evolution of centromeric satellites in Arabi-dopsis thaliana

Arabidopsis centromeres contain large array of satellite repeatsof 178-bp length, which is thought to provide the centromerefunction. It is believed that the satellite repeats in the array arehomogenized by occasional unequal crossing-over, but it is notknown how they are homogenized among chromosomes. It is alsonot known how the copy number of the satellite repeats iscontrolled. Here we report evolution of diverged satellitesequence in one chromosome in one Arabidopsis ecotype. Thepresence of the diverged satellites in that centromere isassociated with increase of satellite repeat number and deletionof significant part of the adjacent heterochromatic region. Wediscuss possible implication of these observations.

2C-02

YOKOTA, Etsuko1,2, SHIBATA, Fukashi1,2,MURATA, Minoru1,2 (1Res. Inst. Biores., OkayamaUniv., 2CREST, JST)

A novel dicentric ring chromosome in Arabidopsis thaliana

The smallest chromosome was found in a transgenic plant ofArabidopsis thaliana. Fluorescence in situ hybridization (FISH)with 18S and 5S rDNAs as probes indicated that this mini-chromosome (2S-D) originated from the short arm of chromosome2. Its size was estimated to be approximately 5 Mb, but notelomere sequences were detected. This indicated that thisminichromosome is a ring chromosome. FISH probed with the180-bp repeats and some BAC clones revealed that there are twocentromeres whose size is ca. 500 kb each, and two copies of theparacentric region from BAC-T5E7 to -F3C11 in the 2S-Dchromosome. This suggests that this dicentric ring chromosomeoriginated from a small ring chromosome (ca. 2.5 Mb) by a singlecrossing-over. Since the centromere-specific protein HTR12 wasfound to localize on both of the 180-bp clusters, those two 180-bpclusters have normal centromere functions. Nevertheless, theminichromosome was stably transmitted to the next generation,so we also analyzed the behaviors of 2S-D at mitosis and meiosis.


2C-03

SHIBATA, Fukashi1,2, YOKOTA, Etsuko1,2,MURATA, Minoru1,2 (1Res. Inst. Biores., OkayamaUniv., 2CREST, JST)

Pseudo-dicentric chromosome in transgenic Araidopsis thali-ana

After transformation experiment using in plant method onArabidopsis thaliana, one plant containing several irregularchromosomes were found. Chromosome complements wereexamined in the offspring of this individual crossing with normalColumbia. Irregular chromosomes were originated in chromo-some 2. Translocation of a fragment of chromosome 2 tochromosome 1 was observed on one chromosome (2S-B). 2S-Bhas two clusters of centromeric repeat, one is normal centromericcluster of chromosome 1 and the other is short fragment ofcentromeric cluster of chromosome 2. The short cluster is only300 kb in length, it is 1/10 times shorter than that of normalcentromeric repeat of chromosome 2. There was no localization ofcentromeric protein HTR12 on the short centromeric repeatobserved on 2S-B and chromosome behaviour of 2S-B in meiosisand mitosis shows that the short centromeric repeat on 2S-Bmight be inactive as centromere.

2C-04

KAWABE, Akira1, CHARLESWORTH, Deborah1

(1Inst. Evolutionary Biol., Univ. Edinburgh)

Centromere location and association of chromosome rear-rangement in A. lyrata and A. thaliana

We tested for linkage and determined the chromosomal positionsof genes in A. lyrata ssp. petraea that are located near thecentromere (CEN) regions of A. thaliana, to analyze chromosomerearrangements and the effect on the centromere regions. Genesfrom each of the ten A. thaliana chromosome arms are alsotightly linked in A. lyrata. Genes from the regions on both sides ofCEN5 have distant map localizations in A. lyrata, whereas genesfrom the other four A. thaliana centromere regions remain linkedin A. lyrata ssp. petraea. This observation suggests that thecentromere positions are conserved between A. thaliana and A.lyrata, even though three centromeres have been lost in A.thaliana, and the core satellite sequences in the two species arevery different. We will discuss karyotype evolution and its effecton the centromere regions in Arabidopsis relatives.

2C-05

NAGAKI, Kiyotaka1,2, KASHIHARA, Kazunari1,2,MURATA, Minoru1,2 (1Res. Inst. Biores., OkayamaUniv., 2Core Research for Evolutional Sci. and Tech.,Japan Sci. Tech. Agency)

Molecular characterization of Luzula nivea holocentricchromosomes with centromere-specific histone H3

The centromere is an essential region for distributing chromo-somes into daughter cells, and only a centromere exists in achromosome at a primary constriction. However, as exceptionalcases, holocentric chromosomes do not have any primaryconstriction, and it looks like entire regions act as a centromere.Luzula species have been known to possess holocentric chromo-somes for a long time, but no critical studies of their kinetochoreproteins have been made. In this study, we successfully isolated acDNA encoding a putative centromere-specific histone H3(LnCENH3) by RT-PCR and RACE-PCR. The deduced aminoacid sequence was then used to raise an anti-LnCENH3 antibody.Immunostaining clearly revealed the diffuse centromere-likestructure that appears in the linear shape at prophase totelophase. Furthermore, it was shown that the amount ofLnCENH3 decreased significantly at interphase. The polar sidepositioning on each chromatid at metaphase to anaphase alsoconfirmed that LnCENH3 represents one of the centromere-specific proteins in L. nivea.

2C-06

ASHIDA, Taizo1, NASUDA, Shuhei1, SATO,Kazuhiro2, ENDO, Takashi R.1 (1Lab. Plant Genet.,Grad. Sch. Agr., Kyoto Univ., 2Res. Inst. Biores.,Okayama Univ.)

PHYSICAL MAPPING OF BARLEY ESTs ON CHROMO-SOME 5H USING STRUCTURAL ABERRATIONS OFCHROMOSOME 5H

In hexaploid wheat, chromosome breakage can be induced by theuse of a gametocidal chromosome derived from Aegilops cylin-drica. When the gametocidal chromosome is present in mono-somic condition, chromosome structural changes occur ingametes lacking it. By using this gametocidal system, we inducedstructural aberrations in barley chromosome 5H added tocommon wheat. Aberrant 5H chromosomes (31 deletions, 83translocations and 10 iso-chromosomes) were detected by acombination of FISH (probe, barley subtelomeric repeatsHvT01) and GISH (probe, total genomic barley DNA). Thebreakpoints of deletion and wheat-barley translocation providenew physical landmarks for barley chromosome. In order toconstruct a physical EST map of barley chromosome 5H, 108barley-specific EST markers that had been assigned to chromo-some 5H were physically mapped to sub-arm regions by using 22aberrant 5H chromosomes in hemizygous or homozygous con-dition.


2C-07

OKUDA, Michiaki1, NASUDA, Shuhei1, ENDO,Takashi R.1 (1Lab. Plant Genet., Grad. Sch. Agr.,Kyoto Univ.)

Analysis of chromosomal localization patterns of modifiedhistones in wheat

In eukaryotes, chromatin structure has an important role inregulation of gene expression and chromosome segregation.Chromatin structure is mainly classified into two types: euchro-matin and heterochromatin. These two states of chromatin arecharacterized by difference in chromatin condensation and intranscriptional activity of the genes in the regions. Recent studiesin mammals revealed that combinations of post-translationalmodifications (methylation, acetylation, and phosphorylation) ofhistone residues determine the state of chromatin structure.However, only a limited information is available in plant species.To investigate chromatin structure in plants, we observedchromosomal localization patterns of several modified histonesin common wheat and its relatives. Our observation suggests thatcombination of histone modifications varies according to chromo-somal regions, and that histone modification is involved information of higher structure of mitotic chromosome.

2C-08

TSUJIMOTO, Hisashi1, HAGRAS, Adel A..1, SATO,Kazuhiro2, KISHII, Masahiro3, DOU, Quanwen1,TANAKA, Hiroyuki1 (1Lab. Plant Genet. and BreedingSci., Faclt. Agr., Tottori Univ., 2Res. Inst. forBioresouces, Okayama Univ., 3Internatl. Maize andWheat Improvement Center (CIMMYT))

Collection and development of genetical resources for wildspecies in tribe Triticeae and their practical application forgenome analysis of a wild species, Hordeum chilense

The variation included in the wild species of tribe Triticeae isconsidered to be the genetical resources for the breeding ofcultivated species in the same tribe as wheat, barley, and rye.However, this variation is not always used efficiently because oflack of enough genetical bases for the wild species. First wecollected and developed wheat strains carrying alien chromo-somes as the bio-resources to make a solid gene bank for the plantmaterials. Next, we developed PCR markers to detect the alienchromosomes added in the wheat genetic background using thegenome resource of barley, i. e., EST markers. We will introducethe activity and report one of our studies using the resources:genome analysis of a wild species, Hordeum chilense.

2C-09

KIKUCHI, Shinji1, TANAKA, Hiroyuki1, TSUJIMOTO,Hisashi1 (1Lab. Plant Genet. and Breeding Sci., Faclt.Agr., Tottori Univ.)

Evolution of genome basic number in the genus Torenia -Does a gene-poor supernumerary chromosome exist? -

In evolution, the variability of the genome basic number isdifferent depending on the taxa. F1 hybrid between Toreniafournieri (TF, 2n = 2x = 18) and Torenia baillonii (TB, 2n = 2x =16) having different genome basic number showed eight bivalentsplus one univalent in the meiotic cells. FISH revealed that theunivalent is of TF and carried no rDNA. This result suggestedthat the addition of a pair of extra chromosomes without manygenes to TB genome produced the TF genome.

2C-10

SAITO, Yoshinaka1, ABE, Tomoko2, KISHII,Masahiro3, TANAKA, Hiroyuki1, TSUJIMOTO,Hisashi1 (1Lab. Plant Genet. and Breeding Sci.,Faclt. Agr., Tottori Univ.,, 2RIKEN, DRI, PlantFunctions Lab, 3International Maize and WheatImprovement Center(CIMMYT))

Estimation of chromosome breakage in wheat by irradiationof heavy ion beam

To develop the method to use heavy ion beams for thechromosome manipulation in wheat, we irradiated the acceler-ated 20Ne beams in dose of 30-50 Gy to the dry seeds of wheatstrains carrying alien (Leymus racemosus) chromosomes. Ge-nomic in situ hybridization (GISH) using the DNA of the alienspecies showed chromosome rearrangements as translocation,insertion, and deletion, in a very high frequency. The analysisrevealed that the frequency of the aberration seen by GISH iseight times more than that observed by ordinary cytologicalmethod with acetocarmine staining.


2C-11

TANABE, Hideyuki1 (1Dept. Biosyst. Sci., Sch. Adv.Sci., The Grad. Univ. Adv. Stud. (Sokendai))

Chromosome territory arrangement of radial and relativepositioning in interphase cell nuclei demonstrated by 3D-FISH technique

Three-dimensional fluorescence in situ hybridization (3D-FISH)technique enabled us to visualize the painted individualchromosome structure, which are highly compartmentalizedcalled “chromosome territories” that are considered as essentialcomponents of the higher order chromatin architecture. Recentstudies in mammals indicated that the radial position of a givenchromosome territory is correlated with its size, its chromatincomposition and replication timing (i.e.; human 18 and 19chromosomes show a different position in lymphocytes andlymphoblastoids; the gene-poor, late replicating chromosome 18territory is preferentially located at the nuclear periphery, whilethe gene-rich and early replicating chromosome 19 territory isfound in the nuclear center). Here we demonstrated by 3D-FISHtechnique to analyze the two aspects of radial and relativepositioning of chromosome territories: human chromosomes 18and 19 territories for evolutionary conservation and for tumori-genesis and human chromosome 2 and its homologs for evolu-tionary translocation in primates, respectively. This newapproach will disclose the functionally relevant principles ofhigher order nuclear architecture.

2C-12

SUGAYA, Kimihiko1, HONGO, Etsuko1, ISHIHARA,Yoshie1, TSUJI, Hideo2 (1Radiation Hazards Res.Group, Natl. Inst. Radiological Sci., 2Low DoseRadiation Effects Research Project, Natl. Inst.Radiological Sci.)

A temperature-sensitive mutation in the WD repeat-contain-ing protein Smu1 is related to maintenance of chromosomeintegrity

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibitschromosomal instability and cell cycle arrest at S and G2 phaseswith decreased DNA synthesis at 39oC. To identify the causativemutation, we fused tsTM18 cells with normal human cells togenerate hybrids carrying fragments of human chromosomes.Analysis of chromosome content of temperature-resistant trans-formants and introduction of a bacterial artificial chromosomecontaining part of human chromosome 9 led to isolation of thehuman SMU1 gene. Comparison of sequences of the Smu1 genefrom wild-type and mutant cells revealed that the mutantphenotype is caused by a G-to-A transition that yields a gly-to-arg substitution at position 489 in hamster Smu1. The sub-stituted glycine is located in the WD-repeat domain of Smu1.Single-stranded DNA accumulated in the nuclei of mutant cellsat 39oC. Furthermore, cdc2 kinase was not activated during G2

phase, and there was no chromosome segregation due toincomplete assembly of the spindle during M phase. Thus,Smu1 appears to be involved directly or indirectly in DNAreplication, activation of cdc2 kinase, spindle assembly, andmaintenance of chromosome integrity.

2C-13

MURAKAMI, Shigefumi1,2, NAKAMURA, Ken-ichi1,2,MASUKATA, Hisao1,2, NAKAGAWA, Takuro2

(1Guraduate School of Frontier Bioscience, OsakaUniv., 2Dept. Biol. Sci., Grad. Sch. Sci., Osaka Univ.)

Isolation of the novel genes that are required for chromosomestability

Maintaining chromosome integrity is crucial for all living cellsand organisms. The gross chromosomal rearrangements (GCRs)can cause cell death or genetic diseases including cancer.However, the underlying mechanism that preserves chromosomeintegrity has not been elucidated. To understand the molecularmechanism, we set up the assay system that can detect GCRs aswell as chromosome loss using fission yeast, S. pombe. The yeastcells were mutagenized by chemical treatment, and the clonesthat exhibit enhanced rates of GCRs and hypersensitivity tohydroxiurea (HU) and/or methyl methanesulfonate (MMS) wereisolated. In this search, we have isolated a mutant of swi1+, thegene product of which is involved in stability of replication forksand in sister chromatid cohesion. In the swi1 mutant, the rates ofGCRs and chromosome loss were increased 90- and 40-fold,respectively, compared to those of wild type. These resultsindicate that Swi1 plays important roles in the genome stability.In addition, we also found a novel mutant that shows increasedGCRs and HU-/MMS-hypersensitivity. The role of the geneproducts in the chromosome maintenance will be discussed.

2C-14

NUNOSHIBA, Tatsuo1, WATANABE, Eri1,TAKAHASHI, Teruhisa1, DAIGAKU, Yasukazu1,2,UI, Ayako2, ISHIKAWA, Satoko3, MOCHIZUKI,Masataka3, ENOMOTO, Takemi2, YAMAMOTO,Kazuo1 (1Grad. Sch. Life Sci.s, Tohoku Univ., 2Grad.Sch. Pharm. Sci., Tohoku Univ., 3Kyoritsu Univ.Pharmacy)

Mechanisms for Loss of Heterozygosity (LOH) induced bynon-mutagenic carcinogens

LOH is one of the important steps for carcinogenesis. We havedemonstrated that DNA lesions induced by mutagenic carcinogenstimulate LOH induction in S. cerevisiae diploid cells predom-inantly caused by gene conversion or allelic crossover. We alsofound that some of non-mutagenic carcinogens such as o-phenylphenol (OPP) and its metabolite dihydroxy biphenyl (DHBP), etc.induced LOH via loss of a chromosome containing non-mutatedallele. To elucidate the mechanisms, we examined effects ofDHBP on yeast cell cycle, on tubulin proteins, and on theirpolyemrization/ depolymerization in vitro. These results suggestthat abnormality of chromosomal segregation, due to partialinhibition of tubulin depolymerization caused by interaction ofsuch chemicals with tubulin, may be an important mechanism forLOH, genetic instability and carcinogenesis induced by non-genotoxic carcinogenesis.


2C-15

KANEKO, Mami1, TAGA, Masatoki1 (1Grad. Sch.Natural Sience and Tech., Okayama Univ.)

Cytogenetic analysis of chromosomes and karyotype in aoomycete Phytophthora infestans

Oomycete is a large group of microbial eukaryotes that has beenintensively studied in mycology and plant pathology. In spite ofits importance, morphology and structure of chromosomes as wellas karyotype of this group remain unknown, including a speciesP. infestans for which genome project is underway. In this study,we observed mitotic chromosomes of P. infestans by lightmicroscopy and attempted karyotyping. For preparing slidespecimens, germinating encysted-zoospores were treated withcolchicine and colcemid to arrest M phase, subjected to the germtube burst method, and then stained with DAPI or Giemsa.Differential stainings including Ag-NOR were also conducted.Chromosomes were composed of coiling sister chromatids, anddistinct constrictions reminiscent of centromeres and AT-richheterochromatin were apparent. The overall morphology of thesechromosomes looked similar to those of higher plants andanimals. In chromosome counting, somewhat bizarre resultswere obtained, that is, chromosome number varied greatly evenin a single strain from 8 to 30, which made determination of bothbasic chromosome number and ploidy level difficult for P.infestans.

3C-01

MIZUTA, Mami1, SATOH, Emi1, NANBA, Michiyo1,SUZUKI, Katsunori1 (1Dept. Biol. Sci., Grad. Sch. Sci.,Hiroshima Univ.)

