EP1104472A1 - Commutateur de gene - Google Patents

Commutateur de gene

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Publication number
EP1104472A1
EP1104472A1 EP99939526A EP99939526A EP1104472A1 EP 1104472 A1 EP1104472 A1 EP 1104472A1 EP 99939526 A EP99939526 A EP 99939526A EP 99939526 A EP99939526 A EP 99939526A EP 1104472 A1 EP1104472 A1 EP 1104472A1
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
plant
seq
target gene
response protein
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.)
Withdrawn
Application number
EP99939526A
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German (de)
English (en)
Inventor
Alberto Zeneca Agrochemicals MARTINEZ
Ian Zeneca Agrochemicals JEPSON
Rupert George University of Nottingham FRAY
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
Syngenta Ltd
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Publication date
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Publication of EP1104472A1 publication Critical patent/EP1104472A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8217—Gene switch
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates inter alia, to the induction of gene expression in a eukaryote by the application of a chemical inducer to the eukaryote and to materials and methods for achieving induction.
  • a chemical inducer to the eukaryote and to materials and methods for achieving induction.
  • Such systems are referred to as "gene switches”.
  • the present invention relates to a method of controlling expression of a target gene in a plant, animal or yeast.
  • Bacterial cells have the ability to respond to the surrounding environment. The response to different environmental cues is essential for survival of bacteria. It is apparent that individual bacteria in a population are also able to sense the density and state of the local bacterial population (the "quorum") of which they are members. That is, an individual bacterium can detect the presence of like bacteria in the surrounding environment. Quorum sensing allows bacteria to synchronise growth, development which when a minimal population level is reached initiates a concerted response from the population.
  • N-(3-oxohexanoyl)-L-homoserine lactone or autoinducer regulates bioluminescence in a cell density-dependent manner.
  • Luxl is the gene involved in the biosynthesis of the homoserine lactone but the mechanism by which this takes place is unclear. It has been proposed that S-adenosylmethionine and coenzymeA or the acyl carrier protein adduct of 3-oxohexanoic acid are substrates for the luxl gene in P. fischeri.
  • LuxR the response regulator or autoinducer receptor
  • OHHL N-(3-oxo)hexanoyl-L-homoserine lactone
  • OHHL N-(3-oxo)hexanoyl-L-homoserine lactone
  • OHHL N-(3-oxo)hexanoyl-L-homoserine lactone
  • LuxR C-terminal end shows amino acid homology to transcriptional activators known as the two component environmental-sensing systems such as UhpA, FixJ and ⁇ arL.
  • LuxR is thought to interact with D ⁇ A as a homodimer to a palindrome within the luxl operator sequence termed lux box.
  • the ⁇ -terminal end of the protein is called the - 9 .
  • Quorum sensing systems are found in other bacteria and may be activated by different homoserine lactones. Such is the case in P. aeruginosa PAO1, Lasl directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone
  • the present invention therefore seeks to provide inter alia, methods and materials for the induction of gene expression in eukaryotes by the application of chemical inducers to the eukaryote.
  • a method of initiating transcription of a target gene in a eukaryotic cell comprising:
  • the eukaryotic cell may already contain the mechanisms to produce the said response protein or may alternatively be provided with them by inserting into the said cell, a polynucleotide which provides for the production of the response protein using techniques well known within the art.
  • the inducible promoter for use in the above mentioned method may comprise the nucleotide sequence depicted as SEQ ID No. 2 and the response protein may comprise the amino acid sequence depicted in SEQ ID No. 16.
  • the inducible promoter may comprise a functional variant such as a polynucleotide which is the complement of one which binds to SEQ ID No. 2 at a temperature of between 60°C and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 % SDS wherein the said polynucleotide is still capable of acting as an inducible promoter upon binding with the said inducing complex.
  • the response protein for use in the method of the present invention may be encoded by the polynucleotide comprises the sequence depicted in SEQ ID No. 5.
  • the polynucleotide encoding the said response protein may comprise the complement of one which binds to SEQ ID No. 5 at a temperature of between 60°C and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS wherein the said polynucleotide still encodes a protein which is capable of forming an inducing complex with the said chemical inducer. It is particularly preferred that the polynucleotide defining the inducible promoter according to the present invention contains the region depicted as SEQ ID No. 21.
  • the method of present invention is particularly applicable to initiating transcription in cells of plants, more particularly in the cells of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus or nut plants.
