01 1a -02 340 - j-stage

WS10-7

FUJITA, Miku1 (1The Third Dept. Agr., Univ. ofHokkaido)

Report of the Activity of iGEM HokkaidoU

Circularization of a protein is one of the ways to enhance itsstability against various temperatures and pH levels. Wechallenged ourselves to circularize proteins using self-assemblingpeptides and linkers. Self-assembling peptide (SAP) is anamphiphilic peptide which self-assembles under physicochemicalconditions. We made SAPs fused to link ends of the protein. Wealso inserted cysteine residues in the linker which is essential forthe circularization so that they form a disulfide bond. This linkeris expected to be applicable to most proteins because it automati-cally adjusts its length. These two elements - the self-assemblingpeptide and the linker - enable the protein to be circularized.

1A-01

Canceled

1A-02

TANAKA, Seiji1,2, MIYAZAWA-ONAMI, Mayumi1,HOSOE, Akari1, ARAKI, Hiroyuki1,2 (1Div. MicrobialGenet., Natl. Inst. Genet., 2SOKENDAI)

Relationship between the initiation reaction of DNA repli-cation and chromatin regulator in eukaryotes

DNA, the carrier of genetic information in organisms, is stored aschromosomes, which is consists from many chromatins, in nucleiin eukaryotes. It is widely known that there are regulatorymechanisms that cooperate chromatin regulating factors andDNA-mediated reactions, such as transcription and DNA repair.This can let us expect that the existence of cooperativemechanism(s) between chromatin regulators and DNA replica-tion, which is a essential process for cell proliferation. However,this is largely unknown, although some study have shown thatthe contribution of chromatin regulators to replication licensingand elongation process of DNA replication. Moreover, thecontribution of chromatin regulators onto the initiation reactionof DNA replication is totally unknown. Here we would like toshow some results indicating a chromatin regulator has a role inthe initiation of DNA replication. Our results reveal novel level ofregulation of DNA replication.

Genes Genet. Syst. (2016) 91

General sessions (1A-01 – 3E-12)

340

1A-03

SASAKI, Mariko1, KOBAYASHI, Takehiko1 (1Lab.Genome Regen., Inst. Molec. Cell. BioSci., Univ.Tokyo)

Understanding the mechanisms that repair DNA double-strand breaks arising from replication fork arrest

Arrested replication forks can lead to DNA double-strand breaks(DSBs). Replication-associated DSBs are a major source ofgenome rearrangements, which are a hallmark of cancer cells,cause human genomic disorders and influence genome diversity.Yet, it remains to be understood how replication-associated DSBsare repaired. In this study, we used the budding yeast ribosomalRNA gene locus to demonstrate that DSBs generated uponreplication fork arrest are normally repaired by the pathwaysindependent of homologous recombination (HR). In markedcontrast, cells lacking a component of replisome Ctf4 requireDSB end resection and subsequent recombination for DSB repair.These observations reveal that repair of DSBs arising fromreplication fork arrest is regulated differently from replication-independent DSBs. Our findings illuminate a critical role of Ctf4in protecting replication-associated DSBs from rearrangement-prone HR pathways.

1A-04

MATSUZAKI, Kenichiro1, BOULTON, Simon2 (1Inst.Prot. Res., Osaka Univ., 2The Francis Crick Inst.)

The Role of Fancj in the maintenance of microsatellites andlymphomagenesis

Maintenance of genome integrity during DNA replication is ofvital importance to ensure that daughter cells inherit an intactcopy of the genetic code. Repetitive DNA sequences are aparticular challenge to genome stability due to their propensityto form secondary structures that hinder replisome progressionand promote template slippage. Microsatellites are repetitivesequences of 1-10 base pairs of DNA and highly prone toexpansion/contraction due to their propensity to form non-B-formDNA structures. Although mismatch repair plays a key role inpreventing microsatellite instability (MSI), activities must alsoexist that unwind secondary structures to facilitate replicationfidelity.??In this study, we found that Fancj-deficient micephenocopy other mouse models of Fanconi anemia (FA). Un-expectedly, Fancj-deficient mice also present with enhancedpredisposition to lymphoma, and cells derived from these miceare hypersensitive to replication inhibitors and show high level ofMSI. These data implicate Fancj as a key factor required tocounteract MSI.?? We propose that FANCJ counteracts theformation of secondary structures that arise during replicationof microsatellite.

1A-05

HASEGAWA, Yuki1, KEYAMURA, Kenji1, HISHIDA,Takashi1 (1Dept. Life Sci., Grad. Sch. Sci., GakushuinUniv.)

The role of Mgs1 in DNA replication stress response inSaccharomyces cerevisiae

Saccharomyces cerevisiae Mgs1 protein possesses DNA bindingand DNA-dependent ATPase activities, which plays a role inmaintaining genome stability during DNA replication. TheMGS1orthologues are highly conserved in prokaryotes and eukaryotes.The yeast Mgs1 protein consists of a eukaryote-specific N-terminal domain containing a ubiquitin binding zinc finger motif,a central core ATP+ATPase domain, and a large C-terminaldomain. In this study, we studied the role of C-terminal domain ofMgs1 by examining the effects of its C-terminal deletions onMgs1 protein functions in vivo. We show that the expression of C-terminal deletions results in higher sensitivity to methylmethanesulfonate compared with wild type Mgs1. This and other datasuggest that the C-terminal deletions of Mgs1 have significantand complex effects on the activity of the protein in vivo.

1A-06

YOSHIYAMA, Kaoru Okamoto1, KAMINOYAMA,Kaori1, SAKAMOTO, Tomoaki1, KIMURA, Seisuke1

(1Life Sci.s, Kyoto Sangyo Univ.)

Mechanism of pathway selection in DNA damage responsesin plants

Being sessile, plants are constantly exposed to various environ-mental stresses, such as ultraviolet from sun and reactive oxygenspecies produced by strong light, high temperature and dryingstresses and so on. It is, thus, imperative that an efficient andspecific DNA damage responses (DDRs) be in place in plants tocope with DNA damage. To understand plant DDRs, we havebeen focusing on Arabidopsis SOG1, a transcription factor,regulates more than 100 genes involved in the DDRs. SOG1works as a master regulator of DDRs as it is required for variousresponses, including; the induction of cell cycle arrest, inductionof DNA repair genes, induction of the endoreduplicative cell cycleand the induction of programmed cell death. However, it isunknown how these responses are regulated by this single masterregulator. We previously showed that SQ motifs of SOG1 arephosphorylated in response to DNA damage and how thephosphorylation is essential for the activation of DDRs. Wewould like to report the relationship between the phosphorylationof SOG1 and the activation of various responses in this meeting.

Genes Genet. Syst. (2016) 91 341

1A-07

HIRAI, Hirohisa1, HIRAI, Yuriko1, KOGA, Akihiko2

(1Mol. Biol. Section, Primate Res. Inst., Kyoto Univ.,2Cell. Biol. Section, Primate Res. Inst., Kyoto Univ.)

De novo chromosomal alterations in interspecies hybrids ofnight monkeys

During chromosome analysis of 16 night (or owl) monkeys inPrimate Res. Inst., Kyoto Univ., we encountered four individualswith unimaginable chromosome changes that are unexpectedfrom a single species. Eventually, they were supposed to bechromosome modification induced by interspecies hybridizationbetween Aotus azarae (2n = 49, 50) and A. grisemembra (2n = 53).They showed diploid chromosome numbers of 51, 52, 52, and 53,respectively. The former two showed chromosome organizationpredictable from their parents, but the latter two contained novelcomplicated chromosome rearrangements, showing trisomies ofX- and 22-chromosomes and a translocation. To figure out thepuzzle, we tried molecular cytogenetic analyses with chromosomepaint and alpha satellite DNA probes.

1A-08

MURATA, Minoru1, KANATANI, Asaka1,KASHIHARA, Kazunari1, NAGAKI, Kiyotaka1

(1Group of Nuclear Genomics, Inst. Plant Sci. andResources, Okayama Univ.)

Transmission control of plant artificial ring chromosome inArabidopsis thaliana

In the model plant, Arabidopsis thaliana, we have generatedartificial ring chromosomes using the Cre/LoxP and Ac/Dssystem. Despite the ring structure, they are stable during mitoticdivisions and are transmissible to the next generation throughmeiosis. One of the artificial ring chromosomes, AtARC1,originated from a centromeric edge of the long arm of chromo-some 2, is approximately 2.85 Mb in size. Although thecentromeric 180-bp repeat array of ARC1 is much shorter thanthat of the original chromosome 2 (0.25 Mb vs. 3.0 Mb), thetransmission rates through the female and male sides were 11%and 28%, respectively, when it was added to wild-type plants(Murata et al. 2103). In this study, ARC1 was transferred to a T-DNA knockout line for Mcm9 (Minichromosome maintenance 9)gene that is expressed preferentially in pollens. As a result, thetransmission of ARC1 increased to 100%, because the Mcm9 geneon ARC1 complemented the recessive allele (mcm9) on chromo-some 2 and restored pollen viability. This suggests that the doseof Mcm9 gene in pollens affects the male-side transmission ofARC1.

1A-09

KAWANO, Shinji1, FUJIMOTO, Kunpei1, YASUDA,Kazushi1, HAMADA, Ami1, TSUTSUI, Ken2,TSUTSUI, Kimiko M.2, IKEDA, Shogo1 (1Dept.Biochem., Faclt. Sci., Okayama Univ. of Sci., 2Dept.Neurogenomics, Grad. Sch. Med., Dent. and Pharm.Sci., Okayama Univ.)

Functional analysis of the C-terminal domain of DNAtopoisomerase II

DNA topoisomerase II (topo II) is an essential enzyme forresolution of topological problems arising in DNA metabolicprocesses such as transcription and replication. We examinedhow the CTD of topo II is associated with DNA catenation activityby comparing WT rat topo II with its truncated mutant. Theresults indicate that DNA binding activity in the CTD of topo IIconcentrates the enzyme in the vicinity of condensed DNA andallows topo II to efficiently form a DNA catenane.

1A-10

OHTA, Shinya1 (1Med. School, Kochi Univ.)

Quantitative proteomics using chicken DT40 cells revealednovel mitotic chromosome scaffold component

Chromosome condensation is essential for proper segregation ofthe genome during mitosis, and gives chromosomes theircharacteristic shape. A classical model proposes that non-histoneproteins act as a structural framework during the formation ofmitotic chromosomes. This framework was suggested to corre-spond to an insoluble biochemical fraction, the chromosomescaffold, which can be derived from chromosomes after solubili-zation of the bulk of the DNA and proteins. Current works haveshown that the components of the chromosomal scaffold fractionhave an essential role in the formation of structurally stablemitotic chromosomes. However we do not yet have clearinformation about the existence of the chromosome scaffold.Here, we determined the protein composition of the chromosomescaffold using mass spectrometry and identified a novel chromo-some scaffold component. This GFP-fusion protein localized to themitotic chromosome axis. Moreover knocking out caused a mitoticprogression error and slightly changed a mitotic chromosomeshape. These results suggested that our novel protein might playa role in the chromosome scaffold for the determination of themitotic chromosome shape.


1B-01

SHIMADA, Makoto K.1 (1Div. Gene Expr. Mech., Inst.Compr. Med. Sci., Fujita Health Univ.)

Learning from the past: comparison among the known locusshowing complex human history of admixture

Genomes of modern human populations include genomic frag-ment introgressed from archaic humans. Details of the intro-gression are still unknown.To show the impact to genetic diversity of modern human (i.e.,Homo sapiens) with examples, I selected 8 loci those had beendiscussed genomic introgression from non-modern humans beforethe publication of the Neanderthal genome, and evaluated themby gene genealogy and the statics for introgression (S* analysis)using sequences of Neanderthal, Denisova, and thousands ofindividuals of modern humans. These analyses show that at leastthree patterns in gene-genealogical topology of introgressedhaplotypes suggested by S* analysis. This may suggest severalevents of introgression from archaic humans before the Out-of-Africa, which resulted in vaster diversity within modern humanthan expected from the introgression from the existing archaichuman sequences.

1B-02

KANAZAWA-KIRIYAMA, Hideaki1,2,8, KURYUKOV,Kirill4, JINAM, Timothy1,2, HOSOMICHI,Kazuyoshi2,5,7, SAO, Aiko3,6, SUVA, Gen3,6, UEDA,Shintaroh3, YONEDA, Minoru6, TAJIMA, Atsushi7,SHINED, Kenichi8, INOUE, Itsuro2,5, SAITOU,Naruya1,2,3 (1Natl. Inst. Genet., 2Dept. Genet.,SOKENDAI, 3Dept. Biol. Sci.s, Grad. Sch. Sci., TheUniv. Tokyo, 4Sch. Med., Tokai Univ., 5Div. Hum.Genet., Natl. Inst. Genet., 6The Univ. Museum, TheUniv. Tokyo, 7Grad. Sch. of Med. Sci., Kanazawa Univ.,8Dept. Anthropol., Natl. Museum of Nature and Sci.)

Determination and analyses of partial nuclear genome of theJomons who lived 3,000 years ago in Sanganji Shell Mound,Fukushima

The Jomon period of the Japanese Archipelago is characterizedby jomon potteries, and abundant human skeletal remains of thisperiod have been excavated. However, the genetic origins of theJomon people and their relationships with modern populationshave not been clarified. We determined a total of 115 million bpnuclear genome sequences from two Jomon individuals (male andfemale each) of the Sanganji Shell Mound (dated 3,000 YBP) withthe Jomon-characteristic mitochondrial DNA haplogroup N9b,and compared these nuclear genome sequences with those ofworldwide populations. We found that the Jomon populationlineage is best considered to have diverged beforediversificationof the present-day East Eurasian populations, with no evidence ofgene flow events detected between the Jomon and the othercontinentalpopulations. This suggests that the Sanganji Jomonpeople descended from anearly phase of population dispersals inEast Asia. We also estimated that themodern mainland Japaneseinherited less than 20% of Jomon peoples’ genomes.

1B-03

FUJITO, Naoko1, SATTA, Yoko1, HANE, Masaya2,MATSUI, Atsushi3, YASHIMA, Kenta1, KITAJIMA,Ken2, SATO, Chihiro2, TAKAHATA, Naoyuki1,HAYAKAWA, Toshiyuki4 (1Sch. Adv. Sci., SOKENDAI(Grad. Univ. Adv. Stud.), 2Biosci. and BioTech. Center,Nagoya Univ., 3Primate Res. Inst., Kyoto Univ., 4Grad.Sch. Systems Life Sci.s, Kyushu Univ.)

The Out of Africa and mental disorders: Adaptation withreduction in STX promoter activity

STX is an enzyme responsible for transfer of polysialic acids toneural cell adhesion molecules and plays an important role inhigher brain function of humans. Curiously, variants of the STXgene are often associated with various mental disorders. Here wefocus on four SNP combinations (promoter types) composing ofthree SNPs (core SNPs) in the promoter region and show that one(“CGC”) type with high frequency (35%) in the Asian populationhas significantly lower promoter activity than the others. Thephylogenetic study, based on haplotype sequences of 63 samplesfrom various ethnic groups, reveals that the ancestral “CGC”emerged between 0.45~0.1 MYA and expanded around 0.1 MYA,which is coincident with the African exodus of modern humans.Further analysis using phased sequences from the 1000 genomeproject data (2504 individuals) reveals that “CGC” has arelatively long homozygosity tract with the average being ~32kb from the core SNPs. This suggests action of positive selectionand is consistent with results of REHH. It is argued that positiveselection has played significant roles in shaping “CGC” in theAsian population throughout the Great Journey of modernhumans.

1B-04

SATO, Daiki1, MAKINO, Takashi1, KAWATA,Masakado1 (1Dept. Ecol. Evolutionary Biol., Grad. Sch.of Life Sci., Tohoku Univ.)

Evolutionary genetic basis underlying social and cognitiveabilities in human

Considering the fact that some psychiatric disorders have seriouseffects on patients’ social or cognitive abilities, the genes under-lying them should be key elements of the unique function inhuman brain. Here we focused on psychiatric disorders relatedgenes and investigated the historical changes of the genespossibly contributing to human specificity by means of evolu-tionary bioinformatic approach. We found two human specificamino acid substitutions (Glu130Gly and Asp136Thr) in solutecareer family 18 member A1 (SLC18A1), which codes vesiculartransporter of monoamine such as serotonin or dopamine, andthese substitutions were predicted to have significant impact onprotein function by both previous descriptions and our estima-tion. Moreover, there was a polymorphism on the 136th site(Thr136Ile), and we detected balancing selection acting on thesite in non-African population. This suggests that the 136th sitewere likely to play important roles at the moment of the out ofAfrica. Our study proposes a new candidate gene putativelyhaving impacts on the evolution of human brain throughmodulation of neurotransmission pathway.


1B-05

WADA, Yoshiko1,2, WADA, Kennosuke1, IWASAKI,Yuki1, KANAYA, Shigehiko2, IKEMURA, Toshimichi1

(1Dept. BioSci, Nagahama Inst. Bio-Sci. Tech., 2NaraInst. Sci. Tech.)

Big data analysis on rapidly evolving zoonotic RNA virusesand its use for establishment of the prediction verificationcycle

Viruses have always posed a significant threat to public health,as exemplified by ebolavirus threat highlighted by recentoutbreaks in West Africa. To face world-wide threats by infectiousviruses (e.g. Ebola, influenza and MERS viruses), we mustinnovate various technologies including big data analysis. Oneimportant characteristics of these viruses is to have RNAgenomes and to have very high evolutionary rates, showingimportance of time-series analysis for developing defense strat-egies against these viruses. The present time-series analysis ofoligonucleotide (e.g. 2~6-mers) composition by word countrevealed directional changes in genome sequences after invasionfrom nonhuman animals, which are predicted to recur afterfuture invasions. This predictability should be due to the fact thatviral growth is dependent on many host factors but human cellsdo not provide a highly suitable growth condition for virusesinvading from nonhumans. Since the time-dependent directionalchanges could be detected even at a month-level, the informationbecomes important for designing diagnostics RT-PCR primers forvirus detection and therapeutic oligonucleotides with longeffectiveness.

1B-06

UCHIDA, Misato1,2, NEVO, Eviatar3, ASADA,Nobuhiko1 (1Dept of Zool., Faclt. Sci., Okayama Univ.Sci., 2Grad. Sch. Biol., Faclt. Sci., Okayama Univ.,3Inst. Evol., Faclt. Sci. and Educ., Univ. of Haifa)

Estimation of viability in Drosophila melanogaster at“Evolution Canyon”, Israel

The enigma of genetic diversity in natural population has beenexplored by using evolutionary analysis. The patterns of micro-scale biodiversity have studied at “Evolution Canyon”, Israel. Iso-female Drosophila melanogaster strains, 2-1 strain from thenorth-facing slope and 6-1 strain from the south-facing slope, and2, 000 egg were prepared in 20 vials. The value of egg-to-adultviability was given in 2-1 strain and 6-1 strain as 0.89 or 0.75,respectively, showing statistically significant difference. Geneticdiversity was higher on the more stressful the south-facing slopethan on the milder the north-facing slope. The inter-slope geneticdifferences were corroborating on the climate selection withviability polygene and in population and stochasticity at the“Evolution Canyon” Israel.

1B-07

MATSUMOTO, Tomotaka1, MISHRA, Neha1,AKASHI, Hiroshi1 (1Natl. Inst. Genet.)

Testing natural selection in Drosophila intron evolution

Pattern of genome evolution is affected by the interacting effect ofmultiple factors as natural selection, mutation, recombinationand so on. Especially, natural selection acting on base composi-tion like codon usage bias can affect evolution in genome widelevel. In this study, we analyzed the variation of base compositionof introns in Drosophila melanogaster genome. We found that thebase composition is highly variable depending on the size ofintron and position within a intron, and the observed hetero-geneity of base composition showed strong negative correlationwith the genetic divergence. Our result suggests that theevolution of base composition of intron in D. melanogaster canbe affected by weak natural selection and therefore, we should becarefully think about using this region as an example of neutralevolution in molecular evolution study.

1B-08

MISHRA, Neha1, MATSUMOTO, Tomotaka1,AKASHI, Hiroshi1 (1Dept. Genet.)

Transcription and within-gene base composition heterogene-ity in D. melanogaster

Evolutionary forces can vary within and across genes andoverlooking their variation causes bias in various molecularevolutionary analyses. We used patterns of base composition inD. melanogaster to reveal variation in evolutionary forces withingenes. Studying base composition variation within gene canprovide insights to the biological processes underlying theevolution of base composition. We found that GC content ofintrons decreases in the direction of transcription. Associationwith RNA polymerase binding levels and transcript abundancesuggest that transcription-associated mutation or selectiongovern intron base composition heterogeneity within gene. Suchvariations in evolutionary forces within genes should be takeninto account while using introns or synonymous sites to testmodels of molecular evolution.


1B-09

HETTIARACHCHI, Nadeeka Nilmini1, SAITOU,Naruya1 (1Natl. Inst. Genet., Mishima, 2Natl. Inst.Genet.)

GC content heterogeneity transition of conserved noncodingsequences occurred at the emergence of vertebrates

Conserved noncoding sequences (CNSs) of Eukaryotes are knownto be significantly enriched in regulatory sequences. CNSs ofdiverse lineages follow different patterns in abundance, sequencecomposition and location. Here we report a thorough analysis ofCNSs in diverse groups of Eukaryotes with respect to GC contentheterogeneity. We examined 24 fungi, 19 invertebrates, and 12non-mammalian vertebrates so as to find lineage specific featuresof CNSs. We found that fungi and invertebrate CNSs arepredominantly GC rich as in plants we previously observed,whereas vertebrate CNSs are GC poor. This result suggests thatthe CNS GC content transition occurred from the ancestral GCrich state of Eukaryotes to GC poor in the vertebrate lineage dueto the enrollment of GC poor transcription factor binding sitesthat are lineage specific. CNS GC content is closely linked withthe nucleosome occupancy that determines the location andstructural architecture of DNAs.

1B-10

RATNAYAKE, Sangeetha Udani1, ENDO, Toshinori1,OSADA, Naoki1 (1Hokkaido Univ., 2Div. Bioeng. andBioinform., Hokkaido Univ.)

Revisiting the Relationship between Evolutionary Conserva-tion and Disease Causality: A Case Study of Beta GlobinGene

Predicting the effect of amino acid mutations in genomes plays avital role in the studies of evolutionary biology and medicalgenomics. In common practice, the level of evolutionary con-servation has been used to infer the biological importance ofamino acids, as predicted by the neutral theory of molecularevolution. There is a doubt came up with, whether thoseconsequences of amino acids are actually applicable with accuratedisease prediction. In order to answer the question, we inves-tigated the relationship between disease causality and evolu-tionary conservation in human beta globin gene (HBB), as it haswell-known protein structure and disease and non-diseasephenotypes are reported for almost all possible amino acidmutations. Unexpectedly, disease causality prediction of HBBusing simple physiochemical grouping performed much betterthan the conservative predictions. Other than the general conceptwhich determines the evolutionary conservation is important, weare looking for possible reasons why there is a considerableamount of variation of molecular evolution rate within HBB.

1B-11

SABER, Morteza Mahmoudi1,2, SAITOU, Naruya1,2,3

(1Dept. Biol. Sci.s, Grad. Sch. Sci., Univ. of Tokyo,2Div. Popul. Genet., Natl. Inst. Genet., 3Dept. Genet.,Sch. Life Sci., Grad. Univ. Adv. Stud. (SOKENDAI))

Evolution of coding and noncoding genomic sequences sharedby humans and great apes

Family Hominidae, which includes humans and great apes, isrecognized for unique complex social behavior and intellectualabilities. Despite the increasing genome data, however, thegenomic origin of its phenotypic uniqueness has remainedelusive. Here we analyzed whole genome sequences of simiansin order to find Hominidae-specific genes and highly conservednoncoding sequences (HCNS). We discovered that Down syn-drome critical region 4 (DSCR4) is the only experimentallyverified gene uniquely present in Hominidae. We showed thatDSCR4 has emerged in several steps through LTR/ERV1, LTR/ERVL retrotransposition, and transversion. Using the genomicdistance as neutral evolution threshold, we identified 1,658Hominidae-specific HCNSs. Polymorphism coverage and derivedallele frequency analysis of Hominidae-specific HCNSs showedthat these HCNSs are under purifying selection, indicating thatthey may harbor important functions. Interestingly, manyancestral sequences of the Hominidae-specific HCNSs showedvery high evolutionary rates. This suggests that new functionsemerged through accelerated evolution, and then purifyingselection started to operate to keep these functions.

1B-12

JINAM, Timothy1, SAITOU, Naruya1 (1Natl. Inst.Genet., Div. Popul. Genet.)

The genetic history of Negrito populations in Southeast Asia

The Negritos are indigenous human populations currently livingin the Philippines, Malay Peninsula and Andaman Islands. Theyhave been of interest to anthropologists due to their uniquephenotype which bears resemblance to African pygmies. Usinggenome-wide SNP analysis, we show their deep divergence fromother East Asians and that shared loci among the Negritos arerelated to some physical features. We also report signals ofadmixture with archaic humans called Denisovans in thePhilippine Negritos. Our results demonstrate the uniqueness ofthese Negrito populations.


1C-01

FUKUDA, Kei1, YUSA, Kosuke2, SHINKAI, Yoichi1

(1Cell. Memory lab., RIKEN, 2Wellcome Trust SangerInst., Hinxton, Cambridge)

Identification of factors involved in provirus silencing byCRISPR-Cas9 system

Proviruses and endogenous retroviruses (ERVs) are frequentlyrepressed in embryonic stem cells (ESCs). It is known thatHistone H3 lysine 9 trimethylation (H3K9me3), mediated bySETDB1, are required for silencing of proviruses and ERVs.However, it remains largely unknown how the silencing state isestablished and maintained. To advance our understanding of thesilencing pathway comprehensively, we developed a powerfulhigh-throughput approach based on a provirus MSCV-GFPreporter and genome-wide genetic screen by CRISPR-Cas9genome editing technology.

