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Analysis of DNA replication initiated from a human cytomegalovirus origin

人类巨细胞病毒起源的 DNA 复制分析

基本信息

批准号：
03454186
负责人：
YAMAGUCHI Nobuo
金额：
$ 2.3万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454186/
关键词：
HCMV DNA replication PCR Microinjection サイトメガロウイルス潜伏感染ウイルスDNA複製

项目摘要

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.

我们开发了一种灵敏的检测哺乳动物细胞中DNA复制的方法，使我们能够检测人巨细胞病毒（HCMV）的复制DNA片段。从1000个显微注射的细胞中提取1/100份DNA，用适当的限制性内切酶消化后进行聚合酶链反应（PCR），以区分复制的质粒和非复制的质粒。将扩增的DNA的一部分进行测序以检测复制的DNA。使用该检测系统分析了HCMV株Towne的HindIII片段A的亚片段，该亚片段含有先前鉴定的DNA复制起点（oriLyt）。一个4.3kb的亚片段（AatII-SacI）与HindIII片段A一样有效地复制。从AatII末端缺失1.3-kb或从SacI末端缺失0.9-kb，丧失了有效的复制能力。我们用一种新的方法分析了由HCMV oriLyt启动的DNA复制的中间产物，该方法包括将模板DNA显微注射到细胞中，0.15%琼脂糖凝胶电泳，在凝胶的部分中进行PCR以检测少量复制的DNA。含有oriLyt的质粒在HCMV感染的细胞中复制，并产生比病毒基因组更大的DNA。单位长度质粒的异质串联聚合物构成质粒的复制中间体。这些结果表明，HCMV oriLyt是在感染的裂解期的DNA复制的滚动循环模式。