Rhythmic Conidiation in Constant Light in Vivid Mutants of Neurospora crassa

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on the surface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constant illumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we report that strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involved in photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvd strains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, and the presence of other mutations. Temperature compensation of the period also depends on light intensity. Dark pulses given in L/L shift the phase of the rhythm. Shifts from L/L to D/D show unexpected after effects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2–3 days in D/D. The rhythm in L/L requires the white collar (wc-1) gene, but not the frequency (frq) gene. FRQ protein shows no rhythm in L/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).     

Temperaure Compensation and Memberane Composition in Neurospora Crassa

The link between temperature compensation of the circadian rhythm and temperature-induced adjustment of membrane composition in Neurospora crassa is briefly reviewed. In common with most organisms, Neurospora responds to changes in growth temperature by adjusting its lipid composition, primarily by increasing the degree of unsaturation of its fatty acids at low temperature. This may result in maintenance of either membrane fluidity or phase transition behavior over a range of temperatures. In Neurospora, there are three mutations (frq, eel, and chol-1) that affect temperature compensation of the circadian rhythm; cel and chol-1 are defective in lipid synthesis, and frq interacts with the other two in double-mutant strains. This suggests that lipid metabolism may play a role in temperature compensation of the rhythm, and that the FRQ gene product may also be involved in membrane function, either in regulating lipid composition or as a sensor responding to changes in lipid composition. (Chronobiology International, 14(5), 445–454, 1997)     

The frq Locus in NEUROSPORA CRASSA: A Key Element in Circadian Clock Organization

Four new circadian clock mutants of Neurospora crassa have been isolated that alter the period length of the circadian conidiation rhythm. Three of these are at the frq locus on linkage group VIIR, where four other clock mutants are located. In contrast to wild type, which has a period length of 21.6 hr, frq-6 has a period length of 19 hr, while frq-7 and frq-8 have period lengths of 29 hr and represent the largest effects of any single gene mutants on circadian periodicity. Thus, seven mutants have now been isolated that map to the frq locus, with period lengths ranging from 16.5 to 29 hr, and each mutant alters clock periodicity by an integral multiple of 2.5 hr. In addition, all frq mutants show incomplete dominance in heterokaryons. The large percentage of clock mutants that map to this locus, coupled with their unique properties, suggests that the frq locus plays an important role in clock organization.—The fourth mutant, designated chrono (chr), has a period length of 23.5 hr, shows incomplete dominance and is unlinked to either of the previously identified clock loci, frq or prd (formerly called frq-5). Double mutants between various combinations of clock mutants show additive effects and indicate no significant gene interaction among mutants at these three loci.     

Complementation Analysis of Linked Circadian Clock Mutants of NEUROSPORA CRASSA

A fourth mutant of Neurospora crassa, designated frq-4, has been isolated in which the period length of the circadian conidiation rhythm is shortened to 19.3 ± 0.3 hours. This mutant is tightly linked to the three previously isolated frq mutants, and all four map to the right arm of linkage group VII about 10 map units from the centromere. Complementation tests suggest, but do not prove, that all four mutations are allelic, since each of the four mutants is co-dominant with the frq⁺ allele—i.e., heterokaryons have period lengths intermediate between the mutant and wild-type—and since heterokaryons between pairs of mutants also have period lengths intermediate between those of the two mutants.     

pH HOMEOSTASIS OF THE CIRCADIAN SPORULATION RHYTHM IN CLOCK MUTANTS OF NEUROSPORA CRASSA

The influence of environmental (extracellular) pH on the sporulation rhythm in Neurospora crassa was investigated for wild-type (frq⁺) and the mutants chr, frq¹, frq⁷, and frq⁸. In all mutants, including wild type, the growth rate was found to be influenced strongly by extracellular pH in the range 4–9. On the other hand, for the same pH range, the period length of the sporulation rhythm is little influenced in wild type, chr, and frq¹. A loss of pH homeostasis of the period, however, was observed in the mutants frq⁷ and frq⁸, which also are known to have lost temperature compensation. Concerning the influence of extracellular pH on growth rates, a clear correspondence between growth rates and the concentration of available H₂PO₄^? ion has been found, indicating that the uptake of H₂PO₄^? may be a limiting factor for growth under our experimental conditions. The loss of pH compensation in the frq⁷ and frq⁸ mutants may be related to less easily degradable FRQ^7,8 proteins when compared with wild-type FRQ. Results from recent model considerations and experimental results predict that, with increasing extra-and intracellular pH, the FRQ⁷ protein degradation increases and should lead to shorter period lengths. (Chronobiology International, 17(6), 733–750, 2000)     

Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock

We report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in Neurospora crassa. This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the frq promoter drives luciferase exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15-h period rhythms at high temperatures, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve.     

Effects of light-dark cycles on the circadian conidiation rhythm inNeurospora crassa

The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq ⁺ and clock mutantsfrq ⁺,frq ³,frq ⁷,frq ⁹ andfrq ¹¹. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq ⁺,frq ³ andfrq ⁷ strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in all of the strains except thefrq ¹¹ strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq ¹ strain in all light-dark cycles examined and in thefrq ⁺ andfrq ³ strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq ⁷ strain was dependent on the light to dark transition when the light period was shorter than 10 hr.     

The circadian conidiation rhythm inNeurospora crassa

The filamentous fungusNeurospora crassais one of the best organisms for analysing the molecular basis of the circadian rhythm observed in asexual spore formation, conidiation. Many clock mutants in which the circadian conidiation rhythm has different characteristics compared to those in the wild-type strain have been isolated since the early 1970s. With the cloning of one of these clock genes,frq, the molecular basis of the circadian clock inNeurosporahas become gradually clearer. Physiological and pharmacological studies have also contributed to our understanding of the physiological basis of the circadian clock inNeurospora. These studies strongly indicate that the circadian clock is based on or is closely related to a network of metabolic processes for cellular activities. Based on these studies, it may be possible to isolate new types of clock mutants which should contribute to a better understanding of the molecular basis of the circadian clock inNeurospora.     

Genetics of Circadian Clocks

The isolation of circadian clock mutants in Neurospora crassa and Drosophila melanogaster have identified numerous genes whose function is necessary for the normal operation of the circadian clock. In Neurospora many of these mutants map to a single locus called frq, whose properties suggest that its gene product is intimately involved in clock function. In Drosophila mutations at the per locus also suggest a significant role for the product of this gene in the insect clock mechanism. The per gene has been cloned and its gene product identified as a proteoglycan, most likely a membrane protein involved in affecting the ionic or electrical properties of cells in which it is located. Future progress in elucidating the mechanisms of circadian clocks are likely to come from continued analysis of clock mutants, both at the genetic and molecular levels.     

Circadian period lengths of lipid synthesis mutants (cel,chol-1) of Neurospora show defective temperature,but intact pH-compensation

The influence of extracellular pH on the circadian sporulation rhythm of Neurospora crassa has been investigated for the mutants chol-1 and cel. Both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. The chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. We investigated the possible correlation between loss of temperature- and pH-compensation in cel and chol-1 similar to the correlation found earlier for the frq⁷ mutant. Our results show that the cel and the chol-1 mutants, although defective in temperature-compensation have an intact pH-compensation of their circadian rhythms. At present, the products of the frq-locus are the only components of the clock that affect the sporulation rhythm of Neurospora both through pH- and temperature-compensation.     

Structure of the frequency‐interacting RNA helicase: a protein interaction hub for the circadian clock

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ‐interacting RNA Helicase (FRH) rhythmically represses gene expression by the white‐collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ‐interacting region at the FRH N‐terminus has variable structure in the absence of FRQ. A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ. A V142G substitution near the N‐terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N‐terminus and KOW module.