Neurospora Crassa Conidia Research Articles

Polysomes in dry- and wet-harvested ungerminated conidia ofNeurospora crassa

Polysomes in dry- and wet-harvested ungerminated conidia ofNeurospora crassa

Studies on phospholipase activities in Neurospora crassa conidia

Phospholipase A activity has been characterized in the conidia of Neurospora crassa. It has been observed that almost the entire phospholipase A activity in the conidia could be leached out by washing these with aqueous solution. The properties of this exocellular phospholipase A have been determined. Two distinct phospholipases have been fractionated from the “conidial water washing” by gel chromatography using Sephadex G-100. Using specific substrate, 1-stearyl-2-oleoyl lecithin, the predominant phospholipase activity in the fraction with higher molecular weight has been found to be due to phospholipase A 1 and that in fraction with lower molecular weight has been found to be due to phospholipase A 2. Properties of these two different enzyme fractions have been studied. The most active fraction in the latter gives a single band in 7.5% polyacrylamide gel. Lysophospholipase activity and detergent-stimulated phospholipase D activity have been detected also in the conidia. The effects of different physical and chemical agents on phospholipase A activity in the “conidial water washing” as well as in the purified fractions have been studied.

Analysis of biological thiols: Derivatization with monobromotrimethylammoniobimane and characterization by electrophoresis and chromatography

A new method for analysis of biological thiols based upon their conversion to fluorescent derivatives by reaction with monobromotrimethylammoniobimane (qBBr) is described. The derivatives are separated by chromatography and by electrophoresis on cellulose thinlayer chromatography plates. The use of two-dimensional mapping makes it possible to differentiate between a wide variety of biological thiols including N-acetylcysteine, CoA, cysteine, cysteinylglycine, cysteamine, ergothioneine, glutathione, γ-glutamylcysteine, homocysteine, mercaptopyrimidine, pantetheine, 4′-phosphopanetheine, thiosulfate, and thiouracil. For applications to biological samples thiols were isolated from crude extracts by binding to a mercuriagarose gel. Following removal from the gel with dithiothreitol, the thiols were derivatized with qBBr. The methods were tested by showing that glutathione is the major thiol in human red blood cells, that glutathione and ergothioneine are the major thiols in Neurospora crassa conidia, and that Bacillus cereus vegetative cells lack glutathione but contain cysteine, pantetheine, and an unidentified thiol in significant amounts.

Regulation of glutamine synthetase messenger ribonucleic acid in conidia of Neurospora crassa

A large quantity of prepackaged mRNA is present in conidia of Neurospora crassa as evidenced by its capacity to direct the synthesis of proteins in two heterologous cell-free systems. With this mRNA no in vitro synthesis of glutamine synthetase could be detected by specific immunoprecipitation. Upon incubation of the conidia under nongrowing conditions, the specific mRNA for glutamine synthetase was evidenced in the polyadenylated RNA and to a lesser degree in the nonpolyadenylated RNA. The amount of specific mRNA correlated well with the specific activity of glutamine synthetase in the conidia, which was dependent on the nitrogen source in the incubation medium. Based on these results we propose that conidia of N. crassa regulate its glutamine synthetase activity at the transcriptional level.

Permeability changes in membranes of Neurospora crassa after freezing and thawing

Conidia of Neurospora crassa which are in different physiological states show different rates of survival after freezing and thawing. [ 14C]adenine uptake by frozen and thawed conidia in different physiological states show a correlation with their survival. The uptake method was extended to study the survival of mycelium in log phase and stationary phase. From the uptake data it appears that log phase mycelium is extremely sensitive to all rates of freezing and thawing studied, while the stationary phase mycelium showed slight tolerance to freezing, if freezing was done at a slow rate. A study of the efflux of labeled compounds from the conidia in various physiological states or from the mycelia after freezing and thawing showed that, although efflux followed the same general trend as survival in conidia, it did not relate to the survival in mycelium, suggesting that the death of conidia or mycelium in the freeze-thaw treatments is not due to efflux of compounds.

Effect of ethylene glycol on transcription of Neurospora crassa conidial genome.

