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)

Bovine thyroglobulin 33-S mRNA has been used as a template for the synthesis of a complementary DNA, using RNA-directed DNA polymerase from the avian myeloblastosis virus. The yield of the reaction was relatively poor and the size of the cDNA did not exceed 10 S. Nevertheless, a copy of high specific radioactivity (approximately 10(7) counts. min-1 microgram-1) could be obtained which hybridized specifically back to its template with an rot1/2 value about 5 times higher than that observed in hybridizations between hemoglobin mRNA (alpha + beta chain) and hemoglobin cDNA. This suggests that thyroglobulin mRNA does not contain extensive internal repetitive sequences. Quantification of thyroglobulin mRNA sequences among various RNA preparations from the beef thyroid was performed using cDNA/RNA hybridizations in RNA excess. The results confirmed that thyroglobulin mRNA represents the large majority of mRNA in membrane-bound polysomes and indicated the virtual absence of thyroglobulin sequences on free polyosomes. The cDNA transcribed from mRNA of bovine origin hybridized efficiently with thyroid RNA from goats, dogs and humans. Although the heterologuous hybrids exhibited the expected decrease in thermal stability, the bovine cDNA provides an appropriate probe for studies dealing with the expression of the thyroglobulin gene in various mammals including man.
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PMID:Reverse transcription of thyroglobulin 33-S mRNA. 7 91

The nature of transcription of the avian retrovirus RNA genome by the alpha form of the viral RNA-directed DNA polymerase has been investigated. Transcription was most efficient when Mn2+ was provided as the divalent metal ion. The patterns of DNA transcription using 70S RNA, 35S RNA-tRNAtrp, or 35S RNA-oligo(dT)12-18 template-primer complexes by the alpha DNA polymerase were essentially identical to those obtained using the alphabeta form. The alpha DNA polymerase appears to be deficient in the synthesis of true duplex DNA but is able to synthesize hairpin-structured DNA initiated at the 5' terminus of the viral genome on the tRNAtrp primer molecule.
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PMID:In vitro transcription of the avian retrovirus genome by the alpha form of the viral RNA-directed DNA polymerase. 7 62

DNA complementary to ovalbumin mRNA and covalently bound to cellulose (cDNA-cellulose) was synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. High concentrations of actinomycin D (200 migrogram/ml) were required to produce 97% inhibition of double-stranded DNA synthesis, but mRNA transcription was only slightly inhibited (14%). The conditions used for binding of mRNA to cDNA-cellulose permitted complete hybridization of ovalbumin mRNA in 10 min while stable poly(A):(dT) hybrids failed to form. The temperature at which 50% of the ovalbumin mRNA activity was eluted from cDNA-cellulose was 62 degrees in 0.01 M Tris.HCl. When a batchwise procedure of hybridization and elution was used, the total recovery of ovalbumin mRNA activity applied to the cDNA-cellulose was greater than 98%, indicating little if any degradation of mRNA. Ovalbumin mRNA activity eluted in each chromatographic run was 50 to 70% of that originally used for the synthesis of the cDNA-cellulose. When total polysomal RNA was subjected to chromatography, the bound fraction consisted of ovalbumin mRNA, rRNA, and material behaving like fragments of ovalbumin mRNA. Applying this fraction to cDNA-cellulose a second time eliminated the rRNA but not the presumptive fragments. Ovalbumin mRNA purified either once or twice was enriched between 43- and 56-fold over polysomal RNA in translational activity.
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PMID:Chromatography of ovalbumin messenger ribonucleic acid on complementary deoxyribonucleic acid-cellulose. 7 8

Particulate DNA polymerase activity that copied poly(2'-O-methylcytidylate) . oligodeoxyguanylate and banded at a density of 1.15 to 1.20 g/ml in sucrose gradients was detected in 8 of 16 human ovary tumors and in 11 of 16 malignant prostate tissues. None of the 10 nonmalignant ovary and prostate tissues examined contained detectable particulate DNA polymerase activity that copied poly(2'-O-methylcytidylate) . oligodeoxyguanylate. Since poly(2'-O-methylcytidylate) . oligodeoxyguanylate is effectively copied by oncornavirus RNA-directed DNA polymerase (reverse transcriptase) although not by the known species of human cell DNA polymerase, these results are interpreted as supporting the concept that some malignant human tissues contain particle-associated reverse transcriptase activity.
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PMID:Detection in human ovary and prostate tumors of DNA polymerase activity that copies poly(2'-O-methylcytidylate) . oligodeoxyguanylate. 7 7

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.
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PMID:Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase. 7 85

The microsomal supernatant fraction obtained from a murine cell line chronically infected with and producing Rauscher leukemia virus (JLSV-10) was found to contain two forms of RNA-directed DNA polymerase (reverse transcriptase). The two enzyme forms, neither of which is detectable in uninfected cells (JLSV-9), were initially partially purified by poly(C)-agarose chromatography, and their separation was achieved by phosphocellulose chromatography. The enzyme form eluting first from phosphocellulose (0.3 M KCl), designated PC I, was found to be identical in all parameters tested to that form isolated directly from purified virions. The second enzyme peak, designated PC II, eluted from phosphocellulose at 0.5 M KCl and was not detectable in purified virions. The PC II enzyme has a molecular weight, determined by velocity sedimentation, of approximately 109,000, as compared with 70,000 for the PC I enzyme, and could not be further dissociated by exposure to high salt or nonionic detergent. Mixing purified virion or PC I DNA polymerase with uninfected cells followed by fractionation did not produce the PC II form, suggesting that it is neither an artifact of purification nor the result of fortuitous complexing of reverse transcriptase with normal cellular component(s). Both PC I and PC II enzyme forms appeared antigenically similar to virion DNA polymerase, demonstrated identical divalent cation requirements for various template-primers, and were capable of copying heteropolymeric regions of rabbit globin mRNA. However, kinetic studies of heat inactivation revealed that the PC II enzyme was far more heat labile than the PC I form, which appeared identical to the virion enzyme in this respect. Furthermore, whereas the PC I and virion-derived reverse transcriptase copied poly(C).(dG)12-18 most efficiently at a template-to-primer molar nucleotide ratio of 25:1, the PC II enzyme preferred a ratio of 5:1 for optimal rates of poly(dG) synthesis. Therefore, by these criteria, there appear to exist two intracellular forms of reverse transcriptase in the JLSV-10 Rauscher leukemia virus-producing murine cell line.
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PMID:Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells. 7 32

