Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Over-representation of Chi sequences caused by di-codon increase in Escherichia coli K-12

Uno, Reina ; Nakayama, Yoichi ; Tomita, Masaru

Gene 380 ( 1 ) 30-37 - 37 2006.09

Research paper (scientific journal), Joint Work, ISSN 0378-1119

Chi sequences (5′-GCTGGTGG-3′) are cis-acting 8 bp sequence elements that enhance homologous recombination promoted by the RecBCD pathway in Escherichia coli. The genome of E. coli K-12 MG1655 contains 1009 Chi sequences and this frequency far exceeds the expected value for occurrence of an 8 bp sequence in a genome of this size. It is generally thought that the over-representation of Chi sequences indicates that they have been selected for during evolution because of their function in recombination. The genes from three E. coli strains (K-12, O157 and CFT) were classified into three categories (island, match to other E. coli, and backbone). Island genes have a different base composition and codon usage in comparison with those in the backbone genes, therefore they were relatively new and not yet adapted to the base composition patterns and codon usage typical of the recipient genome. The over-representation of Chi sequences was examined by comparing Chi frequencies and codon frequencies between island and backbone genes. The difference in the CTGGTG di-codon frequency between the backbone and island genes was correlated with the frequency of Chi sequences which were translated in the Leu-Val (-G|CTG|GTG|G-) reading frame in the K-12 strain. These results suggest that the main reading frame of Chi sequences increased as a result of the di-codon CTG-GTG increasing under a genome-wide pressure for adapting to the codon usage and base composition of the E. coli K-12 strain, and that the RecBCD recombinase might adjust its recognition sequence to a frequently occurring oligomer such as G-CTG-GTG-G. © 2006 Elsevier B.V. All rights reserved.

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Metabolomics approach for enzyme discovery

Saito, Natsumi ; Robert, Martin ; Kitamura, Sayaka ; Baran, Richard ; Soga, Tomoyoshi ; Mori, Hirotada ; Nishioka, Takaaki ; Tomita, Masaru

Journal of Proteome Research 5 ( 8 ) 1979-1987 - 1987 2006.08

Research paper (scientific journal), Joint Work, ISSN 1535-3893

The search for novel enzymes is an important but difficult task in functional genomics. Here, we present a systematic method based on in vitro assays in combination with metabolite profiling to discover novel enzymatic activities. A complex mixture of metabolites is incubated with purified candidate proteins and the reaction mixture is subsequently profiled by capillary electrophoresis electrospray ionization mass spectrometry (CE-MS). Specific changes in the metabolite composition can directly suggest the presence of an enzymatic activity while subsequent identification of the compounds whose level changed specifically can pinpoint the actual substrate(s) and product(s) of the reaction. We first evaluated the method using several Escherichia coli metabolic enzymes and then applied it to the functional screening of uncharacterized proteins. In this manner, YbhA and YbiV proteins were found to display both phosphotransferase and phosphatase activity toward different sugars/sugar phosphates. Our approach should be broadly applicable and useful for enzyme discovery in any system. © 2006 American Chemical Society.

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A simplified method for power-law modelling of metabolic pathways from time-course data and steady-state flux profiles

Kitayama, Tomoya ; Kinoshita, Ayako ; Sugimoto, Masahiro ; Nakayama, Yoichi ; Tomita, Masaru

Theor Biol Med Model 3 24 2006.07

Research paper (scientific journal), Joint Work, ISSN 1742-4682

Background: In order to improve understanding of metabolic systems there have been attempts to construct S-system models from time courses. Conventionally, non-linear curve-fitting algorithms have been used for modelling, because of the non-linear properties of parameter estimation from time series. However, the huge iterative calculations required have hindered the development of large-scale metabolic pathway models. To solve this problem we propose a novel method involving power-law modelling of metabolic pathways from the Jacobian of the targeted system and the steady-state flux profiles by linearization of S-systems. Results: The results of two case studies modelling a straight and a branched pathway, respectively, showed that our method reduced the number of unknown parameters needing to be estimated. The time-courses simulated by conventional kinetic models and those described by our method behaved similarly under a wide range of perturbations of metabolite concentrations. Conclusion: The proposed method reduces calculation complexity and facilitates the construction of large-scale S-system models of metabolic pathways, realizing a practical application of reverse engineering of dynamic simulation models from the Jacobian of the targeted system and steady-state flux profiles. © 2006 Kitayama et al; licensee BioMed Central Ltd.