Ethylene glycol (EG) was found not to alter DNA sequences in Neurospora crassa conidia, though it is believed to be mutagenic in nature. Molecular hybridization revealed 20% increase in whole RNA transcripts in EG-treated conidia, which indicates that while untreated conidia increase RNA synthesis by 2.35fold, treated conidia are inhibited and undergo only a 1.2fold increase. Thus there is an inhibition in potential RNA synthesis, though some RNA synthesis goes on in presence of ethylene glycol.

Effect of the Deprivation of Amino Acids on Conidia of Neurospora crassa

SUMMARY: Arginine and glutamine, which prevents the catabolism of arginine, are accumulated when conidia of auxotrophic mutants of Neurospora crassa are deprived of an amino acid. This accumulation requires sources of carbon and nitrogen, an intact arginine biosynthetic pathway, de novo synthesis of pyrimidines and protein synthesis. There is coordination between the synthesis of glutamine and the synthesis and catabolism of arginine. Conditions which allow these amino acids to accumulate prevent exogenous arginine from being catabolized. Since more than half of the accumulated amino nitrogen is stored as glutamine and arginine, it is proposed that these amino acids are nitrogen and/or carbon reservoirs under non-growth conditions.

Differential deoxyribonucleic acid synthesis during microcycle conidiation in Neurospora crassa

In conidia of Neurospora crassa germinating at 25 degrees C, DNA synthesis measured by incorporation of tritiated adenosine reaches a maximum soon after the outgrowth of the germ tube (6--7 h after inoculation). In conidia heat-treated at 46 degrees C (for 15 h), a maximum of incorporation of the DNA precursor occurs already 1 h after inoculation, then the incorporation progressively declines until the end of the heat-shock. When such conidia are shifted to 25 degrees C, a maximum of DNA synthesis occurs during the development of the presumptive conidiophore as at the outgrowth of normal germ tubes. This wave of DNA synthesis is followed by a second maximum of DNA synthesis, occurring only in the microcyclized cultures, when the premature differentiation of proconidia takes place. Prevention of this second wave of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine respectively reduces or fully inhibits such induced conidial differentiation.

Cyclic AMP and cyclic GMP in germinating conidia of Neurospora crassa

A new method for obtaining synchronous germination allowed accurate time-course studies of endogenous levels of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-phosphate in germinating conidia of Neurospora crassa. The levels of both cyclic nucleotides remained constant throughout germination, showing that they neither signal nor respond to any of the biochemical changes which are known to occur in the germination process. Conidal germination was approximately normal in three cr-1 mutants of Neurospora which have been shown to be deficient in adenylate cyclase activity. Cyclic AMP levels in the mycelia of cr-1 mutants were low, but surprisingly, the levels in conidia were normal.

Role of the carbon source in the activation of ribonucleic acid synthesis during the germination of Neurospora crassa conidia

Neurospora crassa wild-type conidia incubated in minimal media synthesize ribosomal RNA (rRNA), transfer RNA (tRNA), and polyadenylate-containing RNA [poly(A) + -RNA] during the first hour of germination. Likewise, spores incubated in media containing only the carbon source (sucrose-only medium) synthesize a qualitatively similar spectrum of RNA molecules. However, at this stage, a quantitative difference (decrease) in the amount of poly(A) + RNA is detected in spores incubated in sucrose-only medium. At 3 h into germination, spores incubated in a minimal medium continue to synthesize the same spectrum of RNA molecules as at 1 h. Spores incubated for 3 h in a sucrose-only medium exhibit an alteration of the polysome profile and RNA synthesis. In addition, approximately 75% of these spores do not form germ tubes. In those which do form a germ tube the germ tube never elongates beyond one cell diameter.

Inhibition of germ tube formation in Neurospora

Phenethyl alcohol, m-cresol, and related compounds cause inhibition of germ tube formation in conidia of Neurospora crassa. Conidia continue to swell and form large spherical cells that are capable of multiple germ tube formation upon removal of inhibitor.

Isolation and identification of the conidial germination factor of Neurospora crassa.

