吸水链霉菌应城变种10-22中一个复制子的克隆和基本复制区的定位

MOLECULAR CLONING OF A REPLICATION FUNCTION FROM STREPTOMYCES HYGROSCOPICUS STRAIN 10-22 AND LOCALIZATION OF ITS MINIMAL REPLICON

利用由pBR322衍生的链霉菌复制子探针载体pIJ2703,从吸水链霉菌应城变种菌株10-22中克隆了一段13kb的可以自主复制的DNA,制作了携带这段DNA的杂合质粒pHZ54的限制酶图谱。利用缺失、次级克隆等方法将基本复制区确定在2.86kb的范围内。用外切酶Ⅲ顺序缺失法不仅进一步确证了基本复制区的位置,而且将其缩小到2kb的范围内。同时,在上述试验过程中、还组建了一系列既可在大肠杆菌、又可在变铅青链霉菌中复制的双功能质粒,其中有些质粒是有用的穿梭载体。

13 kb DNA carrying a replication function has been isolated from Streptomyces hygroscopicus var. yingchengensis strain 10-22 using a pBR322-derived Streptomyces replication-probe vector pIJ2703. The restrication endonuclease cleavage map of the hybrid plasmid, pHZ54, has been constructed. Following a series of deletion and subcloning experiments, the minimal replication region was located within a 2.86 kb DNA fragment. Subsequently, this region was further shortened into a 2 kb region by exonuclease Ⅲ deletion. A few small bifunctional plasmids which were able to replicate in E. coli as well as in Streptomyces, were obtained during above process. Some plasmids could be used as shuttle vectors.