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GENETICS OF CIRCADIAN
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RHYTHMS Jeffrey
C. Hall
Department of Biology, Brandeis University, Waltham Massachusetts KEY WORDS:
02254
Drosophila . Neurospora , rodents , clock mutants, cycling RNAs and proteins
CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GENETIC VARIANTS USED TO ANALYZE RHyTHMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dros ophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial Organisms . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . M OLECULAR CHRONOBIOLOGY . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . Clone and S eq u ence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclical Expression of Substances Related to Rhythms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION Contemporary genetic studies of biological rhythms involve the isolation and application of mutants as well as the cloning and manipulation of DNA sequences-some but not all of which correspond to loci defined by the "clock mutations." These mutations have, in the main, resulted from screens for strains exhibiting altered circadian rhythms or their apparent absence. Indeed, several mutants with shorter-than-normal or anomalously long circa dian periods, and those that are at least superficially arrhythmic, have been isolated. Other mutants were found by simply noticing something amiss in conjunction with rhythm experiments, or were found by testing the rhythmic ity of certain visible and biochemical mutants.
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One idea behind these genetic approaches is that they could provide one more strategy for understanding what biological clocks consist of. Maybe, then, studies of rhythm mutations, as well as the nucleic acids and encoded proteins defined by them, will increase chronobiologists' chances of elucida ting something about the intriguing, but so far intractable, mechanisms by which so many different kinds of organisms keep time. Some recent investigations in the purely genetic area have expanded the types of rhythm variants available and the kinds of organisms in which single-gene mutations affecting time-dependent phenomena have now been identified. Some of the molecular studies involving rhythms are continuing to analyze mutationally defined clock genes and trying to understand what the products of these loci are doing; other studies in this area are beginning to isolate genes known initially from the standpoint of how their encoded RNAs are expressed. This review discusses recent findings in the genetic area, the molecular one, and where these two intersect chronobiologically. An extensive treat ment of the background information concerning genes and rhythms will be avoided, as this has been covered in several recent articles (27, 31, 43-47, 63, 64, 69, 77, 114, 139, 146-148). GENETIC VARIANTS USED TO ANALYZE RHYTHMS Drosophila PERIOD MUTANTS The most salient clock mutants in these dipteran insects remain, alas, those encoded by mutations at the X-chromosomal period (per) locus of Drosophila melanogaster. The basic features of these genetic vari ants and the phenotypes they cause have been reviewed ad nauseam. The most remarkable aspects of these early studies are that the first set of systematically induced rhythm mutants (in any organism) included a short-period, a long period, and an arrhythmic strain, and that these three independently isolated per mutations were by definition all allelic (65). A large debt is owed by all who work in the area of clock genetics to R. J. Konopka, who led this initial screen for rhythm mutants and has continued to induce and isolate them during the past 20 years. Some of Konopka's per mutants have not (or have barely been) reported. Their basic properties are described here, because they are pertinent to certain fundamental properties of biological clocks: "temperature compensation," on the one hand; and "entrainment" by, or "phase-shift responses" to, environ mental stimuli, on the other. (See 90 or 145 for text-book treatments of these and other details of circadian rhythms.) One of the relatively new per mutants is caused by a long-period allele, perL2 . The circadian locomotor activity rhythms of adults expressing this
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mutation are basically like those affected by perLl (cf 48, 65) . Thus, perLl and perL2 exhibit 28-30 h periodicities ("taus," or 71'», at 25° C. (See 24, 46 for brief accounts or examples of perL2-influenced behavior. ) When such "free-running" behavioral rhythms were monitored in constant darkness (called "DD"), at lower or higher temperatures than the standard 25° C, both perLl (66) and perL2 (33) adults exhibited shortened or lengthened 71'>, respectively. The degrees to which each of these long-period mutants has lost the excellent temperature compensation associated with wild-type rhythms of this poikilothermic organism are very similar (33). A new short-period per mutant has also been found (R. J. Konopka, personal communication). This mutant's free-running period, from the same kinds of activity monitorings referred to above, is an astonishing 16 h, a value 3 h shorter than in the original pers mutant (cf 65 , 151). The latter, whose circadian pacemaker already runs 5 h faster than normal ( 24 h in D. melanogasler), can nevertheless be reset each day to exhibit 24.0 rhythmicity in conditions (called "LD") of cycling light (12 h) vs darkness (12 h). Thus, this altered-T mutant entrains to the LD cycles (see below, Figure 1). Yet, perss behavior in these quasi-natural conditions is not like that of wild-type: The "evening peaks" of locomotor activity are in the daytime, instead of being at "lights-off' as in wild-type (M. Hamblen-Coyle, D. A. Wheeler, M. Rosbash, J. C. Hall, in preparation). Conversely, perLl and perL2 display evening peaks in the night, though these mutants, too, are driven into exhibiting 24.0 h periodicities in 12: 12 LD (Hamblen-Coyle et aI, in prepara tion). It was not necessarily expected that these short- and long-T mutants would be able to be reset, each day, to an LD regime whose cycle durations lie far from the endogenous (free-running) periods specified by these genotypes (also see section below on mammalian mutants). That such mutants can be reset indicates that pers has the phase of rhythm delayed fully 5 h each day, and that the "driven" rhythmicities of perLl and perL2 are phase-advanced by the same amount. These LD phenomena (Figure 1) reflect the underlying "phase-response" system (associated with essentially all pacemakers found in organisms), because they are only circa-dian and hence must be reset daily to respond to the 24 h cycles of the environment. Curiously, the determination of a phase response curve (PRC) for Dro sophila locomotor activity rhythms-now so widely used in studying circa dian rhythms in these species of insect-had never been reported in a primary publication (though see Ref. 44). This has now been remedied (28). As is usual for any eukaryotic organism, part of the wild-type PRC for Drosophila defines a light-insensitive phase in DD, i.e. a l2-h segment of a free-running day during which light pulses lead to no phase shifts; if the flies had been in 12: 12 LD before proceeding into DD, the insensitive portion of the PRC, =
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called the "subjective day," corresponds to when the lights were on during entrainment. Light pulses delivered during approximately the first half of the subjective night led to phase delays, whereas pulses given during late sub jective night induced phase advances (28). per mutations are pleiotropic in their effects on several temporally related phenotypes. These include phenomena whose time-scales are far outside the circadian range, in either direction. In addition, some of the per mutant phenotypes reported (e.g. learning or visual-response abnormalities) do not have any obvious temporal dependence (for review, see 43). The more recently discovered, or revised, elements of per's pleiotropy as it relates to time-based phenotypes warrants some further discussion: per's action during development Developmental timing appears to be altered by peru, pers, and per-zero (perOl) mutations (75). The first of these had the most obvious effects and indeed noticeably lengthened the durations of larval and pupal stages. pers had the opposite effects, though they tended to be less pronounced. perOl erratically lengthened or shortened a given developmental stage compared to the wild-type durations, depending on the rearing conditions (DO, LO, or LL, the last of these meaning constant light). The end of Drosophila's development is its senescence. In this regard, everyone asks (and rightly so) if per mutations affect adult lifespans (e.g. for pers, perhaps the mutant flies live fast, die young). The answer is apparently "no," at least in the sense that, in two sets of studies, there were no systematic lengthenings of per u s lifespan and no shortenings effected by pers (33, 64). At high temperature, however, perOl adults lived a few days fewer than did flies expressing the rhythmic genotypes-the two mutants just noted, as well as coisogenic per + controls (33). This mildly abnormal phenotype, caused by an "arrhythmic" per allele, could, however, be a rather nonspecific viability problem (see Ref. 4 for a general discussion), as opposed to a "time-of-life" deficit. To attempt an interpretation of the positive results obtained from assaying the effects of per mutations on preimaginal stages, Kyriacou et al (75) note that embryonic expression of per mRNA and protein in the central nervous system (CNS)-as demonstrated by James et al (60), Liu et al (81), and Siwicki et al (127); or conceivably that which was detected in the salivary glands, as shown by Bargiello et al (7�ould influence the timing of subsequent developmental stages. In this regard, per expression during the larval period itself is minimal [by Northern blotting, and by in situ detection of the encoded protein in salivary glands only (7)], or has been undetectable (60, 81, 1 27); the same holds for about the first half of pupation (60, 81, 12 0). Pupal expression of per can, with no difficulty, be hypothesized as in volved in the control of periodic ecIosion (for discussion see 8, 81, 120, 127). ,
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Likewise, the gene's adult expression is readily rationalized as one of the factors underlying the fly's locomotor activity rhythm. But is the embryonic expression also important for one or more of the per-influenced phenotypes that occur later in the life cycle? One way to attempt answering this kind of question is to perform temperature-shift and heat-pulse experiments on a conditional mutant. None existed for
per, whereby the gene's function is
"off' at restrictive temperature and "on" at permissive. So a temperature sensitive mutant was created by generating transformants whose transduced
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DNA has a "heat-shock promoter" (hsp) fused to per coding sequences
(34).
