AU647016B2 - Gene switch - Google Patents

Description

OPI DATE 24/08/90 AOJP DATE 27/09/90 APPLN- ID 49476

PCT

PCT NUMBER PCT/GB90/00101 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/08826 C12N 15/54,15/82 Al (43) International Publication Date: 9 August 1990 (09.08.90) (211) International Application Number: PCT/GB90/00101 (74) Agent: HUSKISSON, Frank, Mackie; Imperial Chemical Industries pic, Legal Department, Patents, P.O. Box No.

(22) International Filing Date: 25 January 1990 (25.01.90) 6, Bessemer Road, Welwyn Garden City, Herts AL7 IHD (GB).

Priority data: 8901673.7 26 January 1989 (26.01.89) GB (81) Designated States: AT (European patent), AU, BB, BE (European patent), BF (OAPI patent), BG, BJ (OAPI patent), BR, CF (OAPI patent), CG (OAPI patent), CH (71) Applicant: IMPERIAL CHEMICAL INDUSTRIES PLC (European patent), CM (OAPI patent), DE (European [GB/GB]; Imperial Chemical House, Millbank, London patent), DK (European patent), ES (European patent), SW1P 3JF FI, FR (European patent), GA (OAPI patent), GB (European patent), HU, IT (European patent), JP, KP, KR, (72) Inventors: BRIDGES, Ian, Georges Box 30A, R.R.I, Sla- LK, LU (European patent), MC, MG, ML (OAPI pater, IA 50244 BRIGHT, Simon, William, Jonathan tent), MR (OAPI patent), MW, NL (European patent), 24 Pound Lane, Marlow, Bucks SL7 2AY NO, RO, SD, SE (European patent), SN (OAPI patent), GREENLAND, Andrew, James "The Cabin", Raymill SU, TD (OAPI patent), TG (OAPI patent).

Road East, Maidenhead, Berkshire SL6 8SX (GB).

SCHUCH, Wolfgang, Walter 14 Greenfinch Close, Heathlake Park, Crowthorne, Berkshire RG 1 6TZ Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

647016 (54) Title: GENE SWITCH (57) Abstract A gene promoter sequence which responds to application of an exogenous chemical inducer such as one of the plant herbicide safeners is utilised as a gene switch to enable external control of expression of a gene placed under control of the promoter.

A specific example of the gene switch construct is the maize GSTII (glutathione-S-transferase isoform II) enzyme promoter sequence which is induced by external application of the safener N,N-diallyl-2,2-dichloroacetamide.

WO 90/08826 PCI'/GB90/00101 1 GENE SWITCH This invention relates to a gene promoter sequence which is responsive to an applied exogenous chemical inducer enabling external control of expression of the gene controlled by the promoter. The sequence of the invention is conveniently' referred to as a gene switch.

European Patent Application 332,104 (published 18th September 1989) describes chemically regulatable DNA sequehces which are capable of regulating transcription of an associated DNA sequence in plants or plant tissue. The particular source of the sequences is described as being the PR (pathogenesis-related) protein gene.

According to the invention there is provided a chemically inducible gene promoter sequence isolated from a 27kd subunit of the maize glutathione-S-transferase (GST II) gene.

The invention also provides a chemically switchable gene constructs which include the maize GST II gene promoter operatively linked to a foreign gene or a series of foreign genes whereby expression of said foreign gene or said series of genes may be controlled by application of an effective exogenous inducer.

In practice the chemically inducible promoter of the invention will be inserted as a promoter sequence in a recombinant gene construct destined for use in a plant. The construct will then be WO 90/08826 PCT/GB90/00101 inserted into the plant by transformation.

Expression of protein encoding genes in the construct, being under control of the chemically switchable promoter of the invention, may be controlled by the application of a chemical inducer to the plant.

The method employed for transformation of the plant cells is not especially germane to this invention and any method suitable for the target plant may be employed. Transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation procedures are known from the literature such as agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, microinjection of plant cells and protoplasts, microprojectile transformation and pollen tube transformation, to mention but a few.

Reference may be made to the literature for full details of the known methods.

