JP2000513577A - Fanconi gene ▲ I ▼ - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION Fanconi Gene I The present invention relates to pathophysiologically relevant genes associated with Fanconi anemia (FA), polypeptides encoded thereby, antibodies to the polypeptides, and nucleic acids thereof. , Polypeptides, and antibodies for pharmaceutical use. FA is a rare genetic disease characterized by progressive pancytopenia, congenital malformations, and an increased risk of tumor disease (Glanz and Fraser, J. Med. Genet. 19). 1982) 412-416; Auerbach and Allen, Cancer Genet. Cytogenet. 51 (1991) 1-12). The disease affects about 1 in 300,000 people. The molecular basis of the disease is unknown, but hypersensitivity to the clastogenic effect of DNA crosslinking reagents is an excellent marker of FA genotype (Auerbach and Wolmann, Nature 261 (1976)) 494-496). Cells from FA patients are exposed to numerous chromatid breaks and stains when exposed to mitomycin C (MMC) or diepoxybutane (DEB) at concentrations that show low clastogenic effects on normal cells. 1 shows body substitutions (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 and complete arrest of the G2 phase, revealing defects in the G2 phase of the cell cycle (Kubbies et al., Am. J. Hum. Genet.37 (1985), 1022; Hoehn et al., Fanconi Anaemia, Clinical, Cytogenic and Experimental Aspects (1989), Springer Springer Verlag Berlin-Heidelberg; Seyschab et al., Blood 85 (1995), 2233-2237). It is presently known that there are at least five different complementation groups in 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). Genes for complementation group C have recently been reported (Strathdee et al., Nature 356 (1992), 763, and WO 93/22435), but the type of molecular mechanism of action of the FA-C protein is still unknown. (Gavish et al., Am. J. Hum. Genet. 53 (1993), 685; Yamashita et al., Proc. Natl. Acad. Sci. USA 91 (1994), 6712; Yusufian) ) Et al., J. Biol. Chem. 270 (1995), 9876-9882). Furthermore, two complementary 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). It is an object of the present invention to identify novel genes involved in the cascade of DNA regulation (eg, cell cycle abnormalities, DNA repair, tumorigenesis / tumor progression), and which are associated with the pathophysiological phenotype of Fanconi anemia Was that. The present invention describes the identification, cloning, and characterization of a gene encoding a novel polypeptide, designated Fanconi Gene I. The sequence of this gene was discovered using the differential display method (Liang and Pardee, Science 257 (1992), 967-971) comparing normal and FA fibroblasts. It is a thing. Fanconi gene I is significantly more strongly expressed in FA fibroblasts than in normal fibroblasts. Fanconi gene I, the polypeptide encoded by it, and antibodies to this polypeptide have been shown to cause abnormalities in the cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, and tumor progression. It is suitable as an agent for diagnosing, treating or preventing a directly or indirectly related disease. The subject of the present invention is (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), which is within the scope of the genetic code degeneracy, or (C) a nucleic acid comprising a base sequence that hybridizes under stringent conditions to the sequence of (a) and / or (b). The nucleotide sequence shown in SEQ ID NO: 1 has an open reading frame corresponding to a polypeptide having a length of 72 amino acids. The amino acid sequence of this polypeptide is shown in SEQ ID NO: 2. Human cDNA clones 290799 (Accession No. N71961), 270393 (Accession No. N33066), and 66812 (Accession No. T64946) containing a partial region of the nucleotide sequence shown in SEQ ID NO: 1 are obtained from EMBL EST sequence data bank. Published in. However, no information is given on the complete open reading frame of the displayed sequence, or on the possible biological function. In addition to the base sequence shown in SEQ ID NO: 1 and the base sequence corresponding to this sequence within the scope of the genetic code degeneracy, the present invention also relates to a base sequence that hybridizes with one of the above sequences. In the context of the present invention, the term "hybridization" is defined in Sambrook et al., (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory Press (1989). ), 1.101-1.104). Stringent hybridization refers to washing at 50 ° C., preferably 55 ° C., particularly preferably 62 ° C., and most preferably 68 ° C. for 1 hour with 1 × SSC and 0.1% SDS, especially at 50 ° C., preferably at 50 ° C. It preferably means that a positive hybridization signal is still visible after washing for 1 hour with 0.2 × SSC and 0.1% SDS at 55 ° C., particularly preferably 62 ° C., most preferably 68 ° C. Under such washing conditions, the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence that hybridizes with the nucleotide sequence corresponding to this sequence within the range of degeneracy