Subjecting the developing transformed animals, whose genetic background was perOl, to various hot and cold regimes indicated that the gene's action during preimaginal stages was neither necessary nor sufficient for locomotor activity rhythms to be exhibited by adults. For example, if these transformants developed at low temperature (with per off), and were "LD entrained" under that condition, they were nevertheless rhythmic in their adult behavior if the temperature was turned up once the flies proceeded into constant darkness
(34) . These conditional-mutant experiments have been extended. For example, it is of interest to ask whether a circadian clock is running in these
hsp-per
transformants during the LD cycles that preceed heat treatment and transfer to DD conditions. Recall, as background, experiments showing wild-type flies that were put through their development in DD and then were left in this condition as adults exhibited very weak or no rhythms in their locomotor activity (25). Thus, at least one light-dark transition is necessary to start a strong circadian clock running in this organism, as well as to set its phase. It was hypothesized
(33) that a clock function, as kicked into action by
exposing the flies to LD cycles, could be already operating in animals whose
per allele is off, i.e. in the unheated hsp-per transformants. Thus, when the gene's activity is subsequently switched on, it would be involved in some thing like linking the activity of this pacemaker to "oscillator output," which is part of the pathway mediating the final phenotype (sleep/wake cycles) but is not part of the central clock. This hypothesis about per's action was seriously undermined by the finding that the phase of activity rhythms exhibited by these transformants did
not depend on that of the LD cycle to which the flies
were exposed, prior to turning the gene on at the beginning of DD (33) . Therefore, this gene's action seems to be necessary for pacemaker functioning itself. To bolster the conclusion that per expression is necessary only for the manifestation of the adult fly's rest/activity cycles-and hence is not used at earlier developmental stages for setting up the nervous system to run the rhythms later on-Ewer et al (33) monitored the
hsp-per transformants'
behavior during LD at low temperature and then continued to follow them in DD at high temperature. The question was: Is there any "leakage" of heat\
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Figure 1 Locomotor activity rhythms of adult Drosophila. General movements of these in dividual D. melanogaster flies were monitored automatically (re infrared light-beam breakages) as in Hamblen et al (48). Successive days of the activity events (each such event being represented as a vertical mark, dropping below one of the horizontal lines) are plotted left-to-right and also top-to-bottom (hence, days 1-2 on the top horizontal line, days 2-3 on the next, and so forth). For about the first 10 days of monitoring in each case (LD conditions), the lights cycled on (open portions of horizontal bars on top of each "actogram") and off (filled portions of bars). "Lights-on" times were at noon, i . e. 4 h after the left-most "8" ( = 8 am) in each plot. After the arrows, the lights were turned off (DD conditions). (a) Wild-type actograms, after Dushay et al (28) [left panel] and Hamblen-Coyle et al (49) [right]; each record was from monitoring a male's
CLOCK GENETICS shock-promoted
665
per expression at low temperature, during LD? If so, then
some of these flies, whose genetic background was perOI, should have been somewhat wild-typelike in their LD behavior (cf 28, 49 , 96) , i.e. they would have anticipated the times of L-to-D and D-to-L, by becoming gradually more active in advance of these environmental transitions, and would have been relatively inactive during the middle of the lights-on and lights-off periods. In contrast,
perOl,s behavior in LD seems mostly to be a response to the
environment (Figure l c): rather little activity in the dark, then a rapid (sus
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tained) increase at (and during) the light (see 52 for another viewpoint). The answer from these further
hsp-per experiments was that many of the trans
formed flies that expressed a rhythmic phenotype following a postentrainment temperature-upshift had nevertheless behaved in a notably
pero-like manner
"
"
(cf Figure l c) during the prior monitoring of their activity in LD at low temperature (33). When considering per expression in the developing CNS of embryos from a purely descriptive point of view, note that the in situ localizations of the gene's transcript, its protein, or stainings involving a per-promoted reporter protein have been examined (in published reports) only at low resolution (60, 8 1 , 1 27). Curiously, the regions of such expression (ventral/medial) within a given ganglion are near to those where a "per relative" is also expressed. This is single-minded (sim), defined originally by embryonic lethal mutations causing defective development of the CNS's ventral midline (136) . A stretch of relatively N-terminal, per-encoded amino acids (ca 15% of the total on-paper polypeptide, which is about 1 200 amino acids long) is weakly similar to a relatively N-terminal region (ca 20% of the total) within the sim sequence ( 1 9) . Note (and recall later) that this region of similarity occurs in a region of the per sequence that is highly conserved among Drosophila species ( 1 7 , 135 , 1 44). The sim protein is expressed in nuclei of developing cells of the CNS ( 19),
activity; during
LD, the flies tended t o become active a t least an hour i n advance o f lights-on or DD, the "morning peaks" of behavior largely went away, and the "evening peaks" expanded. (b) Actogram for a elk mutant male (after 28); the fly was "forced" into 24.0 h rhythmicity during LD, in which conditions the evening peaks were earlier than those observed for wild-types; during DD, the "free-running," short-period rhythms caused by this mutation were lights off; during
manifested, wherein the most active portions of a given cycle began and ended at least an hour
(c) Actograms of flies expressing a "per-zero" mutation-left: a male hemizygous for per OJ (after 29); right: a female heterozygous for this mutation and a deletion of the period gene (after 49); in LD, the beginnings and ends of the dense segments of activity markings tend to coincide with the L portions of a given environmental cycle; in subsequent DD, earlier on successive days.
these forced rhythms degenerated into arrhythmicity.
