EP1098985A1 - Genes de la famille des proteines de la boite dead, leurs produits d'expression et leur utilisation - Google Patents

Genes de la famille des proteines de la boite dead, leurs produits d'expression et leur utilisation

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Publication number
EP1098985A1
EP1098985A1 EP99934671A EP99934671A EP1098985A1 EP 1098985 A1 EP1098985 A1 EP 1098985A1 EP 99934671 A EP99934671 A EP 99934671A EP 99934671 A EP99934671 A EP 99934671A EP 1098985 A1 EP1098985 A1 EP 1098985A1
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Prior art keywords
nucleic acid
polypeptide
nucleic acids
rna
seq
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EP99934671A
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German (de)
English (en)
Inventor
Karin Bohnet
Christoph Hüls
Stefan Müllner
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Aventis Research and Technologies GmbH and Co KG
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Aventis Research and Technologies GmbH and Co KG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the provision of new nucleic acids from ciliates coding for expression products, preferably RNA helicases from the DEAD box protein family, and to the use thereof.
  • the modulation of the RNA structure has an essential function in cellular processes such as in pre-mRNA splicing, in RNA transport or in protein translation, since the cellular RNA is present in the cell in different secondary and tertiary structures and, in addition, a large number of RNA-binding proteins ensure further structuring of the RNA. On this
  • the characteristic protein sequences of the DEAD proteins are highly conserved in evolution (see FIG. 1).
  • the DEAD super family is divided into different subfamilies, which are named according to their sequence motif DEAD, DEAH or DExH subfamily. All family members have an ATP binding and RNA binding function as well as an ATP hydrolysis function and mostly an RNA helicase function (FIG. 2).
  • the various members of the DEAD box protein family are characterized by a conserved region that comprises approximately 300 amino acids and is flanked by non-conserved amino acid sequences of varying lengths (Schmid SR, Lindner P., Mol Cell Biol 1991 11: 3463-3471).
  • RNA helicases corresponding to the DEAD homology boxes in cellular processes, preferably in the context of protein biosynthesis, allow these enzymes to be used in a targeted manner with regard to pharmaceutical, agricultural or biotechnical and analytical applications.
  • helicases are mostly essential enzymes, specific inhibition of these enzymes can kill the pathogen (bacteria, fungus, parasite / protozoan, virus). According to Missel et al. in certain protozoa (Trypanosoma, Leishmania, Crithidia), switching off the gene for a DEAD box protein leads to reduced growth (Missel A., Souza AE, Norskau G, Goringer HU, Mol Cell Biol 1997 17: 4895-903).
  • helicases control protein translation, mitosis and DNA repair (Thelu J, Burnod J, Bracchi V, Ambroise-Thomas P, DNA Cell Biol 1994 13: 1109-1115). Helicases are essential for the initiation of translation, in the spliceosome, in the cell cycle and ribosome assembly in yeast. So z. B. the DEAD box protein ROK1 essential for yeast viability, for pre-rRNA processing, and for mitotic growth.
  • RNA viruses whose genomes are sequenced code for at least one putative helicase.
  • examples are NS3 of the hepatitis C virus, helicases of the human coronavirus and the adeno-associated virus, vaccinia virus helicase (Kadare G., Haenni AL, J Virol 1997: 2583-2590)
  • Possible roles for viral helicases are (i) proof reading in replication (ii) initiation of transcription by unraveling the RNA and prevention of loop formation behind the RNA polymerase (iii) initiation Translation:
  • the helicase of the vaccinia virus is essential for the life cycle of the virus and is nucleic acid specific.
  • DEAD box proteins are also described in association with diseases.
  • the ampiification of a specific gene in cancer cells is related to the fact that a DEAD box protein is co-amplified with N-myc, which indicates a role of this protein in the degeneration of
  • Helicase are related to Werner syndrome - early aging - (Yu C, Oshima J., Wijsman E.M., Nakura J. et al., Arn J Hum Genet 1997 60: 330-
  • a connection between defective DNA repair and a mutation in the helica domain of the XNP / ATR-X gene is also known: 149-155).
