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WO1993015213A1 - Regulation de la transcription d'un gene - Google Patents

Regulation de la transcription d'un gene Download PDF

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Publication number
WO1993015213A1
WO1993015213A1 PCT/GB1993/000137 GB9300137W WO9315213A1 WO 1993015213 A1 WO1993015213 A1 WO 1993015213A1 GB 9300137 W GB9300137 W GB 9300137W WO 9315213 A1 WO9315213 A1 WO 9315213A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
tfiid
cdna
sequence
promoter
Prior art date
Application number
PCT/GB1993/000137
Other languages
English (en)
Inventor
Michael Webster Bevan
Michael John Holdsworth
Wolfgang Walter Schuch
Original Assignee
Zeneca 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 Zeneca Limited filed Critical Zeneca Limited
Publication of WO1993015213A1 publication Critical patent/WO1993015213A1/fr

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Classifications

    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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

Definitions

  • the present invention relates to a gene which encodes a factor required for transcription in plant cells and a method for the control of transcription using that gene.
  • RNA polymerase II B
  • General transcription factors are responsible for assembly of the pre-initiation complex at the 'TATA' box, and accurate basal level transcription initiation.
  • Activators are an heterogeneous class of sequence-specific DNA binding proteins that interact with the pre-initiation complex to bring about regulated high level transcription of the associated gene. Interaction of activators with the pre-initiation complex is presumed to be an important level of control for gene expression.
  • TFIID Transcription Factor IID
  • TFIIA/G TFIIB
  • RNA polymerase 2 TFIIE/F
  • TFIIB TFIIB
  • TFIIE/F Transcription Factor IID
  • TBPs Homology between TBPs is at least 80% or greater in the carboxy-terminal 200 amino acids.
  • the amino terminal ends of TBPs are highly disparate, and were initially presumed to play some role in species-specific interactions that regulate transcription initiation. Although it is possible that this does occur other evidence has pointed to functional differences being localised to the highly conserved carboxy-terminal region. Reports to date have focused on analysis of the physical requirements of protein structure for TBP function.
  • An object of the present invention is to provide means for controlling gene transcription in plants.
  • a cDNA having the sequence given in Figure 1 herewith and variations therein permitted by the degeneracy of the genetic code, and any equivalent genomic sequence encoding TFIID TBP-component to which said cDNA hybridises.
  • the invention further provides the cDNA located on plasmid pTFIID which has been deposited, in an Escherichia coli, strain DH5 ⁇ , host, under the terms of the Budapest
  • the invention provides a method of inhibiting the growth of a plant cell comprising stably incorporating into the genome of the said plant by transformation a full or partial length copy of a nucleotide which is antisense to the cDNA defined hereinabove or its genomic equivalent.
  • Such inhibition may also be obtained by stably
  • a method for the isolation of the TFIID promoter comprising probing a genomic library with the cDNA of the invention, isolating a genomic sequence which hybridises to the said cDNA and recovering from the isolated genomic sequence th promoter region lying upstream of the TFIID gene.
  • the invention thus also provides the promoter sequence of the TFIID gene.
  • the invention also provides a method for the induction of constitutive gene expression, comprising placing a selected gene of interest under control of the promoter of the TFIID gene .
  • the invention further provides a recombinant gene construct comprising in sequence a TFIID promoter, a selected gene for expression and a transcription terminator region.
  • TFIID The TFIID gene can be used as a target to inhibit growth of a plant cell by several methods. In addition the gene sequences can be used to isolate the gene in question which is a truly constitutive gene. TFIID is a DNA-binding protein or factor which binds to DNA sequences found on plant promoters such as the "TATA box". Interaction of other factors involved with the initiation of transcription or the enhancement of transcription with the factor is specifically required for gene expression in plants.
  • the cDNA can be used to isolate the gene encoding it. This is described in detail below. This gene is special in that due to the requirement that it be expressed in all tissues of a plant at all times, it is expressed
  • the promoter controlling it is must be a truly constitutive promoter.
  • inhibition of the expression of this gene in specific tissues or at a specific stage during plant development is expected to lead to the inhibition of plant development at that given time. This can be used to inhibit flowering, pollen formation, embryo development and seed formation among others.
