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EP1165607A2 - Proteine (tp) impliquee dans le developpement du systeme nerveux central - Google Patents

Proteine (tp) impliquee dans le developpement du systeme nerveux central

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Publication number
EP1165607A2
EP1165607A2 EP00916770A EP00916770A EP1165607A2 EP 1165607 A2 EP1165607 A2 EP 1165607A2 EP 00916770 A EP00916770 A EP 00916770A EP 00916770 A EP00916770 A EP 00916770A EP 1165607 A2 EP1165607 A2 EP 1165607A2
Authority
EP
European Patent Office
Prior art keywords
protein
gene
sequence
dna sequence
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Withdrawn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
EP00916770A
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German (de)
English (en)
Inventor
Annemarie Poustka
Johannes Coy
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.)
Deutsches Krebsforschungszentrum DKFZ
Original Assignee
Deutsches Krebsforschungszentrum DKFZ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Deutsches Krebsforschungszentrum DKFZ filed Critical Deutsches Krebsforschungszentrum DKFZ
Publication of EP1165607A2 publication Critical patent/EP1165607A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • TP Protein (TP) involved in the development of the nervous system
  • the present invention relates to a protein (T-protein) and related proteins which are involved in the development of the nervous system and are expressed in a tissue and development-specific manner, the variants of these proteins described below and DNA sequences coding for these proteins.
  • the present invention further relates to antibodies or fragments thereof directed against these proteins, and to antisense RNAs or ribozymes directed against the expression of these proteins.
  • the present invention relates to pharmaceuticals and diagnostic methods using the above compounds.
  • the present invention is therefore based on the technical problem of providing means with which disorders in the development and function of the nervous system are diagnosed and can be treated if necessary.
  • the present invention thus relates to a DNA sequence which encodes a protein which is involved in the development and function of the nervous system, in particular the CNS, and which is expressed in a tissue- and development-specific manner, the DNA sequence comprising the following DNA sequences:
  • (k) a DNA sequence that differs from the DNA sequence of (a), (b), (c), (d), (e), (f), (g), (h), (i ) or (j) differs due to the degeneration of the genetic code.
  • the present invention is based on the isolation of a human DNA sequence (called gene "T” or T gene; see Figures 1-8, which encodes the protein TP), it being found that the protein encoded by this DNA sequence is needed in the nervous system. The expression of the gene encoding this protein is increased in the nervous system. The sequence analysis showed that this is a new gene. In addition, other genes could be isolated Have homologies to this gene (murine gene "T”, FIGS. 9 and 10; human gene "T2", FIG. 16; human gene "T3", FIGS. 17 and 18; murine gene T2, FIGS. 12 and 13; murine gene T3, Fig. 19).
  • the T gene, T2 gene and T3 gene are members of the T (gene) family and preferably originate from vertebrates such as humans, mice or rats. Defects in these genes lead to restrictions in the functions of the nervous system, particularly the CNS. Furthermore, these genes have an important function in the control of cell growth and changes in these genes or their expression lead to errors in the control of cell growth, for example also to tumor formation, especially of the neuroblastoma. This cancer almost exclusively affects younger children up to the age of 8. In 25 to 30 percent of cases, the first signs appear within the first 12 months of life. With neuroblastoma, very young cells of the autonomic nervous system degenerate.
  • a neuroblastoma can be diagnosed by the doctor through blood, urine and ultrasound examinations as well as by taking biopsies from the tumor and a bone marrow examination. Once the exact location of the tumor is diagnosed, it is surgically removed. The early formation of metastases is problematic. By isolating and analyzing the T gene, it is now possible to develop novel diagnostic and therapeutic measures for neuroblastoma. This makes it possible to diagnose the cancer at an early stage and to establish forms of therapy that promise improved chances of recovery.
  • T gene family leads to disorders in the development and differentiation of the nervous system, especially the brain. In many cases, this leads to mental illnesses, such as mental retardation or Alzheimer's.
  • the T gene also plays an important role in the interconnection of individual brain areas, for example the forebrain and midbrain. Mutations in this gene in some cases lead to schizophrenic diseases or autism pussy syndromes.
  • human and murine genes important, fundamental conclusions can be drawn about the development of the nervous system and especially the brain. This offers good starting points for researching pathological changes in the nervous system and especially in the brain.
  • the genomic sequences make it easier for patients to be examined for possible mutations.
  • the genomic sequences of the T gene are particularly advantageous when little (tumor) material is available for analysis. This makes it possible, for example, to examine even the smallest tumors for mutations in this gene. Furthermore, it opens up the possibility of checking the success of a therapy (in particular radiation and / or chemotherapy), since tumor cells circulating in the blood with genomic primers which are specific for the genomic DNA are detected by a PCR reaction can.
  • hybridize used in the present invention refers to conventional hybridization conditions, preferably to hybridization conditions in which 5xSSPE, 1% SDS, 1xDenhardts solution is used and the hybridization temperatures between 35 ° C. and 70 ° C., preferably be at 65 ° C.
  • washing is preferably carried out first with 2xSSC, 1% SDS and then with 0, 2xSSC at temperatures between 35 ° C and 70 ° C, preferably at 65 ° C (for the definition of SSPE, SSC and Denhardts solution see Sambrook et al ., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)).
  • variants or fragment used in the present invention encompass DNA sequences which differ from the sequences indicated in the figures by deletion (s), insertion (s), exchange (s) and / or other im Distinguish modifications known in the art or comprise a fragment of the original nucleic acid molecule, the protein or peptide encoded by these DNA sequences still having the properties mentioned above.
  • Functional equivalents, derivatives and precursors are therefore included.
  • Derivatives are, for example, mutation derivatives (generated by, for example, deletions or insertions), fusions, allele variants, muteins and splice variants.
  • FIGS. 14 and 15 Two selected examples of such splice variants are shown in FIGS. 14 and 15. Methods for generating the above changes in the nucleic acid sequence are known to the person skilled in the art and are described in standard works in molecular biology, for example in Sambrook et al. , supra. The person skilled in the art is also able to determine whether a protein encoded by a nucleic acid sequence modified in this way still has the properties mentioned above.
  • the present invention relates to a DNA sequence that encodes a protein that contains the amino acid sequence of FIGS. 1, 9, 11, 12, 13, 14, 15, 16, 17, 18 or 19, wherein the protein has the biological activity defined above.
  • a further preferred embodiment of the present invention relates to antisense RNA, which is characterized in that it is complementary to the above D ⁇ A sequences and the synthesis of the protein encoded by these D ⁇ A sequences can reduce or inhibit and a ribozyme, which is characterized in that it can bind to a part of the above DNA sequences and to the RNA transcribed from these DNA sequences and cleave them, thereby the synthesis of the protein encoded by these D ⁇ A sequences is reduced or inhibited.
  • antisense RNAs and ribozymes are preferably complementary to a coding region of the mRNA.
  • the person skilled in the art is able to prepare and use suitable antisense RNAs based on the disclosed D ⁇ A sequences. Suitable procedures are described for example in EB-Bl 0 223 399 or EP-AI 0 458.
  • Ribozymes are RNA enzymes and consist of a single strand of RNA. These can cleave other RNAs intermolecularly, for example the mRNAs transcribed by the D ⁇ A sequences according to the invention.
  • these ribozymes must have two domains, (1) a catalytic domain and, (2) a domain that is complementary to the target RNA and can bind to it, which is a prerequisite for cleaving the target RNA.
  • a catalytic domain a domain that is complementary to the target RNA and can bind to it, which is a prerequisite for cleaving the target RNA.
  • the D ⁇ A sequences according to the invention or the D ⁇ As coding for the antisense RNAs or ribozymes described above can also be inserted into a vector or expression vector.
