REPLICATION OF AN ORIGIN-CONTAINING DERIVATIVE OF PLASMID RK2 DEPENDENT ON A PLASMID FUNCTION PROVIDED IN TRANS
REPLICATION OF AN ORIGIN-CONTAINING DERIVATIVE OF PLASMID RK2 DEPENDENT ON A PLASMID FUNCTION PROVIDED IN TRANS
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DOI：
10.1073/pnas.76.4.1648
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
HELINSKI, DR
中科院分区：
文献类型：
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作者：
FIGURSKI, DH;HELINSKI, DR
pRK 212.2是广宿主范围质粒RK 2的衍生物，含有2个EcoRI切割片段A和B，它们都不能在大肠杆菌中自身复制。片段A（41.7个酶），而不是片段B（14.4个酶），可以通过插入无关质粒mini-F和ColE 1中来克隆。片段B含有RK 2的复制起点和氨苄青霉素抗性决定子。转化大肠含有mini-F-片段A与片段B DNA的杂交质粒的大肠杆菌细胞导致片段B作为具有RK 2的拷贝数和不相容性的不可移动质粒（pRK 2067）的再环化和复制。片段B不能在缺少片段A的情况下克隆，因为后者抑制由片段B指定的功能，导致宿主细胞活力丧失。片段B的一个小片段（2.4个内切酶）含有RK 2复制起点，但在片段A不存在时不再影响宿主细胞生长，通过插入ColE 1质粒中预先克隆该片段。该杂合质粒命名为pRK 256，将在E. colipolA突变体只有当一个片段A-携带辅助质粒存在。显然，由RK 2的片段B指定的潜在致死功能对于复制不是必需的，并且至少1个反式作用功能直接参与RK 2复制。
pRK212.2, a derivative of the broad host range plasmid RK2, contains 2 EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A was cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. Apparently, the potentially lethal function specified by fragment B of RK2 is not necessary for replication and at least 1 trans-acting function is directly involved in RK2 replication.