CHARACTERIZATION OF THE REPLICATION OF PLASMIDS CONTAINING HR SEQUENCES IN BACULOVIRUS-INFECTED SPODOPTERA-FRUGIPERDA CELLS
CHARACTERIZATION OF THE REPLICATION OF PLASMIDS CONTAINING HR SEQUENCES IN BACULOVIRUS-INFECTED SPODOPTERA-FRUGIPERDA CELLS
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DOI：
10.1006/viro.1993.1529
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发表时间：
1993-10-01
期刊：
影响因子：
3.7
通讯作者：
ROHRMANN, GF
中科院分区：
文献类型：
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作者：
LEISY, DJ;ROHRMANN, GF
研究了一系列含有烟草多核壳核型多角体病毒(AcMNPV)基因组6个同源区中的一个或多个的质粒在感染AcMNPV的果蝇细胞中的复制。虽然包含hr2的结构显示出最有效的复制，但hr区两侧的序列似乎影响复制的水平。带有不含HR的AcMNPV插入物的质粒组与含HR的质粒组相比，复制大大减少。对含有HR的质粒复制的各种特征进行了表征。其中包括质粒复制的时程以及辅助子AcMNPV的感染浓度和感染的多样性对复制效率的影响。经36小时P.I.检测到复制的DNA。并在60小时P.I.之前稳定下来。在本实验条件下，质粒量为0.5~2μg/1.25×10~6个细胞，感染复数为0.1~10倍时，复制效果最好。结果表明，该方法可将病毒DNA复制延迟约12小时。用部分限制性内切酶酶切实验，确定复制的含hR基因的质粒DNA的形式。结果表明，AcMNPV复制的载体是由高分子量的串联体组成的。
The replication of a series of plasmids, each containing one or more of 6 homologous regions (hrs) from theAutographa californicamultinucleocapsid nuclear polyhedrosis virus (AcMNPV) genome, was investigated in AcMNPV-infectedSpodoptera frugiperdacells. Although a construct containinghr2 showed the most efficient replication, the sequences flanking thehrregion appeared to influence the levels of replication. Plasmids with non-hr-containing AcMNPV inserts showed greatly reduced replication relative tohr-containing plasmids. A variety of features ofhr-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 μg/1.25 × 106cells 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 replicatedhr-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.