seminars in THE NEUROSCIENCES, Vol 7, 1995: pp 15-25

The multide roles of beg- in the Drosoj~hila circadian clock I 1

Paul Hardin and Kathleen K. Siwicki*

Behavioral genetic studies have shown that the period (per) gene influences many aspects of Drosophila circadian behavior From these studies, as well as the molecular, neuroanatomical, biochemical and euolutionafy studies r&wed here, the emerging picture of per is that of a temporally regulated, temperature compensated, transcriptional rqtn-essor that controls its own circadian expression and, when expressed in the a#qtniate cells, the circadian behavior of the animal.

Key words: behavioral rhythms /biological clocks /feedback regulation /insects /period gene

JUST BEFORE THE DAWNING of the molecular revolution, the period (per) gene of Drosophila mlanogaster was defined by behavioral genetic studies of three muta- tions that resulted in abnormal circadian rhythms.’ The isolation and sequencing of this gene in the mid- 1980’s2a ushered in the present day multidisciplinary enthusiasm for using this clock gene to probe diverse aspects of Drosophila circadian biology. The per story has been featured in many recent reviews.‘-I6 In this article, we describe how recent work in many dispa- rate disciplines has yielded a new level of under- standing the role of perwithin the circadian clock, and also consider the prospects for further insights into how this gene influences multiple aspects of circadian function.

Behavioral genetics

There are dozens of extant pervariants in D. melanoga- stq including chemically induced mutations, poly- morphisms isolated from natural populations, and innumerable transgenic strains bearing in-vitro muta- tions. Similarly, many diverse aspects of rhythmic

Fmm the Department of Biology and Institute of Biosciences and Technology, Center& Advanced Invertebrate Molecular Sciences, Texas A&M University, College Station, TX 77843-3258, and *Department of Biology, Swarthmore Coh’ege, Swarthmore, PA 19081-1397, USA

01995 Academic Press Ltd 1044-5765/95/010015 + 11$8.00/O

behavior have been characterized, to varying extents, in these different genetic strains. The focus in this section will be the chemically induced per alleles, and aspects of their behavior (see Table 1) which ulti- mately must be explained by any model of this gene’s function in the Drosophila circadian system.

The most familiar behavioral phenotypes of the per mutations are effects on circadian rhythms of eclosion and locomotor activity. The original arrhythmic allele, pep’, is a null mutation phenotypically, in that its effects on circadian behaviors are similar to the effects of per deletions. ‘*“~‘* Another ‘arrhythmic’ allele, pd4, results in minimally detectable circadian func- tion: While the locomotor activities of pep4 flies are arrhythmic by conventional periodogram analysis, higher resolution spectral analyses reveal weak rhythms in a significant fraction of pp flies.lg Most flies home- or hemizygous for the well studied p& mutation have eclosion and locomotor activity rhythms with 29-hour periods at 25”C.‘~18~20 The original short-period allele, pt??, yields eclosion and activity rhythms with periods of 19-20h,‘**’ while two additional short-period alleles, per*and p&& generate behavioral rhythms with periods of 1617hz1 and 22.5h,“*,*s respectively.

In permutant heteozygotes, the periods of circadian rhythms fall between the wild type and homozygous mutant values, thereby demonstrating the semidomi- nance of per mutations. ‘J’*~~**~ In fact, semidomi- nance is a property of most known clock mutations (see articles by Loros, and by Menaker and Takahashi, this issue), which is consistent with the suggestion that the products of these genes might function as oligomers.‘5 Extra copies of wild type per result in shorter periods, and fewer copies yield longer peri- ods.20,24 these dosage effects led to the concept that pi and pef are hypermorphic and hypomorphic alleles, respectively.20v25 More recently, however, stud- ies of the behavioral effects of targeted permutations indicated that shortened circadian periods can result from a variety of single-amino acid substitutions at or near the site of the original p& mutation, or from insertion of a peptide antigen at the N-terminus.26S27 It is difficult to imagine how so many different mutations could enhance PER function, leading to

