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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure using the virus-associated reverse transcriptase was developed for following the kinetics of adsorption, penetration, and uncoating of murine leukemia virus. Viral adsorption to cell membrane was determined by assaying this enzyme activity in isolated debris of mechanically disrupted cells after infection with murine leukemia virus in the presence of actinomycin D. At 37 degrees C, viral adsorption proceeded at a high initial rate, but after 5 min of incubation with the virus, it gradually slowed down. At 4 degrees C, viral adsorption was slower but proceeded at a linear rate. Intracellular virus was determined by centrifuging the cytoplasmic fraction of the disrupted cells at 105,000 x g for 45 min and assaying reverse-transcriptase activity in the high-speed pellet thus obtained. Sucrose gradient analysis of the enzyme activity recovered from the cytoplasm of infected cells indicated that this activity represented intact virus particles. No appreciable amount of such particles was recovered from the cytoplasm of cells infected at 4 degrees C. This indicates that the virions recovered from the cytoplasm of cells infected at 37 degrees C are indeed intracellular virus particles which penetrated into the cells and not just membrane-bound particles mechanically released to the cytoplasmic fraction during cell disruption. By this procedure intracellular virus was found to accumulate in the cytoplasm, reaching a maximal level within 20 min. The accumulated intracellular virus particles gradually disappeared from the cytoplasm, evidently due to their uncoating which was completed within 80 min.
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PMID:Adsorption, penetration, and uncoating of murine leukemia virus studied by using its reverse transcriptase. 9 Jan 59

Upon infection of Chinese hamster ovary cells (CHO), vesicular stomatitis (VSV) virus synthesizes two membrane proteins (the VSV glycoprotein and the VSV matrix or membrane (M) protein) and three nonmembrane proteins (the VSV nucleocapsid, the viral transcriptase, and an NS protein). We have used the VSV-infected cell as a model system for the study of the site of synthesis of these membrane and nonmembrane proteins. We have isolated VSV mRNA from free polyribosomes, membrane-bound polyribosomes, and the postribosomal supernatant, and identified the individual species of VSV mRNA present in each fraction. The mRNA which encodes the VSV glycoprotein is found exclusively on membrane-bound polyribosomes, while the mRNAs which encode the VSV, M, N, and NS proteins are found in free polyribosomes, in the membrane fraction of the cell, and in the postribosomal supernatant. Our results suggest that the VSV glycoprotein is synthesized exclusively on membrane polyribosomes, while at least some of the M, N, and NS proteins are made on free polyribosomes.
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PMID:Site of synthesis of membrane and nonmembrane proteins of vesicular stomatitis virus. 16 63

Polioviral RNA polymerase complex, which consists of enzyme, template, and nascent RNA, is membrane bound in vivo. The solubilized RNA polymerase complex associated spontaneously in vitro with phospholipid bilayer membranes (liposomes) of defined composition. The degree of association at 37 degrees C was greater for those membranes that were more fluid, suggesting that the binding involves the interaction of the RNA polymerase complex with the hydrocarbon chains in the interior of the lipid bilayer. The polymerase activity was not enhanced by addition of the lipid; in fact, the addition of some of the longer-chain lipids resulted in up to a 40% inhibition of the polymerase activity. Spin-label electron paramagnetic resonance experiments, which measured the membrane fluidity, and kinetic experiments on the rate of incorporation of tritiated UTP into RNA by the polymerase were performed as a function of temperature. The results indicated that the activity of the polymerase was not affected by the physical state of the phospholipid membrane and that its active site was not intimately associated with the membrane. Analysis of both the viral and host polypeptides associated with the smooth membrane-bound polymerase indicated that X was the primary viral polypeptide present. In addition, host polypeptides of molecular weight 86,000, 62,000, 54,000, and 46,000 were also present. If the membrane was disrupted with detergent, polypeptide X was released from the polymerase activity, suggesting that X may play a role in binding the polymerase to the membrane. In an analogous manner, polypeptide X associated spontaneously with phospholipid membranes to a greater extent than the capsid polypeptides. Analysis of both the host and viral polypeptides associated with the viral RNA polymerase purified by precipitation in 2 M LiCl indicated that host polypeptides of molecular weight 106,000, 38,000, 33,000, and 14,000 were the major constituents, whereas relatively small amounts of the viral polypeptides were present. It was confirmed that of the viral polypeptides found, polypeptide 4 was present in the largest amount.
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PMID:Association of the polioviral RNA polymerase complex with phospholipid membranes. 18 11

