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

---

Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.
...
PMID:Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation. 255 40

Amplifiable hybridization probes--molecules with a probe sequence embedded within the sequence of a replicatable RNA--will promote the development of sensitive clinical assays. To demonstrate their utility, we prepared a recombinant RNA that contained a 30-nucleotide-long probe complementary to a conserved region of the pol gene in human immunodeficiency virus type 1 (HIV-1) mRNA. Test samples were prepared, each containing a different number of HIV-1 transcripts that served as simulated HIV-1 mRNA targets. Hybridizations were carried out in a solution containing the chaotropic salt, guanidine thiocyanate. Probe-target hybrids were isolated by reversible target capture on paramagnetic particles. The probes were then released from their targets and amplified by incubation with the RNA-directed RNA polymerase, Q beta replicase (EC 2.7.7.48). The replicase copied the probes in an exponential manner: after each round of copying, the number of RNA molecules doubled. The amount of RNA synthesized in each reaction (approximately 50 ng) was sufficient to measure without using radioisotopes. Kinetic analysis of the reactions demonstrated that the number of HIV-1 targets originally present in each sample could be determined by measuring the time it took to synthesize a particular amount of RNA (the longer the synthesis took, the fewer the number of targets originally present). The results suggest that clinical assays involving replicatable hybridization probes will be simple, accurate, sensitive, and automatable.
...
PMID:Quantitative assays based on the use of replicatable hybridization probes. 267 78

A study was made of the fate of parental bluetongue virus (BTV) in infected cells. Within the first hour after infection, the BTV particles are converted to core particles with the loss of major capsid polypeptides P2 and P5. The particles are able to synthesize mRNA in vitro in a transcription reaction characterized by a temperature-dependent inhibition at high core concentrations. From about 6 hr after infection a second uncoating event is observed in which the 470 S core particles are converted to 390 S subcore particles. These particles are morphologically strikingly different from core particles and have a skeletonlike structure with a hexagonal profile and a side to side diameter of 40 nm. These subcore particles contain only one major structural protein, P3, and three minor proteins, P1, P4, and P6. They do, however, contain all 10 double-stranded RNA segments. The results suggest that the characteristic capsomeres on the surface of core particles are composed mainly of P7, the soluble group-specific antigen of BTV. The subcore particles are stable only at very low salt concentrations. Under these conditions no transcriptase activity can be demonstrated.
...
PMID:Uncoating of parental bluetongue virus to core and subcore particles in infected L cells. 302 57

Virus-like particles that contain L-A double-stranded RNA are known to have transcriptase activity whose product is L-A single-stranded plus RNA. In low salt conditions, these particles release their double-stranded RNA and can then use added plus L-A or plus M1 single-stranded RNAs as templates to synthesize their respective double-stranded RNAs. The reaction requires dialyzed L-A virus-like particles as the source of the enzyme, a partially purified cell extract (host factor(s)), added single-stranded RNA as a template, and polyethylene glycol 6000, along with four NTPs. Crude host factor extracts prepared from mak3 or mak10ta mutants also support the reaction as effectively as that from a wild type strain, while a crude extract prepared from a pet18 mutant grown under the nonpermissive conditions is less effective. Template specificity of the in vitro reaction is the same as that expected for the enzyme reaction in vivo. Plus L-A and plus M1 single-stranded RNAs, but not 18 S rRNA, are converted to their respective double-stranded RNAs with net RNA synthesis. The newly synthesized strand of M1 double-stranded RNA is a full-length minus strand. This demonstration of replicase activity in the mature L-A virus-like particles which contain L-A double-stranded RNA is consistent with our previous L-A double-stranded RNA replication model; the difference between the mature L-A virus-like particles and L-A double-stranded RNA-synthesizing particles (expected to be replication intermediates in vivo) is just that the former contain L-A double-stranded RNA, while the latter contain L-A plus single-stranded RNA.
...
PMID:Replicase of L-A virus-like particles of Saccharomyces cerevisiae. In vitro conversion of exogenous L-A and M1 single-stranded RNAs to double-stranded form. 327 47

The ability of the fowl plague virus (FPV) M protein to form a complex with FPV RNP and to inhibit the RNP transcriptase activity in vitro depended on NaCl concentration and did not depend on the concentration of nonionic detergents. The results obtained indicate that the M protein-RNP links formed were of an electrostatic rather than a hydrophobic nature. As demonstrated using individual RNP components, vRNA and RNA-free protein structures, M protein formed complexes only with vRNA, and the complex formation was salt-dependent. Analysis of products formed in the in vitro system containing RNP of FPV in the presence of the M protein showed impairment in the transcription of all RNA segments. The degree of inhibition correlated with the size of a segment, transcription of high molecular weight RNA segments being inhibited significantly more than that of low molecular weight RNA segments.
...
PMID:Interaction of M protein and RNP of fowl plague virus in vitro. 384 Sep 37

