BIOLOGICAL CLOCKS--GENES, BEHAVIOR AND NEUROBIOLOGY

昼夜节律是生物体普遍存在的生物周期 其适当的日常调节与井密切相关 从微生物到哺乳动物都有。 25- 一个长达一年的遗传学方法来理解这些节奏 最近开始揭示时钟机制的元素， 是日常节律的基础 本方案中的子方案 项目申请围绕行为遗传、神经- 遗传学和分子神经生物学的昼夜节律， 果蝇所提出的实验更强调一个~系统~ 而不是一个专注于核心的方法， 分子起搏 因此，行为的神经基质 节奏，以及后期发展的周期性特征，将是 应用节律突变体和转基因技术 携带操纵形式的时钟基因的菌株;这些研究 包括对神经元的解剖输出通路的描述 这些细胞是CNS-起搏细胞的候选细胞， 影响这些细胞的结构和功能的扰动， 结合生物测定的影响， 介导的神经元损伤。中央起搏器的输入路径， 它带来了环境的线索，影响了关键的日常重置， 时钟，将在解剖学和元素方面进行剖析 参与的信号转导途径 处理复位刺激。 输出路径将是 研究，关于不同的生理参数， 被假设为时钟的第一或第二阶段目标 基因功能 后者包括周期性变化的水平， 编码的mRNA和蛋白质;这些分子周期将被 结合生理记录进行跟踪， 干扰，通过应用转基因，在转基因中， 时钟基因（称为周期）已经融合到DNA序列中 编码实时报告基因;这是荧光素酶活性， 最近被证明允许非侵入性监测 活体成年果蝇和预表达组织中的分子节律 从发育后期的动物身上分离出来。 遗传和 分子遗传学研究主要包括两个方面：（1）分析 的行为和神经生物学后果， 操纵一个时钟控制的基因的表达， 同样的神经肽编码基因的扰动 其产物与生物钟基因的一个子集共表达， CNA起搏神经元;（2）CNA起搏神经元的遗传学和生物学研究 节奏突变体，最近分离的缺陷的基础上， 昼夜行为节律：这些表型遗传学研究将 继续克隆由出现的突变所定义的基因， 最有希望的候选人， 果蝇昼夜节律系统的组成部分。

Circadian rhythms are organismically ubiquitous biological cycles whose propr daily regulation is strongly connected to the well being of species ranging from microbes to mammals. The 25- year-old genetic approach toward understanding these rhythms has recently begun to reveal elements of the clock mechanisms that underlie daily rhythmicities. This sub-proposal in a Program Project application revolves around the behavioral genetic, neuro- genetics, and molecular neurobiology of circadian rhythms in Drosophila. The experiments proposed stress more of a ~systems~ approach than one that would concentrate upon the hard core of molecular pacemaking. Thus, the neural substrates of behavioral rhythms, and a periodic feature of late development, will be delved into by application of rhythm mutants and transgenic strains carrying manipulated forms of clock-genes; these studies include descriptions of anatomic output pathways from neurons that are candidate for CNS-pacemaker cells, and selectively effected perturbations of the structure and function of such cells in conjunction with bioassaying the effects of the molecularly mediated neuronal damage. Input paths to the central pacemakers, which bring in environmental cues to effect crucial daily re-sets of the clock, will be dissected both in terms of anatomy and elements of signal-transduction pathways that putatively participate in processing the resetting stimuli. Output pathways will be investigated, with respect to varying physiological parameters that are hypothesize to be the first- or second-stage targets of clock- gene functions. The latter includes cyclically varying levels of the encoded mRNAs and proteins; these molecular cyclings will be tracked in conjunction with physiological recordings and perturbations, by applying a transgene in which portions of a clock gene (called period) have been fused to DNA sequences encoding a real-time reporter; this is luciferase activity, which as recently been shown to permit non-invasive monitoring of molecular rhythms in live adult flies and in per-expressing tissues explanted from animals late in development. Genetic and molecular-genetic studies are proposed in two areas: (1) analysis of the behavioral and neurobiological consequences of manipulating a clock-controlled gene~s expression, and of effecting the same kinds of perturbations of a neuropeptide-encoding gene whose product is co-expressed with clock genes in a subset of the CNA pacemaking neurons; (2) genetic and biological studies of rhythm mutants, recently isolated on the basis of defects in circadian behavioral rhythms: these phenogenetic studies will proceed into cloning of the genes defined by mutations that appear to be the most promising candidates for disrupting important elements of Drosophila~s circadian system.