链霉菌高拷贝质粒pIJ101 DNA的研究 Ⅳ.BclI-E片段上启动子的定位

STUDIES ON THE MULTI-COPY STREPTOMYCES PLASMID pIJ101 Ⅳ.LOCALIZATION OF PROMOTER-ACTIVE REGION ON BclI-E FRAGMENT

将链霉菌高拷贝质粒pIJ101上具有明显启动子活性的BclI—E片段(1.18kb)用MboI酶切后克隆到链霉菌启动子探针载体pIJ425中所做的试验证明,当把这个片段的末端去掉以后,中间的两个次级片段(0.63kb和0.68kb)比完整的BclI—E片段具有更强的(4～6倍)激活指标基因neo表达的能力.Southern印迹杂交试验证明这两个片段均来自于pIJ101的BclI—E片段.这项试验不仅大大缩小了BclI—E片段上的启动子活性区域,而且说明BclI—E片段末端有转录终止子的存在.

The subfragments gererated by Mbo I digestion of 1.18 kb Bell—Efragment of the multi—copy Streptomyces plasmid pIJ101 were cloned into Streptomycespromoter—probe vector pIJ425.Two internal subclones(0.63 kb and 0.68 kb)can activate theexpression of indicator gene(neo)in a level 4—6 times higher than entire Bell—E fragment ineither orientations.Southern hybridization experiment shows that both fragments originated frompIJ101.This experiment not only located promoter—active region more precisely,but alsosuggested that the terminal region of the Bell—E fragment may contain transcriptional terminator(S).