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)

The RNA-directed DNA polymerase of Rous sarcoma virus requires a 4S RNA molecule as primer for the initiation of DNA synthesis on the viral 70S RNA genome. We have now functionally identified primer activity in uninfected cells on the basis of the capacity of cellular 4S RNA to actively participate in the initiation of DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus in vitro. This was accomplished by reconstitution experiments in which 4S RNA from uninfected avian cells was tested for its ability to restore template activity to the viral RNA genome from which all primer had been removed. Similar reconstitution experiments were employed to demonstrate a primer activity in the 4S RNA population of duck, mouse, and human cells. Primer activity appears to be absent in lower eukaryotic or prokaryotic cells. Unambiguous identification of the Rous sarcoma virus primer molecule in uninfected cells was accomplished by directly purifying a 4S RNA molecule from the bulk of host cell transfer RNA and establishing structural similarities between this cellular 4S RNA species and the Rous sarcoma virus primer by two-dimensional paper electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the RNA species. We conclude that the Rous sarcoma virus DNA polymerase can utilize a host cell molecule as primer for the initiation of RNA-directed DNA synthesis in vitro.
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PMID:RNA-directed DNA synthesis by the DNA polymerase of Rous sarcoma virus: structural and functional identification of 4S primer RNA in uninfected cells. 4 51

Nervous system tissues from a number of patients with idiopathic neurological disorders were examined for biochemical evidence of RNA tumor virus infection. RNase-sensitive DNA polymerase activity was found in a cytoplasmic particulate fraction from two patients with Guamanian amyotrophic lateral sclerosis (ALS) but not in brains from two normal U.S. individuals. The buoyant density of the enzyme-containing fraction was 1.16-1.18 g/ml and could be converted to a denser region of the gradient (1.24 g/ml) by treatment with the nonionic surfactant, Sterox. The cation and detergent requirements for the endogenous RNase-sensitive DNA polymerase reaction were determined. The early (5 min) endogenous reverse transcriptase product was analyzed by cesium sulfate gradient centrifugation. RNase- and heat-sensitive RNA-DNA hybrids were detected in the product analysis of two ALS, one Parkinsonism-dementia (PD) brain, and two brains from asymptomatic Chamorros but not in brains from normal U.S. individuals and a number of patients with neuro-psychiatric disorders. The DNA product was a 4.5S heteropolymer that hybridized more extensively to RNA extracted from the enzyme-containing pellet from PD brain as compared to a similar fraction from normal U.S. brain. The DNA product appeared to be unrelated to Rausvher or visna virus 70S RNA as determined by RNA-[-3H]DNA hybridization.
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PMID:RNA-instructed DNA polymerase activity in a cytoplasmic particulate fraction in brains from Guamanian patients. 4 90

A molecular hybridization technique has been used to quantitatively measure the nucleotide sequence relationships of selected mammalian RNA tumor viruses. Reciprocal cross-hybridization tests were done in which a given radioactively labeled, viral genomic RNA species was annealed with an excess of unlabeled, complementary DNA product synthesized in endogenously instructed reverse transcriptase reactions. Hybrid formation was measured with pancreatic RNase A. Three representative mammalian RNA tumor virus groups were examined: murine viruses, simian viruses, and feline viruses. The results of reciprocal cross-hybridization testing have revealed that the murine viruses consist of four distinctly related subgroups: (i) the Friend leukemia virus/Rauscher leukemia virus subgroup, (ii) the Gross leukemia virus subgroup, (iii) the Moloney sarcoma virus subgroup, and (iv) the Kirsten sarcoma virus subgroup. Simian sarcoma virus, the only simian virus examined, appeared to share limited interspecies sequence relationships with members of the other virus groups and in particular with Kirsten sarcoma virus. Of the two members of the feline virus group tested, Rickard feline sarcoma virus and RD-114, each was placed in a separate, unrelated subgroup. Rickard feline sarcoma virus exhibited limited sequence relatedness with members of the other virus groups, whereas RD-114 exhibited none.
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PMID:Quantitative nucleotide sequence relationships of mammalian RNA tumor viruses. 4 42

