UBC Theses and Dissertations
Biochemical changes during sporulation and spore germination of macroconidia in Microsporum gypseum Leighton, Terrance James
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. 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 macroconidia germination response to inhibition, temperature shift, and addition of protease is presented. Microsporum gypseum macroconidia germinated at 37 C possessed from one to eight nuclei per germinated spore compartment. The distribution of nuclei per spore compartment was the result of a random packaging of nuclei from the available nuclear population. Partial germination inhibition by incubation at 25 C or at 37 C in the presence of 10⁻⁴ M PMSF resulted in an enrichment of germinated spores containing high numbers of nuclei per compartment. The selection of higher nuclear numbers was statistically significant. Compartments possessing high numbers of nuclei appeared to be pre-committed for spore germination and were therefore not sensitive to germination inhibition. The relationship of the germination response to temperature shift is discussed with respect to the organism's natural environment. Biochemical events which occur during macroconidial germination have been studied in M. gypseum. The specific activity levels of various metabolic enzymes have been assayed during germination time periods. The accumulated levels of several of these enzymes, as a function of exogenous carbohydrate source, has been investigated. M. gypseum was found to possess a constitutive glyoxalate shunt, a constitutive glucokinase, a fructose PEP transferase and a mannitol PEP transferase. The integration of endogenous reserve utilization during germination is discussed. The assimilation and conversion of ¹⁴C glucose, ¹⁴C amino acids and ¹⁴C uracil into TCA-precipitable material has been studied during early germination time periods. The time course of pool accumulations is also presented. The de novo synthesis of sporulation and spore germination proteins during spore germination is described. The integration of metabolite assimilation and differential synthesis is discussed. Developmental mutants affected in either sporulation or spore germination have been isolated from M. gypseum with the aid of nitrosoguanidine or as spontaneously-occurring mutants. The levels of several developmental proteins have been assayed during sporulation time periods in these mutants. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed. A large scale purification procedure for obtaining highly purified fungal chromatin is described. The isolated chromatin has normal ratios of RNA:DNA and non-basic protein:DNA. However, the ratio of basic protein:DNA was extremely low. The possibility of proteolytic degradation of histone during chromatin isolation was unlikely. Chromatin was 40 - 50% as template active as DNA. It is concluded that basic proteins do not represent a major fraction of M. gypseum chromosomal proteins.
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