US20020164757A1 - Plant proteins - Google Patents

Plant proteins Download PDF

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US20020164757A1
US20020164757A1 US10/036,492 US3649202A US2002164757A1 US 20020164757 A1 US20020164757 A1 US 20020164757A1 US 3649202 A US3649202 A US 3649202A US 2002164757 A1 US2002164757 A1 US 2002164757A1
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sequence
plant
dna
seq
amino acid
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Adriana Hemerly
Paulo Ferreira
Stephane Rombauts
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CropDesign NV
Universidade Federal do Rio de Janeiro UFRJ
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CropDesign NV
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Assigned to UNIVERSIDADE FEDERAL DO RIO DE JANEIRO, CROPDESIGN N.V. reassignment UNIVERSIDADE FEDERAL DO RIO DE JANEIRO ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FERREIRA, PAULO CAVALCANTI GOMES, HEMERLY, ADRIANA S., ROMBAUTS, STEPHANE
Publication of US20020164757A1 publication Critical patent/US20020164757A1/en
Priority to US11/239,325 priority Critical patent/US7622635B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to at least partially purified protein, capable of modulating the DNA replication in plants, muteins thereof, DNA coding therefor and to a method to confer to one or more plant cells the capacity to provide such a protein or mutein.
  • the invention also relates to plants, comprising the said DNA and the progeny thereof.
  • CDC7 kinase Activation of CDC7 as a kinase occurs at the G1/S transition of the cell cycle and is dependent on the binding with another factor, DBF4, at the G1/S transition of the cell cycle, probably by phosphorylating proteins at the origins (Kitada et al, 1992; Genetics 131: 21-29, Lei et al; Genes and Development 11, 3365-3374, 1997).
  • the CDC7 kinase may be a substrate for one or more phosphorylation events.
  • Overexpressed kinase-negative mutants of CDC7 arrest yeast cells in the G1 to S transition and inhibit growth.
  • the complex in yeast is composed of at least 8 proteins, the TPR (tetratricopeptide repeat) containing proteins CDC16, CDC23 and CDC27, and five other subunits named APC1, APC2, APC4, APC5 and APC7 (Peters et al. 1996, Science 274, 1199-1201).
  • the APC targets its substrates for proteolytic degradation by catalyzing the ligation of ubiquitin molecules to these substrates.
  • APC-dependent proteolysis is required for the separation of the sister chromatids at meta- to anaphase transition and for the final exit from mitosis.
  • APC-substrates are the anaphase inhibitor protein Pds1p and mitotic cyclins such as cyclin B, respectively (Ciosk et al. 1998, Cell 93, 1067-1076; Cohen-Fix et al. 1996, Genes Dev 10, 3081-3093; Sudakin et al. 1995, Mol Biol Cell 6, 185-198; Jorgensen et al. 1998, Mol Cell Biol 18, 468-476; Townsley and Ruderman 1998, Trends Cell Biol 8, 238-244).
  • mitotic cyclins such as cyclin B
  • CDC16, CDC23 and CDC27 need to be phosphorylated in the M-phase (Ollendorf and Donoghue 1997, J Biol Chem 272, 32011-32018).
  • Activated APC persists throughout G1 of the subsequent cell cycle to prevent premature appearance of B-type cyclins which would result in an uncontrolled entry into S-phase (Irniger and Nasmyth 1997, J Cell Sci 110, 1523-1531). It has been demonstrated in yeast that mutations in either of at least two of the APC components, CDC16 and CDC27, can result in DNA overreplication without intervening passages through M-phases (Heichman and Roberts 1996, Cell 85, 39-48).
  • CDC16, CDC23 and CDC27 all are tetratricopeptide repeat (TPR) containing proteins.
  • TPR tetratricopeptide repeat
  • a suggested minimal consensus sequence of the TPR motif is as follows: X 3 -W-X 2 -L-G-X 2 -Y-X 8 -A-X 3 -F-X 2 -A-X 4 -P-X 2 (Lamb et al. 1994, EMBO J 13, 4321-4328; X denotes any amino acid, X n a stretch of n of such amino acids).
  • the consensus residues can exhibit significant degeneracy and little or no homology is present in non-consensus residues. The hydrophobicity and size of the consensus residues, rather than their identity, seems to be important.
  • TPR motifs are present in a wide variety of proteins functional in yeast and higher eukaryotes in mitosis (including the APC protein components CDC16, CDC23 and CDC27), transcription, splicing, protein import and neurogenesis (Goebl and Yanagida 1991, Trends Biochem Sci 16, 173-177).
  • the TPR forms a ⁇ helical structure, tandem repeats organize into a superhelical structure ideally suited as interfaces for protein recognition (Groves and Barford 1999, Curr Opin Struct Biol 9, 383-389).
  • Within the ⁇ helix two amphipathic domains are usually present, one at the NH 2 -terminus and the other near the COOH-terminus (Sikorski et al. 1990, Cell 60 ,307-317).
  • the present inventors isolated several novel plant DNA sequences, coding for novel proteins, or novel amino acid sequences thereof involved in the modulation of DNA replication, using degenerated PCR primers based on known genomic or cDNA sequences, e.g. of yeast, mammals and insects.
  • Capable of modulating the DNA replication in plants is to be understood as the capacity of a protein to alter the natural DNA replication mechanism in the said plant, e.g. by up- or down-regulation of the DNA replication in a way, different from the natural situation, or to a higher or lower extent with respect to the natural situation.
  • the natural situation is to be understood as the situation wherein DNA replication takes place in plants, in which the DNA replication machinery is not affected by the introduction of foreign genetic material.
  • Such altering includes mediating e.g. the onset of DNA replication, the rate and extent of DNA replication, the timing of DNA replication in the cell cycle, coupling or uncoupling DNA replication with/from actual subsequent cell division etcetera.
  • novel cDNAs and proteins comprising one or more novel amino acid sequences were found.
  • the present invention therefore relates in the first place to an at least partially purified protein, capable of modulating DNA replication in plants, at least comprising in the amino acid sequence
  • the novel amino acid sequence SEQ ID No 2 (GYGIVYKATRKTDGTEFAIK) is located in two highly conserved domains in protein kinases, Domain I and II (Hawks et al., 1988, Science 241, 42-52).
  • the sequence GYGIV is part of the nucleotide (ATP) binding domain, also known as Domain I in protein kinases.
  • Domain I is part of the catalytic domain of protein kinases.
  • the Glycines (G) are believed to form an elbow around the nucleotide, and the Valine (V) is believed to contribute to positioning of the Glycines.
  • the first Glycine and the Valine are invariant in all protein kinases.
