MOLECULAR-CLONING AND ANALYSIS OF SCHIZOSACCHAROMYCES-POMBE-RAD9, A GENE INVOLVED IN DNA-REPAIR AND MUTAGENESIS
MOLECULAR-CLONING AND ANALYSIS OF SCHIZOSACCHAROMYCES-POMBE-RAD9, A GENE INVOLVED IN DNA-REPAIR AND MUTAGENESIS
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DOI：
10.1007/bf00266239
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发表时间：
1992-04-01
期刊：
影响因子：
--
通讯作者：
CHU, HM
中科院分区：
文献类型：
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作者：
LIEBERMAN, HB;HOPKINS, KM;CHU, HM
突变等位基因rad9-192使裂殖酵母细胞对电离辐射和紫外光敏感。我们已经从S.pombe基因组DNA文库中分离到一种独特的重组质粒，它能够恢复野生型对含有rad9-192的细胞群体的辐射抗性。利用克隆的DNA进行的质粒整合研究，结合交配和四分体分析，表明该分离的DNA含有野生型rad9基因。我们通过单次插入S.pombe ura4基因使克隆片段的修复功能失活。这个非功能片段被用来创造一个可行的中断突变体，从而证明rad9基因不编码必要的细胞功能。此外，rad9-192突变体群体与中断突变体一样对辐射敏感，这表明rad9基因的功能即使不是完全被导致rad9-192表型的分子缺陷所抑制，也是严重的。对rad9的DNA序列分析显示，该基因有一个1,278bp的开放阅读框，分别被53bp、57bp和56bp的三个内含子打断，并以终止密码子标签结束。该基因能够编码426个氨基酸的蛋白质，相应的计算分子量为47,464道尔顿。在rad9基因或其推导的蛋白质序列与以前输入DNA和蛋白质序列数据库的序列之间没有发现显著的同源性。
The mutant allele rad9-192 renders Schizosaccharomyces pombe cells sensitive to ionizing radiation and UV light. We have isolated from a S. pombe genomic DNA library a unique recombinant plasmid that is capable of restoring wild-type levels of radioresistance to a rad9-192-containing cell population. Plasmid integration studies using the cloned DNA, coupled with mating and tetrad analyses, indicate that this isolated DNA contains the wild-type rad9 gene. We inactivated the repair function of the cloned fragment by a single insertion of the S. pombe ura4 gene. This nonfunctional fragment was used to create a viable disruption mutant, thus demonstrating that the rad9 gene does not encode an essential cellular function. In addition, the rad9-192 mutant population is as radiosensitive as the disruption mutant, indicating that rad9 gene function is severely if not totally inhibited by the molecular defect responsible for the rad9-192 phenotype. DNA sequence analysis of rad9 reveals an open reading frame of 1,278 bp, interrupted by three introns 53 bp, 57 bp, and 56 bp long, respectively, and ending in the termination codon TAG. This gene is capable of encoding a protein of 426 amino acids, with a corresponding calculated molecular weight of 47,464 daltons. No significant homology was detected between the rad9 gene or its deduced protein sequence and sequences previously entered into DNA and protein sequence data banks.