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About: Operon is a(n) research topic. Over the lifetime, 14635 publication(s) have been published within this topic receiving 768610 citation(s). more


Open accessJournal ArticleDOI: 10.1073/PNAS.120163297
Kirill A. Datsenko1, Barry L. WannerInstitutions (1)
Abstract: We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells. more

Topics: Plasmid (60%), Recombinase (57%), Operon (54%) more

13,178 Citations

Open accessJournal ArticleDOI: 10.1073/PNAS.89.12.5547
Manfred Gossen1, Hermann Bujard1Institutions (1)
Abstract: Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way. more

Topics: Promoter (57%), Operon (56%), Luciferase (56%) more

5,189 Citations

Open accessJournal ArticleDOI: 10.1128/JB.177.14.4121-4130.1995
Abstract: We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system. more

Topics: PBAD promoter (76%), Arabinose (56%), Operon (52%) more

4,716 Citations

Journal ArticleDOI: 10.1016/0022-2836(80)90283-1
Abstract: Plasmid cloning vectors that enable insertion of DNA fragments between the inducible ara (arabinose) promoter and the lac (lactose) structural genes have been constructed and used for the detection and analysis of signals that control gene transcription. Expression of the lac genes in the absence of the inducer arabinose indicates that transcription originates within the inserted fragment; non-expression of lac with arabinose present indicates that transcription is terminated by the fragment. Using different cloning vectors, DNA fragments generated by a wide variety of restriction endonucleases can be inserted between ara and lac. This procedure has been used to identify and isolate endonuclease-generated DNA fragments from theEscherichia coli chromosome, various R plasmids, bacteriophage T5 and Drosophila melanogaster that contain nucleotide sequences capable of functioning as promoters in E.coli. A characteristic level of lac expression is determined by the amount of transcription that proceeds to the lac genes from a promoter located within each fragment. The effects of genetic regulatory mechanisms acting on a promoter can be assayed by alterations in the level of lac expression. These cloning vectors were also used to bring structural genes located within an inserted DNA fragment under the control of the ara promoter. Insertion of HindIII endonuclease-generated fragments carrying the tetracycline-resistance determinant of pSC101 or the sulfonamide-resistance determinant of the R6-5 plasmid into such vectors resulted in arabinose-induced resistance to tetracycline or sulfonamide. more

Topics: Cloning vector (65%), Lac repressor (64%), Plasmid (59%) more

2,414 Citations

Open accessJournal ArticleDOI: 10.1093/NAR/11.8.2237
Diane K. Hawley1, William R. McClure1Institutions (1)
Abstract: The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence. more

Topics: LacUV5 (66%), Pribnow box (59%), Consensus sequence (58%) more

2,074 Citations

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Topic's top 5 most impactful authors

Charles Yanofsky

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John R. Roth

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