A method for obtaining purified ungerminated macroconidia is described, and a technique for obtaining 85 to 90% germination of macroconidia under normal nutritional conditions is presented.

Germination of Microsporum gypseum macroconidia was accompanied by the release of alkaline protease, calcium ions, and inorganic phosphate into the germination fluid. The rate of germination was greatest during the first 2 hr, decreasing thereafter. This decrease in rate was accompanied by a decrease in protease activity, which was caused by an interaction of the enzyme with the inorganic phosphate released from the spores and accumulated in the germination medium after 2 hr. Germination of high spore densities was regulated by the ratio of released phosphate to protease protein, resulting in a constant percentage of germination at both high and low spore densities. A germination-defective mutant strain failed to germinate normally and released excessively high concentrations of phosphate into the germination medium during the initial 2 hr of incubation. Addition of calcium ions to germination mutant macroconidia stabilized spore morphology, prevented protease inactivation, and allowed normal germ-tube outgrowth. The germination of macroconidia appears to be regulated by the release of phosphate ions, which then inhibit the alkaline protease.

Three types of lysosomes containing either acid protease, alkaline protease, or phosphodiesterase were isolated from a Microsporum gypseum macroconidial homogenate on Ficoll gradients. The acid protease was contained in an assimilative lysosome since its activity was affected by the complexity of the exogenous nitrogen source. Ultracentrifugation and electron microscopy revealed that the alkaline protease-containing vesicles were associated with the spore coat material prior to macroconidial germination. During macroconidial germination, zones of spore coat hydrolysis were seen surrounding these vesicles. Other larger vesicles, believed to contain the phosphodiesterase, were also observed in the spore coat during macroconidial germination.

Interphase chromosomes were isolated in good yield from four species of fungi. In no case does the chromatin contain histones such as are characteristic of the chromosomes of other eukaryotic organisms.That histones are characteristic chromosomal proteins has been demonstrated for a wide variety of eukaryotic creatures. Thus, the chromosomes of higher plants and animals not only contain histones but, in addition, these proteins are similar in number, chemical properties, and even, in some cases, in primary structure (1, 2). Histones chemically similar to those of higher plants and animals have been found in the green alga Chlorella (3) and in the protozoan Tetrahymena (4,5), as well as in a wide variety of invertebrates.There have been several reports that the nuclei and/or chromatin of fungi lack histones, but contain instead chromosomal proteins of a less basic nature (6-9). We have examined the basic chromosomal proteins of several fungi, using what we believe to be the most rigorous of techniques for both chromatin isolation and histone characterization.We describe methods for obtaining purified fungal chromatin that result in the recovery of at least 70% of the DNA present in the homogenate. The possibility of proteolytic degradation of histones (if present) is unlikely. We find that histones analogous to those cf higher eukaryotes are missing in the fungi we have examined. MATERIALS AND METHODS Chromatin isolationAttempts to isolate chromatin from the fungus M4icrosporum gypseum by the methods suggested for liver and pea (10) were not successful. Fungal nuclei are sheared by these methods, and centrifugal forces sufficient to pellet the chromatin result in gross RNA contamination of the nuclear fraction. A more viscous grinding medium should afford greater protection to the nuclei during the cell breakage step. Stern's glycerol grinding medium admirably satisfied this requirement (11).Sporulation and spore purification procedures for Microsporum have been described previously (12, 13). 2-liter Erlenmeyer flasks, containing 600 ml of glucose (1% w/v) and neopeptone (Difco, 1% w/v) (pH 6.5), were inoculated with 106 conidia per ml and shaken at 350 rpm on a New Brunswick controlled environment dry-air shaker (30"C) for 4 days.The mycelia were harvested by suction filtration. The mycelial mat (150 g wet weight) was washed 4 times with 1-liter amounts of ice-cold physiological saline (pH 6.5).The mycelial mat was resuspended in 350 ml of grinding medium (glycerol 50% w/v, 0.5 M sucrose, 0.001 M CaCl2, and 0.05 M Tris, pH 8). The slurry was poured into an aluminum container and frozen by the addition of liquid nitrogen. Several additions of liquid nitrogen were necessary to completely freeze the material. The frozen slurry was ground to a coarse powder and placed into a Waring blendor. When the temperature of the grinding solution reached -30'C, the blendor was turned to full speed (110 V). Homogenization was continued until the temperature reached 0C. The homogenate was again poured into an aluminum c...

Macroconidia of Microsporum gypseum release free amino acids into the medium during germination. A single alkaline protease is also found in the germination supernatant fraction. The purified protease is capable of hydrolyzing isolated spore coats in vitro. Phenyl methyl sulfonyl fluoride (PMSF) is an effective inhibitor of the protease. Incorporation of PMSF at 10-4 M into the germination system inhibits spore germination and the release of free amino nitrogen. Addition of PMSF after germ tube emergence is completed has no effect on subsequent outgrowth. The addition of exogenous purified protease to quiescent spores results in more than a 2.5-fold increase in germinated spores. It is concluded that spore coat proteolysis is an essential event in the germination of dermatophyte macroconidia. A model system to explain macroconidial germination response to inhibition, temperature shift, and addition of protease is presented.