Neither is the plant species into which the chemically inducible promoter is inserted particularly germane to the invention.

Dicotyledonous plant are rel;atively simple to transform whereas the monocotyledonous plants (which include the major cereal crops such as maize) are rather more difficult to transform.

However, this invention may be applied to any plant for which transformation techniques are, or become, available.

Therefore, the invention further provides a plant having a recombinant gene construct which includes the chemically inducible promoter of the invention stably incorporated in its genome by transformation.

WO 90/08826 PC/GB90/001P/I 3 The invention also provides an effective promoter/inducer combination wherein the promoter is the GST II promoter aforesaid and the inducer is N,N-diallyl-2,2-dichloroacetamide (common name: dichloramid) or benzyl-2-chlorp-4-(trifluoromethyl) (common name: flurazole) However, more generically, the present invention comprises a recombinant DNA construct including a gene promoter inducible by application of an exogenous chemical inducer operatively linked to control expression of a target gene. Suitable promoters and specific inducers may be identified by the procedures exemplified hereinbelow in respect of the GST II gene promoter and its inducer. In other words, the invention comprises the use of a gene promoter which is inducible by externally applied chemical to control expression of a gene sequence within a plant genome.

Chemical inducers which are potential inducers of the GSTII 27kd subunit expression include compounds such as: 1. benzyl-2-chloro-4-(trifluoromethyl)-5thiazole-carboxylate; 2. naphthalene-1,8-dicarboxylic anhydride; 3. 2-dichloromethyl-2-methyl-1,3-dioxolane; 4. l-(dichloroacetyl)-hexahydro-3,3,8a-trimethylpyrrole (l,2-a)-pyrimidin-6(2H)-one; 2,2,5-trimethyl-N-dichloroacetyloxazolidine; 6. 1,3-dioxolan-2-ylmethoxyimono(phenyl)benzene acetonitrile; 7. 4,6-dichloro-2-phenyl-pyrimidine; 8. 2,2-dichloro-[N-allyl-N(1,3-dioxalano-2methy)] acetamide; 9. 1-(cyanomethoxy'mino)benzacetonitrile; WO 90/08826 PCr/GB90/00101 4 4'-chloro-2,2,2-trifluoroacetophenone-0-l,3dioxolan- 2 -yl methyloxime; 11. 2,2-dichloro-1-(3,4-dihydro-3-methyl-2H-1,4benzoxazin-4-yl) ethanone; 12. 3-dichloroacetyl-2,2-dimethyloxazolidine; 13. 4-methoxy-3,3-dimethylbenzophenone; 14. 1-cyclohexyl-4,4-dimethyl-2-(1H-1,2,4triazol-1-yl) pent-1-en-3-ol; 2,2-dichlcro-N-(3-methyl-4-thiazolin- 2-ylidene) acetamide; 16. Q,O-diethyl-O-phenyl phosphorothioate; 17. 2,2-spirocyclohexyl-N-dichloroacetyl oxazolidine; 18. N-benzyl-N-ethyl-dichloroacetamide; 19. 3-chloroacetyl-4,4-cyclohexane-spiro-2,2diiaethyl-1,3- oxazolidine; and, spirooxazolidine acetamide.

Glutathione-S-transferazes (GST) are a family of enzymes which catalyse the conjugation of glutathione via the suiphydryl group to a large range of hydrophobic, electroph~lic compounds. The conjugation results in detoxification of these compounds and in insects and mammals, removal from tissue.

GST enzymes have been identified in a range of crop plants including maize, wheat, sorghum and peas. GST's comprise from 1 to 2% of the total soluble protein in etiolated maize seedlings.

The major isoforin of GST can be distinguished in maize tissue. GST I is constitutively expressed and is capable of conjugating glutathione with the pre-emergent herbicides alachlor and atrazine.

Treatment of maize tissues with chemical safeners (for example, N,N-diallyl-2,2-dichloroacetamide) WO 90/08826 PCT/GB90/00101 raises the activity of GST I which participates in the detoxification of the pre-emergent herbicides.

The invention will now be described by way of illustration in the following description.

