Varela-Echavarría A, Prorock C M, Ron Y, Dougherty J P
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
J Virol. 1993 Nov;67(11):6357-64. doi: 10.1128/JVI.67.11.6357-6364.1993.
A protocol was designed to measure the forward mutation rate over an entire gene replicated as part of a Moloney murine leukemia virus-based vector. For these studies, the herpes simplex virus thymidine kinase (tk) gene under the control of the spleen necrosis virus U3 promoter was used as target sequence since it allows selection for either the functional or the inactivated gene. Our results indicate that after one round of retroviral replication, the tk gene is inactivated at an average rate of 0.08 per cycle of replication. Southern blotting revealed that the majority of the mutant proviruses resulted from gross rearrangements and that deletions of spleen necrosis virus and tk sequences were the most frequent cause of the gene inactivation. Sequence analysis of the mutant proviruses suggested that homologous as well as nonhomologous recombination was involved in the observed rearrangements. Some mutations consisted of simple deletions, and others consisted of deletions combined with insertions. The frequency at which these mutations occurred during one cycle of retroviral replication provides evidence indicating that Moloney murine leukemia virus-based vectors may undergo genetic rearrangement at high rates. The high rate of rearrangement and its relevance for retrovirus-mediated gene transfer are discussed.
设计了一种方案来测量作为基于莫洛尼鼠白血病病毒的载体一部分进行复制的整个基因的正向突变率。在这些研究中，受脾坏死病毒U3启动子控制的单纯疱疹病毒胸苷激酶（tk）基因被用作靶序列，因为它允许对功能性或失活基因进行选择。我们的结果表明，经过一轮逆转录病毒复制后，tk基因以平均每个复制周期0.08的速率失活。Southern印迹分析显示，大多数突变前病毒是由大规模重排导致的，并且脾坏死病毒和tk序列的缺失是基因失活的最常见原因。对突变前病毒的序列分析表明，观察到的重排涉及同源和非同源重组。一些突变由简单缺失组成，而其他突变由缺失与插入组合而成。这些突变在逆转录病毒复制一个周期期间发生的频率提供了证据，表明基于莫洛尼鼠白血病病毒的载体可能以高频率发生基因重排。讨论了重排的高频率及其与逆转录病毒介导的基因转移的相关性。
