Genes Genet. Syst. (2009) 84 477

Symposia (S1-1 – S2-10)

3E EMOTO, Kazunori1, WADA, Takeo1, KUTSUKAKE, S1 IKEDA, Masato1 (1Dept. BioSci. and BioTech., Faclt. -11 Kazuhiro1 (1Grad. Sch. Natural Sci. Tech., Okayama -1 Agr., Shinshu Univ.) Univ.) Genome-based strain reconstruction for amino acid produc- Target protein of the RpoS-induced toxin in Salmonella tion enterica serovar Typhimurium The classical approach to strain improvement involves random Although toxin-antitoxin (TA) systems are ubiquitously present mutagenesis and screening, but this technology cannot avoid on bacterial chromosomes, their biological significance has accumulating detrimental or unnecessary mutations. Genome- remained enigmatic. We previously reported that Salmonella based strain reconstruction is a technology that identifies the enterica serovar Typhimurium possesses a toxin gene rsaB, useful mutations in such strains through genome sequencing and which is induced by a stress-specific sigma factor RpoS and then systematically and precisely engineers those mutations into causes growth arrest. Together with its cognate antitoxin gene the wild-type genome, creating a strain with only the useful rsaA, this gene seems to constitute a TA system on the mutations. Strains derived by this reverse engineering method Salmonella chromosome. In this study, as the first step to are expected to be more robust, give higher fermentation yields in elucidate the physiological role of this TA system, we attempted a shorter time and resist stressful conditions. Here we describe to identify a target protein of the toxin. From the cell lysate of the technology by using L-lysine fermentation as a model. Salmonella, an RNA-binding protein CsrA was found to be co- purified with the GST-tagged RsaB protein. Because CsrA is known to be essential for Salmonella growth, this protein is a plausible candidate of the target protein of RsaB. CsrA is known also to inhibit glycogen synthesis through its binding to mRNA of the glgC gene. We showed that RsaB inhibits CsrA from binding to glgC mRNA in vitro. Consistent with this, expression of RsaB within the cell enhanced glycogen synthesis in Salmonella.On the basis of the obtained results, we discuss the physiological role of this TA system.

3E FUKUSHIMA, Tatsuya1, FURIHATA, Isako1, S1 SEKIGUCHI, Junichi1,2, FUKUSHIMA, Tatsuya2, -12 TOKORO, Hideyuki1, SZURMANT, Hendrik2, -2 HASHIMOTO, Masayuki3, YAMAMOTO, Hiroki4 HOCH, James A.2 (1Div. Gene Res., Dept. Life Sci., (1Interdisciplinary Grad. Sch. Sci. and Tech., Shinshu Res. Cent. for Human and Env. Sci.s, Shinshu Univ., Univ., 2Div. Gene Res., Res. Cent. for Human and Env. 2The Scripps Res. Inst.) Sci.s, Shinshu Univ., 3Internatl. Young Researchers Empowerment Center, Shinshu Univ., 4Experimental Activation mechanism of cell division sensor, YycG Farm, Faclt. Textile Sci. and Tech., Shinshu Univ.) The recognition of cell division is very important for the Functions of cell wall binding proteins investigated on the microorganisms. However, the question is how the microorgan- genome analysis isms recognize the timing of cell division. As the answer for this question, recently we demonstrated the essential two-component Among cell wall binding proteins, the largest group consists of system (TCS), YycFG in Bacillus subtilis, senses the cell division. cell wall degrading . In Bacillus subtilis more than 35 Moreover, the inhibitors, YycH and YycI for the TCS, were gene products are predicted to be cell wall degrading enzymes, identified. Now we are studying the activation mechanism of cell and more than 20 products have been analyzed. Some cell wall division sensor, YycG. degrading genes are located in pro-phage regions and Immnofluorescent microscopic observation showed the sensor integrative conjugative element. Although the enzymes are kinase, YycG, localizes at cell division site and the localization is associated with cell lysis, the other physiological functions, such independent of the inhibitors, YycHI. Immunoprecipitation as cell differentiation (spore formation and germination), cell wall experiment demonstrated YycH interacts to YycI without YycG turnover and cell separation during the vegetative cell growth, and the YycH-I complex is associated with YycG in non-dividing have been reported. Various substrate specificities for peptido- cells. Bacterial two-hybrid system for YycG and cell division glycan (PG) are found in cell wall hydrolases: 3 are associated proteins indicates YycG interacts to the cell division proteins. with the glycan portion and 5 are associated with the peptide Since the of the TCS is occurred by cell portion. Recently it was reported the essential two-component division, the switch of inactivation and activation of YycG may be system, YycFG in B. subtilis, positively regulates two PG dependent on the interaction partners, YycHI and division hydrolases (CwlO and LytE) and negatively regulates PG hydro- proteins. lase-inhibitory protein (IseA). In this presentation, we will report the characterization of CwlO and LytE-double mutant and domain exchange of these proteins, and we will discuss with the lateral PG biosynthesis. 478 Genes Genet. Syst. (2009) 84

