Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Proteome-Wide Prediction of Novel DNA/RNA-Binding Proteins Using Amino Acid Composition and Periodicity in the Hyperthermophilic Archaeon Pyrococcus furiosus

Fujishima, K., Komasa, M., Kitamura, S., Suzuki, H., Tomita, M. and Kanai, A.

DNA Research 14 ( 3 ) 91-102 - 102 2007

Research paper (scientific journal), Joint Work, ISSN 13402838

Proteins play a critical role in complex biological systems, yet about half of the proteins in publicly available databases are annotated as functionally unknown. Proteome-wide functional classification using bioinformatics approaches thus is becoming an important method for revealing unknown protein functions. Using the hyperthermophilic archaeon Pyrococcus furiosus as a model species, we used the support vector machine (SVM) method to discriminate DNA/RNA-binding proteins from proteins with other functions, using amino acid composition and periodicities as feature vectors. We defined this value as the composition score (CO) and periodicity score (PD). The P. furiosus proteins were classified into three classes (I-III) on the basis of the two-dimensional correlation analysis of CO score and PD score. As a result, approximately 87 of the functionally known proteins categorized as class I proteins (CO score + PD score > 0.6) were found to be DNA/RNA-binding proteins. Applying the two-dimensional correlation analysis to the 994 hypothetical proteins in P. furiosus, a total of 151 proteins were predicted to be novel DNA/RNA-binding protein candidates. DNA/RNA-binding activities of randomly chosen hypothetical proteins were experimentally verified. Six out of seven candidate proteins in class I possessed DNA/RNA-binding activities, supporting the efficacy of our method. © The Author 2007. Kazusa DNA Research Institute.

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Evolution of ribonuclease H genes in prokaryotes to avoid inheritance of redundant genes

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

BMC Evolutionary Biology 7 128 2007

Research paper (scientific journal), Joint Work

Background. A theoretical model of genetic redundancy has proposed that the fates of redundant genes depend on the degree of functional redundancy, and that functionally redundant genes will not be inherited together. However, no example of actual gene evolution has been reported that can be used to test this model. Here, we analyzed the molecular evolution of the ribonuclease H (RNase H) family in prokaryotes and used the results to examine the implications of functional redundancy for gene evolution. Results. In prokaryotes, RNase H has been classified into RNase HI, HII, and HIII on the basis of amino acid sequences. Using 353 prokaryotic genomes, we identified the genes encoding the RNase H group and examined combinations of these genes in individual genomes. We found that the RNase H group may have evolved in such a way that the RNase HI and HIII genes will not coexist within a single genome - in other words, these genes are inherited in a mutually exclusive manner. Avoiding the simultaneous inheritance of the RNase HI and HIII genes is remarkable when RNase HI contains an additional non-RNase H domain, double-stranded RNA, and an RNA-DNA hybrid-binding domain, which is often observed in eukaryotic RNase H1. This evolutionary process may have resulted from functional redundancy of these genes, because the substrate preferences of RNase HI and RNase HIII are similar. Conclusion. We provide two possible evolutionary models for RNase H genes in which functional redundancy contributes to the exclusion of redundant genes from the genome of a species. This is the first empirical study to show the effect of functional redundancy on changes in gene constitution during the course of evolution. © 2007 Kochiwa et al; licensee BioMed Central Ltd.

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Indeterminacy of Reverse Engineering of Gene Regulatory Networks: The Curse of Gene Elasticity

