Dorrington R A, Bardien S, Rawlings D E
Department of Microbiology, University of Cape Town, Rondebosch, South Africa.
Gene. 1991 Dec 1;108(1):7-14. doi: 10.1016/0378-1119(91)90481-p.
A 3202-bp fragment of plasmid pTF-FC2, cloned into PUC19, had previously been identified as the minimum region required for replication in either Pseudomonas aeruginosa or Escherichia coli polA- mutants. During the course of experiments to construct broad-host-range cloning vectors based on the pTF-FC2 replicon, it was found that the 3202-bp fragment had an absolute requirement for some function of the pUC19 vector. This requirement was eliminated in the presence of co-resident pTF-FC2 derivatives. An additional 1239-bp fragment from pTF-FC2, immediately adjacent to the 3202-bp fragment, was identified which restored the ability of the pTF-FC2 replicon to replicate autonomously. Sequence analysis of the region revealed a single open reading frame encoding a 40-kDa polypeptide, which was synthesised in an in vitro transcription/translation system. A comparison of the amino acid sequence of this protein with sequence data banks revealed limited homology with the RepB' primase of the IncQ plasmid, RSF1010. An M13 delta lac 110 replication-deficient phage system was used to demonstrate that the 40-kDa protein did function as a primase with respect to replication at the origin of replication (vegetative) of pTF-FC2.
克隆到PUC19中的质粒pTF-FC2的一个3202碱基对片段，先前已被确定为在铜绿假单胞菌或大肠杆菌polA-突变体中复制所需的最小区域。在基于pTF-FC2复制子构建广宿主范围克隆载体的实验过程中，发现这个3202碱基对片段对pUC19载体的某些功能有绝对需求。在共驻留的pTF-FC2衍生物存在时，这种需求被消除。从pTF-FC2中鉴定出一个紧邻3202碱基对片段的额外1239碱基对片段，它恢复了pTF-FC2复制子自主复制的能力。对该区域的序列分析揭示了一个编码40 kDa多肽的单一开放阅读框，该多肽在体外转录/翻译系统中合成。将该蛋白质的氨基酸序列与序列数据库进行比较，发现与IncQ质粒RSF1010的RepB'引发酶有有限的同源性。使用M13 δlac 110复制缺陷噬菌体系统证明，就pTF-FC2复制起点（营养型）的复制而言，40 kDa蛋白质确实起到了引发酶的作用。
