Wild-type Neurospora Crassa Research Articles

Neurospora crassa cytoplasmic ribosomes; ribosomal ribonucleic acid synthesis in the wild type.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.

Ageing of Neurospora crassa. II. Organic hydroperoxide toxicity and the protective role of antioxidant and the antioxygenic enzymes

Cumene hydroperoxide and tert.-butyl hydroperoxide at sublethal concentrations initially prevent growth of mycelia of wild-type Neurospora crassa, but after a time the cells grow at a subnormal steady-state rate. The antioxidant nordihydroguaiaretic acid protects unadapted cells from hydroperoxide inhibition, leading to a decrease in the time before growth begins, an increase in steady-state growth rate and an increase in biomass production. The results of growth transfer experiments and enzyme measurements indicated that the acquired resistance to the hydroperoxides is physiological and most likely involves the induction of the synthesis of the antioxygenic enzymes superoxide dismutase, glutathione peroxidase and glutathione reductase. Nordihydroguaiaretic acid normalizes the levels of activities of glutathione peroxidase and glutathione reductase during culture with hydroperoxide. Molecular-induced homolysis of the hydroperoxides, a process that is induced by unsaturated fatty acids of membrane lipids, leads to lipid autoxidation in a chain reaction which produces lipid hydroperoxides, which in turn decomposes to form more free radicals. Nordihydroguaiaretic acid, a well-known free radical scavenger, probably serves to minimize hydroperoxide decomposition, lipid autoxidation and molecular damage from free radicals, whereas the coupled enzyme system glutathione peroxidase and glutathione reductase minimizes these processes by decomposing the hydroperoxides to harmless alcohols. We suggest that either free radicals derived from these processes or some consequent non-radical products may serve as the inducers of this enzyme system, rather than the hydroperoxide substrates.

Mutagenicity of actinomycin D in Neurospora crassa

Actinomycin D is known to bind to native DNA and is widely used as an antineoplastic agent and inhibitor of DNA-dependent RNA and protein synthesis. We report here the induction by actinomycin D of purple adenine-requiring mutants ( ad-3) in wild-type Neurospora crassa. A significant increase in the frequency of ad-3 mutants was evident when the organism was grown vegetatively in the presence of actinomycin D; the mutation frequency was at least 3.6 per 10 6 survivors. The actinomycin D-induced ad-3 mutants were 29% ad-3A and 71% ad-3B. The ad-3B mutants were classed by complementation pattern as 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. The spectrum of complementation types of the actinomycin D-induced mutants most closely parallels that of mutants induced by ICR-170, known to induce base-pair insertions or deletions, or that of X ray-induced or spontaneous mutants. It is significantly different from spectra seen following treatment with nitrous acid or N-methyl- N′-nitrosoguanidine, agents known to induce mainly base-pair substitutions.

Neurospora crassa conidial germination: role of endogenous amino acid pools

The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.

Stimulation of folate metabolism by exogenous glycine in Neurospora crassa wild type

Stimulation of folate metabolism by exogenous glycine in Neurospora crassa wild type

Studies on the binary and ternary complexes formed by a neurospora glutamate dehydrogenase and its substrates

The NADP + specific glutamate dehydrogenase from wild-type Neurospora crassa forms a stable binary complex with NADPH. This can combine with L-glutamate, α-ketoglutarate or the substrate analogue D-glutamate to form ternary complexes which can be distinguished by their different fluorescence properties. The affinity of the enzyme for NADPH diminishes with increases in pH or ionic strength of the solution. Experimental data obtained using modified glutamate dehydrogenases from mutant strains of N. crassa suggest that the reduced-coenzyme binding sites observed fluorimetrically are the same as those observed by enzyme kinetics.

Detection of the ergosterol and episterol isomers lichesterol and fecosterol in nystatin-resistant mutants of Neurospora crassa.

Wild-type Neurospora crassa is completely inhibited by 5 ppm nystatin. Ultraviolet-induced mutants have been isolated that grow in the presence of 60 ppm of the antibiotic. Gas-liquid chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses showed the wild-type sterols to be ergosterol (ergosta-5,7,22-trien-3β-ol) and episterol (ergosta-7,24(28)-dien-3β-ol) in a 3:1 ratio. The mutants contained lichesterol (ergosta-5,8,22-trien-3β-ol) and fecosterol (ergosta-8,24(28)-dien-3β-ol) in a 2:1 ratio, differing from the wild type only in the position of the B-ring unsaturation. A deficiency of an ergosta-8,24 (28)-dien-3β-ol:ergosta-7,24(28)-dien-3β-ol isomerase is indicated.

