Interdisciplinary studies of the Drosophila Circadian Clock

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，我们提出了果蝇时钟的三种跨学科研究。(1)我们将进行协作结构研究，以帮助我们确定特定的调控作用是如何由PER蛋白的某些结构域执行的，这些作用在遗传学和生物化学上是已知的。PER的结构是否表明它如何与TIM和DBT相互作用？这些数据是否表明TIM如何通过DBT抑制PER的磷酸化？假设在PER的n端附近的LOV结构域具有黄素结合结构域吗？(2)我们将通过条件蛋白剪接产生新的突变来分析重要的时钟基因。我们对GSK-3、dbt、vri和Pdp-1的高形态和低形态突变的研究表明，它们都是果蝇生物钟的关键组成部分。然而，由于这些基因的零突变是致命的，因此很难确定它们对生物钟的全部影响。我们正在开发一种化学遗传学，其中可扩散的小分子将可逆地控制活苍蝇中每种重要蛋白质的存在。(3)我们将开发新的微阵列和统计方法来阐明节律性基因组活动的特征，并确定果蝇是否使用单一分子机制来产生所有的昼夜节律。为了开始将生物钟与特定的行为和生理输出联系起来，我们将研究一组由光和生物钟的配对作用调节的新基因。

DESCRIPTION (provided by applicant): We have been studying the molecular control of circadian behavioral rhythms using Drosophila as a model system. Orthologs 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. Here we propose three classes of interdisciplinary investigations of the Drosophila clock. (1) We will conduct collaborative structural studies that can help us determine how specific regulatory actions, previously recognized genetically and biochemically, are performed by certain domains of the PER protein. Does the structure of PER indicate how it interacts with TIM and DBT? Do such data suggest how TIM suppresses PER's phosphorylation by DBT? Does a putative LOV domain near the N-terminus of PER possess a flavin binding domain? (2) We will generate new mutations for the analysis of vital clock genes by Conditional Protein Splicing. Our studies of hyper- and hypo-morphic mutations of GSK-3, dbt, vri, and Pdp-1 indicate that each is a key component of the Drosophila clock. However, because null mutations of these genes are lethal, it has been difficult to determine their full effects on the clock. We are developing a form of chemical genetics in which small diffusible molecules will reversibly control the presence of each vital protein in living flies. (3) We will produce new micro array and statistical approaches to clarify features of rhythmic genome activity and to determine if flies use a single molecular mechanism to generate all circadian rhythms. To begin to link the clock to specific behavioral and physiological outputs, we will investigate a novel set of genes that are regulated by a paired action of light and the circadian clock.