Circadian Clock Genes in Drosophila: Recent Developments

In dian Journ al of Experimental Biology Vo l. 41, August 2003, pp. 797-804

Review Article

Circadian clock genes in Drosophila: Recent developments

P Subramanian', E Balamurugan & G Suthakar

Department of Bi ochemi stry. Faculty o f Science, Annamalai University, Annamalain agar 608002, India

Circadian rh ythms provide a temporal framcwork to li vin g organi sms and are establi shed in a majority of euk aryotes and in a few prokaryotes. The mo lecul ar mechani sms of c ircadian clock is constantly being investi gated in Drosophila melanogaster. The core of th e clock mechan ism was described by a transcription-translati o n feedback loop mode l involving period (per), timeless (t im ), dc/ock and cycle gcnes. However, recent rescarch has idcntified multiple feedback loops cont rolli ng rh ythm generation and expressio n. Novcl mutations o f timeless throw more li ght on thc functions of per and tilll products. Analysis of pdf neuropcptide gene (expressed in c ircadian pacemaker cell s in Drosophila), indicate that PDF acts as the princ ipal c ircadian transmitter and is in vo lved in output path ways. The product of cryptoci!rollle is known to functi on as a circadian photoreceptor as well as component of the circadian clock. This review focuses on thc recent progress in th c fie ld of molecular rhythm research in the fruit n y. The genc(s) andthc gcne product(s) that are in volved in the transmi ssion of environment al information to the clock, as well as the timing signal s from the clock outward to ccllular functi ons are remai n to be determined.

Key words: Circadi an clock genc, C ircadian rh ythms, Drosophila, Molccular rh ythm

The spectrum of biological processes controll ed by Chemical mutagenesis and P-element inserti onal 2 circadian clocks in li vin g organisms range from the mutations have been used -4. The goal of the e daily sleep/wake cycle and levels of vanous investigations is to define the molecul ar machinery enzy mes/hormones to 0 A synthesis and cell that underli es the almost ubiquitous process of di vision!. These circadian (L. circa, about ; dies, a circadian rhythmicity in the fruit fly . The period (per) 2 day) rh ythms indeed have a genetic basis . The and timeless (li/1/ ) genes in Drosophila encode circadian organi zati on of any li vi ng organi sm is mRNAs whose flu ctu ating levels define dail y composed of three broad domains: (i) the input molecul ar rhythms3. The clock proteins also cycle in a 4 pathways - th at transmit the environmental signals 24 hI' period • The cyclic regul ati on of per and rim and (mainly lig ht-dark cycles) to the central the manner in which they generate a molecul ar clock 5 9 oscillator/clock, (ii ) the generation of timing signals have been reviewed in numerous reviews - . The two in the central oscillator/clock, and (i ii ) th e output proteins (PER and TIM) accumulate during the ni ght. pathways - th at transmit these rhythmic signals with Heterodimerization of PER and TIM is required for a 24 hr periodicity to the various clock controlled their translocation into the nucleus, as tertain PER processes whi ch ultimately result in overt rhythms and TIM sequences, cytoplasmic locali zation domain s (Fig. 1). (CLDs), restrict monomers to the cytoplasm !o. Clock genes are beginning to provide an Heterodimeri zation also protects PER from the understanding of the molecul ar mechani sms that activity of a kinase encoded by double-time (dbt). underlie circadi an rh ythms. Over the last few years, DBT promotes phosphorylation and turnover of by geneti c and mQlecular biological approaches novel monomeric PER proteins, which delays cytoplasmic genes (between 8 and 10) and their roles in the accumulation of PERffIM complexes !! ']. The generati on of circadian rhythm were identified in fruit double-time (dbl) gene which encodes a protein fly (Table I). A number of mutants that perturb kinase is responsible for phosphorylating the per gene temporal organi zation in the fly in a variety of ways product. Mutant alleles of dbr are leth al at the pupal have been identified. Extensive coverage of both stage in homozygotes, which suggest some other roles 2 4 recessives and dominants has been achi eved - . of the gene beyond its role in PER phosphorylation and are yet to be identified. The transcription factors (products of dclk and cyc) form heterodimeric *Phone: 04144-238343 Ex tn: 2 12 Fax: 04 144-238080 complexes and bind to per and tim promoters!3. This E-mail: psub @rediffmail.com binding is inhibited by PERffIM complexes. PERITIM 798 INDIAN J EXP BIOL. AUGUST 2003

F.NV IRONMF.NTAL I!"PUT OUTPUT OVERT SIGNALS PATHWAYS PATHWAYS -- RHYTHMS

Fi g. I - The modcl of the circad ian systc m which includcs a ccntral osc illator (c ircadia n clock) that gcncrates rh ythmicit y. input pat hways through which thc osc illator rcce ives light-dark information and output path ways through which the osc illator co ntrols the obse rvable rh ythm s.