The germination-essential substance (germination factor [GF]) that is lost from conidia of Neurospora crassa on exposure to solutions of low water activity has been isolated and identified as a group of iron-transport compounds, or siderochromes. The principal siderochrome of conidia is ferricrocin, a cyclic hexapeptide. A closely related substance, ferrichrome C, is tentatively identified as a minor constituent. The same substances are also present in extracts of mycelium along with small amounts of a third siderochrome, which has not been identified. The GF activity of culture filtrates is due to coprogen, the only siderochrome previously identified with N. crassa.

The physiological response of conidia of neurospora crassa to low temperature in the dehydrated, hydrated, or germinated state

The physiological response of conidia of neurospora crassa to low temperature in the dehydrated, hydrated, or germinated state

Rapid loss of a novel phenylalanyl-tRNA on germination of Neurospora crassa conidia.

SEVERAL theories attempt to explain the control of protein synthesis, and thus development at the level of transfer RNA1,10. We present evidence for changes in the isoaccepting species of phenylalanyl-tRNA during germination and growth of the fungus Neurospora crassa.

Genetic alterations of ribonuclease-sensitive glycoprotein subunits of amino acid transport systems in Neurospora crassa conidia

Neurospora crassa conidia have multiple and constitutive amino acid transport systems. Extraction by KCl releases amino acid-binding glycoproteins which have been purified by arginine affinity chromatography. Disappearance of certain fractions is coordinate with genetic lesions which reduce amino acid transport. Two such affinity fractions contain radioactivity when cells are grown on l-[ 14C]phenylalanine or on [ 14C]uridine, but not when cells are grown on [ 14C ]glucosamine. One purified arginine-binding fraction (B) contains 113 amino acid residues per minimum molecular weight. This glycoprotein also contains eight types of neutral sugar residues. No amino sugars were detected. Electrophoresis of crude extracts reveals five major Coomassie blue-staining species. The number of species is reduced, and the electrophoretic pattern is altered in extracts from transport-deficient strains. Tryptic “fingerprints” of these extracts indicate that mutations that reduce transport result in amino acid substitutions in the extractable glycoproteins. Nondialyzable material which absorbs light in the 260-nm region becomes dialyzable after digestion with RNase. Digestion of conidia with RNase reduces the amount of l-phenylalanine accumulated by the cells after 10 min of incubation with the amino acid.

Conidiation of Neurospora crassa induced by treatment with natrium fluoride in submerged culture.

A transient treatment of pregerminated conidia of Neurospora crassa with NaF induced young, submerged cultures to prematurely differentiate conidia. The inductive treatment decreased the rate of respiration (with lower RQ), reduced the relative concentration of nucleoside triphosphates, and inhibited leucine incorporation into protein and adenosine incorporation into RNA.

Sterols of Neurospora crassa and the pattern of their binding during the growth cycle

1. 1. Mycelium and conidia of Neurospora crassa were extracted with acetone, then saponified with alkaline pyrogallol and extracted with light petroleum. 2. 2. Both extracts (Fractions A and P, respectively) were assayed for sterol. Sterol ester was measured in Fraction A. 3. 3. Ergosterol was the major sterol in each case and there were smaller amounts of C 28 di-unsaturated sterols in each fraction. 4. 4. Cholesterol was present in conidia and young mycelium. 5. 5. Most of the sterol of conidia was in Fraction P (i.e. “tightly bound”). 6. 6. Most of the sterol in rapidly growing mycelium was in Fraction A (“readily extractable”). 7. 7. There was a substantial increase in Fraction P and a decrease in Fraction A at the onset of senescence. 8. 8. Ester content paralleled that for free sterol until day 9 after which there appeared to be a nett hydrolysis of ester. 9. 9. The increase in free ergosterol per culture in the A-fraction was interrupted for 2 days at the time of protoperithecium formation.

Different chromatographic forms of Neurospora crassa nucleases specific for single-stranded nucleic acids.

