The long-term objectives of this project are to identify the circadian clock and understand its mechanism at the cellular level. The circadian (circa, about; ides, one day) biological clock is a physiological mechanism responsible for the timing, on a daily basis, of many fundamental processes, such as gene expression and cell division. In higher organisms, it also regulates blood hormone levels, for example, and more complex phenomena such as activity and alertness. Its importance in human physiology rests on its involvement in the timing of such processes, which can be disrupted environmentally, as in jet lag or shift-work scheduling. Endogenous malfunctions are also hypothesized, and have been associated with the pathology of certain depressions. The biological structure of the circadian clock is not known. We strongly believe that studies of well-chosen unicellular organisms, which can be easily and relatively inexpensively automated to provide statistically meaningful data, may be used to gain basic knowledge of general validity. Clocks in simple organisms share many of the properties of human clocks, and fundamental knowledge from any organism is commonly found to be applicable to others.
The specific aims of this proposal are to elucidate in Gonyaulax polyedra how the effects of (1) light (particularly in relation to color) nd (2) biomolecules affecting circadian rhythms (particularly endogenous substances), are transduced to and interact with the cellular clock mechanism. In the later study, we hope to be able to isolate, identify and locate cellular components of the clock mechanism itself. In order to carry out these projects we plan to develop new and versatile instrumentation allowing for the control of experimental conditions, along with data acquisition and analysis on a long term basis. Action spectra will be determined in several protocols for different kinds of photoresponses of the circadian system: period length, phase shifting by light pulses, self selection of cell position in a light gradient and self selection for phasing in light cycles of different colors. Both narrow band filters and the large spectrograph (Okazaki, Japan) will be used. Creature and gonyauline (a newly) discovered molecule isolated from Gonyaulax) will be studied in relation to their ability to speed up the circadian clock, in view of determining its mechanism. The kinetics of uptake of these molecules will be determined, along with their metabolic fates and associated enzymes. Radiolabeled creatine and gonyauline will be used as probes to identify cellular sites and molecular components of the clock, based on binding studies. We will also screen for other substance found in the cells and medium which may play a role in cell communication, circadian period, and patterns of cellular aggregation and motility.
