Belfort M, Pedersen-Lane J
J Bacteriol. 1984 Oct;160(1):371-8. doi: 10.1128/jb.160.1.371-378.1984.
Random in vitro mutagenesis of the thyA gene is being used to delineate its regulatory elements as well as the functional domains of its product, thymidylate synthase (EC 2.1.1.45). Streamlined procedures have been developed for the isolation and characterization of the mutants. Positive selection for synthase-deficient thyA Escherichia coli permitted the isolation of 400 mutants, which are being categorized by phenotypic and genetic criteria. An in situ 5-fluorodeoxyuridylate binding assay was devised to rapidly probe the substrate binding domain, whereas facile mapping procedures, based on pBR322- or M13-borne thyA deletion derivatives, were developed to localize mutations. The sequence changes of one amber mutation and another mutation that abolishes catalysis while maintaining substrate binding activity are presented. The orientation of the thyA gene on the E. coli chromosome was established.
thyA基因的随机体外诱变正被用于描绘其调控元件以及其产物胸苷酸合酶（EC 2.1.1.45）的功能结构域。已经开发出简化程序用于分离和鉴定突变体。对缺乏合酶的thyA大肠杆菌进行正向选择，从而分离出400个突变体，正根据表型和遗传标准对其进行分类。设计了一种原位5-氟脱氧尿苷酸结合试验来快速探测底物结合结构域，同时基于携带在pBR322或M13上的thyA缺失衍生物开发了简便的定位程序来定位突变。给出了一个琥珀突变以及另一个在维持底物结合活性的同时消除催化作用的突变的序列变化。确定了thyA基因在大肠杆菌染色体上的方向。
