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REVIEWS20 Weller, M. (1979) In Protein Phosphorylation (Lagnado,

J.R., ed.), pp. 112–162, Pion Ltd 21 Karin, M. (1999) How NFkB is regulated: the role of the

IkB kinase (IKK) complex. Oncogene 18, 6867–687422 Wang, D. and Baldwin, A.S. (1998) Activation of nuclear

factor kB-dependent transcription by tumour necrosisfactor-alpha is mediated through phosphorylation ofRelA/p65 on serine 529. J. Biol. Chem. 273,29411–29416

23 Fang, X. et al. (1999) Regulation of BADphosphorylation at Ser112 by the Ras-MAPK signallingpathway by the Ras-mitogen-activated protein kinasepathway. Oncogene 18, 6635–6640

24 Datta, S.R. et al. (1997) Akt phosphorylation of BADcouples survival signals to the cell-intrinsic deathmachinery. Cell 91, 231–241

25 Lizcano, J.M. et al. (2000) Regulation of BAD by cyclicAMP-dependent protein kinase is mediated viaphosphorylation of a novel site, Ser155. Biochem. J.349, 547–557

26 Yaffe, M.B. et al. (1997) The structural basis for 14-3-3:phosphopeptide binding specificity. Cell 91, 961–971

27 Holm, C. et al. (1988) Hormone-sensitive lipase:sequence, expression and chromosomal localisation to19 cent-q13.3. Science 241, 1503–1506

28 Kitamura, T. et al. (1999) Insulin-inducedphosphorylation and activation of cyclic nucleotidephosphodiesterase 3B by the serine/threonine kinaseAkt. Mol. Cell Biol. 19, 6286–6296

29 O’Connell, M.J. et al. (2000) The G2 phase DNA-damage checkpoint. Trends Cell Biol. 10, 296–303

30 Hofmann, I. et al. (1993) Phosphorylation andactivation of human cdc25-C by cdc2-cyclin B and itsinvolvement in the self amplification of MPF at mitosis.EMBO J. 12, 53–63

31 Theroux, S.J. et al. (1992) Signal transduction by theepidermal growth factor receptor is attenuated by aCOOH-terminal domain serine phosphorylation site. J. Biol. Chem. 267, 16620–16626

32 Li, J. et al. (1999) Modulation of insulin receptorsubstrate-1 tyrosine phosphorylation by an Akt-phosphatidylinositol 3-kinase pathway. J. Biol. Chem.274, 9351–9356

33 Williams, M.R. et al. (2000) The role of 3-phosphoinositide-dependent protein kinase 1 inactivating AGC kinases defined in embryonic stemcells. Curr. Biol. 10, 439–448

34 Aguirre, V. et al. (2000) The c-Jun NH2-terminal kinasepromotes insulin resistance during association with

insulin receptor substrate-1 and phosphorylation ofSer307. J. Biol. Chem. 275, 9047–9054

35 Garton, A.J. and Yeaman, S.J. (1990) Identificationand role of the basal phosphorylation site onhormone sensitive lipase. Eur. J. Biochem. 191,245–250

36 Cohen, P. (1999) The Croonian Lecture 1998.Identification of a protein kinase cascade of majorimportance in insulin signal transduction. Phil. Trans.R. Soc. Lond. B 354, 485–495

37 Fiol, C. et al. (1987) Formation of protein kinaserecognition sites by covalent modification of the substrate. Molecular mechanism for thesynergistic action of casein kinase 2 and glycogen synthase kinase 3. J. Biol. Chem. 262,14042–14048

38 Picton, C. et al. (1982) Multisite phosphorylation ofglycogen synthase from rabbit skeletal muscle;phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite forphosphorylation of sites 3 by glycogen synthasekinase-3. FEBS Lett. 150, 191–196

39 Flotow, H. et al. (1990) phosphate groups assubstrate determinants for casein kinase 1 action. J. Biol. Chem. 265, 14264–14269

25 YEARS AGO David Njus described aSearch for the Biochemical Clock in thesepages1. The clock portrayed was the bio-chemical timer for circadian rhythmsand almost everything that we knowabout its molecular inner workings hascome to light since the writing of that ar-ticle. An interesting prediction put for-ward then was that the periodic synthe-sis of a nucleic acid or protein wouldhave no role in timekeeping. Althoughcycling gene and protein activities arenow viewed as central to all circadianclocks, Njus did correctly anticipate that

powerful tools for identifying clock pro-teins would come from newly identifiedmutants of Neurospora and Drosophila.