  • An application of interest for agricultural production is the stimulation of the activity of plant-specific RNA helicases to increase the protein expression of economically relevant proteins.
  • You can either stimulate plant-specific helicases (e.g. by overexpression) or plant-like helicases can be heterologously expressed in useful plants.
  • the present invention therefore relates to nucleic acids coding for RNA helicases, with a nucleic acid sequence according to SEQ ID No. 13 or SEQ ID No. 15 or a functional variant thereof, and parts thereof with at least 8 nucleotides, preferably with at least 15 or 20 nucleotides, in particular with at least 100 nucleotides, especially with at least 300 nucleotides (hereinafter referred to as “nucleic acids according to the invention”).
  • the nucleic acid according to the invention is a DNA or RNA, preferably a double-stranded DNA, and in particular a DNA with a nucleic acid sequence coding for RNA helicases.
  • the term “functional variant” means a nucleic acid that is functionally related to RNA helicases with the described homology boxes.
  • the expression vectors also contain suitable regulatory sequences for the host cell, such as the trp promoter for expression in E. coli (see, for example, EP-B1-0154133) in E. coli, the ADH-2 promoter for expression in yeasts (Rüssel et al. (1983), J. Biol. Chem.
  • the baculovirus polyhedrin promoter for expression in insect cells see, for example, EP-B1-0127839) or the early SV40 promoter or LTR promoters, for example from MMTV (Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 214, 228).
  • MMTV Mammary Tumor Virus
  • virus vectors preferably adenovirus vectors, in particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, e.g. B. a
  • Adeno-associated virus vector consisting exclusively of two inserted terminal repeat sequences (ITR).
  • Suitable adenovirus vectors are described, for example, in McGrory, W.ü. et al. (1988) Virol. 163, 614; Gluzman, Y. et al. (1982) in "Eukaryotic Viral Vectors” (Gluzman, Y. ed.) 187, Cold Spring Harbor Press, Cold Spring Habor, New York; Chroboczek, J. et al. (1992) Virol. 186, 280; Karlsson, S et al. (1986) EMBO J .. 5, 2377 or WO95 / 00655.
  • Suitable adeno-associated virus vectors are described, for example, in Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97; W095 / 23867; Samulski, R.J. (1989) J. Virol, 63, 3822; WO95 / 23867; Chiorini, J.A. et al. (1995) Human Gene Therapy 6, 1531 or Kotin, R.M. (1994) Human Gene Therapy 5, 793.
  • Vectors with gene therapy effects can also be obtained by complexing the nucleic acid according to the invention with liposomes.
  • Lipid mixtures such as those of Feigner, PL et al. (1987) Proc. Natl. Acad. Sei, United States 84, 7413; Behr, JP et al. (1989) Proc. Natl. Acad. Be. USA 86, 6982; Feigner, JH et al. (1994) J. Biol. Chem. 269, 2550 or Gao, X. & Huang, L (1991) Biochim. Biophys. Acta 1189, 195.
  • the DNA is ionically bound to the surface of the liposomes in such a ratio that a positive net charge remains and the DNA is completely complexed by the liposomes.
  • nucleic acids according to the invention can be chemically described, for example, in SEQ ID no. 13 and SEQ ID No. 15 disclosed sequence or using the SEQ ID No. 14 and SEQ ID No. 16 disclosed peptide sequence using the genetic code z. B. can be synthesized by the phosphotriester method (see e.g. Uhlman, E. & Peyman, A. (1990) Chemical Reviews, 90, 543, No. 4).
  • nucleic acids according to the invention itself and variants is isolation from a suitable gene bank using a suitable probe (see, for example, Sambrook, J. et al. (1989) Moiecular Cloning. A laboratory manual. 2nd Edition, Cold Spring Harbor , New York).
  • Suitable as a probe are, for example, single-stranded DNA fragments with a length of approximately 100 to 1000 nucleotides, preferably with a length of approximately 200 to 500 nucleotides, in particular with a length of approximately 300 to 400 nucleotides, the sequence of which is based on the nucleic acid sequence Figures 4 and 6 can be derived.
