Interdisciplinary studies of sleep and circadian rhythms in Drosophila

描述（由申请人提供）：我们之前的工作主要集中在果蝇昼夜节律的分子机制。最初在果蝇中发现的基因的同源物现在已经与脊椎动物（包括鱼、青蛙、老鼠和人类）的节律行为和生理控制联系起来。在这里，我们提出了三类跨学科的研究苍蝇的节律活动/休息行为。(1)我们将使用elav-Gal4驱动来自两个广泛文库的单个UAS-RNAi转基因，以寻找调节睡眠的新遗传途径。我们的目标是使用约19,000个RNAi细胞系来测试果蝇蛋白质编码基因的全部补体。我们将扩展我们对一种新的、睡眠减少的突变体失眠症患者的研究。我们将确定失眠症是否以主动/动态的方式调节睡眠，或者它是否调节了对野生型睡眠水平至关重要的发育途径。我们将测试Insomniac作为Cul3泛素连接酶复合物的底物特异性适配器的假设，并将使用各种生化和分子方法来识别目标底物。(3)我们将进一步确定一小群细胞周期基因CycA及其调节因子（Rca1）、cdk2和cdc42在调节睡眠中的作用。由于表达细胞周期基因的一组神经元的组成性激活会诱导过度睡眠，我们将通过流式细胞术分离这些细胞，以发现这些细胞对睡眠的贡献背后的基因表达模式。

DESCRIPTION (provided by applicant): Our prior work has focused on the molecular mechanisms underlying Drosophila's circadian rhythms. Orthologs of genes initially characterized in Drosophila have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. Here we propose three classes of interdisciplinary investigations of the fly's rhythmic activity/rest behavior. (1) We wil use elav-Gal4 to drive individual, UAS-RNAi transgenes from two extensive libraries in a search for novel genetic pathways regulating sleep. Our objective is to test the full complement of Drosophila's protein-coding genes using ~19,000 RNAi lines. (2) We will extend our studies of a novel, reduced-sleep mutant, insomniac. We will determine whether insomniac regulates sleep in an active/dynamic manner, or whether it regulates a developmental pathway that is essential for wild type levels of sleep. We will test the hypothesis that Insomniac functions as a substrate-specific adapter for the Cul3 ubiquitin ligase complex, and will use a variety of biochemical and molecular approaches to identify a target substrate(s). (3) We will further define the role of a small group of cell cycle genes, CycA, its regulator (Rca1), cdk2 and cdc42, in the regulation of sleep. As constitutive activation of a cluster of neurons expressing cell cycle genes induces excess sleep, we will isolate these cells by flow cytometry to discover patterns of gene expression that may underlie the contribution of these cells to sleep.