WO2001020006A1 - Promoteur inductible - Google Patents

Promoteur inductible Download PDF

Info

Publication number
WO2001020006A1
WO2001020006A1 PCT/GB2000/003476 GB0003476W WO0120006A1 WO 2001020006 A1 WO2001020006 A1 WO 2001020006A1 GB 0003476 W GB0003476 W GB 0003476W WO 0120006 A1 WO0120006 A1 WO 0120006A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
promoter
expression
amdr
target gene
Prior art date
Application number
PCT/GB2000/003476
Other languages
English (en)
Inventor
Ian Jepson
Andrew James Greenland
Alberto Martinez
Mark Xavier Caddick
Arthur Brian Tomsett
Kevan Croft
Angela Tregova
Original Assignee
Syngenta Limited
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 Syngenta Limited filed Critical Syngenta Limited
Priority to AU70273/00A priority Critical patent/AU7027300A/en
Publication of WO2001020006A1 publication Critical patent/WO2001020006A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • 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
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention relates 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 chemically inducible promoters (GENESWITCH is a trademark owned by Zeneca Limited).
  • the present invention relates to a method of controlling expression of a target gene in a plant, mammal or yeast using an inducible system including the amdR gene of Aspergillus nidulans.
  • amdR gene of Aspergillus nidulans is a positive regulatory gene which regulates the expression of four linked structural genes, the catabolism of acetamide (amdS), omega amino acids (gatA and gabA) and lactam (lamA) (Andrianopoulos and Hynes, 1988).
  • the amdS gene encodes an acetamidase which allows use of acetamide as a single source of nitrogen, carbon or both. Mutational analysis has identified both cis and trans-acting mutations. One of the trans-acting mutations is mediated by the amdR regulator gene (Hynes and Pateman, 1970a, Hynes and Pateman, 1970b).
  • lamA is also regulated by AmdR and encodes a lactam permease while gabA encodes a ⁇ - aminobutyric acid (GABA) permease and gatA specifies for an omega amino acid transaminase (Arst, 1976, Arst et al., 1978). All of these genes (amdS, gabA, gatA and lamA) are induced by omega amino acids, GABA and ⁇ -alanine (Andrianopoulos and Hynes, 1988).
  • GABA ⁇ - aminobutyric acid
  • amdR genes from Aspergillus nidulans (Andrianopoulos and Hynes 1988, 1990 EMBL Accession numbers M31517, M3611 1) and Aspergillus oryzae (Accession number M96953) have been identified and sequenced.
  • AmdR comprises at the N-terminal end a cysteine rich "zinc finger" DNA binding motif, followed by four putative acidic transcriptional activation domains or motifs and two regions overlapping the DNA binding region with identity to the simian virus 40 (SV40) larger T antigen nuclear localisation signal (Figure 1) (Andrianopoulos and Hynes, 1990).
  • a missense mutation (Proline 489 to Serine) occurring in the middle of the amdR gene (amdR6 c ) results in AmdR mediated activation of gene expression in the absence of inducer.
  • the mutation is predicted to have a conformational change mimicking that seen in the wild-type amdR in the presence of inducer.
  • Aspergillus result in similar phenotypes to those observed for HapC. In vitro these three factors form a functional complex (Steidl et al 1999). A mdS promoter sequences fused to the minimal promoter CYC 1 -lacZ fusion in yeast leads to Hap complex mediated regulation of gene expression in carbon limiting media (Bonnefoy et al., 1995). In Aspergillus induction of amdS by omega amino acids is abolished in the
  • Arabidopsis genes with equivalent activity to that of the yeast Hap genes have been isolated and thereby plants may have the functional equivalents found in Aspergillus for amdS and gatA gene induction by omega amino acids (Accession number Y13723, YY13724, Y13720, Y13721, Y13722, Y13725 and Y13726).
  • inducible promoter includes promoters which may be induced chemically.
  • Particularly useful promoters are promoter sequences which are controlled by the application of an external chemical stimulus.
  • the external chemical stimulus may be an agriculturally acceptable chemical, the use of which is compatible with agricultural practice and is not detrimental to plants or mammals. This allows particular gene expression to be controlled at particular stages of plant growth or development, by the presence or absence of a chemical which can be applied to the plants or seeds, for example by spraying or using known seed coating techniques.
