NOVEL CIRCADIAN MUTANT OF DROSOPHILA

描述（改编自申请人摘要）：遗传分析 使用果蝇是阐明细胞和分子 生物钟的机制。 在果蝇中的发现，因为 它的遗传顺从性，很可能揭示的机制， 人体生物钟也是如此。 对人类昼夜节律的理解 时钟将有助于治疗睡眠障碍，时差，癌症， 季节性情感障碍（SAD） 此外，在一个 越来越多地必须在白天和黑夜的任何时候发挥作用， 为了了解睡眠/觉醒行为和昼夜节律之间的相互作用， 时钟，如果我们要欣赏这种行为的长期影响， 健康和社会。 在这项拨款申请中提出的工作包括 一种新的昼夜节律的行为，遗传和分子特征 果蝇（Drosophila）的突变体（双倍时间）。 昼夜节律的内源期 在这些突变果蝇中，生物钟从大约24小时减少到18小时。 这项工作将寻求建立分子基础的影响， 双倍的昼夜行为。 特别是，基因产物 受这种新突变的影响， 和无时间性的RNA和蛋白质，核定位，磷酸化状态， 和协会？ 这些振荡被认为是一种反馈 自动调节周期是生物钟的基础。 有可能 双时间突变影响的蛋白质，这也是一个整体 这是这个自动调节周期的组成部分，但也有可能， 影响光影响节律的途径的一个组成部分，或者 生物钟生物化学与行为的耦合途径的组成部分。 工作 这里提出的将完成已经开始的表型表征 双重时间 它对生物钟的反应有什么影响 光，温度，和分子振荡的周期， 永恒的基因产物 受影响基因的分子克隆将允许 确定相应蛋白质的氨基酸序列， 可能暗示了这种蛋白质的功能。 重组蛋白，表达于 原核或真核表达系统，将测试在 体外培养并用于制造抗体。 这些抗体和克隆的DNA 用于确定表达的细胞和亚细胞位点， 这种新的RNA和蛋白质。 这种表情是否显示了昼夜节律的调节， 它是否受到果蝇中任何昼夜节律突变体的影响？ 可能 将用突变体或条件性表达的 转基因果蝇中的蛋白质形式。 最后，分离同源 其他物种的基因，或与之相互作用的分子， 通过对《双时》的规范，为今后的工作提供素材。

DESCRIPTION (Adapted from applicant's abstract): A genetic analysis employing Drosophila melanogaster is elucidating the cellular and molecular mechanisms of the circadian clock. Findings obtained in Drosophila, because of its genetic amenability, are likely to shed light on the mechanisms of the human circadian clock as well. An understanding of the human circadian clock will contribute to treatments for sleep disorders, jet lag, cancer, and Seasonal Affective Disorder (SAD). Moreover, in a society which increasingly must function at all times of day and night, it is imperative to understand interactions between sleep/wake behavior and the circadian clock if we are to appreciate the long-term effects of such behavior on health and society. The work proposed in this grant application encompasses a behavioral, genetic, and molecular characterization of a novel circadian mutant of Drosophila (Double-time). The endogenous period of the circadian clock is reduced from approximately 24 hr to 18 hr in these mutant flies. The work will seek to establish the molecular basis for the effects of Double-time on circadian behavior. In particular, do the gene products affected by this new mutation affect the circadian oscillations of period and timeless RNA and protein, nuclear localization, phosphorylation state, and association? These oscillations are thought to comprise a feedback autoregulatory cycle underlying the circadian clock. It is possible that mutation of Double-time affects a protein which is also an integral component of this autoregulatory cycle, but it is also possible that it affects a component of the pathway by which light affects the rhythms, or a component of the pathways coupling clock biochemistry to behavior. The work proposed here will complete an already initiated phenotypic characterization of Double-time. What are its effects on the responses of the clock to light, to temperature, and on the molecular oscillations of the period and timeless gene products? Molecular cloning of the affected gene will allow the determination of the corresponding protein's amino acid sequence, which may suggest a function of the protein. Recombinant protein, expressed in prokaryotic or eukaryotic expression systems, will be tested for function in vitro and used to make antibodies. These antibodies and the cloned DNA will be used to determine the cellular and subcellular sites of expression for this novel RNA and protein. Does this expression show circadian regulation, and is it affected by any of the circadian mutants in Drosophila? Possible molecular mechanisms will be tested with mutant or conditionally expressed forms of the protein in transgenic flies. Finally, isolation of homologous genes for other species, or of molecules which interact with or are regulated by Double-time, will provide the materials for future work.