Table I - Clock gene mutallls of Drosophila lIIelallogasrer

Gene Clock mutant s Proposed funct ion Rcfe rences

period (per) Pan of th e clock: toge thcr with lilll gcncr:llcs a circad ian fccdback 43 . 44 loop

. 01 . r;1 . U lillll'less (/illl) /1111 • /1111 , 11111 Pan of th e clock toge ther with per gc ncrates a circadian fecdback 17, 18, 40 and rilll.)"/. loop

clock (elk ) or Jerk (jrk) jrk" Part of the clock; activatcs transcription of per, lilll and l'rille gencs: 4S ncgati ve ly regu lates itself gencrating a sccond fecdback loop

n'Cie (eve) Part of the clock: act iva tcs tran sc ription of per. lilll and vrille gcncs 46

dOlible- lillle Pa rt of th c clock : phosph orylates PER and renders it un stabl c in thc II . 12 abscnce of T I M

Negativcly regulatcs per. lilll and pdf gencs: ma y bc a componcn t of IS output pathways err elY " Photic cntrainmcnt. binds TIM in a light-dcpcndcnt man ncr 26.29 pdf pdf" Output frolll latcra l ncurons 16.33 lark liIIl' LARK in vo lvcs in output pa th ways of eclosion rh ythm 47 .48

lokeoll/ Ci rcadian control of fccding bchaviour 24 complexes act as negative autoregul atory co mponent s rh ythm in th e fru it fly; (i) tem perature affects the of th e cloc k directly associating with dCLKJCYC t 3 period of free run nin g rhythms, (ii ) the phase of th e (Fig. 2). rh ythm is often signi ficantly affected by an ambient A key feature in the present model is th at th ere is a temperature level, and (iii ) temperature cycles could lag between the transcripti onal induction of per and entrain th e circadian rhythms. It was found out that I il1l on one hand and th e nu clear translocati on of th e pel and perL flies have properties reciprocal to one repressor proteins that encode on the other. This lag anoth er in regards to temperature and li ght intensity creates a temporal separati on between phases of dependency of their free running periods. The wild induction and repression, which is req uired to type and per mutant flies were clea rl y synchroni zed to generate an osc ill ation. Without the separati on, the 12: 12 hr LD at two different ambient temperature transc ripts (involved in induction and repression) levels of 25° and 30°C (ref. 14). wou ld come to equilibrium. Another important feature is that the half-li ves of the per and lim mRNAs and Vrille - A gene regulating temporal PER and TIM proteins are rather short, hi ghl y regul ated and synthesis tO prect.se I y adapte d .tt to be part 0 f t he tim. e k eepmg. A novel regul atory loop within Drosophila's mechanism. The present feedback loop model (Fig. 2) circadian clock was identified by Blall and Young t5 has engendered wides pread consensus among A screen for cloc -controlled genes recovered vrille researchers, although it has not entirely escaped (vri), a transcription factor essential for embryo ni c criticism (See 'Conclu sions and future perspectives'). development. vri is ex pressed in circadian pacemaker cell s (lateral neurons) of larv al and adult brains. vri Role of lemperalure RNA levels oscill ate with a rh ythm ic periodicity. It is a common understanding that ambi en t Cycling is directly regulated by th e transcripti on temperature has a sign ificant effect on the circadian factors, dCLOCK and CYCLE, which are also SUBRAMANIAN et af.: CIRCADIAN CLOCK GENES IN DROSOPHILA 799

I CYTOSOL I

LIGHT - DARKNESS CUES ~ P 8 ~ --=-!~""" T R : Degradation+ ? T \ dc/k - rvtJU1 o P Oscillating RNAs ~ ~'-t-____.-----,p _e_r __ _ ~ rvtJU1 _

lim ~ ; -rvtJU1 -~ ... vri ~ I -rvtJU1 -~ :_------// ______~ p=df {MfRNA Proteosome - ~ ------~ j PER TIM 1 CYC ClK Degradation ? Y . ccgs ceRE rvtJU1 ______

