The overall objective of the proposed research is to gain insight into molecular components of the circadian clock mechanism in the suprachiasmatic nucleus (SCN) of the hypothalamus. Recent findings have unveiled the identified and distribution of mammalian orthologs of known clock genes in Drosophila, namely mPer1, mPer2 and mPer3. While the specific functions of mammalian Per genes have not been fully defined at the biochemical, physiological, and behavioral level, evidence for their sequence homology and for their rhythmic expression within the SCN has led to the hypothesis that the central organization of the mammalian clock mechanism and its core components coincide with the convergent evolution of circadian clocks in other diverse species. To address this hypothesis, experiments will: 1) utilize cDNA microarrays to obtain a more comprehensive picture of transcripts that are regulated in a circadian fashion (clock and clock-controlled genes); 2) develop real- time, luciferase-reporting of circadian gene expression in the SCN clock mechanism; and 3) examine the roles of Per, Cry, Tim and other candidate clock genes in the SCN clock mechanisms by determining whether rhythmicity in SCN circadian outputs is disrupted when expression of these genes is experimentally fixed at constant levels. Extent immortalized lines of SCN cells developed in my lab will be exploited in analyses throughout this project because their genetic properties convey distinct advantages in identifying and assaying genes that are rhymically regulated. Expression of putative clock elements in immortalized SCN cells will be maintained at constant low or high levels using antisense oligonucleotides or over-expression constructs. By providing a tool for real-time measurements of clock and clock- controlled gene expression and further elucidation of the molecular components of the circadian timekeeping mechanism in mammals, these studies have applied relevance for developing strategies in the treatment diagnosis and understanding of disorders in human mental health and performance that result from internal desynchronization of body processes, including affect disorders, dementia and physiological decline during aging.
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