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)

Plant DNA polymerases and E. coli DNA polymerase I, but not animal DNA polymerases or avian reverse transcriptase, are strongly stimulated by ethidium bromide (EtdBr) when TMP incorporation is followed using a short oligo dT primer at 37 degrees C. The effect is observed with a poly A template in the presence of Mg2+ or Mn2+ and of poly dA template only in the presence of magnesium ions. When a longer primer like poly dT is used, EdtBr inhibited wheat DNA polymerase C activity. This result prompted us to study the effect of the incubation temperature on the drug mediated stimulation. With oligo dT primer the stimulation by EdtBr is not observed at a temperature of incubation lower than 35 degrees C. It is shown that the Tm of poly A-dT12 is around 35 degrees C and that EdtBr will clearly increase this value. The stimulation is lost when the enzyme is preincubated with the primer alone whereas it is not affected when the enzyme is preincubated with the template.
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PMID:Ethidium bromide stimulation of DNA polymerase activity by stabilization of the primer-template duplex. 682 Nov 57

We developed a reverse transcription-polymerase chain reaction (RT-PCR) method which permits the simultaneous amplification of several mRNAs, even though their relative levels may be very different. First-strand cDNAs were synthesized from total RNA by MMLV reverse transcriptase with oligo(dT)15 priming. Analysis was performed in the linear phase of PCR, allowing the detection of the products by polyacrylamide gel electrophoresis followed by ethidium bromide staining. In order to obtain a similar amplification of multiple targets in the same PCR system, it was necessary: (i) to adjust the relative concentration of each set of primers and (ii) to use high-stringency conditions (annealing temperature and addition of organic solvent). These conditions allowed the rapid quantitation of several mRNAs in multiple samples, minimizing experimental variations. The reliability of the method was established by measuring variations of c-Ki-Ras, ornithine decarboxylase, alpha-amylase, and beta-actin mRNA levels during the growth of pancreatic tumoral AR4-2J cells. Glyceraldehyde-6-phosphate dehydrogenase expression showed very small variations which confirm that it represents a reliable internal standard for studies of gene expression. Results from competitive PCR amplification of target cDNA and internal competitive template were in agreement with those of simultaneous amplification, validating the quantitative aspect of the method.
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PMID:Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction. 750 50

Five muscarinic receptor genes (m1-m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase-free DNase to remove any residual genomic DNA. Total RNA (1 microgram) was reverse-transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)-amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR-amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus-hypothalamus and ponsmedulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT-PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS.
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PMID:m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC. 751 60

Sixteen biphenyl derivatives were synthesized and evaluated for their inhibitory activity against HIV-1 replication in acutely infected H9 cells. 3-Bromo- (4) and 3,3'-dibromo-4,4'-dimethoxy-5,6,5',6'-bis(methylenedioxy)-2,2'- bis(methoxycarbonyl)biphenyl (5) demonstrated potent anti-HIV activity with EC50 values of 0.52 and 0.23 micrograms/mL and therapeutic index values of > 190 and > 480, respectively. A comparison of the anti-HIV activity of these biphenyl derivatives suggested that the types of substituents on the phenolic hydroxy groups rather than the number of bromine(s) on the aromatic rings are important to the enhanced anti-HIV activity. Compounds 4 and 5 also showed potent inhibitory activity against HIV-1 reverse transcriptase in a template-primer dependent manner. The site of inhibition of HIV could be related to inhibition of this enzyme. Compounds 4 and 5 did not induce virus expression from the chronic HIV-1-infected cell lines ACH-2 and U1. Furthermore, these two agents did not inhibit an increase in virus production from the chronic HIV-1-infected cell lines when the phorbol ester PMA was present.
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PMID:Anti-AIDS (acquired immune deficiency syndrome) agents. 17. New brominated hexahydroxybiphenyl derivatives as potent anti-HIV agents. 754 78

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.
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PMID:An improved, sensitive, non-radioactive in situ hybridization method for the detection of cytokine mRNAs. 765 59

