Rapid method for isolation and screening of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae.
We describe here a new method for the specific isolation of cytochrome c oxidase-deficient mutants of Saccharomyces cerevisiae. One unique feature of the method is the use of tetramethyl-p-phenylenediamine as a cytochrome c oxidase activity stain for yeast colonies. The staining of yeast colonies by tetramethyl-p-phenylenediamine is dependent upon a functional cytochrome c oxidase and is unaffected by other lesions in respiration. Since the tetramethyl-p-phenylenediamine colony staining reaction is rapid and simple, it greatly facilitates both the identification and characterization of cytochrome c oxidase-deficient mutants. Another feature of the method, which is made possible by the tetramethyl-p-phenylenediamine colony stain, is the use of an op1 parent strain for the isolation of nuclear pet or mitochondrial mit mutants in specific protein-coding genes. A parent strain that carries this marker selects against rho0 or rho- classes of pleiotropic respiratory-deficient mutants, since these are lethal in op1 strains. We have used this method to isolate 123 independently derived cytochrome c oxidase-deficient pet mutants and 300 independently derived mit mutants.
McEwen
J E
JE
Cameron
V L
VL
Poyton
R O
RO
D030401:Cytochrome-c Oxidase Deficiency; D003576:Electron Transport Complex IV / Q000235:genetics*; D005800:Genes, Fungal; D005821:Genetic Techniques; D046911:Macromolecular Substances; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012641:Selection, Genetic
D046911:Macromolecular Substances; D003576:Electron Transport Complex IV