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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 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

Poly(vinylbenzo-18-crown-6), a water-soluble polymer endowed with ion-binding crown moieties as pendent groups, forms insoluble complexes with polyadenylate in the presence of K+; the corresponding monomeric benzo-18-crown-6, does not form a precipitate under the same conditions. In the presence of Na+ and Mn2+ which in aqueous solution complex weakly to crown compounds, no coprecipitation of the crown polymer and polyadenylate occurs; nevertheless, the crown polymer strongly binds to immobilized polyadenylate even under these conditions. The interactions of crown polymer with the poly-nucleotide result in a loss of templating ability of the latter. Using RNA-dependent DNA polymerase of murine leukemia virus it was found that (1) enzymatic action is efficiently inhibited even in the absence of ions which coprecipitate crown polymer and template, (2) inhibition is reversed by addition of excess polynucleotide and (3) monomeric crown does not inhibit the reaction.
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PMID:Ionophorous polymers. Interaction with polynucleotides and effects on RNA-directed DNA polymerase activity. 5 50

Poly(c3A) (poly 3-deazaadenylic acid) and poly(c3I) (poly 3-deazainosinic acid) differ in biological reactivity from their parent compounds poly(A) and poly(I) and from their 7-deaza counterparts poly(c7A) and poly(c7I). Three parameters of biological reactivity were evaluated : (1 degree) interferon induction, (2 degrees) anti-complement activity, (3 degrees) reverse transcriptase inhibition. Unlike poly(A)-poly(U), poly(I)-poly(C) and poly(I)-poly(br5C), the mixtures of poly(c3A) + POLY(U), poly(c3I) + poly(C), and poly(c3I) + poly(br5C) failed to elicit an interferon response in "super-induced" primary rabbit kidney cells; Poly(I) and its analogs poly(c3I) and poly(c7I) inhibited hemolytic complement activity, whereas poly(A) and its analogs poly(c3A) and poly(c7A) failed to do so. Both poly(I) and poly(c7I), but not poly(c3I), lost their anti-complement potency when annealed to either poly(C) or poly(A)-poly(U). Similarly, poly(I) and poly(c7I), but not poly(c3I), suppressed the interferon inducing ability of poly(A)-poly(U), suggesting that both poly(I) and poly(c7I), but not poly(c3I), added to poly(A)-poly(U) to form a triple-helical structure. Poly(I), poly(C7I) and poly(c7A)exerted a distinct inhibitory effect on turine leukemia virus, while under the same conditions poly(c3I) and poly(c3A) showed little, if any, inhibitory effect.
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PMID:Role of purine N-3 in the biologic activities of poly(A) and poly(I). 6 Jul 41

Poly(2'-O-ethylcytidylate) is a poor template-primer for purified avian myeloblastosis virus reverse transcriptase; the relative activities of the template-primers poly(C)-oligo(dG), poly(Cm)-oligo(dG) and poly(Ce)-oligo(dG) are 23:16:1. A mixture of poly(Ce) and poly(dI) is inactive as template-primer, in agreement with the observed inability of these to form a helical complex. By contrast the inactivity of poly(Ce)-poly(I) is shown to be due to the influence of the 2'-O-ethyl residue. Poly(Ce) inhibits poly(A)-oligo(dT)-directed polymerase activity, with Ki = 3 muM, but marked inhibition with poly(A)-poly(dT) occurs only at low concentrations of the latter. Poly(Ce) did not inhibit template-primer activity of poly(C)-poly(dI) and poly(dC)-poly(dI). Qualitative physico-chemical studies show only partial complex formation between oligo(dG) and poly(C) and its 2'-O-alkyl analogues. This is discussed in relation to the widespread use of poly(C)-oligo(dG) as the template-primer for reverse transcriptase.
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PMID:Poly 2'-O-ethylcytidylate, an inhibitor and poor template for AMV reverse transcriptase. 6 Jul 42

Bleomycin inhibits cellular RNA synthesis and the inhibition is nonspecific. The ratio of polyadenylate- [poly(A)] containing RNA to non-poly(A)-containing RNA in the drug-treated human lymphocytic cells, line Wil2, was the same as that in untreated cells. Poly(A) RNA isolated from untreated cells was used as a template for reverse transcriptase to synthesize complementary DNA, which was then used as a probe to assay the sequence diversity of poly(A)RNA's from treated and untreated cells. It was found that essentially all of the poly(A) RNA's in the untreated cells were also present in the treated cells. The effect of bleomycin on the biological activity of messenger RNA (mRNA) was tested with globin mRNA in a wheat germ embryo translation system. Although bleomycin inhibited protein synthesis at high concentrations, the inhibition was not due to a modification of mRNA. This was evidenced by the fact that no decrease in the ability of mRNA to function in the test system was found when globin mRNA was pretreated with high concentrations of bleomycin followed by removal of the drug.
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PMID:Effect of bleomycin on the synthesis and function of RNA. 6 80