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and per also has a nuclear distribution in certain tissues of the adult, such as photoreceptors, cells of the gut, and in the fly's excretory organ (8 1 , 120, 1 27) . In adult neurons, however, the clock gene product appears to be a cytoplasmic protein (127, 1 52). Recent examinations of per-promoted {3galactosidase activity in the "reporter transformants" (cf 81 ) indicate that the fusion protein (approximately the N-terminal half of per, with the C-terminal half replaced by the laeZ gene of E. coli) is similarly expressed throughout the cytoplasm of adult neurons; the stainings seem not to be present in the nuclei of these cell bodies in the brain (X. Liu, personal communication). per signals in tissue types for which the fusion protein is found in nuclei (see above) also seem to be in the cytoplasm of these cells (X. Uu, personal communication). The same reporter transformants exhibit, at embryonic stages, cytoplasmic subcellular localization in the neurons of the developing CNS (S. T. Crews, personal communication). These observations included the demonstration of a separate, nonoverlapping set of CNS cells expressing the per and the sim proteins; moreover, the midline cells removed by the action of a sim mutation left per's cellular expression normal (S. T. Crews, personal communication). Therefore, the per-sim similarity remains an uninterpretable curiosity. This includes the fact that the predicted proteins encoded within these two genes are only somewhat similar to each other, i .e. to nothing else in databases. Finally, in contrast to the case of sim, a per-null genotype allows not only for full viability (all the way to adulthood), but causes no gross morphological defects in the CNS of embryos (or any other stages) .
Salivary gland rhythms A variety of rhythmicities associated with these glands have been reported in Drosophila larvae, albeit with rather scanty data ( 1 04) . One reasonably detailed report showed an apparent circadian rhythm of membrane potential in cells of the larval salivaries, as determined by applica tion of a voltage-sensitive dye ( 143) . Provocatively, this rhythmicity was said to be "damped" or absent in salivary cells of per01 larvae. These phenomena have recently been called into question: Direct recordings of membrane potentials from salivary-gland cells of wild-type larvae, over the course of a given day or even longer, revealed no rhythmic fluctuations in this physiolog ical parameter (G. D. Block & M . W. Young, cited in 57). Another phenotypic effect of per mutations on salivary gland gland physiology , which does not overtly connect to a rhythm, involves intercellular communication among electrically coupled cells within the gland (7). The product of this gene could therefore be a coupling agent. The possible rhythm connection is the following speculation: per-mediated communication among neurons might be the way in which the gene's action participates in building or operating a neural pacemaker (for reviews, see 147 , 148) . Additional findings relevant to this hypothesis involve the 5-1 5 h periodicities that can be extracted by spectral methods from locomotor activity records of pe,o flies
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(e. g. 22, 24, 49) . These hidden rhythmicities in the so-called arrhythmic mutants have been interpreted to mean that the pacemaker underlying these rest/activity cycles is noncircadian and that the per gene product serves to couple the fundamental (relatively high-frequency) oscillator (for review, see 26). It could follow from this view that per promotes clock function but is not per se a component of it. However, what if the hypothetical coupling function occurred within individual neurons, instead of among them? None of the results pertinent to, nor the inferences stemming from, the hidden rhythms of pero demand that the neuronal coupling agent acts intercellularly. It could just as well function within individual neurons, each one of which would be a circadian pacemaker by virtue of per's action . Thus , the gene would be back in the fold as a key component of the central elements of the clock.
Ovarian diapause It has recently been asked if per's influence on temporal ly dependent phenomena extends to helping the flies discriminate short (relatively little daylight in a 24 h cycle) from long days. The latter condition leads to ovarian diapause in many forms, including D . melanogaster, as demonstrated in experiments performed at very low temperature ( 1 22). All the per variants tested for this phenotype did well: Saunders ( 1 2 1 ) found that females expressing either a long (L2)- or a short (S)-period allele were identical to wild-type in their "critical day lengths," or CDLs (whereby 14 h of daylight is "diapause-averting"). The two arrhythmic types that were tested might have been predicted not to be able to detect 14-h (or other) diurnal periods, but they did do. Yet, they were not like wild-type, in that perOJ females had CDLs 2-3 h shorter than normal ( 1 2 1 , 1 23); and per females, who are homozygously deleted of the gene (and, necessarily, of two neigh boring X-chromosomal transcription units as well; 8, 1 03), exhibited 5 h shorter-than-normal CDLs ( 1 2 1 ) . In spite of these mutant phenotypes, the investigators concluded that per expression is not "causally" involved in day or night-length measurements ( 1 2 1 , 1 23), which therefore would be con trolled by the action of a "photoperiodic clock" whose actions are only at best "modulated" by an absence of this clock gene. -
High-frequency rhythms
Certain per mutants have been shown, or sug gested, to cause defects in various high-frequency ultradian rhythms. The fastest such rhythm is the ca 3 Hz heartbeat of 3rd instar larvae (for review, see 109). Heartbeating was said to be erratic inperOJ animals, though this has been reported in abstract form only (23 , 82) . A more recent study of this problem was performed by J. Ewer (personal communication). He found that the heartbeat of a given animal (which was monitored noninvasively, using a method based on light-path interruptions) could be a clean 3 Hz beating, or could consist of periods of regular beating interspersed with periods of erratic signals and interruptions . Both bouts of erratically timed beats and the
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interruptions were of variable, and sometimes quite long, durations. In addi tion, the heartbeat of a given larva could change dramatically over the duration of the recording period (usually 20 min). However, none of these types of records, or temporally dependent trends, were consistently associated with a particular per genotype. This included the fact that there was no more occasional irregularity in perOl or per records than in wild-type larvae (J. Ewer, personal communication). Incidentally, no noticeable expression of per gene products is detectable in the larval heart-or in any larval tissues in one series of studies (8 1 , 1 27); though others (7 , 1 20) report per protein and transcripts in the salivary glands of larvae. In any event, per pleiotropy, in terms of mutational effects, does not extend to the very high frequency range of rhythmicity in the larval heart. There are, on the other hand, many reports (most recently reviewed in 70) of per mutant males exhibiting defects in a rhythm of courtship song, whose ultradian periods in wild-type D . melanogaster are about one minute. Males expressing pers sing with about 40 s rhythms; peru leads to ca 80 s cycles; and perOl males do not exhibit any apparently regular lluctuation of the relevant song parameter (rate of tone pulse production, accompanying the courtship wing vibrations). These mutant and normal phenotypes have been extensively discussed in recent years. One reason for this is that some investigators have attempted to show (20, 35) or argue ( 10) that the singing rhythm is not a feature of these males' wing-vibration-generated sounds. The validity of the song rhythms has recently been reiterated through several experimentally and analytically based arguments (74, 76). Portions of these studies showed that Crossley (20), as well as A. W. Ewing (in some pilot experiments performed by that investigator, prior to his study described in 35), have indeed recorded rhythmic songs; furthermore, the periods were in the usual (wild-type) range for this species. The two reports (74, 76, respec tively) that include reanalyses of Crossley's and Ewing's data also discuss the several technical and algorithmic reasons for these two other investigators' claims that original rhythm results (7 1 ) could not be replicated. (For detailed discussion. see 10, 43, 69, 70, 1 24). If we assume for the time being that the song rhythms are real, there are some new genotype-phenotype correlations. Certain of these stem from the fact that the periods of such rhythms are species-specific, in that D. simulans males vary their rates of song pulse production over cycle durations in the 30-40 second range (7 1 , 73, 144); and the D . yakuba song rhythm period is on the order of 70--80 s ( 1 34). [These three species are all members of the melanogaster subgroup (80) .] With regard to D. simulans and D. melanogaster, the genetic etiology of the song rhythm difference was shown to map solely to the X chromosome, from analyzing the singing of reciprocally hybrid males (73). These studies of the hybrids' behaviors suggested that interspecific differences in the X-
