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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Morser, Michael John
Languages: English
Types: Doctoral thesis
Subjects: QR355
1. The growth cycle of SFV in chick embryo fibroblasts, BHK-2l, HEL and 1-929 cells was described, as were the resultant effects on RNA and protein synthesis. The release of haemagglutinin and LDH from infected ,cells was assayed, and shown to parallel the release')of infectious virus. The effect of altering the temperature of incubation on the growth of the virus, and the concommitant change in macromolecular synthesis , were investigated. There was a substantial inhibition of host cell protein synthesis from .3h after:.1nfection ward , under all the conditions investigated.\ud \ud 2. Five proteins could be readily detected in any of the four cell types when they had been infected with SFV for at least .3h. These five proteins were present throughout the growth cycle, whether the temperature o 0 0 of incubation was .30 C, .37 C or .39 C. Five proteins of the same size were also present in chick cells infected with Sindbis virus. Two of the five were the structural proteins, the other three were NVP 95, NVP 72 and NVP 63.\ud \ud 3. On the basis of its amino acid composition, its carbohydrate composition, and its kinetics of labelling, NVP 6.3 appears to be a precursor of the envelope protein. NVP 95 also contains some carbohydrate.\ud \ud 4. Pulse-chase experiments were carried out in infected chick and BHK- 21 cells. The information from these experiments vas supplemented with results from pulse-chase experiments in which the cells had been treated with TPCK,or NaF, or with amino acid analogues, or subjected to the reversal of a temperature jump. From these results, two further high molecular weight proteins became detectable, NVP 165 and ~1TP 127. It was possible to demonstrate that NVP 165, NVP 127 and NVP 95 and NVP 6.3 were precursors to NVP 72 and the structural proteins. Two models were proposed, based on the presence or absence of a second envelope protein.\ud \ud 5. The proteins present in the plasma membrane and endoplasmic reticulum were investigated The only protein contained in the plasma membrane was the envelope protein, while all the proteins normally found in infected cells were present in the endoplasmic reticulum fraction.\ud \ud 6. On the basis of the proteins synthesised on inhibition with cycloheximide and puromycin, and because of the timing or their formation, it was suggested that none or the proteins so far identified was the RNA polymerase.\ud \ud 7. A method for obtaining a partially purified preparation of the RNA polymerase was described. The RNA and protein species associated with the enzyme were analysed. The M band technique vas also utilised to obtain a further fractionation. By a combination of these methods, it was shown that the replicative intermediate is the first RNA species formed, while only NVP72 vas associated with the RNA polymerase. \ud \ud 8. An in vitro assay for the RNA polymerase was described. The product was replicative intermediate and replicative form. It was shown that the assay was not sensitive to antiserum preparations that neutralised the core or envelope proteins.\ud \ud 9. A method for pulse labelling the RNA was described, using the inhibition of macromolecular synthesis caused by glucosamine. It was demonstrated that the replicative intermediate was the first RNA species formed.\ud
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