Papers - Tomita, Masaru

Validating the significance of genomic properties of Chi sites from the distribution of all octamers in Escherichia coli

Arakawa K., Uno R., Nakayama Y., Tomita M.

Gene 392 ( 1-2 ) 239 - 246 2007.05

ISSN  03781119

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Chi sites (5′-GCTGGTGG-3′) are homologous recombinational hotspot octamer sequences, which attenuate the exonuclease activity of RecBCD in Escherichia coli. They are overrepresented in the genome (1008 occurrences), preferentially located within coding regions (98%), oriented in the direction of replication (75%), and occur most commonly on the mRNA-synonymous sense strand of the double helix (79%). Previous statistical studies of the genome sequence suggested that these genomic properties of Chi sites appear to be related to their role in recombinational repair and therefore to replication and transcription. In this study, we employ three mathematical models to predict the properties of Chi sites from single nucleotide and multi-nucleotide compositions, and validate them statistically using the distribution of all octamer sequences in the entire genome, or exclusively within ORFs. The model based on the overall distribution of all octamers provided better predictions than the single nucleotide composition model, and the ORF and sense strand preference of Chi sites were shown to be within the standard deviation of all octamers. In contrast, the orientation bias of the Chi sites in the direction of replication was significant, although the bias was not as pronounced as with the single nucleotide composition model, suggesting a selective pressure related to the role of RecBCD in replication. © 2007 Elsevier B.V. All rights reserved.

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Roles of hemoglobin allostery in hypoxia-induced metabolic alterations in erythrocytes: Simulation and its verification by metabolome analysis

Kinoshita A., Tsukada K., Soga T., Hishiki T., Ueno Y., Nakayama Y., Tomita M., Suematsu M.

Journal of Biological Chemistry 282 ( 14 ) 10731 - 10741 2007.04

ISSN  00219258

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When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in theT-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [13C]glucose to [13C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O2-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O 2 from the cells. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Simulation study of methemoglobin reduction in erythrocytes: Differential contributions of two pathways to tolerance to oxidative stress

Kinoshita A., Nakayama Y., Kitayama T., Tomita M.

FEBS Journal 274 ( 6 ) 1449 - 1458 2007.03

ISSN  1742464X

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Methemoglobin (metHb), an oxidized form of hemoglobin, is unable to bind and carry oxygen. Erythrocytes are continuously subjected to oxidative stress and nitrite exposure, which results in the spontaneous formation of metHb. To avoid the accumulation of metHb, reductive pathways mediated by cytochrome b5 or flavin, coupled with NADH-dependent or NADPH-dependent metHb reductases, respectively, keep the level of metHb in erythrocytes at less than 1% of the total hemoglobin under normal conditions. In this work, a mathematical model has been developed to quantitatively assess the relative contributions of the two major metHb-reducing pathways, taking into consideration the supply of NADH and NADPH from central energy metabolism. The results of the simulation experiments suggest that these pathways have different roles in the reduction of metHb; one has a high response rate to hemoglobin oxidation with a limited reducing flux, and the other has a low response rate with a high capacity flux. On the basis of the results of our model, under normal oxidative conditions, the NADPH-dependent system, the physiological role of which to date has been unclear, is predicted to be responsible for most of the reduction of metHb. In contrast, the cytochrome b5-NADH pathway becomes dominant under conditions of excess metHb accumulation, only after the capacity of the flavin-NADPH pathway has reached its limit. We discuss the potential implications of a system designed with two metHb-reducing pathways in human erythrocytes. © 2007 The Authors.

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A mathematical model for the Kai-protein-based chemical oscillator and clock gene expression rhythms in cyanobacteria

Miyoshi, Fumihiko ; Nakayama, Yoichi ; Kaizu, Kazunari ; Iwasaki, Hideo ; Tomita, Masaru

JOURNAL OF BIOLOGICAL RHYTHMS 22 ( 1 ) 69-80 - 80 2007.02

Research paper (scientific journal), Joint Work,  ISSN  0748-7304

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In the cyanobacterium, Synechococcus elongatus, most promoters are regulated by a circadian clock under continuous light (LL) conditions. Nevertheless, the basic circadian oscillation is primarily generated by alternating KaiC phosphorylation/dephosphorylation reactions at the posttranslational level. Indeed, the KaiC phosphorylation cycle was recently reconstituted in vitro by incubating KaiA, KaiB, and KaiC proteins with ATP. However, the molecular dynamics of this chemical oscillation and the mechanism that drives the circadian transcription/translation rhythms remain unknown. In this report, the KaiC phosphorylation cycle and the gene regulatory network in the cyanobacterial circadian system have been modeled. The model reproduces the robust KaiC phosphorylation cycle in the absence of de novo gene expression as is observed in vitro, as well as its coupling to transcriptional/translational feedback in LL conditions in vivo. Moreover, the model is consistent with most previous experiments, including various combinations of genetic knockout or overexpression of kai genes. It also predicts that multiple KaiC phosphorylation states and dynamic Kai protein interactions may be required for the cyanobacterial circadian system. © 2007 Sage Publications.

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Noise-reduction filtering for accurate detection of replication termini in bacterial genomes

Arakawa, Kazuharu ; Saito, Rintaro ; Tomita, Masaru

FEBS LETTERS 581 ( 2 ) 253-258 - 258 2007.01

Research paper (scientific journal), Joint Work,  ISSN  0014-5793

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Bacterial chromosomes are highly polarized in their nucleotide composition through mutational selection related to replication. Using compositional skews such as the GC skew, replication origin and terminus can be predicted in silico by observing the shift points. However, the genome sequence is affected by myriad functional requirements and selection on numerous subgenomic features, and elimination of this "noise" should lead to better predictions. Here, we present a noise-reduction approach that uses low-pass filtering through Fast Fourier transform coupled with cumulative skew graphs. It increases the prediction accuracy of the replication termini compared with previously documented methods based on genomic base composition. © 2006 Federation of European Biochemical Societies.

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eXpanda: an Integrated Platform for Network Analysis and Visualization

Negishi, Y., Nakamura, H., Yachie, N., Saito, R. and Tomita, M.

In silico Biology 7 ( 2 ) 141 - 144 2007

ISSN  13866338

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Analysis and visualization of biological networks, such as protein-protein and protein-DNA interactions, are crucially important toward obtaining a thorough understanding of living systems. Here, we present an integrative software platform, eXpanda, which enables an analysis of a very broad range of biological networks, with a special focus on the extraction of characteristic topologies which potentially function as units in the networks. eXpanda is provided as a Perl library which gives full-automatic connections to various biological databases via a Perl programmable interface and can perform topological analysis based on graph theory. The results of these analyses are visualizable by vector graphics. eXpanda is under GNU General Public License. Software package, detailed documentations, source codes, and some sample scripts are downloadable at http://medcd.iab.keio.ac.jp/expanda/. © 2007 IOS Press. All rights reserved.

Alignment-Based Approach for Durable Data Storage into Living Organisms

Yachie, N., Sekiyama, K., Sugahara, J., Ohashi, Y. and Tomita, M.

Biotechnology Progress 23 ( 2 ) 501-505 - 505 2007

Research paper (scientific journal), Joint Work,  ISSN  87567938

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The practical realization of DNA data storage is a major scientific goal. Here we introduce a simple, flexible, and robust data storage and retrieval method based on sequence alignment of the genomic DNA of living organisms. Duplicated data encoded by different oligonucleotide sequences was inserted redundantly into multiple loci of the Bacillus subtilis genome. Multiple alignment of the bit data sequences decoded by B. subtilis genome sequences enabled the retrieval of stable and compact data without the need for template DNA, parity checks, or error-correcting algorithms. Combined with the computational simulation of data retrieval from mutated message DNA, a practical use of this alignment-based method is discussed. © 2007 American Chemical Society and American Institute of Chemical Engineers.

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Dynamic simulation of an in vitro multi-enzyme system

Ishii, N., Suga, Y., Hagiya, A., Watanabe, H., Mori, H., Yoshino, M, and Tomita, M.

FEBS Letters 581 ( 3 ) 413-420 - 420 2007

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

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Parameters often are tuned with metabolite concentration time series data to build a dynamic model of metabolism. However, such tuning may reduce the extrapolation ability (generalization capability) of the model. In this study, we determined detailed kinetic parameters of three purified Escherichia coli glycolytic enzymes using the initial velocity method for individual enzymes; i.e., the parameters were determined independently from metabolite concentration time series data. The metabolite concentration time series calculated by the model using the parameters matched the experimental data obtained in an actual multi-enzyme system consisting of the three purified E. coli glycolytic enzymes. Thus, the results indicate that kinetic parameters can be determined without using an undesirable tuning process. © 2007 Federation of European Biochemical Societies.

