In vitro synthesis of adenovirus core proteins.
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mRNA extracted from polysomes of KB cells at late stages of productive infection with adenovirus type 2 was translated in a cell-free system derived from Krebs II ascites cells. Two of the polypeptides obtained in this reaction corresponded to the adenovirus core protein V and the precursor to core protein VII. The following criteria were used to establish identity between the in vitro products and the virion proteins: comigration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis, cochromatography in sodium dodecyl sulfate-hydroxyapatite, specific immunoprecipitation of the precursor to core protein VII, and tryptic peptide analysis. (+info)
Factors controlling amino acid incorporation by ribosomes from krebs II mouse ascites-tumour cells.
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1. A ribosome-cell sap system capable of supporting the incorporation of (14)C-labelled amino acids into protein has been prepared from Krebs II mouse ascites-tumour cells. The requirements of this system for optimum activity and response to added messenger RNA have been investigated. One such system has been obtained for which amino acid incorporation is almost wholly dependent on the addition of suitable messenger RNA. 2. Ribosomes of widely different but predictable activities in the cell-free system have been prepared from Krebs cells pretreated in a variety of ways. The factors in the pretreatment of the cells responsible for these differences have been investigated. 3. The structural and functional properties of these different ribosome preparations and their response to exogenous messenger RNA have been examined and are discussed in the light of modern concepts of the control of protein synthesis. (+info)
Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.
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Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis. (+info)
Characterization of the polypeptides formed in response to encephalomyocarditis virus ribonucleic acid in a cell-free system from mouse ascites tumor cells.
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The polypeptide products synthesized at different times in a cell-free system from Krebs mouse ascites tumor cells in response to the addition of encephalomyocarditis (EMC) virus ribonucleic acid (RNA) were characterized by electrophoresis on polyacrylamide gels and fingerprint analysis of their tryptic peptides. Translation of the EMC RNA genome with time occurred in a nonrandom fashion in these systems, to yield products containing sequences characteristic of both virion capsid polypeptides and EMC-specific polypeptides present only in the infected cell. The molecular weights of the products fell in a series from 20,000 to 140,000 daltons, although occasionally traces of larger polypeptides were also observed. All of the major polypeptides appeared to arise from partial or complete translation of about 60% of the EMC RNA genome. They were not formed by cleavage of a large precursor molecule. It is suggested that they are artifacts generated by premature "termination" of nascent polypeptide chains at preferred sites. (+info)
Double-stranded RNA as an inhibitor of protein synthesis and as a substrate for a nuclease in extracts of Krebs II ascites cells.
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Concentrations of double-stranded RNA above about 0.1 mug/ml inhibit translation of encephalo-myocarditis viral RNA and mouse globin messenger RNA in extracts of Krebs II ascites cells. Protein synthesis initially proceeds at the control rate, then abruptly shuts off in a manner similar to that observed in reticulocyte lysates [Hunt, T. & Ehrenfeld, E. (1971) Nature New Biol. 230, 91-94]. Substantially higher concentrations of double-stranded RNA are required to give this effect in ascites extracts. Subcellular fractions of Krebs II ascites cells contain a nucleolytic activity capable of digesting several natural and synthetic double-stranded RNAs. This nuclease is most active under conditions of protein synthesis, and part of the activity remains associated with ribosomes upon sedimentation. It is probably because of digestion of double-stranded RNA by this nuclease that higher concentrations of double-stranded RNA are required for inhibition of protein synthesis in Krebs cell extracts than in reticulocyte lysates. (+info)
Discrimination between messenger ribonucleic acids by a mammalian translation initiation factor.
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A factor from rabbit-reticulocyte ribosome, which stimulates by 5-fold initiation of hemoglobin mRNA translation in Krebs ascites cell extracts, was purified to homogeneity. The factor, a protein of about 65,000 molecular weight in sodium dodecyl sulfate, discriminates between various mRNAs: it stimulates translation of alpha globin mRNA and of tobacco mosaic virus RNA, but has only a small effect on beta globin mRNA and no effect at all on Mengo virus RNA translation. (+info)
Translation of vesicular stomatitis messenger RNA by extracts from mammalian and plant cells.
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RNA was isolated from polyribosomes of vesicular stomatitis virus (VSV)-infected cells and tested for its ability to direct protein synthesis in extracts of animal and plant cells. In cell-free, non-preincubated extracts of rabbit reticulocytes, the 28S VSV RNA stimulated synthesis of a protein the size of the vesicular stomatitis virus L protein whereas the 13 to 15S RNA directed synthesis of the VSV M, N, NS, and possibly G proteins. In wheat germ extracts, 13 to 15S RNA also directed synthesis of the N, NS, M, and possibly G proteins. Analysis of extracts labeled with formyl [(35)S]methionine showed that the 28S RNA directed the initiation of synthesis of one protein, whereas the 13 to 15S RNA directed initiation of at least four proteins. It is concluded that the 28S RNA encodes only the L protein, whereas the 13 to 15S RNA is a mixture of species, presumably monocistronic, which code for the four other known vesicular stomatitis virus proteins. (+info)
Studies on the form and synthesis of messenger ribonucleic acid in the rat ventral prostate gland, including its tissue-specific stimulation by androgens.
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1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6-15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5'-fluoro-orotic acid into this 6-15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6-15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6-15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed. (+info)