Sancar A, Kacinski B M, Mott D L, Rupp W D
Proc Natl Acad Sci U S A. 1981 Sep;78(9):5450-4. doi: 10.1073/pnas.78.9.5450.
We have constructed a multicopy plasmid that carries the uvrC gene of Escherichia coli. By inserting the transposon Tn1000 (previously designated gamma delta) into this plasmid, we obtained many derivatives that fail to complement uvrC34. The proteins synthesized by the original plasmid and the uvrC::Tn1000 derivatives were labeled in maxicells and analyzed on gels, demonstrating that a protein of Mr 70,000 encoded by the original uvrC+ plasmid was absent from the mutated noncomplementing derivatives; this protein is presumed to be the uvrC gene product. We found that this protein of Mr 70,000 binds to DNA and have partially purified the uvrC protein by DNA-cellulose chromatography. Because some of the uvrC::Tn1000 derivatives produce truncated polypeptides, the orientation of expression and the location of the promoter were determined by correlating the sizes of the truncated polypeptides with the sites of insertion of Tn1000.
我们构建了一个携带大肠杆菌uvrC基因的多拷贝质粒。通过将转座子Tn1000（先前称为γδ）插入该质粒，我们获得了许多无法互补uvrC34的衍生物。在最大细胞中对原始质粒和uvrC::Tn1000衍生物合成的蛋白质进行标记并在凝胶上分析，结果表明，突变的非互补衍生物中不存在由原始uvrC+质粒编码的70,000道尔顿的蛋白质；该蛋白质被推测为uvrC基因产物。我们发现这种70,000道尔顿的蛋白质与DNA结合，并通过DNA - 纤维素色谱法对uvrC蛋白进行了部分纯化。由于一些uvrC::Tn1000衍生物产生截短的多肽，通过将截短多肽的大小与Tn1000的插入位点相关联，确定了表达方向和启动子的位置。
