"The awesome power of yeast genetics"

Biochemistry and genetics provide a powerful combination for analyzing cell function

Yeast geneticists frequently invoke:

KEGG (Kyoto Encyclopedia of Genes and Genomes – S. cerevisiae metabolic map

Genetics provides a window on cellular reactionsMutations break a link in the 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 screens1. Mutagenized cells are plated under permissive conditions where all cells

grow

2. Replica plates are grown under restrictive conditions to identify mutantsRestrictive conditions allow EITHER parental or mutant cells to grow (NOT

both) Restrictive

Mutant cells don't grow

Original met mutants were isolated in screens where mutants were unable to grow in medium without methionine

Permissive

All cells grow

Mutant cells don't grow without missing nutrient in medium

Three cells acquire mutations

Negative selection

All cells grow on rich media

Only mutant cells grow in presence of toxic analog (selective agent)

Positive selection

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:leu1-1leu1-4leu1-45leu1-∆63 (deletion)

leu1::URA3 (LEU1 gene inactivated by insertion of a wild type URA3+ gene)

Alleles correspond to different mutations in the original LEU1 gene

A primer on gene notation

BY4742 is the parent strain for our met mutant strains

BY4742 and its derivatives have multiple auxotrophies:require histidine, leucine, lysine and uracil to grow

Genotype: MATa his3-∆1 leu2∆0 lys2∆0 ura3∆0

Strain's own ura3 gene is inactive because of a deletion – this will be useful when in our future complementation experiments

Mating type

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 sulfiteCysteine

Methionine

Think of a mutation as a missing arrow in the diagram – which mutants be able to make methionine from the sulfur source?

MET7

aka MET17 and MET15

MET1siroheme synthesis

MET8

aka MET17 and MET15

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

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