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Biomedical subjects

L A Ball

Publications and source records attributed to L A Ball.

88 records · Page 5Linked to original sources
PubMed1977

Transcriptional mapping of vesicular stomatitis virus in vivo.

Synthesis of the proteins of vesicular stomatitis virus (Indiana serotype) was studied in mouse L cells infected with virus that had been exposed to UV radiation. The UV target sizes measured during primary transcription indicated that the five genes occupy a single transcriptional unit. Thus, in infected cells, as a cell-free system, transcription of vesicular stomatitis virus RNA initiates at a single point and proceeds in the order N. NS, M, G, and L.

Cell-Free System↗
PubMed1976

Order of transcription of genes of vesicular stomatitis virus.

The effect of ultraviolet radiation on the expression of the genes of vesicular stomatitis virus (VSV) was studied in a cell-free system which executed coupled transcription and translation of the viral genes. In this system, which contained detergent-activated virus and a cytoplasmic extract of mouse L-cells, three of the five viral proteins (N, NS, and M) were synthesized accurately and efficiently; a putative precursor to the viral glycoprotein (G) was also made, but synthesis of the L protein was not detected. In response to irradiation of VSV, the synthesis of each protein in the coupled system displayed a characteristic single-hit inhibition. This permitted calculation of the apparent target-sizes for expression of the different viral genes relative to the entire genome (3.6 to 4.0 X 10(6) daltons) as measured by loss of infectivity. These are: N, 0.55 X 10(6); NS, 0.83 X 10(6); M, 1.12 X 10(6); G, 1.76 X 10(6) daltons of RNA. Only the N protein gene has a target-size which corresponds to that predicted from the molecular weight of its messenger RNA (molecular weight: 0.55 X 10(6)). The target-sizes for the other three genes are two to four times larger than expected, and are not proportional to the molecular weights of their corresponding messenger RNAs (molecular weights: NS, 0.28 X 10(6); M, 0.28 X 10(6); G, 0.7 X 10(6)). The polar effect of UV irradiation is inconsistent with independent transcription of each of the genes of VSV. Rather, the target-sizes appear to be cumulative, suggesting that trnascription initiates at a single point on VSV RNA and proceeds in the order 3'-N-NS-M-G-(L)-5'.

Cell-Free System↗
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

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↗
PubMedDate not supplied

2',5'-oligoadenylate synthetase in interferon-treated chick cells.

Chick embryo cells respond to interferon by producing very high levels of 2-5A synthetase. From inhibitor studies, it appears that the response involves the controlled derepression of an interferon-inducible gene. Continuous interaction of the cells with interferon seems to be necessary to maintain maximal enzyme induction. Partial purification of the synthetase indicates that it is probably composed of a single polypeptide of 56,000 MW. Upon activation by binding to dsRNA, this polypeptide catalyzes a nucleotidyl transferase reaction in which 5'-AMP residues are transferred from ATP to the 2' position of appropriate acceptors. A number of naturally occurring small molecules (in addition to 2-5A itself) that can function as adenylate acceptors have been identified, but the physiological significance of these adenylylation reactions remain to be established.

2',5'-Oligoadenylate Synthetase↗