Comprehensive screening of budding yeast mutants defectivein recipient ability in trans-kingdom conjugation with donorEschelichia coli

The prokaryotes Escherichia coli and Agrobacterium harboringtra genes can transfer plasmid DNA to yeast cells. Thephenomenon called (Super)kingdom conjugation takes place bythe action of the tra genes in bacteria, while little is known aboutfactors in recipient yeast cells. Because entire plasmid DNA istransfered into yeast cells, the phenomenon might have causedconsiderable effect on evolution of eukaryotic organisms. Purposeof this study is to find out comprehensively genes important forthis phenomenon in recipient yeast. We found 29 mutantsdefective in the transkingdom conjugation ability, after wecarefully examined 4,828 haploid MATa strains among the Yeastdeletion clones constructed by the yeast genome analysisconsortium.

3C-02

KASHIWAZAKI, Jun1, NAKAMURA, Taro1, SHIMODA,Chikashi1 (1Dept. Biol., Grad. Sch. Sci., Osaka CityUniv.)

Two Rab7 orthologs antagonistically control vacuolar mor-phology in fission yeast

A small G protein Rab7 is involved in membrane fusion. S. pombecontains two Rab7 orthologs (Ypt7 and Ypt71), which shareapproximately 60% identical amino acid residues. Probably, theyplay important roles in vacuolar morphology and function,because these proteins localized to vacuolar membrane. Ypt7may promote vacuole fusion, as ypt7D cells had extremely smallvacuoles. In contrast, the ypt71D cells had large vacuoles, andoverexpression of ypt71+ in wild-type cells caused fragmentationof vacuoles. These observations suggest that Ypt71 eitherpromotes vacuole fission or inhibits vacuole fusion. Here wepropose a model that two Rab7 orthologs antagonistically controlvacuolar morphology.


3C-03

ITADANI, Akiko1, NAKAMURA, Taro1, SHIMODA,Chikashi1 (1Grad. Sch. Sci., Osaka City Univ.)

Identification and role of ring components at leading edge offorespore membrane in fission yeast

The nascent plasma membrane of new-born spores, called theforespore membrane (FSM), is constructed within the cytoplasmof diploid zygotes in fission yeast. FSM is initially formed nearthe spindle pole body, a centrosome-equivalent structure, duringmeiosis II. Each haploid nucleus is encapsulated by the extendedFSM. Meu14, a coiled-coil protein localizes to the leading edge ofthe developing FSM to form a ring structure. We identified Act1(actin), Myo1 (type I myosin), Cdc12 (forming) and Cml1 (calm-odulin-like protein) as novel components of the leading edge ring.Phenotypic analysis of the mutants of these proteins revealedthat they play pivotal roles in the formation of the FSM. Wespeculate that the leading edge ring of the FSM may relate to theactomyosin contractile ring in cytokinesis.

3C-04

YAMAMOTO, Satoshi1, KUTSUKAKE, Kazuhiro1

(1Grad. Sch. Natural Sci. Tech., Okayama Univ.)

Salmonella FlgJ protein: role of the N-terminal region in itsexport and function

FlgJ protein of Salmonella enterica serovar Typhimurium iscomposed of 316 amino acids and exported into the periplasmicspace via the flagellum-specific pathway. N-terminal 98 aminoacids of FlgJ is essential for rod formation, whereas C-terminal165 amino acids encodes a flagellum-specific muramidase neededfor efficient penetration of the rod structure into the peptidogly-can layer. In this study, we showed that the polypeptideconsisting of only the C-terminal 165 amino acids of FlgJ couldnot be exported, suggesting that the N-terminal region isessential for export of the protein. This C-terminal 165-amino-acid region was fused to another flagellar protein FlgA, which isknown to be exported via the Sec pathway. The resulting fusionprotein seemed to be able to support efficient penetration of therod structure into the peptidoglycan layer. This result suggeststhat the export via the flagellum-specific pathway per se is notprerequisite for the muramidase domain of FlgJ to function invivo. On the basis of these results and other available informa-tion, we discuss the function of the N-terminal region of FlgJ inflagellar morphogenesis.

3C-05

KUSAKA, Jin1, NISHIBORI, Ayako1, HARA, Hiroshi1,UMEDA, Masato2, MATSUMOTO, Kouji1 (1Dept.Biochem. and Molec. Biol., Faclt. Sci., Univ. Saitama,2Inst. Chem. Res., Kyoto Univ., Uji, Kyoto)

Analysis of the factor involved in the localization of thephospholipids in B. subtilis

We have found that cardiolipin (CL) is localized at the septalmembrane in B. subtilis cells by using the CL-specific fluorescentdye 10-N-nonyl-acridine orange (NAO). And we have foundfurther that phosphatidylethanolamine (PE) is localized mainlyat the septum by using the PE-specific tetracyclic polypeptideprobe Ro09-0198 (Ro) and that most phospholipids synthasesfused to green fluorescence protein (GFP) are also localized at theseptum.Through the examination with Ro conjugated to FITCand that without fixation, we indicated that PE is certainlylocalized at the membranes in the septum. When GFP fusions tolipid synthases were expressed under their own, natural,promoters, we also observed the fluorescence in the septum. Toclarify the region of CL synthase required for targeting to theseptum, we constructed deletion derivatives of ClsA andexamined their localization.

3C-06

KUDOU, Yukari1, KUSAKA, Jin1, HARA, Hirosi1,MATSUMOTO, Kouji1 (1Dept. Biochem. Mol. Biol.,Faclt. Sci., Saitama Univ.)

Analysis of the region of Bacillus subtilis phosphatidylgly-cerophosphate synthase targets to septal membrane

In Bacillus subtilis, it has been demonstrated that cardiolipin(CL) and phosphatidyl ethanolamine(PE) are localized mainly inthe septal membranes. By using GFP fusion, the majority of thelipid synthases were also localized in the septal membranes.Phosphatidylglycerophosphate synthase, PgsA, that catalyzes thecommitted step for the synthesis of the anionic phospholipidconsists of 193 amino acid residues. GFP fusions with a series ofPgsA fragments that progressively deleted from the N-terminusor C-terminus were constructed and their subcellular localizationwas examined. It appears that L10-I107 region of PgsA isrequired to target the enzyme to the septal membranes. Furtherwork for identification of the region of PgsA required for thetargeting is now in progress.


3C-07

KAWAI, Fumitaka1, HARA, Hiroshi1, MATSUMOTO,Kouji1 (1Dept. Biochem. Mol. Biol., Saitama Univ.)

Cardiolipin enrichment in Bacillus subtilis spore membranesand its involvement in germination

Bacillus subtilis undergoes sporulation during nutrient starva-tion from vegetative cells into metabolically dormant andenvironmentally resistant spores. When conditions of its environ-ment are favorable for growth, the spore germinates and goesthrough outgrowth, converted back into growing cells. There arethree sets of nutrient receptors that recognize specific nutrientcalled germinants. Cardiolipin(CL), one of the major acidicphospholipids of B. subtilis membranes, increased, up to 40% ofthe total lipids, in spore membranes, compared with that of themembranes in vegetative cells. Spores of the mutants withdisruptions in all the three paralogous genes of CL synthasecontained a trace amount of CL, suggesting that B. subtilis hasan unknown route of CL synthesis. Spores of the mutantsexhibited no turbidity decrease after exposure to germinants.However, the mutant spores showed normal rate of pyridine-2,6-dicarboxylic acid release after induction with dodecylamine.These results indicate that CL has an important role in earlystep of germination probably related with the germinantreceptors.

3C-08

MATSUNAGA, Sachihiro1, KURIHARA, Daisuke1,KAWABE, Akira1, UCHIYAMA, Susumu1, FUKUI,Kiichi1 (1Dept. BioTech., Grad. Sch. Eng., OsakaUniv.)

Dynamic analyses of plant Aurora kinases during mitosis

Aurora kinases are serine/threonine protein kinases withessential roles in cell division through eukaryotes. They regulatethe interaction between cytoskelton and chromosomes at thekinetochore, the spindle assembly and the cytokinesis. Weidentified three Aurora kinases in Arabidopsis thaliana,AtARK1, AtARK2 and AtARK3. The kinase domain of AtARK1shares 95% and 63% amino acid identity with AtARK2 andAtARK3, respectively. There are two transcriptional forms ofAtARK2. To reveal the localization of AtARKs during plantmitosis, dynamic analyses was performed using GFP-fusedproteins. The localization of AtARK proteins was classified intotwo classes. One class showed subcellular localization of mitoticspindles during cell division. The other class showed the local-ization on chromosomes. The localization of AtARK proteins wasdifferent from that of animal Aurora kinases, reflecting thedifference in cell division between plant and animal. Reference:Kawabe, A. et al. (2005) Characterization of plant Aurora kinasesduring mitosis. Plant Mol. Biol. 58, 1-13.

3C-09

ASHIDA, Yasuyo1, MATSUOKA, Yoshihiro2, NASUDA,Shuhei1, ENDO, Takashi R.1 (1Lab. Plant Genet.,Grad. Sch. Agr., Kyoto Univ., 2Fukui PrefecturalUniv.)

Analysis of meiotic cell division of triploid wheat byimmunostaining of histone H3 phosphorylated at serine 10

Triploid wheat derived from crosses between tetraploid wheat(Triticum turgidum) and Aegilops tauschii produces unreducedgametes. Cytologically, we demonstrated that the unreducedgametes were generated by “single-division meiosis (SDM)”,where only one round of chromosome segregation occurred. Tocharacterize the SDM process, we adopted immunostaining ofhistone H3 phosphorylated at serine 10 (phosH3S10) that showsdifferent chromosomal localization patterns between mitosis andmeiosis. In hexaploid wheat, signals of phosH3S10 were observedat pericentromeric region during metaphase to anaphase inmitotic cells. Signals were detected on entire chromosome duringMI to AI, and were localized at pericentromeric regions duringMII to AII. In triploid wheat, immunostaining signals wereobserved at entire chromosome during prometaphase to meta-phase like first meiotic division in hexaploid wheat. When meioticcells proceeded from metaphase to anaphase, immunosignalswere localized at pericentromeric region and soon disappeared attelophase. Our results suggest that the change of division typesoccurred at metaphase in SDM.

3C-10

SHINOHARA, Miki1, OSHIUMI, Hiroyuki1,SHINOHARA, Akira1,2 (1Div. Integrated ProteinFunctions, Inst. Prot. Res., Osaka Univ., 2Dept. Biol.,Grad. Sch. Sci., Osaka Univ.)

Functional analysis of Spo16, a new component of Synapto-nemal complex in budding yeast

A sporulation specific gene, spo16, was previously isolated, butthe detail of its function has not reported yet. We constructed aspo16mutant strain and analyzed it during meiosis. The spo16mutant generated low viable spores and which showed high 0/2/4-vaiable-spore bias. Then we analyzed the meiotic DSBs andrecombinants in the mutant. The spo16 mutant cells showed adelay in transition of prophase-I to MI with accumulated DSBs,and showed a partial defect in crossover formation. The heteroduplex assay revealed that a ratio of NCR to CR was elevated inspo16 mutant compared to that in WT. The ZMMs (Zip1/2/3,Mer3, Msh4/5) are known as essential components of Synaptne-mal complex formation and crossover regulation. The zmmmutants have already known to show CR specific defect as wellas spo16 mutant. Next, we analyzed Zip1 localization in spo16cells. In wild type cell, Zip1 initially appear as dots, then whichare elongated as lines. In the spo16 mutant cell, initial dots wereobserved, but they were not elongated enough. This resultsuggests that the Spo16 is required for Zip1 function. Now, weconcluded that Spo16 is a new member of ZMMs.


3C-11

MATSUHIRA, Hiroaki1, HARADA, Michiyo1, KUBO,Tomohiko1, MIKAMI, Tetsuo1 (1Lab. Genetic Eng.,Grad. Sch. Agr., Hokkaido Univ.)

Structural polymorphism of mitochondrial metalloprotease-like gene cluster in the restorer-of-fertility, Rf1, locus ofsugar beet.

CMS is a maternally inherited defect in pollen production that isthought to result from the expression of unusual or aberrantmitochondrial genes. The mitochondrial defect can be compen-sated for by specific nuclear genes, termed restorer of fertility (Rf)genes. In order to understand the molecular interaction of Rfwith the sterilizing mitochondrial gene, we have attempted toisolate Rf1 of sugar beet. We have completed the nucleotidesequencing of the region containing Rf1. This result revealed 28ORFs, four of which are found to encode metalloprotease-likeprotein (MPL) having mitochondrial targetting property. Thefour MPLs were organized in a tandem array in Rf1 genomicregion.Interestingly, southern blot analysis suggested that the MPLgene cluster was found to be severely truncated form in the rf1plant. In order to understand the molecular basis of thepolymorphisms, we have determined the nucleotide sequence ofMPL cluster in rf1 plant. From a detailed comparison of the MPLclusters in Rf1 and rf1, we concluded that MPL of rf1 wasgenerated from MPLs of Rf1 by unequal recombination events.

3C-12

SAWAMURA, Kyoichi1, TOMIMURA, Yoshihiko2,SATO, Hajime3, MATSUDA, Muneo3, OGUMA,Yuzuru1 (1Grad. Sch. Life and Env. Sci., Univ.Tsukuba, 2Shiba Gakuen, 3Sch. Med., Kyorin Univ.)

Quasi-linkage detected in the process of establishing inter-specific mosaic genome lines between Drosophila ananassaeand Drosophila pallidosa by means of parthenogenesis

Strong sexual isolation exists between the closely related species,Drosophila ananassae and Drosophila pallidosa, but there is noobvious postmating isolation; both sexes of the hybrids and theirdescendants appear to be completely viable and fertile. Strainsexhibiting parthenogenesis have been derived from wild popula-tions of both species. We intercrossed such strains and estab-lished iso-female lines after the second generation ofparthenogenesis. These lines are clones, carrying homozygouschromosomes that are interspecific recombinants. We established266 such isogenic lines and determined their genetic constitutionby using chromosome and molecular markers. Strong quasi-linkage was seen between loci on the left arm of chromosome 2and on the right arm of chromosome 3; the frequency of inheritingthe two loci from the same species was significantly larger thanexpected. The possible causes, female meiotic bias and geneticincompatibility, are discussed in this presentation. The inter-specific mosaic genome lines reported here will be useful forfuture research identifying genes of speciation and characterevolution.

3C-13

TANEICHI, Takasuke1, KOIDE, Yohei1,NISHIMOTO, Daisuke1, SANO, Yoshio1 (1Lab. PlantBreeding, Grad. Sch. Agr., Hokkaido Univ.)

Hybrid sterility gene S13 found in a distantly related ricespecies, O. longistaminata

The hybrid sterility has been studied in rice to understand thedevelopment of reproductive barriers during speciation. How-ever, information on sterility barriers is still limited in distantlyrelated rice taxa. We examined hybrid sterility genes betweenAsian rice (O. sativa) and African wild species (O. longistamina-ta) with the AA genome. We found that O. longistaminata carriedS13 gene, so called pollen killer, which induced abortion ofmicrospores with the alternative allele (S13a) carried by aJaponica strain (T65), suggesting that gamete eliminator (thestrongest sterility gene) is not necessarily present betweendistantly related taxa. The fine mapping revealed that S13 islinked to A (DFR) with 0.16cM on chromosome 1. Two NILshaving S13 or S13a were used to examine the allelic state in 25Asian strains. Three Japonica strains gave semi-sterile andfertile hybrids in crosses with NILs having S13 and S13a,respectively, while all the other strains including the wildprogenitor gave only fertile hybrids in both crosses. This showsthat most of the strains carried a neutral allele,S13n, suggestingthat S13a has emerged from S13n.

3C-14

OKA, Ayako1, MITA, Akihiko1, TAKAHASHI, Riichi2,AOTO, Toshihiro2, TOTSUKA, Yoshikazu2, UEDA,Masatsugu2, YAMATANI, Noriko1, YAMAMOTO,Hiromi1, TAKAGI, Nobuo3, MORIWAKI, Kazuo4,SHIROISHI, Toshihiko1 (1Lab. Mammalian Genet.,The Natl. Inst. Genet., 2The YS Institute, Inc., 3Dept.economics, Hokusei Gakuen Univ., 4RIKEN BioResourceCenter)

Genetic Analysis of Hybrid Breakdown Caused by Xchromosome Substitution between Two Mouse Subspecies

Reproductive isolation is possibly caused by incompatibilitybetween multiple genes, alleles of which are fixed differentiallyin genetically diverging two populations. Two mouse strains,C57BL/6J (predominantly originated from Mus musculus domes-ticus) and MSM/Ms (M. m. molossinus), are genetically differ-entiated, and succeeding intercrosses result in hybridbreakdown, one form of reproductive isolation. A consomic strain,B6.XMSM, which carries MSM/Ms-derived X chromosome (Chr) inC57BL/6J genetic background, shows male sterility, suggestingincompatibility of MSM/Ms-derived X Chr with other C57BL/6Jchromosomes. In this study, we performed QTL analysis to mapgenes interactive with X Chr. It successfully mapped significantQTLs on Chr1 and Chr11. Males carrying MSM/Ms-derived XChr and the candidate regions of Chr1 and Chr11 partiallyrestored the fertility, indicating that incompatibility of thesegenes causes hybrid breakdown between C57BL/6J and MSM.These genes possibly had a crucial role in mouse subspeciation ofM. m. domesticus and M. m. molossinus.


3C-15

ABE, Kuniya1,2, FUCHIKAMI, Takuya1, SUGIMOTO,Michihiko1, KOBAYAKAWA, Satoru1,2, MISE, Nathan1

(1Mammalian Cellular Dynamics, RIKEN BioResourceCenter, 2Grad. Sch. Life and Env. Sci., Univ. Tsukuba)

Genetic programs for development of mouse embryonic stemcells and primordial germ cells.

