Banerjee S, Kim H Y, Iyer V N
Department of Biology, Carleton University, Ottawa, Ontario, Canada.
Plasmid. 1996 Jan;35(1):58-61. doi: 10.1006/plas.1996.0006.
The essential replicon region of plasmid pCU1 has, within 1.2 kb, two origins of replication that can function in the absence of Escherichia coli DNA polymerase I and one that requires this polymerase. To isolate mutants in the replicon pathway that uses the PolI-dependent origin in the presence of the two other origins, we examined the feasibility of exploiting E. coli strains carrying a polymerase c bypass mutation (pcbA) and which can survive and form colonies with the polymerase activity of polC inactivated at 42 degrees C. The selection scheme that is described was successful and resulted in the isolation of a mutant replicon that is not maintained at 42 degrees C in a PcbA-PolC+tsPolA+ strain. Nucleotide sequencing indentified the mutated region to be within the origin (OriV) that was known to be polA-dependent. Electron microscopy of mutant plasmid molecules replicating in a Pcb+ strain confirmed that OriV is inactivated.
质粒pCU1的基本复制子区域在1.2 kb范围内有两个可在无大肠杆菌DNA聚合酶I的情况下发挥作用的复制起点，以及一个需要该聚合酶的复制起点。为了在存在另外两个复制起点的情况下，分离出在使用依赖PolI的复制起点的复制子途径中的突变体，我们研究了利用携带聚合酶c旁路突变（pcbA）的大肠杆菌菌株的可行性，这些菌株在42℃下polC聚合酶活性失活时能够存活并形成菌落。所描述的选择方案是成功的，结果分离出了一种突变复制子，该复制子在PcbA-PolC+tsPolA+菌株中于42℃下无法维持。核苷酸测序确定突变区域在已知依赖polA的复制起点（OriV）内。对在Pcb+菌株中复制的突变体质粒分子进行电子显微镜观察证实OriV失活。