1C-02

MORI, Ayumi1, SATO, Hiroshi1, KASAI, Megumi1,YAMADA, Tetsuya1, KANAZAWA, Akira1 (1Res.Faclt. Agr., Hokkaido Univ.)

Initiation and spread of RNA silencing in plants depend onthe characteristics of plant architecture

Transgenes integrated into a plant genome are sometimessuppressed by RNA silencing. To understand how spatial andtemporal variation of RNA silencing is generated, we analyzedthe dynamics of RNA silencing in the progeny of a single soybeanplant transformed with the green fluorescent protein (GFP) gene.We found that GFP silencing initiated during plant growth andsubsequently spread to the other part of the plant. Both thepatterns of initiation and spread of GFP silencing varied betweenindividuals. GFP silencing was hardly detectable in the embryos,which is consistent with the notion that RNA silencing is resetduring reproduction. GFP silencing was also absent in particulartissues, suggesting tissue-specific suppression of RNA silencing.This mechanism could be relevant to the lack of RNA silencing inmeristems. Alternatively, inhibition of spread of GFP silencingvia cell-to-cell movement or through the vascular system mayalso account for such phenomena. Overall, our observationssuggest that the induction and/or spread of RNA silencing areinfluenced by the characteristics of plant architecture.

1C-03

NAGAKI, Kiyotaka1, YAMAJI, Naoki1, MURATA,Minoru1 (1Inst. Plant Sci. and Resources, OkayamaUniv.)

Analysis of epigenetic modifications in plant tissues by anovel clearing method

Since investigations of epigenetic status of individual cells intissues can produce not only epigenetic data in different cell-typesbut also positional information of the cells, the investigations areimportant to understand intra- and inter-cell control systems fordevelopments and environmental responses in plants. However,there was no simple way to detect epigenetic modifications ofindividual cells in plant tissues. In this study, we developed aclearing method for immunohistochemical (ePro-ClearSee) toinvestigate epigenetic modifications without sectioning. TheePro-ClearSee method could detect methylated histones, acety-lated histones, methylated DNA and/or centromeric histone H3variants in four dicots and five monocots, and made it possible todetect the immunosignals 200 mm deep in plant tissues for a shortperiod (10 days-3 weeks). The results suggest this method providea simple and universal way to investigate epigenetic modifica-tions in widespread plant species.

1C-04

TANIYAMA, Nobuko1, KOBAYASHI, Kenta1,YAMANE, Junko1, MORI, Tomoya1, YAMASHITA,Jun K.1, FUJIBUCHI, Wataru1 (1Dept. Cell Growthand Differ., Cent. iPS Cell Res. and Application, KyotoUniv.)

Single-cell methylome analysis of mouse blastocyst

As a zygote divides, it generates different cell types and acquirestissue characteristics. As many epigenetic factors are involvedduring its process and have been investigated. However, theanalyses done by cell population provide averaged informationthus masked the feature of each single-cell. Here we performed asingle-cell analysis to find out clues to understand mechanismsregulating tissue specificity and conformation. In this study, wefocused on DNAmethylation, which is known to relate to genomicimprinting and cell identities. Although many DNA methylationanalysis methods are reported, only two are applied to single-cellanalysis. Considering the genome coverage, analytical flexibility,and costs, we selected the scRRBS (single-cell reduced represen-tation bisulfite sequencing) method. This method containsbisulfite conversion step that causes DNA fragmentations, whichdecreases sample yield. Therefore, we developed a new version ofscRRBS method to increase the analytical range. We used mouseblastocysts and checked the effect of our method. Our methodrepresented more CpG sites than the original method withoutchromosome biases.


1C-05

KUBIURA, Musashi1, BOGUTZ, Aaron2, KIMURA,Hironobu3, TAJIMA, Shoji3, LEFEBVRE, Louis2,TADA, Masako4 (1Inst. of Regener. Med. Biofunc.,Grad. Sch. Med. Sci., Tottori Univ., 2Life Sci. Inst.,Univ. of British Columbia, Canada, 3Inst. Prot. Res.,Osaka Univ, 4Chromosome Eng. Res. Cent. (CERC),Tottori Univ)

Functional switching of Dnmt1 during mouse ES cellsdifferentiation

Precise control of DNA methylation (5mC) patterns is essentialfor mammalian embryonic development. Genome-wide DNAactive demethylation occurs in preimplantation embryos. There-after, genomes become highly methylated during epiblastformation. Although DNA methyltransferase (Dnmt) 1/3a/3btriple-knockout (TKO) mouse embryonic stem cells (ESCs) canself-renew in the absence of 5mC, they undergo apoptosis underdifferentiation condition. The major aim of this study was todetermine the mechanism underlying an extensive gain of DNAmethylation and to examine the physiological function of Dnmtsduring mouse epiblast formation. We performed in vitro differ-entiation of epiblast-like cells (EpiLCs) from mouse ESCs lackingmaintenance-type Dnmt1 (1KO ESCs) or a de novo-type Dnmt3aand Dnmt3b (DKO ESCs), and TKO ESCs in which a Dnmt1cDNA transgene was reintroduced (TKO+1). DKO ESCs differ-entiated into EpiLCs, and levels of 5mC increased in these cellsfollowing differentiation. Furthermore, Dnmt1 restores celldifferentiation potency in TKO cells. The results suggest thatDnmt1 possesses a developmentally regulated de novo DNAmethylation activity.

1C-06

TAKUNO, Shohei1, RAN, Jin-Hua2, GAUT, BrandonS.3 (1SOKENDAI, 2Chinese Acad. of Sci., 3Univ.California)

Evolutionary patterns of genic DNA methylation vary acrossland plants

Little is known about patterns of genic DNA methylation acrossthe plant kingdom or about the evolutionary processes that shapethem. To characterize gene-body methylation within exons, wehave gathered single-base resolution methylome data that spanthe phylogenetic breadth of land plants. We find that a basal landplant, Marchantia polymorpha, lacks any evident signal of gene-body methylation, but conifers have high levels of both CG andCHG methylation in expressed genes. To begin to understand theevolutionary forces that shape gene-body methylation, we firsttested for correlations in methylation levels across orthologues.Genic CG methylation levels, but not CHG or CHH levels, arecorrelated across orthologues for species as distantly related asferns and angiosperms. In contrast, genic CHG methylationcorrelates with genome size, suggesting that the host epigeneticresponse to transposable elements also affects genes. Altogether,our data indicate that the evolutionary forces acting on DNAmethylation vary substantially across species, genes and meth-ylation contexts.

1C-07

SAITO, Raku1,2, TAKASHIMA, Kazuya2, TARUTANI,Yoshiaki1,2, KAKUTANI, Tetsuji1,2 (1Dept. Genet.,SOKENDAI, Sch. Life Sci., SOKENDAI, 2Div.Agricltural Genet., Natl. Inst. Genet.)

Specificity and diversity of the transposon-encoded anti-silencing factor

Transposons are usually highly methylated and transcriptionallysilenced. Arabidopsis transposon VANADL21 family harborsanti-silencing factor named VANC21. When VANC21 geneexpressed as transgene, endogenous VANDAL21 sequences arespecifically de-methylated and transcriptionally re-activated.In Arabidopsis genome, there are many VANC-like genes. Bymethylome and transcriptome analysis, it has been suggestedthat each VANDAL family would have different target specificity.We found that VANC6, VANC-like gene harbored by VANDAL6family, induces loss of DNA methylation specifically in endoge-nous VANDAL6 sequences and related families. These highlysequence-specific anti-silencing systems would reduce damage tothe host and contribute long-term advantage for the survival ofVANDAL families.

1C-08

HOSAKA, Aoi1,2, SAITO, Raku1,2, TAKASHIMA,Kazuya2, SASAKI, Taku2, TARUTANI, Yoshiaki1,2,KAKUTANI, Tetsuji1,2,3 (1Div. Agr. Genet., Dept.Genet., SOKENDAI (Grad. Univ. Adv. Stud.), 2Dept.Integr. Genet., Natl. Inst. Genet., 3Dept. Biol. Sci.,Grad. Sch. Sci., The Univ. Tokyo)

Sequence-specific anti-silencing of transposons in Arabidop-sis and its rapid evolution

Transposable elements (TEs) are potentially harmful to the host,and they are silenced by epigenetic mechanisms such as DNAmethylation. However, some TEs counteract host surveillance. Amobile copy of VANDAL21 TEs in Arabidopsis encodes an anti-silencing factor, VANC21. Expression of VANC21 induces loss ofDNA methylation specifically on VANDAL21 copies. The basesfor the sequence specificity and the evolutionary aspects of theanti-silencing mechanisms are largely known.Here we report the binding regions of VANC21 protein by ChIP-seq and discuss bases for the rapid evolution.


1C-09

SASAKI, Taku1, TARUTANI, Yoshiaki1,2, KAKUTANI,Tetsuji1,2,3 (1Natl. Inst. Genet., 2SOKENDAI, 3Univ.Tokyo)

Inactivation mechanism of VANDAL21 transposons inArabidopsis thaliana

VANDAL21 is a Mutator transposon which is transcriptionallyactivated and mobilized in DNA hypomethylation mutant. Anautonomous VANDAL21 copy, named Hiun (Hi), encodes threeORFs: VanA, VanB, and VanC. VanA encodes a putativetransposase but function of proteins encoded by other ORFs isunknown. When VanC sequence is induced as transgene,endogenous Hi and VANDAL21 transposons are specificallyreactivated. Interestingly, if the VanC transgene was segregateapart in the next generation, VANDAL21 transposons areinactivated and re-silenced already in that generation. Here weshow that this resilencing of Hi depends on RNAi machineries.Although the RNAi machinery is not required for keeping Hisilent in wild type, RNAi seems to function efficiently for the denovo silencing.

1C-10

TAIKO, To1, TARUTANI, Yoshiaki2, KATO, Kae2,INAGAKI, Soichi2, ITO, Tasuku2, TAKAHASHI,Mayumi2,3, TOYODA, Atsushi2, FUJIYAMA, Asao2,VINCENT, Colot4, KAKUTANI, Tetsuji1,2 (1Grad. Sch.Sci., Univ. Tokyo, 2Natl. Inst. Genet., 3Cent. EcologicalRes., Kyoto Univ., 4Institut de Biologie (IBENS))

Transgenerational establishment of CG and non-CG DNAmethylation patterns in Arabidopsis

DNA methylation is important for silencing transposable ele-ments (TEs) in eukaryotes. In plant genomes, TEs are heavilymethylated in both CG and non-CG contexts. A central questionstill unanswered is how the TE-specific DNA methylation isestablished. Through genetic and epigenomic analyses, here weexamined transgenerational de novo methylation of TEs afterloss of methylation in CG or non-CG context. RNAi-dependent denovo methylation of TEs proceed efficiently for both CG and non-CG contexts when DNAmethylation of the other context remains.In addition, CG methylation directs precise and efficient recoveryof non-CG methylateon in an RNAi-independent manner.

1C-11

INAGAKI, Soichi1, TAKAHASHI, Mayumi1,KAKUTANI, Tetsuji1,2 (1Dept. Integr. Genet., Natl.Inst. Genet., 2Grad. Sch. Sci., The Univ of Tokyo)

Dynamics of gene-body histone modifications mediatesheterochromatin silencing in Arabidopsis

Transposons are silenced by histone H3 lysine 9 methylation(H3K9me2) and DNA methylation in many eukaryotes. While itis widely accepted that H3K9me2 and DNA methylation aroundpromoter silence the transcription, many silent transposons havethese marks in their transcribed regions (body) and it’s functionis unknown. We are using an Arabidopsis mutant that accumu-lates H3K9me2 ectopically within bodies of transcribed genes andshows developmental abnormalities to clarify the roles of genebody chromatin modifications. Through genetic screenings, wehave found a factor working downstream of the body H3K9me2.

1C-12

KAJITANI, Takuya1, KATO, Hiroaki2, CHIKASHIGE,Yuji3, KIMURA, Hiroshi4, OHKAWA, Yasuyuki5,HERAND, Damien6, MURAKAMI, Yota1 (1Grad. Sch.Sci., Hokkaido Univ., 2Grad. Sch. Med. Res., ShimaneUniv., 3Advanced ICT Res. Inst., Natl. Inst. Inform.and Commu. Tech., 4Grad. Sch. BioSci. and BioTech.,Tokyo Inst. Tech., 5Med. Inst. Bioregulation, KyushuUniv., 6Namur Adv. Res. Coll. (NARC), Namur Univ.)

Phosphorylation at Ser7 of RNA polymerase II CTD limitstranscription speed and transcription elongation

C-terminal domain(CTD) of RNA polymerase II(RNAPII) ishighly phosphorylated in vivo. Phosphorylation at Ser2 andSer5 of CTD is coupled with co-transcriptional events, such asactivating histone modifications and RNA processing. However,the intimate role of phosphorylation at Ser7 has remainedelusive.We found that non-phospho acceptor alanine substitutionmutation (ctdS7A) in fission yeast causes hyperphosphorylationat Ser2 and Ser5 across euchromatic and heterochromaticregions, suggesting hyperactivation of RNAPII. The hyperacti-vated RNAPII in ctdS7A is likely to transcribe rapidly, inferringthat phosphorylation at Ser7 acts as a decelerator of tran-scription. The rapid transcription is facilitated by Paf1.In addition, we found that the rapid transcription in ctdS7Aacross heterochromatin leads to be defective in RNAi-dependentheterochromatin formation. In ctdS7A mutant, Argonaute com-plex could not target to the nascent transcripts. The defects weresuppressed in ctdS7Apaf1DC double mutant. These resultsindicated that at heterochromatic regions, the attenuationfacilitated by Ser7 is prerequisite for exertion of RNAi-dependentheterochromatin formation.


1D-01

KIDERA, Kaho1, ODAJIMA, Takuya1, TONE,Takahiro1, KANESAKI, Tomo2, YOSHIKAWA,Hirofumi3, MAKINO, Osamu1 (1Fac. Sci. and Tech.,Sophia Univ., 2NGRC, Tokyo Univ. Agr., 3Dept.BioSci., Tokyo Univ. Agr.)

Characterization of the rsiV mutations which affect thegrowth of subtilis phageff29

Because only two host factors, the products of mreB and rpoA,have been reported to be essential for the growth of Bacillussubtilis phage 29, we searched for other host factors by theanalysis of Bacillus subtilis spontaneous mutants which affectthe growth of 29. After several screening steps, we sequenced thegenome of 5 candidates and found out 2 clones which hadmutations in rsiV (one was 4 base deletion mutant and anotherwas single nucleotide substitution mutant). Because rsiV is ananti- factor of SigV, we assumed that the increase of SigV regulonexpression inhibited the growth of 29.

1D-02

ASAI, Kei1 (1Grad. Sch. Sci. and Tech., Saitama Univ.)

Genetic analysis of regulation mechanisms for SigM and itscognate anti-sigma factor, YhdL and YhdK, in Bacillussubtilis

In Bacillus subtilis, a Gram-positive sporulating soil bacterium,possesses various kinds of stress response sigma factors. SigM isstrongly involved in response to cell envelope stress. SigMactivity is regulated negatively with anti-sigma factor, YhdLand YhdK. Both of YhdL and YhdK proteins are predicted to bemembrane protein and the N terminal peptide of YhdL is thoughtto bind to SigM, though their molecular mechanism of regulationis not elucidated precisely. The C-terminal portion of YhdL wasgradually deleted. All the deletion leaded to the loss of anti-sigmafunction. The fusion of GFP and the N-terminal domain of YhdL,which is the putative sigma-binding domain, might lead to theloss of YhdL function. A merodiploid strain, which possessed theC-ter-deleted YhdL and GFP-YhdL fusion, was constructed. Thisstrain restored anti-SigM activity, suggesting that both abortivepeptides formed a dimer and compensated lost function (N-ter;sigma-binding and C-ter; signal receiving) with each other. Wealso revealed that so-called transcriptional coupling was requiredfor efficient expression of YhdL and YhdK. Suppressor mutationsof YhdK deletion were obtained and analyzed.

1D-03

YOSHIDA, Saki1, YOSHIKAWA, Hirofumi2, ASAI,Kei1 (1Dept. Biochem. Mol. Biol., Grad. Sch. Sci. Eng.,Saitama Univ., 2Dept. BioSci, Grad. Sch. Agr., TokyoUniv. Agr.)

Research of the representative phenotype and the responsiblegene in Bacillus subtilis SigA only strain

Sigma factors, which are involved in the initiation of tran-scription in bacteria, are one of components of RNA polymeraseholoenzyme. Bacillus subtilis possesses 19 sigma factors, andfunction of alternative sigma factors have been studied mainlyfrom the view of stress response and spore development. In ourformer studies, these alternative sigma factors have beeninactivated simultaneously and finally the strain, SigA only, inwhich only one essential sigma factor, SigA, functioned, wasobtained. The colonies of the strain, SigA only, got transparentand number of the viable cells in colonies decreased rapidly aftergrowing on LB agar plate. Currently, we have succeeded toacquire the suppressor strains, which restored rapid decrease inthe number of the viable cells. From the whole genome sequenceanalysis of these suppressor strains, we tried to find factors,which are responsible for suppressor phenotype. As a result, itwas found that defects in rpsU encoding the 30S ribosomalprotein, S21, are closely related to suppressor phenotype. We arestill elucidating the effect of rpsU deletion.

1D-04

SHIMIZU, Yoko1, YOSIKAWA, Hirofumi2, ASAI, Kei1

(1Regul. Network of Cellular Mol., Mol. Biol., Faclt.Sci., Saitama Univ., 2Faclt. Appl. Bio-Sci., Tokyo Univ.Agr.)

Expression analysis of lysis phenomenon elucidation and SigIby Bacillus subtilis SigI destruction

SigI is an alternative sigma factor that is supposed to involve inheat stress response. We found that the sigI mutant strain waslysed at stationary phase. We thought that SigI might play a rolein regulating maintenance of viability of the cells at stationaryphase. We presumed that some kinds of signal substances areinvolved in this lysis phenomenon, and performed the co-cultureexperiment using the wild-type and the sigI mutant strain.However, the sigI mutant strain was lysed and the wild typestrain was not regardless of each other. Therefore, lysisphenomenon was supposed not to be due to induction with thelysis-inducing factors or cell-cell communication. We constructedthe strain harboring PsigI-GFP fusion and monitored fluores-cence emitted from the individual cells at stationary phase. Flowcytometric analysis showed that PsigI was activated in limitednumber of the cells (around 4%). Cell-staining analysis, by whichthe live cells could be distinguished from the dead cells under thefluorescence microscope, shows that the number of dead cellsincreased in the sigI mutant strain.


1D-05

SEKI, Takahiro1, MATSUOKA, Satoshi1,MATSUMOTO, Kouji1, HARA, Hiroshi1 (1Grad. Sch.Sci. Eng., Saitama Univ.)

Analysis of the activation mechanism of an extracytoplasmicfunction ss factor ssV in the Bacillus subtilis mutant cellslacking glucolipids by using chimera-antisigma factors

The Bacillus subtilis cells lacking glucolipids by disruption of theugtP gene show activation of extracytoplasmic function (ECF) sfactor sV. sV is regulated directly by transmembrane anti-sV

RsiV. The sV is activated specifically by lysozyme. When B.subtilis cells are challenged by lysozyme, RsiV is cleaved byregulated intramembrane proteolysis (RIP). However, we did notobserve the proteolysis of RsiV in the DugtP cells. This resultsuggested that the lack of glucolipids activated the sV by anothermechanism. To elucidate the mechanism, we constructed the twochimera anti-s, RsiVN-RsiWTMC and RsiVNTM-RsiWC, by swap-ping the intracellular N-terminal domain or extracellular C-terminal domain of RsiV and those of RsiW. RsiW is anti-sW, andsW does not respond to the lack of glucolipids. These chimeraanti-s were introduced into the DrsiV and the DrsiV DugtP cells,and the sV activity was measured by monitoring the b-galactosidase activity expressed from PsigV-lacZ. The sV activitydecreased to the wild-type level by expression of RsiVN-RsiWTMC or RsiVNTM-RsiWC in the DrsiV DugtP cells. Thisresult suggested that C-terminal domain of RsiV sensed the lackof glucolipids.

1D-06

MATSUOKA, Satoshi1, MATSUMOTO, Kouji1, ASAI,Kei1, HARA, Hiroshi1 (1Grad. Sch. Sci. and Eng.,Saitama Univ.)

Molecular mechanism of regulation of two component systemWalKR by glucolipids in Bacillus subtilis

Bacillus subtilis cell membranes contain 10% glucolipids. Theyare synthesized by UgtP processively. ugtP mutants are bent anddistended in log phase. This indicates that glucolipids areimportant for the maintenance of cell morphology. In this studywe focused on WalKR two component system and SigI, whichboth are involved in cell wall maintenance. We constructed aseries of sigI promoter which has substitution and/or deletion inits cis element sequences with lacZ transcriptional fusion. Notonly SigI but also WalR was activated by the depletion ofglucolipids resulting from the lacZ assays. Then we constructed aB. sbutilis starain which has walR-hisx12 in its chromosome todetect the degree of phosphorylation. The phosphorylation ofWalR was accelerated by the depletion of glucolipid using thephos-tag method. These suggest that glucolipids may be requiredfor SigI and WalKR functions.

1D-07

IZAWA, Tomoko1, MATSUMOTO, Kouji1,MATSUOKA, Satoshi1, HARA, Hiroshi1 (1Div. LifeSci., Grad. Sch. Sci. Eng., Saitama Univ.)

Function of E.coli RcsF in activation of Rcs signal trans-duction system

The Rcs signal transduction system of E.coli is one of the two-component regulatory systems and regulates the expression ofmany envelope-related genes. These genes respond to the cellsurface stresses and implicated in biofilm formation. Foractivating this system, an outer membrane lipoprotein RcsF isessential. RcsF penetrates the outer membrane through the ?-barrel lumen of OMP and anchors in outer leaflet of the outermembrane by the N-terminal acyl chains. A part of N terminus ofRcsF is exposed on the cell surface (Konovalova et al., 2014).In this study, we focused on the localization of RcsF in variousconditions that activate the Rcs system. We constructed RcsFwith the FLAG tag at various positions. We transformed theseRcsFs to the strains that activate the Rcs system and detected thedomains of these RcsFs that were exposed to the surface. Whenthe Rcs system was activated by gene defects or osmolar stress,RcsF seemed to be kept anchored in the outer leflet of the outermembrane and to change the structure to approach the innermembrane. We supposed that the C-terminal portion of surface-exposed domain of RcsF slid into the lumen of OMP.

1D-08

SATO, Takatsugu1, TAKANO, Akira1, MATSUOKA,Satoshi1, MATSUMOTO, Kouji1, HARA, Hiroshi1

(1Dept. Biochem. Mol. Biol., Grad. Sch. Sci. Eng.,Saitama Univ.)

Visualization of protein-protein interactions among Rcscomponents in Escherichia coli by Periplasmic BiFC

E. coli Rcs system is one of the two-component systems andresponds to various envelope stresses. It regulates envelope-related genes including the genes responsible for colonic acidsynthesis required for biofilm formation. The Rcs system hasmuch more components than typical two-component systems.Minor outer membrane lipoprotein RcsF is one of the essentialcomponents of the system. We speculated that RcsF couldtransmit the signal to histidine kinase RcsC in the cytoplasmicmembrane directly. However, little is known about detailedtransduction mechanism. Here we tried to visualize the directprotein-protein interaction among Rcs components in the peri-plasmic space by Bimolecular Fluorescence Complementation(BiFC). We constructed experimental system for detection ofinteraction in the periplasmic space by fusing the sec signalsequence to BiFC fusion proteins. We could detect interaction ofRcsF with the periplasmic domein of RcsC and YrfF (a negativeregulator of the Rcs system) interaction. This result indicatesRcsF transmit the signal by interaction with two different innermembrane proteins. We are exploring the effects of theseinteractions on Rcs activation.


1D-09

OGAWA, Naoya1, HAYASHI, Hiroki1, KISHI,Tsutomu1 (1Coll. Eng., Nihon Univ.)

Phosphorylation by Pho85/CDK5 triggers degradation of thetranscription factor Swi5 via the SCFCdc4 ubiquitin ligase

Degradation of proteins by the ubiquitin pathway is important forvarious cellular regulatory mechanisms. The SCFCdc4 complex isthe ubiquitin ligase complex that acts as a regulator of cell cycle,signal transduction and transcription. We have previouslyreported that the SCFCdc4 complex regulates S phase initiationthrough degradation of Swi5, the transcription factor required fortranscription of SIC1, the S phase Cdk inhibitor. However, themechanism by which Swi5 is degraded by the SCFCdc4 ubiquitinligase remains unknown. We repot here that Pho85/CDK5 ininvolved in the degradation of Swi5. We used Swi5-ST8A, inwhich S225, S231, S246, 250, S261, S300, T320 and T323 aresubstituted by alanine residues, and is partially stabilized), todemonstrate this. Swi5-ST8A was much more stabilized in pho85mutants than in wild type. The result suggests the presence ofthe phosphorylation site(s) other than the ST8A mutation for thedegradation of Swi5. We identified the serine residue, andsubstitution of this to alanine residue (Swi5-ST9A) stabilizedSwi5-ST9A. In addition, we also identified Pcl (Pho Cyclin)subunits required for Swi5-degradation.

1D-10

HORIKE, Shin-ichi1, MEGURO, Makiko1, NUMATA,Saya1,3, HONDA, Toshiya1,3, OKADA, Gensaku1,YOKOYAMA, Shigeru2, HIGASHIDA, Haruhiro2

(1Adv. Sci. Res. Cent., Kanazawa Univ., 2Res. Cent.Child Mental Dev., Kanazawa Univ., 3Sch. Med.,Kanazawa Univ.)

Identification of novel distant regulatory element in theOXTR gene locus

Characterized by behavioral and cognitive disturbances of child-hood development, autism spectrum disorders (ASD) are cur-rently estimated to affect 1 in 68 children, with a male:femaleratio of 4:1. The etiology of ASD remains unknown, though itlikely involves interactions between multiple genetic and envi-ronmental risk factors. To date, only a small percentage of autismcases (<10%) have been ascribed to single gene disorders such asRett syndrome, tuberous sclerosis complex, and fragile Xsyndrome. However, the genetic alterations of ASD remainelusive in the majority of cases. Current studies suggested thatOXTR and the oxytocin signaling pathway play an important rolein the etiology of ASD. In this study, we applied CRISPR/Cas9genome editing system to generate a deleted allele of theregulatory element of OXTR gene expression. We found thatknockout (KO) allele disrupted the expression of OXTR.

1D-11

AMANO, Takanori1, SHIROISHI, Toshihiko1 (1Natl.Inst. Genet.)

Cis-regulatory polymorphism of Shh is involved in variationof shape and size of the mouse stomach

Polymorphism in cis-regulatory elements can cause differentia-tion of gene regulation, and plays a critical role in divergence ofquantitative traits. Stomach is one of the organs that aremorphologically divergent among vertebrates, implying inter-species variation of expression level of developmental genes.Stomach development may be a good model to know how cis-regulatory variations generate morphological diversity.Shh is expressed in endodermal epithelia during development,and contributes to cell proliferation. Here we found that a 1.7 Kbregion located 100 Kb upstream of Shh contains a stomachenhancer, and named it SLGE.SLGE, which is evolutionally low conserved and found only inrodents, has several SNPs and Indels when comparing C57BL/6(B6) with MSM. Our luciferase assay revealed that MSM SLGEhas higher transcriptional activity than B6 SLGE does. More-over, high expression of Shh was observed in the embryonicstomach of MSM as compared to B6. High regulatory activity ofMSM SLGE is consistent with the fact that MSM has a relativelylarge stomach. Taken together, cis-regulatory variation in SLGEmay play a role in size control of the stomach in rodents.


1E-01

KOKUBA, Shun1, ETO, Satoshi1, ARAKI, Miyuki1,MORITA, Ayaka1, KIMACHI, Yoko1, YAMAMURA,Kenichi1, YOSHINOBU, Kumiko1, ARAKI, Kimi1,ARAKI, Masatake1 (1Inst. Resource Dev. and Anal.,Kumamoto Univ.)

Establishment and analysis of Cre-driver mouse line (Admin-istration of Tamoxifen for Cre-mERT2)

We have developed Database for the Exchangeable Gene TrapClones (EGTC) (http://egtc.jp). In our system, reporter beta-geogene can be exchanged into any other DNA of interest throughCre-mediated recombination. We have produced more than 300Cre driver ES cell lines using EGTC ES cells. Among them, morethan 100 mouse lines could be established. Then we did mating ofCre driver mice with ROSA26R mice, and performed X-galstaining of ROSA26R mice with Cre driver allele.In this study, we used tamoxifen-inducible CremERT2 mouselines. We could demonstrate that administration method ofTamoxifen added to dry non-pelleted feed is better than generaladministration method of Tamoxifen by intraperitoneal injection,concerning to recombinase efficiency, work efficiency and animalwelfare.

1E-02

TAMURA, Masaru1 (1Technol. and Dev. Team forMouse Phenotyping Anal., RIKEN BioRes. Cent.)

Development of a high resolution imaging method for thephenotyping of mouse embryos

The genome editing technologies such as CRISPR/Cas9 systemchanged the concept of functional gene analysis. Using theCRISPR/Cas9, we can generate knockout mice with unprece-dented simplicity and speed. In these circumstances, the develop-ment of high-throughput and high-resolution analysis methods isrequired. Previously, we reported the high throughput and high-resolution soft imaging method using the contrast-enhancedmicro-CT. This method could generate virtual slice images at anyposition and angle from a single sample, and thereby reconstructsthe 3D image. Therefore, the contrast-enhanced micro-CTimaging is a powerful tool to analyze the phenotype of mutantmouse embryo generated by genome editing techniques as a firstscreening of morphological abnormalities. In this study, wereport the improved micro-CT imaging method to obtain morehigh-resolution images of the early mouse embryo such asembryonic day 9 (E9). This new method is possible to takeimages of the microstructure of the developing heart of E9. Inaddition, we will discuss the application and possibilities of thismethod.

1E-03

SHIBUYA, Hirotoshi1, ICHIMURA, Kaoru1, MAENO,Akiteru2, UEDA, Akihiro1, TAMURA, Masaru3,WAKANA, Shigeharu3, SHIROISHI, Toshihiko2,YAMAMOTO, Akitsugu1, YAMAMOTO, Hiroaki1

(1Grad. Sch. Biosci., Nagahama Inst. Bio-Sci. Tech.,2Mammalian Genet. Lab., Natl. Inst. Genet., 3RikenBRC)

Mitf affects the development of mouse eye-balls

Melanocytes are derived from the vertebrate embryo-specificneural crest. Melanocytes localize in various organs, includingnot only the skin but also extracutaneous locations such as thechoroid of eye, inner ear, heart, etc. We have been interested inthe functional differentiation (evolution) of these pigment cells.To approach this question, we need to know what theseextracutaneous melanocytes are doing in such sun-protectedhabitats, still expressing their icon function, i.e., synthesizingmelanin. We first focused on melanocytes in the eye that localizein its melanin rich layer called the “uvea” consisting of choroid,iris, ciliary body. They may still be illuminated by the trans-mitted light through the retinal pigment epithelium (RPE),another type of melanin pigment cells derived from the outerlayer of the optic cup. RPE is believed to mostly absorb sunlight.We used a mouse Mitf mutant allele, Mitfmi-bw. Because Mitfmi-bw

homozygous mice can’t develop melanocytes but RPE, they showa black-eyed white phenotype. Our observation suggests that thechoroidal melanocytes may contribute to support morphogenesisand/or maintenance of the normal vasculature structure.

1E-04

KATAOKA, Taro1,2, TAMURA, Masaru3, MAENO,Akiteru2, SHIROISHI, Toshihiko2 (1Shizuoka Med.Res. Cent. disaster, Juntendo Univ., 2Mamm. Genet.Lab., Genet. Strains Res. Cent., Natl. Inst. Genet.,3Technol. and Dev. Team for Mouse Phenotype Anal.,RIKEN BioRes. Cent.)

Exploration of Genetic Factors Controlling Bone Metabolismof Mouse

Bone volume is controlled by the balance between osteoblasticbone formation and osteoclastic bone resorption, called boneremodeling. To elucidate genetic mechanisms of bone remodelingduring early growth phase, we dissected mouse genome by usingB6-MSM consomic mouse strains. We have reported that thereare multiple QTLs for regulating bone volume on mousechromosome 15.One of those QTLs, it have four candidate genes and wegenerated each candidate gene mutant mouse strains byCRIPR/Cas9 system. This time, we announce the result of bone-related phenotype screening of these mutant strains and itsfunctions as a novel gene for regulating bone volume.


1E-05

SAGAI, Tomoko1, AMANO, Takanori1, MAENO,Akiteru1, MIZUSHINA, Youichi1, NAKAZAWA,Hiromi1, SHIROISHI, Toshihiko1 (1Mamm. Genet.Lab., Natl. Inst. Genet.)

Dosage effect of two Shh enhancers for control of toothnumber in mouse

In mammalian evolution, the tooth number has been reducedfrom ancestral form. In mouse, adult has only one incisor andthree molars in each jaw quadrant, but embryos develop manyvestigial teeth between incisor and the first molar. Notably,mutations of several genes implicated in Wnt, Fgf, Bmp and Shhsignaling pathways cause a supernumerary tooth juxtapose tothe first molar. To elucidate the precise control for normaldentition, identification of the regulatory mechanism required forthese signaling pathways is crucial. Here, we found thatelimination of an epithelium-specific Shh enhancer causes sucha supernumerary teeth at low frequency. Furthermore, incidenceof the abnormal tooth was vastly elevated in the compoundheterozygotes between this knockout (KO) allele and Shh codingKO allele or in the double KO alleles with another epithelium-specific Shh enhancer. It suggests that dose reduction of the Shhsignaling in the dental epithelium is a direct cause of the toothdefect, and two epithelium specific Shh enhancers contribute todose-dependent control of tooth number. Finally, the sequencecomparison identified regulatory cis-motifs shared by the twoenhancers.

1E-06

FURUHATA, Riki1, NAKAHARA, Mai1, ITOU,Haruka1, ARAKI, Masatake2, YOSHINOBU,Kumiko2, YAMAMURA, Ken-ichi3, ARAKI, Kimi1

(1Div. Dev. Genet., Inst. Resource Dev. and Anal.,Kumamoto Univ., 2Div. Bioinfomatics, Inst. ResourceDev. and Anal., Kumamoto Univ., 3Yamamura ProjectLab., Inst. Resource Dev. and Anal., Kumamoto Univ.)

Correlated expression of LincRNA-p21 and p53 in theLincRNA-p21 gene trap mouse line

The LincRNA-p21 gene was recently identified as a long inter-genic non-coding RNA (lincRNA) regulated by p53. Manyinteresting functions of the LincRNA-p21 have been reported,but most of studies were performed using culture cells, therefore,the in vivo function has remained unclear. We have establishedLincRNA-p21 gene trap mouse line (LincRNA-p21gt) andanalyzed the expression pattern and phenotypes. The trap vectorwas inserted in the intron at 12kb downstream of the 1st exon,and the fusion transcript of the 1st exon and the LacZ gene wasproduced. To examine the expression pattern of the LincRNA-p21in adult mice, qRT-PCR and X-gal staining were performed. TheLincRNA-p21 was expressed at low level in almost all tissuesexamined, and relatively higher expression was observed in thecerebellum, pancreas and white adipose tissue. In order to knowwhether the LincRNA-p21 is induced by p53 in vivo, we crossedLincRNA-p21 gene trap line to p53-deficient (p53-/-) mice. InLincRNA-p21gt/+; p53-/- mice, X-gal staining in the cerebellum,pancreas and WAT completely disappeared, indicating that theLincRNA-p21 is regulated by p53 in vivo.

1E-07

SEKI, Ryohei1,2, LI, Cai3, MATSUBARA, Haruka2,KONDO, Mao2, SATO, Tomohiko2, EGAWA, Shiro2,HAYASHI, Shinichi2,4, SAITO, Daisuke2, IRIE,Naoki5, ZHANG, Guojie3, TAMURA, Koji2,SHIROISHI, Toshihiko1 (1Genet. Strains Res. Cent.,Natl. Inst. Genet., 2Grad. Sch. life Sci., Tohoku Univ.,3BGI-Shenzhen, 4Dept. Genet., Cell Biol. and Dev.,Univ. of Minnesota, 5Grad. Sch. Sci., Univ. Tokyo)

Roles of genomic sequences specifically conserved in Aves onmacroevolution of avian-specific morphological features

Birds are characterized by unique morphological traits such asthe wing and feather. Although their developmental mechanismshave been studied, the genomic background underlying theemergence of the traits remains unclear. We hypothesized thatavian-specific highly conserved elements (ASHCEs) should havecrucial functions and identified them by comparing wholegenomes of 48 birds and 9 outgroup vertebrates. We found thatmost ASHCEs are located in non-coding regions, suggesting thatthey play roles in regulating expression of the genes that they areassociated with. To identify such genes, we performed multi-species screening of ASHCEs-associated genes and found thatSim1 was specifically expressed in the region where flightfeathers develop in the chicken embryo. Furthermore, reporterassay analyses revealed that the Sim1-associated ASHCE hadenhancer activity in the endogenous expression domain of Sim1in the chicken wing. Interestingly, similar reporter activity wasalso observed in the mouse forelimb. Taken together, our resultssuggest that acquisition of novel cis-regulatory elements led toavian-specific gene expression, which resulted in creation ofavian-specific traits.

1E-08

MINEI, Ryuhei1, MORI, Tsukasa2, OGURA, Atsushi1

(1Nagahama Inst. Bio-Sci. Tech., 2Dept. Marine Sci.and Resources, Nihon Univ. Coll. of Bioresour. Sci.)

Brain transcriptome analysis of predator-specific stressresponse in Rana pirica

The tadpole of Ezo Brown Frog, Rana pirica, shows predator-specific morphological changes. Larvae of Dragonfly, Aeshnanigroflava, induces heightened tail depth, whereas those ofsalamander, Hynobius retardatus, induces a bulgy morphology.Although, we identified genes related to their predator-specificchanges, it is not understood how R. pirica distinguish A.nigroflava and H. retardatus, and how morphological changesoccur. We hypothesized that the recognition of predators in thebrain is a trigger for this predator-specific morphologicalchanges. Therefore, we analyzed the brain transcriptome byRNA-sequencing to compare the tadpoles of R. pirica under thevarious predator stresses. As a result, we identified several geneclusters corresponding to these predator stresses, which areinvolved in neural network changing and hormone secretion inthe brain. Especially this hormone, secreted from the pituitarygland, is thought to be important cue to induce H. retardatusspecific morphological change, bulgy morphology. We thus foundthat the brain is involved in this predator-specific morphologicaldefense.


1E-09

TAKAHASHI, Toshiki1, MYOSYO, Taijun2, SATO,Tadashi1, SAKAIZUMI, Mitsuru1 (1Grad. Sch. Sci. andTech., Univ. of Niigata, 2Environ. Life Sci., ShizuokaPrefectural Univ.)

Genetic analysis of a XX male in wild medakas fromKyotango

We established an XX strain from a Dmy-negative(XX) malefound in a wild medaka(O.sakaizumii) population at Kohyama,Kumihama, Kyotango. XX males appeared in the F1 progenybetween an XX male of this strain and XX females of an inbredstrain, Hd-rR(O.latipes), suggesting that XX maleness is adominant trait. We obtained backcross progeny from the F1males and Hd-rR females, and mapped the causal region to0Mb~1.1Mb on linkage group 9. Breakpoint analyses narrowedthe causal region to 183kb~540kb. This region included Dmrt1, adownstream gene of Dmy. Amino acid sequences of Dmrt1derived from this XX strain and an inbred strain, HNI-?(O.sakaizumii), were the same. These results suggested that Dmrt1is a prime candidate gene responsible for XX maleness in thisstrain, and that XX maleness could be caused by overexpressionof Dmrt1.

1E-10

TANAKA, Wakana1, HIRANO, Hiro-Yuki1 (1Dept.Biol. Sci.s, Grad. Sch. Sci., Univ. of Tokyo)

Functional analysis of two related genes involved in theaxillary meristem formation in rice

Plant development depends on the function of the meristem.Aerial parts of the plant body are built by the activities of twodistinct meristems, the shoot apical meristem (SAM) and theaxillary meristem (AM). Although a number of studies concerningthe SAM formation have been reported, studies about the AMformation are insufficient.We found that, unlike in Arabidopsis, the AM is formed in astepwise manner in rice. Analysis of the tillers absent1 (tab1)mutant indicated that TAB1 is required for the maintenance ofthe pre-meristem zone (PMZ), the initial stage of this devel-opmental process. TAB1 was expressed in the PMZ, although itsexpression disappeared in the AM after its establishment. Bycontrast, a related WOX gene, WOX4 was expressed in theestablished AM but not in the PMZ. Since WOX4 is required forthe maintenance of the SAM, these results suggest that tworelated WOX genes are sequentially expressed to maintain theundifferentiated state of cells during the AM formation.Lastly, to reveal the difference in the function between TAB1 andWOX4, we generated transgenic rices expressing WOX4 underthe control of the TAB1 promoter and analyzed their phenotypes.

1E-11

MURAI, Koji1 (1Dept. BioSci., Fukui Pref. Univ.)

Cytoplasmic homeosis: pistillody, homeotic transformation ofstamens into pistil-like structures, in alloplasmic wheat lines

The homeotic transformation of stamens into pistil-like struc-tures (pistillody) has been observed in cytoplasmic substitution(alloplasmic) lines of bread wheat (Triticum aestivum) that carrythe cytoplasm of wild relative Aegilops crassa. We showedpreviously that the mitochondrial gene orf260 somehow alteredthe expression patterns of nuclear class B MADS-box genes andTaDL gene, which specify stamens and pistils respectively, toinduce pistillody. Bread wheat cultivar Chinese Spring has afertility restoring gene Rfd1 on chromosome 7BL and did notshow pistillody with Ae. crassa cytoplasm. The ditelo-somic 7BSline which lacks Rfd1 showed pistillody, whereas the monotelo-disomic 7BS line which has one Rfd1 exhibited pistillody in thetwo stamens out of three in each floret. The dosage effect of Rfd1on pistillody induction and transmission of Rfd1 to nextgeneration would be discussed.

1E-12

SHIBUTA, Mio1, AYAKO, Watanabe1, ABE,Mitsutomo1 (1Dept. Biol. Sci.s, Grad. Sch. Sci., Univ.of Tokyo)

FE, Myb-related protein, promotes photoperiodic flowering inArabidopsis

The timely flowering is one of important keys in reproductivesuccess.One of major component of florigen turned out to be a FT protein,and the molecular mechanism of FT transcription in leaves playsan important role as an accumulation point of various informa-tion. In Arabidopsis, CONSTANS (CO) is an essential regulatorof photoperiod-dependent flowering; nevertheless over-expres-sion of CO failed to induce ectopic expression of FT. So we aimedto identify the other FT transcription activator. A classical floralregulator, FE, is a Myb transcription factor. A single amino acidsubstitution mutant, fe-1, exhibited late-flowering phenotypeunder long day conditions, and CO failed to activate FTtranscription in fe-1 background. Conditional over-induction ofFE also activated FT transcription depending on CO activity.Interestingly, over-expression of both FE and CO synergisticallyinduced FT transcription. These results suggest that FE and COinteract each other and regulate the spatiotemporal expression ofFT.


2A-01

HUA, Liu1, ONO, Seijiro1, TSUDA, Katsutoshi1,2,HIRATSUKA, Rie3, FUKAI, Eigo4, OHTANI, Misato5,DEMURA, Taku5, NONOMURA, Kenichi1,2 (1Exp.Farm, Natl. Inst. Genet., 2Grad. Sch. Life Sci.,SOKENDAI/Grad. Univ. Adv. Stud., 3Div. Biol., JikeiUniv. Sch. Med., 4Faclt. Agr., Niigata Univ.rsity,5Grad. Sch. Biol. Sci., Nara Inst. Sci. Tech.)

Germline-subcellular localization of rice Argonaute proteinMEL1, essential for faithful meiosis progression

Argonaute (AGO) is a protein that associates a single-strand 20-30 nucleotides (nt) small RNA as a guide molecule, and functionsin transcriptional (TGS) or posttranscriptional gene silencing(PTGS). A rice protein MEL1 is an AGO protein that isexclusively expressed in male and female germ cells beforemeiosis. MEL1 associates preferentially to 21- and 24-nt siRNAsabundantly expressed in rice anthers. In this study, to ask themolecular function of MEL1, we conducted immunofluorescentstaining of rice germ cells, immuno-electron microscopic obser-vations (immuno-EM), and co-immunoprecipitation (co-IP)/ LC-MS/MS.The co-IP fraction of MEL1 contained a lot of ribosomal subunitproteins, as representing a cytoplasmic role of MEL1 in PTGS.Actually, the immuno-EM suggested that MEL1 could associateribosomes or rough endoplasmic reticulum. Interestingly, thesame fraction also contained histone H2 and H3 variants. Takentogether with the fact that MEL1-GFP fusion protein could locateat the nucleoplasm at the onset of meiosis, the results suggestthat MEL1 plays some important role in TGS in male meiocytenuclei.

2A-02

ONO, Seijiro1, TSUDA, Katsutoshi1,2, TANAKA,Keisuke3, SASAKI, Takuji4, NONOMURA, Ken-ichi1,2

(1Exp. Farm, Natl. Inst. Genet., 2Dept. Life Sci.,SOKENDAI/Grad. Univ. Adv. Stud., 3NODAI GenomeRes. Center, Tokyo Univ. Agr., 4NODAI Res. Inst.,Tokyo Univ. Agr.)

Production of meiotic phasiRNAs is depended on TTMtranscription factor in anther tapetum of rice

It has been poorly understood whether plants have small RNA-mediated silencing systems during meiotic events, like as thepiRNAs in animals. 24-nucleotides (nt) phased small interferingRNAs (phasiRNAs) are the small RNAs which abundantlyexpressed in meiotic anthers of grass species, though theirfunction on reproduction events is largely unknown.In this study, we characterized TTM transcription factor in rice,which specifically expressed in anther tapetum, the somaticnursery cells of male meiocytes, during early meiotic stages. ttmmutant exhibited meiotic retardation and chromosome decon-densation in male meiocytes, while tapetum development itselfseemed normal. TTM simultaneously activated the transcriptionof a number of noncoding precursor loci generating 24-ntphasiRNAs and the DCL3b gene, encoding a dicer-like proteinessential for the 24-nt phasiRNA processing.Our results indicated the biogenesis of 24-nt phasiRNAs wasdepended on TTM transcription factor in meiotic tapetum. Wealso hypothesize that the tapetum-derived 24-nt phasiRNAs haveimportant function in meiosis progression in adjacent meiocytes.

2A-03

HIGASHIDE, Mika1, SHINOHARA, Miki1 (1Lab.Genome and Chromosome Funct., Inst. Prot. Res.,Univ. Osaka)

Slx4, multifunctional scaffold protein, functions for negativeregulation of CO formation specific in centromere-proximalregion and DSB distribution on entire chromosomes

During meiosis, crossover (CO) recombination between homolo-gous chromosomes ensures proper segregation of homologouschromosome in meiosis I. Thus, at least one CO must be formedon each chromosome. In contrast, excessive formation of COs,especially centromere-proximal region, can be topologicallyharmful for chromosome segregation. Then, the number andposition of COs must be strictly controlled during meiosis. MeioticCO formation is induced from programmed double-strand breaks(DSBs) by Spo11 function which is meiosis-specific topoisomer-ase-like transesterase. Actually, many of endonucleases arerequired to generate CO product in each step: DNA end resectionfor homology search, resolution of CO intermediates. In fact,many kinds of endonucleases are available to recognize andmanage intricate DNA structures in vivo. In this study, wefocused on Slx4 which is known as scaffold protein for many ofDNA structure-specific endonucleases activity in vegetative cells.Interestingly, we found that Slx4 is involved in negative controlfor CO formation in centromere-proximal region and in thedistribution for DSB formation independent of CO suppression oncentromere-proximal region. Thus, we conclude Slx4 is multi-functional in meiotic recombination.

2A-04

KE, Li1, SHINOHARA, Miki1,2 (1Lab. Genome-Chromosome Funct, Inst. Prot. Res., Univ. of Osaka,2Dept. Biol. Sci.s, Grad. Sch. Sci., and Faclt. Sci., Univ.Osaka)

Characterization of PP4 Phosphatase Function DuringMeiotic Recombination in Budding Yeast

Meiotic recombination is essential of proper segregation ofchromosomes during meiosis-I by regulating the formation ofChiasma, the physical connection between homologous chromo-somes. The regulation mechanism of meiotic recombination isstill unclear because of its complexly. In this study, I focused onPP4, a Mec1-specific phosphatase, to reveal its function in meioticrecombination. By now, I confirmed PP4 is involved in theregulation of meiosis. Firstly, I found PP4 can regulate thedephosphorylation of Hop1. Then, it is revealed that PP4promotes disassembly of factor from chromosomes, such as Zip1or Rad51. In future, I want to investigate PP4 function duringmeiotic recombination so that it can facilitate revealing themechanism of meiotic recombination.


2A-05

ITO, Kentaro1, MURAYAMA, Yasuto3, TAKAHASHI,Masayuki2, IWASAKI, Hiroshi3 (1Dept. Life Sci., Grad.Sch. BioSci. and BioTech., Tokyo Inst. Tech., 2Schooland Grad. Sch. BioSci. and BioTech., Tokyo Inst.Tech., 3Cell Biol Unit, Inst. innovative Res., TokyoInst. Tech.)

Real-time analysis of DNA strand exchange reaction medi-ated by Rad51

DNA strand exchange reaction is the central phase of homologousrecombination. In eukaryote, Rad51, which is a RecA familyrecombinase binds single-stranded DNA and forms nucleoproteinfilament named presynaptic filament. Presynaptic filamentsearches homologous sequence in double-stranded DNA (dsDNA)and promotes DNA strand exchange within homologous dsDNA.However, to accomplish the reaction, Rad51 requires someaccessary proteins. Schizosaccharomyces pombe Swi5-Sfr1 com-plex is an accessary protein. Previous study demonstrated thatSwi5-Sfr1 stimulates DNA strand exchange activity of Rad51 andstabilizes presynaptic filament active state in the presence ofATP. However, the role of Swi5-Sfr1 after presynaptic phase isunclear. To elucidate activation mechanisms of the reaction bySwi5-Sfr1 in detail, we established real-time observation systemof DNA strand exchange. From our analysis, Swi5-Sfr1 stim-ulates DNA strand exchange reaction after presynaptic phase inthe presence of ATP.

2A-06

IWASAKI, Daichi1,2, HAYASHIHARA, Kayak1,2,SHINOHARA, Miki1,2 (1Inst. Prot. Res., Osaka Univ.,2BioSci., Grad. Sch. Sci., Osaka Univ.)

