Smith, Matthew; (1997) Studies of cyclins in the fission of yeast, Schizosaccharomyces pombe. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The cyclins are a family of proteins that play a key role in the eukaryotic cell cycle through their regulation of cyclin-dependent kinases (CDKs). In the fission yeast, Schizosaccharomyces pombe, the cdc13+ gene encodes a B-type cyclin (p63cdc13) that interacts with the product of the cdc2+ gene (p34cdc2) to regulate entry into mitosis. This p63cdc13/p34cdc2 complex is known to be the central control mechanism in the G2/M transition in all eukaryotic cells. The original aim of this project was, therefore, to crystallize p63cdc13 so that its structure might be determined by X-ray crystallography. Bacterial overexpression of p63cdc13 from an IPTG-inducible promoter resulted in the formation of insoluble inclusion bodies. These were resistant to attempts to solubilise and refold the protein in order to restore its biological activity. However, a truncated form of p63cdc13, cdc13t1, was expressed in the same fashion and found to be soluble. Large amounts of cells induced for cdc13t1 expression were grown by fermentation and the protein purified from whole cell extracts by a five-step column chromatography process. Crystallisation trials on pure cdc13t1 were centred on the "wild screening" method, with promising conditions being investigated more fully using a crystallization robot. Due to the exceptionally high solubility of cdc13t1 no crystals formed, despite an exhaustive search of all precipitating conditions. In higher eukaryotes nine separate cyclin classes have been identified (termed A-l) in association with many CDKs (Cdk 1-8). The formulation of a strategy to purify human cyclin A for X-ray crystallography was started, but abandoned following the publication of its structure by a different group. Studies on human cyclin A were continued in an entirely different manner by investigating the effect of its expression in S. pombe, which has no known homologue. It was discovered that cyclin A severely inhibits fission yeast growth. Cells were found to be unable to undergo mitosis or cytokinesis, yet attempted to rereplicate their DNA. As a result, cells expressing cyclin A were characterized by having an increased size with a single diffuse nucleus. Aberrant microtubule arrays were also evident. Immunoprecipitation experiments did not seem to imply that these results were due to an interaction between cyclin A and p34cdc2.

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