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Query: UNIPROT:P00492 (hypoxanthine-guanine phosphoribosyltransferase)
 2,385 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A significant and reproducible enhancement of purine nucleotide synthesis from hypoxanthine occurs in HAT medium, when communication-competent hypoxanthine-guanine phosphoribosyltransferase (HGPRT+) cells are co-cultured with communication-competent (HGPRT-) LN cells. This enhancement of purine nucleotide synthesis is dependent upon the hypoxanthine concentration and upon the ratio of (HGPRT-): (HGPRT+) cells. Based upon these results a simple biochemical method for the detection of inhibitors of metabolic cooperation between (HGPRT+) cells and (HGPRT-) LN cells is presented. The biochemical method distinguishes inhibitors of metabolic cooperation from inhibitors of hypoxanthine uptake, of hypoxanthine phosphorylation and of nucleic acid synthesis, as well as from general metabolic inhibitors. This method has the advantage that it can be used on a relatively large number of cells, it is simple and not time-consuming, and distinguishes the inhibition of metabolic cooperation by compounds that have a variety of sites of inhibition.
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PMID:Biochemical assay of inhibitors of metabolic cooperation. 685 23

A rapidly-growing, HAT-sensitive cell line LICR-LON-HMy2 has been derived from the ARH-77 human plasma cell leukemia-derived line. It lacks the enzyme hypoxanthine phosphoribosyltransferase (EC 2.4.2.8). Hybrids have been reproducibly made for more than a year and in independent laboratories with lymphocytes from tonsils, lymph nodes and peripheral blood and tonsil lymphocytes cultured with antigens. The parent line and hybrids are very robust in culture and double in 20-30 h. Hybrids clone easily and have stable karyotypes, most with modal numbers in the sixties. Stable Ig secretion patterns have been observed over 20-30 passages, and after cloning. It was estimated that about half the hybrids produce new immunoglobulin chains in addition to the parent cell line's IgG1 (kappa light chain). High, but not limiting, density hybridoma cultures (approximately 5 x 10(5) cells/ml) typically produce 0.25-2 micrograms/ml immunoglobulin per day, but some hybrids produce more. A high-secreting variant of LICR-LON-HMy2 has been derived. The LICR-LON-HMy2 line is human and is distinct from the HAT-sensitive human B cell-lines SKO-007 and GM 1500-6TG Al 2. It is available for distribution.
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PMID:A human-hybridoma system based on a fast-growing mutant of the ARH-77 plasma cell leukemia-derived line. 714 Aug 10

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

RJK39 is a clone of Chinese hamster cells carrying a mutation which inactivates hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and reduces the apparent molecular weight of the enzyme. Using mutagens, we have isolated subclones of RJK39 which will grow in the counterselective HAT medium. Some continue to be HGPRT-deficient and survived the selection because they are resistant to aminopterin. In all but one of the HGPRT-positive revertants, the molecular weight of the enzyme returned to the wild-type value. However, the phenotypes of several of those strains indicate they produce altered forms of HGPRT, and one can conclude that second-site mutations must be able to cause intragenic suppression of the original mutation in RJK39. One of the revertants is pseudotetraploid and functionally heterozygous at the HGPRT locus. Segregation studies with that clone localized the genes for HGPRT, glucose-6-phosphate dehydrogenase, and phosphoglycerate kinase to the short arm of the Chinese hamster X chromosome.
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PMID:Reversion of a mutation affecting the molecular weight of HGPRT: intragenic suppression and localization of X-linked genes. 719 35

Eight Chinese hamster clones (CHO-K1) growing at the 30 mg/ml concentration of 8-azaguanine (AG) were studied. Clones were differentiated by their resistance to AG and to 6-thioguanine, by their plating efficiency on HAT medium, and by the level of hypoxantine incorporation in cells. The differences in phenotypic properties were shown to be associated with variability in hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. HPRT Michaelis constant (KM) for hypoxanthine and phosphoribosylpyrophosphate, and maximal reaction rate (Vm) offered considerable differences between all the resistant clones and sensitive cells. The only possible reason of these differences is a change in the HPRT coding locus. According to the results of the analysis of B15-4b-4 subclones, phenotypic and HPRT activity differences are also connected with each other; however, all subclones have the same KM of HPRT as that of the parental clone. So, differences in HPRT activity (and in Vm) may reflect changes in the HPRT content in cells of subclones. Hence, phenotypic heterogeneity of AG-resistant clones is determined by the interaction of mutational changes in the HPRT locus, and hereditable changes of genetic activity, responsible for variation of HPRT quantity in cells.
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PMID:[Hereditary changes in gene activity as 1 of the causes of phenotypic heterogeneity in 8-azaguanine-resistant Chinese hamster cells (CHO-K1)]. 731 48