  • Another object of the invention is the use of the nucleic acids according to the invention for specifically influencing protein biosynthesis.
  • Protein biosynthesis b) a faster degradation by the degradasome and thus a reduced protein biosynthesis.
  • Inhibition of helicase activity can Inhibit degradation by the degradasome and lead to reduced protein biosynthesis.
  • the basis for this is the finding that important biosynthetic processes can be regulated in a targeted manner by the selective inhibition or activation of helicases.
  • nucleic acids are expressed recombinantly in suitable target organisms as described.
  • the nucleic acids according to the invention preferably Hc2 have proven to be an excellent model for various eukaryotic RNA helicases, preferably from plants.
  • FIG. 3B shows the genetic relationship of some RNA helicases from different organisms in comparison to Hc2.
  • the great structural similarity between Hc2 and RNA helicases from plants is particularly surprising.
  • the recombinant expression of Hc2 enables the use of this new enzyme as a model for research into plant helicases in particular, their structure and function, and for the development of suitable inhibitors or activators of these important enzymes.
  • the nucleic acids according to the invention can be introduced into plants using recombinant DNA technologies.
  • the introduction of the foreign gene with the aid of Agrobacterium tumefaciens can be used as a method.
  • the foreign gene is introduced into the bacterial genome in a known manner.
  • the genes of the bacterium, including the foreign gene are stably integrated into the genome of the plant (Chiiton M.D. et al., Cell 1977 11: 263, Barton K.A. et al., Cell 1983 32: 1033).
  • This method is preferably used for the transformation of dicotelydones.
  • the known methods such as calcium phosphate precipitation, PEG treatment, electroporation, or a can be used to transform monocotelydones
  • Another object of the invention is the use of nucleic acids as selection markers in molecular biology.
  • Conventionally used selection markers are antibiotics.
  • Molecular biologically modified organisms carry a gene that confers resistance to an antibiotic. The organisms are grown in a medium containing antibiotics, so that only the carriers of the resistance gene can develop.
  • helica genes can be used as “resistance genes”. It has been shown (Müilner et al, patent application DPA 19545126.0) that the overexpression of a helica gene in mouse cells gives these cells tolerance to an otherwise toxic substance, leflunomide.
  • nucleic acids as selection markers in molecular biology
  • a suitable vector such as described
  • the nucleic acids according to the invention are to be introduced into cells and selected with a suitable substance, such as leflunomide), against which the cells are tolerant by overexpression of the helicase.
  • Another object of the present invention are also
  • polypeptides and polypeptide fragments which encode Hc1 and Hc2 themselves, with amino acid sequences according to SEQ ID No. 14 and SEQ ID No. 16 or a functional variant thereof, and parts thereof with at least six amino acids, preferably with at least 12 amino acids, in particular with at least 65 amino acids and especially with 257 amino acids Hc1 and 255 amino acids Hc2 (hereinafter referred to as “inventive polypeptides”) 6-12, preferably about 8 amino acid long polypeptide contain an epitope which, after coupling to a support, is used to produce specific poly- or monoclonal antibodies (see, for example, US Pat. No. 5,656,435). Polypeptides with a length of at least about 65 amino acids can also be used directly without carriers for the production of poly- or monoclonal antibodies.
  • this also includes polypeptides, the one
  • Sequence homology in particular a sequence identity of approx. 70%, preferably of approx. 80%, in particular of approx. 90%, especially of approx. 95% to the polypeptides with the amino acid sequences according to FIGS. 5 and 7. Furthermore, this also includes deletion of the polypeptide in the range from about 1-60, preferably from about 1-30, in particular from about 1-15, especially from about 1-5 amino acids.
  • This also includes fusion proteins which contain the inventive polypeptides described above, the Fusion proteins themselves already have the function of an RNA helicase or can only get the specific function after the fusion portion has been split off. Above all, this includes fusion proteins with a proportion of in particular non-human sequences of about 1 to 200, preferably about 1 to 150, in particular about 1 to 100, especially about 1 to 50 amino acids. Examples of non-human
  • Peptide sequences are prokaryotic peptide sequences, e.g. from the E. coli galactosidase or a so-called histidine tag, e.g. a Met-Ala-His6 tag.
