Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RNA-dependent RNA polymerase (replicase) of encephalomyocarditis (EMC) virus was found to be closely associated with the smooth membranes of infected BHK-21 cells. An RNA-dependent EMC replicase was extracted from the membranes with 0.15% sodium dodecyl sulfate (SDS) and 1,1,2-trichlorotri-fluoroethane (Genetron 113) and further purified by high-salt dextran-polyethylene glycol phase separation, sievorptive chromatography, and glycerol gradient sedimentation. The enzyme does not manifest strict specificity toward EMC RNA template. It can use also Qbeta RNA, rRNA of BHK cells, or poly(C). SDS-polyacrylamide gel electrophoresis of purified EMC replicase labeled with radioactive methionine revealed that, of all the stable EMC proteins, the enzyme contains predominantly the 56,000-dalton (E) polypeptide.
 ...
PMID:Isolation and properties of the replicase of encephalomyocarditis virus. 0 14

1. Circular dichroic (CD) spectra of purified intermediate subviral particles of reovirus were determined in the presence of different monovalent cations. 2. The CD spectra reveal that reo intermediate subviral particles can exist in two conformationally different forms. The two forms are readily distinguished by comparison of their ellipticities in the wavelength regions 210 nm and 220 nm, with a Na+-induced form exhibiting a reduced negative ellipticity relative to a Cs+-induced form. 3. The transition between the Na+- and Cs+-induced forms is reversible by manipulation of the species of monovalent cation present and appears to be temperature independent. 4. Temperature variation studies on dilute suspensions of particles indicate that the Na+-induced form is stable, whereas the Cs+-induced from undergoes a second transition, temperature dependent and irreversible, to become a viral core. 5. A model is presented relating these observations to the known properties of reovirus uncoating and transcriptase activation.
 ...
PMID:Circular dichroism of intermediate subviral particles of reovirus. Elucidation of the mechanism underlying the specific monovalent cation effects on uncoating. 6 90

The response of mouse L cells to infection with wild-type (wt) and temperature-sensitive (ts) mutants of vesicular stomatitis virus was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to delineate the synthesis of host cell and viral proteins. Experiments utilized transcriptase mutants of complementation group I (ts114 and ts13), a group IV mutant (ts44) that is restricted in total RNA synthesis (RNA-1) but not in primary transcription, and a group II mutant (ts52) variably restricted in RNA synthesis (RNA +/-). L cells infected with ts mutants at permissive temperature exhibited the wt response of progressive inhibition of host cell protein synthesis accompanied by accumulation of all five viral proteins. Mutant ts44 (IV) also switched off cell protein synthesis at restrictive temperature and accumulated all five viral proteins, but with disproportionate ratios of N and G proteins. At restrictive temperature, cells infected with group I ts mutants failed to accumulate any viral protein and did not exhibit significant reduction in host cell protein synthesis. These data suggest that vesicular stomatitis virus inhibits cell protein synthesis at a stage of viral infection after transcription and possibly translation but preceding replication of progeny viral RNA.
 ...
PMID:Differential inhibition of host protein synthesis in L cells infected with RNA - temperature-sensitive mutants of vesicular stomatitis virus. 17 96

A fraction containing membrane-bound tobacco mosaic virus RNA replicase was isolated form tobacco mosaic virus-infected tobacco callus cultures. The replicase activity reached a maximum 60 h after inoculation and then declined. The enzyme activity was insensitive to actinomycin D and DNase. The corresponding fraction from healthy callus contained essentially no activity. The viral RNA synthesis in vitro proceeded linearly for 30 min and required the four nucleotide triphosphates and Mg2+ ions. Mn2+ was a poor substitute for Mg2+. During RNA synthesis the product was at least 70% resistant to RNase in 2X SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digested by RNase in 0.1X SSC. Analysis of the product by polns) that appeared to be replicative form and a partially RNase-resistant structure similar to replicative intermediate form. Washing the membrane-bound replicase with Mg2+-deficient buffer solubilized enzyme. The solubulized enzyme was further purified by DEAE-Sephadex column chromatography. The DEAE-purified enzyme was nearly completely dependent upon tobacco mosaic virus RNA for activity. Analysis of the product on a sucrose gradient revealed a double-stranded RNA with sedimentation of 16S and smaller heterogeneous RNase-sensitive products.
 ...
PMID:In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: solubilization of membrane-bound replicase and partial purification. 83 35

We have reported that an 11,600-MW (11.6K) protein is coded by region E3 of adenovirus. We have now prepared two new antipeptide antisera that have allowed us to characterize this protein further. The 11.6K protein migrates as multiple diffuse bands having apparent Mws of about 14,000, 21,000, and 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblotting as well as virus mutants with deletions in the 11.6K gene were used to show that the various gel bands represent forms of 11.6K. The 11.6K protein was synthesized in very low amounts during early stages of infection, from the scarce E3 mRNAs d and e which initiate from the E3 promoter. However, 11.6K was synthesized very abundantly at late stages of infection, approximately 400 times the rate at early stages, from new mRNAs termed d' and e'. Reverse transcriptase-polymerase chain reaction and RNA blot experiments indicated that mRNAs d' and e' had the same body (the coding portion) and the same middle exon (the y leader) as early E3 mRNAs d and e, but mRNAs d' and e' were spliced at their 5' termini to the major late tripartite leader which is found in all mRNAs in the major late transcription unit. mRNAs d' and e' and the 11.6K protein were the only E3 mRNAs and protein that were scarce early and were greatly amplified at late stages of infection. This suggests that specific cis- or trans-acting sequences may function to enhance the splicing of mRNAs d' and e' at late stages of infection and perhaps to suppress the splicing of mRNAs d and e at early stages of infection. We propose that the 11.6K gene be considered not only a member of region E3 but also a member of the major late transcription unit.
 ...
PMID:The 11,600-MW protein encoded by region E3 of adenovirus is expressed early but is greatly amplified at late stages of infection. 131 73