In the two decades preceding the 1976article, and in the absence of adequatebiochemical approaches, investigatorshad focused on formal properties of circa-dian rhythms. The rhythms were shownto persist in constant environmental con-ditions, and single-gene mutations wereisolated that altered their period, provingthat circadian rhythms were generated by endogenous, self-sustaining clocks1,2.Many unicellular organisms were shownto have circadian rhythms, so it was con-cluded that timekeeping must emergefrom cellular biochemistry1.

Ingenious studies permitted theunderlying mechanism to be examinedwithout any knowledge of its molecular

composition. When maintaining an or-ganism in constant darkness, it wasfound that a single pulse of light couldadvance, delay or have no effect at all onthe rhythm, depending on the time of ex-posure to light in relation to theendogenous oscillation2. This indicatedordered transitions in a chemicalmechanism that reflected subjective (in-ternal) day and night. Circadianrhythms were also found to be tempera-ture compensated; that is, the period ofthe rhythm was uniform over a range ofambient temperatures2. This means thatthe biochemical mechanism is some-how shielded from the more commontemperature-dependent metabolic activ-ities of the clock-containing cell.

This was a period that produced impor-tant anatomical localizations of circadianclocks in animals2. A circadian rhythm innerve impulses persisted in the isolatedeye of a marine mollusc in vitro. Surgicalablation of the pineal gland in sparrowsand the removal of a portion of the brainin certain insects resulted in behavioralarrhythmias that were reversed by thetransplantation of corresponding donortissues. Within the mammalian hypothala-mus, the suprachiasmatic nucleus (SCN)was found to regulate sleep–wake cycles,temperature and hormonal rhythms. Eachstudy provided evidence that a discretetissue contained a clock but serious diffi-culties remained in identifying biochemi-cal components of a circadian oscillator inany of these cells.

Clock mutantsGenetic screens began to reveal clock

mutants in Drosophila in 1971 and in

Life’s 24-hour clock: molecularcontrol of circadian rhythms in

animal cells

Michael W. YoungOur sleep–wake cycles and many other ~24-hour rhythms of behavior andphysiology persist in the absence of environmental cues. Genetic and bio-chemical studies have shown that such rhythms are controlled by internalmolecular clocks. These are assembled from the cycling RNA and proteinproducts of a small group of genes that are conserved throughout theanimal kingdom.

M.W. Young is at the Laboratory of Geneticsand the NSF Center for Biological Timing, TheRockefeller University, 1230 York Ave, NewYork, NY 10021, USA. Email: young@rockvax.rockefeller.edu

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Neurospora in 19733,4. However, the firstscreens were difficult to implement andlimited in scope. For example, theDrosophila screens examined only ~2000mutagenized X chromosomes and re-quired the assessment of eclosionrhythms (the periodic emergence of theadult from the puparium).

Only one gene was derived from thisearly fly work, period (per), but the threeidentified alleles indicated deep involve-ment in the organization of circadianrhythms. One allele shortened theperiod of the eclosion rhythm to 19 h, asecond lengthened the period to 28 hand a third abolished circadian rhyth-micity3. A similar pattern was found inNeurospora, in which the first isolates(short- and long-period mutations)mapped to a single gene, frequency(frq)4. Molecular cloning of both geneseventually showed that each of the mu-tations affected the encoded proteinsPER and FRQ. Arrhythmic mutationseliminated the proteins whereas period-altering mutations changed the aminoacid sequence3,4.

The sequences of the per and frqgenes did not reveal their function.Instead, important clues came from in-vestigating the temporal patterns of ex-pression of both genes3,4. RNAs pro-duced by per cycled in abundance inconcert with the fly’s behavior. In short-cycle mutants, per RNA production roseand fell within a short period. Long-cycle mutants followed suit, with long-period RNA oscillations, and no per RNAcycling was found in the arrhythmic mu-tant. The mutations affected the struc-ture of the PER protein and so theseobservations suggested that PER wassomehow involved in switching its owngene on and off with a circadian rhythm.Similar conclusions were reached fromstudies of oscillating frq RNA levels inNeurospora.

A mechanism for producing the cy-cling gene activity eventually emergedfrom further work with Drosophila as a re-sult of intense genetic screening. Thistime, many tens of thousands of muta-genized chromosomes were analyzedand a simple system for monitoring loco-motor activity rhythms replaced thecumbersome eclosion assays. The genecount rapidly moved to five. Each newgene was, like per, essential for theproduction of circadian behavioralrhythms. Important conceptual advancescame from a new focus on the molecularinteractions among the clock genes andtheir proteins, and from the discovery oforthologous genes in mammals.

