Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

X chromosome inactivation is associated with a highly asynchronous pattern of DNA replication at most X-linked loci in females. We studied the human HPRT locus, which is subject to X inactivation and expressed from only the active homolog, with the goal of comparing replication properties between the active and inactive homologs in this region using a fluorescence in situ hybridization approach. We found that in normal female lymphoblasts this locus is replicated in a highly asynchronous manner across a broad, discrete 500-600 kb zone with earliest replication appearing at the gene coding sequence. This general timing profile is maintained in normal male lymphoblasts, as well as in hamster x human hybrid cells containing the active human X chromosome. However, the inactive human X chromosome in the hamster cell background does not appear to function in a fully equivalent manner to the normal inactive X chromosome in female cells. Furthermore, reactivation of the inactive human X chromosome in a hamster x human hybrid system by 5-azacytidine treatment and HAT selection restores early replication at the HPRT gene itself, but does not change the overall domain behavior.
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PMID:Replication timing properties of the human HPRT locus on active, inactive and reactivated X chromosomes. 933 Jun 38

The latent effects of radiation-induced damage include "delayed" mutations that arise de novo in the progeny of nonmutant cells. We investigated the early stages of delayed mutagenesis at the HPRT locus of EJ30 human epithelial cells that were exposed to 4 Gy of 137Cs gamma rays. To eliminate directly induced "prompt" HPRT- mutants, cultures were grown in HAT medium before selection in 6-thioguanine was applied. Although irradiated cells were grown in HAT medium throughout the phenotypic expression period, mutant fractions some tenfold above spontaneous levels were observed subsequently; incubation in HAT medium did not cause an increase in mutations in unirradiated cells. We conclude that, in our experimental system, a significant proportion of induced mutation is of a delayed type. We speculate that the delayed induction is caused by an instability process that is a frequent and (typically) transient consequence of exposure of cells to ionizing radiation. The connection, if any, between this process and other manifestations of instability, including the acquisition of a "mutator phenotype," remains to be established.
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PMID:Postirradiation growth in HAT medium fails to eliminate the delayed appearance of 6-thioguanine-resistant clones in EJ30 human epithelial cells. 945 97

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

Variable gene expression amongst transgenic lines occurs due to copy number and to random associations of incoming DNA with chromosomal elements at the site of integration. Here we describe a method of identifying sites permissive for transgene expression and their use for efficient introduction of single copy transgenes by homologous recombination. ES clones were selected in HAT medium for expression of a randomly integrated HPRT marker lying 5' to an Oct4/ lacZ transgene. 794 clones were assessed in vitro for appropriate down-regulation of lacZ following differentiation. Two clones were chosen for further analysis which displayed appropriate and inappropriate gene regulation (clones 710 and 91, respectively). Three developmental promoters (thyroglobulin, Hox2.6 and Myf5) were then sequentially introduced into the original insertion sites in each clone (710 and 91) by homologous recombination, to drive expression of lacZ. Transgenic embryos were assessed for their ability to direct lacZ expression to tissues in which the respective promoter sequences are normally active. The site which appropriately down-regulated lacZ in vitro (710) also showed appropriate in vivo regulation of lacZ from the three developmental promoters. Site 91, however, directed an additional pattern of ectopic expression, which was common to all four promoters. Pre-selection of genomic sites for the introduction of transgenes by gene targeting improves the repeatability of transgene expression and provides an efficient means of single copy transgene introduction by homologous recombination.
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PMID:Pre-selection of integration sites imparts repeatable transgene expression. 1068 42

