On the Mechanism of Temperature Independence in a Biological Clock.

Introduction.-Diurnal rhythms which persist under constant conditions have been found in a variety of plants and animals (see Biinning;l Pittendrigh2). Most of the evidence points to the conclusion that diurnal rhythmicity is endogenous and results from oscillations of physiological processes of one sort or another. An unusual feature of many rhythms, however, is their temperature independence. In instances where study has been directed toward the temperature characteristics (Wahl;3 Brown and Webb;4 Pittendrigh;5 Bruce and Pittendrigh;6 Buihnemann7), it has been found that the period, or duration of a cycle, is unaffected or only slightly affected by temperature changes. The way in which this is achieved in organisms which are otherwise markedly temperature-sensitive is not known. It is possible that some uncontrolled environmental variable might be involved, i.e., that there is an exogenous factor which plays a role in determining the period in rhythmic organisms. Another possibility is that temperature compensation is incorporated as a part of the rhythmic mechanism. In either case, it may be noted that temperature independence constitutes support for the idea that a diurnal rhythm could be related to an accurate biological timing device, or clock. If, in fact, the rhythms are related to a chronometer mechanism, the significance of temperature independence is clear. We have recently demonstrated (Hastings et al.8) that the dinoflagellate Gonyaulax polyedra possesses a diurnal rhythm of luminescence which persists under conditions of constant low light and constant temperature. Since Gonyaulax is a unicellular form which may be grown readily in large quantities and studied biochemically, it is an advantageous material for the study of rhythmicity. In addition, the quantitative measurement of light intensity provides an easy and accurate method for following rhythmic changes. The experiments reported here describe the effect of temperature upon the luminescent rhythm. The results suggest that temperature independence is achieved by means of a compensation mechanism. Materials and Methods.-Gonyaulax polyedra is an armored marine dinoflagellate which is both photosynthetic and luminescent. This organism was isolated from a net sample collected off the pier at Scripps Institution of Oceanography and has been maintained in a unialgal culture in a medium composed of 75 per cent filtered sea water, 2 per cent soil extract, 2 X 10-3 M KNO3, 2 X 10-4 M K2HPO4, 6 X 10-6 Ml FeCl3, and 2.7 X 10-5 M ethylenediamine-tetraacetic acid (EDTA) at pH 8.0-8.2. Under optimal conditions of light and temperature, a growth rate of about one division per day has been measured. Bacteria-free cultures have been found to have a reduced viability and growth rate, apparently due to the lack