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

Mutagenesis in Yeast 31
3.3.12. Glycolytic Cycle Mutants (15):
Alcohol Dehydrogenase Mutants
These include alcohol dehydrogenase tADH), pyruvate carboxylase
(pyc), and phosphoenolpyruvate carboxykinase (PEPCK) mutants, and
also hexokinase and phosphofructokinase mutants and mutants affecting
galactose utilization and phosphogluconate dehydrogenase mutants.
Most of the mutants are isolated by methods based on failure to grow on
glucose, and growth on glycerol or other nonfermentable substrates.
3.3.12.1. ALCOHOL DEHYDROGENASE MUTANTS
These are selected for their resistance to ally1 alcohol. Yeasts producing
normal amounts of alcohol dehydrogenase convert ally1 alcohol to
acrolein, which is toxic, and are killed. Mutants lacking the gene encoding
this enzyme do not convert ally1 alcohol and survive.
1. Either:
a. Mutagemze the yeast strain (petite) and spread lo7 cells/plate on
medium containmg the lowest concentration of ally1 alcohol, followed
by plating the colonies obtained on the medium having the next higher
concentration, until the highest is reached; or
b. Grow the culture in YEP-glucose medium in the turbidostat, and increasing
the concentration of ally1 alcohol from zero, first to 1 O-l 2 mA4, and increa-
sing the concentration stepwise until the desired concentration is reached.
2. Isolate strains resistant to ally1 alcohol, grow, break the cells, and test for
altered forms of ADH using SDS-PAGE electrophoresis (see Note 27).
3.3.12.2. PYRWATE CARBOXYLASE MUTANTS (PYC) (16)
These mutants have nutritionally complex requirements, which makes
it desirable to isolate them in otherwise wild-type strains. They affect the
anapleurotic reactions which supply intermediates to the Kres cycle
reactions. This may make it possible to investigate the interface between
reactions taking place within the mitochondria and those going on in the
cytoplasm. The mutation involved in a deficiency in pyruvate carboxy-
lase is a recessive single-gene nuclear one. These mutants resemble
biotin-deficient mutants in having an aspartic acid requirement; pyru-
vate carboxylase requires biotin as a cofactor.
1. Mutagenize the yeast with EMS (1 h in 3% EMS), recover the cells, wash
first with 6% Na2S203, and then with phosphate buffer.
2. Dilute the suspension and plate on YNELglucose agar, 100-200 viable
cells/plate, to eliminate amino acid auxotrophs.