BioSystems 6 (1975) 188-201 © NORTH-HOLLAND PUBLISHING COMPANY
CIRCADIAN RHYTHMS AND MOLECULAR BIOLOGY* Th&~se Vanden DRIESSCHE Ddpartement de Biologie mol6culaire, Universitd Libre de Bruxelles, 67 rue des Chevaux, 1640 Rhode St. Gen&e, Belgium
It is the purpose of this review article to tentatively pinpoint the main characteristics of circadian rhythms - evidence taken mainly from a few unicellular organisms - and to present the recent theories which should serve as working hypotheses.
1. Introduction Rhythmicity appears to be part of the dynamic state of living matter. Circadian rhythms of 24 hr (in normal conditions) or of about 24 hr (in seemingly constant conditions) are now taken as a regular trait of many functions in eucaryotic organisms. They are not only universal among higher plants and animals (although not all functions vary in a circadian way in a given organism) but now appear to have a far reaching importance in many fields. Mammals such as rats display a variable sensitivity to external agents (E. coli endotoxin for example) and to drugs (reviewed by Reinberg and Halberg, 1971). Since a given medication at fixed dose applied at various times of the day elicit in man responses of variable intensity, it seems possible by an appropriate timing o f the medication to reduce undesirable effects and to enhance the drug efficiency (Reinberg, 1974). Such an achievement has been obtained by Haus et al. (1972) on leukemic mice treated with arabinosylcytosine. In spite of the importance of the matter and although a considerable amount of information on circadian rhythms has accumulated, the mechanism of rhythmicity remains elusive.
2. The circadian rhythms Circadian rhythms are by no means the only oscillations displayed by living matter. Three groups should be considered: high frequency rhythms (the period goes from the fraction of a second to half an hour), median frequency rhythms (from half an hour to two and a half days) and low frequency rhythms (the period is longer than 2.5 days). Circadian rhythms are thus included in the median frequency rhythms, together with the ultradian rhythms (the period goes from half an hour to 20 hours) and the infradian rhythms (from one day to 2.5 days). As previously underlined by Sweeney (1963), the two main characteristics o f the circadian rhythms are the stability of the period and the lability of the phase. In a constant environment, the period differs usually slightly from 24 hr but the difference from 24 hr is constant for any given set
* Invited review. 188
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of conditions and a given organism: the period is species specific. For a given species and a given rhythm, there is a slight but reproducible deviation from 2.4 hr in the period length for any light intensity and any temperature. By "slight" it is meant that the observed period difference is very small in regard with the difference imposed in the external conditions. Bruce and Pittendrigh (1956) reported that the period of the phototactic rhythm of Euglena in constant darkness remains 23 hr whatever the temperature between 33 and 23°C, and increases to 26.2 hr at 16.7°C. In Gonyaulax polyedra (a luminescent protozoan), the period decreases from 26.8 to 22.5 hr when the temperature is lowered from 26.6 ° to 15.9°C (Hastings and Sweeney, 1957). A temperatu~re compensation device is suggested by these observations. Similarly the Gonyaulax displays no change in period length (24.5 hr) in eJ.ther constant darkness or constant light provided that the intensity is of 1200 lux. At 6800 lux however, the period shortens to 22 hr (Hastings, 1959). The usual stability of the period of circadian rhythm contrasts with the highly manipulable frequency of the glycolytic rhythm, which can be changed by temperature as well as by substrate. Pittendrigh and Caldarola (1973) recently devoted an analysis of what they called "circadian rhythms homeostasis". As they pointed out, the existence of mutants which do not compensate for temperature variations is very interesting since it raises the possibility of a regulatory mechanism separated from the rhythm itself. The addition of specific nutrients does not alter the period. But there are examples of effects of nutrients on the expression, duration of maintenance or stability of the oscillations. Cumming (1967) has shown that glucose or sucrose fed to Chenopodium rubrum during the dark period sustains the rhythms over longer periods of time than in the control plants. In fungi Cu ++ sta-
