HIRANO Kazuya Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (80251221)
ITOH Masamitsu Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (80176362)
SUZUKI Norio Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (10010050)
|Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
To isolate and characterize a gene responsible for heat resistance/sensitivity in mammalian cells, hubrid clones were prepared from human glioma A7 cells, which are heat resistant, and Chinese hamster V79 cells, which are relatively heat sensitive. Subcloning of the most resistant hybrids, VA41, was carried out ; a subclone which was as resistant as 47, VA41-4, and a subclone which was as sensitive as V79, VA41-8, were obtained. The number of chromosomes in VA41-4 was larger than that in VA41-8. Human repetitive sequence was found, by Southern analysis, in VA41-4 as well as VA41-8, suggesting that a human gene responsible for heat resistance in VA41 and that VA41-8 became sensitive because it lost the gene during subculture. In order to identify the gene, a DNA transfection experiment, VA41-4 DNA into V79, was carried out. One of the transfectants, I41, was as resistant as A7. A genomic DNA library was prepared in EMBL4 phage vectors. Clones containing human repetitive sequences were found at a frequency of 5 x 10-4 in the library.
Also, cellular heat responses in A7, V79, and the hybrid clones were characterized in order to get clues for possible roles of the heat resistant gene.
The hypersensitivity to heat treatment seemed associated with DNA ladder formation and quick cell death. In heat sensitive V79 and VA41-8 DNA ladder found immediately after heat treatment, and dead cells appeared quickly after heat. On the other hand, heat resistant A7 and VA41-4 cell death process was slower and DNA ladder was not observed. These results suggest that the gene would make cells heat resistant by suppressing DNA ladder formation and cell death process.