Patent Publication Number: US-2003224423-A1

Title: Method of testing for allergic diseases

Description:
FIELD OF THE INVENTION  
       [0001] The present invention relates to a method of testing for an allergic disease and a method of screening for a therapeutic agent for an allergic disease.  
       BACKGROUND OF THE INVENTION  
       [0002] Allergic diseases such as atopic dermatitis are considered to be multifactorial diseases. These diseases are caused by the interaction of many different genes whose expression is independently influenced by multiple environmental factors. Therefore, it is extremely difficult to identify a specific gene causing a specific disease. Furthermore, the expression of mutated or defective genes, or increased or decreased expression of specific genes is envisaged to be associated with allergic diseases. Thus, to reveal the role of gene expression in diseases, it is required to understand how a gene is involved at the onset of a disease and how external stimulus, such as drugs, alters gene expression.  
       [0003] History taking as well as confirmation of the patient&#39;s family history and own anamnesis, are important in general for recent diagnosis of allergic diseases. In addition, for allergy diagnosis based on more objective information, a method of testing patient&#39;s blood samples and a method of observing patient&#39;s immune response to allergen are also performed. Examples of the former method are the allergen-specific IgE test, leukocyte histamine release test and lymphocyte blastogenesis test. The presence of allergen-specific IgE is proof of occurrence of the allergic reaction to the allergen. However, in some patients, allergen-specific IgE may not necessarily be detected. Furthermore, the IgE assay principle requires performing tests for all of the allergens necessary for diagnosis. The leukocyte histamine release test and the lymphocyte blastogenesis test are methods for observing the immune system reaction to allergens in vitro. These methods are complicated to perform.  
       [0004] Another known method is allergy diagnosis based on the immune response observed at the time when a patient is contacted with an allergen. Such a test includes the prick test, scratch test, patch test, intradermal reaction and induction test. These tests allow the direct diagnosis of patient&#39;s allergic reaction while they can be said to be highly invasive tests because patients are actually exposed to allergens.  
       [0005] In addition, test methods for proving the occurrence of allergic reaction caused by any allergen are also attempted. For example, a high serum IgE titer may indicate the occurrence of allergic reaction in the patient. The serum IgE titer corresponds to the total amount of allergen-specific IgE. Though it is easy to determine the total amount of IgE when any allergen is tested, the IgE titer may be reduced in some patients, such as, those with non-atopic bronchitis.  
       [0006] The number of eosinophils and eosinophil cationic protein (ECP) level are diagnostic items for delayed-type reaction following Type I allergy and allergic inflammatory reaction. The number of eosinophils is considered to reflect the progress of allergic symptoms. ECP, a protein contained in eosinophil granules, is also strongly activated in patients with an asthma attack. Indeed these diagnostic items identify allergy symptoms, but they are limited in their scope as diagnostic indicators.  
       [0007] Therefore, a marker (indicator) for an allergic disease that is not only less invasive to patients but also capable of readily providing information necessary for diagnosis would be useful. Since such markers are thought to be deeply involved in disease onset, they may be an important target in the control of allergic symptoms as well as in diagnosis.  
       SUMMARY OF THE INVENTION  
       [0008] An objective of the present invention is to provide an indicator gene for testing an allergic disease. Another objective of the invention is to provide a method of testing for an allergic disease and a method of screening for a therapeutic agent for an allergic disease, both using the expression of the gene as an indicator.  
       [0009] Based on a previously established technique, the “fluorescent differential display method (Fluorescent DD method)” (T. Ito et al. 1994, FEBS Lett. 351: 231-236), the present inventors developed a new DD system capable of analyzing T-cell RNA samples prepared from multiple human blood samples. Using Fluorescent DD method, the present inventors successfully isolated genes whose expression levels change in peripheral blood cells from patients with pollinosis. The genes that were isolated (along with the patent applications filed) are listed below:  
       [0010] Pollinosis-associated gene 373 (WO 00/65046),  
       [0011] Pollinosis-associated gene 419 (WO 00/65045),  
       [0012] Pollinosis-associated gene 513 (WO 00/65049),  
       [0013] Pollinosis-associated gene 581 (WO 00/65048),  
       [0014] Pollinosis-associated gene 795 (WO 00/65050),  
       [0015] Pollinosis-associated gene 627 (WO 00/65051),  
       [0016] Pollinosis-associated gene 441 (WO 00/73435),  
       [0017] Pollinosis-associated gene 465 (WO 00/73439), and  
       [0018] Pollinosis-associated gene 787 (WO 00/73440).  
       [0019] Similarly, genes whose expression levels in peripheral blood differ between patients of allergic diseases and healthy subjects were searched using differential display method. As a result, gene B1153 whose expression level significantly rose in the patient group was discovered and filed as a patent application (WO 02/50269).  
       [0020] The present inventors used the Fluorescent DD method to isolate genes whose expression level is altered in an allergic disease-specific manner. Specifically, first, the present inventors collected blood from normal healthy subjects and patients with allergic diseases (atopic dermatitis and allergic asthma), isolated T cells from the blood, and via the Fluorescent DD method screened for genes whose expression levels differ between the normal healthy subject group and the patient group. As a result, the present inventors succeeded in isolating a gene, “B1799,” that showed significantly higher expression levels in the patient group. The isolated gene was the same as KIAA0603 (GenBank Accession No. AB011175). Genomic sequence information and expression data are available for the mouse homologue of KIAA0603 (GenBank Accession No. AB011175; Genomics 2002 February; 79(2):154-61 Candidate genes required for embryonic development: a comparative analysis of distal mouse chromosome 14 and human chromosome 13q22. Kurihara L J, Semenova E, Miller W, Ingram R S, Guan X J, Tilghman S M. Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, N.J. 08544, USA). According to the report, sequences adjacent to the gene encoding Endothelin B receptor were determined, and the mouse homologue was shown as one of the genes comprised in this adjacent region. Furthermore, since deletion of this region in mice led to embryonic death, these genes were suggested to be related to this incident. No information relating to immunity and allergy is included in the report. The isolated human B1799 gene encodes a protein consisting of 1299 amino acids. This protein has the characteristic of a GTPase activating protein, which indicated that it is involved in cell differentiation and cell cycle control. The expression level of the B1799 gene was higher in atopic dermatitis patients compared to normal healthy subjects, especially higher in patients with moderate symptom. Analysis of the expression of the gene in various types of leukocytes revealed that the expression was high particularly in T cells, more particularly in memory T cells (cells with the phenotypes of CD4 +  and CD45RO + ). Furthermore, the present inventors discovered from the result of in vitro stimulation test that expression of B1799 mRNA is induced during the activated cell death of T cells. Specifically, B1799 mRNA expression was induced under conditions that activated the T cells by stimulus via T cell receptors or stimulus such as calcium ionophores (ionomycins). This relation of B1799 gene expression with the activation of the T cells verifies the involvement of the gene in allergic diseases. Thus, the inventors found that testing for allergic diseases and screening of candidate compounds serving as therapeutic agents for allergic diseases are possible by using the expression level of the gene as an indicator, and completed this invention.  
