WO2004070035A2 - Method for increasing efficiency of homologous recombination in plants - Google Patents

Method for increasing efficiency of homologous recombination in plants Download PDF

Info

Publication number
WO2004070035A2
WO2004070035A2 PCT/EP2004/000964 EP2004000964W WO2004070035A2 WO 2004070035 A2 WO2004070035 A2 WO 2004070035A2 EP 2004000964 W EP2004000964 W EP 2004000964W WO 2004070035 A2 WO2004070035 A2 WO 2004070035A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
caltractin
acid sequence
nucleic acid
plant
Prior art date
Application number
PCT/EP2004/000964
Other languages
French (fr)
Other versions
WO2004070035A3 (en
Inventor
Rocio Sanchez-Fernandez
Barbara Hohn
Jean Molinier
Original Assignee
Sungene Gmbh & Co. Kgaa
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 Sungene Gmbh & Co. Kgaa filed Critical Sungene Gmbh & Co. Kgaa
Publication of WO2004070035A2 publication Critical patent/WO2004070035A2/en
Publication of WO2004070035A3 publication Critical patent/WO2004070035A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination

Definitions

  • the present invention relates to methods for increasing effi- ciency of homologous recombination in plants or plant cells, by decreasing expression or activity of at least one caltractinlike protein in said plant or plant cells.
  • the method may be used for inserting sequences into a distinct locus of the chromosomal DNA.
  • the method may be used for deleting se- quences from the chromosomal DNA flanked by homology sequences by initiating homologous recombination between said homology sequences.
  • the invention furthermore relates to expression constructs suitable for expressing at least part of a nucleic acid sequence encoding for a caltractin-like protein (e.g., in form of antisense or double-stranded RNA) suitable for decreasing expression or activity of at least one caltractin-like protein in plant or plant cells.
  • Object of the invention are furthermore transgenic plants comprising a decreased expression or activity of at least one caltractin-like protein and the use of said transgenic plants for the production of food, feeds, seed, pharmaceuticals or fine chemicals, in particular for the production of oils.
  • the aim of plant biotechnology work is the generation of plants with advantageous novel properties, for example for increasing agricultural productivity, increasing the quality in the case of foodstuffs, or for producing specific chemicals or pharmaceuticals.
  • the production of correspondingly modified eukaryotic organisms can be realised only for a very limited number of organ- isms (like e.g., mice) and only by means of high effort ( anaar R Hoeij akers JH (1997) Genes Funct 1 (3) .165-174) .
  • Exploratory approaches to increase HR in plants comprise expression of proteins like RecA (WO 97/08331) or RecA-homologues derived from other species like e.g., Rad52 (WO 01/68882) or RecA/VirE2 fusion-proteins (WO .01/38504) .
  • Use of poly(ADP- ribose)polymerase inhibitors has demonstrated an increased HR in plants (Puchta H et al . (1995) Plant J 7:203- 210).
  • sequence-unspecific induction of DNA strand breaks is disadvantageous because of the potential mutagenic effect. Sequence-specific induction of DNA strand-breaks may also increase efficiency of HR but is limited to artificial scenarios (Siebert R, Puchta H (2002) Plant Cell 14 (5) : 1121-31) .
  • Caltractin also known as centrin (Cdc31p in Saccharomyces cere- visiae)
  • centrin Cdc31p in Saccharomyces cere- visiae
  • EF-hand calcium-binding
  • Biol 107:133-140 It is a member of the calmodulin superfamily of calcium modulated proteins which include calmodulin, troponin C, parvalbumin, calbindins, and S.-100 protein (Kretsinger RH (1980) CRC Crit Rev Biochem 8:119-174).
  • cal- tractin is most prominently localized to calcium-sensitive contractile fibers, the striated fiber root that connect the basal body complex to the underlying nucleus in interphase cells. It has been suggested that caltractin is a structural component of the cytoskeletal framework of fine filaments that compose these striated fibers (Schulze D et al. (1987) Eur J Cell Biol 45:51- 61) .
  • Caltractin is a protein found in numerous eukaryotic species and is localized in the centrosome (Lee VD et al. (1993) Proc Natl Acad Sci USA 90:11039-11043; Baum P et al . (1988) Mol Cell Biol 8:5386-5397; Levy YY et al . (1996) Cell Motil and Cytoskel 33:298-323) .
  • caltractin may function in a signal transduction pathway that affects cell division (Zhu JK (1992) Plant Physiol 99:1734-1735) .
  • Sequences of plant caltractin-like proteins are deposited in the GeneBank database including sequences from e.g., Arabidopsis thaliana (Gene locus At4g37010; Ace. -No.: NC_003075 and Gene Locus At3g50360; Ace . No. AJ009672) , barley (Hordeum vulgare; GeneBank Ace. -No.: AL505334, AV929632, BF628654, BG343518) , wheat (Triticum aestivum, GeneBank Ac. -No.: BJ292493, BJ298955, BJ253096) corn (Zea mays; GeneBank Ac.
  • the current invention therefore provides a method for further increasing efficiency of HR in plants.
  • the homologous recombination (HR) between A and A' may constitute an intramolecular or an intermolecular recombination event .
  • the two homology-sequences A and A' undergoing homologous recombination may - for example - be both localized on one strand of a chromosomal DNA-sequence, e.g., in form of direct repeats.
  • HR will cause deletion of the sequences localized between A and A' . This may be utilized, e.g., for deleting selection marker sequences from the chromosomal DNA.
  • wo h ⁇ mology-sequences are localized on separate DNA molecules.
  • one of the two homology sequences A and A' undergoing homologous recombination is localized on a chromosomal DNA-sequence.
  • the other sequence may - for example - be localized on a DNA- construct, which may function as a gene targeting construct.
  • mutations are introduced into the chromosomal DNA as a consequence of the HR between A and A' .
  • the invention relates to a method for introducing a mu- tation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell comprising the steps of
  • said DNA-construct comprises at least one homology-sequence A having a sufficient length and homology to at least one part A' of said chromosomal sequence to allow homologous recombination among A and A' , and wherein said DNA-construct introduces said mutation into said chromosomal DNA-sequence in consequence of the homologous recombination between A and A' , and
  • the DNA-construct comprises two homology-sequences A and B having a sufficient length and homology to at least a part A' and B' of said chromosomal sequence, respectively, to allow homologous recombination between A and A' , and B and B' , respectively.
  • a and B additional sequences might be localized (like e.g., expression cassettes, functional elements) which by the homologous recombination are introduced into the chromosomal DNA.
  • the mutation may - for example - be introduced directly by replacement of A by A' , wherein A differs from A' by the mutation to be introduced. If two pairs of homology-sequences (e.'g., A/A' and B/B' ) are used, the mutation may - for example - be introduced by insertion of the sequences comprised between A and B into the chromosomal DNA. Alternatively, the mutation may comprise modification (e.g., base change) of the sequence localized between A' and B' .
  • modification e.g., base change
  • the method of the invention further comprises the step of segregating the mutation introduced by the DNA-construct and the property of a decreased ac- tiyity or expression of at least one caltractin-like protein.
  • the segregation may be carried out by any method known in the art (e.g., crossing and selection), whereby selection may be easily realized by using standard techniques (e.g., PCR or marker technology) to monitor segregation of the mutation.
  • transgenic ex- pression cassettes comprising at least part of a nucleic acid sequence coding for caltractin-like protein under control of a promoter sequence functional in plant cells.
  • Said part may have a length of at least 20 base pair (hereinafter bp) , preferably at least 50 bp, more preferable at least 100 bp, most preferably at least 500 bp.
  • the nucleic acid sequence coding for caltractin-like protein may be orientated in sense and/or antisense direction with regard to the transcription direction of said promoter sequence.
  • the transgenic expression cassette of the invention may cause expression of sense, an- tisense or double-stranded RNA of said nucleic acid sequence coding for caltractin-like protein.
  • Yet another embodiment of the invention relates to transgenic expression vectors comprising at least one transgenic expression cassette of this invention.
  • Yet another embodiment of the invention relates to transgenic organisms comprising at least one transgenic expression vector or transgenic expression cassette of this invention.
  • the organism is a plant organism.
  • Yet another embodiment of the invention related to a plant organism with a decreased activity or expression of at least one caltractin-like protein.
  • said decrease is caused by transformation of said plant with at least one transgenic ex- pression- vector or transgenic expression cassette of this invention or by mutating at least one endogenous gene coding for a caltractin-like protein
  • Yet another embodiment of the invention relates to the use of at least one of the transgenic expression cassettes, transgenic expression vectors, transgenic organisms, or plants with decreased activity or expression of at least one caltractin-like protein in a method for introducing a mutation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell.
  • the caltractin-like proteins are considered key elements for homologous recombination in plants.
  • the method outperforms all those methods in which an increased homologous recom- bination phenotype is realized by overexpressing a HR enhancing protein (like e.g., RecA) .
  • Switching off a gene can be realized without expressing a (foreign) protein.
  • all that is necessary is to deactivate the endogenous gene. This has considerable advantages for approval and acceptance by the con- sumer, who is frequently apprehensive toward plants with foreign proteins .
  • At4g37010 gene product (Arabidopsis thaliana caltractin-like protein) shows 49% identity with the human centrin 2. At4g37010 gene expression - as analyzed by RT-PCR - can be detected in all plant organs : roots, buds, leaves, and flowers.
  • the methods according to the invention can be applied to all plant species, preferably to those in which a cal- tractin-like protein or a functional equivalent thereof is expressed naturally.
  • Caltractin-like proteins can be identified in a large number of plants.
  • the sequences from other plants which are homologous to the caltractin-like protein or nucleic acid sequences disclosed within the scope of the present invention can be found readily for example by database searches or by screening genetic libraries using the caltractin-like protein or nucleic acid sequences as search sequence or probe..
  • caltractin-like protein is intended to include a naturally occurring protein encoded by an amino acid sequence comprising at least one of the following sequence motifs (amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein "X” can stand for any amino acid. Nucleotides, likewise, may be referred to by their commonly accepted single- letter codes) :
  • a first EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of D(T/I)D(G/N)S(G/V) (S/T) IDAXEL, more preferably
  • a second EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of (E/Q) (E/Q)IXX(M/L) (l/M)A(E/D) (V/l)DK, more preferably
  • a third EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of (l/F)DXDX(N/T) GKIS, more preferably (K/R) FX (I/V/L) (I/F)DXDX(N/T) GKIS, most preferably (K/R) AFX(I/V/L) (I/F)DXDX(N/T)GKI-SXX(D/N) (I/L) , wherein said third EF-hand motif is preferably localized between amino acid 85 and 130 of the caltractin-like protein full-length sequence .
  • a fourth EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of AD(R/Q) (D/N)XD(G/R)E, more preferably (I/V)X(E/A)AD(R/Q) (D/N) XD (G/R) E, 'ost preferably (E/D)M(I/V)X(E/A)AD(R/Q) (D/N) D (G/R) E, wherein said fourth EF-hand motif is preferably localized within the last 40 amino acids of the caltractin-like protein full-length se- quence .
  • At least 2 of these motifs occur in a Caltractin-like protein, very especially preferably at least 3 of these motifs, more preferably at least 4 of these mo- tifs, most preferably all motifs a to e.
  • the order of two or more of the motifs a to e may vary but the alphabetical order given above is preferred. Further sequence motifs which are typical for Caltractin-like can be deduced readily by the skilled worker from the sequence alignment of the known Cal- tractin-like proteins, as shown in Fig. 5.
  • Caltractin-like proteins include proteins from the group consisting of :
  • a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34;
  • a functionally equivalent polypeptide molecule comprising an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
  • polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34;
  • polypeptide molecule comprising a fragment of at least 20 consecutive amino acids, preferably 50 consecutive amino acids of at least one of the sequences described under a) or b) exhibiting essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2/ 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • Caltractin-like protein Property or “properties” of a Caltractin-like protein is to be understood in the broad sense and includes for example activity and/or function of said Caltractin-like protein.
  • “Function” is preferably understood to mean the substrate- binding capacity or ligand-binding property of a Caltractin-like polypeptide in an organism, a tissue, a cell or a cell compartment.
  • Suitable substrates are Calcium-ions, but also the DNA or protein interaction partners of a Caltractin-like protein.
  • Binding partners for Caltractin-like proteins can be identified by methods well known to the person skilled in the art, for example by the yeast-2 -hybrid system. Said interaction partners may include but shall not be limited to plant homologues of binding partners of Caltractin-like polypeptides identified in various organism like e.g., XPC (xeroderma pigmentosum factor C; Araki M et al.
  • “Functional equivalent polypeptide” is understood to mean, in particular, natural or artificial mutations of the Caltractinlike polypeptides as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34 and homologous polypeptides from other plants which continue to have essentially the same properties .
  • Homologous polypeptides from the below- described preferred plants are preferred. Mutations encompass substitutions, additions, deletions, inversions or insertions of one or more amino acid residues.
  • the present invention also encompasses those polypeptides which are obtained by modification of a polypeptide as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • Joy substitution (in particular, conservative substitutions) , insertion or deletion have at least 60%, preferably at least 80%, by preference at least 90%, especially preferably at least 95%, very especially preferably at least 98%, homology with one of the polypeptides comprising an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 and exhibit essentially the same properties as the polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • Preferred functional equivalents include conservatively modified variants of the Caltracin-like proteins as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • Constantly modified- variants - applies to both amino acid and nucleic acid sequences.
  • conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al . , J. Biol. Chem. 260: 2605- 2608 (1985); Rossolini et al . , Mol. Cell. Probes 8: 91-98 (1994) ) . Because of the degeneracy of the genetic code, a large number of functionally identical .nucleic acids encode any given protein.
  • the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • Such nucleic acid variations are "silent variations", which are one species of conservatively modified variations . Every nucleic acid sequence recited herein that en- ⁇ codes a polypeptide also describes every possible silent varia- tion of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule.
  • AUG which, along with GUG in some organisms, is ordinarily the only codon for methionine
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and al- leles of the invention.
  • a further embodiment of the invention comprises 'an isolated nucleic acid sequence coding for a caltractin-like protein, wherein said nucleic acid sequence comprises an sequence described by SEQ ID NO: 5, 7, 9, 11, 13, 17,
  • Caltractin-like sequences can be found readily for example by database searches or by screening genetic libraries using the Caltractin-like sequences as search sequence or probe.
  • Additional Caltractin-like proteins can be identified for example from a variety of organisms for which DNA sequences are known, such as, for example, from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Oryza sativa, or Helianthus annuus from databases or homology comparisons.
  • the screening of cDNA libraries or genomic libraries of other organisms, preferably of the plant species which are mentioned further below as hosts for the transformation, using the nucleic acid sequences described under SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 or parts of these as probe is also a method of identifying homologues in other species with which the skilled worker is familiar.
  • the probes derived from the nucleic acid sequences as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 have a length of at least 20 bp, preferably 50 bp, particularly preferably 100 bp, very especially preferably 200 bp, and most preferably 400 bp.
  • a DNA strand which is complementary to the sequences described under SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 may also be employed for screening the libraries.
  • Functional equivalents accordingly, encompass DNA sequences which hybridize under standard conditions with the Caltractinlike nucleic acid sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, with the sequence complementary thereto or parts of the abovementioned and which, as complete sequences, encode proteins which have the same properties as the proteins described under SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • GAP Garnier et al. (1997) Nucleic Acids Res. 25:3389 et seq.), setting the following parameters:
  • Gap weight 50 Length weight: 3
  • sequence which has at least 60% homology with sequence SEQ ID NO: 1 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above program algorithm with the above parameter set, has at least 60% identity.
  • Homology between two polypeptides is understood as meaning the identity of the amino acid sequence over in each case the entire sequence length which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG) , Madison, USA) , setting the following parameters:
  • Gap weight 8 Length weight : 2
  • sequence which has at least 60% homology with sequence SEQ ID NO: 2 at the protein level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 2 by the above program algorithm with the above parameter set, has at least 60% identity.
  • Standard hybridization conditions is to be understood in the broad sense and means stringent or else less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al., in Molecular Cloning (A Laboratory Manual) , 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989) , 6.3.1-6.3.6.
  • the conditions during the wash step can be selected from the range of conditions delimited by low-stringency conditions (approximately 2X SSC at 50°C) and high-stringency conditions (approximately 0.2X SSC at 50°C, preferably at 65°C) (20X SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0).
  • the temperature during the wash step can be raised from low- stringency conditions at room temperature, approximately 22°C, to higher-stringency conditions at approximately 65°C. Both of the parameters salt concentration and temperature can be varied simultaneously, or else one of the two parameters can be kept constant while only the other is varied. Denaturants, for exam- pie formamide or SDS, may also be employed during the hybridization. In the presence of 50% formamide, hybridization is preferably effected at 42°C.
  • Hybridization conditions can be selected, for example, from the following conditions:
  • Wash steps can be selected, for example, from the following conditions :
  • Plant is generally understood as meaning any single- or multi- celled organism or a cell, tissue, part or propagation material (such as seeds or fruit) of same which is capable of photosynthesis. Included for the purpose of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred. Also included are mature plants, seeds, shoots and seedlings, and parts, propagation material (for example tubers, seeds or fruits) and cultures derived from them, for example cell cultures or callus cultures.
  • the term includes the mature plants, seed, shoots and seedlings and their derived parts, propagation material (such as seeds or microspores) , plant organs, tissue, protoplasts, callus and other cultures, for example cell cultures, and any other type of plant cell grouping to give functional or structural units.
  • Mature plants refers to plants at any desired developmental stage beyond that of the seedling. Seedling refers to a young immature plant at an early developmental stage.
  • plant organisms for the purposes of the invention are further organisms capable of being photosynthetically active such as, for example, algae, cyanobacteria and mosses.
  • Preferred algae are green algae such as, for example, algae from the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis is particularly preferred.
  • vascular plants are especially preferred.
  • the term "vascular plant” is intended to include all plants comprising a vascular system in contrast to non-vascular plants like, e.g. Bryophytae.
  • Vascular plants may comprise seedless and seed- carrying vascular plants .
  • Seedless vascular plants are especially plants of the phyla Psilotophyta, Lycophyta, Sphenophyta or Pterophyta
  • Seed-carrying vascular plants are especially plants of the phyla Cycadophyta, Ginkophyta, Coniferophyta, Gnetophyta, Anthophyta.
  • Vascular plant encompasses all annual and perennial monocotyledonous and dicotyledonous plants and includes by way of example but not by limitation those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Tri- folium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bro- mus, Asparagus, Antirrhinum, Hemerocallis, Nemesis, Pelargonium, Panicum, Penniset-um, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Pisum,
  • Preferred plants are those from the following plant families: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaeeae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniaceae, Theaceae, Umbelliferae.
  • Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants such as, for example, the Gramineae family, such as rice, maize, wheat or other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and all grass species.
  • the Gramineae family such as rice, maize, wheat or other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and all grass species.
  • the invention is applied very particularly preferably to dicotyledonous plant organisms.
  • Preferred dicotyledonous plants are selected in particular from the dicotyledonous crop plants such as, for example,
  • Asteraceae such as sunflower, tagetes or calendula and others
  • - Compositae especially the genus Lactuca, very particularly the species sativa (lettuce) and others
  • - Cruciferae particularly the genus Brassica, very particularly the species napus (oilseed rape) , campestris (beet) , oleracea cv Tastie (cabbage) , oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other cabbages
  • the genus Arabidopsis very particularly the species thaliana, and cress or canola and others
  • Cucurbitaeeae such as melon, pumpkin/squash or zucchini and others
  • Rubiaceae preferably the subclass Lamiidae such as, for example Coffea arabica or Coffea liberica (coffee bush) and others ,
  • Solanum very particularly the species tuberosum (potato) and melongena (aubergine) and the genus Capsicum, very particularly the genus annuum (pepper) and tobacco or paprika and others,
  • Sterculiaceae preferably the subclass Dilleniidae such as, for example, Theobroma cacao (cacao bush) and others,
  • Theaceae preferably the subclass Dilleniidae such as, for example, Camellia sinensis or Thea sinensis (tea shrub) and others,
  • Umbelliferae particularly the genus Daucus (very particularly the species carota (carrot) ) and Apium (very particularly the species graveolens dulce (celery) ) and others;
  • Tree species pref- erably comprise plum, cherry, peach, nectarine, apricot, banana, kiwi, papaya, mango, -apple, pear, quince.
  • angiosperms such as, for example, Hepaticae (liverworts) and Musci (mosses) ; pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae, the families of the Rosaceae such as rose, Ericaceae such as rhododendron and azalea, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli, iris, free
  • Preferred within the scope of the invention are those plants which are employed as foodstuffs or feeding stuffs.
  • the method of the invention is suitable to introduce mutations in the chromosomal DNA of a plant cell or a plant organism.
  • Chromosal DNA is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chro- matids, they might be condensed or uncoiled.
  • “Mutation " or “mutated” with respect to a chromosomal DNA- sequence is to be understood in the broad sense and is intended to include substitution, addition, deletion, inversion or insertion of at least one or more base pairs.
  • Said mutation may af- feet the coding region as much as the non-coding region (e.g., the 5' -untranslated, 3' -untranslated, intron or promoter region) of the a gene.
  • Consequences of said mutation may be various and may - for example- cause a decrease or an increase of the amount of mRNA or protein expressed from said gene, or of the function and/or activity of the corresponding gene product.
  • insertion is intended to comprise single base insertion and/or insertion of additional genes or expression cassettes.
  • amount of protein is understood as meaning the amount of protein (e.g., an Caltractin-like polypeptide) in an organism, a tissue, a cell or a cell compartment.
  • Decrease with respect to the amount of mRNA or protein (e.g., expressed from a target gene or coding for a Caltractin-like protein) is to be understood in the broad sense and is intended to include the permanent or temporarily, partially, essentially completely or completely hindering or blocking of the expression of the target gene or the corresponding RNA or mRNA by various cellular mechanisms or of the protein product resulting thereof.
  • RNA or mRNA or protein means the quantitative decrease of the amount of RNA or mRNA or protein, respectively, in an organism, a tissue, a cell or a cell compartment in comparison with the wild type of the same genus and species, to which this method had not been applied, under otherwise identical conditions (such as, for example, culture conditions, plant age and the like) .
  • the decrease amounts to at least 10%, preferably at least 10% or at least
  • Decrease with respect to the function and/or activity of a protein (e.g., expressed from a target gene or coding for a Caltractin-like protein) may include an decrease of the encoding mRNA but may also include a change in the protein sequence of the gene product causing a decreased or abolished function and/or activity.
  • a decrease in the amount of mRNA may be monitored by various techniques well known to the person skilled in the art including but not limited to Northern-hybridization, nuclease protection assay or quantitative reverse transcription PCR (quantitative RT-PCR) .
  • a decrease in the amount of protein (and indirectly the encoding mRNA) may be monitored by various techniques well known to the person skilled in the art including but not limited to the mi- cro-biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218- 222) , the Folin-Ciocalteu-method (Lowry OH et al . (1951) J Biol Chem 193:265-275) or by measurement of* the adsorption of CBB G- 250 (Bradford MM (1976) Analyt Biochem 72:248-254). More specific quantitative analysis may be carried out by methods including but not limited to ELISA ("enzyme linked immunosorbent assay"), Western-blotting, radioimmunoassay (RIA) or other immunoassays .
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • RNA, mRNA or protein e.g., expressed from a target gene
  • activity or function of the corresponding gene product is to be understood in the broad sense and is intended to include the permanent or temporarily initiation, boost, or enhancement of expression from the target gene or the quantity of the RNA or mRNA derived therefrom, or the quantity, activity or function of the corresponding polypeptide encoded thereby by various cellular mechanisms.
  • RNA or mRNA or protein means the quantitative increase of the amount of RNA or mRNA or protein, respectively, in an organism, a tissue, a cell or a cell compartment in comparison with the wild type of the same genus and species, to which this method had not been applied, under otherwise identical conditions (such as, for example, culture conditions, plant age and the like) .
  • the increase amounts to at least 50%, preferably at least 100%, especially preferably at least 500%, very especially preferably at least 1000%.
  • the increase can be measured or monitored by methods known in the art and described in brief above for assessing an decrease in RNA, mRNA or protein.
  • a Caltractin-like activity or a Caltractin-like function is to be interpreted in the wide sense and encompasses the partial or essentially complete inhibition or blocking of the functionality of a Caltractin-like protein in a plant or a part, tissue, organ, cells or seeds thereof, which inhibition or blocking is based on a variety of cytobiological mechanisms.
  • a decrease also encompasses a quantitative decrease of a Caltractin-like protein down to the essential complete absence of the Caltractin-like protein (i.e. lacking detectability of Caltractin-like activity or Caltractinlike function, or lacking immunological detectability of the Caltractin-like protein) .
  • the expression of a particular Caltractin-like protein, or the Caltractin-like activity or Caltractin-like function, in a cell or an organism is preferably reduced by more than 50%, especially preferably by more than 80%, very especially preferably by more than 90%.
  • a decrease of the Caltractin-like activity or the Caltractinlike function is preferably achieved by reduced expression of an endogenous Caltractin-like protein.
  • a decrease of the amount of Caltractin-like protein, the Cal- tractin-like protein activity or the Caltractin-like protein function can be effected - for example - by utilization of at least one of the following methods:
  • RNA of a Caltractin-like protein encoding nucleic acid sequence (Caltractin-like protein dsRNA) or an expression cassette (s) ensuring the expression thereof
  • RNA sequences a Caltractin-like protein encoding nucleic acid sequence or an expression cassette ensuring expression thereof .
  • the antisense nucleic acid sequence is directed against a Caltractin-like gene (i.e. genomic DNA sequences) or a Caltractin-like gene transcript (i.e. RNA sequences), ⁇ -anomeric nucleic acid sequences are also encompassed
  • the sense nucleic acid sequence may be DNA or RNA, but is preferably RNA.
  • Caltractin-like protein encoding gene RNA or Caltractin-like protein or an expression cassette ensuring expression thereof
  • each and every one of these methods may bring about a decrease of the expression of a Caltractin-like protein, activity and/or function of a Caltractin-like protein for the purposes of the invention.
  • a combined use is also feasible.
  • Further methods are known to the skilled worker and can encompass the hindering or prevention of Caltractin-like protein processing, of the Caltractin-like protein or Caltractin-like mRNA transport, inhibition of ribosome attachment, inhibition of RNA splicing, induction of an Caltractin-like protein RNA-degrading enzyme and/or inhibition of translational elongation or termination.
