Tomita H, Soshi T, Inoue H
Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan.
Mol Gen Genet. 1993 Apr;238(1-2):225-33. doi: 10.1007/BF00279551.
A clone containing the DNA repair gene uvs-2 of Neurospora crassa was identified from a Neurospora genomic DNA library using the sib-selection method. Transformants were screened for resistance to methyl methane sulfonate (MMS). A DNA fragment that complements the uvs-2 mutation was subcloned by monitoring its ability to transform the uvs-2 mutant to MMS resistance. Deletion analysis of the cloned DNA indicated that the size of the uvs-2 gene is approximately 1.6 kb. The identity of the uvs-2 gene was verified by restriction fragment length polymorphism (RFLP) mapping. The sensitivity of the transformant to three different mutagens was similar to that of the wild-type strain. Nucleotide sequences of genomic DNA and cDNA of the uvs-2 gene indicated that it has an open reading frame (ORF) of 1572 bp with a 69 bp intron in the middle of the sequence. Two transcription initiation sites located around 73 bp and 290 bp upstream of the translation initiation codon were identified by primer extension experiments. Northern analysis revealed that the nature transcript of the uvs-2 gene was about 1.8 kb long. The uvs-2 gene ORF is deduced to encode a polypeptide of 501 amino acids with a molecular mass of 54 kDa. The proposed polypeptide has 25% identity to the RAD18 polypeptide of Saccharomyces cerevisiae and contains two unique zinc finger motifs for nucleic acid binding. Similarities between the phenotypes of the rad18 and uvs-2 mutants suggest that the uvs-2 gene encodes a protein which is involved in postreplication repair, rather than excision repair.
利用同胞选择法从粗糙脉孢菌基因组DNA文库中鉴定出一个包含DNA修复基因uvs - 2的克隆。通过筛选对甲磺酸甲酯(MMS)具有抗性的转化体来进行研究。通过监测其将uvs - 2突变体转化为对MMS抗性的能力,对与uvs - 2突变互补的DNA片段进行亚克隆。对克隆DNA的缺失分析表明uvs - 2基因的大小约为1.6 kb。通过限制性片段长度多态性(RFLP)图谱验证了uvs - 2基因的身份。转化体对三种不同诱变剂的敏感性与野生型菌株相似。uvs - 2基因的基因组DNA和cDNA的核苷酸序列表明,它有一个1572 bp的开放阅读框(ORF),在序列中间有一个69 bp的内含子。通过引物延伸实验确定了位于翻译起始密码子上游约73 bp和290 bp处的两个转录起始位点。Northern分析显示uvs - 2基因的天然转录本约为1.8 kb长。uvs - 2基因ORF推导编码一个由501个氨基酸组成的多肽,分子量为54 kDa。推测的多肽与酿酒酵母的RAD18多肽有25%的同一性,并含有两个独特的用于核酸结合的锌指基序。rad18和uvs - 2突变体表型之间的相似性表明uvs - 2基因编码一种参与复制后修复而非切除修复的蛋白质。