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Modeling Hsp70-mediated Protein Folding

Hu B, Mayer MP, Tomita M.

Biophysical Journal 91 ( 2 ) 496-507 - 507 2006.07

Research paper (scientific journal), Joint Work, ISSN 00063495

The Hsp70 chaperone system is the major molecular chaperone system that assists protein-folding processes in all cells. To understand these processes, we analyzed the kinetic characteristics of the Escherichia coli homologs of this chaperone system during folding of a denatured protein using computer simulations and compared the results with in vitro refolding experiments. Rate constants used for the model were derived from recent literature or were determined and scrutinized for their applicability to the refolding reaction. Our simulation results are consistent with reported laboratory experiments, not only simulating the refolding reaction of wild-type proteins but also the behavior of mutant variants. Variation of kinetic parameters and concentrations of components of the Hsp70 system demonstrate the robustness of the chaperone system in assisting protein folding. Furthermore, the importance of the synergistic stimulation of the ATPase activity of Hsp70 is demonstrated. The limitations of our kinetic model indicate sore spots in our understanding of this chaperone system. Our model provides a platform for further research on chaperone action and the mechanism of chaperone-assisted refolding of denatured proteins. © 2006 by the Biophysical Society.

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Comparative analysis of base correlations in 5′ untranslated regions of various species

Osada Y., Saito R., Tomita M.

Gene 375 ( 1-2 ) 80 - 86 2006.06

ISSN 03781119

Translational initiation signals, such as Shine-Dalgarno (SD) sequences in bacteria and Kozak consensus sequences in vertebrates, direct ribosomes to initiate protein synthesis from mRNAs. Investigating sequence characteristics of these signals is important, particularly to infer translational initiation mechanisms. Although various statistical analyses of translational initiation signals have been done, few have focused on base correlations that assess base dependencies in the signal sequences. We used relative entropy and mutual information to analyze base conservation and correlation, respectively, in the 5′ UTRs of various species. In eukaryotes, we found peaks of relative entropy at - 3 from the translational start site but no peak of mutual information at that position, indicating that the base at that position (known as the core base of the Kozak sequence) is well conserved but not correlated with neighboring bases and thus functions as a single base. We observed unexpected peaks of mutual information between positions - 2 and - 1 in most eukaryotes. Surprisingly these base correlation also occurred in some bacteria and archaea, although there were no base preferences at neither position. Various dinucleotide patterns existed at these positions, and the correlation between bases at - 2 and - 1 may be relevant to the context of translational initiation. Because dinucleotide patterns of correlated pairs of nucleotides at - 2 and - 1 were not unique within respective organisms, the correlation could not be found when analyzing single-nucleotide conservation. Therefore, mutual information allowed us to discover signals that were not found by simply analyzing base conservation. © 2006 Elsevier B.V. All rights reserved.

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Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption

Soga, Tomoyoshi; Baran, Richard; Suematsu, Makoto; Ueno, Yuki; Ikeda, Satsuki; Sakurakawa, Tadayuki; Kakazu, Yuji; Ishikawa, Takamasa; Robert, Martin; Nishioka, Takaaki; Tomita, Masaru

Journal of Biological Chemistry 281 ( 24 ) 16768-16776 - 16776 2006.06

Research paper (scientific journal), Joint Work, ISSN 0021-9258

Metabolomics is an emerging tool that can be used to gain insights into cellular and physiological responses. Here we present a metabolome differential display method based on capillary electrophoresis time-of-flight mass spectrometry to profile liver metabolites following acetaminophen-induced hepatotoxicity. We globally detected 1,859 peaks in mouse liver extracts and highlighted multiple changes in metabolite levels, including an activation of the ophthalmate biosynthesis pathway. We confirmed that ophthalmate was synthesized from 2-aminobutyrate through consecutive reactions with γ-glutamylcysteine and glutathione synthetase. Changes in ophthalmate level in mouse serum and liver extracts were closely correlated and ophthalmate levels increased significantly in conjunction with glutathione consumption. Overall, our results provide a broad picture of hepatic metabolite changes following acetaminophen treatment. In addition, we specifically found that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress. Ourmethod can thus pinpoint specific metabolite changes and provide insights into the perturbation of metabolic pathways on a large scale and serve as a powerful new tool for discovering low molecular weight biomarkers. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Prediction of non-coding and antisense RNA genes in Escherichia coli with Gapped Markov Model

