IDENTIFICATION OF uvrA GENE MUTATION SITES IN TWO MITOMYCIN-SENSITIVE Deinococcus radiodurans STRAINS

抗辐射菌Ｄｅｉｎｏｃｏｃｕｓｒａｄｉｏｄｕｒａｎｓ具有显著的ＤＮＡ损伤修复能力，包括对丝裂霉素（ＭＣ），紫外线（ＵＶ）及电离辐射等所致的损伤。在用对ＤＮＡ损伤因子具有抗性的野生型抗辐射菌ＫＤ８３０１的基因组ＤＮＡ构建的基因文库中，有四个克隆能够通过其ＤＮＡ转化，使二株对ＭＣ敏感的Ｄｅｉｎｏｃｏｃｃｕｓｒａｄｉｏｄｕｒａｎｓ的突变体－２６２１和３０２１回复对ＭＣ的抗性。克隆了二株突变体中受突变（ｍｔｃＡ或ｍｔｃＢ）影响的基因，并测定了该基因的核苷酸序列。理论上推定抗辐射菌ｕｖｒＡ基因产物的氨基酸序列是由１０３６个氨基酸组成，与许多细菌的ＵｖｒＡ蛋白质具有同源性。用ＰＣＲ技术扩增二株突变体基因组中相应的ＤＮＡ片段，并对其加以测序分析，确定了突变发生的位点。对于突变体３０２１，其ｕｖｒＡ基因中发生了１４４个碱基对（ｂｐ）的缺失突变（缺失部分包括ｕｖｒＡ的起始密码），造成了３０２１的ｕｖｒＡ基因的失活。对于２６２１突变体，其ｕｖｒＡ基因内却发生了一个插入突变，造成了该基因的插入失活。该插入序列由１３２２ｂｐ组成，侧面与１９ｂｐ组成的末端反转重复（ＩｎｖｅｒｔｅｄＴｅｒｍｉｎａｌＲｅｐｅａｔｓ，ＩＴＲ）相连接，并产生?

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C(MC), ultraviolet light(UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC s) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301 which showed the property of wild type phenotype to DNA-damaging agents. Gene affected by mutation ( mtcA or mtcB ) in both mutants was cloned and its nucleotide sequence was determined. The deduced amino acid ( aa ) sequence of Dr uvr A gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites in both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomicDNA of the mutants. A 144-base pairs (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621,suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORF) were found in the IS element. The deduced aa sequences of large and small ORF show homology to a putative transposase found in IS4 of Escherichia coli E. coli ) and to a resolvase found in IS Xc5 of Xanthomonas campestris (Xc) , respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.