The Goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of Neurospora crassa

The Goodwin model is a negative feedback oscillator which describes rather closely the putative molecular mechanism of the circadian clock of Neurospora and Drosophila. An essential feature is that one or two clock proteins are synthesized and degraded in a rhythmic fashion. When protein synthesis in N. crassa (wild-type frq+and long-period mutant frq7) was inhibited by continuous incubation with increasing concentrations of cycloheximide (CHX) the period of the circadian sporulation rhythmicity is only slightly increased. The explanation of this effect may be seen in the inhibition of protein synthesis and protein degradation. In the model, increasing inhibition of both processes led to very similar results with respect to period length. That protein degradation is, in fact, inhibited by CHX is shown by determining protein degradation in N. crassa by means of pulse chase experiments. Phase response curves (PRCs) of the N. crassa sporulation rhythm toward CHX which were reported in the literature and investigated in this paper revealed significant differences between frq+and the long period mutants frq7and csp -1 frq7. These PRCs were also convincingly simulated by the model, if a transient inhibition of protein degradation by CHX is assumed as well as a lower constitutive degradation rate of FRQ-protein in the frq7/ csp -1 frq7mutants. The lower sensitivities of frq7and csp -1 frq7towards CHX may thus be explained by a lower degradation rate of clock protein FRQ7. The phase shifting by moderate temperature pulses (from 25 to 30 degrees C) can also be simulated by the Goodwin model and shows large phase advances at about CT 16-20 as observed in experiments. In case of higher temperature pulses (from 35 to 42 or 45 degrees C=heat shock) the phase position and form of the PRC changes as protein synthesis is increasingly inhibited. It is known from earlier experiments that heat shock not only inhibits the synthesis of many proteins but also inhibits protein degradation. Taking this into account, the Goodwin model also simulates the PRCs of high temperature (heat shock) pulses.     

The wild-type circadian period of Neurospora is encoded in the residual network of the null frq mutants

We model theoretically the response of the widely studied circadian oscillator of Neurospora crassa to inactivation of the frq gene. The resulting organism has been termed "arrhythmic" under constant conditions. Under entrainment to periodic temperature cycles Roenneberg, Merrow and coworkers have shown that the phase angle at which spore formation occurs depends on the entrainment period, curiously even in the null frq mutants (frq9 and frq10). We show that such a response does not imply the presence of a self-sustained free-running oscillator. We derive a simple candidate model (a damped harmonic oscillator) for the null frq mutants that successfully reproduces the observed phase angle response. An endogenous period of 21 h for the damped harmonic oscillator coincides with the endogenous period of wild-type Neurospora. This suggests that the (noise driven) "residual system" present in the mutants may have a significant timekeeping role in the wild-type organism. Our model (with no change of parameters) was then used to investigate spore formation patterns under constant conditions and reproduces the corresponding experimental data of Aronson et al. (Proc. Natl. Acad. Sci. USA 91 (1994) 7683.)     

Modeling Temperature Compensation in Chemical and Biological Oscillators

All physicochemical and biological oscillators maintain a balance between destabilizing reactions (as, for example, intrinsic autocatalytic or amplifying reactions) and stabilizing processes. These two groups of processes tend to influence the period in opposite directions and may lead to temperature compensation whenever their overall influence balances. This principle of “antagonistic balance” has been tested for several chemical and biological oscillators. The Goodwin negative feedback oscillator appears of particular interest for modeling the circadian clocks in Neurospora and Drosophila and their temperature compensation. Remarkably, the Goodwin oscillator not only gives qualitative, correct phase response curves for temperature steps and temperature pulses, but also simulates the temperature behavior of Neurospora frq and Drosophila per mutants almost quantitatively. The Goodwin oscillator predicts that circadian periods are strongly dependent on the turnover of the clock mRNA or clock protein. A more rapid turnover of clock mRNA or clock protein results, in short, a slower turnover in longer period lengths. (Chronobiology International, 14(5), 499–510, 1997)     

sn-1,2-diacylglycerol levels in the fungus Neurospora crassa display circadian rhythmicity

