麦迪霉素产生菌具有启动功能的DNA片段的克隆和分析

CLONING OF DNA FRAGMENTS CONTAINING PROMOTER ACTIVITY FROM STREPTOMYCES MYCAROFACIENS 1748

利用启动子探针质粒载体pIJ486从麦迪霉素产生菌总DNA中克隆得到了一段具有启动功能的DNA片段.通过限制性酶酶切分析,测定插入DNA片段大小为2.3kb.又利用载体pIJ486和pIJ487的新霉素抗性结构基因上游有多酶切点方向相反的性质,分析了插入片段在两个不同方向上的启动能力.结果表明,在两个方向上均有启动功能,但强弱相差六倍.其中在XbaI-HindIII方向上具有较强的启动能力,在变铅青链霉菌中新霉素抗性水平可达20mg/ml以上.进一步对插入片段的三个BamHI小片段进行分析的结果表明,较强启动子区域集中在BamHI-BamHI 0.79kb DNA片段上.

A DNA fragment containing promoter activity has been cloned from midecamycin producing strain (S. mycarofaciens 1748). , using promoter-probe plasmid vector pIJ486. The molecular size of this fragment was 2. 3kb as shown by restriction analysis. A HindIII-HindIII 2. 08KB DNA fragment obtained from the original fragment has been analysed by subcloning it into polylinker of vector pIJ486/7 which have opposite direction. The result showed that HindIII-HiridIII 2. 08kb DNA fragment has promoter activity in both direction. Transformants of plasmid containing this fragment in vector pIJ487 in S. lividans TK24 were resistant to Km in the level of 20mg/ml, but in vector pIJ 486 were resistant to the level of 3mg/ml. It indicated that a rather strong promoter activity region was in the HindIII/XbaI-HindIII direction. BamHI fragments (A-0. 79kg, B-0. 67kb, C-0. 62kb)in 2. 08kb DNA fragment have been studied in regards of their promoter activity. The result suggested that A-0. 79kb region has the same promoter activity as in HindIII-HindIII 2. 08kb DNA fragment.