  • the present invention also includes these D ⁇ A sequences containing vectors or expression vectors.
  • vector refers to a plasmid (eg pUC18, pBR322, pBlueScript), a virus or another suitable vehicle.
  • the D ⁇ A molecule according to the invention is functionally linked in the vector to regulatory elements which allow its expression in prokaryotic or eukaryotic host cells.
  • such vectors typically contain an origin of replication and specific genes which represent the phenotypic Allow selection of a transformed host cell.
  • the regulatory elements for expression in prokaryotes include the lac, trp promoter or T7 promoter, and for expression in eukaryotes the AOXl or GALl promoter in yeast, and the CMV, SV40 , RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells.
  • suitable promoters are the metalothionein I and the polyhedrin promoter.
  • the vector contains the promoter of the human T gene or an orthologist of the T gene. Suitable expression vectors for E.
  • coli include, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred.
  • Vectors suitable for expression in yeast include pYlOO and Ycpadl, pMSXND, pKCR, pEFBOS, cDM8 and pCEV4 for expression in mammalian cells.
  • the expression vectors according to the invention also include vectors derived from baculovirus for expression in insect cells, for example pAcSGHisNT-A.
  • DNA sequences according to the invention can also be inserted in connection with a DNA coding for another protein or peptide, so that the DNA sequences according to the invention can be expressed, for example, in the form of a fusion protein.
  • these other DNAs are reporter sequences encoding a reporter molecule comprising a detectable protein, e.g. a dye, an antibiotic resistance, ß-galactosidase or a substance detectable by spectropshotometric, spectro-fluorometric, luminescent or radioactive assays.
  • the present invention also relates to the above written vectors containing host cells.
  • host cells include bacteria (for example the E. coli strains HB101, DH1, xl776, JM101, JM109, BL21 and SG13009), fungi, for example yeasts, preferably S. cerevisiae, plant cells, insect cells, preferably sf9 cells, and animal cells, preferably vertebrate or mammalian cells.
  • Preferred mammalian cells are CHO, VERO, BHK, HeLa, COS, MDCK, 293 and WI38 cells. Methods for transforming these host cells, for phenotypically selecting transformants and for expressing the DNA molecules of the invention using the vectors described above are known in the art.
  • the genes belonging to the sequences according to the invention can be amplified using suitable primer sequences.
  • the primer sequences indicated in FIG. 20 are particularly suitable for amplifying the T2 and T3 genes.
  • the present invention further relates to proteins encoded by the DNA sequences according to the invention and methods for producing the proteins encoded by the DNA sequences according to the invention.
  • the skilled worker is familiar with conditions for culturing transformed or transfected host cells.
  • the method according to the invention comprises the cultivation of the host cells described above under conditions which allow the expression of the protein (or fusion protein) (preferably stable expression), and the extraction of the protein from the culture or from the host cells. Suitable purification methods (for example preparative chromatography, affinity chromatography, for example immunoaffinity chromatography, HPLC etc.) are generally known.
  • the proteins according to the invention preferably have those in FIGS. 1, 9, 11, 12, 13, 14, 15, 16, 17, 18 or 19 shown amino acid sequences or represent fusions, fragments, derivatives or precursors (bioprecursors) thereof, the above-mentioned properties in the sense of functional equivalents remain.
  • Derivatives are to be understood in particular as those modified proteins or peptides which differ from the sequences shown in the figures by conservative amino acid exchanges or which contain non-conservative amino acid exchanges which do not significantly change the function of the T proteins.
  • the inventors have identified the following amino acid motifs which are suitable for identifying previously unknown proteins which belong to the family of the T / T2 / T3 family according to the invention and a protein superfamily comprising pore membrane proteins and filament-binding proteins. Motif 1:
  • (A, T) means amino acid A or T on this
  • X (2,4) means two to four X's on this
  • a further preferred embodiment of the present invention relates to antibodies against the proteins according to the invention described above or a fragment thereof.
  • This Antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof.
  • fragment means all parts of the monoclonal antibody (for example Fab, Fv or "single chain Fv” fragments) which have the same epitope specificity as the complete antibody. The production of such fragments is known to the person skilled in the art.
  • the antibodies according to the invention are preferably monoclonal antibodies.
  • the antibodies according to the invention can be produced according to standard methods, the protein encoded by the DNA sequences according to the invention or a synthetic fragment thereof serving as an immunogen.
  • Methods for obtaining monoclonal antibodies are known to the person skilled in the art and comprise, for example, as a first step the preparation of polyclonal antibodies using the proteins according to the invention or fragments thereof (for example synthetic peptides) as immunogen for the immunization of suitable animals, for example rabbits or chickens, and the extraction of the polyclonal Antibodies from the serum or egg yolk.
  • Zeil hybrids are produced and cloned from antibody-producing cells and bone marrow tumor cells. A clone is then selected which produces an antibody which is specific for the antigen used. This antibody is then made.
  • Examples of cells that produce antibodies are spleen cells, lymph node cells, B-lymphocytes, etc.
  • animals that can be immunized for this purpose are mice, rats, horses, goats and rabbits.
  • the myeloma cells can be obtained from mice, rats, humans or other sources.
  • Cell fusion can be carried out, for example, by the well-known Köhler and Milstein method.
  • the hybridomas obtained by cell fusion are screened by means of the antigen by the enzyme-antibody method or by a similar method.
  • clones are obtained using the limit dilution method.
  • the clones obtained For example, BALB / c mice are implanted intraperitoneally, the ascites are removed from the mouse after 10 to 14 days, and the monoclonal antibody is purified by known methods (for example ammonium sulfate fractionation, PEG fractionation, ion exchange chromatography, gel chromatography or affinity chromatography).
  • the monoclonal antibody mentioned is an antibody derived from an animal (for example a mouse), a humanized antibody or a chimeric antibody or a fragment thereof.
  • Chimeric, human antibody-like or humanized antibodies have a reduced potential antigenicity, but their affinity for the target is not reduced.
  • the production of chimeras and humanized antibodies or of antibodies similar to human antibodies has been described in detail (see, for example, Queen et al., Proc. Natl. Acad. Sci. USA 86 (1989), 10029, and Verhoeyan et al., Science 239 (1988), 1534).
  • Humanized immunoglobulins have variable scaffold areas, which essentially come from a human immunoglobulin (called acceptor immunoglobulin) and the complementary determinants, which essentially come from a non-human immunoglobulin (e.g. from the mouse) (with the name donor immunoglobulin).
  • acceptor immunoglobulin human immunoglobulin
  • non-human immunoglobulin e.g. from the mouse
  • donor immunoglobulin The constant region (s), if present, originate essentially from a human immunoglobulin.
  • humanized (as well as human) antibodies offer a number of advantages over antibodies from mice or other species: (a) the human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore should Antibody response against such an injected antibody is lower than against a completely foreign mouse antibody or a partially foreign chimeric antibody; (b) since the effector region of the humanized antibody is human, it is likely to interact better with other parts of the human immune system, and (c) Injected humanized antibodies have a half-life that is essentially equivalent to that of naturally occurring human antibodies, allowing smaller and less frequent doses to be administered compared to antibodies from other species.
  • the antibodies according to the invention can be used, for example, for immunoprecipitation of the proteins discussed above, for the isolation of related proteins from cDNA expression banks or for the purposes disclosed below (diagnosis / therapy).
  • the present invention also relates to a hybridoma that produces the monoclonal antibody described above.
  • the present invention relates to antibodies against the individually listed peptides of genes T2 and T3 (cf. FIG. 20).
