WO2000008143A2

WO2000008143A2 - Gene isolated on the short arm of human chromosome 17

Info

Publication number: WO2000008143A2
Application number: PCT/DE1999/002479
Authority: WO
Inventors: Peter Seranski; Annemarie Poustka; Hans Lehrrach; Uwe Radelof
Original assignee: Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts; Max-Planck-Institut Für Molekulare Genetik
Priority date: 1998-08-07
Filing date: 1999-08-06
Publication date: 2000-02-17
Also published as: AU6462999A; WO2000008143A3; DE19835910C1

Abstract

The present invention relates to DNA molecules containing information for a human gene that was isolated on the short arm of chromosome 17. The invention also relates to vectors containing said DNA molecules. In one embodiment, said vectors are suitable for expression of the inventive DNA molecules in prokaryotes or eukaryotes. The invention further relates to medicaments and diagnostic compositions containing said DNA molecules or vectors, the protein coded thereby or an antibody that specifically recognizes said protein.

Description

Gene isolated on the short arm of human chromosome 17

The present invention relates to DNA molecules containing the information for a human gene isolated on the short arm of chromosome 17. The present invention further provides those containing DNA molecules

Vectors ready, wherein in a preferred embodiment these vectors are suitable for expressing the DNA molecules according to the invention in prokaryotes or eukaryotes. Furthermore, the present invention relates to medicaments and diagnostic compositions which contain the above DNA molecules or vectors, the protein encoded thereby or an antibody which specifically recognizes this protein.

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly. The patients affected by this suffer from mental retardation, size growth disorders, abnormalities in the REM sleep phase and abnormal behavior (Hamill et al., Ann. Gent. 31, pp. 36-38 (1990)). It has now been found that SMS is based on an internal deletion / mutation in the p11.2 region of chromosome 17. In general, it was found that other mutations in this region also lead to neurodegenerative diseases, which are partly accompanied by the above-mentioned symptoms.

Since the genes in this region are not yet fully known, the molecular mechanisms leading to the above symptoms, especially in connection with Smith Magenis syndrome, have not yet been elucidated.

The invention is therefore essentially based on the technical problem of creating the molecular basis for elucidating neurodegenerative diseases which produce symptoms which are similar or the same as in Smith's gastric syndrome and for providing suitable diagnostics and / or therapy therefor. This technical problem has been solved by providing the embodiments characterized in the patent claims. A DNA molecule related to SMS could be isolated and identified.

The identification of a gene (HSGT1) that is associated with partial or complete deletion with SMS is of interest, since this helps to clarify the molecular pathogenesis of SMS and thus improved therapies. At the same time, the cloning of the SMS gene forms the basis for the development of diagnostic tests in order to ensure a more reliable and early diagnosis of those affected in the future. In addition, functional analyzes of the protein will undoubtedly contribute to an understanding of neuron development, since the gene is located in a region in the genome (human chromosome 17p11.2; see FIG. 4) that is associated with various neurological diseases. It is therefore considered a candidate gene for studies of the

To look at pathomechanisms that underlie neurodegenerative diseases and developmental disorders in the differentiation of the central nervous system.

Thus, the present invention relates to a DNA molecule (HSGT1) which comprises:

(a) the nucleic acid of FIG. 1, or a DNA or a fragment thereof which differs from it by one or more base pairs, (b) a DNA which hybridizes with the nucleic acid of (a), or

(c) DNA related to the nucleic acid of (a) or (b) via the degenerate genetic code.

The nucleic acid molecules defined under (a) and (c) code for proteins, polypeptides or peptides which still have at least one of the biological activities described below of the protein encoded by the nucleic acid according to FIG. 1, for example as a differentiation factor in neuronal Development and / or intermediary filament in neurons, or whose change or failure leads or can lead to SMS.

