REGULATION OF MAPS IN NERVOUS SYSTEM

MAP1B是一种重要的微管相关蛋白，其表达与微管相关蛋白的表达密切相关。 在生长的轴突中， 在出生后的大脑发育早期。我们对表达的兴趣 MAPs在神经系统中的作用与细胞骨架的作用有关。 在神经元分化过程中的蛋白质，但我们的实验， 建议还将解决有关监管的基本问题， 控制其他大脑特异性基因表达的元件。 本建议中涉及的具体问题包括： 调节MAP1B表达的机制？什么是监管 在转录过程中控制细胞和阶段特异性的元件 MAP1B基因及其对生理信号的响应（例如，NGF， cAMP）？MAP1B在神经元轴突生长过程中的作用是什么？ 第一组实验利用核连续测定来确认 MAP1B表达的控制作用于转录水平， 水平然后可以从研究中确定具体的调节机制 MAP1B启动子的结构和功能。我们假设， 与其他脑特异性基因，MAP1B启动子的基本元件 被限定在起始点上游的相对小的区域内 转录位点。在初步实验中，我们已经分离出 含有部分MAP1B启动子的基因组克隆， 转录起始位点。其他远端元件将 通过神经元细胞的核酸酶超敏性测定鉴定。我们 方法是遵循一组基于体外的实验， 嵌合构建体的测定，其中调节元件与 CAT报告基因，用于分析MAP1B启动子在两种细胞中的功能。 神经元和非神经元细胞。这些实验之后， 通过足迹法对特定调节区域进行物理作图， 甲基化干扰和凝胶迁移分析。附加映射 使用核酸酶超敏性测定的实验将针对 更远的调控元件，并帮助设计启动子构建体 可以在转基因动物体内进行研究。最后 轴突分化期间对MAP1B的需求将通过以下方法进行检查： 用反义核酸抑制原代培养神经元表达 通过微量注射MAP1B肽和抗体 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MAP的启动子，到目前为止，还没有 已经描述了启动子，并且为了分离特异性转录 控制这些基因的因素。这些研究也将成为 用于体内工作和实验工具的开发， 转基因动物的基因表达。最终， 这些基因和它们的启动子将提供对 发育过程中神经元分化的分子途径 神经系统和对环境变化的反应。

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.