Cloning and mapping of a gene for translational initiation factor IF2 in Escherichia coli.

Abstract

A novel method, not relying on genetic complementation of a mutation, was used to clone a gene for translational initiation factor IF2. Two clones from a cosmid library of total E. coli DNA were isolated for their ability to overproduce IF2 in vivo as determined by quantitative immunoblotting. Maxicell analysis of cosmid-encoded proteins and specific immune precipitation of the labeled proteins showed that the structure gene for IF2 (infB) had been cloned. Subcloning fragments from the original cosmids located the infB gene to a 4.8-kilobase pair HindIII/BamHI fragment. This fragment was inserted into an an integration-deficient recombinant .lambda. phage that lysogenizes by homology. By mapping the point of lysogenization on the E. coli chromosome, infB was located at 68 min, very close to argG, nusA, rpsO and pnp. Because the gene for initiation factor IF3 is located at 38 min on the chromosome, the genes for translational initiation factors are not grouped together.