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

A complete deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), in man results in the Lesch-Nyhan (LN) syndrome. Two unrelated patients with the full LN syndrome showed no evidence of a major alteration to the gene encoding HPRT (HPRT) by restriction endonuclease analysis, but exhibited negligible levels of HPRT mRNA on Northern blots. DNA from these patients was characterised further. Amplification, by the polymerase chain reaction (PCR), of individual HPRT-exon fragments from genomic DNA followed by nucleotide (nt) sequence analysis using automated technology, revealed single-base mutations in each patient. One patient has an insertion of a T within exon-2, which places a stop codon in frame, presumably resulting in premature termination of translation of the HPRT mRNA. The other patient has a G----A base substitution at the 5' end of intron-6, at the junction of exon-6 and intron-6. Although dot blot analysis indicated negligible HPRT mRNA in lymphoblast cells from both patients, we were successful in amplifying HPRT cDNA using PCR. Direct nt sequence analysis of the amplified cDNA confirmed the insertion of a T in exon-2 in the one patient and revealed a complete deletion of exon-6 in the other patient, the latter event presumably arising due to aberrant splicing of primary message. Both mutations were also confirmed by hybridisation of amplified genomic DNA with allele-specific oligodeoxyribonucleotide probes. This study illustrates two approaches for analysing DNA mutations at the molecular level and demonstrates the power of PCR technology in the study of genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: two Lesch-Nyhan patients with reduced amounts of mRNA. 184 May 49

Three-allele restriction fragment length polymorphisms (RFLPs) for the restriction endonuclease Bam HI are known at the hypoxanthine-guanine phosphoribosyltransferase (HPRT, E.C.2.4.2.8.) gene locus. The alleles are expressed phenotypically on Southern blots as three distinct pairs of fragments that hybridize to HPRT cDNA: i) a 22-kilobase (kb)/25-kb pair, ii) a 12-kb/25-kb pair, and iii) a 22-kb/18-kb pair. Allele frequencies in 119 unrelated Japanese people were 0.38 for the 22-kb/25-kb allele, 0.43 for the 12-kb/25-kb allele, and 0.19 for the 22-kb/18-kb allele, an average heterozygosity of 66% in Japanese females, a higher rate than in Caucasian females. Five out of nine carriers of partial or complete HPRT deficiency showed heterozygous patterns for Bam HI RFLPs.
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PMID:Bam HI restriction fragment length polymorphisms for hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of carriers and controls of HPRT deficiency in Japan. 197 Feb 11

The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter----3p12::Xq26----Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions (HAT medium) for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. In order to introduce a second selectable genetic marker to the t(X;3) chromosome, A9(GM0439)-1 cells were transfected with pcDneo plasmid DNA. Colonies resistant to both G418 and HAT medium (G418r/HATr) were selected. To obtain A9 cells that contained a t(X;3) chromosome with an integrated neo gene, the microcell transfer step was repeated and doubly resistant cells were selected. G418r/HATr colonies arose at a frequently of 0.09 to 0.23 x 10(-6) per recipient cell. Of seven primary microcell hybrid clones, four yielded G418r/HATr clones at a detectable frequency (0.09 to 3.4 x 10(-6)) after a second round of microcell transfer. Doubly resistant cells were not observed after microcell chromosome transfers from three clones, presumably because the markers were on different chromosomes. The secondary G418r/HATr microcell hybrids contained at least one copy of the human t(X;3) chromosome and in situ hybridization with one of these clones confirmed the presence of a neo-tagged t(X;3) human chromosome. These results demonstrate that microcell chromosome transfer can be used to select chromosomes containing multiple markers.
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PMID:Introduction of new genetic markers on human chromosomes. 199 1

The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of HPRT and ODC.
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PMID:High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase. 200 85

The cytotoxicity and DNA lesions induced by methotrexate (MTX) were compared in wild-type, hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT-) and thymidine-kinase-deficient (TK-) HL-60 cells. TK- and HGPRT- cells were approximately 10 and 3 times more sensitive to MTX than wild-type cells, respectively. Following incubation with 2 microM MTX for 16 hr, TK- cells showed a significantly higher number of DNA strand breaks. Concomitantly, DNA fragmentation at the nucleosomal linker region was detected more prominently in TK- cells. Although MTX tended to decrease TTP pools similarly in all 3 cells types, the initial TTP level in TK- cells was only about one-fifth of that found in the wild type. These results indicate that the thymidine salvage pathway has a pivotal role in mediating MTX-induced toxicity and DNA lesions.
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PMID:Increased methotrexate-induced DNA strand breaks and cytotoxicity following mutational loss of thymidine kinase. 201 62

