Yoon J H, Lee B J, Kang H S
Department of Microbiology, Seoul National University, Korea.
Mol Gen Genet. 1995 Jul 28;248(2):174-81. doi: 10.1007/BF02190798.
The uvsH DNA repair gene of Aspergillus nidulans has been cloned by complementation of the uvsH77 mutation with a cosmid library containing genomic DNA inserts from a wild-type strain. Methylmethane sulfonate (MMS)-resistant transformants were obtained on medium containing 0.01% MMS, to which uvsH mutants exhibit high sensitivity. Retransformation of uvsH77 mutants with the rescued cosmids from the MMS-resistant transformants resulted in restoration of both UV and MMS resistance to wild-type levels. Nucleotide sequence analysis of the genomic DNA and cDNA of the uvsH gene shows that it has an open reading frame (ORF) of 1329 bp, interrupted by two introns of 51 and 61 bp. A 2.4 kb transcript of the uvsH gene was detected by Northern blot analysis. Primer extension analysis revealed that transcription starts at 31 bp upstream from the translation initiation codon. This gene encodes a predicted polypeptide of 443 amino acids, which has two unique zinc finger motifs. The proposed polypeptide displays 39% identity to the Neurospora crassa UVS-2 protein and 24% identity to the Saccharomyces cerevisiae RAD18 protein. The sequence similarity is particularly high in three domains. One zinc finger (RING finger) motif is located in the first domain close to the N-terminus. The other zinc finger motif is in the second domain. In the third domain, the mutation sites in both the uvsH77 and uvsH304 alleles were identified.(ABSTRACT TRUNCATED AT 250 WORDS)
构巢曲霉的uvsH DNA修复基因已通过用含有野生型菌株基因组DNA插入片段的黏粒文库对uvsH77突变进行互补克隆得到。在含有0.01%甲磺酸甲酯(MMS)的培养基上获得了对MMS有抗性的转化体,uvsH突变体对该培养基表现出高敏感性。用从MMS抗性转化体中拯救出的黏粒对uvsH77突变体进行再次转化,导致对紫外线和MMS的抗性恢复到野生型水平。uvsH基因的基因组DNA和cDNA的核苷酸序列分析表明,它有一个1329 bp的开放阅读框(ORF),被两个分别为51 bp和61 bp的内含子打断。通过Northern印迹分析检测到uvsH基因有一个2.4 kb的转录本。引物延伸分析表明转录起始于翻译起始密码子上游31 bp处。该基因编码一个预测的由443个氨基酸组成的多肽,其具有两个独特的锌指基序。所提出的多肽与粗糙脉孢菌的UVS-2蛋白有39%的同一性,与酿酒酵母的RAD18蛋白有24%的同一性。在三个结构域中序列相似性特别高。一个锌指(RING指)基序位于靠近N端的第一个结构域中。另一个锌指基序在第二个结构域中。在第三个结构域中,确定了uvsH77和uvsH304等位基因中的突变位点。(摘要截短至250字)