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)

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.
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PMID:Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction. 128 41

The location of unpaired adenine residues within the secondary structure of rabbit 18S ribosomal RNA was determined by chemical probing. Naked 18S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked 18S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides [Peattie, D. A., & Gilbert, W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4679-4682]. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of 32P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in 18S rRNA determined by comparative sequence analysis [Chan, Y.-L., Gutell, R., Noller, H. F., & Wool, I. G. (1984) J. Biol. Chem. 259, 224-230]. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit 18S rRNA than for E. coli 16S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian 18S rRNA, as compared with prokaryotic 16S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.
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PMID:Chemical probing of adenine residues within the secondary structure of rabbit 18S ribosomal RNA. 334 49

Human epidermal growth factor (EGF) receptor mRNA was detected in cryopreserved tissue sections adherent to whole glass slides using in situ reverse transcriptase polymerase chain reaction. EGF receptor cDNA was synthesized in situ by reverse transcription using an EGF receptor-specific oligonucleotide primer. In situ polymerase chain reaction amplification in the presence of digoxigenin-11-dUTP and subsequent binding with an antidigoxigenin antibody conjugated to alkaline phosphatase allowed direct visualization. Because DNase, RNase, or proteinase K are not required, tissue integrity is maintained. EGF receptor mRNA is expressed in the basal layer of normal human skin epithelium and is significantly overexpressed in squamous cell tumor specimens, which is consistent with conventional analysis of EGF receptor expression. The assay is semiquantitative, quicker, more sensitive, and void of the nonspecific binding associated with in situ hybridization. In situ reverse transcriptase polymerase chain reaction using whole glass slides is ideally suited for detecting moderate to infrequently expressed transcripts in biopsy specimens.
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PMID:Detection of epidermal growth factor receptor mRNA in tissue sections from biopsy specimens using in situ polymerase chain reaction. 808 54

A single-step reverse transcription polymerase chain reaction (SRT-PCR) method was optimized for hepatitis C virus (HCV) RNA detection. Extraction procedures by proteinase K and guanidinium isothiocyanate gave similar results. The optimal MgCl2 concentration for the SRT-PCR method was 2 mM with 10 units of superscript M-MLV RNase H-reverse transcriptase and 1 unit of Taq polymerase. Shorter PCR cycling steps gave a 10-fold-increased PCR product compared with longer cycling steps. Twenty-five anti-HCV-positive sera from chronic hepatitis C patients were positive with SRT-PCR, whereas only 17 out of 25 were positive by dissociated RT and PCR (dRT/PCR). Specificity was assessed by twenty negative controls. SRT-PCR was 5-fold more sensitive (5 HCV RNA copies per assay) than dRT/PCR with an HCV RNA transcript. Our SRT-PCR method for HCV RNA detection appears fully adapted for routine use in a medical virology laboratory.
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PMID:Detection of hepatitis C virus RNA by a reliable, optimized single-step reverse transcription polymerase chain reaction. 857 10

The DNA-dependent DNA polymerase (DDDP) and RNA-dependent DNA polymerase (RDDP) activities of hepadnavirus polymerases are both essential for viral replication. Human hepatitis B virus (HBV) polymerase has been successfully expressed in Escherichia coli as a fusion protein in frame with maltose-binding protein. The present study was undertaken to characterize these two activities and introduce an in vitro assay system. In situ activity gel assays show that the polymerase has both types of activities. One hundred thirty-four kilodaltons of active full-length product was proteolytically cleaved into approximately 73 kDa of active fragment by proteinase K preincubation. Mutation of conserved YMDD motif also confirms that the activities were due to the recombinant polymerase and that this motif is essential to polymerase activity. Two activities of the polymerase show their optima under conditions of 1 mM (DDDP) or 0.25 mM (RDDP) of MnCl2, 400 mM KCl, 37 degrees C (DDDP) or 24 degrees C (RDDP), and pH 7.0-7.7. Substitution of Mg2+ for Mn2+ results in reduction of processivity, which may explain why Mn2+ supports nucleotide incorporation to a higher level than Mg2+. The polymerase is resistant to aphidicolin. Actinomycin D acts selectively on DDDP activity, whereas phosphonoformic acid inhibits both activities. The in vitro HBV polymerase assay system demonstrated herein will be useful for screening potential HBV polymerase inhibitor for the development of anti-HBV drugs.
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PMID:The catalytic properties of human hepatitis B virus polymerase. 867 Feb 70

The detection of persistent bovine viral diarrhea virus (BVDV) infections in cattle by DNA dot blot hybridization was done with a cloned cDNA probe prepared from noncytopathic BVDV (strain NY-1). Due to the variability of specific hybridization results, detection of BVDV by primer-directed polymerase chain amplification was done. Primers were chosen within a reported area of sequence conservation and amino acid homology of Pestiviruses. The amplification region extended from nucleotide 6322 to 7475 and was based on published BVDV sequence data (NADL strain). BVDV RNA was extracted by two methods (proteinase K and guanidinium isothiocyanate) from serum and white blood cell preparations collected from 3 persistently-infected heifers. cDNA was synthesized from extracted BVDV-genomic RNA using reverse transcriptase. Reaction conditions were optimized to amplify the 1153 base pair fragment from the cDNA preparation. Detection of BVDV in the samples by DNA dot blot hybridization using a nucleic acid probe corresponding to the amplified region (6322 to 7475) was compared with polymerase chain reaction assay. The increased sensitivity of the polymerase chain reaction assay provided clearer identification of persistently-infected animals than DNA hybridization under similar conditions.
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PMID:Detection of persistent bovine viral diarrhea virus infections by DNA hybridization and polymerase chain reaction assay. 921 Sep 42

