| Project/Area Number |
61440092
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
分子遺伝学・分子生理学
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| Research Institution | Osaka University, Medical School |
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Principal Investigator |
YOSHIKAWA Hiroshi Osaka Univ., Medical School, Professor, 医学部, 教授 (70019876)
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| Co-Investigator(Kenkyū-buntansha) |
MORIYA Shigeki Osaka Univ., Medical School, Assistant, 医学部, 助手 (40191051)
OGASAWARA Naotake Osaka Univ., Medical School, Lecturer, 医学部, 講師 (10110553)
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| Project Period (FY) |
1986 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥32,200,000 (Direct Cost: ¥32,200,000)
Fiscal Year 1989: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1988: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1987: ¥9,000,000 (Direct Cost: ¥9,000,000)
Fiscal Year 1986: ¥17,000,000 (Direct Cost: ¥17,000,000)
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| Keywords | DnaA protein / DnaA-box / Bacillus subtilis / Pseudomonas putida / Micrococcus luteus / Mycopurasuma capricorum / Chromosomal replication / DnaAーbox / シュードモナス / 染色体の複製開始 / シュードモナス菌 / dnaA遺伝子 / oriC / 細菌染色体 / 複製開始 / DnaA遺伝子 / DnaA・Box配列 / 大腸菌 / P.putida / 塩基配列 |
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| Research Abstract |
1. Conservation of DnaA protein and DnaA-box in bacteria and their evolution (Yoshikawa): (1) Conservation of the combination of dnaA gene and DnaA-boxes was confirmed in Pseudomonas and Micrococcus in addition to E. coli and B. subtilis, suggesting its central role in initiation of chromosomal replication in bacteria. (2) We succeeded in cloning a dnaA homologue from Mycoplasma, cells containing the smallest genome, suggesting ubiquitous presence of DnaA protein in enbacteria. (3) A clone containing a possible dnaA homologue was obtained from yeast, a lower eukaryote.
2. Function of DnaA protein and regulation of its expression (Moriya): (1) Involvement of DnaA protein and DnaA-box in initiation of replication and its regulation was demonstrated using a dnaA-ts mutant cell of B. subthis. (2) DnaA protein was purified, and the mode of its specific binding to DnaA-box was analyzed in vitro. (3) An anti-DnaA antibody was prepared to determine cellular content of DnaA which determines the initiation frequency under certain physiological conditions. (4) In steady state growth, other factors seem to be limiting in the initiation in addition to DnaA.
3. Structure and function of replication machinery in B. subtilis (Ogasawara): (1) Structure of B. subtilis dnaG and dnaH genes were determined to identify them as homologous wtih E. coli dnaN and dnaZX, respectively. They code for two subunits of polymerase III. (2) Structure of a second gene for initiation of replication, dnaB, was determined. Its product protein was purified, and an antibody was prepared. So far, DNA binding activity of the protein was not detected in vitro.
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