Indian Journal of Experimental Biology Vol. 41, August 2003, pp. 797-804

Review Article

Circadian clock genes in Drosophila: Recent developments

P Subramanian', E Balamurugan & G Suthakar

Department of Biochemi stry. Facu lty o f Science, Annamal ai University , Annamalainagar 608002, India

Circadian rhythms provide a temporal framcwork to li ving organi sms and are estab li shed in a majority of eukaryotes and in a few prokaryotes. The mo lecul ar mechani sms of c ircad ian c lock is constantly being investiga ted in Drosophila melanogaster. The core of the clock mechan ism was described by a transcription-translatio n feedback loop mode l involving period (per), timeless (t im ), dc/ock and cycle gcnes. However, recent rescarch has idcntified multipl e feedback loops cont ro lli ng rhythm generation and express io n. Novcl mutations o f timeless throw more li ght on thc function s of per and tilll products. Analys is of pdf neuropcptide gene (ex pressed in c ircadian pacemaker cells in Drosophila), indicate that PDF ac ts as the princ ipal c ircad ian transmitte r and is in vo lved in output path ways. The product of cryptoci!rollle is known to function as a c ircadian photoreceptor as well as component of the c ircad ian c lock. This rev iew focuses on thc recent progress in thc fie ld of molecu lar rhythm research in the fruit n y. The genc(s) andthc gcne product(s) that a re in volved in the transmi ss ion of environmental informat ion to the clock, as well as the timing signal s from the c lock outward to ccllul a r func ti ons are remai n to be determined.

Key words: C ircadi an c lock genc, C ircad ian rhythms, Drosophila , Molccu lar rhythm

The spectrum of biolog ical processes controll ed by circadian clocks in li ving organisms range from the daily sleep/wake cyc le and levels of vanous enzy mes/hormones to 0 A synthes is and cell di visio n!. These circadian (L. circa, about ; dies, a day) rh ythms indeed have a genetic bas is2

. The circadian organi zati o n of any li vi ng organi sm is composed of three broad domains: (i) the input pathways - that transmit the environmental signal s (mainly light-dark cyc les) to the central oscillator/clock, ( ii ) the generation of timing sig nals in the central oscillator/clock, and (i ii ) the output pathways - that transmit these rhythmic signals with a 24 hr periodicity to the various clock controlled processes which ultimate ly result in overt rhythms (Fig. 1).

Clock genes are beginning to provide an understanding of the molecul ar mechani sms that underlie circadian rhythms. Over the last few years, by geneti c and mQlecular biological approaches novel genes (between 8 and 10) and their ro les in the generation of circadian rhythm were identifi ed in fruit fly (Tab le I). A number o f mutants that perturb temporal organization in the fly in a variety o f ways have been identified . Extensive coverage of both recessives and dominants has been achieved2

-4

.

*Phone: 04144-238343 Ex tn: 2 12 Fax: 04 144-238080 E-mail: psub @rediffmail.com

Chemical mutagenes is and P-element inserti onal mutations have been used2

-4. The goal of the e investigations is to define the molecul ar machinery that underlies the a lmost ubiquitous process of c ircadian rhythmic ity in the fruit fly . The period (per) and timeless (li /1/ ) genes in Drosophila encode mRNAs whose fluc tu ating levels define da ily molecular rhythms3. The clock prote ins also cycle in a 24 hI' period4

• The cyclic regul ation of per and rim and the manner in which they generate a molecular clock have been reviewed in numerous rev iews5

-9

. The two proteins (PER and TIM) accumulate during the night. Heterodimerization o f PER and TIM is required for their translocation into the nucleus, as tertain PER and TIM sequences, cytoplasmic localizatio n domains (CLDs), restrict monomers to the cytoplasm!o.

Heterodimerization also protects PER from the activity of a kinase encoded by double-time (dbt ). DBT promotes phosphorylation and turnover of monomeric PER proteins, which delays cytoplasmic accumulation of PERffIM complexes !! ']. The double-time (dbl) gene which encodes a prote in kinase is responsible for phosphorylating the per gene product. Mutant a lle les of dbr are lethal at the pupal stage in homozygotes, which suggest some other ro les 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 complexes and bind to per and tim promoters!3. Thi s binding is inhibited by PERffIM complexes. PERITIM

798 INDIAN J EXP B IOL. AUGUST 2003

F.NV IRONM F.NTAL SIGNALS

I!"PUT PATHWAYS

OUTPUT PATHWAYS -- OVERT

RHYTHM S

Fi g. I - The modcl of the circad ian systcm which includcs a ccntra l osc illator (c ircadian clock) that gcncrates rhy thmicit y. input pathways through whi ch thc osc illator rcce ives li ght-dark informat ion and output path ways through which the osc illator controls the observable rh ythms.

