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)

Somatic cell hybrids were constructed between BALB/c-RAG mouse cells and feline lymphoma cells by the hypoxanthine-aminopterin-thymidine selection scheme. RAG cells spontaneously produce an endogenous B-tropic type C virus. Cat-mouse hybrids preferentially segregate feline chromosomes and retain murine chromosomes-demonstrable by karyotypic and isozyme analyses. Despite the presence of the complete mouse genome, including the viral genome, virus production was diminished to 1-5% of the levels observed in RAG parents based upon particle-associated RNA-dependent DNA polymerase (reverse transcriptase) activity in the culture fluid. Thirty-seven hybrids made on four different occasions had suppressed virus levels, and no hybrids expressed parental virus levels. Reverse selection experiments on 6-thioguanine demonstrated that a restriction gene, tentatively named Bvr-1, was linked to the feline structural genes for hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase; EC 2.4.4.8) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) in cats, probably on the X-chromosome. The genetic mode of action of Bvr-1 is trans dominant in restriction of murine leukemia virus. The restriction locus results in a block late in virus maturation but prior to release, since expression of antigens for viral structural proteins and matrue budding particles is apparent on surfaces of restriced hybrid cells but not in high-speed pellets from culture fluid of restricted cells.
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PMID:Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: identification, location, and phenotypic characterization in cat X mouse somatic cell hybrids. 6 49

To gain insight into the mechanisms by which mutations are induced in human cells by carcinogens, we have determined the kinds and location (spectrum) of mutations induced in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene by (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Individual populations of diploid human fibroblasts were treated with BPDE, or were left untreated (control). After a suitable expression period, the progeny cells were selected for resistance to 6-thioguanine. Individual drug-resistant colonies were isolated, and the mRNA in the lysate of 100-400 cells from each colony was copied directly into cDNA using reverse transcriptase. The cDNA of the HPRT gene of 29 unequivocally independent mutants from BPDE-treated populations and 13 from the control populations was amplified 10(11)-fold, and the product was sequenced directly. Twenty-three of the 29 BPDE-induced mutants examined contained a single base pair substitution; four exhibited two base pair substitutions. Eight out of 13 control mutants exhibited base pair substitutions, and four others were missing a complete exon. Thirty of the 32 base pair substitutions in the BPDE-induced mutants involved G.C base pairs, primarily G.C----T.A transversions. The majority (89%) of the base pair substitutions observed in the mutants from the control population involved an A.T base pair. Base substitutions were found throughout the coding region of the gene, but 41% of those seen in mutants from the BPDE-treated population and 44% of those from the untreated population were located in the first half of exon 3.
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PMID:Kinds and location of mutations induced by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human fibroblasts. 189 56

Human immunodeficiency virus (HIV) infection was studied by means of CD4-expressing human-murine T-cell hybrids, containing a variable amount of human chromosomes. Fusion of the HPRT- murine cell line BW5147 with human T-cell acute lymphoblastic leukemia or normal human blood cells resulted in a panel of human-murine T-cell hybrids. For this study, we used four hybrids containing all or several human chromosomes, which all expressed the CD4 antigen, as assessed by different anti-CD4 monoclonal antibodies (e.g., OKT4A, Leu-3a, and MT151) and, in addition, a variable number of other human T-cell antigens. For infection, HTLV-IIIB-infected H9 cells, pretreated with mitomycin C, and cell-free concentrated supernatants from these cells were used. In cells of inoculated cultures of the CD4+ T-cell hybrids, no viral antigen could be demonstrated. Culture supernatants of inoculated hybrids, except for an initial rise due to the virus inoculum, never showed reverse transcriptase activity above background. Cocultivation of these cell cultures with H9 cells did not result in detectable virus replication. Cocultivation of CD4-expressing hybrid cells with HIV-infected cells did not result in syncytium formation. Moreover, these hybrids were resistent to infection with vesicular stomatitis virus (VSV)-HIV pseudotypes. These findings imply that expression of the CD4 antigen on the cell surface is not sufficient for productive infection with HIV. The infectivity block observed in these hybrids seems to occur at the level of virus penetration, presumably at the stage of membrane fusion events.
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PMID:Human immunodeficiency virus infection studied in CD4-expressing human-murine T-cell hybrids. 246 72