  • dsRNAi double-stranded RNA interference
  • dsRNAi double-stranded RNA interference
  • dsR Ai methods are based on the phenomenon that the simultaneous introduction of complementary strand and counterstrand of a gene transcript causes the expression of the gene in question to be suppressed in a highly efficient manner. The phenotype caused greatly resembles a corresponding knock-out mutant (Waterhouse PM et al. (1998) Proc Natl Acad Sci USA 95:13959-64) .
  • dsRNAi has proved to be particularly effective and advantageous for reducing expression of a Caltractin-like protein.
  • dsRNAi approaches are markedly superior to traditional antisense ap- proaches .
  • the invention therefore furthermore relates to double-stranded RNA molecules (dsRNA molecules) which, upon introduction into a plant (or a cell, tissue, organ or seed derived therefrom) , bring about a decrease of expression of a Caltractin-like protein.
  • dsRNA molecules double-stranded RNA molecules
  • Double-stranded RNA molecule is to be understood to comprise at least one RNA molecule, which is at least theoretically able to form a double-stranded RNA secondary structure by intermo- lecular or intramolecular base pairing.
  • Such secondary structure may be predicted by the base-paring rules of Watson and Crick or by computer algorithms (like e.g., FOLDRNA; Zuker and Stiegler (1981) Nucleic Acids Res 9 (1) :133-48) .
  • the double-stranded RNA molecule for reducing the expression of an Caltractin-like protein comprises,
  • RNA sequence which is essentially iden- tical to at least part of a nucleic acid sequence coding for a Caltractin-like protein
  • second RNA sequence which is essentially complementary to at least part of said first RNA sequence under i) .
  • the double-stranded RNA molecule comprises,
  • RNA sequence which is essentially identical to at least part of a nucleic acid sequence comprising a sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, and
  • RNA sequence which is essentially complementary to at least part of said first RNA sequence under i) .
  • dsRNA sequence can also show insertions, deletions or individual point mutations compared with the target sequence coding for the Caltractin-like protein while still bringing about an effective decrease of the expression.
  • the homology in accordance with the above definition preferably amounts to at least 75%, preferably at least 80%, very especially preferably at least 90%, most preferably 100%, between the sense strand of an inhibitory dsRNA and a part- segment of a nucleic acid sequence encoding a Caltractin-like protein (or between the antisense strand and the complementary strand of a nucleic acid sequence encoding a Caltractin-like protein) .
  • the length of the part-segment amounts to at least 10 bases, preferably at least 25 bases, especially preferably at least 50 bases, very especially preferably at least 100 bases, most preferably at least 200 bases or at least 300 bases.
  • an "essentially identical" dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing with part of a Caltractin-like gene transcript (for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 itiM EDTA at 50°C or 70°C for 12 to 16 h) .
  • the dsRNA can be composed of one- or more strands of polymerized ribonucleotides. Modifications both of the sugar-phosphate backbone and of the nucleosides may be present. For example, the phosphodiester bonds of the. natural RNA can be modified in such a way that they comprise at least one nitrogen or sulfur hetero atom. Bases can be modified in such a way that the activity of, for example, adenosine deaminase is restricted. These and other modifications are described hereinbelow in the methods of stabilizing antisense RNA.
  • the dsRNA can be generated enzymatically or fully or partially synthesized chemically.
  • the double-stranded structure can be formed starting from an individual self-complementary strand or starting from two complementary strands.
  • sense and antisense sequence may be linked by a linking sequence
  • linking sequence can preferably be an intron which is spliced out after the dsRNA has been synthesized.
  • the nucleic acid sequence encoding a dsRNA can comprise further elements such as, for example, transcription termination signals or polyadenyla- tion signals.
  • RNA sequences are to be combined to form a dsRNA in a cell or plant, this can be effected in various ways:
  • the formation of the RNA duplex can be initiated either outside or within the cell.
  • the dsRNA can also encompass a hairpin structure by linking sense and antisense strand by means of a linker (for example an intron) .
  • a linker for example an intron
  • the self- complementary dsRNA structures are preferred since they only require the expression of one construct and always comprise the complementary strands in an equimolar ratio.
  • the expression cassettes encoding the antisense or sense strand of a dsRNA or the self-complementary strand of the dsRNA are preferably inserted into a vector and, using the methods described hereinbelow, stably inserted into the genome of a plant in order to ensure permanent expression of the dsRNA, using se- lection markers for example.
  • the dsRNA can be introduced using a quantity which allows at least one copy per cell. Greater quantities (for example at least 5, 10, 100, 500 or 1000 copies per cell) may bring about a more effective decrease.
  • Caltractin-like proteins from various plant organisms (e.g., Arabidopsis, rice, maize and wheat; see Fig.l) allows the conclusion that this protein is conserved to a high degree within plants, so that the expression of a dsRNA derived from one of the disclosed nucleic acid sequences encoding a Caltractin-like protein as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 appears to have an advantageous effect in other plant species as well.
  • the dsRNA can be synthesized either in vivo or in vitro.
  • a DNA sequence encoding a dsRNA can be brought into an expression cassette under the control of at least one genetic control element (such as, for example, promoter, enhancer, si- lencer, splice donor or splice acceptor or polyadenylation signal) .
  • at least one genetic control element such as, for example, promoter, enhancer, si- lencer, splice donor or splice acceptor or polyadenylation signal.
  • Suitable advantageous constructions are described hereinbelow. Polyadenylation is not required, nor do elements for ini- tiating translation have to be present.
  • a dsRNA can be synthesized chemically or enzymatically.
  • Cellular RNA polymerases or bacteriophage RNA polymerases (such as, for example, T3, T7 or SP6 RNA polymerase) can be used for this purpose. Suitable methods for expression of RNA in vitro are described (WO 97/32016; US 5,593,874; US 5,698,425, US 5,712,135, US 5,789,214, US 5,804,693) .
  • a dsRNA which has been synthesized in vitro chemically or enzymatically can be isolated completely or to some degree from the reaction mixture, for example by extraction, precipitation, electrophoresis, chromatography or combinations of these methods, before being introduced into a cell, tissue or organism.
  • the dsRNA can be introduced directly into the cell or else be applied extracellularly (for example into the interstitial space) .
  • the antisense nucleic acid molecule hybridizes, or binds, with the cellular mRNA and/or genomic DNA encoding the Caltractin-like target protein to be suppressed. This suppresses the transcription and/or translation of the target protein.
  • Hybridization can originate conventionally by the formation of a stable duplex or - in the case of genomic DNA - by the antisense nucleic acid molecule binding to the duplex of the genomic DNA by specific interaction in the major groove of the DNA helix.
  • An antisense nucleic acid sequence suitable for reducing an Caltractin-like protein can be deduced using the nucleic acid se- quence encoding this protein, for example the nucleic acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, following Watson and Crick's base pairing rules.
  • the antisense nucleic acid sequence can be complementary to all of the transcribed mRNA of said protein, be limited to the coding region, or else only be composed of a nu- cleotide, which is partially complementary to the coding or non- coding sequence of the mRNA.
  • the oligonucleo- tide can be complementary to the region encompassing the translation start for said protein.
  • Antisense nucleic acid sequences can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nu- cleotides in length, but may also be longer and comprise at least 100, 200, 500, 1000, 2000 or 5000 nucleotides.
  • Antisense nucleic acid sequences can be expressed recombinantly or synthesized chemically or enzymatically using methods known to the skilled worker. In the case of chemical synthesis, natural or modified nucleotides may be used. Modified nucleotides can im- part an increased biochemical stability to the antisense nucleic acid sequence and lead to an increased physical stability of the duplex formed of antisense nucleic acid sequence and sense target sequence. The following can be used: for example phos- phorothioate derivatives or acridine-substituted nucleotides.
  • a Caltractin-like protein in a further preferred embodiment, can be inhibited by nucleotide sequences which are complementary to the regulatory region of a Caltractin-like gene (for example a Caltractin-like promoter and/or enhancer) and which form triple-helical structures with that DNA double helix so that the transcription of the Caltractin-like gene is reduced.
  • a Caltractin-like gene for example a Caltractin-like promoter and/or enhancer
  • Such methods have been described (Helene C (1991) Anticancer Drug Res 6(6):569-84; Helene C et al. (1992) Ann NY Acad Sci 660:27-36; Maher LJ (1992) Bioassays 14(12) :807- 815) .
  • the antisense nucleic acid molecule can be an ⁇ -anomeric nucleic acid.
  • Such ⁇ -anomeric nucleic acid molecules form specific double-stranded hybrids with complemen- tary RNA in which - as opposed to the conventional ⁇ -nucleic acids - the two strands run parallel to one another (Gautier C et al. (1987) Nucleic Acids Res 15:6625-6641).
  • the antisense nucleic acid molecule can furthermore also comprise 2'-0- methylribonucleotides (Inoue et al . (1987) Nucleic Acids Res 15:6131-6148) or chimeric RNA/DNA analogs (Inoue et al. (1987) FEBS Lett 215:327-330).
  • the above-described antisense strategy can be combined advanta- geously with a ribozyme method.
  • Catalytic RNA molecules or ribozymes can be adapted to suit any target RNA and cleave the phosphodiester backbone at specific positions, functionally deactivating the target RNA (Tanner NK (1999) FEMS Microbiol Rev 23 (3) :257-275) .
  • the ribozyme itself is not modified thereby, but is capable of cleaving further target RNA molecules analogously, thereby assuming the qualities of an enzyme.
  • ribozyme sequences into antisense RNAs confers this enzyme- like RNA-cleaving quality to precisely these antisense RNAs, thus increasing their efficacy in inactivating the target RNA.
  • the generation and the use of such ribozyme antisense RNA molecules is described, for example, in Haseloff et al. (1988) Nature 334: 585-591.
  • ribozymes for example "hammerhead” ribozymes; Haselhoff and Gerlach (1988) Nature 334:585-591
  • the ribozyme technique can increase the efficacy of an antisense strategy.
  • ribozymes can also be identified from a library of diverse ribozymes via a selection process (Bartel D and Szostak JW (1993 ) Science 261 : 1411- 1418 ) .
  • an- Caltractin-like nucleic acid sequence in sense orientation can lead to cosuppression of the corresponding homologous endogenous gene.
  • the expression of sense RNA with homology with an endogenous gene can reduce or switch off the ex- pression of the former, similarly to what has been described for antisense approaches (Jorgensen et al . (1996) Plant Mol Biol 31(5) :957-973; Goring et al . (1991) Proc Natl Acad Sci USA 88:1770-1774; Smith et al . (1990) Mol Gen Genet 224:447-481; Na- poli et al. (1990) Plant Cell 2:279-289; Van der Krol et al .
  • Caltractin-like gene expression may also be reduced using specific DNA-binding factors, for example factors of the zinc finger transcription factor type. These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory regions, and bring about repression of the endogenous gene. The use of such a method makes possible the decrease of the expression of an endogenous Caltractin-like gene without it being necessary to recombinantly manipulate its sequence. Suitable methods for the preparation of suitable factors have been described (Dreier B et al. (2001) J Biol Chem 276 (31) :29466-78; Dreier B et al. (2000) J Mol Biol 303(4) :489-502; Beerli RR et al.
  • Caltractin-like gene This segment is preferably located in the promoter region. For gene suppression, however, it may also be in the region of the coding exons or introns.
  • the segments in question can be obtained by the skilled worker from GeneBank by database search or, starting from an Caltractin-like cDNA whose gene is not present in GeneBank, by screening a genomic library for corresponding genomic clones. The skilled worker is familiar with the methods required therefore.
  • the protein-binding factors can be, for example, aptamers (Famulok M and Mayer G (1999) Curr Top Microbiol Immunol 243:123-36) or antibodies or antibody fragments or single-chain antibodies. Methods for obtaining these factors have been described and are known to the skilled worker.
  • a cytoplasmic scFv antibody was employed to modulate the activity of the phytochrome A protein in genetically modified tobacco plants (Owen M et al . (1992) Biotechnology (N Y) 10 (7) :790-794 ; Franken E et al . (1997) Curr Opin Biotechnol 8 (4) :411-416; Whitelam (1996) Trend Plant Sci 1:286-272).
  • Gene expression may also be suppressed by tailor-made low- molecular-weight synthetic compounds, for example of the polyam- ide type (Dervan PB and B ⁇ rli RW (1999) Current Opinion in Chemical Biology 3:688-693; Gottesfeld JM et al . (2000) Gene Expr 9 (1-2) : 77-91) .
  • These oligomers are composed of the units 3- (dimethylamino)propylamine, N-methyl-3-hydroxypyrrole, N- methylimidazole and N-methylpyrrole and can be adapted to any piece of double-stranded DNA in such a way that they bind into the major groove in a sequence-specific manner and block the expression of these gene sequences.
  • Caltractin-like expression can also be brought about efficiently by inducing the specific Caltractin-like RNA degradation by the plant with the aid of a viral expression system (amplicon) (An- gell, SM et al. (1999) Plant J. 20 (3) :357-362) .
  • amplicon a viral expression system
  • VGS viral induced gene silencing
  • An example of what is used for generating a homologously recombinant organism with reduced Caltractin-like activity is a nucleic acid construct comprising at least part of an endogenous Caltractin-like gene which is modified by a deletion, addition or substitution of at least one nucleotide in such a way that its functionality is reduced or fully destroyed.
  • the modification may also relate to the regulatory elements (for example the promoter) of the gene, so that the coding sequence remains un- modified, but expression (transcription and/or translation) does not take place or is- reduced.
  • the modified region is flanked at its 5' and 3' end by further nucleic acid sequences which must be sufficient in length for making possible recombination. They are, as a rule, in the range of several hundred bases to several kilobases in length (Thomas KR and Capecchi MR (1987) Cell 51:5*03; Strepp et al . (1998) Proc Natl Acad Sci USA 95(8) :4368-4373) .
  • the host organism - for example a plant - is transformed with the recombination construct using the methods described hereinbelow, and clones which' have successfully undergone recombination are selected, for example using a resistance to antibiotics or herbicides.
  • Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations into the host genome predominate .
  • One possibility of eliminating the randomly integrated sequences and thus increasing the number of cell clones with a correct homologous recombination is the use of a sequence-specific recombination system as described in US 6,110,736, by which unspecifically integrated sequences can be deleted again, which simplifies the selection of events which have integrated successfully via homologous recombination.
  • a large number of sequence-specific recombination systems can be used, examples being the Cre/lox system of bacteriophage PI, the FLP/FRT system of yeast, the Gin recombinase of phage Mu, the Pin recombinase from E.coli, and the R/RS system of the pSRl plasmid.
  • the bacteriophage PI Cre/lox and the yeast FLP/FRT system are preferred.
  • the FLP/FRT and cre/lox recombinase system has already been applied to plant systems (Odell et al . (1990) Mol Gen Genet 223: 369-378).
  • Point mutations can also be generated by means of DNA-RNA hybrids also known under the name "chimeraplasty” (Cole-Strauss et al. (1999) Nucl Acids Res 27 (5) : 1323-1330; Kmiec (1999) Gene therapy American Scientist 87 (3) :240-247) .
  • mutations may in induced by transposon mediated mutagenisis (Sundaresan V et al. (1995) Genes Dev. 9(14):1797- 810; Parinov S et al . (1999) Plant Cell 11 (12) :2263-70) .An addi- tional method for inducing mutations in endogenous gene may utilize "Targeting Induced Local Lesions IN Genomes” (TILLING) (McCallum CM (2000) Plant Physiology 123:439-442).
  • PTGS post- transcriptional gene silencing
  • caltractin-like protein e.g., by decreasing activity and/or expression of interaction partners of the caltractin-like protein or other proteins which participate in the same signal transduction pathway than the caltractin-like protein.
  • interaction partners of the caltractin-like protein or other proteins which participate in the same signal transduction pathway than the caltractin-like protein Such proteins may be identified using methods known to the person skilled in the art to elucidate signal transduction pathways like., e.g. interaction* partner network analysis utilizing yeast-two-hybrid or yeast-n-hybrid systems.
  • anti-Caltractin compounds which directly or indirectly bring about a decrease in protein quantity, RNA quantity, gene activity or protein activity of an Caltractin-like protein shall subsequently be combined in the term "anti-Caltractin” compounds.
  • anti-Caltractin compound explicitly includes the nucleic acid sequences, peptides, proteins or other factors employed in the above-described methods.
  • introduction comprises all of the methods which are capable of directly or indirectly introducing an "anti-Caltractin” compound into a plant or a cell, compartment, tissue, organ or seed thereof, or of generating such a compound there .
  • Direct and indirect methods are encompassed.
  • the introduction can lead to a transient presence of an "anti-Caltractin” compound (for example a dsRNA) or else to its stable presence .
  • the "anti-Caltractin” compound can exert its function directly (for example by insertion into an endogenous Caltractin-like gene) .
  • its function can also be exerted indirectly fol- lowing transcription into an RNA (for example in the case of antisense approaches) or following transcription and translation into a protein (for example in the case of binding factors) .
  • the invention encompasses both directly and indirectly acting "anti- Caltractin” compounds .
  • introducing encompasses for example methods such as transfection, transduction or transformation.
  • Anti-Caltractin compounds therefore also encompasses recombi- nant expression constructs which bring about expression (i.e. transcription and, if appropriate, translation) , for example of an Caltractin-like dsRNA or an Caltractin-like "antisense” RNA, preferably in a plant or a part, tissue, organ or seed thereof.
  • the expression construct of the invention comprise at least one nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33;
  • nucleic acid molecule comprising a fragment of at least 20 consecutive nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33;
  • nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least 60% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34;
  • nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, wherein the fragment comprises at least 10 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
  • the DNA-construct utilized to introduce mutations into the chro- mosomal DNA of a plant cell or a plant organism may be of various structural configurations. It may have - for example - a linear, linearized or circular structure. It may be double- stranded or single-stranded (like e.g., T-DNA at a certain point of its transfer) .
  • the DNA-construct is introduced into the cell in form of a linearized double-stranded DNA or in form of a T-DNA.
  • the DNA-construct may be introduced into the plant cells in form of a transposon (comprising the mutation-inducing sequence) .
  • plants are selected in which the transposon had integrated close to the target se- quence .
  • a transposase e.g., introduced by transient expression or crossing
  • the DNA-construct becomes available again for homologous recombination.
  • a homology sequence comprised in a DNA-construct is to be understood to comprise sequences of a length of at least 100 base pair, preferably at least 250 base pair, more preferably at least 500 base pair, especially preferably at least 1000 base pair, most preferably at least 2500 base pair.
  • the term "sufficient homology" with respect to a homology se- quence comprised in a DNA-construct is to be understood to comprise sequences having a homology to the corresponding target sequence comprised in the chromosomal DNA (e.g., the target sequence A' or B' ) of at least 70 %, preferably at least 80 %, more preferably at least 90 %, especially preferably at least 95 %, more especially preferably at least 99 %, most preferably 100 %, wherein said homology extends over a length of at least 50 base pair, preferably at least 100 base pair, more preferably at least 250 base pair, most preferably at least 500 base pair.
  • the DNA-constructs utilized within the method of this invention may comprise additional nucleic acid sequences.
  • Said sequences may be - for example - localized in different positions with respect to the homology sequences.
  • the additional nu- cleic acid sequences are localized between two homology sequences and may be introduced via homologous recombination into the chromosomal DNA,_thereby resembling an insertion mutation of said chromosomal DNA.
  • the additional sequences may also be localized outside of the homology sequences (e.g., at the 5'- or 3 '-end of the DNA-construct) .
  • said sequences are expression cassettes, which may - for example - facilitate expression of selection markers, trait genes, or antisense RNA.
  • said expression cassettes comprise a promoter sequence functional in plant cells opera- tively linked to a nucleic acid sequence which - upon expression - confers an advantageous phenotype to the so transformed plant .
  • the person skilled in the art is aware of numerous sequences which may be utilized in this context, e.g. to increase quality of food and feed or to produce chemicals, fine chemicals or pharmaceuticals (e.g., vitamins, oils, carbohydrates) (Dunwell JM (2000) J Exp Bot 51 Spec No:487-96) .
  • growth. yield, and resistance against abiotic and biotic stress factors may be enhanced.
  • Advantageous properties may be conferred either by overexpressing proteins or by decreasing expression of endogenous proteins by e.g., expressing a corresponding antisense or double-stranded RNA.
  • efficiency of the method of the invention may be further increased by combination with other methods suitable for increasing homologous recombina- tion.
  • Said methods may include for example expression of HR- enhancing proteins (like e.g., RecA; WO 97/08331; Reiss B et al . (1996) Proc Natl Acad Sci USA 93(7) :3094-3098; Reiss B et al . (2000) Proc Natl Acad Sci USA 97(7) :3358-3363) or treatment with PARP inhibitors (Puchta H et al. (1995) Plant J. 7:203-210) .
  • HR- enhancing proteins like e.g., RecA; WO 97/08331; Reiss B et al . (1996) Proc Natl Acad Sci USA 93(7) :3094-3098; Reiss B et al . (2000) Proc Natl Acad Sci USA 97(7) :3358-3363
  • PARP inhibitors
  • PARP inhibitors suitable for use within this invention are known to the person skilled in the art and may include for example preferably 3-Aminobenzamid, 8-Hydroxy-2- methylquinazolin-4-on (NU1025) , 1, llb-Dihydro- [2H]benzopyrano[4,3,2-de] isoquinolin-3-on (GPI 6150), 5- Aminoisoquinolinon, 3 , 4-Dihydro-5- [4- (1-piperidinyl) butoxy] - 1 (2H) -isoquinolinon or compounds described in WO 00/26192, WO 00/29384, WO 00/32579, WO 00/64878, WO 00/68206, WO 00/67734, WO 01/23386 or WO 01/23390.
  • the method may be combined with other methods facilitation homologous recombination and/or selection of the recombinants like e.g., positive/negative selection, excision of illegitimate recombination events or induction of sequence-specific or unspecific DNA double-strand breaks .
  • a nucleic acid molecule whose expression (transcription and, if appropriate, translation) generates an "anti-Caltractin” compound is preferably operably linked to at least one genetic control element (for example a promoter) which ensures expression in an organism, preferably in plants.
  • a genetic control element for example a promoter
  • the expression construct is to be introduced directly into a plant and the "anti-Caltractin" compound (for example the Caltractin-like dsRNA) is to be generated therein in planta, plant- specific genetic control elements (for example promoters) are preferred.
  • the "anti-Caltractin” compound may also be generated in other organisms or in vitro and then be introduced into the plant (as described in Examples 6 and 7) .
  • Preferred in this context are all of the prokaryotic or eukaryotic genetic control elements (for example promoters) which permit the expression in the organism chosen in each case for the preparation.
  • Operable linkage is to be understood as meaning, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example an "anti-Caltractin" compound) and, if appropriate, further regulatory elements such as, for example, a terminator in such a way that each of the regula- tory elements can fulfill its function when the nucleic acid sequence is expressed recombinantly, depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA.
  • direct linkage in the chemical sense is not necessarily required.
  • Genetic control sequences such as, for ex- ample, enhancer sequences, can also exert their function on the target sequence from-positions which are further away, or indeed from other DNA molecules .
  • Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
  • Operable linkage, and an expression cassette can be generated by means of customary recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Ausubel FM et al . (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al . (1990) In: Plant Molecular Biology Manual.
  • sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences.
  • the insertion of sequences may also lead to the expression of fusion proteins.
  • the expression cassette consisting of a linkage of promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a plant genome, for example by transformation.
  • expression cassette also denotes those constructions in which a promoter is positioned behind an endogenous Caltractin- like gene, for example by means of homologous recombination, and the decrease according to the invention of an Caltractin-like protein is brought about by the expression of an antisense Caltractin-like RNA.
  • an "anti-Caltractin" compound for example a nucleic acid sequence encoding an Caltractin-like dsRNA or an Caltractin-like antisense RNA
  • an "anti-Caltractin" compound can be positioned behind an endogenous promoter in such a way that the same effect is manifested. Both approaches lead to inventive expression cassettes.
  • plant-specific promoters is understood as meaning, in principle, any promoter which is capable of governing the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues or plant cultures.
  • expression can be, for example, constitutive, in- ducible or development-dependent.
  • Constant promoters refers to those promoters which ensure expression in a large number of, preferably all, tissues over a substantial period of plant' development, preferably at all times during plant development (Benfey et al.(1989) EMBO J 8:2195- 2202) .
  • a plant promoter or promoter originating from a plant virus is especially preferably used.
  • the promoter of the CaMV (cauliflower mosaic virus) 35S transcript (Franck et al. (1980) Cell 21:285-294; Odell et al . (1985) Nature 313:810-812; Shew aker et al. (1985) Virology 140:281-288; Gardner et al. (1986) Plant Mol Biol 6:221- 228) or the 19S CaMV promoter (US
  • Another suitable constitutive promoter is the Rubisco small subunit (SSU) promoter (US 4,962,028), the leguminB promoter (GenBank Ace. No. X03677) , the promoter of the nopalin synthase from Agrobacterium, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649) , the ubiquitin 1 promoter (Christensen et al .
  • SSU Rubisco small subunit
  • promoters with specificities for seeds such as, for example, the phasedin promoter (US 5,504,200; Bustos MM et al . (1989) Plant Cell 1 (9) : 839-53) , the promoter of the 2S albumin gene (Joseffson LG et al . (1987) J Biol Chem 262:12196- 12201), the legumine promoter (Shirsat A et al. (1989) Mol Gen Genet 215 (2) : 326-331) , the USP (unknown seed protein) promoter (Baumlein H et al.
  • seed-specific promoters are those of the gene encoding high-molecular weight glutenin (HMWG) , gliadin, branching enzyme, ADP glucose pyrophosphatase (AGPase) or starch synthase . Promoters which are furthermore preferred are those which permit a seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like.
  • HMWG high-molecular weight glutenin
  • AGPase ADP glucose pyrophosphatase
  • starch synthase starch synthase
  • the promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzin gene, the prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the easirin gene or the secalin gene) can advantageously be employed.
  • the expression cassettes may also contain a chemically inducible promoter (review article: Gatz et al . (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108), by means of which the expression of the exogenous gene in the plant can be controlled at a particular point in time.
  • a chemically inducible promoter such as, for example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfona ide-inducible promoter (EP 0 388 186) , a tetracyclin-inducible promoter (Gatz et al.
  • an abscisic acid-inducible promoter EP 0 335 528) or an ethanol-cyclohexanone-inducible promoter can likewise be used.
  • an abscisic acid-inducible promoter EP 0 335 528) or an ethanol-cyclohexanone-inducible promoter can likewise be used.
  • the promoter of the glutathione-S transferase isoform II gene (GST-II-27) , which can be activated by exogenously applied safeners such as, for example, N,N-diallyl-2,2-dichloroacetamide (W0 93/01294) and which is operable in a large number of tissues of both monocots and dicots .
  • constitutive promoters are particularly preferred.
  • promoters may be linked operably to the nucleic acid sequence to be expressed, which promoters make possi- ble the expression in further plant tissues or in other organisms, such as, for example, E. coli bacteria.
  • Suitable plant promoters are, in principle, all of the above-described promoters .
  • nucleic acid sequences present in the expression cassettes or vectors according to the invention can be linked operably to further genetic control sequences in addition to a promoter.
  • genetic control sequences is to be understood in the broad sense and refers to all those sequences which have an ef- feet on the materialization or the function of the expression cassette according to the invention.
  • genetic control sequences modify the transcription and translation in pro- karyotic or eukaryotic organisms.
