On the Role of Energy Metabolism in Neurospora Circadian Clock Function

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na₃Vo₄), molybdate (Na₂MoO₄: 2 H₂O), N-ethylmaleimide (NEM), azide (NaN₃), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors.

Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide.

[³¹P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.     

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.     

A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk.

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.     

The Saccharomyces cerevisiae homologue of ribosomal protein S26

The nucleotide sequence of RPS26, the gene encoding a homologue of ribosomal protein small subunit S26 in Saccharomyces cerevisiae, was determined. The deduced amino-acid sequence showed significant identity with its counter- parts from Neurospora crassa, human, rat and Arabidopsis thaliana. Disruption of RPS26 resulted in the formation of micro-colonies, suggesting that it is important for the normal cell growth of S. cerevisiae.     

The 59-kDa polypeptide constituent of 8–10-nm cytoplasmic filaments in Neurospora crassa is a pyruvate decarboxylase

The fungus Neurospora crassa harbors large amounts of cytoplasmic filaments which are homopolymers of a 59-kDa polypeptide (P59Nc). We have used molecular cloning, sequencing and enzyme activity measurement strategies to demonstrate that these filaments are made of pyruvate decarboxylase (PDC, EC 4.1.1.1), which is the key enzyme in the glycolytic-fermentative pathway of ethanol production in fungi, and in certain plants and bacteria. Immunofluorescence analyses of 8–10-nm filaments, as well as quantitative Northern blot studies of P59Nc mRNA and measurements of PDC activity, showed that the presence and abundance of PDC filaments depends on the metabolic growth conditions of the cells. These findings may be of relevance to the biology of ethanol production by fungi, and may shed light on the nature and variable presence of filament bundles described in fungal cells.     

How temperature affects the circadian clock of Neurospora crassa

Light and temperature are major environmental cues that influence circadian clocks. The molecular effects of these zeitgebers on the circadian clock of Neurospora crassa have been studied intensively during the last decade. While signal transduction of light into the circadian clock is quite well characterized, we have only recently begun to understand the molecular mechanisms that underlie temperature sensing. Here we summarize briefly the current knowledge about the effects of temperature on the circadian clock of Neurospora crassa.     

How temperature affects the circadian clock of Neurospora crassa

Light and temperature are major environmental cues that influence circadian clocks. The molecular effects of these zeitgebers on the circadian clock of Neurospora crassa have been studied intensively during the last decade. While signal transduction of light into the circadian clock is quite well characterized, we have only recently begun to understand the molecular mechanisms that underlie temperature sensing. Here we summarize briefly the current knowledge about the effects of temperature on the circadian clock of Neurospora crassa.     

粗糙脉孢菌中甾醇还原酶基因erg24对麦角甾醇合成的影响

粗糙脉孢菌为子囊菌中的高效纤维素降解菌,可以直接以纤维素为营养源进行生长。本研究以粗糙脉孢菌为实验对象,利用基因工程技术构建甾醇还原酶基因erg24的高表达菌株,分别以蔗糖、麦麸、玉米秸秆、小麦秸秆、杨树木屑、水稻秸秆6种物质的粉末为碳源培养野生型粗糙脉孢菌和erg24高表达菌株,利用半定量RT-PCR测定在不同培养条件下erg2、erg24和erg6 3个麦角甾醇合成相关基因的表达水平,采用HPLC方法测定不同培养条件下麦角甾醇的积累量。研究结果表明,分别以玉米秸秆、杨树木屑、水稻秸秆这3种粉末为碳源时,培养物中的erg2、erg24和erg6 3个基因表达量较高。在不同培养条件下erg24高表达菌株合成麦角甾醇量显著高于野生型粗糙脉孢菌的合成量,且以杨树木屑粉末为碳源培养时,所获得的麦角甾醇产量最高,为30.53μg/mg。结果表明erg24基因是粗糙脉孢菌合成麦角甾醇的关键基因之一,利用玉米秸秆、小麦秸秆、杨树木屑或水稻秸秆粉末为碳源培养粗糙脉孢菌时,可获得较高产量的麦角甾醇。研究结果为以农业废弃物为营养源,利用真菌生产麦角甾醇奠定了基础。     

Effect of the homokaryotic or heterokaryotic state of the uvs-2 allele in Neurospora crassa on mitomycin C-induced killing and ad-3 mutation

