University of Groningen

The clock in the cellMadeti Jyothi-Boesl, Cornelia

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Introduction

1CHAPTER

Circadian clocks

Organisms on earth are influenced by the alternation of day and night caused bythe rotation of the earth. Many – if not all - species have developed strategies tocope with daily changes in the environment, which are to a large extent system-atic and predictable. One of the evolved coping strategies is an endogenous,temporal program that regulates daily rhythms of physiology and behavior. Thisprogram functions as a clock to control endogenous daily rhythms with highprecision. The so-called circadian clock (from Latin “circa diem”, about a day) isendogenous, as was proven by experiments that released plants, animals, fungiand cyanobacteria into constant conditions yielding oscillations with an approxi-mately 24 h period. These are called circadian rhythms.

The internal clock, which regulates aspects of molecular biology, physiologyand social interactions across phyla, generates a temporal program that opti-mizes the sequence of daily events and prepares the organism for upcomingevents. Locomotor activity (Pittendrigh & Daan, 1976a) and photoreception areregulated by the circadian system (Freedman et al, 1999) in animals. Leaf move-ment (Darwin, 1880), growth (McClung, 1992; McClung et al, 1992; Quail,2002), opening of stomata, photosynthesis, cell metabolism (Lüttge, 2000) andgene regulation (Bognar et al, 1999) in plants or spore release in fungi (Merrow& Dunlap, 1994; Roenneberg & Merrow, 2001a) are examples of circadian‘behaviour’ in sessile organisms. Even unicellular organisms, e.g. cyanobacteria(Kondo et al, 1994) or the alga Gonyaulax polyedra, show circadian rhythmicity(Roenneberg & Morse, 1993): in a daily repeating cycle Gonyaulax travels fromthe ocean’s surface (during the day to gather photosynthetic energy) to greatdepths (during the night to harvest nutrients).

That the circadian clock also confers an adaptive advantage has beendescribed experimentally (DeCoursey et al, 2000; Johnson & Golden, 1999; Yanet al, 1998). Therefore selective pressure might be the main driving force for theevolution of circadian rhythms.

One of the reasons that circadian biology is a relevant question is that itconcerns our lives in immediately recognizable ways. There are hundreds ofexamples for how circadian rhythms control physiology (and hence potentiallylead to pathology) in humans by influencing circadian variations in hormonelevels, body temperature, mental and physical performance and pharmaco-kinetics (McFadden, 1988; Moore-Ede et al, 1982a; Moore-Ede et al, 1982b;Rocco et al, 1987).

The huge impact that circadian rhythmicity has on human biology and humansociety (Moore, 1997) can be seen in the example of shift work: about 20% of allemployees in developed countries work in night shifts. The problem here arises

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from the fact that during a night shift, the circadian clock-regulated physiology ofthe worker usually remains entrained to local time instead of adjusting to thenight shift. So, while physiology and psychology are saying “sleep!”, shift workersare forced to be active and alert (Scott, 2000; Waterhouse et al, 1997).

Clock mechanisms

Given the pervasive effect of circadian biology on life on earth, there is muchinterest in understanding molecular mechanisms using genetic tools. Though themolecular components of the circadian clock are significantly different amonganimals, plants, fungi and cyanobacteria, important features are common acrossphyla: According to Eskin’s model – which very simply describes circadiansystems for essentially all living things – circadian clocks consist of three majorfunctional components: input pathways, a rhythm generator (central oscillator)and output pathways (Fig. 1.1) (Eskin, 1979). Zeitgeber (German for time-giver)signals (the most important of which are light or/and temperature) are trans-duced to the central oscillator via the input pathway.

One of the most important zeitgebers for the circadian system is light. Whilephotoreception for vision requires high time- and high spatial- resolution, circa-dian photoreception must integrate the amount of light over the course of a day,comparable to a scintillation counter (Roenneberg & Foster, 1997; Roenneberg &Merrow, 2000). The use of additional light qualities to tell time-of-day (e.g.,wavelength/color) has not yet been described, even though both blue and redlight have been shown to feed into the circadian clock of plants as inputs.

