| Project/Area Number |
06680673
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Molecular biology
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| Research Institution | Aichi Cancer Center (1995)
National Institute of Genetics (1994) |
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Principal Investigator |
YAMAGISHI Masahiro Aichi Cancer Center, Laboratory of Cell Biology, Senior Researcher, 生物学部, 主任研究員 (00220252)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUMOTO Kiyohisa Kitasato University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (80092344)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1994: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Capping / Yeast / Guanylyltransferase / Temperature-sensitive mutants / キャッピング / 温度感受性変異株 |
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| Research Abstract |
The yeast mRAN capping enzyme is composed of alpha and beta subunits possessing guanylyltransferase and triphoshatase activities, respectively. In this study, we have isolated 10 recessive temperature-sensitive mutations of the CEG1 gene encoding the alpha subunit and determined the mutation sites. Nine of the mutations(ceg1-1 to ceg1-9)were isolated on a single-copy plasmid-and the remaining one(ceg1-10)on a multi-copy plasmid. The presence of ceg1-10 in multiple copies is essential for the viability of cells carrying the mutation, and a shift to the restrictive temperature resulted in quick growth arrest of ceg1-10 cells while other mutants decreased growth rates gradually upon the temperature up-shift. Intragenic complementation was not observed for pairwise combinations of the mutations.Guanylyltransferase activity was examined for each mutant protein by assaying of a covalent Ceglp-GMP complex formation. While most of the mutant protein showed clear heat-lability, Ceg1-8p and Ceg1-9p maintained substantial activities during incubation at a high temperature. Further characterization was carried out for isolated mutant proteins expressed in E.coli cells.Isolation of extragenic suppressors as well as detailed analyzes of the ceg1 mutant cells are in progress currently.
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