In developing mammalian early embryos, there exist pluripotentstem cells, giving rise to all somatic cells as well as germ linecells. Primordial germ cell (PGC) is the cell-type appeared first inthe germ cell lineage, sharing many features with the embryonicstem cells. Also, the PGCs possess ability to erase epigeneticmodifications on the genome. Despite of this biological impor-tance, molecular nature of the PGCs remains largely unknown.We have established systematic methodologies to analyze PGCsand related embryonic cells, and transcriptome of the PGCs wereexplored by EST analyses and microarray. Analysis of ESTfrequency identified ’signature’ genes for PGCs and showed thatPGCs are more similar to blastocyst rather than undifferentiatedES cells. We compared gene expression profiles of ES, EG, PGC,and PGC-like cells derived from ES cells in vitro. PGCs have anexpression program distinct from ES cells, suggesting thatdynamic changes in gene expression occur during establishmentof germ cell lineage from undifferentiated stem cells. Compar-isons of PGCs and in vitro-formed PGCs identified a set of genesthat characterize PGC development.

1D-01

HIROTSU, Takaaki1,2,3, ISHIHARA, Takeshi1,NISHIDA, Eisuke3, IINO, Yuichi2 (1Dept. Biol.,Faclt. Sci., Kyushu Univ., 2Molec. Genet. Res. Lab.,The Univ. Tokyo, 3Dept. Cell and Dev. Biol., Grad. Sch.BioStud., Kyoto Univ.)

The Ras-MAPK pathway controls neural circuit-dependentodor adaptation by regulating localization of glutamatereceptors in C. elegans

C. elegans shows behavioral plasticity in chemotaxis to volatileodorants. After an exposure to odorants for 5 min, wild-typeworms exhibit decreased chemotaxis to odorants. This plasticity,called early adaptation, requires AIY interneurons, which receivesynaptic inputs from olfactory neurons. Thus early adaptationdepends on the neural network. We have previously reported thatfunctions of the Ras-MAPK pathway in AIY play important rolesfor early adaptation.Here we report that GLR-1, a non-NMDA type glutamatereceptor homolog, is also involved in early adaptation. Rescueexperiments revealed that GLR-1 functions in AIY are requiredfor the adaptation. We found that odorant stimuli causedgathering of the GLR-1 protein at postsynaptic sites in AIYinterneurons. Mutants of the Ras-MAPK pathway exhibitedabnormal localization of GLR-1 in AIY. In vitro phosphorylationanalyses suggested the possibility that MAPK directly phosphor-ylates GLR-1. These results indicate that the Ras-MAPK path-way controls early adaptation by regulating GLR-1 localization inAIY interneurons.

1D-02

KODAMA, Eiji1, KUHARA, Atsushi1, MOHRI, Akiko1,KIMURA, Koutarou1,2, OKUMURA, Masatoshi1,TOMIOKA, Masahiro3, IINO, Yuichi3, MORI, Ikue1,4

(1Lab. Molec. NeuroBiol., Nagoya Univ., 2Presentaddress: Structural Biology Center, Natl. Inst. Genet.,3Molec. Genet. Res. Lab., The Univ. Tokyo, 4Inst. Adv.Res., Nagoya Univ.)

Insulin-like signaling functions in association betweentemperature and feeding state in C. elegans

C. elegans shows behavioral plasticity in response to environ-mental changes. Well-fed animals show attraction but starvedanimals show avoidance to their cultivation temperature on athermal gradient. ins-1 mutants which have a deficit in insulinhomologue migrated to cultivation temperature regardless of thefeeding experience (Aho; Abnormal hunger orientation pheno-type), and were normal in feeding state-dependent locomotoryactivity. These results suggest that ins-1 mutants are likely to bedefective in associating temperature and feeding state. daf-2, age-1 and daf-16 encode homologues of insulin receptor, PI3 kinase,and forkhead-type transcriptional factor, respectively. daf-2mutants and age-1 mutants did not show Aho phenotype, butdaf-16 mutants showed weak Aho phenotype. Further, the daf-2mutation weakly suppressed the ins-1 mutation: ins-1; daf-2double mutants showed weaker Aho phenotype than ins-1mutants. Our results are consistent with the possibility thatins-1 acts upstream of daf-2 and negatively or antagonisticallyaffects on insulin-like signaling pathway required for theassociation mechanism between temperature and feeding state.


1D-03

TACHIKAWA, Sayaka1, KUHARA, Atsushi1, MORI,Ikue1,2 (1Lab. Molec. NeuroBiol., Div. Biol. Sci.,Nagoya Univ., 2Inst. Adv. Res., Nagoya Univ.)

Genetical screen for novel mutants defective in Gaa-mediatedthermosensation

We recently revealed that the loss-of-function mutation in eat-16gene encoding RGS, a negative regulator of Ga, causes hyperactivation of AWC sensory neuron in C. elegans, thereby leadingto cryophilic abnormality in thermotaxis. Interestingly, thisthermotactic abnormality is suppressed by the mutations ingenes involved in G protein-mediated olfactory signaling in AWC.Thus, AWC neurons likely receive both thermal and olfactorystimuli, and transmit and discriminate these sensory signalsthrough the same molecules in G protein-mediated signaling(Okumura et al., submitted).In order to identify new molecular components regulating the Gprotein-mediated thermosensation, we isolated mutants thatsuppress cryophilic phenotype of eat-16 mutants. Of those, nj55mutants showed almost normal thermotaxis and almost normalresponses to an AWC-sensed odorant in the eat-16 background,implying that nj55 mutants have defect in thermosensation, butnot in olfaction. Through the analysis of these mutants, we arehoping to identify new genes required for the Ga-mediatedthermosensation and the discrimination between thermosensoryand olfactory signaling in AWC.

1D-04

GO, Yasuhiro1, SATTA, Yoko1, KUNO, Kaori1,TAKAHATA, Naoyuki1 (1Dept. Biosyst. Sci., The Grad.Univ. Adv. Stud. (Sokendai), Hayama)

Reduced repertoires of olfactory receptor genes in marinemammals

Organisms have increasingly acquired new genes by tandemduplication, transposition, and polyploidization. However, asevolution proceeded, loss of genes has also occurred wheneverthey became dispensable and can occur more rapidly than gain ofnew genes. Here, we hypothesize that the process of acquiringspecies-specificity advances through the environment-drivengenome reconstruction or genome remodeling, mainly by meansof loss of genes (pseudogenization). To substantiate this hypoth-esis, we analyzed the tempo and mode of pseudogenization forolfactory receptor (OR) genes in Cetacea, bottle-nosed dolphins(Tursiops truncates), minke whales (Balaenoptera acutorostrata),and their closest terrestrial mammal, hippopotami (Hippopot-amus amphibious). We found significant differences of functionalconstraints on OR genes not only between Cetacea and hippos butalso within Cetacea, suggesting that adaptation of aquaticenvironments is not only a cause for deterioration of olfactionin marine mammals. We will discuss other causes for olfactorydeterioration in the light of evolution of other sensory systems.

1D-05

NISHI, Akinori1,2, TAKAHASHI, Aki1,2, SHIROISHI,Toshihiko2,3, KOIDE, Tsuyoshi1,2 (1Mouse GenomicsResource Lab., Natl. Inst. Genet., 2SOKENDAI,Genetics, 3Mammalian Genet. Lab., Natl. Inst. Genet.)

Analysis of quantitative trait loci that related to thebehavioral differences between two mouse strains, C57BL/6and MSM/Ms

It is known that there are great differences of behavioral traitsbetween MSM/Ms and C57BL/6, which belong to Mus musculusmolossinus and Mus musculus domesticus, respectively. Toanalyze the quantitative trait loci involved in the difference ofgeneral activity and emotionality between these strains, we usedthe consomic strains established from MSM/Ms and C57BL/6.The consomic strains were established by replacing any onechromosome of C57BL/6 with corresponding chromosome ofMSM/Ms. First, we evaluated the multiple behavioral traits ofconsomic strains. In the result, several consomic strains indicatedlarge varieties of general activity and emotionality. In thesestrains, the behavioral traits of C57BL/6.Chr6CMSM, whichcarries chromosome 6 derived from MSM/Ms were dramaticallydifferent comparing to C57BL/6. For mapping the quantitativetrait loci related to the general activity and emotionality onchromosome 6, we analyzed the phenotype on F2 populationmade between C57BL/6 and C57BL/6.Chr6CMSM. In the result,we obtained two candidate regions, the one associates withemotionality and general activity and the other associates withgeneral activity.

1D-06

UEHARA, Shigeyuki1, IZUMI, Yoshiko1, KUBO,Yuko1, TACHIBANA, Masayoshi2, SHIROISHI,Toshihiko3,4, WANG, Chi chiu5, MINETA, Katsuhiko6,IKEO, Kazuho7, GOJOBORI, Takashi7, YAMAMOTO,Hiroaki1 (1Dept. Dev. Biol. NeuroSci. Grad. Sch. LifeSci., Tohoku Univ., 2Brain Res. Inst., Niigata Univ.,3Functional Genomics Res. Group, RIKEN GenomicSci.s Cent., 4Mammalian Genet. Lab., Genetic StrainsRes. Cent., Natl. Inst. Genet., 5Dept. Obstetrics andGynaecology, The Chinese Univ., 6Bioengineering andBioinform., Grad. Sch. Inform. Sci. Tech., HokkaidoUniv., 7DNA Data Analysis Lab., Center for Inform.Biol., Natl. Inst. Genet.)

Analysis of gene expressions in the mouse inner ear, of whichfunction is affected by melanocytes responsible for hearingacuity

Melanocytes, pigmented cells of mammals originating from theneural crests are distributed throughout the body surface todetermine the coat/skin color. Also, they migrate to the striavascularis in the inner ear cochlea. Loss of them results in loss ofauditory sense, that is, the melanocytes in this region areessential for the normal hearing acuity. Interestingly, pigmenta-tion of these melanocytes is not necessarily required for thisfunction. In order to survey the novel functions of these cells inthe inner ear that are responsible for hearing, we are nowcomparing the gene expressions in the region of C3H/He-Mitfmi-

bw/Mitfmi-bw mice, which don’t have melanocytes and aregenetically deaf, with those of wild type C3H/He mice, whichhave the normal hearing sense. Mitf gene encodes a bHLH-LZtranscription factor and plays an essential role for the melanocytedifferentiation.


1D-07

TSUJIMURA, Taro1, CHINEN, Akito1, KAWAMURA,Shoji1 (1Dept. Integr. BioSci.s, Grad. Sch. Front. Sci.s,Univ. Tokyo)

Identification of a LCR-like distal regulatory region formultiple green-sensitive opsin genes in zebrafish

We previously showed that zebrafish have four green-sensitiveopsin (RH2) genes arrayed in tandem (RH2-1, RH2-2, RH2-3,RH2-4) which differ each other in absorption spectra andexpression area in the retina. In this study, we aimed to identifyregulatory regions of the four genes by exploring genomic regionswhich can induce reporter (GFP) expression when introducedinto zebrafish embryos. Using immediately upstream regions ofthe four genes, proper GFP expression was not induced. Whenthe four RH2 genes were substituted to GFP in a PAC clone(RH2-PAC) containing the four genes, proper GFP expressionwas observed for all the four genes, indicating that the RH2-PACcontains sufficient regulatory regions. By co-injecting restrictionfragments of the RH2-PAC with a GFP construct containingimmediate upstream of RH2-1, we identified a 0.5-kb distalregulatory region located 15-kb upstream of the gene array.When the region was removed from the GFP-substituted RH2-PAC, GFP expression was abolished for all the four RH2 genes.These results indicated that the region is necessary for properexpression of all the four RH2 genes and we designated it RH2-locus control region (LCR).

1D-08

TAKECHI, Masaki1, KAWAMURA, Shoji1 (1Dept.Integr. BioSci.s, Grad. Sch. Front. Sci.s, Univ. Tokyo)

Identification of cis-regulatory regions of blue sensitive visualpigment gene (SWS2) in zebrafish.

Color vision in vertebrates generally stands on a rule: only onetype of visual pigment gene is expressed per one photoreceptorcell. We are trying to understand the mechanisms of the cell-specific expression of visual pigment genes by using zebrafish.Zebrafish retina contains five morphologically distinct classes ofphotoreceptors, each expressing a distinct type of visual pigmentgene. Blue sensitive visual pigment gene, SWS2 (short wave-length sensitive2) is expressed only in long single cones (LSC). Toinvestigate cis-regulatory regions of SWS2, we took a trans-genesis methodology using a green fluorescence protein (GFP) asa reporter. We isolated various upstream regions of the gene andconjugated them to the GFP reporter gene. These constructs wereinjected to zebrafish eggs. We investigated regulatory competenceof the regions by the fluorescence of GFP in larvae. Wedetermined two cis-regulratory regions: one is a region locatedbetween -595 and -404, which promotes SWS2 expression in LSC,the other is a region located -450 and -404, which stronglyrepresses its expression in pineal organ.

1D-09

MATSUMOTO, Yoshifumi1, KAWAMURA, Shoji1

(1Dept. Integr. BioSci.s, Grad. Sch. Front. Sci.s, Univ.Tokyo)

Diversity of visual opsins in spectral sensitivity and expres-sion pattern among congeneric medaka species

We previously showed that Japanese medaka has three green(RH2-A, RH2-B and RH2-C), two blue (SWS2-A and SWS2-B),two red (LWS-A and LWS-B), and a single ultraviolet (SWS1)opsin genes. Importantly, absorption spectra are considerablydifferent between the two blue opsins and among the three greenopsins. Relative gene expression levels in the retina are also verydifferent among these opsin genes. In this study, we isolatedopsin cDNAs by PCR from several congeneric species of medakainhabiting various areas of southern Asia and determined peakabsorption spectra (lmax) of their reconstituted visual pigments.We found a large spectral variation in green opsins amongspecies, especially in RH2-A that is most short-wave-shiftedamong RH2 opsins. Expression patterns of the opsin genes werealso examined in these species by real-time RT-PCR and in situhybridization. Results obtained in this study should be a valuablesource of information to study evolutionary adaptation of medakaspecies to various light environments.

1D-10

KAWAMURA, Shoji1, HIRAMATSU, Chihiro1,TSUTSUI, Toko1, MATSUMOTO, Yoshifumi1 (1Dept.Integr. BioSci.s, Grad. Sch. Front. Sci.s, Univ. Tokyo)

Color vision polymorphism in wild New World monkeys

Color-vision polymorphism of New World monkeys results fromallelic variation of the single-locus red-green opsin gene residingon the X-chromosome. To clarify color-vision variation in naturalpopulations, we analyzed fecal DNA for two wild groups ofcapuchin monkeys (Cebus capucinus) and one group of spidermonkeys (Ateles geoffroyi) inhabiting Santa Rosa National Park,Costa Rica. On the basis of the five-site rule that correlates peakabsorption spectrum (lmax) of an opsin photopigment with itsamino acid composition at five sites, three alleles were found incapuchins (with predicted lmax values 560, 545 and 530 nm) andtwo alleles were found in spider monkeys (560 and 552 nm). Wereconstituted all of these photopigments and measured theirabsorption spectra. While lmax values of the three capuchinalleles were all concordant with the five-site rule, those of spidermonkey alleles considerably violated the rule (553 nm for the 560-nm prediction and 538 nm for the 552-nm prediction). This resultnecessitated us to further investigate the novel spectral mecha-nism evolved in spider monkeys and to survey polymorphism atthe relevant amino acid site(s) in spider monkey populations.


1D-11

KASAGI, Satoshi1, KAWAMURA, Shoji1, SHOJI,Ayako2, KAWATA, Masakado3 (1Dept. Integr. BioSci.s,Grad. Sch. Front. Sci.s, The Univ. Tokyo, 2MitsuiKnowledge Industry, 3Grad. Sch. of Life Sci., TohokuUniv.)

Molecular basis of color vision polymorphism in guppy

In guppy, male coloration is a key for females to choose mates,and preference for male colors varies among females. An earlystudy showed a variety of spectral sensitivity among red-greenphotoreceptor cells in guppy retina. However, genetic basis of thecellular variation remains unknown. In this study, we isolated allvisual opsin genes of guppy. Guppy had all five types of opsingenes as in many other fish. Notably, guppy had multiple red-green (M/LWS) type opsin genes, among which two loci werefound to be highly polymorphic in terms of restriction-fragment-length-polymorphism (RFLP) in genomic Southern hybridization.By contrast, at most only two RFLP alleles were found in otheropsin types, implying that the M/LWS RFLP correlates with thespectral variation. However, there was little variation among theM/LWS RFLP alleles at the five amino acid sites that are knownto be critical in other vertebrates in determining spectralsensitivity of M/LWS opsins, and we have not succeeded inmeasuring their absorption spectra by photopigment reconstitu-tion. The high level of RFLP thus implies that guppy has evolveda novel mechanism to differentiate M/LWS spectral sensitivity.

1D-12

MIYAZAKI, Shinya1, OKANO, Hiroyuki1, HAYASHI,Fumio1, MINAMINO, Tohru2, KOJIMA, Seiji2,NAMBA, Keiichi2,3, OOSAWA, Kenji1 (1Dept. Nano-Material Systems, Grad. Sch. Eng., Gunma Univ.,2Dynamic NanoMachine Project, ICORP, JST, 3Grad.Sch. Frontier BioScience, Osaka Univ.)

