EP0455667A1 - Gene switch - Google Patents

Gene switch

Info

Publication number
EP0455667A1
EP0455667A1 EP90901863A EP90901863A EP0455667A1 EP 0455667 A1 EP0455667 A1 EP 0455667A1 EP 90901863 A EP90901863 A EP 90901863A EP 90901863 A EP90901863 A EP 90901863A EP 0455667 A1 EP0455667 A1 EP 0455667A1
Authority
EP
European Patent Office
Prior art keywords
gst
gene
promoter
inducer
maize
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP90901863A
Other languages
German (de)
French (fr)
Inventor
Ian Georges Bridges
Simon William Jonathan 24 Pound Lane Bright
Andrew James "The Cabin" Greenland
Wolfgang Walter 14 Greenfinch Close Schuch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syngenta Ltd
Original Assignee
Zeneca Ltd
Imperial Chemical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zeneca Ltd, Imperial Chemical Industries Ltd filed Critical Zeneca Ltd
Publication of EP0455667A1 publication Critical patent/EP0455667A1/en
Ceased legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/1085Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088Glutathione transferase (2.5.1.18)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237Externally regulated expression systems
    • C12N15/8238Externally regulated expression systems chemically inducible, e.g. tetracycline

Definitions

  • 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 sequences 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.
  • GST II glutathione-S-transferase
  • the invention also provides a chemically switchable gene constructs which include the maize GST II gene promoter operatively linked to a foreign gene oir 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.
  • 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 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.
  • 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.
  • 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-chloro-4-(trifluoromethyl) -5-thiazole-carboxylate (common name: flurazole)
  • 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.
  • 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:
  • Glutathione-S-transferases are a family of enzymes which catalyse the conjugation of glutathione via the sulphydryl group to a large range of hydrophobic, electrophilic 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 isoform 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.
  • chemical safeners for example, N,N-diallyl-2,2-dichloroacetamide
  • 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
  • FIG. 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.
  • 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
  • Figure 1 shows the results for total GST activity in roots and shoots obtained 23 and 44 hours after treatment as described and Figure 2 shows the separation of the isozymes GST I and GST II.
  • FIG. 5 shows the results using a stem reservoir technique.
  • 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.
  • 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.
  • a cDNA corresponding to a 26 kd subunit of a third, minor component of GST activity in maize seedlings (GST III) has been previously cloned and sequenced.
  • Enzyme activity was measured spectrophotometrically at 340nm using 1-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 8.
  • 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).
  • 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
  • the time course experiments revealed a peak of GST expression at 48 hours after treatement with 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 Hoffraan,1983) . Screening cDNA libraries with antisera to GST I and GST II
  • 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
  • the 5' ends of the oligonucleotides were labelled using polynucleotide kinase as described in the literature.
  • phages containing cDNA were amplified on plates and transferred to nitrocellulose.
  • the filters were hybridised to oligonucleotide probes at temperatures of from 2 to 5°C below the melting temperature calculated for 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 from partial proteolytic cleavage or in the case of genomic DNA, primers based on cDNA sequence determined previously.
  • the isolated cDNA is characterised and subjected to sequencing by one or more of the standard available techniques. Isolation of genomic 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.
  • the PCR method described above may be used to selectively amplify and clone gene fragments.
  • GSTII genes and ther 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 marker genes like GUS and CAT, and testing then in transgenic plants.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Lock And Its Accessories (AREA)

Abstract

On utilise une séquence promotrice de gène répondant à l'application d'un inducteur chimique exogène tel qu'un agent protecteur d'herbicide végétal, comme interrupteur de gène, pour permettre la régulation externe de l'expression d'un gène placé sous la régulation d'un promoteur. Un exemple spécifique de la structure de l'interrupteur de gène est la séquence d'enzyme GSTII (isoforme II de glutathione-S-transférase) du maïs qui est inductrice par application externe de l'agent protecteur N,N-diallyle-2,2-dichloracétamide.A gene promoter sequence responding to the application of an exogenous chemical inducer such as a plant herbicide protective agent is used as a gene switch to allow the external regulation of the expression of a gene placed under the gene. regulation of a promoter. A specific example of the structure of the gene switch is the maize glutathione-S-transferase isoform II (GSTII) enzyme sequence which induces by external application of the protective agent N, N-diallyl-2, 2-dichloracetamide.

