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 extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.
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PMID:The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme. 7 Apr 28

Virions produced by cells in the presence of actinomycin D (Act D virions) contain reverse transcriptase but are deficient in 70S genomic RNA. To assess the role of genomic RNA in encapsidation of a functional reverse transcriptase and to study the interaction of the enzyme and its template in the cores of intact virions, the reverse transcriptase enzymes of normal and Act D virions were compared. The enzymes were indistinguishable by column chromatography, sedimentation velocity, or template/primer preferences. In addition, these enzymes showed equal sensitivity to inactivation by antibodies directed against Rauscher murine leukemia virus DNA polymerase. The enzymes from Act D and normal virions had similar thermal decay rates and were both protected against heat denaturation by natural and synthetic template/primers. By these criteria, the DNA polymerase molecules synthesized and assembled into virions in the absence of genomic RNA are identical to those packaged under normal conditions. Additional studies designed to measure protection of reverse transcriptase by genomic RNA were carried out by comparing the thermal lability of the enzyme in intact Act D and normal virions. The thermal decay rate of reverse transcriptase in Act D virions was identical to that in control virions. In contrast to the lability of the virion-associated enzyme, however, genomic RNA in control virions was stable to heat treatment.
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PMID:Interactions of murine leukemia virus core components: characterization of reverse transcriptase packaged in the absence of 70S genomic RNA. 7 60

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

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

The present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and template-primers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation-velocity measurements of the purified enzymes gave values of 150000, 40000, 100000 and 70000 daltons for DNA polymerase-alpha DNA polymerase-beta, DNA polymerase-gamma and the reverse transcriptase respectively. Serological studies have shown that the reverse transcriptase from human spleen is not antigenically related to cellular DNA polymerase-alpha, -beta or -gamma, but is antigenically related to reverse transcriptase from simian sarcoma virus and gibbon-ape leukaemia virus.
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PMID:Purification, biochemical characterization and serological analysis of cellular deoxyribonucleic acid polymerases and a reverse transcriptase from spleen of a patient with myelofibrotic syndrome. 7 8

Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.
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PMID:Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I. 7 87

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


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