In Vitro Mutagenesis to Define Functional Domains

The identification of protein domains required for function is an important means of defining biochemical roles for a polypeptide. Our studies on regulatory proteins that function during the transition between mitosis and meiosis have extensively relied on targeted in vitro mutagenesis coupled with in vivo genetic assays (1 ,2 ). For example, fission yeast Mei3p is a meiotic activator. It functions by binding to and inhibiting a protein kinase that prevents meiosis (3 ,4 ). To perform structure and function studies, we developed a genetic assay to discriminate between active and inactive mei3 alleles. Systematic deletion mutations were created to define a region of Mei3p that was required for function in vivo (1 ,3 ). Next, site-specific alanine scanning mutagenesis was used to further define important functional residues in the active region of the protein (1 ). This approach led to the identification of a kinase-binding site and has been instrumental in the identification of potential substrates for the kinase. Here, one of the methods used for obtaining site-specific mutations is described.