The endogenous circadian clock present in the suprachiasmatic nucleus (SCN) regulates a number of neuronal, and subsequent locomotor and physiological functions in vertebrates. Factors that determine the synchronization of this clock appear to be transduced to the SCN from a number of neuronal sites, including the Pineal gland. Preliminary experimental studies strongly support a model in which a cAMP-dependent signal transduction pathway regulates the circadian clock by modulating melatonin synthesis in the Pineal gland. However, further studies are required to elucidate the biochemical and molecular basis for integration and feedback regulation of the melatonin and L-Dopa/dopamine pathways involved in neuronal signal transduction. A combination genetic/molecular approach has been successfully exploited to identify and characterize gene products that generate and maintain the circadian clock in Drosophila. The Drosophila """"""""Per"""""""" gene was identified by these genetic methods, and the gene product was subsequently isolated by cDNA cloning techniques. More recently, similar genetic studies have identified a candidate locus in mouse (the """"""""clock"""""""" gene). However, to date, most of the gene products specifying the circadian clock in mouse and other vertebrates have not been isolated, or characterized. Low stringency library screening of rat SCN cDNA libraries with Drosophila """"""""per"""""""" gene product probes, or PCR amplification with degenerate oligonucleotide primers designed using highly conserved sequence information from Drosophila """"""""per"""""""" gene product should result in the isolation of the homologous vertebrate regulatory gene products. PCR- based techniques will be exploited to isolate SCN-specific kinases that most likely regulate the circadian signal transduction pathway. This approach in combination with the cDNA library screening strategies proposed in this study should result in the identification and isolation of a number of the components of the vertebrate (rat) circadian clock.
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