Leisy D J, Rohrmann G F
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301.
Virology. 1993 Oct;196(2):722-30. doi: 10.1006/viro.1993.1529.
The replication of a series of plasmids, each containing one or more of 6 homologous regions (hrs) from the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) genome, was investigated in AcMNPV-infected Spodoptera frugiperda cells. Although a construct containing hr2 showed the most efficient replication, the sequences flanking the hr region appeared to influence the levels of replication. Plasmids with non-hr-containing AcMNPV inserts showed greatly reduced replication relative to hr-containing plasmids. A variety of features of hr-containing plasmid replication were characterized. These included the time course of plasmid replication and the influence of plasmid concentration and multiplicity of infection of the helper AcMNPV on replication efficiency. It was found that replicated plasmid DNA was detectable by 36 hr p.i. and plateaued by 60 hr p.i. Under our experimental conditions, plasmid levels of 0.5-2 micrograms/1.25 x 10(6) cells and multiplicities of infection of 0.1 to 10 produced optimal levels of plasmid replication. The transfection procedure was shown to delay viral DNA replication by about 12 hr. Experiments directed toward determining the form of the replicated hr-containing plasmid DNA were conducted using partial restriction enzyme digestion. The results indicated that the AcMNPV-replicated plasmids were composed of high molecular weight concatemers.
研究了一系列质粒在被苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）感染的草地贪夜蛾细胞中的复制情况，每个质粒都包含AcMNPV基因组6个同源区域（hrs）中的一个或多个。虽然含有hr2的构建体显示出最有效的复制，但hr区域侧翼的序列似乎会影响复制水平。与含有hr的质粒相比，含有非hr的AcMNPV插入片段的质粒复制大大减少。对含有hr的质粒复制的多种特征进行了表征。这些特征包括质粒复制的时间进程以及质粒浓度和辅助AcMNPV的感染复数对复制效率的影响。结果发现，在感染后36小时可检测到复制的质粒DNA，在感染后60小时达到稳定水平。在我们的实验条件下，质粒水平为0.5 - 2微克/1.25×10⁶个细胞，感染复数为0.1至10时，质粒复制达到最佳水平。结果表明，转染过程会使病毒DNA复制延迟约12小时。使用部分限制性内切酶消化进行了旨在确定复制的含hr质粒DNA形式的实验。结果表明，AcMNPV复制的质粒由高分子量的串联体组成。
