Daily and seasonal rhythms are maintained by self-sustaining clocks that are present in many organisms, from bacteria to humans. Disruption of these circadian rhythms can lead to a variety of sleep disorders in humans. The timing mechanism is responsible for maintaining biological rhythms even in the absence of environmental cues. In the fruitfly Drosophila melanogaster examples of behavioral rhythms include (1) eclosion, emergence from the pupal case as adults, and (2) locomotor activity, movement of the fly in its surroundings. Abolishing clock function results in behavioral and molecular arrhythmicities that are not responsive to environmental cycles. Understanding how these rhythms are maintained in flies will shed light on how our own biological clocks contribute to our circadian rhythms and overall homeostasis. The long-term goal of this proposal is to understand the molecular mechanisms involved in the regulation of circadian output. Although many of the pacemaker molecular components and their mutants have been isolated, only a few clock-controlled genes (ccg's) have been identified to date and their function still remains to be resolved. An enhancer trap screen in Drosophila using a P element carrying luciferase (luc+) to assay for bioluminescent rhythmicity should identify novel clock-regulated targets as well as novel accessory molecules to the pacemaker. By including c-myc epitope tags and green fluorescent protein (GFP) upstream of the luc+ cDNA, the spatial expression patterns of any interesting oscillating lines isolated in the screen can be easily determined. Rhythmic lines will be further screened for defects in eclosion or locomotor activity, and dependence upon pacemaker genes for their function. From the most interesting lines the genes will be isolated and their function characterized.