  • a fusion protein with a so-called histidine tag is particularly advantageous for purifying the expressed protein via columns containing metal ions, for example via a Ni2 + -NTA column.
  • NTA stands for the chelator "nitrilotriacetic acid” (Qiagen GmbH, Hilden).
  • the parts of the polypeptide according to the invention represent, for example, epitopes that can be specifically recognized by antibodies.
  • polypeptide according to the invention is obtained, for example, by expression of the nucleic acid according to the invention in a suitable expression system, as already described above, according to the method generally known to the person skilled in the art
  • Another object of the present invention also relates to antibodies which react specifically with the polypeptide according to the invention, the above-mentioned parts of the polypeptide either being themselves immunogenic or by coupling to suitable carriers, such as e.g. bovine serum albumin, immunogenic or can be increased in their immunogenicity.
  • suitable carriers such as e.g. bovine serum albumin
  • the antibodies are either polyclonal or monoclonal.
  • the preparation which is also an object of the present invention, is carried out, for example, by generally known methods by immunizing a mammal, for example a rabbit, with the polypeptide according to the invention or the parts thereof, optionally in the presence of e.g. Freund's adjuvant and / or aluminum hydroxide gels (see e.g. Diamond, B.A. et al. (1981) The New England Journal of Medicine, 1344).
  • the polyclonal antibodies produced in the animal due to an immunological reaction can then be easily isolated from the blood by generally known methods and z. B. clean over column chromatography.
  • Monoclonal antibodies can be produced, for example, by the known method from Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293).
  • Another object of the present invention is also a medicament which contains a nucleic acid or a polypeptide according to the invention and optionally suitable additives or auxiliaries and a method for producing a medicament for the treatment of cancer, autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's Disease, allergies, especially neurodermatitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and / or diabetes and / or to influence cell metabolism, in particular in immunosuppression, especially in transplants and / or hereditary diseases, in particular Werner syndrome, Bloom syndrome, Xeroderma pigmentosa, connective tissue diseases, in which a nucleic acid according to the invention, for example a so-called antisense nucleic acid , or a polypeptide according to the invention is formulated with pharmaceutically acceptable additives and / or auxiliaries.
  • autoimmune diseases in particular multiple sclerosis or rheumatoid arthritis,
  • a drug which contains the nucleic acid according to the invention in naked form or in the form of one of the above-described gene therapy-effective vectors or in a form complexed with liposomes is particularly suitable for gene therapy use in humans.
  • Suitable additives and / or auxiliary substances are, for. B. a physiological saline, stabilizers, proteinase inhibitors, nuclease inhibitors etc.
  • Another object of the present invention is also a diagnostic agent containing the nucleic acids according to the invention, the polypeptides according to the invention or antibodies according to the invention and optionally suitable additives and / or auxiliaries and a method for producing a
  • Diagnostic agent for the diagnosis of cancer autoimmune diseases, in particular multiple sclerosis or rheumatoid arthritis, Alzheimer's disease, allergies, in particular neurodermatitis, type I allergies or type IV allergies, arthrosis, atherosclerosis, osteoporosis, acute and chronic infectious diseases and / or diabetes and / or for Analysis of the cell metabolism, in particular the immune status, especially in the case of transplants and / or for analysis of hereditary diseases, in particular Werner syndrome, Bloom syndrome, Xeroderma pigmentosa, connective tissue diseases, in which a nucleic acid according to the invention, a polypeptide according to the invention or antibodies according to the invention with suitable additives or auxiliary materials are added.
  • a diagnostic agent based on the Polymerase chain reaction PCR diagnostics, for example according to EP-0200362
  • a Northern blot can be produced. These tests are based on the specific hybridization of the nucleic acid according to the invention with the complementary counter strand, usually the corresponding mRNA.