Drug-specific monoclonal antibodies and their antigen-binding Fab fragments reverse acute desipramine toxicity in a rat experimental model by inducing a redistribution of drug from cardiac tissue into serum and extracellular fluid. In order to investigate the use of smaller recombinant antibody fragments such as single chain Fv (sFv) as an antidote, an efficient murine NS/O myeloma expression system was developed. The variable light (VL) and variable heavy (VH) domains of a murine anti-desipramine monoclonal antibody were cloned and sequenced. A 270 amino acid VH-(Gly4Ser)3-VL sFv was prepared by overlapping polymerase chain reaction (PCR) amplification of VH with heavy chain leader peptide, VL, and the linker. This construct was subcloned into a mammalian expression vector which utilizes the SR alpha promoter, a hybrid promoter consisting of the SV40 early promoter with portions of the human T-cell leukemia virus type I long terminal repeat and also containing the Escherichia cloi xanthine-guanine phosphoribosyltransferase gene for selection. NS/O myeloma cells were transfected by electroporation. Stable recombinant NS/O clones were screened for expression of sFv using reverse transcriptase-PCR to detect mRNA and an enzyme-linked immunosorbent assay (ELISA) to detect sFv. Secreted sFv from clones capable of growth to a cell density of 2-4 x 10(6) viable cells/mL was purified in a single step using a desipramine affinity column resulting in 12-39 mg/L of purified sFv. Affinity-purified sFv had comparable desipramine binding activity to Fab when evaluated by competitive ELISA.
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PMID:Cloning, expression, and purification of an anti-desipramine single chain antibody in NS/O myeloma cells. 880 32

The cadherins are a family of calcium-binding membrane glycoproteins. Most cadherins are capable of acting as cell adhesion molecules (CAMs). In order to begin a thorough analysis of the roles of these CAMs in the testis, we employed a reverse transcriptase-polymerase chain reaction (RT-PCR) strategy to identify the cadherins expressed in this tissue at various stages of development. Oligonucleotides encoding amino acid sequences that are conserved among all of the known cadherins were used as primers in the RT-PCR, with cDNA preparations of fetal, newborn, 7-day, 21-day, and adult mouse testes employed as templates. The PCR products were subcloned into a plasmid vector and sequenced. On the basis of the nucleotide sequences of these PCR products, we have determined that five previously characterized cadherins (E-cadherin, N-cadherin, P-cadherin, K-cadherin, and OB-cadherin), as well as two novel cadherins (T1-cadherin and T2-cadherin), are expressed at various stages during testicular development. In order to determine the expression patterns of these cadherins, we ascertained the mRNA levels of each cadherin normalized to the levels of hypoxanthine phosphoribosyltransferase mRNA in fetal, newborn, 7-day, 21-day, and adult mouse testes. We observed that N-cadherin mRNA is expressed at all stages of testicular development, with maximal levels being present in the testes of 21-day-old mice. Furthermore, we found that E-, P-, K-, OB-, and T2-cadherin mRNAs are all expressed in the fetal gonad. The testicular levels of these cadherin mRNAs decreased dramatically after birth. Conversely, T1-cadherin mRNA was not detected in the fetal, newborn, and 7-day-old testes but was present in 21-day-old and adult testes. T1-cadherin levels were 10-fold higher in the testes of adult mice, compared to the levels found in the testes of 21-day-old mice. We speculate that these cadherins will be found to be intimately involved in mediating cell interactions during testicular development.
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PMID:A comprehensive survey of the cadherins expressed in the testes of fetal, immature, and adult mice utilizing the polymerase chain reaction. 887 95

