Van der Avoort H G, Teertstra R, Versteeg R, Weisbeek P J
Biochim Biophys Acta. 1983 Oct 13;741(1):94-102. doi: 10.1016/0167-4781(83)90014-3.
Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.
将噬菌体φX174的DNA片段插入质粒pACYC177和pBR322中，以测试单个噬菌体基因和调控序列的体内效应。通过与噬菌体突变体的重组和互补，随后进行限制性内切酶分析来鉴定φX174插入片段。即使这些病毒基因高效表达，基因B、C、F和G在细胞中仍能稳定维持。只有当这些基因的表达完全被阻断时，带有完整基因D和E的重组质粒才能维持。完整的H和J基因的表达尚未得到证实。完整的基因A显然对宿主细胞具有致死性，因为在重组体中从未发现它。基因F和G即使在它们前面没有特征明确的病毒或质粒启动子序列之一时也能表达。对φX174核苷酸序列的筛选在这两个基因前面刚好给出两个类似启动子的序列。带有复制信号的病毒序列（φX174（+）复制起点、互补链合成起始位点和不相容序列）插入重组质粒时似乎也具有功能。带有φX（+）起点的质粒可被迫进入滚环复制模式。感染噬菌体产生的A蛋白对克隆的病毒起点起反式作用。（-）起点在单链质粒DNA转导过程中可作为互补链合成的起始信号。质粒上不相容序列的细胞内存在会阻止感染噬菌体的繁殖。
