Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous control and organization over time. 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 and to the treatment of mental illness rests on the ubiquity of its influence on human mental and physiological processes. These range from circadian changes in basic physiological functions to the clear involvement of circadian rhythms in human work-rest cycles and sleep. Human psychiatric illnesses known to be a direct result of clock malfunction include common forms of manic-depressive illness and insomnia. Extensive research in the past has demonstrated the extent and significance of clock control of gene expression and enzyme activities, but in general little is known regarding how clocks control the metabolism of the cells in which they operate. One salient aspect of this regulation is clock control of mRNA abundance and protein abundance via circadian regulation of DNA transcription and RNA translation respectively. Molecular genetic techniques have been developed to facilitate the comparison of two nearly identical RNA populations, and the isolation of individual RNA species unique to only one of the two populations. In preliminary experiments, we have used these techniques in the context of circadian control of gene expression to identify three genes that give rise to mRNAs whose levels are unequivocally under circadian clock control. We propose to extend these studies to all phases of the clock cycle in order to evaluate the extent of clock control of gene expression throughout the circadian cycle. Additionally, recent data have established a precedent for clock control of mRNA translation. We propose to evaluate the extent of this type of control by carrying out random translational fusions between genomic DNA and an enzymatic reporter gene, bacterial luciferase. These studies are a first step toward mapping out the topology of circadian regulation of gene expression, and toward developing a system where clock control can be studied on a broad scale at the molecular level.
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