SUPPRESSION OF COLE1 REPLICATION PROPERTIES BY THE INC-P-1 PLASMID-RK2 IN HYBRID PLASMIDS CONSTRUCTED INVITRO
SUPPRESSION OF COLE1 REPLICATION PROPERTIES BY THE INC-P-1 PLASMID-RK2 IN HYBRID PLASMIDS CONSTRUCTED INVITRO
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DOI：
10.1016/0022-2836(79)90395-4
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发表时间：
1979-01-01
影响因子：
5.6
通讯作者：
HELINSKI, DR
中科院分区：
文献类型：
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作者：
FIGURSKI, DH;MEYER, RJ;HELINSKI, DR
通过在它们的EcoRI内切酶切割位点将Inc P-1宽宿主范围质粒RK 2连接到大肠杆菌素原质粒ColE 1，在体外构建杂合质粒。这些质粒对大肠杆菌素E1免疫，不产生大肠杆菌素，并表现出RK 2的所有特征，包括自我传播性。这些联合复制子具有5-7个/染色体的拷贝数，这是RK 2的典型拷贝数，而不是ColE 1的拷贝数。与ColE 1不同，质粒在氯霉素存在下不会复制，并保持在大肠杆菌的DNA聚合酶I突变体中。只有RK 2不相容性的表达，虽然功能ColE 1可以从杂交体中通过EcoRI切割而被拯救。ColE 1拷贝数和不相容性的这种抑制是质粒大小对ColE 1性质的独特影响。氯霉素处理的细胞中ColE 1或ColE 1样质粒复制的抑制是RK 2或RK 2片段（Cri+表型）的特异性效应。这种现象不是质粒大小的函数，并且需要RK 2 DNA与ColE 1的共价连接。RK 2的特异性区域（50.4-56.4 × 10 - 6）是一个特异性区域。103个碱基对）克隆到ColE 1样质粒pBR 313中，携带表达Cri+表型的遗传决定子。
Hybrid plasmids were constructed in vitro by linking the Inc P-1 broad host range plasmid RK2 to the colicinogenic plasmid ColE1 at their EcoRI endonuclease cleavage sites. These plasmids were immune to colicin E1, non-colicin-producing, and to exhibit all the characteristics of RK2 including self-transmissibility. These joint replicons have a copy number of 5-7/chromosome which is typical of RK2, but not ColE1. Unlike ColE1, the plasmids will not replicate in the presence of chloramphenicol and are maintained in DNA polymerase I mutants of Escherichia coli. Only RK2 incompatibility is expressed, although functional ColE1 can be rescued from the hybrids by EcoRI cleavage. This suppression of ColE1 copy number and incampatibility was a unique effect of plasmid size on ColE1 properties. The inhibition of ColE1 or ColE1-like plasmid replication in chloramphenicol-treated cells is a specific effect of RK2 or segments of RK2 (Cri+ phenotype). This phenomenon is not a function of plasmid size and requires covalent linkage of RK2 DNA to ColE1. A specific region of RK2 (50.4-56.4 .times. 103 base-pairs) cloned in the ColE1-like plasmid pBR313 carried the genetic determinant(s) for expression of the Cri+ phenotype.