Krishnan, A., Giuliani, A. and Tomita, M

PLoS ONE 2 ( 6 ) e562 2007

Research paper (scientific journal), Joint Work

Background. Gene Regulatory Networks (GRNs) have become a major focus in interest in recent years. A number of reverse engineering approaches have been developed to help uncover the regulatory networks giving rise to the observed gene expression profiles. However, this is an overspecified problem due to the fact that more than one genotype (network wiring) can give rise to the same phenotype. We refer to this phenomenon as "gene elasticity." In this work, we study the effect of this particular problem on the pure, data-driven inference of gene regulatory networks. Methodology. We simulated a four-gene network in order to produce "data" (protein levels) that we use in lieu of real experimental data. We then optimized the network connections between the four genes with a view to obtain the original network that gave rise to the data. We did this for two different cases: one in which only the network connections were optimized and the other in which both the network connections as well as the kinetic parameters (given as reaction probabilities in our case) were estimated. We observed that multiple genotypes gave rise to very similar protein levels. Statistical experimentation indicates that it is impossible to differentiate between the different networks on the basis of both equilibrium as well as dynamic data. Conclusions. We show explicitly that reverse engineering of GRNs from pure expression data is an indeterminate problem. Our results suggest the unsuitability of an inferential, purely data-driven approach for the reverse engineering transcriptional networks in the case of gene regulatory networks displaying a certain level of complexity. © 2007 Krishnan et al.

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Restauro-G: A Rapid Genome Re-Annotation System for Comparative Genomics

Tamaki, S., Arakawa, K., Kono, N. and Tomita, M.

Genomics Proteomics Bioinformatics 5; 53-58(2007) 5 ( 1 ) 53-58 - 58 2007

Research paper (scientific journal), Joint Work, ISSN 16720229

Annotations of complete genome sequences submitted directly from sequencing projects are diverse in terms of annotation strategies and update frequencies. These inconsistencies make comparative studies difficult. To allow rapid data preparation of a large number of complete genomes, automation and speed are important for genome re-annotation. Here we introduce an open-source rapid genome re-annotation software system, Restauro-G, specialized for bacterial genomes. Restauro-G re-annotates a genome by similarity searches utilizing the BLAST-Like Alignment Tool, referring to protein databases such as UniProt KB, NCBI nr, NCBI COGs, Pfam, and PSORTb. Re-annotation by Restauro-G achieved over 98% accuracy for most bacterial chromosomes in comparison with the original manually curated annotation of EMBL releases. Restauro-G was developed in the generic bioinformatics workbench G-language Genome Analysis Environment and is distributed at http://restauro-g.iab.keio.ac.jp/under the GNU General Public License. © 2007 Beijing Genomics Institute.

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Computational analysis of microRNA-mediated antiviral defense in humans

Watanabe, Y., Kishi, A., Yachie, N., Kanai, A. and Tomita, M.

FEBS Letters 581 ( 24 ) 4603-4610 - 4610 2007

Research paper (scientific journal), Joint Work, ISSN 00145793

Recent studies have proposed the interesting perspective that viral gene expression is downregulated by host microRNAs (miRNAs), small non-coding RNAs well known as post-transcriptional gene regulators. We computationally predicted human miRNA target sites within 228 human-infecting and 348 invertebrate-infecting virus genomes, and we observed that human-infecting viruses were more likely than invertebrate-infecting ones to be targeted by human miRNAs. We listed 62 possible human miRNA-targeted viruses from 6 families, most of which consisted of single-stranded RNA viruses. These results suggest that miRNAs extensively mediate antiviral defenses in humans. © 2007 Federation of European Biochemical Societies.

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Polymer Entrapment in Polymerized Silicate for Preparing Highly Stable Capillary Coatings for CE and CE-MS

Monton, M., Tomita, M., Soga, T. and Ishihama, Y.

Analytical Chemistry 79 ( 20 ) 7838-7844 - 7844 2007

Research paper (scientific journal), Joint Work, ISSN 00032700

An easy-to-implement capillary coating strategy based on polymer entrapment in the network of polymerized silicate is described. In this manner, cationic polymers are tightiy fixed onto the inner wall of the capillary for electroosmotic flow control without necessitating complex surface modification chemistries. The resulting coated capillary exhibited good stability over a wide range of pH, good reproducibility, strong endurance in more than 300 electrophoretic runs, and tolerance of commonly employed organic solvent additives in CE. Applications in CE-MS analysis of biologically important anions as well as sample enrichment are shown. Additionally, it was used as a durable base for attachment of multiple layers of charged polymers on the wall, via electrostatic interaction with the preceding layer. Thus, two novel types of highly stable coated capillaries, one with anodic EOF and the other cathodic, were developed. © 2007 American Chemical Society.