Characterization of mutant flagellar motors defective in theMotA/MotB complex of Salmonella

The flagellar motor of Salmonella rotates by interaction betweena switch complex as rotor and a MotA/B complex as stator and aproton channel. To investigate rotational mechanisms of themotor, we used motile revertants isolated from a mutant whichreleases its flagella and resultingly becomes non-motile in viscousconditions. Ninety-one second-site mutants mapped in motA ormotB gene were measured for their swimming abilities indifferent viscous conditions and compared with it of the wildtype. In the results, many of mutants showed abilities closed tothe wild type in higher viscosity in comparison with lowercondition. We will also report rotational speeds of the mutantmotors in different viscosities.

1D-13

LIU, Qing-xin1, HIRAMOTO, Masaki2, UEDA,Hitoshi3, GOJOBORI, Takashi1, HIROMI, Yasushi2,HIROSE, Susumu3 (1Lab. for DNA Analysis, Centerfor Inform. Biol. and DDBJ, Natl. Inst. Genet., 2Div.Dev. Genet., Natl. Inst. Genet., 3Div. Gene Expression,Natl. Inst. Genet.)

Functional analysis of Drosophila Midline during the neuro-nal development

Formation of the neural network requires coordinated pathfind-ing behavior of many neurons. Each neuron must express axonguidance receptors in their growth cone, and also act as asecretion source of guidance molecules. How neurons orchestratethe expression of multiple guidance genes is poorly understood.Here we show that Drosophila midline encoding a T-box proteincontrols expression multiple axon guidance molecules: Frazzled,ROBO, and Slit. In midline mutant expression of all thesemolecules are reduced, resulting in severe axon guidance defects,whereas misexpression of Midline induces their expression.Midline is present on the promoter of these genes, indicatingthat it directly controls transcription. Our results provideevidence that Midline controls the switch to axon growth.

1D-14

KATSUKI, Takeo1, HIRAMOTO, Masaki1, HIROMI,Yasushi1,2,3 (1Dept. Dev. Genet., Natl. Inst. Genet.,2SOKENDAI, 3CREST, JST)

DROSOPHILA NEURONS POSSESS AN INTRINSIC ABIL-ITY TO GENERATE SUB-AXONAL MEMBRANE COM-PARTMENTS BY A DIFFUSION BARRIER MECHANISM

Membrane compartmentalization is implicated in the controlleddistribution of gene products within a cell. In the nervous systemof Drosophila, the distribution of an axon guidance receptor Roboand its family members (Robo2 and Robo3) is restricted to thedistal segment of axons, which suggests that the axonalmembrane is compartmentalized into distal and proximal seg-ments. Here we show that the compartmentalization of axons isan intrinsic property of neurons, and that the compartmentaliza-tion is achieved by a diffusion barrier mechanism. In primarycultured neurons, Robo2 and Robo3 were both localized to thedistal axon segment without cell-cell contacts, suggesting thatneurons generate the localization of Robo2 and Robo3 through acell-intrinsic mechanism. Furthermore, fluorescent recoveryafter photobleaching (FRAP) analysis revealed that the diffusionof transmembrane molecules is reduced at the border betweendistal and proximal axon segments, suggesting that the borderacts as a diffusion barrier for transmembrane molecules. Wepropose that the regulated distribution of molecules in neuronsmay be based on the intrinsic sub-axonal membrane compart-mentalization.


2D-01

KAWAI, Mikihiko1,2, UCHIYAMA, Ikuo3, KOBAYASHI,Ichizo2,4,5 (1Grad. Sch. Med., Univ. Tokyo, 2Inst. Med.Sci., Univ. Tokyo, 3Res. Cent. Computational Sci.,Okazaki National Res. Inst.s, 4Dept. Medical GenomeSci., Grad. Sch. Front. Sci., Univ. Tokyo, 5GraduateProgram in Biophysics and Biochem., Grad. Sch. Sci.,Univ. Tokyo)

Reconstruction of pathway of genome rearrangement ofgenus Neisseria by in silico genome comparison

By comparison of closely related genome sequences, we can findthe genome rearrangements and consider how those structurewere made. We compared the genome sequences of four bacterialstrains, belonging to genus Neisseria. We compared the genomesglobally to find genome rearrangements and compared tonucleotide level. We found genome rearrangement not reportedpreviously and reconstructed the possible mechanism leading tothe structure.

2D-02

KOJIMA, Kenji1, FUJIWARA, Haruhiko2 (1Bioinform.Cent., Inst. Chem. Res., Kyoto Univ., 2Dept. Integr.BioSci.s, Grad. Sch. Front. Sci.s, Univ. Tokyo)

Dualen: An extraordinary non-LTR retrotransposon familyencoding dual endonucleases

Non-long terminal repeat (non-LTR) retrotransposons are mobileelements that transpose via RNA intermediate. Encodingendonuclease besides reverse transcriptase (RT) characterizestheir transposition mechanism called target-primed reversetranscription. Despite the necessity, non-LTR retrotransposonsonce exchanged their endonuclease domains. Early branchednon-LTR retrotransposons encode a restriction-like endonuclease(RLE) and recently branched retrotransposons encode an apur-inic/apyrimidinic-like endonuclease (APE). In this study, wecharacterized an unusual non-LTR retrotransposon family Dual-en, from the genome of the green alga Chlamydomonasreinhardtii. Dualen encodes dual endonucleases, RLE and APE,in addition to RT. Dualen also encodes RNase H and cysteineprotease. In the phylogenetic tree based on the RT sequences,Dualen is positioned at the midpoint between the RLE-encodingand the APE-encoding groups. The phylogenies of APE andRNase H are consistent with the RT phylogeny. The domainstructure and the phylogenies imply that Dualen is a retro-transposon conserving the domain structure just after theacquisition of APE by an RLE-encoding non-LTR retrotranspo-son.

2D-03

MIURA, Ikuo1, TARIQ, Ezaz2, MINEYAMA, Masaru1,OHTANI, Hiromi1, JENIFFER, Graves2 (1Inst.Amphibian Biol., Grad. Sch. Sci., Hrioshima Univ.,2Comparative Genomics Groups, Research School ofBiol. Sci.s, The Australian National Univ.)

Transoposon-like sequences in the sex chromosomes ofJapanese frog Rana rugosa: - Change of physical locationduring the W chromosome differentiation -

To find sex-linked DNA markers in the frog Rana rugosa, we haveamplified the genomic DNA of gynogenetic female WW and maleZZ using degenerate oligonucleotide-primed PCR (DOP-PCR).The DOP-PCR identified a W-linked DNA fragment of around0.5kbp (designated as WW-0.5), which showed partially sequencesimilarity to the transposon DNA of the European frog Ranatemporaria. Genomic southern hybridization elucidated that theWW-0.5 sequence is shared by the autosomes, Z and W sexchromosomes. By FISH, strong hybridization signals wereobserved on the telomeric regions of autosomes 3, 10 and 11and on the long arm telomeric region of the Z chromosome,suggesting their clustering on the terminal regions. On the otherhand, a few comparatively weaker signals were observed in theintermediate regions of the W chromosome but none on theterminal regions. These results, together with previous informa-tion about the structural change of the W chromosome, suggestthat the transposon-like sequence was originally clustered on theterminal region of the proto-W chromosome, and later dispersedthroughout the W chromosome by some structural changeassociated with a pericentric inversion.

2D-04

FUKUI, Tomokazu1, ITOH, Masanobu2,3 (1Div.Applied Science for Functionality, Grad. Sch. Sci. andTech., Kyoto Inst. Tech., 2Dept. Appl. Biol., Faclt.Textile Sci.s, Kyoto Inst. Tech., 3Insect BiomedicalResearch Center, Kyoto Inst. Tech.)

Inactivated P elements in M’ strain genome of Drosophilamelanogaster

Drosophila melanogaster has many copies of P elements in theirgenomes. Full-size P and KP elements predominate, but some ofthem appear to be inactive in wild populations. In order toevaluate determination of the P-M system phenotype, weexamined position effects of P element activity. We cloned Pelements from genomic library of one of wild M’ strains (OM-5)from Chichijima of Bonin Islands. Each of P element clones weredetermined their genomic positions and transcriptional activity.We found one full-size P element and 18 KP elements and 3others. We did not find any mutations (nucleotide substitution orin/del) in the full-size P and KP elements. Most of the P elementswere inserted into inside or near active gene. Expression of eachP element was also identified. These results suggest thatinactivation of the P elements in OM-5 genome is independentof position effect.


2D-05

KOGA, Akihiko1 (1Dept. Biol., Nagoya Univ.)

Highly frequent body color mutation caused by transposableelement excision in the medaka fish

The i locus of the medaka fish represents the tyrosinase gene thatis essential for biosynthesis of the black pigment melanin. Thegene for the ib mutant allele carries an insertion of the Tol2transposabe element in the promoter region, exhibiting a weakalbino phenotype. It can be expected that excision of this elementleads to the recovery of the wild-type body color, but such aphenomenon has not been observed for more than ten years. Wemade repeated one-pair matings of fish of the ib / ib genotype andscreened embryos for phenotype recovery. Highly frequentmutations to the wild-type and novel body colors occurred insome of the sublines that had diverged in the course ofinbreeding. PCR analysis of new mutant fish provided evidencefor precise and imprecise excision of the Tol2 element. Diversi-fication of phenotypes due to highly frequent excision of DNA-based transposable elements have been reported in manyorganisms, but not in vertebrates. Our results are the firstexample of this phenomenon in a vertebrate. Occurrence in someof the sublines suggests the presence of a genetic factorcontrolling the transposition frequency.

2D-06

HIRAMOTO, Tetsuya1, NITASAKA, Eiji1 (1Dept. Biol.Sci., Grad. Sch. Sci., Kyushu Univ.)

The responsible gene for petaloid mutation is a weak allele ofDUPLICATED in the Japanese morning glory

Many floricultural plants have similar mutants but the mecha-nism of petaloidy is not well understood since there is no relatedmutant in Arabidopsis and Antirrhinum. In petaloid (pt) mutantsof the Japanese morning glory, stamens (especially anther) areconverted to petal-like organs. The tip of pistil shows sepal-likestructure in strong pt mutants, suggesting that pt is weakmutant of C-function MADS-box gene. This was confirmed by acomplementation test between pt and duplicated (dp), a strongmutant of C-funtion MADS-box gene DP. Molecular analysis of ptmutant showed that a full length En/Spm-related transposableelement, Tpn-109 is inserted in the DP second intron. Moreover,we identified new dp alleles, dp-2 and dp-3, and it is consideredthat all dp alleles were derived from pt mutants, and the Tpnelement in the dp alleles are products of deletion and/ orduplication of Tpn109. pt mutants show fluctuate phenotypesthat the degree of petaloidy changes weaker as plant growth inthe same plants. Analyses of genomic structure and transcriptionlevel of pt showed that this phenotypic change is caused by theactivation of DP promoter during the plant growth.

2D-07

YOKOYAMA, Kouhei1, NITASAKA, Eiji1 (1Dept. Biol.Sci., Grad. Sch. Sci., Kyushu Univ., 2Dept. Biol. Sci.,Grad. Sch. Kyushu Univ.)

Identification of autonomous element of En/Spm-relatedtransposable element, Tpn1 family in the Japanese morningglory

In the Japanese morning glory (Ipomoea nil), analyses ofresponsible genes for unstable mutant phenotypes revealed thatthese were induced by insertions of En/Spm-related transposableelements, Tpn1 family. These mutants show somatic and germ-line reversions caused by excision of Tpn elements. However, allof identified Tpn elements are non-autonomous, and theirinternal regions contain captured host genes. Thus an unidenti-fied autonomous Tpn element should drive these non-autono-mous Tpns. Our preliminary trials to detect transposase-relatedsequence using degenerated PCR were failed because there aremany subfamilies of En/Spm-related transposons in genome. Acomparison of transcripts from different Tpn elements indicatedthat they have the common, conserved 3’-most exon and use theidentical acceptor site. We succeeded to isolate transcriptspossibly derived from autonomous Tpn element with primerscomplementary to this common exon and conserved domains oftransposases of En/Spm-related transposon. Using this tran-script as a probe, genomic structure of autonomous Tpn elementwere characterized.

2D-08

UCHIYAMA, Takako1, SAITO, Yumiko1, HASHIDA,Shin-nosuke2, MIKAMI, Tetsuo3, SANO, Yoshio1,KISHIMA, Yuji1 (1Lab. Plant Breeding, Grad. Sch.Agr., Hokkaido Univ., 2Inst. Molec. Cell. BioSci.s,Univ. Tokyo, 3Lab. Genetic Eng., Grad. Sch. Agr.,Hokkaido Univ.)

Commitment to the Tam3 transposase for the transpositionalregulation in Antirrhinum.

The transposon Tam3 from Antirrhinum majus undergoes low-temperature-dependent transposition (LTDT): low growing tem-peratures around 15˚C permit the transposition, while hightemperatures around 25˚C strongly suppress transposition. Itwas suggested that LTDT of Tam3 is regulated by temperature-dependent nuclear localization of the Tam3 transposase. In thisstudy, we confirmed that the nuclear extract from the plantsgrown at 15˚C bound to a Tam3 sequence, but that from theplants grown at 25˚C did not. On the other hand, Antirrhinumpossesses two independent Stabiliser genes, which specificallysuppress the Tam3 transposition. Here we also examined thatwhether these Stabiliser genes are involved in the Tam3transposase expression and/or the nuclear localization, or theother mechanisms.


2D-09

OHIRA, Takayuki1, MIKI, Daisuke2, TSUCHIMOTO,Suguru1, OSAWA, Isaku1, SHIMAMOTO, Ko2,OHTSUBO, Eiichi1, OHTSUBO, Hisako1 (1Inst. Molec.Cell. BioSci.s, Univ. Tokyo, 2The Grad. Sch. Biol. Sci.s,Nara Inst. of Sci. and Tech.)

Involvement of siRNA in repression and DNA methylation ofthe rice retroposon p-SINE1.

The rice retroposon p-SINE1 is highly methylated, and itstranscripts are processed into siRNA. Rice has five Dicer-likegenes (OsDCL1 - OsDCL5), which are supposed to producesiRNA. To elucidate involvement of siRNA in repression andDNA methylation of p-SINE1, we used five lines of cultured cellsin which each OsDCL genes are silenced. We found that theamount of p-SINE1 transcripts obviously increased in cells whereOsDCL3 or OsDCL5 was silenced, and DNA methylation of p-SINE1 dramatically decreased in cells where OsDCL4 wassilenced. These results suggest that siRNA involved in repressionand that involved in DNA methylation are produced by differentDCL proteins.

2D-10

MIURA, Asuka1, KAKUTANI, Tetsuji1 (1Dept.Integrated Genetics, Natl. Inst. Genet.)

Mechanisms to immobilize CACTA transposons in Arabidop-sis by DNA methylation

An important role of DNA methylation in eukaryotic genome is tosuppress deleterious movement of transposons. An Arabidopsistransposon CACTA1 is epigenetically immobilized in wild typeplants. The immobilization requires DNA methylation, as thistransposon is mobilized in the mutants of the chromatin-remodeling gene DDM1 (decrease in DNA methylation 1) or ofthe DNA methyltransferases MET1 and CMT3 (Miura et al 2001Nature, Kato et al 2003 Curr Biol). The immobilization ofCACTA1 by the DNA methylation may operate through trans-acting factors (such as repression of transposase expression) orcis-limitted factors (such as inhibition of transposase access dueto heterochromatin formation). We distinguished the trans andcis requirements of the CACTA activation using autonomous andnon-autonomous CACTA copies. Their mobility was examinedafter independently exposing to the ddm1 mutation. The resultssuggest that not only trans-acting but also cis-limitted factorscontribute to the epigenetic immobilization.

2D-11

KINOSHITA, Yuki1, MIURA, Asuka1, KINOSHITA,Tetsu1, KAKUTANI, Tetsuji1 (1Dept. IntegratedGenetics, Natl. Inst. Genet.)

Role of SINE-related direct repeats of the imprinted FWAgene in Arabidopsis thaliana

FWAdisplays imprinted expression in endosperm (Kinoshita etal. 2004 Science). This gene contains hypermethylated direct-repeats at the 5’ region, which is similar to retrotransposonSINE3 (Lippman et al. 2004 Nature). Although the geneexpression correlates to DNA methylation status of the repeats(Soppe et al 2000 Molecular Cell), there is no evidence that FWAis controlled by DNA methylation of the repeats. To understandrole of this transposon-related repeats and potential impact to thegene expression by DNA mathylation, we performed targeted denovo methylation to a hypomethylated epi-allele by use of doublestranded RNA producing constructs. We will report the resultsand discuss the role of SINE-related direct repeats.

2D-12

SAZE, Hidetoshi1, KAKUTANI, Tetsuji1 (1Dept.Integrated Genetics, Natl. Inst. Genet.)

Loss-of-funation epigenetic mutation derived from ddm1(decrease in DNA methylation) mutant of Arabidopsis

Arabidopsis ddm1 (decrease in DNA methylation) mutationinduces a variety of developmental abnormalities throughheritable changes in other loci. One of the abnormilities, whichwe named bonsai, was heritable as an unstable recessive traiteven in the presence of the wild type DDM1 gene. Throughlinkage analysis, we identified a gene silenced only in bonsailines. The bonsai phenotype was reproduced by knocking-down ofthe identified gene by the dsRNA. Although no change innucleotide sequence was identified, bisulfite-sequencing analysisrevealed that the repression of BONSAI transcription in this lineis associated with hyper-methylation of DNA in whole BONSAIregion.