of the gene code is the nucleotide sequence according to the present invention. is there. The base sequence according to the present invention is preferably DNA. However, nucleic acid analogs such as RNA or peptide nucleic acids can also be included. The nucleic acid according to the present invention is particularly preferably a protein coding portion of the nucleotide sequence shown in SEQ ID NO: 1, or the nucleotide sequence shown in SEQ ID NO: 1, or preferably at least 20% of this sequence. It comprises bases, and particularly preferably at least 50 bases in length, and sequences having a homology higher than 80%, preferably higher than 90%, most preferably higher than 95%. Yet another subject of the present invention is a polypeptide encoded by a nucleic acid as described above. The polypeptide preferably has (a) the amino acid sequence set forth in SEQ ID NO: 2, or (b) the amino acid sequence set forth in SEQ ID NO: 2 with greater than 70%, preferably 80%, %, Particularly preferably greater than 90%. The nucleic acids according to the invention can preferably be obtained from mammals, in particular from humans. These can be isolated by a known technique using a short fragment of the nucleotide sequence shown in SEQ ID NO: 1 as a probe and / or primer for hybridization by a known method. Further, the nucleic acid according to the present invention is also prepared by chemical synthesis in which a modified base structural unit such as a 2′-0-alkylated base structural unit can be selectively used instead of a normal nucleotide structural unit. be able to. Nucleic acids composed partially or completely of modified base building blocks can be used as therapeutic agents such as, for example, antisense nucleic acids or ribozymes. The present invention also includes nucleic acid analogous compounds such as peptidic nucleic acids having a base sequence corresponding to the nucleic acid according to the present 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 may be any desired prokaryotic or eukaryotic vector, preferably one in which the DNA sequence according to the invention is under the control of an expression signal (promoter, operator, enhancer, etc.). Examples of prokaryotic vectors include 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 present invention is particularly preferably a eukaryotic vector such as a vector for yeast, or a vector (for example, a plasmid vector, a virus vector, or a plant vector) suitable for cells of higher organisms. Such vectors are known to those of skill in the field of molecular biology and will not be described in further detail herein. In this regard, reference is made in particular to Sambrook et al., Supra, Chapter 16, above. In addition to the polypeptide set forth in SEQ ID NO: 2, the invention also relates to muteins, variants, and fragments thereof. These are understood as sequences that differ from the amino acid sequence set forth in SEQ ID NO: 2 by substitution, deletion and / or insertion of each amino acid or short fragment of amino acids. The term "variant" includes natural allelic or splice variants of Fanconi polypeptide I with in vitro mutagenesis using recombinant DNA techniques, especially using chemically synthesized oligonucleotides. ) Produced proteins that substantially match the protein set forth in SEQ ID NO: 2 with respect to biological and / or immunological activity. The term also includes chemically modified polypeptides. These include polypeptides whose terminal and / or reactive amino acid side groups have been modified by acylation, such as acetylation or amidation. The present invention also relates to a vector comprising at least a 20 nucleotide long part of the sequence shown in SEQ ID NO: 1. This portion preferably has a nucleotide sequence obtained from the protein coding region of the sequence shown in SEQ ID NO: 1 or a nucleotide sequence obtained from a region essential for protein expression. These nucleic acids are particularly suitable for producing antisense nucleic acids, preferably up to 50 nucleotides, which can be used for therapy. A further subject of the invention is a cell transformed by a nucleic acid according to the invention or a vector according to the invention. The cells may be eukaryotic or prokaryotic. Methods for transforming cells with nucleic acids are common in the art and will not be described in further detail. Examples of preferred cells are eukaryotic cells, especially 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 producing antibodies. In this case, an antibody can be produced in a conventional manner by immunizing a laboratory animal with the full-length polypeptide or a fragment thereof and isolating the resulting polyclonal antiserum. Monoclonal antibodies can be obtained in a known manner from antibody-producing cells of laboratory animals by cell fusion according to the method of Koehler and Milstein or a further improvement thereof. Also, human monoclonal antibodies can be produced by known methods. Recombinant Fanconi