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.     

The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3. Received: 17 October 1996 / Accepted: 19 November 1996     

A ras homologue of Neurospora crassa regulates morphology

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa. Received: 1 October 1996 / Accepted: 9 December 1996     

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.     

ENTRAINMENT ELICITS PERIOD AFTEREFFECTS IN NEUROSPORA CRASSA

Circadian clocks continue to oscillate in constant conditions with their own period (τ) and entrain to a cyclic environment by adjusting their intrinsic period to that of the zeitgeber. When circadian clocks are released from entrained to constant conditions, the τ of their initial free-run often depends on the nature of the prior zeitgeber. These postentrainment effects on period (τ-aftereffects) have predominantly been reported for animals but, so far, not fungi. The authors therefore investigated τ aftereffects in the classic circadian model system Neurospora crassa. The standard laboratory strain frq⁺, the short-period mutant frq¹, and the long-period mutant frq⁷ were entrained to 11 different photoperiods in a 24-h day (2–22?h) and to zeitgebers with six different T (16–26?h), and then released to constant darkness. τ-Aftereffects in response to different photoperiods correlated weakly with prior photoperiod in frq⁺ and were unsystematic in both period mutant strains. Strength and direction of the τ-aftereffect in zeitgeber cycles with different T depended on their length and on the strain, showing a negative correlation with zeitgeber length in frq⁺ and positive correlations in frq¹ and frq⁷. It has been proposed that τ-aftereffects are based on interactions of oscillators within a cellular network. The present findings in Neurospora, which grows as a syncytium, suggest that τ-aftereffects also exist in circadian systems based on multioscillatory networks organized at the molecular level. (Author correspondence: roenneberg@lmu.de)     

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)     

Organisation of the bph gene cluster of transposon Tn 4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds

  Tn4371 is a 55 kb transposon which encodes enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds into benzoate and 4-chlorobenzo-ate derivatives. We constructed a cosmid library of Tn4371 DNA. The bph genes involved in biphenyl/4-chlorobiphenyl degradation were found to be clustered in the middle of the transposon. Sequencing revealed an organisation of the bph genes similar to that previously found in Pseudomonas sp. KKS102, i.e. the bphEGF genes are located upstream of bphA1A2A3 and bphA4 is separated from bphA1A2A3 by bphBCD. Consensus sequences for σ54-associated RNA polymerase were found upstream of bphA1 and bphEGF. Plasmid RP4::Tn4371 was transferred into a mutant of Alcaligenes eutrophus H16 lacking σ54. In contrast to wild-type H16 exconjugants, the σ54 mutant exconjugants could not grow on biphenyl, indicating the dependence of Tn4371bph gene expression on σ54. The Tn4371-encoded bph pathway was activated when biphenyl and various biphenyl-like compounds were present in the growth medium. Preliminary observations indicate the presence of a region outside the catabolic genes downstream of bphA4 which is involved in mediating at least the basal expression of BphC. Received: 13 May 1996 / Accepted: 16 September 1996     

Sequential functioning of Sym-13 and Sym-31 , two genes affecting symbiosome development in root nodules of pea ( Pisum sativum L.)

Two Fix⁻ mutants of pea (Pisum sativum L.) which are unable to fix molecular nitrogen, E135f (sym-13) and Sprint-2Fix⁻ (sym-31), were crossed to create the doubly homozygous recessive line, named RBT (sym-13, sym-31). The ultrastructural organization of nodules of the RBT line was compared with that of each of the two parental mutant lines and with the original wild-type genotypes of the cultivars Sparkle and Sprint-2. It was shown that the RBT line is similar to the mutant line Sprint-2Fix⁻ in having abnormal symbiosome composition and bacteroids with relatively undifferentiated morphology. Because the phenotypic manifestation of the sym-31 mutant allele suppresses the phenotypic manifestation of the sym-13 mutant allele, it is concluded that the function of the gene Sym-31 (which is mutated in the Sprint-2Fix⁻ line) is necessary at an earlier stage of symbiosome development than the gene Sym-13 (which is mutant in the E135f line). Received: 28 October 1996 / Accepted: 22 January 1997     

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.     

The heterocyst-specific fdxH gene product of the cyanobacterium Anabaena sp. PCC 7120 is important but not essential for nitrogen fixation

To clarify the role of the heterocyst-specific [2Fe-2S] ferredoxin in cyanobacterial nitrogen fixation, mutational analysis of the Anabaena 7120 fdxH gene region was carried out. First, the DNA sequence of the wild-type 3509-bp EcoRI fragment downstream of the fdxH gene was determined. Genes homologous to ORF3 from the fdxH gene regions of A. variabilis and Plectonemaboryanum, the mop genes of Clostridiumpasteurianum encoding molybdo-pterin binding proteins, and ORF3 from the A. variabilis hydrogenase gene cluster were identified within the sequenced region. For mutational analysis the Anabaena 7120 mutant strains LAK4, BMB92, and KSH10 were constructed. In LAK4 the fdxH coding region is disrupted by an interposon, whereas BMB92 is deleted for a 2799-bp NheI fragment encompassing fdxH, ORF3, mop, ORF4, and ORF5. Mutant strain KSH10 is a derivative of BMB92, complemented for fdxH but not for the other genes located further downstream. Analysis of the Nif phenotype of these mutant strains showed that FdxH is necessary for maximum nitrogenase activity and optimal growth under nitrogen-fixing conditions, but not absolutely essential for diazotrophic growth. The role of alternative electron donors for nitrogenase, which might substitute for FdxH, is discussed. Iron concentrations (1μM Fe) sufficient to induce synthesis of the vegetative cell flavodoxin did not stimulate diazotrophic growth of the fdxH mutant strains, suggesting that FdxH was not replaced by a NifJ-flavodoxin system. Comparison of LAK4 and BMB92 indicated that one of the genes located downstream of fdxH might also play a (minor) role in nitrogen fixation. Received: 28 May 1996 / Accepted: 4 October 1996     

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.