JP2000513577A - Fanconi gene▲I▼ - Google Patents

Abstract

(57) [Abstract] The present invention relates to pathophysiologically relevant genes associated with Fanconi anemia (FA), genes encoded thereby, antibodies against said polypeptides, and pharmaceutical uses of said nucleic acids, polypeptides, and antibodies.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The Fanconi Gene I The present invention relates to a pathophysiologically relevant gene associated with Fanconi anemia (FA), a polypeptide encoded thereby, antibodies against the polypeptide, and pharmaceutical uses of the nucleic acids, polypeptides, and antibodies. FA is a rare genetic disease characterized by progressive pancytopenia, congenital malformations, and a high risk of neoplastic disease (Glanz and Fraser, J. Med. Genet. 19 (1982) 412-416; Auerbach and Allen, Cancer Genet. Cytogenet. 51 (1991) 1-12). The disease affects approximately 1 in 300,000 people. Although the molecular basis of the disease is unknown, hypersensitivity to the clastogenic effects of DNA cross-linking agents is an excellent marker of the FA genotype (Auerbach and Wolmann, Nature 261 (1976) 494-496). Cells from patients with FA show numerous chromatid breaks and chromatid substitutions when contacted with concentrations of mitomycin C (MMC) or diepoxybutane (DEB) that have low clastogenic effects on normal cells (Sasaki and Tonomura, Cancer Res. 33 (1973), 1829-1836, and Auerbach, Exp. Hematol. 21 (1993), 731). Incubation of FA lymphocytes and fibroblasts with MMC reveals a delay in the G2 phase transition and a complete arrest, revealing defects in the G2 phase of the cell cycle (Kubbies et al., Am. J. Hum. Genet. 37(1985), 1022; Hoehn et al., Fanconi Anemia, Clinical, Cytogenic and Experimental Aspects (1989), Springer Verlag Berlin-Heidelberg; Seyschab et al., Blood 85(1995), 2233-2237). It is now known that there are at least five different complementation groups within the FA population (Duckworth-Rysiecki et al., Somatic Cell Mol. Genet. 11 (1985), 35; Strathdee et al., Nature 356 (1992), 763; Joenje et al., Blood 86 (1995), 2156-2160). Although genes for complementation group C have recently been reported (Strathdee et al., Nature 356 (1992), 763, and WO 93/22435), the type of molecular mechanism of action of the FA-C proteins remains unclear (Gavish et al., Am. J. Hum. Genet. 5 3 (1993), 685; Yamashita et al., Proc. Natl. Acad. Sci. USA 91 (1994), 6712; Youssoufian et al., J. Biol. Chem. 270 (1995), 9876-9882). Furthermore, two complementation groups, FAA and FAD, have been localized on the chromosome (Pronk et al., Nature Genet. 11 (1995), 338-340; Whitney et al., Nature Genet. 11 (1995), 341-343). The objective of the present invention was to identify novel genes involved in DNA regulatory cascades (e.g. cell cycle abnormalities, DNA repair, tumorigenesis/progression) and associated with the pathophysiological phenotype of Fanconi anemia. The present invention describes the identification, cloning, and characterization of a novel polypeptide-encoding gene, designated Fanconi gene I. The sequence of this gene was discovered using differential display (Liang and Pardee, Science 257 (199 2), 967-971) comparing normal and FA fibroblasts. Fanconi gene I is significantly more highly expressed in FA fibroblasts than in normal fibroblasts. Fanconi gene I, the polypeptide encoded thereby and antibodies against this polypeptide are suitable as agents for diagnosing, treating or preventing diseases directly or indirectly related to abnormalities in cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis and tumor progression. The subject of the present invention is a nucleic acid comprising: (a) the nucleotide sequence shown in SEQ ID NO: 1, or a protein-coding portion thereof; (b) a nucleotide sequence corresponding to the sequence of (a) within the scope of the genetic code degeneracy; or (c) a nucleotide sequence hybridizing to the sequence of (a) and/or (b) under stringent conditions. The nucleotide sequence shown in SEQ ID NO: 1 has an open reading frame corresponding to a polypeptide 72 amino acids in length. The amino acid sequence of this polypeptide is shown in SEQ ID NO: 2. Human cDNA clones 290799 (accession number N71961), 270393 (accession number N33066) and 66812 (accession number T64946), which contain partial regions of the nucleotide sequence shown in SEQ ID NO: 1, have been published in the EMBL EST sequence data bank. However, no