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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer. Mutagenesis of the protease-sensitive region (residues 430-440), which is part of the crystallized domain, indicates that any alteration of the wild-type sequence results in increased proteolysis of the p66 subunit. A model of asymmetric processing in HIV-1 reserve transcriptase which involves partial unfolding of the RNase H domain is proposed based on these results and the recently reported three-dimensional structure of this domain.
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PMID:Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase. 171 88

Phosphonoformate, an inhibitor of reverse transcriptase in a number of retroviruses, was shown to have a dose-related inhibitory effect on human T-cell lymphotropic virus type III (HTLV-III) replication in the H9 cell line in vitro. HTLV-III replication was eliminated at a concentration of 680 mcgmol, a noncytotoxic dose. A lower dose of 132 mcgmol inhibited HTLV-III replication by more than 98%, as measured by reverse transcriptase activity, compared with untreated infected cultures. Reverse transcriptase activity in HTLV-III particles was completely inhibited by 5 mcgmol of phosphonoformate. Growth of uninfected H9 cells was not affected by the concentration of the drug. In clinical trials to treat cytomegalovirus infection in immunocompromised patients, constant serum levels of between 100-450 mcgmol of phosphonoformate have been achieved in 140 subjects. Further studies are recommended to evaluate the potential of phosphonoformate in patients infected with HTLV-III. It may be the least toxic of the antiviral agents that have been shown to have anti-HTLV-III activity.
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PMID:Inhibition of human T-cell lymphotropic virus type III in vitro by phosphonoformate. 240 14

Reverse transcriptase of murine retroviruses is a monomeric protein of approximately 80,000 daltons, which is encoded by the central portion of the viral pol gene. To prepare large quantities of the enzyme, we have constructed gene fusions between the trpE gene and portions of the pol gene of Moloney murine leukemia virus. The inserted pol gene sequences include the entire coding region for the mature enzyme and various amounts of additional coding sequences. Many of these constructs express high levels of reverse transcriptase activity even though the NH2 and COOH termini of the protein product only approximate the correct termini of the authentic protein.
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PMID:Expression of enzymatically active reverse transcriptase in Escherichia coli. 241 Sep 10

Reverse transcriptase activity was found in rat liver enclosed in virus-like particles. Through hybridization with DNA probes of A- and C-type retroviruses and with the help of electron microscopy the virus-like particles have been identified as endogenous retroviruses related to the mouse intracisternal A-particles. Blot hybridization revealed the provirus DNA in the rat genome. The enzyme was isolated from the virus-like particles, purified and characterized. The main properties of the enzyme resemble those of the mammalian retrovirus reverse transcriptase.
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PMID:Reverse transcriptase of rat liver associated with the endogenous retrovirus related to the mouse intracisternal A-particles. 241 55

The NusA protein of Escherichia coli is a factor which mediates termination of transcription. In this paper, we demonstrate that the NusA protein can bind in vitro to a specific site on the mRNA of bacteriophage lambda. Several RNAs were synthesized by in vitro transcription of truncated lambda DNA templates, and the activity of NusA binding to these RNAs was examined by a Millipore filter-binding assay. RNAs containing the sequence immediately upstream of the boxA site were trapped on the filter by association with the NusA protein, but those lacking the site were not. Anti-NusA antibody inhibits this binding. To determine the binding site precisely, we developed a new method which we have named 'reverse-transcriptase mapping'. The RNA transcribed from the pL promoter was incubated with 32P-labelled DNA primer and NusA, and the primer-extension reaction was started by adding the reverse transcriptase. In this way, the primer extension was blocked at the position G of the boxA RNA sequence (5'CGCUCUUA 3'), indicating that the NusA-protection site is immediately upstream of boxA and includes the 5'-end C. The NusA protein purified from a temperature-sensitive nusA mutant defective in transcription termination showed reduced and thermolabile RNA-binding activity, suggesting that the RNA-binding activity is related to the physiological function of NusA.
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PMID:E. coli NusA protein binds in vitro to an RNA sequence immediately upstream of the boxA signal of bacteriophage lambda. 241 64

Reverse transcriptase activity of the human immunodeficiency virus (HIV) was blocked in vitro by immunoglobulin G (IgG) derived from certain individuals infected with this retrovirus. A heterogeneous immune response for inhibition of enzyme function was noted. Catalytic activity was depressed by 50% or more with the use of 10 micrograms of IgG from 11 of 16 HIV-seropositive asymptomatic carriers, but from 0 of 8 seronegative controls and 2 of 12 patients with acquired immune deficiency syndrome (AIDS) or the AIDS-related complex (ARC). The inhibitor was confined to the F(ab')2 fragment. It was not directed against the poly(rA) X oligo(dT) template, nor against major envelope or structural viral antigens, and did not cross-react with bacterial, avian, or other mammalian DNA polymerases. It did not correlate with recognition of polymerase antigens by radioimmunoprecipitation. Loss of this inhibitor may be associated with development of clinical disease. Ten asymptomatic HIV-seropositive carriers with high titers of IgG antibodies to reverse transcriptase were followed for a mean of 3 years. All of four lost inhibitory capability prior to development of AIDS or ARC, while titers persist in the six who remain clinically healthy.
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PMID:Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity. 243 4

An attempt to study the functional role of one of the most conservative domains found in all RNA-dependent RNA and DNA polymerases of plant and animal viruses (the so called "DD-domain") was made. A structure similar to the "DD-domain" was found in a minor T7 phage tail protein--gpII. Antibodies against this phage protein have been raised and used to probe "DD-domain" in molecules of avian myeloblastose virus reverse transcriptase and E. coli RNA-dependent RNA polymerase. The antibodies are shown to inhibit the activity of these enzymes under certain conditions. At the same time inhibition of the reverse transcriptase reaction causes the decrease in length of the most high molecular cDNA-products as well. The experimental data obtained are discussed in view of the suggested hypothesis on the probable functional role of the "DD-domain" of RNA-dependent polymerases.
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PMID:[Possible functional role of the DD-domain of RNA-dependent polymerases]. 243 38

3'-Azido-3'-deoxythymidine triphosphate (erythro) and its threo isomer were synthesized and investigated for their inhibition of HIV reverse transcriptase from virus isolate U 937/HTLV-III. The erythro isomer was a competitive inhibitor of the reaction directed by (rA)n(dT)12-18 and the Ki value was 0.0022 microM. The threo isomer was at least 100-fold less active. The inhibition was specific for dTMP incorporation, and dGMP incorporation using (rC)n(dG)12-18 as template/primer was not affected. The Km value for dTTP varied between 0.7 microM and 1.7 microM. Reverse transcriptase from nine HIV isolates were tested for inhibition by the erythro isomer and only slight differences in sensitivity were observed.
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PMID:Inhibition of the reverse transcriptase from HIV by 3'-azido-3'-deoxythymidine triphosphate and its threo analogue. 244 Mar 80

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

A mechanism to explain somatic hypermutation in immunoglobulin variable region genes is proposed employing polynucleotide information transfer through the error prone DNA----RNA----DNA loop. During transcription of the rearranged V-region, the primary transcript undergoes either inappropriate termination, cleavage, or reverse transcriptase priming allowing a V-region specific reverse-transcriptase-integrase complex to synthesize a DNA copy of the rearranged V-region and integrate it, by homologous recombination, back into the normal chromosomal site. Some consequences and predictions of the hypothesis are discussed.
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PMID:Hypothesis: somatic hypermutation by gene conversion via the error prone DNA----RNA----DNA information loop. 244 41


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