RESUMO
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.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Regulação da Expressão Gênica , Genes Bacterianos , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Sequência de Bases , Enzimas de Restrição do DNA/metabolismo , Genes Reguladores , Histidina , Peso Molecular , Óperon , PlasmídeosRESUMO
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.
Assuntos
Escherichia coli/genética , Histidina/biossíntese , Óperon , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , Escherichia coli/metabolismo , Genes Bacterianos , Teste de Complementação Genética , Plasmídeos , Transcrição Gênica , Transformação BacterianaRESUMO
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 lambda 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.