Journal of Experimental Botany, Vol. 53, No. 376, pp. 1871-1877,
September 1, 2002
© 2002 Oxford University Press
Differential Top10 promoter regulation by six tetracycline analogues in plant cells
Received 28 February 2002; Accepted 4 June 2002
1 Department of Plant Sciences, University of Cambridge, Downing Site, Cambridge CB2 3EA, UK
2 Department of Botany, North Carolina State University, Raleigh, NC 27695-7612, USA
3 Allgemeine und Entwicklungsphysiologie, Albrecht-von-Haller-Institut, Universität Göttingen, Untere Karspüle 2, D-7073 Göttingen, Germany
4 To whom correspondence should be addressed. E-mail: john.love{at}plantsci.cam.ac.uk
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The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l–1. Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l–1 and was easily washed out to restore Top10-driven expression in 12–24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.
Key words: Key words: Tetracycline analogues, tobacco, Top10 promoter activity, tissue culture.
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Stringent control of expression by inducible promoters is essential for studying transgenes that may have deleterious effects on transformed organisms. Artificial promoters that combine tight transcriptional control and induction by low concentrations of chemical ligands have been developed from bacterial repressor- or animal steroid-binding elements. These promoters include the tetracycline-inducible ‘Triple-Op’ promoter (Gatz and Quail, 1988), the glucocorticoid-induced ‘GAL4-UAS’ promoter (Aoyama and Chua, 1997), the ethanol-inducible ‘alcA’ promoter (Caddick et al., 1998), the ecdysone-inducible ‘GRH’ promoter (Martinez et al., 1999), and the tetracycline-repressible ‘Top10’ promoter (Weinmann et al., 1994). The Top10 promoter has certain advantages compared to other chemically regulated promoters: the regulatory molecule, tetracycline, has no naturally occurring analogue in animal or plant cells. Transgene expression is induced by removal of tetracycline, thereby minimizing the possibility of secondary physiological effects that may be caused by its presence. Micromolar levels of tetracycline repress Top10 activity and have no discernible pleiotropic effects on transformant organisms (Gossen and Bujard, 1992; Weinmann et al., 1994). Most importantly, transcriptional control by the Top10 promoter is extremely tight, with no detectable background transgene expression (Love et al., 2000). The Top10 promoter incorporates two transgenic elements and therefore requires two transformation events to function. The first transformation yields material expressing the tetracycline trans-activator (tTA) polypeptide, which is a chimeric fusion between the tetracycline repressor DNA binding domain (TetR) and the VP16 enhancer domain of Herpes simplex virus (Postle et al., 1984; Triezenberg et al., 1988). The second transformation introduces into the tTA background the Top10 promoter sequence (Weinmann et al., 1994) fused to the transgene of interest. In the absence of tetracycline, the TetR domain of the tTA binds to the Top10 promoter and the VP16 domain drives transgenic transcription (Hinrichs et al., 1994). When tetracycline is present, it binds to the TetR causing a conformational change that prevents the interaction with the Top10 promoter (Lederer et al., 1995) and inhibits Top10-driven transcription. The Top10 promoter has been used to regulate transgene expression in bacteria (Geissendörfer and Hillen, 1990), animal cells (Saez et al., 1997) and in several plant species including Nicotiana tabacum (Weinmann et al., 1994), Physcomitrella patens (Zeidler et al., 1996) and Arabidopsis thaliana (Love et al., 2000). These studies, however, focused on transforming a functional Top10 promoter system into the plant species in question and offered only limited information regarding the practicalities of its application. For example, in animal cells, tetracycline, anhydrotetracycline or doxycycline, are routinely selected to regulate Top10 promoter activity (Gossen et al., 1995; A-Mohammadi et al., 1997), whereas in plants, tetracycline has been the exclusive choice. Tetracycline, however, is light labile and may not be the best candidate to regulate the Top10 promoter in autotrophic organisms. We therefore investigated whether it was possible to regulate the level of Top10-controlled transgene expression in plant cells more precisely by using various tetracycline analogues (Valcavi, 1981) to control Top10 promoter activity.