I protein or peptide fragments, especially the N-terminal or C-terminal peptides thereof, are preferred as immunogens. Thus, a further subject of the present invention is an antibody against the Fanconi I protein or a variant thereof, preferably an antibody that does not show cross-reactivity with another Fanconi-related protein such as the FAC protein. This antibody is preferably produced against a full-length polypeptide or a peptide sequence corresponding to amino acids 1 to 20 or 53 to 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 the antibodies thereto provide the necessary conditions for specifically searching for effectors of this protein. Substances having an inhibitory or activating effect on the polypeptide according to the invention can selectively influence the cellular functions regulated by this polypeptide. As a result, they can be used to treat relevant clinical features such as, for example, cytopenia and tumors. Therefore, the present invention includes a method for identifying an effector of the Fanconi I protein, which comprises contacting a cell expressing this protein with various substances that may be effectors, such as low-molecular substances. Also included are methods for analyzing whether the cell has undergone a change, such as, for example, cell activation, cell inhibition, cell proliferation, and / or a genetic change in the cell. In this way, the binding target of the Fanconi I protein can also be identified. In the case of a clinical picture caused by a deletion of the Fanconi I protein, gene therapy including transferring the nucleic acid encoding the Fanconi I protein into an appropriate target tissue, for example, by a vector such as a viral vector, may be performed. It is possible. On the other hand, pathologies resulting from an inability to control the expression of Fanconi I protein can be treated by gene therapy that blocks this expression. Furthermore, the presented results also provide the basis for target diagnosis of diseases that are causally or indirectly associated with altered activity of Fanconi I protein. These tests can be performed using a specific nucleic acid probe for detection at the nucleic acid level such as the gene or transcription level, or an antibody against the Fanconi I protein for detection at the polypeptide level. . As described above, the present invention relates to a pharmaceutical composition containing the above-described nucleic acid, vector, cell, polypeptide, and antibody as an active ingredient. The pharmaceutical composition according to the present invention may further comprise general pharmaceutical carrier substances, auxiliary substances and / or additives and, optionally, an active ingredient. The pharmaceutical composition is particularly useful for diagnosing cell cycle abnormalities, abnormal cell activation, abnormal cell cycle progression, abnormal DNA repair, cytopenia, tumorigenesis, and / or diseases associated with tumor progression. It can be used for treatment or prevention. Furthermore, the compositions according to the invention can also be used to diagnose an individual's predisposition to such a disease, in particular the risk of cytopenia and / or tumor disease. Further, a further aspect of the present invention provides that a sample, such as a patient or a sample of body fluid or tissue obtained from the patient, is contacted with a pharmaceutical composition according to the present invention, and the nucleotide sequence and / or expression of a nucleic acid according to the present invention is determined. This is a method for diagnosing the above diseases, which is measured qualitatively or quantitatively. For example, these measurement methods can be performed at the nucleic acid level by using a probe for nucleic acid hybridization or by the reverse transcription / PCR method, or at the protein level by an antibody using a cytochemical or histochemical method. it can. The pharmaceutical composition is particularly preferably used as a marker for confirming the onset of cytopenia, tumor, or other growth-related diseases, or the presence or absence of a predisposition for the pathophysiological change. Finally, the present invention also relates to a method for treating or preventing one of the above-mentioned diseases, wherein a pharmaceutical composition according to the present invention is administered to a patient, comprising a dose effective for the disease. About the method. Specific 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 clarified by the following Examples and Sequence Listing. SEQ ID NO: 1 shows a nucleotide sequence containing gene information encoding Fanconi gene I, and SEQ ID NO: 2 shows an amino acid sequence of an open reading frame of the nucleotide sequence shown in SEQ ID NO: 1. SEQ ID NO: 3 shows the nucleic acid primer SP1, SEQ ID NO: 4 shows the nucleic acid primer SP2, SEQ ID NO: 5 shows the nucleic acid primer SP3, SEQ ID NO: 6 , Shows the nucleic acid primer SP4, and SEQ ID NO: 7 shows the