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)

We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)). The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea. RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit. Cleavage or modification sites were detected by primer extension with reverse transcriptase. We present a three-dimensional model derived from mapping experiments and graphic modeling. Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove. The resulting helix is slightly unwound. Comparative analysis of probing experiments leads to several conclusions. (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs. (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment. (3) The fragment contains the complete information required for S8 binding. (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites. (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.
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PMID:Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA. A model for the interaction and the tertiary structure of the RNA binding site. 332 31

RNases and chemical probes were used to study the accessibility of each nucleotide of 5S RNA in the native and reconstituted 7S particle from Xenopus laevis oocytes. RNase or chemically treated 5S RNA from intact 7S particles was isolated and analysed using an oligodeoxynucleotide primer and reverse transcriptase. The results were superimposed on a cylindrical projection of an RNA double helix and the protection effects were shown to cluster at two regions on the molecular surface. A three-dimensional model is proposed for the 7S particle in which protein-RNA contacts occur mainly in the major groove of 5S RNA.
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PMID:Xenopus transcription factor IIIA binds primarily at junctions between double helical stems and internal loops in oocyte 5S RNA. 358 66

Pichinde virus, a member of the arenavirus group, was examined for polymerase activity. Purified virus was found to contain RNA-dependent RNA polymerase but not RNA-dependent DNA polymerase activity. Since RNase but neither DNase nor actinomycin D inhibited the endogenous polymerase reaction, RNA of the virus appeared to be used as the template. The divalent cations Mg(2+) and Mn(2+) were required for optimal reactivity. The RNA product was partially resistant to RNase and the resistant portion had a sedimentation coefficient of 22 to 26S in sucrose gradients.
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PMID:Polymerase activity of Pichinde virus. 413 69

An RNA-dependent DNA polymerase associated with intracisternal A particles has been characterized. The enzyme required Mg(2+) or Mn(2+), dithiothreitol and the presence of all four deoxyribonucleoside triphosphates for the expression of maximal activity. Sensitivity of the endogenous RNA-dependent DNA polymerase activity to a low concentration of pancreatic ribonuclease in the presence of a high concentration of NaCl suggested that the enzyme might be utilizing the A particle endogenous RNA as template. Evidence in support of this was provided by analyses of early and late DNA products of the endogenous reaction by Cs(2)SO(4) isopycnic gradient centrifugation and hybridization of purified 60 to 70S and 35S RNAs of A particles with the purified DNA product.
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PMID:Characterization of an endogenous RNA-dependent DNA polymerase associated with murine intracisternal A particles. 413 71

An RNA-directed DNA polymerase was isolated from the peripheral blood leukocytes of a patient with acute myelomonocytic leukemia by successive purification of a particulate cytoplasmic fraction with endogenous, ribonuclease-sensitive DNA polymerase activity. Like RNA-directed DNA polymerase from mammalian type-C virus, the human leukemic cell enzyme efficiently utilized (A)(n).(dT)(12-18) and (C)(n).(dG)(12-18) and had an approximate molecular weight of 70,000. Further, the leukemic cell enzyme was strongly inhibited by antisera to RNA-directed DNA polymerase of primate type-C virus in a fashion similar to that noted with an extensively purified RNA-directed DNA polymerase from a person with acute myelogenous leukemia [Todaro, G.J. & Gallo, R.C. (1973), Nature 244, 206]. By these biochemical and immunological results the leukemic cell enzyme could be differentiated from all other known cellular DNA polymerases but could not be distinguished from RNA-directed DNA polymerase of primate type-C virus. We interpret these data, combined with observations published elsewhere, to indicate that human acute myelogenous leukemia cells contain components related to primate type-C virus. The parameters used in this study may provide the specificity and sensitivity required for determining the presence or absence and (if present) the relatedness of RNA-directed DNA polymerase in other cases and types of human leukemia.
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PMID:Relationship between RNA-directed DNA polymerase (reverse transcriptase) from human acute leukemic blood cells and primate type-C viruses. 413 50

