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)

We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [(3)H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S.
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PMID:Virus-specific messenger RNA and nascent polypeptides in polyribosomes of cells replicating murine sarcoma-leukemia viruses. 435 69

Exposure in vitro of various mammalian retroviruses to the chelating agents EDTA or EGTA in millimolar concentrations resulted in partial disintegration of viral membranes as measured by accessibility or even release of reverse transcriptase, an internal viral protein, without any other treatment usually required. Among the viruses responding to chelators were mammalian type C viruses, primate type D viruses and bovine leukemia virus. The effect was dose-dependent. The avian type C virus AMV, however, was found to be not susceptible to the agents. Rauscher mouse leukemia virus treated in vitro with EDTA or EGTA showed reduced infectivity in mice. The results are considered as evidence for some association of divalent cations with membranes of mammalian retroviruses. The disintegrating activity of EGTA suggests that Ca2+ is an integral constituent of viruses but Mg2+ may also be involved. These cations seem to be responsible for maintaining integrity of retroviral membranes which, after chelation of ions, are either disrupted or become permeable for the exogenous template of reverse transcriptase. In addition, the disintegrating activity of trifluoperazine may indicate that a calmodulin-like protein occurs in retroviral membranes.
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PMID:Disintegration of retroviruses by chelating agents. 681 93

We describe a rapid fluorometric assay for reverse transcriptase (RT) activity. After RT is incubated in the presence of poly(A).oligo(dT) and dTTP for up to 1 h, the reaction is stopped with EDTA and aliquots are added to cuvettes containing 4',6-diamidino-2-phenylindole (DAPI). DAPI fluorescence, which is increased upon binding the RNA.DNA heteroduplex, is measured after 30 min and is linearly dependent on the enzymatic reaction time and the amount of active RT added to the enzyme assay. The increased fluorescence correlates well with the incorporation of [alpha-32P]dTTP into DNA (r2 = 0.986). However, similar assays with the Klenow fragment using poly(dA).oligo(dT) did not result in increased fluorescence under conditions wherein incorporation of [alpha-32P]dTTP into DNA was documented. Thus, the poly(A).poly(dT) [RNA.DNA] heteroduplex must differ from the poly(dA).poly(dT) [DNA.DNA] duplex in a manner that allows for a perturbation of DAPI fluorescence. The relative specific activities of RT in crude preparations measured with the fluorometric assay were comparable to conventional isotopic enzyme assays as were determinations for the type of inhibition and the kinetic constants of purified RT with inhibitors such as zidovudine 5'-triphosphate, nevirapine, and oltipraz.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluorometric measurement of reverse transcriptase activity with 4',6-diamidino-2-phenylindole. 753 86

Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and EDTA.Fe(II) were used to investigate the structure of Drosophila melanogaster ribosomes. Cleavage reactions were performed on intact ribosomes in cell lysates in vitro and analyzed by primer extension with reverse transcriptase using oligodeoxynucleotide primers. Regions of 18S and 28S ribosomal RNAs (rRNAs) which are accessible to MPE.Fe(II) and EDTA.Fe(II) are located almost exclusively within expansion segments. The accessibility of these regions to cleavage indicates that they are likely exposed on the surface of eukaryotic ribosomes. These results provide information about the overall tertiary structure of rRNA in ribosomes.
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PMID:Mapping RNA regions in eukaryotic ribosomes that are accessible to methidiumpropyl-EDTA.Fe(II) and EDTA.Fe(II). 806 Sep 91

A microtiter well-based quantitative reverse transcriptase-PCR assay for determination of BCR-ABL mRNA, which relies on coamplification of the target with an RNA internal standard (IS), was developed. The hapten digoxigenin (Dig) is incorporated during PCR. Target RNA and IS contain identical primer recognition sites and generate same-sized amplification products distinguishable by hybridization with probes specific to the molecules' central part. The hybrids are determined with an anti-Dig-alkaline phosphatase conjugate with fluorosalicylphosphate as substrate. Fluorescent complexes of fluorosalicylate-Tb(III)-EDTA are measured by time-resolved fluorometry. The ratio of fluorescence values for target and IS is linearly related to initial target RNA in the range of 1000 to 200000 molecules. Samples containing K562 total RNA amidst 1 microgram of RNA from normal cells give fluorescence ratios that are linearly related to 30-10000 K562 cells. CVs for 30, 200, and 900 K562 cells are approximately 11%.
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PMID:Quantitative RT-PCR combined with time-resolved fluorometry for determination of BCR-ABL mRNA. 896 27