  • the nucleic acid according to the invention can also be modified here, as described, for example, in EP0063879.
  • a DNA fragment according to the invention is preferably labeled using suitable reagents, for example radioactive with ⁇ -P32-dATP or non-radioactive with biotin, according to generally known methods and incubated with isolated RNA, which was preferably bound to suitable membranes made of cellulose or nylon, for example .
  • RNA it is also advantageous to separate the isolated RNA prior to hybridization and binding to a membrane, for example by means of agarose gel electrophoresis. With the same amount of RNA examined from each tissue sample, the amount of mRNA that was specifically labeled by the probe can thus be determined.
  • Another diagnostic agent contains the polypeptide according to the invention or the immunogenic parts thereof described in more detail above.
  • the polypeptide or parts thereof which are preferably attached to a solid phase, e.g. B. from nitrocellulose or nylon, for example, with the body fluid to be examined z. As blood, are brought into contact in vitro, for example with
  • the antibody-peptide complex can then be detected, for example, using labeled anti-human IgG or anti-human IgM antibodies.
  • the label is, for example, an enzyme, such as peroxidase, that catalyzes a color reaction. The presence and the amount of autoimmune antibodies present can thus be easily and quickly detected via the color reaction.
  • Another diagnostic agent contains the antibodies according to the invention themselves. With the aid of these antibodies, for example, a tissue sample from humans can be easily and quickly examined to determine whether the polypeptide in question is present.
  • the antibodies according to the invention are labeled, for example, with an enzyme, as already described above.
  • the specific one Antibody-peptide complex can be detected easily and just as quickly via an enzymatic color reaction.
  • Another object of the present invention also relates to a test for the identification of functional interactors, such as e.g. Inhibitors or stimulators containing a nucleic acid according to the invention, a polypeptide according to the invention or the antibodies according to the invention and, if appropriate, suitable additives and / or auxiliaries.
  • functional interactors such as e.g. Inhibitors or stimulators containing a nucleic acid according to the invention, a polypeptide according to the invention or the antibodies according to the invention and, if appropriate, suitable additives and / or auxiliaries.
  • a suitable test for identifying functional interactors is e.g. B. the so-called "two-hybrid system" (Fields, S. & Sternglanz, R. (1994) Trends in Genetics, 10, 286).
  • a cell for example a yeast cell
  • one or more expression vectors or transfected which express a fusion protein which contains the polypeptide according to the invention and a DNA binding domain of a known protein, for example from Gal4 or LexA from E. coli, and / or express a fusion protein which contains an unknown polypeptide and a transcription activation domain, for example of Gal4, herpesvirus VP16 or B42, and the cell also contains a reporter gene, for example the LacZ gene from E.
  • the unknown polypeptide is encoded, for example, by a DNA fragment that comes from a gene bank, for example from a human gene bank immediately produced a cDNA library using the expression vectors described in yeast so that the test can be carried out immediately thereafter.
  • the nucleic acid according to the invention is cloned in a functional unit to the nucleic acid coding for the lexA-DNA binding domain, so that a fusion protein from the polypeptide according to the invention and the LexA-DNA binding domain is expressed in the transformed yeast.
  • cDNA fragments from one Cloned cDNA library in functional unit to the nucleic acid coding for the Gal4 transcription activation domain, so that a fusion protein from an unknown polypeptide and the Gal4 transcription activation domain is expressed in the transformed yeast.
  • the yeast transformed with both expression vectors, which is for example Leu2- additionally contains a nucleic acid which codes for Leu2 and is controlled by the LexA promoter / operator.
  • the Gal4 transcription activation domain binds to the LexA promoter / operator via the LexA-DNA binding domain, whereby the latter is activated and the Leu2 gene is expressed.
  • the Leu2 yeast can grow on minimal medium that does not contain leucine.
  • the activation of the transcription can be demonstrated by the formation of blue or green fluorescent colonies.
  • the blue or fluorescent staining can also be done easily quantify in the spectrophotometer eg at 585 nM in the event of a blue color.