The DNA molecules defined under (a) also include DNA molecules which differ from the sequence given in FIG. 1 by deletion (s), insertion (s),

Exchange (s) and / or other modifications known in the art, e.g. alternative splicing, distinguish or comprise a fragment of the original nucleic acid molecule, the protein encoded by these DNA molecules also having one or more of the biological activities described above. This also includes allele variants. 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., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989) . 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 one of the biological activities of HSGT1. This can be done, for example, by examining whether the modified protein takes on the same functions in cell culture models as the wild-type HSGT1 protein.

The term “hybridizing DNA” refers to a DNA that hybridizes with a DNA of (a) under customary conditions, in particular at 20 ° C. below the melting point of the DNA. The term "hybridize" refers to conventional hybridization conditions, preferably to hybridization conditions in which 5xSSPE, 1% SDS, IxDenhardts solution is used and the hybridization temperatures between 35 ° C and 70 ° C, preferably at 65 ° C lie. 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 ., supra). Stringent hybridization conditions, as described, for example, in Sambrook et al., Supra, are particularly preferred. In a particularly preferred embodiment, the DNA molecule according to the invention is a cDNA.

In a further preferred embodiment, the DNA molecule according to the invention is a genomic DNA which is preferably derived from a mammal, for example a human. Screening methods based on nucleic acid hybridization permit the isolation of the genomic DNA molecules according to the invention from any organism or derived genomic DNA banks, wherein probes are used which contain the nucleic acid sequence shown in FIG. 1 or a part thereof. The genomic DNA, for example from a human, can be used to identify further mutations which are involved in the development of neurodegnerative diseases, in particular SMS, for example mutations which lead to amino acid exchanges, inhibition of splicing or faulty splicing.

Interestingly, a polymorphism was found in the HSGT1 gene, which is located in the protein-coding region of the gene and which codes for a polyglutamine accumulation, i.e. there is a variable repetition of the sequence CAG (see FIG. 1). Polyglutamine accumulations are already in various genes related to neuropathological diseases, e.g. Chorea

Huntington.

The nucleic acids according to the invention can also be inserted into a vector or expression vector. Thus, the present invention also encompasses vectors containing these nucleic acid molecules. Examples of this are the

Known specialist. In the case of an expression vector for E. coli, these are e.g. pGEMEX, pUC derivatives (e.g. pUC8), pBR322, pBlueScript, pGEX-2T, pET3b and pQE-8. For expression in yeast e.g. to call pY100 and Ycpadl, while for expression in animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 must be specified. The is particularly suitable for expression in insect cells

Baculovirus expression vector pAcSGHisNT-A. In a preferred embodiment, the nucleic acid molecule according to the invention is in the vector with regulatory Functionally linked elements that 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 allow the phenotypic selection of a transformed host cell. The regulatory elements for the

Expression in prokaryotes, for example E. coli, include the lac, trp promoter or T7 promoter, and for expression in eukaryotes the AOX1 or GAL1 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 metallothionein I and the polyhedrin promoter.

Suitable vectors include in particular T7-based expression vectors for expression in bacteria (Rosenberg et al., Gene 56 (1987), 125) or pMSXND for expression in mammalian cells (Lee and Nathans, J.Biol.Chem. 263 (1988) , 3521).

In a preferred embodiment, the vector containing the DNA molecules according to the invention is a virus, for example an adenovirus, vaccinia virus or an AAV virus, which is useful in gene therapy. Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV,

RSV or GaLV. For the purposes of gene therapy, the DNA molecules according to the invention can also be transported to the target cells in the form of colloidal dispersions. These include, for example, lipososms or lipoplexes (Mannino et al., Biotechniques 6 (1988), 682).

General methods known in the art can be used to construct expression vectors which contain the DNA molecules according to the invention and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as are described, for example, in Sambrook et al., Supra. The present invention also relates to host cells containing the vectors described above. These host cells include bacteria, yeast, insect and animal cells, preferably mammalian cells. The E. coli strains HB101, DH1, x1776, JM101, JM109, BL21, XLIBIue and SG 13009, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero, HeLa and the insect cells sf9 are preferred. 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.

