MOLECULAR CHARACTERIZATION OF A NEURAL PROTEIN

神经蛋白的分子表征

• 批准号: 3412408
• 负责人: SUSANNAH S CHANG
• 金额: $ 21.15万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1994-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3412408
• 关键词: animal tissue; axon; cell adhesion; cell adhesion molecules; cell cell interaction; chick embryo; dendrites; developmental neurobiology; gene expression; growth cones; immunocytochemistry; laboratory rabbit; membrane proteins; molecular cloning; neuronal guidance; neurons; northern blottings; polymerase chain reaction; protein purification; southern blotting; tissue /cell culture

The accurate extension of neuronal processes during development is essential to the proper functioning of the mature nervous system. The long term goal of this research is to understand how molecules on axons promote, and direct, the extension of other axons. The specific objective of this proposal is to study the molecular and functional properties of an axonal surface molecule that may function in promoting neurite extension. A novel cell surface molecule has been identified and named DM-RASP. I have immunopurified DM and it is a single protein species of 95 kD. Its amino terminal protein sequence has been determined, and by comparison to the Genpept database it is unique. Oligonucleotide probes designed from the amino acid analysis have identified five clones from an E19 chick brain CDNA library. One of these clones has been partially sequenced, and the deduced amino acids from the partial sequence show that DM-RASP is a member of the immunoglobulin superfamily of neural cell adhesion molecules. Northern analysis of chick brain RNA using as a probe the CDNA insert from one of the clones reveals two DM-RASP messages. Antibodies against DM-RASP reduce neurite extension on axons, suggesting that its function is to support axon extension. DM-RASP is expressed on the surface of some but not all early axons in the developing chick embryo. Thus it is possible that DM-RASP may be involved in helping axons make pathway specific choices. The first objective is to completely sequence the message for DM-RASP. Southern analysis will show whether the DM-RASP MRNAS are the result of two different genes or the result of alternate processing of one gene. A developmental profile of DM-RASP gene expression will be obtained by Northern analysis, and this will also show whether the multiple messages are differentially regulated. The second objective is to study the functional properties of DM-RASP. The amount of DM-RASP required to support neurite extension will be determined. The neuronal cell types which extend upon DM-RASP will be identified. Neuronal growth cones will be cultured on alternate stripes of DM-RASP and other proteins, to test if growth cones show a preference for one substrate molecule versus another. If a cell specific choice to extend upon DM-RASP is observed, then it could be that DM-RASP acts to specifically direct neurite extension. The third objective is to identify the mouse homologue of DM-RASP and other related proteins. Since we have identified one vertebrate cell surface molecule with a restricted distribution in the nervous system, we may now be able to identify others.

开发过程中神经元过程的准确扩展是 对于成熟神经系统的正确功能至关重要。 这 这项研究的长期目标是了解轴突上的分子 促进和指导其他轴突的延​​伸。 该提议的具体目的是研究分子和 轴突表面分子的功能特性，可能在 促进神经突延伸。 一个新型的细胞表面分子已经 识别并命名为DM-Rasp。 我有免疫疗法的DM，这是一个 95 kd的单蛋白质。 其氨基末端蛋白序列 已经确定，并且通过与Genpept数据库进行比较是 独特的。 由氨基酸分析设计的寡核苷酸探针 已经从E19 Chick Brain cDNA文库中鉴定出五个克隆。 一 这些克隆已被部分测序，并推导的氨基 部分序列的酸表明DM-RASP是 神经细胞粘附分子的免疫球蛋白超家族。 北方 使用探针对鸡脑RNA进行分析 这些克隆揭示了两个DM-RASP消息。 针对DM-RASP的抗体减少轴突上的神经突延伸，表明 它的功能是支持轴突扩展。 DM-RASP在 发育中的小鸡的某些但不是所有早期轴突的表面 胚胎。 因此，DM-RASP可能参与帮助 轴突做出途径特定的选择。 第一个目的是完全对DM-RASP的消息进行序列。 南方分析将显示DM-RASP mRNA是否是 两个不同的基因或一个基因替代加工的结果。 DM-RASP基因表达的发展曲线将通过 北方分析，这还将显示多个消息是否 受差异调节。 第二个目标是研究DM-RASP的功能特性。 支持神经突扩展所需的DM-RASP量将是 决定。 在DM-RASP上延伸的神经元细胞类型将是 确定。 神经元生长锥将在交替条纹上培养 DM-RASP和其他蛋白质，以测试生长锥是否显示出偏好 一个底物分子与另一个底物分子。 如果特定于细胞的选择 观察到DM-RASP上的延伸，然后可能是DM-RASP作用到 特别是直接神经突延伸。 第三个目标是确定DM-RASP的小鼠同源物和 其他相关蛋白质。 由于我们已经确定了一个脊椎动物细胞 表面分子在神经系统中具有限制分布， 我们现在可能能够识别他人。