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 utilized an AFP-HPRT transgene, i.e. the HPRT coding sequences under the regulation of AFP enhancer and promoter sequences, to localize the AFP extinguisher locus in intertypic somatic cell hybrids (hepatoma X fibroblast). This hybrid gene construct, which directly links AFP regulation to a reversibly selective gene, enabled the selection of stably transfected cells which express AFP, as well as cells showing extinction of AFP. Mouse hepatoma cells stably transfected with and expressing the transgene were fused to human fibroblasts, and the resulting somatic cell hybrids were characterized using Southern, Northern and karyotypic analyses. That several hybrids exhibited the proper extinction of AFP, AFP-HPRT and albumin suggests coregulation of these genes by an extinguisher. Segregant lines derived from these hybrids were selected for the loss of extinguisher activity and for reexpression of the transgene. Karyotypic analysis of hybrid and segregant lines, exhibiting proper AFP, albumin and AFP-HPRT phenotypes, revealed that the presence of human chromosome 7 was most closely associated with the AFP-extinguished state. The hybrids generated in these studies now make it possible to isolate the sequences responsible for AFP and albumin extinction.
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PMID:Characterization of somatic cell hybrids exhibiting extinction of AFP, albumin and an AFP-HPRT transgene. 878 91

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

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

The REV1 gene encodes a Y-family DNA polymerase that has been postulated to have both catalytic and structural functions in translesion replication past UV photoproducts in mammalian cells. To examine if REV1 is implicated in DNA damage tolerance mechanisms after exposure of human cells to a chemical carcinogen, we generated a plasmid expressing REV1 protein fused at its C-terminus with green fluorescent protein (GFP). In transient transfection experiments, virtually all of the transfected cells had a diffuse nuclear pattern in the absence of carcinogen exposure. In contrast, in cells exposed to benzo[a]pyrenediolepoxide, the fusion protein accumulated in a focal pattern in the nucleus in 25% of the cells, and co-localized with PCNA. These data support the idea that REV1 is present at stalled replication forks. We also examined the mutagenic response at the HPRT locus of human cells that had greatly reduced levels of REV1 mRNA due to the stable expression of gene-specific ribozymes, and compared them to wild-type cells. The mutant frequency was greatly reduced in the ribozyme-expressing cells. These data indicate that REV1 is implicated in the mutagenic DNA damage tolerance response to BPDE and support the development of strategies to target this protein to prevent such mutations.
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PMID:REV1 accumulates in DNA damage-induced nuclear foci in human cells and is implicated in mutagenesis by benzo[a]pyrenediolepoxide. 1552 96

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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