       [0021] The present invention relates to a method of testing for allergic diseases and a method of screening for therapeutic agents for allergic diseases based on the expression level of an indicator gene, the B1799 gene, which shows a high expression in allergic disease patients. Specifically, the present invention relates to the following testing methods for allergic diseases, kits therefor, and methods of screening for therapeutic agents for allergic diseases:  
       [0022] (1) A method of testing for an allergic disease, the method comprising the steps of:  
       [0023] (a) measuring the expression level of an indicator gene in a biological sample from a test subject;  
       [0024] (b) comparing the expression level with that of the indicator gene in a biological sample from a healthy subject; and  
       [0025] (c) judging the test subject to have an allergic disease when the expression level of the indicator gene in the biological sample from the test subject is found to be significantly elevated, wherein the indicator gene is B1799 gene.  
       [0026] (2) The testing method according to (1), wherein the allergic disease is atopic dermatitis.  
       [0027] (3) The testing method according to (1), wherein the gene expression level is measured via cDNA PCR.  
       [0028] (4) The testing method according to (1), wherein the gene expression level is measured by detecting a protein encoded by the gene.  
       [0029] (5) The testing method according to (1), wherein the biological sample contains peripheral blood T cells.  
       [0030] (6) A reagent for diagnosis of an allergic disease, said reagent comprising a polynucleotide that comprises at least 15 continuous nucleotide sequence of B1799 gene or a complementary sequence thereof;  
       [0031] (7) A reagent for testing for an allergic disease, said reagent comprising an antibody that binds to a polypeptide consisting of the amino acid sequence encoded by B1799 gene;  
       [0032] (8) A method for screening a therapeutic agent for an allergic disease, wherein said method comprises the steps of:  
       [0033] (a) contacting a candidate compound with a cell expressing an indicator gene;  
       [0034] (b) measuring the expression level of said indicator gene; and  
       [0035] (c) selecting a compound which decreases the expression level of the indicator gene as compared to a control where said candidate compound has not been contacted,  
       [0036] wherein the indicator gene is B1799 gene.  
       [0037] (9) The method according to (8), wherein the cell is T cell.  
       [0038] (10) A method of screening for a therapeutic agent for an allergic disease, the method comprising the steps of:  
       [0039] (a) administering a candidate compound to a test animal;  
       [0040] (b) measuring the expression level of an indicator gene in leukocytes of the test animal; and  
       [0041] (c) selecting a compound which decreases the expression level of the indicator gene compared to a control where the candidate compound has not been administered,  
       [0042] wherein the indicator gene is B1799 gene.  
       [0043] (11) A method of screening for a therapeutic agent for an allergic disease, the method comprising the steps of:  
       [0044] (a) contacting a candidate compound with cells containing a reporter gene linked under the control of transcriptional regulatory region of an indicator gene;  
       [0045] (b) measuring the expression level of the reporter gene; and  
       [0046] (c) selecting a compound which decreases the expression level of the reporter gene compared to a control where the candidate compound has not been contacted,  
       [0047] wherein the indicator gene is B1799 gene.  
       [0048] (12) A method of screening for a therapeutic agent for an allergic disease, the method comprising the steps of:  
       [0049] (a) contacting a candidate compound with a protein encoded by an indicator gene or a gene functionally equivalent thereto;  
       [0050] (b) measuring the activity of the protein; and  
       [0051] (c) selecting a compound which decreases the activity of the protein compared to a control where the candidate compound has not been contacted,  
       [0052] wherein the indicator gene is B1799 gene.  
       [0053] (13) A therapeutic agent for an allergic disease comprising as the main ingredient a compound obtainable by the screening method according to any one of (8), (10), (11), and (12).  
       [0054] (14) A therapeutic agent for an allergic disease, which comprises as a main ingredient an antisense DNA that contains a sequence complementary to a sequence comprising at least 15 continuous nucleotides of the sense strand sequence of an indicator gene, wherein the indicator gene is B1799 gene.  
       [0055] (15) A therapeutic agent for an allergic disease, which comprises as a main ingredient an antibody which binds to a protein encoded by an indicator gene, wherein the indicator gene is B1799 gene.  
       [0056] (16) An animal model of allergic disease, wherein said animal is a transgenic nonhuman vertebrate, in which the expression level of an indicator gene, or a gene functionally equivalent thereto, has been increased in T cells, wherein the indicator gene is B1799 gene.  
       [0057] (17) A kit for screening for a therapeutic agent for an allergic disease, the kit comprising a polynucleotide comprising at least 15 continuous nucleotide sequence of an indicator gene or its complementary sequence, wherein the indicator gene is B1799 gene.  
       [0058] (18) A kit for screening for a therapeutic agent for an allergic disease, the kit comprising an antibody which binds to a polypeptide consisting an amino acid sequence encoded by an indicator gene and cells expressing the indicator gene, wherein the indicator gene is B1799 gene.  
       [0059] The present invention also relates to a method of treating an allergic disease, which comprises the step of administering a compound of any one of the following (A) to (C). In addition, the present invention relates to the use of a compound of any one of the following (A) to (C) for producing a therapeutic agent for an allergic disease:  
       [0060] (A) a compound obtainable by the screening method according to any one of (8), (10), (11), and (12) above;  
       [0061] (B) an antisense DNA having a sequence complementary to a sequence comprising at least 15 continuous nucleotides of the sense strand sequence of B1799 gene; and  
       [0062] (C) an antibody which binds to a protein encoded by B1799 gene.  
       [0063] Furthermore, the present invention relates to a method of producing an allergic disease animal model, the method comprising the step of increasing the expression level of B1799 gene or a gene functionally equivalent thereto in T cells in a nonhuman vertebrate. In addition, the present invention relates to the use of a transgenic nonhuman vertebrate, in which the expression level of B1799 gene or a gene functionally equivalent thereto has been increased, as an allergic disease animal model. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0064]FIG. 1 shows the amount of expression of B1799 in atopic dermatitis patient samples.  
     [0065]FIG. 2 shows the amount of expression of B1799 in leukocytes in peripheral blood.  
     [0066]FIG. 3 shows the amount of expression of B1799 in T cell subgroups derived from peripheral blood.  
     [0067]FIG. 4 shows the (a) the change in the rate of dead cells during T cell activated cell death, and (b) the change in the expression of B1799 during T cell activated cell death.  
     [0068]FIG. 5 shows the change in the expression of B1799 due to T cell stimulation.  
     [0069]FIG. 6 shows the structure of B1799.  
