Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hypoxanthine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes conversion of hypoxanthine and guanine to their respective mononucleotides. Because of its ubiquitous nature, HPRT is known as a "housekeeping" gene and has been frequently used as an internal control in reverse transcriptase-polymerase chain reaction (RT-PCR) quantification of cytokine mRNA. Cloning and sequencing of the gerbil HPRT cDNA sequence is an important step in the development of RT-PCR procedures in this model. Two forms of gerbil HPRT cDNA were isolated and molecularly characterized.
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PMID:Hypoxanthine phosphoribosyltransferase cDNA in gerbils (Meriones unguiculatus). 1009 10

In the human glutathione S-transferase (GST) mu gene family, homozygous deletion of GSTM1 is the null phenotype (frequency of approximately 50% in Caucasians). In the current study, GSTM1 status was determined in human cell lines using reverse transcriptase, polymerase chain reaction, and immunochemistry. Cell lines were challenged with a range of doses of styrene-7,8-oxide (SO) and then toxicity and genotoxicity were monitored. Toxicity was determined by growth in flasks and genotoxicity by cloning in microplates in the presence/absence of 6-thioguanine, to detect mutations at the hypoxanthine phosphoribosyltransferase (hprt) locus. A SO concentration-dependent decrease in survival was observed for all cell lines, with GSTM1-deficient lines being more sensitive. The IC(50)s of deficient and proficient cell lines were 0.45 and 0.55 mM SO, respectively. The difference between survival of GSTM1-deficient and -proficient cell lines approached statistical significance. The background mutation frequency of GSTM1-deficient cell lines was 2 x 10(-5), and that of GSTM1-proficient cell lines was 3 x 10(-6). GSTM1-deficient cell lines were significantly more sensitive than GSTM1-proficient cell lines to mutation induction for concentrations up to 2.5 mM SO (P < 0.001, regression analysis). These results suggest that cell lines containing metabolically competent GSTM1 are able to efficiently use GSTM1 to conjugate SO and reduce its hazard. This supports the epidemiological evidence that GSTM1 influences sensitivity to chemical carcinogenesis and subsequent risk of cancer induction.
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PMID:Role of glutathione S-transferase mu (GSTM1) in styrene-7,8-oxide toxicity and mutagenicity. 1142 77

Quantification of mRNAs from extremely small human samples remains a challenge. Requiring minimal amounts of tissue and no post-reaction manipulation, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is an attractive method to quantitatively assess the expression of rare mRNAs. We evaluated the applicability of the technique on RNA extracted from human endomyocardial biopsies and isolated cardiomyocytes, and compared the technique to the RT-competitive PCR approach. Primers and probes were designed to amplify the three subtypes of human beta -adrenoceptors (beta1-, beta2- and beta3 AR), as well as reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the oncogene ABL by real-time RT-PCR. Specific primers and a deleted competitor were synthetized to compare the quantitation of the beta 3 AR mRNA expression by RT-competitive PCR. We validated the technique on human cardiomyocytes either freshly isolated or selectively excised from fixed sections of human myocardium by Laser Capture Microdissection. The standard curves obtained for the cDNA's analysed showed mean slopes comprised between -3.3 and -3.7. Inter- and intra-assay variability of gene quantitation was reflected by mean values of the variance coefficients of Ct of 4.84+/-1.13% and 2.73+/-0.39% or 3.32+/-1.03% and 2.21+/-0.24% (corresponding to percent variances of copy numbers of 83.07+/-12.72% and 34.45+/-9.03% or 47.40+/-8.59% and 23.83+/-3.16%) for human beta3 AR and GAPDH genes, respectively. The expression of GAPDH, HPRT and ABL mRNA was characterized by a very low dispersion of individual values across cardiac pathologies, suggesting that these genes may be used as reference genes in quantitative PCR studies. Finally, we applied the technique to detect rare mRNAs, such as beta -AR mRNAs, from small human endomyocardial biopsies and even isolated cardiomyocytes. Real-time RT-PCR is appropriate to quantitate rare messenger RNAs, including in extremely small human tissue samples. This method appears very promising for futures studies of gene expression in several pathophysiological conditions, including heart failure.
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PMID:Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies. 1173 59

Enhanced linear growth, hyperplasia, and tumorigenesis are well-known characteristics of "viable yellow" agouti A(vy)/- mice (Wolff GL, Roberts DW, Mountjoy KG. Physiol Genomics 1:151-163, 1999); however, the functional basis for this aspect of the phenotype is unknown. In the present study, we ascertained whether agouti signaling protein (ASIP) levels in A(vy)/a or a/a livers are associated with hepatocyte proliferation as a possible factor in promotion of hepatocellular tumor formation. Proliferating cell nuclear antigen (PCNA) assays and quantitative real-time reverse transcriptase polymerase chain reaction assays were performed on liver samples from mottled yellow A(vy)/a, pseudoagouti A(vy)/a, and black a/a VY mice to determine mitotic indices and expression levels of A(vy )and a in relation to the expression level of the housekeeping gene hprt. We found that ASIP levels were approximately 100-fold higher in yellow than in pseudoagouti or black mice and that the proportion of PCNA-positive hepatocytes was greater (P < 0.001) in yellow than in pseudoagouti or black mice.
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PMID:Agouti signaling protein stimulates cell division in "viable yellow" (A(vy)/a) mouse liver. 1795 45

Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5' end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays.
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PMID:Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR. 2621 21


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