The effects of five readily available tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline and tetracycline, on Top10-driven transgene expression in transformed Nicotiana tabacum tissue culture cells (NT1 cells) in the light and in darkness were tested. The maximum concentration at which NT1 cells grew normally, the minimum concentration that repressed Top10 promoter activity, the degree to which transgene expression levels are regulated, and the kinetics of transgene de-repression were determined for each compound. The suitability for plant research of GR 33076X, a novel tetracycline analogue that has been shown to promote, rather than repress, Top10 promoter activity in animal and bacterial cells was also studied (Chrast-Balz and Hooft van Huijsduijnen, 1996). Transformed NT1 cells were cultured in media containing anhydrotetracycline, doxycycline or tetracycline at concentrations that inhibited Top10 promoter-driven transgene expression. The addition of GR 33076X to NT1 cells that were cultured with anhydrotetracycline or doxycycline present had no significant effect on Top10-driven translation. However, in the presence of tetracycline, GR 33076X de-repressed Top10 promoter activity to over 60% of that observed in the total absence of tetracycline-induced inhibition. Different tetracycline analogues can therefore be used to regulate Top10 promoter activity in plant cells and this property may be exploited in planning an optimum course of transgene regulation.
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Materials and methods |
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NT1 cell culture and transformation
Nicotiana tabacum tissue culture (NT1) cells were maintained by weekly subculture of 0.5 ml cell suspension into 5 ml NT1 culture medium (1x Murashige and Skoog Salt Base (Gibco), 30 g l–1 sucrose, 0.18 g l–1 KH2PO4, 0.1 g l–1 myo-inositol, 1 mg l–1 thiamine HCl, 0.2 mg l–1 2,4-D, pH 5.5–5.7) and incubated at 27 °C, with orbital shaking at 190 rpm, in darkness (Persson et al., 2002). Cells that were cultured in constant light were exposed to 25 µmol s–1 m–2 fluorescent, warm white light.
NT1 cells were transformed using Agrobacterium tumefaciens (strain LBA-4404) mediated gene transfer as described in Persson et al. (2001). Cells were initially transformed with the binary vector plasmid ptetVP16 (Weinmann et al., 1994) that contains, between the left and right T-DNA borders, a plant kanamycin selectable marker and the tetracycline trans-activator (tTA) coding sequence under control of the cauliflower mosaic virus 35S promoter. Independent, transgenic NT1 microcalli were picked, suspended in 1 ml NT1 culture medium containing 50 µg l–1 kanamycin and 200 µg ml–1 timetin and were incubated for 7 d at 27 °C, with orbital shaking at 190 rpm, in darkness. Cells grown from these cultures were transferred to 5 ml NT1 culture medium containing 50 µg ml–1 kanamycin and the lines maintained as described above. Cultures that expressed high levels of tTA mRNA were identified by Northern Blot analysis with an antisense RNA probe to the TetR sequence. A single culture that expressed high levels of the tTA was selected and transformed a second time using A. tumefaciens containing the plasmid pBin-Hyg Top10GUS (Weinmann et al., 1994) or pBinHTop10mGFP5 (Love et al., 2000). Each plasmid carried a hygromycin phosphotransferase plant selectable marker. pBin-Hyg Top10GUS carries the coding sequence for ß-glucuronidase (GUS) driven by the Top10 promoter and upstream of the nos terminator. pBinHTop10mGFP5 carries the coding sequence for ER-targeted GFP (Haselhoff et al., 1997) under Top10 promoter control and upstream of the ocs terminator. Double-transgenic microcalli were screened for GUS expression by incubating approximately 10 mg of freshly harvested cells in 200 µl of a 2 mM solution of 5-bromo-4-chloro-3-indolyl