     [0070]FIG. 7 shows the predicted tertiary structure model of B1799 protein. Arg343 of Gyp1p and Arg973 of B1799 are shown by arrows. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0071] The present invention relates to a method of testing for allergic diseases using the expression level of B1799 gene as an indicator in leukocytes of a test subject. According to the present invention, the B1799 gene was demonstrated to show a higher expression level in a patient group with allergic diseases as compared to the group of healthy subjects. Therefore, allergic diseases can be tested using the expression level of the B1799 gene as an indicator. Herein, the B1799 gene is referred to as “the indicator gene.” The term “allergic disease” used herein is a general term for diseases involving allergic reactions. More specifically, this term is defined as a disease for which an allergen is identified, a strong correlation between exposure to the allergen and the onset of the pathological change is demonstrated, and the pathological change has been proven to have an immunological mechanism. Herein, an immunological mechanism means that leukocytes show an immune response to allergen stimulation. Examples of allergens are mite antigens, pollen antigens, etc.  
     [0072] Representative allergic diseases include atopic dermatitis, allergic rhinitis, pollinosis, bronchial asthma and insect allergy. Allergic diathesis is a genetic factor that is inherited from allergic parents to children. Familial allergic diseases are also called atopic diseases, and their causative factor that can be inherited is atopic diathesis. Among allergic diseases, asthma is a general term for diseases accompanied with respiratory organ symptoms.  
     [0073] As used herein, the term “B1799 gene” refers to human B1799 gene identified in Examples and a gene located in the same locus or a gene located in a homologous locus in another organism. For example, in the present invention, the B1799 gene includes an arbitrary allele in the same locus as or homologous locus to that of the human B1799 gene identified in Examples. The nucleotide sequence of the transcription product of the human B1799 gene identified in Examples and the amino acid sequence of a protein encoded by the gene is shown in SEQ ID Nos: 1 and 2, respectively. Specifically, in the present invention, the B1799 gene includes the human B1799 gene (SEQ ID NO:1) identified in Examples, its polymorphic variants (including SNPs), mutants, splicing variants, and homologues from other organisms. Since the expressional control for these genes are presumed to be substantially identical or similar to that of the human B1799 gene shown in SEQ ID NO:1, tests for allergic diseases according to the present invention can be carried out by detecting their expression.  
     [0074] Specifically, in the present invention, the B1799 gene is substantially identical to an endogenous gene that comprises the following nucleic acid. The endogenous gene refers to an artificially unmodified gene contained in an organism in nature, and a gene that comprises the same nucleotide sequence is substantially identical to the endogenous gene. Furthermore, herein the term “gene” refers to a transcription product or nucleic acid (e.g., DNA, RNA, etc.) encoding the same.  
     [0075] (a) A nucleic acid that comprises a nucleotide sequence selected from the nucleotide sequence of SEQ ID NO:1.  
     [0076] (b) A nucleic acid comprising a nucleotide sequence of a coding sequence of SEQ ID NO:1, which contains one or more nucleotide substitutions, deletions and/or insertions.  
     [0077] (c) A nucleic acid encoding at least 15 continuous amino acids of the amino acid sequence of SEQ ID NO:2.  
     [0078] (d) A nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:2 containing one or more amino acid substitutions, deletions and/or insertions.  
     [0079] (e) A nucleic acid which hybridizes under a stringent condition to a nucleic acid containing at least 50 continuous nucleotides of SEQ ID NO:1 but no nucleotide sequence derived from any sequence other than SEQ ID NO:1.  
     [0080] The nucleic acid according to (a) includes those containing preferably at least 40, more preferably 60, 100, 200, 500, and 1,000 or more continuous nucleotides of the nucleotide sequence of SEQ ID NO:1 (more preferably the coding region of SEQ ID NO:1; nt 348 to nt 4244), and most preferably its 5922 nucleotides (i.e., the whole length). Such nucleic acids include polymorphic variants, mutants, and splicing variants of the gene primarily within the same species. Examples of the nucleic acid according to (a) are those containing the nucleotide sequences of at least 30, more preferably 35, 40, 45, and 50 or more continuous nucleotides of the nucleotide sequence of SEQ ID NO:1. Nucleotide sequence is preferably selected from the coding region of SEQ ID NO:1 (nt 348 to nt 4244), and any desirable numbers of nucleotide sequences may be selected. In the case of selecting a plurality of nucleotide sequences, it is desirable to select nucleic acids comprising the continuous nucleotide sequences at 2 or more, preferably 3, 4, and 5 or more different sites so that the nucleic acids do not overlap.  
     [0081] The nucleic acid according to (b) includes those containing the nucleotide sequences of the coding region of SEQ ID NO:1 (nt 348 to nt 4244 in SEQ ID NO:1) in which preferably 15% or less, more preferably 10%, 8%, 5%, and 1% or less of the total nucleotides is substituted, deleted, and/or inserted. Such nucleic acids may include counterpart genes of other close relative species, polymorphic variants, mutants, and splicing variants thereof. Such nucleic acids are those containing nucleotide sequences having the sequence identity of preferably 85% or more, more preferably 90%, 92%, 95%, and 99% or more to the coding region set forth in SEQ ID NO: 1.  
     [0082] Nucleic acid or amino acid sequence identity can be determined using, for example, a BLAST program (Altschul, S. F. et al., 1990, J. Mol. Biol. 215: 403-410). More specifically, nucleotide sequence identity is determined using the blastn program, while amino acid sequence identity using blastp program, and sequence identity search is conducted, for example, using default parameters with all filters containing Low complexity off at the BLAST website of the National Center for Biotechnology Information (NCBI) (Altschul, S. F. et al. (1993) Nature Genet. 3: 266-272; Madden, T. L. et al. (1996) Meth. Enzymol. 266: 131-141; Altschul, S. F. et al. (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. and Madden, T. L. (1997) Genome Res. 7: 649-656). For example, using the blast2sequences program (Tatiana, A. et al. (1999) FEMS Microbiol Lett. 174: 247-250) for comparing two sequences, the two sequences can be aligned to determine the sequence identity. In this case, gaps are similarly treated as mismatches to calculate the identity value for the entire coding region of SEQ ID NO:1.  
     [0083] The nucleic acid according to (c) includes those encoding preferably at least 20, more preferably 30, 50, 100, 300, and 500 or more continuous amino acids of the amino acid sequence of SEQ ID NO:2, and most preferably its 1299 amino acids (i.e., the whole length). Such nucleic acids include, similarly as in the above-described (a), polymorphic variants, mutants, and splicing variants of genes primarily within the same species of organism. Furthermore, the nucleic acid according to (c) includes those containing partial nucleotide sequences encoding the amino acid sequence of at least 8, more preferably 10, 15, 20, and 25 or more continuous amino acids of SEQ ID NO:2 at 2 or more, preferably 3 or more, more preferably 4 and 5 or more sites so that each nucleic acids do not overlap.  
     [0084] The nucleic acid according to (d) includes those encoding the amino acid sequence of SEQ ID NO:2 in which preferably 15% or less, more preferably 10%, 8%, 5%, and 1% or less of the whole number of its amino acid residues are substituted, deleted, and/or inserted. These nucleic acids may contain untranslated sequences. Such nucleic acids include a counterpart gene of other close relative species, polymorphic variants, mutants, and splicing variants. Such nucleic acids are those encoding proteins comprising amino acid sequences having the sequence identity of preferably 85% or more, more preferably 90%, 92%, 95%, and 99% or more to the amino acid sequence of SEQ ID NO:2. Treating gaps similar to mismatches, the identity value relative to the whole amino acid sequence of SEQ ID NO:2 is calculated. Amino acid sequence identity can be determined according to the above-described method.  
