Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous temporal control and organization. The cellular machinery that generates this ability is known collectively as the biological clock. The importance of a detailed understanding of the circadian clock to our understanding of physical and mental health rests on the ubiquity of its influence on cellular and organismal processes. The long term goal of this work is to be able to describe, in the language of genetics and biochemistry, the feedback cycle that comprises this ubiquitous and important aspect of cellular regulation. Historically, clocks have been difficult to study, in part because so many factors that affect the clock do so through pleiotropic rather than direct means. Additionally in terms of """"""""clock biochemistry"""""""", there is a problem in distinguishing between the clock mechanism and clock regulated processes, i.e. between the gears of the clock and the hands of the clock. Thus, a major goal in chronobiology has long been to unequivocally establish the identity of a component of the clock. By doing so, one could then begin to dissect the feedback cycle from the inside. During the course of the past granting period we succeeded in identifying a component of the clock, the frq gene. The level of the frq transcript follows a rhythm of period length defined by the allelic state of frq itself (it is self-regulatory), stopping the frq transcript cycling stops the clock, and releasing frq restarts the rhythm from the point defined by a negative feedback cycle wherein frq represses it's own synthesis. These data, in the context of frq genetics, establish it as a gear in the clock. We now hope to unravel the feedback loop by studying the regulation of frq - finding and characterizing the cellular factors that regulate frq will identify factors upstream from frq in the feedback loop, and finding the immediate factors with which FRQ interacts will identify the downstream factors. Specifically, (1) we will use a combination of genetic and biochemical methods to determine how transcriptional regulation of frq is effected, both autonomously via the clock and exogenously by factors such as light that reset the clock. We will characterize these factors and study their regulation. (2) We will use yeast two-hybrid technology to identify and characterize genes encoding the protein partners with which FRQ interacts in the operation of the clock, and we will continue to characterize the domain structure and cell biology of FRQ itself through reverse genetics and the use of FRQ-specific antisera.
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