Circadian rhythms in Neurospora crassa: biochemistry and genetics.

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conidia from aerial hyphae and from each 0 t h ~ ; ’conse~ quently, conidia remain attached to aerial hyphae, and the plates or tubes are unable to self-inoculate during handling.20 The e m (easily wettable) mutant2’ has a similar phenotype and cpuld also be used. It has the advantage that conidial suspensions for the uniform inoculation of liquid cultures can be prepared from this mutant while they cannot be prepared from strains bearing csp-1 or csp-2.

flGURE 1. The Neurospora conidiation rhythm is expressed as arcs or conidiation by a culture which had been inoculated on agar medium at the perimeter of the petri plate.

petri plates. Cultures growing in constant darkness are monitored for growth at frequent intervals, and the position of the growing edge of each culture is marked on the bottom of the petri dish or race tube under red light, which has no effect on rhythmicity in Neurosporu. When the cultures have finished growing, the positions of the clearly visible conidiation bands, relative to the marked growth fronts, allow the calculation of the period and phase of the rhythm. For monitoring rhythmicity, a mutant strain of Neurosporu, band (bd),which allows the expression of the conidiation rhythm in closed petri dishes or growth tubesI5 is used. Although the primary biochemical defect is not known, the physiological basis of the mutant phenotype appears to be a resistance of growth and conidiation to C0,.l6 Cultures of Neurosporu accumulate CO, near the surface of the culture, and this accumulated CO, normally inhibits conidiation unless it is removed by blowing fresh air across the colony. The bd strain, in addition to its C0,-resistant conidiation, also grows approximately 30% slower than bd’. Biotin starvation produces a phenotype similar to that of the bd mutation,” which suggests that the bd mutation affects a biotin-containing carboxylase complex. Other mutant strains of Neurosporu also express the conidiation rfiythm,ls and the range of expression runs from very weak (wild-type) to quite strong (bd). Mutations that affect conidial separation, csp-1 or csp-2, are also often employed in monitoring rhythmicity. These strains are deficient in the formation of cross walls separating the

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C. Monitoring Rhythmicity in Liquid Cultures 1. Mycelial Disk Cultures Rhythmicity in a liquid culture system was first demonstrated by Perlman et al.’ using a pantothenate-requiring strain (bd pun-2). Disks were cut from a mycelial mat grown in a liquid medium in constant light and transferred to shaking liquid culture in constant darkness in a medium lacking pantothenate so that little, if any, growth occurred. When subsequently transferred, in darkness, from the shaker to fully supplemented agar medium at various times, the disks began to grow and to produce conidial bands. The timing of the bands indicated that circadian rhythmicity had been initially triggered by the transfer of the cut disks to darkness and had continued cryptically in the shaking disk culture. The disk cultures remained rhythmic for approximately 60 to 70 h, although they remained viable for longer periods. An improvement on this systems uses low levels of carbon source to limit growth, rather than pantothenate starvation, allowing the monitoring of rhythmicity in strains which do not carry thepun-2 mutation. Monitoring rhythmicity in shaking disk culture eliminates the complication of biochemical changes associated with the conidiation process, and is also useful for studies of the effects on the rhythm of pulses of chemicals.* 2. Other Liquid Culture Systems Attempts to develop rhythmic, growing cultures of Neurospora in liquid medium have focused on two approaches. The first has been to grow Neurosporu in the dark from a conidial inoculum in the presence of sorbose, a nonmetabolizable sugar.= Sorbose inhibits hyphal elongation and produces highly branched mycelia, which, in liquid culture, form small spheres. These spheres grow slowly, but resume normal growth when sorbose is removed. When these spheres were individually transferred to growth tubes in the dark, the timing of the appearance of the bands indicated that the circadian clock had been running in these ~ p h e r e s . The ~ ~ .second ~ ~ approach has been to attempt to develop rhythmicity in a fast-growing shaker culture.25Although the cultures were synchronized by 12 to 16 h of light after inoculation and were placed in constant darkness, no sign of a circadian rhythm could be detected on transfer to agar medium. The growth rate of these cultures was rapid at 22OC, with a doubling time of 4 to 6 h. Most microorganisms do not exhibit circadian rhythms when they are grown under conditions where their doubling time is less than 24 h.26Growth of

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Microbiology day. In recognition of the role of circadian rhythmicity as a sensory mechanism, we have divided this review into three main sections - “Input”, “The Oscillator”, and “Output” - along with introductory and concluding sections.

Circadian Rhythms in Neurospora crassa: Biochemistry and Genetics Patricia L. Lakin-Thomas, Gary G. Cote, and Stuart Brody ’


1. INTRODUCTION A. Definitions Circadian rhythms are biological rhythms with innate penodicities of roughly 1 d. The term comes from circa diem, meaning “about a day.”’ Such rhythms are widespread throughout the plant and animal kingdoms and among the eucaryotic microorganisms and have been reported among prok a r y ~ t e s . ~The - ~ manifestations of this rhythmicity in the metabolism of cells and organisms range from the “gating” of cell division to complex behavioral changes. Sexual and asexual reproduction, bioluminescence, hormonal levels, and activityhest cycles are a few of the many additional biological phenomena under the control of circadian rhythms. Despite the large amount of phenomenological data about observed rhythms, there is little biochemical knowledge about the underlying mechanism. This review focuses on the circadian clock of one organism, the ascomycete fungus, Neurospora crassa, and discusses various approaches being employed in its study. Circadian rhythmicity in Neurospora was the subject of an excellent, comprehensive review by Feldman and Dunlap in 1983,6 to which the reader is referred for a complete coverage of older material. Certain basic properties are common to the circadian rhythms of most organisms:’ (1) the rhythm is endogenous and selfsustaining; (2) the period of the rhythm is close to but not equal to 24 h; (3) the period of the rhythm varies little with temperature, a property called temperature compensation (see “Input: Temperature Effects”); and (4) the phase of the rhythm can be changed by pulses of light or temperature, or entrained to follow lightldark or temperature cycles (see “Input”). Under constant conditions, circadian rhythms persist and are said to free run. The phase of a free-runningrhythm is measured in circadian time, abbreviated CT. The units of circadian time are circadian hours, equal to 1/24 the free-running period. CT 12 is defined as the time of release into constant darkness from a light-dark cycle or from constant light. In a free-running cycle, the subjective day is the period from CT 0 to CT 12, and the subjective night is the period from CT 12 to CT 24. The circadian rhythm mechanism is, in a sense, a biological sensory apparatus able to receive input from the environment, process it through a central oscillator, and generate an output. It is different from other biological sensors in that it distinguishes changes, in particular, repetitive rhythmic changes, in the environment. Similarly, the output is not just a biochemical, developmental, or behavioral response, but the rhythmic restriction of such a response to a particular, appropriate time of 1990

B. Neurospora Conidiation Rhythm 1. The Rhythm The circadian rhythm in Neurospora is routinely assayed by its expression as a conidiation (asexual spore formation) rhythm on agar media. This complex developmental process is apparently triggered by the clock at some phase of the circadian cycle. Although the conidiation process is closely tied to the oscillator mechanism, several observations suggest that this linkage is one way: (1) biochemical rhythms persist in the absence of the conidiation rhythm (see “Output”); (2) cultures growing in liquid medium, which conidiate poorly, if at all, are apparently rhythmic, based on the phase of the conidiation rhythm which starts upon transfer from liquid culture to agar r n e d i ~ m ;and ~ . ~(3) morphological mutations affecting conidiation do not change the underlying periodicity of the circadian rhythm.” Therefore, the conidiation process itself is not part of the mechanism involved in generating the oscillation. The conidiation process involves the formation of new structures, such as aerial hyphae and conidia, and as expected, a considThe erable number of genes are activated in this region of conidiation is also characterized by more dense mycelial mass (measured as mass per unit area) than the nonconidiating region, increased branching of the mycelia, and aerial outgrowths from the mycelia from which conidia are formed. These changes result in a clearly discernable area called a “band”. The regions between the bands, called “interbands”, are characterized by a less dense mycelial growth and few conidia. Although the interband areas continue to increase in mass,” conidiation does not occur in these regions. These alternating band and interband regions leave a “fossil” record of the rhythm (see Figure 1). 2. Monitoring the Rhythm In practice, cultures for monitoring rhythmicity are grown on agar media in petri plates or growth tubes. The latter are cylindrical glass tubes, also called “race tubes”, usually 30 to 40 cm,6 but sometimes up to 60 cm long,14 which allow monitoring more cycles of the rhythm than is possible with

P. L.Lakim-Thomas earned her Ph.D. at the University of California, San Diego, La Jolla. Dr. Lakin-Thomas is a postdoctoral Research Fellow in the Department of Botany, University of Cambridge, Cambridge, United Kingdom. G. G. Cot6 earned his Ph.D. at the University of California, San Diego. Currently, Dr. Cot6 is apst-doctoral Research Assistant, Department of Molecular and Cell Biology, University of Connecticut, Stom. S. Brody earned his Ph.D. at Stanford University, Stanford, California. Presently, Dr. Brody is a Professor, Department of Biology, University of California, San Diego, La Jolla.

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Neurospora on an agar surface is also very rapid, with a mass doubling time of about 6 h.I3 These rapidly growing surface cultures express the circadian rhythm, which suggests that Neurospora may not follow the general rule, and that lack of detectable rhythrmcity in fast-growing shaker cultures must result from some other factor. However, it is difficult to estimate the length of the cell cycle at the growing front of cultures on agar, and these cultures may not be subject to the same restraints on rhythmicity as are logarithmically growing liquid cultures.

D. Other Methods of Assaying Rhythmicity In addition to the visible conidiation rhythm, the rhythmic production of CO, could also be monitored,’” biochemical rhythms could be monitored (see “Output”), and the ascospore ejection rhythm in the sexual cycle of Neurospora could be measured. The CO, rhythm has been detected in growth tube cultures’8 and in liquid shaker cultures which have stopped Although this assay is nondestructive to the culture, it is inconvenient since each culture requires periodic measurement of its CO, effluent with an infrared analyzer. Even if a number of cultures are monitored simultaneously with appropriate switching manifolds, it would still be far less convenient than assaying the visible conidiation rhythm. Monitoring the circadian rhythm by periodic assays of a biochemical oscillation (see “Output”) is possible, but would be extremely tedious. Furthermore, in order for it to be a useful measurement, it would require greater precision of measurement than is currently available for any known biochemical rhythm. Analysis of the rhythm in the sexual cycle is described in the section on “Output”.

II. INPUT A. introduction Study of environmental input to the circadian oscillator is of interest not only in itself, but also for the clues which may be provided as to the nature of the oscillator. Starting with a given environmental input, it is potentially possible to trace the input pathway to the oscillator itself by identifying the receptor for the input, second messengers triggered by the activated receptor, other messengers triggered by these and so on, although it is possible that second messengers may so multiply that the oscillator mechanism becomes hidden in the altered physiology of the organism. One example for this approach of tracing the pathway from input towards the oscillator is the work of Eskin and ~ o l l e a g u e s ~studying ” - ~ ~ input from the photoreceptor to the circadian oscillator in the retina of the sea hare, ApIysia californica. In nature, the most obvious circadian-rhythmic environmental variables are temperature and illumination, and both of these variables have been extensively studied with respect to their effects on rhythmicity in Neurospora. Again the review of Feldman and Dunlap6is recommended, and we also discuss

input of both temperature and light signals in detail below. The environment also presents numerous other daily rhythms, such as of humidity, ultraviolet irradiation, color of ambient illumination, magnetic field strength, and activity of other organi s m in the ecological community, even of atmospheric electrical p~tential.~’ Changes in magnetic field strength apparently do not affect the Neurospora rhythm$, however, which of the other environmental signals above may affect circadian rhythmicity, or even be detected, has been little studied in Neurospora. Gravity is also rhythmic, due to the tidal influences of the sun and moon, but this also has not been studied with Neurospora. However, the effect of removing Neurospora to an environment of microgravity aboard the space shuttle was examined by Sulzman et al.” Neurospora remained rhythmic in microgravity and the period was unchanged compared to controls maintained on earth. There was a loss of clarity in the expression of rhythmicity as conidial banding which gave the appearance of damping the rhythm; however, the cultures aboard the shuttle were sealed in a foam package for protection whereas the control cultures on the ground were not, and it is possible that the effects on conidiation were due to buildup of carbon dioxide in the sealed package. This possibility is supported by the clear rhythmic banding which followed the unpacking of the cultures for observation during space flight. In discussing the effects of environmental variables on rhythmicity in the following sections, the concept of a phase response curve will occur repeatedly. Phase response curves will also appear in the “Oscillator” section of this review when the effects of drugs, inhibitors, and other treatments on the oscillator are discussed. The reader is referred to Tyson et al.” for a concise discussion of the mathematical background of phase response curves, and to Winf~-ee~~ for the classic extended treatment of the meaning of phase shifts. Winfree’s full-color versiod6 is highly recommended, especially for those of us willing to admit to “math anxiety”. See also Glass and M a ~ k e especially y~~ Chapters 2 and 6, for a concise discussion of phase resetting in a variety of biological oscillators. Many treatments and insults, when applied as discrete pulses, induce a stable change in the phase of observed rhythms, presumably either through specific signal transduction mechanisms or through direct effects on the oscillator. In either case, the central oscillator itself is altered, and the new phase to which the oscillator is shifted by a pulse depends on the old phase at which the pulse was applied. Traditionally, the “phase shift”, that is, the new phase minus the old phase, is plotted against the old phase. This plot is referred to as a phase response curve or PRC (see Figures 2A and 2B). Positive phase shifts are called “advances” and negative phase shifts are called “delays”; this terminology, however, is ambiguous since, for example, a 14-h advance is indistinguishablefrom a 10-h delay. W i r ~ f r e ehas ~ ~suggested an alternative way of plotting phaseresetting, plotting new phase vs. old phase (see Figures 2C and 2D).

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biochemical significance in terms of the mechanism of the oscillator, it is a useful landmark for comparing PRCs produced by different treatments, providing phase shifts are limited to & 12 h since the phase of the crossover point will be different if,other conventions are adopted. In certain mathematical models of the oscillator, notably socalled simple the concepts of advances and delays have actual meaning in terms of the mechanism of phase shifting; the clock can speed up, slow down, even run backwards. In other models, however, advances and delays have no objective meaning and are merely useful bookkeeping conventions. The use of advance and delay terminology in the traditional PRC format thus implies greater knowledge of the mathematical structure of the oscillator than is currently available, unless the terminology is clearly understood to be only a bookkeeping convention. The traditional PRC format has the further disadvantage of introducing an additional source of error through the necessity of calculating phase shifts from the measured old and new phases. We, therefore, recommend plotting phaseshift data in Winfree’s format where these extra calculations and the potentially misleading advance and delay terminology are avoided. However, in this review, we continue to use traditional PRC terminology where convenient, rather than replotting all the data in the literature in the new format.

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FIGURE 2. Hypothetical examples of resetting behavior. A and B are traditional PRCs while C and D show the same data plotted in Winfree’P new phase vs. old phase format. A and C show type 1 resetting, while B and D show type 0 resetting. Although a l l four graphs appear rectangular, it is important to remember that they are actually toroidal or doughnut shaped,Is since on both the new phase and old phase axes CT 0 and CT 24 are the same phase, and on the phase shift axis 12 and - 12 are the same phase shift. To properly visualize this, imagine the curves rolled up vertically to bring CT 0 and CT 24, or 12 and 12 together on the y axis, and the resulting tube then joined at its ends. (Figure adapted from Winfree, A. T., The Geometry of Biological Time, 1980.)

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There are two types of resetting behavior found in circadian systems: type 1 or “weak” resetting, and type 0 or “strong” resetting. In type 1 resetting, phase shifts are typically (but not necessarily) small. In the traditional PRC (Figure 2A), the curve wiggles around the zero-phase-shift axis, crossing it twice. In Winfree’s format (Figure 2C), the curve covers the entire cycle of new phases, wiggling around the new phase = old phase diagonal. In type 0 resetting, phase shifts are always large at some values of old phase. In the traditional format (Figure 2B), the curve crosses the x-axis only once and appears discontinuous; however, this is an illusion since the curve is perfectly continuous at the hole of the doughnut where the 12 and - 12 phase shifts come together. In Winfree’s format (Figure 2D), the new phases are restricted to a narrow range and the curve wiggles around the x-axis or a line parallel to it. The apparent discontinuity which is seen with type 0 resetting in the traditional PRC format is called a “crossover point” or “breakpoint”. Although this crossover point may have no

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B. Responses to Light 1. Introduction Two environmental variables are likely to be the physiologically relevant inputs to the circadian oscillator: light and temperature. Of these two, the effects of light have been most extensively studied in Neurospora, and have been reviewed by Feldman and Dunlap,6 and more recently by Rensing and S ~ h i l lE. ~ d r~n u n d ~also ~ ~ summarizes this information and makes comparisons to other organisms. The following section has been divided into five topics: a brief summary of the known light responses in Neurospora; the effects of light on the phase of the oscillator; light-insensitive mutants; characterization of the photoreceptor; and the search for the intracellular phototransduction mechanism.

2. Light Responses There are six distinct in vivo responses to light which have been described in Neurospora: (I) induction of carotenoid synthesis in hyphaef’ (2) induction of protoperithecia; 41 (3) phototropism of perithecial beaks;” (4) induction of conidiation in starved cultures;43(5) suppression of circadian conidiation by constant light;1s.” and ( 6 ) phase shifting and entrainment of the circadian rhythm by light p ~ 1 ~ eA ~seventh . ’ ~response, ~ ~ ~ initiation of the circadian rhythm in DD by light pulses,* may be another aspect of the phase-shifting/entrainment response. All are classified as blue-light responses since they are induced by blue (or white) light but not by red light, and in cases where an action spectrum has been determined, the major peak is


Microbiology around 465 nm. Action spectra have been determined for the suppression of circadian conidiation by constant light,@for the induction of carotenoid synthesis4 and for phase shifting,4s and the spectra are similar to each other. UV light at 254 nm also phase-shifts the rhythm.47 Although these six responses are similar to each other with respect to their sensitivity to blue light, there may be more than one blue-light receptor in Neurospora. There may also be more than one intracellular signal produced by a single photoreceptor and more than one target for each signal. Studies on the wc mutant (as described below) indicate that the photoreceptor and signal transduction pathway leading to the oscillator may be shared with other blue light responses. (See Schmidt48 and Sengef’ for general reviews of blue-light responses). The light responses which are relevant to a discussion of the circadian oscillator are suppression of rhythmic conidiation by constant light, phase shifting and entrainment, and initiation of the rhythm. The “suppression” of circadian conidiation in constant light? has sometimes been taken to indicate that constant light “stops” the clock, but as Paietta and Sargent suggest in their study of light-insensitive mutants” (see below), this “suppression” might be more precisely described as photoinduction of conidiation. Continuous conidiation induced by constant light might thus mask the persisting rhythm. The question of whether or not constant light “holds” the oscillator at one phase was addressed by Gooch” who suggested that the oscillator continues to operate in constant light. His argument is discussedbelow under “The Oscillator:Mathematical Models.” This leaves only phase shifting and initiation of the rhythm as light responses related directly to the mechanism of the oscillator, and there is good reason to suspect that they are merely two aspects of the same phenomenon. Russoa has demonstrated that although bd cultures germinated in the dark do not express circadian conidiation, a brief pulse of blue light will initiate rhythmicity. This phenomenon has been reported for other organisms as well. Most recently, Dowse5*reported that dark-reared Drosophila individuals are arrhythmicand suggested that this arrhythmicity results from asynchrony in a population of cellular oscillators. The Neurospora mycelium may also consist of a population of oscillators (see below under “The Oscillator: Mathematical Models”); if so, then initiation of circadian conidiation by blue light could result from synchronization of these individual oscillators through the phaseshifting effects of light. In contrast to Russo’s results, Winfree and Twaddle53noted that under their conditions, dark-reared mycelia do show circadian conidiation, but the phases of individual cultures are random and unrelated to other cultures germinated at the same time. It could be interesting to investigate the reason for this discrepancy: perhaps the conidial suspension used by Russo results in the asynchronous germination of many conidia which subsequently fuse to produce an asYnchronOUS mycelium while Winfree and Twaddle may have used inoculation methods and strains (which carried CSP and an unidentified “stopper” mutation) which result in either syn-

chronous germination or the preferential outgrowth of a single synchronous mycelium. In conclusion, the effects of light on the circadian oscillator of Neurospora may all be different aspects of the phase-shifting response. 3. Effects of Ligbt on the Phase of the Oscillator Light phase shifting in Neurospora was fmt demonstrated by Sargent et a l . I 5 and shown to be similar to that in many other organisms. This property allows the clock to be entrained by 24-h light-dark cycles, and by single light pulses every 24 h.6 It can also be entrained to cycles of other than 24 h, but the limits of entrainment have not been determined.6 Unlike some organisms such as D r o ~ o p h i l aNeurospora ,~~ does not exhibit large transients after a phase shift; that is, the new phase is apparent in the first conidiation band formed after the stimulus and only small changes in phase are seen in subsequent bands.I5 The Neurospora oscillator is capable of type 0 resetting by light, as has been demonstrated in several laboratorie~.~~ An example is shown in Figure 3, plotted in both the traditional PRC format with an apparent breakpoint at the phase of maximum phase shifts and in Winfree’s new phase vs. old phase format to show the continuity of the data. The existence of type 0 resetting implies the existence of a singularity, a combination of stimulus magnitude and old phase at which the new phase is ambiguous.35Singularitieshave been defined for other circadian oscillators but not yet for Neurospora. Unfortunately, knowing the topology of the phase-resetting behavior of the oscillator provides few clues as to the biochemical mechanism; it merely allows us to eliminate as possibilities the class of oscillators described as single “simple clocks” (see “The Oscillator: Mathematical Models” for further discussion). 4. Ligbt-Insensitive Mutants Several mutants with reduced sensitivity to light have been identified: [poky], rib (riboflavin requiring), lis (light insensitive) and wc (white collar). The first such mutant to be reportedSS was [poky], which is resistant to light-induced suppression of banding. Brain et al.55demonstrated that [poky] continues to express circadian conidiation under light intensities two orders of magnitude greater than required to suppress the rhythm in wild type. This mutant is defective in mitochondrial protein and is therefore deficient in some cytochromes and in mitochondrial respiration. The insensitivity to light is not a consequence of [pokyl’s respiratory deficiency, as a respiratory mutant rsp-2 shows normal light suppression of the rhythm. Schulz et aL5’ have examined phase shifting in [poky] and found that maximmum phase shifts by light are smaller in [poky] than in wild type, although phase shifting by cycloheximide is normal. This indicates that the oscillator itself is not affected, but only the light signal transduction pathway. Paietta and Sargent” described reduced responses to light in riboflavin-requiring mutants rib. When flavin deficiency is induced by growth at the restrictive temperature (for the tem-

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FIGURE 3. Type 0 phase resetting in Neurospora. Strain bd csp-l in1 was inoculated onto race tubes containing Vogel’s medium plus 25 p.44 inositol. Cultures were exposed to white fluorescent light for 24 h, then transferred to DD, at 22°C. Every 2 h from 12 to 32 h after transfer to DD, four cultures were given a pulse of 1 e n of whjte lighc in a second experiment, four cultures were given a light pulse at 1-h intervals from 27 to 32 h after DD. Four control cultures were used to calculate an average circadian time of each light pulse (“old phase”). New phase was determined for each experimental culture by calculating the circadian time of the light pulse using a regression line for all bands after the light pulse. In A, the difference between new phase and old phase is plotted as “phase shift”, and phase shifts are arbitrarily limited to 12. In B, new phase is plotted directly. The data cover only one circadian cycle, but have been double plotted on both axes for clarity. Each point represents one race tube.

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perature-sensitive mutant rib- 1) or on limiting riboflavin (for rib-2), these mutants show reduced photosuppression of banding, light phase shifting, and light-induced carotenoid synthesis. Normal light responses are restored in the mutants under flavin-sufficient conditions.