  • the present invention is therefore not only intended for a method for finding polypeptide-like interactors, but also extends to a method for finding substances which are identical to those described above Protein-protein complex can interact.
  • Such peptide-like as well as chemical interactors are therefore referred to in the context of the present invention as functional interactors which can have an inhibiting or a stimulating effect.
  • Figure 1 shows schematically the conserved areas (homology boxes) of the proteins from the DEAD protein superfamily. The numbers between the areas indicate the distances in amino acids between the homoboxes.
  • Figure 2 schematically describes the conserved areas and their known functions of the expressed proteins according to Guer Pace, F.V. (1994) supra.
  • FIGS. 3A and 3B describe the phylogenetic trees of Hc1 and Hc2 and establish the evolutionary relationships. These figures were created with the program: Lasergene (module MegAlign 3.1.7) from the company: DNASTAR Inc .; using the Clustal algorithm (Higgins D.G, Sharp P.M., CABIOS (1989), Vol. 5, no.2, 151-153).
  • SEQ ID No. 13 shows the nucleic acid sequence of Hc1.
  • SEQ ID No. 14 shows the amino acid sequence corresponding to SEQ No. 13th
  • SEQ ID No. 15 shows the nucleic acid sequence of Hc2.
  • SEQ ID No. 16 shows the amino acid sequence corresponding to SEQ No. 15.
  • the practical work that has led to the present invention is mainly based on established, known methods of microbiology, molecular biology and recombinant DNA technology.
  • RNA pellet is obtained by centrifugation (10 min, 10,000xg, 4 ° C). The pellet is washed two more times, dried and taken up in DEPC water. After a 10-15 minute incubation at 55-60 ° C the RNA can be stored at -80 ° C. From the total RNA, mRNA is purified using oligo (dT) -sepharose (Clontech mRNA Separator Kit # K1040-2) Example 3: Preparation of cDNA
  • PCR polymerase chain reaction
  • PCR program was 5 min 95 ° C, 95 ° C / 37s - 50 ° C / 37s - 72 ° C / 37s - 30 cycles.
  • Example 5 Cloning and sequencing of the fragments After PCR, the PCR product is separated on a 1% agarose gel. The specific fragments are cut out and purified using the QIAGEN Gel Extraction Kit. The purified fragments are used directly for cloning (Invitrogen Original TA Cloning Kit # K2000-01). Positive clones are grown in shake culture and the plasmid DNA is purified using the QIAGEN Maxi Prep Kit. Sequencing is performed using the AbiPrism Model 377 automated sequencer.
  • Hc1 and Hc2 are cloned into a suitable vector, preferably pGEX-4T-1 GST fusion vector (Pharmacia Biotech).
  • a suitable vector preferably pGEX-4T-1 GST fusion vector (Pharmacia Biotech).
  • Hc1 and Hc2 are prepared from Tetrahymena thermophila cDNA using suitable primers using PCR.
  • a standard PCR approach contains 10 mM Tris-HCl, pH 8.3, 50mM KCI, 1.5mM MgCl2, 0.001% gelatin, 75 ⁇ M dNTP, 0.3ng of each primer, 0.5 ⁇ l cDNA, 0.5U Taq polymerase.
  • the primers (2A) 5 ' ATAAGAATGCGGCCGCTGTTCTACCGATTCTGTGAATATA 3 ' (3A) 5 ' CGCGGATCCTC ACT GGT TCG GGT AAG ACT GCT ACT TTC TCT 3 were used to amplify the fragment Hc1, and the primers TACTACATAATAATAATCATAATAATAAGAATAATCATAAGAATAATCATAAGAATAATCATAAGAATAATCAGAATAAGAATAAGAATAATCAGAATAAGAATAAGAATAATCAGAATAAGAATCAGAATAAGAAT (3) (TAGGATAAGAATCATAAGAAT) were used to amplify the fragment Hc2 '(8A) 5' ATAAGAATGCGGCCGCGTTCTACCGATTCTGTGGACATAG 3 'used.
  • Primer (2A) contains a NotI interface, (3A) a BamHI interface, (7A) an EcoRI and (8A) a NotI interface.
