| Project/Area Number |
07558288
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
環境影響評価(含放射線生物学)
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| Research Institution | University of Tokyo |
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Principal Investigator |
MITANI Hiroshi University of Tokyo School of Science, Dept.Biol.Sciences, Associate Professor, 大学院理学系, 助教授 (70181922)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1996: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Medaka / DNA repair / UV / Photolyase / mutation / 光回復酸素 / トランスジェニック / PCR / トランスジェニック魚類 |
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| Research Abstract |
We constructed a eukaryotic expression plasmid of the Medaka photolyase gene and introduced it into Medaka cells in vitro and in vivo. The expression plasmid contains a cytomegalovirus enhancer and a thymidine kinase promoter to overexpress the photolyase gene of the Medaka. First, we transfected this construct into cultured Medaka cells and established several lines of transfectant. Every transfectant showed enhanced ability of pyrimidine dimer repair in the presence of fluorescent light. In the transfectant that showed the most enhanced ability of photorepair, the augmented transcription of photolyase gene was observed compared with that of progenitor OL32 cells. In this transfectant, we also observed an enhanced rate of UV survival with 20 min of fluorescent light treatment after irradiation with a 400 J/m2 UV sunlamp. Next, the expression construct was microinjected into the embryos of the Medaka at the one cell stage. Compared with the nontreated counterparts, the overexpression of a photo-lyase gene was detected in the microinjected embryos, but we failed to detect a significant increase in photoreactivability of death at the midblastula stage. Next, we tried to detect gamma-ray induced delection mutants of CPD-photolyase (phr) gene and p53 gene of the Medaka by a PCR system utilizing their DNA polymorphism between different populations. Males from the northern population were irradiated with 4.75 Gy gamma-rays and from 1 day to 12 days after irradiation they were mated with females from the southern population.
We got 2182 normally hatched embryos and 313 abnormally developed embryos from gamma-irradiated sperm or spermatid. We found 4 phr mutants our of 313 and a p53 mutant among abnormal embryos. This mutation rate is close to previously reported one determined by a SLT system or a AP-PCR system. The loss of linkage markers to suggested that the deletion region induced by gamma-rays should be more than 40 cM in abnormally developed embryos.
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