| Project/Area Number |
10680679
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | National Institute of Genetics |
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Principal Investigator |
NISHIMURA Akiko National Institute of Genetics, Associate Professor, 系統生物研究センター, 助教授 (20142002)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Cell division / Ap4A / apaH / glyS / glycylation / Ap4A sinthesis / E.coli / Ap4A binding protein / 分子生物学 / 細胞分裂 / グリシルtRNA合成酵素 / Ap4A分解酵素 / cfcA遺伝子 / cfcB遺伝子 |
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| Research Abstract |
We found previously that cfc mutation uncouples DNA replication and cell division, and elevates the frequency of cell division. We further analyzed the structure and the role of the cfc genes. The cfc mutants divide before they reach the size at which cfc^+ cells divide, and produce many small cells-each with a single nucleoid. The mutations affect the timing of cell division, but not other processes of cell cycle, such as the length of a cell cycle and the initiation mass for chromosome replication. CfcA has a missence mutation in glySa which encodes the α-subunit of glycyl-tRNA synthetase, and cfcB1 has an IS2 insertion in apaH which encodes Ap4A hydrolase. The Cfc properties of both cfc mutants were suppressed by a multicopy plasmid carrying apaH^+, and the intracellular level of Ap4A in cfcA was 15-fold higher, and cfcB was 100-fold higher than their parent. Experiments using a wild-type cell showed that a high level of Ap4A caused early cell division, and a low level of Ap4A caused delayed cell division. we have purified the GlyS-6xHis tagged proteins from cfc^+ and cfc^- strains and analysed the catalytic activity in vitro for Ap4A synthesis and kinetic constants of tRNA aminoacylation catalyzed by GlyS.Mutant type GlyS synthesized more Ap4A than wild type GlyS but showed lower degradation activity of Ap4A to ADP than wild type GlyS.Catalytic activity for glycylation (Km/Kcat) of GlyS from cfcA is 20〜100 times higher than that from wild type. Therefore, I conclude that Ap4A is a signal for induction of cell division. High level of Ap4A is responsible for the initiation of cell division. The glyS mutation allows efficient synthesis of Ap4A.We also identified novel Ap4A binding protein A (AbpA) and analyzed N terminal sequence of amino acid. Conditional null mutation of abpA induced delayed cell division, and overproduction of abpA induced Cfc phenotype.
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