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.

https://www.pnas.org/content/pnas/89/12/5547.full.pdf

Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.

The therapeutic properties of light have been known for thousands of years, but it was only in the last century that photodynamic therapy (PDT) was developed. At present, PDT is being tested in the clinic for use in oncology--to treat cancers of the head and neck, brain, lung, pancreas, intraperitoneal cavity, breast, prostate and skin. How does PDT work, and how can it be used to treat cancer and other diseases?

Photodynamic therapy for cancer

5.1. Detection Formats 475 5.2. Food Quality and Safety Analysis 477 5.2.1. Pathogens 477 5.2.2. Toxins 479 5.2.3. Veterinary Drugs 479 5.2.4. Vitamins 480 5.2.5. Hormones 480 5.2.6. Diagnostic Antibodies 480 5.2.7. Allergens 481 5.2.8. Proteins 481 5.2.9. Chemical Contaminants 481 5.3. Medical Diagnostics 481 5.3.1. Cancer Markers 481 5.3.2. Antibodies against Viral Pathogens 482 5.3.3. Drugs and Drug-Induced Antibodies 483 5.3.4. Hormones 483 5.3.5. Allergy Markers 483 5.3.6. Heart Attack Markers 484 5.3.7. Other Molecular Biomarkers 484 5.4. Environmental Monitoring 484 5.4.1. Pesticides 484 5.4.2. 2,4,6-Trinitrotoluene (TNT) 485 5.4.3. Aromatic Hydrocarbons 485 5.4.4. Heavy Metals 485 5.4.5. Phenols 485 5.4.6. Polychlorinated Biphenyls 487 5.4.7. Dioxins 487 5.5. Summary 488 6. Conclusions 489 7. Abbreviations 489 8. Acknowledgment 489 9. References 489

Surface Plasmon Resonance Sensors for Detection of Chemical and Biological Species

Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

The optimism of the early period of antimicrobial discovery has been tempered by the emergence of bacterial strains with resistance to these therapeutics. Today, clinically important bacteria are characterized not only by single drug resistance but also by multiple antibiotic resistance--the legacy of past decades of antimicrobial use and misuse. Drug resistance presents an ever-increasing global public health threat that involves all major microbial pathogens and antimicrobial drugs.

/pdf/antibacterial-resistance-worldwide-causes-challenges-and-1cbvr301rt.pdf

Antibacterial resistance worldwide: causes, challenges and responses.

A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.

https://science.sciencemag.org/content/sci/268/5218/1766.full.pdf

Transcriptional activation by tetracyclines in mammalian cells

Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of Ptet promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2S-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.

https://www.pnas.org/content/pnas/97/14/7963.full.pdf

Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity

Tetracycline-resistance determinants encoding active efflux of the drug are widely distributed in gram-negative bacteria and unique with respect to genetic organization and regulation of expression. Each determinant consists of two genes called tetA and tetR, which are oriented with divergent polarity, and between them is a central regulatory region with overlapping promoters and operators. The amino acid sequences of the encoded proteins are 43-78% identical. The resistance protein TetA is a tetracycline/metal-proton antiporter located in the cytoplasmic membrane, while the regulatory protein TetR is a tetracycline inducible repressor. TetR binds via a helix-turn-helix motif to the two tet operators, resulting in repression of both genes. A detailed model of the repressor-operator complex has been proposed on the basis of biochemical and genetic data. The tet genes are differentially regulated so that repressor synthesis can occur before the resistance protein is expressed. This has been demonstrated for the Tn10-encoded tet genes and may be a common property of all tet determinants, as suggested by the similar locations of operators with respect to promoters. Induction is mediated by a tetracycline-metal complex and requires only nanomolar concentrations of the drug. This is the most sensitive effector-inducible system of transcriptional regulation known to date. The crystal structure of the TetR-tetracycline/metal complex shows the Tet repressor in the induced, non-DNA binding conformation. The structural interpretation of many noninducible TetR mutants has offered insight into the conformational changes associated with the switch between inducing and repressing structures of TetR. Tc is buried in the core of TetR, where it is held in place by multiple contacts to the protein.

Mechanisms underlying expression of tn10 encoded tetracycline resistance

Carbon catabolite repression in bacteria.

The tetracycline repressor (TetR) regulates the most abundant resistance mechanism against the antibiotic tetracycline in grain-negative bacteria. The TetR protein and its mutants are commonly used as control elements to regulate gene expression in higher eukaryotes. We present the crystal structure of the TetR homodimer in complex with its palindromic DNA operator at 2.5 A resolution. Comparison to the structure of TetR in complex with the inducer tetracycline-Mg2+ allows the mechanism of induction to be deduced. Inducer binding in the repressor core initiates conformational changes starting with C-terminal unwinding and shifting of the short helix a6 in each monomer. This forces a pendulum-like motion of helix a4, which increases the separation of the attached DNA binding domains by 3 A, abolishing the affinity of TetR for its operator DNA.

Wolfgang Hillen

Papers

Transcriptional activation by tetracyclines in mammalian cells

Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity

Mechanisms underlying expression of tn10 encoded tetracycline resistance

Carbon catabolite repression in bacteria.

Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system.