Guanosine Phosphoribosyl Transferase Research Articles

Characterization of the murine cytomegalovirus m136 gene

The 230-kbp murine cytomegalovirus (MCMV) genome is predicted to encode 182 open reading frames (orfs). One gene whose functional role is not known is encoded by the 762-bp m136 orf. Sequence analysis of rat cytomegalovirus (RCMV) strains Maastricht and English revealed homologous orfs, pr136, and ORF HJ4, respectively. Conservation of these orfs suggested that m136 and the RCMV homologs might play a role during virus replication. Expression of an epitope tagged form of m136 (m136-V5) yielded a polypeptide of 34 kDa that localized to the perinuclear region of transfected mouse 3T3 fibroblasts. Three independently generated MCMV m136 mutants were isolated and characterized. Mutations were introduced into the m136 orf by inserting either a beta-glucuronidase (m136-beta-gluc) or a guanosine phosphoribosyl transferase (m136-gpt) expression cassette into a unique BglII site, or by inserting a gpt cassette into a deleted region (Deltam136) of m136. No differences were observed in viral yield, plaque size, and plaque morphology between the parental strain and any of the m136 mutant viruses. In vivo analysis using a SCID mouse virulence model showed a consistently measurable attenuated phenotype for all three m136 mutants. The results showed that although the m136 gene was not essential for replication in vitro or in vivo, an intact m136 gene was necessary to yield wild type virulence during infection of the host.

DNA Methylation Precedes Chromatin Modifications under the Influence of the Strain-Specific Modifier Ssm1

Ssm1 is responsible for the mouse strain-specific DNA methylation of the transgene HRD. In adult mice of the C57BL/6 (B6) strain, the transgene is methylated at essentially all CpGs. However, when the transgene is bred into the DBA/2 (D2) strain, it is almost completely unmethylated. Strain-specific methylation arises during differentiation of embryonic stem (ES) cells. Here we show that Ssm1 causes striking chromatin changes during the development of the early embryo in both strains. In undifferentiated ES cells of both strains, the transgene is in a chromatin state between active and inactive. These states are still observed 1 week after beginning ES cell differentiation. However, 4 weeks after initiating differentiation, in B6, the transgene has become heterochromatic, and in D2, the transgene has become euchromatic. HRD is always expressed in D2, but in B6, it is expressed only in early embryos. The transgene is already more methylated in B6 ES cells than in D2 ES cells and becomes increasingly methylated during development in B6, until essentially all CpGs in the critical guanosine phosphoribosyl transferase core are methylated. Clearly, DNA methylation of HRD precedes chromatin compaction and loss of expression, suggesting that the B6 form of Ssm1 interacts with DNA to cause strain-specific methylation that ultimately results in inactive chromatin.

Mutagenicity of Tocopheryl Quinones: Evolutionary Advantage of Selective Accumulation of Dietary a-Tocopherol

We have shown that phenolic antioxidant tocopherols are oxidized to nonarylating α-tocopheryl quinone (α-TQ) and arylating γ- and δ-TQ electrophiles. The arylating quinones stimulate apoptosis and are highly cytotoxic in mammalian cells. Some xenobiotic phenolic antioxidants are mutagens, and it has been suggested that their arylating quinone metabolites are the active agents in mutagenesis related to carcinogenesis. We found that neither α- nor γ-TQ was directly genotoxic in supercoiled-to-nicked circular DNA conversions, but these agents interacted with the cytomegalovirus reporter-driven plasmid and enhanced luciferase transfection, with γ-TQ >?α-TQ. The Ames test, using γ-TQ and a number of Salmonella strains, showed no evidence of bacterial mutagenesis. γ-TQ was highly cytotoxic and α-TQ slightly cytotoxic in eukaryocyte AS52 cells. A guanosine phosphoribosyltransferase gene assay showed that γ-TQ was highly mutagenic and α-TQ slightly mutagenic in AS52 cells. A review of the literature identified associations where a decrease in dietary γ-tocopherol (γ-T) diminishes and an increase in dietary γ-T and its quinone enhances carcinogenicity. Humans and other omnivores selectively accumulate α-tocopherol, even though γ-T is their principal dietary tocopherol. We suggest that this selectivity confers an evolutionary advantage by limiting tissue γ-T, a putative precursor of the mutagen γ-TQ.

Selectable insertion and deletion mutagenesis of the human cytomegalovirus genome using the Escherichia coli guanosine phosphoribosyl transferase (gpt) gene.

We describe the mutagenesis of the IRSI-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 (beta) 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.

Recombinant retroviral systems for the analysis of drug resistant HIV.

