EP1165607A2 - Protein (tp) that is involved in the development of the nervous system - Google Patents

Abstract

Disclosed are a protein (TP) and proteins which are related thereto. Said proteins are involved in the development of the nervous system, especially the central nervous system, and are expressed in a tissue-specific and development-specific manner. The invention also relates to DNA sequences that code said proteins and antibodies or fragments thereof which are directed against said proteins. The invention further relates to antisense RNA or ribozymes which are directed against the expression of the proteins. Disclosed are medicaments and diagnostic processes in which the above-mentioned compounds are used. The invention further relates to a non-human mammal whose TP-coding gene is modified.

Description

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. Finally, the present invention relates to pharmaceuticals and diagnostic methods using the above compounds.

Mutations in genes that play a role in the formation and maintenance of the nervous system are of the greatest knowledge and economic importance, since diseases of the nervous system, especially the CNS, are very common, often characterized by a severe, sometimes fatal course of the disease and so far can only be treated to a very limited extent. The increase in life expectancy is associated with a drastic increase in neurological and mental illnesses. These cause a severe reduction in the quality of life of the people concerned and considerable costs for both those affected and for society.

The isolation and analysis of genes specific for the nervous system offers a good opportunity to prevent diseases such as To be able to examine and finally treat schizophrenia, Alzheimer's, autism, manic depression and mental retardation.

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.

This technical problem is solved by providing the embodiments characterized in the claims.

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:

(a) the DNA sequence of Figures 1, 2, 3, 4, 5, 6, 7 or 8;

(b) the DNA sequence of Figure 9 or 10;

(c) the DNA sequence of Figure 11;

(d) the DNA sequence of Figure 12 or 13;

(e) the DNA sequence of FIG. 14 or 15,

(f) the DNA sequence of Fig. 16,

(g) the DNA sequence of Fig. 17 or 18, (h) the DNA sequence of Fig. 19,

(i) a DNA sequence hybridizing with (a), (b), (c), (d), (e), (f), (g) or (h);

(j) Variants, derivatives, precursors or fragments of the DNA sequence of (a), (b), (c), (d), (e), (f), (g), (h) or (i) ; or

(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, as designated below, 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. Because these nerves run along the back of the abdomen and chest, most neuroblastomas occur in the abdomen, pelvis, chest, or neck. More than half of the diseases originate from the adrenal medulla, which is also formed by nerve cells. Signs that may indicate a neuroblastoma in toddlers are knots, swelling, bone pain, limping, tiredness, fever, pallor, sweating, persistent cough, bruising around the eye. 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.

Furthermore, mutations in genes in the T gene family lead 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. With the help of the 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.

The term "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. After hybridization, 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)). Stringent hybridization conditions, as described, for example, in Sambrook et al. , supra. The terms “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 (bioprecursors) are therefore included. Derivatives are, for example, mutation derivatives (generated by, for example, deletions or insertions), fusions, allele variants, muteins and splice variants. 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.

In a preferred embodiment, 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.

By lowering or inhibiting the expression of the DNA sequences described above, the synthesis of the proteins encoded by them, for example the T protein, can be reduced or eliminated, which is desirable, for example, in certain disease states. Therefore, a further preferred embodiment of the present invention relates to antisense RNA, which is characterized in that it is complementary to the above DΝA sequences and the synthesis of the protein encoded by these DΝA 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 DΝA sequences is reduced or inhibited. These 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 DΝA 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 DΝA sequences according to the invention. In principle, 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. Based on procedures described in the literature, it is now possible to construct specific ribozymes that cut a desired RNA at a specific, pre-selected location (see, for example, Tanner et al., In: Antisense Research and Applications, CRC Press, Inc. (1993 ), 415-426).

The DΝA sequences according to the invention or the DΝAs coding for the antisense RNAs or ribozymes described above can also be inserted into a vector or expression vector. Thus, the present invention also includes these DΝA sequences containing vectors or expression vectors. The term "vector" refers to a plasmid (eg pUC18, pBR322, pBlueScript), a virus or another suitable vehicle. In a preferred embodiment, the DΝA molecule according to the invention is functionally linked in the vector to regulatory elements which allow its expression in prokaryotic or eukaryotic host cells. In addition to the regulatory elements, for example a promoter, 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, for example E. coli, 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. Further examples of suitable promoters are the metalothionein I and the polyhedrin promoter. In a preferred embodiment, 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.

General methods known in the art can be used to construct expression vectors containing the DNA sequences of the invention and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as described, for example, in Sambrook et al. , supra. The 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. Preferably 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. These 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. With regard to the definitions of these terms, reference is made to the respective explanations above. 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) (I, P, V) (L, T) (G, A, Q) (L, V) XXX (L, V) Motif 2: IYTDWAN Motif 3:

AXXXXXXXXXGXXXXXXAXXXXXXXXXXXXXXXXXXQ Motif 4:

SXXXXDX (12, 20) KX (17, 22) AXXXXXXXXL style 5:

IYTDWANXXLX (K, R) Motif 6:

KX (18, 21) AXXXXXXXXLX (15, 24) S Motif 7: NX (3, 11) SXXXAXXXXXXXL

Explanation: X means every amino acid

(A, T) means amino acid A or T on this

Job

X (2,4) means two to four X's on this

Job

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. In this context, the term "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.