bilizes the rhythm in Leptosphaeria michotii (Jerebzoff et al., 1969) and rhythms of various periodicities can be expressed at certain glucose concentrations only (Aspergillus niger, Jerebzoff et al., 1974).Noteworthy also is the effect of acetate on the period of the phototactic rhythm of Euglena (Feldman and Bruce, 1972). Phase, on .the other hand, is labile. It is obvious that any circadian modulation of adaptive value should be phase sensitive to external synchronizing agents in order to be efficient. The phase of a rhythm can be shifted by the displacement in time of the alternation of light and dark. Let us take an organism submitted to light from 9 to 21 and to dark from 21 to 9 and let us change the timing in such a way that night replaces day and vice versa. After a few transients, the phase is reset and the phase relationship with respect to the exogenous entraining cycle is restored. In addition, there is a well delineated circadian rhythm in light sensitivity: a light stimulus will elicit a phase shift, either an advance or a delay of different amounts. This can be also represented along the time scale as a phase response curve (Pittendrigh, 1965). This phenomenon is examplified by Gonyaulax (Hastings, 1959). Kept in constant darkness, the luminescent rhythm is expressed. If a light signal of 6 hr is given entirely before the appearance of the peak (acrophase of the rhythm), it will induce a delay of the peak. In contrast, the signal given just at the time of the peak will produce an advance. The phase response curve obtained in this way is very asymetrical which shows that the system is endowed of polarity. It should also be noted that the circadian modulation has a real adagtative value since it is initiated in advance, in such a way that the organism will have reached the optimum performance state when the external peculiar conditions will be obtained.
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Free running oscillations can be phaseshifted also by temperature steps and pulses. An interesting feature is that temperature step-up always generate advances whereas temperature step-down generate only delays (Zimmerman et al., 1968). Not only many functions are susceptible of circadian modulations, not only superior organisms display circadian rhythmicity but unicellular eucaryotic organisms are endowed with several oscillating functions. Gonyaulax
, , ......
Fig. 1. Time map o f circadian functions in Acetabularia. Upper part: photosynthesis: solid line; chloroplast shape: broken line with open triangles; RNA synthesis: broken line with open circles; ATP content: dotted line with +. Lower part: photosynthesis: solid line; polysaccharide content: broken line with open circles; number of chloroplasts containing one granule: broken line with open triangles; n u m b e r of chloroplasts containing three or more granules: dotted line with x; plastid division: solid line with x.
has a luminescent rhythm in both glow and stimulated luminescence, in photosynthesis, in division (Hastings, 1959). Euglena (Edmunds et al., 1974) and Acetabularia have even a greater number of oscillating functions. The temporal morphology o f the latter is graphically represented in Fig. 1: ttle value of the various functions at a certain time of the day is calculated as percent of the mean of the day in order to allow comparison o f the different functions: photosynthesis, chloroplast RNA synthesis, chloroplast polysaccharide content (biochemical determination or number of granules), chloroplast ATP content, division of chloroplast (not represented on fig. 1), and shape of the organelles in longitudinal view (the chloroplasts are more elongated in the middle of the light period than they are in the middle of the dark period). In addition the ultrastructure of the chloroplasts appears quite different at 9 a.m., 3 p.m., 6 p.m. or 3 a.m. (light being given from 9 a.m. to 9 p.m.): both the aspect and the location of the thylakoids are variable, the density of the stroma changes and, as indicated above, the number of carbohydrate granules. All these variations are maintained at least during a few cycles in constant conditions, even if the mean level of the function (its base line) is dramatically changed. This is the case for all functions when the algae are submitted to continuous illumination of intensity equal to that given normally during 12 hr only (i.e. the light period of the cycle). In fact, in Acetabularia, as in most organisms, high light intensity abolishes rhythmicity. To summarize, circadian rhythms have a very stable period, which calls for compensatory mechanisms, the natural period being species specific, and a labile phase which is entrained by external agents. These can shift the phase more or less efficiently depending on the circadian internal phase of the organism at which the perturbation is imposed. Circadian rhythms are of general occurrence
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in eucaryotic organisms. Even unicellular organisms have an elaborated temporal morphology and display numerous oscillating functions.