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Paietta and Sargent” selected mutants showing Circadian conidiation in constant light, and described three nonallelic light-insensitive (lis) mutants. Although these mutants have reduced sensitivity to light suppression of banding, they are nomal with respect to phase shifting, entrainment, and induction of carotenoid synthesis. This might indicate either a separate photoreceptor for the photosuppression process, or else a single photoreceptor coupled to divergent signal transduction pathways. Results reported with the wc mutants indicate that at least some component(s) of the phototransduction mechanism must be common to most blue-light responses in Neurospora. These mutants were originally described as deficient in light-induced carotenoid synthesis in the hyphae (but not deficient in constitutive synthesis in conidia) and were proposed to be regulatory mutants blocked in p h o t o i n d ~ c t i o n .A ~ ~total * ~ of 11 wc mutants have been isolated, and all fall into only two complementation g r o ~ p s . ~These ’ mutants have also been shown to be insensitive to the effects of light on phototropism of perithecial beaks“ and induction of protoperithecia.61Russoa has demonstrated that the wc mutants are blind for the induction of rhythrmcity in dark-grown cultures as well. The wc mutants are therefore defective in every light response tested so far. If induction of rhythrmcity depends on the phase-shifting response, then it would be predicted that the wc mutants should also be insensitive to phase shifting by pulses of light. As pointed out by R U S S Othe , ~ difficulty in attempting to test this prediction is that the wc mutants cannot be induced to express circadian banding in continuous darkness with the usual lightto-dark transition. An obvious choice for an alternate inducer would be a temperature pulse, which might synchronize an asynchronous mycelium through its phase-shifting effects (see “Temperature Effects” below). For example, Paietta and SargenP8successfully used a temperature pulse rather than light to synchronize their flavin-deficient cultures. If circadian conidiation can be induced in wc mutants by temperature pulses (or other treatments), it would suggest a screening method for isolating new mutants defective in light signal transduction to the circadian oscillator: such mutants would be arrhythmic in DD following a light-to-dark transition unless induced to express circadian banding by a temperature pulse. If mutations are found which show normal carotenoid induction and therefore do not mimic the white-collar phenotype, they would be good candidates for defects in a phototransduction pathway specific to the circadian oscillator. If the wc mutants prove to be insensitive to phase shifting by light, it would lend additional weight to the proposal that the two wc genes code for the photoreceptor linked to the circadian oscillator or for components of the signal transduction pathway.62The blue-light induction of conidiation in starved cultures described by N i ~ m e m a nappears ~~ to use a different photoreceptor from the other blue-light responses (see below), and if so, it can be predicted that wc mutations would not block this response. The in vivo absorption spectrum was determined

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Microbiology for two wc mutants by Horwitz et al.63 who found no difference from the wild-type spectrum, indicating either that the photoreceptor is present in wc or that the photoreceptor cannot be detected by this method even in the wild type. Another aspect of wc mutations is that they block the production of protoperithecia in the dark as well as induction of protoperithecia by light,61indicating a common regulatory process for induction of sexual differentiation and for light responses. htoperithecial formation can be induced in wc mutants as in wild type by treatment with conidia,& demonstrating that wc does not affect sexual differentiation itself but only one of the induction pathways. It could be argued that this makes it less likely that the wc gene products are components of the photoreceptor itself, but rather some components of the signal transduction mechanism which are common to several induction pathways; several possibilities suggest themselves, such as shared G-proteins or protein kinases. The wc genes deserve intensive study, and are good candidates for the molecular biology approach: cloning these genes could provide valuable tools for dissecting signal transduction mechanisms in Neurospora. ,


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5. Characterizationof the Photoreceptor It is not yet known whether a single blue-light receptor mediates all the blue-light responses in Neurospora. Therefore, in discussing “the” photoreceptor, it should be remembered that data relevant to one light response may not apply to all responses. It is now fairly well established that the photoreceptor for the circadian oscillator in Neurospora must consist of a flavin linked to a b-type cyt~chrome.~’.~-Initial evidence for this has come from the action spectrum for phase shifting4’ which is similar to the action spectra for blue-light responses in many organisms and is also similar to the absorption spectra of both flavins and carotenoids. That the photoreceptor is not a carotenoid is suggested by Russo’s results6’ with a strain carrying three albino mutations al-1, al-2, aI-3 which together reduce the carotenoid levels to less than 0.5% of wild type. This strain was shown to have the same threshold for blue-light induction of protoperithecia as the wild type. This eliminates the possibility that the bulk of the carotenoids act as the photoreceptor, but cannot exclude a role for trace carotenoids. Genetic evidence from the light-insensitive mutants described above implicates both flavins and cytochromes as components of the photoreceptor. The [poky] mutant is deficient in cytochromes, and has reduced light responses.” Riboflavinrequiring mutants rib show reduced light responses under flavindeficient condition~.’~ Suppression of banding is affected to a greater degree than either phase shifting or carotenoid induction in the rib mutants, leading these authorsS8to suggest that there may be more than one flavin-containing photoreceptor. In addition, Paietta and SargenP showed that photosuppression of the rhythm and phase shifting can be made sensitive to light

at 540 nm by supplementing a rib mutant with riboflavin analogs which have absorption maxima near 540 nm. Taken together, the data on [poky] and the rib mutants provide genetic evidence that the photoreceptor for the circadian oscillator includes a flavixdcytochromecomplex. ‘ Further evidence for a role for flavins in photoreception comes from the work of Fritz et al.69 They have extended Paietta and Sargent’s work on the rib mutants by looking at the effects of excess flavin on sensitivity to light phase shifting. The sensitivity of the bd rib-2 strain was shown to be proportional to the concentration of riboflavin in the medium, and sensitivity was found to be correlated with the levels of intracellular riboflavin. The photosensitive compound is apparently riboflavin, and not its derivatives: intracellular levels of FMN and FAD do not correlate with light sensitivity, and analogues of riboflavin which cannot be phosphorylated can confer light sensitivity. In addition, it seems unlikely that the photosensitive flavin is bound to protein: the phase-shifting response does not saturate at concentrations of riboflavin up to 250 times the optimal concentration for growth, and riboflavin analogues with bulky side chains are effective in confemng sensitivy. Absorbed light can change the properties of the photoreceptor pigment. Therefore, one method for identifying photoreceptors is to look for light-induced absorbance changes which have an action spectrum similar to that of the response. Such blue-light induced absorbance changes have been reported in N e u r o s p ~ r a ~and ~ . indicate ~ * ~ ~ the reduction of a b-type cytochrome by a flavin. Borgeson and Bowman7’have examined the subcellular distribution of blue-light induced absorbance changes and have found absorbance changes with spectra characteristic of b-type cytochromes in the plasma membrane (PM), endoplasmic reticulum (ER), and mitochondria. The mitochondrial spectrum is similar to that of the cytochrome b of the respiratory chain. The ER spectrum resembles nitrate reductase, which is known to contain a blue-light reducible flavoproteidcytochrome b complex,72and the absorbance change is increased on medium which induces nitrate reductase activity. The PM spectrum is distinct from both the ER and mitochondrial spectra. These light-induced absorbance changes also display differential sensitivity to inhibitors: photoreduction of the PM cytochrome is more sensitive to inhibition by both azide and SHAM than is photoreduction of either the ER or mitochondrial cytochromes. Azide has been reported to quench photoexcited f l a v i n ~ and , ~ ~ it can inhibit light phase shifting in Neurospora (see below). Thus, Borgeson and Bowman’s fractionation study indicates that a PM cytochrome may be a blue-light photoreceptor, while the cytochromes found in the mitochondria and ER are probably the respiratory cytochrome and nitrate reductase, respectively. However, the possibility of a minor component in the ER or in the mitochondria acting as the photoreceptor is not ruled out. Light-induced absorbance changes have been examined in a number of mutants. The [poky] mutant has normal photored-


Critical Reviews In ucible b-type cytochromes in the ER and PM, but excess cytochrome c is found in the plasma membrane fraction.74It has been proposed that this excess cytochrome c channels electrons away from the photoreceptor flavin resulting in the impaired light responses in [poky].74Blue-light reducible cytochromes in the mutants uf-1, wc-1 , rib-1 , qf-4 (which is defi~ient'~ in cytochromes uuj and b), and prd- 1 are essentially the same as in wild type.76Infiq-1 the absorbance peak of the blue-light reducible cytochrome is shifted to a lower wavelength, and in fiq-9 the ER cytochrome is not reducible by blue light.76 Physiological roles for the molecules which are identified with these absorbance changes have not been determined, and the photoreceptor for the circadian oscillator may not be responsible for these absorbance changes. Klemm and N i n n e m a ~ ~found n ~ ~ that in starved mycelia, light-induced absorbance changes do not correlate with light phase shifting. Over the course of several hours of starvation, the light-induced absorbance changes increase in size while the light-induced phase shifts decrease in size. Light-induced absorbance changes do correlate with the induction of conidiation in starved mycelia, and N i ~ e m a n has n ~ reported ~ evidence that this response may use nitrate reductase as the photoreceptor. That the photoreceptor for the circadian oscillator is not nitrate reductase was demonstrated by Paietta and Sargent," who reported that nitrate reductase-deficient mutants nit-1 , nit-2, and nit-3 are competent for light phase shifting as well as photosuppression.

6. The Phototransduction Mechanism The evidence that light sensitivity can be decreased or blocked in the [poky] and rib mutants without destroying rhythmicity indicates that the photoreceptor itself is not an integral part of the oscillator. The effects of light on the receptor must therefore be relayed to the oscillator through a signal transduction mechanism. Two approaches have been used in attempts to identify the intracellular events induced by light in Neurosporu: searching for treatments or drugs which block or inhibit the light signal, and searching for light-induced changes in intracellular molecules. Several factors have been tested for their effects on the phaseshifting response to light, and the results are summarized in Table 1. Nakashima. and Feldmanso reported that the size of light phase shifts is temperature sensitive. Phase shifts are reduced in size at 30°C (as compared to 25°C) and nearly disappear at 34"C,indicating either that the phototransduction pathway includes a temperature-sensitive component, or that the amount of photoreceptor (or some other component of the signaling pathway) is reduced by growth at high temperatures. Brodyw has investigated the effects on light phase shifting of various media components. Phase shifting is not blocked by substitution of KNO, for NH4N0, or by replacing Vogel's salts with Fries' salts. Using the liquid-culture disk system,' Nakashima" found that light phase shifting can be blocked by treatment of mycelial

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disks with azide, diethylstilbestrol (DES), DCCD, or ethanol, but only in mycelia cultured at pH 6.7; light phase shifting is not blocked by these treatments in mycelia cultured at the standard medium pH of 5.7. Vanadate, venturicidin, and oligomycin do not block light phase shifting at either pH. Mycelia cultured at pH 6.7 are eight times less sensitive to light than cultures at 5.7, as determined by the irradiation time required for maximal phase shifting. Light phase shiftlng is also blocked by removal of NH4+ from the medium, but again only in cultures at pH 6.7.83These effects at neutral pH require that the mycelial disks be cultured at this pH for 48 h before the light pulse, and do not depend on the pH of the medium in which the light signal is given. This does not support the idea that proton translocation is involved in light phase shifting, but may indicate that long-term changes (perhaps in membrane properties?) occur during the 48 h at neutral pH. Most recently, Nakashimae2 has shown that the effects of DES on light phase shifting (at neutral pH) and on growth rate can be overcome by addition of unidentified low-molecularweight compounds found in yeast extract. DES and several related compounds have been shown to induce phase-shifts when applied at concentrations higher than those needed to block light phase ~hifting,'~ but this phase shifting property of DES is not overcome by yeast extract.82 Although DES and related compounds inhibit the plasma membrane ATPase in v i t r ~ and , ~ ~inhibit respiration in v i ~ o , ~ ~ NakashimaS2did not find a correlation between their relative effectiveness in blocking light phase shifting and their effects on either plasma membrane ATPase or respiration. It should be noted that the effects of the inhibitors on the plasma membrane ATPase were tested in a purified preparation and the results may not reflect their activity in vivo, leaving the possibility that their effects on light phase shifting may correlate with their in vivo effects on the plasma membrane ATPase. As with many inhibitor studies, the target for DES could prove to be very difficult to identify. DES has been reported to have nonspecific detergent-like effects on membrane proteinss6 and may disrupt membrane structure. However, the identification of the active substance(s) in yeast extract could provide a valuable clue. These conditions which block light phase shifting could be useful tools in studies aimed at identifying the intracellular messengers used by the phototransduction pathway (see below). A putative messenger proposed as both necessary and sufficient for phase shifting could be eliminated from consideration if it is found to be unaffected by these blocking treatments. The search for the signal transduction pathway used by the blue-light photoreceptor in Neurosporu can be guided by the progress made in other systems in which signal transduction mechanisms have been identified: for example, in animal cells stimulated by hormones, growth factors, or neurotransmitters, and in vertebrate rod cells and invertebrate photoreceptor cells

Volume 17, Issue 5

Microbiology Table 1 Inhibitors of Phase Shifting by Light Ref.

Comments Mutations Inhibition [poky1 rib-1 , rib-2 wc-1, wc-2


No effect lis-1, Xis-2, lis-3 nit-1 , nir-2, nit-3 cr- I Treatments Inhibition High temperature Culture ,at pH 6.7

DES Azide DCCD Ethanol NH,-free medium No effect Vanadate Venturicidin Oligomycin Substitution of KNO, for NH,NO, Substitution of Fries’ salts for Vogel’s salts

Altered cytochrome content Less sensitive when riboflavin deficient Blind for initiation of rhythm and other light responses, not tested for phase shifting

57 58 42,59-6 1

Insensitive for photoinduction of conidiation Nitrate reductase deficient Adenylate cyclase and CAMP deficient

50 78 79

Assayed in htb mutants 8 Times less sensitive than at pH 5.7 Only when cultured at pH 6.7, overcome by yeast extract Only when cultured at pH 6.7 Only when cultured at pH 6.7 Only when cultured at pH 6.7 Only when cultured at pH 6.7

80 81

stimulated by light. The known mechanisms can be grouped into four categories: (1) activation of phospholipase C, breakdown of inositol phospholipids, and release of inositol phosphates and diacylglycerol, with subsequent mobilization of calcium and activation of protein kinase C; (2) modulation of the activity of nucleotide cyclases, or cyclic nucleotide phosphodiesterases, with subsequent effects on CAMPand/or cGMP levels; (3) activation of protein kinase activity of the receptor itself; d-ct modulation of ion channels. There is as yet no direct evidence that inositol phospholipid turnover (mechanism 1) may be involved in light transduction in Neurosporu. No blue-light effects on inositol phospholipid turnover or on calcium fluxes have been reported, although there is some evidence for the involvement of calcium in the clock mechanism (discussed below under “Oscillator - Biochemical Approach”). The role of protein kinase activation in the blue-light response is under investigationby Rensing and co-workers8’ who have reported changes in protein phosphorylation after bluelight irradiation. However, there is no information yet as to whether the kinases involved are protein kinase C (mechanism l), CAMP-dependent protein kinase (mechanism 2), Ca2+-calmodulin dependent protein kinase (mechanism 1 or 4),or other protein kinases (mechanism 3).

8132

81 81 81 83

81 81 81 84

I

84

Light-induced ion fluxes, which might result from mechanism 4,have not been directly demonstrated in Neurospora. Sat0 et aLB8found rhythms in the Na+ and KC contents of mycelia (see “Output”), but found no rapid changes in these ions following a light exposure. Kritsky and co-workers89~90 have investigated the effects of blue light on the electrophysiological properties of the plasma membrane, and found a bluelight induced hyperpolarization.This hyperpolarization is probably due to an activation of the electrogenic H+-ATPase, which generates the largest component of the membrane potential,” but the long lag time between onset of illumination and the maximum response (about 20 to 25 min) argues against a direct activation of the proton pump by light. A more rapid response is an increase in input resistance,goreaching a maximum after 2 to 5 min of illumination, but no mechanism for this change has been proposed. A we mutation blocks the light-induced changes in both membrane potential and input re~istance,’~ indicating that these electrophysiological changes are induced by events in the blue-light signal transduction process. Lightinduced ion fluxes deserve further investigation, and several techniques are available which could be applied to the problem in Neurospora. Ion-selective electrodes have been used in plant cells to detect light-induced calcium fluxes associated with photosyr~thesis’~ and the technique can be readily adapted to


Critical Reviews In Neurosporu. Another promising method is patch clamping, which has been applied to plant cell protoplasts to detect bluelight induced H+ extrusion and K + a c c ~ m u l a t i o n . ~ . ~ ~ More attention has been focused on the possible role of cyclic nucleotides in blue-light effects (mechanism 2). Kritsky et al.” observed a light-induced decrease in the level of CAMP, but no change in the level of cGMP. Sokolovsky and Kritskyg7 reported a rapid (within 1 min) light-induced activation of CAMP phosphodiesterase and an accompanying decrease in CAMP; the activation was also observed in cell-free extracts. Shaw and Hardir~g,~’ using rapid freeze-clamping methods and improved assays, reexamined the effects of blue light on cyclic nucleotides and could not find any light-induced changes in either CAMPor cGMP. It should be noted that both of these groups looked for cyclic nucleotide effects in relation to photoinduction of carotenoid synthesis, not phase shifting, and were not concerned with controlling for the circadian phase of their cultures. If the effect of light on cyclic nucleotides is phase dependent, differences in protocols between laboratories could give very different results. Light induced decreases in CAMPlevels have been reported by Hasunuma et The light-induced effects on cAMP are relatively large and are rapid, with the maximum decrease reached within 1 min. These effects may be phase dependent: the largest decrease in CAMP is seen at 12 h after the light-todark transition, at which time a peak in CAMPlevels is also observed in the dark controls, while little change in CAMPis seen at 18 h after the transition. However, the peak in cAMP levels at 12 h does not seem to replicate between experiments. Hasunuma et al.w also report light-induced decreases in cGMP levels, but these changes are small and may not be significant. In one experiment, the effects of continuous white light on cyclic nucleotides were examined, and although the authors claim a rapid oscillation in CAMPwith a period of 60 to 90 min, the sampling interval of 30 min makes it impossible to distinguish the “oscillations” from random noise. In any case, the oscillations cannot be attributed to the effects of continuous light since similar fluctuations are seen in the dark control. (See “Cyclic AMP” below for further discussion of Hasunuma’s work). The hypothesis that CAMP directly participates in the transduction mechanism for light-induced phase shifting is difficult to reconcile with the observation that the crisp-1 mutant, which is completely deficient in CAMP, can be entrained to LD cycles (see “Cyclic AMP”).As suggested by Rensing and S ~ h i 1 1changes ,~~ in CAMPphosphodiesterase activity could be mediated by changes in calcium levels: calmodulin can either activatelW or inhibit”’ cyclic phosphodiesterases. Light-induced changes in cyclic nucleotides could therefore be secondary to an effect of light on calcium and could be signals for light effects other than phase shifting, although the results of Shaw and Harding9’ discussed above argue against a role in the induction of carotenoid synthesis. 374

Many signal transduction systems use GTP-binding proteins as transducers between the receptor and the effector,Io2and it is reasonable to suppose that G-proteins may be involved in the light response in Neurosporu. Hasunuma et a l . I o 3 have reported preliminary evidence that GTP-binding proteins might be present in Neurosporu and have suggested their involvement in light signal transduction. There is as yet no evidence linking G-proteins to any light response in Neurosporu, although the question warrants further investigation. C. Temperature Effects . 1. Introduction With few exceptions organisms are subjected to large variations in environmental temperature, often within a single day. There is a twofold relationship between this variation in environmental temperature and circadian rhythmicity. On the one hand, the period of the rhythm must be conserved despite temperature variation since a clock which runs faster or slower depending on the temperature would not keep accurate time. This conservation of period length despite variation in temperature is termed temperature compensation.’04On the other hand, changes in temperature can provide time cues to synchronize biological rhythms to environmental rhythms. That organisms take advantage of these cues is indicated by phaseshifting responses to temperature changes and by entrainment to temperature cycles. Neurosporu shows both temperature compensation of period and responsiveness to temperature changes. 2. Temperature Compensation a. INTRODUCTION

The term, temperature compensation, was chosen to describe the conservation of period despite changes in temperature in order to emphasize that rhythmicity is not temperature independent since conservation of period is not perfect and since temperature changes can phase shift the rhythm.’05 It is not known whether temperature compensation is truly an input process, that is, whether some sensor measures temperature and inputs a rate-adjusting signal to the oscillator, or whether temperature compensation is an intrinsic property of the oscillator mechanism itself. For convenience, we discuss it in this section along with other temperature effects on rhythmicity. In biology, temperature effects are often quantified using the concept of Qlo, which is defined as the ratio of the rate of a process at a certain temperature to its rate at a temperature 10°C colder. The rate of a rhythm is its frequency, the inverse of the period. Thus, the Qlo for circadian rhythmicity is the ratio of frequency at one temperature to frequency at a temperature 10°C colder, or the mathematically equivalent ratio of the lower-temperature period to the higher-temperature period. The term “Ql0 of the rhythm” is preferred over the inaccurate term “Ql0 of the period length”, sometimes used in the literature, since only rhythms, not periods or frequencies,

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Microbiology can have rates. While most biological and chemical processes have Ql:s of 2 to 3 ,Io6 most circadian rhythms are temperaturecompensated and have Ql;s close to 1.0. The circadian conidiation rhythm of Neurosporu is strongly temperature compensated below 30°C and weakly temperature compensated above this "breakpoint".s0 The Qloof the rhythm is approximately 1.O below 30"C, and approximately 1.3 above 30°C. At high temperatures, conidiation banding is not clearly expressed; however, using mutants in which banding is clear even at high temperatures (lugh-temperature banding mutants hrb- 1 and htb-2), Nakashima and FeldmansOdemonstrated that the period could be as short as 15 to 16 h at 36°C. This pattern of temperature compensation is puzzling given the ecology of Neurosporu. Its natural habitat is in exposed situations following forest and brush-fires in tropical and semitropical regionsIm where temperatures certainly exceed 30°C a good portion of the time. Perhaps accurate timing is less important in the high temperature daytime than it is in the cooler nighttime. b. MUTATIONS AFFECTING TEMPERATURE COMPENSATION

Temperature compensation in Neurosporu can be altered by mutation. The chrono (chr)mutant,79which has a longer period than wild type, is strongly temperature compensated even at temperatures above 3OoC,'Oswhere the wild type is only weakly compensated. The prd-3 also has a longer period than wild type; its rhythm is overcompensated - the period increases with increasing temperature. lo8 The prd-4 muwhich has a shorter period than wild type, has a temperature-dependence pattern in which the Qloof the rhythm changes several times between 18 and 34"C.'08 Mutations can also partially or completely abolish temperature compensation. Mutations at the frequency (frq)"o*l" locus that lengthen the period (but not those that shorten the period) exhibit weaker temperature compensation at lower temperatures than does the wild type. ma Strains carrying the frq3 mutation, for example, exhibit weak compensation above 25"C, while strains carrying thefiq-7 allele exhibit weak compensation, with a Ql0 of the rhythm about 1.3 throughout the tested temperature range of 16 to 34"C, although the Qlo is somewhat closer to 1.O below 25°C than above it. The growth rates of all the f i q 'mutants, however, show the same temperature dependence as that of the wild type.Io8 Another allele at the fiq locus, frq-9,Il2 has lost even the weak temperature compensation offrq-7; the Qloof the rhythm is approximately 2 over the range 18 to 30°C.'4 The period of frq-9 is also sensitive to nutritional conditions, and varies with carbon source and nitrogen source; however, the temperature dependence of the period is similar on all carbon and nitrogen sources tested.14 The cel (chain elongation)mutant, which is defective in fatty acid synthesis, also has altered temperature compensation.20 Although temperature compensation of the conidiation rhythm is normal at temperatures from 22 to 30"C, below about 21"C,

the Qlo is 3.3.*O Thus, at low temperatures the period of cel has an even greater temperature dependence than does that of frq-9. The period of cel, like that offrq-9, is also sensitive to the composition of the growth medium, particularly to supplemental fatty acids.20*113.114 Studies on the effects of temperature, carbon source, and supplemental fatty acids on the rhythmicity of the cel mutant have provided a wealth of data which is discussed in detail later in this review. Thefrq-9 and cei mutants of Neurospora are the only known mutants which have essentially lost the property of temperature compensation. Both, however, have a normal circadian period at a temperature near the middle of the temperature range permitting growth, around 20 to 26°C'4*20(the exact temperature being dependent on nutritional conditions hfrq-9). Both mutants have rhythms which are light sensitive, and both can be entrained by lighvdark ~ y c l e s . ' ~ *Hence, ' ' ~ in both mutants, temperature compensation has been lost independently of other properties of rhythmicity.20 Although thefiq-9 mutant has lost temperature compensation at all temperatures, below 20°C the Qlo is noticeably greater than at higher temperatures.I4Thefrq-9 mutant thus resembles the cei mutant in having a breakpoint temperature below which the period of the rhythm exhibits increased temperature dependence. It has been suggested that frq-9 may be a null mutation for temperature compensation, that is, a mutation causing complete loss of gene function.14The cel mutant would similarly appear to be a temperature-sensitive mutant with a null phenotype at low temperature. However, it cannot be concluded that these mutants completely lack the temperature compensation mechanism or any component thereof since it is not known what the Qlo of a rhythm lacking this mechanism would be, or even if such a rhythm could be maintained. Furthermore, since fiq9 and cel have different Qlo'sfor rhythmicity, they cannot both be exhibiting a null phenotype. c. IS TEMPERATURE COMPENSATION AN ADAPTIVE PROPERTY?

Although it is logical to assume that circadian rhythrmcity is temperature compensated to provide accurate time keeping at all temperatures likely to be encountered in the environment, this assumption has never been tested. It is also possible that temperature compensation is a nonadaptive side effect of the intrinsic nature of the circadian oscillator itself. Experiments monitoring the effects of hypothermia on mammals demonstrate that even homeothemic organisms have temperaturecompensated rhyt"s'15-''7 which suggests that temperature compensation is an intrinsic property of the oscillator itself. The imperfect temperature compensationof Neurospora at temperatures to be expected in its natural environment further supports this possibility. On the other hand, certain tropical plants"' and marine organisms119have been found to lack temperature compensation of their circadian rhythms, which suggests that temperature compensation may be an adaptive

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Critical Reviews In


property of the rhythm which can be lost in environmentswhere it is no longer beneficial. The cel andfrq-9 strains, lacking temperature compensation, could be used to test whether this property is important for accurate time keeping. Both strains entrain to light/dark cycles,'4*113 but this has not been tested at different temperatures. The phase of the conidiation rhythm relative to the light/ dark cycle should be determined at different temperatures to test whether loss of temperature compensation leads to abnormal timing of conidiation. The phase of conidiation in these strains could also be compared to that of wild type under ambient light/dark and temperature cycles approximatingNeurospora's natural habitat.

d. THE MECHANISM OF TEMPERATURE COMPENSATION

Tempemhue compensation is not unique to circadian rhythms. The metabolism of many poikilotherms, particularly those of intertidal habitat, is temperature compensated.'06The mechanism of this temperature compensation appears to result from temperature-dependent changes in the kinetic parameters of enzymes, such as the binding constants for substrates, inhibitors, and activators. '06 Isozymes with different temperature responses have also been implicated.'06In light of this, it seems likely that the mechanism of temperature compensation may reside in the kinetic properties of one or a few enzymes in the oscillator mechanism. Altering these kinetic properties by mutation could alter or abolish temperature compensation as well as alter the period. A number of models have been proposed to explain temperature compensation of circadian rhythms. Some of these postulate temperature-dependent processes which inhibit the oscillator mechanism, 1 M ~ 1 2or 0 a temperature-dependentreduction in the levels of enzymes involved in periodicity.12' Another localizes oscillator processes in membranes and bases temperature compensation on temperature-dependent adjustment of membrane composition. Still another postulates counterbalancing temperature-sensitivities in different parts of the circadian cycle so that, as the temperature increases, one portion of the cycle shortens while the other lengthens.'= Recently a model based on a temperature-dependent inhibitor of the oscillator'"'' has been used as the foundation of a more detailed model explaining many of the properties of thefrq-7 mutant. 124 These models are all plausible, but with the exception of the membrane model, there is no evidence to support any of them. As is discussed later in this review, the cel mutant, which has lost temperature compensation, has also lost the ability to maintain normal membrane fatty acid composition,125which suggests that the maintenance of an appropriate membrane composition may be important for temperature compensation. Models for temperature compensation based on membrane properties and/or the known mechanisms of temperature compensation in poikilotherm metabolism would seem the most promising.