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Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-modified Metal Oxide Chromatography for Nano-LC-MS/MS in Proteomics Applications

Sugiyama, N., Masuda, T., Shinoda, K., Nakamura, A., Tomita, M. and Ishihama, Y.

Mol Cell Proteomics 6 ( 6 ) 1103-1109 - 1109 2007

Research paper (scientific journal), Joint Work, ISSN 15359476

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and β-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and β-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Statistical Analysis of Gene Expression in Innate Immune Responses: Dynamic Interactions between MicroRNA and Signaling Molecules

Piras, V., Selvarajoo, K., Fujikawa, N., Choi, S., Tomita, M., Giuliani, A. and Tsuchiya, M.

Genomics and Informatics 5 107-112 2007

Research paper (scientific journal), Joint Work

Multiple high throughput analyses monitor the response of E. coli to perturbations

Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., Hirasawa, T., Naba, M., Hirai, K., Hoque, A., Yee, Ho, Pei., Kakazu, Y., Sugawara, K., Igarashi, S., Harada, S., Masuda, T., Sugiyama, N., Togashi, T., Hasegawa, M., Takai, T., Yugi, K., Arakawa, K., Iwata, N., Toya, Y., Nakayama, Y., Nishioka, T., Shimizu, K., Mori, H. and Tomita, M.

Science 316 ( 5824 ) 593-597 - 597 2007

Research paper (scientific journal), Joint Work, ISSN 00368075

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

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Network scaling invariants help to elucidate basic topological principles of proteins

Krishnan, A., Giuliani, A., Zbilut, J.P. and Tomita, M.

Journal of Proteome Research 6 ( 10 ) 3924-3934 - 3934 2007

Research paper (scientific journal), Joint Work, ISSN 15353893

The structural architecture of proteins continues to be an area of active research. Despite the difference in models dealing with the way proteins fold into their tertiary structures, it is recognized that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits. However, there are a number of different definitions of what comprises an independent subunit. In the belief that an unequivocal definition of a domain must be based on the most fundamental property of protein 3D structure, namely, the adjacency matrix of inter-residues contact, we adopt a network representation of the protein. In this work, we used a well-established, global method for identifying modules in networks, without any specific reference to the kind of network being analyzed. The algorithm converges toward the maximization of the modularity of the given protein network and, in doing so, allows the representation of the residues of the protein in terms of their intramodule degree, z, and participation coefficient, P. We demonstrate that the labeling of residues in terms of these invariants allows for information-rich representations of the studied proteins as well as to sketch a new way to link sequence, structure, and the dynamical properties of proteins. We discovered a strong invariant character of protein molecules in terms of P/z characterization, pointing to a common topological design of all protein structures. This invariant representation, applied to different protein systems, enabled us to identify the possible functional role of high P/z residues during the folding process. Additionally, we observe a hierarchical behavior of protein structural organization that provides a sequence-secondary-tertiary structure link. The discovery of similar and repeatable scaling laws at different level of definitions going from hydrophobicity patterning along the sequence up to the size of an autonomous folding unit (AFU) and general contact distribution of the entire molecule suggest a hierarchical-like behavior of protein architecture. This implies the possibility to select different privileged scales of observation for deriving useful information on protein systems. © 2007 American Chemical Society.

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Inferring rules of Escherichia coli translational efficiency using an artificial neural network

Mori, K., Saito, R., Kikuchi, S. and Tomita, M.