  • the second Glycine is almost invariant.
  • the sequence AIK in the same peptide is also highly conserved and it is located in Domain II, which is also part of the catalytic domain.
  • the Alanine (A) and the Lysine (K) are invariant in all kinases, and the Isoleucine is highly conserved.
  • the Lysine residue appears to be involved in mediating the phosphotransfer reaction (Hawks et al, 1988).
  • the novel exon encoded by amino acid sequence SEQ ID No 3 (DVIEKKDGPCSGTKGFRAPE) is part of Domain VIII of protein kinases. Mutagenesis has implicated a role of this domain in the catalytic activity (Hawks et al., 1988).
  • the amino acids Threonine (T), Phenylalanine and Alanine (A) are highly conserved, and the Glutamic Acid (E) is invariant.
  • the novel exon encoded by amino acid sequences SEQ ID No 4 (NIKDIAQLRGSEELWEVAKLHNRESSFPK) is located in Domain XI of protein kinases, and that in the peptide, the first Leucine (L), and the second Lysine (K) are highly conserved and therefore are believed to be quite important for the correct activity of the protein.
  • CDC27A1 Arabidopsis thalian cDNA sequence termed CDC27A1 was found, which upon comparison in the above mentioned databanks, showed high homology with an Arabidopsis thalian genomic DNA sequence (accession number AC 001645).
  • the coding sequence of CDC27A1 (SEQ ID NO 9), found by the present inventors, indicated the presence of two additional coding regions in the Arabidopsis CDC27, the gene, corresponding with the amino acid sequences given by SEQ ID NOS 6 and 7.
  • novel DNA replication modulating proteins in plants were found, comprising one or more of the above mentioned novel amino acid sequences.
  • the novel exon encoded by amino acid sequence SEQ ID No 6 (VNLQLLARCYLSNQAYSAYYILK) is part of a unique NH 2 -terminal domain conserved in CDC27 homologues of different origin.
  • the unique domain is located upstream of the NH 2 -terminal TPR unit of CDC27 (Tugendheim et al. 1993, Proc Natl Acad Sci USA 90, 10031-10035).
  • the role of this domain is currently not known, but its conservation suggests that it is indispensable for CDC27 function.
  • the NH 2 -terminal TPR of CDC27 is not tandemly repeated and spans the amino acid residues 174 to 202 in SEQ ID No 5.
  • Proteins, comprising this novel exon sequence according to the invention may therefore promote APC-substrate action and therewith allowing DNA-replication.
  • a peptide comprising the novel exon sequence may be used to occupy the binding region of the substrates for the APC complex, and therewith inhibiting the complex-substrate interactions, resulting in inactivation of APC and to polyploiddization/endoreduplication.
  • the novel amino acid sequence SEQ ID No 7 (AYMERLILPDELVTEENL) is located just after the last (10th) TPR of CDC27 spanning the amino acid residues 670-703 in SEQ ID No 5. Carboxy-terminal extensions downstream from this 10 th TPR and variable in length and sequence are common in all known CDC27 proteins. However, the sequence SEQ ID No 7 shows 50 and 55% homology to the corresponding regions of the CDC27 homologues of Schizosaccaromyces pombe and Aspergillus nidulans, respectively.
  • RLI three consecutive core amino acids of this TPR, RLI, are also present in SEQ ID No 7 and, although very limited, some further homology can be discovered.
  • SEQ ID No 7 is part of a truncated TPR.
  • the block of tandemly repeated TPRs in CDC27 should be extended from 9 (spanning amino acids 406 to 703 in SEQ ID No 5) to 10 (amino acids 704 to 728 in SEQ ID No 5).
  • a dimer of the basic 34 amino acid TPR repeat is the more common evolutionary unit (Sikorski et al. 1990, Cell 60, 307-317).
  • CDC27A2 is characterized in that a fragment of 33 nucleotides present in CDC27A1 (nucleotides 1029-1061 of SEQ ID NO 9) is missing in CDC27A2.
  • the nucleotide sequence of the CDC27A2 cDNA is given in SEQ ID NO 14, the corresponding amino acid sequence of the CDC27A2 protein is defined in SEQ ID NO 11.
  • SEQ ID NO 11 is different from SEQ ID NO 5 in that the amino acid sequence ‘AIPDTVTLNDP’ (SEQ ID NO 12) present in CDC27A1 is absent in CDC27A2.
  • CDC27B has GenBank accession number AC006081 and is annotated as CDC27. However, upon isolation and characterization of the corresponding cDNA, the present inventors noticed that the amino acid sequence predicted and presented in GenBank is lacking the stretch of 161 NH 2 -terminal amino acids as given in SEQ ID NO 10.
  • the cDNA sequence of CDC27B is defined in SEQ ID NO 15 and the derived amino acid sequence of the CDC27B protein is given in SEQ ID NO 13.
  • the full-length CDC27B protein comprises a peptide 75% identical to the peptide as defined in SEQ ID NO 6.
  • SEQ ID NO 6, and thus also SEQ ID NO 10 are part of a unique NH 2 -terminal domain conserved in CDC27 homologues of different origin.
  • Any erroneous modulation of APC activity e.g. by mutations in SEQ ID No 6 as part of a conserved sequence in CDC27 proteins and/or SEQ ID No 7 being a putative novel truncated TPR motif in CDC27, will likely result in loss of control over normal DNA replication cycles via the mechanisms described above. Mutations in CDC27 can indeed trigger DNA overreplication and thus the generation of polyploid cells (Heichmann and Roberts 1996, Cell 85, 39-48). Such endoreduplication might be related to cell expansion (Traas et al. 1998, Curr Opin Plant Biol 1, 498-503) and, thus, a higher storage capacity in such polyploid cells. This advantageous property is highly desired in crop plants or parts of plants such as seeds, roots, tubers and fruits.
  • DNA replication modulating proteins according to the present invention comprising one or more of the above mentioned amino acid sequences, or having 80% amino acid identity therewith, may originate from plant species as well as from other species as long as the said proteins are capable of modulating DNA replication in one or more plant species.
  • protein is to be understood as any amino acid sequence having a biological function, optionally modified by e.g. glycosylation.
  • the protein according to the present invention preferably comprises one or more of the amino acid sequences according to c) or d), the respective amino acid identity preferably being at least 50%.
  • protein includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means.
  • polypeptide includes peptides of two or more amino acids in length, typically having more than 5, 10 or 20 amino acids.
  • amino acid sequences of the invention are not limited to the sequences obtained from the particular protein but also include homologous sequences obtained from any source, for example related plant proteins, cellular homologues and synthetic peptides, as well as variants or derivatives thereof.
