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

大肠杆菌K12菌株KS40和质粒pKY241已被设计用来方便地筛选质粒中的supF突变。KS40具有耐甲苯二酸(Nal)突变(GyrA)和链霉素(Sm)耐药突变(RpsL)。利用体外诱变技术，将琥珀色突变引入到克隆的大肠杆菌rpsL结构基因中，获得了携带gyrAam突变的pKY241的衍生物pOF105。当含有pOF105的KS40(记为KS40/pOF105)被转化到含有supF基因的质粒上时，KS40/pOF105产生了对NaL和Sm敏感的菌落。如果质粒上的supF基因携带失活突变，则KS40/pOF105将形成耐NAL和抗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、易于制备单链质粒DNA的M13复制起始点和supF基因的载体质粒，并利用该系统对氧诱导的突变进行了鉴定。在EDTA存在下用亚铁离子处理载体质粒时，主要观察到G：C到C：G颠换突变，其次是G：C到T：A颠倒突变。亚铁离子导致DNA中8-羟基脱氧鸟嘌呤(8-oxodG)的生成增加。因此，我们可以推测，Fe^&lt；2&gt；诱导的G：C到T：A的颠换是8-oxodG；G：C到C：G的来源不明。当用钴(II)直接处理该质粒时，主要观察到8到50个碱基的缺失，然后是移码突变。缺失连接部位有重复序列。因此，DNA-钴复合体可以干扰DNA复制，促进异常滑移错配DNA复制形成缺失突变。大肠杆菌宿主和载体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.