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)

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.
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PMID:Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. 171 65

A recombinant DNA polymerase derived from the thermophilic eubacterium Thermus thermophilus (Tth pol) was found to possess very efficient reverse transcriptase (RT) activity in the presence of MnCl2. Many of the problems typically associated with the high degree of secondary structure present in RNA are minimized by using a thermostable DNA polymerase for reverse transcription, and predominantly full-length products can be obtained. The cDNA can also be amplified in the polymerase chain reaction (PCR) with the same enzyme. The Tth pol was observed to be greater than 100-fold more efficient in a coupled RT/PCR than the analogous DNA polymerase from Thermus aquaticus (Taq pol). The sensitivity of the reactions performed by Tth pol allowed for the detection of ethidium bromide stained products starting with as little as 100 copies of synthetic cRNA. Similar results were also obtained with RNA from a Philadelphia-chromosome positive cell line. Detection of IL-1 alpha mRNA was possible starting with 80 pg of total cellular RNA. The ability of Tth pol to perform both reverse transcription and DNA amplification will undoubtedly prove useful in the detection, quantitation, and cloning of cellular and viral RNA.
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PMID:Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. 171 96

Twenty different isolates of the soil bacterium Myxococcus xanthus were examined for the presence of multicopy single-stranded DNA (msDNA)-producing retroelements, or retrons. Each strain was analyzed by ethidium bromide staining for msDNA, 32P labeling of the msDNA molecule by the reverse transcriptase (RT) extension method, and DNA hybridization experiments with probes derived from two retrons, Mx162 and Mx65, previously cloned from M. xanthus DZF1. These analyses revealed that all M. xanthus strains contain an msDNA very similar to Mx162 msDNA, and 13 strains also contain a second smaller msDNA very similar to Mx65 msDNA. In addition, the strains contained retron-encoded genes msr and msd, which code for msDNA, and a gene for RT responsible for the synthesis of msDNA. These genes show greater than 80% nucleotide sequence similarity to retrons Mx162 or Mx65. The near-ubiquitous occurrence of msDNA retrons among M. xanthus strains and their homogeneous nature are in marked contrast to the highly diverse but rarely occurring msDNA-producing elements of Escherichia coli. The possible origin and evolution of RT and retron elements is discussed in view of these findings.
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PMID:Survey of multicopy single-stranded DNAs and reverse transcriptase genes among natural isolates of Myxococcus xanthus. 171 54

Protein extracts from the protozoan ciliate Paramecium tetraurelia revealed high levels of RNA-dependent DNA polymerase activity (reverse transcriptase). Stable and constant during the somatic phase of the cell cycle, the reverse transcriptase activity quickly diminished following the completion of the sexual phases of the cell cycle: conjugation and autogamy. The Paramecium reverse transcriptase presented a number of common features with retroviral polymerases: ability to copy synthetic templates such as poly(rCm).oligo(dG) as well as mRNA; sensitivity to various reverse transcriptase inhibitors such as HPA 23, suramin, phosphonoformate and ethidium bromide; insensitivity to the action of other DNA and RNA polymerase inhibitors and, finally, the requirement for divalent cations before the enzyme can function: either magnesium or manganese. Although the reverse transcriptase activity was not proven to be independent from one of the DNA polymerases in paramecia, its high activity predicts a role in the paramecia cell cycle. From what we are able to conceive today two possible roles could be envisaged. Participation in the anlage macronucleus formation: micronuclear sequences are first transcripted and, after rearrangements of the RNA molecules, these are retrotranscribed into the macronuclear DNA molecules or association with retrotransposons that participate in the movement of certain macronuclear sequences into the germ-line micronucleus.
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PMID:RNA-dependent DNA polymerase activity in Paramecium tetraurelia: what for? 243 48

Poly (2'-O-methylcytidylic acid) is recognized as a template in reactions catalyzed by RNA-dependent DNA polymerases in the presence of Mn2+ as divalent cation. We report that kinetic data obtained for dGTP and template under optimal experimental conditions in the reaction catalyzed by reverse transcriptase showed some similarities between the poly (2'-O-methylcytidylic acid)/Mn2+ and polyribocytidylic acid/Mg2+ systems. The reaction was inhibited by the action of N-ethylmaleimide and novobiocin, and to a lesser extent by ethidium bromide and tetramethyl ethidium bromide.
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PMID:Kinetic properties of poly (2'-O-methylcytidylic acid)-directed reverse transcriptase reaction. 246 2

