Molecular cloning of sequence-specific DNA binding proteins using recognition site probes

Abstract

Genes encoding sequence-specific DNA binding proteins can be isolated by screening lambda gt11 expression libraries with recognition site DNAs. This strategy is derived from that developed for the isolation of genes using antibody probes. Many different genes encoding transcriptional regulatory proteins have been cloned using this strategy. The DNA binding domains of these regulatory proteins contain different structural motifs including the helix-turn-helix, the "zinc finger" and the "leucine zipper". Various aspects of the screening strategy are evaluated and a detailed protocol is provided. In addition to binding site DNAs, protein and nucleotide probes have been successfully used to screen expression libraries. Therefore ligand based expression screening may be quite general in scope.