The Lesch-Nyhan syndrome is an X-linked disorder caused by a virtually complete absence of the key enzyme of purine recycling, hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is characterized by uric acid overproduction and severe neurological dysfunction. No treatment is yet available for the latter symptoms. A possible long-term solution is gene therapy, and recombinant adenoviruses have been proposed as vectors for gene transfer into postmitotic neuronal cells. We have constructed an adenoviral vector expressing the human HPRT cDNA under the transcriptional control of a short human cytomegalovirus major immediate early promoter (RAd-HPRT). Here we show that infection of human 1306, HPRT-negative cells with RAd-HPRT, expressed high enough levels of HPRT enzyme activity, as to reverse their abnormal biochemical phenotype, thus enhancing hypoxanthine incorporation and restoring purine recycling, increasing GTP levels, decreasing adenine incorporation, and allowing cell survival in HAT medium in which only cells expressing high levels of HPRT can survive. Infection of murine STO cells, increased hypoxanthine incorporation and restored purine recycling, thus allowing cell survival in HAT medium, and reduced de novo purine synthesis. Although both cells were able to survive in HAT medium post infection with RAd-HPRT, some of the biochemical consequences differed. In summary, even though adenoviral vectors do not integrate into the genome of target HPRT-deficient human or murine cells, RAd-HPRT mediated enzyme replacement corrects abnormal purine metabolism, increases intracellular GTP levels, and allows cells to survive in a negative selection medium.
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PMID:Adenoviruses encoding HPRT correct biochemical abnormalities of HPRT-deficient cells and allow their survival in negative selection medium. 1085 May 48

Marked differences between the mutagenic efficiency of N-methyl-N-nitrosourea (MNU), a potent carcinogen, methyl methane sulphonate (MMS) and dimethyl sulphate (DMS), both weak carcinogens, have been reported at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and ouabain loci in V79 cells. Differences in levels of O6-guanine methylation produced by these alkylating agents, has been interpreted as indicating that O6-methylguanine is the DNA lesion specifically responsible for their mutagenic and carcinogenic effects. Because of the heterogeneity of molecular events which can result in forward mutation this conclusion seems unjustified. The development and characterisation of a reverse assay from 6-thioguanine resistance and HAT medium sensitivity (TG(R) and HAT(S)), to 6-thioguanine sensitivity and HAT medium resistance (TG(S) and HAT(R)) HGPRT(-)-->HGPRT+ in V79 cells, has allowed us to test the above hypothesis in a more specific way. Ethyl methane sulphonate, a weak carcinogen and MNU, both of which produce significant levels of O atom alkylation, were similarly effective mutagens in the reverse direction. At equitoxic doses, DMS was 40-60 fold less efficient. There was however, no quantitative correlation between numbers of revertants induced and measured levels of O6-alkylguanine. From these and other observations it is concluded that O6-alkylguanine is not the only potentially mutagenic lesion in mammalian cells.
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PMID:Evidence for the involvement of lesions other than O6-alkylguanine in mammalian cell mutagenesis. 1121 71

We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing alpha-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both alpha-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.
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PMID:Stable gene expression from a mammalian artificial chromosome. 1157 Dec 65

Exchange plasmid pF-HPRT-F3 and Flp expression plasmid pCMV-Flp were constructed and then introduced using electroporation system into F18 ES cell line, which have an exchange cassette F-Neo-F3 at HPRT locus. After HAT selection, HAT resistant clones were obtained. Then G418 sensitivity test and Southern blotting were carried out to screen RMCE recombinants. The results indicated that RMCE had taken place in three of 12 HAT resistant clones. The frequency is 25%. The result demonstrates that it is realizable to introduce transgene to HPRT locus by using Flp recombinase mediated cassette exchange reaction.
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PMID:[The study of a HPRT locus-specific transgenic method based on FLP recombinase mediated cassette exchange]. 1254 6

Primary mouse brain cells were cultured with HPRT (hypoxanthine phosphoribosyl transferase)-deficient ES (embryonic stem) cells to see if the ES cells could provide cues sufficient to reprogram a pluripotential state. After 5 days of coculture, HPRT-deficient ES cells were killed by selection in HAT (hypoxanthine, aminopterin, thymidine) medium. We observed islands of HAT-resistant ES-like cells surrounded by differentiated cells. Cell lines generated from three such "islands" proved to be spontaneous, pluripotential ES-neural hybrids, and gave rise to a chimera following blastocyst injection. Re-expression of the ES-specific gene Foxd3 from somatic-derived chromosomes suggested that the somatic nucleus had been reprogrammed. Our results raise the intriguing possibility that ASCs shown to contribute to multiple tissues in blastocyst-injection studies may not contribute as a result of pluripotency. Instead contributions may arise from spontaneous fusion events in which phenotype is determined by either cytoplasmic dominance, nuclear reprogramming, or both.
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PMID:Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro. 1263 Apr 12


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