The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT- Chinese hamster V79 cells. Several gpt- cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. Each cell line exhibits a characteristic spontaneous mutation frequency (10(-5) to 10(-2)) in 6-thioguanine (6TG) selection. While spontaneous mutagenesis to gpt- occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency (approximately 3 x 10(-5)), was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and beta-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The gpt locus of the g12 transfectants, however, is two to three times more sensitive to UV and 2.5-4.5 times more sensitive to X-ray mutagenesis than the endogenous hprt of wild-type V79 cells. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus. Future studies with these transgenic cells and other transgenic lines are planned to compare the mutability and repair of the same gene (gpt) at different integration sites in mammalian cells.
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PMID:Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis. 219 84

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of Plasmodium falciparum plays a key role in the salvage of preformed purine nucleotides from parasite-infected erythrocytes. Since P. falciparum cannot synthesize purines de novo, development of inhibitors specific for the parasite HGPRT should be an effective method of chemotherapy. To provide sufficient amounts of HGPRT for biochemical and crystallographic analysis, we have isolated the P. falciparum HPRT cDNA sequence and expressed it in an Escherichia coli strain deficient for both de novo purine synthesis and guanine utilization (strain GP120). GP120 cells containing the P. falciparum HPRT plasmid vector (pRD500), when grown in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) which induces the tac promoter of the expression vector, produce a novel protein of 26 kDa, which is in agreement with the predicted Mr deduced from the HPRT cDNA open reading frame. In addition, we have demonstrated significant HGPRT activity in cell-free extracts of GP120[pRD500] cultures grown in minimal medium containing xanthine, as the sole source of purines, and IPTG.
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PMID:Cloning and expression in Escherichia coli of a hypoxanthine-guanine phosphoribosyltransferase-encoding cDNA from Plasmodium falciparum. 220 41

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.
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PMID:In vivo hprt mutant frequencies in T-cells of normal human newborns. 230 72

We have used the pZipHprtNeo shuttle vector to determine the types of DNA sequence alterations induced by a potent carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P2). The shuttle vector contains a human cDNA hprt as the target gene and is stably integrated into a chromosome of the mouse cell line VH12. After Trp-P2 treatment, 59 independent HPRT- mutant clones of VH12 were isolated and altered sequences of the mutant hprt- cDNA genes were determined. Mutations induced by Trp-P2 comprised a variety of events; base substitutions, frameshifts, deletions and complex. Frameshifts were the most frequent mutational events (51%), and base substitutions were the next most frequent (30%) followed by deletions (14%). Examination of the DNA sequence context in the mutant genes revealed that approximately 70% of mutations induced by Trp-P2 occurred at G:C sites and thymine residues were the suggested target for the remainder of mutations. The results seem consistent with the previously reported finding that in vivo, metabolically activated Trp-P2 specifically binds to the C8 position of guanine residues in DNA to form C8G-Trp-P2 adducts (Hashimoto et al., Mutat, Res., 105, 9-13, 1982). As for molecular mechanisms, we showed that slippage and slippage misalignment could predict the generation of a large portion of Trp-P2-induced mutations found in the cDNA gene.
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PMID:Mutational specificity of the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]-indole in mammalian cells. 233 11

Methods for measuring somatic mutation and chromosome aberration in humans are currently advancing and provide important new opportunities for biologic dosimetry of nuclear workers. Methods to test somatic mutation in four human genes (hprt, hla-a, glycophorin A, and beta globin) are reviewed briefly and evaluated for their applicability to biological radiation dosimetry of nuclear workers. Two somatic mutation tests can be currently recommended: an HPRT method applied to recently exposed workers and the glycophorin A method applied to workers exposed over their working lifetime. A new method of chromosome analysis using DNA hybridization with chromosome-specific gene libraries allows one to paint single or multiple chromosome pairs in standard metaphase preparations. This method is ideal for rapid and reliable detection of reciprocal translocations, the key lesion for the evaluation of long-term radiation exposure. Both mutational and aberrational approaches should be fostered in the expectation that they will complement other forms of dosimetry and will improve our ability to clarify whether or not significant health effects are dosimetrically related.
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PMID:New approaches for biological monitoring of radiation workers. 235 56


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