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)

We have characterized a DNA-protein interaction within a sequence element distal from the site of transcription initiation within the mouse housekeeping gene (HPRT) promoter region. This interaction occurs within a 35-base pair regulatory element which confers cell type-specific gene transcription, designated as the HPRT cis-acting regulatory element (HCRE). Competition analysis by gel mobility shift electrophoresis indicates that this DNA-protein interaction is novel and not related to many transcription factors previously reported. Cell cycle synchronization experiments and gel mobility shift assays have demonstrated that within the HCRE a specific DNA-protein complex responds to G1 activation of the cell cycle. Experiments to purify specific DNA-binding proteins that interact with the HCRE has resulted in the purification of one sequence-specific DNA-binding protein of approximately 66 kDa. To determine the putative DNA-binding sequence, footprinting analysis has mapped the protection from DNase I hydrolysis which confers a core sequence of GTCTGGGT using both affinity purified protein and crude nuclear extract. This DNA motif represents a novel protein-binding sequence. Interestingly, data base searches have identified the same or homologous sequences of this DNA motif in additional genes, potentially related to cellular growth and proliferation. This consensus was most notable within a region 5' upstream of the ornithine decarboxylase gene. The unique cell type-specific regulation of the HPRT gene in the intestinal mucosa is not completely understood at this time but because of the relationship of ornithine decarboxylase expression to cell proliferation and more specifically, to mucosal cell renewal in the intestine, the function of DNA-protein interactions within the consensus sequence may prove analogous. This may account for the cell type-specific and cell-cycle responsive gene regulation previously demonstrated with HPRT. Identification of one sequence-specific DNA-binding protein within the HCRE suggest that this protein contributes to the trans-activation of specific genes during the immediate-early response of the cell cycle.
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PMID:Characterization of DNA-protein interactions within a distal regulatory element upstream of a mammalian housekeeping gene promoter. 155 10

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other "housekeeping genes," thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3-0.8 pg/micrograms DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8-1.2 pg/microgram DNA, which corresponds to 6-10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5-10 h before the onset of DNA synthesis, and a steady-state level was reached after 2-3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (Lesch-Nyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.
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PMID:Levels of hypoxanthine phosphoribosyltransferase RNA in human cells. 168 3

To determine the methylation status of female germ cells in reference to the programmed reversal of X chromosome inactivation in these cells, we examined human fetal ovaries at developmental stages from the time germ cells initiate meiosis to when they cease to synthesize DNA (8-21 weeks gestation). Using methylation-sensitive restriction enzymes, we analyzed 57 MspI sites (32 sites in the CpG islands, and 25 nonclustered sites) from five X-linked housekeeping genes (HPRT, G6PD, P3, PGK, and GLA) and two tissue specific genes (X-linked F9 and autosomal EPO). Methylation patterns were compared to those of male germ cells, sperm, and somatic tissues of both sexes. All 32 MspI sites in CpG islands were unmethylated in germ-cell fractions of fetal ovary and adult testes, which could explain the reversibility of X inactivation in these tissues. However, whereas male meiotic germ cells were extensively methylated outside the islands (in the body of genes) and the methylation patterns resembled those of most somatic tissues, none of the 25 nonclustered CpGs was methylated in DNA contributed by the germ-cell component of fetal ovaries. The presence of faint MspI-like fragments in HpaII digests of fetal testes as well as fetal ovary prior to the onset of meiosis suggests that DNA of primordial germ cells is unmethylated in both sexes. Our observations of meiotic germ cells suggest that the female germ cells remain unmethylated, but that methylation in male germ cells occurs postnatally, prior to or during the early stages of spermatogenesis. In any event, the striking sex difference in methylation status of endogenous single-copy genes in meiotic germ cells could provide a molecular basis for parental imprinting of the mammalian genome.
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PMID:Sex difference in methylation of single-copy genes in human meiotic germ cells: implications for X chromosome inactivation, parental imprinting, and origin of CpG mutations. 169 9

Studies from several laboratories worldwide have developed a large database for in vivo hypoxanthine-guanine phosphoribosyltransferase gene mutations in human T-lymphocytes. Sufficient differences have been found thus far between the spectrum for spontaneous mutations in adults and that observed in the fetus to suggest fundamental differences in in vivo mutagenic mechanisms at these two life stages. In adults, only approximately 15% of hypoxanthine-guanine phosphoribosyltransferase mutations have structural alterations on Southern blots, while in the fetus 75% of mutations show alterations of which one-half are deletions of exons 2 and 3. We have now sequenced the breakpoint sites for these specific deletions in 18 mutant lymphocyte clones isolated from 13 normal newborns. Three classes of deletions were found. Each class had the same intron 1 breakpoint but a different intron 3 breakpoint. These mutations have all the signatures of a V(D)J recombinase-mediated event (a 5' consensus heptamer, 3' consensus heptamer and nonamer, nibbling, non-germline-encoded nucleotides, P-nucleotides). At the 3' breakpoint of the most common class (comprising 83% of the mutants) a perfect heptamer can be created by postulating a hairpin loop which could attain a Z-DNA configuration. This feature may indicate recombinase preference for certain DNA structures. These results implicate the V(D)J recombinase in illegitimate events causing mutation in this housekeeping gene during T-cell development. Inactivation of genes involved in the control of growth and differentiation (e.g., tumor suppressor genes) by this mechanism may have important implications for cancer development.
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PMID:V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes. 193 63