  • the inducible promoter may be one which allows particular gene expression in other eukaryotes such as yeast and mammals. Inducible promoters are known, examples of which include the Ale A/ R switch system described in our International Publication No. WO.
  • a method of controlling expression of a target gene in a eukaryote comprising integrating, preferably stably integrating, within the genome of the eukaryote an expression system comprising:
  • a first polynucleotide comprising said target gene operably linked to and under the control of a promoter comprising an acetamide-, omega amino acid- or ⁇ -alanine-regulated promoter;
  • a second polynucleotide comprising a DNA sequence encoding a regulator protein; whereby expression of the target gene depends upon the presence of both the regulator protein and an inducer of the regulated promoter.
  • amdR The stable expression of amdR in plants will produce a protein which is capable of interacting with an amdR inducer such as acetamide, ⁇ -amino butyric acid (GABA) or ⁇ -alanine. Exogenous application of inducer compound will lead to inducible expression of the target gene.
  • an amdR inducer such as acetamide, ⁇ -amino butyric acid (GABA) or ⁇ -alanine.
  • GABA ⁇ -amino butyric acid
  • inducer compound Exogenous application of inducer compound will lead to inducible expression of the target gene.
  • amdR inducers An advantage of using amdR inducers over other inducers is that these compounds are likely to be plant systemic, non-phytotoxic and mobile. Thus, the expression of the target gene is controllable by application of exogenous amdR inducers. Examples of such inducers include acetamide, omega- amino acids such as GABA.
  • 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.
  • the target gene may encode at least part of a functional protein or an antisense sequence.
  • 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.
  • promoter is used herein to refer to a full promoter or, alternatively, to a minimal promoter sequence linked to an enhancer or operator protein. A wide variety of promoters is well known to those skilled in the art.
  • substantially identity covers identity with respect to at least the essential nucleic acids/ nucleic acid residues of a polynucleotide sequence providing the sequence retains the function of that polynucleotide sequence e.g. functions as a regulator protein in the inducible system of the present invention.
  • identity is shown when 60% or more of the nucleotides are common with the polynucleotide of the present invention, more typically 65%, preferably 70%, more preferably 75%, even more preferably 80% or 85% and, especially preferred are 90%, 95%m 98% or 99% or more identity.
  • BESTFIT both from the Wisconsin Genetics Computer Group (GCG) software package
  • BESTFIT compares two sequences and produces an optimal alignment of the most similar segments.
  • GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate.
  • the comparison is made by alignment of the sequences along their whole length.
  • a variant thereof with reference to the present invention means any substitution of, variation of , modification of , replacement of, deletion of or the addition of one or more nucleic acid(s) from or to a polynucleotide providing the resultant sequence exhibits the same function as that polynucleotide.
  • the term also includes DNA which substantially hybridises to the DNA of the present invention and which codes for at least part of that polynucleotide. Preferably, such hybridisation occurs at, or between, low and high stringency conditions.
  • low stringency conditions can be defined as 3 x SSC at about ambient temperature to about 65 °C
  • high stringency conditions as 0.1 x SSC at about 65 °C.
  • SSC is the name of the buffer of 0.15M NaCl, 0.015M trisodium citrate. 3 x SSC is three times as strong as SSC and so on. Also provided is the use of sequences according to the present invention in the production of other variants using molecular evolution and/ or
  • the promoter will usually be an acetamide- or GABA-regulated promoter such as lamA, amdS, gabA or gatA.
  • the first polynucleotide may comprise a 5' region which is regulatable by the response regulator protein. This may, for example, be a promoter region such as the gatA or amdS promoter regions or a specific response element. Response elements are well known to those skilled in the art.
  • the first polynucleotide may also comprise a 3 ' terminal region.