complete open reading frame of the sequences shown or any information on possible biological functions has been given. In addition to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequences which correspond to this sequence within the scope of the degeneracy of the genetic code, the present invention also relates to nucleotide sequences which hybridize with one of the above sequences. In the present invention, the term "hybridization" is used in the same way as in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), pp. 1.101-1.104). Stringent hybridization preferably means that a positive hybridization signal is observed even after washing for 1 hour at 50°C, preferably 55°C, particularly preferably 62°C and most preferably 68°C with 1xSSC and 0.1% SDS, in particular after washing for 1 hour at 50°C, preferably 55°C, particularly preferably 62°C and most preferably 68°C with 0.2xSSC and 0.1% SDS. A nucleotide sequence which hybridizes under such washing conditions with the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence corresponding to this sequence within the scope of the degeneracy of the genetic code is a nucleotide sequence according to the invention. The nucleotide sequence according to the invention is preferably DNA. However, it may also include RNA or nucleic acid analogues such as peptidic nucleic acids. The nucleic acids according to the invention particularly preferably comprise the protein-coding part of the base sequence shown in SEQ ID NO:1 or a sequence which has a homology of more than 80%, preferably more than 90%, most preferably more than 95% with the base sequence shown in SEQ ID NO:1 or with a part of this sequence, preferably at least 20 bases long and particularly preferably at least 50 bases long. A further subject of the invention is a polypeptide encoded by the above-mentioned nucleic acid. This polypeptide preferably has (a) the amino acid sequence shown in SEQ ID NO:2 or (b) a homology of more than 70%, preferably more than 80%, particularly preferably more than 90% with the amino acid sequence shown in SEQ ID NO:2. The nucleic acids according to the invention can preferably be obtained from mammals, in particular from humans. They can be isolated by known techniques using short fragments of the base sequence shown in SEQ ID NO:1 as probes and/or primers for hybridization according to known methods. Furthermore, the nucleic acids according to the invention can also be prepared by chemical synthesis, in which modified base building blocks, such as for example 2'-0-alkylated base building blocks, can be used selectively instead of the normal nucleotide building blocks. Nucleic acids composed of partially or completely modified base building blocks can be used as therapeutic agents, for example as antisense nucleic acids or ribozymes. The invention also includes nucleic acid analogues, such as peptidic nucleic acids, whose base sequences correspond to the nucleic acids according to the invention. A further subject of the invention is a vector comprising at least one copy of the nucleic acid according to the invention. This vector can be any desired prokaryotic or eukaryotic vector, preferably one in which the DNA sequence according to the invention is under the control of expression signals (promoter, operator, enhancer, etc.). Examples of prokaryotic vectors are chromosomal vectors, such as bacteriophages, and extrachromosomal vectors, such as plasmids, with circular plasmids being particularly preferred. Suitable prokaryotic vectors are described, for example, in Sambrook et al., supra, chapters 1-4. The vector according to the invention is particularly preferably a eukaryotic vector, such as a yeast vector or a vector suitable for cells of higher organisms, such as a plasmid vector, a viral vector, a plant vector. Such vectors are known to those skilled in the art of molecular biology and will not be specified in more detail here. In this regard, reference is made in particular to Sambrook et al., supra, chapter 16. In addition to the polypeptide shown in SEQ ID NO:2, the present invention also relates to mutant proteins, variants and fragments thereof. These are understood as sequences which differ from the amino acid sequence shown in SEQ ID NO:2 by substitution, deletion and/or insertion of individual amino acids or short fragments of amino acids. The term "variants" includes naturally occurring allelic variants or splice variants of Fanconi