| Project/Area Number |
04454598
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | The Institute of Phisical and Chemical Research (RIKEN) |
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Principal Investigator |
HANAOKA Fumio RIKEN, Cell. Physiol. Lab., Chief Scientist, 細胞生理学研究室, 主任研究員 (50012670)
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| Co-Investigator(Kenkyū-buntansha) |
MASUTANI Chikahide RIKEN, Cell. Physiol. Lab., Special Researcher, 細胞生理学研究室, 基礎特研 (40241252)
SUGASAWA Kaoru RIKEN, Cell. Physiol. Lab., Research Scientist, 細胞生理学研究室, 研究員 (70202124)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1993: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1992: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | DNA polumerase alpha / ts mutant / growth-revertants / primase / gene expression / overexpression / DNAポリメラーゼalpha / 細胞周期 / 温度感受性突然変異株 / PCR-SSCP法 / 遺伝子発現 |
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| Research Abstract |
We isolated cDNA of the catalytic subunit of DNA polymerase alpha from tsFT20 cells, a temperature-sensitive mutant cell line derived from mouse FM3A cells. DNA sequence analysis revealed that the cDNA has a single mutation, a cytosine to thymine substitution that changes amino acid 1180 from serine to phenylalanine. We have also shown that tsFT20 cells could be rescued by transfection with the wild-type cDNA.In addition, we detected mutation sites in one spontaneous and six N-methyl-N'-nitro-N-nitrosoguanidine-induced growth revertants of tsFT20 cells. All revertant cell lines had a second point mutation adjacent to the first mutation site in tsFT20 cells.
During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In order to analyze how the expression of these genes are controlled, we have isolated upstream region of these genes and determined their nucleotide sequence. There are 1-2 E2F-binding sequence and 10-14 dp palindromic sequence, and one AP1-binding motif in each upstream region.
We overexpressed cDNAs of the DNA polymerase alpha-primase complex in E.coli or insect cells, purified the gene products and raised the specific antibodies against them. Co-precipitation experiments showed that 54K subunit has affinity to all other three subunits. We have also shown that 180K subunit has DNA polymerase activity and 46K subunit has primase activity.
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