2D-13

OHTSUBO, Hisako1,2, TAGUCHI, Yuu1, TAMURA,Koichiro2, OHTSUBO, Eiichi1 (1The Institute ofMolecular and Cellular Biosciences, Univ. Tokyo,2Tokyo Metropolitan Univ., Faculty of Urban LiberalArts)

Identification and characterization of self-priming LTRretrotransposons in plants

LTR-retrotransposons are ubiquitous components and comprisethe large fraction of eukaryote genome. Recently, we found a newgroup of LTR-retrotransposons in rice as well as in Arabidopsis,which does not employ tRNA as a primer for the initiation ofreverse transcription. RIRE5 is the one of the members of thisgroup. RIRE5 has a sequence, called cPBS, which is homologousto the PBS in downstream of the promoter region in its own 5’-LTR region. The presence of cPBS suggests that RIRE5 mayemploy a self-priming mechanism by using cPBS instead of tRNAfor its initiation of reverse transcription. We analyzed the 5’-terminus of the transcripts of RIRE5 in Oc suspension culturedcell under the presence of 5-azacytidine, a DNA methylationinhibitor. The transcript analysis revealed that there was a cPBSat the region of 150bp downstream of the transcription start site.This suggests that RIRE5 employs the self-priming mechanism inthe initiation of its reverse transcription. We also determined theinsertion dates for the elements of RIRE5 by comparison of LTRdivergences with the divergence between adh1 gene in rice andmaize.

2D-14

KITAMURA, Tomoko1, NASUDA, Shuhei1, NOMURA,Taiji1, OGIHARA, Yasunari2, ENDO, Tkashi R.1

(1Lab. Plant Genet., Grad. Sch. Agr., Kyoto Univ.,2Lab. Genetic Eng., Grad. Sch. Agr., Kyoto Pref. Univ.)

High level of nucleotide substitutions between wheat culti-vars found near a Copia-type retrotransposon adjacent to aTaBx3 gene on chromosome 5A.

We found restriction fragment length polymorphisms (RFLPs)between deletion and normal chromosomes 5A of common wheatby hybridizing the TaBx3 probe. We addressed in the lastmeeting that these RFLPs were caused not by an insertion or adeletion of a novel sequence but by nucleotide substitutions. Byexploiting the complete nucleotide sequence of a transformation-competent artificial chromosome (TAC) clone carrying the TaBx3gene, we compared nucleotide sequences of the region surround-ing the TaBx3 gene between normal Chinese Spring (CS) and itsdeletion lines. Nucleotide substitutions were found through the58 kb interval defined by the TAC clone. Pedigree analysisrevealed that the origin of the variant sequence is a commonwheat cultivar Jones Fife (JF) that was crossed in the course ofestablishing deletion lines. The nucleotide substitutions betweenCS and JF were not evenly distributed through the 58 kbinterval, and highly populated in the region of a Copia-typeretrotransposon insertion located 850 bp upstream of the TaBx3gene. We will discuss the substitution rates, their distribution,and the pattern of nucleotide substitution in the 58 kb sequence.

3D-01

YAMAMOTO, Shouji1, KUTSUKAKE, Kazuhiro1,2

(1Grad. Sch. Natural Sci. Tech., Okayama Univ.,2Dept. Biol., Faclt. Sci., Okayama Univ.)

Post-transcriptional control of the phase-1 flagellin gene fliCby the FljA protein in flagellar phase variation of Salmonella

Phase variation of Salmonella enterica is a phenomenon that twodifferent flagellin genes, fliC (phase 1) and fljB (phase 2), areexpressed alternately. This is controlled by inversion of a DNAsegment containing the fljB promoter. The fljB gene constitutesan operon with the fljA gene, which encodes a negative regulatorfor the fliC expression. It has long been believed that the fliCrepression by FljA should occur at a transcriptional level.However, recent results reported by us and others suggestedthat a post-transcriptional control is more profound in thisrepression. In this study, we examined in vivo and in vitro themechanism of fliC repression by FljA more carefully. The half lifeof fliC mRNA was found to be much shorter in the phase-2 cellsthan in the phase-1 cells. FljA-mediated reduction of fliC mRNAstability was not observed in an E. coli rne mutant, suggestingthat RNase E may be involved in the degradation of fliC mRNA.Purified His-tagged FljA protein was shown to bind specifically tofliC mRNA and inhibit its translation in vitro. We propose that inphase-2 cells FljA binds to fliC mRNA and inhibits its translation,which in turn facilitates its degradation.

3D-02

ONO, Katsuhiko1, ABO, Tatsuhiko1 (1Grad. Sch.Natural Sci. Tech., Okayama Univ.)

Isolation of E.coli mutants which show synthetic lethality incombination with ssrA deletion.

Bacterial SsrA RNA has both tRNA and mRNA functions andpromotes the special type of translation which is called trans-translation. In trans-translation, the ribosomes stalled on themRNA are released and nascent polypeptides are C-terminallytagged for subsequent proteolysis. Though literally all thebacterial strains whose genome sequence have been so farreported have ssrA gene, some bacterial strains require it fornormal growth and some do not, and the biological significance oftrans-translation is still to be unveiled. In order to elucidate thebiological significance of trans-translation, we performed trans-poson mutagenesis to screen the E. coli mutants which are lethalor sick in the absence of SsrA. In our screening system, based onthe system reported by Bernahrdt and de Boer (Mol. Microbiol.52:1255-69), ssrA-carrying unstable plasmid is used to identifythe mutants which require SsrA to grow normally. Sixteenmutants have been shown to require SsrA to grow and theirgrowth defect phenotype were not uniform, suggesting that trans-translation is required in more than one aspect.


3D-03

IWAMOTO, Akira1, YONESAKI, Tetsuro1 (1Dept.Biol. Sci.,Grad. Sch. Sci., Osaka Univ.)

Characterization of RnlA essential for Escherichia coli RNaseLS

In order to elucidate the mechanism for control of mRNAdegradation, we have been studying with bacteriophage T4 andEscherichia coli. E. coli RNase LS cleaves mRNA and triggersdegradation of various mRNAs. Remarkably, this RNase has ananti-T4 phage effect. That is, RNase LS rapidly degrades T4mRNAs and leads to the silencing of late genes, thus blocking T4propagation. The dmd gene,one of T4 early genes, inhibits RNaseLS activity. When this gene is normal, T4 propagation isnormalbecause of complete repression of the anti-T4 phage effect.From the analysis of E. coli mutants which lost the RNase LSactivity, we identified yfjN as a causal gene, and named this genernlA. RNase LS is suggested to consist of multiple components forthe function because of its Sedimentation rate through a sucrosegradient. Here, we attempted to identify candidates for thecomponents of RNase LS using Immunoprecipitation andanalyses of N-terminal primary sequences. As a result, we foundthat triose phosphate isomerase was essential for RNase LS. Inaddition, we also found that Dmd associated with RnlA directly.

3D-04

KOSEKI, MAIKO1, GOTO, KAZUNORI1, MASUTA,Chikara1, KANAZAWA, Akira1 (1Grad. Sch. Agr.,Hokkaido Univ.)

The sequence-specific degradation of the chalcone synthaseRNA induces star-type flower color pattern in petunia

Petunia hybrida Red Star is a variety whose flowers exhibit astar-type red and white bicolor pattern. We conducted experi-ments to determine whether RNA silencing is involved in theformation of flower color pattern. We compared the mRNA levelsof six genes involved in the anthocyanin biosynthesis in the redand white sectors of the petals using real-time RT-PCR. Only thelevel of CHS-A mRNA was depressed in the unpigmented flowersectors. Nuclear run-on transcription assay indicated that theCHS-A gene is transcribed in both white and red sectors of theflower. A Northern blot analysis of the small RNA fractionrevealed that short interfering RNAs (siRNAs) of CHS-A geneaccumulated in the white, but not the red, sectors of the flower.Infection of plants by a virus carrying a suppressor of sequence-specific RNA degradation blocked the generation of CHS-A-silenced sectors. These results indicate that sequence-specificdegradation of the CHS-A RNA is the primary cause of theformation of white sectors in the Red Star flowers. Our findingssuggest that common mechanisms are involved in the inductionof CHS-A RNA silencing in both transgenic and non-transgenicpetunias.

3D-05

URASAKI, Akihiro1, KAWAKAMI, Koichi1 (1Div.Molec. and Dev. Biol., Natl. Inst. Genet.)

Remobilization of the Tol2 transposable element in zebrafish

We have been developing the gene trap method using the Tol2transposon in zebrafish. To date, we have identified andcharacterized 102 unique GFP expression patterns. 75% of thegenes trapped by the transposon insertions turned out to bepreviously uncharacterized genes, indicating that the gene trapmethod is useful to identify novel developmental genes. Cur-rently, chromosomal insertions of Tol2 are created by plasmidinjection. To develop a novel method to create insertions, weinjected the transposase mRNA into embryos carrying a singletransposon insertion in the hoxc cluster and tested whether theintegrated genomic copy could be mobilized. 40 injected fish weremated and the F1 progeny was analyzed. In F1 fish, we identified10 new GFP expression patterns, different from that of theoriginal insertion. Molecular characterization of these fishrevealed that the integrated transposon was excised from theoriginal locus efficiently and integrated in new loci located ondifferent chromosomes. Thus, we successfully developed amethod for remobilization by which revertants of existinginsertions and insertions of new loci can be created efficiently.

3D-06

KOBAYASHI, Satoshi1, KAWAMOTO, Shyoko1,MIZUTA, Yoko1, DEMIYA SVEN, Minoru1, MULJADI,Hendry3, SUZUKI, Satoshi3, ABE, Takashi4, ARAKI,Jiro6, SIRAI, Yasuyuki6, ITO, Takehiko6, KONDO,Takashi6, KITAMOTO, Asanobu2, MIYAZAKI, Satoru5,GOJOBORI, Takashi4, SUGAWARA, Hideaki4,TAKEDA, Hideaki3, FUJIYAMA, Asao1 (1Natl. Inst.Inform. Research Inform. Research Div., 2Natl.Inst. Inform. Infrastructuer Systems Res. Div., 3Natl.Inst. Inform. Res. Cent. Testbeds Prototyping, 4Natl.Inst. Genet., 5Tokyo Univ. Sci. Faclt. Pharm. Sci.,6Mitsubishi Res. Inst.)

The development of a new generation biotechnology portal foreducation and genetics

Today’s scientific education is facing tough problems. For onething, a large number of biological terms are being used in themedia which go beyond the level of high school education andtherefore are not easy to learn. For another thing, people,especially the younger generation, are takingless and lessinterest in science, which is in urgent need of a solution. Wechallenge this situation.We are developing a website, theBioportal, with the aim of bridging the gap between professionalbiologists and the general public to promote ‘scientific communi-cation’ in the Japanese society. Our website is designed to meetthe needs of both the‘non-specialist’ and the‘specialist’ in differ-ent ways. For the non-specialist, biological knowledge is providedthrough the description of biological terms, whereas for thespecialist, practical functions such as genomic information searchand document retrieval are offered. The Bioportal website is opento public at the following URL (http://www.bioportal.jp/).


3D-07

ONAI, Kiyoshi1,2, MORISHITA, Megumi1, ISHIURA,Masahiro1,2,3,4 (1Cent. Gene Res., Nagoya Univ., 2Bio-oriented Tech. Res. Adv. Inst. (BRAIN), 3Div. Biol. Sci.,Grad. Sch. Sci., Nagoya Univ., 4Aichi Sci. Tech.Found.)

Cryopreservation of cyanobacteria.

Routine subculturing is not a practical method to maintainmicroorganisms as a reliable source. It is very time-consumingand liable to cause genetic and physiological changes in cells andcontamination. In contrast, cryopreservation, which is a methodfor long-term storage of living cells at a very low-temperature, isvery effective to maintain cells in the same state as before it wasstored. However, there are few literatures concerning thecryopreservation of cyanobacteria.In this study, we determinedthe optimum conditions for cryopreservation of typical fourcyanobacterial strains, Synechocystis sp. strain PCC 6803,Synechococcus sp. strain PCC 7942, Anabaena sp. strain PCC7120, and Thermosynechococcus elongatus BP-1. Efficient sur-vival rates were obtained by examining the growth phase of cells,cooling rate, and the concentration of methanol as a cryoprotec-tant.

3D-08

OKAMOTO, Kazuhisa1,2,3, ONAI, Kiyoshi1,3,MATSUO, Takuya1, KUCHO, Ken-ichi1, IWASE, Ryo1,4,MORISHITA, Megumi1, ISHIURA, Masahiro1,2,3,4

(1Cent. Gene Res., Nagoya Univ., 2Aichi Sci. Tech.Found., 3Bio-oriented Tech. Res. Adv. Inst. (BRAIN),4Div. Biol. Sci., Grad. Sch. Sci., Nagoya Univ.)

Development of a new continuous-culture system, which canmonitor cell growing rate and bioluminescence.

Continuous-cell-culture system to keep culture at constant cellgrowth conditions is necessary for the time course sampling ofcells. That system allows us high reliability result from experi-ment. There are many problems in previous systems as follows:(1) previously systems can not monitor cell growing rate; but also(2) the bioluminescence from a bioluminescence reporter strain inreal time. Therefore, it is difficult evaluate the condition ofculturing cells in real time; (3) They also can not automaticallystore sampled cells under low temperature conditions.To solvethese problems, we developed a new continuous, cell-culturesystem for liquid culture. Using the system, we cultured twostrains of cyanobacteria, a thermophilic cyanobacterium, and thegreen alga Chlamydomonas, and utilized the cultured cells tostudy circadian rhythm.

3D-09

MATSUO, Takuya1, ONAI, Kiyoshi1,2, OKAMOTO,Kazuhisa1,2,3, MINAGAWA, Jun4, ISHIURA,Masahiro1,2,3,5 (1Cent. Gene Res., Nagoya Univ.,2Bio-oriented Tech. Res. Adv. Inst. (BRAIN), 3AichiSci. Tech. Found., 4Inst. Low Temperature Sci., Univ.Hokkaido, 5Div. Biol. Sci., Grad. Sch. Sci., NagoyaUniv.)

Nuclear regulation of circadian gene expression in thechloroplast

The circadian clock regulates the expression of many genes. Thecircadian regulation extends not only to the nuclear genome butalso to the chloroplast genome. Until now, several studies havesuggested that circadian gene expression in the chloroplast maybe regulated by factors encoded in the nuclear genome. However,it is unclear to what extent the nucleus contributes to chloroplastcircadian gene expression. In this study, we developed a real-timebioluminescence monitoring system for chloroplast circadiangene expression in the unicellular green alga Chlamydomonasreinhardtii and provided direct evidences that the chloroplastcircadian gene expression is under the control of the nucleus-encoded circadian oscillator.


1E-01

TAKEZAKI, Naoko1, NAKAMURA, Shinichi2, OKABE,Akinobu3, MATSUSHITA, Osamu3 (1Inform. Tech.Cent., Kagawa Univ., 2Faclt. Med., Kanazawa Univ.,3Faclt. Med., Kagawa Univ.)

Frequent domain organization changes in the evolution ofclostridial collagenases

Clostridia, gram-positive endospore-forming anaerobic bacteria,include various pathogens, and their collagenases are assumed tobe involved in their pathogenesis. In previous studies three typesof domains were identified in the enzymes, i.e., catalytic domain(metalloprotease family M9B), PKD domain (PKD), and collagen-binding domain (CBD). Duplicated PKD or CBD were found insome of them. In this study, knowledge on their primarysequences was expanded by a novel affinity purification, rapidcloning, and direct sequencing. Our analyses showed that lossand duplication of PKD and CBD in the clostridial enzymes havehappened frequently. By contrast, the domain organization ofBacillus collagenases has been conserved, though there are threeparalogs in most of the species. In addition to Firmicutes(Clostridia and Bacilli), our extensive phylogenetic analysisrevealed that divergent species of Proteobacteria, as well asActinobacteria and Spirochetes, harbor M9 peptidases of variousdomain organization and varying numbers of paralogs.

1E-02

SU, Zhi-hui1,2, AZUMA, Hiroshi3, NAKAMURA,Keiko3 (1JT Biohistory Res. Hall, 2Grad. Sch. Sci.,Osaka Univ., 3Grad. Sch. Sci., Kyoto Univ.)

Molecular phylogeny of Japanese figs and fig pollinators

Figs (Ficus, Moraceae) are classified into 4 subgenera: Ficus,Urostiguma, Pharmacosycea and Sycomorus, and consists ofsome 750 species, which are distributed mainly in tropical andsubtropical regions on the world. There are 13 Ficus speciesdistributed in Japanese Islands, and 3 species endemic toOgasawara Islands. Figs developed a unique pollination systemwith fig wasps. The interaction between figs and fig-pollinatingwasps (pollinator) is one of the most specialized cases ofpollination mutualism known, and considered to be “one to one”rule. Our purpose is to understand the patterns and mechanismsof speciation and evolution of figs and fig wasps. In this study, weanalyzed the phylogenetic relationships using chloroplast DNAand nuclear ITS sequences for Ficus species, and mitochondrialCOI gene and nuclear rDNA for pollinators. The trees showedthat the phylogenetic relationships of Japanese figs and fig-pollinators are mostly consistent with each other, but the obviousdifferences have also been found. This result suggests that thehost change of pollinators would have occurred in the co-evolutionary process of figs and fig pollinators.

