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The tim gene, which we recently cloned (1), is essential for the production of circadian rhythms in Drosophila (2, 3). Molecular data indicated that TIM protein may be required at a specific time of day to allow accumulation and nuclear localization of the PER protein (2-4), so we determined if expression of the tim gene showed temporal regulation. We examined the expression in heads of adult Drosophila for the following reasons: (i) The clock is known to be located in the head (5). (ii) Oscillation of per RNA was first demonstrated in adult heads, although subsequently it was shown to occur in most body tissues as well (6, 7). (iii) All effects of tim on per RNA and PER protein have been studied in adult heads (2-4).
Adult flies were maintained in the presence of 12-hour light: 12-hour dark cycles (LD 12:12), and the amount of tim RNA in the heads was measured at 4-hour intervals over 4 days (Fig. 1). (Figure 1 omitted) We found that tim RNA quantities oscillated during the course of LD12:12, with peak quantities corresponding to the end of the day and lowest quantities to dawn. Oscillations of per RNA have the same phase (2, 6, 7). The amplitude of the variation in tim RNA quantities appeared similar to that reported for Per (2, 6). On some days there was as much as a 15-fold difference between peak and trough amounts (Fig. 1B, day 1).
Oscillations of per RNA persist in constant darkness and are, therefore, considered a circadian rhythm (6), and oscillations of the RNA encoded by the frequency (frq) clock gene in Neurospora also persist in the absence of environmental signals (8). We studied the expression of tim and per RNA in wild-type and per sup S (short-period mutant) flies under free-running conditions and found that oscillations of both per and tim RNA persist in constant darkness with indistinguishable periods, phases, and amplitudes (Fig. 2). (Figure 2 omitted) The plot of tim RNA oscillations is essentially superimposed on the curve displaying per RNA cycling. Whereas both RNAs cycle with a ==23-hour periodicity in the wild type, they cycle in per sup S with a 17- to 18-hour period (Fig. 2). The amplitude of the oscillations in wildtype and...