研究者詳細 - 冨田　勝

論文 - 冨田　勝

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Cooperative regulation for Okazaki fragment processing by RNase HII and FEN-1 purified from a hyperthermophilic archaeon, Pyrococcus furiosus

Sato, A., Kanai, A., Itaya, M. and Tomita, M

Biochemical and Biophysical Research Communications 309 （ 1 ） 247 - 252 2003年

研究論文（学術雑誌）, 共著, ISSN 0006291X

A reconstitution system that recapitulates the processing of Okazaki-primer RNA was established by the heat-stable recombinant enzymes RNase HII and FEN-1 (termed Pf-RNase HII and Pf-FEN-1, respectively) prepared from a hyperthermophilic archaeon, Pyrococcus furiosus. A 35-mer RNA-DNA/DNA hybrid substrate mimicking an Okazaki fragment was used to investigate the properties of the processing reaction in vitro at 50°C. Pf-RNase HII endonucleolytically cleaves the RNA primer region, but does not cut the junction between RNA and DNA. Removal of the RNA of the RNA-DNA junction was brought about by Pf-FEN-1 after Pf-RNase HII digestion. In the presence of 0.25-5mM MnCl2, Pf-FEN-1 alone weakly cleaved the junction. The addition of Pf-RNase HII to the reaction mixture increased removal efficiency and optimal Pf-FEN-1 activity was achieved at an equal amount of the two enzymes. These results indicate that there are at least two steps in the degradation of primer RNA requiring a step-specific enzyme. It is likely that Pf-RNase HII and Pf-FEN-1 cooperatively process Okazaki fragment during lagging-strand DNA replication. © 2003 Elsevier Inc. All rights reserved.

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Computational analysis of stop codon readthrough in D.melanogaster

Sato, M., Umeki, H., Saito, R., Kanai, A. and Tomita, M.

Bioinformatics 19 （ 11 ） 1371 - 1380 2003年

研究論文（学術雑誌）, 共著, ISSN 13674803

Motivation: Readthrough is an unusual process in which a stop codon is misread or skipped. Recently it has been shown that some translation is regulated by the readthrough reactions although the complete mechanism is not clear. Therefore, the discovery of 'readthrough genes' is important for further investigation of their cellular roles, which may provide additional insights into the mechanism of translational regulation. Results: We constructed a system that lists candidates of readthrough genes based on the existence of a 'protein motif' at the 3′ untranslated region (UTR). Using this system, we extracted 85 candidates from 4082 nucleic acid sequences of Drosophila melanogaster in GenBank database. The sequences of these candidates had a slightly more stable secondary structure and different base preferences compared to the non-candidates. As these features are known to have an effect on readthrough events, we would like to suggest that these candidates contain actual readthrough genes.

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Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection

Numata K., Kanai, A., Saito, R., Kondo, S., Adachi, J., Wilming, L.G., Hume, D.A., Hayashizaki, Y. and Tomita, M. ;RIKEN GER Group; GSL Members. Hayashizaki, Y. and Tomita, M.

Genome Research 13 （ 6 B ） 1301 - 1306 2003年

研究論文（学術雑誌）, 共著, ISSN 10889051

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products - has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

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Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Okazaki Y., Furuno M., Kasukawa T., Adachi J., Bono H., Kondo S., Nikaido I., Osato N., Saito R., Suzuki H., Yamanaka I., Kiyosawa H., Yagi K., Tomaru Y., Hasegawa Y., Nogami A., Schönbach C., Gojobori T., Baldarelli R., Hill D.P., Bult C., Hume D.A., Quackenbush J., Schriml L.M., Kanapin A., Matsuda H., Batalov S., Beisel K.W., Blake J.A., Bradt D., Brusic V., Chothia C., Corbani L.E., Cousins S., Dalla E., Dragani T.A., Fletcher C.F., Forrest A., Frazer K.S., Gaasterland T., Gariboldi M., Gissi C., Godzik A., Gough J., Grimmond S., Gustincich S., Hirokawa N., Jackson I.J., Jarvis E.D., Kanai A., Kawaji H., Kawasawa Y., Kedzierski R.M., King B.L., Konagaya A., Kurochkin I.V., Lee Y., Lenhard B., Lyons P.A., Maglott D.R., Maltais L., Marchionni L., McKenzie L., Miki H., Nagashima T., Numata K., Okido T., Pavan W.J., Pertea G., Pesole G., Petrovsky N., Pillai R., Pontius J.U., Qi D., Ramachandran S., Ravasi T., Reed J.C., Reed D.J., Reid J., Ring B.Z., Ringwald M., Sandelin A., Schneider C., Semple C.A.M., Setou M., Shimada K., Sultana R., Takenaka Y., Taylor M.S., Teasdale R.D., Tomita M., Verardo R., Wagner L., Wahlestedt C., Wang Y., Watanabe Y., Wells C., Wilming L.G., Wynshaw-Boris A., Yanagisawa M.

