Fu J F, Chang H C, Chen Y M, Chang Y S, Liu S T
Molecular Genetics Laboratory, Chang-Gung College of Medicine and Technology, Taiwan.
Mol Gen Genet. 1996 Apr 10;250(6):699-704. doi: 10.1007/BF02172981.
A 1.6-kb DNA region required for the replication of pSW500 from Erwinia stewartii SW2 has been identified. DNA sequencing analysis revealed that this DNA fragment consists of a DnaA box, seven 16-bp direct repeats, and a 1005-bp open reading frame. The seven direct repeats have been demonstrated to mediate the incompatibility function of the plasmid. Primer extension analysis showed that the 1005-bp ORF is transcribed in vivo and the +1 site of the transcript is located 113 bp upstream from the translation initiation codon of the ORF. Complementation studies showed that this ORF is required for the replication of the plasmid and may encode a replication protein, RepA. Gene fusion studies revealed that the expression of repA is autoregulated by RepA. We also found that the pSW500 replicon has a copy number of approximately two and that the plasmid is stably maintained in Escherichia coli, thus demonstrating that the replicon contains all the elements required for copy number control and plasmid stability in E. coli. Curing of pSW500 from E. stewartii SW2 revealed that loss of pSW500 did not have any obvious effect on morphology or physiology of the cells, suggesting that pSW500 does not encode a function that is indispensable for the survival of the organism.
已鉴定出来自斯氏欧文氏菌SW2的pSW500复制所需的1.6kb DNA区域。DNA测序分析表明，该DNA片段由一个DnaA框、七个16bp的直接重复序列和一个1005bp的开放阅读框组成。七个直接重复序列已被证明介导质粒的不相容性功能。引物延伸分析表明，1005bp的开放阅读框在体内被转录，转录本的+1位点位于开放阅读框翻译起始密码子上游113bp处。互补研究表明，该开放阅读框是质粒复制所必需的，可能编码一种复制蛋白RepA。基因融合研究表明，repA的表达受RepA自身调节。我们还发现，pSW500复制子的拷贝数约为两个，并且该质粒在大肠杆菌中稳定维持，从而证明该复制子包含大肠杆菌中拷贝数控制和质粒稳定性所需的所有元件。从斯氏欧文氏菌SW2中消除pSW500表明，pSW500的缺失对细胞的形态或生理没有任何明显影响，这表明pSW500不编码对该生物体生存不可或缺的功能。
