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:2.7.7.49 (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

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

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

In this work, we present evidence that enriched human peripheral blood T lymphocytes, depleted of contaminating monocytes, rapidly express tumor necrosis factor alpha (TNF-alpha) mRNA when exposed to low doses of gamma-irradiation. In total PBL, TNF-alpha mRNA accumulation increased threefold as early as 30 minutes following exposure to 4 Gy and then declined to the baseline level by 3-5 h, as measured by the reverse transcriptase-polymerase chain reaction (RT-PCR). The increase in TNF-alpha mRNA was also observed in populations of enriched T cells and decreased when the dose of irradiation was increased to 10 Gy, strongly suggesting that T lymphocytes, the most radiosensitive cells of the body, contributed directly to the increase of TNF-alpha mRNA. A good correlation was found between mRNA expression and TNF-alpha protein secretion. Interestingly, a eightfold increase in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA accumulation was also detected in both PBL and enriched T cells irradiated at 4 Gy for 3 h compared with unirradiated cells. This irradiation effect was almost completely abolished, however, following exposure to 10 Gy. Together these data suggest that T cells are responsible for the irradiation-induced expression of TNF-alpha and GAPDH.
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PMID:Induction of tumor necrosis factor alpha expression in human T lymphocytes following ionizing gamma irradiation. 872 80

It is important to determine sensitive biomarkers for both exposure and susceptibility since differences in individual susceptibility to potentially hazardous chemicals may represent a major variable in the assessment of risk. Induction of cytochrome P450IA1 (CYP1A1) may be a measure of environmental exposure to aromatic hydrocarbons in cigarette smoke. This study investigated the use of reverse transcriptase-polymerase chain reaction (RT-PCR) to detect constitutive levels of CYP1A1 mRNA in the peripheral lymphocytes of a population of smokers and non-smokers as a potential marker of exposure. In addition, the presence of an Msp 1 restriction fragment length polymorphism was analyzed using a simple PCR method as a biomarker for susceptibility. DNA and RNA were isolated from the peripheral lymphocytes of 20 smokers and a matched group of non-smokers. RT-PCR was used to detect the endogenous levels of CYP1A1 mRNA with glyceraldehyde-3-phosphate dehydrogenase as a control gene. The 3'-region of CYP1A1 gene was amplified by PCR and underwent restriction digestion with Msp 1 to detect the polymorphism. The endogenous CYP1A1 expression as detected by RT-PCR was very low and variable and there was a slight but not significant increase in the smokers by comparison with non-smokers. Thirty-two of the volunteers were homozygous for the normal allele while 8 were heterozygous for the uncommon Msp 1 allele and none was homozygous for the polymorphism. The allele frequency (0.1) was found to be in Hardy-Weinberg equilibrium. Since only a slight increase was seen in endogenous CYP1A1 mRNA levels in the peripheral lymphocytes of smokers by comparison with non-smokers, the effect may have been diluted by variation in sensitivity to dose, a threshold of exposure effect, or the return of mRNA to baseline between exposures. The wide variation in mRNA levels may reflect the influence and exposure of different environmental factors. The sensitivity of PCR-based methods suggests that they may have an important role in future overall biomonitoring of exposure and susceptibility to environmental chemicals.
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PMID:Detection of CYP1A1 mRNA levels and CYP1A1 Msp polymorphisms as possible biomarkers of exposure and susceptibility in smokers and non-smokers. 879 34

A competitive reverse transcriptase-PCR (RT-PCR) assay has been developed for the quantification of particular mRNA species in human articular cartilage. Competitor RNA species were synthesized that differed from the amplified target sequence only by the central insertion of an EcoRI restriction site. By using known amounts of synthetic target and competitor RNA, it was shown that competitor RNA molecules designed in this way are reverse-transcribed and amplified with equal efficiency to the target of interest. Furthermore quantification could be performed during the plateau phase of the PCR, which was necessary when using ethidium bromide fluorescence as a detection system. The inhibition of aggrecan and link-protein mRNA expression by interleukin 1 or tumour necrosis factor in monolayers of human articular chondrocytes quantified by this competitive RT-PCR method compared favourably with Northern hybridization studies. The main advantage of this technique is that it can be used to quantify levels of mRNA with RNA extracted directly from 100 mg wet weight of human articular cartilage. Age-related changes in aggrecan and link-protein mRNA were therefore quantified in human articular cartilage directly after dissection from the joint. The concentration of link-protein mRNA was higher in immature cartilage than in mature cartilage when expressed relative to the amount of glyceraldehyde-3-phosphate dehydrogenase mRNA, but no age-related changes were observed in aggrecan mRNA expression. The ratio of aggrecan to link-protein mRNA was higher in mature cartilage than in immature tissue. These age-related differences in the molecular stoichiometry of aggrecan and link-protein mRNA might have implications with respect to the regulation of the formation and the stability of the proteoglycan aggregates in cartilage.
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PMID:Quantification of aggrecan and link-protein mRNA in human articular cartilage of different ages by competitive reverse transcriptase-PCR. 891 86