ß-D-glucuronide (X-Gluc., Molecular Probes, USA) for 12 h at 37 °C (Jefferson, 1987). To measure the effects of tetracycline analogues on cell growth and Top10-controlled GUS expression, transgenic NT1 cells were cultured in media containing titred concentrations of anhydrotetracycline (Clontech, USA), doxycycline, minocycline, oxytetracycline, and tetracycline (all from Sigma, USA) or GR 3376X (Chrast-Balz and Hooft van Huijsduijnen, 1996). N,N dimethylformamide (Sigma, USA) was used as a carrier for the analogues and its final concentration was less than 0.5% v/v unless otherwise indicated. Experiments were replicated at least four times. To measure the kinetics of Top10 promoter de-repression in vivo, NT1 cells that expressed the tTa were transformed with pBinHTop10mGFP5 and a transgenic line that showed similar Top10 promoter regulation as the Top10::GUS line was selected. NT1 cells were cultured for 5 d in media containing doxycycline or tetracycline to repress the activity of the Top10 promoter. Cells were washed three times and transferred to fresh NT1 cell culture medium. Cells were observed under the fluorescence microscope at 6, 12, 18, 24, 36, and 48 h after transfer, following the protocol described by Scott et al. (1999).
RNA extraction and Northern blot
NT1 cells were harvested by filtration through 42.5 µm filter papers and frozen in liquid N2. Total RNA was extracted from approximately 0.5 g of NT1 cells following the protocol described in Thompson et al. (1983). 5 µg RNA was separated by agarose-gel electrophoresis (Sambrook et al., 1989), blotted and fixed onto a GeneScreen hybridization transfer membrane. Northern blots were incubated overnight, with 32P-UTP-radiolabelled antisense RNA probes. Membranes were then washed, dried and autoradiographed.
GUS activity assays
GUS activity assays were performed using the Plant GUS-LightTM kit (Tropix Inc., Perkin Elmer Biosystems). NT1 cells were ground under liquid N2. 10 mg of pulverized cells were suspended in 300 µl Tropix lysis buffer, vortexed for 15 s, and centrifuged at 15 000 rpm for 3 min. 20 µl of the cleared lysate was transferred to a 96 well opaque microtitre plate (Costar). A diluted series of recombinant ß-glucuronidase (Sigma) and an extract from wild-type NT1 cells were included on each plate. Plates were introduced into a LUMIstar microtitre plate reader (BMG Lab Technologies, Inc.) and 70 µl Tropix GUS reaction buffer containing Tropix glucuron substrate was added to each sample by the machine. Following 20 min incubation at room temperature, 100 µl Tropix light accelerator was added to each sample. Plates were shaken for 2 s and the light emission of each sample recorded for 10 s. Light emission was related to GUS activity using the standards included on each plate. The total protein content of each sample was determined using the BioRad Bradford Assay.
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Selection of double-transgenic NT1 cells
NT1 tobacco tissue culture cells are particularly suitable to investigate Top10 promoter regulation in plant cells, as a large number of independently transformed lines can be produced. Moreover, cells are maintained in liquid culture, allowing maximum exposure between the cells and the dissolved chemicals. The transgenes that make up the Top10 promoter system were united by generating a parent line that reliably expressed the tTA and subsequently transforming cells from this line with the Top10-driven transgenes. Although simultaneous transformations are possible, combining the tTA and the Top10-driven gene on the same vector may result in transcriptional interference between the two transgenes (Thompson and Myatt, 1997). Moreover, the tTA is toxic at high cellular concentrations (Shockett et al., 1995; Shockett and Schatz, 1996), therefore a transformed line in which tTA expression is characterized is advantageous.