Poly (2-azaadenylic acid) [(aza2A)n] and poly(2-azainosinic acid [(aza2I)n], two newly synthesized analogues of (A)n and (I)n, in which CH-2 of the purine ring is replaced by a nitrogen atom, have been evaluated in various biological assay systems. (Aza2A) n formed a complex with (U)n and (br5U)n, and (aza2I)n formed a complex with (C)n and (br5C)n, but these complexes were markedly destabilized relative to the corresponding (A)n or (I)n complexes. The (aza2A)n-and (aza2I)n-derived complexes failed to stimulate the production of interferon in primary rabbit kidney cells and human diploid fibroblasts, under conditions (A)n. (U)n, (I)n. (C)n and (I)n. (br5C)n induced high amounts of interferon. both (aza2A)n and (aza2I)n exerted a marked inhibitory effect on the endogenous RNA directed DNA polymerase (reverse transcriptase) activity associated with murine leukemia virus. They caused a relatively mild inhibition of complement activity in an hemolytic assay system.
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PMID:Biologic activities of poly (2-azaadenylic acid) and poly (2-azainosinic acid). 7 66

Poly(A)-containing encephalomyocarditis virus RNA functions as an excellent template for cDNA synthesis in vitro with an RNA-dependent DNA polymerase in the presence of an oligothymidylate primer. Under appropriate conditions, discrete transcripts of increasing chain length were obtained, suitable for sequence analysis. A limited cDNA fragment of 36 nucleotides, primer (dT)10 included, was synthesized when dGTP was omitted from the reaction mixture and its primary structure was elucidated using direct DNA-sequencing methods. The complement corresponds to the 3' end of encephalomyocarditis RNA. The hexanucleotide (5'-3')(A-A-U-A-A-A) found in this sequence is also present in all 3' non-coding regions of poly(A)-containing eukaryotic mRNAs studied until now, in nearly identical positions relative to the poly(A) tail. The possible biological significance of this structural homology is discussed.
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PMID:The 3'-Terminal nucleotide sequence of encephalomyocarditis virus RNA. 7 34

Poly (2-methylthioinosinic acid) [poly(ms2I)] was found to markedly inhibit the RNA directed DNA polymerase (reverse transcriptase) activity of murine (Moloney, Rauscher) leukemia virus and murine (Moloney) sarcoma virus, while under the same conditions the unsubstituted parent compound poly(I) showed little, if any, inhibitory effect. Copolymers of inosinic acid (I) and 2-methylthioinosinic acid2(ms2I) showed an intermediary effect, depending on the I:ms2I ratio. Poly(ms2I) also inhibited the transformation of normal cells by murine (Moloney) sarcoma virus, as assessed by an infectious center assay.
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PMID:Inhibition of oncornavirus functions by poly (2-methylthioinosinic acid). 7 96

Total poly(A)-mRNA from polyribosomes of MOPC 21 mouse myeloma were investigated. Poly(A)-mRNA was released by two successive chromatography on oligo (dT)-cellulose. A 14S fraction of total poly(A)-mRNA was obtained and partially purified by sucrose gradient centrifigation followed by acrylamide gel electrophoresis. As estimated from the electrophoretic analysis, the 14S mRNA has three components, one of which appears to be 18S rRNA and two others--mRNAs with molecular weight of 5.2.10(5) and 3.8.10(5), respectively. Total poly(A)-mRNA and partially purified 14S mRNA were active when employed as a template in a reverse transcription and cell-free system from wheat germ. DNA complementary to the 14S mRNA was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA was heterogeneous in size with the average size of about 800 nucleotides when analyzed by gel electrophoresis in 98% formamide. The maximal length was about 1100 nucleotides that consistent with full template length. About half of the translation product directed by the 14S mRNA migrated as mature L-chain Ig (upon polyacrylamide gel electrophoresis in sodium dodecylsulfate). The presented data suggested that 14S mRNA species contain mRNA L-chain Ig.
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PMID:[mRNA of mouse plasmacytoma. Reverse transcription and translation in cell-free systems]. 8 67

Poly(A) containing rat liver 21S RNA homogeneous in polyacrylamide gel electrophoresis under denaturing conditions and stimulating the synthesis of ceruloplasmin in a cell-free proteinsynthesizing system, was used as a template for reverse transcription in the presence of T10 primer and highly purified reverse transcriptase from avian myeloblastosis virus. The cDNA made this way was characterized by means of hybridization kinetics with mRNA, by melting of the hybrids formed and by chain length measurements. To increase the degree of representativity, the ceruloplasmin mRNA was fragmented by mild alkaline treatment, enzymatically polyadenylated and transcribed. The cDNA made was fully characterized and the kinetic complexity measured by hybridization with the mRNA was found to be equal to 2300 nucleotides as compared with the value of 3000 nucleotides is expected from gel electrophoresis data. The observed difference may indicate the presence of repeated sequences in the given mRNA. The sufficient representativitness of the synthesized cDNA and its specificity with respect to ceruloplasmin mRNA allows to use it as a molecular probe to study the ceruloplasmin gene structure.
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PMID:[Enzymatic synthesis and characterization of DNA complementary to ceruloplasmin mRNA from rat liver]. 9 44


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