Mayall, Stephen James; (1996) Cyclins in the cellular slime mould Dictyostelium Discoideum. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

I expressed Xenopus laevis cyclin A1 in the cellular slime mould Dictyostelium discoideum to investigate the currently unclear role of A-type cyclins. Expression of this cyclin in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe causes a cell cycle arrest. However, overexpression of wild-type or an 'indestructible' form of the protein had no effect upon the growth of Dictyostelium vegetative cells or their subsequent development when starved. This may be explained by the inability of Xenopus cyclin A1 to bind to the two cloned Dictyostelium cyclin dependent kinases (CDKs) in vitro. A single B-type cyclin has been previously cloned in Dictyostelium. In this thesis, I describe the identification of two new cyclins from this organism. I constructed a cDNA library from growing Dictyostelium amoebae and then isolated clones able to complement the defect of the (clnl, cln2, cln3) strain of S. cerevisiae. The rescuing clones fell into three classes. One encoded the already characterised B-type cyclin. The second showed extensive homology to mitotic cyclins from other species and also contained large runs of asparagine repeats in non-conserved regions of the protein. The final class of clone showed weak homology to the S. cerevisiae Cln3, Pho80 and Pell cyclin-like proteins. Antibodies were raised against the mitotic cyclin protein overexpressed in bacteria. A polypeptide of the correct size was recognised in Dictyostelium extracts. The cyclin had no effect upon growth or development when overexpressed in Dictyostelium cells in vivo in either a wild-type or a N-terminally truncated form.

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