The entire histidine operon of Escherichia coli K-12 was cloned in the vector plasmid pBR313, and a complete restriction map of the operon was determined. By using subclones, complementation tests, and enzyme assays, we were able to make a correlation between the physical map and the genetic map of the operon. We determined the sequence of a fragment of DNA 665 base pairs long, comprising the distal portion of the hisC gene, the proximal portion of the hisB gene, and the internal transcription initiation site hisBp. The efficiency of this promoter was assessed under different physiological conditions by cloning the DNA fragment in a recombinant vector system used to study transcriptional regulatory signals. The precise point at which transcription initiates was determined by S1 nuclease mapping.

The operator-distal genes hisBHAFI(E) of the Escherichia coli K-12 histidine operon were mapped on a DNA fragment 4,500 base pairs long. This fragment, originally present in a X transducing phage, was cloned in the vector plasmid pBR313. A restriction map was determined, allowing identification of the orientation of the genes in the fragment. The cloned genes were expressed in appropriate hosts, independent of the orientation of the DNA fragment, as shown by transformation tests and by enzyme assays of one of the gene products, hisB, histidinol phosphatase. An internal transcription initiation site was identified by isolation of the cellular RNA, hybridization to specific DNA probes, and mapping by S1 nuclease.With the development of recombinant DNA technology, the histidine operon of the enterobacteria Escherichia coli and Salmonella typhimurium has been the subject of extensive studies (9). Most of the work has been concerned with the elucidation of the mechanisms of operon regulation. Expression of this biosynthetic operon in both species is regulated at the transcriptional level by an overall mechanism termed attenuation (9, 48). Recent studies from our and other laboratories have been concerned with the genetic analysis of the regulatory region (28), DNA sequencing of the regulatory region (4, 20, 44) and of regulatory mutants (29), and cloning and expression (transcription and translation) of the proximal part of the operon (5, 14, 23). There are several other aspects of the his operon organization which are of potential interest: internal promoters (1, 21), intercistronic regions (40), and multifunctional gene products (42). As a prerequisite to studying some of these features, we report here the cloning of the distal portion of the E. coli K-12 his operon, a restriction map of this region, the orientation and expression of its genes, and the mapping of an internal transcription initiation site.MATERIALS AND METHODS Bacterial strains, phage, and plasmids. Bacterial strains used are listed in Table 1. Strain FB251 was constructed by selecting a spontaneous thy mutant with thymidine-trimethoprim selection (36) in strain FB186 and mating this derivative with an Hfr strain thy' recA56 (N1200), selecting for thy' recombinants, and scoring for UV sensitivity (14). Phage lysates and transduction tests were performed as previously de-scribed (2). Details of the construction of histidine recombinant plasmids are given below.Media, growth conditions, and enzyme assays. Liquid media were LB broth (36) and minimal medium (45) supplemented with 0.5% glucose. Solid media contained 1.2% agar (Difco) and were nutrient broth (36) and minimal medium (45) supplemented with 0.5% glucose. Amino acids were added at 0.5 mM; Lhistidine was added at 0.1 mM, and histidinol was added at 1.0 mM. Tetracycline and ampicillin were added to both liquid and solid media at 25 and 50 ,ug/ ml, respectively. Strains for enzyme assays or for RNA preparation were grown in minimal medium to an absorbancy at 650 nm of 0.8. Assay procedures for th...

We used an expression vector plasmid containing the Escherichia coli K-12 histidine operon regulatory region to subclone the E. coli hisC gene. Analysis of plasmid-coded proteins showed that hisC was expressed in minicells. A protein with an apparent molecular weight of 38,500 was identified as the primary product of the hisC gene. Expression was under control of the hisGp promoter and resulted in very efficient synthesis (over 100-fold above the wild-type levels) of imidazolylacetolphosphate:L-glutamate aminotransferase, the hisC gene product. The complete nucleotide sequence of the hisC gene has been determined. The gene is 1,071 nucleotides long and codes for a protein of 356 amino acids with only one histidine residue.