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)

The effects of Mg++, Mn++, and KCl addition, individually and in combination, on the rate of DNA- and RNA-primed DNA synthesis by avian myeloblastosis virus DNA polymerase (reverse transcriptase) using a variety of natural and synthetic template-primer combinations were examined. Optimal divalent cation concentrations were found to vary by as much as 10-fold depending upon the template-primer used to direct synthesis. Addition of KCl to reaction mixtures containing optimal divalent cation concentrations produced stimulation or inhibition of DNA synthesis which was also template-specific. DNA synthesis on the modified template poly (2'-0-methylcytidylate) was uniquely stimulated by combinations of divalent cations. With Mg++ as divalent cation, deviations from classical Michaelis-Menten kinetics of substrate saturation were observed with all template-primers tested.
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PMID:Observations on template-specific conditions for DNA synthesis by avian myeloblastosis virus DNA polymerase. 6 Jul 40

A simple and rapid solid-phase reverse transcriptase assay was developed based on the use of poly(rA):oligo(dT)12-18 as template primer immobilized on DEAE cellulose paper squares to detect human immunodeficiency virus (HIV) and/or other retroviruses in cell culture supernatants. It was found that PEG (per se) -up to 4% concentrations (w/v)--did not inhibit reverse transcriptase activity. Optimal conditions of the assay were determined. This solid-phase technique is much faster and more convenient than the methods described previously.
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PMID:A solid phase reverse transcriptase micro-assay for the detection of human immunodeficiency virus and other retroviruses in cell culture supernatants. 169 Dec

Optimal management of human immunodeficiency virus type 1 (HIV-1) infections may require combinations of anti-HIV-1 agents. Zidovudine (AZT, 3'-azido-3'-deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-alpha A (rIFN-alpha A) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZT-resistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-1 were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFN-alpha A in two-drug regimens against AZT-resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT-containing regimens may involve inhibition of AZT-sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro.
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PMID:Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro. 171 49

Murine leukemia (Rauscher and Moloney strains) and sarcoma (Kirsten strain) virions, as well as the mammary tumor virus of mice, contain an RNA-dependent DNA polymerase. Optimal incorporation of deoxyribonucleoside triphosphates occurs at a critical detergent (Triton X-100) concentration (0.010-0.014%). At higher than optimal detergent concentrations the virion is seen to be disrupted and enzyme activity is lost. The virion, enzymatic activity, and newly synthesized DNA all cosediment in a sucrose gradient. Thus far the enzymatic activity has been found only in RNA viruses that have oncogenic properties.
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PMID:DNA synthesis by RNA-containing tumor viruses. 433 15

Optimal conditions for detecting reverse transcriptase activity in human placental extracts are described. They vary with the state of the placenta at birth and are influenced by relative amounts of detergent, monovalent cation, and protein in the reaction mixture. Demonstrating activity of the placental enzyme requires detergent, but the enzyme is sensitive to high detergent concentrations. This sensitivity can be be altered by lowering the monovalent cation concentration from 0.154 to 0.034 M and by adding protein to the reaction mixture. The detection of reverse transcriptase in Rauscher murine leukemia virus and baboon endogenous type C virus, but not in the Mason-Pfizer monkey type D virus, shows similar requirements.
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PMID:Optimal conditions for detection of reverse transcriptase activity in human placentas. 620 94

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.
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PMID:Continuous in vitro evolution of bacteriophage RNA polymerase promoters. 752 54

Reovirus type 3 has been implicated in the origin and pathogenesis of extrahepatic biliary atresia and idiopathic neonatal hepatitis, but routine detection of this virus in hepatobiliary tissues from affected infants by culture and histological techniques has been unsuccessful. In this study, oligonucleotide primers specific to the M3 genome segment of reovirus 3 (Dearing) were used in a reverse transcriptase-mediated polymerase chain reaction technique to develop a sensitive and specific assay for the detection of reovirus 3 RNA in formalin-fixed, paraffin-embedded patient samples. Optimal reaction conditions were determined by testing infected murine tissues and preserved human liver tissue supplemented with reovirus 3. Archival specimens from 50 infants, including 14 with extrahepatic biliary atresia, 20 with idiopathic neonatal hepatitis, and 16 age-matched controls, were evaluated. Successful amplification of human albumin complementary DNA from the preserved tissues confirmed the presence of intact RNA in every patient specimen tested. Analysis of the amplification reactions by agarose gel electrophoresis and Southern blot hybridization detected the presence of reoviral RNA only once in a single patient sample. These results do not support a strong role for reovirus 3 in the development of neonatal cholestatic liver disease. The recent association of other RNA viruses of the Reoviridae family with murine liver disease and human extrahepatic biliary atresia indicates that continued investigation into a viral cause for idiopathic neonatal hepatobiliary disease is warranted.
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PMID:Reovirus 3 not detected by reverse transcriptase-mediated polymerase chain reaction analysis of preserved tissue from infants with cholestatic liver disease. 753 24

Although detection of hepatitis C virus RNA with polymerase chain reaction has become the standard for diagnosis, extensive application has been thwarted by polymerase chain reaction's labor intensiveness, risk of false-positive results through contamination and time required for individual assays. To minimize these limitations, we developed and validated a one-step hepatitis C virus RNA polymerase chain reaction assay. The one-step method was compared with traditional hepatitis C virus RNA polymerase chain reaction using primers from the highly conserved 5' untranslated region of the hepatitis C virus genome. Variables studied in the one-step method included the source and quantity of reverse transcriptase (RTase), the concentration of MgCl2 and the duration of reverse transcription and complementary DNA amplification cycles. Optimal conditions for the one-step method were obtained with 25 U of reverse transcriptase and 2 mmol/L MgCl2. The one-step method substantially reduced the time required for analysis. The sensitivity of the one-step method was comparable to that of traditional hepatitis C virus RNA polymerase chain reaction using serially diluted RNA extracted from the serum of a hepatitis C virus-infected patient. The specificity of the one-step method was confirmed on Southern-blot hybridization. The results exhibited 100% concordance with results of traditional hepatitis C virus RNA polymerase chain reaction in 50 serum samples, including those of positive and negative controls. In addition, 100% concordance was observed between the two methods' results when sera containing low levels of hepatitis C virus RNA were used.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:One-step RNA polymerase chain reaction for detection of hepatitis C virus RNA. 768 79

We report the design of hybrid molecules to bind in the minor groove of B-DNA, which combine DNA alkylating and cross-linking ability for increased chemotherapeutic efficacy, with sequence specificity, to minimize side effects. Optimal linkage geometries have been determined for the synthesis of bis-anthramycin and anthramycin-netropsin hybrid molecules. Earlier studies on linked drugs have typically been based on molecular mechanics calculations. This work, in contrast, uses the observed crystal structures of a netropsin/DNA complex and a new anthramycin/DNA complex to determine the exact spacing between two individual drugs when bound in the minor groove of B-DNA. Molecular linkers then are designed and tested between these two experimental positions, to form a chimeric or bis-linked compound molecule. A linked anthramycin-netropsin molecule has been designed specifically to target the polypurine tract second-strand primer site of the reverse transcriptase of HIV-1.
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PMID:Design of B-DNA cross-linking and sequence-reading molecules. 776 21

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


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