  • the DNA sequence of the second polynucleotide preferably comprises the amdR sequence and may also comprise a 5' promoter region operably linked to and controlling the DNA sequence encoding the AmdR regulator protein. A 3 ' terminal region may also be present.
  • the amdR sequence is preferably that encoded by the sequence shown in SEQ. ID. No. 2 or a sequence having substantial identity therewith or a variant or fragment thereof.
  • the eukaryote into which the expression system is integrated may be a plant, preferably a transgenic plant, a mammal or yeast.
  • the regulator protein When the eukaryote is a plant, the regulator protein (i.e.AmdR) may be expressed under the control of a constitutive promoter, that is, a promoter driving the expression in all cell and tissue types throughout the life cycle of the plants. Examples of such promoters are CaMV35S, maize Ubiquitin and Arabidopsis Ubiquitin 3. Alternatively, the regulator protein expression may be regulated by a tissue specific promoter.
  • a constitutive promoter that is, a promoter driving the expression in all cell and tissue types throughout the life cycle of the plants. Examples of such promoters are CaMV35S, maize Ubiquitin and Arabidopsis Ubiquitin 3.
  • the regulator protein expression may be regulated by a tissue specific promoter.
  • the polynucleotides used in the method of the first aspect of the invention themselves form further aspects of the present invention.
  • a polynucleotide comprising a DNA sequence comprising a target gene operably linked to and under the control of a promoter comprising an acetamide-, omega amino acid- or ⁇ -alanine-regulated promoter and wherein the polynucleotide further comprises a promoter comprising an acetamide-, omega amino acid- or ⁇ -alanine-regulated promoter and wherein the polynucleotide further comprises a
  • the amdR inducible promoter of the present invention may be used in a method which comprises stacking it with other inducible promoters to control a number of traits independently of each other.
  • This use of the different chemistries offers the advantages of different application methods and targets.
  • inducible promoters include the Ale A/R switch system described in our International Publication No. WO 93/21334, the GST switch system described in our International Publication Nos WO 90/08826 and WO 93/031294, the ecdysone switch described in our International Publication No. WO 96/37609.
  • the polynucleotide of the second aspect of the invention may additionally comprise one or more regions each of the regions expressing a protein and each being operably linked to and under the control of separate inducible promoter regions.
  • the purpose of the method of the first aspect of the invention is to induce expression of a target gene in a eukaryote and, therefore, in a third aspect of the invention there is provided the use of the polynucleotide of the second aspect in the inducible expression of a target gene, wherein the protein encoding regions are expressed in a eukaryote.
  • Preferred features of the second and third aspects of the invention are as for the first aspect.
  • eukaryotic organism tissue transformed by the polynucleotide of the second aspect or material derived from the transformed tissue.
  • the eukaryote may be a plant, mammal or yeast.
  • the inducible promoter of the present invention is particularly useful for mono- or di- cotyledonous plants in which it is desired to introduce a chemically regulated trait. Therefore, the tissue or material derived from the tissue will generally be plant tissue or material. In a further aspect of the invention, there is provided morphologically fertile whole plant comprising this tissue or material.
  • 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), MS14,18,10 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.
  • genes 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.
  • control of expression of the regulator protein may also be developmentally or temporally controlled by using a specific promoter such as those controlling expression of genes required during seed formation, germination such as cysteine proteinases (as specified in our International Publication No WO 97/35983 ) and malate synthase.
  • a specific promoter such as those controlling expression of genes required during seed formation, germination such as cysteine proteinases (as specified in our International Publication No WO 97/35983 ) and malate synthase.
