Daily fluctuations in complex behavior is influenced by the rhythmicity of endogenous circadian (approximately 24 hr) clocks that respond to external time cues. Malfunctions in the human circadian timing system are implicated in the manifestation of several disease states, including manic-depression and seasonal affective disorders. A better understanding of the time-keeping mechanism should lead to the development of more efficient treatments to combat disorders associated with altered clock function. Studies in different model systems have greatly contributed to our knowledge of the basic properties that govern human circadian rhythms. Collectively, studies on a variety of organisms suggest that similar macromolecular pathways are required for the proper functioning of clocks. Despite these important findings, in no case is the time-keeping mechanism known. A significant limitation is the lack of identified clock components. Recent evidence indicates that the Drosophila period (per) protein (PER) is a bona fide """"""""gear"""""""" in a circadian clock. The main focus of this proposal is to elucidate how PER activity contributes to the mechanism that measures or tells time. The experimental strategy is based on our recent studies demonstrating that PER is (i) progressively phosphorylated through a circadian cycle and (ii) contains a novel dimerization motif termed PAS common to several transcription factors. First, the levels of the different isoforms of PER will be precisely measured as a function of time. Furthermore, the relationship between PER phosphorylation and dimerization will be investigated. These studies will also include a collaborative effort to investigate the components that interact with PER in wild type flies and in the recently identified arrhythmic mutant timeless. Second, the phosphorylated status of PER will be determined after perturbing the phase of the clock. In related studies, we will explore the hypothesis that temperature influences the dynamics of PER oligomerization. Third, phosphorylation sites on PER and kinases that phosphorylate PER will be identified. Site-directed mutagenesis of these sites should result in animals with altered behavioral rhythms. It is anticipated that elucidation of the biochemical activities that underlie Drosophila circadian clocks will provide a better framework for understanding and manipulating human clocks.
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