Twenty independent NT1 cell lines that were transformed with the ptetVP16 plasmid (Weinmann et al., 1994) and were resistant to kanamycin were generated. From these lines, a single, transformant NT1 cell line was selected that consistently expressed the tTA at high levels. Cells from this line were transformed, a second time, with pBIN-Hyg Top10GUS. Eighty stable, double-transformant microcalli were selected based on kanamycin and hygromycin resistance. Cells from each of these 80 cell lines were cultured in the absence of any tetracycline analogue and stained for GUS with a 2 mM solution of X-Gluc (Jefferson, 1987). Of these 80 lines, 55 (69%) exhibited the dark blue stain that is diagnostic of GUS, indicating that the Top10 promoter was active in these cells (compare the staining of cells from lines 12 and 16 to that of the other cell lines in Fig. 1A). Cells from each of these 55 lines were cultured either in the absence of tetracycline analogue or in media containing 2 µmol l–1 tetracycline and samples stained for GUS expression. 41 cell lines, i.e. 51% of the retrieved double-transformant microcalli, stained positive for GUS, indicating that the Top10 promoter was not inhibited by tetracycline in these cell lines (note lines 8 and 14 in Fig. 1A). The remaining 14 cell lines, representing 17.5% of the original double-transformant microcalli, showed no GUS staining in cells cultured with tetracycline. When these 14 cell lines were cultured in media containing graded tetracycline concentrations and subsequently stained for GUS, only 6, i.e. 7% of the retrieved, double-transformant microcalli, showed GUS staining that was proportional to the concentration of applied tetracycline, indicative of a functional Top10 promoter. Moreover, GUS staining was relatively similar between these independent lines (lines 25, 34 and 49 of Fig. 1A). This low yield of functional, double-transgenic cell lines may reflect the complexity of the Top10 system that relies, not only on two independent transgenic events, but also on the subsequent in vivo interaction between the regulatory molecule, the tTA, and the Tet.Op sequences of the Top10 promoter. It is therefore essential to select a sufficient number of independent transformants in order to obtain a satisfactory number of individuals or cell lines possessing the desired Top10-regulated phenotype.
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(Set 3). (C, D, E, F, G) Relative GUS activity of double-transformant NT1 cells from line 49, incubated for 7 d with varying concentrations of tetracycline (C), doxycycline (D) minocycline (E), anhydrotetracycline (F), and oxytetracycline (G). The total level of Top10-driven GUS expression for each cell culture was determined as a measure of GUS activity using the GUS-light system from Tropix, and normalized to the total amount of cellular protein. Total GUS activity was related to that of the control sample containing no tetracycline to yield the Relative GUS Activity of the sample. Black bars represent cells cultured in darkness and grey bars are for cells cultured in the light. Bars represent the mean of four (cultures in darkness) or seven (cultures in the light) independent replicates with standard errors shown. Bars labelled WT indicate wild-type NT1 cells. (H) Relative GUS activity of double-transformant NT1 cells from line 49 that were incubated for 7 d with 10 nmol l–1 tetracycline and varying concentrations of GR 33076X. The total level of Top10-driven GUS expression for each cell culture was determined as a measure of GUS activity using the GUS-light system from Tropix, and normalized to the total amount of cellular protein. The relative GUS activity of each cell culture was determined as a percentage of the GUS activity of control samples that contained containing no tetracycline. Bars represent the mean of nine independent replicates with standard errors shown. (I) Fluorescent images of double-transformant NT1 cells that constitutively express the tTA and ER-targeted GFP under control of the Top10 promoter. Cells were incubated for 5 d in 10 nmol l–1 tetracycline, washed and imaged at the indicated times thereafter. The control was cultured in the absence of tetracycline. After 12 h GFP fluorescence is visible and reaches a level comparable to the control at 24 h. All images were acquired using the same optical and electronic parameters. Scale bars represent 20 µm.Effects of tetracycline analogues on NT1 cell growth and Top10 promoter activity
The effects of anhydrotetracycline, doxycycline, minocycline, oxytetracycline, tetracycline, and GR 33076X on cell growth and on Top10-controlled GUS expression in NT1 cell line 49 that showed no GUS expression following incubation with tetracycline but high levels of GUS activity in its absence, were investigated.
