REGULATION OF MAP2 IN BRAIN

微管相关蛋白2（Microtubule Associated protein 2，MAP 2）是一种复杂的蛋白质， 与神经元细胞骨架的微管共同纯化，并且似乎 在微管的组织中起着重要作用， 与其他蛋白质和细胞器的相互作用。 MAP 2特别 对大脑感兴趣是因为它的丰富性，它在大脑中的特定定位， 树突及其在脑发育过程中的表达调控 和神经元分化。 MAP 2在脑和PC 12细胞中表达的分子机制， 在蛋白质合成、mRNA和编码基因的水平上进行研究 对于这种蛋白质。 为此，将通过以下方法分离MAP 2 cDNA： 用高特异性多克隆筛选λ gt 11脑文库， 克隆抗体 克隆身份将通过杂交确认 选择，并通过探测来自不同组织和细胞的RNA 的cDNA。 最初的观察显示，MAP 2水平的显著诱导， 用NGF处理PC 12细胞。 这种诱导的机制将是 通过MAP 2合成速率（通过 脉冲标记）、MAP 2 mRNA的翻译活性（体外 翻译）和mRNA的稳态水平（通过北方印迹）。 类似的技术将被用来跟踪发育调节 MAP 2同种型（MAP 2a和MAP 2b）在大鼠脑中的表达;具体而言，这些 不同的同种型是前体加工或翻译的产物， 两份不同的抄本 MAP 2的遗传复杂性将是 通过Southern印迹检测，以确定MAP 2基因是否是 共享结构和功能域的家族。 最后 MAP 2 mRNA的出现和定位，通过原位测量 杂交，将比较免疫细胞化学分布 树突中的MAP 2，因此，测试了位于树突附近的多聚核糖体的假设。 树突棘可能参与局部蛋白质合成。 本研究的目的是了解 细胞骨架蛋白在神经元分化过程中的作用， 在与许多神经系统疾病相关的形态学异常中发挥作用， 特别是与树突营养不良相关的疾病， 智力迟钝。

Microtubule Associated protein 2 (MAP2), is a complex protein, which co-purifies with microtubules of the neuronal cytoskeleton, and seems to have an important role in the organization of microtubules and their interactions with other proteins and organelles. MAP2 is of particular interest in brain because of its abundance, its specific localization in dendrites, and the regulation of its expression during brain development and neuronal differentiation in culture. The molecular mechanism of MAP2 expression in brain and in PC12 cells, will be studied at the level of protein systhesis, mRNA and the genes that code for this protein. For that purpose, MAP2 cDNA will be isolated, by screening lambda gt11 brain libraries with highly specific polyclonal and monoclonal antibodies. The clone identity will be confirmed by hybrid selection, and by probing RNA derived from different tissues and cells lines with the cDNA. Initial observations showed a significant induction of MAP2 levels during treatment of PC12 cells with NGF. The mechanism of this induction will be analyzed by the temporal correlation between the rate of MAP2 synthesis (by pulse labeling), the translational activity of MAP2 mRNA (by in vitro translation), and the steady state levels of the mRNA (by Northern blots). Similar techniques will be used to follow the developmental regulation of MAP2 isotypes (MAP2a and MAP2b) in rat brain; specifically, whether these different isotypes are the product of precursor processing, or translation of two distinct transcripts. The genetic complexity of MAP2 will be examined by Southern blots, to determine whether the MAP2 gene is part of a family that shares structural and functional domains. Finally, the appearance and localization of MAP2 mRNA, measured by in situ hybridization, will be compared to the immunocytochemical distribution of MAP2 in dendrites, thus, testing the hypothesis that polysomes located near dendritic spines may be engaged in local protein synthesis. The objective of this research is to understand the regulation of cytoskeletal proteins during neuronal differentiation, and the role they play in the morphological abnormalities associated with many neurological disorders, particularly, in relation to dendrite dystrophy associated with mental retardation.