Foley S, Bron S, Venema G, Daly C, Fitzgerald G F
Department of Microbiology, University College, Cork, Ireland.
Plasmid. 1996 Sep;36(2):125-41. doi: 10.1006/plas.1996.0040.
The replication origin region, ori, of the Lactococcus lactis subsp. lactis plasmid pCI305 contains three-and-one-half directly repeated 22-bp sequences and two inverted repeat sequences, IR1 and IR2. These inverted repeat sequences overlap the promoter of the repB gene, which encodes a protein (RepB) essential for plasmid replication. Gel retardation assays, using lactococcal crude cell extracts in which RepB was overproduced, were used to demonstrate that the replication protein interacts with DNA sequences within the origin region. IR1 was identified as a RepB binding site. The -35 region of the repB promoter is contained within the loop of the potential stem-loop structure of IR1, suggesting autoregulation of repB. The pCI305 RepB failed to interact with DNA sequences within the minimal replicons of nine other members of the pCI305 family of plasmids and it was concluded that this DNA-protein interaction was replicon specific. In vivo studies were performed to determine the role of the three-and-one-half copies of the 22-bp sequences. When this sequence was provided in trans on a compatible vector, it resulted in the loss of pCI305 from the cell population (incompatibility).
乳酸乳球菌亚种乳酸乳球菌质粒pCI305的复制起始区域ori包含三个半直接重复的22碱基对序列以及两个反向重复序列IR1和IR2。这些反向重复序列与repB基因的启动子重叠，repB基因编码一种对质粒复制至关重要的蛋白质（RepB）。利用过量表达RepB的乳球菌粗细胞提取物进行凝胶阻滞试验，以证明复制蛋白与起始区域内的DNA序列相互作用。IR1被鉴定为RepB结合位点。repB启动子的-35区域包含在IR1潜在茎环结构的环内，提示repB的自我调节。pCI305 RepB未能与pCI305质粒家族其他九个成员的最小复制子内的DNA序列相互作用，由此得出结论，这种DNA-蛋白质相互作用具有复制子特异性。进行了体内研究以确定22碱基对序列三个半拷贝的作用。当该序列通过相容载体反式提供时，导致细胞群体中pCI305丢失（不相容性）。
