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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Authors present a procedure for the determination of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes which exhibits high sensitivity and requires low quantities of lymphocytes. 5 normal subjects and 4 patients affected by chronic lymphocytic leukemia (CLL) were considered. Human lymphocytes were prepared and treated as previously reported. To the incubation mixtures buffered with 50 mM TRIS-HCl pH 7.4 either 14C-adenine or 14C-hypoxanthine was added: after deproteinization and neutralization we followed the formation of either 14C-adenylic acid (AMP) or 14C-inosinic acid (IMP) by HPLC. A Supelcosil C18 5 microns (250 X 4.5 mm) column was used: IMP was eluted with 20 mM KH2PO4 pH 5.5 while AMP with a linear gradient to 40% B in 20 min., where A was 20 mM KH2PO4 pH 5.5 and B methanol/water 60:40. Evaluation of AMP and IMP formed was carried out by determination of the radioactivity of the collected peaks. The values of APRT in leukemic patients were enhanced when referred to the proteins and those of HGPRT decreased: the Authors propose to complete the study evaluating the intracellular content of adenine and hypoxanthine.
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PMID:[Behavior of the enzymes of the salvage pathway of purine bases in leukemia lymphocytes]. 239 7

5-Azacytidine (5-AzaC) induced mutation in the TK+/- human lymphoblastoid line, TK6, at both the thymidine kinase (tk) locus as measured by resistance to trifluorothymidine (F3TdR), and the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, as measured by resistance to 6-thioguanine (6TG). F3TdRR and 6TGR mutant fractions induced by 5-AzaC were observed after a normal phenotypic expression time and remained stable. Interestingly, 5-AzaC was 5-10 times more mutagenic at the tk locus than the hgprt locus. However, F3TdRR colonies from 5-AzaC-treated cultures behaved like TK-deficient mutants induced by other chemical mutagens. The TK or HGPRT phenotype had no effect on the toxicity of 5-AzaC, thus eliminating differential toxicity as a potential cause for the observed higher mutability at the tk locus. 5-AzaC did not induce F3TdRR cells in the parental TK+/+ lymphoblastoid line, indicating that 5-AzaC-induced F3TdRR variants were not due to a dominant alteration in gene expression. 5-AzaC did not induce chromosomal aberrations in TK6 cells, eliminating clastogenic events as a potential cause for the higher mutability at the tk locus. 5-AzaC was also found to be mutagenic in a forward mutation assay to 8-azaguanine resistance in Salmonella typhimurium.
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PMID:Studies of mutagenicity and clastogenicity of 5-azacytidine in human lymphoblasts and Salmonella typhimurium. 242 Nov 58

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).
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PMID:Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation. 243 Dec 68

The exact role of adenosine in the adenosine deaminase (EC 3.5.4.4) deficiency-related severe combined immunodeficiency disease has not been ascertained. We analysed the effects of adenosine, in the presence of the adenosine deaminase inhibitor, deoxycoformycin, on cell growth, cell phase distributions and intracellular nucleotide concentrations of cultured human lymphoblasts. Adenosine had a biphasic effect on cell growth and cell cycle distribution of a partial hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) deficient MOLT-HPRT cell line. After 24 h of incubation, 60 microM adenosine inhibited cell growth more extensively than did 100 and 200 microM adenosine. The distribution of the MOLT-HPRT cells in the various phases of the cell cycle showed a similar biphasic pattern. Adenosine concentrations in the medium below 10 microM caused accumulation of adenine ribonucleotides and depletion of phosphoribosylpyrophosphate, UTP and CTP in the cells. This was associated with inhibition of cell growth. Medium adenosine concentrations above 10 microM neither resulted in accumulation of adenine ribonucleotides nor in inhibition of cell growth.
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PMID:Inhibition of lymphoid cell growth by adenine ribonucleotide accumulation. The role of phosphoribosylpyrophosphate-depletion induced pyrimidine starvation. 243 39

Deficiencies of HPRT are usually associated with increased concentrations of PRPP and increased levels of APRT activity in erythrocytes. We report the case of a male with a partial deficiency of HPRT in whom these two parameters were normal. The clinical features of this patient were those associated with severe hyperuricaemia and gout. Studies of intact erythrocytes showed rates of incorporation of [14C]hypoxanthine and of [14C]adenine into purine nucleotides which were almost indistinguishable from normal. However, HPRT activity in erythrocyte lysates was only 9% of normal. In cell extracts of cultured lymphoblasts, the HPRT activity was 20% of control values and the APRT activity was normal. The PRPP concentration and the rate of de novo purine synthesis in cultured lymphoblasts were both intermediate between controls and lymphoblasts from patients with the Lesch-Nyhan syndrome.
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PMID:HPRT-deficiency associated with normal PRPP concentration and APRT activity. 243 88

