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)

Yeast poly(adenylic acid)-containing messenger RNA was isolated from total cellular RNA by affinity chromatography on poly(uridylic acid)-cellulose. The relative complexity of the isolated yeast mRNA was assessed by hybridization analysis with complementary DNA synthesized from the isolated messenger RNA (mRNA) with viral reverse transcriptase. Approximately 25% of the mRNA hybridized at an apparent Crt1/2 of 5 X 10(-3) mol sl.(-1), while the remainder hybridized at an average Crt1/2 of 10(-1) mol sl.-1. Poly(adenylic acid)-containing yeast mRNA was translated in vitro in a wheat germ cell-free extract, and the major polypeptides synthesized have the same molecular weight as the major proteins present in the cell. Four of these proteins were identified by coelectrophoresis and immune precipitation to be pyruvate kinase, enolase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase. These data demonstrate in agreement with the hybridization results that yeast contains major mRNA species and that some of the glycolytic enzyme mRNAs make up part of the major fraction. A procedure is outlined for the preparation of yeast mRNA which is essentially free of ribosomal RNA contamination and is further enriched in the major mRNAs present in the cell.
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PMID:Characterization of purified poly(adenylic acid)-containing messenger ribonucleic acid from Saccharomyces cerevisiae. 31 54

The modulation of the rat cortical m1 muscarinic receptor mRNA was studied with a method of quantitation using the polymerase chain reaction after conversion to complementary DNA (cDNA) with AMV reverse transcriptase (RT/PCR). Primers specific to the C3 region of the m1 mRNA were employed. The y-intercepts from the linear regions of semilogarithmic plots of PCR product versus cycle number were used as measures of the levels of m1 muscarinic mRNA-measured relative to that of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (the 'ratio' method). Alternatively, m1 mRNA in total cortical RNA samples was quantitated from the increase in product elicited by adding a known amount of exogenous m1 muscarinic cDNA sequence (the 'spiking' method). This allowed calculation of absolute level of m1 mRNA, which was 4.1 pg/micrograms total RNA. We measured the level of the rat cortical m1 mRNA after 1 week of chronic receptor blockade with atropine, showing upregulation of 154% by the GAPDH/m1 ratio method and 145% by the spiking method. That this transcriptional alteration was specific was indicated by the finding that the level of GAP-43 mRNA was not affected by atropine treatment.
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PMID:Chronic atropine administration up-regulates rat cortical muscarinic m1 receptor mRNA molecules: assessment with the RT/PCR. 137 72

We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.
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PMID:RNA abundance measured by a lysate RNase protection assay. 138 Nov 96

Endothelin-1, a 21-amino acid peptide secreted by endothelial cells, has constrictor and mitogenic activity for vascular smooth muscle cells, and its mitogenic activity is synergistic with that of platelet-derived growth factor. Endothelial cell-derived endothelin-1 might therefore contribute to intimal hyperplasia in reendothelialized segments of vascular grafts or of endarterectomy and angioplasty sites. Because intimal hyperplasia occurs most often at sites with disordered flow patterns and lower fluid shear stress, we tested the effects of static culture versus high laminar shear stress (25 dyne/cm2) on endothelin-1 precursor (preproendothelin) gene mRNA transcript levels and endothelin-1 peptide release in cultured human endothelial cells. Primary cultures of human umbilical vein endothelial cells were subjected to controlled levels of shear stress in parallel plate flow chambers for 24 hours. To detect preproendothelin mRNA we applied a linked reverse transcriptase-polymerase chain reaction (RT/PCR) to RNA extracted from cultures. Southern blots of RT/PCR reaction products were hybridized with radioactive phosphorous (32P) labeled probes for the amplified preproendothelin complementary deoxyribonucleic acid (cDNA). Detection by RT/PCR of mRNA for glyceraldehyde 3-phosphate dehydrogenase was used to measure a constitutively expressed control signal. Endothelin-1 release into culture medium was measured by radioimmunoassay. Application of 25 dyne/cm2 of shear stress for 24 hours sharply reduced endothelial cell levels of precursor preproendothelin mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluid flow decreases preproendothelin mRNA levels and suppresses endothelin-1 peptide release in cultured human endothelial cells. 206 49

