Stewart, Elspeth Charlotte; (1995) Analysis of Xenopus cyclin A. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Maturation Promoting factor is the enzyme that catalyses the entry of cells into mitosis or meiosis. It consists of at least two subunits: a protein kinase encoded by a member of the cyclin dependent kinase (cdk) family and a regulatory cyclin subunit. This thesis describes studies on the functional domains of Xenopus cyclin A1. I have made a series of deletion and point mutants of Xenopus cyclin A1 and developed assays to test the properties of these altered proteins. I have shown that deletion of as few as 14 residues from the C-terminus of cyclin A, or mutation of highly conserved residues within its 'cyclin box', gives proteins that are severely impaired in their ability to bind to p34cdc2 or p33cdk2. An important property of mitotic cyclins is their rapid destruction at the end of M phase, I have demonstrated that those mutant cyclin Al subunits that cannot bind efficiently to p34cdc2 or p33cdk2 cannot be destroyed at the end of M-phase. Some mutants of cyclin A1 that contained deletions within the N-terminus of the protein also resisted normal destruction. After showing that the deletion of two conserved motifs from these cyclin A mutants was not responsible for their stability, I concluded that it was due to conformational changes within their N- termini. Mutation of the sequence motif in cyclin A1 that shows homology to the 'destruction box' of cyclin B, also gives a protein that is unable to be proteolysed. Wild-type cyclin A1 is normally a nuclear protein in interphase. I have shown that those cyclin A1 mutants that cannot bind to p34cdc2 are excluded from the nucleus in tissue culture cells, and that the N-terminus of cyclin A is not required for the correct subcellular localisation of this protein.

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