| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1987: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1986: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Research Abstract |
Many studies in cell culture system suggested that aging in vivo is based on cellular aging, and related with mutation. The process in DNA replication and repair takes a chief part on mutation. In this work we studied mutation mechanism for DNA replication in aphidicolin resistency as a basal phenomenon for cellular aging. The aphidicolin resistant clones originated from the human cell line were newly established. Mutagenized with ethyl methansulfonate, one clone was obtained that was confirmed to proliferate in /.1 <micrn>g/ml of aphidicolin. And further mutagenized with UV-light, five clones were established that was possible to proliferate in 0.3 <micrn>g/ml of this drug. Four clones were shown to be prolonged cell cycle, resistant to hydroxyurea in different survival curves each other and normally sensitive to 5-fluorodeoxyuridine. So, these clones were thought to be independent mutants for ribonucleotide reductase gene. And the analysis in genetic level revealed decreased or increased transcription of ribonucleotide reductase gene. These mutants would be useful for analyses of cell growth and DNA replication mutant.
The fibroblast cell line ( 1629SV14) derived from Cockayne syndrome patient was already known to be UV-sensitive. We found that this cell was also sensitive to thymidine and aphidicolin. And in the course of DNA replication after UV-irradiation, dNTP-pool and DNA polymerase <alpha> activity showed the same manner as normal, but the uptake of thymidine and the activity of ribonucleotase did not recovered. Hybridization with mouse cell allowed this cell th recover the sensitivity to aphidicolin as well as to UV, partially. And the introduction of ribonucleotide reductase gene was correlated with aphidicolin resistency. As ribonucleotide reductase is the key enzyme for deoxyribonucleotides synthesis, this enzyme was thought to be closely related to cellular aging and mutation.
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