Lysates of Moloney murine sarcoma-leukemia virus [M-MSV(MLV)], a virus complex grown in the rat cell line 78A-1, were found to contain three RNase H species separable by polycytidylic acid[poly(C)]-agarose chromatography. RNase H activity (RNase H I) associated with RNA-directed DNA polymerase eluted at 0.23 M KCI from poly(C)-agarose. RNase H II, which eluted from poly(C)-agarose at 0.12 M KCI and was not associated with DNA polymerase activity, was shown to be identical to an RNase H species (designated RNase H II) previously isolated from M-MSV(MLV) by a different procedure (G. F. Gerard and D. P. Grandgenett, J. Virol. 15:785-797, 1975). M-MSV(MLV) RNase H II was established to be a random exohybridase that requires free-chain termini in its hybrid substrate for activity. Lysates of Rickard feline leukemia virus also contained RNase H activity not associated with DNA polymerase activity that eluted from poly(C)-agarose at 0.12 M KCl. A third species of enzyme from M-MSV(MLV) lysates, called RNase H III, did not bind to poly(C)-agarose in 0.06 M KCl. RNase H III was purified from lysates of M-MSV(MLV) and M-MLV (grown in mouse cells) by sequential chromatography on poly(C)-agarose, DEAE-cellulose, phosphocellulose, and polyuridylic acid-Sepharose. Purified RNase H III (i) was free of any associated DNA polymerase activity, (ii) had an apparent molecular weight of 30,000 determined by Sephadex G-100 gel filtration, (iii) had an absolute requirement for Mn2+ (1 mM optimum) for the degradation of [3H](A)n.(dT)n, (iv) was inhibited by the presence of any salt in reaction mixtures, and (v) was endoribonucleolytic in its mode of action as indicated by the size distribution of limited degradation products of [3H](A)n.(dT)n. RNase H III was inhibited by antisera prepared against Rauscher MLV and simian sarcoma virus reverse transcriptase, and the quantity of RNase H III and RNase H I present in lysates of M-MLV were reduced and increased proportionately if virus was lysed in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. These results indicate that RNase H III is a proteolytic cleavage product of DNA polymerase-RNase H. Substantial RNase H activity that did not bind to poly(C)-agarose in 0.06 M KCl was also found in lysates of Harvey MSV(MLV), Rauscher MLV, and Rickard feline leukemia virus, but not in lysates of avian myeloblastosis virus.
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PMID:Multiple RNase H activities in mammalian type C retravirus lysates. 7 33

The avian retrovirus RNA-directed DNA polymerase contains an activity that is capable of removing hydrogen bonds from duplex nucleic acid molecules. This "unwinding-like" activity appears to be specific in its action, affecting RNA.DNA and DNA.DNA duplex molecules but not RNA.RNA duplexes. Studies with defined RNA.DNA hybrid molecules (e.g., Rous sarcoma virus RNA and complementary DNAs representing specific regions of the Rous sarcoma virus genome) and DNA.DNA duplexes indicate that, although this activity can remove a portion of the hydrogen bonds from these double-stranded structures, complete separation of complementary strands is not accomplished. The unwinding-like activity exhibits sensitivities to temperature and monovalent and divalent cation concentrations. It can also remove a specific large oligonucleotide from the 5' end of the viral genome subsequent to RNase H hydrolysis of viral RNA complexed to DNA present at that terminus. This reverse transcriptase-associated unwinding-like activity is discussed with respect to recently proposed models of retrovirus proviral DNA synthesis.
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PMID:Unwinding-like activity associated with avian retrovirus RNA-directed DNA polymerase. 7 11

The reaction scheme, developed for the synthesis of the gliotoxin analogue 2, was found to be of general applicability for analogues with varying substituents at N(1) and C(2). Analogues 11b-g prepared by this method are inhibitors of reverse transcriptase (RNA-directed DNA polymerase). Their inhibitory activity seems to be related to the lipophilicity of the effector molecules: the most lipophilic compound is the most active inhibitor. The techniques of reversed-phase thin-layer chromatography with silylated, precoated plates as well as reversed-phase high-performance liquid chromatography were used to measure the relative lipophilicities; both techniques gave analogous results.
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PMID:Gliotoxin analogues as inhibitors of reverse transcriptase. 1. Effect of lipophilicity. 8 Apr 49

Type C RNA virus(es) was readily released from primary lymphosarcoma cell cultures of WH/J rabbits after induction with halogenated pyrimidines. The virus contained an RNA-directed DNA polymerase and the p30 structural protein. The rabbit virus RNA-directed DNA polymerase and p30 protein shared antigenic homologies with other mammalian type C oncornaviruses but appear to also possess unique antigenic determinants. Normal rabbit liver contained DNA homologous to a 3H-labeled complementary DNA transcript of the rabbit viral genome, indicating that type C viral genetic information is present in at least the WH/J strain of rabbits.
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PMID:Induction of type C RNA virus from cultured rabbit lymphosarcoma cells. 8 Apr 60


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