S1 SASAKI, Hiroyuki1 (1Dept. Integrated Genetics, Natl. S1 SHIRAHIGE, Katsuhiko1, BANDO, Masashige1, -3 Inst. Genet., Research Org. of Inform. and Systems) -5 SAKAI, Akiko1, KATOU, Yuki1, ITOH, Takehiko1 (1Dept. Biol. Sci.s, Tokyo Inst. Tech.) Identification and characterization of functional small by next generation sequencers Genomic Analyses of Cohesinopathy Functional small RNAs are highly conserved through evolution Cohesin regulates sister chromatid cohesion during the mitotic and have important roles in various biological processes, such as cell cycle. In addition to this role, cohesin has also been cell differentiation and development, defense against viruses and demonstrated to play a critical role in regulation of gene other parasitic agents, and heterochromatin formation. The small expression in nondividing cells as a transcriptional insulator. RNAs are classified into three major categories, i.e. miRNA, Heterozygous mutations in the cohesin loader NIPBL or cohesin siRNA and piRNA, depending on the biogenesis pathway and structural components result in the multisystem developmental interacting factors. The emergence of the next generation disorder Cornelia de Lange Syndrome (CdLS). Genome-wide sequencers now enables us to know the composition of small assessment of transcription in 16 mutant cell lines from severely RNA repertoire in a given cell, mode of their biogenesis, target affected CdLS probands has identified a unique profile of genes, and presence/absence of RNA editing. In this symposium, I dysregulated gene expression. In B cell lines established from will present some of our recent results and discuss the power of CdLS patients, 60% of cohesin binding sites were lost compared the next generation sequencers in small RNA studies. to healthy control B cell lines, suggesting that 40% of cohesin sites are sufficient for mitotic function of cohesin. Cohesin localization analysis also demonstrates a preference for inter- genic regions suggesting a cis-regulatory function mimicking that of a boundary/insulator interacting protein. However, the binding sites are enriched within the promoter regions of the dysregu- lated genes and are significantly decreased in CdLS proband, indicating an alternative role of cohesin as a transcription factor.

S1 SUZUKI, Yutaka1 (1Dept. of Med. Genome Sci., Grad. S1 GOJOBORI, Takashi1 (1Natl. Inst. Genet.) -4 Sch. of Sci, The Univ. Tokyo) -6 Integrated Analysis of Human Alternative Promtoers using Database in the time of Next Generation Sequencers Next Generation Sequencer We carried out genome-wide analysis of the putative alternative promoters in human genes by multifaceted use of massive paralleled sequencer. We characterized 130 million transcrip- tional start site (TSS) sequence tags which were identified by TSS Seq analysis in 12 human cell types. Although the number of the TSS tag clusters (TSCs) is generally large throughout the cell types, TSCs showing significant expression levels were relatively rare and had distinctive several sequence features from other lowly expressed TSS tag clusters. ChIP-Seq and Nucleosome-Seq analyses revealed highly-ordered nucleosome structure and strong signals of the binding of polymerase II predominantly around the TSCs of the former group, possibly allowing their deterministic transcriptions. Evaluation of 16080 previously sequenced and 846 newly shotgun-sequenced complete cDNA sequences also showed that transcripts associated with those TSCs are more likely to be translated. By RNA Seq analysis using polysome fraction, we directly observed at least one-third of those transcript products are incorporated in translating ribosome. Genes Genet. Syst. (2009) 84 479

