SHIMAZU Yoshie National Institute of Radiological Sciences, Division of Chemical Pharmacology,, 薬理化学研究部, 主任研究員 (10162687)
MORIMYO Mitsuoki National Institute of Radiological Sciences, Division of Genetics, Head, 遺伝研究部, 室長 (00166430)
|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1993 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1991 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Active oxygen species are assumed to be an important cause of mutagenesis, tumorigenesis, diseases and aging. Although the defense mechanisms and responses of prokaryotic cells to toxic radicals have almost been reserved, those of eukaryotic cells remain unsolved. The mutants of yeast and mammalian cells sensitive to active oxygen generating agents, plumbagin(PG), were isolated and genetic analyzes including gene cloning were performed.
1) Isolation and Analysis of S.pombe mutants sensitive to active oxygen
S.pombe, G408 cells were mutagenized with EMS and about 400 mutants sensitive to PG were isolated. These mutants were classified into 3 groups ; (〕SY.encircled .〔)1 only sensitive to PG, (〕SY.encircled .〔)2 cross-sensitive to X-ray and/or UV, (〕SY.encircled .〔)3 cross-sensitive to oxygen. These results suggested that the defense mechanisms of yeast against active oxygen toxicity are attained by the elimination of active oxygen species and by DNA repair mechanisms. This is a good agree
ment with the results of E.coli. Among 50 radiation-sensitive mutants, M12 was a new one, and the relevant gene radM12 was cloned. The gene was a new stress-response gene located on chromosome 1 of S.pombe consisted of 474bp encoding (157 amino acids) a new uniquitine-conjugating enzyme E2.
2) Analysis of adaptive mechanism of S.pombe against active oxygen
To clone directly stress-inducible genes, the fusion DNA library was made by ligation of cloning vector carrying reporter gene lacZ with fractionated S.pombe DNA.These DNA were transformed into a wild type S.pombe strain, and the colonies of fusion transformants exhibiting increased expression of lacZ by stress such as UV, X-ray, heat, and oxygen radicals were isolated. Plasmids were extracted from 71 candidates and 26 genes were identified from DNA sequence. Analyzes of genes are now in progress.
3) CHO-K1 cell mutants sensitive to active oxygen-generating agents
Nine mutants isolated from EMS-treated CHO-K1 cells with increased sensitivity to the lethal effect of PG, were classified into 5 groups A-E, by the sensitivity to PG and methyl viologen(MV). The strains of B and C groups were rather sensitive to both agents. Two mutants of group B (pa13, pb4) were sensitive to both drugs, and 2 mutants of group C(pa14, pa15) were sensitive to PG, extremely sensitive to MV.Genetic complementation analyzes of these 4 mutants were carried out using MV sensitivity. Group B was divided into 2, I and II.Pa14 and pa15 in group C belonged to the same complementation group III.These 4 mutants were also sensitive to mitomycin C (MMC) classified into 3 complementation groups as to MMC-sensitivity. As pa13 and pb4 were also sensitive to cis-diaminedichloroplatinum II, they may have a defect in the repair of DNA-crosslinks induced by these active oxygen generating agents. Less