Novel Circadian Mutant of Drosophila

描述（由申请人提供）：本申请旨在确定酪蛋白激酶在生物钟机制中的作用。生物钟通常与光和温度的日常环境周期同步，但在许多生物体中是内源性产生的。在人类中，生物钟控制着睡眠-觉醒行为以及其他生理波动。生物钟的紊乱会导致睡眠障碍、时差反应和全身不适。许多人被迫轮班工作，而轮班工作与他们的生物钟不一致，从而导致身体和认知缺陷，影响个人的健康和工作质量。因此，对昼夜节律机制的理解将有助于我们控制生物钟的能力，以造福于受影响的个人和社会。我们目前关于生物钟机制的知识大多来自果蝇的遗传分析，果蝇的生物钟机制与人类的生物钟机制非常相似。例如，双倍时间酪蛋白激酶（DBT）被认为调节果蝇和人类中另一种时钟蛋白（PER）的细胞质和细胞核积累，本文提出的工作旨在确定DBT的作用是否受其他因素的调节，以及它的活性如何有助于PER的积累。在第一个具体目标中，将进一步研究几种dbt突变改变昼夜节律周期的机制。在第二个具体目标中，DBT影响PER稳定性的方式将通过在果蝇细胞系（S2）中表达野生型和突变的DBT和PER来解决。PER的磷酸化以及DBT和PER相互作用因子的存在也将通过体外和蛋白质组学方法进行评估，包括从S2细胞分离的蛋白质的质谱分析。最后，在第三个具体目标中，PER和DBT的突变形式将作为转基因在果蝇中表达，以阐明DBT在体内的作用。脊椎动物酪蛋白激酶I将沿着果蝇DBT用于特定目的2 - 3

DESCRIPTION (provided by applicant): This application seeks to determine the role of casein kinase in the mechanism of the circadian clock. Circadian clocks, while typically in synchrony with the daily environmental cycles of light and temperature, are generated endogenously in many organisms. In humans, the clocks control sleep-wake behavior as well as other physiological fluctuations. Disruptions of the circadian clock lead to sleep disorders, jet lag and a general malaise. Much of the population is forced to work shifts that are not in phase with their circadian clocks, thereby leading to physical and cognitive deficits that impact the health of the individuals and the quality of their work. Therefore, an understanding of the circadian mechanism will contribute to our ability to control the circadian clock for the benefit of affected individuals and of society. Much of our current knowledge about the mechanism of the circadian clock has come from a genetic analysis in fruit flies, which have a circadian mechanism quite similar to the human mechanism. For instance, the doubletime casein kinase (DBT) is thought to regulate the cytoplasmic and nuclear accumulation of another clock protein (PER) in both fruit flies and man. The work proposed here seeks to determine whether DBT's effects are regulated by other factors, and how its activity contributes to the accumulation of PER. In the first specific aim, the mechanism for the alteration of circadian period by several dbt mutations will be further investigated. In the second specific aim, the way in which DBT affects the stability of PER will be addressed by expressing wild type and mutated DBT and PER in a Drosophila cell line (S2). The phosphorylation of PER and the presence of DBT- and PER-interacting factors will also be assessed with in vitro and proteomic approaches, including a mass spectrometry analysis of proteins isolated from S2 cells. Finally, in a third specific aim, mutant forms of PER and DBT will be expressed as transgenes in flies, in order to elucidate the role of DBT in vivo. A vertebrate casein kinase I will be employed along with Drosophila DBT for specific aims 2 -3