15

I? Hardin and K. K. Siruicki

Table 1. Behavorial phenotypes of per mutations

Allele Locomotor activity Temperature LD locomotor Courtship song References and eclosion period depenclance ZlCtiVi~ period

at 25°C of period

Wiid type 24h constant (15+29”C) morning Se evening peaks 50-60 s 1, 28, 29, 33 pefll Arrhythniic - active all day arrhythmic 1, 19, 32-33 per04 Arrhythmic - morning & evening peaks - 19 pe+ 29 h 20% longer (15+29”(Z) 70-90 s 1, 28, 29, 33 pers 19-20 h constant (17-29°C)

late evening peak early evening peak 38-45 s 1, 28. 29, 33

5% longer (at 15°C) per= 16-17 I1 8% shorter (15-+29”C) very early evening peak - peGk 22.5 h - early evening peak variable 22T123

the conclusion that j4x? almost certainly causes a decrement of function in this region of the protein.*@‘.

One of the defining features of circadian rhythms is temperature compensation, that is, the stability of period over a wide range of temperatures. In some per mutant strains, however, circadian period is tem- perature-dependent. The periods of wild type rhythms are constant at temperatures ranging from 15°C to 29°C; pe? periods also are constant (although shorter than wild type) from 17°C to 29”C, and very slightly longer at 15”C.‘8~2g Yet, the periods of pe# and pe? mutant rhythms are significantly influenced by tem- perature: pe# rhythms become longer (by about 20%) ,*8,*g and pe? rhythms become shorter (by about S%)*’ as the temperature is increased from 15°C to 29°C. Another per variant which renders the rhythms temperature-sensitive is a molecularly engineered mutation that deletes sequences encoding a stretch of repeated Thr-Gly pairs.2g*so Thus, in addition to being required for freerunning behavioral rhythms, and acting as a sensitive regulator of their periods, it seems that another function of @-in the circadian clock is to compensate for temperature fluctuations.

While most of circadian behavioral analysis con- cerns free-running rhythms in constant darkness (DD), circadian systems function in nature to allow organisms to anticipate daily environmental cycles. The effects of per mutations on the patterns of fruit fly activity in light:dark cycles (LD) have been analysed in detai1.‘g~2’~22~3’ Wild type D. melanogaster are most active at dawn and dusk, with the morning and evening bouts of activity beginning a few hours before lights-on and lights-off, respectively.s1~32 Period-short- ening mutations advance the phase of the evening activity peak in LD, while mutations that lengthen freerunning periods delay the onset of evening activity. 21*22,3’ These predictable effects on the phase of-evening activity in LD contrast with the relative stability of the phase of morning activity in period-

altered mutants. Arrhythmic pet”’ mutants tend to be more active when lights are on, and less active in the dark, with behavior apparently changing in response to (rather than in anticipation of) the light cycle transitions.‘“*“’ The LD activity patterns of most pdJ mutants, however, are more like wild type than peg”,” another indication that this mutation retains some minimal level of per function.

Another role for per (albeit not in the circadian clock) is indicated by its influence on the ‘song’ produced by males engaged in courtship behavior. Kyriacou and Halls3 reported that the period of this song rhythm, which is about 55 set in wild type males, is shortened in per’ males and lengthened in p&’ males. Despite some controversy,34*3” these appear to be real and reproducible phenotypes.g*36*37 In fact, the 35sec period of the Drosophila simulans courtship song rhythm was transferred along with its pn-gene to D. meZan.ogaster transformants. While the molecular and biochemical studies described in subsequent sections are beginning to unveil some of the mecha- nisms by which per regulates daily and circadian rhythms, the mechanism of its effects on the courtship song rhythm remains as mysterious as ever.