The fungus Neurospora crassa is a model organism for investigating the biochemical mechanism of circadian (daily) rhythmicity. When a choline-requiring strain (chol-1) is depleted of choline, the period of the conidiation rhythm lengthens. We have found that the levels of sn-1,2-diacylglycerol (DAG) increase in proportion to the increase in period. Other clock mutations that change the period do not affect the levels of DAG. Membrane-permeant DAGs and inhibitors of DAG kinase were found to further lengthen the period of choline-depleted cultures. The level of DAG oscillates with a period comparable to the rhythm of conidiation in wild-type strains, choline-depleted cultures, and frq mutants, including a null frq strain. The DAG rhythm is present at the growing margin and also persists in older areas that have completed development. The phase of the DAG rhythm can be set by the light-to-dark transition, but the level of DAG is not immediately affected by light. Our results indicate that rhythms in DAG levels in Neurospora are driven by a light-sensitive circadian oscillator that does not require the frq gene product. High levels of DAG may feed back on that oscillator to lengthen its period.     

Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency.

The Neurospora crassa frequency locus encodes a 989 amino acid protein that is a central component, a state variable, of the circadian biological clock. We have determined the sequence of all or part of this protein and surrounding regulatory regions from additional fungi representing three genera and report that there is distinct, preferential conservation of the frequency open reading frame (ORF) as compared with non-coding sequences. Within the coding region, many of the domain hallmarks of the N. crassa protein are highly conserved, especially an internal region bearing the causative mutations in frq1 and frq7, the most extreme alleles in the frequency allelic series. Despite considerable diversity among the strains analyzed in terms of morphology, growth, circadian clock output and frq sequence, the ORF from the most distantly related fungus included in this study (Sordaria fimicola) rescues rhythmicity in a N.crassa frequency null strain. Both sequence conservation, and the ability of frequency from a genus displaying one developmental program to complement circadian defects in a separate genus with a distinct, clock-regulated developmental program, are consistent with a central role of the frequency gene product in a general circadian oscillator capable of controlling diverse outputs in a variety of systems.     

Genetic interactions between clock mutations in Neurospora crassa: can they help us to understand complexity?

Recent work on circadian clocks in Neurospora has primarily focused on the frequency (frq) and white-collar (wc) loci. However, a number of other genes are known that affect either the period or temperature compensation of the rhythm. These include the period (no relationship to the period gene of Drosophila) genes and a number of genes that affect cellular metabolism. How these other loci fit into the circadian system is not known, and metabolic effects on the clock are typically not considered in single-oscillator models. Recent evidence has pointed to multiple oscillators in Neurospora, at least one of which is predicted to incorporate metabolic processes. Here, the Neurospora clock-affecting mutations will be reviewed and their genetic interactions discussed in the context of a more complex clock model involving two coupled oscillators: a FRQ/WC-based oscillator and a 'frq-less' oscillator that may involve metabolic components.     

Loss of temperature compensation of circadian period length in the frq-9 mutant of Neurospora crassa

A new circadian clock mutant of Neurospora crassa has been isolated, whose most distinctive characteristic is the complete loss of temperature compensation of its period length. The Q10 of the period length was found to be equal to about 2 in the temperature range from 18 degrees to 30 degrees C. The period length was also found to be dependent on the composition of the medium, including the nature and concentration of both the carbon source and the nitrogen source. Although the rate of the clock and the growth rate were directly related when affected by varying the temperature, they were inversely related when altered by changing the composition of the medium. Therefore, the mutation has not simply coupled clock rate to growth rate in this strain. The mutation maps to the frq locus, where seven other clock mutations previously studied also map. Therefore, this mutant has been called frq-9. Since several of the other frq mutants show partial loss in temperature compensation, it is suggested that the frq gene or its product is closely related to the temperature compensation mechanism of the circadian clock of Neurospora.     

