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)

To study Moloney murine leukemia virus (M-MulV) proteins associated with the integration of proviral DNA into the host chromosome, we isolated endonuclease activities from purified virion preparations of the wild type and two of its replication mutants. A major endonuclease activity was identified in virions of M-MuLV; the enzyme catalyzed nicks in double-stranded DNA in the presence of either Mn2+ or Mg2+ and was stimulated by ATP. The endonuclease nicked DNA adjacent to all four nucleotides with some preference for G and C. The same enzyme, and in comparable amounts, was isolated from two virus replication mutants: dl2905, deficient in the processing of Pr65gag and Pr200gag-pol, and dl50401, deficient for the virus integration function. In the process of these experiments, the residual reverse transcriptase in mutant dl2905 was shown to be the mature size, implying that the uncleaved precursor lacks enzymatic activity. It appears that the major endonuclease activity found in virions of M-MuLV is not encoded by either the gag or pol genes.
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PMID:Characterization of endonuclease activities in Moloney murine leukemia virus and its replication-defective mutants. 303 6

Preinitiation complexes (complex 0) or complexes which either made 2 or an average of 10 phosphodiester bonds (complexes 2 and 10, respectively) were assembled in vitro on the adenovirus 2 major late promoter. Each of the complexes was digested extensively with DNase I; the protected DNAs were purified and hybridized in a series of end-labeled oligonucleotides homologous to sequences on the coding or noncoding strands near the initiation site. The hybrids were then extended with reverse transcriptase to map the extent of template protection conferred by proteins in the complex. The downstream protection edge revealed by this approach was approximately +30, +25, and +35 for complexes 0, 2, and 10, respectively. We subsequently found that the apparent inward movement of the downstream protection boundary on initiation could be produced by satisfying the energy requirement for transcription initiation (i.e., by treating with ATP or dATP). The downstream boundary change occurred as rapidly as we could perform the test (less than 60 s) and was not blocked by alpha-amanitin. DNAs from trimmed complexes 0, 2, or 10 all supported extension to a single upstream edge at about position -42. Upstream protection was stable in the preinitiation complex, but when postinitiation complexes were incubated for extended periods, protection of the entire upstream region was lost. This decay of upstream protection, like the movement of the downstream boundary, was found to result from exposure to ATP or dATP. Unlike the downstream boundary movement, however, the upstream change was relatively slow; about 15 min was required to lose one-half of the protection.
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PMID:Variations in template protection by the RNA polymerase II transcription complex during the initiation process. 368 85

Globin messenger RNA, isolated from human peripheral blood reticulocytes, was transcribed into complementary DNA by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus. The complementary DNA was then transcribed into (32)P-labeled complementary RNA by E. coli RNA polymerase in the presence of alpha-(32)P-labeled ribonucleoside triphosphates. The fingerprint pattern obtained from ribonuclease T1 digests of human globin complementary RNA was specific and reproducible. Different patterns were obtained from digests of duck, mouse, and rabbit globin complementary RNA. The fingerprint patterns obtained from digests of purified natural human 10S globin messenger RNA, labeled in vitro with (125)I or with [gamma-(32)P]ATP and polynucleotide kinase, were similar to that of the complementary RNA but contained some additional oligonucleotides. Sufficient nucleotide sequence information has been obtained from about 50% of the intermediate sized oligonucleotides (8-14 base residues long), to make possible examination of correspondence between these nucleotide sequences and globin amino-acid sequences. Approximately 70% of these oligonucleotide sequences can be matched to unique amino-acid sequences in the alpha- or beta-globin chains. The other 30% do not match known amino-acid sequences and presumably correspond to untranslated portions of the mRNA; some of these sequences, however, can be matched to amino-acid sequence in the abnormally long segment of the alpha chain of hemoglobin Constant Spring, which is thought to result from a chain-termination mutation.
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PMID:Nucleotide sequences of human globin messenger RNA. 413 9

Influenza B/LEE/40, B/Rome/1/67, B/Hong Kong/8/73, and B/Victoria/98926/70 viruses have a similar polypeptide composition as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These viruses are composed of six or seven polypeptides, depending on whether one or two high-molecular-weight polypeptides are resolved, ranging in molecular weights from 27,000 to 90,400. Three of these polypeptides, namely the heavy and light hemagglutinin chains and the neuraminidase, have attached carbohydrate. Highly purified influenza B/LEE/40 and B/Rome/1/67 virus preparations have RNA-dependent RNA polymerase activity equivalent to the incorporation of 100 and 30 pmol, respectively, of (3)H-UMP per mg of virus protein per h at 37 C, which is demonstrated only in detergent-treated virus suspensions. However, no RNA-dependent DNA polymerase enzyme activity was detected in the two viruses although virus suspensions were "activated" by heat, alpha-chymotrypsin, and detergents. Other enzymatic activities were associated with purified preparations of influenza B virus and were attributed to minor contamination of virus with host cell enzymes. Thus, nucleoside and deoxynucleoside phosphohydrolase enzymes were active in the absence of detergents and catalyzed the release of 1,200 and 1,800 nmol of P(i) per mg of virus protein in 30 min at 37 C from ATP and dATP substrates. Thin-layer chromatography indicated that the products of the phosphohydrolase enzymes of influenza B/LEE/40 were mainly nucleoside diphosphate and monophosphate. The latter enzymes were tightly bound to influenza B/LEE/40 virus and could not be removed completely by repeated centrifugation, including centrifugation of the virus to equilibrium in density gradients of 25 to 40% (wt/vol) cesium chloride. A low degree of RNase (approximately 0.01 mug% contamination) and phosphatase (10-30 nmol of P(i) released per mg of virus protein per 30 min) activity was detected in some, but not all, influenza B/LEE/40 virus preparations.
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PMID:Polypeptide composition of Influenza B viruses and enzymes associated with the purified virus particles. 435 55

