Isolation and Identification of Yeasts from Natural ... - Library Science

Mutagenesis in Yeast 19
2.3. Isolation of Particular Classes of Mutants
2.3.1. Induction of Mitochondrial Mutations
2.3.1.1. PETITE COLONIE MUTANTS
1. Tubes of YEPD broth, 5 ml/tube.
2. Plates of YEPD agar.
3. Acriflavin solutron, 0.4%, or ethidium bromide solution, 1% (stock solutron).
4. Plates of YEPG (1% yeast extract, 1% peptone, 4% glycerol v/v) agar.
2.3.1.2. MIT- MUTANTS
1. Yeast culture, haploid, carrying the pet9 mutation (a nuclear mutatron con-
ferrmg respiratory deficiency) and one other auxotrophic marker, but other-
wise being rho+mit+ (i.e., wild-type with respect to mtDNA), and also a strain
of apet rho” tester strain (rho’ meaning lacking detectable mtDNA).
2. Medmm containing 10% glucose, 1% yeast extract, 1% peptone. Both
broth and agar.
3 350 mM Sterile MnC& solution.
4. YEPG agar (ethanol may be substituted for glycerol).
5. YNBD agar.
2.3.1.3. ANTIBIOTIC-RESISTANT MUTANTS
1, Yeast cultures, respiratory competent, 48 h, in YEPD broth. These may be
haploid or diploid, but must be respiratory competent.
2. YEPG plates, containing antibiotics, such as chloramphenicol or erythro-
mycin (1.5-2.5 mg/mL) or oligomycin (2.5 pg/mL).
3. 350 m&I Sterile MnCl* solution.
4. Tubes of 5 mL sterile YEPD.
2.3.2. Mutator Mutants
1. Yeast cultures (auxotrophic for lysine), 48 h, m YEPD medium. The pres-
ence of other auxotrophic markers is desirable.
2. Plates of synthetic complete medtum (YNBD, plus amino acids as
required), containing 1 and 20 pg/mL of lysme.
3. Sterile water for washing cells.
2.3.3. Determination of Revertant Frequency
1. Flask of limiting medium, at least 1 L (the low-lysine medium described
above; 1 yglmL lysine) containing all other requirements for the yeast
strain used. Place a magnetic stirring bar in the medium before sterihzmg.
2. Yeast strain, grown m normal medium.
3. Multiwell plates, presterilized, 2 mL/well capacity, 1000 wells/yeast stram.
4. Automatic pipetor, to deliver 1 mL medium/well.
5. Magnetic stirrer