We have investigated three aspects of RNA turmor virus replication and cell transformation: (1) the properties of the purified avian and mammalian viral RNA-directed DNA polumerase, (2) some characteristics of the viral 60-70S RNA genome, 30-40S RNA subunits and intracellular viral RNA species, and (3) the interaction of the viral DNA polymerase with its RNA template early during infection and cell transformation by the murine sarcoma-leukemia virus (MSV[MLV]). Avian myeloblastosis virus (AMV) contains two forms of RNA-directed DNA polymerase, alpha, consisting of a single polypeptide of molecular weight 65,000, and alphabeta, consisting of two polypeptides of molecular weights 65,000 and 105,000. The alpha and alphabeta forms of AMV DNA polymerase both possess RNase H activity that requires free end termini on the ribopolymer and can degrade the RNA of the RNA-DNA hybrid in the 3' to 5' and 5' to 3' directions. But, alpha and alphabeta possess a different mode of exoribonuclease activity. While alphabeta RNase H is a processive exoribonuclease that degrades the polynucleotide chain to a core residue before attacking a second chain, alpha RNase H is a random exoribonuclease that releases the polynucleotide after each scission. Highly purified Moloney-MSV(MLV) DNA polymerase has both RNase H activity and the ability to read viral 60-70S RNA. These activities comigrate through five different steps of purification and are present at levels comparable to those found in purified AMV DNA polymerase. The MSV(MLV) 60-70S RNA genome and 35S RNA subunits were shown by periodate oxidationtritiated borohydride reduction to contain adenosine as the major 3'-terminal nucleoside. Poly (A) segments were isolated from viral 60-70S and 35S RNA by treatment with RNase A or RNase T1 and purified by afinity chromatography and gel electrophoresis. Viral poly(A) was shown to be present at the 3' terminus as -G(C,U)A190AOH. The similar sequence reported for poly(A) present in mammalian mRNA suggests that similar mechanisma are involved in the transcription and processing of both cellular and viral DNA sequences. Within transformed cells replicating MSV(MLV), viral 35S and 20S RNA were found in membrane-bound polyribosomes, whereas only 35S RNA was detected in free polyribosomes. The origin and function of 20S RNA is unknown. The early events during rapid infection and cell transformation of mouse 3T6 cells by the Harvey strain of MSV(MLV) were studied. By both autoradiographic analysis and molecular hybridization, viral DNA synthesis was detected in the cytoplasm by 1 hour after infection, reached a maximum at 2 hours, and subsequently decreased. Cytological chase experiments produced evidence that cytoplasmic viral DNA was transported to the nucleus. In situ hybridization experiments using radioactive viral DNA product as a probe demonstrated the rapid association of viral DNA sequences with the chromocenters of interphase nuclei and with the centromeric heterochromatin regions of some chromosomes.
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PMID:Properties of oncornavirus RNA-directed DNA polymerase, the RNA template, and the intracellular products formed early during infection and cell transformation. 5 Sep 2

Reticuloendotheliosis viruses (REV) contain an endogenous RNA-directed DNA polymerase activity. The endogenous DNA polymerase activity can be elicited in purified preparations of REV by treatment with nonionic detergents. The enzyme activity has a strong preference for manganous ions. Therefore, appreciable endogenous DNA polymerase activity can be demonstrated only if the reaction mixture contains appropriate concentrations of manganous ions. Enzyme activity can be inhibited by pretreatment with RNase or deletion of one or more deoxyribonucleoside triphosphates from the reaction mixture. In contrast, actinomycin D has little effect in initial DNA synthesis. The results from both velocity and equilibrium centrifugation indicate that the nascent chains of product DNA are associated with 60S viral RNA. The DNA product of the endogenous DNA polymerase reaction is hybridizable to REV RNA, but not to avian leukosis virus RNA.
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PMID:Characterization of endogenous RNA-directed DNA polymerase activity of reticuloendotheliosis viruses. 5 36

Particles with the density and enzymatic activity characteristic of known oncornavirus have been previously described in bone marrow cells from patients with leukemia in relapse and in remission. We have confirmed these findings and studied two patients in whom preleukemia was among the diagnostic considerations. Following cultivation of bone marrow from these patients for 1 week in conditioned media with dexamethasone, a high-speed pellet of the supernatant fluid and disrupted cells was prepared and analyzed on a sucrose gradient for enzymatic activity characteristic of RNA-directed DNA polymerase (reverse transcriptase). Peaks of endogenous DNA polymerase activity showing ribonuclease sensitivity and/or stimulation with the synthetic template poly(rC)-(dG)12-18 were demonstrated in both patients at densities of 1.15 to 1.19 and 1.21 to 1.24 g/ml. Subsequently, diagnosis 2 and 4 months after initial evaluation revealed acute myelogenous leukemia and malignant histiocytosis, respectively. Prior studies have suggested a possible etiological significance of such particles in human leukemia. The demonstration of similar particles preceding clinically overt disease in these patients supports this hypothesis and offers the possibility of early diagnosis and treatment.
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PMID:Oncornavirus-like particles from cultured bone marrow cells preceding leukemia and malignant histiocytosis. 5 58