  • the method of the present invention may be used to initiate transcription in a variety of tissues, including roots, leaves, stems and reproductive tissues.
  • the chemical inducer which may be used in the above mentioned method is N-(3-oxo)hexanoyl-L-homoserine lactone or a functional equivalent thereof and may be applied to the plant cell as part of an agriculturally acceptable formulation.
  • the chemical inducer may be produced within the plant by inserting into the genome of the plant a polynucleotide encoding a protein which provides for the production of the chemical inducer within the plant.
  • This polynucleotide may, for example, be under the transcriptional control of a constitutive promoter, a gene switch (such as the alcA/R, Heliothis ecdysone and GST-27 gene switch), wound inducible, tissue or temporal promoters.
  • a gene switch such as the alcA/R, Heliothis ecdysone and GST-27 gene switch
  • promoter sequence or a part thereof may obtainable from the vanl gene of Vibiro anguillarum and the response protein encoded by the vanR gene may be used with the chemical N-(3-oxo-decanoyl)-L-homoserine lactone (ODHL) or a functional variant thereof.
  • ODHL chemical N-(3-oxo-decanoyl)-L-homoserine lactone
  • the polynucleotide encoding the said response protein in the method referred to above may be bounded by a promoter and a terminator sequence and in particular the promoter may be inducible such as the Ale A/ R switch system, the GST switch system and the ecdysone switch.
  • the promoter may be constitutive such as cauliflower mosaic virus 35S/19S, maize Ubiquitin and Arabidopsis Ubiquitin 3 or may be developmentally regulated specific promoter such as those controlling expression of gene required during seed formation, germination such as cysteine proteinases (as specified in our International Publication No WO 97/35983 ) and malate synthase.
  • the target gene in the method referred to above may be any gene of interest, for example, ⁇ -glucuronidase; Bacillus thuringenesis toxin; barnase or barstar.
  • a method of providing plants containing an inducible target gene comprising: (a) inserting into a plant cell which cell provides for production of a response protein a polynucleotide defining an inducible promoter sequence operably liked to said target gene; and (b) regenerating morphologically normal fertile plants thereof; and (c) applying to the population of regenerants a chemical inducer or a functional variant thereof capable of binding to said response protein, whereby said chemical inducer binds to said response protein to form an inducing complex which binds to and induces said inducible promoter thereby initiating transcription of said target gene; and (d) selecting those plants which are expressing the said target gene.
  • the inducible promoter sequence referred to in the preceding paragraph may comprise the nucleotide sequence depicted as SEQ ID No. 2 or a functional variant thereof and the response protein comprises the amino acid sequence depicted as SEQ ID No 16 or a functional variant thereof and the said chemical inducer is N-(3-oxo)hexanoyl-L- homoserine lactone or a functional equivalent thereof.
  • the inducible promoter sequence may contain the nucleotide sequence depicted as SEQ ID No.
  • the response protein may comprises the amino acid sequence depicted as SEQ ID No 17 or a functional variant thereof and the said chemical inducer may be N-(3-oxo)dodecanoyl-L-homoserine lactone or a functional equivalent thereof.
  • the said inducible promoter sequence may contain the nucleotide sequence depicted as SEQ ID No. 12 or a functional variant thereof and the response protein may comprise the amino acid sequence depicted as SEQ ID No 18 or a functional variant thereof and the said chemical inducer may be N-(3- oxo)octanoyl-L-homoserine lactone or a functional equivalent thereof.
  • the present invention also provides plants produced according to the method of the preceding paragraph which plants may be selected from the group consisting of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus or nut plants.
  • transgenic plants are obtained by regeneration from the transformed cells.
  • Numerous transformation procedures are known from the literature including agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, micro injection or plants cells and protoplasts, microprojectile transformation. All of these methods are well known within the art.
  • the present invention may also be applied to any plant for which transformation techniques are, or become, available.
  • a DNA construct which comprises a first polynucleotide region which comprises an inducible promoter sequence operably liked to and capable when induced of initiating transcription of a target gene and a second polynucleotide region which provides for the production of a response protein wherein upon contact of the said response protein with a chemical inducer the said response protein binds the chemical inducer to produce an inducer complex which binds the said inducible promoter sequence thereby initiating transcription of the said target gene.