  • the present invention covers variants, homologues or derivatives of the amino acid sequences of the present invention, as well as variants, homologues or derivatives of the nucleotide sequence coding for the amino acid sequences of the present invention.
  • a homologous sequence is taken to include an amino acid sequence which is at least 50, 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level over at least 18, preferably all amino acids within the sequences as shown in SEQ ID Nos 2, 3, 4, 6 and 7 in the sequence listing herein.
  • homology should typically be considered with respect to those regions of the sequence known to be essential for the above discussed functions of the novel amino acid sequences rather than non-essential neighbouring sequences.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
  • Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
  • % Homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • variant in relation to the amino acid sequences of the present invention includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence has similar activity as the polypeptides presented in the sequence listings.
  • sequences of the invention may be modified for use in the present invention. Typically, modifications are made that maintain the activity of the sequence.
  • Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified sequence retains the relevant activity.
  • the kinase activity should be maintained in such a variant of a peptide according to the invention comprising SEQ ID NO 2.
  • Amino acid substitutions may include the use of non-naturally occurring analogues, for example to increase blood plasma half-life of a therapeutically administered polypeptide.
  • Proteins of the invention are typically made by recombinant means. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins of the invention may also be produced as fusion proteins, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6 ⁇ His, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the function of the protein of interest sequence.
  • Proteins of the invention may be in a substantially isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as substantially isolated.
  • a protein of the invention may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a protein of the invention.
  • the protein according to the present invention comprises the amino acid sequence as given in SEQ ID NO 1 or NO 5 or NO 11 or NO 13, or has at least 50%, preferably at least 60%, more preferably at least 70, still more preferably 80% and most preferably at least 90% amino acid identity with one of the said sequences.
  • SEQ ID NO 1 relates to the complete amino acid sequence (889 AA) of the novel CDC7 protein according to the present invention comprising SEQ ID NOS 2, 3 and 4 (AA 411-430, 710-729, 767-795).
  • SEQ ID NO 5 is the complete amino acid sequence (727 AA) of the novel plant CDC27A1 comprising SEQ ID NOS 6 and 7 and 12 (AA 37-60 and AA 711-727 and AA 344-354 respectively).
  • SEQ ID NO 11 is the complete amino acid sequence (716 AA) of the novel plant CDC27A2 comprising SEQ ID NOS 6 and 7 (AA 37-60 and AA 700-716, respectively) but lacking SEQ ID NO 12.
  • SEQ ID NO 13 is the complete amino acid sequence (739 AA) of the novel plant CDC27B comprising SEQ ID NO 10 (AA-1-161) which itself comprises a peptide 75% identical to SEQ ID NO 6 (AA 36-59).
  • the proteins according to the present invention may be of non-plant origin, as is indicated above, the protein according to the present invention is preferably a plant protein, more preferably a CDC7 or CDC27 protein, or a functional analogue thereof.
  • a functional analogue is to be understood as any protein or peptide having similar biological effects as a plant CDC7 protein or a CDC27 protein, irrespectively of the origin thereof.
  • the present invention relates to a mutein of the protein according to the present invention, said mutein comprising at least one amino acid substitution, deletion or addition, affecting the DNA replicative effect of the said protein.
  • the proteins according to the present invention are of high interest for an improvement of e.g. agricultural crops or parasite resistance.
  • the modulating effect thereof can be affected, which may lead to desirable or improved properties of the protein.
  • DNA replication modulating proteins according to the invention may be activated or deions or additions may be situated within or flanking the amino acid sequence, as given by SEQ ID NOS 2, 3, 4, 6, 7, 10 or 12 (or having at least 50% amino acid identity therewith).
  • DNA replicating modulating proteins according to the invention may also comprise one or more tetratricopeptide repeat (TPR) domains.
  • TPR tetratricopeptide repeat
  • Such domains have been identified in CDC27 (amino acid regions 174-202, 403-431, 432-465, 466-499, 500-533, 534-567, 568-601, 602-635, 636-669, 670-703 in SEQ ID No 5; delineation of regions based on the yeast CDC27 homologue; Lamb et al. 1994, EMBO J 13, 4321-4328) as well as in CDC16, CDC23 and many other proteins (Goebl and Yanagida 1991, Trends Biochem Sci 16, 173-177).
  • TPR domains The function of these TPR domains is to enable the protein to interact with other proteins in the anaphase promoting complex (APC).
  • APC anaphase promoting complex
  • CDC27 protein a novel TPR or TPR-like domain has been identified which includes SEQ ID No 7. Mutation analysis in TPR domains of yeast CDC27 has revealed that intact TPRs are necessary for CDC27 function (Lamb et al. 1984, EMBO J 13, 4321-4328) and, thus, also for a functional APC. In the absence of CDC27 function, DNA synthesis becomes uncoupled from cell cycle progression resulting in the establishment of polyploid cells (Heichman and Roberts 1996, Cell 85, 39-48).
  • the present invention relates to a peptide, comprising
  • These peptides are or maybe part of important regulatory sites for binding cellular factors or being a substrate for activating/deactivating mechanisms, such as phosphorylation.
  • the present invention relates to antibodies specifically recognizing a cell cycle interacting protein according to the invention or parts, i.e. specific fragments or epitopes, of such a protein.
  • the antibodies of the invention can be used to identify and isolate other cell cycle interacting proteins and genes in any organism, preferably plants.
  • These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc.
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfré, J. Meth. Enzymol.
  • antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of proteins according to the invention as well as for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention.
  • surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies selections, yielding a high increment of affinity from a single library of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In many cases, the binding phenomena of antibodies to antigens is equivalent to other ligand/anti-ligand binding.
  • the present invention relates to a non-genomic DNA sequence, coding for a protein or mutein or peptide according to the present invention, or a DNA sequence having a sequence homology of at least 75% with the said sequence, or to the complementary sequence thereof. Also DNA sequences having at least 75% homology with the above mentioned DNA sequences are encompassed within the invention. These sequences are particularly useful in the generation of DNA vectors to multiply the DNA sequence or to introduce the said sequence in a host organism, in order to obtain the encoded protein. Further said sequences or parts thereof are advantageously used to identify and isolate homologous sequences from other biological species.
  • the DNA sequence is preferably substantially free of sequences intervening the coding sequence, and is preferably cDNA.
  • DNA-sequences of the invention comprise nucleic acid sequences encoding the amino acid sequences of the invention. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.
  • Polynucleotides of the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.