S1 UCHIYAMA, Ikuo1 (1Natl. Inst. Basic Biol., Natl. S1 HATTORI, Masahira1, OSHIMA, Kenshiro1, KIM, -7 Institutes of Natural Sci.) -9 Seokwon1, KUROKAWA, Ken2, TOH, Hidehiro3, TAYLOR, Todd3 (1Center for Omics and Bioinform., Comparative genome informatics in the era of high-through- Grad. Sch. Front. Sci.s, The Univ. Tokyo, 2Dept. Biol. put sequencing Inform., Tokyo Inst. Tech., 3RIKEN Adv. Sci. Inst.) Recent development of massively parallel sequencing technolo- Metagenomics and genomics decoding human gut micro- gies leads us to a new era of high-throughput sequencing, and is biomes revolutionizing biological research based on genomics. Compara- tive genomics is a useful approach to integrate various data for Intestinal microbes are taxonomically complex and constitute an better understanding of biological phenomena from the viewpoint ecologically dynamic microbiota that has long been believed to of evolution. We are developing a system for comparative possess a strong impact on human physiology. Furthermore, they genomics based on automated ortholog identification, named are heavily involved in the maturation and proliferation of RECOG. RECOG covers wide range of comparative genome human intestinal cells, helping to maintain their homeostasis, analysis including closely related genome comparison, distantly and can be causative of various diseases such as inflammatory related genome comparison, and metagenome analysis. We are bowel disease and obesity. We performed a metagenomic analysis also trying to implement functions supporting various strategies of fecal samples from 13 healthy Japanese individuals (DNA Res. in practical comparative genomics projects. 14:169, 2007), in which about 660,000 microbial genes were predicted from 479 Mb of unique metagenomic sequence data. Among them, we identified 315 gene families commonly enriched in the all samples and 647 new gene families exclusively present in human intestinal microbiomes. We have extended this research by performing further analysis of the predicted genes, 16S ribosomal RNA gene analysis, mapping of metagenomic data, and genomic sequencing of human microbes to more deeply evaluate their genetic and functional natures. This work is being conducted as part of the International Human Microbiome Project that was launched in 2008 as an international collabo- rative research effort.

S1 KOBAYASHI, Ichizo1 (1Dept. Medical Genome Sci.s, -8 School of Front. Sci., Univ. Tokyo) Strategy for comparison of multiple complete genome sequences within a species or a population Comparison of complete genome sequences within a species or a population allows recognition of a dynamic picture of genome evolution. Especially in the prokaryotes, we can trace horizontal gene transfer, which are frequent and contributes to adaptation, as well as mutagenesis and genome rearrangements. I introduce analysis of Japanese strains of Helicobacter pylori. Collaborators: Takeshi Azuma, Masaru Yoshida (Kobe University); Mikihiko Kawai, Yoshikazu Furuta, Takeshi Tsuru, Naofumi Handa, Noriko Takahashi, Masahira Hattori, Kenshiro Oshima (Univer- sity of Tokyo); Mikihiko Kawai, Ikuo Uchiyama (NIBB); Koji Yahara (Kurume University). 480 Genes Genet. Syst. (2009) 84

