reproduced verbatim): One of the most intriguing questions in biology is how organisms keep circadian time in the absence of daily environmental cues. Essentially all organisms (microbes, plants, and animals) use an endogenous timekeeping mechanism, or clock, to activate various physiological and behavioral rhythms at the appropriate time of day. Our understanding of the clock is particularly important for human health and well being since basic physiological activities including sleep, endocrine and cardiovascular function and drug tolerance are rhythmically controlled. Clock dysfunction is related to several common mental disorders such as manic-depressive illness, seasonal affective disorder and insomnia. Extensive research has shown that the timekeeping mechanism of the clock, called a circadian oscillator, is comprised of an autoregulatory feedback loop in gene expression. One of the most extensively characterized feedback loop oscillators is that from the fruit fly Drosophila melanogaster. Since the Drosophila feedback loop oscillator uses essentially the same set of genes as the mammalian circadian feedback loop oscillator, what we learn about the mechanism underlying the Drosophila feedback loop function may be directly applicable to function of the mammalian feedback loop. We recently showed that the Drosophila circadian oscillator is comprised of two interlocked negative loops: the well studied per/tim feedback loop in which per and tim transcription is activated by dCLK-CYC and repressed by PER-TIM, and a novel dClk feedback loop in which dClk transcription is repressed (directly or indirectly) by dCLK-CYC and de-repressed via PER-TIM interactions with dCLK-CYC. From this data, we make several predictions about the molecular interactions needed to regulate rhythmic dClk expression. These predictions form the basis for the specific aims of this application.: 1) identifying the factor(s) responsible for dClk activation, 2) determining how dCLK and CYC repress dClk transcription and 3) determining whether PER-TIM interactions with dCLK-CYC are capable of de-repressing dClk transcription.
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