Then, for example, 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. Examples of 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. For example, 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).

In a particularly preferred embodiment, 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). The constant region (s), if present, originate essentially from a human immunoglobulin. When administered to human patients, 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.

In a preferred embodiment, the present invention relates to antibodies against the individually listed peptides of genes T2 and T3 (cf. FIG. 20).

It has been found that the following peptide can be used specifically for the generation of antibodies to the protein T. The amino acid sequence of the suitable peptide is:

EKGEDPETRRMRTVKNIAD

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. With a DNA sequence according to the invention or probes or primers derived therefrom, 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. To this end, 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. For example, the protein according to the invention can be introduced into mammals, in particular humans, by customary measures. For this purpose, it can be advantageous to couple the protein to a protein that is not considered foreign by the respective body, for example transferrin or bovine serum albumin (BSA). A DNA sequence, antisense RNA or ribozyme according to the invention can also be introduced and expressed in mammals, in particular humans. With an antibody according to the invention, 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. In a preferred embodiment, the vector containing the nucleic acids described above is a virus, for example an adenovirus, vaccinia virus or adenovirus. Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. For the purposes of gene therapy, the 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).

Finally, 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. Depending on the configuration of the diagnostic kit, 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). After determining the expression of the T genes, 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. By detecting the expression of the T genes thus the bandung can be adapted very quickly and efficiently to the respective requirements and thus contributes significantly to the improved therapy.

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.

The term "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.

The expression “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. This can be done, inter alia, by inserting a sequence coding for the GFP (green fluorescent protein) protein within one Exons or as an independent exon. These methods are generally described in Schwartzberg et al. , Proc. Natl. Acad. Be. USA, Vol. 87, pp. 3210-3214, 1990, to which reference is made here.

In particular, the change can be carried out as described below. 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. In this way, 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. 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 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.

In a further preferred embodiment, 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. This transforms the original mouse T2 gene into a fusion protein that carries the EGFP protein at the C-terminus. In this way, a mouse can be generated which allows the expression of the T2 gene to be followed 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 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. Furthermore, 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. In this way, the original mouse T3 gene becomes a fusion protein that carries the EGFP protein at the C-terminus. In this way, 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. 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 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;

(b) preparation of embryonic stem cells from a non-human mammal (preferably mouse);

(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),

(d) culturing the cells of step (c;

(e) selection of the cultured cells from step (d) for the presence of the heterologous sequence, in particular the selectable marker,

(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.

In step (c) the mechanism of homologous recombination (cf. RM Torres, R. Kühn, 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. With this method, 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.

The term "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.

The term "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. Such a marker is e.g. the loxP / tk neo-cassette, which can be removed from the genome again using the Cre / loxP system.

Furthermore, the person skilled in the art knows conditions and materials in order to carry out steps (a) - (f).

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.

It was found by the inventors in the sequence analysis that 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.

All three genes of the T family play a major role in tumors. 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.

In many tumor diseases, the T2 gene is also affected by genomic rearrangements and an altered expression can be demonstrated in tumors. For example, 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).

Through the isolation and precise analysis of the T gene, 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 unexpected discovery that genes that are so different Performing tasks within the cell, belonging to a common protein superfamily, is extremely surprising and, at first glance, even contradictory. However, if one analyzes the functions of the individual genes, two main functions of these proteins can be derived. The CLIP-170 protein binds to microtubules, the newly isolated T proteins and the POM121 protein are located in the nuclear pore complex. Because of the properties of these proteins, the inventors propose to refer to this protein superfamily as the POMIC protein superfamily. POMIC is said to stand for pore and / or microtubule binding protein. Within the framework of the isolation and analysis of the T gene, two paralogues of the T gene could be isolated, the T2 and T3 genes described in more detail above. 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. In contrast, 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. In contrast, 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. By inserting about 400 amino acids, 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.

