MOLECULAR GENETIC ANALYSIS OF FUNGAL CIRCADIAN RHYTHMS

从单细胞到哺乳动物的复杂生物中都有生物钟的描述，其功能是控制细胞活动和行为的日常节奏。详细了解生物钟的重要性可以通过它的无处不在和它在人类生理学中的作用来理解，包括内分泌功能、睡眠/觉醒周期、精神疾病以及药物耐受性和有效性。此外，许多人体组织中的细胞分裂是受生物钟调节的，这为癌症化疗提供了有希望的新方法。我们的长期目标是了解昼夜节律的分子和生化基础。所有生物体的时钟都是在细胞内组装的，时钟组成部分是进化保守的；因此，简单的真核生物提供了适当的实验系统来研究时钟机制并有效地实现这些目标。昼夜节律的一个重要方面是基因表达的时钟控制。然而，对于这种调节是如何发生的，以及细胞中发出时间信息信号的成分，人们知之甚少。为了回答这些问题，我们将重点研究模型系统粗神经孢子虫生物钟控制基因的生化功能和调控。在具体目标1中，我们将使用生化技术分离与ccg-2基因中正顺式作用时钟元件结合的反式作用因子，这对于节律性是必要和充分的。编码该因子的基因将被克隆并分析其在昼夜节律输出通路中的作用。在Specific Aim 2中，我们将根据野生型与无时钟型菌株中时钟控制基因的差异表达，对参与昼夜节律输出调节的新基因进行基因突变选择。将两个时钟控制基因的启动子（一个由FRQ通路正调控，一个由FRQ通路负调控）与可选择标记mtr融合，将允许分离导致嵌合体不适当表达的突变体。在Specific Aim 3中，我们将使用暴力筛选新的时钟输出信号突变体，以及时钟和时钟本身的环境输入途径中的突变体。在突变体选择和筛选中发现的基因座将被分析其在昼夜节律中的作用，克隆和重要基因将用于启动哺乳动物同源物的搜索，如Specific Aim 4所述。总之，这些实验将使我们更详细地了解细胞是如何随着时间的推移而组织起来的。

Circadian clocks have been described in organisms ranging in complexity from unicells to mammals and function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology including endocrine function, sleep/wake cycles, psychiatric illness, as well as drug tolerances and effectiveness. Additionally, cell division in many human tissues is clock-regulated, providing the basis for promising new approaches to cancer chemotherapy. Our long term goals are to understand the molecular and biochemical basis for circadian rhythmicity. The clock in all organisms is assembled within the cell and clock components are evolutionarily conserved; thus, simple eukaryotes provide appropriate experimental systems to investigate clock mechanisms and to efficiently achieve these goals. An important aspect of circadian rhythmicity is clock control of gene expression. However, little is known about how this regulation takes place or of the components that signal time information in the cell. To answer these questions, we are focusing our studies on the biochemical function and regulation of clock-controlled genes in the model system Neurospora crassa. In Specific Aim 1 we will use biochemical techniques to isolate the trans-acting factor(s) that bind to a positive cis-acting clock element in the ccg-2 gene which is both necessary and sufficient for rhythmicity. The gene(s) encoding the factor(s) will be cloned and analyzed with respect to their role in circadian output pathways. In Specific Aim 2 we will carry out a genetic mutant selection for novel genes involved in the regulation of circadian output based on differential expression of the clock-controlled genes in a wild type versus a clock-null strain. Fusion of the promoter of two clock-controlled genes, one positively and one negatively regulated by a pathway involving FRQ, to the selectable marker mtr will permit the isolation of mutants that result in improper expression of the chimera. In Specific Aim 3 we will use a brute force screen for novel clock output signaling mutants, as well as for mutants in environmental input pathways to the clock and the clock itself. The loci identified in the mutant selections and screens will be assayed for their role in circadian rhythmicity, cloned, and important genes will be used to initiate a search for mammalian orthologs as described in Specific Aim 4. Together, these experiments will permit a more detailed understanding of how the cell is organized as a function of time.