  • the present invention also provides a plant having stably incorporated in its genome by transformation a gene construct carrying a tissue-specific or a development-specific promoter which operates in the cells of the target plant tissue and a DNA binding protein gehe construct which is capable, when expressed, of inhibiting transcription in the cells of the said target tissue resulting in death of the cells.
  • One preferred application of this invention is in the creation of male sterile plants for use in the production of hybrids.
  • Our International Patent application WO 90/00110 incorporated herein by reference, relates to the production of such male sterile plant lines.
  • our invention the subject of that International Application, is a plant gene construct comprising a disrupter gene encoding a protein which is capable of disrupting the biogenesis of viable pollen, and a gene regulatory sequence and which includes a promoter sequence inducible by external
  • the construct comprises:
  • the disrupter protein examples include; the mammalian uncoupling protein (UCP) gene, a mutated form of the gene for the ⁇ -subunit of F 1 -ATPase which has sequences added or deleted such that these changes result in the retention of the ability to assemble with other subunits but interfere with function as an ATP synthase, a mutated, synthetic for of the oli 1 gene encoding subunit 9 of the F o -ATPase, a mutated form of a mitochondrial transit pre-sequence which malfunctions during transfer resulting in the disruption of protein transport to mitochondria, and, a gene construct carrying a fusion between the ⁇ -subunit gene from yeast and the ⁇ -glactosidase gene from E. coli, resulting in expression of a disrupting fusion protein.
  • the mammalian uncoupling protein (UCP) gene a mutated form of the gene for the ⁇ -subunit of F 1 -ATPase which has sequences added or deleted such
  • the present invention provides an alternative means of disrupting pollen formation comprising inhibition of the TFIID gene.
  • the invention also provides a plant, particularly a monocotyledonous plant, and more particularly a corn plant, having stably incorporated within its genome a gene
  • cell-specific promoters particular cells or tissue may be targeted and destroyed within complex organisms.
  • One particular application could be the destruction of cells essential for male flower development, leading to male sterility or the inhibition of seed development leading to plants which do not carry seeds.
  • the invention therefore provides a method of preventing or inhibiting growth and development of plant cells based on gene constructs which inhibit transcription and gene
  • the technique has wide application in a number of crops where inhibition of particular cells or tissue is required.
  • Transgenic plants are obtained by regeneration from the transformed cells.
  • a cDNA library was constructed in Lambda gt-11 using polyadenylated RNA obtained from young potato ( Solanum tuberosum L.cv. Desiree) tubers according to the
  • the cDNA library (10 recombinant phage) was screened with a TBP cDNA clone from Arabidopsis thaliana L, and the inserts from
  • a TBP cDNA clone from Arabidopsis thaliana L was used to screen a cDNA library from potato tubers. From an initial screen of 10 clones from an amplified portion of this library one clone was isolated (ST-1), and from a subsequent screen of 10 clones from the primary library a further three clones were isolated (ST-2, ST-3, ST-4). The complete nucleotide sequence of ST-2 (representing the largest spliced cDNA insert) was determined ( Figure 1).
  • This DNA sequence was compared to those available in the EMBL-database and indicated a high level of homology between ST-2 and TBPs from both human and yeast sources.
  • the cDNA ST-2 contains an open reading from
  • Potato TBP also shares over 80% homology with the carboxy-terminal region of TBP from humans, yeast and Drosophila melanogaster.
  • a nested set of 3' truncations of the TBP cDNA were constructed by digestion of plasmids with suitable
  • ESAs electrophoretic mobility shift assays
  • Reactions were carried out in 10 ⁇ l volumes containing 0.1 ⁇ l reticulocyte-lysate translation product as previously described (20) using radiolabelled probe DNA (290pM, 4000cpm) and 333ng (63.4nM) polydGdC.dGdC
  • Double-stranded oligonucleotides used in competition experiments were of the following structure (top-strand shown):
  • the copolymer polyGdC.dGdC (used as non-specific DNA in EMSA experiments) was a very inefficient competitor for TBP binding to the patatin promoter (Figure 4). Specificity of DNA binding for the TATA box was demonstrated in competitio experiments in which an oligonucleotide containing the wild type TATA motif from the patatin gene (see Figure 3) was shown to compete effectively with the patatin gene promoter for ST-2 binding, whereas an oligonucleotide containing a mutated TATATATA box (replaced with TAGGGGTA) was competed at a very much lower level.
  • optimised reaction conditions for potato TBP binding to the patatin promoter TATA-box was calculated from the binding data ( Figure 5d), and in four independent experiments gave value 2 . 4x10 9 M -1 ⁇ 1.1 ⁇ 10 11 .
  • K S the specific equilibrium constant
  • K n the non-specific
  • TBP is encoded as a single gene.
  • TBP from Arabidopsis is present in at least two copies.
  • a genomic library of potato was screened using the cD clone.
  • a genomic clone can be isolated using this approach.

