Molecular & Cellular Mechanisms of Circadian Rhythms

Hastings, J.

Abstract

The long-term objective of this research is to elucidate the basic cellular and biochemical mechanism(s) responsible for endogenous daily (circadian) rhythms. Such rhythms may continue under constant conditions for weeks (or even longer). Circadian control is therefore is not due simply to day and night cycles, even though the biological clock is reset by light and dark (as when we cross time zones). The circadian clock controls fundamental phenomena, such as gene expression, cell division and motility; it times bodily functions, sleep-wake cycles, and is involved in drug tolerance and drug efficacy as well as jet lag and aging. The circadian system is thus of fundamental importance in human physiology and medicine. Many basic questions relevant to our understanding the clock in human physiology can be addressed in lower organisms, including the unicellular dinoflagellate, Gonyaulax, where the bioluminescence rhythm is explicitly mirrored by the activities and amounts of two proteins involved in luminescence: luciferase and luciferin binding protein (LBP), which are contained in specific organelles. We found that the synthesis of LBP is under circadian control, and that the number of the organelles varies with the time of day.
The specific aims of the project are: (1) To determine the mechanism of circadian control of the synthesis of one of these proteins, LBP. We will look for a time-specific cytoplasmic factor affecting the rate of in vitro translation of the LBP message and, through the isolation and sequencing of a full length LBP cDNA, seek information concerning its binding site. The cellular localization of this message will be examined by in situ hybridization. (2) To determine the mechanism of circadian regulation of the degradation of LBP. A specific protease active in breaking down this protein will be isolated, purified and characterized with regard to substrate specificity and circadian control. The circadian activation of proteolytic activity, as well as the daily disappearance of the bioluminescent organelle by some timed cytoplasmic signal will be explored. (3) To isolate and determine structures of genomic regions corresponding to the LBP cDNA. (4) To identify and characterize the nature of circadian control for another night phase protein, a day phase protein, and one or more whose activity is not circadian regulated. Control mechanisms for these proteins will be studied following the approach used for LBP.