nucleic acid primer SP5. Examples Example 1: Cell culture diploid early human fibroblasts H94-38 and H94-17 were isolated from fetal lung tissue and provided by D. Schindler (University of Wuerzburg) . H94-38 cells are diagnosed as having a Fanconi anemia phenotype by cell cycle analysis, prolonging the G2 phase and adding MMCs show increased G2 arrest. Complementation studies indicate that the H94-38 cells do not belong to the Fanconi complementation groups A, B, C, and D, but probably belong to complementation group E. H94-17 control cells do not show increased MMC sensitivity. Eel salts (BRL Inc., Gettysburg, MD, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). The cells were cultured in MEM medium containing Gaithersburg, MD, US) at 37 ° C., 7% CO ↓ 2 and 95% humidity. To prepare RNA, cells were synchronized by reducing serum (0.1%) and stimulated 48 hours later with 10% fetal calf serum. After an additional 30 hours, cells were semi-confluent and harvested for RNA isolation. After a 30-hour culture period in medium containing BrdU, a portion of these cultured cells were harvested for analysis of cell cycle status in a proliferation assay. Springer Verlag, Berlin-Heidelberg, described by Kubbies (Radbruch, A. (eds.) Flow Cytometry and Cell Sorting). Heidelberg) 1992, pp 75-85), cell number in the cell cycle G0 / G1, S and G2 / M phases was determined by the high resolution flow cytometry BrdU Hoechst quench method. Example 2 mRNA Differential Display An RNA kit from Gen Hunter (Brooklyn, Mass., USA) was used for mRNA differential display. 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 minutes. The RNA sample was quantified by measuring the absorbance at 260 nm and analyzed on an agarose gel. For reverse transcription, 0.2 μg of total RNA was used. A total of 8 μg of total RNA was isolated from 1 × 10 6 fibroblasts. Reverse transcription of RNA was carried out 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, in each case 20 μl of Two reaction mixtures were performed. The solution was heated at 65 ° C. for 5 minutes, cooled at 37 ° C. for 10 minutes, and then 100 U of Moloney murine leukemia virus (MMLV) reverse transcriptase was added. After incubation at 37 ° C. for 1 hour, the mixture was heated at 75 ° C. for 5 minutes and then stored at −20 ° C. PCR was performed using 1/10 volume of the reverse transcription mixture, 2 μM dNTP, 0.2 μM of each T ↓ 11N primer, 0.2 μM of an arbitrary sequence primer, 10 μCi of α [35S] dATP, and 1 U of Taq-DNA polymerase. (Boehringer, Mannheim, GmbH). PCR was performed using a Perkin-Elmer 2400 Gene Amp for 40 cycles of 94 ° C for 30 seconds, 40 ° C for 2 minutes, 72 ° C for 30 seconds, and finally for 5 minutes at 72 ° C. . Gen Hunter (13-mer with HindIII restriction enzyme site), Operon (Allameda, CA, USA, USA), and Genosys (Texas, USA) Various random primers from The Woodlands (TX, USA) (in each case a 10-mer primer with a GC content of 60-70%) were used. Samples were denatured in a sequencing gel running buffer at 80 ° C. for 2 minutes before separation on a 5-6% denaturing polyacrylamide sequencing gel. For each sample, PCR experiments were performed twice, and they were sequentially separated on the same polyacrylamide gel. Dried gels were analyzed by autoradiography to analyze differentially expressed genes. Reproducible bands corresponding to differentially expressed genes were excised from the gel. The cDNA was eluted from the gel slice by boiling in 100 μl of sterile water for 15 minutes. The DNA in the supernatant was collected by ethanol precipitation in the presence of glycogen. The DNA was then reamplified using the corresponding primers and PCR conditions as above, except that the reaction mixture was free of radioisotope, using a dNTP concentration of 20 μM. The PCR amplified fragment thus obtained was separated on an agarose gel, and the corresponding gel slice was eluted by centrifugation using a 0.45 μm Millipore Durapore membrane tube. This sample was stored at −20 ° C. for Northern analysis. In this way, a total of 60 bands were obtained from the 106 combinations of different primers, 43 of which were reamplified bands, which were differentially expressed in FA and control cells. Corresponds to the expressed gene. This differential expression was reproducible. Example 3: Northern Analysis Sambrook (Sambrook) et al. ((1989) supra) by standard methods and subjected to Northern blot analysis. Nucleic acids were transferred and cross-linked onto positively charged nylon membranes (Boehringer Mannheim GmbH) using 10xSSC by downward directed capillary transfer. Using a hexamer primer having an arbitrary sequence, labeling with a Hi-Primer labeling kit (Boehringer, Mannheim, GmbH), directly from the reamplification mixture of PCR Specific probes were labeled. Free nucleotides were separated on a G50 Sephadex spin column. The probe thus produced was hybridized with total RNA. After hybridization at 42 ° C. for 16-20 hours, the filters were washed twice with 1 × SSC, 0.1% SDS