Molecular biology of per

In the early 1980’s genetic mapping studies had localized the per gene to a small segment of the X chromosome at position 3Bl-2.“.‘” This region of the genome was isolated by the Young group at Rock- efeller University2 and the Hall and Rosbash groups at Brandeis University.4 The boundaries of chromoso- mal deletions that delimited per’s position were located within the cloned genomic region, and transcripts mapping within this interval were tested to determine which were specifically altered by chromo- somal aberrations that disrupted rhythmic behavior. Only one transcript of 4.5kb met this criterion,2 and

16

The Drosophila circadian cloth

Lhus was considered to be the po- Lranscripl. Its iclentity was confirmed functionally when genomic DNA fragments conLaining all or part of the 45kh RNA transcription unit were Lransfornied in10 /I&” flies and tested for behavioral rhythmicity; all geno- mic fragments containing Lhe entire protein coding region of the 4.5kb transcripL were capable of rescuing behavioral rhyLhmicity, confirming that lhis was indeed Lhe />fl producL. ‘H.+0~4 ’

A screen for cDNA clones representing the pn transcript yielded several isolales, which were grouped into three structurally distinct classes.” The most frequently (n = 4) recovered variant was Lype A, which has eighL exons and conLains a 1218-amino acid open reading frame (Figure 1). Two cDNAs, representing type B, differed from type A by having an additional intron in the 3’ untranslated region (S’UTR) as well as an unusually spliced intron which removes 96 amino acids within exon 5. A single cDNA representing type C was found in which only the first four introns are removed, thereby changing the last 149 amino acids to a unique 107 amino acid sequence. These pnsplice variants, which were all recovered from a head cDNA

library, would each encode a different protein. Recent resulLs, based on direct measurements of head RNA, confirm that type A is indeed the most abundant per Lranscript and that Lranscripts containing the type B intron in the S’UTR are present, but contrast with earlier results in that neither transcripts having the type B-specific intron in exon 5 nor transcripts corresponding to type C are detected (Y, Cheng and P. Harclin, unpublished results). T~LIS, these recent findings suggest Lhat only 2 per mRNA types, which would encode the same PER polypeptide, are present in the fly head.

Of course, there has been considerable interest in identif+ng the function of PER proLein. The initial searches of protein sequence databases only revealed similarity between a series of Threonine-Glycine (Thr-Gly) repeats of PER and sequences of Serine- Glycine repeats in some proteoglycan core proteins.“.” Thus began the widely recognized but ill-fated hypothesis that PER is a proteoglycan.3*5V4’ More recently, extensive biochemical studies have shown that PER is not a proteoglycan.“” Although databank searches failed to provide useful insights into the

per gene 5’ m 3’ A

per RNA’s

“L ,’ per peP1 pe2 , . (243) (464) (589)

PER protein NH; COOH-

1218

Figure 1. Structure and organization of the pergene and its products. The structure of the pergene which gives rise to the most abundant per RNA type (type A) consists of eight exons, which are divided into untranslated (white boxes) and translated (black boxes) sequences, and seven introns (black lines). Another major splice variant (type B) is derived from splicing of an intron in the 3’ untranslated region (grey lines). Both of these per mRNA splice variants have the same coding region (twisted black line). The PER protein is 1218 amino acids from N-terminus (NH,+) to C-terminus (COOH). Three regions, Cl, C2 and C3 (white hatched boxes), are conserved between Dipterans and Lepidopterans. The PAS domain (black hatched box) is involved in PER dimerization. The Thr-Gly (TG) repeat region (black box) is involved in the courtship song rhythm and temperature compensation of circadian rhythms. The amino acid numbers definin the limits of each region are indicated below the protein, and the sites of the pd, pd’, and p 9 mutations are shown above the protein. The arrhythmic pet” mutation is a stop codon; the long-period pe# and short-period pa’ mutations result in single amino acid substitutions at the indicated positions.

17

P Hardin and K. K. Siwicki

biochemical function of per; the nature and positions of the pns and pe# mutations helped to identify functional domains of the protein. By combining transformations and sequencing of per DNA from the mutants, pe? and pe# were shown to be due to missense mutations at amino acids 589 and 243, respectively, while perD’ was found to be a stop codon at position 464 (Figure 1).s4*& Although the sequences surrounding pe? and PI& were not note- worthy at the time, they have subsequently yielded some insights on function (see later).