CLOCK OUTPUT

OVERT C1RCARDIAN RHYTHMS Fig. 2 - Molecul ar compo nents of the circadian clock in Drosophila melanogaster. PER and TIM are negative elements and CLK and CYC are positivc elements of the autoregulatory loop (involving E-box target sequences). PER monomers are unstable when Ih ey are phosphorylated by DBT. CRY (The circadian photoreceptor) interacts with TIM and TrM is degraded by proteasomc. PER-TIM complex acts as reprcssor (R) of dclk gene which also has activators (A) which are yet to be determined. Positive and negative slemcnts also regulate vri exprcssion. VRI in turn controls this loop. At present. it is unknown whether VRI regulates per and tim expression indepcndentl y of PDF or via PDF. PDF is shown to be involved in the output regulating pathways. PER-TIM-CYC-CLK tetrameric complex could acti va te circadian clock regulatory elements (CCRE) of clock controlled genes (ccgs) which would lead to th e expression of circadian rhythmicities via th e clock output pathways. Activating and inhibiting pathways are indicaled by solid and broken lines respectivel y. required for oscillations of period and timeless RNA. PDF function and VRI could regulate per and lim Eliminating the normal vri cycle suppresses period expression independently of PDF or via PDF'5.16 and timeless expression and causes long-period (Fig. 2). circadian rhythms and arrhythmicity, indicating that cycling vri is required for a functional Drosophila Timeless gene-Novel mutants and functions clock'5. Three alleles of tim have been reported recently Further, PER and TIM are additionally involved (limO! was found to exist a decade ago) tim,i" a (alongwith dCLOCK and CYCLE) in a second temperature sensitive, long period (26-30 hr) allele ' 7, autoregulatory loop, whereby they directly mediate timSL, which was found as a suppressor of perL, and uL the cycling expression of VRlLLE (VRl). VRl lim , which is known to generate rhythms with oscillations are required for per and tim expression, periods of around 33 hr' 8. Characterization of these for overt circadian rhythmicity and for accumulation tim mutations showed that the Drosophila pacemaker of PDF (pigment dispersing factor), a neuropeptide can support very long period rhythms that are supposed to regulate output pathways of rhythms'6. remarkably stable. How TIM is degraded was a Continuous vri expression suppresses PDF levels, mystery until the recent demonstration that indicating that cycling VRI is required for wild type proteasomes are involved in TIM degradation 19. TIM ~o o INDIAN J EXP BIOl, AUGUST 2003 was found to be phosphorylated, ubiquitinated and compl ex may not repress as effici entl y as wild type then targeted to the proteasome for degradation In PERrrIM, or (2) th e PERrrlM complex may not be · h II) l R 2o response to IIg t . th e only repressor. These findin gs . also lead to the The free- running peri od (free run- persistence of interpretation that TIM uL region may contribute to the i rhythms und er constant conditi ons) of lin/ ' (a clock association of full -length PER and TIM protein s or mutation on th e second chromosome) mutants is all ow association of unidenti fied factors in Iluenci ng drasticall y length ened in a temperature-dependent PER/TIM heterod imeri zati on/di ssociation in vivo. l7 manner . PER and TIM protein levels become lower TIM-independent reg ulatory activity of PER was in lin/' mutants as temperature becomes hi gher. This also suggested by in vitro cell culture studi es IX. In mutation red uces per and not lim mRN A abundance. absence of TIM. PER alone co uld repress In addition, PER constituti ve ly dri ven by the dCLOCK/CYCLE mediated transcri pt ion of per in rhodopsin I promoter is lowered in ril mutants, the cultured Drosophila S2 cells. Thus, in th e nu cleus, indicating that li,,/' mainly affects the per feedback conversion of PERrrlM heterodimers to PER