  • the present invention therefore provides an inducible promoter 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 inducible promoter 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.
  • progeny of plants according to the present invention which progeny comprises the polynucleotide described above stably incorporated into its genome, the seeds of such plants and such progeny.
  • 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. Reference may be made to the literature for full details of the known methods.
  • Dicotyledonous and monocotyledonous plants can be transformed.
  • the present invention may be applied to any plant for which transformation techniques are, or become, available.
  • the present invention can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, and cotton; cereals such as wheat, barley, rice, maize, and sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melons; and vegetables such as potato, carrot, lettuce, cabbage and onion.
  • field crops such as canola, sunflower, tobacco, sugarbeet, and cotton
  • cereals such as wheat, barley, rice, maize, and sorghum
  • fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melons
  • vegetables such as potato, carrot, lettuce, cabbage and onion.
  • the inducible promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues.
  • a plant may contain an AmdR inducible promoter in conjunction those including the Ale A/ R switch system, the GST switch system and the ecdysone switch.
  • the polynucleotide sequences according to the present invention may also be used in the production of other variants using molecular evolution and/ or DNA shuffling techniques. Such techniques are described, for example, in US Patent No. 5 605 793, US Patent No. 5 811 238 and US Patent No. 5 830 721. In essence this technique involves expression of a gene in a microbial expression system such as
  • Escherichia coli The particular system selected must be validated and calibrated to ensure that biologically active peptides are expressed, which may be readily achieved using an in vivo bioassay.
  • the gene or preferably a collection of related genes from different species, may be subject to mutagenic polymerase chain reaction (PCR) as is known in the art. Fragmentation of the products and subsequent repair using PCR leads to a series of chimaeric sequences reconstructed from parental variants. These chimaeras are then expressed in the microbial system which can be screened in the usual way to determine active mutants, which may then be isolated and sequenced. Reiteration of this molecular evolution DNA shuffling cycle may lead to progressive enhancement of the desired properties.
  • PCR polymerase chain reaction
  • any variants produced will have improved chemically inducibility compared to known promoter sequences. Accordingly, in yet another aspect of the present invention, there is provided the use of the polynucleotide of the second aspect in the production of other chemically- inducible promoter variants using molecular evolution and/ or other DNA shuffling techniques.
  • the inducible promoter 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.
  • 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.
  • a polynucleotide of the second aspect of the invention for use in medicine, particularly in gene therapy and the treatment of cancer.
  • a further aspect of the invention provides the use of a polynucleotide of the second aspect of the invention in the preparation of an agent for use in gene therapy or for the treatment of cancer.
  • Model plants such as tobacco or Arabidopsis plants, expressing the amdR (or improved versions of the amdR gene) and reporter gene cassette may be used to screen the activity of compounds for e.g. improved activity, mobility or stability.
  • Compounds may affect the AmdR protein in two ways. The first is an inhibitory effect or antagonistic activity and the second is a stimulatory effect or agonistic activity.
  • the screen can be based on monitoring a reporter gene activity elicited by the different compounds being tested.
  • a method of screening compounds for bioactivity comprising monitoring the activity of a reporter gene driven by amdR response element in response to exposure to said compounds. 2.
  • Plants transformed with the amdR mutant form amdR6 c and a downstream trait gene will result in constitutive expression of the trait gene.
  • the expression may be brought about by an inducer of amdR gene expression and inhibited by an antagonist of the amdR.
