REGULATION OF MAPS IN NERVOUS SYSTEM

神经系统地图的调节

• 批准号: 2265335
• 负责人: Itzhak Fischer
• 金额: $ 17.98万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2265335
• 关键词: DNA footprinting; PC12 cells; antibody; antisense nucleic acid; axon; brain; cell differentiation; cyclic AMP; developmental neurobiology; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; laboratory rabbit; laboratory rat; microinjections; microtubule associated protein; molecular cloning; neoplastic cell culture for noncancer research; nervous system; neurogenesis; neurotrophic factors; nucleic acid sequence

MAP1B is a prominent microtubule associated protein whose expression is developmentally regulated, showing the highest levels in growing axons during early postnatal brain development. Our interest in the expression of MAPs in the nervous system has been related to the role of cytoskeletal proteins during neuronal differentiation, but the experiments that we propose will also address fundamental problems concerning the regulatory elements that control the expression of other brain- specific genes. Specific questions addressed in this proposal include: What are the mechanisms that regulate MAP1B expression? What are the regulatory elements that control the cell and stage-specificity during transcription of the MAP1B gene and its response to physiological signals (e.g., NGF, cAMP)? What is the role of MAP1B during axonal growth in neurons? The first set of experiments utilizes nuclear run-on assays to confirm that the control of MAP1B expression is exerted at the transcriptional level. Specific regulatory mechanisms can then be identified from studies of the structure and function of MAP1B promoter. We hypothesize that, as with other brain-specific genes, the basic elements of the MAP1B promoter are defined within a relatively small region upstream from the initiation site of transcription. In preliminary experiments we have already isolated genomic clones that contain part of the MAP1B promoter and determined the initiation site of transcription. Additional distal elements will be identified by nuclease hypersensitivity assays of neuronal cells. Our approach is to follow a set of experiments that are based on in vitro assays of chimeric constructs in which regulatory elements are linked to a reporter CAT gene for functional analysis of MAP1B promoter in both neuronal and non-neuronal cells. These experiments will be followed by physical mapping of specific regulatory regions by footprinting, methylation interference, and gel shift assays. Additional mapping experiments using nuclease hypersensitivity assays will be directed at more distal regulatory elements and help to design promoter constructs that can be studied in vivo with transgenic animals. Finally, the requirement for MAP1B during axonal differentiation will be examined by suppression of its expression in cultured primary neurons using antisense oligonucleotides and by microinjection of MAP1B peptides and antibodies prepared against selected regions of the molecule. These strategies can be extended to study promoters of other MAPs, for which, thus far, no promoters have been described, and to isolate specific transcription factors that control these genes. These studies will also become the basis for in vivo work and development of experimental tools for manipulation of gene expression in transgenic animals. Ultimately, the characterization of these genes and their promoters will provide an understanding of the molecular pathways of neuronal differentiation during development of the nervous system and in response to changes in the environment.

Map1b是一种突出的微管相关蛋白，其表达是 在开发监管上，显示出生长轴突的最高水平 在产后早期发育期间。我们对表达的兴趣 神经系统中的地图与细胞骨架的作用有关 神经元分化过程中的蛋白质，但是我们的实验 建议还将解决有关监管的基本问题 控制其他脑特异性基因表达的元素。 本提案中解决的具体问题包括： 调节MAP1B表达的机制？什么是监管 转录过程中控制细胞和阶段特异性的元素 MAP1B基因及其对生理信号的反应（例如NGF， 营）？ MAP1b在神经元中轴突生长中的作用是什么？ 第一组实验利用核跑步测定来确认 MAP1B表达的控制在转录上施加 等级。然后可以从研究中确定特定的调节机制 MAP1B启动子的结构和功能。我们假设这是 与其他大脑特异性基因，MAP1B启动子的基本元素 在起始上游的相对较小的区域内定义 转录部位。在初步实验中，我们已经隔离了 包含MAP1B启动子的一部分并确定的基因组克隆 转录的启动点。其他远端元素将是 通过神经元细胞的核酸酶超敏反应鉴定。我们的 方法是遵循一组基于体外的实验 嵌合构建的测定，其中调节元素与 用于MAP1B启动子功能分析的记者CAT基因 神经元和非神经元细胞。这些实验将随后 通过足迹对特定监管区域的物理映射， 甲基化干扰和凝胶转移测定法。附加映射 使用核酸酶高敏分析的实验将针对 更远端的监管要素，并有助于设计启动子结构 可以用转基因动物在体内研究。最后， 轴突分化过程中对MAP1B的要求将通过 使用反义抑制其在培养的原代神经元中的表达 寡核苷酸和通过对MAP1B肽和抗体的显微注射 针对分子的选定区域制备。这些策略可以是 扩展到研究其他地图的启动子，到目前为止， 已经描述了启动子，并分离了特定的转录 控制这些基因的因素。这些研究也将成为基础 用于体内工作和开发实验工具，以操纵 转基因动物中的基因表达。最终，特征 这些基因及其促进者将提供对 神经元分化的分子途径 神经系统和对环境变化的反应。