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Yeast geneticists frequently invoke:
"The awesome power of yeast genetics"
Biochemistry and genetics provide a powerful
combination for analyzing cell function
Genetics provides a window on cellular reactions
Mutations break a link in the metabolic map
KEGG (Kyoto Encyclopedia of Genes and Genomes – S. cerevisiae metabolic map
Cysteine and methionine play
central roles in metabolism
Mutant strains unable to grow in
the absence of methionine were
instrumental in identifying the
steps involved in methionine
synthesis
How is methionine synthesized in yeast?
How are mutants isolated?
How are auxotrophic mutants used to study methionine synthesis?
How is methionine synthesized in yeast?
How are mutants isolated?
How are auxotrophic mutants used to study methionine synthesis?
How are yeast mutants isolated?
Mutants are isolated in genetic screens in which investigators
look for particular phenotypes that occur at low frequencies
Investigators use irradiation or chemical
mutagens to increase the spontaneous
rate of mutation by orders of magnitude
(>50% cells may die…....)
Spontaneous DNA mutations occur with a
rate of ~10-8/generation
Wild type yeast do not require methionine to grow – screening for
mutants who require methionine to grow could provide insights into the
gene products required for methionine synthesis
Genetic screens
1. Mutagenized cells are plated under permissive conditions where all cells grow
2. Replica plates are grown under restrictive conditions to identify mutants
Restrictive conditions allow EITHER parental or mutant cells to grow (NOT both)
Permissive
Restrictive
All cells grow
Mutant cells don't grow
Original met mutants were isolated in screens where mutants were
unable to grow in medium without methionine
Mutant cells don't grow without
missing nutrient in medium
Three cells acquire
mutations
All cells grow on rich media
Only mutant cells grow in presence
of toxic analog (selective agent)
A primer on gene notation
Gene names are ITALICIZED
Dominant genes begin with a capital letter (in S. cerevisiae, all three
letters are capitalized, but not in other species)
LEU1
MET3
URA3
Mutant versions of the genes are italicized, but in lower case
leu1
met3
ura3
When possible, information about the mutant allele is noted:
leu1-1
Alleles correspond to
leu1-4
different mutations in
leu1-45
the original LEU1 gene
leu1-∆63 (deletion)
leu1::URA3 (LEU1 gene inactivated by insertion of a wild type URA3+ gene)
BY4742 is the parent strain for our met mutant strains
Genotype: MATa his3-∆1 leu2∆0 lys2∆0 ura3∆0
Mating
type
Strain's own ura3 gene is inactive
because of a deletion – this will be
useful when in our future
complementation experiments
BY4742 and its derivatives have multiple auxotrophies:
require histidine, leucine, lysine and uracil to grow
met mutants have metX::KANR alleles
How is methionine synthesized in yeast?
How are mutants isolated?
How are auxotrophic mutants used to study methionine synthesis?
Auxotrophic mutant strains
Carry mutations that render them unable to synthesize some
molecules required for viability
Grow in rich media
Do not grow in defined media lacking essential molecules that
they cannot synthesize
Auxotrophs have many uses in genetics - e.g. often used as hosts
for plasmids
Selective plating can
provide some clues
Which sulfur source can
replace methionine in
supporting the growth of a
met mutant?
You will first compare the
growth of strains with met
mutations on defined media
containing three different
sulfur sources:
Sodium sulfite
Cysteine
Methionine
Think of a mutation as a
missing arrow in the
diagram – which mutants be
able to make methionine
from the sulfur source?
siroheme
synthesis
aka MET17 and MET15
MET7
MET1
MET8
If sulfite was used as the
sulfur source, met3, met14,
and met16 (sulfate
assimilation) mutants would
grow
Mutations affecting sulfite
reductase (met5, met10,
met1 and met8) or
homocysteine synthase would
not grow
Selective plating provides some
clues, but may not discriminate
between mutants
MET1
MET8
aka MET17 and MET15
Differential media can also be used to place genes in the pathway
H2S forms a dark precipitate on BiGGY agar
BiGGY (Bismuth Glucose Glycine Yeast agar)
BiGGY contains sulfite and 0.1% yeast extract, a source of methionine