Novel Circadian Mutant of Drosophila

果蝇的新型昼夜节律突变体

• 批准号: 6824196
• 负责人: JEFFREY L PRICE
• 金额: $ 25.99万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6824196
• 关键词: Drosophilidae; arthropod genetics; biological clocks; casein kinase; cell line; chemical stability; circadian rhythms; enzyme activity; enzyme mechanism; gene expression; gene mutation; genetic regulation; genetically modified animals; mass spectrometry; molecular biology; phenotype; phosphorylation; protein localization; protein protein interaction; proteomics; transcription factor

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

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。生物钟虽然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，这与他们的生物钟不同步，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，了解昼夜节律机制将有助于我们控制生物钟的能力，以造福受影响的个人和社会。我们目前关于生物钟机制的大部分知识都来自对果蝇的遗传分析，果蝇的昼夜节律机制与人类机制非常相似。例如，双倍酪蛋白激酶（DBT）被认为可以调节果蝇和人类中另一种时钟蛋白（PER）的细胞质和核积累。本文提出的工作旨在确定 DBT 的作用是否受到其他因素的调节，以及其活性如何促进 PER 的积累。在第一个具体目标中，将进一步研究几种 dbt 突变改变昼夜节律的机制。在第二个具体目标中，将通过在果蝇细胞系（S2）中表达野生型和突变型 DBT 和 PER 来解决 DBT 影响 PER 稳定性的方式。 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，包括对从 S2 细胞中分离的蛋白质进行质谱分析。最后，在第三个具体目标中，PER和DBT的突变形式将在果蝇中表达为转基因，以阐明DBT在体内的作用。脊椎动物酪蛋白激酶 I 将与果蝇 DBT 一起用于特定目标 2 -3