Published papers

Multivariate analyses are often used to identify major trends of variation in synonymous codon usage among genes. These analyses need to be performed on properly normalized codon usage data to avoid biases masking this synonymous variation, i.e., gene length, amino acid usage, and codon degeneracy; however, previous studies have failed to do so. In this paper, we demonstrate that the use of alternative normalized data (called 'relative adaptiveness' in the literature) can avoid all these biases and furthermore, can identify more trends of variation among genes, including GC-ending codon usage, GT-ending codon usage, and gene expression level. © 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

The dynamics of the intracellular metabolite concentrations were investigated for the wild type and pykA gene knockout mutant Escherichia coli in responses to the glucose pulse addition during glucose-limited continuous culture and in responses to the ammonia pulse addition during ammonia-limited continuous culture. For this, we developed a new automated rapid sampling device, which enables us to take samples rapidly within a second. The intracellular concentrations of G6P, F6P, 2PG, PEP, OAA, 6PG, Ribu5P, E4P and NADPH were higher for pykA mutant as compared with the wild type under both limited continuous cultures, and the concentrations of PYR, ATP and acetate were much lower for pykA mutant than those of the wild type. These phenomena reflected the fact that the accumulation of PEP caused the increased flux from PEP to OAA and that the accumulated PEP inhibited pfk, which caused the accumulation of G6P and F6P, which in turn increased the flux toward pentose phosphate (PP) pathway and increased the PP pathway metabolite concentrations. Oxygen uptake rate (OUR) was a little higher for pykA mutant as compared with that of its wild type, while CO2 production rate (CER) shows the reverse trend. OUR and CER were much less for NH3-limited condition than that of NH3-rich condition. The intracellular concentrations of PEP, ATP and PYR decreased rapidly within several seconds, whereas the concentrations of G6P, F6P FBP, 6PG, ADP, NADH and NADPH increased after glucose pulse addition during glucose-limited condition for both wild type and pykA knockout mutant. Initial decrease in PEP concentration was considered to be due to PTS system. The intracellular concentration of NADPH decreased after NH 3 pulse addition under NH3-limited condition for both strains, which is due to the fact that NADPH is utilized through glutamate production under NH3 addition. © 2005 Elsevier B.V. All rights reserved.

Background: A GC-compositional strand bias or GC-skew (=(C-G)/(C+G)), where C and G denote the numbers of cytosine and guanine residues, was recently reported near the transcription start sites (TSS) of Arabidopsis genes. However, it is unclear whether other eukaryotic species have equally prominent GC-skews, and the biological meaning of this trait remains unknown. Results: Our study confirmed a significant GC-skew (C > G) in the TSS of Oryza sativa (rice) genes. The full-length cDNAs and genomic sequences from Arabidopsis and rice were compared using statistical analyses. Despite marked differences in the G+C content around the TSS in the two plants, the degrees of bias were almost identical. Although slight GC-skew peaks, including opposite skews (C < G), were detected around the TSS of genes in human and Drosophila, they were qualitatively and quantitatively different from those identified in plants. However, plant-like GC-skew in regions upstream of the translation initiation sites (TIS) in some fungi was identified following analyses of the expressed sequence tags and/or genomic sequences from other species. On the basis of our dataset, we estimated that >70 and 68% of Arabidopsis and rice genes, respectively, had a strong GC-skew (>0.33) in a 100-bp window (that is, the number of C residues was more than double the number of G residues in a +/-100-bp window around the TSS). The mean GC-skew value in the TSS of highly-expressed genes in Arabidopsis was significantly greater than that of genes with low expression levels. Many of the GC-skew peaks were preferentially located near the TSS, so we examined the potential value of GC-skew as an index for TSS identification. Our results confirm that the GC-skew can be used to assist the TSS prediction in plant genomes. Conclusion: The GC-skew (C > G) around the TSS is strictly conserved between monocot and eudicot plants (ie. angiosperms in general), and a similar skew has been observed in some fungi. Highly-expressed Arabidopsis genes had overall a more marked GC-skew in the TSS compared to genes with low expression levels. We therefore propose that the GC-skew around the TSS in some plants and fungi is related to transcription. It might be caused by mutations during transcription initiation or the frequent use of transcription factor-biding sites having a strand preference. In addition, GC-skew is a good candidate index for TSS prediction in plant genomes, where there is a lack of correlation among CpG islands and genes. © 2005 Fujimori et al; licensee BioMed Central Ltd.

We developed a computational technique to assist in the huge-scale identification of charged metabolites. The electrophoretic mobility of metabolites in capillary electrophoresis-mass spectrometry (CE-MS) was predicted from their structure, using an ensemble of artificial neural networks (ANNs). Comparison between relative migration times of 241 various cations measured by CE-MS and predicted by a trained ANN ensemble produced a correlation coefficient of 0.931. When we used our technique to characterize all metabolites listed in the KEGG ligand database, the correct compounds among the top three candidates were predicted in 78.0% of cases. We suggest that this approach can be used for the prediction of the migration time of any cation and that it represents a powerful method for the identification of un characterized CE-MS peaks in metabolome analysis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.