Graduate Thesis Or Dissertation
 

Regulation of late gene expression in vaccinia virus

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  • The prototypal poxvirus vaccinia is a cytoplasmic-replicating DNA virus which contains a large genome with the capacity to encode 200 polypeptides. Tight temporal regulation coordinates the expression of this large number of genes throughout a relatively short replication cycle. As a first step towards identifying and understanding the regulatory mechanisms involved in gene expression of this virus, I undertook a project designed to localize, sequence, and determine the expression profile of genes representing the late temporal class. Using greater than 50% of the viral genome represented by an available plasmid library, I carried out hybrid-selected translation to identify the genomic loci of major late gene products. After determining the preliminary map positions of a number of late gene products, I chose 65,000 and 32,000 dalton molecular weight late proteins for further analysis due to their abundance in infected cells, suggesting their importance. I then carried out a series of S1 nuclease mapping experiments that led to identification of the 5' ends of transcripts originating from the genomic region of interest. After closing in on these late genes by translational and transcriptional mapping techniques, I determined the nucleotide sequence of 5.1 kilobases of DNA from that region of the genome. The late genes of interest were found to reside within a tandemly-oriented cluster of at least six closely-spaced late genes. The sequence information enabled me to construct probes necessary for the precise mapping of the 5' ends for the corresponding mRNAs. I extended these 5' S1 nuclease mapping analyses to include RNA I had isolated from various times after infection. The results indicated that the late gene cluster is non-coordinately regulated. Not only did I find that these genes were turned on at different times, but also that the qualitative profiles of steady state mRNAs were different. The available nucleotide sequences, RNA start sites, and temporal regulatory schemes of this late gene cluster should now facilitate a directed approach to determining control signals responsible for regulating late gene transcription in vaccinia virus.
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