Description

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 sequences 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 oir 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 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. 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-chloro-4-(trifluoromethyl) -5-thiazole-carboxylate (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)-5- thiazole-carboxylate;
2. naphthalene-1,8-dicarboxylic anhydride; 3. 2-dichloromethyl-2-methyl-l,3-dioxolane;
4. l-(dichloroacetyl)-hexahydro-3,3,8a-trimethyl- pyrrole (l,2-a)-pyrimidin-6(2H)-one;
5. 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-2- methy) ] acetamide;
9. l-(cyanomethoxyimino)benzacetonitrile; 10. 4'-chloro-2,2,2-trifluoroacetophenone-0-1,3- dioxolan- 2-yl methyloxime;
11. 2,2-dichloro-l-(3,4-dihydro-3-methyl-2H-l,4- benzoxazin-4-yl) ethanone; 12. 3-dichloroacetyl-2,2-dimethyloxazolidine;
13. 4-methoxy-3,3-dimethylbenzophenone;
14. l-cγclohexyl-4,4-dimethyl-2-(1H-1,2,4- triazol-1-yl) pent-l-en-3-ol;
15. 2,2-dichloro-N-(3-methyl-4-thiazolin- 2-ylidene) acetamide;
16. 0,0-diethyl-O-phenyl phosphorothioate;
17. 2,2-spirocyclohexyl-N-dichloroacetyl oxazolidine;
18. N-benzyl-N-ethyl-dichloroacetamide; 19. 3-chloroacetyl-4,4-cyclohexane-spiro-2,2- dimethyl-1,3- oxazolidine; and, 20. spirooxazolidine acetamide.
Glutathione-S-transferases (GST) are a family of enzymes which catalyse the conjugation of glutathione via the sulphydryl group to a large range of hydrophobic, electrophilic 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 isoform 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) 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
(0.003 to 30 pp ) 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 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 μq 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 activity in maize seedlings (GST III) has been previously cloned and sequenced. Enzyme Assay
Enzyme activity was measured spectrophotometrically at 340nm using 1-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 7.5% polyvinylpyrrolidone in a pestle and mortar, at 4°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 (17.5%, 30:0.174 acrylamide: bisacrylamide) of GST I or GST II was achived by concentrating pure GST samples using Amicon 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 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 Hoffraan,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 5°C below the melting temperature calculated for 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 from 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 genomic 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 used to selectively amplify and clone gene fragments. GSTII genes and ther 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 marker genes like GUS and CAT, and testing then in transgenic plants.

Claims

1. A chemically inducible gene promoter sequence isolated from a 27kd subunit of the maize glutathione-S-transferase (GST II) gene
A chemically switchable gene 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 transforma ion.
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¬ methyl)-5-thiazole-carboxylate.
A promoter/inducer combination wherein the promoter is the GST II promoter aforesaid and the inducer is N,N-diallyl-2,2-dichloro- acetamide.
EP90901863A 1989-01-26 1990-01-25 Gene switch Ceased EP0455667A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB898901673A GB8901673D0 (en) 1989-01-26 1989-01-26 Gene switch
GB8901673 1989-01-26

Publications (1)

Publication Number Publication Date
EP0455667A1 true EP0455667A1 (en) 1991-11-13

Family

ID=10650613

Family Applications (1)

Application Number Title Priority Date Filing Date
EP90901863A Ceased EP0455667A1 (en) 1989-01-26 1990-01-25 Gene switch

Country Status (6)

Country Link
EP (1) EP0455667A1 (en)
AU (1) AU647016B2 (en)
CA (1) CA2008695C (en)
GB (1) GB8901673D0 (en)
WO (1) WO1990008826A1 (en)
ZA (1) ZA90604B (en)