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...
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PMID:On the DNA polymerase III of mouse myeloma: partial purification and characterization. 23 42

Dimethylnitrosamine (DMNA) strongly inhibited RNA synthesis in mouse liver under conditions when the nucleotide pattern, rate of nucleotide synthesis and phosphorylation ratio were unaffected. (An unidentified, probably non-nucleotide, component in the acid-soluble liver fraction was selectively reduced.) The inhibition of RNA synthesis was associated with a decrease in the RNA polymerase activity of isolated liver nuclei, well established already 45 min after DMNA administration. The reduced activity included both Mg2+- and Mn2+/(NH4)2SO4-stimulated polymerase functions. The inhibition in vivo involved the whole complement of RNA, including poly (A)-containing RNA and isolated poly(A) sequences. The transfer of labelled RNA from the nucleus to the cytoplasm was not impaired. There was no detachment of poly(A)-containing RNA from the microsomes, and the proportion of tightly membrane-bound microsomal RNA and poly(A) sequences was not reduced as determined by use of a flotation technique. No breakage or shortening of the poly(A) chains was indicated by sedimentation analysis.
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PMID:RNA metabolism and poly(A) distribution in mouse liver following administration of dimethylnitrosamine. 66 22

Effect of amphotericin B and nistatin on template activity of nuclear membrane-bound (DNPm) and free (DNPo) dog kidney chromatin after intravenous injections of antibiotics and after the incubation of isolated kidney cell nuclei with the antibiotics is studied. It is found that injections of amphotericin B and nistatin resulted in the increase of DNPo template activity in RNA polymerase system, the stimulating effect of nistatin being higher than that of amphotericin B. Injections of nistatine stimulated also template activity of DNPm, while amphotericin B produced no effect on DNPm. When studing the effect of polyene antibiotics on template activity of DNPo and DNPm in vitro, it is found that the intensity of RNA synthesis after incubation of isolated nuclei with antibiotics is considerably increased, and stimulating effect of nistatin is higher than of amphotericin B. Both antibiotics produced no effect on template activity of DNP in vitro. Thus, comparative analysis of changes in template activity of dog kidney chromatin under the effect of polyene antibiotics in vivo and in vitro has revealed the similarity of these drugs and draws to the conclusion that nistatin and amphotericin B produce a direct effect on template activity of chromatin.
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PMID:[The change of template activity of dog kidney chromtin by polyene antibiotics in vivo and in vitro]. 103 Jun 44

The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.
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PMID:Synthesis of the complete 200K polyprotein encoded by cowpea mosaic virus B-RNA in insect cells. 143 6

The protein-coding capacities of rat and human catechol O-methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate. Two types of clones corresponding to the structures of human placental cDNA clones were used. The shorter clones, containing the 663-residue open reading frame for the soluble COMT (S-COMT), produced 24-kDa (rat) and 26-kDa (human) polypeptides. Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino-terminal extensions to the S-COMT polypeptides, yielded 28-kDa (rat) and 30-kDa (human) putative membrane-bound COMT (MB-COMT) polypeptides as the main products. These clones also yielded low amounts of the S-COMT polypeptides. Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S-COMT AUG codon. In accordance with this postulation, the relative amount of S-COMT could be affected by changing the translation initiation contexts preceding the first AUG codon. The 28-kDa and 30-kDa products, but not the 24-kDa and 26-kDa products, associated with microsomal membranes cotranslationally, indicating that the amino-terminal extensions were functional signal sequences. However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels. The MB-COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion. These results indicate that MB-COMT synthesized in vitro is an integral membrane protein having an amino-terminal signal-anchor sequence.
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PMID:Cell-free synthesis of rat and human catechol O-methyltransferase. Insertion of the membrane-bound form into microsomal membranes in vitro. 176 63

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al. (1984) J. Biol. Chem. 259, 1807].
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PMID:Melibiose permease and alpha-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system. 215 86

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.
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PMID:Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes. 283 22


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