Transcript Power Point
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