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 size of the DNA product synthesized by RNA-directed DNA polymerase (isolated from avian myeloblastosis virus) was found to be important for complementary DNA (cDNA)-mRNA hybridization reactions. Incomplete cDNA to rabbit reticulocyte globin mRNA formed poor hybrids and presumably lacked sequences needed for hybridization. The size of the cDNA synthesized was influenced by the reaction conditions used. The complementary DNA product contained 10 S material when synthesis was done at high deoxynucleoside triphosphate concentrations (greater than 50 muM) while the product was smaller than the template when synthesis was at lower concentrations. The concentration and size (oligo(dT)6 to (dT)10) of primer had little or no effect on the product size. Increasing the concentration of 10 S globin mRNA caused the cDNA product to contain more small material. The cDNA synthesized at high deoxynucleoside triphosphate concentrations was fractionated into heavy, medium, and light fractions by alkaline sucrose density centrifugation. All hybridized to globin mRNA. The larger cDNAs had a higher TM when hybridized to globin mRNA, a lower dTMP/dCMP ratio (indicating that the poly(dT) region constituted a smaller fraction of the molecule), and gave increased protection of 125I-labeled mRNA from nuclease digestion. The full size cDNA was especially useful for studying the RNA transcribed from chromatin by RNA polymerase. The complement of the 5' end of the mRNA is contained only in full size cDNA; the 5' end is the part of the mRNA first transcribed by the RNA polymerase assuming correct transcription. Thus, full size cDNA can hybridize more effectively to the short RNA transcripts that are obtained than partial cDNA. RNA transcribed from rabbit bone marrow chromatin by Escherichia coli RNA polymerase hybridized twice as efficiently to complete cDNA as it did to partial cDNA demonstrating the usefulness of full size cDNA.
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PMID:Importance of full size complementary DNA in nucleic acid hybridization. 5 64

In the post-mitochondrial fraction of murine LBN/b leukemic cells, four fractions with DNA polymerase activity (I, II, III, IV) were found. On the basis of ion exchanger affinity and poly(A), poly(C) and poly(Cm) replication ability, fraction I was classified as RNA-directed DNA polymerase of viral origin. On the basis of the differences in the ion exchanger affinity, molecular weight, template requirement, pH-dependence of enzymatic activity and NaCl concentration, divalent ion requirements and susceptibility to N-ethylmaleimide inhibition, fractions II, III and IV were classified as DNA-directed DNA polymerases beta, alpha and gamma, respectively. Three fractions, i.e. reverse transcriptase, and DNA-directed DNA polymerases beta and gamma, were found to incorporate dTMP on a poly(A)-oligo(dT) template-primer. Despite the similarity of the reaction of DNA polymerases beta and gamma with poly(A)-oligo(dT), some other properties of these enzymes suggest that they represent distinct enzymatic entities.
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PMID:DNA polymerases of murine LBN/b leukemic cells. 5

The nascent DNA transcript of intracisternal A particle RNA-directed DNA polymerase appeared to be covalently linked to an RNA primer. Fidelity of transcription is evident since the DNA transcript hybridized specifically back to 35-70 S RNA of intracisternal A particles but not with heterologous RNAs. This DNA transcript has an approximate molecular weight of 145 000, estimating 350 polynucleotides and base ratios with an excess of dGMP.
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PMID:Physicochemical analysis of the deoxyribonucleic acid product of murine intracisternal A particle RNA-directed DNA polymerase. 6 65

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.
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PMID:Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus. 6 60

Type C oncornavirus isolation was attempted from cell cultures of tissues from 7 patients with systemic lupus erythematosus. Detection was based on the characteristic sedimentation of 3H-uridine-labelled virions at a density of 1-16 g/ml. Cultures positive by this method were negative by two other criteria for type C viruses: characteristic virions by electron microscopy and the viral enzyme RNA-directed DNA polymerase. The positive results were probably due to cellular damage by prolonged radiolabelling, with release of organelles containing labelled RNA sedimenting at the same density as type C viruses.
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PMID:Type C oncornavirus isolation studies in systemic lupus erythematosus. I. Attempted detection by isopycnic sedimentation of 3H-uridine-labelled virions. 6 58

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

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.
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PMID:Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe. 6 86

Radioactively labelled DNAs (5 X 10(6) cpm/mug) complementary to human 18 S and 28 S ribosomal RNA were synthesized using RNA-directed DNA polymerase (EC 2.7.7.7). These complementary DNAs were used to measure human ribosomal gene numbers by two independent methods, both of which indicated numbers at least four-fold lower than those previously reported. First, the kinetics of the annealing of the complementary DNAs with total human placental DNA indicated that the number of both 18-S and 28-S ribosomal genes per haploid genome is approximately 50. Second, saturation experiments in which a constant amount of DNA was annealed with increasing amounts of complementary DNA also indicated that the number of 28 S ribosomal RNA genes in human placental and spleen DNA is is about 50 per haploid genome.
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PMID:A new estimate of human ribosomal gene number. 6 94

A RNA-directed DNA polymerase associated with particles that band at a density characteristic of type C RNA viruses was found in normal rabbit placental and uterine tissues taken during the early stages of gestation. That the rabbit RNA-directed DNA polymerase is distinct from the known cellular DNA polymerases and similar to the RNA-directed DNA polymerase of mammalian type C RNA viruses is shown by column chromatographic characteristics, template primer preferences, molecular weight determination, and an absolute requirement for the divalent cations.
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PMID:Evidence for a particle-associated RNA-directed DNA polymerase in rabbit placental and uterine tissues. 6 25

Rabbit lymphosarcoma tissues contain 70 S RNA and RNA-directed DNA polymerase encapsulated in particulate components that band in the density region of type C RNA viruses. RNA-directed DNA polymerase associated with the particles could be distinguished from cellular DNA polymerases by salt elution from phosphocellulose. The enzyme preferred the template primers poly(rA)-(dT)12-18 and poly(rC)-(dG)12-18 over other synthetic template primers and also utilized viral 70 S RNA as template; these properties are not observed with the known cellular DNA polymerases.
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PMID:Presence of a high-molecular-weight RNA and RNA-directed DNA polymerase in rabbit hereditary lymphosarcoma. 6 26


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