  • the said first polynucleotide of the DNA construct may comprise the nucleotide sequence depicted as SEQ ID No.
  • the second polynucleotide region may comprise a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 16 or a functional variant thereof.
  • the said first polynucleotide may comprise the nucleotide sequence depicted as SEQ ID No. 10 or a functional variant thereof and the second polynucleotide region may comprise a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 17 or a functional variant thereof.
  • the said first polynucleotide may comprise the nucleotide sequence depicted as SEQ ID No.
  • the second polynucleotide region may comprise a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 18 or a functional variant thereof.
  • the said second polynucleotide region referred to above may comprise a promoter operably linked to the polynucleotide encoding the said response protein.
  • the said promoter may be inducible and constitutive or developmentally controlled.
  • the present invention still further provides the use of a DNA construct referred to above in the production of a plant containing a target gene of which the expression may be controlled by the application of a chemical inducer to the said plant.
  • a method of screening compounds in a bioassay comprising applying an amount of a chemical to a plant which plant contains a polynucleotide comprising an inducible promoter operably linked to a region encoding a reporter protein and which plant is also capable of producing a response protein and testing the said plant for the production of the said reporter protein.
  • the chemical could be applied to the plant and the activity of the reporter gene monitored for e.g. improved activity, mobility or stability or to assess if the chemical had an inhibitory effect on the response protein which resulted in a decrease in activity of the reporter gene.
  • a method of providing a plant which contains a target gene which is inducibly controlled comprising: (a) inserting into a first plant cell a polynucleotide comprising a first inducible promoter operably linked to a target gene and regenerating a first morphologically normal fertile plant therefrom;
  • the term "functional variant" with respect to a polynucleotide encoding the inducible promoter of the present invention includes variant sequences which are the complement of a sequence which hybridises to the inducible promoter sequence at a temperature of between 60°C and 65°C in 0.3 strength citrate buffered saline containing 0.1 %> SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 %> SDS and which are still capable of acting as an inducible promoter.
  • the term "functional variant" with respect to the response protein includes variant proteins obtained by conservative substitutions within the amino acid sequence which substitutions do not significantly adversely affect the ability of the response protein to bind the chemical inducer. In particular substitutions may be made between the following amino acid groups viz.
  • transgenic in relation to the present invention does not include a wild type regulator promoter in its natural environment in combination with its associated functional gene in its natural environment.
  • target gene means any gene of interest.
  • a target gene can be any gene that is either foreign or natural to the eukaryote in question.
  • construct which is synonymous with terms such as “cassette”, “hybrid” and “conjugate” - includes a target gene directly or indirectly attached to the regulator promoter, such as to form a cassette.
  • An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence intermediate the promoter and the target gene.
  • fused in relation to the present invention which includes direct or indirect attachment.
  • constructs also include plasmids and phage which are suitable for transforming a cell of interest.
  • expression system means that the system defined above can be expressed in an appropriate organism, tissue, cell or medium. The system may comprise one or more constructs and may also comprise additional components that ensure to increase expression of the target gene by use of the regulator promoter.
  • the inducible promoters of the present invention is in the control of male sterility.
  • the anther is the site of male reproductive processes in flowering plants. It is composed of several tissues and cell types and is responsible for producing pollen grains that contain the sperm cells.
  • the tapetum is a specialised tissue which plays a critical role in pollen formation. It surrounds the pollen sac early in pollen development, degenerates during the latter stages of development and is not present in an organised form in the mature anther. The tapetum produces a number of compounds which aid pollen development or are incorporated into the pollen outer wall and it has been demonstrated that many of the natural male sterility mutations have impaired tapetum differentiation or function.
  • Tapetal tissue is therefore critical to the formation of functional pollen grains.
  • a number of genes have been identified and cloned that are specifically expressed in tapetal tissue. They include Osg6B, Osg4B (Tsuchiya et al. 1994, Yokoi, S et al. 1997), pEl, p T72 (WO9213957), p CA55 corn (WO92/13956) , TA29, TA13,(Seurinck et al. 1990), RST2 corn ( WO9713401), MS 14, 18, 10 and A6, A9 from Brassica napus (Hird et al. 1993). Anther specific clones have been isolated from a number of species Bp4A and C (Albani et al.