  • variant in relation to the nucleotide sequence of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a polypeptide, preferably having at least the same activity as sequences presented in the sequence listings.
  • sequence homology preferably there is at least 75%, more preferably at least 85%, more preferably at least 90% homology to the sequences shown in the sequence listing herein. More preferably there is at least 95%, more preferably at least 98%, homology.
  • Nucleotide homology comparisons may be conducted as described above.
  • a preferred sequence comparison program is the GCG Winsconsin Bestfit program described above.
  • the default scoring matrix has a match value of 10 for each identical nucleotide and ⁇ 9 for each mismatch.
  • the default gap creation penalty is ⁇ 50 and the default gap extension penalty is ⁇ 3 for each nucleotide.
  • the present invention also encompasses nucleotide sequences that are capable of hybridising selectively to the sequences presented herein, or any variant, fragment or derivative thereof, or to the complement of any of the above.
  • Nucleotide sequences are preferably at least 15 nucleotides in length, more preferably at least 20, 30, 40 or 50 nucleotides in length.
  • hybridization shall include “the process by which a strand of nucleic acid joins with a complementary strand through base pairing” as well as the process of amplification as carried out in polymerase chain reaction technologies.
  • Preferred polynucleotides of the invention will comprise regions preferably at least 80 or 90% and more preferably at least 95% homologous to nucleotides (1229-1291), (2126-2187) or (2298-2385) of SEQ ID No 8 or (109-181) or (2125-2181) or (1029-1061) of SEQ ID No 9; or (109-181) or (2092-2148) of SEQ ID NO 14; or (1-483) of SEQ ID NO 15.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego Calif.), and confer a defined “stringency” as explained below.
  • Maximum stringency typically occurs at about Tm-5° C. (5° C. below the Tm of the probe); high stringency at about 5° C. to 10° C. below Tm; intermediate stringency at about 10° C. to 20° C. below Tm; and low stringency at about 20° C. to 25° C. below Tm.
  • a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.
  • both strands of the duplex are encompassed by the present invention.
  • the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.
  • Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of ways.
  • Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other viral/bacterial, or cellular homologues particularly cellular homologues found in plant cells may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein.
  • Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing such libraries with probes comprising all or part of SEQ ID Nos 8 or 9 or 14 or 15. This may be useful where for example under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • polynucleotides may be obtained by site directed mutagenesis of characterised sequences, such as SEQ ID No 8 or 9. This may be useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • Polynucleotides of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.
  • Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
  • Polynucleotides such as a DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.
  • primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the lipid targeting sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA.
  • the primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
  • the DNA sequence according to the invention it may in some instances be advantageous to incorporate one or more intervening sequences (introns) in the sequence coding for the protein to be expressed, as in some expression systems, one or more splicing events must take place in order to obtain high expression rates (e.g. for expression of a barley thionin in transgenic tobacco; Carmona et al. 1993, Plant J 3, 457-462).
  • intervening sequences introns
  • the coding sequence i.e. the cDNA
  • the proper regulatory elements such as promotor and terminator sequences
  • the invention in a special embodiment (referring to FIGS. 1 and 2), relates to a cDNA sequence, comprising the DNA sequence as given by SEQ ID NO 8 or SEQ ID NO 9 or SEQ ID NO 14 or SEQ ID NO 15, or having a sequence homology with SEQ ID NO 8 or SEQ ID NO 9 or SEQ ID NO 14 or SEQ ID NO 15 of at least 75% or is the complementary sequence thereof.
  • SEQ ID NO 8 is the cDNA sequence of CDC7 of Arabidopsis thaliana ,comprising the coding sequence for the newly identified amino acid sequences (SEQ ID NOS 2, 3 and 4) as are discussed above.
  • SEQ ID NO 9 is the cDNA sequence of CDC27 of Arabidopsis thaliana ,includes the sequences coding for the newly identified amino acid sequences (SEQ ID NOS 6 and 7 and 12) as discussed above.
  • SEQ ID NO 14 is the cDNA sequence of CDC27A2 of Arabidopsis thalian and includes the sequences coding for the newly identified amino acid sequences (SEQ ID Nos 6 and 7) as discussed above but lacks the sequence coding for the newly identified amino acid sequence (SEQ ID NO 12).
  • SEQ ID NO 15 is the cDNA sequence of CDC27B of Arabidopsis thalian and includes the sequences coding for the newly identified amino acid sequence (SEQ ID NO 10) as discussed above.
  • DNA replication modulating proteins in particular in CDC7 and CDC27 respectively
  • the sequences according to SEQ ID NOS 8 and 9 and 14 and 15, or parts thereof can advantageously be used to isolate and identify homologntary sequence thereof.
  • Such a DNA sequence codes for an amino acid sequence that till now was not known to be part of DNA replication modulating proteins, in particular of CDC7 and CDC27. It was now found, that DNA sequences, corresponding to the nucleotides 1229-1291, 2126-2187 and 2298-2385 of SEQ ID NO 8 code for new amino acid sequences of plant CDC7.
  • the DNA sequence, corresponding to nucleotides 109-181 and 2125-2148 of SEQ ID NO 9 code for novel amino acid sequences of plant CDC27A1, of Arabidopsis thaliana .
  • the DNA sequence, corresponding to nucleotides 109-181 and 2092-2148 of SEQ ID NO 14 code for novel amino acid sequences of plant CDC27A2 of Arabidopsis thaliana .
  • the DNA sequence, corresponding to nucleotides 1-483 of SEQ ID NO 15 codes for novel amino acid sequence of plant CDC27B of Arabidopsis thaliana.
  • Said DNA sequences may therefore in particular be used to identify and isolate genes or gene fragments from other plants or organisms that are homologous to the CDC7 or CDC27 sequence discussed above.
  • the DNA sequences according to the invention may be used as primers for use in a nucleic acid amplification technique.
  • Said primers can be used in a particular amplification technique to identify and isolate substantially homologous nucleic acid molecules from other plant species.
  • the design and use of said primers is known by the person skilled in the art.
  • amplification primers comprise a contiguous sequence of at least 6 nucleotides, in particular 13 nucleotides, preferably 15 to 25 nucleotides or more, identical or complementary to the nucleotide sequence encoding the amino acid sequence of SEQ ID Nos 1-7 and 10-13.
  • Another application is the use as a hybridization probe to identify nucleic acid molecules hybridizing with a nucleic acid molecule of the invention by homology screening of genomic DNA or cDNA libraries.
  • an appropriate marker for specific applications, such as for the detection of the presence of a nucleic acid molecule of the invention in a sample derived from an organism, in particular plants.
  • Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
  • the nucleic acid sequence for a protein of the invention can also be used to generate hybridization probes for mapping the naturally occurring genomic sequence.
  • the sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridization to chromosomal spreads, flow-sorted chromosomal preparations, or artificial chromosome constructions such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries as reviewed in Price (Blood Rev. 7 (1993), 127-134) and Trask (Trends Genet. 7 (1991), 149-154).
  • Polynucleotides of the invention can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell.
  • the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell.
  • Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.
  • a polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • operably linked means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • Vectors of the invention may be transformed or transfected into a suitable host cell as described below to provide for expression of a protein of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and optionally recovering the expressed protein.
  • the vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used, for example, to transfect or transform a host cell.
  • Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in.
  • promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters which are functional in mammalian, cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used.
  • the promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). Tissue-specific promoters specific for selected plant tissue cells are particularly preferred, see below in section “transgenic plants”.
  • the promoters may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the invention relates to DNA vectors, particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that comprise a DNA sequence according to the invention.
  • Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors: see for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Habor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994).
  • Said vector further preferably comprises a promoter, functional in plant cells, operably linked to the DNA sequence, according to the invention. With such a vector, the DNA sequence according to the invention can be expressed in plant cells and may modulate the DNA replication in the said cells.
  • the present invention relates to a method for identifying and/or obtaining proteins capable of modulating the DNA replication in plants, comprising a two-hybrid screening assay, using CDC27 or CDC7 polynucleotide sequences as a bait and a cDNA library of a cell suspension culture as prey.
  • the yeast two-hybrid assay is a genetic strategy developed to identify proteins (encoded by the cDNAs, the ‘preys’) able to interact in vivo with a known protein (the ‘bait’). Interactions between proteins are detected through the reconstitution of the activity of a transcription activator and the subsequent expression of a reporter gene.
  • the cell culture may be from any organism possessing cell cycle interacting proteins such as animals, preferably mammals. Particularly preferred are plant cell suspension cultures such as from Arabidopsis.
  • the nucleic acid molecules encoding proteins or peptides identified to interact with CDC7 or CDC27 in the above mentioned assay can be easily obtained and sequenced by methods known in the art. Therefore, the present invention also relates to a DNA sequence encoding a cell cycle interacting protein obtainable by the method of the invention.
  • transformed plants can be made using the nucleotide sequences according to the invention.
  • Such a transformation of the new gene(s), proteins or inactivated variants/muteins thereof will either positively or negatively have an effect on cell division.
  • Methods to modify the expression levels and/or the activity are known to persons skilled in the art and include for instance overexpression, co-suppression, the use of ribozymes, sense and anti-sense strategies, gene silencing approaches.
  • Sense strand refers to the strand of a double-stranded DNA molecule that is homologous to a mRNA transcript thereof.
  • the “anti-sense strand” contains an inverted sequence which is complementary to that of the “sense strand”.
  • the nucleic acid molecules according to the invention are in particular useful for the genetic manipulation of plant cells in order to modify the characteristics of plants and to obtain plants with modified, preferably with improved or useful phenotypes.
  • the invention can also be used to modulate the cell division and the growth of cells, preferentially plant cells, in in vitro cultures.
  • a transformed plant can thus be obtained by transforming a plant cell with a gene encoding a polypeptide concerned or fragment thereof alone or in combination.
  • tissue specific promoters in one construct or being present as a separate construct in addition to the sequence concerned, can be used.
  • the present invention relates to a method for the production of transgenic plants, plant cells or plant tissue comprising the introduction of a nucleic acid molecule or vector of the invention into the genome of said plant, plant cell or plant tissue.
  • the invention further relates to a method for modulating DNA replication in plant cells, plant parts or plants by conferring to one or more plant cells the capacity to provide a protein, or a mutein thereof according to the invention, in an amount sufficient to modulate DNA replication and/or to block mitosis of the said cells.
  • the said capacity is conferred to one or more plant cells, by
  • [0118] c) incubating the cells, plant parts or plants at conditions, allowing expression of the DNA according to claim 11 or 12, to produce a protein according to the invention or a mutein thereof according to the invention.
  • the molecules are placed under the control of regulatory elements which ensure the expression in plant cells.
  • regulatory elements may be heterologous or homologous with respect to the nucleic acid molecule to be expressed as well with respect to the plant species to be transformed. In general, such regulatory elements comprise a promoter active in plant cells.
  • constitutive promoters are used, such as the 35 S promoter of CaMV (Odell, Nature 313 (1985), 810-812) or promoters of the polyubiquitin genes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689).
  • tissue specific promoters see, e.g., Stockhaus, EMBO J. 8 (1989), 2245-2251).
  • promoters which are specifically active in tubers of potatoes or in seeds of different plants species, such as maize, Vicia, wheat, barley etc. Inducible promoters may be used in order to be able to exactly control expression.
  • inducible promoters are the promoters of genes encoding heat shock proteins. Also microspore-specific regulatory elements and their uses have been described (WO96/16182). Furthermore, the chemically inducible Tet-system may be employed (Gatz, Mol. Gen. Genet. 227 (1991); 229-237). Further suitable promoters are known to the person skilled in the art and are described, e.g., in Ward (Plant Mol. Biol. 22 (1993), 361-366).
  • the regulatory elements may further comprise transcriptional and/or translational enhancers functional in plants cells. Furthermore, the regulatory elements may include transcription termination signals, such as a poly-A signal, which lead to the addition of a poly A tail to the transcript which may improve its stability.
  • Methods for the introduction of foreign DNA into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes , the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, biolistic methods like particle bombardment, pollen-mediated transformation, plant RNA virus-mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus and other methods known in the art.
  • the plants which can be modified according to the invention and which either show overexpression of a protein according to the invention or a reduction of the synthesis of such a protein can be derived from any desired plant species.
  • They can be monocotyledonous plants or dicotyledonous plants, preferably they belong to plant species of interest in agriculture, wood culture or horticulture interest, such as crop plants (e.g. maize, rice, barley, wheat, rye, oats etc.), potatoes, oil producing plants (e.g. oilseed rape, sunflower, pea nut, soy bean, etc.), cotton, sugar beet, sugar cane, leguminous plants (e.g.
  • the invention further relates to progeny of such plants and to plant material such as roots, flowers, fruit, leaves, pollen, seeds, seedlings or tubers, obtainable from the plant according to the invention.
  • the invention further relates to a plant cell, transformed with a vector according to the present invention, or comprising DNA according to the present invention.
  • the invention also relates to plants, obtainable by the method according to the present invention and to progeny of such a plant and to plant material, such as roots, flowers, fruit, leaves, pollen, seeds, seedlings or tubers, obtainable from the plant according to the invention.