  • the expression cassettes according to the invention encompass a promoter func- tional in plants r 5 '-upstream of the nucleic acid sequence in question to be expressed recombinantly, and 3' -downstream a terminator sequence as additional genetic control sequence and, if appropriate, further customary Regulatory elements, in each case linked operably to the nucleic acid sequence to be expressed recombinantly.
  • Genetic control sequences also encompass further promoters, promoter elements or minimal promoters, all of which can modify the expression-governing properties.
  • tissue-specific expression may additionally depend on certain stress- ors, owing to genetic control sequences.
  • Such elements have been described, for example, for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266(26): 17131 -17135) and heat stress (Schoffl F et al . , Molecular & General Genetics 217(2- 3) :246-53, 1989) .
  • control sequences are, for example, the Gram-positive promoters amy and SP02, and the yeast or fungal promoters ADC1, MFa , AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
  • Genetic control sequences furthermore also encompass the 5'- untranslated regions, introns or noncoding 3' -region of genes, such as, for example, the actin-1 intron, or the Adhl-S introns 1, 2 and 6 (general reference: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)) . It has been demonstrated that they may play a significant role in the regulation of gene expression. Thus, it has been demonstrated that 5 ' -untranslated sequences can enhance the transient expression of heterologous genes. Examples of translation enhancers which may be mentioned are the tobacco mosaic virus 5' leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like. Furthermore, they may promote tissue specificity (Rouster J et al . (1998) Plant J 15:435-440).
  • the expression cassette may advantageously comprise one or more of what are known as enhancer sequences, linked operably to the promoter, which make possible an increased recombinant expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, may also be inserted at the 3 ' end of the " nucleic acid sequences to be expressed recombinantly. One or more copies of the nucleic acid sequences to be " expressed recombinantly may be present in the gene construct .
  • Polyadenylation signals which are suitable as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of the T-DNA (octopin synthase) of the Ti plasmid pTiACHS (Gielen et al . (1984) EMBO J 3:835 et seq.) or functional equivalents thereof.
  • terminator sequences which are especially suitable are the OCS (octopin synthase) terminator and the NOS (nopalin synthase) terminator.
  • Control sequences are furthermore to be understood as those which make possible homologous recombination or insertion into the genome of a host organism or which permit removal from the genome.
  • homologous recombination for example the natural promoter of a particular- gene may be exchanged for a promoter functional in plants .
  • Methods such as the cre/lox technology permit a tissue-specific, if appropriate inducible, removal of the expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14 (4) : 381-92) .
  • specific flanking sequences (lox sequences) , which later allow removal by means of ere recombinase, are attached to the target gene.
  • an expression cassette and the vectors derived from it may com- prise further functional elements.
  • the term functional element is to be understood in the broad sense and refers to all those elements which have an effect on the generation, amplification or function of the expression cassettes, vectors or transgenic organisms according to the invention.
  • the following may be men- tioned by way of example, but not by limitation:
  • Selection marker are useful to select and separate successfully transformed or homologous recombined cells.
  • Selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or gly- phosate) .
  • a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or gly- phosate) .
  • a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin,
  • Phosphinothricin acetyltransferases also named Bialophos ® resistance; bar; de Block et al . (1987) EMBO J 6: 2513-2518
  • EPSPS 5-enolpyruvylshikimate-3 -phosphate synthase
  • Glyphosate ® degrading enzymes (Glyphosate ® oxidoreductase; gox)
  • acetolactate syn- thases for example mutated ALS variants with, for example, the S4 and/or Hra mutation
  • NPTII G418- resistence genes coding e.g., for neomycin phosphotransferases
  • aadA gene which confers resistance to the antibiotic spectinomycin
  • SPT streptomycin phosphotransferase
  • HPT hygromycin phosphotransferase
  • Negative selection marker are especially suitable to select organisms with defined deleted sequences comprising said marker (Koprek T et al . (1999) Plant J 19(6) : 719-726).
  • Examples for negative selection marker comprise thy idin kinases (TK) , cyto- sine deaminases (Gleave AP et al . (1999) Plant Mol Biol.
  • Reporter genes encode readily quantifiable proteins and, via their color or enzyme activity, make possible an assessment of the transformation efficacy, the site of expression or the time of expression.
  • genes encoding reporter proteins such as the green fluorescent protein (GFP) (Sheen et al.(1995) Plant Journal 8 (5) : 777-784 , Haseloff et al. (1997) Proc Natl Acad Sci USA 94(6) :2122-2127; Reichel et al.(1996) Proc Natl Acad Sci USA 93 (12) :5888-5893 ,
  • ⁇ -galactosidase encoding a protein which regulates the production of anthocyanin pigments (red coloring) in plant tissue and thus makes possible the direct analysis of the promoter activity without addition of further auxiliary substances or chromogenic substrates; Dellaporta et al . , In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, 11:263-282, 1988), with ⁇ - glucuronidase being very especially preferred (Jefferson et al., EMBO J. 1987, 6, 3901-3907) .
  • Origins of replication which ensure amplification of the expression cassettes or vectors according to the invention in, for example, E. coli.
  • Examples which may be mentioned are ORI (origin of DNA replication) , the pBR322 ori or the P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .
  • a selectable marker which confers resistance to a biocide (for example herbicide) , a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic to the cells which have successfully undergone recombination.
  • a biocide for example herbicide
  • a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic to the cells which have successfully undergone recombination.
  • the selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Reports 5:81-84).
  • an expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably into plants or plant cells, tissue, organs, parts or seeds) can be effected advantageously using vectors which comprise the expression cassettes.
  • the expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction cleavage site.
  • the plasmid formed is first introduced into E. coli. Correctly transformed E. coli are selected, grown, and the recombinant plasmid is obtained by the methods familiar to the skilled worker. Restriction analysis and sequencing may serve to verify the cloning
  • vectors may be plasmids, cosmids, phages, viruses or else agrobacteria.
  • the expression cassette is introduced by means of plasmid vectors.
  • Preferred vectors are those which make possible stable integration of the expression cassette into the host genome.
  • the invention furthermore relates to transgenic plant organisms or tissues, organs, parts, cells or propagation material thereof which comprise a transgenic expression cassette for "anti- Caltractin" compound or a transgenic vector encompassing such an expression cassette.
  • Such a transgenic plant organism is generated, for example, by means of transformation or transfection by means of the corresponding proteins or nucleic acids .
  • the generation of a transformed organism requires introducing the DNA in question (for example the expression vector) , RNA or protein into the host cell in question.
  • transformation or transduction or transfection
  • the DNA or RNA can be introduced directly by microinjec- tion or by bombardment with DNA-coated microparticles.
  • the cell can be permeabilized chemically, for example using polyeth- ylene glycol, so that DNA can enter the cell by diffusion.
  • the DNA can also be introduced by protoplast fusion with other DNA- containing units such as minicells, cells, lysosomes or liposomes.
  • Another suitable method of introducing DNA is electroporation, where the cells are permeabilized reversibly by an elec- trical pulse.
  • Suitable methods have been described (for example by Bilang et al. (1991) Gene 100:247-250; Scheid et al . (1991) Mol Gen Genet 228:104-112; Guerche et al . (1987) Plant Science 52:111-116 ' ; Neuhause et al . (1987) Theor Appl Genet 75:30-36; Klein et al . (1987) Nature 327:70-73; Howell et al .
  • Suitable methods are especially protoplast transformation by polyethylene-glycol- induced DNA uptake, the biolistic method with the gene gun, what is known as the particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution, and microinjection. injection.
  • transformation can also be effected by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Agrobacterium-mediated transformation is best suited to dicotyledonous plant cells. The methods are described, for example, by Horsch RB et al . (1985) Science 225: 1229f.
  • the expression cassette is integrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector. If a Ti or Ri plasmid is to be used for the transformation, at least the right border, but in most cases the right and left border, of the Ti or Ri plasmid T-DNA is linked to the expression cassette to be introduced in the form of a flanking region.
  • Binary vectors are preferably used.
  • Binary vectors are capable of replication both in E. coli and in Agrobacterium.
  • they comprise a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border sequence. They can be transferred directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187) .
  • the selection marker gene permits the selection of transformed Agrobacteria and is, for example, the nptll gene, which confers resistance to kanamycin.
  • the Agrobacterium which acts as host organism in this case should already contain a plasmid with the vir region. The latter is required for transferring the T-DNA to the plant cell.
  • An Agrobacterium transformed in this way can be used for transforming plant cells .
  • the use of T-DNA for transforming plant cells has been studied and described intensively (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al . (1985) EMBO J 4:277-287).
  • Various binary vectors are known, some of which are commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA) .
  • the plasmid used does not need to meet any particular additional requirements in the case of the injection or electroporation of DNA or RNA into plant cells. Simple plasmids such as those of the pUC series can be used. If complete plants are to be regenerated from the transformed cells, it is necessary for an additional selectable marker gene to be located on the plasmid.
  • Stably transformed cells i.e. those which contain the introduced DNA integrated into the DNA of the host cell, can be selected from untransformed cells when a selectable marker is part of the DNA introduced.
  • genes which can act as mark- ers are all those which are capable of conferring resistance to antibiotics or herbicides are given above .
  • Transformed cells which express such marker genes are capable of surviving in the presence of concentrations of a corresponding antibiotic or herbicide which kill an untransformed wild type.
  • the resulting plants can be bred and hybridized in the customary fashion. Two or more generations -should be grown in order to ensure that the genomic integration is stable and hereditary.
  • a complete plant can be obtained using methods known to the skilled worker. For example, callus cultures are used as starting mate- rial. The development of shoot and root can be induced in this as yet undifferentiated cell biomass in a known fashion. The shoots obtained can be planted out and bred.
  • Transgenic for example regarding a nucleic acid sequence, an expression cassette or a vector comprising said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression cassette or vector, refers to all those constructs originating by recombinant methods in which either
  • nucleic acid sequence coding for at least part of a Cal- tracin-like protein, antisense RNA, or double stranded RNA, or
  • nucleic acid sequence a) for example a promoter, or
  • Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, very especially preferably at least 5000 bp, in length.
  • non-natural, synthetic "artificial" meth- ods such as, for example, muta.genization.
  • the invention also relates to transgenic organisms transformed with at least one of the nucleic acid sequences according to the invention, expression cassette according to the invention or vector according to the invention, and to cells, cell cultures, tissues, parts - such as, for example, leaves, roots and the like in the case of plant organisms - or propagation material derived from such organisms.
  • organism is to be understood in the broad sense and refers to prokaryotic and eu- karyotic organisms, preferably bacteria, yeasts, fungi, non- human animal organisms and plant organisms. Preferred plant organisms are indicated above.
  • Host or starting organisms which are preferred as transgenic organisms are mainly plants in accordance with the above defini- tion. Included within the scope of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Furthermore included are the mature plants, seed, shoots and seedlings, and parts, propagation material and cultures derived therefrom, for example cell cultures. Mature plants refers to plants at any developmental stage beyond that of the seedling. The term seedling refers to a young immature plant in an early developmental stage.
  • the transgenic organisms can be generated with the above- described methods for the transformation or transfection of organisms .
  • the invention furthermore relates to the use of the transgenic organisms according to the invention and of the cells, cell cul- tures, parts - such as, for example, roots, leaves and the like in the case of transgenic plant organisms - derived from them, and to transgenic propagation material such as seeds or fruits, for the production of foodstuffs or feeding stuffs, pharmaceuticals or fine chemicals.
  • a method for the recombinant production of pharmaceuticals or fine chemicals in host organisms wherein a host organism is transformed with one of the above-described expression cassettes and this expression cassette comprises one or more structural genes which encode the desired fine chemical or catalyze the biosynthesis of the desired fine chemical, the transformed host organism is cultured, and the desired fine chemical is isolated from the culture medium.
  • This method can be applied widely to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, and natural and synthetic flavorings, aroma substances and colorants.
  • tocopherols and tocotrienols and carotenoids are especially preferred.
  • the transformed host organisms are cultured and the products are isolated from the host organisms or the culture medium by methods known to the skilled worker.
  • Another embodiment of the invention is directed to a process for facilitating plant breeding utilizing a plant organism having a decreased activity or expression of at least one caltractin-like protein.
  • the plant of the invention having said decreased activity or expression of at least one caltractin-like protein exhibits an enhanced rate of meiotic recombination.
  • This property may, for example, be utilized to facilitate breeding programs by e.g., allowing faster segregation of advantageous and disadvantageous traits. For example, in case particular traits are to be crossed into an elite line and subsequently the unwanted traits of the non-elite plant (e.g., a wild relative) have to be crossed out, this goal can be faster achieved utilizing the plants of the invention.
  • SEQ ID NO: 1 Nucleic acid sequence encoding Caltractinlike protein from Arabidopsis thalina
  • SEQ ID NO: 2 Amino acid sequence encoding Caltractinlike protein from Arabidopsis thalina
  • SEQ ID NO: 3 Nucleic acid sequence encoding Caltractin- " like protein from Arabidopsis thalina
  • SEQ ID NO : 4 Amino acid sequence encoding Caltractinlike protein from Arabidopsis thalina
  • SEQ ID NO: 5 Nucleic acid sequence encoding Caltractinlike protein from Brassica napus (partial)
  • SEQ ID NO: 7 Nucleic acid sequence encoding Caltractinlike protein from barley (Hordeum vulgare)
  • SEQ ID NO: 8 Amino acid sequence encoding Caltractin- like protein from barley (Hordeum vulgare)
  • SEQ ID NO: 9 Nucleic acid sequence encoding Caltractinlike protein from rice (Oryza sativa; partial)
  • SEQ ID NO: 10 Amino acid sequence encoding Caltractinlike protein from rice (Oryza sativa,- partial)
  • SEQ ID NO: 11 Nucleic acid sequence encoding Caltractinlike protein from rice (Oryza sativa,- partial)
  • SEQ ID NO: 12 Amino acid sequence encoding Caltractin- like protein from rice (Oryza sativa; partial) 13. SEQ ID NO: 13 Nucleic acid sequence encoding Caltractinlike protein from wheat (Triticum aestivum)
  • SEQ ID NO: 14 Amino acid sequence encoding Caltractin- like protein from wheat (Triticum aestivum)
  • SEQ ID NO: 15 JSTucleic acid sequence encoding Caltractinlike protein from wheat (Triticum aestivum; partial)
  • SEQ ID NO: 16 Amino acid sequence encoding Caltractinlike protein from wheat (Triticum aestivum; partial)
  • SEQ ID NO: 17 Nucleic acid sequence encoding Caltractinlike protein from linseed (partial)
  • SEQ ID NO: 18 Amino acid sequence encoding Caltractinlike protein from linseed (partial)
  • SEQ ID NO: 19 Nucleic acid sequence encoding Caltractinlike protein from corn (Zea mays)
  • SEQ ID NO: 20 Amino acid sequence encoding Caltractin- like protein from corn (Zea mays)
  • SEQ ID NO: 21 Nucleic acid sequence encoding Caltractinlike protein from sunflower (partial)
  • SEQ ID NO: 22 Amino acid sequence encoding Caltractinlike protein from sunflower (partial)
  • SEQ ID NO: 23 Nucleic acid sequence encoding Caltractinlike protein from Atriplex nummularia
  • SEQ ID NO: 24 Amino acid sequence encoding Caltractinlike protein from Atriplex nummularia
  • SEQ ID NO: 25 Nucleic acid sequence encoding Caltractin- like protein from Marsilea vestita 26.
  • SEQ ID NO: 26 Amino acid "sequence encoding Caltractinlike protein from Marsilea vestita
  • SEQ ID NO: 27 Nucleic acid sequence encoding Caltractin- like protein from Soybean (Glycine max)
  • SEQ ID NO: 28 Amino acid sequence encoding Caltractinlike protein from Soybean (Glycine max)
  • SEQ ID NO: 29 Nucleic acid sequence encoding Caltractinlike protein from potato (Solanum tuberosum)
  • SEQ ID NO: 30 Amino acid sequence encoding Caltractin- like protein from potato (Solanum tuberosum)
  • SEQ ID NO: 31 Nucleic acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
  • SEQ ID NO: 32 Amino acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
  • SEQ ID NO: 33 Nucleic acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
  • SEQ ID NO: 34 Amino acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
  • Fig. 1 Homologous recombination frequencies (HRF; Fold change) in 3 different P 24 plants (24.4, 24.6, 24.10). HRF was measured in a batch of 2 week old T2 plants (>50 plants/replicate). The figure shows heterozygous T2 plants. Ho- mozygous plants exhibited the same fold change.
  • Fig. 2 Displays scheme of At4g37010 gene locus disrupted by T-DNA tagging (gray boxes: Exons of At4g37010 gene; white box: pSKI015 T-DNA; arrow indicated transcription direction from 35S enhancer) . Due to the orientation of the T-DNA, the four copies of the 35S enhancer present near the right border could drive the anti-sense expression of at least part of the At4g37010 gene .
  • Fig. 3 mRNA steady state level of centrin determined by RT-PCR in a batch of 2 week old T2 plants. Demonstrated is a significantly reduced expression of the At4g37010 derived mRNA in the heterozygous knockout plant in comparison to wild-type plants.
  • Fig. 4 Homologous recombination frequencies (fold change respect to the untransformed parental line) in different IC9 (Fig.4B) and IC6 (Fig. 4A) reporter lines (A,E,F, I, ,M,N,0, and 2,3,10,11,17,18 respectively) having intergrated the pOEXhpCEN construct ..
  • Fig. 5 Alignment of Caltractin-like proteins from various plant species.
  • 1 Arabidopsis 1 (Arabidopsis thaliana, Gene Locus At4g37010)
  • 2 Arabidopsis 2 (Arabidopsis thaliana, corresponding to GeneBank Ace. -No.: AJ009672; Gene Locus At3g50360)
  • 3 Brassica napus
  • 4 Marsilea vestitia (corresponding to GeneBank Ace. -No.: U92973)
  • 5 Atriplex nummularia (corresponding to GeneBank Ace.
  • oligonucleotides can be synthesized chemically in the known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897) .
  • the cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoreses, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, multiplication of phages and sequence analysis of recombinant DNA, are carried out as decribed by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules were sequenced using an ABI laser fluorescence DNA sequencer following the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74:5463-5467) .
  • the plant Arabidopsis thaliana belongs to the higher plants (flowering plants) . This plant is closely related to other plant species from the Cruciferae family such as, for example,
  • Arabidopsis thaliana can be employed as model plant for other plant species.
  • Binary vectors such as pBinAR can be used for the transformation of plants (H ⁇ fgen und Willmitzer (1990) Plant Science 66: 221- 230) .
  • the binary vectors can be constructed by ligating the cDNA into T-DNA in sense and antisense orientation. 5' of the cDNA, a plant promoter activates the transcription of the cDNA. A polyadenylation sequence is located 3' of the cDNA. Tissue-specific esqpression can be achieved using a tissue- specific promoter.
  • seed-specific expression can be achieved by cloning in the napin or the LeB4- or the USP promoter 5' of the cDNA. Any other seed-specific promoter element can also be used.
  • the CaMV 35S promoter can be used for constitutive expression in the whole plant.
  • Agrobacterium-mediated plant transformation can be carried out for example using the Agrobacterium tumefaciens strains GV3101 (pMP90) (Koncz und Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404 (Clontech) . Standard transformation techniques may be used for the transformation (Deblaere et al. (1984) Nucl Acids Res 13:4777-4788) .
  • Agrobacterium-mediated plant transformation can be effected using standard transformation and regeneration techniques (Gelvin SB, Schilperoort R, Plant Molecular Biology Manual, 2nd ed. , Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuch Universitye Signatur: BT11-P ISBN 0-7923-2731-4; Glick BR, Thompson JE, Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pp.', ISBN 0-8493-5164-2).
  • oilseed rape can be transformed by cotyledon or hypocotyl transformation (Moloney et al. (1989) Plant Cell Report 8:238-242; De Block et al.(1989) Plant Physiol 91: 694-701).
  • the use of antibiotics for the selection of agrobacteria and plants depends on the binary vector used for the transformation and the agrobacterial strain.
  • the selection of oilseed rape is usually carried out using kanamycin as selectable plant marker.
  • Agrobacterium-mediated gene transfer into linseed can be carried out for example using a technique described by Mlynarova et al. (1994) Plant Cell Report 13:282- 285. Soya can be transformed for example using a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, US 5,376,543, US 5,169,770 (University of Toledo) .
  • Example 5 Studying the expression of a recombinant gene product in a transformed organism
  • the activity of a recombinant gene product in the transformed host organism was measured at the transcription and/or translation level . ⁇
  • a suitable method for determining the level of transcription of the gene is to carry out a Northern blot as described hereinbelow (for reference see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York, or the above examples section) , where a primer which is designed such that it binds to the gene of interest is labeled with a detectable label (usually a radiolabel or chemiluminescent label) so that, when the total RNA of a culture of the organism is extracted . , separated on a gel, transferred to a stable matrix and incubated with this probe, binding and the extent of binding of the probe indicates the presence and the amount of mRNA for this gene.
  • a detectable label usually a radiolabel or chemiluminescent label
  • Cellular total RNA can be prepared from cells, tissues or organs using several methods, all of which are known in the art, for example the method B ⁇ rmann ER et al. (1992) Mol. Microbiol. 6:317-326.
  • RNA hybridization 20 ⁇ g of total RNA or 1 ⁇ g of pol (A) + RNA are separated by means of gel electrophoresis in 1.25% strength agarose gels using formaldehyde and following the method described by Amasino (1986, Anal. Biochem. 152, 304), transferred to positively charged nylon membranes (Hybond N+, Amersham, Brunswick) by capillary force using 10 x SSC, immobilized by UV light and prehybridized for 3 hours at 68°C using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl, 1 % SDS, 100 mg herring sperm DNA) .
  • the DNA probe is labeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization step, using alpha- 32 P-dCTP (Amersham Pharmacia, Brunswick, Germany) .
  • Hybridization is carried out overnight at 68°C after addition of the labeled DNA probe in the same buffer.
  • the wash steps are carried out twice for 15 minutes using 2 X SSC and twice for 30 minutes using 1 X SSC, 1% SDS, at 68°C.
  • the sealed filters are exposed at -70°C for a period of 1 to 14 days.
  • the Arabidopsis line IC9 homozygous for a single copy of the intermolecular recombination substrate GRU'S'G'U', was used as reporter in a genetic screen aimed at identifying plants with increased frequency of homologous recombination.
  • the plasmid pGRU'S'G'U' was constructed as follows : The 3 'end of the GUS gene (fragment US) was digested from pUS ( Tinland B (1994) Proc Natl Acad Sci USA 91(17) :8000-8004) using Xbal and Hindlll, blunted with Klenow and subsequently cloned into the Hindi site of pUC19.
  • the resulting plasmid, pU'S' was digested with Xbal and HindiII in order to excise the U'S' fragment, which was then cloned between the Xbal and HindiII sites of the plasmid pG'U' (Tinland et al . , 1994).
  • the construct obtained is the recombination substrate plasmid pGRU'S'G'U', which carries on its T-DNA two non-functional regions of the uidA (GUS) gene (namely U'S' and G'U') where U' represents 1213 bp of repeated identical sequence in direct orientation.
  • Example 7 Screening for hyper-recombination phenotype
  • the activation tagging plasmid pSKI015 ( ' Weigel D et al. (2000) Plant Physiol. 122:1003-1014), containing four repetitions of the 35S enhancer close to the right border, was mobilised into Agrobacterium and used to transform the homozygous IC9 line by the in planta transformation method. Plants were cultured in a growth chamber under a 16h/8h photoperiod (20°C/16°C) . Seeds from these plants were germinated on soil, and Basta-resistant plants, having integrated the pSKI015 plasmid, were selected. Three or four leaves from 3 week-old individual primary trans- formants (TI) were harvested and used for histochemical GUS as- say.
  • TI primary trans- formants
  • the number of GUS+ spots per plant representing the number of homologous recombination events per plant, was determined. Plants having at least 2 GUS+ sectors on independent leaves were considered putative mutants and analyzed further. These plants were selfed, and >30 plants from the T2 generation were analysed for homologous recombination frequency (HRF; defined as the average number of GUS+ spots in a population of plant siblings) . Experiments were at least duplicated. One of the plants ana- lyzed, called P24, showed a s'ignificant increase of HRF when compared to the IC9 parental line, and was therefore considered as a hyper-recombination mutant (Fig. 1) .
  • HRF homologous recombination frequency
  • Genomic DNA of TI P24 plants was prepared using the DNeasy extraction kit (Qiagen) , and the right border-genomic DNA junction was analysed by plasmid rescue (Weigel et al . 2000).
  • the T-DNA from pSKI015 was found to be integrated at the 3' end of the At4g37010 gene, disrupting the stop codon and the 3' UTR of this gene (Fig. 2) . Due to the orientation of the pSKI015 T-DNA, the four copies of the 35S enhancer present near the right border could drive the anti-sense expression of at least part of the At4g37010 gene.
  • the reverse transcrip- tion reaction was performed using using 3 ⁇ g of total RNA, oligo-dT primers and the Reverse transcription kit (Amersharm) .
  • the PCR reaction was performed in a final volume of 50 ⁇ l, containing l ⁇ l of the RT reaction mixture, 1.25U of ExTaq (Takara) , 1.5 mM MgCl 2 , 200 ⁇ M of each dNTP and 2 ⁇ M of gene-specific primers
  • CEN958 (SEQ ID NO: 35): 5' -CAAATAAACGAATTGATGGCAG-3 ' and CEN3' (SEQ ID NO: 36): 5 ' -CCATTACTAATTGATTTATACTTAGC-3 ' .
  • an At4g37010 RNAi construct that was created in the following way: the vector pPZP200 (Hajdukiewicz P et al. (1994) Plant Mol Biol. 25 (6) : 989-94) was digested with Ascl and Xbal in order to clone the nos terminator. The vector obtained was digested with EcoRI and Ascl in order to clone the selection marker cassette [mas promoter (1' -2' ) -sull gene-35S terminator].
  • a multi- cloning site (Ncol, BstXI, Hindlll, BsrGI, Avrll) was inserted.
  • the resulting plasmid was digested with Hindlll and Avrll, and the fad2 intron (Smith, N.A. et al . (2000) Nature 407:319-320), previously amplified by PCR in order to add the required re- striction sites, was cloned there.
  • the vector obtained was called pOEXhp, and the second exon of the At4g37010 gene (165 bp) was cloned into it between the Ncol and Xhol sites in sense orientation and between the BsrGI and Avrll sites in antisense orientation to generate the RNAi construct pOEXhpCEN.
  • the frag- ment for sense orientation was produced by PCR using the primers CENNcoF (5' -GGCCGGCCATGGCAGCAAATAAACGAATTGATGGCAG-3' ; SEQ ID NO: 37) and CENXhor (5' -GGCCGGCTAGAGATTATTGTCGTGGTCAATGATC-3 ' ; SEQ ID NO: 38) , which added to the resulting PCR fragment the restriction sites Ncol and Xhol respectively.
  • the fragment for antisense orientation was produced by PCR using the primers CENBsrGr (5' -GGCCGGTGTACAATTATTGTCGTGGTCAATGATC-3' ; SEQ ID NO: 39) and CENAvrf (5' -GGCCGGCCTAGGCAGCAAATAAACGAATT GATGGCAG-3' ,- SEQ ID NO: 40), which added to the resulting PCR fragment the restriction sites BsrGI and Avrll.
  • PCR reactions were performed in a final vlume of 50 ⁇ l, containing 100 ng of Arabidopsis genomic DNA, 1.25U of ExTaq (Takara) , 1.5 mM MgCl 2 , 200 ⁇ M of each dNTP and 2 ⁇ M of the required primers.
  • Initial denaturation was done at 95°C for 1 min, then a plifica- tion was done for 30 cycles with a denatura.tion time of 30 sec at 94°C, followed by annealing for 30 sec at 58°C and extension for 30 sec at 72°C.
  • the resulting pOEXhpCEN plasmid was mobilised into Agrobacterium and used to transform homozygous IC9 plants (carrying the intermolecular recombination substrate) .
  • HRF was measured in the T2 generation in a batch of >60 plants and found to be significantly increased (Fig. 4B) .
  • Fig. 4B The RNAi construct was also transformed into another reporter line (IC5 ; Fig. 4A) , which differs from IC9 only in the genome location of GRU'S'G'U'.
  • HRF was measured in 2 week old plants (at least 50 plants per replicate) and HRF was also found to be increased.
  • the steady state level of centrin mRNA in pOEXhpCEN plants was determined by RT-PCR as describe in the previous paragraph, an for both IC9 and IC6, the steady state level of the At4g37010 mRNA was significantly reduced.
  • the mutant P24 showing a hyper-recombinogenic phenotype, was isolated using a genetic screen based on the GRU'S'G'U' recombination substrate.
  • the hyper-recombinogenic phenotype is due to lower mRNA steady state level of the At4g37010 gene.
  • the At4g37010 gene product is similar to human centrin 2.

Landscapes

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

Abstract

The present invention relates to methods for increasing efficiency of homologous recombination in plants or plant cells, by decreasing expression or activity of at least one caltractinlike protein in said plant or plant cells. The method may be used for inserting sequences into a distinct locus of the chromosomal DNA. Furthermore the method may be used for deleting sequences from the chromosomal DNA flanked by homology sequences by initiating homologous recombination between said homology sequences. The invention furthermore relates to expression constructs suitable for-expressing at least part of a nucleic acid sequence encoding for a caltractin-like protein (e.g., in form of antisense or double-stranded RNA) suitable for decreasing expression or activity of at least one caltractin-­like protein in plant or plant cells. Object of the invention are furthermore transgenic plants comprising a decreased expression or activity of at least one caltractin-like protein and the use of said transgenic plants for the production of food, feeds, seed, pharmaceuticals or fine chemicals, in particular for the production of oils.

Description

Method for increasing efficiency of homologous recombination in plants
The present invention relates to methods for increasing effi- ciency of homologous recombination in plants or plant cells, by decreasing expression or activity of at least one caltractinlike protein in said plant or plant cells. The method may be used for inserting sequences into a distinct locus of the chromosomal DNA. Furthermore the method may be used for deleting se- quences from the chromosomal DNA flanked by homology sequences by initiating homologous recombination between said homology sequences. The invention furthermore relates to expression constructs suitable for expressing at least part of a nucleic acid sequence encoding for a caltractin-like protein (e.g., in form of antisense or double-stranded RNA) suitable for decreasing expression or activity of at least one caltractin-like protein in plant or plant cells. Object of the invention are furthermore transgenic plants comprising a decreased expression or activity of at least one caltractin-like protein and the use of said transgenic plants for the production of food, feeds, seed, pharmaceuticals or fine chemicals, in particular for the production of oils.
The aim of plant biotechnology work is the generation of plants with advantageous novel properties, for example for increasing agricultural productivity, increasing the quality in the case of foodstuffs, or for producing specific chemicals or pharmaceuticals.
Gene targeting technologies - achieving a directed integration into the chromosomal DNA of a host organism by homologous recombination - only have a satisfying efficiency in prokaryotes and yeast. The production of correspondingly modified eukaryotic organisms can be realised only for a very limited number of organ- isms (like e.g., mice) and only by means of high effort ( anaar R Hoeij akers JH (1997) Genes Funct 1 (3) .165-174) . The low efficiency of homologous recombination (HR) which is for mice in the range of about 1:1x10s can only be compensated by the use of work- and time-intensive selection techniques (like e.g., the embryonic stem "ES" cell technology) . For other species - especially for plants - corresponding equivalent routine techniques do not exist (Mengiste T & Paszkowski J (1999) Biol Chem. 380:749-758; Vergunst AC & Hooykaas PJJ (1999) Crit Rev Plant Sci 18:1-31; Puchta H (1999) Met ods Mol Biol 113:447-451; Hohn B & Puchta H (1999) Proc Natl Acad Sci USA 96:8321-8323) .
Attempts to achieve HR in plants lead ' in most of the cases to a random, non-homologous ("illegitimate") insertion into the host cell DNA (Roth DB & Wilson JH (1988) Illegitimate recombination in mammalian cells. In" "Genetic recombination", R. Kucherlapati and G.R. Smith Eds., American Society of Microbiology, Washington, USA; S.621-635; Puchta H und Hohn B (1996) Trends Plant Sci 1:340-348) . This is caused in plants by highly inefficient HR.
The reasons therefore are the object of ongoing research (review article: Mengiste T & Paszkowski J (1999) Biol Chem 380(7- 8) : 749-58) . In consequence, increasing HR in plants is considered a long-felt need in the field of plant biotechnology.
Exploratory approaches to increase HR in plants comprise expression of proteins like RecA (WO 97/08331) or RecA-homologues derived from other species like e.g., Rad52 (WO 01/68882) or RecA/VirE2 fusion-proteins (WO .01/38504) . Use of poly(ADP- ribose)polymerase inhibitors has demonstrated an increased HR in plants (Puchta H et al . (1995) Plant J 7:203- 210).
Initiation of sequence-unspecific DNA double-strand breaks was also found to increase efficiency of HR in plants (Puchta H et al. (1995) Plant J 7 (2) , 203-210 ; Lebel EG et al . (1993) Proc
Natl Acad Sci USA 90 (2) :422-6) . However, sequence-unspecific induction of DNA strand breaks is disadvantageous because of the potential mutagenic effect. Sequence-specific induction of DNA strand-breaks may also increase efficiency of HR but is limited to artificial scenarios (Siebert R, Puchta H (2002) Plant Cell 14 (5) : 1121-31) .
Selection of HR events from events of illegitimate recombination can be facilitated by certain combinations of positive and nega- tive selection techniques (WO 99/20780) .
Caltractin, also known as centrin (Cdc31p in Saccharomyces cere- visiae) , is a 20-kD calcium-binding (EF-hand) protein which was originally cloned at the DNA level from the unicellular green alga Chlamydomonas reinhardtii (Huang B et al. (1988) J Cell
Biol 107:133-140). It is a member of the calmodulin superfamily of calcium modulated proteins which include calmodulin, troponin C, parvalbumin, calbindins, and S.-100 protein (Kretsinger RH (1980) CRC Crit Rev Biochem 8:119-174). In algal cells, cal- tractin is most prominently localized to calcium-sensitive contractile fibers, the striated fiber root that connect the basal body complex to the underlying nucleus in interphase cells. It has been suggested that caltractin is a structural component of the cytoskeletal framework of fine filaments that compose these striated fibers (Schulze D et al. (1987) Eur J Cell Biol 45:51- 61) .
Caltractin is a protein found in numerous eukaryotic species and is localized in the centrosome (Lee VD et al. (1993) Proc Natl Acad Sci USA 90:11039-11043; Baum P et al . (1988) Mol Cell Biol 8:5386-5397; Levy YY et al . (1996) Cell Motil and Cytoskel 33:298-323) .
Within the plant kingdom caltractin/centrin-like proteins are described for Dunaliella salina (Ko JH and Lee SH (1996) Plant Physiol. 112: 445; GenBank Ace . -No . U53812) , Arabidopsis thaliana (Cordeiro MCR et al (1998) FEBS Lett 434: 387; GenBank Ace. -No. AJ009672) and tobacco (Stopping-Mellet V et al . (1999) Eur J Cell Biol 78: 842; GenBank Ace . -Nos . AF072519 and AF072520) . The function of caltractin in plants is not clear. It is discussed that - like its yeast homologue, the CDC31 gene product - it is probably required for the function of the micro- tubule organizing center (MTOC) , and has a role in cell division. As a calcium -binding component of the cytoskeletal network that presumably transduces the changes of cytosolic calcium concentrations, caltractin may function in a signal transduction pathway that affects cell division (Zhu JK (1992) Plant Physiol 99:1734-1735) .
Sequences of plant caltractin-like proteins are deposited in the GeneBank database including sequences from e.g., Arabidopsis thaliana (Gene locus At4g37010; Ace. -No.: NC_003075 and Gene Locus At3g50360; Ace . No. AJ009672) , barley (Hordeum vulgare; GeneBank Ace. -No.: AL505334, AV929632, BF628654, BG343518) , wheat (Triticum aestivum, GeneBank Ac. -No.: BJ292493, BJ298955, BJ253096) corn (Zea mays; GeneBank Ac. -No.: AW065528, BI478579, BI674782) , Atriplex nummularia (GeneBank Ac. -No.: M90970) , and Marsilea vestita (GeneBank Ac. -No.: U92973) . Most of these se- quences are only partial (EST; expressed sequence tag) sequences without any annotation.
' Despite the above described first progress in increasing HR in plants, the currently available methods are far from achieving an efficiency allowing an economically satisfying success rate. The current invention therefore provides a method for further increasing efficiency of HR in plants.
A first embodiment of the invention relates to a method for introducing a mutation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell comprising the steps of
a) providing one or more plant cells comprising
i) a decreased activity or expression of at least one caltractin-like protein, and
ii)at least one pair of DNA homology-sequences A and A' having a sufficient length and homology between each other to allow homologous recombination among A and A' , wherein at least one of said sequences A or A' is part of the chromosomal DNA of said plant cell,
and
b) selecting one or more plant cells comprising a mutation in said chromosomal DNA-sequence in consequence of the homologous recombination between A and A' .
The homologous recombination (HR) between A and A' may constitute an intramolecular or an intermolecular recombination event .
For an intramolecular HR event, the two homology-sequences A and A' undergoing homologous recombination may - for example - be both localized on one strand of a chromosomal DNA-sequence, e.g., in form of direct repeats. In a case like that, HR will cause deletion of the sequences localized between A and A' . This may be utilized, e.g., for deleting selection marker sequences from the chromosomal DNA. For an intermolecular HR event, wo hϊmology-sequences (e.g., A and A') are localized on separate DNA molecules. For example, one of the two homology sequences A and A' undergoing homologous recombination is localized on a chromosomal DNA-sequence. The other sequence may - for example - be localized on a DNA- construct, which may function as a gene targeting construct. In such a case, mutations are introduced into the chromosomal DNA as a consequence of the HR between A and A' . In a preferred embodiment the invention relates to a method for introducing a mu- tation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell comprising the steps of
a) providing one or more plant cells comprising a decreased activity or expression of at least one caltractin-like pro- tein
b) introducing at least one DNA-construct into said plant cells, wherein said DNA-construct comprises at least one homology-sequence A having a sufficient length and homology to at least one part A' of said chromosomal sequence to allow homologous recombination among A and A' , and wherein said DNA-construct introduces said mutation into said chromosomal DNA-sequence in consequence of the homologous recombination between A and A' , and
c) selecting one or more cells comprising said mutation.
In a preferred embodiment the DNA-construct comprises two homology-sequences A and B having a sufficient length and homology to at least a part A' and B' of said chromosomal sequence, respectively, to allow homologous recombination between A and A' , and B and B' , respectively. Between A and B additional sequences might be localized (like e.g., expression cassettes, functional elements) which by the homologous recombination are introduced into the chromosomal DNA.
If one pair of homology-sequences (e.g., A/A') is used, the mutation may - for example - be introduced directly by replacement of A by A' , wherein A differs from A' by the mutation to be introduced. If two pairs of homology-sequences (e.'g., A/A' and B/B' ) are used, the mutation may - for example - be introduced by insertion of the sequences comprised between A and B into the chromosomal DNA. Alternatively, the mutation may comprise modification (e.g., base change) of the sequence localized between A' and B' .
In another preferred embodiment, the method of the invention further comprises the step of segregating the mutation introduced by the DNA-construct and the property of a decreased ac- tiyity or expression of at least one caltractin-like protein.
This will produce a plant with normal activity and expression of said caltractin-like protein (s) comprising the mutation introduced by the DNA-construct. The segregation may be carried out by any method known in the art (e.g., crossing and selection), whereby selection may be easily realized by using standard techniques (e.g., PCR or marker technology) to monitor segregation of the mutation.
Another embodiment of the invention relates to transgenic ex- pression cassettes comprising at least part of a nucleic acid sequence coding for caltractin-like protein under control of a promoter sequence functional in plant cells. Said part may have a length of at least 20 base pair (hereinafter bp) , preferably at least 50 bp, more preferable at least 100 bp, most preferably at least 500 bp. The nucleic acid sequence coding for caltractin-like protein may be orientated in sense and/or antisense direction with regard to the transcription direction of said promoter sequence. In consequence, the transgenic expression cassette of the invention may cause expression of sense, an- tisense or double-stranded RNA of said nucleic acid sequence coding for caltractin-like protein.
Yet another embodiment of the invention relates to transgenic expression vectors comprising at least one transgenic expression cassette of this invention.
Yet another embodiment of the invention relates to transgenic organisms comprising at least one transgenic expression vector or transgenic expression cassette of this invention. Preferably, the organism is a plant organism. Yet another embodiment of the invention related to a plant organism with a decreased activity or expression of at least one caltractin-like protein. Preferably, said decrease is caused by transformation of said plant with at least one transgenic ex- pression- vector or transgenic expression cassette of this invention or by mutating at least one endogenous gene coding for a caltractin-like protein
Yet another embodiment of the invention relates to the use of at least one of the transgenic expression cassettes, transgenic expression vectors, transgenic organisms, or plants with decreased activity or expression of at least one caltractin-like protein in a method for introducing a mutation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell.
Owing to the findings within the invention, the caltractin-like proteins (e.g., AT4g37010) are considered key elements for homologous recombination in plants. Moreover, the method outperforms all those methods in which an increased homologous recom- bination phenotype is realized by overexpressing a HR enhancing protein (like e.g., RecA) . Switching off a gene can be realized without expressing a (foreign) protein. In a preferred case, all that is necessary is to deactivate the endogenous gene. This has considerable advantages for approval and acceptance by the con- sumer, who is frequently apprehensive toward plants with foreign proteins .
The At4g37010 gene product (Arabidopsis thaliana caltractin-like protein) shows 49% identity with the human centrin 2. At4g37010 gene expression - as analyzed by RT-PCR - can be detected in all plant organs : roots, buds, leaves, and flowers.
In principle, the methods according to the invention can be applied to all plant species, preferably to those in which a cal- tractin-like protein or a functional equivalent thereof is expressed naturally. Caltractin-like proteins can be identified in a large number of plants. The sequences from other plants which are homologous to the caltractin-like protein or nucleic acid sequences disclosed within the scope of the present invention can be found readily for example by database searches or by screening genetic libraries using the caltractin-like protein or nucleic acid sequences as search sequence or probe..
As used herein, the term "caltractin-like protein" is intended to include a naturally occurring protein encoded by an amino acid sequence comprising at least one of the following sequence motifs (amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein "X" can stand for any amino acid. Nucleotides, likewise, may be referred to by their commonly accepted single- letter codes) :
a) A first EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of D(T/I)D(G/N)S(G/V) (S/T) IDAXEL, more preferably
LFD(T/I)D(G/N)S(G/V) (S/T) IDAXEL (N/K) VAMR, most preferably LFD(T/l)D(G/N)S(G/V) (S/T) IDAXEL (N/K)VAMR,(A/S)LGFE wherein said first EF-hand motif is preferably localized in the N- terminal region of the caltractin-like protein, more pref- erably between amino acid 1 and 60 of the caltractin-like protein full-length sequence.
b) A second EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of (E/Q) (E/Q)IXX(M/L) (l/M)A(E/D) (V/l)DK, more preferably
(E/Q) (E/Q)IXX(M/L) (l/M)A(E/D) (V/l) D (D/N) XS (G/A) XID (F/Y) , most preferably (E/Q) (E/Q) IXX (M/L) (l/M)A(E/D) (V/I)DK(D/N) XS (G/A)XID (F/Y) (D/E) (D/E)F, wherein said second EF-hand motif is preferably localized between amino acid 45 and 95 of the caltractin-like protein full-length sequence.
c) A third EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of (l/F)DXDX(N/T) GKIS, more preferably (K/R) FX (I/V/L) (I/F)DXDX(N/T) GKIS, most preferably (K/R) AFX(I/V/L) (I/F)DXDX(N/T)GKI-SXX(D/N) (I/L) , wherein said third EF-hand motif is preferably localized between amino acid 85 and 130 of the caltractin-like protein full-length sequence .
d) A fourth EF-hand motif preferably localized in an amino acid sequence having a consensus sequence of AD(R/Q) (D/N)XD(G/R)E, more preferably (I/V)X(E/A)AD(R/Q) (D/N) XD (G/R) E, 'ost preferably (E/D)M(I/V)X(E/A)AD(R/Q) (D/N) D (G/R) E, wherein said fourth EF-hand motif is preferably localized within the last 40 amino acids of the caltractin-like protein full-length se- quence .
e) A sequence element having the consensus sequence
(E/D) (F/Y)XX(M/I)MX(K/R)T, wherein said motif is preferably localized within the last 20 amino acids of the caltractin- like protein full-length sequence.
Especially preferably, at least 2 of these motifs (a to e) occur in a Caltractin-like protein, very especially preferably at least 3 of these motifs, more preferably at least 4 of these mo- tifs, most preferably all motifs a to e. The order of two or more of the motifs a to e may vary but the alphabetical order given above is preferred. Further sequence motifs which are typical for Caltractin-like can be deduced readily by the skilled worker from the sequence alignment of the known Cal- tractin-like proteins, as shown in Fig. 5.
More preferably Caltractin-like proteins include proteins from the group consisting of :
a) a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; and
b) a functionally equivalent polypeptide molecule comprising an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, or 34 and exhibits essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; and
c) a polypeptide molecule comprising a fragment of at least 20 consecutive amino acids, preferably 50 consecutive amino acids of at least one of the sequences described under a) or b) exhibiting essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2/ 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
"Property" or "properties" of a Caltractin-like protein is to be understood in the broad sense and includes for example activity and/or function of said Caltractin-like protein.
"Function" is preferably understood to mean the substrate- binding capacity or ligand-binding property of a Caltractin-like polypeptide in an organism, a tissue, a cell or a cell compartment. Suitable substrates are Calcium-ions, but also the DNA or protein interaction partners of a Caltractin-like protein. Binding partners for Caltractin-like proteins can be identified by methods well known to the person skilled in the art, for example by the yeast-2 -hybrid system. Said interaction partners may include but shall not be limited to plant homologues of binding partners of Caltractin-like polypeptides identified in various organism like e.g., XPC (xeroderma pigmentosum factor C; Araki M et al. (2001) J Biol Chem 276 (22) : 18665-72) , the Kiclp kinase (Sullivan DS et al. (1998) J Cell Biol 143 (3) :751-65) , the Karl (karyogamyl) protein (Vallen EA et al. (1994) Genetics 137 (2) :407-22; Geier BM et al . (1996) J Biol Chem 271 (45) :28366- 74) , the heterotrimeric G-protein transducin (Wolfrum (1995) Cell Motil Cytoscheleton 32: 55-64, Pulvermuller et al (2002) J Cell Biol 22:2194-2203).
"Functional equivalent polypeptide" is understood to mean, in particular, natural or artificial mutations of the Caltractinlike polypeptides as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34 and homologous polypeptides from other plants which continue to have essentially the same properties . Homologous polypeptides from the below- described preferred plants are preferred. Mutations encompass substitutions, additions, deletions, inversions or insertions of one or more amino acid residues. Thus, the present invention also encompasses those polypeptides which are obtained by modification of a polypeptide as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
Functional equivalent polypeptide derived from one of the polypeptides comprising an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 according to the invention Joy substitution (in particular, conservative substitutions) , insertion or deletion have at least 60%, preferably at least 80%, by preference at least 90%, especially preferably at least 95%, very especially preferably at least 98%, homology with one of the polypeptides comprising an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 and exhibit essentially the same properties as the polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
Functional equivalents derived from the nucleic acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 according to the invention by substitution, insertion or deletion have at least 60%, preferably at least
80%, by preference at least 90%, especially preferably at least 95%, very especially preferably at least 98%, homology with one of the nucleic acid sequences as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 according to the invention and encode polypeptides having essentially the same properties as a polypeptide comprising an amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 2'6, 28, 30, 32, or 34.
Preferred functional equivalents include conservatively modified variants of the Caltracin-like proteins as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34. "Conservatively modified- variants"- applies to both amino acid and nucleic acid sequences. With respect to particular nu- cleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al . , J. Biol. Chem. 260: 2605- 2608 (1985); Rossolini et al . , Mol. Cell. Probes 8: 91-98 (1994) ) . Because of the degeneracy of the genetic code, a large number of functionally identical .nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations", which are one species of conservatively modified variations . Every nucleic acid sequence recited herein that en- ■ codes a polypeptide also describes every possible silent varia- tion of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which, along with GUG in some organisms, is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accord- ingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and al- leles of the invention.
Same of the above specified sequences coding for Caltractin-like proteins are for the first time provided within this invention. The were identified using an intensive screening for Caltractinlike protein homologues especially in agronomically important plants. These sequences facilitate carrying out the invention in said crops. Therefore, another embodiment of the invention com- prises an isolated polypeptide sequence coding for a caltractinlike protein, wherein said polypeptide sequence comprises an amino acid sequence described by SEQ ID NO: 6, 8, 10, 12, 14,
18, 20, 22, 28, or 30. A further embodiment of the invention comprises 'an isolated nucleic acid sequence coding for a caltractin-like protein, wherein said nucleic acid sequence comprises an sequence described by SEQ ID NO: 5, 7, 9, 11, 13, 17,
19, 21, 27, or 29.
The sequences from other plants which are homologous to the Caltractin-like sequences disclosed within the scope of the present invention can be found readily for example by database searches or by screening genetic libraries using the Caltractin-like sequences as search sequence or probe. Additional Caltractin-like proteins can be identified for example from a variety of organisms for which DNA sequences are known, such as, for example, from Arabidopsis thaliana, Brassica napus, Nicotiana tabacum, Solanum tuberosum, Oryza sativa, or Helianthus annuus from databases or homology comparisons.
The screening of cDNA libraries or genomic libraries of other organisms, preferably of the plant species which are mentioned further below as hosts for the transformation, using the nucleic acid sequences described under SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 or parts of these as probe is also a method of identifying homologues in other species with which the skilled worker is familiar. In this context, the probes derived from the nucleic acid sequences as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 have a length of at least 20 bp, preferably 50 bp, particularly preferably 100 bp, very especially preferably 200 bp, and most preferably 400 bp. A DNA strand which is complementary to the sequences described under SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 may also be employed for screening the libraries.
Functional equivalents, accordingly, encompass DNA sequences which hybridize under standard conditions with the Caltractinlike nucleic acid sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, with the sequence complementary thereto or parts of the abovementioned and which, as complete sequences, encode proteins which have the same properties as the proteins described under SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
Homology between two nucleic acid sequences is understood as meaning the identity of the nucleic acid sequence over in each case the entire sequence length which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25:3389 et seq.), setting the following parameters:
Gap weight: 50 Length weight: 3
Average match: 10 Average mismatch: 0
For example a sequence which has at least 60% homology with sequence SEQ ID NO: 1 at the nucleic acid level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 1 by the above program algorithm with the above parameter set, has at least 60% identity.
Homology between two polypeptides is understood as meaning the identity of the amino acid sequence over in each case the entire sequence length which is calculated by comparison with the aid of the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG) , Madison, USA) , setting the following parameters:
Gap weight : 8 Length weight : 2
Average match: 2,912 Average mismatch: -2, 003
For example a sequence which has at least 60% homology with sequence SEQ ID NO: 2 at the protein level is understood as meaning a sequence which, upon comparison with the sequence SEQ ID NO: 2 by the above program algorithm with the above parameter set, has at least 60% identity.
"Standard hybridization conditions" is to be understood in the broad sense and means stringent or else less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al., in Molecular Cloning (A Laboratory Manual) , 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989) , 6.3.1-6.3.6.
For example, the conditions during the wash step can be selected from the range of conditions delimited by low-stringency conditions (approximately 2X SSC at 50°C) and high-stringency conditions (approximately 0.2X SSC at 50°C, preferably at 65°C) (20X SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature during the wash step can be raised from low- stringency conditions at room temperature, approximately 22°C, to higher-stringency conditions at approximately 65°C. Both of the parameters salt concentration and temperature can be varied simultaneously, or else one of the two parameters can be kept constant while only the other is varied. Denaturants, for exam- pie formamide or SDS, may also be employed during the hybridization. In the presence of 50% formamide, hybridization is preferably effected at 42°C. Some examples of conditions for hybridization and wash step are shown hereinbelow:
(1) Hybridization conditions can be selected, for example, from the following conditions:
a) 4X SSC at 65°C, b) 6X SSC at 45°C, c) 6X SSC, 100 μg/ml denatured salmon sperm DNA at 68°C, with optionally 0.5% SDS, d) 6X SSC,' 0.5% SDS, 100 μg/ml denatured fragmented salmon sperm DNA, 50% formamide at 42°C, e) 50% formamide, 4X SSC at 42°C, f) 2X or 4X SSC at 50°C (low-stringency condition) , or g) 30 to 40% formamide, 2X or 4X SSC at 42°C (low- stringency condition) .
(2) Wash steps can be selected, for example, from the following conditions :
a) 0.1X SSC at 65°C. b) 0.1X SSC, 0.5 % SDS at 68°C. c) 0.1X SSC, 0.5% SDS, 50% formamide at 42°C. d) 2X SSC at 65°C (low-stringency condition) . The decrease of the expression of an Caltractin-like protein, the Caltractin-like activity or the Caltractin-like function can be realized in many ways .
"Plant" is generally understood as meaning any single- or multi- celled organism or a cell, tissue, part or propagation material (such as seeds or fruit) of same which is capable of photosynthesis. Included for the purpose of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred. Also included are mature plants, seeds, shoots and seedlings, and parts, propagation material (for example tubers, seeds or fruits) and cultures derived from them, for example cell cultures or callus cultures. The term includes the mature plants, seed, shoots and seedlings and their derived parts, propagation material (such as seeds or microspores) , plant organs, tissue, protoplasts, callus and other cultures, for example cell cultures, and any other type of plant cell grouping to give functional or structural units. Mature plants refers to plants at any desired developmental stage beyond that of the seedling. Seedling refers to a young immature plant at an early developmental stage.
Furthermore, plant organisms for the purposes of the invention are further organisms capable of being photosynthetically active such as, for example, algae, cyanobacteria and mosses. Preferred algae are green algae such as, for example, algae from the genus Haematococcus, Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis is particularly preferred.
Among plants vascular plants are especially preferred. The term "vascular plant" is intended to include all plants comprising a vascular system in contrast to non-vascular plants like, e.g. Bryophytae. Vascular plants may comprise seedless and seed- carrying vascular plants .
"Seedless vascular plants" are especially plants of the phyla Psilotophyta, Lycophyta, Sphenophyta or Pterophyta "Seed-carrying vascular plants" are especially plants of the phyla Cycadophyta, Ginkophyta, Coniferophyta, Gnetophyta, Anthophyta.
"Vascular plant" encompasses all annual and perennial monocotyledonous and dicotyledonous plants and includes by way of example but not by limitation those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Tri- folium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bro- mus, Asparagus, Antirrhinum, Hemerocallis, Nemesis, Pelargonium, Panicum, Penniset-um, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea and Populus.
Preferred plants are those from the following plant families: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaeeae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniaceae, Theaceae, Umbelliferae.
• Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants such as, for example, the Gramineae family, such as rice, maize, wheat or other cereal species such as barley, millet and sorghum, rye, triticale or oats, and sugar cane, and all grass species.
The invention is applied very particularly preferably to dicotyledonous plant organisms. Preferred dicotyledonous plants are selected in particular from the dicotyledonous crop plants such as, for example,
- Asteraceae such as sunflower, tagetes or calendula and others,
