NOVEL CIRCADIAN MUTANT OF DROSOPHILA

果蝇的新型昼夜节律突变体

基本信息

批准号：
2675609
负责人：
JEFFREY L PRICE
金额：
$ 9.33万
依托单位：
WEST VIRGINIA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-04-01 至 2002-03-31
项目状态：
已结题

项目摘要

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.

描述（改编自申请人的摘要）：遗传分析利用黑腹果蝇阐明了细胞和分子生物钟的机制。在果蝇中获得的发现，因为其遗传适应性，可能会揭示其机制人体生物钟也是如此。了解人体昼夜节律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）。而且，在一个社会中，越来越需要在白天和晚上的任何时间运行，这是势在必行的了解睡眠/觉醒行为与昼夜节律之间的相互作用如果我们要了解这种行为对我们的长期影响健康与社会。本拨款申请中提议的工作包括新型昼夜节律的行为、遗传和分子特征果蝇突变体（双倍）。昼夜节律的内生周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。特别是基因产物受这种新突变的影响会影响周期的昼夜节律振荡和永恒的RNA和蛋白质、核定位、磷酸化状态、和协会？这些振荡被认为包含反馈生物钟的自动调节周期。有可能的是 Double-time 的突变会影响蛋白质，该蛋白质也是一个整体这种自动调节循环的组成部分，但也有可能影响光影响节律的途径的一个组成部分，或者将时钟生物化学与行为耦合的途径的组成部分。工作这里提出的将完成已经启动的表型表征的双倍时间。它对时钟的响应有什么影响光，温度，以及周期和分子振荡永恒的基因产品？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，可能表明蛋白质的功能。重组蛋白，表达于原核或真核表达系统，将测试其功能体外并用于制造抗体。这些抗体和克隆的 DNA 将用于确定细胞和亚细胞的表达位点这种新的RNA和蛋白质。这个表情是否显示了昼夜节律调节，它是否受到果蝇昼夜节律突变体的影响？可能的将用突变体或条件表达来测试分子机制转基因果蝇中蛋白质的形式。最后，分离同源物其他物种的基因，或与其他物种相互作用或与之相互作用的分子的基因由Double-time监管，将为今后的工作提供素材。