Repression of the branched-chain amino acid biosynthetic enzymes in developing conidia and mycelia of Neurospora.

During germination of the conidia of wild type Neurospora crassa, the enzymes of the isoleucine–valine pathway increase in activity. This increase is partially repressed in the presence of a combination of exogenous isoleucine, leucine, and valine. Repressed levels of enzymic activity are also observed in mycelium during the logarithmic phase of growth. In both cases, isoleucine and leucine together are almost as effective without valine as when the three are present together. The synthesis of these enzymes does appear to be affected by their end products but not to the extent exhibited in bacteria.

Hexagonal inclusions in an ergosterol-free mutant of Neurospora crassa

Refractive hexagonal inclusions in hyphae of wild-type Neurospora crassa were compared with those in two mutants deficient in or lacking ergosterol. The mutants contained more inclusions than the wild type and there were no discernible morphological differences among inclusions of the three strains. It is concluded that these inclusions do not consist of ergosterol as previously reported.

Effect of Tungsten and Vanadium on the in Vitro Assembly of Assimilatory Nitrate Reductase Utilizing Neurospora Mutant nit-1

Abstract Tungsten or vanadium in a nutrient medium containing nitrate, but not ammonia, as the sole nitrogen source inhibited the formation of assimilatory NADPH-nitrate reductase (but not nitrate-inducible NADPH-cytochrome c reductase) and growth of wild type Neurospora crassa. Inhibition was prevented by high levels of molybdate in the nutrient medium but was not reversed by adding molybdate to subsequently prepared cell-free enzyme preparations. Neither tungsten nor vanadium was inhibitory when added to partially purified wild type Neurospora NADPH-nitrate reductase. Cell-free preparations of uninduced wild type grown in increasing concentrations of tungsten or vanadium showed progressive inhibition of ability to assemble nitrate reductase in vitro (with extracts of nitrate-induced Neurospora mutant nit-1) which was partially or entirely restored by subsequent addition of molybdate to the extracts of the tunsgten- or vanadium-grown uninduced wild type. Tungsten or vanadium added to acid-treated bovine milk xanthine oxidase or to extracts of uninduced wild type also inhibited in vitro enzyme assembly, with added molybdate partially preventing inhibition. The tungsten or vanadium analogue of nitrate reductase formed in vivo or by in vitro assembly is considerably more labile than its molybdenum counterpart as indicated by the complete loss of 185W or 48V from the enzyme as a result of trichloroacetic acid precipitation or heat treatment. Sucrose density gradient profiles of the Neurospora NADPH-nitrate reductase formed in vitro with 185W- or 48V-grown uninduced wild type or induced nit-1 showed, as in the case of 99Mo, that uninduced wild type and not nit-1 can be the source of tungsten or vanadium in the in vitro assembly of the enzyme. Exogenous 185W added to cell-free extracts of uninduced wild type or acid treated bovine milk xanthine oxidase prior to in vitro assembly of nitrate reductase using nit-1 failed to appear in the subsequently formed enzyme, whereas 48V experienced a small but consistent incorporation only with acid-treated xanthine oxidase. There was no in vitro exchange or incorporation of 185W or 48V into the already formed wild type enzyme. The data implicate tungsten and vanadium as competitive inhibitors of molybdenum in nitrate reductase structure and function.

Methylation and processing of mitochondrial ribosomal RNAs in poky and wild-type Neurospora crassa

Mitochondrial ribosomal RNAs (25 S and 19 S) are produced by cleavage of a common high molecular weight precursor (32 S), which is a transcript of mitochondrial DNA. The poky strain in Neurospora crassa is characterized by a deficiency of mitochondrial small-subunit ribosomes. Pulse-labeling studies were carried out in poky indicating that the maturation and cleavage steps, which lead to the production of mitochondrial ribosomal RNAs, are abnormal. Studies of RNA methylation in methionine-requiring strains were also made. In control cells, the time course of incorporation of [C 3H 3]methionine label into mitochondrial RNAs, and the contents of methyl groups in the RNAs suggest that 32 S RNA is the major site for methylation. In poky, mitochondrial 25 S and 19 S RNAs have methyl contents of approximately 70% and 55%, respectively, of control values. The cytoplasmic ribosomal RNAs and mitochondrial 4 S RNAs, by contrast, show no differences between poky and control cultures. It is concluded that in poky, most of the putative 19 S RNA is degraded before it is incorporated into small ribosomal subunits. This defect could be a consequence of undermethylation, or both defects could result from a primary abnormality yet undefined.

