酵母DNA修复基因RAD16温度敏感突变株的分离及人类互补基因的鉴定

Isolation of Temperature-sensitive Mutant of Yeast Saccharamyces cerevisae RAD16 Gene and Identification of Human Homolog

用PCR介导的基因重组法敲除酵母RAD16基因,用羟胺处理酵母RAD16基因表达质粒,获得RAD16基因突变库,并将其转化RAD16基因敲除的酵母细胞.用平皿复制法获得温度敏感突变株rad16-ts2,经互补试验证实,该突变表型为RAD16基因突变所致.将人cDNA表达文库转化rad16-ts2后筛选温度敏感拯救(rescue)表型,回收拯救表型酵母细胞中的质粒.测序结果表明,所获得的人cDNA克隆为HLTF/Zbu1/SMACA3基因.比较分析显示,人类该基因编码蛋白质氨基酸序列与酵母Rad16的同一性为32%,相似性则达50%.人HLTF/Zbu1/MACA3基因在HeLa细胞中过表达,可显著抑制过氧化氢损伤和UV照射诱导的细胞凋亡.

PCR-mediated recombination was used to delete RAD16 gene of yeast Saccharamyces cerevisae. Plasmids containing full length RAD16 gene was treated with hydroxylamine to obtain RAD16 mutant library. The mutant RAD16 gene library was transformed into RAD16 deletion strain （radl6 △ ） to obtain the temperature-sensitive mutant of RAD16 gene by plate-replicating. A mutant, radl6-ts2, was successfully identified and human cDNA expression library was transformed into this strain. By rescue of radl6-ts2 growth at 37℃, the human homolog of yeast RAD16 was identified and it turns out to be HLTF/Zbul/SMARCA3 by sequencing analysis. Overexpression of human HLTF in HeLa cells can significantly inhibit apoptosis induced by hydrogen peroxide treatment or UV-irradiation.