  • expression of dominant negative mutants of CDC7 or CDC27 are used to modulate DNA replication in plant cells, plant tissues, plant organs and/or whole plants. These embodiments involve the overexpression of a mutein or mutant gene according to the present invention which will inhibit the function of a wild-type allele when expressed in the same cell, thereby the phenotype of a transgenic plant, plant organ or plant cell expressing the mutant will be that of a blocked cell cycle progression.
  • Herskowitz Nature 329: 219-222 (1987), reviews the inactivation of genes by interference at the protein level, which is achieved through the expression of specific genetic elements encoding a polypeptide comprising both intact, functional domains of the wild type protein as well as nonfunctional domains of the same wild type protein. Such peptides are known as dominant negative mutant proteins.
  • a DNA vector comprises DNA, coding for a mutein according to the present invention, that is operably linked to a nematode-induced promoter, said promoter functional in plant cells.
  • Nematode infection of plants may cause severe problems to plant growth and crop generation. After penetrating the roots of their hosts, nematodes induce, at the infection sites, the development of feeding cells, specialised in the uptake of solutes from the vascular system of the plant. These infection sites are of crucial importance for the development for the parasite. In this way, root-knot nematodes induce multinucleated giant cells in the infected plant with highly elevated DNA contents.
  • a CDC7 mutein which is not further capable to induce the onset of the DNA synthesis, e.g. by loss of one or more phosphorylation sites or loss of binding function to a plant homolog of yeast DBF4 (Jackson et al 1993 Mol Cell Biol 13, 2899-2908) could, when present in sufficient amounts, block the onset of the DNA synthesis.
  • DNA coding for such a mutein, and under the control of a promoter, functional in plant cells and inducible by the presence of nematodes in or in the vicinity of the plant cells, is comprised in the plant cells
  • the mutein can be expressed in the presence or vicinity of nematodes. This may lead to a DNA synthesis block, therewith avoiding further nematode development.
  • the advantage of such a system is the fact that the plant is not producing any heterologous nematocide, that may be harmful for the plant itself.
  • Such a system is not restricted to CDC7.
  • the person, skilled in the art, aware of this application, will be well aware of the possibilities to take other DNA replication modulating proteins, such as CDC27 for developing an analogous anti-nematode system.
  • a further embodiment of the invention involves the down regulation of CDC27.
  • a further embodiment of the invention involves the downregulation of CDC27 resulting in suppression of the APC complex, modulation of DNA replication and/or blocking mitosis. This can be achieved by expression of CDC27 point mutants.
  • An alternative strategy can be envisaged involving a CDC27 mutein consisting of a block of TPR tandem repeats. Such a mutein is still likely to interact with other TPR-containing proteins from the APC such as CDC16 and CDC23 or APC regulator proteins such as PP5. As such, APC component proteins or APC regulator proteins would probably be titrated out and normal APC function be prevented.
  • a CDC27 mutein wherein the SEQ ID No 7 has been mutated, leading to the incapability of this mutein to bind with other factors of the APC can be mentioned.
  • the mutated protein would be still able to interact with the substrate, therewith titrating out the APC, abolishing or at least seriously reducing the APC-function, leading to the formation of polyploid cells.
  • mutations in SEQ ID No 6 or 10 could render the mutein incapable of interacting with the substrate but still capable of binding with the other factors of the APC-complex. The result is the generation of a dominant negative, as the complex will not be able to drive the destruction of key components of the cell cycle machinery, responsible to control the number of DNA-replication cycles.
  • DNA sequences, vectors or proteins, regulatory sequences or recombinant DNA molecules of the invention can be used to modulate, for instance, endoreduplication in storage cells, storage tissues and/or storage organs of plants or parts thereof.
  • Preferred target storage organs and parts thereof for the modulation of endoreduplication are, for instance, seeds (such as from cereals,als, oilseed crops), roots (such as in sugar beet), tubers (such as in potato) and fruits (such as in vegetables and fruit species). Furthermore it is expected that increased endoreduplication in storage organs and parts thereof correlates with enhanced storage capacity and as such with improved yield.
  • a plant with modulated endoreduplication in the whole plant or parts thereof can be obtained from a single plant cell by transforming the cell, in a manner known to the skilled person, with the above-described means.
  • Another embodiment of the invention relates to a method for modulating DNA replication and the resultant generation of male or female sterile plants. This would be achieved by the expression of dominant negative mutants of either cdc7 or cdc27 under the control of very specific promoters—either from male or female gametophytes—to block cell division and disrupt meiosis. The resulting plants would be naturally sterile.
  • Another embodiment of the invention relates to a method for the generation of plant cells, plant tissues, plant organs, or whole plants with the capacity for the overexpression of CDC7 in combination with a plant homolog of Dbf4 thereby modulating DNA replication.
  • Results in yeast indicate that the association of Dbf4 with CDC7 is essential for the G1 to S transition, namely DNA synthesis (Ohtoshi A, Miyake T, Arai K, Masai H; Mol Gen Genet 254(5): 562-70 May 20, 1997). Therefore in the present invention, by overexpressing both CDC7 and Dbf4 proteins, one can activate, stimulate or initiate DNA synthesis in cells where DNA synthesis does not normally take place, such as cells that have already gone through the cell cycle. As a consequence the amount of DNA is increased in the cell therewith manipulating the level of endoreduplication as is outlined above.
  • Another embodiment of the invention relates to the generation of polyploid plant cells, plant parts or plants.
  • plant cells are transformed with a vector, comprising the coding sequence of plant CDC27, according to the present invention, under the control of a suitable promotor and optionally other expression controlling elements, these plant cells may produce CDC27.
  • a suitable promotor and optionally other expression controlling elements these plant cells may produce CDC27.
  • extra rounds of DNA replication may take place before mitosis, leading to polyploid cells.
  • FIGS. 1 and 2 and 5 The architecture of the CDC7 and CDC27 genes are illustrated in FIGS. 1 and 2 and 5 .
  • FIG. 1 illustrates the genomic architecture of the Arabidopsis CDC7 gene, wherein the exons are boxed. The numbers above the box indicate the length of the exon, the number below and between two boxes indicates the length of the intron.
  • the total length of the coding sequence is 2667 nucleotides, coding for 889 amino acids.
  • the fifth, eleventh and thirteenth exons comprise novel coding sequence; in FIG. 1, the corresponding boxes are black. It is to be understood, and obvious to a skilled person, that the first and the last triplet of the coding sequence of an exon, may partially be encoded by the last two or one nucleotide(s) from the adjacent downstream exon, and, accordingly, by the first two or one nucleotide(s) of the adjacent upstream exon.
