Identification of gene products from cloned fragments of the left arm of λdapB2

Abstract

A set of recombinant plasmids encompassing the bacterial substitution in λdapB2 (Mackie, G. A. (1980) J. Biol. Chem. 255, 8928–8935) has been characterized genetically by complementation and biochemically by an analysis of the proteins encoded by individual plasmids in vitro. In addition to a cluster of four genes which includes the dapB locus, a total of seven gene products has been identified. One of these is ribosomal protein S20, whose gene (rpsT) has been localized to a 0.56-kilobase segment of DNA bounded by HindIII and HindII sites. Positive identification of this gene on plasmids pGM9 and pGP2 has been achieved by analysis of the products encoded by these plasmids in vitro and by the ability of pGM9 to complement a strain lacking S20 among its 30S subunit proteins. A second gene is that for isoleucyl tRNA synthetase (ileS). Its presence on pGM21 has been ascertained by the latter's ability to direct the synthesis of a protein in vitro with the size anticipated for this gene product. Extracts of cells harbouring this plasmid also exhibit greater isoleucyl tRNA synthetase activity than parental extracts. A third gene is at least 1.3 kilobases distant from rpsT and encodes a protein of 24 000 molecular weight, of unknown function. This locus is in turn about 6.5 kilobases from λ gene E. The latter gene, and several others, all of which likely derive from λ DNA are carried on a 5.2-kilobase fragment inserted in the piasmid pGM11 which includes the junction between phage and bacterial sequences on the left side of λdapB2. This fragment of DNA is inserted into the vector in a manner which permits the coupled transcription and translation of λ genes D and E in vitro, despite the absence of their natural promoter. The most striking feature of the organization of the bacterial genes on λdapB2 is the clustering of genes for polypeptides in the right half of the bacterial substitution in contrast to the apparent minimal use of the coding potential in the left half of the substitution.