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.     

Gene topography and function. I. Gene expression in germinating conidia of Neurospora crassa

In an attempt to find clues for the significance of the gene ordering along the eukaryotic chromosome, a system consisting of germinating conida of Neurospora crassa was studied. Thirteen enzyme activities corresponding to genes widely distributed on five chromosomes were determined in dormant and in germinating conidia. Ten of these enzymes showed lower activities in the resting state, and the time patterns of their increase were determined during germination. The results obtained do not support a scheme of sequential expression of genes during the emergence from dormancy as a counterpart of the sequence of the corresponding genes along the chromosome. Two of the loci studied were analyzed both in the normal (wild-type) ordering and in a translocated position in which the two genes are located in an inverted order respective to the centromere and farther apart from it. The altered order of the genes did not influence significantly the time and pattern of increase in the activities of the corresponding enzymes.     

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.     

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.     

Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa

Abstract Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa . Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia.     

Cryobiology of Neurospora crassa. II. Alteration in freeze response of Neurospora crassa conidia by additives

Neurospora crassa conidia in aqueous suspensions were frozen and thawed in the presence of various agents. Colony counts with these treatments were compared with those of the following (a) unfrozen, agent-treated, (b) unfrozen water suspended, and (c) frozen, water suspended. It was found that dimethyl sulfoxide (0.5–20%) resulted in total protection against freeze damage. Glycerol and calcium chloride decreased survival as much as 90% with fast freeze. The latter agents have properties which should decrease the rate of outflow of cellular water during temperature lowering. The results are consistent with the proposal that intracellular ice crystal growth to membrane rupturing dimensions is the damaging freeze mechanism under these conditions.     

Changes in the glutathione thiol-disulfide status of Neurospora crassa conidia during germination and aging.

Improved methods were developed for the determination of reduced glutathione (GSH), glutathione disulfide (GSSG), and protein-glutathione disulfide (PSSG) and applied to determine the glutathione status at various stages of the asexual life cycle for the band strain of Neurospora crassa. The GSH-GSSG ratio in freshly harvested dry conidia was found to be about 150 but decreased to around 6 when dryconidia were aged (stored) for 10 days after harvest. When conidia were germinated, this ratio increased to about 300 during the first 10 min of the 6-h germination process. In mycelia, during log-phase growth, the ratio was about 10-3. Changes in the ratio occurred primarily through changes in the GSSG content, which ranges from about 0.023 (mycelia) to 2(10-day aged conidia) mumol per g (dry weight) of residue, whereas GSH levels varied by a factor of about two. The PSSG content varied from 0.02 (mycelia) to 0.6 (10-day aged conidia) mumol per g (dry weight) of residue and generally paralleled the GSSG content. The results demonstrate the potential importance of thiol-disulfide reactions as a mechanism for the control of physiological properties associated with dormancy, and the observed changes in GSSG level are found to be compatible with the view that GSSG plays a role in the regulation of protein synthesis through control of polysome formation.     

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.     

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.     

Preparation of immobilized NAD glycohydrolase from Neurospora crassa conidia by hydrophobic interaction--characteristics of the enzyme derivative

NAD glycohydrolase from Neurospora crassa conidia has been immobilized by hydrophobic interaction on Sepharose 4B beads coated with propyl residues through CNBr activation. The bond resulted stable under a wide range of conditions (ionic strength, temperature, pH). As a result of immobilization the pH optimum for catalytic activity shifted by about 0.2 pH unit in the acidic direction, to lie between 7.5 and 7.3. The stability of the enzymatic activity was largely enhanced by effect of immobilization but the Km value towards NAD+ was increased compared with that of the free enzyme (1 X 10(-3) and 2 X 10(-4) M respectively).