Localizing per expression and the circadian pacemaker

In adult flies, per is expressed in the nervous system and in several nonneural tissues including the gut, Malpighian tubules, and the gonads.455”g Within the central nervous system, PER protein is concentrated in a few clusters of neurons in the protocerebrum, as well as in the nuclei of glial cells dispersed throughout the optic lobes, the brain and the thoracic gan- glion. 45*4s50 In the peripheral nervous system, per is expressed in photoreceptor cells of the compound eyes and ocelli, in the ring gland and in peripheral nerves. 4s47 Some aspects of the adult expression pattern have been detected in mid-to-late pupal stages. 46*47 The gene is expressed transiently in the central nervous system of developing embryos, and is generally not detectable in larval tissues,45*47.51 although there was one unconfirmed report of expression in larval salivary glands.42.

Among the most impressive and heroic efforts in the history of Drosophila rhythms have been those directed towards determining which of the many sites of per expression are essential for controlling circa- dian behavior. Based on the effects of manipulating per expression using a heat inducible per gene, per expression in adult flies was found to be both necessary and sufficient for locomotor activity rhythms.*’ The adult brain was implicated in control- ling behavioral rhythms when transplantation of pe? brains into p&’ flies yielded animals with short-period activity rhythms. 52 The analyses of genetic mosaics were equally impressive. First, the locus of per-function was mapped to the head with externally marked mosaic flies.53 Then Ewer and coworkers carried out an extensive analysis of internally marked mosaics,4g and found that the control of circadian behavior could not be mapped to a single discrete focus within the head. Weak rhythms were present in several

mosaics with only small patches of per+ expression in putative glial cells in the ventral brain, suggesting that neuronal per expression is not necessary for circadian rllythms.4” Nonetheless, they found that rhythms were more robust in mosaics containing per’ in lateral brain regions, where some neurons express the gene.‘”

There are three bilateral clusters of pwexpressing neurons in wildtype brains: (1)6-g dorsal neurons (DNs) that lie in the dorsal protocerebrum, (2) a group of 3-7 lateral neurons (LNd or LNl) that lie dorsal and anterior to (3) a separate group of 4-10 lateral cells (LNv or LN2) located between the medulla and the lateral protocerebrum at the level of the esophagus.45*49*54*55 The hypothesis that per; expressing lateral neurons might control behavioral rhythmsJ5 was substantiated by recent evidence that, in certain strains of per transgenic flies which exhibit nearly normal circadian behavioral rhythms, the lateral neurons are the only cells in which PER protein is detectable55 (I. Edery, personal commu- nication). Thus, while per expression in either glial cells or lateral neurons can produce rhythmic behav- ior, the evidence suggests that rhythms resulting from lateral neuron expression are stronger. In fact, the presence of lateral neurons may be required to produce even the weak activity rhythms observed in the ‘glial only’ mosaics described above,4g since robust (and rhythmic) glial per expression is not sufficient to generate free-running behavioral rhythms in disco mutant flies, whose lateral neurons are missing due to developmental defects.48~5” It is possible that per- expressing lateral neurons and glial cells represent redundant circadian pacemakers in the fly nervous system, reminiscient of the multiple pacemakers in the hypothalamus, pineal, and retina of lower vertebrates.57-5g

The projections from one group of lateral neurons have been revealed in spectacular detail in recent anatomical studies.54*56 Although antibodies used to detect PER or PER-kgalactosidase fusion proteins did not reveal the projections of these cells,4548*50 evi- dence from other insects suggested that neurons whose cell bodies and projections stained with an antibody to pigment dispersing hormone (PDH) might be circadian pacemaker cells.60V61 This inspired Helfi-ich-Forster’s double-labelling studies in Droso- philaF4 which clearly demonstrated that the ventral group of pmxpressing lateral neurons (LNv or LN2) overlaps completely with a cluster of 8-10 PDH- immunoreactive neurons at the anterior margin of the medulla. There are four large (diameter 8-12 pm)

18

The Drosophila circadian clock

and four small (diameter 4-6 urn) PER-PDH-contain- ing neurons. Processes of the large LNv’s project ventrally into the posterior optic tract, then to the contralateral optic lobes, where they arborize exten- sively across the distal surface of the medulla.56 The small LNv’s project dorsally to the lateral horn of the protocerebrum. There are other weakly PDH-immu- noreactive cells in this dorsal location,56 as well as the PER-containing DNs, but the question of their identity was not determined in the double-labelling experi- ments.54 The LNd clusters were not stained with anti- PDH. The projections of the LNv’s, as revealed by PDH-immunoreactivity, define, at least in part, the anatomy of the circadian system in Drosophila.

