MOLECULAR GENETIC ANALYSIS OF FUNGAL CIRCADIAN RHYTHMS

生物钟已经在从单细胞到哺乳动物的复杂生物体中被描述，并且其功能是控制细胞活动和行为的日常节律。 详细了解生物钟的重要性可以通过其普遍存在及其在人类生理学中的既定参与来理解，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时钟调节的，为有前途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物化学基础。所有生物的时钟都是在细胞内组装的，并且时钟组件在进化上是保守的;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，很少有人知道这种调节是如何发生的，或者是细胞中发出时间信息的组件。 为了回答这些问题，我们将研究重点放在模式系统粗糙脉孢菌中生物钟控制基因的生化功能和调控上。在具体目标1中，我们将使用生物化学技术分离反式作用因子，该因子与ccg-2基因中的正顺式作用时钟元件结合，这对于节律性是必要的和足够的。 将克隆编码因子的基因，并分析其在昼夜输出途径中的作用。 在具体目标2中，我们将进行遗传突变体选择的新基因参与调节昼夜节律输出的基础上的差异表达的时钟控制的基因在野生型与时钟无效菌株。 两个时钟控制基因的启动子（一个受涉及FRQ的途径正调控，一个受涉及FRQ的途径负调控）与选择标记mtr的融合将允许分离导致嵌合体不适当表达的突变体。 在具体目标3中，我们将使用蛮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突变体。在突变体选择和筛选中鉴定的基因座将测定其在昼夜节律中的作用，克隆，并且重要基因将用于启动哺乳动物直系同源物的搜索，如具体目标4中所述。 总之，这些实验将允许更详细地了解细胞如何组织为时间的函数。

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.