GENE EXPRESSION IN THE MAMMALIAN CIRCADIAN SYSTEM

Dartmouth College, Hanover NH

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Abstract

DESCRIPTION (Applicant's abstract): Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous temporal control and organization known as a circadian rhythm. 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 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 has demonstrated the significance of clock control of gene expression: the circadian timing loop itself comprises a transcription-translation based feedback loop and also a major aspect of circadian OUTPUT from the clock includes clock-controlled gene expression. In Specific Aim 1 we will array the BMAP UniGene set, isolate brain tissues corresponding to those used for the BMAP at subjective dawn, noon, dusk, and midnight, and use these RNA to make probes and determine using microarrays the global changes in gene expression occurring in large brain regions as a function, of time-of day. The dominant circadian pacemaker in mammals is in the anterior hypothalamus, in paired bundies of neurons located on top of the optic chiasm, the suprachiasmatic nuclei or SCN. In mice this clock tissue occupies about 1 ul, but it is sufficiently well defined that it can be identified in non-fixed tissue. We have isolated SCN tissue at different times of day and have generated time-of-day specific libraries from the mouse SCN. In Specific Aim 2 we will carry out suppression subtractive hybridization to enrich for the contribution of rare cDNAs from rare transcripts, and use microarrays to compare the genes in the UniGene set with those found exclusively in the SCN. This will allow the identification of SCN-specific genes if any exist and allow the creation of SCN-gene chips. In Specific Aim 3, using these SCN and BMAP UniGene microarrays, we will to characterize the time-of-day specific gene expression pattern in the SCN. Recognizing that there are anatomically discrete and functionally described microscopic subregions within the SCN, in Specific-Aim 4 we will assess the feasibility of combining Laser Capture Microscopy, T7 RNA amplification, and microarrays to examine clock-controlled gene expression in the dorsomedial vs. ventrolateral and anterior vs. posterior subregions of the SCN.