DEVELOPMENTAL EFFECTS OF GROWTH DELAY IN FIDGET MICE

描述(改编自调查人员摘要)：这是第一次 RO1的竞争性续订，该RO1建议表征 新分离的基因，在烦躁的小鼠中发生突变，并被PI命名为 坐立不安。在之前的资金周期中，PI通过以下方式识别该基因 位置克隆是一类分化的蛋白质AAA的新成员 组，共享ATPase域，并且通常可以在 蛋白质折叠和解折叠的过程。尽管这种基因广泛存在于 表达出来，它的功能丧失导致了一组非常具体的 其分子和细胞基础是 默默无闻。当前的应用程序将通过以下方式调查这些基础 用LacZ融合蛋白确定fidgetin的亚细胞定位 由基因打靶产生，表位标记的构建物被引入培养 细胞，并通过产生抗血清来定位内源蛋白 免疫细胞化学(目标1)。将鉴定与fidgetin相互作用的蛋白质 在目标2中，使用酵母双杂交筛选，然后在体内表达和 免疫共沉淀研究。在目标3中，仔细分析fidgetin RNA 在正常和各种突变小鼠胚胎发育过程中的表达 在假设Fidgetin要么在上游要么在 先前显示的基因下游，当突变时会导致类似于 坐立不安。最后，目标4将尝试深入了解可能的冗余 以及烦躁相关基因在表达研究中的作用，修饰基因 作图和基因打靶。

DESCRIPTION (Adapted from investigator's abstract): This is the first competitive renewal of an RO1 that proposes to characterize the function of a newly isolated gene that is mutated in fidget mice, and which the PI has named fidgetin. In the previous funding cycle, the PI identified the gene by positional cloning as a novel member of a divergent class of proteins, the AAA group, that share an ATPase domain, and which may function generally in processes of protein folding and unfolding. Although the gene is widely expressed, it's loss of function gives rise to a set of remarkably specific developmental phenotypes whose molecular and cellular underpinnings are obscure. The current application will investigate these underpinnings by determining the subcellular location of fidgetin with a lacZ fusion protein generated by gene targeting, epitope-tagged constructs introduced into culture cells, and generating antisera to localize the endogenous protein by immunocytochemistry (Aim 1). Fidgetin-interacting proteins will be identified in Aim 2 using a yeast two-hybrid screen followed by in vivo expression and co-immunoprecipitation studies. In Aim 3, careful analyses of fidgetin RNA expression during embryonic development of normal and various mutant mice will be carried out in the context of hypotheses that fidgetin is either upstream or downstream of genes previously shown when mutated to cause defects similar to fidget. Finally, Aim 4 will attempt to gain insight into possible redundancy and action of fidget-related genes by expression studies, modifier gene mapping, and gene targeting.