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)

A protein kinase associated with purified virions of avian myeloblastosis BAI strain A was partially purified by ion-exchange chromatography and gel filtration. The transfer of phosphate catalyzed by this enzyme required a divalent metal ion and ATP as phosphate donor. GTP could not be substituted for ATP, and the reaction was unaffected by either cyclic AMP or beef-heart protein-kinase inhibitor. Of the virus and nonvirus proteins tested as phosphate acceptors, only acidic proteins were phosphorylated. In particular, purified preparations of reverse transcriptase from avian myeloblastosis virus did not accept phosphate. The enzyme is a basic protein (pI = 9.3), and, on the basis of molecular sieving through Sephadex G-200 and velocity sedimentation on glycerol gradient, the protein kinase has a molecular weight of 45,000.
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PMID:Protein kinase from avian myeloblastosis virus. 2 25

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety.
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PMID:Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine. 127 98

Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir, NSC 614846) is an anti-retroviral agent that may be useful in the treatment of AIDS. We have examined the ability of (-)-enantiomeric carbovir triphosphate to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49). A comparison of inhibition kinetics was made with 3'-azido-2',3'-dideoxythymidine triphosphate and phosphonoformate. Inhibition of the reverse transcriptase was evaluated using poly(rA).oligo(dT)12-18, poly(rC).oligo(dG)12-18, or influenza virion RNA template with a specific oligodeoxynucleotide as primer. (-)-Carbovir 5'-triphosphate was shown to be a potent inhibitor of HIV-1 reverse transcriptase with an apparent Ki similar to that of 3'-azido-2',3'-dideoxythymidine triphosphate. Chain elongation studies utilizing an MS2 RNA template showed that (-)-carbovir 5'-triphosphate terminated transcription at positions identical to those where dideoxy-GTP terminated. This indicates that (-)-carbovir 5'-monophosphate is incorporated into the newly synthesized DNA and terminates transcription at that point. We conclude that (-)-carbovir 5'-triphosphate is a potent inhibitor of the HIV-1 reverse transcriptase enzyme and that (-)-carbovir most likely inhibits HIV by activity at the triphosphate level by a combination of direct competition for binding of the natural deoxynucleoside triphosphates to the reverse transcriptase and chain termination.
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PMID:DNA chain termination activity and inhibition of human immunodeficiency virus reverse transcriptase by carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine triphosphate. 137 Dec 85

The 47-kDa lipoprotein is an abundant integral membrane protein and dominant immunogen of Treponema pallidum subsp. pallidum. Previous DNA sequencing of the 47-kDa-lipoprotein gene did not reveal consensus features representative of other bacterial lipoprotein genes; this prompted further analyses with emphasis on elucidation of the N terminus of the molecule. To assist in localizing start signals for the protein, the transcription initiation site for the 47-kDa-antigen gene was determined. RNA isolated from both T. pallidum and recombinant Escherichia coli expressing the 47-kDa antigen was used as a template in reverse transcriptase primer extension. Upon analysis of cDNA products, transcription initiation was localized to one nucleotide in T. pallidum and to two adjacent nucleotides in E. coli. When various primers were used in DNA sequencing reactions for these analyses, a previously undetected nucleotide (G) was found in the purported 5' untranslated region; this altered the upstream reading frame to reveal plausible sites for ribosome binding (GGAGG), translation initiation (GTG start codon), and signal peptidase II processing (Val-Val-Gly-Cys). Discounting acylation, the molecular weight of the mature polypeptide is 45,756 (approximately 46,600 with acylation). To derive nonacylated 47-kDa antigen for further structure-function studies, the 47-kDa-antigen gene was subcloned without acylation signals as a genetic construct encoding a glutathione S-transferase fusion protein; following cleavage with thrombin, the nonacylated 47-kDa protein was hydrophilic rather than amphiphilic but retained its antigenicity when tested against 116 human serum samples from patients with various stages of syphilis.
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PMID:Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum. 137 97