Yachie, Nozomu ; Numata, Koji ; Saito, Rintaro ; Kanai, Akio ; Tomita, Masaru

Gene 372 ( 1-2 ) 171-181 - 181 2006.05

Research paper (scientific journal), Joint Work, ISSN 0378-1119

A new mathematical index was developed to identify and characterize non-coding RNA (ncRNA) genes encoded within the Escherichia coli (E. coli) genome. It was designated the GMMI (Gapped Markov Model Index) and used to evaluate sequence patterns located at the separate positions of consensus sequences, codon biases and/or possible RNA structures on the basis of the Markov model. The GMMI was able to separate a set of known mRNA sequences from a mixture of ncRNAs including tRNAs and rRNAs. Consequently, the GMMI was employed to predict novel ncRNA candidates. At the beginning, possible transcription units were extracted from the E. coli genome using consensus sequences for the sigma70 promoter and the rho-independent terminator. Then, these units were evaluated by using the GMMI. This identified 133 candidate ncRNAs, which contain 29 previously annotated small RNA genes and 46 possible antisense ncRNAs. Furthermore 12 transcripts (including five antisense RNAs) were confirmed according to the expression analysis. These data suggests that the expression of small antisense RNAs might be more common than previously thought in the E. coli genome. © 2006 Elsevier B.V. All rights reserved.

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Large-scale identification of protein-protein interaction of Escherichia coli K-12

Arifuzzaman, M. ; Maeda, M. ; Itoh, A. ; Nishikata, K. ; Takita, C. ; Saito, R. ; Ara, T. ; Nakahigashi, K. ; Huang, H. C. ; Hirai, A. ; Tsuzuki, K. ; Nakamura, S. ; Altaf-Ul-Amin, M. ; Oshima, T. ; Baba, T. ; Yamamoto, N. ; Kawamura, T. ; Ioka-Nakamichi, T. ; Kitagawa, M. ; Tomita, M. ; Kanaya, S. ; Wada, C. ; Mori, H. ; Tomita, M

Genome Research 16 ( 5 ) 686-691 - 691 2006.05

Research paper (scientific journal), Joint Work, ISSN 1088-9051

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni 2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology. ©2006 by Cold Spring Harbor Laboratory Press.

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Parameter estimation for stiff equations of biosystems using radial basis function networks

Matsubara, Yoshiya ; Kikuchi, Shinichi ; Sugimoto, Masahiro ; Tomita, Masaru

BMC Bioinformatics 7 11 2006.04

Research paper (scientific journal), Joint Work, ISSN 1471-2105

Background: The modeling of dynamic systems requires estimating kinetic parameters from experimentally measured time-courses. Conventional global optimization methods used for parameter estimation, e.g. genetic algorithms (GA), consume enormous computational time because they require iterative numerical integrations for differential equations. When the target model is stiff, the computational time for reaching a solution increases further. Results: In an attempt to solve this problem, we explored a learning technique that uses radial basis function networks (RBFN) to achieve a parameter estimation for biochemical models. RBFN reduce the number of numerical integrations by replacing derivatives with slopes derived from the distribution of searching points. To introduce a slight search bias, we implemented additional data selection using a GA that searches data-sparse areas at low computational cost. In addition, we adopted logarithmic transformation that smoothes the fitness surface to obtain a solution simply. We conducted numerical experiments to validate our methods and compared the results with those obtained by GA. We found that the calculation time decreased by more than 50% and the convergence rate increased from 60% to 90%. Conclusion: In this work, our RBFN technique was effective for parameter optimization of stiff biochemical models. © 2006 Matsubara et al; licensee BioMed Central Ltd.

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GEM System: automatic prototyping of cell-wide metabolic pathway models from genomes

Arakawa, Kazuharu ; Yamada, Yohei ; Shinoda, Kosaku ; Nakayama, Yoichi ; Tomita, Masaru

BMC Bioinformatics 7 11 2006.03

Research paper (scientific journal), Joint Work, ISSN 1471-2105

Background: Successful realization of a "systems biology" approach to analyzing cells is a grand challenge for our understanding of life. However, current modeling approaches to cell simulation are labor-intensive, manual affairs, and therefore constitute a major bottleneck in the evolution of computational cell biology. Results: We developed the Genome-based Modeling (GEM) System for the purpose of automatically prototyping simulation models of cell-wide metabolic pathways from genome sequences and other public biological information. Models generated by the GEM System include an entire Escherichia coli metabolism model comprising 968 reactions of 1195 metabolites, achieving 100% coverage when compared with the KEGG database, 92.38% with the EcoCyc database, and 95.06% with iJR904 genome-scale model. Conclusion: The GEM System prototypes qualitative models to reduce the labor-intensive tasks required for systems biology research. Models of over 90 bacterial genomes are available at our web site. © 2006 Arakawa et al; licensee BioMed Central Ltd.