polypeptide I, as well as proteins produced by recombinant DNA techniques, in particular by in vitro mutagenesis using chemically synthesized oligonucleotides, which essentially correspond to the protein shown in SEQ ID NO:2 in terms of biological and/or immunological activity. The term also includes chemically modified polypeptides. These include polypeptides whose termini and/or reactive amino acid side groups have been modified by acylation, such as acetylation or amidation. The present invention also relates to vectors comprising a portion of the sequence shown in SEQ ID NO:1, which is at least 20 nucleotides long. This part preferably has a base sequence taken from the protein coding region of the sequence shown in SEQ ID NO: 1 or from a region essential for the expression of the protein. These nucleic acids are particularly suitable for the production of antisense nucleic acids, preferably of up to 50 nucleotides, which can be used for therapeutic purposes. A further subject of the invention is a cell transformed with a nucleic acid according to the invention or with a vector according to the invention. This cell can be a eukaryotic or prokaryotic cell. The method of transforming a cell with a nucleic acid is common in the art and will not be specified in more detail. Examples of preferred cells are eukaryotic cells, in particular animal cells, particularly preferably mammalian cells. A further subject of the invention is the use of a polypeptide according to the invention, or a fragment of this polypeptide, as an immunogen for the production of antibodies. In this case, the antibodies can be produced in the usual manner by immunizing a laboratory animal with the full-length polypeptide or a fragment thereof and then isolating the resulting polyclonal antisera. Monoclonal antibodies can be obtained in the known manner from antibody-producing cells of a laboratory animal by cell fusion according to the method of Koehler and Milstein or further improvements thereof. Human monoclonal antibodies can also be produced by known methods. Recombinant Fanconi I protein or peptide fragments, in particular N-terminal or C-terminal peptides thereof, are preferred as immunogens. Thus, a further subject of the invention are antibodies against the Fanconi I protein or variants thereof, which preferably do not cross-react with other Fanconi-related proteins, such as the FAC protein. The antibodies are particularly preferably raised against the full-length polypeptide or against a peptide sequence corresponding to amino acids 1-20 or 53-72 of the amino acid sequence shown in SEQ ID NO:2. The preparation of the Fanconi I protein, the nucleic acid encoding it and antibodies thereagainst provides the necessary conditions for a specific search for effectors of this protein. Substances having an inhibitory or activating effect on the polypeptide according to the invention can selectively affect the cellular functions regulated by this polypeptide. As a result, they can be used to treat associated clinical conditions, such as, for example, cytopenias or tumors. The subject matter of the invention therefore also includes a method for identifying effectors of the Fanconi I protein, in which cells expressing this protein are exposed to various potential effector substances, for example small molecule substances, and the cells are analyzed for changes, for example cell activation, cell inhibition, cell proliferation and/or genetic changes in the cells. In this way, binding targets of the Fanconi I protein can also be identified. In the case of clinical conditions caused by a deficiency of the Fanconi I protein, gene therapy can be performed, which involves the transfer of a nucleic acid encoding the Fanconi I protein into suitable target tissues, for example by a vector, such as a viral vector. On the other hand, pathologies caused by an uncontrolled expression of the Fanconi I protein can be treated by gene therapy that blocks this expression. Furthermore, the presented results provide the basis for targeted diagnosis of diseases that are causally or indirectly related to a change in the activity of the Fanconi I protein. These tests can be performed using specific nucleic acid probes for detection at the nucleic acid level, such as the gene or transcript level, or antibodies against the Fanconi I protein for detection at the polypeptide level. Thus, the present invention relates to pharmaceutical compositions comprising