  • amino acid sequence of the suitable peptide is:
  • the present invention makes it possible to investigate disorders of the development and function of the nervous system at the genetic level. These include neurological and psychiatric diseases (including Alzheimer's, Parkinson's disease, schizophrenia, manic-depressive diseases, autism, mental retardation), injuries to the nervous system, with congenital damage to the nervous system or with degenerative diseases of the nervous system. Furthermore, the invention enables the treatment of cancer, including tumors of the nervous system, such as neuroblastoma, astrocytoma, glioblastoma, medulloblastoma. This diagnosis can be not only postnatal, but also already happen prenatally.
  • neurological and psychiatric diseases including Alzheimer's, Parkinson's disease, schizophrenia, manic-depressive diseases, autism, mental retardation
  • injuries to the nervous system with congenital damage to the nervous system or with degenerative diseases of the nervous system.
  • the invention enables the treatment of cancer, including tumors of the nervous system, such as neuroblastoma, astrocytoma, glioblastoma,
  • DNA sequence according to the invention it can be determined in mammals, in particular humans, whether they contain a gene which codes and / or expresses the protein according to the invention or whether this gene forms a mutated form of the protein leads that is no longer biologically active.
  • the person skilled in the art can carry out conventional methods such as reverse transcription, PCR, LCR, hybridization and sequencing.
  • the antibodies according to the invention are also suitable for diagnostics, ie for example for the detection of the presence and / or the concentration of the protein according to the invention, a shortened or extended form of the protein etc. in a sample.
  • the antibodies can be bound, for example, in liquid phase immunoassays or to a solid support.
  • the antibodies can be labeled in different ways. Suitable markers and labeling methods are known in the art. Examples of immunoassays are ELISA and RIA.
  • the present invention thus also relates to a diagnostic method for the detection of a disturbed expression of the protein according to the invention or for the detection of a modified form of this protein, in which a sample is brought into contact with the DNA sequences according to the invention or the antibody or fragment thereof according to the invention and then wise directly or indirectly determines whether the concentration of the protein and / or its amino acid sequence differ from a protein obtained from a healthy patient.
  • the present invention also allows therapeutic measures to be carried out for the disorders discussed above, ie the above-described DNA sequences according to the invention, antisense RNAs, ribozymes and antibodies can also be used to prepare a medicament, for example to control the expression of the protein according to the invention or to Exchange of a mutated form of the gene can be used for a functional form and thus also for the manufacture of a medicament for the prevention or treatment of diseases of the nervous system, in particular tumor diseases of the CNS.
  • the protein according to the invention can be introduced into mammals, in particular humans, by customary measures.
  • a DNA sequence, antisense RNA or ribozyme according to the invention can also be introduced and expressed in mammals, in particular humans.
  • the expression of the protein (TP) according to the invention or the related proteins can be controlled and regulated.
  • the present invention thus also relates to a medicament which contains the DNA sequences described above, antisense RNA, the ribozyme, the expression vector, the protein according to the invention or the antibody or the fragment thereof.
  • This drug may also contain a pharmaceutically acceptable carrier.
  • Suitable carriers and the formulation of such medicaments are known to the person skilled in the art. Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, surfactants, sterile solutions, etc.
  • the medicaments can be administered orally or parenterally.
  • Methods for parenteral administration include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intravenous, intraperitoneal, or intranasal.
  • the appropriate dosage is determined by the attending physician and depends on various factors, for example the age, gender, weight of the patient, the stage of the disease, the type of administration, etc.
  • the nucleic acids described above are preferably inserted into a vector suitable for gene therapy and introduced into the cells, for example under the control of a tissue-specific vector.
  • the vector containing the nucleic acids described above is a virus, for example an adenovirus, vaccinia virus or adenovirus. Retroviruses are particularly preferred.
  • nucleic acids according to the invention can also be transported to the target cells in the form of colloidal dispersions. These include, for example, liposomes or lipoplexes (Mannino et al., Biotechniques 6 (1988), 682).
  • the present invention relates to a diagnostic kit for carrying out the diagnostic method described above, which contains a DNA sequence according to the invention or the above-described antibody according to the invention or a fragment thereof.
  • a diagnostic kit for carrying out the diagnostic method described above, which contains a DNA sequence according to the invention or the above-described antibody according to the invention or a fragment thereof.
  • the DNA sequence or the antibody or the fragment thereof can be immobilized.
  • Sequences of the T genes can be applied to nylon membranes or glass slides and hybridized with complex cDNA samples from tumors and associated normal tissues, or sick and associated healthy tissues. This enables the (fully automated) detection of the expression of these genes.
  • the sequences used for this can be, for example, the entire cDNA sequence or short sequence sections, for example 10-15 bp oligomers (see, inter alia, FIG. 20).
  • the therapy including cancer therapy, can then be specifically selected or adapted according to the individual situation of the patient. Genes whose altered expression are already influencing the treatment of the patient are, for example, the N-Myc gene in neuroblastoma.
  • the isolation and characterization of the human gene according to the invention and in particular the mouse homologue thereof furthermore allow the establishment of an animal model, which is very valuable for the further study of diseases of the nervous system and cancer at the molecular level.
  • the present invention thus also relates to a non-human mammal whose T gene or T2 or T3 gene is changed, e.g. by inserting a heterologous sequence, in particular a selection marker sequence.
  • non-human mammal includes any mammal whose T gene, or T2 or T3 gene, may be altered. Examples of such mammals are mouse, rat, rabbit, horse, cattle, sheep, goat, monkey, pig, dog and cat, with mouse being preferred.
  • T gene or T2 or T3 gene that has been changed means that the gene which occurs naturally in the non-human mammal is modified by standard methods by means of standard methods to change the gene structure or the gene sequence. This can be achieved, inter alia, by introducing a deletion of approximately 1-2 kb, in the place of which a heterologous sequence, for example a construct for mediating antibiotic resistance (for example a “neo-cassette”), is introduced . Furthermore, heterologous sequences can be introduced into the T gene, which allow time and tissue-specific deletions to be carried out in vivo. Furthermore, heterologous sequences can be introduced into the T gene, which make it possible to follow the expression of the T gene in vivo.
  • GFP green fluorescent protein
  • Figure 9 represents part of the cDNA sequence of the mouse T gene.
  • Figure 10 shows an intron sequence of the mouse T gene flanked by two exons. These murine sequences can now be used for the targeted modification of the mouse T gene. For example, the splicing sequences of the intron can be deleted or changed so that the T gene is no longer spliced correctly. Furthermore, by inserting a splice acceptor sequence of another exon of the mouse T gene into the intron sequence, a sequence can be inserted into this intron which is recognized as an exon and which is spliced to the exons of the T gene located in front of it.
  • This inserted sequence can be, for example, an exon that encodes the EGFP protein (Enhanced Green Fluorescent Protein).
  • This turns the original mouse T gene into a fusion protein that contains the EGFP protein.
  • a mouse can preferably be generated which allows the expression of the T gene to be monitored in vivo.
  • the inserted sequence can be designed in the end (eg polyA signal, splice signals, etc.) such that no further exons of the T gene are spliced to the inserted exon or the spliced exon is no longer translated. This results in a deletion of the mouse T protein at the C-terminal end or a premature termination of the reading frame and an (at least partial) inactivation of the protein function of the mouse T gene can be achieved.
  • sequences can also be inserted as new exon sequences which result in an mRNA sequence in which this new mRNA sequence is located at the 3 'end. Suitable sequences can then be used to change the stability of the mRNA or to change its location in the cell. The associated phenotype of the mice modified in this way can then provide important conclusions about the Function of the T gene. These mice can then also be used to find new active substances that compensate for the loss of function of the T gene.
  • the sequence from FIG. 13 is used to produce a knock-out mouse.
