This proposal addresses the molecular control of biological clocks in individual organisms, tissues and single cells. Circadian rhythms have been demonstrated to regulate many processes in all organisms in which they have been examined. Recent progress in several model systems has identified the presence of """"""""clock"""""""" genes and proteins that represent the molecular components of these ubiquitous biological clocks. This proposal is concerned with understanding how these clock genes and proteins are regulated and how they contribute to the circadian organization of the organism at the tissue, cellular and molecular level. We plan to employ model systems that are tractable both genetically and molecularly in order to gain novel insights into the generation and utilization of circadian clocks. Given the ubiquitous nature of these clocks, knowledge gained in one particular system will have a broad impact. We have generated strains of transgenic Drosophila that contain the period clock gene fused to the firefly luciferase gene. We have used these strains to create an assay where we can monitor per gene transcription for the first time in a living animal, and this assay has already revealed novel features of per transcription that were previously unappreciated. We propose to employ this powerful assay to investigate the pattern of per transcription in live animals and to identify different tissues that contain autonomous circadian clocks. Furthermore, we will generate single cells from circadian tissues and use these to analyze clock regulation at the single cell level. These experiments will provide substantial insight into the circadian organization throughout the animals, as well as how circadian information is generated and transduced. We will gain novel information on the interaction between phototransduction and circadian-regulatory pathways, which in turn will broaden our understanding of how circadian clocks are integrated into cellular regulatory networks. Given the ubiquity of circadian-regulated physiology, the identification of common clock components and pathways will have a significant impact on understanding the pacemaker mechanisms and malfunctions associated with known features of human well-being.
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