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.