A clone which codes for a nucleic acid according to the invention was obtained from DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, MascheroderWeg 1b, 38124 Braunschweig in accordance with the Budapest Treaty under the deposit number DSM 12357 (filing date: July 31, 1998) or DSM 12472 (deposit date: September 16, 1998) [= E. coli / PSB8.7-l 13281].

The present invention also relates to a method for producing a protein which is encoded by the above nucleic acid, comprising the cultivation of the host cells described above under conditions which allow the expression of the

Allow protein (preferably stable expression), and recovery of the protein from the culture. Suitable methods for the recombinant production of the protein are generally known (see for example Holmgren, Annu.Rev.Biochem. 54 (1985), 237; LaVallie et al., Bio / Technology 11 (1993), 187; Wong, Curr.Opin.Biotec - h.6 (1995), 517; Romanos, Curr.Opin.Biotech. 6 (1995), 527; Williams et al., Curr.

Opin. Biotech. 6 (1995), 538; and Davies, Curr. Opin.Biotech.6 (1995), 543. Suitable purification methods (for example preparative chromatography, affinity chromatography, for example immunoaffinity chromatography, HPLC etc.) are also generally known.

In a further embodiment, the present invention relates to a DNA molecule (HSGT1) encoded according to the invention or according to the protein obtained in the current process. The amino acid sequence of such a protein is shown in Fig. 2. In this connection it should be mentioned that the protein according to the invention can be modified according to customary methods known in the art. These modifications include exchanges, insertions, or deletions of amino acids that modify the structure of the protein while essentially maintaining its biological activity. The exchanges preferably include "conservative" exchanges of amino acid residues, ie exchanges for biologically similar residues, for example the substitution of a hydrophobic residue (for example isoleucine, valine, leucine, methionine) for another hydrophobic residue, or the substitution of one polar residue for another polar residue (eg arginine against lysine, glutamic acid against aspartic acid etc.). Deletions can lead to the generation of molecules which are significantly smaller in size, ie which lack amino acids at the N or C terminus, for example. In order to still perform the function of a protein according to the invention, one should be added to the sequence of FIG. 2 at the amino acid level

Homology of at least 50% is present.

The present invention also relates to antibodies which specifically recognize the protein HSGT1 described above. The antibodies can be monocional, polyclonal or synthetic antibodies or fragments thereof, for example Fab, Fv or scFv fragments. These are preferably monocional antibodies. For the production it is favorable to immunize animals, in particular rabbits or chickens for a polyclonal and mice for a monoclonal antibody, with an above (fusion) protein or fragments thereof. Further "boosters" of the animals can be carried out with the same (fusion) protein or fragments thereof. The polyclonal antibody can then be obtained from the serum or egg yolk of the animals. The antibodies according to the invention can be produced according to standard methods, the protein encoded by the DNA molecules according to the invention or a synthetic fragment thereof serving as an immunogen. Monocional antibodies can be produced, for example, by the method described by Köhler and Milstein (Nature 256 (1975), 495) and Galfre (Meth. Enzymol. 73 (1981), 3). where mouse myeloma cells are fused with spleen cells derived from immunized mammals. These antibodies can be used, for example, for immunoprecipitation of the proteins discussed above or for the isolation of related proteins from cDNA expression banks. 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 further relates to the use of the DNA molecules, vectors, proteins and / or antibodies described above. These are preferably used for the production of a medicament for the prevention or treatment of neurodegenerative diseases, in particular SMS. The drug can be used in gene therapy, the methods or vectors described above for introducing the inventive

DNA molecules can be used. On the other hand, the protein encoded by the DNA molecules according to the invention can be administered directly so as to provide sufficient amounts of the biologically active protein in cells which no longer have functional copies of the gene according to the invention. The medicament according to the invention is preferably used when biologically active protein is not formed or is formed in too small amounts in the patient concerned. There are indications that this gene plays a role in the development of autism.

