Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive
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© Springer-Verlag 1997
O R I G I N A L PA P E R
J. Ramón De Lucas · Susana Valenciano · Ana I. Domínguez · Geoffrey Turner · Fernando Laborda
Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method
Received: 16 April 1997 / Accepted: 26 July 1997
Abstract Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3amino-1,2,4-triazole. From approximately 5 × 107 viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole–) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, L-glutamate, or L-proline). The Ole– mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim– (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 × g supernatant, but produced wildtype levels of penicillin. Key words Aspergillus nidulans · Peroxisomes · Oleate mutants · per mutants · pas mutants · Pim– mutants · acu mutants · Peroxisome biogenesis · Penicillin
J. R. De Lucas · S. Valenciano · A. I. Domínguez · F. Laborda (Y) Departamento de Microbiología y Parasitología, Facultad Farmacia, Universidad de Alcalá de Henares, Ctra. Madrid-Barcelona Km. 33, E-28871 Alcalá de Henares (Madrid), Spain Tel. +34-1-8854621; Fax +34-1-8854621 e-mail: [email protected] G. Turner Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Western Bank, Sheffield S10 2UH, UK
Introduction Peroxisomes (microbodies) are multipurpose organelles found in almost all eukaryotic organisms, including fungi; they are bounded by a single membrane, and compartmentalize a broad spectrum of metabolic processes [see Van Den Bosch et al. (1992) and Subramani (1993) for reviews]. They perform functions common to most eukaryotic cells. In fungi, besides carrying out the β-oxidation of fatty acids and the glyoxylate cycle, they are involved in methanol and alkane assimilation in some yeasts (Veenhuis et al. 1981; Tanaka and Ueda 1993) and in karyogamy in Podospora anserina (Berteaux-Lecellier et al. 1995). In addition, they contain the enzyme that catalyzes the last step of the biosynthesis of penicillin in Penicillium chrysogenum (Müller et al. 1991) When a fatty acid such as oleic acid is the sole carbon source for the yeast Sac
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