Tomizawa J I, Ohmori H, Bird R E
Proc Natl Acad Sci U S A. 1977 May;74(5):1865-9. doi: 10.1073/pnas.74.5.1865.
Cleavage maps of colicin E1 plasmid DNA and its smaller derivative, pNT1 DNA, were constructed by using restriction endonucleases. The nucleotide sequence of a region that contains the orgin of replication was determined. The site of the nucleotide from which DNA replication is initiated was determined with 6S L-fragments, the DNA fragment first made on colicin E1 plasmid DNA. The fragments were labeled with [gamma-32P]ATP and polynucleotide 5'-hydroxyl-kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) at the 5'-OH groups which were uncovered by alkali treatment. The site is one of three consecutive nucleotides, dA, dA, and dC, located at a unique position. One or a few rA residues were found to be attached to some of the DNA molecules. The transition from the primer RNA to DNA occurs in a region consisting of a segment of five A residues. Both sides of this segment are rich in G and C.
通过使用限制性内切酶构建了大肠杆菌素E1质粒DNA及其较小的衍生物pNT1 DNA的切割图谱。测定了包含复制起点区域的核苷酸序列。利用6S L片段确定了DNA复制起始的核苷酸位点，6S L片段是在大肠杆菌素E1质粒DNA上首先产生的DNA片段。这些片段用[γ-32P]ATP和多核苷酸5'-羟基激酶（ATP：5'-去磷酸多核苷酸5'-磷酸转移酶，EC 2.7.1.78）在经碱处理后暴露的5'-OH基团处进行标记。该位点是位于一个独特位置的三个连续核苷酸dA、dA和dC之一。发现一些DNA分子上连接有一个或几个rA残基。从引物RNA到DNA的转变发生在由五个A残基组成的一段区域内。该片段的两侧富含G和C。
