Proc. Nati. Acad. Sci. USA Vol. 85, pp. 1394-1397, March 1988 Biochemistry TnlO-encoded tet repressor can regulate an operator-containing plant promoter (cauliflower mosaic virus 35S promoter/electroporation/transient chloramphenicol acetyltransferase assays) CHRISTIANE GATZ* AND PETER H. QUAILt Departments of Botany and Genetics, University of Wisconsin, Madison, WI 53706 Communicated by Folke Skoog, October 26, 1987 (receivedfor review July S, 1987) ABSTRACT The TnlO-encoded tet repressor-operator The TnlO-encoded tet repressor regulates the expression system was used to regulate transcription from the cauliflower of the Tc resistance operon by binding to nearly identical mosaic virus (CaMV) 35S promoter. Expression was moni- operator sequences that overlap with three divergent pro- tored in a transient assay system by using electric field- moters (14, 15). The genes of the tet operon are only mediated gene transfer ("electroporation") into tobacco pro- transcribed in the presence of the inducer Tc, which pre- toplasts. The tet repressor, being expressed in the plant cells vents the repressor from binding to its operator sequences. under the control of eukaryotic transcription signals, blocks The tet repressor was chosen for regulating a plant promoter transcription of a CaMV 35S promoter chloramphenicol ace- for two reasons. (i) With a native molecular mass of 48 kDa, tyltransferase (cat) fusion gene when the two tet operators diffusion into the nucleus seemed likely (16). (ii) The high flank the "TATA" box. In the presence of the inducer equilibrium association constant of the repressor-inducer tetracycline, expression is restored to full activity. Location of complex ensures efficient induction at sublethal Tc concen- the operators 21 base pairs downstream of the transcription trations (17), thus making the system useful as an on/off start site does not significantly affect transcription in the switch for the specific regulation of transferred genes. presence of the repressor. These experiments show that a prokaryotic regulatory protein can function in plant cells. The MATERIALS AND METHODS tet repressor-operator complex may be useful for specifically inducing transferred genes at different stages of plant devel- Electric Field-Mediated Gene Transfer ("Electroporation") and Transient Assay Conditions. Expression was measured in opment. electroporated Nicotiana tabacum (gift from T. Bradshaw, University of Washington, Seattle) protoplasts. For the In prokaryotes as well as in eukaryotes, regulation of tran- preparation of protoplasts, suspension cultured cells were scription initiation is mediated by proteins that recognize taken on the third day after being subcultured. Culture specific DNA sites, thereby influencing RNA polymerase conditions were as follows. Cells were maintained in 4.3 g of activity (1). One of the well-characterized negative control Murashige-Skoog (MS) salts per liter, 30 g of sucrose per mechanisms in Escherichia coli involves repressor proteins liter, 1 mg of thiamine per liter, 1 g of myo-inositol per liter, binding to operator DNA, thus preventing RNA polymerase 2 g of KH2PO4 per liter, and 0.2 mg of 2,4-dichlorophenox- from binding (2). In eukaryotes, however, the position of the yacetic acid (2,4-D) per liter at 280C with shaking at 150 rpm regulatory DNA binding sites is not limited to the region near and were subcultured every week with a 5% inoculum. Cells the start site of transcription. Negative (3-5) as well as were washed twice with the culture medium containing 0.4 positive (6) control sequences function somewhat indepen- M mannitol (MSM medium) and then incubated in this dently of their positions and orientations with respect to the medium with 500 mg of cellulase and 50 mg of macerozyme regulated promoter. In spite of these fundamental differ- per 50 ml of cell culture for 2 hr. Cells were pelleted, washed ences between eukaryotic and prokaryotic control mecha- twice with MSM medium, washed once in electroporation nisms, the E. coli lexA protein is able to repress gene buffer (10 mM Hepes, pH 7.2/150 mM NaCl/4 mM expression in yeast (7). Recently, Hu and Davidson (8) have CaCl2/0.4 M mannitol), and resuspended in the same buffer shown that a suitably engineered lac repressor-operator at 4 x 106 protoplasts per ml. After mixing 0.5 ml of the system is functional in mouse cells. By using the TnlO- protoplast-containing solution with 0.5 ml of the same solu- encoded tet repressor-operator interaction, we have ap- tion containing 200 ,ug of supercoiled plasmid DNA, electro- proached the question of whether the plant eukaryotic poration was done as described (18). The electric pulse was transcription machinery can be inhibited by the same mech- delivered from a 490-,uF capacitor charged to 340 V with a anism as is E. coli RNA polymerase-namely, by steric 3-msec resistance-capacitance time