Molecular cloning of the Escherichia coli gene for diadenosine 5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase

Abstract

A clone overproducing diadenosine tetraphosphatase (diadenosine 5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase) activity was isolated from an Escherichia coli cosmid library. Localization of the DNA region responsible for stimulation of this activity was achieved by deletion mapping and subcloning in various vectors. Maxicell experiments and immunological assays demonstrated that a 3.5-kilobase-pair DNA fragment carried the structural gene apaH encoding the E. coli diadenosine tetraphosphatase. The DNA coding strand was determined by cloning this fragment in both orientations in pUC plasmids. It was also shown that the overproduction of diadenosine tetraphosphatase decreased the dinucleoside tetraphosphate concentration in E. coli by a factor of 10.