GENETICS & BIOCHEMISTRY OF NEURONAL PAS DOMAIN PROTEINS

这项研究的目的是解决生物 表达的两种转录因子的作用和生化活性 在哺乳动物的大脑中。 这两种蛋白质都是bHLH-PAS的成员 家族，分别命名为神经元PAS结构域蛋白1（NPAS1） NPAS2。 NPAS1仅在脑神经元中表达， 脊髓 NPAS2主要在脑和脊髓中表达， 而且在与肠道，子宫， 乳腺和前列腺。 将采用三管齐下的方法 来研究这些转录因子的生物学和生物化学。 第一种攻击方法是有针对性地破坏基因 在实验室小鼠中编码NPAS 1和NPAS 2。 传统的方法有 已用于统一消除NPAS1功能。 此类小鼠 一个可行的和适当大小的殖民地正在培育的行为 和神经解剖学研究。 NPAS2函数将有条件地 在每个已知表达该转录因子的组织中消除。 在特定组织中缺乏NPAS2的小鼠将在 脑组织的行为、神经解剖和生理研究 对全身缺血和兴奋性药物的反应。 第二 所提议的研究的目标将需要努力确定NPAS1 和NPAS2靶基因。 培养的神经元细胞将被编程为 有条件地表达NPAS1或NPAS2，以响应定义的 刺激。 信使RNA将从这些细胞中制备， 在每个转录因子的诱导之后。 这种材料将 进行代表性差异分析（RDA），以便 寻找那些被激活或被抑制的基因， 诱导NPAS1或NPAS2。 RDA方法还将用于 分析从来自突变体的脑组织制备的信使RNA 缺乏NPAS1或NPAS2的小鼠。 一旦假定的靶基因 已确定的分子分析方法将被 用于搜索NPAS1和NPAS2响应元件。 第三和 最后的实验方法将涉及生物化学和生物物理 研究集中于调节NPAS2活化的机制。 NPAS2 存在于大脑皮层锥体神经元中的一种潜在的细胞质状态， 海马和大脑皮层。 因此，我们有理由预计， 这种蛋白质作为一种功能性转录因子的活性 将需要特定的感应事件。 生物化学研究 重组NPAS2揭示了血红素辅基的存在 与PAS域相关。 生物化学、分子生物学和 生物物理学研究将被用来研究功能相关性 血红素辅基的作用，以及它在控制 锥体神经元中NPAS2的活性。

The objective of the proposed research is to resolve the biological roles and biochemical activities of two transcription factors expressed in the mammalian brain. Both proteins are members of the bHLH-PAS family, respectively designated neuronal PAS domain protein 1 (NPAS1) and NPAS2. NPAS1 is expressed exclusively in neurons of the brain and spinal cord. NPAS2 is expressed predominately in the brain and spinal cord but also in epithelial cell associated with the gut, uterus, mammary gland and prostate. A three-pronged approach will be employed to study the biology and biochemistry of these transcription factors. The first prong of attack will involve targeted disruption of the genes encoding NPAS1 and NPAS2 in laboratory mice. Conventional methods have already been used to uniformly eliminate NPAS1 function. Such mice are viable and an appropriately sized colony is being bred for behavioral and neuro-anatomical studies. NPAS2 function will be conditionally eliminated in each tissue known to express this transcription factor. Mice lacking NPAS2 in specific tissues will be investigated in behavioral, neuro-anatomical and physiological studies of brain tissue in response to both global ischemia and excitotoxic drugs. The second objective of the proposed research will entail efforts to identify NPAS1 and NPAS2 target genes. Cultured neuronal cells will be programmed to conditionally express either NPAS1 or NPAS2 in response to defined stimuli. Messenger RNA will be prepared from such cells before and after induction of each transcription factor. This material will then be subjected to representational difference analysis (RDA) in order to search for genes that are either activated or repressed following induction of NPAS1 or NPAS2. The RDA method will also be utilized to analyze messenger RNA prepared from brain tissue derived from mutant mice lacking either NPAS1 or NPAS2. Once putative target genes have been identified, established methods of molecular analysis will be employed to search for NPAS1 and NPAS2 response elements. The third and final experimental approach will entail biochemical and biophysical studies focused on the mechanisms regulating activation of NPAS2. NPAS2 exists in a latent, cytoplasmic state in pyramidal neurons of the hippocampus and cerebral cortex. As such, there is reason to anticipate that the activity of the protein as a functional transcription factor will require a specific inductive event. Biochemical studies of recombinant NPAS2 have revealed the presence of a heme prosthetic group associated with the PAS domain. Biochemical, molecular biological and biophysical studies will be employed to study the functional relevance of the heme prosthetic group as well as its role in controlling the activity of NPAS2 in pyramidal neurons.