Molecular rhythms and the feedback loop

Even though the per gene was in hand, and several years of studies had succeeded in defining its protein sequence and developmental expression patterns, there had been little real progress in elucidating the mechanisms by which it influenced circadian timing. This situation began to change when rhythmic fluctu- ations in the intensity of PER immunostaining were observed during the course of experiments directed towards defining the tissue distribution of PER.45 These protein rhythms initially were observed in photoreceptors and optic lobe glial cells,45 but later were seen in all perexpressing cells in the head including central brain neurons and glial cells.4s In each of these different cell types, PER immuno- reactivity was most intense near the end of the night (ZT21), and barely detectable at the end of the day (ZT12) in LD conditions, and these fluctuations persisted during constant darkness4* (see Figure 2). Recent immunoblotting studies have confirmed that these rhythms in staining intensity represent true fluctuations in the abundance of PER, with an amplitude of approximately 10-fold.4s*62 Rhythmic fluctuations were also observed for PERS protein, but the peak occurred earlier than for wild type PER in LD, and the period of the rhythm in DD was about 20 h 43.48

The level at which these rhythms in PER protein were regulated was a mystery, particularly since earlier reports (based on Northern assays of RNA prepared from whole flies) asserted that per RNA did not exhibit rhythmic changes in abundance.4*6s Subsequent measurement of RNA from fly heads, where PER protein both cycles4’ and functions to control beha- vioral rhythms,4g*53 showed that per mRNA does

19

indeed exhibit circadian fluctuations in abundance.64 The RNA cycles 6-8 hours in advance of the protein in LD, where RNA starts to accumulate during the middle of the day (ZT6) and peaks early in the night (ZT15), and also continues during constant dark- ness64 (see Figure 2). In the pe? mutant, the phase of the RNA oscillation is shifted several hours earlier than in wild type in LD, and the period is shortened to -20 h in DD, while no oscillations are seen in the pep’ mutant regardless of environmental condi- tions.64 Taken together, the relative phases of PerRNA and protein cycling and the effects of pep’ and pe? on these cycles suggest that changes in @RNA levels give rise to changes in PER protein, and that the protein feeds back to influence the timing of its own RNA’s rhythm (Figure 2). The parallel effects of the point mutations on both molecular and behavioral cycles, along with the results of experiments using heat inducible per genes to produce stable phase shifts in behavioral rhythms,65 suggest that this feedback loop in perexpression may be an integral component of the Drosophila circadian clock.

Since per has such a diverse expression pattern, it was of interest to know whether this feedback loop operates in all per=expressing tissues, or whether it is restricted to certain cells in the nervous system that mediate behavioral rhythms. The compound eyes, which are not required for either entrainment or circadian activity rhythms,66.67 nonetheless show robust per RNA’2.62 and protein48s2 cycling in the same phase as the rest of the head during LD cycles. Likewise, body tissues (with the exception of the ovary) exhibit per RNA cycling in phase with that of the head during LD conditions, but do not manifest any known circadian rhythms.68 In fact, the inability to detect per RNA cycling in early Northern assays of whole fly RNA4*6s may be explained by the large proportion of non-cycling per RNA in ovaries.68 During constant darkness, the amplitude of per RNA cycling in bodies dampens much more quickly than in heads.“’ Also, PER protein is not readily detectable in glial cells in constant darkness, but continues to cycle prominently in lateral neurons and photoreceptors4’ (&vi&i, unpublished observations). Thus, whiie per expression is rhythmic in many cells and tissues in ID, some of these oscillators seem to be more robust than others under freerunning DD conditions.

