链霉菌高拷贝质粒pIJ101 DNA的研究 Ⅱ.在大肠杆菌中具有终止子活性片段的克隆和分析

STUDIES ON THE MULTICOPY STREPTOMYCES PLASMID pIJl01 I.Cloning and analysis of the terminator-active fragments in Escherichia coli

在大肠杆菌的终止子探针载体pIJ667上,neo基因的正常表达可使寄主产生较强的卡那霉索抗性。然而,把链霉菌高拷贝质粒pIJ101上的BclⅠ片段克隆到这个载体的BglⅡ位点,使neo的结构基因与其启动子隔开所做的克隆试验显示,有两个插入片段,BclⅠ-A和BclⅠ-E,可以有效地终止neo基因的转录,使大肠杆菌寄主对卡那霉素呈现敏感。这两个片段在pIJ101的限制性内切酶图谱上已得到了定位,其活性区域的大小分别为2.9和1.19kb。把pIJ101衍生的小质粒pIJ350上BclⅠ片段克隆到pIJ667中,不仅进一步暗示出pIJ101 BclⅠ-A片段上终止子功能的存在,而且把这个区域缩小到1.32kb的范围内。此外,这些终止子活性的表达显示出明显的方向性。

pIJ667 is a Escherichia coli terminator-probe vector, the expression of its neo genecan confer highly resistance to kanamycin on its host. However, the experiments to sepa-rate the neo structure gene and its promoter by cloning in its BglⅡ site the Bcl I fra-gments of the multi-copy Streptomyces plasmid pIJ101 reveraled that, two Bcl I fragme-nts, Bcl I-A and Bcl I-E, can efficiently terminate the transcripton of the neo gene,making their hosts highly sensitive to kanamycin. The two fragments have been locatedon the restriction endonuclease cleavage map of pIJ101. The size of the terminator-activeregions is 2.9 kb and 1.19 kb, respectively. The cloning of the smaller Bcl I fragmentfrom plJ101-derived plasmid plJ350 into the Bgl Ⅱ site of plJ667 not only give the sameindication for the presence of terminator activity on Bcl I-A fragment, but also narrowedhe active region down to 1.32 kb. In addition, the apparent orientation-dependenoe ofhe trminator function was clearly shown by these experiments.