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chromosomal period genes' informational content or expression could be the factor(s) underlying the presumed evolutionary divergence of this behavioral phenotype (which, incidentally, has adaptive significance in terms of repro ductive isolation) (72, 73). All this has been confirmed by some clone and sequence experiments augmented by interspecific gene transfers. Wheeler et al (144) isolated per+ DNA from D . simulans (via homology to D . melanogaster probes) and introduced this material into D . melanogaster, by creating the appropriate germ-line transformants (cf 16, 150); the genetic background of these barely partially hybrid males included hemizygosity for perOI. The flies sang as if they were D . simulans (144) in that they expressed rather fast-running "song clocks," i.e. the periodicities were squarely in the 30-40 s simulans range that for melanogaster being 50-65 s. Therefore, the source of this behavioral difference between these two species of Drosophila resides at only one locus. Sequence comparisons of portions of simulans per to the corresponding regions of the melanogaster gene showed minimal divergence, except within a subset of the gene's largest exon (which corresponds to ca 570 amino acids, i.e. about 45% of the total protein). The ca 360-bp segment encodes a series of alternating threonine-glycine pairs, which is (at least descriptively) a hallmark of this gene's informational content in some, but not all, Drosophila species (e.g. 17, 58, 102). The Thr-Gly repeat, moreover, has been im plicated as being especially important in the control of song, but not circa dian, rhythms (150). Thus, further interspecific gene transfer experiments were performed to produce transformed males carrying per of D . melanogas ter from which 700 bp (including the aforementioned 360) had been removed and replaced by the corresponding cassette from D . simulans; the reciprocal chimeric transformant type was also generated. The former males sang as if the whole per gene was from D . simulans and the latter as if they carried a per + DNA fragment entirely from D. melanogaster ( 144). Finally, this study showed that intra- and interspecific variations in the length of the Thr-Gly repeat were probably not responsible for the single-gene control of the song rhythm difference between melanogaster and simulans; but instead that 1-4 amino acid substitutions (just C-terminal to the repeat region) have occurred over evolutionary time and define the intragenic etiology of these species specific courtship song rhythm differences. CLOCK MUTATIONS AT LOCI OTHER THAN PER
Certain engineered per mutants are song rhythm variants only (cf 150). Indeed, all the transfonned types noted above exhibited similar circadian rhythms of locomotor activity; the same is found when comparing the behavior of wild-type D . melanogaster and D . simulans adults ( 144). Reciprocal types of rhythm mutants are now known: The X-chromosomal Clock mutation, isolated (64) by virtue of a ca 1 .5 h shortening of the circadian rhythms of locomotor activity (Figure Ib),
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leads to essentially normal one-minute song rhythms when elk is hemizygous in males (28) . Also, disconnected mutants (so named because of their eye brain disconnections; 132) sing in an essentially normal manner (70); yet their locomotor activity is largely arrhythmic (29), i .e. similar to that of per o adults (Figure l c). These findings on the elk mutant could mean that the fly's song and circadian clocks share some but not all mechanistic components (and of course these pacemaker mechanisms could not be the self-same entities , given that the relevant periodicities differ by 103). disco's rhythm defect implies that the eNS abnormalities, which are probably caused by these mutations (see below), are more important in the brain than in more posterior portions of the adult nervous system. This inference is based on the following: (a) Eye-brain connectivity, or even the presence of eyes, is largely irrelevant to the fly's circadian system (reviewed in 43); it follows that this aspect of the disco phenotype is not responsible for the mutant's circadian-arrhythmic behavior (see discussion in 29); thus, other features of the fly's head neuroanatomy are likely to be deranged in disco. (b) The pacemaker for the courtship song-as defined by the focus of a per-mutation's effects on the behavior of genetic mosaics-seems to be in the male's thoracic nervous system (R.I. Konopka, C. P. Kyriacou, 1. C. Hall, unpublished data, cited in 42), whereas the circadian pacemaker is almost certainly in the head (given the mosaic results reported in 68). The recently determined details of rhythm defects caused by elk were accompanied by a fairly fine-level localization of the mutation responsible for these phenotypes. elk may not define a novel gene because it maps so very near the per locus (28) . Whether this variant is mutated in a close neighbor of per (and there are some molecular candidates, cf. 8, 83, 103), or within the confines of that locus remains undetermined. If the latter is true, where and how elk is mutated would be interesting, given that it could be: (a) associated with amino acid substitution in an informatively different location from that responsible for the other intragenically mapped short-period mutant, i.e. the pers-defined serine (which became substituted by asparagine on induction of this mutation; 9 , 151); or (b) a "hyper-expression" regulatory variant, based on the negative correlation of per + dosage with circadian period (18, 130, 152); and (c) instructive about the control of circadian and song rhythms which are usually affected in parallel by per variants (the other exception, if "perC1k" turns out to be one too, appeared in 150). Another X-chromosomal rhythm mutation, Andante (And), is like per in that it affects circadian rhythms (67) and probably leads to abnormal song cycling as well. Thus, And causes a "moderately slow" circadian clock (by definition) and slightly longer-than-normal song rhythm periodicities in males hemizygous for this mutation (70). And's basic phenotype (with respect to which the mutant was isolated by Konopka et al.; 67) is that, for circadian
CLOCK GENETICS
67 1
rhythms of either eclosion or adult locomotor activity, the periods are 1 - 1 .5 h longer than normal. The mutation is semidominant, as are essentially all
And is also associated with a melanogaster, called dusky (dy):
circadian rhythm mutations (summarized in 27). pigmentation mutation, long known in D.
And map to the dy locus (by recombination and using chromo dy mutant was found to exhibit normal circadian rhythms (67) . However, some newly induced dys (59; J. M. Ringo, H. B. Dowse, cited in 57) do lead to 25-26 h rhythms, which are essentially the same as those observed in tests of the original And mutant (67). On the other hand, one new dy exhibits short-period circadian rhythmicity (1. M. Ringo, H. B. Dowse, personal communication), and still others (57 , 59) have normal rhythms (cf. 67) .
Not only did
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somal aberrations), but it is per se dusky-winged (67). Yet, a classical
Despite the genetic complexities implied by these results, the rhythm and pigment functions defined by these mutations could be the same, or at least subsets of a "complex locus" of related (primary) gene actions. Is
And-dy,
then, interesting, or could a biochemical defect (related to biogenic amines, hence body color and neurochemistry; cf. 107) indirectly affect rhythms in a manner destined not to be incisively informative? Alternatively, perhaps the relevant amine-containing substance (if any) is involved in actual pacemaker functioning. Both possibilities should be kept in mind with regard to some recent findings involving
ebony (e) body color mutants in D. melanogaster: Flies
homozygous for a given mutant allele exhibit a variety of locomotor rhythm anomalies, both in DD and in LD (94). Interestingly, eclosion rhythms, determined for
e cultures, were indistinguishable from wild-type (94), so that e
these mutants are the most specific yet in their rhythm abnormalities. Yet,
mutants have long been known to be pleiotropically defective in other adult behaviors, which are not apparently related to clock functions; and there are some data on e-induced "amine abnormalities" as well (see 94 and 1 07 for background literature). The classical visual-response defects exhibited by
e
flies were suggested to be involved in the rhythm defects, because blockage of
no e double mutants-partly suppressed the
all light input through external photoreceptors--
Further
Quick links to online content Annu. Rev. Genet. 1990. 24:659-97 Copyright © 1990 by Annual Reviews Inc. All rights reserved
GENETICS OF CIRCADIAN
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RHYTHMS Jeffrey
C. Hall
Department of Biology, Brandeis University, Waltham Massachusetts KEY WORDS:
02254
Drosophila . Neurospora , rodents , clock mutants, cycling RNAs and proteins
CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GENETIC VARIANTS USED TO ANALYZE RHyTHMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dros ophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial Organisms . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . M OLECULAR CHRONOBIOLOGY . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . Clone and S eq u ence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclical Expression of Substances Related to Rhythms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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INTRODUCTION Contemporary genetic studies of biological rhythms involve the isolation and application of mutants as well as the cloning and manipulation of DNA sequences-some but not all of which correspond to loci defined by the "clock mutations." These mutations have, in the main, resulted from screens for strains exhibiting altered circadian rhythms or their apparent absence. Indeed, several mutants with shorter-than-normal or anomalously long circa dian periods, and those that are at least superficially arrhythmic, have been isolated. Other mutants were found by simply noticing something amiss in conjunction with rhythm experiments, or were found by testing the rhythmic ity of certain visible and biochemical mutants.