- Compositae, especially the genus Lactuca, very particularly the species sativa (lettuce) and others, - Cruciferae, particularly the genus Brassica, very particularly the species napus (oilseed rape) , campestris (beet) , oleracea cv Tastie (cabbage) , oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other cabbages; and the genus Arabidopsis, very particularly the species thaliana, and cress or canola and others,
- Cucurbitaeeae such as melon, pumpkin/squash or zucchini and others,
- Leguminosae, particularly the genus Glycine, very particularly the species max (soybean) , soya, and alfalfa, pea, beans or peanut and others,
- Rubiaceae, preferably the subclass Lamiidae such as, for example Coffea arabica or Coffea liberica (coffee bush) and others ,
- Solanaceae, particularly the genus Lycopersicon, very particularly the species esculentum (tomato) , the genus
Solanum, very particularly the species tuberosum (potato) and melongena (aubergine) and the genus Capsicum, very particularly the genus annuum (pepper) and tobacco or paprika and others,
- Sterculiaceae, preferably the subclass Dilleniidae such as, for example, Theobroma cacao (cacao bush) and others,
- Theaceae, preferably the subclass Dilleniidae such as, for example, Camellia sinensis or Thea sinensis (tea shrub) and others,
- Umbelliferae, particularly the genus Daucus (very particularly the species carota (carrot) ) and Apium (very particularly the species graveolens dulce (celery) ) and others;
and linseed, cotton, hemp, clover, flax, cucumber, spinach, carrot, sugar beet, red pepper, beet, radish, sweet potato, cucumber, chicory, cauliflower, broccoli, asparagus, onion, gar- lie, strawberry, raspberry, blackberry, pineapple, avocado and the various tree, nut and grapevine species. Tree species pref- erably comprise plum, cherry, peach, nectarine, apricot, banana, kiwi, papaya, mango, -apple, pear, quince.
Also encompassed are ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or turf. Plants which may be mentioned by way of example but not by limitation are angiosperms, bryophytes such as, for example, Hepaticae (liverworts) and Musci (mosses) ; pteridophytes such as ferns, horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgo and Gnetatae, the families of the Rosaceae such as rose, Ericaceae such as rhododendron and azalea, Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceae such as African violet, Balsaminaceae such as touch-me-not, Orchidaceae such as orchids, Iridaceae such as gladioli, iris, freesia and crocus, Compositae such as marigold, Geraniaceae such as geranium, Liliaceae such as dracena, Moraceae such as ficus, Araceae such as cheeseplant and many others.
Preferred within the scope of the invention are those plants which are employed as foodstuffs or feeding stuffs.
The method of the invention is suitable to introduce mutations in the chromosomal DNA of a plant cell or a plant organism.
"Chromosomal DNA" is to be understood as the genomic DNA of the cellular nucleus independent from the cell cycle status. Chromosomal DNA might therefore be organized in chromosomes or chro- matids, they might be condensed or uncoiled.
"Mutation " or "mutated" with respect to a chromosomal DNA- sequence is to be understood in the broad sense and is intended to include substitution, addition, deletion, inversion or insertion of at least one or more base pairs. Said mutation may af- feet the coding region as much as the non-coding region (e.g., the 5' -untranslated, 3' -untranslated, intron or promoter region) of the a gene. Consequences of said mutation may be various and may - for example- cause a decrease or an increase of the amount of mRNA or protein expressed from said gene, or of the function and/or activity of the corresponding gene product. The term "insertion" is intended to comprise single base insertion and/or insertion of additional genes or expression cassettes. "Amount of protein" is understood as meaning the amount of protein (e.g., an Caltractin-like polypeptide) in an organism, a tissue, a cell or a cell compartment.
"Decrease" with respect to the amount of mRNA or protein (e.g., expressed from a target gene or coding for a Caltractin-like protein) is to be understood in the broad sense and is intended to include the permanent or temporarily, partially, essentially completely or completely hindering or blocking of the expression of the target gene or the corresponding RNA or mRNA by various cellular mechanisms or of the protein product resulting thereof. More preferred, "decrease" of the amount of RNA or mRNA or protein means the quantitative decrease of the amount of RNA or mRNA or protein, respectively, in an organism, a tissue, a cell or a cell compartment in comparison with the wild type of the same genus and species, to which this method had not been applied, under otherwise identical conditions (such as, for example, culture conditions, plant age and the like) . The decrease amounts to at least 10%, preferably at least 10% or at least
20%, especially preferably at least 40% or 60%, very especially preferably at least 70% or 80%, most preferably at least 90% or 95%. Furthermore a complete inactivation of expression and/or gene function is preferred ("knock-out").
"Decrease" with respect to the function and/or activity of a protein (e.g., expressed from a target gene or coding for a Caltractin-like protein) may include an decrease of the encoding mRNA but may also include a change in the protein sequence of the gene product causing a decreased or abolished function and/or activity.
A decrease in the amount of mRNA may be monitored by various techniques well known to the person skilled in the art including but not limited to Northern-hybridization, nuclease protection assay or quantitative reverse transcription PCR (quantitative RT-PCR) .
A decrease in the amount of protein (and indirectly the encoding mRNA) may be monitored by various techniques well known to the person skilled in the art including but not limited to the mi- cro-biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218- 222) , the Folin-Ciocalteu-method (Lowry OH et al . (1951) J Biol Chem 193:265-275) or by measurement of* the adsorption of CBB G- 250 (Bradford MM (1976) Analyt Biochem 72:248-254). More specific quantitative analysis may be carried out by methods including but not limited to ELISA ("enzyme linked immunosorbent assay"), Western-blotting, radioimmunoassay (RIA) or other immunoassays .
"Increase" with respect to the amount of RNA, mRNA or protein (e.g., expressed from a target gene) or the activity or function of the corresponding gene product is to be understood in the broad sense and is intended to include the permanent or temporarily initiation, boost, or enhancement of expression from the target gene or the quantity of the RNA or mRNA derived therefrom, or the quantity, activity or function of the corresponding polypeptide encoded thereby by various cellular mechanisms.
More preferred, "increase" of the amount of RNA or mRNA or protein means the quantitative increase of the amount of RNA or mRNA or protein, respectively, in an organism, a tissue, a cell or a cell compartment in comparison with the wild type of the same genus and species, to which this method had not been applied, under otherwise identical conditions (such as, for example, culture conditions, plant age and the like) . The increase amounts to at least 50%, preferably at least 100%, especially preferably at least 500%, very especially preferably at least 1000%. The increase can be measured or monitored by methods known in the art and described in brief above for assessing an decrease in RNA, mRNA or protein.
"Decrease" or "decreasing" in connection with a Caltractin-like protein, a Caltractin-like activity or a Caltractin-like function is to be interpreted in the wide sense and encompasses the partial or essentially complete inhibition or blocking of the functionality of a Caltractin-like protein in a plant or a part, tissue, organ, cells or seeds thereof, which inhibition or blocking is based on a variety of cytobiological mechanisms.
For the purposes of the invention, a decrease also encompasses a quantitative decrease of a Caltractin-like protein down to the essential complete absence of the Caltractin-like protein (i.e. lacking detectability of Caltractin-like activity or Caltractinlike function, or lacking immunological detectability of the Caltractin-like protein) . In this context, the expression of a particular Caltractin-like protein, or the Caltractin-like activity or Caltractin-like function, in a cell or an organism is preferably reduced by more than 50%, especially preferably by more than 80%, very especially preferably by more than 90%.
A variety of strategies for reducing the expression of a Caltractin-like protein, the Caltractin-like activity or Caltractin-like function are encompassed in accordance with the in- vention. The skilled worker is aware of a series of different methods being available for influencing the expression of a Caltractin-like protein, the Caltractin-like activity or the Caltractin-like function in the desired manner.
A decrease of the Caltractin-like activity or the Caltractinlike function is preferably achieved by reduced expression of an endogenous Caltractin-like protein.
A decrease of the amount of Caltractin-like protein, the Cal- tractin-like protein activity or the Caltractin-like protein function can be effected - for example - by utilization of at least one of the following methods:
a) Introduction of a double-stranded RNA of a Caltractin-like protein encoding nucleic acid sequence (Caltractin-like protein dsRNA) or an expression cassette (s) ensuring the expression thereof
b) Introduction of an antisense RNA of a Caltractin-like protein encoding nucleic acid sequence or an expression cassette ensuring expression thereof . Encompassed are those methods in which the antisense nucleic acid sequence is directed against a Caltractin-like gene (i.e. genomic DNA sequences) or a Caltractin-like gene transcript (i.e. RNA sequences), α-anomeric nucleic acid sequences are also encompassed
c) Introduction of an antisense RNA of a Caltractin-like protein encoding nucleic acid sequence in combination with a ribozyme or an expression cassette ensuring expression thereof
d) Introduction of a sense nucleic acid sequence of a Caltractin-like protein encoding nucleic acid sequence for in- ducing co-suppression or an expression cassette ensuring expression thereof. The sense nucleic acid sequence may be DNA or RNA, but is preferably RNA.
e) Introduction of a DNA- or protein-binding factor against a
Caltractin-like protein encoding gene, RNA or Caltractin-like protein or an expression cassette ensuring expression thereof
f) Introduction of a viral nucleic acid sequence causing degradation of the RNA encoding a Caltractin-like protein or an expression cassette ensuring expression thereof
g) Introduction of constructs for inducing a homologous recombi- nation on at least one endogenous Caltractin-like gene, for example for generating knock-out mutants
h) Introduction of at least one mutation into an endogenous gene encoding a Caltractin-like protein for generating a loss of function (for example generation of stop codons, reading- frame shifts and the like)
In this context, each and every one of these methods may bring about a decrease of the expression of a Caltractin-like protein, activity and/or function of a Caltractin-like protein for the purposes of the invention. A combined use is also feasible. Further methods are known to the skilled worker and can encompass the hindering or prevention of Caltractin-like protein processing, of the Caltractin-like protein or Caltractin-like mRNA transport, inhibition of ribosome attachment, inhibition of RNA splicing, induction of an Caltractin-like protein RNA-degrading enzyme and/or inhibition of translational elongation or termination.
The individual processes which are preferred may be described in greater detail hereinbelow:
a) Introduction of a double-stranded RNA
The method of regulating genes by means of double-stranded RNA ("double-stranded RNA interference"; dsRNAi) has been described repeatedly for animal and plant organisms (for example Matzke MA et al. (2000) Plant Mol Biol 43:401-415; Fire A. et al (1998) Nature 391:806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364) . Express reference is made to the processes and methods described in the above references. Effective gene suppression can also be demonstrated upon transient expression or following transient transformation for example as the consequence of biolistic transformation (Schweizer P et al. (2000) Plant J 2000 24: 895-903) . dsR Ai methods are based on the phenomenon that the simultaneous introduction of complementary strand and counterstrand of a gene transcript causes the expression of the gene in question to be suppressed in a highly efficient manner. The phenotype caused greatly resembles a corresponding knock-out mutant (Waterhouse PM et al. (1998) Proc Natl Acad Sci USA 95:13959-64) .
The dsRNAi method has proved to be particularly effective and advantageous for reducing expression of a Caltractin-like protein. As described, inter alia, in WO 99/32619, dsRNAi approaches are markedly superior to traditional antisense ap- proaches .
The invention therefore furthermore relates to double-stranded RNA molecules (dsRNA molecules) which, upon introduction into a plant (or a cell, tissue, organ or seed derived therefrom) , bring about a decrease of expression of a Caltractin-like protein.
"Double-stranded RNA molecule" is to be understood to comprise at least one RNA molecule, which is at least theoretically able to form a double-stranded RNA secondary structure by intermo- lecular or intramolecular base pairing. Such secondary structure may be predicted by the base-paring rules of Watson and Crick or by computer algorithms (like e.g., FOLDRNA; Zuker and Stiegler (1981) Nucleic Acids Res 9 (1) :133-48) .
The double-stranded RNA molecule for reducing the expression of an Caltractin-like protein comprises,
i) at least one first RNA sequence which is essentially iden- tical to at least part of a nucleic acid sequence coding for a Caltractin-like protein, and ii) at least one second RNA sequence which is essentially complementary to at least part of said first RNA sequence under i) .
Preferably, the double-stranded RNA molecule comprises,
i) at least one first RNA sequence which is essentially identical to at least part of a nucleic acid sequence comprising a sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, and
ii) at least one second RNA sequence which is essentially complementary to at least part of said first RNA sequence under i) .
"Essentially identical" means that the dsRNA sequence can also show insertions, deletions or individual point mutations compared with the target sequence coding for the Caltractin-like protein while still bringing about an effective decrease of the expression. The homology in accordance with the above definition preferably amounts to at least 75%, preferably at least 80%, very especially preferably at least 90%, most preferably 100%, between the sense strand of an inhibitory dsRNA and a part- segment of a nucleic acid sequence encoding a Caltractin-like protein (or between the antisense strand and the complementary strand of a nucleic acid sequence encoding a Caltractin-like protein) . In particular, to avoid non-specific action of the dsRNA, the length of the part-segment amounts to at least 10 bases, preferably at least 25 bases, especially preferably at least 50 bases, very especially preferably at least 100 bases, most preferably at least 200 bases or at least 300 bases. As an alternative, an "essentially identical" dsRNA can also be defined as a nucleic acid sequence which is capable of hybridizing with part of a Caltractin-like gene transcript (for example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 itiM EDTA at 50°C or 70°C for 12 to 16 h) .
Also encompassed is the use of the dsRNA molecules according to the invention in the methods for introducing mutations into the chromosomal DNA of a plant cell or a plant organism. The dsRNA can be composed of one- or more strands of polymerized ribonucleotides. Modifications both of the sugar-phosphate backbone and of the nucleosides may be present. For example, the phosphodiester bonds of the. natural RNA can be modified in such a way that they comprise at least one nitrogen or sulfur hetero atom. Bases can be modified in such a way that the activity of, for example, adenosine deaminase is restricted. These and other modifications are described hereinbelow in the methods of stabilizing antisense RNA. The dsRNA can be generated enzymatically or fully or partially synthesized chemically.
The double-stranded structure can be formed starting from an individual self-complementary strand or starting from two complementary strands. In a single self-complementary strand, sense and antisense sequence may be linked by a linking sequence
("linker") and can form for example a hairpin structure. The linking sequence can preferably be an intron which is spliced out after the dsRNA has been synthesized. The nucleic acid sequence encoding a dsRNA can comprise further elements such as, for example, transcription termination signals or polyadenyla- tion signals.
If the two RNA sequences are to be combined to form a dsRNA in a cell or plant, this can be effected in various ways:
a) transformation of the cell or plant with a vector comprising both expression cassettes,
b) cotransformation of the cell or plant with two vectors, one of them comprising the expression cassettes with the sense strand and the other comprising the expression cassettes with the antisense strand,
c) hybridizing two plants, each of which has been transformed with one vector, one of the vectors comprising the expression cassettes with the sense strand and the other comprising the expression cassettes with the antisense strand.
The formation of the RNA duplex can be initiated either outside or within the cell. Like in WO 99/53050, the dsRNA can also encompass a hairpin structure by linking sense and antisense strand by means of a linker (for example an intron) . The self- complementary dsRNA structures are preferred since they only require the expression of one construct and always comprise the complementary strands in an equimolar ratio.
The expression cassettes encoding the antisense or sense strand of a dsRNA or the self-complementary strand of the dsRNA are preferably inserted into a vector and, using the methods described hereinbelow, stably inserted into the genome of a plant in order to ensure permanent expression of the dsRNA, using se- lection markers for example.
The dsRNA can be introduced using a quantity which allows at least one copy per cell. Greater quantities (for example at least 5, 10, 100, 500 or 1000 copies per cell) may bring about a more effective decrease.
As already described, 100% sequence identity between dsRNA and a gene transcript encoding a Caltractin-like protein is not necessarily required in order to bring about an effective decrease of the Caltractin-like expression. Accordingly, there is the advantage that the method is tolerant with regard to sequence deviations as may exist as the consequence of genetic mutations, polymorphisms or evolutionary divergence. Thus, for example, it is possible to use the dsRNA generated on the basis of the Cal- tractin-like sequence of one organism to suppress the Caltractin-like expression in another organism. The high sequence homology between the Caltractin-like proteins from various plant organisms (e.g., Arabidopsis, rice, maize and wheat; see Fig.l) allows the conclusion that this protein is conserved to a high degree within plants, so that the expression of a dsRNA derived from one of the disclosed nucleic acid sequences encoding a Caltractin-like protein as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33 appears to have an advantageous effect in other plant species as well.
The dsRNA can be synthesized either in vivo or in vitro. To this end, a DNA sequence encoding a dsRNA can be brought into an expression cassette under the control of at least one genetic control element (such as, for example, promoter, enhancer, si- lencer, splice donor or splice acceptor or polyadenylation signal) . Suitable advantageous constructions are described hereinbelow. Polyadenylation is not required, nor do elements for ini- tiating translation have to be present.
A dsRNA can be synthesized chemically or enzymatically. Cellular RNA polymerases or bacteriophage RNA polymerases (such as, for example, T3, T7 or SP6 RNA polymerase) can be used for this purpose. Suitable methods for expression of RNA in vitro are described (WO 97/32016; US 5,593,874; US 5,698,425, US 5,712,135, US 5,789,214, US 5,804,693) . A dsRNA which has been synthesized in vitro chemically or enzymatically can be isolated completely or to some degree from the reaction mixture, for example by extraction, precipitation, electrophoresis, chromatography or combinations of these methods, before being introduced into a cell, tissue or organism. The dsRNA can be introduced directly into the cell or else be applied extracellularly (for example into the interstitial space) .
However, it is preferred to transform the plant stably with an expression construct which brings about the expression of the dsRNA. Suitable methods are described hereinbelow.
b) Introduction of a Caltractin-like antisense nucleic acid sequence
Methods for suppressing a specific protein by preventing its mRNA from accumulating by means of antisense technology have been described in many instances, including in the case of plants (Sheehy et al. (1988) Proc Natl Acad Sci USA 85: 8805- 8809; US 4,801,340; Mol JN et al . (1990) FEBS Lett 268(2) :427- 430) . The antisense nucleic acid molecule hybridizes, or binds, with the cellular mRNA and/or genomic DNA encoding the Caltractin-like target protein to be suppressed. This suppresses the transcription and/or translation of the target protein. Hybridization can originate conventionally by the formation of a stable duplex or - in the case of genomic DNA - by the antisense nucleic acid molecule binding to the duplex of the genomic DNA by specific interaction in the major groove of the DNA helix.
An antisense nucleic acid sequence suitable for reducing an Caltractin-like protein can be deduced using the nucleic acid se- quence encoding this protein, for example the nucleic acid sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, following Watson and Crick's base pairing rules. The antisense nucleic acid sequence can be complementary to all of the transcribed mRNA of said protein, be limited to the coding region, or else only be composed of a nu- cleotide, which is partially complementary to the coding or non- coding sequence of the mRNA. Thus, for example, the oligonucleo- tide can be complementary to the region encompassing the translation start for said protein. Antisense nucleic acid sequences can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nu- cleotides in length, but may also be longer and comprise at least 100, 200, 500, 1000, 2000 or 5000 nucleotides. Antisense nucleic acid sequences can be expressed recombinantly or synthesized chemically or enzymatically using methods known to the skilled worker. In the case of chemical synthesis, natural or modified nucleotides may be used. Modified nucleotides can im- part an increased biochemical stability to the antisense nucleic acid sequence and lead to an increased physical stability of the duplex formed of antisense nucleic acid sequence and sense target sequence. The following can be used: for example phos- phorothioate derivatives or acridine-substituted nucleotides.
In a further preferred embodiment, the expression of a Caltractin-like protein can be inhibited by nucleotide sequences which are complementary to the regulatory region of a Caltractin-like gene (for example a Caltractin-like promoter and/or enhancer) and which form triple-helical structures with that DNA double helix so that the transcription of the Caltractin-like gene is reduced. Such methods have been described (Helene C (1991) Anticancer Drug Res 6(6):569-84; Helene C et al. (1992) Ann NY Acad Sci 660:27-36; Maher LJ (1992) Bioassays 14(12) :807- 815) .
In a further embodiment, the antisense nucleic acid molecule can be an α-anomeric nucleic acid. Such α-anomeric nucleic acid molecules form specific double-stranded hybrids with complemen- tary RNA in which - as opposed to the conventional β-nucleic acids - the two strands run parallel to one another (Gautier C et al. (1987) Nucleic Acids Res 15:6625-6641). The antisense nucleic acid molecule can furthermore also comprise 2'-0- methylribonucleotides (Inoue et al . (1987) Nucleic Acids Res 15:6131-6148) or chimeric RNA/DNA analogs (Inoue et al. (1987) FEBS Lett 215:327-330). c) Introduction of a Caltractin-like" antisense nucleic acid sequence in combination with a ribozyme
The above-described antisense strategy can be combined advanta- geously with a ribozyme method. Catalytic RNA molecules or ribozymes can be adapted to suit any target RNA and cleave the phosphodiester backbone at specific positions, functionally deactivating the target RNA (Tanner NK (1999) FEMS Microbiol Rev 23 (3) :257-275) . The ribozyme itself is not modified thereby, but is capable of cleaving further target RNA molecules analogously, thereby assuming the qualities of an enzyme. The incorporation of ribozyme sequences into antisense RNAs confers this enzyme- like RNA-cleaving quality to precisely these antisense RNAs, thus increasing their efficacy in inactivating the target RNA. The generation and the use of such ribozyme antisense RNA molecules is described, for example, in Haseloff et al. (1988) Nature 334: 585-591.
In this manner, ribozymes (for example "hammerhead" ribozymes; Haselhoff and Gerlach (1988) Nature 334:585-591) can be used catalytically to cleave the mRNA of an enzyme to be suppressed, for example Caltractin-like, and to prevent translation. The ribozyme technique can increase the efficacy of an antisense strategy. Methods of expressing ribozymes for reducing specific proteins are described in (EP 0 291 533, EP 0 321 201,
EP 0 360 257) . The expression of ribozyme in plant cells has also been described (Steinecke P et al. (1992) EMBO J 11(4) :1525-1530; de Feyter R et al . (1996) Mol Gen Genet. 250 (3) :329-338) . Suitable target sequences and ribozymes can be determined as described for example by "Steinecke P, Ribozymes, Methods in Cell Biology 50, Galbraith et al . eds, Academic Press, Inc. (1995), pp. 449-460" by calculating the secondary structure of ribozyme RNA and target RNA as well as by their interaction (Bayley CC et al . (1992) Plant Mol Biol. 18(2) :353- 361; Lloyd AM and Davis RW et al . (1994) Mol Gen Genet.
242 (6) :653-657) . For example, derivatives of the Tetrahymena. L- 19 IVS RNA with regions which are complementary to the mRNA of the Caltractin-like protein to be suppressed can be constructed (see also US 4,987,071 and US 5,116,742). As an alternative, such ribozymes can also be identified from a library of diverse ribozymes via a selection process (Bartel D and Szostak JW (1993 ) Science 261 : 1411- 1418 ) .
d) Introduction of a Caltractin-like sense nucleic acid sequence for inducing cosuppression
The expression of an- Caltractin-like nucleic acid sequence in sense orientation can lead to cosuppression of the corresponding homologous endogenous gene. The expression of sense RNA with homology with an endogenous gene can reduce or switch off the ex- pression of the former, similarly to what has been described for antisense approaches (Jorgensen et al . (1996) Plant Mol Biol 31(5) :957-973; Goring et al . (1991) Proc Natl Acad Sci USA 88:1770-1774; Smith et al . (1990) Mol Gen Genet 224:447-481; Na- poli et al. (1990) Plant Cell 2:279-289; Van der Krol et al . (1990) Plant Cell 2:291-99). In this context, the homologous gene to be reduced can be repressed either fully or only in part by the construct introduced. The possibility of translation is not required. The application of this technique to plants is described, for example, by Napoli et al . (1990) The Plant Cell 2: 279-289 and in US 5,034,323.
e) Introduction of DNA- or protein-binding factors against Caltractin-like genes, Caltractin-like RNAs or Caltractin-like proteins
Caltractin-like gene expression may also be reduced using specific DNA-binding factors, for example factors of the zinc finger transcription factor type. These factors attach to the genomic sequence of the endogenous target gene, preferably in the regulatory regions, and bring about repression of the endogenous gene. The use of such a method makes possible the decrease of the expression of an endogenous Caltractin-like gene without it being necessary to recombinantly manipulate its sequence. Suitable methods for the preparation of suitable factors have been described (Dreier B et al. (2001) J Biol Chem 276 (31) :29466-78; Dreier B et al. (2000) J Mol Biol 303(4) :489-502; Beerli RR et al. (2000) Proc Natl Acad Sci USA 97 (4) : 1495-1500; Beerli RR et al. (2000) J Biol Chem 275 (42) : 32617-32627; Segal DJ and Barbas CF 3rd. (2000) Curr Opin Chem Biol 4(1) :34-39; Kang JS and Kim JS (2000) J Biol Chem 275 (12) : 8742-8748 ; Beerli RR et al . (1998) Proc Natl Acad Sci USA 95 (25) : 14628- 14633; Kim JS et al . (1997) Proc Natl Acad Sci USA 94(8):3616 -3620; Klug A (1999) J Mol Biol 293 (2) :215-218; Tsai SY et al . (1998) Adv Drug Deliv Rev 30 (1-3) :23-31; Mapp AK et al . (2000) Proc Natl Acad Sci USA 97(8) :3930-3935; Sharrocks AD et al . (1997) Int J Biochem Cell Biol 29 (12) :1371-1387; Zhang L et al . (2000) J Biol Chem 275 (43) :33850-33860) .
These factors can be selected using any desired portion of an Caltractin-like gene. This segment is preferably located in the promoter region. For gene suppression, however, it may also be in the region of the coding exons or introns. The segments in question can be obtained by the skilled worker from GeneBank by database search or, starting from an Caltractin-like cDNA whose gene is not present in GeneBank, by screening a genomic library for corresponding genomic clones. The skilled worker is familiar with the methods required therefore.
Furthermore, it is possible to introduce, into cells, factors which inhibit the Caltractin-like target protein itself. The protein-binding factors can be, for example, aptamers (Famulok M and Mayer G (1999) Curr Top Microbiol Immunol 243:123-36) or antibodies or antibody fragments or single-chain antibodies. Methods for obtaining these factors have been described and are known to the skilled worker. For example, a cytoplasmic scFv antibody was employed to modulate the activity of the phytochrome A protein in genetically modified tobacco plants (Owen M et al . (1992) Biotechnology (N Y) 10 (7) :790-794 ; Franken E et al . (1997) Curr Opin Biotechnol 8 (4) :411-416; Whitelam (1996) Trend Plant Sci 1:286-272).
Gene expression may also be suppressed by tailor-made low- molecular-weight synthetic compounds, for example of the polyam- ide type (Dervan PB and Bύrli RW (1999) Current Opinion in Chemical Biology 3:688-693; Gottesfeld JM et al . (2000) Gene Expr 9 (1-2) : 77-91) . These oligomers are composed of the units 3- (dimethylamino)propylamine, N-methyl-3-hydroxypyrrole, N- methylimidazole and N-methylpyrrole and can be adapted to any piece of double-stranded DNA in such a way that they bind into the major groove in a sequence-specific manner and block the expression of these gene sequences. Suitable methods have been de- scribed (see, inter alia, Bremer RE et al. (2001) Bioorg Med Chem. 9 (8) :2093-103; Ansari AZ et al . (2001) Chem Biol. 8(6):583-92; Gottesfeld JM et al . (2001) J Mol Biol. 309(3) :615- 29; Wurtz NR et al . (2001) Org Lett 3 \8) :1201-3 ; Wang CC et al . (2001) Bioorg Med Chem 9(3) :653-7; Urbach AR and Dervan PB (2001) Proc Natl Acad Sci USA 98(8) : 343-8; Chiang SY et al . (2000) J Biol Chem. 275 (32) :24246-54) .
g) Introduction of viral nucleic acid sequences and expression constructs which cause Caltractin-like RNA degradation
Caltractin-like expression can also be brought about efficiently by inducing the specific Caltractin-like RNA degradation by the plant with the aid of a viral expression system (amplicon) (An- gell, SM et al. (1999) Plant J. 20 (3) :357-362) . These systems - also termed "VIGS" (viral induced gene silencing) - introduce, into the plant, nucleic acid sequences with homology to the transcripts to be suppressed, with the aid of viral vectors.
