RUI: Control of DNA Replication and Mitotic Checkpoint Function by the Aspergillus nidulans nim0 Gene

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9507485 James The goal of this project is to define the function of the Aspergillus nidulans cell cycle regulator nimO. In particular, the experiments will address the role of nimO in controlling the initiation of DNA replication and in establishing the checkpoint that prevents mitosis from occurring until DNA has been replicated. The specific aims of the research are as follows: I. nimO function will be defined through a combination of molecular and phenotypic analyses, with the following specific objectives: (a) Define precisely when nimO function is required during the cell cycle using in vivo DNA labelling and double reciprocal shift experiments. (b) Determine the consequences for cell growth and cell cycle progression of a disruption of the nimO gene. (c) Map functional domains of the nimO gene by site-directed mutagenesis and overexpression of mutant nimO alleles. In particular, analyze the function of the novel Cys2-His2 zinc finger domain collaboration . (d) Characterize the abnormal mitotic induction of nimO18 mutants using immunocytological methods. (e) Determine the temporal expression of the nimO gene during the cell cycle by Northern blot analysis. II. Identify genes whose products interact with the nimO protein in order to mediate cell cycle control. A genetic approach will be used to isolate extragenic suppressors of the nimO18 mutation. Alternatively, a two-hybrid approach may be used to identify nimO-interacting proteins. %%% Control of the eukaryotic cell cycle, the carefully orchestrated series of events in which the chromosomes are replicated and then segregated into identical daughter nuclei, is an issue of fundamental importance in cell biology.The proposed molecular and genetic analyses of the nimO gene of the fungus Aspergillus nidulans will enhance the understanding of eukaryotic cell cycle regulation by characterizing functionally important regions of the protein and by delineating the role of this gene in cell cycle progression and mitotic checkpoint functi on. Given the scientific excellence of this project and the investigator's deep commitment to involving undergraduates in it, this project is an example of the type which will raise the scientific literacy of the population. ***