Zagursky R J, Berman M L
Gene. 1984 Feb;27(2):183-91. doi: 10.1016/0378-1119(84)90139-2.
We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.
我们构建了嵌合质粒载体，将M13噬菌体的复制起点和基因间区域克隆到pBR322的PvuII（pZ145）和AhaIII（pZ150、pZ152）位点。在没有M13噬菌体的情况下，这些质粒像任何其他源自ColE1的质粒一样复制，并赋予氨苄青霉素和四环素抗性（Amp、Tet）。用M13噬菌体感染后，质粒上存在的病毒复制起点允许噬菌体指导的质粒复制，并在M13样颗粒中产生高产率的单链（ss）质粒DNA。这种仅代表质粒一条链的ssDNA，可用作通过Sanger等人（1977年）描述的双脱氧测序方法进行快速DNA序列测定的底物。由于这些质粒含有完整的pBR322，基因间区域可以转移到大多数pBR322衍生物上，以产生ss质粒DNA，而不会影响受体质粒以供进一步研究。我们还构建了pZ145的缺失衍生物质粒pZ146，它不会对M13辅助噬菌体的生长产生干扰，尽管该质粒被包装到噬菌体颗粒中。这一结果证实了这样一种理论，即基因间区域由两个结构域组成：一个结构域是参与噬菌体形态发生的片段，另一个结构域是功能性复制起点区域，它会干扰M13复制。
