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  • 1
    Electronic Resource
    Electronic Resource
    Molecular Genetic Approaches to the Study of Fungal Pathogenesis* (1989)
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Phytopathology 27 (1989), S. 463-481 
    Details
    ISSN: 0066-4286
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Biology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1146/annurev.py.27.090189.002335
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  • 2
    Electronic Resource
    Electronic Resource
    Probing the active site of mitogillin, a fungal ribotoxin[The first ] (1998)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 29 (1998), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are highly specific ribonucleases, which inactivate the ribosome enzymatically by cleaving the eukaryotic 28S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326, which are present in a 14-base sequence (the α-sarcin loop) conserved among the large subunit rRNAs of all living species. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing point mutations using hydroxylamine into a recombinant Met-mature mitogillin (mitogillin with a Met codon at the N-terminus and no leader sequence) gene constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an Escherichia coli expression vector, the proteins overexpressed and purified to homogeneity and their activities examined by in vitro ribonucleolytic assays. These studies identified the His-49Tyr, Glu-95Lys, Arg-120Lys and His-136Tyr mutations to have a profound impact on the ribonucleolytic activities of mitogillin. We conclude that these residues are key components of the active site contributing to the catalytic activities of mitogillin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00987.x
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  • 3
    Electronic Resource
    Electronic Resource
    Construction and pathogenicity of Aspergillus fumigatus mutants that do not produce the ribotoxin restrictocin (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA), produces a potent cytotoxjn called restrictocin. To investigate the role of restrictocin in (PA, we have constructed fungal strains in which the res gene has been inactivated by gene disruption. These disruptants lack the specific extracellular ribonucleolytic activity associated with restrictocin, as measured by an in vitro rabbit reticulocyte lysate assay. Western blot analysis of one drsruptant, using an anti-restrictocin monoclonal antibody, confirmed that the toxin is not produced. The growth characteristics of the disruptants could not be distinguished from those of their parental isolates on a variety of culture media. The pathogenicity of two disruptants was assessed in a murine model of IPA. There were no significant differences in mortality when these strains were compared with the parental isolates and an ectopic transformant. In addition, histological examination of infectediung tissue did not reveal any obvious differences in the number or size of fungal colonies or in the polymorphonuclearleucocyte response. Our results demonstrate that restrictocin is not an important virulence factor in this model of IPA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1993.tb01236.x
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  • 4
    Electronic Resource
    Electronic Resource
    Biochemical characteristics of sporelings of Puccinia graminis f. sp. tritici and protoplasts from wheat isolines of potential significance in specificity (1984)
    Oxford, UK : Blackwell Publishing Ltd
    Plant pathology 33 (1984), S. 0 
    Details
    ISSN: 1365-3059
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: The affinity of various lectins for protoplasts from isolines of wheat differing in their reaction to Puccinia graminis Pers. f. sp. tritici, Ericks and E. Henn. race 21 and for infection structures of the fungus was studied. Protoplasts of both isolines were agglutinated by concanavalin A and soybean lectin and lysed by wheat germ agglutinin. Pokeweed mitogen and wheat germ agglutinin bound to fungal germ tubes and appressoria but not to uredospore walls, substomatal vesicles or infection hyphae.Viability of protoplasts from either isoline was not affected when they were incubated with uredospores, germlings with and without infection structures or with an extract of the susceptible line, heavily infected by the fungus. No differences between polypeptide fractions prepared from protoplasts of the isolines were detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-3059.1984.tb02645.x
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  • 5
    Electronic Resource
    Electronic Resource
    An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 6 (1992), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Invasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus, is a life-threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase-chain-reaction-based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS-polyacrylamide gel etectrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild-type cells, the mutant had undetectable activity on azocollagen and elastin-Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus, and that the mutant strain will be useful in assessing the role of AFAlp in pathogenicity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1992.tb00891.x
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  • 6
    Electronic Resource
    Electronic Resource
    Insertional mutagenesis of Aspergillus fumigatus (1998)
    Springer
    Molecular genetics and genomics 259 (1998), S. 327-335 
    Details
    ISSN: 1617-4623
    Keywords: Key words Insertional mutagenesis ; Aspergillus fumigatus ; Electroporation ; Restriction enzyme-mediated integration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380050819
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  • 7
    Electronic Resource
    Electronic Resource
    Comparison of Virulence between Clinical and Environmental Isolates of Aspergillus fumigatus (1998)
    Springer
    European journal of clinical microbiology & infectious diseases 17 (1998), S. 778-780 
    Details
    ISSN: 1435-4373
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Using a mixed infection model of murine invasive pulmonary aspergillosis, the comparative virulence of three clinical and four environmental isolates of Aspergillus fumigatus was investigated after intranasal inoculation. Coloured conidiospore mutants were first derived from clinical strains by ultraviolet mutagenesis and then compared with the parental strains and environmental strains. When the slight reductions in virulence associated with the spore colour mutations were taken into account, some environmental strains were shown to be less virulent than their corresponding clinical strains. It has yet to be determined whether these differences can account for the observation that many patients with invasive pulmonary aspergillosis appear to be infected with a single strain of Aspergillus fumigatus.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s100960050184
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  • 8
    Electronic Resource
    Electronic Resource
    Molecular epidemiological study of invasive pulmonary aspergillosis in a renal transplantation unit (1994)
    Springer
    European journal of clinical microbiology & infectious diseases 13 (1994), S. 318-321 
    Details
    ISSN: 1435-4373
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The molecular epidemiology of an outbreak of invasive pulmonary aspergillosis which occurred on a renal transplantation unit was investigated. Restriction fragment length polymorphism and random amplified polymorphic DNA analyses were used to characterise the isolates ofAspergillus fumigatus from the patients (2 isolates from each of 2 patients) and from the environment (11 isolates). While the isolates from the environment could be readily distinguished from each other by both methods, each patient appeared to be infected with a single strain. These findings lend support to the suggestion that certain isolates ofAspergillus fumigatus are more pathogenic than others.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01974610
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  • 9
    Electronic Resource
    Electronic Resource
    The role of the Aspergillus fumigatusareA gene in invasive pulmonary aspergillosis (1998)
    Springer
    Molecular genetics and genomics 258 (1998), S. 553-557 
    Details
    ISSN: 1617-4623
    Keywords: Key words invasive pulmonary aspergillosis ; areA ; nitrogen utilisation ; fungal pathogenicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The areA gene of Aspergillus nidulans is a positive-acting transcriptional factor required for the expression of genes involved in the utilization of a broad range of nitrogen sources other than ammonium and glutamine. We have investigated the role in pathogenesis of the corresponding gene (AfareA) of Aspergillus fumigatus, a causative agent of invasive pulmonary aspergillosis. Stable and unstable AfareA − strains were constructed and tested for altered virulence in mice on the basis of host survival, mixed infection experiments and genetic reversion studies. These showed that the AfareA gene contributes to, but is not essential for, virulence. Strains carrying extragenic mutations that partially suppress the AfareA − phenotype for growth on different nitrogen sources were tested for altered virulence in mixed infection studies. One of these was found to be more virulent than the parental AfareA − strain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380050767
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