Then, transcription is switched off, probably mediated by plant defense mechanisms against viruses. Suitable techniques and methods have been described (Ratcliff F et al . (2001) Plant J 25(2) :237-45; Fagard M and Vaucheret H (2000) Plant Mol Biol 43(2-3) :285-93; Anandalakshmi R et al . (1998) Proc Natl Acad Sci USA 95 (22) :13079-84; Ruiz MT (1998) Plant Cell 10(6): 937-46).
h) Introduction of constructs for inducing homologous recombination on endogenous Caltractin-like genes, for example for generating knock-out mutants
An example of what is used for generating a homologously recombinant organism with reduced Caltractin-like activity is a nucleic acid construct comprising at least part of an endogenous Caltractin-like gene which is modified by a deletion, addition or substitution of at least one nucleotide in such a way that its functionality is reduced or fully destroyed. The modification may also relate to the regulatory elements (for example the promoter) of the gene, so that the coding sequence remains un- modified, but expression (transcription and/or translation) does not take place or is- reduced.
In the case of conventional homologous recombination, the modified region is flanked at its 5' and 3' end by further nucleic acid sequences which must be sufficient in length for making possible recombination. They are, as a rule, in the range of several hundred bases to several kilobases in length (Thomas KR and Capecchi MR (1987) Cell 51:5*03; Strepp et al . (1998) Proc Natl Acad Sci USA 95(8) :4368-4373) . For homologous recombination, the host organism - for example a plant - is transformed with the recombination construct using the methods described hereinbelow, and clones which' have successfully undergone recombination are selected, for example using a resistance to antibiotics or herbicides.
Homologous recombination is a relatively rare event in higher eukaryotes, especially in plants. Random integrations into the host genome predominate . One possibility of eliminating the randomly integrated sequences and thus increasing the number of cell clones with a correct homologous recombination is the use of a sequence-specific recombination system as described in US 6,110,736, by which unspecifically integrated sequences can be deleted again, which simplifies the selection of events which have integrated successfully via homologous recombination. A large number of sequence-specific recombination systems can be used, examples being the Cre/lox system of bacteriophage PI, the FLP/FRT system of yeast, the Gin recombinase of phage Mu, the Pin recombinase from E.coli, and the R/RS system of the pSRl plasmid. The bacteriophage PI Cre/lox and the yeast FLP/FRT system are preferred. The FLP/FRT and cre/lox recombinase system has already been applied to plant systems (Odell et al . (1990) Mol Gen Genet 223: 369-378).
i) Introduction of mutations into endogenous Caltractin-like genes for generating a loss of function (for example generation of stop codons, reading-frame shifts and the like)
Further suitable methods for reducing the Caltractin-like activity are the introduction of nonsense mutations into endogenous Caltractin-like genes, for example by introducing RNA/DNA oli- gonucleotides into the plant (Zhu et al . (2000) Nat Biotechnol 18(5) :555-558) and the generation of knock-out mutants with the aid of, for example, T-DNA mutagenesis (Koncz et al . (1992) Plant Mol Biol 20 (5) : 963-976) , ENU (N-ethyl-N-nitrosourea) or EMS (ethyl methane sulfonate) mutagenesis or homologous recombination (Hohn B and Puchta H (1999) Proc Natl Acad Sci USA 96:8321-8323.) . Point mutations can also be generated by means of DNA-RNA hybrids also known under the name "chimeraplasty" (Cole-Strauss et al. (1999) Nucl Acids Res 27 (5) : 1323-1330; Kmiec (1999) Gene therapy American Scientist 87 (3) :240-247) . In addition, mutations may in induced by transposon mediated mutagenisis (Sundaresan V et al. (1995) Genes Dev. 9(14):1797- 810; Parinov S et al . (1999) Plant Cell 11 (12) :2263-70) .An addi- tional method for inducing mutations in endogenous gene may utilize "Targeting Induced Local Lesions IN Genomes" (TILLING) (McCallum CM (2000) Plant Physiology 123:439-442).
The methods of dsRNAi, cosuppression by means of sense RNA and "VIGS" ("virus induced gene silencing") are also termed "post- transcriptional gene silencing" (PTGS) . PTGS methods, like the decrease of the Caltractin-like function or activity with dominant-negative Caltractin-like variants, are especially advantageous because the demands regarding the homology between the en- dogenous gene to be suppressed and the sense or dsRNA nucleic acid sequence expressed recombinantly (or between the endogenous gene and its dominant-negative variant) are lower than, for example, in the case of a traditional antisense approach. Such criteria with regard to homology are mentioned in the descrip- tion of the dsRNAi method and can generally be applied to PTGS methods or dominant-negative approaches. Owing to the high degree of homology between the Caltractin-like proteins from maize, rice and barley, a high degree of conservation of this protein in plants can be assumed. Thus, using the Caltractin- like nucleic acid sequences from barley, maize or rice, it is presumably also possible efficiently to suppress the expression of homologous Caltractin-like proteins in other species without the isolation and structure elucidation of the Caltractin-like homologues occurring therein being required. Considerably less labor is therefore required. Similarly, the use of dominant- negative variants of- an Caltractin-like protein from rice, maize or barley can presumably also efficiently reduce or suppress the function/activity of its homologue in other plant species.
The person skilled in the art may easily identify equivalent ways to achieve the same effect than achieved by decreasing activity and/or expression of a caltractin-like protein, e.g., by decreasing activity and/or expression of interaction partners of the caltractin-like protein or other proteins which participate in the same signal transduction pathway than the caltractin-like protein. Such proteins may be identified using methods known to the person skilled in the art to elucidate signal transduction pathways like., e.g. interaction* partner network analysis utilizing yeast-two-hybrid or yeast-n-hybrid systems.
All of the substances and compounds which directly or indirectly bring about a decrease in protein quantity, RNA quantity, gene activity or protein activity of an Caltractin-like protein shall subsequently be combined in the term "anti-Caltractin" compounds. The term "anti-Caltractin" compound explicitly includes the nucleic acid sequences, peptides, proteins or other factors employed in the above-described methods.
For the purposes of the invention, "introduction" comprises all of the methods which are capable of directly or indirectly introducing an "anti-Caltractin" compound into a plant or a cell, compartment, tissue, organ or seed thereof, or of generating such a compound there . Direct and indirect methods are encompassed. The introduction can lead to a transient presence of an "anti-Caltractin" compound (for example a dsRNA) or else to its stable presence .
Owing- to the different nature of the above-described approaches, the "anti-Caltractin" compound can exert its function directly (for example by insertion into an endogenous Caltractin-like gene) . However, its function can also be exerted indirectly fol- lowing transcription into an RNA (for example in the case of antisense approaches) or following transcription and translation into a protein (for example in the case of binding factors) . The invention encompasses both directly and indirectly acting "anti- Caltractin" compounds .
The term "introducing" encompasses for example methods such as transfection, transduction or transformation.
"Anti-Caltractin" compounds therefore also encompasses recombi- nant expression constructs which bring about expression (i.e. transcription and, if appropriate, translation) , for example of an Caltractin-like dsRNA or an Caltractin-like "antisense" RNA, preferably in a plant or a part, tissue, organ or seed thereof.
More preferably the expression construct of the invention comprise at least one nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33;
b) a nucleic acid molecule comprising a fragment of at least 20 consecutive nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33;
c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least 60% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; and
d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, wherein the fragment comprises at least 10 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
The DNA-construct utilized to introduce mutations into the chro- mosomal DNA of a plant cell or a plant organism may be of various structural configurations. It may have - for example - a linear, linearized or circular structure. It may be double- stranded or single-stranded (like e.g., T-DNA at a certain point of its transfer) . Preferably, the DNA-construct is introduced into the cell in form of a linearized double-stranded DNA or in form of a T-DNA. In addition, the DNA-construct may be introduced into the plant cells in form of a transposon (comprising the mutation-inducing sequence) . Preferably, plants are selected in which the transposon had integrated close to the target se- quence . By action of a transposase (e.g., introduced by transient expression or crossing) the DNA-construct becomes available again for homologous recombination.
The term "sufficient length" with respect to a homology sequence comprised in a DNA-construct (e.g., the homology sequence A or B) is to be understood to comprise sequences of a length of at least 100 base pair, preferably at least 250 base pair, more preferably at least 500 base pair, especially preferably at least 1000 base pair, most preferably at least 2500 base pair.
The term "sufficient homology" with respect to a homology se- quence comprised in a DNA-construct (e.g., the homology sequence A or B) is to be understood to comprise sequences having a homology to the corresponding target sequence comprised in the chromosomal DNA (e.g., the target sequence A' or B' ) of at least 70 %, preferably at least 80 %, more preferably at least 90 %, especially preferably at least 95 %, more especially preferably at least 99 %, most preferably 100 %, wherein said homology extends over a length of at least 50 base pair, preferably at least 100 base pair, more preferably at least 250 base pair, most preferably at least 500 base pair.
The DNA-constructs utilized within the method of this invention may comprise additional nucleic acid sequences. Said sequences may be - for example - localized in different positions with respect to the homology sequences. Preferably, the additional nu- cleic acid sequences are localized between two homology sequences and may be introduced via homologous recombination into the chromosomal DNA,_thereby resembling an insertion mutation of said chromosomal DNA. However, the additional sequences may also be localized outside of the homology sequences (e.g., at the 5'- or 3 '-end of the DNA-construct) . In cases where the additional sequence resembles a negative selection marker this may allow a distinction of illegitimate insertion events from correct insertion events mediated by homologous recombination. Corresponding negative markers are described below and suitable methods are well known in the art (WO 99/20780) .
Preferably, said sequences are expression cassettes, which may - for example - facilitate expression of selection markers, trait genes, or antisense RNA. Preferably said expression cassettes comprise a promoter sequence functional in plant cells opera- tively linked to a nucleic acid sequence which - upon expression - confers an advantageous phenotype to the so transformed plant . The person skilled in the art is aware of numerous sequences which may be utilized in this context, e.g. to increase quality of food and feed or to produce chemicals, fine chemicals or pharmaceuticals (e.g., vitamins, oils, carbohydrates) (Dunwell JM (2000) J Exp Bot 51 Spec No:487-96) . Furthermore, growth. yield, and resistance against abiotic and biotic stress factors may be enhanced. Advantageous properties may be conferred either by overexpressing proteins or by decreasing expression of endogenous proteins by e.g., expressing a corresponding antisense or double-stranded RNA.
In a preferred embodiment of the invention, efficiency of the method of the invention may be further increased by combination with other methods suitable for increasing homologous recombina- tion. Said methods may include for example expression of HR- enhancing proteins (like e.g., RecA; WO 97/08331; Reiss B et al . (1996) Proc Natl Acad Sci USA 93(7) :3094-3098; Reiss B et al . (2000) Proc Natl Acad Sci USA 97(7) :3358-3363) or treatment with PARP inhibitors (Puchta H et al. (1995) Plant J. 7:203-210) . Various PARP inhibitors suitable for use within this invention are known to the person skilled in the art and may include for example preferably 3-Aminobenzamid, 8-Hydroxy-2- methylquinazolin-4-on (NU1025) , 1, llb-Dihydro- [2H]benzopyrano[4,3,2-de] isoquinolin-3-on (GPI 6150), 5- Aminoisoquinolinon, 3 , 4-Dihydro-5- [4- (1-piperidinyl) butoxy] - 1 (2H) -isoquinolinon or compounds described in WO 00/26192, WO 00/29384, WO 00/32579, WO 00/64878, WO 00/68206, WO 00/67734, WO 01/23386 or WO 01/23390. Furthermore, the method may be combined with other methods facilitation homologous recombination and/or selection of the recombinants like e.g., positive/negative selection, excision of illegitimate recombination events or induction of sequence-specific or unspecific DNA double-strand breaks .
In an expression construct, a nucleic acid molecule whose expression (transcription and, if appropriate, translation) generates an "anti-Caltractin" compound is preferably operably linked to at least one genetic control element (for example a promoter) which ensures expression in an organism, preferably in plants. If the expression construct is to be introduced directly into a plant and the "anti-Caltractin" compound (for example the Caltractin-like dsRNA) is to be generated therein in planta, plant- specific genetic control elements (for example promoters) are preferred. However, the "anti-Caltractin" compound may also be generated in other organisms or in vitro and then be introduced into the plant (as described in Examples 6 and 7) . Preferred in this context are all of the prokaryotic or eukaryotic genetic control elements (for example promoters) which permit the expression in the organism chosen in each case for the preparation.
Operable linkage is to be understood as meaning, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example an "anti-Caltractin" compound) and, if appropriate, further regulatory elements such as, for example, a terminator in such a way that each of the regula- tory elements can fulfill its function when the nucleic acid sequence is expressed recombinantly, depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for ex- ample, enhancer sequences, can also exert their function on the target sequence from-positions which are further away, or indeed from other DNA molecules . Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
Operable linkage, and an expression cassette, can be generated by means of customary recombination and cloning techniques as are described, for example, in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Silhavy TJ, Berman ML and Enquist LW (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) , in Ausubel FM et al . (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience and in Gelvin et al . (1990) In: Plant Molecular Biology Manual. However, further sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences. The insertion of sequences may also lead to the expression of fusion proteins. Preferably, the expression cassette, consisting of a linkage of promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a plant genome, for example by transformation.
However, expression cassette also denotes those constructions in which a promoter is positioned behind an endogenous Caltractin- like gene, for example by means of homologous recombination, and the decrease according to the invention of an Caltractin-like protein is brought about by the expression of an antisense Caltractin-like RNA. Analogously, an "anti-Caltractin" compound (for example a nucleic acid sequence encoding an Caltractin-like dsRNA or an Caltractin-like antisense RNA) can be positioned behind an endogenous promoter in such a way that the same effect is manifested. Both approaches lead to inventive expression cassettes.
The term plant-specific promoters . is understood as meaning, in principle, any promoter which is capable of governing the expression of genes, in particular foreign genes, in plants or plant parts, plant cells, plant tissues or plant cultures. In this context, expression can be, for example, constitutive, in- ducible or development-dependent.
The following are preferred:
a) Constitutive promoters
"Constitutive" promoters refers to those promoters which ensure expression in a large number of, preferably all, tissues over a substantial period of plant' development, preferably at all times during plant development (Benfey et al.(1989) EMBO J 8:2195- 2202) . A plant promoter or promoter originating from a plant virus is especially preferably used. The promoter of the CaMV (cauliflower mosaic virus) 35S transcript (Franck et al. (1980) Cell 21:285-294; Odell et al . (1985) Nature 313:810-812; Shew aker et al. (1985) Virology 140:281-288; Gardner et al. (1986) Plant Mol Biol 6:221- 228) or the 19S CaMV promoter (US
5,352,605; WO 84/02913; Benfey et al . (1989) EMBO J 8:2195-2202) are especially preferred. Another suitable constitutive promoter is the Rubisco small subunit (SSU) promoter (US 4,962,028), the leguminB promoter (GenBank Ace. No. X03677) , the promoter of the nopalin synthase from Agrobacterium, the TR dual promoter, the OCS (octopine synthase) promoter from Agrobacterium, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649) , the ubiquitin 1 promoter (Christensen et al . (1992) Plant Mol Biol 18:675-689; Bruce et al . (1989) Proc Natl Acad Sci USA 86:9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (US 5,683,439), the promoters of the vacuolar ATPase subunits, the promoter of the Arabidopsis thaliana nitrilase-1 gene (GenBank Ace. No.: U38846, nucleotides 3862 to 5325 or else 5342) or the promoter of a proline-rich protein from wheat (WO 91/13991) , and further promoters of genes whose constitutive expression in plants is known to the skilled worker. The CaMV 35S promoter and the Arabidopsis thaliana nitrilase-1 promoterare particularly preferred.
b) Tissue-specific promoters
Furthermore preferred are promoters with specificities for seeds, such as, for example, the phasedin promoter (US 5,504,200; Bustos MM et al . (1989) Plant Cell 1 (9) : 839-53) , the promoter of the 2S albumin gene (Joseffson LG et al . (1987) J Biol Chem 262:12196- 12201), the legumine promoter (Shirsat A et al. (1989) Mol Gen Genet 215 (2) : 326-331) , the USP (unknown seed protein) promoter (Baumlein H et al. (1991) Mol Gen Genet 225 (3) :459-67) , the napin gene promoter (US 5,608,152; Stalberg K et al. (1996) Planta 199:515-519), the promoter of the sucrose binding proteins (WO 00/26388) or the legumin B4 promoter (LeB4; B umlein H et al. (1991) Mol Gen Genet 225: 121-128; Baumlein et al. (1992) Plant Journal 2(2):233-9; Fiedler U et al . (1995) Biotechnology (NY) 13 (10) : 1090f) , the Arabidopsis oleosin promoter (WO 98/45461) , and the Brassica Bce4 promoter (WO 91/13980) .
Further suitable seed-specific promoters are those of the gene encoding high-molecular weight glutenin (HMWG) , gliadin, branching enzyme, ADP glucose pyrophosphatase (AGPase) or starch synthase . Promoters which are furthermore preferred are those which permit a seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like. The promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzin gene, the prolamin gene, the gliadin gene, the glutelin gene, the zein gene, the easirin gene or the secalin gene) can advantageously be employed. c) Chemically inducible promoters
The expression cassettes may also contain a chemically inducible promoter (review article: Gatz et al . (1997) Annu Rev Plant Physiol Plant Mol Biol 48:89-108), by means of which the expression of the exogenous gene in the plant can be controlled at a particular point in time. Such promoters such as, for example, the PRP1 promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesulfona ide-inducible promoter (EP 0 388 186) , a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J 2:397-404), an abscisic acid-inducible promoter EP 0 335 528) or an ethanol-cyclohexanone-inducible promoter (WO 93/21334) can likewise be used. Also suitable is the promoter of the glutathione-S transferase isoform II gene (GST-II-27) , which can be activated by exogenously applied safeners such as, for example, N,N-diallyl-2,2-dichloroacetamide (W0 93/01294) and which is operable in a large number of tissues of both monocots and dicots .
Particularly preferred are constitutive promoters.
Furthermore, further promoters may be linked operably to the nucleic acid sequence to be expressed, which promoters make possi- ble the expression in further plant tissues or in other organisms, such as, for example, E. coli bacteria. Suitable plant promoters are, in principle, all of the above-described promoters .
The nucleic acid sequences present in the expression cassettes or vectors according to the invention can be linked operably to further genetic control sequences in addition to a promoter. The term "genetic control sequences" is to be understood in the broad sense and refers to all those sequences which have an ef- feet on the materialization or the function of the expression cassette according to the invention. For example, genetic control sequences modify the transcription and translation in pro- karyotic or eukaryotic organisms. Preferably, the expression cassettes according to the invention encompass a promoter func- tional in plants r 5 '-upstream of the nucleic acid sequence in question to be expressed recombinantly, and 3' -downstream a terminator sequence as additional genetic control sequence and, if appropriate, further customary Regulatory elements, in each case linked operably to the nucleic acid sequence to be expressed recombinantly.
Genetic control sequences also encompass further promoters, promoter elements or minimal promoters, all of which can modify the expression-governing properties. Thus, for example, the tissue- specific expression may additionally depend on certain stress- ors, owing to genetic control sequences. Such elements have been described, for example, for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266(26): 17131 -17135) and heat stress (Schoffl F et al . , Molecular & General Genetics 217(2- 3) :246-53, 1989) .
Further advantageous control sequences are, for example, the Gram-positive promoters amy and SP02, and the yeast or fungal promoters ADC1, MFa , AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
In principle, all natural promoters with their regulatory se- quences like those mentioned above may be used for the method according to the invention. In addition, synthetic promoters may also be used advantageously.
Genetic control sequences furthermore also encompass the 5'- untranslated regions, introns or noncoding 3' -region of genes, such as, for example, the actin-1 intron, or the Adhl-S introns 1, 2 and 6 (general reference: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)) . It has been demonstrated that they may play a significant role in the regulation of gene expression. Thus, it has been demonstrated that 5 ' -untranslated sequences can enhance the transient expression of heterologous genes. Examples of translation enhancers which may be mentioned are the tobacco mosaic virus 5' leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like. Furthermore, they may promote tissue specificity (Rouster J et al . (1998) Plant J 15:435-440).
The expression cassette may advantageously comprise one or more of what are known as enhancer sequences, linked operably to the promoter, which make possible an increased recombinant expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, may also be inserted at the 3 ' end of the" nucleic acid sequences to be expressed recombinantly. One or more copies of the nucleic acid sequences to be"expressed recombinantly may be present in the gene construct .
Polyadenylation signals which are suitable as control sequences are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of the T-DNA (octopin synthase) of the Ti plasmid pTiACHS (Gielen et al . (1984) EMBO J 3:835 et seq.) or functional equivalents thereof. Examples of terminator sequences which are especially suitable are the OCS (octopin synthase) terminator and the NOS (nopalin synthase) terminator.
Control sequences are furthermore to be understood as those which make possible homologous recombination or insertion into the genome of a host organism or which permit removal from the genome. In the case of homologous recombination, for example the natural promoter of a particular- gene may be exchanged for a promoter functional in plants . Methods such as the cre/lox technology permit a tissue-specific, if appropriate inducible, removal of the expression cassette from the genome of the host organism (Sauer B (1998) Methods. 14 (4) : 381-92) . In this method, specific flanking sequences (lox sequences) , which later allow removal by means of ere recombinase, are attached to the target gene.
An expression cassette and the vectors derived from it may com- prise further functional elements. The term functional element is to be understood in the broad sense and refers to all those elements which have an effect on the generation, amplification or function of the expression cassettes, vectors or transgenic organisms according to the invention. The following may be men- tioned by way of example, but not by limitation:
1. Selection markers
Selection marker are useful to select and separate successfully transformed or homologous recombined cells.
1.1 Positive selection markers Selection markers confer a resistance to a biocidal compound such as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO 98/45456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin) or herbicides (e.g., phosphinothricin or gly- phosate) . Especially preferred selection markers are those which confer resistance to herbicides. Examples which may be mentioned are:
- Phosphinothricin acetyltransferases (PAT; also named Bialophos ®resistance; bar; de Block et al . (1987) EMBO J 6: 2513-2518)
- 5-enolpyruvylshikimate-3 -phosphate synthase (EPSPS) conferring resistance to Glyphosate® (N- (phosphonomethyl) glycine) ,
- Glyphosate® degrading enzymes (Glyphosate® oxidoreductase; gox) ,
- Dalapon® inactivating dehalogenases (deh)
- sulfonylurea- and imidazolinone-inactivating acetolactate syn- thases (for example mutated ALS variants with, for example, the S4 and/or Hra mutation)
- Bromoxynil® degrading nitrilases (bxn)
- Kanamycin- or. G418- resistence genes (NPTII; NPTI) coding e.g., for neomycin phosphotransferases,
- 2-Desoxyglucose-6-phosphate phosphatase (D0GR1-Gene product; WO 98/45456; EP 0 807 836) conferring resistance against 2- desoxyglucose (Randez-Gil et al. (1995) Yeast 11:1233-1240) .
Additional suitable positive selection marker are the aadA gene, which confers resistance to the antibiotic spectinomycin, the streptomycin phosphotransferase (SPT) gene, which allows resistance to streptomycin and the hygromycin phosphotransferase (HPT) gene, which mediates resistance to hygromycin.
1.2) Negative selection marker Negative selection markers are especially suitable to select organisms with defined deleted sequences comprising said marker (Koprek T et al . (1999) Plant J 19(6) : 719-726). Examples for negative selection marker comprise thy idin kinases (TK) , cyto- sine deaminases (Gleave AP et al . (1999) Plant Mol Biol.
40 (2) :223-35; Perera RJ et al . (1993) Plant Mol. Biol 23(4): 793-799; Stougaard J; (1993) Plant J 3:755-761), cytochrom P450 proteins (Koprek et al. (1999) Plant J 16:719-726), haloalkan dehalogenases (Naested H (1999) Plant J 18:571-576), iaaH gene products (Sundaresan V et al. (1995) Genes & Development 9:1797- 1810) , cytosine deaminase codA (Schlaman HRM and Hooykaas PJJ (1997) Plant J 11:1377-1385), or tms2 gene products (Fedoroff NV & Smith DL (1993) Plant J 3:273- 289) .
2) Reporter genes
Reporter genes encode readily quantifiable proteins and, via their color or enzyme activity, make possible an assessment of the transformation efficacy, the site of expression or the time of expression. Very especially preferred in this context are genes encoding reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13 (1) :29-44) such as the green fluorescent protein (GFP) (Sheen et al.(1995) Plant Journal 8 (5) : 777-784 , Haseloff et al. (1997) Proc Natl Acad Sci USA 94(6) :2122-2127; Reichel et al.(1996) Proc Natl Acad Sci USA 93 (12) :5888-5893 ,
Tian et al . (1997) Plant Cell Rep 16:267-271; WO 97/41228; Chui WL et al. (1996) Curr Biol 6:325-330; Leffel SM et al . (1997) Biotechniques . 23 (5) : 912-8) , chlora phenicol transferase, a luciferase (Ow et al . (1986) Science 234:856-859; Millar et al. (1992) Plant Mol Biol Rep 10:324-414), the aequorin gene
(Prasher et al . (1985) Biochem Biophys Res Commun 126(3) :1259- 1268) , β-galactosidase, R locus gene (encoding a protein which regulates the production of anthocyanin pigments (red coloring) in plant tissue and thus makes possible the direct analysis of the promoter activity without addition of further auxiliary substances or chromogenic substrates; Dellaporta et al . , In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium, 11:263-282, 1988), with β- glucuronidase being very especially preferred (Jefferson et al., EMBO J. 1987, 6, 3901-3907) . 3) Origins of replication, which ensure amplification of the expression cassettes or vectors according to the invention in, for example, E. coli. Examples which may be mentioned are ORI (origin of DNA replication) , the pBR322 ori or the P15A ori (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .
4) Elements which are necessary for Agrobacterium-mediated plant transformation, such as, for example, the right or left border of the T-DNA or the vir region.
To select cells which have successfully undergone homologous recombination, or else to select transformed cells, it is, also typically necessary to introduce a selectable marker, which confers resistance to a biocide (for example herbicide) , a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic to the cells which have successfully undergone recombination. The selection marker permits the selection of the transformed cells from untransformed ones (McCormick et al. (1986) Plant Cell Reports 5:81-84).
The introduction of an expression cassette according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably into plants or plant cells, tissue, organs, parts or seeds) can be effected advantageously using vectors which comprise the expression cassettes. The expression cassette can be introduced into the vector (for example a plasmid) via a suitable restriction cleavage site. The plasmid formed is first introduced into E. coli. Correctly transformed E. coli are selected, grown, and the recombinant plasmid is obtained by the methods familiar to the skilled worker. Restriction analysis and sequencing may serve to verify the cloning
S G ■
Examples of vectors may be plasmids, cosmids, phages, viruses or else agrobacteria. In an advantageous embodiment, the expression cassette is introduced by means of plasmid vectors. Preferred vectors are those which make possible stable integration of the expression cassette into the host genome. The invention furthermore relates to transgenic plant organisms or tissues, organs, parts, cells or propagation material thereof which comprise a transgenic expression cassette for "anti- Caltractin" compound or a transgenic vector encompassing such an expression cassette.
Such a transgenic plant organism is generated, for example, by means of transformation or transfection by means of the corresponding proteins or nucleic acids . The generation of a transformed organism (or a transformed cell or tissue) requires introducing the DNA in question (for example the expression vector) , RNA or protein into the host cell in question.