  • Figure 13 describes a mouse sequence of the T2 gene. Switching off the mouse T2 gene can be achieved in different ways. For example, the splicing sequence (GT) (underlined in FIG. 13) can be deleted or changed so that the T2 gene is not spliced correctly anymore. Furthermore, by inserting a splice acceptor sequence of another exon of the mouse T2 gene into the subsequent intron sequence, a sequence can be inserted into this intron which is recognized as an exon and which is spliced to the exons of the T2 gene located in front of it. This inserted exon can be, for example, an exon that encodes the EGFP protein.
  • the inserted sequence can be designed in the end (eg polyA signal, etc.) in such a way that no further exons are spliced from the T2 gene to the inserted exon. This results in a deletion of the mouse T2 protein at the C-terminal end and an (at least partial) inactivation of the protein function of the mouse T2 gene can be achieved. Furthermore, such sequences can also be inserted as new exon sequences which result in an mRNA sequence in which this new mRNA sequence is located at the 3 'end.
  • Suitable sequences can then be used to change the stability of the mRNA or to change its location in the cell.
  • the associated phenotypes of the mice modified in this way can then provide important conclusions about the function of the T2 gene.
  • These mice can then also be used to find new active substances that compensate for the loss of function of the T gene.
  • a mammal can be generated that has a change in the T3 gene.
  • the sequence in FIG. 19 represents part of the murine cDNA sequence of the T3 gene.
  • Targeted changes to the mouse T3 gene can be achieved by deletions or insertions.
  • the inserted sequence can be, for example, an exon that encodes the EGFP protein.
  • the original mouse T3 gene becomes a fusion protein that carries the EGFP protein at the C-terminus.
  • a mouse can be generated which allows the expression of the T3 gene to be monitored in vivo.
  • the inserted sequence can be designed in the end (eg polyA signal, etc.) in such a way that no further exons are spliced from the T3 gene to the inserted exon. This results in a deletion of the mouse T3 protein at the C-terminal end and an (at least partial) inactivation of the protein function of the mouse T3 gene can be achieved.
  • sequences can also be inserted as new exon sequences which result in an mRNA sequence in which this new mRNA sequence is located at the 3 'end.
  • Suitable sequences can then be used to change the stability of the mRNA or to change its location in the cell.
  • the associated phenotype of the mice modified in this way can then provide important conclusions about the function of the T3 gene. These mice can then also be used to find new active substances that compensate for the loss of function of the T3 gene.
  • Another object of the present invention are cells obtained from the above non-human mammal. These cells can be in any form, e.g. in a primary or long-term culture.
  • a non-human mammal according to the invention can be provided by conventional methods.
  • a method is favorable which comprises the following steps: (a) Production of a DNA fragment, in particular a vector, containing an altered T, T2 or T3 gene, the gene having been altered by inserting a heterologous sequence, in particular a selectable marker;
  • step (c) transforming the embryonic stem cells from step (b) with the DNA fragment from step (a), the T gene in the embryonic stem cells being changed by homologous recombination with the DNA fragment from (a),
  • step (d) culturing the cells of step (c;
  • step (e) selection of the cultured cells from step (d) for the presence of the heterologous sequence, in particular the selectable marker,
  • step (f) Generating chimeric non-human mammals from the cells of step (e) by injecting these cells into mammalian blastocysts (preferably mouse blastocysts), transferring the blastocysts into pseudo-pregnant female mammals (preferably mouse) and analyzing the progeny obtained for a change in the T gene.
  • mammalian blastocysts preferably mouse blastocysts
  • pseudo-pregnant female mammals preferably mouse
  • step (c) the mechanism of homologous recombination (cf. RM Torres, R. kuhn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997) is used to transfect embryonic stem cells.
  • the homologous recombination between the DNA sequences present in a chromosome and new, added cloned DNA Sequences allows a cloned gene to be inserted into the genome of a living cell instead of the original gene.
  • embryonic germ cells can be used to obtain via chimeras animals that are homozygous for the desired gene or the desired gene part or the desired mutation.
  • embryonic stem cells refers to any embryonic stem cells from a non-human mammal that are suitable for mutating the T gene.
  • the embryonic stem cells are preferably from the mouse, in particular the cells E14 / 1 or 129 / SV.
  • vector encompasses any vector which, by recombination with the DNA of embryonic stem cells, enables a change in the T, T2 or T3 gene.
  • the vector preferably has a marker which can be used to select for existing stem cells in which the desired recombination has taken place.
  • a marker is e.g. the loxP / tk neo-cassette, which can be removed from the genome again using the Cre / loxP system.
  • the present invention provides a non-human mammal whose T, T2 or T3 gene is altered. This change can be a switching off of the gene expression regulating function. With such a mammal or cells from it, the gene expression-controlling function of the TP protein can be investigated selectively. It is also possible to find substances, drugs and therapeutic approaches that can be used to selectively influence the controlling function.
  • the present invention therefore provides a basis for responding to a wide variety of diseases. to act. Such diseases are, for example, restrictions on the CNS functions, which extend to mental retardation, or the induction of cancer due to errors in the control of cell proliferation.
  • the T2 gene in the coding region of the cDNA sequence contains CGG trinucleotides which are known to be sensitive to methylation.
  • the T2 gene therefore has a methylation-sensitive and unstable sequence in the coding region (N-terminal region of the protein which has no homology to Protein T or Protein T3), which leads to the failure of the gene with accompanying mental retardation and uncontrolled cell growth, like cancer.
  • the T gene is affected by genomic rearrangements in many tumors. For example, in neuroblastoma, genomic changes in the DNA of tumors compared to the DNA of the associated healthy tissue can be detected. Furthermore, the expression of the T gene is e.g. changed in brain tumors. In glioblastomas in advanced stages, among other things, a strongly changed expression can be determined. In meningiomas, tumor-specific changes in the expression of the T gene and the appearance of the T protein can also be detected.
  • the T2 gene is also affected by genomic rearrangements and an altered expression can be demonstrated in tumors.
  • genomic rearrangements of the T2 gene can be identified in melanoma and lung tumors. Differences in expression are also detectable, for example, in gliomas, glioblastomas, astrocytomas and PNETs (primitive neuro-ectodermal tumors).
  • the T3 gene is also affected by genomic rearrangements and expression changes in many tumors. In the case of colon carcinomas, for example, rearrangements can be demonstrated. Differences in expression can be found in gliomas, glioblastomas, astrocytomas and PNETs (primitive neuro-ectodermal tumors).
  • the inventors have now discovered that the T protein has a certain relationship to proteins that perform very different functions in the cell. Sequence analysis of these proteins showed that the genes encoding these proteins are probably due to a common or similar precursor gene. Proteins such as the POM121 protein (Hallberg et al., J. Cell Biol. 122, pp. 513-522, 1993) belong in these protein superfamilies. It is one of two known core pore membrane proteins in vertebrates. This family also includes the CLIP-170 protein, which binds vesicles and other organelles within the cell to microtubules (Pierre et al., Cell 70, pp. 887-900, 1992).
  • the T protein family is evolutionarily and functionally between the CLIP (cytoplasmic linker protein-170) and the POM121 protein. This intermediate position is also determined by the sequence analysis and the putative protein structure underpinned.
  • the nuclear pore membrane protein POM121 has no pronounced coiled-coil structure, whereas the CLIP-170 protein shows a very pronounced coiled-coil structure between the N and C terminus (cf. FIG. 29).
  • the family of T proteins contains coiled-coil structures, which are, however, significantly less pronounced than with CLIP-170.
  • the family of T proteins takes a similar intermediate position with regard to the presence of hydrophobic domains.
  • the POM121 protein has a hydrophobic domain at the N-terminus, which is embedded in the core membrane and positions the protein in the core pore.
  • the CLIP-170 protein has no pronounced hydrophobic domain.
  • the T protein and the T3 protein have a hydrophobic domain with three hydrophobic partial regions (cf. FIG. 30).