TIMELESS, a partner for PEROne of the four new Drosophila clock

genes, timeless (tim), encodes a proteinthat physically associates with PER3,4.Both proteins are produced with arhythm that, in part, reflects cyclinggene activity. The per and tim genes arecoordinately expressed so that theirRNA levels rise and fall together. A muta-tion of any Drosophila clock gene thatadjusts the period length of the behav-ioral rhythm produces correlated shiftsin the phases and periods of both RNArhythms3,4.

In wild-type flies, PER and TIM accu-mulate in nuclei but the loss of eitherprotein restricts the accumulation of theremaining protein to the cytoplasm andeliminates the rhythms of both per andtim transcription3,4. Thus, the proteinsmust heterodimerize to move to the nu-cleus, and nuclear translocation is re-quired to influence per and tim express-ion3,4. Nuclear localization signals havebeen found in both PER and TIM, ashave sequences mediating the for-mation of PER–TIM complexes3.

For PER, heterodimerization dependson two domains: PAS, a ~260 amino acidsequence found in many proteins (seebelow), and PER-CLD, a unique se-quence of ~60 amino acids. PER-CLDpromotes the cytoplasmic localizationof monomeric PER proteins, so its physi-cal association with TIM appears to per-mit nuclear translocation of thePER–TIM complex3. A single amino acidsubstitution in the PAS domain of PER,perL, has been shown to lower the affin-ity of PER for TIM and to delay nuclearlocalization of PER–TIM complexes. Thismutation also produces long-periodmolecular and behavioral rhythms3. Thechange in period length is substantial(~4 h), indicating that the rates of asso-ciation of PER and TIM help to establishmolecular oscillations that occur withina circadian rather than higher-fre-quency range. A similar conclusion canbe drawn from studies of timL1, a long-period mutation affecting one of twoPER-interacting sequences in the TIMprotein5.

dCLOCK and CYCLETwo mutations in Drosophila and a

third in the mouse identified activatorsof per and tim transcription. A behav-ioral screen in the mouse produced asemidominant long-period mutant,Clock (Clk). Homozygotes generate long-period locomotor activity rhythms orare arrhythmic4. Sequencing revealedthat Clock encodes a transcription

factor of the bHLH–PAS family4. Clockis therefore related to PER by virtue of the protein-interaction domain PAS.Inclusion of bHLH sequences allowsdirect binding of CLOCK to DNA, a prop-erty not shared by PER.

Of the two new Drosophila mutations,one affected the orthologous fly genedClock (dClk) and the second affected agene encoding another bHLH–PAS fam-ily protein, referred to as CYCLE (CYC).Either Drosophila mutation produces ar-rhythmicity when homozygous4. Whenproduced in cultured Drosophila cells,the dCLK and CYC proteins will physi-cally associate and activate transcrip-tion from reporters containing the perand tim promoters4,6,7. If the PER andTIM proteins are also both produced toallow their nuclear localization in thesecells, transcriptional activation by dCLKand CYC is suppressed4,6. Complexescontaining various combinations of thefour proteins have been observed invivo, and PER and TIM block DNA bind-ing by dCLK and CYC in vitro7. In cul-tured mammalian cells, CLK and a CYCortholog (BMAL1) activate transcriptionfrom promoter sequences found inmammalian per (Ref. 4), pointing to sig-nificant conservation of an autoregula-tory mechanism from flies to mammals.

Further work with Drosophila mu-tants has indicated an additional role forthe PER–TIM complex. After trans-location to nuclei, strong inhibition ofdCLK–CYC activity requires the produc-tion of nuclear PER that is free of TIM8.The need to assemble, translocate andthen disassemble these complexes de-lays the transition from transcriptionalactivation of per and tim to repression.This conclusion is supported by long-period (33 h) mutations of tim that pro-long PER–TIM associations by ~12 h8.Because PER–TIM complexes are associ-ated with nuclei for ~6–8 h in wild-typeflies, these studies again show how cer-tain mutants expose steps in the mol-ecular cycle that influence its circadianfrequency (Fig. 1).

Molecular basis for entrainmentHow do environmental light–dark

cycles affect this molecular oscillator?That is, how do light and dark changeits phase and period (entrain it)? Lightrapidly eliminates the TIM protein3,4,which appears to be phosphorylatedand subsequently ubiquitinated in re-sponse to light, then degraded by theproteasome9. Light-dependent turnoverof TIM requires the function of aflavoprotein, CRYPTOCHROME (CRY).

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The CRY proteins of plants and an-imals are thought to have evolvedfrom light-activated DNA-repairenzymes, the photolyases10,11. Thephotolyases promote redox reac-tions in response to light, so it isunclear how CRY influences thephosphorylation and/or ubiquiti-nation of TIM.

