用于在大肠杆菌中对克隆DNA进行正性半乳糖抗性选择的质粒载体。

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli.

作者信息

Ahmed A

出版信息

Gene. 1984 Apr;28(1):37-43. doi: 10.1016/0378-1119(84)90085-4.

Abstract

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.

摘要

将携带来自λgal⁺的部分半乳糖操纵子的HindIII - EcoRI片段插入pBR322中，产生了一种质粒（pAA3），该质粒对缺失半乳糖操纵子的大肠杆菌菌株赋予强烈的半乳糖敏感性。对半乳糖的敏感性是由位于pBR322的tet基因中的启动子表达激酶和转移酶（但不是表异构酶）基因引起的。在HindIII连接处插入携带Tn9的DNA片段会阻断半乳糖表达并产生半乳糖抗性表型。因此，半乳糖抗性可用于筛选在HindIII位点克隆的DNA片段。该系统有效地用于克隆λ、酵母和人类DNA。通过检测四环素抗性可以直接筛选克隆片段中是否存在启动子。或者，这些质粒可以用作黏粒，在多个位点克隆大片段DNA。描述了几种相关载体的构建。