     [0085] The nucleic acid according to (e) includes those hybridizing to nucleic acids comprising preferably at least 80, more preferably 100, 120, and 200 or more continuous nucleotides of SEQ ID NO:1 (more preferably its coding region; nt 348 to nt 4244), and substantially containing no other nucleotide sequence than that of SEQ ID NO:1 (more preferably its coding region; nt 348 to nt 4244) under the stringent condition. Such nucleic acids may be those hybridizing under the stringent condition to probes that have been prepared, for example, using the nucleic acid containing the nucleotide sequence of SEQ ID NO:1 as a template. Probes of 50 to several hundreds of nucleotides long can be synthesized, for example, by the DNA synthesis method, or by the random prime method, nick translation method and PCR method.  
     [0086] The stringent condition for the nucleic acid according to (e) is that hybridization is carried out in a hybridization solution preferably containing NaCl in the range of about 0.5 to about 0.9 M at 60° C., preferably at 62° C., more preferably at 65° C., and then washing is performed at the same temperature as for hybridization in 1×SSC, preferably in 0.5×SSC, more preferably in 0.2×SSC, still more preferably in 0.1×SSC for 1 h. In this case, the temperature conditions of hybridization and washing that greatly influence the stringency can be adjusted according to the melting temperature (Tm) of the probe, which depends on the ratio of constituent nucleotides of the probe to the base pairs to which the probe hybridizes, the length of the probe, and the composition (concentrations of salts and formamide) of a hybridization solution. Considering these conditions, those skilled in the art can empirically set up the appropriate condition to confer the equivalent stringency. As a hybridization solution, for example, 4×SSC, preferably ExpressHyb™ Hybridization Solution (Clontech) etc. may be used.  
     [0087] The B1799 gene preferably encodes a GTPase activating protein (GAP). GAP is a protein binding to the GTP binding protein. The GTPase activity of the GTP binding protein is enhanced by the interaction with GAP (Scheffzek, K. et al., Trends Biochem. Sci., 1998, 23(7): 257-262). The GTP binding protein that plays an important role in signal transduction, is activated when GTP binds to it and inactivated when the bound GTP is converted to GDP. The GTP binding protein itself has GTPase activity, and GAP enhances its GTPase activity. GAP controls signal transduction by assisting conversion of the GTP binding protein to the inactivated form. Furthermore, the B1799 gene of the present invention preferably encodes a protein having a phosphotyrosine interaction domain (PID) that is involved in the binding between proteins at the phosphotyrosine binding domain and a TBC domain. The PID domain is conserved in proteins interacting with phophotyrosine, and is found in a group of proteins involved in various biological processes comprising signal transduction mediated by cell surface receptors or occurring during protein transport (Bork, P. and Margolis, B., 1995, Cell 80(5): 693-694). The TBC domain was discovered in Tbc1 (Richardson, P. M. and Zon, L. I., 1995, Oncogene 11(6): 1139-1148), and is commonly seen in oncogene tre-2 and yeast cell cycle regulating factor BUB2 and cdc16. The TBC domain exists in Gyp6 and Gyp7, and is considered to be a domain widely distributed in GAPs of Rab-like GTPases (Neuwald, A. F., 1997, Trends Biochem. Sci. 22(7): 243-244).  
     [0088] In a preferred embodiment of the present invention, the B1799 gene is a nucleic acid encoding a transcription product, which is transcribed from a promoter regulating the transcription of a genomic DNA that encodes mRNA comprising the nucleic acid of SEQ ID NO: 1 in a native human cell or from the counterpart of the promoter in other species.  
     [0089] Specifically, the method of testing for allergic diseases according to the present invention includes the steps of: (a) measuring the expression level of the indicator gene (i.e., B1799 gene) in a biological sample from a test subject; (b) comparing the expression level with that in a corresponding sample from a healthy subject; and (c) judging the test subject as being affected with an allergic disease if the expression level of the indicator gene in the biological sample of the test subject is enhanced compared to that in the healthy subject. Since the B1799 gene can be used as an indicator in the present invention, the B1799 gene is herein also called simply an indicator gene. As described above, the B1799 gene of the present invention includes, beside the human gene, homologues of other species. Therefore, unless otherwise stated, B1799 gene of species other than human means endogenous homologues in those species. If the expression level of the B1799 gene in a test subject is higher than that in a healthy subject, the test subject is judged to have an allergic disease. Alternatively, a standard value may be set in advance based on the expression level of the B1799 gene in a healthy subject, and the expression level in a test subject can be compared to the standard value. Methods to set a standard value and acceptable range based on the measured value of the indicator gene are well known in the art. For example, a range of ±2 S.D. can be used as the acceptable range. If the expression level of the indicator gene in the test subject is within the acceptable range, the test subject is suggested to be normal with respect to allergic diseases; and if the expression level is over the range, the subject is judged to have an allergic disease.  
     [0090] The present testing method can be conducted by using not only the B1799 gene alone but its combination with other genes that serve as an indicator for allergic diseases. The accuracy of the test can be improved by detecting the expression of multiple genes in combination. Generally, allergic disease patients form a heterogeneous population, and thus, a more accurate diagnosis is enabled by using multiple genes as indicators.  
     [0091] In this invention, expression of the indicator gene includes both transcription and translation. That is, the expression level of the indicator gene can be the level of mRNA or protein corresponding to the gene. Therefore, the method of testing for allergic diseases of the present invention may be performed based on comparing the level of mRNA corresponding to the indicator gene, or the level of the protein encoded by the gene. The measurement of the mRNA level can be carried out according to known genetic analysis methods. Specifically, one can use, for example, a hybridization technique using the nucleic acids that hybridize to the indicator gene as probes, or a gene amplification technique using DNAs that hybridize to that gene as primers.  
     [0092] The probes or primers used for the testing of the present invention can be designed based on the nucleotide sequence of the indicator gene. For example, the nucleotide sequence of the human B1799 gene identified in Example and the amino acid sequence encoded by the gene are described in SEQ ID NOs: 1 and 2, respectively. In general, genes of higher animals are often accompanied by polymorphism. Many molecules produce isoforms comprising different amino acid sequences from each other during the splicing process. Any genes can be used as the indicator gene of the present invention, even though they differ from the above-mentioned gene in the nucleotide sequence due to polymorphism, alternative splicing, and such.  