BioSystems 90 ( 2 ) 414-420 - 420 2007

Research paper (scientific journal), Joint Work, ISSN 03032647

Although the machinery for translation initiation in Escherichia coli is very complicated, the translational efficiency has been reported to be predictable from upstream oligonucleotide sequences. Conventional models have difficulties in their generalization ability and prediction nonlinearity and in their ability to deal with a variety of input attributions. To address these issues, we employed structural learning by artificial neural networks to infer general rules for translational efficiency. The correlation between translational activities measured by biological experiments and those predicted by our method in the test data was significant (r = 0.78), and our method uncovered underlying rules of translational activities and sequence patterns from the obtained skeleton structure. The significant rules for predicting translational efficiency were (1) G- and A-rich oligonucleotide sequences, resembling the Shine-Dalgarno sequence, at positions -10 to -7; (2) first base A in the initiation codon; (3) transport/binding or amino acid metabolism gene function; (4) high binding energy between mRNA and 16S rRNA at positions -15 to -5. An additional inferred novel rule was that C at position -1 increases translational efficiency. When our model was applied to the entire genomic sequence of E. coli, translational activities of genes for metabolism and translational were significantly high. © 2006 Elsevier Ireland Ltd. All rights reserved.

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Computational Methods for MicroRNA Target Prediction

Watanabe, Y., Tomita, M. and Kanai, A.

Methods in Enzymology 427 65-86 - 86 2007

Research paper (scientific journal), Joint Work, ISSN 00766879

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Large numbers of miRNAs have been identified in a wide range of species, and most of them are known to downregulate translation of messenger RNAs (mRNAs) via imperfect binding of the miRNA to a specific site or sites in the 3′ untranslated region (UTR) of the mRNA. Identification of genes targeted by miRNAs is widely believed to be an important step toward understanding the role of miRNAs in gene regulatory networks. As part of the effort to understand interactions between miRNAs and their targets, computational algorithms have been developed based on observed rules for features such as the degree of hybridization between the two RNA molecules. These in silico approaches provide important tools for miRNA target detection, and together with experimental validation, help to reveal regulated targets of miRNAs. Here, we summarize the knowledge that has been accumulated about the principles of target recognition by miRNAs and the currently available computational methodologies for prediction of miRNA target genes. © 2007 Elsevier Inc. All rights reserved.

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Selection effects on the positioning of genes and gene structures from the interplay of replication and transcription in bacterial genomes

Arakawa, K. and Tomita, M.

Evolutionary Bioinformatics 3 279-286 - 286 2007

Research paper (scientific journal), Joint Work

Bacterial chromosomes are partly shaped by the functional requirements for effi cient replication, which lead to strand bias as commonly characterized by the excess of guanines over cytosines in the leading strand. Gene structures are also highly organized within bacterial genomes as a result of such functional constraints, displaying characteristic positioning and structuring along the genome. Here we analyze the gene structures in completely sequenced bacterial chromosomes to observe the positional constraints on gene orientation, length, and codon usage with regard to the positions of replication origin and terminus. Selection on these gene features is different in regions surrounding the terminus of replication from the rest of the genome, but the selection could be either positive or negative depending on the species, and these positional effects are partly attributed to the A-T enrichment near the terminus. Characteristic gene structuring relative to the position of replication origin and terminus is commonly observed among most bacterial species with circular chromosomes, and therefore we argue that the highly organized gene positioning as well as the strand bias should be considered for genomics studies of bacteria.

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In silico screening of archaeal tRNA-encoding genes having multiple introns with bulge-helix-bulge splicing motifs

Sugahara, J., Yachie, N., Arakawa, K. and Tomita, M.

RNA 13 ( 5 ) 671-681 - 681 2007

Research paper (scientific journal), Joint Work, ISSN 13558382

In archaeal species, several transfer RNA genes have been reported to contain endogenous introns. Although most of the introns are located at anticodon loop regions between nucleotide positions 37 and 38, a number of introns at noncanonical sites and six cases of tRNA genes containing two introns have also been documented. However, these tRNA genes are often missed by tRNAscan-SE, the software most widely used for the annotation of tRNA genes. We previously developed SPLITS, a computational tool to identify tRNA genes containing one intron at a noncanonical position on the basis of its discriminative splicing motif, but the software was limited in the detection of tRNA genes with multiple introns at noncanonical sites. In this study, we initially updated the system as SPLITSX in order to correctly predict known tRNA genes as well as novel ones with multiple introns. By a comprehensive search for tRNA genes in 29 archaeal genomes using SPLITSX, we listed 43 novel candidates that contain introns at noncanonical sites. As a result, 15 contained two introns and three contained three introns within the respective putative tRNA genes. Moreover, the candidates completely complemented all the codons of two archaeal species of uncultured methanogenic archaeon, RC-I and Thermofilum pendens Hrk 5, with novel candidates that were not detectable by tRNAscan-SE alone. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 RNA Society.