as active ingredients the above-mentioned nucleic acids, vectors, cells, polypeptides and antibodies. The pharmaceutical compositions according to the invention may also comprise common pharmaceutical carrier substances, auxiliary substances and/or additives and, optionally, active ingredients. The pharmaceutical compositions can be used to diagnose, treat or prevent diseases, in particular those related to cell cycle abnormalities, cell activation abnormalities, cell cycle progression abnormalities, DNA repair abnormalities, cytopenia, tumor formation and/or tumor progression. Furthermore, the compositions according to the invention can also be used to diagnose an individual's predisposition to such diseases, in particular the risk of cytopenia and/or tumor disease. Furthermore, a further subject of the present invention is a method for diagnosing the above-mentioned diseases, in which a patient or a sample, such as a body fluid or tissue sample obtained from a patient, is contacted with the pharmaceutical composition according to the invention and the base sequence and/or expression of the nucleic acid according to the invention is qualitatively or quantitatively determined. These methods of determination can be carried out, for example, at the nucleic acid level using nucleic acid hybridization probes or by reverse transcription/PCR methods, or at the protein level by antibodies using cytochemical or histochemical methods. This pharmaceutical composition is particularly preferably used as a marker for identifying the onset of cytopenias, tumors or other proliferation-related diseases or the presence or absence of a predisposition to said pathophysiological changes. Finally, the present invention also relates to a method for treating or preventing one of the above-mentioned diseases, comprising administering to a patient a pharmaceutical composition according to the present invention, comprising an effective dose of an active ingredient against the disease. Examples of pharmaceutical compositions suitable for therapeutic purposes are, for example, bispecific antibodies, and antibody toxins or antibody-enzyme conjugates. Further preferred pharmaceutical compositions for therapeutic purposes are antisense nucleic acids, gene therapy vectors or other low molecular weight activators or inhibitors. The present invention will be further elucidated by the following examples and sequence listing. SEQ ID NO:1 shows a nucleotide sequence containing genetic information encoding Fanconi gene I; SEQ ID NO:2 shows an amino acid sequence of the open reading frame of the nucleotide sequence shown in SEQ ID NO:1; SEQ ID NO:3 shows nucleic acid primer SP1; SEQ ID NO:4 shows nucleic acid primer SP2; SEQ ID NO:5 shows nucleic acid primer SP3; SEQ ID NO:6 shows nucleic acid primer SP4; and SEQ ID NO:7 shows nucleic acid primer SP5. EXAMPLES Example 1: Cell Culture Diploid primary human fibroblasts H94-38 and H94-17 were isolated from fetal lung tissue and provided by D. Schindler (University of Wuerzburg). H94-38 cells were diagnosed as Fanconi anemia phenotype by cell cycle analysis, showing a prolonged G2 phase and increased G2 arrest upon addition of MMC. Complementation studies show that H94-38 cells do not belong to Fanconi complementation groups A, B, C, and D, but probably to complementation group E. H94-17 control cells show no increased MMC sensitivity. Cells were cultured in MEM medium containing Eale's salts (BRL, Gaithersburg, MD, US) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37°C, 7% CO2, and 95% humidity. For RNA preparation, cells were synchronized by serum reduction (0.1%) and stimulated after 48 h with 10% fetal bovine serum. After an additional 30 h, cells were semiconfluent and harvested for RNA isolation. After a 30-h incubation period in medium containing BrdU, a portion of these cultures was taken to analyze the cell cycle status by proliferation assay. The number of cells in the G0/G1, S and G2/M phases of the cell cycle was measured by high-resolution flow cytometric BrdU Hoechst quench method as described by Kubbies (Radbruch, A. (Ed.) Flow Cytometry and Cell Sorting, Springer Verlag Berlin-Heidelberg, 1992, pp75-85). Example 2: mRNA Differential Display For mRNA differential display, an RNA kit from Gen Hunter (Brooklyn, MA, USA) was used. Total RNA was isolated from synchronized cell cultures using Tripure reagent (Boehringer, Mannheim, GmbH, GER) according to the manufacturer's