Molecular Genetic Analysis of Fungal Circadian Rhythms

描述(申请人提供)：从细菌到人类的生物体都使用生物钟来控制日常的生化、生理和行为节奏。这种时钟会影响人类的生理，而正常时钟功能的中断会导致精神健康问题，包括躁郁症。通过对脉孢子虫和果蝇的研究，我们开始了解时钟的分子机制。然而，时钟调节基因表达的方式在很大程度上是未知的。我们已经鉴定了四类脉孢子虫时钟控制基因，它们的mRNA水平在黎明、中午、黄昏和午夜达到峰值。人类的生物钟还调节基因的表达在不同的时间达到峰值。因此，一个关键的问题是，时钟如何发出信号，让基因在一天中的不同时间表达。一种假设是，时钟的单一输出路径包含时间延迟，以触发不同基因集的顺序表达。第二个假设是，时钟有特定的输出路径，每个路径调节一个基因类别。我们已经建立了一种基因选择来识别输出途径基因，并发现了四个基因(COP-1、COP-2、COP-3和NRC-2(COP-5))，这些基因是一个或多个时钟控制基因节律性所必需的。为了验证这两个假设，我们将克隆这四个基因，并确定时钟如何影响它们的mRNA和蛋白质产物的表达、定位和/或修饰。为了定位输出通路，我们将确定四个基因中任何一个的突变如何影响不同类别的时钟调控基因的表达，以及其他COP基因和COP蛋白的表达水平、定位和/或修饰。利用生物化学和遗传学方法，我们将确定已知的时钟成分是否与COP蛋白相互作用，以向下游基因发送时间信息。我们的研究将对理解生物钟如何控制明显的节律性产生重大影响，并可能为治疗由昼夜节律障碍引起的人类疾病提供新的方法。

DESCRIPTION (provided by applicant): Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. This clock affects human physiology, and disruptions of normal clock function can cause mental health problems, including manic depression. From studies of Neurospora and Drosophila we are beginning to understand the molecular mechanism of the clock. However, the way in which the clock regulates gene expression is largely unknown. We have identified four classes of Neurospora clock-controlled genes whose mRNA levels peak at dawn, noon, dusk and midnight. The clock in humans also regulates genes whose expression peaks at different times. Thus, a critical question is how the clock signals genes to be expressed at different times of day. One hypothesis is that a single output pathway from the clock incorporates time delays to trigger sequential expression of the different gene sets. A second hypothesis is that there are time-of-day-specific output pathways from the clock, with each pathway regulating one of the gene classes. We have developed a genetic selection to identify output-pathway genes and have found four genes (cop-1, cop-2, cop-3 and nrc-2 (cop-5)) required for rhythmicity of one or more clock-controlled genes. To test the two hypotheses, we will clone the four genes and determine how the clock affects expression, localization and/or modification of their mRNA and protein products. To map the output pathways, we will determine how mutations in any one of the four genes affects the expression of the different classes of clock-regulated genes, and the expression level, localization and/or modification of the other cop genes and COP proteins. Using biochemical and genetic methods, we will determine if known clock components interact with the COP proteins to signal time-of-day information to downstream genes. Our studies will have a significant impact on understanding how circadian clocks control overt rhythmicity and will potentially provide novel approaches for therapies for human diseases that result from circadian dysfunction.