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Functional analysis of RNA polymerase II responsible for induction of sister chromatid exchange.

负责诱导姐妹染色单体交换的 RNA 聚合酶 II 的功能分析。

基本信息

批准号：
06640804
负责人：
TSUJI Hideo
金额：
$ 1.34万
依托单位：
National Institute of Radiological Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06640804/
关键词：
Sister chromatid exchange RNA polymerase II Temperature-sensitive metant FISH Mutation cDNA Gene cDNA RNA polymerase

项目摘要

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.

温度敏感的CHO-K1细胞突变株tsTM 4在非允许温度（39 ℃）下表现出姐妹染色单体交换（SCE）沿着异常诱导和S期细胞DNA合成减少。这些片段的DNA序列分析显示，它是人RNA聚合酶II最大亚基基因，HSRPIILS，用荧光原位杂交法将HSRPIILs基因定位于17 p13.1。将含有HSRPIILs基因编码区的克隆基因组DNA片段转染tsTM 4细胞，可使tsTM 4细胞恢复异常的SCE诱导表型。北方分析表明，该HSRPIILs基因与正常对照组相比，具有较高的SCE诱导表型。在39 ℃和33.5 ℃下转录，这表明RPIILS基因的缺陷是导致tsTM 4突变体中SCE异常诱导的原因。当通过Northevn分析检测转录的RNA量时，即使在非允许温度下，tsTM 4细胞的转录活性相对于精细基因也是正常的。这表明RPIILS基因的缺陷仅限于转录一个为了确定RPIILS基因的突变位点，我们从中国仓鼠细胞和tsTM 4细胞中分离了RPIILS基因的cDNA，并进行了序列测定，发现了几个可能的突变位点。