EP1362059A2 - Method for identifying herbicidally active substances - Google Patents

Method for identifying herbicidally active substances

Info

Publication number
EP1362059A2
EP1362059A2 EP02726107A EP02726107A EP1362059A2 EP 1362059 A2 EP1362059 A2 EP 1362059A2 EP 02726107 A EP02726107 A EP 02726107A EP 02726107 A EP02726107 A EP 02726107A EP 1362059 A2 EP1362059 A2 EP 1362059A2
Authority
EP
European Patent Office
Prior art keywords
seq
activity
nucleic acid
protein
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02726107A
Other languages
German (de)
French (fr)
Inventor
Gunnar Plesch
Astrid Blau
Klaus DÄSCHNER
Mathieu Klein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF Metabolome Solutions GmbH
Original Assignee
Metanomics GmbH
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
Priority claimed from DE10107843A external-priority patent/DE10107843A1/en
Priority claimed from DE10125537A external-priority patent/DE10125537A1/en
Application filed by Metanomics GmbH filed Critical Metanomics GmbH
Publication of EP1362059A2 publication Critical patent/EP1362059A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—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
    • C12N15/8274—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 herbicide resistance
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • New herbicides should be characterized by the broadest possible range of activity, ecological and toxicological harmlessness and low application rates.
  • SEQ ID NO: 102 SEQ ID NO: 106 or SEQ ID NO 108
  • SEQ ID NO: 101 SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109;
  • cc nucleic acid sequence which is a derivative or a fragment of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ ID NO: 11,
  • SEQ ID NO: 94 SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108, and at least 60% homology has nucleic acid level; •
  • dd nucleic acid sequence which is suitable for derivatives or fragments of the polypeptides with those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29 ", SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 ' , SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID'NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,
  • SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 encoded amino acid sequences shown that have at least 50% homology at the amino acid level;
  • nucleic acid sequence which codes for a fragment or a 5 epitope of a polypeptide which binds specifically to an antibody, the antibody specifically binding to a polypeptide which is derived from that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 ,. SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
  • SEQ ID NO: 26 SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
  • SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 is encoded;
  • nucleic acid sequence which codes for a fragment of a nucleic acid shown in aa) and which has an m ⁇ A methyl transferase activity, a DNA binding activity or "DNA repair” activity, e.g. as in
  • RAD 54 a thioredoxin activity, a VAV2 activity, a fructokinase activity, a zinc finger protein activity, a LYTB activity, a crepopin activity, a leucine protein activity, a DNAJ activity, a CRSI activity , an alanyl tRNA synthetase- '
  • Propeptide isolog activity a 26S proteoso subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, a cecropin family signature, ftsH has chloroplast protease activity, an AIM1 activity, 0 a UDP-glucuronyl transferase activity, an FPFl-
  • “Expression” is understood to mean the new synthesis in vitro and in vivo of nucleic acids and proteins encoded by nucleic acids, in particular that of the above-mentioned nucleic acid and amino acid sequences.
  • expression encompasses all biosynthetic steps leading up to the mature protein or its degradation, e.g. Transcription, translation, modification or processing of nucleic acids and proteins, e.g. pre- or post-transcriptional processing steps or post-translational modifications, e.g. Splicing, editing, polyadenylation, capping, modifications of amino acids, e.g. Glycosylation, methylation, acetylation, binding of coenzymes, phosphorylation, ubiquitation, binding of fatty acids, signal peptide processing, etc.
  • transcription means RNA synthesis with the aid of an RNA polymerase in the 5 '->3' direction using a DNA template.
  • Translation means the biosynthesis of proteins in vitro and in vivo. Every molecule and every substance is stand, due to the expression, for example the transcription or translation of a nucleic acid, for example a DNA or RNA, e.g. '. of a gene arises, the term also encompassing the following processing products, such as, for example, after splicing or modification.
  • a processed RNA for example a catalytic RNA, a functional RNA such as tRNAs or rRNAs, or. understood a coding RNA, such as mRNA.
  • a protein is synthesized, which is also understood as a "gene product”. Proteins can 'during and after translation "different mit ists- steps be subjected, as enumerated above as an example.
  • the term" activity of the gene product is the biological activity or function of an RNA or a protein, such as the Enzy atic activity, the receptor binding property, the ability to bind certain proteins, nucleic acids or metabolites, for example in protein complexes, that is to say for example the regulative property or the transporter function of the protein or the RNA, as it naturally occurs in the organism.
  • Reduction of the activity of the gene product is a reduction in the biological activity compared to the natural activity of the gene product of at least 10%, advantageously at least 20%, preferably at least 30%, particularly preferably by at least 50% and very particularly preferably by at least 70%.
  • Blocking the activity of the gene product means the whole che, that is 100% inhibition of
  • Activity or the partial blocking of the activity preferably an at least 80%, particularly preferably at least 90%, very particularly preferably at least 95% blocking of the biological activity.
  • the activity of the gene product can also be reduced indirectly, for example by inhibiting the formation or activity of interaction partners, for example by influencing the metabolic chain in which the gene product is incorporated.
  • an enzyme or protein in the same metabolic chain can be inhibited, which leads to a blocking of the following, previous or another enzyme involved and thus the gene product described herein, for example by substrate or product inhibition .
  • Such reductions by indirectly influencing the activity of an enzyme have been described in detail, for example for the interaction of the glycolysis proteins and metabolites, and are easily transferable to other metabolic pathways in which the gene products described herein play a role.
  • a gene product used according to the invention can be reduced or inhibited in its activity by reducing the activity of interaction partners, for example other proteins a protein complex is reduced or inhibited with the gene product described herein. This can lead to the fact that the entire complex is no longer activated or does not arise, or only partially arises or can no longer be regulated. Examples of such influencing of the activity are described, for example, for spliceosomes, polymerases, ribosomes etc.
  • “Fragment” is understood to mean a partial sequence of a sequence described here which comprises fewer nucleotides or amino acids than the sequences described here.
  • a fragment can e.g. 1%, 5%, 10%, 30%, 50%, 70%, 90% of the original sequence.
  • a fragment preferably comprises 100, more preferably 50, even more preferably less than 20 amino acids of the corresponding nucleic acids.
  • One embodiment thus relates to a method according to the invention, the expression or the activity of the nucleic acids or amino acids mentioned being reduced or blocked by reducing or blocking the transcription, translation, processing and / or modification of at least one of the nucleic acid sequence or amino acid sequence according to the invention.
  • one, two, three or more sequences can be reduced or blocked in their activity.
  • the method according to the invention can be carried out in individual separate method approaches or advantageously in a high-throughput screening (HTS) and can be used to identify substances with herbicidal activity or by antagoriists.
  • HTS high-throughput screening
  • substances can also advantageously be identified that interact with the above-mentioned nucleic acids or with their gene products. These substances are potential herbicides which can be further improved in their action via classic chemical synthesis.
  • Substances identified or selected according to the method can advantageously be placed on a plant in order to test the herbicidal activity of the substances. Those substances are selected which show herbicidal activity.
  • the substances can also be identified in an in vitro test in addition to the aforementioned in vivo test method. On Such an in vitro test with the nucleic acids according to the invention or their gene products has the advantage that the substances can be quickly and easily screened for their biological effects. Such tests are also advantageous for 5 the so-called HTS.
  • the method can be carried out with free nucleic acid such as DNA or RNA, free gene products or advantageously in an organism, bacteria, yeasts, fungi or advantageously plants being used as the organism.
  • free nucleic acid such as DNA or RNA
  • free gene products or advantageously in an organism, bacteria, yeasts, fungi or advantageously plants being used as the organism.
  • Conditional or natural mutants which have the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ are advantageously used as organisms ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, 5 SEQ ID ⁇ NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,
  • SEQ ID NO: 40 SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
  • Conditional mutants are to be understood as meaning 5 mutants which have a reduction in expression, for example transcription or translation, of the aforementioned nucleic acids or of the gene products encoded by them only after induction.
  • An example of such conditional mutants are mutants in which the nucleic acids are located behind a 0 temperature-sensitive promoter which is not functionally at higher temperatures, that is to say the transcription in higher: emperaturen for example above 37 ° C prevented.
  • Expression regulation by an effector molecule is also possible, for example when the expression is controlled by a regulatable promoter, such as, for example, the Tet systems.
  • Another embodiment according to the invention is a method for identifying an antagonist of proteins, which is used by a nucleic acid sequence as used in the method according to the invention, in particular selected from the group:
  • SEQ ID NO: 11 SEQ ID NO: 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO 30, SEQ ID NO 32,
  • SEQ ID NO: 34 SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO 40,
  • SEQ ID NO: 44 SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
  • SEQ ID NO: 60 SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
  • SEQ ID NO: 76 SEQ ID NO: 78, ' SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,
  • SEQ ID NO: 100 SEQ ID NO: 102, SEQ ID NO: 106 or
  • SEQ ID NO: 108 shown sequence, '
  • nucleic acid sequence which, on the basis of the degenerate genetic code, results from the back-translation of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
  • SEQ ID NO: 41 SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
  • SEQ ID NO: 67 SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,
  • Nucleic acid sequence which is a derivative or a fragment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 , SEQ ID-NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO :.
  • SEQ ID NO: 58 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: '70, SEQ ID NO: 72 , SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: - 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 nucleic acid sequences shown, and has at least 60% homology at the nucleic acid level;
  • SEQ ID NO: 6 SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
  • Antibody binds - where the antibody specifically binds to a
  • Polypeptide binds that the in SEQ ID NO: 1, SEQ ID NO: 3,
  • SEQ ' ID NO: 13 SEQ ID NO: 15, SEQ ID NO:: 17, SEQ ID NO: 26., SEQ ID NO: - 28, SEQ ID NO: 30, SEQ ID NO:: 32, SEQ ID NO : 34.
  • SEQ ID NO: 46 SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,
  • SEQ ID NO: 54 SEQ ID NO: 56, SEQ ID NO:: 58, SEQ ID NO: 60,
  • nucleic acid sequence ' which is encoded for a fragment of a nucleic acid shown in aa) and which has a m6A methyltransferase activity, a DNA binding activity or "DNA repair” activity, for example as in RAD 54 , a thioredoxin
  • VAV2 activity fructokinase activity, zinc finger protein activity, LYTB activity, crepopin activity, leucine protein activity, DNAJ activity, CRSl activity, alanyl tRNA synthetase Activity, an OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, a crepopin activity, a leucine protein activity, a DNAJ activity CRSl activity, an alanyl tRNA synthetase activity, an OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, one Cecropin family signature, ftsH has chloroplast protease activity, AIMl activity, UDP-glucuronyl transferase activity, FPFl activity, SHI-like
  • 2, 6-diaminopimelate ligase activity (murE), a ß-glucoside activity, a hydroxymethylglutaryl-CoA reductase, a GDSL motif lipase / hydroxylase-like protein activity, a cellulose synthase-like protein activity, a tRNA gluta insynthetase, a Has exonuclease-like protein activity, a sec-independent Translocase protein TATC activity or a selenium binding protein-like protein activity; and or
  • SEQ ID NO: 94 encoded amino acid sequences which has at least 20% homology at the amino acid level and has an equivalent biological activity; or
  • Ii) describes the testing of one of the biological activities described above, e.g. an enzyme activity as given in the examples or a bond, preferably a strong bond between protein and candidate substance.
  • the antagonist (s) identified under letter iii) is / are placed on a plant in order to test its herbicidal activity and the antagonist (s) are selected that show herbicidal activity.
  • the method according to the invention can be carried out in separate separate method approaches in vivo or in vitro and / or advantageously together or particularly advantageously in a high-throughput screening and can be used for the identification of substances with herbicidal activity or of antagonists.
  • the nucleic acid sequences identified or selected in the method according to the invention are essential for the growth and development of higher plants.
  • the substances identified in the process according to the invention are therefore suitable as herbicides in agriculture.
  • SEQ ID NO: 1 The nucleic acids SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 , SEQ ID NO: 76, SEQ ID NO: 78, SEQ
  • SEQ ID NO: 109 The 1 genomic DNA of the sequences is in each case in SEQ ID NO: 19 (genomic DNA from line P95), SEQ ID NO: 20 (genomic DNA from line P9), SEQ ID NO: 21. (genomic DNA from line P38), SEQ ID NO: 22 (genomic DNA from line P44), SEQ ID NO: 23 (genomic DNA from line P77), SEQ ID NO: 24 (genomic DNA from line P102), SEQ ID No: 43 (genomic DNA from line A 301034) and SEQ ID NO: 42 (genomic DNA from line A 300857).
  • SEQ ID NO: 19 genomic DNA from line P95
  • SEQ ID NO: 20 Genomic DNA from line P9
  • SEQ ID NO: 21. Genomic DNA from line P38
  • SEQ ID NO: 22 Genomic DNA from line P44
  • SEQ ID NO: 23 Genomic DNA from line P77
  • SEQ ID NO: 24 Genomic DNA from line P102
  • the blast comparison shows similarities to thio-redoxins of various origins (Fraser et al., Nature 390 (6660), 580-586, 1997; Reith et al., Plant Mol. Biol. Rep., 13, 333-335, 1995).
  • SEQ ID NO: 11 encodes a protein (accession number BAB09578.1) which has weak homologies with various zinc finger proteins. It shows weak homology to zinc finger proteins and ⁇ DNA binding proteins which, such as. Fingerpr the zinc. otein 265 from Rat (Karginova, EA-., Am. J. Physiol. 273 (5 Pt 2), F731-F738 (1997)) for which a function for the regulation of transcription and / or splicing is assumed. , In vitro transcription or splicing assays have been widely described and are known to the person skilled in the art.
  • the protein encoded by SEQ ID NO: 13 (AB36712.1) has similarities to LYTB proteins especially to LYTB SYNY3 from Synechocystis (Q55643).
  • the gene or the protein encoded by the gene is located on • Chromosome V (Accession number AB006706).
  • ESTs gb: Z34640, Z30476, AA605545, Z34228, H76883, Z26425 are known within the sequence of SEQ ID O: 13.
  • SEQ ID NO: 15 codes for a hypothetical protein (CAB81447,1) which contains the crepropin family signature and has weak homologies to yc proto-oncogenes.
  • the protein (AAF23295) encoded by SEQ ID NO: 17 (see also ESTAV528166) has a certain homology to a leucine-rich protein from humans (Accession number P42704, Wang et al., In vitro Cell Dev. Biol. Amin. 1994, 30A (2): 111-114).
  • LRP130 leucine-rich protein
  • SEQ ID No: 26 encodes a protein that has homology to various DNAJ chaperone proteins (Heat Shock Protein 40) for a range of 40 amino acids, e.g. for DNAJ protein (Q9UXR9) from Methanosarcina thermophila (Hoffmann-Bang, Gene 238 (2), 387-395,
  • SEQ ID No: 28 encodes a hypothetical protein (CAC01859.1).
  • the derived protein sequence shows clear homologies to the CRS1 gene product from maize (AAG00595), which is required for 20 splicing of the group II intron of the chloroplast gene atpF.
  • SEQ ID No: 30 presumably codes for an alanyl tRNA synthesis (BAB10601.1).
  • SEQ ID No: 32 codes for a protein which has strong homology to the "chloroplast outer envelope 86 protein" OEP86 from pea P. sativum, GenBank Accession number Z31581 and has an ATP / GTP binding site motif (P-loop).
  • ORF CAB80744.1
  • SEQ ID No: 34 codes for a protein whose derived amino acid sequence (AAF25967.1) shows clear similarity to an "FMRF amide propeptide isolog (gij 1871179) from Arabidopsis. 5
  • SEQ ID No: 36 encoding an unknown protein (BAB02572.1) ⁇ with weak homology to protein proteosome 26S PROTEASOME SUBUNIT S5B, (Deveraux, 1995, J. Biol Che 270 (40), the 23,726th..
  • SEQ ID NO: 38 encodes an unknown protein.
  • SEQ ID NO: 40 encodes protein that has homology to a geranylgeranyl pyrophosphate synthase (Bartley, Plant Physiol. 104, 1469-1470, 1994).
  • 5 SEQ ID NO: 44 codes for a hypothetical protein of ORF AT4g28590, which has a "Cecropin" family signature (AA237-245).
  • SEQ ID NO: 46 encodes a putative ftsH chloroplast protease of ORF At2g30950.
  • SEQ ID NO: 48 is similar to the "AIMl” protein from Arabidopsis (CAB43915.1).
  • ORF ORF19B15.40
  • GB Z31666
  • gb Z33957
  • Z31666 This protein is a peroxisomal tetrafunctional enzyme of fatty acid metabolism.
  • SEQ ID NO: 50 encodes a UDP-glucuronyltransferase-like protein of ORF K21H1.19. By incorporating the T-DNA in this position, the transcription is very likely to be changed or prevented and the function of the gene is thereby destroyed.
  • SEQ ID NO: 52 encodes a protein of unknown function of the ORF At2gl5820.
  • SEQ ID NO: 54 encodes a protein of the ORF ATF12B17_20, an FPF1-like (flowering promoting factor1) protein.
  • SEQ ID NO: 56 encodes a protein of the ORF ATF12B17_10 with similarity to KIAA1038 protein from Homo sapiens.
  • the SEQ ID NO: 58 encodes a protein of ORF F24P17.10 with unconstrained 'known per function. In the blastp comparison with standard settings, clear homoligies to a nodulin / gluta ate- - ammonia ligase - like protein are shown.
  • SEQ ID NO: 60 encodes a SHI-like zinc finger protein (short internodes) of ORF K1L20.13.
  • SEQ ID NO: 62 encodes a protein similar to crpl from Zea mays, PIR: T01685 (ORF F4P12_400). This ORF also includes the ESTs gb: Al999771.1, T45254, AA713158 *.
  • SEQ ID NO: 64 encodes a putative protein with similarities to hypothetical proteins from Arabidopsis. The blastp analysis also shows a clear homology to CRS1 from Zea mays Accession AAG00595, which is a Group II intron splicing factor (Till, B et al., RNA 7 (9), 1227-1238 (2001)) ORF (T21H19_100). SEQ ID NO: 66 encodes a protein of unknown function of the At5g24315 gene.
  • SEQ ID NO: 68 encodes a protein ORF " (T20O10_10) with high similarity to translation releasing factor RF-1 from Synechocystis (PIR: S76914).
  • the deduced amino acid sequence z contains a prokaryotic type I pepeptide chain detachment factor motif, AA280 -296.
  • SEQ ID NO: '70 encodes a protein with high similarity to an allergen ( "minor allergen") from Alternaria alternata (PIR2: S43111).
  • ESTS gb: R64949, AA651052 have also already been found for ORF (C7A10.610).
  • SEQ ID -NO: 72 encodes a protein similar to the ' alpha subunit of a putative signal sequence receptor (ORF At2gll60).
  • SEQ ID NO: 74 encodes a protein of unknown function of ORF AT4g01220, which ESTs gb: AA597894,. AA597304 includes.
  • SEQ ID NO: 76 shows a similarity to oxidoreductases in the blastp analysis with standard settings. The insertion of the T-DNA at this position interrupts the ORF F13011.11. The cellular function of the encoded proteins is unknown.
  • SEQ ID NO: 78 codes for the protein of ORF F25L23_240 * a farnesyltransferase subunit A.
  • SEQ ID NO: 80 codes for an ATP-dependent copper transporter RANl-like protein (ORF T19K24.18).
  • SEQ ID NO: 82 encodes the protein of ORF F19B15.50- and has similarities to proteins rich in glycine.
  • the ORF includes the ESTs gb: Z29181, T42831, Z34138, Z33797, Z30844.
  • SEQ ID NO: 84 encodes a protein unknown function (ORF K21H1.18).
  • SEQ ID NO: 86 encodes a protein that also shows from plants in the blastp comparison under default high homologies to various syntaxins and syntaxinähnlichen proteins.
  • ORF F309.4 gb
  • SEQ ID NO: 88 encodes the protein of ORF AT2g31830-, a putative inositol polyphosphate 5 'phosphatase.
  • SEQ ID NO: 90 encodes the protein of ORF F24D7.13, which encodes 5 for a putative UDP-N-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase (murE).
  • SEQ ID NO: 92 encodes the protein of ORF MRC8.5 . which codes for a beta-glucosidase. 10
  • SEQ ID NO: 94 codes for the protein of ORF F15M4.1, which codes for a hydroxymethylglutaryl-CoA reductase.
  • SEQ ID NO: 96 encodes the protein of the ORF MRN17.4. This 15 codes for a GDSL motif lipase / hydrolase-like protein.
  • SEQ ID NO: 98 encodes the protein of ORF dl3705c, which encodes a cellulose synthase-like protein.
  • SEQ ID NO: 100 codes for the protein of ORF K5J14.11, which codes for a protein similar to the crpl protein from maize.
  • SEQ ID NO: 102 encodes the protein of ORF F4F7.26.
  • This ORF codes for a putative t-RNA glutamine synthetase and has, in particular, homology to tRNA glutamine synthetase GI: 2995454 from Lupinus luteus.
  • SEQ ID NO: 104 encodes the protein of ORF MFB13.17, which is a . Exonuclease-like protein encoded. 30
  • SEQ ID NO: 106 encodes the protein of ORF At2g01110. This codes for a putative "sec-independent" translocase protein TATC (putative sec-independent protein translocase protein TATC).
  • SEQ ID NO: 108 codes for the protein of ORF F28J12.180, which codes for a putative protein.
  • ORF F28J12.180 codes for a putative protein.
  • the derived amino acid sequence shows, in addition to clear homologies to various hypothetical and putative proteins, also strong similarity to selenium binding - 0 protein-like proteins.
  • Plant calli or plant cells that contain the sequences described according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID MO: 11, SEQ ID NO : 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, - SEQ ID NO: 28, SEQ ID NO: 30 SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 , SEQ ID NO: 38 SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ' ID NO: 48 SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO; 62, SEQ ID NO: 64 SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO; 70, SEQ ID NO: 72 SEQ ID NO: 74, SEQ.
  • Chemical compounds which reduce the biological activity, the growth or the vitality of the organisms are to be understood as compounds which prefer the biological activity, the growth or the vitality of the organisms by at least 10%, advantageously by at least 30% inhibit by at least 50%, particularly preferably by at least 70%, very particularly preferably by at least 90%, ie reduce or block.
  • a substance that is particularly advantageous is the. Damage cell lines with low activity or, preferably, that is lethal, but does not damage or is lethal to cell lines that have a higher activity of the gene product.
  • lines of organisms can be used in the method mentioned which express the sequences according to the invention and in particular the gene products which are encoded by nucleic acids according to the invention, but which are not recombinant as long as a line has a higher gene expression or activity of the gene product than one other line.
  • Such lines can of course occur or be generated by mutagenesis.
  • Assay systems which suppress the identification of substances which suppress the formation of the gene products and / or the functions exercised by the gene products or the activity of the gene products in intact plants, plant parts, plant tissues or plant cells are known to the person skilled in the art.
  • An example is here Test systems for the inhibition of enzymes such as fructokinase activity as described by Tangney et al. (J. Mol. Microbiol. Biotechnol., 2 (1), 2000: 71-80), Martinez-Barajas et al. (Protein Expr. Purif., 1997, 11 (1), 41-46), Kanaya a et al.
  • test systems can be used advantageously, for example, for so-called inhibition assays for clone P77, for example.
  • FCS fluorescence correlation spectroscopy
  • the molecules to be examined are fluorescence-labeled and, for example, a defined volume is placed in microtiter plates. The fluctuation of the molecules is shown in the samples
  • the binding of the molecules can be determined or quantified by changing the diffusion coefficient.
  • This method can be used to measure advantageously in a wide concentration range.
  • the method is advantageously suitable for the measurement of recombinant
  • angiotensin II 35 angiotensin II, NEN ® Life Science Products, Boston, MA, USA.
  • the compound or substance to be examined is then added to the protein in excess.
  • the diffusion of the protein labeled in this way is finally carried out using an FCS system (e.g. ConfoCor2 with LSM 510, Carl Zeiss microscope, Jena, Germany).
  • SELDI ProteinChip ® surface enhanced laser desorption ionization
  • the protein is immobilized on the SELDI Protein-Chips ® , for example via the His tags already used for cleaning or via ion or hydrophobic interactions with the chip.
  • the ligands are then placed on this chip prepared in this way using, for example, an autosampler. After one or more washing steps with buffers of different ionic strength, the bound ligands are analyzed with the LDI laser. , The binding strength of the ligands is determined after each washing step.
  • Biacore so-called Biacore method
  • SPR so-called plasma resonance
  • the refractive index change which is determined for a change in the mass concentration at the surface, is the same for all proteins or polypeptides, that is to say this method can advantageously be used for a wide variety of proteins.
  • recombinantly expressed proteins are also advantageously used here, which are bound to the Biacore chip (Upsala, Sweden), for example via histidine residues (e.g. His-Tag).
  • the chip thus produced is again connected to the ligands, e.g. with an autosampler and binding via a detection system sold by Biacore using the SPR signal i.e. measured by changing the refractive index.
  • substances that are particularly specific with e.g. bind a protein or protein fragment which is encoded by a nucleic acid, the expression of which is essential for the growth of the plants can be isolated using the methods mentioned.
  • This enables a simplified identification of possible inhibitors, the proteins, e.g. inhibit in their enzyme properties, binding properties or other activities, e.g. also by inhibiting their processing, as described above, or preventing their transport within the cell or import and export from organelles or cells.
  • the substances identified in this way can also be applied to plants in a further step in screening processes, as are known to the person skilled in the art, and their influence on growth and development can be examined.
  • a selection is made from the infinite number of chemical compounds that would be suitable for a screening process, which makes it considerably easier for the person skilled in the art to identify herbicidal substances.
  • Specific binding means the specificity of interactions between two partners, for example proteins with one another or between protein (enzyme) and substrate (substrate specificity). It is based on a certain molecular spatial structure. If it destroys, it is called denaturation, which is often irreversible and can go so that the 'specificity lost mostly. This biological activity is strongly dependent on the environmental conditions (buffer, temperature, contacts to non-physiological surfaces such as glass or missing cofactors). With enzyme substrate or cofactor, with receptor ligand or with antibody-antigen bonds one speaks of specific bonds. The enzyme-substrate interaction is described thermodynamically in the simplest case using the Michaelis-Menten equation.
  • the identified substances can then be applied to plants, microorganisms or cells, e.g. on plant cells, and then the influence on the metabolism of these plants can be observed, e.g. Enzy - activities, photosynthetic activities, metabolic activity,
  • Fixation rate, gas exchange, DNA synthesis, growth rates are suitable for examining the viability of cells.
  • Substances that inhibit growth, e.g. of cells, in particular plant cells, reduce, in particular block, are then preferably suitable as a selection for herbicidal compositions.
  • Another object of the invention is a. Process for identifying inhibitors of plant proteins which are ' encoded by the nucleic acid sequences used in the process according to the invention, with potentially herbicidal activity by cloning the gene products, overexpressing them in a suitable expression cassette - for example in insect cells -, opening the cells and use the cell extract directly or after enrichment or isolation of the protein in a test system for measuring biological activity in the presence of low molecular weight chemical compounds.
  • the invention therefore furthermore relates to substances identified by the processes according to the invention, the substance having a molecular weight of less than 1000 daltons, advantageously less than 900 daltons, preferably less than 800, particularly preferably less than 700, very particularly preferably less than 600 daltons, a ki Value less than 1 mM, and preferably less than three Has hydroxyl groups on a ring containing carbon atoms or the substance is a proteinogenic substance, an antisense RNA, an inhibitory or an interfering RNA (RNAi).
  • RNAi interfering RNA
  • the term “sense” refers to the strand of a double-stranded DNA that is homologous to the mRNA transcript.
  • the "anti-sense” strand contains an inverted sequence that is complementary to that of the "sense” strand.
  • An antisense nucleic acid molecule comprises, for example, a nuclide sequence that is ' complementary to the w sense ' nucleic acid molecule which encodes a protein or an active RNA, for example' complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • an antisense nucleic acid molecule can hydrogen bond to a sense nucleic acid molecule.
  • the antisense nucleic acid molecule can be complementary to, or only a part of, any coding strand shown here.
  • the term "coding region” refers to the region of one Nucleic acid sequence whose codons are translated into amino acids. "The antisense nucleic acid molecule can also be complementary to" non-coding regions "of the coding strand of the nucleic acid molecules shown.
  • the term" non-coding region refers to 5'- and 3 ' - sequences that flank the coding region and that are not in a polypeptide can be translated (eg also referred to as 5 ' ⁇ and 3' untranslated regions).
  • the nucleic acid molecule which comprises an antisense sequence can also comprise further elements which are important for the expression and stability of the molecule, for example capping structures, poly A-tails etc.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but can also be an oligonucleotide which? is complementary to only a portion of the coding or non-coding region of the mRNA.
  • an antisense oligonucleotide can be complementary to the region that encompasses or surrounds the translation start of the mRNA.
  • An antisense oligonucleotide can be, for example, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid molecule can be produced by chemical synthesis and enzymatic ligation according to methods known to the person skilled in the art.
  • An antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or nucleotides modified in various ways so that the biological stability of the molecules is increased or the physical stability of the duplex that forms between the antisense and sense nucleic acids , is enhanced, for example, phosphorothioate derivatives and acridine-substituted nucleotides be used.
  • modified nucleotides that can be used for the production of antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthines, xanthines, 4-acetylcytosines, 5- (carboxyhydroxylmethyl) uracil , 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyl-auanine, 2-methyl , 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil,
  • 5-methoxyaminomethyl-2-thiouracil beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine Thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thio- •. uracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2, 6-diaminopurines.
  • antisense nucleic acid molecules can be produced biologically using expression vectors into which polynucleotides have been cloned, whose orientation is opposite (so that RNA, transcribed from the inserted poly- nucleotide, in an antisense orientation to a target polynucleotide as follows) has been described above).
  • the antisense nucleic acid molecule can also be an " ⁇ -anomeric" nucleic acid molecule.
  • An " ⁇ -anomeric" nucleic acid molecule forms specific double-stranded hybrids with complementary RNAs, in which, in contrast to ordinary ß-units,. the strands run parallel to each other.
  • the antisense nucleic acid molecule can be 2-0-methylribo ⁇ ucleotide or chimeric RNA-DNA analogues. include.
  • the antisense nucleic acid molecule can be a ribozyme.
  • Ribozymes are catalytic RNA molecules with a ribonuclease activity that are able to cut single-stranded nucleic acids, such as mRNA, to which they have a complementary region. Ribozymes (eg hammerhead ribozymes) can be used to catalytically or non-catalytically cut the mRNA of the sequences described herein and thus to prevent translation of the mRNA.
  • a ribozyme which is specific to one of the nucleic acid sequences mentioned herein can be constructed on the basis of the cDNA sequences shown here or on the basis of heterologous sequences which can be identified by the methods described herein.
  • a derivative of the Tetrahymena L-19 IVSRNA can be produced by the The nucleotide sequence of the active region is complementary to the nucleotide sequence that is cut in a coding mRNA.
  • one of the coding or non-coding sequences described herein or an mRNA thereof can also be used to select a catalytic RNA from a pool of RNAs (see, for example, Bartel, 1993, Science, 261,. 1411).
  • nucleotide sequences which are complementary to a regulatory region of the nucleic acid sequences described here form a triple-helical structure which prevents transcription of the following gene (for example Helene, 1991, Anticance-Drug Des. 6, 596; Helene, 1992, Ann. " NY Acad. Sci. 660, 27, or Mower, 1992, Bioassays, 14, 807.
  • Antibodies are understood to mean, for example, polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies or else synthetic antibodies.
  • Antibodies according to the invention or their fragments are in principle to be understood as meaning all immunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or their subclasses such as the subclasses of the IgG or their mixtures.
  • IgG and its subclasses such as IgGi, IgG 2 , IgG 2a , IgG, IgG 3 or IgGii are preferred.
  • the IgG subtypes IgG ⁇ or IgG b are particularly preferred.
  • All shortened or modified antibody fragments with one or two binding sites complementary to the antigen such as antibody parts with a binding site corresponding to the antibody of light and heavy chain, such as Fv, Fab or F, are to be considered fragments (from ') 2 fragments or single-strand fragments.
  • Shortened double-strand fragments such as Fv, Fab or F (ab ') are preferred.
  • These fragments can be obtained, for example, enzymatically by cleaving off the Fc part of the antibodies with enzymes such as papain or pepsin, by chemical oxidation or by genetic engineering manipulation of the antibody genes. Genetically manipulated, unabridged fragments can also be used advantageously.
  • the antibodies or fragments can be used alone or in mixtures. Antibodies can also be part of a fusion protein.
  • the substances identified can be chemically synthesized or microbiologically produced substances and can occur, for example, in cell extracts from, for example, plants, animals or microorganisms. Furthermore, the substances mentioned may be known in the prior art, but have not hitherto been known as a herbicide.
  • the reaction mixture can be a cell-free extract or can comprise a cell or cell culture. Suitable methods are known to the person skilled in the art and are described, for example, in general in Alberts, Molecular Biology the cell, 3rd Edition (1994), for example Chapter 17.
  • the substances mentioned may be eg added to the reaction mixture or the culture medium or the cells are injected or sprayed onto a plant. '
  • a sample containing a substance active according to the method according to the invention has been identified, then it is either possible to isolate the substance directly from the original sample or the sample can be divided into different groups, e.g. if they are from a variety of 'different
  • Components exist so as to reduce the number of different substances per sample and then to repeat the method according to the invention with such a "sub-sample" of the original sample.
  • the steps described above can be repeated several times, preferably until the sample identified according to the method according to the invention only comprises a small number of substances or only one substance.
  • the substance or derivative thereof identified according to the method according to the invention is preferably further formulated in such a way that it is suitable for use in plant breeding or plant cell or tissue culture.
  • the substances that have been tested and identified according to the method according to the invention can be, for example: Expression libraries, e.g. cDNA expression libraries,
  • Inhibitors or activators Methods for the production of chemical derivatives or analogs are known to the person skilled in the art. The derivatives and analogues mentioned can be tested according to methods according to the prior art. Furthermore, computer-aided design or peptidomimetics can be used to produce suitable derivatives and analogues.
  • the cell or tissue which can be used for the method according to the invention is preferably a host cell, plant cell or a plant tissue according to the invention, as described in the above-mentioned embodiments.
  • the derivatives (s) (the plural and the singular are equivalent for this application and their definitions) of the nucleic acids used in the methods according to the invention include, for example, functional homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 , SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, • SEQ ID NO: 68, SEQ ID
  • SEQ ID NO: 108 encoded proteins or their biological activity, that is proteins which have the same biological reactions as those of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO : 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID ⁇ NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: .46, SEQ ID NO: 48, SEQ ID NO: 50 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:
  • the 'sequences according to the invention described herein encode homologs of the proteins described in the examples, and preferably have the activities specified for the homologs.
  • SEQ ID NO: 1 codes for a protein which is similar to an “m6A ⁇ methyltransferase. The protein sequence is reproduced in SEQ ID NO: 2.
  • SEQ ID NO: 3 codes for a so-called X DNA repair protein RAD 54 like protein homolog ", the protein sequence of which can be found in SEQ ID NO: 4.
  • SEQ ID NO: ' 5 can code for a thioredoxin, the protein sequence is shown in SEQ ID NO: 6.
  • SEQ ID NO: ' 7 codes for an unknown protein, the sequence of which is shown in SEQ ID NO: 2.
  • SEQ ID NO: 9 codes for a fructokinase, the protein sequence of which can be found in SEQ ID NO: 10.
  • SEQ ID NO: 11 codes for a protein which has weak homologies with various zinc finger proteins.
  • the protein sequence can be found in SEQ ID NO: 12.
  • SEQ ID NO: 13 encodes a protein that is similar to LYTB proteins.
  • SEQ ID NO: 14 represents the protein sequence.
  • SEQ ID NO: 15 contains a so-called crepropin family signature and has weak homologies to myc proto-oncogenes.
  • SEQ ID NO: 17 encodes a protein that has some homology to a leucine-rich human protein.
  • SEQ ID NO: 18 shows the protein sequence.
  • SEQ ID NO 32 SEQ ID NO: " 34, SEQ ID NO: 36, SEQ ID NO 38,
  • SEQ ID NO 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 1 106 or SEQ ID NO: 108 encode homologues or have similarity to proteins, the activity or function of which is shown above or in the examples.
  • the protein sequences are each in 20 SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
  • SEQ ID NO: 45 SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO: 51,
  • SEQ ID NO: 101 SEQ ID NO: 103, SEQ ID NO: 105.
  • SEQ ID NO: 107 30 or SEQ ID NO: 109.
  • Derivatives are also understood to mean those peptides which have homology to the polypeptides with the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: ' 9, 5 SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID ' NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
  • SEQ ID NO: 108 sequences of at least 20%, preferably 30%, more preferably 50%, still more preferably 70%, more
  • amino acid sequences are at least 40, 50, 60 or 70%, more preferably 80%, even more preferably 90%, most preferably 95% or more identical.
  • sequences of the invention are homologous to Nu leinklaebene at least 45 or 55 "%, preferably at least 60 or 65%,
  • fragment means a shortened sequence of the original sequence.
  • the shortened sequence (nucleic acid or protein) can have different lengths, the minimum sequence length - is a sequence length that has at least one comparable function, e.g.
  • nucleic acids that encode a fragment 35 or an epitope of a polypeptide that specifically binds to an antibody that specifically binds to a polypeptide described as ⁇ according to the invention, in particular that of one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, 40 SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, ' SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 , SEQ ID NO: 62, 45 SEQ ID NO: 64, SEQ ID NO: 64
  • Fragments or epitopes of a polypeptide that interact specifically with such an antibody have a significant homology in the spatial structure to the polypeptides described here, at least in some areas. Preferably 'also they have a high homology at the amino acid level to the said sequences, preferably 20%, are more preferably 40%, more preferably 60%, even more 80%, are most preferred 90% or more.
  • the spatial structure of a polypeptide is essentially responsible for the interactions of the polypeptide with other compounds and, if necessary, for its enzymatic activity.
  • fragments according to the invention whose sequence has only a low homology to the polypeptides described, but whose spatial structure has a high homology to the polypeptides described, that is to say those which contain epitopes of the sequences described here
  • Fragments which comprise epitopes of the polypeptides according to the invention can also be used to "occupy" the interaction partners of the polypeptides according to the invention, ie to prevent their interaction with the polypeptides according to the invention. For this it is advantageous if the fragments have a higher affinity for a binding partner have ypeptid than the naturally occurring Pol '.
  • fragments which are encoded by nucleic acids according to the invention and include one of the biological activities "referred to above.
  • Allelic variants include, in particular, functional variants which, by deletion, insertion or substitution of nucleotides from the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5; SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
  • SEQ ID NO: 32 SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,
  • SEQ ID NO: 40 SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
  • SEQ ID NO: 50 SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,
  • SEQ ID NO: 58 SEQ ID NO: • 60, SEQ ID NO: 62, SEQ ID NO: 64,
  • SEQ ID NO: 90 SEQ ID NO: 92-, SEQ ID NO: 94, SEQ ⁇ D NO: 96,
  • SEQ ID NO: 98, SEQ ID NO: 100 SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 are available, the biological, for example enzymatic activity or binding properties Shafts of the derived synthesized proteins are preserved.
  • DNA sequences can be generated with the aid of the nucleic acid sequences according to the invention, for example starting from those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 .
  • SEQ ID NO: 26 SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: .36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72.
  • SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 described DNA sequences or parts of these sequences, for example with conventional hybridization methods or the PCR technique from other eukaryotes such as, for example, microorganisms such as yeasts, fungi, ciliates, plants such as algae, moss or other plants.
  • These DNA sequences hybridize with the sequences mentioned under standard conditions.
  • short oligonucleotides for example the conserved or other, are advantageously used .
  • DNA hybrids are approximately 10 ° C lower 'ls those of DNA: RNA hybrids Long.
  • DNA hybrids are advantageously 0.1 ⁇ SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C.
  • the hybridization conditions are advantageously at 0, 1 x SSC and temperatures between about 30 ° C to 55 ° C, preferably between about 45 ° C to 55 ° C.
  • These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide.
  • the experimental conditions for DNA hybridization are in relevant textbooks of genetics such as Sambrook et al. , "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G "+ C content.
  • homologs of the sequence SEQ ID No: 1 are among derivatives,
  • SEQ ID NO: 34 SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50
  • SEQ ID NO: 52 SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58
  • SEQ ID NO: 60 SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66
  • SEQ ID NO: 68 SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74
  • SEQ ID NO: 76 SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90
  • SEQ ID NO: 100 SEQ ID NO: 102, SEQ ID NOir 106 or
  • SEQ ID NO: 108 for example, shortened eukaryotic homologs
  • SEQ ID NO: 34 SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40,
  • SEQ ID NO: 44 SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
  • SEQ ID NO: 60 SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
  • nucleic acids used in the method according to the invention in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
  • SEQ ID NO: 30 SEQ ID NO: 32, SEQ D NO 34, SEQ ID NO 36-,
  • SEQ ID NO: 38 SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO 46,
  • SEQ ID NO: 48 SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO 60, SEQ ID NO 62,
  • SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 1 ⁇ 8 and their fragments and derivatives are therefore advantageously suitable for isolating further essential, new genes from other organisms, preferably plants.
  • nucleic acid sequences according to the invention in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
  • SEQ ID NO: 17 SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38
  • SEQ ID NO: 40 SEQ ID NO 44, SEQ ID NO: 46, SEQ ID NO 48
  • SEQ ID NO: 66 SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO 72 SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO: 78, SEQ ID NO 80
  • SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 whose gene products encoded by them are used in the method according to the invention can be synthetically produced or naturally obtained or contain a mixture of synthetic and natural DNA components, and consist of different heterologous gene segments from different organisms.
  • synthetic nucleotide sequences with codons are generated, which are preferred by the corresponding host organisms, for example plants. This generally leads to optimal expression of the heterologous genes.
  • These plant preferred codons can be determined from the highest protein frequency codons expressed in most interesting plant species.
  • Corynebacterium glutamicum is given in: Wada et al. 10 (1992) Nucleic Acids Res. 20: 2111-2118). Such experiments can be carried out using standard methods and are known to the person skilled in the art.
  • Functionally equivalent sequences which code for the nucleic acids used in the method according to the invention are those derivatives of the sequences according to the invention which, despite a different nucleotide sequence, still have the desired functions, that is to say the biological activity of the proteins.
  • Functional equivalents thus include naturally occurring variants of the 20 sequences described herein as well as artificial, e.g. artificial nucleotide sequences obtained by chemical synthesis, in particular adapted to the codon use of a plant.
  • Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable.
  • the specific codon usage can be determined by a specialist familiar with plant genetic methods by computer evaluations of other
  • amino acid sequences which are one in the sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, 45 SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO 45, SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO : 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO: 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 77, SEQ ID NO: 79, SEQ ID NO
  • SEQ ID NO: 12 SEQ ID NO: 14, SEQ ID NO 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO 31, SEQ ID NO: 33,
  • SEQ ID NO: 61 SEQ ID NO: 63, SEQ ID NO 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: '71, SEQ ID NO 73, SEQ ID NO: 75,
  • SEQ ID NO: 101, SEQ ID NO :; 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 is retained or is not significantly reduced. Not significantly reduced means all proteins which still have at least 10%, preferably 20%, particularly preferably 30%, 50%, 70%, 90% or more of the biological activity of the starting protein.
  • certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.).
  • arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues.
  • one or more amino acids can also be interchanged, added or removed in their order, or several of these measures can be combined with one another.
  • Derivatives are also to be understood as functional equivalents which in particular also include natural or artificial mutations in the nucleic acid sequences used SEQ ID NO: 1, SEQ ID NO: 3,
  • SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO 34 SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO 44
  • SEQ ID NO: 46 SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52
  • SEQ ID NO: 76 SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO 94, SEQ ID NO: 96, SEQ ID NO: 98,
  • SEQ ID NO: 100 SEQ ID NO: 102, SEQ ID NO: 106 or
  • SEQ ID NO: 41 SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
  • SEQ ID NO: 105 SEQ ID NO: 107 or SEQ ID NO: 109 derived amino acid sequences
  • SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 is the nucleic acid sequences shown, and have at least 60% homology at the nucleic acid level; or
  • nucleic acid sequence which is suitable for derivatives or fragments of the polypeptides with the in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
  • SEQ ID NO: 109 encoded amino acid sequences which have at least 50% homology at the amino acid level
  • nucleic acid sequence which codes for a fragment or an epitope of a polypeptide which specifically binds to an antibody, the antibody specifically binding to a polypeptide which the one in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
  • SEQ ID NO: 13 SEQ. ID NO: 15, SEQ ID NO: 17, SEQ I NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 , SEQ ID NO: .58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO .: 70, SEQ ID NO: 72 , SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78 ,.
  • nucleic acid sequence which encodes a fragment of a nucleic acid shown in a) and which has an m6A methyl transferase activity, a DNA-binding activity or "DNA repair" activity, for example as in RAD 54, a thioredoxin Activity, a VAV2 activity, a fructokinase activity, a zinc finger protein activity, a LYTB activity, a crepopin activity, a leucine protein activity, a DNAJ activity, a CRSl activity, an alanyl tRNA activity Synthetase activity, an OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity or a geranylgeranyl pyrophosphate synthase activity, a crepopin activity, a leucine protein activity, a DNAJ activity, a CRSl activity, an alanyl tRNA synthetase activity, an OEP86 activity, an FMRF amide pro
  • nucleic acid sequence which is suitable for derivatives of the polypeptides with the in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
  • SEQ ID NO 18 SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ • ID NO 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO 63, SEQ ID NO 65,
  • SEQ ID NO 67 " SEQ ID NO: 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO: 77, SEQ ID NO 79, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO: 85, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO 91, SEQ ID NO: 93, SEQ ID NO 95, SEQ ID NO 97,
  • SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 encoded amino acid sequences, which has at least 20% homology at the amino acid level and an equivalent possesses biological activity;
  • nucleic acid sequence is linked to one or more regulatory signals.
  • the aforementioned terms have the meaning given above.
  • nucleic acids according to the invention e.g. those in SEQ ID NO: 1,
  • SEQ ID NO 3 SEQ ID NO: 5
  • SEQ ID NO: 7 SEQ ID NO: 9
  • SEQ ID NO 26 SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO 32,
  • SEQ ID NO 34 SEQ ID NO: 36 SEQ ID NO: 38, SEQ ID NO 40, SEQ ID NO 44, SEQ ID NO: 46 SEQ ID NO: 48, SEQ ID NO 50,
  • SEQ ID NO: 108 sequences which are to be understood as the result of the genetic code and / or their functional or non-functional derivatives which have been functionally linked to one or more regulatory signals advantageously for regulating, in particular increasing, the gene expression and which Control expression of the coding sequence in the host cell.
  • These regulatory sequences are said to. enable the targeted expression of the genes or the proteins. This may for example each represent the host organism, that the gene " ⁇ expressed only after induction and / or ated überexpri, or that it is constitutively expressed and / or overexpressed.
  • these regulatory sequences are sequences to which inductors or repressors bind • and thus regulate the expression of the nucleic acid in addition to these new regulatory sequences or instead of these
  • nucleic acid construct according to the invention can also advantageously consist only of the natural, genetically modified regulatory region at the 5 'and / or 3' end.
  • the gene construct can, however, also have a simpler structure, ie no additional regulatory signals have been inserted in front of the nucleic acid sequence or its derivatives and the natural promoter with its regulation has not been removed. Instead, the natural. Regulation sequence mutated so that regulation no longer takes place and / or gene expression is increased.
  • the gene construct can also advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences.
  • the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • the expression can also be specifically reduced or blocked.
  • promoters which can advantageously control the expression of foreign genes in organisms in plants or fungi are suitable as promoters in the expression cassette.
  • a plant promoter or promoters derived from a plant virus are preferably used.
  • Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacis - T7, T5, T3 , gal, trc, ara, SP6, ⁇ -p R - or contained in the ⁇ -Pjy promoter, which are advantageously used in gram-negative bacteria.
  • the expression cassette can also contain a chemically inducible promoter through which the expression of the nucleic acid sequences in the nucleic acid construct according to the invention in the
  • Organisms can be advantageously controlled in the plants at any given time.
  • Such advantageous plant promoters are, for example, the PRPl promoter [Ward et al., Plant. Mol. Biol. 22 (1993), 361-366], one that is inducible by benzenesulfonamide (EP 388186), one that is inducible by tetracycline (Gatz et al., (1992) Plant J. 2,397-404), one that is inducible by salicylic acid Promoter (WO 95/19443), a promoter inducible by abscisic acid (EP 335528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334).
  • Further plant promoters are, for example, the promoter of the cytosolic FBPase from potato, the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445-245), the promoter of the phosphoribosyl pyrophosphate A idotransferase from glycine ax (see Genbank Accession number U87999) or a node-specific promoter as in EP 249676 can also be used advantageously.
  • DNA fragments When preparing an expression cassette, various DNA fragments can be manipulated to produce a nucleotide sequence. to obtain, which reads expediently in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • the promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter • can be both native or homologous as well as foreign or heterologous to the host organism, for example to the host plant.
  • the expression cassette contains in the 5 '-3' transcription direction the promoter, a DNA sequence which codes for the proteins used in the method according to the invention and a region for the transcriptional termination. Different termination areas can advantageously be interchanged.
  • Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as ' transitions and transversions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be used. at suitable manipulations, such as restriction, -chewing-back- or filling of overhangs for -bluntends-, complementary ends of the fragments can be made available for the ligation.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) or corresponding functional equivalents.
  • An expression cassette is produced by fusing a suitable promoter with a suitable nucleic acid sequence and a polyadenylation signal using common recombination and cloning techniques, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • nucleic acid sequences used in the method according to the invention contain all the sequence features which are necessary in order to achieve a localization which is correct for the location of the biological action or activity. Therefore no further targeting sequences per se are necessary. However, such a localization can be desirable and advantageous and can therefore be artificially changed or strengthened so that such fusion constructs are a preferred advantageous embodiment of the invention.
  • sequences that ensure targeting in plastids are advantageous.
  • targeting in other compartments e.g. in the vacuole, in the mitochondrion, in the endoplasmic reticulum (ER), peroxisomes, lipid body or, due to the lack of corresponding operative sequences, it is desirable to remain in the compartment of formation, the cytosol.
  • the nucleic acid sequences according to the invention are advantageously cloned together with at least one reporter gene into an expression cassette which is introduced into the organism via a vector or directly into the genome.
  • This reporter gene should enable easy detection via a growth, fluorescence, chemo-, bioluminescence or resistance assay or via a photometric measurement.
  • These genes enable the transcription activity and thus the expression of the genes to be measured and quantified easily. This enables genome sites to be identified which show different productivity.
  • an expression cassette comprises upstream, ie at the 5 'end of the coding sequence, a promoter and downstream, ie at the 3' end, a polyadenylation signal and optionally further regulatory elements which coincide with the coding in between sequence are linked to the used in the method of this invention operatively proteins'.
  • operative linkage is to the sequential arrangement of promoter, encoding sequence, terminator and, if necessary, further regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the
  • An expression cassette can contain, for example, a constitutive promoter, for example the 35S promoter, the gene to be expressed and the ER retention signal.
  • the amino acid sequence KDEL lysine, aspartic acid, glutamic acid, leucine
  • KDEL amino acid sequence KDEL
  • the expression cassette is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host organism.
  • a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host organism.
  • Suitable plasmids are, for example, in E.
  • Advantageous yeast promoters are, for example, 2 ⁇ M, pAG-1, YEp6, YEpl3 or pEM-BLYe23.
  • Examples of algae or plant promoters are pLGV23, pGHlac + , pBINl9, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer , L., 1988)
  • the above-mentioned vectors or derivatives of the above-mentioned vectors represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds.
  • vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phas ide, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally. Chromosomal replication is preferred. Functional and non-functional vectors are included.
  • the nucleic acid construct according to the invention can also advantageously be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA can consist of a linearized plasmid or only of the nucleic acid construct as a vector or the nucleic acid sequences used.
  • nucleic acid sequences used in the method according to the invention can also be introduced into an organism alone.
  • nucleic acid sequences in addition to the nucleic acid sequences, further genes are to be introduced into the organism, they can all be introduced into the organism together with a reporter gene in a single vector or each individual gene with or without a reporter gene in each vector, the different vectors being introduced simultaneously or successively can be.
  • the vector advantageously contains at least one copy of the nucleic acid sequences used and / or the nucleic acid constructs according to the invention.
  • the nucleic acid construct can be incorporated into the tobacco transformation vector pBinAR and be under the control of the 35S promoter or the USP promoter.
  • phosphinotricin bar resistance
  • methionine sulfoximine methionine sulfoximine
  • sulfonyl urine Substance
  • ilv resistance ind S. cerevisiae ilv2
  • the glyphosate or clearfield resistance AHAS resistance
  • advantageous vectors can contain sequences for integration into the genome of the organisms, preferably of the plants. Examples of such sequences are the so-called T-DNA borders.
  • advantageous vectors may also contain promoters and terminators, such as those described above. So-called polyA sequences can also be contained in the vector.
  • Advantageous vectors ⁇ are for example 1, 2 and remove. 3 SEQ ID NO: 25 shows the advantageous sequence of the vector pMTX la300.
  • Fusion vectors used in prokaryotes frequently use inducible systems with and without fusion proteins or fusion oligopeptides, it being possible for these fusions to take place both at the N-terminal and at the C-terminal or other usable domains of a protein.
  • Such fusion vectors usually serve: i.) To increase the expression rate of the RNA ii.) To increase the achievable protein synthesis rate, iii.) To increase the solubility of the protein, iv. ) or to simplify purification by means of a binding sequence that can be used for affinity chromatography.
  • proteolytic cleavage sites are also introduced via fusion proteins, which enables a part of the fusion protein to be split off, also for purification.
  • recognition sequences for proteases are e.g. Factor Xa, thrombin and enterokinase.
  • Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which contains glutathione S-transferase (GST), maltose binding protein, or protein A.
  • GST glutathione S-transferase
  • E. coli expression vectors are pTrc [Amann et al. , (1988) Gene 69: 301-315] and pET vectors [Studier et al. ' , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Stratagene, Amsterdam, Netherlands].
  • vectors for use in yeast are pYep-Secl (Baldari, et al., (1987) E bo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES derivatives (Invitrogen Corporation, San Diego, CA).
  • Vectors for use in filamentous mushrooms are described in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., Eds., P. 1-28, Cambridge University Press: Cambridge.
  • insect cell expression vectors can also be used advantageously, e.g. for expression, in Sf .9 cells. These are e.g. the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • plant cells or algal cells can advantageously be used for gene expression.
  • plant expression vectors can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium, vectors for plant transformation", Nucl. Acid: Res. 12: '8711-8721.
  • nucleic acid sequences according to the invention can be expressed in mammalian cells.
  • Examples of corresponding expression vectors are pCDM ⁇ and pMT2PC mentioned in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EMBO J. 6: 187-195).
  • promoters to be used are preferably of viral origin, such as promoters of polyoma, adenovirus 2, cytomegalovirus or Simian virus 40.
  • Further prokaryotic and eukaryotic expression systems are mentioned in chapters 16 and 17 in Sambrook et al. , Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Further advantages adhesive vectors are described in Hellens et al. (Trends in plant science, 5, 2000).
  • nucleic acids according to the invention can be introduced into organisms, for example in plants, by all methods known to the person skilled in the art.
  • transformation The transfer of foreign genes into the genome of a plant " is referred to as transformation.
  • the methods described here for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun - known as the particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution, DIE microinjection and the Agrobacterium-mediated gene transfer of gene transfer takes place advantageously in the present invention, with examples' for example the Agrobacterium tumefaciens strain GV 3101 pMP90, The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol.
  • the construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).
  • Agrobacteria transformed with such a vector can then be used in a known manner for transforming plants, in particular crop plants, such as tobacco plants, for example, by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the introduced nucleic acid or DNA will be cloned into special Pl 'asmide, namely either into an intermediate vector or into a binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA.
  • Intermediate vectors cannot replicate in agrobacteria.
  • the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation).
  • Binary vectors can replicate in both E.
  • coli and agrobacteria contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187). Serving as host cell should contain a plasmid of Agrobacterium carrying a vir region '. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and is sufficiently described in EPA-0 120 516; , Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters BV, Alblasserdam (1985), Chapter V; Fraley et al. , Crit. Rev. Plant. Sci., 4: 1-46 and An et al. EMBO J. "4 (1985), 277-287.
  • plant explants can expediently with Agrobacterium tumefaciens or Agrobacterium rhizogenes are co-cultured.
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes are co-cultured.
  • From the infected plant material (cultured suspension, for example, pieces of leaf, stem segments, roots, but also protoplasts or plant 'cells) may then in a suitable medium, which anti-' may contain biotics or biocides for selecting transformed cells whole plants be regenerated ,
  • the plants thus obtained can then be examined for the presence of the introduced DNA.
  • Other ways of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf., for example Willmitzer, L., 1993 Transgenic plants.
  • Agrobacteria transformed with a vector according to the invention can also be used in a known manner to transform plants such as test plants such as Arabidopsis or crop plants such as cereals, corn, oats, rye, barley, wheat, soybeans, rice, tree
  • 35 wool, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot, paprika, rapeseed, tapioca, cassava, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and wine species are used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and
  • the genetically modified plant cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by SD Kung and 45 R. Wu, Potrykus or Höfgen and Willmitzer. Plants in the sense of the invention are understood to mean plant cells, tissue, organs or whole plants such as seeds, tubers, flowers, pollen, fruits, seedlings, roots, leaves, stems or other parts of plants. In addition, plants are understood to mean propagation material such as seeds, fruits, seedlings, cuttings, tubers, cuts or rhizomes.
  • Suitable organisms or host organisms for the nucleic acid according to the invention, the expression cassette or the vector are in principle advantageously all organisms which are able to express the nucleic acids used according to the invention or are suitable for the expression of recombinant genes.
  • plants such as Arabidopsis, Asteraceae such as Calendula or crops such as soybean, peanut, castor oil, sunflower, - maize, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean ' , microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae or protozoa such as dinoflagellates such as Crypthecodinium.
  • organisms that naturally synthesize oils in large quantities such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, calendula, peanut, cocoa bean or sunflower.
  • transgenic animals are also suitable as host organisms, for example C. elegans.
  • transgenic plants which contain a functional or non-functional nucleic acid construct according to the invention or a functional or non-functional vector according to the invention.
  • functional is to be understood to mean that the nucleic acids used in the method are expressed alone or in the nucleic acid construct or in the vector and a biologically active gene product is produced.
  • Non-functional in the sense of the invention means that the nucleic acids used in the method alone or in the nucleic acid construct or in the vector are not transcribed, are not expressed and / or a biologically inactive gene product is produced.
  • the so-called antisense RNAs are also non-functional nucleic acids or, when inserted into the nucleic acid construct or the vector, a non-functional nucleic acid construct or non-functional vector.
  • Both the nucleic acid construct according to the invention and the vector according to the invention can be used advantageously for the production of transgenic organisms, preferably plants.
  • transgene in the sense of the invention is to be understood that the nucleic acids used in the method are not in their natural place in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously.
  • Tansgen also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been changed.
  • Transgenic is preferably to be understood as meaning the expression of the nucleic acids at a non-natural site in the genome, that is to say there is homologous or preferably heterologous expression of the nucleic acids. The same applies to the nucleic acid construct according to the invention or the vector.
  • Usable expression strains for example, the 1 have a lower protease activity, are described in such: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.
  • the invention also includes the use of the nucleic acids according to the invention, for example those under SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO : 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, 'SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO
  • SEQ ID NO: 92 SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 nucleotide sequences set forth for the creation of genetically modified plants, which are characterized in that they are modified proteins of the SEQ ID NO: 1, 'SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:.
  • SEQ ID NO: 13 SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO
  • SEQ ID NO: 108 encoded proteins, - which are characterized in that they have a very much lower interaction with the herbicide or their activity is not impaired by the herbicide.
  • nucleic acids used in the method according to the invention in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 , SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID ⁇ NO: 30, SEQ ID NO: 32, SEQ .ID NO: 34, SEQ ID NO: 36 , SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, - SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ " ID NO: 54 , SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO
  • SEQ ID NO: 11 SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO 40,
  • SEQ ID NO: 44 SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO 50,
  • SEQ ID NO: 60 SEQ ' ID NO: 62, SEQ ID NO: 64, SEQ ID NO 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO 74,
  • SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 " or SEQ ID NO: 108 are suitable target proteins for newly developed herbicides.
