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Title: Genetic and biochemical analysis of Survivin, a chromosomal passenger protein, in chicken and human cells
Author: Yue, Zuojun
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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I successfully generated Survivin conditional knockout DT40 cell lines rescued by tetO: survivin under the regulation of tTA2 driven by the cloned Kif4 promoter. SurvivinOFF cells could be completely rescued by chicken or human Survivin fused to GFP. My experiments demonstrated that cells lacking Survivin (SurvivinOFF) could enter mitosis, and activate the spindle checkpoint in the absence of tension. However SurvivinOFF cells failed to complete cytokinesis, although initial formation of the cleavage furrow and ingression were normal. SurvivinOFF cells became multinucleated and multipolar before undergoing apoptosis during interphase. SurvivinOFF cells were not more sensitive to etoposide, staurosporine or taxol than wild type or SurvivinON cells. Two mutants reported in the literature to be dominant negative, Survivin D55A (a mutant reported to be pro-apoptotic) and T36A/E (lacking a reportedly essential Cdk1 phosphorylation site) can rescue the knockout cells. In contrast, the C59A and C86A mutants which destroy Zinc finger motifs in the BIR domain could not. Linker region mutants L98AV100A and L104AL106A (TS mutants) could rescue Survivin knockout at 39*C and 39*C, respectively, and target correctly in SurvivinOFF cells. By contrast, these mutants do not localize and rescue life under certain conditions at 41*C. Knockout cells stably expressing tagged Survivin were used for tandem affinity purification of Survivin and its interacting proteins. Mass spectrometry analysis of these samples identified several potential interactors. My results showed that Survivin is essential for the completion of mitosis, but that the activation of the spindle checkpoint in the absence tension does not need Survivin. Our experiments failed to provide additional evidence for a role of Survivin as an essential anti-apoptotic factor.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available