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)

Adriamycin inhibited the endogenous RNA-, poly (A)-d(T)12-, and calf thymus DNA-catalyzed reaction of reverse transcriptase from AKR mouse murine leukemia virus (AKR-MLV). This inhibition was found at the reaction levels of endogenous RNA-directed and subsequent DNA-directed DNA synthesis. Although adriamycin and actinomycin D significantly reduced the growth of AKR mouse cells (K3b), the treatment with adriamycin could bot inhibit the AKR-MLV production in these cells. Actinomycin D inhibited AKR-MLV production completely in the same experimental condition. In adriamycin-resistant K3b/Am cells, which were isolated by intermittent treatment of K3b cells with adriamycin, persistence of AKR-MLV was demonstrated. K3b/Am cells showed some altered characteristics such as reduced growth rate and tumorigenicity.
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PMID:Effects of adriamycin on the reverse transcriptase and the production of murine leukemia virus. 6 7

Based upon our previous report indicating the presence of retrovirus-like particles in human gastric cancer cells, we analyzed the putative endogenous reverse transcriptase activity these particles should have. To evaluate the specificity of reverse transcription over that displayed by normal cellular DNA polymerases, the following discriminatory criteria were used: 1) resistance to high concentrations of Actinomycin D; 2) sensitivity to preincubation with ribonuclease A; 3) behavior in cesium sulfate isopycnic gradients and 4) size-shifting of putative template-product complexes after RNase exposure in agarose gel electrophoresis. We report a significant endogenous reverse transcriptase activity associated with membrane-encapsidated particles from terminally-illed patients but not in normal counterparts. Although these structures closely resemble retro viruses, a new model is proposed to explain our findings.
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PMID:Further characterization of RNA-dependent-DNA polymerase activity in human gastric cancer. 128 60

Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts. Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000. Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity. The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer. Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length. Synthesis of long cDNA required 8 mM Mg2+, 4 mM Mn2+, 2 mM dNTPs, and saturating levels of enzyme. Actinomycin D efficiently limited the enzyme to the first strand synthesis. Additional characteristics of the fusion protein are described.
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PMID:Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli. 241 Apr 13

Actinomycin D, known for its suppression of cellular RNA synthesis and for the reduction of the rate of synthesis of double-stranded DNA by the RNA tumor virus RNA-dependent DNA polymerase, was found to interact with single-stranded DNA in such a way as to inhibit DNA . DNA and DNA . RNA hybridizations. This finding is discussed in the light of the observation that DNA elongation during DNA synthesis of RNA tumor viruses is blocked in vitro in the presence of actinomycin D. It thus supports the model that hybridization is a necessary step during RNA tumor virus DNA synthesis.
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PMID:Effect of actinomycin D on nucleic acid hybridization: the cause of erroneous DNA elongation during DNA synthesis of RNA tumor viruses in vitro. 626 Jan 49

The aim of this study was to examine the expression of interleukin-1 receptor antagonist (IL-1ra) in human polymorphonuclear cells (PMN) treated with transforming growth factor-beta 1 (TGF beta 1). TGF beta 1 induced IL-1ra transcripts in human circulating PMN and the induction was not blocked by protein synthesis inhibitors. Actinomycin D blocked induction by TGF beta 1 of IL-1ra transcripts, suggesting the involvement of gene transcription. The half life of IL-1ra transcripts was prolonged by TGF beta 1. By reverse transcriptase-polymerase chain reaction, TGF beta 1 was found to augment the transcripts coding for both the intracellular (keratinocyte type) and the secreted form of IL-1ra. TGF beta 1 induced the production of IL-1ra in PMN. Induction of IL-1ra by TGF beta 1 in PMN may represent a further mechanism by which this molecule can counteract the potent pro-inflammatory properties of IL-1.
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PMID:Induction by transforming growth factor-beta 1 of the interleukin-1 receptor antagonist and of its intracellular form in human polymorphonuclear cells. 780 48

We recently identified a gene that is induced by lymphocyte activation (ILA). The sequence of the full-length 1.4-kb cDNA characterized ILA as a new member of the nerve growth factor/tumor necrosis factor (NGF/TNF) receptor family and the human homologue of the murine T-cell-specific receptor 4-1BB. The present study demonstrates ILA mRNA isoforms at 4.4, 4.0, and 1.8 kb in poly-A+ RNA from activated, but not from resting human peripheral blood T lymphocytes. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to study tissue distribution and regulation of ILA expression. The gene was induced in T lymphocytes by phytohemagglutinin (PHA), phorbol myristate acetate (PMA), and antibody to CD3, in B lymphocytes by PMA and antibodies to cell surface Ig, and in blood monocytes by interleukin-1 beta (IL-1 beta), lipopolysaccharide (LPS), and PMA. In T lymphocytes, ILA mRNA was detectable 1.5 hours after stimulation, reached maximal levels at 8 hours, and declined to background levels by 48 hours. Induction of ILA mRNA required protein synthesis and was primarily due to increased transcription. Actinomycin D reduced ILA mRNA levels in activated lymphocytes 50% within 30 minutes, demonstrating a relatively short half-life of this mRNA. Analysis of nonlymphoid cells showed that ILA mRNA was not detectable in resting cells. However, in contrast to the lymphoid-specific expression of the murine 4-1BB gene, ILA was detected in nonlymphoid cells, including epithelial and hepatoma cells after stimulation with IL-1 beta. ILA was not detectable in several brain derived cell lines. The ILA cDNA encodes a 30-kD protein as demonstrated by in vitro translation, and this protein is immunoprecipitated by antisera that were raised against ILA peptides or a glutathione-S-transferase fusion protein. Flow cytometry showed expression of ILA protein on a subset of activated T or B lymphocytes. In conclusion, activation-dependent expression of ILA is found not only in T lymphocytes, but also in B lymphocytes, monocytes, and diverse nonlymphoid cell types.
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PMID:ILA, the human 4-1BB homologue, is inducible in lymphoid and other cell lineages. 784 93