Changes in free and membrane-bound ribosomes during the vegetative growth of Neurospora crassa.

SUMMARY: Free and membrane-bound ribosomes were isolated from wild-type Neurospora crassa containing the genetic backgrund Em 5297a at 6, 12, 18, 24, 36, 48 and 72 h of growth. Log phase began at 8 h and continued to the end of 24 h, the beginning of the stationary phase. The ratio of free to membrane-bound ribosomes remained relatively constant during log phase. During stationary phase, the ratio of free to bound ribosomes rose sharply because of a decrease in the concentration of bound ribosomes.

The neutral carotenoids of wild-type and mutant strains of Neurospora crassa

The neutral carotenoids of wild-type Neurospora crassa and of carotenoid mutants at four discrete genetic loci were isolated using gradient elution chromatography on deactivated alumina columns. Carotenoids were identified by absorption spectrophotometry and thin layer cochromatography with carotenoid standards. Phytoene, phytofluene, ζ-carotene, β-carotene, neurosporene, torulene, lycopene, and 3,4-dehydrolycopene were isolated from wild type. Phytoene, phytofluene, ζ-carotene, β-carotene, neurosporene, γ-carotene, lycopene, and one unknown carotenoid, tentatively identified as 15,15′-cis-β-carotene, were isolated from a yellow mutant, ylo-1. ylo-1 also contained residual carotenoids having similar absorption spectra to, but very different chromatographic behavior from, phytofluene, ζ-carotene, β-carotene, and lycopene. Albino and colored al-1 mutants contained large amounts of phytoene and only traces of other neutral carotenoids. Albino al-2 and al-3 mutants contained only traces of neutral carotenoids.

Changes in enzyme activity of germinating conidia of Neurospora crassa

The conidia of wild-type Neurospora crassa are shown to have a drastically lower activity for three enzymes of the isoleucine-valine pathway—acetohydroxy acid synthetase, dihydroxy acid dehydratase, and aminotransferase—than the actively growing mycelium. Lower activity was also found in the conidia for ornithine transcarbamylase and aspartate transcarbamylase. Lower activity (10- to 100-fold) was found for the overall synthesis of valine from pyruvate in the conidia as compared to the mycelium as expected. In addition it is also apparent that the distribution of the isoleucine-valine enzymes is different in conidia from the mycelium as regards activity in the mitochondria as compared to the cytosol. In conidia their activity in the mitochondria is lower than in the cytosol, but the opposite holds in the mycelium. These differences are also reflected in the overall activity. Cycloheximide inhibits the increase in total activity of the acetohydroxy acid synthetase and the dehydratase during germination of the conidia.

Electron microscopy of selectively stimulated microconidiogenesis in wild type Neurospora crassa.

Microconidiogenesis in Neurospora crassa wild type is selectively stimulated by iodoacetate. The ultrastructural features of various stages of microconidiogenesis have been studied. The conidiogenous cells or phialides first bear a protuberance which then erupts into a conspicuous collarette surrounding, above a lamellated cushion, the successively budded, often elongated microconidia.

Acetohydroxy acid synthetase with a pH optimum of 7.5 from Neurospora crassa mitochondria: characterization and partial purification.

An acetohydroxy acid synthetase (AAS) has been found associated with the mitochondrial fraction of wild-type Neurospora crassa. It has a pH optimum of 7.5 and is presumed to be homologous to the pH 8.0 AAS that synthesizes the valine and isoleucine precursors in bacteria and yeast. The enzyme was characterized and purified 30- to 60-fold. The AAS activity of intact mitochondria requires thiamine pyrophosphate (TPP), Mn(2+) or Mg(2+), and flavine adenine dinucleotide (FAD), and is sensitive to end product inhibition by l-valine. This inhibition is pH-dependent and noncompetitive with respect to pyruvate. Activity is slightly repressed during exponential growth in the presence of valine, isoleucine, and leucine. Extraction of the AAS from the mitochondria has a profound influence on the following properties: pH optimum, sensitivity to l-valine, response to FAD, binding of TPP, apparent K(m), and stability at 0 to 4 C. The catalytic properties of the partially purified enzyme are described. Two forms of the partially purified AAS can be isolated from preparative Sephadex G-200 chromatographic columns. Both forms are electrophoretically and antigenically similar but one form has an estimated molecular weight of 110,000 to 120,000 whereas the predominant form is a much larger and more buoyant molecule.