     [0093] As a primer or probe can be used a polynucleotide comprising at least 15, preferably at least 20, more preferably at least 30, and much more preferably at least 35 continuous nucleotides of the nucleotide sequence of the B1799 gene or its complementary sequence. Herein, the term “complementary sequence” refers to the nucleotide sequence of one strand of a double stranded polynucleotide, which is composed of A:T (or A:U) and G:C base pairs, to the other strand. Furthermore, such polynucleotides may comprise nucleotide sequences other than a part of the nucleotide sequence of B1799 or its complementary sequence. In addition, polynucleotides that are at least 70%, preferably 80% or more, more preferably 90% or more, and much more preferably 95% or more identical to the nucleotide sequence of at least 20, preferably 30, more preferably 35, and much more preferably 40 continuous nucleotides of the B1799 gene or its complementary sequence can be used as reagents for testing allergic diseases of the present invention. The degree of homology between nucleotide sequences can be determined by an algorithm, such as BLAST. The detection of the indicator gene is enabled by specific hybridization of the above-mentioned polynucleotides to the gene. A specific hybridization can be confirmed if there is no significant cross-hybridization with DNA and/or RNA encoding other genes under the above-mentioned stringent conditions.  
     [0094] Such polynucleotides are useful as probes to detect the indicator gene, and as primers to amplify the indicator gene. When used as a primer, those polynucleotides have a chain length of usually 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, DNAs comprising the whole sequence of the indicator gene (or its complementary strand), or its partial sequence that contains at least 15-bp, are used. When used as a primer, its 3′ region must be complementary to the indicator gene, while the 5′region can be linked to a restriction enzyme-recognition sequence or tag.  
     [0095] “Polynucleotides” as used in the present invention may be either DNA or RNA. These polynucleotides may be either synthetic or naturally-occurring. Also, DNA used as a probe for hybridization is usually labeled. Examples of labeling methods are described below. Herein, the term “oligonucleotide” refers to polynucleotides with relatively low degree of polymerization. A chain length of an oligonucleotide is, for example, within 100 nucleotides. Oligonucleotides are included in polynucleotides. The labeling methods are as follows:  
     [0096] nick translation labeling using DNA polymerase I;  
     [0097] end labeling using polynucleotide kinase;  
     [0098] fill-in end labeling using Klenow fragment (Berger S L, Kimmel A R. (1987) Guide to Molecular Cloning Techniques, Methods in Enzymology, Academic Press; Hames B D, Higgins S J (1985) Genes Probes: A Practical Approach. TRL Press; Sambrook J, Fritsch E F, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press);  
     [0099] transcription labeling using RNA polymerase (Melton D A, Krieg P A, Rebagkiati M R, Maniatis T, Zinn K, Green M R. (1984) Nucleic Acid Res., 12, 7035-7056); and  
     [0100] non-radioisotopic labeling of DNA by incorporating modified nucleotides (Kricka L J. (1992) Nonisotopic DNA Probing Techniques. Academic Press).  
     [0101] For testing for allergic diseases using hybridization techniques, for example, Northern hybridization, dot blot hybridization, or DNA microarray technique may be used. Furthermore, gene amplification techniques, such as RT-PCR method may be used. In RT-PCR, RNA is extracted from a biological sample, cDNA is prepared by its reverse transcription, and PCR is conducted using the obtained cDNA as the template to determine the gene expression level. By using the PCR amplification monitoring method during the gene amplification step in RT-PCR, one can achieve more quantitative analysis for the gene expression of the present invention.  
     [0102] In the PCR gene amplification-monitoring method, the detection target (DNA or reverse transcript of RNA) is hybridized to probes that are dual-labeled at both ends with different fluorescent dyes whose fluorescence cancels each other out. When the PCR proceeds and Taq polymerase degrades the probe as a result of its 5′-3′ exonuclease activity, the two fluorescent dyes become distant from each other and the fluorescence is detected. The fluorescence is detected in real time. By simultaneously measuring a standard sample in which the copy number of the target is known, it is possible to determine the copy number of the target in the subject sample with the cycle number where PCR amplification is linear (Holland P. M. et al., 1991, Proc. Natl. Acad. Sci. USA 88: 7276-7280; Livak K. J. et al., 1995, PCR Methods and Applications 4(6): 357-362; Heid C. A. et al., 1996, Genome Research 6: 986-994; Gibson E. M. U. et al., 1996, Genome Research 6: 995-1001). For the PCR amplification-monitoring method, for example, ABT PRISM7700 (PE Biosystems) may be used.  
     [0103] The method of testing for allergic diseases of the present invention can be also carried out by detecting a protein encoded by the indicator gene. Hereinafter, a protein encoded by the indicator gene is described as an indicator protein. For example, for such test methods Western blotting, immunoprecipitation method, and ELISA method may be employed using an antibody that binds to the indicator protein.  
     [0104] Antibodies that bind to the indicator protein used in the detection may be produced by techniques well known to those skilled in the art. Antibodies used in the present invention may be polyclonal or monoclonal antibodies (Milstein C. et al., 1983, Nature 305 (5934): 537-40). For example, polyclonal antibodies against the indicator protein may be produced by collecting blood from mammals sensitized with the antigen, and separating the serum from this blood using known methods. As polyclonal antibodies, serum containing polyclonal antibodies may be used. If desired, a fraction containing polyclonal antibody can be further isolated from this serum. Alternatively, monoclonal antibodies may be obtained by isolating immune cells from mammals sensitized with the antigen, fusing these cells with myeloma cells, and such, cloning the obtained hybridomas, and collecting the antibodies from the culture of the hybridomas.  
     [0105] For detecting an indicator protein, these antibodies may be appropriately labeled. Alternatively, instead of labeling the antibody, a substance that specifically binds to the antibody, for example, protein A or protein G, may be labeled to indirectly detect the indicator protein. Specifically, one example of a detection method is ELISA.  
     [0106] A protein or its partial peptide used as an antigen may be obtained, for example, by inserting a gene encoding the protein or its portion into an expression vector, introducing the vector into an appropriate host cell to produce a transformant, culturing the transformant to express the recombinant protein, and purifying the expressed recombinant protein from the culture or the culture supernatant. Alternatively, oligopeptides consisting of a partial amino acid sequence of the protein can be chemically synthesized to be used as the immunogen.  
     [0107] Furthermore, the method of testing for allergic diseases of the present invention can be conducted using the activity of indicator protein in leukocytes as an indicator. The activity of indicator protein refers to the biological activity possessed by the protein. More specifically, activity such as GAP activity, i.e. activation of the GTP binding protein, can be mentioned as the activity of the indicator protein of the present invention. Alternatively, its interaction with the GTP binding protein can be used as the index of the activity. Detection of the activity of the indicator protein can be preformed based on conventional methods. More specifically, the GAP activity can be determined by measuring the GTPase activity of a specific GTP binding protein on which this protein acts. For example, Gyp6 protein specifically acts on Ypt6 protein, a GTP binding protein, and enhances its GTPase activity. Therefore, by detecting the increase in GTPase activity of Ypt6 protein, the activity of Gyp6 protein can be measured (Strom M. et al., 1993, Nature 361(6414): 736-739). Thus, by measuring the activity of a specific GTP binding protein on which the indicator protein B1799 of the present invention acts, the activity of the indicator protein can be measured.  