Primed synthesis sequencing requires a partially single-stranded molecule in which a specific polynucleotide primer with a free 3' hydroxyl terminus is base paired to a single-stranded template. The template and primer strands can be DNA or RNA in any combination. A polymerase, either DNA polymerase for DNA/DNA or RNA/DNA primer-template combinations or reverse transcriptase for a DNA/RNA primer-template molecule, is used to copy the template by chain extension. In the chain termination method of sequencing (see Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463; 1977), nucleotide-specific chain terminators (dideoxy ATP(ddA), ddC, ddG, and ddT) are used to promote a nucleotide specific termination in the chain extension, so that some of the chains terminate at every position at which that nucleotide occurs in the sequence. By using each of the nucleotide specific-chain terminators in separate chain extension reactions and on the same primed template, the sequence of the template can be determined. This method is simple, rapid, and accurate and has been used to determine the sequences of whole DNA viruses (phi X174 and G4), plasmid cloned DNA, eukaryote mRNAs, and RNA viral genomes. These primed synthesis methods of sequencing are now being developed into methods for nucleotide mutagenesis.
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PMID:Primed synthesis methods of sequencing DNA and RNA. 615 73

Triphosphorylated 5'-end fragments about 100 nucleotides long were prepared from purified nuclear pre-mRNA using a modified hydroxyapatite method. These fragments as well as fragments of total pre-mRNA were polyadenylated by ATP:RNA adenyl-transferase and used as templates for the synthesis of [32P]cDNA by reverse transcriptase in the presence of an oligo(dT)-primer, cDNA transcribed from total fragments of pre-mRNA and from 5'-end fragments (5'-cDNA) were hybridized with excess of nuclear pre-mRNA. The extent of hybridization was 65-70 and 80-85% in different experiments. 18% of total cDNA and 35% of 5'-cDNA hybridized with mRNA from polysomes. A high homology between mRNA and triphosphorylated 5'-ends of pre-mRNA may be explained in the terms of splicing. The sequences adjacent to the triphosphorylated 5'-ends of pre-mRNA represent a specific class with complexity about 2.10(5) nucleotides. Less than 30% of 5'-cDNA hybridized with intermediately repetitive DNA, while the main portion hybridized with unique DNA sequences. About 15% of 5'-cDNA contain oligo (dA) sequences, originated from oligo (U) in the pre-mRNA.
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PMID:[Structure of nuclear pre-mRNA. XIII. Hybridization properties of triphosphorylated 5'-end fragments]. 616 15

The Mn2+-dependent endonuclease activity associated with the avian myeloblastosis virus RNA-directed DNA polymerase has been shown to be activated by ATP in the presence of Mg2+. In the presence of Mn2+ the endonucleolytic activity was stimulated about 3-fold by the addition of ATP. The earlier identified Mr = 40,000 Friend murine leukemia virus (F-MuLV)-associated endonuclease which functions in the presence of both Mg2+ and Mn2+ has also been shown to be similarly stimulated by ATP. For both endonuclease activities stimulation was only observed at ATP concentrations above 0.5 mM, and it did not increase upon elevating the ATP concentration above 2.5 mM. ADP and dATP also stimulated both activities, although not to the same extent as ATP. GTP had no apparent effect and AMP seemed to inhibit both activities. The effect ATP analogs had on the F-MuLV associated endonuclease activity could suggest that the endonuclease reaction in the presence of ATP might involve the cleavage of beta-gamma phosphate bonds in ATP. Neither adenyl-5'-yl imidodiphosphate nor (beta, gamma-methylene)adenosine 5'-triphosphate stimulated the activity, whereas significant stimulation was observed in the presence of (alpha, beta-methylene)adenosine 5'-triphosphate. Although no ATPase activity could be detected in the purified F-MuLV endonuclease preparation, the data do not exclude the possibility that ATP may be cleaved in amounts which are equivalent to the number of nicks introduced into DNA by the virus-associated endonuclease. In the presence of ATP and Mg2+ the F-MuLV-associated endonuclease nicked both supercoiled and linear DNA duplexes extensively, although the former was nicked more readily than the latter. Single-stranded DNA functioned poorly as a substrate. The nicks introduced by the enzyme contained a 5'-phosphoryl terminus and a 3'-hydroxyl group.
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PMID:Effect of ATP on the Friend Murine leukemia virus-associated endonuclease activity and the endonuclease activity of the avian myeloblastosis virus RNA-directed DNA polymerase. 616 71

