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Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A general method for constructing and selecting recombinant vaccinia viruses with insertions, deletions, or mutations in any gene that is similar in principle to one originally devised for Saccharomyces cerevisiae (S. Scherer and R. W. Davis, Proc. Natl. Acad. Sci. USA 76:4951-4955, 1979) is described. The selectable marker used, Escherichia coli guanine phosphoribosyltransferase, is not retained within the final recombinant virus, and hence, this procedure may be used serially to introduce several foreign genes or to make multiple site-directed mutations.
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PMID:Transient dominant selection of recombinant vaccinia viruses. 215 65

The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.
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PMID:Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes. 217 May 78

We have developed a procedure for the selection of recombinant vaccinia viruses with applicability to poxvirus mutagenesis studies and to the use of vaccinia virus as an expression vector. The method depends on the specific inability of a recombinant vaccinia virus expressing the Escherichia coli guanine phosphoriboxyltransferase gene (gpt) to form plaques on a hypoxanthine-guanine phosphoribosyltransferase-negative line of mouse fibroblasts in the presence of 6-thioguanine. Recombinant viruses that have the gpt removed can form plaques under selection conditions, thus providing a simple and efficient selection protocol. We have demonstrated the method by isolating a pseudo-wild type revertant virus and a simple deletion mutant virus from a recombinant vaccinia virus with gpt inserted into the vaccinia virus gene encoding the major 35,000-Da secretory protein.
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PMID:Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses. 221 14

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.
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PMID:Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors. 313 Apr 92

A transient expression system has been developed to compare the relative efficiency of expression of various vaccinia virus DNA sequences containing transcriptional regulatory elements. A plasmid vector was constructed containing both the Escherichia coli galactokinase gene (galK) and the guanine phosphoribosyltransferase gene (gpt). To direct the expression of gpt within this vector, a vaccinia virus promoter region was isolated from the HindIII-F fragment of the genome and inserted 5' to gpt coding sequence. Four unique cloning sites in front of galK allow simple and precise fusion of various vaccinia virus DNA fragments that contain the regulatory site of interest to galK. Sequences containing promoter regions were ligated to the coding segment of the galK to create four recombinant plasmids, which were introduced into vaccinia virus-infected cells by transfection. Both galK and gpt were thus expressed under the control of vaccinia virus transcriptional units, and the enzymatic activities were measured in the same cell extract with a filter-binding assay. The major advantage of this transient expression system is that the variations in galK expression are always measured relative to the internal gpt standard. Changes in the galK/gpt ratio resulting from different vaccinia promoters of galK are thus a quantitative measurement of promoter strength.
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PMID:Transient expression system to measure the efficiency of vaccinia promoter regions. 332 Nov 10

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.
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PMID:A poxvirus-encoded uracil DNA glycosylase is essential for virus viability. 847 56

Recombinant vaccinia viruses based on the highly attenuated Modified Vaccinia Ankara (MVA) strain expressing HIV-1 antigen genes were constructed by a novel procedure involving the transient use of two marker genes. The selectable markers used, the Escherichia coli guanine phosphoribosyltransferase (gpt) and the beta-galactosidase (lacZ) genes, are not retained within the final recombinant virus. The transient marker stabilisation (TMS) procedure allows the generation of marker-free recombinant viruses in a series of simple plaque purification steps. HIV-1 gag pol genes were inserted into two loci of vaccinia MVA demonstrating the efficiency of the procedure.
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PMID:Transient marker stabilisation: a general procedure to construct marker-free recombinant vaccinia virus. 957 48

Avipoxviruses show an abortive replication phenotype in mammalian cells and are under evaluation as safe vectors for vaccination. Non-essential gene sequences located in highly conserved regions of virus genomes are considered particularly useful to integrate heterologous DNA. Fowlpox virus F11L orthologue is described in this paper as a suitable locus for insertion into fowlpox virus genome. Disruption of the F11L coding sequence by integration of an expression cassette for the Escherichia coli lacZ and guanine phosphoribosyltransferase marker genes resulted in the isolation of replication competent knockout viruses. Growth of F11L-knockout viruses in primary chicken embryo fibroblasts was unimpaired in comparison to wild type-virus. To test the generation of vector viruses, an insertion plasmid was constructed that contains F11L-specific sequences for homologous recombination, the E. coli lacZ and gpt genes as transient selectable marker, and the vaccinia virus early/late promoter P7.5 for transcriptional control of target gene expression. The coding sequence of the melanoma-associated antigen tyrosinase was chosen as model recombinant gene. Isolation of tyrosinase-recombinant viruses, which produced stably the insert, demonstrated the usefulness of the F11L-insertion site for the generation of fowlpox vectors. Rapid isolation of those recombinants was achieved by using a double selective system and linearising the vector plasmid before transfection.
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PMID:Generation of recombinant fowlpox virus using the non-essential F11L orthologue as insertion site and a rapid transient selection strategy. 1236 39

The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.
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PMID:Construction of Deletion-knockout Mutant Fowlpox Virus (FWPV). 2725 12