MOLECULAR GENETIC ANALYSIS OF FUNGAL CIRCADIAN RHYTHMS

生物钟在从单细胞到哺乳动物的各种复杂生物体中都有描述，并具有控制细胞活动和行为的日常节奏的功能。对时钟的详细了解的重要性可以通过它的普遍存在以及它对人类生理学的既定参与来认识，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，许多人类组织中的细胞分裂是受时钟调节的，这为有希望的癌症化疗新方法提供了基础。我们的长期目标是了解昼夜节律的分子和生化基础。所有生物体中的时钟都是在细胞内组装的，时钟成分在进化上是保守的；因此，简单的真核生物提供了合适的实验系统来研究时钟机制并有效地实现这些目标。昼夜节律性的一个重要方面是对基因表达的时钟控制。然而，关于这种调节是如何发生的，或者细胞中发出时间信息的成分，人们知之甚少。为了回答这些问题，我们正在重点研究模式系统粗糙脉孢菌中时钟控制基因的生化功能和调控。在特定的目标1中，我们将使用生化技术分离与CCG-2基因中的正顺作用时钟元件结合的反式作用因子(S)，这是节律性的必要条件和充分条件。编码因子(S)的基因(S)将被克隆并分析它们在昼夜输出途径中的作用。在特定的目标2中，我们将根据时钟控制基因在野生型和非钟型菌株中的差异表达，对参与昼夜节律输出调节的新基因进行遗传突变选择。将两个时钟控制基因的启动子(一个受FRQ途径正向调控，另一个受FRQ途径负调控)与可选择标记MTR融合，将允许分离导致嵌合体不正确表达的突变体。在具体目标3中，我们将使用暴力筛选新的时钟输出信号突变体，以及环境中到时钟和时钟本身的输入路径中的突变体。在突变选择和筛选中确定的基因座将被分析它们在昼夜节律中的作用，克隆和重要的基因将被用来启动特定目的4中描述的哺乳动物同源基因的搜索。这些实验将允许更详细地了解细胞是如何随着时间的变化而组织的。

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.