  • nucleic acids mentioned are overexpressed in an advantageous embodiment and the following process steps are advantageously carried out:
  • SEQ ID NO: 28 SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO: 34,
  • SEQ ID NO: 36 SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO: 44,
  • SEQ ID NO: 46 SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO: 52,
  • SEQ ID NO: 54 SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO: 60,
  • SEQ ID NO: 62 SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO: 68,
  • SEQ ID NO: 70 SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO: 76,
  • SEQ ID NO: 78 SEQ ID NO 80, SEQ ID NO 82, SEQ ID NO: 84,
  • SEQ ID NO: 102 SEQ ID NO: 106 or SEQ ID NO: 108 or from a nucleic acid sequence which, on the basis of the degenerate genetic code, can be derived from the back-translation of those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID'NO: 8,
  • SEQ ID NO 10 SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO 31, SEQ ID NO SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO 39, SEQ ID NO SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO SEQ ID NO: 61, SEQ ID NO: 63, ⁇ SEQ ID NO 65,
  • SEQ ID NO: 83 SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO 89,
  • SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 can be derived, or from derivatives or fragments of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO ;: 11
  • SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
  • SEQ ID NO: 36 SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
  • SEQ ID NO: 46 SEQ ID NO: 48, • SEQ ID NO: 50, SEQ ID NO: 52,
  • SEQ ID NO: 54 SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
  • SEQ ID NO: 62 SEQ ID NO: 64, SEQ ID NO ': 66, SEQ ID NO: 68,
  • SEQ ID NO: 70 SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
  • SEQ ID NO: 78 SEQ ID NO: 80, SEQ ID NO: 82 or
  • SEQ ID NO: 108 nucleic acid sequences shown for polypeptides with the in SEQ ID NO: 2, SEQ ID NO: 4,
  • SEQ ID NO 47 SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,
  • SEQ ID NO: 109 encode amino acid sequences shown and encode at least 50%, 60%, preferably 70%, 80%, 90% or more homology at the amino acid level
  • Proteins in a heterologous system for example one
  • Microorganism such as a bacterium of the genus Escherichia such as E. coli XLl-Red or in a cell free system
  • the modified protein or the modified nucleic acid obtained in this way is advantageously transferred, for example that under SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68 ⁇ , SEQ ID NO: 70, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, S
  • a further embodiment of the invention is a method for producing modified gene products which, from the nucleic acid sequences according to the invention described herein, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: '15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 , SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72
  • SEQ ID NO: 5 SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,
  • SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
  • SEQ ID NO: 36 SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
  • SEQ ID NO: 46 SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,
  • SEQ ID NO: 54 SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,
  • SEQ ID NO: 70 SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
  • sequences selected by the method described above can advantageously be introduced into an organism.
  • a further subject of the invention is therefore an organism produced by this method, the organism preferably being a plant.
  • the method is also suitable for gene expression of the above-mentioned biologically active derivatives and fragments.
  • Modified proteins and / or nucleic acids which can impart resistance to herbicides in plants can be obtained from the sequences according to the invention described herein, in particular from the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO : 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
  • SEQ ID NO: 15 SEQ ID NO: 17, SEQ ID NO 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO 34, SEQ ID NO: 36,
  • SEQ ID NO: 38 SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO: 46,
  • SEQ ID NO: 48 SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58, SEQ ID NO: 60 SEQ ID NO 62,
  • This mutagenesis can, for example, the stability and / or enzymatic activity of enzymes or the properties such as binding of low molecular weight compounds with less than 1000 daltons, advantageously less than 900 daltons, preferably less than 800, particularly preferably less than 700, very particularly preferably less than 600 daltons, of proteins or improve or change antisense RNA in a very targeted manner.
  • mutagenesis and selection are, for example, methods such as the in vivo mutagenesis of seeds or pollen and selection of resistant alleles in the presence of the inhibitors according to the invention, followed by genetic and molecular ones Identification of the modified, resistant allele.
  • mutagenesis and selection of resistances in cell culture by increasing the culture in the presence of successively increasing concentrations of the inhibitors according to the invention.
  • the increase in the spontaneous mutation rate can be exploited by chemical / physical mutagenic treatment.
  • modified genes can also be isolated with microorganisms which have an endogenous or recombinant activity of the proteins coded by the nucleic acids used in the method according to the invention and which are sensitive to the inhibitors identified according to the invention.
  • the cultivation of the microorganisms on media with increasing concentrations of inhibitors according to the invention allows the selection and evolution of resistant variants of the targets according to the invention.
  • the frequency of the mutations can in turn be increased by mutagenic treatments.
  • Another object of the invention is a method for creating nucleotide sequences which code for gene products which have a changed biological activity, the biological activity being changed in contrast to the fact that there is increased activity.
  • Increased activity is to be understood as meaning an activity which is at least 10%, preferably at least 30%, particularly preferably at least 50% or 70%, very particularly preferably at least 100% higher than that of the starting organism or of the starting gene product.
  • the biological activity may have been changed so that the substances and / or agents according to the invention no longer or no longer bind correctly to the nucleic acid sequences and / or the gene products encoded by them.
  • no longer or no longer correctly means that the substances have at least 30%, preferably at least 50%, particularly preferably at least 70%, very particularly preferably at least 80% or not at all of the modified nucleic acids and / or bind gene products in comparison to the starting gene product or the starting nucleic acids.
  • Yet another aspect of the invention therefore relates to a transgenic plant genetically modified by the method according to the invention described above.
  • nucleic acids used in the method according to the invention in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
  • SEQ ID NO: 13 SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: -40, SEQ ID NO: "44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66-, SEQ ID NO: 68, SEQ ID NO : 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, " SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90
  • a further subject of the invention is a method for producing transgenic plants which are resistant to substances found by a method according to the invention, characterized in that in these plants Nucleic acids according to the invention with one of the described biological activis act, in particular with the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: -34, SEQ ID NO: 36 / SEQ ID NO : 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ' ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64
  • a new protein / new RNA is introduced, which is characterized in that the chemical structure of the protein or nucleic acid, such as RNA or DNA, which is responsible for the herbicidal action of the low molecular weight substance is changed in such a way that the modified structure no longer has a herbicidal action, or the herbicide in the modified plant is inactivated or modified, for example is broken down, not taken up or not transported or transported into the vacuum, etc., i.e. the interaction of the herbicide with the target location can no longer take place.
  • a new nucleic acid for example a gene, which is introduced into the plant, the nucleic acid coding for a gene product whose function is separated less or not at all from the herbicidal substance.
  • a so-called “alternative pathway” can be created.
  • that the function of the target is taken over by another gene present in the plant or introduced into the plant or its gene product.
  • the present invention therefore also includes the use of plants which genes hit by the T-DNA insertion with the nucleic acid sequences used in the method according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, ⁇
  • SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
  • SEQ ID NO: 36 SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
  • SEQ ID NO: 46 SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,
  • SEQ ID NO: 54 SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
  • SEQ ID NO: 62 SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,
  • SEQ ID NO: 70 SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
  • SEQ ID NO: 78 SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84,
  • Alternative methods for identifying homologous nucleic acids, for example in other plants with sequences similar to those using transposons, are known to the person skilled in the art.
  • This invention therefore also relates to the use of alternative insertion mutagenesis methods for inserting foreign nucleic acid into the nucleic acid sequences according to the invention described herein, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
  • SEQ ID NO: 40 SEQ ID NO 44, ' SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56,
  • SEQ ID NO: 58 SEQ ID NO 60, SEQ ID NO: 62., SEQ ID NO: 64,
  • SEQ ID NO: 98 SEQ ID NO 100, SEQ IDNO: 102, SEQ ID O: 106 or SEQ ID NO: 108 in sequences derived from these sequences on the basis of the genetic code and / or their derivatives or fragments, for example from other plants.
  • a further embodiment are agents comprising a growth-regulating amount of at least one substance identified by the method according to the invention or an antagonist identified by a method according to the invention and at least one inert liquid and / or solid carrier and optionally at least one surface-active substance.
  • These substances or compositions according to the invention with their herbicidal action can be used as defoliants, desiccants, herbicides and in particular as weed killers. Weeds in the broadest sense are understood to mean all plants that grow up in places where they are undesirable. Whether the substances or active substances found with the aid of the methods according to the invention act as total or selective herbicides depends, inter alia, on the amount used, their selectivity and other factors. The substances can be used against the following weeds, for example:
  • Echinochloa Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Braehiaria, Lolium, Bromus, Ayena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Sphenumum, Ischaemum octenium, agrostis, alopecurus, apera.
  • the substances identified or agents containing them in the process according to the invention can advantageously also be used in a further number of crop plants for eliminating undesired plants.
  • the following crops are considered, for example:
  • Carthamus tinetorius Carya illinoinensis, Citrus limon, Citrus sinensis, Coffea arabica (Coffea canephora, Coffea liberica), Cucumis sativus, Cynodon dactylon, Daucus carota, Elaeis guineensis, Fragaria vesca, Glycine max, Gossypium hirsutum, (Gossypium arboreum, Gossypium herbaceum, Gossypium vitifoliu), Helianthus annuus, Hevea brasiliensis, Hordeulus lupusasus, Hordeulus lupusasus, Hordeulus lupusis, Hordeulus lupusia Lens culinaris, Linum usitatissimum, Lycopersicon lycopersicum, Malus spec, Manihot esculenta, Medicago s
  • the substances found by the process according to the invention can advantageously also be used in crops which are tolerant to the action of herbicides by breeding, including genetic engineering methods.
  • the substances according to the invention or the herbicidal compositions comprising them can be sprayed, for example, in the form of directly sprayable aqueous solutions, powders, suspensions, including high-strength aqueous, oily or other suspensions or dispersions, emulsions, oil dispersions, pastes, dusts, sprinkling agents or granules , Atomizing, dusting, scattering or pouring can be used.
  • the application forms depend on the purposes; in any case, they should ensure the finest possible distribution of the active compounds according to the invention.
  • Suitable inert liquid and / or solid carriers liquid additives such as mineral oil fractions of medium to high boiling point, such as kerosene or diesel oil, 'furthermore coal tar oils and oils of vegetable or animal origin, aliphatisehe, cyclic and aromatic hydrocarbons, for example paraffin, tetrahydronaphthalene,. alkylated naphthalenes or their derivatives, alkylated benzenes or their derivatives, alcohols such as methanol, ethanol, propanol, butanol, cyclohexanol, ketones such as cyclohexanone or strongly polar solvents, for example amines such as N-methylpyrrolidone or water.
  • liquid additives such as mineral oil fractions of medium to high boiling point, such as kerosene or diesel oil, 'furthermore coal tar oils and oils of vegetable or animal origin, aliphatisehe, cyclic and aromatic hydrocarbons, for example par
  • aqueous forms of use such as emulsion concentrates, suspensions, pastes, wettable powders or water-dispersible granules, which can be prepared, for example, by adding water.
  • emulsion concentrates such as emulsion concentrates, suspensions, pastes, wettable powders or water-dispersible granules
  • the substances and / or agents, the so-called substrates as such or dissolved in an oil or solvent can be homogenized in water by means of wetting agents, adhesives, dispersants or emulsifiers.
  • concentrates which consist of an active substance, wetting agent, tackifier, dispersant or emulsifier and possibly solvent or oil and which are suitable for dilution with water.
  • Suitable surfactants are the alkali metal ', alkaline earth metal and ammonium salts of aromatic Sulfo ⁇ klareh, for example ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and' from fatty acids, alkyl- and alkylarylsulfonates, alkyl, lauryl "ether and fatty alcohol sulfates, as well as salts of sulfated hexa-, hepta- and octadecanols and of fatty alcohol glycol ether, condensation products of sulfonated naphthalene and its derivatives with formaldehyde, condensation products of naphthalene or naphthalenesulfonic acids with phenol and pormaldehyde, polyoxyethylene octylphenol ether, ethoxylated isooctyl, phenyl, octyl, o
  • Powders, materials for broadcasting and dusts can advantageously be produced as solid carriers by mixing or grinding the active substances together with a solid carrier.
  • Granules e.g. Coating, impregnation and homogeneous granules can be produced by binding the active ingredients to solid carriers.
  • Solid carriers are, for example, mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bolus, loess, clay, dolomite, diatomaceous earth, calcium and magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate , Ureas and vegetable products such as flour, tree bark, wood and nutshells, cellulose powder or other solid carriers.
  • mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bolus, loess, clay, dolomite, diatomaceous earth, calcium and magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as am
  • the concentrations of the substances and / or agents according to the invention in the ready-to-use preparations can be varied within a wide range.
  • the formulations generally contain 0.001 to 98% by weight, preferably 0.01 to 95% by weight, of at least one active ingredient.
  • the active ingredients are in one Purity from 90% to 100%, preferably 95% to 100% (according to the NMR spectrum).
  • the herbicidal compositions or the substances can be applied pre- or post-emergence. If the active ingredients are less compatible for certain crop plants, application techniques can be used in which the herbicidal compositions or substances are sprayed with the aid of sprayers in such a way that the leaves of the sensitive crop plants are not struck wherever possible, while the active ingredients are applied to the leaves below them unwanted plants or the uncovered floor area (post-directed, lay-by).
  • the substances and / or agents according to the invention can be mixed with numerous representatives of other herbicidal or growth-regulating active ingredient groups and applied together.
  • substances and / or agents according to the invention may also be useful to apply the substances and / or agents according to the invention, alone or in combination with other herbicides, mixed with other crop protection agents, for example with agents for controlling pests or phytopathogenic fungi or bacteria.
  • miscibility with mineral salt solutions which are used to remedy nutritional and trace element deficiencies.
  • Non-phytotoxic oils and oil concentrates can also be added.
  • a further subject of the invention is the use of a substance identified by one of the "methods or compositions of the invention comprising these substances as a herbicide or for regulating the growth of plants.
  • the invention also relates to a kit comprising the nucleic acid construct according to the invention, the substances according to the invention, e.g. identifies the antibody according to the invention, the antisense nucleic acid molecule according to the invention and / or an antagonist and / or a herbicidal substance according to the methods according to the invention and the composition described below.
  • a further subject of the invention is a composition comprising the substance according to the invention, the antibody according to the invention, the antisense nucleic acid construct according to the invention and / or an antagonist according to the invention and / or a substance according to the invention identified by a method according to the invention.
  • Wild-type Arabidopsis plants of ecotype C24 were transformed using a modified "in planta" transformation protocol (Bechthold et al., 1992; Clough and Bent, 1998) and transgenic FL plants were selected using antibiotic or herbicide resistance (including Clearfield).
  • T2 seeds from these lines were placed on sterile medium and on soil and visually examined after 7 days of growth under standard conditions for the occurrence of dying seedlings. In particular, changes in pigmentation to its complete absence and morphological anomalies were observed. Only those lines for which a segregation ratio of approx. 2: 1-3: 1, and therefore two to three times the amount of resistant plants to sensitive plants, were determined in a parallel investigation. This ratio is indicative of a single integration site that causes resistance.
  • Example 1 Identification and analysis of line P9, which segregates for a lethal mutation
  • Line P9 (see SEQ ID NO: 3) was identified as described above as a line that segregates for a keellethal mutation. The exact count of the cleavage showed that 25% of the offspring showed the albino phenotype, 25% of the offspring showed sensitivity to the selection and 50% of the offspring showed resistance to the selection. This cleavage ratio is expected when only the homozygously resistant seedlings are homozygous for the mutation, thus showing the recessive phenotype, so that the T-DNA insertion is very closely linked to the lethal mutation. The coupling was further checked in a cosegregation analysis. The descendants of 34 wild-type, resistant plants of the P9 line were analyzed. In all cases, albinos were found in the offspring.
  • genomic DNA was isolated from about 50 mg of tissue from these plants using standard products and methods (columns from Qiagen, Hilden, Germany, or Phytopure-Kit from Amersham Pharmacia, Freiburg, Germany) and gel-electrophoretic separation for integrity and Quantity examined.
  • the genomic sequences adjacent to the T-DNA were amplified using a modified adapter-PCR protocol (Spertini et al., 1999).
  • the PCR was carried out under standard conditions for 7 cycles at an annealing temperature of 72 ° C and for 32 at an annealing temperature of 65 ° C carried out in 25 ⁇ l reaction volume,
  • the amplicon was diluted 1:50 in H0 and ul 'of this dilution for a second round of amplification (5 cycles of' an annealing temperature of 67 ° C and 28 cycles at an annealing temperature of 60 ° C otherwise standard PCR conditions) 5 used.
  • broehene ORF shows a high homology to the DNA repair protein RHP54 "from yeast (Muris et al., J. Cell. Sei., 1996) as well as similarities to a number of other DNA-binding proteins.
  • Line P38 was identified as described above as a line that segregates for a seedling lethal mutation. The exact count of the split showed that 25% of the offspring
  • primers for the predicted 5 'and 3' end were synthesized and used for a standard PCR with Arabidopsis cDNA.
  • the mRNA was isolated from seedlings by means of Dynal oligo-dT Dynabeads, and the cDNA was produced therefrom using a Gibco-BRL cDNA synthesis kit and an oligo-dT primer.
  • the PCR product was' into a TA vector (pCRScriptll, Invitrogen) and sequenced. The sequence showed complete agreement with the predicted sequence and gene structure for this ORF.
  • the inventors hereby document for the first time the experimental confirmation for the existence of this ORF in the predicted structure.
  • Example 3 Identification and analysis of line P44 that segregates for a lethal mutation
  • the line . P44 was identified as a line that segregates for a seedling lethal mutation as described above. The exact count of the cleavage showed that 25% of the offspring showed the albino phenotype, 25% of the offspring showed sensitivity to the selection and 50% of the offspring showed resistance to the selection. This cleavage ratio is expected when only the homozygously resistant seedlings show the phenotype, so the T-DNA insertion is very closely linked to the lethal mutation. The coupling was further checked in a cosegregation analysis. 34 wild-type, resistant plants of the P44 line analyzed the offspring. In all cases, albinos were found in the offspring.
  • Example 4 Identification and analysis of line P77, which segregates 35 for a lethal mutation
  • Line P77 was identified as described above as a line that segregates for a seedling lethal mutation. The exact count of the split showed that 25% of the offspring
  • the T-DNA was inserted in position 35442 of BACT5L19 (Accession number AL049481) of chromosome IV from Arabidopsis.
  • the T-DNA was inserted at line P98 in position 54861 of the PI clone MVA3 (Accession number: AB006706) of the chromosome V.
  • the insertion of the T-DNA was carried out at of line P99b in position 66042 of BAC F10M10 (AL035521) localized on chromosome IV.
  • the insertion of the T-DNA on chromosome IV was localized in the region of the contig fragment 69 in position 46342-46355 (AL 161573).
  • Line P103 had an insertion of the T-DNA in position 57314 of BAC F11F8 (AC016661) of chromosome I.
  • Example 6 Identification and analysis of lines P91 and P99a that segregate for a lethal mutation
  • clones P91 and P99a were identified as lines which segregate for seedling lethal mutations.
  • the molecular biological analyzes were carried out as described in Examples 1 to 4.
  • Line A300364 (SEQ ID NO: 26 [nucleic acid] and 27 [protein]) was identified analogously to the examples mentioned above.
  • Line A300364 cleaves for an embroyletal mutation.
  • a 2: 1 cleavage was observed.
  • the T-DNA is inserted in position 39517 of chromosome II (EMBLJ C004238) in these lines. There the insertion interrupts and thus inactivates an ORF (At2g34860) which codes for an unknown protein (AAC12826.1).
  • the protein shows homology to various DNAJ chaperone protein (Heat Shock Protein 40) over a range of 40 amino acids, for example the DNAJ protein (Q9UXR9) from Methanasarcina thermophila (Hoffmann-Bang et al., Gene 238 (2), 387-395 (1999)).
  • DNAJ protein Q9UXR9 from Methanasarcina thermophila (Hoffmann-Bang et al., Gene 238 (2), 387-395 (1999)).
  • Line A301034 (SEQ ID NO: 28 [nucleic acid] and 29 [protein]) was identified analogously to the aforementioned examples.
  • Line A3-01034 cleaves for an embroyletal mutation.
  • a 2: 1 cleavage was observed.
  • the T-DNA is inserted on chromosome V in position 25928 of the BAC T21H19 (EMBL
  • the derived protein sequence shows clear homologies to the CRSl gene product from maize (AAG00595), which for splicing of the group II intron of the chloroplast gene atpF is required.
  • SEQ ID NO: 43 shows the genomic sequence of line A301034 from start to stop codon, including introns.
  • the activity can be / tested, for example, in assays as described in Bock, Nucleic Acids Res., 1995, 23, 2544-7.
  • Line A300377 (SEQ ID NO: 30 [nucleic acid] and 31 [protein]) was identified analogously to the examples mentioned above.
  • Line A300377 cleaves for an embroyletal mutation.
  • a 2: 1 cleavage was observed.
  • the T-DNA is inserted in position 14509 of the Pl clone MRNl7 (AB005243) .. and therefore very likely in the 3 'untranslated region of an alanyl tRNA synthesis (BAB10601.1).
  • line A300841 (SEQ ID No: 32 [nucleic acid] and 33 [protein]) was identified as essential.
  • Line A300841 cleaves for an embryoletal 5 mutation.
  • a 2: 1 cleavage was observed.
  • ⁇ In the study of 35 lines an absolute co-segregation between the T-DNA and leading to the albino mutation was observed.
  • the T-DNA is inserted in position 3183 of BAC T14P8, which corresponds to position 143432 in Contigfrag . 6 0 of chromosome IV corresponds.
  • OEP86 The activity of an OEP86 can e.g. as in Muckel, J. Biol. 0 Chem., 1996, 271, 23846-52, Young, Plant Physiol., 1999, 121, ⁇ 237-44, or in the Review Keegstra, Curr. Opin. Plant Biol., 1999, 2, 471-6, assays described or cited.
  • Line 2266c (SEQ ID No: 34 [nucleic acid] and 35 [protein]) was identified analogously to the abovementioned examples. 5 ' Line 2266c cleaves for an embryoletal mutation. A 2: 1 cleavage can be observed.
  • the T-DNA is inserted in position 26501 of BAC F6N18 (AC017118) of chromosome I. There, the insertion interrupts an ORF whose derived amino acid sequence (AAF25967.1) shows clear similarity to an "FMRF amide 10 propeptide isolog (gi
  • Line P61 (SEQ ID 15 NO: 36 [nucleic acid] and 37 [protein]) was identified analogously to the abovementioned examples. Line P61 cleaves for an embryoletal mutation. A 2: 1 cleavage was observed. When 35 lines were examined, an absolute cosegregation between the T-DNA and the mutation leading to angry albino phenotype was observed. The T-DNA is inserted in position 20 28640 of the BAC F4B12 (EMBLNEW
  • the insertion inhibits the expression of an ORF that begins in position 28705 and for an unknown protein (BAB02572.1) with weak homology to proteosome protein 26S PROTEASOME SUBUNIT S5B, (Deveraux, Q., Jensen, C. and Rechsteiner, 25 M., Molecular cloning and expression of a 26 S protease subunit. Enriched in dileucine repeats, J. Biol. Chem. 270 (40), 23726-23729 (1995).
  • Line A300857 (SEQ ID No: 38 [nucleic acid] and 39 [protein]) was identified analogously to the abovementioned examples. - A300857 cleaves for an embroyletal mutation. When examining 35 lines, an absolute cosegregation between
  • the T-DNA is inserted in position 51122 of BAC T10O24 of chromosome I (EMBL: AC007067). There the insertion interrupts and thus inactivates an ORF (T10O24.14) which codes for an unknown protein (AAD39574.1).
  • SEQ ID NO: 42 shows the associated genomic sequence.
  • Line A300367 45 (SEQ ID NO: 40 [nucleic acid] and 41 [protein]) was identified analogously to the abovementioned examples.
  • A300367 cleaves for an embroyletal mutation.
  • the T-DNA is inserted in position 31058 "Contig fragments" 86 (EMBL: ATCHRIV86) of chromosome IV.
  • EMBL ATCHRIV86
  • chromosome IV By inserting a few base pairs upstream of the start codon (51073) of a geranylgeranyl pyrophosphate synthase (Bartley and Scolnik, 1994) (Plant Physiol.
  • geranylgeranyl pyrophosphate synthase can e.g. as described in Zhu et al., Plant Cell Physiol., 1997, 38, 337-61, or Okada, Plant Physiol., 2000, 122, '1045-56.
  • Line 305735 (SEQ ID NO: 44 [nucleic acid] and SEQ ID NO: 45 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 305735 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is integrated in position 46571 of the sequence ATCHRIV69, accession number AL161573. The insertion at this position disrupts the ORF AT4g28590, which codes for a hypothetical protein which has a "Cecropin" family signature (A ⁇ 237-245).
  • Line 303726 (SEQ ID NO: 46 [nucleic acid] and SEQ ID NO: 47 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 303726 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 51568 of BACs AC004669 on chromosome 2. The insertion at this point very likely prevents or affects the transcription and thus the function of the ORF At2g30950. This ORF codes for a putative ftsH chloroplast protease.
  • Line 304249 (SEQ ID NO: 48 [nucleic acid] and SEQ ID NO: 49 [protein encoded by the above nucleic acid]).
  • Line 304249 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • Several ESTs have already been described for this ORF, GB: Z31666, gb: Z33957, Z31666. This protein is a peroxisomal tetrafunctional enzyme of fatty acid metabolism.
  • Line 304264 (SEQ ID NO: 50 [nucleic acid] and SEQ ID NO: 51 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 304264 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is inserted in position 63762 of BAC ⁇ B020742.
  • the installation site is approx. 240 base pairs upstream of the start codon for an ORF K21H1.19, which codes for a UDP-glucuronyl transferase-like protein.
  • Line 304485 (SEQ ID NO: 52 [nucleic acid] and SEQ ID NO: 53 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 304485 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 25034 of BACs AC006438 on chromosome 2. By integrating at this point, the T-DNA destroys the ORF At2gl5820, which codes for an unknown protein.
  • Line 304652 (SEQ ID NO: 54 '[nucleic acid] and SEQ ID NO: 55 [protein encoded by the above nucleic acid]) and SEQ ID NO: 56 were analogous to the abovementioned examples
  • Line 304652 segregates for an albinolethal mutation that cosegregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 6309 of BACs ATF12B17, Accession AL353995, on chromosome 5. Two different open reading grids are annotated for the neighboring region.
  • Line 304656 (SEQ ID NO: 58 [nucleic acid] and SEQ ID NO: 59. [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 304656 segregates for an albinolethal mutation that. with the resistance marker and thus the T-DNA cosegregated.
  • the T-DNA is inserted in position 35169 of BAC F24P17 (Accession AC011623) on chromosome 3. There, the insertion interrupts and thus destroys an ORF F24P17.10, which codes for an unknown protein.
  • a blastp comparison with standard settings shows clear homoligues to a nodulin / glutamate-ammonia ligase - like protein.
  • Line 302192 (SEQ ID NO: 60 [nucleic acid] and SEQ ID NO: 61 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 302192 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 43178 of BAC AB022211 on chromosome 5.
  • Line 302636 (SEQ ID NO: 62 [nucleic acid] and SEQ ID NO: 63 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • the line 302636 segregates for an albinolethal mutation with the resistance marker and thus the T-DNA.
  • the T-DNA is (sion Acces': AL132966) at position 141,376 of the BACs ATF4P12 on chromosome 3 inserted.
  • the integration of the T-DNA at this position interrupts and thus inactivates the ORF F4P12_400, which codes for protein similar to crpl au Zea mays, PIR: T01685.
  • This ORF also includes the ESTs gb: AI999771.1, T45254, AA713158 ".
  • Line 302894 (SEQ ID NO: 64 [nucleic acid] and SEQ ID NO: 65 (protein encoded by the above nucleic acid)) was identified analogously to the abovementioned examples.
  • Line 302894 segregates for a albinolethale mutation cosegregates with 'the resistance marker and thus the T-DNA.
  • the T-DNA is inserted in position 23970 of the BACs ATT21H19 (Accession: AL391148) on chromosome 5.
  • the insertion of the T-DNA at this point interrupts an ORF (T21H19_100) which codes for a putative protein with similarities to hypothetical proteins from Arabidopsis.
  • the blastp analysis also shows clear homology.
  • CRS1 from Zea mays Accession AAG00595 ' , which is a Group II intron splicing factor (Till, B et al., RNA 7 (9), 1227-1238 (2001)).
  • Line 305146 (SEQ ID NO: 66 [nucleic acid] and SEQ ID NO: 67 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 305146 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 65706 of the PI clone MOP9 (accession: AB0067.01) on chromosome 5. The insertion of the T-DNA at this point disrupts the ORF of the At5g24315 gene, which codes for an unknown protein.
  • Line 305156 (SEQ ID NO: 68 [nucleic acid] and SEQ ID NO: 69 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 305156 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 898 of the BAC ATT20O10 M0P9 (Accession: AL163816) on chromosome 3.
  • the insertion of the T-DNA on this enjoy interrupts an ORF (T20O10_10) which shows a protein with a high similarity to translation releasing factor RF-1 from Synechocystis (PIR: S76914).
  • the deduced amino acid sequence contains a prokaryotic Type I pepeptide chain detachment factor motif, AA280-296
  • Line 304044 (SEQ ID NO: 70 [nucleic acid] and SEQ ID NO: 71 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 304044 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is inserted in position 44121 of the BACs ATAP22 MOP9 (Accession: Z99708) on chromosome 4.
  • the insertion of the T-DNA at this point interrupts an ORF (C7A10.610) which is for a protein with high similarity to an allergen ("inor allergen") from Alternaria alternata (PIR2: S43111).
  • ESTS gb: R64949., ⁇ A651052 have also already been found for this ORF.
  • Line 140412 (SEQ ID NO: 72 [nucleic acid] and SEQ ID NO: 73 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 140412 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 68520 of sequence AC006264 on 'chromosome 2.
  • the insertion of the T-DNA breaks the 3'UTR of the gene At2g21160 and thus prevents • very likely the function of the ORFs by Bestabilmaschine the transcript.
  • the ORF At2g21160 codes for the alpha subunit of a putative signal sequence receptor.
  • the line 159012 was - identified.
  • Line 159012 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 127261 of the sequence ATCHRIV3 (frag ent No. 3), Accession AL161491 on chromosome 4.
  • the insertion of the T-DNA interrupts the ORF AT4g01220, - which contains the ESTs gb: AA597894, AA597304 and codes for unknown protein.
  • Line 106037 (SEQ ID NO: 76 [nucleic acid] and SEQ ID NO: 77 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 106037 segregates for an albinolethal mutation with the resistance marker. and thus the T-DNA cosegregates.
  • the T-DN ⁇ is inserted in position 50359 of sequence AC006193 on chromosome 1.
  • the insertion of the T-DNA at this position interrupts the ORF F13011.ll, which codes for an unknown protein.
  • the blastp analysis with standard settings shows a similarity to oxidoreductases.
  • Line 126905 (SEQ ID NO: 78 [nucleic acid] and ' SEQ ID NO: 79 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 126905 segregates for an embryolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted in position 71928 of BACs ATF25L23, Accession AL356014, on chromosome 3. The insertion of the T-DNA at this position interrupts the ORF ' F25L23_240 "which codes for a farnesyl transferase subunit A.
  • Line 12.7458 (SEQ ID NO: 80 [nucleic acid] and SEQ ID NO: 81 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 127458 segregates for an embryolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is inserted in position 45352 of BACs T19K24, Accession AC002342, on chromosome 5. The insertion of the T-DNA at this position interrupts the ORF T19K24.18, which codes for the ATP-dependent copper transporter RANl.
  • Line 304249b (SEQ ID NO: 82 [nucleic acid] and SEQ ID NO: 83 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 304249b segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • line 304264b (SEQ ID " NO: 84 [nucleic acid] and SEQ ID NO: 85 [protein encoded by the above nucleic acid]) was identified.
  • Line 304264b segregates for an albinolethal mutation associated with the resistance marker and thus The T-DNA is cosegregated
  • the T-DNA is inserted in position 63762 of the BACs AB020742
  • the installation parts are located approximately 340 base pairs upstream from the start codon for an ORF K21H1.18 which is similar to unknown proteins.
  • Line 192813 (SEQ ID NO: 86 [nucleic acid] and SEQ ID NO: 87 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 192813 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted on chromosome 1 in position 9869 of the BACs-F309 with the accession AC006341. By inserting approx.
  • Line 203521 (SEQ ID NO: 88 [nucleic acid] and SEQ ID NO: 89 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • the Line 203521 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is inserted on Chromsom 2, Section 179 of 255 Accession AC006533. The insertion destroys the ORF AT2g31830, which codes for a putative inositol polyphosphate 5 'phosphatase.
  • line 206462 (SEQ ID NO: 90 [nucleic acid] and SEQ ID NO: 91 [protein encoded by the above nucleic acid]) was identified.
  • Line 206462 segregates for an albinolethal mutation, -with the resistance marker and The T-DNA is inserted on chromosome 1 in position 53577-53600 of BAC F24D7, accession AC011622.
  • the ORF F24D7.13 which is responsible for a putative UDP-N- Acetyl-muramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase (urE) coded, destroyed.
  • Line 216642 (SEQ ID NO: 92 [nucleic acid] and SEQ ID NO: 93 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 216642 segregates for an albinolethal mutation, which segregates with the resistance marker and thus the T-DNA cos.
  • the T-DNA is inserted on chromosome 3 in position 18529 of PI clone MRC8, Accession-AB020749. The insertion at this point destroys the ORF MRC8.5, which codes for a beta-glucosidase.
  • Line 219902 (SEQ ID NO: 94 - [nucleic acid] and SEQ ID NO: 95 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 219902 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted on chromosome 1 in position 7740 of BAC F15M4, Accession AC012394. The insertion at this point destroys the ORF F15M4.1, which codes for a hydroxymethylglutaryl-CoA reductase.
  • Line 220801 (SEQ ID NO: 96 [nucleic acid] and SEQ ID NO: 97 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 220801 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is inserted on chromosome 5 in position 15447-15472 of Pl clone MRN17, accession AB005243. With this insertion approx. 580bp upstream from the start codon, at least the
  • Line 224933 (SEQ ID NO-: 98 [nucleic acid] and SEQ ID NO: 99 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 224933 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is on chromosome 4, ⁇ SSA I FCA Contig fragment No. 3., Accession Z97338, in position 107932-107997. This insertion destroys the functionality of the ORF dl3705c, which codes for a cellulose synthase-like protein.
  • Line 229091 (SEQ ID NO: 100 [nucleic acid] and SEQ ID NO: 101 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 229091 segregates for an albinolethal mutation which co-segregates with the resistance marker and thus with the " T-DNA.
  • the T-DNA is inserted on position 5, TAC clone: K5J14, accession AB023032, in position 55778. By insertion on this The functionality of the ORF K5J14.11 is destroyed, which codes for a protein similar to the crpl protein from maize.
  • Line 246473 (SEQ ID NO: 102 [nucleic acid] and SEQ ID NO: 103 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 246473 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted on chromosome 1, BAC F4F7, accession AC079374, in position 17376.
  • the insertion at this point, approx. 7 bp downstream of the ORF F4F7.26, is highly likely to The transcription or transcript stability and thus the functionality for this open reading frame is destroyed.
  • This ORF codes for a putative t-RNA glutamine synthetase and in particular has homology to tRNA glutamine synthetase GI: 2995454 from Lupinüs luteus.
  • Line 304139 (SEQ ID NO: 104 [nucleic acid] and SEQ ID NO: 105 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 304139 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA.
  • the T-DNA is inserted on chromosome 5, P 1 clone MFB 13, accession AB010073, in position 49311-49335. By insertion at this point, approx. 25 bp downstream of the ORF MFB13.17, the transcription or the transcript stability and thus the functionality for this open reading frame, which codes for an exonuclease-like protein, is destroyed with high probability.
  • Line 304886 (SEQ ID NO: 106 [nucleic acid] and SEQ ID NO: 107 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples.
  • Line 304886 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is inserted on chromosome 2, BAC clone F23H14, section 1 of 255, accession AC006837, in position 84045.
  • the ORF At2g01110 is interrupted and deactivated by the insertion at this point. This codes for a putative "sec-independent" trans-locase protein TATC (putative sec-independent protein ' t r ah S iocase protein TATC). The sequence is also described in WO 144277.
  • Line 306053 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA.
  • the T-DNA is on chromosome 4, BAC clone F28J12, accession AGAL021710, in position 74806-74828. The insertion at this point interrupts and inactivates the ORF F28J12.180, which codes for a putative protein.
  • the deduced amino acid sequence shows In addition to clear homologies to various hypothetical and putative proteins, there is also strong similarity to proteins similar to selenium binding proteins.

Abstract

The invention relates to genes and methods for isolating genes coding for gene products, the lack of said gene products leading to significantly delayed growth and/or completely stunted growth at the embryonic stage of Arabidopsis thaliana. The invention relates to the use of said genes and the gene products coded thereby for discovering novel herbicides. The invention also relates to the use of the genes and gene products identified here for producing plants which are resistant to herbicides. The invention further relates to a method for identifying herbicidally active substances, substances identified according to said method and the use of said substances as herbicides. Furthermore, the invention relates to nucleic acid constructs, vectors, organisms containing said nucleic acid constructs and/or vectors, and especially transgenic plants containing said nucleic acid constructs and/or vectors. Disclosed is also a method for identifying antagonists and the use thereof, antibodies and antisense RNA molecules, and agents containing a herbicide substance which is isolated according to the inventive methods. Finally, the invention relates to compositions containing the antibodies, the antisense nucleic acids and/or the antagonists.

Description

Verfahren zur Identifizierung von Substanzen mit herbizider Wirkung Method of identifying substances with herbicidal activity
Beschreibungdescription
Eine moderne Landwirtschaft ist ohne den Einsatz von Herbiziden nicht denkbar. Gegenwärtig wird der Wert der auf der gesamten Welt' eingesetzten Herbizide auf ca. 30 Mrd. DM geschätzt. Obwohl bereits eine ganze Reihe von hoch wirksamen und ökologisch unbedenklichen Herbiziden auf dem Markt sind, ergibt sich die Notwendigkeit für neue Herbizide zum einen aus der Tatsache, dass immer wieder Unkräuter eine Resistenz gegen bereits eingesetzte Herbizide entwickeln und diese somit zum Teil nicht mehr ein- gesetzt werden können, zum anderen aus der Tatsache, dass ein Teil der Herbizide ökologische Nachteile aufweist. Zum heutigen Zeitpunkt werden in vielen Fällen noch Herbizide als Mischungen eingesetzt werden, die mehrere aktive Wirkstoffkomponenten enthalten, was ökologisch wenig vorteilhaft ist und zudem besondere Anforderungen an die Formulierung stellt.Modern agriculture is inconceivable without the use of herbicides. Currently, the value of the herbicides' used throughout the world is estimated at about 30 billion. Although a whole series of highly effective and ecologically harmless herbicides are already on the market, the need for new herbicides arises on the one hand from the fact that weeds repeatedly develop resistance to herbicides already used and some of them are no longer used. can be set, on the other hand from the fact that some of the herbicides have ecological disadvantages. At the present time, herbicides are still used in many cases as mixtures which contain several active ingredient components, which is not ecologically advantageous and also places special demands on the formulation.
Neue Herbizide sollten sich durch einen möglichst breiten Wirkungsbereich, ökologische und toxikologische Unbedenklichkeit sowie geringe Aufwandmengen auszeichnen.New herbicides should be characterized by the broadest possible range of activity, ecological and toxicological harmlessness and low application rates.
Das bisherige Vorgehen bei der Identifizierung und Entwicklung neuer Herbizide ist durch die Applikation von potentiellen Wirkstoffen direkt auf geeignete Testpflanzen gekennzeichnet . Dieses Verfahren weist den Nachteil auf, dass für die Tests relativ große Substanzmengen erforderlich sind. Dies ist im Zeitalter der kombinatorischen Chemie mit seiner in einer' äußerst großen Vielfalt, aber nur in geringen Mengen herstellbaren Substanzen jedoch selten gegeben und stellt von daher eine wichtige Limitierung bei der Entwicklung neuer Herbizide dar. Zum anderen Werden bei der direkten Applikation auf die zu testenden Pflanzen bereits im ersten Screeningschritt äußerst hohe Anforderungen an die Substanz gestellt, da nicht nur die Inhibierung oder sonstige Modulation der Aktivität eines zellulären Zieles (in der Regel • .ein Protein oder Enzym) erforderlich sind, sondern die Substanz dieses Ziel zunächst überhaupt erreichen muss, was bereits in diesem ersten Schritt Anforderungen an die Testsubstanz in Bezug auf Aufnahme durch die Pflanze; Permeabilität durch die verschiedenen Zellwände und Membranen, Persistenz zur Erreichung des gewünschten Effektes, und schließlich Inhibierung/Veränderung der Aktivität des gewünschten Zielenzyms erfordert. Es ist angesichts dieser Erfordernisse daher nicht überraschend, dass zum einen die Identifizierung neuer Wirkstoffe immer höhere Kosten verursacht, zum anderen immer weniger Wirkstoffe entdeckt werden.The previous procedure for identifying and developing new herbicides is characterized by the application of potential active ingredients directly to suitable test plants. This method has the disadvantage that relatively large amounts of substance are required for the tests. This is rarely the case in the age of combinatorial chemistry with its ' extremely large variety, but only in small quantities, and therefore represents an important limitation in the development of new herbicides provided test plants in the first screening step extremely high demands on the substance, as not only the inhibition or other modulation of the activity of a cellular target are required (usually •. a protein or enzyme), but the substance of this target first ever reach must what is already in this first step requirements for the test substance in relation to absorption by the plant; Permeability through the various cell walls and membranes, persistence to achieve the desired effect, and finally inhibition / change in the activity of the desired target enzyme is required. In view of these requirements, it is therefore not surprising that, on the one hand, the identification of new active substances causes ever higher costs and, on the other hand, fewer and fewer active substances are discovered.
Es war deshalb Aufgabe der vorliegenden Erfindung, Targets für die Identifizierung neuer Herbizide, sowie neue Herbizide und deren Verwendung zur Verfügung zu stellen. Diese Aufgabe wurde durch ein Verfahren zur Identifizierung von Substanzen mit herbizider Wirkung gelöst, dadurch gekennzeichnet, dassIt was therefore an object of the present invention to provide targets for the identification of new herbicides and new herbicides and their use. This object was achieved by a method for identifying substances with a herbicidal action, characterized in that
a) die Expression oder die Aktivität des Genprodukts einer Nukleinsäure oder eines Gens umfassend:a) the expression or the activity of the gene product of a nucleic acid or a gene comprising:
aa) Nukleinsäuresequenz mit der in SEQ ID NO: 1,aa) nucleic acid sequence with that in SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 /SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 /
SEQ ID NO: 9, SEQ ID NO: • 11, SEQ ID NO: 13,SEQ ID NO: 9, SEQ ID NO: • 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32
SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38
SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64
SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70
SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88
SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94
SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100,SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100,
SEQ ID NO: 102 , SEQ ID NO : 106 oder SEQ ID NO 108 dargestellten SequenzSEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO 108
bb) Nukleinsäuresequenz , die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID.NO: 12,bb) Nucleic acid sequence which, on the basis of the degenerate genetic code, results from the back-translation of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID. NO: 12,
SEQ ID NO: 14, SEQ ID NO 16, SEQ ID NO: 18,SEQ ID NO: 14, SEQ ID NO 16, SEQ ID NO: 18,
SEQ ID NO: 27, SEQ ID NO 29, SEQ ID NO: 31,SEQ ID NO: 27, SEQ ID NO 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO 35, SEQ ID NO: 37,SEQ ID NO: 33, SEQ ID NO 35, SEQ ID NO: 37,
SEQ ID NO: 39, SEQ ID NO 41, SEQ ID NO: 45,SEQ ID NO: 39, SEQ ID NO 41, SEQ ID NO: 45,
SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO: 51,SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO 55, SEQ ID NO: 57,SEQ ID NO: 53, SEQ ID NO 55, SEQ ID NO: 57,
SEQ ID NO: 59, SEQ ID NO 61, SEQ ID NO: 63,SEQ ID NO: 59, SEQ ID NO 61, SEQ ID NO: 63,
SEQ ID NO: 65, SEQ ID NO 67, SEQ ID NO: 69,SEQ ID NO: 65, SEQ ID NO 67, SEQ ID NO: 69,
SEQ ID NO: 71, SEQ ID NO 73, SEQ ID NO: 75,SEQ ID NO: 71, SEQ ID NO 73, SEQ ID NO: 75,
SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO 85, SEQ ID NO 87, SEQ ID NO: 89, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO: 95, SEQ ID NO 97, SEQ ID NO 99,SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO 85, SEQ ID NO 87, SEQ ID NO: 89, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO: 95, SEQ ID NO 97, SEQ ID NO 99,
SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen ableiten lässt;SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109;
cc) Nukleinsäuresequenz , die ein Derivat oder ein Fragment der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ ID NO: 11,cc) nucleic acid sequence which is a derivative or a fragment of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO: 17,SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO: 17,
SEQ ID NO 26, SEQ 'ID NO 28, SEQ ID NO: 30,SEQ ID NO 26, SEQ ' ID NO 28, SEQ ID NO: 30,
SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO: 36,SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO: 36,
SEQ D NO 38, SEQ ID NO 40, SEQ ID NO: 44,SEQ D NO 38, SEQ ID NO 40, SEQ ID NO: 44,
SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO: 50,SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO: 50,
SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO: 56,SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO: 56,
SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO: 62,SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO: 62,
SEQ ID NO 64, SEQ ID NO 6 666,, SEQ ID NO: 68,SEQ ID NO 64, SEQ ID NO 6 666 ,, SEQ ID NO: 68,
SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO: 74,SEQ ID NO 70, SEQ ID NO 72, SEQ ID NO: 74,
SEQ ID NO 76, SEQ ID NO 7 788,, SEQ ID NO: 80,SEQ ID NO 76, SEQ ID NO 7 788 ,, SEQ ID NO: 80,
SEQ ID NO 82, SEQ ID NO 84, SEQ ID NO: 86,SEQ ID NO 82, SEQ ID NO 84, SEQ ID NO: 86,
SEQ ID NO 88, SEQ ID NO 90, SEQ ID NO: 92,SEQ ID NO 88, SEQ ID NO 90, SEQ ID NO: 92,
SEQ ID NO: 94, SEQ ID NO 96 , SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO : 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Nukleinsäuresequenzen ist, und mindestens 60 % Homologie auf Nukleinsäureebene aufweist; • SEQ ID NO: 94, SEQ ID NO 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108, and at least 60% homology Has nucleic acid level; •
dd) Nukleinsäuresequenz, die für Derivate oder Fragmente der Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29", SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35', SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID'NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,dd) nucleic acid sequence which is suitable for derivatives or fragments of the polypeptides with those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29 ", SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 ' , SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID'NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,
SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,
SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91,
SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97,SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97,
SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO : 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen codiert, die mindestens 50 % Homologie auf Aminosäureebene aufweisen;SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 encoded amino acid sequences shown that have at least 50% homology at the amino acid level;
ee) Nukleinsäuresequenz, die für ein Fragment oder ein 5 Epitope eines Polypeptides codiert, das spezifisch an einem Antikörper bindet, wobei der Antikörper spezifisch an ein Polypeptid bindet, das von der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,. SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,ee) Nucleic acid sequence which codes for a fragment or a 5 epitope of a polypeptide which binds specifically to an antibody, the antibody specifically binding to a polypeptide which is derived from that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 ,. SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
10. SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, 10th SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
15 ' -SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ -ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,15 'SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66 , SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
20 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: .86,20 SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: .86,
SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Sequenz codiert wird;SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 is encoded;
25 ff) Nukleinsäuresequenz, die für ein Fragment einer in aa) dargestellten Nukleinsäure codiert und das eine mδA-Methyltransferase-Aktivität, eine DNA-bindende- Aktivität oder "DNA-repair"-Aktivität, z.B. wie bei25 ff) Nucleic acid sequence which codes for a fragment of a nucleic acid shown in aa) and which has an mδA methyl transferase activity, a DNA binding activity or "DNA repair" activity, e.g. as in
30 RAD 54, eine Thioredoxin-Aktivität, eine VAV2-Aktivität, eine Fructokinase-Aktivität, eine Zinkfingerprotein- Aktivität, eine LYTB-Aktivität, eine Crepopin-Aktivität, eine Leucin-Protein-Aktivität, eine DNAJ-Aktivität, ein CRSl-Aktivität, eine Alanyl-tRNA-Synthetase-' 30 RAD 54, a thioredoxin activity, a VAV2 activity, a fructokinase activity, a zinc finger protein activity, a LYTB activity, a crepopin activity, a leucine protein activity, a DNAJ activity, a CRSI activity , an alanyl tRNA synthetase- '
35 Aktivität, eine OEP86-Aktivität, eine FMRF-Amid-35 activity, an OEP86 activity, an FMRF amide
Propeptid-Isolog-Aktivität, eine 26S Proteoso subunit S5B-Aktivität, eine Geranylgeranylpyrophosphatsynthase- Aktivität, eine Cecropin-Familiensignatur aufweißt, ftsH Cloroplast-Protease-Aktivität hat, eine AIM1-Aktivität, 0 eine UDP-glucuronyltransferase-Aktivität, eine FPFl-Propeptide isolog activity, a 26S proteoso subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, a cecropin family signature, ftsH has chloroplast protease activity, an AIM1 activity, 0 a UDP-glucuronyl transferase activity, an FPFl-
Aktivität, eine SHI-ähnliche Zinkfingerprotein-Aktivität hat, eine Crpl-Aktivität, eine CRSl-Aktivität, eine translation releasing factor RF-1-Aktivität, eine Farnesyltransferase Untereinheit A-Aktivität oder eine 5 ATP-abhängige Kupfertransporter RANl-Aktivität hat; und/oder gg) Nukleinsäuresequenz, die für Derivate der Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,Activity, SHI-like zinc finger protein activity, Crpl activity, CRSl activity, translation releasing factor RF-1 activity, farnesyltransferase subunit A activity or 5 ATP-dependent copper transporter RANl activity; and or gg) Nucleic acid sequence which is suitable for derivatives of the polypeptides with the in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,
SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, -SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99,- SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: .105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Amino- säuresequenzen codiert, die mindestens 20 % Homologie auf Aminosäureebene aufweist und eine äquivalente biologischeSEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, -SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO : 97, SEQ ID NO: 99, - SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO:. 105, SEQ ID NO: 107 or SEQ ID NO: 109 encoded amino acid sequences, which has at least 20% homology at the amino acid level and an equivalent biological
Aktivität besitzt; oderHas activity; or
b) die Expression oder Aktivität einer Aminosäuresequenz, die . von einer Nukleinsäuresequenz nach aa) bis gg) codiert wird,b) the expression or activity of an amino acid sequence which. is encoded by a nucleic acid sequence according to aa) to gg),
beeinflusst wird und solche Substanzen ausgewählt werden, die die Expression oder die Aktivität reduzieren oder blockieren.is influenced and those substances are selected which reduce or block expression or activity.
Unter "Expression" wird die Neusynthese in vitro und in vivo von Nukleinsäuren und durch Nukleinsäuren codierten Proteinen verstanden, insbesondere die der obengenannten Nukleinsäure- und Aminosäuresequenzen. Der Begriff "Expression" umfasst alle bis zum reifen Protein oder dessen Abbau führenden Biosyntheseschritte, z.B. Transkription, Translation, Modifizierung oder Prozessierung von Nukleinsäuren und Proteinen, z.B. prae- oder posttranskriptionelle Prozessierungsschritte oder post- translationale Modifikationen, z.B. Splicing, Editing, Poly- adenylierung, Capping, Modifikationen von Aminosäuren, z.B. Glykosilierung, Methylierung, Acetylierung, Bindung von Coenzymen, Phosphorylierung, Ubiquitation, Bindung von Fettsäuren, Signalpeptidprozessierung, etc."Expression" is understood to mean the new synthesis in vitro and in vivo of nucleic acids and proteins encoded by nucleic acids, in particular that of the above-mentioned nucleic acid and amino acid sequences. The term "expression" encompasses all biosynthetic steps leading up to the mature protein or its degradation, e.g. Transcription, translation, modification or processing of nucleic acids and proteins, e.g. pre- or post-transcriptional processing steps or post-translational modifications, e.g. Splicing, editing, polyadenylation, capping, modifications of amino acids, e.g. Glycosylation, methylation, acetylation, binding of coenzymes, phosphorylation, ubiquitation, binding of fatty acids, signal peptide processing, etc.
Wobei im Sinne der Erfindung unter "Transkription" die RNA- Synthese mithilfe einer RNA-Polymerase in 5'—> 3 ' -Richtung anhand einer DNA-Matrize zu verstehen ist. Unter "Translation" ist die Biosynthese von Proteinen in vitro und in vivo zu verstehen. Unter Genprodukt ist jedes Molekül und jede Substanz zu ver- stehen, die aufgrund der Expression, z.B. der Transkription oder Translation einer Nukleinsäure, z.B. einer DNA oder RNA, z.B'. eines Gens, entsteht, wobei der Begriff auch die folgenden Prozessierungsprodukte, wie z.B. nach Splicing oder Modifizie- rung, umfasst. So wird' unter Genprodukt z.B. eine prozessierte RNA, z.B. eine katalytische RNA, eine funktioneile RNA, wie tRNAs oder rRNAs, oder. eine codierende RNA, wie mRNA, verstanden. Als Folge der Translation einer mRNA wird ein Protein synthetisiert, das ebenfalls als "Genprodukt" verstanden wird. Proteine können ' während und nach der Translation" verschiedenen Prozessierungs- schritten unterworfen sein, wie oben beispielhaft aufgezählt. Unter "Aktivität des Genprodukts" ist die biologische Aktivität bzw. Funktion einer RNA oder eines Proteins, wie beispielsweise die enzy atische Aktivität, die Rezeptorbindungseigenschaft, die Fähigkeit, bestimmte Proteine, Nukleinsäuren oder Metaboliten zu binden, z.B. in Proteinkomplexen, das heißt z.B. die regulative Eigenschaft oder die Transporterfunktion des Proteins oder der RNA, zu verstehen, wie sie im Organismus natürlicherweise vorkommt. Unter "Reduktion der Aktivität des Genprodukts" ist eine Verringerung der biologischen Aktivität im Vergleich zur natürlichen Aktivität des Genprodukts von mindestens 10 %, vorteilhaft mindestens 20 %, bevorzugt mindestens 30 %, besonders bevorzugt um mindestens 50 % und ganz besonders bevorzugt um mindestens 70 % zu verstehen. Blockierung der Aktivität des Genprodukts bedeutet die gänzliche, das heißt 100 %ige Inhibierung derIn the context of the invention, "transcription" means RNA synthesis with the aid of an RNA polymerase in the 5 '->3' direction using a DNA template. "Translation" means the biosynthesis of proteins in vitro and in vivo. Every molecule and every substance is stand, due to the expression, for example the transcription or translation of a nucleic acid, for example a DNA or RNA, e.g. '. of a gene arises, the term also encompassing the following processing products, such as, for example, after splicing or modification. For example, a processed RNA, for example a catalytic RNA, a functional RNA such as tRNAs or rRNAs, or. understood a coding RNA, such as mRNA. As a result of the translation of an mRNA, a protein is synthesized, which is also understood as a "gene product". Proteins can 'during and after translation "different Prozessierungs- steps be subjected, as enumerated above as an example. The term" activity of the gene product "is the biological activity or function of an RNA or a protein, such as the Enzy atic activity, the receptor binding property, the ability to bind certain proteins, nucleic acids or metabolites, for example in protein complexes, that is to say for example the regulative property or the transporter function of the protein or the RNA, as it naturally occurs in the organism. "Reduction of the activity of the gene product" is a reduction in the biological activity compared to the natural activity of the gene product of at least 10%, advantageously at least 20%, preferably at least 30%, particularly preferably by at least 50% and very particularly preferably by at least 70%. Blocking the activity of the gene product means the whole che, that is 100% inhibition of
Aktivität oder die teilweise Blockierung der Aktivität, bevorzugt eine mindestens 80 %ige, besonders bevorzugt mindestens 90%ige, ganz besonders bevorzugt mindestens 95 %ige Blockierung der biologischen Aktivität.Activity or the partial blocking of the activity, preferably an at least 80%, particularly preferably at least 90%, very particularly preferably at least 95% blocking of the biological activity.
Eine Reduktion der Aktivität des Genprodukts kann auch indirekt erfolgen, z.B. in dem die Bildung oder Aktivität von Interaktionspartnern gehemmt wird, z.B. in dem die Stoffwechselkette, in die das Genprodukt eingebunden ist, beeinflusst wird. Z.B. kann eine Hemmung •nicht nur des fraglichen Enzyms, sondern auch eines in derselben Stoffwechselkette stehenden Enzyms oder Proteins erfolgen, die zu einer Blockierung des folgenden, vorhergehenden oder eines anderen involvierten Enzyms führt und somit des hierin beschriebenen Genproduktes, z.B. durch Substrat- oder Produkthemmung. Solche Reduktionen durch indirekte Beeinflussung der Aktivität eines Enzyms ist z.B. für die Wechselwirkung der Glykolyseproteine und -metaboliten ausführlich beschrieben worden und leicht übertragbar auf andere Stoffwechselwege, in denen die hierin beschriebenen Genprodukte eine Rolle spielen. Genauso kann ein erfindungsgemäß verwendetes Genprodukt in seiner Aktivität reduziert oder gehemmt werden, in dem die Aktivität von Interaktionspartnern, z.B. anderen Proteinen in einem Proteinkomplex mit dem hierin beschriebenen Genprodukt reduziert oder gehemmt wird. Das kann dazu führen, dass der gesamte Komplex nicht mehr aktiviert oder nicht oder nur teilweise entsteht oder nicht mehr regulierbar ist. Beispiele für solche Beeinflussung der Aktivität sind z.B. für Spleißosomen, Poly- merasen, Ribosomen etc. beschrieben.The activity of the gene product can also be reduced indirectly, for example by inhibiting the formation or activity of interaction partners, for example by influencing the metabolic chain in which the gene product is incorporated. For example, not only the enzyme in question, but also an enzyme or protein in the same metabolic chain can be inhibited, which leads to a blocking of the following, previous or another enzyme involved and thus the gene product described herein, for example by substrate or product inhibition , Such reductions by indirectly influencing the activity of an enzyme have been described in detail, for example for the interaction of the glycolysis proteins and metabolites, and are easily transferable to other metabolic pathways in which the gene products described herein play a role. In the same way, a gene product used according to the invention can be reduced or inhibited in its activity by reducing the activity of interaction partners, for example other proteins a protein complex is reduced or inhibited with the gene product described herein. This can lead to the fact that the entire complex is no longer activated or does not arise, or only partially arises or can no longer be regulated. Examples of such influencing of the activity are described, for example, for spliceosomes, polymerases, ribosomes etc.
Unter "Fragment" wird eine Teilsequenz einer hierin beschriebenen Sequenz verstanden, die weniger Nukleotide oder Aminosäuren u - fasst, als die hierin beschriebenen Sequenzen. Ein Fragment kann z.B. 1 %, 5 %, 10 %, 30 %, 50 %, 70 %, 90 % der ursprünglichen Sequenz umfassen. Vorzugsweise umfasst ein Fragment 100, mehr bevorzugt 50, noch mehr bevorzugt weniger as 20 Aminosäuren der • entsprechenden Nukleinsäuren.“Fragment” is understood to mean a partial sequence of a sequence described here which comprises fewer nucleotides or amino acids than the sequences described here. A fragment can e.g. 1%, 5%, 10%, 30%, 50%, 70%, 90% of the original sequence. A fragment preferably comprises 100, more preferably 50, even more preferably less than 20 amino acids of the corresponding nucleic acids.
Die Bedeutung der einzelnen Biosyntheseschritten ist dem Fachmann bekannt und kann z.B. in "Molecular Biology of the cell", Alberts, New York, 1998, "Biochemie" Stryer, 1988, New York, "Biochemieatlas", Michal, Heidelberg, 1999 oder in "Dictionary of Biotechnology" , Coombs, 1992, nachgelesen werden.The meaning of the individual biosynthesis steps is known to the person skilled in the art and can e.g. in "Molecular Biology of the cell", Alberts, New York, 1998, "Biochemie" Stryer, 1988, New York, "Biochemieatlas", Michal, Heidelberg, 1999 or in "Dictionary of Biotechnology", Coombs, 1992.
Eine Ausführungsform betrifft somit ein erfindungsgemäßes Verfahren, wobei die Expression oder die Aktivität der genannten Nukleinsäuren oder Aminosäuren dadurch reduziert oder blockiert wird, dass die Transkription, Translation, Prozessierung und/oder Modifikation mindestens einer der erfindungsgemäßen Nukleinsäuresequenz oder Aminosäuresequenz reduziert oder blockiert wird. Erfindungsgemäß können ein, zwei, drei oder mehr Sequenzen in ihrer Aktivität reduziert oder blockiert werden.One embodiment thus relates to a method according to the invention, the expression or the activity of the nucleic acids or amino acids mentioned being reduced or blocked by reducing or blocking the transcription, translation, processing and / or modification of at least one of the nucleic acid sequence or amino acid sequence according to the invention. According to the invention, one, two, three or more sequences can be reduced or blocked in their activity.
