ANALYSIS OF GENES ESSENTIAL FOR NEURONAL DIFFERENTIATION

神经元分化必需基因的分析

• 批准号: 6481919
• 负责人: KALPANA P WHITE
• 金额: $ 25.41万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6481919
• 关键词: Drosophilidae; RNA binding protein; RNA splicing; amyloid proteins; biological signal transduction; cell differentiation; confocal scanning microscopy; developmental genetics; developmental neurobiology; gene deletion mutation; gene expression; genetic regulatory element; genetically modified animals; immunocytochemistry; neurogenetics; neurons; protein structure function; receptor; reporter genes; tissue /cell culture

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.

提出的实验将阐明两个泛池的功能 神经表达的蛋白质，Elav和Appl，分化 和所有神经元的维护。研究的长期目标 与ELAV有关的是了解神经元特异性的机制 替代剪接。 神经元特异性剪接的机制将 使用神经元特异性RNA结合Elav进行研究 蛋白质，下游确定的靶标，调节的内含子 基因neuroglian。 具体而言，这些研究将阐明 NRG神经元特异性替代剪接的机制 通过定义与调节的涉及的顺式元素通过定义的MRNA 结合Elav和其他核因素的内含子，并评估其 体内意义。 调节的其他神经元特异性因素 将确定此过程。 这些研究将使用记者 体内和细胞培养分析中的转基因以评估剪接， 研究RNA - 蛋白质相互作用的生化方法， 识别其他因素的遗传方法。 果蝇应用 蛋白质是β-淀粉样蛋白前体蛋白（APP）的成员 蛋白质家族。 这个家庭的第一个确定成员， APP被暗示是在阿尔茨海默氏病中发挥作用。 尽管这种蛋白质家族在 神经系统，这些蛋白质的正常生理作用 不太了解。 Apph蛋白在中的假设 果蝇神经元功能SAS A细胞表面受体调节 将测试神经元过程和突触。 其他蛋白质 将确定参与应用程序介导的过程的。 实验设计将广泛使用分子和 果蝇中可能的遗传方法：使用野生型和 体外诱变的转基因和转基因动物，并靶向 表达，分析体内功能。 免疫细胞化学 技术和共聚焦显微镜将用于研究神经元 将进行研究和神经生理研究 突触功能。 由于Elav和Appl都属于 进化保守的蛋白质家族这种分析将 有助于对功能的机械理解 人类中的相关蛋白质。