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)

From lots of 20 to 30 g of avian myeloblastosis virus RNA-dependent DNA polymerase was obtained in preparations of purity greater than 95% by using a two-step column chromatographic procedure employing DEAE (DE 52) and carboxymethylcellulose (CM 52.). Yields of RNA-dependent DNA polymerase varied from approximately 20,000 to 35,000 U/g of virus. Specific activity of the enzyme was about 35,000 to 60,000 U/mg of protein. Free of detectable RNase activity, the product exhibited a molecular weight of about 160,000, an isoelectric point of 6.5, and approximately 2 mol of fatty acid per mol of enzyme.
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PMID:Reverse transcriptase from avian myeloblastosis virus. 8 21

We have investigated the use of oligodeoxycytidylic acid [oligo(dC)] as a primer for the initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro, employing the viral RNA genome as template. The addition of oligo(dC)(12-18) to viral 35S RNA results in a stimulation of DNA synthesis by the viral RNA-directed DNA polymerase comparable to that observed when oligo(dT) is employed as a primer. Under similar conditions neither oligo(dA)(12-18) nor oligo(dG)(12-18) was active as primer for transcription of the avian retrovirus genome. Several different approaches have been employed to localize the oligo(dC)(12-18) binding site on the viral genome, including isolation of poly(A)-containing fragments, competition hybridization, and RNase H hydrolysis. These analyses indicate that oligo(dC)(12-18) binds to a site approximately 2,000 to 3,000 nucleotides from the 3' terminus of the genome of transforming strains of avian sarcoma viruses and approximately 700 to 1,000 nucleotides from the 3' terminus of nontransforming avian retroviruses. Therefore, the major site of initiation of DNA synthesis by oligo(dC)(12-18) appears to be in the vicinity of the 3' end of the env gene and the 5' end of the src gene, although the presence of minor initiation sites located elsewhere on the viral genome cannot be excluded by these data. Characterization of oligonucleotides after pancreatic RNase hydrolysis and poly(C)-Sepharose chromatography of viral RNA directly demonstrates the presence of oligoguanylic acid residues in the avian sarcoma virus genome. DNA sequences transcribed from the oligo(dC) primer appear to be conserved in all of the avian leukosis-sarcoma viruses tested. The use of oligo(dC) as a tool for the production of specific complementary DNA probes is discussed.
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PMID:Initiation of DNA synthesis by the avian retrovirus reverse transcriptase in vitro: nature and location of the oligodeoxycytidylic acid primer binding site. 9 Jan 58

The genetic compositions of two independently derived preparations of the Bratislava-77 strain (B77) of Rous sarcoma virus were analyzed after each was passaged seven or more times in duck embryo fibroblasts. RNase, T1-resistant oligonucleotide fingerprint analysis of virion RNA from both preparations of duck-passaged B77 revealed the presence of two large noncontiguous deletions. Approximately 75% of the RNAs contained a deletion which spans oligonucleotides 304 to 4 on the viral genome (about 3,500 nucleotides) and encompasses all of the B77 polymerase gene. More than 90% of the RNAs also contained a deletion which spans src-specific oligonucleotides 6 and 5(about 2,200 nucleotides) and is identical to the deletion observed in transformation-defective B77. Virion RNA from duck-passaged B77 also contained two oligonucleotides (D1 and D2) not observed in the RNA of B77 virus grown on chicken embryo fibroblasts. Analysis of the virion RNA of duck-passaged B77 by denaturing agarose gel electrophoresis revealed four major subunits with molecular weights of 3.40 x 10(6), 2.65 x 10(6), 2.25 x 10(6), and 1.55 x 10(6). Whereas the 3.40- and 2.65-megadalton (Mdal) RNA species comigrated with the nondefective and transformation-defective RNAs of B77 propagated on chicken embryo fibroblasts, no counterparts to the 2.25- and 1.55-Mdal RNAs were observed in the RNA of B77 grown on chicken embryo fibroblasts. Oligonucleotide fingerprint analysis of these RNA species revealed that the 2.65-Mdal RNA contains the src-specific deletion and that 2.25-Mdal RNA contains the polymerase region deletion; both of these deletions were observed in the 1.55-Mdal RNA, which was the major RNA subunit species detected in duck-passaged B77. The new oligonucleotides (D1 and D2) observed in the duck-passaged virus were present in the 2.25- and 1.55-Mdal RNA species in vitro and in vivo and directs the synthesis of a 130,000-dalton protein (p130). p130 contains antigenic determinants specific for p27 (gag gene) and gp85 (env gene) but does not contain sequences which cross-react with antisera directed against the alpha beta form of RNA-dependent DNA polymerase (pol gene). This RNA, therefore, is generated by a fusion of the gag and env genes of Rous sarcoma virus B77.
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PMID:Deletion mutant of the Bratislava-77 strain of Rous sarcoma virus containing a fusion of the group-specific antigen and envelope genes. 9 86