  • Figure 1 shows a diagram of the amdR open reading frame.
  • Figure 2 shows a plasmid map of pSANl
  • Figure 3 shows a plasmid map of pS AN2
  • Figure 4 shows a plasmid map of pAT31-1
  • Figure 5 shows a plasmid map of pSAN3
  • Figure 6 shows a plasmid map of pSAN4
  • Figure 7 shows a plasmid map of p221.9
  • Figure 8 shows a plasmid map of p221.9gatAl merA
  • Figure 9 shows a plasmid map of pACN6
  • Figure 10 shows a plasmid map of pACN6gatAl-mer
  • Figure 11 shows a plasmid map of p221.9AmdSl-mer
  • Figure 12 shows a plasmid map of p221.9AmdS3-merA
  • Figure 13 shows a plasmid map of pACN ⁇ amdS 1 -mer
  • Figure 14 shows a plasmid map of pACN6amdS3-mer
  • FIG. 15 shows how AmdR activates reporter gene expression in Arabidopsis
  • Figure 16 shows transient expression of AmdR activates reporter gene expressing in bombarded tobacco leaf cells
  • Figure 17 shows a plasmid map of pMOG888(Pat)
  • Figure 18 shows a plasmid map of pSAN2:221amdSl
  • Figure 19 shows a plasmid map of pSAN2:221amdS3
  • Figure 20 shows a plasmid map of pSAN2:221gatAl
  • Figure 21 shows a plasmid map of pSAN4:221gatAl
  • Figure 22 shows a plasmid map of pSAN2:ACN6amdS2
  • Figure 23 shows a plasmid map of pSAN2:ACN6amdS4
  • Figure 24 shows a plasmid map of pSAN2:ACN6gatA
  • Figure 25 shows a plasmid map of pSAN4:ACN6gatA
  • 26 is the amdR sequence in pSANl (SEQ. ID NO. 1)
  • Figure 27 is the amdR sequence in pSAN2 (SEQ. ID NO. 2)
  • Figure 28 is the amdR sequence in pSAN3 (SEQ. ID NO. 3)
  • Figure 29 is the amdR sequence in pSAN4 (SEQ. ID NO. 4)
  • the cloning vector pSLNl is a modified pUC 8 vector where the polylinker region contains the 35S CaMV promoter and Nos terminator flanked by EcoRI at the 5' end of the 35S CaMV promoter and Hindlll at the 3' end of the Nos terminator. Between the promoter and Nos terminator Spel, Notl, Sail, Sphl, Xhol, Kpnl, Eagl and Fsel the unique cloning restriction sites were incorporated.
  • the plasmid pJMBI (a pUC8 based plasmid with the following insert HindlIVSphl/PstV35SCaMY promoter
  • /Xhol/amdR/BamHVSmal/Kpnl/SaclfNos terminator/Ecoi?I contains the amdR fragment from Aspergillus nidulans and can be excised from the plasmid by digesting with Sphl and Kpnl. The fragment was purified and introduced into a pSLNI Sphl/Kpnl vector to produce pSANl ( Figure 2). The sequence of the amdR gene in pSANl is given in Figure 26.
  • Example 2 Construction of transient expression plasmid containing the amdR open reading frame and intron 2 for expression in plants (pSAN2)
  • the introduction of the intronB sequence into the amdR ORF was carried out by taking advantage of the unique Sphl site in exon2 and a unique Xhol site in exon 4.
  • Oligonucleotide were designed to include these sites and a 500bp fragment was amplified using an Aspergillus nidulans genomic library.
  • the PCR was carried out with pfu polymerase and using the manufacturer's buffer conditions.
  • the PCR fragment was purified and digested with Sphl and Xhol.
  • pSANl was restriction enzyme digested with Sphl and Xhol to drop out the intronless region between exon2 and exon 3 and replace it with the sequenced PCR fragment containing exon2 -Intron B-exon3 and exon4 .
  • the resulting plasmid was named pSAN2 ( Figure 3).
  • the sequence of the ⁇ mdR gene in pSAN2 is given in Figure 27.
  • the amdr ⁇ c mutation is a single C to T transition at position 1867 located I exon 4. This causes a proline to serine missence mutation at amino acid 489 of the amdR protein.
  • the plasmid pAT31-l (Andrianopoulos and Hynes, 1988, Figure 4) was restriction enzyme digested with XhollPstl to drop out the region in exon4 containing the mutation at position 1867 of the amdR gene. This fragment was cloned into the equivalent sites of the pSANl XhollPstl vector to produce pSAN3 ( Figure 5).
  • the sequence of the amdR gene in pSAN3 is given in Figure 28.
  • p221.9amdSl and p221.9amdS3 were constructed using two oligonucleotides asmdsl 5' TCGAGTTCGGCGAAGCCAGCCAATATTCAGCG 3'(SEQ. ID. No. 7) and amds2 5' TCGACGCTGAATATTGGCTGGCTTCGCCGAAC 3' (SEQ. ID. No. 8). which were mixed in equimolar ratios and re-annealed prior to introducing into p221.9 Xhol/Sall vector to produce p221.9amdSl ( Figure 1 1), p221.9amdS3 ( Figure 12), pACN6amds2 ( Figure 12) and pACN6amdS4 ( Figure 14).
  • Example 6 Assessment of amdR expression and reporter vectors in the presence of amdR inducer compound in stable transformed Aspergillus nidulans.