3. phase Shifting by Temperature Steps and Pulses a. PHASE SHlmNG OF CULTURES ON SOLID MEDIUM

Although the periods of circadian rhythms are generally temperature compensated, their phases can be shifted by changes in temperature. It is not known if such phase shifts result from temporary failure of temperature compensation or if they are mediated by specific sensory and message transduction systems. One fonn of temperature change which has been studied is the temperature step, in which temperature is abruptly raised or lowered to a new value, in a rough mimicry of the temperature changes which occur naturally at dawn or dusk. Francis and SargentIZ6have found that both temperature increases and temperature decreases phase shift the rhythm of the bd strain of Neurospora growing on agar in race tubes. The size of the observed phase shift is a function of the circadian time at which the temperature step occurred, of the magnitude and direction of the temperature change, and of the starting temperature. Temperature increases (from 25.5 to 305°C)yield mostly phase advances, up to 15 h, with some small phase delays while temperature decreases (from 30.5 to 25.5"C) yield mostly phase delays, up to 15 h, with some small phase advances. The PRCs for temperature increases and decreases are not mirror images. After a temperature step, the new phase appears to be stably attained immediately, that is, all conidiation cycles after the temperature pulse show the same phase shift relative to the Since a phase delay of greater than 12 h is equivalent to a phase advance and vice versa, the data of Francis and Sargent can be replotted as typical type 0 PRCs with maximum phase shifts (12 h) for both temperature increases and decreases occurring in the early subjective night. The data of Francis and Sargent can also be replotted in Winfree's format, as new phase vs. old phase (see Introduction to "Input"). Both temperature increases and decreases phase shift the rhythm to a narrow range of new phases (see Figures 4A and 4B), typical of type 0 phase response curves. Regardless of the original phase, temperature increases shift the phase to the subjective afternoon (roughly CT 6 to 8) whereas temperature decreases shift the phase to the subjective morning (roughly CT 0 to 6). Thus, temperature increases and decreases reset the clock, respectively, to the warmer and cooler portions of the subjective day. Francis and Sargent126also have found that temperature pulses, in which the temperature is raised or lowered for only a few hours and then returned to its original value, phase shift the rhythm of the bd strain of Neurospora growing on agar in race tubes. The size of the phase shift is a function of the circadian time of the pulse, the magnitude and direction of the temperature change, and the duration of the pulse. Although in other organisms, such as Drosophila rnelanoga~ter,'~'the magnitude of the phase shift resulting from a temperature pulse can be predicted from the magnitudes of the phase shifts of the equivalent temperature steps UP and steps down, this is not true of Neurospora.

Microbiology

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temperature difference of as little as 2°C (23.5125.5”C) was sufficient to entrain the rhythm. This supports the possibility that phase shifting induced by temperature changes acts to synchronize circadian rhythms to the day/night cycle in nature.

A

20

P

z3

16 ’

12

2 8 4

0 24


20

5I

12

2 8 4

0 0

4

8

12

Old Phase

16

20

24

0

4

8

I2

16

20

24

Old Phase

FIGURE 4. Phase shifting of the Neurosporu conidiation rhythm induced by temperature changes. The data of Francis and SargentLZ6 for phase shifting induced in the conidiation rhythm of the bd strain of Neurospora by temperature steps and pulses were replotted in Winfree’s format (i.e., new phase vs. old phase). A, temperature increases (from 25.5 to 30.5”C).B, temperature decreases (from 30.5 to 25.5’C). C,heat pulses (from 25.5 to 303°C).D,cold pulses (from 25.5 to 20.5’C).

As for temperature steps, the phase change following a temperature pulse is apparently stably attained immediately. This is in contrast to the case in other organisms, such as Drosophilu melanoguster54 in which the first cycle or cycles following a temperature pulse show transient phase shifts different from the stable phase shift ultimately attained. If the data for 6-h temperature pulses are replotted to eliminate phase shifts greater than 12 h, the heat pulse PRC for 5°C pulses (from a 25.5”C baseline) is a typical type 0, or strong PRC with maximum shifts (12 h) at about CT 18, the middle of the subjective night. The cold pulse PRC for 5°C pulses (baseline again 25.5”C) appears, however, to be a type 1, or weak PRC,with maximum phase advances of about 4 h and maximum delays of about 5 h. When replotted in Winfree’s format (Figure 4C), heat pulses give new phases restricted to roughly the region of CT 0 to CT 10, the subjective day, while cold pulses (Figure 4D)give new phases which range over the cycle of phases, as is typical of type 1 resetting. Apparently the phase of the rhythm is less sensitive to cold pulses than to equivalent heat pulses. Francis and Sargent also found that a 24-h cycle of alternating temperature steps up and steps down entrained the conidiation rhythm of Neurospora in complete darkness.126A 1990

b. PHASE SHIFTING OF CULTURES IN LIQUID MEDIUM

Phase shifting of Neurospora in liquid culture by heat and cold pulses has been investigated by Nakashima, 128.129 who used pulses of greater magnitude (up 9°C or down 11°C from a 26°C baseline), but shorter duration (3 h vs. 6 h) than did Francis and Sargent above. The effects of a heat pulse are qualitatively similar to those obtained by Francis and Sargent with Neurospora on agar medium. Inspection of the PRCs reported indicates that resetting is type 0, with maximum phase shifting in the mid to late subjective night. Replotting the data in Winfree’s format (not shown) confirms type 0 resetting and shows that the new phases after resetting are restricted to the subjective midday (CT 2 to CT 10). However, the effects of a cold pulse in liquid culture differed from the results of Francis and Sargent with Neurosporu on agar medium.lZ6The PRC reported by Nakashima128.’29 appears to be type 0, or strong, with maximum phase shifts in the early subjectivenight, unlike the apparent type 1, or weak PRC on agar medium. Replotting in Winfree’s format (not shown) reveals that the new phases after resetting all fell within 4 h before or after subjectivedawn. This stronger response to cold of Neurospora in liquid culture compared to Neurosporu on agar medium could result from the larger magnitude of the cold pulse employed by Nakashima. Using a system almost identical to that of Nakashima, Rensing and c o - w ~ r k e r s ’examined ~~ the phase shifting effects of 3-h heat pulses of 30, 35, or 40°C (baseline temperature = 25°C). The different magnitude pulses give similar PRCs with, in general, larger phase shifts resulting from the larger magnitude pulses. However, the PRCs are somewhat unusual. Whereas most PRCs, including the heat-pulse PRCs of Nakashima128*129 and of Francis and Sargent,lZ6show gradual changes from advances to delays, those of Rensing and coworkers show gradual changes from delays to advances. When replotted in Winfree’s format (not shown), it is unclear whether resetting is type 1 or type 0. This discrepancy in results between laboratories using the liquid culture system has been attributed to general variability in phase shifting and to slight differences between laboratories in the handling of the cultures.13oHowever, it is difficult to imagine a major change in the shape of the PRC arising from slight differences in culture handling. A potential criticism of the work of both N a k a ~ h i m a ~ * ~ J ~ ~ and Rensing and co-workerslMis that to measure phase, they both transferred the mycelial disks from liquid culture to solid medium precisely at the end of the temperature pulse. Although it has been shown that the transfer from one medium to another causes minimal phase shifting itself,g it is possible that the combined effects of simultaneous temperature change and medium transfer may differ from the effects of either treatment alone. It might be preferable to transfer the mycelial disks to

377


Critical Reviews In agar medium several hours after the end of the temperature pulse. Temperature-pulse phase shifting experiments with Neurospora have been performed almost exclusively with the bd strain. However, some studies have also been performed on thefrq mutants, using the liquid culture system for assaying phase shifts. Rensing and c o - ~ o r k e r s reported ’~~ that the PRCs of frq-1 and frq-7 differed from those of frq’ in the size of the phase shifts and in the phase of the breakpoint; however, they did not present the actual PRCs of the mutants, and did not report whether these PRCs were type 1 or type 0, so it is not clear whether the PRCs of these mutants can be validly compared to that of the wild type. Naka~hima,’*~ on the other hand, has found that the heat-pulse (26 to 35°C) PRC offrs7 is identical to that offrq+ while the cold-pulse (26 to 15°C) PRC offrq-7 differs from that of the wild type. While, as noted above, the wild-type cold-pulse PRC appears to be type 0, the PRC reported by Nakashima forfrq-7 appears to be type 1, and replotting the data in Winfree’s format (not shown) conf m s this. These results with thefrq mutants must be considered skeptically until discrepancies between laboratories using the liquid culture system for measuring phase shifts are resolved. Naka~hirna’*~ has concluded, based on a comparison of the cold-pulse PRCs of frq-7 and wild type, that rhythrmcity is altered in thefrq-7 strain in the time range between CT 9 and CT 14, wherefrq-7 is apparently less sensitive to cold, and he has proposed a simple model to explain this difference. This model is based on the dubious assumption that the Neurospora rhythm is a simple clock (see “Oscillator: Mathematical Models”) in which the rhythm can be sliced up into discrete time ranges. However, since the wild type apparently shows strong resetting to cold pulses while frq-7 apparently shows weak resetting, the phase of thefrq-7 rhythm is apparently less sensitive to cold than that of the wild type, as suggested by Nakashima, although this sensitivity cannot be localized to any particular time range. C.

PHASE SHIFTING AND THE HEAT SHOCK RESPONSE

Many organisms, when subjected to sudden temperature increases, exhibit what is called a heat-shock response, in which the synthesis of specifjc heat-shock proteins is increased while total protein synthesis de~lines.’~~-”~ Rensing and co-workers’m have suggested that phase shifting induced by heat pulses may be part of this heat-shock response. They have found that the maximum advances and delays from heat-pulse PRCs, and the induction of heat-shock proteins and reduction of total protein synthesis resulting from a heat pulse, all share a similar linear dependence on the magnitude of the temperature change during the heat pulse: Although suggestive, these results do not necessarily implicate heat-shock response in mediating high-temperature phase shifting. Cornelius and Rensing’” have found a circadian rhythm of heat-shock protein inducibility. Maximum inducibility of heat-shock occurred at approximately CT 10 to 11, close to the circadian time of minimal

378

phase shifting induced by heat pulses, 126~129~130 which suggests that heat-shock proteins do not mediate phase shifting. Rensing and co-workers have proposed that heat-shock proteins might be important to the functioning of the clock.130 They reason that if a protein is important in rhythmicity, inducing the synthesis of that protein at a phase where its normal rhythmic synthesis is low would cause large phase shifts while inducing its synthesis at a phase where normal rhythmic synthesis is high should have little effect. Since their phase response curves to heat pulses show maximal and minimal phase shifting at phases where the rhythm of heat-shock protein inducibility’” shows minimal and maximal synthesis, respectively, they concluded that these heat-shock proteins might be involved in rhythrmcity. However, their logic requires a protein that shows a rhythm of uninduced synthesis and a phase-independent level of induced synthesis. A rhythm of uninduced synthesis of heat-shock proteins has not been demonstrated, and the induced synthesis of these proteins is not phase independent. 4. Suppression of Rhythmicity By Cold A final effect of temperature on circadian rhythmicity in Neurospora has been noted. Prolonged exposure to 4”C, or even 10.5”C, appears to suppress rhythmicity, apparently “stopping” the clock at about CT 22.lZ6The center of the first band after return to normal temperature thus always occurs 22 to 24 h later. Surprisingly, short exposures to these temperatures have little phase-shifting effect. lZ6 An alternative explanation of the idea of “stopping” has been proposeds’ based on limit cycle theory, in which rhythmicity continues in the cold, but with different values of the basic rhythmic parameters (whatever they may be). This model is discussed further under ‘‘Oscillator: Mathematical Models” below.

-

5. Temperature Effects Conclusions Neurospora, like other organisms studied, has a circadian rhythm with a temperature-compensated period, yet which is potentially capable of responding to temperature changes as time cues. The mechanism of temperature compensation is unknown. The frq-9 and cel mutants, which have essentially lost temperature compensation, are potential research tools for investigating this phenomenon. Research on the biochemistry of the cel mutant, which is discussed below, implicates mitochondrial and membrane involvement. The sequence of events in response to temperature steps and pulses likewise is unknown. We do not even know whether these responses stem from temporary failure of temperature compensation or whether they are mediated by specific sensory and signal transduction systems. A preliminary attack on this question would involve analyzing the phase-shifting effects of temperature steps and pulses on the cel andfrq-9 mutants since these mutants appear to have lost temperature compensation. In addition, known signal transduction mechanisms, such as those involving CAMPor inositol phospholipids, could be stud-

Volume 17, Issue 5

Microbiology ied with regard to possible involvement in transducing temperature information to the clock of Neurosporu.


111. THE OSCILLATOR: STRATEGIES AND EXPERIMENTS A. Introduction To date, there is no solid biochemical data which would allow identification of the components of the oscillator mechanism in Neurosporu or in any other organism. Therefore, a review can only focus on strategies and techniques while discussing experiments which may or may not have any direct bearing on the identity of these unknown components. The basic strategies have been: ( 1 ) perturbing an input pathway and tracing this path to the oscillator; (2) perturbing an output pathway and tracing this path back to the oscillator; or (3) perturbing the oscillator directly. The strategies can be further classified according to whether the perturbation is by mutation (the genetic approach) or with drugs and inhibitors (the biochemical approach). Input and output strategies are dealt with in Sections II and IV whereas this section deals with direct perturbations of the oscillator by mutation or with chemicals. B. The Genetic Approach 7. Strategies The basic genetic strategies for studying rhythmicity have been the isolation of mutants with altered periods and the screening of existing mutants for alterations in period since any mutation which alters the period must have some effect, direct or indirect, on the oscillator mechanism. Mutations which directly affect clock components might alter either the actual molecules whose levels oscillate, or components necessary for the oscillations, but whose levels do not actually oscillate. Mutations which indirectly affect the oscillator would include any that alter gene products which normally are not part of the clock mechanism, but, because their levels or activities are changed by the mutation, now have some effect on the clock. The oscillator could also be indirectly affected by mutations that alter components of input pathways in such a way that the oscillator receives erroneous input which drives it at an altered period. At present, none of the mutants known to alter the period of Neurosporu can definitively be described as either direct or indirect. Because of the uncertainty as to whether an actual clock component is affected by a mutation, it might be useful to describe all clock mutations as clock-affecting to avoid any semantic arguments. Once isolated, mutations can be studied biochemically, or with classic genetic techniques, such as determination of dominance and studies of interactions between different mutations. Isolation of mutants also opens the way for the application of the techniques of molecular biology.

2. Clock-Affecting Mutations a. MUTATIONS DETECTED BASED ON CHANGES IN PERIOD

The isolation of clock-affecting mutants provides just one half of the structure-function relationship, in that these mutants have alterations in a known function but a defect in an unknown ‘ structure. Techniques for tracking down the unknown primary defects in such mutants can involve several different kinds of screening procedures. For example, one could screen particular kinds of low molecular weight compounds for changes in their levels, or screen the protein population by two-dimensional electrophoresis. One could also clone and sequence the genes in question. The use of several approaches is desirable, and it should be kept in mind that proteins communicate with each other through small molecules, so that even after the successful identification of a primary biochemical defect, one must analyze the resulting changes in metabolism. Seven loci have been identified based on their ability to alter the period of the free-running rhythm of Neurosp0ru:frq (frequency),l10 chr ( C ~ O ~ prd-1 O ) , (~p ~e r i ~ d ) , ’ ~ .r~d~-~2 ,prd’~ 3,’09 prd-4,’09 and clu-1 (clock affe~ting).”~ These map at seven different locations in the genome of Neurospora. With the exception of thefiq locus, the loci are known by one mutant allele each. Except for clu-I, which is a chromosomal rearrangement,13’ it is not known whether these mutations are point mutations, deletions, insertions, or other rearrangements. Some properties of these mutants are listed in Table 2. Feldman and colleagues also discuss the frq, chr, and prd loci.6*109 These clock-affecting mutations were isolated by screening mutagenized conidia for altered period, or were fortuitiously discovered among the progeny of a cross. One strategy for isolating mutants that has not yet been used in studying rhythmicity in Neurosporu is the screening of chromosomal rearrangements. This strategy has been employed to isolate new mutations in Drosophila melunogaster.144*145 Viable duplications could be used to screen for genes which, when present in an extra dose, lead to alterations in period. This screening procedure has the advantage of being able to detect loci which would be lethal if mutated. One could also screen chromosomal rearrangements for changes in period resulting firom break points within loci, or from changes in the proximity of different genes. While hundreds of chromosomal rearrangements are known in Neurosporu,146the clu-1 mutation is the only chromosomal rearrangement currently known to affect rhyth~nicity;’~’it has a period of approximately 28 h.’36 Chromosomal rearrangements could also be used to produce partial diploids of clockaffecting loci and these could be used to test dominance of mutant alleles and to measure the dependence of period on gene dosage. The most intensively studied clock-affecting mutation in Neurosporu is the frq locus. Eight independentfrq mutations have been isolated, and the evidence is convincing that the different isolates are allelic to each other. ‘Iz These alleles are

1990

379

Critical Reviews In Table 2 Summary of Period Changes in Neurospora Mutants Period (h)


Shorter period arg- 13 cys-4 cys- 12 frq-1’ frq-2frq-4 fi9-B Pq-Y (above 26°C) glP-3 [mi-3] and other mitochondrial mutantsb 0117

phe- 1 prd-4 Longer period cef (below 22°C) cel (at 22°C) chr cla- 1 frq-3’ frq-7fTq-8’ frq-9’ (below 26°C) prd- 1 prd-2 prd-3

Dominance

19 19 19 16 19 17-21 19 18-20

NT NT NT Codominant Codominant Recessive NT NT

142 143 1 43 110

18-19 19 18

Codominant NT Dominant

138, 139 141 79, 109

25-40 2042’ 23.5 27 24 29 21-35 26 25 25

NT Recessive Codominant Codominant Codominant Codominant Recessive Recessive Recessive Recessive

20 113, 114 79 136 110 111 112 19, 135 79, 109 79, 109

Note: NT = not tested a

Ref.

The fiq mutants are apparently allelic. See Table 3 for further information on these mutants. ‘ Period is sensitive to fatty acid supplements in the medium.

110, 1 1 1

112 140 140

sation whilefrq-9 is lacking this feature altogether (see “Input: Temperature Effects’’). The altered temperature compensation of frq-3,fiq-7, and frq-9 has raised the interesting possibility’“ that thefrq gene is involved in temperature compensation. Temperature compensation may be an integral property of the oscillator, or it may be a separate mechanism which has an input to the oscillator. Therefore, thefrq mutations could affect either the oscillator mechanism itself, or a separate temperature-compensation mechanism. One possibility is that thefrq gene product is a temperature Sensor which adjusts the rate of the oscillator according to the temperature. The mutations might lead to proteins which “misread” the temperature; for example, at 22”C, the mutant might produce the response more appropriate for 30°C. Temperature compensation would be preserved by mutant proteins which misread the temperature similarly at all temperatures while it would be lost by mutant proteins for which the misreading is temperature sensitive. The frq gene product might alternatively be part of the oscillator mechanism itself, with the mutations affecting a protein or part of a protein that interfaces with the input from the temperature-compensation mechanism. This interface could be as simple as the hydrophobic area of a protein that is sensitive to its lipid environment, or a binding site for an allosteric effector. Since the temperature-compensation mechanism is also altered in a mutant defective in fatty acid biosynthesis (see “Studies on the cel Strain’’ below), the phospholipid fatty acid composition of the frq mutants was examined. No large differences in gross fatty acid composition were detected in the different frq 147. I48

Thefrq gene has been cloned by Dunlap and c o - ~ o r k e r s ’ ~ ~ who undertook a chromosomal walk from the closely linked frq-1 (16.5-h period);”Ofrq-2, frq-4, and frq-6 (19 h);llO.l’l olP gene. Cosmids containing thefrq region transform strains containing the recessivefrq-9 allele to normal period and norfrq-3 (24 h);”O frq-7 and frq-8 (29 h);ll’ and frq-9 (variable mal temperature compensation.149 The sequence of the genomic The period of the wild type,frq+, is 21.5 h. It has region encoding thefrq gene has one large open reading frame been ~ ~ ~ t e d that ~ * the ~ frq ~ . alleles * ~ . constitute ~ ~ ~ an delic (Owof approximately 800 amino acids, and two transcripts, series whose members have periods differing from that of the of approximately 1.5 and 5 kb, are found which hybridize to wild type by multiples of 2.5 h, which suggests that there could DNA from this region.149There is a region in thefrq sequence be a repeating quantal element in the organization of the clock which closely matches the eukaryotic consensus ribosome bindmechanism. However, the 2.5-h period interval is only obing site.’” served at approximately 25°C since temperature-compensation Although the amino acid sequence predicted from the ORF varies in the different alleles. lo* in thefrq region does not match any currently known protein Thefrq-9 allele is unusual in that its period is dependent on sequence, a region of giycine-threonine repeats has been the composition of the growth medium.14 Under certain conwhich shows some sequence similarity to a region in the Droditions, this mutant has a null phenotype and appears arsophila clock gene, per.151-152 This region may allow some rhythmic.I4 However, it is not a null mutant for the oscillator type of posttranslational modification of the frq and per gene since altering the composition of the growth medium allows products. 149 Glycosylation is a good candidate for such a modthe expression of the conidiation rhythm,14 although it could ification, and there are several consensus sites for N-linked be a null mutant for the frq gene product. l4 The frq-7 mutant and O-linked glycosylation. A similar glycine-threonine repeat is also interesting; it possesses a circadian rhythm mechanism that is resistant to phase shifting by cycloheximide p ~ 1 s e s . I ~ ~ region has also been found in a gene for a K’ channel protein in Drosophila, the Shaker locus.153The frq region also has This is discussed below under “Protein Synthesis”. Frq-3 and sequences in the large ORF which, at the amino acid level, frq-7 are also unusual in having altered temperature-compen-


Microbiology match some of the consensus sites for protein phosphorylation by protein kinases A and C.’49The presence of potential kinase sites suggests the possibility of metabolic regulation of the activity of this protein by other proteins, and that this could be related to input from signals such as light or temperature. The possibility that the Neurosporafrq gene and the Drosophilu per gene might code for similar gene products has been suggested previously.109.154~155There are, however, differences in the properties of these mutants: thefrq mutants affect temperature compensation, while per mutants affect this property only slightly,’54 and no true null allele comparable to p e p allele’56has been found forfrq. Furthermore, the evidence is becoming stronger that the per gene product may play a role in coupling rhythmic cells into a rhythmic t i s ~ u e . ’ ~It~would -’~ thus be a component of what has been termed the intercellular clock, as distinct from the intracellular clock which generates rhythrmcity within single cells. 157 Since Neurospora would be expected to possess only the components of the intracellular clock, it would be expected to have no counterpart to the per gene. Nonetheless, the sequence similarity between frq and per argues for some similarity in function. The cloning of thefrq gene paves the way for monitoring transcription and translation of its gene product, localization of the gene product, determinationof the changes in the various f i q mutants, and directed mutagenesis of thefrq locus. Molecular biological analysis of other clock-affecting mutants is also possible. The prd-4 gene has been cloned, and transformation with this clone is currently being attempted.16’Clearly, the molecular biological approach has much potential for contributing to our understanding of circadian rhythmicity in Neurospora. b. CLOCK-AFFECTING MUTATIONS DISCOVERED BY SCREENING OTHER MUTATIONS

The rationale of this strategy is straightforward: to screen mutants with known biochemical defects for any altered clock function. Two types of information can be obtained from this approach: either the pathway affected by the mutation has an influence on the clock, directly or indirectly, or the flux through the pathway does not affect the clock mechanism. Although the screening of biochemical and auxotrophic mutants appears to be a type of “fishing expedition”, it has eliminated a number of metabolic pathways from consideration as components of the oscillator, and it has identified mutations with significant clock effects (see “Studies on the cei Strain” below). In practice, the biochemical mutants are generally crossed into the bd strain to allow the expression of the rhythm. With auxotrophs, these double mutant strains are tested on media supplemented with high and low levels of the appropriate supplement. A total of 85 biochemical and morphological mutants, representing a significant portion of the known mutants in Neurospora, have been screened for changes in period; these are listed in Table 3. Seventy-threeof these have shown no changes in period at the screening temperature, although they have not

been screened for changes in period at other temperatures, that is, for changes in temperature compensation. Most of them have also not been tested for any effects on phase shifting. These 73 mutations with normal period affect a wide range of metabolic reations, including amino acid synthesis, lipid synthesis, assimilation of alternate carbon and nitrogen sources, and mitochondrial metabolism, as well as mycelial morphology and resistance to inhibitors. In the case of many auxotrophs, the mutation could only be screened in the presence of the required supplement because growth or conidiation was poor without supplement. One cannot conclude, therefore, that rhythmicity does not require the end product of the mutated pathway, but only that the flux through that pathway is unnecessary for normal period. The screened mutations include a variety of different morphological mutants which were screened to determine whether the introduction of a gene causing slow growth would change any clock property and whether defects in the conidiation process would lead to changes in any clock property. Since these morphological mutations did not affect the period, both answers are negative, at least with respect to the clock period. The 12 mutations which do alter period are discussed further below. Three of them affect amino acid synthesis, two affect lipid synthesis, one affects carbon assimilation, and six affect mitochondrial energy metabolism. A complete list of all Neurospora loci known to 1982 has been compiled by Perkins et al.,I7O and the strains which are discussed in the following sections are described therein. A small percentage of auxotrophic mutations in Neurospora are associated with chromosomal rearrangements,’* and the altered period exhibited by certain auxotrophic mutants could result from such a rearrangement and not from the mutation itself. Such mutations should be tested for chromosomal rearrangements, and several independent isolates of the mutations should be screened for period effects, if possible. Mutations affecting amino acid synthesis Three mutations affecting amino acid synthesis slightly shorten the period. These are cys-4 and cys-12 (cysteine req~iring),’~~ and arg-13 (arginine requiring).’” The arginine mutant effects are puzzling since other auxotrophsfor this amino acid have normal periods.’42Mutations affecting other amino acid pathways are available, but have not yet been tested. Mutations affecting lipid metabolism - Two mutations affecting lipid synthesis affect the period. The phe-1 (phenylalanine requiring) mutant has a 19-h period, even in the presence of high levels of phenylalanine.”’ It has been speculated’71 that this mutant is blocked in an early step in the synthesis of certain sterols, and that high levels of phenylalanine, which it requires for growth, allow the synthesis of sterols by an alternate catabolic pathway. This has been confirmed by the patterns of incorporation of radioactive phenylalanine and acetate into sterols in the mutant.’” The basis for the shortened period is not known, but could result from an effect on membrane composition since sterols are integral constituents of membranes.

