Hilton Sally, Winstanley Doreen
Warwick HRI, The University of Warwick, Wellesbourne, Warwick CV35 9EF, UK.
J Gen Virol. 2007 May;88(Pt 5):1496-1504. doi: 10.1099/vir.0.82760-0.
The entire genome of Cydia pomonella granulovirus (CpGV) was systematically screened for origins of DNA replication, using an infection-dependent DNA replication assay in the granulovirus-permissive Cydia pomonella cell line, Cp14R. All seven cosmids in an overlapping library that covered the CpGV genome were found to replicate in the assay. A genomic library of 32 overlapping plasmids was subsequently screened. Plasmids that replicated were in turn subcloned into 1-2 kbp overlapping fragments. Eleven subclones replicated, each containing at least one of the 13 single-copy 74-76 bp imperfect palindromes, previously identified in the CpGV genome as possible origins of replication. Genome fragments of 156 bp, each containing one of the 13 palindromes, were cloned to verify replication and provided confirmation that these 13 palindromes are the only origins of replication in the genome. A real-time PCR method was developed for the quantification of DNA replication, which eliminated the need for Southern blotting and hybridization. A set of deletion clones allowed further quantitative characterization of one of the palindromes. The previously proposed non-homologous region origin of replication did not replicate in the assay.
利用在颗粒体病毒允许的苹果蠹蛾细胞系Cp14R中进行的依赖感染的DNA复制测定法，对苹果蠹蛾颗粒体病毒（CpGV）的全基因组进行了DNA复制起始位点的系统筛选。发现在覆盖CpGV基因组的重叠文库中的所有七个黏粒在该测定中都能复制。随后筛选了一个由32个重叠质粒组成的基因组文库。将能够复制的质粒依次亚克隆成1 - 2 kbp的重叠片段。11个亚克隆能够复制，每个亚克隆都包含先前在CpGV基因组中鉴定为可能的复制起始位点的13个单拷贝74 - 76 bp不完全回文序列中的至少一个。克隆了包含13个回文序列之一的156 bp基因组片段以验证复制，并证实这13个回文序列是基因组中仅有的复制起始位点。开发了一种实时PCR方法用于DNA复制的定量分析，从而无需进行Southern印迹和杂交。一组缺失克隆允许对其中一个回文序列进行进一步的定量表征。先前提出的非同源区域复制起始位点在该测定中不能复制。
