The yeast Malassezia furfur is a natural inhabitant of the human skin microflora that induces an allergic reaction in atopic dermatitis. To identify allergens of M. furfur, we separated a crude preparation of M. furfur antigens as discrete spots by 2‐D PAGE and detected IgE‐binding proteins using sera of atopic dermatitis patients. We identified the known allergens, Mal f 2 and Mal f 3, and determined N‐terminal amino acid sequences of six new IgE‐binding proteins including Mal f 4. The cDNA and genomic DNA encoding Mal f 4 were cloned and sequenced. The gene was mitochondrial malate dehydrogenase and encoded Mal f 4 composed of 315 amino acids and a signal sequence of 27 amino acids. We purified Mal f 4, which had a molecular mass of 35 kDa from a membrane fraction of a lysate of cultured cells. Thirty of 36 M. furfur‐allergic atopic dermatitis patients (83.3%) had elevated serum levels of IgE to purified Mal f 4, indicating that Mal f 4 is a major allergen. There was a significant correlation of the Phadebas RAST unit values of Mal f 4 and the crude antigen, but not between Mal f 4 and the known allergen Mal f 2.
We show a new transformation system for prototrophic yeast strains including those of Saccharomyces cerevisiae, Kluyveromyces lactis, K. marxianus, and Candida glabrata. This system is composed of an antibiotic, aureobasidin A (AbA), and its resistance gene AUR1-C as a selection marker. Southern analysis of genomic DNAs of the transformants indicated that the copy number of the plasmid increased from one to more than four, depending on the concentration of AbA used for selection of the transformants. The AUR1-C gene was also effective as a selection marker for gene disruption, and was able to disrupt both copies of the gene on homologous chromosomes of diploid cells by a single round of transformation. This system has a broad application in the transformation and gene disruption of prototrophic strains of a variety of yeast species.z 1998 Federation of European Biochemical Societies.
To study the mechanism of action of the antibiotic aureobasidin A (AbA) on yeasts, we isolated a dominant mutant of Schizosaccharomyces pombe which gave high resistance to AbA. From a genomic library of the mutant, an aur1R mutant gene conferring AbA resistance was isolated. One amino-acid mutation, a substitution of glycine with cysteine at residue 240, was responsible for the acquisition of AbA resistance. The wild-type aur1+ gene was essential for viability, and its over-expression enhanced significant resistance to AbA. The predicted protein of S. pombe aur1R was highly homologous in primary structure and hydropathy profile with that of Saccharomyces cerevisiae AUR1R isolated as an AbA-resistance gene. To analyze a role in cell growth of S. pombe aur1+, temperature-sensitive mutants (aur1ts) were obtained by random mutagenesis procedures using a modified PCR. The aur1ts mutation caused a defect in cell elongation at the non-permissive temperature and finally led to cell death. These results suggest that Aur1p was a target of the antibiotic AbA and was required in the cell elongation of cell-end tips and in the viability of S. pombe.
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