Three fractions with single-strand-specific nuclease activity have been isolated by chromatography on phosphocellulose and hydroxyapatite from Neurospora crassa conidia. Two of the fractions containing, respectively, 9% and 7% of the total nuclease activity recovered had the same chromatographic and enzymological properties as the endonuclease and exonuclease previously reported. The third fraction, containing 84% of the total nuclease activity recovered, was shown to contain a mixture of single-strand-specific endonuclease and exonuclease activities enzymologically indistinguishable from the activities previously described. The two components of this fraction were resolved by polyacrylamide gel electrophoresis. A fraction with the same chromatographic properties was also isolated from N. crassa mycelia in which single-strand-specific endonuclease, but no exonuclease activity, was detected. The two mycelial endonuclease fractions were enzymologically indistinguishable. The results were shown not to be due to artifacts of chromatography. Different batches of N. crassa conidia have been found to be heterogeneous with respect to contents of endonuclease and exonuclease.

The kinetics of l-aspartate transport in Neurospora crassa conidia

Transport of the acidic amino acids by Neurospora crassa conidia is shown to be mediated by neutral and general amino acid transport systems. The neutral transport system is shown to possess “high affinity”, but low transport capacity for the acidic amino acids. The general transport system is shown to have “low affinity” and high transport capacity for these amino acids. The non-linearity of a Lineweaver-Burk plot of aspartate transport by the wild type has been shown to result from an overlapping of two transport systems for accumulation of this amino acid. Removal, by mutation, of the neutral transport system restores the transport kinetics to a linear function. The combined, and separate, transport activities are shown to be stereospecific, preferring the l-stereoisomeric form over the d-form, and to transport the neutrally charged species of l-aspartate and l-glutamate. The transport activity is dependent on pH, and the V, but not the C 1 2 , constant of the general transport system is pH dependent. Two models for amino acid transport are presented as possible mechanisms to explain the observed kinetics. One model involves three genetically distinct transport systems. The second model utilizes a single “allosteric permease” to account for the kinetic data.

Mutation induction in Neurospora crassa incubated in mice and rats.

When Neurospora crassa conidia were injected into the peritoneal cavity of untreated mice or rats and kept there for more than 24 hours, the ad-3 mutation frequency among the surviving conidia increased sharply, more so in rats than in mice. This increase in the ad-3 mutation frequency was attributed both to direct cellular contact between conidia and mammalian cells and to macromolecules already present in untreated animals. Conidia enclosed in dialysis tubing or in diffusion chambers placed surgically in the peritoneal cavity had a much lower frequency of ad-3 mutations than conidia injected into the peritoneal cavity as a suspension. This was interpreted as indicating that the major fraction of mutations are mediated through a cellular contact. To determine whether the dialysis bags were permeable to mutagens, a comparison was made between the mutation frequencies obtained with conidia placed in dialysis bags and with conidia distributed at random throughout the peritoneal cavity in host animals treated with methyl methanesulfonate (MMS). MMS (100 mg/kg) was injected into the tail vein 8 hours after the conidia were placed in the animals. Ten hours after the injection of the mutagen, the conidia were recovered and analyzed for the induction of ad-3 mutations. The MMS-induced mutation frequency was the same among both groups of conidia, demonstrating that the dialysis bags did not become impermeable to small molecules during the time of incubation in the animal. In the host-mediated assay an indiactor organism is injected into the peritoneal cavity of an animal which is then treated with a chemical or its metabolites, to assay for mutagenicity. The present experiments show that the increased mutation frequency induced in the indicator organism after intraperitoneal injection and incubation may give false positive results in the host-mediated assay unless a comparison is made with suitable untreated controls. Autopsies of animals 24 days after intraperitoneal inoculation with Neurospora conidia, and sectioning and staining of various organs (Malling and Cosgrove, 1970) showed that some conidia were still localized in various organs, even though essentially all of them had been inactivated 96 hours after injection. The inactivation of these fungal spores may result from an enzymatic degradation of their DNA, mediated by the host, and halting this process prematurely may result in the induction of recessive-lethal mutations. Thus these studies also suggest that one of the important defense mechanisms of higher animals against infectious organisms may be the induction of mutations.