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Critical Reviews In Table 3 Mutants Screened for Effects on Period Locus


Amino acid synthesis arg-13 w-4 cys- 12 am am;en-(am)-1 arg- 10 asp gln- I his-3 lys- 1 met-7 met-9 phe-2 ser-3 ser-5 trp-3 tyr- 1 Lipid metabolism cel phe- 1 chol-2 erg-1 erg-3 in1 ufa- 1 Vitamin synthesis nic-2 nic-3 nt pan- 1 pan-2 pdr- 1 rib- 1 rib-2 Other auxotrophs ace- 1 ace-3 ace4 ace-7 ad-4 . cr-1 SUC

Carbon, nitrogen or phosphate assimilation glp-3 W--1) acu- 1 acu-3 acu-5 acu-6 acu-7 cpd- 1 cpd-2 glp-2 inv nit-1 nit-3 ota

382

Description

Period (h)

Ref.

Arginine requirer Cysteine requirer Cysteine requirer Amination deficient Amination deficient; enhancer Arginine requirer Aspartate requirer Glutamine requirer Histidine requirer Lysine requirer Methionine requirer Methionine requirer Phenylalanine requirer Serine requirer Serine requirer Tryptophan requirer Tyrosine requirer

19 19 19 NC NC NC NC NC NC NC NC NC NC NC NC NC NC

142 143 143 6, 84 84 142 84 84 10 84 6, 142 84 84 6 6 10 10

Palmitate requirer, fatty acid synthetase deficient Phenylalanine requirer, ergosterol synthesis deficient Choline requirer Ergosterol synthesis deficient Ergosterol synthesis deficient Inositol requirer

20-40 19 NC NC NC NC

Oleate reqUirer, fatty acid desaturation deficient

NC

20 141 10 162 162 10, 139, 162 136

Nicotinate requirer Nicotinate requirer Nicotinate requirer Pantothenate requirer Pantothenate requirer Pyridoxine requirer Riboflavin requirer Riboflavin requirer

NC NC NC NC NC NC NC NC

10 10 10 6 9 10 66 66

Acetate requirer Acetate requirer, pyruvate dehydrogenase deficiency Acetate requirer, pyruvate dehydrogenase deficiency Acetate requirer Adenine requker Adenylate cyclase deficient Acetate or succinate requirer

NC NC NC NC NC NC NC

163 164 164 163 84 79 84

Enhanced glycerol utilization Acetate nonutilizer Acetate nonutiIizer, glyoxylate shunt deficient Acetate nonutilizer Acetate nonutilizer, gluconeogenesis deficient Acetate nonutilizer, oxoglutamte dehydrogenase deficient Repressible acid phosphatase deficient Repressible acid phosphatase deficient Glycerol nonutilizer Sucrose nonutilizer, invertase deficient Nitrate reductase deficient Nitrate reductase deficient Ornithine transaminase deficient

19 NC NC NC NC NC NC NC NC NC NC NC NC

140

Volume 17, Issue 5

165 165 163, 165 165 164, 165 166 166 163 18 18 18 84

Microbiology Table 3 (continued) Mutants Screened for Effects on Period Description

Period (h)

Ref.

Benomyl resistant, p-tubulin affected Cycloheximide resistant, 80s ribosomes affected Cycloheximide resistant, 80s ribosomes affected Chloramphenicol sensitive

NC NC NC NC

84 168 168 84

Colonial Colonial, temperature sensitive Conidiation delayed Conidial separation deficient Conidial separation deficient Conidial separation deficient Aconidiate Osmotic pressure sensitive Spreading growth Spreading colonial Spreading colonial

NC NC NC NC NC NC NC NC NC NC NC

84 84 10 169 84 84 10 84 10 10 10

Cytochrome oxidase subunit 1 deficient (allelic)

18-19

84, 140

Presumptive ATPase subunit 6 deficient, temperame sensitive Mitochondrial ribosome defect

NC

84

NC

55

Cytochrome au3 deficient Cytochrome 6 deficient Cytochrome b deficient, temperature sensitive Cytochrome b and au3 deficient Oligomycin resistant, ATPase subunit 9 affected Antimycin-sensitive, alternate oxidasc deficient Cytochrome b and au3 deficient

19 18 20 20 18-20 NC NC

140 140

Albino Cold-sensitive ribosome synthesis Light-insensitive for suppression of rhythm Light-insensitive for suppression of rhythm Light-insensitive for suppression of rhythm NADase deficient

NC NC NC NC NC NC


Locus Inhibitor resistance Bmf Cyh-1 cyh-2 cpl-1 Morphological mutants col-4 Cot-3 cr-3 csp- 1 csp2 ear

fr OS-4

Pi spco-6 spco-9 Mitochondrial energy metabolism Maternally inherited [m’-2] “‘-31 [mi-51 [C-931 @bI([mi-ll) Nuclear inherited cya-5 ~yb-2 ~yb-3 Cyt-4 011’

ANT-1 cyt- 1 Miscellaneous al-2 crib-I lis- 1

lis-2 lis-3

Mda

84 84

138, 139 140 84 10 25 50 50 50 84

‘Note: NC = no change in period.

The cel (fatty acid chain elongation defective) mutant, which requires saturated fatty acids for has lost temperature compensation, as is discussed above under “Input: Temperature Effects”. The period of cel is also altered by the addition of naturally occurring fatty acids to the growth medium, and effects of these compounds are relatively This mutant has a deficiency in fatty acid ~ynthetase”~ and is therefore blocked in the synthesis of saturated fatty acids. The ufa-1 (unsaturated-fatty acid requiring) mutant, blocked at a later stage of fatty acid synthesis, the desaturation of stearic does not affect the period of the circadian rhythm at 22”C, or

at lower temperatures, or in the presence of the fatty acid, linoleic acid. 136 This suggests that it is a block in the early part of the fatty acid synthesis pathway that leads to the loss of temperature compensation. However, the flux through the pathway synthesizing sahuated fatty acids is not itself necessary for normal rhythmicity since the cel strain supplemented with palmitic acid has an apparently wild-type rhythm.Considerable research has focused on the biochemistry of the cel mutant, and this is discussed in depth below. Mutations affecting assimilation of carbon, phosphate, or nitrogen The glp-3 mutant, which is allelic tofi-1 (fe-

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383


Critical Reviews In male fertility can grow well on glycerol as a carbon source, compared to the poor growth of wild type.175This mutant has a 19-h period at 22"C,I4O but has not been characterized further. Other mutants involving carbon, phosphate, or nitrogen metabolism were also analyzed. These strains will all grow reasonably well on a minimal-glucose medium, but are unable to grow on specific alternate sources of carbon, nitrogen, or phosphate. The lack of period effects in these mutants indicates that the flux through certain pathways can be tentatively eliminated as important to the biological clock mechanism; these are the glyoxylate pathway (defective in the acu-3 mutant), the TCA cycle (defective in the ucu-7 mutant), gluconeogenesis (defective in the acu-6 mutant), and the pathway that catabolizes argirrine and omithine (defective in the ofu mutant). Of course, these pathways were assumed to be irrelevant to the clock since normal rhythmicity is observed under growth conditions where these pathways are repressed. The cpd mutants, like bd, exhibit rhythmic conidiation, but they do not affect the circadian period. They are discussed below under "Cyclic AMP",

Mutations affecting mitochondrial energy metabolism -

The mitochondrial mutants that have been tested to date for clock effects either have faster clocks, with periods in the 18to 20-h range, or have normal periods. No mutations with longer period have yet been found. Several alleles are known at the oh7 (oIigomycin resistance) locus which have shorter periods, and the decrease in period roughly correlates with the degree of resistance to oligomycin. 138~139The oh7 mutation affects the DCCD-binding subunit of the mitochondrial ATP synthetase, and the amino acid substitutions of each allele have been determined. 138*176 The temperature-sensitive mutant [C931, which is maternally inherited, is also a presumptive ATPase mutant.'" It has a normal period at 22°C but at 32°C or higher, where the mutation is expressed, the banding pattern is not visible and therefore the period has not been determined." Several mutations which affect mitochondrial cytochromes lead to shorter periods. These include qb-2" and ~yb-3,84which are deficient in cytochrome b;178,179 cya-5,IN which is deficient in cytochrome U U , ; ' ~ and ~ ~ y t - 4which , ~ is defective in processing mitochondrial rRNA75and is deficient in both cytochrome b and cytochrome UU,."~ The cyb-3 mutant is particularly interesting, as it is temperature sensitive and does not clearly express the cytochrome deficiency below about 38°C,'79 although the period effect is seen at 22"C.84A 19-h period is also shown by a maternally inherited mutant140 represented by the isolates, [rni-2], [rni-3], and [mi-5].'*' Initial studies'82J83on the oft7, [mi], and cya-5 mutants have shown that they have slower growth rates than the wild type and have twofold more-mitochondrial protein per total protein in mycelial extracts; whether this increased mitochondrial dosage is the cause of the faster clock rate is an open question. Thus, a variety of mitochondrial defects produce both 19-h periods and an increase in the relative level of mitochondrial protein. 384

3. Studies on the eel Strain a. INTRODUCTION

The cef mutant was first isolated based on its requirement for the fatty acid 18:0.Iw (Fatty acids are designated in this section by the x:y notation where x = number of carbons and y = number of double bonds.) It was shown to require saturated fatty acids for optimal however, the requirement is not absolute, as it grows, albeit slowly, without supplement. 173 The mutant exhibits reduced fatty acid synthesis resulting from levels of 4'-phosphopantetheine cofactor bound to the fatty acid synthetase which are less than 2% of wildtype levels. 173 Lakin-ThomasIa found that several Neurospora proteins could be labeled by added radioactive pantothenic acid, and that the cef mutant was deficient in the labeling of only one of these proteins, which had a molecular weight consistent with it being the fatty acid synthetase. This suggests that the defect in cef is either in the fatty acid synthetase itself or in a cofactor-attaching enzyme which is specific for the fatty acid synthetase. The loss of temperature compensation in the cef mutant at temperatures below 22°C is described above (see "Input: Temperature Effects"). Above this breakpoint temperature, the rhythm has apparently normal temperature compensation. The cef mutant is temperature sensitive in another respect. At high temperatures, growth rate ceases to increase with temperature. Like loss of temperature compensation, this phenomenon exhibits a breakpoint; below 23"C, the Qlofor growth is normal, approximately 2.6, while above 23°C it is 1.0." b. EFFECTS OF SUPPLEMENTAL FATTY ACIDS ON cel

The period of the conidiation rhythm of the cef mutant is sensitive to certain supplemental fatty acids in the growth medium while the period of the wild type is not affected by any supplemental fatty acid. Although saturated fatty acids such as 16:O and 18:O permit optimal growth of the mutant without affecting the period at 22°C the unsaturated fatty acids, 18:1, 18:2, and 18:3, lengthen the period to as long as 26, 40 and 33 h, respectively, at 22°C.'13This observation is particularly interesting since the active compounds are not exotic antibiotics or inhibitors, but normal constituents of Neurospora hyphae. ' I 3 This initial observation has been extended by the finding that saturated fatty acids, 8 to 13 carbons long, also lengthen the period of cel at 22"C.'I4 Fatty acids 14 or 16 carbons long, however, not only do not lengthen the period, they reduce or cancel the effects of period-lengthening fatty acids like 18:2 when the two species are added to the growth medium together. *I4 Mattern et aL20 showed that the effects of the fatty acids, lS:l, 18:2, 18:3, and 12:0, on the period of cel could be explained by a change in the breakpoint temperature below which the rhythm is not temperature compensated. These fatty acids raise the value of the breakpoint temperature to about 26°C with the result that the period is lengthened at 22°C.

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Microbiology tion is lost. It is furthermore not known whether fatty acids Conversely, the fatty acids 14:O and 16:0, which cancel the which fail to lengthen the period are simply not taken up by effects of period-lengthening fatty acids, lower the breakpoint Neurospora, or are taken up, but rapidly converted to inactive temperature to about 18"C, with the result that the period is metabolites. Conversely, it is not known whether fatty acids normal down to this temperature. It has not been demonstrated, , which do lengthen the period are active themselves, or whether however, whether all fatty acids which lengthen the period at they are rapidly converted to active metabolites. 22°C raise the temperature compensation breakpoint, nor has it been demonstrated whether other fatty acids, reportedly "inactive" at 22"C, also lower the temperature-compensation c. REVERSAL OF FAlTY ACIDINDUCED PERIOD LENGTHENING breakpoint. Adding still another layer of complexity to the phenomenon Interestingly, some of the fatty acids which alter the breakof the cef mutation is the finding that period lengthening by point temperature below which normal temperature compenfatty acids may be reversed by certain environmental condisation of rhythrmcity is lost, also alter the breakpoint temperature tions. Some of these environmental conditions have already above which normal temperature-dependence of growth rate is been alluded to above. At elevated temperatures, for example, lost. The fatty acids 12:O and 18:1, for example, reduce the period is normal in the presence of period-lengthening fatty temperature range of normal temperature responses by raising acid supplements,20 so heat can be considered to reverse the the breakpoint value for loss of temperature compensation, and fatty acid effects. Similarly, the fatty acids 16:O and 14:0, lowering the breakpoint value for loss of growth-rate temperwhich partially restore temperature Compensation, reverse the ature dependence while the fatty acids, 140 and 16:0, broaden effects of fatty acids which lengthen the period at 22°C.z0*113 the range of normal temperature responses by lowering the Certain carbon sources also reverse period lengthening by supbreakpoint value for loss of temperature compensation and plemental fatty acids.182J9z The carbon sources found to be raising the breakpoint value for loss of growth-rate temperature effective acetate, glycerol, glutamate, casamino acids, pydependence.*O Any explanation for these breakpoints in temruvate, and ethanol are a l l nonfermentable carbon sources perature effects on period and growth rate must explain the which are of necessity metabolized in the mitochondria. Tween fact that the breakpoints for period effects and for growth 40 and tween 80, as carbon sources, also reverse period lengtheffects are different; thus, for example, they cannot be due to ening. 192 These compounds are water-soluble esters of 16:O a hypothetical phase transition in the membrane lipids of cel. and 18:1, respectively, fatty acids which are oxidized to acetyl A variety of fatty acids, both natural and synthetic, have CoA which is then metabolized in the mitochondria. Connow been tested for ability to lengthen the period of the cel versely, none of the sugars tested, including both pentoses and strain at 22°C. The results are summarized in Table 4. Saturated hexoses, both monosaccharides and disaccharides, and both fatty acids 8 to 13 carbons long lengthen the period, while ketoses and aldoses, reversed fatty acid-induced period lengthshorter (6 carbons) or longer (14 to 24 carbons) do The ening. 192 Acetate, at least, may exert its period-stabilizing efunsaturated fatty acids, 18:1, 18:2, and 18:3, which occur fects by lowering the breakpoint temperature for loss of naturally in Neurospora, and y-18:3, which does not, all temperature compensation since the period of cel is circadian lengthen the p e r i ~ d " ~while * ' ~ ~other monounsaturated fatty at 18°C on acetate as a carbon acids do not. lS6 A cis double bond is not necessary for period There are a number of biochemical differences which would lengthening since trans, trans 18:2 lengthens the period,Is6as be expected during growth on a nonfermentable carbon source does the triple bond analog of 18:l (9-octadecynoic acid).Is8 relative to growth on sugars. The glyoxylate cycle must be Very unusual analogs in which the double bonds of 18:1, induced to provide two-carbon units for biosynthesis, and the 18:2, or 18:3 have been replaced variously with bromine, epoxy, monophosphate shunt must also be induced to provide methylene, or alkoxy substituents have also been t e ~ t e d . ' ~ ~ " ~pentose ~ cytoplasmic reducing equivalents. Similarly, glycolysis would some lengthen the period while others do not, and there appears shut down for lack of substrate and gluconeogenesis would be to be no simple way to predict the effect of any given analog. For example, 9,lO-dibromostearic acid and 9,10,12,13,15,16required to provide monosaccharides. Increased levels of mihexabromostearic acid lengthen the period while 9,10,12,13tochondrial enzymes and increased flux through mitochondrial tetrabromostearic acid does not.Is9 energy pathways would also be expected. The finding that These experiments with both natural and synthetic fatty acids nonfermentable carbon sources reverse period lengthening by provide a wealth of data which must be interpreted only with fatty acids suggests that some aspect of metabolism, induced, great caution. In most cases, all that is known is that the period or at least altered, by growth on nonfermentable carbon souces, of the rhythm of the cel mutant at 22°C is lengthened in the blocks the effects of supplemental fatty acids on temperature compensation in cel. The possibility that mitochondrial mePresence of the fatty acid in question. It is not known whether all fatty acids which lengthen the period do so by the same tabolism in the presence of these carbon sources may be important for reversal of period lengthening is supported by the mechanism, in Particular, whether they do so by altering the finding that antimycin, an inhibitor of mitochondrial cytobreakpoint temperature below which temperature compensa-

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Critical Reviews In Table 4 Ability of Fatty Acids to Lengthen the Period of the cel Strain at 22°C Active Saturated fatty acids

8:O 9:O 10:0 11:O 12:O 13:O

cis-Monounsaturated

A9 18:l AIO 11:l

cis-Polyunsaturated

18:2 18:3 A6.9.12 18:3 r-A9J2 18:2

Ref.

Inactive

6:O 14:O 16:O 17:O 18:O 20:O 22:O 24:O A9 16:l A6 18:l A l l 18:l All 201 A" 22:l

113, 114

113, 186, 187

113, 186


A9.12.15

mum-Unsaturated AlkynYl Brominated'

Cyclopropylb

r-Ag 16:l r-A9 18:l Br. 18:O

186 188 189, 190

(CHZ-),

189

9-octadecynoic

11-Br 1l:O 12-Br12:O Br, 18:O Br, 18:O (-CH,-)18:O

1810

(-CHz-)J 18:O OH 18:O

HYbXy'

Epoxyb

(-0-) 18:O

(-O-)z 18:O

Akoxy'

Me0 18:O EtO 18:O

(MeO), 18:O

(-0-)a

189 189

18:O 189,191

(MeO), 18:O

Pro 18:O iBuO 18:O The di-, tetra-, and hexa-bromo fatty acids were synthesized by bromination of A9 18:1, A9.12 18:2, and A9.12,u 18:3,respectively, and thus represent the 9,lO-;9,10,12,13-; and 9,10,12,13,15,1 &isomers, respectively. The mono-, di-, and hi-substituted fatty acids in these groups were synthesized by conversion of the double bonds of A9 18:1,AgJ2 18:2, and AgJZJ518:3, respectively, to ring structuxes. They thus represent the 9:lO-;9:10, 12:13-;and 910, 1213, 15:16isomers, respectively. The mono-, di-, and hi-substituted fatty acids in these groups were synthesized by reduction of the double bonds of A9 18:l. A9.12.18:2, and A9J2J5 18:3, respectively. They thus represent mixtures of isomers with the substituent(s) at the 9 or 10; 9 or 10 and 12 or 13; and 9 or 10, 12 or 13,and 15 or 16 positions, respectively.

chrome c reductase, also reverses period lengthening by fatty acids. Three loci are known at which secondary mutations reverse period lengthening by fatty acids in the cel mutant and are thus epistatic to the cel mutation with respect to this phenotype. These are the 0lP locus,193the maternally inherited locus (two alleIes, [mi-2]and [mi-5]),1'10 and theprd-1 Mutations at the oll7 locus alter subunit 9 of the mitochondrial ATP synthetase (the DCCD-binding protein), conferring oligomycin r e ~ i s t a n c e . ~Several ~~.~~ alleles ~ of this mutation are known, altering different amino acids of subunit 9, and confemng Reversal of period different degrees of resistance. 138~176~196 lengthening by different alleles correlates with oligomycin resistance in that the least resistant allele is unable to reverse period lengthening by 18:2 while the most resistant alleles restore circadian period in the presence of 18:2 even at 18°C.L94 It is interesting that 18:2, one of the fatty acids whose period

lengthening is reversed by the 0lF mutation, itself confers some degree of oligomycin resistance.193The [mi-21 and [mi-5]mutations are mutations of the mitochondrial genome which lead to defects in subunit 1 of the mitochondrial cytochrome c o x i b e . 181.197 The prd- 1 mutation also reverses period lengthening by fatty acids. It was isolated on the basis of altered circadian rhythmicity; its period is lengthened to 25.6 h.135The primary defect is unknown, although the mutant apparently produces 2 to 2.5 times as much mitochondrial protein per unit mycelial mass as does the wild type.182The prd-1 mutant also has a bulk phospholipid fatty acid composition different from that of prd+, both in liquid culture and on plates.147However, the phospholipid fatty acid composition of the mitochondria is apparently normal. 192 A number of factors which reverse period lengthening in cel also shorten the period of wild type to 18 to 20 h and lead to

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Microbiology


increased levels of mitochondrial protein relative to total pro~ , and rrni-51 mutations,182 tein. These include the d ~[rni-2], and continuous exposure to a n t i m y ~ i n . 'The ~ ~ prd-1 mutation also increases levels of mitochondrial protein relative to total protein, lS2 although it does not shorten the period, but lengthens it. These results suggest that the effect of the cel mutant may be mediated through mitochondrial metabolism, although a trivial explanation is also possible. Mitochondria possess a fatty-acid synthesizing system which may be distinct from the cytoplasmic fatty acid ~ynthetase.'~~ If increased levels of mitochondrial proteins correlate with increased mitochondrial fatty acid synthesis, this could have the effect of suppressing the primary defect in fatty acid synthesis resulting from the cel mutation. d. POSSIBLE MECHANISMSOF FAlTY ACID ACTION