S2 YOSHIKAWA, Hiroshi1 (1Professor Emeritus, Osaka S2 ITOH, Tateo1 (1Dept. Biol., Faclt. Sci., Shinshu Univ.) -1 Univ. and Nara Inst. of Sci. and Tech.) -3 Replication: from DNA to genome Initiation and its control of the ColE-type plasmids revisited Following the discoveries of double strand structure, semi- The in vitro replication of closed circular double-stranded DNA conservative replication and DNA polymerase, biochemistry was first demonstrated for the ColE1 plasmid by using a cell became the major issue of DNA replication studies in 1960s. On extract of E. coli. Initiation of replication requires only three host the other hand, the size of DNA molecule of phage and bacteria proteins. Replication starts from a unique position on the plasmid was a hot topic at that time and one genome–one DNA molecule DNA, the origin. The primer precursor synthesized by RNA was hypothesized. We attempted to solve this problem by polymerase is longer than 555 nt and forms a persistent hybrid studying the mode of genome replication and discovered a with the template DNA near the origin. The hybrid is cleaved at sequential replication of B. subtilis chromosome and identified the origin by RNase H and then the cleaved RNA is used as a a unique replication origin, forks and terminus of the genome. primer for DNA synthesis by DNA polymerase I. Replication is This finding led to the concept of Replicon. Thirty years later, we controlled by a small RNA, that is complementary to the 5’ end met a similar situation in eucaryotes. While biochemical studies region of the primer precursor. Interaction of the small RNA with on replication were in bloom, we turned into an essence of genome the elongating primer precursor inhibits the formation of a replication: mechanism and biological significance of replication persistent hybrid between the primer precursor and the template of multi-replicons. I will talk on our double challenges on genome DNA. This was the first example of the antisense RNA replication. regulation. For the formation of a persistent hybrid between the primer precursor and the template DNA a specific tertiary structure of the primer precursor is essential and the interaction of the antisense RNA inhibits its formation. A mechanism of evolution of a functional structure of RNA has been proposed.

S2 ARAI, Ken-Ichi1 (1Professor Emeritus, The Univ. S2 MATSUKAGE, Akio1 (1Former Chief, Aichi -2 Tokyo) -4 Ctr. Res. Inst. and former Professor, Japan Women’s Univ.) From Resolution and Reconstitution of DNA replication in vitro to Signal Transduction by Cytokines Animal cell DNA polymerases and their expressional regu- lation The major tasks in DNA replication were to elucidate the mechanism of initiation, elongation and termination. In 1960’s, We found DNA polymerase (pol) g in 1973 in addition to formally characterized properties of DNA Polymerase I. found pols a and b. Three pols were purified to make antibodies. In 1970’s, by employing phage M13, G4 and fX174 templates, his Using this, cDNA for pol b was cloned, and this enzyme was group identified the components required for driving replication expressed in E. coli. Antibodies were also used to detect pols in fork such as DNA polymerase III holoenzyme, primase, and DNA chick embryos. ‘Pol a, in contrast to pol b, is sharply repressed at helicase etc. In collaboration with Dr. Yoshito Kaziro, I studied the onset of differentiation. We started to clarify mechanisms the role of GTP in protein synthesis and demonstrated that a governing the expression of DNA replication-related genes using molecular switch is involved in the basic process of energy D. melanogaster. to find a common element DRE in the promoters transduction. This switch is turned on and off via ligand induced of pol a and PCNA genes, then, regulatory factor DREF. DRE/ conformational change and is used in driving replication fork as DREF is now known as a master key for many cell proliferation- well as signal transduction regulated by ATP and/or GTP. related genes. Interestingly, DREF is a target of many differ- entiation and chromatin-related regulators. Genes Genet. Syst. (2009) 84 481

