Student Theses and Dissertations

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RU Laboratory

Nurse Laboratory


fission yeast, S-phase, M-phase, single cell assays, cyclin dependent kinase


Nearly forty years ago, cell fusion experiments revealed two tenets of our understanding of the cell cycle today. One, controls maintain ordered progression of the cell cycle. Two, diffusible factors in the cytoplasm promote cell cycle transitions. The studies presented here stem from those principles. In fission yeast, Schizosaccharomyces pombe, we have investigated the controls over S-phase within the cell cycle and over S-phase and mitosis among multiple nuclei within a common cytoplasm. To achieve faithful replication of the genome in each cell cycle, re-initiation of S-phase is prevented in G2 and origins are restricted from re-firing within S-phase. Failure in these controls could lead to polyploidy and local gene amplification contributing to genome instability. To investigate the block to re-replication during G2 we used single cell assays (BrdU pulse labeling and live cell fluorescence microscopy) and DNA microarray analysis. Depletion of the mitotic cyclin induces periodic S-phases correlated with G1/S gene expression, cell volume doubling, and uses mostly mitotic S-phase origins to replicate the genome evenly. We conclude that cyclin dependent kinase (CDK) inhibits re-initiation of a mostly normal S-phase program during G2. To identify features of replication origins important for amplification, we investigated origin firing and local genome amplification in the presence of excess helicase loaders using our single cell assays and microarrays. Coordination of origin firing is lost and specific origins are necessary for local amplification but act only within a permissive chromosomal context. Origins associated with amplification are highly AT-rich, fire early during mitotic S-phase, and are located in large intergenic regions. We propose that these features predispose replication origins to re-fire within a single S-phase, or to remain active after passive replication. Finally, we aimed to distinguish whether the decision to undergo S-phase and M-phase is nuclear autonomous or cytoplasmically driven. We demonstrate using our single-cell assays that multiple nuclei in the same cell can undergo DNA synthesis or nuclear division out of synchrony in the absence of key CDK inhibitors. We conclude that these activities are nuclear autonomous in multinucleate fission yeast cells, and propose that inhibition of CDK activity by Rum1 and Wee1 may coordinate and synchronize these cell cycle events in the common cytoplasm.


A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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