The genome of the Australian marsupial Macropus robustus contains a highly conserved processed hypoxanthine phosphoribosyltransferase homologue, HPRT-2. Using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and protein isoelectric focusing (IEF) we have shown this processed gene to be fully functional, but liver specific. In contrast, the unprocessed X-linked parent gene HPRT-1 was expressed in all somatic tissues. Expression of the HPRT-2 gene effectively doubles the total HPRT enzyme activity in liver compared to other tissues. Analysis of the 5'-flanking sequence of HPRT-2 revealed regions with homology to the liver-specific regulatory motifs C/EBP, NF-IL6, LF-A1 and LF-B1, although the functional significance of these regions remains unknown. Consistent with X chromosome inactivation in female mammals, transcript levels of the unprocessed X-linked gene HPRT-1 were similar in males and females in all tissues examined. No HPRT-2 activity was detected in testes, indicating that this gene does not compensate for sex chromosome inactivation during spermatogenesis. Moreover, the demonstration of very high HPRT-1 enzyme levels in testes indicated that such a compensatory mechanism may not be required. Phylogenetic analyses attribute considerable antiquity to the processed gene and PCR with conserved primers spanning exons 4-8 of genomic DNA from several different kangaroo species inferring the existence of a conserved processed HPRT-2 homologue in these marsupial species. However, no such conserved PCR product was obtained with DNA from eutherian species, suggesting that integration of HPRT-2 occurred after the separation of the metatherian and eutherian lineages.
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PMID:Identification of a novel tissue-specific processed HPRT gene and comparison with X-linked gene transcription in the Australian marsupial Macropus robustus. 904 50

Expression and androgen regulation of the gene for neuronal nitric oxide synthase (NOS I) were examined in neurons of the major pelvic ganglia in male rats. Some of these postganglionic neurons innervate the penis and produce nitric oxide, which is believed to play a major role in penile erection. Rats were either castrated or sham operated and implanted with SILASTIC brand capsules filled with powdered testosterone (T) or 5alpha-dihydrotestosterone (5alphaDHT) or left empty. After 4 days, the number of neurons intensely stained for NADPH-diaphorase as well as those giving a NOS I signal in in situ hybridization experiments increased in castrated rats treated with testosterone by 31% and 42%, respectively, relative to those in untreated castrated rats. This suggests that the increase in NADPH-diaphorase activity resulted from enzyme synthesis and was due to a modification of NOS I messenger RNA (mRNA) accumulation. After 7 days, Northern blot analysis showed that castration produced a decrease in the amount of NOS I mRNA relative to that of ribosomal RNA. This decrease was almost prevented by T treatment. No significant differences were observed by reverse transcriptase-PCR between 7-day and 28-day treatments. However, in 7-day castrated rats treated with 5alphaDHT, NOS I signals relative to those of hypoxanthine phosphoribosyltransferase, taken as reference, were significantly higher than those in castrated rats and resembled those in sham-castrated rats, suggesting that 5alphaDHT was probably more potent than testosterone in preventing the decrease in NOS I mRNA levels elicited by castration. These results show that NOS I can be positively regulated by androgens and are consistent with the suggestion that these steroids play a role in the physiological processes of penile erection.
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PMID:Androgens modulate nitric oxide synthase messenger ribonucleic acid expression in neurons of the major pelvic ganglion in the rat. 923 55

We measured the frequency of mutant (MF) lymphocytes at the hprt locus in a population of 43 coke-oven workers exposed to PAH and in a group of 26 non-exposed workers. A non-significant increase in MF in the exposed group (19.0 +/- 16.3) compared to the non-exposed group (15.8 +/- 14.6) was observed. Moreover, when we considered smoking habits for the overall population, the MF values were higher, although not significantly, in smokers than in non-smokers. For some T-cell mutant clone structural alterations, splicing and coding errors were detected by PCR-based methods. We analysed 161 HPRT- clones, derived from exposed and non-exposed workers by multiplex-PCR and 56 HPRT- clones by reverse transcriptase-PCR. Overall, the percentages of the different types of gene alterations were similar in exposed and non-exposed subjects. Only the frequency of splice mutations in mutant clones derived from coke-oven workers was higher (22%) than in non-exposed donors (11%).
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PMID:Determination of HPRT mutant frequency and molecular analysis of T-lymphocyte mutants derived from coke-oven workers. 953 72

Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.
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PMID:Regulation of hypoxanthine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNA expression in porcine immune cells and tissues. 967 36

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.
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PMID:Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum. 980 51


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