Tolmachev O E, Gusiatiner M M
Mol Gen Mikrobiol Virusol. 1991 Nov(11):3-8.
Construction of the shuttle cloning vectors for Escherichia coli-Brevibacterium flavum system is described. Expression of the Sp/Sm resistance determinant derived from the Corynebacterium plasmid pCG4 was registered in Escherichia coli cells. The genetic determinant for Sp/Sm resistance was shown to be located in a 2.2 kb PstI-SphI fragment by the deletion analysis mapping in Escherichia coli cells. Using Escherichia coli as a host we cloned the unique 0.8 kb EcoRI-EcoRI fragment of Brevibacterium flavum bacteriophage phi BSh6 in the plasmids with dual replication origins. Blocking of the shuttle vector transfer to Brevibacterium flavum by the insertion of bacteriophage phi BSh6 DNA was observed. The deletion of entire phage fragment or a specific part of it made it possible introduction of plasmids harboured by Escherichia coli cells into Brevibacterium flavum. A potential vector for homologous DNA cloning in Brevibacterium flavum was constructed.
本文描述了用于大肠杆菌-黄色短杆菌系统的穿梭克隆载体的构建。源自棒状杆菌质粒pCG4的Sp/Sm抗性决定簇在大肠杆菌细胞中得以表达。通过在大肠杆菌细胞中的缺失分析定位，表明Sp/Sm抗性的遗传决定簇位于一个2.2 kb的PstI-SphI片段中。以大肠杆菌作为宿主，我们将黄色短杆菌噬菌体phi BSh6的独特0.8 kb EcoRI-EcoRI片段克隆到具有双复制起点的质粒中。观察到通过插入噬菌体phi BSh6 DNA可阻止穿梭载体转移至黄色短杆菌。删除整个噬菌体片段或其特定部分使得大肠杆菌细胞携带的质粒能够导入黄色短杆菌。构建了一种用于黄色短杆菌中同源DNA克隆的潜在载体。
