Generation of astaxanthin mutants in Xanthophyllomyces dendrorhous using a double recombination method based on hygromycin resistance.
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.
Centro de Biotecnología y Dpto. Cs. Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.
Mauricio
M
Niklitschek
Marcelo
M
Baeza
María
M
Fernández-Lobato
Víctor
V
Cifuentes
D001487:Basidiomycota / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics; D016681:Genome, Fungal / Q000235:genetics; D006921:Hygromycin B / Q000494:pharmacology; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011995:Recombination, Genetic; D014158:Transcription, Genetic / Q000235:genetics; D014170:Transformation, Genetic; D024341:Xanthophylls / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics
D004271:DNA, Fungal; D020648:Peptide Elongation Factor 1; D024341:Xanthophylls; D006921:Hygromycin B; C005948:astaxanthine