Tetracycline operator

Blaauw et al. [84] adapted a state-of-the-art Tet° system known from gene expression systems in mammalian cell fines (see Part IV, Chapter 1). Plasmid 1 (response plasmid) remains extrachromosomal, and has a hsr selection cassette and a tetracycline-responsive minimal promoter consisting of seven copies of the tetracycline operator sequence (tetO) upstream of an actl5 minimal promoter this is used... 

P = constitutive promoter, tetR = TETrepressor gene,TETR = TET repressor protein, tetO = TET operator sequence, = tetracycline, ere/CRE = Cre recombinase gene/protein, PLEA = late embryogenesis abundant promoter, RIP = gene for ribosome inactivating protein, shaded blocks are loxP sites in orientation shown by solid triangle. After [48]... 

Various microbiological processes are used in the production of a number of antibiotics, for instance penicillins, tetracyclines, chloramphenicol and streptomycins. The major areas of such operations being ... 

The operational stability of the IME was determined by the performance of the IME in a real cellulose hydrolysis reactor. 50-mL tubes were charged with 2.5 g a-cellulose (50 g/L) in 10 mM sodium acetate pH 4.8. Tetracycline and cycloheximide... 

Unlike the Gre/loxP system, the tetR/O switch is not dependent on enzyme-based DNA rearrangements but is controlled by the small synthetic compound doxycycline (Dox). In the absence of Dox the tetracycline repressor (tetR), expressed from a constitutive promoter, binds to the tet operator (tetO) within a modified HI promoter and blocks shRNA transcription (Fig. 3b). Exposure to Dox dissociates the tetR from tetO which enables shRNA expression from the HI promoter (22). Thus, gene silencing is induced by Dox addition (20, 22, 23). This system is successfully in use to validate the potential of candidate drug targets in vivo (24-28). A commercial service for the production of Dox inducible knockdown mice and rats is available from Taconic (see www. taconic.com). 

Fig. 1. Molecular mechanism of the tetracycline-regulated shRNA transcription, (a) In the absence of doxycycline, the constitutively expressed repressor homodimerizes and then binds to Tet operator 2 (TetO ) sequences in the inducible expression vector preventing shRNA transcription, (b) When tetracycline is added to the medium, it causes a conformational change in the repressor, leading to its detachment from the Tet operator sequences and shRNA transcription induction.

CZE is the most widely used mode due to its simplicity of operation and its versatility. Selectivity can be most readily altered through changes in running buffer pH or by use of buffer additives such as surfactants or chiral selectors. The major drawback with CZE is that it deals with aqueous electrolytic systems, whereas components can only be separated if they are charged and soluble in water. CZE separation of various antibacterials including penicillins, tetracyclines, and macrolides has been reported (86). Determination of cefixime, an oral cephalosporin antibiotic, and its metabolites in human urine has been also successfully carried out with CZE (87). 

Fig. 5.3. Schematic representation of die display vector pASKIntlOO. fl, fl replication origin cat, chloramphenicol resistance marker tetR, tetracycline repressor encoding gene tetP/O, tetracycline promotor/operator region colEl, ColEl replication origin intimin, truncated eaeA gene of EHEC 0157 H7. Unique Ava I (Sma I, Xma I) and Bam HI restriction sites allow die in-frame fusion of genes encoding various passenger domains, as described in furdier detail in Wentzel et al. [7].

Figure 2.3 Tetracycline regulated gene expression (Adapted from Applied Molecular Genetics, R.Miesfeld, Wiley publishing, New York, 1999, p. 190). Terminology CMV (cytomegalovirus) VP (activating domain of viral protein VP 16) fefR (Tet represser) tTA (tetracycline-regulated trans-activator protein) rtTA (reverse let transactivator protein) (tel())- (repeat of 7 tet operator sequences) TATA (transcription initiation consensus sequence).

The fusion protein, or tTA, binds to the tet operator sequences in the absence of tetracycline and stimulates transcription. When tetracycline is added, it binds to the tTA and prevents it from binding to the tet operator sequences. Thus, transcription is repressed in the presence of tetracycline, as depicted in Figure 2.3. The gene of interest can be turned off and on simply by the administration of tetracycline, which is nontoxic to mammalian cells and mice (Shin, 2000). 

This system is advantageous since tetR has high specificity for both its operator sequences and tetracycline (Hillen and Wissmann, 1989 Takahashi et al., 1986). This allows the system to function at tetracycline concentrations well below toxic levels in mammalian cells (Gossen et al., 1995). Tetracycline-derivatives, such as doxycycline, have been shown to be even better inducers than tetracycline. This is due to their increased affinity for tetR and high functional stability (Gossen et al., 1995 Gossen and Bujard, 1993). This system is commonly referred to as Tet-off since the gene is turned off in the presence of tetracycline. 

Crystal structures of repressors suggest a common helix-turn-helix motif for DNA binding. Repressors are protein molecules which bind to and block specific nucleotide sequences in DNA (operators), thereby regulating gene expression. Some of the repressors act without co-factors, others need a co-repressor to bind to DNA, like the tryptophan (trp) repressor, or they do not bind to the DNA operator in presence of a co-repressor like tetracyclin (TET) repressor. 

Bowman RJ, Sillah A. Operational comparison of single-dose azithromycin and topical tetracycline for trachoma. Invest Ophthalmol Vis Sci 2000 41 4074-4079. 

All tetracycline, 6-demethyltetracycline, declomycin, and chlortetracycline standards were provided by Lederle Laboratories as the hydrochloride salts. All fast atom bombardment spectra were obtained with a VG (Manchester, UK) ZAB SE equipped with a cesium ion gun operated at 30 kV. The optimum matrix was "magic bullet" with a methanol solvent. (Structure2)... 

The reverse phenomenon, decreased enzyme synthesis, can also be the mechanism of drug resistance. The antimetabolite pro-drug 6-mercaptopurine (6MP) is activated to its nucleotide by inosine-5 -phosphate pyrophosphorylase. The enzyme is deleted in resistant neoplastic cells. Resistance to 5-fluorouracil similarly develops by deletion of the enzyme converting this pro-drug to its active nucleotide. A mechanism of resistance by which a drug is excluded from its site of action can also be operative. This has been established for tetracycline antibiotics. Here the permeability of the cellular membrane to the drug is altered so that it cannot penetrate and accumulate within the target cell. Similarly, it has been demonstrated with such a membrane modification in MTX-resistant leukemia cells in mice. 

Resistance is widespread and often is indncible. The three main resistance mechanisms are (1) decreased accumulation of tetracycline (decreased antibiotic influx or acquisition of an energy-dependent efflux pathway) (2) production of a ribosomal protein that displaces tetracycline from its target, a protection that also may occur by mutation and (3) enzymatic inactivation of tetracyclines. Cross-resistance amongst tetracyclines depends on which mechanism is operative. Tetracycline resistance due to a ribosomal protection mechanism produces cross-resistance to doxycycUne and minocycline because the target site protected is the same for all tetracyclines. 

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