Table I - Clock gene mutallls of Drosophila lIIelall ogasrer

Gene Clock mutants Proposed funct ion Rcfe rences

period (per) Pan of the clock: togethcr w ith lilll gcncr:llcs a circad ian fccdback 43. 44 loop

lillll'less (/i lll ) . 01 . r;1 . U

/1111 • /1111 , 11111 Pan of the clock together w ith per gcncrates a circadian fecdback 17, 18, 40 and rilll .)"/. loop

clock (elk ) or Jerk (j rk) 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 transcription of per. lilll and vrille gcncs 46

dOlible- lillle Part of thc clock : phosphorylates PER and renders it unstabl c in thc II . 12 abscnce of T I M

Negativcly regulatcs per. lilll and pdf gencs: may bc a componcn t of IS output pathways

err " elY Photic cntrainmcnt. binds TIM in a light -dcpcndcnt man ncr 26.29

pdf pdf"

liIIl'

Outpu t frolll latcra l ncurons 16.33

lark LARK in volvcs in output path ways of eclosion rhy thm 47.48

lokeoll/ Ci rcadian control of fccding bchaviour 24

complexes act as negative autoregul atory co mponents of the clock directly associating with dCLKJCYC t 3

(Fig. 2). A key feature in the present model is that there is a

lag between the transcripti onal induction of per and I il1l on one hand and the nuclear transloca ti on of the repressor proteins that encode on the other. This lag creates a temporal separati on between phases of induction and repression, which is req uired to generate an osc ill ation. Without the separati on, the transcripts (involved in induction and repression) wou ld come to equilibrium. Another important feature is that the half-li ves of the per and lim mRNAs and proteins are rather short, hi ghly regulated and

. I d tO tt b f h . k . prectse y adapte . to e part 0 t e time eepmg mechanism. The present feedback loop model (Fig. 2) has engendered widespread consensus among researchers, although it has not entirely escaped crit icism (See 'Conc lusions and future perspectives').

Role of lemperalure It is a common understanding that ambien t

temperature has a sign ificant effect on the circad ian

rh ythm in the fru it fly; (i) tem perature affects the period of free run ning rhythms, (ii ) the phase of the rhythm is often signi ficantly affected by an ambient temperature level, and (iii ) temperature cycles could entrain the circad ian rhythms. It was found out that pel and perL flies have properties reciprocal to one another in regards to temperature and li ght intensity dependency of their free running periods. The wild type and per mutant flies were clearl y synchroni zed to 12: 12 hr LD at two different ambient temperature levels of 25° and 30°C (ref. 14).

Vrille - A gene regulating temporal PER and TIM synthesis

A novel regul atory loop within Drosophila's circad ian clock was identified by Blall and Young t5

A screen for cloc -controll ed genes recovered vrille (vri), a transcription factor essential for embryonic development. vri is expressed in circadian pacemaker cell s (lateral neurons) of larval and adult brains. vri RNA levels oscill ate with a rhythm ic periodicity. Cycling is direct ly regulated by the transcripti on factors, dCLOCK and CYCLE, which are also

SUBRAMANIAN et af.: CIRCADIAN CLOCK GENES IN DROSOPHILA 799

LIGHT -DARKNESS CUES

~ --=-!~" "" T R :

? T dc/k

I CYTOSOL I

~ P 8

+ Degradation \ o - rvtJU1 .-- Osci llating P RNAs ~

~'-t-____ ---,p_e_r __ _ rvtJU1 _ ~

lim ~ ; -rvtJU1 -~ ... vri ~

I -rvtJU1 -~

Proteosome

j

:_------------------------------------------// ______ ~p=df {MfRNA - ~ --------------------------------------~

