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In a search for new inhibitors of the TNF-alpha promoter activity, a new spiro-compound, designated oxaspirodion, was obtained as a mixture of four isomers from fermentations of the ascomycete Chaetomium subspirale. The structure was determined by a combination of spectroscopic techniques.
Strains of three species of the ascomycetous yeasts Hanseniaspora guilliermondii, Torulaspora delbrueckii, and Debaryomyces hansenii, isolated from the above-ground parts of plants in similar biocenoses of distant geographic regions (Moscow and Novosibirsk regions), have been investigated. The strains in each species were indistinguishable with respect to phenotypic features and general DNA characteristics as determined by restriction analysis. However, comparison of the strains using PCR analysis with nonspecific primers revealed considerable intraspecific variability. From their electrophoretic patterns, the strains of the three species studied were found to cluster in accordance with the region of isolation. This phenomenon is interpreted as an example of the existence of geographical races in the major eurytopic species of yeasts.
Superficial fungal infections, including oral thrush, often affect aged full denture wearers and many individuals over 65 years old. The aim of this study was to examine the ultrastructural morphology of a very rare yeast, named Hanseniaspora uvarum/guillermondi, member of the Ascomycetes family, whose pathogenesis and behaviour is not widely known. The yeast was isolated from whitish lesions of the buccal mucosa of an 70 years old woman. The specimen was collected with a mouth swab and cultured in Sabourauds-Dextrose agar. The identification of the organism was performed on the Api 20C Aux system. The yeast colonies, after fixation in glutaraldehyde 3% for 1 hour were immersed in OsO4 1% solution for 1 hour and were "in tissue" stained with uranyl acetate. Ultrathin sections, were observed with TEM Jeol C x 100. Our ultrastructural observations showed that this yeast had a thick cell wall in which the outer surface appeared fuzzy. In some yeasts we observed multilayered intracytoplasmic membrane a figure which is not described as far as we know in any yeast. Many vacuoles were frequently observed in the cytoplasm and especially in the center of the oval shaped cells. Bilateral budding which form ascospores is identical for the morphology of this yeast.
In the course of our search for new biologically active metabolites, lachnellin A (1), a metabolite with high cytotoxic and antimicrobial activities, the structurally related lachnellins B, C and D (3, 4, 7), and naphthalene-1,3,8-triol (8), an inhibitor of malate synthase (EC 4.1.3.2), were isolated from submerged cultures of the ascomycete Lachnellula sp. A 32-89. The antimicrobial, cytotoxic and phytotoxic activities of lachnellin A depended on its reactivity and could be abolished by the addition of cysteine. The enzyme inhibiting activity of (8) was due to reactive intermediates during melanization and was no longer observed in the presence of serum albumin. In addition, rac-scytalone (9), (+)-trans-3,4-dihydro-3,4,8-trihydroxy-1 (2H)-naphthalenone (10), 2,5-dihydroxytoluene (11), and (R)-(-)-5-methylmellein (12) were obtained from the same source and biologically characterized.
Cloning of mating type (MAT) genes from ascomycetes has been hampered by low conservation among them. One of the pair of MAT genes, represented by MAT-2 of Cochliobolus heterostrophus (a loculoascomycete) and mt a of Neurospora crassa (a pyrenomycete), encodes a protein with a conserved DNA binding motif called the high mobility group (HMG) box. PCR with primer pairs corresponding to the borders of the C. heterostrophus and the N. crassa HMG boxes generated an approximately 0.3-kb product from genomic DNAs of MAT-2 and mt a strains, respectively, but not from MAT-1 and mt A strains. The C. heterostrophus primers amplified approximately 0.3-kb products from DNA of most loculoascomycete genera tested but not from DNA of pyrenomycete genera; this specificity was reversed with the N. crassa primers. The validity of the PCR procedure was documented by near sequence identity between the C. heterostrophus MAT-2 HMG box and PCR products from several Cochliobolus spp. and by cosegregation of the PCR product with mating type in progeny of Setosphaeria turcica and of Cryphonectria parasitica. Regions of the MAT locus flanking the HMG box were readily cloned using the TAIL-PCR procedure with a combination of random and specific primers.
Cloning of mating type (MAT) genes from ascomycetes has been hampered by low conservation among them. One of the pair of MAT genes, represented by MAT-2 of Cochliobolus heterostrophus (a loculoascomycete) and mt a of Neurospora crassa (a pyrenomycete), encodes a protein with a conserved DNA binding motif called the high mobility group (HMG) box. PCR with primer pairs corresponding to the borders of the C. heterostrophus and the N. crassa HMG boxes generated an approximately 0.3-kb product from genomic DNAs of MAT-2 and mt a strains, respectively, but not from MAT-1 and mt A strains. The C. heterostrophus primers amplified approximately 0.3-kb products from DNA of most loculoascomycete genera tested but not from DNA of pyrenomycete genera; this specificity was reversed with the N. crassa primers. The validity of the PCR procedure was documented by near sequence identity between the C. heterostrophus MAT-2 HMG box and PCR products from several Cochliobolus spp. and by cosegregation of the PCR product with mating type in progeny of Setosphaeria turcica and of Cryphonectria parasitica. Regions of the MAT locus flanking the HMG box were readily cloned using the TAIL-PCR procedure with a combination of random and specific primers.
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The influence of glucose concentration and other carbohydrates (monosaccharides: fructose, galactose, mannose; polyols: mannitol and sorbitol; disaccharides: lactose, sucrose and commercial sucrose; and industrial sugarcane molasses) were compared as sole carbon sources for the production of Botryosphaeran, an exopolysaccharide (EPS) produced by Botryosphaeria sp. The optimum glucose concentration for EPS production was 50 g l(-1). With the exception of mannitol, the fungus produced EPS on all carbon sources studied, with highest yields occurring with sucrose followed by glucose. All EPS showed exclusively glucose after acid hydrolysis and monosaccharide analysis. FTIR spectroscopy demonstrated the presence of beta-anomers indicating that all the EPS produced by Botryosphaeria sp. on the different carbon sources were essentially of the beta-D-glucan type.
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Supernumerary chromosomes, termed "conditionally dispensable" (CD) chromosomes, are known in Nectria haematococca. Because these CD chromosomes had been revealed solely by pulsed-field gel electrophoresis, their morphological properties were unknown. In this study, we visualized a 1.6-Mb CD chromosome of this fungus by three different types of fluorescence in situ hybridization. The CD chromosome at mitotic metaphase was similar in its appearance to the other chromosomes in the genome. Heterochromatic condensation was not distinct in the CD chromosome, suggesting that it is primarily euchromatic. It was also evident that the CD chromosome is unique and not a duplicate of other chromosomes in the genome. At interphase and prophase, the CD chromosome was not dispersed throughout the nucleus, but occupied a limited domain. Occasionally, occurrence of two distinct unattached copies of the CD chromosome were observed during interphase and metaphase.
Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.
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DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII. PKII activity is stimulated by Ca2+ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 microM-free Ca2+ and 1 microgram/ml of the modulator. The stimulatory effect of the Ca(2+)-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80. PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca(2+-)-calmodulin dependent protein kinase, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.