Detecting Mutations and Recombination in Yeast

Derivatives of E134 were plated on media that allowed us to detect new mutations: CAN medium is a standard minimal medium that lacks arginine and contains canavanine, a toxic agent that is an analog to arginine. If loss of function mutations occur in the CAN1 gene, the transporter loses its function, preventing the toxic canavanine from entering the cell and allowing the mutated cells to grow on this medium. The LYS2 locus of E134-derived strains contains an insertion of 14 adenine residues, which causes auxotrophy to lysine. Frameshift mutations (+1 or −2) can restore the Lys+ phenotype. SD-Lys is a standard-defined medium that lacks lysine. The trp1-289 mutation of E134-derived strains contains a single nucleotide change, which causes auxotrophy to tryptophan. Reversion to the original nucleotide allows the cells to grow on an SD-Trp plate, which is a standard-defined medium that lacks tryptophan.
Derivatives of BLS2 were plated on media that allowed us to detect new sister chromatid recombination: Strain BLS2 contains 5′ ade3 and 3′ ade3 fragments that are separated by URA3 marker. This construct allows the detection of new unequal sister chromatid recombination (USCR) by plating the cells on the appropriate medium (SD-HIS) [32 (link)]. Mutation and USCR rates were calculated as described in [33 (link)].