Das erfindungsgemäße Verfahren kann in einzelnen getrennten Verfahrensansätzen oder vorteilhaft in einem High-Throughput- Screening (HTS) durchgeführt werden und zur Identifizierung von Substanzen mit herbizider Wirkung oder von Antagoriisten verwendet werden. Im vorgenannten Verfahren können vorteilhaft auch Substanzen identifiziert werden, die mit den oben genannten Nukleinsäuren bzw. mit deren Genprodukten interagieren, diese Substanzen sind potentielle Herbizide, die über die klassische chemische Synthese in ihrer Wirkung weiter verbessert werden können.The method according to the invention can be carried out in individual separate method approaches or advantageously in a high-throughput screening (HTS) and can be used to identify substances with herbicidal activity or by antagoriists. In the above-mentioned method, substances can also advantageously be identified that interact with the above-mentioned nucleic acids or with their gene products. These substances are potential herbicides which can be further improved in their action via classic chemical synthesis.
Nach dem Verfahren identifizierte bzw. ausgewählte Substanzen können vorteilhaft auf eine Pflanze verbracht werden, um die herbizide Aktivität der Substanzen zu testen. Es werden solche Substanzen auswählt, die eine herbizide Aktivität zeigen. In einer weiteren vorteilhaften Ausführungsform des Verfahrens können die Substanzen auch neben dem vorgenannten in vivo-Test- verfahren auch in einem in vitro Test identifiziert werden. Ein derartiger in vitro Test mit den erfindungsgemäßen Nukleinsäuren bzw. deren Genprodukten hat den Vorteil, dass die Substanzen rasch und einfach auf ihre biologische Wirkung hin gescreent werden können. Derartige Test eignen sich auch vorteilhaft für 5 das sog. HTS.Substances identified or selected according to the method can advantageously be placed on a plant in order to test the herbicidal activity of the substances. Those substances are selected which show herbicidal activity. In a further advantageous embodiment of the method, the substances can also be identified in an in vitro test in addition to the aforementioned in vivo test method. On Such an in vitro test with the nucleic acids according to the invention or their gene products has the advantage that the substances can be quickly and easily screened for their biological effects. Such tests are also advantageous for 5 the so-called HTS.
Das Verfahren kann mit freien Nukleinsäure wie DNA oder RNA, freien Genprodukten oder vorteilhaft in einem Organismus durchgeführt werden, wobei als Organismus Bakterien, Hefen, Pilze oder 0 vorteilhaft Pflanzen verwendet werden. Als Organismen werden vorteilhaft konditionale oder natürliche Mutanten verwendet, die die Sequenzen SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, 5 SEQ ID~NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,The method can be carried out with free nucleic acid such as DNA or RNA, free gene products or advantageously in an organism, bacteria, yeasts, fungi or advantageously plants being used as the organism. Conditional or natural mutants which have the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ are advantageously used as organisms ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, 5 SEQ ID ~ NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,
SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
SEQ- ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: .56,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: .56,
■ SEQ ID NO: '58, SEQ ID. NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, ■ SEQ ID NO: '58, SEQ ID. NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID-NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, 0 SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 betreffen. Unter konditionalen Mutanten sind 5 Mutanten zu verstehen, die erst nach Induktion eine Reduktion der Expression, z.B. der Transkription oder der Translation der vorher genannten Nukleinsäuren oder der durch sie codierten Genprodukte, aufweisen. Ein Beispiel derartiger konditionaler Mutanten sind Mutanten, in denen die Nukleinsäuren hinter einem 0 temperatursensitiven Promotor liegen, der bei höheren Temperaturen nicht funktioneil ist, das heißt der die Transkription bei höhere : emperaturen beispielsweise oberhalb 37°C verhindert. Ebenfalls möglich ist eine Expressionsregulation durch ein Effektor-Molekül, z.B. bei Kontrolle der Expression durch einen regulierbaren Promotor, wie z.B. die Tet-Systeme.SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, 0 SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108. Conditional mutants are to be understood as meaning 5 mutants which have a reduction in expression, for example transcription or translation, of the aforementioned nucleic acids or of the gene products encoded by them only after induction. An example of such conditional mutants are mutants in which the nucleic acids are located behind a 0 temperature-sensitive promoter which is not functionally at higher temperatures, that is to say the transcription in higher: emperaturen for example above 37 ° C prevented. Expression regulation by an effector molecule is also possible, for example when the expression is controlled by a regulatable promoter, such as, for example, the Tet systems.
Eine weitere erfindungsgemäße Ausführungsform ist ein Verfahren zur Identifikation eines Antagonisten von Proteinen, die durch eine Nukleinsäuresequenz wie sie in dem erfindungsgemäßen Ver- 0 fahren eingesetzt wird, insbesondere ausgewählt aus der Gruppe:Another embodiment according to the invention is a method for identifying an antagonist of proteins, which is used by a nucleic acid sequence as used in the method according to the invention, in particular selected from the group:
a) einer Nukleinsäuresequenz mit der in SEQ ID NO: 1,a) a nucleic acid sequence with that in SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO 30, SEQ ID NO 32,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO 30, SEQ ID NO 32,
SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO 40,SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO 40,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,
SEQ ID NO: 76, SEQ ID NO: 78,' SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,SEQ ID NO: 76, SEQ ID NO: 78, ' SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,
SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oderSEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or
SEQ ID NO: 108 dargestellten .Sequenz,'SEQ ID NO: 108 shown sequence, '
b) einer Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,b) a nucleic acid sequence which, on the basis of the degenerate genetic code, results from the back-translation of the sequences in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57,.' SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57 ,. '
SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, 'SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, '
SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,
SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,
SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ' ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten AminosäureSequenzen ableiten lässt,SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ 'ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 derived amino acid sequences,
c) Nukleinsäuresequenz, die ein Derivat oder ein Fragment der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID-NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:. 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: '70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO:- 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Nukleinsäuresequenzen ist, und mindestens 60 % Homologie auf Nukleinsäureebene aufweist;c) Nucleic acid sequence, which is a derivative or a fragment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO : 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 , SEQ ID-NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO :. 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: '70, SEQ ID NO: 72 , SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: - 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 nucleic acid sequences shown, and has at least 60% homology at the nucleic acid level;
d) Nukleinsäuresequenz , die ' für Derivate oder Fragmente der Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4,d) nucleic acid sequence 'for derivatives or fragments of the polypeptides in the SEQ ID NO: 4: 2, SEQ ID NO
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO': 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103 , SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen codiert, die mindestens 50 % Homologie auf Aminosäureebene aufweisen;SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO ' : 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 encoded amino acid sequences which encode at least 50% homology Have amino acid level;
e) Nukleinsäuresequenz, die für ein Fragment oder ein Epitope eines Polypeptides codiert, das spezifisch an eineme) Nucleic acid sequence which codes for a fragment or an epitope of a polypeptide which is specific to a
Antikörper bindet,- wobei der Antikörper spezifisch an einAntibody binds - where the antibody specifically binds to a
Polypeptid bindet, das der in SEQ ID NO: 1, SEQ ID NO: 3,Polypeptide binds that the in SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ'ID NO: 13, SEQ ID NO: 15, SEQ ID NO: : 17, SEQ ID NO: 26., SEQ ID NO:- 28, SEQ ID NO: 30, SEQ ID NO: : 32, SEQ ID NO: 34.SEQ ' ID NO: 13, SEQ ID NO: 15, SEQ ID NO:: 17, SEQ ID NO: 26., SEQ ID NO: - 28, SEQ ID NO: 30, SEQ ID NO:: 32, SEQ ID NO : 34.
SEQ ID NO: 36, SEQ ID NO: 3 388,, SEQ ID NO: : 40, SEQ ID NO: 44SEQ ID NO: 36, SEQ ID NO: 3 388 ,, SEQ ID NO:: 40, SEQ ID NO: 44
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: : 50, SEQ ID NO: 52,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:: 50, SEQ ID NO: 52,
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: : 58, SEQ ID NO: 60,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: : 6 666,, SSEEQQ IIDD N NOO:: 6 688,, • SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: : 7 744,, SSEEQQ IIDD N NOO:: 7 766,,SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO:: 6 666 ,, SSEEQQ IIDD N NOO :: 6 688 ,, • SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO:: 7 744 ,, SSEEQQ IIDD N NOO :: 7 766 ,,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: : 8 822,, SSEEQQ IIDD N NOO:: 8 844.,SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:: 8 822 ,, SSEEQQ IIDD N NOO :: 8 844.,
SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: : 9 900,, SSEEQQ IIDD N NOO:: 9 922,,SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO:: 9 900 ,, SSEEQQ IIDD N NOO :: 9 922 ,,
SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: : 9 988,, SSEEQQ IIDD N NOO:: 1 10010, - SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID D NNOO:: 108 dargestellten Sequenz codiert wird;SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO:: 9 988 ,, SSEEQQ IIDD N NOO :: 1 10010 SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID D NNOO :: 108 the sequence shown is encoded;
f) Nukleinsäuresequenz,' die für ein Fragment einer in aa) dargestellten Nukleinsäure codiert wird und das eine-- m6A-Methyl- transferase-Aktivität, eine DNA-bindende-Aktivität oder "DNA- repair"-Aktivität, z.B. wie bei RAD 54, eine Thioredoxin-f) Nucleic acid sequence ' which is encoded for a fragment of a nucleic acid shown in aa) and which has a m6A methyltransferase activity, a DNA binding activity or "DNA repair" activity, for example as in RAD 54 , a thioredoxin
Aktivität, eine VAV2-Aktivität, eine Fructokinase-Aktivität , eine Zinkfingerprotein-Aktivität, eine LYTB-Aktivität, eine Crepopin-Aktivität, eine Leucin-Protein-Aktivität, eine DNAJ- Aktivität, ein CRSl-Aktivität, eine Alanyl-tRNA-Synthetase- Aktivität, eine OEP86-Aktivität, eine FMRF-Amid-Propeptid- Isolog-Aktivität, eine 26S Proteosom subunit S5B-Aktivität, eine Geranylgeranylpyrophosphatsynthase-Aktivität, eine Crepopin-Aktivität, eine Leucin-Protein-Aktivität, eine DNAJ- Aktivität, ein CRSl-Aktivität, eine Alanyl-tRNA-Synthetase- Aktivität, eine OEP86-Aktivität, eine FMRF-Amid-Propeptid- Isolog-Aktivität, eine 26S Proteosom subunit S5B-Aktivität, eine Geranylgeranylpyrophosphatsynthase-Aktivität, eine Cecropin-Familiensignatur aufweißt, ftsH Cloroplast-Protease- Aktivität hat, eine AIMl-Aktivität, eine UDP-glucuronyl- transferase-Aktivität, eine FPFl-Aktivität, eine SHI-ähnliche Zinkfingerprotein-Aktivität hat, eine Crpl-Aktivität, eine CRSl-Aktivität, eine translation releasing factor RF-1-Aktivität , eine Farnesyltransferase Untereinheit A-Aktivität, eine ATP-abhängige Kupf r ransporter RAN1-Aktivität , eine Syntaxin oder Syntaxin-ähnliche Proteinaktivität, eine Inositol-Polyphosphat-5 ' -Phosphatase- Aktivität, eine UDP-N-Acetylmuramoylalanyl-D-glutamat-Activity, VAV2 activity, fructokinase activity, zinc finger protein activity, LYTB activity, crepopin activity, leucine protein activity, DNAJ activity, CRSl activity, alanyl tRNA synthetase Activity, an OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, a crepopin activity, a leucine protein activity, a DNAJ activity CRSl activity, an alanyl tRNA synthetase activity, an OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, one Cecropin family signature, ftsH has chloroplast protease activity, AIMl activity, UDP-glucuronyl transferase activity, FPFl activity, SHI-like zinc finger protein activity, Crpl activity, CRSl activity , a translation releasing factor RF-1 activity, a farnesyltransferase subunit A activity, an ATP-dependent copper transporter RAN1 activity, a syntaxin or syntaxin-like protein activity, an inositol polyphosphate 5 'phosphatase activity, a UDP-N-D-glutamate Acetylmuramoylalanyl-
2, 6-Diaminopimelat-ligaseaktivität (murE) , eine ß-Glucosi- dase-Aktivität, eine Hydroxymethylglutaryl-CoA-Reductase, eine GDSL-Motif-Lipase/Hydroxylase-ähnliche Proteinaktivität, eine Zellulosesynthase ähnliche Proteinaktivität, eine tRNA Gluta insynthetase, eine Exonuklease-ähnliche Proteinaktivität, eine sec-unabhängige Translocase-Protein TATC- Aktivität oder eine Selenium-Bindungsprotein ähnliche Proteinaktivität hat; und/oder2, 6-diaminopimelate ligase activity (murE), a ß-glucoside activity, a hydroxymethylglutaryl-CoA reductase, a GDSL motif lipase / hydroxylase-like protein activity, a cellulose synthase-like protein activity, a tRNA gluta insynthetase, a Has exonuclease-like protein activity, a sec-independent Translocase protein TATC activity or a selenium binding protein-like protein activity; and or
g) Nukleinsäuresequenz, die für Derivate der Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID'NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO:, 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,' SEQ ID NO: 92, SEQ ID NO:. 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Aminosäuresequenzen codiert, die mindestens 20 % Homologie auf Aminosäureebene aufweist und eine äquivalente biologische Aktivität besitzt; oderg) Nucleic acid sequence which is suitable for derivatives of the polypeptides with those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID ' NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 , SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, ' SEQ ID NO: 92, SEQ ID NO :. 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 encoded amino acid sequences which has at least 20% homology at the amino acid level and has an equivalent biological activity; or
codiert werden indem folgende Verfahrensschritte durchlaufen werdenare encoded by going through the following process steps
i) Inkontaktbringen von Zellen, die das Protein exprimieren, oder des Proteins mit einem Kandidatenstoff;i) contacting cells that express the protein or the protein with a candidate substance;
ii) Testen der biologischen Aktivität des Protein; iii) Vergleichen der biologischen Aktivität des Proteins mit einer Standardaktivität in Abwesenheit des Kandidatenstoffs, wobei eine ■ Verringerung der biologischen Aktivität des Proteins anzeigt, dass der Kandidatenstoff ein Antagonist ist.ii) testing the biological activity of the protein; iii) comparing the biological activity of the protein with a standard activity in the absence of the candidate substance, a decrease in the biological activity of the protein indicating that the candidate substance is an antagonist.
Unter ii) wird das Testen einer der oben beschriebenen biologischen Aktivitäten beschrieben, z.B. eine Enzymaktivität, wie sie in den Beispielen angegeben ist oder eine Bindung, vorzugsweise eine starke Bindung zwischen Protein- und Kandidatenstoff.Ii) describes the testing of one of the biological activities described above, e.g. an enzyme activity as given in the examples or a bond, preferably a strong bond between protein and candidate substance.
In einer vorteilhaften Ausführungsform des oben beschriebenen Verfahrens wird/werden der/die -unter Buchstabe, iii) identifizierte (n) Antagonist (en) auf eine Pflanze verbracht, um seine/ ihre herbizide Aktivität zu testen und der/die Antagonist (en) ausgewählt, die eine herbizide Aktivität zeigen.In an advantageous embodiment of the method described above, the antagonist (s) identified under letter iii) is / are placed on a plant in order to test its herbicidal activity and the antagonist (s) are selected that show herbicidal activity.
Das erfindungsgemäße Verfahren kann in einzelnen getrennten Verfahrensansätzen in vivo oder in vitro und/oder vorteilhaft gemeinsam oder besonders vorteilhaft in einem High-Throughput- Screening durchgeführt werden und zur Identifizierung von Substanzen mit herbizider Wirkung oder von Antagonisten verwendet werde .The method according to the invention can be carried out in separate separate method approaches in vivo or in vitro and / or advantageously together or particularly advantageously in a high-throughput screening and can be used for the identification of substances with herbicidal activity or of antagonists.
Die im erfindungsgemäßen Verfahren identifizierten bzw. selektierten Nukleinsäuresequenzen - sind für das Wachstum und die Entwicklung von höheren Pflanzen essentiell. Die Unterdrückung der Bildung der Genprodukte, d.h. der Expression, z.B. durch die spezifische Beeinflussung von z.B. Translation, Transkription oder Prozessierung und/oder der Unterdrückung der von den codierten Genprodukten ausgeübten Funktion bzw. biologischen Aktivität in intakten Pflanzen durch Substanzen vorteilhaft niedermolekularen Substanzen mit einem Molekulargewicht von kleiner 1000 Dalton, vorteilhaft kleiner 900 Dalton, bevorzugt kleiner 800, besonders bevorzugt kleiner 700,' ganz besonders bevorzugt kleiner 600 Dalton, vorteilhaft mit einem Ki-Wert kleiner 1 mM und vorteilhaft mit weniger als drei Hydroxylgruppen an einem Kohlenstoffatom-enthaltenden Ring oder proteinogenen Substanzen oder einer Sense- oder Antisense-RNA oder einem Antikörper oder Antikörperfragment führt daher zu vorteilhafterweise massiven Veränderungen des Wachstums und der Entwicklung der betroffenen Pflanzen. Die im erfindungsgemäßen Verfahren identifizierten Substanzen sind deshalb in der Landwirtschaft 'als Herbizide geeignet.The nucleic acid sequences identified or selected in the method according to the invention are essential for the growth and development of higher plants. The suppression of the formation of the gene products, ie the expression, for example by specifically influencing, for example, translation, transcription or processing and / or the suppression of the function or biological activity carried out by the coded gene products in intact plants by substances, advantageously low-molecular substances with a molecular weight of less than 1000 daltons, advantageously less than 900 daltons, preferably less than 800, more preferably less than 700, 'most preferably less than 600 Dalton, preferably with a Ki value of less than 1 mM and advantageously with less than three hydroxy groups on one carbon atom-containing ring, or proteinogenic substances or a sense or antisense RNA or an antibody or antibody fragment therefore advantageously leads to massive changes in the growth and development of the plants concerned. The substances identified in the process according to the invention are therefore suitable as herbicides in agriculture.
Die in den erfindungsgemäßen Verfahren verwendeten Nukleinsäuren SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,- SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID'NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 sind essentiell für Organismen, bevorzugt für Pflanzen. Einige Genprodukte der genannten Sequenzen sind z.B. den Polypeptiden der Sequenzen SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID~NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO:' '47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ'ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO:.97, SEQ ID NO: 99, SEQ IDNO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oderThe nucleic acids SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74 , SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID ' NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 are essential for Organisms, preferably for plants. Some gene products of the sequences mentioned are, for example, the polypeptides of the sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID ~ NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: '' 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ' ID NO : 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: .97, SEQ ID NO: 99, SEQ IDNO: 101, SEQ ID NO: 103, SEQ ID NO : 105, SEQ ID NO: 107 or
SEQ ID NO: 109 zu entnehmen. Die1 genomische DNA der Sequenzen wird jeweils 'in SEQ ID NO: 19 (genomische DNA von Linie P95) , SEQ ID NO: 20 (genomische DNA von Linie P9) , SEQ ID NO: 21. (genomische DNA von Linie P38) , SEQ ID NO: 22 (genomische DNA von Linie P44) , SEQ ID NO: 23 (genomische DNA von Linie P77), SEQ ID NO: 24 (genomische DNA von Linie P102) , SEQ ID No: 43 (genomische DNA von .Linie A 301034) und SEQ ID NO: 42 (genomische DNA von Linie A 300857) wiedergegeben. Für Linie A 300841 und A 300367 ist die genomische Sequenz identisch zu' SEQJ ID No: 32 und SEQ ID NO: 40, da kein Intron gefunden wurde.SEQ ID NO: 109. The 1 genomic DNA of the sequences is in each case in SEQ ID NO: 19 (genomic DNA from line P95), SEQ ID NO: 20 (genomic DNA from line P9), SEQ ID NO: 21. (genomic DNA from line P38), SEQ ID NO: 22 (genomic DNA from line P44), SEQ ID NO: 23 (genomic DNA from line P77), SEQ ID NO: 24 (genomic DNA from line P102), SEQ ID No: 43 (genomic DNA from line A 301034) and SEQ ID NO: 42 (genomic DNA from line A 300857). For lines A 300841 and A 300367 the genomic sequence is identical to 'SEQJ ID No: 32 and SEQ ID NO: 40 since no intron was found.
SEQ ID NO: 1 (= Klon P95) codiert für ein Protein (PID: g2460037) , das Ähnlichkeiten zu verschiedenen m6A-Methyl- transferasen unter anderem aus Maus (NP_062695) und Mensch (AAB71850) aufweist.SEQ ID NO: 1 (= clone P95) codes for a protein (PID: g2460037) which has similarities to various m6A methyl transferases from, inter alia, mouse (NP_062695) and human (AAB71850).
SEQ ID NO: 3 (= Klon P9) codiert für ein "DNA repair protein RAD54-like" Protein-Homolog, das auf Chromosom 3 (EMBL/AP000419) von Arabidopsis liegt. Es hat Ähnlichkeiten im Blast-Vergleich zu einer Reihe von DNA-bindenden Proteinen wie Helicasen, Trans- activatoren etc. (Emery et al . , Gene 104 (1)', 103-106, 1991). Eine besonders hohe Ähnlichkeit liegt zu dem DNA-Repair-Protein RHP 54 aus Schizosaccharomyces po be vor (Muris et al . , J. Cell. Sei. 109 (Pt 1), 73-81, 1996).SEQ ID NO: 3 (= clone P9) codes for a "DNA repair protein RAD54-like" protein homolog which is located on chromosome 3 (EMBL / AP000419) from Arabidopsis. It has similarities in blast comparison to a number of DNA-binding proteins such as helicases, trans-activators etc. (Emery et al., Gene 104 (1) ' , 103-106, 1991). There is a particularly high similarity to the DNA repair protein RHP 54 from Schizosaccharomyces po be (Muris et al., J. Cell. Sci. 109 (Pt 1), 73-81, 1996).
SEQ ID NO: 5 (= Klon P38) codiert für einen ORF auf Chromosom 3 (EMBLNEW/AC023912) , der möglicherweise für ein Thioredoxin codiert. Im Blast-Vergleic zeigen sich Ähnlichkeiten zu Thio- redoxinen verschiedener Herkunft (Fräser et al., Nature 390 (6660), 580-586, 1997; Reith et al . , Plant Mol. Biol. Rep. , 13, 333-335, 1995).SEQ ID NO: 5 (= clone P38) codes for an ORF on chromosome 3 (EMBLNEW / AC023912), which may code for a thioredoxin. The blast comparison shows similarities to thio-redoxins of various origins (Fraser et al., Nature 390 (6660), 580-586, 1997; Reith et al., Plant Mol. Biol. Rep., 13, 333-335, 1995).
Der Klon P44 (= SEQ"1 ID NO: 7) codiert für ein Protein des TAC Klons K15C23 (EMBLALERT/AB024024) ohne eindeutige Homologie in der Blast-Suche. Sequenzvergleiche auf Aminosäureebene zeigen schwache Homologien zu VAV2-Proteinen (kleines ras-ähnliches GTP- bindende Protein, Swissprot Q60992, Schuebel et al . , Oncogene 13 (2), 363-371, 1996; Henske et al . , Ann. Hum. Genet. 59 (Pt 1), 25-37, 1995) . - ' . The clone P44 (= SEQ " 1 ID NO: 7) codes for a protein of the TAC clone K15C23 (EMBLALERT / AB024024) without clear homology in the blast search. Sequence comparisons at the amino acid level show weak homologies to VAV2 proteins (small ras-like GTP-binding protein, Swissprot Q60992, Schuebel et al., Oncogene 13 (2), 363-371, 1996; Henske et al., Ann. Hum. Genet. 59 (Pt 1), 25-37, 1995). ' .
SEQ ID NO: 9 (= Klon P77) codiert wahrscheinlich für eine Fructo- kinase des BAC Klons F24B22 des Chromosoms 3 (EMBL/ATF24B22) . Blast-Vergleiche bestätigen diese Homologie (swissprot P37829, Smith et al., Plant Physiol., 102 (3), 1043, 1993; Blatch et al., Gene, 95 (1), 17-23, 1990).SEQ ID NO: 9 (= clone P77) probably codes for a fructokinase of BAC clone F24B22 of chromosome 3 (EMBL / ATF24B22). Blast comparisons confirm this homology (swissprot P37829, Smith et al., Plant Physiol., 102 (3), 1043, 1993; Blatch et al., Gene, 95 (1), 17-23, 1990).
Von SEQ ID NO: 11 wird ein Protein (Accession number BAB09578.1) codiert, das schwache Homologien zu- verschiedenen Zinkfinger- Proteinen hat . Es zeigt schwache Homologien zu Zinkfingerproteinen und ■DNA-bindendenen Proteinen, wie bspw. dem Zink- fingerpr.otein 265 aus Ratte (Karginova, E.A-. , Am. J. Physiol. 273 (5 Pt 2), F731-F738 (1997)) für welches eine Funktion für die Regulation der Transkription und/oder des Splicings angenommen wird.. In vitro Transkriptions- oder Splicing-Assays sind vielfach beschrieben und dem Fachmann bekannt .SEQ ID NO: 11 encodes a protein (accession number BAB09578.1) which has weak homologies with various zinc finger proteins. It shows weak homology to zinc finger proteins and ■ DNA binding proteins which, such as. Fingerpr the zinc. otein 265 from Rat (Karginova, EA-., Am. J. Physiol. 273 (5 Pt 2), F731-F738 (1997)) for which a function for the regulation of transcription and / or splicing is assumed. , In vitro transcription or splicing assays have been widely described and are known to the person skilled in the art.
Das durch SEQ ID NO: 13 codierte Protein (AB36712.1) hat Ähnlichkeiten zu LYTB-Proteinen speziell zu LYTB SYNY3 aus Synechocystis (Q55643) . Das Gen bzw. das durch das Gen codierte Protein liegt auf • Chromosom V (Accession number AB006706) . Innerhalb der Sequenz von SEQ ID O: 13 sind einige ESTs (gb:Z34640, Z30476, AA605545, Z34228, H76883, Z26425) bekannt.The protein encoded by SEQ ID NO: 13 (AB36712.1) has similarities to LYTB proteins especially to LYTB SYNY3 from Synechocystis (Q55643). The gene or the protein encoded by the gene is located on • Chromosome V (Accession number AB006706). Several ESTs (gb: Z34640, Z30476, AA605545, Z34228, H76883, Z26425) are known within the sequence of SEQ ID O: 13.
SEQ ID NO: 15 codiert für ein hypothetisches Protein (CAB81447,1) , das die Crepropin-Familien-Signatur enthält und schwache Homologien zu yc-Protoonkogenen aufweist. Das von SEQ ID NO: 17 (siehe auch ESTAV528166) codierte Protein (AAF23295) , hat eine gewisse Homologie zu einem Leucin-reichen Protein aus dem Mensch (Accession number P42704, Wang et al . , In vitro Cell Dev. Biol. Amin. 1994, 30A(2) : 111-114). Es zeigt eine 5 substanzielle Homologie zu einem leucinreichen- Protein (LRP130) aus Mensch dessen Funktion unbekannt ist, aber auf Aminosäure- ebene schwache Homologien zu den Consensusequenzen der ATP- Bindungssteilen in ATP-abhängigen Kinasen und der Proteinkinase C-Phosphorylierungsstelle des epidermal growth factor Rezeptors .SEQ ID NO: 15 codes for a hypothetical protein (CAB81447,1) which contains the crepropin family signature and has weak homologies to yc proto-oncogenes. The protein (AAF23295) encoded by SEQ ID NO: 17 (see also ESTAV528166) has a certain homology to a leucine-rich protein from humans (Accession number P42704, Wang et al., In vitro Cell Dev. Biol. Amin. 1994, 30A (2): 111-114). It shows a 5 substantial homology to a leucine-rich protein (LRP130) from humans whose function is unknown, but weak homologies at the amino acid level to the consensus sequences of the ATP binding parts in ATP-dependent kinases and the protein kinase C phosphorylation site of the epidermal growth factor Receptor.
1010
SEQ ID No: 26 codiert für ein Protein, das für einen Bereich von 40 Aminosäuren Homologie zu verschiedenen DNAJ Chaperon-Proteinen (Heat Shock Protein 40) hat, z.B. zu DNAJ Protein (Q9UXR9) aus Methanosarcina thermophila (Hoffmann-Bang, Gene 238 (2), 387-395,SEQ ID No: 26 encodes a protein that has homology to various DNAJ chaperone proteins (Heat Shock Protein 40) for a range of 40 amino acids, e.g. for DNAJ protein (Q9UXR9) from Methanosarcina thermophila (Hoffmann-Bang, Gene 238 (2), 387-395,
15 1999) T15 1999) T
SEQ ID No: 28 codiert für ein hypothetisches Protein (CAC01859.1) . Die abgeleitete Proteinsequenz zeigt deutliche Homologien zum CRS1 Genprodukt aus Mais (AAG00595) , welches für 20 das Splicen des Gruppe II Introns des Chloroplastengens atpF benötigt wird.SEQ ID No: 28 encodes a hypothetical protein (CAC01859.1). The derived protein sequence shows clear homologies to the CRS1 gene product from maize (AAG00595), which is required for 20 splicing of the group II intron of the chloroplast gene atpF.
SEQ ID No: 30 codiert vermutlich für eine Alanyl-tRNA-Syntethase (BAB10601.1) .SEQ ID No: 30 presumably codes for an alanyl tRNA synthesis (BAB10601.1).
2525
SEQ ID No: 32 codiert für ein Protein, das eine starke Homologie zum "chloroplast outer envelope 86 protein" OEP86 aus Erbse P. sativum, GenBank Accession number Z31581 und besitzt ein ATP/ GTP-Bindungsstellenmotiv (P-loop) . Für diesen ORF (CAB80744.1)SEQ ID No: 32 codes for a protein which has strong homology to the "chloroplast outer envelope 86 protein" OEP86 from pea P. sativum, GenBank Accession number Z31581 and has an ATP / GTP binding site motif (P-loop). For this ORF (CAB80744.1)
30 sind bereits ESTs (EST gb:AI998804.1, R90258, AA651438) bekannt.30 ESTs (EST gb: AI998804.1, R90258, AA651438) are already known.
SEQ ID No: 34 codiert für ein Protein, dessen abgeleitete Aminosäuresequenz (AAF25967.1) deutliche Ähnlichkeit zu einem "FMRF- Amid Propeptid-Isolog (gij 1871179) aus Arabidopsis zeigt. 5SEQ ID No: 34 codes for a protein whose derived amino acid sequence (AAF25967.1) shows clear similarity to an "FMRF amide propeptide isolog (gij 1871179) from Arabidopsis. 5
SEQ ID No: 36 codiert für ein unbekanntes Protein (BAB02572.1) ■ mit schwacher Homologie zu Proteosomenprotein 26S PROTEASOM SUBUNIT S5B, (Deveraux, 1995, J. Biol. Che . 270 (40), 23726.SEQ ID No: 36 encoding an unknown protein (BAB02572.1) ■ with weak homology to protein proteosome 26S PROTEASOME SUBUNIT S5B, (Deveraux, 1995, J. Biol Che 270 (40), the 23,726th..
0 SEQ ID NO: 38 codiert für ein unbekanntes Protein.0 SEQ ID NO: 38 encodes an unknown protein.
SEQ ID NO: 40 codiert für Protein, das Homologie zu einer Geranylgeranylpyrophosphatsynthase (Bartley, Plant Physiol. 104, 1469-1470, 1994) hat. 5 SEQ ID NO: 44 codiert für ein hypothetisches Protein des ORF AT4g28590, welches eine "Cecropin" Familiensignature (AA237-245) aufweist .SEQ ID NO: 40 encodes protein that has homology to a geranylgeranyl pyrophosphate synthase (Bartley, Plant Physiol. 104, 1469-1470, 1994). 5 SEQ ID NO: 44 codes for a hypothetical protein of ORF AT4g28590, which has a "Cecropin" family signature (AA237-245).
SEQ ID NO: 46 codiert für eine putative ftsH Chloroplast-Protease des ORFs At2g30950.SEQ ID NO: 46 encodes a putative ftsH chloroplast protease of ORF At2g30950.
SEQ ID NO: 48 hat Ähnlichkeit mit dem "AIMl" Protein aus Arabidopsis (CAB43915.1) . Zu diesem ORF (F19B15.40) sind bereits mehrere ESTs, GB:Z31666, gb:Z33957, Z31666, beschrieben. Bei diesem Protein handelt es sich um ein peroxisomales tetrafunktionales Enzym des Fettsäurestof wechsels.SEQ ID NO: 48 is similar to the "AIMl" protein from Arabidopsis (CAB43915.1). Several ESTs have already been described for this ORF (F19B15.40), GB: Z31666, gb: Z33957, Z31666. This protein is a peroxisomal tetrafunctional enzyme of fatty acid metabolism.
SEQ ID NO: 50 codiert für ein UDP-glucuronyltransferase-ähnliches Protein- des ORFs K21H1.19. Durch den Einbau der T-DNA in diese Position wird sehr wahrscheinlich die Transkription verändert oder unterbunden und damit die Funktion des Gens zerstört.SEQ ID NO: 50 encodes a UDP-glucuronyltransferase-like protein of ORF K21H1.19. By incorporating the T-DNA in this position, the transcription is very likely to be changed or prevented and the function of the gene is thereby destroyed.
SEQ ID NO: 52 codiert ein Protein unbekannter Funktion des ORF At2gl5820.SEQ ID NO: 52 encodes a protein of unknown function of the ORF At2gl5820.
SEQ ID NO: 54 codiert ein Protein des ORFs ATF12B17_20, ein FPF1-ähnliches (flowering promoting factorl) Protein.SEQ ID NO: 54 encodes a protein of the ORF ATF12B17_20, an FPF1-like (flowering promoting factor1) protein.
SEQ ID NO: 56 codiert ein Protein des ORFs ATF12B17_10 mit Ähnlichkeit zu KIAA1038 Protein aus Homo sapiens.SEQ ID NO: 56 encodes a protein of the ORF ATF12B17_10 with similarity to KIAA1038 protein from Homo sapiens.
Die SEQ ID NO: 58 codiert ein Protein des ORF F24P17.10 mit unbe-' kannter Funktion. Im blastp-Vergleich mit Standardeinstellungen zeigen sich deutliche Homoligien zu einem nodulin / gluta ate- - ammonia ligase - like protein.The SEQ ID NO: 58 encodes a protein of ORF F24P17.10 with unconstrained 'known per function. In the blastp comparison with standard settings, clear homoligies to a nodulin / gluta ate- - ammonia ligase - like protein are shown.
SEQ ID NO: 60 codiert ein SHI-ähnliches Zinkfingerprotein (short internodes) des ORFs K1L20.13.SEQ ID NO: 60 encodes a SHI-like zinc finger protein (short internodes) of ORF K1L20.13.
SEQ ID NO: 62 codiert ein Protein mit Ähnlichkeit zu crpl aus Zea mays, PIR:T01685 (ORF F4P12_400) . Dieser ORF beinhaltet zudem die ESTs gb:Al999771.1, T45254, AA713158*.SEQ ID NO: 62 encodes a protein similar to crpl from Zea mays, PIR: T01685 (ORF F4P12_400). This ORF also includes the ESTs gb: Al999771.1, T45254, AA713158 *.
SEQ ID NO: 64 codiert ein putatives Protein mit Ähnlichkeiten zu hypothetischen Proteinen aus Arabidopsis. In der blastp-Analyse zeigt sich zudem eine deutliche Homologie zu CRS1 aus Zea mays Accession AAG00595, wobei es sich um einen Gruppe II Intron Splicingfaktor handelt (Till,B et al., RNA 7 (9), 1227-1238 (2001)) ORF (T21Hl9_100) . SEQ ID NO: 66 codiert für ein Protein unbekannter Funktion des Gens At5g24315.SEQ ID NO: 64 encodes a putative protein with similarities to hypothetical proteins from Arabidopsis. The blastp analysis also shows a clear homology to CRS1 from Zea mays Accession AAG00595, which is a Group II intron splicing factor (Till, B et al., RNA 7 (9), 1227-1238 (2001)) ORF (T21H19_100). SEQ ID NO: 66 encodes a protein of unknown function of the At5g24315 gene.
SEQ ID NO: 68 codiert ein Protein ORF "(T20O10_10) mit hoher Ähn- lichkeit zu translation releasing factor RF-1 aus Synechocystis (PIR:S76914) zeigt. Dazu enthält die abgleitete Aminosäureseque z ein prokaryotische Type Klasse I Pepetidkettenablösungsfaktor- Motiv, AA280-296.SEQ ID NO: 68 encodes a protein ORF " (T20O10_10) with high similarity to translation releasing factor RF-1 from Synechocystis (PIR: S76914). In addition, the deduced amino acid sequence z contains a prokaryotic type I pepeptide chain detachment factor motif, AA280 -296.
SEQ ID NO:' 70 codiert ein Protein mit hoher Ähnlichkeit zu einem Allergen ( "minor allergen" ) aus Alternaria alternata (PIR2:S43111) . Zu dem ORF (C7A10.610) wurden zudem bereits die ESTS gb:R64949, AA651052 gefunden.SEQ ID NO: '70 encodes a protein with high similarity to an allergen ( "minor allergen") from Alternaria alternata (PIR2: S43111). ESTS gb: R64949, AA651052 have also already been found for ORF (C7A10.610).
SEQ ID -NO: 72 codiert für ein Protein mit Ähnlichkeit zur' Alpha-Untereinheit eines putativen Signalsequencereceptors (ORF At2gll60) .SEQ ID -NO: 72 encodes a protein similar to the ' alpha subunit of a putative signal sequence receptor (ORF At2gll60).
SEQ ID NO: 74 codiert für ein Protein unbekannter Funktion des ORF AT4g01220, der die ESTs gb:AA597894, . AA597304 beinhaltet.SEQ ID NO: 74 encodes a protein of unknown function of ORF AT4g01220, which ESTs gb: AA597894,. AA597304 includes.
SEQ ID NO: 76 zeigt in der Blastp-Analyse mit Standardeinstellungen eine Ähnlichkeit zu Oxidoreduktasen. Die Insertion der T-DNA an dieser Position unterbricht den ORF F13011.11. Die zelluläre Funktion der codierten Proteine ist nicht bekannt .SEQ ID NO: 76 shows a similarity to oxidoreductases in the blastp analysis with standard settings. The insertion of the T-DNA at this position interrupts the ORF F13011.11. The cellular function of the encoded proteins is unknown.
SEQ ID NO: 78 codiert für das Protein des ORF F25L23_240* eine Farnesyltransferase Untereinheit A.SEQ ID NO: 78 codes for the protein of ORF F25L23_240 * a farnesyltransferase subunit A.
SEQ ID NO: 80 codiert für ein ATP-abhängigen Kupfertransporter RANl-ähnliches Protein (ORF T19K24.18).SEQ ID NO: 80 codes for an ATP-dependent copper transporter RANl-like protein (ORF T19K24.18).
SEQ ID NO: 82 codiert das Protein des ORF F19B15.50- und hat Ähnlichkeiten zu glyzinreichen Proteinen. Der ORF beinhaltet die ESTs gb:Z29181, T42831, Z34138, Z33797, Z30844.SEQ ID NO: 82 encodes the protein of ORF F19B15.50- and has similarities to proteins rich in glycine. The ORF includes the ESTs gb: Z29181, T42831, Z34138, Z33797, Z30844.
SEQ ID NO: 84 codiert für ein Protein- unbekannter Funktion (ORF K21H1.18) .SEQ ID NO: 84 encodes a protein unknown function (ORF K21H1.18).
SEQ ID NO: 86 codiert' ein Protein, welches im blastp-Vergleich unter Standardeinstellung hohe Homologien zu verschiedenen Syntaxinen und syntaxinähnlichen Proteinen auch aus Pflanzen zeigt. Für den ORF F309.4 sind den bereits mehrere ESTs identifiziert (gb|F15498, gb|H37515, gb|T41906, gb|T22448, gb|W43356, gb|T20739) . SEQ ID NO: 88 codiert für das Protein des ORF AT2g31830-, eine putative Inositol-polyphosphate-5 ' -Phosphatase .SEQ ID NO: 86 'encodes a protein that also shows from plants in the blastp comparison under default high homologies to various syntaxins and syntaxinähnlichen proteins. Several ESTs have already been identified for ORF F309.4 (gb | F15498, gb | H37515, gb | T41906, gb | T22448, gb | W43356, gb | T20739). SEQ ID NO: 88 encodes the protein of ORF AT2g31830-, a putative inositol polyphosphate 5 'phosphatase.
SEQ ID NO: 90 codiert für das Protein des ORF F24D7.13, der 5 für eine putative UDP-N-Acetylmuramoylalanyl-D-glutamate-2, 6-Diaminopimelate-ligase (murE) codiert.SEQ ID NO: 90 encodes the protein of ORF F24D7.13, which encodes 5 for a putative UDP-N-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase (murE).
. SEQ ID NO: 92 codiert, das Protein des ORF MRC8.5., der für eine Beta-Glucosidase codiert. 10, SEQ ID NO: 92 encodes the protein of ORF MRC8.5 . which codes for a beta-glucosidase. 10
SEQ ID NO: 94 codiert für das Protein des ORF F15M4.1, der für eine Hydroxymethylglutaryl-CoA-Reductase codiert .SEQ ID NO: 94 codes for the protein of ORF F15M4.1, which codes for a hydroxymethylglutaryl-CoA reductase.
SEQ ID NO: 96 codiert für das Protein des ORFs MRN17.4. Dieser 15 codiert—für ein GDSL-Motif Lipase/Hydrolase-like Protein.SEQ ID NO: 96 encodes the protein of the ORF MRN17.4. This 15 codes for a GDSL motif lipase / hydrolase-like protein.
SEQ ID NO: 98 codiert für das Protein des ORFs dl3705c, der für eine Zellulose-Synthase-ähnliches Protein codiert.SEQ ID NO: 98 encodes the protein of ORF dl3705c, which encodes a cellulose synthase-like protein.
20. SEQ ID NO: 100 codiert für das Protein des ORFs K5J14.11, der für ein dem crpl Protein aus Mais ähnliches Protein codiert.20. SEQ ID NO: 100 codes for the protein of ORF K5J14.11, which codes for a protein similar to the crpl protein from maize.
SEQ ID NO: 102 codiert für das Protein des ORF F4F7.26. Dieser ORF codiert für eine• putative t-RNA Glutaminsynthetase und weist 25 insbesondere Homologie zur tRNA Glutaminsynthetase GI:2995454 aus Lupinus luteus auf.SEQ ID NO: 102 encodes the protein of ORF F4F7.26. This ORF codes for a putative t-RNA glutamine synthetase and has, in particular, homology to tRNA glutamine synthetase GI: 2995454 from Lupinus luteus.
SEQ ID NO: 104 codiert für das Protein des ORF MFB13.17, der ein . Exonuklease-ähnliches Protein codiert. 30SEQ ID NO: 104 encodes the protein of ORF MFB13.17, which is a . Exonuclease-like protein encoded. 30
SEQ ID NO: 106 codiert für das Protein des ORF At2g01110. Dieser codiert für ein putatives "sec-unabhängiges" Translokase-Protein TATC (putative sec-independent protein translocase protein TATC) .SEQ ID NO: 106 encodes the protein of ORF At2g01110. This codes for a putative "sec-independent" translocase protein TATC (putative sec-independent protein translocase protein TATC).
5 SEQ ID NO: 108 codiert für das Protein des ORF F28J12.180, welcher für ein putatives Protein codiert. In blastp-Analysen mit Standardeinstellung- zeigt die abgeleitete Aminosäuresequenz, neben deutliche Homologien zu verschiedenen hypothetischen und pu- tativen Proteinen auch starke Ähnlichkeit zu Selenium-Bindung- 0 sprotein ähnlichen Proteinen.5 SEQ ID NO: 108 codes for the protein of ORF F28J12.180, which codes for a putative protein. In blastp analyzes with standard settings - the derived amino acid sequence shows, in addition to clear homologies to various hypothetical and putative proteins, also strong similarity to selenium binding - 0 protein-like proteins.
Die genannten Sequenzen wurden alle in Arabidopsis identifiziert.The sequences mentioned were all identified in Arabidopsis.
Die Unterdrückung der Bildung der Genprodukte bzw. Unterdrückung 5 der von den codierten Genprodukten ausgeübten Funktion oder Aktivität in intakten Pflanzen durch eine niedermolekulare Substanz führt zur Reduzierung, bevorzugt zur Unterdrückung Suppression of the formation of the gene products or suppression 5 of the function or activity carried out by the coded gene products in intact plants by means of a low-molecular substance leads to a reduction, preferably to suppression
Stamm, Pflanzensamen, . Pflanzenkalli oder Pflanzenzellen, die die erfindungsgemäß beschriebenen Sequenzen, insbesondere SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID MO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,- SEQ ID NO: 28, SEQ ID NO: 30 SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ' ID NO: 48 SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO; 62, SEQ ID NO: 64 SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO; 70, SEQ ID NO: 72 SEQ ID NO: 74, SEQ. ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID-NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 SEQ ID NO: 90, .SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96 SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 oder Derivate oder Fragmente davon, die die biologische Aktivität dieser Sequenzen besitzen, funktional exprimieren;Stem, plant seeds,. Plant calli or plant cells that contain the sequences described according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID MO: 11, SEQ ID NO : 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, - SEQ ID NO: 28, SEQ ID NO: 30 SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 , SEQ ID NO: 38 SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ' ID NO: 48 SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO; 62, SEQ ID NO: 64 SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO; 70, SEQ ID NO: 72 SEQ ID NO: 74, SEQ. ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 SEQ ID-NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 SEQ ID NO: 90, .SEQ ID NO : 92, SEQ ID NO: 94, SEQ ID NO: 96 SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 or derivatives or fragments thereof functionally express the biological activity of these sequences;
b) Zugabe von chemischen Verbindungen (die auf ihre Herbizid- aktivität getestet werden sollen) zu den Linien mit den unterschiedlichen Expressions- oder Aktivitätsleveln des Genp'rodukts , z.B. zu den unter a) genannten rekombinanten Organismen und nicht-rekombinanten Ausgangsorganismen mit einem anderen, vorzugsweise geringeren Expreεsions- oder Aktivitätslevel des Genprodukts ,-b) addition of chemical compounds (which are tested activity on their herbicide should) to the lines with the different expression or activity levels of GENP 'roductS, for example to the in a) said recombinant organisms and non-recombinant parental organisms other with one, preferably lower level of expression or activity of the gene product,
c) - Bestimmung der biologischen Aktivität, beispielsweise der enzymatischen Aktivität, des Wachstums oder' der Vitalität der beiden Linien, z.B. der rekombinanten Organismen, im Ver- gleich zu den nich -rekombinanten Ausgangsorganismen, nach Zugabe von chemischen Verbindungen gemäß Punkt b) ; undc) - Determination of the biological activity, for example the enzymatic activity, the growth or the ' vitality of the two lines, for example the recombinant organisms, in comparison to the non-recombinant starting organisms, after adding chemical compounds in accordance with point b); and
d) Selektion der chemischen Verbindungen, die die biologische Aktivität, beispielsweise die enzymatische Aktivität, das Wachstum oder die Vitalität der Linie mit der- geringeren Aktivität reduziert oder vollständig hemmt bzw. blockiert, z.B. die die biologische Aktivität, das Wachstum oder die Vitalität der nicht rekombinanten Organismen, der gemäß Punkt c) bestimmten chemischen Verbindungen, im Vergleich zu den behandelten rekombinanten Organismen reduzieren oder vollständig hemmen bzw. blockieren. Ein in der Landwirtschaft nutzbares Herbizid kann auch identifiziert werden, wenn die oben in a) erzeugten rekombinanten Organismen in einem Verfahren getestet werden, das folgende Schritte umfasst:d) Selection of the chemical compounds which reduce or completely inhibit or block the biological activity, for example the enzymatic activity, the growth or the vitality of the line with the lower activity, for example the biological activity, the growth or the vitality of the line recombinant organisms, the chemical compounds determined in accordance with point c), reduced or completely inhibited or blocked in comparison to the treated recombinant organisms. An agricultural herbicide can also be identified if the recombinant organisms generated in a) above are tested in a method comprising the following steps:
(b) Zugabe von chemischen Verbindungen, die auf ihre(b) adding chemical compounds based on their
Herbizidaktivität getestet werden sollen, zu den unter (a) genannten rekombinanten Organismen; undHerbicidal activity to be tested, to the recombinant organisms mentioned under (a); and
(c) Bestimmung der biologischen Aktivität, beispielsweise der enzymatischen Aktivität, des Wachstums oder der Vitalität der rekombinanten Organismen nach Zugabe von chemischen Verbindungen- gemäß (b) im Vergleich zu denselben nicht behandelten rekombinanten Organismen; und(c) determination of the biological activity, for example the enzymatic activity, the growth or the vitality of the recombinant organisms after addition of chemical compounds according to (b) in comparison to the same untreated recombinant organisms; and
(d) Selektion der chemischen Verbindung, die die biologische Aktivität, z.B. die enzymatische Aktivität, das Wachstum oder die Vitalität der behandelten Organismen im Vergleich zu den unbehandelten Organismen reduziert oder vollständig hemmt oder blockiert.(d) selection of the chemical compound that exhibits biological activity, e.g. the enzymatic activity, the growth or the vitality of the treated organisms is reduced or completely inhibited or blocked in comparison to the untreated organisms.
Unter chemischen Verbindungen, die die biologische Aktivität, das Wachstum oder die Vitalität der Organismen reduzieren, sind Verbindungen zu verstehen, die die biologische Aktivität, das .Wachs- tum oder die Vitalität der Organismen mindestens um 10 %, vorteilhaft um mindestens 30 %, bevorzugt um mindestens 50 %, besonders bevorzugt um mindestens 70 %, ganz besonders bevorzugt um mindestens 90 % hemmen, d.h. reduzieren oder blockieren.Chemical compounds which reduce the biological activity, the growth or the vitality of the organisms are to be understood as compounds which prefer the biological activity, the growth or the vitality of the organisms by at least 10%, advantageously by at least 30% inhibit by at least 50%, particularly preferably by at least 70%, very particularly preferably by at least 90%, ie reduce or block.
Vorteilhaft ist insbesondere eine Substanz, die die. Zelllinien mit geringer Aktivität schädigen oder, vorzugsweise, die letal ist, jedoch nicht Zelllinien, die eine höhere Aktivität des Genproduktes aufweisen, schädigt oder für diese letal ist.A substance that is particularly advantageous is the. Damage cell lines with low activity or, preferably, that is lethal, but does not damage or is lethal to cell lines that have a higher activity of the gene product.
Allgemein können in dem genannten Verfahren Linien von Organismen eingesetzt werden, die die erfindungsgemäßen Sequenzen und insbesondere die Genprpdukte, die durch erfindungsgemäße Nukleinsäuren codiert werden, exprimieren, die jedoch nicht reko binant sind, solange eine Linie eine höhere Genexpression oder Aktivität des Genprodukts aufweist als eine andere Linie. Solche Linien können natürlich auftreten oder durch Mutagenesen erzeugt werden.In general, lines of organisms can be used in the method mentioned which express the sequences according to the invention and in particular the gene products which are encoded by nucleic acids according to the invention, but which are not recombinant as long as a line has a higher gene expression or activity of the gene product than one other line. Such lines can of course occur or be generated by mutagenesis.
AssaySysteme,, die die Identifizierung von Substanzen, die die Bildung der Genprodukte und/oder die von den Genprodukten aus- geübten Funktionen oder die Aktivität der Genprodukte in intakten Pflanzen, Pflanzenteilen, -geweben oder Pflanzenzellen unterdrücken, sind dem Fachmann bekannt. Beispielhaft sei hier auf Testsysteme für die Inhibierung von Enzymen wie der Fructo- kinaseaktivität wie von Tangney et al. (J. Mol. Microbiol. Bio- technol., 2 (1), 2000: 71-80), Martinez-Barajas et al. (Protein Expr. Purif., 1997, 11 (1), 41-46), Kanaya a et al . (Plant 5 Physiol., 1997,.113 (4), 1379-1384) oder von Veiga-da-Cunha et al. (Protein Expr. Purif., 19 (1), 2000: 48-52) beschrieben, verwiesen. Diese Testsysteme können beispielsweise vorteilhaft für sog. Inhibierungsassays für beispielsweise den Klon P77 verwenden.Assay systems which suppress the identification of substances which suppress the formation of the gene products and / or the functions exercised by the gene products or the activity of the gene products in intact plants, plant parts, plant tissues or plant cells are known to the person skilled in the art. An example is here Test systems for the inhibition of enzymes such as fructokinase activity as described by Tangney et al. (J. Mol. Microbiol. Biotechnol., 2 (1), 2000: 71-80), Martinez-Barajas et al. (Protein Expr. Purif., 1997, 11 (1), 41-46), Kanaya a et al. (Plant 5 Physiol., 1997, .113 (4), 1379-1384) or by Veiga-da-Cunha et al. (Protein Expr. Purif., 19 (1), 2000: 48-52). These test systems can be used advantageously, for example, for so-called inhibition assays for clone P77, for example.
1010
Weitere vorteilhafte Assaysysteme sind beispielsweise die Fluoreszenz Kprrelationsspectroskopie (= FCS) . Mit Hilfe der FCS (Brock et al., PNAS, 1999, 96, 10123-10128; Lamb et al., J. Phys. Org. Chem., 2000, .13654-658) ist es möglich die zeitlicheFurther advantageous assay systems are, for example, fluorescence correlation spectroscopy (= FCS). With the help of FCS (Brock et al., PNAS, 1999, 96, 10123-10128; Lamb et al., J. Phys. Org. Chem., 2000, .13654-658) it is possible to determine the time
15 Diffusion von Molekülen zu messen bzw. die Differenz der gebundenen gegenüber den freien Molekülen zu ermitteln. Hierzu werden die zu untersuchenden Moleküle fluoreszenzmarkiert und beispielsweise ein definiertes Volumen in Mikrotiterplatten gegeben. Die Fluktuation der Moleküle wird in den Proben dabei15 Measure the diffusion of molecules or determine the difference between the bound and the free molecules. For this, the molecules to be examined are fluorescence-labeled and, for example, a defined volume is placed in microtiter plates. The fluctuation of the molecules is shown in the samples
20 von der Braunschen Molekularbewegung getrieben. Durch einen in der Probe fokusierten Laser lassen sich die translateralen bzw. rotationale Diffusion und Konformationsänderungen der Moleküle verfolgen und über eine Korrelation analysieren. Durch Bindung an andere Substanzen ändert sich der Diffusionskoeffizient der Mole-20 driven by the Braun molecular movement. Using a laser focused in the sample, the translational or rotational diffusion and changes in the conformation of the molecules can be tracked and analyzed using a correlation. Binding to other substances changes the diffusion coefficient of the mole
25 küle. Mit Hilfe verschiedener Algorithmen lässt sich die Bindung - der Moleküle über die Änderung des Diffusionskoeffizienten, ermitteln bzw. quantifizieren. Mit dieser Methode kann in einem breiten Konzentrationsbereich vorteilhaft gemessen werden. Die Methode eignet sich vorteilhaft für die Messung von rekombinanten25 coolers. With the help of various algorithms, the binding of the molecules can be determined or quantified by changing the diffusion coefficient. This method can be used to measure advantageously in a wide concentration range. The method is advantageously suitable for the measurement of recombinant
30 Proteinen, die vorteilhaft mit einem sog. His-Tag zur leichteren Aufreinigung über handelsübliche Chromatographie-Säulen (Porath et al., Nature 1975, 258, 598-599) versehen sind. Das so gereinigte Protein wird schließlich mit einem Fluoreszenzmarker wie z.B. Carboxytetramethylrhodamin oder BODIPY® (z.B. BÖDIPY 576/58930 proteins, which are advantageously provided with a so-called His tag for easier purification on commercially available chromatography columns (Porath et al., Nature 1975, 258, 598-599). The protein purified in this way is finally treated with a fluorescent marker such as carboxytetramethylrhodamine or BODIPY ® (eg BÖDIPY 576/589
35 Angiotensin II, NEN® Life Science Products, Boston, MA, USA) versehen. Die zu untersuchende Verbindung bzw. Substanz wird zu dem Protein anschließend in einem Überschuss zugegeben. Die Diffusion des so markierten Proteins wird schließlich mit einem FCS-System (z.B. ConfoCor2 mit LSM 510, Carl Zeiss Mikroskop, Jena, Deutsch-35 angiotensin II, NEN ® Life Science Products, Boston, MA, USA) provided. The compound or substance to be examined is then added to the protein in excess. The diffusion of the protein labeled in this way is finally carried out using an FCS system (e.g. ConfoCor2 with LSM 510, Carl Zeiss microscope, Jena, Germany).
40.land) ermittelt .40.land) determined.
Eine weitere vorteilhafte Detektions-Methode für das erfindungsgemäße Verfahren ist die sog. "Surface Enhanced Laser Desorption Ionisation"-Methode (= SELDI ProteinChip®) . Diese Methode wurde 45 von Hutchens und Yip (1980) erstmals beschrieben. Mit dieser Methode, die für die reproduzierbare, gleichzeitige Identifizierung von Biomarkern oder Antigenen entwickelt wurden (Hutchens und Yip, Rapid Commun. Mass Spectrom, 1993, 7, 576-580), kann die Ligand-Protein-Bindung über Massenspektrometrie analysiert werden. Dabei erfolgt die Detektion über normale TOF-Detektion (= time of flight)-. Auch bei dieser Methode können rekombinant exprimierte Proteine wie oben beschrieben exprimiert und gereinigt werden. Zur Messung wird das Protein auf den SELDI Protein- Chips® immobilisiert, beispielsweise über die schon zur Reinigung verwendeten His-Tags oder über Ionen- oder hydrophobe Wechselwirkungen mit dem Chip. Auf diesen so vorbereiteten Chip werden anschließend die Liganden mit beispielsweise einem Autosampier gegeben. Nach einem oder mehreren .Waschschritten mit Puffern verschiedener Ionenstärke werden die gebundenen Liganden mit dem LDI-Laser analysiert., Dabei wird die Bindungsstärke der Liganden nach jedem Waschschritt ermittelt.Another advantageous detection method for the method according to the invention is the so-called "surface enhanced laser desorption ionization" method (= SELDI ProteinChip ® ). This method was first described by Hutchens and Yip (1980) 45. Using this method, which was developed for the reproducible, simultaneous identification of biomarkers or antigens (Hutchens and Yip, Rapid Commun. Mass Spectrom, 1993, 7, 576-580), the ligand-protein binding can be analyzed via mass spectrometry. The detection takes place via normal TOF detection (= time of flight). With this method, recombinantly expressed proteins can also be expressed and purified as described above. For the measurement, the protein is immobilized on the SELDI Protein-Chips ® , for example via the His tags already used for cleaning or via ion or hydrophobic interactions with the chip. The ligands are then placed on this chip prepared in this way using, for example, an autosampler. After one or more washing steps with buffers of different ionic strength, the bound ligands are analyzed with the LDI laser. , The binding strength of the ligands is determined after each washing step.
Als weitere vorteilhafte Dektionsmethode sei die sog. Biacore- Methode genannt, bei der der Refraktionsindes an der Oberfläche bei Bindung von Liganden and das an der Oberfläche gebundene Protein analysiert wird. Bei dieser Methode wird eine Kollektion von kleinen Liganden sequentiell in eine Messzelle mit dem gebundenen Protein gegeben. Die Bindung an der Oberfläche wird über eine erhöhte sog. "Plasmon-Resonanz" (= SPR) über die Aufzeichnung der Laserrefraktion von der Oberfläche ermittelt . Im allgemeinen ist die Refraktionsindexänderung, die für eine Änderung der Massenkonzentration an der Oberfläche, ermittelt wird für alle Proteine oder Polypeptide gleich, das heißt diese •Methode kann vorteilhaft für die verschiedensten Proteine verwendet werden. (Liedberg et al . , Sens. Actuators, 1984, 4, 299-304) . Wie oben beschrieben werden auch hier vorteilhaft rekombinant exprimierte Proteine verwendet, die an den Biacore Chip (Upsala, Schweden) beispielsweise über Histidin-Reste (z.B. His-Tag) gebunden werden. Der so hergestellte Chip wird wieder mit den Liganden in - Verbindung gebracht z.B. mit einem Autosampier und die Bindung über ein von Biacore vertriebenes Detektionssystem mit Hilfe des SPR-Signals d.h. über die Änderung des RefraktionsIndex gemessen.Another advantageous deciding method is the so-called Biacore method, in which the refractive index on the surface is analyzed when ligands are bound to the protein bound to the surface. With this method, a collection of small ligands is sequentially placed in a measuring cell with the bound protein. The binding to the surface is determined by means of an increased so-called "plasmon resonance" (= SPR) by recording the laser refraction from the surface. In general, the refractive index change, which is determined for a change in the mass concentration at the surface, is the same for all proteins or polypeptides, that is to say this method can advantageously be used for a wide variety of proteins. (Liedberg et al., Sens. Actuators, 1984, 4, 299-304). As described above, recombinantly expressed proteins are also advantageously used here, which are bound to the Biacore chip (Upsala, Sweden), for example via histidine residues (e.g. His-Tag). The chip thus produced is again connected to the ligands, e.g. with an autosampler and binding via a detection system sold by Biacore using the SPR signal i.e. measured by changing the refractive index.