  • cloned genes which are applicable as target genes in the present invention include ⁇ glucanase (Ori et al. 1990), pectate lyase ( Budelier et al. 1990) and chitinase (Lotan et al. 1989) which are expressed in the transmitting tissue and a proteinase inhibitor (Atkinson et al. 1993) which are expressed in the style.
  • Others are pathogenesis related or are homologues of genes involved in the cleavage of glycosidic bonds. These enzymes may facilitate pollen tube growth by digesting proteins in the tissue through which the pollen tube grows. A number of female sterile mutants have been identified in Arabidopsis.
  • the present invention therefore provides a gene switch which is operably linked to a foreign gene or a series of foreign genes whereby expression of said foreign gene or said series of foreign genes may be controlled by application of an effective exogenous inducer.
  • the gene switch of the present invention therefore, when linked to an exogenous or foreign gene and introduced into a eukaryote by transformation, provides a means for the external regulation of expression of that foreign gene.
  • a plant may contain, for example, an inducible promoter according to the present invention in conjunction with other switch type mechanisms examples of which include inducible promoters include the Ale A/ R switch system described in International Publication No. WO. 93/21334, the GST switch system described in International Publication Nos WO 90/08826 and WO 93/031294 and the ecdysone switch described in our International Publication No. WO 96/37609.
  • the methods and products of the present invention may also be used to control expression of foreign proteins in eukaryotes such as yeast and mammalian cells. Many heterologous proteins for different applications may be produced by expression in such eukaryotic cells.
  • the present invention is advantageous in that it provides control over the expression of foreign genes in such cells. It also provides a further advantage, particularly in yeast and mammalian cells, where accumulation of large quantities of a heterologous protein can damage the cells, or where the heterologous protein is damaging such that expression for short periods of time is required in order to maintain the viability of the cells.
  • the inducible system of the present invention also has applicability in gene therapy as it allows the timing of the therapeutic gene to be controlled.
  • the present invention is therefore not only advantageous in transformed mammalian cells but also to mammals per se. Furthermore, the present invention may be used to switch on genes which produce potentially damaging or lethal proteins. Such a system may be employed in the treatment of cancer in which cells are transformed with genes which express proteins which are lethal to the cancer. The timing of the action on such proteins on the cancer cells may be controlled using the switch of the present invention.
  • Figure 1 shows a schematic representation of the general structure of the response regulator protein of the bacterial quorum sensing system.
  • Figure 2 shows a schematic representation of the homoserine lactone gene switch system.
  • Figure 3 is a plasmid map of the reporter gene construct, p221.91ux6.
  • Figure 4 is a plasmid map of p221.91ux2.
  • Figure 7 Plasmid map of p221.9Lpro.
  • Figure 8 Depicts the expression vector containing the LuxR gene in the pDH51LuxR plasmid.
  • Figure 9 Shows the expression cassette containing the enhanced N-terminal fusion protein of SV40-NLS-Gal4-LuxR in the plasmid pDH51SVLuxR.
  • Figure 10 Shows the expression cassette containing the enhanced C-terminal fusion of LuxR-SV40-NLS-Gal4 in the plasmidDH5 lLuxRSV.
  • Figure 1 1 is the plasmid map of p221.91asbox. Containing one copy of the LasR Box.
  • Figure 12 is a plasmid map of the transient expression construct containing LasR, pSinLASR.
  • Figure 13 expression plasmid pFunLuxR for expression in monocotyledon protoplasts.
  • Figure 14 expression plasmid pFunLasR for expression in monocot protoplasts.
  • Figure 15 expression plasmid pFunTraR for expression in monocot protoplasts.
  • Figure 17 reporter plasmid p221.9trabox2 containing tra box of sequence 2 described by Zhu and Winans, (1999).
  • Figure 18 is a plasmid map of binary vectors containing the effector and reporter cassettes, pAHLl.
  • Figure 19 is a plasmid map of binary vectors containing the effector and reporter cassettes, pAHL2.
  • Figure 20. is a plasmid map of binary vectors containing the effector and reporter cassettes, pAHL3.
  • Figure 21 is a plasmid map of binary vector containing luxl gene, pBDHELI.
  • Figure 22 is a plasmid map of pSB401 containing the Lux operon.
  • Figure 23 shows the root of a transgenic tobacco plant that was grown in tissue culture under aseptic conditions was placed on an LB agar plate and overlain with top agar containing E. coli with the plasmid pSB401 ( Figure 22). This strain expresses the lux operon and will bioluminesce in response to OHHL and HHL. Bioluminescence in the region around the root was clearly seen with the naked eye.
