Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
 2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine if expression of genes on the inactive X is inducible in human cells, we looked for reactivation events in a clone of fibroblasts transformed with origin-defective SV40. The karyotype of these cells was grossly heteroploid so that the aneuploidy associated with SV40 transformation occurs even in the absence of viral replication. This transformed clone, heterozygous for hypoxanthine phosphoribosyltransferase (HPRT), lacks HPRT activity, as the mutant allele is on the active X and the normal allele on the inactive X. Reactivation of the HPRT+ allele on the inactive X was observed at a frequency of 6 X 10(-5) per cell and increased approximately eightfold following treatment with the cytidine analogs 5-azacytidine (5azaC) and 5-azadeoxycytidine. The fact that spontaneous reactivation is detectable in some clones, but not all, suggests that the environment of the SV40-transformed cell, although not sufficient to induce generalized derepression, increases the frequency of rare reactivation events. The methylation pattern at the HPRT locus revealed transformation-associated alterations that may have predisposed these cells to reactivation events, spontaneous as well as 5azaC-induced.
Somat Cell Mol Genet 1986 Nov
PMID:Reactivation of X-linked genes in human fibroblasts transformed by origin-defective SV40. 302 33

Long-range-acting gene activator elements were randomly isolated from the human genome by functional selection. HeLa cells were transfected with an enhancer trap, a plasmid containing an enhancerless xanthine-guanosine phosphoribosyltransferase (gpt) gene transcribed from the simian virus 40 early promoter, and stably transformed GPT+ cells were selected. From several transformants, human DNA sequences flanking the enhancer trap were cloned. Two gene activators (GA1 and GA2) were found in the cloned human DNAs. GA1 and GA2 showed strong enhancer activity both in a stable transformation assay and in a transient expression assay. They had functional properties similar to those of other known enhancers: GA1 and GA2 activated the expression of a linked gene over distances of at least 5 kilobases both upstream and downstream in an orientation-independent fashion. GA1 may be required for the initial establishment of gene activation but was not essential for the maintenance of active expression. GA1 and GA2 were active not only in HeLa cells but also in other types of human cells, such as neuroblastoma cells. This indicates a limited but relatively broad cell type specificity. The HeLa genome contains multiple copies of GA1, while GA2 exists once in the genome.
Mol Cell Biol 1986 Dec
PMID:Random isolation of gene activator elements from the human genome. 302 43

DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GC----AT transitions, 4/30 were AT----GC transitions, 3/30 were AT----TA transversions, and 1/30 was an AT----CG transversion. We observed that 37/40 HENU-induced mutations were GC----AT transitions and that the remaining 3/40 were AT----GC transitions. A majority of the GC----AT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5'-GG(A or T)-3'; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the G----A changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The AT----GC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5'-NTC-3'. A strand preference was not apparent for these mutations.
Mol Gen Genet 1988 Nov
PMID:Mutation spectra of N-ethyl-N'-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli. 306 48

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.
Mol Cell Biol 1987 Feb
PMID:Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses. 310 47

The metabolic activation of promutagens by freshly isolated and cryopreserved rat hepatocytes was compared using the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyltransferase assay (CHO/HGPRT). Cryopreserved rat hepatocytes were equivalent to freshly isolated hepatocytes in their ability to metabolize dimethylbenz(a)anthracene (DMBA) and dimethylnitrosamine (DMN) to active mutagens. Similar dose-response curves were observed using either freshly isolated or cryopreserved hepatocytes as activating systems after treatment with DMBA (0.1-1 micrograms/ml) and DMN (0.075-0.6 mg/ml). Our results suggest that cryopreserved hepatocytes are similar to freshly isolated hepatocytes as an experimental system for studies on promutagen activation.
Environ Mol Mutagen 1988
PMID:Promutagen activation by freshly isolated and cryopreserved rat hepatocytes. 313 8

