Llanes C, Gabant P, Couturier M, Bayer L, Plesiat P
Laboratoire de Bacteriologie, Faculté de Medecine, Université de Franche-Comté, Besançon, France.
Plasmid. 1996 Jul;36(1):26-35. doi: 10.1006/plas.1996.0028.
RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1. The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b). In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1. Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA). ORF2 was not essential for replication in Escherichia coli and its function remains to be determined. Using complementation experiments, the replication origin (ori) of RepA/C was defined. The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats. To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed. Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons.
RepA/C是IncA/C不相容群质粒特有的复制子，最近从质粒RA1中分离得到。该自主复制区域的序列已确定；它包含13个重复序列，表明该复制子利用迭代子来控制其拷贝数。该序列包含两个开放阅读框，一个可能编码33 kDa的蛋白质（ORF1），另一个可能编码14 kDa的蛋白质（ORF2）（Llanes等人，1994b）。在这项研究中，我们使用体外转录/翻译系统检测到一种多肽，其大小与ORF1推导产物的大小非常吻合。缺失和插入突变分析表明，ORF1对复制至关重要；它编码一种起始蛋白（称为RepA）。ORF2对大肠杆菌中的复制不是必需的，其功能仍有待确定。通过互补实验，确定了RepA/C的复制起点（ori）。ori位于repA下游的一个600 bp片段中，包含10个正向重复序列。为了研究repA表达的调控，构建了转录融合体PrepA::lacZ。对其分析表明，repA与大多数由迭代子控制的复制子的repA基因一样，受到转录自调控。