The MRX complex ensures NHEJ fidelity through Xrs2-FHA?dependent Tel1 activation

Because DNA double-strand breaks (DSBs) is the most cytotoxicDNA lesion and often cause genomic instability, precise repair ofDSBs is essential for maintenance of genomic stability. In yeastXrs2, an ortholog of human Nbs1, is a multi-functional regulatorysubunit of Mre11-Rad50-Xrs2 (MRX) complex, and its function iscritical for DSB repair, whether by homologous recombination(HR) or non-homologous end joining (NHEJ). Previously, wereported that the Xrs2 FHA domain is required for NHEJthrough the physical interaction with DNA ligase IV complex.Here we show that the Xrs2 FHA domain is required both tosuppress of imprecise repair of DSBs, as well as to promote therobust activation of Tel1 in the DNA damage response pathway.The role of the FHA domain in Tel1 activation is independent ofthe Tel1-binding activity of the Xrs2 C-terminus, which mediatesthe recruitment of Tel1 to DSB ends. Both the Xrs2 FHA domainand Tel1 were required for the timely removal of the Ku complexfrom DSB ends to suppress imprecise repair. Thus, the Xrs2 FHAdomain is involved not only in promotion of precise NHEJ, butalso in suppression of imprecise repair to maintain DSB repairfidelity.

2B-01

SATO, Tadashi2, HAMAGUCHI, Satoshi2,SAKAIZUMI, Mitsuru2, KOBAYASHI, Tohru1,MYOSHO, Taijun1 (1The Dept. Sch. Food andNutritional Sci., Shizuoka Univ., 2Inst. Sci. Tech.,Niigata Univ.)

The sex-determining gene Dmy and a novel sex-determininggene GsdfneoY coexist in wild medaka population in Nagasaki

A conserved sex-determining gene Dmy have been identified inJapanese medaka. However, males without Dmy and femaleswith Dmy were found in wild population. Recently, we found asex-reversed XX male without Dmy in a wild population atNagasaki, Japan. Sex-linkage analysis suggested that theresponsible gene of XX male was Gsdf on linkage group 12. Thenull mutants with a frameshift mutation in GsdfneoY (Gsdf on theneoY chromosome) coding region developed as sex-reversedfemales (XY females), indicating that GsdfneoY is the novel sex-determining gene. In 2014, we also found 34 males with Dmy and8 males GsdfneoY in a wild population of medaka at Nagasaki.Together, we revealed that two sex-determining genes, Dmy andGsdfneoY, coexist in a wild medaka population at Nagasaki.

2B-02

WATADA, Masayoshi1, YOSHIMURA, Kanamu1,KAGAYAMA, Daisuke2 (1Grad. Sch. Sci. and Eng.,Ehime Univ., 2Inst. AgroBiol. Sci., NARO)

Genetic analysis of the new sex ratio disorder found inDrosophila

One strain of Drosophila biauraria collected in Hokkaido severalyears ago showed sex ratio disorder, which produced only femalesevery generation. This phenomenon is not reproduction byparthenogenesis, and females of the disorder strain need to bemated by males of another strains for production of females.Among 55 strains of D. biauraria collected in 2015, we found thatthree strains of the species had the same characteristic, and thisresult showed that this phenomenon was polymorphic in naturalenvironment in Hokkaido. We studied symbiotic infection by PCRmethod using standard primers of Wolbachia and Spiroplasma,and also checked the infection by antibiotic treatment. Thesestudies cleared that the disorder was not caused by the infectionof these symbionts. In addition, we injected body fluid of thedisorder strain into the normal strain that produce females andmales, and got strains that produced only females among thefemales of the normal strain, suggesting that this sex ratiodisorder was caused by the cytoplasmic factor other thanbacterial symbionts. In the present study, we showed that thefactor seemed to be a RNA virus by the study of RNA-seqanalysis.


2B-03

YOSHIDA, Kohta1, KITANO, Jun1 (1Div. EcologicalGenet., Natl. Insitute Genet.)

A probabilistic model for karyotype evolution

Recent several reports suggest that karyotype evolution playsimportant roles in speciation and adaptive evolution. To inves-tigate the functions and mechanisms of karyotype evolution, aprobabilistic model applicable for phylogenetic comparativemethods for karyotype evolution is needed. We established aprobabilistic model based on centric fusions and fissions andpericentric inversions, which are major events of karyotypeevolution. This model is useful for the phylogenetic comparativeanalyses of karyotype evolution of large taxonomic groups.

2B-04

MATSUMOTO, Tomotaka2, YOSHIDA, Kohta1,KITANO, Jun1,2 (1Div. Ecological Genet., Natl. Inst.Genet., 2Div. Evolutionary Genet., Natl. Inst. Genet.)

Gene Flow and Sex Chromosome Evolution

Alleles that benefit one sex but harm the other (sex-antagonisticalleles) can induce structural changes in sex chromosomes.However, if regulatory mutations resolve sexual antagonismand restrict gene expression in one sex, these alleles becomeneutral in the other and are easily fixed. Fixed alleles willtherefore no longer generate selective pressures for sex chromo-some structural changes. Using analytical approximations andindividual-based simulations, we show that gene flow of delete-rious alleles into a population can promote the establishment ofY-autosome fusions even after regulatory mutations resolveantagonistic effects. This is because fused Y-chromosomes thatcapture alleles beneficial for local males have a higher meanfitness compared to unfused Y in the presence of deleterious geneflow. Our results demonstrate that spatial variation in sex-specific selection should be considered as a factor affecting theprocess of sex chromosome evolution.

2B-05

SEIKE, Taisuke1, NIKI, Hironori1 (1Microbial Genet.Lab., Genet. Strains Res. Center, Natl. Inst. Genet.)

Mechanism of pheromone diversification in yeasts

Reproductive isolation is a key process of speciation. Recentstudies have revealed that a few key mutations in pheromone/receptor can give rise to a new species in the fission yeastSchizosaccharomyces pombe (Seike et al., Genetics, 2012;PNAS, 2015), but the mechanism of pheromone diversificationin nature is unclear.S. pombe has two mating types, Minus (M) and Plus (P). Matingis controlled by mating pheromones, M-factor and P-factor,secreted by M- and P-type cells, respectively. M-factor is encodedby three redundant genes, whereas P-factor is located at a singlelocus that encodes four copies of pheromone ORFs. Here, weexamined 53 strains of S. pombe whose origins differ from that ofthe laboratory strain. Notably, all M-factor genes were highlyconserved. In contrast, the P-factor gene was very diverse,resulting that a few amino acid substitutions and increasednumbers of copies (>5 copies) in the mature P-factor-codingregion. Thus, recognition specificity of the M-factor is tight,whereas that of the P-factor is loose. Provably, the biased systemfor pheromone recognition might allow both tight recognition forpartners and flexible adaptation in yeasts.

2B-06

MUTO, Leona1, KAMIMURA, Yoshitaka2, TANAKA,Kentaro1, TAKAHASHI, Aya1,3 (1Dept. Biol. Sci.,Tokyo Metropolitan Univ., 2Dept. Biol., Keio Univ.,3Res. Cent. Genomics and Bioinform, TokyoMetropolitan Univ.)

Coevolution of genitalia between male and female inDrosophila suzukii : Effect of morphological diversificationin ovipositor

Drosophila suzukii females have uniquely modified ovipositors,which have enabled them to lay eggs inside the hard skin of freshfruits. Their ovipositors have more elongated but less curvedshape compared to those of a closely related species, D.subpulchrella. In Drosophila spp., the ovipositor is also knownto play an important role in copulation. During the copulation,female ovipositor is usually grasped by male genitalia. From adetailed observation of the genitalia of copulating pairs, it wasconfirmed that male claspers hold the posterior areas of femaleovipositor in both species. Also, we found that a pair of maleparameres with hook-shaped tips stab the female body duringcopulation in D. subpulchrella, but they do not reach the femalebody in D. suzukii. This suggests that D. suzukii may have lost thefunction of parameres due to elongation of the ovipositor in thisspecies. Our observations suggest an intriguing possibility thatcoevolution in genitalia morphology between male and female inD. suzukii has been driven by changes in female ovipositor shapedue to a shift in host fruit preference to fruits with much harderskins.


2B-07

SAWAMURA, Kyoichi1, WANG, Zhuo2, MIURA,Kohei2, HAYASHI, Takaaki2, HIRAI, Kazuyuki3,AWASAKI, Takeshi3, WADA, Moe4, KEIRA, Yoko4,ISHIKAWA, Hiroyuki O.4 (1Faclt. Life and Env. Sci.s,Univ. Tsukuba, 2Grad. Sch. Life and Env. Sci., Univ.Tsukuba, 3Sch. Med., Kyorin Univ., 4Grad. Sch. Sci.,Chiba Univ.)

Interaction among nucleoporins and reproductive isolation inDrosophila

In diverse organisms, intracellular localization of nuclear porecomplex is determined by the interaction between Nup107-160subcomplex (Y-complex Nups) and ELYS/MEL-28. UsingCRISPR/Cas9, we disrupted the CG14215 gene, an X-linked Elyshomolog of Drosophila melanogaster. Homozygous and hemi-zygous Elys mutants were viable but female sterile. Mutantfemales laid fertilized eggs but the eggs never developed. AndNup160, Nup96, and Nup37, but not other Y-complex Nups, werehaplo-insufficient in the Elys mutation background. In otherwords, Elys/Y; Nup/+ and Elys/Elys; Nup/+ were lethal at thepupal stage. Furthermore, positive natural selection was detectedin the Elys-like domain of the gene, especially in the branch for D.simulans, by the Ka/Ks ratio test. Based on the phenotypicsimilarity to interspecific hybrid or introgression female sterilityand inviability, we speculate the possible involvement of Elys-Nups interaction in reproductive isolation between D. mela-nogaster and D. simulans.

2B-08

KUEI-YUEH, Ko1, HUANG, Hsuan-Cheng3, JUAN,Hsueh-Fen2 (1Genome & Systems Biol. DegreeProgram, National Taiwan Univ., 2Dept. Life Sci.,National Taiwan Univ., 3Inst. BioMed. Inform.,National Yang-Ming Univ.)

Phylotranscriptomic patterns of network stochasticity andpathway dynamics during embryogenesis

Various evo-devo models have been proposed to describe thesimilarities and differences among developmental processes ofdifferent species. Although pieces of evidence indicate an evolu-tionary constraint during mid-developmental stage, its explan-ation and molecular mechanism are not fully understood yet.In our study, we aimed to tackle the problem via the tools ofnetwork and pathway analysis. Using the protein-proteininteraction network information and gene expression data, wefirst measured the stochasticity within the biological networkduring the developmental process. Such analysis enables us totrace and compare the changes of network randomness amongdevelopmental processes of different species. In our result, anetwork reformation is observed during the development. Thecomplexity of interconnections in the intermediate stages ishigher than that in the early and late stages. Next, we sketchedout the pathway dynamics during developmental process in orderto narrow down our study from global network connections todetailed molecular regulations. With the systematic networkview of developmental process, our results provided an alter-native aspect of comparative embryology.

2B-09

CHIH-KUAN, Chen1 (1Program in Inst. Ecol.Evolutionary Biol., National Taiwan Univ.)

Regulatory Differences in Natal Down Development betweenAltricial Zebra Finch and Precocial Chicken

Birds are the most diversified terrestrial vertebrates, largelythanks to the diversification of feather. The hatchlings of altricialbirds are almost naked, whereas those of precocial birds arecovered with natal down. This difference is thought to reflectadaptation to different environments. However, the molecularbasis is unclear. In zebra finch hatchlings, natal down partiallycovers the posterior dorsal (PD) skin, but is suppressed in theanterior dorsal (AD) skin. After analyzing the transcriptomes ofAD and PD skins over three developmental stages, I found thatthe feather growth promoter SHH was expressed higher in PDskin than in AD skin. Moreover, the data suggested that the FGF/MAPK signaling pathway is involved in natal down growthsuppression and that FGF16 is an upstream signaling suppres-sor. Ectopic expression of FGF16 on chicken skin suppressed thefeather bud elongation, similar to the phenotype found in zebrafinch hatchlings. I proposed that FGF16-related signals cause thenaked AD skin in zebra finch. My study provides insights into theregulatory divergence in natal down formation between precocialand altricial birds.

2B-10

CHERIF, Amira3, MEDIOUNI BEN JEMAA, Jouda3,TAYLOR, Demar1 (1Lab. Appl. Entomol. and Zool.,Univ. Tsukuba, 2Natl. Agronomic Inst. Tunisia(INAT), 3Lab. Biotechnol. Appl. to Agric., Natl. Agric.Res. Inst. of Tunisia (INRAT))

Molecular Characterization of Three Mayetiola Species(Diptera: Cecidomyiidae)

Cereal midges are considered as the most destructive pests ofwheat, barley and oat worldwide. Species from the genusMayetiola have been observed in the main cereal cultures ofTunisia. Some studies have studied M. destructor that attackswheat and M. hordei that attacks barley. However, a thirdimportant species M. avenae, observed in oat, has never beenstudied and is not well documented in Tunisia.This study aimed to distinguish between the 3 species of midgesby molecular characterization of a region of the cytochromeoxidase subunit I (COI) gene commonly used to reveal geneticdiversity between gall midges. A set of 30 individuals, 10 fromeach species, was used and three primer sets tested. Only oneprimer pair allowed us to distinguish between the differentspecies. The phylogenetic tree clearly showed significant separa-tion of the three different species into dissimilar clades. Eachclade contained only specimens from the same species. Ourresults indicate this area of the COI gene can be used as a markerto reveal genetic diversity between species of Mayetiola.


2C-01

ZHOU, Hua1, HIRATA, Megumi1, OSAWA, Ryo1,FUJINO, Kaien1, KISHIMA, Yuji1 (1Hokkaido Univ.;Res. Faclt. Agr.; Lab. Plant Breeding)

Epigenetic-independent regulation of Tam3 in Antirrhinummajus detains transposase to the plasma membrane bytargeting its BED-zinc finger domain

Transposable elements (TEs) are considered to be parasites ofhost genomes as they act as powerful mutagens. If not kept incheck, they can cause gene disruption, genome rearrangement,and genomic takeover. Hence, activities of TEs are under rigidcontrol of hosts. Up to now, all discovered TE regulations havebeen epigenetic-dependent, with the exception of the DNAtransposon Tam3. Blocking nuclear translocation of Tam3 trans-posase (TPase) is consistent with the suppression of Tam3 inAntirrhinum majus. Our data uncovered that epigenetic-inde-pendent regulation of Tam3 is mediated by the BED-zinc fingerdomain of Tam3 TPase. The host targets the N-terminus of theBED-zinc finger domain, which contains two highly conservedaromatic amino acids, to detain Tam3 TPase to the plasmamembrane, and to silence Tam3. Zinc finger proteins performbroader functions in transcriptional regulation through theirDNA binding ability. Here, our results revealed epigenetic-independent silencing against TE activity in a post-translationalmanner was due to the protein binding ability of BED-zinc fingerdomain.

2C-02

MAKIYA, Kayo1, EBINUMA, Izuru1, ASANO,Masahiro1, TATEO, Taichi1, KASAHARA, Tomo1,NAKAYAMA, Yuya1, UCHIYAMA, Takako1, KISHIMA,Yuji1 (1Grad. Sch. Agr., Univ. of Hokkaido)

The analysis of comprehensive transition motion of trans-poson Tam3 in Nivea locus of Antirrhinum majus

We have studied transposition of DNA transposon Tam3 inAntirrhinum. We tested nivearecurrens:Tam3(nivrec) allele,which is derived from a pigmentation locus called Nivea encodingchalcone synthase in Antirrhinum. This allele has Tam3 in thepromotor region, and causes variegation spots in flower petals.Tam3 shows low temperature dependent transposition(LTDT)atlow temperature(15C), but not at high temperature(25C). In thisstudy, Tam3 transpositions from nivrec were examined onfrequency and repaired sequences(footprint), and compared:somatic cell vs germline cell, homozygous of nivrec vs hemizygouswith different alleles. The results obtained were summarized asfollows: 1) more than a quarter of somatic transpositions of Tam3left no footprints and reverted the sequence of nivrec to the WT.2) frequencies of germline transpositions were lower than those ofthe somatic transpositions. 3) footprints of somatic transpositionswere hardly transmitted to the germline cells. 4) the footprintsfrom the nivrec homozygous allele tended to be smaller sequencechanges, compared with those from the hemizygous nivrec allelescombined with the allele without Tam3.

2C-03

TSUGANE, Kazuo1,2, TSUGANE, Mika3, MAEKAWA,Masahiko4 (1Natl. Inst. Basic Bio, 2SOKENDAI,3Nagoya Univ., 4Okayama Univ.)

The gain-of-function mutations in rice caused by insertion ofthe DNA transposon, nDart1

DNA transposons used cut and paste manner for transposition,occupied 13 % of rice genome. The endogenous non-autonomoustransposon, nDart1elements tend to insert into genic regionsunder natural growth conditions. The dominant mutants wereoccasionally isolated from the nDart1-promoted tagging line.Efficient selection and analysis of dominant mutants to analyzethe gene functions in rice is very useful. A semidominant mutant,Bushy dwarf tiller1 (Bdt1), which has the valuable agronomictraits of multiple tillering and dwarfism, was obtained from thetagging line. The Bdt1 mutant carried a newly inserted nDart1 at38-bp upstream of the transcription initiation site of a non-protein-coding gene, miR156d. This insertion caused an upstreamshift of the miR156d transcription initiation site and, conse-quently, increased the functional transcripts producing maturemicroRNAs. These results indicate that the total amount ofmiR156d is controlled not only by transcript quantity but also bytranscript quality. This study demonstrates the ability of nDart1to produce gain-of-function mutants as well as further insightsinto the function of transposable elements in genome evolution.

2C-04

HAYASHI, Yui1, MASUDA, Yukari2, KATO, Atsushi2,ITO, Hidetaka2 (1Grad. Sch. Life Sci., Hokkaido Univ.,2Faclt. Sci., Hokkaido Univ.)

A transpositional regulation of a heat-activated retrotrans-poson in Arabidopsis

ONSEN“ is a heat-activated retrotransposon in Arabidopsisthaliana. Previously we showed that transcript level of ONSENwas increased by heat stress and transposition of ONSEN wasobserved in the progeny of stressed plants that deficient in siRNAbiosynthesis. In this study


2C-05

NOZAWA, Kosuke1, MASUDA, Seiji1, KATO,Atsushi2, ITO, Hidetaka2 (1Grad. Sch. Life Sci.,Hokkaido Univ., 2Faclt. Sci., Hokkaido Univ.)

Analysis of transcriptional regulation of heat-activatedretrotransposon

Transposable elements are major components of plant genomesand most of them are silenced by epigenetic modifications.However, recent studies showed the environmental stressinduces some transpositions. ONSEN is an Ty1/copia-typeretrotransposon in Arabidopsis. ONSEN became transcription-ally active and synthesized extrachromosomal DNA subjected toheat stress. Moreover, high frequency of new insertions wasobserved in the progenies of stressed mutants that deficiency ofsiRNA biosynthesis. In Arabidopsis, ONSEN is conserved amongaccessions. We found that a transcript level of the heat-activationof ONSENwas various among accessions. The copy number and acis element of the heat-activation of ONSEN were analyzedamong accessions. We report the result of a comparative analysisof the transcriptional regulation of ONSEN in the accession.

2C-06

IRITANI, Akihide1, NGUYEN, Quyet1, VAN VU, Ba1,OHKITA, Shuhei1, YOKOYA, Kana1, IKEDA,Ken-ichi1, NAKAYASHIKI, Hitoshi1 (1Grad. Sch. Agr.Sci., Kobe Univ.)

An Argonaute gene in the fungus Magnaporthe oryzaefunctions as a negative regulator of RNA silencing

In the genome of the rice blast fungus, Pyricularia oryzae, threeArgonaute-like genes were identified, which we designatedMoAgo1, MoAgo2 and MoAgo3, respectively. Gene knockout(KO) studies revealed that MoAgo1 and MoAgo2 were redun-dantly involved in hairpin RNA- and retrotransposon-triggeredRNA silencing in P. oryzae. Surprisingly, the KO mutants ofMoAgo3 (Dmoago3) showed elevated levels of gene silencing whentriggered either by hairpin RNA or the LTR retrotransposonMAGGY, suggesting that MoAgo3 functioned as a negativeregulator of RNA silencing in P. oryzae. FLAG-tagged Agoproteins were used to pull down their associated sRNAs for deepsequencing analysis. sRNA species associated with the three Agoproteins were partly specific to each protein but largely over-lapped with each other. The biological role of MoAgo3 is ofinterest but remains elusive. MoAgo3 might contribute tomaintain transposable elements moderately active for generatingnew genetic variations in this phytopathogenic fungus.

2C-07

INOUE, Kota2, ICHIYANAGI, Kenji1, FUKUDA, Kei3,GLINKA, Michael4, SASAKI, Hiroyuki2 (1Grad. Sch.Bioagricultural Sci., Nagoya Univ., 2Med. Inst.Bioregulation, Kyushu Univ., 3Cell memory lab.,RIKEN, 4Faclt. Sci., Univ. of Bristol)

Switching from PTGS to TGS for epigenetic regulation ofretrotransposons during male germ cell development in mice

During male germ cell development in mice, both DNAmethylation and PIWI-interacting small RNAs (piRNAs) areinvolved in regulation of retrotransposon activity. The piRNAsystem destroys retrotransposon-derived RNAs and guides denovo DNA methylation at some retrotransposon promoters inprospermatogonia. However, it remains unclear whether DNAmethylation contributes to retrotransposon silencing in this celltype. We performed DNA methylome and transcriptome analysison developing male germ cells from Pld6 and Dnmt3l knockoutmice, which are defective in piRNA biogenesis and de novo DNAmethylation, respectively. The results revealed that, in sperma-togonia, DNA methylation plays a very minor role in retro-transposon regulation, while the piRNA system plays animportant role by a post-transcriptional mechanism. However,DNA methylation had a long-term effect: hypomethylationcaused by the Dnmt3l mutation resulted in retrotransposonactivation in later stages of development (spermatocytes), andtranscriptional silencing by DNA methylation became moreimportant in spermatocytes, indicating a developmentally pro-gramed switch of mechanisms to combat retrotransposons.

2C-08

NAKAGAWA, So1,2, TAKAHASHI, Mahoko Ueda2

(1Dept. Molec. Life Sci., Tokai Univ. Sch. Med., 2Micro/Nano Technol. Center, Tokai Univ.)

gEVE, genome-scale endogenous viral element database, andits applications

In mammals, approximately 10% of genome sequences corre-spond to endogenous viral elements (EVEs) including endogenousretroviruses (ERVs), which are thought to be derived from viralinfections in germ cells. Although most EVEs have beeninactivated by insertions, deletions, substitutions, and/or epige-netic modifications, a few open reading frames (ORFs) of EVEsare still active and express viral proteins in the hosts. Indeed,several genes derived from EVEs have been found to befunctional for host species, such as syncytins for placentadevelopment in various mammals. However, no databases ofEVE ORFs are available, and therefore molecular functions andevolutionary pathways of EVEs have not yet been understoodcomprehensively. Hereby, I developed EVE ORF databases for 19mammalian species (http://geve.med.u-tokai.ac.jp) containing atotal of 736,771 non-overlapping EVE ORFs. The gEVE databaseprovides nucleotide and amino acid sequences, genomic loci andfunctional annotations of EVE ORFs. In this talk, I will introducethe gEVE database as well as its application to the varioustranscriptome analyses with various large-scale sequencing data.


2C-09

NISHIHARA, Hidenori1 (1Dept. Life Sci. and Technol.,Tokyo Inst. Tech.)

Evolution of cis-regulatory network involving transposableelements in mammals

It is largely unknown how much transposable elements (TEs)have involved in the morphological evolution of mammals. In thepresent work, I analyzed the ChIP-seq data of human to revealthe binding sites of ER-alpha and several transcription factorsthat are responsible for the development of mammary gland inmammals, and compared those overlapping with TEs. It isrevealed that the binding sites of these factors are significantlybiased within several kinds of TEs which contain the bindingmotif in the consensus sequences. Genomic regions where the TE-derived putative functional elements locate in the higher densityare the promoter regions of several genes, most of which areknown to be involved in the development of mammary gland. Inaddition, two waves of retrotransposition bursts had producedthe major TE-derived functional elements during mammalianevolution. Therefore, these TE families had propagated thesources of the transcription factor binding sites via retrotrans-position and have contributed to increase the cis-regulatoryelements involved in mammary gland evolution.

2C-10

KOGA, Akihiko1, HIRAI, Hirohisa1 (1Primate Res.Inst., Kyoto Univ.)

Rapid replacement of centromeric DNA by another sequence:events observed in gibbons and owl monkeys

Centromeres usually contain large amounts of tandemly repeatedsequences as their DNA components. The nucleotide sequences ofthese repetitive DNAs differ from taxon to taxon. It is widelythought be a main cause of such differences that centromericDNA undergoes frequent reduction and increase in the copynumber followed by bottleneck effects. Another possible causewould be the neocentromere formation and subsequent amplifi-cation of sequences seeded there. We have observed replacementof centromeric DNA in primates, which may be useful indetermining which cause is more likely to be. One example is ingibbons, in which centromeres of most chromosomes had beenreplaced by retrotransposon-related tandem repeats. Anotherexample is in owl monkeys, where the centromeres of allchromosomes had been replaced by a partially deleted variantof the original centromeric DNA.

2D-01

YOSHIMI, Kazuto1, IMAI, Yuji1, TANAVE, Akira1,MASHIMO, Tomoji2, KOIDE, Tsuyoshi1 (1MouseGenomics Resource Lab., Natl. Inst. Genet., 2Inst. Exp.Anim. Sci., Grad. Sch. Med., Osaka Univ.)