Two recombinant retroviral systems are described that can be used to analyze antiretroviral drug activity and HIV breakthrough (replication in the presence of the drug). The first system utilizes a recombinant HIV encoding beta-galactosidase as a reporter gene (HIV-LacZ). The defective HIV-LacZ virus is produced in COS cells after co-transfection of a plasmid encoding the HIV-LacZ genome with a plasmid encoding HIV proteins necessary for packaging and infectivity. Subsequent infection of CD4+ target cells, followed by assay for LacZ expression, permits the rapid identification of individual virus-infected cells. This system can be used to quantitate the inhibition of early events in the HIV replicative cycle and is suitable for the screening of compounds for anti-HIV activity. However, this system cannot be used to analyze HIV drug resistance because of the limited genetic heterogeneity of the virus that is produced in COS cells. To circumvent this problem, a second system has been developed in which heterogenous recombinant HIV is produced by rescue with replication-competent 'helper' HIV. This system required the production of CD4+ cell lines containing defective proviruses encoding either LacZ or guanosine phosphoribosyl transferase (gpt). The defective proviruses are rescued by infection of the cell lines with 'helper' HIV and used to infect target cells in the presence of antiretroviral agents. Subsequent reporter gene assay is used to identify virus-infected cells. This system has been used to detect rare HIV breakthrough infection of cells in the presence of the non-nucleoside reverse transcriptase inhibitor TIBO R82150. Similar analyses with other antiretroviral agents, alone and in combination, may help identify therapeutic strategies that minimize breakthrough replication of HIV.

New parental cell lines for generating human hybridomas

In contrast to the success achieved with the production of hybridomas in the mouse system, creating human hybridomas is problematic. The reason is believed to be a lack of suitable malignant human cell lines. The work presented here demonstrates the establishment of three human parent cell lines - two of which are of T cell origin - by installing hypoxanthine guanosine phosphoribosyl transferase (HGPRT) and/or thimidine kinase (TK) deficiencies into the leukemic cell lines REH, 1301 and SKW-3. In order to isolate true hybridomas, selection procedures must guarantee complete death of the enzyme-deficient malignant parent cells. In this respect sublines with a combined HGPRT and TK deficiency proved to be superior to those with only one enzyme deficiency, especially in combination with the newly developed hypoxanthine/aminopterine/thymidine/azaserine (HATA) selection medium. However, selection media should be of low nonspecific toxicity. This was shown to be a particular property of the thymidine-free azaserine/hypoxanthine (AH) selection medium. Preliminary data show an extraordinary ability of one subclone of the hypoxanthine guanosine phosphoribosyl transferase-negative T cell line SKW-3 to generate human T-T hybridomas. They are of a stable, nearly tetraploid karyotype and express new surface antigens, thus providing new possibilities for the investigation of human T lymphocyte function by means of hybridoma technology.

Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence

Poxviruses are known to contain a large number of open reading frames, particularly near the termini of the viral genome, that are not required for growth in tissue culture. However, many of these these gene products are believed to play important roles in determining the virulence of the virus by modulating the host immune response to the infection. Recently it has been shown that Shope fibroma virus encodes, within the terminal inverted repeats, a protein (T2) related to the cellular tumor necrosis factor receptor (TNFR) and which specifically binds both TNFa and TNFβ We have sequenced the terminal regions of two other Leporipoxviruses (myxoma virus and malignant rabbit fibroma virus) that are extremely invasive and capable of inducing extensive immunosuppression in rabbits and demonstrate that they also encode a closely related T2 homolog with all the structural motifs predicted for a secreted TNF binding protein. To investigate the biological role of the T2 protein, we have inactivated the myxoma virus T2 gene within each copy of the viral TIR by the insertion of a dominant selectable marker ( Escherichia coli guanosine phosphoribosyltransferase) and selection of the recombinant virus in the presence of mycophenolic acid. The success of the inactivation of both copies of T2 was confirmed by the loss a broad protein band (52–56 kDa) of the predicted size for T2 from the profile of proteins secreted from mutant virus-infected BGMK cells at earlytimes after infection. Although the T2-minus recombinant myxoma virus grew normally in tissue culture, upon infection of susceptible rabbits the viral disease was observed to be significantly attenuated. The majority of infected rabbits were able to mount an effective immune response to the infection and completely recovered. These survivor rabbits became immune to subsequent challenge with wild type myxoma virus. We conclude that the T2 viral protein is an important secreted virulence factor and that it in all likelihood functions by compromising the antiviral effects of TNF. We propose the term “viroceptor” to describe viral-encoded homologs of cellular lymphokine receptors whose function is to intercept the activity of the cognate lymphokine in order to short circuit the host immune response to the viral infection.

Preparation of Isotope-labeled Dihydroneopterin 3'-Triphosphate with High Specific Activity

SummaryIsotope-labeled dihydroneopterin 3'-triphosphate with 3H at positions C-1' and C-2', respectively, has been prepared from isotope-labeled glucose as starting material. Glucose was first converted enzymatically to ribose 5-phosphate. GMP was subsequently obtained by the action of phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase. It was subsequently phosphorylated to GTP in two steps using adenylate kinase and guanylate kinase. Dihydroneopterin triphosphate was prepared from GTP by the action of recombinant GTP-cyclohydrolase I from Escherichia coli. The method allows the incorporation of 3H and 14C isotope labels into any desired position of dihydroneopterin triphosphate. Rapid purfication procedures for phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase as well as HPLC assays for their determinations are described.

Biochemistry and metabolism of Giardia.

Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Parnas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphoribosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Deoxycytidine on Mycoplasma-Associated Inhibition of Thymidine Incorporation and Growth in Antifolate-Containing Media

Several mycoplasma species markedly inhibit lymphokine- and mitogen-induced 3H-thymidine incorporation in cultured lymphoid cells, but have negligible short-term effects on cellular DNA synthesis as assessed by cytofluorography or by cell counts. The deoxyribonucleotide precursor deoxycytidine (dC) reverses this inhibition, but has little effect on isotope incorporation in uninfected cultures. Human lymphoblastoid leukemia cell lines contaminated with mycoplasma and hypoxanthine guanosine phosphoribosyl transferase (HGPRT)-deficient subclones do not grow in conventional HAT medium, but the unselected parent lines proliferate when dC is included in the culture medium. The beneficial effect of dC on the growth of contaminated cultures in selection medium is amplified by the addition of the cytidine deaminase inhibitor tetrahydrouridine (THU). These observations and corroborating nucleotide pool analysis suggest that dC may exert its beneficial effects on cellular proliferation and isotope utilization by inhibiting a mycoplasma-associated enzyme, thymidine phosphorylase. The data also suggest that the conversion of dC to dU by the cellular enzyme cytidine deaminase reduces the ability of dC to salvage contaminated cultures in the presence of an antifolate. The addition of dC to the culture medium in various 3H-thymidine incorporation assays makes possible the detection of stimulatory lymphokines despite the presence of mycoplasma contamination of the indicator cells. The normalization of nucleotide pools and cellular growth of mycoplasma-infected HGPRT (+) human leukemic cell lines with the addition of dC to HAT selection medium has made possible the use of infected HGPRT-deficient subclones as fusion partners in the generation of T-T hybridomas. Our studies also suggest that the ability of cells to grow in HAT medium only when dC is included is presumptive evidence for mycoplasma infection.

Fast non-chromatographic method to assay for xanthine-guanosine phosphoribosyl transferase.

Fast non-chromatographic method to assay for xanthine-guanosine phosphoribosyl transferase.

I region-restricted antigen presentation by B cell-B lymphoma hybridomas.

The activation of T lymphocytes for many responses requires interaction with specialized antigen presenting cells (APC) which express I region associated (Ia) antigens. Cells of the monocyte-macrophage lineage and the recently described dendritic cell have been shown to be effective APC. Antigen-specific T cells appear to recognize a complex of antigen and Ia molecules on the surface of the APC and such joint recognition is the basis of the restriction of T-cell activation due to the major histocompatibility complex. The Ia molecules appear to be Ir gene products. However the biochemical nature of antigen presentation has been difficult to approach largely because of problems in obtaining sufficient numbers of highly purified APC. Although several Ia antigen-positive B lymphoma cell lines, which have proved very effective APC, have been recently described, they have all been of the same H-2d haplotype. We report here on hybridomas produced by fusion of normal C57BL/6 (B6) B cells (H-2b) to a hypoxanthine guanosine phosphoribosyl transferase (HGPRT) deficient variant of one of these lymphoma cell lines. The hybridomas appear to express functional Ia molecules of both parental cell haplotypes. Such cloned somatic cell hybrids should prove useful in studying the properties of Ir genes and Ia molecules in antigen presentation.

The metabolism of adenosine and distribution of adenosine receptor lymphocytes in two human circulating T cell subsets.

Human circulating E rosette forming cells (ERFC), rerosetted with sheep erythrocytes in the presence of adenosine, yielded two T-lymphocyte subpopulations: a major fraction forming E-rosettes (E resistant = ER) and a minor non-rosetting fraction (E sensitive - ES). Both T cell subpopulations converted adenosine mainly into inosine. However, ES cells metabolized adenosine more extensively than ER cells. Adenosine deaminase (ADA) activity was significantly higher in ES cells. Purine nucleoside phosphorylase (PNP) activity, as well as hypoxanthine guanosine phosphoribosyl transferase (HGPRT) activity were similar in both T cell subsets. The ratio of ADA/PNP in ES cells relative to ER cells was 1.8 suggesting that ES cells are at an earlier stage of differentiation. Enrichment of lymphocytes bearing a receptor for adenosine was demonstrated in ES cells.

The effects of topoinhibition and cytochalasin B on metabolic cooperation

Metabolic cooperation was used to investigate the ability of 3T3 cells to form intercellular junctions under conditions of topoinhibition and topostimulation, respectively. A transformed BHK cell lacking hypoxanthine guanosine phosphoribosyl transferase, which was used as an indicator, was found to incorporate 3H-hypoxanthine when in prolonged (20 hr) or brief (1 hr) contact with donor 3T3 cells, whether the latter were in a quiescent layer or in the region of stimulation at the edge of a wound. Cytochalasin B (1 μg/ml) prevented the development of metabolic cooperation and abolished most preexisting cooperation between donor 3T3 cells and recipient BHK cells. Cooperation was reestablished by removal of the drug. Cytochalasin B inhibited cooperation by the donor 3T3 cells in both the layer and wound edges, irrespective of topoinhibition and topostimulation. This provides further evidence that alteration in capacity to form stable intercellular junctions is not a necessary feature of the topoinhibition phenomenon.