Genetic Complementation Protocols

Genetic selection provides a powerful tool for the study of cellular processes. It is particularly useful in analyzing protein sequence constraints when used in conjunction with directed molecular evolution. Our lab has used this approach to analyze the function of enzymes involved in DNA metabolism, to study the mutability of protein domains, and to generate mutant proteins possessing properties different from those selected by natural evolution (1 –4 ). To illustrate the concept, this chapter discusses genetic complementation of an E. coli strain defective in expression of the small subunit of ribonucleotide reductase (NrdB). Wild-type NrdB, in trans , is used to complement the hydroxyurea hypersensitivity of the defective strain. Cloning of the wild-type gene, expression, and complementation methods are discussed. The principles used for complementation with ribonucleotide reductase should be applicable to other enzymes for which a complementation system can be established.