at room temperature for 15 minutes, and then washed with 1 × SSC, 0.1% SDS at 50 ° C. for 1 hour. . The film was examined by autoradiography. Northern analysis indicated that the approximately 480 bp long PCR fragment had differential expression. Northern analysis in cultured cell and tissue samples reveals a major band with a length of <1 Kb and more frequently a band with a length of approximately 1.5-2.5 kb, probably due to splice variants. Was. For example, no bands were seen in tumor cells such as HeLa, Raji, and K562. In embryonic tissue, only a relatively large band was found in pigmented cartilage of the eye. Example 4 Characterization of DD-PCR Fragments Corresponding to Differentially Expressed mRNA Species PCR fragments which were found to be different by Northern blot were subjected to TA cloning kit (INVITROGEN) according to the manufacturer's instructions. This construct was used to transform E. coli strain INVαF ′ after ligation to pCR ™ 2.1 using. Clones containing plasmids containing differential fragments and vectors were cloned from Sambrook et al. (1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The plasmid was isolated by culturing according to standard methods according to (State). The sequencing primer M13 / pUC (Boehringer Mannheim cat. No .: 1010 077) and the reverse sequencing primer M13 / pUC (Boehringer Mannheim cat. No .: 1010 093) The nucleotide sequence of the inserted sequence was determined using an automated DNA sequencer 370A and a DNA sequencing kit (part number: 402079) purchased from Applied Biosystems Company. The identified 5 'region of the nucleotide sequence was used as a sequence-specific primer SP1: 5' ACT CT C CAG GAA ATC 3 '(SEQ ID NO: 3), SP2: 5' GAC AGT GCA GAT CAT CTG TAC CA A3 5 ′ / 3′RACE kit (Boehringer Mannheim) catalog using '′ (SEQ ID NO: 4) and SP3: 5 ′ GGC AAT TGT AAG CAA ACA GTA TCA 3 ′ (SEQ ID NO: 5) No .: 1734 792), and ligated to pCR 2.1 as above, and sequenced. The fact that both fragments belong to the same mRNA is due to the fact that there is an overlapping region of 120 bases having the same nucleotide sequence, and that the primer (SP4: 5'AAG CCA CAG GCT GGG TGG CTG 3 ′ (SEQ ID NO: 6)) and a primer (SP5: 5 ′ TTT TCT GAC ACA TGG ATA AAC 3 ′ (SEQ ID NO: 7)) located at the 3 ′ end of the differential display fragment. The sequence of the Fanconi gene I. The thus obtained cDNA of Fanconi gene I was ligated to pCR 2.1 and sequenced in the same manner as described above. Differential expression of the entire mRNA of Fanconi gene I was revealed by Northern blot analysis as described in Example 3. Example 5 Preparation of Expression Construct and Expression The FA-1 gene described in Example 4 was prepared by standard methods (Sambrook, Fritsch, Maniatis, Molecular cloning: Experimental Manual ( Molecular Cloning. A Laboratory Manual), 2nd edition, Cold Spring Harbor, 1989) and recloned into the eukaryotic cell expression vector pCDNA3 (Invitrogen), which expresses the CMV promoter / The Neo gene was used (under control of the SV40 expression cassette) as a selection gene, and a stable FA-1 expressing cell line was prepared using CHO cells. For this purpose, 20 μg of lipofectamine (Gibco; in 750 μl of MEM-alpha medium) were replaced with 10 μg of DNA (750 μl of MEM-alpha). Medium) and incubated at room temperature for 45 minutes, then diluted with 6 ml of MEM-alpha medium, and mixed with MEM-alpha medium (Gibco) from a T75 cell culture bottle (Nunc). (Gibco) 5 × 10 6 CHO cells for 6 h. Incubation was performed at 37 ° C. The cells were then washed with MEM-alpha / 10% FCS (fetal bovine serum). The cells were cultured in fresh medium for 48 hours at 37 ° C. Subsequently, 1 mg / ml neomycin (G418) (Boehringer, Mannheim) was added and selective pressure was applied. (Becton Dickinson) cloned as a single cell in a 96-well culture plate (Nunc) containing fresh medium to establish a stably transfected CHO clone. G418 selection pressure until The use of DHFR as. Selection for gene further cultured over, a stably can be, in addition to obtaining a transfected CHO cell line carrying out gene amplification of the FA-1 gene. Example 6 : BALB / c mice were immunized intraperitoneally with antibody-producing recombinant FA-1 protein (prepared in CHO cells), priming was performed in Freund's complete adjuvant, and all boosters were immunized with Freund's Performed in incomplete adjuvant. The dose was 50-100 μg. Booster immunizations were performed at approximately 4 week intervals until the serum titer reached 1: 50,000. The spleen cells of 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 plated in 24-well cell culture dishes (Nunc) in RPMI / 10% FCS based HA medium (Boehringer Mannheim). Two weeks after fusion, the positive primary culture was plated in a 96-well cell culture plate (Nunc) in RPM1 / 10% FCS (Boehringer, Mannheim). Individual cells were cloned by the FACS method (Becton Dickinson). The hybridoma cell clones thus obtained were expanded in vivo to obtain monoclonal antibodies. For this, 5 × 10 6 hybridoma cells were inoculated intraperitoneally into mice previously treated with pristane (Sigma Chemical Company). After 10-21 days, 2-3 ml of ascites was removed from each mouse and monoclonal antibodies were isolated therefrom by routine methods.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (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 countries EP (AT, BE, CH, DE , DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR) , NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, 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, FI, 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 Planitzel Simon Germany Berlin Pasingel Strat C 54