The mechanism for establishing the DNA methylation patterns observed in adult mammalian tissues is not well understood. To determine when adult patterns are established for housekeeping genes, we examined the clustered CpGs in genes on the human active X chromosome (PGK, G6PD, P3, GdX, HPRT) and the autosomal gene, DHFR. We find unique methylation patterns present at the P3 locus in all tissues analyzed from 6- to 9-week fetal specimens, and at the HPRT locus in adrenal gland DNA at this stage of development. Adult patterns are established subsequently by demethylating specific CpGs. Our results show that demethylating events affecting CpG islands are programmed during mammalian fetal development. They suggest that the process of de novo methylation in the fetus methylates at least some sites in the 3' region of the CpG islands in active genes and that adult patterns are established at 6-14 weeks developmental age by sequence-specific demethylation.
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PMID:Programmed demethylation in CpG islands during human fetal development. 201 94

Part of the higher-order structure of chromatin is achieved by constraining DNA in loops ranging in size from 30 to 100 kilobase pairs; these loops have been implicated in defining functional domains and replicons and possibly in facilitating transcription. Because the human active and inactive X chromosomes differ in transcriptional activity and replication, we looked for differences in their chromatin loop structures. Since the islands of CpG-rich DNA at the 5' ends of X-linked housekeeping genes are the regions where functional differences in DNA methylation and nuclease sensitivity are found, we looked for scaffold association of these sequences after extraction of histones with lithium diiodosalicylate. Specifically, we examined the 5' CpG islands within the hypoxanthine phosphoribosyltransferase, glucose 6-phosphate dehydrogenase, P3, GdX, phosphoglycerate kinase type 1, and alpha-galactosidase loci in human lymphoblasts obtained from individuals with 1 to 4 X chromosomes. Although we detected no scaffold-associated regions near these genes, we found several such regions at the ornithine transcarbamylase and blood clotting factor IX loci. Our results suggest that the CpG islands are excluded from the nuclear scaffold and that even though transcriptionally active, housekeeping genes are less likely than X-linked tissue-specific genes to be scaffold associated. In all cases, the pattern of scaffold association was the same for loci on active and inactive X chromosomes.
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PMID:Chromatin loop structure of the human X chromosome: relevance to X inactivation and CpG clusters. 276 35

The mouse hypoxanthine phosphoribosyltransferase gene, like several other housekeeping genes, lacks many of the features associated with promoters of RNA polymerase II-transcribed genes. HPRT transcripts have multiple initiation sites and an HPRT minigene was used to show that only 49 bases of 5' flanking sequence was necessary for normal expression in cultured cells. The essential region, which occurs within a complex series of direct repeats, is homologous to sequences upstream of other housekeeping genes. When this sequence was deleted, cryptic upstream initiation sites were revealed. Similar aberrant patterns of initiation were seen with all minigenes assayed in Xenopus oocytes. We speculate that this region of the HPRT promoter is involved in a different interaction with the transcriptional machinery to that occurring at more conventional promoters.
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PMID:Expression of the mouse HPRT gene: deletional analysis of the promoter region of an X-chromosome linked housekeeping gene. 345 94

To explore the molecular basis of X chromosome inactivation, we have examined the human locus for glucose-6-phosphate dehydro-genase (G6PD) in various human tissues. Studies of DNA from males and females and from somatic cell hybrids with active or inactive X chromosomes, show that two remarkably dense clusters of CpG dinucleotides in the 3' coding sequences are hypomethylated in active G6PD genes but extensively methylated in inactive ones. Reacquisition of G6PD activity, either spontaneous or induced by 5-azacytidine, is accompanied by demethylation of both clusters; however, the clusters remain methylated in reactivants that express HPRT but not G6PD. Our observations implicate these 3' CpG clusters in the transcription of G6PD and in maintenance of dosage compensation for X linked housekeeping genes.
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PMID:Complete concordance between glucose-6-phosphate dehydrogenase activity and hypomethylation of 3' CpG clusters: implications for X chromosome dosage compensation. 651 79

To explore the role of DNA methylation in maintaining dosage compensation of X chromosome-linked genes and in regulating the transcriptional activity of "housekeeping" genes, we characterized DNA methylation of active, inactive, and derepressed alleles at the locus for hypoxanthine phosphoribosyltransferase (HPRT) on the human X chromosome. The methylation of Hpa II and Hha I sites in HPRT alleles on the active X chromosome was the same in all tissues. The consensus pattern includes hypomethylation of 5' clustered sites and extensive methylation of the 3' sequence. The striking feature of methylation of inactive X-chromosome alleles is nonuniformity and less extensive hypomethylation of the 5' cluster. Analysis of HPRT alleles reactivated in response to 5-azacytidine showed at least partial restoration of the consensus pattern. These observations indicate that methylation of housekeeping genes on the X chromosome is the same as that of autosomal ones and that the overall pattern and methylation of multiple sites within a cluster may cooperate to facilitate transcription. Furthermore, the fidelity of methylation of the active allele and the extensive drift in methylation of the inactive allele suggest that mechanisms involved in X-chromosome dosage compensation may be directed at the active rather than inactive X chromosome.
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PMID:Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation. 658 29

Competitive polymerase chain reaction (PCR) is a sensitive method for quantification of cytokine mRNA expression. Co-amplification of an internal standard serves as control for comparing the efficiency of PCR in different samples. We have developed a novel control fragment for multiple analyses of rat cytokine gene expression containing primers for IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, TGF-beta 1, IFN-gamma and MIP-2. Additional primers were incorporated to analyse the content of T cells (CD3), activated T cells (CD25) and housekeeping genes (beta-actin and HPRT). As an example we demonstrate analysis of IL-2 mRNA expression in small pieces of kidney tissue obtained from rats after kidney allotransplantation. The IL-2 expression decreased tenfold during treatment with an anti-rat CD4 monoclonal antibody as compared to untreated animals.
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PMID:A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rats. 782 31


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