Tang R S, Barton D J, Flanegan J B, Kirkegaard K
Department of Microbiology and Immunology, Stanford University School of Medicine, California 94305-5402, USA.
RNA. 1997 Jun;3(6):624-33.
Poliovirus RNA has been shown to undergo homologous genetic recombination at a high frequency in infected human cells. Recently it has become possible to mimic the entire intracellular replicative cycle of poliovirus replication in cytoplasmic extracts prepared from HeLa cells, resulting in the generation of infectious poliovirions. The mechanism of poliovirus RNA recombination has been shown previously to be coupled to RNA replication, presumably by template switching during the replication of parental RNAs. Experiments were designed to test whether recombinant poliovirus RNA molecules are produced in a cell-free environment. Recombinant molecules generated bear marker sequences that can be detected physically by reverse transcription and PCR. We report here successful detection of poliovirus RNA recombination in a cell-free replication system. The frequency measured for cell-free RNA recombination between two polymorphic marker loci 656 nt apart was between 10(-2) and 10(-3) recombinants/genome, a frequency comparable to or slightly higher than that measured for RNA recombination in infected cells.
脊髓灰质炎病毒RNA已被证明在受感染的人类细胞中会高频发生同源基因重组。最近，在从HeLa细胞制备的细胞质提取物中模拟脊髓灰质炎病毒复制的整个细胞内复制周期成为可能，从而产生有感染性的脊髓灰质炎病毒粒子。脊髓灰质炎病毒RNA重组机制先前已被证明与RNA复制相关联，推测是在亲代RNA复制过程中通过模板转换实现的。设计实验以测试重组脊髓灰质炎病毒RNA分子是否在无细胞环境中产生。所产生的重组分子带有可通过逆转录和PCR进行物理检测的标记序列。我们在此报告在无细胞复制系统中成功检测到脊髓灰质炎病毒RNA重组。在两个相距656个核苷酸的多态性标记位点之间，无细胞RNA重组的频率为每基因组10^(-2)至10^(-3)个重组体，该频率与在受感染细胞中测量的RNA重组频率相当或略高。
