title: Rapid localization and characterization of random mutations within the 2 micron circle site-specific recombinase: a general strategy for analysis of protein function.
abstract: A method for rapidly localizing and characterizing mutations within the 2 micron circle site-specific recombinase enzyme (FLP) is described. The strategy consists of dividing the gene 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, mutagenizing the engineered gene and selecting mutants in vivo, localizing each mutation to one of the gene segments by an in vivo complementation assay, and characterizing the mutation by 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.
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000502:physiology; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology; D005813:Genes, Synthetic; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation; D010957:Plasmids; D012150:Polymorphism, Restriction Fragment Length; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase