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

一个温度敏感的CHO-K1细胞突变体tsTM4在非允许温度（39℃）下表现出异常诱导姐妹染色单体交换（ses）和DNA合成减少。我们通过将人类基因组DNA转染到tsTM4细胞中，并从次级转化子中收集人类DNA片段，克隆了一个人类基因，弥补了tsTM4细胞的遗传缺陷。这些片段的DNA序列分析显示，它是人类RNA聚合酶II最大的亚基基因HSRPIILS，有29个外显子，长度为31 kbp。通过荧光原位杂交将HSRPIILS基因定位到17p13.1。将含有HSRPIILS整个编码区的克隆基因组DNA片段转染到tsTM4细胞中，可挽救其异常的SCE诱导声型。Northern分析表明，在转化过程中，HSRPIILS基因在39个和33.5个接合点上转录，这表明RPIILS基因的缺陷是导致tsTM4突变体异常诱导sce的原因。Northevn分析显示，即使在非允许温度下，tsTM4细胞对精细基因的转录活性也正常。这表明RPIILS的缺陷仅限于部分基因的转录或负责DNA代谢的另一功能。为了确定RPIILS基因的突变位点，我们从野生型中国仓鼠细胞和tsTM4细胞中分离RPIILS cdna，并对其进行测序。发现了几个候选突变位点。

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.