Cellular resistance to 6-thioguanine is almost always associated with a complete loss of hypoxanthine phosphoribosyltransferase (HPRT) activity, while resistance to 8-azaguanine has frequently been shown to occur independently of any changes in the HPRT activity. As a result, mutant cells selected for resistance to 6-thioguanine are also resistant to 8-azaguanine, but cells selected for 8-azaguanine resistance are not necessarily cross-resistant to 6-thioguanine. Our previous studies demonstrated that this difference is due to differential utilization of the two purine analogs by HPRT in the cells. In this paper we describe a novel selective procedure for the systematic isolation of cellular mutants that contain wild-type levels of HPRT, are sensitive to 6-thioguanine, and yet are able to survive in medium containing both HAT and a high level of 8-azaguanine. We also present evidence which shows that such mutants arise through a mutation that specifically alters the HPRT molecule so that the enzyme no longer recognizes 8-azaguanine as a substrate, while remaining catalytically functional with hypoxanthine and 6-thioguanine. Mutants of this type may be useful as a marker in gene-transfer experiments.
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PMID:Isolation of somatic cell mutants with specified alterations in hypoxanthine phosphoribosyltransferase. 739

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT Utrecht or mutant cDNA with HPRT Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.
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PMID:Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line. 811 42

Chinese hamster cell clones of independent origin, which were resistant to purine base analogs and induced by the activated c-Ha-ras1 oncogene, were isolated. It was shown that the isolated clones stably retained resistance after cultivation on a medium without an analog, confirming mutational nature of the resistance. Most of the clones are able to grow on the HAT medium, retaining partial activity of the hypoxanthine phosphoribosyltransferase enzyme (HPRT); i.e., they are leaky mutants. Analysis by blot-hybridization did not reveal the presence of human ras-sequences in any of the mutants studied. Evidently, the mutagenic action of the oncogene is not insertional, and resistance is not linked to the stably integrated oncogene. The mutagenic effect of c-Ha-ras1 is likely to be of the "hit-and-run" type.
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PMID:[Characteristics of mutants induced by the c-Ha-ras1 oncogene and the nature of the oncogene's mutagenic action]. 860 5

Mitotic recombination is believed to play an important role in the development of many cancers. An improved system has been developed to detect reversion of an intragenic DNA duplication, as a model for intrachromosomal homologous recombination. The 'LNtd' strain of human fibroblasts, derived from a Lesch-Nyhan donor, produces no detectable hypoxanthine phosphoribosyltransferase (HPRT) activity due to a 13.7-kilobase-pair DNA insertion duplicating exons 2 and 3 of the HPRT locus. These cells are therefore sensitive to selection in HAT medium, against cells lacking functional HPRT enzyme. Clonal reversion to HAT resistance occurs spontaneously at 1-3 x 10(-5)/cell/generation, and can be induced by brief exposure to a variety of carcinogenic agents. Six known carcinogens, including two (diethylstilbestrol and nickel chloride) which were non-mutagenic in Salmonella by Ames HIS-reversion tests, showed dose-dependent induction of LNtd reversion by a maximum of 2.4- to > 11-fold over controls (each p < 0.01). In contrast, 5 non-carcinogenic agents, including two 'Ames-positive' chemicals, sodium azide and 8-hydroxyquinoline, evoked no more than a 1.7-fold increase in reversion (not significant). The molecular events associated with reversion to HAT-resistance were characterized, relative to the parental strain, in HATR clones derived from either untreated or carcinogen-treated cells. Both the intron-3:intron-1 junction situated between the duplicated HPRT segments in LNtd cells (amplified by polymerase chain reaction), and a restriction fragment corresponding to the duplicated HPRT DNA (assessed by Southern-blot hybridization), were lost from the majority of HATR revertant clones, whether they arose spontaneously or following exposure to Cr(VI) or ultraviolet light. These results imply that HATR reversion is induced in LNtd cells by carcinogenic treatments, through a mechanism consistent with homologous recombination, and is highly concordant with induction of in vivo carcinogenesis by the same agents.
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PMID:Carcinogens stimulate intrachromosomal homologous recombination at an endogenous locus in human diploid fibroblasts. 950 87

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


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