6.1: Cells require sulfur-containing amino acids

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Mutant organisms provide powerful tools to study biochemical pathways in living cells. This semester, we are working with yeast strains that are unable to synthesize methionine (Met) or cysteine (Cys) because one of the genes involved in the biosynthetic pathway has been inactivated. Met and Cys are essential amino acids for all organisms. The sulfur atoms in their side chains impart distinctive chemistries to Met and Cys, which has important implications for protein function. Unlike us, wild type yeast are able to synthesize both Met and Cys, using only inorganic sulfate as a sulfur source. Each of the met mutant strains that we are using this semester is missing a single MET gene, and this deletion prevents the strain from growing on media containing only sulfate as the sulfur source. The deleted MET genes in the strains have been replaced with a bacterial kanamycin resistance (KAN^R ) gene by homologous recombination (Winzeler et al., 1999). Depending on the exact met mutation, the strains may be able to synthesize Met and Cys from other sulfur sources that they transport into the cell and convert to Met or Cys. In this lab, you will use selective media containing various sulfur sources and differential media to distinguish between three met mutants. In the next lab, you will use the polymerase chain reaction (PCR) to more conclusively identify the mutant met strains.