In order to clarify the biological activities of (-)-oxetanocin G, and (-)-oxetanocin A and its carbocyclic analogue, (-)-carboxetanocin G, the inhibitory effects of triphosphate derivatives of these compounds (OXT-GTP, OXT-ATP, and C-OXT-GTP) on eukaryotic and viral DNA polymerases were examined. DNA polymerase alpha purified from calf thymus was weakly inhibited by OXT-GTP and OXT-ATP but strongly by C-OXT-GTP, the Ki value being 0.22 microM. On the other hand, rat DNA polymerase beta was not affected by these analogues. DNA polymerase gamma purified from bovine testes was very weakly inhibited by OXT-GTP and OXT-ATP, but not by C-OXT-GTP. DNA polymerase from herpes simplex virus type-II (HSV-II) was strongly inhibited by all three analogues, the Ki values ranging from 0.5 to 1.0 microM. Human immunodeficiency virus-encoded reverse transcriptase (HIV RT) was also strongly inhibited by these three analogues, the Ki value of C-OXT-GTP being slightly smaller than that of OXT-GTP or OXT-ATP. Analysis of products synthesized on singly primed M13 single-stranded DNA by DNA polymerase alpha, HSV-II DNA polymerase or HIV RT in the presence of the analogues revealed that OXT-GTP and C-OXT-GTP were incorporated into DNA and caused chain termination mainly at sites one or two nucleotides beyond the cytosine bases on the template.
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PMID:Inhibitory effects of triphosphate derivatives of oxetanocin G and related compounds on eukaryotic and viral DNA polymerases and human immunodeficiency virus reverse transcriptase. 138 92

The expression and biological function of interleukin-6(IL-6), and its receptor mRNA, were studied in a human megakaryocytic cell line (CMK). IL-6 possessed stimulatory effects on the DNA synthesis as well as colony formation of CMK cells. The IL-6 receptor mRNA could be detected by the method of reverse transcriptase polymerase chain reaction (RT-PCR) but not Northern blotting. On the contrary, IL-6 mRNA was detected by the method of RT-PCR, and its expression induced by the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) could be clearly shown by Northern blotting. These findings indicate that IL-6 and its receptor mRNA should be analyzed by both methods, and the growth and differentiation of CMK cells may be controlled by an IL-6 autocrine loop. Next, the expression and biological role of low molecular GTP-binding proteins (smg p21A and -B) mRNAs were examined in CMK cells. Both the smg p21A and -B mRNAs were detected in CMK cells using Northern blotting, and their levels were markedly elevated by TPA treatment. The mRNA level of glycoprotein IIb, a typical marker of the megakaryocytes, was increased by TPA, but the time course of the increase in the smg p21 mRNA levels was more rapid that that in the GPIIb mRNA level. These findings suggest that smg p21s play an important role during the TPA-induced differentiation of CMK cells.
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PMID:[Expression and detection of platelet specific genes in human megakaryocytic cells]. 177 68

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensitive events. Other G alpha-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to Gi-2 alpha and Gs alpha protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G alpha gene family, at least two other G alpha subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G alpha subunits were isolated and characterized. The results indicate that this G alpha subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.
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PMID:Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells. 190 75

Four flavonoids (i.e., baicalein, quercetin, quercetagetin, and myricetin), known to be inhibitors of HIV-reverse transcriptase, have been shown to be more or less inhibitory to the activities of various cellular DNA and RNA polymerases. The degree of the inhibition varied depending on the combination of the flavonoid and the enzyme species: baicalein was moderately inhibitory to DNA polymerase gamma and E. coli DNA polymerase I; quercetin was strongly inhibitory to DNA polymerase beta and E. coli RNA polymerase and moderately inhibitory to DNA polymerase I; quercetagetin was a potent inhibitor for all of DNA polymerases alpha, beta, gamma, and I and RNA polymerase; myricetin was a strong inhibitor of DNA polymerases alpha and I and RNA polymerase. However, terminal deoxynucleotidyltransferase was virtually insensitive to inhibition by these flavonoids. The inhibition by the flavonoids was due to competition with the template.primer in the case of the DNA polymerases, whereas the inhibition was due to competition with the triphosphate substrate (GTP) in the case of RNA polymerase. The Ki values of these flavonoid inhibitors for DNA and RNA polymerases was determined.
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PMID:Mechanisms of inhibition of various cellular DNA and RNA polymerases by several flavonoids. 229 90

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.
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PMID:Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. 244 94

Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system. Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis. The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data. Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits. Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop. This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site. Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding. Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV. Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites. alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain. Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.
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PMID:Domain VI of Escherichia coli 23 S ribosomal RNA. Structure, assembly and function. 246 15


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