Conidial repression by phenethyl alcohol and its serological consequences in Neurospora crassa.

Phenethyl alcohol (PEA) at concentrations ranging from 0.04 to 0.08% (v/v) repressed conidial differentiation (C) and its associated carotenogenesis in wild type neurospora crassa. With 0.16–0.32%, most of the conidia germinate into swollen, yeast-like cells (Y) producing buds instead of mycelial filaments (M). Incubation with PEA increased alcoholic fermentation and sharply reduced the immunoelectrophoretic diagram. The step-wise reduction of Neurospora morphogenesis (C→M→Y) reveals the trimorphic potentiality of its hyphal system.

Purification and Partial Characterization of Glucose 6-Phosphate Dehydrogenase from Neurospora crassa

Abstract A procedure is described for the purification of glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP oxidoreductase, EC 1.1.1.49) from wild type Neurospora crassa. A 2400-fold purification was achieved with a 10% yield and an average specific activity of 470. The glucose 6-phosphate and NADP Michaelis constants were determined, as well as the optimal pH for activity. The analogues, 2-deoxyglucose 6-phosphate and galactose 6-phosphate, were utilized as substrates in place of glucose 6-phosphate. No reaction was observed when NAD was substituted for NADP. The amino acid composition and extinction coefficient are given.

A paracrystalline inclusion in Neurospora crassa. Induction by ethidium and acridine, isolation, and characterization.

A paracrystal indistinguishable from the one which occurs in the mitochondrial mutant abnormal-1 can be induced in wild-type Neurospora crassa after growth in either ethidium or euflavine. This paracrystal has been isolated and partially characterized. It appears to be composed of a single polypeptide (mol wt 68,000) which can be reversibly crystallized and dissociated by changes in the pH and ionic strength. When aggregated, the polypeptide forms oligomers which are arranged end-to-end into fibers. During the characterization of the polypeptide, it was found that the polypeptide's electrophoretic and immunological properties could be used as assays. Using these methods it was found that the polypeptide normally accumulates in a soluble form in the cytoplasm of wild-type Neurospora at the end of the log-phase of growth.

A Derepressible Active Transport System for Glucose in Neurospora crassa

Abstract Two glucose uptake systems have been detected in ungerminated conidia of wild type Neurospora crassa, strain 74A; the high affinity system has been characterized. Uptake and substrate competition experiments showed that the high affinity system was relatively specific for d-glucose but also transported the nonmetabolizable analogues 6-deoxy-d-glucose, 2-deoxy-d-glucose, and 3-O-methyl-d-glucose. The system exhibited a Km of 0.07 mm and a Vmax of 4 mµmoles per mg, dry weight, per min for 3-O-methyl-d-glucose uptake. The nonmetabolizable analogues were accumulated against concentration gradients; chromatographic analysis showed that the material accumulated in each case was the free sugar. Uptake by the high affinity system was inhibited by azide and dinitrophenol. These results were consistent with an active transport mechanism for uptake. Two alternative mechanisms, group translocation and coupled transport, were considered unlikely because no transitory substrate derivative could be detected during uptake and because influx of 3-O-methyl-d-glucose was not coupled to efflux of any organic metabolite. The high affinity system was repressed when the conidia were germinated on glucose medium but was restored when the glucose was either depleted or removed from the medium. This restoration was inhibited by cycloheximide. Two additional kinetic features were of importance in interpreting 3-O-methyl-d-glucose transport. First, previously accumulated substrate stimulated the subsequent influx of 3-O-methyl-d-glucose. The stimulation was proportional to the internal substrate concentration and was inhibited by azide. Second, efflux appeared to be a carrier-mediated process, but, in contrast to the high affinity uptake system, it was not inhibited by azide or by sulfhydryl reagents.