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Characteristics and clustering of human ribosomal protein genes

Ishii, Kyota, Washio, Takanori, Uechi, Tamayo, Yoshihama, Maki, Kenmochi, Naoya, Tomita, Masaru

Bmc Genomics 7 37 2006.02

Research paper (scientific journal), Joint Work, ISSN 1471-2164

Background: The ribosome is a central player in the translation system, which in mammals consists of four RNA species and 79 ribosomal proteins (RPs). The control mechanisms of gene expression and the functions of RPs are believed to be identical. Most RP genes have common promoters and were therefore assumed to have a unified gene expression control mechanism. Results: We systematically analyzed the homogeneity and heterogeneity of RP genes on the basis of their expression profiles, promoter structures, encoded amino acid compositions, and codon compositions. The results revealed that (1) most RP genes are coordinately expressed at the mRNA level, with higher signals in the spleen, lymph node dissection (LND), and fetal brain. However, 17 genes, including the P protein genes (RPLP0, RPLP1, RPLP2), are expressed in a tissue-specific manner. (2) Most promoters have GC boxes and possible binding sites for nuclear respiratory factor 2, Yin and Yang 1, and/or activator protein 1. However, they do not have canonical TATA boxes. (3) Analysis of the amino acid composition of the encoded proteins indicated a high lysine and arginine content. (4) The major RP genes exhibit a characteristic synonymous codon composition with high rates of G or C in the third-codon position and a high content of AAG, CAG, ATC, GAG, CAC, and CTG. Conclusion: Eleven of the RP genes are still identified as being unique and did not exhibit at least some of the above characteristics, indicating that they may have unknown functions not present in other RP genes. Furthermore, we found sequences conserved between human and mouse genes around the transcription start sites and in the intronic regions. This study suggests certain overall trends and characteristic features of human RP genes. © 2006 Ishii et al; licensee BioMed Central Ltd.

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Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein

Kanai A., Sato A., Imoto J., Tomita M.

Biochemical Journal 393 ( 1 ) 373 - 379 2006.01

ISSN 02646021

Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75°C) with an apparent dissociation constant of 0.6 μM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them. © 2006 Biochemical Society.

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Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirements

Kochiwa H., Itaya M., Tomita M., Kanai A.

FEBS Journal 273 ( 2 ) 420 - 429 2006.01

ISSN 1742464X

Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA-RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg2+ ions, Caenorhabditis elegans has four RNase H1-related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1-related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene-specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg2+ ions, C. elegans has another enzyme that shows preference for Mn2+ ion as a cofactor. We characterized this Mn 2+-dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism. © 2005 FEBS.

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Computational analysis of microRNA targets in Caenorhabditis elegans

Watanabe, Yuka, Yachie, Nozomu, Numata, Koji, Saito, Rintaro, Kanai, Akio, Tomita, Masaru

Gene 365 ( 1-2 SPEC. ISS. ) 2-10 - 10 2006.01

Research paper (scientific journal), Joint Work, ISSN 0378-1119

MicroRNAs (miRNAs) are endogenous ∼ 22-nucleotide (nt) non-coding RNAs that post-transcriptionally regulate the expression of target genes via hybridization to target mRNA. Using known pairs of miRNA and target mRNA in Caenorhabditis elegans, we first performed computational analysis for specific hybridization patterns between these two RNAs. We counted the numbers of perfectly complementary dinucleotide sequences and calculated the free energy within complementary base pairs of each dinucleotide, observed by sliding a 2-nt window along all nucleotides of the miRNA-mRNA duplex. We confirmed not only strong base pairing within the 5′ region of miRNAs (nts 1-8) in C. elegans, but also the required mismatch within the central region (nt 9 or nt 10), and we found weak binding within the 3′ region (nts 13-14). We also predicted 687 possible miRNA target transcripts, many of which are thought to be involved in C. elegans development, by combining the abovementioned hybridization tendency with the following analyses: (1) prediction of the miRNA-mRNA duplex with free-energy minimization; (2) identification of the complementary pattern within the miRNA-mRNA duplex; (3) conservation of target sites between C. elegans and C. briggsae, a related soil nematode; and (4) extraction of mRNA candidates with multiple target sites. Rigorous tests using shuffled miRNA controls supported these predictions. Our results suggest that miRNAs recognize their target mRNAs by their hybridization pattern and that many target mRNAs may be regulated through a combination of several specific miRNA target sites in C. elegans. © 2005 Elsevier B.V. All rights reserved.

