Retroviral recombination is temperature dependent.
Retroviral recombination is temperature dependent.
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逆转录病毒重组具有温度依赖性。
DOI：
10.1099/0022-1317-82-6-1359
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Zhang,Jiayou
中科院分区：
文献类型：
--
作者：
Li,Ting;Zhang,Jiayou
两个相互矛盾的体外观察表明逆转录病毒重组是温度依赖性的。Ouhammouch & Brody （Nucleic Acids Research20, 5443-5450, 1992）认为逆转录病毒重组率应随着温度升高而升高。然而，Shimomaye & Salvato （Gene Analysis Techniques6, 25 - 28,1989）和Brookset al. （Biotechniques19, 806 - 812,814 - 815,1985）发现，在低温下，RNA紧密折叠的结构可能会阻碍逆转录沿着RNA模板进行，从而增加其与模板分离的机会；因此，提高反应温度是克服模板二级结构，防止cDNA合成过早终止的最简单方法。本文构建了两种基于小鼠白血病病毒（MLV）的载体。第一个基因包含两个突变的gfpgene串联位置。上游gfpgene在其3 ‘端编码突变，而下游gfpgene在其5 ’端编码突变。发生在两个突变gfpgene之间的重组恢复了一个功能性的gfpgene。在荧光显微镜下观察含有functionalgfpgene的细胞呈绿色。第二个MLV载体含有一个功能基因，两端各有两个相同的序列。在两个相同序列之间发生的重组导致gfp基因的缺失。在显微镜下观察含有gfp缺失载体的细胞无色或清晰。使用这两种载体，我们已经证明逆转录病毒重组是温度依赖性的，并且随着温度从31°C升高到43°C，重组率降低。
Two conflictingin vitroobservations suggest that retroviral recombinations are temperature dependent. Ouhammouch & Brody (Nucleic Acids Research20, 5443–5450, 1992) suggested that retroviral recombination rates should increase as temperature increases. However, Shimomaye & Salvato (Gene Analysis Techniques6, 25–28, 1989) and Brookset al. (Biotechniques19, 806–812, 814–815, 1985) found that at low temperature the tightly folded structure of RNAs may hinder reverse transcription proceeding along the RNA template, which increases its chance of dissociating from the template; therefore, raising the reaction temperature was the simplest way to overcome template secondary structure and prevent premature termination of cDNA synthesis. In this report, two vectors based on murine leukaemia virus (MLV) were constructed. The first contained two mutatedgfpgenes in tandem positions. The upstreamgfpgene encoded a mutation at its 3′ end, while the downstreamgfpgene encoded a mutation at its 5′ end. The recombination that occurred between the two mutatedgfpgenes restored a functionalgfpgene. The cells that contained the functionalgfpgene were green when observed under a fluorescence microscope. The second MLV vector contained a functionalgfpgene with two identical sequences flanking either end. A recombination that occurred between the two identical sequences resulted in deletion of thegfpgene. Cells containing the vector with thegfpdeletion were colourless or clear when observed under the microscope. Using these two vectors, we have demonstrated that retroviral recombination is temperature dependent and the rate of recombination decreases as temperature is raised from 31 to 43 °C.
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发表时间：
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