1E-03

TSUJINO, Fumi1, MONTANO, Adriana1, TAKAHATA,Naoyuki1, SATTA, Yoko1 (1Dept. Biosyst. Sci., theGrad. Univ. Adv. Stud. (Sokendai))

Evolution of peptidoglycan recognition proteins in the innateimmune system of metazoa

Peptidoglycan recognition proteins (PGRPs) are pattern recog-nition proteins in the innate immune system that recognizebacterial peptidoglycans and mediate host responses to bacterialinfection. PGRPs are found in both vertebrates and invertebrates.Here we investigated the birth and death process of PGRPsinvolved in each vertebrate and invertebrate innate immunesystem. Based on the amino acid sequences of PGRP domainsthat are common to one to 13 PGRP paralogs observed for eachspecies in metazoa, we examined the origin and subsequentevolution of PGRPs by gene or domain duplication. The numberof PGRPs tends to vary even among jawed vertebrates, despitethe fact that these organisms share the acquired immune systemof common origin. We were particularly concerned with thedifferences about the duplication pattern of PGRPs betweenjawed vertebrates and insects, both of which have evolved onalmost the same evolutionary time scale. All vertebrate speciesexamined here possess human PGRP orthologs only, whereasinsects appear to possess their own sets of PGRPs. Evolution ofPGRPs in a wide taxonomic group of metazoa is furtherdiscussed.

1E-04

ISHIWATA, Keisuke1, SASAKI, Go2, MIYATA,Takashi1,2,3, SU, Zhi-hui1,2 (1Dept. Biol. Sci.s, Grad.Sch. Sci., Osaka Univ., 2JT Biohistory Res. Hall,3Grad. Sch. Sci. Eng., Waseda Univ.)

Phylogenetic analysis for investigating the origin of wingedinsects (Pterygota, Insecta)

Hexapods (Hexapoda) consist of four orders (Protura, Collembola,Diplura and Thysanura) of apterous insects and many orders ofwinged insects. It has been considered that the apterygotesemerged first, and the winged insects diverged from theapterygotes and then developed wings. In recent years, however,molecular evidence has largely changed the phylogenetic rela-tionships of arthropods, and furthermore, suggested that Hex-apoda are not monophyletic. Morphological and molecularphylogenetic analyses have also suggested a closer affinitybetween Thysanura and winged insects, but it is still unclearthat which lineage of Thysanura the winged insects branched offfrom. To investigate the origin of the winged insects, in thisstudy, we used several nuclear genes to analyze the phylogeneticrelationships of apterous and winged insects.


1E-05

SASAKI, Go1, ISHIWATA, Keisuke2, MIYATA,Takashi1,2,3, SU, Zhi-hui1,2 (1JT Biohistory Res. Hall,2Grad. Sch. Sci., Osaka Univ., 3Grad. Sch. Sci. Eng.,Waseda Univ.)

Is Hexapoda monophyletic? - Phylogenetic position of wing-less insects (Apterygota)

Hexapods can be divided into two groups, winged (pterygotes)and wingless (apterygotes) insects. It has been thought thatHexapoda is monophyletic and closely related with Myriapoda.Recentry, molecular phylogenetic analyses revealed that Hex-apoda and Crustacea form a cluster, Pancrustacea. Furthermore,phylogenetic analysis with mitochondrial proteins suggests abasal position of Collembola (wingless insetcts) within Pancrus-tacea. This is inconsistent with monophyly of insects. However,the phylogenetic relationships within Pancrustacea and theorigin of Hexapoda remain to be resolved. It is important thatphylogenetic analyses are conducted with multiple, especiallynuclear DNA-coded genes to resolve this problem. To clarify thephylogenetic relationships among pancrustacean, we sequencedseveral nuclear DNA-coded genes from insects, crustacean,chelicerates and myriapods. On the basis of these sequences,together with those published in databases, we carried outphylogenetic analysis based on the maximum likelihood (ML)method.

1E-06

WADA, Hiroshi1, MURAI, Miho2, YONEDA,Masahiko2, NAKAMURA, Toshiya3, KURATANI,Shigeru4, KUBOKAWA, Kaoru5, ZHANG, Shicui6,SATOH, Noriyuki7 (1Grad. Sch. Life and Env. Sci.,Univ. Tsukuba, 2Aichi Prefectural College of Nursingand Health, 3Dept. Medicine, Hirosaki Univ., 4CDB,Riken, Kobe Japan, 5Inst. Oceanology, Univ. Tokyo,6Dept. Marinebiology, Ocean Univ. Qingdao, 7Grad.Sch. Sci., Kyoto Univ.)

Molecular evolutionary history of vertebrate cartilage matrixprotein complex

We traced the molecular evolutionary history of the matrixcomponents of vertebrate cartilage: hyaluronic acid, aggrecan,and link protein. Hyaluronic acid synthase (HAS): enzymeresponsible for biosyntheses of hyaluronic acid was not found inthe ascidian genome, or in any other metazoan for which thegenome sequence has been analyzed. On the other hand, thevertebrate HAS showed sequence similarity with prokaryotegenes that catalyze the same biochemical reaction. Therefore, it islikely that HAS was acquired by lateral gene transfer fromprokaryotes in the vertebrate ancestor. By contrast, genescontaining the link module that binds aggrecan and link proteinto hyaluronic acid were found in ascidians and amphioxus. Theirmolecular structure and expression were more similar to those ofvertebrate CD44, which is involved in lymphocyte migration. Wepresent evidence that the ascidian link module can bind anotherglycosaminoglycan, heparin. Therefore, the link module acquiredspecificity for hyaluronic acid after the lateral gene transfer.Subsequently, aggrecan and the link protein have evolved as anovel component of the extracellular matrix by exon shuffling.

1E-07

NIIMURA, Yoshihito1 (1Dept. Bioinform., Medical Res.Inst., Tokyo Medical and Dental Univ.)

Evolutionary Dynamics of Olfactory Receptor Genes inVertebrates

Mammals have ~1,000 olfactory receptor (OR) genes, which formthe largest known multigene family. To understand the evolu-tionary dynamics of OR genes in vertebrates, we conducted aphylogenetic analysis of all OR genes identified from the genomesequences of zebrafish, pufferfish, frogs, chickens, humans, andmice. We found the following things: (1) The most recent commonancestor between fishes and tetrapods had at least nine ancestralOR genes and all OR genes identified were classified into ninegroups each of which originated from one ancestral gene. (2)Although the number of OR genes is much smaller in fishes(~100) than in mammals, the diversity is larger in fishes. Fisheshave eight different group genes, while in mammals, only twogroup genes were found and ~90% of the gene family belong toone group (g). (3) OR gene groups present in mammals or birdsare nearly absent in fishes and vice versa. (4) The OR gene familyin frogs is as diverse as that in fishes, but ~90% of the frog genesbelong to group g, as is observed in mammals. All theseobservations can be explained by the environmental change thatorganisms have experienced at the time of terrestrial adaptation.

1E-08

KOYANAGI, Kanako1, ITOH, Takeshi2,3, HAGIWARA,Masato4,5, GOJOBORI, Takashi3,6, IMANISHI,Tadashi1,3 (1Grad. Sch. Inform. Sci. Tech., HokkaidoUniv., 2Genome Res. Dept., Natl. Inst. AgroBiol. Sci.,3Biol. Inform. Res. Cent., Natl. Inst. Adv. Industr. Sci.Tech., 4Japan Biol. Inform. Res. Cent., Japan Biol.Inform. Consortium, 5AxioHelix, Co. Ltd., 6Center forInform. Biol. and DDBJ, Natl. Inst. Genet.)

On the origin of the vertebrate-specific genome structure

Although human genes appear to be randomly distributed acrossthe genome, analyses of the closely located adjacent gene pairshave suggested that the bidirectional pairs were enriched in thehuman genome and these pairs tended to be coexpressed bysharing promoter sequences. Here, using the H-Invitationaldataset consisting of 41,118 human full-length cDNAs, wecompared this biased organization found in the human genomewith those in the genomes of nine other eukaryotes to revealwhen and how the biased organization had evolved. As a result,the organization was found only in mammals, but not in othereukaryotes. Interestingly, it turned out that many of these genesin the bidirectional arrangement were highly conserved amongvarious species. These results imply that the bidirectionalarrangement of these pairs had arisen by utilizing already-existing genes in the lineage leading to mammals recently, noearlier than the vertebrate-ascidian divergence. Since the novelbidirectional arrangement could result in novel co-regulatedtranscription, our results provide evidence that shows how atranscriptional regulation system has evolved through changes inthe genome organization.


1E-09

SASAKI, Takeshi1, NIKAIDO, Masato2, WADA,Shiro3, YAMADA, Tadasu4, GOTO, Mutsuo5, PASTENE,Luis5, CAO, Ying6,7, HASEGAWA, Masami6,7, OKADA,Norihiro1,2 (1Div. Speciation Mechanism, Natl. Inst.Basic Biol., 2Grad. Sch. BioSci. and BioTech., TokyoInst. Tech., 3National Res. Inst. of Fisheries Sci.,Fisheries Res. Agency, 4Natl. Sci. Museum, 5The Inst.Cetacean Research, 6Inst. Statistical Mathematics,7Dept. Biosyst. Sci., Grad. Univ. Adv. Stud.)

Molecular phylogenetics and evolutionary history of thenewly discovered baleen whale (Balaenoptera omurai)

Balaenoptera omurai, formerly classified as a small form ofBryde’s whale, was recently reclassified as a new baleen whalespecies of the family Balaenopteridae. Although researchers haveinvestigated the evolutionary history of Balaenopteridae andtheir relatives using molecular phylogenetic methods, thetaxonomy of the Bryde’s whale related species (B. brydei, B.omurai and B. edeni) remains unclear. Until recently, there waslittle consensus regarding baleen whale phylogeny, the resolutionof which has been complicated by the extensive radiation of thesewhales. Moreover, research on B. omurai has made Balaenopter-idae phylogeny more complex in terms of whether this whale isevolutionarily related to Bryde’s whales. We have used completemtDNA sequences and SINE insertion patterns to construct theevolutionary history of B. omurai. The combined results demon-strate that B. omurai diverged from a common ancestor of B.brydei, B. edeni and sei and blue whales early in theBalaenopteridae-Eschrichtiidae clade. Our data suggest that B.omurai evolved as an ancient independent lineage that divergedmuch earlier than sei whales, B. brydei and B. edeni.

1E-10

KIM, Hyung-cheol1, SUMIYAMA, Kenta1, SAITOU,Naruya1 (1Div. Popul. Genet., Natl. Inst. Genet. &Dept. Genet., Sch. Life Sci., The Grad. Univ. Adv.Stud.)

Screening of upstream factors regulating branchial archexpression of mouse Dlx3 gene

The mouse Distal-less (Dlx) genes, organized as tail-to-tail bigeneclusters, show a nested expression pattern in the branchialarches along proximal–distal axis. Dlx1/2 shows an early andbroad expression in both the maxillary and mandibular arches.Dlx5/6 is expressed exclusively in the distal portion of themandibular arch. Dlx3/7 is also expressed in the mandibulararch, but the pattern is more restricted distally and laterally thanother clusters. Dlx bigene clusters contain cis-acting intergenicsequences that are involved in their regulation. Functional cis-acting element sequences have been reported in the element I37-2 evolutionarily conserved in the Dlx3/7 cluster.The expression mechanism of Dlx genes is not understood well. Inorder to identify upstream factors, yeast one-hybrid system withmouse embryonic branchial arch cDNA library was screenedusing I37-2 cis-element as a binding target. Positive clones werefurther tested by electrophretic mobility shift assay (EMSA). Sofar several transcriptional factors were isolated. These factorsinteract with I37-2 region that may actually encode a complexregulator of transcription.

1E-11

OOTA, Hiroki1,2, DUNN, Casey W.3, SPEED, WilliamC.2, PAKSTIS, Andrew J.2, KIDD, Judith R.2, KIDD,Kenneth K.2 (1Dept. Integr. BioSci.s, Grad. Sch. Front.Sci.s, Univ. Tokyo, 2Dept. Genet., Yale Univ. Sch.Med., New Haven, CT, USA, 3Dept. Ecology andEvolutionary Biol., Yale Univ.)

Evolution of the Class I ADH gene cluster in primates

The alcohol dehydrogenase (ADH) gene family exists widely inthe genomes of many organisms, and provides a fine model forevolution of gene families. The seven human ADH genes, locatedon Chr. 4 in tandem, are classified into five classes. Mice have sixADH genes located on Chr. 3 in the same order as humans. Theinteresting difference between humans and mice is that micehave only one Class I gene. The three Class I genes in humansshow very high nucleotide similarity both in exons (~90%) andintrons (~70%), suggesting duplication events occurred probablyafter primates diverged from rodents. We screened the BAClibraries of three great apes, three old world monkeys, two newworld monkeys, two prosimians, and bat, and are sequencing theADH cluster clones in order to obtain nucleotide sequence for thewhole region of the gene cluster in those primates and bats.Initial analyses of the three Class I genes show no indication ofany gene conversions among human, chimpanzee, bonobo, gorilla,orangutan, and baboon. Here we present the progress of thesequencing and the preliminary analysis on the ADH gene clusterevolution in primate lineage. [Supported by NIH grant AA09379]

1E-12

MASUYAMA, Waka1, SAITO, Naruya1, SUMIYAMA,Kenta1, OKAMURA, Yasushi2 (1Dept. Genet., theGrad. Univ. Adv. Stud. (SOKENDAI), 2Okazaki Inst.for Integ. Biosci.)

Molecular evolution of the TPTE gene family: a domainfusion in the common ancestor of chordates and complex geneduplications in primates.

Gene duplication and domain fusion may contribute to thephenotypic evolution of chordates. Recently, a novel gene (Ci-VSP) was identified. This gene has strong similarity to both avoltage sensor domain of the ion channel genes and a phospha-tase domain (Murata et al. 2005 435:1239-43). To know theevolutionary history of this interesting gene, blast search to theentire database and phylogenetic tree analysis were conducted.Vertebrate counterpart of Ci-VSP gene turned out to be the TPTEgene family, but there was no such gene outside of chordates.Interestingly, only primates have multiple TPTE genes. Thisresult raises the possibility that TPTE gene increased its copynumber as the genomic system became more complex.


1E-13

SAKATE, Ryuichi1,2, SATO, Yoshiharu2, MATSUYA,Akihiro3, FUJII, Yasuyuki1,2, ITOH, Takeshi4,GOJOBORI, Takashi2,5, IMANISHI, Tadashi2 (1JapanBiol. Inform. Res. Cent., Japan Biol. Inform.Consortium, 2Biol. Inform. Res. Cent., Natl. Inst. Adv.Industr. Sci. Tech., 3Hitachi Co., Ltd., 4Genome Res.Dept., Natl. Inst. AgroBiol. Sci., 5Cent. Inform. Biol.DNA Data Bank Japan, Natl. Inst. Genet.)

Partitioning the human genome into differently conservedregions obtained by whole genome comparison betweenhuman, chimpanzee, mouse and rat lineages

In order to elucidate the evolutionary process of human genomeorganization we compared human, chimpanzee, mouse and ratgenomes and built genome alignments that originated from non-repetitive sequences. We detected evolutionary conserved regionsbetween the four species then partitioned the human genomewith the patterns of conservation. We discovered that 19.9% ofthe human genome sequences were conserved among all fourspecies while 43.9% of them were conserved specifically betweenhuman and chimpanzee lineages. The results imply that primate-specific genome regions in the human genome, which may havebeen obtained after the divergence of primates and rodents, arelarger than expected. Additionally, it provides important clues forunderstanding the evolution of the human genome from themammalian lineage.

1E-14

MISAWA, Kazuharu1 (1CREATE, Chiba Industry andAdv. Cent., 2Kazusa DNA Research Insititute)

A Statistical Test for Long Branch Attraction

Long Branch Attraction (LBA) is the phenomenon that longbranches cluster together when genealogical tree reconstructionis performed using the maximum parsimony (MP) method. LBAis caused by parallel and backward substitutions that take placemostly with a long branch. In this study, we investigated theproportion of sites which support LBA tree by assuming a simplesubstitution model for DNA and amino acid sequences. We alsodeveloped a test statistics that allows us to test the proportion ofsites which support a certain tree is significantly greater than theproportion of sites that support the LBA tree or not. The testindicated that the previous studies that showed the basal positionof rodents among mammals were not caused by LBA.

2E-01

ENDO, Kingo1, DAIGAKU, Yasukazu1, MAMAMOTO,Kazuo1 (1Grad. Sch. Life Sci.s, Tohoku Univ.)

Spontaneous mutagenesis in haploid and diploid Saccharo-myces cerevisiae.

To understand the mechanisms involved in spontaneous muta-genesis in Saccharomyces cerevisiae, we constructed RAD52,REV3, and RAD52 REV3 mutations and estimated the rate andspecificity of endogenous CAN1 to CanR mutations. Disruption ofthe RAD52 results in increase in the mutation rate withessentially the same spectrum of mutation. Disruption of REV3results in decrease in mutation rate but increase in recombina-tion rate. Disruption of both RAD52 and REV3 results in decreasein mutation rate. These results indicate that RAD52 is involvedin the recombinational repair of spontaneous DNA damage that,if left unrepaired, can give rise to spontaneous mutation viaREV3-dependent processing. Possible model for distinct RAD52and REV3 stalled replication fork re-start is presented.

2E-02

SAKAI, Akiko1 (1The Grad. Sch. Biol. Sci.s, NAIST)

Impact of active oxygen species on spontaneous mutagenesisin E.coli.