Long single-stranded oligonucleotide-mediated knock-in withCRISPR/Cas9 in rodents

Genome editing technologies have enabled generating geneticallymodified animals within the past few years. Development of anefficient knock-in (KI) technology will facilitate easy and flexiblegenome engineering to introduce precise mutations or geneticmodifications at any target sites in many species.We have reported the efficient generation of several types of KIrats using single-stranded oligonucleotides (ssODNs), such asSNP substitution, small DNA fragment insertion, and DNAfragment elimination of a 7 kb retrotransposon element (YoshimiK et al., Nat Commun 2014). It is easy to design and synthesizessODNs as a DNA donor, while the limitation of ssODN-mediatedKI is the maximum length of the insert DNA, which is less thanalmost 200 bp.In this study, we developed a new strategy to generate longssODN fragments (lsODNs) using nicking endonucleases. Micro-injection of a synthesized lsODN with gRNA and Cas9 mRNAproduced several types of KI rodents, such as GFP-KI rats atThy1 locus and floxed-KI mice at Tyr locus. LsODN-mediated KItechnologies can apply to any target site of any species, thussimplifying genome engineering in living organisms.

2D-02

TESHIGAWARA, Rika1,2, HIRANO, Kunio1,NAGATA, Shogo1, AINSCOUGH, Justin3, TADA,Takashi1 (1Dept. Stem Cell Eng., Field of Stem CellRes. Inst. Front. Med. Sci., Kyoto Univ.,, 2Grad. Sch.Med., Kyoto Univ., 3Dept. Biol., Univ. of York)

OCT4 activity during conversion of human intermediatelyreprogrammed stem cells to iPSCs through mesenchymal-epithelial transition

To facilitate understanding the mechanisms of somatic reprog-ramming to human induced pluripotent stem cells (iPSCs), wehave established intermediately reprogrammed stem cells(iRSCs), human mesenchymal cells that express exogenousOct4, Sox2, Klf4 and c-Myc (OSKM) and endogenous SOX2 andNANOG. iRSCs can be stably maintained at low density. At highdensity, however, they are induced to enter mesenchymal-epithelial transition (MET), resulting in reprogramming to aniPSC state. Morphological changes through MET correlate withsilencing of exogenous OSKM, and upregulation of endogenousOCT4. A CRISPR/Cas9-mediated GFP knock-in visualized thetemporal regulation of endogenous OCT4 in cells converting fromiRSC to iPSC state. OCT4 activation coincident with silencing ofOSKM occurred prior to entering MET. Notably, OCT4 insta-bility was frequently observed in cells of developing post-METcolonies until a late stage (>200 cells), demonstrating that OCT4-activated post-MET cells switched from asymmetric to symmetriccell division in late stage reprogramming.


2D-03

MAKINO, Shigeru1, GONDO, Yoichi1 (1RIKENBioRes. Cent.)

Mechanisms of translation reinitiation by frameshiftedmutant alleles

Post-termination ribosomes occasionally reinitiate translationfrom an AUG codon downstream of the termination codon. It hasbeen, however, unknown what extent translation reinitiationgenerally occurs. We generated mutant alleles carrying out-of-frame indel mutations by CRISPR-Cas9 in the mouse NIH3T3cell line. Unexpectedly, we found that the translation wasreinitiated from an AUG codon other than the native translationstart codon in all these frameshifted alleles that we established.In this study, we found that translation reinitiation is not limitedto the mouse cells but may occur universally irrespective of thespecies difference in the presence of a premature terminationcodon.Translation reinitiation also occurs in wild-type genes as uORFs,which are found in almost half of human transcripts. However,the functions of uORFs have not been understood except severalcases. Detailed analysis of the frameshifted cell lines reveals acritical regulatory role of translation reinitiation in geneexpression as a new paradigm in the central dogma.

2D-04

IWAMA, Hisakazu1, FUJITA, Koji2, IMACHI,Hitomi3, MURAO, Koji3, MASAKI, Tsutomu2 (1LifeSci. Res. Cent., Kagawa Univ., 2Dept. Gastroenterol.Neurol, Faclt. Med., Kagawa Univ., 3Dept. EndocrinolMetab, Faclt. Med., Kagawa Univ.)

Relationship of expression intensities between human micro-RNAs and their targets in view of microRNAs’ ages

To elucidate the interrelation of microRNAs’ (miRNA) targetingspecificity with the evolutionary age of the miRNA, we usedmultiple RNA-seq data sets for human miRNAs and protein-coding genes that were built separately. Then we investigated therelationship of the expression intensity of the miRNAs with thatof the protein coding genes. As a result, we found that the tissue-specifically expressed miRNAs showed a characteristic pattern ofavoidance and preference according to the targets’ genesexpression intensities in the same tissue as the miRNA. Thistargeting pattern was shown to become more distinct as themiRNAs’ ages get older.

2D-05

KISHI, Nobuyuki1, HARA, Satoshi2, TAKADA, Shuji2,GOTO, Yuji1, KUBOTA, Souichirou1 (1Lab. Molec.Genet. and CytoGenet., Dept. Biol., Faclt. Sci., TohoUniv., 2Dept. Systems BioMed., Natl. Res. Inst. forChild Health and Dev.)

Small RNA profiling and detection of piRNA from testiculargerm cells in the inshore hagfish

It is known that chromosome elimination occurs during earlyembryogenesis in eight hagfishes. Previous studies indicated thechromosomes restricted to germ cells in these hagfishes aregenerally heterochromatic and mosaics of various highly repet-itive DNA. Recently, Piwi-interacting RNAs (piRNAs), a class ofsmall RNA of 24-30 nucleotides long that are predominantlytranscribed in germ cells of broad species from arthropods tomammals, were involved in heterochromatinization with hetero-chromatin protein 1 in Drosophila. Thus we profiled the smallRNAs expressed in testis and liver of inshore hagfish, Eptatretusburgeri using next generation sequencer. We obtained 91,000unique sequences of 26-30 nucleotide small RNAs. Most of thoseshowed the several features of piRNAs, although they had noobvious homology with known small RNAs. By semi-quantitativeRT-PCR analysis using testis and liver, 16 of top 20 piRNAcandidates were exclusively transcribed in testis. Some of thesecandidates showed partial homology to germline-restricted highlyrepetitive sequences, implying piRNA-mediated heterochromati-nization of the chromatin harboring those repetitive sequences inthe hagfishes.

2D-06

KITANO, Shohei1,2, AIZAWA, Yasunori3,4 (1Dept. LifeSci., 2Educ. Academy of Computational Life Sci., TokyoInst. Technol., 3Grad. Sch. Life Sci. and Technol.,Tokyo Inst. Tech., 4Cent. Biol. Resources and Inform.)

Functional analysis on a highly conserved and upstreamORF-encoded protein in mice

Mammalian mRNAs are believed to be monocistronic for decades.Recent proteomic studies however revealed that thousands ofopen reading frames located on the 5’ untranslated regions(upstream ORFs, uORFs) are translated in vivo. However, nouORF that encodes bioactive protein has been discovered yet. Inthis study, we discovered an uORF-derived small protein that ishighly conserved among vertebrates and specifically expresses inbrain. In this presentation, we will discuss functionality of thisuORF-derived protein by showing the latest data on our mousemodel.


2D-07

NAGAO, Michiaki1, TSUCHIYA, Fumina2,MOTOHASHI, Reiko3, ABO, Tatsuhiko4,5 (1Grad.Sch. Sci. and Tech., Okayama Univ., 2Dept. Biol. Env.Sci., Faclt. Agr., Shizuoka Univ., 3Lab. MolecularBreeding, Coll. of Agric., Shizuoka Univ., 4Grad. Sch.Sci. and Tech., Okayama Univ., 5Dept. Biol., Faclt.Sci., Okayama Univ.)

Release factor homologs of Arabidopsis thaliana

Non-stop mRNAs, which lack in-frame stop codon, frequentlyoccur in cells. When it is translated, ribosome stalls at the end ofit to form non-productive translation complex (NTC). Accumu-lation of NTCs is supposed to be deleterious, and bacteria areknown to have at least one of three ribosome rescue factors,tmRNA, ArfA or ArfB, to dissolve them. Among these, ArfB is ahomolog of class I release factors. Chloroplasts and mitochondriaare thought to originate in bacteria, and have bacteria-liketranslation system. While no functional tmRNA or ArfA homologshave been so far reported in both organelles, ICT1, humanmitochondrial ArfB homolog, is essential for human cells,suggesting that ArfB homolog is the primary ribosome rescuefactor in organelle. Here, we surveyed ArfB homologs fromArabidopsis thaliana genome. One of the RF homologs surveyedis highly homologous to ArfB and ICT1 and has their character-istic features such as GGQ motif and unstructured C terminalregion rich in basic amino acids. Moreover, it showed ribosomerescue activity in Escherichia coli cell. These suggest that itfunctions as the ribosome rescue factor in plant organelles.

2D-08

KUTSUNA, Shinsuke1, SEGAWA, Yuki1, KOIKE,Anna1, MUROYA, Mitsuhiko1, ONAI, Kiyoshi2,ISHIURA, Masahiro2, MANABE, Katsushi1 (1BionanoSystem Sci., Yokohama City Univ., 2Gene Res.,Nagoya Univ.)

Analysis of flower movement mutants and seedling clockmutants

Previous reports indicates that the circadian clock regulates thediurnal petal movement in several plant species, such as morningglory and kalanchoe. However, the relationship between the clockand the diurnal movement is remained to be determined atMolec. Genet. level. In Arabidopsis thaliana, we had isolatedseveral flower movement mutants named as Y-shaped curvatureat flower closure. These show low amplitude flower opening andclosure. Here, we will report that the flower movement mutantsycu1, 2, and 3 exhibit several circadian phenotypes undercontinuous light condition. The first, their seedlings shows lossof sustainability of the petiol circadian rhythm. In addition, geneexpression profiles in flower organ in the mutants lose circadianrhythmisity. These results suggest that deep linkage between themolecular circadian gene expression and diurnal movements inthe plant. In this meeting, we would like to discuss about theclock gene expressions and the movements.

2D-09

NISHIZAKI, Eri1, MORITA, Akitsugu1, YAMAGUTI,Akimitsu1, KITAYAMA, Yoko2, OYAMA, Tokitaka3,KONDO, Takao2, KUTSUNA, Shinsuke1 (1Grad. Sch.NanoBiosci., Yokohama City Univ., 2Div. BiologicalSci., Grad. Sch. Sci., Nagoya Univ., 3Div. Biol. Sci.,Grad. Sch. Sci., Kyoto Univ.)

Spatiotemporal analysis of circadian clock protein homolog inthe cyanobacterium Prochlorococcus marinus str. NATL1A

Many organisms on the earth has circadian clock of about 24-hperiod. They control their biological activities by circadian clockand fit a day round environmental change. Cyanobacteria arephotosynthetic prokaryotes known to have a circadian clock.Synechococcus elongatus PCC 7942 is the kind of cyanobacteriafor which most bacterial circadian research has been conducted.Many studies of them show that thier circadian clock consists ofthree clock protein, KaiA, KaiB, and KaiC. Marine cyanobacteria,Prochlorococcus marinus str. NATL1A (hereafter P. marinus),used in this research. They are the most abundant photosyntheticorganisms on Earth. They lost KaiA in the process of theevolution. Homologs of kaiB and kaiC is maintained in thegenome of many kinds of Prochlorococcus, but it’s still unclearwhether P. marinus has the circadian rhythm. In this study, weanalyze the spatiotemporal expression of P. marinus KaiB andKaiC proteins by Western blot. As a result, it is suggested thatboth KaiB and KaiC expression place in the cell moves in a day.

2D-10

KOBAYASHI, Yuto1, RYO, Masashi1, OSHIKOSHI,Yuta1, IDEGUCHI, Yamato1, KUWANO, Takashi1,TEZUKA, Takafumi1, AOKI, Setsuyuki1 (1TheComplex Systems Dept., Grad. Sch. Inform. Sci.,Nagoya Univ., 2Sch. Inform. and Sci.s, Nagoya Univ.)

Bioluminescence bursts in Escherichia coli luciferase report-er strains

We introduced the firefly luciferase gene as a transcriptionalreporter into an Escherichia coli WT strain. All the resultingreporter strains exhibited an abrupt and large increase ofbioluminescence immediately before the onset of the stationarygrowth phase. This “bioluminescence burst” was not promoter-specific and did not correlate with changes in the levels ofluciferase mRNAs or luciferase proteins. Therefore, the bio-luminescence burst is generated by reflecting changes in a post-translational process(es). We are currently studying the changesin some metabolites and bioluminescence profiles of some gene-knockout reporter strains, which lack genes potentially involvedin the bioluminescence reaction by the luciferase protein.


2E-01

SONODA, Satoru1, OHTA, Akane1, MARUO, Ayana1,UJISAWA, Tomoyo1, KUHARA, Atsushi1 (1Lab. Mol.and Cell. Regul., Faclt. Sci. Eng., & Inst. IntegrNeurobiol, Konan Univ.)

Feedback system between sperm and temperature sensing-neuron in cold acclimation of C. elgans

Temperature acclimation in animals is important mechanism forsurvival and proliferation. To investigate temperature acclima-tion, we are using cold acclimation in Caenorhabditis elegans. 20˚C-cultivated animals did not survival at 2˚C. In contrast, 15˚Ccultivated animals can survive. Previously reported, this coldacclimation is regulated by ASJ temperature sensing-neuron,which releases insulin that is received by intestine and neuron(Ohta, Ujisawa et al., Nature commun, 2014). To identify genes indownstream of insulin-signaling, we performed DNA microarrayanalysis. We found that expressions of sperm genes were changedin insulin receptor mutants, and sperm mutants showedabnormal cold acclimation. Unexpectedly, genetic epistasisanalysis suggested that abnormal cold acclimation of spermmutant was suppressed by the mutations in ASJ temperaturesensing-neuron. Calcium imaging analysis showed that ASJneuronal activity in response to temperature was decreased insperm mutant gsp-4, and rescued by expressing gsp-4 gene insperm. Thus, we propose a novel feedback between sperm andASJ temperature sensing-neuron in cold acclimation of C. elgans.

2E-02

OKAHATA, Misaki1, OHTA, Akane2, MINAKUCHI,Yohei3, TOYODA, Atsushi3, KUHARA, Atsushi2

(1Dept. Biol., Grad. Sch. Natural Sci., Konan Univ.,2Inst. Integr Neurobiol, Konan Univ., 3Natl. Inst.Genet.)

Genetic analysis of natural variation and potassium channelin cold acclimation

Temperature is important environmental factor for animals. Weare studying cold acclimation of natural variants in C. elegans.AB1 from Australia can rapidly acclimate to new temperature,whereas CB4856 from Hawaii can slowly acclimate. To identifythe responsible gene polymorphism, we decoded genome sequenceof three natural variants including AB1. We made manyrecombinants by crossing AB1 with CB4856. We performedSNP and phenotype analysis of these RI lines. Responsiblepolymorphism was mapped between 1.1 and 5.1cM. We are goingon another study to identify new genes involved in coldacclimation, by using the information of previous DNA micro-array analysis. So far, mutant defective in kqt-2 encodingpotassium channel showed abnormal cold acclimation. Theexpression of KQT-2 was observed in a pair of head sensoryneurons and intestine.

2E-03

IOROI, Makoto1, FUJII, Satoko2, INOUE, Tomoka2,KUHARA, Atsushi2, OHTA, Akane2 (1Dept. Biol.,Grad. Sch. Natural Sci., Konan Univ., 2Inst. Integr.Neurobiol., Konan Univ.)

CRH-1/CREB and NCS-1 regulate cold acclimation speed inC. elegans

Temperature acclimation is important for life and proliferation ofanimals. C. elegans changes their cold tolerant status dependingon previous ambient temperature. We found that the coldtolerant status is overwritten in only three hours through CRH-1/CREB in neurons. We call this phenomenon as a coldacclimation. Our calcium imaging implies that ASJ temperaturesensoryneuron itself alters temperature response depending oncultivation temperature, which is modulated by CREB. In orderto identify the site of action of CREB in cold acclimation, weexpressed CREB cDNA in a various sets of neurons in CREBmutant. As a result, abnormal delay of cold acclimation in CREBmutant was rescued by the expression of CREB cDNA in bothASJ sensoryneuron and RMG interneuron. Recently, we foundthat ncs-1 mutant defective in Neuronal Calcium Sensor (NCS)showed abnormal delay of cold acclimation. NCS-1 is expressed inthirteen head neurons including RMG interneuron. Geneticepistasis analysis indicates that CRH-1/CREB and NCS-1accelerate cold acclimation in the different pathways. We arenow trying to identify whether function of NCS-1 in RMG isnecessary for cold acclimation.

2E-04

TAKAGAKI, Natsune1, OHTA, Akane2, MINAKUCHI,Yohei3, TOYODA, Atsushi3, KUHARA, Atsushi2

(1Dept. Biol., Grad. Sch. Natural Sci., Konan Univ.,2Inst. Integr. Neurobiol, Konan Univ., 3Natl. Inst.Genet.)

Xanthine metabolism regulates cold acclimation of C. elegans

We are aiming at the elucidation of temperature acclimationmechanism. Wild-type animals can survive at 2 degree aftercultivation at 15 degree, however, 25 degree-cultivated animalscan not survive at 2 degree. We found that a novel mutation,chr1, causes abnormal cold acclimation. To identify responsiblegene of this mutation, we performed SNP analysis by using nextgeneration sequencer. As a result, F55B11.1 gene encodingXanthine dehydrogenase (XDH) was the responsible gene fordefective cold acclimation, and F55B11.1(ok3234) null mutantshowed defect in cold acclimation. Defective cold acclimation ofF55B11.1(ok3234) mutants were rescued by F55B11.1 genomicgene. Furthermore, F55B11.1::VENUS reporter gene was ex-pressed in socket glial cells in the head (OLQso), excretory celland intestine. We are now introducing a series of cell-specificrescue experiments. Since Xanthine dehydrogenase (XDH) isknown to produce uric acid that is an antioxidant of xanthine,F55B11.1 mutant probably reduces capacity to detoxify ROS. Wetherefore hypothesizes that cell membranes are damaged and celldeath increases, so F55B11.1 mutant might become sensitive tocold stimulation.


2E-05

ARAI, Mary1, INOUE, Akitoshi1, ISHIHARA,Takeshi1 (1Dept. Biol., Faclt. Sci., Kyushu Univ.)

Analyses of the regulation of forgetting by the food signals inthe olfactory learning of C. elegans

In C. elegans, TIR-1/JNK-1 pathway in AWC sensory neuronsaccelerates the forgetting of the adaptation to diacetyl, which issensed by AWA sensory neurons. In wild-type animals, theadaptation is sustained for less than 4 hours on food afteradaptation, whereas, in tir-1 loss-of-function mutant, the adap-tation persists more than a day in the same condition. However,when, after conditioned with diacetyl, animals are incubatedwithout food, both wild-type and tir-1 (lf) animals are recoveredfrom the adaptation within 4 hours. Therefore, we postulatedthat food signals, which are inhibited by TIR-1/JNK-1 pathway inAWC, inhibit forgetting. To elucidate the mechanisms for theregulation of the forgetting by the food signals, we carried out twodifferent screenings from tir-1 (lf). In one screening, we searchedfor the mutants showing the normal forgetting even whenanimals were cultivated on food after conditioning. In anotherscreening, we searched for the mutants showing the prolongedadaptation even when animals were incubated without food.From these two screenings, we isolated 10 mutant lines ascandidates.

2E-06

HINO, Takahiro1, FUJIWARA, Manabi2, ISHIHARA,Takeshi2 (1Grad. Sch. Systems Life Sci.s, KyushuUniv., 2Faclt. Sci., Dept. Biol., Kyushu Univ.)

The analysis of the developmental changes in the chemotaxisof C. elegans

Many animal species change their behavioral patterns duringgrowth to behave appropriately for their developmental stage.However, the molecular mechanisms which give rise to thebehavioral changes depending on the development are unclear.Here we explore the mechanism of the behavioral changes duringthe development with C. elegans. We have shown that C. eleganslarvae show different chemotactic behavior from adults.To analyze the mechanism which regulates such a developmentalchange, firstly we developed a new chemotaxis assay paradigmwhich is suitable for larvae. With this assay paradigm, weconfirmed that larvae show different chemotactic behavior fromadults. By analyzing the correlation between the chemotaxis andthe moving speed, we confirmed the chemotactic difference is notdue to their difference of moving speed. Furthermore, to revealthe molecular mechanism, we screened for mutants, whose larvaeshow the chemotactic behavior like adults, and isolated onecandidate mutant. To analyze the role of the gene responsible forthe phenotype, we have tried to identify this gene. We hope touncover the molecular mechanism which regulates behaviorsdepending on the development.

2E-07

MURAKAMI, Hinata1, TANAKA, Ryoya1, HIGUCHI,Tomohiro1, ITO, Hiroki1, SATO, Kosei1, YAMAMOTO,Daisuke1 (1Div. NeuroGenet., Dept. Dev. Biol.NeuroSci. Grad. Sch. Life Sci., Tohoku Univ.)

Attempts at producing genetic tools for the analysis ofDrosophila subobscura-specfic courtship behaviors

In two species belonging to the genus Drosophila, Drosophilasubobscura exhibits a particularly exotic behavior; a male giveshis digests to a potential mate as a “nuptial gift”, a behavior notobserved in D. melanogaster. For the understanding of the neuralmechanism underlying such a unique component in courtshipbehavior, we attempt to generate transgenic D. subobscura flies,in which neuronal cluster-specific expression of the thermosensi-tive dTrpA1 channels can be induced with GAL4/UAS and FLP/FRT systems. In D. melanogaster, the fruitless (fru) gene isconsidered as the master regulator of the male courtship circuitryformation. On the assumption that fru plays a similar role in D.subobscura, we are generating several transgenic flies in D.subobscura that include: fru-GAL4, UAS-dTrpA1, UAS>-stop>dTrpA1 and hs-FLP. We have succeeded in generating thefruitless-GAL4 line by means of the CRISPR/Cas9 system. Inaddition, we already have multiple attP lines in D. subobscura, sothat UAS-dTrpA1, UAS>stop>dTrpA1 and hs-FLP can beintegrated individually into an attP docking site.

2E-08

UEYAMA, Morio1,2, ISHIGURO, Taro2,3,4, GENDRON,Tania F.4, FUJIKAKE, Nobuhiro2, KONNO, Takuya5,KOYAMA, Akihide5, ONODERA, Osamu5,ISHIKAWA, Kinya3, WADA, Keiji2, PETRUCELLI,Leonard4, NAGAI, Yoshitaka1,2 (1Dept.Neurotherapeutics, Osaka Univ. Grad. Sch. Med.,2Dept. Degenerative Neurological Diseases, Natl. Inst.NeuroSci., Natl. Cent. Neurol and Psychiatry, 3Dept.Neurol and Neurol. Sci., Grad. Sch. Med. and DentalSci, Tokyo Med. and Dental Univ., 4Dept. NeuroSci.,Mayo Clinic, 5Dept. Neurol., Clinical NeuroSci.Branch, Brain Res. Inst., Niigata Univ.)

Toxicity of dipeptide repeat proteins in C9 ALS/FTD model fly

An abnormal expansion of G4C2 repeat within the C9ORF72gene has been found to be the most common genetic mutation foramyotrophic lateral sclerosis (ALS)/ frontotemporal dementia(FTD). In the brain of C9 ALS/FTD patients, the expanded repeatRNA transcribed from mutated gene leads to the formation ofRNA aggregates, namely RNA foci, and is also translated intodipeptide repeat (DPR) proteins by the repeat associated non-ATG (RAN) translation. Although these RNA foci and/or DPRproteins are involved in the neurodegeneration, the molecularmechanisms leading to ALS/FTD pathogenesis remain unclear.To dissect the pathogenic roles of expanded repeat RNA and DPRproteins, we established novel Drosophila models of ALS/FTDexpressing both expanded G4C2 repeat RNA and DPR proteins,or expressing only expanded G4C2 repeat RNA. We found thatflies expressing both expanded repeat RNA and DPR proteins inthe eye or in neurons showed severe rough eye phenotype ormotor dysfunction, respectively, while flies expressing onlyexpanded repeat RNA exhibited normal eye or normal motorfunction, respectively. These results indicate that DPR proteinsmainly contribute to the neurodegeneration.


2E-09

LEON, Julio1, MORIOKA, Noriko1, SHENG, Zijing1,NAKABEPPU, Yusaku1 (1Div. NeurofunctionalGenomics, Med. Inst. Bioregulation, Kyushu Univ.)

Mechanisms of Neurodegeneration induced by a DNA repairenzyme, MUTYH

We have shown that 8-oxoguanine accumulation in nuclear ormitochondrial DNA in brains under oxidative conditions is highlyassociated with neurodegeneration, and that MTH1 (8-oxo-dGTPase) and OGG1 (8-oxoguanine DNA glycosylase) protectbrains by preventing the 8-oxoguanine accumulation, whileMUTYH (adenine DNA glycosylase) accelerates neurodegenera-tion. Isolated neurons from wild-type and MTH1/OGG1-deficientmice exhibited efficient neuritogenesis in the presence ofantioxidants, however, in the absence of antioxidants MTH1/OGG1-deficient neurons increased 8-oxoguanine accumulation intheir mitochondrial DNA, and exhibited significantly poorneuritogenesis, indicating that MUTYH induces neuronal dys-function when 8-oxogianine accumulated in mitochondrial DNAin neurons. Oxidative stress markedly activated microgliaisolated from wild-type but not MUTYH-deficient brains, andonly the former significantly induced neuronal death in co-culture experiments. These results indicate that MUTYH inducesneuronal dysfunction and microglial activation, independently,and thus accelerates neurodegeneration.

2E-10

OKADA, Norihiro1 (1Found. Adv. InterNatl. Sci.,2National Cheng Kung Univ.)