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Zeitgebersignal

Inputpathway

Rhythmgenerator

Outputpathway

Figure 1.1 Cartoon of a basic circadian system, that can be described as input pathways thatperceive and transduce external, entraining signals from a zeitgeber (e.g. light, tempera-ture), and a rhythm generator or central oscillator that generates rhythms and regulatesvarious output pathways creating overt rhythms. (re-drawn after Roenneberg and Merrow,1998; Eskin, 1979).

Photoreceptors are the best characterized components of circadian inputpathways. They will either deliver a signal directly (e.g., WC-1 in Neurosporacrassa) or indirectly (e.g., melanopsin in mice) to the rhythm generator/centraloscillator. Concerning cellular clocks (the former case), there will be diversestrategies to carry signals that hold exogenous time information to the endoge-nous circadian program. Plants and fungi and some examples of transparentanimals (like Drosophila) can harvest light intracellularly, allowing for an effi-cient (i.e. comprising only few steps) transfer of information downstream.Concerning complex, hierarchical clocks (the latter example), animals receivelight exclusively through the eyes, and must send signals via the retino-hypothal-amic tract to the circadian pacemaker in the brain, called the suprachiasmaticnucleus (SCN) (Berson et al, 2002; Menaker, 2003). These examples show howbroadly the Eskin model applies to circadian systems.

The SCN is made up of neurons that display a circadian rhythm in geneexpression and neurophysiology, even when dissociated into single cells (Welshet al, 1995). Thus, the complexity of the system is revealed: the SCN functions asone oscillator that orchestrates others such as liver and lung clocks, but it itself ismade up of individual cells that show self-sustained free running and entrainablecircadian rhythms. Hence, the cellular system (input-oscillator-output) parallelsthat of the organism.

Concerning the molecular oscillator mechanism, animals with altered circa-dian properties were generated in mutagenesis experiments, eventually leadingto the discovery of clock genes. The first clock genes were discovered inDrosophila (Konopka & Benzer, 1971), with Neurospora following soon after(Feldman & Hoyle, 1973). Mammalian clock genes were finally revealed, too(King et al, 1997; Lowrey et al, 2000; Ralph & Menaker, 1988). The discoveredcomponents were modeled into a network based on genetic experiments. Thecartoons drawn of circadian networks (Reppert & Weaver, 2002; Schwartz et al,2001) reveal the complexity of circadian systems.

The central oscillator generates self-sustained rhythmicity (see below) of theclock, and then it is the job of output pathways to transduce this oscillatorysignal downstream. One mechanism is via gene expression (transcriptional regu-lation). This was first shown for Neurospora, specifically with the discovery ofclock controlled genes (ccg’s) (Loros et al, 1989). Microarray studies helped toidentify 145 ccg’s, whose predicted or known functions in development, metabo-lism, cell signaling and stress responses suggest a contribution of the circadianclock in a wide range of cell processes (Correa et al, 2003). An example of anoutput pathway in mammals is the one that leads to induction of the vip (vasoac-tive intestinal peptide)-gene (Hurst et al, 2002; Silver et al, 1999). This genetogether with the genes coding for other neuropeptides (vasopressin, cholecys-

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tokinin and substance P) are used as molecular readouts for circadian rhythmsand represent examples of ccg’s in mammals. Furthermore, more than a hundredgenes have been shown to be under direct clock control (Oishi et al, 2003), andmany more might be, as many microarray analyses in mammals (Delaunay &Laudet, 2002; Duffield, 2003), but also in Arabidopsis (Schaffer et al, 2001), andDrosophila (McDonald & Rosbash, 2001) suggest.

Clock properties

By analyzing the behavior of organisms, properties of their clocks can bededuced, shared features defined and characteristics of circadian clocks ingeneral described (Gwinner, 1986; Pittendrigh, 1960; Roenneberg & Merrow,1998). These include at least the following:● Rhythmicity.

There must be a quantifiable ‘up’ and ‘down’.● Circadian range.

