Effects of Choline Depletion on the Circadian Rhythm in Neurospora crassa

One approach to identifying components of the circadian oscillator is to screen for clock defects in mutants with known biochemical lesions. The chol-1 mutant of Neurospora crassa is defective in the first methylation step of phosphatidylcholine synthesis, the conversion of phosphatidylethanolamine to phosphatidylmonomethylethanolamine, and requires choline for normal growth. Choline depletion of this mutant inhibits growth and lengthens the period of the rhythm of conidiation. On high levels of choline (above 20 µM), the growth rate and the period of the rhythm are normal. Below about 10 µM choline, the growth rate and period length depend on the choline concentration, and the period is about 58 h on minimal medium without choline. Choline depletion decreases period stability, and replicate cultures do not remain in phase due to variability in period within each culture. At intermediate levels of choline (around 10 µM) cultures are often arrhythmic. The choline requirement for growth can be met by the phosphatidylcholine precursors monomethylethanolamine and dimethylethanolamine, and these supplements also restore a normal period. Choline depletion of the chol-1 strain exaggerates the rhythm in growth rate previously reported in a chol + strain. Growth rate during formation of a conidial band (measured as forward advance of the mycelial front) is less than half of the maximum rate during non-conidiating interband formation. Choline-depleted cultures can be entrained to light/dark (LD) cycles with periods near to their free-running periods. Cultures on 10 µM choline (with a free-running period of about 25 h) can be entrained to a 24 h (12:12) LD cycle, but not to a 36 h (18:18) or 48 h (24:24) LD cycle. Cultures on 0.5 µM choline (free-running period of about 52 h) or minimal medium (free-running period of about 58 h) can be entrained to 18:18 and 24:24 LD cycles, but not a 12:12 cycle. The phase relationship of the conidiation rhythm to the zeitgeber for low-choline cultures in LD 24:24 is similar to high choline cultures in LD 12:12. Continuous light abolishes rhythmicity in choline-depleted cultures. These results may indicate a role for membrane phospholipids, and the metabolites of phosphatidylcholine in particular, in the control of the period of the circadian oscillator in Neurospora.     

Effects of Choline Depletion on the Circadian Rhythm in Neurospora crassa

One approach to identifying components of the circadian oscillator is to screen for clock defects in mutants with known biochemical lesions. The chol-1 mutant of Neurospora crassa is defective in the first methylation step of phosphatidylcholine synthesis, the conversion of phosphatidylethanolamine to phosphatidylmonomethylethanolamine, and requires choline for normal growth. Choline depletion of this mutant inhibits growth and lengthens the period of the rhythm of conidiation. On high levels of choline (above 20 µM), the growth rate and the period of the rhythm are normal. Below about 10 µM choline, the growth rate and period length depend on the choline concentration, and the period is about 58 h on minimal medium without choline. Choline depletion decreases period stability, and replicate cultures do not remain in phase due to variability in period within each culture. At intermediate levels of choline (around 10 µM) cultures are often arrhythmic. The choline requirement for growth can be met by the phosphatidylcholine precursors monomethylethanolamine and dimethylethanolamine, and these supplements also restore a normal period. Choline depletion of the chol-1 strain exaggerates the rhythm in growth rate previously reported in a chol + strain. Growth rate during formation of a conidial band (measured as forward advance of the mycelial front) is less than half of the maximum rate during non-conidiating interband formation. Choline-depleted cultures can be entrained to light/dark (LD) cycles with periods near to their free-running periods. Cultures on 10 µM choline (with a free-running period of about 25 h) can be entrained to a 24 h (12:12) LD cycle, but not to a 36 h (18:18) or 48 h (24:24) LD cycle. Cultures on 0.5 µM choline (free-running period of about 52 h) or minimal medium (free-running period of about 58 h) can be entrained to 18:18 and 24:24 LD cycles, but not a 12:12 cycle. The phase relationship of the conidiation rhythm to the zeitgeber for low-choline cultures in LD 24:24 is similar to high choline cultures in LD 12:12. Continuous light abolishes rhythmicity in choline-depleted cultures. These results may indicate a role for membrane phospholipids, and the metabolites of phosphatidylcholine in particular, in the control of the period of the circadian oscillator in Neurospora .     