Fluid shear stress can stimulate secretion of tissue plasminogen activator (tPA) by cultured human endothelial cells, while plasminogen activator inhibitor type-1 secretion remains unstimulated. To determine whether hemodynamically induced changes in tPA messenger RNA (mRNA) levels also occur, primary cultures from the same harvest of primary human umbilical vein endothelial cells were either maintained in stationary culture or exposed to arterial levels of shear stress (25 dynes/cm2) for 24 hours. Total cellular RNA was isolated from the shear stressed and stationary cultures and the relative levels of tPA mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were determined using a coupled reverse transcriptase/polymerase chain reaction method. As indicated by the amount of amplification product, tPA mRNA levels were many fold higher (greater than 10) in endothelial cells subjected to shear stress for 24 hours than in stationary controls. In contrast, mRNA levels for GAPDH were similar in control and shear stressed cells. The constancy of the measured GAPDH signal indicated that the tPA response was a selective effect of fluid shear stress. When a similar polymerase chain reaction method was used, the mRNA levels of basic fibroblast growth factor (bFGF) were found not to vary in comparison to GAPDH mRNA after 24 hours of shear stress. These results indicate that enhancement of the fibrinolytic potential of endothelial cells in response to hemodynamic forces could involve transcriptional events.
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PMID:Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. 211 Jan 69

The effects of 3-methylcholanthrene (3-MC) and pyridine on rat renal cytochrome P450 (CYP) 1A1 and 1A2 mRNA expression have been examined by Northern-blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by Southern-blot analysis of the PCR products. Northern-blot hybridization and RT-PCR analyses of kidney poly(A)+ RNA revealed that 3-MC treatment produced a time-dependent increase in the renal CYP1A1 and 1A2 mRNA levels, with CYP1A1 and 1A2 mRNA levels maximally increased at 24 and 18 hr, respectively, after treatment. These data were confirmed via RT-PCR analysis using a subsaturating level of cDNA template and by Southern-blot analysis of the PCR products. This approach served as the foundation for examining the effects of pyridine on CYP1A1 and 1A2 expression in renal tissue. RT-PCR analysis of renal CYP1A1 and 1A2 poly(A)+ RNA levels after treatment with pyridine (200 mg/kg/day for 3 consecutive days) revealed that CYP1A1 mRNA levels were maximally elevated approximately 10-fold after pyridine treatment for 2 consecutive days, whereas CYP1A2 mRNA levels were maximally elevated approximately 3-fold at 24 hr after treatment. The mRNA levels of glyceraldehyde 3-phosphate dehydrogenase, which served as an internal control, remained constant after 3-MC or pyridine treatment. These results show that expression of CYP1A1 and 1A2 mRNAs is enhanced in renal tissue after exposure to 3-MC or pyridine, and that constitutive expression of CYP1A1 seems to be greater than that of CYP1A2 in renal tissue.
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PMID:3-Methylcholanthrene and pyridine effects on CYP1A1 and CYP1A2 expression in rat renal tissue. 749 48

To assess regulation of constitutive prostaglandin G/H synthase (PGHS-1) by interleukin-1 (IL-1) in osteoblastic MC3T3-E1 cells, we compared analysis by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with Northern blot analysis. Using RT-PCR, IL-1 increased PGHS-1 mRNA levels by 1.84 +/- 0.10 or 2.07 +/- 0.17, depending on the method of calculation. Using Northern blot analysis, the effect of IL-1 on PGHS-1 mRNA levels was more variable, and the variability was increased by normalization of PGHS-1 mRNA levels to the housekeeping genes, beta-actin and glyceraldehyde phosphate dehydrogenase (GAPDH), because their mRNA levels were also regulated by IL-1. We conclude that competitive RT-PCR is a reproducible and accurate method for studying small changes in mRNA levels.
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PMID:Measurement of interleukin-1 stimulated constitutive prostaglandin G/H synthase (cyclooxygenase) mRNA levels in osteoblastic MC3T3-E1 cells using competitive reverse transcriptase polymerase chain reaction. 752 76