Nature 420 （ 6915 ） 563-573 - 573 2002年12月

研究論文（学術雑誌）, 共著, ISSN 0028-0836

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

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Comprehensive sequence analysis of translation termination sites in various eukaryotes

Ozawa Y., Hanaoka S., Saito R., Washio T., Nakano S., Shinagawa A., Itoh M., Shibata K., Carninci P., Konno H., Kawai J., Hayashizaki Y., Tomita M.

Gene 300 （ 1-2 ） 79-87 - 87 2002年10月

研究論文（学術雑誌）, 共著, ISSN 0378-1119

Recent investigations into the translation termination sites of various organisms have revealed that not only stop codons but also sequences around stop codons have an effect on translation termination. To investigate the relationship between these sequence patterns and translation as well as its termination efficiency, we analysed the correlation between strength of consensus and translation efficiency, as predicted according to Codon Adaptation Index (CAI) value. We used RIKEN full-length mouse cDNA sequences and ten other eukaryotic UniGene datasets from NCBI for the analyses. First, we conducted sequence profile analyses following translation termination sites. We found base G and A at position +1 as a strong consensus for mouse cDNA. A similar consensus was found for other mammals, such as Homo sapiens, Rattus norvegicus and Bos taurus. However, some plants had different consensus sequences. We then analysed the correlation between the strength of consensus at each position and the codon biases of whole coding regions, using information content and CAI value. The results showed that in mouse cDNA, CAI value had a positive correlation with information content at positions +1. We also found that, for positions with strong consensus, the strength of the consensus is likely to have a positive correlation with CAI value in some other eukaryotes. Along with these observations, biological insights into the relationship between gene expression level, codon biases and consensus sequence around stop codons will be discussed. © 2002 Published by Elsevier Science B.V.

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Towards Computer Aided Design (CAD) of Useful Microorganisms

Tomita, M.

Bioinformatics 17 1091-1092 2002年

研究論文（学術雑誌）, 単著

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Characterization of the aryl hydrocarbon receptor repressor gene and association of its Pro185Ala polymorphism with micropenis

Fujita H, Hasegawa T, Takahashi T, Matsuo N, and Kosaki K, Tomita M.

Teratology 65 （ 1 ） 10 - 18 2002年

共著, ISSN 00403709

Background: Genetic background of a fetus contributes to the abnormal development after teratogen exposure. In rodents, in utero exposure to dioxins affects male external genital development. The effects of dioxins are mediated via the aryl hydrocarbon receptor (AHR) and its binding protein, aryl hydrocarbon receptor nuclear translocator (ARNT). In mice, aryl hydrocarbon receptor repressor (AHRR), which binds to ARNT in competition with AHR, plays a critical negative regulatory role in AHR signaling. We attempt to characterize the human AHRR gene and investigate the relationship between AHRR polymorphisms and the incidence of micropenis, a phenotype of undermasculinization. Methods: We identified and characterized the human homolog of mouse AHRR, taking advantage of the publicly available draft version of the human genome sequence. After detecting an AHRR protein polymorphism by the direct sequencing of pooled human genomic DNA, we evaluated the association between the polymorphism and the presence or absence of micropenis (<-2.5 SD) in patients with micropenis and control subjects. Results: The deduced sequence for human AHRR (715 residues) and the mouse AHRR protein exhibited 81% sequence homology to each other. The Pro185Ala polymorphism was identified between the PAS-A region and the highly conserved arginine/cysteine-rich RCFRCRL/VRC region. Forty-six percent (27/59) of patients with micropenis and 27% (22/80) of the controls were homozygous for 185Pro; this difference in frequencies was significant (P = 0.03). Conclusions: Homozygosity for the 185Pro allele of AHRR may increase the susceptibility of a fetus to the undermasculinizing effects of dioxin exposure in utero, presumably through the diminished inhibition of AHR-mediated signaling. © 2002 Wiley-Liss, Inc.

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Causal analysis of CpG suppression in the Mycoplasma genome

Goto, M., Washio, T. Tomita M.

Microbial and Comparative Genomics 5 51-58 2002年

共著

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Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry

Soga, T. Ueno, Y., Naraoka, H., Ohashi, O., Nishioka, T. Tomita M.