instructions. RNA was stored at -80°C as isopropanol-precipitated RNA covered with 70% ethanol until use. DNAse I (Boehringer Mannheim, GmbH) was added to 1-5 μg of total RNA in 1× DNAse I reaction buffer and incubated at 37°C for 30 min. The RNA samples were quantified by measuring the absorbance at 260 nm and analyzed on agarose gels. 0.2 μg of total RNA was used for reverse transcription. A total of 8 μg of total RNA was isolated from 1×10 6 fibroblasts. Reverse transcription of RNA was performed in duplicate in 20 μl reaction mixtures in 1× reverse transcription buffer, 20 μM of each dNTP, and 2 μM of each single-base anchor primer, T↓11A, T↓11G, or T↓11C. The solution was heated to 65°C for 5 min and cooled to 37°C for 10 min before adding 100 U of Moloney murine leukemia virus (MMLV) reverse transcriptase. After incubation at 37°C for 1 h, the mixture was heated to 75°C for 5 min and stored at −20°C. PCR was performed in a reaction solution containing 1/10 the reverse transcription mixture, 2 μM dNTPs, 0.2 μM of each T↓11N primer, 0.2 μM of the arbitrary sequence primer, 10 μCi of α[35S]dATP, and 1 U of Taq-DNA polymerase (Boehringer, Mannheim, GmbH). PCR was performed in a Perkin-Elmer 2400 Gene Amp with 40 cycles of 94°C for 30 s, 40°C for 2 min, 72°C for 30 s, and a final cycle of 72°C for 5 min. Various random primers (in all cases 10-mer primers with GC content of 60-70%) from Gen Hunter (13-mer with HindIII restriction site), Operon (Allameda, CA, USA), and Genosys (The Woodlands, TX, USA) were used. Samples were denatured in sequencing gel running buffer at 80°C for 2 min before separation on a 5-6% denaturing polyacrylamide sequencing gel. PCR experiments were performed in duplicate for each sample and sequentially separated on the same polyacrylamide gel. To analyze the differentially expressed genes, the dried gels were analyzed by autoradiography. Reproducible bands corresponding to the differentially expressed genes were excised from the gel. The cDNA was eluted from the gel slices by boiling in 100 μl of sterile water for 15 min. The DNA in the supernatant was collected by ethanol precipitation in the presence of glycogen. The DNA was then re-amplified using the corresponding primers and PCR conditions as described above, except that a dNTP concentration of 20 μM was used and the reaction mixture was radioisotope-free. The PCR-amplified fragments thus obtained were separated on an agarose gel and the corresponding gel slices were eluted by centrifugation in 0.45 μm Millipore Durapore membrane tubes. The samples were stored at −20° C. for Northern analysis. In this way, a total of 60 bands were obtained from 106 different primer combinations, of which 43 were re-amplified bands, corresponding to genes differentially expressed in FA cells and control cells. This differential expression was reproducible. Example 3: Northern analysis Northern blot analysis was performed by the standard method of Sambrook et al. (1989, supra). Nucleic acids were transferred onto a positively charged nylon membrane (Boehringer Mannheim GmbH) by downward capillary migration with 10x SSC and crosslinked. Specific probes were labeled directly from the PCR reamplification mixture using hexamer primers of arbitrary sequence and labeled with the Hi-Primer labeling kit (Boehringer, Mannheim, GmbH). Free nucleotides were separated by G50 Sephadex spin columns. The probes thus generated were hybridized with total RNA. After 16-20 hours of hybridization at 42°C, the filters were washed twice with 1x SSC, 0.1% SDS for 15 minutes at room temperature, followed by 1x SSC, 0.1% SDS for 1 hour at 50°C. The membranes were then examined by autoradiography. Northern analysis showed differential expression of a PCR fragment approximately 480 bp in length. Northern analysis of cultured cells and tissue samples revealed a major band <1 kb in length and more frequent bands of approximately 1.5-2.5 kb in length, possibly due to splice variants. No bands were seen in tumor cells, e.g., HeLa, Raji, and K562. In embryonic tissues, only one relatively large band was found in the pigmented integumentary cartilage of the eye. Example 4: Characterization of DD-PCR fragments corresponding to differentially expressed mRNA species. PCR fragments found to be differential on Northern blots were ligated into pCR2.1 using the TA cloning kit (INVITROGEN) according to the manufacturer's instructions, and the construct was then transformed