CD44 is a polymorphic family of immunologically related cell surface proteoglycans and glycoproteins implicated in cell-cell and cell-matrix adhesion interactions, lymphocyte activation and homing, cell migration, and tumor metastasis. CD44 exists as a standard form and as multiple isoforms, each generated by alternative splicing of up to 10 variant exons (termed v1-v10) encoding parts of the extracellular domain. Using semiquantitative reverse transcriptase-PCR and Southern hybridization, alternative CD44 mRNA splicing was examined in 10 papillary thyroid carcinomas, 8 nodular goiters, 9 adenomas, 2 cases of thyroiditis, and 3 histologically normal thyroid controls. The amount of input cDNA for the CD44 PCRs was standardized against an internal control gene (glyceraldehyde phosphate dehydrogenase). Four papillary carcinomas showed significant overexpression of CD44 transcripts migrating between 750 and 1000 bp. These cases demonstrated reduced levels of the 482-bp standard isoform transcript. In six papillary cancers, we found a prominent v6-containing isoform at 750 bp that was present in only trace amounts in normal thyroid tissue. It is of interest that similar findings were seen in the majority of the goiters and adenomas but not in the cases of thyroiditis. These results show that deregulation of alternative CD44 splicing is a common feature of disordered thyroid follicular cell growth, both in neoplastic and nonneoplastic lesions. The data imply an important role for CD44, including CD44v6, in the pathogenesis of various thyroid lesions.
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PMID:Deregulated alternative splicing of CD44 messenger RNA transcripts in neoplastic and nonneoplastic lesions of the human thyroid. 755 35

To examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic gene expression in vitro, freshly isolated rat thymocytes were incubated with 10 nM TCDD, and reverse transcriptase polymerase chain reaction experiments were performed using primers specific for prostaglandin G/H synthase (PGHS) and glyceraldehyde 3-phosphate dehydrogenase. TCDD selectively repressed PGHS gene expression, with maximal inhibition occurring within 60 min. Gel retardation assays demonstrated that dioxin transiently induced binding of the ubiquitous transcription factor NF kappa B to its cognate response element at early time points. However, TCDD had little ability to induce transformation of the Ah receptor to the xenobiotic responsive element in thymic cytosol. These results indicate that TCDD exerts changes in thymocyte gene expression prior to inducing toxicity.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated gene expression in the immature rat thymus. 782 58

Peptide YY (PYY) is a 36-amino-acid peptide known to inhibit pancreatic and gastrointestinal secretion. Immediately following small bowel resection, intestinal PYY mRNA and plasma PYY levels rise. The purpose of this study was to determine whether PYY expression changes in the pancreas during the adaptive period after extensive small bowel resection. Female Sprague-Dawley rats (250 g) underwent 70% small intestinal resection or transection alone as control. Animals were sacrificed at 6 hr, 24 hr, 1 week, or 2 weeks following operation (N = 5/time group). Pancreatic tissue was harvested and RNA was isolated by the guanididium-thiocyanate method. PYY mRNA was analyzed by reverse transcriptase PCR, standardized to glyceraldehyde-3-phosphate dehydrogenase, and semiquantitated by Southern blotting and 32P cpm. Ribonuclease protection assay was used to confirm PCR results. PYY mRNA expression was increased 9 1/2-fold beginning 6 hr after resection compared to transection (P < 0.05). PYY mRNA levels remain elevated, 2 1/4-fold greater than control after 2 weeks (P < 0.05) as analyzed by reverse transcriptase PCR and ribonuclease protection assay. Quantitation by ribonuclease protection assay reveals a gradual elevation of PYY mRNA levels in transected animals compared to a nonoperated rat starting at 1 and 2 weeks. Pancreatic PYY mRNA levels increase rapidly after extensive intestinal resection and remain elevated 2 weeks postoperatively. These results confirm for the first time that the increase in PYY seen after extensive intestinal resection also occurs in extraintestinal sites. In the pancreas, elevated PYY levels may inhibit exocrine secretion, reducing luminal volume, and thereby facilitating intestinal adaptation.
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PMID:Pancreatic peptide YY mRNA levels increase during adaptation after small intestinal resection. 783 Apr 8


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