A multiplicity of methods are available for this procedure, which is termed transformation (or transduction or transfection) (Keown et al. (1990) Methods in Enzymology 185:527-537) . For example, the DNA or RNA can be introduced directly by microinjec- tion or by bombardment with DNA-coated microparticles. Also, the cell can be permeabilized chemically, for example using polyeth- ylene glycol, so that DNA can enter the cell by diffusion. The DNA can also be introduced by protoplast fusion with other DNA- containing units such as minicells, cells, lysosomes or liposomes. Another suitable method of introducing DNA is electroporation, where the cells are permeabilized reversibly by an elec- trical pulse. Suitable methods have been described (for example by Bilang et al. (1991) Gene 100:247-250; Scheid et al . (1991) Mol Gen Genet 228:104-112; Guerche et al . (1987) Plant Science 52:111-116'; Neuhause et al . (1987) Theor Appl Genet 75:30-36; Klein et al . (1987) Nature 327:70-73; Howell et al . (1980) Science 208:1265; Horsch et al.(1985) Science 227:1229-1231; De- Block et al. (1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press Inc. (1989)).
In plants, the above-described methods of transforming and regenerating plants from plant tissues or plant cells are exploited for transient or stable transformation. Suitable methods are especially protoplast transformation by polyethylene-glycol- induced DNA uptake, the biolistic method with the gene gun, what is known as the particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution, and microinjection. injection.
In addition to these "direct" transformation techniques, transformation can also be effected by bacterial infection by means of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The Agrobacterium-mediated transformation is best suited to dicotyledonous plant cells. The methods are described, for example, by Horsch RB et al . (1985) Science 225: 1229f.
When Agrobacteria are used, the expression cassette is integrated into specific plasmids, either into a shuttle or intermediate vector, or into a binary vector. If a Ti or Ri plasmid is to be used for the transformation, at least the right border, but in most cases the right and left border, of the Ti or Ri plasmid T-DNA is linked to the expression cassette to be introduced in the form of a flanking region.
Binary vectors are preferably used. Binary vectors are capable of replication both in E. coli and in Agrobacterium. As a rule, they comprise a selection marker gene and a linker or polylinker flanked by the right and left T-DNA border sequence. They can be transferred directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187) . The selection marker gene permits the selection of transformed Agrobacteria and is, for example, the nptll gene, which confers resistance to kanamycin. The Agrobacterium which acts as host organism in this case should already contain a plasmid with the vir region. The latter is required for transferring the T-DNA to the plant cell. An Agrobacterium transformed in this way can be used for transforming plant cells . The use of T-DNA for transforming plant cells has been studied and described intensively (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al . (1985) EMBO J 4:277-287). Various binary vectors are known, some of which are commercially available such as, for example, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA) .
Further promoters which are suitable for expression in plants have been described (Rogers et al . (1987) Meth in Enzymol 153:253-277; Schardl et al . (1987) Gene 61:1-11; Berger et al . (1989) Proc Natl Acad Sci USA 86:8402-8406). Direct transformation techniques are 'suitable for any organism and cell type.
The plasmid used does not need to meet any particular additional requirements in the case of the injection or electroporation of DNA or RNA into plant cells. Simple plasmids such as those of the pUC series can be used. If complete plants are to be regenerated from the transformed cells, it is necessary for an additional selectable marker gene to be located on the plasmid.
Stably transformed cells, i.e. those which contain the introduced DNA integrated into the DNA of the host cell,, can be selected from untransformed cells when a selectable marker is part of the DNA introduced. Examples of genes which can act as mark- ers are all those which are capable of conferring resistance to antibiotics or herbicides are given above . Transformed cells which express such marker genes are capable of surviving in the presence of concentrations of a corresponding antibiotic or herbicide which kill an untransformed wild type. The resulting plants can be bred and hybridized in the customary fashion. Two or more generations -should be grown in order to ensure that the genomic integration is stable and hereditary. Corresponding methods are described, for example, in Jenes B et al.(1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, En- gineering and Utilization, edited by SD Kung and R Wu, Academic Press, pp. 128-143 and in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225) . The construct to be expressed is preferably cloned into a vector which is suitable for the transformation of Agrobacterium tumefaciens, for example pBinl9 (Bevan et al . (1984) Nucl Acids Res 12:8711f).
As soon as a transformed plant cell has been generated, a complete plant can be obtained using methods known to the skilled worker. For example, callus cultures are used as starting mate- rial. The development of shoot and root can be induced in this as yet undifferentiated cell biomass in a known fashion. The shoots obtained can be planted out and bred.
The skilled worker is familiar with such methods of regenerating intact plants from plant cells and plant parts. Methods to do so are described, for example, by Fennell et al . (1992) Plant Cell Rep. 11: 567-570; Stoeger et al • (199≤) Plant Cell Rep. 14:273- 278; Jahne et al . (1994) Theor Appl Genet 89:525-533.
"Transgenic", for example regarding a nucleic acid sequence, an expression cassette or a vector comprising said nucleic acid sequence or an organism transformed with said nucleic acid sequence, expression cassette or vector, refers to all those constructs originating by recombinant methods in which either
a) the nucleic acid sequence coding for at least part of a Cal- tracin-like protein, antisense RNA, or double stranded RNA, or
b) a genetic control sequence linked operably to said nucleic acid sequence a) , for example a promoter, or
c) (a) and (b)
is not located in its natural genetic environment or has been modified by recombinant methods, an example of a modification being a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, very especially preferably at least 5000 bp, in length. A naturally occurring expression cassette - for example the naturally occurring combination of the Caltractin-like promoter with the corresponding Caltractin-like gene - becomes a transgenic expression cassette when it is modified by non-natural, synthetic "artificial" meth- ods such as, for example, muta.genization. Such methods have been described (US 5,565,350; WO 00/15815; also see above).
The invention also relates to transgenic organisms transformed with at least one of the nucleic acid sequences according to the invention, expression cassette according to the invention or vector according to the invention, and to cells, cell cultures, tissues, parts - such as, for example, leaves, roots and the like in the case of plant organisms - or propagation material derived from such organisms. The term organism is to be understood in the broad sense and refers to prokaryotic and eu- karyotic organisms, preferably bacteria, yeasts, fungi, non- human animal organisms and plant organisms. Preferred plant organisms are indicated above.
Host or starting organisms which are preferred as transgenic organisms are mainly plants in accordance with the above defini- tion. Included within the scope of the invention are all genera and species of higher and lower plants of the Plant Kingdom. Furthermore included are the mature plants, seed, shoots and seedlings, and parts, propagation material and cultures derived therefrom, for example cell cultures. Mature plants refers to plants at any developmental stage beyond that of the seedling. The term seedling refers to a young immature plant in an early developmental stage.
The transgenic organisms can be generated with the above- described methods for the transformation or transfection of organisms .
The invention furthermore relates to the use of the transgenic organisms according to the invention and of the cells, cell cul- tures, parts - such as, for example, roots, leaves and the like in the case of transgenic plant organisms - derived from them, and to transgenic propagation material such as seeds or fruits, for the production of foodstuffs or feeding stuffs, pharmaceuticals or fine chemicals.
Furthermore preferred is a method for the recombinant production of pharmaceuticals or fine chemicals in host organisms, wherein a host organism is transformed with one of the above-described expression cassettes and this expression cassette comprises one or more structural genes which encode the desired fine chemical or catalyze the biosynthesis of the desired fine chemical, the transformed host organism is cultured, and the desired fine chemical is isolated from the culture medium. This method can be applied widely to fine chemicals such as enzymes, vitamins, amino acids, sugars, fatty acids, and natural and synthetic flavorings, aroma substances and colorants. Especially preferred is the production of tocopherols and tocotrienols and carotenoids. The transformed host organisms are cultured and the products are isolated from the host organisms or the culture medium by methods known to the skilled worker. The production of pharmaceuticals such as, for example, antibodies or vaccines, is described by Hood EE, Jilka JM. Curr Opin Biotechnol. 1999 Aug; 10(4) :382- 6; Ma JK, Vine ND. Curr Top Microbiol Immunol. 1999; 236:275-92.
Another embodiment of the invention is directed to a process for facilitating plant breeding utilizing a plant organism having a decreased activity or expression of at least one caltractin-like protein. The plant of the invention having said decreased activity or expression of at least one caltractin-like protein exhibits an enhanced rate of meiotic recombination. This property may, for example, be utilized to facilitate breeding programs by e.g., allowing faster segregation of advantageous and disadvantageous traits. For example, in case particular traits are to be crossed into an elite line and subsequently the unwanted traits of the non-elite plant (e.g., a wild relative) have to be crossed out, this goal can be faster achieved utilizing the plants of the invention.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof can be suggested by persons skilled i the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes .
Sequences
1. SEQ ID NO: 1 Nucleic acid sequence encoding Caltractinlike protein from Arabidopsis thalina
2. SEQ ID NO: 2 Amino acid sequence encoding Caltractinlike protein from Arabidopsis thalina
3. SEQ ID NO: 3 Nucleic acid sequence encoding Caltractin- "like protein from Arabidopsis thalina
4. SEQ ID NO : 4 Amino acid sequence encoding Caltractinlike protein from Arabidopsis thalina
5. SEQ ID NO: 5 Nucleic acid sequence encoding Caltractinlike protein from Brassica napus (partial)
6. SEQ ID NO : 6 Amino acid sequence encoding Caltractinlike protein from Brassica napus (partial)
7. SEQ ID NO: 7 Nucleic acid sequence encoding Caltractinlike protein from barley (Hordeum vulgare)
8. SEQ ID NO: 8 Amino acid sequence encoding Caltractin- like protein from barley (Hordeum vulgare)
9. SEQ ID NO: 9 Nucleic acid sequence encoding Caltractinlike protein from rice (Oryza sativa; partial)
10. SEQ ID NO: 10 Amino acid sequence encoding Caltractinlike protein from rice (Oryza sativa,- partial)
11. SEQ ID NO: 11 Nucleic acid sequence encoding Caltractinlike protein from rice (Oryza sativa,- partial)
12. SEQ ID NO: 12 Amino acid sequence encoding Caltractin- like protein from rice (Oryza sativa; partial) 13. SEQ ID NO: 13 Nucleic acid sequence encoding Caltractinlike protein from wheat (Triticum aestivum)
14. SEQ ID NO: 14 Amino acid sequence encoding Caltractin- like protein from wheat (Triticum aestivum)
15. SEQ ID NO: 15 JSTucleic acid sequence encoding Caltractinlike protein from wheat (Triticum aestivum; partial)
16. SEQ ID NO: 16 Amino acid sequence encoding Caltractinlike protein from wheat (Triticum aestivum; partial)
17. SEQ ID NO: 17 Nucleic acid sequence encoding Caltractinlike protein from linseed (partial)
18. SEQ ID NO: 18 Amino acid sequence encoding Caltractinlike protein from linseed (partial)
19. SEQ ID NO: 19 Nucleic acid sequence encoding Caltractinlike protein from corn (Zea mays)
20. SEQ ID NO: 20 Amino acid sequence encoding Caltractin- like protein from corn (Zea mays)
21. SEQ ID NO: 21 Nucleic acid sequence encoding Caltractinlike protein from sunflower (partial)
22. SEQ ID NO: 22 Amino acid sequence encoding Caltractinlike protein from sunflower (partial)
23. SEQ ID NO: 23 Nucleic acid sequence encoding Caltractinlike protein from Atriplex nummularia
24. SEQ ID NO: 24 Amino acid sequence encoding Caltractinlike protein from Atriplex nummularia
25. SEQ ID NO: 25 Nucleic acid sequence encoding Caltractin- like protein from Marsilea vestita 26. SEQ ID NO: 26 Amino acid "sequence encoding Caltractinlike protein from Marsilea vestita
27. SEQ ID NO: 27 Nucleic acid sequence encoding Caltractin- like protein from Soybean (Glycine max)
28. SEQ ID NO: 28 Amino acid sequence encoding Caltractinlike protein from Soybean (Glycine max)
29. SEQ ID NO: 29 Nucleic acid sequence encoding Caltractinlike protein from potato (Solanum tuberosum)
30. SEQ ID NO: 30 Amino acid sequence encoding Caltractin- like protein from potato (Solanum tuberosum)
31. SEQ ID NO: 31 Nucleic acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
32. SEQ ID NO: 32 Amino acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
33. SEQ ID NO: 33 Nucleic acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
34. SEQ ID NO: 34 Amino acid sequence encoding Caltractinlike protein from tobacco (Nicotiana tabacum)
35. SEQ ID NO: 35 Oligonucleotide primer CEN958 5 ' -CAAATAAACGAATTGATGGCAG-3
36. SEQ ID NO: 36 Oligonucleotide primer CEN3 '
5 ' -CCATTACTAATTGATTTATACTTAGC-3
37. SEQ ID NO: 37 Oligonucleotide primer CENNcoF
5 ' -GGCCGGCCATGGCAGCAAATAAACGAATTGATGGCAG-3 ' 38. SEQ ID NO: 38 Oligonucleotide 'primer CENXhor
-5' -GGCCGGCTAGAGATTATTGTCGTGGTCAATGATC-3 '
39. SEQ ID NO: 39 Oligonucleotide primer CENBsrGr 5 ' -GGCCGGTGTACAATTATTGTCGTGGTCAATGATC-3 '
40. SEQ ID NO: 40 Oligonucleotide primer CENAvrf
5' -GGCCGGCCTAGGCAGCAAATAAACGAATTGATGGCAG-3 '
Figures
Fig. 1: Homologous recombination frequencies (HRF; Fold change) in 3 different P 24 plants (24.4, 24.6, 24.10). HRF was measured in a batch of 2 week old T2 plants (>50 plants/replicate). The figure shows heterozygous T2 plants. Ho- mozygous plants exhibited the same fold change.
Fig. 2: Displays scheme of At4g37010 gene locus disrupted by T-DNA tagging (gray boxes: Exons of At4g37010 gene; white box: pSKI015 T-DNA; arrow indicated transcription direction from 35S enhancer) . Due to the orientation of the T-DNA, the four copies of the 35S enhancer present near the right border could drive the anti-sense expression of at least part of the At4g37010 gene .
Fig. 3: mRNA steady state level of centrin determined by RT-PCR in a batch of 2 week old T2 plants. Demonstrated is a significantly reduced expression of the At4g37010 derived mRNA in the heterozygous knockout plant in comparison to wild-type plants.
Fig. 4: Homologous recombination frequencies (fold change respect to the untransformed parental line) in different IC9 (Fig.4B) and IC6 (Fig. 4A) reporter lines (A,E,F, I, ,M,N,0, and 2,3,10,11,17,18 respectively) having intergrated the pOEXhpCEN construct ..
Fig. 5: Alignment of Caltractin-like proteins from various plant species. 1: Arabidopsis 1 (Arabidopsis thaliana, Gene Locus At4g37010) ; 2: Arabidopsis 2 (Arabidopsis thaliana, corresponding to GeneBank Ace. -No.: AJ009672; Gene Locus At3g50360) ; 3: Brassica napus; 4: Marsilea vestitia (corresponding to GeneBank Ace. -No.: U92973) ; 5: Atriplex nummularia (corresponding to GeneBank Ace. -No.: M90970) ; 6: Olyci'ne max ,- 7: Hordeum vulgare; 8: Lycopersicum e (Licopersicum esculentum; tomato): 9: Linseed (Linus usitatissimum) ; 10: Nicotiana tabacum 1 (corresponding to GeneBank Ace. -No.: AF072519) ; 11: Nicotiana tabacum 2 (corre- sponding to GeneBank Ace. -No.: AF072520) ; 12: Oryza sativa 1; 13: Oryza sativa 2; 14: Potato (Solanum tuberosum); 15: Triticum a 1 (Triticum aestivum, wheat); 16: Triticum a (Triticum aestivum, wheat, corresponding to GeneBank Ace. -No.: BJ253096; incomplete); 17: Zea mays; 18: Sunflower (Helianthus annuus) . The consensus sequence indicating regions of homology is given below the individual sequences.
Examples
General methods :
Unless otherwise specified, all chemicals were from Fluka
(Buchs) , Merck (Darmstadt) , Roth (Karlsruhe) , Serva (Heidelberg) and Sigma (Deisenhofen) . Restriction enzymes, DNA-modifying enzymes and molecular biological kits were from Amersham- Pharmacia (Freiburg) , Biometra (Gδttingen) , Roche (Mannheim) , New England Biolabs (Schwalbach) , Novagen (Madison, Wisconsin, USA) , Perkin-Elmer (Weiterstadt) , Qiagen (Hilden) , Stratagen (Amsterdam, Netherlands) , Invitrogen (Karlsruhe) and Ambion (Cambridgeshire, United Kingdom) . The reagents used were employed in accordance with the manufacturer's instructions.
For example, oligonucleotides can be synthesized chemically in the known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897) . The cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoreses, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, multiplication of phages and sequence analysis of recombinant DNA, are carried out as decribed by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules were sequenced using an ABI laser fluorescence DNA sequencer following the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74:5463-5467) .
Example 1 : General methods
The plant Arabidopsis thaliana belongs to the higher plants (flowering plants) . This plant is closely related to other plant species from the Cruciferae family such as, for example,
Brassica napus, but also to other families of dicotyledonous plants . Owing to the high degree of homology of its DNA sequences or its polypeptide sequences, Arabidopsis thaliana can be employed as model plant for other plant species.
Culture of Arabidopsis plants The plants are grown either on Murashige-Skoog medium supplemented with 0.5 % sucrose (Ogas et al. (1997) Science 277:91-94) or in soil (Focks & Benning (1998) Plant Physiol 118:91-101) . To achieve uniform 'germination and flowering times, the seeds are first placed on medium or scattered on the soil and then stratified for two days at 4°C. After flowering, the pods are labeled. According to the labels, pods aged 6 to 20 days post-anthesis are then harvested.
Example 2: Plasmids for the transformation of plants
Binary vectors such as pBinAR can be used for the transformation of plants (Hόfgen und Willmitzer (1990) Plant Science 66: 221- 230) . The binary vectors can be constructed by ligating the cDNA into T-DNA in sense and antisense orientation. 5' of the cDNA, a plant promoter activates the transcription of the cDNA. A polyadenylation sequence is located 3' of the cDNA. Tissue-specific esqpression can be achieved using a tissue- specific promoter. For example, seed-specific expression can be achieved by cloning in the napin or the LeB4- or the USP promoter 5' of the cDNA. Any other seed-specific promoter element can also be used. The CaMV 35S promoter can be used for constitutive expression in the whole plant.
Example 3 : Transformation of Agrobacterium
Agrobacterium-mediated plant transformation can be carried out for example using the Agrobacterium tumefaciens strains GV3101 (pMP90) (Koncz und Schell (1986) Mol Gen Genet 204: 383-396) or LBA4404 (Clontech) . Standard transformation techniques may be used for the transformation (Deblaere et al. (1984) Nucl Acids Res 13:4777-4788) .
Example 4 : Transformation of plants
Agrobacterium-mediated plant transformation can be effected using standard transformation and regeneration techniques (Gelvin SB, Schilperoort R, Plant Molecular Biology Manual, 2nd ed. , Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuch Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick BR, Thompson JE, Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 pp.', ISBN 0-8493-5164-2).
The transformation of Arabidopsis thaliana by means of Agrobacterium was carried out by the method of Bechthold et al . , 1993 (CR. Acad. Sci. Ser. Ill Sci. Vie., 316, 1194-1199).
For example, oilseed rape can be transformed by cotyledon or hypocotyl transformation (Moloney et al. (1989) Plant Cell Report 8:238-242; De Block et al.(1989) Plant Physiol 91: 694-701). The use of antibiotics for the selection of agrobacteria and plants depends on the binary vector used for the transformation and the agrobacterial strain. The selection of oilseed rape is usually carried out using kanamycin as selectable plant marker.
Agrobacterium-mediated gene transfer into linseed (Linum usitatissimum) can be carried out for example using a technique described by Mlynarova et al. (1994) Plant Cell Report 13:282- 285. Soya can be transformed for example using a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, US 5,376,543, US 5,169,770 (University of Toledo) .
The transformation of plants using particle bombardment, polyethylene glycol mediated DNA uptake or via the silicon carbonate fiber technique is described, for example, by Freeling and Walbot "The Maize Handbook" (1993) ISBN 3-540-97826-7, Springer Verlag New York) .
Example 5 : Studying the expression of a recombinant gene product in a transformed organism
The activity of a recombinant gene product in the transformed host organism was measured at the transcription and/or translation level . ■
A suitable method for determining the level of transcription of the gene (which indicates the amount of RNA available for translating the gene product) is to carry out a Northern blot as described hereinbelow (for reference see Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York, or the above examples section) , where a primer which is designed such that it binds to the gene of interest is labeled with a detectable label (usually a radiolabel or chemiluminescent label) so that, when the total RNA of a culture of the organism is extracted., separated on a gel, transferred to a stable matrix and incubated with this probe, binding and the extent of binding of the probe indicates the presence and the amount of mRNA for this gene. This information indicates the degree of transcription of the transformed gene. Cellular total RNA can be prepared from cells, tissues or organs using several methods, all of which are known in the art, for example the method Bόrmann ER et al. (1992) Mol. Microbiol. 6:317-326.
Northern hybridization:
To carry out the RNA hybridization, 20 μg of total RNA or 1 μg of pol (A) + RNA are separated by means of gel electrophoresis in 1.25% strength agarose gels using formaldehyde and following the method described by Amasino (1986, Anal. Biochem. 152, 304), transferred to positively charged nylon membranes (Hybond N+, Amersham, Brunswick) by capillary force using 10 x SSC, immobilized by UV light and prehybridized for 3 hours at 68°C using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl, 1 % SDS, 100 mg herring sperm DNA) . The DNA probe is labeled with the Highprime DNA labeling kit (Roche, Mannheim, Germany) during the prehybridization step, using alpha-32P-dCTP (Amersham Pharmacia, Brunswick, Germany) . Hybridization is carried out overnight at 68°C after addition of the labeled DNA probe in the same buffer. The wash steps are carried out twice for 15 minutes using 2 X SSC and twice for 30 minutes using 1 X SSC, 1% SDS, at 68°C. The sealed filters are exposed at -70°C for a period of 1 to 14 days.
To study the presence or the relative amount of protein translated from this mRNA, standard techniques such as a Western blot may be employed (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York) . In this method, the cellular total proteins are extracted, separated by means of gel electrophoresis, transferred to a matrix like nitrocellulose and incubated with a probe such as an antibody which binds specifically to the desired protein. This probe is usually provided with a chemiluminescent or colorimetric label which can be detected readily. The presence and the amount of the label observed indicates the presence and the amount of the desired mutated protein which is present in the cell.
Example 6 : Plant material and activation-tagging plasmid
The Arabidopsis line IC9, homozygous for a single copy of the intermolecular recombination substrate GRU'S'G'U', was used as reporter in a genetic screen aimed at identifying plants with increased frequency of homologous recombination. The plasmid pGRU'S'G'U' was constructed as follows : The 3 'end of the GUS gene (fragment US) was digested from pUS ( Tinland B (1994) Proc Natl Acad Sci USA 91(17) :8000-8004) using Xbal and Hindlll, blunted with Klenow and subsequently cloned into the Hindi site of pUC19. The resulting plasmid, pU'S', was digested with Xbal and HindiII in order to excise the U'S' fragment, which was then cloned between the Xbal and HindiII sites of the plasmid pG'U' (Tinland et al . , 1994). The construct obtained is the recombination substrate plasmid pGRU'S'G'U', which carries on its T-DNA two non-functional regions of the uidA (GUS) gene (namely U'S' and G'U') where U' represents 1213 bp of repeated identical sequence in direct orientation.
Example 7: Screening for hyper-recombination phenotype
The activation tagging plasmid pSKI015 ('Weigel D et al. (2000) Plant Physiol. 122:1003-1014), containing four repetitions of the 35S enhancer close to the right border, was mobilised into Agrobacterium and used to transform the homozygous IC9 line by the in planta transformation method. Plants were cultured in a growth chamber under a 16h/8h photoperiod (20°C/16°C) . Seeds from these plants were germinated on soil, and Basta-resistant plants, having integrated the pSKI015 plasmid, were selected. Three or four leaves from 3 week-old individual primary trans- formants (TI) were harvested and used for histochemical GUS as- say. The number of GUS+ spots per plant, representing the number of homologous recombination events per plant, was determined. Plants having at least 2 GUS+ sectors on independent leaves were considered putative mutants and analyzed further. These plants were selfed, and >30 plants from the T2 generation were analysed for homologous recombination frequency (HRF; defined as the average number of GUS+ spots in a population of plant siblings) . Experiments were at least duplicated. One of the plants ana- lyzed, called P24, showed a s'ignificant increase of HRF when compared to the IC9 parental line, and was therefore considered as a hyper-recombination mutant (Fig. 1) .
Example 8: Molecular analysis of the P24 hyper-recombination mutant
Genomic DNA of TI P24 plants was prepared using the DNeasy extraction kit (Qiagen) , and the right border-genomic DNA junction was analysed by plasmid rescue (Weigel et al . 2000). The T-DNA from pSKI015 was found to be integrated at the 3' end of the At4g37010 gene, disrupting the stop codon and the 3' UTR of this gene (Fig. 2) . Due to the orientation of the pSKI015 T-DNA, the four copies of the 35S enhancer present near the right border could drive the anti-sense expression of at least part of the At4g37010 gene.
The effect of the a&tivation tagging T-DNA on At4g37010 was determined by RT-PCR. Total RNA was prepared from T2 P24 plants using the RNeasy extraction kit (Qiagen) . The reverse transcrip- tion reaction was performed using using 3 μg of total RNA, oligo-dT primers and the Reverse transcription kit (Amersharm) . The PCR reaction was performed in a final volume of 50 μl, containing lμl of the RT reaction mixture, 1.25U of ExTaq (Takara) , 1.5 mM MgCl2, 200 μM of each dNTP and 2 μM of gene-specific primers
CEN958 (SEQ ID NO: 35): 5' -CAAATAAACGAATTGATGGCAG-3 ' and CEN3' (SEQ ID NO: 36): 5 ' -CCATTACTAATTGATTTATACTTAGC-3 ' .
Initial denaturation was done at 95°C for 1 min, and then amplification was performed for 25-35 cycles with a denaturation time of 1 min at 94°C, followed by annealing for 1 min at 58 °C and extension for 30 sec at 72°C. Different dilutions of the RT reaction mixture and number of PCR cycles were used to confirm that the PCR amplifications were within the linear range. According to these analysis, the P24 mutant (both homozygous and heterozygous forms) showed a lower At4g37010 mRNA steady state level when compared to wild type plants (Fig. 3) . This dominant negative effect can be explained by to antisense translation of the At4g37010 gene from the four copies of the 35S promoter present near the right border of the pSKI015 T-DNA. Example 9: Phenotype reproduction
In order to check if the hyper-recombinogenic phenotype showed by the P24 plant was due to downregulation of the At4g37010 gene, we tried to reproduce the phenotype using pOEXhpCEN, an At4g37010 RNAi construct that was created in the following way: the vector pPZP200 (Hajdukiewicz P et al. (1994) Plant Mol Biol. 25 (6) : 989-94) was digested with Ascl and Xbal in order to clone the nos terminator. The vector obtained was digested with EcoRI and Ascl in order to clone the selection marker cassette [mas promoter (1' -2' ) -sull gene-35S terminator]. After that, a multi- cloning site (Ncol, BstXI, Hindlll, BsrGI, Avrll) was inserted. The resulting plasmid was digested with Hindlll and Avrll, and the fad2 intron (Smith, N.A. et al . (2000) Nature 407:319-320), previously amplified by PCR in order to add the required re- striction sites, was cloned there. The vector obtained was called pOEXhp, and the second exon of the At4g37010 gene (165 bp) was cloned into it between the Ncol and Xhol sites in sense orientation and between the BsrGI and Avrll sites in antisense orientation to generate the RNAi construct pOEXhpCEN. The frag- ment for sense orientation was produced by PCR using the primers CENNcoF (5' -GGCCGGCCATGGCAGCAAATAAACGAATTGATGGCAG-3' ; SEQ ID NO: 37) and CENXhor (5' -GGCCGGCTAGAGATTATTGTCGTGGTCAATGATC-3 ' ; SEQ ID NO: 38) , which added to the resulting PCR fragment the restriction sites Ncol and Xhol respectively.
The fragment for antisense orientation was produced by PCR using the primers CENBsrGr (5' -GGCCGGTGTACAATTATTGTCGTGGTCAATGATC-3' ; SEQ ID NO: 39) and CENAvrf (5' -GGCCGGCCTAGGCAGCAAATAAACGAATT GATGGCAG-3' ,- SEQ ID NO: 40), which added to the resulting PCR fragment the restriction sites BsrGI and Avrll. The PCR reactions were performed in a final vlume of 50 μl, containing 100 ng of Arabidopsis genomic DNA, 1.25U of ExTaq (Takara) , 1.5 mM MgCl2, 200 μM of each dNTP and 2 μM of the required primers. Initial denaturation was done at 95°C for 1 min, then a plifica- tion was done for 30 cycles with a denatura.tion time of 30 sec at 94°C, followed by annealing for 30 sec at 58°C and extension for 30 sec at 72°C. The resulting pOEXhpCEN plasmid was mobilised into Agrobacterium and used to transform homozygous IC9 plants (carrying the intermolecular recombination substrate) . HRF was measured in the T2 generation in a batch of >60 plants and found to be significantly increased (Fig. 4B) . Experiments were a least duplicated. The RNAi construct was also transformed into another reporter line (IC5 ; Fig. 4A) , which differs from IC9 only in the genome location of GRU'S'G'U'. Experiments were performed as described for line IC9, and HRF was measured in 2 week old plants (at least 50 plants per replicate) and HRF was also found to be increased. The steady state level of centrin mRNA in pOEXhpCEN plants was determined by RT-PCR as describe in the previous paragraph, an for both IC9 and IC6, the steady state level of the At4g37010 mRNA was significantly reduced.
Conclusions
The mutant P24, showing a hyper-recombinogenic phenotype, was isolated using a genetic screen based on the GRU'S'G'U' recombination substrate. The hyper-recombinogenic phenotype is due to lower mRNA steady state level of the At4g37010 gene. The At4g37010 gene product is similar to human centrin 2.