  • Figure 24 Shows a leaf of a transgenic tobacco plant that was grown in tissue culture under aseptic conditions. The leaf was excised from the plant and placed on an LB agar plate and overlaid with top agar containing Chromobacterium violaceum indicator strain. The violet colour detected represent the induction of the indicator stain gene showing the extrusion of OHHL from the plan tissue into the bacterial medium. Sequences
  • SEQ ID No. 1 is the Lux/ box promoter region.
  • SEQ ID No. 2 is the Luxl promoter region.
  • SEQ ID No. 3 & 4 are the LuxRBavnhl fragment.
  • SEQ ID No. 5 & 6 are the LuxR sequence/protein sequence.
  • SEQ ID No. 7 & 8 are the NVLuxR fusion flanked by BamHI and Pstl sites/protein sequence.
  • SEQ ID No. 9 & 10 are the LuxRNV sequence/protein sequence.
  • SEQ ID No. 11 is a TraRl fragment.
  • SEQ ID No. 12 is the TraR2 fragment.
  • SEQ ID No. 13 is the LasBoxl region.
  • SEQ ID No. 14 is the LasBox2 region.
  • SEQ ID No. 15, 16, 17 and 18 are the TraBoxl,2,3,4 regions respectively.
  • SEQ ID No. 19 is the LuxR response protein sequence.
  • SEQ ID No. 20 is the LasR response protein sequence.
  • SEQ ID No. 21 is the TrasR response protein sequence.
  • SEQ ID No. 22 & 23 are the open reading frames of LasR and TraR respectively.
  • SEQ ID No.24 is the Lux Box promoter region.
  • p221.9 plasmid contains a -60CaMV minimal promoter fused to GUS downstream of the Hin ⁇ lll and Sail unique cloning sites.
  • a recombinant plasmid was identified and named p221.91ux6 ( Figure 3).
  • the same oligonucleotide was used to generate p221.9LuxR2 ( Figure 4), p221.9LuxR3 ( Figure 5) and p221.9LuxC ( Figure 6).
  • p221.91pro was generated using the bacterial promoter containing the Lux box sequences ( Figure 7 Sequence ID 2).
  • lux receptor was altered at both ends of the coding sequence.
  • a plant Kozac consensus sequence was placed with an Ncol site at the ATG start of the coding sequence. Upstream from the Kozac consensus sequence a BamHI unique site was introduced using PCR.
  • the sense oligonucleotide was luxrbamhl 5' CCCGGATCCTAACAATGGGTATGAAAGACATAAATG 3' (Sequence ID.3 )and the antisense primer was luxrbamh2 5'
  • CGAACTCGAGTCATGATTTTAAAGTATGGGCAA-TCAATTG 3' (Sequence ID.4 ).
  • the PCR reaction was carried out using Tag polymerase (2.5 U) in a reaction buffer containing lOOng of template DNA, 100 ng of each oligonucleotide, 20 mM TRIS-HC1 pH 8.4, 50 mM KC1, 10 mM MgCl 2 , 50 mM dNTPs and using hot start conditions followed by 15 cycles of denaturing (94°C for 1 minute), annealing (66°C for 1 min) and synthesis (72°C for 3 min).
  • the fragment was purified and digested using BamHVXhol and was introduced into pDH51 BamHl/Sall vector to give pDH511uxR (Xhol and Sa l restriction enzymes produce compatible ends) ( Figure 8).
  • the sequence of the insert was determined and compared to the published LuxR sequence (Sequence ID. 5) (Devine et al, 1988).
  • Tobacco shoot cultures cv. Samsun were maintained on solidified MS medium + 3% sucrose in a controlled environment room (16 hour day / 8 hour night at 25°C, 55%o R.H), were used as the source material for protoplasts. Leaves were sliced parallel to the mid-rib, discarding large veins and the slices were placed in CPW13M (13%) mannitol, pH5.6, 860mmol/kg) for 1 hour to pre-plasmolyse the cells.
  • This solution was replaced with enzyme mixture (0.2%> cellulase R10, 0.05%> macerozyme R10 in CPW9M (CPW13M but 9% mannitol), pH5.6, 600mmol/kg) and incubated in the dark at 25°C overnight (16 hours).