The model of a molecular feedback loop has provided a framework for subsequent studies of per function, with many focused on determining how PER influences the levels of its own RNA. A series of experiments with constructs containing per upstream

l? Hardin and K, K. Siwicki

sequences fused to a reporter gene showed that pn RNA cycling is regulated at the level of RNA synthe- sis.6g The clear implication from this result is that PER regulates of its own gene’s transcription. Correlated with this is the fact that PER is nuclear in all tissues except the ovary,50 and per mRNA expression is not rhythmic in the ovary,” suggesting that the pn feedback loop operates only in tissues where PER is nuclear. Since per RNA levels are low when the protein levels are high (see Figure 2)) PER would be predicted to inhibit its own gene’s transcription. This prediction was tested by overexpressing PER at high levels in photoreceptors, under the control of the Drosophila ninaE (Rhl) promoter, resulting in the complete

repression of endogenous per RNA expression in those cells.“’ Thus, PER so~ml~ow represses its own RNA’s synthesis.

In order for the negative feedback loop to operate with a 24hr period, it is crucial that the phase lag between the rise in peg RNA (late day) and the accumulation of nuclear PER protein (middle of night) be precisely regulated. There presently is little evidence pertaining to how this phase lag might be determined. It has been suggested that a new circa- dian rhythm mutant called timeless (tim), might inter- act with PER in this phase of the feedback loop70371 (see article by Sehgal, this issue). Also, since this is the phase of the circadian cycle when light pulses cause

State of the

Levels of per RNA

and protein

Figure 2. Relationship between the state of the per feedback loop and the cycling of per RNA and protein. The states of the feedback loop at different times of day are shown within 4 cells, where the gene (hatched box) within the nucleus (dark grey) is transcribed to give rise to per RNA (twisted white line), which is translated in the cytoplasm (light grey) into PER protein (tangled black line), which then feeds back to inhibit per gene transcription. The arrows connecting the gene, the RNA, and the protein represent maximal (thickest arrows) to minimal (dashed thin arrows) effects of the different components of the feedback loop. The times illustrated are 4 11, 10 h, 16 h and 22 h after lights on (Zeitgeber Time) or subjective lights on (Circadian Time), for flies entrained to cycles of 12 h light (white bar): 12 h dark (black bar). The relative abundance of per RNA u and protein l are shown for the same four timepoints, and range Erom baseline (CT 4 for RNA and CT 10 for protein) to maximal levels (CT 16 for RNA and CT 22 for protein).

20

Tile Drosophila ciwadian clock

delays,” it seems likely that light-activated signal transduction pathways have access to the feedback loop at this phase

All data accumulated thus far are consistent with a role for PER in transcriptional regulation. This model is further supported by the finding that PER shares a sequence motif, termed a PAS domain (Figure l), with three other transcription factors: the human aryl hydrocarbon receptor nuclear translocator (ARNT) ,72 (MR) ,7:3.74

the murine aryl hydrocarbon receptor and the singhwninded (sim) gene of Droso-

phila. The PAS domains of these four proteins contain two 51 amino-acid repeats near the ends of-a 250-300 amino acid stretch of similarity.72*73 ARNT, AhR and Sim each contain a separate basic-helix-loop- helix (bHLH) domain that mediates DNA binding and protein dimerization.‘“*77 The lack of this bHLH domain (or any other known DNA binding domain) in PER suggests that PER does not bind to DNA directly. Important insights into the specific role PER plays in regulating transcription and circadian phe- nomena have come from its biochemical analysis.

Biochemical properties of PER

When the PAS domain was discovered,7’*7s nothing was known about its biochemical function. The first suggestion that it might be a dimerization domain was based on the functional relationship between the Ah receptor and ARNT, which are thought to dimerize, enter the nucleus, and activate transcription.73 Recent experiments indicate that this domain is involved in protein-protein dimerization, as complete or partial PER proteins containing this domain can mediate homotypic or heterotypic interactions both in vitro and in vivo.7g The pe# mutation, which resides in the PAS domain, weakens its ability to dimerize.7g This finding suggests that PAS dimerization may contribute to those aspects of circadian function which are altered by pe# (i.e. freerunning period, temperature compensation and phase in LD), and also may explain the semidominance of per mutations.7g Although PER contains no known DNA binding motifs, its abilities to dimerize and to repress per transcription suggest that it binds to (and inhibits) a transcriptional activator in much the same way that the inhibitor of DNA binding (Id) and the extramacrochetae proteins inhibit the MyoD and daughterbss transcriptional activators, respectively. 8o,*1

Another aspect of PER biochemistry that has received considerable attention recently is phosphor-