The DNA product of the endogenous reverse transcriptase reaction of Gibbon ape lymphoma virus has been analyzed and characterized. Data show that in simultaneous detection assays in which the type and/or concentration of divalent cation is varied the best yield of rapidly-sedimenting DNA was obtained from reactions containing 1.5 mM Mn2+. This yield is ten-fold better than the yield observed at the optimal Mg2+ concentration (5.0mM). Evidence is presented to show that DNA synthesized at the optimal concentration of either of these cations consists of large pieces varying in size from 4 to 12S. This DNA hybridizes efficiently to homologous viral RNA (greater than 60 percent annealing) and protects at least two-thirds of GALV 70S [32P]RNA from ribonuclease digestion. The hybrids formed with homologous viral RNA are stable as evidenced by their thermal elution patterns from hydroxylapatite columns. In contrast, DNA synthesized in reactions in which the concentration of Mn2+ or Mg2+ was greater than optimal was predominantly 4S or smaller in size and displayed a low level of hybridization (less than 10 percent) to homologous viral RNA.
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PMID:The endogenous reverse transcriptase activity of Gibbon ape lymphoma virus: characterization of the DNA product. 5 76

Measurement of DNA polymerase in leukaemic guinea-pig plasms reveals the presence of low levels of sedimentable and non-sedimentable enzymic activities. Since the sedimentable DNA polymerase is ribonuclease sensitive, uses poly(C).oligo(dG) as template, and bands in a sucrose density gradient at 1-17 g/ml it is thought to be the GPLV-associated reverse transcriptase. The soluble DNA polymerase is stimulated by ribonuclease and is probably of cellular origin.
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PMID:DNA polymerase activity in plasma from leukaemic guinea-pigs. 5 90

Intracisternal A particles from the FLOPC-1 line of BALB/c myeloma have been shown to contain high-molecular-weight RNA (60 to 70S) that is sensitive to RNase, alkali degradation, and heat but resistant to Pronase treatment. The intracisternal A-particle RNA contains tract of poly (A) approximately 180 nucleotides long. As shown in a reconstitution experiment, by antigenic analysis of A-particle preparation and the SC cytopathogenicity assay, the 70S RNA was not due to contamination by type C virus particles. The FLOPC-1 intracisternal A particles also possess an endogenous RNA-dependent DNA polymerase. The enzyme required Mn2+ or Mg2+, dithiothreitol, detergent, and four deoxyribonucleoside triphosphates for maximum activity. Enzymatic activity was maximally stimuated by poly (rC)-oligo (dG)12-18 and less with poly (rG)-oligo (dC)10 or poly (rA)-oligo (dT)12-18 as compare with synthetic DNA/DNA duplex templates such as poly (dA)-oligo (dT)12-18. The enzyme can utilize the A-particle endogenous RNA as template as shown by analysis of the early and late DNA products of the endogenous reaction by CsSO4 isopycnic gradient centrifuation and hybridization of purified 70S or 35S A-particle RNA with the purified complementary DNA product. Approximately 50% of the A-particle complementary DNA also hybridized with oncornavirus RNA.
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PMID:Intracisternal A particles from FLOPC-1 BALB/c myeloma: presence of high-molecular-weight RNA and RNA-dependent DNA polymerase. 5 76

An RNA-directed DNA polymerase associated with transformation-defective (td) segregant of Rous sarcoma virus (RSV) has been characterized. The enzyme required both a monovalent and a divalent cation, a sulfhydryl reducing agent, and all four deoxyribonucleoside triphosphates for the expression of maximal activity. Sensitivity of the endogenous RNA-directed DNA polymerase activity to a low concentration of pancreatic RNase indicated that the enzyme utilized the td virus endogenous RNA as template. Maximal DNA synthesis was observed in a reaction mixture of pH 8 - 8.5 at 45 C with a manganese concentration of 1 mM. The enzyme of the td virus responded to exogenous template-primers in a manner characteristic of DNA polymerase of RNA tumor viruses, and the response became substantially greater when noncomplementary precursors were omitted from the reaction mixture. The endogenous reaction kinetics were examined. Three phases of DNA synthesis could be distinguished. Evidence was obtained showing that during the third and slowest phase of DNA synthesis the reaction mixture was not depleted of precursors and that the enzyme was fully active to initiate DNA synthesis with newly-added viral or synthetic RNA templates. Comparison of TMP and dAMP incorporation kinetics suggested that at the initial phase the enzyme preferentially copies A-rich region(s) of viral RNA. A comparison was also made between the endogenous reaction of the td virus and that of its parent sarcoma virus. The pH optimum, metal ion requirements, effect of sulfhydryl agents, response to exogenous template-primers, and kinetics of DNA synthesis, were all compared. No significant difference between the reaction of the td virus and its sarcomatogenous counterpart could be demonstrated.
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PMID:Endogenous DNA polymerase of a transformation-defective rous sarcoma virus: characterization and comparison with the enzyme of the non-defective parent. 6 91


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