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The period gene (per) is required for Drosophila melanogaster to manifest circadian (congruent to 24 hr) rhythms. We report here that per protein (PER) undergoes daily oscillations in apparent molecular mass as well as abundance. The mobility changes are largely or exclusively due to multiple phosphorylation events. The temporal profile of the classic short-period form of PER (PERS) is altered in a manner consistent with the mutant strain's behavioral phenotype. As changes in abundance and phosphorylation persist under constant environmental conditions, they reflect or contribute to a free-running rhythm. We suggest that the phosphorylation status of PER is an important determinant in the Drosophila clock's time-keeping mechanism.
We report the in vivo characterization of the Drosophila CLOCK protein (dCLOCK), a transcription factor that is required for the expression of the circadian clock genes period (per) and timeless (tim). dCLOCK undergoes circadian fluctuations in abundance, is phosphorylated throughout a daily cycle, and interacts with PER, TIM, and/or the PER-TIM complex during the night but not during most of the day. Our results suggest that PER and TIM participate in transcriptional autoinhibition by physically interacting with dCLOCK or a dCLOCK-containing complex. Nevertheless, in the absence of PER or TIM, the levels of dCLOCK are constitutively low, indicating that PER and TIM also act as positive elements in the feedback loop by stimulating the production of dCLOCK.
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