  • the exchange of the N-terminus in the T2 protein in comparison to the basic evolutionary form led to the loss of this pronounced hydrophobic domain.
  • all three T-proteins have in common the very similar structure of the C-terminus.
  • the T3 protein is most similar to the T protein within the T protein family. But the T3 protein has also undergone a change in the course of evolution.
  • the N-terminus was changed compared to the T-protein. This insertion resulted in a further coiled-coil structure compared to the otherwise very similar T protein.
  • the T protein and T3 protein perform functions in the nuclear membrane localized form that are similar to that of the POM121. Interestingly, part of the C-terminus in the POM121 protein was lost in the course of evolution. Compared to the POM121 protein, the T proteins have a longer C terminus. This longer C-terminus enables many interactions with other proteins. It is also worth mentioning that a leucine zipper structure was discovered in the T protein, which facilitates interactions with other proteins.
  • the T protein family plays an important role in mediating interactions between cell organelles and filaments, including microtubules.
  • Microtubules play an important role in nerve cells, for example; for axons, for example, the plus ends of the microtubules point from the body away, while the microtubules in dendrites have both orientations. This cell polarity is of great importance for the functioning of a cell or living being. Furthermore, microtubules open up efficient organelle transport and they are essential for the general organization of membrane structures in a cell.
  • the T proteins play an important mediating role between membrane structures and microtubules.
  • the T gene and the T3 gene perform their function in particular as membrane proteins in the core pore, while the T2 protein acts in particular as a cytoplasmic protein.
  • the T gene and the T3 gene are part of the nuclear pore complex.
  • Nuclear pore complexes are extremely complex structures that mediate the bidirectional transport of macromolecules between the nucleus and the cytoplasm.
  • the nuclear pore complex is embedded in the nuclear envelope and surrounds a central channel with a structure that has so far been insufficiently defined.
  • Peripheral structures, short cytoplasmic filaments and a basket-like structure are attached to both sides of the central core pore complex. This basket-like structure interacts with molecules that pass through the nuclear pore complex. The mechanism of opening the nuclear pore complex has so far been poorly understood.
  • the nuclear envelope is deliberately dissolved and its components, including the nuclear pore proteins, are distributed within the mitotic cytoplasm. At the end of mitosis, all of these components are reused to form the core shells of the daughter cells.
  • the N-terminal half of the T protein has poor homology to the pore membrane protein POM121. The homology extends over the entire area of the POM121 protein and has an identity of approx. 18% at the protein level, so that the DNAs on which these proteins are based do little even stringent conditions should not hybridize with each other.
  • the protein T according to the invention plays a very fundamental role with regard to the formation and structure of the core pore.
  • a detailed analysis of the protein revealed a lipophilic domain at the N-terminus of the T protein.
  • this sequence has no homology to the lipophilic sequence of the POM121 protein.
  • a short section of amino acids which may serve as a signal sequence.
  • various constructs of the T gene were produced.
  • Various parts of the N-terminus of the T-protein were fused with the EnhancedGreenFluorescentProtein (EGFP).
  • EGFP EnhancedGreenFluorescentProtein
  • the fusion protein which contained the unchanged N-terminus of the T protein (putative signal sequence with lipophilic membrane domain), was actually embedded in the nuclear membrane.
  • the fusion construct in which the putative signal sequence and the lipophilic domain are missing, was not embedded in the core membrane and accumulated in the cytoplasm. This showed that the N-terminus of the T protein is necessary and sufficient to lead to localization within the nuclear membrane.
  • Antibodies against a peptide sequence of the T protein were generated to show that the T protein is actually located in the nuclear membrane. With these antibodies, immuno-isotochemical studies were carried out on human, mouse and rat tissues. It was found that the antibody detects a protein that is located in the core membrane.
  • the results of the analysis of the expression at the protein level with the aid of the antibody are in very good agreement with the results of the analysis of the RNA expression.
  • the mouse ortholog of the T gene was used in the RNA in situ analyzes. With the help of the human T gene cD ⁇ A clones, murine cD ⁇ A cones of the mouse ortholog were first isolated and sequenced. Sequence analysis confirmed that the isolated cD ⁇ A clones were the mouse ortholog. Such a murine cD ⁇ A clone of the T gene was then used for the R ⁇ A in situ hybridization (cf. FIGS. 25, 26, 27, 28). An expression analysis of the mouse T gene was then possible with the aid of this technique.
  • T gene plays a crucial role in the development, formation and maintenance of the nervous system in vertebrates.
  • pc post coneeptionem
  • no expression can be seen yet.
  • pc post coneeptionem
  • an expression in the ventral mesencephalon and in the telencephalon is detectable.
  • pc expression of the T gene can be determined in the telencephalon, in the ventral mesencephalon and in the myelencephalon.
  • T gene or T2 or T3 gene were carried out in order to find out where the T gene or T2 or T3 gene are expressed.
  • the T gene is predominantly expressed in the brain, hardly or not at all in the heart, lungs, placenta, liver, skeletal muscle, kidney or pancreas (regardless of adult or fetal tissue).
  • the T2 gene on the other hand, is hardly expressed in the brain, but rather in the heart (adult and fetal), adult liver, adult skeletal muscle and adult kidney.
  • the T3 gene is expressed in all tissues tested (adult and fetal heart, brain, liver, kidney; placenta, adult skeletal muscle, adult pancreas), except in the fetal lung.
  • the bidirectional transport of molecules through the nuclear membrane is of crucial importance for the function of every eukaryotic cell.
  • the information that is stored in the nucleus in the form of DNA (chromosomes) is transcribed into RNA, however, the information is only translated into protein in the cytoplasm. If the transcribed information (mRNA) does not get into the cytoplasm, the information is lost and there can be dramatic disturbances within the cell. But this transprot is not a one-way street; it is just as important that certain substances and proteins get into the nucleus so that cell function can be maintained.
  • T protein is also embedded in the core membrane.
  • the T protein is almost twice as large as the POM121 protein, ie it has a much larger binding capacity than the POM121 protein.
  • the T protein is therefore very well suited to isolating possible binding partners that attach to the T protein, in particular the C terminus of the T protein.
  • the tissue-specific expression of the T gene shows impressively that nuclear pore proteins (in particular nuclear pore membrane proteins) do not have to be expressed in all cells and at all times like 'housekeeping' genes.
  • the predominant expression of the T gene in the nervous system shows that the T protein has a very specific function in the nervous system.
  • the predominant expression of the T gene in the nervous system can now be used for the development of new drugs and new drug classes.
  • new substances can now be isolated which specifically influence the bidirectional transport in the core pores of the nervous system.
  • the localization of the T protein within the nuclear membrane is of great advantage.
  • chemical compounds can be tested become. Many pharmaceutical companies have suitable screening procedures in which more than 200,000 chemicals can be tested.
  • reporter assays eg GFP fusion proteins, colored substances, etc.
  • GFP fusion proteins eg GFP fusion proteins, colored substances, etc.
  • new active substances can be isolated which specifically influence the transport of molecules in nuclear pores, in particular those of the nervous system.
  • T proteins according to the invention T, T2, T3 protein
  • possible binding partners which can represent active substances in the above-mentioned sense
  • T, T2, T3 protein T proteins according to the invention
  • binding partners which can represent active substances in the above-mentioned sense
  • Yeast Two- Hybrid system This system is based on the discovery that cellular transcription activators, such as GAL4 or lexA from yeast, can be broken down into two independent functional domains. Both domains are normally part of a protein in the nucleus of the yeast cell, which binds to certain activating sequences of different target genes and regulates their transcription.
  • the DNA binding domain (BD) specifically binds to a specific DNA target sequence (upstream activating sequence) in the vicinity of the target gene promoter.
  • the other domain, the activation domain (AD) increases the transcription rate of the target gene by interacting with the transcription initiation complex which is bound to the promoter of the target gene.
