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Department of Microbiology and Immunology, Fairchild Building, Stanford University School of Medicine, Stanford, CA 94305-5402, USA
We describe the mutagenesis of the IRS1-US5 region of the human cytomegalovirus genome, demonstrating the potential of the E. coli guanosine phosphoribosyl transferase (gpt) gene as a selectable marker for insertion 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 this gene with a gpt insert had no effect on growth of any of these strains of virus in resting or dividing human fibroblasts, or in human thymus plus liver implants in SCID-hu mice. Transcripts of the gpt gene, under control of the herpes simplex virus thymidine kinase promoter adjacent to the US3 enhancer in the viral genome, accumulated with delayed early (
) kinetics. Mutants with deletions in the IRS1 and US3-US5 regions were isolated by back-selection against gpt with the drug 6-thioguanine by growing virus in human Lesch-Nyhan (hypoxanthine-guanine 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.
* Author for correspondence. Fax +1 415 723 1606. e-mail mk.esm@forsythe.stanford.edu
Present address: Department of Medicine, Cambridge University, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK.
Present address: Virology Division, Department of Medicine, University of Washington, Pacific Medical Center, Seattle, WA 98144, USA.
Received 13 February 1995;
accepted 15 May 1995.
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