     [0108] Normally, in the testing method of this invention, a biological sample from a subject is used as a test sample. A biological sample such as peripheral blood T cells can be used. Leukocytes used for the test may be purified or partially purified. Preferably, leukocytes prepared from peripheral blood, more preferably T-cells, of subjects are used as the test sample. T-cells can be prepared from peripheral blood by known methods. Specifically, for example, heparinized blood is collected, diluted, fractionated by centrifugation using Ficoll to separate PBMC. The separated PBMC may be used as it is as the sample for the test for allergic diseases of the present invention. PBMC contains lymphocytes and monocytes. As shown in Examples, among these blood cells, the indicator gene is highly expressed in T cells. The expression level of the indicator gene can be measured without great influence of cells other than T cells. Direct analysis of not a purified T-cell fraction but a lymphocyte fraction as a test sample enables a convenient bed-side test. Alternatively, T cells can be isolated by allowing them to be specifically adsorbed by microbeads to which the anti-CD3 antibody has been immobilized.  
     [0109] The intracellular mRNA of the indicator gene or the indicator protein can be measured in disintegrated blood cells. It is also possible to measure the indicator protein in blood. For the preparation of T cell lysate and extraction of mRNA, kits such as an RNeasy Mini™ (Qiagen) and ISOGEN™ (Nippon Gene) on the market may be conveniently used.  
     [0110] The measured value of expression level of the indicator gene in test biological samples can be corrected by known methods. The changes in the gene expression level in the biological samples can be compared using the corrected values. The measured value of the expression level of the genes that are to be used as indicators in the present invention may be corrected based on the measured value of the expression level of the genes (such as housekeeping genes) that are expressed in the biological samples and do not largely fluctuate in their expression levels regardless of the condition of the cell. Examples of such genes are β-actin gene and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.  
     [0111] According to the test method of the present invention, the expression level of the indicator gene in leukocytes from a test subject and the expression level in the corresponding leukocytes from a healthy subject are measured. It is examined whether the expression level in the leukocytes of the test subject is increased compared to that in the healthy subject. The increase in the expression level of the indicator gene in the leukocytes from the test subject is correlated with allergic diseases.  
     [0112] The testing for an allergic disease according to the present invention includes, for example, those as described below. Even a patient who cannot be judged as having an allergic disease by the ordinary testing in spite of showing allergic disease-like symptoms can be easily judged as an allergic disease patient by the testing of this invention. More specifically, the elevation of the indicator gene expression in patients with symptoms suspect of an allergic disease indicates that the symptoms would probably be those of an allergic disease. Allergic disease-like symptoms are exemplified by dermatitis (itching, flare), rhinitis (nasal congestion, running nose, sneeze), and asthma (stridor, dyspnea). The method of testing for allergic diseases according to the present invention includes tests for determining whether such allergic disease-like symptomsare caused by allergic reaction. Although these symptoms are also observed in xeroderma, cold syndrome (coryza), bronchitis, and such, it is possible to determine whether these symptoms are caused by allergic reaction or not, according to the test method of the present invention. In addition, the method of testing for allergic diseases of the present invention includes tests to determine whether a subject has allergic diathesis or not. Specifically, the test is conducted for a subject without noticeable symptom, and the subject is determined to have allergic diathesis if an increase in the expression level of the indicator gene is detected. For treatment of an allergic disease-like symptom, it is a very important process to determine whether allergic reaction is the cause of the symptom or not. The testing method of this invention can provide extremely important information for identifying the cause of diseases. Alternatively, even without referring to information of symptoms, allergic disease patients can be screened by testing samples and by screening samples that highly express the indicator gene.  
     [0113] In addition, the testing method is useful to judge whether allergic symptoms are getting ameliorated or not. The expression level of the indicator gene of this invention increases in T cells which is contained in PBMC of patients with an allergic disease. T cells play a central role in immune response. Therefore, the decreased expression level of the indicator gene in patients diagnosed as having an allergic disease indicates amelioration of allergic symptoms, and the increased expression level indicates that the allergic symptoms would be in progress.  
     [0114] Furthermore, the expression of the indicator gene B1799 has been revealed to be high in T cells among leukocytes, especially in T cells having the phenotypes of CD4 +  and CD45RO +  (i.e., memory T cells). Therefore, the indicator gene can be used as a marker for these T cells. Moreover, the expression of the indicator gene is significantly induced in activated cell death of T cells. In addition, the expression is also induced by activation stimuli by a stimulant such as ionomycin. Thus, since the expression of the indicator gene reflects the state of T cell activation, the activation level of T cells may be determined by measuring the expression of the indicator gene in T cells.  
     [0115] The present invention also relates to the use of a transgenic nonhuman animal, in which the expression level of the indicator gene B1799 or a gene functionally equivalent thereto is elevated in T cells, as an allergic disease model animal. The allergic disease model animal is useful in clarifying changes in vivo in allergy. Furthermore, the allergic disease model animal of this invention is useful in the assessment of therapeutic agents for an allergic disease.  
     [0116] The present invention demonstrated the elevated expression level of the indicator gene in T cells. Therefore, animals in which the expression level of the B1799 gene or a gene functionally equivalent thereto is artificially elevated in T cells can be utilized as the allergic disease model animal. Herein, transgenic animals refer to animals genetically modified by insertion, substitution, and/or deletion of one or more nucleotides in their genomic DNAs. This allergic disease model animal is useful in clarifying changes in vivo in allergy. Furthermore, the allergic disease model animal of this invention is useful in assessing and screening for therapeutic agents for an allergic disease. In this case, the increased expression level in T cells includes the increased expression level of the indicator gene in the whole blood cells. That is, the expression level elevation of the indicator gene includes that in not only T cells but also blood cells as a whole or the whole body.  
     [0117] Genes functionally equivalent to the B1799 gene used in the present invention refer to those encoding proteins having the activity equivalent to that of the protein (indicator protein) encoded by the indicator gene B1799. Such genes include artificial variants of the human B1799 gene or its counterparts from other organisms. For example, in the case of inserting a gene into an expression vector, deletion of nucleotides corresponding to several amino acid residues at the N- or C-terminus, and addition of nucleotides corresponding to a tag peptide sequence or other amino acid sequence derived from the expression vector are frequently performed. The resulting proteins are different in the amino acid sequence from the endogenous protein in natural cells, but their activities are substantially equivalent to that of the endogenous protein. Herein, genes encoding such proteins are referred to as genes functionally equivalent to the endogenous gene.  
     [0118] One example of the activities of the indicator protein used in this invention is the GAP activity. For example, a gene encoding the human B1799 protein of SEQ ID NO:2 in which one or more amino acids are deleted, inserted, and/or substituted and which maintains the GAP activity, can be used to prepare the transgenic model animal of this invention. Such modified proteins should have a homology of, for example, 90% or more, preferably 95% or more and, still more preferably 99% or more to the amino acid sequence of the human B1799 protein. Furthermore, genes which comprises nucleotide sequences hybridizing to the sequence of SEQ ID NO:1 under stringent conditions, and encode proteins having the GAP activity, can also be used as genes functionally equivalent to the B1799 gene.  
