Function of theperiod and thetimeless genes in the Drosophila circadian clock control

摘要

The period (per) gene is a central component in the circadian pacemaker of Drosophila melanogaster. per undergoes circadian oscillations at the molecular levels of both mRNA and protein. The period protein (PER) may be involved in the feedback regulation of its own gene's oscillatory transcription. The newly identified timeless (tim) gene may be a second clock component of Drosophila. The biochemical function of both PER and TIM remains elusive.;Chapter 2 of this dissertation reports that constitutive overexpression of PER through a heterogeneous promoter represses the endogenous per gene's transcription and its cycling, providing direct evidence in supporting the autoregulatory function of PER and suggesting that the autoregulation may be a negative feedback loop. This feedback loop is likely to be cell autonomous, as cycling of the endogenous per gene is only inhibited in the cells expressing this transgene but not in other cell types.;Posttranscriptional mechanisms may regulate PER's biochemical function. PER undergoes progressive phosphorylation during its circadian cycling. The phosphorylation could be a regulatory mechanism for PER's biochemical activity, nuclear translocation, and/or protein stability. Chapter 3 contains some characterizations of PER's phosphorylation, in the attempt to map the time-specific phosphorylation sites on PER.;It has been difficult to find clock mutants by genetic screens, due to the nonspecific effects of developmental and behavioral deficit. Thus I took a biochemical approach in searching for new clock genes, particularly those that may interact with PER physically. Chapter 4 reports the identification of TIM as the major PER-associating protein in fly extracts, the stoichiometric characterization of the PER-TIM heterodimer, the characterization of TIM's circadian cycling and phosphorylation, and the discovery of a selective degradation of TIM by light treatment and the subsequent release of PER from the dimeric complex. The results indicate that the PER-TIM heterodimer is an important functional unit of the clock mechanism and it can explain the reciprocal autoregulation of both proteins on both gene's transcription. The results also suggest that light-induced TIM degradation may be responsible for the entraining and phase-resetting effects of light on the Drosophila circadian clock.