Die erfindungsgemäßen Verfahren haben eine Reihe von Vorteilen wie beispielsweise:The methods according to the invention have a number of advantages such as:
* neue potentielle Angriffsorte für herbizide Wirkstoffe können identifiziert werden,* new potential targets for herbicidal agents can be identified,
* Identifizierung von Herbiziden, die eine möglichst umfassende Pflanzenspezies unabhängige Wirkung haben, * Substanzen, die mittels der kombinatorischen Chemie erzeugte wurden, und die sich durch eine hohe Vielfalt, aber geringe zur Verfügung stehende Mengen auszeichnen können effizient auf Inhibitoren der neu identifizierten Angriffsorte geprüft werden* Identification of herbicides that have the most comprehensive possible plant species independent effect, * Substances that have been generated using combinatorial chemistry and that are characterized by a large variety but small amounts available can be efficiently tested for inhibitors of the newly identified attack sites
* sie erlauben, landwirtschaftlichen Nutzpflanzen im Fall von z.B. sehr breite Wirksamkeit aufweisenden Herbiziden (Totalherbiziden oder auch selektiven Herbiziden) Resistenz gegen- über diesen zu vermitteln (siehe Beschreibung im folgenden)* they allow agricultural crops in the case of e.g. to impart very broad activity to herbicides (total herbicides or selective herbicides) which are resistant to them (see description below)
Z.B. können Substanzen, die besonders spezifisch mit z.B. einem Protein oder Proteinfragment binden, das von einer Nukleinsäure codiert wird,, deren Expression essentiell für das Wachstum der Pflanzen ist, mit den genannten Verfahren isoliert werden. Dies ermöglicht eine vereinfachte Identifikation möglicher Inhibitoren, die Proteine, z.B. in ihren Enzymeigenschaften, Bindeeigenschaften oder sonstigen Aktivitäten hemmen, z.B. auch durch die Inhibierung ihrer Prozessierung, wie oben beschrieben, oder ihren Transport innerhalb der Zelle oder Im- und Export aus Organellen oder Zellen verhindern. Die so identifizierten Substanzen können auch in einem weiteren Schritt in Screening- Verfahren, wie sie dem Fachmann bekannt sind, auf Pflanzen aufgebracht werden und auf ihre Beeinflussung des Wachstums und der Entwicklung hin untersucht werden. Somit wird eine Auswahl aus der unendlichen Zahl chemischen Verbindungen, die sich für ein Screeningverfahren eignen würden, getroffen, die es dem Fachmann wesentlich erleichtert, herbizide Substanzen zu identifizieren.For example, substances that are particularly specific with e.g. bind a protein or protein fragment which is encoded by a nucleic acid, the expression of which is essential for the growth of the plants, can be isolated using the methods mentioned. This enables a simplified identification of possible inhibitors, the proteins, e.g. inhibit in their enzyme properties, binding properties or other activities, e.g. also by inhibiting their processing, as described above, or preventing their transport within the cell or import and export from organelles or cells. The substances identified in this way can also be applied to plants in a further step in screening processes, as are known to the person skilled in the art, and their influence on growth and development can be examined. Thus, a selection is made from the infinite number of chemical compounds that would be suitable for a screening process, which makes it considerably easier for the person skilled in the art to identify herbicidal substances.
Unter "spezifische Bindung" versteht man die Spezifität von Interaktionen zwischen zwei Partnern, z.B. Proteinen untereinander oder von Protein (Enzym) und Substrat (Substratspezifität) . Sie beruht auf einer bestimmten molekularen räumlichen Struktur. Wird sie zerstört, spricht man von Denaturierung, die oftmals irreversibel ist und wodurch die' Spezifität meistens verloren gehen kann. Diese biologische Aktivität ist stark abhängig von den Umgebungsbedingungen (Puffer, Temperatur, Kontakte zu unphysiologische Oberflächen wie Glas oder fehlende Cofaktoren) . Bei Enzym-Substrat oder Cofaktor, bei Rezeptor-Ligand oder bei Antikörper-Antigen Bindungen spricht man von spezifischen Bindungen. Die Enzym-Substrat Wechselwirkung wird ther o- dynamisch im einfachsten Fall mit der Michaelis-Menten-Gleichung beschrieben. Sie beschreibt die Enzymaktivität über die sog. Michaelis -Menten-Konstante, die wiederum die Kinetik wieder- spiegelt . Diese Konstante ist auch die Maßeinheit für die Enzymaktivität, die wiederum die Spezifität wiederspiegelt. Definition der Enzymaktivitätseinheit (nach IUB) : Eine Einheit U entspricht derjenigen Enzymmenge, welche die Umsetzung von einem Mikromol Substrat pro Minute unter genau festgelegten Versuchsbedingungen katalysiert. Die spezifische Aktivität wird meist in U/mg angegeben.“Specific binding” means the specificity of interactions between two partners, for example proteins with one another or between protein (enzyme) and substrate (substrate specificity). It is based on a certain molecular spatial structure. If it destroys, it is called denaturation, which is often irreversible and can go so that the 'specificity lost mostly. This biological activity is strongly dependent on the environmental conditions (buffer, temperature, contacts to non-physiological surfaces such as glass or missing cofactors). With enzyme substrate or cofactor, with receptor ligand or with antibody-antigen bonds one speaks of specific bonds. The enzyme-substrate interaction is described thermodynamically in the simplest case using the Michaelis-Menten equation. It describes the enzyme activity using the so-called Michaelis-Menten constant, which in turn reflects the kinetics. This constant is also the unit of measure for enzyme activity, which in turn reflects specificity. Definition of the enzyme activity unit (according to IUB): One unit corresponds to U the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute under precisely defined test conditions. The specific activity is usually given in U / mg.
In einem weiteren Schritt können dann die identifizierten Substanzen auf Pflanzen, Microorganismen oder Zellen aufgebracht werden, z.B. auf Pflanzenzellen, und dann die Beeinflussung des Stoffwechsels dieser Pflanzen beobachtet werden, z.B. Enzy - aktivitäten, Photosyntheseaktivitäten, Stoffwechselaktivität,In a further step, the identified substances can then be applied to plants, microorganisms or cells, e.g. on plant cells, and then the influence on the metabolism of these plants can be observed, e.g. Enzy - activities, photosynthetic activities, metabolic activity,
Fixierungsrate, Gasaustausch, DNA-Synthese, Wachstumsraten. Diese und viele andere dem Fachmann bekannte Methoden eignen sich, um die Viabilität von Zellen zu untersuchen. Substanzen, die das Wachstum, z.B. von Zellen, insbesondere Pflanzenzellen, reduzier-en, insbesondere blockieren, eignen sich dann bevorzugt als Auswahl für herbizide Mittel.Fixation rate, gas exchange, DNA synthesis, growth rates. These and many other methods known to the person skilled in the art are suitable for examining the viability of cells. Substances that inhibit growth, e.g. of cells, in particular plant cells, reduce, in particular block, are then preferably suitable as a selection for herbicidal compositions.
Weiterhin können schon in einem sehr frühen Stadium Untersuchungen zu den Aufwandmengen der gefundenen Herbizide gemacht werden. Außerdem kann die hohe Spezifität und Effizienz gegenüber Unkräutern leicht ermittelt werden.Furthermore, investigations into the application rates of the herbicides found can be carried out at a very early stage. In addition, the high specificity and efficiency against weeds can be easily determined.
Mit Hilfe des erfindungsgemäßen Verfahrens kann eine Vielzahl von chemischen Verbindungen schnell und einfach auf herbizide Eigenschaften überprüft werden. Das Verfahren gestattet es, reproduzierbar aus einer großen Anzahl von Substanzen gezielt solche mit großer Wirkstärke auszuwählen, ' um mit diesen Substanzen anschließend weitere, dem Fachmann geläufige vertiefte Prüfungen' durchzuführen.With the aid of the method according to the invention, a large number of chemical compounds can be checked quickly and easily for herbicidal properties. The method allows reproducible from a large number of substances specifically select those with high potency, 'in order to then further, with these substances known to those skilled in-depth tests "carried out.
Weiterer Gegenstand der Erfindung ist ein. Verfahren zur Identifizierung von Inhibitoren pflanzlicher Proteine, die ' durch die im erfindungsgemäßen Verfahren verwendeten'Nuklein- Säuresequenzen codiert werden, mit potentiell herbizider Wirkung indem man die Genprodukte kloniert, in einer geeigneten Expressionskassette - beispielsweise in Insektenzellen - zur Überexpression bringt, die Zellen öffnet und den Zellextrakt direkt bzw. nach Anreicherung oder Isolierung des Proteins in einem Testsystem zur Messung der biologischen Aktivität in Gegen- wart von niedermolekularen chemischen Verbindungen einsetz .Another object of the invention is a. Process for identifying inhibitors of plant proteins which are ' encoded by the nucleic acid sequences used in the process according to the invention, with potentially herbicidal activity by cloning the gene products, overexpressing them in a suitable expression cassette - for example in insect cells -, opening the cells and use the cell extract directly or after enrichment or isolation of the protein in a test system for measuring biological activity in the presence of low molecular weight chemical compounds.
Ein weiterer Gegenstand der Erfindung sind deshalb Substanzen identifiziert nach den erfindungsgemäßen Verfahren, wobei die Substanz ein Molekulargewicht von kleiner 1000 Dalton, vorteil- haft kleiner 900 Dalton, bevorzugt kleiner 800, besonders bevorzugt kleiner 700, ganz besonders bevorzugt kleiner 600 Dalton, einem Ki-Wert kleiner 1 mM, und vorzugsweise weniger als drei Hydroxylgruppen an einem Kohlenstoffatom-enthaltenden Ring hat bzw. wobei es sich bei der Substanz um eine proteinogene Substanz, um eine Antisense-RNA, eine inhibierende oder eine interferierende RNA (RNAi) handelt.The invention therefore furthermore relates to substances identified by the processes according to the invention, the substance having a molecular weight of less than 1000 daltons, advantageously less than 900 daltons, preferably less than 800, particularly preferably less than 700, very particularly preferably less than 600 daltons, a ki Value less than 1 mM, and preferably less than three Has hydroxyl groups on a ring containing carbon atoms or the substance is a proteinogenic substance, an antisense RNA, an inhibitory or an interfering RNA (RNAi).
Der Begriff "sense" bezieht sich auf den Strang einer doppel- strängigen DNA der homolog zu dem mRNA-Transkript ist. Der "Anti-' sense"-Strang enthält eine invertierte Sequenz, die komplementär zu der des "Sense"-Strangs ist. Ein Antisense-Nukleinsäuremolekül umfasst z.B. eine Nukletidsequenz, die' komplementär zu dem wSense"-Nukleinsäuremolekül ist, das ein Protein oder eine aktive RNA codiert, z.B.' komplementär zu dem codierenden Strang eines doppelsträngigen cDNA-Moleküls oder komplementär zu einer mRNA- Sequenz . Folglich kann ein Antisense-Nukleinsäuremolekül- Wasser- stoffbrückenbindungen zu einem Sense-Nukleinsäuremolekül ausbilden. Das Antisense-Nukleinsäuremolekül kann komplementär zu jedem hier gezeigten codierenden Strang sein, oder nur zu einem Teil davon. Der Begriff "codierende Region" bezieht sich auf die Region einer Nukleinsäuresequenz, deren Codone in Aminosäuren translatiert werden. "Auch kann das Antisense-Nukleinsäuremolekül komplementär zu "nicht-codierenden Regionen" des codierenden Strangs der gezeigten Nukleinsäuremoleküle sein.. Der Begriff "nicht-codierende Region" bezieht sich auf 5'- und 3 '-Sequenzen, die die codierende Region flankieren und die nicht in ein Polypeptid translatiert werden (z.B. auch bezeichnet als 5'~ und 3 '-nicht-translatierte Regionen). Das Nukleinsäuremolekül, das eine Antisensese uenz umfasst, kann auch weitere für die Expression und Stabilität des Moleküls wichtige Elemente umfassen, z.B. Capping-Strukturen, poly A-tails etc.The term “sense” refers to the strand of a double-stranded DNA that is homologous to the mRNA transcript. The "anti-sense" strand contains an inverted sequence that is complementary to that of the "sense" strand. An antisense nucleic acid molecule comprises, for example, a nuclide sequence that is ' complementary to the w sense ' nucleic acid molecule which encodes a protein or an active RNA, for example' complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Thus, an antisense nucleic acid molecule can hydrogen bond to a sense nucleic acid molecule. The antisense nucleic acid molecule can be complementary to, or only a part of, any coding strand shown here. The term "coding region" refers to the region of one Nucleic acid sequence whose codons are translated into amino acids. "The antisense nucleic acid molecule can also be complementary to" non-coding regions "of the coding strand of the nucleic acid molecules shown. The term" non-coding region "refers to 5'- and 3 ' - sequences that flank the coding region and that are not in a polypeptide can be translated (eg also referred to as 5 '~ and 3' untranslated regions). The nucleic acid molecule which comprises an antisense sequence can also comprise further elements which are important for the expression and stability of the molecule, for example capping structures, poly A-tails etc.
Das Antisense-Nukleinsäuremolekül kann komplementär zu der gesamten codierenden Region einer mRNA sein, aber kann auch ein Oligonukleotid sein, welches? komplementär zu nur einem Teil der codierenden oder nicht-codierenden Region der mRNA ist. Z.B. kann ein Antisense-Oligonukleotid komplementär zu der Region sein, die den Translationsstart der mRNA umfasst oder umgibt . Ein Antisense-Oligonukleotid kann z.B. 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45- oder 50-Nukleotide lang sein. Ein Antisense-Nukleinsäuremolekül kann durch chemische Synthese und enzymatische Ligation nach dem Fachmann bekannten Verfahren hergestellt werden. Eine Antisense-Nukleinsäuremolekül kann chemisch synthetisiert werden unter Verwendung von natürlich vorkommenden Nukleotiden oder auf verschiedene Arten modifizierten Nukleotiden, so daß die biologische Stabilität der Moleküle erhöht ist oder die physi- kaiische Stabilität des Duplex, die sich zwischen der Antisense- und Sense-Nukleinsäure bildet, verstärkt ist, z.B. können Phosphorothioatderivate und Acridin-substituierte Nukleotide verwendet werden. Beispiele für modifizierte Nukleotide, die für die Herstellung von Antisense-Nukleinsäuren verwendet werden können, umfassen 5-Fluorouracil, 5-Bromouracil, 5-Chloro- uracil, 5-Iodouracil, Hypoxanthine, Xanthine, 4-Acetylcytosine, 5- (Carboxyhydroxylmethyl)uracil, 5-Carboxymethylaminomethyl-2 -thiouridine, 5-Carboxymethylaminomethyluracil, Dihydrouracil, Beta-D-galactosylqueosine, Inosine, N6-Isopentenyladenine, 1-Methylguanine, 1-Methylinosine, 2, 2-Dimethylguanine, 2-Methyl- adenine, 2-Methylguanine, 3-Methylcytosine, 5-Methylcytosine, N6-Adenine, 7-Methylguanine, 5-Methylaminomethyluracil,The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but can also be an oligonucleotide which? is complementary to only a portion of the coding or non-coding region of the mRNA. For example, an antisense oligonucleotide can be complementary to the region that encompasses or surrounds the translation start of the mRNA. An antisense oligonucleotide can be, for example, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid molecule can be produced by chemical synthesis and enzymatic ligation according to methods known to the person skilled in the art. An antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or nucleotides modified in various ways so that the biological stability of the molecules is increased or the physical stability of the duplex that forms between the antisense and sense nucleic acids , is enhanced, for example, phosphorothioate derivatives and acridine-substituted nucleotides be used. Examples of modified nucleotides that can be used for the production of antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthines, xanthines, 4-acetylcytosines, 5- (carboxyhydroxylmethyl) uracil , 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyl-auanine, 2-methyl , 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil,
5-Methoxyaminomethyl-2-thiouracil, Beta-D-mannosylqueosine, 5 '-Methoxycarboxymethyluracil, 5-Methoxyuracil, 2-Methylthio- N6-isopentenyladenine, Uracil-5-oxyacetic- acid (v) , Wybutoxosine, Pseudouracil, Queosine, 2-Thiocytosine, 5-Methyl-2-thiouracil, 2-Thiouracil, 4-Thiouracil, 5-Methyluracil, Uracil-5-oxyaceti- cacidmethylester, Uracil-5-oxyacetic acid (v) , 5-Methyl-2-thio- •. uracil, 3-(3-Amino-3-N-2-carboxypropyl)uracil, (acp3)w, und 2 , 6-Diaminopurine.5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine Thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thio- •. uracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2, 6-diaminopurines.
Alternativ können Antisense-Nukleinsäuremoleküle biologisch hergestellt werden unter Verwendung von Expressionsvektoren, in - welche Polynukleotide kloniert wurden, deren Orientation gegenläufig ist (so daß RNA, transkribiert von dem inserierten Poly- • nukleotid, in- einer Antisenseorientierung zu einem Zielpoly- nukleotid wie es weiter oben beschrieben wurde, ist) .Alternatively, antisense nucleic acid molecules can be produced biologically using expression vectors into which polynucleotides have been cloned, whose orientation is opposite (so that RNA, transcribed from the inserted poly- nucleotide, in an antisense orientation to a target polynucleotide as follows) has been described above).
Das Antisense-Nukleinsäuremolekül kann auch ein "α-Anomeric" Nukleinsäuremolekül sein. Ein "α-Anomeric" Nukleinsäuremolekül . formt spezifische Doppelstranghybride mit komplementären RNAs, in denen, im Gegensatz zu gewöhnlichen ß-Eihheiten,. die Stränge parallel 'zueinander laufen. Das Antisense-Nukleinsäuremolekül kann 2-0-Methylriboήukleotide oder chimäre RNA-DNA-Analoge . umfassen.The antisense nucleic acid molecule can also be an "α-anomeric" nucleic acid molecule. An "α-anomeric" nucleic acid molecule . forms specific double-stranded hybrids with complementary RNAs, in which, in contrast to ordinary ß-units,. the strands run parallel to each other. The antisense nucleic acid molecule can be 2-0-methylriboήucleotide or chimeric RNA-DNA analogues. include.
Weiterhin kann das Antisense-Nukleinsäuremolekül ein Ribozym sein. Ribozyme sind katalytische RNA-Moleküle mit einer Ribo- nukleaseaktivität, die dazu fähig sind, einzelsträngige Nukleinsäuren, wie z.B. mRNA, zu denen sie eine komplementäre Region haben, zu schneiden. Ribozyme (z.B. Hammerheadribozyme) können dazu verwendet werden katalytisch oder nichtkatalytisch mRNA der hierin beschriebenen Sequenzen zu schneiden und somit die Translation der mRNA zu verhindern. Ein Ribozym, das zu einer der hierin genannten Nukleinsäurese uenzen spezifisch ist, kann aufgrund der hier gezeigten cDNA-Sequenzen konstruiert werden oder auf Basis von heterologen Sequenzen, die nach den hierin beschriebenen Methoden identifiziert werden können. Z.B. kann ein Derivat der Tetrahymena L-19 IVSRNA hergestellt werden, indem die Nukletidsequenz der aktiven Region komplementär zu der Nukleotid- sequenz ist, die in einer codierenden mRNA geschnitten wird. Alternativ kann auch eine der hierin beschriebenen codierenden oder nicht-codierenden Sequenzen oder einer mRNA davon verwendet werden, um eine katalytische RNA aus einem Pool von RNAs auszuwählen (s. z.B. Bartel, 1993, Science, 261,. 1411). Alternativ kann die Expression auch inhibiert werden, indem Nukleotid- sequenzen, die komplementär - zu einer regulatorischen Region der hierin beschriebenen Nukleinsäuresequenzen ist (z.B. ein Promotor oder Enhancer) eine triple-helikale Struktur bildet, die die Transkription des folgenden Gens verhindert (z.B. Helene, 1991, Anticance-Drug Des. 6, 596; Helene, 1992, Ann. "NY Acad. Sei. 660, 27, oder Mäher, 1992, Bioassays, 14, 807.Furthermore, the antisense nucleic acid molecule can be a ribozyme. Ribozymes are catalytic RNA molecules with a ribonuclease activity that are able to cut single-stranded nucleic acids, such as mRNA, to which they have a complementary region. Ribozymes (eg hammerhead ribozymes) can be used to catalytically or non-catalytically cut the mRNA of the sequences described herein and thus to prevent translation of the mRNA. A ribozyme which is specific to one of the nucleic acid sequences mentioned herein can be constructed on the basis of the cDNA sequences shown here or on the basis of heterologous sequences which can be identified by the methods described herein. For example, a derivative of the Tetrahymena L-19 IVSRNA can be produced by the The nucleotide sequence of the active region is complementary to the nucleotide sequence that is cut in a coding mRNA. Alternatively, one of the coding or non-coding sequences described herein or an mRNA thereof can also be used to select a catalytic RNA from a pool of RNAs (see, for example, Bartel, 1993, Science, 261,. 1411). Alternatively, the expression can also be inhibited in that nucleotide sequences which are complementary to a regulatory region of the nucleic acid sequences described here (for example a promoter or enhancer) form a triple-helical structure which prevents transcription of the following gene (for example Helene, 1991, Anticance-Drug Des. 6, 596; Helene, 1992, Ann. " NY Acad. Sci. 660, 27, or Mower, 1992, Bioassays, 14, 807.
Unter "Antikörpern" sind beispielsweise polyklonale, monoklonale, humane oder humanisierte oder rekombinante Antikörper oder Fragmente davon, single chain Antikörper oder auch synthetische Antikörper zu verstehen. Unter erfindungsgemäßen Antikörpern oder deren Fragmente sind prinzipiell alle Immunoglobulinklassen wie IgM, IgG, IgD, IgE, IgA oder ihre Subklassen wie die Subklassen des IgG oder deren Mischungen zu verstehen. Bevorzugt sind IgG und seine Subklassen wie beispielsweise IgGi, IgG2, IgG2a, IgG , IgG3 oder IgGii. Besonders bevorzugt sind die IgG Subtypen IgGχ oder IgGb- Als Fragmente seien alle verkürzten oder veränderten Antikörperfragmente mit einer oder zwei dem Antigen komplementären Bindungsstellen, wie Antikörperteile mit einer den Antikörper entsprechenden von leichter und schwerer Kette gebildeten Bindungsstelle wie Fv-, Fab- oder F(ab' )2-Fragmente oder Einzelstrangfragmente, genannt. Bevorzugt sind verkürzte Doppelstrang- fragmente wie Fv-, Fab- oder F(ab') . Diese Fragmente können beispielsweise auf enzymatischem Wege durch Abspaltung des Fc- Teils der Antikörper mit Enzymen wie Papain oder Pepsin, durch chemische Oxidation oder durch gentechnische Manipulation der Antikörpergene erhalten werden. Auch genmanipulierte nicht- verkürzte Fragmente können vorteilhaft verwendet werden. Die Antikörper oder Fragmente können allein oder in Mischungen verwendet werden. Antikörper können auch Teil eines Fusionsproteins sein.“Antibodies” are understood to mean, for example, polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies or else synthetic antibodies. Antibodies according to the invention or their fragments are in principle to be understood as meaning all immunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or their subclasses such as the subclasses of the IgG or their mixtures. IgG and its subclasses such as IgGi, IgG 2 , IgG 2a , IgG, IgG 3 or IgGii are preferred. The IgG subtypes IgGχ or IgG b are particularly preferred. All shortened or modified antibody fragments with one or two binding sites complementary to the antigen, such as antibody parts with a binding site corresponding to the antibody of light and heavy chain, such as Fv, Fab or F, are to be considered fragments (from ') 2 fragments or single-strand fragments. Shortened double-strand fragments such as Fv, Fab or F (ab ') are preferred. These fragments can be obtained, for example, enzymatically by cleaving off the Fc part of the antibodies with enzymes such as papain or pepsin, by chemical oxidation or by genetic engineering manipulation of the antibody genes. Genetically manipulated, unabridged fragments can also be used advantageously. The antibodies or fragments can be used alone or in mixtures. Antibodies can also be part of a fusion protein.
Die identifizierten Substanzen können chemisch synthetisierte oder mikrobiologisch produzierte Stoffe sein und z.B. in Zellextrakten von z.B. Pflanzen, Tieren oder Mikroorganismen auftreten. Weiterhin können die genannten Stoffe zwar im Stand der Technik bekannt sein, aber bisher nicht bekannt sein als Herbizid. Das Reaktionsgemisch kann ein zellfreier Extrakt sein oder eine Zelle oder Zellkultur umfassen. Geeignete Methoden sind dem Fachmann bekannt und werden z.B. allgemein beschrieben in Alberts, Molecular Biology the cell, 3rd Edition (1994), z.B. Kapitel 17. Die genannten Stoffe können z.B. zu dem Reaktionsgemisch oder dem Kulturmedium zugegeben werden oder den Zellen injiziert werden oder auf eine Pflanze gesprüht werden.' The substances identified can be chemically synthesized or microbiologically produced substances and can occur, for example, in cell extracts from, for example, plants, animals or microorganisms. Furthermore, the substances mentioned may be known in the prior art, but have not hitherto been known as a herbicide. The reaction mixture can be a cell-free extract or can comprise a cell or cell culture. Suitable methods are known to the person skilled in the art and are described, for example, in general in Alberts, Molecular Biology the cell, 3rd Edition (1994), for example Chapter 17. The substances mentioned may be eg added to the reaction mixture or the culture medium or the cells are injected or sprayed onto a plant. '
Wenn eine Probe, die ein nach der erfindungsgemäßen Methode aktive Substanz beinhaltet, identifiziert wurde, dann ist es entweder möglich, den Stoff direkt von der ursprünglichen Probe zu- isolieren, oder man kann die Probe in verschiedene Gruppen teilen, z.B. wenn sie aus einer Vielzahl von' verschiedenenIf a sample containing a substance active according to the method according to the invention has been identified, then it is either possible to isolate the substance directly from the original sample or the sample can be divided into different groups, e.g. if they are from a variety of 'different
Komponenten besteht, um so die Zahl der verschiedenen Substanzen pro Probe zu reduzieren und dann das erfindungsgemäße Verfahren mit einer solchen "Unterprobe" der ursprünglichen Probe zu wiederholen. Abhängig von der Komplexität der Probe können die oben beschriebenen Schritte .mehrmals wiederholt werden, vorzugsweise bis die gemäß der erfindungsgemäßen Methode identifizierte Probe nur noch eine geringe Anzahl von Substanzen oder nur noch eine Substanz umfasst. Vorzugsweise wird der gemäß, der erfindungsgemäßen Methode identifizierte Stoff oder Derivate davon weiter formuliert, so, dass er für die Anwendung in der Pflanzenzüchtung oder Pflanzenzell- oder Gewebekultur geeignet ist.Components exist so as to reduce the number of different substances per sample and then to repeat the method according to the invention with such a "sub-sample" of the original sample. Depending on the complexity of the sample, the steps described above can be repeated several times, preferably until the sample identified according to the method according to the invention only comprises a small number of substances or only one substance. The substance or derivative thereof identified according to the method according to the invention is preferably further formulated in such a way that it is suitable for use in plant breeding or plant cell or tissue culture.
Die Stoffe, die gemäß dem erfindungsgemäßen Verfahren getestet und identifiziert wurden, können beispielsweise sein: Expressionsbibliotheken, z.B. cDNA-Expressionsbibliotheken,The substances that have been tested and identified according to the method according to the invention can be, for example: Expression libraries, e.g. cDNA expression libraries,
Peptide, Proteine, Nukleinsäuren, Antikörper, kleine organische Stoffe, Hormone, PNAs oder ähnliches (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell. 79 (1994) , 193-198 und darin zitierte Referenzen) . Diese Stoffe könne auch funktioneile Derivate oder Analogon der bekanntenPeptides, proteins, nucleic acids, antibodies, small organic substances, hormones, PNAs or the like (Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995), 237-245; Gibbs, Cell. 79 ( 1994), 193-198 and references cited therein). These substances can also be functional derivatives or analogs of the known ones
Inhibitoren oder Aktivatoren sein. Verfahren zur Herstellung von chemischen Derivaten oder Analogon sind dem Fachmann bekannt . Die genannten Derivate und Analogon können gemäß Verfahren nach dem Stand der Technik getestet werden. Weiterhin kann computer- gestütztes Design oder Peptidomimetics zur Herstellung geeigneter Derivate und Analogon verwendet werden. Die Zelle oder das Gewebe, die/das für das er indungsgemäße Verfahren verwendet werden kann, ist vorzugsweise eine erfindungsgemäße Wirtszelle, Pflanzenzelle oder ein Pflanzengewebe, wie in den oben genannten Ausführungsformen beschrieben.Inhibitors or activators. Methods for the production of chemical derivatives or analogs are known to the person skilled in the art. The derivatives and analogues mentioned can be tested according to methods according to the prior art. Furthermore, computer-aided design or peptidomimetics can be used to produce suitable derivatives and analogues. The cell or tissue which can be used for the method according to the invention is preferably a host cell, plant cell or a plant tissue according to the invention, as described in the above-mentioned embodiments.
Unter Derivate (n) (der Plural und der Singular seien für diese Anmeldung und deren Definitionen äquivalent) der in den erfindungsgemäßen Verfahren verwendeten Nukleinsäuren sind beispielsweise funktioneile Homologe der von SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, • SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98 , SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oderThe derivatives (s) (the plural and the singular are equivalent for this application and their definitions) of the nucleic acids used in the methods according to the invention include, for example, functional homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 , SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, • SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76 , SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or
SEQ ID NO: 108 codierten Proteine oder deren biologischer Aktivität, das heißt Proteine, die dieselben biologischen Reaktionen wie die von SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID~NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO:.46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, 'SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 codierten Proteine ausführen, zu verstehen. Diese Derivate bzw. Gene sind ebenfalls als herbizide Targets geeignet .SEQ ID NO: 108 encoded proteins or their biological activity, that is proteins which have the same biological reactions as those of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO : 9, SEQ ID No: 11, SEQ ID No: 13, SEQ ID No: 15, SEQ ID ~ NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: .46, SEQ ID NO: 48, SEQ ID NO: 50 , SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO : 84, 'SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 encoded proteins. These derivatives or genes are also suitable as herbicidal targets.
Die 'hierin erfindungsgemäß beschriebenen Sequenzen codieren für Homologe zu den in den Beispielen beschriebenen Proteinen und haben vorzugsweise die für die Homologe angegebenen Aktivitäten. •The 'sequences according to the invention described herein encode homologs of the proteins described in the examples, and preferably have the activities specified for the homologs. •
SEQ ID NO: 1 codiert für ein Protein, das zu einer" m6A~Methyl- transferase Ähnlichkeiten aufweist. Die Proteinsequenz ist in SEQ ID NO: 2 wiedergegeben ist. SEQ ID NO: 3 codiert für ein sog. XDNA repair protein RAD 54 like Protein-Homolog" , dessen Proteinsequenz SEQ ID NO: 4 zu entnehmen ist. SEQ ID NO:' 5 kann für ein Thioredoxin codieren, die Proteinsequenz ist in SEQ ID NO: 6 dargestellt. SEQ ID NO: '7 codiert für ein unbekanntes Protein, dessen Sequenz in SEQ ID NO: 2 wiedergegeben ist. SEQ ID NO: 9 codiert für vorzugsweise eine Fructokinase, deren Proteinsequenz SEQ ID NO: 10 zu entnehmen ist. SEQ ID NO: 11 codiert für ein Protein, das schwache Homologien zu verschiedenen Zinkfinger- Proteinen aufweist. Die Proteinsequenz ist SEQ ID NO: 12 zu ent- nehmen. Von SEQ ID NO: 13 wird ein Protein codiert, das Ähnlichkeiten mit LYTB-Proteinen hat. SEQ ID NO: 14 gibt die Proteinsequenz wieder. Für ein Protein, mit der in SEQ ID NO: 16 ge- zeigten Sequenz, codiert die Sequenz SEQ ID NO: 15. Dieses durch SEQ ID NO: 15 codierte hypothetische Protein enthält eine sog. Crepropin-Fa ilien-Signatur und hat schwache Homologien zu myc- Protoonkogenen. SEQ ID NO: 17 codiert für ein Protein, das eine gewisse Homologie zu einem Leucin-reichen Protein aus Mensch hat. SEQ ID NO: 18 ist die Proteinsequenz zu entnehmen. DieSEQ ID NO: 1 codes for a protein which is similar to an “m6A ~ methyltransferase. The protein sequence is reproduced in SEQ ID NO: 2. SEQ ID NO: 3 codes for a so-called X DNA repair protein RAD 54 like protein homolog ", the protein sequence of which can be found in SEQ ID NO: 4. SEQ ID NO: ' 5 can code for a thioredoxin, the protein sequence is shown in SEQ ID NO: 6. SEQ ID NO: ' 7 codes for an unknown protein, the sequence of which is shown in SEQ ID NO: 2. SEQ ID NO: 9 codes for a fructokinase, the protein sequence of which can be found in SEQ ID NO: 10. SEQ ID NO: 11 codes for a protein which has weak homologies with various zinc finger proteins. The protein sequence can be found in SEQ ID NO: 12. SEQ ID NO: 13 encodes a protein that is similar to LYTB proteins. SEQ ID NO: 14 represents the protein sequence. For a protein with which in SEQ ID NO: 16 showed sequence, encoded the sequence SEQ ID NO: 15. This hypothetical protein encoded by SEQ ID NO: 15 contains a so-called crepropin family signature and has weak homologies to myc proto-oncogenes. SEQ ID NO: 17 encodes a protein that has some homology to a leucine-rich human protein. SEQ ID NO: 18 shows the protein sequence. The
Nukleinsäuren SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,Nucleic acids SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO 32, SEQ ID NO: "34, SEQ ID NO: 36, SEQ ID NO 38,SEQ ID NO 32, SEQ ID NO: " 34, SEQ ID NO: 36, SEQ ID NO 38,
S SEEQQ IIDD NNOO: 4 400,, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48,S SEEQQ IIDD NNOO: 4 400 ,, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO 48,
1 100 SSEEQQ IIDD NNOO: 5 500,, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO 56,1 100 SSEEQQ IIDD NNOO: 5 500 ,, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO 56,
S SEEQQ IIDD NNOO: 5 588,, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO 64,S SEEQQ IIDD NNOO: 5 588 ,, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO 64,
S SEEQQ IIDD NNOO: 6 666,, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO 72,S SEEQQ IIDD NNOO: 6 666 ,, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO 72,
SEQ ID NO 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO 8 800,SEQ ID NO 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO 8 800,
SEQ ID NO 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO 8888,SEQ ID NO 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO 8888,
15 SEQ ID NO 90, SEQ ID NO: 92, SEQ ID NO: 94/ SEQ ID NO 9966,15 SEQ ID NO 90, SEQ ID NO: 92, SEQ ID NO: 94 / SEQ ID NO 9966,
SEQ ID NO 98, SEQ ID NO: 100 , SEQ ID NO : 102, SEQ ID NO: 1 106 oder SEQ ID NO: 108 codieren Homologe oder- haben Ähnlichkeit zu Proteinen, deren Aktivität oder Funktion oben oder in den Beispielen wiedergegeben ist. Die Proteinsequenzen sind jeweils in 20 den SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,SEQ ID NO 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 1 106 or SEQ ID NO: 108 encode homologues or have similarity to proteins, the activity or function of which is shown above or in the examples. The protein sequences are each in 20 SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO 39, SEQ ID NO: 41,SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO 39, SEQ ID NO: 41,
SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO: 51,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO: 59,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO 65, SEQ ID NO: 67,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO 65, SEQ ID NO: 67,
25 SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO 7 733,, SSEEQQ IIDD NNOO:: 7755,,25 SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO 7 733 ,, SSEEQQ IIDD NNOO :: 7755 ,,
SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO 8811,, SSEEQQ IIDD NNOO:: 8833,,SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO 8811 ,, SSEEQQ IIDD NNOO :: 8833 ,,
SEQ ID NO: 85, SEQ IDNO: 87, SEQ ID NO 8899,, SSEEQQ IIDD NNOO:: 9911,,SEQ ID NO: 85, SEQ IDNO: 87, SEQ ID NO 8899 ,, SSEEQQ IIDD NNOO :: 9911 ,,
SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO 9977,, SSEEQQ IIDD NNOO:: 9999,,SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO 9977 ,, SSEEQQ IIDD NNOO :: 9999 ,,
SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105,. SEQ ID NO: 107 30 oder SEQ ID NO: 109 dargestellt.SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105. SEQ ID NO: 107 30 or SEQ ID NO: 109.
Unter Derivaten werden auch solche Peptide verstanden, die eine Homologie zu den Polypeptiden mit den in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: '9, 5 SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID'NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,Derivatives are also understood to mean those peptides which have homology to the polypeptides with the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: ' 9, 5 SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID ' NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, 0 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, 0 SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,
SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ' ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, 5 SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oderSEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ 'ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, 5 SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or
SEQ ID NO: 108 gezeigten Sequenzen von mindestens 20 %, vorzugsweise 30 %, mehr bevorzugt 50 %, noch mehr bevorzugt 70 %, mehr SEQ ID NO: 108 sequences of at least 20%, preferably 30%, more preferably 50%, still more preferably 70%, more
SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49 , SEQ ID NO : 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57 , SEQ ID NO : 59 , SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65 , SEQ ID NO : 67 , SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73 , SEQ ID NO : 75 , 5 SEQ ID NO: 77, SEQ ID'NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 zu entnehmen. Unter Homologie ist IdentitätSEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, 5 SEQ ID NO : 77, SEQ ID'NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 remove. Under homology is identity
10 zu verstehen, das heißt die Aminosäuresequenzen sind zu mindestens 40, 50, 60 oder 70 %, mehr bevorzugt 80 %, noch mehr bevorzugt 90 %, am meisten bevorzugt 95 % oder mehr identisch. Die erfindungsgemäßen Sequenzen sind auf Nu leinsäureebene mindestens 45 oder 55 "% homolog, bevorzugt mindestens 60 oder 65 %,10 to understand, that is, the amino acid sequences are at least 40, 50, 60 or 70%, more preferably 80%, even more preferably 90%, most preferably 95% or more identical. The sequences of the invention are homologous to Nu leinsäureebene at least 45 or 55 "%, preferably at least 60 or 65%,
15 besonders .bevorzugt 75 %, ganz besonders bevorzugt mindesten 80 % noch mehr bevorzugt 90 der 95 %. oder mehr homolog.15 especially . preferably 75%, very particularly preferably at least 80%, even more preferably 90 of the 95% . or more homologous.
Weiterhin umfasst der Begriff Derivate sowie der Begriff "Fragmente" auch Teilbereiche oder Fragmente der aufgeführtenFurthermore, the term derivatives and the term “fragments” also include partial areas or fragments of the listed ones
20 Sequenzen oder deren homologen Sequenzen von mindestens 50 Aminosäuren, vorteilhaft von mindestens 40 Aminosäuren, bevorzugt von mindestens 30 Aminosäuren, besonders bevorzugt von mindestens 20 Aminosäuren, ganz besonders bevorzugt von mindestens 10 Aminosäuren, die es ermöglichen, selektiv interagierende Substanzen20 sequences or their homologous sequences of at least 50 amino acids, advantageously of at least 40 amino acids, preferably of at least 30 amino acids, particularly preferably of at least 20 amino acids, very particularly preferably of at least 10 amino acids, which enable selectively interacting substances
25 zu identifizieren. Der Begriff "Fragment", ."Sequenzfragment" - oder "Teilsequenz" bedeutet eine verkürzte Sequenz der Original- sequenz . Die verkürzte Sequenz (Nukleinsäure oder Protein) kann unterschiedliche Längen haben, die minimale Sequenzlänge- ist eine Sequenzlänge, die wenigstens eine vergleichbare Funktion, z.B.25 to identify. The term "fragment", "sequence fragment" - or "partial sequence" means a shortened sequence of the original sequence. The shortened sequence (nucleic acid or protein) can have different lengths, the minimum sequence length - is a sequence length that has at least one comparable function, e.g.
30. Bindungseigenschaften, oder Aktivität der Originalsequenz hat. Entsprechende Verfahren sind z.B. wie oben beschrieben SELDI, FCS oder Biocore und sind dem Fachmann bekannt.30. Has binding properties, or activity of the original sequence. Appropriate methods are e.g. as described above, SELDI, FCS or Biocore and are known to the person skilled in the art.
Ebenfalls umfasst sind somit Nukleinsäuren, die ein Fragment 35. oder ein Epitope eines Polypeptides codieren, das spezifisch an einem Antikörper bindet, der spezifisch an eine, als ■ erfindungsgemäß beschriebenes Polypeptid bindet, insbesondere das von einer der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, 40 SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,' SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, 45 SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID' NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Sequenz codiert wird. Fragment oder Epitope eines Polypeptides , die mit einem solchen Antikörper spezifisch interagieren, weisen eine signifikante Homologie in der räumlichen Struktur zu den hierin beschriebenen Polypeptiden, zumindest in Teilbereichen,- auf. Vorzugsweise' besitzen sie ebenfalls eine hohe Homologie auf Aminosäureebene zu den genannten Sequenzen, vorzugsweise 20 %, mehr bevorzugt sind 40 %, mehr bevorzugt 60 %, noch mehr 80 %, am meisten bevorzugt sind 90 % oder mehr. Die räumliche Struktur eines Polypeptides ist jedoch im wesentlichen mitverantwortlich für die Interaktionen des Polypeptides mit anderen Verbindungen sowie ggf . für seine enzymatische Aktivität . Folglich können - in den erfindungsgemäßen Verfahren oder Fragmenten eingesetzt werden, deren Sequenz nur eine geringe Homologie zu den beschriebenen Polypeptiden aufweist, deren räumliche Struktur jedoch eine hohe Homologie zu den beschriebenen Polypeptiden aufweist., also solche, die Epitope der hierin beschriebenen Sequenzen enthalten, um Interaktionspartner zu finden, die dann die hierin beschriebenen Polypeptide inhibieren oder inaktivieren. Fragmente, die Epitope der erfindungsgemäßen Polypeptide umfassen, können auch verwendet werden, um die Interaktionspartner der erfindungsgemäßen Polypeptide zu "besetzen", d.h. ihre Inter- aktion mit den erfindungsgemäßen Polypeptiden zu verhindern. Hierzu ist es dann vorteilhaft, wenn die Fragmente eine höhere Affinität zu einem Bindungspartner haben als das natürlich vorkommende Pol'ypeptid. Ebenfalls umfasst sind Fragmente, die von erfindungsgemäßen Nukleinsäuren codiert werden und eine der oben genannten biologischen Aktivitäten" umfassen.Also included are nucleic acids that encode a fragment 35 or an epitope of a polypeptide that specifically binds to an antibody that specifically binds to a polypeptide described as ■ according to the invention, in particular that of one of those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, 40 SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, ' SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO : 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60 , SEQ ID NO: 62, 45 SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID ' NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 encoded sequence. Fragments or epitopes of a polypeptide that interact specifically with such an antibody have a significant homology in the spatial structure to the polypeptides described here, at least in some areas. Preferably 'also they have a high homology at the amino acid level to the said sequences, preferably 20%, are more preferably 40%, more preferably 60%, even more 80%, are most preferred 90% or more. However, the spatial structure of a polypeptide is essentially responsible for the interactions of the polypeptide with other compounds and, if necessary, for its enzymatic activity. Consequently, - can be used in the methods or fragments according to the invention whose sequence has only a low homology to the polypeptides described, but whose spatial structure has a high homology to the polypeptides described, that is to say those which contain epitopes of the sequences described here To find interaction partners who then inhibit or inactivate the polypeptides described herein. Fragments which comprise epitopes of the polypeptides according to the invention can also be used to "occupy" the interaction partners of the polypeptides according to the invention, ie to prevent their interaction with the polypeptides according to the invention. For this it is advantageous if the fragments have a higher affinity for a binding partner have ypeptid than the naturally occurring Pol '. Also included are fragments which are encoded by nucleic acids according to the invention, and include one of the biological activities "referred to above.
- Allelvarianten umfassen insbesondere funktioneile Varianten, die durch Deletion, Insertion oder Substitution von Nukleotiden aus der in SEQ ID NO: 1, SEQ ID-NO: 3, SEQ ID NO: 5; SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,- Allelic variants include, in particular, functional variants which, by deletion, insertion or substitution of nucleotides from the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5; SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,
SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,
SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,
SEQ ID NO: 58, SEQ ID NO: •60, SEQ ID NO: 62, SEQ ID NO: 64,SEQ ID NO: 58, SEQ ID NO: • 60, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID NO: 66 , SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,
SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88,SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88,
SEQ ID NO: 90, SEQ ID NO: 92-, SEQ ID NO: 94, SEQ ΪD NO: 96,SEQ ID NO: 90, SEQ ID NO: 92-, SEQ ID NO: 94, SEQ ΪD NO: 96,
SEQ ID NO: 98, SEQ ID NO: 100 SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Sequenz erhältlich sind, wobei die biologische, z.B. enzymatische Aktivität oder Bindungseigen- Schäften der abgeleiteten synthetisierten Proteine erhalten bleibt.SEQ ID NO: 98, SEQ ID NO: 100 SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 are available, the biological, for example enzymatic activity or binding properties Shafts of the derived synthesized proteins are preserved.
Solche DNA-Sequenzen lassen sich mit Hilfe der erfindungs- gemäßen Nukleinsäuresequenzen, z.B. ausgehend von den in SEQ ID NO: 1, SEQ ID NO: 3 , SEQ ID NO: 5 , SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17., SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO:.36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72. SEQ ID NO: 74, SEQ ID NO:. 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:- 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ. ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 beschriebenen DNA-Sequenzen oder Teilen dieser Sequenzen, beispielsweise mit üblichen Hybridisierungs- verfahren oder der PCR-Technik aus anderen Eukaryonten wie beispielsweise Mikroorganismen wie Hefen, Pilzen, Ciliaten, Pflanzen wie Algen, Moosen oder sonstigen Pflanzen, isolieren. Diese DNA- Sequenzen hybridisieren unter Standardbedingungen mit den genannten Sequenzen. Zur Hybridisierung werden vorteilhaft kurze Oligo- nukleotide, beispielsweise der konservierten oder sonstigen ' Such DNA sequences can be generated with the aid of the nucleic acid sequences according to the invention, for example starting from those in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 . , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: .36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72. SEQ ID NO: 74, SEQ ID NO: , 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: - 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92 , SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ. ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 described DNA sequences or parts of these sequences, for example with conventional hybridization methods or the PCR technique from other eukaryotes such as, for example, microorganisms such as yeasts, fungi, ciliates, plants such as algae, moss or other plants. These DNA sequences hybridize with the sequences mentioned under standard conditions. For the hybridization, short oligonucleotides, for example the conserved or other, are advantageously used .
Bereiche, die über Vergleiche mit anderen verwandten Genen in dem Fachmann bekannter Weise ermittelt werden können, verwendet. Es können aber auch längere Fragmente der erfindungsgemäßen Nukleinsäuren oder die vollständigen Sequenzen für die Hybridisierung verwendet- werden. Je nach der verwendeten Nukleinsäure: Oligo- nukleotid, längeres Fragment oder vollständige Sequenz oder je nachdem welche Nukleinsäureart DNA oder RNA für die Hybridisierung verwendet werden, variieren diese Standardbedingungen. So liegen beispielsweise die Schmelztemperaturen für DNA:DNA- Hybride ca 10°C niedriger' ls die von DNA:RNA-Hybriden gleicher Läng .Areas used which can be determined by comparison with other related genes in a manner known to the person skilled in the art. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization. These standard conditions vary depending on the nucleic acid used: oligonucleotide, longer fragment or complete sequence or depending on the type of nucleic acid DNA or RNA used for the hybridization. Thus, for example, the melting temperatures for DNA: DNA hybrids are approximately 10 ° C lower 'ls those of DNA: RNA hybrids Long.
Unter Standardbedingungen sind beispielsweise je nach Nukleinsäure Temperaturen zwischen 42 und 58°C in einer wässrigen Puffer- lösung mit einer Konzentration zwischen 0,1 bis 5 x SSC (1 X SSC = 0,15 M NaCl, 15 mM Natriumeitrat, pH 7,2) oder zusätzlich in . Gegenwart von 50 % For amid wie beispielsweise 42°C in 5 x SSC, 50 % Formamid zu verstehen. Vorteilhafterweise liegen die Hybridisierungsbedingungen für DNA:DNA-Hybride bei 0,1 x SSC und Temperaturen zwischen etwa 20°C bis 45°C, bevorzugt zwischen etwa 30°C bis 45°C. Für DNA:RNA-Hybride liegen die Hybridisierungsbedingungen vorteilhaft bei.0 , 1 x SSC und Temperaturen zwischen etwa 30°C bis 55°C, bevorzugt zwischen etwa 45°C bis 55°C. Diese angegebenen Temperaturen für die Hybridisierung sind beispielhaft kalkulierte Schmelztemperaturwerte für eine Nukleinsäure mit einer Länge von ca. 100 Nukleotiden und einem G + C-Gehalt von 50 % in Abwesenheit von Formamid. Die experimentellen Bedingungen für die DNA-Hybridisierung sind in einschlägigen Lehrbüchern der Genetik wie beispielsweise Sambrook et al . , "Molecular Cloning" , Cold Spring Harbor Laboratory, 1989, beschrieben und lassen sich nach dem Fachmann bekannten Formeln beispielsweise abhängig von der Länge der Nukleinsäuren, der Art der Hybride oder dem G" + C- Gehalt berechnen. Weitere Informationen zur Hybridisierung kann der Fachmann folgenden Lehrbüchern entnehmen: Ausubel et al . (eds) , 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, Nucleic Acids Hybridisation: A Practical Approach, IRL Press at Oxford -Uni- versity Press, Oxford; Brown (ed) , 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford Univ'ersity Press,_ Oxford.Under standard conditions, for example, depending on the nucleic acid, temperatures are between 42 and 58 ° C. in an aqueous buffer solution with a concentration between 0.1 and 5 × SSC (1 × SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2 ) or additionally in. Understanding the presence of 50% for amide such as 42 ° C in 5 x SSC, 50% formamide. The hybridization conditions for DNA: DNA hybrids are advantageously 0.1 × SSC and temperatures between approximately 20 ° C. to 45 ° C., preferably between approximately 30 ° C. to 45 ° C. For DNA: RNA hybrids, the hybridization conditions are advantageously at 0, 1 x SSC and temperatures between about 30 ° C to 55 ° C, preferably between about 45 ° C to 55 ° C. These specified temperatures for the hybridization are, for example, calculated melting temperature values for a nucleic acid with a length of approx. 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are in relevant textbooks of genetics such as Sambrook et al. , "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G "+ C content. Further information on hybridization can be obtained from Those skilled in the art can read the following textbooks: Ausubel et al. (Eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Harnes and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford -University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University 's press, _ Oxford.
Weiterhin sind unter Derivaten Homologe der Sequenz SEQ ID No: 1,Furthermore, homologs of the sequence SEQ ID No: 1 are among derivatives,
SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9,SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32
SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40 SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50
SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58
SEQ ID NO: 60., SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66
SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74
SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90
SEQ ID NO:- 92, SEQ ID NO: 94, SEQ .ID NO: 96, SEQ ID NO: 98SEQ ID NO: - 92, SEQ ID NO: 94, SEQ .ID NO: 96, SEQ ID NO: 98
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NOir 106 oderSEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NOir 106 or
SEQ ID NO: 108 beispielsweise eukaryontische Homologe, verkürzteSEQ ID NO: 108, for example, shortened eukaryotic homologs
Sequenzen, Einzelstrang-DNA der codierenden und nichtcodierenden DNA-Sequenz oder RNA der codierenden und nichtcodierenden DNA- Sequenz zu verstehen.To understand sequences, single-stranded DNA of the coding and non-coding DNA sequence or RNA of the coding and non-coding DNA sequence.
Außerdem sind unter Homologen der Sequenzen SEQ ID NO: 1,In addition, among homologs of the sequences SEQ ID NO: 1,
SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9,SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:.15, SEQ ID NO: 17,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: .15, SEQ ID NO: 17,
SEQ ID NO: 26, SEQ .ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,SEQ ID NO: 26, SEQ .ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,
SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 oder SEQ ID NO: 40,SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66,
SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,
SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 Derivate wie beispielsweise Varianten aus anderen Organismen beispielsweise anderen Pflanzen zu verstehen.' Diese Varianten können durch ein oder mehrere Nukleotidaustausche, durch Insertion(en) und/oder. Deletion(en) verändert sein, ohne dass aber die Funktionalität bzw. die biologische Aktivität der Varianten beeinträchtigt wird. Vorzugsweise haben sie eine Homologie von mindestens 20 % und eine äquivalente biologische Aktivität.SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 to understand derivatives such as, for example, variants from other organisms, for example other plants. ' These variants can be replaced by one or more nucleotides, by insertion (s) and / or. Deletion (s) must be changed without impairing the functionality or the biological activity of the variants. They preferably have a homology of at least 20% and an equivalent biological activity.
Die im erfindungsgemäßen Verfahren verwendeten Nukleinsäuren, insbesondere SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID No : 5, SEQ ID No: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,The nucleic acids used in the method according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 26, SEQ ID NO: 28,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 26, SEQ ID NO: 28,
SEQ ID NO: 30, SEQ ID NO: 32, SEQ D NO 34, SEQ ID NO 36-,SEQ ID NO: 30, SEQ ID NO: 32, SEQ D NO 34, SEQ ID NO 36-,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO 46,SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO 46,
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO 60, SEQ ID NO 62,SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO 60, SEQ ID NO 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO 68, SEQ ID' NO 70,SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO 68, SEQ ID ' NO 70,
SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NOSEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO
SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO 84, SEQ ID NOSEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO 84, SEQ ID NO
SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO SEQ ID NO: 96, SEQ ID NO: 98, SEQ- ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 1Ö8 und deren Fragmente und Derivate sind deshalb vorteilhaft dafür geeignet um weitere essentielle, neue Gene aus anderen Organismen bevorzugt Pflanzen zu isolieren.SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 1Ö8 and their fragments and derivatives are therefore advantageously suitable for isolating further essential, new genes from other organisms, preferably plants.
Die erfindungsgemäßen Nukleinsäuresequenzen,- insbesondere SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No : 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,The nucleic acid sequences according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID No: 5, SEQ ID No: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30 SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38
SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO: 46, SEQ ID NO 48SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO: 46, SEQ ID NO 48
SEQ ID NO: .50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO 56SEQ ID NO: .50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO 56
-SEQ ID NO: 58, SEQ ID NO 60, SEQ ID NO: 62, SEQ ID NO 64-SEQ ID NO: 58, SEQ ID NO 60, SEQ ID NO: 62, SEQ ID NO 64
SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO: 70, SEQ ID NO 72 SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO: 78, SEQ ID NO 80SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO: 70, SEQ ID NO 72 SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO: 78, SEQ ID NO 80
SEQ ID NO: 8 822,, SSEEQQ IIDD NNOO: 84, SEQ ID NO: 86, SEQ ID NO 88SEQ ID NO: 8 822 ,, SSEEQQ IIDD NNOO: 84, SEQ ID NO: 86, SEQ ID NO 88
SEQ ID NO: 9 900,, SSEEQQ IIDD NNOO: 92, SEQ ID NO: 94, SEQ ID NO 96SEQ ID NO: 9,900 ,, SSEEQQ IIDD NNOO: 92, SEQ ID NO: 94, SEQ ID NO 96
SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 deren durch sie codierte Genprodukte im er- findungsgemäßen Verfahren verwendeten werden, können synthetisch hergestellt oder natürlich gewonnen sein oder eine Mischung aus synthetischen und natürlichen DNA-Bestandteilen enthalten, sowie aus verschiedenen heterologen Genabschnitten verschiedener Organismen bestehen. Im allgemeinen werden synthetische Nukleo- tid-Sequenzen mit Codons erzeugt, die von den entsprechenden Wirtsorganismen beispielsweise Pflanzen bevorzugt werden. Dies 5 führt in der Regel zu einer optimalen Expression der heterologen Gene. Diese von Pflanzen bevorzugten Codons können aus Codons mit der höchsten Proteinhäufigkeit bestimmt werden, die in den meisten interessanten Pflanzenspezies exprimiert werden. Ein Beispiel für Corynebacterium glutamicum ist gegeben in: Wada et al . 10 (1992) Nucleic Acids Res. 20:2111-2118). Die Durchführung solcher Experimente sind mit Hilfe von Standardmethoden durchführbar und sind dem Fachmann auf dem Gebiet bekannt.SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 whose gene products encoded by them are used in the method according to the invention can be synthetically produced or naturally obtained or contain a mixture of synthetic and natural DNA components, and consist of different heterologous gene segments from different organisms. In general, synthetic nucleotide sequences with codons are generated, which are preferred by the corresponding host organisms, for example plants. This generally leads to optimal expression of the heterologous genes. These plant preferred codons can be determined from the highest protein frequency codons expressed in most interesting plant species. An example of Corynebacterium glutamicum is given in: Wada et al. 10 (1992) Nucleic Acids Res. 20: 2111-2118). Such experiments can be carried out using standard methods and are known to the person skilled in the art.
Funktioneil äquivalente Sequenzen, die für die im erfindungs- 15 gemäßen—Verfahren verwendeten Nukleinsäuren codieren, sind solche Derivate der erfindungsgemäßen Sequenzen, welche trotz abweichender Nukleotidsequenz noch- die gewünschten Funktionen, das heißt die biologische Aktivität der Proteine besitzen. Funktionelle Äquivalente umfassen somit natürlich vorkommende Varianten der 20 hierin beschriebenen Sequenzen sowie künstliche, z.B. durch chemische Synthese erhaltene, insbesondere an den Codon-Gebrauch einer Pflanze angepasste, künstliche Nukleotid-Sequenzen.Functionally equivalent sequences which code for the nucleic acids used in the method according to the invention are those derivatives of the sequences according to the invention which, despite a different nucleotide sequence, still have the desired functions, that is to say the biological activity of the proteins. Functional equivalents thus include naturally occurring variants of the 20 sequences described herein as well as artificial, e.g. artificial nucleotide sequences obtained by chemical synthesis, in particular adapted to the codon use of a plant.
Außerdem sind artifizielle DNA-Sequenzen geeignet, solange sie,In addition, artificial DNA sequences are suitable as long as they
25 wie oben beschrieben, zu Produkten führen, die die oben genannten Aktivitäten oder die gewünschte Eigenschaft, beispielsweise die Bindung an einen Rezeptor oder die enzymatische Aktivität vermitteln. Solche artifiziellen DNA-Sequenzen können beispielsweise durch Rückübersetzung mittels Molecular Modelling konstruierter25, as described above, lead to products which mediate the abovementioned activities or the desired property, for example binding to a receptor or the enzymatic activity. Such artificial DNA sequences can be constructed, for example, by back-translation using molecular modeling
30 Proteine oder durch in vitro-Selektion ermittelt werden. Mögliche Techniken zur- in vitro-Evolution von DNA zur Veränderung bzw. Verbesserung der DNA-Sequenzen sind beschrieben bei Patten, P.A. et al., Current Opinion in Biotechnology 8,, 724-733(1997) oder bei Moore,. J.C. et • al . , Journal of Molecular Biology 272,30 proteins or can be determined by in vitro selection. Possible techniques for the in vitro evolution of DNA to change or improve the DNA sequences are described in Patten, PA et al., Current Opinion in Biotechnology 8 ,, 724-733 (1997) or in Moore ,. JC et • al. , Journal of Molecular Biology 272,
35 336-347 ( 1997). Besonders geeignet sind codierende DNA-Sequenzen, die durch Rückübersetzung einer Polypeptidsequenz gemäß der für die Wirtspflanze spezifischen Kodon-Nutzung erhalten- erden. Die spezifische Kodon-Nutzung kann ein mit pflanzengenetischen Methoden vertrauter Fachmann durch Computerauswertungen anderer,35 336-347 (1997). Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable. The specific codon usage can be determined by a specialist familiar with plant genetic methods by computer evaluations of other
40 bekannter Gene der zu transformierenden Pflanze leicht ermitteln.Easily identify 40 known genes of the plant to be transformed.