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One idea behind these genetic approaches is that they could provide one more strategy for understanding what biological clocks consist of. Maybe, then, studies of rhythm mutations, as well as the nucleic acids and encoded proteins defined by them, will increase chronobiologists' chances of elucida ting something about the intriguing, but so far intractable, mechanisms by which so many different kinds of organisms keep time. Some recent investigations in the purely genetic area have expanded the types of rhythm variants available and the kinds of organisms in which single-gene mutations affecting time-dependent phenomena have now been identified. Some of the molecular studies involving rhythms are continuing to analyze mutationally defined clock genes and trying to understand what the products of these loci are doing; other studies in this area are beginning to isolate genes known initially from the standpoint of how their encoded RNAs are expressed. This review discusses recent findings in the genetic area, the molecular one, and where these two intersect chronobiologically. An extensive treat ment of the background information concerning genes and rhythms will be avoided, as this has been covered in several recent articles (27, 31, 43-47, 63, 64, 69, 77, 114, 139, 146-148). GENETIC VARIANTS USED TO ANALYZE RHYTHMS Drosophila PERIOD MUTANTS The most salient clock mutants in these dipteran insects remain, alas, those encoded by mutations at the X-chromosomal period (per) locus of Drosophila melanogaster. The basic features of these genetic vari ants and the phenotypes they cause have been reviewed ad nauseam. The most remarkable aspects of these early studies are that the first set of systematically induced rhythm mutants (in any organism) included a short-period, a long period, and an arrhythmic strain, and that these three independently isolated per mutations were by definition all allelic (65). A large debt is owed by all who work in the area of clock genetics to R. J. Konopka, who led this initial screen for rhythm mutants and has continued to induce and isolate them during the past 20 years. Some of Konopka's per mutants have not (or have barely been) reported. Their basic properties are described here, because they are pertinent to certain fundamental properties of biological clocks: "temperature compensation," on the one hand; and "entrainment" by, or "phase-shift responses" to, environ mental stimuli, on the other. (See 90 or 145 for text-book treatments of these and other details of circadian rhythms.) One of the relatively new per mutants is caused by a long-period allele, perL2 . The circadian locomotor activity rhythms of adults expressing this
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mutation are basically like those affected by perLl (cf 48, 65) . Thus, perLl and perL2 exhibit 28-30 h periodicities ("taus," or 71'», at 25° C. (See 24, 46 for brief accounts or examples of perL2-influenced behavior. ) When such "free-running" behavioral rhythms were monitored in constant darkness (called "DD"), at lower or higher temperatures than the standard 25° C, both perLl (66) and perL2 (33) adults exhibited shortened or lengthened 71'>, respectively. The degrees to which each of these long-period mutants has lost the excellent temperature compensation associated with wild-type rhythms of this poikilothermic organism are very similar (33). A new short-period per mutant has also been found (R. J. Konopka, personal communication). This mutant's free-running period, from the same kinds of activity monitorings referred to above, is an astonishing 16 h, a value 3 h shorter than in the original pers mutant (cf 65 , 151). The latter, whose circadian pacemaker already runs 5 h faster than normal ( 24 h in D. melanogasler), can nevertheless be reset each day to exhibit 24.0 rhythmicity in conditions (called "LD") of cycling light (12 h) vs darkness (12 h). Thus, this altered-T mutant entrains to the LD cycles (see below, Figure 1). Yet, perss behavior in these quasi-natural conditions is not like that of wild-type: The "evening peaks" of locomotor activity are in the daytime, instead of being at "lights-off' as in wild-type (M. Hamblen-Coyle, D. A. Wheeler, M. Rosbash, J. C. Hall, in preparation). Conversely, perLl and perL2 display evening peaks in the night, though these mutants, too, are driven into exhibiting 24.0 h periodicities in 12: 12 LD (Hamblen-Coyle et aI, in prepara tion). It was not necessarily expected that these short- and long-T mutants would be able to be reset, each day, to an LD regime whose cycle durations lie far from the endogenous (free-running) periods specified by these genotypes (also see section below on mammalian mutants). That such mutants can be reset indicates that pers has the phase of rhythm delayed fully 5 h each day, and that the "driven" rhythmicities of perLl and perL2 are phase-advanced by the same amount. These LD phenomena (Figure 1) reflect the underlying "phase-response" system (associated with essentially all pacemakers found in organisms), because they are only circa-dian and hence must be reset daily to respond to the 24 h cycles of the environment. Curiously, the determination of a phase response curve (PRC) for Dro sophila locomotor activity rhythms-now so widely used in studying circa dian rhythms in these species of insect-had never been reported in a primary publication (though see Ref. 44). This has now been remedied (28). As is usual for any eukaryotic organism, part of the wild-type PRC for Drosophila defines a light-insensitive phase in DD, i.e. a l2-h segment of a free-running day during which light pulses lead to no phase shifts; if the flies had been in 12: 12 LD before proceeding into DD, the insensitive portion of the PRC, =
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called the "subjective day," corresponds to when the lights were on during entrainment. Light pulses delivered during approximately the first half of the subjective night led to phase delays, whereas pulses given during late sub jective night induced phase advances (28). per mutations are pleiotropic in their effects on several temporally related phenotypes. These include phenomena whose time-scales are far outside the circadian range, in either direction. In addition, some of the per mutant phenotypes reported (e.g. learning or visual-response abnormalities) do not have any obvious temporal dependence (for review, see 43). The more recently discovered, or revised, elements of per's pleiotropy as it relates to time-based phenotypes warrants some further discussion: per's action during development Developmental timing appears to be altered by peru, pers, and per-zero (perOl) mutations (75). The first of these had the most obvious effects and indeed noticeably lengthened the durations of larval and pupal stages. pers had the opposite effects, though they tended to be less pronounced. perOl erratically lengthened or shortened a given developmental stage compared to the wild-type durations, depending on the rearing conditions (DO, LO, or LL, the last of these meaning constant light). The end of Drosophila's development is its senescence. In this regard, everyone asks (and rightly so) if per mutations affect adult lifespans (e.g. for pers, perhaps the mutant flies live fast, die young). The answer is apparently "no," at least in the sense that, in two sets of studies, there were no systematic lengthenings of per u s lifespan and no shortenings effected by pers (33, 64). At high temperature, however, perOl adults lived a few days fewer than did flies expressing the rhythmic genotypes-the two mutants just noted, as well as coisogenic per + controls (33). This mildly abnormal phenotype, caused by an "arrhythmic" per allele, could, however, be a rather nonspecific viability problem (see Ref. 4 for a general discussion), as opposed to a "time-of-life" deficit. To attempt an interpretation of the positive results obtained from assaying the effects of per mutations on preimaginal stages, Kyriacou et al (75) note that embryonic expression of per mRNA and protein in the central nervous system (CNS)-as demonstrated by James et al (60), Liu et al (81), and Siwicki et al (127); or conceivably that which was detected in the salivary glands, as shown by Bargiello et al (7�ould influence the timing of subsequent developmental stages. In this regard, per expression during the larval period itself is minimal [by Northern blotting, and by in situ detection of the encoded protein in salivary glands only (7)], or has been undetectable (60, 81, 1 27); the same holds for about the first half of pupation (60, 81, 12 0). Pupal expression of per can, with no difficulty, be hypothesized as in volved in the control of periodic ecIosion (for discussion see 8, 81, 120, 127). ,
CLOCK GENETICS
663
Likewise, the gene's adult expression is readily rationalized as one of the factors underlying the fly's locomotor activity rhythm. But is the embryonic expression also important for one or more of the per-influenced phenotypes that occur later in the life cycle? One way to attempt answering this kind of question is to perform temperature-shift and heat-pulse experiments on a conditional mutant. None existed for
per, whereby the gene's function is
"off' at restrictive temperature and "on" at permissive. So a temperature sensitive mutant was created by generating transformants whose transduced
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DNA has a "heat-shock promoter" (hsp) fused to per coding sequences
(34).