The oscillation has a free-running period (FRP) in the circadian range inconstant conditions, i.e. one full cycle takes approximately 24 hours

● Robustness of the amplitude.The amplitude of the oscillation has to be sufficiently robust to drive outputrhythms

● Self-sustainment. In constant conditions (without zeitgebers) the rhythmicity continuesunabated, and is therefore self-sustained and endogenous. In some organismsthe endogenous circadian rhythmicity can continue over years. (Gwinner,1986; Richter, 1978)

● Entrainability. Circadian systems must be synchronizable to zeitgeber cycles, a propertycalled entrainment (Roenneberg et al, 2003). Hereby, the organism entrainswith a specific relationship, the phase angle, to external cues (like naturallight and temperature cycles, but also food or certain chemicals) keeping itsphysiological functions synchronized with the environment. Circadiansystems are able to entrain to cycle lengths different from 24 hours, but onlywithin a certain range. This property is called the range of entrainment,defined by the minimum and the maximum cycle length (called ‘T’) to whichthe system is still able to entrain. Being exposed to very short or long cyclelengths, an organism can show a frequency demultiplication (e.g., only oneconidial band every two 12 h cycles in Neurospora (Merrow et al., 1999) or afrequency multiplication ((Pittendrigh & Daan, 1976b), e.g., two conidial

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bands per cycle)). Entrainment differs from driven-ness (a reaction to a zeit-geber stimulus that is uniform in different zeitgeber conditions and does notnecessarily require a circadian clock) in being an active process where theinfluence of timing information on the circadian clock depends on the state ofthe circadian clock at the time of exposure.

● Temperature compensation.Circadian rhythms are highly temperature compensated, i.e. the period isroughly unaltered even when the (constant) temperatures applied vary over arather wide (10°C difference or more) range (Pittendrigh, 1954). Thisphenomenon extends to other parameters like pH inside a cell, nutrition andsocial interaction, as well, and could therefore be termed noise compensation.

The TTO (Transcription-Translation-Oscillator) as a model todescribe molecular clock mechanisms

As mentioned above, clock genes have been identified through mutant screensand they have been constructed in various configurations based largely on molec-ular genetic and genetic experiments. The predominant theory explaining themolecular mechanism of circadian rhythms is that of a Transcription-Translation-regulated-Oscillator (TTO). According to this theory some of the so-called“canonical clock genes” are rhythmically transcribed. Their protein productsnegatively feed back to regulate their own transcription (see Fig. 1.2). InNeurospora crassa, the negative element FREQUENCY (FRQ) feeds back via theWHITE-COLLAR-COMPLEX (WCC) to an element within the promoter region ofthe frq gene (Loros & Dunlap, 2001a). Via posttranslational protein modifica-tions, additional interlocked loops and nuclear import the molecular feedbackprocess is slowed down to occur once per circa 24h period (Lakin-Thomas,2006b). Recent modeling efforts (Roenneberg & Merrow, 2002) show that afreerunning period (FRP) of around 24 hours can be achieved by forming anetwork of several interconnected short-period TTOs. This model furthermoremimics all of the circadian clock properties mentioned above, suggesting that thisis one possibility for how molecular clocks are put together.

The TTO model, as it stands, still fails to explain much of the circadian mech-anism. Anomalies have been accumulating over the last years, including thedemonstration of rhythmicity in organisms with constant clock gene transcrip-tion, and rhythmicity in clock gene knock-out mutants (Bell-Pedersen et al, 2005;Loros & Feldman, 1986; Yang & Sehgal, 2001). It has been suggested thatrhythmic transcription may have other functions in the circadian system (e.g.participating in input and output pathways and providing robustness to the oscil-

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lations) and that circadian systems might use a non-circadian oscillator consistingof metabolic feedback loops, which acquires its circadian properties from addi-tional regulatory molecules such as the products of canonical clock genes (Lakin-Thomas, 2006b). Rhythmic de-/phosphorylation of clock components, a hypoth-esized ‘phoscillator’, might be common to all circadian systems, as suggested bythe pervasive and prominent role played by kinases and phosphatases in eukary-otic clocks (Merrow et al., 2006).