  • Aspergillus nidulans protoplasts were prepared and transformed as described by Tilburn et ⁇ /.(1983). Protoplasts were transformed with a positive control alone, with a reporter construct alone or with a reporter construct with an effector construct (pSANl, 2, 3 or 4). Colonies of resistant Aspergillus were isolated and tested by PCR to insure the presence of the transformed DNA. Positive clones were grown up and genomic DNA extractions carried out which were used in Southern blots (Sambrook et ⁇ /.,1987) to insure that DNA was integrated. The genomic DNA was isolated from mycelium grown overnight at 37°C in 200 ml of nutrient media. The mycelium was harvested by passing the media through sterile muslin.
  • the harvested mycelium was washed thoroughly with distilled water and gently blotted dry and paced in liquid nitrogen.
  • the mycelium wa ground up in liquid nitrogen and transferred to a sterile tube where 500 ⁇ l of RNAseA (lOmg/ml) and 1.6 ml of DNA extraction buffer (300mM Tris-HCl pH 8.5, 375 mM NaN 2 , 37.5 mM EDTA and 3% (w/v) Sarkosyl) were added per gram of mycelium.
  • the mixture was incubated at 37°C for one hour after which a 1 : 1 mixture of phenol: chloroform was added.
  • the tube was gently mixed for 10 minutes and centriftiged for 20 minutes at 12000 X g.
  • the aqueous layer was successively extracted with phenol: chloroform (1 :1) and chloroform prior to precipitation of the genomic DNA using 1/10 volume of sodium acetate pH 5.2 and
  • the leaf tissue was bombarded as follows. HeliosTM Gene Gun system was used with gold particles. The particles were prepared as described by the manufacturer (Biorad). 100 ⁇ g of plasmid DNA in 50mM Spermidine was added to 50mg Gold particles per
  • Example 8 Induction of reporter gene expression upon transient transformation into Tobacco leaf tissue of amdR expression vectors and reporter vectors in the presence of amdR inducer compound.
  • Example 9 Vector construction for stable transformation of tobacco plants.
  • the generation of the binary vectors was carried out in the same way for all combinations of the effector cassette (cassette containing the amdR) and the reporter cassette containing the minimal 35S promoter region.
  • the binary vector used contained the Pat selectable cassette and is called pMOG888(pat) ( Figure 18).
  • the pMOG888(pat) plasmid was digested with Hindlll and EcoRI to which the Hindlll/EcoRI reporter cassette fragment was ligated. Clones containing the reporter cassette within pMOG888(Pat) were digested with EcoRI/Sgfl.
  • the vector was ligated with the EcoRI/Sgfl DNA fragment containing the amdR expression cassette to generate the binary vectors containing both reporter and effector cassettes. These were named as follows: pSAN2:221amdS2 ( Figure 18), pSAN2:221amdS4 ( Figure 19), pSAN2:221gatA ( Figure 20) and pSAN4:221gatA ( Figure 21).
  • pSAN2:221amdS2 Figure 18
  • pSAN2:221amdS4 Figure 19
  • pSAN2:221gatA Figure 20
  • pSAN4:221gatA Figure 21.
  • a similar strategy was followed for the production of the binary vectors containing the effector cassette and the reporter cassette containing the minimal promoter derived from the alcohol dehydrogenase gene of Aspergillus nidulans.
  • PMOG888(Pat) was digested with EcoRI/Sgfl and was ligated to the EcoRI/Sgfl amdR expression cassette to generate the binary plasmid containing the effector cassette.
  • This newly produced plasmid was digested with EcoRI into which the EcoRI reporter cassette fragment was introduced.
  • the resulting plasmid generated binary vectors for transformation of Agrobacterium containing the amdR expression cassette and reporter cassette in one vector. These are named as follows: pSAN2:ACN6amdsl ( Figure 22), pSAN2:ACN6amds3 ( Figure 23), pSAN2:ACN6gatA ( Figure 24) and pSAN4:ACN6gatA ( Figure 25).
  • Transgenic tobacco and Arabidopsis plants are generated via Agrobacterium mediated transformations. For each construct a population of 50 individual transgenic events is produced. The transgenic plants are screened via PCR for the presence of both effector and reporter cassettes. Plants containing both components of the inducible promoter are transferred to the greenhouse where selfed seed is collected. A preliminary assay of the primary transgenic plants is carried out by growing plants propagated by cloning in media containing 5 and 15mM GABA ( ⁇ -aminobutyric acid) and leaf tissue is collected at 0, 24, 48, 72 and 96 hours to monitor for GUS reporter gene activity.
  • GABA ⁇ -aminobutyric acid

