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

Mutagenesis in Yeast 21
2.3.9. Antibiotic-Sensitive Mutants:
“Kamikaze” Mutants (7)
1. YEPD plates and YEPD broth.
2. Yeast strain BL15 (kamikaze), which grows normally at 32OC but dies
after 40 min exposure to 42°C.
3. Antibiottcs, as mentioned earlier, and trimethoprtm if desired.
2.3.10. PEP4 Mutants
1 Haploid yeast strain.
2. Solutions of N-acetylphenylalanine+naphthyl ester (APE) and fast gar-
net (GBC), which gives a red color with the naphthol released.
3. Hide powder azure, for detection of proteinase B-deficient mutants.
2.3.11. Inositol-Requiring Mutants
1. Mutagenized (EMS) haploid yeast strain, having any required auxotrophic
or other mutations to be used as markers.
2. Media: YEPD agar plates, and minimal medium (YNBD), plus any ammo
acids and other factors required for growth of the yeast strain, but without
inositol.
3. Minimal medium as described, + mositol, 2 yglmL.
2.3.12. Inositol-Excreting Mutants
1. Mutagenized yeast strain, as described.
2. Media: YEPD, minimal medium (YNBD plus adenine and any ammo acids
required by the indicator yeast strains).
3. Indicator yeast strains, auxotrophic for both adenine and inositol.
2.3.13. Sterol-Requiring Mutants
1. Wild-type, haploid cells, grown for 48 h at 30°C m YEPD.
2. YEPD with 40 pg/mL ergosterol and 15-20 ug/mL nystatin.
3. Equipment for UV-irradiation.
2.3.14. Mutants Auxotrophic
for 2’-Deoxythymidine 5’-Monophosphate (dTMP)
1. Haploid yeast strain, wild-type.
2. Mimmal medium (yeast nitrogen base without 0.67% amino acids) contain-
ing either glucose (2%) or glycerol (4% v/v) as sole carbon source, plus sulfa-
nilamide (5 mg/mL), ammopterin (100 pg/mL), vitamin-free casamino
acids (0.15%), adenine (30 pg/mL), and dTMP (100 and 10 pg/mL).
3. YEPD plates, with and without dTMP (100 pg/mL rf used).
4. Phosphate buffer, 0. IM (pH 7.1), for washing cells.