NOVEL CIRCADIAN MUTANT OF DROSOPHILA

描述(摘自申请者的摘要)：一项基因分析 利用黑腹果蝇阐明了细胞和分子 生物钟的机制。在果蝇身上的发现，因为 对其遗传亲和性的研究，很可能有助于揭示 人类生物钟也是如此。对人类生理节律的理解 时钟将有助于治疗睡眠障碍、时差、癌症、 和季节性情感障碍(SAD)。此外，在一个 日夜不停地工作，这是势在必行的 了解睡眠/清醒行为与昼夜节律之间的相互作用 如果我们要意识到这种行为对人类健康的长期影响 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 果蝇突变体(双倍体)。昼夜节律的内生期 这些突变果蝇的时钟从大约24小时减少到18小时。 这项工作将寻求建立影响的分子基础 昼夜节律行为的双倍时间。尤其是基因产品 受这一新突变的影响，周期的昼夜波动 和永恒的RNA和蛋白质，核定位，磷酸化状态， 那联想呢？这些振荡被认为是一种反馈 生物钟背后的自动调节周期。有可能是 双倍时间的突变影响一种蛋白质，该蛋白质也是一个整体 是这个自动调节循环的组成部分，但也有可能它 影响光影响节律的途径的一个组成部分，或者 连接生物钟生物化学和行为的通路的组成部分。这项工作 将完成已经启动的表型表征 两倍的时间。它对时钟的响应有什么影响 光，温度，以及周期和周期的分子振荡 永恒的基因产品？对受影响基因的分子克隆将使 相应蛋白质氨基酸序列的测定，这是 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，将在 体外培养并用来制造抗体。这些抗体和克隆的DNA将 用来确定细胞和亚细胞的表达部位 这是一种新型的RNA和蛋白质。这个表达方式是否显示出昼夜节律， 它是否受到果蝇中任何昼夜节律突变的影响？可能的 分子机制将通过突变或条件性表达进行测试 转基因果蝇体内蛋白质的不同形式。最后，同源基因的分离 其他物种的基因，或与之相互作用的分子或 受双重时间规范，将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.