Cell growth over the 7 d culture period was used as an indicator of the toxicity of the tetracycline analogues to the NT1 cells in culture. The carrier molecule, N,N, dimethylformamide (DMF) had no effect on cell growth at a concentration of 1% v/v or lower. For all experiments, DMF concentration was maintained at 0.5% v/v. Of the six tetracycline analogues investigated in this study, minocycline had the most potent inhibitory effect on NT1 cell growth, reducing yield by 50% at a concentration of 1.5 µmol l–1. A 50% decrease in cell growth was observed at 3 µmol l–1 doxycycline, 10 µmol l–1 oxytetracycline, 12 µmol l–1 tetracycline, 20 µmol l–1 anhydrotetracycline, and at 20 µmol l–1 GR33076X.
Transcription of Top10-driven GUS was determined by Northern blot, and GUS expression levels were quantified using the GUS-light assay (Tropix). In the absence of any tetracycline analogue, the transformed NT1 cells expressed high levels of GUS as shown by a strong mRNA band that hybridized to the antisense GUS RNA probe (Fig. 1B) and high GUS-light emission. When the transgenic NT1 cells were cultured in darkness, Top10-driven GUS expression was completely repressed by 4 nmol l–1 doxycycline (Fig. 1C), or by 10 nmol l–1 tetracycline (Fig. 1D), or by 40 nmol l–1 minocycline (Fig. 1E), or by 150 nmol l–1 to 200 nmol l–1 anhydrotetracycline (Fig. 1F), or by oxytetracycline concentrations in excess of 200 nmol l–1 (Fig. 1G). Conversely, GR 33076X had no inhibitory effect on Top10-controlled GUS expression. For each analogue, the degree of Top 10 promoter inhibition could not necessarily be predicted by direct comparison with bacterial or animal systems, or from in vitro binding affinity to the TetR. The most striking example is anhydrotetracycline: in animal cells, anhydrotetracycline inhibits Top10 promoter activity at concentrations ranging from 10 nmol l–1 (Chrast-Balz and Hooft van Huijsduijnen, 1996) to 200 nmol l–1 (A-Mohammadi et al., 1997), whereas tetracycline is effective in the same cell types at the higher concentration range of 100 nmol l–1 to 400 nmol l–1. Moreover, the KA of anhydrotetracycline for the TetR is, at approximately 100x109 mol–1, 30-fold higher than that of tetracycline (Lederer et al., 1996). One might therefore predict that anhydrotetracycline would be a more potent inhibitor of Top10 promoter activity than is tetracycline in NT1 cells. Instead, the opposite is true; tetracycline is 10–20-fold more potent an inhibitor of the Top10 promoter than is anhydrotetracycline.
10 nmol l–1 doxycycline or 20 nmol l–1 tetracycline repressed Top10 activity in cells that were cultured in constant light (Fig. 1C, D). The inhibitory concentration of either compound was approximately 2-fold higher than that required to repress the Top10 promoter in darkness, and is probably due to photolytic degradation. However, the effective concentration remains extremely low and approximately 3 orders of magnitude below the half-lethal concentration for either compound.
The level of Top10-controlled GUS expression is exquisitely sensitive to varying concentrations of each inhibitory tetracycline analogue in both the light and the dark. This property allows the titration of transgene expression at levels intermediate between the on and off states and allows comparisons between the effects of different levels of transgene expression in the same transgenic organism.