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SPLITS: a new program for predicting split and intron-containing tRNA genes at the genome level

Sugahara, Junichi ; Yachie, Nozomu ; Sekine, Yasuhiko ; Soma, Akiko ; Matsui, Motomu ; Tomita, Masaru ; Kanai, Akio

In Silico Biology 6 ( 5 ) 411-418 - 418 2006

Research paper (scientific journal), Joint Work, ISSN 1386-6338

In the archaea, some tRNA precursors contain intron(s) not only in the anticodon loop region but also in diverse sites of the gene (intron-containing tRNA or cis-spliced tRNA). The parasite Nanoarchaeum equitans, a member of the Nanoarchaeota kingdom, creates functional tRNA from separate genes, one encoding the 5'-half and the other the 3'-half (split tRNA or trans-spliced tRNA). Although recent genome projects have revealed a huge amount of nucleotide sequence data in the archaea, a comprehensive methodology for intron-containing and split tRNA searching is yet to be established. We therefore developed SPLITS, which is aimed at searching for any type of tRNA gene and is especially focused on intron-containing tRNAs or split tRNAs at the genome level. SPLITS initially predicts the bulge-helix-bulge splicing motif (a well-known, required structure in archaeal pre-tRNA introns) to determine and remove the intronic regions of tRNA genes. The intron-removed DNA sequences are automatically queried to tRNAscan-SE. SPLITS can predict known tRNAs with single introns located at unconventional sites on the genes (100%, tRNAs with double introns (85.7%), and known split tRNAs (100%). Our program will be very useful for identifying novel tRNA genes after completion of genome projects. The SPLITS source code is freely downloadable at http://splits.iab.keio.ac.jp/. © 2006 IOS Press. All rights reserved.

Genome-wide screening of dioxin-responsive genes in fetal brain: bioinformatic and experimental approaches

Fujita, Hideki ; Samejima, Hazuki ; Kitagawa, Noriyuki ; Mitsuhashi, Takayuki ; Washio, Takanori ; Yonemoto, Junzo ; Tomita, Masaru ; Takahashi, Takao ; Kosaki, Kenjiro

Congenital Anomalies 46 ( 3 ) 135-143 - 143 2006

Research paper (scientific journal), Joint Work, ISSN 0914-3505

Many of the effects of dioxins, which are potent environmental pollutants and teratogens, are mediated through the aryl hydrocarbon receptor, also known as the dioxin receptor. The purpose of the present study was to characterize dioxin-responsive genes in a comprehensive manner using two complementary approaches: bioinformatic analysis and microarray analysis. First, we characterized the overall distribution of the cis-regulatory element for the dioxin-responsive element sequence (DRE) 'gcgtg' within putative promoter regions. We assembled the upstream sequences 10 kb from the transcription start site and evaluated their location and frequency in the human and mouse genomes. Second, we characterized the expression profile of mouse embryonic day 12 fetal brain exposed to 2,3,7,8-tetrarchlorodibenzo-p-dioxin. The distributions of 26 680 DREs among 2843 human genes and 98 711 DREs among 18 541 mouse genes were examined. In both species, the DREs tended to be located close to the transcription start site. Forty genes exhibited significant induction or repression following dioxin exposure in fetal mice. The set of genes exhibited a strong functional coherence, with statistically significant enrichment in organogenesis and the DNA-dependent regulation of transcription, according to Gene Ontology annotations. In both humans and mice, DREs were preferentially distributed close to transcription start sites. Evolutionary conservation of this unique DRE distribution pattern suggests that DREs may be involved in transcriptional regulation. In mice, prenatal dioxin exposure altered the expression of 10 transcription factors, many of which have been documented to play a role in organogenesis. These genes may represent potential mediators of dioxin's effects in fetal tissues. © 2006 The Authors.