proteins loop at a posttranscriptional level. An excess of I'er+ appears to be required to complete transcripti onal lX gene dosage can ameliorate all ril phenotypes, repression and terminate each molecular cyc le • including th e weak nuclear locali zati on of PER, Ea rli er studies suggested th at the stabi li ty of PER in suggestin g that lilll ,.i, afrects circadian rh ythms by the nu cleus is also a control point that influences reducing PER abundance and it s subsequent peno. d I engt I1 II . H owever, t he present eV I'd ences IX transportation into nu clei as temperature increases 17. indi cated th at dissociation of PERITIM compl ex to form nucl ear PER is necessary step in the molec ul ar In dependent r e}!, lIlaLO I)' a ·ti vily of PER regulation of period len gth of th e ex pressed rh ythms The molecul ar ph enotype of lill/L (mutation and in tilll UL mutant, interference with thi s step leading to amino ac id substitution at positi on 260) is produces severely altered circadian rh ythms. prolonged nuclear locali za tion of the PERITIM uL complex, but, p er and lim RNA levels remain Period alld clock inf711 ence temporal patlems q{ moderately hi gh for an ex tended time (- 10 hr)l x. hundreds of I l"anscripls uL Removi ng TIM with li ght brings the p er and lim Of lat e, period gene was found to influence th e RNAs to a lower level, co nsistent with the view th at ex pression pattern s of over 600 non- oscillating 2 1 PER alone may be suffi cient for transcriptional transcripts • Temporal ex pression levels of several repression of per and tilll genes IH. Although the hundred genes also differed significantly between TIM uL region of the TIM protein has not been wild type fli es kept in LO versus 00 but di ffered characteri zed fun cti onally. th e mutation does not minimally between pel' flies kept in the sa me two affect any of the protein fragments that were found in conditions (LO and 00). This indicates th at th e prior studies to independently bind to PER 18.20. period-dependent circadian cl ock regulates onl y a The lin/" phase response curve (c hronobiological novel set of rhythmically ex pressed tran scripts. jargon used to represent the displacement of circadian However, period regulates basal and lig ht-regulated rh ythms by short perturbations of li ght) indicated that gene ex pression to a very broad ex tent. Recent studi es the circadi an oscill ator has been altered with respect also showed that cycling in most of the genes was lost to the functions occurring at late ni ght. It was proved in arrhythmic clock mutants under light-dark uL 22 th at PER and TIM are physically associated during conditions . Hence, expression of periodica ll y the interval of prolonged protein accumulation and regulated genes may be coordinated loca ll y 0 11 immunocytochemical analysis showed that TIM uL chromosomes where . mall clusters of genes are persists in ph otoreceptor cell nuclei for an extended regulated jointly. These results further suggest th at ls UL time . Thus, th e tilll mutation prolongs the duration many genes in vo lved in diverse fun ctions are under of nuclear locali zati on of th e PERrrlMuL complex circadian control and also reveal th e complexity of uLl 8 and affects li ght-independent degradation of TIM . circadian gene ex pression in Drosophila. Even though the PERrrlMuL complex stays in th e The data indicating that all oscillating gene nucleus mu ch longer th an the wild-type complex ex pression is under Clk control, led to a hypothesis does, Rothenfluh PI a i . lx, observed prolonged that Drosophila has no Clk-independent circadian derepression of per and tim RNA. There are two systems. A larger number of genes is affected in Clk- poss ibl e ex planations for thi s: ( I) the PERrrlMuL mutant fl le· s t h an per mutant fl 'l es 21.22 , suggestin. g ttl1 at SUBRA M AN IAN eT al.: CIRCADIAN CLOCK GENES IN DROSOPHILA 80 1

