Cloning and expression of mammalian DNA ligase gene
| Project/Area Number |
61580164
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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 |
代謝生物化学
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
TERAOKA Hirobumi Medical Research Institute, Tokyo Medical & Dental University, 難治疾患研究所, 助教授 (30019137)
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| Co-Investigator(Kenkyū-buntansha) |
HORIKAWA Saburo Medical Research Institute, Tokyo Medical & Dental University, 難治疾患研究所, 助手 (10127136)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | DNA ligase / DNA replication / DNA repair / nene expression / cDNA cloning / cDNAクローニング |
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| Research Abstract |
DNA ligase II (Mr = 68 kDa) has been purified from calf thymus and the rabbit antiserum was obtained. Immunochemical analysis with anti(calf thymus DNA ligase I)IgG and anti(DNA ligase II)IgG has implied that two independent forms fo DNA ligase exist in mammalian cells and tissues. DNA ligase I (Mr = 200 kDa) seems to play a role in DNA replication, Whereas DNA ligase II may be involved in DNA repair. Since there is no direct evidence for the presence in vivo of these two forms of DNA ligase, a powerful approach to answering questions about the existence, expression and function of two DNA ligases is through gene cloning. In order to isolate cDNA clones of mammalian DNA ligase with synthetic probes, amino-terminal sequence of calf thymus DNA ligase II was examined, and the amino-terminal proved to be blocked. Then amino-acid sequence of isolated peptides after chemical and enzymatic clevages of DNA ligase II was determined, but no reliable result was obtained mainly because of the small amount of the pure enzyme obtained. Recently two independent cDNA clones have been isolated from a calf thymus cDNA library with a synthetic 30-mer complementary to the nucleotide sequence at the putative active site in the deduced amino-acid sequences of Saccharomyces cerevisiae and S. pombe DNA ligases. The DNA fragments from the cDNAs were subcloned into pUC118/119 for sequencing. When the sequencing of cDNAs reveals the site homologous to the putative active site of yeast DNA ligase, the products of cDNAs expressed in Escherichia coli JM109 will be detected with the antibodies against calf thymus DNA ligases.
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Report
(2 results)
Research Products
(15 results)