We have used a simple method for detecting HIV-1 in the serum of infected individuals using the polymerase chain reaction (PCR). This is useful if only serum, or other specimens which would not be expected to harbour proviral DNA, is available for diagnostic testing. Viral RNA present in serum is bound to silica particles in the presence of a high concentration of guanidinium thiocyanate (GuSCN) which denatures any proteins present, specifically ribonucleases. After washing the RNA/silica pellet, the RNA is eluted in water and reverse transcribed using random primers and Moloney murine leukaemia virus reverse transcriptase in the presence of a modified PCR buffer. The resultant cDNA is amplified using nested PCR and the products of amplification are detected by gel electrophoresis and ethidium bromide staining. The identity of bands on the gel is confirmed using a digoxigenin-labelled oligomer probe. The method is a general one applicable to amplification of any RNA species.
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PMID:Detection of HIV-1 in serum, using reverse transcription and the polymerase chain reaction (RT-PCR). 768 79

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

We have developed a method to monitor mRNA expression that is based upon the reverse transcriptase-polymerase chain reaction (RT-PCR) and includes multiple sets of primer pairs in coamplification reactions. To observe relative changes in mRNA steady-state levels, each target in a multiplex reaction was amplified to within a predetermined range by using PCR cycle numbers specific for each target. Optimal PCR cycle numbers for target templates were determined by preliminary titration experiments performed using the "primer-dropping" method. By employing this method, the overall amplification reaction was limited, permitting the PCR products to remain within the exponential range of the amplification curve and yet be detectable on ethidium bromide-stained gels. We demonstrated the utility of this method by monitoring the expression kinetics of cyclins A, B1, D1, and E, and of the immediate-early genes c-fos, c-myc, and beta-actin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the multiplex reactions as an endogenous internal standard to control for variations in product abundances due to differences in individual RT and PCR reaction efficiencies. Changes in gene expression of less than twofold to greater than 75-fold were readily distinguished.
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PMID:Monitoring mRNA expression by polymerase chain reaction: the "primer-dropping" method. 788 71

The aim of the present study was to determine, in a population of 70 HIV-1 infected patients with antibodies to HCV, the percentages of individuals with an active replication of HIV-1, HCV or both. During a one year follow-up of these patients at different stages of disease, blood samples were regularly collected for determination of transaminase, beta 2 microglobulin and CD4+ lymphocytes. Total RNAs were extracted from the sera, retrotranscribed with MoMuLV reverse transcriptase and nested PCR assays were carried out separately with sets of primers homologous to the 5' non-translated region of HCV and in HIV-1 gag. The amplified products were subjected to electrophoresis and observed under u.v. illumination after staining with ethidium bromide. For some samples, the identity of the amplified products was confirmed by Southern blotting by hybridization with enzyme-labelled probes. A total of 57% of the patients were found to produce HIV-1 RNA and 62% HCV RNA, while 34% produced both. HIV-1 RNA production was correlated with beta 2 microglobulin and CD4+ levels; active replication of HCV was correlated with hepatitis but not with CD4+ levels nor with HIV-1 RNA synthesis.
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PMID:HIV-1 and HCV co-infected patients: detection of active viral expression using a nested polymerase chain reaction. 790 23

A sensitive, highly resolvable, and quantitative method was designed to analyse the diversity of polymerase chain reaction (PCR)-amplified transcripts which possess length polymorphism. A reverse transcriptase-PCR technique was used to amplify rearranged T cell antigen receptor (TCR) transcripts isolated from human blood. Oligonucleotide primers specific for conserved TCR V and C region sequences were used in PCR, with one of the primers end-labeled with 32P. Amplified cDNA products were analysed by polyacrylamide sequencing gel electrophoresis with an M13mp18 sequencing ladder as a size marker. 32P-labeled products were detected by either autoradiography or PhosphorImager. The method allowed determination of the sizes of PCR products with the precision of one nucleotide. The resolution using this technique was much higher than by electrophoresis in agarose gel with ethidium bromide staining. The sizes of PCR products determined by sequencing gel electrophoresis were consistent with the lengths of nucleotide sequences obtained after subcloning PCR products in competent bacterial cells. Analysis of PCR products by sequencing gel electrophoresis was more rapid and as accurate as nucleotide sequence analysis in determining the relative ratios of TCR mRNA in mixtures of T cell clones. The method is applicable for analysis of both rearranged TCR and immunoglobulin genes.
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PMID:Analysis of diversity of T cell antigen receptor genes using polymerase chain reaction and sequencing gel electrophoresis. 793 Jun 51


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