Mitomycin C (MC) was tested for its killing and mutagenic activities in the ad-3 forward-mutation test in Neurospora crassa. The test was conducted in 4 dikaryons of N. crassa in order to determine the effect of the uvs-2 allele, which causes a defect in nucleotide excision repair, on MC-induced killing and ad-3 mutation. These dikaryons were homokaryotic for uvs-2⁺ (H-12), homokaryotic for uvs-2 (H-59), and heterokaryotic for uvs-2/uvs-2⁺ (H-70 and H-71). MC induced killing and ad-3 mutation in H-12, but the presence of uvs-2 in the homokaryotic state (H-59) resulted in a great increase in the killing and mutagenic activities of MC. This increased sensitivity to MC-induced killing and mutation conferred by uvs-2 in the homokaryotic state (H-59 vs. H-12) is a different effect than that noted by others for a defect in nucleotide excision-repair in Escherichia coli and Salmonella typhimurium or in human cells. The dikaryons heterokaryotic for uvs-2/uvs-2⁺ had the same sensitivity to MC as H-12, indicating that for MC-induced killing and ad-3 mutation uvs-2 is recessive to uvs-2⁺.     

Purification, N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

A novel type of FKBP in the secretory pathway of Neurospora crassa

FKBPs define a subfamily of peptidyl-prolyl cis/trans isomerases (PPIases). PPIases are known to play roles in cellular protein folding, protein interactions and signal transduction. Here we describe NcFKBP22 from Neurospora crassa, a novel type of FKBP. NcFKBP22 is synthesized as a precursor protein with a cleavable signal sequence. In addition to a typical FKBP domain in the amino-terminal part mature NcFKBP22 contains a novel second domain which is unique amongst all known FKBPs. The amino acid composition of this carboxy-terminal domain is highly biased. Secondary structure predictions suggest that this domain may form an amphipathic -helix. The carboxy-terminus of NcFKBP22 is –HNEL, a potential endoplasmic reticulum (ER) retention signal, suggesting that NcFKBP22 is a resident protein of the ER.     

Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals.

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3B^R mutations. The percentages of ad-3B^R mutations showing allelic complementation with polarized or nonpolirized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation.

The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains.

Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced d-3     

Heat Shock Proteins and Circadian Rhythms

Significant circadian rhythms in heat shock gene expression were observed in a prokaryotic species (Synechocystis). In eukaryotes, in contrast, several heat shock genes (constitutive and inducible) were shown to be constantly expressed. A few cases of circadian expression of heat shock proteins (HSPs), however, have been reported. Significant circadian changes of thermotolerance were observed in yeast and several plant species. Higher thermo-tolerance can be attributed to a higher abundance of HSPs, but also to other adaptive mechanisms. Zeitgeber effects of temperature changes can be explained on the basis of their direct effects on the state variables of the clock gene (per, frq) expression and its negative feedback loop. Effects of increased HSP concentrations, as observed after heat shock, but also after light and serotonin (5HT), appear possible, in particular with respect to nuclear localization of the clock (PER) protein, but these effects have not been documented yet. Thus, the role of HSPs in the circadian clock system is little understood and, from our point of view, deserves more attention. (Chronobiology International, 13(4), 239-250, 1996)     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. II. More extensive genetic tests reveal an unexpectedly high frequency of multiple-locus mutations

More extensive genetic tests have been performed on a series of 832 X-ray-induced specific-locus mutations in the ad-3 region of a 2-component heterokaryon (H-12) of Neurospora crassa, reported earlier (Webber and de Serres 1965). Using a new tester strains and techniques for performing large-scale genetic tests (heterokaryon, dikaryon and trikaryon) to characterize ad-3 mutants induced in 2-component heterokaryons, new data have been obtained on this sample of X-ray-induced ad-3 mutants. These new data show that unexpectedly high frequencies of both single-locus (gene/point) mutations and multilocus deletions in the ad-3 region have additional, but separate, sites of resessive lethal (RL^CL) damage in the immediately adjacent genetic regions. The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory and classical models of chromosome structure during interphase. Current models of interphase chromosome structure in higher eukaryotes as revealed by chromosome “painting” offer a possible explanation of the Neurospora data.