Cloning of mutH and identification of the gene product

Abstract

A specialized lambda transducing phage carrying the mutH gene and several deletion derivatives of this phage were characterized by restriction enzyme analysis. This analysis localized the mutH gene to a small region of bacterial DNA on the transducing phage and facilitated the subsequent cloning of this gene into the multicopy plasmid pBR322. The mutH gene is contained entirely on a 1.5-kb HindIII fragment as judged by the ability of plasmids carrying this fragment to complement mutH- alleles on the bacterial chromosome. Using recombinant plasmids containing the 1.5-kb HindIII fragment, we identified an Mr 25000 protein as the product of the mutH gene in an in vitro transcription-translation system as well as in maxicells. Various deletion derivatives of the mutH-containing plasmids that exhibit a Mut- phenotype also have lost the Mr 25000 protein.