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Comparative analysis of cis-encoded antisense RNAs in eukaryotes

Numata, K., Okada, Y., Saito, R., Kiyosawa, H., Kanai, A. and Tomita, M.

Gene　392 392 ( 1-2 ) 134-141 - 141 2007

Research paper (scientific journal), Joint Work,  ISSN  03781119

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Recent large-scale transcriptomic analyses have identified numerous endogenously encoded cis-antisense RNAs that are thought to play important roles in diverse cellular processes although comprehensive comparative studies among multiple species have yet to be performed. To investigate conserved genomic features across various species that may be related to sense-antisense regulation, we performed comparative analysis of approximately 1000-2000 cis-encoded antisense RNA pairs from five model eukaryotes (Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana, and Oryza sativa). Analysis of overlapping patterns relative to the exon-intron structure revealed that the number of pairs sharing the 3′ part of the transcripts was larger than that of the 5′-sharing pairs except in rice. Moreover, most of the well-conserved sense-antisense pairs between human and mouse exhibited 3′-overlaps, suggesting that regulatory mechanisms involving these regions may be important in sense-antisense transcription. Functional classification using Gene Ontology revealed that genes related to catalytic activity, nucleotide binding, DNA metabolism, and mitochondria were preferentially distributed within the set of exon-overlapping sense-antisense genes compared to the non-exon-overlapping group in animals. Despite the numerous sense-antisense pairs identified in human and mouse individually, the number of conserved pairs was extremely small (6.6% of the entire set). Whereas both genes of most of the conserved sense-antisense pairs had protein-coding potential, nearly half of the non-conserved pairs included a non-coding RNA, suggesting that non-coding sense-antisense RNAs may function in species-specific regulatory pathways. © 2006 Elsevier B.V. All rights reserved.

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The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

Hu, B. and Tomita, M.

Systems and Synthetic Biology 1 ( 1 ) 47-58 - 58 2007

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

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Hsp70 chaperones assist protein folding by cycling between the ATP-bound T state with low affinity for substrates and the ADP-bound R state with high affinity for substrates. The transition from the T to R state is catalyzed by the synergistic action of the substrate and DnaJ cochaperones. The reverse transition from the R state to the T state is accelerated by the nucleotide exchange factor GrpE. These two processes, T-to-R and R-to-T conversion, are affected differently by temperature change. Here we modeled Hsp70-mediated protein folding under permanent and transient heat shock based on published experimental data. Our simulation results were in agreement with in vitro wild-type Escherichia coli chaperone experimental data at 25°C and reflected R-to-T ratio dynamics in response to temperature effects. Our simulation results suggested that the chaperone system evolved naturally to maintain the concentration of active protein as high as possible during heat shock, even at the cost of recovered activity after return to optimal growth conditions. They also revealed that the chaperone system evolved to suppress ATP consumption at non-optimal high growing temperatures. © Springer Science + Business Media B.V. 2007.

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The GC skew index: a measure of genomic compositional asymmetry and the degree of replicational selection

Arakawa, K. and Tomita, M.

Evolutionary Bioinformatics 3   159 - 168 2007

Research paper (scientific journal), Joint Work

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Circular bacterial chromosomes have highly polarized nucleotide composition in the two replichores, and this genomic strand asymmetry can be visualized using GC skew graphs. Here we propose and discuss the GC skew index (GCSI) for the quantifi cation of genomic compositional skew, which combines a normalized measure of fast Fourier transform to capture the shape of the skew graph and Euclidean distance between the two vertices in a cumulative skew graph to represent the degree of skew. We calculated GCSI for all available bacterial genomes, and GCSI correlated well with the visibility of GC skew. This novel index is useful for estimating confi dence levels for the prediction of replication origin and terminus by methods based on GC skew and for measuring the strength of replicational selection in a genome.

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Robust effects of Tsr-CheBp and CheA-CheYp affinity in bacterial chemotaxis

Matsuzaki, Y., Kikuchi, S. and Tomita, M.