The drawings which accompany this application are as follows: Figure 1 Figure 1 shows the results for total GST activity in roots and shoots obtained 23 and 44 hours after treatment with R25 as described below; Figure 2 shows the chromatographic separation of the isozymes GST I and GST II.

Figure 3 shows GST I activity present in untreated anther tissue; Figure 4 shows the stimulation of GST II activity after treatment with R25 as described below; Figure 5 shows the results using a stem reservoir technique; Figure 6 shows the results with the application by spray; and, Figure 7 is a time course graph generated in the manner described below.

Safener treatment of corn tissue For treatment of young maize seedlings, seeds were germinated on moist filter paper. After germination and growth (up to one week) the safener N,N-diallyl-2,2- dichloroacetamide (hereinafter referred to as R25) was added to the water in the filter paper to give a range of concentrations (C.003 to 30 ppmy and the seedlings grown for a further 23 to 44 hours before harvesting of root and shoot tissue. Figure 1 shows the results for total GST activity in roots and shoots obtained 23 WO 90/08826 PCT/GB90/00101 6 and 44 hours after treatment as described and Figure 2 shows the separation of the isozymes GST I and GST II.

For treatment of maize tassel and anther tissue, a solution of 800 pg of R25 was injected into the node directly below the developing tassel.

Uptake then continued for a further 48 to 72 hours.

Figure 3 shows that only GST I activity was present in untreated anther tissue and Figure 4 shows the stimulation of GST II activity after treatment as described.

Alternatively a 100 ppm solution of R25 was supplied from a glass reservoir attached to the exposed stem immediately below the developing tassel. Figure 5 shows the results using a stem reservoir technique.

Additionally, R25 was applied as a 100 ppm spray directly on to the exposed developing tassel.

Figure 6 shows the results with the application by spray.

Both GST proteins have a native molecular weight of approximately 50 kd. As in mammals, maize GST's are dimeric; GST I has apparently identical subunits of 29 kd, whereas GST II is a heterodimer of a 29 kd subunit similar to that found in GST I and a novel 27 kd subunit which is only present in tissue treated with safener except in seedling root where it is constitutively expressed, but still can be induced by safener treatment.

A cDNA and a gene corresponding to the 29 kd subunit of GST I have been cloned previously and sequenced. In addition, a cDNA corresponding to a 26 kd subunit of a third, minor component of GST WO 90/08826 PCrGB90/00101 7 activity in maize seedlings (GST III) has been previously cloned and sequenced.

Enzyme Assay Enzyme activity was measured spectrophotometrically at 340nm using l-chloro- 2,4-dinitrobenzene (CDNB) as a substrate. The reaction buffer contained 0.1M EDTA, 0.001M CDNB and 0.0025M glutathione.

Preparation of extracts and enzyme purification Tissue was homogenised in 0.05M Tris.HCl, pH 7.8; 0.001M EDTA; 0.001M DTT; and polyvinylpyrrolidone in a pestle and mortar, at 4 0 C, and centrifuged at 30,000g to obtain a crude extract.

Separation of the GST isoforms from the crude extract was achieved as follows: the crude extract was applied to a DEAE Sepharose column and washed with 0.01M Tris.HCl, pH 7.8; 0.001M EDTA; and 0.001M DTT. The bound GST was eluted with 0.3M potassium chloride. Fractions containing GST activity were combined and desalted using PD10 gel filtration columns. Separation of the GST I and GST II isoforms was achieved by FPLC on a mono-Q column and a zero to 0.4M potassium chloride concentration gradient.

Pure samples of GST I and GST II were obtained by applying desalted fractions of GST I and GST II from the FPLC to a glutathione-S-sepharose affinity column equilibrated with 0.05M phosphate buffer at pH 7.3. After washing with buffer, bound GST was eluted with 0.005M glutathione.

SDS-PAGE 30:0.174 acrylamide: bisacrylamide) of GST I or GST II was achived by concentrating pure GST samples using Amicon WO 90/08826 PCr/GB90/00101 8 Centricon 10 Microconcentrations (Trade Mark), denaturing samples in mercaptoethanol containing Laemmli buffer, and staining the gels with Coomassie Blue.

