1987 Fiscal Year Final Research Report Summary
Base changes in the replication of DNA induced by nucleotide analogs and the use for in vitro mutagenesis
| Project/Area Number |
61480430
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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 |
Biological pharmacy
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| Research Institution | Okayama University |
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Principal Investigator |
HAYATSU Hikoya Faculty of Pharmaceutical Sciences, Okayama University, 薬学部, 教授 (10012593)
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| Co-Investigator(Kenkyū-buntansha) |
NEGISHI Kazuo Faculty of Pharmaceutical Sciences, Okayama University, 薬学部, 助手 (70116490)
WATAYA Yusuke Faculty of Pharmaceutical Sciences, Okayama University, 薬学部, 助教授 (90127598)
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| Project Period (FY) |
1986 – 1987
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| Keywords | N^4-Aminocytidine / transition mutation / replication error / M13mp2,FM3A cell / V79 cell / トランジション変異 / 複製 |
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| Research Abstract |
The moecular mechanism of N^4-aminocytidine-mediated mutagenesis was investigated. A summary of findings achieved is as follows. (1) A study of in vitro DNA synthesis using E. coil DNA polymerase I large fragment as the enzyme showed that N^4-aminodeoxycytidine 5'-triphosphate (dC^<am>TP) can serve as a substrate for the enzyme and can be incorporated int onewly formed DNA. The efficiency of incorporation was about 50% of the natural substrate dCTP, and this nucleotide analog can be used as a substitute for dCTP and dTTP. (2) Use of dC^<am>TP in the M13mp2 DNA synthesizing system, followed by transfection of the resulting DNA to E. coli allowed production of mutant phages. More than 100 mutant clones were spearated, the DNA was isolated and their nucleotide sequences determined. The results indicated that all possible transitions, A to G, G to A, C to T and T to C, and only these transitions, have occurred. This result is consistent with a mechanism in which tautomeric shift in N^4-aminocytosine between the amino and the imino forms is the cause of the mutation. (3) Mutations were induced in Drosophila when the larve were fed with N^4-aminocytidine. (4) An improved method to synthesize N^4-aminocytidine was established.
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