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Biomedical subjects

I M Kerr

Publications and source records attributed to I M Kerr.

At least 127 records · Page 7Linked to original sources
PubMed1976

Interferon-induced inhibition of protein synthesis in L-cell extracts: an ATP-dependent step in the activation of an inhibitor by double-stranded RNA.

The translation of encephalomyocarditis virion RNA in extracts from interferon-treated L-cells is inhibited by the addition of double-stranded RNA (dsRNA) at 400 ng/ml. A similar inhibition in response to dsRNA is seen in control cell extracts supplemented with small amounts of a postribosomal supernatant fraction from interferon-treated cells (interferon cell sap): Neither interferon cell sap nor dsRNA alone is inhibitory in control systems. The inhibition is much reduced if translation is carried out at low ATP concentrations.Conversely, the inhibitory capacity of the interferon cell sap is increased 100-fold if it is preincubated with dsRNA and ATP prior to its addition to the protein-synthesizing system. After this preincubation all detectable dsRNA can be removed without any diminution of the inhibitory activity of the cell sap. These results are compatible with a two-step model for the inhibition in which a pre-inhibitor is activated by dsRNA, the activated inhibitor then interacting with the protein synthesis system to inhibit translation.

Adenosine Triphosphate↗
PubMed1976

Translation of murine leukemia virus RNA in cell-free systems from animal cells.

The virion RNA of Moloney murine leukemia virus (MuLV) has been translated in eukaryotic cell-free systems derived from mouse L- and human HeLa cells. In both systems at least three polypeptides, approximately 60,000, 70,000, and 180,000 in apparent molecular weight, were formed in response to the added 35S MuLV RNA. All three polypeptides were precipitable with antiserum to detergent-disrupted MuLV. Fingerprint analysis of tryptic digests indicated that all three contain anino acid sequences in common with each other and with the major methionine-containing structural proteins of the virion.

Amino Acid Sequence↗
PubMed1974

Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNA(F).

The translation of encephalomyocarditis virus (EMC) RNA is markedly inhibited in cell-free systems from interferon-treated, vaccinia virus-infected L-cells (10, 11). The polypeptide products synthesized in response to EMC RNA in cell-free systems from these and untreated infected cells have been analyzed by electrophoresis on polyacrylamide gels. Qualitatively, the same EMC-specific polypeptides were synthesized throughout. In experiments using preincubated microsomes from normal Krebs cells to assay cell sap from L-cells which had been exposed to interferon prior to infection, only the amount of the EMC-specific polypeptide products was reduced. This result suggests that there is an inhibition very early in translation in interferon-treated, infected cells. Initiation seems a priori the more attractive site for this inhibition, but an effect shortly after initiation cannot be excluded. With unfractionated cell-free systems from interferon-treated infected L-cells, however, there appeared to be an additional minor inhibitory effect on polypeptide chain elongation, in that the EMC-specific polypeptides synthesized showed not only a reduction in amount but also a bias towards lower molecular weight. The formylated methionyl initiator tRNA (Fmet-tRNA(F)) was used as a further probe into the apparent effect on intiation. With this reagent we have confirmed that there is one major initiation site for the translation of EMC RNA in these cell-free systems. In addition, the results have shown that EMC-specific polypeptide chains initiated with Fmet escape the major interferon-mediated inhibition at or shortly after initiation.

Journal Article↗
PubMed1972

Characterization of the polypeptides formed in response to encephalomyocarditis virus ribonucleic acid in a cell-free system from mouse ascites tumor cells.

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.

Animals↗
PubMed1972

Mechanism of interferon action: inhibition of viral messenger ribonucleic acid translation in L-cell extracts.

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.

Amino Acids↗
PubMed1971

Virus protein synthesis in animal cell-free systems: nature of the products synthesized in resonse to ribonucleic acid of encephalomyocarditis virus.

Ribonucleic acid (RNA) from encephalomyocarditis (EMC) virus stimulates the incorporation of amino acids into protein in cell-free protein-synthetic systems derived from Krebs mouse ascites tumor cells and chick embryo fibroblasts; the mouse system is the more responsive to the viral RNA. The greater part of this difference in activity can be ascribed to the cell sap, but the origin of the ribosomes also has a marked effect. The nature of the polypeptides formed in these cell-free systems was investigated by electrophoresis on polyacrylamide gels and by fingerprint analysis of tryptic digests. The same product in part appears to be synthesized in response to the EMC RNA in both systems. It was not detected if the EMC RNA was partly degraded (</=4S) or replaced by other species of RNA, including that from influenza virus. The results suggest that EMC RNA is partially translated in these systems to yield virus-specific polypeptides.

Amino Acids↗
PubMed1971

Protein synthesis in cell-free systems: an effect of interferon.

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.

Amino Acids↗
PubMed1971

Synthesis of capsid and noncapsid viral proteins in response to encephalomyocarditis virus ribonucleic acid in animal cell-free systems.

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of (35)S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.

Animals↗
PubMed1970

Proein-synthetic activity of ribosomes from interferon-treated cells.

Cell-free protein-synthetic systems from normal and interferon-treated chick cells were compared. No difference was found in the amino acid incorporation activities of such ribosome-cell sap systems or in their response to polyuridylic acid. Throughout a variety of experiments we failed to detect the formation of a discrete peak of virus-specific polysomes, when ribosome monomers and subunits (from interferon-treated or control cells) were incubated with labeled Sindbis or Semliki Forest virus ribonucleic acid (RNA). Some binding of viral RNA did occur, but the complexes formed were evident in sucrose gradients as a broad, rapidly sedimenting shoulder on the ribosome monomer peak. Interferon pretreatment of cells did not affect the formation of these complexes in vitro, nor did it alter their rate of breakdown on incubation under amino acid incorporation conditions. Experiments with inhibitors of protein synthesis showed that such "breakdown" was not dependent upon amino acid incorporation and was not an index of translation. In these respects, our results are in marked contrast to those of Marcus and Salb. These results, together with our failure to detect any significant change in the protein composition of ribosomes from interferon-treated cells, suggest that such treatment does not result in a modification of the ribosome per se. They do not, however, rule out the involvement of a factor(s) required for ribosomes and viral RNA to function in viral protein synthesis. Indeed, it remains likely that interferon acts through such a mechanism, although the precise level at which the inhibition occurs remains to be elucidated.

Amino Acids↗
PubMed1966

Factors controlling amino acid incorporation by ribosomes from krebs II mouse ascites-tumour cells.

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.

Adenosine Triphosphate↗