Based on the inventors' findings, the T gene and the T3 gene are part of the nuclear pore complex. Nuclear pore complexes (NPCs) 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. Furthermore, observations of cells undergoing mitosis have so far shown that 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. Through detailed analysis of the T gene, the inventors found that 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. However, this sequence has no homology to the lipophilic sequence of the POM121 protein. In front of the lipophilic domain in the T protein is a short section of amino acids, which may serve as a signal sequence. In order to determine whether this putative signal sequence and the lipophilic domain are involved in the incorporation into the nuclear membrane in vivo, various constructs of the T gene were produced. Various parts of the N-terminus of the T-protein were fused with the EnhancedGreenFluorescentProtein (EGFP). The EGFP was fused to the C-terminus of the T protein. 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. In contrast, 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. Since it is difficult to distinguish with the aid of a light microscope whether the protein is located in the nuclear membrane or in the nucleus itself, further analyzes were carried out using the high-resolution method of electron microscopy. This made it possible to clearly show that the T protein is localized in the core membrane. A second antibody was used as the detection reaction, to which the enzyme radish pedodixase was coupled and which led to a color reaction (DAB). The dye formed can only be seen on the electron micrographs on the cytoplasmic side of the core membrane. This indicates that the antibody recognizes an epitope of the T protein that is accessible to the antibody from the cytoplasmic side. Analysis of the immunohistochemical sections also showed that the antibody recognizes neurons very specifically (cf. FIG. 24). 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 cDΝA clones, murine cDΝA cones of the mouse ortholog were first isolated and sequenced. Sequence analysis confirmed that the isolated cDΝA clones were the mouse ortholog. Such a murine cDΝA clone of the T gene was then used for the RΝA 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. The exact analysis of the spatio-temporal expression course showed that the T gene plays a crucial role in the development, formation and maintenance of the nervous system in vertebrates. During early mouse embryogenesis on day 9.5 post coneeptionem (pc = post coneeptionem), no expression can be seen yet. On day 10.5 pc an expression in the ventral mesencephalon and in the telencephalon is detectable. At this stage there is also strong expression in the area of the connection of the mesencephalon and the telencephalon (forbrain-midbrain). On day 11.5 pc expression of the T gene can be determined in the telencephalon, in the ventral mesencephalon and in the myelencephalon. On day 12.5 pc an expression in neurons of the mantle zone of the developing brain and in the nuclei of the peripheral nerves is visible. Furthermore, there is an expression in the myelencephalon, spinal cord, and spinal ganglia. A low expression can be detected in the mesencephalon and telencephalon. No expression is detectable, for example, in proliferating neurons in the subventricular layer or in migrating neurons of the ^λ intermediate 'zone. On day 14.5 pc there is also one Expression in mesenchymal tissues, eg around the spine or in the area of developing bones, visible. On day 16.5 pc a strong expression can be found in all parts of the brain and the peripheral nervous system (eg spinal ganglia and nerve fibers of the tail). Furthermore, expression in differentiating neurons of the mantle zone of the telencephalon can be detected. Expression in neurons of the spinal cord and spinal ganglia is also detectable. In the development of the brain after birth, expression in the olfactory bulb (ol factory bulb), in the cerebral cortex and in the developing hippocampus can be demonstrated. A low expression, however, can be found in the coliculus and the developing cerebellum. There is a similar expression pattern in the fully developed brain.

Northern blots (see FIG. 23) 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 discovery of the T gene and the detailed analysis of this gene with all the information obtained from it created the basis for developing completely new types of drugs and classes of active ingredients. 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. If, for example, a transcription factor that - like the other proteins in the cytoplasm - does not get into the cell nucleus, it cannot trigger the transcription of the other genes. This is often associated with dramatic disturbances in cell activity, including the death of the cell or the organism. This makes it clear that nuclear pore proteins have an extremely important function within the cell. The analysis of the T gene has now shown that the T protein is also embedded in the core membrane. Interestingly, 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. With the help of the T protein, 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. With the help of automated tests chemical compounds can be tested become. Many pharmaceutical companies have suitable screening procedures in which more than 200,000 chemicals can be tested. For this purpose, for example, reporter assays (eg GFP fusion proteins, colored substances, etc.) can be used, which indicate the successful transport of a molecule into the nucleus or into the cytoplasm. In this way, new active substances can be isolated which specifically influence the transport of molecules in nuclear pores, in particular those of the nervous system.

The identification and analysis of interactions between the T proteins according to the invention (T, T2, T3 protein) or peptides or fragments thereof and possible binding partners, which can represent active substances in the above-mentioned sense, can be done, for example, with the "Yeast Two- Hybrid system "(Fields, Nature 340, pp. 245-247 (1989)). 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. One domain, 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. In the "Yeast Two Hybrid System" this structure of transcription factors is used in a modified form. 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. This creates a constellation in which the bait protein or peptide is exposed in the immediate vicinity of the reporter gene promoter. 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. If 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. For this purpose, 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. For injuries to the nervous system, for congenital damage to the 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. In particular, 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. Furthermore, 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. An important application of this drug is among other things the prevention of the growth and spread of tumor cells. In addition, the identified active ingredients are used as medicines that specifically stimulate the growth of certain cells. This then makes it possible for cells or structures of the nervous system to damaged by injury or degenerative processes. The 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. Furthermore, 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. In this way, 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. For example, 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.

Furthermore, 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),

(b) contacting a cell extract with the antibody and identifying the antibody / protein complex,

(c) analyzing the complex to identify a protein that has bound to the protein of the complex and is not an antibody, and

(d) optionally repeating steps (a) to (c) in order to identify further proteins of this function.