Generation of antibodies to the enzyme Sufficient protein to enable the immunisation of rabbits is obtained by pooling the isolated enzyme subunit isolated as described above from a number of separate experiments. The 27 kD GST II polypeptide is subsequently purified to apparent homogeneity by electroelution from polyacrylamide gel slices. Antisera are prepared against the 27 kd polypeptide. The immunisation of'rabbits is carried out essentially according to Mayer and Walker (1978).

N-terminal sequence analysis The amino terminal sequence of the intact 27 kd subunit of GST II or partial proteolytic cleavage products was determined by sequential Edman degradation and subsequent amino acid analysis by HPLC.

Time Course A time course experiment was carried out to examine the expression of GST's after safener treatment. A 30 ppm solution of R25 was applied to three-day old seedling roots and tissue harvested after various time intervals following safener treatment. Samples were tested for GST activity using the enzyme assay described above. The results of this experiment are presented graphically in Figure 7.

Synthesis of cDNA libraries The time course experiments revealed a peak of GST expression at 48 hours after treatement with WO 90/08826 P(rGB90/00101 9 safener. Therefore, two cDNA libraries were constructed from RNA extracted from tissue at 24 and 48 hours after safener treatment. -To ensure that the induction procedure had been successful, a one gram sample of 24 hour induced tissue was taken and assayed for GST II. This experiment revealed that the tissue used to construct the cDNA library had indeed been successfuly induced as GST II accounted for 45.5% of the total GST activity.

Double-stranded cDNA was prepared from oligo dT-cellulose-purified RNA by a method employing RNaseH and E.coli DNA polymerase I in the synthesis of the second strand, without prior purification of single-stranded cDNA (Gubler and Hoffman,1983).

Screening cDNA libraries with antisera to GST I and GST II In order to identify a cDNA clone encoding maize tassel GST enzyme, bacteriophage from the amplified cDNA library are screened with anti-maize GST enzyme serum. The clones producing the strongest signals are re-screened.

Screening cDNA libraries using oligo probes Mixtures of synthetic oligonucleotides based on the amino acid sequence determined above were prepared by phosphoramidite chemical synthesis.

The 5' ends of the oligonucleotides were labelled using polynucleotide kinase as described in the literature.

Approximately 40,000 phages containing cDNA were amplified on plates and transferred to nitrocellulose. The filters were hybridised to oligonucleotide probes at temperatures of from 2 to 0 C below the melting temperature calculated for 10 the lowest melting point probe in the mixture. Hybridising plaques were selected and rescreened through two or more rounds exactly as described above but at lower densities.

Isolation of cDNA gene sequences by the PCR method cDNA or DNA sequences are isolated from the libraries described using oligo primers based on the amino acid sequence obtained form partial proteolytic cleavage or in the case of genomic DNA, primers based on cDNA sequence determined previously.

Characterisation and sequence analysis of GST cDNA clones The isolated cDNA is characterised and subjected to sequencing by one or more of the standard available techniques.

Isolation of qenomic sequences An existing genomic library of fragments of total maize DNA cloned into XEMBL3 is used to isolate clones that hybridise to the cDNA clones isolated as described above.

Alternatively, the PCR method described above may be 20 used to selectively amplify and clone gene fragments.

GSTII genes and their promoter sequences can then be isolated, and characterised using established techniques.

It can be demonstrated that the GSTII promoter sequences mediate safener-induced gene activity by fusing them to 25 marker genes like GUS and CAT, and testing then in transgenic plants.

A method for the isolation of the genomic GSTII clone, which includes the promoter sequence, is described below.

The cDNA for GST-II-27 was utilised to design a gene probe for the isolation of a corresponding genomic sequence which included the promoter region.