3. Mechanisms of rhythmicity
3.1. Hereditary rhythms Circadian rhythms must be inherited since the natural period (that expressed in the absence of synchronizers) is species specific. This is explicitely demonstrated by the genetic experiments per:formed by Biinning on Phaseolus (1935) anti by Konopka and Benzer on Drosophila (1971). These authors have identified a small region of a few cistrons only on the X chromosome of Drosophila responsible for the circadian rhythms of both activity and emergence and for the length of their natural periods. On Acetabularia we have shown that grafts of stalks and rhizoids between a strain endowed or devoid c,f rhythmicity display a rhYthm of photosynthesis after 3 weeks when (and only when) they have the nucleus containing rhizoid originated from the rhythmic strain (Vanden Drie,;sche, 1967). Not only is the expression of rhythmicity dependent on the nucleus but also 1:he phase of the rhythms. Schweiger and her collaborators (1964) performed transplantation and implantation experiments with algae whose stalk and rhizoid were in opposite lighting regime, submitted thereafter to continuous light and they observed, after a few days, a rhythm in phase with the regime previously entraining the rhythm of the nucleus.
3.2. Dependance on transcription Actinomycin D is a very specific inhibitor of transcription of RNA from the DNA tem-
plate. In its presence transcription is reduced to a few percent of the value of the controls. Acetabularia submitted during one week to this inhibitor displays a circadian rhythm of reduced amplitude (photosynthesis however is almost unaltered) (Vanden Driessche, 1966). In addition, the more concentrated is the actinomycin D (0.27 27 ~g/ml), the stronger is the inhibition. After 2 weeks all rhythms are abolished, even at very low concentrations. Transcription thus is not an everyday requirement but is needed within 7-- 15 days in both LD conditions (Vanden Driessche, 1966) and in LL conditions (Mergenhagen and Schweiger, 1971). Some other organisms however express their rhythms only if RNA synthesis remains unimpaired, Aplysia for example (Salfinki et al., 1971). RNAs might have an important role in rhythmicity since there is a parallelism between their amount and the amplitude of the oscillation. A treatment with RNAse, which breaks the RNA molecules, results paradoxically in an increase in RNA content (Brachet and Six, 1966). Measurements of photosynthesis, not during the treatment (during this time, photosynthesis is too low to allow the determination of a rhythm), but after a recovery period, show an increased amplitude of the rhythm. A similar conclusion can presumably be drawn from fragmentation experiments: the removal of the apex results also in an augmentation of the amplitude of the rhythm and the greater the removed fragment, the more important the amplitude (Vanden Driessche, 1967). Anucleate Acetabularia can be easily obtained and kept up to 4 or 5 weeks. In such algae, the rhythm remains unaltered for extended periods of time (up to 40 days) (Sweeney and Haxo, 1961; Richter, 1963; Schweiger et al., 1964; Vanden Driessche, 1966). This seems to stand in contrast with the effects of actinomycin D. The variance is
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explained, however, on the assumption that the RNAs become stabilized in some way in the anucleate cells (the RNAs involved in rhythmicity, Vanden Driessche, 1967, as well as the RNAs or some RNAs involved in morphogenesis, Brachet, 1968; Schweiger, 1970; Zetsche et al., 1972). The RNAs implicated in rhythmicity are of nuclear origin since (1) anucleate A cetabularia treated with RNAse recover the photosynthetic capacity in normal culture medium but not their rhythms, (2) anucleate Acetabularia treated with actinomycin D are not affected by the inhibitor, at least during 7 - 1 5 days (Vanden Driessche, 1966; Sweeney et al., 1967) and (3) anucleate Acetabularia treated with rifampicin, a potent inhibitor of chloroplastic transcription in this alga show no sign of decreased amplitude in their rhythms in 48 hr (Vanden Driessche et al., 1970). The necessity of transcription for the maintenance of the rhythm in Acetabularia seems thus to be the means of transfer of the nuclear information to the cytoplasm, according to the classic scheme DNA --, RNA. The increase in amplitude of the rhythm when the RNA content is augmented is not clear. The life-time of the RNAs is very long in Acetabularia (still longer in anucleate cells) but could be much shorter in other systems.