Claims

We claim:
1. A method for introducing a mutation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell com- prising the steps of
a) providing one or more plant cells comprising
i) a decreased activity or expression of at least one caltractin-like protein, and
ii) at least one pair of DNA homology-sequences A and A' having a sufficient length and homology between each other to allow homologous recombination among A and A' , wherein at least one of said sequences A or A' is part of the chromosomal DNA of said plant cell,
and
b) selecting one or more plant cells comprising a mutation in said chromosomal DNA-sequence in consequence of the homologous recombination between A and A' .
2. A method as claimed in claim 1, wherein the homologous recom- bination between A and A' constitutes an intramolecular or an intermolecular recombination event .
3. A method as claimed in claim 1 or 2, wherein two homology- sequences A and A' are localized on one strand of a chromoso- mal DNA-sequence as direct repeats and wherein homologous recombination causes deletion of the sequences localized between A and A' .
4. A method as claimed in claim 1 or 2, wherein each of the two sequences A and A' undergoing homologous recombination is localized on a separate DNA molecule .
5. A method for introducing a mutation of at least one base pair in at least one chromosomal DNA-sequence of a plant cell co - prising the steps of
a) providing a plant cell comprising a decreased activity or expression of at least one caltractin-like protein b) introducing at least one DNA-construct into said plant cell, wherein said DNA-construct comprises at least one homology-sequence A having a sufficient length and homology to at least one part A' of said chromosomal sequence to fa- cilitate homologous recombination among A and A' , and wherein said DNA-construct introduces said mutation into said chromosomal DNA-sequence in consequence of the homologous recombination between A and A' , and
c) selecting cells comprising said mutation.
6. A method as claimed in claim 5, wherein the DNA-construct comprises two homology-sequences A and B having a sufficient length and homology to at least a part A' and B' of said chromosomal sequence, respectively, to allow homologous recombination between A and A' , and B and B' , respectively.
7. A method as claimed in claim 5 or 6, wherein the DNA- construct comprises additional sequences localized between the homology sequences A and B, wherein said additional sequences are introduced into the chromosomal DNA upon homologous recombination between A and A' and B and B' , respectively.
8. A method as claimed in any of claim 5 to 7 , wherein said method further comprises the steps of
d) segregating the mutation and/or additional sequences introduced by the DNA-construct and the property of an de- creased activity or expression of at least one caltractinlike protein, and
e) produce a plant with normal activity and expression of at said caltractin-like protein (s) comprising the mutation and/or additional sequences introduced by the DNA- construct .
9. A method as claimed in any of claim 1 to 8, wherein the caltractin-like protein is encoded by an amino acid sequence comprising at least one of the following sequence motifs:
a) D(T/I)D(G/N)S (G/V) (S/T) IDAXEL, b) (E/Q) (Ξ/Q)IXX(M/L) (l/M)A(E/D) (V/l)DK, c) (I/F)DXDX(N/T)GKIS, d) AD (R/Q) (D/N) XD (G/R) E, e) (E/D) (F/Y) XX (M/I) MX (K/R) T .
10. A method as claimed in any of claim 1 to 9, wherein the cal- tractin-like protein is encoded by an amino acid sequence selected from the group consisting of :
a) a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or
34; and
b) a functional equivalent polypeptide molecule comprising an amino acid sequence which is at least 60% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34 and exhibits essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or
34 ; and
c) a polypeptide molecule comprising a fragment of at least
20 consecutive amino acids, preferably 50 consecutive amino acids of at least one of the sequences described under a) or b) exhibiting essentially the same properties as a polypeptide molecule comprising an amino acid sequence described by the amino acid sequence of SEQ ID NO: 2, 4, 6, '8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
11.A method as claimed in any of claim 1 to 9, wherein the decrease of the amount of Caltractin-like protein, the Caltractin-like activity or the Caltractin-like function is ef- fected using at least one of the following methods:
a) Introduction of a double-stranded RNA of a Caltractinlike protein encoding nucleic acid sequence (Caltractinlike dsRNA) or an expression cassette (s) ensuring the expression thereof,
b) Introduction of an antisense RNA of a Caltractin-like protein encoding nucleic acid sequence or an expression cassette ensuring expression thereof,
c) Introduction of an antisense RNA of a Caltractin-like protein encoding nucleic acid sequence in combination with a ribozyme or an expression cassette ensuring expression thereof,
d) Introduction of a sense nucleic acid sequence of a Caltractin-like protein encoding nucleic acid sequence for inducing co-suppression or an expression cassette ensuring expression thereof
e) Introduction of a DNA- or protein-binding factor against aCaltractin-like protein encoding gene, RNA or Cal- tractin-like protein or an expression cassette ensuring expression thereof
f) Introduction of viral nucleic acid sequence causing degradation of the RNA encoding a Caltractin-like protein or an expression cassette ensuring expression thereof
g) Introduction of construct for inducing a homologous recombination on an endogenous Caltractin-like protein encoding gene for generating knock-out mutants
h) Introduction -of at least one mutation into an endogenous gene encoding a Caltractin-like protein for generating a loss of function.
12.An isolated polypeptide sequence coding for a caltractin-like protein, wherein said polypeptide sequence comprises an amino acid sequence described by SEQ ID NO: 6, 8, 10, 12, 14-, 18, 20, 22, 28, or 30.
13.An isolated nucleic acid sequence coding for a caltractinlike protein, wherein said nucleic acid sequence comprises an sequence described by SEQ ID NO: 5, 7, 9, 11, 13, 17, 19, 21, 27, or 29.
14. A double-stranded RNA molecule for reducing the expression of a Caltractin-like protein comprising,
i) at least one first RNA sequence which is essentially identical to at least part of a nucleic acid sequence coding for a Caltractin-like protein, and
ii) at least one second RNA sequence which is essentially complementary to at least part of said first RNA se- quence under i) .
15. A double-stranded RNA molecule of claim 14, wherein said double-stranded RNA molecule comprises,
i) at least one first RNA sequence which is essentially identical to at least part of a nucleic acid sequence comprising a sequence described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33, and
ii) at least one second RNA sequence which is essentially complementary -to at least part of said first RNA sequence under i) .
16. A transgenic expression cassette comprising at least part of a nucleic acid sequence coding for a caltractin-like protein under control of a promoter sequence functional in plant cells.
17. A transgenic expression cassette comprising a nucleic acid sequence as claimed in claim 13 or a nucleic acid sequence encoding a double-stranded RNA molecule as claimed in claim 14 or 15, in operable linkage with a promoter functional in plant cells.
18. A' transgenic expression cassette of claim 16 or 17, wherein the nucleic acid sequence coding for a caltractin-like protein is selected from the group consisting of:
a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33;
b) a nucleic acid molecule comprising a fragment of at least 20 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, or 33; c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% identical to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34; and
d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34, wherein the fragment comprises at least
10 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
19. A transgenic expression cassette of any of claim 16 to 18, wherein the nucleic acid sequence coding for caltractinlike protein is be orientated in sense and/or antisense direction with regard to the transcription direction of said promoter sequence.
20. A transgenic expression cassette of any of claim 16 to 19, wherein the expression cassette is capable to cause expression of sense, antisense or double-stranded RNA of said nucleic acid sequence coding for a caltractin-like protein.
21. A transgenic expression vectors comprising at least one nucleic acid sequence of claim 13, or a transgenic expression cassette of any of claim 16 to 20.
22. A transgenic organisms comprising at least one transgenic expression vector of claim 21 or a transgenic expression cassette of any of claim 16 to 20 or a nucleic acid sequence of claim 13,
23. A transgenic organisms of claim 22, wherein said organism is a vascular plant.
24. Transgenic plant organism having a decreased activity or expression of at least one caltractin-like protein.
25. Transgenic plant organism of claim 24, wherein said decrease is caused by transformation of said plant with at least one transgenic expression vector of claim 21 or transgenic expression cassette of any of claim 16 to 20 or by mutating at least one endogenous gene coding for a caltractin-like protein.
26. Use of at least one isolated nucleic acid sequence of claim 13, double-stranded RNA of claim 14 or 15, transgenic -ex-
' ression cassette of any of claim 16 to 20, transgenic expression vectors of claim 21, transgenic organism of claim 22 to 25, in a method for introducing a mutation of at least one base pair in at least one chromosomal DNA- sequence of a plant cell.
27. Processes for facilitating plant breeding utilizing a plant organism having a decreased activity or expression of at least one caltractin-like protein.
28. Processes of claim 27, in which the plant organism is a plant organism of any of claim 23 to 25.
PCT/EP2004/000964 2003-02-03 2004-02-03 Method for increasing efficiency of homologous recombination in plants WO2004070035A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US44456003P 2003-02-03 2003-02-03
US60/444,560 2003-02-03

Publications (2)

Publication Number Publication Date
WO2004070035A2 true WO2004070035A2 (en) 2004-08-19
WO2004070035A3 WO2004070035A3 (en) 2004-12-16

Family

ID=32850886

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2004/000964 WO2004070035A2 (en) 2003-02-03 2004-02-03 Method for increasing efficiency of homologous recombination in plants

Country Status (1)

Country Link
WO (1) WO2004070035A2 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
EP2612918A1 (en) 2012-01-06 2013-07-10 BASF Plant Science Company GmbH In planta recombination
CN108345219A (en) * 2018-03-01 2018-07-31 东华大学 Fypro production technology based on class brain memory GRU
US10280428B2 (en) * 2011-08-12 2019-05-07 Life Technologies Corporation Molecular biology tools for algal engineering
CN110684806A (en) * 2012-09-07 2020-01-14 美国陶氏益农公司 FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8691555B2 (en) 2006-09-28 2014-04-08 Dsm Ip Assests B.V. Production of carotenoids in oleaginous yeast and fungi

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024890A2 (en) * 2000-09-18 2002-03-28 Pioneer Hi-Bred International, Inc. Rice mlh1 ortholog and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024890A2 (en) * 2000-09-18 2002-03-28 Pioneer Hi-Bred International, Inc. Rice mlh1 ortholog and uses thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BHATTACHARYA D ET AL: "Molecular cloning and evolutionary analysis of the calcium-modulated contractile protein, centrin, in green algae and land plants" PLANT MOLECULAR BIOLOGY, NIJHOFF PUBLISHERS, DORDRECHT, NL, vol. 23, no. 1, 1993, pages 1243-1254, XP002266444 ISSN: 0167-4412 *
CORDEIRO M C R ET AL: "Characterization of early induced genes in Arabidopsis thaliana responding to bacterial inoculation: identification of centrin and of a novel protein with two regions related to kinase domains" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 434, no. 3, 4 September 1998 (1998-09-04), pages 387-393, XP004258807 ISSN: 0014-5793 *
MENGISTE T ET AL: "PROSPECTS FOR THE PRECISE ENGINEERING OF PLANT GENOMES BY HOMOLOGOUS RECOMBINATION" BIOLOGICAL CHEMISTRY, XX, XX, vol. 380, no. 7/8, 1999, pages 749-758, XP000885763 ISSN: 1431-6730 *
STOPPIN-MELLET VIRGINIE ET AL: "Characterization of microsome-associated tobacco BY-2 centrins" EUROPEAN JOURNAL OF CELL BIOLOGY, vol. 78, no. 11, November 1999 (1999-11), pages 842-848, XP009031813 ISSN: 0171-9335 *
VEERARAGHAVAN SUDHA ET AL: "Structural independence of the two EF-hand domains of caltractin" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 32, 9 August 2002 (2002-08-09), pages 28564-28571, XP002283043 ISSN: 0021-9258 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7851199B2 (en) 2005-03-18 2010-12-14 Microbia, Inc. Production of carotenoids in oleaginous yeast and fungi
US9909130B2 (en) 2005-03-18 2018-03-06 Dsm Ip Assets B.V. Production of carotenoids in oleaginous yeast and fungi
US10280428B2 (en) * 2011-08-12 2019-05-07 Life Technologies Corporation Molecular biology tools for algal engineering
EP2612918A1 (en) 2012-01-06 2013-07-10 BASF Plant Science Company GmbH In planta recombination
WO2013102875A1 (en) 2012-01-06 2013-07-11 Basf Plant Science Company Gmbh In planta recombination
CN110684806A (en) * 2012-09-07 2020-01-14 美国陶氏益农公司 FAD2 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
CN108345219A (en) * 2018-03-01 2018-07-31 东华大学 Fypro production technology based on class brain memory GRU

Also Published As

Publication number Publication date
WO2004070035A3 (en) 2004-12-16

Similar Documents

Publication Publication Date Title
Gidoni et al. Novel cis-acting elements in Petunia Cab gene promoters
WO2015171894A1 (en) Methods for selecting plants after genome editing
US20090083877A1 (en) Transcription Factors, DNA and Methods for Introduction of Value-Added Seed Traits and Stress Tolerance
EP4234701A2 (en) Regeneration of genetically modified plants
CN105859860A (en) Application of disease resistance-related protein to regulation and control of plant disease resistance
EP3757219A1 (en) Enhanced plant regeneration and transformation by using grf1 booster gene
EP4045522A1 (en) Enhanced disease resistance of crops by downregulation of repressor genes
CN113845578B (en) MYB transcription factor for regulating and controlling synthesis of plant procyanidine, and coding gene and application thereof
CN111727257A (en) Compositions and methods for increasing crop yield through trait stacking
US7267979B2 (en) Method of controlling gene silencing using site specific recombination
CN117106820A (en) Method for creating few lateral branches of tomatoes through genome editing and application of method
WO2004070035A2 (en) Method for increasing efficiency of homologous recombination in plants
CN106047833A (en) OsCIPK31 and application of coding gene thereof in regulation of herbicide resistance of plants
AU2004267161B2 (en) Expression cassettes for the bi-directional transgenic expression of nucleic acids in plants
CA2978099A1 (en) Modulation of dreb gene expression to increase maize yield and other related traits
US7265264B2 (en) Gene for a Dof transcription factor capable of altering the size and stature of a plant
CN106279386A (en) A kind of Rice Panicle grown on top development associated protein and encoding gene thereof and application
CN105820220A (en) Stress resistance relevant protein and application of coding gene in regulating alkali resistance of plants
KR101329224B1 (en) 1 Cys-Prx promoter from Oryza sativa L.
CN105985418A (en) Application of growth-related protein GRP4 in regulation of plant growth
CN109811005A (en) The application of plant type GAP-associated protein GAP OsSLA1 and its encoding gene in adjusting and controlling rice Leaf inclination
US7179957B1 (en) Method for altering nitrogen or oil content of seeds by down regulating AGL11 expression or activity
DE10212893A9 (en) Process for increasing the oil content in plants
WO2004085644A2 (en) Method for producing recombinant organisms
WO2003018818A2 (en) Method and means for modulating plant cell cycle proteins and their use in plant cell growth control

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG 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 NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

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

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)