30 Clk affects a wide range of genetic networks. Further, TIM . This interaction was specific to functional most of the circadian gene network is indirectly CRY, since cr/, a mutation that leaves CRY partially regulated by Clk23. inactive is unable to interact with TIM or PER-TIM3!. Further, CR Y was found to undergo a photochemical Takeout-gene controlling circadian feeding change all owin g it to interact with TiM both in the behaviour cytoplasm and nucleus. This interaction represents the So el al. 24 reported identificati on and characteri ­ initial step in circadian phototransduction, and renders zati on of a new clock-regulated gene, takeout (to). 10 the PERffIM complex inactive and unable to is implicated in circadian contro l of feeding partiCipate. . .III t he negat.iv e Clee db ac k27 '.28 However, behaviour. Its expression is down regulated in the how CRY transduces li ght signals to the molecular clock mutants (per and Clk) tested. In wild type fli es, pacemaker that generates circadian rhythms IS 10 mRNA ex hibits daily cycling pattern with a ilOvel unknown at present. phase, show ing delays relative to those of better characterized clock mRNAs (per and lim). The E-box PDF and pacemaker cells of the fly containing 10 promoter seq uence revealed a The circadian organi zation of locomotor activity remarkable sequence identity with the E-box region of and eclosion is govern ed by a pacemaker within a the per and tim promoters. However, unlike per and discrete set of neurons in th e Drosophila brain. lilli , the E-box of 10 is not in vo lved in the amplitude Nevertheless, molecular studies have es tablished th e and phase of transcripti onal cyclin g of 10. Hence, it presence of autonomous circadian clocks in isolated cou ld be concluded th at the circadian delayed appendages and excretory stru ctures32 ; the molecular transcriptional phase of to is most likely the result of mechanisms underl ying clock function in these tissues indirect regulation through unknown tran sc ripti on may not be identi cal. These peripheral clocks may facto rs. coordinate the physiological processes, whi ch could ultimately result as overt temporal expressions Cl'yptoch.-ome - circadian photoreceptor and (locomotion and eclosion). component The pacemaker cell s (lateral neurons in the brain ) Cryptochrome is a flavin containing protein, express hi gh levels of clock genes (at least per and presumably derived from photolyase, which is lim) and are implicated in the circadian control of recently reported to med iate circadian photoreception activity rh ythms. These cell s could potentially 25 in an im als incl uding Drosophila . It is coded by cry communicate with the oth er ti ssues of the tly by gene-'?6 ?7- and t he gene pro d uct can p hYS .lca II y associ. ate electri cal, chemical or hormonal signals - or a 28 with TIM and appears to be an important regulator combinati on of these3!. The neuropeptide pigment of li ght-dependent TIM degradation in some cell dispersing factor (PDF) is one likel y candidate for types. In uy" mutants the stabiliLy of PER and TIM is pacemaker cell output. PDF levels normally cycle