  • the enzyme mixture was passed through a 75 ⁇ m sieve and the filtrate was centrifuged at 600rpm for 3.5 minutes, discarding the supernatant.
  • the pellet was resuspended in 0.6M sucrose solution and centrifuged at 600rpm for 10 minutes.
  • the protoplasts were removed and diluted with CPW9M (pH5.6, 560mmol/kg) and pelleted by centrifuging at 600rpm for 3.5 minutes.
  • the protoplasts were resuspended in CPW9M, counted, diluted to 2xl0 6 /ml in MaMg medium (Negrutui et al., 1987) and aliquoied at 4xl0 5 protoplasts per treatment. 20 ⁇ g each of effector and reporter plasmid DNA (lmg/ml) were added followed by 200 ⁇ l PEG solution (Negrutui et al., 1987).
  • the protoplasts were incubated at room temperature for 10 minutes before addition of 5ml MSP19M medium (MS medium, 3%> sucrose, 9% mannitol, 2mg/l NAA, 0.5mg/l BAP, pH5.6, 700mmol/kg) in the presence or absence of ligand (N-(-3-oxohexanoyl)-L-homoserine lactone (OHHL)).
  • MSP19M medium MS medium, 3%> sucrose, 9% mannitol, 2mg/l NAA, 0.5mg/l BAP, pH5.6, 700mmol/kg
  • ligand N-(-3-oxohexanoyl)-L-homoserine lactone (OHHL)
  • Protoplasts were isolated from Nicotiana plumb aginifolia suspension cells. Suspension cells were sub-cultured once a week in Np suspension medium in 250 ml Erlenmeyer flasks that were shaken at 100 RPM , at 25°C with 16h 8h light/dark regime. Protoplasts were isolated 2 or 3 days after subculture. Two or 2.5 g fresh weight of cells were incubated with 20 ml filter-sterilised enzyme solution and shaken at 40 rpm at 25°C.
  • the enzyme solution comprised 1% cellulysin (Calbiochem 219466), 1 % Macerozyme RIOTM (Yakult, Tokyo), and 1% Driselase (SigmaTM D- 9515) dissolved Artificial Sea Water Mannitol. After 3 h cell digestion in enzyme solution, they were washed through 100-, 50-um diameter sieves with W5 solution before being collected by centrifugation at 80 x g for 4 min. This method is also used for the isolation of wheat protoplasts.
  • Protoplasts were resuspended in W5 medium at densities ranging from 0.1 to 0.2 x 10 6 /ml and sedimented for 4 min at 80 g. They were then taken up in 0.2 to 0. 5 ml of: 0.4 M mannitol, 15 mM MgCl 2 , 0.1 % MES, 2 % glucose, pH 5.6 containing 15 ⁇ g plasmid DNA. Five minutes later poly-ethylene glycol (PEG) 4000 (FlukaTM) at 40%) (wlv) in 0.4 M mannitol and 0. 1 M Ca(N0 3 ).4H 2 0 pH 9.0 was added to give final PEG concentrations of 20%.
  • PEG poly-ethylene glycol
  • Transient transformed tobacco protoplasts were harvested by centrifugation at 3000 rpm. by collecting them by centrifugation at 3000 rpm. The supernatant was discarded and the protoplasts resuspended in GUS extraction buffer (Jefferson et al., 1987) for preparation of ⁇ -glucoronide extracts. The tubes were vortexed for 1 minute and then spun at 13000rpm for 2 minutes. The supernatant was collected and placed in a fresh eppendorf tube. 20 ⁇ l of the extract were used in the GUS assays.
  • Fluorometric assays for GUS activity were performed with the substrate 4- methylumbelliferyl-D-glucuronide (SigmaTM) using a Perkin-ElmerTM LS-35 fluorometer (Jefferson et al., 1987). Protein concentration of tissue homogenates were determined by the Bio-RadTM protein assay (Bradford, 1976).
  • the transient expression experiments were carried out as described above.
  • the same reporter plasmid was used but different effector constructs were produced.
  • a strong activator such as VP 16 would enhance transcriptional efficacy of the receptor, it was introduced in both the N- and C- terminal ends of LuxR.
  • the VP16 was also fused to the nuclear localisation signal (NLS) from SV40. Both the NLS of SV40 and VP16 were obtained from the yeast two hybrid plasmid pPC86 as a fused fragment which meant that it could be fused as one on to LuxR.