Subjecting the developing transformed animals, whose genetic background was perOl, to various hot and cold regimes indicated that the gene's action during preimaginal stages was neither necessary nor sufficient for locomotor activity rhythms to be exhibited by adults. For example, if these transformants developed at low temperature (with per off), and were "LD entrained" under that condition, they were nevertheless rhythmic in their adult behavior if the temperature was turned up once the flies proceeded into constant darkness
(34) . These conditional-mutant experiments have been extended. For example, it is of interest to ask whether a circadian clock is running in these
hsp-per
transformants during the LD cycles that preceed heat treatment and transfer to DD conditions. Recall, as background, experiments showing wild-type flies that were put through their development in DD and then were left in this condition as adults exhibited very weak or no rhythms in their locomotor activity (25). Thus, at least one light-dark transition is necessary to start a strong circadian clock running in this organism, as well as to set its phase. It was hypothesized
(33) that a clock function, as kicked into action by
exposing the flies to LD cycles, could be already operating in animals whose
per allele is off, i.e. in the unheated hsp-per transformants. Thus, when the gene's activity is subsequently switched on, it would be involved in some thing like linking the activity of this pacemaker to "oscillator output," which is part of the pathway mediating the final phenotype (sleep/wake cycles) but is not part of the central clock. This hypothesis about per's action was seriously undermined by the finding that the phase of activity rhythms exhibited by these transformants did
not depend on that of the LD cycle to which the flies
were exposed, prior to turning the gene on at the beginning of DD (33) . Therefore, this gene's action seems to be necessary for pacemaker functioning itself. To bolster the conclusion that per expression is necessary only for the manifestation of the adult fly's rest/activity cycles-and hence is not used at earlier developmental stages for setting up the nervous system to run the rhythms later on-Ewer et al (33) monitored the
hsp-per transformants'
behavior during LD at low temperature and then continued to follow them in DD at high temperature. The question was: Is there any "leakage" of heat\
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Figure 1 Locomotor activity rhythms of adult Drosophila. General movements of these in dividual D. melanogaster flies were monitored automatically (re infrared light-beam breakages) as in Hamblen et al (48). Successive days of the activity events (each such event being represented as a vertical mark, dropping below one of the horizontal lines) are plotted left-to-right and also top-to-bottom (hence, days 1-2 on the top horizontal line, days 2-3 on the next, and so forth). For about the first 10 days of monitoring in each case (LD conditions), the lights cycled on (open portions of horizontal bars on top of each "actogram") and off (filled portions of bars). "Lights-on" times were at noon, i . e. 4 h after the left-most "8" ( = 8 am) in each plot. After the arrows, the lights were turned off (DD conditions). (a) Wild-type actograms, after Dushay et al (28) [left panel] and Hamblen-Coyle et al (49) [right]; each record was from monitoring a male's
CLOCK GENETICS shock-promoted
665
per expression at low temperature, during LD? If so, then
some of these flies, whose genetic background was perOI, should have been somewhat wild-typelike in their LD behavior (cf 28, 49 , 96) , i.e. they would have anticipated the times of L-to-D and D-to-L, by becoming gradually more active in advance of these environmental transitions, and would have been relatively inactive during the middle of the lights-on and lights-off periods. In contrast,
perOl,s behavior in LD seems mostly to be a response to the
environment (Figure l c): rather little activity in the dark, then a rapid (sus
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tained) increase at (and during) the light (see 52 for another viewpoint). The answer from these further
hsp-per experiments was that many of the trans
formed flies that expressed a rhythmic phenotype following a postentrainment temperature-upshift had nevertheless behaved in a notably
pero-like manner
"
"
(cf Figure l c) during the prior monitoring of their activity in LD at low temperature (33). When considering per expression in the developing CNS of embryos from a purely descriptive point of view, note that the in situ localizations of the gene's transcript, its protein, or stainings involving a per-promoted reporter protein have been examined (in published reports) only at low resolution (60, 8 1 , 1 27). Curiously, the regions of such expression (ventral/medial) within a given ganglion are near to those where a "per relative" is also expressed. This is single-minded (sim), defined originally by embryonic lethal mutations causing defective development of the CNS's ventral midline (136) . A stretch of relatively N-terminal, per-encoded amino acids (ca 15% of the total on-paper polypeptide, which is about 1 200 amino acids long) is weakly similar to a relatively N-terminal region (ca 20% of the total) within the sim sequence ( 1 9) . Note (and recall later) that this region of similarity occurs in a region of the per sequence that is highly conserved among Drosophila species ( 1 7 , 135 , 1 44). The sim protein is expressed in nuclei of developing cells of the CNS ( 19),
activity; during
LD, the flies tended t o become active a t least an hour i n advance o f lights-on or DD, the "morning peaks" of behavior largely went away, and the "evening peaks" expanded. (b) Actogram for a elk mutant male (after 28); the fly was "forced" into 24.0 h rhythmicity during LD, in which conditions the evening peaks were earlier than those observed for wild-types; during DD, the "free-running," short-period rhythms caused by this mutation were lights off; during
manifested, wherein the most active portions of a given cycle began and ended at least an hour
(c) Actograms of flies expressing a "per-zero" mutation-left: a male hemizygous for per OJ (after 29); right: a female heterozygous for this mutation and a deletion of the period gene (after 49); in LD, the beginnings and ends of the dense segments of activity markings tend to coincide with the L portions of a given environmental cycle; in subsequent DD, earlier on successive days.
these forced rhythms degenerated into arrhythmicity.
