Rosen J, Ryder T, Inokuchi H, Ohtsubo H, Ohtsubo E
Mol Gen Genet. 1980;179(3):527-37. doi: 10.1007/BF00271742.
The nucleotide sequence of the entire region required for autonomous replication and incompatibility of an R100 plasmid derivative, pSM1, has been determined. This region includes the replication region and all plasmid encoded information required for replication. Numerous reading frames for possible proteins can be found in this region. The existence of one of these proteins called RepA1 (285 amino acids; 33,000 daltons) which is encoded within the region known by cloning analysis to be required for replication is supported by several lines of evidence. These include an examination of the characteristic sequences on the proximal and distal ends of the coding region, a comparison of the sequence of the replication regions of pSM1 and the highly related R1 plasmid derivative Rsc13 as well as other biochemical and genetic evidence. The existence of two other proteins, RepA3 (64 amino acis; 7000 daltons) and RepA2 (103 amino acids; 11,400 daltons) is also consistent with most of the criteria mentioned above. However, the region encoding RepA3, which by cloning analysis is within the region responsible for both replication and incompatibility, has never been demonstrated to produce a 7,000 dalton polypeptide. Since a large secondary structure can be constructed in this region, it is possible that the region contains structure or other information that is responsible for incompatibility. RepA2, encoded entirely within the region identified by cloning analysis to be responsible for incompatibility but not for replication can be visualized in vivo and in vitro. However, the nucleotide sequence of the region encoding RepA2 is completely different in mutually incompatible plasmid derivatives of R1 and R100. It is therefore unlikely that RepA2 plays a major role in incompatibility. Thus, we predict that RepA2 is required to initiate DNA synthesis at the replication origin and that the region proximal to RepA2 either encodes a gene product or structure information that is responsible for incompatibility.
已确定了R100质粒衍生物pSM1自主复制和不相容性所需的整个区域的核苷酸序列。该区域包括复制区域以及复制所需的所有质粒编码信息。在该区域中可找到许多可能蛋白质的阅读框。通过克隆分析确定为复制所需区域内编码的一种名为RepA1（285个氨基酸；33,000道尔顿）的蛋白质的存在得到了多条证据的支持。这些证据包括对编码区域近端和远端特征序列的检查、pSM1和高度相关的R1质粒衍生物Rsc13以及其他生化和遗传证据的复制区域序列的比较。另外两种蛋白质RepA3（64个氨基酸；7000道尔顿）和RepA2（103个氨基酸；11,400道尔顿）的存在也与上述大多数标准一致。然而，通过克隆分析编码RepA3的区域在负责复制和不相容性的区域内，从未被证明能产生7000道尔顿的多肽。由于该区域可构建一个大的二级结构，该区域可能包含负责不相容性的结构或其他信息。RepA2完全在通过克隆分析确定为负责不相容性而非复制的区域内编码，可在体内和体外观察到。然而，在R1和R100相互不相容的质粒衍生物中，编码RepA2的区域的核苷酸序列完全不同。因此，RepA2不太可能在不相容性中起主要作用。因此，我们预测RepA2是在复制起点启动DNA合成所必需的，并且RepA2近端区域要么编码一种基因产物，要么编码负责不相容性的结构信息。