What is fighting? Analysis of its neurogenomic state

We are going to elucidate what the fighting is from a viewpoint ofmolecular biology by using so-called fighting fish (Betta splen-dens). In this case, we mean the fighting between the samespecies, but not between different species. The fighting betweenthe same species is territorial behavior and is believed to beconserved during evolution. The advantage of the fighting fish isthat the fighting continues for more than one hound sometimesup to 2 hours, facilitating us to analyze the transcriptome offighting. Accordingly, in the case of fighting of fighting fish, thenew transcriptome that characterizes fighting appears duringfighting, which is so-called the neurogenomic state of fighting.According to our preliminary analysis of RNA-Seq of thetranscriptome, there are many transcripts that show the differ-ence by 4 folds expression between before fighting and duringfighting. About after 20 mins of fighting, IEGs (immediate EarlyGenes) expression can be observed. These expressions arebelieved to be needed for consolidation of memory. Therefore,this is a very good system to analyze what kind of geneexpressions are required to consolidation of memory of fighting.

3A-01

HAYASHI, Masafumi1, KEYAMURA, Kenji1,HISHIDA, Takashi1 (1Dept. Life Sci., Grad. Sch. Sci.,Gakushuin Univ.)

Identification of phosphorylation-dependent regulatorymechanism for DNA damage tolerance in Saccharomycescerevisiae

DNA Damage Tolerance (DDT) pathway, comprises many of theubiquitin conjugation factor, promotes mono- and polyubiquiti-nation of proliferating-cell nuclear antigen, which facilitatesbypass of lesions by replicating across the damaged templatethrough the error-prone DNA polymerases or using the undam-aged sister chromatid as a template. These ensure the completionof replication without removing the lesions, therefore enablingcells to repair them at a later time. Thus, DDT pathway plays animportant role in the maintenance of genome integrity duringreplication. In this study, we expressed epitope-tagged DDTproteins and examined phosphorylation status of them in thepresence or absence of DNA damage. Using Phos-tag SDS-PAGE,we found that Rad5 was detected as at least two distinct bands,and slower-migrating bands were disappeared after treatmentwith lambda phosphatase. We also showed that Rad5 phosphor-ylation was not enhanced by DNA damage, but become moreprominent in the S/G2/M phase cells than in G1 cells. Theseresults suggest that Rad5 is phosphorylated in cell-cycle depend-ent manner, although a mechanism that is at present unclear.

3A-02

MASUDA, Yuji1,2, KANAO, Rie1, MASUTANI,Chikahide1 (1Dept. Genome Dynamics, Res. Inst. ofEnv. Medicine, Nagoya Univ., 2Dept. Toxicogenomics,Nagoya Univ. Grad. Sch. Med.)

Biochemical analysis of HLTF, a human homologue of SWI/SNF-related ubiquitin ligase RAD5

DNA is constantly damaged by a wide variety of exogenous andendogenous agents and most of such DNA lesions inhibit DNAsynthesis. To cope with such problems during replication, cellshave molecular mechanisms to continue DNA replication in thepresence of DNA lesions. Such DNA damage tolerance mecha-nisms are divided into two pathways, translesion DNA synthesisand template switch (TS), and they are activated by mono- andpoly-ubiquitination of PCNA, respectively. In yeast, RAD5, aubiquitin ligase, plays a role for the poly-ubiquitination of PCNA,and it has been believed that HLTF, a human homologue ofRAD5, has the equivalent function in humans. However,molecular mechanisms for the stimulation of RAD5/HLTF atthe damage site under the DNA replication remain obscure. Wefound previously that the ligase activity of HLTF is DNA-dependent. Here, we report biochemical data about the ligaseactivity of HLTF and discuss regulatory mechanisms of HLTF.


3A-03

NISHIDA, Hirokazu1,2, TAKUYA, Yoda3, TANABE,Maiko2, TSUJI, Toshiyuki4, YODA, Takao4, SHIRAI,Tsuyoshi4, TAKEYAMA, Haruko3, ISHINO,Yoshizumi5 (1New Energy and Ind. Tech. Dev.Organization, 2Hitachi, Ltd. Res. & Dev. Group, 3Dept.Life Sci. and Med. Biosci., Waseda Univ., 4Dept. Comp.BioSci., Nagahama Inst. Bio-Sci. Tech., 5Dept. BioSci.and BioTech., Faclt. Agr., Kyushu Univ.)

Exonuclease processivity of archaeal replicative DNA poly-merase in association with PCNA is expedited by mismatchesin DNA

Replicative DNA polymerase comprises two independent do-mains, Polymerase (DNA-synthesis) and Exonuclease (DNA-correction (degradation)) domains, and switches the DNAinteracting domain by altering the relative arrangement toPCNA molecule.In the two DNA polymerase/PCNA/DNA complex structures inpolymerase and exonuclease modes, PCNA seems to hold theDNA substrate, whereas the DNA polymerase dynamically altersits relative arrangement to the others. In this study, weconstructed several alanine-substituted mutants of the basicresidues inside the PCNA ring presumably responsible for theDNA interaction, and examined the polymerase and exonucleasereactions performed by DNA polymerase with PCNA. As a result,the alanine-substituted mutants of PCNA exhibited the sub-stantial enhancement only in the exonuclease reaction. On theother hand, when the mismatch-induced DNA were employed forthe substrates, in which the PCNA-DNA interactions are notexpected to be retained, the exonuclease reaction was facilitated.Taken together, PCNA-DNA interaction formed between thebasic residues inside PCNA and normal dsDNA may act as thesuppressor of the exonuclease reaction.

3A-04

TAKATO, Yokoi1, KOJIMA, Yuna1, TANAKA,Shuitsu1, HATAKEYAMA, Shin1 (1Lab. Genet., Grad.Sch. Sci. Eng., Saitama Univ.)

Function of Neurospora crassa MSH1 in the maintenance ofmitochondria DNA

The MutS protein, which recognizes the misincorporated nucleo-tide in the newly synthesized DNA strand in E. coli, is also foundin eukaryotic cells as MSH (MutS homolog) proteins, MSH1 toMSH6. Among these, MSH1 is conserved exclusively in fungi, sothis protein implied to have unique role in fungal cell. Knockoutof the msh1 gene in Neurospora caused severe phenotypes, suchas early cease of hyphal growth, and showing mutagen sensitivityand deletion of mitochondrial DNA (mtDNA). By fusing with theGFP, MSH1 protein revealed its localization in mitochondria.Further, endogenic expression of this protein without mitochon-drial localization signal, which found in the N-terminus ofNeurospora MSH1 polypeptide, showed similar phenotypes tothat of the msh1 knock out mutant. These results indicate thatthe MSH1 protein may function in the maintenance of mtDNA,which is indispensable to sustain mitochondrial function one ofwhich correlates to energy supply for the elongation of hyphal tip.

3A-05

MIYAJI, Masahiro1, KATO, Yuichi1, HAYASHI,Yuichiro1, ZHANG-AKIYAMA, Qiu-mei1 (1Div. Biol.Sci., Grad. Sch. Sci., Kyoto Univ.)

Phenotypic analysis of base excision repair deficient nem-atode

To be submitted later

3A-06

TAKANO, Noriko1, OHNO, Mizuki1, SASAKI,Fumiko1, NAKATSU, Yoshimichi1, TSUZUKI,Teruhisa1 (1Dept. Med. Biophys. and Radiat. Biol.,Faclt. Med. Sci., Kyushu Univ.)

Mismatch DNA repair system effectively maintain theintegrity of genome in somatic and germline cells

Mismatch DNA repair (MMR) system is required for the accurateDNA replication. Deficiency of MMR leads to a hyper mutatorphenotype and consequently increases a tumor incidence. MMR-defect is well known as the cause of the hereditary colorectalcancer in human.To clarify the role of MMR in mammalian germline mutationrate, we analyzed de novo germline mutations by whole exomesequencing using parent-child-trio sample ofMsh2 deficient mice.We found an increased incidence of indel mutations of 1-2 units intandem repeats (TR) in addition to single base substitutionmutations. These results indicate that MMR play an importantrole in the maintenance of genome integrity of germline cells inmice. It suggests the efficiency of MMR directly influences to therate of genetic diversity.


3A-07

HAYASHIDA, Genki1, NAKATSU, Yoshimichi2,HIDAKA, Kyoko3, FUJIKANE, Ryosuke4, HIDAKA,Masumi4, TSURIMOTO, Toshiki5, TSUZUKI,Teruhisa2 (1Grad. Sch. of Systems of Life Sci.s,Kyushu Univ., 2Dept. Med. Biophys. and Radiat. Biol.,Faclt. Med. Sci., Kyushu Univ., 3Cent. FundamentalEduc., KitaKyushu Univ., 4Dept. Odontology,Fukuoka Dental Coll., 5Dept. Biol., Faclt. Sci.s,Kyushu Univ.)

Isolation of mismatch repair factor MSH2 variants found inLynch syndrome with CRISPR/Cas9 system

DNA mismatch repair (MMR) system maintains genome integ-rity by correcting replication errors. MMR is also known toinvolve in apoptotic responses to DNA damaging agents. Germ-line mutations in MMR genes cause hereditary nonpolyposiscolorectal cancer (Lynch syndrome). The somatic mutations alsocause sporadic colorectal cancers. In mice, a mutation in ATPasedomain, G674A, is reported to cause deficiency in MMR but not inapoptotic induction, suggesting that the induction of apoptosis isindependent from the repair reaction. We developed a newsystem to characterize MSH2 variants found in Lynch syndromepatients using the Epstein-Barr virus-based expression vector.Exogenously expressed G674A mutant protein (G674A) success-fully interacted with its endogenous partner, MSH6. Theresulting mutant MutS? could bind DNA with G/T mismatch,but failed to form a sliding clamp on DNA. Contrary to the resultsof G674A knock-in mutant mice, HeLa cells expressing G674Awere deficient in apoptosis induction. In order to confirm theseresults, we isolated mutant HeLa cell lines harboring G674mutations using genome editing with CRISPR/Cas9 system, andcurrently characterize these cell lines.

3A-08

SHIOIRI, Takuma1, KEYAMURA, Kenji1, HISHIDA,Takashi1 (1Dept. Life Sci., Grad. Sch. Sci., GakushuinUniv.)

Mechanism of cellular tolerance to chronic low-dose UVirradiation in nucleotide excision repair-deficient cells inSaccharomyces cerevisiae

Genome DNA is constantly damage by exogenous and endoge-nous DNA damaging agents. The major source of exogenous DNAdamage is ultraviolet (UV) radiation present in sunlight. Itcauses replication-blocking lesions such as pyrimidine dimers,which are primarily repaired by the nucleotide excision repair(NER) pathway. Our previous studies demonstrated that whilechronic low-dose UV light (CLUV) does not impair the growth ofNER-deficient rad14D yeast cells during 16 hours, despite theobservation that DNA lesions continued to accumulate withCLUV exposure. In this study, rad14D cells were grown forseveral days by diluting the cultures into fresh medium every 24hours under CLUV irradiation. The result shows that long-termexposure to CLUV induces homologous recombination to conferCLUV-resistant phenotype in rad14D cells. These results suggestthat the homologous recombination pathway is important togenomic integrity and survival in mitotically growing cell whenirreparable DNA damages are accumulated.

3A-09

FUNAKOSHI, Masafumi1, MATSUDA, Ryousuke1,ZHANG-AKIYAMA, Qiu-Mei1 (1Stress Response Biol.,Div. Biol. Sci., Grad. Sch. Sci., Kyoto Univ.)

The role of Ciona intestinalis’ AP endonuclease, CiAPEX1,CiAPEX2 and CiP0 in AP site repair

In this study, we focused on Apurinic/apyrimidinic (AP) sites asDNA damages which induce genomic instability. AP sites arewell-known DNA damages, this is because these damages aremost frequently generated in living body and AP sites accumu-lation are known to induce severe effects to organisms. APendonuclease play an important role in AP site repair and threetypes of AP endonuclease have been reported, APEX1, APEX2and P0. APEX1 is the major AP endonuclease and repair almostall the AP sites in mammals. On the other hand, APEX2 and P0have unique characteristics. APEX2 are expected to contribute tothe mitochondrial genome integrity and P0 is a ribosomal protein.However it is not known why organisms have AP endonucleasemore than one and how these AP endonucleases contribute to thegenome integrity. In this study, to solve these problems, wecompare the function of all the AP endonuclease in Cionaintestinalis, which is a model organism in developmental biology.

3A-10

LE, Lan Anh Thi1, MAKI, Hisaji1 (1Lab. Microb.Molec. Genet., Draduate Sch. Biol. Sci.s, Nara Inst.Sci. Tech.)

Oxidative DNA damage is a major cause of spontaneousoccurrence of homologous DNA recombination

Reactive oxygen species are a major cause of spontaneousmutations in aerobic organisms by attacking DNA to producemany kinds of oxidative DNA damages which induce variousmutations and chromosomal aberrations. Recently, our labora-tory found that environmental factors such as nutrient, oxygenconcentration and pH have a significant effect on cellular level ofoxidative DNA damages. However, it was also shown thatspontaneous base substitution mutations are not affected bysuch environmental factors. Here we show that the RecA-dependent chromosomal rearrangements are affected by theenvironmental factors which vary the cellular level of oxidativeDNA damages. In order to investigate spontaneous recombina-tion events, a strain containing two copies of rpsL genes wasconstructed, and allelic recombination events between two rpsLgenes were detemined. The result shows that environemtalfactors promoted spontaneous recombination events throughthe production of oxidative DNA damages.


3A-11

ZHANG, Yingbiao1, MATSUZAKA, Tomoyuki2, YANO, Hirokazu3,NAKANO, Toshiaki4, ISHIKAWA, Ken5, FUKUYO, Masaki6,TAKAHASHI, Noriko7, SUZUKI, Yutaka8, SUGANO, Sumio8,IDE, Hiroshi9, KOBAYASHI, Ichizo10 (1Univ. Tokyo, 2Dept. Math.and Life Sci.s, Grad. Sch. Sci., Hiroshima Univ., 3Dept. Comput.Biol. and Med. Sci. (formerly Dept. Med. Genome Sci.), Grad. Sch.Front. Sci., Univ. of Tokyo, 4Dept. Math. and Life Sci.s, Grad. Sch.Sci., Hiroshima Univ., 5Natl. Cancer Inst., Natl. Inst. Health,6Dept. Mol Oncol., Grad. Sch. Med., Chiba Univ., 7Dept. Comput.Biol. and Med. Sci. (formerly Dept. Med. Genome Sci.), Grad. Sch.Front. Sci.s, Univ. of Tokyo, 8Dept. Comput. Biol. and Med. Sci.(formerly Dept. Med. Genome Sci.), Grad. Sch. Front. Sci.s, Univ.of Tokyo, 9Dept. Comput. Biol. and Med. Sci. (formerly Dept. Med.Genome Sci.s), Grad. Sch. Front. Sci., Univ. Tokyo, 10Dept. Math.and Life Sci.s, Grad. Sch. Sci., Hiroshima Univ., 11Dept. Comput.Biol. and Med. Sci. (formerly Dept. Med. Genome Sci.), Grad. Sch.Front. Sci., Univ. Tokyo)

Restriction glycosylases: involvement of endonuclease activitiesin the restriction process

All the examined restriction enzymes were phosphodiesterasesgenerating 3’ OH and 5’ P ends, but a restriction enzyme (restrictionglycosylase), R.PabI from a hyperthermophile, ascribed the breakageto high temperature while the other report identified its weak APlyase activity generating atypical ends. Here we addressed this issuein mesophiles. We first purified R.PabI homologs from Campylobactercoli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstratedtheir DNA cleavage, DNA glycosylase and AP lyase activities in vitroat 37oC. AP lyase is more coupled with glycosylase in R.CcoLI than inR.PabI. R.CcoLI or R.PabI expression caused restriction of incomingbacteriophage/plasmid DNA and endogenous chromosomal DNAwithin E. coli at 37oC. The R.PabI-mediated restriction was promotedby AP endonuclease action in vivo or in vitro. These results revealedthe role of endonuleolytic DNA breakage in the restriction and yetdiversity in the endonucleases involved. The cleaved ends are difficultto repair in vivo, which may signify their biological significance. Suchdifficulty may be useful in application.

3A-12

HAYASHI, Yuichiro1, QIU-MEI, Zhang-Akiyama1

(1Lab. Stress Response Biol., Grad. Sch. Sci., KyotoUniv.)

The involvement in genomic stability maintenance of KsgA,DNA glycosylase in E.coli, and its human homologs

Base Excision Repair (BER) is a system repairing damaged bases.DNA glycosylase works at the first step in BER. Once, werevealed that KsgA, a dimethyltransferase in E.coli, is a bifunc-tional DNA glycosylase removing cytosine paired with thymine-glycol and 5-formyluracil. We also elucidated that KsgAsuppresses spontaneous mutation frequency. Furthermore, weconfirmed that human homologs of KsgA, DIMT1L and TFB1M,have DNA glycosylase and AP lyase activities and suppressspontaneous mutation frequency as well as KsgA. We researchthe involvement in maintenance of genome stability of KsgA andits human homologs now.

3B-01

YUKUHIRO, Kenji1, SAKAGUCHI, Hiroki2, ITOH,Masanobu2, KOMOTO, Natuo1, TOMITA, Shuichiro1

(1NARO, Inst. AgroBiol. Sci., 2Kyoto Inst. Tech.)

Genetic differentiation between Japanese population of themulberry silkworm, Bombyx mandarina by the biaseddistribution of three SNPs on COI gene

Three out of 135 SNP sites (SNP156, 325 and 666) on COI geneplayed a crucial role in dividing Japanese populations of Bombyxmandarina. In SNP325, three variable forms were observed. Twoof them are highly frequent and distribute biasedly: SNP325T isfixed in populations present in districts west to Hyogo prefecture.Populations in Hyogo as well as two northern Kyoto populationsshow polymorphic state of both forms. In eastern populations,SNP325C were fixed or nearly fixed except for some populationsin Kanto districts. These populations included variable levels ofSNP325T, especially SNP325T is fixed in Kimitsu population.SNP156T is predominated in 10 populations in the northern partof Japan, including Hakodate, Hokkaido to Tsuruoka-Asahi,Yamagata prefecture. High frequencies of SNP666A are limitedlydetected in two populations, Daisen, Tottori prefecture and Gozu-Izumo, Shimane prefecture. SNP666A and SNP666G are segre-gated in two populations, Takahashi-Makabe, Okayama prefec-ture, and Mihara, Hiroshima prefecture. It is notable that thesethree sites are fixed in Bombyx mori, Chinese B. mandarina andB. huttoni.

3B-02

MORIGUCHI, Natsuki1, UCHIYAMA, Kentaro2,MIYAGI, Ryutaro3, TAKAHASHI, Aya3, TAMURA,Koichiro3, TSUMURA, Yoshihiko4, TESHIMA, KosukeM.6, KUSUMI, Junko5, TACHIDA, Hidenori6

(1Evolutionary Genet. Lab., Dept. Systems Life Sci.s,Kyushu Univ., 2Dept. Forest Genet., Forestry ForestProducts Res. Inst., 3Evolutional Genetic Lab., Dept.Biol. Sci.s, Tokyo Metro. Univ., 4Faclt. Life and Env.Sci.s, Univ. Tsukuba, 5Dept. Env. Changes Faclt.Social and Cultural Studies, Kyushu Univ., 6Dept.Biol., Faclt. Sci.s, Kyushu Univ.)

Analysis of population structure of Cryptomeria japonicabased on amplicon sequenced data

Cryptomeria japonica is a coniferous tree species with a broad naturaldistribution in the Japanese archipelago. There are two varietiesdistinguished by their morphological traits: the ura-sugi variety (C.japonica var. radicans) has slender branchlets with soft leaves and ismainly distributed on the Japan Sea side, while the omote-sugivariety (C. japonica) has rough branchlets with hard leaves and isdistributed on the Pacific Ocean side. Because each variety consists oftwo groups of populations, C. japonica comprises 4 groups ofpopulations. In this study, 94 samples from 4 natural populations(Ajigasawa, Bijodaira, Shimowada, Yakushima), each representingone of the 4 groups, were used to determine sequences at 142 nucleargenes using next generation sequencing. Three clusters wereidentified by a PCA analysis, Yakushima, Omote-sugi and Ura-sugiclusters. The phylogenic tree estimated by software TreeMixsuggested the Yakushima population first diverged from the otherpopulations and later the Ura-sugi populations originated from theOmote-sugi population. An application of software BayeScan to the142 genes data revealed no candidate genes under natural selection.


3B-03

KAWAMURA, Sayu1, TESHIMA, Kosuke1 (1Dept.Biol., Faclt. Sci.s, Kyushu Univ.)

Study of population differentiation measures

FST is one of the most widely used measurements to assessgenetic differentiations. Many researchers arranged calculationmethods and proposed relating measures. To understand theproperties of these FST related measures, we conducted coales-cent simulation, and calculated FST-related measures. As geneticmarkers, DNA sequences, SNPs and microsatellites were consid-ered.

Judging from the pattern of the values, the measures wereclassified into three groups. This classification agrees with thegrouping taken into account their definitions: measures dividedby between-population-diversity, divided by total-diversity, andothers. It was also found that measures in the same groupconverged to the same value regardless of marker types as long aswe can use enough markers. We also evaluated the effect of theascertainment bias on the distribution of FST calculated fromSNP typing data. The effects of the ascertainment appeared asthe shift of the distributions, but the direction of the shiftdepends on the types of ascertainment schemes for SNP discoveryprocesses. We should be careful about the calculation method ofmeasures and the ascertainment bias when we use genotypingdata.

3B-04

BESSHO, Kazuhiro1,2, OTTO, Sarah P.3 (1Dept.Evolutionary Studies of Biosyst., Sch. Adv. Sci.,SOKENDAI (Grad. Univ. Adv. Stud.) ShonanVillage,2JSPS Res. Fellow, 3The Univ. of British Columbia)

Fixation probability in a mixed population of gametophytesand sporophytes

Among many terrestrial plants and macroalgae, the haploidgametophyte and the diploid sporophyte alternate throughmeiosis and syngamy (haploid-diploid life cycles). However,classical population genetic theories generally assume either aspecies with fully haploid (haplont) or fully diploid (diplont) lifecycles. To understand the evolution of haploid-diploid organisms,we modeled the fixation probability of selected alleles in apopulation with free-living gametophytes and sporophytes.Analyzing the model by a branching process approximation anddiffusion approximation, we find the fixation probability ofselected alleles analytically, allowing for different fecunditiesand mortalities in the different ploidy stages. Because the neteffect of selection is a key determinant of the fixation probability,a mutation that is deleterious at one ploidy stage but not theother can have a net beneficial effect. When one ploidy phasepredominates in the population, the fixation probability differssubstantially from either haplont or diplont species because of theskew in the effective population size.

3B-05

HARADA, Ko1, DWIYANTI, Fifi Gus1, KAMIYA,Koichi1, SIREGAR, Iskandar1, DEWAY, Bibian1,CHONG, Lucy1 (1Faclt. Agr., Ehime Univ.)

Phylogeography of Dryobalanops aromatica (Dipterocarpa-ceae) in Sundaic region

Two hundreds and twenty three Leaf samples of Dryobalanopsaromatica(Dipterocarpaceae) were collected from 9 populations inthe tropical rain forests of Sundaic region in Southeast Asia.Genetic variation was examined by using 8 microsatellite loci.The Bayesian model based cluster analysis showed the bestnumber of K=2 indicating these populations were grouped intotwo in Sumatra-Malay and Borneo. By using IM (Isolation withMigration) simulation, these groups were found to be separated7200 years ago. It was also shown that the ancestral populationwas twenty times larger than the present populations as a whole.These suggest the tropical rain forest was expanded much largerthan today’s at the last glacial maximum (LGM), which occurredtwenty thousand years ago. Considerable amount of migrationsbetween the two groups also suggests that the hypotheticalsavanna corridor on the center of the exposed Sunda Shelf at theLGM was disconnected by forest belts.

3B-06

YASHIMA, Akiko Sato2, INNAN, Hideki1 (1Dept.Math. Eng., Musashino Univ., 2Dept. EvolutionaryStudies of Biosyst., Sch. Adv. Sci., The Grad. Univ.Adv. Stud. (SOKENDAI))

Genetic Variations of the marine fishes and the freshwaterfishes

Understanding the patterns of genetic variation within andbetween populations is important in ecology, evolution, conser-vation and population genetics. Although tremendous amounts ofeffort have been made to evaluate microsatellite variation innatural populations for over 5,000 species so far, there have beenvery few works that compared multiple species. We analyzedmicrosatellite variation in more than 200 fish species to explorewhat determine the species-wide pattern of genetic variation. Wefound that their migration ability and preference (e.g., freshwatervs marine environment) is the major factor to specify the patternof genetic variation.


3B-07

OKA, Ayako1, SHIROISHI, Toshihiko1 (1MammalianGenet. Lab., Natl. Inst. Genet.)

Polymorphism in the cis-regulatory elements and its impacton the reproductive isolation

Improper gene regulation is implicated in reproductive isolation,but its genetic bases are unknown. We previously reported thatmouse inter-subspecific X chromosome substitution strains (B6-ChrXMSM and B6-XTMSM) show reproductive isolation character-ized by male-specific sterility due to disruption of meiotic entryduring the spermatogenesis. Transcriptome analysis of thesestrains revealed that a gross aberration in gene expression on thesubstituted X chromosome in testis. The misregulation of X-linked genes showed asymmetry; more genes were disproportion-ally downregulated rather than upregulated. This suggests thatgenetic incompatibility between cis-regulatory elements andtrans-acting regulatory factors may occur in the transcriptionalregulation of genes on the substituted X chromosome. To clarifythe genetic basis of cis-trans incompatibility, we identifiedenhancers for Taf7l gene that is a candidate gene responsiblefor the small testes of B6-XTMSM strain. The result from theluciferase assay showed allelic differences of enhancer activities.

