| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Effects of a defect in oxidative phosphorylation (F_1-ATPase defect) on Escherichia coli cells was investigated by proteome and transcriptome analyses.
(1) Sapmple preparation : E. coli K-12 strain W1485 and its F_1-ATPase defective mutant ware cultured in glucose in limited chemostat and used as samples for the analyses.
(2) Proteome analysis : All the expressed proteins were separated by 2 dimentional gel electrophoresis, and the protein spots that showed difference in their densities between the two strains were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.
(3) Transcriptome analysis : Total RNA was extracted from both strains, and ^<30>P-labeled cDNA wsa prepared. DNA array was probed with the prepared cDNA, and the image analysis of the hybridized DNA array was conducted for the analysis of the difference in the gene expression between the two strains.
[Results] Among enzymes involved in glycolysis, several enzymes (phosphoglycerate kinase, enolase, pyruvate kinase I, pyruvate dehydrogenase) showed increased expression both in the level of enzyme activities and in protein amounts (proteome analysis) in the mutant. However, transcriptome analysis showed only pyruvate dehydrogenase expression was increased in the mutant, and no difference was observed in the transcription of the rest of the glycolytic enzymes. Many TCA cycle enzymes (citrate synthase, succinyl-CoA synthetase, malate dehydrogenase, isocitrate lyase, malate synthase) were found to be down-regulated in tha mutant in the levels of enzyme activities, proteome and transcriptome analyses. However, succinate dehydrogenase and fumarate hydratase were found to be up-regulated only in the level of enzyme activities and in proteome analysis. In respiratory chain, expression of NADH dehydrogenase-2 and cytochrome bd oxidase were found to be up-regulated in transcriptome analysis.
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