Ribonuclease and chemical probes were used to investigate the binding sites of ribosomal protein L18 on Escherichia coli 5 S RNA using both end-labelling and reverse transcriptase procedures. The results, together with earlier data, were superimposed on a cylindrical projection of RNA double helices and most of the protection effects were found to cluster in the major groove at two sites located on one side of the RNA at the junctions of helix II with the adjoining internal loops A and B. The loop A/helix II junction was investigated using 5 S RNA mutants, produced by site-directed mutagenesis, that exhibited altered binding properties to L18. These results, together with those from a circular dichroism study of L18 complexed with the wild-type and different mutant RNAs, enabled us to assign an L18-induced conformational change to loop A. We infer that this change contributes to the co-operative binding of L5 to helix I, which may be reinforced by the binding of the very basic N-terminal peptide of L18 within the minor groove of helix I. A psoralen derivative formed a mono-addition product with U25 within loop B in the free RNA but not in the L18 complex. Moreover, the modified molecules were selected against in L18 binding experiments. Protection effects that occurred within the adjoining helix III and loop C were compatible with a tertiary interaction between loop C and loop B/helix III that could be stabilized by the L18 binding to the junction of helix II and loop B. Further support for a bipartite binding site derived from the finding that ethidium bromide molecules that are displaced from E. coli 5 S RNA by L18 intercalate both at the loop A/helix II junction and in loop B at the binding site of the psoralen derivative.
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PMID:Protein L18 binds primarily at the junctions of helix II and internal loops A and B in Escherichia coli 5 S RNA. Implications for 5 S RNA structure. 247 86

To test the utility of the polymerase chain reaction in identifying single base mutations in a gene known to give rise to an altered enzyme and drug resistance phenotype, a human colon adenocarcinoma cell line resistant to methotrexate, with a known single base mutation (Srimatkandada et al., J. Biol. Chem. 264:3524, 1989) was examined. Poly A+ RNA was used for cDNA synthesis with reverse transcriptase, deoxynucleoside triphosphates, and 5 microM 3' primer that anneals outside the coding region of the human dihydrofolate reductase. The RNA:DNA hybrid was used as a template for the polymerase chain reaction with the addition of a 5' primer and Thermus aquaticus (Taq)I DNA polymerase. These primers flank the coding region of the human dihydrofolate reductase and define a region of 650 bases. The polymerase chain reaction was carried out for 40 cycles resulting in full length transcripts in microgram amounts clearly visible by ethidium bromide staining on agarose gels. DNA was isolated by standard methods, and double-stranded DNA was sequenced by the chain-termination method using TaqI DNA polymerase. A single point mutation was discovered at position 91 (T----C) resulting in a substitution of serine for phenylalanine at codon 31, as determined previously by classical cDNA cloning and sequencing. Sequence analysis indicated that this base transition resulted in the loss of Eco RI and Xmn I sites and the gain of a HinfI site in the cDNA, which were confirmed by restriction digests.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Detection of a single base mutation in the human dihydrofolate reductase gene from a methotrexate-resistant cell line using the polymerase chain reaction. 264 Jan 57

The avian retrovirus pp32 DNA endonuclease and the beta polypeptide of the reverse transcriptase contain the same three phosphoserine (p-Ser) tryptic peptides. At least 95% of the Pi label is nearly equally distributed between two major p-Ser tryptic peptides derived from either beta or pp32. These polymerase gene-derived proteins were metabolically labeled with various radioactive amino acids or Pi, and the purified protein was subjected to cyanogen bromide or hydroxylamine cleavage. The results indicated that the two major p-Ser tryptic peptides map to the COOH-termini of both proteins. The two major p-Ser tryptic peptides isolated from Pi-labeled pp32 were subjected to proteolysis by three separate specific proteases. Analysis of the data suggested that these p-Ser are located on pp32 at amino acid positions 262 and 282 from the amino terminus of pp32 (286 amino acids in length). At present, we cannot exclude the possibility that one or both p-Ser peptides map between amino acid positions 124 to 150. The role of this site-specific phosphorylation of pp32 and beta is also discussed.
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PMID:Avian retrovirus pp32 DNA endonuclease is phosphorylated on Ser in the carboxyl-terminal region. 283 11

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.
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PMID:Purification of avian myeloblastosis virus DNA polymerase by affinity chromatography on polycytidylate-agarose. 413 57

Spumavirinae or foamy viruses have been shown to have a characteristic RNA-dependent DNA polymerase activity. We demonstrate here the existence of an RNase H activity that copurifies with the 81-kilodalton monomeric polypeptide, which carries the RNA-dependent DNA polymerase activity of simian foamy virus type 1. RNase H degrades RNA hybrid substrates; however, it does not solubilize single-stranded RNAs. Inactivation assays with heat, high levels of bivalent cations, ethidium bromide, and sodium fluoride suggest that the RNase H catalytic site could be topologically independent from the DNA polymerase catalytic site.
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PMID:Characterization of RNase H activity associated with reverse transcriptase in simian foamy virus type 1. 619 Oct 42


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