The aim of this study was to examine the expression of interleukin-1 receptor antagonist (IL-1ra) in human myelomonocytic cells treated with IL-13. IL-13 induced IL-1ra transcripts in human circulating monocytes and polymorphonuclear cells (PMN). Induction by IL-13 was not blocked, but rather superinduced, in the presence of the protein synthesis inhibitor cycloheximide. Actinomycin D blocked induction, suggesting involvement of gene transcription. The half-life of IL-1ra transcripts was prolonged by IL-13 from 1.3 hours to 4.5 hours in monocytes and to 12 hours in PMN. By reverse transcriptase-polymerase chain reaction, IL-13 was found to augment the transcripts coding for the soluble form of IL-1ra, but also to induce the expression of the intracellular (keratinocyte) form of IL-1ra, the latter being extremely low or undetectable in myelomonocytic cells. IL-13 induced production of IL-1ra in myelomonocytic cells, augmenting both cell-associated and released protein. Induction of IL-1ra by IL-13 may represent a further mechanism by which this molecule can counteract the potent proinflammatory properties of IL-1.
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PMID:Interleukin-13 induces the production of interleukin-1 receptor antagonist (IL-1ra) and the expression of the mRNA for the intracellular (keratinocyte) form of IL-1ra in human myelomonocytic cells. 790 31

pS2 is an estrogen-induced mRNA species that was originally identified in the breast cancer cell line MCF-7. Exposure of the cells to basic fibroblast growth factor (bFGF) at the concentration of 10-100 ng/ml for 48-72 h resulted in a marked increase in the concentration of pS2 protein in the medium. The polymerase chain reaction with reverse transcriptase revealed that bFGF increased the amount of intracellular pS2 mRNA: immunocytochemical studies showed that exposure to the factor increased the amount of intracellular pS2 protein. Simultaneous addition of cycloheximide with bFGF completely abolished induction of pS2 protein, although it did not affect the induction of pS2 mRNA. Actinomycin D did not affect the stimulatory effect of bFGF on synthesis/secretion of pS2 protein. bFGF effectively abolished decay of the pS2 mRNA level caused by actinomycin D. These results suggest that the induction of the synthesis/secretion of pS2 protein by bFGF occurs at the post-transcriptional level, most probably due to the stabilization of pS2 mRNA. Another finding, that bFGF and estradiol have a synergistic effect on induction of pS2 protein, suggests the possibility that these two inducers act by a different but partly overlapping mechanism.
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PMID:Effect of basic fibroblast growth factor on synthesis/secretion of pS2 protein by human breast cancer cells (MCF-7). 795 94

Primer extension assays using recombinant templates constructed to contain all 256 possible base quartets in a minimum length sequence were used to examine binding of the anticancer drug actinomycin D to single-stranded DNA. Single-stranded templates were generated by digestion of linearized plasmid with the double-strand-specific T7 gene 6 exonuclease. Actinomycin D formed high-affinity, kinetically stable complexes that paused primer elongation at specific sites by HIV-1 reverse transcriptase, Sequenase (modified T4 DNA polymerase), the Klenow fragment of Escherichia coli DNA polymerase, and Vent (exo-) DNA polymerase. Pauses occurred most commonly near G+C-rich nucleotide clusters, including GpC steps, the preferred sites of double-stranded DNA binding. Complexes were stable for several minutes at temperatures over 50 degrees C as determined by their abilities to pause Vent polymerase at elevated temperatures. Significant variations were noted in pause patterns of different polymerases, demonstrating differential responses of polymerases to a bound actinomycin. Covalent adducts formed on template DNA by a photoaffinity analog of actinomycin D completely stopped primer extension. These results support the possibility that actinomycin D inhibits transcription elongation by complexing single-stranded DNA in the open transcription complex. Single-stranded DNA binding by actinomycin D or analogs may also provide routes for combating HIV or other viruses which replicate through single-stranded intermediates.
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PMID:Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptase and other polymerases. 863 3

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


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