A Drosophila cry mutant (cryb)blocks light-dependent TIM degra-dation in a cell-autonomous fash-ion4,6,12. In cultured Drosophilacells, light induces a physical asso-ciation of CRY and TIM, whichcould form part of the intracellularmechanism that marks TIM fordegradation6,13. CRY appears to bethe only photoreceptor dedicatedto the regulation of circadianbehavioral rhythms in Drosophilabecause, although wild-type fliesbecome arrhythmic in constantlight owing to the continuousdegradation of TIM, cryb flies re-main rhythmic in constant light14.

The sensitivity of TIM to lightsuggests a molecular basis for the synchronization of circadianrhythms to the phase and periodof environmental light cycles. RNAfrom per and tim can accumulatein the presence of light but pat-terns of PER–TIM complex for-mation will be set by the frequencyof light-to-dark transitions. The re-sponse of the oscillator to pulses oflight can also be understood. A lightpulse administered shortly after sunsetwill produce phase delays because theformation of PER–TIM complexes andnuclear translocation will be retarded.A light pulse delivered just before dawnwill prematurely release PER from nu-clear PER–TIM complexes and advancethe timing of maximal transcriptionalrepression. With regard to early formalmodels of the endogenous oscillator inDrosophila, subjective day now corre-sponds to that part of the molecularcycle during which TIM proteins are notpresent to be perturbed by light.

Double-time allows the rhythm to besustained in constant darkness

The response of TIM to light guaran-tees molecular oscillations in the pres-ence of light–dark cycles but how arecycles of gene activity generated in theabsence of environmental cues? As de-scribed above, the rates of associationof PER and TIM have been shown to in-fluence the time of nuclear localization.After their movement to the nucleus,

PER–TIM complexes persist for severalhours before PER is released to providea key repressor. These delays will pro-mote a cycling mechanism by separat-ing times of transcriptional activation ofper and tim from times of transcriptionalrepression.

Another element in this regulation isdouble-time (dbt), which encodes a ki-nase that is closely related to human ca-sein kinase Ie. DBT is constitutively pro-duced and binds to PER, regulating itsaccumulation. This association stimu-lates PER phosphorylation in both thenucleus and the cytoplasm15,16. In theabsence of DBT, PER is stable both as amonomer and in PER–TIM complexes.However, in the presence of the kinase,PER is degraded unless it is bound toTIM15,16. DBT-dependent phosphoryl-ation appears to retard cytoplasmic PERaccumulation until high levels of perRNA and TIM protein have been pro-duced. In the nucleus, after disassemblyof PER–TIM complexes, DBT will deter-mine the rate of turnover of the free PERrepressor and thus the timing of the endof the cycle (Fig. 1).

Mammalian clocksOrthologs of each of the

Drosophila clock genes have beenrecovered from mice and humans(Fig. 2). In mice, these include:three period genes, mPer1, mPer2and mPer3, which express cyclingRNAs in the SCN4; two cryp-tochrome genes, mCry1 and mCry2,which also oscillate in sometissues17,18; and (as mentionedabove) the mPer activators Clockand bmal1 (Ref. 4).

Gene knockouts have shownthat mPer2 is required for circadianbehavioral rhythms in the mouse19,whereas only subtle effects havebeen observed for loss of mPer1 ormPer3 alone20 (Fig. 2). mCry knock-outs reveal a role for these genes inmice that differs from that inDrosophila. Although there is someevidence that mCry1 and mCry2might influence responses tolight11,21, the most prominent func-tion for the mCRY proteins appearsto be in the organization of theclock itself: loss of both genes re-sults in arrhythmicity22. The ex-pression of mCry1 cycles with a cir-cadian rhythm in the SCN17,18, andmCry2 has been shown to cycle inmuscle18. The mCRY1 and mCRY2proteins act as negative transcrip-tional regulators of mCry1, mCry2,mPer1, mPer2 and mPer3 in cul-tured cells18,23. These effects are

likely to be mediated by direct physicalinteractions with CLK–BMAL1complexes20,23.

A single Tim gene has been identifiedin mice and humans4. This gene is mostclosely related to a Drosophila paralogof timeless of unknown function20,24,25. Amouse knockout generates embryoniclethality that has precluded behavioraltesting24. Although mTIM can regulatemPer expression4, there are importantdifferences from Drosophila. First, mTimRNA and protein are found in the SCNbut do not cycle4,26. Second, in SCN cells,the mTIM and mPER proteins are physi-cally associated with the mCRY proteinsbut the mPER proteins have not been re-covered with mTIM26. Third, light doesnot effect mTIM degradation26. Instead,light induces expression of mPer1 andmPer2 by an undetermined mechanism4.

