| Project/Area Number |
03454553
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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 |
放射線5生物学
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
YAMAIZUMI Masaru Kumamoto Univ. Sch. of Medicine, Professor, 医学部, 教授 (70107093)
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| Co-Investigator(Kenkyū-buntansha) |
TATEISHI Satoshi Kumamoto Univ. Sch. of Medicine, Research Associate, 医学部, 助手 (00227109)
SUGANO Tatsuo Kumamoto Univ. Sch. of Medicine, Research Associate, 医学部, 助手 (00211300)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 1992: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1991: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Keywords | Xeroderma Pigmentosum / DNA Repair / XP Complementing Factors / Group B XP / Group C XP / in vitro Repair System |
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| Research Abstract |
A peptide corresponding to the C-terminal 12 amino acids of XP-B complementing factor was chemically synthesized and conjugated with hemocyanin through disulfide linkage. Antisera against this peptide were raised in rabbits and specific IgG was purified by affinity chromatography using EAE-Sepharose 4B coupled with the peptide. The antibody was confirmed to react with the peptide by western blotting. To test whether this antibody reacts with a native form of XP-B complementing factor, we employed two alternative methods. In the first method, this antibody was microinjected into nuclei of normal human cells, and the effect on unscheduled DNA synthesis in these microinjected cells was examined. In the second method, this antibody was mixed with a HeLa cell extract containing an activity to correct the defect of XP-B cells, and the mixture was microinjected into the cytoplasm of XP-B cells. If the antibody blocks the activity of XP-B complementing factor, unscheduled DNA synthesis in these microinjected cells is expected to be suppressed. In both cases, the results were negative, demonstrating that our antibody reacts with XP-B complementing factor only in a denaturing condition. Because our purpose is to examine the interaction between XP-B complementing factor and XP-C complementing factor, we need antibodies reactive with native forms of these proteins. Fot this we obtained cDNA fragments spanning N-terminal one-thirds of XP-B and XP-C cDNAs by PCR method using a cDNA library constructed from fractionated mRNA derived from HeLa cells. These cDNA fragments were ligated with a qprokaryotic expression vector, pET23a, at its polylinker site. Fusion proteins overexpressed in E. coli were recovered in insoluble fraction and purified by metal-chelating chromatography. Rabbits are now being immunized with these proteins.
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