Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Expression plasmids containing the human alpha 1-antitrypsin (alpha 1 AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT- HPRT- rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes in cis, as only three of 20 clones tested were affected for expression of alpha 1AT mRNA. In contrast, double selection yielded predominantly trans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal alpha 1AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1 trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.
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PMID:Direct selection of hepatoma cell variants deficient in alpha 1-antitrypsin gene expression. 133 97

The measurement of the activity of the X-linked enzyme HPRT has been widely used as an indicator of X-chromosome activity during preimplantation development in the mouse. More recently, the concomitant measurement of the activity of the autosomally-encoded enzyme APRT has been used in an attempt to decrease the variability inherent in the measurement of enzyme activity from minute samples such as preimplantation embryos. In this study the use of the HPRT-deficient mouse mutant, Hprtb-m3, allowed the unequivocal identification of the parental origin of HPRT activity measured in embryos derived from crosses between wild-type mice, and mice which were homozygous or hemizygous for the Hprtb-m3 allele. Results were similar to those of a previous study, where oocyte-encoded HPRT activity accounted for about 10% of total HPRT activity at 76 hours post human chorionic gonadotrophin injection and the paternally-derived Hprt allele was shown to be transcriptionally active by the late 2-cell stage. In contrast to other studies, differential expression of the two Hprt alleles was detected during the preimplantation period, in embryos derived from crosses between wild-type and HPRT-deficient mice. Evidence was also found for the existence of an X-linked locus which influences the amount of APRT activity in the unfertilized oocyte. We propose that the expression pattern of this locus may be influenced by its parental origin.
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PMID:Imprinting of phosphoribosyltransferases during preimplantation development of the mouse mutant, Hprtb-m3. 145 55

We have analyzed the adenine phosphoribosyltransferase (APRT) enzyme from Chinese hamster ovary cells through the study of mutants that are able to grow in the presence of the toxic adenine analogue 8-azaadenine. The distribution of the amino acid alterations was analyzed in terms of the binding regions for the purine and phosphoribosylpyrophosphate substrates and a comparison was made with mutants known in human APRT and human, mouse and hamster hypoxanthine-guanine phosphoribosyltransferase. A number of mutants were found to cluster in several regions of the amino acid sequence. Residual enzyme activity with adenine was determined and this was correlated with substrate binding regions. A model of the secondary structure features is proposed.
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PMID:Mutational analysis of the structure and function of the adenine phosphoribosyltransferase enzyme of Chinese hamster. 171 94

The initiation of carcinogenesis by carcinogens such as 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) is thought to involve the formation of DNA adducts. However, the diastereomeric diol epoxide, 7r,8t-dihydroxy-9,10c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-II), also forms DNA adducts but is inactive in standard carcinogenesis models. We have measured the formation and loss of DNA adducts derived from BPDE-II in a DNA-repair-proficient line of Chinese hamster ovary (CHO) cells, AT3-2, and in two derived mutant cell lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. BPDE-II adducts were lost from cellular DNA in AT3-2 cells with a half-life of 13.8 h; this was about twice the rate found for BPDE-I adducts. BPDE-II adducts were also lost from DNA in UVL-1 and UVL-10 cells, but at a much slower rate. When purified DNA was modified in vitro with BPDE-II and then held at 37 degrees C, DNA adducts were removed at a rate identical to that seen in UVL-1 and UVL-10 cells, suggesting that the loss in these cells was not due to enzymatic DNA-repair processes but to chemical lability of the adducts. Mutant frequencies at the APRT and HPRT loci were measured at BPDE-II doses that resulted in greater than 20% survival, and were found to increase linearly with dose. In the DNA-repair-deficient cells, the HPRT locus was moderately hypermutable compared with AT3-2 cells (about 5-fold); the APRT locus was extremely hypermutable, giving about 25-fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial levels of binding. When we compared the mutational efficiency of BPDE-II at both loci in AT3-2 cells (the mutant frequency in mutants/10(6) survivors at a dose that resulted in one adduct per 10(6) base pairs) with our previous studies of BPDE-1, we found that BPDE-II was 4-5 times less efficient as a mutagen than BPDE-I. This difference in mutational efficiency could be explained in part by the increased rate of loss of BPDE-II adducts from the cellular DNA, part of which was due to an increased rate of enzymatic removal of these lesions compared with the removal of BPDE-I adducts.
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PMID:Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzo[a]pyrene diol epoxides in CHO cells. 172 82

Chinese hamster ovary (CHO) cells were subjected to electroporation in the presence of 5-methyl deoxycytidine-triphosphate. This treatment increases by 10 to 100-fold the frequency of cells lacking thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, or adenine phosphoribosyltransferase. The inactivation of the genes coding for these enzymes is thought to occur following the direct incorporation of the methylated nucleotide triphosphate into DNA. The enzyme-deficient clones were stable, but almost all were reactivated at high frequency by the demethylating agent 5-azacytidine, to produce derivatives with enzyme activity. The results indicate that there is a direct relationship between DNA methylation and gene silencing.
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PMID:Gene silencing in mammalian cells by uptake of 5-methyl deoxycytidine-5'-triphosphate. 172 91

A novel method for measuring AMP-deaminase activity in human erythrocytes is presented, based on the determination of the reaction product, IMP, using high performance liquid chromatography. IMP formation was found to be proportional both to the incubation time and the amount of haemolysate over a wide range. The minimal detectable AMP-deaminase activity was more than 1000 times lower than the mean activity found in healthy controls (1083 nmol/h/mg Hb). No marked difference of activity was found in the patients with the following inherited purine disorders: familial juvenile gouty nephropathy and deficiencies of adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase or adenine phosphoribosyltransferase. The activity in the erythrocytes of patients with chronic renal failure was also similar to controls. The existence of subjects with low erythrocyte AMP-deaminase activity in the population has been confirmed.
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PMID:A high performance liquid chromatographic assay for AMP-deaminase activity in the erythrocytes of healthy subjects and patients with inherited purine disorders. 191 25

The uptake of purine nucleosides (guanosine and hypoxanthine) and bases (guanine, hypoxanthine and adenine) and their incorporation into nucleotides were studied in enterocytes isolated from fed and 3-day fasted guinea pig jejunum. Both total uptake and synthesis of nucleotides were greater for these purines in the fasted, as compared to the fed state for the first 5 min, when the initial substrate concentration in the medium was 10 microM. Increased uptake did not result from a change in the relative distribution of synthesized nucleotides between the fed and fasted states. Reduced catabolism was observed in the medium by enterocytes from fasted as compared to fed animals after 1 min of incubation with both inosine and guanosine. Preincubation of enterocytes with allopurinol (a xanthine oxidase inhibitor) decreased total uptake but increased the formation of IMP from hypoxanthine. Xanthine oxidase activity measured in mucosa from fasted guinea pigs was lower than that from fed animals (6.29 vs. 9.30 nmol/min per mg protein, respectively). However, activities of the salvage enzymes adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase were not significantly different between the fed and fasted states. These data show that allopurinol treatment, and mucosal atrophy resulting from fasting, decrease xanthine oxidase activity and increase nucleotide synthesis from exogenous substrates in enterocytes from the guinea-pig small intestine, suggesting a regulatory function of mucosal xanthine oxidase in purine salvage by the small intestine.
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PMID:The effect of nutritional state and allopurinol on nucleotide formation in enterocytes from the guinea pig small intestine. 200 79

Reports describing short (less than 20 bp) gene deletions causing human genetic disease were collated in order to study underlying causative mechanisms. Deletion breakpoint junction regions were found to be non-random both at the nucleotide and dinucleotide sequence levels, an observation consistent with an endogenous sequence-directed mechanism of mutagenesis. Direct repeats of between 2bp and 8bp were found in the immediate vicinity of all but one of the 60 deletions analysed. Direct repeats are a feature of a number of recombination, replication or repair-based models of deletion mutagenesis and the possible contribution of each to the spectrum of mutations examined was assessed. The influence of parameters such as repeat length and length of DNA between repeats was studied in relation to the frequency, location and extent of these deletions. Findings were broadly consistent with a slipped mispairing model but the predicted deletion of one whole repeat copy was found only rarely. A modified version of the slipped mispairing hypothesis was therefore proposed and was shown to possess considerable explanatory value for approximately 25% of deletions examined. Whereas the frequency of inverted repeats in the vicinity of gene deletions was not significantly elevated, these elements may nevertheless promote instability by facilitating the formation of secondary structure intermediates. A significant excess of symmetrical sequence elements was however found at sites of single base deletions. A new model to explain the involvement of symmetric elements in frameshift mutagenesis was devised, which successfully accounted for a majority of the single base deletions examined. In general, the loss of one or a few base pairs of DNA was found to be more compatible with a replication-based model of mutagenesis than with a recombination or repair hypothesis. Seven hitherto unrecognized hotspots for deletion were noted in five genes (AT3, F8, HBA, HBB and HPRT). Considerable sequence homology was found between these different sites, and a consensus sequence (TGA/GA/GG/TA/C) was drawn up. Sequences fitting this consensus (i) were noted in the immediate vicinity of 41% of the other (sporadic) gene deletions, (ii) were found frequently at sites of spontaneous deletion in the hamster APRT gene, (iii) were found to be associated with many larger human gene deletions/translocations, (iv) act as arrest sites for human polymerase alpha during DNA replication and (v) have been shown by in vitro studies of human polymerase alpha to be especially prone to frameshift mutation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment. 201 84

This paper (1) presents an analysis of published data on the molecular nature of spontaneously arising and radiation-induced mutations in mammalian somatic cell systems and (2) examines whether the molecular nature and mechanisms of origin of radiation-induced mutations, in mammalian in vivo and in vitro systems, as currently understood, are consistent with expectations based on the biophysical and microdosimetric properties of ionizing radiation. Depending on the test system (CHO cells, human T lymphocytes and human lymphoid cell line TK6), 80-97% of spontaneous HPRT mutations show normal Southern patterns; the remainder is due to gross changes, predominantly partial (intragenic) deletions. Total gene deletions at the HPRT locus are rare except in the TK6 cell line. At the APRT locus in CHO cells, 80-97% of spontaneous mutations are due to base-pair changes, the remainder being, mostly, partial deletions. The latter can extend upstream in the 5' direction but not beyond the APRT gene in the 3' direction. At the human HLA-A locus (T lymphocytes), the percentage of mutations with normal Southern patterns is lower than that for HPRT, and in the range of 50-60%. At the HLA-A locus, mitotic recombination contributes substantially to the mutation spectrum (approximately 30% of mutations recovered) and this is likely to be true of the TK locus in the TK6 cell line as well. With a few exceptions, most of the radiation-induced mutations show altered Southern patterns and are consistent with their being deletions and/or other gross changes (HPRT, 70-90% (CHO); 50-85% (TK6); 50-75% (T lymphocytes); TK, 60-80% (TK6); HLA-A, 80% (T lymphocytes); DHFR, 100% (CHO]. The exceptions are APRT mutations in CHO cells (16-20% of mutants with deletions or other changes) and HPRT mutations in T lymphocytes from A-bomb survivors (15-25%); the latter finding is consistent with the occurrence of in vivo selection against HPRT mutant cells. In cases of HPRT intragenic deletions analyzed (CHO cells and V79 Chinese hamster cells), there is evidence for a non-random distribution of breakpoints. The spontaneous mutation frequencies vary widely, from about 0.04/10(6) cells (sickle cell mutations at the human HBB locus) to 30.8/10(6) cells (HLA-A mutations in T lymphocytes) and are dependent on the locus, the system employed and a number of other factors. Those for the other loci fall between these limits.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Ionizing radiation and genetic risks. III. Nature of spontaneous and radiation-induced mutations in mammalian in vitro systems and mechanisms of induction of mutations by radiation. 202 1

This study was designed to simulate purine nucleoside phosphorylase (PNP) deficiency by preincubating with guanosine (Guo) to minimize PNP activity while investigating the metabolism of [14C] deoxyguanosine (dGuo) at physiologic concentrations (10 microM) by unstimulated thymocytes, tonsil-derived T and B lymphocytes, and peripheral blood cells over short time periods. GTP was the principal metabolite formed from dGuo by all cell types with functional PNP and hypoxanthine-guanine phosphoribosyltransferase, confirming formation via degradation to guanine with subsequent salvage by hypoxanthine-guanine phosphoribosyltransferase. Thymocytes also formed a small amount of deoxyguanosine triphosphate (dGTP), presumably through direct phosphorylation by deoxycytidine kinase. Incorporation of dGuo into GTP was effectively inhibited in all instances under PNP deficiency conditions and dGTP levels increased up to 10-fold in thymocytes, but tonsil-derived B or T lymphocytes and unfractionated PBL still accumulated no detectable dGTP. E and platelets formed low amounts of dGTP under these conditions. Preincubation with adenine (50 microM) to reverse any Guo-induced toxicity reduced the incorporation of dGuo into GTP without inhibitor in all cell types with intact adenine phosphoribosyltransferase, but had no effect on dGTP accumulation in thymocytes, with or without inhibitor, thus excluding any indirect formation of dGTP via the de novo route. The rapid metabolism of dGuo to GTP, in the absence of PNP inhibition and subsequent effects of the altered GTP concentrations on cellular metabolism, may account for the differing responses reported by investigators with the use of low dGuo concentrations (enhancing), compared with high (inhibitory), concentrations in mitogen-stimulated lymphocyte studies. The exclusive ability of thymocytes to accumulate significant amounts of dGTP, and inability of B cells to do so, provides a logical explanation for the selective T cell immunodeficiency in PNP deficiency.
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PMID:Mechanisms of deoxyguanosine lymphotoxicity. Human thymocytes, but not peripheral blood lymphocytes accumulate deoxy-GTP in conditions simulating purine nucleoside phosphorylase deficiency. 210 95


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