Last, nuclear translocation of mPERappears to be influenced by het-erodimerization of the different mPerproteins and by mPer interactions withthe mCRYs rather than with mTIM18,27

(Fig. 2). Because mCRY regulates mPer

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Figure 1The Drosophila clock. The per and tim genes are coor-dinately activated by dCLK (C) and CYC (B)4,6. The perand tim RNAs (~) are translated to form PER (P) and TIM(T) proteins that must heterodimerize to enter the nu-cleus3,4. Nuclear PER–TIM complexes and nuclear PERcause weak and strong suppression of dCLK–CYC activ-ity, respectively3,4,8, and thus autoregulate per and timtranscription; PER and TIM also promote dClk gene ex-pression28,29. Cytoplasmic heterodimerization of PER andTIM is retarded by the kinase DBT (D), which promotesthe phosphorylation (orange circle) and degradation ofPER proteins15,16. Unlike PER, PER–TIM complexes arestable in the presence of DBT. Kinase-dependent delaysin PER accumulation are required for cycling expressionof per and tim15. DBT also regulates PER phosphorylationand degradation in the nucleus. TIM proteins are de-graded in the presence of light, which entrains molecularoscillations to the phase and period of environmentallight–dark cycles3,4,6. The effects of light on TIM are me-diated by the flavoprotein CRY in a cell-autonomous fash-ion6. Dashed arrows show delays.

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transcription, binds mPER andstimulates mPER nuclear localiza-tion, which are all roles of TIM inflies, the function of TIM in mam-mals might have been supplantedby the CRY proteins. Perhaps physi-cal association of the CRY and TIMproteins, a partnership that hasbeen conserved from insects tomammals, has fostered an evolu-tionary substitution of function.Alternatively, because a paralog ofTIM has now been recognized bycomplete sequencing of theDrosophila genome, a paralog ofmTim might be recovered in mam-mals whose function more closelymatches that of the Drosophilaclock protein.

Differences in the behavior ofthe mouse Clk and bmal1 genesalso distinguish them from theirorthologs in flies. In Drosophila,dClk expression cycles with a cir-cadian rhythm and the oscillationseems to be regulated by levels ofthe PER–TIM complex28,29. In thisadded feedback loop, dCLK pro-tein appears to inhibit dClk RNAsynthesis, and physical associ-ations of PER, TIM and dCLK arelikely to inhibit this autoregulatoryactivity. In mice, however, Clk ex-pression does not cycle but bmal1transcription does. Expression ofbmal1 is, in this case, positivelyregulated by mPer2 (Ref. 20).Therefore, in spite of these mecha-nistic differences, per can be seento regulate the cycling levels of its tran-scriptional activator, the CLK–BMALcomplex, in both flies and mice.

Only two circadian mutants havebeen recovered by behavioral screeningin mammals, Clock from mice (as de-scribed above) and tau, a mutation inthe Syrian hamster. Homozygous taumutants have locomotor activityrhythms with a period of 20 h (Ref. 30).Remarkably, cloning of the tau locus hasrevealed that the mutation affects thesequence of hamster casein kinase Ie(Ref. 31). Thus, tau is an ortholog of theDrosophila clock gene double-time. Thehamster tau mutation is caused by anamino acid substitution and the mutantkinase is defective in binding and phos-phorylating mammalian PER proteins31.

In human cultured cell assays, co-expression of human casein kinase Iepromotes phosphorylation of PER andinhibits PER nuclear translocation32. Inthis case, phosphorylation blocks thefunction of a PER nuclear localization

signal; the kinase also decreases PERstability33. Because TIM stimulates PER nuclear localization and protectsPER from DBT-regulated phosphoryl-ation and turnover in flies, it will be in-teresting to determine whether regu-lation of PER nuclear localization by CRYin mammals also involves effects of CRYon the activity of casein kinase Ie. In anycase, much of the function of this kinaseappears to have been conserved amongDrosophila, mice and humans (Fig. 2).

Peripheral clocksThe clock genes of flies and mammals

are expressed in many non-neuronaltissues. Although behavioral rhythmic-ity depends on expression in defined re-gions of the brain, the peripheral acti-vities of clock genes contribute toself-sustaining clocks. For Drosophila,these dispersed clocks are indepen-dently photosensitive. Cells from thewings, legs and antennae continue toproduce cycling per and tim RNA after

they are explanted from the hostand maintained in culture, and thephase of the molecular rhythmscan be reset in culture by alteringthe timing of the light–darkcycle4,6. These autonomous, dis-persed clocks can be quite resist-ant to the influences of other clocktissues in the same organism. Forexample, in the absence of furtherlight–dark cues, transplanted ex-cretory organs maintained theiroriginal phase rather than shiftingto the previously entrainedrhythm of the host34.