Any hypothesis of the mechanism whereby fatty acids affect the period of the cel mutant must explain why the cel mutant is affected while eel+ is not. As there is no evidence that any protein other than the fatty acid synthetase is altered in cel, this protein apparently must play an integral role in sensitizing the rhythm of cel. Either the fatty acid synthetase itself must be part of the oscillator, which seems unlikely, or else reduced activity of this enzyme must trigger other changes in lipid metabolism, which, in turn,make components of the oscillator sensitive to the effects of exogenous fatty acids. One possibility is that changes in lipid metabolism in cel alter the metabolism of the exogenous fatty acids. Metabolism of exogenous radioactive fatty acids by cel and by eel+ has been studied.192Although no qualitative differences between the strainshave been found, the cel mutant both oxidizes exogenous fatty acids and incorporates them into lipids to a greater extent than does the wild type. Another possibility is that altered lipid metabolism in cel might lead to altered membrane composition which could sensitize the cel mutant to the effects of exogenous fatty acids if these effects are mediated through membranes or membranelocalized processes. The phospholipid fatty acid composition of the cel mutant and of the wild type have been examined in liquid culture at 22°C.125 The phospholipid fatty acid composition of both strains varies during logarithmic growth under these conditions, but the phospholipid fatty acid composition of cel varies in a way much different than that of cel+ . Notably, the level of 18:2 increases with growth in cel+ but is constant in cel while the level of 16:O is constant in cel+ but increases with growth in cel. The reason for abnormal variation in fatty acid composition in the unsupplemented cel strain is unknown. One possibility is that reduced activity of the fatty acid synthetase complex differentially induces other enzymes of fatty acid synthesis so that fatty acids are not produced in appropriate ratios. IU Three general mechanisms by which fatty acids could lengthen the period of the sensitized cel strain can be suggested fatty acids could affect membrane properties; they could affect spe-

cific proteins; or they could affect general cell metabolism through their own metabolism. The first major mechanism by which fatty acids might act is through effects on membrane properties. One way this could occur is through incorporation of fatty acids into membrane lipids. Many of the fatty acids which lengthen the period are known to disrupt membranes and increase their fluidity when incorporated into membrane lipids, including cis-unsaturated fatty acids, short-chain saturated fatty acids, and fatty acids with bulky akoxy side chains. This has led to the hypothesis that fatty acids might lengthen the period of cel through effects on bulk fluidity of cellular m e r n b r a n e ~ . " ~ *This ' ~ ~ hypoth*~~~ esis predicts that certain fatty acids, such as the positional isomers of 18:l and certain analogs with bulky side chains, which do not lengthen the period, should do so; however, these fatty acids might not be incorporated into membranes, or might be rapidly converted to inactive metabolites. Similarly, trans, wum 18:2, which does lengthen the period, would be predicted not to do so. It is difficult to envision metabolism which could convert this fatty acid into a membrane-disruptive fatty acid, but such metabolism has not been ruled out. The uptake of saturated fatty acids, 6 to 24 carbons long, into mycelia of the cel mutant was measured by gas chromatography of fatty acids extracted from mycelia of cel cultures grown with the fatty acid supplement.11* This procedure does not distinguish between free fatty acids and those incorporated into lipids. Only supplemental fatty acids which do not occur naturally in Neurosporu lipids could be analyzed, and, of these, only 17:O and 20:0, which do not lengthen the period, could be detected. Furthermore, IT-radiolabeled 12:0, a periodlengthening fatty acid, was rapidly taken up by the cel strain, but was mostly oxidized, the label being released as radioactive carbon dioxide. l4 Further evidence against fatty acids acting by incorporation into lipids comes from experiments with fatty acids having methoxy side chains. While only the monomethoxy derivative lengthened the period, the dimethoxy and trimethoxy derivatives were incorporated into the lipids of the cel strain to an extent five to nine times greater than the monomethoxy derivative.IS9Based on these and other observations on the incorporation of fatty acids into cel, Mattern114J86-189 has ruled out the hypothesis that fatty acids lengthen the period by directly altering the bulk fluidity of cellular membranes. However, localized incorporation into and fluidization of specific membranes cannot be ruled out. Fatty acids could also change membrane properties by directly partitioning into membranes. This mechanism has been proposed to explain the inhibition by certain fatty acids of capping in mouse spleen lymphocytes,199although this explanation has been disputed.2w In the lymphocyte system, only cis-unsaturated fatty acids, and not saturated or trans-unsaturated fatty acids, are effective. This has been explained as resulting from partitioning of the cis-unsaturated fatty acids

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Critical Reviews In into fluid domains in the membrane while saturated and transunsaturated fatty acids partition into gel Although the levels of free fatty acids partitioned into lipids of cel and cel+ growing with exogenous fatty acids have not been measured, measurement of total fatty acids taken up by cel mycelia114indicates that the short-chain saturated fatty acids which lengthen the period are not present in the membranes of the cel strain to a detectable level. The fact that transunsaturated fatty acids are effective in lengthening the period of cel is further evidence against this mechanism of fatty acid action. Exogenous fatty acids could also affect membranes by altering membrane composition through changes in lipid metabolism. Both supplemental 16:O and supplemental 18:2 have been found to alter the phospholipid fatty acid composition pattern of cel while having no effect on the wild type.'= Surprisingly, 16:O-supplemented cultures of cel have relatively less 16:O than unsupplemented cultures, as if cel overcompensated for the presence of the exogenous fatty acid. Both 16:O and 18:2 alter the phospholipid fatty acid composition pattern in a similar manner, making it more like the pattern of the wild type;however, 18:2, which lengthens the period of cel, makes the phospholipid fatty acid composition pattern of cel even more like that of the wild type than does 16:0, which does not lengthen the period. Thus, the observed changes in fatty acid composition cannot explain the different effects of these fatty acids on period. The phospholipid fatty acid composition of the cel strain on solid medium, supplemented with either 16:O or 18:2, also markedly differed from that of cel+ Again, the cel strain appears to overcompensate for exogenous 16:O and has less of this fatty acid when grown on solid medium supplemented with it. With either 16:O or 18:2 supplement, the 18:2 level was significantly higher in cel than in cel+ while the 16:O level was significantly lower; thus, again, the period-lengthening effects of the fatty acids do not correlate with their compositionaltering effects. The second general mechanism by which fatty acids might act is through specific effects on specific proteins in the cel strain.192At least some of the fatty acids which affect the period of cel, 16:1, 18:1, 18:2, trans, rrans-18:2, y-18:3, and 20:3, confer resistance to the inhibitor oligomycin in both cel and cel+ .193.201 That this results from specific interaction with the mitochondrial ATP synthetase is suggested by the finding that DCCD covalently cross-links 18:2 to the ATP synthetase.202 However, the short-chain fatty acid, 12:0, which does lengthen the period of cel does not affect oligomycin resistance.*Ol Furthermore, fatty acids confer oligomycin resistance in both cel and cel+ ,so it seems unlikely that fatty acids affect rhythrmcity through effects on the ATP synthetase. Uncoupling of oxidative phosphorylation has been suggested as a possible mechanism by which fatty acids might lengthen the period.1'3.J92Such uncoupling may result from Specific

388

interaction between fatty acids and either the mitochondrial ATP ~ y n t h e t a s e or ~ ~the ~ . adenine ~~ carrier protein.z05However, both saturated and unsaturated fatty acids are effective uncouplers, which suggests that fatty acids do not affect rhythmicity by this mechanism. Fatty acids have been shown to activate mammalian protein kinase C in vitro in the absence of calcium, phospholipids, or diacylglycerol. The cis-unsaturated fatty acids, 204, 18:1, 18:2, and 18:3, are the most effective,2o6and these l8-carbon unsaturated fatty acids are also among the most effective in lengthening the period of cel(20:4 has not been tested for effects on periodicity). As with period lengthening, the trans isomer of 18:l is essentially ineffective at activating protein kinase C.206.M The even-carbon saturated fatty acids of 4 to 18 carbons do not activate protein b a s e C,- although some of them lengthen the period in cel (1O:O and 12:O). The activation of protein kinase C by fatty acids is not due to their detergent action, but to interaction of the protein with individual fatty acid molecules.206This suggests that free fatty acids might also affect protein kinase C in vivo. A protein kinase similar to mammalian protein kinase C has been isolated from Neurospora,zo8and the possibility that fatty acids may lengthen the period of cel by activating this enzyme is worth further study. The apparent sensitivity of cel might be explained if the normal metabolism of exogenous fatty acids in the wild type prevents exposure of protein kinase C to significant levels of the fatty acid. Alternatively, changes in the membrane composition of cel might make the protein kinase C more sensitive to exogenous fatty acids than in the wild type. Fatty acids can also affect other properties of protein kinase C without necessarily activating the enzyme; the fatty acids, 18:0, 18:1, and 20:0, and to a lesser extent 18- and 20-carbon unsaturated fatty acids, facilitate the binding of phorbol esters to protein kinase C.zo9If fatty acids affect periodicity through protein kinase C, similar interactions of the enzyme with the saturated fatty acids 16:O and 14:O might explain the periodstabilizing effect of these compounds. All the unsaturated fatty acids which lengthen the period of cel have double bonds, either cis or trans, at the A9 position whereas, with few exceptions, the unsaturated fatty acids which do not lengthen the period do not have double bonds at this position.186It has been suggested that period lengthening depends on the presence of a cloud of electrons at this position along the acyl chain,Ig6 and one could further speculate that this localized electron cloud is involved in specific interactions with specific proteins. Period lengthening by the triple bond analog of 18:l is consistent with the apparent requirement for A9unsaturation. Is8 The fatty acids which have A9double bonds, but do not lengthen the period, cis 16:l and trans 16:1, might not be incorporated into lipids by Neurospora, or, if incorporated, might be rapidly metabolized, perhaps by elongation to A11 18: Period lengthening by at least some of the shortchain saturated fatty acids might be explained by desatmtion

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Microbiology and elongation to 18:3,which has a A9 double bond;'14 enzymes desaturating 12:O to 12:3, and elongating 12:3 to 18:3 have been demonstrated in spinach chloroplasts,2'0*2''and in Penicillium chrysogenum. However, period lengthening by fatty acid analogs with bromine, methoxy, methylene, or other s~bstituents'~~ is inconsistent with the apparent requirement for Ag unsaturation since these compounds lack welectron clouds at this position, and no known metabolic reactions can convert these to unsaturated fatty acids. The third general mechanism by which fatty acids could act is by affecting general cellular metabolism by virtue of their own metabolism. Metabolism of these fatty acids could differ in cel and cel+, which would explain the sensitivity of cel to the exogenous fatty acid^.'^'.'^^ As mentioned earlier, the cel strain both oxidizes and incorporates exogenous fatty acids to a greater extent than does the wild type; however, this differential metabolism is true of both 18:2, which lengthens the so it cannot period of cel at 22"C, and 16:0, which does explain the effects of fatty acids. In summary, it is not known how fatty acids affect the period of the cef mutant. Three possible general mechanisms have been described above. .Although none currently explains all of the data, none can be definitively ruled out either. 8.

CONCLUSION

though not necessarily valid. The experiments using cultures of cel on solid medium might not be relevant either since it has not been demonstrated that rhythmicity persists in older areas of cel cultures on agar, as it does in the wild type,169or that this rhythmicity is sensitive to temperature and to fatty 'acids. Thus, rhythrmcity is known to occur only in a narrow strip of mycelium at the growing front, the fatty acid composition of which cannot be measured. Nonetheless, it is research on the lipid and mitochondrial biochemistry of cel that promises the greatest chance of understanding altered temperature compensation in this mutant. Many gaps in our knowledge of this biochemistry remain. We do not know the metabolic fate of most of the fatty acids which lengthen the period. We do not know the effect on the phospholipid fatty acid composition of cel of any fatty acid other than 16:O and 18:2. Finally, we do not know what effect environmental conditions and secondary mutations which prevent fatty acid effects may have on either the metabolic fate of fatty acids or on phospholipid fatty acid composition. Such data are necessary before the mechanism of temperature compensation and the nature of its loss in the cel mutant may be elucidated.

- MEMBRANES AND MITOCHONDRIA

The cel mutant is the only mutant with a known primary defect in which temperature compensation is lost. If the nature of this loss can be elucidated, it would provide invaluable insight into the mechanism of circadian rhythmicity. Research with the cel mutant has implicated the participation of membrane properties in maintaining temperature compensation. Evidence for this includes the altered phospholipid fatty acid composition of the mutant, the lability of the phospholipid fatty acid composition to supplemental fatty acids, and the epistasis over cel of prd-1, which is also altered in fatty acid composition. Involvement of mitochondria in maintaining temperature compensation is similarly indicated by the reversal of fatty acid-induced period lengthening by nonfermentable carbon sources, by mitochondrial inhibitors, by the ohT, [rni-23, and [rni-5] mutations, and by the prd-1 mutation which may lead to increased expression of mitochondrial proteins. Despite the evidence implicating membranes and mitochondria, the mechanism of temperature compensation in Neurospora remains unknown. Although a wealth of data has been collected on the cel mutation, as yet no testable hypothesis has been developed which is consistent with all of this data. Much of the data is phenomenological-i.e., measurement of period rather than biochemical, and much of the biochemical data may not be relevant since it is based on cultures grown in liquid medium in constant light, where rhythmicity is not expressed. These experiments have been based on the assumption that if a major defect in lipid metabolism in cel results in altered rhythmicity, this defect might be detected even when rhythmicity is not expressed. This is a reasonable assumption, al-

-

l 990

4. Genetic and Environmental Manipulations of Clock-Affecting Mutants A variety of strategies can be employed to study clockaffecting mutants.

1. 2.

3. 4.

5.

Two different alleles of the same gene can be tested against each other for dominance in heterocaryons. Two different alleles can be tested against each other for dominance in partial diploids; this strategy has not yet been applied to Neurospora clock mutants. Two different mutations can be tested for interactions in double mutant strains. A mutation can be probed with chemicals which alter the period or the phase of the rhythm. Finally, a mutation can be studied with respect to its response to varied environmental input; an example is the determination of the light and temperature phaseresponse curves for the& mutants, which is discussed below under "Mathematical Models".

a. STUDIES USING HETEROCARYONS

The use of heterocaryons for clock studies has been helpful in establishing dominance-recessive relationshipsfor clock mutants. In Neurospora, heterocaryons are produced by inducing the fusion of mycelia of two different genotypes so that different alleles at a locus are in different nuclei sharing a common cytoplasm. Unless the gene product is an RNA confined to the nucleus, the results with heterocaryons are presumably comparable to results with heterozygotes or partial diploids, which are used to study dominance in other organisms. To date, most clock mutants in Neurosporu have been found to be either codominant or recessive (Table 2) while preliminary evidence

389


Critical Reviews In indicates prd-4 may be dominant. Plotting period against the nuclear ratios in heterocaryons of variousfrq allele^^^^^^'^ shows that there is a linear relationship between period and nuclear ratio. This codominance suggests that the product of thefrq gene plays an important role in determining the rate of the clock p r o c e s ~ . ~The . ~ ~occurrence ~ ~ ’ ~ of codominant clock mutations could be interpreted in either of two ways. Codominant mutations could affect reactions which cany a large share of the control of the flux through the oscillator mechanism. A discussion of the important role of codominant genes in the control of flux through metabolic pathways is given by Kacser and Alternatively, codominant mutations could affect noncatalytic gene products or oligomeric protein complexes. For example, the codominance of the 01z7mutation with respect to oligomycin resistance can be explained by the interaction of many subunits in the F, portion of the mitochondrial ATPase.13’ b. STUDIES USING DOUBLE MUTANTS

Double mutant strains have been used to detect interactions between the gene products of different clock-affecting loci; the best-studied locus from this standpoint is thefrq locus. Table 5 shows the double mutant combinations tested to date. The results from these double mutant studies fall into two general classes: either there is no interaction between the two mutations, and the period is approximately as expected from two mutations affecting the period independently, or some type of interaction is observed. The expected period of a double mutant in which there is no interaction can be predicted by a simple model,

71.2

=

7172

70

where q, is the wild-type period, and T~ are the mutant ~ predicted double mutant period. This periods, and T ~ is. the phenotype will be described as multiplicative.’%In earlier papers, the independent action of two genes was described by an additive rule:79*11**135 each mutation was assumed to add to or subtract from the period a fixed number of hours. The data on a number of double mutant strains fit the multiplicative rule somewhat better than the additive model, although with mutants having periods deviating little from that of the wild type, both the additive and multiplicative models predict the double mutant period within the limits of experimental accuracy.194 Interactions between mutants can be further classified as either partially or completely epistatic. Complete epistasis is the situation where the double mutant has a phenotype identical to that of one .or the other of the single mutant parents. Partial epistasis is the situation where the double mutant period is intermediate between the predictions of complete epistasis and the multiplicative model, and is a catch-all category which refers to any interaction which is not complete epistasis. A

Table 5 Interactions between Clock-Affecting Mutations

Note: M = Multiplicative, PE = Partial Epistasis, and CE = complete epistasis. The references cited are those which present the original data upon which the conclusions regarding type of interaction arc based. *

Interactions with the cel mutation were always studied in the presence of supplemental linoleic acid.

more complete analysis and discussion is given by LakinThomas and Brody.‘% (Note, however, that in this paper, complete epistasis was termed “epistasis” while partial epistasis was termed “interaction”.) Although the basis for interactive behavior is not known for any of the mutant combinations which are discussed here, it might be analyzed in light of pathway control theory (see, for example, Kacser and which relates the change in the activity of one step in a metabolic pathway to the resulting change in the total flux through the pathway. If the effect of one mutation on the phenotype is altered by another mutation, these two mutations would be expected to affect the same general pathway while independent effects of two mutations would be equated with unrelated pathways. The mutations listed across the top of Table 5 and listed on the left in the top half of the table are those mutations which show partial epistasis with each other in double mutant combinations. Double mutants betweenfrq and cel show partial epistasis with the effect of cel depending on the period of the frq allele.194Double mutants betweenfrq and chr show partial epistasis when the double mutant periods are plotted against thefrq mutant period,’% although any given double mutant period is not very different from that predicted by the multiplicative model. Unpublished show that the chr cel double mutant has a 35-h period when supplemented with linoleic acid as compared to the expected period on this medium of 44 h, again indicating partial epistasis. The prd-3 cel and prd-4 cel double mutants both have 35-h periods,216although the expected periods are 47 and 33 h, respectively. Similarly, the prd-3 frq-3 double mutant has a period 2.7 h longer than


Microbiology the predicted multiplicative value of 28.0 h.79 All of these mutants are known to affect temperature compensation, and their effects are described above under “Input: Temperature Effects”. The interactions between these mutations suggest that they all affect the same pathway (or component of the oscillator), and that this pathway is part of the temperature-compensation mechanism. The prd-4 mutant seems to be an exception: this mutation also affects temperature compensation, but in combination withfrq-1 orfrq-2, it shows no interaction.79This may indicate that prd-4 af€ects some component of the temperature-compensation mechanism which is independent of the component affected by the mutations in the top half of the table. The lower half of the table summarizes data on double mutant combinations which do not show partial epistasis. Of the mutations listed on the left in the lower half of the table, prd-l and prd-2 are known to have little or no effect on temperature compensation10gwhile 0lP and [mi-2] have not been tested for temperature compensation effects. In the combinations tested to date, these mutations show multiplicative, noninteractive behavior withfrq and chr, but are completely epistatic to cel. An example of the latter phenomenon is the suppression by oh7 of the 40-h period of cel on linoleic acid supplement.’94A prediction arising fiom the patterns in this table is that mutations which are epistatic to cel might be epistatic to thefrq-9 mutation, which is also defective in temperature compensation. Several [mi-2]frq-9 isolates have been constructed and tested?” and the results clearly indicate that these double mutants do not show complete epistasis, and are still lacking temperature compensation. They have shorter periods thanfrq-9 at all temperatures and slightly slower growth rates. These findings indicate that [mi-2] andfrq, like olzv andfrq, andprd-1 andfrq, have independent effects on the clock. These results suggest that the complete epistasis seen with the cel mutant may not be related to temperature compensation, but may be somehow involved with saturated fatty acid synthesis. A mutation which leads to an increase in the production of saturated fatty acids might be epistatic to cel, but not have any effect on& or chr. The recent that Neurosporu mitochondria can synthesize saturated fatty acids independently of the cytoplasmic fatty acid synthetase suggests that the oh7 and [mi]mutations, which increase the amount of mitochondrial protein relative to total protein,’82could lead to an increase in the mitochondrial synthesis of those saturated fatty acids which reverse the loss of temperature compensation in cel. Based on these double mutant studies, we suggest that at least three independent pathways can be defined: temperature compensation I (affected byfrq, chr, cel, and prd-3), temperature compensation II (affected by prd-4), and another pathway, possibly related to fatty acid synthesis (affected by prd1 olzr, [mi-21, and cel). The prd-2 mutant might be included in the third groUping, or it might define a fourth category, depending on whether it is found to be completely epistatic to

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cel. It has also not yet been determined whether the mutations which are completely epistatic to cel interact with each other; for example, do prd-1 and ol? interact? This scheme of classification based on interactions would allow one to classify new mutants or chemical treatments into groups according to their interactions with known mutants, although, obviously, a considerable amount of serious biochemistry, and more genetics, will be needed to determine whether this is a workable classification scheme. c. STUDIES OF INHIBITOR EFFECTS ON MUTANT STRAINS

The rationale of these studies is to ask whether any particular clock-affecting mutation causes a changed sensitivity to an inhibitor and whether this information could provide any biochemical clues to the nature of the mutation. One could classlfy some of the known chemical effectors of the Neurosporu clock in a manner similar to that of the mutant classification described above. One effector that shows an interaction with the temperature compensation system is cycloheximide since the frq7 clock is relatively insensitive to its phase-shifting action.!” On the other hand, the clock effector, theophylline, and the related compound, aminophylline, would be predicted not to affect temperature compensation since their effects on period show multiplicative behavior with frq-1, frq-2, and frq-7.Iw A third effector, antimycin, would also be predicted not to affect temperature compensation, but rather the pathway possibly linked to fatty acid synthesis since the antimycin period effect shows interactive behavior with the prd-1 mutation.218 Obviously, more data are needed to assess t h i s classification scheme, and studies on other period and phase effectors would be welcome. C. The Biochemical Approach 1. Strategies a. THE USE OF CHEMICALS TO PROBE THE OSCILLATOR The rationale for this approach is straightforward, although the interpretation of the results may not be. The objective is to try to idenhfy biochemical reactions or pathways that are components of the biological clock by treating Neurosporu with specific chemical effectors of specific reactions or pathways and studying the effect on the clock. If these compounds have effects on rhythmicity, the affected area of metabolism would be implicated in the mechanism of circadian rhythmicity. The compounds may be applied continuously and the rhythm monitored for changes in period since such a change would signal an effect on the oscillator itself. Alternatively, the compounds may be applied in discrete pulses, and the rhythm monitored for changes in phase since a stable change in phase would also signal an effect on the oscillator. The compound may be applied in discrete pulses at various phases of the circadian cycle to produce a phase-response curve, as described in the introduction to “Input”. Compounds tested for effects on phase or period in Neurosporu are listed in Table 6.

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Critical Reviews In Table 6 The Effects of Chemicals on the Neurospora Rhythm Phase shifts

Compound


Macromolecular synthesis inhibitors Anisomycin DChIoramphenicol Cycloheximide 6-Methyl purine Tetracycline Mitochondrial inhibitors Antimycin A Aurovertin hide CCCP Cyanide NEM Oligomycin Ionophores A23 187 Nystatin Valinomycin Calcium metabolism Dantroiene. Trifluoperazine and related compounds CAMPand cGMP Metabolism CAMP analogs cGMP analogs

Forskolin Quinidine Theophylline and related compounds Miscellaneous Amiloride L-Chloramphenicol D*0 DCCD Diethylstilbestrol and related compounds Fluoroleucine Lithium Molybdate 2-Phenylethanol Phenylmethanol 3-Phenyl-1-pmpanol Tetradecanoylphorbol acetate Vanadate

Cytoplasmic protein synthesis inhibitor Mitochondrial protein synthesis inhibitor Cytoplasmic protein synthesis inhibitor RNA synthesis inhibitor Protein synthesis inhibitor Electron transport inhibitor ATPase inhibitor Cytochrome oxidase inhibitor Mitochondrial uncoupler Cytochrome oxidase inhibitor ATPasdphosphatase inhibitor ATPase inhibitor

Period

J

6

J J

124, 220 6

J

221,222

J J J J J

57, 222

J J

19

219

NC

219

19 19

140 223

NC 21-25

227 227, 228

19 24

219 229

24

23 1

18 NC NC

232 232 232

57

57

J

22 1 224 224

Calcium transport inhibitor Calmodulin antagonists

J J

38 225

J J Adenylate cyclase activator Adenylate cyclase inhibitor CAMPphosphodiesterase inhibitors

J J J

226 226 226 227 6, 227

Na+/H+ antiport inhibitor Inactive isomer of D-chloramphenicol Heavy water Proton ATPase inhibitor Plasma membrane ATPase inhibitors Amino acid analog

J

38

J

57 85 230

ATPasdphosphatase inhibitor Alters lipid composition Alters lipid composition Alters lipid composition Protein kinase C activator ATPase/phosphatase inhibitor

J

57

J J

Ref.

222 57, 222

Calcium ionophore Potassium ionophore Potassium ionophore

J J

01)

Ref.

38 57

Note: NC = no change in period

There are a number of problems in the interpretation of the

data gathered by this approach. (1) Nonspecific effects of the compound: compounds affecting metabolism are rarely specific in their action; it has been noted that the specificity of such a compound is often an inverse function of the length of time it has been in use by b i o ~ h e m i s t s .Mutants ~~ resistant to the primary effects of a compound may be used to rule out side effects. As discussed in the appropriate sectionsbelow, mutants resistant to cycloheximide, oligomycin, and nystatin have been used to demonstrate that the phase-shifting effects of these compounds must be mediated through their primary metabolic

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effect. (2) Secondary effects of the compound even if the primary effect is very specific, the secondary effects that are subsequently produced can ripple out to many areas of metabolism. For example, although oiigomycin acts on the clock through the mitochondrial ATPase, this does not necessarily implicate the ATPase complex itself as a component of the oscillator: the secondary effects of oligomycin on cellular ATP levels, for example, could affect many other processes. The question of primary vs. secondary effects on the clock mechanism has never been resolved for any specific chemical. (3) Indirect effects on the oscillator: it is very difficult to tell

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Microbiology whether the effect of a chemical on the clock is due to a direct effect on the oscillator or to an effect on an input pathway. In particular, phase-shifting effects may result from the activation of steps in the light or temperature signal input pathways. Phase-shifting experiments have additional problems associated with them. Removing the drug at the end of the pulse is not always straightforward, and it can be difficult to determine the exact phase when the drug is effective. For example, Olesiak and co-workers234have demonstrated that the effects of protein synthesis inhibitors in Gonyuulax can persist for hours after they are washed out, making it impossible to determine the duration of the treatment. There are additional problems with interpreting drug phase response curves. While it is tempting to conclude that phases of maximal or minimal phase-resetting activity correspond to phases of maximal or minimal sensitivity of the oscillator to the drug, this may be an erroneous assumption. Phase is a complex, unknown function of the state of all clock components, and the maximum sensitivity of phase need not correlate with the maximum sensitivity of any one of these components. Similarly, the phases of the crossover points or breakpoints may have no biochemical meaning, and will, in fact, migrate if conventions other than limiting maximal phase shifts to 2 1 2 h are adopted. See "Mathematical Models" below for further discussion of these points. In addition, the attractiveness of this approach has become somewhat tarnished now that so many different chemicals have been found to shift the phase. The significance of the large number of compounds that affect phase may not be fully understood until a mass of data has accumulated to indicate what classes of chemicals do not lead to phase shifts. Many compounds have been tested for ability to cause phase shifts, period changes, or both, based on their ability to inhibit pathways postulated to have a clock function. Other compounds have been tried just because they were available. We have compiled the following list of properties of the ideal clockaffecting compound, or "chnobiotic", partly in the hope that it could be used as a guide in the selection of specitic chemicals to be tested, but mainly to stimulate discussion on this subject and to act as a standard for evaluating the literature. 1.