3B-08

MATSUMOTO, Yuki1,2,3, NISHINO, Jo4, NAKAOKA,Hirofumi5, GOTO, Tatsuhiko6, KOIDE, Tsuyoshi1,2

(1Grad. Sch. Life Sci., SOKENDAI, 2Mouse GenomicsResource Lab., Natl. Inst. Genet., 3JSPS Res. Fellow,4Grad. Sch. Med., Nagoya Univ., 5Div. Hum. Genet.,Natl. Inst. Genet., 6Sch. Life Sci., The Univ. ofNottingham)

Identification of genetic loci associated with tameness usingselection and association mapping in mice

Tame behavior is one of the major elements in domestication anddefined as increased interaction of animals with human. Toidentify genes associated with active tameness which is definedas contacting human hand (contacting), we performed selectivebreeding for contacting using wild-derived heterogeneous stock(WHS), which is a mixed population derived from 8 wild mousestrains originated in various geographic regions. Following theselective breeding, we used selection mapping as well asassociation mapping and found two candidate loci on Chromo-some 11 potentially associated with contacting.

3B-09

GONDO, Yoichi1, FUKUMURA, Ryutaro1,ISHITSUKA, Yuichi1, MAKINO, Shigeru1, MORI,Kazuki2, KUHARA, Satoru2, TOYODA, Atsushi3,FUJIYAMA, Asao3 (1RIKEN BioRes. Cent., 2Faclt.Agr., Kyushu Univ., 3Cent. Inform. Biol., Natl. Inst.Genet.)

Detection and analysis of spontaneous mutations in standardinbred mice: whole genome sequencing (WGS) and singlenucleotide variation (SNV) call

We have accumulated spontaneous mutations in C57BL/6JJclinbred mice for 4 generations by complete outbreeding method.We have generated 8 independent pedigrees, the fourth-gener-ation females of which were subjected to WGS and SNV calls. Themutations were accumulated in the complete outbreeding schemeso that de novo mutations should be found only in one fourth-generation female as heterozygotes. Within non-repetitive ge-nomic sequences with sufficient read coverage, a total of 17,325SNV candidates were primarily called. Among them, 1,874 SNVwere homozygous commonly found in all the eight fourth-generation females; thus, they are fixed SNPs. The number ofunique heterozygous SNVs found in only one of the 8 fourth-generation females was 2,069 that encompass the de novomutations. Seven hundreds and twenty one candidates wereun-fixed SNPs. Peculiarly, the remaining 12,661 candidates werefound in all the 8 fourth-generation females as heterozygotes.Such a large number of false-positive SNV calls strongly indicatesome problems of the current mouse genome reference sequencelike segmental duplications.

3B-10

FUKUMURA, Ryutaro1, KOTAKI, Hayato1,ISHITSUKA, Yuichi1, MAKINO, Shigeru1, NAKAI,Yuji1, MORI, Kazuki2, KUHARA, Satoru2, TOYODA,Atsushi3, FUJIYAMA, Asao3, GONDO, Yoichi1

(1RIKEN BioRes. Cent., 2Faclt. Agr., Kyushu Univ.,3Cent. Inform. Biol., Natl. Inst. Genet.)

Detection and analysis of spontaneous mutations in standardinbred mice: validation and tracing of SNV (single nucleotidevariation) candidates

We have extracted 2,069 SNV candidates by WGS from eight G5female genomes in which independent spontaneous mutationshad been accumulated for 4 generations (from G1 to G5generation) with no selection to recessive deleterious mutations(Gondo et al. This meeting). We have validated 1,649 SNVcandidates from 6 out of the 8 genomes and 1329 have so far beenexperimentally confirmed to be true mutations. Among them,1074 SNV were also found in a few of the eight G1 mice; thus,they are newly arisen rare SNPs in the breeder’s colony. Theremaining 255 SNV were de novo mutations during the fourgenerations. Roughly three quarters of the mutations occurred onGC pairs. By tracing 23 confirmed de novo mutations in thepedigrees, 6, 8, 6 and 3 were derived from G2, G3, G4 and G5mice, respectively by Mendelian inheritance. It was completed toaccumulate spontaneous mutations for four generations in a yearin the mouse. This mutation analysis scheme is universallyapplicable to any species that proliferate by sexual reproduction.


3B-11

OSADA, Naoki3, MIYAGI, Ryutaro1, TAKAHASHI,Aya1,2 (1Dept. Biol. Sci.s, Tokyo Metro. Univ., 2Res.Cent. Genomics and Bioinform., Tokyo MetropolitanUniv., 3Grad. Sch. Inform. Sci. Tech., Hokkaido Univ.)

Genome-wide analysis of cis- and trans-regulatory effects ongene expression using allele samples from a natural pop-ulation of Drosophila melanogaster

Quantifying the relative contributions of cis- and trans-regu-latory variations on intraspecific gene expression is an essentialstep to understanding the evolution of transcriptome. Wequantified the genome-wide allele specific expression (ASE)variations in F1s between 18 different Drosophila melanogasterstrains sampled from the Drosophila Genetic Reference Panel(DGRP) and a reference strain from another population. Headand body samples of the F1 adult females were subjected to RNA-seq and the subsequent ASE quantification. A linear modelapproach revealed that the degree of relative cis-regulatorycontribution on gene expression variation is affected moststrongly by local polymorphism level in both head and bodysamples. The degree of relative trans-regulatory contribution wasstrongly affected by local recombination rate in head and byfemale-bias in expression in body. Analysis of the effects ofproximal transposon element (TE) insertions on ASE revealedthat TE insertions affected transcription levels of ovary-ex-pressed genes more pronouncedly than genes that are notexpressed in ovary.

3B-12

TAKADA, Yasuaki1, TAKAHASHI, Aya2, MIYAGI,Ryutaro2, ENDO, Toshinori1, OSADA, Naoki1 (1Grad.Sch. Inform. Sci. Tech., Hokkaido Univ., 2Dept. Biol.Sci.s, Tokyo Metro. Univ.)

The cause of gene expression changes in reciprocal crosses ofDrosophila melanogaster by measuring allele-specific geneexpression

Elucidating how gene expression is regulated by intercellularsignals is an important subject in genetics. There are three majorfactors affecting the allele-specific gene expression (ASE), cis-regulatory effect, imprinting effect and maternal effect. However,methods to evaluate these three effects has not been establishedbecause the three factors are highly confounding. Here, wepropose a method to evaluate these effects on the ASE anddecomposing these factors. We estimated the ASE from RNA-seqdata obtained from reciprocally-crossed adult fruit flies (Droso-phila melanogaster), and applied the new method. As a result,only the cis-regulatory effect were detected and there was nodetectable imprinting and maternal effect in adult flies. Incontrast, analysis of mouse trophoblast stem cells showed thatthe cis-regulatory effect was the strongest, the imprinting effectwas the next, and the maternal effect was the weakest in mice.

3C-01

NAKAGAWA, Takuro1, ONAKA, Atsushi1,TAKAHASHI, Tatsuro S.1, MASUKATA, Hisao1

(1Dept. Biol. Sci.s, Grad. Sch. Sci., Osaka Univ.)

Rad51 and Rad54 promote noncrossover recombinationbetween centromere repeats to prevent isochromosomeformation

Non-allelic recombination between repetitive DNA elements inthe genome can result in gross chromosomal rearrangements(GCRs). Our previous studies have shown that Rad51 suppressesthe formation of isochromosomes that are produced by recombi-nation between inverted repeats in the centromere that isessential for proper segregation of chromosomes. However, itremains unclear how Rad51 suppresses such homology-mediatedGCRs. Here, we found in fission yeast that Rad51 and Rad54,which binds and regulates Rad51, promote a noncrossover type ofrecombination between centromere repeats, thereby suppressingcrossover recombination that leads to the isochromosome for-mation. Mutations in Rad51 and Rad54 severely reduced non-crossover recombination between inverted repeats in thecentromere, and greatly increased the rate of the isochromosomeformation. Deletion of Mus81 endonuclease that is required forcrossover recombination reduced both crossovers between in-verted repeats in the centromere (i.e. inversions) and isochromo-somes. Thus, we propose that homologous recombinationcatalyzed by Rad51 and Rad54 preferentially promote noncross-over thereby suppressing homology-mediated GCRs.

3C-02

ARAKI, Masatake1, TAKEDA, Iyo1, OHGA,Toshinori1, ETO, Satoshi1, NAKAHARA, Mai1,YOSHINOBU, Kumiko1, ARAKI, Kimi1 (1Inst.Resource Dev. and Anal., Kumamoto Univ.)

Functional analysis of chromosome specific clustered trapregion (CSCT) 13

We developed the Database for the Exchangeable Gene TrapClones (EGTC) (http://egtc.jp). During the annotation of trapclones, we found new genome element CSCT (ChromosomeSpecific Clustered Trap region). There were 39 EGTC clonesmapped in the CSCT region, they were distinguished in CSCT2,CSCT4, CSCT12 or CSCT13. Using CRISPR/Cas9 system,CSCT13 region (1.6 Mbp) was deleted in the mouse ES cells(CSCT KO). We could establish CSCT KO mouse line. Matingbetween heterozygotes gave apparently normal homozygotes.However, mating between homozygotes gave relatively smallnumber of pups. Moreover, CSCT13 KO mice showed compara-tively low rate of homologous recombination during meiosis forthe region corresponding to CSCT13. On the other hand, outsideof this region showed up-regulation of homologous recombinationduring meiosis. This study suggest that CSCT13 might be relatedwith the early embryogenesis and homologous recombinationduring meiosis.


3C-03

OHGA, Toshinori1, TAKEDA, Iyo1, ETO, Satoshi1,NAKAHARA, Mai1, YOSHINOBU, Kumiko1, ARAKI,Kimi1, ARAKI, Masatake1 (1Inst. Resource Dev. andAnal., Kumamoto Univ.)

Establishment of a mouse line deleted CSCT2 region (3.1Mbp)

We found new genome element CSCT (Chromosome SpecificClustered Trap region), and studied CSCT13 region exist inchromosome 13. In the analysis of CSCT13 region, we areobtaining interesting results suggesting the important role in theearly embryogenesis and homologous recombination duringmeiosis. So this time, we focused on the CSCT2 region (presentin chromosome 2) and decided to produce a knockout mouse lineto know if this region has similar function with CSCT13, or not.By using the knock-in vector comprising a left arm (1.7kbp) and aright arm (1.3kbp), and pX459 to express puro transiently, it wasdeleted the entire region (3.1 Mbp). It’s efficiency was approx-imately 15%. We could establish mouse lines from the ES cellsand have started phenotype analysis.

3C-04

MOURI, Kousuke1, SAGAI, Tomoko1, AMANO,Takanori1, SHIROISHI, Toshihiko1 (1Natl. Inst.Genet.)

Enhancer rearrangement causes regulatory change of geneexpression

Genomic structural mutation produces new regulation of geneexpression via rearrangements of cis regulatory elements. Wefound that Hammer toe (Hm), a mouse syndactyly mutant, has a150kb translocation from chr14 to chr5, the upstream of Sonichedgehog (Shh). Though this translocated fragment contained nocoding sequences, BAC transgenic reporter assay showed thisfragment had an enhancer activity in interdigital region. EctopicShh expression in autopod and interdigital region was seen byqPCR.Syndactyly phenotype of Hm was not shown when the trans-location was placed at a cis-position relative to a Shh knockoutallele. It was also not shown when we remove translocatedfragment by CRISPR/Cas9. From these results, we concludedthat translocated fragment is the cause of syndactyly phenotypeby acting as a new enhancer of Shh.An inversion of translocated fragment which was induced bygenome editing enhanced syndactyly phenotype and the level ofectopic Shh expression. On the other hand, Rnf32, which locatesat the opposite side of the translocation from Shh, was reduced itsectopic expression by inversion. Translocated fragment is likelyto change its activity with its orientation.

3C-05

FUJIMOTO, Akihiro1,2, FURUTA, Mayuko2, TOTOKI,Yasushi3, TSUNODA, Tatsuhiko2, KATO, Mamoru3,HIROKI, Yamaue4, KAZUAKI, Chayama5, MIYANO,Satoru6, ABURATANI, Hiroyuki7, SHIBATA,Tatsuhiro3, NAKAGAWA, Hidewaki2 (1Dept. DrugDiscovery Med., Grad. Sch. Med., Kyoto Univ., 2IMS,RIKEN, 3Natl. Cancer Center Res. Inst., 4WakayamaMed. Univ., 5Hiroshima Univ. Sch. Med., 6Inst. Med.Sci., The Univ. Tokyo, 7Res. Cent. Adv. Sci. Tech., TheUniv. Tokyo)

Whole genome mutational landscape and characterization ofnon-coding and structural mutations in liver cancer

Liver cancer, which is most often associated with virus infection,is prevalent worldwide, and its underlying etiology and genomicstructure are heterogeneous. Here we provide a whole-genomelandscape of somatic alterations in 300 liver cancers fromJapanese individuals. Our comprehensive analysis identifiedpoint mutations, structural variations (STVs), and virus integra-tions, in noncoding and coding regions. We discovered recurrentlymutated coding and noncoding regions, such as long intergenicnoncoding RNA genes (NEAT1 and MALAT1), promoters, andregulatory regions. STV analysis found a significant associationwith replication timing and identified known (CDKN2A, CCND1,APC, and TERT) and new (ASH1L, NCOR1, and MACROD2)cancer-related genes that were recurrently affected by STVs,leading to altered expression. These results emphasize the valueof whole-genome sequencing analysis in discovering cancer drivermutations and understanding comprehensive molecular profilesof liver cancer, especially with regard to STVs and noncodingmutations.

3C-06

KRYUKOV, Kirill1, IMANISHI, Tadashi1 (1TokaiUniv. Sch. Med., BioMed. Inform. Lab.)

Human Contamination in Genome Assemblies

Contamination in genome assembly can lead to wrong orconfusing results when using such genome as reference insequence comparison. Although the possibility of bacterialcontamination is well known, the problem of human-originatedcontamination received little attention. In this study we system-atically surveyed 45,735 available genome assemblies for evi-dence of human contamination. We determined lineage specificityof individual genome fragments, and used it to distinguishbetween contamination and conservation. Non-human genomefragments that show high specificity to primates were selected aslikely human originated sequences. We found that 154 genomeassemblies contain fragments that with high confidence originateas contamination from human DNA. We found that majority ofcontaminating human sequences were present in the referencehuman genome assembly for over a decade. We’d like to highlightthe importance of contamination detection when performinggenome assembly. We recommend that existing contaminatedgenomes should be revised to remove contaminated sequences,and that new assemblies should be checked for presence ofhuman DNA before submitting them to public databases.


3C-07

KAMEDA, Masahiro1, TESHIGAWARA, Rika1, SUN,Liang Tso1, CHO, Junkwon1, TADA, Takashi1 (1Dept.Stem Cell Eng., Inst. Front. Med. Sci., Kyoto Univ.)

Effect of Anti-aging Hormone ADIPONECTIN on Survival ofHuman Pluripotent Stem Cell

Human Adiponectin (APN) containing the C1Q and collagendomains is an adipocyte-secreting protein.APN functions as a key player of anti-aging in anti-arterio-sclerosis, and anti-diabetes.Notably, pluripotent and adult stemcells play an important role in anti-aging by maintenance ofhomeostasis through adequate replacement of old tissues withregenerated new tissues.However, it remains unknown whetherAPN is involved in stem cell property. Here, we explored functionof APN in survival of human induced pluripotent stem (iPS) cells.Interestingly, survival rate of iPS cells 48 hours after subculturesignificantly increased by addition of APN in culture medium.Similar results were obtained with different iPS cell lines, anddifferent assay systems.More detailed analysis demonstrated that effect of APN onimprovement of survival rate of iPS cells are obvious in 2-4-cellcolonies, but not more than 8-cell colonies, suggesting that APN isinvolved in survival of iPS cells, prior to establishment of E-cadherin-dependent cell-cell adhesion.

3C-08

KOSAKA, Kengo1, MIURA, Shiroh2, SANO, Ken2,FUJIOKA, Ryuta3, SAITSU, Hirotomo4, TANIWAKI,Takayuki2, YAMAMOTO, Ken5, SHIBATA, Hiroki1

(1Div. Human Molec. Genet., Med. Inst. Bioregulation,Kyushu Univ., 2Div. Respirology, Neurol andRheumatol., Dept. Med., Kurume Univ. Sch. Med.,3Dept. Food and Nutrition, Beppu Univ. Jounior Coll.,4Dept. Med. Chem., Hamamatsu Univ. Sch. Medicine,5Dept. Med. Chem., Kurume Univ. Sch. Med.)

Identification of causal genetic variants associated with aclinically new type of hereditary motor and sensory neuro-pathy by Linkage analysis and Exome sequencing

We previously reported a clinically new type of autosomaldominant disorders of motor and sensory neuropathy withproximal dominancy in the lower extremities, urinary disturb-ance, and paroxysmal dry cough (Miura et al. J Neurol Sci 2008).To identify the causative mutation for the disease, we studied thispedigree consisting of 19 family members including 9 patients in5 generations. On a multipoint linkage analysis, we observed asingle peak of LOD scores on 1p13.3-q23 (LOD = 2.24). The exomesequencing revealed 1,335 patient-specific single nucleotidevariants (SNVs). Through the validation, we confirmed cosegre-gation of 2 candidate SNVs in the pedigree, and through thefunctional predictions of the cosegregated SNVs, we conduced theSNV located in IQGAP3 is highly likely to be the causativevariant for the disease. This SNV is located within 5’ splice siteconsensus sequence in intron 27 in IQGAP3, potentially affectingthe IQGAP3 splicing. The SNV is also located within a non-codingRNA (ENST00000476565). The effect of the SNV is underconsideration through detecting splice variants or examiningthe function of the non-coding RNA.

3C-09

MORIKAWA, Takuya1, MIURA, Shiroh2, FUJIOKA,Ryuta3, KOSAKA, Kengo1, YAMADA, Kohei1,HATTORI, Gohsuke4, MOTOMURA, Manabu5,TANIWAKI, Takayuki2, SHIBATA, Hiroki1 (1Div.Genomics, Med. Inst. Bioregulation, Kyushu Univ.,2Div. Respirol., Neurol, and Rheumatol., Dept. Med.,Kurume Univ. Sch. Med., 3Dept. Food and Nutr.,Beppu Univ. Junior Coll., 4Dept. Neurosurg., KurumeUniv. Sch. Med., 5Dept. Internal Med., NagasakiYurino Hosp.)

Homozygous 4-bp deletion in the DDHD1 gene, resulting thecomplete deletion of DDHD domain, as a causative variant ina SPG28 patient

Spastic paraplegia (SPG) type 28 is an autosomal recessivehereditary SPG caused by mutations in the DDHD1 gene. Weexamined a Japanese 54-years-old male patient with autosomalrecessive SPG. By the exome sequencing analysis, we identified17,248 homozygous nucleotide variants in the patient. Throughthe examination of 47 candidate genes known to be responsiblefor autosomal recessive SPG, we identified a novel homozygous 4-bp deletion c914_917delGTAA,Ser305Ilefs*2 in exon2 of theDDHD1 encoding phosphatidic acid-preferring phospholipaseA1 (PA-PLA1). The mutation is expected to cause a frameshiftgenerating premature stop codon at 3-bp downstream from thedeletion. In consequence, the DDHD domain that is known to becritical for PLA1 activity is completely depleted in the mutatedDDHD1 protein, resulting the practically a null mutation of theDDHD1. By Sanger sequencing, we confirmed that both parentsare heterozygous for the mutation. This variation was notdetected in 474 Japanese control subjects. We conclude that thenovel mutation in DDHD1 is the causative variant for the SPG28patient that is the first record of the disease in Asian population.

3C-10

IKEUCHI, Tatsuro1 (1Formerly, Med. Res. Inst.,Tokyo Med. and Dental Univ.)

High school textbooks of “Seibutsu-kiso (Basis of Biology)”under the MEXT New Course of Study: with special referenceto the new edition after the 2016 examination procedure byMEXT

There have been two main problems to be improved in the highschool “biology” textbooks under the new education guidelinesoffered in 2012 by the Ministry of Education, Culture, Sports, andtechnology (MEXT). One is that the topic pertaining to the“Mendel’s laws of inheritance” was moved from the high schoolsto the junior high school curriculums, and is now not included inhigh school biology textbooks. The second is the insufficientdescription for explaining the mechanism of human heredity.Recently, the new editions of high school textbooks of “Seibutsu-kiso (Basis of Biology)” were presented after the 2016 examina-tion procedure by MEXT. Contrary to our expectation there wereno sufficient improvements about the above mentioned problems.Outlines of the new edition were introduced. It should be againemphasized that the Mendel’s laws of inheritance is fundamentaland indispensable for understanding the modern genetics, andthat a basic knowledge of human heredity is essential for facing itin the “personal genome era” that we are now entering.


3D-01

SEINO, Kenji1, SETO, Yousuke1, TAMURA, Kouitiro1

(1Life Sci., Grad. Sch. Sci. and Eng., TokyoMetropolitan Univ.)

Changes in gut microbiota at fungal infection in theDrosophila melanogaster species group

Microbial resistance of Drosophila species varies among differentspecies as Drosophila adapted to various microbial environmentsspecies by species. For example, D. virilis inhabits fungi richenvironments, whereas D. melanogaster inhabits yeast richenvironments. Accordingly, D. virilis survives longer in a culturewith Penicillium fungi than D. melanogaster does. However,overproduction of antimicrobial peptides that play an importantrole in microbial resistance in gut alters gut microbiota to shortenthe host fly longevity, suggesting that stability of normal gutmicrobiota is responsible for the microbial resistance. In thisstudy, to clarify whether the gut microbiota is responsible for themicrobial resistance, we examined gut microbiota for fourDrosophila species, sequencing 16S ribosomal DNA and asurvival rate with Penicillium sp. As a result, we found thatgut microbiota in all four species was distorted by the ingestion ofPenicillium fungi to decline the survival rate during lava to adult.Therefore, the maintenance of gut microbiota by antimicrobialpeptides is expected to be a critical factor of the survival ofDrosophila species.

3D-02

SETO, Yosuke1, TAMURA, Koichiro1 (1Dept. Biol.Sci.s, Sch. Sci. Eng., Tokyo Metropolitan Univ.)

Comparative gene expression study of antifungal immuneresponse among Drosophila species

Drosophila flies are exposed to infection of various microorgan-isms from their digestive organs, as they feed and breed onfermented fruits and sap, where a variety of fungi propagate.They defend themselves from invading microorganisms withinnate immune system. In this study, we use three Drosophilaspecies which show different food preference. Drosophila mela-nogaster, D. virilis and D. brachynephros utilize fermented fruits,sap and mushrooms, respectively. To clarify the antifungalimmune system responsible for the difference of food preference,we compared gene expression patterns in gut and in fat bodybetween the three species in response to the infection ofPenicillium fungus. As the results, antimicrobial peptides andlysozymes synergistically act against the infected fungi in D.virilis and D. brachynephros unlike the antifungal immuneresponse in the fat body of D. melanogaster. Additionally,expressions of many serine protease genes were up-regulated inboth the infected D. virilis and D. brachynephros. These resultsindicate that the immune system has been substantially differ-entiated between Drosophila species having different ecologicalcharacters.

3D-03

TATSUMOTO, Shoji1, TOYODA, Atsushi4, IMAI,Hiroo5, HIRAI, Hirohisa5, YAMAMORI, Tetsuo6, ISA,Tadashi7, FUJIYAMA, Asao4, GO, Yasuhiro1,2,3

(1Dept. Brain Sci.s, Cent. Novel Sci. Initiatives, Natl.Inst. Natural Sci., 2Dept. System NeuroSci., Natl. Inst.Physiological Sci., 3Dept. Physiol. Sci., Grad. Sch. LifeSci., SOKENDAI (Grad. Univ. Adv. Stud.), 4Comp.Genomics Lab., Natl. Inst. Genet., 5Mol. Biol. Section,Primate Res. Inst., Kyoto Univ., 6Mol. Anal. for HigherBrain Funct., Brain Sci. Inst., RIKEN, 7Dept.NeuroSci., Grad. Sch. Med. and Faclt. Med., KyotoUniv.)

Population exome analysis in macaques reveals evolutionarytrajectories of Japanese monkeys

To reveal evolutionary histories of Japanese monkeys, weperformed whole exome sequences (WES) in 77 Japanesemonkeys as well as 13 Chinese and 8 Indian rhesus monkeys asoutgroups. We obtained the high-quality reads that cover 95.5%of the target region (53.7 Mb) with at least one read and identifiedabout 345K SNPs. Principal component analysis (PCA) clearlyshows three distinct clusters of Japanese and two populations(Chinese and Indian) of rhesus monkeys. Moreover, a clusteringanalysis shows that Chinese and Indian rhesus monkeys aremaking sister groups, which is not consistent with previousworks that insisted more closely relationships between Japaneseand Chinese rhesus monkeys. In addition, we found thatJapanese monkey evolutionary and functionally specific genesare enriched in the genes involved in the fatty acid synthesis,skeletal development, brain function, immune response, spermmortality, and chemosensory perception.

