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

温度敏感型CHO-K1细胞突变体tsTM4在不允许的温度(39ﾟC)下，在滞留于S期的细胞中表现出姐妹染色单体互换(SCE)的异常诱导和DNA合成的降低。我们通过将人类基因组DNA导入TsTM4细胞并从二次转化子中收集人类DNA片段，克隆了一个弥补TsTM4细胞遗传缺陷的人类基因。序列分析表明，HSRPIILS是人类RNA聚合酶II最大亚基基因，有29个外显子，全长31kb.HSRPIILS基因经荧光原位杂交定位于17p13.1。将含有HSRPIILS全部编码区的克隆DNA片段导入TTM4细胞，可挽救其异常的诱导音型。Northern分析表明，HSRPIILS基因在39ﾟC和33.5ﾟC处转录。通过Northevn分析，发现RPIILS基因缺陷仅限于部分基因的转录，或与DNA代谢的其他功能有关。为了确定RPIILS基因的突变位点，从中国仓鼠野生型细胞和tsTM4细胞中分离了RPIILS基因的cDNAs，并对其进行了测序。

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.