Cell-specific selection of mutants of a herpes simplex virus recombinant carrying deletions.
Herpes simplex virus 1 (HSV-1) recombinant R316 was constructed so as to convert the thymidine kinase (TK), a beta gene, into an alpha-regulated gene by insertion of the BamHI N fragment in the proper transcriptional orientation into the BglII cleavage site of the TK gene (L. E. Post, S. Mackem, and B. Roizman, Cell 24, 555-565 (1981).) The BamHI N fragment contains the promoter and regulatory domains of the alpha 4 gene in addition to an origin of viral DNA synthesis and the complete domain of the alpha 22 gene. Passage of the R316 virus in HEp-2 or in human embryonic lung (HEL) cells resulted in rapid accumulation of mutants carrying approximately 4.4-kbp deletions in the insert. No appreciable accumulation of the deletions was observed upon passage of R316 virus in Vero cells. The accumulation of deletions in HEp-2 and HEL cells could not be attributed to the fusion of the TK gene with the alpha 4 gene promoter, to the presence of an origin of DNA replication, or to overexpression of any of the genes whose domain is contained entirely in the HSV-1 Bam HI N fragment; these conclusions are based on the observations that deletions did not accumulate in HEL or HEp-2 cells infected with recombinant R315, containing BamHI N inserted in an inverted orientation, or with recombinant R314, carrying an alpha 4-TK chimera constructed by insertion of the HSV-1 BamHI Z fragment into the BglII cleavage site in the TK gene. The BamHI Z fragment also contains a functional origin of DNA synthesis. The hypothetical models which could explain the host range-specific accumulation of deletions are discussed.
Sears
A E
AE
Roizman
B
B
D000818:Animals; D002460:Cell Line; D002872:Chromosome Deletion; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D015245:Deoxyribonuclease BamHI; D005809:Genes, Regulator; D005814:Genes, Viral; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D013937:Thymidine Kinase / Q000235:genetics; D014779:Virus Replication
D004274:DNA, Recombinant; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI