Rechinskiĭ V O, Savochkina L P, Bibilashvili R Sh
Mol Biol (Mosk). 1981 Jul-Aug;15(4):950-6.
A new plasmid vector, designated pBRS188 has been constructed for cloning of promoter-containing DNA fragments. This plasmid is a derivative of the E. coli drug-resistance plasmid pBR322 in which a small region (13 base pairs long) within the Tc promoter is eliminated. As a result of the alteration pBRS188 has lost the ability to confer Tc resistance to the host strain. Cloning of foreign DNA fragments, carrying promoters for E. coli RNA polymerase, into the unique EcoRI site of pBRS188 allows to isolate the recombinant TcR transformants. Our construction required the use of new techniques, involving partial hydrolysis of DNA fragments by E. coli DNA polymerase I in the presence of one deoxyribonucleosidetriphosphate and by nuclease S1. An important feature of this method is the ability to regenerate restriction endonuclease recognition sites at junctions of DNA fragments.
一种名为pBRS188的新型质粒载体已被构建用于克隆含启动子的DNA片段。该质粒是大肠杆菌耐药性质粒pBR322的衍生物，其中Tc启动子内的一个小区域（13个碱基对长）被删除。由于这种改变，pBRS188失去了赋予宿主菌株Tc抗性的能力。将携带大肠杆菌RNA聚合酶启动子的外源DNA片段克隆到pBRS188的独特EcoRI位点，可分离出重组TcR转化体。我们的构建需要使用新技术，包括在一种脱氧核糖核苷三磷酸存在的情况下，利用大肠杆菌DNA聚合酶I对DNA片段进行部分水解，以及利用核酸酶S1进行水解。该方法的一个重要特点是能够在DNA片段的连接处再生限制性内切酶识别位点。
