Insertion and deletion mutagenesis of the human cytomegalovirus genome.
Insertion and deletion mutagenesis of the human cytomegalovirus genome.
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人类巨细胞病毒基因组的插入和缺失诱变。
DOI：
10.1073/pnas.84.20.7213
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发表时间：
1987
影响因子：
11.1
通讯作者：
Mocarski,ES
中科院分区：
文献类型：
--
作者：
Spaete,RR;Mocarski,ES
人类巨细胞病毒（CMV）的研究一直受到限制，缺乏分子遗传学技术可用于操纵病毒基因组。我们已经开发了利用修饰的大肠杆菌lacZ基因作为遗传标记的CMV的位点特异性插入和缺失突变的方法。将lacZ基因置于主要β基因调控信号的控制下，并通过同源重组插入病毒基因组中，破坏CMV DNA的L-组分重复序列中该β基因的两个拷贝之一。我们观察到高水平表达的β-半乳糖苷酶的重组在时间上真实的方式，这种酶的水平接近1%的总蛋白在感染的细胞。因此，CMV是在人细胞中高水平表达外源基因产物的有效载体。在存在显色底物5-溴-4-氯-3-吲哚基β-D-半乳糖苷的情况下，使用lacZ缺陷型病毒的回选，我们产生了随机终点缺失突变体。这些突变体的分析表明，CMV DNA序列侧翼插入已被删除，从而建立这种方法作为一种手段，以确定是否侧翼的lacZ插入序列是病毒生长的抑制剂。在该方法的初始测试中，我们已经表明，可以删除一个拷贝的L-组分重复序列的7800个碱基对，而不影响人成纤维细胞中的病毒生长。
Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. We have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major beta gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this beta gene within the L-component repeats of CMV DNA. We observed high-level expression of beta-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside, we generated random endpoint deletion mutants. Analysis of these mutants revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, we have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.