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Title: Kinetics of RNA synthesis in Rotavirus infected cells
Author: Johnson, Moira A.
ISNI:       0000 0001 3591 2489
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1988
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A method has been developed to allow the independent quantification of the level of synthesis of each strand of different viral genes during a synchronous infection of tissue culture cells. It was developed and applied to the UK tissue culture adapted bovine Rotavirus whose genome consists of 11 discrete segments of monocistronic double stranded RNA (dsRNA). Cloned copies of the 11 genes of the UKtc Bovine Rotavirus have been sub-cloned into the polylinker region of the pGEM1 and pGEM2 transcription vectors. Using the T7 and SP6 promotors flanking this region, high specific activity RNA probes to either strand of the gene have been generated and used in Solution Hybridisations to quantitate the transcription and replication of each gene. Quantification of the plus-strand (m-RNA) synthesis at hourly intervals throughout the growth cycle provided evidence for both quantitative and qualitative regulation of transcription. Quantitative control of transcription was demonstrated by the accumulation of much higher levels of m-RNA for some viral genes (eg:-2 and 7) than others (eg:-4 and 6). The relative molar amounts of the different viral proteins was measured at 6.5 hours post-infection and showed that they did not directly reflect the accumulation of their encoding mRNAs, providing evidence for the existence of translational control of gene expression. For example the high levels of VP6 and VP10 produced was due primarily to high translational frequency, whereas the high level of VP8 could be correlated to a high level of accumulation of its encoding m-RNA. The existence of qualitative control of viral transcription was demonstrated by the finding that the transcription of 4 genes (5,6,7 and 9) occurred independently of protein synthesis in infected cells. These data suggested that the proteins encoded by these genes may have a regulatory role in the replication cycle. Study of the minus-strand synthesis for each viral gene indicated that the RNA replication for Rotaviruses is probably the same as that of mammalian Reo viruses. The method described in this thesis is a generic one since with the availability of suitable c-DNA clones it could be used to achieve qualitative and quantitative analysis of viral gene expression in any viral system.
Supervisor: Not available Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology