Circadian rhythms in Neurospora crassa: clock mutant effects in the absence of a frq-based oscillator

In Neurospora, the circadian rhythm is expressed as rhythmic conidiation driven by a feedback loop involving the protein products of frq (frequency), wc-1 (white collar-1), and wc-2, known as the frq/wc (FWC) oscillator. Although strains carrying null mutations such as frq(10) or wc-2Delta lack a functional FWC oscillator and do not show a rhythm under most conditions, a rhythm can be observed in them by the addition of geraniol or farnesol to the media. Employing this altered media as an assay, the effect of other clock mutations in a frq(10)- or wc-2Delta-null background can be measured. It was found that the existing clock mutations fall into three classes: (1) those, such as prd-3 or prd-4 or frq(1), that showed no effect in a clock null background; (2) those, such as prd-1 or prd-2 or prd-6, that did have a measurable effect in the frq(10) background; and (3) those, such as the new mutation ult, that suppressed the frq(10) or wc-2Delta effect, i.e., geraniol/farnesol was not required for a visible rhythm. This classification suggests that some of the known clock mutations are part of a broader multioscillator system.     

Circadian rhythms in Neurospora crassa: membrane composition of a mutant defective in temperature compensation

The cel mutant of Neurospora, partially blocked in fatty acid synthesis and lacking temperature compensation of its circadian rhythm below 22 degrees C, had a phospholipid fatty acid composition in liquid shaker culture distinctly different from that of a cel+ control strain. During growth, cel+ exhibited a reproducible increase in its linoleic acid level from about 32 to a plateau at 63 mol%, and a corresponding decrease in its linolenic acid level from about 40 to a plateau at 10 mol%. The level of palmitic acid was constant at 19 mol%. In the cel strain, the linoleic acid level was constant at 54 mol% while the palmitic acid level increased from about 12 to about 23 mol%. Supplementation with palmitic or linoleic acids altered the patterns of fatty acid composition of cel, but did not affect the pattern of cel+. Altered fatty acid composition cosegregated with the cel marker. The mitochondrial phospholipids of cel in liquid culture also had abnormal fatty acid composition, as did the whole mycelial phospholipids on solid medium. These results are consistent with the involvement of membrane homeostasis in the temperature compensation of circadian rhythms.     

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

Employing a fatty acid-requiring strain (bd csp cel) of Neurospora crassa, the 21.5-h period of the circadian spore-forming rhythm was manipulated by fatty acid supplementation. The addition to the medium of an unsaturated fatty acid (oleic, linoleic, or linolenic acid) lengthened the period to 26, 40, or 33 h, respectively. Ther period-lengthening effect of linoleic acid was proportional to its concentration up to 1.3 X 10(-4) M, and also was reversed by the addition to the medium of a saturated fatty acid, palmitic acid. None of these period-lengthening effects was observed in the prototrophic strain (bd csp cel+).     

Circadian rhythms of performance: new trends

This brief review is concerned with how human performance efficiency changes as a function of time of day. It presents an overview of some of the research paradigms and conceptual models that have been used to investigate circadian performance rhythms. The influence of homeostatic and circadian processes on performance regulation is discussed. The review also briefly presents recent mathematical models of alertness that have been used to predict cognitive performance. Related topics such as interindividual differences and the postlunch dip are presented. (Chronobiology International, 17(6), 719-732, 2000)     

Circadian rhythms in Neurospora crassa: Dynamics of the clock component frequency visualized using a fluorescent reporter

The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organism’s circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ–mCherryNC (FRQ–mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100×). Fluorescence of FRQ–mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150–200 μm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ protein’s nuclear trafficking may be more complex than previously realized.     

Circadian rhythms in Neurospora crassa: oscillation in the level of an adenine nucleotide.

In a mutant strain (bd) of Neurospora, the biological clock is visibly expressed at the growing front of a mycelial mat by sequential periods of conidiating (spore-forming) and non-conidiating growth. The edges (8 mm) of the mycelium at different ages were sampled during a 31 h period, and the adenine nucleotide levels were enzymatically assayed. In the edge region, the total adenosine 5'-monophosphate (AMP) level showed an oscillation, with a minimum of 0.5 mumol/g (residual dry weight) and a maximum of 6.0 mumol/g. The total adenosine 5'-triphosphate level and the total adenosine 5'-diphosphate level showed no obvious oscillation. The oscillation in AMP content had many of the properties of a circadian rhythm. Its period was about 22 h long, it was phase-shifted by light, and it was damped out by continuous illumination. The oscillation in AMP level led to an oscillation in the overall cellular energy charge from 0.65 to 0.93. However, the energy charge calculation does not take into account any possible compartmentalization of AMP, and therefore must be interpretated cautiously. It is suggested that the underlying cause of the oscillation in AMP level could be a rhythmic, partial uncoupling of mitochondrial oxidative phosphorylation.     