Degradation

PER TIM 1 CYC ClK

? 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 e lements and CLK and CYC are positivc eleme nts o f the autoregulatory loop (involving E-box target sequences). PER monomers are unstable when Ihey are phosphorylated by DBT. CRY (The c ircadian photoreceptor) interacts with TIM and TrM is degraded by proteasomc. PER-TIM complex ac ts 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 invo lved in the output regulating pathways. PER-TIM -CYC-CLK tetrameric complex could ac ti va te circadian c lock regulatory elements (CCRE) of clock controlled genes (ccgs) which would lead to the expression of circadian rhythmicities via the c lock output pathways. Activating and inhibiting pathways are indicaled by solid and broken lines respectivel y.

required for oscillations of period and timeless RNA. Eliminating the normal vri cycle suppresses period and timeless express ion and causes long-period circadian rhythms and arrhythmicity , indicating that cycling vri is required for a functional Drosophila clock' 5.

Further, PER and TIM are additionally involved (alongwith dCLOCK and CYCLE) in a second autoregulatory loop, whereby they directly mediate the cycling expression of VRlLLE (VRl). VRl oscillations are required for per and tim expression, for overt circadian rhythmicity and for accumulation of PDF (pigment dispersing factor) , a neuropeptide supposed to regulate output pathways of rhythms' 6. Continuous vri expression suppresses PDF levels, indicating that cycling VRI is required for wild type

PDF function and VRI could regulate per and lim expression independently of PDF or via PDF'5.16

(Fig. 2) .

Timeless gene-Novel mutants and functions Three alleles of tim have been reported recently

(limO! was found to exist a decade ago) tim,i" a temperature sensitive, long period (26-30 hr) allele ' 7,

timSL, which was found as a suppressor of perL, and limuL

, which is known to generate rhythms with periods of around 33 hr ' 8. Characterization of these tim mutations showed that the Drosophila pacemaker can support very long period rhythms that are remarkably stable. How TIM is degraded was a mystery until the recent demonstration that proteasomes are involved in TIM degradation 19. TIM

~oo INDIAN J EXP BIOl, AUGUST 2003

was found to be phosphorylated, ubiquitinated and then targeted to the proteasome for degradation In

I· h II) response to Ig t . The free- running period (free run- persistence of

rhythms under constant co nditi ons) of lin/i' (a clock

mutation on the second chromosome) mutants is drasticall y lengthened in a temperature-dependent manner l7. PER and TIM protein levels become lower in lin/' mutants as temperature becomes hi gher. Thi s mutation red uces per and not lim mRN A abundance. In addition , PER constituti vely dri ven by the rhodopsin I promoter is lowered in ril mutants, indicating that li,, /' mainly affects the per feedback loop at a posttranscriptional level. An excess of I'er+ gene dosage can ameliorate all ril phenotypes, including the weak nuclea r locali zati on of PER, sugges ting that lilll ,.i, afrects circad ian rhythms by reducing PER abundance and it s subsequent transportation into nuclei as temperature increases 17.

Independent r e}!, lIlaLOI)' a ·ti vily of PER The molecul ar phenotype of lill/L (mutation

leading to amino ac id substitution at positi on 260) is prolonged nuclear locali za tion of the PERITIM uL

complex , but, per and lim RNA levels remain moderately hi gh for an ex tended time (- 10 hr)l x. Removi ng TIM uL with light brings the p er and lim RNAs to a lower level, co nsistent with the view that PER alone may be suffi cient for transcriptional repression of per and tilll genes IH. Although the TIM uL reg ion of the TIM protein has not been characteri zed functionally. the mutati on does not affect any of the protein fragments that were found in prior studies to independently bind to PER 18.20.

The lin/" phase response curve (chronobi ological jargon used to represent the displacement of circadian rhythms by short perturbations of li ght) indicated that the circadi an oscill ator has been altered with respect to the functions occu rring at late night. It was proved th at PER and TIM uL are physically associated during the interval of prolonged protein accumulation and immunocytochemical analysis showed that TIM uL

persists in photoreceptor ce ll nuclei for an extended time ls. Thus, the tilllUL mutation prolongs the duration of nuclear localizati on of the PERrrlM uL complex and affec ts li ght-independent degradation of TIM uLl 8

.