. 1 affected. cry RNA cycles and hi ghest levels are found with a circadian rhythm!6.3 3, and are altered in a 9 . in the early part of th e da/ . This cycl in g persists in number of clock gene mutants! 5. Although, PDF is constant darkness and is dependent on PER, TIM, considered as a prime candidate for an output 2X 30 CLK and CYC - . In per and lim null mutants, cry molecule, a large proportion of output genes is RNA levels are constantly low and in elk and eye specific for a single overt rhythm. These genes may mutants the levels are constantly hi gh, indi cating also be restricted in their expression to specific central po iti ve reg ul at ion by the former and negative or peripheral ti ssues that contain oscill ators. regu Iatl·o n b y t Ile I atter two components2 629. . Howcver, Levels of pdf mRNA do not cycle. Ex pression of CRY protein cycles only in the presence of li ght:dark the protein is also non-cyclic in the cell bodies of cycles. Apparently this cycl in g is driven by li ght­ lateral neurons, but cycles at their axon terminals, 3 sensiti vity of the protei n because DD (constant dark) which may be in ac tive of cyclic release 1.34 . It was 26 29 conditi ons lead to constan t accumul ation of CRy . . found that PDF protein accumulates in the lateral Yeast two-hybrid assays showed th at CRY neurons (circadian pacemaker cell s) with a circadian 3o interacts with PER , TiM and PER-TIM complex in rhythm showing th e hi ghest accumul ation in the earl y presence of li ght but not in dark28. Further analyses part of the day and lowest at night. Overexpression of indi cated that C terminus of CRY is responsible for PDF lateral neurons does not eliminate its cycling or the li ght dependence of the interactions with PER and behavioural rhythms, suggestin g that the mcchanisms 802 INDIAN J EXP BIOL, AUGUST 2003 that mediate its cyclic release are not easily phase during development. Once set, the pacemaker saturated 16 .3 I. can control the rhythmic output of later events that Further, the peri od of this accumulation rhythm is will occur at a particular time of day. Recent lines of shortened by the pel's mutation and continuous evidences support a ' time-memory' clock. Extra­ accumulati on of PDF in the dorsal brain is associated retinal vi sual structures have been proposed to pl aya with arrhythmia and a variety o f period changes in role in both larval and adult circadi an 37 38 adult locomotor acti vity. PDF could couple the photoreception . . Malpel et al. 39 , analysed th e molecul ar clock to timed behavi our and vri conveys interactions between extra-retinal structures of the 7 8 15 essential regulatory signals from the clock to POF . . visual system and the clock neurons during brain (Fi g. 2). development. Larval optic nerv e has contacts with the Analysis of the null mutation of pigment-dispersing lateral neurons in the embryoni c brain . Analyses of factor gene, p((r demonstrates that pdf is indeed visual system-defecti ve genotypes showed that the in volved in the circadian output regul ation of absence of the afferent optic nerve resulted in 39 circadi an overt rhythms 16. Recent research also defective lateral neurons formati on . showed that some of the clock genes affect pdf Since no overt circadi an rhythmicity of behaviour expression. Mutati ons in dclk or cyc reduce PDF or physiology has been detected in larv ae of D. 35 staining in lateral neurons and disruption of vri meianogaster , there are no known circadi an outputs OSCI' 11 atlon' s a I so resu Its 1Il' PDF sta..llllll g"151 1. at this developmental stage, except a hypotheti cal one from the larval LNs to the antiphase neurons, whi ch Postembryonic development of circadian clock may be in part responsible for the synzhroni zati on of 35 functions the molecul ar rhythms of the latter . However, The clock genes, period and timeless, are expressed circadian clock is operating in larvae, under the cyclically in the larval central nervous system of control of at least one of the key clock genes (per). Drosophila and dail y oscillations of per expression Since a 'time-memory clock' does not need an output persist throughout metamorphosis in lateral neurons pathway, until circadian rhythmicity is finall y 35 of larvae . PER and TIM cyclings in these neurons expressed in adults, the pacemaker neurons in larvae may be responsible for the phenomenon of ' larval probably would not require an anatomical target. time memory' . Kaneko et al.35 further showed that phase shifts of molecul ar oscillations during the larval Conclusions and future perspectives stage were small er th an those measured by adult The first problem with the current model is the behaviour, suggesting mo lecularly transient responses time-delay questi on: all of the processes in this casual during development. chain (Fig. 2) are normall y carried out rapidly, and A li ght pulse given to larvae was found to phase some transcription factors can be transported into the shift adult behaviour. These phase shifts were nucleus and be acting on the nuclear targets within di fferent in directi on and magnitude between pel and minutes. There is a necessity of 'time delays' at one wild type, indicating that per is involved in larv al or more time points in the chain (to account fo r the time memory. Further, hi stological experiments degradation of clock gene products at specific ti me suggested that li ght stimuli can set thi s larv al clock by points) to make this loop take 24 hr to complete, but at least two input pathways, one disrupted by a at present evidences (kineti c data) are lacking to mutation (cr/) in the putative blue-li ght receptor indicate how these delays could be accompli shed. 26 cryptochrome , the other by a mutation of the norpA Further, if the per gene is transcri bed from a gene, which eliminates certain larval responses to constitutive promoter, it can rescue rh ythmicity in 35 40 li ght , as well as li ght responses of adult compound arrhythmic null mutant per fli es and the RNA and 36 eyes and ocelli , protein are found to be rhythmicall y abundant, in spite 4o The adult activity and eclosion rhythms of fli es can of the constant rate of transcription . It has also been be synchronized by li ght-dark (LO) cycles observed that expression of tim cONA transgene could 40 experienced during the larval stages, although the also rescue rhythmicity in arrhythmic null mutant lirn animals were kept in constant darkness. This larv al fli es, even though the l im RNA is not rh ythmic in 41 pacemaker responsible for the synchronization of abundance . Hence, it appears that fl ies can be circadian rhythms is a 'time-memory' clock, whi ch rh ythmic if either per or tim is consti tutively expressed; takes in environmental stimuli necessary to set its however, at present, it is unknown how they are. SUBRAMANIAN et al.: CIRCADIAN CLOCK GENES IN DROSOPHILA 803