The invention is further described with reference to the figures, which show:

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

16: partial human cDNA sequence (gene T2) with a deduced amino acid sequence

17: partial human cDNA sequence (gene T3; protein isoform 1) with a deduced amino acid sequence

18: partial human cDNA sequence (gene T3; protein isoform 2) with a deduced amino acid sequence 19: partial murine cDNA sequence with deduced amino acid sequence (gene T3)

20: oligonucleotides and peptides (T gene)

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 JFC277 (DSM12371); human cDNA; represents the human cDNA sequence of bp 1218-3690

Clone JFC405 (DSM12372); human cDNA; represents the human cDNA sequence of Bp 1-1891

Clone JFC601 (DSM12373); murine cDNA; represents the murine cDNA sequence of bp 225-3026

Clone JFC950 (DSM12374); human genomic clone; represents human genomic sequence

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.

On February 2, 1999 the following clone was deposited with the DSMZ in accordance with the Budapest Treaty:

Clone JFC-BN27 (DSM 12659); contains the sequence of Bp 4370-8690 of the human cDNA sequence

On February 19, 1999 the following clone was deposited with the DSMZ in accordance with the Budapest Treaty:

Clone JFC-BN20 (DSM 12698); 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 invention is further described on the basis of the exemplary embodiment below.

EXAMPLES

With regard to the methods used, Sambrook, J., Fritsch, E.F. and Maniatis, T. (Molecular cloning; a laboratory manual; second edition; Cold Spring Harbor Laboratory Press, 1989) and Current Protocols in Molecular Biology (John Wiley and Sons, 1994-1998), using the techniques mentioned below, in particular preparation of DNA or RNA or Northern blot are sufficiently known to the person skilled in the art and are mastered.

Before the implementation of the experiments is described in detail, the work strategy should be explained in the next section. In search of genes which, in the mutated state, trigger diseases of the CNS (eg neurodegenerative diseases, mental retardation, tumor diseases of the CNS), 23 cDNA clones were developed from a human fetal brain cDNA library (from Stratogen, Heidelberg) isolated. A human fetal brain cDNA library was used as the starting material, since it was assumed that genes which play a role in the development of the CNS and in particular the brain are present in a fetal brain cDNA library. However, since so-called household genes (genes that are expressed in most tissues) are also expressed in the CNS, it has now been tested whether the selected cDNA clones originate from genes that have CNS-specific expression. For this purpose, 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. Using this cDNA clone, 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.

EXAMPLE 1: Identification of the T genes 1. Titration of the cDNA libraries

In order to ensure an effective infection, it was first necessary to produce phage-competent bacteria in an overnight culture. The magnesium ions contained in the medium induce the bacterial maltose receptor to which the phage binds to infect the bacterium.

Execution :

Prepare 50 μl E. coli XLl-Blue in 50 ml LB medium, adding MgSO ₄ in a concentration of 10 mM to the medium. Incubate overnight at 30 ° C and 220 rpm. Centrifuge the bacteria at 4 ° C and 1000 xg for 10 min. Resuspend in 25 ml of 10 mM MgS0 ₄ . The phage-competent bacteria produced in this way could be stored at 4 ° C for up to one week.

2. Plating out the cDNA libraries

The Baltimore Biological Lab. (BBL) agar plates, and BBL top agarose can be prepared. The phages (human or murine cDNA library, Stratagene) were diluted 1:10 ³ and 1:10 ⁴ with SM medium in order to obtain individual plaques after plating.

Execution :

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. Place 15 μl of the diluted phage solution as specified above and 250 μl of the competent XL-1 bacteria in a 15 ml Falcon tube. 20 min. incubate at room temperature. Add 10 ml BBL-Top agarose, swirl and put on the warmed agar plate. After about 20 minutes the top agarose layer is solid and the plates can be stacked with the agar side up. Incubation takes place overnight at 37 ° C. After the incubation period has elapsed, the plates can be stored at 4 ° C or can be used directly to transfer the phage plaques. to Store the plates tightly together with a chloroform-soaked cloth in plastic bags. The chloroform prevents the growth of cold-loving bacteria and fungi.

3. In vivo execution

The cDNA libraries used (human and murine fetal brain cDNA library; from Stratagene, Heidelberg) were cloned in the vector λ-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 λ-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).

In particular, 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.

Execution :

Add 100 μl of a single phage λ-ZAPII clone with 200 μl XL1 bacteria and 2 μl helper phage (included in the Stratagene kit). 15 minutes. Shake at 37 ° C and 80 rpm, the specific attachment of both phage types to the host bacterium. Add 3 ml LB medium. Incubate for 2 h at 37 ° C and 200 rpm. During this time, DNA replication of the plasmid contained in the 1-ZAPII vector, its Circularization, as well as packaging in envelope proteins and removal from the bacteria instead. At 70 ° C for 20 min. heat. Then 15 min. Centrifuge at 4000g. This kills the remaining bacteria and separates their fragments from the plasmids present in the phage shell, which are in the supernatant. Add 1 ul of this to 200 ul SOLR host cells, 15 min. Incubate at 37 ° C. Plate 100 μl on LB / Amp plates. Store at 37 ° C overnight. The bacterial clones that have now grown contain the plasmid with the corresponding cDNA insert. A mini-prep DNA preparation was carried out in each case.

