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?

Spontaneous DNA mutations occur with a rate of ~10-8/generation 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 Permissive All cells grow Restrictive 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 All cells grow Restrictive 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 Negative selection Positive selection 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 leu1-4 leu1-45 leu1-∆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

BY4742 is the parent strain for our met mutant strains 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 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?

MET7 aka MET17 and MET15 MET1 siroheme synthesis MET8 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?

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 MET1 MET8 aka MET17 and MET15 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