Cryobiology of neurospora crassa. I. Freeze response of Neurospora crassa conidia

Neurospora crassa conidia were frozen and thawed in water suspensions at various rates and with different minimum temperatures. Colony counts of the experimental conidia were compared with those of controls, which were taken as 100% survival. The data revealed that (1) survivals were near 100% after fast thaw (400 °/min) regardless of the freeze rate, (2) percentage of survival was inversely related to freeze rate when combined with slow thaw, (3) slow thaw (0.5 °/min) was damaging, and (3) the rates of freeze-thaw affected the system only in the −5 to −20 ° interval. The damaging freeze conditions were those which favor ice crystal growth. It is suggested that rupture of the membrane by ice crystals seems to be the plausible mechanism of damage in freezing and thawing N. crassa conidia.     

Glutamine metabolism in nitrogen-starved conidia of Neurospora crassa.

During nitrogen deprivation, de novo synthesis of glutamine synthetase was induced in non-growing conidia of Neurospora crassa. When ammonia or glutamine was added to conidia which had been deprived of nitrogen, glutamine and arginine accumulated at a higher rate than in condia not deprived of nitrogen. The degradation of exogenous glutamine to glutamate is apparently a necessary step in the accumulation of glutamine and arginine within the conidia. In non-growing conidia, a cycle probably operates in which glutamine is degraded and resynthesized. The advantages of such a cycle would be that the carbon and nitrogen could be used to synthesize amino acids in general, as well as for the synthesis and accumulation of arginine and/or glutamine in particular.     

Nuclear division cycle in germinating conidia of Neurospora crassa.

A temperature-sensitive mutant has been shown to be blocked at a specific point in the nuclear division cycle: just before the initiation of DNA synthesis at the time when the spindle pole bodies have duplicated but not separated. The metabolic activities of conidia of this mutant strain at the nonpermissive temperature have led us to conclude that the nuclei in a population of dormant conidia are arrested at various points in the nuclear division cycle. This conclusion is substantiated by the activities of conidia in the presence of the inhibitory drugs cycloheximide and hydroxyurea. In each inhibitory situation we observed that some, but not all, of the conidia were able to accomplish DNA synthesis and/or nuclear division.     

An inducible acetate transport system in Neurospora crassa conidia.

Neurospora crassa conidia possess an active transport system for the uptake of acetate. This system was characterized as: (a) energy dependent; (b) taking place against a concentration gradient; (c) saturating at higher substrate concentrations and (d) competitively inhibited by propionate. Activity of the acetate transport system can be further enhanced by preincubating conidia in 1 mM acetate medium for 180 min (the inducible transport system). The conidial system and the inducible system have similar properties. The development of the inducible transport was dependent on RNA and protein synthesis. A genetic control of this system was further confirmed by isolating a mutant acp-i acetate permease, inducible) that fails to develop the inducible transport system.     

Studies on phospholipase activities in Neurospora crassa conidia

Cytosol retinyl ester lipoprotein complex from rat liver was capable of transferring its unesterified retinol component to serum aporetinol-binding protein. In the presence of serum albumin and aporetinol-binding protein, 68% of retinyl ester was hydrolyzed and up to 30% of unesterified retinol was transferred from cytosol retinyl ester lipoprotein complex to serum aporetinol-binding protein in 24 h at 30 °C. The reconstituted retinol-retinol-binding protein complex showed biochemical and biophysical properties similar to native retinol-retinol-binding protein. Both native and reconstituted retinol-retinol-binding proteins had identical uv, CD, and fluorescence spectra as well as binding affinity to prealbumin. Treatment of cytosol retinyl ester lipoprotein with sulfhydryl reagent, with 1 n NaCl, or with diisopropyl fluorophosphate (0.14 mm) abolished the hydrolysis of retinyl ester; however, the activity of retinol transfer from cytosol retinyl ester lipoprotein complex to serum retinol-binding protein was still unaffected. The activity of retinol transfer was proportional to the amount of retinol content in the complex and the amount of aporetinol-binding protein. These experiments suggest that the cytosol retinyl ester lipoprotein complex serves three major functions: (i) as a storage form of retinyl ester and retinol; (ii) as an enzyme for hydrolyzing its own retinyl ester ligand; and (iii) as a medium for transfer of unesterified retinol to serum retinol-binding protein.     

Conserved mRNA from the conidia of Neurospora crassa

Summary Species of RNA showing the characteristics of mRNA have been isolated from ungerminated conidia and from mycelia of Neurospora crassa grown for 8, 16 and 24 hours. Molecular hybridization between such RNA species and DNA together with hybridization competition between mRNA from ungerminated conidia and from growth periods of 8, 16 and 24 hours, showed that conidia contain conserved mRNA. Such mRNA may participate in protein synthesis taking place up to 30 minutes of incubation of the conidia.     

