Michaels M L, Kim C W, Matthews D A, Miller J H
Molecular Biology Institute, University of California, Los Angeles 90024.
Proc Natl Acad Sci U S A. 1990 May;87(10):3957-61. doi: 10.1073/pnas.87.10.3957.
By using site-directed oligonucleotide mutagenesis, amber nonsense stop codons (5'-TAG-3') have been introduced at 20 sites in the Escherichia coli thymidylate synthase gene. By transforming the thyA mutant plasmids into 13 strains, each of which harbor different amber suppressor tRNAs, we were able to generate over 245 amino acid substitutions in E. coli thymidylate synthase (EC 2.1.1.45). Growth characteristics of these mutants have been studied, yielding a body of information that includes some surprising results in light of the recently published crystal structure of the enzyme.
通过使用定点寡核苷酸诱变技术，在大肠杆菌胸苷酸合成酶基因的20个位点引入了琥珀色无义终止密码子（5'-TAG-3'）。通过将thyA突变体质粒转化到13个菌株中，每个菌株都携带不同的琥珀色抑制tRNA，我们能够在大肠杆菌胸苷酸合成酶（EC 2.1.1.45）中产生超过245个氨基酸取代。对这些突变体的生长特性进行了研究，根据最近发表的该酶晶体结构，得到了一系列包含一些惊人结果的信息。
