Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus
Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus
复制标题
单纯疱疹病毒胸苷激酶缺陷缺失突变体的体外构建及拯救
作者：
J. Smiley
在体外，在DNA分子上预先选定的位置引入特定突变的能力，使人们能够测试关于特定DNA序列功能的假设。单纯疱疹病毒1型(HSV-1)的基因组由150个碱基对组成，可能太大了，无法在体外直接引入突变。因此，为了将这些技术应用于HSV基因的研究，我们研究了两步法的可行性。首先将已克隆在大肠杆菌pBR322中的一小段HSV-1 DNA中引入一个明确的突变，然后通过DNA介导的标记REASE将该突变整合到感染性HSV-1的基因组中。该方法是使用胸苷激酶(TK)1-3的病毒基因进行测试的。选择TK有几个原因。首先，在大多数细胞培养条件下，TK对于病毒的感染性是不必要的，对于TK和TK+表型1，3都有有效的选择系统。这有助于TK缺乏突变的恢复，并允许快速和详细的遗传分析3，4。第二，Stow等人5已经证明TK DNADNA序列可以很容易地从−片段中拯救到传染性病毒中。第三，纯化的HSV-1 TK DNA能够将缺乏TK的哺乳动物细胞转化为TK+表型6，从而可以在存在和不存在其他调节病毒基因产物的情况下研究病毒基因的表达。因此，TK基因为研究特定DNA序列在真核细胞中基因表达中的作用提供了一个有利的系统。结果表明，在体外产生的TK基因座800碱基缺失突变被高频率挽救，产生了HSV-1的TK缺失缺失突变。
The ability to introduce defined mutations at pre-selected sites on DNA molecules in vitro allows one to test hypotheses about the function of specific DNA sequences. The genome of herpes simplex virus type 1 (HSV-1), consisting of 150 kilobase pairs, is probably too large to allow the direct introduction of mutations in vitro. Therefore, to apply these techniques to the study of HSV genes, the feasibility of a two-step procedure was examined. A defined mutation was first introduced into a small segment of HSV-1 DNA which had previously been cloned in the Escherichia coli plasmid pBR322; the mutation was then incorporated into the genome of infectious HSV-1 by DNA-mediated marker rescue. The approach was tested using the viral gene for thymidine kinase (TK)1–3. TK was chosen for several reasons. First, TK is dispensable for viral infectivity in most conditions of cell culture, and efficient selection systems are available for both the TK− and TK+ phenotypes1,3. This facilitates the recovery of TK-deficient mutants and allows rapid and detailed genetic analysis3,4. Second, Stow et al.5 have shown that TK− DNA sequences are readily rescued from DNA fragments into infectious virus. Third, purified HSV-1 TK DNA is able to convert TK-deficient mammalian cells to a TK+ phenotype6, so that the expression of the viral gene can be studied in both the presence and absence of other regulatory viral gene products. Thus, the TK gene provides a favourable system for an examination of the role of specific DNA sequences in the expression of genes in eukaryotic cells. The results presented here demonstrate that an 800-base pair deletion mutation at the TK locus, generated in vitro, was rescued at a high frequency, producing a TK-deficient deletion mutant of HSV-1.