Time Flies for Drosophila

sequence of per was diminished. Were PER and TIM Circadian rhythms, based upon a 24 hr cycle, function acting as competitive inhibitors of heterodimerization by in both eukaryotes and prokaryotes to keep the organism sequestering dCLK, or were they preventing the dCLKin tune with its environment. Endogenous cell-autonoCYC heterodimer from binding DNA? Lee et al. found mous clocks control daily molecular cycles, which manithat when dCLK and CYC were mixed together with PER fest themselves as physiological and behavioral oscillaand/or TIM in vitro, the same amount of CYC was bound tions. Environmental stimuli such as light–dark (LD) or to dCLK as when PER and TIM were not present. These temperature cycles entrain, or synchronize, these celluresults suggest that PER and TIM do not disrupt the lar clocks to the outside world. The molecular basis for dCLK-CYC complex, but instead associate with dCLKcircadian clocks in various organisms has been reviewed CYC to form trimers or tetramers that have a diminished extensively (Dunlap, 1999). Transcriptional feedback ability to bind DNA. As PER or TIM can interfere with loops have emerged as a common theme in molecular the dCLK-CYC complex individually, it is possible that clocks from cyanobacteria to mice. This minireview foPER or TIM monomers have a role in regulation of the cuses on the recent progress in the field of clock refeedback loop once inside the nucleus. Little is known search in Drosophila melanogaster, which has been the about the state or fate of these proteins following nuclear most productive for circadian studies to date. The rate translocation. Furthermore, the precise cellular expresof discovery is likely to increase dramatically with the sion pattern of dCLK or CYC is not known, and it would long-awaited release of the fly genome sequence and be reassuring to find that they are present in the same the availability of genomic tools such as DNA microarcells as PER and TIM, and more importantly in cells rays, which will allow for comprehensive definitions of known to control rhythmic behavior. clock-controlled gene expression. Recent research usAnother interesting question is how PER and TIM coning Drosophila has identified several components of the tribute to the antiphasic cycling of dClk. Recent progress molecular clock and, of course, raised many questions toward answering this has come from examining dClk regarding their mechanism of action. The circadian clock mRNA levels in various clock mutant backgrounds, as appears to be composed of multiple feedback loops, dClk cycling may also be regulated by an interplay of and the search for proteins that transmit environmental PER, TIM, CYC, and dCLK. dClk mRNA is upregulated information to the clock, as well as the timing signals from in dClk or Cyc single mutants, downregulated in per null the clock outward to cellular functions, is underway. flies, and upregulated again in per and dClk or Cyc Interlocked Feedback Loops double mutants (Glossop et al., 1999). This suggests For a few years the clock looked relatively simple: the that dClk may act as its own repressor. However, it is core transcription/translation feedback loop is depennot known whether dClk represses its own transcription dent upon levels of period (per) and timeless (tim) mRNA directly, or whether the repressor is an unidentified facrising during the day and peaking in the early evening. tor whose expression is dependent upon the dCLK-CYC Transcription of per and tim is activated by the heteroheterodimer (Glossop et al., 1999). There must also be dimer of dCLOCK (dCLK) and CYCLE (CYC), two basic a separate dClk activator(s) that is independent of PER, helix-loop-helix-PAS (PER-ARNT-SIM) transcription facdCLK, and CYC (see Figure 1); otherwise, high levels of tors (Darlington et al., 1998; Hogenesch et al., 1998). dClk mRNA would not occur in the absence of PER and In the evening, rising levels of PER-TIM heterodimers dCLK or CYC. Intriguingly, expression of cryptochrome translocate into the nucleus and downregulate their own (cry), which is involved in entrainment of behavorial activation, thus establishing oscillating levels of PER rhythms by light, cycles in phase with dClk, and cry and TIM (see Dunlap, 1999 and references therein). As (Emery et al., 1998) and dClk (Glossop et al., 1999) mRNA PER and TIM are not likely to directly bind DNA, this levels are affected the same way in various clock mutant deactivation presumably occurs by inhibiting the dCLKcombinations. Therefore, cry is likely to be regulated by CYC heterodimer. Precisely how PER and TIM inhibit the same mechanism as dClk. their own transcriptional activation by the dCLK-CYC New Spoke in the Wheel complex has been unclear until recently. Now that your head is spinning thinking about the interWork by Lee et al. (1999) has begun to fill in the gaps locked feedback loops within the circadian oscillator, it in our understanding of the actual mechanics of the is time to add a new player: vrille (vri). Most of the fly dCLK-CYC/PER-TIM feedback loop. Previous research clock components have been identified in forward gesuggested that dCLK and CYC activate per and tim by netic screens, but genomics approaches are now con-