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PositiveElement

RNA

Clock protein B

Clock gene B

Clock gene A

NegativeElement

Clock protein A

RNA

Figure 1.2 A simplified circadian transcription/translation oscillator (TTO) model with 2interlocked loops: Clock gene A is transcribed into RNA and translated into protein. Clockprotein B positively regulates transcription of clock gene A. Clock protein A negatively regu-lates its own transcription by interfering with the positive effect of clock protein B. Clockprotein A also positively regulates production of clock protein 2, via either transcription ortranslation. Biosynthetic pathways are shown as solid lines with arrowheads. Positive influ-ence is shown as a circle with plus sign. Negative influence is shown as a dashed linetogether with a square filled with a minus sign. Nuclear/cytoplasmic compartmentation,phosphorylation, degradation pathways, environmental inputs, and outputs to clock-controlled genes and observed rhythms have been omitted. (re-drawn after Lakin-Thomas,2006)

The biology of Neurospora crassa

This thesis employs the model genetic organism Neurospora crassa as an experi-mental tool. Here, I describe basic features of Neurospora ecology.

The filamentous fungus N. crassa belongs to the phylum Ascomycota or ‘sacfungi’, due to the sac-like ascospore containers that are built during sexual propa-gation. Depending on environmental conditions this fungus can propagate asexu-ally or reproduce sexually. Most Neurospora species are haploid and spend mostof their life cycle in this state, because the diploid nuclei formed during thesexual phase are only transient (information taken from http://www.fgsc.net/Neurospora/sectionB2.htm). In its asexual stage, Neurospora forms a mycelium,a network of tubular filaments with multiple haploid nuclei (syncytial hyphae),whereas macroconidia (hereafter called conidia) are formed from aerial hyphae.Conidia do not survive for a long time in nature, but allow for rapid spreadingdue to their huge number. Upon environmental signals (e.g., hydration) conidiagerminate to form hyphae, which grow by tip extension and branch to formmycelia.

Historically, the genus Neurospora was thought to be predominant in moist,tropical and subtropical areas (information taken from http://www.fgsc.net/Neurospora/sectionB4.htm), but recent collection initiatives revealed thatNeurospora even habituates many temperate zones as far North as Alaska(Jacobson et al, 2004). In nature, Neurospora is one of the first colonists in areasof burnt-over vegetation (Jacobson et al, 2006; Perkins & Turner, 1988), and hasas such been described already in 1925 after the fire of Tokyo (Kitazima, 1925).It grows easily indoors on food or food waste, accounting for its commonly usedname “red bread mold”.

Evolving in and adapting to an exposed natural habitat, Neurospora hasdeveloped a variety of light responses including mycelial carotenoid production(Harding & Turner, 1981), formation of sexual structures (perithecia), theirphototropism (Degli-Innocenti et al, 1984; Harding & Melles, 1984), gene expres-sion (Arpaia et al, 1993; Collett et al, 2002; Crosthwaite et al, 1995a; Li & Schmid-hauser, 1995; Sommer et al, 1989) and entrainment of its circadian rhythm.

Neurospora crassa as a tool to study the circadian clock

Neurospora, which spawned the “One gene - One enzyme” hypothesis in the early1940s, is an excellent research tool for several reasons: ● It exists predominantly in a haploid state, e.g. no backcross is needed to

screen Neurospora progeny, which makes reverse and forward genetics easier.

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● It has a fully sequenced genome (Galagan et al, 2003) making molecularresearch systematic.

● A wealth of genetic and biochemical tools are available from the decades ofbasic genetics work that it has been used for.

● Additionally, Neurospora has a short generation time of a few weeks, andpotentially many progeny and much tissue can be grown in a few days.

However, the key for circadian research is the easily detectable circadian outputbehavior of Neurospora. The standard phenotypic assay to assess circadian rhyth-micity in Neurospora is the ‘race tube assay’, where cultures are grown on solidagar media in glass tubes. Thus grown, N. crassa shows a free running circadianrhythm in conidia formation (banding) of about 22h in darkness. The bands areeasily visualized on solid agar medium (Pittendrigh et al, 1959). Underentraining conditions (e.g., light or temperature cycles), the bands show adistinct phase relationship to external time (Chang & Nakashima, 1997; Lakin-Thomas & Brody, 2000; Merrow et al, 1999b); Roenneberg and Merrow, 2001).In constant light, discrete banding is mostly absent, with conidia being producedcontinuously (Roenneberg & Merrow, 2001; Pittendrigh et al, 1959).