Top10 promoter de-repression by GR 33076X
GR 33076X has been shown activate the Top10 promoter in the presence of repressive concentrations of anhydrotetracycline or doxycycline in HeLa cells (Chrast-Balz and Hooft van Huijsduijnen, 1996). Therefore, the effect of GR 33076X on Top10-driven GUS expression was investigated in the presence of 4 nmol l–1 doxycycline, 10 nmol l–1 tetracycline or 150 nmol l–1 anhydrotetracycline, all of which repress Top10 promoter activity in the transgenic NT1 cells. Minocycline, which had the most potent toxic effect of all the analogues investigated, and oxytetracycline, which had the least potent inhibitory effect on Top10-driven GUS expression, were omitted. Unlike the situation observed in HeLa cells, GR 33076X did not de-repress Top10-driven GUS expression in NT1 cells that were incubated with either 4 nmol l–1 doxycycline or 150 nmol l–1 anhydrotetracycline. Conversely, in the presence of 10 nmol l–1 tetracycline, GR 33076X de-repressed Top10-controlled GUS expression at an concentration optimum between 4 µmol l–1 and 6 µmol l–1 (Fig. 1H), i.e. at a 400:1 and 600:1 molar ratio of GR 33076X:tetracycline. The levels of Top10-regulated GUS expression attained following de-repression with GR 33076X was approximately 60% of that observed in the absence of tetracycline. Furthermore, increasing the concentration of tetracycline in the medium decreased the effectiveness of GR 33076X, even at a GR 33076X:tetracycline molar ratio that de-repressed Top10 activity at the lower tetracycline concentration.
Kinetics of Top10 promoter de-repression by washing and by GR 3376X
Cells expressing Top10-driven mgfp-5 were cultured in 4 nmol l–1 doxycycline, 10 nmol l–1 tetracycline or 150 nmol l–1 anhydrotetracycline for 4 d, washed, transferred to fresh NT1 culture medium and GFP fluorescence imaged at specified times thereafter. For cells that had been cultured with tetracycline or anhydrotetracycline prior to washing, GFP fluorescence was initially undetectable and increased over a 24 h period, when it attained levels similar to controls grown without any tetracycline analogue (Fig. 1I). This time-course reflects a combination of events including depletion of the inhibitory compound from its nuclear targets, transcription of the Top10::mGFP5 transgene, GFP translation, and post-translational formation of the GFP fluorophore which takes approximately 4 h (Heim et al., 1994; Kain and Kitts, 1997). Contrary to tetracycline, cells that were cultured in doxycycline showed no significant GFP signal over 48 h. The difficulty in reversing Top10 inhibition by doxycycline may therefore limit its application to inactivating the Top10 promoter, for example, in studies of RNA stability.
GR 33076X enables Top10-driven translation without the requirement for washing cells. However, GR 3376X is strongly fluorescent and is applied to the NT1 cell cultures at a concentration (4 µmol l–1) that precluded the use of the GFP-based induction assay described above. Instead, cells expressing Top10::GUS were cultured in medium containing 10 nmol l–1 tetracycline for 4 d and GR 33706X added to a final concentration of 4 µmol l–1. Cells were sampled and tested for GUS, but showed no detectable GUS expression, even 48 h after addition of GR 33076X. Although the observations described herein may limit the practical use of GR 33076X in plants, many tetracycline analogues have been generated (Valcavi, 1981), and their properties may be readily tested in vivo.