into E. coli strain INVαF'. Clones carrying plasmids containing the differential fragments and vector were grown and the plasmids isolated by standard methods according to Sambrook et al. (1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The insert was sequenced using sequencing primer M13/pUC (Boehringer Mannheim catalog number: 1010 077) and reverse sequencing primer M13/pUC (Boehringer Mannheim catalog number: 1010 0 93) with an automated DNA sequencer 370A and a DNA sequencing kit (part number: 4020 79) purchased from Applied Biosystems Company. The 5' region of the revealed base sequence was identified using sequence-specific primers SP1: 5'ACT CT C CAG GAA ATC 3' (SEQ ID NO: 3), SP2: 5'GAC AGT GCA GAT CAT CTG TAC CA A 3' (SEQ ID NO: 4), and SP3: 5'GGC AAT TGT AAG CAA ACA GTA TCA 3' (SEQ ID NO: 5) with a 5'/3' RACE kit (Boehringer Mannheim catalog number: 1734 792), and ligated into pCR 2.1 as described above and sequenced. The fact that both fragments belong to the same mRNA was demonstrated by the presence of an overlapping region of 120 bases with identical nucleotide sequence and by the generation of the sequence of Fanconi gene I by PCR using a primer located outside the 5' end of the RACE fragment (SP4: 5'AAG CCA CAG GCT GGG TGG CTG 3' (SEQ ID NO: 6)) and a primer located at the 3' end of the differential display fragment (SP5: 5'TTT TCT GAC ACA TGG ATA AAC 3' (SEQ ID NO: 7)). The cDNA of Fanconi gene I thus obtained was ligated into pCR2.1 and sequenced as described above. The differential expression of the entire Fanconi gene I mRNA was demonstrated by Northern blot analysis as described in Example 3. Example 5 Preparation and Expression of Expression Constructs The FA-1 gene described in Example 4 was recloned into the eukaryotic expression vector pCDNA3 (Invitrogen) by standard methods (Sambrook, Fritsch, Maniatis, (Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989). The expression was controlled by the CMV promoter/enhancer and the BGH polyA signal. As a selection gene, the Neo gene was used (under the control of an SV40 expression cassette). A stable FA-1 expressing cell line was prepared using CHO cells. For this, 20 μg of Lipofectamine (Gibco; in 750 μl MEM-alpha medium) was added to 10 The mixture was mixed with 1 μg of DNA (in 750 μl MEM-alpha medium), incubated for 45 min at room temperature, and then diluted with 6 ml MEM-alpha medium. This mixture was added to 5 × 10↑6 CHO cells in MEM-alpha medium (Gibco) in T75 cell culture flasks (Nunc) for 6 h. Incubation was performed at 37°C. The cells were then washed with MEM-alpha/10% FCS (fetal calf serum) and cultured in fresh medium for 48 h at 37°C. Selection pressure was then applied by adding 1 mg/ml neomycin (G418) (Boehringer, Mannheim). Surviving cells were identified by FACS (Becton Dickinson). The FA-1 cells were cloned as single cells in 96-well culture plates (Nunc) with fresh medium by ELISA (Dickinson) and further cultured under G418 selection pressure until stably transfected CHO clones were established. The use of DHFR as a selection gene allows gene amplification of the FA-1 gene in addition to obtaining stably transfected CHO cell lines. Example 6: Antibody Production BALB/c mice were immunized intraperitoneally with recombinant FA-1 protein (prepared in CHO cells). Primary immunization was performed in complete Freund's adjuvant and all boosts were performed in incomplete Freund's adjuvant. The dose was 50-100 μg. Boosts were performed at approximately 4-week intervals until the serum titer was 1:50,000. Spleen cells from the immunized animals were then immortalized with the myeloma cell line P3X63.Ag8.653. Fusion was performed by standard methods (J. Immunol. Methods 39 (1980), 285-308). The fusion ratio of spleen cells to myeloma cells was 1:1. The fusion products were seeded in 24-well cell culture dishes (Nunc) in HA medium (Boehringer Mannheim) based on RPM1/10% FCS. Two weeks after fusion, positive primary cultures were cloned individually by FACS (Becton Dickinson) in 96-well cell culture plates (Nunc) in RPM1/10% FCS (Boehringer, Mannheim). The hybridoma cell clones thus obtained were expanded in vivo to obtain monoclonal antibodies. For this, 5 x 10^6 hybridoma cells were inoculated intraperitoneally into mice pretreated with pristane (Sigma Chemical Company). After 10-21 days, 2-3 ml of ascites fluid was removed from each mouse, from which monoclonal antibodies were routinely isolated.