4. "random primed" DNA labeling

The 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.

Execution :

For a typical labeling approach, dissolve 100 ng DNA in a volume of 12 μl H ₂ 0. Heating for 10 minutes at 95 ° C causes denaturation of the DNA in single strands. Store the batch on ice to prevent reassociation of the two complementary DNA strands. Complete the reaction mixture with 4 μl OLB (oligo labeling buffer, 1 μl Klenow (1U) as well as 2.5 μl a- ³² P-dCTP and 2.5 l a- ³² P-dATP. Incubate overnight at room temperature. While During this time, the complementary strand, starting from the hexanucleotides attached to a single strand, takes place through the Klenow fragment of E. coli DNA polymerase I. The DNA is radiolabelled by the incorporation of the a- ³² P-dCTP and the a- ³² P-dATP.

5. Separation of unincorporated radioactive nucleotides

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.

Execution :

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 μl TE. Discard the solution that has passed through the column. Place the Eppendorf tube under the column. Add 350 μl TE. Collect the radioactive solution that has passed through the column.

6. Plaαue "blot"

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.

Execution :

Place a labeled, marked Hybond-N membrane free of air bubbles on the plate with the phage plaques for 1 min. The marking pattern has been transferred. Place on Whatman paper soaked with denaturing solution (0.5 M NaOH; 1.5 M NaCl) for 10 min, with the plaque side up. 10 min. neutralize in 50 mM phosphate buffer. The remaining remains of the bacterial lawn are wiped off with a little pressure using a Kleenex cloth soaked in phosphate buffer. The filters are designed to dry at room temperature. The filters were then baked at 90 ° C. for 1 hour. 7. Hybridization

The hybridization is based on the binding of complementary, single stranded nucleic acids. For this purpose, 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. In the hybridization, 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.

Execution :

First the DNA filter in hybridization solution (0.5 M NaPi

(pH 7.2); 7% SDS; 0.2% BSA; 0.2% PEG 6000; 0.05% polyvinyl pyrrolidone 360,000; 0.05% Ficoll 70000; Prehybridize 0.5% dextran sulfate) at 0.1 ml / cm ² at 65 ° C. Incubate the filters in a plastic box in a shaking water bath for at least 1 h at 65 ° C. Discard the prehybridization solution. The radioactively labeled sample (see 4 and 5 above) with 0.5 ml / cπr hybridization solution

Apply (65 ° C) to the filters. The activity of the sample should not fall below 50 cpm, measured at a distance of 40 cm. Hybridization takes place overnight at 65 ° C. (human cDNA library) or 55 ° C. (interspecies hybridizations human-mouse and for the isolation of the homologous genes). The filters twice for 30 min with about 500 ml wash buffer in a shaking bath at 65 ° C

Wash (55 ° C). An autoradiography was then carried out. 8. Autoradiography

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.

With the help of the above-mentioned techniques, the complete mRNA coding for the protein of the T gene could be isolated. Furthermore, using cDΝA 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.

EXAMPLE 2: Northern Blot

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. For the T3 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.

a) Random priming:

For a typical labeling approach, dissolve 100 ng DNA in a volume of 12 μl. Heating for 10 minutes at 95 ° C causes the denaturation of the DNA in single strands. Approach on ice to reassociate the two complementary To prevent DNA strands. Complete the reaction mixture with 4 μl OLB, 1 μl Klenow (1U) as well as 2.5 μl a- ³² P- dCTP and 2.5 μl a- ³² P- dATP. Incubate overnight at room temperature. During this time, the complementary strand, starting from the hexanucleotides attached to a single strand, takes place through the Klenow fragment of E. coli DNA polymerase I. The DNA is radiolabelled by incorporating the a- ³² P-dCTP and the a- ³² P-dATP.

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. 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 radioactive labeling approach. Add 320 μl TE. Discard the solution that has passed through the column. Place the Eppendorf tube under the column. Add 350 μl TE. Collect the radioactive solution that has passed through the column.

b) Hybridization:

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

Incubated for 6 h at 65 ° C.

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.

Water) in a shaking bath at 65 ° C.

c) Autoradiography:

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.

The results of the Northern blots performed are shown in FIG. 23.

EXAMPLE 3: RNA in situ hybridization

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 μm 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 / μg. For this purpose, 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. For hybridization, 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.

The results of the RNA in situ hybridization are shown in Figs. 25, 26, 27 and 28.