Analytical Chemistry 74 （ 10 ） 2233-2239 - 2239 2002年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00032700

A method for simultaneous determination of anionic metabolites based on capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry is described. To prevent current drop by the system, electroosmotic flow (EOF) reversal by using a cationic polymer-coated capillary was indispensable. A mixture containing 32 standards including carboxylic acids, phosphorylated carboxylic acids, phosphorylated saccharides, nucleotides, and nicotinamide and flavin adenine coenzymes of glycolysis and the tricarboxylic acid cycle pathways were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. Key to the analysis was EOF reversal using a cationic polymer-coated capillary and an electrolyte system consisting of 50 mM ammonium acetate, pH 9.0. The relative standard deviations of the method were better than 0.4% for migration times and between 0.9% and 5.4% for peak areas. The concentration detection limits for these metabolites were between 0.3 and 6.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL); i.e., mass detection limits ranged from 9 to 200 fmol, at a signal-to-noise ratio of 3. This method was applied to the comprehensive analysis of metabolic intermediates extracted from Bacillus subtilis, and 27 anionic metabolites could be directly detected and quantified.

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Integrative modeling of gene expression and metabolism with E-CELL simulation system

Hashimoto, K., Seno, S., Dhar, P.K. and Tomita, M

Artificial Life and Robotics 6 99-107 2002年

研究論文（学術雑誌）, 共著, 査読有り

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Proposition of cortical connectivity between 30 cytoarchitectural areas of human cerebral cortex

Fukuda R., Hara J., Shankle W., Tomita M.

Neurocomputing 32-33 （ 33 ） 749-755 - 755 2002年

研究論文（学術雑誌）, 共著, ISSN 09252312

Connectivity between 30 cytoarchitectural areas covering almost the whole region of neocortex has been proposed. The data source provided by J.L. Conel was used for the analyses. The analyses consisted of computing correlation coefficients between each cortical layer of one cortical area to each cortical layer of another cortical area over the eight age-points and comparison of the time of the first appearance of myelinated axons per layer per area. Derived connectivity supports some interesting findings reported recently by others and is in good agreement with neural network model deduced from neuroimaging and psychological experiments. (C) 2000 Elsevier Science B.V. All rights reserved.

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Pressure-assisted capillary electrophoresis electrospray ionization mass spectrometry for analysis of multivalent anions

Soga, T., Ueno, Y., Naraoka, H., Matsuda, K., Tomita, M., and Nishioka, T

Analytical Chemistry 74 （ 24 ） 6224-6229 - 6229 2002年

研究論文（学術雑誌）, 共著, ISSN 00032700

We describe a method, based on pressure-assisted capillary electrophoresis coupled to electrospray ionization mass spectrometry (PACE/ESI-MS), that allows the simultaneous and quantitative analysis of multivalent anions, such as citrate isomers, nucleotides, nicotinamideadenine dinucleotides, and flavin adenine dinucleotide, and coenzyme A (CoA) compounds. Key to the analysis was using a noncharged polymer, poly(dimethylsiloxane), coated to the inner surface of the capillary to prevent anionic species from adsorbing onto the capillary wall. It was also necessary to drive a constant liquid flow toward the MS by applying air pressure to the inlet capillary during electrophoresis to maintain a conductive liquid junction between the capillary and the electrospray needle. Although theoretical plates were inferior to those obtained by CE/ESI-MS using a cationic polymer-coated capillary, the PACE/ESI-MS method improved reproducibility and sensitivity of these anions. Eighteen anions were separated by PACE and selectively detected by a quadrupole mass spectrometer with a sheath-flow electrospray ionization interface. The relative standard deviations (n = 6) of the method were better than 0.6% for migration times and between 1.4% and 6.2% for peak areas. The detection limits for these species were between 0.4 and 3.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL), that is, mass detection limits calculated in the range from 12 to 110 fmol at a signal-to-noise ratio of 3. The utility of the method was demonstrated by analysis of Citrate isomers, nucleotides, dinucleotides, and CoA compounds extracted from Bacillus subtilis cells.

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On biased distribution of introns in various eukaryotes

Sakurai, A., Fujimori, S., Kochiwa, H., Kitamura-Abe, S., Washio, T., Saito, R., The RIKEN Genome Exploration Research Group Phase II Team, Carninci, P., Hayashizaki, Y., and Tomita, M.