Transcriptase activity was dissociated from vesicular stomatitis virions by highionic-strength buffer containing Triton X-100. Considerable enzyme activity could be restored by recombining inactive sedimentable and nonsedimentable virion fractions. Reconstituted transcriptase activity was dependent on the presence of all four nucleoside triphosphates and the concentration of heat-labile molecules in both supernatant and pellet fractions. Lower NaCl concentrations removed approximately 46% of virion protein, but did not release transcriptase activity from the pellet fraction, nor could incorporation of (3)H-uridine-5'-triphosphate by complete virions be increased by adding soluble transcriptase. Evidence that the virion nucleocapsid is the transcription template was provided by finding that the pellet contained predominantly virion core nucleoprotein, ribonucleic acid, and homogeneous nucleocapsid coils when viewed by electron microscopy. Removal of envelope G and M proteins by Triton and low-salt buffer without decreasing nucleocapsid polymerase activity indicates that neither G nor M protein is necessary for transcription. Additional data are required to determine whether the minor nucleocapsid proteins L or NSl, or both, which are at least partially solubilized in high-salt buffer, are the transcriptase. Preliminary data suggest that the major N nucleoprotein, which was not solubilized by high-salt buffer, is also required for transcription. Defective T virions contained at least as much transcriptase per weight as did B virions, as determined by restoration with T supernatant fluids of transcription function to B nucleocapsid template. However, the T nucleocapsid would not serve as template for B or T transcriptase, a finding which is interpreted as evidence of T template defectiveness. The presence of defective T nucleocapsids did not interfere with B or T transcriptase function reconstituted with B template.
...
PMID:Dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis B and T virions. 434 47

The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis.
...
PMID:L protein requirement for in vitro RNA synthesis by vesicular stomatitis virus. 435 10

The ribonucleoprotein-dependent RNA transcriptase in vesicular stomatitis B virions of four temperature-sensitive (ts) mutants belonging to complementation group I was analyzed in vitro at permissive (31 C) and restrictive (39 C) temperatures. The RNA-synthesizing activity of all four ts mutants was more labile at 39 C than was the transcriptive activity of wild-type (wt) virions. In order to locate the temperature-sensitive transcription defect in the mutants, wt and ts mutant virions were fractionated by Triton X-100-high salt solubilizer into a sedimentable ribonucleoprotein template and a nonsedimentable enzyme fraction, each of which alone had little or no transcriptive activity. The template- and enzyme-containing fractions of wt virions were then tested for their capacity to restore transcriptive activity at 39 C to corresponding template and enzyme preparations of ts mutant virions. Recombination of wt template and ts enzymes resulted in no significant restoration of capacity to synthesize RNA at restrictive temperature. In contrast, transcriptive function at 39 C was reconstituted by recombining the wt enzyme with the template component of ts mutants. It appears, therefore, that the enzyme, rather than the template, is the temperature-sensitive component of the transcription complex of group I vesicular stomatitis virus mutants.
...
PMID:Location of the transcription defect in group I temperature-sensitive mutants of vesicular stomatitis virus. 435 28

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride-1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.
...
PMID:Nucleic acid enzymology of extremely halophilic bacteria. Gel-filtration and density-gradient-centrifugation studies of the molecular weights of Halobacterium cutirubrum polynucleotide phosphorylase and deoxyribonucleic acid- and ribonucleic acid-dependent ribonucleic acid polymerases. 511 75

A large-particle fraction obtained from reovirus-infected L cells contained both replicase and transcriptase activity. The in vitro replicase reaction slowed down soon after initiation, whereas the transcriptase reaction proceeded at an unabated rate. The replicase and transcriptase were both template-bound and could be separated from one another by controlled chymotryptic digestion followed by centrifugation in a CsCl gradient. The transcriptase was recovered as a sharp band (rho = 1.43) and resembled virus core derived from mature virions. In contrast, replicase activity was distributed throughout the gradient, indicating that replicase is associated with structures of various density in CsCl. In subsequent experiments, the replicase product was found to be indistinguishable from the double-stranded ribonucleic acid (RNA) reovirus genome with respect to its buoyant density in cesium-salt gradients and denaturation-annealing characteristics. A "hybridization-competition" experiment in which the replicase product was denatured and annealed in the presence of an excess of plus-RNA indicated that the in vitro replicase reaction proceeded by means of a unilateral synthesis of minus-RNA upon a preexisting plus-RNA template, presumably of single-stranded form.
...
PMID:Unilateral synthesis of reovirus double-stranded ribonucleic acid by a cell-free replicase system. 511 15


<< Previous 1 2 3 4 5 6 7 8 9 Next >>

---