2. 3. 4.

5.

that is being inhibited if one can determine what reverses or suppresses the effect. The compound should have a measurable effect on metabolism, and this effect should be measured in experiments employing the compound, so that a correlation can be made between effects on the clock and effects on metabolism. This would ensure that the compound is in fact affecting its assumed target under the conditions tested.

b. BIOCHEMICAL CHARACTERIZATIONOF CLOCK-AFFECTING

MUTANTS

An alternative biochemical approach is to compare the metabolism of clock-affecting mutants to that of the wild type in hopes of correlating changes in metabolism with changes in rhythmicity. This approach is best exemplified by research on the eel mutant, which has been discussed in detail above. Preliminary studies of fatty acid synthesis have also been performed with thefiq and prd-1 mutants. While thefrq mutants have normal gross fatty acid composition^,^^*^" prd-1 has an abnormal fatty acid composition in liquid culture and on agar medium. c. MEDIA EFFECTS

It is noteworthy that the period is little affected by changes in the growth medium.23s Various sources of organic and inorganic nitrogen may be supplied with no effects on the peThe period is similarly unaffected by different formulations of trace minerals,= or by changes in the content of phosphate,= or Different sources of agar affect the clarity of conidial band formation by the cel mutant, but do not affect the period.= Various sugars may be used as carbon sources without effects on the period.16JnOn the other hand, nonfermentable carbon sources, such as glycerol or acetate, shorten the period to about 20 h.'= While no definite clues as to the oscillator mechanism have come out of this study of the media used for growth, several possibilities have been tentatively eliminated from consideration: in particular, nitrate reductase, which is repressed by ammonium salts as the nitrogen source; the enzymes of glycolysis, which are repressed or inhibited on acetate media; and the enzymes of gluconeogenesis, which are repressed or inhibited on glucose media.

Either the compound should be highly specific in its action, or else mutants resistant to the primary effect of the compound should be available for testing to rule out the possibility that effects on rhythmicity result from side effects of the compound. The compound should be easily washed out so that it is possible to do a clean pulse. The amount of period change or phase shift should be proportional to the concentration of the compound, within reasonable limits. The clock effects should be reversible, either by the addition of another compound or by changes in the environment since one can often better understand the function

2. Protein Synthesis

Protein synthesis inhibitors have been widely studied for effects on rhythmicity in many organisms (reviewed by Edmunds39).In Neurosporu, the inhibitor, cycloheximide, has strong phase-shifting effects, and inhibition of protein synthesis by this drug has been quantitated.u0 Cycloheximide-resistant mutants have been used to demonstrate that the phase-shifting effects are mediated by the primary effect of the drug on protein synthesis on 80s ribosome^.^^' Initial interpretations of the stressed the potential role of protein synthesis itself

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Critical Reviews In in the clock mechanism. However, the discovery that protein synthesis in thefrq-7 strain is sensitive to cycloheximide, while the clock in this strain is not,’” has led to consideration of alternative possibilities. Dunlap and Feldman124have proposed that the wild-typefrq protein normally has a short half-life, and that cycloheximide pulses therefore reduce the level of the frq protein. In their hypothesis, thefrq-7 mutation has increased the level or stability of the protein so that inhibitor pulses do not lower its activity below a critical level. They have devised a model’24 based on this hypothesis which accounts for the long period, loss of temperature compensation, and cycloheximide resistance of frq-7. Iffrq-7 does increase the halflife of thefrq-protein, it could do so by altering the primary structure of the protein so as to reduce its rate of proteolytic degradation. Alternatively,frq-7 could lead to changes in posttranslational modification, such as phosphorylationor acylation of thefrq protein, which could increase its half-life. Another possibility is that the product of thefrq locus might not be the actual protein with a short half-life, but rather an enzyme that influences the half-life of that protein by catalyzing its posttranslational modification. The cycloheximide resistance of the clock in frq-7 raises several new experimental questions. Sincefrq-7 is deficient in temperature compensation, it might be interesting to measure the sensitivity of cel and frq-9 to phase-shifting by cycloheximide since these mutants appear to completely lack temperature compensation. Studies comparing protein half-lives of frq-7 and the wild type might also be enlightening. If the cloned frq gene can be used to prepare antibody to thefrq protein, the half-life of this protein might be specifically studied infrq7 and the wild type. However, as attractive as the idea is that frq-7 affects the half-life of a protein, alternatives are still possible. One of these, the idea that frq-7 could affect the amplitude of the oscillator, is discussed in “Mathematical Models” below. 3. Mitochondria1Energy Metabolism As can be seen in Table 6, several inhibitors of mitochondrial metabolism have chronobiotic effects. Two of these inhibitors, antimycin A’4oand aurovertin,= lead to shortened, 19-h periods. These inhibitors. have different primary sites of action: antimycin A blocks electron transport to cytochrome c , while aurovertin inhibits binding of ADP to the F, portion of the ATP synthetase. Chloramphenicol, an inhibitor of protein synthesis on mitochondrial ribosomes, also shortens the period to 19 h.,I9 However, the effect of chloramphenicol on the period may have been caused by secondary effects since both optical isomers were effective, yet only the D isomer inhibited protein synthesis.219These compounds all inhibit the growth rate of Neurospora. Other inhibitors, SHAM and rotenone, have little effect on period,236but since they do not inhibit the growth of Neurospora,236they may simply not affect the mitochondria of this organism. As previously noted, periods of 18 to 20 h are also produced by 011’ and several other mitochondrial mu-

tations, and by growing wild-type Neurospora on nonfermentable carbon sources (which would be expected to alter mitochondrial metabolism relative to growth on sugars). Thus, a number of factors affecting mitochondrial metabolism all decrease the period. The oh7 mutation and other mitochondrial mutations increase levels of mitochondrial proteins relative to total protein (see “The Genetic Approach” above). Similar increases in the levels of mitochondrial proteins result from treatment of Neurospora with chloramphenicol or antimycin ALES which suggests that shortened periods may be related to increased levels of mitochondrial proteins. The oh7 and [mi] mutations, growth on nonfermentable carbon sources, and chronic treatment with antimycin also abolish the periodlengthening effects of fatty acids on the cel mutant (see “Studies on the cel Strain” above). As Table 6 also shows, a number of mitochondrial inhibitors produce phase shifts. NakashimaU2has reported dose-response curves for phase shifting by CCCP, antimycin, azide, and cyanide, and has also assayed 0, consumption of mycelia and ATP levels under the same experimental conditions. No correlation is found between the size of the phase shift and the depletion of ATP or the inhibition of 0, consumption. Particularly interesting is the finding that cyanide at 0.5 mM almost completely depletes ATP without inducing a phase shift. In an earlier paper, NakashimaEsinvestigated the effects of an inhibitor of the plasma membrane ATPase, diethylstilbestrol, and related compounds on phase shifting and found no correlation between the phase shifting effects of the compounds and their effects on the activity of the plasma membrane ATPase. Because diethylstilbestrol can also inhibit mitochondrial functions, Nakashima assayed the effects of DES and related compounds on oxygen consumption by mycelia. The correlation between phase shifting and inhibition of oxygen consumption was better than the correlation between phase shifting and inhibition of the plasma membrane ATPase, but there were still anomalies which led Nakashima to conclude that the inhibition of oxygen consumption was not the direct cause of phase shifting. Problems with the use of diethylstilbestrol are discussed above under “Input: Light, The Phototransduction Mechanism”. Schulz et al.” have also investigated energy metabolism and phase shifting, and found similar phase shifts induced by vanadate and molybdate (inhibitors of ATPases and phosphatases) as well as by NEM, cyanide, azide, and oligomycin. DCCD also induced phase shifts, but the PRC differed from those of the others. The levels of various organic phosphates, inorganic phosphate, and inorganic polyphosphate were assayed by N M R during treatment with molybdate, cyanide, and azide, all of which decreased the adenine nucleotide peak, and during treatment with DCCD, which had no effect on the NMR spectrum. n u s , the ability to reduce adenine nucleotide levels correlated with a particular shape of the PRC, but not with the ability to phase shift the rhythm per se. These studies all indicate that there is not a simple correlation between phase and total ATP


Microbiology levels, or between phase and mitochondrial 0, consumption. Experiments involving mitochondrial inhibitors are difficult to interpret for two reasons. First, some of these compounds, such as DCCD and cyanide, are known to affect processes outside the m i t o c h ~ n d r i a , ~ while ' * ~ ~ secondary effects have not been ruled out for others, such as a i d e and antimycin A. The question of side effects has only been examined for oligomycin: mycelial disk cultures of an ohT strain were found to be resistant to phase shifting with oligomycin, even at 100fold higher than the normal doses used for phase shifting.239 The second difficulty with the use of mitochondrial inhibitors is that many of them are not easily washed out; most have high affinities for their site of inhibition,24oand some are not water soluble. DCCD is well known to cross-link to many proteins at glutamate residues,"' and one can expect that some of it will never be washed out, Nakashima* has assayed the recovery of ATP levels after the end of a cyanide or antimycin pulse and has found that the levels increase slowly over the course of several hours, indicating that the inhibitors are still present at significant levels long after the end of the pulse. Because of these difficulties, caution is required in interpreting PRCs generated with inhibitors of mitochondrial metabolism. The effects of mitochondrial inhibitors on the period and phase of the rhythm suggest a role for mitochondria in the Neurosporu circadian rhythm mechanism. This is further supported by the period-altering effects of mutations which affect mitochondrial proteins. In addition, rhythms in energy charge have been r e p ~ r t e d . ~ ~(see . " ~"Output" for further discussion). Although a variety of interpretations of these observations can be made, these interpretations can be roughly grouped as follows. (1) Mitochondria might play a "supporting role"; that is, they provide the energy to run the cl&k. At certain phases of the rhythm, the failure of this energy supply could have effects, such as a drop in plasma membrane potential, which could lead to phase-shifts; however, the lack of correlation between ATP levels and phase shifting argues against this role. (2) Mitochondria could play a more direct, but secondary role in the clock mechanism through their contribution to determining ion concentrations in the cytoplasm and ion gradients across the mitochondrial membrane. (3) The oscillator could be partly or wholly composed of oscillating components that are localized to the mitochondria. 4. IonophoredCalcium Metabolism The evidence for the involvement of calcium and other ions in the clock of Neurosporu is similar to that found for other organisms: ionophores221.u4 and calmodulin inhibitors2" affect the phase, and lithium affects the period.231Phase shifting by the potassium ionophores, nystatin and valinomycin, has been demonstrated by K ~ y a m a The . ~ ~erg-1 and e r g 3 mutants, which lack ergosterol and are thus resistant to growth inhibition by nystatin, are also resistant to phase shifting by this compound, demonstrating that phase shifting is mediated through

nystatin's interaction with plasma membrane sterols.224 Lithium effects on the period of the clock have been reported for many organisms and recently for Neurosporu as well;=' lithium affects ion transport in animal cells243and also has effects on the inositol phosphate signaling system which regulates the second messenger role of calcium.2a246 Nakashima has been investigating the role of calcium in the clock mechanism, and has reportedu1 phase shifting by the calcium ionophore A23187. Phase shifts are induced by the ionophore in a calcium-free medium and are blocked by high extracellular calcium. Antimycin A increases the size of the A23187-induced phase shifts and also increases the calcium concentration needed to block the phase shifts. A23187 in a calcium-free medium would be expected to deplete intracellular calcium, but neither cytoplasmic free-calcium levels nor the contents of intracellular calcium pools were assayed in this study. A number of secondary effects on calcium-sensitive cellular processes could be expected from prolonged calcium depletion, so it is difficult to interpret these results. An additional complication is the associated pH effect: A23187 is a calcium/proton exchanger, as is the calcium export system in the Neurosporu plasma membrane,247and therefore the effects of calcium depletion could be due to cytoplasmic acidification. Clearly, much more needs to be done to characterize the effects of A23187 on calcium pools and on other ion gradients in Neurosporu before any conclusions can be reached concerning the effect of this ionophore on the clock. The effects of calcium on cellular processes are mediated in many cases by calmodulin, and Nakashima has reported phase shifting by calmodulin antagonists in Neurosporu.225Several calmodulin antagonists have been assayed for phase-shifting effects, and their relative effectiveness at phase shifting correlates with published data on their relative effectiveness as calmodulin inhibitors in a rat-brain preparation. Homologs of several of the inhibitors which are ineffective as calmodulin antagonists are ineffective at phase shifting, supporting the claim that the phase shifts are mediated through calmodulin and not through the side effects of the inhibitors. Nakashima has proposed'28-221**that calcium accumulation by mitochondria may be important in the clock mechanism. Calcium transport by intracellular organelles has not been adequately characterized in lower eukaryotes, but if mitochondrial calcium transport in Neurosporu is similar to that in vertebrates, it is unlikely that mitochondrial calcium accumulation plays an important role. At normal cytoplasmic freecalcium levels, vertebrate mitochondria do not accumulate calcium to any extent, and the mitochondrial calcium pool merely reflectsthe cytoplasmic Phase shifting experiments with ionophores and calmodulin inhibitors are subject to the usual problems of side effects, and the persistence of the compounds long after the end of the pulse. Secondary effects can result from the interactions between Various ion gradients through membrane potentials and

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Critical Reviews In through common ion transport proteins. In addition, ionophores may have immediate effects on energy metabolism as well since energy is needed to establish and maintain ion gradients and since ion gradients are involved in energy conservation. Calcium may also interact with other ion gradients by activating or inactivating ion transport proteins; for example, high cytosolic calcium levels activate the electrogenic proton ATPase of Neurospora.249 Several models exist for self-sustaining circadian oscillations, based on protons,z0 potassium ions,”’ and calcium ions.“’ Models based on calcium ions deserve special comment since calcium ion concentration is kept at low levels and is carefully regulated in cells, affects many enzymes, and is involved in transduction of environmental signals. In some systems, high-frequency oscillations in calcium ion levels are part of a mechanism that transduces the information about a hormone level into frequency inf~rmation.”~ Whether the kinetics of ion transport systems could be modulated appropriately to produce a 24-h oscillation is an open question. The involvement of ions, and calcium in particular, in signal transduction suggests a possible role for ions in the input mechanisms of the clock. For example, the collapse of an ion gradient by the opening of a light-activated ion gate is an attractive mechanism for a phase shift. 5. Cyclic AMP Cyclic nucleotides have been proposed as components of the circadian oscillator; early work in Neurospora on this hypothesis has been discussed by Feldman and Dunlap.6 Briefly, Feldman2” found that inhibitors of cyclic AMP (CAMP)phosphodiesterase (PDE), including theophylline, aminophylline, and caffeine, can lengthen the period by 2 to 4 h. However, normal levels of CAMP are apparently not required for the operation of either the oscillator or the phototransduction pathway: the crisp-1 (cr-1) mutant, which has been reported to completely lack adenylate ~ y c l a s e ~ and ~ . ”to~ have low levels of endogenous CAMP,^^.^^ expresses a normal period and can be entrained by a 12:12 L:D cycle.79 Feldman and Dunlap6 also reviewed unpublished work by Perlman on period changes and phase shifts by various drugs which affect CAMPmetabolism. Inhibitors of PDE, which would be predicted to increase cAMP levels, lengthen the period, while quinidine, an inhibitor of adenylate cyclase which would be expected to decrease CAMP levels, has no effect on the period. Although drugs which inhibit either PDE or adenylate cyclase have different effects on the period, both classes of drugs give similar phaseresponse curves. RensingZ6 also reports “moderate” phase shifts induced by PDE inhibitors as well as by exogenous CAMP.Feldman and Dunlap6 discussed various interpretations of this data, but, as they pointed out, in the absence of any data on cAMP levels in the presence of these drugs, no consistent hypothesis can be formulated to explain how changes in CAMPmight affect the oscillator. Furthermore, as with all drug studies, there remains the possibility that these agents

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affect the clock through unknown side effects rather than through the (assumed) primary effects. Recent reports from Hasunuma’s laboratory have revived interest in the role of cyclic nucleotides. While investigating phosphate metabolism in Neurospora, Has~numa”’.”~isolated mutants (cpd) which may define regulatory genes controlling several enzymes involved in phosphate m e t a b o l i ~ m .The ~~.~~ cpd mutants are deficient in a cyclic nucleotide phosphodiesterase (cPDase). This enzyme is found in the culture medium as well as intracellularly, hydrolyzes both cAMP and cGMP, and is repressed by high phosphate media. It is apparently identical to the previously described, repressible acid phosphatase.260Hasunuma proposedzw that cPDase might regulate intracellular cAMP levels and consequently regulate conidiation. However, cPDase has a pH optimum of 3.2 to 4.0 and is inactive at neutral intracellular pH.2w Moreover, because it is repressed by phosphate and is exported to the medium, it is more likely to be an extracellular degradative enzyme involved in phosphate assimilation. The cpd mutants express a banding growth pattern on solid medium,166and this growth pattern has been shown to be circadian: the period in DD is approximately 22 h, the rhythm entrains to a 24-h 9:15 L:D cycle, and the free-running period is temperature compensated between 17 and 33°C. These mutants also express a banding pattern in standing liquid culture, although no evidence has been provided as to whether this banding pattern is also circadian. The similarity in banding growth on solid medium between the cpd mutants and bd, and the low levels of CAMPin cpd,”8 led Hasunuma to suggestZ6l that cAMP plays a central role in the circadian oscillator. However, it has been generally assumed that bd affects only the expression of the oscillator by permitting conidiation under high CO, concentrations in closed culture vessels. Wild-type strains will express banding growth if the CO, concentration is maintained at a low level by blowing air over the cultures,’6 and there is no evidence that CAMPlevels are decreased under these conditions. Hasunuma et al. assayed levels of various PDE activities, adenylate cyclase, and cyclic nucleotides in a number of mutants including bd,”9.262and on this basis, bd was claimed to be a regulatory gene controlling CAMPlevels.263 The differences found between bd and wild type are small, however, and may not be statistically significant; in later reports,% the “low” levels of cAMP measured in bd are higher than in the earlier r e p ~ r t s . ~ ~ . ~ ~ ~ Hasunuma et al.% assayed CAMP and cGMP across two or three circadian cycles in several strains and reported fluctuations in the levels of these compounds, although the claim that these fluctuations are circadian oscillations is not supported by the data (see “Output”). Light-induced changes in cAMP and cGMP were reported by Hasunuma (see above under “Input: Light”). Phase shifts by exogenous cAMP and cGMP were also reported and put forth as evidence that cyclic nucleotides are components of the circadian oscillator. The method used for assaying phase shifts was previously described,264and it

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Microbiology kinase. The fmt question to test is whether or not this mutant exhibits a circadian rhythm, and if so, whether it has an altered period or is defective in light phase shifting. This mutant could also be used to test whether the phase-shifting effects of PDE and adenylate cyclase inhibitors and of exogenous cyclic nucleotides are mediated through changes in cAMP levels since such effects should be absent in this mutant.

was shown that the experimental manipulations alone induce 5- to 6-h phase shifts in control cultures at the single time point for which cyclic nucleotide phase-shifting data are reported. These control phase shifts were subtracted from the phase shifts induced by cyclic-nucleotide treatment, leaving 1- to 2-h phase differences between them, but the large phase shifts in the controls cast doubt on the significance of these small differences. Other laboratories using the same liquid culture system as Hasunuma report only small phase shifts in control cult u r e ~ . An ~ . ~additional problem with this experimental procedure is the evidence from Pall’s laboratory265that Neurosporu may not be very permeable to exogenous cyclic nucleotides: the growth of cr-1 can be stimulated by exogenous CAMP,but only at concentrations of 5 mM or above and temperatures above 25°C while Hasunuma used 0.5 mM CAMPat 25°C in his phase-shifting experiments. On the whole, the work from Hasunuma’s laboratory is difficult to evaluate, because insufficient data are provided for the statistical analysis necessary in these types of experiments. For example, Hasunuma reports small differences in cyclic nucleotide and enzyme levels between strain^,"^.^^^ but since the data are derived from duplicate determinations on single cultures, the significance of these small differences cannot be established. Confidence in this work is also undermined by inconsistencies between Hasunuma’s results and those of other laboratories: for example, cr-1 has been shown to have undetectably low levels of CAMPand c G M P and less than 2% wild-type levels of adenylate ~ y c l a s e , ~ .in” ~contrast to Hasunuma’s assay of 20% wild-type levels of adenylate cylase."^ Hasunuma’s assay methods also cast doubt on the reliability of much of the data: his extraction method2* for cyclic nucleotides involves an unconventional 4- to 1-d extraction in TCA at O’C, although rapid freeze clamping is usually required when assaying these unstable and rapidly changing r n e t a b o l i t e ~ . ~ ~ . ~ ~ ~ Hasunuma has proposed a modelm for the circadian oscillator which invokes well-known regulatory relationships between second messengers, including cyclic nucleotides, inositol phosphates, diacylglycerol, intracellular calcium levels, and all the associated synthetic and hydrolytic enzymes, protein b a s e s , G-proteins, etc. While such a model certainly has many attractive features, there is as yet no definitive evidence that any of these components are involved in the mechanism of circadian rhythrmcity. The cpd mutants do appear to be similar to bd, and an analysis of the control of conidiation in these mutants might tell us something about the output of the oscillator. However, the absence of any serious defects in the circadian rhythm of cr-1, which is deficient in adenylate cyclase, is still a substantial obstacle to any suggestion of a direct participation of CAMPin the mechanism of the clock. The recent isolation of a mutant (cpk) deficient in CAMPdependent protein suggests another test for detemining the role of CAMP in light transduction and/or the oscillator since the effects of cAMP are believed to be mediated by this

6. Membranes Membranes have been frequently proposed as components of the circadian lock.^^*^^^ In Neurosporu, the evidence that membranes play a role in rhythmicity consists of the phaseshifting effects of ionophores (see ‘‘IonophoredCalcium Metabolism” above); the effects of substances, such as D20, which affect membrane properties; the loss of temperature compensation in the cel mutant, which is defective in fatty acid synthesis; the sensitivity of the period of this mutant to exogenous fatty acids (see “Studies on the cel Strain” above); the altered phospholipid fatty acid composition of cef‘” and of the clock-affecting mutant prd-1;’47 and the rhythm of fatty acid composition of the phospholipids of Neurospor~.~~’ Interpretation of the ionophore and D20 data is complicated by the usual questions of direct vs. secondary effects, and the fatty acid composition rhythm may represent output of the clock, rather than its actual mechanism (see “Output”). However, the various lines of evidence involving the cel mutant and the altered fatty acid composition of prd-1 remain currently the strongest correlation between rhythmicity and any biochemical function. In particular, evidence from studies on the cel mutant, which is discussed in detail above, strongly implicates membranes in the mechanism of temperature compensation. Nakashima has presented data which could be interpreted to rule out membranes as components of rhythmicity.232He has found that the alcohols, phenyhnethanol, 2-phenylethanol, and 3-phenyl-1-propanol, all have similar effects on phospholipid fatty acid composition and phospholipid head group composition. However, of these alcohols, only 2-phenylethanol affects the period, shortening it to about 18 h. These results clearly indicate that there is no simple correlation between fatty acid composition and period. The further argument could be made that since membrane composition is altered by two of these alcohols while clock functioning is unimpaired, membranes must not be involved in clock function. However, organisms are known to adjust their membrane composition to maintain normal functioning of membranes despite changes in the environment, particularly temperature changes, which would otherwise alter membrane f u n ~ t i o n , ” ~and . ~ ~Neurosporu ~ is no Thus, it could equally be argued that the changes in membrane composition seen on treatment with these alcohols compensate for the direct effects of these alcohols on membranes, thus preserving membrane properties rather than altering them. In this case, if membranes play a role in rhythmicity, the alcohols would have no effect on the clock mediated through membrane changes, and the period-altering effects of 1990

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Critical Reviews In . 2-phenylethanol would be attributed to secondary effects. In any case, in these experiments clock effects and phospholipid composition were monitored under very different conditions: solid acetate medium in constant darkness for monitoring clock effects and liquid glucose medium, with a different salt composition, in constant light for monitoring phospholipid composition. The possibility exists that the metabolic effects of the alcohols could be very different under the different culture conditions. The evidence implicating membranes in the clock mechanism can be interpreted in several ways: (1) membranes could play a passive, supporting role in the clock mechanism, providing the physical structure or compartmentation necessary for the functioning of clock components which are not membrane-bound; (2) membranes could play a more active role in mediating temperature-compensation through changes in lipid composition; (3) clock components could be integral membrane proteins; or (4) membranes could be integral clock components, the structure or composition of membranes themselves oscillating. Because membranes are intimately involved in most cellular processes, it is not surprising that there is evidence that membranes play some role in clock processes. Nevertheless, the exact nature of this role is still not apparent. The experiments that have sought to correlate membrane composition with rhythmicity have looked at composition of the entire mycelium, and perhaps this is too gross a level to examine in order to perceive clock functioning. It is possible that only certain cellular membranes, or only certain domains of membranes, or only certain classes of lipids play a role in clock functioning.

