Construction and expression of hybrid plasmids containing Escherichia coli K-12 uxu genes

Abstract

The three genes of the Escherichia coli K-12 uxu region (uxu operon and regulatory gene) were isolated on a ColE1-uxu hybrid plasmid from the bank of Clarke and Carbon, and a restriction map of this region was established. In vitro recombination techniques were used to subclone the uxu restriction fragments into the plasmid vector pBR322 or pBR325. The various chimeric plasmids obtained were analyzed by restriction mapping and characterized genetically by introducing them in uxu mutant or wild-type strains. Differential rates of synthesis of the enzymes coded for by the uxu region were measured in the plasmid-containing strains; amplification of the products of the cloned genes was up to 40-fold the level found in haploid strains. The enzymes coded for by uxuA and uxuB were synthesized in vitro in a coupled transcription-translation system, confirming the results of the cloning experiments. The restriction analysis also suggests that the transcriptional direction of the uxu operon is from uxuA to uxuB and that the order of the loci in this region is: uxuR (regulatory gene), uxuB, uxuA, uxuAp (promoter), uxuAo (operator).

Analysis of theEscherichia coligenome VI: DNA sequence of the region from 92.8 through 100 minutes

“…We therefore identify these ORFs as uxuA, B and R, respectively. The identification is supported by identity of the restriction maps for the whole operon (22) and mapping data (23), but the whole operon is in the opposite orientation to that shown on the current genetic map (10). cst.…”

Genetic and physical characterization of a plasmid carrying the E. coli kdgA gene

“…The isolation of plasmid pAL1 completes the molecular cloning of the genes involved in the hexuronate-catabolizing system [20][21][22]. This plasmid bears the kdgA and edd genes.…”

Cloning and endonuclease restriction analysis of uidA and uidR genes in Escherichia coli K-12: determination of transcription direction for the uidA gene.

“…Further purification of the DNA was done by dye-buoyant centrifugation in CsCl gradients containing ethidium bromide (24). Restriction endonuclease digestion and DNA ligation were carried out as described previously (25). E. coli cells were prepared for transformation with plasmid DNA by the method of Mandel and Higa (16), as modified by Wensink et al (33).…”

“…Less than 1.0 Md separates uidA and uidR since BamHI-1 and BamHI-2 lie within uidA and Wuid, respectively. uidR is probably very close to uidA, as exuR is close to the exuT operon (18) and uxuR is close to the uxuA-uxuB operon (25).…”