Poliovirus RNA encodes an RNA-dependent RNA polymerase, designated 3Dpol, that catalyzes the synthesis of both plus and minus strand viral RNA. This enzyme was purified to near homogeneity from poliovirus-infected HeLa cells, recombinant baculovirus-infected insect cells, and from Escherichia coli transformed with an expression plasmid containing poliovirus 3D sequences. The two recombinant expression systems produced significantly higher yields of active enzyme than could be attained from virus-infected HeLa cells. All preparations contained a 52-kDa protein, recognized by antisera raised against 3D expressed as a fusion protein in E. coli. Immunoreactive protein resolved into 3-4 species on isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional gels. Efforts to demonstrate that the multiple spots resulted from phosphorylation were negative. Furthermore, no evidence for autophosphorylation of purified 3Dpol was obtained. Purified 3Dpol from recombinant sources manifested the same specific activities as enzyme from poliovirus-infected HeLa cells in both a poly(A)-dependent poly(U) polymerase assay and a poliovirus RNA-dependent RNA polymerase assay. The products of the latter reaction reached the length of the template (7.5 kilobases) in 20-30 min, indicating an elongation rate of approximately 300 nucleotides/min at 30 degrees C. No products exceeded the length of the template. Intermediate length products were detected, which presumably resulted from pauses in transcription due to template structure. All transcription was dependent on primer. The kinetic parameters of all three purified enzyme preparations were the same; the Km for UTP was 2.4 +/- 0.1 microM in an RNA polymerase activity assay. Product formation was linear for up to 45 min, except for a 3-5-min lag before synthesis began. The lag was independent of enzyme concentration, and independent of the template used. The lag was eliminated by preincubating enzyme, template, primer, and three of the four nucleotide triphosphates, but not by preincubating any subset of these components. This suggested that a preinitiation complex must form as a prerequisite to RNA synthesis. Partially purified preparations of 3Dpol from the three sources showed significant differences in activities and products, including the appearance of primer-independent polymerase activity and production of dimer-length RNA products. These variable properties are likely due to different contaminating activities provided by the different cellular hosts, since upon further purification, all three enzymes exhibited identical properties.
 ...
PMID:Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources. 166 Aug 94

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.
 ...
PMID:Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase. 185 68

Gene P1 of Mycoplasma pneumoniae, which codes for a major adhesin, is flanked by two sequences with open reading frames designated ORF4 and ORF6 (Inamine et al., 1988b). In order to identify proteins translated from those ORFs, gene fusions between the N-terminus of the RNA replicase of the Escherichia coli bacteriophage MS2 and selected regions of ORF4 and ORF6 were constructed. The corresponding fusion proteins synthesized in Escherichia coli were used to immunize mice. Antisera directed against ORF4-related sequences did not recognize M. pneumoniae antigens in Western blot analysis, but antisera directed against ORF-6-derived fusion proteins reacted with two M. pneumoniae proteins of 40 kDa and 90 kDa. In addition, some of the antisera also recognized proteins that formed in a sodium dodecyl sulphate/polyacrylamide gel a protein ladder between 115 and 145 kDa.
 ...
PMID:Identification of gene products of the P1 operon of Mycoplasma pneumoniae. 190 24

Transplantable erythroblastic leukemia was induced by 300-rad irradiation of C3H mice. Conditions for in vitro growth of the leukemic cells were studied. None of interleukin-3, granulocyte/macrophage colony-stimulating factor and erythropoietin could support the growth of the cells in vitro. In contrast, the leukemic cells grew into a stroma-dependent cell line, ELM-D, in close contact with the stromal cell layer of 900-rad-irradiated long-term bone marrow culture. A stroma-independent cell line, termed ELM-I-1, was further established from the non-adherent population in the co-culture of the leukemic cells, ELM-D, with stromal cells. Reverse transcriptase activity was not detectable in ELM-D or ELM-I-1 cells. Studies on binding and cross-linking of 125I-erythropoietin showed that ELM-I-1 cells had erythropoietin receptors, and two major radiolabeled protein products with molecular weights of 120 kDa and 140 kDa were detected on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions.
 ...
PMID:Stromal cell-dependent growth of leukemic cells from murine erythroblastic leukemia. 246 Apr 23

The genome of infectious bursal disease virus (IBDV) consists of two segments of double-stranded (ds)RNA with molecular weights of 2.2 X 10(6) and 1.9 X 10(6) Da, respectively. After treatment of IBDV particles with proteinase K in the presence of sodium dodecyl sulfate (SDS), linear dsRNA molecules are released from the virus particles. However, after heating of virus particles at 100 degrees for 3 min in 1.5% SDS, without the protease, dsRNA-protein complexes can be seen under the electron microscope: Knob-like proteinaceous structures are linked to the ends of the dsRNA molecules of either size class which are circularized to form individual rings. A 90,000-Da IBDV structural polypeptide, the only protein encoded by the smaller genome segment, has been demonstrated to remain firmly linked to the IBDV genome under these conditions. No functional data exist about this circularizing protein; it is a probable candidate for an RNA-dependent RNA polymerase or an assembly protein for the two dsRNA segments. At high particle concentrations, or when the preparations are allowed to stand for several hours before spreading, these complexes tend to aggregate to form flower-like structures.
 ...
PMID:The two segments of the infectious bursal disease virus genome are circularized by a 90,000-Da protein. 303 77


1 2 3 4 5 6 7 8 9 10 Next >>