Mott J E, Van Arsdell J, Platt T
Nucleic Acids Res. 1984 May 25;12(10):4139-52. doi: 10.1093/nar/12.10.4139.
We have cloned the gene for the lac operon repressor (lacI) of Escherichia coli into the M13 related phage f1. Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptase. LacI mutants are found at a frequency of 1 in 10(4) using a genetic screen in vivo. For two-thirds of the 60 mutants, lesions were identified within the first 400 bases of lacI, by dideoxy sequencing. An unexpectedly wide range of different lesions were observed, including transitions, transversions, and deletions (of which the most common were the removal of single base pairs). The replacement of dTTP by dBrUTP in the filling reaction resulted in a doubling of deletions in the sample population as well as the anticipated T to C and C to T transitions. Although the lacI gene has been extensively studied in vivo, the power of this technique for mutagenesis in vitro is demonstrated by the generation of three previously undescribed lacI mutations.
我们已将大肠杆菌乳糖操纵子阻遏物（lacI）的基因克隆到与M13相关的噬菌体f1中。通过用禽成髓细胞瘤病毒（AMV）逆转录酶填补lacI基因上有缺口的双链DNA分子，在体外对lacI基因进行诱变。利用体内遗传筛选，以10⁴分之一的频率发现了LacI突变体。通过双脱氧测序，在60个突变体中的三分之二里，在lacI的前400个碱基内鉴定出了损伤。观察到了意外广泛的不同损伤类型，包括转换、颠换和缺失（其中最常见的是单个碱基对的去除）。在填补反应中用dBrUTP替代dTTP，导致样本群体中的缺失增加了一倍，同时出现了预期的T到C和C到T的转换。尽管lacI基因已在体内得到广泛研究，但通过产生三个先前未描述的lacI突变，证明了该体外诱变技术的强大功能。