The PER protein is nuclear and its levels are 8 Panda S. Hogenesch 1 B & Kay S A, Circad ian rhythms fro m rhythmic in the lateral neurons but PER is available fli es to hu man, Narure, 41 7 (2002) 329. 9 Hall 1 C. Geneti cs and mo lecular biology of rh ythms in widely in other fly tissues, and in the ovaries the Drosophila and other insects. Adv Gener. 48 (2003) 2 17. 4t protein is neither nuclear nor rhythmic • Furthermore, 10 Saez L & Young M W. Reg ulated nuclear ent ry of the the per gene affects the period of the courtship-song Drosophila clock protein s PE RI OD and TIMELESS, rh ythm in Drosophila, even though this rhythm has a Nellroll , 17(1996)9 11. II Price 1 L, Blau 1, Rothenflu h A, Abodeely M, Kloss B & period of 1 min, which is obviously very rapid to Young M W, double- rillle is a new Drosophila clock ge nc depend on a transcription-translation feedback loop. th at regulates PERI OD protein accumul ati on, Cell , 94 (1998) This raises the possibility that these clock proteins 83. may have a primary function that is as yet unknown 12 Kl oss B. Price 1, Sala L, Blau 1. Rothenfluh A, Wesley C S and that their effects on circadian clocks may be & Young M W, The Drosophila clock ge ne dO ll ble- rime encodes a protein closely rclated to human casein kin ase h:. secondary . Additionall y, the avail able evidences led Cell. 94 (1998) 97 . to a hypothesis that there may be additional proteins, 13 Lee C, Bae K & Edery I, PER and TIM inh ib it the DNA which could modi fy the action(s) of clock gene bindin g ac ti vity of a dCLOCK-CYC/dB MAL I heterodilller products th at have already been identified42 and there without disru pting formati on of the heterod imer: A basi s fo r circadian transc ri ption, Mol Cell Bioi, 19 (1999) 53 16. may also be additional transcription factors regul ating 14 Tami oka K, Light and temperature cooperate to regulate the clock gene activities. circadian locomotor rhyth m of wil d type and mu tants of The understanding of molecular organi zati on of Drosophila meianogasrer, J In secr Physiol, 44 (1998) 587. circadian clock is not yet complete. Many of the IS Blau 1 & Young M W, Cycling vrille ex pression is req ui red details of clock input, output pathways of for a full y functi onal Drosophila clock, Cell, 99 (1999) 66 1. 16 Renn S C P, Park 1 H, Rosbas h M. Hall 1 C & Taghert P H. synchronizati on and detail s of passing the timing A pdf neuropeptide gene mutati on and ab lation of PDF signal to vari ous body ti ssues-have yet to be neurons each cause severe abnormaliti es of behavioural completely worked out. Understanding the molecul ar circadian rhythms in Drosophila, Cell, 99 ( 1999) 791. bases of th e clock (from input to output) in the 17 Matsumoto A, Tomi oka K. Chiba Y & Tanimura T, rim'il lengthens circadian period in a temperature-dependent amenable system of Drosophila will shed li ght on the manner through suppression of PERI OD pro tein cyclin g and workings of the clock and its influence on the nuclear localizati on, Mol Cell Bioi. 19 ( 1999) 4343. behaviour and physiology of hi gher mammals and 18 Rothenfl uh A, Yo un g M W & Saez L. A TIM ELESS­ humans. in dependent fun cti on fo r PERI OD protein s in th e Drosophila • clock, Neuron, 26 (2000) 505. Acknowledgement 19 Na idoo N, Song W, Hunter-Ensor M & Sehgal A. A role for th e proteasome in the li ght response of the rilll eless clock Fin ancial support from DST, New Delhi in the protein. Science. 285 (1999) 1737. form of a research project to PS is gratefull y 20 Sangoram A. Saez L. Antoc h M, Gekakis N, Stankis D, acknowledged . Whiteley A. Fruechte E, Vitatema M. Shimomura K & Ki ng D. Mammali:lI1 circadian au toregu latory loop: A rillleless onholog and mPE R I interact and negati vely autoregul ale CLOCK­ References BMA LI induced transcripti on, Neuron, 21 (1998) 11 0 I. Moore-Ede M C, Sul zman F M & Fu ll er C A, The c10rks thar 2 1 Lin Y, Han M, Shimada B, Wang L, Gibler T M. Amarakone time us: Physiology of rh e circadian tillling sysrem (Harvard A, Awad T A, Stormo G D, Va n Gelder R N & Toghert P H. Uni ve rsit y Press, Cambridge) 1982. In fl uence of the period-dependent circadian clock on diu rn al 2 Ed munds L N 1r, Cellular and lIIolecular bases of biological circadian and aperiodic ge ne expression in Drosophila clocks (Springer- Verl ag, New York ) 1988. l1I elallogasrer, Proc Narl Acad Sci (USA), 99 (2002) 9562. 3 Sehgal A, Rothenfluh-Hil fiker A, Hunter-Ensor M, Chen Y, 22 Ueda H R, Matsum oto A, Kawamura M, Lino M, Tanilllura Myers M P & Young M W, Rh ythmic expression of timeless: T & Hashi moto S, Genome-wide transcri ptio nal A basis fo r promoting circadi an cycles in period gene orchestrati on of circadi an rh yt hms in Drosophila, J Bioi autoregul at ion, Science, 270 (1995) 808. Chem. 277 (2002) 14048. 4 Dunl ap 1 C, Molecul ar bases for circadian clocks, Cell, 96 23 McDonald M 1 & Rosbash M, Microarray analysis and (I 999) 27 1. organi zation of ci rcadi an gene ex pression in Drosophila, 5 Su bramani an P & Lakh ot ia S C, Molecular rh ythms th at Cell , 107(2001)567. regul ate rh yt hm genes in Drosophila, Cu,.,. Sci, 77 ( 1999) 24 So W V, Sarov-Blat L, Kotarski C K, McDonald M 1. Allada 11 65. R & Rosbash M, rakeour, a novel Drosophila gene under 6 Wi ll iams 1 A & Sehga l A, Molecul ar compo nent s of the circadian clock transc ri pti onal regulation, Mol Cell Bioi. 20 circadian system in Drosophila. Ann Rev Physiol. 63 (200 I) (2000) 6935. 729. 25 Sanc ar A, Cryptochrome: The second photoacti ve pigment in 7 Harmer S L, Panda S & Kay S A, Molecul ar bases of th e cye and its ro le in circadian photorecepti on, At/n Rev ci rcadi an rhy th ms , Anll Rev Cell Dev Bioi, 17 (200 I) 2 15. Biochem. 69 (2000) 3 1. 804 I DIAN J EXP BIOL, AUGUST 2003