The behavior of peripheral insectclocks differs from that of dispersedmammalian clocks. In rats, mPeroscillations detected in explantedmuscle, lung and liver are notphotoresponsive. In the intact or-ganism, however, they do appear torespond to photoentrainment ofthe SCN, which is innervated byprojections from the retina.Significantly, the timing of re-sponses of muscle, lung and liverdiffer from each other, so that reset-ting of the SCN can precede realign-ment of the phase of the peripheraloscillations by several days35. Thecontrast with Drosophila is striking:peripheral clocks throughoutDrosophila are uniformly and inde-pendently phase-shifted by light4,6.Evidently mammals have adoptedstrategies for the humoral coordina-tion of dispersed clocks because,unlike Drosophila, they are not

translucent. This conclusion is bolsteredby the recent finding of directly photo-entrainable clocks in the kidney and heartof zebrafish36.

The variety of tissues containing cir-cadian clocks in mammals is impressiveand might include most organsystems37. Cycling clock-gene express-ion has even been demonstrated in im-mortalized fibroblasts that have beenmaintained in culture for over twodecades37. In addition to raising impor-tant questions about mechanisms forcoordinating and entraining dispersedclock activities, mammalian studies ofperipheral clocks have shown a strikingpervasiveness of circadian regulation inanimal biology.

Connecting molecular clocks to behaviorHow do cycles of clock gene expression

influence timed physiology and behavior?Work in Drosophila has been stimulatedby the discovery of several clock-regulated behavioral and physiological

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Figure 2The mammalian clock. Depending on the cell type18, upto three per genes and two cry genes are activated byCLK (C) and BMAL1 (B). Heterodimerization of PER (P)proteins or the interaction of the PER and CRY proteinsfacilitates PER nuclear localization18,27. PER and CRYproteins suppress CLK–BMAL1 activity4,18,23,27 and atleast one PER protein (PER2) modestly promotes bmal1gene expression20. Although CLK–BMAL1 suppressionby a PER–CRY heterodimer is shown here, it is not clearhow many classes of PER- and/or CRY-containing com-plexes are active in vivo18,20,23,27. The possibly overlap-ping roles of CRY and PER proteins also complicate gen-etic analysis of the individual contributions of PER1,PER2, PER3, CRY1 and CRY2 (Refs 11,19,20). Nuclearlocalization of PER is retarded in cultured cells by ca-sein kinase Ie (TAU) dependent phosphorylation (orangecircle), which affects the PER nuclear localizationsignal32 (NLS). This kinase is an ortholog of DrosophilaDBT and is affected by the hamster circadian mutanttau 31. PER’s phosphorylation by casein kinase Ie alsoappears to foster PER degradation33.

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programs that might be suitablefor molecular dissection. Theserange from olfactory tuning38 tothe circadian organization of feed-ing behavior39.

Most is known about the mol-ecular control of Drosophila’s loco-motor (running) activity by arhythmically accumulating neuro-peptide, pigment dispersing factor(PDF). This neuropeptide is pro-duced by lateral neurons (LNs),which are the circadian pacemakercells of the Drosophila centralbrain. Oscillating PDF immuno-reactivity is seen in projectionsconnecting the LNs to dorsal re-gions of the Drosophila brain,which might form a nucleus for theregulation of motor activity40.

Mutants that fail to produce PDFshow rhythmic locomotion in thepresence of light–dark cycles. Themutant flies are largely active dur-ing the day and inactive at night butbecome arrhythmic when trans-ferred to constant darkness41.Injection of a pulse of PDF intoareas of the cockroach brain associ-ated with pacemaker cell functionshifts the phase of the locomotoractivity rhythm in a fashion that dependson the time of day of the injection42.Continuous expression of PDF in thepacemaker area of the Drosophila brainalso alters locomotor activity rhythms43.These observations indicate a role forPDF in establishing the phase of themolecular rhythm, or possibly in coordi-nating the rhythmic activity of individualLNs.