  • the PCR program was 5 min 95 ° C, 95 ° C / 37s - 50 ° C / 37s - 72 ° C / 37s - 30 cycles.
  • the fragments to be cloned are purified using a 1% agarose gel (QIAgen gel extraction kit) and the ends to be cloned are prepared by digestion with NotI and BamHI (Hc1) or EcoRI and Notl (Hc2).
  • the vector pGEX-4T-1 is also prepared by digestion with Notl and BamHI (Hc1) or EcoRI and Notl (Hc2).
  • the vector and insert are ligated overnight at 16 ° C., the ligation batches are used to transform competent TOP10F '(Invitrogen) E.coli cells. Positive clones are picked and used for protein expression.
  • the construct pGEX-Hd or pGEX-Hc2 allows the translation of a fusion protein consisting of 257 amino acids (28.3 kDa) from Hc1 or 255 amino acids (28.1 kDa) from Hc2 and glutathione-S-transferase (24 kDa).
  • the fusion protein contains all homology boxes (DEAD, SAT, ...) which distinguish the members of the protein family.
  • an overnight culture is induced with IPTG and the fusion protein from the supernatant is purified in a batch process with glutathione Sepharose 4B or via a glutathione Sepharose 4B column.
  • the glutathione-S-transferase is cleaved with thrombin.
  • z. B 100 ug GST fusion protein with a unit of thrombin proteinase in 1x PBS at 22 ° C for 16 hours.
  • the gene product from Hc1 or Hc2 is separated off by gel filtration, e.g. B. with a Superdex 200 HR 10/30 column (Pharmacia Biotech). Biorad's Gel Filtration Chromatography Standard (Ref. 151-1901) can be used as a standard.
  • Example 6 ATPase activity
  • the reaction mixture for measuring the ATPase activity contains 150 mM NaCI, 5 mM KCI, 1.5 mM MgCI2, 20 mM Hepes / KOH, pH 7, 1 mM dithiothreitol, 1 mM PMSF, 10 ⁇ M ATP and 0.2 ⁇ l 32P-ATP in one Total volume of 50 ⁇ l.
  • the reaction mixture is heated to 37 ° C. and Hc1 or Hc2 is added. After 30 min at 37 ° C., the reaction is stopped by adding 400 ⁇ l of 7% activated carbon in 50 mM HCl and 5 mM H3P04.
  • the samples are mixed and centrifuged at 13,000 rpm for 15 min. The radioactivity released is measured with a scintillation counter in the supernatant.
  • the helicase activity of Hc1 or Hc2 can be followed by dissociation of double-stranded RNA.
  • the substrate can be any RNA oligomer that is labeled on a strand, e.g. with 32P.
  • the reaction mixture contains 32P-labeled helicase substrate, Hc1 or Hc2 in various concentrations, 2 mM ATP, 5 mM dithiothreitol and 50 ⁇ g bovine serum albumin in 20 mM Tris-HCl.
  • the reaction is carried out at 37 ° C for 30 min and stopped by heating.
  • reaction mixture is applied to a 16 cm X 18 cm 12% non-denaturing polyacryamide gel and separated at a constant current of 25 mA.
  • the gel is dried under vacuum and exposed (e.g. Kodak RPXRP-5 film, -70 ° C).
  • Homologs can be used as antisense strands.
  • a plasmid is constructed which carries the desired antisense sequence, as well as Selection markers, eg neomycin, a promoter which controls the expression of the antisense RNA and RNA-stabilizing sequences.
  • Selection markers eg neomycin
  • the transcribed sequences are transcribed in the cell and the transcript hybridizes with the target DNA.
  • sequences synthesized in vitro can also be brought into the cell by microinjection.
  • Example 9 Gene probe for finding new members of the DEAD box protein family.
  • the fragments Hc1 and Hc2 are used to isolate new DEAD box proteins from suitable organisms.
  • the specific gene fragments are amplified using the polymerase chain reaction (PCR) and simultaneously labeled with digoxigenin, according to Boehringer Mannheim PCR DIG Probe Synthesis Kit # 1636 090. Plasmid DNA of the cloned fragments Hc1 or Hc2 is used as a template .
