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

这个项目的目标是定义细粒曲霉细胞周期调节剂nimO的功能。特别是，实验将解决nimO在控制DNA复制起始和建立检查点中的作用，该检查点阻止有丝分裂发生，直到DNA被复制。研究的具体目的如下：I. nimO功能将通过分子和表型分析的结合来定义，具体目标如下：(a)使用体内DNA标记和双倒移实验精确定义细胞周期中何时需要nimO功能。(b)确定nimO基因破坏对细胞生长和细胞周期进程的影响。(c)通过位点定向诱变和突变的nimO等位基因的过表达来绘制nimO基因的功能域。特别分析了新型Cys2-His2锌指结构域协同的功能。(d)利用免疫细胞学方法表征nimO18突变体的异常有丝分裂诱导。(e) Northern blot分析细胞周期中nimO基因的时间表达。2。鉴定其产物与nimO蛋白相互作用以介导细胞周期控制的基因。将采用遗传方法分离nimO18突变的基因外抑制子。另外，双杂交方法可用于鉴定nimo相互作用蛋白。真核细胞周期是一系列精心安排的事件，在这些事件中染色体被复制，然后分离成相同的子核。对真核细胞周期的控制是细胞生物学中一个至关重要的问题。拟对真菌中性曲霉的nimO基因进行分子和遗传分析，通过表征该蛋白的重要功能区域，并通过描绘该基因在细胞周期进程和有丝分裂检查点功能中的作用，将增强对真核细胞周期调控的理解。鉴于该项目在科学上的卓越性，以及研究者对本科生参与其中的深刻承诺，该项目是提高人口科学素养的一个例子。* * *

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. ***