3. 3. Rhythmicity and translation Inhibitors of the synthesis of proteins do not seem to affect rhythmicity, in some species at least, since they do not produce any change in the phase or in the period of the oscillation in free running conditions. Sweeney and her collaborators (1967) have demonstrated that chloramphenicol does not affect the photosynthetic rhythm of A cetabularia although the base line is greatly lowered as a consequence of the reduction of the photosynthetic rate. In Gonyaulax, chlorampheni-
col brings about the appearance of a secondary peak and an increase in amplitude of the rhythm in luminescence (maybe due to an accumulation of RNA) whereas puromycin totally inhibits luminescence (Hastings, 1960). In contrast, prolonged exposure to cycloheximide affects the phototactic rhythm of Euglena, whose period is increased in a dose-dependent way (Feldman, 1967; Feldman and Stevens, 1973). Short exposures (less than 24 hr) however do not affect the mechanism of rhythmicity. After removal of the inhibitor, alanine dehydrogenase activity resumes its oscillation with no change in phase relative to that of the control (Sulzman and Edmunds, 1973). The mechanism of rhythmicity therefore cannot be looked at as a straight forward process involving RNA and protein synthesis.
3.4. Metabolic rhythms in Crassulaceae Plants of this family are characterized by their ability, in certain conditions to accumulate malic acid at night. Studying one of them, Kalanchoe, Queiroz (1970) demonstrated that there is a slow shift from one metabolic steady state to another when the plants are transferred from a long day regime to a short day one. Under short day conditions, the activities of the enzymes phosphoenolpyruvate carboxylase (PEPCase) and malic enzyme (ME) increase progressively. These two enzymes catalyse the following pathways from phosphoenolpyruvate (PEP) to oxaloacetate (OAA), malate and pyruvate (pyr)
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PEPCase PEP + CO 2
aspartate A remarkable feature of this shift is that the enzymatic activities not only increase but initiate circadian oscillations. Let us first analyze the increase in PEF'Case. Its induction is dependent on phytochrome (the membranar pigment sensitive to the photoperiodic signals) (Queiroz, 1968) and is regulated by feed-back control by malate and by an inhibitor which is present under long days only and disappears progressively during the 37 first days of short day regime (Morel et al., 1972). The authors emphasized the anaplerotic nature of PEPCase, an enzyme wttich is able to shift from a discrete activity level to a very high one, under the influence of external factors, bringing about a large metabolic change. It results from a change in both activity and enzymatic capacity (population of enzymes) (Morel et al., 1972). The progressive increase in phosphoenol pyruvate carboxylase activity proceeds up to the 60th day, time at which an abrupt decrease is initiated. Simultaneously, circadian oscillations are observed in malic enzymes and in four enzymes which have been studied in detail by Queiroz and his collaborators (1972) up to the 80th day after transition from long days to short days. The instabilities might involve metabolites of some cellular compartments but not necessarily (see Morel et al., 1972: for comments). Queiroz (1970) underlined the significance of "active" and "inactive" pools which have been determined, for another Crassulaceae, Bryophyllum, to be respectively 30 and 70% of the carboxylic acids (Mac Lennan, Beevers and Harley, in Queiroz, 1970). The "inactive" pool (as regards the metabolic syntheses)
, pyruvate| + C O 2 NADPH
-alanine might have a key regulatory role, inducing gene expression (as in Bacteria). The four enzymes are PEPCase, ME, GOT (aspartate aminotransferase) and GPT (alanine aminotransferase). Since malate accumulates (the "acid metabolism" of the Crassulaceae), an asymetry is created between the portion of the pathway catalyzed by PEPCase-(GOT)MDH and the portion catalyzed by ME-GPT. All four enzymes oscillate in a circadian way. The amplitudes of the four oscillations are independent from one another and increase with the number of short days (Queiroz et al., 1972).