These drugs 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, wetting agents, sterile solutions, etc. The medicaments can be administered orally or parenterally. Methods for parenteral administration include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal or intranasal administration. 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 present invention further relates to a diagnostic composition which contains the above-described DNA molecule or the antibody or combinations thereof, optionally together with a suitable detection means.

The DNA molecule according to the invention, as defined above, can also be used as a probe to isolate DNA molecules which originate, for example, from another species or another organism and encode a protein with the same biological activity. For this purpose, the probe preferably has a length of at least 10, particularly preferably at least 15 bases. Suitable detection methods based on hybridization are known to the person skilled in the art. Suitable labels for the probe are also known to the person skilled in the art and include, for example, labeling with radioisotopes, bioluminescent, chemiluminescent, fluorescent labels, metal chelates, enzymes, etc.

In addition, this can also by a PCR (Wiedmann et al., PCR Methods Appl. 3, pp. 551-564 (1994); Saiki et al., Nature 324, pp. 163-166 (1986)) or "Ligase chain reaction "(LCR) (Taylor et al., Curr. Opin. Biotechnol. 6, pp. 24-29 (1995); Rouwendal et al., Methods Mol. Biol. Pp. 149-156 (1996)), the Primers are derived from the sequence in FIG. 1 and suitable primers (in terms of length, complementarity to the matrix, the area to be amplified, etc.) can be designed by a person skilled in the art according to customary methods.

The methods described above also allow the identification of others

Mutations that are related to neurodegenerative diseases, with the sequence of a patient with SMS or related symptoms healthy patients is compared.

In addition, the present invention relates to a method for diagnosing neurodegenerative diseases, in particular SMS, in vitro, comprising the following steps:

Isolation of nucleic acid from the patient, performing an LCR or PCR with suitable primers or a hybridization analysis with one or more suitable probes,

Comparison of the lengths of the amplified or hybridizing fragments with the lengths of control fragments from the non-mutated HSGT1 gene, or sequencing of the amplified fragments and comparison with the sequence of control fragments from the non-mutated

HSGT1 gene, wherein length differences or mutations which lead to substitutions, deletions or additions of amino acids or to premature termination of translation are indicative of neurodegenerative diseases, in particular SMS.

Alternatively, the diagnosis can also be made by isolating lymphoblasts and producing metaphase preparations which are subjected to a FISH analysis in order to determine whether certain areas of the genome are present at all.

In the above-mentioned method, methods known to the person skilled in the art regarding the preparation of DNA or RNA from biological samples, the restriction digest of the DNA, the separation of the restriction fragments on size-separating gels, for example agarose gels, the preparation and

Labeling of the probe and detection of the hybridization, for example via "Southem blot"; be applied. The above detection can also be carried out using PCR or LCR. Primers are used which flank the sequence according to the invention or suitable subregions. Diagnostically important are amplification products of DNA from the tissue in question, which differ, for example in terms of their length and / or sequence, from the amplification products of DNA from healthy tissue.

In an alternative embodiment, a method can be used which comprises the following steps:

Isolation of RNA from the patient,

Performing a Northem analysis with one or more suitable probes,

Comparison of the concentration and / or length of HSGT1 mRNA of the patient sample with an mRNA of a healthy person, with length differences or the absence or a lower concentration of the HSGT1 mRNA (for example due to mutations in regulatory sequences which are related to transcription) in comparison to the control mRNA is indicative of a neurogenerative disease, in particular for SMS.

In this method, methods known to the person skilled in the art can be used with regard to the preparation of total RNA or poly (A) + RNA from biological samples, the separation of the RNAs on size-separating gels, for example denaturing agarose gels, the production and labeling of the probe and the detection be applied via "Northern blot".

In a further alternative embodiment, a possible disease can also be diagnosed by a method which comprises the following steps:

Obtaining a cell sample from the patient, Bringing the cell sample thus obtained into contact with the above-described antibody according to the invention as a probe under conditions which allow the binding of the antibody, non-occurring or weak binding of the antibody indicating a lack of or decreased expression of HSGT1, which is indicative of a disease.

