MOLECULAR ANALYSIS OF RETINAL/PINEAL MELATONIN SYNTHESIS

该项目的主要目标是确定 视网膜/松果体基因、N-乙酰转移酶（NAT），并检测NAT基因 在视觉和神经传导中表达。这项工作的主要目的是 五年的研究计划是为了回答这个问题： 视网膜/松果体NAT酶是昼夜驱动振荡器的关键成分 是视网膜/松果体NAT酶，受环境线索控制， 分子水平？该项目将调查一个具体阶段， 这种内源性的节奏和夹带的光：黑暗周期的 速率限制酶，合成褪黑激素。 松果体是一个很好的系统来研究神经化学的所有步骤 转导，包括配体-受体相互作用，第二信使 调节和特定基因的选择性调节。两个特点 研究松果体神经传导的分子机制有：（1） 由涉及α-和β-受体的交感神经纤维的神经支配;以及 (2)生物化学活动中的高幅度昼夜节律，包括 一些对已知的分子触发物有快速反应。虽然 视网膜与松果体共享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。实验将 表征编码NAT的视网膜/松果体组织特异性基因。 将在视网膜和松果体中分析正常组织特异性表达 老鼠和近亲繁殖的老鼠。 原代松果体细胞培养也将用于 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并表征编码 视网膜/松果体酶，NAT，通过筛选大鼠松果体表达文库。 2.产生针对重组融合的免疫探针的抗血清 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探针定位， 描述和测量昼夜节律调节和环境变化 调节其mRNA和蛋白质的NAT。 所描述的实验将使用免疫和重组DNA 研究环境因素对基因表达影响的工具 在松果体和视网膜中。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本分子的研究 神经系统中的昼夜节律的本质。生物昼夜节律 节奏不仅在我们的日常生活中很重要， 一生健康。对基因正常结构的了解 在松果体和视网膜中的表达可能会导致更好地了解 褪黑激素合成酶参与视觉和神经 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.