Synthesis of mRNA is Required for Light-Induced Phase Shifting of the Circadian Conidiation Rhythm in Neurospora crassa

The process of light-induced phase shifting was investigatedin Neurospora crassa using a liquid culture system and a combinationof treatment with a nucleoside analogue and light. 5-Azacytidineinhibited the light-induced phase shifting at all phases thatwere sensitive to light. Electrophoresis of proteins that weresynthesized in a translation system in vitro showed that 5-azacytidineinhibited the synthesis of most mRNAs. The inhibition of mRNAsynthesis was correlated with the inhibition of light-inducedphase shifting. An excess of cytidine completely overcame theinhibition by 5-azacytidine of both light-induced phase shiftingand mRNA synthesis. Other analogues, namely, 6-azauridine and6-methylpurine, failed to inhibit either the light-induced phaseshifting or the synthesis of mRNA. Two-dimensional gel electrophoresisshowed that the levels of expression of nine mRNAs were affectedby light within 30 min after irradiation. By contrast, the oscillatorof the circadian clock was not affected by pulse treatment with5-azacytidine alone because such treatment failed to shift thephase of the circadian rhythm at any phase. These results indicatethat newly synthesized mRNA(s) is required during the processof signal transduction, from the light-perceiving system tothe circadian clock, for light-induced phase shifting in Neurospora. (Received October 17, 1994; Accepted January 23, 1995)     

Calcium Inhibits Phase Shifting of the Circadian Conidiation Rhythm of Neurospora crassa by the Calcium Ionophore A23187

Effects of the calcium ionophore, A23187, and antimycin A on the circadian conidiation rhythm of Neurospora crassa were examined. A23187 at a concentration of 1 mum in medium not containing divalent cations delayed the phase by 10 hours at CT 10 and advanced it by 5 hours at CT 14 (CT 12 corresponds to the time that discs are transferred from light to dark). This phase shifting was completely inhibited by addition of 0.1 millimolar CaCl(2) but not by MgCl(2) at any concentrations examined.Antimycin A inhibited respiration by 90% at a concentration of 0.2 micrograms per milliliter and lowered the ATP content by 85%. Antimycin A alone caused small phase advances but in combination with A23187 resulted in a large phase delay at CT 10. This phase shifting was not reversed by addition of CaCl(2) lower than 10 millimolar.     

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).     

Rhythms of Enzyme Activity Associated with Circadian Conidiation in Neurospora crassa

The mycelial growth front of the band strain of Neurospora grown on a solid surface exhibits a circadian rhythm of conidiation. Enzyme assays on extracts from that mycelium have shown that the activities of 6 of 13 enzymes (nicotinamide adenine dinucleotide nucleosidase, isocitrate lyase, citrate synthase, glyceraldehydephosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase) and soluble-protein content oscillate with the visible morphological change. The rhythmic enzymes associated with the Krebs and glyoxylate cycles are more active during conidiogenesis, whereas the activities of the rhythmic enzymes of glycolysis and the hexose monophosphate shunt are reduced during that phase. The absence of enzyme oscillations in wild-type and fluffy strains which do not form conidia under the conditions employed suggests that the enzyme fluctuations are associated with conidiogenesis itself. Oscillations of enzyme activity as a function of time are restricted to the growth front. A permanent record of rhythmicity associated with conidial and nonconidial regions does, however, exist in the mycelial mat behind the growth front. The activities of three enzymes (nicotinamide adenine dinucleotide nucleosidase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase) are not directly influenced by CO(2) concentration, but are correlated with the prescence or absence of conidiation which is controlled by CO(2) concentration. In contrast, citrate synthase and malate dehydrogenase activities are correlated with changes in CO(2) concentration.     

Cold-sensitive Mutants of Neurospora crassa

Cold-sensitive mutants of the eucaryote Neurospora crassa have been isolated by a modification of the filtration-enrichment technique of Catcheside. The mutants include osmotic remedial, auxotrophic, transport, and incorporation deficient isolates.     

Circadian Nature of a Rhythm Expressed by an Invertaseless Strain of Neurospora crassa

A new strain of Neurospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth tube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is about 24 hours (22.7 hours at 25 degrees ); B) the period is relatively temperature-independent (Q(10) is between 0.95 and 1.21 for temperature range of 18 to 35 degrees ); C) the rhythm persists in continuous darkness at constant temperature for a minimum of 14 days without damping out; and D) the phase of the rhythm can be shifted by a single brief exposure to light. The sensitivity of this strain to light has been demonstrated further by the entrainment of the rhythm to a period of 24.0 hours using a suitable light-dark regime, and by the inhibition by light of the appearance of a rhythm; i.e., continuous conidiation occurs when the strain is subjected to continuous light. The new strain is compared to 2 other strains of Neurospora which also express a rhythm, patch and clock.     