Families Citing this family (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8901677D0 (en) 1989-01-26 1989-03-15 Ici Plc Hybrid seed production
US5907086A (en) * 1991-05-01 1999-05-25 Pioneer Hi-Bred International, Inc. Plant promoter sequences
GB9114259D0 (en) * 1991-07-02 1991-08-21 Ici Plc Plant derived enzyme and dna sequences
WO1993001290A1 (en) * 1991-07-11 1993-01-21 International Flower Developments Pty. Ltd. Genetic sequences encoding flavonoid pathway enzymes and uses therefor
ATE207962T1 (en) 1992-04-13 2001-11-15 Syngenta Ltd DNA CONSTRUCTIONS AND PLANTS CONTAINING THEM
GB9223332D0 (en) * 1992-11-06 1992-12-23 Ici Plc Production of polyhydroxyalkanoate in plants
GB9223454D0 (en) * 1992-11-09 1992-12-23 Ici Plc Novel plants and processes for obtaining them
US6066456A (en) * 1993-12-30 2000-05-23 Zeneca Limited Plant-derived enzyme and DNA sequences and uses thereof
EG23907A (en) * 1994-08-01 2007-12-30 Delta & Pine Land Co Control of plant gene expression
US5723765A (en) * 1994-08-01 1998-03-03 Delta And Pine Land Co. Control of plant gene expression
JPH11506319A (en) 1995-05-26 1999-06-08 ゼネカ・リミテッド Gene switch containing ecdysone receptor
US5859338A (en) * 1995-06-06 1999-01-12 California Institute Of Technology Plant clavata1 nucleic acids, transformed plants, and proteins
AU715535B2 (en) * 1995-09-22 2000-02-03 Syngenta Limited Plant glutathione S-transferase promoters
US6610828B1 (en) 1996-05-24 2003-08-26 Syngenta Limited Heliothis ecdysone receptor
EP1017830A1 (en) * 1997-07-30 2000-07-12 Zeneca Limited Genetic method for controlling sprouting
HU228267B1 (en) 1998-02-20 2013-02-28 Syngenta Ltd Method for producing hybrid seed, expression casettes usable in the method, and plant tissuses or plants containing them
SI1068311T1 (en) 1998-04-08 2011-07-29 Commw Scient Ind Res Org Methods and means for obtaining modified phenotypes
EP1029923A1 (en) 1999-01-27 2000-08-23 D.J. Van Der Have B.V. Method for conveying BNYVV resistance to sugar beet plants
EP1780284B1 (en) 1999-12-16 2009-10-28 CropDesign N.V. Optimized T-DNA transfer and vectors therefor
EP2119786A1 (en) 2008-05-13 2009-11-18 Expressive Research B.V. Increased production of health-promoting compounds in plants
US20130055471A1 (en) 2009-12-15 2013-02-28 Edwin Henricus Antonius HOLMAN Transgenic Ozone-Resistant Plants
NL2004624C2 (en) 2010-04-28 2011-11-01 Stichting Dienst Landbouwkundi A new glycosyltransferase protein and its role in the metabolism of phenylpropanoid volatiles in tomato.
WO2012151254A1 (en) 2011-05-02 2012-11-08 Board Of Regents Of The University Of Nebraska Plants with useful traits and related methods
US9612235B2 (en) * 2012-04-05 2017-04-04 Koch Biological Solutions, Llc Herbicidal compound screening
US10767188B2 (en) 2013-09-25 2020-09-08 Nutech Ventures Methods and compositions for obtaining useful plant traits
WO2018037123A1 (en) 2016-08-26 2018-03-01 Lesaffre Et Compagnie Improved production of itaconic acid
EP3533878A1 (en) 2018-02-28 2019-09-04 Dutch DNA Biotech B.V. Process for producing citramalic acid employing aspergillus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9008826A1 *

Also Published As

Publication number Publication date
AU4947690A (en) 1990-08-24
AU647016B2 (en) 1994-03-17
GB8901673D0 (en) 1989-03-15
WO1990008826A1 (en) 1990-08-09
CA2008695A1 (en) 1990-07-26
ZA90604B (en) 1990-11-28
CA2008695C (en) 1999-03-16

Similar Documents

Publication Publication Date Title
CA2008695C (en) Gene switch
US5866792A (en) Plant-derived enzyme and DNA sequences and uses thereof
US5608143A (en) External regulation of gene expression
US6172279B1 (en) Plant gene construct encoding a protein capable of disrupting the biogenesis of viable pollen
KR100225352B1 (en) Chemically regulatable dna sequences and genes and uses thereof
KR100212691B1 (en) Chimeric dna construction useful for producing transgenic disease-resistant plants, transgenic plants containing them and process for their preparation
US5633437A (en) Gene exhibiting resistance to acetolactate synthase inhibitor herbicides
CA1338902C (en) Plant cells resistant to herbicidal glutamine synthetase inhibitors
US5145777A (en) Plant cells resistant to herbicidal glutamine synthetase inhibitors
EP0200746B1 (en) Plant cells resistant to herbicidal glutamine synthetase inhibitors
US6730828B1 (en) Isolated nucleic acids encoding a wheat glutathione transferase, plants transformed therewith and methods of use thereof
US6740749B1 (en) Ozone-induced gene expression in plants
US7091312B2 (en) Biocidal protein
US6066456A (en) Plant-derived enzyme and DNA sequences and uses thereof
US20020108145A1 (en) Constitutive disease resistance (CDR1) gene and methods of use thereof
AU609391C (en) Plant cells resistant to herbicidal glutamine synthetase inhibitors
JP2003009690A (en) Method of producing plant having increased resistance to substance hindering growth of the same

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19910711

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: ZENECA LIMITED

17Q First examination report despatched

Effective date: 19940928

APAB Appeal dossier modified

Free format text: ORIGINAL CODE: EPIDOS NOAPE

APAB Appeal dossier modified

Free format text: ORIGINAL CODE: EPIDOS NOAPE

APAD Appeal reference recorded

Free format text: ORIGINAL CODE: EPIDOS REFNE

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: SYNGENTA LIMITED

APAB Appeal dossier modified

Free format text: ORIGINAL CODE: EPIDOS NOAPE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20010517

APAF Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNE