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Title: Molecular genetic studies of the Prp8 splicing factor
Author: Brown, Andrew James
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Prp8 is a component of the U5 and snRNP important for entry of U5 and other snRNPs into the spliceosome, and is present in the active site(s) at the times when the splicing reactions occur. Genes encoding this protein have been isolated from yeast and other eukaryotes and show that Prp8p is extraordinary well conserved, consistent with its multiple roles. This thesis focuses on the only region of yeast Prp8p not known to be common to other eukaryotes: a repetitive acidic and proline-rich domain at the N-terminus. Removal of part or all of this domain inhibits function, but I have found that cells lacking this domain are viable if the truncated protein is overproduced. A reconstruction approach suggests that proline is the most important feature of this domain, and thus function may be analogous to proline-rich regions of other proteins which in general complex assembly. The phenotype of truncation mutants is consistent with this. Spliceosome components are present in the yeast nucleus at much lower concentration than in other eukaryotes, suggesting a reason as to why this otherwise highly conserved protein possesses the extra domain. This thesis also describes the analysis of several mutants of yeast PRP8 which have the highly unexpected phenotype of a block to cell cycle progression. I show that these mutants also affect splicing. The growth defect of one of them (dbf3-1) is suppressed by a cDNA copy of the TUB1 gene. TUB1 contains an intron and encodes a microtubule monomer (α-tubulin) functional in M-phase. Suppression separates the cell cycle and splicing defects, as the splicing defect is unaffected by suppression. These data strongly support the hypothesis that the cell cycle defect is a secondary consequence of a splicing defect, the link being a gene (TUB1) which functions in the cell cycle and which contains an intron. The cell cycle block is observed because the splicing defect is mild: except for the TUB1 intron, splicing is sufficient to maintain growth.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available