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NT1 cell lines expressing a functional, tetracycline-regulated Top10 promoter system were obtained with a frequency of 7% following re-transformation of NT1 cells from a parent line that reliably expressed the tTA. The level of cellular toxicity, the degree of Top10-regulated transgene expression and the kinetics of transgene de-repression were determined for five tetracycline analogues. The effect that each analogue had on the Top 10 promoter in NT1 cells could not be predicted by direct comparison with bacterial or animal systems, nor from the in vitro binding affinity to the tetracycline repressor. Anhydrotetracycline, the most potent inhibitor of Top10-driven expression in animal cells, was 10- or 20-fold less effective in NT1 compared to tetracycline or doxycycline. Doxycycline, at 4 nmol l–1, had the most potent inhibitory effect on Top10 promoter activity, but the inhibition was not easily reversed. Tetracycline was the most versatile of the analogues tested, inhibiting the Top10 promoter at 10 nmol l–1 and being easily washed out to restore Top10-driven expression completely after approximately 24 h. The level of Top10-controlled GUS expression was exquisitely sensitive to varying concentrations of each inhibitory tetracycline analogue, allowing the titration of transgene expression and, therefore, the direct comparison of transgene effects over a range of expression levels in the same transgenic plant or cell line. Finally, the novel tetracycline analogue GR 33076X was shown to antagonize the repression of the Top10 promoter in the presence of inhibitory concentrations of tetracycline, but not of doxycycline or of anhydrotetracycline. However, washing the cells free of tetracycline induced transgene expression faster than by adding GR 33076X to the cell cultures, which may limit the practical use of GR 33076X in plants.
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Acknowledgements |
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We are grateful to Dr Rob Hooft van Huijsduijnen of the Geneva Biomedical Research Institute, Switzerland, for his kind gift of compound GR 33076X in the initial stages of this study and to Dr Li Zhu of Clontech Laboratories Inc., USA who contracted the synthesis of GR 33076X from High Force Research, UK. We also acknowledge the technical assistance of Ms Emilie Holton and of Mr Darnell Graham. This research was supported by the NASA Specialized Center of Research and Training at NC State University, award number NAGW-4984 and by a Broodbank Research Fellowship at the University of Cambridge.
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References |
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A-Mohammadi S, Alvarez-Vallina L, Ashworth LJ, Hawkins RE. 1997. Delay in resumption of the activity of tetracycline-regulatable promoter following removal of tetracycline analogues. Gene Therapy 4, 993–997.[Web of Science][Medline]
Aoyama T, Chua N-H. 1997. A glucocorticoid-mediated transcriptional induction system in transgenic plants. The Plant Journal 11, 605–612.[Web of Science][Medline]
Chrast-Balz J, Hooft van Huijsduijnen R. 1996. Bi-directional gene switching with the tetracycline repressor and a novel tetracycline antagonist. Nucleic Acids Research 24, 2900–2904.
Caddick MX, Greenland AJ, Jepson I, Krause K-P, Qu N, Riddell KV, Salter MG, Schuch W, Sonnewald U, Tomsett AB. 1998. An ethanol inducible gene switch for plants used to manipulate carbon metabolism. Nature Biotechnology 16, 177–180.[Web of Science][Medline]
Gatz C, Quail PH. 1988. Tn-10 encoded tet repressor can regulate an operator containing plant promoter. Proceedings of the National Academy of Sciences, USA 85, 1394–1397.
Geissendörfer M, Hillen W. 1990. Regulated expression of heterologous genes in Bacillus subtilis using the Tn10- encoded tet regulatory elements. Applied Microbiology and Biotechnology 33, 657–663[Web of Science][Medline]
Gossen M, Bujard H. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences, USA 89, 5547–5551.
Gossen M, Freundlieb S, Bender G, Müller G, Hillen W, Bujard H. 1995. Transcriptional activation of tetracyclines in mammalian cells. Science 268, 1766–1769.
Haselhoff J, Siemering KR, Prasher DC, Hodge S. 1997. Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proceedings of the National Academy of Sciences, USA 94, 2122–2127.
Heim R, Prasher DC, Tsien RY. 1994. Wavelength mutations and post-translational autoxidation of green fluorescent protein. Proceedings of the National Academy of Sciences, USA 91, 12501–12504.
Hinrichs W, Kisker C, Düvel M, Müller A, Tovar K, Hillen W, Saenger W. 1994. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Science 264, 418–420.
Jefferson RA. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporter 5, 387–405.
Kain SR, Kitts P. 1997. Expression and detection of green fluorescent protein (GFP). Methods in Molecular Biology 63, 305–324.