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Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

Baba, Tomoya ; Ara, Takeshi ; Hasegawa, Miki ; Takai, Yuki ; Okumura, Yoshiko ; Baba, Miki ; Datsenko, Kirill A. ; Tomita, Masaru ; Wanner, Barry L. ; Mori, Hirotada

Molecular Systems Biology 2 2006.0008. 2006

Research paper (scientific journal), Joint Work, ISSN 1744-4292

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants - the 'Keio collection' - provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/). © 2006 EMBO and Nature Publishing Group.

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GPAC: benchmarking the sensitivity of genome informatics analysis to genome annotation completeness

Arakawa, Kazuharu ; Nakayama, Yoichi ; Tomita, Masaru

In Silico Biology 6 ( 1-2 ) 49-60 - 60 2006

Research paper (scientific journal), Joint Work, ISSN 1386-6338

In view of the recent explosion in genome sequence data, and the 200 or more complete genome sequences currently available, the importance of genome-scale bioinformatics analysis is increasing rapidly. However, computational genome informatics analyses often lack a statistical assessment of their sensitivity to the completeness of the functional annotation. Therefore, a pre-analysis method to automatically validate the sensitivity of computational genome analyses with regard to genome annotation completeness is useful for this purpose. In this report we developed the Gene Prediction Accuracy Classification (GPAC) test, which provides statistical evidence of sensitivity by repeating the same analysis for five different gene groups (classified according to annotation accuracy level), and for randomly sampled gene groups, with the same number of genes as each of the five classified groups. Variability in these results is then assessed, and if the results vary significantly with different data subsets, the analysis is considered "sensitive" to annotation completeness, and careful selection of data is advised prior to the actual in silico analysis. The GPAC test has been applied to the analyses of Sakai et al., 2001, and Ohno et al., 2001, and it revealed that the analysis of Ohno et al. was more sensitive to annotation completeness. It showed that GPAC could be employed to ascertain the sensitivity of an analysis. The GPAC benchmarking software is freely available in the latest G-language Genome Analysis Environment package, at http://www.g-language.org/. © 2006 IOS Press. All rights reserved.

MEGU: pathway mapping Web-service based on KEGG and SVG

Kono, Nobuaki ; Arakawa, Kazuharu ; Tomita, Masaru

In Silico Biology 6 ( 6 ) 621-625 - 625 2006

Research paper (scientific journal), Joint Work, ISSN 1386-6338

Scientific visualization is a key technology to enhance our understanding of huge masses of omics data, especially for cell-wide, high-throughput measurements of the physiological networks. We have developed a web application that visualizes complex omics data of multiple layers simultaneously, including transcriptome, proteome, and metabolome, onto an integrated pathway diagram derived by connecting the individual KEGG pathway maps; the mapped images are generated in Scalable Vector Graphics for easy editing by hand or with computer programs or drawing software. The web application is available at http://megu.iab.keio.ac.jp/. © 2006 IOS Press. All rights reserved.

Identification and expression analysis of putative mRNA-like non-coding RNA in Drosophila

Inagaki S., Numata K., Kondo T., Tomita M., Yasuda K., Kanai A., Kageyama Y.

Genes to Cells 10 ( 12 ) 1163 - 1173 2005.12

ISSN 13569597

One of the most surprising results to emerge from mammalian cDNA sequencing projects is that thousands of mRNA-like non-coding RNAs (ncRNAs) are expressed and constitute at least 10% of poly(A)+ RNAs. In most cases, however, the functions of these RNA molecules remain unclear. To clarify the biological significance of mRNA-like ncRNAs, we computationally screened 11 691 Drosophila melanogaster full-length cDNAs. After eliminating presumable protein-coding transcripts, 136 were identified as strong candidates for mRNA-like ncRNAs. Although most of these putative ncRNAs are found throughout the Drosophila genus, predicted amino acid sequences are not conserved even in related species, suggesting that these transcripts are actually non-coding RNAs. In situ hybridization analyses revealed that 35 of the transcripts are expressed during embryogenesis, of which 27 were detected only in specific tissues including the tracheal system, midgut primordial cells, visceral mesoderm, germ cells and the central and peripheral nervous system. These highly regulated expression patterns suggest that many mRNA-like ncRNAs play important roles in multiple steps of organogenesis and cell differentiation in Drosophila. This is the first report that the majority of mRNA-like ncRNAs in a model organism are expressed in specific tissues and cell types. © 2005 Blackwell Publishing Ltd.

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