Cloning and mapping of the manganese superoxide dismutase gene (sodA) of Escherichia coli K-12

Abstract

An Escherichia coli gene bank composed of large DNA fragments (about 40 kilobases) was constructed by using the small cosmid pHC79. From it, a clone was isolated for its ability to overproduce superoxide dismutase. The enzyme overproduced was manganese superoxide dismutase, as determined by electrophoresis and antibody precipitation. Maxicell analysis and two-dimensional O'Farrell polyacrylamide gel electrophoresis demonstrated that the structural gene, sodA, of manganese superoxide dismutase was cloned. Subcloning fragments from the original cosmid located the sodA gene within a 4.8-kilobase EcoRI-BamHI fragment. This fragment was inserted into a lambda phage which was deleted for the att region and consequently could only lysogenize by recombination between the cloned bacterial DNA insertion and the bacterial chromosome. Genetic mapping of the prophage in such lysogens indicated that the chromosomal sodA locus lies near 87 min on the E. coli map.