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Inhibitory effect of phosphoenolpyruvate on glycolytic enzymes in Escherichia coli

Ogawa, T., Mori, H., Tomita, M. and Yoshino, M.

Research in Microbiology 158 ( 2 ) 159-163 - 163 2007

Research paper (scientific journal), Joint Work, ISSN 09232508

For analyzing the control of energy metabolism in Escherichia coli, we carried out kinetic analyses of glycolytic enzymes purified from the overexpressing clones of E. coli K12 W3110 that were constructed with the vector pCA24N. Phosphoenolpyruvate (PEP) acted as an effective inhibitor of enzymes of the preparatory phase in glycolysis. Glucokinase was potently inhibited by PEP in a competitive manner with respect to ATP: the Ki value for PEP was 0.1 mM. PEP further inhibited phosphoglucoisomerase to a lesser extent, and phosphofructokinase A and aldolase A with 10-fold the Ki values of glucokinase and phosphoglucoisomerase. Glucose is incorporated into E. coli through two pathways: the PTS (PEP-dependent phosphotransferase system) and the glucokinase reaction. PEP, a potent inhibitor of E. coli glucokinase, unlike most eukaryotic hexokinases, can act as a signal molecule controlling glucose uptake and glycolytic flux in cells. © 2006 Elsevier Masson SAS. All rights reserved.

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Simulation of developmental changes in action potentials with ventricular cell models

Itoh, H., Naito, Y. and Tomita, M.

Systems and Synthetic Biology 1 ( 1 ) 11-23 - 23 2007

Research paper (scientific journal), Joint Work, ISSN 18725325

During cardiomyocyte development, early embryonic ventricular cells show spontaneous activity that disappears at a later stage. Dramatic changes in action potential are mediated by developmental changes in individual ionic currents. Hence, reconstruction of the individual ionic currents into an integrated mathematical model would lead to a better understanding of cardiomyocyte development. To simulate the action potential of the rodent ventricular cell at three representative developmental stages, quantitative changes in the ionic currents, pumps, exchangers, and sarcoplasmic reticulum (SR) Ca2+ kinetics were represented as relative activities, which were multiplied by conductance or conversion factors for individual ionic systems. The simulated action potential of the early embryonic ventricular cell model exhibited spontaneous activity, which ceased in the simulated action potential of the late embryonic and neonatal ventricular cell models. The simulations with our models were able to reproduce action potentials that were consistent with the reported characteristics of the cells in vitro. The action potential of rodent ventricular cells at different developmental stages can be reproduced with common sets of mathematical equations by multiplying conductance or conversion factors for ionic currents, pumps, exchangers, and SR Ca 2+ kinetics by relative activities. © Springer Science + Business Media B.V. 2007.

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Prediction of liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome usina artificial neural networks

Shinoda K., Sugimoto M., Yachie N., Sugiyama N., Masuda T., Robert M., Soga T., Tomita M.

Journal of Proteome Research 5 ( 12 ) 3312 - 3317 2006.12

ISSN 15353893

We developed a computational method to predict the retention times of peptides in HPLC using artificial neural networks (ANN). We performed stepwise multiple linear regressions and selected for ANN input amino acids that significantly affected the LC retention time. Unlike conventional linear models, the trained ANN accurately predicted the retention time of peptides containing up to 50 amino acid residues. In 834 peptides, there was a strong correlation (R2= 0.928) between measured and predicted retention times. We demonstrated the utility of our method by the prediction of the retention time of 121 273 peptides resulting from LysC-digestion of the Escherichia coli proteome. Our approach is useful for the proteome-wide characterization of peptides and the identification of unknown peptide peaks obtained in proteome analysis. © 2006 American Chemical Society.