Treatment of human cells with 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.
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PMID:Targeting RNA decay with 2',5' oligoadenylate-antisense in respiratory syncytial virus-infected cells. 905 Aug 83

The expression of the mitochondrial benzodiazepine receptor gene was assayed by a semi-quantitative non-radioactive reverse transcriptase polymerase chain reaction (RT-PCR) assay. The level of amplified mitochondrial benzodiazepine receptor mRNA was expressed as a ratio of either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-actin mRNA co-amplified in the same RT-PCR assay. The relative amounts of mitochondrial benzodiazepine receptor RNA in several rat tissues were found to be similar to the previously reported relative amount of mitochondrial benzodiazepine receptor binding sites. The level of these binding sites has also been reported to be altered by stress stimuli. In this study we specifically measured the effect of stress on the mRNA levels of the mitochondrial benzodiazepine receptor as an alternative method to the binding assay in an attempt to understand the mechanism by which stress alters binding. Sprague-Dawley male rats were either forced to swim for 15 min in 18 degrees C water or restrained in a plastic cylinder for 45 min either once, or twice daily for 7 days. Neither the swim stress, nor acute or chronic restraint stress, caused a measurable statistically significant relative change in mitochondrial benzodiazepine receptor mRNA in the adrenal gland, kidney, testis and olfactory bulb. However, daily treatment of rats for 7 days with 4 mg/kg of dexamethasone caused a significant decrease in mitochondrial benzodiazepine receptor gene expression in adrenal glands. This finding and the measurement of the relative levels of mitochondrial benzodiazepine receptor mRNA in the various tissues indicate that mitochondrial benzodiazepine receptor density is regulated to some extent at the gene expression level. However, the lack of detectable stress-induced changes in mRNA levels for this receptor seem to indicate that either mRNA changes were below detectable levels or that other mechanisms may be involved in the previously reported stress-induced changes of mitochondrial benzodiazepine receptor density. Because the focus of this work was on the regulation of mitochondrial benzodiazepine receptor gene expression, ligand binding studies to determine changes in receptor densities were not performed.
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PMID:Dexamethasone, but not stress, induce measurable changes of mitochondrial benzodiazepine receptor mRNA levels in rats. 927 84

Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway. Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis. We have cloned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encoded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide-binding domain and a catalytic domain, which was highly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment of the isolated cDNA was hybridized to a 1.3-kb mRNA transcript. There was a marked increase in Tthgapdh mRNA level at the mid-exponential phase, followed by a gradual decrease. Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.
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PMID:Cloning and sequencing of a cDNA encoding glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena thermophila: growth-associated changes in its mRNA expression. 930 12

Nitric oxide (NO), a potent and versatile free radical, is synthesized in leukocytes by the inducible form of NO synthase (iNOS). In this study, leukocytes in pregnant mouse uterus were investigated for expression of the iNOS gene. Inducible NOS mRNA, which was identified by reverse transcriptase polymerase chain reaction, was high relative to an invariant mRNA, glyceraldehyde-3-phosphate dehydrogenase, in midgestation uteri (gestation days [g.d.] 10, 12, and 14) but was low in late-gestation uteri (g.d. 16 and 18). Inducible NOS protein, identified immunohistochemically in paraformaldehyde-fixed uteri taken from g.d. 6 through 18 using rabbit antibodies generated to mouse carboxyl terminus iNOS peptides, was prominent in a few myometrial mast cells at early stages and was strongly expressed from g.d. 6 through g.d. 14 in myometrial macrophage-like cells. Inducible NOS protein was first detected in uterine (u) natural killer (NK) cells at g.d. 8. Signals peaked in this lineage at g.d. 10 and declined thereafter. Uterine leukocytes cultured in vitro expressed the iNOS gene; a hybridoma cell line derived from mouse uNK cells (GWM1-2) contained iNOS mRNA, and cells migrating from mouse metrial gland explants included iNOS/ leukocytes. Large, granular iNOS + uNK cells were absent from the uteri of homologously mated pregnant TgE26 mice, an NK cell-deficient transgenic mouse strain, but immunoreactive iNOS was detectable in trophoblast, a cell lineage that did not contain immunoreactive iNOS in NK cell-competent Swiss-Webster mice. In TgE26 mothers gestating normal embryos, the same pattern was observed. Collectively, the results of this study demonstrate that iNOS is present in mouse uterine leukocytes including mast cells, macrophage-like cells, and uNK cells, and suggest that in the absence of uNK cells, the placenta synthesizes iNOS. These findings are consistent with the postulate that leukocyte NO contributes importantly to events associated with successful pregnancy that are likely to include relaxation of vascular smooth muscle.
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PMID:Expression of the inducible nitric oxide synthase gene in mouse uterine leukocytes and potential relationships with uterine function during pregnancy. 931 87


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