───────────────────────────────────────────────────────── Continued from the front page (51)Int.Cl. ⁷ identification symbol FI Theme code (for reference) A61P 7/00 A61P 7/00 35/00 35/00 43/00 111 43/00 111 C07K 14/47 C07K 14/47 16/18 16/18 C12N 5/10 C12Q 1/68 A C12Q 1/68 G01N 33/53 D G01N 33/53 C12N 5/00 B (81)Designated States EP(AT,BE,CH,DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, GH, HU, ID, IL, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Simon Planitzer 54 Pasinger Strasse, Berlin, Germany

Claims (1)

[Claims] 1. A nucleic acid comprising (a) the nucleotide sequence shown in SEQ ID NO:1 or a portion thereof encoding a protein thereof, (b) a nucleotide sequence corresponding to the sequence of (a) within the degeneracy of the genetic code, or (c) a nucleotide sequence which hybridizes to the sequences of (a) and/or (b) under stringent conditions. 2. A nucleic acid according to claim 1, comprising a protein-coding portion of the nucleotide sequence shown in SEQ ID NO:1. 3. A nucleic acid according to claim 1, having a homology of greater than 80% to the nucleotide sequence shown in SEQ ID NO:1, or a portion thereof. 4. A modified base or nucleic acid analogue comprising the nucleotide sequence according to any one of claims 1 to 3. 5. A vector comprising at least one copy of the nucleic acid according to any one of claims 1 to 3 or a portion thereof. 6. A vector according to claim 5, capable of expressing the nucleic acid in a suitable host cell. 7. A cell transformed with a nucleic acid according to any one of claims 1 to 3 or a vector according to claims 5 or 6. 8. A polypeptide encoded by a nucleic acid according to any one of claims 1 to 3. 9. A polypeptide according to claim 8, having (a) the amino acid sequence shown in SEQ ID NO:2, or (b) a homology of more than 70% to the amino acid sequence shown in SEQ ID NO:2. 10. Use of a polypeptide according to claim 8 or 9, or a fragment of said polypeptide, as an immunogen for the production of antibodies. 11. An antibody against a polypeptide according to claim 8 or 9. 12. An antibody according to claim 11 against a full-length polypeptide or a peptide sequence corresponding to amino acids 1 to 20 or 53 to 72 of SEQ ID NO:2. 13. A modified polypeptide comprising the amino acid sequence according to claim 8 or 9. 14. A pharmaceutical composition comprising as an active ingredient (a) a nucleic acid according to any one of claims 1 to 4, (b) a vector according to claim 5 or 6, (c) a cell according to claim 7, (d) a polypeptide according to claim 8, 9 or 13, and/or (e) an antibody according to claim 11 or 12. 15. The composition according to claim 14, further comprising a common pharmaceutical carrier substance, auxiliary substance, and/or additive. 16. Use of the composition according to claim 14 or 15 for diagnosing a disease associated with abnormalities in cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, and/or tumor progression. 17. Use of the composition according to claim 14 or 15 for diagnosing a predisposition to a disease associated with abnormalities in cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, and/or tumor progression. 18. Use of the composition according to claim 14 or 15 for the manufacture of a medicament for diagnosing a disease associated with abnormalities in cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumor formation, and/or tumor progression, or for diagnosing a predisposition to such diseases. 19. Use of the composition according to claim 14 or 15 for the manufacture of a medicament for treating or preventing a disease associated with abnormalities in cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumor formation, and/or tumor progression. 20. Use according to claim 19 for the manufacture of a medicament for gene therapy. 21. A method for diagnosing a disease associated with abnormalities in cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumor formation, and/or tumor progression, or for diagnosing a predisposition thereto, comprising contacting a patient or a sample taken from a patient with the composition according to claim 14 or 15 and measuring the base sequence and/or expression of the nucleic acid according to claim 1. 22. A method for treating or preventing a disease associated with abnormalities in cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumor formation, and/or tumor progression, comprising administering to a patient a composition according to claim 14 or 15, comprising an effective amount of an active ingredient against such a disease. 23. A method for identifying an effector of the protein according to claim 8 or 9, comprising contacting a cell expressing the protein with various substances that may be effectors and analyzing changes in the cell.