25: Expression of the mouse T gene during mouse embryogenesis. Bright field (a, c, e, g) and dark field (b, d, f, h) images of horizontal (a, b) and sagittal sections (ch) through a 10.5 (a, b), 12.5 ( c, d), 14.5 (e, f) and 16.5 (g, h) dpc embryo (dpc = days post coneeptionem) which were hybridized with an antisense riboprobe of the mouse T gene, dec = Decidua , g = intestine, he = heart, lab = labyrinth, li = liver, me = myelcephalon, sc = spinal cord, sga = spinal ganglia, sb = tooth bud, te = telencephalon. Bar = 1 mm

Fig. 26: Expression of the mouse T gene in the postnatal brain. Bright field (a, d) and dark field images (b, c, e, f) of horizontal sections through a 1 wpn (weeks post natalis) and 6 wpn head, which were combined with a T-gene antisense (b, e) and sense sample ( c, f) were hybridized, ce = cerebellum, cor = cortex, cos = Colliculuε, ey = eye, hi = hippocampus, ne nasal epithelium, whether = olfactory bulb. Bar = 1 mm

Fig. 27: Magnification of the 10.5 dcp embryo of Fig. 25 a, b. The arrows point to an area of low expression in the somites (arrows in b). A strong expression can be seen in the region between mesencephalon and telencephalon ("Forbrain-Midbrain-Junction"). Aod = dorsal aorta, me = mesencephalon, sc = spinal cord, te = telencephalon. Bar = 100

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, vn = ventricle. Bar = 100 μm.

EXAMPLE 4: Preparation of Antibodies

Animals are immunized with a synthetically produced peptide of the sequence "EKGEDPETRRMRTVKNIAD" to generate antibodies against the T protein as follows:

Immunization protocol for polyclonal antibodies in the

Rabbits

600 μg of purified KLH are coupled per immunization

Peptide in 0.7 ml PBS and 0.7 ml complete or incomplete Freund's adjuvant used.

Day 0: 1st immunization (complete Freund's adjuvant) Day 14: 2nd immunization (incomplete Freund's adjuvant; icFA)

Day 28 3rd Immunization (icFA) Day 56 4th Immunization (icFA) Day 80 Bleed

The rabbit's serum is tested in an immunoblot. For this purpose, 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. For this purpose, 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 (36μM 5 'bromo-4-chloro-3-indolylphosphate, 400μM nitroblue tetrazolium, 100mm Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl ₂ ) at room temperature until bands become visible.

It turns out that polyclonal antibodies according to the invention can be produced.

Immunization protocol for polyclonal antibodies in chicken 100 μg of purified KLH-coupled peptide in 0.8 ml of PBS and 0.8 ml of complete or incomplete Freund's adjuvant are used per immunization. Day 0. 1. Immunization (complete Freund's adjuvant)

Day 28: 2nd immunization (incomplete Freund's adjuvant; icFA)

Day 50: 3rd immunization (icFA)

Antibodies are extracted from egg yolk and tested in a Western blot. Polyclonal antibodies according to the invention are detected.

Immunization protocol for mouse monoclonal antibodies Pro immunization uses 250 μg of purified KLH-coupled peptide in 0.25 ml of PBS and 0.25 ml of complete or incomplete Freund's adjuvant; at the 4th immunization the peptide is dissolved in 0.5 ml (without adjuvant).

Day 0. 1. Immunization (complete Freund's adjuvant)

Day 28: 2nd immunization (incomplete Freund's adjuvant; icFA)

Day 56: 3rd immunization (icFA)

Day 84: 4. Immunization (PBS) Day 87: Fusion

Supernatants from hybridomas are tested in a Western blot. Monoclonal antibodies according to the invention are detected.

EXAMPLE 5: Immunohistochemical Assays

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.

1st day

Section thickness 6-10 μm, usual fixation on slides,

Storage up to about 2 months in -80 ° C Take the cuts out the evening before and let them dry overnight at RT

Rinse slides in PBS, tip over, rinse again, then

Let stand in PBS for 10 min, remove slides and around the tissue

Wipe off the liquid with a cloth.

Circle with PAP-PEN (protein glycerin; Dako) that none

Liquid can run out more.

Add 100 μl peroxidase blocking solution (from Dako, Hamburg), incubate for 20 min

Rinse slides in PBS, tip, rinse again, then

Let stand in PBS for 10 min.

Remove the slide and the tissue around the tissue

Wipe off the liquid with a cloth. Dilute normal (sheep) serum 1:10 with PBS (e.g. sheep Dako

X0503, from Dako Hamburg), add 100 μl of it and incubate for 20 min

Rinse slides in PBS, tip over, rinse again, then

Let stand in PBS for 10 min, remove slides and around the tissue

Wipe off the liquid with a cloth.

Add first antibody in a dilution of 1: 100.

Add 100 μl of first antibody (in PBS) and incubate in the refrigerator in a moist chamber overnight. Control: without first antibody

2 day

Take the moist chamber out of the refrigerator and let it stand at RT. Rinse slides in PBS, tip over, rinse again, then leave to stand in PBS for 10 min, if many slides are analyzed, wash twice with PBS.

Remove the slide and wipe the liquid around the tissue with a cloth.

Dilute the second antibody 'Antirabbit-Biotinylated' 1: 100 (Amersham, Braunschweig) in PBS and add 100 μl. Incubate for 45 min in a moist chamber at RT. Rinse slides in PBS, tip over, rinse again, then leave to stand in PBS for 10 min.