3.4.1. The phase shifting The PEPCase peak shifts only very slightly during the period of time between transition (from long days to short days) and day 30. The shift (a delay) proceeds steadily between day 30 and day 60, time at which a phase jump is observed. A steady shift is again observed in the following days. (Queiroz et al. discussed the theoretical implications of these observations on the basis of a model, showing that it is consistent with populations of oscillating enzymes, with feed back, combined with cumulative effects of short days.) Since there is an accumulation of malic acid (the characteristic feature of Crassulaceae), the activity peak of ME, which is not in phase with that of PEPCase, must be expressed with a delay. However, the phase shifting advances progressively the ME peak and brings it slightly in advance relative to that of PEPCase on day 50. In contrast, no accumulation of intermediary products occurs
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between PEPCase and GOT and the latter activity peak is in advance with respect to the PEPCase one, up to the 60th day. GOT and GPT have approximately the same phase, in spite of the dissymetry of the pathway. It is suggested that it is due to the coupling of the two reactions by ~-cetoglutamate (Queiroz et al., 1972). The phase shifts of the circadian oscillations of GOT and GPT proceed also in pace. A reversal of phase relationship occurs consequently between ME and GPT at about the 60th day. The authors considered several hypotheses in order to explain how the circadian variations of PEPCase are elicited and shifted. The latter process seems to be due to iterative effects of thermoperiodism in combination with the short day metabolic status. As concerns ME, it appears likely that malate is the coupling agent between the oscillations of PEPCase and ME and that it progressively surpasses the direct entrainment of the ME oscillation by the signal " d a w n " (when the concentration of malate reaches a threshold value as a result of the increased activity of PEPCase) until the phase jump. Moreover, the induction of ME could depend on malate and its synthesis be inhibited by an excess of malate. When the peaks of the two enzymes PEPCase and ME are almost in coincidence, the amplitude of the oscillation should be reduced, which is observed. The abruptness of the j u m p is thus tentatively attributed to the occurrence of a critical phase angle between oscillations of PEPCase and ME. Queiroz et al. (1972) underlined the fact that all enzymes are oscillating, although the regulation of the whole system lies upon the first enzyme of the pathway. The shifts in the phases determine the progressive changes between day metabolism and night metabolism which progressively develops.
3. 5. Membranes and circadian rhythms Several substances have been shown to affect the rhythms: ethanol, theophyllin, digitonin increase (the two first ones) or decrease (the last one) the period of the circadian rhythms in constant conditions (Btinning and Moser, 1973). D 2 0 also increases the period, and this has now been experimented in numerous species (see Enright, 1971, for a review). In its presence, however, in L:D conditions, entrainment is no more possible. These substances are not specific but their common denominator is an action on membranes. The effects of valinomycin are more conclusive since it acts on membrane permeability and specifically interfers with the transport of potassium ions. Biinning and Moser (1972) exposed leaves of Phaseolus multiflorus to this agent through the transpiration stream. Depending on the time at which valinomycin is introduced (being kept for a given period of time), different phase shifts are observed and a phase response curve has been drawn, similar, to a large extent, to the phase response curve obtained for wilting. Both processes involve permeability. The response curves also have some common features with that obtained with light signals. Recently it has been shown that these conclusions can be extended to another species at least (Sweeney, 1974). Finally, in higher animals, it has been shown that hormones can phase shift a rhythm. Andrews (1968) succeeded in phase shifting the secretory rhythms of adrenals in organ cultures with ACTH. The site of action of the hormone, in target cells, is associated with membranes (and results in the production of cAMP which might be brought in parallel with the experiments with theophyllin reported above). It cannot be decided however what role membranes might have in the operation of circadian rhythmicity. In summarizing the pre-
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vailing hypotheses on its mechanism, it will be shown that two possible roles have been attributed to membranes: pace-maker and gate or synchronizer.
4.1. Exogenous control o f circadian rhythmicity: Brown's theory In Brown's opinion, there is no special internal device providing circadian oscillations (1969, 1 9 7 0 ) . He considers that several external agents interact: light, temperature and geophysical factors. The organisms handle the signals in a species specific way. He called this "autophasing". In normal conditions, the alternation of light and dark prevails upon other factors, which explains the usual 24 hr cycle. In contrast, in seemingly constant conditions, the period deviates from 24 hr because geophysical factors are the synchronizing agents and because the various organisms interpret the signals in slightly different ways.
4.2. Endogenous rhythmicity
4.2.1. The biological clock theories For the time being, several authors consider that a central mechanism underlies rhythmicity since (1) circadian rhythms are hereditary (the location of the particular gene has been determined on one of the chromosomes of Drosophila) and (2) it has not been possible, so far, to dissociate the various rhythms in the unicellular organism Gonyaulax. With these considerations in mind and taking into account the probable key role of membranes, two models have recently been proposed.
4. 2.1.1. Acetabularia as a model for circadian rhy th micity Sweeney developped this model at the Second International Symposium on Acetabularia (abstract, 1972 and fully developed model, 1974b). Sweeney assumes that two small non protein molecules X and Y migrate from the organelles (nucleus and chloroplasts) to the cytoplasm and vice versa and generate circadian oscillations due to the change in their intracellular distribution. The latter controls the membrane permeability of the organelles (providing a feed back loop) and c o n s e q u e n t ly influences the rate at which the distribution of the molecules can be equilibrated between organelles and cytoplasm. The detailed operation is the following: At phase 0 ° the molecules X, which are unstable, are inside the organelles for which they have a great affinity. These X molecules induce the synthesis of Y molecules within the organelles (all organelles are known to contain DNA and a protein synthetizing machinery). The X molecules make the membrane impermeable to both X and Y. Since the X molecules are unstable, their concentration decreases and the permeability increases. The Y molecules diffuse into the cytoplasm where, in turn, they induce the synthesis of the X molecules. At phase 180 °, the X molecules are thus predominantly in the cytoplasm. Since they have a special affinity for the organelles, whose membrane is, at this time, permeable to X and Y, the newly synthetized molecules accumulate within the organelles, rendering their membranes again impermeable. In this way they are trapped in the organelles at phase 0 °. Sweeney assumes that the role of the nucleus on circadian rhythms is not effected through RNAs since, paradoxically, anucleation does not affect the rhythm, but actinomycin D does. Its large volume accounts for
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the experimental results obtained with transplantation and implantation experiments. However, a molecule which can be synthetized both in the nucleus and the chloroplasts is unknown. As underlined by the author, the model readily explains the light effects. Since several papers have established that light modifies the properties of the membranes of Acetabularia, Sweeney logically assumes that high light intensities keeps the membrane in a "frozen" state (either open or closed to the X and Y molecules) preventing thus the occurrence of the oscillations (as observed, see section "Circadian rhythms").
22.214.171.124. Model of N/us, Sulzmann and Hastings A second model has been recently elaborated by Njus, Sulzmann and Hastings (1974). They identify the master oscillation with ions and membranes. According to their hypothesis, circadian rhythms result from temporal variations of membrane properties due to lateral movement of some membrane particles; owing to the variations in the membrane properties, their ion transport capability would also vary. In turn, ions influence the conformational state of the membrane. Cooperative phenomena could be implicated. Three series of experiments should be underlined in this context: (a) The effects of valinomycin, which interfers specifically with the transport of K + (see above), (b) The phase shifting effect of K + pulses on isolated Aplysia eyes (Eskin, in Njus et al., 1974), (c) The lengthening effect of LiC1 on the period of Kalanchoe petals (Engelmann, 1972). The authors speculate that ion pulses ineffectiveness in many systems should be attributable to regulatory mechanisms. However, if a general circadian oscillator exists, it is not clear why it escapes the regulatory mechanism in some systems and not in others and, in the former case, why the mechanism nullifies the
effect of a K + pulse but not the effect of K + concentration resulting from the circadian oscillation. Another question arises: why does the circadian variation, embodied by lateral migration of membrane molecules, cover a period of 24 hr whereas Frye and Edinin (1970) evaluated to 40 min the thorough redistribution of membrane markers in mammalian cells in culture. Njus, Sulzman and Hastings propose that photoreceptors, also localized in the membrane are photosensitive ion gates and provide coupling between external agents and the oscillator since they regulate the K + flux (the experiments with valinomycin seem to indicate that K ÷ is specifically implicated). Consequently, light can induce phase shifts and synchronization as well as control of the freerunning period length. This