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)

A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a Delta hpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of Delta proC Delta hpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of beta-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina: A >5,000-fold range of promoter activities could be measured by using uidA: the methyl-coenzyme M reductase operon fusion displayed approximately 300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.
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PMID:Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea. 1500 62

Gene expression changes are used with increasing frequency to assess the effects of exposure to environmental agents. Housekeeping (Hk) genes are essential in these analyses as internal controls for normalizing expression levels evaluated with Real-Time PCR (RT-PCR). Ideal Hk genes are constitutively expressed, do not respond to external stimuli and exhibit little or no sample-to-sample or run-to-run variation. Previous studies indicate that some commonly used Hk genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin have differential expression in various cell lines. Here we examine the expression of 11 Hk genes in four normal human lymphoblastoid cell lines and one T-cell leukemia (Jurkat) cell line following exposure to graded doses of ionizing radiation or to varying ratio concentrations of phytohemagglutinin (PHA) and phorbol myristate acetate (PMA). PHA and PMA are known to have synergistic effects on the expression of some genes and have very different effects from those of radiation. There has been no systematic study performed to ascertain the best control genes for radiation and/or PHA/PMA exposures in lymphoblastoid cells. Using a two-step reverse-transcriptase RT-PCR protocol we show that following radiation doses ranging from 0 to 400 cGy, 18S rRNA, acidic ribosomal protein, beta-actin, cyclophilin, GAPDH, phosphoglycerokinase, beta-2 microglobulin (B2M), beta-glucuronidase, hypoxanthine phosphoribosyltransferase and transferrin receptor showed no significant variation in expression in normal lymphoblastoid cells. In contrast, only 18S rRNA levels were unchanged in Jurkat cells. After PHA/PMA treatment of the same normal cell lines, B2M showed no significant variation and 18S rRNA, GAPDH and transcription binding protein (TBP) were minimally responsive, whereas in Jurkat cells all these genes were unresponsive. While our results suggest that the utility of a particular Hk gene should be determined for each experimental condition, 18S rRNA and B2M appear to be excellent candidates for use as internal controls in RT-PCR in human lymphoblastoid cells because they have the most constant levels of expression across cell lines following exposure to ionizing radiation as well as to PHA/PMA.
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PMID:Evaluation and validation of housekeeping genes in response to ionizing radiation and chemical exposure for normalizing RNA expression in real-time PCR. 1790 13

The selection of stable endogenous control genes is critical for normalization of quantitative real-time PCR (qPCR) data. In this study, we aimed to identify a suitable set of control genes to be used as endogenous references for gene expression evaluation in human peripheral blood samples among coronary artery disease patients. The expression levels of 12 endogenous control genes procured from TATAA Biocenter (Goteborg, Sweden) were measured in five acute coronary syndrome patients and five chronic stable angina patients. Gene expression stability was analyzed using two different software applications i.e geNorm and NormFinder. Results suggested that beta-glucuronidase is the most stable endogenous control, followed by hypoxanthine-guanine phosphoribosyltransferase. The NormFinder analysis further confirmed that beta-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase were on the first rank order with the most stable expression among endogenous control genes analyzed and 60S acidic ribosomal protein P0. Besides this, the expression levels of 18S rRNA were revealed to be highly variable between coronary heart disease patients. We thus recommend the use of beta-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase as reference genes for accurate normalization of relative quantities of gene expression levels in coronary artery disease patients using qPCR. Also the use of 18S rRNA as a control gene should be avoided.
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PMID:[Identification of endogenous control genes for gene expression studies in peripheral blood of patients with coronary artery disease]. 2380 54