Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus
Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus
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DOI：
10.1128/jvi.76.18.9323-9334.2002
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发表时间：
2002-09-01
影响因子：
5.4
通讯作者：
Passarelli, AL
中科院分区：
文献类型：
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作者：
Crouch, EA;Passarelli, AL
已知在构建用于外源基因表达的重组病毒期间，杆状病毒感染促进其基因组与质粒DNA之间的高频重组。然而，对促进同源重组（HR）所必需的病毒基因知之甚少。我们开发了一种检测方法来鉴定刺激HR所必需的病毒基因。在该检测中，我们使用了两种含有广泛序列同源性的质粒，如果HR发生，则产生可见和可定量的表型。质粒含有在N或C末端突变的绿色荧光蛋白基因（gfp）和DNA复制的病毒起点。当将含有这些突变gfp基因的质粒单独或一起转染到昆虫细胞中时，观察到很少的绿色荧光蛋白（GFP）阳性细胞，证实了单独的宿主细胞机制不能促进高水平的HR。然而，如果将病毒DNA或参与DNA复制的病毒基因与含有突变gfp的质粒一起沿着转染到细胞中，观察到GFP阳性细胞的显著增加。发现病毒基因ie-1、ie-2、lef-7和p35在所有其他DNA复制基因存在下对于有效HR是重要的。然而，ie-1和ie-2足以在没有其他病毒基因的情况下促进HR。缺乏病毒复制起点的扩增底物对重组有相似的遗传要求，但对ie-1的依赖性较低。有趣的是，即使HR是由DNA复制的病毒起点的存在下刺激的，病毒刺激的HR可以在DNA合成抑制剂阿非迪可林的存在下进行。
It is known that baculovirus infection promotes high-frequency recombination between its genomes and plasmid DNA during the construction of recombinant viruses for foreign gene expression. However, little is known about the viral genes necessary to promote homologous recombination (HR). We developed an assay to identify viral genes that are necessary to stimulate HR. In this assay, we used two plasmids containing extensive sequence homology that yielded a visible and quantifiable phenotype if HR occurred. The plasmids contained the green fluorescent protein gene (gfp) that was mutated at either the N or the C terminus and a viral origin of DNA replication. When the plasmids containing these mutant gfp genes were transfected into insect cells alone or together, few green fluorescent protein (GFP)-positive cells were observed, confirming that the host cell machinery alone was not able to promote high levels of HR. However, if viral DNA or viral genes involved in DNA replication were cotransfected into cells along with the mutant gfp-containing plasmids, a dramatic increase in GFP-positive cells was observed. The viral genes ie-1, ie-2, lef-7, and p35 were found to be important for efficient HR in the presence of all other DNA replication genes. However, ie-1 and ie-2 were sufficient to promote HR in the absence of other viral genes. Recombination substrates lacking a viral origin of replication had similar genetic requirements for recombination but were less dependent on ie-1. Interestingly, even though HR was stimulated by the presence of a viral origin of DNA replication, virally stimulated HR could proceed in the presence of the DNA synthesis inhibitor aphidicollin.