Claims (1)

[Claims] 1． (A) Coding the nucleotide sequence shown in SEQ ID NO: 1 or its protein Part to be loaded, (B) a nucleotide sequence corresponding to the sequence of (a) within the range of degeneracy of the genetic code, Or (C) hybridizes to the sequences (a) and / or (b) under stringent conditions Soybean base sequence A nucleic acid comprising: 2． Claims: It comprises a protein coding portion of the nucleotide sequence shown in SEQ ID NO: 1. The nucleic acid according to 1. 3． A base sequence represented by SEQ ID NO: 1 or a part thereof, having a phase higher than 80% 2. The nucleic acid according to claim 1, having the same sex. Four. A modified base or nucleic acids comprising the base sequence according to any one of claims 1 to 3. Similar compounds. Five. At least one copy of the nucleic acid according to any one of claims 1 to 3 or a part thereof. A vector that also contains one. 6． The vector according to claim 5, wherein the nucleic acid can be expressed in a suitable host cell. - 7． The nucleic acid according to any one of claims 1 to 3, or the vector according to claim 5 or 6. Cells that have been transformed by the 8． Polypeptide encoded by the nucleic acid according to any one of claims 1 to 3. Chid. 9． (A) having the amino acid sequence of SEQ ID NO: 2, or (B) has a homology of more than 70% to the amino acid sequence shown in SEQ ID NO: 2 9. The polypeptide according to claim 8. Ten. The polypeptide of claim 8 or 9, or a fragment of the polypeptide, Use as an immunogen for antibody production. 11． An antibody against the polypeptide according to claim 8 or 9. 12． A full-length polypeptide, or amino acids 1-20 or 53-72 of SEQ ID NO: 2 12. The antibody according to claim 11, against a peptide sequence corresponding to a amino acid. 13. A modified polypeptide comprising the amino acid sequence according to claim 8 or 9. 14. (A) the nucleic acid according to any one of claims 1 to 4, (B) the vector according to claim 5 or 6, (C) the cell of claim 7, (D) the polypeptide of claim 8, 9 or 13, and / or (E) the antibody according to claim 11 or 12; A pharmaceutical composition comprising, as an active ingredient. 15． Further comprising common pharmaceutical carrier substances, auxiliary substances, and / or additives, 15. The composition according to claim 14. 16． Cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, And / or diagnosing a disease associated with abnormal tumor progression. Or use of the composition of claim 15. 17． Cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, And / or to diagnose a predisposition to a disease associated with abnormal tumor progression Use of the composition according to claim 14 or 15. 18． Cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, And / or a drug for diagnosing a disease associated with abnormal tumor progression; Is intended for the manufacture of a medicament for diagnosing a predisposition to those diseases Use of the composition according to 14 or 15. 19. Cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, And / or a drug for treating or preventing a disease associated with abnormal tumor progression Use of the composition according to claim 14 or 15 for the manufacture of an agent. 20. 20. Use according to claim 19 for the manufacture of a medicament for gene therapy. twenty one. Cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, And / or have a predisposition to a disease associated with abnormal tumor progression Method for diagnosing a disease associated with The sample thus obtained is brought into contact with the composition according to claim 14 or 15, and the nucleus according to claim 1. A method for measuring the base sequence and / or expression of an acid. twenty two. Cell cycle, cell activation, cell cycle progression, DNA repair, cytopenia, tumorigenesis, And / or to treat or prevent a disease associated with abnormal tumor progression The method according to claim 14 or 15, comprising an effective amount of the active ingredient against such a disease. A method of administering a composition as described to a patient. twenty three. A method for identifying an effector of the protein according to claim 8 or 9. Thus, cells expressing the protein can be transformed into various effector A method of contacting with a substance and analyzing a change in the cell.