We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.
Mol Cell Biol 1985 May
PMID:Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells. 315 4

A series of stable mutants bearing nuclear genetic markers were developed from the established chicken cell line DU24. The mutants were obtained after mutagenesis of DU24 cells with ethyl methanesulfonate (EMS) or arose spontaneously when plated in the appropriate selective medium. Clones resistant to 5-bromodeoxyuridine (BrdU) were obtained following a two-step selection procedure and analyzed. The BrdUr cells were found to be deficient in thymidine kinase activity and were HAT sensitive. Molecular characterization of these mutants revealed no deletions or other rearrangements, but methylation of some cytosine residues was decreased in the mutants. A similar restriction profile was seen in a series of mutants made resistant to BrdU after cultivation of DU24 cells in increasing concentrations of the drug over a period of six months. Selection of EMS-treated BrdUr cells in 10 microM ouabain gave rise to a clone resistant to both drugs and which was still HAT sensitive. Clones resistant to 6-thioguanine were also isolated, but showed wild-type hypoxanthine phosphoribosyltransferase activity and were HAT resistant. A number of the cell lines isolated were found to be suitable for fusion experiments with both chicken cells and cells from other vertebrate species.
Somat Cell Mol Genet 1988 May
PMID:Development and characterization of mutant chicken cell lines for somatic cell genetics studies. 316 27

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.
Mol Cell Biol 1988 Oct
PMID:Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells. 318 46

The pathways of adenine nucleotide catabolism were investigated in cultured beating cardiomyocytes. The activity of the enzymes involved in AMP degradation was assayed in cell extracts. Fluxes of label from ATP to the various purine derivatives were measured in intact cells. Under physiological conditions, cells degraded AMP through deamination to IMP. IMP was rapidly degraded to inosine, hypoxanthine, xanthine and uric acid, which were effluxed from the cells. This is in accord with the fact that the activity of AMP deaminase (EC 3.5.4.6) was 7-fold that of AMP 5'-Nucleotidase (EC 3.1.3.5). Mild ATP-degradation, induced by inhibition of glycolysis by iodoacetate, caused no alterations in the degradation pathways (more than 85% through deamination to IMP). However, fast ATP-degradation (83% of adenine nucleotides/10 min), induced by simultaneous inhibition of glycolysis and electron transport (by antimycin A), caused increased dephosphorylation of AMP to adenosine (50% of total AMP-degradation). The cardiomyocyte extracts were found to contain a significant activity of purine nucleoside phosphorylase (EC 2.4.2.1). Despite the presence of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), salvage of hypoxanthine to IMP, both at physiological as well as at conditions associated with ATP degradation, was slow. The salvage of adenosine appeared to be efficient at physiological conditions, but not at fast rates of ATP degradation.
J Mol Cell Cardiol 1988 Jan
PMID:Pathways of adenine nucleotide catabolism in primary rat cardiomyocyte cultures. 325 63

The localization of the A-raf cellular oncogene on the mouse X chromosome has been determined using Xbal-restricted DNAs prepared from progeny of an interspecies backcross between the B6.CBA.R1 and the Spe/Pas mouse strains. This localization to the proximal part of the mouse X chromosome has been confirmed by the use of somatic cell hybrids, carrying partially deleted X chromosomes and suggests that the A-raf oncogene localizes to a region lying some 10-17 centimorgans proximal to the hypoxanthine phosphoribosyltransferase (Hprt) gene between the locus DXPas4 and the locus DXPas7 defined by the cross-reacting human X chromosome-specific probe DXS32 (M2C). This localization on the mouse X chromosome is compatible with the presence of the A-raf oncogene on the short arm of the human X chromosome between the centromere and Xp21.
Somat Cell Mol Genet 1987 May
PMID:A-raf oncogene localizes on mouse X chromosome to region some 10-17 centimorgans proximal to hypoxanthine phosphoribosyltransferase gene. 329 48


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