RAPID LOCALIZATION AND CHARACTERIZATION OF RANDOM MUTATIONS WITHIN THE 2-MU-CIRCLE SITE-SPECIFIC RECOMBINASE - A GENERAL STRATEGY FOR ANALYSIS OF PROTEIN FUNCTION

A method for rapidly localizing and characterizing mutations within the 2.mu. circle site-specific recombinase enzyme (FLP) is described. The strategy consists of (1) dividing the range coding for FLP (FLP) by artificially introduced unique restriction enzyme sites (that do not alter the amino acid sequence of the protein) into segments of 150-200 bp, (2) mutagenizing the engineered gene and selecting mutagenesis in vivo, (3) localizing each 8 mutation to one of the range segments by an in vivo complementation assay, and (4) characterizing the mutation sequencing of only this DNA segment. The experimental designs embodied by this method should be of wide application in investigations of protein function in general and of DNA-protein interactions in particular.