3D-04

MATSUSHITA, Yuka1, TAKEZAKI, Naoko2, MELIN,Amanda Dawn3, KAWAMURA, Shoji1 (1Dept. Integr.BioSci.s, Grad. Sch. Front. Sci.s, The Univ. Tokyo,2Life Sci. Res. Cent., Kagawa Univ., 3Dept. Anthropol.& Archaeol., Univ. of Calgary)

L-M opsin nucleotide divergence depicts contrasting selectivemodes between trichromatic New World howler monkey andOld World primates

In New World monkeys, howler monkeys (Alouatta) are the onlygenus having L and M opsin genes juxtaposed on the Xchromosome. It is reported that they also have a high frequencyof L/M hybrid opsin genes, which diverge from normal L and Mopsins in absorption spectra. A previous study showed that in OldWorld primates, excepting humans, the spectral differencebetween L and M opsins is maintained by purifying selectionagainst homogenization due to gene conversion in central exons.However, it is not known whether such homogenization andpurifying selection are also operating in Alouatta. Here, we usetarget capture and next-generation sequencing (NGS) to examinenucleotide divergence between Alouatta L and M opsin genesincluding all introns. Contrasting to Old World primates, the Land M opsin genes showed high nucleotide divergence through-out the gene region including introns. Thus, homogenization dueto gene conversion is not obvious. Nevertheless, Alouatta specieshave L/M hybrid opsin genes at high frequency. This suggests adistinct mode of selection in Alouatta from Old World primatesallowing color vision variation as in other New World monkeys.


3D-05

KAWAMURA, Shoji1, KASAGI, Satosi1,2, KASAI,Daisuke1, TEZUKA, Ayumi3, SHOJI, Ayako3,TAKAHASHI, Akiyoshi2, IMAI, Hiroo4, KAWATA,Masakado3 (1Dept. Integr. BioSci.s, Grad. Sch. Front.Sci.s, The Univ. Tokyo, 2Sch. Marine Biosci.s, KitasatoUniv., 3Grad. Sch. life Sci., Tohoku Univ., 4PrimateRes. Inst., Kyoto Univ.)

Spectral sensitivity of guppy visual pigments reconstituted invitro to resolve association of opsins with cone cell types

The guppy (Poecilia reticulate) shows remarkable variation ofphotoreceptor cells in the retina. Microspectrophotometry (MSP)has revealed varying green, green-yellow and yellow cone cellsamong guppies in Trinidad and Venezuela (Cumana). In theguppy genome, there are four long-wave opsin loci. Two LWS-1alleles have potentially differing spectral sensitivity. However,the absorption spectra of these photopigments have not beenmeasured directly and the association of cell types with theseopsins remains speculative. In the present study, we reconsti-tuted these opsin photopigments in vitro. The wavelengths ofmaximal absorbance (max) of LWS-3 was much shorter than thevalue expected from the five-sites“ rule. The two LWS-1 allelescould explain difference of the reported MSP max values for theyellow cone class between Trinidad and Cumana guppies.Absence of the short-wave-shifted LWS-3 and the green-yellowcone in the green swordtail supports the hypothesis that this cellclass of the guppy co-expresses the LWS-1 and LWS-3. Theseresults reveal the basis of variability in the guppy color vision.”

3D-06

IIZUKA, Tomoyo1, NOZAWA, Masafumi2, IKEO,Kazuho1,3 (1Dept. Genet., SOKENDAI, 2TokyoMetropolitan Univ., 3Natl. Inst. Genet.)

Prediction of the responsible gene that determines themorphological characters of septal pore cap, the specificstructure in Agaricomycetes

Identifying the gene that corresponds to the morphologicaldiversity is important to know the genetic basis of evolution ofmorphology. In this study, we focus on the morphologicalcharacters of septal pore cap (SPC); component of cell-to-celljunction of Agaricomycotina(Fungi).The phylogenetic tree shows that “vesiculate” SPC (vSPC) is mostancestral type of SPC and the perforate“ SPC (pSPC) hademerged multiple times from ”imperforate“ SPC (iSPC).

3D-07

OOTA, Satoshi1 (1RIKEN BioRes. Cent.)

ThreeTree: a metric-based method to reconstruct phyloge-netic trees

Reconstruction of phylogenetic trees is equivalent to map theevolutionary distance space to the two-dimensional tree graphspace. It is well known that the pair-wise evolutionary distancespace often violates metricity. This is one of the main causes formisinference of phylogenetic trees. Meanwhile, virtually allexisting phylogenetic reconstruction methods follow the mini-mum evolution (ME) principle. The ThreeTree method is a newkind of approach to reconstruct phylogenetic trees based on theoptimal metricity of the evolutionary distance space rather thanthe ME principle. This method can neatly find evolutionarilyplausible topologies even from phylogenetically ill-conditioneddata.

3D-08

IWAMOTO, Eisuke1, TAMURA, Koichiro1,2 (1Dept.Biol. Sci.s, Tokyo Metro. Univ., 2Res. Cent. Genomicsand Bioinformatics)

Sorting and filtering sequence data makes phylogenomic treeestimationbetter

In molecular phylogenetic analyses, a phylogenetic tree isreconstructed from molecular sequence data based on a mathe-matical model of molecular evolution. Today, most of the tree-reconstruction methods assume that the pattern of evolution isconstant through all the concatenated genes. When this assump-tion is violated, the accuracy of phylogenetic tree reconstructionmay deteriorate. In this study, we developed a method to improvethe accuracy of phylogenetic tree reconstruction by removing thegenes that cause errors in phylogenetic tree reconstruction.We first sorted genes by means of the similarities betweendistance matrices of the genes, and then decided the amount ofdata removed according to the difference between the observeddistance matrix and the patristic distance matrix obtained fromthe inferred phylogenetic tree. We tested this method by usingthe honey bee dataset (Woodard et al. 2011), which was reportedto cause erroneous phylogenetic tree-reconstruction by basecomposition bias (Romiguier et al. 2016). We found that themethod improved the accuracy of phylogenetic tree reconstruc-tion.


3D-09

ENDO, Toshinori1 (1Grad. Sch. Inform. Sci. Tech.,Hokkaido Univ.)

Extraction and comparative analysis of genes over levels oftaxonomic classification in common

To address commonalities and lineage-specificities of gene setsacross eukaryotes, comprehensive classification and analysis wasconducted on the genomes for 10 phyla, 24 classes, 99 orders, 189genus and 393 species publicly available at NCBI repository. Thetotal number of sequence used accounts for 9,114,517 entries. Forprimary gene identification across species, provided annotationdata are utilized by extracting stem functional information. Allthe taxa information were manually curated for classificationpurpose, based on Taxonomy database and other resources for theclassification in question. This is because some NCBI is notauthority of taxonomy and some taxonomic information did notseem reliable. Based on above efforts, genes are classified on thecommonalities across each level of taxa, and then by commonalityspectrum. In the presentation, I report the resulted pivotal genesshared across levels of taxa to discuss their roles in evolutionarypaths.

3D-10

TAKEZAKI, Naoko1, NISHIHARA, Hidenori2 (1LifeSci. Res. Cent., Kagawa Univ., 22Grad. Sch. BioSci.and BioTech., Tokyo Inst. Tech.)

Phylogenetic analysis of coelacanth, lungfish, and tetrapodswith slowly evolving ray-finned fish as the outgroup

In our previous analysis of the relationships of coelacanths,lungfishes and tetrapods, with cartilaginous fish as the outgroup,the sister relationship between lungfish and tetrapods wasconstructed with high statistical support. However, with ray-finned fish as the outgroup, the sister relationship of coelacanthand tetrapods was mostly constructed, though the statisticalsupport tended to be low. Our study indicated that the treetopology was likely to be distorted because of the high divergenceof the ray-finned fish sequences. This study shows that with ray-finned fish with slow evolutionary rate, gar and bowfin, as theoutgroup, the sister relationship of lungfishes and tetrapods wasreconstructed with high statistical support.

3D-11

WATABE, Dai1, YUASA, Hiroshi2, ENDO, Toshinori1

(1Grad. Sch. Inform. Sci. Tech., Hokkaido Univ., 2TheRes. Inst. of Evolutionary Biol.)

Estimation of the bottle gourd propagation route by molec-ular analysis

Bottle gourds distribute worldwide. It is considered one of theoldest cultivated plants, probably native to Africa where theirwild species solely exists. In this study, we analyzed DNAdiversity of regional specimens of bottle gourds collected world-wide to clarify the propagation route. For this purpose, wedetermined sequences of three chloroplast and four nuclear DNAregions from 60 samples. The results revealed that two INDELsand three SNPs in chloroplast consistently separated African andAsian types except Ethiopian being classified as Asian. For twoINDELs, two previous studies contradicted. Our results sup-ported Erickson et al. (2005) who suggested American bottlegourds arrived via Asia. The nuclear variations also separatedAfrican and Asian consistent with the chloroplast results.Interestingly, a Guatemalan sample belonged to the Asianvariation and the rest of American samples were hybrids. Ourresults supported that the ancient propagation was via Asiabased on DNA typing.

3D-12

TAKEMURA, Toshihiko1 (1Takemura Lab.)

On the influence of the water molecular undulation for thecreation of the macromolecules in the lives body

From the great many hydrogen bonds which are led in the manyparts of the lives body created the adsorbed heavy water [H3O]what lead the LASER gravity through a series of the energyaccumulating reactions, which are the antagonistic rays of theincreased intermolecular gravity and the scatter rays as theincident rays of the resonance rays of the hydrogen which by thereflected repulsion of the hydrogen in the reverse distributionspace, what are strong coherent force and diffuse so reflect frominner to the body cuticle repeatedly so diffused all over the bodyand the body become the light reverse distribution space, whatcreate the great many virtual images on the line of the eachradiation and filled up the proper energy levels so recreate thegreat many hydrogen bond colonies scattering in the lives body.These great many hydrogen bonds colonies on the virtual imagesfilled up the many matters and become the many macromoleculesin the lives body, what reaction are led through the wateradsorbed the matter (M-H2O)by the substitution.For reference the T. Takemura scattering.


3E-01

YOSHIDA, Takanori1, KAWABE, Akira1 (1Faclt. LifeSci.s, Kyoto Sangyo Univ.)

De novo genome assembly of tower mustard, Turritis glabra

Genome rearrangements such as inversions and chromosomefusions are one of the major driving forces of genome evolutions.Model plant Arabidopsis thaliana and its relative species hadexperienced several chromosome rearrangements (Lysak et al.2006). Although the outline of rearrangements have beeninvestigated by comparative chromosome paintings, the detailsof breakpoints are still not clear for many relative species becauseof the lack of precise genome assemblies. The genome assemblybased on the single-molecule real-time sequencing can improvedraft genomes and provide many insights into the details ofchromosome rearrangements. We conducted sequencing byPacBio sequencer and de novo assembly of Turritis glabra, arelative of A. thaliana. Compared with ancestral karyotype, T.glabra have reduced number of chromosomes because ofchromosome rearrangements (Lysak et al. 2016). The draftgenome had 594 contigs with N50 of 563 Kb. We found majorbreakpoints of rearrangement at chromosome 2 and 4 bycomparing with synteny block of A. thaliana. In future, themolecular evolution of flanking genes should be analyzed toreveal the effect of chromosome rearrangements.

3E-02

NAITO, Ken1, SAKAI, Hiroaki1, TAKAHASHI, Yu1,ISEKI, Kohtaro2, MUTO, Chiaki1, KAGA, Akito1,TOMOOKA, Norihiko1 (1NATO Genetic ResourceCenter, 2Japan InterNatl. Res. Cent. of AgriculturalSci.)

De novo assembly of whole genome sequences of the wildspecies of genus Vigna by PacBio sequencer

Genus Vigna consists of legume crops such as azuki bean as wellas a number of wild species. One of the most attractive features ofthe genus Vigna is the great adaptability of the wild species toharsh environmental conditions. By figuring out the mechanismsof how they adapt to such environments, it would be possible togenerate novel crops tolerant to the stress conditions. Genomeinformation is of great importance to promote such researches.Thus, in order to establish the genome basis of genus Vigna, welaunched the Vigna genome project.In this study, we adopted single molecule real time (SMRT)sequencing because of its read length. We sequenced the 10 wildspecies which has great tolerance against stresses of high salt,acidic soil, alkaline soil, drought or flooding. The genome sizesranged from 480 Mb to 780 Mb, the sequencing depth rangedfrom 20x to 60x. The assembly stats achieved contig N50 of morethan 3.0 Mb in several species.

3E-03

NAKAMURA, Chiharu1, TAKENAKA, Shotaro1

(1Dept. Plant Life Sci., Faclt. Agr., Ryukoku Univ.)

Cytoplasmic diversity affecting submergence stress responsesin wheat

Plant cells possess three different genomes, i.e, nuclear, mito-chondrial and chloroplast genomes, in their respective cellularcompartments. These genomes have been known to interact tomaintain plant homeostasis during its whole developmentalstages. In the Triticum-Aegilops complex, cytoplasmic genomediversity and nucleus-cytoplasm (NC) genome interactionsaffecting various phenotypes can effectively be studied using aseries of NC hybrids in which particular nuclear genomes ofwheat are combined with various cytoplasmic genomes ofAegilops species. We evaluated submergence stress responses in39 NC hybrids at germination and the early seedling stages.Bioassays revealed a large variability among them. SomeAegilops cytoplasms either enhanced or reduced the sensitivityto submergence stress of NC hybrids as compared with that oftheir nuclear donor. Because all NC hybrids possessed anidentical nuclear genome, observed variability can be ascribedto cytoplasmic genome differences and further suggests aninvolvement of retrograde regulation by that different cytoplas-mic genomes regulate expression of the donor nuclear genomeunder this important abiotic stress in wheat.

3E-04

TAKADA, Toyoyuki1, KONDO, Shinji2, YASAKA,Taku3, ABE, Takashi3, KIYOSAWA, Hidenori4,SUZUKI, Yutaka5, ATSUSHI, Toyoda6, FUJIYAMA,Asao2,6, SHIROISHI, Toshihiko1 (1Mammalian Genet.Lab., Genetic Strains Res. Cent., Natl. Inst. Genet.,Mishima, 2Cent. Genome Inform, Res. Org. Inform.Systems, 3Dept. Inform Eng., Niigata Univ.rsity,4Dept. Environ. Med., Kochi Med. School, Kochi Univ.,5Dept. Comput. Biol. and Med. Sci., Grad. Sch. Front.Sci.s, The Univ. Tokyo, 6Comp. Genomics Lab., Natl.Inst. Genet.)

Comparative analysis of gene expression polymorphismsbased on mouse inter-subspecific genome divergence

Mammalian complex traits are determined by genetic andenvironmental factors along with sex and aging of the individ-uals. The genetic factors include variation of gene expressionlevels as well as amino acid substitutions. We intend to surveythe intersubspecific differences of the gene expression levelsusing Japanese wild mice-derived inbred strain MSM/Ms andmost commonly used inbred strain C57BL/6J. MSM/Ms strainshows highly extent of phenotypic variations in many complextraits and vast amount of genome diversity for C57BL/6J. At thebeginning, we have started genome-wide transcriptome analysisof liver samples, because it is inferred that wild-derived MSM/Msstrain has regionally adapted unique energy metabolic pathwaybetween commonly used inbred strains. In this talk, we will showrecent results of liver transcriptome analysis of C57BL/6J, MSM/Ms and experimental cross strains generated from them.


3E-05

FURUYAMA, Masahiro2, SATO, Tadashi1, MYOSHO,Taijun1, SAKAIZUMI, Mitsuru1 (1Faclt. Sci., NiigataUniv., 2Grad. Sch. Sci. and Tech., Niigata Univ.)

Gene-centromere mapping in the medaka fish using half-tetrad analysis

Gene-centromere mapping in medaka using half-tetrad analysisMasahiro Furuyama, Tadashi Sato, Taijun Myosyo, MitsuruSakaizumiAlthough gene-centromere mapping provides important resour-ces for genetics and comparative genomic studies, it has not beenreported in medaka. Medaka has 24 pairs of chromosomesconsisting of 10 pairs of biarmed and 14 pairs of monoarmedchromosomes. Fluorescent in situ hybridization (FISH) analyseshave shown that linkage group (LG)1, 9 and 18 are biarmed(Matsuda et al., 2002, Brunner et al., 2001 and Kondo et al., 2002)and LG11 and 13 are monoarmed (Myosho et al., 2012). In thisstudy we performed half-tetrad analysis using F1 hybrid femalesbetween two inbred strains, Hd-rR (Oryzias latipes) and HNI (O.sakaizumii) and males of an inbred strain of O. luzonensis. Atotal of 141 DNA markers were selected across 24 LGs.Genotyping of 112 triploid hybrids obtained by inhibiting therelease of the second polar body of oocytes localized all 24centromeres onto their respective LGs according to their closestmarkers. We propose here that 10 LGs (1, 2, 4, 9, 12, 14, 18, 19, 21and 23) are biarmed and the remaining 14 LGs are monoarmedchromosomes.

3E-06

NAGAOKA, Haruna1, MYOSHO, Taijun3, SATO,Tadashi2, SAKAIZUMI, Mitsuru2 (1Grad. Sch. Sci. andTech., Niigata Univ., 2Inst. Sci. Tech., Acad. Assembly,Niigata Univ.rsity, 3Sch. Food and Nutritional Sci.,Univ. of Shizuoka)

Lethality Caused by Substitution of the Chromosome Be-tween Medaka Strains

It lies at the heart of speciation to reveal hybrid incompatibilitybetween related species. Japanese medakas, Oryzias latipes andO. sakaizumii, have parapatric distribution in Japan. Inbredstrains have been established from both species, such as Hd-rRfrom O. latipes and HNI from O. sakaizumii. They can mate andproduce fertile progeny. Consomic embryos, Hd-rR-Chr23HNI

which carry the HNI Chr23 in the Hd-rR background, developabnormally, and die before hatch. This suggests a geneticincompatibility of the HNI Chr23 and Hd-rR backgroundchromosome (s). A causal factor on Chr23 was mapped near atelomere. To search for responsible factors in the Hd-rR back-ground, we crossed Hd-rR-Chr23HNI/Hd-rR, which is heterozygousfor Chr23, to (HNI×Hd-rR)F1, and obtained 28 progenyhomozygous for HNI Chr23. All the 28 progeny had heterozygousgenotype for a centromeric marker on Chr20, suggesting anotherresponsible factor on Hd-rR Chr20. Recombinant breakpointanalyses restricted the region of two responsible factors to23.86Mbp~end of Chr23 and to 0~3.1Mbp of Chr20 on themedaka physical map.

3E-07

KINJO, Sonoko1, YAGUCHI, Shunsuke2, YAMAMOTO,Takashi3, KIYOMOTO, Masato4, IKEO, Kazuho1

(1Cent. Inform. Biol., Natl. Inst. Genet., 2ShimodaMarine Res. Cent., Univ. of Tsukuba, 3Dept. Math. andLife Sci.s, Grad. Sch. Sci., Hiroshima Univ.,4Tateyama Marine Lab., Marine and Coastal Res.Center, Ochanomizu Univ.)

A genome and transcriptome database of a sea urchinHemicentrotus pulcherrimus created by NGS data analysisplatform, MASER

We developed and utilized a next-generation sequencing (NGS)data analysis platform, MASER (Management and AnalysisSystem for Enormous Reads) for analyzing genome and tran-scriptome data of sea urchin Hemicentrotus pulcherrimus whichhas a long history as a model organism in the field ofdevelopmental and cell biology since the mid 1900s. MASERallows us not only to analyze NGS data but also share theresultant data with public users or limited users for some ofanalyses. By applying this system to a genome of H. pulcherri-mus, we obtained a total of 16,251 scaffold sequences with N50length of 143k bp and identified approximately 25,000 genes. Thesize of genome was estimated to be around 800M bp. Theresultant sequences and annotation information are deposited inthe web site. This study is a first model case for de novo assemblyand public release of eukaryotic genome easily and effectively byusing MASER (http://cell-innovation.nig.ac.jp/public/contents/service_en.html).

3E-08

MASUYA, Hiroshi1, TAKATSUKI, Terue1, SAITO,Mikako1, TAKAYAMA, Eiki1, OHSHIMA, Kazuya1,TANAKA, Nobuhiko1 (1Tech. and Dev. unit forknowledge base of mouse phenotype, RIKEN BioRes.Cent.)

J-phenoeme: a database portal for phenotype information

In the use of information in the medical science, utility of theconcept of “phenotype” becomes realized. For example, disease-gene relationships will be enriched by the enhancement ofmutation-phenotype relationship across species, because diseasecomposed of multiple phenotypes. We are working on the trial ofthe integration and wider dissemination of phenotype data fromdomestic bio-resource centers, such as mouse strains from RikenBRC, consomic mouse strains from NIG, NBRP rat, NBRPmedaka and data of International Mouse Phenotyping Consor-tim. These data are linked from the database portal, J-phenome(http://jphenome.jp). In the integrated database, strains, pheno-type data and mutations are described with IDs of world-standard and can be used in linked data from these IDs.


3E-09

UENO, Michimune1, MATSUMOTO, Kouji1,MATSUOKA, Satoshi1, HARA, Hiroshi1 (1Dept.Biochem. Mol. Biol., Programs in Life Sci., Grad. Sch.Sci. Eng., Saitama Univ.)

An attempt to search B.subtilis gene that rescues lethality ofE.coli strain lacking PE/CL

Escherichia coli membranes consist of three kinds of majormembrane phospholipids: phosphatidylethanolamine (PE), phos-phatidylglycerol (PG), and cardiolipin (CL). Previous studiesreported that an E.colimutant lacking PE or lacking both PG andCL is viable. However an E.coli mutant lacking PE and CL, thatis, a mutant having only PG as a major membrane lipid isinviable. By contrast, Bacillus subtilis strain that has only PG asa major membrane lipid is viable. Therefore we attempted tosearch for a B. subtilis gene that rescued lethality of the E. colistrain lacking PE and CL, because of the function of the gene mayreveal essential roles of major membrane phospholipids in E.colicells.

3E-10

IMAI, Yukiko1,2, MATSUOKA, Satoshi1, HARA,Hiroshi1, MATSUMOTO, Kouji1 (1Dept. Biochem. Mol.Biol., Faclt. Sci., Saitama Univ., 2Inst. Hum. Genet.)

Septal localization by membrane targeting sequences at theCOOH terminus of Bacillus subtilis cardiolipin synthase

The acidic phospholipid cardiolipin (CL) is localized on polar andseptal membranes and plays an important physiological role inBacillus subtilis cells. ClsA, the enzyme responsible for CLsynthesis, is also localized on septal membranes. We found thatGFP fusions of the enzyme with NH2-terminal and internaldeletions retained septal localization. However, derivatives withdeletions starting from the COOH-terminus (Leu482) ceased tolocalize to the septum once the deletion passed the Ile at 448,indicating that the sequence responsible for septal localization isconfined within a short distance from the COOH-terminus. Twosequences, Ile436-Leu450 and Leu466-Leu478, are predicted toindividually form an amphipathic -helix. This configuration isknown as a membrane targeting sequence (MTS) and we refer tothem as MTS2 and MTS1, respectively. Either one has the abilityto effect septal localization and each of these by itself localizes tothe septum. Membrane association of the constructs of thisenzyme containing the MTSs was verified by subcellularfractionation of the cells. A model of diffusion and capture ofClsA to synthesize CL on the septal membrane is discussed.

3E-11

KANAZAWA, Miaki1, TANAKA, Naoyuki1,MATSUSHIMA, Yumeka1, TAKAHASHI, Yasuhiro1

(1Dept. Biochem. Mol. Biol., Grad. Sch. Sci. Eng.,Saitama Univ.)

Genetic analysis of suppressor mutations that bypass theessential components of iron-sulfur cluster biosynthesissystem

Biological assembly of iron-sulfur (Fe-S) cluster is mediated bycomplex systems consisting of multiple proteins. E.coli possessestwo distinct systems called the ISC and SUF machineriesencoded by iscSUA-hscBA-fdx-iscX and sufABCDSE, respec-tively. Deletion of both pathways is lethal due to the lack of thebiosynthetic apparatus for Fe?S clusters. Here we report thatmodification of isoprenoid biosynthetic pathway can bypass theindispensability of the Fe?S cluster biosynthetic systems, andshow that the ?isc ?suf double mutants can grow withoutdetectable Fe-S cluster-containing proteins. We also constructeda series of mutants in which each isc gene was disrupted in thedeletion background of sufABCDSE. Phenotypic analysis of themutants revealed that Fdx and IscA are not indispensable underanaerobic conditions. Furthermore, we found that suppressormutations in IscU, an Fe-S scaffold protein, could bypass theessential role of the chaperone system HscA and HscB that bindspecifically to IscU. The suppressor mutations are distributedthroughout the IscU molecule, suggesting that they induceconformational change of IscU and mimic the role of HscA/HscBchaperone system.

3E-12

SATO, Mutsumi1, MAHANNA, Sally1, IMANAKA,Takanobu2, ARAKI, Masatake1, OHMURAYA,Masaki2, ARAKI, Kimi1 (1Inst. Resource Dev. andAnal., Kumamoto Univ., 2Dept. Genet., Hyogo Coll. ofMed.)

The roles of cathepsin B, D, and L in pancreatic acinar cells

Cathepsin B, D, and L (CB, CD, and CL) are the major lysosomalproteases and are widely distributed in the cells of variousmammalian tissues. These cathepsins participates in variousphysiological events such as regulation of programmed cell death,activation of enzymatic precursors, and metabolic degradation ofintracellular proteins through autophagy. Autophagy is a majorpathway for degradation of cytoplasmic proteins and organelles,and has been implicated in the pathogenesis of acute and chronicpancreatitis.To investigate the role of CB, CD, and CL in pancreatic acinarcells, we generated and examined mice specifically deficient foreach of CB, CD, and CL in pancreatic acinar cells. CB–/– , CD–/– ,and CL–/–mice showed normal pancreatic development andautophagic activity in both physiological and pancreatitisconditions. Although CB–/–L–/– and CD–/–L–/– mice were almostnormal in physiological condition, the autophagic activity wasimpaired in the pancreas of CB–/–D–/– mice, and similar toautophagy deficient mice (Atg5–/–). We therefore conclude thatCB and CD can implicated in autophagic activity within thepancreatic acinar cells.


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