Starting with mutant screening for strains with altered free running periods(FRP’s) in Neurospora, the first Neurospora clock gene, frequency (frq), was foundin the early 1970s (Feldman & Hoyle, 1973). Genetic analyses resulted in thedescription of more than 30 mutant alleles influencing the clock (Feldman &Dunlap, 1983; Lakin-Thomas et al, 1990). Further screening showed that sevenof these 30 mutants were alleles of the frq gene, conferring shorter (e.g. frq1,FRP=16.5 h) or longer period lengths (e.g. frq7, FRP=27 h) than the normal22h. Also, arrhythmic strains, like frq9, carrying a recessive, loss-of-functionmutation, were found (Loros & Feldman, 1986) or subsequently generated (e.g.,frq10, where almost the whole open reading frame (ORF) of the frq-gene isremoved). In the FRQ-deficient mutants light entrainment of the conidiationrhythm is impaired, indicating an additional role for the FRQ-protein in the lightinput system (Merrow et al, 1996; Merrow et al., 2003).

The expression of frq is regulated through transcriptional and posttranscrip-tional control mechanisms (Fig. 1.3). Briefly, the transcription of frq is positivelyregulated by the WHITE-COLLAR-1 (WC-1) and WHITE-COLLAR-2 (WC-2)proteins, and the FRQ protein feeds back negatively on its own transcription(Aronson et al, 1994; Crosthwaite et al, 1997a). Nuclear localization of FRQ isessential for rhythmicity. FRQ enters the nucleus as it is made and represses accu-mulation of frq mRNA (Luo et al, 1998). As mentioned, phosphorylation is acrucial player in the generation of circadian rhythms and FRQ is progressivelyphosphorylated throughout the day and controls the activity of WC-1 and WC-2

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by regulating their phosphorylation states (Schafmeier et al, 2005). If this phos-phorylation is inhibited experimentally, then the rate of FRQ turnover decreasesand period length increases (Liu et al, 2000). Several kinases, e.g CASEINKINASEs I and II or the calcium/calmodulin-dependent kinase (CAMK) as well asPROTEIN PHOSPHATASE 2A (PP2A) and PROTEIN PHOSPHATASE 1 (PP1),regulate the stability of the FRQ protein and the length of the free-running period(Görl et al, 2001; Liu et al, 2000; Yang et al, 2002).

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ccgs and otheroutput genes

frq

FRQWC-1 WC-2

VVD

WCC

FLO

wc-1 wc-2

vvd

Figure 1.3 The molecular circadian clock mechanism in Neurospora crassa. frq, wc-1, wc-2,vvd and their gene products form interconnected transcription/translation feedback loopsthat are essential for normal circadian behaviour in Neurospora. The levels of frequency (frq)RNA and FRQ protein depend on WHITE-COLLAR-1 (WC-1) and WHITE-COLLAR-2 (WC-2),which heterodimerize to form the White Collar Complex (WCC). WC-1 levels depend onFRQ. In constant darkness, expression of FRQ protein results in reduced frq RNA accumula-tion. The net effect is two interlinked regulatory loops. Light (shown as flashes) reaches thesystem through the WCC, which is essential for light responses in Neurospora. VVD gates thelight input to the system by interaction with WC-1. ccgs are clock-controlled-genes, some ofwhich are light-induced. The FLO (frq-less-oscillator) has been shown to exist, but compo-nents have not been described yet (Merrow et al., 1999; Loros & Feldman, 1986). Biosyntheticpathways are shown as solid lines with arrowheads. Positive influence is shown as a circle withplus sign. Negative influence is shown as a dashed line together with a square filled with aminus sign. (Re-drawn from (Merrow & Roenneberg, 2001; Heintzen et al, 2001).

