MOLECULAR ANALYSIS OF RETINAL/PINEAL MELATONIN SYNTHESIS

该项目的主要目标是定义 视网膜/松果体N-乙酰转移酶(NAT)基因及其检测 在视觉和神经传导中的表达。这样做的主要目标是 五年研究建议是要回答这样一个问题： 视网膜/松果体NAT酶是昼夜节律驱动振荡器的关键成分 以及视网膜/松果体NAT酶是否受环境线索控制 在分子水平上？这个项目将调查一个特定的阶段 这种内生节奏和光的夹带：黑暗的循环 速率=合成褪黑素的限制酶。 松果体是研究神经化学所有步骤的极好系统。 转导，包括配体-受体相互作用，第二信使 对特定基因的调节和选择性调节。两个特点 研究松果体神经传导的分子机制有(1) α受体和β受体的交感纤维的神经支配； (2)生化活动中的高幅度昼夜节律，包括 其中一些对已知的分子触发反应迅速。尽管 视网膜与松果体有着相似的蛋白质和生化途径， 褪黑激素在视网膜中的作用尚不清楚。这些实验将会 描述编码NAT的视网膜/松果体组织特异性基因。 正常组织特异性表达将在视网膜和松果体中进行分析 老鼠和近亲交配的小鼠。松果体细胞原代培养也将用于 研究肾上腺素能调节在基因表达中的作用。 该项目将有三个具体目标： 1.分离全长cDNA并鉴定编码该基因的cDNAs 视网膜/松果体酶，NAT，通过筛选大鼠松果体表达文库。 2.制备抗重组融合免疫探针的抗血清 从翻译的cDNA中鉴定的蛋白质和合成肽 编码NAT。 3.应用这些重组和免疫学探针定位， 描述和衡量昼夜节律和环境的变化 它们的mRNA和蛋白对NAT的调节。 描述的实验将使用免疫学和重组dna。 检测环境线索对基因表达的影响的工具 在松果体和视网膜。松果体和视网膜被广泛地 通过生理学、药理学和生物化学方法进行研究。这些 研究将扩展这些关于基础分子的研究 神经系统昼夜节律的本质。生物节律 节奏不仅在我们的日常生活中很重要，而且在维持我们的 终身健康。关于基因正常结构的知识 在松果体和视网膜中的表达可能有助于更好地理解 褪黑素合成酶在视觉和神经中的作用 转导。

The major goal of this project is to define the structure of the retinal/pineal gene, N-acetyltransferase (NAT) and to examine NAT gene expression in visual and neural transduction. The primary objective of this five year research proposal is to answer the question: Is the retinal/pineal NAT enzyme a key component of a circadian driving oscillator and is the retinal/pineal NAT enzyme controlled by environmental cues at the molecular level? This project will investigate one specific phase of this endogenous rhythm and the entrainment by the light:dark cycle of the rate=limiting enzyme that synthesizes melatonin. The pineal is an excellent system to study all steps in neurochemical transduction, including ligand-receptor interactions, second messenger regulation, and selective regulation of specific genes. Two characteristics for studying molecular mechanisms of pineal neurotransduction are (1) innervation by sympathetic fibers involving alpha- and beta-receptors; and (2) high amplitude circadian rhythms in biochemical activities, including some that are rapidly responsive to known molecular triggers. Although the retina shares similar proteins and biochemical pathways with the pineal, the role of melatonin in the retina is not understood. The experiments will characterize the retinal/pineal tissue-specific genes coding for NAT. Normal tissue-specific expression will be analyzed in retinas and pineals of rats and inbred mice. Primary pineal cell cultures will also be used to study the role adrenergic regulation has in gene expression. The project will have three specific aims: 1. Isolate full-length cDNAs and characterize cDNA probes encoding the retinal/pineal enzyme, NAT, by screening a rat pineal expression library. 2. Generate antisera for immunological probes against recombinant fusion proteins and synthesized peptides identified from the translated cDNAs encoding NAT. 3. Apply these recombinant and immunological probes to localize, characterize and measure changes in circadian regulation and environmental modulation of their mRNA and protein for NAT. The experiments described will employ immunological and recombinant DNA tools to examine the influence of environmental cues on the genes expressed in the pineal and retina. The pineal and retina have been extensively studied by physiological, pharmacological and biochemical approaches. These studies will extend these studies concerning the fundamental molecular nature of circadian rhythms in the nervous system. Biological circadian rhythms are important not only in our daily lives, but in maintaining our health throughout life. Knowledge of the normal structure of genes expressed in the pineal and retina may lead to a better understanding the involvement of the melatonin synthesizing enzyme in visual and neural transduction.