Remove the slide and wipe the liquid around the tissue with a cloth.

Dilute streptavidin peroxidase (Streptavidin-horseraddish) (from Amersham, Braunschweig) 1: 100 with PBS and add 100 μl.

Incubate for 45 min in a moist chamber at RT. Rinse slides in PBS, tip over, rinse again, then leave to stand in PBS for 10 min.

Remove the slide and wipe the liquid around the tissue with a cloth.

Staining: A drop of chromogen per ml of buffer shortly before

Admit usage. Vortex and darken.

Add 100 μl of staining solution (Dako, Hamburg). Finally the

Color control. Incubate for approximately 2 minutes.

Incubate slides in water. Look under the microscope.

Place 1-2 drops of Crystal Mount on the cut. If there is an air bubble, vacuum with a tissue. The rest of the slide is wiped with HCl-EtOH to remove the color.

Put a line of glue (eu putty) on the cover gals Press the coverslip onto the slide without creating air bubbles.

The enzyme on the second antibody leads to the formation of a dye (DAB), whereby the T protein can be detected.

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.

Claims

claims

1. DNA sequence which encodes a protein which is involved in the development 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:

(a) the DNA sequence of FIGS. 1, 2, 3,

4, 5, 6, 7 or 8;

(b) the DNA sequence of Figure 9 or 10;

(c) the DNA sequence of Figure 11;

(d) the DNA sequence of Figure 12 or 13; (e) the DNA sequence of FIG. 14 or 15,

(f) the DNA sequence of Fig. 16,

(g) the DNA sequence of Fig. 17 or 18, (h) the DNA sequence of Fig. 19,

(i) a DNA sequence hybridizing with (a), (b), (c), (d), (e), (f), (g) or (h);

(j) Fragments, variants, functional equivalents, derivatives or precursors of the DNA sequence of (a), (b), (c), (d), (e), (f), (g), (h) or (i); or (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.

2. DNA sequence according to claim 1, which encodes a protein or peptide which contains the amino acid sequence of FIGS. 1, 9, 11, 12, 13, 14, 15, 15 16, 17, 18 or 19, wherein the protein or peptide has the biological activity defined in claim 1.

3. Antisense RNA, characterized in that it is complementary to the DΝA sequence of claim 1 or 2 and may reduce or inhibit the synthesis of the protein encoded by this DNA sequence.

Ribozyme, characterized in that it is complementary to the DNA sequence of claim 1 or 2 and can specifically bind to and cleave the RNA transcribed from this DNA sequence, thereby reducing or inhibiting the synthesis of the protein encoded by this DNA sequence becomes.

5. Expression vector containing the DΝA sequence according to claim 1 or 2 or the antisense RNA according to claim 3 or the ribozyme according to claim 4.

6. Expression vector according to claim 5, which additionally contains the promoter of the human T gene or an orthologist of the T gene.

7. Expression vector according to claim 5 or 6, which the T, T2 or T3 protein or fragments thereof in the form of a

Reporter fusion protein encoded.

8. host cell transformed with the expression vector according to any one of claims 5-7.

9. Protein which is encoded by the DΝA sequence according to claim 1 or 2 and which is involved in the development of the nervous system and is expressed in a tissue- and development-specific manner or fusion proteins, fragments, variants, derivatives or precursors of the protein.

10. Protein according to claim 9, which has one of the following motifs:

Motif 1 (A, T) (I, P, V) (L, T) (G, A, Q) (L, V) XXX (L, V) Motif 2 IYTDWAN Motif 3 AXXXXXXXXXGXXXXXXAXXXXXXXXXXXXXXXXXXXXQ Motif 4 SXXXXDX (12, 20 ) KX (17, 22) AXXXXXXXXL style 5 IYTDWANXXLX (K, R) Motif 6: KX (18.21) AXXXXXXXXLX (15.24) S Motif 7: NX (3.11) SXXXAXXXXXXXL

with: X = each amino acid (A, T) = amino acid A or T at this point

X (number 1, number 2) = number 1 to number 2 X's at this point

11. A method for producing the protein of claim 9, which comprises culturing the host cell of claim 8 under suitable conditions and recovering the protein from the cell or the growth medium.

12. Antibody directed against the protein of claim 9, or fragment thereof.

13. The antibody of claim 12, which is obtained by immunizing animals with a peptide having the sequence "EKGEDPETRRMRTVKNIAD".

14. Use of the DNA sequence according to claim 1 or 2, the antisense RNA according to claim 3, the ribozyme according to claim 4, the expression vector according to one of claims 5-7, the protein according to claim 9 or the antibody or the fragment thereof Claim 12 or 13 for the prevention or treatment of diseases of the nervous system, in particular the CNS.

15. Use according to claim 14, wherein the disease of the nervous system is a tumor disease of the CNS.

16. Use according to claim 14, wherein it is in the treatment of diseases of the nervous system to promote neuronal regeneration in injuries to the nervous system and in degenerative diseases of the

Nervous system hand11.