The cycling expression of PDF is in-directly controlled by the molecularclock. Accumulation of pdf RNA requiresthe presence of CLK and CYC but, para-doxically, pdf RNA levels do notcycle40,44. Rather, oscillating productionof PDF peptide from constitutively pro-duced pdf RNA depends on the cyclingof a transcription factor of the PAR-domain family, vrille (vri)44. The stepsconnecting vri and PDF have not beenestablished but the regulatory path be-tween the molecular clock and cyclingvri expression is understood. The vripromoter contains binding sites fordCLK–CYC and is therefore activated bythese proteins and repressed byPER–TIM44. Cycling vri expression mustregulate critical elements of theDrosophila clock in addition to PDF be-cause constitutive vri expression elimi-nates circadian rhythmicity and entrain-ment to light–dark cycles. This is a more

severe behavioral phenotype than lossof pdf alone (Fig. 3).

Earlier studies showed that there is asimilar strategy in mammals. Oscillatingexpression of the neuropeptide vaso-pressin is regulated by CLK–BMAL1binding of the vasopressin promoter45.Thus, for both flies and mammals, theautoregulatory elements PER, TIM(CRY),CLOCK and BMAL1 have a double signifi-cance: a molecular oscillation is pro-duced and sustained by this small groupof transcription factors and these inturn begin to assemble the output of acircadian clock.

Looking beyond the animal kingdomWith the identification of orthologs of

the fly and mammalian clock genes infish and amphibia, a substantially con-served mechanism appears to governcircadian rhythms throughout the ani-mal kingdom. Autoregulatory mecha-nisms have also been uncovered inmolecular studies of bacterial, fungaland plant clocks but, so far, these in-volve genes that are, at best, distantlyrelated to those of animals. Possibly thehighest homology is represented by twotranscription factors, WC-1 and WC-2,which activate the clock gene frq inNeurospora. The FRQ protein sup-presses frq gene activation, presumably

by interfering with the functions ofWC-1 and WC-2, to produce oscil-lations in frq expression4,46. TheWC genes encode PAS-domain-con-taining proteins and thus showlimited sequence similarity to PER,CLK and CYC–BMAL1. No signifi-cant frq homologies have beendetected within the Animalia.

Although it is still too soon to at-tempt comparisons with plantclocks, work with the cyanobac-terium Synechococcus is quite ad-vanced47,48. Close to 80% of its genesare regulated with a circadianrhythm. Three relevant loci havebeen identified by genetic screens(kaiA, kaiB and kaiC) whose pro-teins physically associate and auto-regulate gene expression to producecircadian molecular cycling. None ofthese genes has any detectable simi-larity to the components of animalor fungal clocks. This suggests thatcircadian clockworks have evolvedindependently at least twice andpossibly three times to generate themechanisms found in cyanobac-teria, in Neurospora and throughoutthe animal kingdom. Yet it isstartling to see a common strategy

emerge each time a new mechanism is as-sembled. Cycling gene expression andautoregulation always provide the molec-ular foundation for circadian rhythms.This evolutionary convergence could indi-cate that natural selection has failed to un-cover an alternative scheme for building acircadian clock.

References1 Njus, D. (1976) The search for the biochemical clock.

Trends Biochem. Sci. 1, 79–802 Takahashi, J.S. and Zatz, M. (1982) Regulation of

circadian rhythmicity. Science 217, 1104–11113 Young, M.W. (1998) The molecular control of circadian

behavioral rhythms and their entrainment inDrosophila. Annu. Rev. Biochem. 67, 135–152

4 Dunlap, J.C. (1999) Molecular bases for circadianclocks. Cell 96, 271–290

5 Rothenfluh, A. et al. (2000) Isolation and analysis of sixtimeless alleles that cause short- or long-period circadianrhythms in Drosophila. Genetics 156, 665–675

6 Scully, A.L. and Kay, S.A. (2000) Time flies forDrosophila. Cell 100, 297–300

7 Lee, C. et al. (1999) PER and TIM inhibit the DNAbinding activity of a Drosophila CLOCK-CYC–dBMAL1heterodimer without disrupting formation of theheterodimer: a basis for circadian transcription. Mol.Cell. Biol. 19, 5316–5325

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9 Naidoo, N. et al. (1999) A role for the proteasome inthe light response of the TIMELESS clock protein.Science 285, 1737–1741

10 Cashmore, A.R. et al. (1999) Cryptochromes: blue lightreceptors for plants and animals. Science 284, 760–765

11 Sancar, A. (2000) CRYPTOCHROME: the secondphotoactive pigment in the eye and its role in circadianphotoreception. Annu. Rev. Biochem. 69, 31–67

12 Stanewsky, R. et al. (1998) The cryb mutation identifiescryptochrome as a circadian photoreceptor inDrosophila. Cell 95, 681–692

