Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An established Chinese hamster cell line was fused with microcells isolated from phenotypically stable transferent mouse cells which contained a mouse transgenome coding for an abnormal form of mouse hypoxanthine phosphoribosyltransferase (HPRT, EC. No. 2.4.2.8) (Willecke et al. 1979). Two hybrids were isolated which expressed the abnormal form of mouse HPRT but no mouse alpha-galactosidase (GALA, EC. No. 3.2.1.22). In one of these microcell hybrids the abnormal HPRT activity segregated under counter-selective conditions with mouse chromosome 3. No mouse chromosome or additional mouse gene marker was found in the second microcell hybrid, possibly because of breakage and/or rearrangement of the integrated transgenome during the isolation of this hybrid. We conclude from these results that the transferred mouse HPRT gene is a phenotypically stable clone is not integrated at its homologous site on the host X chromosome. Rather, the transgenome is probably integrated into mouse chromosome 3, possibly due to homologies in repeated DNA sequences which may occur in the transgenome and which are interspersed at many sites in the host genome.
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PMID:DNA-mediated transfer of the mouse gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells: no integration of the transferred gene at its homologous site in the host genome. 694 9

Mouse A9 cells, L-cell-derived mutants deficient in hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) were found to be incapable of binding (125)I-labeled epidermal growth factor (EGF) to the cell surface. The A9 cells were fused with human diploid fibroblasts (WI-38) possessing EGF-binding ability, and human-mouse cell hybrids (TA series) were isolated after hypoxanthine/aminopterin/thymidine/ouabain selection. Analyses of isozyme markers and chromosomes of four representative clones of TA hybrids indicated that the expression of EGF-binding ability is correlated with the presence of human chromosome 7 or 19. Four subclones were isolated from an EGF-binding-positive line, TA-4, and segregation of EGF-binding was found to be concordant with the expression of human mitochondrial malate dehydrogenase (MDHM; L-malate:NAD(+) oxidoreductase, EC 1.1.1.37), a marker for chromosome 7, but not with glucosephosphate isomerase (GPI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), a marker for chromosome 19. Furthermore, evidence from 27 clones of AUG hybrids that were produced between A9 and another human fibroblast line, GM1696, carrying an X/7 chromosome translocation indicated that EGF-binding ability segregates together with human MDHM and two X-linked markers, HPRT and glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP(+) 1-oxidoreductase, EC 1.1.1.49), that are located on the translocation chromosome 7p(+). These results permit assignment of the gene, designated EGFS, which is associated with the expression of EGF-binding ability, to human chromosome 7 and its localization to the p22-qter region. Because the EGF receptor is reported to be a glycoprotein the EGFS could be either a structural gene(s) for receptor protein or a gene(s) for modifying the receptor protein through glycosylation.
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PMID:Genetics of cell surface receptors for bioactive polypeptides: binding of epidermal growth factor is associated with the presence of human chromosome 7 in human-mouse cell hybrids. 696 72

Mouse teratocarcinoma cells (OTT6050) deficient for thymidine kinase were fused with rat hepatoma cells ( Fu5AH ) deficient for hypoxanthine phosphoribosyltransferase using inactivated Sendai virus. The hybrid cells were selected and cultured in the presence of HAT medium. A clonally established hybrid cell line ( As3 ), which in addition to its mouse genome contains several rat chromosomes, expresses rat specific enzyme variants and produces large primarily undifferentiated tumors, with some hepatoma characteristics in athymic nude mice. To reveal the in vivo developmental potential of these cells and to determine whether, under different experimental conditions, they are capable of participating in tissue differentiation, the As3 cells were injected into mouse blastocysts from the C57BL/6 strain. The experimental blastocysts were then transferred into the uteri of pseudopregnant foster mothers to allow further development. From a total of 212 blastocysts transplanted, 61 fetuses developed and were analysed for As3 contributions between the 10th and 18th day of gestation. Four fetuses at day 18 showed hybrid cell participation in their livers and a few organs of only endo-mesodermal origin, as judged from the presence of rat-specific enzyme variants. The enzymes were organ-specifically expressed (e.g., lactate dehydrogenase) or appeared newly during in situ differentiation while being absent in the original hybrid cells (e.g., glycerol-3-phosphate dehydrogenase). During short in vitro culture of the chimaeric organs, it was possible to select for the hybrid cells which reverted to an enzyme pattern simiar to but not identical with the As3 cell line and different to that observed in situ.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue preference and differentiation of malignant rat x mouse hybrid cells in chimaeric mouse fetuses. 718 53

