Molecular analysis of the replication elements of the broad-host-range RepA/C replicon.
Molecular analysis of the replication elements of the broad-host-range RepA/C replicon.
复制标题
广泛宿主范围 RepA/C 复制子复制元件的分子分析。
DOI：
10.1006/plas.1996.0028
复制
发表时间：
1996
期刊：
影响因子：
2.6
通讯作者：
P. Plésiat
中科院分区：
文献类型：
--
作者：
C. Llanes;P. Gabant;M. Couturier;L. Bayer;P. Plésiat
RepA/C 是 IncA/C 不相容性质粒组特异的复制子，最近从质粒 RA1 中分离出来。该自复制区域的序列已确定；它包含 13 个重复，表明复制子使用 iterons 来控制其拷贝数。该序列包含两个 ORF，一个可能编码 33 kDa 蛋白质 (ORF1)，第二个可能编码 14 kDa 蛋白质 (ORF2)（Llanes 等人，1994b）。在这项工作中，我们使用体外转录/翻译系统检测到了一种多肽，其大小与ORF1的推导产物的大小非常吻合。删除和插入突变分析表明ORF1对于复制至关重要；它编码一种起始蛋白（称为 RepA）。 ORF2对于大肠杆菌中的复制并不是必需的，其功能仍有待确定。使用互补实验，定义了 RepA/C 的复制起点 (ori)。 ori位于repA下游的600bp片段中，包含10个同向重复序列。为了研究repA表达的控制，构建了转录融合体PrepA::lacZ。其分析表明，repA 是转录自动调节的，复制子的大多数 repA 基因也是由 iteron 控制的。
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.