Establishment of Environmental Mutagen Screening System with Reference of DNA Sequence Alteration

大肠杆菌K12菌株KS40和质粒pKY241先前设计用于容易筛选质粒中的supF突变。KS40具有耐钠二酸（Nal） （gyrA）和耐链霉素（Sm） （rpsL）突变。利用体外诱变技术，将一个琥珀色突变引入克隆的大肠杆菌rpsL结构基因中，得到了携带gyrAam突变的pKY241的衍生物pOF105。将含有pOF105的KS40（命名为KS40/ pOF105）与含有supF基因的质粒转化后，KS40/ pOF105产生nal敏感菌落和sm敏感菌落。如果质粒上的supF基因携带失活突变，则KS40/ pOF105将形成耐nal和耐sm的菌落。我们进一步构建了携带pBR322复制起始点、β -内酰胺酶基因、M13复制起始点（易于制备单链质粒DNA）和supF基因的载体质粒。利用该系统对氧诱导突变进行了表征。在EDTA存在的情况下，亚铁离子处理载体质粒，主要观察到G: C到C: G的翻转突变，其次是G: C到T: A的翻转突变。亚铁离子导致质粒DNA中8-羟基脱氧鸟嘌呤（8-oxodG）的形成增加。因此，我们可以指出，Fe^<2+>-诱导的G: C到T: A翻转的起源是8-oxodG；G: C到C: G的来源不明。当质粒被认为与DNA结合的钴（II）直接处理时，主要观察到8到50个碱基缺失，然后是移码突变。缺失连接位点具有重复序列。因此，DNA-钴复合物可以干扰DNA复制，促进异常的滑错配对DNA复制形成缺失突变。大肠杆菌宿主和载体质粒的设计将建立环境诱变剂的筛选体系。

Escherichia coli K12 strain KS40 and plasmid pKY241 had been previously designed for easy screening of supF mutations in plasmid. KS40 has mutation of nalidixic acid (Nal) -resistant (gyrA) and streptomycin (Sm) -resistant (rpsL) mutations. Using in vitro mutagenesis, an amber mutation was introduced into the cloned rpsL structural gene, of E.coli, to give pOF105, a derivative of pKY241 which carries gyrAam mutation. When KS40 containing pOF105 (designated KS40/ pOF105) is transformed with plasmid having supF gene, KS40/ pOF105 give rise to Nal-sensitive and Sm-sensitive colonies. If the supF gene on the plasmid carries an inactivating mutation, then KS40/ pOF105 will form Nal-resistant and Sm-resistant colonies. We have further constructed a vector plasmid carrying the pBR322 replication origin, beta-lactamase gene, M13 replication origin which permits easy preparation of single-stranded plasmid DNA,and supF gene.By using the system, oxygen species induced mutations were characterized. When the vector plasmid was treated with ferrous ion in the presence of EDTA,G : C to C : G transversion mutations were predominantly observed followed by G : C to T : A transversion. Ferrous ion resulted in increased formation of 8-hydroxydeoxyguanine (8-oxodG) in plasmid DNA.We can therefore indicate that the origin of Fe^<2+>-induced G : C to T : A transversion is 8-oxodG ; the origin of G : C to C : G is unidentified. When the plasmid was directly treated with cobalt (II), which is assumed to bind DNA,8 to 50 base deletions were predominantly observed followed by frameshift mutations. The sites of deletion junction have repeated sequences. Therefore, DNA-cobalt complex can interfere DNA replication, facilitating aberrant slip-mispairing DNA replication forming deletion mutations.The design of E.coli host as well as vector plasmid will establish the screening system for environmental mutagens.