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Figure 3Rhythmic expression of factors mediating circadian be-havior are controlled by oscillating activities of dCLKand CYC. The neuropeptide PDF influences the express-ion of locomotor activity rhythms in Drosophila41. PDFaccumulates with a circadian rhythm in nerve pro-cesses formed by pacemaker cells of the fly brain40.Constant production of PDF interferes with rhythmic be-havior43 but pulses of PDF can reset the phase of thebehavioral rhythm42, suggesting roles for PDF in bothbehavioral output and entrainment of the molecularclockworks (black arrows). Cycling vri expression is re-quired for PDF accumulation and for the molecularoscillations of per and tim, indicating a role for VRI in the oscillator itself (red line) and in the oscillator’soutput and entrainment through effects on PDF44

(black line). Cycling vri transcription is generated bydCLK–CYC binding of the vri promoter and periodicsuppression by PER and TIM44. In mammals, oscillat-ing expression of vasopressin and Dbp, a distantrelative of vri, are similarly regulated by inter-actions of CLK–BMAL1 with the vasopressin and Dbppromoters45,49,50.

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17 Miyamoto, Y. and Sancar, A. (1998) Vitamin B2-basedblue-light photoreceptors in the retinohypothalamictract as the photoactive pigments for setting thecircadian clock in mammals. Proc. Natl. Acad. Sci. U. S. A. 95, 6097–6102

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19 Zheng, B. et al. (1999) The mPer2 gene encodes afunctional component of the mammalian circadianclock. Nature 400, 169–173

20 Shearman, L.P. et al. (2000) Interacting molecularloops in the mammalian circadian clock. Science 288,1013–1019

21 Okamura, H. et al. (1999) Photic induction of mPer1and mPer2 in cry-deficient mice lacking a biologicalclock. Science 286, 2531–2534

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26 Field, M.D. et al. (2000) Analysis of clock proteins inmouse SCN demonstrates phylogenetic divergence of

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27 Yagita, K. et al. (2000) Dimerization and nuclear entryof mPER proteins in mammalian cells. Genes Dev. 14, 1353–1363

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31 Lowrey, P.L. et al. (2000) Positional syntenic cloningand functional characterization of the mammaliancircadian mutation tau. Science 288, 483–492

32 Vielhaber, E. et al. (2000) Nuclear entry of thecircadian regulator mPER1 is controlled by mammalian casein kinase I e. Mol. Cell. Biol.20, 4888–4899

33 Keesler, G.A. et al. (2000) Phosphorylation anddestabilization of human period I clock protein byhuman casein kinase I e. Neuroreport 11, 951–955

34 Giebultowicz, J.M. et al. (2000) TransplantedDrosophila excretory tubules maintain circadian clockcycling out of phase with the host. Curr. Biol. 10, 107–110

35 Yamazaki, S. et al. (2000) Resetting central andperipheral circadian oscillators in transgenic rats.Science 288, 682–685

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38 Krishnan, B. et al. (1999) Circadian rhythms inolfactory responses of Drosophila melanogaster.Nature 400, 375–378

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40 Park, J.H. et al. (2000) Differential regulation ofcircadian pacemaker output by separate clock genes inDrosophila. Proc. Natl. Acad. Sci. U. S. A. 97,3608–3613

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42 Petri, B. and Stengl, M. (1997) Pigment-dispersinghormone shifts the phase of the circadian pacemakerof the cockroach Leucophaea maderae. J. Neurosci.17, 4087–4093

43 Helfrich-Forster, C. et al. (2000) Ectopic expression ofthe neuropeptide pigment-dispersing factor altersbehavioral rhythms in Drosophila melanogaster. J. Neurosci. 20, 3339–3353

44 Blau, J. and Young, M.W. (1999) Cycling vrilleexpression is required for a functional Drosophilaclock. Cell 99, 661–671

45 Jin, X. et al. (1999) A molecular mechanism regulatingrhythmic output from the suprachiasmatic circadianclock. Cell 96, 57–68

46 Iwasaki, H. and Dunlap, J.C. (2000) Microbial circadianoscillatory systems in Neurospora and Synechococcus:models for cellular clocks. Curr. Opin. Microbiol. 3, 189–196

47 Kondo, T. and Ishiura, M. (2000) The circadian clock ofcyanobacteria. BioEssays 22, 10–15

48 Johnson, C.H. and Golden, S.S. (1999) Circadianprograms in cyanobacteria: adaptiveness andmechanism. Annu. Rev. Microbiol. 53, 389–409

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50 Yamaguchi, S. et al. (2000) Role of DBP in thecircadian oscillatory mechanism. Mol. Cell. Biol. 20, 4773–4781