A new circadian clock mutant of Neurospora crassa has been isolated, whose most distinctive characteristic is the complete loss of temperature compensation of its period length. The Q10 of the period length was found to be equal to about 2 in the temperature range from 18 degrees to 30 degrees C. The period length was also found to be dependent on the composition of the medium, including the nature and concentration of both the carbon source and the nitrogen source. Although the rate of the clock and the growth rate were directly related when affected by varying the temperature, they were inversely related when altered by changing the composition of the medium. Therefore, the mutation has not simply coupled clock rate to growth rate in this strain. The mutation maps to the frq locus, where seven other clock mutations previously studied also map. Therefore, this mutant has been called frq-9. Since several of the other frq mutants show partial loss in temperature compensation, it is suggested that the frq gene or its product is closely related to the temperature compensation mechanism of the circadian clock of Neurospora.

Temperature compensation of circadian period length in 12 clock mutants of Neurospora crassa has been examined at temperatures between 16 and 34°C. In the wild-type strain, below 300C (the "breakpoint" temperature), the clock is well-compensated (Qlo -1), while above 30°C, the clock is less well-compensated (Qlo -1.3). For mutants at the frq locus, mutations that shorten the circadian period length (frq-4,frq-2,frq4, and frq-6) do not alter this temperature compensation response. In long period frq mutants (frq-3, frq-7, frq-8) 2 To whom reprint requests should be sent. the rhythm is less than one. An "over-compensation" explanation seems the most plausible for this fact (10). (b) Circadian systems can be phase-shifted, or reset, by temperature steps or temperature pulses (23). This indicates that the clock recognizes temperature differences and adapts to new steady-state temperatures. In this regard, Pavlidis et a!. (15) have proposed a mathematical model for temperature compensation which also accounts for a large pan of the data on phase shifting by temperature steps and pulses. (O) For a number of organisms, such as Euglena (2) and Neurospora (19), the apparent temperature independence exists only within a limited temperature range, and outside this range the circadian system becomes significantly more temperature-dependent. This suggests that a temperature compensation mechanism works efficiently only within certain physiological or biochemical limits.In recent years, several laboratories have turned to genetic analysis to help elucidate the molecular mechanisms underlying circadian clocks. Single gene mutants that alter the free-running period length of the rhythm have now been found in four organisms-Drosophila melanogaster (12), Drosophila pseudoobscura (17), Chlamydomonas reinhardi (1), and Neurospora crassa (6). It is of interest to determine whether the mutations that alter period length in these strains also affect temperature compensation of period length. In C reinhardi, the mutants have normal temperature compensation (1) while in D. melanogaster some small differences in the Qlo values have been found (11).In N. crassa, several analyses of the temperature responses of the wild-type (ie. band) strain have been carried out. Temperature steps and pulses produce phase shifts similar to those seen in other organisms (8). In addition, the period length of the rhythm is temperature-compensated, but only within a limited temperature range. Sargent et al. (19) found that between 18 and 250C, the Neurospora clock has a Qlo of 0.95, i.e. it is slightly over-compensated, while in the range of 25 to 350C, the Qlo increases to 1.2.This distinction between a low temperature range, in which the clock is well-compensated, and a high temperature range, in which it is less well-compensated, has recently been confirmed (13), although small differences in the "break-point" temperature between the high and low range (30 versus 250C) and the Qlo in the high temperature range (1.3 versus 1.2) were observed.Th...

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Neurespora c issa ha been utlzed extensively in the study of (5,9,13), and the genetic analysis of mutations affecting period lengths (6-8). In addition, circadian rhythms have been found in a variety of biochemical parameters, including pyrdine nucleotides (1), 5'-AMP levels (2), fatty acids (12), RNA and DNA synthesis (11), and several enzymes (10).The present study arose from the need to define the Neurospora system more fully and to develop new techniques for further analysis of the clock. Difficulties in obtaining sufficient culture 'This work was supported by National Institute of General Medical Sciences Grant GM-22144.material for the assay of certain biochemical parameters, the need to develop a reliable method of pulsing drugs, and the need to know more about the physiology of the Neurospora clock system were among the factors considered in developing this investigation. Specifically, we wished to answer the questions of whether it was possible to determine the phase of the clock in a small portion of the mycelium rather than in the entire culture, and additionally, whether there is a functional clock in hyphae behind the growing front.The latter question is significant in light ofreports ofNeurospora aging (20, 21), which have indicated that the growing tips of a culture are metabolically active, contain large numbers of nuclei and active mitochondria, and show considerable cytoplasmic streaming, while, even a short distnce behind this growing front (1-2 cm), the hyphae are metabolically less active, filled with fat globules and vacuoles, contain few nuclei or mitochondria, and show little cytoplasmic streaming. Because the growing front must differentiate to form aerial hyphae and conidia at the appropriate time in the circadian cycle, it is evident that temporal information is available in this region. The region behind the growing front remains essentially unchanged, undergoing no significant differentiation during subsequent circadian cycles. Therefore, the existence or nature of temporal information in this older portion of the culture cannot be directly determined simply by observing the culture.Preliminary studies in this laboratory indicated that small portions of mycelium cut from the growing front and transferred to fresh medium showed a circadian rhythm with a phase essentially the same as that shown by the growing front of the intact original culture (3). We report here a method for assaying the phase of mycelium in any region of the culture and a detailed analysis of the results of such assays. MATERIALS AND METHODSStains. The band,csp-l strain of Neurospora crassa (obtained from S. Brody) was used in all experiments. The csp-l mutation prevents the separation of conidia from the hyphae and thus prevents spreading of conidia during transfer operations (18).Medium. Acetate-casamino acids medium (Vogel's [19] salts, 1.2% sodium acetate, 0.05% casamino acids, 2% agar) was added to race tubes 220 mm long and 14 mm in diameter (8.3 ml/tube) and to Petri dishes 150 mm in diameter a...