Für das erfindungsgemäße Verfahren sind vorteilhaft Aminosäuresequenzen zu verstehen, die eine in den Sequenzen SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, 45 SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO 45, SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO: 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO: 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 77, SEQ ID NO: 79, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO 85, SEQ ID NO: 87, SEQ ID NO 89, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO: 9 955,, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellte Aminosäuresequenz oder eine daraus durch Substitution, Inversion, Insertion oder Deletion von einem oder mehreren Aminosäureresten erhältliche Sequenz enthalten, wobei die biologische Aktivität des in SEQ ID NO 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,For the method according to the invention, amino acid sequences which are one in the sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, 45 SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO 45, SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO 59, SEQ ID NO 61, SEQ ID NO : 63, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 69, SEQ ID NO: 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 77, SEQ ID NO: 79, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO 85, SEQ ID NO: 87, SEQ ID NO 89, SEQ ID NO 91, SEQ ID NO 93, SEQ ID NO: 9 955 ,, SEQ ID NO 97, SEQ ID NO 99, SEQ ID NO 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 or an amino acid sequence shown or a sequence obtainable therefrom by substitution, inversion, insertion or deletion of one or more amino acid residues, where the biological activity of the in SEQ ID NO 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO 31, SEQ ID NO: 33,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, 'SEQ ID NO 39, SEQ ID NO: 41,SEQ ID NO: 35, SEQ ID NO: 37, 'SEQ ID NO 39, SEQ ID NO: 41,
SEQ ID NO: 45, SEQ ID NO: SEQ ID NO 49, SEQ ID NOT -51,SEQ ID NO: 45, SEQ ID NO: SEQ ID NO 49, SEQ ID NOT -51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO: 59,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:' 71, SEQ ID NO 73, SEQ ID NO: 75,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: '71, SEQ ID NO 73, SEQ ID NO: 75,
SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO 81, SEQ ID NO: 83,SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO 81, SEQ ID NO: 83,
SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO 89, SEQ ID NO: 91,SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO 89, SEQ ID NO: 91,
SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99,SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99,
SEQ ID NO: 101, SEQ ID NO:; 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Proteins erhalten bleibt bzw. nicht wesentlich reduziert wird. Unter nicht wesentlich reduziert sind alle Proteine zu verstehen, die noch mindestens 10 %, bevorzugt 20 %, besonders bevorzugt 30 %, 50 %, 70 %, 90 % oder mehr, der biologischen Aktivität des Ausgangsproteins aufweisen. Dabei können beispielsweise bestimmte Aminosäuren durch solche mit ähnlichen physikochemisehen Eigenschaften (Raumerfüllung, Basizität, Hydrophobizität etc.) ersetzt werden. Beispielsweise werden Argi- ninreste gegen Lysinreste, Valinreste gegen Isoleucin'reste oder Asparaginsäurereste gegen Glutaminsäurereste ausgetauscht . Es können aber auch ein oder mehrere Aminosäuren in ihrer Reihenfolge vertauscht, hinzugefügt oder entfernt werden, oder es können mehrere dieser Maßnahmen miteinander kombiniert werden.SEQ ID NO: 101, SEQ ID NO :; 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 is retained or is not significantly reduced. Not significantly reduced means all proteins which still have at least 10%, preferably 20%, particularly preferably 30%, 50%, 70%, 90% or more of the biological activity of the starting protein. For example, certain amino acids can be replaced by those with similar physicochemical properties (space filling, basicity, hydrophobicity, etc.). For example, arginine residues are exchanged for lysine residues, valine residues for isoleucine residues or aspartic acid residues for glutamic acid residues. However, one or more amino acids can also be interchanged, added or removed in their order, or several of these measures can be combined with one another.
Unter Derivaten sind auch funktionelle Äquivalente zu verstehen, die insbesondere auch natürliche oder künstliche Mutationen der verwendeten Nukleinsäuresequenzen SEQ ID NO: 1, SEQ ID NO: 3,Derivatives are also to be understood as functional equivalents which in particular also include natural or artificial mutations in the nucleic acid sequences used SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO 9, SEQ ID NO: 11,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO 9, SEQ ID NO: 11,
SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 26SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 26
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO 34 SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO 44SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO 34 SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO 44
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO 60 rf^SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO 60 rf ^
IL^IL ^
SEQ ID NO: 76, SEQ ID NO 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO 94, SEQ ID NO: 96, SEQ ID NO: 98,SEQ ID NO: 76, SEQ ID NO 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO 94, SEQ ID NO: 96, SEQ ID NO: 98,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oderSEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or
SEQ ID NO: 108 dargestellten Sequenz, oderSEQ ID NO: 108 sequence shown, or
b) einer Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes aus den .durch Rückübersetzung der in SEQ ID NO: 2, SEQ ID NO 4, SEQ ID NO: 6, SEQ ID NO: 8, ■ SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,b) a nucleic acid sequence which, owing to the degeneracy of the genetic code from the .by back-translation of in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, ■ SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
'SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,'SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO:- 35, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 33, SEQ ID NO: - 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57,
SEQ.. .ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65,SEQ ... ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65,
SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,
SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,
SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97,SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97,
SEQ ID NO: 99 , SEQ ID NO: 101 , SEQ ID NO : 103,SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103,
SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen ableiten lässt,SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 derived amino acid sequences,
- c) Nukleinsäuresequenz, die ein Derivat oder ein Fragment der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:.26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:' 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,. SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ" ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ' ID NO: 88, . SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96,- c) Nucleic acid sequence which is a derivative or a fragment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: .26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: '56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 . SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ "ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ' ID NO: 88,. SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96,
SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Nukleinsäuresequenzen ist, und mindestens 60 % Homologie auf Nukleinsäureebene aufweisen; oderSEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 is the nucleic acid sequences shown, and have at least 60% homology at the nucleic acid level; or
d) Nukleinsäuresequenz , die für Derivate oder Fragmente der Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59 , SEQ ID NO : 61 ,d) nucleic acid sequence which is suitable for derivatives or fragments of the polypeptides with the in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 , SEQ ID NO : 69 ,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69,
SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75 , SEQ ID NO : 77 ,SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83 , SEQ ID NO : 85 ,SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91 , SEQ ID NO : 93 ,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93,
SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99 , SEQ ID NO : 101 ,SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101,
SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oderSEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or
SEQ ID NO: 109 dargestellten Aminosäuresequenzen codiert, die mindestens 50 % Homologie auf Aminosäureebene aufweisen;SEQ ID NO: 109 encoded amino acid sequences which have at least 50% homology at the amino acid level;
e) Nukleinsäuresequenz, die für ein Fragment oder ein Epitope eines Polypeptides codiert, das spezifisch an einem Antikörper bindet, wobei der Antikörper spezifisch an ein Polypeptid bindet, das der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,e) Nucleic acid sequence which codes for a fragment or an epitope of a polypeptide which specifically binds to an antibody, the antibody specifically binding to a polypeptide which the one in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO: 13, SEQ. ID NO: 15, SEQ ID NO: 17, SEQ I NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:.58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO.: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78,. SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder ' SEQ ID NO: 108 dargestellten Sequenz codiert wird;SEQ ID NO: 13, SEQ. ID NO: 15, SEQ ID NO: 17, SEQ I NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO : 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56 , SEQ ID NO: .58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO .: 70, SEQ ID NO: 72 , SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78 ,. SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or 'SEQ ID NO: 108;
f) Nukleinsäuresequenz, die ein Fragment einer in a) dar- gestellten Nukleinsäure codiert und das eine m6A-Methyl- transferase-Aktivität, eine DNA-bindende-Aktivität oder "DNA- repair"-Aktivität, z.B. wie bei RAD 54, eine Thioredoxin- Aktivität, eine VAV2-Aktivität, eine Fructokinase-Aktivität, eine Zinkfingerprotein-Aktivität, eine LYTB-Aktivität, eine Crepopin-Aktivität, eine Leucin-Protein-Aktivität, eine DNAJ- Aktivität, ein CRSl-Aktivität, eine Alanyl-tRNA-Synthetase- Aktivität, eine OEP86-Aktivität, eine FMRF-Amid-Propeptid- Isolog-Aktivität, eine 26S Proteosom subunit S5B-Aktivität oder eine Geranylgeranylpyrophosphatsynthase-Aktivität, eine Crepopin-Aktivität, eine Leucin-Protein-Aktivität, eine DNAJ- Aktivität, ein CRSl-Aktivität, eine Alanyl-tRNA-Synthetase- Aktivität, eine OEP86-Aktivität, eine FMRF-Amid-Propeptid- Isolog-Aktivität, eine 26S Proteosom subunit S5B-Aktivität, eine Geranylgeranylpyrophosphatsynthase-Aktivität, eine Cecropin-Familiensignatur aufweißt, ftsH Cloroplast-Protease- Aktivität hat, eine AIMl-Aktivität, eine UDP-glucuronyltrans- ferase-Aktivität, eine FPFl-Aktivität, eine SHI-ähnliche Zinkfingerprotein-Aktivität hat, eine Crpl-Aktivität , eine CRSl-Aktivität, eine translation releasing factor RF-1-Aktivität, eine Farnesyltransferase Untereinheit A-Aktivität, eine ATP-abhängige Kupfertransporter RANl-Aktivität hat, eine Syntaxin oder Syntaxin-ähnliche Proteinaktivität, eine Inositol-Polyphosphat-5 ' -Phosphatase- Aktivität, eine UDP-N-Acetylmuramoylalanyl-D-glutamat- 2, 6-Diaminopimelat-ligaseaktivität (murE) , eine ß-Gluco- sidase-Aktivität, eine Hydroxymethylglutaryl-CoA-Reductase, eine GDSL-Motif-Lipase/Hydroxylase-ähnliche Proteinaktivität, eine Zellulosesynthase ähnliche Proteinaktivität, eine tRNA Glutaminsynthetase, eine Exonuklease-ähnliche Proteinaktivität, eine sec-unabhängige Translocase-Protein TATC- Aktivität oder eine Selenium-Bindungsprotein ähnliche Proteinaktivität; und/oderf) Nucleic acid sequence which encodes a fragment of a nucleic acid shown in a) and which has an m6A methyl transferase activity, a DNA-binding activity or "DNA repair" activity, for example as in RAD 54, a thioredoxin Activity, a VAV2 activity, a fructokinase activity, a zinc finger protein activity, a LYTB activity, a crepopin activity, a leucine protein activity, a DNAJ activity, a CRSl activity, an alanyl tRNA activity Synthetase activity, an OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity or a geranylgeranyl pyrophosphate synthase activity, a crepopin activity, a leucine protein activity, a DNAJ activity, a CRSl activity, an alanyl tRNA synthetase activity, an OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, a cecropin family sig Naturally, ftsH has chloroplast protease activity, AIMl activity, UDP-glucuronyltransferase activity, FPFl activity, SHI-like activity Zinc finger protein activity, Crpl activity, CRSl activity, translation releasing factor RF-1 activity, farnesyltransferase subunit A activity, ATP-dependent copper transporter RANl activity, syntaxin or syntaxin-like protein activity, an inositol polyphosphate 5 'phosphatase activity, a UDP-N-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase activity (murE), a β-glucosidase activity, a hydroxymethylglutaryl-CoA reductase, a GDSL-Motif lipase / hydroxylase-like protein activity, a cellulose synthase-like protein activity, a tRNA glutamine synthetase, an exonuclease-like protein activity, a sec-independent Translocase protein TATC activity or a selenium binding protein-like protein activity; and or
g) Nukleinsäuresequenz, die für Derivate der Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4,- SEQ ID NO: 6, SEQ ID NO: 8,g) nucleic acid sequence which is suitable for derivatives of the polypeptides with the in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16,
SEQ ID NO 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ • ID NO 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO 59, SEQ ID NO: 61, SEQ ID NO 63, SEQ ID NO 65,SEQ ID NO 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ • ID NO 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO 59, SEQ ID NO: 61, SEQ ID NO 63, SEQ ID NO 65,
SEQ ID NO 67", SEQ ID NO: 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO: 77, SEQ ID NO 79, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO: 85, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO 91, SEQ ID NO: 93, SEQ ID NO 95, SEQ ID NO 97,SEQ ID NO 67 " , SEQ ID NO: 69, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO: 77, SEQ ID NO 79, SEQ ID NO 81, SEQ ID NO 83, SEQ ID NO: 85, SEQ ID NO 87, SEQ ID NO 89, SEQ ID NO 91, SEQ ID NO: 93, SEQ ID NO 95, SEQ ID NO 97,
SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen codiert, die mindestens 20 % Homologie auf Aminosäureebene aufweist und eine äquivalente biologische Aktivität besitzt;SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 encoded amino acid sequences, which has at least 20% homology at the amino acid level and an equivalent possesses biological activity;
wobei die Nukleinsäuresequenz mit einem oder mehreren Regulationssignalen verknüpft ist. Die vorhergenannten Begriffe haben die oben genannte Bedeutung.wherein the nucleic acid sequence is linked to one or more regulatory signals. The aforementioned terms have the meaning given above.
Unter dem erfindungsgemäßen Nukleinsäurekonstrukt sind die erfindungsgemäßen Nukleinsäuren, z.B. die in SEQ ID NO: 1,Under the nucleic acid construct according to the invention, the nucleic acids according to the invention, e.g. those in SEQ ID NO: 1,
SEQ ID NO 3, SEQ ID NO: 5, . SEQ ID NO: 7 , SEQ ID NO: 9SEQ ID NO 3, SEQ ID NO: 5,. SEQ ID NO: 7, SEQ ID NO: 9
SEQ ID NO 11, SEQ ID NO: 13 SEQ ID NO: 15, SEQ ID NO 17,SEQ ID NO 11, SEQ ID NO: 13 SEQ ID NO: 15, SEQ ID NO 17,
SEQ ID NO 26, SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO 32,SEQ ID NO 26, SEQ ID NO: 28 SEQ ID NO: 30, SEQ ID NO 32,
SEQ ID NO 34, SEQ ID NO: 36 SEQ ID NO: 38, SEQ ID NO 40, SEQ ID NO 44, SEQ ID NO: 46 SEQ ID NO: 48, SEQ ID NO 50,SEQ ID NO 34, SEQ ID NO: 36 SEQ ID NO: 38, SEQ ID NO 40, SEQ ID NO 44, SEQ ID NO: 46 SEQ ID NO: 48, SEQ ID NO 50,
SEQ ID NO 52, SEQ ID NO: 54 SEQ ID NO: 56, SEQ ID NO 58,SEQ ID NO 52, SEQ ID NO: 54 SEQ ID NO: 56, SEQ ID NO 58,
SEQ ID NO 60, SEQ ID NO: 62 SEQ ID NO: 64, SEQ ID NO 66, SEQ ID NO": 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oderSEQ ID NO 60, SEQ ID NO: 62 SEQ ID NO: 64, SEQ ID NO 66, SEQ ID NO " : 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO : 100, SEQ ID NO: 102, SEQ ID NO: 106 or
SEQ ID NO: 108 genannten Sequenzen, die sich als Ergebnis des genetischen Codes und/oder deren funktionellen oder nicht funktioneilen Derivate zu verstehen, die mit einem oder mehreren Regulationssignalen vorteilhafterweise zur Regulation, ins- besondere Erhöhung, der Genexpression funktioneil verknüpft wurden und welche die Expression der codierenden Sequenz in der Wirtszelle steuern. Diese regulatorischen Sequenzen sollen . die gezielte Expression der Gene bzw. der Proteine ermöglichen. Dies kann beispielsweise je nach Wirtsorganismus bedeuten, dass das Gen"~erst nach Induktion exprimiert und/oder überexpri iert wird, oder dass es konstitutiv exprimiert und/oder überexprimiert wird. Beispielsweise handelt es sich bei diesen regulatorischen Sequenzen um Sequenzen an die Induktoren oder Repressoren binden • und so die Expression der Nukleinsäure regulieren. Zusätzlich zu diesen neuen Regulationssequenzen oder , anstelle dieserSEQ ID NO: 108 sequences which are to be understood as the result of the genetic code and / or their functional or non-functional derivatives which have been functionally linked to one or more regulatory signals advantageously for regulating, in particular increasing, the gene expression and which Control expression of the coding sequence in the host cell. These regulatory sequences are said to. enable the targeted expression of the genes or the proteins. This may for example each represent the host organism, that the gene "~ expressed only after induction and / or ated überexpri, or that it is constitutively expressed and / or overexpressed. By way of example, these regulatory sequences are sequences to which inductors or repressors bind • and thus regulate the expression of the nucleic acid in addition to these new regulatory sequences or instead of these
Sequenzen kann die natürliche Regulation dieser Sequenzen vor den eigentlichen Strukturgenen noch vorhanden sein und gegebenenfalls genetisch verändert worden sein, so dass die natürliche Regulation ausgeschaltet und die Expression der Gene erhöht wurde. Das erfindungsgemäße Nukleinsäurekonstrukt kann auch vorteilhaft nur aus der natürlichen gentechnologisch veränderten Regulationsregion am 5'- und/oder 3 '-Ende bestehen. Das Gen- konstrukt kann aber auch einfacher aufgebaut sein, das heißt es wurden keine zusätzlichen RegulationsSignale vor die Nuklein- säuresequenz oder dessen Derivate inseriert und der natürliche Promotor mit seiner Regulation wurde nicht entfernt. Stattdessen wurde die natürliche. Regulationssequenz so mutiert, dass keine Regulation mehr erfolgt und/oder die Genexpression gesteigert wird-. Diese veränderten Promotoren können in Form von Teil- Sequenzen (= Promotor mit Teilen der erfindungsgemäßen Nuklein- säuresequenzen) auch allein vor das natürliche Gen zur Steigerung der Aktivität gebracht werden. Das Genkonstrukt kann außerdem vorteilhafterweise auch eine oder mehrere sogenannte "enhancer Sequenzen" funktionell verknüpft mit dem Promotor enthalten, die eine erhöhte Expression der Nukleinsäuresequenz ermöglichen. Auch am 3 '-Ende der DNA-Sequenzen können zusätzliche vorteilhafte Sequenzen inseriert werden wie weitere regulatorische Elemente oder Terminatoren. Die im erfindungsgemäßen Verfahren verwendeten Nukleinsäuresequenzen können in einer oder mehreren Kopien in der Expressionskassette (= Genkonstrukt) enthalten sein. Die regulatorischen Sequenzen bzw. Faktoren können dabei wie oben beschrieben vorzugsweise die Genexpression der eingeführten Gene positiv beeinflussen und dadurch erhöhen. So kann eine Verstärkung der regulatorischen Elemente vorteilhafterweise auf der Transkriptionsebene erfolgen, indem starke Transkriptionssignale wie Promotoren und/oder "Enhanσer" verwendet werden. Daneben ist aber auch eine Verstärkung der Translation möglich, indem beispielsweise die Stabilität der mRNA verbessert wird. In einer anderen vorteilhaften Ausführungsform kann die Expression jedoch auch gezielt reduziert oder blockiert werden.Sequences, the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation was switched off and the expression of the genes increased. The nucleic acid construct according to the invention can also advantageously consist only of the natural, genetically modified regulatory region at the 5 'and / or 3' end. The gene construct can, however, also have a simpler structure, ie no additional regulatory signals have been inserted in front of the nucleic acid sequence or its derivatives and the natural promoter with its regulation has not been removed. Instead, the natural. Regulation sequence mutated so that regulation no longer takes place and / or gene expression is increased. These modified promoters can also be brought in the form of partial sequences (= promoter with parts of the nucleic acid sequences according to the invention) in front of the natural gene to increase the activity. The gene construct can also advantageously contain one or more so-called "enhancer sequences" functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences. The nucleic acid sequences used in the method according to the invention can be contained in one or more copies in the expression cassette (= gene construct). As described above, the regulatory sequences or factors can preferably have a positive influence on the gene expression of the introduced genes and thereby increase it. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". In addition, an increase in translation is also possible, for example, by improving the stability of the mRNA. In another advantageous embodiment, however, the expression can also be specifically reduced or blocked.
Als Promotoren in der Expressionskassette sind grundsätzlich alle Promotoren geeignet, die die Expression von Fremdgenen in Organismen vorteilhaft in Pflanzen oder Pilzen steuern können. Vorzugsweise verwendet man insbesondere ein pflanzliche Promotoren oder Promotoren, die aus einem Pflanzenvirus entstammen. Vorteilhafte Regulationssequenzen für das erfindungsgemäße Verfahren sind beispielsweise in Promotoren wie cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacis-- T7-, T5-, T3-, gal-, trc-, ara-, SP6-, λ-pR- oder im λ-Pjy-Promotor enthalten, die vorteilhafterweise in gram-negativen Bakterien Anwendung finden. Weitere vorteilhafte Regulationssequenzen sind beispielsweise in den gram-positiven Promotoren amy und SP02, in den Hefe- oder Pilzpromotoren ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28 , ADH oder in den Pflanzenpro otoren wie CaMV/35S [Franck et al., Cell 21(1980) 285-294], SSU, OCS, lib4, STLS1, B33, nos (= Nopalin Synthase Promotor) oder im Ubiquitin-Pro otor enthalten-. Die Expressionskassette kann auch einen chemisch induzierbaren Promotor enthalten, durch den die Expression der Nukleinsäure- Sequenzen im erfindungsgemäßen Nukleinsäurekonstrukt in denIn principle, all promoters which can advantageously control the expression of foreign genes in organisms in plants or fungi are suitable as promoters in the expression cassette. In particular, a plant promoter or promoters derived from a plant virus are preferably used. Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacis - T7, T5, T3 , gal, trc, ara, SP6, λ-p R - or contained in the λ-Pjy promoter, which are advantageously used in gram-negative bacteria. Further advantageous regulatory sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters such as CaMV / 35S [ Franck et al., Cell 21 (1980) 285-294], SSU, OCS, lib4, STLS1, B33, nos (= nopalin synthase promoter) or contained in the ubiquitin promoter. The expression cassette can also contain a chemically inducible promoter through which the expression of the nucleic acid sequences in the nucleic acid construct according to the invention in the
Organismen vorteilhaft in den Pflanzen zu einem bestimmten Zeitpunkt gesteuert werden kann. Derartige vorteilhafte Pflanzenpromotoren sind beispielsweise der PRPl-Promotor [Ward et al., Plant. Mol. Biol. 22(1993), 361-366], ein durch Benzensulfonamid- induzierbarer (EP 388186) , ein durch Tetrazyklin-induzierbarer (Gatz et al., (1992) Plant J. 2,397-404), ein durch Salizylsäure induzierbarer Promotor (WO 95/19443), ein durch Äbscisinsäure- induzierbarer (EP 335528) bzw. ein durch Ethanol- oder Cyclo- hexanon-induzierbarer (WO 93/21334) Promotor. Weitere Pflanzen- promotoren sind beispielsweise der Promotor der cytosolischen FBPase aus Kartoffel, der ST-LSI Promotor aus Kartoffel (Stockhaus et al., EMBO J. 8 (1989) 2445-245), der Promotor der Phosphoribosylpyrophosphat A idotransferase aus Glycine ax (siehe auch Genbank Accession Nummer U87999) oder ein Nodien- spezifischen Promotor wie in EP 249676 können vorteilhaft verwandt werden. In der Expressionskassette (= Genkonstrukt, Nukleinsäurekonstrukt) können wie oben beschrieben noch weitere Gene, die - in die Organismen eingebracht werden sollen, enthalten sein. Diese Gene können unter getrennter Regulation oder unter der gleichen Regulationsregion wie die im Verfahren verwendeten Nukleinsäuresequenzen liegen. Bei diesen Genen handelt es sich beispielsweise um Biosynthesegene des Stoffwechsels wie der Fettsäure-, der Aminosäure- oder der Vitaminbiosynthese oder Regulationsgene um nur einige zu nennen.Organisms can be advantageously controlled in the plants at any given time. Such advantageous plant promoters are, for example, the PRPl promoter [Ward et al., Plant. Mol. Biol. 22 (1993), 361-366], one that is inducible by benzenesulfonamide (EP 388186), one that is inducible by tetracycline (Gatz et al., (1992) Plant J. 2,397-404), one that is inducible by salicylic acid Promoter (WO 95/19443), a promoter inducible by abscisic acid (EP 335528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334). Further plant promoters are, for example, the promoter of the cytosolic FBPase from potato, the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445-245), the promoter of the phosphoribosyl pyrophosphate A idotransferase from glycine ax (see Genbank Accession number U87999) or a node-specific promoter as in EP 249676 can also be used advantageously. As described above, the expression cassette (= gene construct, nucleic acid construct) can also contain further genes which are to be introduced into the organisms. These genes can be under separate regulation or under the same regulatory region as the nucleic acid sequences used in the method. These genes are, for example, biosynthetic genes of metabolism such as fatty acid, amino acid or vitamin biosynthesis or regulatory genes to name but a few.
Prinzipiell können alle natürlichen Promotoren mit ihren Regulationssequenzen wie die oben genannten für die erfindungsgemäße Expressionskassette und das erfindungsgemäße Verfahren, wie unten beschrieben, verwendet werden. Darüberhinaus können auch synthe- tische Promotoren vorteilhaft verwendet werden.In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the expression cassette according to the invention and the method according to the invention, as described below. In addition, synthetic promoters can also be used advantageously.
Bei der Präparation einer Expressionskassette können verschiedene DNA-Fragmente manipuliert werden, um eine Nukleotid-Se uenz . zu erhalten, die zweckmäßigerweise in der korrekten Richtung liest und die mit einem korrekten Leseraster ausgestattet ist, Für die Verbindung der DNA-Fragmente (= erfindungsgemäße Nukleinsäuren) miteinander können an die Fragmente Adaptoren oder Linker angesetzt werden.When preparing an expression cassette, various DNA fragments can be manipulated to produce a nucleotide sequence. to obtain, which reads expediently in the correct direction and which is equipped with a correct reading frame. To connect the DNA fragments (= nucleic acids according to the invention) to one another, adapters or linkers can be attached to the fragments.
Zweckmäßigerweise können die Promotor- und die Terminator- Regionen in Transkriptionsrichtung mit einem Linker oder Polylinker, der eine oder mehrere Restriktionsstellen für die Insertion dieser Sequenz enthält, versehen werden. In der Regel hat der Linker 1 bis 10, meistens 1 bis 8, vorzugsweise 2 bis 6 Restriktionsstellen. Im allgemeinen hat der Linker innerhalb der regulatorischen Bereiche eine Größe von weniger als 100 bp, häufig weniger als 60 bp, mindestens jedoch 5 bp. Der Promotor • kann sowohl nativ bzw. homolog als auch fremdartig bzw. heterolog zum Wirtsorganismus beispielsweise zur Wirtspflanze sein. Die Expressionskässette beinhaltet in der 5 '-3 '-Transkriptionsrichtung den Promotor, eine DNA-Sequenz, die für die im erfindungsgemaßen Verfahren verwendeten Proteine codiert und eine Region für die transkriptionale Termination. Verschiedene Terminationsbereiche können vorteilhaft gegeneinander aus- getauscht werden.The promoter and terminator regions can expediently be provided in the transcription direction with a linker or polylinker which contains one or more restriction sites for the insertion of this sequence. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. In general, the linker has a size of less than 100 bp, often less than 60 bp, but at least 5 bp within the regulatory ranges. The promoter • can be both native or homologous as well as foreign or heterologous to the host organism, for example to the host plant. The expression cassette contains in the 5 '-3' transcription direction the promoter, a DNA sequence which codes for the proteins used in the method according to the invention and a region for the transcriptional termination. Different termination areas can advantageously be interchanged.
Ferner können Manipulationen, die passende Restriktionsschnittstellen bereitstellen oder die überflüssige DNA oder Restriktionsschnittstellen entfernen, eingesetzt werden. Wo Insertionen, Deletionen oder Substitutionen wie z.B.' Transitionen und Transversionen in Frage kommen, können in vitro-Mutagenese, -primerrepair-, Restriktion oder Ligation verwendet werden. Bei geeigneten Manipulationen, wie z.B. Restriktion, -chewing-back- oder Auffüllen von Überhängen für -bluntends-, können komplementäre Enden der Fragmente für die Ligation zur Verfügung gestellt werden.Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as ' transitions and transversions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be used. at suitable manipulations, such as restriction, -chewing-back- or filling of overhangs for -bluntends-, complementary ends of the fragments can be made available for the ligation.
Von Bedeutung für eine vorteilhafte hohe Expression kann u.a. das Anhängen des spezifischen ER-Retentionssignals SEKDEL sein (Schouten, A. et al- , Plant Mol. Biol. 30 (1996), 781-792), die durchschnittliche Expressionshöhe wird damit verdreifacht bis vervierfacht. Es können auch andere Retentionssignale, die natürlicherweise bei im ER lokalisierten pflanzlichen und tierischen Proteinen vorkommen, für den Aufbau der Kassette eingesetzt werden.Of importance for an advantageous high expression can i.a. the attachment of the specific ER retention signal SEKDEL (Schouten, A. et al-, Plant Mol. Biol. 30 (1996), 781-792), the average expression level is tripled to quadrupled. Other retention signals, which occur naturally in plant and animal proteins located in the ER, can also be used to construct the cassette.
Bevorzugte Polyadenylierungssignale sind pflanzliche Poly- adenylierungssignale, vorzugsweise solche, die im wesentlichen T-DNA-Polyadenylierungssignale aus Agrobacterium tumefaciens , insbesondere des Gens 3 der T-DNA (Octopin Synthase) des Ti- Plasmids pTiACH5 entsprechen (Gielen et al., EMBO J. 3 (1984), 835 ff) oder entsprechende funktioneile Äquivalente.Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984), 835 ff) or corresponding functional equivalents.
Die Herstellung einer Expressionskassette erfolgt durch Fusion eines geeigneten Promotors mit einer geeigneten Nukleinsäuresequenz sowie einem Polyadenylierungssignal nach gängigen Rekombinations- und Klonierungstechniken, wie sie beispielsweise in T. Maniatis, E.F. Fritsch und J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) sowie in T.J. Silhavy, M.L. Berman und L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) und in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987) beschrieben werden.An expression cassette is produced by fusing a suitable promoter with a suitable nucleic acid sequence and a polyadenylation signal using common recombination and cloning techniques, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience (1987).
Bei der Präparation einer Expressionskassette können verschiedene DNA-Fragmente manipuliert werden, •um eine Nukleotid-Se uenz zu - erhalten, die zweckmäßigerweise in der korrekten Richtung liest urid die mit einem korrekten Leseraster ausgestattet ist. Für die Verbindung der DNA-Fragmente miteinander können an die, Fragmente Adaptoren oder Linker angesetzt werden.When preparing an expression cassette, various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and is equipped with a correct reading frame. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments.
Die im erfindungsgemäßen Verfahren verwendeten Nukleinsäurese- quenzen beinhalten alle Sequenzmerkmale, die notwendig sind, um eine für den Ort der biologischen Wirkung bzw. Aktivität korrekte Lokalisation zu erreichen. Daher sind keine weiteren Targeting- Sequenzen per se notwendig. Allerdings kann eine solche Lokalisation wünschenswert und vorteilhaft sein und daher künstlich verändert oder verstärkt werden, so dass auch solche Fusions- konstrukte eine bevorzugte vorteilhafte Ausführungsform der Erfindung sind.The nucleic acid sequences used in the method according to the invention contain all the sequence features which are necessary in order to achieve a localization which is correct for the location of the biological action or activity. Therefore no further targeting sequences per se are necessary. However, such a localization can be desirable and advantageous and can therefore be artificially changed or strengthened so that such fusion constructs are a preferred advantageous embodiment of the invention.
Vorteilhaft sind dafür beispielsweise Sequenzen, die ein Targeting in Plastiden gewährleisten. Unter, bestimmten Umständen kann auch ein Targeting in andere Kompartimente (referiert: Kermode, Grit. Rev. Plant Sei. 15, 4 (1996), 285-423) z.B. in in die Vakuole, in das Mitochondrium, in das Endoplas atische Retikulum (ER) , Peroxisomen, Lipidkörper oder durch ein Fehlen entsprechender operativer Sequenzen ein Verbleib im Kompartiment des Entstehens, dem Zytosol, wünschenswert sein.For example, sequences that ensure targeting in plastids are advantageous. Under certain circumstances, targeting in other compartments (ref .: Kermode, Grit. Rev. Plant Sei. 15, 4 (1996), 285-423) e.g. in the vacuole, in the mitochondrion, in the endoplasmic reticulum (ER), peroxisomes, lipid body or, due to the lack of corresponding operative sequences, it is desirable to remain in the compartment of formation, the cytosol.
Vorteilhafterweise werden die erfindungsgemäßen Nukleinsäure- sequenzen zusammen mit mindestens einem Reportergen in eine Expressionskassette kloniert, die in den Organismus über einen Vektor oder direkt in das Genom eingebracht wird. Dieses Reportergen sollte eine leichte Detektierbarkeit über einen Wachstums-, Fluoreszenz-, Chemo-, Biolumineszenz- oder Resistenz- assay oder über eine photometrische Messung ermöglichen. Bei- spielhaft seien als Reportergene Antibiotika-oder Herbizidresistenzgene, Hydrolasegene, Fluoreszenzproteingene, Biolumineszenzgene, Zucker- oder Nukleotidstoffwechselgene oder Biosynthesegene wie das Ura3-Gen, das Ilv2-Gen, das Luciferase- gen, 'das ß-Galactosidasegen, das gfp-Gen, das 2-Desoxyglucose- 6-phosphat-Phosphatasegen, das ß-Glucuronidase-Gen, ß-Lactamase- gen, das Neomycinphosphσtransferasegen, das Hygromycinphospho- transferasegen oder das BASTA (= Gluphosinatresistenz) -Gen genannt. Diese Gene ermöglichen eine leichte Messbarkeit und Quantifizierbarkeit der Transcriptionsaktivität und damit der Expression der Gene. Damit lassen sich Genomstellen identifizieren, die eine unterschiedliche- Produktivität zeigen.The nucleic acid sequences according to the invention are advantageously cloned together with at least one reporter gene into an expression cassette which is introduced into the organism via a vector or directly into the genome. This reporter gene should enable easy detection via a growth, fluorescence, chemo-, bioluminescence or resistance assay or via a photometric measurement. Examples are gen as reporter genes antibiotic or herbicide resistance genes, hydrolase, fluorescence protein genes, bioluminescence, sugar or nucleotide or biosynthesis genes such as the Ura3 gene Ilv2 gene luciferase way of example, 'the ß-galactosidase gene, the gfp gene, called the 2-deoxyglucose-6-phosphate-phosphatase gene, the β-glucuronidase gene, β-lactamase gene, the neomycin phosphotransferase gene, the hygromycin phosphotransferase gene or the BASTA (= gluphosinate resistance) gene. These genes enable the transcription activity and thus the expression of the genes to be measured and quantified easily. This enables genome sites to be identified which show different productivity.
Gemäß einer bevorzugten Ausführungsform umfasst eine".Expressions- kassette stromaufwärts, d.h. am 5 '-Ende der codierenden Sequenz, einen Promotor und stromabwärts, d.h. am 3 '-Ende, ein Poly- adenylierungssignal und gegebenenfalls weitere regulatorische Elemente, welche mit der dazwischenliegenden codierenden Sequenz für die im erfindungsgemäßen Verfahren verwendeten Proteinen operativ' verknüpft sind. Unter einer operativen Verknüpfung ver- steht man die sequenzielle Anordnung von Promotor, codierender Sequenz , Terminator und ggf . weiterer regulativer Elemente derart, dass jedes der regulativen Elemente seine Funktion bei der Expression der codierenden Sequenz bestimmungsgemäß erfüllen kann. Die zur operativen Verknüpfung bevorzugten Sequenzen sind Targeting-Sequenzen zur Gewährleistung der subzellulären Lokalisation in Plastiden. Aber auch Targeting-Sequenzen zur Gewährleistung der εubzellulären Lokalisation im Mitochondrium, im Endoplasmatischen Retikulum (= ER) , im Zellkern, in Öl- körperchen oder anderen Kompartimenten sind bei Bedarf einsetzbar sowie Translationsverstärker wie die 5 ' -Führungssequenz aus dem Tabak-Mosaik-Virus (Gallie et al . , Nucl. Acids Res . 15 (1987), 8693-8711).According to a preferred embodiment, an expression cassette comprises upstream, ie at the 5 'end of the coding sequence, a promoter and downstream, ie at the 3' end, a polyadenylation signal and optionally further regulatory elements which coincide with the coding in between sequence are linked to the used in the method of this invention operatively proteins'. comparable operative linkage is to the sequential arrangement of promoter, encoding sequence, terminator and, if necessary, further regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the The sequences preferred for operative linking are targeting sequences to ensure subcellular localization in plastids, but also targeting sequences to ensure subcellular localization in the mitochondrion, in the endoplasmic reticulum (= ER), in the cell nucleus, in oil corpuscles or other compartments can be used if necessary, and translation enhancers such as the 5 'guiding sequence from the tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 ( 1987), 8693-8711).
Eine Expressionskassette kann beispielsweise einen konstitutiven Promotor beispielsweise den 35S-Promotor, das zu exprimierende Gen und das ER-Retentionssignal enthalten. Als ER-Retentions- signal wird bevorzugt die Aminosäuresequenz KDEL (Lysin, Asparaginsäure, Glutaminsäure, Leucin) verwendet.An expression cassette can contain, for example, a constitutive promoter, for example the 35S promoter, the gene to be expressed and the ER retention signal. The amino acid sequence KDEL (lysine, aspartic acid, glutamic acid, leucine) is preferably used as the ER retention signal.
Die Expressionskassette wird zur Expression in einem prokaryon- tischen oder eukaryontisehen WirtsOrganismus beispielsweise einem Mikroorganismus wie einem Pilz oder einer Pflanze vorteilhafterweise in einen Vektor wie beispielsweise einem Plasmid, einem Phagen oder sonstiger DNA inseriert, der eine optimale Expression der Gene im Wirtsorganismus ermöglicht. Geeignete Plasmide sind beispielsweise in E. coli pLG338, pACYC184, pBR-Serie wie z.B. pBR322, pUC-Serie wie pUC18 oder pUC19, Mll3mp-Serie, pKC30, pRep4, pHSl, pHS2 , pPLc236, pMBL24, pLG200, pUR290, pIN-III113-Bl, λgtll oder pBdCI, in Streptomyces pIJlOl, pIJ364, pIJ702 oder pIJ361, in Bacillus pUBllO, pCl94 oder pBD214, in Corynebacterium pSA77 oder pAJ667, in Pilzen pALSl, pIL2 oder pBBH6, weitere vorteilhafte Pilzvektoren werden von Romanos, M.A. et al.,For expression in a prokaryotic or eukaryotic host organism, for example a microorganism such as a fungus or a plant, the expression cassette is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host organism. Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR series such as pBR322, pUC series such as pUC18 or pUC19, Mll3mp series, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113 -Bl, λgtll or pBdCI, in Streptomyces pIJlOl, pIJ364, pIJ702 or pIJ361, in Bacillus pUBllO, pCl94 or pBD214, in Corynebacterium pSA77 or pAJ667, in mushrooms pALSl, pIL2 or pBzBH6, others are advantageous by al .,
[(1992) "Foreign gene expression in yeast: a review" , Yeast 8: 423-488] und von van den Hondel, C.A.M.J.J. et al . [(1991) "Heterologous gene expression in filamentous fungi] sowie in More Gene Manipulations in Fungi [J.W. Bennet & L.L. Lasure, eds., p. 396-428: Academic Press: San Diego] und in "Gene transfer Systems and vector development for filamentous fungi" [van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. l-2'8, Cambridge University Press: Cambridge] beschrieben. Vorteilhafte Hefe- promotoren sind beispielsweise 2 μM, pAG-1-, YEp6, YEpl3 oder pEM- BLYe23. Beispiele für Algen- oder Pflanzenpromotoren sind pLGV23, pGHlac+, pBINl9, pAK2004, pVKH oder pDH51 (siehe Schmidt, R. and Willmitzer, L., 1988). Die oben genannten Vektoren oder Derivate der vorstehend genannten Vektoren stellen eine kleine Auswahl der möglichen Plasmide dar. Weitere Plasmide sind dem Fachmann wohl bekannt und können beispielsweise aus dem Buch Cloning Vectors (Eds. Pouwels P. H. et al . Elsevier, Amsterdam-New York-Oxford, 1985 , ISBN 0 444 904018) entnommen werden. Geeignete pflanzliche Vektoren werden unter anderem in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Kap. 6/7, S.71-119 be- schrieben. Vorteilhafte Vektoren sind sog. shuttle-Vektoren oder binäre Vektoren, die in E. coli und Agrobacterium replizieren.[(1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488] and by van den Hondel, CAMJJ et al. [(1991) "Heterologous gene expression in filamentous fungi] and in More Gene Manipulations in Fungi [JW Bennet & LL Lasure, eds., P. 396-428: Academic Press: San Diego] and in" Gene transfer Systems and vector development for filamentous fungi "[van den Hondel, CAMJJ & Punt, PJ (1991) in: Applied Molecular Genetics of Fungi, Peberdy, JF et al., eds., p. l-2'8, Cambridge University Press: Cambridge] Advantageous yeast promoters are, for example, 2 μM, pAG-1, YEp6, YEpl3 or pEM-BLYe23. Examples of algae or plant promoters are pLGV23, pGHlac + , pBINl9, pAK2004, pVKH or pDH51 (see Schmidt, R. and Willmitzer , L., 1988) The above-mentioned vectors or derivatives of the above-mentioned vectors represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier , Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018) Suitable plant vectors are described, inter alia, in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, p.71-119 wrote. Advantageous vectors are so-called shuttle vectors or binary vectors which replicate in E. coli and Agrobacterium.
Unter Vektoren sind außer Plasmide auch alle anderen dem Fach- mann bekannten Vektoren wie beispielsweise Phagen, Viren wie SV40, CMV, Baculovirus, Adenovirus, Transposons, IS-Elemente, Phas ide, Phagemide, Cosmide, lineare oder zirkuläre DNA zu verstehen. Diese Vektoren können autonom im Wirtsorganismus repliziert oder chromosomal repliziert werden bevorzugt ist eine chromosomale Replikation. Umfasst sind funktioneile und nicht-funktionelle Vektoren.In addition to plasmids, vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus, adenovirus, transposons, IS elements, phas ide, phagemids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally. Chromosomal replication is preferred. Functional and non-functional vectors are included.
In einer weiteren Ausgestaltungsform des Vektors kann die erfindungsgemäße Nukleinsäurekonstrukt auch vorteilhafterweise in Form einer linearen DNA in die Organismen eingeführt werden und über heterologe oder homologe Rekombination in das Genom des Wirtsorganismus integriert werden. Diese lineare DNA kann aus einem linearisierten Plasmid oder nur aus dem Nukleinsäurekonstrukt als Vektor oder den verwendeten Nukleinsäuresequenzen bestehen.In a further embodiment of the vector, the nucleic acid construct according to the invention can also advantageously be introduced into the organisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination. This linear DNA can consist of a linearized plasmid or only of the nucleic acid construct as a vector or the nucleic acid sequences used.
In einer weiteren vorteilhaften Ausführungsform können die im erfindungsgemäßen Verfahren verwendeten Nukleinsäuresequenzen auch alleine in einen Organismus eingebracht werden.In a further advantageous embodiment, the nucleic acid sequences used in the method according to the invention can also be introduced into an organism alone.
Sollen neben den Nukleinsäuresequenzen weitere Gene in den Organismus eingeführt werden, so können alle zusammen mit einem Reportergen in einem einzigen Vektor oder jedes einzelne Gen mit oder ohne einem Reportergen in je einem Vektor in den Organismus eingebracht werden, wobei die verschiedenen Vektoren gleichzeitig oder sukzessive eingebracht werden können.If, in addition to the nucleic acid sequences, further genes are to be introduced into the organism, they can all be introduced into the organism together with a reporter gene in a single vector or each individual gene with or without a reporter gene in each vector, the different vectors being introduced simultaneously or successively can be.
Der Vektor enthält vorteilhaft mindestens, eine Kopie 'der verwendeten Nukleinsäuresequenzen und/oder des erfindungsgemäßen Nukleinsaurekonstrukte.The vector advantageously contains at least one copy of the nucleic acid sequences used and / or the nucleic acid constructs according to the invention.
Beispielhaft kann das Nukleinsäurekonstrukt in den Tabak-Transformationsvektor pBinAR eingebaut werden und unter der Kontrolle des 35S-Promotor oder des USP-Promotor stehen.For example, the nucleic acid construct can be incorporated into the tobacco transformation vector pBinAR and be under the control of the 35S promoter or the USP promoter.
Alternativ kann ein rekombinanter Vektor (= Expressionsvektor) auch in-vitro transkribiert und translatiert werden, z.B. durch Nutzung des T7 Promotors und der T7 RNA Polymerase.Alternatively, a recombinant vector (= expression vector) can also be transcribed and translated in vitro, e.g. by using the T7 promoter and the T7 RNA polymerase.
Weitere vorteilhafte Vektoren enthalten in Pflanzen oderOther advantageous vectors contained in plants or
Pflanzenkulturen nutzbare Resistenzen wie die Phosphinotricin- (= bar-Resistenz) , die Methioninsulfoximin-, die Sulfonylharn- stoff- (= ilv-Resistenz, ind S. cerevisiae ilv2) , die Phenoxy- phenoxyherbizid- (= ACCase-Resistenz) , die Glyphosate- oder Clearfieldresistenz (AHAS-Resistenz) bzw. die Gene, die für diese Resistenzen codieren. Diese Resistenzen sind in ganzen Pflanzen zur Selektion von transgenen Pflanzen nutzbar. Nur Pflanzen, die über eine Transformation, diese Resistenzen erhalten haben, können in Gegenwart der selektierenden Substanz wachsen. In Zellkulturen auf Agarplatten können nach Transformation in planta - beispielsweise Infiltration der Samenvorläuferzellen - auch 0 beispielsweise Kanamycin oder Hygromycin als selektierendes Agenz verwendet werden. Darüber hinaus können vorteilhafte Vektoren Sequenzen für die Integration in das Genom der Organismen bevorzugt der Pflanzen enthalten. Beispiele für derartige Sequenzen sind die sog. T-DNA-Borders . Außerdem können vorteilhafte Vektoren noch -Promotoren und Terminatoren, wie beispielsweise die oben beschriebenen, enthalten. Auch sogenannte PolyA- Sequenzen können im Vektor enthalten sein. Vorteilhafte Vektoren ■ sind beispielsweise Figur 1, 2 und 3 zu entnehmen. SEQ ID NO: 25 gibt die vorteilhafte Sequenz des Vektors pMTX la300 wieder. Dieser enthält eine Kanamycin-Resistenz (Nukleotid 4922-5713), eine Phosphinotricin-Resistenz (Nukleotid 6722-7288) , das LacZalpha-Fragment (Nukleotid 7630-7864), einen Teil von pVSlsta (Nukleotid 945-1945), einen Teil von pBR322bom (Nukleotid 3948-4208), eine T-Border-Sequenz (links, Nukleotid 6138-6163), eine T-Border-Sequenz (rechts, Nukleotid 7924-7949), ein PolyA- Teil (Nukleotid 7292 - 7503), den mas2 ' 1' -Promotor (Nukleotid 6241-6718) und zwei Replikation-s-Origins pVSlrep (Nukleotid 6241-6718) sowie pBR322ori (Nukleotid 43-4628) .Resistant crops, such as phosphinotricin (= bar resistance), methionine sulfoximine, sulfonyl urine, Substance (= ilv resistance, ind S. cerevisiae ilv2), the phenoxyphenoxyherbicide (= ACCase resistance), the glyphosate or clearfield resistance (AHAS resistance) or the genes coding for these resistances. These resistances can be used in whole plants for the selection of transgenic plants. Only plants that have received these resistances through transformation can grow in the presence of the selecting substance. After transformation in planta - for example infiltration of the seed precursor cells - 0 for example kanamycin or hygromycin can also be used as a selecting agent in cell cultures on agar plates. In addition, advantageous vectors can contain sequences for integration into the genome of the organisms, preferably of the plants. Examples of such sequences are the so-called T-DNA borders. In addition, advantageous vectors may also contain promoters and terminators, such as those described above. So-called polyA sequences can also be contained in the vector. Advantageous vectors ■ are for example 1, 2 and remove. 3 SEQ ID NO: 25 shows the advantageous sequence of the vector pMTX la300. This contains a kanamycin resistance (nucleotide 4922-5713), a phosphinotricin resistance (nucleotide 6722-7288), the LacZalpha fragment (nucleotide 7630-7864), part of pVSlsta (nucleotide 945-1945), part of pBR322bom (Nucleotide 3948-4208), a T-border sequence (left, nucleotide 6138-6163), a T-border sequence (right, nucleotide 7924-7949), a polyA part (nucleotide 7292 - 7503), the mas2 '1' promoter (nucleotide 6241-6718) and two replication s-origins pVSlrep (nucleotide 6241-6718) and pBR322ori (nucleotide 43-4628).
In Prokaryoten verwendete Expressionsvektoren nutzen häufig induzierbare Systeme mit und ohne Fusionsproteinen bzw Fusions- oligopeptiden, wobei diese Fusionen sowohl N-terminal als auch C- terminal oder anderen nutzbaren Domänen eines Proteins erfolgen können. Solche Fusionsvektoren dienen in der Regel dazu: i.) die Expressionsrate der RNA zu erhöhen ii.) die erzielbare Protein- sytheserate zu erhöhen, iii.) die Löslichke t des Proteins zu erhöhen, iv. ) oder die Reinigung durch einen für die Affinitätschromatographie nutzbare Bindesequenz zu vereinfachen. Häufig werden auch proteolytische Spaltstellen über Fusionsproteine eingeführt, was die Abspaltung eines Teils des Fusionsproteins auch der Reinigung ermöglicht. Solche ErkennungsSequenzen für Proteasen erkennen sind z.B. Faktor Xa, Thrombin und Enterokinase .Expression vectors used in prokaryotes frequently use inducible systems with and without fusion proteins or fusion oligopeptides, it being possible for these fusions to take place both at the N-terminal and at the C-terminal or other usable domains of a protein. Such fusion vectors usually serve: i.) To increase the expression rate of the RNA ii.) To increase the achievable protein synthesis rate, iii.) To increase the solubility of the protein, iv. ) or to simplify purification by means of a binding sequence that can be used for affinity chromatography. Frequently, proteolytic cleavage sites are also introduced via fusion proteins, which enables a part of the fusion protein to be split off, also for purification. Such recognition sequences for proteases are e.g. Factor Xa, thrombin and enterokinase.
Typische vorteilhafte Fusions- und Expressionsvektoren sind pGEX [Pharmacia Biotech Ine; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40], pMAL (New England Biolabs , Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) welches Glutathion S-transferase beinhaltet (GST) , Maltose Bindeprotein, oder Protein A.Typical advantageous fusion and expression vectors are pGEX [Pharmacia Biotech Ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40], pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which contains glutathione S-transferase (GST), maltose binding protein, or protein A.
Weitere Beispiele für E. coli Expressionsvektoren sind pTrc [Amann et al . , (1988) Gene 69:301-315] und pET Vektoren [Studier et al.', Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Stratagene, Amsterdam, Niederlande] .Further examples of E. coli expression vectors are pTrc [Amann et al. , (1988) Gene 69: 301-315] and pET vectors [Studier et al. ' , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; Stratagene, Amsterdam, Netherlands].
Weitere vorteilhafte Vektoren zur Verwendung in Hefe sind pYep- Secl (Baldari, et al . , (1987) E bo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES-Derivate (Invitrogen Corporation, San Diego, CA) . Vektoren für die Nutzung in filamen- tosen Pilzen sind beschrieben in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge .Further advantageous vectors for use in yeast are pYep-Secl (Baldari, et al., (1987) E bo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES derivatives (Invitrogen Corporation, San Diego, CA). Vectors for use in filamentous mushrooms are described in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al., Eds., P. 1-28, Cambridge University Press: Cambridge.
Alternativ können auch vorteilhaft Insektenzellexpressions- vektoren genutzt werden z.B. für die Expression, in Sf .9 Zellen. Dies sind z.B. die Vektoren der pAc Serie (Smith et al. (1983) Mol . Cell Biol . 3:2156-2165) und der pVL series (Lucklow and Summers (1989) Virology 170 : 31-39) .Alternatively, insect cell expression vectors can also be used advantageously, e.g. for expression, in Sf .9 cells. These are e.g. the vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
Des weiteren können zur Genexpression vorteilhaft Pflanzenzellen oder Algenzellen genutzt werden. Beispiele für Pflanzenexpressionsvektoren finden sich in Becker, D. , et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol . Biol . 20: 1195-1197 oder in Bevan, M.W. (1984) "Binary Agrobacterium, vectors for plant transformation" , Nucl . Acid: Res. 12 :' 8711-8721.Furthermore, plant cells or algal cells can advantageously be used for gene expression. Examples of plant expression vectors can be found in Becker, D., et al. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197 or in Bevan, M.W. (1984) "Binary Agrobacterium, vectors for plant transformation", Nucl. Acid: Res. 12: '8711-8721.
Weiterhin können die erfindungsgemäßen Nukleinsäuresequenzen in Säugerzellen exprimiert werden. Beispiel für entsprechende Expressionsvektoren sind pCDMδ und pMT2PC genannt in: Seed, B. (1987) Nature 329:840 oder Kaufman et al . (1987) EMBO J. 6:187-195). Dabei sind vorzugsweise • zu nutzende Promotoren viralen Ursprungs wie z.B. Promotoren des Polyoma, Adenovirus 2, Cytomegalovirus oder Simian Virus 40. Weitere prokaryotische und eukaryotische Expressionssysteme sind genannt in Kapitel 16 und 17 in Sambrook et al . , Molecular Cloning: A Laboratory Manual . 2nd, ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Weitere vorteil- hafte Vektoren werden in Hellens et al . (Trends in plant science, 5, 2000) beschrieben.Furthermore, the nucleic acid sequences according to the invention can be expressed in mammalian cells. Examples of corresponding expression vectors are pCDMδ and pMT2PC mentioned in: Seed, B. (1987) Nature 329: 840 or Kaufman et al. (1987) EMBO J. 6: 187-195). In this context, promoters to be used are preferably of viral origin, such as promoters of polyoma, adenovirus 2, cytomegalovirus or Simian virus 40. Further prokaryotic and eukaryotic expression systems are mentioned in chapters 16 and 17 in Sambrook et al. , Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Further advantages adhesive vectors are described in Hellens et al. (Trends in plant science, 5, 2000).
Das Einbringen der erfindungsgemäßen Nukleinsäuren, der 5 Expressionskassette oder des Vektors in Organismen beispielsweise in Pflanzen kann prinzipiell nach allen dem Fachmann - bekannten Methoden erfolgen.In principle, the nucleic acids according to the invention, the expression cassette or the vector can be introduced into organisms, for example in plants, by all methods known to the person skilled in the art.
Für Mikroorganismen kann der Fachmann entsprechende Methoden den 0 Lehrbüchern von Sambrook, J. et al. (1989) Molecular cloning: A laboratory anual, Cold Spring Harbor Laboratory Press, von F.M. Ausubel et al. (1994) Current protocols in molecular bio- logy,' John Wiley and Sons, von D.M. Glover et al., DNA Cloning Vol.l, (1995), IRL Press (ISBN 019-963476-9), von Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press oder Guthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press entnehmen.For microorganisms, the person skilled in the art can use the corresponding textbooks from Sambrook, J. et al. (1989) Molecular cloning: A laboratory anual, Cold Spring Harbor Laboratory Press, by FM Ausubel et al. (1994) Current protocols in molecular biology, ' John Wiley and Sons, by DM Glover et al., DNA Cloning Vol. 1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in Yeast Genetics, Cold Spring Habor Laboratory Press or Guthrie et al. See Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, 1994, Academic Press.
Die Übertragung von Fremdgenen in das Genom einer Pflanze "wird als Transformation bezeichnet. Es werden dabei die beschriebenen Methoden zur Transformation und Regeneration von Pflanzen aus Pflanzengeweben oder Pflanzenzellen zur transienten oder stabilen Transformation genutzt. Geeignete Methoden sind die Protoplasten- transformation durch Polyethylenglykol-induzierte DNA-Aufnahme, das biolistische Verfahren mit der Genkanone - die sogenannte particle bombardment Methode, die Elektroporation, die Inkubation trockener Embryonen in DNA-haltiger Lösung, die- Mikroinjektion und der durch Agrobacterium vermittelte Gentransfer vorteilhaft erfolgt der Gentransfer in der vorliegenden Erfindung mit bei- ' spielsweise dem Agrobacterium tumefaciens Stamm GV 3101 pMP90. Die genannten Verfahren sind beispielsweise in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, herausgegeben von S.D. Kung und R.. Wu, Academic Press (1993) 128-143 sowie in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol. 42 (1991) 205-225) beschrieben. Vorzugsweise wird das zu exprimierende Konstrukt in einen Vektor kloniert, der geeignet ist, Agrobacterium tumefaciens zu transformieren, beispielsweise pBinl9 (Bevan et al . , Nucl. Acids Res . 12 (1984) 8711) . Mit einem solchen Vektor transformierte Agro- bakterien können dann in bekannter Weise zur Transformation von Pflanzen, insbesondere von Kulturpflanzen, wie z.B. von Tabakpflanzen, verwendet werden, indem beispielsweise verwundete Blätter oder Blattstücke in einer Agrobakterienlösung gebadet und anschließend in geeigneten Medien kultiviert werden. Die Transformation von Pflanzen mit Agrobacterium tumefaciens wird beispielsweise von Höfgen und Willmitzer in Nucl . Acid Res . (1988) 16, 9877 beschrieben oder ist unter anderem bekannt aus F.F. White, Vectors for Gene Transfer in Higher Plants;' in Trans- genic Plants, Vol. 1, Engineering and Utilization, herausgegeben von S .O. Kung und R. Wu, Academic Press, 1993, S. 15-38.The transfer of foreign genes into the genome of a plant " is referred to as transformation. The methods described here for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic method with the gene gun - known as the particle bombardment method, electroporation, incubation of dry embryos in DNA-containing solution, DIE microinjection and the Agrobacterium-mediated gene transfer of gene transfer takes place advantageously in the present invention, with examples' for example the Agrobacterium tumefaciens strain GV 3101 pMP90, The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, published by SD Kung and R. Wu, Academic Press (1993) 128-143 as well e in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol. 42 (1991) 205-225). The construct to be expressed is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with such a vector can then be used in a known manner for transforming plants, in particular crop plants, such as tobacco plants, for example, by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media. The transformation of plants with Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known from, among others FF White, Vectors for Gene Transfer in Higher Plants; ' in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S .O. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
Im folgenden ist eine vorteilhafte Ausführungsform wiedergegeben. Werden für die Transformation Agrobakterien verwendet, wird die einzuführende Nukleinsäure bzw. DNA in spezielle Pl'asmide cloniert werden, und zwar entweder in einen intermediären Vektor oder in einen binären Vektor. Die intermediären Vektoren können aufgrund von Sequenzen, die homolog zu Sequenzen in der T-DNA sind, durch homologe Rekombination in das Ti- oder Ri-Plasmid der Agrobakterien integriert werden. Dieses enthält außerdem die für den Transfer der T-DNA notwendige vir-Region. Intermediäre Vektoren können nicht in Agrobakterien replizieren. Mittels eines Helferplasmids kann der intermediäre Vektor auf Agrobacterium tumefaciens übertragen werden (Konjugation) . Binäre Vektoren können sowohl in E.coli als auch in Agrobakterien replizieren. Sie enthalten ein Selektionsmarker-Gen und einen Linker oder Polylinker, welche von der rechten und linken T-DNA Grenzregion eingerahmt werden. Sie können direkt in die Agrobakterien transformiert werden (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187) . Das als Wirtszelle dienende Agrobakterium soll 'ein Plasmid, das eine vir-Region trägt, enthalten. Die vir-Region ist für den Transfer der T-DNA in die Pflanzenzelle notwendig. Zusätzliche T-DNA kann vorhanden sein. Das derartig transformierte Agrobakterium wird zur Transformation von Pflanzenzellen verwendet.An advantageous embodiment is shown below. Be used for the transformation of Agrobacteria, the introduced nucleic acid or DNA will be cloned into special Pl 'asmide, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in both E. coli and agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187). Serving as host cell should contain a plasmid of Agrobacterium carrying a vir region '. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells.
