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Title: Characterisation of Schizosaccharomyces pombe wis2+, a cyclophilin-40 homologue
Author: Gaskell, Terri Louise
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2000
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The work described in this thesis is concerned with further genetic and molecular analysis of wis2+ The aim of this work was to establish a cellular function for wis2+ and to explain the effect of overexpression in the triple mutant strain described above. Using an in vitro binding assay, a number of proteins that show a physical interaction with Wis2 were isolated. These proteins were identified by peptide sequencing. Five were found to be ribosomal proteins. Another, ~85kD, was identified as S. pombe Hsp90; the intensity of the interacting band is increased, and the interaction is more consistent, if the extract is made from S. pombe cells which have previously been heat shocked. Genetic analysis revealed that overexpressing of wis2+ lowers the restrictive temperature of a number of cdc2 mutant alleles which arrest at G2-M. This effect of enhancing a cell cycle block is in contrast to the situation in the cdc25-22 wee1-10 win1-1 background where the G2-M block is relieved by wis2+ overexpression. An extensive genetic screen was conducted to isolate mutations that are synthetically lethal with wis2D, with the aim of identifying functionally redundant genes or downstream targets of wis2. No synthetic lethal interactions were identified. Indirect immunofluorescence studies established that Wis2 is localised exclusively in the cytoplasm. Consistent with this, a consensus nuclear export signal was identified in the C-terminus of Wis2. Structure-function analysis showed that overexpression of the C-terminal domain of Wis2 alone has the same activity as full length Wis2 in vivo. Overexpression of the N-terminus alone had no observable effect in the genetic backgrounds tested. The C-terminal domain of Wis2 was shown to be responsible for the interaction with Hsp90 in the in vitro binding experiment; this is consistent with results from other systems where the TPR domain was found to be responsible for Hsp90 binding.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available