Even though the PERrrlM uL complex stays in the nucleus much longer than the wild-type complex does, Rothenfluh PI a i . lx, observed prolonged derepression of per and tim RNA. There are two poss ibl e ex planations for this: ( I) the PERrrlM uL

complex may not repress as effici entl y as wild type PERrrIM , or (2) the PERrrlM complex may not be the only repressor. These findin gs l R

.2o also lead to the

interpretation that TIM uL region may con tribute to the association of full -length PER and TIM proteins or allow association of unidenti fied factors in Iluenc i ng PER/TIM heterod imeri zati on/di ssociation in vivo.

TIM-independent reg ulatory activity of PER was also suggested by in vitro cell culture studi es IX. In absence of TIM. PER alone co uld repress dCLOCK/CYCLE mediated transcri pt ion of per in the cultured Drosophila S2 cells. Thus, in the nucleus, conversion of PERrrlM heterodimers to PER proteins appears to be required to complete transcripti onal repression and terminate each molecu lar cyc le lX

•

Earli er studies suggested that the stabi li ty of PER in the nucleus is also a control point that influences

. d I I II H h 'd IX peno engt 1 . owever, t e present eV I ences indi cated that dissociation of PERITIM compl ex to form nucl ear PER is necessary step in the molec ul ar regulation of period length of the ex pressed rhythms and in tilll UL mutant , interference with thi s step produces severely altered circadian rhythms.

Period alld clock inf711ence temporal patlems q{ hundreds of I l"anscripls

Of late, period gene was found to influence the ex press ion patterns of over 600 non-osc illating transcripts2 1

• Temporal ex pression levels of several hundred genes also differed significantly between wild type fli es kept in LO versus 00 but di ffered minimall y between pel' flies kept in the sa me two conditions (LO and 00). This indicates that the period-dependent circadian clock regulates onl y a novel set of rhythmically ex pressed transcripts. However, period regulates basal and light-regulated gene ex pression to a very broad ex tent. Recent studi es also showed that cycling in most of the genes was los t in arrhythmic clock mutants under light-dark conditions22

. Hence, expression of period ically regulated genes may be coordinated loca ll y 0 11

chromosomes where . mall clusters of genes are regulated jointly. These results further suggest that many genes in vo lved in diverse functions are under circadian control and also reveal the complexity of circadian gene ex pression in Drosophila .

The data indicating that all oscillating gene ex press ion is under Clk control , led to a hypothesis that Drosophila has no Clk-independent circadian systems. A larger number of genes is affected in Clk-

fl · h fl ' 2122 . t mutant les t an per mutant les . , suggesting tl1at

. 1 .

SUBRAM AN IAN eT al.: CIRCADIAN CLOCK GENES IN DROSOPHILA 80 1

Clk affects a wide range of genetic networks . Further, most of the circadian gene network is indirectly regulated by Clk23.

Takeout-gene controlling circadian feeding behaviour

So el al. 24 reported identificati on and characteri ­zati on of a new clock-regulated gene, takeout (to). 10

is implicated in circadian contro l of feeding behav iour. Its expression is down regulated in the clock mutants (per and Clk) tested. In wild type fli es, 10 mRNA ex hibits daily cycling pattern with a i lOvel phase, showing delays relative to those of better characterized clock mRNAs (per and lim) . The E-box containing 10 promoter seq uence revealed a remarkable sequence ident ity with the E-box region of the per and tim promoters. However, unlike per and lilli , the E-box of 10 is not in vo lved in the amplitude and phase of transcriptional cycling of 10. Hence, it cou ld be concluded that the circadian delayed transcriptional phase of to is most likely the result of indirect regulation through unknown transcripti on facto rs.

Cl'yptoch.-ome - circadian photoreceptor and component

Cryptochrome is a flavin contain ing protein, presumably derived from photolyase, which is recently reported to med iate circad ian photoreception in an imals incl uding Drosophila25

. It is coded by cry ?6 ?7 d h d h . II . gene-'- an t e gene pro uct can p YS lca y associate

with TIM28 and appears to be an important regulator of li ght-dependent TIM degradation in some cell types. In uy" mutants the stabiliLy of PER and TIM is affected. cry RNA cyc les and highest levels are found in the early part of the da/9

. This cycl ing persists in constant darkness and is dependent on PER, TIM, CLK and CYC2X

-30. In per and lim null mutants, cry

RNA levels are constantly low and in elk and eye mutants the levels are constantly hi gh, indicating po iti ve regul at ion by the former and negative

I · b I I 2629 H regu atlon y t le atter two components . . owcver, CRY protein cycles only in the presence of light:dark cycles. Apparently this cycl ing is driven by light­sensiti vity of the protei n because DD (constant dark) conditions lead to constan t accumul ation of CRy26.29

.

Yeast two-hybrid assays showed that CRY interacts with PER3o

, TiM and PER-TIM complex in presence of light but not in dark28. Further analyses indicated that C terminus of CRY is responsible for the light dependence of the interact ions with PER and

TIM30. This interaction was spec ific to function al CRY, since cr/, a mutation that leaves CRY partially inactive is unable to interact with TIM or PER-TIM3!. Further, CR Y was found to undergo a photochemical change allowing it to interact with TiM both in the cytoplasm and nucleus. This interaction represents the initial step in circadian phototransduction, and renders the PERffIM complex inactive and unable to

. . . h . C db k27 28 H partiCipate III t e negative lee ac '. owever, how CRY transduces li ght signals to the molecular pacemaker that generates circad ian rhythms IS

unknown at present.

PDF and pacemaker cells of the fly The circad ian organi zation of locomotor activity

and eclosion is governed by a pacemaker within a discrete set of neurons in the Drosophila brain. Nevertheless, molecular studies have es tablished the presence of autonomous circad ian clocks in isolated appendages and excretory st ructures32 ; the molecular mechanisms underlying clock function in these tissues may not be identical. These peripheral clocks may coord inate the physiological processes, which could ultimately result as overt temporal expressions (locomotion and eclosion).

The pacemaker cells (latera l neurons in the brain) express high levels of clock genes (at least per and lim) and are implicated in the circadian control of activ ity rhythms. These cell s cou ld potentially communicate with the other ti ssues of the tly by electrical, chemical or hormonal signals - or a combination of these3!. The neuropeptide pigment dispersing factor (PDF) is one likel y candidate for pacemaker cell output. PDF leve ls normally cycle with a circadian rhythm! 6.33, and are altered in a number of clock gene mutants! 5. Although, PDF is considered as a prime candidate for an output molecule, a large proportion of output genes is spec ific for a sing le overt rhythm. These genes may also be restricted in their expression to specific central or peripheral ti ssues that contain osc illators.

Levels of pdf mRNA do not cycle. Expression of the protein is also non-cyclic in the cell bodies of lateral neurons, but cycles at their axon terminal s, which may be inac tive of cyclic release3

1.34 . It was found that PDF protein accumulates in the latera l neurons (circadian pacemaker cell s) with a circadian rhythm showing the highest accumul ation in the earl y part of the day and lowest at night. Overexpression of PDF lateral neurons does not eliminate its cycling or behavioural rhythms, suggesting that the mcchanisms

802 INDIAN J EXP BIOL, AUGUST 2003

that mediate its cyclic re lease are not easily saturated 1 6.3 I .

Further, the pe riod of thi s accumulation rhythm is shortened by the pel's mutation and continuous accumulation o f PDF in the dorsal brain is associated with arrhythmi a and a variety o f period changes in adult locomotor acti vity . PDF could couple the molecul ar clock to timed behavio ur and vri conveys essential regulatory signals from the c lock to POF7

.8

.15

(Fig. 2). Analysis of the null mutation o f pigment-dispersing

facto r gene, p((r demonstrates that pdf is indeed in volved in the c ircadian output regulation o f c ircadi an overt rhythms 16. Recent research also showed that some of the c lock genes affect pdf express ion. Mutatio ns in dclk or cyc reduce PDF staining in latera l neuro ns and disruptio n o f vri

' 11 ' I I ' PDF .. 151 1 OSC I atlons a so resu ts 1Il sta llllllg" .

Postembryonic development of circadian clock functions

The clock genes, period and timeless, are expressed cycl ically in the larva l centra l nervous system of Drosophila and da il y oscillations o f per express ion pers ist throughout metamorphosis in latera l neurons o f larvae35

. PER and TIM cyclings in these neurons may be responsible for the phenomenon of ' larval time memory' . Kaneko et al.35 further showed that phase shifts of molecular oscillations during the larval stage were smalle r than those measured by adult behaviour, suggesting mo lecularly transient responses during development.