  • FIGS. 2 and 5 the genomic architecture of the CDC27A1 and CDC27B genes, respectively, of Arabidopsis thalian are depicted as explained for FIG. 1.
  • the second and the sixteenth (last) exon (black in FIG. 2) comprise novel coding sequences and were not identified in the known genomic CDC27A1 sequence of Arabidopsis thalian (see text).
  • the entire sequence comprises 2184 nucleotides, corresponding to 727 amino acids.
  • the first 5 exons (black in FIG. 5) and part of the 6 th exon (black in FIG. 5) comprise novel coding sequences and were not identified in the known genomic CDC27B sequence of Arabidopsis thalian (see text).
  • the entire sequence comprises 2151 nucleotides, corresponding to 716 amino acids.
  • FIGS. 3 and 4 the complete cDNA sequence of CDC7 and CDC27A1, respectively, according to the present invention are depicted, with the respective encoded amino acid sequence therebelow.
  • Vertical lines in the nucleotide sequence indicate the exon boundaries, i.e. 2
  • the exon boundaries are derived from genomic CDC7 and CDC27A1 sequences (see examples 1 and 2 respectively).
  • Such lines are also drawn in the amino acid sequence, although, as is indicated above, the amino acids, flanking such a vertical line, may be partially encoded by the adjacent exon. Exact positioning of the vertical line is in such a case not possible and is set at the left or the right of such an amino acid in an arbitrary manner. See examples 1 and 2 for further details.
  • FIGS. 7 and 8 the expression of CDC27A and CDC27B genes is illustrated.
  • FIG. 7A shows expression of CDC27A genes (both CDC27A1 and CDC27A2 are detected; indicated by the arrows) in several Arabidopsis thalian tissues: 1-etiolated seedlings; 2-flowers; 3-buds; 4-stems; 5-leaves; 6-roots; siliques; ⁇ negative control.
  • FIG. 7A shows expression of CDC27A genes (both CDC27A1 and CDC27A2 are detected; indicated by the arrows) in several Arabidopsis thalian tissues: 1-etiolated seedlings; 2-flowers; 3-buds; 4-stems; 5-leaves; 6-roots; siliques; ⁇ negative control.
  • FIG. 7A shows expression of CDC27A genes (both CDC27A1 and CDC27A2 are detected; indicated by the arrows) in several Arabidopsis thalian tissues: 1-etiola
  • FIG. 7B shows the expression of CDC27A genes in Arabidopsis thalian root cultures treated with different substances: 1-abscisic acid (ABA); 2-2,4-dichlorophenoxyacetic acid (2,4-D); 3-hydroxyurea; 4-kinetin; 5-kinetin+1-naphthaleneacetic acid (NAA); 6-NAA; 7-oryzalin; 8-starvation; 9-untreated control roots; ⁇ negative control.
  • FIG. 8A shows the expression of the CDC27B gene in several Arabidopsis thaliana tissues as outlined in FIG. 7A.
  • FIG. 7B illustrates the expression of the CDC27B gene in Arabidopsis root cultures treated with different substances as outlined in FIG. 7B.
  • oligonucleotides were as follows: 1 (sense): 5′ AAA/G ATA/C/T GGA/C/G/T GAA/G GGA/C/G/T ACA/ C/G/T TT 3′ 2 (sense): 5′ ATA/C/T ATA/C/T CAC/T AGA/G GAA/G ATA/C/T AA 3′ 3 (antisense): 5′ AG C/TTC A/C/G/TGG A/C/G/TGC C/TCT A/GAA A/C/ G/TC 3′ 4 (antisense): 5′ AC A/C/G/TCC A/C/G/TA/GC A/GCT CCA A/C/G/TAT A/GTC 3′
  • This fragment was then used to screen a lambda gt10 cDNA library prepared from total Arabidopsis plants.
  • This Arabidopsis cDNA contains an open reading frame encoded encoding a polypeptide of 384 amino acids (amino acid 473 to amino acid 856 in FIG. 3).
  • the SRS search program the EMBL and EMBLnew databanks were screened for gene sequences designated or annotated with the term cdc7.
  • One genomic sequence from Arabidopsis thalian was found (accession number Z97342).
  • This submitted genomic sequence comprised a predicted gene, indicated as “having similarity to protein kinase HSK of fission yeast”, having 11 exons and coding for a protein having 829 amino acids.
  • the said genomic sequence was compared with the identified partial cDNA sequence, using the “best-fit program”.
  • the identified cDNA-sequence covered nucleotides 119827 to 121978 of the genomic sequence of Z97342.
  • the identified cDNA-sequence did not correspond with the complete coding sequence of the predicted gene on the Z97342 sequence.
  • two additional coding sequences were identified, namely nucleotides no 120770-120709 and 120350-120263 of Z97342, coding for the amino acid sequences of SEQ ID NOS 3 and 4 respectively.
  • the fragments were eluted from agarose gel and cloned using standard techniques and sequenced.
  • the deduced amino acid sequence encoded by the PCR fragment showed clear homology to the yeast published CDC7 sequences and matched with an the above mentioned Arabidopsis genomic sequence.
  • the DNA-fragment, comprising the missing 5′ terminal sequence comprised an additional coding sequence of 63 nt (nrs 122340 to 122278 in Z97342) not identified in Z97342, coding for the amino acid sequence of SEQ ID NO 2.
  • the presently identified CDC7 cDNA comprises additional novel coding sequences, corresponding to novel exons (nos 5, 11 and 13 in FIG. 3), that were not identified in Z97342, and codes for a protein of 890 amino acids.
  • oligonucleotides were as follows: 1 (sense): 5′ TGG GTA/C/G/T TTA/G GCA/C/G/T A/CAA/G GG 3′ 2 (sense): 5′ ATG GAA/C/G/T G/ATT/C/A TA/TC/T AGA/C/G/T AC 3′ 3 (antisense): 5′ AGA/G CAT/C TAT/C AAT/C GCA/C/G/T T GG 3′ 4 (antisense): 5′ TA T/A/G AC/T CAT A/C/G/TCC C/TAA A/C/G/CC A/ GAA
  • First strand cDNA prepared from flower buds was used as template in nested PCR reactions.
  • the first reaction was carried using the pair of oligos 1 and 4, and the second reaction used oligos 2 and 3.
  • PCR conditions were as described (Ferreira et al., 1991, Plant Cell 3, 531-540), except that the annealing temperature of the first reaction was 45 C, and for the second reaction, 37 C was used.