Lederer T, Takahashi M, Hillen W. 1995. Thermodynamic analysis of tetracycline-mediated induction of Tet repressor by a quantitative methylation protection assay. Analytical Biochemistry 232, 190–196.[Web of Science][Medline]
Lederer T, Kintrup M, Takahashi M, Sum P-E, Ellestad GA, Hillen W. 1996. Tetracycline analogs affecting binding to Tn10-encoded Tet repressor trigger the same mechanism of induction. Biochemistry 35, 7439–7446.[Medline]
Love J, Scott AC, Thompson WF. 2000. Stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system. The Plant Journal 21, 579–588.[Web of Science][Medline]
Martinez A, Sparks C, Drayton P, Thompson J, Greenland A, Jepson I. 1999. Creation of ecdysone receptor chimeras in plants for controlled regulation of gene expression. Molecular and General Genetics 261, 546–552.
Persson SH, Wyatt SE, Love J, Thompson WF, Robertson D, Boss WF. 2001. The Ca2+ status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants. Plant Physiology 126, 1092–1104.
Persson SH, Love J, Tsou P-L, Robertson D, Thompson WF, Boss WF. 2002. When a day makes a difference. Interpreting data from endoplasmic reticulum-targeted green fluorescent protein fusions in cells grown in suspension culture. Plant Physiology 128, 341–344.
Postle K, Nguyen TT, Bertrand KP. 1984. Nucleotide sequence of the repressor gene of the Tn10 encoded tetracycline resistance determinant. Nucleic Acids Research 12, 4849–4963.
Saez E, No D, West A, Evans RM. 1997. Inducible gene expression in mammalian cells and transgenic mice. Current Opinion in Biotechnology 8, 608–616.[Web of Science][Medline]
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press.
Scott A, Wyatt S, Tsou P-L, Robertson D, Strömgren-Allen N. 1999. Model system for plant cell biology: GFP imaging in living onion epidermal cells. Biotechniques 26, 1125–1132.[Web of Science][Medline]
Shockett P, Fillipantino M, Hellman H, Schatz D. 1995. A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proceedings of the National Academy of Sciences, USA 92, 6522–6526.
Shockett PE, Schatz DG. 1996. Diverse strategies for tetracycline-regulated inducible gene expression. Proceedings of the National Academy of Sciences, USA 93, 5173–5176.
Thompson AJ, Myatt SC. 1997. Tetracycline-dependent activation of an upstream promoter reveals transcriptional interference between tandem genes within T-DNA in tomato. Plant Molecular Biology 34, 687–692.[Web of Science][Medline]
Thompson WF, Everett M, Polans NO, Jorgensen RA, Palmer JD. 1983. Phytochrome control of RNA levels in developing pea and mung bean leaves. Planta 158, 487–500.[Web of Science]
Triezenberg SJ, Kingsbury RC, McKnight SL. 1988. Functional dissection of VP16, the trans-activator of Herpes simplex virus immediate early gene expression. Genes and Development 2, 718–729.
Valcavi U. 1981. Tetracyclines: chemical aspects and some structure-activity relationships. In: Grassi G, Sabath LD, eds. New trends in antibiotics: research and therapy. Elsevier/North Holland Biochemical Press, 3–25.
Weinmann P, Gossen M, Hillen W, Bujard H, Gatz C. 1994. A chimeric transactivator allows tetracycline-responsive gene expression in whole plants. The Plant Journal 5, 559–569.[Web of Science][Medline]
Zeidler M, Gatz C, Hartmann E, Hughes J. 1996. Tetracycline-regulated reporter gene expression in the moss Physcomitrella patens. Plant Molecular Biology 30, 199–205.[Web of Science][Medline]
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  W. Tang and R. J. Newton Regulated gene expression by glucocorticoids in cultured Virginia pine (Pinus virginiana Mill.) cells J. Exp. Bot., July 1, 2004; 55(402): 1499 - 1508. [Abstract] [Full Text] [PDF]
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