Molecular and Cellular Studies of Circadian Rhythms

描述（由申请人提供）：我们一直在研究以果蝇为模型系统的昼夜行为节律的分子控制。最初在苍蝇身上发现的基因同源物，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。果蝇生物钟的一个核心组成部分是一个反馈回路，其中两个生物钟蛋白PERIOD （PER）和TIMELESS （TIM）抑制它们自己的转录。这种反馈的时间延迟促进了振荡性基因表达。我们最近发现了控制这种延迟的新的细胞特征。另外的研究发现了影响生物钟周期性的新基因和蛋白质。在本提案中，我们将(1)研究特异性DBT定向磷酸化对不同PER功能的影响，如转录抑制、稳定性和定时核积累。(2) PER和TIM似乎通过它们在细胞质中的物理相互作用而被修饰，从而允许它们随后独立的核积累。我们将研究DBT和CK2两种激酶以及磷酸酶PP2a在这一调控中的作用。(3)我们将确定新发现的PER/TIM细胞质间隔计时器是否有助于昼夜节律钟的温度补偿。(4)我们将对培养细胞中调节PER和TIM定时核积累的新基因和蛋白进行高通量筛选。(5)一项涉及数百种转基因RNAi的运动活动筛选显示，特定核细胞蛋白的减少大大延长了果蝇时钟的周期。该蛋白对节律性贡献的分子途径将在果蝇和S2细胞中进行探索。候选基因方法起源于果蝇的正向遗传筛选，允许人类经期蛋白和酪蛋白激酶1的突变同源物与先天性睡眠错误联系起来。对果蝇中这些基因和蛋白质的早期功能研究也被用作探索人类睡眠异常模式的特定机制的基础。我们相信，我们提出的果蝇生物钟的遗传、生物物理和生化研究将继续揭示新的组织和功能原理，从而促进对人类昼夜节律的理解。

DESCRIPTION (provided by applicant): We have been studying the molecular control of circadian behavioral rhythms using Drosophila as a model system. Homologues of genes initially characterized in the fly, have now been linked to the control of rhythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. A central component of the fly clock is a feedback circuit in which two clock proteins, PERIOD (PER) and TIMELESS (TIM), repress their own transcription. Temporal delays in this feedback promote oscillatory gene expression. We have recently discovered novel cellular features controlling one such delay. Additional studies have identified new genes and proteins affecting periodicity of the circadian clock. In this proposal we will (1) examine the effects of specific DBT- directed phosphorylations on distinct PER functions such as transcriptional repression, stability and timed nuclear accumulation. (2) PER and TIM appear to be modified by their physical interaction in the cytoplasm, allowing their subsequent, independent nuclear accumulation. We will study the roles of two kinases, DBT and CK2, and a phosphatase, PP2a, in this regulation. (3) We will determine whether a newly discovered, PER/TIM cytoplasmic interval timer contributes to temperature compensation of the circadian clock. (4) We will conduct a high-throughput screen for new genes and proteins regulating the timed nuclear accumulation of PER and TIM in cultured cells. (5) A locomotor activity screen involving several hundred transgenic RNAi stocks has shown that reduction of a specific karyopherin substantially lengthens the period of the fly clock. The molecular pathway underlying this protein's contribution to rhythmicity will be explored in flies and S2 cells. PUBLIC HEALTH RELEVANCE Candidate gene approaches, originating in the forward genetic screens of Drosophila, allowed mutant orthologs of human PERIOD protein and casein kinase 1 to be connected to inborn errors of sleep. The early functional studies of these genes and proteins in Drosophila have also been used as the basis for exploring specific mechanisms underlying aberrant patterns of human sleep. We believe our proposed genetic, biophysical, and biochemical studies of Drosophila's circadian clock will continue to reveal new principles of organization and function that promote an understanding of human circadian rhythms.