21

ylation. Mutations in the dun.ce gene, which lead to elevated CAMP levels, cause shortened freerunning periods of about 23 h, and have abnormal phase response curves,” suggesting that something is awry in their circadian pacemakers. Protein kinase A (PKA)-deficient Dco mutants were tested for effects on locomotor activity rhythms. The majority of Dco mutants with 15-30% of normal PKA activity were arrhythmic, and those individuals that were rhythmic had abnormal rhythms,” suggesting that phosphor- ylation by PKA may play some role in the circadian clock. One possible target for such a kinase is PER itself, which is phosphorylated in a time dependent manner.“” As PER accumulates during the night it undergoes a progressive increase in phosphorylation, until it is abundantly phosphorylated as its levels decline around dawn.“3 This temporal pattern of PER phosphorylation was seen under LD and DD condi- tions, and was reported to occur earlier during an LD- cycle in per? 43 Although we do not yet know how or even if PER phosphorylation is involved in the rhythm generating mechanism, the dramatic effects of Dco mutations on behavioral rhythms suggest that it will be important to characterize this phenomenon in more detail.

Evolution

Behavioral genetic and biochemical studies of the PAS domain and the Thr-Gly repeat region were both rooted in and substantiated by phylogenetic compar- isons. Such comparisons also have begun to reveal aspects of clock function by telling us how per has evolved to meet the needs of different species.

Although efforts to isolate mammalian genes homologous to ps either by homology or PCR-based approaches, have not been productive to date, these strategies have yielded authentic per homologues from several Drosophila species,38*83*84 and more recently, from other insect familiess5 and orderss6 A direct comparison of per sequences of Drosophila to those of the silkrnoth Antheraea pernyi indicates that the encoded protein sequences show 39% overall identity, and confirms the general pattern of interspersed conserved and non-conserved regionss6 first observed among per genes of different Drosophila species.8s Of the six regions conserved in Ih-osofhiZa (cl-c6), three are also well conserved between fly and silkmoth per (see Figure 1). A stretch of 77 amino acids at the N-terminus (cl) is 57% identical between fly (D. vitilis) and moth (A. peny~J .86 The longest conserved

I? Hardin and K. K. Siwicki

stretch (~2) extends from the PAS domain to 48 amino acids downstream of the pe? site; this region is 46% identical between moth and fly.86 The strongest homology in the entire gene occurs at the pe? end of the c2 region, with 73% identity in a stretch of 51 amino acids. This includes the ‘peptide-S’ sequence used to generate anti-PER antibodies, which labeled pacemaker structures in molluscan eyes and rodent brains.87*88

Another short stretch of 38 amino acids in the c3 region is 50% identical between fly and moth.86 Between the p& domain and the c3 region is an extremely variable domain, which includes the notori- ous Thr-Gly repeats of D. meZanogaster@r (Figure 1). The rapid evolution of this repeat region has been extensively documented by Kyriacou and colleagues. Using PCR with primers from the conserved flanking sequences, they have cloned the repeat region from a plethora of individual flies. Their sequences have revealed (1) wide variations between species in the length and sequence composition of this region,8g*g0 and (2) considerable polymorphism in the number of Thr-Gly repeats among natural populations of D.meZa- nogas&g1*g2 which also had been observed among laboratory strains.sO Rather than dismissing the poly- morphic region as functionally uninteresting, they have analysed the patterns of occurrence of the various alleles in European and North African popula- tions of D. melanogasti The results revealed a strong latitudinal cline in the length of the Thr-Gly repeat, with a predominance of longer (Thr-Gly)sO alleles in the north and a higher frequency of shorter (Thr- Gly) r7 alleles in the south. ‘* While allelic variations in the number of Thr-Gly repeats might be expected on the basis of the relatively high mutation rates of repetitive DNA sequences,” the presence of a latitu- dinal cline rather than a random distribution of alleles suggests some selection for longer repeats at higher latitudes. ‘* Although the allele frequencies did not correlate with any known environmental variables, the authors suggested that the Thr-Gly repeat region might be subject to selection for temperature com- pensation of circadian rhythm period.‘* This sugges- tion was based on studies of the locomotor activity rhythms of transgenic flies lacking the Thr-Gly region, 3o which were found to be abnormal in their temperature compensation.*’ Recent work in Kyr- iacou’s lab supports the view that there is a relation- ship between Thr-Gly length and temperature com- pensation (C.P. Kyriacou, personal communication).