Artificial intelligence in medicine 41 ( 2 ) 145-150 - 150 2007

Research paper (scientific journal), Joint Work,  ISSN  09333657

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Objective: Cell motility and chemotaxis play a role in the virulence of pathogenic bacteria, such as escape from host immune responses. Escherichia coli chemotaxis provides a well-characterized model system for the bacterial chemotaxis network. Two features of E. coli chemotaxis include signal amplification and robustly accurate adaptation. Recent simulation studies with models considering the effects of other receptors have suggested possible mechanisms for signal amplification. Although precise adaptation to aspartate has been explained by conventional kinetic models, the adaptation behavior of models incorporating the effects of other receptors remains unclear. Methods: We concentrated on how receptor crosstalk affects minimization of adaptation error and compared models in which the contribution of other receptors varied. Results: We demonstrated that the model is adaptable to attractant concentrations ranging from 0.1μ M to 10 mM with a decreased error rate (from 8% to 2%) when the kinetic constant of CheA and phosphorylated CheY dissociation is increased. Conclusion: The results suggest that accurate adaptation is maintained through control of both the interaction of cytoplasmic Che proteins and the activity of the receptor complex. © 2007 Elsevier B.V. All rights reserved.

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Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation

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

Rapid Communications in Mass Spectrometry 21 ( 22 ) 3579 - 3584 - 3584 2007

Research paper (scientific journal), Joint Work,  ISSN  09514198

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We have fabricated a coaxial sheath liquid flow microelectrospray ionization (μESI) interface for capillary electrophoresis coupled with mass spectrometry (CE/MS). The ESI interface, which features a reduced probe diameter (130 μm i.d. x 174 μm o.d.) with a nebulizer-free format, can relatively easily electrospray a large amount of make-up sheath liquid (5-10 μL/min) over the long term (more than 80 runs) with a high degree of stability. The interface also provides higher separation qualities and improved detection sensitivities compared with a conventional ion spray (IS) interface. Copyright © 2007 John Wiley & Sons, Ltd.

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Permuted tRNA Genes Expressed via a Circular RNA Intermediate in Cyanidioschyzon merolae

Soma, A., Onodera, A., Sugahara, J., Kanai, A., Yachie, N., Tomita, M., Kawamura, F., Sekine, Y.

Science 318 ( 5849 ) 450-453 - 453 2007

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

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A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified 11 transfer RNA (tRNA) genes in which the 3′ half of the tRNA lies upstream of the 5′ half in the genome. We verified that these genes are expressed and produce mature tRNAs that are aminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual processing pathway in which the termini of the tRNA precursor are ligated, resulting in formation of a characteristic circular RNA intermediate that is then processed at the acceptor stem to generate the correct termini.

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Model based definition of population heterogeneity and its effects on metabolism in sporulating Bacillus subtilis

Morohashi, M., Ohashi, Y., Tani, S., Ishii, K., Itaya, M., Nanamiya, H., Kawamura, F., Tomita, M. and Soga, T.

Journal of Biochemistry 142 ( 2 ) 183-191 - 191 2007

Research paper (scientific journal), Joint Work,  ISSN  0021924X

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The soil bacterium Bacillus subtilis forms dormant, robust spores as a tactic to ensure survival under conditions of starvation. However, the sporulating culture includes sporulating and non-sporulating cells, because a portion of the cell population initiates sporulation in wild-type strain. We anticipated that the population effect must be considered carefully to analyse samples yielding population heterogeneity. We first built a mathematical model and simulated for signal transduction of the sporulation cue to see what mechanisms are responsible for generating the heterogeneity. The simulated results were confirmed experimentally, where heterogeneity is primarily modulated by negative feedback circuits, resulting in generation of a bistable response within the sporulating culture. We also confirmed that mutants relevant to negative feedback yield either sporulating or non-sporulating subpopulations. To see the effect of molecular mechanism between sporulating and non-sporulating cells in distinct manner, metabolome analysis was conducted using the above mutants. The metabolic profiles exhibited distinct characteristics with time regardless of whether sporulation was initiated or not. In addition, several distinct characteristics of metabolites were observed between strains, which was inconsistent with previously reported data. The results imply that careful consideration must be made in the interpretation of data obtained from cells yielding population heterogeneity. © 2007 The Japanese Biochemical Society.

