| Project/Area Number |
06640804
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
遺伝
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| Research Institution | National Institute of Radiological Sciences |
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Principal Investigator |
TSUJI Hideo National Institute of Radiological Science, 2nd Research Group, Senior Researcher, 第2研究グループ, 主任研究官 (40163795)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1994: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Sister chromatid exchange / RNA polymerase II / Temperature-sensitive metant / FISH / Mutation / cDNA / Gene / cDNA / RNA polymerase |
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| Research Abstract |
A temperature-sensitive CHO-K1 cell mutant, tsTM4, exhibited abnormal induction of sister chromatid exchages (SCEs) along with decreased DNA synthesis in the cells arrested in the S phase at the nonpermissive temperature (39ﾟC).We have cloned a human gene that complemented the genetic defect of tsTM4 cells by transfecting human genomic DNA into them and collecting the human DNA fragments from the secondary transformants. DNA seguence analysis of these fragments revealed that it is the human RNA polymerase II largest subunit gene, HSRPIILS,with 29 exons spanning 31 kbp.The HSRPIILS gene was mapped to 17p13.1 by fluorescence in situ hybridization.Transfection of cloned genomic DNA fragments containing the entire coding regions of HSRPIILS into tsTM4 cells rescued their abnormal SCE induction phonotype.The Northern analysis showed that the HSRPIILS was transcribed at 39ﾟC and 33.5ﾟC in the transformant, suggesting that a defect in the RPIILS gene is responsible for the abnormal induction of SCEs in the tsTM4 mutant.Transcriptional activity of tsTM4 cells was normal with respect to fine genes even at the nonpermissive temperature when the amounts of transcribed RNA were examined by Northevn analysis.This suggests that a defect in RPIILS is limited to transcription of only a part of genes or responsible for another function in DNA metabolism.To identify mutational sites of RPIILS gene, RPIILS cDNAs were isolated from the wild-type Chinese hamster cells and tsTM4 cells, and sequenced.Several candidate sites for mutation were found.
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