  • the DNA binding domain (BD) of GAL4 or lexA is expressed there as a fusion protein with a "bait protein or peptide" (here: T, T2 or T3 protein / peptide) in yeast cells.
  • This fusion protein also has a nuclear localization signal through which it is transported into the cell nucleus of the yeast.
  • the bait fusion protein binds there to a target sequence (UAS) which is located in the yeast strain used in the vicinity of the promoters of two reporter genes (eg auxotrophic markers (HIS3) and enzymatic marker (lacZ)) is located.
  • UAS target sequence
  • HIS3 auxotrophic markers
  • lacZ enzymatic marker
  • a second fusion protein is now additionally expressed in the same yeast cell. This consists of the activation domain (AD) of GAL4 or lexA and a prey protein or peptide. It also has a core localization signal.
  • the prey fusion protein is also transported in the cell nucleus of the yeast.
  • the prey protein and the bait protein exposed at the UAS enter into a physical interaction with one another, then the statistical probability increases that the activation domain is in the vicinity of the reporter gene promoter. This leads to an increase in the transcription of the reporter genes, the extent of which is proportional to the strength of the interaction between bait and prey protein.
  • a cDNA library or a combinatorial peptide library can be used as prey proteins.
  • the present invention also relates to a method for identifying inhibitors or enhancers of the T protein family according to the invention.
  • the nucleic acid sequences or parts of these sequences which are part of the T gene or its paralogues or orthologs are inserted into suitable vectors and used for the transfection or transformation of cells, tissues or organisms.
  • These modified cells, tissues or organisms are then used to identify inhibitors or enhancers of the T protein or its paralogenous or orthologous proteins (for example T2 and T3) or proteins which interact directly or indirectly with these proteins.
  • the inhibitors or enhancers identified by this approach can be used for active pharmaceutical ingredients or medicaments or for their production and can be used for the treatment of diseases such as cancer, neurological and psychiatric disorders and injuries to the nervous system.
  • the nervous system or in the case of degenerative diseases of the nervous system can, among other things, be targeted to promote neuronal regeneration or to improve the interconnection of individual nerve areas (application including Alzheimer's disease, Parkinson's disease, schizophrenia, manic-depressive diseases, autism, mental retardation).
  • the present invention makes it possible to test which substances or therapeutic agents are suitable for increasing or reducing the action of the T protein or the family of T proteins.
  • the changed core pore properties which are influenced by the proteins T and T3, can be detected by suitable screening methods. These include, for example, the visualization of the bidirectional transport through the nuclear pore or the detection of an altered transcription of cellular or reporter genes.
  • substances or therapeutic agents can be identified that inhibit or promote the action of proteins that are directly or indirectly involved in the action of the T protein or the family of T proteins.
  • Substances or therapeutic agents that show an increase or decrease in the activity of the T protein (or T2 or T3) in the above-mentioned screening methods can be used to determine whether the increase or decrease in the effect of the T protein is too therapeutically desirable Effects. Above all, this includes inhibiting the growth or spread of tumor cells or promoting neuronal regeneration, for example after nerve injuries (including paraplegia and craniocerebral trauma).
  • the identified substances can then be used as drugs or for the manufacture of these drugs. These drugs then make it possible to inhibit or block the spread of the disease-inducing cells and thus to contain or cure the disease as a whole.
  • T protein (or T2 or T3) can also be used in those screening processes which not only allow the detection of the changed nuclear pore properties but also to identify upstream or downstream or parallel signal cascades. This makes it possible, for example, to identify tyrosine kinases or tyrosine phosphatases that regulate proteins, which in turn directly or indirectly influence the action of the T protein (or T2 or T3). In this way, suitable targets for positively influencing cell events can be identified and characterized.
  • the T protein although it occurs as a nuclear pore protein, plays an important role in the interactions and interactions with filaments of the cell, for example microtubules and actin. These interactions can now be examined, for example by fusion proteins of the T protein with the EGFP protein.
  • Cells that have been stably or transiently transformed or transfected with constructs for such fusion reporter proteins can be incubated with substances or pharmaceuticals in order to identify substances that can interact with the T protein with filaments such as the actin filaments or the Increase or decrease microtubules.
  • active substances can be isolated which, among other things, positively influence the growth of nerve cells or the inhibition of the growth of tumor cells.
  • immunoprecipitation should be mentioned as a method for identifying such possible active substances. It can be used to isolate proteins that bind to the T protein family. With these proteins, further immunoprecipitations can then be carried out in order to isolate new proteins which then no longer interact directly with the T protein.
  • the present invention relates to a method for the identification of further proteins which play a role in the development and function of the nervous system and / or are a nuclear pore protein, the method comprising the following steps: (a) producing an antibody against a protein of the T family (T, T2 or T3 protein),
  • Figure 1 human cDNA sequence (gene T) and deduced amino acid sequence
  • FIG. 2 human genomic DNA sequence (gene T)
  • FIG. 3 human genomic DNA sequence (gene T)
  • FIG. 4 human genomic DNA sequence (gene T)
  • FIG. 5 human genomic DNA sequence (gene T)
  • FIG. 6 human genomic DNA sequence (gene T)
  • FIG. 7 human genomic DNA sequence (gene T)
  • FIG. 8 human genomic DNA sequence (gene T)
  • Figure 9 partial murine cDNA sequence (gene T) and deduced amino acid sequence
  • FIG. 10 partial murine genomic DNA sequence (gene T)
  • Figure 11 partial human cDNA sequence (gene T2) and deduced amino acid sequence
  • Figure 12 partial murine cDNA sequence (gene T2) and deduced amino acid sequence
  • Figure 13 partial murine cDNA sequence (gene T2) and deduced amino acid sequence
  • Figure 14 Splice variant of the human T gene with a deduced amino acid sequence
  • Figure 15 Splice variant of the human T gene with a deduced amino acid sequence
  • Fig. 21 Sequence comparison within the T family
  • Fig. 22 Protein alignment of POM121 protein and T protein
  • Fig. 23 Northern blot analysis
  • Fig. 24 Immunohistochemical examinations and electron micrographs
  • Fig. 25 In situ hybridization with embryonic RNA
  • Fig. 26 In situ hybridization with RNA from the brain
  • Fig. 27 In situ hybridization with RNA from fetal brain
  • Fig. 28 In situ hybridization with RNA from mouse nerve tissue
  • Fig. 29 Comparison of the coiled-coil regions between CLIP protein, T protein and POM121
  • Fig. 30 Hydrophobicity blots for POM 121, T protein and T3 protein The following clones were deposited in accordance with the Budapest Treaty with the DSMZ (German Collection for Microorganisms and Cell Cultures GmbH), Mascheroder Weg 1b, Braunschweig, on August 18, 1998:
  • Clone JFC955 (DSM12375); human genomic clone; represents human genomic sequence; includes start of the cDNA sequence
  • Clone JFC N2112 (DSM12376); human genomic clone; has been fully sequenced. The sequence is shown in Figure 2 and contains the sequence of bp 1756-4228 of the human cDNA sequence.
  • Clone JFC-BN27 (DSM 12659); contains the sequence of Bp 4370-8690 of the human cDNA sequence
  • Clone JFC-BN20 contains the sequence of bp 2025-6280 of the human cDNA sequence On February 1, 2000, the following clone was deposited with the DSMZ in accordance with the Budapest Treaty:
  • cDNA clone pL70 (DSM13270); represents essential parts of the T3 gene.
  • the sequences shown in Figs. 2-8 come from the clones JFC955 (DSM 12375) and JFC950 (DSM 12374).
  • the sequence shown in Fig. 1 comes from the clones JFC277 (DSM 12371), JFC405 (DSM 12372) and JFC-BN27 (DSM 12659) and JFC-BN20 (DSM 12698).
  • the sequence shown in Fig. 9 comes from the clone JFC610 (DSM12373).
  • the selected cDNA clones originate from genes that have CNS-specific expression.
  • the cDNA pieces ('inserts') contained in the individual cDNA clones were isolated and used for hybridization with Northern blots.
  • the Northern blots used included polyA-RNA from various human tissues (e.g. brain, skeletal muscle, liver and kidney) and various stages of development (fetal and adult tissues). As not only brain-specific genes are expressed in the fetal brain, as mentioned above, hybridization with the Northern blots was used to identify cDNA clones that are expressed primarily in the brain and less in other tissues.
  • This differential analysis identified a cDNA clone that has a brain-specific expression pattern.
  • the entire mRNA sequence for the new protein encoded therein could be isolated and decrypted (gene T with protein TP encoded therein) by repeated hybridization of the fetal cDNA library.
  • the Baltimore Biological Lab. (BBL) agar plates, and BBL top agarose can be prepared.
  • the phages human or murine cDNA library, Stratagene
  • the phages were diluted 1:10 3 and 1:10 4 with SM medium in order to obtain individual plaques after plating.
  • BBL agar For the BBL agar (pH 7.2), 10 g of BBL trypticase, 5 g of NaCl and 10 g of select agar are weighed in and made up to 1 1 with H 2 0. The agar is released by autoclaving. After cooling to approx. 60 ° C, pour the plates. The plates are preheated to 37 ° C before use to prevent premature solidification of the top agarose.
  • the BBL top agarose (pH 7.2) was made up to 1 1 H 2 0 with 10 g BBL trypticase, 5 g NaCl, 6.5 g agarose and 10 ml IM MgS0 4 solution. Dissolve by autoclaving and prepare in a water bath at 41 ° C.
  • the cDNA libraries used (human and murine fetal brain cDNA library; from Stratagene, Heidelberg) were cloned in the vector ⁇ -ZAPII. This made it possible to avoid subcloning the phage insert into a plasmid vector.
  • This protocol allows cDNA, which is an insert in the ⁇ -ZAPII vector, to be converted in a simple manner by an in vivo approach into an insert which is now in the plasmid bluescript SK (-).
  • the principle of this approach is that a helper phage introduces information for proteins that only allow DNA amplification in the area of the phage genome that has the genetic information for the plasmid with cDNA insert. It was largely carried out according to the manufacturer's protocol (Stratagene).
  • plating was carried out in such a way that individual phage plaques were on the plate.
  • the in vivo exeption test okol 1 was then carried out with these individual plaques.
  • the plasmid DNA and its plasmid inserts were isolated from the bacterial clones and then hydrided with Northern blots. The selection of the clones to be investigated was based on the expression pattern in the Northern blots.
  • radioactive labeling of the double-stranded insert DNA of the cDNA clone was carried out as follows for the further isolation of overlapping cDNA clones.
  • the unincorporated nucleotides were separated using a self-made Sephadex G-50 column.
  • the separation principle of the column is based on the Exclusion chromatography.
  • the smaller unincorporated nucleotides fit into small pores of the column material, while the DNA remains excluded from them.
  • the volume in which the nucleotides can move is therefore larger than the volume available to the DNA. If you now carry a mixture of DNA and nucleotides on the column, the DNA runs through the column faster than the nucleotides. This allows the unincorporated nucleotides to be separated.
  • a Pasteur pipette was sealed with a small glass bead. Fill the Pasteur pipette with Sephadex G-50 ("Fine") dissolved in water until the filling material is 5 cm below the top edge of the Pasteur pipette. 2x rinsing the column with TE. Apply the above radioactive labeling approach. Add 320 ⁇ l TE. Discard the solution that has passed through the column. Place the Eppendorf tube under the column. Add 350 ⁇ l TE. Collect the radioactive solution that has passed through the column.
  • Sephadex G-50 Sephadex G-50
  • the plaque "blot” was carried out for the analysis of the cDNA library in order to make the cDNA located in phage clones accessible for hybridization.
  • the hybridization is based on the binding of complementary, single stranded nucleic acids.
  • the DNA to be examined was immobilized on a membrane and hybridized with a radioactively labeled probe.
  • the complementary binding is retained even after washing off the non-specific adherent probes and can be made visible by autoradiography.
  • single-stranded molecules were incubated under salt and temperature conditions, which favor the formation of base-paired double strands.
  • a crucial factor in association and dissociation kinetics are the hydrogen bonds between the base pairs G-C and A-T.
  • the hybridization reaction is influenced by changes in the temperature and the salt and sample concentration.
  • the filters were packed in cling film.
  • the autoradiography was carried out at -80 ° C. in an X-ray cassette which contained an intensifying film made of calcium tungstate. The exposure lasted 30 minutes to a few days, depending on the strength of the signal.
  • the complete mRNA coding for the protein of the T gene could be isolated. Furthermore, using cD ⁇ A clones of this newly isolated gene T, two further genes (T2 and T3) could be isolated, which have pronounced homologies with this gene. The techniques mentioned above were used again for this. In order to isolate the related genes T2 and T3, the hybridization temperature was reduced to 55 ° C.
  • the multiple tissue Northern blots were purchased from CLONTECH (Palo Alto, California, USA) and used according to the manufacturer's instructions.
  • the respective DNA samples of the genes T, T2 and T3 were radioactively labeled and hybridized with the Northern.
  • the sequence of bp 1-4200 of FIG. 1 was used for the analysis of the expression pattern at the Northern blot level for the T gene.
  • the sequence of bp 1310-4870 of FIG. 17 was used for hybridization.
  • the sequence of bp 3120-4230 of FIG. 16 was used for the gene T2.
  • the "random priming" method was used for the radioactive labeling of double-stranded DNA.
  • the unincorporated nucleotides were separated using a self-made Sephadex G-50 column.
  • the separation of the column is based on the exclusion chromatography.
  • the smaller unincorporated nucleotides fit into small pores of the column material, while the DNA remains excluded from them.
  • the volume in which the nucleotides can move is therefore larger than the volume available to the DNA. If you now carry a mixture of DNA and nucleotides on the column, the DNA runs through the column faster than the nucleotides. This allows the unincorporated nucleotides to be separated. To do this, a Pasteur pipette is sealed with a small glass bead.
  • the Northern blots were hybridized as described below. First, the Northern blots were pre-hybridized in 10 ml hybridization solution (350 ml 20% SDS, 500 ml IM phosphate buffer, pH 7.2; 150 ml distilled water) at 65 ° C. The Northern blots were used in a Glass tube in a hybridization roller oven for the duration of
  • the pre-hybridization solution was discarded.
  • the radiolabelled sample was added to the filters with 10 ml hybridization solution (65 ° C).
  • Hybridization took place overnight at 65 ° C. The filters were then washed twice for 30 min with about 500 ml wash buffer (80 ml
  • IM phosphate buffer pH 7.2; 100 ml 20% SDS, 1820 ml dist.
  • the filters were sealed in plastic film.
  • the autoradiography was carried out at -80 ° C. in an X-ray cassette which contained an intensifying film made of calcium tungstate. The exposure lasted 1-4 days depending on the strength of the signal.
  • Embryos at various stages of development have been isolated from pregnant NMRI mice.
  • the embryos and other tissue samples were fixed overnight with 4% paraformaldehyde in PBS at 4 ° C. 10 ⁇ m frozen sections of the embryos were transferred to slides coated with 3-aminopropyltriethoxysilane.
  • Sense strand and antisense samples were prepared by in vitro transcription with a- 35 S-UTP, with a specific activity of> 10 9 decays per minute / ⁇ g.