  • a PCR approach contains Expand TM High Fidelity Buffer (Boehringer Mannheim # 1636 090), 200 uM dATP, 200 uM dGTP, 200 uM dCTP, 130 uM dTTP, 70 uM DIG-11-dUTP, 0.3 ng of each primer, 100 pg Plasmid DNA, 2.6 U Taq polymerase.
  • the primers were used to amplify the Hc1 fragment
  • the antibody solution is placed on the column and allowed to drip through at 4 ° C. at a flow rate of -100 ml / h. Then the column is washed several times: with 250 ml buffer A (plus 0.5% NP40), then with 125 ml 0.1 M borate buffer, pH 9.0, then with 125 ml borate buffer pH ⁇ .O, then with 125 ml 0.2 M triethanolamine, pH 8.2.
  • the Fc region of the antibody is coupled to the protein A-Sepharose via cross-linking.
  • cell lysate of suitable organisms is placed on the antibody column.
  • the column is washed several times and the bound proteins with a suitable buffer, e.g. Glycine, pH 3, eluted in Tris-HCl, pH 8.
  • Example 13 Diagnostic probes The Hc1 or Hc2 gene fragments or homologous gene fragments or parts thereof
  • Containing homology boxes can be used as diagnostic probes.
  • the DNA fragments are immobilized on a suitable matrix (e.g.
  • a patient's mRNA is purified and reverse transcribed, e.g. with MMLV reverse transcriptase, 2 h at 37 ° C, transcribed into cDNA.
  • the cDNA is labeled, e.g. with 32P or digoxigenin.
  • the cDNA is in a suitable hybridization buffer, e.g. DIG EasyHyb
  • the gene fragments Hc1 and Hc2 can be used as selection markers in molecular biology. It has been shown that overexpression of an RNA helicase in mouse cells makes it tolerant of the substance leflunomide (Müllner patent).
  • an expression vector is constructed which contains Hc1 or Hc2 and a gene to be expressed.
  • the gene to be expressed can lie next to or between the helicase gene. Suitable host cells are transformed with the vector (e.g. cloning into a pGEX vector and introduction into E. coli). If the gene to be expressed lies next to the Hc gene, the transformants become tolerant to leflunomide if the vector is successfully introduced.
  • the success of the ligation must be e.g. B. Check via blue-white screening. If the gene to be expressed is in the Hc gene, the Hc gene is destroyed if the ligation is successful and the transformants lose their tolerance to leflunomide if the vector is successfully introduced.

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Abstract

La présente invention concerne la préparation de deux nouveaux acides nucléiques (Hc1 et Hc2) qui constituent des homologues de protéines de la boîte DEAD connues, correspondent aux ARN hélicases et peuvent être obtenus à partir de ciliés. L'invention concerne également l'introduction des nouveaux acides nucléiques, selon des technologies de recombinaison d'ADN, dans des cellules cibles appropriées, et leur utilisation pour la régulation de la transcription et de la traduction. L'invention concerne en outre la transcription in vitro ou in vivo des nouveaux acides nucléiques pour la production de nouvelles protéines, ainsi que leur utilisation pour la thérapie et le diagnostic.
EP99934671A 1998-07-22 1999-07-10 Genes de la famille des proteines de la boite dead, leurs produits d'expression et leur utilisation Withdrawn EP1098985A1 (fr)

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CN1315540A (zh) * 2000-03-24 2001-10-03 上海博德基因开发有限公司 一种新的多肽——人解螺旋酶13和编码这种多肽的多核苷酸
CN1315543A (zh) * 2000-03-27 2001-10-03 上海博德基因开发有限公司 一种新的多肽——人rna解旋酶10和编码这种多肽的多核苷酸
CN1324946A (zh) * 2000-05-19 2001-12-05 上海博德基因开发有限公司 一种新的多肽——人atp依赖的rna解螺旋酶11和编码这种多肽的多核苷酸

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