Presence of an actin-like protein in mycelium of Neurospora crassa.

Addition of ATP, CaCl2, and KCl to supernatants prepared from mycelia of Snowflake (strain 507), a morphological mutant of Neurospora crassa, results in the formation of filaments 70 nm in diameter. The "decorated" appearance of these filaments after incubation with heavy meromyosin from rabbits suggests they are actin-like.

Localization of trehalase in the ascospores of Neurospora: relation to ascospore dormancy and germination.

An association of trehalase with the innermost wall (endosporium) of ascospores of Neurospora is suggested, because this enzyme could be lyophilized in the presence of various wall components and heated in this dried state at 65 C without loss of activity. Ground ascospore walls, purified mycelial walls, a wall fraction consisting of protein, glucan and polygalactosamine, or bovine serum albumin stabilize trehalase under these conditions. No other substances tested protected as well as the above materials. Immunofluorescent labeling of trehalase shows that it is localized in the endosporium. Therefore, it is most probable that in dormant ascospores of Neurospora, trehalase, and its substrate, trehalose, are physically separated. Trehalose is located in the cytoplasm, whereas trehalase resides within the protein and carbohydrate matrix of the innermost major cell wall layer of the ascospore. The association with the cell wall protects the enzyme against the heating which is necessary to activate germination. Activation, whether by heat or chemical treatment (furfural), probably involves an increase in the permeability of the ascospore plasma membrane allowing trehalose to diffuse to the vicinity of its hydrolase, thereby providing the energy and intermediates for germination.

Activity and heat stability of trehalase from the mycelium and ascospores of Neurospora.

Trehalases from the ascospores of Neurospora tetrasperma and the mycelium of N. crassa were compared. Enzymes from both sources have identical electrophoretic mobilities, K(m)'s, responses to pH, immunological reactions, and activities in low-molarity buffers. Because both enzymes are so similar, conclusions about the properties of the ascospore enzyme may, be made by studying mycelial trehalase. Mycelial trehalase is most active and stable in low-molarity buffers. The enzyme exists in at least three species; the smallest has a molecular weight between 105,000 and 125,000 and is predominant in low-molarity buffers at 37 C. The stability of trehalase to heating at 65 C can be increased by increasing enzyme concentration and by the addition of polyols. Ascospores contain large amounts of trehalose, which protects trehalase from heat inactivation at 65 C. The importance of this phenomenon in vivo and its relationship to the localization of trehalase in ascospores is discussed.

Identification of a self-inhibitor from spores of Dictyostelium discoideum.

A self-inhibitor of spore germination has been isolated from spores of Dictyostelium discoideum, a cellular slime mold, and chemically characterized as 2-dimethylamino-6-oxypurineriboside.

Isolation, mapping, and characterization of trehalaseless mutants of Neurospora crassa.

Mutant strains of Neurospora crassa that lack trehalase and are unable to grow on trehalose were isolated, and the gene (tre) was positioned on the right arm of linkage group I. Maltase and beta-galactosidase activities are almost identical in tre(-) strains, whereas that of invertase was reduced by more than half and those of acid phosphatase and amylase were somewhat increased. Heterocaryons between standard and trehalaseless strains yield less than one-tenth the activity of the former. In addition, strains with duplications heterozygous for trehalase produce less than 1% of the activity of the standard strain. An inhibitor of trehalase has been found in tre(-) strains; its sensitivity to heat and proteolysis, and its nondialyzability suggest that this substance is a protein. The mig gene, which determines the rate of migration of trehalase on acrylamide gels, has been shown to be less than 1 map unit away from the tre gene.

The Nature of Cold-induced Dormancy in Urediospores of Puccinia graminis tritici.

When air-dry urediospores of the wheat stem rust, Puccinia graminis f. sp. tritici, are exposed to temperatures below freezing, their germinability is markedly reduced, even after prolonged thawing at room temperature. Germinability is fully restored by a brief heat-shock or by vapor phase hydration. We have found that this "cold dormancy" cannot be reversed once the spores contact liquid water. Enhanced loss of metabolites occurs immediately upon suspension of cold-dormant urediospores in liquid without a prior heat-shock. Such leakage is two to three times greater than from untreated or heatshocked cold-dormant spores and accounts for up to 70% of the soluble pool of metabolites normally present in germinating urediospores. Respiratory activity of cold-dormant urediospores declines rapidly during incubation in liquid. Incorporation of isotopic carbon into cold-dormant urediospores is only a fraction of that of untreated or heat-activated spores. Thus, cold shock transforms the spores into a state of supersensitivity to liquid water, which is reversed by heat-shock or slow hydration by vapor phase equilibration. The primary cause of damage to cold-dormant cells exposed to liquid water appears to be irreversible permeability damage, followed by metabolic injury.

Conversion of furfural to furoic acid and furfuryl alcohol by Neurospora ascospores.

The metabolism of furfural was studied with regard to possible mechanisms by which the chemical induces germination in ascospores. Incubation of ascospores in furfural resulted in the uptake of a small percent of the furfural, and the conversion of the bulk of it to furoic acid which was in turn converted to furfuryl alcohol. Conversion also occurred in Neurospora mycelium and conidia with the order being furfural to furfuryl alcohol to furoic acid. Conversion appears to be a noninducible enzymatic process localized on the outer surface of the cell. Conversion was completely inhibited without preventing germination indicating that conversion is not involved in the breaking of dormancy in Neurospora ascospores.

Furfural uptake by Neurospora ascospores.

Furfural uptake was studied with regard to possible mechanisms of inducing germination in ascospores. Uptake was found to involve a large, weakly bound reversible component and a small tightly bound irreversible component. Localization experiments indicate that almost all of the furfural removed from the media is bound to the spore wall. However, a small amount may penetrate into the cytoplasm. The results so far suggest that furfural induces germination by solubilizing or activating a bound or compartmentalized enzyme(s) on the cell membrane or other diffusion barrier of the cell.

Mechanisms of protection of trehalase against heat inactivation in Neurospora.

The half-life of trehalase and invertase at 65 and 60 C was found to be much greater when intact ascospores of Neurospora tetrasperma were heated, as compared with extracts. By contrast, no protection was afforded these enzymes when they were heated in intact conidia and mycelium of N. crassa or N. tetrasperma. The protective effect of ascospores for trehalase was further investigated by heating ascospore extracts before and after dialysis. The removal of small molecules by dialysis lowered the heat resistance of trehalase significantly in such extracts. When the dialysate from extracts of mycelium, conidia, or ascospores was added to dialyzed enzyme extracts, that from ascospores was by far the most active. However, the same dialysates had only a small protective effect on invertase. The addition of ashed dialysates did not protect trehalase, and trehalose and glucose protected less effectively than the dialysate.