  • the N-terminal fusion was constructed by isolating and introducing into pBluescript a ClallNoil fragment of pPC86 containing the SV40 NLS and Gal4 activation domain.
  • the resulting pBluescriptSV40V was digested with Hindlll and filled in followed by Bgl ⁇ l digestion.
  • the fragment was introduced into SmallBamHl pDH51 luxR vector (BamHI and Bgl ⁇ l enzymes produce compatible ends) to produce the N-terminal fusion in LuxR and yield plasmid pDH51NVLuxR ( Figure 10).
  • the methionine start site is provided by the SV40 fragment.
  • the C-terminal end fusion was carried out by placing at the Real site in LuxR the blunted fragment of NLS/VP16.
  • pDH51LuxR was digested with Real, blunt ended and ligated to the ClallNoil SV40-Gal4 blunt ended fragment.
  • the fusion resulted in plasmid pDH5 ILuxRNV which contains four linking amino acids, C A K L, between the end of LuxR and the start of the nuclear locating sequence.
  • Example 3 Transient expression of LuxR, LasR and TraR in monocot protoplasts.
  • the vector of choice for expression in monocot transient system was pFun which contains ubiquitin promoter region and the Nos terminator. In between the two there is multiple unique cloning sites.
  • LuxR was transferred into pFun as a BamHI fragment.
  • pLasR was transferred as a BamHI/Kpnl fragment.
  • TraR was isolated from Agrobacterium tumefaciens by PCR using oligonucleotides TraRl 5' AATTGGTACCCACC ATGCAGC ACTGGCTGGACAAGTTGACC 3 ' (Sequence ID. 11) and TraR2 5' AATTGGATCCCAGATCAGCTTTCTTCTGCTTGGCGAGG 3' (Sequence ID 12).
  • the purified fragment was restriction enzyme digested with BamHI and introduced into pFun BamHI vector.
  • the expression vectors were named as follows: pFunLuxR ( Figure 13), pFunLasR ( Figure 14) and pFunTra ( Figure 15).
  • Reporter plasmid used with the expression vector encoding for LuxR is the same as that used in the transient experiments carried out in Tobacco (Examples 1 and 2).
  • the parental plasmid p221.9 was used.
  • LasBox and the two TraBoxes were synthetically made.
  • the LasBox oligonucleotides are as follows LasBox 1 5' TCGACACCTGCGAGTTCTCCGAGGTG 3' (Sequence ID 13) and LasBox2 is 5' TCGACACCTCGGAGAACTCGCAGG TG 3' (Sequence ID 14).
  • the TraBoxes as described by Zhu and Winans, (1999) were used and the oligos are as follows, TraBoxl 5 ' TCGACTACACGTCTAGACGTGTAGG 3'
  • the reporter cassette was isolated from p221.91ux6 by digesting with EeoRI and H dIII to yield a 2.0kb fragment. The fragment was purified and introduced into pBin 19 Eco RI/H/ ⁇ dlll vector to produce pBinrepA ⁇ L.
  • the three different variants of the LuxR receptor, that is LuxR and the two enhanced versions were restriction enzyme digested with EcoRI (site flanking both sides of the effector cassette) and introduced into a dephosphorylated EcoRI pBinl9repA ⁇ L vector to produce either pAHLl (LuxR) ( Figure 18), pAHL2 (SVLuxR) ( Figure 19) or pAHL3 (LuxRSV) ( Figure 20).
  • the plant transformation construct pAHL 1 ( Figure 18), pAHL2 ( Figure 19) and pAHL3 ( Figure 20), containing LuxR or chimeric LuxR and a reporter gene cassette, were transformed into Agrobacterium tumefaciens LBA4404 using the freeze/thaw method described by Holsters et al. (1978). Tobacco (Nicotiana tabacum cv Samsun) transformants were produced by the leaf disc method (Horsch et al., 1988). Shoots were regenerated on medium containing 1 OOmg/1 kanamycin. After rooting, plantlets were transferred to the glasshouse and grown under 16 h light 8 h dark conditions
  • OHHL was dissolved in methanol (Sigma) and the stock maintained at -20°C. The compound was diluted in growth media used to germinate seedlings. Uninduced seedlings were treated with equivalent amount of methanol. Seeds were germinated in MS media supplemented with 0.8%> (w/v) agar. Seedlings were collected 2 days post-germination (two cotyledon stage).