Light reception in Neurospora

Several mutations have been reported to affect light responsiveness inNeurospora crassa (Linden et al, 1997). For example mutations in the whitecollar-1 and white collar-2 (wc-1 and wc-2) genes have been shown to impairlight-regulated carotenogenesis. Many other light responses are also abolished inthese mutants (Ninnemann, 1991; Perkins et al, 1982; Russo, 1988), whichmade the WC-1 and WC-2-proteins possible candidates for photoreceptors(Harding & Shropshire, 1980). That wc-1- and wc-2-mutants are also clockmutants was shown later (Crosthwaite et al., 1997) and makes any light inputpathway mutant potentially interesting to use for understanding the mechanismsof the circadian clock in Neurospora. An interesting case is the cytoplasmic bluelight photoreceptor and flavoprotein VIVID (VVD) (Schwerdtfeger & Linden,2003): although the mutant does not display a difference in free running periodin several conditions (Shrode et al, 2001), it regulates entrainment (Elvin et al,2005; Heintzen et al, 2001; Madeti, unpublished data), apparently through itsimpact on photoadaptation.

In addition to VVD, WC-1 and WC-2, the fully sequenced and annotatedgenome (Galagan et al, 2003) of Neurospora crassa (available at:http://www.broad.mit.edu/annotation/genome/neurospora/Home.html)provides additional photoreceptor candidates:● A possible green light photoreceptor protein with high homology to bacteri-

orhodopsin, novel opsin-1 (nop-1), was identified (Bieszke et al, 1999a;Bieszke et al, 1999b). NOP-1 binds retinal and forms a photochemically activepigment (Brown et al, 2001) but neither the physiological function, ingeneral, nor the involvement of this fungal opsin in the circadian system isknown.

● The same is true for the homolog to archaean rhodopsins, ORP-1 (OPSIN-RELATED-PROTEIN 1). Being regulated by heat-shock, it appears to beinvolved in responses to pH, organic solvents and stress (Nemcovic &Borkovich, 2003).

● The genome sequence also reveals a cryptochrome homologue (Daiyasu et al,2004) and two homologues of bacterial phytochromes (Catlett et al, 2003),possible candidates for Red/Far Red photoreceptor genes. phy1 mRNA levelshave been described to be under clock control (Froehlich et al, 2005),whereas knockouts of phy-1 and phy-2 are described not to have an effect onany -so far- known photoresponses. In the same publication, the putativeNeurospora blue light photoreceptor protein CRYPTOCHROME (nCRY) is saidto be photo-regulated by the WC-Complex (Froehlich et al, 2005), but itsfunction is not known yet.

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● Also, a homolog of the Aspergillus nidulans gene velvet is present inNeurospora. In Aspergillus, this gene is involved in the signal transduction ofboth red and blue light (Yager et al, 1998). The presence of three genes(velvet, phy-1 and phy-2) possibly involved in red light photoreception issurprising given the fact that red light responses have not been described yetin Neurospora – it is thought to be blind for red light (e.g.(Dharmananda,1980; Froehlich et al, 2005) - and suggests that the photobiology inNeurospora might be more complex than recognized, so far.

Action spectra show how much light of a given wavelength is required forsynthesis of mycelial carotenoids (De Fabo et al., 1976), phase shifting of theconidiation rhythm (Dharmananda, 1980), photosuppression of self-sustainedrhythmicity in conidial band formation (Sargent and Briggs, 1967) and the invitro light induced binding of the WC-1 protein to the promotor of the frq gene(Froehlich et al., 2002). In Neurospora crassa, the aforementioned responsescould not be stimulated by wavelengths longer than 520 nm and all of themshow a maximal activity around 460 nm with sensitivity extending into the UVAregion (De Fabo et al, 1976; Froehlich et al, 2002; Sargent & Briggs, 1967). Allthis indicates that flavins or carotenoids are involved as chromophores. However,since a triple albino mutant (al-1, al-2, al-3) containing less than 0.5% of wtcarotenoids is still able to exhibit normal sensitivity for other light responses,photoreception in N. crassa is probably not based on carotenoids (Russo, 1988).Furthermore, mutants deficient in biosynthesis of riboflavin exhibit a decreasedlight sensitivity, which makes flavin species (as have been identified cofactors forWC-1 and VVD) the best candidates for Neurospora photoreceptor chromophores.