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and per also has a nuclear distribution in certain tissues of the adult, such as photoreceptors, cells of the gut, and in the fly's excretory organ (8 1 , 120, 1 27) . In adult neurons, however, the clock gene product appears to be a cytoplasmic protein (127, 1 52). Recent examinations of per-promoted {3galactosidase activity in the "reporter transformants" (cf 81 ) indicate that the fusion protein (approximately the N-terminal half of per, with the C-terminal half replaced by the laeZ gene of E. coli) is similarly expressed throughout the cytoplasm of adult neurons; the stainings seem not to be present in the nuclei of these cell bodies in the brain (X. Liu, personal communication). per signals in tissue types for which the fusion protein is found in nuclei (see above) also seem to be in the cytoplasm of these cells (X. Uu, personal communication). The same reporter transformants exhibit, at embryonic stages, cytoplasmic subcellular localization in the neurons of the developing CNS (S. T. Crews, personal communication). These observations included the demonstration of a separate, nonoverlapping set of CNS cells expressing the per and the sim proteins; moreover, the midline cells removed by the action of a sim mutation left per's cellular expression normal (S. T. Crews, personal communication). Therefore, the per-sim similarity remains an uninterpretable curiosity. This includes the fact that the predicted proteins encoded within these two genes are only somewhat similar to each other, i .e. to nothing else in databases. Finally, in contrast to the case of sim, a per-null genotype allows not only for full viability (all the way to adulthood), but causes no gross morphological defects in the CNS of embryos (or any other stages) .
Salivary gland rhythms A variety of rhythmicities associated with these glands have been reported in Drosophila larvae, albeit with rather scanty data ( 1 04) . One reasonably detailed report showed an apparent circadian rhythm of membrane potential in cells of the larval salivaries, as determined by applica tion of a voltage-sensitive dye ( 143) . Provocatively, this rhythmicity was said to be "damped" or absent in salivary cells of per01 larvae. These phenomena have recently been called into question: Direct recordings of membrane potentials from salivary-gland cells of wild-type larvae, over the course of a given day or even longer, revealed no rhythmic fluctuations in this physiolog ical parameter (G. D. Block & M . W. Young, cited in 57). Another phenotypic effect of per mutations on salivary gland gland physiology , which does not overtly connect to a rhythm, involves intercellular communication among electrically coupled cells within the gland (7). The product of this gene could therefore be a coupling agent. The possible rhythm connection is the following speculation: per-mediated communication among neurons might be the way in which the gene's action participates in building or operating a neural pacemaker (for reviews, see 147 , 148) . Additional findings relevant to this hypothesis involve the 5-1 5 h periodicities that can be extracted by spectral methods from locomotor activity records of pe,o flies
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(e. g. 22, 24, 49) . These hidden rhythmicities in the so-called arrhythmic mutants have been interpreted to mean that the pacemaker underlying these rest/activity cycles is noncircadian and that the per gene product serves to couple the fundamental (relatively high-frequency) oscillator (for review, see 26). It could follow from this view that per promotes clock function but is not per se a component of it. However, what if the hypothetical coupling function occurred within individual neurons, instead of among them? None of the results pertinent to, nor the inferences stemming from, the hidden rhythms of pero demand that the neuronal coupling agent acts intercellularly. It could just as well function within individual neurons, each one of which would be a circadian pacemaker by virtue of per's action . Thus , the gene would be back in the fold as a key component of the central elements of the clock.
Ovarian diapause It has recently been asked if per's influence on temporal ly dependent phenomena extends to helping the flies discriminate short (relatively little daylight in a 24 h cycle) from long days. The latter condition leads to ovarian diapause in many forms, including D . melanogaster, as demonstrated in experiments performed at very low temperature ( 1 22). All the per variants tested for this phenotype did well: Saunders ( 1 2 1 ) found that females expressing either a long (L2)- or a short (S)-period allele were identical to wild-type in their "critical day lengths," or CDLs (whereby 14 h of daylight is "diapause-averting"). The two arrhythmic types that were tested might have been predicted not to be able to detect 14-h (or other) diurnal periods, but they did do. Yet, they were not like wild-type, in that perOJ females had CDLs 2-3 h shorter than normal ( 1 2 1 , 1 23); and per females, who are homozygously deleted of the gene (and, necessarily, of two neigh boring X-chromosomal transcription units as well; 8, 1 03), exhibited 5 h shorter-than-normal CDLs ( 1 2 1 ) . In spite of these mutant phenotypes, the investigators concluded that per expression is not "causally" involved in day or night-length measurements ( 1 2 1 , 1 23), which therefore would be con trolled by the action of a "photoperiodic clock" whose actions are only at best "modulated" by an absence of this clock gene. -
High-frequency rhythms
Certain per mutants have been shown, or sug gested, to cause defects in various high-frequency ultradian rhythms. The fastest such rhythm is the ca 3 Hz heartbeat of 3rd instar larvae (for review, see 109). Heartbeating was said to be erratic inperOJ animals, though this has been reported in abstract form only (23 , 82) . A more recent study of this problem was performed by J. Ewer (personal communication). He found that the heartbeat of a given animal (which was monitored noninvasively, using a method based on light-path interruptions) could be a clean 3 Hz beating, or could consist of periods of regular beating interspersed with periods of erratic signals and interruptions . Both bouts of erratically timed beats and the
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interruptions were of variable, and sometimes quite long, durations. In addi tion, the heartbeat of a given larva could change dramatically over the duration of the recording period (usually 20 min). However, none of these types of records, or temporally dependent trends, were consistently associated with a particular per genotype. This included the fact that there was no more occasional irregularity in perOl or per records than in wild-type larvae (J. Ewer, personal communication). Incidentally, no noticeable expression of per gene products is detectable in the larval heart-or in any larval tissues in one series of studies (8 1 , 1 27); though others (7 , 1 20) report per protein and transcripts in the salivary glands of larvae. In any event, per pleiotropy, in terms of mutational effects, does not extend to the very high frequency range of rhythmicity in the larval heart. There are, on the other hand, many reports (most recently reviewed in 70) of per mutant males exhibiting defects in a rhythm of courtship song, whose ultradian periods in wild-type D . melanogaster are about one minute. Males expressing pers sing with about 40 s rhythms; peru leads to ca 80 s cycles; and perOl males do not exhibit any apparently regular lluctuation of the relevant song parameter (rate of tone pulse production, accompanying the courtship wing vibrations). These mutant and normal phenotypes have been extensively discussed in recent years. One reason for this is that some investigators have attempted to show (20, 35) or argue ( 10) that the singing rhythm is not a feature of these males' wing-vibration-generated sounds. The validity of the song rhythms has recently been reiterated through several experimentally and analytically based arguments (74, 76). Portions of these studies showed that Crossley (20), as well as A. W. Ewing (in some pilot experiments performed by that investigator, prior to his study described in 35), have indeed recorded rhythmic songs; furthermore, the periods were in the usual (wild-type) range for this species. The two reports (74, 76, respec tively) that include reanalyses of Crossley's and Ewing's data also discuss the several technical and algorithmic reasons for these two other investigators' claims that original rhythm results (7 1 ) could not be replicated. (For detailed discussion. see 10, 43, 69, 70, 1 24). If we assume for the time being that the song rhythms are real, there are some new genotype-phenotype correlations. Certain of these stem from the fact that the periods of such rhythms are species-specific, in that D. simulans males vary their rates of song pulse production over cycle durations in the 30-40 second range (7 1 , 73, 144); and the D . yakuba song rhythm period is on the order of 70--80 s ( 1 34). [These three species are all members of the melanogaster subgroup (80) .] With regard to D. simulans and D. melanogaster, the genetic etiology of the song rhythm difference was shown to map solely to the X chromosome, from analyzing the singing of reciprocally hybrid males (73). These studies of the hybrids' behaviors suggested that interspecific differences in the X-
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Annu. Rev. Genet. 1990.24:659-697. Downloaded from www.annualreviews.org by University of Sussex on 10/17/12. For personal use only.
