ANALYSIS OF GENES ESSENTIAL FOR NEURONAL DIFFERENTIATION

这些实验将阐明两个泛- 分化过程中神经表达蛋白ELAV和APPL 以及维持所有神经元的功能研究的长期目标 与ELAV相关的是了解神经元特异性 选择性剪接 神经元特异性剪接的机制将 使用ELAV进行研究，ELAV是一种神经元特异性RNA结合蛋白， 蛋白质及其下游识别的靶点--调控内含子 神经胶质细胞的基因。 具体来说，这些研究将阐明 神经元特异性选择性剪接的机制 通过定义参与调控的顺式元件， 结合ELAV和其他核因子内含子，并评估其 体内意义。 其他调节神经元特异性因子 将确定这一进程。 这些研究将使用报告者 体内和细胞培养测定中的转基因以评估剪接， 研究RNA-蛋白质相互作用的生物化学方法，以及 遗传学方法来确定其他因素。 果蝇APPL 蛋白质是β淀粉样前体蛋白（APP）的成员 蛋白质家族 第一个被确认身份的家庭成员， APP在阿尔茨海默病中起作用。 虽然这个蛋白质家族在大肠杆菌中广泛表达， 神经系统，这些蛋白质的正常生理作用 并没有得到很好的理解。 假设APPL蛋白在 果蝇神经元功能作为细胞表面受体调节 将测试神经元过程和突触。 其他蛋白质 参与APPLE介导的过程。 实验设计将广泛使用分子和 果蝇可能的遗传学方法：野生型和 体外诱变的转基因和转基因动物， 表达，以测定体内功能。 免疫细胞 技术和共聚焦显微镜将用于研究神经元 将进行乔木和神经生理学研究 突触功能 由于ELAV和APPL都属于 进化上保守的蛋白质家族，这种分析将 有助于机械理解的功能， 人类的相关蛋白质。

The proposed experiments will elucidate functions of two pan- neurally expressed proteins, ELAV and APPL, in differentiation and maintenance of all neurons. The long term objective of studies related to ELAV is to understand mechanism of neuron-specific alternative splicing. Mechanisms of neuron-specific splicing will be investigated using ELAV, a neuron-specific RNA binding protein, and it~s downstream identified target, the regulated intron of the gene neuroglian. Specifically, these studies will elucidate the mechanism of neuron-specific alternative splicing of the nrg pre-mRNA by defining cis-elements involved in the regulated intron that bind ELAV and other nuclear factors, and assess their in vivo significance. Other neuron-specific factors that regulate this process will be identified. These studies will use reporter transgenes in vivo and in cell culture assays to assess splicing, biochemical approaches to study RNA-protein interactions, and genetic methods to identify other factors. The Drosophila APPL protein is a member of the beta amyloid precursor protein (APP) family of proteins. The first identified member of this family, APP, is implicated to have a role in Alzheimer~s disease. Although this family of proteins is widely expressed within the nervous system, the normal physiological roles of these proteins are not well understood. The hypothesis that the APPL protein in Drosophila neuron function sas a cell surface receptor to regulate neuronal processes and synapses will be tested. Other proteins that are involved in APPL-mediated processes will be identified. The experimental design will make extensive use of molecular and genetic approaches possible in Drosophila: use of wild type and in vitro mutagenized transgenes and transgenic animals, and targeted expression, to assay in vivo function. Immunocytochemical techniques and confocal microscopy will be used to study neuronal arbors and neurophysiological studies will be conducted study synaptic function. Since ELAV and APPL both belong to evolutionarily conserved protein families this analysis will contribute to the mechanistic understanding of the function of related proteins in humans.