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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)

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.
Mol Cell Biol 1988 Jul
PMID:Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells. 284 88

As part of our mouse model of somatic mutation, we have begun to characterize spontaneously occurring hypoxanthine phosphoribosyltransferase (HPRT) -deficient mouse lymphocytes. Lymphocytes were cloned by in vitro exposure of spleen cells from male C57B1/6 mice to the mitogen concanavalin A, conditioned medium containing lymphocyte growth factors, and thioguanine (TG), in a limiting dilution assay. The 17 TG-resistant clones recovered were all highly deficient in HPRT activity and were found by analysis of surface antigens to be representative of the major subclasses of T lymphocytes. Southern analysis of lymphocyte genomic DNA detected alterations of the hprt gene in 12/17 of the HPRT-deficient lymphocyte clones. Of these 12, 2/17 were lacking the entire hprt locus, 7/17 lacked part of the locus, and 3/17 had other, unidentified alterations.
Somat Cell Mol Genet 1987 Jul
PMID:Cloned mouse lymphocytes permit analysis of somatic mutations that occur in vivo. 284 75

X8/6T2, a hamster-human hybrid cell line which contains an inactive human X chromosome, was treated with 5-azacytidine and selected for derepression of hypoxanthine-guanine phosphoribosyltransferase. Clones were examined for coreactivation of the phosphoglycerate kinase gene (Pgk). Of 68 of these hybrids, approximately 20% expressed measurable human phosphoglycerate kinase (PGK) activity. A 600-base-pair region of the Pgk 5' CpG cluster was examined for the methylation status of eight CCGG sites (site 1 being 5'-most) in a number of PGK-negative and PGK-positive cell lines. The inactive X chromosome is normally methylated at all eight sites, and this was also true for the majority of X8/6T2 cells. However, several PGK-negative hybrids were demethylated in the site 3 to site 6 region. PGK activity correlated with demethylation at both sites 6 and 7. The data for PGK-positive and -negative hybrids indicate that demethylation at or near site 7 was necessary for reactivation of Pgk. Chromatin sensitivity to MspI digestion in the nuclei of male lymphoblastoid cells and several PGK-positive and PGK-negative hybrids was examined. PGK-positive cell lines were hypersensitive to digestion, while PGK-negative hybrids were resistant. Cleavage at sites 6 and 7 was observed in all PGK-positive cell lines at each MspI concentration examined. Sites 7 and 8 were less accessible to digestion than site 6. Cleavage in the site 2 to site 5 region was observable at the lowest MspI concentration. In most PGK-positive hybrids, a nonspecific endogenous nuclease detected the presence of a hypersensitive region spanning at least 450 base pairs, bounded at the 3' end near HpaII site 6. Nuclease hypersensitivity appears to be related to promoter activity, because sites 7 and 8 are in transcribed regions of the gene. These data indicate that specific sites within the CpG cluster have a dominant controlling influence over the Pgk promoter conformation and the transcriptional activation of Pgk.
Mol Cell Biol 1988 Nov
PMID:Demethylation of specific sites in the 5' region of the inactive X-linked human phosphoglycerate kinase gene correlates with the appearance of nuclease sensitivity and gene expression. 285 Apr 67

The enzymes that catalyse the salvage of purines in Entamoeba histolytica trophozoites have been surveyed. Adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), adenine phosphoribosyltransferase (PRTase) (EC 2.4.2.7), xanthine PRTase (EC 2.4.2.22) and hypoxanthine PRTase (EC 2.4.2.8) were all detected in cell homogenates but only at low activities, whereas AMP deaminase (EC 3.5.4.6) and guanine PRTase (EC 2.4.2.8) were not found. Phosphorylases (EC 2.4.2.1) active in both anabolic and catabolic directions were present and all nucleosides tested were phosphorylated by kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73). 3'-Nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were found, the former being mainly particulate. Nucleotide interconversion enzymes (adenylosuccinate lyase, EC 4.3.2.2; adenylosuccinate synthetase, EC 6.3.4.4; IMP dehydrogenase, EC 1.2.1.14; GMP synthetase, EC 6.3.5.2 and GMP reductase, EC 1.6.6.8) were not detected. The results suggest that in E. histolytica the main route of nucleotide synthesis is from the individual bases through the actions of phosphorylases and kinases.
Mol Biochem Parasitol 1986 Apr
PMID:Purine-metabolising enzymes in Entamoeba histolytica. 287 91

Studies on a cell line with amplified copies of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene and HPRT gene transfer experiments revealed the existence of a nonfunctional HPRT-related sequence in the mouse genome. This sequence was isolated and found to be a processed HPRT pseudogene. With the exception of a small internal deletion, the pseudogene is believed to comprise a complete reverse transcript of HPRT mRNA, although the 3' end of the pseudogene was lost in the cloning process. A probe from a region flanking the mouse pseudogene was used to investigate the evolutionary relationships of mammalian HPRT pseudogenes. The pseudogenes in mouse and Chinese hamster appear to have a common origin, but no homology to any of the four known human HPRT pseudogenes was detected. A pseudogene-linked restriction fragment length polymorphism was used to map the pseudogene to the distal end of mouse chromosome 17.
Somat Cell Mol Genet 1988 Jul
PMID:Characterization, evolutionary relationships, and chromosome location of processed mouse HPRT pseudogene. 289 12