32P-labeled light chain messenger RNA was prepared from mouse MOPC 21 myeloma cells. The messenger RNA was hybridized to purified repetitive nuclear DNA and both the hybridized (repetitive 32P-RNA) and nonhybridized (nonrepetitive 32P-RNA) fractions were isolated. Only the nonhybridized RNA gave a T1 ribonuclease fingerprint showing oligonucleotides derived from the variable and constant regions of the light chain messenger RNA. In addition, this fingerprint showed oligonucleotides derived from the untranslated regions of the light chain messenger RNA. The nonrepetitive 32P-RNA was shown to rehybridize only with the unique fraction of total nuclear DNA. The rapidly hybridizing part of the unfractionated 32P-RNA preparation, therefore, is not a component of the light chain messenger RNA itself. Complementary DNA was prepared with reverse transcriptase using unlabeled light chain messenger RNA as template, and the transcripts were fractionated into various size classes. Complementary DNA molecules greater than 900 bases in length hybridized with both the initial messenger RNA and with the nonrepetitive 32P-RNA but failed to hybridize with excess purified repetitive 32P-RNA. The rapidly hybridizing component of the messenger RNA fraction, therefore, does not appear to be transcribed by reverse transcriptase. It is concluded that, under the experimental conditions used, the light chain messenger RNA hybridizes exclusively with unique DNA.
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PMID:Demonstration that a mouse immunoglobulin light chain messenger RNA hybridizes exclusively with unique DNA. 80 42

The molecular weight of fibroin messenger RNA has been measured by its contour length in the electron microscope and its electrophoretic mobility under conditions of complete denaturation. These methods give values of 5.6 times 10-6 and 6.0 times 10-6 daltons, respectively. The average molecular weight of 5.8 times 10-6 (1.6 times 10-4 base residues) would be expected to code for a protein of 414,000 daltons, a value about 12 percent larger than the most recently published size for fibroin. When the mRNA is prepared carefully to avoid shear breakage, it is as homogeneous in size as ribosomal RNA. Between 43 percent and 81 percent of the mRNA molecules bind to oligo(dT)-cellulose and are presumed to contain a poly(A) sequence. This sequence, isolated after ribonuclease digestion, comprises about 0.6 percent of the molecule, or an estimated 100 nucleotides. The poly(A) has been localized at the end of the molecule by hybridizing with a small poly (dT:dA) piece and subsequent visualization by electron microscopy. This observation, along with the ability of reverse transcriptase to transcribe fibroin mRNA with an oligo(dT) primer, demonstrates that the poly(A) is at the 3' end of the mRNA.
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PMID:The size of fibroin messenger RNA and its polyadenylic acid content. 112 53

Two DNA strand transfer reactions occur during retroviral reverse transcription. The mechanism of the first, minus strand strong-stop DNA, transfer has been studied in vitro with human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT) and a model template-primer system derived from the HIV-1 genome. The results reveal that HIV-1 RT alone can catalyze DNA strand transfer reactions. Two kinetically distinct ribonuclease (RNase) H activities associated with HIV-1 RT are required for removal of RNA fragments annealed to the nascent DNA strand. Examination of the binding of DNA.RNA duplex and single-stranded RNA to HIV-1 RT during strand transfer supports a model where the enzyme accommodates both the acceptor RNA template and the nascent DNA strand before the transfer event is completed. The polymerase activity incorporated additional bases beyond the 5' end of the RNA template, resulting in a base misincorporation upon DNA strand transfer. Such a process occurring in vivo during retroviral homologous recombination could contribute to the hypermutability of the HIV-1 genome.
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PMID:Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. 127 6

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.
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PMID:Mechanism and fidelity of HIV reverse transcriptase. 128 79

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

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.
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PMID:Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. 137 Oct 14

Primer tRNA regions involved in the interactions between human immunodeficiency virus reverse transcriptase (HIV RT) and tRNA(Lys) were studied by digestion of primer with pancreatic ribonuclease in the presence or absence of HIV RT. The acceptor stem of tRNA(Lys) is not noticeably protected against nuclease action in the presence of HIV RT, while this enzyme clearly protects part of the anticodon and dihydrouridine loops of tRNA(Lys). The acceptor stem of primer tRNA was digested by RNase A only in the presence of the retroviral enzyme, suggesting a partial destabilization of this region by the HIV RT. Synthetic oligoribonucleotides, corresponding to the anticodon and the dihydrouridine loops, inhibited strongly reverse transcription, confirming the strong interaction of these tRNA regions with the enzyme.
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PMID:Preferential interaction of human immunodeficiency virus reverse transcriptase with two regions of primer tRNA(Lys) as evidenced by footprinting studies and inhibition with synthetic oligoribonucleotides. 137 51


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