Reactive oxygen species (ROS), including hydrogen peroxide(H2O2), and hydroxyl radical (OH-), can damage DNA and thusare possibly involved in spontaneous mutagenesis. Nevertheless,no direct evidence for the link between ROS and spontaneousmutations has been reported. To elucidate how much extent ofspontaneous mutations is derived from spontaneous DNAdamages caused by ROS, we assessed spontaneous mutationsoccurring within rpsL gene in E. coli cells grown under aerobicand anaerobic conditions and in the cells treated with H2O2.Hotspot types of base substitutions (hotspot) occurring in aerobi-cally grown cells were sharply suppressed in wild-type cells (WT)grown under the anaerobic condition, while treatment of the WTwith H2O2 further increased the frequency. Furthermore, weobserved an increased frequency of the hotspot in a fur- strainthat produces OH- more efficiently than WT. These data stronglysuggest that oxidative DNA lesions formed spontaneously in E.coli cells caused the hotspot.


2E-03

HASEGAWA, Kimiko1, YOSHIYAMA, Kaoru1, MAKI,Hisaji1 (1Grad. Sch. Biol. Sci.s, NAIST, Nara, JAPAN)

Spontaneous mutagenesis associated with nucleotide excisionrepair in E. coli

Nucleotide excision repair (NER) is a general repair system thateliminates various lesions from DNA. Therefore, the NER seemedto efficiently remove various kinds of spontaneous DNA damages.To investigate roles of the NER in spontaneous mutagenesis, wehave analyzed a large number of base substitutions occurringspontaneously within rpoB gene in NER-proficient, -defecientand -overexpressed E. coli cells were analyzed. However, our datasuggested that the spontaneous mutagenesis was induced ratherthan suppressed by NER under the normal growth condition. Tothis unexpected result, we reasoned that NER-like DNAmetabolism may occur on undameged or spontaneously damagedgenome, leading to a significant level of errors during the repairDNA synthesis by DNA polymerase I(pol I). To examine thishypothesis, we constructed and anlyzed E. coli strains that carryan editing-exonuclease deficient polA mutation. From these data,we concluded that a significant portion of spontaneous mutationsare due to the action of NER, and such spontaneous mutationsare derived from replication errors during the repair DNAsynthesis.

2E-04

YAMAMOTO, Ayumi1, YAMAMOTO, Kazuo1,HIROUCHI, Tokuhisa1, MORI, Tamiki1 (1Dept.Molecular Biosciences, Grad. Sch. Life Sci., TohokuUniv.)

Characterization of the Soybean classPP CPD photolyase

The UV-B radiation induces the formation of two classes ofphotoproducts in DNA-the cyclobutane pyrimidine dimer (CPD)and the pyrimidine[6-4]pyrimidone photoproduct (6-4 product).Many orgamisms produce enzymes, termed photolyase, whichspecifically bind and repair these lesions via a UV-A/blue light-dependent mechanism. CPD photolyases are classified into 2types (classI and classP). The gene for class P CPD photolyasehas been cloned from higher plants such as Arabidopsis,cucumbers, rice and spinacia. Here we report the isolation andcharacterization of the soybean classPCPD photolyase cDNA.The sequence of amino acids predicted from the cDNA sequencewas highly homologous to that of higher plants classP CPDphotolyases. When the CPD photolyase cDNA was expressed in aphotolyase-deficient Escherichia coli, photoreactivation activitywas restored by the illumination with fluorescent light.

2E-05

YASUDA, Gentaro1,2,3,4, NISHI, Ryotaro1,2,3,4,WATANABE, Eriko1, MORI, Toshio5, IWAI,Shigenori6, SUGASAWA, Kaoru1,4, HANAOKA,Fumio1,3,4 (1Cell. Physiol. Lab., RIKEN, 2Grad. Sch.Pharm. Sci., Osaka Univ, 3Grad. Sch. Front. Biosci,Osaka Univ, 4SORST, JST, 5Nara Med. Univ, 6Grad.Sch. Engineer. Sci., Osaka Univ)

Functional abnormalities of a pathogenic XPC variant thathas a single amino acid substitution

Xeroderma pigmentosum (XP) is a human autosomal recessivegenetic disorder, which is characterized by hypersensitivity to UVlight and a predisposition to skin cancer. One of the XP-responsible gene products, XPC protein, is involved in DNAdamage recognition for global genome nucleotide excision repair(GG-NER). Two amino acid substitution mutations have been sofar reported, and functional abnormalities of these mutant XPCproteins remained to be elucidated.Here we show evidence that one of the point mutations (W690S),which was identified in the XP13PV patient, results in destabi-lization of the XPC protein in vivo. Furthermore, the recombinantmutant XPC protein appears to lack the damaged DNA-bindingactivity in vitro. We also examined effect of the W690S mutationon in vivo dynamics of the XPC protein in locally UV-irradiatedcells. Although the mutant XPC protein accumulated in thedamaged areas in a UV-DDB-dependent manner, recruitment ofthe XPA protein that is required for later stages of the GG-NERreaction is impaired. Our result indicate that the W690Smutation results in both quantitative and functional defects ofthe XPC protein, which contribute to occurrence of XP.

2E-06

SHIOMI, Tadahiro1, SHIOMI, Naoko1, NOSHIRO,Katsuko1, AIZAWA, Shiro2 (1Low Dose RadiationResearch Projects, Res. Cent. Radiation Safety, Natl.Inst. Radiological Sci., 2Radiation Hazards Res. Group,Res. Cent. Radiation Safety, Natl. Inst. RadiologicalSci.)

Charactrization of xeroderma pigmentosum group G bonemarrow chimera mice

In addition to xeroderma pigmentosum (XP), mutations in thehuman XPG gene cause early onset of Cockayne syndrome (CS) insome patients with characteristics such as growth retardationand short life span. Xpg null mice exhibited growth retardationand a short life span as human patients do. We generated fourmutant Xpg mouse strains with different mutations in Xpg geneto identify the Xpg region that causes onset of the CS phenotype.We found that the deletion of C terminal 183 amino acids resultsin the CS phenotype. The primary embryonic fibroblasts isolatedfrom Xpg-deficient mice underwent premature senescence andexhibited the early onset of immortalization. If chimera mice inwhich some tissues are replaced with Xpg-deficient cells ortissues could be generated, it seems to be very interesting toanalyze the fate of such chimera mice and/or Xpg-deficient cellsin such chimera mice. So we have generated bone marrowchimera mice in which normal blood cells were replaced withXpg-deficient cells and examined their life span, shortening of lifespan by X-irradiation and class switching of immunoglobulinheavy chain.


2E-07

ICHIISHI, Akihiko1, SOTANI, Sayuri1, HOSHINO,Masayuki1 (1Faclt. Life Sci.s, Toyo Univ.)

Analysis of Saccharomyces cerevisiae RAD23 homolog inNeurospora crassa

In Saccharomyces cerevisiae, the complex of Rad4 and Rad23 hasbeen suggested to function in the damage recognition step ofnucleotide excision repair (NER). In order to study NER inNeurospora crassa, we isolated and characterized a mutant of theS. cerevisiae RAD23 homolog gene in N. crassa. In BLASTsearches of the Neurospora genome database using amino acidsequence of the S. cerevisiae Rad23 protein, we found a candidate(NCU07542.2) and named it ncRAD23. We constructed ncRAD23gene disruption mutant using a homologous recombination andtested for UV sensitivity together with mus-38 and mus-18mutant strains. The mus-38 gene encodes a homologue of S.cerevisiae Rad1, and mus-18 gene encodes a homologue ofSchizosaccharomyces pombe Uvde. UV sensitivity of ncRAD23deletion mutant was similar to that of the wild type. ThencRAD23 mus-38 double mutant showed same sensitivity of themus-38 mutant. In contrast, the ncRAD23 mus-18 double mutantwas more sensitive to UV irradiation than the mus-18 mutant.These observations suggest that ncRAD23 is involved in NER butnot in the mus-18 pathway.

2E-08

KAWABATA, Tsuyoshi1, SUZUKI, Keiichiro1, INOUE,Hirokazu1 (1Dept. Regulation Biol., Faclt. Sci., Univ.Saitama)

Genetic relationship between genes participate in translesionsynthesis and two DNA repair genes mus-46 and mus-47,homologs of Saccaromyces cevisiae UBC13 and MMS2

In this study, we characterized Neurospora crassa mus-46 andmus-47, homologous to UBC13 and MMS2 of Saccharomycescerevisiae respectively. The mus-46 and mus-47 genes were foundin searching genome database of N. crassa and cDNA sequenceswere determined. Deduced amino acid sequences were comparedwith homologs of human and S. cerevisiae. MUS46 and MUS47exhibited approximately 70% and 40% identity to each homolog,respectively. A physical interaction between MUS46 and MUS47was investigated by yeast two hybrid system, resulting in positivereactions and indicating that these two proteins interact eachother in N. crassa as well as other organisms. We disrupted thesetwo genes in N. crassa by gene replacement. Sensitivity tomutagens of these disruptants was tested. Both of these strainsshowed higher sensitivity to MMS, MNNG and UV than wildtype. Extent of sensitivities in mus-46 and mus-47 mutants wasnearly equal. Furthermore, we will also report about epistaticanalyses of mus-46 to other DNA repair mutations indicatingthat mus-46 and mus-47 have genetic interactions with genesparticipate in translesion synthesis.

2E-09

ISHIBASHI, Toru1,5, HAYAKAWA, Hiroshi2, ITO,Riyoko3, MIYAZAWA, Masayuki4, YAMAGATA,Yuriko4, SEKIGUCHI, Mutsuo1,5 (1BiomolecularEngineering Res. Inst., 2Dept. Med. Biochem., Grad.Sch. Med. Sci., Kyushu Univ., 3Dept. Physiological Sci.and Molec. Biol., Fukuoka Dental College, 4Grad. Sch.Pharm. Sci., Kumamoto Univ., 5Front. Res. Cent.,Fukuoka Dental College)

Human enzymes for preventing transcriptional errors causedby oxidative stress

An oxidized form of guanine, 8-oxo-7,8-dihydroguanine (8-oxoguanine), is produced in cell by reactive oxygen speciesformed during normal cellular metabolism. This oxidized basecan pair with both adenine and cytocine and, thus, thepersistence of this base in messenger RNA would cause transla-tional errors. Here we show that among human MutT-relatedproteins, NUDT5 and MTH1 have abilities to prevent transla-tional errors caused by oxidative damage. Expression of cDNA forNUDT5 or MTH1 in Escherichia coli mutT-deficient cells reducedthe level of production of erroneous proteins to the wild type one.Both NUDT5 and MTH1 can hydolyze 8-oxo-GDP, an oxidizedform of GDP, to the corresponding monophosphate. Moreover,MTH1, but not NUDT5, possesses an activity to degrade 8-oxo-GTP to the monophosphate. The elimination of 8-oxoguanine-containing ribonucleotides from the RNA precursor pool isimportant to secure the accurate protein synthesis and bothNUDT5 and MTH1 may be involved in this process in humancells.

2E-10

TAKAGI, Yasumitsu1, HIDAKA, Masumi2, KOMORI,Kayoko2, SANADA, Masayuki3, YOSHIDA, Hiroki4,SEKIGUCHI, Mutsuo5 (1Front. Res. Cent., Fukuokadental college, 2Biomolecular Eng. Research Insutitute,3Dept. Physiological Sci. and Molec. Biol., Fukuokadental college, 4Dept. Biomolecular Sci., Saga MedicalSchool, 5Front. Res. Cent., Fukuoka dental college)

Signaling pathway in O6-methylguanine-induced apoptosis

Modified DNA bases which do not prevent progression of thereplication fork but cause mispairing are troublesome, since theyyield mutation. To prevent such outcomes, multicellular organ-isms are equipped with a system to eliminate cells with suchmispairs by inducing apoptosis. O6-methylguanine(O6mG)-medi-ated killing pathway, induced by alkylating agents, provides anexcellent model to reveal this apoptotic defense system. We haveshown that blocking of the mismatch recognition (MMR) pathwaycan dissociate killing and tumorigenic effects of an alkylatingagent.To understand the mechanism to transmit this deathsignal, we have investigated the roles of caspase-3 and Apaf1proteins in alkylation-induced apoptosis, by using primary andestablished cell lines derived from knockout mice with combina-tions of Mgmt (O6mG-DNA methyltransferase, a repair enzymefor O6mG), Mlh1 (an essential component of the MMR system)and/or Apaf1 gene defects.


2E-11

KOMORI, Kayoko1, TAKAGI, Yasumitsu2, TAKANO,Tomoko1, SEKIGUCHI, Mutsuo1,2, HIDAKA,Masumi1 (1Dept. Molec. Biol., Biomolecular Eng. Res.Inst. (BERI), 2Front. Res. Cent., Fukuoka DentalCollege)

An isolation and characterization of genes involved in DNAdamage-inducible apoptotic pathway

The genomic DNA in cell is continuously undergoing damage asconsequences of endogenous processes and exposure to exogenousagents. The resulting lesions of the DNA can be repaired byvarious systems or induce cell death to preserve genomeintegrity. Simple alkylating agents are known to induce mod-ifications of bases, among which O6-metylguanine which cancause GC ! AT transition mutation, is most notabel. This lesionis specifically repaired by O6-metylguanine-DNA methyltransfer-ase (MGMT). In cell lines lacking the MGMT activity, even lowlevels of alkylating agents cause cell death that depends onmismatch recognition proteins. However, the exact mechanism ofthis apoptotic pathway has been less understood. To investigatethe molecular mechanism of alkylated DNA-inducible apoptoticpathway, we carried out random mutagenesis by gene-traptechnique on the established mouse Mgmt -/- cell line and isolatedabout 50 mutants, which acquired increased resistance toalkylating agent, and then identified 27 disrupted genes. Insome mutant clones, reduced levels of caspase-3 activation andincreased mutant frequencies by MNU-treatment were observedas compared with un-trapped cells.

2E-12

ITO, Riyoko1, HAYAKAWA, Hiroshi2, SEKIGUCHI,Mutsuo3,4, ISHIBASHI, Toru3,4 (1Dept. PhysiologicalSci. and Molec. Biol., Fukuoka Dental College, 2Dept.Med. Biochem., Grad. Sch. Med. Sci., Kyushu Univ.,3Front. Res. Cent., Fukuoka Dental College, 4Bio-molecular Eng. Res. Inst.)

Multiple enzyme activities of Escherichia coli MutT proteinfor sanitization of nucleotide pool

8-OxoGua (8-oxo-7,8-dihydroguanine) is produced in nucleic acidsas well as in nucleotide pools of cells, by reactive oxygen speciesnormally formed during cellular metabolic processes. MutTprotein of Escherichia coli specifically degrades 8-oxoGua-containing deoxyribo- and ribonucleoside triphosphates to corre-sponding nucleoside monophosphates, thereby preventing mis-incorporation of 8-oxoGua into DNA and RNA, which would causemutation and phenotypic suppression, respectively. Here, wereport that the MutT protein has additional activities for cleaningup the nucleotide pools to ensure accurate DNA replication andtranscription. It hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP with aKm value considerably lower than that for its normal counter-part, dGDP. Furthermore, the MutT possesses an activity todegrade 8-oxo-GDP to the related nucleoside monophosphatewith a Km value 8,000 times lower than that for GDP. The Kmvalues for 8-oxo-dGTP and 8-oxo-GTP were also very low. Thesemultiple enzyme activities of the MutT protein would facilitatethe high fidelity of DNA and RNA syntheses.

2E-13

HIDAKA, Masumi1, TAKAGI, Yasumitsu2, TAKANO,Tomoko1, KOMORI, Kayoko1, SEKIGUCHI, Mutsuo1,2

(1Dept. Molec. Biol., Biomolecular Eng. Res. Inst.,2Front. Res. Cent., Fukuoka Dental College)

Mismatch repair proteins sequentially recognize alkylatedDNA through the action of PCNA and induce apoptosis

Mismatched bases, such as O6-methylguanines, are produced incells exposed to alkylating agents and cause apoptosis. In humancells damaged by an alkylating agent, we detected a proteincomplex composed of MutSa, MutLa and PCNA by immunopre-cipitation method using chromatin extracts, in which protein-protein interactions were stabilized by chemical cross-linking.Time course experiments revealed that MutSa, consisting ofMSH2 and MSH6 proteins, and PCNA bind to the DNA to forman initial complex, and MutLa, composed of MLH1 and PMS2,binds to the complex when the DNA is damaged. Reduction of thePCNA content by siRNA or inhibition of DNA replication byaphidicolin significantly reduced the levels of the PCNA-MutSa-MutLa complex and also suppressed an increase in the caspase-3activity, a hallmark for the induction of apoptosis. Theseobservations imply that the induction of apoptosis is coupledwith the progression of DNA replication through the action ofPCNA.

2E-14

KITAMURA, Maki1, INOUE, Hirokazu1 (1Lab.Genetics, Dept. Regulation Biol., Faclt. Sci., SaitamaUniv.)

Analysis of a poly(ADP-ribose) polymerase (PARP) in Neuro-spora crassa

Poly ADP-ribosylation is a post-translational modification ofnuclear proteins and plays important roles in DNA repair,apoptosis and telomere maintenance. Poly(ADP-ribose) polymer-ase (PARP) catalyses the formation of this polymers using NAD+

as substrate. In human, PARPs constitute a large family of 18proteins. But, much of these function are unknown. Theseproteins have PARP catalytic domain on C-terminal region,which is highly conserved during family. In this study, wesearched the genes that encode proteins PARP homologue inNeurospora crassa using N.crassa genome database. We identi-fied only one ORF and named the gene ncPARP. To analyze thisgene function, an ncparp null mutant was generated by genereplacement. We show that the ncparp mutant was not sensitiveto methyl methanesulphonate (MMS) although PARP-1 null miceare sensitive. And the ncparp mutant indicated the defect of ahyphal elongation.


