Cloning of the Structural Genes of the Escherichia coli Adenosinetriphosphatase Complex

The structural genes (unc genes) encoding the eight proteins of the adenosine triphosphatase complex of Escherichia coli are arranged in an operon at about 83.5 min on the E. coli chromosome. Five of these genes, uncA, D, G, H, and C, code for the α, β, γ, δ and ϵ subunits of the soluble portion of the complex (F₁-ATPase), respectively, and the remaining three genes, uncB, E, and F, encode the insoluble portion (F₀). A number of approaches to the problem of cloning the structural genes of the unc operon have been used. A simple approach to cloning the unc structural genes uses an F′ plasmid isolation as the enriched DNA source for insertion of the operon into a multicopy plasmid vector using standard restriction and ligation procedures. A partial diploid (strain AN862) that duplicates the entire unc operon of E. coli is used as a source of the DNA to be cloned. The F′ plasmid in strain AN862 can be isolated and used as an enriched source of the unc genes. After restriction of the isolated F′ plasmid and ligation with the cloning vector, the ligation mix is used to transform an appropriate recipient strain, and selection is made for the antibiotic resistance determinant of the plasmid vector and for the ability to grow on succinate minimal medium.