Selection of Respiratory Mutants of Neurospora crassa

A method is described that permits the isolation of mutants that are defective in mitochondrial respiration. The techniques of inositol-less death and overlay with 2,3,5-triphenyl-2H-tetrazolium chloride are utilized to select for mutant colonies. Colonies that survive inositol-less death and fail to reduce the tetrazolium dye are then tested polarographically for cyanide-sensitive respiration. A preliminary characterization of three mutants obtained by this method is presented. The mutants have been characterized by their cyanide-sensitive respiration rate, growth rate, the state III respiration rate of isolated mitochondria, inhibition of the respiration of isolated mitochondria by cyanide and antimycin A, and cytochrome spectra. All of the mutants described differ from the parent strain in some of these aspects.     

Mutation of the Cys-9 Gene, Which Encodes Thioredoxin Reductase, Affects the Circadian Conidiation Rhythm in Neurospora Crassa

Ten cysteine auxotrophs of Neurospora crassa were examined with regard to the period lengths of their circadian conidiation rhythms. One of the these cysteine auxotrophs, cys-9, showed dramatic changes in the circadian conidiation rhythm. At 10 μM methionine, the cys-9 mutant had a period length that was 5 hr shorter than that of the wild-type strain during the first 3 days after transfer to continuous darkness. At this concentration of methionine, the period length was unstable after the fourth day and varied widely from 11 to 31 hr. In contrast, other cysteine auxotrophs did not show such instability of the period length at any of the concentrations of methionine tested. Furthermore, only the cys-9 mutant exhibited partial loss of the capacity for temperature compensation of the period length. With regard to cold-induced phase-shifting of the circadian conidiation rhythm, the cys-9 mutant was more sensitive than the wild-type strain to low temperature. The cys-9(+) gene was cloned and was found to encode NADPH-dependent thioredoxin reductase. These results indicate that mutation of the gene for thioredoxin reductase results in abnormal expression of the circadian conidiation rhythm in N. crassa.     

Circadian Rhythms in Neurospora crassa: Effects of Saturated Fatty Acids

To assess their effects on the conidiation rhythm in Neurospora, 14 saturated fatty acids from 6 to 24 carbons long were used to supplement the bd csp and bd csp cel strains. Both strains express a circadian spore-forming rhythm when grown on solid media; the cel mutation confers a partial fatty acid requirement. Fatty acid supplements from 8 to 13 carbons long lengthened the free-running period of bd csp cel compared with the control value of 21 h; the maximal effect (33 h) was obtained with nonanoic acid (9:0) at a concentration of 5 x 10(-4) M. In contrast, the period of bd csp remained unchanged under all experimental conditions. The short-chain fatty acids (<14 carbons) reduced the rate of advance of the growth front in both strains, compared with unsupplemented controls. However, this inhibition did not appear to be responsible for the lengthened periods in bd csp cel. Nor was direct incorporation of the short-chain (period-lengthening) fatty acids into mycelial total lipids responsible, since such incorporation was not observed. In fact, extensive metabolic conversion of these supplements by both strains was indicated by the disappearance of short-chain fatty acids from the agar media coupled with their absence in mycelial lipids, and by the liberation of (14)CO(2) from cultures supplemented with [1-(14)C]lauric acid (12:0).     

Mutants affecting thymidine metabolism in Neurospora crassa

When (14)C-thymidine labeled only in the ring is administered to Neurospora crassa, the majority of the recovered label is found in the ribonucleic acid (RNA). Three mutants were isolated in which different steps are blocked in the pathway that converts the pyrimidine ring of thymidine to an RNA precursor. Evidence from genetic, nutritional, and accumulation studies with the three mutants shows the pathway to proceed as follows: thymidine --> thymine --> 5-hydroxymethyluracil --> 5-formyluracil --> uracil --> uridylic acid. A mutant strain in which the thymidine to thymine conversion is blocked is unable to metabolize thymidine appreciably by any route, including entry into nucleic acids. This suggests that Neurospora lacks a thymidine phosphorylating enzyme. A second mutation blocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step, whereas a third prevents utilization of uracil and all compounds preceding it in the pathway. The mutant isolation procedures yielded three other classes of mutations which are proposed to be affecting, respectively, regulation of the thymidine degradative pathway, transport of pyrimidine free bases, and transport of pyrimidine nucleosides.     