  • a fragment of approximately 300 bp was eluted from agarose gel and cloned in pGEM-T.
  • the Arabidopsis CDC27A1 cDNA contains one open reading frame, encoding a polypeptide of 727 amino acids (FIG. 4).
  • the databanks EMBL and EMBL new were screened for gene sequences, homologous to the present CDC27 cDNA sequence.
  • a genomic sequence from Arabidopsis thalian (accession number AC001645) was found, comprising 14 exons, coding for a protein of 727 AA.
  • the present cDNA-sequence was compared with the said genomic Arabidopsis sequence (1) using the “best fit”-program. It appeared that the present cDNA comprised additional coding information for two novel exons, namely the second and last exon of the Arabidopsis CDC27-gene (exons 2 and 16 in FIG. 4).
  • amino acid sequences encoded by the second and last exon are depicted in SEQ ID NOS 6 and 7 respectively.
  • Dominant negative mutants of CDC7 are constructed by creating substitution mutations including amino acid residues 1 (G), 5 (V), 18 (A) and 20 (K) of SEQ ID No2; amino acid residues 13 (T), 16 (F), 18 (A) and 20 (E) of SEQ ID No3; amino acid residues 7 (L) and 18 (K) of SEQ ID No4. Substitutions are not conservative. Expression of a CDC7 DN in a whole plant, a plant tissue, a plant organ or a plant cell results in cell cycle arrest at G1/S.
  • the CDC7 DN mutants can be obtained by site-directed mutagenesis using the ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Fidelity of the mutagenesis are confirmed by sequencing.
  • the point mutants in (1) are obtained by site-directed mutagenesis using the ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Fidelity of the mutagenesis are confirmed by sequencing.
  • Deletion mutants in (2), (3) and (4) are obtained by high-fidelity PCR (Expand High Fidelity PCR System, Boehringer, Mannheim) using primers designed to amplify the desired stretches of the CDC27 nucleotide sequence.
  • Primers include extensions recognized by restriction endonucleases to allow easy cloning in a vector such as pUC18. Amplified sequences are checked by nucleotide sequence determination.
  • the CDC7 DN coding sequence is operably linked to a plant promoter responsive to nematode infection and to the NOS polyadenylation site.
  • the ARM1 or Att0728 promoters can be used (Barthels et al. 1997, Plant Cell 9, 2119-2134).
  • the CDC7 DN expression cassette is subsequently transferred to a binary vector such as pGSC1704 and the resulting vector electroporated into Agrobacterium tumefaciens C58C1RifR (pGV2260). Transformants are selected on streptomycin/spectinomycin containing medium and checked for the presence of the integral transformed binary vector.
  • Arabidopsis thaliana Col-0 is transformed with the selected A. tumefaciens strain by the floral dip method (Clough and Bent 1998, Plant J 16, 735-743).
  • Transgenic plants are selected after seed germination in the presence of hygromycin. Selected transgenic lines and untransformed control lines are infected with root knot or cyst nematodes. Successfulness of infection is scored visually two weeks after inoculation (in vitro infection) or six weeks after inoculation (infection of soil-grown plants). Transgenic lines are considered resistant relative to control plants when they display a significant decrease in the number of females or cysts on roots and/or a significantly reduction in nematode feeding sites and/or egg production and/or viable nematodes in the eggs.
  • any of the muteins are operably linked to a constitutive promoter such as the CaMV 35S promoter (Kay et al. 1987, Science 236, 1299-1302) or to a seed endosperm-specific promoter such as from a 2S albumin seed storage protein (Guerche et al. 1990, Plant Cell 2, 469-478) or to the BLZ2 promoter (Carbonero et al, 1999 in press) and to a polyadenylation signal.
  • a constitutive promoter such as the CaMV 35S promoter (Kay et al. 1987, Science 236, 1299-1302) or to a seed endosperm-specific promoter such as from a 2S albumin seed storage protein (Guerche et al. 1990, Plant Cell 2, 469-478) or to the BLZ2 promoter (Carbonero et al, 1999 in press) and to a polyadenylation signal.
  • Such expression cassettes are transferred to A. thaliana as described
  • Selected transformant lines have a general higher rate of endoreduplicating cells (CaMV 35S promoter) and/or produce seeds with a higher amount of polyploid endosperm cells (2S albumin promoter). Endoreduplication or polyploidism is assessed in several ways.
  • the DNA content of plant cells is measured by flow cytometry (Galbraith et al. 1991, Plant Physiol 96, 985-989).
  • the cyclin B-degrading activity of the APC is determined as described by King et al. (1995, Cell 91, 279-288).
  • First-strand cDNA was prepared from RNA isolated from different Arabidopsis thalian tissues (etiolated seedlings, flowers, flower buds; stems; leaves; roots; siliques) and from Arabidopsis thalian root cultures treated for 48 h with different chemical substances (10 ⁇ 6 M abscisic acid; 10 ⁇ 7 M 2,4-dichllorophenoxyacetic acid; 100 mM hydroxyurea; 10 ⁇ 6 M kinetin; 10 ⁇ 6 M kinetin+10 ⁇ 6 M 1-naphthaleneacetice acid; 10 ⁇ 6 M 1-naphthaleneacetic acid; 2% (w/v) oryzalin).
  • PCR was performed with these cDNAs using CDC27A-specific primers (sense primer 51′ CCG TAG TGC TAG AAT AGC A 3′ and antisense primer 5′ AGT CAG CGT TGA AGT c3′) or CDC27B-specific primers (sense primer 5′ TCT CTC GAG GAA GAA AGG CAA CAA 3′ and antisense primer 5′ GGT TCT TGG AGT AGC TAT GGT TTC 3′).
  • the resulting fragments generated by PCR were seperated in an agarose gel, blotted to a nylon membrane and hybridized with an 32 P labeled CDC27A or CDC 27B DNA probe. Results are shown in FIG.
  • FIG. 8 illustrates the results for CDC27B.
  • FIGS. 7 and 8 are representative of 3 independent experiments. Both genes are expressed in all plant tissues, but at reduced levels in open flowers an siliques. Expression of both genes is not drastically affected by hormone treatments, except for a reduction in expression levels observed when roots were incubated with 2,4-D (2,4-dichlorophenoxyacetic acid).
  • the primers used to clone the open reading frame were: sense primer 5′ TCT CTC GAG GAA GAA AGG CAA CAA 3′ and antisense primer 5′ GGT TCT TGG AGT AGC TAT GGT TTC 3′.
  • the new Arabidopsis CDC27 homologue is referred to as CDC27B.

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