Experimental Mycology最新文献

Heise, N., Travassos, L. R., and Cardoso de Almeida, M. L. 1995. Paracoccidioides brasiliensis expresses both glycosylphosphatidylinositol-anchored proteins and a potent phospholipase C. Experimental Mycology 19, 111-119. This study reports, for the first time, the detection of glycosylphosphatidylinositol (GPI) membrane anchors in proteins of a pathogenic fungus, Paracoccidioides brasiliensis. Taking into account that fungal antigens are found in the sera of paracoccidioidiomycosis patients and that cleavage of this glycolipid by phospholipases is a means of selective protein release, the presence of an enzyme with this property has also been investigated. Using a methodological approach in which the proteins were immobilized on nitrocellulose, treated with phospholipase C of Trypanosoma brucei and then probed with antibodies which recognize the 1,2-cyclic-phosphate inositol moiety formed as a reaction product in proteins bearing the glycolipid anchor, it was possible to detect a major glycoprotein in the 80- to 90-kDa range, as well as two other minor species of 66 and 43 kDa. All of them bind to Concanavalin-A and are also substrates of a very potent fungal phospholipase C which is inhibited by p -chloromercuri-phenylsulfonic acid and is insensitive to EDTA. The integrity of glycosylphosphatidylinositol anchors in proteins of P. brasiliensis is impaired by 0.1 M NaOH, a finding indicative of a diacyl glycerolipid moiety which is quite surprising since it is, with the exception of African trypanosomes surface proteins and Torpedo acetylcholinesterase, an uncommon feature among GPIs in general. The present findings may have implications in the pathology of paracoccidiodomycosis.

Templeton, M. D., Greenwood, D. R., and Beever, R. E. 1995. Solubilization of Neurospora crassa rodlet proteins and identification of the predominant protein as the proteolytically processed eas (ccg-2) gene product. Experimental Mycology 19, 166-169. Proteins from conidial rodlet preparations of Neurospora crassa were solubilized in trifluoroacetic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized rodlets revealed a predominant protein of approximately 7 kDa. This protein was absent from preparations of N. crassa cultures carrying the eas mutation. The protein was purified by reverse-phase high-performance liquid chromatography and the N-terminal amino acid sequence of the purified protein was found to be identical to an internal portion of the deduced amino acid sequence of eas. Comparison of the sequences indicates a 29-amino-acid leader which is cleaved to generate the mature protein.

Amir, R., Steudle, E., Levanon, D., Hadar, Y., and Chet, I. 1995. Turgor changes in Morchella esculenta during translocation and sclerotial formation. Experimental Mycology 19, 129-136. Turgor pressure was measured during six stages of growth and pseudosclerotial formation in Morchella esculenta indirectly (by thermocouple psychrometer) and directly (by cell pressure probe). The fungus was grown on a split plate, enabling separation between mycelium growing on defined medium (water potential -0.5 MPa) and sclerotia which formed on glucose noble agar (water potential -2.1 MPa). Under these conditions, nutrients were translocated from the mycelium to the developing sclerotia. Direct turgor potential measurements showed that the gradient between the mycelium and the sclerotia increases during sclerotial development (reaching a maximum of 0.53 MPa), thereby suggesting that translocation is a turgor-driven mass flow. During sclerotial development, the turgor potential in the peripheral tips of the sclerotial hyphae must be high enough to bring about the growth of the numerous hyphae, which comprise the sclerotium, and simultaneously low enough in the primary hyphae, which carry the stream of nutrients, to attract translocation from the mycelium. Since sclerotial hyphae are too small for direct measurement by cell pressure probe, a psychrometer was used, revealing high turgor in the sclerotial tissue (1.2 MPa) during selerotial development. Direct measurement in the primary hyphae at this time gave a value of 0.7 MPa. Taken together, these measurements indicate the presence of a turgor gradient inside the sclerotial tissue, from the primary hyphae to the peripheral cells. The present study is the first to make use of a cell pressure probe to measure turgor gradients in a fungus during translocation followed by sclerotial morphogenesis.

Kawasaki, L., Farrés, A., and Aguirre, J. 1995. Aspergillus nidulans mutants affected in acetate metabolism isolated as lipid nonutilizers. Experimental Mycology 19, 81-85. Interest in extracellular fungal lipases has increased mainly because of their industrial applications. However, no studies have been done on a genetically well characterized filamentous fungus like Aspergillus nidulans. Here we show that A. nidulans produces an extracellular lipase when grown in solid or liquid cultures containing lipids as carbon source. This lipase is glucose-repressed in a creA-independent fashion. Seven mutants isolated by their inability to utilize lipids as sole carbon source were also unable to utilize acetate as sole carbon source. Representative mutants from each of three complementation groups were tested for allelism with strains carrying well known mutations affecting acetate metabolism. They were found to contain acuD (Isocitrate lyase), acuF (PEP carboxykinase), and acuE (Malate synthase) alleles. Screening of lipid nonutilizing mutants for growth in acetate provides a method for the isolation of both lipase minus and new acetate metabolism mutants.

Gargas, A., and Taylor, J. W. 1995. Phylogeny of discomycetes and early radiations of the apothecial Ascomycotina inferred from SSU rDNA sequence data. Experimental Mycology 19, 7-15. We used nucleotide sequences of the small subunit ribosomal genes (SSU rDNA) to examine evolutionary relationships of apothecial ascomycetes (division Ascomycota; class Discomycetes sensu ), commonly known as the cup fungi. The apothecial ascomycetes include both lichen-forming and free-living fungi. We sequenced the SSU rDNA from representatives of 10 fungal genera from four orders: Pezizales (Ascobolus lineolatus, Morchella elata agg., Peziza badia); Leotiales (Leotia lubrica, Sclerotinia sclerotiorum); Caliciales (Calicium tricolor, Mycocalicium albonigrum, Sphaerophorus globosus); and Lecanorales (Lecanora dispersa, Porpidia crustulata). Of these, C. tricolor, S. globosus, L. dispersa, and P. crustulata are lichen-forming fungi. Based on parsimony analyses of approximately 1750 aligned nucleotides of their SSU rDNA, we determined a most parsimonious tree (MPT). This hypothesis suggests that the apothecial ascomycetes are a paraphyletic assemblage, basal to other groups of filamentous ascomycetes including representatives of the perithecial fungi and cleistothecial fungi. The most parsimonious tree produced using this dataset supported the monophyly of the orders Pezizales, Leotiales, and Lecanorales. However, there was no support for monophyly of the representative Caliciales; S. globosus had affinities with members of the Lecanorales. This phylogenetic hypothesis recognizes Pezizales as basal and supports Nannfeldt's hypothesis (1932) of a primitive apothecial ascomata with subsequent evolution of perithecial and cleistothecial forms. This MPT provides a foundation for understanding evolution of the ascomycetous fungi.

Yang, H., Shen, G-P., Park, D.C., Novotny, C. P., and Ullrich, R. C. 1995. The Aα mating-type transcripts of Schizophyllum commune. Experimental Mycology 19, 16-25. Aα1, Aα3, and Aα4 ds- and ss-DNA probes from the polymorphic Aα mating-type locus of Schizophyllum commune were used to probe Northern blots of poly(A⁺) RNA extracted from strains of various Aα mating types. The purpose of these experiments was to identify, map, and characterize the transcripts produced from the regions of the Aα locus. The transcripts unique to Aα mating type map colinear with the open reading frames identified from DNA sequence and are encoded within the fragments which activate the A developmental pathway in transformation. These data confirm the existence and structure of the previously hypothesized Y and Z Aα mating-type genes. Transcripts from the Y and Z genes are present in vegetative cells of homokaryons and dikaryons and in cells of the fruiting bodies. The presence of the transcripts throughout the life cycle is consistent with the model of Y and Z proteins as "master switches" of A-regulated development.

Hosking, S. L., Robson, G. D., and Trinci, A. P. J. 1995. Phosphoinositides play a role in hyphal extension and branching in Neurospora crassa. Experimental Mycology 19, 71-80. Three phosphoinositide turnover inhibitors, lysocellin (8 μM), piericidin B₁N -oxide (11 μM), and (2S, 3R, 5R)-3-azido-2-benzoyloxy-5-hydroxycyclohexanone, an inositol analogue (8 μM), were found to act as paramorphogens, inhibiting hyphal extension and increasing hyphal branching of Neurospora crassa without affecting specific growth rate. At higher concentrations (lysocellin, 50 μM; piericidin B₁N-oxide, 500 μM; inositol analogue, 50 μM) the compounds inhibited spore germination. At 1.6 μM, lysocellin reduced the level of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate in N. crassa by 65 and 90%, respectively. The site of action of lysocellin was shown to be phosphatidylinositol kinase. At 1.3 μM, lysocellin completely inhibited phosphatidylinositol kinase activity in vitro, a figure comparable to the concentration at which it inhibits phosphoinositide turnover in mammalian cells.

Wu, B.C., and Magill, C. W. 1995. Spontaneous mutations at fingerprint loci in clonal lineages of the rice blast fungus. Experimental Mycology 19, 86-90. When successive asexual generations of monospore-derived cultures from the Tm5 strain of Magnaporthe grisea were subjected to DNA fingerprint analysis with an anonymous genomic DNA probe, gain or loss of hybridizing fragments was common. Three of 24 isolates examined in the first asexual generation gained a BamHl fragment. In the next generation six cultures derived from two of these isolates all gained or lost up to three fragments. By comparison, only 1 of 28 isolates from a different culture showed a fingerprint band change, which was the loss of one fragment in the second generation. Where tested, changes in one isolate were also present in its progeny, and since 28 fragments could be scored in the original cultures, the overall patterns remained distinct. However, at the rate of change seen in the Tm5 isolates, fingerprint patterns have the potential to diverge rapidly.

Polizeli, M. L. T. M., Noventa-Jordāo, M. A., Marques da Silva, M., Jorge, J. A., and Terenzi, H. F. 1995. (1,3)-β-D-Glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa. Experimental Mycology 19, 35-47. The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-βD-glucan synthase activity. fz, sg, os-1 segregants from slime × wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, K_m app, V_max, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate × wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.

Gunde-Cimerman, N., and Cimerman, A. 1995. Pleurotus fruiting bodies contain the inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase-lovastatin. Experimental Mycology 19, 1-6. In the fruiting bodies of the fungus Pleurotus ostreatus, also called the oyster mushroom, we found a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase—lovastatin. The appearance of the inhibitor during the development of fruiting bodies was followed and lovastatin determined in the vegetative mycelium, in the primordia, as well as in different parts of sporocarps of different sizes. Less lovastatin was found in stipes as compared to pili or in mature stages in the lamellae and basidiospores.