Die Verwendung von T-DNA für die Transformation von Pflanzen- zellen ist intensiv untersucht und ausreichend in EPA-0 120 516; .Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B.V. , Alblasserdam (1985), Chapter V; Fraley et al . , Crit. Rev. Plant. Sei., 4: 1-46 und An et al . EMBO J." 4 (1985), 277-287 beschrieben worden.The use of T-DNA for the transformation of plant cells has been intensively investigated and is sufficiently described in EPA-0 120 516; , Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters BV, Alblasserdam (1985), Chapter V; Fraley et al. , Crit. Rev. Plant. Sci., 4: 1-46 and An et al. EMBO J. "4 (1985), 277-287.
Für' den Transfer der DNA in die Pflanzenzelle können Pflanzen- Explantate zweckmäßigerweise mit Agrobacterium tumefaciens oder Agrobacterium rhizogenes kokultiviert werden. Aus dem infizierten Pflanzenmaterial (z.B. Blattstücke, Stengelsegmente, Wurzeln, aber auch Protoplasten oder Suspensions-kultivierte Pflanzen- ' zellen) können dann in einem geeigneten Medium, welches Anti- ' biotika oder Biozide zur Selektion transformierter Zellen enthalten kann, wieder ganze Pflanzen regeneriert werden. Die so erhaltenen Pflanzen können dann auf Anwesenheit der eingeführten DNA untersucht werden. Andere Möglichkeiten der Einführung fremder DNA unter Verwendung des biolistischen Verfahrens oder durch Protoplastentransformation sind bekannt (vergl. z.B. Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H.J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim- New York-Basel-Cambridge) . ) 5For 'the transfer of the DNA into the plant cell, plant explants can expediently with Agrobacterium tumefaciens or Agrobacterium rhizogenes are co-cultured. From the infected plant material (cultured suspension, for example, pieces of leaf, stem segments, roots, but also protoplasts or plant 'cells) may then in a suitable medium, which anti-' may contain biotics or biocides for selecting transformed cells whole plants be regenerated , The plants thus obtained can then be examined for the presence of the introduced DNA. Other ways of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf., for example Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (HJ Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge). ) 5
Auch die Transformation monokotyler Pflanzen mittels Agrobacterium basierender Vektoren wurde beschrieben (Chan et al, Plant Mol. Biol. 22(1993), 491-506; Hiei et al, Plant J. 6" (1994) 271-282; Deng et al; Science in China 33 (1990), 28-34; WilminkThe transformation of monocotyledonous plants using Agrobacterium-based vectors has also been described (Chan et al, Plant Mol. Biol. 22 (1993), 491-506; Hiei et al, Plant J. 6 " (1994) 271-282; Deng et al; Science in China 33 (1990), 28-34; Wilmink
10 et al, Plant Cell Reports 11, (1992) 76-80; May et al; Biotechnology 13 (1995) 486-492; Conner und Domisse; Int. J. Plant Sei. 153 (1992) 550-555; -Ritchie et al; Transgenic Res. (1993) 252-265) . Alternative Systeme zur Transformation von monokotylen Pflanzen sind die Transformation mittels des biolistischen10 et al, Plant Cell Reports 11, (1992) 76-80; May et al; Biotechnology 13 (1995) 486-492; Conner and Domisse; Int. J. Plans Be. 153: 550-555 (1992); -Ritchie et al; Transgenic Res. (1993) 252-265). Alternative systems for the transformation of monocotyledonous plants are the transformation using the biolistic
15 Ansatzes (Wan und Lemaux; Plant Physiol. 104 (1994), 37-48; Vasil et al; Biotechnology 11 (1992), 667-674; Ritala et al, Plant Mol. Biol 24, (1994) 317-325; Spencer et al, Theor. Appl . Genet . 79 (1990), 625-631) die Protoplastentransformation, die Elektro- poration von partiell permeabilisierten Zellen; die Einbringung15 approach (Wan and Lemaux; Plant Physiol. 104 (1994), 37-48; Vasil et al; Biotechnology 11 (1992), 667-674; Ritala et al, Plant Mol. Biol 24, (1994) 317-325; Spencer et al, Theor. Appl. Genet. 79 (1990), 625-631) the protoplast transformation, the electroporation of partially permeabilized cells; the contribution
20 von DNA mittels Glasfasern. Insbesondere die Transformation von Mais wurde in der Literatur mehrfach beschrieben (vgl. z.B. WO 95/06128; EP 0513849 AI; EP 0465875 AI; EP 0292435 AI; Fromm et al, Biotechnology 8 (1990), 833-844; Gordon-Kamm et al, Plant Cell 2 (1990), 603-618; Koziel et al, Biotechnology 11(1993)20 of DNA using glass fibers. In particular, the transformation of maize has been described several times in the literature (see, for example, WO 95/06128; EP 0513849 AI; EP 0465875 AI; EP 0292435 AI; Fromm et al, Biotechnology 8 (1990), 833-844; Gordon-Kamm et al, Plant Cell 2 (1990), 603-618; Koziel et al, Biotechnology 11 (1993)
25 194-200; Moroc et al, Theor Applied Genetics 80 (190) 721-726).25 194-200; Moroc et al, Theor Applied Genetics 80 (190) 721-726).
Auch die erfolgreiche Transformation anderer Getreidearten wurde bereits beschrieben z.B. für Gerste (Wan und Lemaux, s.o.; Ritala et al, s.o.; Weizen (Nehra et al, Plant J. 5(1994) 285-297).The successful transformation of other cereals has also been described, e.g. for barley (Wan and Lemaux, see above; Ritala et al, see above; wheat (Nehra et al, Plant J. 5 (1994) 285-297).
3030
Mit einem erfindungsgemäßen Vektor transformierte Agrobakterien können ebenfalls in bekannter Weise zur Transformation von Pflanzen wie Testpflanzen wie Arabidopsis oder Kulturpflanzen wie Getreide, Mais, Hafer, Roggen, Gerste, Weizen, Soja, Reis, Baum-Agrobacteria transformed with a vector according to the invention can also be used in a known manner to transform plants such as test plants such as Arabidopsis or crop plants such as cereals, corn, oats, rye, barley, wheat, soybeans, rice, tree
35 wolle, Zuckerrübe, Canola, Sonnenblume, Flachs, Hanf, Kartoffel, Tabak, Tomate, Karotte, Paprika, Raps, Tapioka, Maniok, Pfeilwurz, Tagetes, Alfalfa, Salat und den verschiedenen Baum-, Nuss- und Weinspezies verwendet werden, z.B. indem verwundete Blätter oder Blattstücke in einer Agrobakterienlösung gebadet und an-35 wool, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot, paprika, rapeseed, tapioca, cassava, arrowroot, tagetes, alfalfa, lettuce and the various tree, nut and wine species are used, e.g. by bathing wounded leaves or leaf pieces in an agrobacterial solution and
40 schließend in geeigneten Medien kultiviert werden.40 finally be cultivated in suitable media.
Die genetisch veränderten Pflanzenzellen können über alle dem Fachmann bekannten Methoden regeneriert werden. Entsprechende Methoden können den oben genannten Schriften von S.D. Kung und 45 R. Wu, Potrykus oder Höfgen und Willmitzer entnommen werden. Unter Pflanzen im Sinne der Erfindung sind Pflanzenzellen, -gewebe, -Organe oder ganzen Pflanzen wie Samen, Knollen, Blüten, Pollen, Früchte, Sämlinge, Wurzeln, Blätter, Stengel oder sonstige Pflanzenteile zu verstehen. Außerdem ist unter Pflanzen Vermehrungsmaterial wie Samen, Früchte, Sämlinge, Stecklinge, Knollen, Schnitte oder Wurzelstöcke zu verstehen.The genetically modified plant cells can be regenerated using all methods known to the person skilled in the art. Appropriate methods can be found in the above-mentioned writings by SD Kung and 45 R. Wu, Potrykus or Höfgen and Willmitzer. Plants in the sense of the invention are understood to mean plant cells, tissue, organs or whole plants such as seeds, tubers, flowers, pollen, fruits, seedlings, roots, leaves, stems or other parts of plants. In addition, plants are understood to mean propagation material such as seeds, fruits, seedlings, cuttings, tubers, cuts or rhizomes.
Als Organismen bzw. Wirtsorganismen für die erfindungsgemäße Nukleinsäure, die Expressionskassette oder den Vektor eignen - sich prinzipiell vorteilhaft alle Organismen, die in der Lage sind die erfindungsgemäß verwendeten Nukleinsäuren zu exprimieren bzw. für die Expression rekombinanter Gene geeignet sind. Beispielhaft seien Pflanzen wie Arabidopsis, Asteraceae wie Calendula oder Kulturpflanzen wie Soja, Erdnuss, Rizinus, Sonnenblume,- Mais, Baumwolle, Flachs, Raps, Kokosnuss, Ölpalme, Färbersaflor (Carthamus tinctorius) oder Kakaobohne', Mikroorganismen wie Pilze beispielsweise die Gattung Mortierella, Saprolegnia oder Pythium, Bakterien wie die Gattung Escherichia, Hefen wie die Gattung Saccharomyces , Cyanobakterien, Ciliaten, Algen oder Protozoen wie Dinoflagellaten wie Crypthecodinium genannt. Beispielsweise seien Organismen, die natürlicherweise Öle in größeren Mengen synthetisieren wie Soja, Raps, Kokosnuss, Ölpalme, Färbersaflor, Rizinus, Calendula, Erdnuss, Kakaobohne oder Sonnenblume genannt. Prinzipiell sind als Wirtsorganismen auch transgene Tiere geeignet beispielsweise C. elegans.Suitable organisms or host organisms for the nucleic acid according to the invention, the expression cassette or the vector are in principle advantageously all organisms which are able to express the nucleic acids used according to the invention or are suitable for the expression of recombinant genes. Examples include plants such as Arabidopsis, Asteraceae such as Calendula or crops such as soybean, peanut, castor oil, sunflower, - maize, cotton, flax, rapeseed, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa bean ' , microorganisms such as fungi, for example the genus Mortierella, Saprolegnia or Pythium, bacteria such as the genus Escherichia, yeasts such as the genus Saccharomyces, cyanobacteria, ciliates, algae or protozoa such as dinoflagellates such as Crypthecodinium. For example, organisms that naturally synthesize oils in large quantities, such as soybean, rapeseed, coconut, oil palm, safflower, castor bean, calendula, peanut, cocoa bean or sunflower. In principle, transgenic animals are also suitable as host organisms, for example C. elegans.
Bevorzugt sind transgene Pflanzen, die ein erfindungsgemäßes funktionelles oder nicht funktionelles Nukleinsäurekonstrukt oder einen erfindungsgemäßen -funktionellen oder nicht funktionellen Vektor enthalten. Unter funktioneil ist im -Sinne der Erfindung zu verstehen, dass die im Verfahren verwendeten Nukleinsäuren allein oder im Nukleinsäurekonstrukt oder im Vektor exprimiert werden und ein biologisch aktives Genprodukt hergestellt wird. Unter nicht funktionell ist im Sinne der Erfindung zu verstehen, dass die im Verfahren verwendeten Nukleinsäuren allein oder im Nukleinsäurekonstrukt oder im Vektor nicht transcribiert, nicht exprimiert werden und/oder ein biologisch inaktives Genprodukt hergestellt wird. In diesem Sinne handelt es sich bei den sogenannten Antisense-RNAs auch um nicht funktioneile Nuklein- säuren bzw. bei Insertion in das Nukleinsäurekonstrukt oder den Vektor um ein nicht funktionelles Nukleinsäurekonstrukt oder nicht funktionellen Vektor. Sowohl das erfindungsgemäße Nukleinsäurekonstrukt als auch der erfindungsgemäße Vektor kann zur Herstellung von transgenen Organismen bevorzugt Pflanzen vorteilhaft verwendet werden. Unter transgen im Sinne der Erfindung ist zu verstehen, dass die im Verfahren verwendeten Nukleinsäuren nicht an ihrer natürlichen Stelle im Genom eines Organismus sind, dabei können die Nukleinsäuren homolog oder heterolog exprimiert werden. Tansgen bedeutet aber auch, dass die erfindungsgemäßen Nukleinsäuren an ihrem natürlichen Platz im Genom eines Organismus sind, dass jedoch die Sequenz gegenüber der natürlichen Sequenz verändert wurde und/oder das die RegulationsSequenzen, der natürlichen Sequenzen verändert wurden. Bevorzugt ist unter transgen die Expression der Nukleinsäuren an nicht natürlicher Stelle im Genom zu verstehen', das heißt eine homologe oder bevorzugt heterologe Expression der Nukleinsäuren liegt vor. Gleiches gilt für das erfindungsgemäße Nukleinsäurekonstrukt oder den Vektor.Preferred are transgenic plants which contain a functional or non-functional nucleic acid construct according to the invention or a functional or non-functional vector according to the invention. In the sense of the invention, functional is to be understood to mean that the nucleic acids used in the method are expressed alone or in the nucleic acid construct or in the vector and a biologically active gene product is produced. Non-functional in the sense of the invention means that the nucleic acids used in the method alone or in the nucleic acid construct or in the vector are not transcribed, are not expressed and / or a biologically inactive gene product is produced. In this sense, the so-called antisense RNAs are also non-functional nucleic acids or, when inserted into the nucleic acid construct or the vector, a non-functional nucleic acid construct or non-functional vector. Both the nucleic acid construct according to the invention and the vector according to the invention can be used advantageously for the production of transgenic organisms, preferably plants. By transgene in the sense of the invention is to be understood that the nucleic acids used in the method are not in their natural place in the genome of an organism, and the nucleic acids can be expressed homologously or heterologously. However, Tansgen also means that the nucleic acids according to the invention are in their natural place in the genome of an organism, but that the sequence has been changed compared to the natural sequence and / or that the regulatory sequences of the natural sequences have been changed. Transgenic is preferably to be understood as meaning the expression of the nucleic acids at a non-natural site in the genome, that is to say there is homologous or preferably heterologous expression of the nucleic acids. The same applies to the nucleic acid construct according to the invention or the vector.
Nutzbare Wirtszellen sind weiterhin genannt in: Goeddel, GeneUsable host cells are also mentioned in: Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) .Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
Verwendbare Expressionsstamme z.B. solche, die1 eine geringere Proteaseaktivität aufweisen sind beschrieben in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.Usable expression strains, for example, the 1 have a lower protease activity, are described in such: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128.
Weiterhin umfasst die Erfindung auch die Verwendung der erfindungsgemäßen Nukleinsäuren, z.B. der unter den SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, ' SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID. O: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 dargelegten Nukleotidsequenzen zur Erstellung von genetisch veränderten Pflanzen, die dadurch gekennzeichnet sind, dass sie veränderte Proteine, der von den SEQ ID NO: 1, ' SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:.13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oderFurthermore, the invention also includes the use of the nucleic acids according to the invention, for example those under SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO : 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, 'SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID. O: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 nucleotide sequences set forth for the creation of genetically modified plants, which are characterized in that they are modified proteins of the SEQ ID NO: 1, 'SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:. 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or
SEQ ID NO: 108 codierten Proteine enthalten, - die dadurch gekennzeichnet sind dass sie eine sehr viele geringere Interaktion mit dem Herbizid aufweisen bzw. in ihrer Aktivität nicht durch das Herbizid beeinträchtigt werden.SEQ ID NO: 108 encoded proteins, - which are characterized in that they have a very much lower interaction with the herbicide or their activity is not impaired by the herbicide.
Die" im erfindungsgemäßen Verfahren verwendeten Nukleinsäuren, insbesondere SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID~NO: 30, SEQ ID NO: 32, SEQ .ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44 , - SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ" ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108, deren aufgrund des degenerierten genetischen Codes abgeleiteten Sequenzen sowie deren Derivate wurden aus einer Populationen transgener Pflanzen identifiziert, die zum einen dadurch gekennzeichnet war, dass sie mittels des Agrobacterium transformiert worden sind und im Rahmen dieses Prozesses neue DNA im Chromosomen an zufälliger Stelle integriert worden war. Durch Rückkreuzungen wurden schließlich Pflanzen isoliert, die die identifizierten Nukleinsäuren auf beiden homologen Chromosomen enthalten. Darüber- hinaus sind diese Pflanzen dadurch gekennzeichnet, dass sie im' Verlauf des Screenings' als für lethale Mutationen segregierende Linien identi iziert worden sind. Diese Pflanzen weisen als Ergebnis der Integration der neuen DNA starke Behinderungen im Wachstum und oder Entwicklung auf. Es ist davon auszugehen, dass diese Wachstums- und Entwicklungsbehinderungen darauf zurückzuführen sind, dass die neu inserierte DNA in für Wachstum und Entwicklung wichtige Gene" integriert hat, wodurch deren biologische Funktion eingeschränkt oder blockiert wird. Dieses bedeutet, dass diese Gene sowie deren aufgrund des degenerierten genetischen Codes abgeleiteten Sequenzen sowie deren Derivate für Proteine codieren, welche in analoger Weise wie für die in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,The nucleic acids used in the method according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 , SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID ~ NO: 30, SEQ ID NO: 32, SEQ .ID NO: 34, SEQ ID NO: 36 , SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, - SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ " ID NO: 54 , SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO : 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108, whose sequences derived on the basis of the degenerated genetic code and their derivatives have been identified from a population of transgenic plants which, on the one hand, dad was characterized by the fact that they had been transformed by means of the Agrobacterium and in the course of this process new DNA had been integrated into the chromosome at a random point. Backcrossing finally isolated plants that contain the identified nucleic acids on both homologous chromosomes. In addition, these plants are characterized in that they have been identified in the 'course of the screening' as lines which segregate lethal mutations. As a result of the integration of the new DNA, these plants have severe impediments to growth and or development. It can be assumed that these growth and developmental hindrances are due to the fact that the newly inserted DNA has "integrated into genes which are important for growth and development", as a result of which their biological function is restricted or blocked. This means that these genes and their degenerate genetic codes derived sequences and their derivatives for proteins encoding, which in an analogous manner as for SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO 40,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO 40,
SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO 50,SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO 50,
SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58,
- SEQ ID NO: 60, SEQ' ID NO: 62, SEQ ID NO: 64, SEQ ID NO 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO 74,- SEQ ID NO: 60, SEQ ' ID NO: 62, SEQ ID NO: 64, SEQ ID NO 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO 74,
SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO 82,SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO 82,
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO 90,SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO 90,
SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO 98,SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO 98,
SEQ ID NO: 100 , SEQ ID NO : 102, SEQ ID NO: 106 "oder SEQ ID NO: 108 beschrieben, geeignete Zielproteine für neu zu entwickelnde Herbizide darstellen.SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 " or SEQ ID NO: 108, are suitable target proteins for newly developed herbicides.
Zur Erzeugung derartiger veränderter Proteine werden in einer vorteilhaften Ausführungsform die genannten Nukleinsäuren über- exprimiert und, folgende Prozessschritte werden vorteilhaft durchlaufen:To produce such modified proteins, the nucleic acids mentioned are overexpressed in an advantageous embodiment and the following process steps are advantageously carried out:
a) Expression der von den SEQ ID NO: 1, SEQ ID NO: 3,a) expression of those of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO: 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO: 26,SEQ ID NO: 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO: 34,SEQ ID NO: 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO: 44,SEQ ID NO: 36, SEQ ID NO 38, SEQ ID NO 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO: 52,SEQ ID NO: 46, SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO: 52,
SEQ ID NO: 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO: 60,SEQ ID NO: 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO: 68,SEQ ID NO: 62, SEQ ID NO 64, SEQ ID NO 66, SEQ ID NO: 68,
SEQ ID NO: 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO: 76,SEQ ID NO: 70, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO 80, SEQ ID NO 82, SEQ ID NO: 84,SEQ ID NO: 78, SEQ ID NO 80, SEQ ID NO 82, SEQ ID NO: 84,
SEQ ID NO: 86, SEQ ID NO 88, SEQ ID NO 90, SEQ ID ' NO : 92,SEQ ID NO: 86, SEQ ID NO 88, SEQ ID NO 90, SEQ ID ' NO: 92,
SEQ ID NO: 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO: 100,SEQ ID NO: 94, SEQ ID NO 96, SEQ ID NO 98, SEQ ID NO: 100,
SEQ ID NO: 102 SEQ ID NO: 106 oder SEQ ID NO: 108 oder von einer Nukleinsäuresequenz, die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID'NO: 8,SEQ ID NO: 102 SEQ ID NO: 106 or SEQ ID NO: 108 or from a nucleic acid sequence which, on the basis of the degenerate genetic code, can be derived from the back-translation of those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID'NO: 8,
SEQ ID NO 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO 31, SEQ ID NO SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO 39, SEQ ID NO SEQ ID NO: 45, SEQ ID NÖ: 47, SEQ ID NO 49, SEQ ID NO SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO SEQ ID NO: 61, SEQ ID NO: 63, ■ SEQ ID NO 65,SEQ ID NO 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO 31, SEQ ID NO SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO 39, SEQ ID NO SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO 49, SEQ ID NO SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO 57, SEQ ID NO SEQ ID NO: 61, SEQ ID NO: 63, ■ SEQ ID NO 65,
SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO 73,SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO 81,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO 81,
SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO 89,SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO 89,
SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO 97,SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO 97,
SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103,SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103,
SEQ ID NO: 105, SEQ ID NO : 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen ableiten lässt, oder von Derivaten oder Fragmenten der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO;: 11SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 can be derived, or from derivatives or fragments of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO ;: 11
SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, • SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 46, SEQ ID NO: 48, • SEQ ID NO: 50, SEQ ID NO: 52,
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO': 66 , SEQ ID NO: 68,SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO ': 66, SEQ ID NO: 68,
SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 oderSEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 or
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90,
SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO; 96 , SEQ ID NO: 98,SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO; 96, SEQ ID NO: 98,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106,SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106,
SEQ ID NO: 108 dargestellten Nukleinsäuresequenzen, die für Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4,SEQ ID NO: 108 nucleic acid sequences shown for polypeptides with the in SEQ ID NO: 2, SEQ ID NO: 4,
SEQ ΪD NO 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ΪD NO 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:' 27,SEQ ID NO 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: '27,
SEQ ID NO 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
SEQ ID NO 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45,SEQ ID NO 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45,
SEQ ID NO 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,SEQ ID NO 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,
SEQ ID NO 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 6-9,SEQ ID NO 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 6-9,
SEQ ID NO 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,SEQ ID NO 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO 79, SEQ ID NO: 81, EQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO 79, SEQ ID NO: 81, EQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO:' 93,SEQ ID NO 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: '93,
SEQ ID NO 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101,SEQ ID NO 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101,
SEQ ID NO 103 , SEQ ID NO : 105, SEQ ID NO: 107 oderSEQ ID NO 103, SEQ ID NO: 105, SEQ ID NO: 107 or
SEQ ID NO: 109 dargestellten Aminosäuresequenzen codieren und mindestens 50 %, 60 %, vorzugsweise 70 %, 80 %, 90 % oder mehr Homologie auf Aminosäureebene aufweisen, codiertenSEQ ID NO: 109 encode amino acid sequences shown and encode at least 50%, 60%, preferably 70%, 80%, 90% or more homology at the amino acid level
Proteine in einem heterologen System beispielsweise einemProteins in a heterologous system, for example one
Mikroorganismus wie einem Bakterium der Gattung Escherichia wie E. coli XLl-Red oder in einem ZeilfreiensystemMicroorganism such as a bacterium of the genus Escherichia such as E. coli XLl-Red or in a cell free system
b) Randomisierte oder gerichtete Mutagenese des Proteins durch Modifikation der Nukleinsäure,b) randomized or directed mutagenesis of the protein by modification of the nucleic acid,
c) Messung der Interaktion oder der biologischen Aktivität des • veränderten Proteins mit dem Herbizid bzw. in 'Gegenwart des Herbizids,c) measuring the interaction or biological activity of the modified protein with the herbicide • or in 'the presence of the herbicide,
d) Identifizierung von Derivaten des Proteins die eine geringere Interaktion oder eine wenig beeinflusste biologische Aktivität aufweisen,d) identification of derivatives of the protein which show less interaction or little influenced biological activity,
e) Testung der biologischen Aktivität des Proteins nach Applikation des Herbizides . Vorteilhaft erfolgt die Überführung des so erhaltenen veränderten Proteins bzw. der veränderten Nukleinsäure, z.B. der unter SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68~, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ IDJSTO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 aufgeführten Sequenzen sowie der weiteren oben beschriebenen erfindungsgemäßen Sequenzen, z.B. Derivate und Fragmente, z.B. von anderen Pflanzen, in einen Organismus vorteilhaft in eine Pflanze, bevorzugt pflanzliche Zellen. e ) testing the biological activity of the protein after application of the herbicide. The modified protein or the modified nucleic acid obtained in this way is advantageously transferred, for example that under SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68 ~ , SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO : 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ IDJSTO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 and the other sequences according to the invention described above, for example derivatives and fragments, for example from others ren plants, in an organism advantageously in a plant, preferably plant cells.
Eine weitere Ausführungsform der Erfindung ist ein Verfahren zur Erzeugung veränderter Genprodukte, die von den hierin beschriebenen erfindungsgemäßen Nukleinsäuresequenzen, insbesondere SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:' 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 codiert werden, dadurch gekennzeichnet, dass es folgende Prozessschritte umfasst:A further embodiment of the invention is a method for producing modified gene products which, from the nucleic acid sequences according to the invention described herein, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: '15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 , SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO : 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 , SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 can be coded, characterized in that it has the following process step te includes:
a) Expression der von den SEQ ID NO: 1, SEQ ID NO: 3,a) expression of those of SEQ ID NO: 1, SEQ ID NO: 3,
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,
SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 codierten Proteine oder Derivate oder Fragmente, z.B. von anderen Pflanzen, davon in einem heterologen System oder in einem ' ZeilfreiensystemSEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 encoded proteins, or derivatives or fragments, eg, from other plants, including in a heterologous system or in a 'Zeilfreiensystem
b) Randomisierte oder gerichtete Mutagenese des Proteins durch Modifikation der Nukleinsäure,b) randomized or directed mutagenesis of the protein by modification of the nucleic acid,
c) Messung der Interaktion des veränderten Genprodukts mit dem Herbizid oder der biologischen Aktivität des veränderten Genprodukts in Gegenwart des Herbizids,c) measuring the interaction of the modified gene product with the herbicide or the biological activity of the modified gene product in the presence of the herbicide,
d) Identifizierung von Derivaten des Proteins die eine geringere Interaktion aufweisen oder in ihrer Aktivität weniger beein- flusst sind,d) identification of derivatives of the protein which have less interaction or are less influenced in their activity,
e) Testung der biologischen Aktivität des P oteins nach Applikation des Herbizides,e) testing the biological activity of the protein after application of the herbicide,
f) Auswahl der Nukleinsäuresequenzen, die oder deren 'Genprodukte eine veränderte biologische Aktivität gegenüber dem Herbizid, vorzugsweise eine verringerte Hemmung durch das Herbizid oder ' geringere Interaktion mit dem Herbizid, aufweisen.f) selection of the nucleic acid sequences or their 'gene a modified biological activity toward the herbicide, preferably a decreased inhibition by the herbicide or' lower interaction with the herbicide exhibit.
Die nach dem oben beschriebenen Verfahren nach- ausgewählten Sequenzen können vorteilhaft in einen Organismus eingebracht werden. Deshalb ist ein weiterer' Erfindungsgegenstand ein nach diesem Verfahren hergestellter Organismus, bevorzugt ist der Organismus eine Pflanze. Das Verfahren eignet sich auch für die Genexpression der oben genannten biologisch aktiven Derivate und Fragmente .The sequences selected by the method described above can advantageously be introduced into an organism. A further subject of the invention is therefore an organism produced by this method, the organism preferably being a plant. The method is also suitable for gene expression of the above-mentioned biologically active derivatives and fragments.
Anschließend erfolgt die Regeneration ganzer Pflanzen und Überprüfung der Resistenz gegenüber dem Herbizid in intakten Pflanzen.Then whole plants are regenerated and the resistance to the herbicide in intact plants is checked.
Veränderte Proteine und/oder Nukleinsäuren, die in Pflanzen Resistenz gegen Herbizide vermitteln können, können aus den hierin beschriebenen, erfindungsgemäßen Sequenzen, insbesondere aus den Sequenzen SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,Modified proteins and / or nucleic acids which can impart resistance to herbicides in plants can be obtained from the sequences according to the invention described herein, in particular from the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO : 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO 34, SEQ ID NO: 36,SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO 34, SEQ ID NO: 36,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO: 46,SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO 44, SEQ ID NO: 46,
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58, SEQ ID NO: 60 SEQ ID NO 62,SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO 58, SEQ ID NO: 60 SEQ ID NO 62,
SEQ ID NO: 64/ SEQ ID NO 66, SEQ ID NO: 68 SEQ ID NO 70,SEQ ID NO: 64 / SEQ ID NO 66, SEQ ID NO: 68 SEQ ID NO 70,
SEQ ID NO: 72, SEQ ID NO 74, SEQ ID NO: 76 SEQ ID NO 78,SEQ ID NO: 72, SEQ ID NO 74, SEQ ID NO: 76 SEQ ID NO 78,
SEQ ID NO: 80, SEQ ID NO 82, SEQ , ID NO : 84 SEQ ID NO 86,SEQ ID NO: 80, SEQ ID NO 82, SEQ , ID NO: 84 SEQ ID NO 86,
SEQ ID NO: 88, SEQ ID NO 90, SEQ ID NO: 92 SEQ ID NO 94,SEQ ID NO: 88, SEQ ID NO 90, SEQ ID NO: 92 SEQ ID NO 94,
SEQ ID NO: 96, SEQ ID NO 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 oder deren Derivaten aus anderen Pflanzen auch über die sogenannte "site directed muta- genesis" hergestellt werden. Durch diese Mutagenese kann beispielsweise die Stabilität und/oder enzymatische Aktivität von Enzymen oder die Eigenschaften wie Bindung von niedermolekularen Verbindungen mit kleiner 1000 Dalton, vorteilhaft kleiner 900 Dalton, bevorzugt kleiner 800, besonders bevorzugt kleiner 700, ganz besonders bevorzugt kleiner 600 Dalton, von Proteinen oder Antisense-RNA sehr gezielt verbessern bzw. verändert werden.SEQ ID NO: 96, SEQ ID NO 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 or their derivatives from other plants also via the so-called "site directed mutagenesis " getting produced. This mutagenesis can, for example, the stability and / or enzymatic activity of enzymes or the properties such as binding of low molecular weight compounds with less than 1000 daltons, advantageously less than 900 daltons, preferably less than 800, particularly preferably less than 700, very particularly preferably less than 600 daltons, of proteins or improve or change antisense RNA in a very targeted manner.
Weiterhin können Veränderungen über die von Spee et al . (Nucleic Ac'ids Research, Vol. 21, No. 3, 1993: 777-78) beschriebenen PCR-Methode unter Verwendung von dITP zur zufälligen Mutagenese erzielt werden oder durch die von Rellos et al. (Protein Expr. Purif., 5, 1994: 270-277) weiter verbessert Methode.Furthermore, changes over the Spee et al. (Nucleic Ac ' ids Research, Vol. 21, No. 3, 1993: 777-78) can be achieved using the PCR method using dITP for random mutagenesis or by the method described by Rellos et al. (Protein Expr. Purif., 5, 1994: 270-277) further improved method.
Eine weitere Möglichkeit zur Herstellung dieser veränderten Proteine und/oder von Nukleinsäuren ist eine von Stemmer et al. (Proc. Natl. Acad. Sei. USA, Vol. 91, 1994: 10747-10751) beschriebene "in vitro" Rekombinationstechnik für die molekulare Evolution oder die von Moore et al. (Nature Biotechnology Vol. 14, 1996: 458-467) beschriebene Kombination der PCR- und Rekombinationsmethode .Another possibility for producing these modified proteins and / or nucleic acids is one of Stemmer et al. (Proc. Natl. Acad. Sci. USA, Vol. 91, 1994: 10747-10751) described "in vitro" recombination technique for molecular evolution or that described by Moore et al. (Nature Biotechnology Vol. 14, 1996: 458-467) described combination of the PCR and recombination method.
Ein weiterer Weg zur Mutagenese von Nukleinsäuren bzw. Proteinen wird von Greener et al. in Methods in Molecular Biology (Vol. 57, 1996: 375-385)" beschrieben. In EP-A-0 909 821,wird eine Methode zur Veränderung von Proteinen unter Verwendung des Mikroorganismus E. coli XL-1 Red beschrieben. Dieser Mikroorganismus erzeugt bei der Replikation Mutationen in den eingeführten Nukleinsäuren und führt so zu einer Veränderung der genetischen Information. Über Isolierung der veränderten Nukleinsäuren bzw. der veränderten Proteine und Testung auf Resistenz lassen sich leicht vorteilhafte Nukleinsäuren und die durch sie codierten Proteine und umgekehrt identifizieren. Diese können dann nach Einbringen in Pflanzen dort die Resistenz ausprägen und so zur Resistenz gegen die Herbizide führen.Another route to mutagenesis of nucleic acids or proteins is described by Greener et al. in Methods in Molecular Biology (Vol 57, 1996:. 375-385). described "In EP-A-0909821, a method for modifying proteins using the microorganism E. coli is described XL-1 Red This microorganism. generates mutations in the inserted nucleic acids during replication and thus leads to a change in the genetic information By isolating the changed nucleic acids or the changed proteins and testing for resistance, it is easy to identify advantageous nucleic acids and the proteins encoded by them and vice versa then develop resistance there after introduction into plants and thus lead to resistance to the herbicides.
Weitere Methoden der Mutagenese und Selektion sind beispiels- weise Methoden wie die in vivo Mutagenese von Samen oder Pollen und Selektion resistenter Allele in Anwesenheit der erfindungsgemäßen Inhibitoren, gefolgt von genetischer und molekularer Identifizierung des veränderten, resistenten Allels . Weiterhin die Mutagenese und Selektion von Resistenzen in der Zell- .kultur durch Vermehrung der Kultur in Anwesenheit von sukzessiv steigenden Konzentrationen der erfindungsgemäßen Inhibitoren. Dabei kann die Erhöhung der spontanen Mutationsrate durch chemische/physikalische mutagene Behandlung ausgenutzt werden. Wie vorgehend beschrieben lassen sich auch mit Mikroorgansimen, die eine endogene oder rekombinante Aktivität der durch die im erfindungsgemäßen- Verfahren verwendeten Nukleinsäuren codierten Proteine haben, und die gegenüber den erfindungsgemäß identifizierten Inhibitoren sensitiv sind, veränderte Gene isolieren. Die Anzucht der Mikroorgansimen auf Medien mit steigenden Konzentrationen von erfindungsgemäßen Inhibitoren erlaubt die Selektion und Evolution von resistenten Varianten der erfindungsgemäßen Targets-.- Die Frequenz der Mutationen kann wiederum durch muta- genen Behandlungen erhöht werden.Further methods of mutagenesis and selection are, for example, methods such as the in vivo mutagenesis of seeds or pollen and selection of resistant alleles in the presence of the inhibitors according to the invention, followed by genetic and molecular ones Identification of the modified, resistant allele. Furthermore, mutagenesis and selection of resistances in cell culture by increasing the culture in the presence of successively increasing concentrations of the inhibitors according to the invention. The increase in the spontaneous mutation rate can be exploited by chemical / physical mutagenic treatment. As described above, modified genes can also be isolated with microorganisms which have an endogenous or recombinant activity of the proteins coded by the nucleic acids used in the method according to the invention and which are sensitive to the inhibitors identified according to the invention. The cultivation of the microorganisms on media with increasing concentrations of inhibitors according to the invention allows the selection and evolution of resistant variants of the targets according to the invention. The frequency of the mutations can in turn be increased by mutagenic treatments.
Daneben stehen Verfahren zur gezielten Veränderungen von Nukleinsäuren zur Verfügung (Zhu et al. Proc. Natl. Acad. Sei. USA, Vol. 96, 8768-8773 und Beethem et al., Proc. Natl. Acad. Sei. USA, Vol 96, 8774-8778) . Diese Methoden ermöglichen es, in den Proteinen solche Aminosäuren, die für die Bindung von Inhibitoren von Bedeutung sind, durch funktionell äquivalente Aminosäuren zu ersetzen, die jedoch die Bindung des Inhibitors verhindern.In addition, methods for the targeted modification of nucleic acids are available (Zhu et al. Proc. Natl. Acad. Sei. USA, Vol. 96, 8768-8773 and Beethem et al., Proc. Natl. Acad. Sei. USA, Vol 96 , 8774-8778). These methods make it possible to replace in the proteins those amino acids which are important for the binding of inhibitors by functionally equivalent amino acids which, however, prevent the binding of the inhibitor.
Ein weiterer Erfindungsgegenstand ist weiterhin ein Verfahren zur Erstellung von Nukleσtidsequenzen welche für Genprodukte codieren, die eine veränderte biologische Aktivität aufweisen, wobei die biologische Aktivität dahingegen verändert wurde, dass eine erhöhte Aktivität vorliegt. Unter erhöhter Aktivität ist eine gegenüber dem Ausgangsorganismus bzw. gegenüber dem Ausgangsgenprodukt um mindestens 10 %, bevorzugt um mindestens 30 %, besonders bevorzugt um mindestens 50 % oder 70 %, ganze besonders bevorzugt um mindestens 100 % höhere Aktivität zu verstehen. Weiterhin kann die biologische Aktivität dahingegen verändert worden sein, dass die erfindungsgemäßen Substanzen und/oder Mittel nicht mehr oder nicht mehr richtig an die Nukleinsäuresequenzen und/oder die durch sie codierten Genprodukte binden. Unter nicht mehr oder nicht mehr richtig ist im Sinne der Erfindung zu verstehen, dass die Substanzen um mindestens 30 %, bevorzugt mindestens 50 %, besonders bevorzugt um mindestens 70 %, ganz besonders bevorzugt um mindestens 80 % oder gar nicht mehr an die veränderten Nukleinsäuren und/oder Genprodukte im Vergleich zum Ausgangsgenprodukt oder den Ausgangsnukleinsäuren binden. Noch ein weiterer Aspekt der Erfindung betrifft deshalb eine nach dem oben beschriebenen erfindungsgemäßen Verfahren genetisch veränderte transgene Pflanze.Another object of the invention is a method for creating nucleotide sequences which code for gene products which have a changed biological activity, the biological activity being changed in contrast to the fact that there is increased activity. Increased activity is to be understood as meaning an activity which is at least 10%, preferably at least 30%, particularly preferably at least 50% or 70%, very particularly preferably at least 100% higher than that of the starting organism or of the starting gene product. Furthermore, the biological activity may have been changed so that the substances and / or agents according to the invention no longer or no longer bind correctly to the nucleic acid sequences and / or the gene products encoded by them. For the purposes of the invention, no longer or no longer correctly means that the substances have at least 30%, preferably at least 50%, particularly preferably at least 70%, very particularly preferably at least 80% or not at all of the modified nucleic acids and / or bind gene products in comparison to the starting gene product or the starting nucleic acids. Yet another aspect of the invention therefore relates to a transgenic plant genetically modified by the method according to the invention described above.
Genetisch veränderten transgene Pflanzen, die gegen die nach den erfindungsgemäßen Verfahren gefundenen Substanzen und/oder Mittel enthaltend diese Substanzen resistent sind, können auch durch Überexpression der in den erfindungsgemäße Verfahren verwendeten Nukleinsäuren, insbesondere SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,Genetically modified transgenic plants which are resistant to the substances and / or agents containing these substances found by the method according to the invention can also be overexpressed by the nucleic acids used in the method according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,
SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: -40, SEQ ID NO:" 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66-, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80," SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, "SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 erzeugt werden. -Deshalb ist ein weiterer Erfindungsgegenstand ein Verfahren zur Erzeugung transgener Pflanzen, die gegen Substanzen, die nach einem erfindungsgemäßen Verfahren gefunden wurden, resistent sind, dadurch gekennzeichnet, dass in diesen Pflanzen erfindungsgemäße Nukleinsäuren mit einer der beschriebenen biologischen Aktivität, insbesondere mit den Sequenzen SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: -34, SEQ ID NO: 36/ SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ' ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: -86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 überexprimiert werden. Ein ähnliches Verfahren wird beispielhaft in Lermantova et al., Plant Physiol., 122, 2000: 75-83 beschrieben. Natürlich können auch die hierin genannten Derivate und Fragmente, z.B. aus anderen Pflanzen, verwendet werden, die die gewünschte Aktivität aufweisen. Die oben beschriebenen erfindungsgemäßen Verfahren zur Erzeugung resistenter Pflanzen ermöglichen die Entwicklung neuer Herbizide, die eine möglichst umfassende Pflanzenspezies unabhängige Wirkung aufweisen (sog. Totalherbizide) , in Kombination mit der Ent- wicklung von gegenüber dem Totalherbizid resistenten Nutzpflanzen. Gegenüber Totalherbiziden resistente Nutzpflanzen sind bereits verschiedentlich beschrieben worden. Dabei können mehrere Prinzipien zur Erzielung einer Resistenz unterschieden werden:SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: -40, SEQ ID NO: "44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66-, SEQ ID NO: 68, SEQ ID NO : 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, " SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, " SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102 , SEQ ID NO: 106 or SEQ ID NO: 108. Therefore, a further subject of the invention is a method for producing transgenic plants which are resistant to substances found by a method according to the invention, characterized in that in these plants Nucleic acids according to the invention with one of the described biological activis act, in particular with the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: -34, SEQ ID NO: 36 / SEQ ID NO : 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ' ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: -86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO : 108 overexpressed. A similar method is described by way of example in Lermantova et al., Plant Physiol., 122, 2000: 75-83. Of course, the derivatives and fragments mentioned here, for example from other plants, which have the desired activity can also be used. The methods according to the invention for producing resistant plants described above enable the development of new herbicides which have the most comprehensive possible plant species independent action (so-called total herbicides), in combination with the development of useful plants which are resistant to the total herbicide. Useful plants resistant to total herbicides have already been described in various ways. There are several principles for achieving resistance:
a) Resistenzerzeugung in einer Pflanze über Mutationsverfahren oder gentechnische" Verfahren, indem das als Zielort für das Herbizid dienende Protein deutlich überproduziert wird und indem auf Grund des großen Überschusses des als Zielort für das"Herbizid dienende Protein, die von diesem Protein in der Zelle ausgeübte Funktion auch nach -Applikation des Herbizides beibehalten wird.a) Generation of resistance in a plant via mutation methods or recombinant "process by which a destination serving for the herbicide protein is significantly overproduced and by due to the large excess of as target for the" serving herbicide protein, the force exerted by this protein in the cell Function is maintained even after application of the herbicide.
b) Veränderung der Pflanze dahingehend, dass eine modifizierte Version des als Zielort des Herbizid fungierenden Proteins eingeführt wird und dass das neu eingeführte modifizierte Protein vom Herbizid nicht in seiner Funktion beeinträchtigt wird.b) Modification of the plant in such a way that a modified version of the protein which acts as the target of the herbicide is introduced and that the function of the newly introduced modified protein is not impaired by the herbicide.
c) Veränderung der Pflanze dahingehend, dass ein neues Protein/ eine neue RNA eingeführt wird, welche (s) dadurch gekennzeichnet ist, dass die für die herbizide Wirkung der niedermolekularen Substanz verantwortliche chemische Struktur des Proteins oder der Nukleinsäure, wie der RNA oder der DNA, so verändert wird, dass durch die veränderte Struktur keine herbizide Wirkung mehr entfaltet- werden kann, oder das Herbizid in der veränderten Pflanze inaktiviert oder modifiziert wird, z.B. abgebaut wird, nicht aufgenommen oder nicht transportiert wird oder in die Vakuόle transportiert wird, etc., d.h. die Interaktion des Herbizids mit dem .Zielort nicht mehr erfolgen kann.c) Modification of the plant such that a new protein / new RNA is introduced, which is characterized in that the chemical structure of the protein or nucleic acid, such as RNA or DNA, which is responsible for the herbicidal action of the low molecular weight substance is changed in such a way that the modified structure no longer has a herbicidal action, or the herbicide in the modified plant is inactivated or modified, for example is broken down, not taken up or not transported or transported into the vacuum, etc., i.e. the interaction of the herbicide with the target location can no longer take place.
d) dass die Funktion des Targets durch eine neue in die Pflanze eingebrachte Nukleinsäure, z.B. ein Gen ersetzt wird, wobei die Nukleinsäure für ein Genprodukt codiert, das geringer oder gar nicht von der herbiziden Substanz in seiner Funktion getrennt wird. So kann z.B. ein sogenannter "alternativer Pathway" geschaffen werden. e) dass die Funktion des Targets durch ein anderes in der Pflanze vorhandenes oder in die Pflanze eingebrachtes Gen bzw. dessen Genprodukt übernommen wird.d) that the function of the target is replaced by a new nucleic acid, for example a gene, which is introduced into the plant, the nucleic acid coding for a gene product whose function is separated less or not at all from the herbicidal substance. For example, a so-called "alternative pathway" can be created. e) that the function of the target is taken over by another gene present in the plant or introduced into the plant or its gene product.
Die vorliegende Erfindung umfasst daher weiterhin die Verwendung von Pflanzen, die durch die T-DNA Insertion getroffene Gene mit den in dem erfindungsgemäßen Verfahren verwendeten Nukleinsäuresequenzen, insbesondere SEQ ΪD NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, ■The present invention therefore also includes the use of plants which genes hit by the T-DNA insertion with the nucleic acid sequences used in the method according to the invention, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, ■
SEQ ID NO: 13, SE -Q ID NO: 1"5"", SEQ ID NO : 17, SEQ ID NO: 26,SEQ ID NO: 13, SE -Q ID NO: 1 "5"" , SEQ ID NO: 17, SEQ ID NO: 26,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,
SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,
SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84,SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84,
SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92,SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92,
SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100,SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100,
SEQ ID NO: 102- , SEQ ID NO : 11006 oder SEQ ID NO: 108 oder die weiteren genannten Sequenzen, z.B. Fragmente und Derivate, z.B. aus anderen Pflanzen, enthalten, zur Entwicklung von neuen Herbiziden. Dem Fachmann sind alternative Verfahren zur Identifizierung von homologen Nukleinsäuren beispielsweise in anderen Pflanzen mit ähnlichen Sequenzen wie unter Verwendung von Trans- posons, bekannt. Gegenstand dieser Erfindung ist daher auch die Verwendung von alternativen Insertionsmutageneseverfahren zur Insertion von fremder Nukleinsäure in die hierin beschriebenen, erfindungsgemäßen Nukleinsäuresequenzen, insbesondere SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 102-, SEQ ID NO: 11006 or SEQ ID NO: 108 or the other sequences mentioned, e.g. Fragments and derivatives, e.g. from other plants, included, for the development of new herbicides. Alternative methods for identifying homologous nucleic acids, for example in other plants with sequences similar to those using transposons, are known to the person skilled in the art. This invention therefore also relates to the use of alternative insertion mutagenesis methods for inserting foreign nucleic acid into the nucleic acid sequences according to the invention described herein, in particular SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO:' 11, SEQ ID NO: 13, SEQ ID NO: 15,SEQ ID NO: 9, SEQ ID NO: '11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO 26, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 17, SEQ ID NO 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO 34, SEQ ID NO: 36, .SEQ ID NO: 38,SEQ ID NO: 32, SEQ ID NO 34, SEQ ID NO: 36, .SEQ ID NO: 38,
SEQ ID NO: 40, SEQ ID NO 44,' SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 40, SEQ ID NO 44, ' SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO 52, SEQ ID NO: 54, SEQ ID NO: 56,
SEQ ID NO: 58, SEQ ID NO 60, SEQ ID NO: 62., SEQ ID NO: 64,SEQ ID NO: 58, SEQ ID NO 60, SEQ ID NO: 62., SEQ ID NO: 64,
SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 66, SEQ ID NO 68, SEQ ID NO: 70, SEQ ID NO: 72,
SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO: 78, SEQ ID NO: 80,SEQ ID NO: 74, SEQ ID NO 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO 92, SEQ ID NO: 94, SEQ ID NO: 96,SEQ ID NO: 82, SEQ ID NO 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO 92, SEQ ID NO: 94, SEQ ID NO: 96,
SEQ ID NO: 98, SEQ ID NO 100, SEQ IDNO: 102, SEQ ID O: 106 oder SEQ ID NO: 108 in von diesen Sequenzen aufgrund des genetischen Codes abgeleitete Sequenzen und/oder deren Derivate oder Fragmente z.B. aus anderen Pflanzen. ω ω toSEQ ID NO: 98, SEQ ID NO 100, SEQ IDNO: 102, SEQ ID O: 106 or SEQ ID NO: 108 in sequences derived from these sequences on the basis of the genetic code and / or their derivatives or fragments, for example from other plants. ω ω to
LΠ O in o o σ LΠLΠ O in o o σ LΠ
Eine weitere Ausführungsform sind Mittel, enthaltend eine das Wachstum regulierende Menge mindestens einer Substanz identifiziert nach den erfindungsgemäßen Verfahren oder eines- Anta- gonisten identifiziert nach einem erfindungsgemäßen Verfahren und mindestens einen inerten flüssigen und/oder festen Trägerstoff sowie gegebenenfalls mindestens einen oberflächenaktiven Stoff.A further embodiment are agents comprising a growth-regulating amount of at least one substance identified by the method according to the invention or an antagonist identified by a method according to the invention and at least one inert liquid and / or solid carrier and optionally at least one surface-active substance.
Diese erfindungsgemäßen Substanzen oder Mittel mit ihrer her- biziden Wirkung können als Defoliants, Desiccants, Kraut- abtötungsmittel und insbesondere- als Unkrautvernichtungsmittel verwendet werden. Unter Unkraut im weitesten Sinne sind alle Pflanzen zu verstehen, die an Orten aufwachsen, an denen sie unerwünscht sind. Ob die mit Hilfe der erfindungsgemäßen Verfahren gefundenen Substanzen bzw. Wirkstoffe als totale oder selektive Herbizide wirken, hängt unter anderem von der angewandten Menge, ihrer Selektivität und weiteren Faktoren ab. Die Substanzen können beispielsweise gegen folgende Unkräuter verwendet werden:These substances or compositions according to the invention with their herbicidal action can be used as defoliants, desiccants, herbicides and in particular as weed killers. Weeds in the broadest sense are understood to mean all plants that grow up in places where they are undesirable. Whether the substances or active substances found with the aid of the methods according to the invention act as total or selective herbicides depends, inter alia, on the amount used, their selectivity and other factors. The substances can be used against the following weeds, for example:
Dikotyle Unkräuter der Gattungen:Dicot weeds of the genera:
Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, -Taraxacum.Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippernia, Rorippa, Rorippa, Rorippa Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, -Taraxacum.
Monokotyle Unkräuter der Gattungen:Monocot weeds of the genera:
Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Braehiaria, Lolium, Bromus, Ayena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyl- octenium, Agrostis, Alopecurus, Apera.Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Braehiaria, Lolium, Bromus, Ayena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Sphenumum, Ischaemum octenium, agrostis, alopecurus, apera.
In Abhängigkeit von der jeweiligen Applikationsmethode können die im erfindungsgemäßen Verfahren identifizierten Substanzen bzw. sie enthaltende Mittel vorteilhaft noch in einer weiteren Zahl von Kulturpflanzen zur Beseitigung unerwünschter Pflanzen eingesetzt werden. In Betracht kommen beispielsweise folgende Kulturen:Depending on the respective application method, the substances identified or agents containing them in the process according to the invention can advantageously also be used in a further number of crop plants for eliminating undesired plants. The following crops are considered, for example:
Allium cepa, Ananas comosus, Arachis hypogaea, Asparagus officinalis, Beta vulgaris spec. altissima, Beta vulgaris spec. rapa, Brassica napus var. napus, Brassica napus var. napo- brassica, Brassica rapa var. silvestris, Camellia sinensis,Allium cepa, Ananas comosus, Arachis hypogaea, Asparagus officinalis, Beta vulgaris spec. altissima, Beta vulgaris spec. rapa, Brassica napus var. napus, Brassica napus var. napos-brassica, Brassica rapa var. silvestris, Camellia sinensis,
Carthamus tinetorius, Carya illinoinensis, Citrus limon, Citrus sinensis, Coffea arabica (Coffea canephora, Coffea liberica) , Cucumis sativus, Cynodon dactylon, Daucus carota, Elaeis guineensis, Fragaria vesca, Glycine max, Gossypium hirsutum, (Gossypium arboreum, Gossypium herbaceum, Gossypium vitifoliu ) , Helianthus annuus, Hevea brasiliensis, Hordeum vulgäre, Humulus lupulus, Ipomoea batatas, Juglans regia, Lens culinaris, Linum usitatissimum, Lycopersicon lycopersicum, Malus spec, Manihot esculenta, Medicago sativa, Musa spec., Nicotiana tabacum (N. rustica) , Olea europaea, Oryza sativa, Phaseolus lunatus, Phaseolus vulgaris, Picea abies, Pinus spec, Pisum sativum, Prunus avium, Prunus persica, Pyrus communis, Ribes sylestre, Ricinus communis, Saccharum officinarum, Seeale cereale, Solanum tuberosum, Sorghum bicolor (s. vulgäre), Theobroma cacao, Tri- folium pratense, Triticum aestivum, Triticum durum, Vicia faba, Vitis vinifera, Zea mays.Carthamus tinetorius, Carya illinoinensis, Citrus limon, Citrus sinensis, Coffea arabica (Coffea canephora, Coffea liberica), Cucumis sativus, Cynodon dactylon, Daucus carota, Elaeis guineensis, Fragaria vesca, Glycine max, Gossypium hirsutum, (Gossypium arboreum, Gossypium herbaceum, Gossypium vitifoliu), Helianthus annuus, Hevea brasiliensis, Hordeulus lupusasus, Hordeulus lupusasus, Hordeulus lupusis, Hordeulus lupusia Lens culinaris, Linum usitatissimum, Lycopersicon lycopersicum, Malus spec, Manihot esculenta, Medicago sativa, Musa spec., Nicotiana tabacum (N. rustica), Olea europaea, Oryza sativa, Phaseolus lunatus, Phaseolus vulgaris, Picea abies, Pinus spec., Pisumativ , Prunus avium, Prunus persica, Pyrus communis, Ribes sylestre, Ricinus communis, Saccharum officinarum, Seeale cereale, Solanum tuberosum, Sorghum bicolor (see vulgar), Theobroma cacao, Trifolium pratense, Triticum aestivum, Triticum durum Vitis vinifera, Zea mays.
Die nach dem erfindungsgemäßen Verfahren gefundenen Substanzen können vorteilhaft auch in Kulturen verwandt werden, die durch Züchtung einschließlich gentechnischer Methoden gegen die Wirkung von Herbiziden tolerant sind.The substances found by the process according to the invention can advantageously also be used in crops which are tolerant to the action of herbicides by breeding, including genetic engineering methods.
Die erfindungsgemäßen Substanzen bzw. die sie enthaltenden herbiziden Mittel können beispielsweise in Form von direkt versprühbaren wässrigen Lösungen, Pulvern, Suspensionen, auch hochprozentigen wässrigen, öligen oder sonstigen Suspensionen oder Dispersionen, Emulsionen, Öldispers.ionen, Pasten, Stäubemitteln, Streumitteln oder Granulaten durch Versprühen, Vernebeln, Verstäuben, Verstreuen oder Gießen angewendet werden. Die Anwendungsformen richten sich nach den Verwendungszwecken; sie sollten in jedem Fall möglichst die feinste Verteilung der erfindungsgemäßen Wirkstoffe gewährleisten.The substances according to the invention or the herbicidal compositions comprising them can be sprayed, for example, in the form of directly sprayable aqueous solutions, powders, suspensions, including high-strength aqueous, oily or other suspensions or dispersions, emulsions, oil dispersions, pastes, dusts, sprinkling agents or granules , Atomizing, dusting, scattering or pouring can be used. The application forms depend on the purposes; in any case, they should ensure the finest possible distribution of the active compounds according to the invention.
Als inerte flüssige und/oder feste Trägerstoffe kommen flüssige Zusatzstoffe wie Mineralölfraktionen von mittlerem bis hohem Siedepunkt, wie Kerosin oder Dieselöl,' ferner Kohlenteeröle sowie Öle pflanzlichen oder tierischen Ursprungs, aliphatisehe, cyclische und aromatische Kohlenwasserstoffe, z.B. Paraffin, Tetrahydronaphthalin, . alkylierte Naphthaline oder deren Derivate, alkylierte Benzole oder deren Derivate, Alkohole wie Methanol, Ethanol, Propanol, Butanol, Cyclohexanol, Ketone wie Cyclohexanon oder stark polare Lösungsmittel, z.B. Amine wie N-Methyl- pyrrolidon oder Wasser in Frage .Suitable inert liquid and / or solid carriers, liquid additives such as mineral oil fractions of medium to high boiling point, such as kerosene or diesel oil, 'furthermore coal tar oils and oils of vegetable or animal origin, aliphatisehe, cyclic and aromatic hydrocarbons, for example paraffin, tetrahydronaphthalene,. alkylated naphthalenes or their derivatives, alkylated benzenes or their derivatives, alcohols such as methanol, ethanol, propanol, butanol, cyclohexanol, ketones such as cyclohexanone or strongly polar solvents, for example amines such as N-methylpyrrolidone or water.
Weitere vorteilhafte Anwendungsformen der erfindungsgemäßen Substanzen und/oder Mittel sind Wässrige Anwendungsformen wie Emulsionskonzentrate, Suspensionen, Pasten, netzbaren Pulvern oder wasserdispergierbaren Granulaten, die beispielsweise durch Zusatz von Wasser bereitet werden können. Zur Herstellung von Emulsionen, Pasten oder Öldispersionen können die Substanzen und/oder Mittel die sog. Substrate als solche oder in einem Öl oder Lösungsmittel gelöst, mittels Netz-, Haft-, Dispergier- oder Emulgiermittel in Wasser homogenisiert werden. Es können aber auch aus wirksamer Substanz, Netz-, Haft-, Dispergier- oder Emulgiermittel und eventuell Lösungsmittel oder Öl bestehende " Konzentrate hergestellt werden, die zur Verdünnung mit Wasser geeignet sind.Further advantageous forms of use of the substances and / or agents according to the invention are aqueous forms of use, such as emulsion concentrates, suspensions, pastes, wettable powders or water-dispersible granules, which can be prepared, for example, by adding water. For production of Emulsions, pastes or oil dispersions, the substances and / or agents, the so-called substrates as such or dissolved in an oil or solvent, can be homogenized in water by means of wetting agents, adhesives, dispersants or emulsifiers. However, it is also possible to prepare concentrates which consist of an active substance, wetting agent, tackifier, dispersant or emulsifier and possibly solvent or oil and which are suitable for dilution with water.
Als oberflächenaktive Stoffe kommen die Alkali-', Erdalkali-, Ammoniumsalze von aromatischen Sulfoήsäureh, z.B. Lignin-, Phenol-, Naphthalin- und Dibutylnaphthalinsulfonsäure, sowie ' von Fettsäuren, Alkyl- und Alkylarylsulfonaten, Alkyl-, Lauryl-„ ether- und Fettalkoholsulfaten, sowie Salze sulfatierter Hexa-, Hepta- und Octadecanolen sowie von Fettalkoholglykolether, Kondensationsprodukte von sulfonierte Naphthalin und seiner Derivate mit Formaldehyd, Kondensationsprodukte des Naphthalins bzw. der Naphthalinsulfonsäuren mit Phenol und Pormaldehyd, Poly- oxyethylenoctylphenolether, ethoxyliertes Isooctyl-, Octyl- oder Nonylphenol, Alkylphenyl-, Tributylphenylpolyglykolether, Alkyl- arylpolyetheralkohole, Isotridecylalkohol, Fettalkoholethylen- oxid-Kondensate, ethoxyliertes Rizinusöl, Polyoxyethylenalkyl- ether oder Polypxypropylenalkylether, Laurylalkoholpolyglykol- etheracetat, Sorbitester, Lignin-Sulfitablaugen oder Methyl- cellulose in Betracht .Suitable surfactants are the alkali metal ', alkaline earth metal and ammonium salts of aromatic Sulfoήsäureh, for example ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and' from fatty acids, alkyl- and alkylarylsulfonates, alkyl, lauryl "ether and fatty alcohol sulfates, as well as salts of sulfated hexa-, hepta- and octadecanols and of fatty alcohol glycol ether, condensation products of sulfonated naphthalene and its derivatives with formaldehyde, condensation products of naphthalene or naphthalenesulfonic acids with phenol and pormaldehyde, polyoxyethylene octylphenol ether, ethoxylated isooctyl, phenyl, octyl, octyl, phenyl -, Tributylphenyl polyglycol ether, alkyl aryl polyether alcohols, isotridecyl alcohol, fatty alcohol ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ether or polypxypropylene alkyl ether, lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin sulfite lye or methyl cellulose.