regulatory mechanism might be exerted either directly (in unicellular organisms) or indirectly, being then mediated by hormones. The transmembrane ion fluxes should be mediated by transport proteins, and the variation in transport rate involves activation or inhibition of these proteins due to changes in both their distribution and their volume. The lateral migration is dependent on the fluidity of the lipids. The temperature-compensating mechanism suggested for the maintenance of their fluidity is assumed to account for the temperature independence of circadian rhythms. This attractive suggestion could, as pointed out by the authors, account for some peculiarities of Euglena temperature compensation in different developmental states; it does not, however, explain the change in sign of temperature compensation of Gonyaulax versus Euglena. The two models have several common features: both involve compartmentation and transmembrane transport of small molecules which provide a feed-back loop by regulating their own transport by means of their concen-
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tration. They accommodate well the eucaryotic occurrence of circadian rhythms and account for light effects. Sweeney's model postulates synthesis o f small non protein molecules in organeltes. The model of Njus et al. specifically implicates ions and membrane-bound ion transport molecules. Both of them assume that membranes take a part in the generation of oscillations. It should be noted that Queiroz et al. (195'2) have also considered, among others, a very similar model and discussed its possible relevance in the acid metabolism of Crassulaceae. The model of Pavlidis however -- discussed below - seems to explain better some features of the circadian systems.
4. 2. 2. Cybernetic theory This theory mainly derives from mathematical models, one of the most satisfactory, from the biological standpoint, being that o f Pavlidis ( 1969, 1971 ). Pavlidis presented a model-system in which a population of interacting oscillators results in a general period much greater than the individual periods of tile various oscillations ( o f the same or different frequency). There exists a strong inhibitory coupling between the oscillators. Computerized, the model simulates phase-shifts, phase response curves and temperature compensation. It has been shown that the model is compatible with biological evidence (Vanden Driessche, 1973). Specifically biochemical oscillations arise from instabilities due to temporary depletion or lowered level o f :some metabolite or factor within a cell compartment. Various oscillations are likely to be coupled the ones with the others, either directly, in the same way as glycolytic oscillations are*, or indirectly by * The two reactions fruc~:ose - 6 phosphate ~ fructose diphosphate and phosphoglyceraldehyde--+ diphosphoglyceric acid are coupled by means of both the glycolytic chain and the adenylate system (Chance et al., 1967; Betz, 1967; Hess and Boiteux, 1973).
means of interactions with membranes, eventually involving pleiotropic effects. Similarly their maintenance implicate defined concentrations of metabolites or factors. Although better understood than any other one, the glycolytic oscillations are by no means exceptions. To take just two examples of metabolic oscillations of high frequency, let us cite the ATP oscillations in Acetabularia (von Klitzing, 1969) and those of creatine kinase activity (Chetverikova, 1973). Relevant also are the fluctuating reservoir sizes of some compounds of photosynthesis described by Wilson and Calvin (1955) under limiting CO 2 pressure. Membranes are assumed to have a dual function. First, they create compartmentation and, consequently, allow some metabolites or ions to reach some low or high threshold value (within the particular compartment), level at which biochemical oscillations arise. This however is not a necessary prerequisite (see metabolic rhythms in Crassulaceae). Second, certain enzymes are part of the membrane and have a variable activity depending on the concentration of some ions or factors. In addition, membranes might be the coupling agents between oscillations from two different compartments, eventually shifting the phase. This stands in agreement with the experiments of Andrews (1968) on the ACTH effects on the adrenal secretory pattern. Whereas only freeze-etched microscopy is hoped to give some direct information concerning possible changes in membrane configuration, the drastic modifications of ultrastructural appearance o f all c o m p o n e n t parts of the chloroplasts of Acetabularia at different times of the day are suggestive of variations of more than one constituant. The cybernetic theory, although it awaits experimental demonstration, applies very well to the experimental evidence provided by circadian rhythms. It has been previously ana-