Circadian rhythms in Neurospora crassa: a clock mutant, prd-1, is altered in membrane fatty acid composition

The fatty acid compositions of the phospholipids of Neurospora crassa mutants with altered periods were determined to test the possibility that some of these mutants might have altered membrane composition. In liquid shaker culture in constant light the bd (band) strain, which has a normal period (21.6 h), exhibited a growth-dependent increase in linoleic acid content and a decrease in linolenic acid content during early log phase growth. By late log phase, fatty acid composition was essentially constant. The phospholipid fatty acid compositions of bd strains containing mutations at the frq (frequency) and chr (chrono) loci were indistinguishable from that of the bd strain under the conditions used. However, a bd strain containing a mutation at the prd-1 (period) locus, as well as prd-1 segregants from a cross of this strain to a bd strain, had altered patterns of growth-dependent fatty acid composition; linoleic and linolenic acid contents changed more slow than in the bd strain and continued to change throughout growth. In addition, the fatty acid composition of a bd prd-1 strain on solid medium differed from that of the bd strain. It is proposed that the prd-1 mutation leads to altered membrane homeostasis, which in turn affects circadian rhythmicity because some or all components of the rhythm-generating system are membrane-localized.     

Circadian rhythms in plants: a millennial view

Circadian rhythms are endogenous rhythms with periods of approximately 24 h. These rhythms are widespread both within any given organism and among diverse taxa. As genetic and molecular biological studies, primarily in a subset of model organisms, have begun to identify the components of circadian systems, there is optimism that we will soon achieve a detailed molecular understanding of circadian timing mechanisms. Although plants have provided many examples of rhythmic outputs, and our understanding of photoreceptors of circadian input pathways is well-advanced, plants have lagged behind other groups of organisms in the identification of components of the central circadian oscillator. However, there are now a number of promising candidates for components of plant circadian clocks, and it seems probable that we will soon know the details of a plant central oscillator. Moreover, there is also accumulating evidence that plants and other organisms house multiple circadian clocks, both in different tissues and, quite probably, within individual cells. This provides an unanticipated level of complexity with the potential for interaction among these multiple oscillators.     

Circadian rhythms: perturbing a food-entrained clock

When food is scarce, a food-entrainable circadian clock coordinates mammalian activity rhythms with a predictable daily mealtime. Neural and molecular substrates of this circadian function have long eluded localization, but new studies suggest a critical role for a familiar circadian clock gene.     

Circadian rhythms in cockroaches

Summary The driving oscillator, which mediates circadian locomotor rhythms in cockroaches, appears to reside in the protocerebrum of the brain. The evidence indicates that the optic lobes are crucial elements in this circadian system, and that control of rhythmicity is mediated through electrical, rather than hormonal, channels. Lesions were placed at various sites within the optic lobes in order to localize the areas controlling rhythmicity. It appears that the two innermost synaptic areas (the lobula and the medulla) constitute the crucial optic lobe elements. The outer synaptic area of the optic lobe (the lamina) is not necessary for the expression of rhythmicity, but does function as a coupling through which light cycles, transduced by the compound eyes, entrain the circadian clock.I would like to thank both Dr. Sue Binkley for her helpful comments in the preparation of this report, and Mr. Eli Levine for his assistance in photography. Support for this research was provided by a grant from the National Science Foundation (NS GB-30497).     

Circadian rhythms in Neurospora crassa: the effects of point mutations on the proteolipid portion of the mitochondrial ATP synthetase

Summary Five oligomycin-resistant (oli ^r) mutant strains of Neurospora crassa were analyzed for their growth rate and for the periodicity of their circadian rhythm. The most resistant strains had periods of 18–19 h while the least resistant strain had a normal period of 21.0 h. There was a rough correlation between the in vivo degree of oligomycinresistance and the amount of change in the period. Several of the oli ^r mutations have been previously described by Sebald et al. (1977) in terms of known amino acid changes in the primary structure of the proteolipid, or DCCD-binding protein, found in the F₀ membrane portion of the mitochondrial ATP synthetase. Amino acid changes in the structure of this protein are reported here for two other oli ^r mutations. The proteolipid isolation procedures were slightly modified to include a delipidation step, and an HPLC procedure was developed to separate the hydrophobic peptides of this protein. Analysis of heterocaryons carrying both the oli ^r and oli ^s markers indicated that the oli ^r and oli ^s mutations were codominant to each other in terms of period and growth rate. The changes in the primary structure of this DCCD-binding protein reported here are the first known examples of changes in the primary structure of a protein which alter the period of a circadian rhythm.     

Circadian complexes: Circadian rhythms under common gene control

Summary Circadian rhythms for food and water consumption were measured in five inbred strains of mice under a photoperiod of 16 h light and 8 h dark (16:8 LD), and under constant light (LL).Significant strain differences were observed which indicate that a common gene difference, or set of differences inMus musculus influences both the phase angle () associating the rhythms with the light-dark cycle, and the periods (_LL) of circadian rhythms for food and water consumption. The biological clock mechanism influenced by this genetic variance is common to both food and water circadian rhythms, and differs among the five inbred strains. A positive genetic correlation was observed between the phase angle () and the period (_LL) of each rhythm. This observation can be understood in terms of a functional relationship between phase and period proposed by Pittendrigh and Daan (1976b) for the entrainment of a circadian oscillator by a light-dark cycle in nocturnal rodents.These results suggest that circadian rhythms for food and water consumption in mice are regulated by a common physiological mechanism, and would respond to natural selection as a single circadian complex under common gene control.     

Circadian rhythms,light, and photoperiodism: A re-evaluation

Senkado-Sancho (1799): “Schocho Shiyo Momochidori”, 90pp. Edo (Tokyo). (Rearing Methods of Various Birds) The long-day treatment with lamp stands for hastening the twittering in early spring to the Japanese bush warbler (Common manners in high class peoples in Tokugawa Era).