Riaz, Abida; (1997) Cyclin B in fission yeast mitosis and meiosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The fission yeast, Schizosaccharomyces pombe can follow two alternative developmental pathways; either it can enter the mitotic cell cycle and undergo proliferation, or it can conjugate and go through meiosis and sporulation. Using different methods of synchronisation we have investigated the pattern of synthesis and destruction of p63cdc13, cyclin B, during both of these fundamental cellular processes. Centrifugal elutriation is the method of choice for fission yeast synchronization as it is results in minimal physiological disturbance for the cells. The major disadvantage of this method is that culture size is limited by the volume of the elutriation chamber and a mitotic index greater than 30% is rarely achieved. Induction synchrony methods produced much higher levels of synchrony allowing changes in p63cdc13 and p34cdc2 levels to be detected through the cell cycle. In mitosis, the level of cyclin B showed characteristic cell cycle oscillations, accumulating during interphase and undergoing rapid degradation at mitosis. No significant change in p34cdc2 was observed, nor of p31, a recently described fission yeast PSTAIRE-related protein. Meiosis is the key step of sexual reproduction in eukaryotic organisms. In order to study the variation in cyclin B through the meiotic cell cycle, a novel method of synchronisation was used. S. pombe cells carrying the temperature-sensitive pat 1.114 allele are capable of initiating meiosis irrespective of nutritional conditions, a feature that was exploited to produce synchronous meiotic cultures. Exponentially growing pat 1.114 diploids at 25°C were incubated at 34°C for 4 hr during which time cells progressed through meiosis I and II. Upon return to the permissive temperature, cells initiated sporulation in a synchronous manner. p63cdc13 accumulated through meiosis I, declined and then showed an increase as cells progressed to meiosis II. A striking feature of Western blots of meiotic cells using three independently isolated monospecific anti-cdc13 antibodies was the appearance of a 130 k Da band that accumulated through the second meiotic division. There was no detectable variation in p34cdc2 through the meiotic cycle. An increase in p34cdc2-associated histone H1 kinase activity was observed in meiosis II, but not in meiosis I. Sporulating cells showed no variation in the level of p34cdc2, but the level of p63cdc13 decreased as cells began to form spores. Suppressors of cyclin B were isolated and characterised by classical genetics as a first step in the identification of genetic interactions with the fission yeast cyclin B.

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