Telomerase is a unique reverse transcriptase involved in the maintenance of genomic integrity. In an attempt to understand the properties of this enzyme and to study the effect of deoxynucleoside analogues, we have isolated and partially purified telomerase from the blast cells of a patient with acute myelogenous leukemia. During the course of purification of telomerase, three characteristic forms of this enzyme activity were separated. Two processive forms and one less processive form were noted. All forms of the enzyme activities could be abolished by RNase A and proteinase K treatments, implying that they are ribonucleoproteins. The major form of telomerase was characterized with respect to divalent ion requirements, effect of salt and nonionic detergents. The Km of deoxynucleoside triphosphates was determined with a modified telomerase repeat array protocol assay. Studies with deoxynucleoside analogues indicated that 3'-azido-3'deoxythymidine triphosphate is much more inhibitory than 2',3'-dideoxy 2',3'didehydrothymidine triphosphate, and the cytidine analogue ddCTP was not inhibitory. ddGTP was the most potent inhibitor among all dideoxynucleosides studied.
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PMID:Telomerase from human leukemia cells: properties and its interaction with deoxynucleoside analogues. 958 32

A rapid and sensitive protocol for the detection and amplification of infectious bursal disease virus (IBDV) RNA in the bursa of Fabricius was developed. By digestion with proteinase K and subsequent extraction with phenol and chloroform, IBDV RNA was efficiently released from a single bursa. IBDV RNA extraction time was shortened to 4 hr, compared with 24 hr with the traditional method, and only one bursa was needed instead of five. This more simplified procedure resulted in a significant reduction in costs due to less labor and the reduction in expensive chemicals and reagents. Four primers were selected from the sequence of a hypervariable region in VP2 genes. For the amplification of genomic IBDV RNA, the product (643 bp) of reverse transcriptase-polymerase chain reaction (RT-PCR) was reamplified and double checked by a nested PCR amplifying a 491-bp cDNA. The sensitivity of nested PCR was at least 100 times greater than RT-PCR as determined by dilution of the bursal homogenate. The fidelity of the nested PCR product was confirmed by Southern blotting, demonstrating specificity to the VP2 gene of IBDV. The simplified sample processing, shortened procedure time, and technical ease of this nested PCR render it more suitable for implementation in routine RT-PCR with restriction fragment length polymorphism testing for the detection and differentiation of IBDV RNA, especially for studies of IBDV infections of individual chickens.
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PMID:Simplified sample processing combined with a sensitive nested polymerase chain reaction assay for detection of infectious bursal disease virus in the bursa of Fabricus. 977 48

A rapid and simplified protocol for in situ hybridization (ISH) with polymerase chain reaction (PCR)-derived single-stranded DNA probes and S1 nuclease revealed transcripts of bone matrix proteins on decalcified skeletal bone specimens. Mouse bone tissue was fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin. Each pair of primers for reverse transcriptase -PCR was designed to amplify a 280-bp DNA fragment from the coding region of the mature protein of mouse osteonectin (ON) and a 320-bp fragment from the coding region of mouse osteopontin (OP). Initial PCR products were eluted, purified, and reamplified by unidirectional PCR in the presence of the digoxigenin (DIG)-labeled dUTP. ISH was carried out by proteinase K treatment, hybridization, and washing. The unhybridized single-stranded DNA probe was selectively removed by S1 nuclease treatment. Hybridized probes were visualized with the alkaline phosphatase-conjugated anti-DIG antibody. The transcripts of ON and OP were clearly detected on the thin sections of the decalcified bone. Because this protocol does not require cloning or in vitro transcription, reliable and stable ISH can be done in an ordinary laboratory equipped with a thermal cycler.
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PMID:In situ hybridization with polymerase chain reaction-derived single-stranded DNA probe and S1 nuclease. 993 Aug 78

Endothelins (ETs) are a family of potent vasoconstrictor and comitogenic polypeptides consisting of 21-amino acids. Using in situ hybridization, ET-1 mRNA has previously been localized to neuronal cell bodies in fourteen human brain regions. However, because in situ hybridization has a limited detection sensitivity of 20 mRNA copies per cell, ET-1 mRNA may be present in previously undetected areas. Hence, our objective was to localize ET-1 mRNA in specific human brain regions and astrocytic tumours using the more sensitive in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR). Human brain autopsy tissue and surgical cerebral tumour tissue were treated with proteinase K and DNase, followed by RT-PCR using primers specific for ET-1 mRNA and digoxygenin-labelled dUTP in the PCR mixture. The DIG-dUTP was localized with an immunodetection system. We demonstrate ET-1 mRNA labelling in twenty two of the twenty four brain regions studied including those regions in which ET-1 mRNA has been observed by in situ hybridization. In addition, the localization of ET-1 mRNA observed in astrocytomas suggests a role for ET-1 in tumour pathogenesis. In situ RT-PCR has proven to be highly sensitive in its ability to detect low mRNA expression at the cellular level. Our results confirm a role for ET-1 in the human nervous system.
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PMID:Cellular distribution of endothelin-1 mRNA in human brain by in situ RT-PCR. 1176 33


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