Identification and Analysis of Clock Controlled Genes

描述（由申请人提供）：几乎所有适当检查的真核生物都显示出具有内源性时间控制和组织的能力，称为昼夜节律。负责产生这种能力的细胞机制统称为生物钟。详细了解生物钟对我们理解身心健康和治疗精神疾病的重要性在于它对人类心理和生理过程的影响无处不在。从基本生理功能的昼夜节律变化到人类工作、休息周期和睡眠的节律变化。已知的人类精神疾病是生物钟故障的直接结果，包括睡眠阶段障碍、躁狂抑郁症和失眠症。 广泛的研究已经证明了基因表达的时钟控制的重要性，但关于时钟如何控制它们在其中运行的细胞的行为却知之甚少。在所有真核生物中，这种调节的一个突出方面是多个反馈回路的相互作用，以控制细胞生理学的许多方面，包括mRNA丰度。我们已经确定了几个这样的时钟互连回路，它们将输出耦合到输入，并将输出耦合回时钟本身。我们将使用遗传和生物化学的方法来确定调解这种时钟调节的顺式和反式作用因子。在具体目标1中，我们将跟踪脉孢菌中的发现，即一个关键的细胞周期调节器是时钟控制的，并且对于耦合到时钟环路的输出振荡器是必不可少的。此外，我们将克隆和表征在遗传筛选中已经确定的其他时钟基因。在具体目标2中，我们将研究VVD在影响输出以及时钟中的输入和PAS激酶方面的作用，以及对影响输出的因素进行基于生物发光的筛选。我们已经在哺乳动物昼夜节律生物学方面建立了坚实的概念和实践基础，并已开始通过微阵列阐明哺乳动物细胞振荡器中的时钟调节基因表达模式。在具体目标3中，我们将利用哺乳动物生物钟调控基因的特性来识别控制昼夜节律细胞行为的调控途径。这些方法将通过我们的工作与一个简单的真核生物模型系统和哺乳动物细胞培养模型的时钟提供的见解照亮。

DESCRIPTION (provided by applicant): Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous temporal control and organization known as a circadian rhythm. The cellular machinery responsible for generating this ability is collectively known as the biological clock. The importance of a detailed understanding of the circadian clock to our understanding of physical and mental health and to the treatment of mental illness rests on the ubiquity of its influence on human mental and physiological processes. These range from circadian changes in basic physiological functions to the clear involvement of rhythms in human work rest cycles and sleep. Human psychiatric illnesses known to be a direct result of clock malfunction include forms of sleep phase disorders, manic-depressive illness and insomnia. Extensive research has demonstrated the significance of clock control of gene expression, but little is known regarding how clocks control the behavior of the cells in which they operate. One salient aspect of this regulation in all eukaryotes is the interplay of multiple feedback loops to control many aspects of cell physiology, including mRNA abundance. We have identified several such clock-interconnected loops that couple output to input and output back to the clock itself. We will use genetic and biochemical approaches to identify the cis- and trans-acting factors mediating this clock regulation. In Specific Aim 1 we will follow up the finding in Neurospora that a critical cell cycle regulator is clock-controlled and is essential for an output oscillator coupled to the clock loop. Additionally we will clone and characterize additional clock genes already identified in a genetic screen. In Specific Aim 2 we will examine the role of VVD in influencing output as well as input and PAS kinases in the clock, as well as carrying out a bioluminescence-based screen for factors affecting output. We have developed a strong conceptual and practical base in mammalian circadian biology and have begun the elucidation, via microarrays, of clock-regulated gene expression patterns in a mammalian cellular oscillator. In Specific Aim 3 we will exploit the identities of mammalian clock-regulated genes to identify regulatory pathways controlling circadian cell behavior. These approaches will be illuminated by the insights afforded through our work with both a simple eukaryotic model system and a mammalian cell culture model of the clock.