17. Use according to claim 14, wherein it is the Treatment of diseases of the nervous system involves the restoration of the neural connections and the restoration of congenital and acquired malfunctions of the nervous system.

18. Use according to claim 15 for inhibiting the growth and spread of tumor cells.

19. Diagnostic method for the detection of a disturbed expression of the protein according to claim 9 or for the detection of an altered form of this protein, in which a sample with the DNA sequence according to claim 1 or 2 or the antibody or the fragment thereof according to claim 12 or 13 in Touches and then 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.

20. Diagnostic kit for performing the method according to claim 19, which contains the DNA sequence according to claim 1 or 2 and / or the antibody or the fragment thereof according to claim 12 or 13.

21. Non-human mammal, the naturally occurring T, T2 or T3 gene of which is a change in

Has gene structure or the gene sequence.

22. Non-human mammal, wherein a change in the gene structure of the T, T2 or T3 gene in the mammal is achieved by the introduction of a deletion, in the place of which a homologous or heterologous sequence is introduced.

23. Non-human mammal, wherein a change in the gene structure of the T, T2 or T3 gene is achieved by inserting a homologous or heterologous sequence into the corresponding gene naturally occurring in the mammal.

24. The non-human mammal of claim 22 or 23, wherein the heterologous sequence is a selection marker sequence.

25. The non-human mammal of claim 24, wherein the selection marker sequence mediates resistance to neomycin.

26. A method for producing a non-human mammal according to any one of claims 21-25, characterized by the following steps:

(a) Production of a DNA fragment, in particular a vector, containing an altered T, T2 or T3 gene, the T, T2 or T3 gene being replaced by

Insertion of a heterologous sequence, in particular a selectable marker, has been changed;

(b) preparation of embryonic stem cells from a non-human mammal (preferably mouse); (c) Transformation of 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), (d) culturing the cells from step (c) ,

(e) selection of the cultured cells from step (d) for the presence of the heterologous sequence, in particular the selectable marker,

(f) Generating chimeric non-human mammals from the cells of step (e) by injection

Cells in mammalian blastocysts (preferably mouse blastocytes), transferring the blastocysts into pseudo-pregnant female mammals (preferably mouse) and analysis of the progeny obtained for a change in the T, T2 or T3 gene.

27. A transgenic cell or tissue capable of producing a T protein or part of the T protein To express orthologes of it.

28. Use of the non-human mammal according to any one of claims 21-25 or the transgenic cell or transgenic tissue according to claim 27 for the analysis of the

Function of the T gene family.

29. Use of the non-human mammal according to one of claims 21-25 or the transgenic cell or the transgenic tissue according to claim 27 for the identification of inhibitors and enhancers of the T gene family.

30. Vertebrate gene as well as functional equivalent, derivative or a bioprecursor thereof, which code for a protein that is statistically significant

Amino acid sequence homology to the T gene, T2 gene or T3 gene according to one of the figures FIG. 1, 9, 11, 12, 13, 14, 15, 16, 16 17, 18 or 19.

31. T-Gen and its Ver br t e n o r t h o 1 o g e and vertebrate paralogues encoding a nuclear pore protein.

32. Vertebrate protein which has an amino acid sequence according to FIG. 1 or an amino acid sequence which differs from that

Amino acid sequence in Figure 1 differs in one or more amino acids.

33. Vertebrate T, T2 or T3 gene and the protein encoded therein, in all its naturally occurring allelic and mutant forms.

34. Medicament containing a protein according to claim 9 or a functional equivalent, a fragment or a bioprecursor thereof in combination with a pharmaceutically suitable carrier.

35. Methods of identification of substances that have a reinforcing or inhibiting influence on the action of T-protein, T2-protein or T3-protein, by means of

Determination of the bidirectional transport through the nuclear pores, determination of the binding to filaments of the cell (e.g.

Actin filaments and microtubules), or determining the increased or decreased transcription of cellular or reporter genes.

36. Method for the identification of substances which have a reinforcing or inhibiting influence on the action of proteins which are functionally linked to the T-protein in a direct or indirect manner, or which represent parallel signal or function paths, by means of - determination of the bidirectional transport through the

Nuclear pores,

Determination of the p h o r y 1 i e r u n g and dephosphorylation of proteins,

Determination of the binding of the T protein to filaments of the cell (e.g. actin filaments and microtubules), or

Determination of the increased or decreased transcription of cellular or reporter genes.

37. The method according to claim 35 or 36, wherein the altered transcription with reporter molecules, preferably the occurrence of certain mRNAs or the EGFP protein, is detected.

38. A method for identifying further proteins that 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 according to claim 9,

(b) contacting a cell extract with the antibody and identifying the antibody / protein complex, (c) analyzing the complex to identify a protein that has bound to the protein of the complex and is not an antibody, and

(d) optionally repeating steps (a) to (c) in order to identify further proteins of this function.