Gene 300 （ 1-2 ） 89-95 - 95 2002年

研究論文（学術雑誌）, 共著, ISSN 03781119

We conducted comprehensive analyses on intron positions in the Mus musculus genome by comparing genomic sequences in the GenBank database and cDNA sequences in the mouse cDNA library recently developed by Riken Genomic Sciences Center. Our results confirm that introns have a tendency to be located toward the 5′ end of the gene. The same type of analysis was conducted in the coding region of seven eukaryotes (Saccharomyces cerevisiae, Plasmodium falciparum, Caenorhabditis elegans, Drosophila melanogaster, M. musculus, Homo sapiens, Arabidopsis thaliana). Introns in genes with a single intron have a locational bias toward the 5′ end in all species except A. thaliana. We also measured the distance from the start codon to the position of the intron, and found that single introns prefer the location immediately after the start codon in S. cerevisiae and P. falciparum. We discuss three possible explanations for these findings: (1) they are the consequence of intron loss by reverse-transcriptase; (2) they are necessary to accommodate the function; and (3) they are concerned with the mechanism of pre-mRNA splicing. © 2002 Elsevier Science B.V. All rights reserved.

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Inferring alternative splicing patterns in mouse from a full-length cDNA library and microarray data

Kochiwa, H., Suzuki, R., Washio, T., Saito, R., The RIKEN Genome Exploration Research Group Phase II Team, Bono, H., Carninci, P., Okazaki, Y., Miki, R., Hayashizaki, Y., and Tomita, M.

Genome Research 12 （ 8 ） 1286-1293 - 1293 2002年

研究論文（学術雑誌）, 共著, ISSN 10889051

Although many studies on alternative splicing of specific genes have been reported in the literature, the general mechanism that regulates alternative splicing has not been clearly understood. In this study, we systematically aligned each pair of the 21,076 cDNA sequences of Mus musculus, searched for putative alternative splicing patterns, and constructed a list of potential alternative splicing sites. Two cDNAs are suspected to be alternatively spliced and originating from a common gene if they share most of their region with a high degree of sequence homology, but parts of the sequences are very distinctive or deleted in either cDNA. The list contains the following information: (1) tissue, (2) developmental stage, (3) sequences around splice sites, (4) the length of each gapped region, and (5) other comments. The list is available at http://www.bioinfo.sfc.keio.ac.jp/ intron. Our results have predicted a number of unreported alternatively spliced genes, some of which are expressed only in a specific tissue or at a specific developmental stage. ©2002 by Cold Spring Harbor Laboratory Press.

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Computational challenges in cell simulation: A software engineering approach

Takahashi, K., Yugi, K., Hashimoto, K., Yamada, Y., Pickett, C., and Tomita, M

IEEE Intelligent Systems 17 （ 5 ） 64-71 2002年

研究論文（学術雑誌）, 共著

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Complete mutation analysis panel of the 39 human HOX genes

Kosaki K, Takahashi T, Sasaki K, Tomita M, McGinnis W, and Matsuo N.

Teratology 65 （ 2 ） 50-62 - 62 2002年

研究論文（学術雑誌）, 共著, ISSN 00403709

Background: The HOX gene family consists of highly conserved transcription factors that specify the identity of the body segments along the anteroposterior axis of the embryo. Because the phenotypes of mice with targeted disruptions of Hox genes resemble some patterns of human malformations, mutations in HOX genes have been expected to be associated with a significant number of human malformations. Thus far, however, mutations have been documented in only three of the 39 human HOX genes (HOXD13, HOXA13, and HOXA11) partly because current knowledge on the complete coding sequence and genome structure is limited to only 20 of the 39 human HOX genes. Methods: Taking advantage of the human and mouse draft genome sequences, we attempted to characterize the remaining 19 human HOX genes by bioinformatic analysis including phylogenetic footprinting, the probabilistic prediction method, and comparison of genomic sequences with the complete set of the human anonymous cDNA sequences. Results: We were able to determine the full coding sequences of 19 HOX genes and their genome structure and successfully designed a complete set of PCR primers to amplify the entire coding region of each of the 39 HOX genes from genomic DNA. Conclusions: Our results indicate the usefulness of bioinformatic analysis of the draft genome sequences for clinically oriented research projects. It is hoped that the mutation panel provided here will serve as a launchpad for a new discourse on the genetic basis of human malformations. © 2002 Wiley-Liss, Inc.

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Computer analysis of potential stem structures of rRNA operons in various procaryote genomes

Saito, R., Ozawa, Y., Kuzuno, N., Tomita M.