This detection can also be performed using standard techniques known to those skilled in the art. These are also known cell disruption methods which allow the isolation of the protein in such a way that it can be brought into contact with the antibody. The detection of the bound antibody is preferably carried out via immunoassays, for example Western blot, ELISA or RIA.

The diagnostic methods according to the invention can also be carried out as prenatal diagnostic methods according to techniques known to the person skilled in the art.

Finally, the present invention relates to kits for carrying out the diagnostic methods according to the invention which contain the antibody according to the invention or a fragment thereof, a DNA molecule according to the invention as a probe or for

PCR or LCR suitable primer pair based on the sequence of the DNA molecule according to the invention, optionally in combination with a suitable detection means.

Depending on the design of the kit to be carried out with the kit according to the invention

In the diagnostic method, the DNA molecules, antibodies or fragments thereof contained in the kit can be immobilized on a suitable carrier.

The invention will now be further described with reference to the figures, which show:

Fig. 1: Nucleic acid sequence of the cDNA of the HSGT1 gene

The CAG trinucleotide repeat is shown in bold. The primer bin Parts of the PCR amplification that amplify a polymorphism are underlined.

Fig. 2: Amino acid sequence derived from Fig. 1

Figure 3: Northern blot analysis using RNAs from different tissues. An RT-PCR product from fetal brain RNA from the 5 'end including the polymorphic repeats was used as the hybridization sample.

Fig. 4: Genome map with the exact location of the HSGT1 gene

The invention will now be described below with reference to the examples.

example 1

Northern blots

Northern blots with RNAs from various tissues were overnight at 42 ° C in a hybridization buffer with 2xSSC, Denhardt's solution, formamide and herring sperm DNA and the specific probe (RT-PCR product from fetal brain RNA from the 5'- Hybridized at the end including the polymorphic repeat). The

Filters were washed two to three times at 65 ° C in 2xSSC / 0.1% SDS and exposed to a Kodak film or Fuji film at -70 ° C for 24 to 48 hours.

The result of the Northern blot is shown in Fig. 3. It can be seen that the HSGT1 gene has an 8 kB transcript and is ubiquitously weakly expressed.

Example 2: Production and purification of an HSGT1 protein according to the invention

To produce an HSGT1 protein according to the invention, the DNA from FIG. 1 is provided with Barn HI linkers, cut with Barn HI and cut into Barn Hl cleaved expression vector pQE-8 (Diagen) inserted. The expression plasmid pQ / HSGT1 is obtained. Such a code for a fusion protein of 6 histidine residues (N-terminus partner) and the HSGT1 protein according to the invention from FIG. 2 (C-terminus partner). pQ / HSGT1 is used to transform E.coli SG 13009 (see. Gottesmann, S. et al., J. Bacteriol. 148, (1981), 265-273). The bacteria are cultivated in an LB medium with 10 μg / ml ampicillin and 25 μg / ml kanamycin and induced for 4 h with 60 μM isopropyl-β-D-thiogalactopyranoside (IPTG). By adding 6 M guanidine hydrochloride, the bacteria are lysed, and then the lysate is used for chromatography (Ni-NTA resin) in the presence of 8 M urea in accordance with the instructions of the

Manufacturer (Diagen) of the chromatography material performed. The bound fusion protein is eluted in a pH 3.5 buffer. After neutralization, the fusion protein is subjected to an 18% SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue (cf. Thomas, J.O. and Kornberg, R.D., J. Mol.Biol. 149 (1975), 709-733).

It has been shown that a (fusion) protein according to the invention can be produced in highly pure form.

Example 3:

Production and detection of an antibody according to the invention

A fusion protein according to the invention from Example 3 is subjected to an 18% SDS polyacrylamide gel electrophoresis. After staining the gel with 4 M sodium acetate, an approximately 200 kD band is cut out of the gel and in

Phosphate buffered saline incubated. Pieces of gel are sedimented before the protein concentration of the supernatant is determined by SDS-polyacrylamide gel electrophoresis, which is followed by a Coomassie blue staining. Animals are immunized with the gel-purified fusion sport as follows: Immunization protocol for polyclonal antibodies in rabbits

35 μg of gel-purified fusion protein in 0.7 ml of PBS and 0.7 ml of complete or incomplete Freund's adjuvant are used per immunization.

