MOLECULAR CHARACTERIZATION OF NEURONAL NUCLEAR ANTIGENS

神经元核抗原的分子表征

基本信息

批准号：
3417984
负责人：
CHARLES R. BUCK
金额：
$ 5.6万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-04-07 至 1994-07-31
项目状态：
已结题

项目摘要

One of the hallmark features of the nervous system is the dramatic variety of cell types and connections formed between cells. The overall goal of this research is to identify molecular regulators of the processes of development and differentiation in this system. Mechanisms are sought whereby this cell-type specificity and connectivity is generated and functions to maintain the dramatic plasticity of the system throughout life. Two anti-mouse monoclonal antibodies which specifically recognize neuronal nuclear proteins, called NeuN and F41, have been generated. The nervous system of mice develops in a manner similar to humans but over a greatly condensed time span so that these animals are an excellent model of human nervous system development and function. Indeed, the monoclonals described herein cross react with human neuronal nuclear antigens. The pattern of expression and biochemical properties of these mouse neuronal nuclear antigens suggest that they may be intimately involved in the regulation of nervous system differentiation. For example, the antigens display different but overlapping neuronal cell-type specificities. The genes encoding these antigens have been identified by screening recombinant cDNA expression libraries with the monoclonal antibodies. Full nucleotide sequence of the mRNA encoding these antigens will be obtained and exhaustively compared with known genetic sequence databases to determine if related genes have been described and to provide insight into the function of these antigens. Purified protein will be subjected to limited amino acid sequence analysis to confirm the cDNA sequence of these genes. Antisense oligonucleotides or cDNA constructs will be introduced to cell lines or primary neuronal cultures in an attempt to eliminate or reduce these proteins to directly assay protein function. In addition, cDNA clones will be used to explore the function of these antigens by introducing them into heterologous cell types. Factors such as phenotypic differentiation and changes in transcriptional activity will be assessed in the transfected cells. The possibility that the antigens bind to DNA specifically and regulate the expression of other genes will also be explored using antigen protein binding to known or putative regulator sequences. Antigen proteins will be further characterized biochemically by analyzing electrophoretic mobilities and the state of phosphorylation and glycosylation of the proteins. The antigenic protein will be purified using immunoaffinity chromatography and HPLC. These novel, nervous system specific nuclear proteins will illuminate mechanisms of regulation of the neuronal differentiation and development.

神经系统的标志性特征之一是戏剧性细胞之间形成多种细胞类型和连接。总体这项研究的目的是确定分子调节因子该系统中的发展和差异化过程。机制寻求这种细胞类型的特异性和连接性是生成和功能以维持系统的戏剧性可塑性一生。两种抗小鼠单克隆抗体识别称为NEUN和F41的神经元核蛋白已是生成。小鼠的神经系统以类似的方式发展人类，但在大量凝结的时间范围内，以使这些动物是人类神经系统发展和功能的出色模型。实际上，本文描述的单克隆交叉与人神经元反应核抗原。表达和生化特性的模式这些小鼠神经元核抗原可能是密切参与了神经系统分化的调节。例如，抗原显示不同但重叠的神经元细胞类型的特异性。编码这些抗原的基因已经通过筛选重组cDNA表达文库的确定单克隆抗体。 mRNA编码的全核苷酸序列这些抗原将被详尽地与已知的遗传序列数据库以确定相关基因是否已经描述并提供对这些抗原功能的见解。纯化的蛋白质将受到有限的氨基酸序列分析以确认这些基因的cDNA序列。反义寡核苷酸或cDNA构建体将被引入细胞系或原发性神经元培养试图消除或减少这些培养蛋白质直接测定蛋白质功能。另外，cDNA克隆将通过引入来探索这些抗原的功能它们成为异源细胞类型。表型等因素将评估分化和转录活性的变化在转染的细胞中。抗原与DNA结合的可能性具体并调节其他基因的表达也将是使用抗原蛋白与已知或推定调节剂结合进行探索序列。抗原蛋白将在生化上进一步表征通过分析电泳迁移率和磷酸化状态和蛋白质的糖基化。抗原蛋白将是使用免疫亲和力色谱和HPLC纯化。这些小说，神经系统特定的核蛋白将照亮神经元分化和发育的调节。