S2 HIRAGA, Sota1 (1Former Professor, Kumamoto Univ. S2 KATAYAMA, Tsutomu1 (1Dept. Molec. Biol., Grad. -5 and Researcher, Kyoto Univ.) -7 Sch. Pharm. Sci., Kyushu Univ.) From isolation of oriC plasmids in E. coli to study of partition Structure, function and regulation of DnaA, the initiator of E. mechanism coli I though, 24 years ago, that isolation of replication origin was The finding of the E. coli dnaA gene was a crucial progress for essential for study on mechanism of replication initiation. I indicating the presence of the initiator that had been predicted by actually isolated F’ plasmids carrying the replication origin (oriC) the replicon model. Hirota and Kohiyama made major contribu- DNA segment of E. coli chromosome (Hiraga, 1976). Further- tion to this finding in the Jacob laboratory in Pasteur Institute. more, we found the partition system of F plasmid (Ogura and The replicator oriC was isolated using independent method- Hiraga, 1983). Although the molecular mechanism of F partition ologies by three groups of Hirota et al., Hiraga et al. and Nagata had been unclear for long time, we recently proposed the new et al. Nucleotide sequence and basic functional structure of oriC paradigm that F partition is performed by the reaction-diffusion were revealed by Takamani et al. Exact nucleotide sequence of system of SopA and SopB proteins (Adachi et al., 2006). Moreover dnaA was revealed by a group of Ohmori, Nagata, Sakakibara et we have found the MukB operon that is essential for proper al. Moreover, an oriC replication system reconstituted with partitioning of sister chromosomes (Niki et al., 1991). purified proteins was constructed in the Kornberg laboratory, where Ogawa et al. played a crucial role. Also, Sekimizu et al. found that DnaA is activated by ATP binding. These findings are most important progresses for understanding the regulation of replicational initiation. Based on these, researches on structure and functional mechanisms of initiation complexes and their regulatory mechanisms are rapidly progressing. I would like to summarize these progresses for discussion of the future study.

S2 TSURIMOTO, Toshiki1 (1Lab. of Chromo. Func., Dep. S2 SHIRAHIGE, Katsuhiko1, KATOU, Yuki1, TANAKA, -6 of Biol., Fac. of Sci. Kyushu Univ.) -8 Hirokazu1, KOMATA, Makiko1, SUTANI, Takashi1 (1Dept. Biol. Sci.s, Tokyo Inst. Tech.) Studies on molecular interactions and reconstitutions for understanding of the replcation mechanism Links between Chromosomal DNA replication and other chromosomal functions-A Genomic View of Chromosomal DNA replication reaction progresses through highly specific DNA replication- interactions by multiple replication factors. My first replication research was to identify a protein specifically interacting with the Our main interest is to know the molecular mechanisms for the bacteriphage l ori. Using this strategy, I was able to purify l O maintenance of genome integrity. For this purpose, we are protein as the first purified protein among bacterial initiators, studying the process of chromosome metabolsims using genomic and to develop the in vitro system of l ori dependent DNA approaches.Genetic and biochemical approaches identified hun- replication with O protein. Subsequently, I continued my dreds of proteins which have something to do with chromosome replication research with SV40 virus DNA system at CSHL, metabolisms so far. Now, genomic approaches let us know how and engaged in identification of novel eukaryotic replication these proteins are actually integtated into the process of factors. I was fortunate to purify a novel protein, replication chromosome metabolic pathways, and how each pathway is factor C (RFC), which functions, along with PCNA, to assemble connected to make a huge network for the faithful maintenance of the eukaryotic replication fork complex. We are continuing genome. Our recent trial for the understanding of links between dissections and reconstitutions of the functional network of DNA replication and DNA replication checkpoint will be replication factors to elucidate molecular dynamics of the presented. replication reaction. 482 Genes Genet. Syst. (2009) 84

Mini symposia (MS1-1 – MS2-7)

