SELECTABLE INSERTION AND DELETION MUTAGENESIS OF THE HUMAN CYTOMEGALOVIRUS GENOME USING THE ESCHERICHIA-COLI GUANOSINE PHOSPHORIBOSYL TRANSFERASE (GPT) GENE
Rf. Greaves et al., SELECTABLE INSERTION AND DELETION MUTAGENESIS OF THE HUMAN CYTOMEGALOVIRUS GENOME USING THE ESCHERICHIA-COLI GUANOSINE PHOSPHORIBOSYL TRANSFERASE (GPT) GENE, Journal of General Virology, 76, 1995, pp. 2151-2160
We describe the mutagenesis of the IRS1-US5 region of the human cytome
galovirus genome, demonstrating the potential of the E. coli guanosine
phosphoribosyl transferase (gpt) gene as a selectable marker for inse
rtion and deletion mutagenesis of high passage (AD169, Towne) as well
as low passage (Toledo) strains of virus. Despite evidence suggesting
that the US3 gene product may play a regulatory role, disruption of th
is gene with a gpt insert had no effect on growth of any of these stra
ins of virus in resting or dividing human fibroblasts, or in human thy
mus plus liver implants in SCID-hu mice. Transcripts of the gpt gene,
under control of the herpes simplex virus thymidine kinase promoter ad
jacent to the US3 enhancer in the viral genome, accumulated with delay
ed early (beta) kinetics. Mutants with deletions in the IRS1 and US3-U
S5 regions were isolated by back-selection against gpt with the drug 6
-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthineguanin
e phosphoribosyl transferase deficient) skin fibroblasts immortalized
with human papillomavirus oncogenes. Thus, we demonstrate a dependable
method for insertion and deletion mutagenesis that can be applied to
any region of the viral genome.