Electron microscopy of the rodlet layer of Neurospora crassa conidia.

Neurospora crassa macroconidia possess a regularly arranged layer of small fibers (rodlets) near the spore surface. The structure and location of this layer were studied by making surface replicas, by negative staining, by freeze-fracturing and deep-etching, and by thin sectioning. When conidia were shaken vigorously in water, the layer fragmented and became separated from the surface in sheets. Negative staining of such sheets showed that the individual rodlets have a hollow central core. When conidia were shaken gently in water or fixative, large fragments of the rodlet layer often remained on the conidial surface. The fragments tended to fold back on each other such that multiple layers were sometimes seen in thin sections. It is concluded that in dry conidia the rodlets are located on the extreme outside of the spore where they form a monolayer with only occasional regions of overlap.     

Alteration of Tryptophan-mediated Regulation in Neurospora crassa by Indoleglycerol Phosphate

The accumulation of imidazoleglycerol phosphate during growth of Neurospora crassa in the presence of 3-amino-1,2,4-triazole was found to cause derepression of tryptophan synthetase and to inhibit the induction of kynureninase. Accumulation of indoleglycerol phosphate in response to growth in the presence of indole acrylic acid or anthranilic acid was also accompanied by derepressed synthesis of tryptophan synthetase. Enzyme synthesis in mutants (his-7 and trp-4) unable to form these intermediates was not altered under similar conditions. The rate of formation of tryptophan synthetase and kynureninase was found to differ in the presence of tryptophan and indole.     

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.     

Inactivation of Neurospora crassa conidia by singlet molecular oxygen generated by a photosensitized reaction.

Photodynamic damage of Neurospora crassa conidia was studied in the presence of the photosensitizing dye, toluidine blue O. Conidia which germinated to form colonies decreased in number as irradiation time became longer. The photoinactivation of conidia was suppressed by azide, bovine serum albumin, and histidine, and was stimulated in deuterium oxide. Wild-type conidia were less sensitive to the irradiation than albino conidia. In the wild-type, carotenoid-enriched conidia were more resistant against the lethal damage than the conidia which contained small amounts of carotenoids. These results suggest that singlet molecular oxygen causes photodynamic lethal damage to N. crassa conidia and that singlet molecular oxygen is quenched by endogenous 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.     

Ultraviolet-inactivation of conidia from heterokaryons of Neurospora crassa containing uv-sensitive mutations.

The effect of three UV-sensitive mutations of Neurospora crassa, upr-I, uvs-4 and uvs-6, on the ultraviolet-inactivation of conidia from two-component heterokaryons was investigated. In two-component heterokaryons with wild-type sensitivity to radiation inactivation, all three conidial fractions exhibited similar ultraviolet-inactivation curves. Each UV-sensitive mutation studied uniquely modified the ultraviolet-inactivation curves of conidia from two-component heterokaryons. In heterokaryons heterokaryotic for upr-I, the upr-I mutation was recessive and the repair function determined by the wild type allele was functional to some degree in homokaryotic upr-I conidia. All three conidial fractions of heterokaryons containing upr-I in both components showed increased sensitivity to ultraviolet light. The uvs-4 mutation was recessive and resulted in conidia with increased UV-sensitivity only when included in both components of a heterokaryon. Homokaryotic uvs-4 conidia, which arose from heterokaryons containing both uvs-4 and wild-type components, exhibited wild-type survival. Therefore, as with upr-I, there was a carryover the repair capability to conidia which were genetically UV-sensitive. The uvs-6 mutation, when included in one component of a two-component heterokaryon, resulted in increased UV-sensitivity of both heterokaryotic and homokaryotic uvs-6 conidia. When both components contained uvs-6, the UV-sensitivity of all three conidial fractions was increased and all showed similar inactivation curves. Thus, as with upr-I and uvs-4, there was a carryover of the wild-type repair capability to genetically uvs-6 conidia. Heterokaryon tests for complementation between two non-allelic UV-sensitive mutations showed that in heterokaryotic conidia, complete complementation occurred between upr-I and uvs-4.     

Inhibition of adenine and hypoxanthine uptake by guanosine in conidia of Neurospora crassa

Guanosine competitively inhibits the uptake of adenine and hypoxanthine by the general purine-base permease in conidia of Neurospora crassa. There is no reciprocal effect of adenine or hypoxanthine on guanosine uptake so it is suggested that guanosine can bind to the general purine-base permease to cause this inhibition but is not transported through this system. It is known that guanosine is transported by two separate nucleoside transport sites. Guanosine also noncompetitively inhibits the specific adenine uptake system of germinating conidia.