Production of particles with the ultrastructural appearance of C-type virions persisted for at least 6 h in actinomycin D-treated cells infected with murine leukemia virus. This phenomenon occurred despite severe inhibition of viral RNA synthesis. Virus particles present in a 6-h harvest sedimented in sucrose gradients with the buoyant density characteristic of RNA tumor viruses (1.16 g/cm(3)) and exhibited high levels of reverse transcriptase activity in response to the exogenous template polyriboadenylic acid.oligo deoxythymidylic acid in the range of untreated controls. However, RNase-sensitive endogenous activity was only (1/5) the level found in controls. This observation correlated with a marked reduction in infectivity. Kinetic studies on the appearance of labeled RNA in banded virions revealed that within the first hour after addition of actinomycin D, particles contained 60 to 70S RNA and two low-molecular-weight RNA species corresponding to 8 and 4S RNA. After approximately 1 h of incubation with actinomycin D, 60 to 70S RNA could not be detected and 4S RNA was the predominant species. These findings suggest that murine leukemia virus particles assembled in the presence of actinomycin D are deficient in 60 to 70S viral RNA.
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PMID:Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D. 413 68

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of (32)P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T(1) and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the alpha or beta globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants.
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PMID:Nucleotide sequence analysis of RNA synthesized from rabbit globin complementary DNA. 413 14

The DNA polymerase of the Prague strain of Rous sarcoma virus of subgroup C and of the Schmidt-Ruppin strain of subgroup A has been solubilized. DNA polymerase purified by sucrose gradient sedimentation and chromatography on DEAE-cellulose represented less than 2% of the soluble [(14)C]protein of the virus. The enzyme was separated from 90% of the viral glycoprotein; it is probably different from the viral group-specific antigen. The sedimentation coefficient (s(20, w)) of the soluble DNA polymerase was 8 S before, and 6 S after, incubation with pancreatic RNase. The molecular weight of the 8S DNA polymerase was estimated to be about 170,000, and that of the 6S DNA polymerase to be about 110,000. Purified DNA polymerase had a high activity with 60-70S viral RNA or salmon DNA as template, but it had a low activity with heat-dissociated 60-70S RNA, influenza virus RNA, or the RNA of tobacco mosaic virus as template. Neither the 8S nor the 6S DNA polymerase had endogenous template activity. The DNA-dependent and the RNA-dependent DNA polymerase activities of the Prague strain coincided in sucrose gradients, both in the 8S and the 6S form. It is concluded that the RNA-dependent and the DNA-dependent DNA polymerase activities of the avian tumor viruses are probably due to the same enzyme.
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PMID:Properties of a soluble DNA polymerase isolated from Rous sarcoma virus. 432 88

Early chicken embryos that are either positive or negative for group-specific antigens of avian leukosis viruses contained endogenous RNA-directed DNA polymerase activity. This endogenous DNA polymerase activity was not increased after mixture of soluble DNA polymerases isolated from chicken embryos with disrupted chicken embryo cells. The endogenous activity was resistant to treatment with deoxyribonuclease, and the initial rate of DNA synthesis was partially resistant to actinomycin D. In contrast, over 90% of the endogenous polymerase activity was destroyed by ribonuclease in medium with high salt concentration. The DNA product of the endogenous DNA polymerase activity from chicken embryos did not hybridize with RNA of Rous sarcoma virus or reticuloendotheliosis virus, whereas about 40% of this DNA product hybridized with the RNA from the same chicken-cell fraction. Antibody against DNA polymerase of avian myeloblastosis virus did not neutralize the chicken endogenous DNA polymerase activity. These results demonstrate that uninfected chicken embryo cells contain endogenous RNA-directed DNA polymerase activity that is not derived from avian leukosis or reticuloendotheliosis viruses.
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PMID:Endogenous RNA-directed DNA polymerase activity in uninfected chicken embryos. 433 97

The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.
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PMID:Strandedness and complementarity of DNA from long-term RNA-dependent DNA polymerase reactions of Soehner-Dmochowski murine sarcoma virus. 435 60


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