Bhanot O S, Ray A
Proc Natl Acad Sci U S A. 1986 Oct;83(19):7348-52. doi: 10.1073/pnas.83.19.7348.
A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75%. When repair of O6 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.
本文介绍了一种基于噬菌体φX174的位点特异性诱变系统，用于研究致癌物诱导的DNA修饰在体内的诱变频率和特异性。将在特定位点含有O6-甲基鸟嘌呤的（-）链引物与φX缺口双链DNA的基因G中的单链区域杂交。该杂交体经酶促转化为复制型DNA，并用于转化大肠杆菌细胞。通过基因互补挽救了由该修饰产生的所有基因G突变体。复制型DNA（+）链的裂解基因E中的琥珀突变阻止了源自该链的野生型噬菌体的裂解生长。在由转化细胞产生的每个含突变体的感染中心中，与野生型相比，基因G突变体噬菌体的比例为3:1。因此，在体内，复制型φX DNA中的O6-甲基鸟嘌呤具有75%的诱变频率。当发生O6-甲基鸟嘌呤的修复时，是在复制前进行的。这些突变完全是由于胸腺嘧啶的错误掺入。