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Promoteur inductible et procédé permettant de réguler l'expression d'un gène cible dans un eucaryote, qui consiste à intégrer dans le génome de l'eucaryote, de préférence de manière stable, un système d'expression comprenant (i) un premier polynucléotide comportant ledit gène cible lié de manière opérationnelle à un promoteur et commandé par ce dernier, ledit promoteur comprenant un promoteur à régulation acétamide, acide aminé oméga ou β-alanine et (ii) un second polynucléotide comportant une séquence d'ADN codant une protéine régulatrice liée de manière opérationnelle à un autre promoteur et commandée par ce dernier. L'expression du gène cible dépend de la présence tant de la protéine régulatrice que d'un inducteur. La protéine régulatrice est de préférence la protéine AmdR.
PCT/GB2000/003476 1999-09-15 2000-09-12 Promoteur inductible WO2001020006A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU70273/00A AU7027300A (en) 1999-09-15 2000-09-12 Chemically inducible promoter and method for controlling the expression of a target gene in an eukaryote

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9921825.7 1999-09-15
GBGB9921825.7A GB9921825D0 (en) 1999-09-15 1999-09-15 A gene switch

Publications (1)

Publication Number Publication Date
WO2001020006A1 true WO2001020006A1 (fr) 2001-03-22

Family

ID=10860976

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2000/003476 WO2001020006A1 (fr) 1999-09-15 2000-09-12 Promoteur inductible

Country Status (4)

Country Link
AU (1) AU7027300A (fr)
GB (1) GB9921825D0 (fr)
ID (1) ID27618A (fr)
WO (1) WO2001020006A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021334A1 (fr) * 1992-04-13 1993-10-28 Zeneca Limited Produits de recombinaison d'adn et plantes les incorporant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993021334A1 (fr) * 1992-04-13 1993-10-28 Zeneca Limited Produits de recombinaison d'adn et plantes les incorporant

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANDRIANOPOULOS A ET AL: "CLONING AND ANALYSIS OF THE POSITIVELY ACTING REGULATORY GENE AMDR FROM ASPERGILLUS-NIDULANS", MOLECULAR AND CELLULAR BIOLOGY, vol. 8, no. 8, 1988, pages 3532 - 3541, XP000979208, ISSN: 0270-7306 *
ANDRIANOPOULOS A ET AL: "SEQUENCE AND FUNCTIONAL ANALYSIS OF THE POSITIVELY ACTING REGULATORY GENE AMDR FROM ASPERGILLUS-NIDULANS", MOLECULAR AND CELLULAR BIOLOGY, vol. 10, no. 6, 1990, pages 3194 - 3203, XP002158728, ISSN: 0270-7306 *
JEPSON I ET AL: "CHEMICAL-INDUCIBLE GENE EXPRESSION SYSTEMS FOR PLANTS - A REVIEW", PESTICIDE SCIENCE,GB,ELSEVIER APPLIED SCIENCE PUBLISHER. BARKING, vol. 54, no. 4, December 1998 (1998-12-01), pages 360 - 367, XP000804296, ISSN: 0031-613X *

Also Published As

Publication number Publication date
GB9921825D0 (en) 1999-11-17
ID27618A (id) 2001-04-19
AU7027300A (en) 2001-04-17

Similar Documents

Publication Publication Date Title
EP0329308B1 (fr) Systèmes de contrôle de la pollinisation utilisant des gènes antisens pour la production de semences hybrides
US6559357B1 (en) Methods for altering mass and fertility in plants
KR20010041129A (ko) 화분 특이적 프로모터
EP0706570B1 (fr) Vegetaux presentant une reponse modifiee a l'ethylene
AU2018220084A1 (en) Method for plant improvement
US6787687B1 (en) Rin gene compositions and methods for use thereof
CA2372323C (fr) Procedes pour accroitre la proliferation de cellules vegetales par inhibition fonctionnelle d'un gene inhibiteur de la cycline vegetale
CA2258571A1 (fr) Reductases de sterol de plantes et leurs utilisations
US20220275383A1 (en) Sterile genes and related constructs and applications thereof
WO1997048793A9 (fr) Reductases de sterol de plantes et leurs utilisations
AU5379699A (en) Gene switch
US6762347B1 (en) NOR gene compositions and methods for use thereof
WO2001014561A1 (fr) Compositions de gene nor et leurs procedes d'utilisation
US7071379B2 (en) Methods for altering organ mass in plants
CN114573669A (zh) 蛋白质Ghd7在调控植物抗低氮性中的应用
US20080216200A1 (en) Alternative splicing factors polynucleotides, polypeptides and uses thereof
WO2001020006A1 (fr) Promoteur inductible
AU765413B2 (en) Light-repressible promoters
US20020199219A1 (en) Modulation of abscisic acid signal transduction in plants
CN110959043A (zh) 利用bcs1l基因和向导rna/cas核酸内切酶系统改良植物农艺性状的方法
EP1203086B1 (fr) Element similaire de mite et element d'activation de transcription
US7199285B2 (en) Modulation of abscisic acid signal transduction in plants
US20040172685A1 (en) Method of using MAPK4 and orthologues thereof to control plant disease resistance and plant growth
WO2001004315A2 (fr) Compositions de genes rin et leurs procedes d'utilisation
CN116836249A (zh) 与小麦产量相关的三个同源基因及相关蛋白质

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP