Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.
Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.
D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D009173:Mycophenolic Acid / Q000494:pharmacology*; D010430:Pentosyltransferases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012641:Selection, Genetic; D014616:Vaccinia virus / Q000235:genetics*; D014779:Virus Replication / Q000187:drug effects; D001616:beta-Galactosidase / Q000235:genetics
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D001616:beta-Galactosidase; D009173:Mycophenolic Acid