Analysis of DNA replication initiated from a human cytomegalovirus origin

我们开发了一种在哺乳动物细胞中复制DNA的灵敏方法，使我们能够检测到人类巨细胞病毒(HCMV)的复制DNA片段。从1000个显微注射细胞中提取1/100的DNA，用适当的限制性内切酶消化后进行聚合酶链式反应(PCR)，以区分复制的和非复制的质粒。部分扩增的DNA被电泳法检测复制的DNA。用该检测系统分析了人巨细胞病毒Towne株HindIII片段A的亚片段，该片段包含一个已知的DNA复制起点(OriLyt)。4.3kb的亚片段(AatII-Saci)的复制效率与HindIII片段A的复制效率一样高。从AatII端缺失1.3kb或从SacI端缺失0.9kb，丧失了有效的复制能力。这些结果表明，巨细胞病毒Towne株的oriLyt位于4.3kb片段的1.3区和0.9区。我们用一种新的方法分析了由HCMV oriLyt引发的DNA复制的中间产物，该方法包括向细胞内微量注射模板DNA，0.15%琼脂糖凝胶电泳法，以及凝胶部分的PCR检测少量复制的DNA。含有oriLyt的质粒会在感染HCMV的细胞中复制，并产生比病毒基因组更大的DNA。单位长度的多相串联聚合物构成了该质粒的复制中间体。这些结果表明，在感染的裂解期，HCMV oriLyt参与了DNA复制的滚动环状模式。

We developed a sensitive assay for DNA replication in mammalian cells that enable us to detect replicated DNA fragments of human cytomegalovirus (HCMV). A 1/100 portion of DNA extracted from 1000 microinjected cells was subjected to polymerase chain reaction (PCR) after digestion with appropriate restriction enzymes to differentiate replicated from nonreplicated plasmids. A portion of the amplified DNA was electrophoresed to detect replicated DNA. Subfragments of the HindIII fragment A of HCMV strain Towne, which contains a previously identified origin of DNA replication (oriLyt), were analyzed with the assay system. A 4.3-kb subfragment (AatII-SacI) replicated as efficiently as the HindIII fragment A. Efficient replication ability was lost with a 1.3-kb deletion from the AatII end or a 0.9-kb deletion from SacI end. These results suggest that the boundaries of oriLyt of HCMV strain Towne lie within the 1.3- and the 0.9- regions of the 4.3-kb fragment.We analyzed intermediates of DNA replication initiated from HCMV oriLyt by a novel method which consists of a combination of microinjection of template DNA into cells, 0.15% agarose gel electrophoresis, and PCR in the fraction of the gel to detect a small amount of replicated DNA. A plasmid containing oriLyt replicated in the HCMV-infected cells and produced DNA larger than the viral genome. Heterogeneous tandem polymers of a unit length plasmid constituted the replicating intermediates of the plasmid. These results suggest that HCMV oriLyt is involved in a rolling circular mode of DNA replication during the lytic phase of infection.