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Multiresolution analysis uncovers hidden conservation of properties in structurally and functionally similar protiens

Chua G.H., Krishnan A., Li K.B., Tomita M.

Journal of Bioinformatics and Computational Biology 4 ( 6 ) 1245 - 1267 2006.12

ISSN 02197200

Physicochemcial properties of amino acids are important factors in determining protein structure and function. Most approaches make use of averaged properties over entire domains or even proteins to analyze their structure or function. This level of coarseness tends to hide the richness of the variability in the different properties across functional domains. This paper studies the conservation of physicochemical properties in a functionally similar family of proteins using a novel wavelet-based technique known as multiresolution analysis. Such an analysis can help uncover characteristics that can otherwise remain hidden. We have studied the protein kinase family of sequences and our findings are as follows: (a) a number of different properties are conserved over the functional catalytic domain irrespective of the sequence identities; (b) conservation of properties can be observed at different frequency levels and they agree well with the known structural/ functional properties of the subdomains for the protein kinase family; (c) structural differences between the different kinase family members are reflected in the waveforms; and (d) functionally important mutations show distortions in the waveforms of conserved properties. The potential usefulness of the above findings in identifying functionally similar sequences in the twilight and midnight zones is demonstrated through a simple prediction model for the protein kinase family which achieved a recall of 93.7% and a precision of 96.75% in cross-validation tests. © Imperial College Press.

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HybGFS: a hybrid method for genome-fingerprint scanning

Shinoda, Kosaku ; Yachie, Nozomu ; Masuda, Takeshi ; Sugiyama, Naoyuki ; Sugimoto, Masahiro ; Soga, Tomoyoshi ; Tomita, Masaru

BMC Bioinformatics 7 8 2006.10

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

Background: Protein identification based on mass spectrometry (MS) has previously been performed using peptide mass fingerprinting (PMF) or tandem MS (MS/MS) database searching. However, these methods cannot identify proteins that are not already listed in existing databases. Moreover, the alternative approach of de novo sequencing requires costly equipment and the interpretation of complex MS/MS spectra. Thus, there is a need for novel high-throughput protein-identification methods that are independent of existing predefined protein databases. Results: Here, we present a hybrid method for genome-fingerprint scanning, known as HybGFS. This technique combines genome sequence-based peptide MS/MS ion searching with liquid-chromatography elution-time (LC-ET) prediction, to improve the reliability of identification. The hybrid method allows the simultaneous identification and mapping of proteins without a priori information about their coding sequences. The current study used standard LC-MS/MS data to query an in silico-generated six-reading-frame translation and the enzymatic digest of an entire genome. Used in conjunction with precursor/product ion-mass searching, the LC-ETs increased confidence in the peptide-identification process and reduced the number of false-positive matches. The power of this method was demonstrated using recombinant proteins from the Escherichia coli K12 strain. Conclusion: The novel hybrid method described in this study will be useful for the large-scale experimental confirmation of genome coding sequences, without the need for transcriptome-level expression analysis or costly MS database searching. © 2006 Shinoda et al; licensee BioMed Central Ltd.

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Evaluation of metabolic alteration in transgenic rice overexpressing dihydroflavonol-4-reductase

Takahashi, Hideyuki ; Hayashi, Mitsunori ; Goto, Fumiyuki ; Sato, Shigeru ; Soga, Tomoyoshi ; Nishioka, Takaaki ; Tomita, Masaru ; Kawai-Yamada, Maki ; Uchimiya, Hirofumi

Annals of Botany 98 ( 4 ) 819-825 - 825 2006.10

Research paper (scientific journal), Joint Work, ISSN 0305-7364

Background and Aims: Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars. Methods: Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified. Key Results: In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6- bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased. Conclusions: The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells. © The Author 2006. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

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