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lyzed in detail (Vanden Driessche, 1973). Especially relevant is the frequency demultiplication, a corollary of the theory of the population of oscillators: in several systems both circadian rhythms and oscillations of higher frequency are observed. Such is the case of one genotype of Chenopodium (Cumming, 1967), Gonyaulax treated with chloramphenicol (Hastings, 1960), Acetabularia (von Klitzing, 1969; Vanden Driessche, 1970), Euglena (Walther and Edmunds, 1973), Aspergillus (Jerebzoff et al., 1974) and Myrothocium (in Nguyen van Huong, 1967). As development proceeds in higher organisms, the rhythmic pattern evolves. Hellbrii.gge (1960) studying human beings demonstrated that progressively, from one week to another, then from one month to another, and, later, from one year to another, the pattern of the rhythm of body temperature evolves and becomes that of adults. Other rhythms similarly change with age. The same has been found true for avians (Petr~n and Sollberger, t967). Although the composition of membranes is not likely to change much, new functions are operated and new steady states are reached. The shift in metabolism of the Crassulaceae transferred from long days to short days results probably of a comparable process. The analysis of the evolution of the oscillatory pattern of several enzymes could be indicative of the complexity of the system, it stands in agreement with Pavlidis hypothesis and their study might provide a better understanding of circadian rhythms. Prevention of rhythmicity, such as that encountered usually in plants submitted to high light intensities could be readily explained by the fact that the concentrations of metabolites are too high to allow oscillations, especially sustained oscillations. Alternatively (or in addition), undamped oscillations could depend on coupling. Such a case has been encountered by Landahl and Licko ( 1 9 7 3 ) i n
computer simulation. Since almost the same period is maintained in all permissive conditions, Pittendrigh and Caldarola (1973) recently spoke of the "homeostasis of the frequency of circadian oscillations" and analyzed the process in biological and mathematical terms. The significant differences in longevity of Drosophila observed by Pittendrigh and Minis (1972) when the insects are reared in various cycles different from 24 hr strongly suggest the existence of a population of oscillators. Are the differences in the number of consecutive hours of illumination sufficient to produce differences in membrane composition? A large gap however exists between the period of tile biochemical oscillations, usually shorter than a few minutes and that of circadian rhythms. Mathematicians consider that such an important frequency reduction is not insuperable. Experimental arguments supporting the theory of the population of oscillators (including a very important frequency reduction) come from the work of neurophysiologists. In a penetrating review, Hugelin (1972) emphasized (1) the general occurrence of coupling between oscillators in the generation of rhythmic patterns in neurones systems and (2) the gradation in complexity of the systems in the course of evolution. Hugelin (1972) reviewed the rather simple neurone circuits generating patterns in oligosynaptic systems (the pattern depends on which interneurone is stimulated). In his work with TycDumont, Hugelin has established that nystagmus results from the competition of two inhibitory systems and one reexcitable system. In coupled oscillators, such as Aplysia retinal cells, Jacklet and Geronimo ( 1972, in Hugelin, 1972) succeeded in reducing the period of the activity rhythm from 24 to 7.50 hr. Finally in the brain respiratory center, a complex oscillator, Hugelin and Bertrand identified four types of neurones organized in two feed-back
T. Vanden Driessche, 63rcadian rhythms and molecular biology
circuits involving additional positive and negative controls. It thus appears that in the course of evolution more and more complex circuits have evolved but that even the rather simple ones involve circuits. There is evidence for lengthening of the periods and for a role of the quality of excitable cells (at least in some systems, see also Hinkle and Camhi, 1972).
5. Conclusion Circadian rhythms are a remarkable evolutionary achievement, enabling organisms, unicellular or possessing many differentiated organs, to synthetize substances or to activate biochemical pathways in the most appropriate rate for the time of the day. They have a very stable period but, ill unfavourable conditions, rhythmicity might not be expressed. Moreover the circadian pattern is subjected to developmental regulati on. Rhythmicity is encoded in the genome but apparently does not involve a unique straight forward D N A - R N A - p r o t e i n chain. Three devices have been proposed' autophasing in Brown's exogenous hypothesis, small molecules and their membrane-bound transport in both Sweeney's and Njus', Sulzman's and Hastings' hypotheses and the population of intercoupled oscillators arising from biochemical oscillations.
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