     [0119] The model animal of this invention can be used as a model for an allergic disease, for example, in a method comprising the steps of: (a) detecting a phenotype of the model animal, and (b) correlating the difference of the phenotype of said model animal from the corresponding phenotype of a control animal, whose expression level of the indicator gene in T cells is lower compared to that of said model animal, with an allergic disease. Examples of the control animals include non-transgenic animal having the same genetic background as that of said model animal and a transgenic animal in which an empty vector has been introduced. The phenotype may be any desired phenotypes including allergic symptoms such as dermatitis, rhinitis, and asthma, or the activation of immunocytes or changes in gene expression.  
     [0120] In the present invention, it is highly significant to assess roles of the indicator gene B1799 and effects of drugs targeting this gene using transgenic animals in which the expression level of the indicator gene is elevated in T cells, as the allergic disease model animal. Furthermore, the allergic disease model animals according to the present invention are useful not only in screening for pharmaceutical agents for the treatment or prophylaxis of an allergic disease as described below but also in elucidating the mechanism of allergic diseases, and, furthermore, testing the safety of screened compounds. For example, if the allergic disease model animals according to this invention either develop clinical manifestations of atopic dermatitis or allergic asthma, or show changes in measured values associated with any allergic diseases, it is possible to construct a system for screening for compounds that can cure the allergic conditions.  
     [0121] Herein, the elevation of the expression level refers to any of the states that: a target gene introduced as an exogenous gene is allowed to be expressed; transcription of a gene inherent in the host and/or its translation into protein are/is increased; and, decomposition of the protein that is a translation product is suppressed. The gene expression level can be confirmed by, for example, quantitative PCR as described in Examples. Furthermore, the expression level or activity of the translation product protein can be confirmed by comparing it with that in a normal state.  
     [0122] A typical transgenic animal is an animal which has been transfected with a gene of interest and allowed to express the gene. In another type of transgenic animals, the half-life of mRNA may be extended by removing a sequence from the untranslated region (UTR) of mRNA, which renders RNA unstable. Furthermore, in another type of transgenic animals, a mutation is introduced into the coding region of the indicator gene to increase its activity or modify the amino acid sequence of the gene product protein so as to be hardly decomposed. Examples of mutation in the amino acid sequence are substitution, deletion, insertion, or addition of amino acid residues. In addition, mutation in the transcriptional regulatory region of the indicator gene also enables to enhance the expression of the gene.  
     [0123] Methods for obtaining transgenic animals targeting a specific gene are well-known in the art. That is, a transgenic animal can be obtained by a method where the gene and ovum are mixed and treated with calcium phosphate; a method where the gene is directly introduced into pronuclei of oocyte under a phase contrast microscope using a micropipette (microinjection method, U.S. Pat. No. 4,873,191); a method where the gene is introduced into embryonic stem cells (ES cells), etc. Furthermore, other methods include a method where ovum is infected with a gene-inserted retroviral vector and a sperm vector method where a gene is introduced into ovum mediated by sperm. The sperm vector method is a gene recombination technique for introducing an exogenous gene and performed by allowing an exogenous gene to adhere to a sperm or to be incorporated in a sperm by the electroporation method or such and fertilizing ovum with the sperm (M. Lavitranoet, et al., Cell, 57, 717, 1989).  
     [0124] Transgenic animals used as the allergic disease model animal of the present invention can be produced using all the vertebrates except for humans. More specifically, transgenic animals in which various genes have been introduced and their expression levels are modified are produced using vertebrates such as mice, rats, rabbits, miniature pigs, goats, sheep, monkeys, and cattle.  
     [0125] Furthermore, the present invention relates to methods of screening for therapeutic agents for allergic diseases. According to the present invention, the indicator gene shows a statistically significant increase in its expression level in the case of allergic diseases. Therefore, it is possible to obtain a therapeutic agent for an allergic disease by selecting a compound capable of reducing the expression level of this gene. Herein, compounds that reduce the expression level of a gene refer to those having inhibitory effects on any steps of the transcription or translation of the gene, stability of a transcript (mRNA) or a translation product (protein), and the expression of a gene function such as protein activity.  
     [0126] A method of screening for a therapeutic agent for allergic diseases of the present invention can be carried out either in vivo or in vitro. The in vivo screening method can be carried out, by the steps of:  
     [0127] (a) administering a candidate compound to a test animal;  
     [0128] (b) measuring the expression level of the indicator gene in leukocytes of the test animal; and  
     [0129] (c) selecting the compound that reduces the expression level of the indicator gene, compared to a control without administration of the compound.  
     [0130] As a test animal in the screening method of the present invention, any desirable animal that expresses the indicator gene of the present invention may be used. Specifically, such animals include, for example, mice, rats, guinea pigs, rabbits, cats, pigs, miniature pigs, goats, cattle, sheep, and monkeys. Preferably animals with elevated expression of the indicator gene are used. Furthermore, by using animals that exhibit allergic symptoms, the effect of candidate compounds on the symptoms can be evaluated.  
     [0131] The effect of a candidate drug compound can be detected by administering the candidate drug compound to a test animal and monitoring the action of the compound on the expression level of the indicator gene in leukocytes of the animal. The change in the expression level of the indicator gene in leukocytes of the test animal can be monitored by the same method as the above-mentioned test method of the present invention. Based on the detection result, candidate drug compounds for allergic diseases can be screened by selecting candidate drug compounds that reduce the expression level of the indicator gene.  
     [0132] More specifically, the screening of the present invention can be carried out by collecting leukocytes from a test animal to which a candidate compound has been given, and comparing the expression level of the indicator gene with that in corresponding leukocytes collected from an animal without administration of the candidate compound. Leukocytes used for the test may be purified or crude samples and include PBMC and purified T cells. Preferably, purified T-cells are used. Methods for collecting and preparing these biological samples are known in the art.  
     [0133] These screening methods enable the selection of drugs relating to the expression of the indicator gene in various ways. Specifically, for example, drug candidate compounds having the following activity can be found:  
     [0134] reduce the transcriptional activity of the indicator gene;  
     [0135] reduce the translation level from the transcript of the indicator gene;  
     [0136] inhibit the activity of translation product of the indicator gene; and  
     [0137] reduce the stability of the transcript of the indicator gene or accelerate its decomposition.  
     [0138] The above-described screening method of the present invention also includes a method comprising the step of stimulating test animals with an allergen before and/or after the administration of a candidate compound. When the allergen stimulation is performed prior to the administration of a candidate compound, it is possible to detect the activity of a candidate compound that inhibits immune response occurring after the allergen stimulation. Compounds obtainable by this screening method are expected to have therapeutic effects on an allergic disease. When allergen stimulation is conducted after the candidate compound administration, it is possible to detect the activity of a candidate compound that suppresses initiation of immune response occurring by the allergen stimulation. Compounds obtainable by this screening method are expected to have prophylactic effects on an allergic disease. An allergen usable in the screening method of this invention includes allergenic substances known in the art. More specifically, allergenic substances well-known in the art include mite, house dust, plant pollen, proteins derived from diverse foods, etc. as the These allergens may be derived from the nature or synthesized by gene recombination technique and the like method. Furthermore, allergens may be protein fragments. Methods for preparing a purified allergen are also well-known in the art.  