2E-15

TORI, Kazuo1, ISHINO, Yoshizumi1 (1Dept. GeneticResources Tech., Univ. Kyushu)

Identification and characterization of a novel deoxyribonu-clease in Archaea

Deoxyribonucleases (DNases) play very important roles in allDNA transactions including DNA replication, repair, andrecombination. Many kinds of DNases have been identified fromEukarya and Bacteria, and their functions have been charac-terized in detail to date.In Archaea, the third domain of life, several DNases includingstructural homologs of eukaryotic enzymes and archaea-specificenzymes are known. However, as compared with eukarya andbacteria, a few DNases have been characterized in Archaea. It isimportant to identify all of the DNases in the cells to understandthe molecular mechanisms of DNA transactions in Archaea.In this study, we identified a novel DNases from the hyper-thermophilic archaeon, Pyrococcus furiosus, and analyzed itsfunctions. Thus, current our results will be presented.

3E-01

BAUTISTA-ZAPANTA, J-ney1, HASSAN ARAFAT,Hussam1, TANAKA, Katsuyuki1, SAWADA,Hiroyuki2, SUZUKI, Katsunori1 (1Dept. BiologoicalSci., Grad. Sch. Sci., Hiroshima Univ., 2Natl. Inst.Agro-Env. Sci.)

Variation of Intervening sequence at 23S rRNA and 16S-23SITS sequence among biovar 1, 2 and 3 Agrobacterium strains

To give an insight to genetic diversity relationship of Agro-bacterium, rDNA sequences were investigated in 33 strainsbelonging to three Agrobacterium biovars: 7 strains of biovar 1(Rhizobium radiobacter), 17 strains of biovar 2 (R. rhizogenes)and 9 strains of biovar 3 (R. vitis). Nucleotide sequences of the16S rRNA gene, 16S-23S ITS and intervening sequence (IVS) inthe 23S rRNA gene were determined. Phylogenetic analysisbased on the highly conserved 16S rRNA gene sequencesconfirmed that all strains studied belong to each of the biovars.The 16S-23S ITS region and IVS were variable even in strainsbelonging to the same biovar. In all biovar 2 strains studied, therewas only a single type of 16S-23S ITS and IVS observed in eachgenome while some members of the biovar 1 and 3 strains couldhave at least two types of 16S-23S ITS and IVS in each genome.There are 6, 7 and 4 16S-23S ITS types and 3, 2 and 4 IVS typesobserved in biovar 1, 2 and 3 strains, respectively. These resultsindicate that rDNA diversity is narrower in biovar 2 than inbiovar 1 and 3.

3E-02

HANDA, Hirokazu1,2 (1Natl. Inst. AgroBiol. Sci.,2Grad. Sch. Life and Env. Sci., Tsukuba Univ.)

Origin and differentiation of cytoplasm of Japanese rapeseedvarieties revealed from mitochondrial DNA analysis

Using about 60 varieties of rapeseed bred in Japan after the Meijiperiod, I investigated a type of mitochondrial genome andpresence of mitochondrial plasmid, and carried out a comparativeanalysis with the breeding history of Japanese rapeseedvarieties. Cytoplasmic genome of rapeseed was classified roughlyinto two types, type I and type II. And type II cytoplasm ofrapeseed was closely resembled with that of B. rapa, which is oneof parents of rapeseed, amphidiploid species between B. rapa andB. oleracea. In this study, I showed that all of varieties with typeII mitochondria were originated from interspecies crossesbetween rapeseed and B. rapa. This indicates that type IIcytoplasm is introduced to rapeseed by a process of breeding. Onthe other hand, it was known that mitochondrial plasmid wastransmitted from pollen parent to the progeny. Participation ofnuclear and/or mitochondrial genome was suggested for thetransmission of plasmid.


3E-03

WATADA, Masayoshi1, NAGASHIMA, Kumiko1

(1Dept. Biol., Faclt. Sci., Ehime Univ.)

Molecular evolution and horizontal transfer of P elements inDrosophila

P elements are mobile genetic sequences that were first describedin Drosophila melanogaster and one of the best-characterizedtransposable elements in Drosophila. However, most of the Pelements have been detected in the subgenus Sophophora and Pelements in the other genera have not been studied extensively.In the present study, we surveyed the distribution of the Pelements in several genera including the primitive groups ofDrosophila. We found P elements in some genera including theprimitive groups such as Amiota. However, P elements found inthe present study have a similarity to the P elements in thesubgenus Sophophora with one exception. In addition, our resultsreveal a P elements phylogeny that is inconsistent with thephylogeny of the species themselves. These results suggest thatmost of P elements reported in the present study have been comefrom the P elements of Sophophora although the horizontaltransfer events have not occurred recently.

3E-04

NOZAWA, Masafumi1, KUMAGAI, Masahiko2,AOTSUKA, Tadashi3, TAMURA, Koichiro3 (1Dept.Biol. Sci.s, Grad. Sch. Sci., Tokyo Metro. Univ., 2Dept.Biol., Faclt. Sci., Tokyo Metropolitan Univ., 3Dept.Biol., Faculty of Urban Liberal Arts, Tokyo MetropolitanUniv.)

Genome-wide expansion of a repeat sequence during theevolution of the Drosophila ananassae subgroup

Repeat sequences occupy substantial amount of a genomesequence (e.g., ca. 50% of the human genome sequence),addressing important issues, i.e., why and how repeat sequenceshave accumulated in a genome through evolution. A newlydiscovered repeat sequence in the genomes of the Drosophilaananassae subgroup provided us a glimpse of evolutionaryprocess of repeat sequences, because its recent birth makes uspossible to trace back to the origin. We obtained the followingresults. (1) More than 10,000 copies of the repeat sequences weredetected in species of the D. bipectinata complex belonging to theD. ananassae subgroup by quantitative DNA hybridizationtechniques. (2) Wide distribution of the repeat sequences amongeuchromatic regions were shown by the polytene chromosome insitu hybridization of D. parabipectinata as well as by BLASTsearch in the draft genome sequence of D. ananassae. (3) Morethan half of the repeat loci formed arrays of several unitsequences by the BLAST search. These results suggest that therepeat sequences are categorized into neither tandem repeats nortransposable elements.

3E-05

YAMAZAKI, Ryo1, TERAI, Yohey1, OTA, Ryoko1,OKADA, Norihiro1,2 (1Grad. Sch. BioSci. and BioTech.,Tokyo Inst. Tech., 2Natl. Inst. Basci Biology)

The role of c-type lysozyme gene during adaptive radiation ofEast African cichlid fishes

Lakes Victoria, Malawi and Tanganyika in the East African RiftValley harbor roughly 500, 500, and 250 endemic species ofcichlid fishes, respectively, and these fishes provide a spectacularexample of the explosive adaptive radiation of living vertebrates.The fishes are ecologically and morphologically highly diverseand they are adapted to various environments. In these fishes, itis postulated that lysozyme acts as a part of innate immunesystem by its bacteriolysis activity. Here, we studied theevolution of the lysozyme gene in cichlids. We isolated c-typelysozyme gene from several cichlid species. Southern blot andsequence analysis showed that c-type lysozyme gene haveduplicated several times and have evolved at an accelerated rateby positive selection during the adaptive radiation of cichlids inthe Great Lakes. The diversification of this gene may have playedan important role in host defense during the process ofadaptation to various environments.

3E-06

SAWAI, Hiromi1, SATTA, Yoko1, TAKAHATA,Naoyuki1 (1Dept. Biosyst. Sci., The Grad. Univ. Adv.Stud. (Sokendai))

A large ancestral population size and the origin of domesticchickens

Domestic chickens have a large amount of biochemical andmorphological variations, a basis of the genetic response tointensive artificial selection throughout the domestication proc-ess. The phylogenetic analysis of mitochondrial DNA sequencesrevealed that the most recent common ancestor (MRCA) ofdomestic chickens goes back to 0.7-0.9 million years (myr) ago.The recent SNP data of three domestic chickens also consistentlyrevealed a rather ancient origin of the nucleotide polymorphism.To investigate the origin of nuclear genetic diversity of chickensin more detail, we first estimated the nucleotide substitution rateat 33 introns in domestic chickens, red junglefowls, turkeys andquails. The calibrated rate becomes faster than that in higherprimates. Second, we examined the nucleotide divergence at thesame set of introns among two domestic chickens and redjunglefowls. This sequence information suggests that the MRCAof domestic chickens goes back to 2.6-3.0 myr ago. High degrees ofpolymorphism in domestic chickens are attributed to either alarge founding population size or repeated introgression of redjunglefowls or both.


3E-07

KANEKO, Satoko1, TAKAHATA, Naoyuki1, SATTA,Yoko1 (1Dept. Biosyst. Sci.s, The Grad. Univ. Adv.Stud. (SOKENDAI))

Enhanced neutral substitution rates in processed pseudo-genes

While we studied the processed pseudogene of Makorin1-p1 inmice (Kaneko et al. Annual meeting in Osaka 2004, MBE in NewZealand 2005), we noted that the substitution rate is significantlyenhanced relative to the rate either at the synonymous sites or inintrons of functional Makorin1. It turned out that the enhancedrate in the processed pseudogene is largely attributed to frequentsubstitutions at CpG dinucleotides, although these sites aregenerally under-represented as in other genomic regions. Herewe propose our idea that processed pseudogenes can provide anideal case for studying the substitution process at CpG sites. Theidea is based on the premise that a processed pseudogene hasbeen non-functional since its origin, a situation different fromthat in duplicated genes. We then generalize the idea to accountfor the rate variation at seemingly neutral synonymous, intronicand intergenic sites and provide discussion in particular relationto two recent papers by Nachman and Crowell (Genetics 2000)and Subramanian and Kumar (Genome Research 2004).

3E-08

SAKAI, Hiroaki1,2,3, KOYANAGI, Kanako O.4,IMANISHI, Tadashi4,5, ITOH, Takeshi1,5,GOJOBORI, Takashi2,5,6 (1Genome Res. Dept., Natl.Inst. AgroBiol. Sci., 2Japan Biol. Inform. Res. Cent.,Japan Biol. Inform. Consortium, 3Kyowa Hakko KogyoCo. Ltd., 4Grad. Sch. Inform. Sci. Tech., HokkaidoUniv., 5Biol. Inform. Res. Cent., Natl. Inst. Adv.Industr. Sci. Tech., 6Center for Inform. Biol. andDDBJ, Natl. Inst. Genet.)

Molecular evolutionary analysis of processed pseudogenes inthe human and mouse genomes

Despite the wide distribution of processed pseudogenes inmammalian genomes such as human and mouse, little wasknown about their roles in molecular evolution. Here, to elucidatethe quantitative and qualitative contribution of processedpseudogenes to the mammalian genome evolution, we attemptedto detect processed pseudogenes by extensively mapping themRNAs to the human and mouse genomes and estimated the rateof their origin. As a result, we could detect 7348 and 6188processed pseudogenes in the human and mouse genomes,respectively and revealed that the rate was more than 2% pergene per million years, which was markedly higher than the rate(0.9%) of duplication in the human genome. Furthermore, about1% of the processed pseudogenes seemed to be reinvigorated bypost-retrotransposition transcription. Since the expression pat-terns of transcribed pseudogenes in mouse were biased to testisand quite different from those in human, their emergence mighthave led to species-specific evolution. Our results indicate thatthe generation of processed pseudogenes was not futile but hasbeen an indispensable resource driving dynamic evolution of themammalian genomes.

3E-09

IWAMA, Hisakazu1, TAKEZAKI, Naoko1, GOJOBORI,Takashi2,3 (1Inform. Tech. Cent., Kagawa Univ.,2Cent. Inform. Biol. DNA Data Bank Japan, Natl. Inst.Genet., Res. Org. Inform. Systems, 3Biol. Inform. Res.Cent., Natl. Inst. Adv. Industr. Sci. Tech.)

High Degree of Upstream Sequence Conservation for Tran-scription Factor Genes by Comparative Analysis of Human-Mouse Orthologues

Detecting evolutionarily conserved sequences is a useful way toidentify regulatory elements in genomic sequences. In this study,with the aim of elucidating the feature of the regulatory network,we systematically collected 3,750 pairs of human-mouse ortho-logues and identified more than 100,000 conserved sequencesbased on the alignments of the 8kb-upstream genomic sequencesof those gene pairs. As a result, we found that the genes with highdegree of the upstream sequence conservation are predominantlytranscription factor (TF) genes. In particular, TF genes that arerelated to developmental processes showed further higher degreeof the upstream conservation than the other TF genes. Theseresults suggest that regulatory networks with potentially highconnectivity have been preferentially conserved among TF genes,in particular, TF genes related to developmental processes.

3E-10

IWASE, Minyo1, SATTA, Yoko1, TAKAHATA,Naoyuki1 (1Dept. Biosyst. Sci., Grad. Univ. Adv. Stud.(Sokendai))

Ectopic sequence exchanges in the Kallmann syndrome 1gene region of primate sex chromosomes

The sequence comparison of the distal half of the human X shortarm with the Y counterpart shows that the per-site sequencedifferences (p) are ca. 10% on average. Despite this rather highextent of sex chromosomal differentiation, there are a number ofsmall regions in which thep value is significantly lower than theaverage. One such region is located within the Kallmannsyndrome 1 gene on the X chromosome (KAL1 or KALX). Toinvestigate the evolutionary history of KALX of 210-kb lengthand gain some insight of the cause of Kallmann syndrome, wepartially sequenced KALX and its gametologous pseudogene onthe Y chromosome (KALY) for various primate species. Thesequence analysis reveals a number of independent exondeletions on the Y chromosome as well as gene conversion-likeevents between KALX and KALY in diverse primate lineages.Taken together, the distal half of the X short arm appears toectopically pair with gametologous regions of the Y chromosomeeven though they have differentiated since some tens of millionyears ago. It is argued that gene conversion-like events fromKALY to KALX can dysfunction the KALX gene and in humansthey are manifested as the Kallmann syndrome.


3E-11

YOSHIHARU, Sato1, SAKATE, Ryuichi1,2,MURAKAMI, Katsuhiko1,2, MATSUYA, Akihiro3,TAKEDA, Jun-ichi1, FUJII, Yasuyuki1,2, ITOH,Takeshi4, GOJOBORI, Takashi1,5, IMANISHI,Tadashi1 (1Biol. Inform. Res. Cent., Natl. Inst. Adv.Industr. Sci. Tech., 2Japan Biol. Inform. Res. Cent.,Japan Biol. Inform. Consortium, 3Hitachi Co., Ltd.,4Genome Res. Dept., Natl. Inst. AgroBiol. Sci., 5Cent.Inform. Biol. DNA Data Bank Japan, Natl. Inst.Genet.)

Comprehensive analysis of segmental duplications in humangenome

We analyzed the segmental duplications thoroughly at the wholegenome level in order to assess the role of segmental duplicationin the genome and gene evolution. First, we performed a self-alignment of NCBI build 35 human genome (~3 billion bp). Usingthe genome alignment software BLASTZ, we searched forsegmental duplications in the human genome. As a result, wefound that 5.2% of the human genome contains segmentalduplications, and ~40% (2.1% of the human genome) of whichwere intra-chromosomal. Moreover, we deduced the divergencetime of segmental duplications and the duplicated genesembedded in them. Then we classified the duplicated genesaccording to theiracquisition dates and predicted functions.

3E-12

BHOWMICK, Bejon1, TAKAHATA, Naoyuki1, SATTA,Yoko1 (1Dept. Biosyst. Sci., The Grad. Univ. Adv. Stud.(Sokendai))

Evolution of male-specific Y (MSY) genes in humans

To elucidate the evolutionary tempo and mode of nine malespecific Y (MSY) genes, we carried out phylogenetic analyses witha silent clock rate. The rate was calibrated as 1.4 x 10-9/site/yearover those genes for which chimpanzee or other primate orthologsare available. In addition to functional copies, MSY genes oftenpossess two or more pseudogenes on the Y chromosome.Interestingly, some pseudogenes are human specific while othersare of ancient origins. We discuss the role of locus duplication,retrotransposition, translocation and gene conversion in evolu-tion of primate MSY genes.

3E-13

SHIBATA, Hiroki1, GOTO, Hiroki1, TANAKA, Kunika1,KUROKI, Yoko2, TOYODA, Atsushi2, HATTORI,Masahira2,3, SAKAKI, Yoshiyuki2, FUJIYAMA, Asao2,4,FUKUMAKI, Yasuyuki1 (1Div. Disease Genes, ResearchCenter for Genetic Information, Med. Inst.Bioregulation, Kyushu Univ., 2Sequence Tech. Team,Genome Core Tech. Facilities, RIKEN Genomic Sci.sCent., 3Lab. Genome Information, Kitasato Inst. LifeSci., Kitasato Univ., 4Research Information Res. Div.,Natl. Inst. Inform.)

Molecular evolution of the glutamate receptor gene familysince the human-chimpanzee divergence.

Along with the rapid enlargement of the brain in the humanlineage, the information processing within the brain should havebeen dynamically developed so that the “humanness” phenotypessuch as technology and language could have evolved. Glutamateis one of the major neurotransmitters in the primate brain.Multiple recent genetic studies indicate the involvement of theglutamate transmission system in schizophrenia where theinformation processing is disorganized within the brain. Toclarify the contribution of glutamate receptor genes to the humanevolution since the human-chimpanzee divergence, we plannedcomparative analyses of the complete set of the glutamatereceptor gene family between human and chimpanzee. Sincethe public data of chimpanzee is still premature, we havedetermined nucleotide sequences of most of the chimpanzeeglutamate receptor genes. We will report on molecular evolu-tionary analyses of these data comparing with the public humanand murine sequences.


80-6abstracts 425..498 - J-Stage

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