A light pul se given to larvae was found to phase shift adult behaviour. These phase shifts were di fferent in directio n and magnitude between pel and wild type, indicating that per is invo lved in larval time memory . Further, histo logical experiments suggested that li ght stimuli can set thi s larval c lock by at least two input pathways, o ne disrupted by a mutation (cr/) in the putative blue- light receptor c ryptochrome26

, the other by a mutation o f the norpA gene, which e liminates certain larval responses to light35

, as well as light respo nses of adult compound eyes and ocelli 36

,

The adult activity and eclosio n rhythms of fli es can be synchronized by light-dark (LO) cycles experienced during the larval stages, although the animals were kept in constant darkness. Thi s larval pacemaker responsible for the synchronization of circadian rhythms is a ' time-memo ry' clock, which takes in environmental stimuli necessary to set its

phase during development. Once set, the pacemaker can control the rhythmic output of later events that will occur at a particul ar time of day. Recent lines of evidences support a ' time-memory ' c lock. Extra­retinal vi sual structures have been proposed to playa role in both larval and adul t c ircadi an photoreception37

.38

. Malpel et al. 39 , analysed the interactions between extra- re tinal structures of the visual system and the clock neuro ns during brain development. Larval opti c nerve has contacts with the latera l neurons in the embryoni c brain . Analyses of visual system-defecti ve genotypes showed that the absence o f the afferent optic nerve resulted in defec tive late ra l neurons fo rmation39

.

S ince no overt c ircadi an rhythmi city of behav io ur or physiology has been detected in larvae of D. meianogaster35

, there are no known circadi an outputs at this developmental stage, except a hypothetical one from the larval LN s to the antiphase neurons, which may be in part respo nsible for the sy nzhroni zation of the molecular rhythms o f the latte r35

. However, c ircadian clock is operating in larvae, under the co ntrol of at least one of the key clock genes (per). Since a ' time-memory clock' does not need an output pathway, until c ircadian rhythmicity is finally expressed in adults, the pacemaker neurons in larvae probably would not require an anatomical target.

Conclusions and future perspectives The first problem with the current model is the

time-delay questio n: a ll o f the processes in thi s casual chain (Fig. 2) are normally carried out rapidly, and some transcription factors can be transported into the nucleus and be acting on the nuclear targets within minutes. There is a necess ity of ' time delays' at one or more time po ints in the chain (to account fo r the degradation o f clock gene products at specific ti me points) to make thi s loop take 24 hr to complete, but at present evidences (kine ti c data) are lacking to indicate ho w these delays could be accompli shed.

Further, if the per gene is transcri bed from a constitutive promoter, it can rescue rhythmicity in arrhythmic null mutant per40 fli es and the RNA and prote in are found to be rhythmically abundant, in spite of the constant rate of transcription4o. It has a lso been observed that expression o f tim cONA transgene could also rescue rhythmicity in arrhythmic null mutant lirn40

fli es, even though the l im RNA is not rhythmic in abundance41

. Hence, it appears that fl ies can be rhythmic if either per or tim is consti tutively ex pressed; 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 rhythmic in the lateral neurons but PER is available widely in other fly tissues, and in the ovaries the prote in is neither nuclear nor rhythmic4t

• Furthermore, the per gene affects the period of the courtship-song rhythm in Drosophila , even though thi s rhythm has a period of 1 min, which is obviously very rapid to depend on a transcription-translation feedback loop. This raises the poss ibility that these clock prote ins may have a primary function that is as yet unknown and that their effects on circadian clocks may be secondary . Additi onall y, the ava ilable evidences led to a hypothes is that there may be additional prote ins, which could modi fy the actio n(s) o f c lock gene products that have a lready been identified42 and there may also be additional transcriptio n fac tors regulating clock gene activities.

The understanding o f molecular organi zation of circadian clock is not yet complete. Many of the details of c lock input, output pathways of synchronizati on and detail s of passing the timing signal to various body ti ssues-have yet to be completely worked out. Understanding the molecular bases o f the clock (from input to output) in the amenable system of Drosophila will shed light on the workings o f the c lock and its influence on the behavio ur and phys io logy of higher mammals and humans.

Acknowledgement Financial support from DST, New Delhi in the

form of a research project to PS is gratefull y acknowledged .

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