Sugimoto K, Kohara Y, Okazaki T
Proc Natl Acad Sci U S A. 1987 Jun;84(12):3977-81. doi: 10.1073/pnas.84.12.3977.
Initiation sites of T7 phage DNA replication in the presence and absence of T7 phage gene 4 primase have been analyzed by using Escherichia coli cells infected with T7 phage amber mutants, T73,6 and T73,4,6, respectively. Restriction analysis of the [3H]thymidine-labeled DNA, synthesized by the T73,4,6 phage-infected cells in the presence of 2',3'-dideoxy-3'-azidothymidine, has shown that only the light (L) strand of T7 DNA has been synthesized from the primary origin area to the right. Transition sites from RNA to DNA have been located precisely in the primary origin region of the T7 phage genome. In the gene 4- condition, greater than 20 transition sites have been detected only in the L strand. They scattered widely downstream from the phi 1.1 promoters and mostly downstream from the phi 1.3 promoter. The same transition sites have been detected in the gene 4+ condition, suggesting that the transcripts started from these promoters are used as primers of the rightward L-strand DNA synthesis in the gene 4+ condition. In addition, many heavy (H)- and L-strand transition sites have been detected at gene 4 primase sites in the gene 4+ condition. The relative roles of T7 phage RNA polymerase and primase at the primary origin have been discussed.
分别使用感染了T7噬菌体琥珀突变体T73,6和T73,4,6的大肠杆菌细胞，分析了存在和不存在T7噬菌体基因4引发酶时T7噬菌体DNA复制的起始位点。对在2',3'-二脱氧-3'-叠氮胸苷存在下由T73,4,6噬菌体感染的细胞合成的[3H]胸苷标记的DNA进行限制性分析，结果表明，T7 DNA只有轻（L）链是从初级起始区域向右合成的。RNA到DNA的转换位点已精确定位在T7噬菌体基因组的初级起始区域。在基因4缺陷的情况下，仅在L链中检测到20多个转换位点。它们广泛分布在phi 1.1启动子下游，且大多在phi 1.3启动子下游。在基因4正常的情况下也检测到了相同的转换位点，这表明从这些启动子起始的转录本在基因4正常的情况下用作向右L链DNA合成的引物。此外，在基因4正常的情况下，在基因4引发酶位点还检测到许多重（H）链和L链转换位点。本文还讨论了T7噬菌体RNA聚合酶和引发酶在初级起始位点的相对作用。
