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)

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as a template for in vitro synthesis of 32P-labeled RNA and deoxysubstituted RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. In addition, the 3' nearest neighbor was determined for several fragments resulting from digestion with T1 ribonuclease. The utility of the deoxysubstitution technique was demonstrated by the ease with which the sequences of pyrimidine-rich fragments could be determined. Many sequences thus determined were long enough to fit uniquely with the alpha- or beta-globin amino acid sequences. The positions of these fits were found to be clustered, leading us to believe that only certain regions of the complementary DNA are transcribed by Escherichia coli RNA polymerase. Other unique characteristics of RNA synthesis from a complementary DNA template include a high yield of free poly(A) and the fact that one must use low rather than high salt buffers to obtain transcripts of high molecular weight.
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PMID:Rabbit globin mRNA: analysis of T1 RNAse digestion fragments. 6 35

The sequence of 129 nucleotides next to the poly(A) tail of encephalomyocarditis virus RNA has been determined by rapid gel sequencing of cDNA synthesized with DNA polymerase I or reverse transcriptase and a phasing primer, [5'-32P]p(dT)8dC. The sequence is in accord with (a) the pyrimidine tracts which were mapped in blocks along the cDNA, (B) the sequences of seven characteristic T1 RNase oligonucleotides in the RNA transcribed from the cDNA with RNA polymerase, and (c) a limited amount of sequence deduced by partial spleen phosphodiesterase digestion and depurination of endonuclease IV oligonucleotides. The 3' end shows little secondary structure on its own. Ten nonsense codons block all three reading frames such that at least 26 nucleotides do not code for protein. The possible function of a homology A-A-U-A-A-A with other polyadenylated RNAs is discussed.
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PMID:Sequence of 129 nucleotides at the 3'-terminus of encephalomyocarditis virus RNA. 7 85

In view of recent developments, the structural determinants of the interferon inducing activity of polynucleotides have been (re)evaluated. To induce interferon, the polynucleotide should be sufficiently large and double-stranded, although not necessarily double-stranded over its whole length. It should be sufficiently stable to both thermal denaturation and hydrolysis by nucleases. It should also contain a particular steric conformation. This conformation is most regularly ensured by the presence of 2'-hydroxyl in the ribose moieties and intact purine-pyrimidine base pairs in the interior of the double helix. Other biologic activities of polynucleotides, such as anti-complement activity and inhibition of reverse transcriptase (RNA-directed DNA polymerase) activity, depend on structural requirements which are rather antagonistic to those governing the interferon response.
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PMID:Interferon induction by polynucleotides: structure-function relationship. 35 56

A series of pyrimidine nucleoside analogues containing [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino- 1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-ribofuranose as the pentose were found to inhibit human immunodeficiency virus type 1 [HIV-1(IIIB)] replication at a concentration of 0.06-0.8 microM but were not cytotoxic at a 1000- to 10,000-fold higher concentration. These nucleoside derivatives were also effective against various other HIV-1 strains, including those resistant to 3'-azido-3'-deoxythymidine, but not against HIV-2, simian immunodeficiency virus, Moloney murine sarcoma virus, or other RNA or DNA viruses. They proved to be highly specific inhibitors of the RNA-dependent DNA polymerase function of the HIV-1 reverse transcriptase, showing no marked inhibition of the HIV-1 reverse transcriptase-associated DNA-dependent DNA polymerase activity, HIV-2 reverse transcriptase, DNA polymerase alpha, herpes simplex virus 1 DNA polymerase, or Thermus aquaticus DNA polymerase.
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PMID:2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2'-dioxide)pyrimidine (TSAO) nucleoside analogues: highlyselective inhibitors of human immunodeficiency virus type 1 that are targeted at the viral reverse transcriptase. 137

We have adopted the in vitro hepatocyte culture system of the duck infected with duck hepatitis B virus (HDBV) to an anti-viral assay system. Using this method, we found that 2',3'-dideoxy-3'-azidothymidine (N3dT) and 2',3'-dideoxy-3'-O-methylthymidine (OMeT) had antiviral effects against DHBV replication in the concentrations of 20-50 mumol/l and 4-40 mumol/l, respectively. The N3dT inhibited the single strand DNA formation (negative strand), which is an intermediate of virus replication. However, the inhibition of single strand DNA synthesis by OMeT was relatively weak. These two compounds may have different mechanisms of DHBV DNA replication inhibition. Two other 3'-substituted pyrimidine analogues tested were very weak inhibitors. Antiviral agents that inhibit the reverse transcriptase activity of the hepadnavirus DNA polymerase could be potential candidates for the chemotherapy of these viruses.
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PMID:Inhibition of duck hepatitis B virus replication in vitro by 2',3'-dideoxy-3'-azidothymidine and related compounds. 168 76

A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.
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PMID:Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. 168 52