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT- human lymphoblast colonies induced by eight repetitive 150 microM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli, DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism.
Environ Mol Mutagen 1988
PMID:Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli. 290 Jul 62

Hypervariable DNA sequences may be used as probes to derive DNA "finger-prints" for individuals. To assess the use of the human 33.15 and 33.6 probes (isolated by Jeffreys and coworkers) for characterizing cell lines of nonhuman origin, DNA from different stocks of Chinese hamster (CH) cells was screened. All CHO (ovary) sublines could be readily distinguished from CH-V79 sublines by their fingerprints, but where two stocks had been derived recently from the same line, their fingerprints were nearly identical. Similarly fingerprints of HPRT-deficient mutants derived from one cell stock were identical. A V79 x CHO fusion hybrid showed equal fingerprint band-sharing with each parent line, while early-passage diploid CH cells had a fingerprint closer to CHO than to V79. Thus these data introduce a simple means of typing cell lines to avoid cross-contamination, of checking cell hybrids, and of assessing the divergence of cell stocks from one another.
Somat Cell Mol Genet 1988 Nov
PMID:Fingerprinting cell lines: use of human hypervariable DNA probes to characterize mammalian cell cultures. 290 77

X-chromosome inactivation was investigated in human chorionic villi in the first trimester of pregnancy and cultured cells established from them. Expression of glucose-6-phosphate dehydrogenase (G6PD) was evaluated in these extraembryonic cells from four females heterozygous for the electrophoretic variants (AB) of G6PD. In each case the uncultured villi as well as derived cultured cells expressed the AB phenotype for G6PD with about equal intensity for the A and B bands. Single-cell-derived clones established from two of the four cases expressed either G6PD A or B. One clone expressing G6PD B was fused with mouse cells, and a hybrid clone retaining the inactive human X chromosome was isolated; there was no evidence of human G6PD expression in this clone retaining an inactive human X. DNA methylation in the first intron of the human gene for hypoxanthine phosphoribosyltransferase (HPRT) was evaluated in the four pairs of cultured villi and fetal cells. No differences were detected between the cultured villi and fetal cells as they all showed bands characteristic of an inactive X from somatic cells. These results show that there is no preferential inactivation of an X in the majority of cells that constitute human tertiary chorionic villi or in cultured cells derived from them. Long-term cultures established from chorionic villi appear to be no different from somatic cells with respect to X-chromosome inactivation.
Somat Cell Mol Genet 1989 Mar
PMID:X-chromosome inactivation in cultured cells from human chorionic villi. 292 38

Insulin gene transcription relies on enhancer and promoter elements which are active in pancreatic beta cells. We showed that adenovirus type 5 infection of HIT T-15 cells, a transformed hamster beta cell line, represses insulin gene transcription and mRNA levels. Using expression plasmids transiently introduced into HIT T-15 cells, we showed that adenovirus type 5 E1a transcription regulatory proteins repress insulin enhancer-promoter element activity as assayed with a surrogate xanthine-guanine phosphoribosyltransferase gene. We relate E1a repression of the insulin gene to other examples of repression of enhancer-dependent genes by E1a and discuss the possible relationship of this repression to insulin gene regulation.
Mol Cell Biol 1987 Mar
PMID:Repression of insulin gene expression by adenovirus type 5 E1a proteins. 295 90

With the aim of producing nonviral shuttle vectors for mammalian cells, we have constructed mouse mitochondrial DNA derivatives comprising the xanthine-guanine phosphoribosyltransferase gene as a selectable marker. Complete or subcomplete mitochondrial genomes were inserted into the plasmid pBB3 and transferred into hepatoma cells in order to generate, in vivo, new recombinant molecules. A second- and a third-generation vector, p12.2b and p delta respectively, were thus isolated for their ability to shuttle from mammalian cells to recA+ E. coli. Transfection of rodent fibroblasts and hepatoma cells showed that, contrary to our expectations, p12.2b and p delta are not self-replicating episomes; their shuttling from mammalian cells to recA+ E. coli is mediated by tandem integrated copies. The relevant property of p12.2b and p delta is a ubiquitous propensity to form head-to-tail multimeric structures when they integrate into mammalian host chromosomes. This ability is missing in pBB3 and appears only following the insertion of various mitochondrial or nuclear DNA fragments into the plasmid. These data are discussed in terms of homologous recombination and shuttling of integrated vectors.
Somat Cell Mol Genet 1985 May
PMID:Shuttling of integrated vectors from mammalian cells to E. coli is mediated by head-to-tail multimeric inserts. 298 36


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