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

Abstract

Le facteur de transcription IID (TFIID) d'un gène de plante code la protéine de liaison à l'ADN nécessaire à la transcription de tous les gènes structurels. La régulation négative de l'expression de TFIID, par l'intermédiaire de la transformation au moyen d'une copie messagère ou anti-messagère du gène de TFIID, empêche l'expression du gène, provoque un défaut de transcription, et, par conséquent, la mort de la cellule. On peut cibler cette régulation négative vers des types de cellules particuliers ou vers des étapes de développement particulières au moyen de la sélection de promoteurs appropriés. Etant donné que TFIID est exprimé simultanément dans toutes les cellules, l'effet de son promoteur est réellement constitutif et peut s'utiliser pour provoquer l'expression constitutive de gènes étrangers situés en aval dudit promoteur.
PCT/GB1993/000137 1992-01-24 1993-01-22 Regulation de la transcription d'un gene WO1993015213A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB929201549A GB9201549D0 (en) 1992-01-24 1992-01-24 Control of gene transcription
GB9201549.4 1992-01-24

Publications (1)

Publication Number Publication Date
WO1993015213A1 true WO1993015213A1 (fr) 1993-08-05

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AU (1) AU3362293A (fr)
GB (1) GB9201549D0 (fr)
WO (1) WO1993015213A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044465A1 (fr) * 1996-05-20 1997-11-27 Monsanto Company Procede de regulation de la germination de graines au moyen de sequences d'acyl-coa oxydase du soja
WO1998042851A1 (fr) * 1997-03-26 1998-10-01 Cambridge University Technical Services Ltd. Plantes presentant une croissance modifiee
EP0881288A1 (fr) * 1997-05-26 1998-12-02 Hoechst Aktiengesellschaft Purification de complexes transcriptionnels d'ordre supérieur à partir d'animaux transgéniques non humains
WO1999004004A1 (fr) * 1997-07-18 1999-01-28 E.I. Du Pont De Nemours And Company Homologue vegetal de l'ada2 de la levure, un adaptateur de transcription
EP0834566A3 (fr) * 1996-09-20 1999-02-03 Nisshinbo Industries, Inc. Gène de coton
WO1999009175A1 (fr) * 1997-08-15 1999-02-25 E.I. Du Pont De Nemours And Company Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription
WO1999022002A1 (fr) * 1997-10-24 1999-05-06 Cropdesign N.V. Nouveau cycline mitogene et son utilisation
WO1999013083A3 (fr) * 1997-09-05 1999-05-06 Cropdesign Nv Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
WO1999014331A3 (fr) * 1997-09-16 1999-06-10 Cropdesign Nv Inhibiteurs de la kinase cycline-dependante et utilisations de ceux-ci
WO2002016644A1 (fr) * 2000-08-18 2002-02-28 Massachusetts Institute Of Technology Inhibition de l'interaction proteine-proteine
US6710227B1 (en) 1998-09-16 2004-03-23 Cropdesign N.V. Cyclin-dependent kinase inhibitors and uses thereof
US7265267B1 (en) 1997-09-16 2007-09-04 Cropdesign N.V. Cyclin-dependent kinase inhibitors and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EMBL SEQUENCE DATABASE, HEIDELBERG, FRG. RELEASE 30, 31 OCT. 1991. ACCESSION NO. X62494 TATA-BINDING PROTEIN. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044465A1 (fr) * 1996-05-20 1997-11-27 Monsanto Company Procede de regulation de la germination de graines au moyen de sequences d'acyl-coa oxydase du soja
US6169174B1 (en) 1996-09-20 2001-01-02 Nisshinbo Industries, Inc. Cotton plant gene
EP0834566A3 (fr) * 1996-09-20 1999-02-03 Nisshinbo Industries, Inc. Gène de coton
WO1998042851A1 (fr) * 1997-03-26 1998-10-01 Cambridge University Technical Services Ltd. Plantes presentant une croissance modifiee
US6559358B1 (en) 1997-03-26 2003-05-06 Cambridge University Technical Services, Ltd. Plants with modified growth
EA003425B1 (ru) * 1997-03-26 2003-04-24 Кембридж Юниверсити Текникал Сервисиз Лимитед Химерный ген, обеспечивающий экспрессию в растениях циклина d-типа, и способ получения растений с измененными ростовыми свойствами при использовании указанного гена
EP0881288A1 (fr) * 1997-05-26 1998-12-02 Hoechst Aktiengesellschaft Purification de complexes transcriptionnels d'ordre supérieur à partir d'animaux transgéniques non humains
WO1999004004A1 (fr) * 1997-07-18 1999-01-28 E.I. Du Pont De Nemours And Company Homologue vegetal de l'ada2 de la levure, un adaptateur de transcription
WO1999009175A1 (fr) * 1997-08-15 1999-02-25 E.I. Du Pont De Nemours And Company Genes de codage de dr1 et drap1, complexes represseurs globaux de la transcription
WO1999013083A3 (fr) * 1997-09-05 1999-05-06 Cropdesign Nv Methode et dispositif de modulation de proteines de cycle cellulaire vegetal et leur utilisation dans la regulation de la croissance de cellules vegetales
WO1999014331A3 (fr) * 1997-09-16 1999-06-10 Cropdesign Nv Inhibiteurs de la kinase cycline-dependante et utilisations de ceux-ci
US7265267B1 (en) 1997-09-16 2007-09-04 Cropdesign N.V. Cyclin-dependent kinase inhibitors and uses thereof
WO1999022002A1 (fr) * 1997-10-24 1999-05-06 Cropdesign N.V. Nouveau cycline mitogene et son utilisation
US6710227B1 (en) 1998-09-16 2004-03-23 Cropdesign N.V. Cyclin-dependent kinase inhibitors and uses thereof
WO2002016644A1 (fr) * 2000-08-18 2002-02-28 Massachusetts Institute Of Technology Inhibition de l'interaction proteine-proteine

Also Published As

Publication number Publication date
AU3362293A (en) 1993-09-01
GB9201549D0 (en) 1992-03-11

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