The metabolism of adenosine and its effects on phosphoribosylpyrophosphate, PP-ribose-P, dependent nucleotide synthesis were studied using erythrocytes from patients with adenosine deaminase and hypoxanthine phosphoribosyltransferase deficiency as models. The phosphorylation of adenosine was progressively inhibited by concentrations of adenosine greater than 1 mumol L-1 for control and ADA deficient erythrocytes. There was essentially no initial rate of phosphorylation at 30 mumol L-1 adenosine. Adenosine, 1 mumol L-1, also caused a 60% reduction in PP-ribose-P concentration in ADA deficient erythrocytes. For HPRT deficient erythrocytes in which ADA activity was blocked by coformycin, 10 mumol L-1 inosine stimulated PP-ribose-P dependent nucleotide synthesis from adenine, whereas, 10 mumol L-1 adenosine inhibited nucleotide synthesis. These observations suggest that adenosine phosphorylation and PP-ribose-P dependent nucleotide synthesis are inhibited under conditions in which adenosine accumulates, such as in hereditary or pharmacologically induced ADA deficiency.
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PMID:Substrate inhibition of adenosine phosphorylation in adenosine deaminase deficiency and adenosine-mediated inhibition of PP-ribose-P dependent nucleotide synthesis in hypoxanthine phosphoribosyltransferase deficient erythrocytes. 245 96

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.
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PMID:Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells. 247 Oct 66

The C86 line of female embryonal carcinoma cells contains one active and one inactive X chromosome. Following methylnitrosourea mutagenesis, a clone called C86AGM2 was isolated that carries a mutated hprt gene on the active X chromosome. This hprtm allele encodes an HPRT enzyme that has less than 1% normal enzyme activity, is thermolabile, and has an altered isoelectric point. Following treatment with drugs that demethylate DNA, the hprt+ gene from the inactive X chromosome in C86AGM2 cells became active as determined by the appearance of HPRT activity with the thermodenaturation and electrofocusing characteristics of the normal enzyme. No expression of this hprt+ gene occurred if C86AGM2 cells were induced to differentiate prior to DNA demethylation. Stable lines of C86AGM2 cells expressing both the hprtm and hprt+ genes did not inactivate either gene following differentiation.
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PMID:Reactivation of hprt on the inactive X chromosome with DNA demethylating agents. 247 61

Treatment with 5-azacytidine, a potent inhibitor of DNA methylation, was used to induce activation of the selectable hprt gene on the inactive X chromosome in a diploid female Chinese hamster cell line. The transformed, stably diploid cell line F3B was selected in media containing the lethal purine analogue 6-thioguanine, to generate a phenotypically HPRT- mutant, F3BT1, of presumed genotype hprt-/hprt(+), where (+) represents the presumably wild-type allele on the inactive X chromosome. Treatment of F3BT1 with 5-azacytidine resulted in phenotypic reversion to HPRT+ at a frequency greater than 10(-3). Similar treatment of 6-thioguanine-resistant control lines derived from male cells, or from CHO (which has no inactive X chromosome), had no effect on the frequency of phenotypic reversion, indicating that activation of the hprt(+) allele, rather than reversion of the hprt- is responsible. This conclusion is substantiated by documentation of the low mutagenic capacity of 5-azacytidine in this system. Proof that the hprt(+) allele can be activated by 5-azacytidine treatment was obtained in somatic cell hybrids in which hprt gene products from the active and inactive X chromosomes could be distinguished by isoelectric focusing. Our results demonstrate that X-linked gene activation associated with generalized DNA demethylation occurs with high frequency in transformed diploid Chinese hamster cells.
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PMID:Activation of the hprt gene on the inactive X chromosome in transformed diploid female Chinese hamster cells. 248 Sep 66

The isoenzyme of hypoxanthine-guanine phosphoribosyltransferase (HPRT, E.C.2.4.2.8) functions in the metabolic salvage of purines. Partial HPRT deficiency is associated with gouty arthritis, while absence of activity results in Lesch-Nyhan (LN) syndrome. We characterized five unrelated patients with HPRT deficiency to understand the spectrum of molecular defects using Southern and Northern blot, polymerase chain amplification of HPRT mRNA and DNA sequencing, and oligonucleotide hybridization analysis of the HPRT gene. Southern blot analysis of DNA indicated that mutations leading to HPRT deficiency in our five patients were not the result of major chromosomal rearrangements or deletions. Sequencing analysis of the amplified DNA from three different patients with HPRT deficiency implied three unique molecular abnormalities: 1) one single-base substitution at codon 54 (from ATG to CTG) resulting in the replacement of methionine with leucine in an LN patient, 2) two single-base substitutions at codon 179 (from GTT to GGT) and at codon 180 (from GGA to AGA) resulting in the replacement of valine with glycine and glycine with arginine in a gouty patient, and 3) 51 nucleotide deletion between nucleotides 747 and 797 resulting in the formation of shorter sized HPRT mRNA and putative two amino-acid deleted HPRT protein in another gouty patient. These results are the direct molecular evidence of genetic heterogeneity in mutant HPRT.
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PMID:Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase mutations in five unrelated Japanese patients. 257 41


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