Molecular and Cellular Studies of Circadian Rhythms

描述(申请人提供)：我们一直在以果蝇为模型系统研究昼夜行为节律的分子控制。最初以苍蝇为特征的基因的同源基因，现在已经与控制包括鱼、青蛙、老鼠和人类在内的脊椎动物的节律行为和生理有关。飞行时钟的一个中心组成部分是一个反馈电路，在这个电路中，两个时钟蛋白，周期蛋白(PER)和永恒时钟蛋白(TIM)，抑制自己的转录。这种反馈中的时间延迟促进了基因的振荡表达。我们最近发现了控制其中一种延迟的新的细胞特征。更多的研究发现了影响生物钟周期的新基因和蛋白质。在这项建议中，我们将(1)研究特定的DBT指导的磷酸化对不同的PER功能的影响，如转录抑制、稳定性和定时核积累。(2)PER和TIM似乎被它们在细胞质中的物理相互作用所修饰，从而允许它们随后独立的核积累。我们将研究两个激酶，DBT和CK2，以及一个磷酸酶，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时钟的温度补偿。(4)我们将进行高通量筛选，寻找调控PER和TIM在培养细胞中定时核积累的新基因和蛋白。(5)涉及数百个转基因RNAi群体的运动活动筛查表明，特定核粘附素的减少显著延长了飞行时钟的周期。这种蛋白质对节律性的作用的分子途径将在果蝇和S2细胞中进行探索。与公共健康相关的候选基因方法起源于果蝇的正向遗传筛查，允许人类周期蛋白和酪蛋白激酶1的突变同源基因与先天性睡眠错误有关。这些基因和蛋白质在果蝇中的早期功能研究也被用作探索人类睡眠异常模式背后的特定机制的基础。我们相信，我们提出的对果蝇生物钟的遗传、生物物理和生化研究将继续揭示新的组织和功能原理，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.