26 Stanewsky R, Kaneko M , Emery P. Berella B. Wage r-Smith 37 Blanchardon E. GrimJ B, Klarsfeld A. Chebt A, Hardin P E. K. Kay S, Rosbash M & Hall J C. The cr/' Illutation Preat T & Rouyer F, Defining th e role of Drosophila lateral identifies cryptochrome as a circadian ph otorece ptor in neurons in the comrol of circadian activity and ec los ion Dro.wphiln, Cell, 95 (1998) 6R I. rh ythms by targe ted ge neti c ablation and PER IOD protein 27 Hall J C. Cryptochromes: Sen so ry recep tion . tran sducti on overex prcss ion, Ellr J Nellrosci. 13 (200 I ) 871 . and cloc k fun cti ons subserl'i ng circadian sy stems. Cllrr Opill 38 Helfrich-Forster C. Wimer C. Hofbaucr A. Hall J C & Nellrobiol. 10 (2 000) 456. Stancwsky R. The circadian clock of fruit fli es is blind after 28 Ce ri ani M F, Darlington T K. Stakni s D, Mas P. Pelli A A. elimination of all known ph otorece ptors. Nell roll. 30 (200 I ) Weitz C J & Kay S A. Light-dependelll sequ estration of 249. TIMELESS by CRYPTOCHROME, Sciell ce. 285 ( 1999) 39 M alpel S, Klarsfeld A & Rou ye r F. Larve l opti c nerve and 553 . adult extra-retinal ph otorece ptors sequentiall y assoc iate with 29 Emery P, So W V, Kaneko M. Hall J C & Rosbash M . CRY, clock neurons during Drosophila brain development. a Drosophila clock and light -regulated crypt oc hrome. is a Del'l'loplllelll , 129 (2002) 1443. major contrihutor to circadian rh ythm re selling and 40 M ye rs M P. Wager-Sm ith K. Wes ley C S, Young M W & ph otosensit ivity, Cell. 95 ( 1998) 669. Se hgal A. Pos itional cloning and sequence analysis of th e 30 Rosato E & Kyri acou P, Flies, cloc k and evoluti on. Phil Drosophila mutant lilllele.\·.\·, Sciell ce 263 ( 1994) 1603. TrailS R Soc LOlld 13 356 (2 00 I) 1769. 41 Hall J C. Genetics of biolog ical rh ythms in Drosophila. Adl' 3 1 Helfri ch-Forster C. Tauber M . Park J H. Verse n M M . Cellel 38 ( 1998) 135 . Sch neuw ly S & Hofbau er A. Ectopi c express ion of th e 42 M ye rs E M . Yu J & Sehga l A. Circadian control of ec los ion: neuropeptide pi gment dispersing fa ctor alters be hav ioral Interac ti on betwee n a celllral and periphera l clock in rh ythm s in Drosophila lIl elallogasler. 20 (2000) 3339. Drosophila lII e!wwgasler. Cllrr /3iol, 13 (2003) 1426. 32 Roe nn ebe rg T & M errow M , Circadian systems: Different 43 Konopka R J & Ben zer S. Cloc k mutants of Drosophila le ve ls of complexity. Phil Trail S R Soc LOll d LJ, 356 (2 001 ) lII elallogasler. Pmc NaIl Acari Sci (USA), 68 ( 197 1) 2 11 2. 1687. 44 Bargiello T A. Jackson F R & Young M W, Res toration of 33 Park J H. Helfrich-Forster C. Lee G, Liu L. Roshash M & circad ian behavioural rh ythms by gene' tran sfer in Hall J C. Differential regu lati on circadian pa ce make r output Drosophila. Natllre. 3 12 ( 1984) 752. by se parate clock genes in DrosOIJilila, Proc Nar l Acad Sci 45 Darlington T K, Wager-Smith K. Cerian i M F. Stankis D, USA. 97 (2000) 3608. 34 Kaneko M , Hamblen M J & Hall J C. In vo lvement of th e Gekasis N. Steeves T D L, Weitz C J. Taka hashi J S & Kay S A. Clos ing the circadian loop: CLOCK induced transc ription IJe riod gene in developmental time-memory: EITec L of th e or its own inhibitors. per and lilll. Sciell ce. 2RO ( 1998) 1599. PI'I-' mutation on pha se shits induced by light pu lses del ivered to Drosophila larvae, J Bioi Rhwhllls. 15 (2 000) 46 Rutila J E, Suri V. Le M. Venu s So W, Rosbash M & I-I all J 13. C. CYCLE is a second bHLI-I-PAS clock protein e>senti al for 35 Bu sto M . Iye ngar B & Campos A R, Genetic dissec tion of ci rcadian rh ythmicity and tran sc ription of Drosophila period be hav iour: Modulation of locomoti on by light in th e and lillleless, Cell. 93 ( 1998) 80S. Drosophila IIl ciallogasler larva requires genetically distinct 47 Newby L M & Jackson F R, A ncw biologica l rh ythm mutant visual system functi ons. J Nellrosci. 19 ( 1999) 3337. of Drosophila lII e/(lliOM esler th at identi fies a gene with an 36 Pea rn M T , Rand all L L, Shonridge R D, Burg M G & Pak esse ntial embryonic functi on. Cellelics. 135 ( 1993 ) 1077. W L, Molecul ar, biochemica l and electrophysiological 48 McNeil G P, Zhang X, Genova G & Jackso n F R, A chJracterization of Drosophila norA mutants. J Bioi Chelll , molec ular rh yt hm mediating circadian cloc k output in 27 1 ( 1996)4937. Drosophila, Neuroll . 20 ( 1998) 297.