All eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous control and organization over time. The cellular machinery that generates this ability is known collectively as the biological clock. The importance of a detailed understanding of the circadian clock rest on the apparent ubiquity of its influence on cellular and organismal processes. Phylogenetically this ranges from control of cell division and enzyme activities in unicells, to a multiplicity of human systems including endocrine function, work rest cycles and sleep, and drug tolerances and effectiveness. The long term goal of the proposed work is to be able to describe, in the language of genetics and biochemistry, the feedback cycle that comprises the mechanism of the circadian biological clock. Historically, a major goal in the study of the clock has been to establish the identity of a """"""""gear or cog"""""""" in the clock mechanism. There is much reason to believe that such components act primarily at the level of intracellular regulation so that lower eukaryotes may be valid model systems even for vertebrate cellular clocks. Detailed genetic, biochemical and pharmacological dissection of the circadian clock mechanism has been seriously pursued in only two organisms, Drosophila and the haploid ascomycete fungus Neurospora crassa. In each organism research has revealed the existence of a few key components in the feedback loop. Two of these in Neurospora are encoded by the genetic loci frq and prd-4. We have now cloned these genes so that they can be examined and manipulated at the molecular level. We will carry out molecular genetic analyses of frq and prd-4. We will complete the characterization of the transcription units of these genes, characterize the expression as a function of time of day and nutritional state, and determine the molecular nature of the mutant phenotypes. We will complete a phylogenetic analysis of the frq and prd-4 genes at the level of nucleic acid hybridization. We will carry out genetic analyses of frq and prd-4 in which we will examine the dosage dependence of the clock on these genes. We will determine the null phenotype for frq and prd-4, and will undertake, through both classical and reverse genetics, the production of new alleles of both genes. We will carry out biochemical analyses of frq and prd-4 in which we will identify the protein products encoded by these genes. We will raise antibodies to both clock gene products, and will use these antibodies to determine the turnover characteristics of each protein, to follow the level of each protein as a function of time of day, and to determine the intracellular localization of the proteins by light and immune-electron microscopy.
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