UBC Theses and Dissertations
Involvement of CDK/cyclin motif in ciliate cell cycle regulation Zhang, Hong
The eukaryotic cell cycle is controlled by oscillation of the activity of cyclindependent protein kinases (Cdks). Ciliates, with their elaborate cellular structures and unusual cell cycle organization, present interesting subjects for cell cycle studies. A novel Cdk termed PtCdk2 was isolated in Paramecium tetraurelia, and with PtCdkl and PtCdk3 forms the P. tetraurelia Cdk family. The lack of affinity of PtCdk2 for yeast p13[sup suc1] protein distinguishes it from PtCdk3, the only Paramecium Cdk so far displaying p13[sup suc1] binding. PtCdk2 displays a cell cycle-stage dependent histone HI kinase activity, peaking at the end of the vegetative cell cycle. Coincidence of the kinase activity peaks of PtCdkl, PtCdk2 and PtCdk3 with initiation of macronuclear DNA synthesis (IDS), cell division and point of commitment to cell division (PCD), respectively, suggests they play different roles in cell cycle control. Consistent with the conservation of cyclin-binding domains in the P. tetraurelia Cdks, two mitotic cyclin homologues, PtCycl and PtCyc2, have been identified. This is the first time that cyclin genes have been cloned in ciliates. Both PtCycl and PtCyc2 proteins show characteristic patterns of accumulation and destruction during the vegetative cell cycle, with PtCycl peaking at the PCD, and PtCyc2 reaching the maximal level at the end of the cell cycle. Results of coimmunoprecipitation experiments indicate that PtCycl and PtCyc2 are associated with PtCdk3 and PtCdk2, respectively. Study on the subcellular localization of TtCdkl in Tetrahymena thermophila by immunofluorescence microscopy reveals its association with the membrane-skeletal domains that surround mature but not nascent basal bodies in the cell cortex, suggesting that TtCdkl plays a role in the regulation of formation of the complex membrane-skeletal layer of this cell during division-related cortical morphogenesis. A partial TtCDKl knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei. My work not only extends the ubiquity of Cdk/cyclin motif in the eukaryotic cell cycle regulation to ciliates, but also reveals interesting and unique roles of these molecules in cell cycle control in these organisms.
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