Total deficiency of hypoxanthine phosphoribosyltransferase (HPRT) in humans causes the neurological disorder Lesch-Nyhan syndrome. The HPRT gene is expressed at basal levels in all tissues but at higher levels in the brain, the relevance and mechanism of which is unknown. To determine if cis-acting DNA elements play a role in the tissue-differential pattern of expression, we generated transgenic mice carrying different sequences of the human HPRT (hHPRT) promoter fused to the bacterial lacZ gene. We show that a 1.6 kb fragment of the hHPRT promoter contains essential information to direct beta-galactosidase expression preferentially to the basal ganglia, cerebral cortex, hippocampus, and several other areas of the forebrain. At least two elements within the 1.6 kb fragment appear to be required for neuronal expression. A 182 bp element (hHPRT-NE) represents one of these sequences and is involved not only in conferring neuronal specificity but also in repressing transgene expression in non-neuronal tissues. These studies provide molecular insight into the mechanism of increased HPRT expression in the brain.
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PMID:5'-flanking sequences of the human HPRT gene direct neuronal expression in the brain of transgenic mice. 752 86

Hypoxanthine phosphoribosyltransferase-deficient (HPRT-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41-43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the "pluripotent" HM-1 genome with the "somatic" spleen cell genome during hybrid cell formation and the presence of the "somatic" X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the "somatic" X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype.
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PMID:In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes. 959 May 28

The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells.
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PMID:Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells. 1067 84

In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor kappaB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.
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PMID:A new method for the selection of protein interactions in mammalian cells. 1083 90

Mutant mitochondrial (mt) DNA variants are related to human disease and have been investigated using cytoplasmic hybrid (cybrid) cells generated from human tumor cells in which mutant mt maintenance depends on the cell line. It is, however, unclear whether human intercellular fusion of non-tumorous cells influences the maintenance of disease-related mutant mt. A preliminary experiment of cell-cell fusion between a human skin fibroblast cell line from a Lesch-Nyhan syndrome patient and an osteosarcoma cybrid cell line harboring the mitochondrial tRNALeu(UUR)A3243G mutation showed a decrease of A3243G mutant mtDNA in fused cells during passages. In order to confirm the decrease of mutant mtDNA, we performed cell-cell fusion experiments using another human lung fibroblastic cell line. When the hygromycin-resistant osteosarcoma cybrid cell line was fused with the fibroblasts without any A3243G mtDNA mutations, the proportion of A3243G mutant mtDNA in the hybrid cells gradually decreased during cell culture and almost completely disappeared in all hybrid clones at the end of 15 passages. These results indicated that A3243G mutant specific mtDNA decreases in the hybrid background when normal fibroblast-derived cell contents, including the nucleus and mt, were introduced. Thus, we are hypothesizing that the non-tumorigenic fibroblast cellular components induce a difference in replication efficacy between the mtDNAs with and without the A3243G mutant sequence, which may be related to the decrease of disease-related mutant mtDNA in the hybrid cells.
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PMID:Loss of mutant mitochondrial DNA harboring the MELAS A3243G mutation in human cybrid cells after cell-cell fusion with normal tissue-derived fibroblast cells. 1995 14

Lesch-Nyhan disease (LND) is a severe and incurable X-linked genetic syndrome caused by the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT), resulting in severe alterations of central nervous system, hyperuricemia and subsequent impaired renal functions. Therapeutic options consist in supportive care and treatments of complications, but the disease remains largely untreatable. Enzyme replacement of the malfunctioning cytosolic protein might represent a possible therapeutic approach for the LND treatment. Protein transduction domains, such as the TAT peptide derived from HIV TAT protein, have been used to transduce macromolecules into cells in vitro and in vivo. The present study was aimed to the generation of TAT peptide fused to human HPRT for cell transduction in enzyme deficient cells. Here we document the construction, expression and delivery of a functional HPRT enzyme into deficient cells by TAT transduction domain and by liposome mediated protein transfer. With this approach we demonstrate the correction of the enzymatic defect in HPRT deficient cells. Our data show for the first time the feasibility of the enzyme replacement therapy for the treatment of LND.
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PMID:HIV-1 TAT-mediated protein transduction of human HPRT into deficient cells. 2412 87

Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.
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PMID:Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells. 2866 80


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