  • Example 5 Stable constitutive expression of yenl gene in tobacco plants
  • pBDHELI ( Figure 21) was constructed by fusing the alfalfa mosaic virus (AMV) translation enhancer sequence from pBi526 (Datla et al., Plant Science 94,
  • the assay consists of placing a leaf of the transgenic plant on an agar plate overnight. The leaf was then removed and the evil mutant of Chromobacterium violaceum spread over the plate. Violacein (a purple pigment) production by the bacterium can be seen when OHHL had diffused out of the leaf and into the agar ( Figure 24).
  • Example 6 LuxR is activated by compounds produced by yenl plants.
  • Example 7 Cross of LuxR tobacco high expressor plant with yenl tobacco plant.
  • Plants are produced by crossing the yenl expressing plant with the AHL switch plants.
  • the seed of the progeny was collected and assayed for GUS activity as described above.
  • GUS activity was assayed for in all tissues and different ages as the expectation was that GUS protein would be present in all tissues expressing the yenl gene, yenl Is under control of the 35S CaMV promoter as is the LuxR protein in the AHL switch.
  • progeny seedlings were grown in 1/2MS and were collected 2 days post-germination or when cotyledons were fully extended.
  • Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

Abstract

L'invention a trait, entre autres, à un procédé d'initiation de transcription d'un gène cible dans une cellule eucaryote, qui comprend les étapes consistant à: (a) prévoir une cellule eucaryote capable de produire une protéine de réaction; (b) insérer dans le génome de ladite cellule un polynucléotide définissant une séquence de promoteur inductible liée de façon exploitable audit gène cible, et capable après induction d'initier la transcription de celui-ci; et (c) appliquer à ladite cellule un inducteur chimique capable de se lier à ladite protéine de réaction, ce qui entraîne la liaison dudit inducteur chimique à ladite protéine de réaction pour former un complexe inducteur qui se lie audit promoteur inductible, et déclenche ainsi l'initiation de transcription dudit gène cible.
EP99939526A 1998-08-13 1999-08-12 Commutateur de gene Withdrawn EP1104472A1 (fr)

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GBGB9817704.1A GB9817704D0 (en) 1998-08-13 1998-08-13 Gene switch
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US7189506B1 (en) 1999-03-03 2007-03-13 Genelabs Technologies, Inc. DNA binding compound-mediated molecular switch system
AU3513000A (en) * 1999-03-03 2000-09-21 Genelabs Technologies, Inc. Dna binding compound-mediated molecular switch system
JP2003504032A (ja) 1999-07-01 2003-02-04 カルジーン エルエルシー 真核細胞中の遺伝子発現の調節
EP1507010B1 (fr) * 1999-07-01 2009-08-12 Calgene LLC Régulation d'expression génétique dans des cellules eucaryotes
GB9927191D0 (en) * 1999-11-17 2000-01-12 Angeletti P Ist Richerche Bio Methods and means for regulation of gene expression
WO2001053479A2 (fr) * 2000-01-24 2001-07-26 Sangamo Biosciences, Inc. Sequences activatrices ii
WO2010089387A1 (fr) 2009-02-09 2010-08-12 Morphosys Ag Production de mélanges oligoclonaux d'immunoglobulines dans des cellules individuelles
ITMI20090946A1 (it) * 2009-05-28 2010-11-29 Novartis Ag Espressione di proteine ricombinanti
WO2022118972A1 (fr) * 2020-12-04 2022-06-09 国立大学法人神戸大学 Commutateur génique doté de propriétés élevée en termes d'expression et de régulation
CN115873125A (zh) * 2021-09-29 2023-03-31 中国科学院深圳先进技术研究院 用于哺乳动物体系的嵌合型别构转录因子、调控元件组和诱导表达系统

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US5196318A (en) * 1990-06-26 1993-03-23 The Texas A&M University System Precisely regulated expression of deleterious genes
JPH11506319A (ja) * 1995-05-26 1999-06-08 ゼネカ・リミテッド エクジソンレセプターを含む遺伝子スイッチ
GB9516241D0 (en) * 1995-08-08 1995-10-11 Zeneca Ltd Dna constructs
US5759798A (en) * 1995-12-08 1998-06-02 Woods Hole Oceanographic Institution Assays, test kits and bacteria for detection of autoinducers

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