7. Correlation of Period Effects and Metabolic Effects When considering period changes in experiments using chemical effectors, it would be useful to have a quantitative measure of the effectiveness of the chemical in altering period. To do this, one must not only measure the period change, but must also know the primary target of the chemical and quantitatively measure its effect on this target. Although many possible formulas correlating these two phenomena could be employed, we suggest.comparing the percentage change in the period produced by the compound to the percentage change in the enzyme activity or flux through the metabolic pathway that it affects. This could be expressed simply as the coefficient,

where 7 stands for the period and E stands for the activity of an enzyme or-the flux through a metabolic pathway. C would be the “clock coefficient”, and its value would indicate the sensitivity of period to changes in the activity of a given enzyme or in the flux through a given pathway. This simple formula is analogous to the sensitivity coefficient of Kacser and Bums214

which relates the percent change in overall flux through a pathway to the percent change in the activity of a particular enzyme. The clock coefficient would allow one to assess the relative sensitivity of the clock to various chemicals. For example, if the 90% inhibition of a particular metabolic pathway only produced a 1-h period change (approximately 4%), then the clock would be relatively insensitive to this change (C equals approximately 0.05),and one could conclude that the metabolic pathway is, at least, not a major player in the clock. The concept of clock coefficients could be employed for mutants and for gene dosage (see “The Genetic Approach” above), as well as for inhibitors. Of course, it may very well be that many components are involved in the clock mechanism, and that no one of them has-more than a small share in the control of the period of the clock. This may be merely a restatement, in kinetic terms, of Pittendrigh’~’~~ assertion that the clock is buffered against changes in metabolism.

D. Mathematical Models 1. Introduction Although no solid biochemical evidence is available as to the identity of the components of the clock in any organism, there has been no shortage of models proposed in attempts to simulate the properties of various clocks, including that of Neurosporu. Models for circadian oscillators can be classified according to the biochemical processes which are proposed as components of the clock, and E d r n ~ n d provides s~~ a thorough review of current biochemical models relevant to several organisms. Clock models can also be classified according to their mathematical properties, characterized by the number of oscillating variables which the model employs (and therefore the number of dimensions required to map the trajectory of the system as it changes with time), and by the way the model responds to phase-resetting perturbations. One of these mathematical models is the “limit cycle”; this term is often found in the circadian rhythm literature, and has recently been applied to the Neurosporu In the following discussion, we contrast this model with the more intuitive “simple clock” model and point out the usefulness of the limit cycle model in interpreting phase-shift data in Neurospora and in designing informative experiments. 2. Simple and Nonsimple Clocks A few comments about oscillator terminology may be useful, taking Winfree’s classic work3s as a guide. A “simple clock” (Winfree, Chapter 3) is a one-dimensional oscillator which needs only one variable (phase) to describe its state; a simple clock advances through an unvarying succession of phases, and its phase can be reset only by making it run faster or slower through the cycle. An example of a simple clock is a relaxation oscillator, also known as the “integrate and fire” model of Glass and M a ~ k e yin , ~which ~ the variable rises from its baseline value to a threshold and then instantaneously resets back to the baseline. This model can be successfully applied to some

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Microbiology types of biological oscillators such as neural activity. Another example of a simple clock would be a developmental cycle consisting of a succession of developmental stages: the completion of one stage would initiate the beginning of the next stage and so on until the last stage reinitiates the first. Many biological oscillators cannot be described by one-dimensional simple clocks, but require more than one variable. An example would be a negative feedback loop in which an enzyme produces a product which inhibits the activity of the enzyme. If there is a time delay before the inhibition takes effect, this system can oscillate, and two variables are needed to describe its state: the enzyme activity and the product (inhibitor) concentration. A “nonsimple clock” (Winfree, Chapter 5 ) is therefore defined as an oscillator which requires two or more variables to describe its state, and this state traces a path through a two-or-more dimensional state space. It can be reset by pushing it off its unperturbed path into a new area of state space, from which it will move along a new trajectory; it may or may not return eventually to its original path. A limit cycle (or attracting cycle in Winfree’s terminology) is defined by Winfree as a nonsimple oscillator which has “a preferred amplitude from which it can be perturbed and to which it regulates back again” (Winfree, p. 146); it eventually returns to its preferred path. Limit cycles are thus only one type of nonsimple oscillator. Figure 5 presents an idealized model of a limit cycle in which two variables are required to determine the state of the oscillator. Winfree’s results with phase resetting in Drosophila (Winfree,Chapter 7 ) have demonstrated that the Drosophila clock may not be a single limit cycle oscillator since it does not always return to a preferred amplitude. It may be one of a number of other things (Winfree, Chapter 7): a different type of single nonsimple oscillator, a pair of independent limit cycle oscillators, or a population of simple or nonsimple oscillators. The term “limit cycle” cannot therefore be automatically applied to any nonsimple circadian oscillator in the absence of detailed information about the oscillator’s behavior. Nevertheless, the limit cycle is a useful model which satisfactorily simulates (so far) what is known about the phaseresetting behavior of the Neurospora oscillator.

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X FIGURE 5. (A) An idealized two-dimensional limit cycle. X and Y are the state variables; it is only necessary to know the values of X and Y to completely describe the state of the system. X and Y interact with each other in a regulatory loop such that the rate of change of one continuously affects the rate of change of the other. The thick circle is the limit cycle which describes how X and Y change with time in an unperturbed system. The thin arrows are sample trajectories which the system will follow in returning to the limit cycle after a perturbation. For example, if the system is perturbed from point A to point B, it will follow the BC trajectory. (B) Isochrons. The limit cycle in A has been divided into 24 circadian hours. The straight lies are isochrons, or lines of equal phase. When the system is perturbed, it will return to the limit cycle along a trajectory as in A but with a new phase determined by the isochron on which it finds itself after the perturbation. For example, three different stimuli (shown by arrows) were given at CT 2, 10, and 19; all will reset the oscillator to CT 3, although in each case the system will follow a different trajectory in returning to the cycle. (C) Type 0 phase resetting. A large stimulus is given to the system at many old phases, and the new phases are determined by the isochrons to which the system is shifted. New phase is confined to a narrow range of isochrons, in this example CT 5-7. Compare to Figure 2D under “Input-Introduction”, in which new phase is confined to CT 21-3.

3. Simple-Clock.Models in Neurospora The simple-clock model has often been applied to circadian rhythms (as reviewed by Winfree, pp. 92-94). The Neurospora clock is capable of type 0 resetting (as described above under “Input”), and therefore, according to topological arguments (Winfree, Chapter 3), cannot be a single simple clock. Nevertheless, the simple-clock model seems to be maintaining its hold in spite of its inappropriateness. For example, Nakashima’s,recent work on PRCS’*~ seems to be inspired by an implicit “tape-reading” model of the oscillator. PRCs for a number of treatments (including light pulses, temperam pulses, and inhibitors) were compared by determining the crossover point between maximum phase delays and maximum phase advances. These crossover points were used by Nakashima to 1990

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Critical Reviews In assign sensitive phases to each treatment, and to “estimate the time-order of metabolic reactions which comprise or influence clock function”. This analysis is apparently based on the assumption that the circadian cycle consists of a succession of discrete biochemical events and is therefore a one-dimensional simple clock. Crossover points are often used to describe phase-resetting behavior, and as mentioned above under “Input”, can be useful landmarks for comparison. However, in the case of type 0 resetting, the crossover point may have no physiological significance, and is an artifact of the method of plotting data as phase shifts. “Crossover points” or “breakpoints” at maximum phase shifts are said to define the phase at which the oscillator is most sensitive to the phase-shifting treatment, and similarly “dead zones” or “refractory periods” with minimal phase shifts are said to indicate the phase at which the oscillator is insensitive. However, if the oscillator is not a simple clock but is instead a limit cycle oscillator, these inferences about sensitive and insensitive phases may be meaningless in biochemical terms. For example, in Figure 5C, a stimulus at old phase 6 would produce no phase shift (the system would remain on isochron 6) while a stimulus at old phase 18 would produce a 12-h shift (to isochron 6). Yet in both cases, a large increase in variable X has been produced by the phase-shifting treatment. It is therefore meaningless to ask at what phase the components of the oscillator are most sensitive or insensitive to the stimulus, and meaningless to conclude that X is “important” at phase 18 and “unimportant” at phase 6. If X were suddenly removed from the system at phase 6, its “importance” would become obvious. Perhaps for type 0 resetting, it would be more informative to replot the data in Winfree’s new phase vs. old phase format and compare the new phases to which the oscillator is reset by various treatments. For example, in Figure 5C, if several different treatments all reset the phase to around CT 24, it could be concluded that they must all cause a large increase in variable Y. This means, of course, that they will all have a crossover point at CT 12 if their effects are plotted as phase shifts. Because the crossover point can be derived from the new phase vs. old phase plot, it can be used for the same type of analysis. Rensing and SchilP have collated the phases of maximum advance and maximum delay for a number of phase-resetting treatments in Neurosporu, and they suggest that there are two clusters of phases which could correspond to two state variables or opposite effects on one state variable. Their analysis uses phase advances and delays, and might be more easily interpreted if the PRCs were replotted in Winfree’s format and the new phases compared. We have replotted Nakashima’s data’2s in the new phase vs. old phase format and find that light and high temperatlire pulses reset the oscillator to around CT 510; cycloheximide, diethylstilbestrol, antimycin, and trifluoperazine reset it to around CT 15-20; and low temperature and A23187 reset it to around CT 20-5. As expected from the graphical relationship between PRCs and Winfree’s plots, the

crossover points reported by Nakashima are about 12 h, or 180°, away from the new phases. Another example of the use of simple-clock models in Neurosporu is the analysis of thefrq mutants. Feldman and Dunlap6 invoked a type of “tape-reading model” by suggesting that the differences in period lengths between thefrq mutants indicate the existence of a “2.5 hour quantum element”. suggested on the basis of his analysis of light phase resetting that only one part of the circadian cycle is affected by thefrq mutants and that this segment is shortened or lengthened by the various frq mutations. N a k a ~ h i m a ~ ~ ~ J ~ ~ has continued t h i s line of thought by determining the PRCs for several treatments for thefrq-7 mutant and comparing these to the equivalent PRCs of the wild type to determine “the phases in which individual clock genes function.”12s Nakashima’s conclusion is thatfrq-7 differs from the wild type only during the “frq phase” from CT9 to CT14,129which is approximately the same phase (CT8 to CT14) proposed by Dha~mananda~~ to be affected byfrq (see “Input, Phase-Shifting by Temperature Steps and Pulses” for further discussion). We have replotted Dharmananda’s original data4’ on light phase resetting using Winfree’s format and have compared the frq mutants with respect to the ranges of new phases to which the clock is reset by the light pulse. This range is narrow for frq-1, the mutant with the shortest period: new phase is confined to a band between CT6 and CT13. The range increases with increasing period length of the frq mutant: for frq-2 and frq-4 the range is CT3 to CT13, forfrq-3, it is CT4 to CT16, and forfrq-7 andfrq-8, it is CT4 to CT17. The most striking feature of the new phase vs. old phase plots is not that any particular old phase differs betweenfrq mutants, but that the entire plot changes gradually from type 0 infrq- 1 to something closer to type 1 infrq-7 andfrq-8 (see Figure 6). This indicates that the light pulse is perceived as a strong signal by the shortperiod frq’s Vig-1, frq-2, and frq-4) and as a weaker signal by the long-period frq’s (frq-3, frq-7, and frq-8). It might, therefore, be possible to obtain afrq-l-type resetting curve for frq-7 andfrq-8 by using a larger dose of light. On the basis of this analysis, we suggest that the simple-clock mode112s~129 is inappropriate in this case: thefrq genes cannot be said to affect only one phase of the circadian cycle. 4. Limit Cycles in Neurospora The difference in phase resetting behavior betweenfrq-7 and frq-1 could be described as an increased resistance of frq-7 to perturbations:frq-7 is resistant to phase resetting by light, cold pulses (see “Input, Phase-Shifting by Temperature Steps and Pulses”), and cycloheximide (see above). We suggest that if the Neurosporu clock behaves as a limit cycle, then the long period of frq-7 and its resistance to phase-resetting perturbations could both be explained by an increase in the amplitude of the oscillator (see Figure 7). This mathematical model for the effect offrq-7 is compatible with the biochemical model proposed by Dunlap and Feldman:124if thefrq-7 gene product

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Old Phase flGURE 6. Phase resetting by light infrq mutants. Data from Dha~mananda'~ have been replotted in Winfree's format. (A)frq-1. ( B ) f r q + . (C)frq-7 andfrq8. The resetting curve for the combined data forfrq-2,frq-4, andfrq-6 (not shown) is intermediate in shape betweenfrq-1 andfrq+, and that forfrq-3 (not shown) is intermediate betweenfrq+ and frq-7.

has a longer half-life, its increased amount could be responsible for the increased amplitude of the oscillator. However, there is another possible explanation which does not require a change in the stability of a labile protein: if thefrq-7 mutation increases the amplitude by some unknown mechanism, then any phaseshifting perturbation, including cycloheximide treatment, will be perceived as a weaker signal by the oscillator. Changes in the amplitude of a limit cycle oscillator could be invoked to explain several other observations about the Neurospora rhythm. Thefrq-1 mutant is apparently more sensitive thanfig+ to phase resetting by cycloheximide, and the

frq-4and frq-3 mutants are similar to frq' This indicates a rough correlation between increased period and decreased sensitivity to phase shifting by both light and cycloheximide in the series offrq alleles. The long-period mutant, prd-1, is much less sensitive to light phase shifting than the wild type.45 When the cef mutant is grown in the presence of linoleic acid, which induces a long period, phase shifting by both light and high temperature pulses is reduced in comparison with the same strain grown on palmitic acid with a normal period.278In all of these cases, an increased (or decreased) amplitude could account for both the increase (or decrease) in period and the 1990

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X FIGURE 7. Amplitude changes in a limit cycle oscillator. (A) The idealized limit cycle and its isochrons from Figure 5 are repeated. The period of the rhythm is assumed to be proportional to the circumference of the limit cycle. The effects of a phase-resetting pulse are shown by the shifted circle as in Figure 5C. A strong, Type 0 response is shown. (B) The amplitude of the limit cycle has been increased, either by mutation (as postulated forfrq-7). or by environmentalfactors (as postulated for linoleic acid with the cel mutant). The increased circumference indicates an increase in period. The same phaseresetting pulse as in A has been applied, and it is assumed to produce the same absolute change in the state variable X. Note that the response is now a weak, type 1 response. A n y phase-resetting perturbation which has the same absolute effect on the state variables of both the mutant and wild type wiIl produce a weaker resetting response in the mutant.

decrease (or increase) in sensitivity to phase-shifting perturbations. However, not all changes in period can be accounted for by a change in amplitude: for example, the long-period mutant prd-3 is very similar to wild type in its phase-resetting response to light.” Similarly, not all changes in phase resetting by light can be accounted for by changes in amplitude: the [poky] mutant is insensitive to phase resetting by light, but is very similar to wild type in phase resetting by cy~loheximide.’~ Two recent papers have addressed the question of limit cycles in Neurosporu. Nakashima and Hastings*= have updated Woodward’s mixing experiments (cited by Feldman and Dunlap6) in which heterocaryons are formed between two out-

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of-phase cultures and the resulting new phase is determined. These experiments use Nakashima’s liquid culture system to estabIish out-of-phase mycelial disks, and these are then inoculated in pairs onto race tubes. In contrast to Woodward’s results, no “dominant” phase is found: in many cases when &e difference in phase between the parents is small, the new phase is the average of the parental phases, or corresponds to the phase of one of the parents. Analysis of nuclear ratios suggested that new phase tends to correspond to the parent which contributed the greater proportion of nuclei. In some cases, however, when the difference in phase between the parents is large, the new phase is not related in any obvious way to the phase of the parents. Nakashima and Hastings suggest that this may be related to the properties of the oscillator, and that a limit cycle model could account for these results. GoochS*has investigated the effects of light and temperature pulses of various durations on the Neurospora clock. In these experiments, cultures were entrained to two 12:12 L:Dcycles, exposed to either light periods or temperature pulses of various durations, released into DD, and monitored for determination of the new phase. This experimental protocol determines new phase for a series of stimuli of different strengths given at one old phase, and can be plotted as one plane in a “time crystal” as described by Winfree.35 For short stimulus durations, the observed phase resetting is similar to pulse experiments. For long durations, the oscillator is apparently “held” at one phase until released by the end of the pulse. This “holding” is not perfect, however, and new phase shows a 4-h variation depending on the time of the end of the pulse. This “wiggle” can be modeled by a limit cycle which continues to oscillate during the stimulus but within new parameters: this would be equivalent to shoving the whole system in Figure 5C very far to the right during the stimulus and shoving it back to the left when the stimulus ends. This limit cycle model satisfactorily accounts for Gooch’s results, but, as with the work of Nakashima and ha sting^,^^^ it is not clear that these results necessarily require a limit cycle model and could not be modeled by another nonsimple oscillator or by a population of oscillators. The mycelium of Neurospora is coenocytic, with many nuclei sharing a common cytoplasm, and this ought to allow free communication (at least of small molecules) between all areas. Because the Neurosporu mycelium is not a population of individual cells, it may seem peculiar to suggest that Neurospora might consist of a population of oscillators, yet such a population model was suggested by Feldman and Dunlap6 in discussing Woodward’s mixing experiments. This model requires individual oscillators to be somehow localized to separate compartments within the coenocytic mycelium, and there is evidence in the literature to support this. Dhmananda and F e l d m a ~ demonstrated ’~~ that in cultures growing on solid medium, the clock continues to run in old areas Of the Culture behind the growing front, but that the phase lags behind the growing front by about 1.5 Wd. This phase gradient could be produced by an increase in period in older areas behind the

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Microbiology front of cultures on solid medium, although it has been shown169 that the circadian oscillator continues to run in old areas of the culture behind the growing front. The phase of the oscillator determines which developmental program will be followed by the new growth at the front, and once the binary decision has been made (to conidiate or not to conidiate), that area of the culture carries out the program and presumably becomes permanently differentiated. The oscillation between conidiating and nonconidiating growth at the growing front produces the visible morphological rhythms in production of aerial hyphae, production of conidiospores, and synthesis of carotenoids. Spore formation in microorganisms is generally considered to be a “catabolic” event, and the morphological rhythm expressed by Neurosporu on the surface of uniform medium implies that the oscillator must ovemde the usual developmental signals. Either the cultures must be continuously in an anabolic state and the oscillator induces the developmental program during band phase, or there is local starvation in the few millimeters just behind the growth front where conidiation takes place and the oscillator suppresses conidiation in this area during the interband phase. A rhythm in total mass, measured as dry weight, can also be observed at the growing front.” The sexual cycle of Neurospora has also been investigated for the expression of circadian rhythmicity, and Brody has demonstrated a rhythm in ascospore ejection.28’This investigation was initiated for several reasons:

growing front; clearly there is not perfect phase communication from front to back. Neither is there communication across the gowing front: Winfree and Twaddle53induced phase discontinuities and phase gradients at the growing front of cultures by shadowing part of the culture while the other part received a phase-resetting light treatment, and found that discontinuities persist for many days. This indicates a complete lack of transfer of phase information between adjacent areas of the growing suggests front and, as discussed by Winfree and that the clock mechanism may be compartmentalized in large organelles or in membranes. Another example of the compartmentation of phase information has been seen in the cel strain which, when grown on petri plates supplemented with 18:2, can express different phases in adjacent areas of the culture.279These findings raise the intriguing problem of what the “unit oscillator” might be in Neurosporu, and what barriers to communication might create the compartmentalization which allows the culture to behave as a population of oscillators.

IV. OUTPUT A. Introduction The function of a circadian oscillator, if it has a function at all, must be to supply timing information to other processes in the cell, and therefore to “drive” these processes in a rhythrmc manner. In the absence of any information about the identity of the oscillator itself, we can only Observe its workings indirectly by observing its effects on these driven rhythms; therefore, all information about the oscillator and its input comes from observing its output. The most obvious and most easily assayed output in Neurosporu is the conidiation rhythm (described in the Introduction), and by using this assay the fundamental properties of the Neurosporu oscillator have been described (see Introduction for further details): 1.

2. 3. 4.

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To monitor the rhythm, cultures were inoculated onto crossing medium and allowed to form protoperithecia in the light. The cultures were then fertilized with the opposite mating type, placed in constant darkness, and inverted over a narrow opening which allowed ejected spores to fall onto a motor-driven circular grid. The grid was slowly rotated beneath the opening and ascospores were collected over a 72-h period in constant darkness. The apparatus for monitoring the rhythm was supplied by Perkins. A clear rhythm in the number of ascospores per unit grid was found, with a period of about 22 h. The rhythm persisted for 3 d with diminishing amplitude. The ascospore ejection rhythm from a&- 1 x fiq- 1 cross had a period of about 17 h, similar to the period of the conidiation rhythm in this mutant. In the absence of the initial light stimulation, no rhythm was found and few total ascospores were ejected.

It persists in constantdarkness and at constant temperature. The phase is set by the light-to-dark transition and can be reset by light or temperature pulses. It is temperature compensated. The period is under genetic control.

Any rhythmic process in the cell which is driven by the oscillator will share these properties, and before any fluctuating variable can be classified as genuine circadian output, at least some of these criteria must be met. The known Neurosporu rhythms have been thoroughly reviewed by Feldman and M a p 6 (up to 1983) and by Edmunds” (1987), but a few additional comments can be added. Table 7 summarizes the variables which have been assayed for circadian rhythmicity in Neurospora.

B. Rhythmic Variables 1. Morphology The conidiation rhythm is expressed only at the growing

Other fungal species have been shown to have such a rhythm.289 In Neurosporu, the perithecia (sexual fruiting bodies) are phototropic and will bend toward blue light.42 Perkins290has observed that large numbers of ascospores are ejected by the perithecia within a few hours of the light-to-dark transition.

2. Conidiation-AssociatedEnzymes The developmental program leading to conidiation clearly requires changes in the activities of a number of enzymes. 1990

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Critical Reviews In Table 7 Variables Assayed for Circadian Rhythmicity REFERENCES RhythmiC


CA' Morphology Conidiation Mass Ascospore ejection Macromolecules DNA and RNA content Total RNA and rRNA Uridine incorporation Gene transcription Total soluble protein Rotein synthesis Inhibition of protein synthesis by cycloheximide Induction of heat-shock protein synthesis Calmodulin content Enzymes NADase, citrate synthase, isocitrate lyase Glutamate dehydrogenase, malate dehydrogenase, three phosphatases Glyceraldehyde-phosphate dehydrogenase, phosphogluconate dehydrogenase, glucosed-phosphate dehydrogenase Energy metabolism ATP and ADP AMP NAD(P)(H) redox ratio CO, production Ions Cytoplasmic pH K+/Na+ ratio Small molecules 18:2/18:3 ratio Galactosamine, glucosamine

NCA

CL

NCL

SD

CA

Nonrhythmic NCA NCL

280 13 28 1 282

282 29 1

282 291 283 227 124, 220 134 225 283 283

283 283

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242 285 286

286 287

88 271

271 288

Culture systems are indicated by the following codes: CA. cultures on agar medium undergoing conidiation, NCA: nonconidiating strains (wild-type or fluffy) on agar medium, CL: cultures in liquid medium undergoing conidiation, NCL: cultures in liquid medium not conidiating, SD:sexually differentiated cultures on agar crossing medium.

Hochberg and SargentB3assayed the activities of enzymes related to conidiation at the growing front of cultures growing on solid medium. Rhythmic activity (per milligram dry weight of mycelium) was found during the first 2 d of growth for NADase, citrate synthase, isocitrate lyase, glyceraldehydephosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase. A rhythm in total soluble protein (per milligram dry weight) was also found. Two of the enzymes were assayed again over days 4 to 6, and the rhythms were shown to persist, thus satisfying the first criterion for establishing the circadian nature of the rhythms. None of the other criteria were demonstrated, but the close association of these rhythms with the conidiation rhythm makes it likely that they represent genuine circadian ouput. These rhythms were shown to be related to conidiation by two criteria: (1) the activities do not oscillate in old areas of the culture, 404

but instead reflect the permanent developmental state (band or interband), and (2) none of these activities oscillate in the wild type or fluffy (fZ) strains which undergo little or no conidiation. The rhythm in total soluble protein was not assayed in the nonconidiating strains. A potential criticism of Hochberg and Sargent's results is that the presence of NADase in the extracts used to assay the activity of the dehydrogenases may invalidate these assays. The assays for glyceraldehyde-phosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase use either NAD or NADP as substrates, and these substrates might be hydrolyzed by NADase. The observed rhythm in NADase might therefore also produce an apparent but artifactual rhythm in dehydrogenases, with the peak of NADase producing a trough in the apparent dehydrogenase activities. Hochberg and Sargent answered this criticism

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Microbiology by reporting that the initial reaction rates were not affected by increasing the NAD(P) concentration in the assays, and that the peak of NADase activity did not always correspond to the trough of dehydrogenase activity. BrodyZwhas reexamined this problem and found rhythms in NADase and dehydrogenases in the bd strain similar to Hochberg and Sargent’s results. in contrast, however, Brody found that the peak of NADase coincided with the troughs of dehydrogenase activity, and that the reaction rate increased when additional NAD(P) was added to the assays. Furthermore, mixing extracts from band and interband gave less than additive results, as expected if high NADase levels in either extract hydrolyzed the NAD(P). Brody also assayed these enzymes in the bd nada strain, which is deficient in NADase, and found no rhythms in dehydrogenases. Citrate synthase and isocitrate lyase were not assayed in this strain. These results indicate that a rhythm in NADase may be responsible for the apparent rhythm in dehydrogenases. Although Hochberg and SargentZs3did not find rhythms in glutamate dehydrogenase or malate dehydrogenase using NAD(P)-linked assays, these enzymes may have higher affinity for NAD(P) than the other dehydrogenases assayed. The results reported by Hochberg and SargenPS3using the nonconidiatingfl and wild type strains highlight the difficulties in looking for rhythms which are part of the oscillator mechanism in cultures expressing the developmental conidiation rhythm: it is necessary to demonstrate the persistence of the rhythm in the absence of morphological change. The persistence of a rhythm in a nonconidiating strain such a s p may not necessarily indicate that the rhythmic variable must be unrelated to conidiation;j7 does show a rhythm in aerial hyphae production under some conditions.2B2Rhythms observed in this strain might therefore be related to the early stages of the developmental program which lead to conidiation, even if the final stages of conidiogenesis are not completed. Anyone searching for rhythms directly related to the mechanism of the oscillator would be well advised to choose a culture system in which morphological rhythms do not occur, such as the liquid culture system developed by Nal~ashima.~ As shown in Table 7, very few variables have yet been demonstrated to be rhythmic in nonconidiating culture systems. 3. Macromolecules

Martens and SargentZs2assayed the nucleic acid content of cultures growing on solid medium and found rhythms in both DNA and RNA content @er milligram mycelium); these rhythms have not been demonstrated to be circadian, but are synchronous with the conidiation rhythm. Since the amplitudes are lower in wild type. and thefl strain, these rhythms seem to be related to conidiation. No nucleic acid rhythms were found in three types of liquid cultures (shaking, bubbling, or standing). A rhythm in uridine incorporation was observed by transfemng samples of the growth front from solid medium to liquid medium containing [3H]-uridine. This rhythm could result from

rhythmic polymerase activity, nucleic acid degradation, or changes in the rate of uridine uptake or of uridine pool sizes. A rhythm in the induction of heat-shock protein synthesis was described by Cornelius and Rensing’” using a standing liquid culture system. Heat-shock protein synthesis was induced at various times during one circadian cycle, and the rates of synthesis of all three major heat-shock proteins were found to peak at the same time. The assay protocol eliminated the possibility of age-related changes by shifting cultures into the dark at various times after inoculation and assaying all cultures at the same age (but different circadian phases). This culture system apparently undergoes conidiation, so it is not clear whether this observed rhythm is related to the conidiation programRecent work by Loros et al.291has demonstrated the application of molecular biology techniques to the analysis of circadian output. They have isolated clock-controlled genes by screening both cDNA and genomic libraries with cDNA probes enriched in poly(A)+ RNA sequences expressed at two different circadian times, CT 1 and CT 13 (“morning” and “evening”). Two “morning-specific” genes were identified, and their expression was shown to be rhythmic when assayed across two circadian cycles by Northern analysis of total cellular RNA extracted from cultures at various phases. Expression was assayed in two strains, frq+ andfrq-7, with conidiation rhythm periods of 21.5 and 29 h, respectively. These two morningspecific genes are rhythrmcally expressed with periods appropriate to the strains from which the RNA was derived, thus providing strong evidence that these genes are controlled by the circadian oscillator. Expression of these genes is apparently controlled at the level of transcription.29zIt might be of interest to determine how transcription of these genes is affected by a phase-shifting light pulse. In contrast to the results of Martens and Sargent,282Loros et al.291found no evidence of circadian changes in total RNA, or in rRNA, although total RNA decreases with age. They used the liquid culture disk system in which no morphological changes occur, which indicates that the nucleic acid rhythms seen by Martens and SargexP on solid medium may be related to conidiation. 4. Small Molecules A rhythm in CO, production was observed by Woodward and SargentZs6in cultures on solid media and in standing liquid cultures. The CO, rhythm in liquid culture has a 22-h period and is seen only after the culture reaches stationary phase and growth stops.z7Two short-periodfiq mutants have short-period CO, rhythms, which indicates true circadian control (cited by Feldman and Dunlap6). Sat0 et measured the content of Na+ and K+ in the growth front of cultures on solid medium and found oscillations in both ions, with the maxima at opposite phases, and consequently a rhythm in the K+/Na+ ratio, This rhythm was shown to be circadian by several criteria: the period is approximately 21 h; the phase is set by the light-to-dark transition


Critical Reviews In (rather than the time of inoculation); constant light abolishes the rhythm; and a phase-shifting light pulse produces a corresponding change in the ion contents. This rhythm is likely to be associated with conidiation: the K+/Na+ ratio increases as conidia develop in the band region, and the peak of the ion ratio rhythm coincides with conidiation. Although changes in the ratio at the growth front can be detected 8 h after a phaseshifting light pulse, no changes can be detected after 1 h, indicating that fluxes of Na+ and K’ are not immediate effects of a light pulse. Oscillations in adenine nucleotides have been reported by two laboratories, but with conflicting results. Delmer and BrodyZ4’sampled the growing front of cultures on solid medium and found a rhythm in AMP, but no obvious rhythms in ATP or ADP, resulting in a rhythm in total adenylate energy charge. The rhythm appears to be Circadian by several criteria: the period is about 22 h; the phase of the rhythm is set by the light-to-dark transition rather than the time of inoculation; and the rhythm is absent in constant light. No evidence was provided to indicate whether the rhythm is associated with conidiation or if it is independent of morphologicalchanges. Schulz et al.” addressed this question by assaying adenine nucleotides in the liquid culture system in the absence of morphological rhythms and reported an oscillation in energy charge; however, the maximum occurs at approximately the same circadian time as the minimum in Delmer and Brody’s report. The original data for individual nucleotides were not presented, although the authors stated that ATP and ADP show a circadian pattern with ADP at a maximum 6 h before ATP, again in contrast to Delmer and Brody’s results indicating no rhythm in ATP or ADP. Schulz et al. provided no indication of the statistical error in their assays, making it difficult to evaluate the significance of the reported oscillation. No evidence has been provided to indicate whether this oscillation is circadian, such as a demonstration that the phase of the peak is set by the lightto-dark transition and is not related to the time of transfer to starvation conditions. Whether the energy charge follows a circadian rhythm in the absence of morphological change remains an open question. In view of the critical role played by the adenine nucleotide pool in the regulation of many cellular processes, this is an important point which deserves further work, as does the question of whether the levels of any other nucleotides oscillate. Oscillationsin GTP would be particularly interesting, given the important role of GTP in regulating cellular processes through GTP-binding proteins. Using nonconidiating stationary liquid cultures, Hasunuma et al.99.261 assayed CAMPacross two or three circadian cycles in DD in wild type, bd, frq-l,frq-2, pho-2, Cpd-1, and CPd2 strains and-found large-amplitude fluctuations in CAMPin some strains. Two peaks of CAMPper cycle were found, but the sampling interval of 3 h makes it impossible to determine the period with any accuracy. The data which are reported are apparently the results of duplicate assays from single cultures, 406

and the statistical significanceof these oscillations has not been established. No evidence has been provided in either paper to indicate whether the light-to-dark transition would reliably establish the phase of the peaks or whether these fluctuations are related merely to aging or starvation; for example, Terenzi et al.= found large transient increases in CAMP levels which correlate with the onset of stationary phase and starvation in liquid culture. Hasunuma et al.w also assayed cGMP, and although they claim to see circadian oscillations, the peaks are small and fall at exactly the same time in all the strains tested, indicating the possibility of artifacts. It would seem premature to conclude from these reports that circadian changes in cyclic nucleotides do occur. 5. Fatty Acids A rhythm in fatty acid composition was described by Roeder et al.’” using cultures on solid medium. This rhythm was seen in the mole percentages of two unsaturated fatty acids, linoleic (18:2) and linolenic (18:3), in both the total lipids and the phospholipids of the mycelia, and the differences between m a ima and minima were shown to be statistically signifcant. The peak in the level of one fatty acid coincides with the trough in the level of the other. The oscillation has a period of about 20 h, and the phase of the oscillation is set by the light-todark transition rather than the total growth time, indicating a true circadian rhythm. The rhythm persisted in the bd’ csp-1 strain which shows no gross morphological changes. C. Nonrhythmic Variables It is instructive to note that not everything in Neurospora is driven by the circadian oscillator. This implies that either ( 1 ) the rhythmic variables are specifically linked to regulation by the clock, and if we could identify the regulatory signals, we could follow them to their origin and identify the oscillator, or (2) the regulatory signals have so many pleiotropic effects that nearly everything in the cell tends to oscillate, and the nonrhythrmc variables are specificallybuffered against change. The relatively few variables which have been assayed and can be assigned to rhythmic or nonrhythmic categories so far do not fall into any clear patterns, and what little is known about their various regulatory mechanisms cannot yet distinguish between these two possibilities. In their study of enzymes associated with conidiation, Hochberg and SargentzE3found several enzyme activities which are not rhythmic, even in the presence of the conidiation rhythm: glutamate dehydrogenase, malate dehydrogenase, repressible alkaline phosphatase, constitutive alkaline phosphatase, and constitutiveacid phosphatase. Brody and Harris293assayed pyridine nucleotides in band and interband regions behind the growing front of cultures on solid media and found differences demin the levels of NAD(P) and NAD(P)H. DieckmannZE5 onstrated that changes in nucleotides at the growing front are small and the redox ratio does not vary with circadian time;

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Microbiology therefore, the differences in old areas of the culture reported by Brody and Harris are age related and are not due to a rhythm in nucleotide levels. Nakashima* assayed calmodulin content in liquid culture at two phases and found no change over two cycles. Schmit and BrodyBS assayed the levels of galactosamine and glucosamine in band and interband regions of cultures on solid medium and found no rhythm in either sugar, although the level of galactosamine increased with age. P e r h a P assayed the relative rates of synthesis of more than 100 proteins by labeling cultures in liquid medium with [35S]-methionineat two different phases and analyzing the labeled proteins by two-dimensional gel electrophoresis, and no differences were found between the two phases. However, as with Nakashima’s calmodulin assays,= it is conceivable that a genuine rhythm could have been missed by assaying only two points per cycle. Dunlap and F e l d n ~ a n assayed ’~~ the inhibition of protein synthesis by cycloheximide at a number of phases and found the same inhibition at all times assayed. This is in agreement with the earlier observation by Nakashima et al.= that the dose response curves for protein synthesis inhibition by cycloheximideare the same at two different phases. Johnsonzs7 looked at intracellular pH in Neurospora and found large changes which proved to be noncircadian. Using a radioactively labeled probe for intracellular pH, Johnson found a large increase and a subsequent decrease in pH after transfer of mycelial disks in liquid culture to starvation medium. The timing of the peak was found to be the same in fi4+ and f.s1 despite their diffexent circadian periods; and in cultures shifted to constant dark 12 h apart, the peak occurs at the same time after media transfer regardless of the circadian phase. The pH fluctuation is, therefore, not driven by the circadian oscillator, but may be related to the onset of starvation. This work provides a good example of the kind of critical analysis needed to distinguish between circadian and noncircadian fluctuations. D. Analysis of the Output Pathway One strategy for identifying the mechanism of circadian rhythmicity is to follow the transduction pathway backwards from the observed output to the driving oscillator. This approach is being used by several laboratories working on organisms other than Neurospora. For example, the control of rhythmic bioluminescence in Gonyaular has been traced to rhythms in translation of luciferin binding protein and luciferand rhythms in NAD(P)-dependent enzymes in Lemnucan be attributed to rhythms in NAD kinase and NADP phosphatase which in turn might be controlled by a rhythm in calcium/calmodulin.295 This approach has not yet progressed very far in Neurospora, although there are several likely candidates for analysis. The rhythmic gene transcription reported by Laos et al.291may be the most promising system since it is amenable to all the methods in the molecular biologist’s repertoire for analyzing the control of transcription. The fatty acid desaturation rhythm

reported by Roeder et aLZ7l could provide the starting point for an analysis of the control of desaturase activity. Much is known about the induction of heat shock proteins in other organisms, and this background could be applied to an analysis of the rhythm of heat shock protein induction in Neurospora.’”

E. “Hands” vs. “Gears” At every stage of output analysis, once a rhythmic variable has been demonstrated to be truly circadian, the question immediately arises as to whether the observed rhythm is in fact part of the oscillator itself, or is another output, driven by the oscillator. To use the terminology of Goto et al.,s2 is it a “gear” in the clock mechanism, or merely a “hand” on the clockface? Several criteria have been proposed for distinguishing between the two. Engelmann and SchrempPO listed a number of experimental approaches, with specific reference to membranes; Roeder et al.”’ expanded this list of criteria, and ~ ~ redefined “gears” in terms Goto et al.252and E d m u n d ~have of state variables and parameters characterizing the oscillator. A consensus list of criteria would look something l i e this: a circadian rhythm in the level or activity of some biochemical component is likely to be a “gear” and not a “hand” if (1) treatments or mutations which change phase or period of observed rhythms also change the properties of the component (where ‘‘properties” can mean amount, activity, location, etc.); (2) perturbations in the component within physiological limits, by chemical intervention or mutation, cause changes in period or phase of all observed rhythms; (3) both increases and decreases in the component affect the observed rhythm, and in opposite ways; and (4) in any model which successfully simulates all the known properties of the oscillator, the component must appear as a state variable or parameter. By these criteria, none of the observed rhythms in Neurospora has yet been shown to be a gear and not just a hand. The principal difficulty seems to be our inability to specifically modify the activity or level of a putative gear without simultaneously affecting many other processes in the cell. The dissection of the circadian oscillator thus shares the same methodological difficulties as many other problems in cell biology. Our tools are often too blunt and clumsy to tease apart the many strands of a complex regulatory network.

V. SUMMARY A. Introduction Our goal in writing this review has been to demonstrate how genetic and biochemical approaches are being used to probe the mechanism of the circadian rhythm in Neurospora crmsa. The ultimate goal of this line of research, from our perspective, is nothing less than a complete description at the molecular level of the workings of the circadian oscillator and its input and output pathways. In this final section, we briefly summarize what is known about the Neurospora clock and give

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Critical Reviews In the reader some idea of how far we are from that ultimate goal. We therefore present two lists: the first is a compilation of the few hard facts and the promising new developments, and the second is a “wish list” of things we need to know and would like to have. Cross-references to relevant sections of the review are indicated in parentheses (e.g., Section III.B.2.a).

2.

B. Established Evidence and Current Interpretations 3.


1. Input 1.

2.

3.

4.

5.

The Neurosporu oscillator is sensitive to and can be phase shifted by both light pulses (Section II.B.3.) and temperatme changes (Section II.C.3.a.). Under appropriate conditions, both light and temperature are capable of strong (type 0) resetting (Section LA.). The Neurosporu oscillator is therefore similar to many other circadian clocks. Some of the probable components of the photoreceptor have been identified. The action spectrum for phase shifting, and genetic evidence from the [poky] (cytochromedeficient), rib (riboflavin-requiring), and ul (albino) mutants implicate a flavinkytochrome complex (Section II.B.5.). Several conditions have been identified which inhibit light phase shifting. High temperature, neutral pH during growth, and several compounds added during growth at neutral pH can decrease or block light phase shifting (Section II.B.6.). Havin deficiency (in the rib mutants) or cytochrome abnormalities (in the [poky] mutant) can also inhibit light phase shifting (Section II.B.4.). The light input pathway can be disrupted without affecting the oscillator itself and must therefore be separate from the oscillator mechanism. The wc (white-collar) mutants are good candidates for mutants defective in light signal transduction. These mutants are defective in all light responses tested so far, but have not yet been tested for phase shifting of the oscillator (Section II.B.4.). Several mutants have been shown to affect temperature compensation. Frq-9 (frequency) and cel (deficient in fatty acid synthesis) have apparently lost temperature compensation, indicating that temperature compensation can be disrupted without destroying rhythmicity (Section II.C.2.b.). Chr (chmno), prd-3 (period), prd-4, and some of the otherfrq alleles (particularlyfrq-7) all differ subtly from wild type (and from each other) in their temperature compensation (Section II.C. 2. b. ) .

2. Oscillator 1.

5.

6.

7.

8.

9.

Six loci have been identified as clock-affecting genes by selecting for altered periods (Section III.B.2.a.):frq, chr, prd- 1, prd-2, prd-3, and prd-4. The chromosomal rear-

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4.

rangement, cla-1 (clock-affecting) also has an altered period. Mutations have been isolated which alter the period without altering the growth rate. The variousfrq alleles have periods either faster or slower than wild type but normal growth rates, indicating that the oscillator can be altered without affecting processes required for growth (Section III.B.2.a.). The most interesting clock-affecting gene, frq, has been cloned and sequenced, and the prd-4 gene has also been cloned. The cloning of clock-affecting genes paves the way for the use of molecular biology techniques to discover the biochemical functions of these genes (Section III.B.2.a.). The mechanism of protein synthesis has been eliminated as a potential component of the oscillator. The frq-7 mutant is resistant to the phase shifting effects of cycloheximide, although its protein synthesis machinery is fully sensitive (Section III.C.2.). A number of other biochemical pathways have also been eliminated as components of the oscillator. The absence of clock defects in a number of mutants defective in amino acid synthesis, some aspects of lipid synthesis, vitamin synthesis, and several other pathways implies that flux through these pathways is not essential to the clock mechanism (Section III.B.2.b.). This rules out the “holistic” model which proposes that the oscillator mechanism is the entire cell, and makes it more likely that a discrete clock mechanism can be identified eventually. Twelve mutants with known biochemical defects have been shown to affect the period. These mutants affect amino acid synthesis, lipid synthesis, glycerol utilization, and mitochondrial functions (Section III.B.2.b.). A number of clock-affecting compounds (‘ ‘chronobiotics”) have been shown to alter period or phase. These compounds affect a variety of cellular processes: protein synthesis (Section III.C.2.), mitochondrial energy metabolism (Section III.C.3.), cyclic AMP (Section III.C.5.), membranes (Section III.C.6.), ion fluxes (Section III.C.4.), and calcium metabolism (Section m.C.4.). There is evidence that the maintenance of normal membrane composition is important for normal clock functioning. The cel mutant has an altered membrane fatty acid composition and is defective in temperature compensation (Section III.B.3.). The prd-1 mutant has altered fatty acid composition and an abnormal period (Section III.C.1.b.). There is evidence for a role for mitochondria in the clock mechanism. Mitochondrial mutants can change the period (Section III.B.2.b.), and mitochondrial inhibitors can change the period and induce phase shifts (Section III.C.3.). In addition, mitochondrial mutants and inhib-

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MlCroDlology itors can modulate the effects of exogenous fatty acids on the cel mutant (Section III.B.3.c.). 10. Clock-affecting mutants and chronobiotics can be grouped into classes by looking for interactions between mutations in double-mutant strains and between chronobiotics and mutations. This strategy has the potential for identifying mutants and chronobiotics which affect the same clock component (Section III .B .4.b .). 11. The Neurospora oscillator is capable of type 0 resetting and therefore cannot be a simple clock. In contrast, the limit cycle model is, so far, an adequate mathematical model for the Neurosporu clock (Section III.D.3.). 12. Phase shifting by light, temperature, and cycloheximide are all affected similarly byfrq mutations. If the oscillator is a limit cycle, then the changes in period and the altered effects of all phase shifting perturbations in these mutants could be explained by changes in the amplitude of the limit cycle (Section III.D. 4. ).

5.

2. Oscillator 1.

2.

3. output 1.

2.

There are a number of rhythmic variables which show true circadian rhythmicity and which may be driven by the oscillator. Although the majority of these variables appear to be linked to conidiation, a few have been shown to persist in its absence: gene transcription (Section IV.B.3.),fatty acid desaturation (Section IV.B.5.), CO, output (Section IV.B.4.), and possibly ATP/ADP ratios (Section IV.B.4.). Two of the genes showing rhythmic transcription have been cloned. These genes could provide tools for using molecular biology techniques to dissect output pathways (Section IV.B.3.).

3.

4.

5.

C. Goals Yet to be Accomplished 1. Input 6. 1.

2.

3. 4.

No input mechanism, either for light or for temperature, has yet been identified, and there are no strong candidates for intracellular signals produced by either light or temperature (Sections II.B.6. and II.C.5.). The subcellular localization of the photoreceptor has not been established. The photoreceptor is assumed to be membrane bound, but so far it cannot be unequivocally localized to a particular membrane fraction (Section II.B.5.). The photoreceptor complex itself has not been identified. The flavidcytochrorne complex has not been purified or characterized biochemically (Section II.B.5.). There is no evidence for or against a specific ‘‘temperature sensor” for phase shifting. Light responses have a discrete photoreceptor, but the target for temperature input may be either a discrete receptor or a more gener-

alized effect on the reaction lunetics of the oscillator (Section II.C.5.). The mechanism of temperature compensation is unknown, and there are few testable clues other than the possible involvement of membrane composition. There is no evidence whether temperature compensation is an input mechanism separate from the oscillator or is an integral feature of the oscillator itself (Section II.C.2.).

7.

8.

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No component of the oscillator has yet been identified. Several criteria for identifying a clock component have been proposed, but as yet no putative component satisfies these criteria (Section N.E.). The primary biochemical defects in clock-affecting mutants isolated by changes in period have not been identified. Nothing is known about the biochemical basis of the clock defects in these mutants (Section III.B.2.a.). It is not known whether any of the mutations affecting the period do so by direct or indirect effects on the oscillator or by effects on input pathways (Sectionm.B. 1.). New strategies are needed to idenhfy clock-affecting mutants. One promising strategy being used by Dunlap and co-workers2%is to couple the promoters from rhythmically transcribed RNAs to easily screenable or selectable markers and to transformNeurosporu with these chimeric genes. Such transfomants could easily be screened for mutations altering period which would express the marker out of phase with the wild type. Rigorous biochemical studies are needed to identify the intracellular targets of chronobiotics. Effects of chronobiotics on their assumed targets have rarely been assayed, and much more needs to be done to establish whether changes in period or phase are correlated with changes in the assumed targets (Section III.C.1.a.). Better quantification of the effects of metabolic perturbations on the period is needed. This would require assaying the effects of chemicals and mutations on specific metabolic functions and comparing these effects to the corresponding change in period. Effects on period could be quantitated through the use of a “clock coefficient” (Section III.C.7.). More work is needed to define the effective durations of pulses of phase shifting inhibitors. The intracellular effects of such inhibitors may persist long after they are washed out of the extracellular medium, and therefore it is almost never known how “clean” the reported pulse durations are (Section III.C.1.a.). The use of more inhibitor-resistant mutants is needed to help idenufy the targets of chronobiotics. In most cases, it is not clear whether the effects of chronobiotics on the clock are due to their primary action on the known target,

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Critical Reviews In


9.

10.

11.

or to side effects, and mutants similar to the cycloheximide-resistant, nystatin-resistant, and oligomycin-resistant mutants can be used to answer this question (Section III.C. 1.a.). It would be useful to have a truly arrhythmic mutant, analogous to the pef‘ mutant in Drosophilu, or a treatment which would produce true arrhythmicity. However, such a situation may not be viable in lower eukaryotes like Neurospora: if the cellular oscillator is composed of essential metabolic pathways, the complete loss of rhythmicity could be lethal. A true temperature-sensitive mutant might be informative. If a mutant was available with a temperature-sensitive clock (which might, for example, be arrhythmic at high temperature), it would provide the opportunity of identifying the thermolabile protein as part of the oscillator. A systematic search for “negative data” could be useful. Now that a number of phase-shifting and period-altering treatments are known, it would be helpful to put their significance into perspective by defining areas of metabolism which can be perturbed by mutation and/or chemicals without affecting the clock (Section III.C. 1.a.).

3. output 1.

2.

3.

The output pathway has not yet been identified for any observed rhythm (Section IV.). There has been no systematic attempt to study the control of any observed rhythm as a strategy for identifying the oscillator. It should be possible to work backwards from an observed rhythm to the driving oscillator by identifying the regulatory signals which control the observed rhythm (Section N.D.). It has not been demonstrated whether any observed rhythm is an output driven by the oscillator or is a component of the oscillator itself (Section IV.E.).

ACKNOWLEDGMENTS We thank numerous investigators in this field for sending us reprints, preprints, and unpublished data for our utilization, Richard Crain of the University of Connecticut and Art Winfree of the University of Arizona for critically reading portions of the manuscript, and Debbie David and Lyn Alkan for invaluable secretarial assistance.

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Volume 17, Issue 5

Circadian rhythms in Neurospora crassa: the role of mitochondria.

Circadian rhythms in Neurospora crassa: effects of saturated fatty acids.

Genetics of circadian rhythms.

Circadian rhythms in Neurospora crassa on a polydimethylsiloxane microfluidic device for real-time gas perturbations.

Biochemical genetics of Neurospora crassa conidial germination.

Synchronizing stochastic circadian oscillators in single cells of Neurospora crassa.

Genetics and physiology of Neurospora crassa glutamine auxotrophs.

Epigenetic and Posttranslational Modifications in Light Signal Transduction and the Circadian Clock in Neurospora crassa.

Cellulase of Neurospora crassa.

Effect of pH and biotin on a circadian rhyythm of conidiation in Neurospora crassa.

Dawn- and dusk-phased circadian transcription rhythms coordinate anabolic and catabolic functions in Neurospora.

Glutamine metabolism and cycling in Neurospora crassa.

Modulation of Circadian Gene Expression and Metabolic Compensation by the RCO-1 Corepressor of Neurospora crassa.

Suppressing the Neurospora crassa circadian clock while maintaining light responsiveness in continuous stirred tank reactors.

Beta-galactosidases from Neurospora crassa.

Isolation of Neurospora crassa bradytrophs.

Phase resetting of the Neurospora crassa circadian oscillator: effects of inositol depletion on sensitivity to light.

Regulation of thymidine metabolism in Neurospora crassa.

Ageing and Circadian rhythms.

Heterochromatin controls γH2A localization in Neurospora crassa.

Multiple intracellular peptidases in Neurospora crassa.

Specificity of nucleoside transport in Neurospora crassa.

Circadian Rhythms in Cyanobacteria.

The aging biological clock in Neurospora crassa.