S2 ARAKI, Hiroyuki1,2 (1Div. Microbial Genetics, Natl. MS1 UEHARA, Hiroshi1, ABE, Takashi1, NAKAIZUMI, -9 Inst. Genet., 2Dept. Genet., SOKENDAI) -1 Yuki1, WADA, Chieko1, INOKUCHI, Hachiro1, IKEMURA, Toshimichi1 (1Nagahama Inst. Bio-Sci. Isolation of new replication factors by genetic screenings in Tech.) budding yeast and their characterization: approach to elucidation of molecular mechanism in the initiation of The hunting Genes contributing to “Human Health” and eukaryotic DNA replication “Sustainable World” as the educational program for under- graduate and high-school students The initiation of DNA replication requires several consecutive reactions. In eukaryotic cells, the initiation process is separated As the educational program of bioinformatics, databases for genes into two steps. First, the MCM complex (MCM2-7), a DNA that may contribute to “human health” or “sustainable world” are helicase, is loaded onto origins that bind to Origin Recognition constructed. Specifically, students set themes on their own Complexes (ORC), to form the pre-Replicative Complex (pre-RC) initiative and search for such genes that may contribute to in G1 phase. Then, other replication factors including DNA “human health” or “sustainable world” from vast quantities of polymerases are loaded to the pre-RC formed origins when genomic sequences that were obtained by ongoing large-scale Cyclin-Dependent Kinase (CDK), a key regulatory factor of the sequencing of mixed environmental biological samples (metage- cell cycle, is activated at G1/S boundary. Using budding-yeast nomic analysis) and have been stored in the International genetic screenings, we isolated many replication factors, which Nucleotide Sequence Databanks (DDBJ/EMBL/GenBank) pri- associate with replication origins when CDK is activated. Of marily without functional annotation. Many candidate genes these factors, Sld2 and Sld3 proteins are phosphorylated by CDK which have the potentiality for production of pharmaceuticals, and bind to the Dpb11 protein in a phosphorylation-dependent physiologically functional food, bio-ethanol, etc., have been found manner. These phosphorylation-dependent interactions enhance out. complex formation of replication proteins. This complex forma- We have already started to go to high-schools to have lessons for tion seems to promote loading of replication proteins to origins, students. Then, if the students find out new useful candidate such as GINS complex. The GINS complex forms an active gene, we can register the genes in “Database for Genes for helicase complex, together with MCM and Cdc45 proteins. We Contributing to Human Health and Sustainable World” publi- thus propose that the CDK-dependent interactions promote cized by us with their names under the Integrated Data Project of formation of active helicase complex. DBCLS. We can provide an educational program which enables high school students to make new knowledge discovery from the massive genome information.

S2 MASAI, Hisao1 (1The Tokyo Metro. Inst. Med. Sci.) MS1 BONO, Hidemasa1 (1Database Center for Life Sci. -10 -2 (DBCLS), Research Org. of Inform. and Systems (ROIS)) Regulation of DNA replication program: genetic determi- nants and plasticity How to make full use of the integrated database for life science in undergraduate and high schools DNA replication program which defines the selection of sites and the timing of its activation may be genetically determined. The advent of DNA microarray and next-generation DNA However, significant level of plasticity is observed in eukaryotic sequencing has changed the lives of life scientists. They now systems in the execution of replication program. For examples, must biologically interpret a bunch of their data making full use although replication origins are fired with 100% efficiency in the of what we call ’public databases’. Meanwhile, Ministry of prokaryotic replicions, firing of each origin may be less efficient in Education, Culture, Sports, Science and Technology (MEXT) of eukaryotes but multiple origins may complement with each other Japan launched the Integrated Database Project (2006-2011) to to ensure the replication of the entire genome. This also enables maintain the central repository of database for life science in the eukaryotic replicons to adopt dramatically altered replication Japan. As a central hub of the project, Database Center for Life programs under some specific conditions. By using fission yeast Science (DBCLS) was established in Research Organization of as a model, we have identified multiple positive and negative Information and Systems (ROIS) as one of national research factors which may modulate the processes of origin selection and center in Japan. The project and DBCLS will be introduced in my activation timing. We will discuss potential mechanisms of plastic talk. regulation of eukaryotic replication programs with these factors.