Pulver-, Streu- und Stäubemittel können als feste Trägerstoffe vorteilhaft durch Mischen oder gemeinsames Vermhlen der wirksamen Substanzen mit einem festen Trägerstoff hergestellt werden.Powders, materials for broadcasting and dusts can advantageously be produced as solid carriers by mixing or grinding the active substances together with a solid carrier.
Granulate, z.B. Umhüllungs-, Iprägnierungs- und Homogengranulate können- durch Bindung der Wirkstoffe an feste Trägerstoffe hergestellt werden. Feste Trägerstoffe sind beispielsweise Mineralerden wie Kieselsäuren, Kieselgele, Silikate, Talkum, Kaolin, Kalkstein, Kalk, Kreide, Bolus, Löß, Ton, Dolomit, Diatomeenerde, Calcium- und Magnesiutαsulfat, Magnesiumoxid, gemahlene Kunststoffe, Düngemittel, wie Ammoniumsulfat, Ammoniumphosphat, Ammoniurαnitrat, Harnstoffe und pflanzliche Produkte wie Getreidemehl, Baumrinden-, Holz- und Nussschalen ehl, Cellulosepulver oder andere feste Trägerstoffe .-Granules, e.g. Coating, impregnation and homogeneous granules can be produced by binding the active ingredients to solid carriers. Solid carriers are, for example, mineral earths such as silicas, silica gels, silicates, talc, kaolin, limestone, lime, chalk, bolus, loess, clay, dolomite, diatomaceous earth, calcium and magnesium sulfate, magnesium oxide, ground plastics, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate , Ureas and vegetable products such as flour, tree bark, wood and nutshells, cellulose powder or other solid carriers.
Die Konzentrationen der erfindungsgemäßen Substanzen und/oder Mittel in den anwendungsfertigen Zubereitungen können in weiten Bereichen variiert werden. Die Formulierungen enthalten im all- gemeinen 0,001 bis 98 Gew.-%, vorzugsweise 0,01 bis 95 Gew.-%, mindestens eines Wirkstoffs. Die Wirkstoffe werden dabei in einer Reinheit von 90 % bis 100 %, vorzugsweise 95 % bis 100 % (nach NMR-Spektrum) eingesetzt.The concentrations of the substances and / or agents according to the invention in the ready-to-use preparations can be varied within a wide range. The formulations generally contain 0.001 to 98% by weight, preferably 0.01 to 95% by weight, of at least one active ingredient. The active ingredients are in one Purity from 90% to 100%, preferably 95% to 100% (according to the NMR spectrum).
Die Applikation der herbiziden Mittel bzw. der Substanzen kann im Vorauflauf- oder im Nachauflaufverfahren erfolgen. Sind die Wirkstoffe für gewisse Kulturpflanzen weniger verträglich, so können Ausbringungstechniken angewandt werden, bei welchen die herbiziden Mittel oder Substanzen mit Hilfe der Spritzgeräte so gespritzt werden, dass die Blätter der empfindlichen Kulturpflanzen nach Möglichkeit nicht getroffen werden, während die Wirkstoffe auf die Blätter darunter wachsender unerwünschter Pflanzen oder die unbedeckte Bodenfläche gelangen (post-directed, lay-by) .The herbicidal compositions or the substances can be applied pre- or post-emergence. If the active ingredients are less compatible for certain crop plants, application techniques can be used in which the herbicidal compositions or substances are sprayed with the aid of sprayers in such a way that the leaves of the sensitive crop plants are not struck wherever possible, while the active ingredients are applied to the leaves below them unwanted plants or the uncovered floor area (post-directed, lay-by).
Zur Verbreiterung des WirkungsSpektrums und zur Erzielung syneirgistischer Effekte können die erfindungsgemäßen Substanzen und/oder Mittel mit zahlreichen Vertretern anderer herbizider oder wachstumsregulierender Wirkstoffgruppen gemischt und gemeinsam ausgebracht werden. Beispielsweise kommen als Mischungs- partner 1, 2, 4-Thiadiazole, 1, 3, 4-Thiadiazole, Amide, Amino- phosphorsäure und deren Derivate, Aminotriazole, Anilide, (Het) -Aryloxyalkansäure und deren Derivate, Benzoesäure und deren Derivate, Benzothiadiazinone, 2-Aroyl-l, 3-cyclohexandione, Hetaryl-Aryl-Ketone, Benzylisoxazolidinone, Meta-CF3-phenyl- derivate, Carbamate, Chinolinsäure und deren Derivate, Chlor- acetanilide, Cyclohexan-1, 3-dionderivate, Diazine, Dichlor-" propionsäure und deren Derivate, Dihydrobenzofurane, Dihydro- furan-3-one, Dinitroaniline, Dinitrophenole, Diphenylether, Dipyridyle, Halogencarbonsäuren und deren Derivate, Harnstoffe, 3-Phenyluracile, Imidazole, Imidazolinone, N-Phenyl-3,4, 5, 6- tetrahydrophthalimide, Oxadiazole, Oxirane, Phenole, Aryloxy- oder Heteroaryloxyphenoxypropionsäureester, Phenylessigsäure und deren Derivate, Phenylpropionsäure und deren Derivate, Pyrazole, Phenylpyrazole, Pyridazine, Pyridincarbonsäure und deren Derivate, Pyrimidylether, Sulfonamide, Sulfonylharnstoffe, . Triazirie, Triazinone, Triazolinone, Triazolcarboxamide, Uracile in Betracht.In order to broaden the spectrum of activity and to achieve synergistic effects, the substances and / or agents according to the invention can be mixed with numerous representatives of other herbicidal or growth-regulating active ingredient groups and applied together. For example, 1, 2, 4-thiadiazoles, 1, 3, 4-thiadiazoles, amides, aminophosphoric acid and their derivatives, aminotriazoles, anilides, (het) -aryloxyalkanoic acid and their derivatives, benzoic acid and their derivatives, benzothiadiazinones , 2-aroyl-l, 3-cyclohexanediones, hetaryl aryl ketones, benzylisoxazolidinones, meta-CF3-phenyl derivatives, carbamates, quinolinic acid and their derivatives, chloroacetanilides, cyclohexane-1, 3-dione derivatives, diazines, dichloro- " propionic acid and its derivatives, dihydrobenzofurans, dihydrofuran-3-ones, dinitroanilines, dinitrophenols, diphenyl ethers, dipyridyls, halocarboxylic acids and their derivatives, ureas, 3-phenyluracils, imidazoles, imidazolinones, N-phenyl-3,4, 5, 6 - tetrahydrophthalimide, oxadiazole, oxirane, phenol, aryloxy- or heteroaryloxyphenoxypropionic acid ester, phenylacetic acid and its derivatives, phenylpropionic acid and its derivatives, pyrazoles, phenylpyrazoles, pyridazines, pyridinecarboxylic acid and their derivatives, pyrimidyls ether, sulfonamides, sulfonylureas,. Triazirie, Triazinone, Triazolinone, Triazolcarboxamide, Uracile into consideration.
Außerdem kann es von Nutzen sein, die erfindungsgemäßen Substanzen und/oder Mittel allein oder in Kombination mit anderen Herbiziden auch noch mit weiteren Pflanzenschutzmitteln gemischt, gemeinsam auszubringen, beispielsweise mit Mitteln zur Bekämpfung von Schädlingen oder phytopathogenen Pilzen bzw. Bakterien. Von Interesse ist ferner die Mischbarkeit mit Mineralsalzlösungen, welche zur Behebung von Ernährungs- und Spurenelementmängeln eingesetzt werden. Es können auch nichtphytotoxische Öle und Ölkonzentrate zugesetzt werden. Die Aufwandmengen an Wirkstoff (= Substanzen und/oder Mittel) betragen je nach Bekämpfungsziel, Jahreszeit, Zielpflanzen und WachstumsStadium -0,001 bis 3,0, vorzugsweise 0,01 bis 1,0 kg/ha aktive Substanz .It may also be useful to apply the substances and / or agents according to the invention, alone or in combination with other herbicides, mixed with other crop protection agents, for example with agents for controlling pests or phytopathogenic fungi or bacteria. Also of interest is the miscibility with mineral salt solutions, which are used to remedy nutritional and trace element deficiencies. Non-phytotoxic oils and oil concentrates can also be added. The application rates of active ingredient (= substances and / or agents) are -0.001 to 3.0, preferably 0.01 to 1.0 kg / ha of active substance, depending on the control target, season, target plants and growth stage.
Ein weiterer erfindungsgemäßer Gegenstand ist die Verwendung einer Substanz identifiziert nach einem der erfindungsgemäßen" Verfahren oder Mittel enthaltend diese Substanzen als Herbizid oder zur Wachstumsregulierung von Pflanzen.A further subject of the invention is the use of a substance identified by one of the "methods or compositions of the invention comprising these substances as a herbicide or for regulating the growth of plants.
Außerdem betrifft die Erfindung ein Kit, umfassend das erfindungsgemäße Nukleinsäurekonstrukt, der erfindungsgemäßen Substanzen, z.B. den erfindungsgemäßen Antikörper, das erfindungsgemäße Antisense-Nukleinsäuremolekül und/oder einen Antagonisten und/oder eine herbizide Substanz identifiziert gemäß den erfindungsgemäßen Verfahren sowie die unten beschriebene Zusammensetzung.The invention also relates to a kit comprising the nucleic acid construct according to the invention, the substances according to the invention, e.g. identifies the antibody according to the invention, the antisense nucleic acid molecule according to the invention and / or an antagonist and / or a herbicidal substance according to the methods according to the invention and the composition described below.
Ein weiterer erfindungsgemäßer Gegenstand ist eine Zusammen- Setzung, enthaltend die erfindungsgemäße Substanz, den erfindungsgemäßen Antikörper, das erfindungsgemäße Antisense- Nukleinsäurekonstrukt und/oder einen erfindungsgemäßen Antagonisten und/oder eine erfindungsgemäße Substanz identifiziert nach einem erfindungsgemäßen Verfahren.A further subject of the invention is a composition comprising the substance according to the invention, the antibody according to the invention, the antisense nucleic acid construct according to the invention and / or an antagonist according to the invention and / or a substance according to the invention identified by a method according to the invention.
Die Erfindung wird durch die nachstehenden Beispiele weiter veranschaulicht, die nicht als beschränkend aufgefasst werden sollten.The invention is further illustrated by the following examples, which should not be construed as limiting.
Beispiele:Examples:
a) Molekularbiologische Methodena) Molecular biological methods
Molekularbiologische Methoden wie sie hier eingesetzt wurden entsprechen dem Stand der Technik und sind an verschiedenen Stellen, wie bspw. Sambrock et al., Molecular Cloning, eds . , _ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), Reiter et al., Methods in Arabidopsis Research, World Sientific Press (1992), Schultz et al., Plant Molecular' Bio- logy Manual, Kluwer Academic Publishers (1998) und Martinez- Zapater und Salinas, Methods in Molecular Biology, Vol. 82: Arabidopsis Protocols eds., Humana Press Inc., Totowa, NJ. Diese Referenzen beschreiben die gängigen Ständardmethoden für die Produktion, Identifizierung und Klonierung von durch T-DNA-Insertionen hervorgerufenen Mutanten. Zusätzlich wurde für die Identifizierung von Insertionsstellen auf eine weitere gängige Methode, wie sie u.a. von Spertini et al., Biotechniques 27: 308-314 (1999) beschrieben wurde, zurückgegriffen. Die Sequenzierungen erfolgten durch die Firma AGOWA (Berlin) .Molecular biological methods as used here correspond to the state of the art and can be found at various points, such as Sambrock et al., Molecular Cloning, eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), Reiter et al., Methods in Arabidopsis Research, World Sientific Press (1992), Schultz et al., Plant Molecular 'Biological Manual, Kluwer Academic Publishers (1998) and Martinez-Zapater and Salinas, Methods in Molecular Biology, Vol. 82: Arabidopsis Protocols eds., Humana Press Inc., Totowa, NJ. These references describe the common standard methods for the production, identification and cloning of mutants caused by T-DNA insertions. In addition, another common method was used for the identification of insertion sites, as described, inter alia, by Spertini et al., Biotechniques 27: 308-314 (1999). The sequencing was carried out by AGOWA (Berlin).
b) Materialb) material
. Die verwendeten Chemikalien wurden, wenn im Text nicht gesondert angegeben, in p.A. -Qualität von den Firmen Fluka (Neu-Ulm) , Merck (Darmstadt) , Roth (Karlsruhe) , Serva (Heidelberg) und Sigma (Deideshofen) bezogen. Lösungen wurden unter Verwendung von reinem pyrogenfreiem Wasser, im Text als 1110 bezeichnet, aus einer Ionenaustauschanlage der Firma (TKA, Niederelbert) hergestellt. Restriktionsnukleasen, DNA-modifizierende Enzyme und molekularbiologische Kits und Oligonukleotide wurden bezogen von den Firmen Amersham, The chemicals used were, unless otherwise specified in the text, in p.A. -Quality sourced from the companies Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deideshofen). Solutions were prepared using pure pyrogen-free water, referred to as 1110 in the text, from an ion exchange system from the company (TKA, Niederelbert). Restriction nucleases, DNA-modifying enzymes and molecular biological kits and oligonucleotides were obtained from Amersham
Pharmacia (Freiburg) , Biometra (Göttirigen) , Dynal (Hamburg) , Gibco-BRL (Gaithersburg, MD., USA), lnvitrogen (Groningen, Niederlande), MBI Fermentas (St. Leon Rot), New England Biolabs (Schwalbach, Taunus) , Novagen (Madispn, Wisconsin, USA) , Qiagen (Hilden) , Röche Diagnostics (Mannheim) , StratagenePharmacia (Freiburg), Biometra (Göttirigen), Dynal (Hamburg), Gibco-BRL (Gaithersburg, MD., USA), lnvitrogen (Groningen, Netherlands), MBI Fermentas (St. Leon Rot), New England Biolabs (Schwalbach, Taunus ), Novagen (Madispn, Wisconsin, USA), Qiagen (Hilden), Röche Diagnostics (Mannheim), Stratagene
(Amsterdam, Niederlande) , TTB-Molbiol (Berlin) . Falls nicht anders beschrieben wurden die Produkte nach den Angaben der Hersteller eingesetzt.(Amsterdam, Netherlands), TTB-Molbiol (Berlin). Unless otherwise described, the products were used according to the manufacturer's instructions.
c) Herstellung und Identifizierung von Linien, die für lethale Mutationen segregierenc) Generation and identification of lines that segregate for lethal mutations
Wildtypische Arabidopsis-Pflanzen des Ökotyps C24 wurden mittels 'eines modifizierten "in planta"Transformations- Protokoll transformiert (Bechthold et al., 1992; Clough and Bent, 1998) und transgene Fl Pflanzen mittels Antibiotikaöder Herbizidresistenzen (u.a. Clearfield) selektioniert . T2-Samen dieser Linien wurden auf Sterilmedium und auf Erde ausgelegt und nach 7 Tagen Wachstum unter Standardbedingungen auf das Vorkommen von absterbenden Keimlingen visuell hin untersucht . Insbesondere wurden Veränderungen der Pigmentierung his hin zu dere völligen Fehlen sowie morphologische Anomalien beobachtet. Es wurden nur solche Linien weiter untersucht, für die in einer Paralleluntersuchung eine , Segregationsverhältnis von ca. 2:1-3:1, daher die zwei bis dreifache Menge resistenter Pflanzen gegenüber sensitiven Pflanzen bestimmt wurden. Dieses Verhältnis ist indikativ für einen einzelnen Integrationsort, der die Resistenz bewirkt.Wild-type Arabidopsis plants of ecotype C24 were transformed using a modified "in planta" transformation protocol (Bechthold et al., 1992; Clough and Bent, 1998) and transgenic FL plants were selected using antibiotic or herbicide resistance (including Clearfield). T2 seeds from these lines were placed on sterile medium and on soil and visually examined after 7 days of growth under standard conditions for the occurrence of dying seedlings. In particular, changes in pigmentation to its complete absence and morphological anomalies were observed. Only those lines for which a segregation ratio of approx. 2: 1-3: 1, and therefore two to three times the amount of resistant plants to sensitive plants, were determined in a parallel investigation. This ratio is indicative of a single integration site that causes resistance.
Verschiedene Linien, die für eine letale Mutation segregieren, wurden gefunden. Die molekularbiologischen Analysen wurden wie unter den Beispielen 1 bis 4 beschrieben, durchgeführt. Genannt ist die zu den Linien gehörenden SEQ ID NO., die die jeweils mutierten Sequenzen beschreiben, ab Beispiel 7.Various lines that segregate for a lethal mutation have been found. The molecular biological analyzes were carried out as described in Examples 1 to 4. Called is the SEQ ID NO. belonging to the lines, which describe the mutated sequences, from Example 7.
Beispiel 1: Identifizierung und Analyse der Linie P9, die für eine lethale Mutation segregiertExample 1: Identification and analysis of line P9, which segregates for a lethal mutation
Die Linie P9 (siehe SEQ ID NO: 3) wurde wie oben beschrieben als Linie, die für eine kei lingslethale Mutation segregiert, identifiziert. Die genaue Auszählung der Spaltung ergab, dass 25 % der Nachkommen den Albinophänotyp, 25 % der Nachkommen Sensitivität gegenüber der Selektion und 50 % der Nachkommen Resistenz gegenüber der Selektion zeigten. Dieses SpaltungsVerhältnis wird erwartet, wenn ausschließlich die homozygot resistenten Keimlinge homozygot für die Mutation sind, somit den recessiven Phänotyp zeigen, daher die T-DNA-Insertion mit der lethalen Mutation sehr eng gekoppelt ist . Die Kopplung wurde weiterhin in einer Cose- gregationsanalyse überprüft. Dazu wurden von 34 wildtypischen, resistenten Pflanzen der Linie P9 die Nachkommen analysiert. In allen Fällen konnten wiederum Albinos in der Nachkommenschaft gefunden werden. Dieser Umstand lässt den Schluss zu, dass die Resistenz-vermittelnde T-DNA-Insertion und die Mutation immer zusammen vererbt werden und damit (mit sehr hoher Wahrscheinlichkeit) übereinstimmen. Daraufhin wurde die Linie molekularbiologisch untersucht, um den T-DNA-Integrationsort genau zu bestimmen. Dazu wurde aus ca. 50 mg Gewebe dieser Pflanzen genomische DNA mittels Standardprodukten und Methoden (Säulen der Firma Qiagen, Hilden, Germany, oder Phytopure-Kit der Firma Amersham Pharmacia, Freiburg, Germany) isoliert und anhand gel- elektrophoretischer Auftrennung auf Integrität- und Menge hin untersucht. Die Amplifikation der zu der T-DNA benachbarten genomischen Sequenzen erfolgte mittels eines modifizierten Adaptor-PCR-Protokolls (Spertini et al., 1999). Etwa 50 bis 100 ng der DNA wurden jeweils für eine kombinierte Restriktion und Adaptorligation mit den Restriktionsenzymen, Bglll, Muni, Spei, Pspl406I/Bspl991, Aflll und dem aus den annealten Oligos 5 ' CTAATACGACTCACTATAGGGCTCGAGCGGCCGGGCAGGT-3 ' und 5 'NN(2_4)AC- CTGCCCAA-3 ' bestehenden Adaptor', wobei 5'NN(2_4) den zum jeweiligen Enzym passenden Überhang repräsentieren, unterworfen. Ein μl dieser mit Adaptoren versehenen genomischen DNA wurde für eine Amplifikation der zu den T-DNAs flankierend liegenden Sequenzen unter Verwendung eines adaptorspezifischen (5'GGATC- CTAATACGACTCACTATAGGGC-3 ' und eines genspezifischen Primers (LB1: 5 ' TGACGCCATTTCGCCTTTTCA-3 ' für die "Left border" und RB 1: 5 ' CAACTTAATCGCCTTGCAGCACA-3 ' für die "Right bordet v ) eingesetzt . Die PCR wurde unter Standardbedingungen für 7 Zyklen bei einer Annealingtemperatur von 72°C und für 32 bei einer Annealing- te peratur von 65°C in 25 μl Reaktionsvolumen durchgeführt, Das erhaltene Amplifikat wurde 1:50 in H0 verdünnt und ein μl 'dieser Verdünnung für eine Zweite Amplifikationsrunde (5 Zyklen bei 'einer Annealing-Temperatur von 67°C und 28 Zyklen bei einer Annealingtemperatur von 60°C ansonsten Standard-PCR-Bedingungen) 5 eingesetzt. Hierzu werden "eingerückte" daher auf dem PCR-Produkt weiter innenliegende Primer (5 ' -TATAGGGCTCGAGCGGC-3 ' für den Adaptor, LB2 : 5 'CAGAAATGGATAAATAGCCTTGCTTCC-3 ' für die "Left • border" und RH2 : 5 'AGCTGGCGTAATAGCGAAGAG-3 ' für die "Right border") eingesetzt, wodurch die Spezifität und SelektivitätLine P9 (see SEQ ID NO: 3) was identified as described above as a line that segregates for a keellethal mutation. The exact count of the cleavage showed that 25% of the offspring showed the albino phenotype, 25% of the offspring showed sensitivity to the selection and 50% of the offspring showed resistance to the selection. This cleavage ratio is expected when only the homozygously resistant seedlings are homozygous for the mutation, thus showing the recessive phenotype, so that the T-DNA insertion is very closely linked to the lethal mutation. The coupling was further checked in a cosegregation analysis. The descendants of 34 wild-type, resistant plants of the P9 line were analyzed. In all cases, albinos were found in the offspring. This fact leads to the conclusion that the resistance-mediating T-DNA insertion and the mutation are always inherited together and therefore match (with a very high probability). The line was then examined in terms of molecular biology in order to precisely determine the T-DNA integration site. For this purpose, genomic DNA was isolated from about 50 mg of tissue from these plants using standard products and methods (columns from Qiagen, Hilden, Germany, or Phytopure-Kit from Amersham Pharmacia, Freiburg, Germany) and gel-electrophoretic separation for integrity and Quantity examined. The genomic sequences adjacent to the T-DNA were amplified using a modified adapter-PCR protocol (Spertini et al., 1999). About 50 to 100 ng of DNA were respectively for a combined restriction and adapter ligation with restriction enzymes, BglII, Muni, SpeI Pspl406I / Bspl991, AflII and from the annealed oligos 5 'CTAATACGACTCACTATAGGGCTCGAGCGGCCGGGCAGGT-3' and 5 'NN (2 _ 4 ) AC-CTGCCCAA-3 'existing adapter', where 5'NN ( 2 _ 4 ) represent the overhang suitable for the respective enzyme. One μl of this genomic DNA provided with adapters was used for an amplification of the sequences flanking the T-DNAs using an adapter-specific (5'GGATC-CTAATACGACTCACTATAGGGC-3 'and a gene-specific primer (LB1: 5' TGACGCCATTTCGCCTTTTCA-3 'for the " Left border "and RB 1: 5 'CAACTTAATCGCCTTGCAGCACA-3' for the" Right bordet v ). The PCR was carried out under standard conditions for 7 cycles at an annealing temperature of 72 ° C and for 32 at an annealing temperature of 65 ° C carried out in 25 μl reaction volume, The amplicon was diluted 1:50 in H0 and ul 'of this dilution for a second round of amplification (5 cycles of' an annealing temperature of 67 ° C and 28 cycles at an annealing temperature of 60 ° C otherwise standard PCR conditions) 5 used. For this purpose, "indented" primers located further inside on the PCR product (5 '-TATAGGGCTCGAGCGGC-3' for the adapter, LB2: 5 'CAGAAATGGATAAATAGCCTTGCTTCC-3' for the "Left • border" and RH2: 5 'AGCTGGCGTAATAGCGAAGAG-3' used for the "Right border"), increasing the specificity and selectivity
10 der Amplifikation erhöht wird. Von dem in 50 μl Reaktionsvolumen erhaltenen Amplifikaten wurde ein Aliquot einer gelelektro- phoretischen Analyse unterzogen. Für- die Linie P9 wurde für, die Enzymkombination Pspl406I/BspII9I ein 1350 bp-großes Fragment für die linke T-DNABorder und ein 750 bp-großes Fragment für10 of the amplification is increased. An aliquot of the amplificates obtained in 50 μl reaction volume was subjected to a gel electrophoretic analysis. For line P9, the enzyme combination Pspl406I / BspII9I was a 1350 bp fragment for the left T-DNA border and a 750 bp fragment for
15 die rechte Border identifiziert . Die Produkte wurden mittels der Primer RBseq und Lbseq (5 ' -CAATACATTACACTAGCATCTG-3 ' ) sequenziert. Beide Produkte identifizierten den identischen Bereich im Genom von Arabidopsis. Die erfolgreiche Identifizierung wurde durch eine PCR-Reaktion mit einem für die abgeleitete flankieren-15 identified the right border. The products were sequenced using the primers RBseq and Lbseq (5 '-CAATACATTACACTAGCATCTG-3'). Both products identified the identical region in the Arabidopsis genome. Successful identification was confirmed by a PCR reaction flanked by a
20 den Sequenz und einem für die T-DNA-spezifischen Primer, RBl verifiziert. Der Erhalt des PCR-Produkts der vorhergesagten Größe spezifisch für diese Linie bestätigte die erfolgreiche Identifizierung der Insertionssteile der T-DNA. Die Blast-Analyse der isolierten Sequenzen (BLASTN, Altschul -,et al., 1990) J Mol . Biol.20 the sequence and one for the T-DNA specific primer, RBl verified. Obtaining the PCR product of the predicted size specific to this line confirmed the successful identification of the insertion parts of the T-DNA. Blast analysis of the isolated sequences (BLASTN, Altschul -, et al., 1990) J Mol. Biol.
25 215:403-410) demonstrierte die Insertion der linken Border in25 215: 403-410) demonstrated the insertion of the left border in
Position 53498 und der rechten Border in Position 54283 des Klons MVIIl des ' Chromosoms 111 (EMBL| AP000419 ) . In diesem Bereich ist ein ORF (MVI11.13) annotiert, der für ein "DNA Repair protein RAD-54-like"-Protein codiert. Dieser durch die Insertion unter-Position 53498 and the right border in position 54283 of clone MVIIl of ' chromosome 111 (EMBL | AP000419). In this area an ORF (MVI11.13) is annotated which codes for a "DNA Repair protein RAD-54-like" protein. This through the insertion
30 broehene ORF zeigt eine hohe Homologie zu dem DNA Repair Protein RHP54"aus Hefe (Muris et al., J. Cell. Sei., 1996) sowie Ähnlichkeiten zu einer Reihe weiterer DNA-bindender Proteine.30 broehene ORF shows a high homology to the DNA repair protein RHP54 "from yeast (Muris et al., J. Cell. Sei., 1996) as well as similarities to a number of other DNA-binding proteins.
Beispiel 2: Identifizierung und Analyse der Linie P38, die 35 für eine 'lethale MutationExample 2: Identification and analysis of line P38, 35 for a lethal mutation
Die Linie P38 wurde wie oben beschrieben als Linie, die für eine keimlingslethale Mutation segregiert, identifiziert. Die genaue Auszählung der Spaltung ergab, dass 25 % der NachkommenLine P38 was identified as described above as a line that segregates for a seedling lethal mutation. The exact count of the split showed that 25% of the offspring
40 den Albinophänotyp, 25 % der Nachkommen Sensitivität gegenüber der Selektion und 50 % der Nachkommen Resistenz gegenüber der Selektion zeigten. Dieses Spaltungsverhältnis wird erwartet, wenn ausschließlich die homozygot resistenten Keimlinge den Phäno- typ zeigen, daher die T-DNA-Insertion mit der lethalen Mutation40 showed the albino phenotype, 25% of the offspring showed sensitivity to the selection and 50% of the offspring showed resistance to the selection. This cleavage ratio is expected when only the homozygously resistant seedlings show the phenotype, hence the T-DNA insertion with the lethal mutation
45 sehr eng gekoppelt ist. Mit der Cosegregationsanalyse soll die Kopplung weiterhin überprüft werden. Dazu wurden von 34 wildtypischen, resistenten Pflanzen der Linie P'38 die Nachkommen analysiert. In allen Fällen konnten wiederum Albinos in der _Nachkommenschaft gefunden werden. Dieser Umstand lässt den Schluss zu, dass die Resistenz-vermittelnde TJ-DNA-Insertion und die Mutation immer zusammen vererbt werden und damit (mit sehr hoher Wahrscheinlichkeit) übereinstimmen. Die molekularbiologische. Analyse wurde wie für Beispiel 1 beschrieben durchgeführt . Für die Linie P38 wurde für das Enzym Muni ■ ein 350 bp-großes Fragment für die linke T-DNA-Border identifiziert. Das Produkt wurde mittels der Primer Lbseq sequenziert und zeigte die erwartet Identität zu einem Bereich des Arabidopsis-Genoms .' Die erfolgreiche Identifizierung wurde durch eine PCR-Reaktion mit einem für die abgeleitete flankierenden Sequenz und einem für die T-DNA-spezifischen Primer, LBl, verifiziert. Der Erhalt des PCR- Produkts der vorhergesagten Größe spezifisch für diese Linie bestätigte die erfolgreiche Identifizierung der Insertionsstelle der T-DNA. Die Blast-Analyse der isolierten Sequenz (BLASTN, Altschul et al . , 1990) J Mol. Biol. 215:403-410) demonstrierte die Insertion der T-DNA in Position 42163 des BAC-Klons F3E22 des Chromosoms 111 (EMBLNEW|AC023912) . Nach der Annotation dieses Bereichs ist die Integration in einen vorhergesagten ORF (F3E22.13), der signifikante Ähnlichkeit zu verschiedenen Thio- redoxinen neigt, erfolgt. Daraufhin wurden Primer für das vorhergesagte 5 ' - und 3 ' Ende synthetisiert und für eine Standard-PCR mit cDNA von Arabidopsis eingesetzt. Die mRNA wurde mittels Oligo-dT Dynabeads von Dynal aus Keimlingen isoliert, die cDNA daraus unter Verwendung eines cDNA-Synthese-Kits der Firma Gibco-BRL und einem Oligo-dT-Primen hergestellt. Das erhaltene PCR-Produkt wurde ' in einen TA-Vektor (pCRScriptll, Invitrogen) ligiert und sequenziert. Die Sequenz zeigte vollständige Über- einstim ung mit der vorhergesagten Sequenz und Genstruktur für diesen ORF. Demnach dokumentieren die Erfinder hiermit erstmals die experimentelle Bestätigung für die Existenz dieses ORFs in der vorhergesagten Struktur.45 is very closely coupled. The coupling should continue to be checked with the cosegregation analysis. For this, the offspring were of 34 wild-type, resistant plants of the line P '38 analyzed. In all cases, albinos could be found in the progeny. This fact leads to the conclusion that the resistance-mediating TJ-DNA insertion and the mutation are always inherited together and therefore match (with a very high probability). The molecular biological. Analysis was carried out as described for Example 1. A 350 bp fragment for the left T-DNA border was identified for the P38 line for the Muni enzyme. The product was sequenced using the primer Lbseq and showed the expected identity to a region of the Arabidopsis genome. ' Successful identification was verified by a PCR reaction with one for the derived flanking sequence and one for the T-DNA-specific primer, LB1. Obtaining the PCR product of the predicted size specific to this line confirmed the successful identification of the insertion site of the T-DNA. Blast analysis of the isolated sequence (BLASTN, Altschul et al., 1990) J Mol. Biol. 215: 403-410) demonstrated the insertion of the T-DNA in position 42163 of BAC clone F3E22 of chromosome 111 (EMBLNEW | AC023912 ). After the annotation of this area, the integration into a predicted ORF (F3E22.13), which tends to be significantly similar to various thio-redoxins, took place. Then primers for the predicted 5 'and 3' end were synthesized and used for a standard PCR with Arabidopsis cDNA. The mRNA was isolated from seedlings by means of Dynal oligo-dT Dynabeads, and the cDNA was produced therefrom using a Gibco-BRL cDNA synthesis kit and an oligo-dT primer. The PCR product was' into a TA vector (pCRScriptll, Invitrogen) and sequenced. The sequence showed complete agreement with the predicted sequence and gene structure for this ORF. The inventors hereby document for the first time the experimental confirmation for the existence of this ORF in the predicted structure.
Beispiel 3 : Identifizierung und Analyse der Linie P44 die für eine lethale Mutation segregiertExample 3: Identification and analysis of line P44 that segregates for a lethal mutation
Die Linie . P44 wurde wie oben beschrieben als Linie, die für eine keimlingslethale Mutation segregiert, identifiziert. Die genaue Auszählung der Spaltung ergab,, dass 25 %' der Nachkommen den Albinophänotyp, 25 % der Nachkommen Sensitivität gegenüber der Selektion und 50 % der Nachkommen Resistenz gegenüber der Selektion zeigten. Dieses SpaltungsVerhältnis wird erwartet, wenn ausschließlich die homozygot resistenten Keimlinge den Phäno- typ zeigen, daher die T-DNA-Insertion mit der lethalen Mutation sehr eng gekoppelt ist. Die Kopplung wurde weiterhin in einer Cosegregationsanalyse überprüft Dazu wurden von 34 wildtypischen, resistenten Pflanzen der Linie P44 die Nachkommen analysiert. In allen Fällen konnten wiederum Albinos in der Nachkommenschaft gefunden werden. Dieser Umstand lässt den Schluss zu, dass die Resistenz-vermittelnde T-DNA-Insertion und die Mutation immer 5 zusammen vererbt werden und damit (mit sehr hoher Wahrscheinlichkeit) übereinstimmen. Die molekularbiologische Analyse wurde wie . für Beispiel 1 beschrieben durchgeführt . Für die Linie P44 wurde ' tUr das Enzym Muni ein 350 bp-großes und für das Enzym Bglll ein 500 bp-großes Fragment jeweils für die linke T-DNA-BordenThe line . P44 was identified as a line that segregates for a seedling lethal mutation as described above. The exact count of the cleavage showed that 25% of the offspring showed the albino phenotype, 25% of the offspring showed sensitivity to the selection and 50% of the offspring showed resistance to the selection. This cleavage ratio is expected when only the homozygously resistant seedlings show the phenotype, so the T-DNA insertion is very closely linked to the lethal mutation. The coupling was further checked in a cosegregation analysis. 34 wild-type, resistant plants of the P44 line analyzed the offspring. In all cases, albinos were found in the offspring. This fact leads to the conclusion that the resistance-mediating T-DNA insertion and the mutation are always inherited together 5 and therefore match (with a very high probability). The molecular biological analysis was like. described for Example 1 performed. For line P44 'TUR, the enzyme was Muni a 350 bp large and for the enzyme Bgl II, a 500 bp fragment for the left T-DNA Borden
10 identifiziert. Die Produkte wurden mittels des Primers Lbseq sequenziert und definierten die identische Position im Arabi- dopsis-Genoms . Die erfolgreiche Identifizierung wurde durch eine PCR-Reaktion reit einem für die abgeleitete flankierenden Sequenz und einem für die T-DNA-spezifischen Primen, LB1, ver-10 identified. The products were sequenced using the primer Lbseq and defined the identical position in the Arabidopsis genome. Successful identification was confirmed by a PCR reaction using one for the derived flanking sequence and one for the T-DNA-specific primes, LB1.
15 ifiziert. Der Erhalt des PCR-Produkts der vorhergesagten Grölte spezifisch für diese Linie bestätigte die erfolgreiche Identifizierung der Insertionsstelle der T-DNA. Die Blast-Analyse der isolierten Sequenz (BLASTN, Altschul et al., 1990) J Mol. Biol. 215:403-410) demonstrierte die Insertion der T-DNA in Position15 ifified. The receipt of the PCR product of the predicted size specifically for this line confirmed the successful identification of the insertion site of the T-DNA. Blast analysis of the isolated sequence (BLASTN, Altschul et al., 1990) J Mol. Biol. 215: 403-410) demonstrated the insertion of the T-DNA in position
20 66762 des TAC-Klons K15C23 (EMBLALERT|AB024024) . Nach' der20 66762 of the TAC clone K15C23 (EMBLALERT | AB024024). After
Annotation dieses Bereichs ist die Integration in einen vorhergesagten ORF (K15C23.10). erfolgt, dessen abgeleitete Aminosäuresequenz keine signifikanten Homologien .zeigt . Schwache Homologien findet man dagegen zu VAV2 Proteine aus Maus (AccessionAnnotation of this area is the integration into a predicted ORF (K15C23.10). whose deduced amino acid sequence shows no significant homologies. On the other hand, weak homologies can be found with VAV2 proteins from mice (accession
25 Q60992) und Mensch (Accession: P52735) . Daraufhin wurden Primer für das vorhergesagte 5'- und 3'- Ende des ORFs synthetisiert und für eine Standard-PCR mit cDNA von Arabidopsis eingesetzt. Die cDNA wurde wie in Beispiel 3 beschrieben hergestellt . Die Sequenz zeigte vollständige Übereinstimmung mit der vorher-25 Q60992) and human (Accession: P52735). Then primers for the predicted 5 'and 3' ends of the ORF were synthesized and used for a standard PCR with Arabidopsis cDNA. The cDNA was prepared as described in Example 3. The sequence showed complete agreement with the previous
30 gesagten Sequenz und Genstruktur für diesen ORF. Demnach dokumentieren die' Erfinder hiermit erstmals die experimentelle Bestätigung für die Existenz dieses ORFs in der vorhergesagten Struktur.30 said sequence and gene structure for this ORF. The inventors hereby document for the first time the experimental confirmation for the existence of this ORF in the predicted structure.
Beispiel 4: Identifizierung und Analyse der Linie P77, die 35 für eine lethale Mutation segregiertExample 4: Identification and analysis of line P77, which segregates 35 for a lethal mutation
Die Linie P77 wurde wie oben beschrieben als Linie, die für eine keimlingslethale Mutation segregiert, identifiziert. Die genaue Auszählung der Spaltung ergab, dass 25 % der NachkommenLine P77 was identified as described above as a line that segregates for a seedling lethal mutation. The exact count of the split showed that 25% of the offspring
40 den Albinophänotyp, 25 % der Nachkommen Sensitivität gegenüber der Selektion und 50 % der Nachkommen Resistenz gegenüber der Selektion zeigten. Dieses SpaltungsVerhältnis wird erwartet, wenn ausschließlich die homozygot resistenten Keimlinge den Phänotyp zeigen, daher die T-DNA-Insertion mit der lethalen40 showed the albino phenotype, 25% of the offspring showed sensitivity to the selection and 50% of the offspring showed resistance to the selection. This cleavage ratio is expected when only the homozygously resistant seedlings show the phenotype, hence the T-DNA insertion with the lethal one
45 Mutation sehr eng gekoppelt ist. Die Kopplung wurde weiterhin in einer Cosegregationsanalyse überprüft. Dazu wurden von 34 wildtypischen, resistenten Pflanzen der Linie P77 die Nachkommen analysiert. In allen Fällen konnten wiederum Albinos in der Nachkommenschaft gefunden werden. Dieser Umstand lässt den Schluss zu, dass die Resistenz-vermittelnde T-DNA-Insertion und die Mutation immer zusammen vererbt werden und damit (mit sehr hoher Wahrscheinlichkeit) übereinstimmen. Die molekularbiologische Analyse wurde wie' für Beispiel 1 beschrieben durchgeführt. Für die Linie P77 wurde für die Enzymkombination Pspl4061/Bspll9I ein 650 bp-großes und für die linke T-DNA-Border identifiziert. Die Produkte wurden mittels des Primers Lbseq sequenziert. Die erfolgreiche Identifizierung wurde durch eine PCR-Reaktion mit einem für die abgeleitete flankierenden Sequenz und einem für die T-DNA-spezifischen Primer, LBI, verifiziert. Der Erhalt des PCR- Produkts der vorhergesagten Größe spezifisch für diese Linie bestätigte die erfolgreiche Identifizierung der Insertionsstelle der T-DNA. Die Blast-Analyse der. isolierten Sequenz (BLASTN, Altschul et al., 1990) J Mol. Biol. 215:403 410) zeigte eine absolute Übereinstimmung der Sequenz mit einem Abschnitt auf dem Chromosom III von Arabidopsis und demonstrierte die Insertion der T-DNA in Position 13436 des BAC-Klons F24B22 (EMBLATF24B22) und unterbricht ein vorhergesagtes offenes Lesesraster- (F24B22.50), welches für ein Protein mit hoher Ähnlichkeit zu verschiedenen Fructokinases bspw. aus Kartoffel (Solanum tuberosum, Accession: P37829) oder aus dem Bakterium Vibrio alginolyticus (Accession: P22824) codiert. Daraufhin wurden Primer für das vorhergesagte 5'- und 3'- Ende des ORFs synthetisiert und für eine Standard-PCR mit cDNA von Arabidopsis eingesetzt. Die cDNA wurde wie in Beispiel 3 beschrieben hergestellt. Die Sequenz zeigte vollständige Übereinstimmung mit der vorhergesagten Sequenz und Genstruktur für diesen ORF. Demnach dokumentieren die Erfinder hiermit erst- mals die experimentelle Bestätigung für die Existenz dieses ORFs in der vorhergesagten Struktur.45 mutation is very closely coupled. The coupling was further checked in a cosegregation analysis. The descendants of 34 wild-type, resistant plants from the P77 line were added analyzed. In all cases, albinos were found in the offspring. This fact leads to the conclusion that the resistance-mediating T-DNA insertion and the mutation are always inherited together and therefore match (with a very high probability). The molecular biological analysis was carried out as described for Example 1. For line P77, a 650 bp size was identified for the enzyme combination Pspl4061 / Bspll9I and for the left T-DNA border. The products were sequenced using the primer Lbseq. Successful identification was verified by a PCR reaction with one for the derived flanking sequence and one for the T-DNA specific primer, LBI. Obtaining the PCR product of the predicted size specific to this line confirmed the successful identification of the insertion site of the T-DNA. The blast analysis of the. isolated sequence (BLASTN, Altschul et al., 1990) J Mol. Biol. 215: 403 410) showed an absolute match of the sequence with a section on the chromosome III of Arabidopsis and demonstrated the insertion of the T-DNA in position 13436 of the BAC Clones F24B22 (EMBLATF24B22) and interrupts a predicted open reading grid (F24B22.50), which for a protein with high similarity to various fructokinases e.g. from potato (Solanum tuberosum, Accession: P37829) or from the bacterium Vibrio alginolyticus (Accession: P22824) coded. Then primers for the predicted 5 'and 3' ends of the ORF were synthesized and used for a standard PCR with Arabidopsis cDNA. The cDNA was prepared as described in Example 3. The sequence showed complete agreement with the predicted sequence and gene structure for this ORF. Accordingly, the inventors hereby document for the first time the experimental confirmation for the existence of this ORF in the predicted structure.
Beispiel 5: Identifizierung und Analyse der Linien P95, P98,Example 5: Identification and Analysis of Lines P95, P98,
P99b, P102 und P103, die für eine lethale Mutation segregierenP99b, P102 and P103, which segregate for a lethal mutation
Analog den vorgenannten Beispielen 1 bis 4 wurden die Klone P95, P98, P99b, P102 und P103 als Linien identifiziert, die für keim- lingslethale Mutationen segregieren. Die molekularbiologischen Arbeiten bzw. Analysen wurden wie unter Beispiel 1 bis 4 beschrieben durchgeführt .Analogously to the above-mentioned Examples 1 to 4, the clones P95, P98, P99b, P102 and P103 were identified as lines which segregate for seedling-lethal mutations. The molecular biological work and analyzes were carried out as described in Examples 1 to 4.
Bei der Linie P95 erfolgte die Insertion der T-DNA in Position 35442 des BACT5L19 (Accession number AL049481) des Chromosoms IV von Arabidopsis . Die Insertion der T-DNA erfolgte bei der Linie P98 in Position 54861 des Pl-Klons MVA3 (Accession number: AB006706) des Chromosoms V. Die Insertion der T-DNA wurde bei der Linie P99b in Position 66042 des BACs F10M10 (AL035521) auf Chromosom IV lokalisiert. Bei Klon P102 wurde die Insertion der T-DNA auf Chromosom IV im Bereich des Contigfragments 69 in Position 46342-46355 (AL 161573) lokalisiert. Die Linie P103 wies eine Insertion der T-DNA in Position 57314 des BACs F11F8 (AC016661) des Chromosoms I auf.In line P95, the T-DNA was inserted in position 35442 of BACT5L19 (Accession number AL049481) of chromosome IV from Arabidopsis. The T-DNA was inserted at line P98 in position 54861 of the PI clone MVA3 (Accession number: AB006706) of the chromosome V. The insertion of the T-DNA was carried out at of line P99b in position 66042 of BAC F10M10 (AL035521) localized on chromosome IV. In the case of clone P102, the insertion of the T-DNA on chromosome IV was localized in the region of the contig fragment 69 in position 46342-46355 (AL 161573). Line P103 had an insertion of the T-DNA in position 57314 of BAC F11F8 (AC016661) of chromosome I.
Beispiel 6: Identifizierung und Analyse der Linien P91 und P99a, die für eine lethale Mutation segregierenExample 6: Identification and analysis of lines P91 and P99a that segregate for a lethal mutation
Analog den vorgenannten Beispielen wurden die Klone P91 und P99a als Linien identifiziert, die für keimlingsletale Mutationen segregieren. Die molekularbiologischen Analysen wurden wie unter den Beispielen 1 bis 4 beschrieben, durchgeführt.Analogously to the above examples, clones P91 and P99a were identified as lines which segregate for seedling lethal mutations. The molecular biological analyzes were carried out as described in Examples 1 to 4.
Beispiel 7Example 7
Analog zu den vorgenannten Beispielen wurde die Linie A300364 (SEQ ID NO: 26 [Nukleinsäure] und 27 [Protein]) identifiziert. Linie A300364 spaltet für eine embroyletale Mutation. Es wurde eine 2:1 Spaltung beobachtet. Bei der Untersuchung von 35 Linien wurde eine absolute Cosegregation zwischen der T-DNA und der zum Albinophänotyp führenden Mutation beobachtet. In dieser Linien ist die T-DNA in Position 39517 des. Chromosoms II (EMBLJ C004238) inseriert . Dort unterbricht und damit inaktiviert die Insertion einen ORF (At2g34860), der für ein unbekanntes Protein (AAC12826.1) codiert. In Blastp-Vergleichen mit Standardeinstellungen zeigt das Protein über einen Bereich von 40 Aminosäuren Homologie zu verschiedenen DNAJ Chaperon-Protein (Heat Shock Protein 40) bspw. dem DNAJ Protein (Q9UXR9) aus Methano- sarcina thermophila (Hoffmann-Bang et al., Gene 238 (2), 387-395 (1999)).Line A300364 (SEQ ID NO: 26 [nucleic acid] and 27 [protein]) was identified analogously to the examples mentioned above. Line A300364 cleaves for an embroyletal mutation. A 2: 1 cleavage was observed. When 35 lines were examined, absolute cosegregation between the T-DNA and the mutation leading to the albino phenotype was observed. The T-DNA is inserted in position 39517 of chromosome II (EMBLJ C004238) in these lines. There the insertion interrupts and thus inactivates an ORF (At2g34860) which codes for an unknown protein (AAC12826.1). In blastp comparisons with standard settings, the protein shows homology to various DNAJ chaperone protein (Heat Shock Protein 40) over a range of 40 amino acids, for example the DNAJ protein (Q9UXR9) from Methanasarcina thermophila (Hoffmann-Bang et al., Gene 238 (2), 387-395 (1999)).
Beispiel 8Example 8
Analog zu den vorgenannten Beispielen wurde die Linie A301034 (SEQ ID NO: 28 [Nukleinsäure] und 29 [Protein]) identifiziert. Linie A3-01034 spaltet für eine embroyletale Mutation. Es wurde eine 2:1 Spaltung beobachtet. Bei der Untersuchung von 35 Linien wurde eine absolute Cosegregation zwischen der T-DNA und der zum Albinophänotyp führenden Mutation beobachtet . Die T-DNA ist auf Chromosom V in Position 25928 des BACs T21H19 (EMBL|ATT21H19) inseriert. Die Insertion an dieser Stelle .unterbricht ein Gen (T21H19_100) , welches für ein putatives Protein (CAC01859.1) codiert. Dieses zeigt Homologie zu anderen putativen Proteinen aus Arabidopsis. Die abgeleitete Proteinsequenz zeigt deutliche Homologien zum CRSl Genprodukt aus Mais (AAG00595) , welches für das Splicen des Gruppe II Introns des Chloroplastengens atpF benötigt wird. SEQ ID NO: 43 zeigt die genomische Sequenz der Linie A301034 vom Start- bis Stopcodon, inklusive Introns.Line A301034 (SEQ ID NO: 28 [nucleic acid] and 29 [protein]) was identified analogously to the aforementioned examples. Line A3-01034 cleaves for an embroyletal mutation. A 2: 1 cleavage was observed. When 35 lines were examined, absolute cosegregation between the T-DNA and the mutation leading to the albino phenotype was observed. The T-DNA is inserted on chromosome V in position 25928 of the BAC T21H19 (EMBL | ATT21H19). The insertion at this point interrupts a gene (T21H19_100) which codes for a putative protein (CAC01859.1). This shows homology to other putative proteins from Arabidopsis. The derived protein sequence shows clear homologies to the CRSl gene product from maize (AAG00595), which for splicing of the group II intron of the chloroplast gene atpF is required. SEQ ID NO: 43 shows the genomic sequence of line A301034 from start to stop codon, including introns.
5 Die Aktivität kann z.B. in Assays, wie sie in Bock, Nucleic Acids Res., 1995, 23, 2544-7 beschrieben sind/ getestet werden.5 The activity can be / tested, for example, in assays as described in Bock, Nucleic Acids Res., 1995, 23, 2544-7.
Beispiel 9Example 9
0 Analog zu den vorgenannten Beispielen wurde die LinieA300377 (SEQ ID NO: 30 [Nukleinsäure] und 31 [Protein]) identifiziert. Linie A300377 spaltet für eine embroyletale Mutation. Es wurde eine 2:1 Spaltung beobachtet. Bei der Untersuchung von 35 Linien wurde eine absolute Cosegregation zwischen der T-DNA und der zum Albi- 5 nophänotyp führenden Mutation beobachtet. Die T-DNA ist in Position 14509 des Pl Klons MRNl7 (AB005243).. und damit sehr wahrscheinlich im 3 ' -nicht translatierten Bereich einer Alanyl-tRNA- Syntethase (BAB10601.1) inseriert.Line A300377 (SEQ ID NO: 30 [nucleic acid] and 31 [protein]) was identified analogously to the examples mentioned above. Line A300377 cleaves for an embroyletal mutation. A 2: 1 cleavage was observed. When 35 lines were examined, an absolute cosegregation between the T-DNA and the mutation leading to the albino phenotype was observed. The T-DNA is inserted in position 14509 of the Pl clone MRNl7 (AB005243) .. and therefore very likely in the 3 'untranslated region of an alanyl tRNA synthesis (BAB10601.1).
0 Beispiel 100 Example 10
Analog zu den vorgenannten Beispielen wurde die Linie A300841 (SEQ ID No: 32 [Nukleinsäure] und 33 [Protein]) als essentiell identifiziert. Linie A300841 spaltet für eine embryoletale 5 Mutation. Es wurde eine 2:1 Spaltung beobachtet. Bei der ■ Untersuchung von 35 Linien wurde eine absolute Cosegregation zwischen der T-DNA und der zum Albinophänotyp führenden Mutation beobachtet. Die T-DNA ist in dieser Linie in Position 3183 des BACs T14P8 inseriert, was der Position 143432 in Contigfrag ent .6 0 des Chromosoms IV entspricht. Durch die Insertion in dieserAnalogous to the aforementioned examples, line A300841 (SEQ ID No: 32 [nucleic acid] and 33 [protein]) was identified as essential. Line A300841 cleaves for an embryoletal 5 mutation. A 2: 1 cleavage was observed. ■ In the study of 35 lines an absolute co-segregation between the T-DNA and leading to the albino mutation was observed. The T-DNA is inserted in position 3183 of BAC T14P8, which corresponds to position 143432 in Contigfrag . 6 0 of chromosome IV corresponds. By inserting in this
Position -wird ein offenes Leseraster für ein putatives "chloro- plast outer envelope 86-like" Protein zerstört. Für diesen ORF (CAB80744.1) sind bereits ESTs in den Datenbanken vorhanden (EST gb:Al998804.1, R90258, AA651438). Das codierte Protein zeigt eine 5 stark Homologie zu dem chloroplast outer envelope 86 protein" OEP86 aus Erbse P. sativum, GenBank Accessionnummer Z31581 und. besitzt ein ATP/GTP-Bindungsstellenmotiv (P-loop) .Position - an open reading frame for a putative "chloroplast outer envelope 86-like" protein is destroyed. ESTs are already available in the databases for this ORF (CAB80744.1) (EST gb: Al998804.1, R90258, AA651438). The encoded protein shows a strong homology to the chloroplast outer envelope 86 protein "OEP86 from pea P. sativum, GenBank accession number Z31581 and. Has an ATP / GTP binding site motif (P-loop).
Die Aktivität eines OEP86 kann z.B. wie in Muckel, J. Biol. 0 Chem., 1996, 271, 23846-52, Young, Plant Physiol., 1999, 121, ■ 237-44, oder in dem Review Keegstra, Curr. Opin. Plant Biol., 1999, 2, 471-6, beschriebenen oder zitierten Assays getestet werden.The activity of an OEP86 can e.g. as in Muckel, J. Biol. 0 Chem., 1996, 271, 23846-52, Young, Plant Physiol., 1999, 121, ■ 237-44, or in the Review Keegstra, Curr. Opin. Plant Biol., 1999, 2, 471-6, assays described or cited.
5 Beispiel 115 Example 11
Analog zu den vorgenannten Beispielen wurde die Linie 2266c .(SEQ ID No: 34 [Nukleinsäure] und 35 [Protein]) identifiziert. 5' Linie 2266c spaltet für eine embryoletale Mutation. Es kann eine 2:1 Spaltung beobachtet werden. Die T-DNA ist in Position 26501 des BAC F6N18 (AC017118) des Chromosoms I inseriert. Dort unter- - bricht die Insertion einen ORF, dessen abgeleitete Aminosäuresequenz (AAF25967.1) deutliche Ähnlichkeit zu einem "FMRF-Amid 10 Propeptid-Isolog (gi| 1871179) aus Arabidopsis zeigt.Line 2266c (SEQ ID No: 34 [nucleic acid] and 35 [protein]) was identified analogously to the abovementioned examples. 5 ' Line 2266c cleaves for an embryoletal mutation. A 2: 1 cleavage can be observed. The T-DNA is inserted in position 26501 of BAC F6N18 (AC017118) of chromosome I. There, the insertion interrupts an ORF whose derived amino acid sequence (AAF25967.1) shows clear similarity to an "FMRF amide 10 propeptide isolog (gi | 1871179) from Arabidopsis.
Beispiel 12Example 12
Analog zu den vorgenannten Beispielen wurde die Linie P61 (SEQ ID 15 NO: 36 [Nukleinsäure] und 37 [Protein]) identifiziert. Linie P61 spaltet für eine embryoletale Mutation. Es wurde eine 2:1 Spaltung beobachtet. Bei der Untersuchung von 35 Linien wurde eine absolute Cosegregation zwischen der T-DNA und der zürn Albinophänotyp führenden Mutation beobachtet. Die T-DNA ist in Position 20 28640 des BACs F4B12 (EMBLNEW|AP001299) auf Chromosom III inseriert . Die Insertion unterbindet die Expression eines ORFs , der in Position 28705 beginnt und für ein unbekanntes Protein (BAB02572.1) mit schwacher Homologie zu Proteosomenprotein 26S PROTEASOME SUBUNIT S5B, (Deveraux, Q . , Jensen, C. and Rechsteiner, 25 M. , Molecular cloning and expression of a 26 S protease subunit . enriched in dileucine repeats, J. Biol. Chem. 270 (40), 23726-23729 (1995) codiert.Line P61 (SEQ ID 15 NO: 36 [nucleic acid] and 37 [protein]) was identified analogously to the abovementioned examples. Line P61 cleaves for an embryoletal mutation. A 2: 1 cleavage was observed. When 35 lines were examined, an absolute cosegregation between the T-DNA and the mutation leading to angry albino phenotype was observed. The T-DNA is inserted in position 20 28640 of the BAC F4B12 (EMBLNEW | AP001299) on chromosome III. The insertion inhibits the expression of an ORF that begins in position 28705 and for an unknown protein (BAB02572.1) with weak homology to proteosome protein 26S PROTEASOME SUBUNIT S5B, (Deveraux, Q., Jensen, C. and Rechsteiner, 25 M., Molecular cloning and expression of a 26 S protease subunit. Enriched in dileucine repeats, J. Biol. Chem. 270 (40), 23726-23729 (1995).
Beispiel 13Example 13
3030
Analog zu den vorgenannten Beispielen wurde die Linie- A300857 (SEQ ID No: 38 [Nukleinsäure] und 39 [Protein]) identifiziert. - A300857 spaltet für eine embroyletale Mutation. Bei der Untersuchung von 35 Linien wurde eine absolute Cosegregation zwischenLine A300857 (SEQ ID No: 38 [nucleic acid] and 39 [protein]) was identified analogously to the abovementioned examples. - A300857 cleaves for an embroyletal mutation. When examining 35 lines, an absolute cosegregation between
35 der T-DNA und der zum Albinophänotyp führenden Mutation beobachtet. In dieser Linie ist die T-DNA in Position 51122 des BACs T10O24 des Chromosoms I (EMBL:AC007067) inseriert. Dort unterbricht und damit inaktiviert die Insertion einen ORF (T10O24.14) der für ein unbekanntes Protein (AAD39574.1) codiert.35 of the T-DNA and the mutation leading to the albino phenotype were observed. In this line, the T-DNA is inserted in position 51122 of BAC T10O24 of chromosome I (EMBL: AC007067). There the insertion interrupts and thus inactivates an ORF (T10O24.14) which codes for an unknown protein (AAD39574.1).
40 SEQ ID NO: 42 zeigt die zugehörige genomische Sequenz.40 SEQ ID NO: 42 shows the associated genomic sequence.
Beispiel 14Example 14
Analog zu den vorgenannten Beispielen wurde die Linie A300367 45 (SEQ ID NO: 40 [Nukleinsäure] und 41 [Protein]) identifiziert. A300367 spaltet für eine embroyletale Mutation. Bei der Untersuchung von 35 Linien wurde eine absolute Cosegregation zwischen der T-DNA und der zum Albinophänotyp führenden Mutation beobachtet. In dieser Linie ist die T-DNA in Position 31058 "Contig fragments" 86 (EMBL:ATCHRIV86) des Chromosoms IV inseriert. Durch ie Insertion wenige Basenpaare stromaufwärts des Startcodons (51073) einer Geranylgeranylpyrophόsphatsynthase (Bartley and Scolnik, 1994) (Plant Physiol. 104, 1469-1470, 1994), Accession L25813 wird mit sehr hoher Wahrscheinlichkeit die Funktionalität des Gens durch Störung der Transkription, der Transkriptstabilität oder zumindest der Translation zerstört. So beschreibt Okada, Plant Physiol., 2000, 122, 1045-56, in Arabidopsis fünf verschiedene GGPPs," die in drei verschiedenen Kompartimenten lokalisiert sind. Überraschenderweise' ist das hier gezeigte Protein oder Transkript, vermutlich eine GGPP, essentiell.Line A300367 45 (SEQ ID NO: 40 [nucleic acid] and 41 [protein]) was identified analogously to the abovementioned examples. A300367 cleaves for an embroyletal mutation. When examining 35 lines, an absolute cosegregation between the T-DNA and the mutation leading to the albino phenotype. In this line, the T-DNA is inserted in position 31058 "Contig fragments" 86 (EMBL: ATCHRIV86) of chromosome IV. By inserting a few base pairs upstream of the start codon (51073) of a geranylgeranyl pyrophosphate synthase (Bartley and Scolnik, 1994) (Plant Physiol. 104, 1469-1470, 1994), Accession L25813, the functionality of the gene is very likely to be impaired by transcription interference Transcript stability or at least the translation destroyed. Thus, Okada, Plant Physiol., 2000, 122, 1045-56, describes five different GGPPs in Arabidopsis, " which are located in three different compartments. Surprisingly, the protein or transcript shown here, presumably a GGPP, is essential.
Die Aktivität einer Geranylgeranylpyrophosphatsynthase kann z.B. wie in Zhu et al., Plant Cell Physiol., 1997, 38, 337-61, oder Okada, Plant Physiol., 2000, 122,' 1045-56, beschriebenen Testsystemen getestet werden.The activity of a geranylgeranyl pyrophosphate synthase can e.g. as described in Zhu et al., Plant Cell Physiol., 1997, 38, 337-61, or Okada, Plant Physiol., 2000, 122, '1045-56.