  • the linearized mouse T gene cDNA clone from FIG. 9 was transcribed with T7 or Sp6 RNA polymerase. The sample length was reduced to 150-200 nucleotides by alkaline lysis.
  • the slides were in a solution containing 50% formamide, 10% dextran sulfate, 0.3 M NaCL, 10 mM Tris, 10 mM sodium phosphate pH 6.8, 20 mM dithiothreitol, 0.2% Denhardt's solution, 0.1 Triton X-100, 0.1 mg / ml Escherichia coli RNA and 0.1 mM non-radioactive aS-UTP, prehybridized at 54 ° C.
  • the 35 S-labeled sample (8 x 10 4 decays per minute per ml) was added to the hybridization mix and the hybridization then continued for 16 h at 54 ° C. in a moist chamber.
  • the slides were then washed in the hybridization solution for 2 hours.
  • the remaining non-hybridized RNA sample was then digested with RNase A.
  • the slides were then washed for 30 min at 37 ° C with 2x SSC, 0.1% SDS and for 30 min with 0.1x SSC, 0.1% SDS.
  • the slides were then dehydrated with increasing ethanol concentrations.
  • the slides were then covered with Ilford K5 autoradiography emulsion. After 1-2 weeks of exposure at 4 ° C, the slides were incubated in Kodak Dl9b developer and then stained with Giemsa.
  • the sections were analyzed in dark and bright field illumination using a Zeiss SV8 stereomicroscope and an Axiophot microscope and photographed with an Agfa Ortho black and white film.
  • Fig. 26 Expression of the mouse T gene in the postnatal brain.
  • Fig. 28 Expression of the T gene during the development of the nervous system. Expression of the T gene in neurons in the mantle zone of the developing brain and in nuclei of peripheral nerves (arrow in b). No expression is visible in proliferating neurons in the subventricular layer or in migrating neurons in the intermediate zone (c, d). On day 16.5 dcp a strong expression in differentiating neurons of the mantle zone of the telencephalon is visible (e, d). Expression in neurons of the spinal cord and spinal ganglia is also visible (g, h). Furthermore, low expression is visible in a single layer under the skin (g, h).
  • iz intermediate zone
  • mz mantle zone
  • sc spinal cord
  • sga spinal ganglia
  • sk skin
  • svl subventricular layer
  • the rabbit's serum is tested in an immunoblot.
  • the peptide used for immunization is subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1984), 203-209).
  • Western blot analysis was performed as in Bock, C.-T. et al. , Virus Genes 8, (1994), 215-229.
  • the nitrocellulose filter is incubated for one hour at 37 ° C. with a first antibody. This antibody is rabbit serum (1: 10000 in PBS). After several washing steps with PBS, the nitrocellulose filter is incubated with a second antibody.
  • This antibody is an alkaline phosphatase-linked monoclonal goat anti-rabbit IgG antibody (Dianova) (1: 5000) in PBS. After 30 minutes of incubation at 37 ° C, there are several washing steps with PBS and then the alkaline phosphatase detection reaction with developer solution (36 ⁇ M 5 'bromo-4-chloro-3-indolylphosphate, 400 ⁇ M nitroblue tetrazolium, 100mm Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 ) at room temperature until bands become visible.
  • developer solution 36 ⁇ M 5 'bromo-4-chloro-3-indolylphosphate, 400 ⁇ M nitroblue tetrazolium, 100mm Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl 2
  • Antibodies are extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention are detected.
  • the immunohistochemical tests shown in FIG. 24 were carried out with an affinity-purified polyclonal rabbit antibody against the T protein (hereinafter referred to as first antibody) prepared as above.
  • Mouse brain was removed and treated as follows.
  • Liquid can run out more.
  • the enzyme on the second antibody leads to the formation of a dye (DAB), whereby the T protein can be detected.
  • DAB dye
  • Fig. 24 (ad): light microscopic images showing that the T protein is located in or on the nucleus of the cell.
  • the electron microscopic image in e shows that the T protein is not located in the nucleus but in the membrane.
  • the images are in very good agreement with a function as a membrane-bound nuclear pore protein.
  • the arrows in e show the dye formed, which can be seen on the cytoplasmic side of the core membrane.

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  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Protéine (TP) et protéines apparentées qui sont impliquées dans le développement du système nerveux, en particulier du système nerveux central, et qui sont exprimées d'une manière spécifique aux tissus et spécifique au développement. La présente invention concerne également des séquences d'ADN qui codent ces protéines, ainsi que des anticorps ou fragments desdits anticorps dirigés contre ces protéines et de l'ADN antisens ou des ribozymes dirigés contre l'expression desdites protéines. Elle concerne encore des médicaments et des méthodes de diagnostic dans lesquels sont utilisés les composés susmentionnés. Elle concerne enfin un mammifère non humain dont le gène codant pour la TP est modifié.
EP00916770A 1999-02-26 2000-02-28 Proteine (tp) impliquee dans le developpement du systeme nerveux central Withdrawn EP1165607A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19908423A DE19908423A1 (de) 1999-02-26 1999-02-26 An der Entwicklung des ZNS beteiligtes Protein (TP)
DE19908423 1999-02-26
PCT/DE2000/000583 WO2000050451A2 (fr) 1999-02-26 2000-02-28 Proteine (tp) impliquee dans le developpement du systeme nerveux central

Publications (1)

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EP1165607A2 true EP1165607A2 (fr) 2002-01-02

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EP (1) EP1165607A2 (fr)
AU (1) AU3801800A (fr)
DE (1) DE19908423A1 (fr)
WO (1) WO2000050451A2 (fr)

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Publication number Priority date Publication date Assignee Title
DE10042609A1 (de) * 2000-08-30 2002-03-28 Deutsches Krebsforsch Verwendung von T-Proteinen zur differentiellen Charakterisierung und Therapie von Verletzungen und Tumoren des Nervensystems
WO2003085134A2 (fr) * 2002-04-05 2003-10-16 The University Of Tokyo Methodes de diagnostic et de traitement du cancer colorectal
ES3015712T3 (en) 2019-11-04 2025-05-07 Ummon Healthtech Method of, and computerized system for labeling an image of cells of a patient
WO2024215528A1 (fr) 2023-04-13 2024-10-17 Ventana Medical Systems, Inc. Dosage de prolifération pour des tumeurs solides fixes

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WO1993016178A2 (fr) * 1992-02-12 1993-08-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Sequences caracteristiques du produit de transcription des genes humains
ES2239819T3 (es) * 1993-11-04 2005-10-01 Innogenetics N.V. Epitopos inmunodominantes de celulas t humanas del virus de la hepatitis c.
WO1995014772A1 (fr) * 1993-11-12 1995-06-01 Kenichi Matsubara Signature genique
GB9510944D0 (en) * 1995-05-31 1995-07-26 Bogaert Thierry Assays and processes for the identification of compounds which control cell behaviour,the compounds identified and their use in the control of cell behaviour
EP0825198A1 (fr) * 1996-08-22 1998-02-25 K.U. Leuven Research & Development Famille de gènes Plag et tumorigénèse
GB9625283D0 (en) * 1996-12-04 1997-01-22 Janssen Pharmaceutica Nv Vertebrate homologues of unc-53 protein of c.elegans or functional eqivalents thereof and cdna sequences coding for said homologue
US6103886A (en) * 1997-04-11 2000-08-15 Whitehead Institute For Biomedical Research Genes in the non-recombining region of the Y chromosome
GB9811962D0 (en) * 1998-06-03 1998-07-29 Janssen Pharmaceutica Nv Vertebrate homologue of UNC-53 protein of C.elegans

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Title
See references of WO0050451A2 *

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WO2000050451A3 (fr) 2001-08-02
DE19908423A1 (de) 2000-08-31
AU3801800A (en) 2000-09-14

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