The dialdehyde derivative of ATP inhibits DNA synthesis by AMV reverse transcriptase, while the polymerase-associated ribonuclease H activity is significantly resistant to this reagent. Neither ATP nor its dialcohol form effectively block DNA synthesis, indicating that the aldehyde moiety is required for inhibition. The nature of the reactivity of dialdehyde-ATP with AMV reverse transcriptase has been examined and we find that: (a) inhibition is non-competitive with respect to substrate deoxynucleoside triphosphate concentration, suggesting that dialdehyde-ATP does not react at the substrate binding site; (b) pretreatment of enzyme with dialdehyde-ATP or sulfhydryl group binding reagents results in the complete loss of its template binding activity; however, treatment of preformed enzyme-template-primer complex with both inhibitors did not dissociate this complex; (c) the inhibitory effect of dialdehyde-ATP was completely reversed upon addition of reducing agents, such as dithiothreitol and sodium borohydride, indicating that dialdehyde-ATP reacts with the sulfhydryl groups present in AMV reverse transcriptase; (d) comparative studies carried out with the classical sulfhydryl reagent, dithiobisnitrobenzoic acid, revealed a remarkable similarity in its action to that of dialdehyde-ATP. We therefore conclude that the dialdehyde-ATP-mediated inhibition of AMV DNA polymerase is effected via blockage of essential sulfhydryl groups present in the enzyme protein.
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PMID:The mechanism of inhibition of avian myeloblastosis virus reverse transcriptase by a dialdehyde derivative of ATP. Inactivation of essential sulfhydryl group function. 618 18

The p30 antigen from Rauscher leukemia virus (R-MuLV) was separated into two fractions by chromatography on either phosphocellulose or DEAE-cellulose. The p30-I and p30-II were indistinguishable immunologically or by isoelectrofocusing and gel electrophoresis. An ATPase activity was tightly associated with p30-II that could not be separated by ion-exchange chromatography, isoelectrofocusing, or glycerol velocity gradient sedimentation. The ATPase hydrolyzed the gamma phosphate from only ATP or dATP. Immunoglobulin directed against R-MuLV p30 completely inhibited the p30-II associated ATPase. Glycerol velocity gradient analysis showed that p30-I sedimented as a 30-kDa species while the p30-II and its associated ATPase sedimented as a 60-kDa species. The p30-II was converted entirely to a 30-kDa form by treatment with 0.2% (w/v) lithium dodecyl sulfate, suggesting that it represented a complexed species of p30. Finally, p30-II was found to stimulate the activity of R-MuLV reverse transcriptase, but p30-I had no effect on the activity of the enzyme. These results suggested the existence of at least two different forms of p30 in R-MuLV.
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PMID:Characterization of a p30 fraction from Rauscher leukemia virus which has an associated ATPase activity. 620 91

A cell line, TBSV7, that produces noninfectious murine sarcoma virus (MuSV) in the absence of helper MuLV was isolated from TB cells infected with the supernatant of MuSV349 cells. These noninfectious MuSV particles with "immature" C-type virus morphology contain a 2.2 X 10(6)-Da genomic RNA and an uncleaved 62,000-Da gag precursor protein (Pr62). Neither viral envelope proteins (gp70, p15E, p12E) nor reverse transcriptase were detected in these virus particles. Pr62 was found to be phosphorylated in vivo and it could be phosphorylated in vitro with [gamma-32P]ATP, indicating that protein kinase was packaged in these noninfectious virions. In vitro processing of Pr62 to smaller molecular weight proteins could be achieved by the addition of Mo-MuLV and Nonidet P-40. The initial cleavage products were proteins with molecular weights of 38K (Pr38) and 27K (Pr27). Under optimum conditions Pr38 was cleaved to p30 and a protein band migrating with MuLV-p10, while Pr27 was cleaved to a 17,000-Da protein that migrated slower than MuLV-p15 and a protein band migrating with MuLV-p12. Pulse-chase experiments performed on TBSV7 cells superinfected with Mo-MuLV indicated that intracellular processing of Pr62 was much slower than that of Pr65. Cleavage protein products of Pr62 similar in size to the in vitro protein products were also detected in TBSV7 cells superinfected with MuLV.
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PMID:Isolation and characterization of a Mo-MuSV-transformed TB cell line that produces noninfectious MuSV particles with uncleaved gag protein which is processed in the presence of Mo-MuLV. 632 21


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