Day 0: 1st immunization (Freund's complete adjuvant)

Day 14: 2nd immunization (incomplete Freund's adjuvant; icFA)

Day 28: 3rd immunization (icFA)

Day 56: 4th immunization (icFA) Day 80: Bleeding

The rabbit's serum is tested in an immunoblot. For this purpose, a fusion protein according to the invention from Example 3 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). The Western Blot

Analysis is carried out as in Bock, C.-T. et al., Virus Genes 8, (1994), 215-229. For this purpose, the nitrocellulose filter is incubated with a first antibody at 37 ° C. for 1 h. 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 a monoclonal goat anti-rabbit IgG antibody (Dianova) (1: 5000) coupled with alkaline phosphatase in PBS. After 30 minutes of incubation at 37 ° C, there are several washing steps with PBS and then the alkaline phosphatase detection reaction with developer solution (36 μm 5 'bromo-4-chloro-3-indoiyl phosphate, 400 μM nitroblue tetrazolium, 100 mM tris HCl, pH 9.5, 100 mM NaCI, 5 mM MgCI ₂ ) at room temperature until bands become visible.

It turns out that polyclonal antibodies according to the invention can be produced. Immunization protocol for chicken polyclonal antibodies

40 μg of gel-purified fusion protein in 0.8 ml of PBS and 0.8 ml of complete or incomplete Freund's adjuvant are used per immunization.

Day 0: 1st immunization (Freund's complete adjuvant)

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

Day 50: 3. 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

For each immunization, 12 μg gel-purified fusion protein in 0.25 ml PBS and 0.25 ml complete or incomplete Freund's adjuvant are used; at the 4th immunization the fusion protein is dissolved in 0.5 ml (without adjuvant).

Day 0: 1st immunization (Freund's complete adjuvant)

Day 28 2nd immunization (Freund's incomplete adjuvant; icFA) Day 56 3rd immunization (icFA) Day 84: 4th immunization (PBS) Day 87: Fusion

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

Claims

claims

1. Nucleic acid coding for the protein HSGT1, the nucleic acid comprising:

(a) the nucleic acid of FIG. 1 or a nucleic acid or a fragment thereof that is different from one or more base pairs,

(b) a nucleic acid hybridizing with the nucleic acid of (a) or

(c) a nucleic acid related to the nucleic acid of (a) or (b) via the degenerate genetic code.

2. The nucleic acid of claim 1, being a cDNA or genomic DNA.

3. A vector comprising the nucleic acid of claim 1.

4. Vector according to claim 3, wherein the nucleic acid with regulatory

Functionally linked elements that allow expression in prokaryotic or eukaryotic host cells.

5. The vector of claim 3 or 4, which is an RNA virus or retrovirus.

6. transformant containing the expression plasmid according to claim 3.

7. The transformant of claim 6 which is a bacterium, a yeast, insect, animal or mammalian cell.

8. Protein comprising the amino acid sequence of FIG. 2 or a protein different therefrom by one or more amino acid exchanges or a fragment of these proteins.

9. A method for producing the protein according to claim 8, comprising culturing the transformant according to claim 6 or 7 under conditions which allow expression of the protein, and recovering the protein from the culture.

10. Antibody directed against the protein according to claim 8.

11. Use of the protein according to claim 8 as a reagent for the diagnosis and / or therapy of neurodegenerative diseases, in particular the Smith Magenis syndrome.

12. Use of the nucleic acid according to claim 1 or 2 as a reagent for the diagnosis and / or therapy of neurodegenerative diseases, in particular the Smith Magenis syndrome.

13. Use of the antibody according to claim 10 as a reagent for diagnosis and / or therapy of neurodegenerative diseases, in particular the Smith Magenis syndrome.