Beispiel 15Example 15
Analog zu den vorgenannten Beispielen wurde die Linie 305735 (SEQ ID NO: 44 [Nukleinsäure] und SEQ ID NO: 45 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 305735 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 46571 der Sequenz ATCHRIV69, Accession number AL161573, integriert. Durch die Insertion an dieser Position wir der ORF AT4g28590 unterbrochen, der für ein hypothetisches Protein codiert, welches eine- "Cecropin" Familiensignature (AÄ237-245) aufweist.Line 305735 (SEQ ID NO: 44 [nucleic acid] and SEQ ID NO: 45 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 305735 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is integrated in position 46571 of the sequence ATCHRIV69, accession number AL161573. The insertion at this position disrupts the ORF AT4g28590, which codes for a hypothetical protein which has a "Cecropin" family signature (AÄ237-245).
Beispiel 16Example 16
Analog zu den vorgenannten Beispielen wurde die Linie 303726 (SEQ ID NO: 46 [Nukleinsäure] und SEQ ID NO: 47 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 303726 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 51568 des BACs AC004669 auf Chromsom 2 inseriert. Durch die Insertion an dieser Stelle wird sehr wahrscheinlich die Transkription und damit die Funktion des ORFs At2g30950 verhindert oder beeinträchtigt Dieser ORF codiert für eine putative ftsH Chloroplast-Protease. Beispiel 17Line 303726 (SEQ ID NO: 46 [nucleic acid] and SEQ ID NO: 47 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 303726 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 51568 of BACs AC004669 on chromosome 2. The insertion at this point very likely prevents or affects the transcription and thus the function of the ORF At2g30950. This ORF codes for a putative ftsH chloroplast protease. Example 17
Analog zu den vorgenannten. Beispielen wurde die Linie 304249 (SEQ ID NO: 48 [Nukleinsäure] und SEQ ID NO: 49 [durch vor- stehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304249 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die Insertion in Position 13004 des BACs ATF19B15, Accession AL078470, zerstört den ORF F19B15.40 der für das "AIMl" Protein aus Arabidopsis (CAB43915.1) codiert. Zu diesem ORF sind bereits mehrere ESTs, GB:Z31666, gb:Z33957, Z31666, beschrieben. Bei diesem Protein handelt es sich um ein peroxisomales tetrafunktionales Enzym des Fettsäurestoffwechsels .Analogous to the above. Examples have identified line 304249 (SEQ ID NO: 48 [nucleic acid] and SEQ ID NO: 49 [protein encoded by the above nucleic acid]). Line 304249 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The insertion in position 13004 of the BACs ATF19B15, Accession AL078470, destroys the ORF F19B15.40 which codes for the "AIMl" protein from Arabidopsis (CAB43915.1). Several ESTs have already been described for this ORF, GB: Z31666, gb: Z33957, Z31666. This protein is a peroxisomal tetrafunctional enzyme of fatty acid metabolism.
Beispiel 18Example 18
Analog zu den vorgenannten Beispielen wurde die Linie 304264 (SEQ ID NO: 50 [Nukleinsäure] und SEQ ID NO: 51 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304264 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 63762 des BACs ÄB020742 inseriert. Die Einbaustelle befindet sich ca. 240 Basenpaare stromaufwärts vom Startcodon für einen ORF K21H1.19, der für eine UDP-glucuronyltrans- ferase-ähnliches Protein codiert. Durch den Einbau der T-DNA in diese Position wird sehr wahrscheinlich die Transkription verändert oder unterbunden und damit die Funktion des Gens zerstört.Line 304264 (SEQ ID NO: 50 [nucleic acid] and SEQ ID NO: 51 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 304264 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is inserted in position 63762 of BAC ÄB020742. The installation site is approx. 240 base pairs upstream of the start codon for an ORF K21H1.19, which codes for a UDP-glucuronyl transferase-like protein. By incorporating the T-DNA in this position, the transcription is very likely to be changed or prevented and the function of the gene is thereby destroyed.
Beispiel 19Example 19
Analog zu den vorgenannten Beispielen wurde die Linie 304485 (SEQ ID NO: 52 [Nukleinsäure] und SEQ ID NO: 53 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304485 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 25034 des BACs AC006438 auf Chromsom 2 inseriert. Durch die Integration an dieser Stelle zerstört die T-DNA den ORF At2gl5820, der für- ein unbekanntes Protein codiert.Line 304485 (SEQ ID NO: 52 [nucleic acid] and SEQ ID NO: 53 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 304485 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 25034 of BACs AC006438 on chromosome 2. By integrating at this point, the T-DNA destroys the ORF At2gl5820, which codes for an unknown protein.
Beispiel 20Example 20
Analog zu den vorgenannten Beispielen wurde die Linie 304652 (SEQ ID NO: 54' [Nukleinsäure] und SEQ ID NO: 55 [durch vor- stehende Nukleinsäure codiertes Protein]) sowie SEQ ID NO: 56Line 304652 (SEQ ID NO: 54 '[nucleic acid] and SEQ ID NO: 55 [protein encoded by the above nucleic acid]) and SEQ ID NO: 56 were analogous to the abovementioned examples
[Nukleinsäure] und SEQ ID NO: 57 [durch vorstehende Nukleinsäure codiertes Protein] identifiziert. Die Linie 304652 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 6309 des BACs ATF12B17, Accession AL353995, auf Chromsom 5 inseriert. Für die benachbarte Region sind zwei verschiedene offene Lese- raster annotiert. Durch die Integration ca. 382 Basenpaare stromaufwärts der ORFs ATF12B17_20 bzw. ATF12B17_10 wird sehr wahrscheinlich die Transkription und damit die Funktion der Gene, welche für ein FPFl-ähnliches (flowering promoting factorl) Protein (ATF12B17_20, SEQ ID NO: 54 bzw. SEQ ID NO: 55) bzw. ein Protein (ATF12B17_10, SEQ ID NO: 56 bzw. SEQ ID NO: 57) mit Ähnlichkeit zu KIAA1038 Protein aus Homo sapiens, gestört oder unterbunden.[Nucleic acid] and SEQ ID NO: 57 [protein encoded by the above nucleic acid]. Line 304652 segregates for an albinolethal mutation that cosegregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 6309 of BACs ATF12B17, Accession AL353995, on chromosome 5. Two different open reading grids are annotated for the neighboring region. By integrating approx. 382 base pairs upstream of the ORFs ATF12B17_20 and ATF12B17_10, it is very likely that the transcription and thus the function of the genes which are responsible for an FPFl-like (flowering promoting factorl) protein (ATF12B17_20, SEQ ID NO: 54 or SEQ ID NO: 55) or a protein (ATF12B17_10, SEQ ID NO: 56 or SEQ ID NO: 57) with similarity to KIAA1038 protein from Homo sapiens, disturbed or prevented.
Beispiel 21Example 21
Analog zu den vorgenannten Beispielen wurde die Linie 304656 (SEQ ID NO: 58 [Nukleinsäure] und SEQ ID NO: 59. [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304656 segregiert für eine albinolethale Mutation, die. mit dem Resistenzmarker und damit der T-DNA cosegregiert .' Die T-DNA ist in Position 35169 des BACs F24P17 (Accession AC011623) auf Chromsom 3 inseriert. Dort unterbricht und damit zerstört die Insertion einen ORF F24P17.10, der für ein unbekanntes Protein codiert. Im blastp-Vergleich mit Standardeinstellungen zeigen sich deutliche Homoligien zu einem nodulin / glutamate-ammonia ligase - like protein.Line 304656 (SEQ ID NO: 58 [nucleic acid] and SEQ ID NO: 59. [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 304656 segregates for an albinolethal mutation that. with the resistance marker and thus the T-DNA cosegregated. ' The T-DNA is inserted in position 35169 of BAC F24P17 (Accession AC011623) on chromosome 3. There, the insertion interrupts and thus destroys an ORF F24P17.10, which codes for an unknown protein. A blastp comparison with standard settings shows clear homoligues to a nodulin / glutamate-ammonia ligase - like protein.
Beispiel 22Example 22
Analog zu den vorgenannten Beispielen wurde die Linie 302192 (SEQ ID NO: 60 [Nukleinsäure] und SEQ ID NO: 61 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 302192 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert . Die T-DNA ist in Position 43178 des BACs AB022211 auf Chromsomen 5 inseriert. Durch die Integration ca. 454 Basenpaare 'stromaufwärts des ORFs K1L20.13 wird sehr wahrscheinlich des Transkription und damit die Funktion des Gens, welches für ein SHI-ähnliches Zinkfingerprotein (short internodes) codiert, gestört oder unter- bunden..Line 302192 (SEQ ID NO: 60 [nucleic acid] and SEQ ID NO: 61 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 302192 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 43178 of BAC AB022211 on chromosome 5. By integrating approx. 454 base pairs ' upstream of the ORF K1L20.13, it is very likely that the transcription and thus the function of the gene which codes for an SHI-like zinc finger protein (short internodes) will be disturbed or prevented.
Beispiel 23Example 23
Analog zu den vorgenannten Beispielen wurde die Linie 302636 (SEQ ID NO: 62 [Nukleinsäure] und SEQ ID NO: 63 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 302636 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 141376 des BACs ATF4P12 (Acces'sion: AL132966) auf Chromosom 3 inseriert . Die Integration der T-DNA an dieser Position unterbricht und damit inaktiviert den ORF F4P12_400, welcher für Protein.mit Ähnlichkeit zu crpl au Zea mays, PIR:T01685 codiert. Dieser ORF beinhaltet zudem die ESTs gb:AI999771.1, T45254, AA713158".Line 302636 (SEQ ID NO: 62 [nucleic acid] and SEQ ID NO: 63 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. The line 302636 segregates for an albinolethal mutation with the resistance marker and thus the T-DNA. The T-DNA is (sion Acces': AL132966) at position 141,376 of the BACs ATF4P12 on chromosome 3 inserted. The integration of the T-DNA at this position interrupts and thus inactivates the ORF F4P12_400, which codes for protein similar to crpl au Zea mays, PIR: T01685. This ORF also includes the ESTs gb: AI999771.1, T45254, AA713158 ".
Beispiel 24Example 24
Analog zu den vorgenannten Beispielen wurde die Linie 302894 (SEQ ID NO: 64 [Nukleinsäure] und SEQ ID NO: 65 (durch vorstehende Nukleinsäure codiertes Protein)) identifiziert. Die Linie 302894 segregiert für eine albinolethale Mutation, die mit' dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 23970 des BACs ATT21H19 (Accession: AL391148) auf Chromosom 5 inseriert. Die' Insertion der T-DNA an dieser Stelle unterbricht einen ORF (T21H19_100) der für ein putatives Protein mit Ähnlichkeiten zu hypothetischen Proteinen aus Arabidopsis codiert. In der blastp-Analyse zeigt sich zudem eine deutliche Homologie zu. CRS1 aus Zea mays Accession AAG00595', wobei es sich um einen Gruppe II Intron Splicingfaktor handelt (Till,B et al., RNA 7 (9), 1227-1238 (2001)).Line 302894 (SEQ ID NO: 64 [nucleic acid] and SEQ ID NO: 65 (protein encoded by the above nucleic acid)) was identified analogously to the abovementioned examples. Line 302894 segregates for a albinolethale mutation cosegregates with 'the resistance marker and thus the T-DNA. The T-DNA is inserted in position 23970 of the BACs ATT21H19 (Accession: AL391148) on chromosome 5. The insertion of the T-DNA at this point interrupts an ORF (T21H19_100) which codes for a putative protein with similarities to hypothetical proteins from Arabidopsis. The blastp analysis also shows clear homology. CRS1 from Zea mays Accession AAG00595 ' , which is a Group II intron splicing factor (Till, B et al., RNA 7 (9), 1227-1238 (2001)).
Beispiel 25Example 25
Analog zu den vorgenannten Beispielen wurde die Linie 305146 (SEQ ID NO: 66 [Nukleinsäure] und SEQ ID NO: 67 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 305146 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 65706 des Pl-Klons MOP9 (Accession: AB0067.01) auf Chromosom 5 inseriert. Die Insertion der T-DNA an die'ser Stelle unterbricht den ORF des Gens At5g24315, der für ein unbekanntes Protein codiert.Line 305146 (SEQ ID NO: 66 [nucleic acid] and SEQ ID NO: 67 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 305146 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 65706 of the PI clone MOP9 (accession: AB0067.01) on chromosome 5. The insertion of the T-DNA at this point disrupts the ORF of the At5g24315 gene, which codes for an unknown protein.
Beispiel 26Example 26
Acc : AL163816 Name : ATT20O10 .Acc: AL163816 Name: ATT20O10.
Analog zu den vorgenannten Beispielen wurde die Linie 305156 (SEQ ID NO: 68 [Nukleinsäure] und SEQ ID NO: 69 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 305156 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 898 des BACs ATT20O10 M0P9 (Accession: AL163816) auf Chromosom 3 inseriert . Die Insertion der T-DNA an dieser Stelle unterbricht einen ORF (T20O10_10) der für ein Protein mit hoher Ähnlichkeit zu translation releasing factor RF-1 aus Synechocystis (PIR:S76914) zeigt. Dazu enthält die abgleitete Aminosäuresequenz ein prokaryotische Type Klasse I Pepetidketten- ablösungsfaktor-Motiv, AA280-296Line 305156 (SEQ ID NO: 68 [nucleic acid] and SEQ ID NO: 69 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 305156 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 898 of the BAC ATT20O10 M0P9 (Accession: AL163816) on chromosome 3. The insertion of the T-DNA on this Stelle interrupts an ORF (T20O10_10) which shows a protein with a high similarity to translation releasing factor RF-1 from Synechocystis (PIR: S76914). For this purpose, the deduced amino acid sequence contains a prokaryotic Type I pepeptide chain detachment factor motif, AA280-296
Beispiel 27Example 27
Analog zu den vorgenannten Beispielen wurde die Linie 304044 (SEQ ID NO: 70 [Nukleinsäure] und SEQ ID NO: 71 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304044 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 44121 des BACs ATAP22 MOP9 (Accession: Z99708) auf Chromosom 4 inseriert . Die Insertion der T-DNA an dieser Stelle unterbricht einen ORF (C7A10.610) der für ein Protein mit hoher Ähnlichkeit zu einem Allergen (" inor allergen") aus Alternaria alternata (PIR2 :S43111) . Zu diesem ORF wurden zudem bereits die ESTS gb:R64949., ÄA651052 gefunden.Line 304044 (SEQ ID NO: 70 [nucleic acid] and SEQ ID NO: 71 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 304044 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is inserted in position 44121 of the BACs ATAP22 MOP9 (Accession: Z99708) on chromosome 4. The insertion of the T-DNA at this point interrupts an ORF (C7A10.610) which is for a protein with high similarity to an allergen ("inor allergen") from Alternaria alternata (PIR2: S43111). ESTS gb: R64949., ÄA651052 have also already been found for this ORF.
Acc: Z99708 Name: ATAP22Acc: Z99708 Name: ATAP22
Beispiel 28Example 28
Analog zu den vorgenannten Beispielen wurde die Linie 140412 • (SEQ ID NO: 72 [Nukleinsäure] und SEQ ID NO: 73 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 140412 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 68520 der Sequenz AC006264 auf ' Chromosom 2 inseriert. Die Insertion der T-DNA unterbricht den 3'UTR des Gens At2g21160 und unterbindet • somit sehr wahrscheinlich die Funktion des ORFs durch die Bestabilisierung des Transkripts . 'Der ORF At2g21160 codiert für die Alpha-Untereinheit eines putativen Signalsequencereceptors .Line 140412 • (SEQ ID NO: 72 [nucleic acid] and SEQ ID NO: 73 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 140412 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 68520 of sequence AC006264 on 'chromosome 2. The insertion of the T-DNA breaks the 3'UTR of the gene At2g21160 and thus prevents • very likely the function of the ORFs by Bestabilisierung the transcript. 'The ORF At2g21160 codes for the alpha subunit of a putative signal sequence receptor.
Acc: AC006264 Name: AC006264Acc: AC006264 Name: AC006264
Beispiel 29Example 29
Analog zu den vorgenannten Beispielen wurde die Linie 159012 (SEQ-ID NO: 74 [Nukleinsäure] und SEQ ID NO: 75 [durch vor- ■ stehende Nukleinsäure codiertes Protein])- identifiziert. Die Linie 159012 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 127261 der Sequenz ATCHRIV3 (frag ent No. 3), Accession AL161491 auf Chromosom 4 inseriert. Die Insertion der T-DNA unterbricht den ORF AT4g01220,- der die ESTs gb:AA597894, AA597304 beinhaltet und für unbekanntes Protein codiert.(74 [nucleic acid] and SEQ ID NO: SEQ ID NO 75 [Related by upstream ■ nucleic acid encoded protein]) in analogy to the above examples, the line 159012 was - identified. Line 159012 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 127261 of the sequence ATCHRIV3 (frag ent No. 3), Accession AL161491 on chromosome 4. The insertion of the T-DNA interrupts the ORF AT4g01220, - which contains the ESTs gb: AA597894, AA597304 and codes for unknown protein.
Acc: AL161491 Name: ATCHRIV3 (Fragment No. 3]Acc: AL161491 Name: ATCHRIV3 (Fragment No. 3)
Beispiel 30Example 30
Analog zu den vorgenannten Beispielen wurde die Linie 106037 (SEQ ID NO: 76 [Nukleinsäure] und SEQ ID NO: 77 [durch vor- stehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 106037 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker. und damit der T-DNA cosegregiert. Die T-DNÄ ist in Position 50359 der Sequenz AC006193 auf Chromosom 1 inseriert. Die Insertion der T-DNA an dieser Position unterbricht den ORF F13011.ll, der für ein unbekanntes Protein codiert. Im der der Blastp-Analyse mit Standardeinstellungen zeigen sich eine - Ähnlichkeit zu Oxidoreduktasen.Line 106037 (SEQ ID NO: 76 [nucleic acid] and SEQ ID NO: 77 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 106037 segregates for an albinolethal mutation with the resistance marker. and thus the T-DNA cosegregates. The T-DNÄ is inserted in position 50359 of sequence AC006193 on chromosome 1. The insertion of the T-DNA at this position interrupts the ORF F13011.ll, which codes for an unknown protein. The blastp analysis with standard settings shows a similarity to oxidoreductases.
Acc: AC006193 Name: AC006193Acc: AC006193 Name: AC006193
Beispiel 31Example 31
Analog zu den vorgenannten Beispielen wurde die Linie 126905 (SEQ ID NO: 78 [Nukleinsäure] und 'SEQ ID NO: 79 [durch vor- stehende Nukleinsäure codiertes Protein]) identifiziert. DieLine 126905 (SEQ ID NO: 78 [nucleic acid] and ' SEQ ID NO: 79 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. The
Linie 126905 segregiert für eine embryolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 71928 der BACs ATF25L23, Accession AL356014,- auf Chromosom 3 inseriert. Die Insertion der T-DNA an dieser Position unterbricht den ORF' F25L23_240" der für eine Farnesyltransferase Untereinheit A codiert.Line 126905 segregates for an embryolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted in position 71928 of BACs ATF25L23, Accession AL356014, on chromosome 3. The insertion of the T-DNA at this position interrupts the ORF ' F25L23_240 "which codes for a farnesyl transferase subunit A.
Acc: AL356014 Name: ATF25L23Acc: AL356014 Name: ATF25L23
Beispiel 32Example 32
Analog zu den vorgenannten Beispielen wurde die Linie 12.7458 (SEQ ID NO: 80 [Nukleinsäure] und SEQ ID NO: 81 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 127458 segregiert für eine embryolethale Mutation, die mit .dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 45352 der BACs T19K24, Accession AC002342, auf Chromosom 5 inseriert. Die Insertion der T-DNA an dieser Position unterbricht den ORF T19K24.18, der für den ATP-abhängigen Kupfer- transporter RANl codiert.Line 12.7458 (SEQ ID NO: 80 [nucleic acid] and SEQ ID NO: 81 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 127458 segregates for an embryolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is inserted in position 45352 of BACs T19K24, Accession AC002342, on chromosome 5. The insertion of the T-DNA at this position interrupts the ORF T19K24.18, which codes for the ATP-dependent copper transporter RANl.
Acc: AC002342 Name: ATAC002342 Beispiel 33Acc: AC002342 Name: ATAC002342 Example 33
Analog zu den vorgenannten Beispielen wurde die Linie 304249b (SEQ ID NO: 82 [Nukleinsäure] und SEQ ID NO: 83 [durch vor- stehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304249b segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die- Insertion in Position 17105 des BACs ATF19B15, Accession AL078470, zerstört den ORF F19B15.50, der Ähnlichkeiten zu glyzinreichen Proteinen zeigt und die ESTs gb:Z29181, T42831, Z34138, Z33797, Z30844, beinhaltet.Line 304249b (SEQ ID NO: 82 [nucleic acid] and SEQ ID NO: 83 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 304249b segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The insertion in position 17105 of BACs ATF19B15, Accession AL078470, destroys ORF F19B15.50, which shows similarities to glycine-rich proteins and which contains ESTs gb: Z29181, T42831, Z34138, Z33797, Z30844.
Beispiel 34Example 34
Analog zu den vorgenannten Beispielen wurde die Linie 304264b (SEQ ID"NO: 84 [Nukleinsäure] und SEQ ID NO: 85 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304264b segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist in Position 63762 des BACs AB020742 inseriert. Die Einbau- steile befindet sich ca. 340 Basenpaare stromaufwärts vom Startcodon für einen ORF K21H1.18, der Ähnlichkeit zu unbekannten Proteinen aufweist.Analogously to the abovementioned examples, line 304264b (SEQ ID " NO: 84 [nucleic acid] and SEQ ID NO: 85 [protein encoded by the above nucleic acid]) was identified. Line 304264b segregates for an albinolethal mutation associated with the resistance marker and thus The T-DNA is cosegregated The T-DNA is inserted in position 63762 of the BACs AB020742 The installation parts are located approximately 340 base pairs upstream from the start codon for an ORF K21H1.18 which is similar to unknown proteins.
Beispiel 35Example 35
Analog zu den vorgenannten Beispielen wurde die Linie 192813 (SEQ ID NO: 86 [Nukleinsäure] und SEQ ID NO: 87 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 192813 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist -auf Chromsom 1 in Position 9869 des BACs- F309 mit der Accession AC006341 inseriert. Durch die Insertion ca .- 323 bp ■ stromaufwärts von dem Startcodon für einen ORF, F309.4, für den bereits mehrere ESTs identifiziert wurden (gb|F15498, gb|H37515, gb|τ41906, gb|T22448, gb|W43356, gb|T20739), wird mit hoher Wahrscheinlichkeit die Transkription und damit die Funktion des Gens für diesen ORF inhibiert. Der ORF codiert für ein Protein, welches im blastp-Vergleich unter Ständardeinstellung hohe Homologien zu verschiedenen. Syntaxinen und syntaxinähnlichen Proteinen auch aus Pflanzen zeigt.Line 192813 (SEQ ID NO: 86 [nucleic acid] and SEQ ID NO: 87 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 192813 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted on chromosome 1 in position 9869 of the BACs-F309 with the accession AC006341. By inserting approx. 323 bp ■ upstream of the start codon for an ORF, F309.4, for which several ESTs have already been identified (gb | F15498, gb | H37515, gb | τ41906, gb | T22448, gb | W43356, gb | T20739), it is highly likely that the transcription and thus the function of the gene for this ORF will be inhibited. The ORF codes for a protein which, in a blastp comparison under standard setting, has high homologies to different. Syntaxins and syntaxin-like proteins also from plants shows.
Beispiel 36Example 36
Analog zu den vorgenannten Beispielen wurde die Linie 203521 (SEQ ID NO: 88 [Nukleinsäure] und SEQ ID NO: 89 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 203521 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert . Die T-DNA ist auf Chromsom 2, Section 179 von 255 Accession AC006533 inseriert. Durch die Insertion wird der ORF AT2g31830, der für eine putative Inositol-polyphosphate-5 '-Phosphatase codiert, zerstört.Line 203521 (SEQ ID NO: 88 [nucleic acid] and SEQ ID NO: 89 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. The Line 203521 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is inserted on Chromsom 2, Section 179 of 255 Accession AC006533. The insertion destroys the ORF AT2g31830, which codes for a putative inositol polyphosphate 5 'phosphatase.
Beispiel 37Example 37
Analog zu den vorgenannten Beispielen" wurde die Linie 206462 (SEQ ID NO: 90 [Nukleinsäure] und SEQ ID NO: 91 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 206462 segregiert für eine albinolethale Mutation, -die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist. auf Chromsom 1 in Position 53577-53600 des BACs F24D7, Accession AC011622 inseriert. Durch die Insertion ah dieser Stelle wird der ORF F24D7.13, der für eine putative UDP-N-Acetyl- muramoylalanyl-D-glutamate-2 , 6-Diaminopimelate -ligase ( urE) codiert, zerstört.Analogously to the abovementioned examples " , line 206462 (SEQ ID NO: 90 [nucleic acid] and SEQ ID NO: 91 [protein encoded by the above nucleic acid]) was identified. Line 206462 segregates for an albinolethal mutation, -with the resistance marker and The T-DNA is inserted on chromosome 1 in position 53577-53600 of BAC F24D7, accession AC011622. By inserting ah here, the ORF F24D7.13, which is responsible for a putative UDP-N- Acetyl-muramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase (urE) coded, destroyed.
Beispiel 3ΪExample 3Ϊ
Analog zu den vorgenannten Beispielen wurde die Linie 216642 (SEQ ID NO: 92 [Nukleinsäure] und SEQ ID NO: 93 [durch vor- stehende Nukleinsäure codiertes Protein]) identifiziert. DieLine 216642 (SEQ ID NO: 92 [nucleic acid] and SEQ ID NO: 93 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. The
Linie 216642 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA -cosegregiert. Die T-DNA ist auf Chromsom 3 in Position 18529 des PI Klons MRC8, Accession- AB020749 inseriert. Durch die Insertion an dieser Stelle wird der ORF MRC8.5, der für eine Beta-Glucosidase codiert, zerstört.Line 216642 segregates for an albinolethal mutation, which segregates with the resistance marker and thus the T-DNA cos. The T-DNA is inserted on chromosome 3 in position 18529 of PI clone MRC8, Accession-AB020749. The insertion at this point destroys the ORF MRC8.5, which codes for a beta-glucosidase.
• Beispiel 39 • Example 39
Analog zu den vorgenannten Beispielen wurde die Linie 219902 (SEQ ID NO: 94 -[Nukleinsäure] und SEQ ID NO: 95 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 219902 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 1 in Position 7740 des BACs F15M4, Accession AC012394 inseriert. Durch die Insertion an dieser Stelle wird der ORF F15M4.1, der für eine Hydroxymethylglutaryl-CoA-Reductase codiert, zerstört. Beispiel 40Line 219902 (SEQ ID NO: 94 - [nucleic acid] and SEQ ID NO: 95 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 219902 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted on chromosome 1 in position 7740 of BAC F15M4, Accession AC012394. The insertion at this point destroys the ORF F15M4.1, which codes for a hydroxymethylglutaryl-CoA reductase. Example 40
Analog zu den vorgenannten Beispielen wurde die Linie 220801 (SEQ ID NO: 96 [Nukleinsäure] und SEQ ID NO: 97 [durch vor- stehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 220801 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 5 in Position 15447-15472 des Pl Klons MRN17 , Accession AB005243 inseriert. Durch diese Insertion ca. 580bp stromaufwärts vom Startcodon wird vermutlich zumindest dieLine 220801 (SEQ ID NO: 96 [nucleic acid] and SEQ ID NO: 97 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 220801 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is inserted on chromosome 5 in position 15447-15472 of Pl clone MRN17, accession AB005243. With this insertion approx. 580bp upstream from the start codon, at least the
Transkription und damit die Funktion des ORFs MRN17.4 zerstört. Dieser codiert für ein GDSL-Motif Lipase/Hydrolase-like Protein.Transcription and thus the function of the ORF MRN17.4 destroyed. This codes for a GDSL-Motif lipase / hydrolase-like protein.
Beispiel 41Example 41
Analog zu den vorgenannten Beispielen wurde die Linie 224933 (SEQ ID NO-: 98 [Nukleinsäure] und SEQ ID NO: 99 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 224933 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 4, ΞSSA I FCA Contig-Fragment No. 3., Accession Z97338, in Position 107932-107997 inseriert .. Durch diese Insertion wird die Funktionalität des ORFs dl3705c, der für ein Zellulose-Synthase-ähnliches Protein codiert, zerstört.Line 224933 (SEQ ID NO-: 98 [nucleic acid] and SEQ ID NO: 99 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 224933 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is on chromosome 4, ΞSSA I FCA Contig fragment No. 3., Accession Z97338, in position 107932-107997. This insertion destroys the functionality of the ORF dl3705c, which codes for a cellulose synthase-like protein.
Beispiel 42Example 42
Analog zu den vorgenannten Beispielen wurde die Linie 229091 (SEQ ID NO: 100 [Nukleinsäure] und SEQ ID. NO: 101 [durch vor- stehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 229091 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit "der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 5, TAC-Klon:K5J14, Accession AB023032, in Position 55778 inseriert. Durch die Insertion an dieser Stelle wird die Funktionalität des ORFs K5J14.11 zerstört, - der für ein dem crpl Protein aus Mais ähnliches Protein codiert..Line 229091 (SEQ ID NO: 100 [nucleic acid] and SEQ ID NO: 101 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 229091 segregates for an albinolethal mutation which co-segregates with the resistance marker and thus with the " T-DNA. The T-DNA is inserted on position 5, TAC clone: K5J14, accession AB023032, in position 55778. By insertion on this The functionality of the ORF K5J14.11 is destroyed, which codes for a protein similar to the crpl protein from maize.
Beispiel 43Example 43
Analog zu den vorgenannten Beispielen wurde die Linie 246473 (SEQ ID NO: 102 [Nukleinsäure] und SEQ ID NO: 103 [durch vorstehende Nukleinsäure codiertes Protein] ) identifiziert. Die Linie 246473 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 1, BAC F4F7 , Accession AC079374, in Position 17376 inseriert. Durch die Insertion an dieser Stelle, ca. 7 bp stromabwärts von dem ORF F4F7.26 wird mit hoher Wahrscheinlich- keit die Transkription bzw. die Transkriptstabilität und damit die Funktionalität für dieses offenen Leseraster zerstört. Dieser ORF codiert für eine putative t-RNA Glutaminsynthetase und weist insbesondere Homologie zur tRNA Glutaminsynthetase GI: 2995454 aus Lupinüs luteus auf .Line 246473 (SEQ ID NO: 102 [nucleic acid] and SEQ ID NO: 103 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 246473 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted on chromosome 1, BAC F4F7, accession AC079374, in position 17376. The insertion at this point, approx. 7 bp downstream of the ORF F4F7.26, is highly likely to The transcription or transcript stability and thus the functionality for this open reading frame is destroyed. This ORF codes for a putative t-RNA glutamine synthetase and in particular has homology to tRNA glutamine synthetase GI: 2995454 from Lupinüs luteus.
Beispiel 44Example 44
Analog zu den vorgenannten Beispielen wurde die Linie 304139 (SEQ ID NO: 104 [Nukleinsäure] und SEQ ID NO: 105 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304139 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 5, P 1 Klon MFB 13, Accession AB010073, in Position 49311-49335 inseriert. Durch die insertion an dieser Stelle, ca. 25 bp stromabwärts von dem ORF MFB13.17 wird mit hoher Wahrscheinlichkeit die Transkription bzw. die Transkriptstabilität und damit die Funktionalität für dieses offene Leseraster zerstört, welches für ein Exonuklease-ähnliches Protein codiert.Line 304139 (SEQ ID NO: 104 [nucleic acid] and SEQ ID NO: 105 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 304139 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus with the T-DNA. The T-DNA is inserted on chromosome 5, P 1 clone MFB 13, accession AB010073, in position 49311-49335. By insertion at this point, approx. 25 bp downstream of the ORF MFB13.17, the transcription or the transcript stability and thus the functionality for this open reading frame, which codes for an exonuclease-like protein, is destroyed with high probability.
Beispiel 45Example 45
Analog zu den vorgenannten Beispielen wurde die Linie 304886 (SEQ ID NO: 106 [Nukleinsäure] und SEQ ID NO: 107 [durch vorstehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 304886 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 2, BAC Klon F23H14, Section 1 von 255, Accession AC006837, in Position 84045 inseriert. Durch die Insertion an dieser Stelle wird der ORF At2g01110 unterbrochen und inaktiviert. Dieser codiert für ein putatives "sec-unabhängiges" Trans- lokase-Protein TATC (putative sec-independent protein' trahSiocase protein TATC) . Die Sequenz wird auch in WO 144277 beschrieben.Line 304886 (SEQ ID NO: 106 [nucleic acid] and SEQ ID NO: 107 [protein encoded by the above nucleic acid]) was identified analogously to the abovementioned examples. Line 304886 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is inserted on chromosome 2, BAC clone F23H14, section 1 of 255, accession AC006837, in position 84045. The ORF At2g01110 is interrupted and deactivated by the insertion at this point. This codes for a putative "sec-independent" trans-locase protein TATC (putative sec-independent protein ' t r ah S iocase protein TATC). The sequence is also described in WO 144277.
Beispiel 46Example 46
Analog zu den vorgenannten Beispielen wurde die Linie 306053 (SEQ ID NO: 108 [Nukleinsäure] und SEQ ID NO: 109 [durch vor-' stehende Nukleinsäure codiertes Protein]) identifiziert. Die Linie 306053 segregiert für eine albinolethale Mutation, die mit dem Resistenzmarker und damit der T-DNA cosegregiert. Die T-DNA ist auf Chromsom 4, BAC-Klon F28J12, Accession AGAL021710, in Position 74806-74828. Durch die Insertion an dieser Stelle wird der ORF F28J12.180, welcher für ein putatives Protein codiert, unterbrochen und inaktiviert. In blastp-Analysen mit Standardeinstellung zeigt die abgeleitete Aminosäureseguenz , neben deutliche Homologien zu verschiedenen hypothetischen und putativen Proteinen auch starke Ähnlichkeit zu Selenium-Bindungs- protein ähnlichen Proteinen. (: 108 [nucleic acid] and SEQ ID NO: SEQ ID NO 109 [encoded by upstream 'standing nucleic acid protein]) in analogy to the above examples, the line 306053 was identified. Line 306053 segregates for an albinolethal mutation that co-segregates with the resistance marker and thus the T-DNA. The T-DNA is on chromosome 4, BAC clone F28J12, accession AGAL021710, in position 74806-74828. The insertion at this point interrupts and inactivates the ORF F28J12.180, which codes for a putative protein. In blastp analyzes with standard settings, the deduced amino acid sequence shows In addition to clear homologies to various hypothetical and putative proteins, there is also strong similarity to proteins similar to selenium binding proteins.

Claims

Patentansprüche claims
1. Verfahren zur Identifizierung von Substanzen mit herbizider Wirkung, dadurch gekennzeichnet, dass:1. A method for identifying substances with a herbicidal action, characterized in that:
a) die Expression oder die Aktivität des Genprodukts einer Nukleinsäure oder eines Gens umfassend:a) the expression or the activity of the gene product of a nucleic acid or a gene comprising:
aa) Nukleinsäuresequenz mit der in SEQ ID NO: 1,aa) nucleic acid sequence with that in SEQ ID NO: 1,
SEQ ID NO 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO 9, SEQ ID NO: 11, SEQ ID NO: 13,SEQ ID NO 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO 15, SEQ ID NO: 17, SEQ ID NO: 26SEQ ID NO 15, SEQ ID NO: 17, SEQ ID NO: 26
SEQ ID NO 28, SEQ ID NO: 30,- SEQ ID NO: 32SEQ ID NO 28, SEQ ID NO: 30 SEQ ID NO: 32
SEQ ID NO 34, SEQ ID NO: 36, SEQ ID NO: 38SEQ ID NO 34, SEQ ID NO: 36, SEQ ID NO: 38
SEQ ID NO 40, SEQ ID NO: 44, SEQ ID NO: 46SEQ ID NO 40, SEQ ID NO: 44, SEQ ID NO: 46
SEQ ID NO 48, SEQ ID NO: 50, SEQ ID NO: 52,SEQ ID NO 48, SEQ ID NO: 50, SEQ ID NO: 52,
SEQ' ID NO 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ' ID NO 54, SEQ ID NO: 56, SEQ ID NO: 58,
SEQ ID NO 60, SEQ ID NO: 62,' SEQ ID NO:.64,SEQ ID NO 60, SEQ ID NO: 62, ' SEQ ID NO: .64,
SEQ ID NO 66, SEQ ID NO: 68, SEQ ID NO: 70,SEQ ID NO 66, SEQ ID NO: 68, SEQ ID NO: 70,
SEQ ID NO 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO 72, SEQ ID NO: 74, SEQ ID NO: 76,
SEQ ID NO 78, SEQ ID NO: 80,' SEQ ID NO: 82SEQ ID NO 78, SEQ ID NO: 80, 'SEQ ID NO: 82
SEQ ID NO 84, SEQ ID NO: '86, SEQ ID NO: 88,SEQ ID NO 84, SEQ ID NO: '86, SEQ ID NO: 88,
SEQ ID NO 90, SEQ ID NO: 92, SEQ ID NO: 94,SEQ ID NO 90, SEQ ID NO: 92, SEQ ID NO: 94,
SEQ ID NO 96, SEQ ID NO: 98, SEQ ID NO: 100,SEQ ID NO 96, SEQ ID NO: 98, SEQ ID NO: 100,
SEQ ID NO 102 , SEQ ID NO: 106 oder SEQ ID NO: 108 dargestellten Sequenz ; oderSEQ ID NO 102, SEQ ID NO: 106 or SEQ ID NO: 108 sequence shown; or
bb) Nukleinsäuresequenz, .die sich aufgrund des degenerierten genetischen Codes aus den durch Rückübersetzung der in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:' 12, SEQ ID NO: 14, SEQ ID NO:' 16,bb) nucleic acid sequence NO: '12, SEQ ID NO: 14, SEQ ID NO:' 16,
SEQ TD NO: 18, SEQ ID NO: 27, SEQ ID NO: 29,SEQ TD NO: 18, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,
SEQ ID NO: 45, SEQ ID NO: 47,' SEQ ID NO: 49,SEQ ID NO: 45, SEQ ID NO: 47, ' SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,
SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61,
SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73,
SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,
Fig. + Sequ. SEQ ID NO: 81, SEQ ID NO 83, SEQ ID NO: 85,Fig. + Sequ. SEQ ID NO: 81, SEQ ID NO 83, SEQ ID NO: 85,
SEQ ID NO: 87, SEQ ID NO 89, SEQ ID NO: 91,SEQ ID NO: 87, SEQ ID NO 89, SEQ ID NO: 91,
SEQ ID NO: 93, SEQ ID NO 95, SEQ ID NO: 97,SEQ ID NO: 93, SEQ ID NO 95, SEQ ID NO: 97,
SEQ ID NO: 99, SEQ ID NO 101 , SEQ ID NO: 103,SEQ ID NO: 99, SEQ ID NO 101, SEQ ID NO: 103,
SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen ableiten lässt;SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 derived amino acid sequences;
cc) Nukleinsäuresequenz, die ein Derivat oder ein - Fragment der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ. ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100 , SEQ ID NO : 102, SEQ ID NO: 106 oder SEQ ID N NOO: : ιoε $ dargestellten Nukleinsäuresequenzen ist, uunndd mindestens 60 % Homologie auf Nukleinsäureebene aufweist;cc) Nucleic acid sequence which is a derivative or a - fragment of the SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 SEQ ID NO: 9, SEQ. ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO : 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52 , SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO : 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102 , SEQ ID NO: 106 or SEQ ID N NOO:: ιoε $ nucleic acid sequences shown, and has at least 60% homology at the nucleic acid level;
dd) Nukleinsäuresequenz, die für Derivate oderdd) nucleic acid sequence which is suitable for derivatives or
Fragmente der Polypeptide mit den in SEQ ID NO: 2, SEQ ID NO: 4, SEQ- ID NO: 6, SE ID NO: 8,Fragments of the polypeptides with those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SE ID NO: 8,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO 14,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO 14,
SEQ ID NO: 16, SEQ ID NO: 18,. SEQ ID NO ' 27,SEQ ID NO: 16, SEQ ID NO: 18. SEQ ID NO '27,
SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO ' 33,SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO '33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO 39,SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO 39,
SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO 47,SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO 47,
SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO 53,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO 53,
SEQ ID NO:- 55, SEQ ID NO: 57, SEQ ID NO 59,SEQ ID NO: - 55, SEQ ID NO: 57, SEQ ID NO 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO 65,SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO 65,
SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO 71,SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO 71,
SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO 77,SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO 83,SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO 83,
SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO 89,SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO 89,
SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO 95,SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO 95,
SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO 101,SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO 101,
SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestellten Aminosäuresequenzen codiert, die mindestens 50 % Homologie auf Amino- säureebene aufweisen;SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109 amino acid sequences shown encoded that have at least 50% homology at the amino acid level;
ee) Nukleinsäuresequenz, die für ein Fragment oder ein Epitope eines Polypeptides codiert, das spezifisch an einem Antikörper bindet, wobei der Antikörper spezifisch an ein Polypeptid bindet, das von der in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,ee) Nucleic acid sequence which codes for a fragment or an epitope of a polypeptide which specifically binds to an antibody, the antibody specifically binding to a polypeptide which is derived from that described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO : 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:" 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: "48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,
SEQ ID NO: 58, SEQ ID NO: 60, SEQ " ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,SEQ ID NO: 58, SEQ ID NO: 60, SEQ " ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,
SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96 , SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108. dargestellten Sequenz codiert wird;SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108 . the sequence shown is encoded;
ff) Nukleinsäuresequenz, die für ein Fragment einer in aa) dargestellten Nukleinsäure codiert und das eine m6A-Methyltransferase-Aktivität, eine DNA- binde'nde-Aktivität oder "DNA-repair"-Aktivität, z.B. wie bei RAD 54, eine Thioredoxin-Aktivität, eineff) nucleic acid sequence, a DNA-binding a fragment of an in aa) illustrated nucleic acid encoding and a m6A methyltransferase activity 'hands activity or "DNA repair" activity, such as with wheel 54, a thioredoxin Activity, a
VÄV2-Aktivität, eine Fructokinase-Aktivität , eine Ziήkfingerprotein-Aktivität, eine LYTB-Aktivität, eine Crepopin-Aktivität, . eine Leucin-Protein- Aktivität, eine DNAJ-Aktivität, ein CRSl-Aktivität, eine Alanyl-tRNA-Synthetase-Aktivität, eineVÄV2 activity, a fructokinase activity, a finger finger protein activity, a LYTB activity, a crepopin activity,. a leucine protein activity, a DNAJ activity, a CRS1 activity, an alanyl tRNA synthetase activity, a
OEP86-Aktivität, eine FMRF-Amid-Propeptid-Isolog- Aktivität, eine 26S Proteosom subunit S5B-Aktivität, eine Geranylgeranylpyrophosphatsynthase-Aktivität, eine Cecropin-Familiensignatur aufweißt, ftsH Cloro- plast-Protease-Aktivität hat, eine AIMl-Aktivität, eine UDP-glucuronyltransferase-Aktivität, eine FPFl-Aktivität, eine SHI-ähnliche Zinkfingerprotein- Aktivität hat, eine Crpl-Aktivität, eine CRSl- Aktivität, eine translation releasing factor RF-1-Aktivität, eine Farnesyltransferase Untereinheit A-Aktivität, eine ATP-abhängige Kupfertransporter RANl-Aktivität , eine Syntaxin oder Syntaxin- ähnliche Proteinaktivität, eine Inositol-Poly- phosphat-5 ' -Phosphatase-Aktivität , eine UDP-N-Acetyl- muramoylalanyl-D-glutamat-2 , 6-Diaminopimelat-ligase- aktivität (murE) , eine ß-Glucosidase-Aktivität, eine Hydroxymethylglutaryl-CoA-Reductase, eine GDSL-Motif-OEP86 activity, an FMRF amide propeptide isolog activity, a 26S proteosome subunit S5B activity, a geranylgeranyl pyrophosphate synthase activity, a Cecropin family signature, ftsH chloroplas protease activity, an AIMI activity, a UDP-glucuronyltransferase activity, FPFl activity, SHI-like zinc finger protein activity, Crpl activity, CRSl activity, translation releasing factor RF-1 activity, farnesyltransferase subunit A activity, ATP- dependent copper transporter RANl activity, a syntaxin or syntaxin similar protein activity, an inositol polyphosphate 5 'phosphatase activity, a UDP-N-acetyl muramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase activity (murE), a β-glucosidase activity, a hydroxymethylglutaryl-CoA reductase, a GDSL motif
Lipase/Hydroxylase-ähnliche Proteinaktivität, eine Zellulosesynthase ähnliche Proteinaktivität, eine tRNA Glutaminsynthetase, eine Exonuklease-ähnliche Proteinaktivität, eine sec-unabhängige Translocase- Protein TATC-Aktivität oder eine Selenium-Bindung- sprotein ähnliche Proteinaktivität hat; und/oderHas lipase / hydroxylase-like protein activity, a cellulose synthase-like protein activity, a tRNA glutamine synthetase, an exonuclease-like protein activity, a sec-independent Translocase protein TATC activity or a selenium binding-protein-like protein activity; and or
gg) Nukleinsäuresequenz, die für Derivate der Polypeptide mit den in SEQ ID NO: 2, SEQ ID- NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, .SEQ ID NO: 12,gg) Nucleic acid sequence which is suitable for derivatives of the polypeptides with those in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, .SEQ ID NO: 12,
-SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: -31, - SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45-, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,-SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: -31, - SEQ ID NO: 33, SEQ ID NO: 35 , SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 45-, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,
SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,
SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 oder SEQ ID NO: 109 dargestelltenSEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107 or SEQ ID NO: 109
Aminosäuresequenzen codiert, die mindestens 20 % Homologie auf Aminosäureebene aufweist und eine äquivalente biologische Aktivität besitzt; oderEncoded amino acid sequences that have at least 20% homology at the amino acid level and have an equivalent biological activity; or
b) die Expression oder Aktivität einer Aminosäuresequenz, die von einer Nukleinsäuresequenz nach aa) bis gg) codiert wird, •b) the expression or activity of an amino acid sequence which is encoded by a nucleic acid sequence according to aa) to gg),
beeinflusst wird und solche Substanzen ausgewählt werden, die die Expression oder die Aktivität reduzieren oder blockieren. is influenced and those substances are selected which reduce or block expression or activity.
2. Verfahren nach Anspruch 1, wobei die Expression oder die Aktivität der Nukleinsäure oder Aminosäure dadurch reduziert oder blockiert wird, dass die2. The method according to claim 1, wherein the expression or the activity of the nucleic acid or amino acid is reduced or blocked by the
a) Transcription, b) Translation, c) Prozessierung und/oder d) Modifikationa) Transcription, b) Translation, c) Processing and / or d) Modification
der Nukleinsäuresequenz oder Aminosäuresequenz in Anspruch 1 reduziert oder blockiert wird.the nucleic acid sequence or amino acid sequence in claim 1 is reduced or blocked.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass die Identifizierung der Substanzen in einem High- Throughput-Screening (HTS) durchgeführt wird.3. The method according to claim 1 or 2, characterized in that the identification of the substances is carried out in a high-throughput screening (HTS).
4. Verfahren nach einem der Ansprüche 1 bis 3,. dadurch gekennzeichnet, dass man die ausgewählten Substanzen ■ auf eine Pflanze verbringt, um die herbizide Aktivität der Substanzen zu testen und die Substanzen auswählt, die eine herbizide Aktivität zeigen.4. The method according to any one of claims 1 to 3 ,. characterized in that the selected substances are ■ placed on a plant in order to test the herbicidal activity of the substances and the substances which have a herbicidal activity are selected.
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass das Verfahren in einem Organismus durch- geführt wird.5. The method according to any one of claims 1 to 4, characterized in that the method is carried out in an organism.
6. Verfahren nach einem der Ansprüche 1 bis 5 , dadurch gekennzeichnet, dass als Organismus Bakterien, Hefen, Pilze oder Pflanzen verwendet werden.6. The method according to any one of claims 1 to 5, characterized in that bacteria, yeast, fungi or plants are used as the organism.
7. Verfahren nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, dass ein Organismus verwendet wird, der eine konditionale oder natürliche Mutante einer der in Anspruch 1 beschriebenen Sequenzen ist .7. The method according to any one of claims 1 to 6, characterized in that an organism is used which is a conditional or natural mutant of one of the sequences described in claim 1.
8. Nukleinsäurekonstrukt enthaltend eine Nukleinsäuresequenz , dargestellt in Anspruch 1, wobei die Nukleinsäuresequenz mit einem oder mehreren Regulationssignalen verknüpft ist.8. A nucleic acid construct containing a nucleic acid sequence, shown in claim 1, wherein the nucleic acid sequence is linked to one or more regulation signals.
9. Substanz identifiziert nach einem Verfahren nach einem der Ansprüche 1 bis 7, wobei die Substanz ein Molekulargewicht von kleiner 1000 Dalton, einem Ki-Wert kleiner 1 mM und weniger als drei Hydroxylgruppen an einem Kohlenstoffatom- • enthaltenden Ring hat . 9. Substance identified by a method according to any one of claims 1 to 7, wherein the substance has a molecular weight of less than 1000 daltons, a Ki value of less than 1 mM and less than three hydroxyl groups on a ring containing carbon atoms.
10. Substanz identifiziert nach einem Verfahren nach einem der Ansprüche 1 bis 7, wobei es sich bei der Substanz um eine proteinogene Substanz oder um eine Antisense-RNA handelt .10. Substance identified by a method according to any one of claims 1 to 7, wherein the substance is a proteinogenic substance or an antisense RNA.
5 11. Substanz nach Anspruch 10, wobei es sich um einen Antikörper gegen das durch eine der in Anspruch 8 dargestellten Sequenz codiertes Protein handelt .11. The substance of claim 10, which is an antibody against the protein encoded by one of the sequences shown in claim 8.
12. Nukleinsäurekonstrukt nach Anspruch 8, wobei im Nukleinsäure- 10 konstrukt zusätzliche weitere Nukleinsäuresequenzen enthalten sind.12. Nucleic acid construct according to claim 8, wherein additional nucleic acid sequences are contained in the nucleic acid construct.
13. Vektor enthaltend ein Nukleinsäurekonstrukt gemäß Anspruch 8 oder 12. 1513. Vector containing a nucleic acid construct according to claim 8 or 12. 15
14. Organismus enthaltend mindestens ein Nukleinsäurekonstrukt gemäß Anspruch 8 oder 12 oder -mindestens einen Vektor gemäß14. Organism containing at least one nucleic acid construct according to claim 8 or 12 or at least one vector according to
Anspruch 13.Claim 13.
20 15. Organismus nach Anspruch 14, wobei es sich. bei dem Organismus um eine Pflanze, einen Mikroorganismus oder ein Tier handelt.15. The organism of claim 14, wherein it is . the organism is a plant, a microorganism or an animal.
16. Transgene Pflanze enthaltend ein funktionelles oder nicht funktionelles Nukleinsäurekonstrukt gemäß Anspruch 8 oder 1216. Transgenic plant containing a functional or non-functional nucleic acid construct according to claim 8 or 12
25 oder einen Vektor gemäß Anspruch 13.25 or a vector according to claim 13.
17. Verwendung eines Nukleinsäurekonstrukt gemäß Anspruch 8 oder 12 öder eines Vektors gemäß Ansprüche 13 zur Herstellung von transgenen' Pflanzen.17. Use of a nucleic acid construct according to claim 8 or 12 or a vector according to claim 13 for the production of transgenic ' plants.
3030
18. Verfahren zur Identifikation eines Antagonisten von Proteinen, die durch eine Nukleinsäuresequenz gemäß Anspruch' 8 oder 12 codiert werden indem folgende-Verfahrens- schritte durchlaufen werden18. A method for identifying an antagonist of proteins that are 8 or 12 encoded by a nucleic acid sequence according to claim 'are traversed by the following steps-process
35 i) Inkontaktbringen von Zellen, die das Protein exprimieren, oder des Proteins mit einem Kandidatenstoff;35 i) contacting cells that express the protein or the protein with a candidate substance;
ii) Testen der biologischen Aktivität des Protein;ii) testing the biological activity of the protein;
40 iii) Vergleichen der biologischen Aktivität des Proteins mit einer Standardaktivität in Abwesenheit des Kandidatenstoffs, wobei eine Verringerung der biologischen Aktivität des Proteins anzeigt, dass der Kandidaten- 45 stoff ein Antagonist ist. Iii) comparing the biological activity of the protein with a standard activity in the absence of the candidate substance, a decrease in the biological activity of the protein indicating that the candidate substance is an antagonist.
19. Verfahren nach Anspruch 18, dadurch gekennzeichnet, dass man den nach Anspruch 18 Buchstabe iii) identifizierten Anta- gonisten auf eine Pflanze verbringt, um seine herbizide Aktivität zu testen und die Antagonisten auswählt, die eine 5 herbizide Aktivität zeigen.19. The method according to claim 18, characterized in that the antagonist identified according to claim 18 letter iii) is placed on a plant in order to test its herbicidal activity and the antagonists are selected which show 5 herbicidal activity.
20. Verfahren zur Bekämpfung unerwünschten Pflanzenwuchses, dadurch gekennzeichnet, dass man eine herbizid wirksame Menge einer Substanz identifiziert nach einem Verfahren- gemäß20. A method for controlling undesirable plant growth, characterized in that a herbicidally effective amount of a substance is identified according to a method
10 Anspruch 1 bis 7 oder eines Antagonisten identifiziert nach einem Verfahren gemäß Anspruch 18 oder 19 auf Pflanzen oder deren Lebensraum oder auf Pflanzen und deren Lebensraum einwirken lässt.10 claims 1 to 7 or an antagonist identified by a method according to claim 18 or 19 on plants or their habitat or on plants and their habitat.
15 21. Verwendung einer Substanz identifiziert nach einem Verfahren nach, einem der Ansprüche 1 bis 7 oder eines- Antagonisten identifiziert nach einem Verfahren gemäß Anspruch 18 oder 19 als Herbizid oder zur Wachstumsregulierung von Pflanzen.15. Use of a substance identified by a method according to one of claims 1 to 7 or an antagonist identified by a method according to claim 18 or 19 as a herbicide or for regulating the growth of plants.
20 22. Verfahren zur Erzeugung veränderter Genprodukte, die von den Nukleinsäuresequenzen SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,22. A method for producing modified gene products which are derived from the nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,
25 SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,25 SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO : 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,
30 SEQ ID NO: 78', SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 oder SEQ ID NO: 108 codiert werden, dadurch gekennzeichnet, dass es folgende Prozess-30 SEQ ID NO: 78 ' , SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 106 or SEQ ID NO: 108, characterized in that it follows Process-
35 schritte umfasst:35 steps includes:
a) Expression der von den SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID. O: 15,a) Expression of those of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID , O: 15,
40 SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28,40 SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 28,
SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:' 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: '44 , SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54,
45 SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,45 SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60,
SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ-ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100,. SEQ ID NO: 102,SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100 ,. SEQ ID NO: 102,
SEQ ID NO: 106 oder SEQ ID NO: 108 codierten Proteine in einem heterologen System oder in einem Zeilfreien SystemSEQ ID NO: 106 or SEQ ID NO: 108 encoded proteins in a heterologous system or in a cell-free system
b) Randomisierte oder gerichtete Mutagenese des Proteins durch Modifikation der Nukleinsäure,b) randomized or directed mutagenesis of the protein by modification of the nucleic acid,
c) Messung der Interaktion des veränderten Genprodukts mit dem Herbizid oder der biologischen Aktivität des veränderten Genproduktes in Gegenwart des Herbizids,c) measuring the interaction of the modified gene product with the herbicide or the biological activity of the modified gene product in the presence of the herbicide,
d) Identifizierung von Derivaten des Proteins die eine geringere Interaktion aufweisen oder in ihrer Aktivität weniger beeinflusst sind,d) identification of derivatives of the protein which have less interaction or are less influenced in their activity,
e) Testung der biologischen Aktivität des Proteins nach Applikation des Herbizides,e) testing the biological activity of the protein after application of the herbicide,
f) Auswahl der Nukleinsäuresequenzen, die oder deren Gen- produkte eine veränderte biologische Aktivität gegenüber dem Herbizid aufweisen.f) Selection of the nucleic acid sequences which or whose gene products have a modified biological activity towards the herbicide.
23. Verfahren nach Anspruch 22, dadurch gekennzeichnet, dass die gemäß Anspruch 22 f) ausgewählten Sequenzen in einen - Organismus eingebracht werden.23. The method according to claim 22, characterized in that the sequences selected according to claim 22 f) are introduced into an organism.
24. Verfahren zur Erzeugung transgener Pflanzen, die gegen-' Substanzen gefunden nach einem Verfahren nach einem der Ansprüche I bis 7 oder einem Verfahren gemäß Anspruch 18 oder .19 resistent sind, dadurch gekennzeichnet, dass in diesen Pflanzen Nukleinsäuren mit den in Anspruch 1 beschriebenen Sequenzen überexprimiert werden.24. A method for producing transgenic plants, the counter- ' substances found by a method according to any one of claims I to 7 or a method according to claim 18 or . 19 are resistant, characterized in that nucleic acids with the sequences described in claim 1 are overexpressed in these plants.
■25. Organismus hergestellt nach einem Verfahren gemäß Anspruch 22 oder 23 oder einem Verfahren gemäß Anspruch 24.■ 25th Organism produced by a method according to claim 22 or 23 or a method according to claim 24.
26. Mittel, enthaltend eine herbizid wirksame Menge mindestens einer Substanz identifiziert nach einem Verfahren nach einem der Ansprüche 1 bis 7 oder eines Antagonisten identifiziert nach einem Verfahren gemäß Anspruch 18 oder 19 und mindestens einen inerten flüssigen und/oder festen Trägerstoff sowie gegebenenfalls mindestens einen oberflächenaktiven Stoff.26. Agent containing a herbicidally effective amount of at least one substance identified by a method according to any one of claims 1 to 7 or an antagonist identified by a method according to claim 18 or 19 and at least an inert liquid and / or solid carrier and optionally at least one surfactant.
27. Mittel, enthaltend eine das Wachstum regulierende Menge mindestens einer Substanz identifiziert nach einem Verfahren nach einem der Ansprüche 1 bis 7 oder eines Antagonisten identifiziert nach einem Verfahren gemäß Anspruch 18 oder 19 und mindestens einen inerten flüssigen und/oder festen Trägerstoff sowie gegebenenfalls mindestens einen ober- flächenaktiven Stoff.27. Agent containing a growth-regulating amount of at least one substance identified by a method according to one of claims 1 to 7 or an antagonist identified by a method according to claim 18 or 19 and at least one inert liquid and / or solid carrier and optionally at least one surfactant.
28. Zusammensetzung, enthaltend die Substanz nach einem der Ansprüche 9 bis 11 oder einen Antagonisten identifiziert gemäß Anspruch 18.28. A composition containing the substance according to any one of claims 9 to 11 or an antagonist identified according to claim 18.
29. Kit, umfassend das Nukleinsäurekonstrukt nach Anspruch 8 oder 12, die Substanz nach einem der Ansprüche 9 bis 11, einen Antagonisten identifiziert gemäß Anspruch 18 oder die Zusammensetzung nach Anspruch 28. 29. A kit comprising the nucleic acid construct according to claim 8 or 12, the substance according to one of claims 9 to 11, an antagonist identified according to claim 18 or the composition according to claim 28.
EP02726107A 2001-02-16 2002-02-13 Method for identifying herbicidally active substances Withdrawn EP1362059A2 (en)

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DE10107843A DE10107843A1 (en) 2001-02-16 2001-02-16 Identifying herbicides and plant growth regulators, from ability to inhibit specific genes, also use of these genes to prepare herbicide-resistant transgenic plants
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DE10125537 2001-05-23
DE10125537A DE10125537A1 (en) 2001-05-23 2001-05-23 Identifying herbicides and plant growth regulators, from ability to inhibit specific genes, also use of these genes to prepare herbicide-resistant transgenic plants
PCT/EP2002/001466 WO2002066660A2 (en) 2001-02-16 2002-02-13 Method for identifying herbicidally active substances

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CA2437937A1 (en) 2002-08-29
WO2002066660A3 (en) 2003-08-28
AR032709A1 (en) 2003-11-19
US20050246784A1 (en) 2005-11-03
WO2002066660A2 (en) 2002-08-29

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