Daily rhythms of physiology and behavior in most life forms are driven by internal circadian pacemakers which use the physically fluctuating stimuli to measure the passage of time. Disruptions of biological clock cause severe health problems in our society, including jet-lag, insomnia, and shiftwork-related illnesses. Similarities in molecular mechanisms underlying central pace-making functions in mammals and fruit flies (Drosophila melanogaster) suggest that Drosophila is an excellent model system to study the biological clock. However, the mechanisms by which the central oscillators regulate the overt physiological and behavioral rhythms (output pathways) are largely unknown. Our recent studies identified the first circadian messenger; it is a neuropeptide, pigment-dispersing factor (PDF). Interestingly, the core-clock transcription factors activate pdf expression in the pacemaker cells. These studies indicate that any factors affecting normal PDF production can cause aberrant rhythms. Given the importance of the PDF in the regulation of clock output, the objectives of this application are to examine transcriptional regulatory mechanisms of the pdf gene in Drosophila by performing the following specific aims: 1. Dissect pdf promoter. This will be accomplished by site-directed mutagenesis of the pdf enhancer and subsequent functional in vivo assay. Yeast one-hybrid and EMS-mutagenesis screen will be employed to find pdf transcriptional regulators. 2. Characterize a new rhythm mutant. The gene disrupted by a P-element insertion will be isolated and characterized molecularly. 3. Clone the pdf gene in D. virilis (Dv-pdf). Using RT-PCR or library screening, we will isolate the Dv-pdf gene and its regulatory elements. The Dv-pdf promoter will be assayed heterologously in D. melanogaster.