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chromosomal period genes' informational content or expression could be the factor(s) underlying the presumed evolutionary divergence of this behavioral phenotype (which, incidentally, has adaptive significance in terms of repro ductive isolation) (72, 73). All this has been confirmed by some clone and sequence experiments augmented by interspecific gene transfers. Wheeler et al (144) isolated per+ DNA from D . simulans (via homology to D . melanogaster probes) and introduced this material into D . melanogaster, by creating the appropriate germ-line transformants (cf 16, 150); the genetic background of these barely partially hybrid males included hemizygosity for perOI. The flies sang as if they were D . simulans (144) in that they expressed rather fast-running "song clocks," i.e. the periodicities were squarely in the 30-40 s simulans range that for melanogaster being 50-65 s. Therefore, the source of this behavioral difference between these two species of Drosophila resides at only one locus. Sequence comparisons of portions of simulans per to the corresponding regions of the melanogaster gene showed minimal divergence, except within a subset of the gene's largest exon (which corresponds to ca 570 amino acids, i.e. about 45% of the total protein). The ca 360-bp segment encodes a series of alternating threonine-glycine pairs, which is (at least descriptively) a hallmark of this gene's informational content in some, but not all, Drosophila species (e.g. 17, 58, 102). The Thr-Gly repeat, moreover, has been im plicated as being especially important in the control of song, but not circa dian, rhythms (150). Thus, further interspecific gene transfer experiments were performed to produce transformed males carrying per of D . melanogas ter from which 700 bp (including the aforementioned 360) had been removed and replaced by the corresponding cassette from D . simulans; the reciprocal chimeric transformant type was also generated. The former males sang as if the whole per gene was from D . simulans and the latter as if they carried a per + DNA fragment entirely from D. melanogaster ( 144). Finally, this study showed that intra- and interspecific variations in the length of the Thr-Gly repeat were probably not responsible for the single-gene control of the song rhythm difference between melanogaster and simulans; but instead that 1-4 amino acid substitutions (just C-terminal to the repeat region) have occurred over evolutionary time and define the intragenic etiology of these species specific courtship song rhythm differences. CLOCK MUTATIONS AT LOCI OTHER THAN PER
Certain engineered per mutants are song rhythm variants only (cf 150). Indeed, all the transfonned types noted above exhibited similar circadian rhythms of locomotor activity; the same is found when comparing the behavior of wild-type D . melanogaster and D . simulans adults ( 144). Reciprocal types of rhythm mutants are now known: The X-chromosomal Clock mutation, isolated (64) by virtue of a ca 1 .5 h shortening of the circadian rhythms of locomotor activity (Figure Ib),
Annu. Rev. Genet. 1990.24:659-697. Downloaded from www.annualreviews.org by University of Sussex on 10/17/12. For personal use only.
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leads to essentially normal one-minute song rhythms when elk is hemizygous in males (28) . Also, disconnected mutants (so named because of their eye brain disconnections; 132) sing in an essentially normal manner (70); yet their locomotor activity is largely arrhythmic (29), i .e. similar to that of per o adults (Figure l c). These findings on the elk mutant could mean that the fly's song and circadian clocks share some but not all mechanistic components (and of course these pacemaker mechanisms could not be the self-same entities , given that the relevant periodicities differ by 103). disco's rhythm defect implies that the eNS abnormalities, which are probably caused by these mutations (see below), are more important in the brain than in more posterior portions of the adult nervous system. This inference is based on the following: (a) Eye-brain connectivity, or even the presence of eyes, is largely irrelevant to the fly's circadian system (reviewed in 43); it follows that this aspect of the disco phenotype is not responsible for the mutant's circadian-arrhythmic behavior (see discussion in 29); thus, other features of the fly's head neuroanatomy are likely to be deranged in disco. (b) The pacemaker for the courtship song-as defined by the focus of a per-mutation's effects on the behavior of genetic mosaics-seems to be in the male's thoracic nervous system (R.I. Konopka, C. P. Kyriacou, 1. C. Hall, unpublished data, cited in 42), whereas the circadian pacemaker is almost certainly in the head (given the mosaic results reported in 68). The recently determined details of rhythm defects caused by elk were accompanied by a fairly fine-level localization of the mutation responsible for these phenotypes. elk may not define a novel gene because it maps so very near the per locus (28) . Whether this variant is mutated in a close neighbor of per (and there are some molecular candidates, cf. 8, 83, 103), or within the confines of that locus remains undetermined. If the latter is true, where and how elk is mutated would be interesting, given that it could be: (a) associated with amino acid substitution in an informatively different location from that responsible for the other intragenically mapped short-period mutant, i.e. the pers-defined serine (which became substituted by asparagine on induction of this mutation; 9 , 151); or (b) a "hyper-expression" regulatory variant, based on the negative correlation of per + dosage with circadian period (18, 130, 152); and (c) instructive about the control of circadian and song rhythms which are usually affected in parallel by per variants (the other exception, if "perC1k" turns out to be one too, appeared in 150). Another X-chromosomal rhythm mutation, Andante (And), is like per in that it affects circadian rhythms (67) and probably leads to abnormal song cycling as well. Thus, And causes a "moderately slow" circadian clock (by definition) and slightly longer-than-normal song rhythm periodicities in males hemizygous for this mutation (70). And's basic phenotype (with respect to which the mutant was isolated by Konopka et al.; 67) is that, for circadian
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rhythms of either eclosion or adult locomotor activity, the periods are 1 - 1 .5 h longer than normal. The mutation is semidominant, as are essentially all
And is also associated with a melanogaster, called dusky (dy):
circadian rhythm mutations (summarized in 27). pigmentation mutation, long known in D.
And map to the dy locus (by recombination and using chromo dy mutant was found to exhibit normal circadian rhythms (67) . However, some newly induced dys (59; J. M. Ringo, H. B. Dowse, cited in 57) do lead to 25-26 h rhythms, which are essentially the same as those observed in tests of the original And mutant (67). On the other hand, one new dy exhibits short-period circadian rhythmicity (1. M. Ringo, H. B. Dowse, personal communication), and still others (57 , 59) have normal rhythms (cf. 67) .
Not only did
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somal aberrations), but it is per se dusky-winged (67). Yet, a classical
Despite the genetic complexities implied by these results, the rhythm and pigment functions defined by these mutations could be the same, or at least subsets of a "complex locus" of related (primary) gene actions. Is
And-dy,
then, interesting, or could a biochemical defect (related to biogenic amines, hence body color and neurochemistry; cf. 107) indirectly affect rhythms in a manner destined not to be incisively informative? Alternatively, perhaps the relevant amine-containing substance (if any) is involved in actual pacemaker functioning. Both possibilities should be kept in mind with regard to some recent findings involving
ebony (e) body color mutants in D. melanogaster: Flies
homozygous for a given mutant allele exhibit a variety of locomotor rhythm anomalies, both in DD and in LD (94). Interestingly, eclosion rhythms, determined for
e cultures, were indistinguishable from wild-type (94), so that e
these mutants are the most specific yet in their rhythm abnormalities. Yet,
mutants have long been known to be pleiotropically defective in other adult behaviors, which are not apparently related to clock functions; and there are some data on e-induced "amine abnormalities" as well (see 94 and 1 07 for background literature). The classical visual-response defects exhibited by
e
flies were suggested to be involved in the rhythm defects, because blockage of
no e double mutants-partly suppressed the
all light input through external photoreceptors--