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Variation in the Correlation of G + C Composition with Synonymous Codon Usage Bias among Bacteria

Suzuki H, Saito R, Tomita M.

EURASIP J Bioinform Syst Biol. 2007 ( 61374 )  2007

Research paper (scientific journal), Joint Work,  ISSN  16874145

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G + C composition at the third codon position (GC3) is widely reported to be correlated with synonymous codon usage bias. However, no quantitative attempt has been made to compare the extent of this correlation among different genomes. Here, we applied Shannon entropy from information theory to measure the degree of GC3 bias and that of synonymous codon usage bias of each gene. The strength of the correlation of GC3 with synonymous codon usage bias, quantified by a correlation coefficient, varied widely among bacterial genomes, ranging from -0.07 to 0.95. Previous analyses suggesting that the relationship between GC3 and synonymous codon usage bias is independent of species are thus inconsistent with the more detailed analyses obtained here for individual species.

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Visualization of three-way comparisons of omics data

Baran, R., Robert, M., Suematsu, M., Soga, T. and Tomita, M.

BMC Bioinformatics 8   72 2007

Research paper (scientific journal), Joint Work

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Background: Density plot visualizations (also referred to as heat maps or color maps) are widely used in different fields including large-scale omics studies in biological sciences. However, the current color-codings limit the visualizations to single datasets or pairwise comparisons. Results: We propose a color-coding approach for the representation of three-way comparisons. The approach is based on the HSB (hue, saturation, brightness) color model. The three compared values are assigned specific hue values from the circular hue range (e.g. red, green, and blue). The hue value representing the three-way comparison is calculated according to the distribution of three compared values. If two of the values are identical and one is different, the resulting hue is set to the characteristic hue of the differing value. If all three compared values are different, the resulting hue is selected from a color gradient running between the hues of the two most distant values (as measured by the absolute value of their difference) according to the relative position of the third value between the two. The saturation of the color representing the three-way comparison reflects the amplitude (or extent) of the numerical difference between the two most distant values according to a scale of interest. The brightness is set to a maximum value by default but can be used to encode additional information about the three-way comparison. Conclusion: We propose a novel color-coding approach for intuitive visualization of three-way comparisons of omics data. © 2007 Baran et al; licensee BioMed Central Ltd.

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Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis

Toya, Y., Ishii, N., Hirasawa, T., Tomita, M. and Soga, T.

Journal of Chromatography A 1159 ( 1-2 ) 134–141 - 141 2007

Research paper (scientific journal), Joint Work,  ISSN  00219673

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We have developed a metabolic flux analysis method that is based on 13C-labeling patterns of the intracellular metabolites directly measured by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The flux distribution of the central carbon metabolism in Escherichia coli was determined by this new approach and the results were compared with findings obtained by conventional GC-MS analysis based on isotopomer of the proteinogenic amino acids. There were some differences in estimation results between new approach using CE-TOFMS and conventional approach using GC-MS. These were thought to be attributable to variations in measured mass distributions between amino acids and the corresponding precursors and to differences in the sensitivity of the exchange coefficients to mass distributions. However, our CE-TOFMS method facilitates high-throughput flux analysis without requiring complicated sample preparation such as hydrolysis of proteins and derivatization of amino acids. © 2007 Elsevier B.V. All rights reserved.

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Analysis of nucleotides by pressure-assisted capillary electrophoresis　mass spectrometry using silanol mask technique

Soga, T., Ishikawa, T., Igarashi, S., Sugawara, K., Kakazu, Y. and Tomita, M.

Journal of Chromatography A 1159 ( 1-2 ) 125–133 - 133 2007

Research paper (scientific journal), Joint Work,  ISSN  00219673

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A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis-electrospray ionization mass spectrometry (PACE-MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide-adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n = 6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme. © 2007 Elsevier B.V. All rights reserved.

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Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector

Kuroki, A., Ohtani, N., Tsuge, K., Tomita, M. and Itaya, M.

Gene 399 ( 1 ) 72-80 - 80 2007

Research paper (scientific journal), Joint Work,  ISSN  03781119

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The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges. © 2007 Elsevier B.V. All rights reserved.

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