constant. Chloramphen- interference with a repressor protein. Introducing the tet icol acetyltransferase (CAT) activity was measured after 24 regulatory elements that respond to the inducer tetracycline hr of incubation (18). Equal amounts of protein were added (Tc) (9, 10) into plant cells provides a unique tool to in each assay. The amount of acetylated chloramphenicol specifically regulate expression of transferred genes. By was determined by cutting the reaction product from a silica using this system rather than plant promoters regulated by plate and counting the radioactivity in a liquid scintillation light (11), stress (12), or hormones (13), one has the advan- spectrometer. tage that only the expression of the transferred gene will be affected by the inducer. This contrasts with the regulatory Abbreviations: CAT, chloramphenicol acetyltransferase; CaMV, factors mentioned above, where pleiotropic effects on the cauliflower mosaic virus; 2,4-D, 2,4-dichlorophenoxyacetic acid; plant are expected. Tc, tetracycline; 01 and 02, operators 1 and 2. *Present address: Institut fuer Genbiologische Forschung, Ihnestr 33, 1 Berlin 63, F.R.G. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at the present payment. This article must therefore be hereby marked "advertisement" address: Plant Gene Expression Center, 800 Buchanan Street, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Albany, CA 94710. 1394 Downloaded by guest on September 30, 2021 Biochemistry: Gatz and Quail Proc. Natl. Acad. Sci. USA 85 (1988) 1395 Construction of Recombinant Plasmids. For constructing region of the tet repressor (tetR) gene was inserted as an pTET7 the cauliflower mosaic virus (CaMV) 35S promoter EcoRI fragment [from pWH305 (24)] between the CaMV 35S fragment [nucleotides 7017-7437 (19)] was subcloned from promoter and the nos poly(A) signal by replacing the nptII an intermediate construct containing these promoter se- gene of pCaMVNEO (20). pTETO contains the same EcoRI quences between the HindIII and the Pst I sites ofthe PiAN7 fragment in the reverse polarity and was used for control (Biolabs, Northbrook, IL) polylinker inserted into pUC18 experiments. (20). The promoter was fused to the cat gene by cloning the former as a Sma I/Bgl II fragment into pGA582 (21) that had been digested with Hpa I and Bgl II. This construct contains RESULTS nucleotides - 390 to + 1 relative to the start site oftranscrip- We inserted the tet operators (01 02) into selected sites of tion of the CaMV 35S promoter, 50 base pairs (bp) between the CaMV 35S promoter whose functional domains have + 1 and the start site ofcat translation, the cat coding region, been characterized by 5' deletions (25, 26). pTET8 (see Fig. and the nos poly(A) signal site. The promoter-cat-nos con- 1) contains the operators between the transcription start site struct was subsequently inserted as a HindIII/Sal I fragment and the initiation codon, with the first base of the left into pUC19 to yield pTET7. Plasmid pTET8 contains an operator (O1) being base + 22 of the cat-encoded mRNA. 80-bp-long EcoRI fragment with the two tet operator sites The inserted operator fragment contains one ATG initiation (14, 22) cloned into the Bgl II site ofpTET7 after filling in the codon that is in frame with the ATG of the cat gene, so that protruding ends. Plasmid pTET8 contains 3 bp of the TnJO- expression was not reduced by an upstream out-of-frame encoded sequence flanking operator 1 (01) and 20 bp flank- open reading frame (27). In pTET14 (Fig. 1A) 01 is posi- ing operator 2 (02). For constructing pTET14, two comple- tioned between the CAAT box and the TATA box and 02 is mentary oligonucleotides containing the "TATA" box ofthe between the TATA box and the start site of transcription. CaMV 35S promoter between the two operators were syn- We chose the position between the consensus boxes under thesized with Hga I and Sau III ends. The distance between the assumption that the CaMV 35S promoter has the same the "CAAT" box and the TATA box was maintained as in characteristics as the thymidine kinase promoter (28). the wild-type promoter. The spacing between the operators Linker scanning of this promoter has shown that the se- was 11 bp as in the E. coli operon. Because of multiple Hga quences between the CAAT and the TATA boxes can be I sites in pUC19, the plasmid was constructed by ligating an changed without eliminating promoter activity. Taking into isolated promoter fragment from HindIII to Hga I ofpTET7, account that spacing between the TATA box and the up- with the hybridized oligonucleotides and the HindIII/Bgl II stream element (in this case, the CAAT box) is important for vector fragment of pTET7. The correct insertion of the maximal promoter activity, at least in simian virus 40 early operators into pTET8 and pTET14 was confirmed by sub- promoter (29), we maintained the CaMV wild-type spacing.