Imanaka T, Ishikawa H, Aiba S
Mol Gen Genet. 1986 Oct;205(1):90-6. doi: 10.1007/BF02428036.
The 2.6 kb kanamycin-resistant (Kmr) plasmid, pRAT11, was constructed using both the replication determinant (repA) region of the 10.8 kb tetracycline-resistant (Tcr) low copy number plasmid pTB52 and another fragment (0.9 kb) that contained solely the Kmr gene of pUB110. The complete nucleotide sequence of this plasmid was determined. The repA region contained a large open reading frame encoding RepA protein (396 amino acid residues). In vitro transcription and translation of the repA gene were confirmed. RepA protein was shown to be indispensable for plasmid replication, and acted in trans on DNA. The part of the repA gene encoding the specific recognition region of the RepA protein was located and contained 3.5 direct repeats of 24 bp (GGTTTCAAAAATGAAACGGTGGAG). Upstream and downstream of the direct repeats were the recognition sequence (TTATCCACA) of the Escherichia coli DnaA protein and an AT-rich region, respectively. The replication control mechanism of the low copy number Bacillus plasmid is discussed.
2.6 kb的卡那霉素抗性（Kmr）质粒pRAT11是利用10.8 kb的四环素抗性（Tcr）低拷贝数质粒pTB52的复制决定簇（repA）区域和另一个仅包含pUB110的Kmr基因的片段（0.9 kb）构建而成的。测定了该质粒的完整核苷酸序列。repA区域包含一个编码RepA蛋白（396个氨基酸残基）的大开放阅读框。证实了repA基因的体外转录和翻译。RepA蛋白被证明对质粒复制是必不可少的，并且在DNA上起反式作用。确定了repA基因中编码RepA蛋白特异性识别区域的部分，其包含24 bp（GGTTTCAAAAATGAAACGGTGGAG）的3.5个直接重复序列。直接重复序列的上游和下游分别是大肠杆菌DnaA蛋白的识别序列（TTATCCACA）和一个富含AT的区域。讨论了低拷贝数芽孢杆菌质粒的复制控制机制。
