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. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Allocation of blade surface area to meiospore production was quantified for semelparous and iteroparous representatives of the genus Laminaria (Phaeophyta: Laminariales) at each of two sites in Barkley Sound, Vancouver Island, B.C. The annual semelparous sporophyte Laminaria ephemera produced sori between April and July; a maximum mean percentage of 31.7% of total vegetative blade area was devoted to sorus production, and 100% of the individuals were reproductive within at least one sample. The perennial iteroparous sporophyte Laminaria setchellii produced sori throughout the year; a maximum mean percentage of 30.4% of total blade area was devoted to sorus production, and a maximum of 54% of the individuals were reproductive within any sample. These results are discussed in the context of life history evolution. Concentric rings are visible in cross-section of the stipes of Laminaria setchellii. These rings were demonstrated to form annually, and thereby permitted estimation of individual age and of age structure among populations of L. setchellii at two sites in Barkley Sound. Age structures were dissimilar between populations, and showed no evidence of stability. The twelve year age class was most abundant at one site (Wizard Rock), and the two and three year age classes were most abundant at a second site (Execution Bay). / Science, Faculty of / Botany, Department of / Graduate

Zoospores of 17 species in 14 genera and sperm of 13 species in 11 genera of Laminariales were studied by electron microscopy. The zoospores are unique in the brown algae in lacking both an eyespot in the single chloroplast and an associated swelling at the base of the shorter, posterior flagellum. Spores of all species possess a distal whiplash on the longer, mastigoneme-bearing anterior flagellum; although it is only seldom preserved for electron microscopy, this appendage may sometimes be as long as the mastigoneme-bearing portion of the flagellum. A microtubular cytoskeleton with connections to the flagellar basal bodies is responsible for maintaining the shape of the zoospore. The sperm are also unique in the brown algae. They are elongate and possess two to three plastids and several mitochondria, but lack an eyespot. Their most distinctive feature is the long posterior flagellum which tapers distaliy as the doublet microtubules of the axoneme are transformed into singlets and then decrease in number. The sperm also bear a distal whiplash on the mastigoneme-bearing anterior flagellum. These laminarialean zoospores and sperm are ultrastructurally distinct from those known in other orders of brown algae, and from Chorda of the Laminariales, which confirms that Chorda is primitive. The longer posterior flageilum of the sperm recalls similar modifications in sperm of other oogamous brown algae, and suggests a common functional significance. / Science, Faculty of / Botany, Department of / Graduate

The development of sporangia and sporangioles is described in the mucoralean genera Choanephora, Blakeslea, Cunninghamella, and Mycotypha. The fine-structure of spore development is examined with transmission and scanning electron microscopy. Cytoplasmic cleavage in multisporous sporangia of Choanephora and Blakeslea involves fusion of cleavage vesicles to form a cleavage apparatus. Similar cleavage events occur in trisporous sporangioles of Blakeslea but the cleavage apparatus is oriented with the three longtitudinal "suture" lines of the sporangiole wall. The outer layer of spore walls is derived from a fibrous coating on the membranes of the cleavage apparatus. The inner wall layer is formed after deposition of the outer layer and is associated with the presence of granular vesicles in the spore cytoplasm. In monosporous sporangioles of Choanephora, Cunninghamella, and Mycotypha no cleavage apparatus is produced. Spores of all genera studied possess a bilayered wall with the outer layer demonstrating much greater electron-density than the inner layer. The relative thicknesses of the two wall layers varies greatly among genera. The outer layer is deposited in ridges and furrows in spores of Choanephora and Blakeslea. The structure of bipolar spore appendages is identical in these two genera. The ultrastructure of spines on sporangial surfaces is described. Spines on sporangioles of Cunninghamella exhibit a fine-structure different from that of Choanephora and Blakeslea. No spines are produced on spore walls of any of the genera studied. The identification of cellular components is examined using plastic-section histochemistry and light-microscopy. The cleavage apparatus initially contains carbohydrate which disappears as the spores mature. Bipolar spore appendages are shown to be composed of carbohydrate, with their bases staining for protein. In all spores, the cytoplasm shows intense staining for protein and weak staining for carbohydrates. Spore walls are difficult to stain with any of the procedures used. Based on the observed comparative ultrastructural development of asexual spores, recommendations and comments are made concerning the taxonomy of the Mucorales. / Science, Faculty of / Botany, Department of / Graduate

Thesis (Ph. D.)--University of Wisconsin--Madison, 1940. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 50-53).

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The fine structure of the basidiospores of Pluteus cervinus and Agrocybe acericola is described using TEM, SEM, and light microscopy.The basidiospore wall of Pluteus cervinus is bipartite. No surface ornamentation or germ pore is present. The protoplasm is surrounded by a typical membrane which lacks distinct invaginations. Spores contain much stored lipid, which is centrally located. Spores are uninucleated with the nucleus closely appressed to the cell membrane, located at the base of the spore near the hilar appendage. Mitochondria with few, well-delineated plate-like cristae are present. Endoplasmic reticulum (ER) is scant. Ribosomes occur regularly attached to the ER and outer mitochondrial membrane, as well as being densely packed throughout the cytoplasm. Various sized vacuoles containing an electron dense material are present. Microbody-like organelles are observed, which are probably glyoxysomes, since assays of malate synthase, a maker enzyme of the glyoxylate cycle, are positive.The basidiospore wall of Agrocybe acericola is composed of two distinct layers, both layers are continuous around the spores, at the germ pore the outer wall is very thin and the inner wall becomes thicker. The plasma membrane is appressed to inner wall and lacks invaginations. The protoplasm is densely packed with small granules, probably ribosomes and/or glycogen. Spores contain very little lipid with bipolar distribution. Mitochondria are well-defined and distributed in cytoplasm. Spores are binucleate with the two nuclei lying on a line nearly perpendicular to the long axis of the cell. Various size single membrane-bound vacuoles are widely distributed in the cytoplasm. These vacuoles were shown to contain acid phosphatase indicating lysosomal activity. Microbodies, probably glyoxysomes, are observed. Malate synthase assays are positive indicating the occurrence of the glyoxylate cycle. / Department of Biology

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LD2668 .T4 1954 H8 / Master of Science

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