A Failure to Detect an Influence of Magnetic Fields on the Growth Rate and Circadian Rhythm of Neurospora crassa

Low strength magnetic fields, 6.36 and 32.25 gauss, were found to have no effect, with one questionable exception, on the circadian rhythm and growth rate of Neurospora crassa. This was true whether the fields were continuous, pulsed 20 minutes daily, or on a 12: 12, on-off cycle.     

Circadian timekeeping in Neurospora crassa and Synechococcus elongatus

At first, the saprophytic eukaryote Neurospora crassa and the photosynthetic prokaryote Synechococcus elongatus may seem to have little in common. However, in both organisms a circadian clock organizes cellular biochemistry, and each organism lends itself to classical and molecular genetic investigations that have revealed a detailed picture of the molecular basis of circadian rhythmicity. In the present chapter, an overview of the molecular clockwork in each organism will be described, highlighting similarities, differences and some as yet unexplained phenomena.     

Effects of Membrane ATPase Inhibitors on Light-Induced Phase Shifting of the Circadian Clock in Neurospora crassa

Effects of several membrane ATPase inhibitors on light-induced phase shifting of the circadian conidiation rhythm in Neurospora crassa were examined using mycelial discs in liquid culture. Suppression of phase shifting by the inhibitors was strongly dependent on the pH of the liquid medium in which the discs were cultured during the time from light-dark transition (beginning of free-run) to light irradiation. When discs were cultured in pH 6.7 medium, azide, the inhibitors of plasma membrane ATPase (diethylstilbestrol and N, N′-dicyclohexylcarbodiimide), and ethanol completely suppressed the effect of light on the clock. In contrast, mycelial discs cultured in pH 5.7 medium were fully phase-shifted by light in the presence of the same and even higher concentrations of the chemicals. However, sensitivity to light of the discs cultured in relatively acidic medium was eight times higher than that of the discs cultured at neutral pH. Oligomycin and venturicidin, inhibitors of mitochondrial ATPase, did not suppress phase shifting by light at either pH.     

Isolation and Characterization of Low-Kynureninase Mutants of Neurospora crassa

Two kynureninase activities are known in Neurospora crassa, one of which (kynureninase I) is inducible, the other (kynureninase II) being constitutive. A method is described for the isolation of low-kynureninase mutants of N. crassa. When grown on an inducer, the mutants show significantly less kynureninase I activity compared with wild type, whereas constitutive kynureninase II activity is unaffected. Since a low level of kynureninase I activity remains in the mutants examined, the mutations may be in a regulatory gene or genes. Other experiments are described concerning the molecular weights of the two enzymes and the intracellular localization and specificity of kynureninase II.     

Circadian regulation of the light input pathway in Neurospora crassa

FREQUENCY (FRQ) is a critical element of the circadian system of Neurospora. The white collar genes are important both for light reception and circadian function. We show that the responsiveness of the light input pathway is circadianly regulated. This circadian modulation extends to light-inducible components and functions that are not rhythmic themselves in constant conditions. FRQ interacts genetically and physically with WHITE COLLAR-1, and physically with WHITE COLLAR-2. These findings begin to address how components of the circadian system interact with basic cellular functions, in this case with sensory transduction.     

Altered Fatty Acid Distribution in Mutants of Neurospora crassa

Morphological mutants of Neurospora with decreased levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide ad enine dinucleotide (NADH) contained only 20% as much of a polyunsaturated fatty acid (linolenic acid) as the wild type in both the phospholipid and neutral lipid fractions. There was an excellent correlation between linolenic acid levels and morphological appearance as a function of total NADPH content, but no correlation with NADH content. The linolenic acid deficiency was balanced by a relative increase in the amounts of the less unsaturated fatty acids (oleic and linoleic acids), but the level of three other fatty acids did not appear to be changed. This accumulation of these two precursors suggests that the NADPH deficiency preferentially affected the final desaturation step, i.e., the conversion of linoleic to linolenic acid. The NADPH needed for this reaction in vivo was probably generated by the pentose phosphate shunt, since mutations affecting the shunt lead to the decreased levels of linolenic acid. It is not clear whether the changes in fatty acid distribution affect the morphogenesis of Neurospora, or if these changes are just part of the NADPH-deficiency syndrome.