The genome of human immunodeficiency virus (HIV) and especially the envelope gene are mutated with unusually high frequency during in vivo replication. Recent studies indicate that HIV reverse transcriptase (RT) is unusually error prone and that the number of generated mutations is disproportionately high within repetitive base sequences. To study the ability of recombinant and wild-type HIV RT to traverse specific homo-oligomeric stretches, we used bacteriophage M13 DNA templates that contain different oligo(purine) and oligo(pyrimidine) inserted tracts. The progress of HIV RT along these templates was potently inhibited from further progression only at a (dA)16 insert. Comparison with other polymerases indicates that the almost complete blockage of polymerization beyond an oligo(dA) insert is unique to HIV RT and Moloney murine leukemia virus RT, which has high sequence homology with HIV RT. The extent of termination of HIV RT at the oligo(dA) run is not affected by alterations in the concentration of KCl, Mg2+, dNTP, or by a decrease in pH. Obstruction of HIV RT opposite the oligo(dA) insert is not alleviated by moving the primer position further upstream from the oligo(dA) insert. Lastly, HIV RT purified directly from virions is also specifically arrested at an oligo(dA) tract. Competition experiments indicate that the concentration of active HIV RT in the presence of M13(dA)16 DNA is similar to that observed in the presence of M13(dG)16 DNA. In addition, preincubation of M13(dA)16 DNA with HIV RT does not subsequently inhibit avian myeloblastosis virus RT from successfully traversing the (dA)16 insert. Therefore, it appears that the blockage of chain elongation of HIV RT at the (dA)16 insert is not the result of trapping the enzyme at this site.
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PMID:Synthesis of DNA by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts. 169 89

Human immunodeficiency virus type 1 reverse transcriptase (EC 2.7.7.49), a heterodimer consisting of two polypeptide chains of molecular weights 66,000 and 51,000, fluoresces due to the presence of 36 tryptophan residues with an emission peak centered at 338 nm. The association of 2'-deoxynucleoside 5'-triphosphates with the enzyme results in a decrease in the intensity of the tryptophan emission spectrum, which can be used to calculate apparent dissociation constants. The Kd values determined for binding of the four natural 2'-deoxynucleoside 5'-triphosphates to the free enzyme range from 36.7 +/- 1.8 microM for dTTP to 47.3 +/- 3.9 microM for dATP. The 5'-triphosphate of zidovudine has a Kd of 54.1 +/- 1.3 microM. The enzyme shows no preference for purine or pyrimidine nucleotides. Hill coefficients and the results of dual ligand titration experiments demonstrate that the free enzyme possesses a single dNTP binding site for which the four natural substrates and the 5'-triphosphate of zidovudine compete. The presence of homopolymeric template-primers does not result in selective binding of the complementary 2'-deoxynucleoside 5'-triphosphate, indicating that Watson-Crick base pairing is not involved in the initial binding reaction. The major force driving the association of the ligands with the binding site is hydrophobic. Approximately 14% of the binding energy is derived from electrostatic interactions. Although Mg2+ is required for catalytic activity, it is not absolutely required for initial binding.
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PMID:Initial binding of 2'-deoxynucleoside 5'-triphosphates to human immunodeficiency virus type 1 reverse transcriptase. 171 63

The general physical characteristics and replication of retroviruses are considered, along with assays for viral products. The specific agent for acquired immunodeficiency syndrome, the human immunodeficiency virus (HIV), is characterized as a lentivirus causing persistent, lifelong infection. While human immunodeficiency virus retroviruses share many of the same properties as other replication-competent viruses, genetic variability occurs among HIV isolates, and this variability may have a considerable effect on the virus' virulence, cell type specificity, viral susceptibility to antiviral compounds, clinical presentation, and disease progression. The most notable difference between HIV replication and other retroviruses is the intricate control of HIV gene expression by viral and cellular factors. Possible mechanisms by which HIV kills infected cells include the formulation of multinucleate syncytia; cytopathic components within the virions themselves; and interaction between viral envelope proteins and the CD4 molecule on the cell surface. Agents shown to inhibit viral replication at the level of the reverse transcriptase are phosphonoformate, sulfated polysaccharides, rifabutin, and nucleoside analogs, as well as purine and pyrimidine analogs. To date, only one nucleoside analog, zidovudine, has demonstrated clear clinical benefit and anti-HIV activity.
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PMID:Biology of retroviruses: detection, molecular biology, and treatment of retroviral infection. 219 45

We have carried out a systematic investigation of the efficiency of misincorporation by Avian Myeloblastosis Virus reverse transcriptase with all possible combinations of dNTP substrate, template nucleotide, and the nucleotide at the 3' terminus of the primer. A series of synthetic oligonucleotide primers were annealed to single stranded M13 DNA templates, and a single dNTP was misincorporated at the primer 3' end using AMV reverse transcriptase. The proportion and pattern of misincorporation and incorporation in all 64 situations was assayed using [5'-32p] labelled primers, and the products were separated on denaturing polyacrylamide gels. Correct incorporations occurred more readily than misincorporations. The efficiency of misincorporation depended on the individual primer, but, comparing primers, a clear dependence on the template nucleotide was observed for the preferential misincorporation of different dNTPs. The exact combination of template and dNTP was important; although purine:pyrimidine (dNTP substrate:template nucleotide) and pyrimidine:purine misincorporations occurred comparatively readily, some pyrimidine:pyrimidine and purine:purine reactions were equally efficient and yet others were never seen to occur. Some misincorporations were facilitated by subsequent correct incorporations, but despite this our results suggest that the level of misincorporation is limited by the rate of reaction and enzyme inactivation rather than by exonuclease activity.
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PMID:Misincorporation by AMV reverse transcriptase shows strong dependence on the combination of template and substrate nucleotides. 242 57


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