Other than a small effect on temperature com- pensation, the circadian behavior of flies deleted of

the Thr-Gly repeat region is essentially norma1.2g*30 Yet, this mutation has a profound affect on the courtship song rhythm, specifically shortening its peri0d.s’ Moreover, replacement of the D. melanoga- ster Thr-Gly region with that of D. simulans, followed by reintroduction of this chimeric per gene into D. melanogast~ yields D. m.elanogaster males which pro- duce a D. simulans courtship song.38 Thus, it seems that one of the functions of this region is to confer species specificity to the courtship song rhythm.“.

Comparisons among the repeat regions of several Drosophila species have revealed an intriguing correla- tion between the length of the Thr-Gly repeat and the constitution of the immediately flanking sequences.“*” ‘Mix and match’ experiments, where the repeat region from one species is fused with the flanking region of another, indicate that the Thr-Gly repeats and their flanking sequences are indeed coevolving, and further imply that these evolutionary dynamics are a result of selection for temperature compensation (C.P. Kyriacou, personal communica- tion). This may explain why different Thr-Gly lengths have different selective values at different latitudes in D. melanogaster (with no polymorphisms in the flank- ing sequences), whereas in D. simuluns, the three major Thr-Gly length variants all have flanking changes associated with them, and appear to be selectively equal, as there is no latidudinal cline in D. simulans repeat length.g4 These analyses of pervariants have provided one of the few cases in the field of molecular evolution where genotypic variations can been correlated with a specific phenotype of an organism, and thus provide a uniquely accessible system for testing evolutionary mechanisms.

Conclusions

The emerging picture of per is of a gene whose product regulates its own transcription by a negative feedback loop. The state of the molecular feedback loop oscillates between high levels of mRNA and high levels of protein with a 24 h period, and thereby generates a circadian rhythm. Certainly other pro- teins, encoded by other genes, interact with PER in this molecular clock, but the more we learn about this first clock gene, the more circadian phenomena it seems to influence, and thus it continues to conform to our initially naive expectations that it would be a central component of the fly’s circadian system. While PER does not bind DNA directly, it can dimerize, via the PAS domain, with itself or other PAS-containing

22

The Drosophila circadian clock

transcriptional regulators. A mutation in the PAS domain (p&) interferes with dimerization and lengthens circadian period, presumably by delaying PER’s ability to inhibit transcription during the dark phase. Mutations in the most highly conserved region of pm; which includes ix??, quicken the pace of both behavioral and molecular oscillations, but the bio- chemical basis of this effect is obscure.

Inevitably, the wealth of new information concern- ing per function has created more questions than it has answered. These questions, which concern all aspects of per function, include: determining how pe? and the new per’ allele accelerate the clock’s pace, identifying PER’s dimerization partner, determining how environmental LD cycles determine the phase of the molecular oscillations, defining how per oscillators in different tissues are coupled, revealing the con- tribution of phosphorylation to PER function, and determining how PER buffers its activity against temperature changes. The answers to these questions should go a long way towards determining how per produces rhythmic modulations of behavior, or at least to crystallize a new set of questions which will bring us closer to this goal.

While the traditional approach to studying clock mechanisms has been to work inward towards a pacemaker, either forward from entraining stimuli or backwards from rhythmic outputs, Konopka’s beha- vioral mutants cut straight to the heart of the pacemaker. Molecular genetic analysis revealed a novel and mysterious protein which, after a few false starts and initial frustrations, has begun to reveal the inner workings of a biological clock to a fascinated scientific audience. Now, ironically, we can begin to think about deciphering the inputs and outputs of the Drosophila clock by working outwards from the core of the pacemaker.

Acknowledgements

We thank Jeff Hall and Bambos Kyriacou for critical comments on the manuscript. Supported by NIH NS31214 (I?H.) and NSF IBN-9310256 (ILK-S.).

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