Abstract
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.
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Smiley, J. Construction in vitro and rescue of a thymidine kinase-deficient deletion mutation of herpes simplex virus. Nature 285, 333–335 (1980). https://doi.org/10.1038/285333a0
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DOI: https://doi.org/10.1038/285333a0
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