Human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma are currently widely used clinically for prognostication and in monitoring antiretroviral therapy. Accurate and reproducible results are critical for patient management. To determine the effects of specimen collection and handling procedures on quantitative measurement of HIV-1 RNA, we compared anticoagulants and sample processing times. Whole blood was collected from 20 HIV-1-infected patients in EDTA, acid citrate dextrose (ACD), and heparin tubes, aliquoted, and stored at room temperature. Plasma was separated from whole-blood aliquots prepared at < or =1, 3, 6, 24, and 48 h postcollection and then stored at -70 degrees C until use. HIV-1 RNA levels were determined by the AMPLICOR HIV-1 MONITOR assay. Heparinized plasma samples, which were pretreated with heparinase prior to analysis, had the lowest baseline HIV-1 RNA levels. In the first 6 h, HIV-1 RNA levels decreased by 10, 20, and 31% in EDTA, ACD, and heparin tubes, respectively. From 6 to 48 h postcollection, HIV-1 RNA levels decreased in all anticoagulants, albeit at a slower, more consistent rate. Our results indicate that EDTA should be the anticoagulant of choice for plasma HIV-1 RNA measurement by reverse transcriptase PCR, but ACD tubes are acceptable if the plasma is separated within 6 h of blood collection. Caution must be applied in the interpretation of absolute HIV-1 RNA copy number values obtained with suboptimal specimen collection and processing procedures.
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PMID:Optimization of specimen-handling procedures for accurate quantitation of levels of human immunodeficiency virus RNA in plasma by reverse transcriptase PCR. 954 39

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

The effect of 44 different metal ions (Ag+, Al3+, As(O-)2, Au3+, Ba2+, Be2+, Bi3+, Cd2+, Ce3+, CO2+, Cr(O2-)4, Cr3+, Cs+, Cu2+, Fe3+, Fe2+, Ga3+, Ge4+, Hg2+, Ir4+, La3+, Li+, Mn2+, MO6+, Ni2+, OS4+, Pb2+, Pt4+, Rb+, Rh3+, Sb5+, Se(O2-)4, Se(O2-)3, Sn2+, Sr2+, Th4+, T1+, U(O2+)2, V(O-)3, VO2+, W(O2-)4, Y3+, Zn2+, and Zr4+) on the activity of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV-1) was investigated in vitro. For this study, the RT activity assay was carried out by means of an enzyme-linked immunosorbent assay (ELISA) kit, using the template/primer hybrid poly(A) oligo(dT)15, which required some modifications: (1) possible interfering metal chelators (such as EDTA) in the original lysis buffer were avoided, and a new buffer (50 mM Tris-NO3, pH 7.8) was used throughout; (2) an amount of 2 ng of RT per well was considered to be optimal after checking the linearity of the reaction with increasing amounts of enzyme; (3) an incubation temperature of 37 degrees C and an incubation time of 1 h were chosen after preliminary studies in a wide range of temperature and time. At an incubation temperature > or = 40 degrees C, there was a dramatic loss of enzymatic activity. In addition, when RT alone was preincubated for 1 h at 5 degrees C, 25 degrees C, and 37 degrees C, there was a large (83%) loss of activity at 37 C as compared to that at 5 degrees C. These results are indicative of enzyme thermolability, which is higher in the absence of substrates. The effect of metal ions on RT activity was tested using two different metal salt concentrations (10(-4) M and 10(-5) M). Under such experimental conditions, the presence of five metal ions (Pt4+, Ag+, Rh3+, Zn2+, and Hg2+) decreased the RT activity in a dose-response fashion. The observed order of effectiveness with respect to inhibition was Pt4+ > Ag+ > Rh3+ > Zn2+ = Hg2+. Estimated mean inhibitory concentrations (IC50) were 7.8 microM for (NH4)2PtCl6, 14.1 microM for AgNO3, 46.8 microM for RhCl3, 53.7 microM for Zn(SO)4, and 56.2 microM for Hg(NO3)2. Because these data are of the same order of magnitude as the corresponding values related to other RT inhibitors used in anti-AIDS therapy, metal compounds or their derivatives could give an interesting contribution in the development of new RT inhibitors for clinical use.
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PMID:Effects of trace metal compounds on HIV-1 reverse transcriptase: an in vitro study. 1032 22

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was after 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ml). Only 4% of samples processed after 6 h lost more than 50% (>/=0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4(+)-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4(+)-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.
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PMID:Effects of anticoagulant, processing delay, and assay method (branched DNA versus reverse transcriptase PCR) on measurement of human immunodeficiency virus type 1 RNA levels in plasma. 1040 79

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.
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PMID:Molecular, biochemical and immunological studies of hen pancreatic deoxyribonuclease I. 1060 24


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