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Generally two selection markers are required to obtain homozygous mutations in a diploid background, one for each gene copy that is interrupted. In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of the Xanthophyllomyces dendrorhous yeast, using hygromycin B resistance as the only selection marker. To accomplish this, in a first step, a heterozygous hygromycin B-resistant strain is obtained by a single process of transformation (carrying the inserted hph gene). Following, the heterozygous mutant is grown in media with increasing concentrations of the antibiotic. In this way, the strains that became homozygous (by mitotic recombination) for the antibiotic marker would able to growth at higher concentration of the antibiotic than the heterozygous. The method can be potentially applied for obtaining double mutants of other diploid organisms. © 2012 Springer Science+Business Media New York., This work was supported by Fondecyt 1100324, Innova CORFO 07CN13PZT-17, CICYT BIO2010-20508-C04-01/04, and by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa.

Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that may play important roles in the evolution of eukaryote genomes and may be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is usually a laborious process, especially when trying to analyze a large number of samples. In this chapter, we describe a simple method to isolate dsRNA elements from yeasts using low amounts of starting material, and their application to different Xanthophyllomyces dendrorhous strains. Furthermore, the methodologies for enzymatic and hybridization characterizations, and quantification of relative dsRNA abundance are detailed. © 2012 Springer Science+Business Media New York.