Barany F
Proc Natl Acad Sci U S A. 1985 Jun;82(12):4202-6. doi: 10.1073/pnas.82.12.4202.
An efficient method for introducing two codons into a cloned gene has been applied to studying functional regions of the pBR322-encoded tetracycline-resistance gene and beta-lactamase (ampicillin-resistance) gene. Single-stranded hexameric linkers are inserted into a preexisting cohesive end restriction site to create a new (six-base recognition) restriction site. Insertion mutations are enriched by using biochemical selection or are selected by using a kanamycin-resistance cassette (biological selection). Phenotypes of insertion mutations isolated in the tetracycline-resistance gene support the hypothesis that it is comprised of two domains connected by a central hinge. Mutations in the beta-lactamase gene are temperature sensitive and demonstrate altered sensitivity to various beta-lactams and inhibitors.
一种将两个密码子引入克隆基因的有效方法已被应用于研究pBR322编码的四环素抗性基因和β-内酰胺酶（氨苄青霉素抗性）基因的功能区域。单链六聚体接头被插入到预先存在的粘性末端限制性位点中，以创建一个新的（六碱基识别）限制性位点。插入突变通过生化选择得以富集，或通过使用卡那霉素抗性盒进行选择（生物学选择）。在四环素抗性基因中分离出的插入突变的表型支持了这样一种假说，即它由通过中央铰链连接的两个结构域组成。β-内酰胺酶基因中的突变对温度敏感，并表现出对各种β-内酰胺类药物和抑制剂的敏感性改变。