Prospect of this thesis

As a young student I was fascinated by fungi and was inspired by a lecture ofProfessor Agerer from the Botanical Institute of the University of Munich. Hedescribed the complex factors interacting to make mushrooms grow in theautumn. Still, I would have never imagined writing a thesis on fungi (especiallythe „red bread mold“ Neurospora crassa) and the complex factors that influencetheir daily timing system. Even more improbable – even though my family tookme on mushroom collection trips as a child - was that one day I would be part ofan international team searching for the first wild Neurospora isolates intemperate climates in Europe.

In my first months as a doctoral student, I had the rare opportunity to see theobject of my further studies in nature after the huge and devastating fires inEurope in 2003. Neurospora crassa is a colonist found on burnt trees after forest

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fires and was, until 2003, apart from reports from French bakeries, not describedin temperate climates, but rather designated to be an inhabitant of the tropicsand subtropics. Chapter 2 of this thesis describes the findings of the 2003 collec-tion trip in Europe and compares the strain prevalence and growth patterns tothose of the previously known strains.

Chapter 3 describes chronobiological experiments done with a collection ofwild type Neurospora crassa strains from the whole world. These experimentsinclude the newly collected strains from Europe together with the older onesfrom the rest of the world. Strains collected from different latitudes were used toassess the correlations of latitude-of-origin and phase of entrainment or freerunning period.

Chapter 4 also characterizes clock properties in wild type strains, but thistime, with an eye to genetics. Two wild type strains were crossed and 200 to 500progeny were selected for a quantitative trait analysis experiment. While ourcollaborators at U. C. Berkeley generated genetic markers for the strains, weworked on phenotyping them for the circadian clock. Earlier QTL-studies in miceand Arabidopsis indicated that more genes than expected were involved in theclock quantitative traits phase and free-running period. Given this and Neurosporacrassa’s optimal prerequisites for a QTL-study, it is surprising that no earlierstudies existed.

The following chapters set up much of the remainder of the thesis. Whereasthe work with wild type strains, especially using QTL, is one approach to findnovel clock genes, using entrainment for phenotyping a mutant is another way toreach the same end. This approach is ongoing in the lab. For my thesis, however,I took this a step further, combining a functional genetics approach with usingentrainment to reveal new clock genes.

Chapter 5 describes assaying a cryptochrome mutant in Neurospora crassa. Itdisplays the same free running period as the wild type/background strain bdA,but it shows differences in the phase of entrainment in blue or white light cycles.What does this tell us about entrainment and the dogma of „longer period-laterentrainment and shorter period –earlier entrainment“?

In Chapter 6 formal entrainment properties of (a lab strain of) Neurosporacrassa are discussed.

In Chapter 7, I describe the entrainment of N. crassa in a ‚circadian surface’using temperature as a zeitgeber.

Chapter 8 reviews a recent publication describing the cloning of the ‚band’gene. Having been used as a basically universal standard and background strainfor circadian experiments, the discovery of its identity as a ras-1-mutant raisesmany questions about its applicability. The most important –and still not solved-question is: is bd/ras-1 a clock mutant itself?

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Chapter 9, a review on circadian clocks in human fibroblasts gives an insightinto relatively new circadian research in human tissues. Do in vitro experimentsusing human fibroblasts together with questionnaires have the potential to makethe challenging bunker or constant routine experiments unneccessary? IsNeurospora a parallel relative to human fibroblasts, with respect to circadiansystems? Much of the work that was built up using Neurospora was done beforetissue culture systems were developed for mammalian cells.

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Berson DM, Dunn FA, Takao M (2002) Phototransduction by retinal ganglion cells that setthe circadian clock. Science 295: 1070-1073

Bieszke JA, Braun EL, Bean LE, Kang S, Natvig DO, Borkovich KA (1999a) The nop-1 gene ofNeurospora crassa encodes a seven transmembrane helix retinal-binding protein homolo-gous to archaeal rhodopsins. Proc Natl Acad Sci USA 96(14): 8034-8039

Bieszke JA, Spudich EN, Scott KL, Borkovich KA, Spudich JL (1999b) A eukaryotic protein,NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactivepigment. Biochem 38(43): 14138-14144

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