The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa

The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature.     

A Recessive Circadian Clock Mutation at the frq Locus of NEUROSPORA CRASSA

A circadian clock mutant of Neurospora crassa, the most distinctive characteristic of which is the complete loss of temperature compensation of its period length, maps to the frq locus where seven other clock mutants have previously been mapped. This mutant, designated frq-9, is recessive to the wild-type allele and to each of the other frq mutants; thus, it differs from the other mutants, which show incomplete dominance to wild type and to each other. Complementation analysis suggests either that the frq locus is a single gene or that frq-9 is a deletion that overlaps adjacent genes. Preliminary efforts at fine structure mapping have indicated that recombination between certain pairs of frq mutations is less than 0.005%, a distance consistent with the locus being a single gene. The recessive nature of frq-9, coupled with complete loss of temperature compensation, suggests that this mutant may represent the null phenotype of the locus and that the frq gene is involved in the temperature compensation mechanism of the clock.--Genetic mapping studies have placed the frq locus on linkage group VIIR, midway between oli (oligomycin resistance) and for (formate auxotrophy), about 2 map units from each, and clearly indicate that frq and oli are separate genes.     

The Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity. We conclude that there may be many gene products involved in the clock mechanism, there may be multiple interacting oscillators comprising the clock mechanism, there may be feedback from output pathways onto the oscillator(s) and from the oscillator(s) onto input pathways, and there may be several independent clocks coexisting in one organism. Thus even a relatively simple lower eukaryote can be used to address questions about a complex, networked circadian system.     

Isolation and Characterization of a Temperature-Sensitive Circadian Clock Mutant of Neurospora Crassa

A new circadian clock mutant has been isolated in Neurospora crassa. This new mutation, called period-6 (prd-6), has two features novel to known clock mutations. First, the mutation is temperature sensitive. At restrictive temperatures (above 21°) the mutation shortens circadian period length from a wild-type value of 21.5 hr to 18 hr. At permissive temperatures (below 21°) the mutant has a 20.5-hr period length close to that of the wild-type strain. Second, the prd-6 mutation is epistatic to the previously isolated clock mutation period-2 (prd-2). This epistasis is unusual in that the prd-2 prd-6 double mutant strain has an 18-hr period length at both the restrictive and permissive temperatures. That is, the temperature-sensitive aspect of the phenotype of the prd-6 strain is lost in the prd-2 prd-6 double mutant strain. This suggests that the gene products of the prd-2 and prd-6 loci may interact physically and that the presence of a normal prd-2(+) protein is required for low temperature to ``rescue' the prd-6 mutant phenotype. These results, combined with our recent finding that prd-2 and some alleles of the frq gene show genetic synergy, suggest that it may be possible to establish a more comprehensive model of the Neurospora circadian clock.     

A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora crassa.

FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identified Neurospora homologs of casein kinase 1 epsilon. CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frq Delta PEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frq Delta PEST-1 RNA and FRQ Delta PEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components.     

Epistatic and synergistic interactions between circadian clock mutations in Neurospora crassa

We identified a series of epistatic and synergistic interactions among the circadian clock mutations of Neurospora crassa that indicate possible physical interactions among the various clock components encoded by these genes. The period-6 (prd-6) mutation, a short-period temperature-sensitive clock mutation, is epistatic to both the prd-2 and prd-3 mutations. The prd-2 and prd-3 long-period mutations show a synergistic interaction in that the period length of the double mutant strain is considerably longer than predicted. In addition, the prd-2 prd-3 double mutant strain also exhibits overcompensation to changes in ambient temperature, suggesting a role in the temperature compensation machinery of the clock. The prd-2, prd-3, and prd-6 mutations also show significant interactions with the frq(7) long-period mutation. These results suggest that the gene products of prd-2, prd-3, and prd-6 play an important role in both the timing and temperature compensation mechanisms of the circadian clock and may interact with the FRQ protein.