The GST-II-27 cDNA sequence was examined to identify a specific region which does not occur in other maize GSTs or other plant/animal/viral/vector sequences, and hence would be suitable as a specific gene probe. A 208 bp S sequence was isolated by PCR from the 3' end of the cDNA Ssequence of GST-II-27 and purified by acrylamide gel r P 9 9 electrophoresis. This PCR probe was random prime labelled 1Oa and used to screen 5x106 recombinants from a maize genomic library (p rtial MboI digested D4A with an average insert size of The positive clones were plaque-purified with the 3' PCR probe and a 5' oligo (130 bp from the 5' end of the cDNA). These genomic clones were then mapped to identify fragments running from the 5' end of the cDNA into the promoter region. An EcoRI fragment containing around 4kB of promoter region was isolated. This fragment was subcloned into a pBS vector, designated plasmid pGIE7, which was deposited in the National Collections of Industrial and Marine Bacteria (NCIMB), Aberdeen, with the accession number NCIMB 40426.

Primer extension analysis (as described by Ausubel et al, 1987, Current Protocols in Molecular Biology) was used to map the transcription start point (TSP) to 16 bp from the end of the cDNA. Sequence analysis of the promoter sublone pGIE7 revealed putative TATA and CAAT boxes -29 bp and -110 bp from the predicted TSP respectfully. The TSP site, TATA box and intron/exon boundary regions fit the plant consensus sequences, confirming that the isolatsd sequence is indeed the promoter sequence.

S* SEQUENCING OF THE GST-II-27 PROMOTER FRAGMENT 25 PCR and restriction analysis of the genomic clones indicated that two introns convering 300 bp are present in the region corresponding to the 5' end of the cDNA, and Sthat one of the introns contains an EcoRI cleavage site.

Plasmid pGIE7 contains the EcoRI-EcoRI fragment which covers the GST-II-27 promoter region plus some coding sequence.

Geron:~c ublOnes pGIE7 and pGIS15 were used to sequence 5.4 Kb of the GST--II-27 gene and promoter. The majority was sequenced on both strands.

GST-II-27 PROMOTER CONSTRUCTS Using a combination of fine mapping of promoter subclones and sequence data, appropriate restriction sites were chosen for promoter constxucts. A variety of '9 transient assay vectors for protoplast systems (pPUG) and lOb stable transformation vectors for tobacco (pGSTTAK) ind Zea mays (pZM/RMS-3) were constructed. All recombinant plasmids were identified by hybridisation probes, their integrity and orientation checked by restriction mapping and boundaries of completed vectors analysed by sequencing.

A variety of transient and stable transformation GUS vectors were constructed using approximately 3.8 Kb of the GST-II-27 promoter. Nde I was used to cut the GST-II-27 promoter at the ATG and 4 Kb upstream. This fragment was cut with EcoRI, blunted and cloned into Sma I site of pTAK (a Binl9) based promoterless GUS construct). The GST GUS cassette from pGSTTAK was then cloned into the transient assay vector pPUG (3.8 GST promoter and GUS). The same GST GUS cassette was also cloned into a pUC derived vector containing the BAR selectable cassette giving pZM/RMS-3.

5306N 0..

Claims (5)

1. A chemically inducible gene promoter sequence isolated from a 27kd subunit of the maize glutathione-S-transferase (GST.II) gene.

2. A chemically switchable gee construct which includes the maize GST II gene promoter operatively linked to a gene or a series of genes whereby expression of said gene or said series of genes may be controlled by application of an effective exogenous inducer.

3. A plant having a construct as claimed in claim 2 stably incorporated in its genome by transformation.

4. A promoter/inducer combination wherein the promoter is the GST II promoter aforesaid and the inducer is N,N-diallyl-2,2-dichloro- acetamide or benzyl-2-chloro-4-(trifluoro- A promoter/inducer combination wherein the promoter is the GST II promoter aforesaid and the inducer is N,N-diallyl-2,2-dichloro- acetamide.

6. A method for the selective contro, of gene expression in a transgenic plant comprising placing a protein encoding DNA sequence under control of the promoter of the 27kd sub-unit of the maize glutathione-S-transferase gene and applying to the said plant an effective concentration of a chemical inducer of that promoter. DATED: 26 November 1993 PHILLIPS ORMONDE FITZPATRICK Attorneys for: IMPERIAL CHEMICAL INDUSTRIES PLC 4863N 3 39 WDN 12