Gene 259 （ 1-2 ） 217-222 - 222 2001年12月

研究論文（学術雑誌）, 共著, ISSN 03781119

The processing of 16S rRNA and 23S rRNA by RNase III in E.coli is known to involve stem structures formed by both ends of the rRNA. Indeed, complementary nucleotide sequences are usually found at both ends of 16S rRNA and 23S rRNA. However, whether or not this phenomenon exists in various other bacteria has not yet been adequately studied. We have conducted computer analyses of potential stem structures of rRNA operons in 12 bacterial and 3 archaeal genomes, and compared characteristics of the stem structures among these species. We systematically computed free energy values by exhaustively 'annealing' sequences around the 5′ end and sequences around the 3′ end of both 16S rRNA and 23S rRNA genes, in order to predict potential stem structures. The results suggest that rRNAs in most species form stem structures at both ends. Some species, such as A.aeolicus, seem to form unusually stable stem structures. On the other hand, some rRNAs, such as rRNAs of D.radiodurans, seem not to form solid stem structures. This suggests that rRNA processing in those species must employ a reliable targeting mechanism other than recognizing stem structures by RNase III. © 2000 Elsevier Science B.V.

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Gene: Foreword

Bernardi G., Macaya G., Tomita M.

Gene 276 （ 1-2 ） 2001年10月

ISSN 03781119

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Correlation between sequence conservation of the 5′ untranslated region and codon usage bias in Mus musculus genes

Sakai H., Washio T., Saito R., Shinagawa A., Itoh M., Shibata K., Carninci P., Konno H., Kawai J., Hayashizaki Y., Tomita M.

Gene 276 （ 1-2 ） 101 - 105 2001年10月

ISSN 03781119

The codon adaptation index (CAI) values of all protein-coding sequences of the full-length cDNA libraries of Mus musculus were computed based on the RIKEN mouse full-length cDNA library. We have also computed the extent of consensus in flanking sequences of the initiator ATG codon based on the 'relative entropy' values of respective nucleotide positions (from -20 to +12 bp relative to the initiator ATG codon) for each group of genes classified by CAI values. With regard to the two nucleotides positions (-3 and +4) known to be highly conserved in Kozak's consensus sequence, a clear correlation between CAI values and relative entropy values was observed at position -3 but this was not significant at position +4, although a significant correlation was found at position -1 of the consensus sequence. Further, although no correlation was observed at any additional positions, relative entropy values were very high at positions -4, -6, and -8 in genes with high CAI values. These findings suggest that the extent of conservation in the flanking sequence of the initiator ATG codon including Kozak's consensus sequence was an important factor in modulation of the translation efficiency as well as synonymous codon usage bias particularly in highly expressed genes. © 2001 Published by Elsevier Science B.V.

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Functional annotation of a full-length mouse cDNA collection

Kawai J., Shinagawa A., Shibata K., Yoshino M., Itoh M., Ishii Y., Arakawa T., Hara A., Fukunishi Y., Konno H., Adachi J., Fukuda S., Aizawa K., Izawa M., Nishi K., Kiyosawa H., Kondo S., Yamanaka I., Saito T., Okazaki Y., Gojobori T., Bono H., Kasukawa T., Saito R., Kadota K., Matsuda H., Ashburner M., Batalov S., Casavant T., Fleischmann W., Gaasterland T., Gissi C., King B., Kochiwa H., Kuehl P., Lewis S., Matsuo Y., Nikaido I., Pesole G., Quackenbush J., Schriml L.M., Staubli F., Suzuki R., Tomita M., Wagner L., Washio T., Sakai K., Okido T., Furuno M., Aono H., Baldarelli R., Barsh G., Blake J., Boffelli D., Bojunga N., Carninci P., De Bonaldo M.F., Brownstein M.J., Bult C., Fletcher C., Fujita M., Gariboldi M., Gustincich S., Hill D., Hofmann M., Hume D.A., Kamiya M., Lee N.H., Lyons P., Marchionni L., Mashima J., Mazzarelli J., Mombaerts P., Nordone P., Ring B., Ringwald M., Rodriguez I., Sakamoto N., Sasaki H., Sato K., Schönbach C., Seya T., Shibata Y., Storch K.F., Suzuki H., Toyo-Oka K., Wang K.H., Weitz C., Whittaker C., Wilming L., Wynshaw-Boris A., Yoshida K., Hasegawa Y., Kawaji H., Kohtsuki S., Hayashizaki Y.

Nature 409 （ 6821 ） 685-690 - 689 2001年02月

研究論文（学術雑誌）, 共著, ISSN 0028-0836

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

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