     [0139] For example, as a model closely resembling human atopic dermatitis, a spontaneous dermatitis model using NC/Nga mouse has been reported. Administration of the mite antigen (5 μg/ear) into the auricle of this mouse 8 times in total at 2 to 3 days intervals enables the induction of symptoms that closely resemble human atopic dermatitis after two weeks. The screening according to this invention can be conducted by administering a candidate compound to this system and monitoring changes in the expression level of the indicator gene of this invention.  
     [0140] In vitro screening can be performed, for example, by a method in which a candidate compound is contacted with cells expressing the indicator gene to select compounds that reduce the expression level of the indicator gene. The method may be carried out, for example, by the following steps of:  
     [0141] (a) contacting a candidate compound with cells that express the indicator gene;  
     [0142] (b) measuring the expression level of the indicator gene; and  
     [0143] (c) selecting the compound that reduces the expression level of the indicator gene, compared to a control with which the compound has not been contacted.  
     [0144] Peripheral blood leukocytes and cell lines derived from leukocytes can be used as indicator gene-expressing cells. Specifically, T cells can be exemplified as the leukocytes. T cell lines include Molt4 cells and Jurkat cells. Human acute leukemia T cell line Jurkat (ATCC Number TIB-152) can be obtained from ATCC.  
     [0145] In the screening method of the present invention, first a candidate compound is added to the cell. Then, the expression level of the indicator gene in the cell is measured to select the compound that reduces the expression level of the gene compared to that of a cell which has not been contacted with the candidate compound (control).  
     [0146] In the screening method of the present invention, expression level of the indicator gene can be compared by detecting the mRNA level transcribed from the gene or protein level encoded by the gene. When comparing the expression level using mRNA, an mRNA sample is prepared from the cells as described above. When comparing the expression level using protein, a protein sample is prepared from cells. Detection of mRNA and protein can be performed by known methods as described above.  
     [0147] Furthermore, based on the disclosure of this invention, it is possible to obtain the transcriptional regulatory region for the indicator gene of this invention to construct a reporter assay system. A reporter assay system means a assay system for screening for compounds that regulate the transcriptional activity of a transcriptional regulatory region using the expression level of a reporter gene localized downstream of the transcriptional regulatory region as an indicator. Specifically, such screening comprises the steps of:  
     [0148] (a) contacting a candidate compound with cells comprising a reporter gene that is linked to function under the control of a transcriptional regulatory region of the indicator gene;  
     [0149] (b) measuring the expression level of the reporter gene; and  
     [0150] (c) selecting the compound that decreases the expression level of the reporter gene, compared to a control with which the candidate compound has not been contacted.  
     [0151] The DNA region of about 0.5 kb to about 5 kb upstream from the transcription initiation point can be used as a transcriptional regulatory region. Transcriptional regulatory regions may include known transcriptional factor binding sequences, transcriptional regulating elements, and furthermore, CAAT box and TATA box, which are normally seen in the promoter region. Examples of the reporter genes include chloramphenicol acetyltransferase (CAT) gene, luciferase gene, growth hormone genes. The expression level of these reporter genes may be measured by detecting the activity of the expressed protein. Methods for detecting the activity of the proteins are well known in the art. A candidate compound that decreases the expression level of the reporter gene compared to that in a cell without having been contacted with the candidate compound, is selected.  
     [0152] The genomic sequence encoding the human B1799 gene (GenBank Ac. No. AL139230) has been revealed. Based on the sequence, the transcription initiation point and the upstream promoter sequence can be determined. A reporter construct can be prepared by amplifying the promoter region of B1799 gene utilizing PCR and such based on the sequence information, and linking a reporter gene downstream of the promoter region. Specifically, for example, a transcriptional regulatory region used for the screening of the present invention can be obtained as follows. First, screening is performed by a method that uses PCR or hybridization based on the nucleotide sequence of the indicator gene disclosed in the present invention or the sequence of the promoter region of the B1799 gene, to obtain a genomic DNA clone containing the cDNA sequence from a human genome DNA library, such as BAC library and YAC library. Based on the obtained genomic DNA sequence, the transcriptional regulatory region is obtained from upstream of the cDNA disclosed in the present invention. A reporter construct is constructed by cloning the obtained transcriptional regulatory region so that it is positioned upstream of the reporter gene. The resulting reporter construct is transformed into a cultured cell strain to prepare a transformant for screening. By contacting candidate compounds with this transformant, compounds that regulate the expression of reporter genes can be screened.  
     [0153] Furthermore, cells containing a reporter gene linked so as to function under the control of the transcriptional regulatory region of the indicator gene may be cells of so-called knockin animal. Knockin animal refers to a transgenic animal in which an exogenous gene has been inserted into the protein-coding region of the endogenous target gene. Since this knocked-in exogenous gene is inserted downstream of the transcriptional regulatory region of the target gene, it is expressed under a similar expression control to that for the endogenous target gene. It is possible to carry out the screening using the knockin animal or cells obtained from it using the reporter gene as a knockin gene. A screening method using a knockin animal comprises the steps of: (a) administering a candidate compound to an animal in which a reporter gene has been knocked-in to the indicator gene site, (b) measuring the expression level of the reporter gene, and (c) selecting a compound capable of reducing the expression level of the reporter gene compared to a control in which the candidate compound has not been administered. For example, the expression level of the reporter gene in leukocytes, more preferably in T cells of the knockin animal to which a candidate compound is administered or not administered, are measured to select a compound capable of reducing the expression level. Furthermore, a screening method using cells from the knockin animal comprises the steps of: (a) contacting a candidate compound with cells from an animal in which a reporter gene has been knocked-in to the indicator gene site, (b) measuring the expression level of the reporter gene, and (c) selecting a compound capable of reducing the expression level of the reporter gene compared to that in a control in which the candidate compound has not been contacted. For example, the expression level of the reporter gene in leukocytes, more preferably in T cells obtained from a knockin animal are measured in the presence or absence of a candidate compound to select a compound capable of reducing the expression level. These screenings are included in the screening method of the present invention. In the screening using an individual animal in particular, it is preferable to perform the screening using a knockin heterozygote in which one allele is left intact so as to express the indicator gene, while the reporter gene has been knocked in the other allele, to prevent the complete deletion of the function intrinsic to the indicator gene.  
     [0154] Alternatively, screening based on the activity of the indicator protein can be employed as the in vitro screening method of the present invention. More specifically, the present invention relates to a method of screening for therapeutic agents for allergic diseases comprising the steps of:  
     [0155] (a) contacting a candidate compound with a protein encoded by the indicator gene of the present invention or a gene functionally equivalent thereto;  
     [0156] (b) measuring the activity of the protein; and  
     [0157] (c) selecting the compound that reduces the activity of the protein as compared to a control with which the candidate compound has not been contacted.  
     [0158] Herein, genes functionally equivalent to the indicator gene include endogenous B1799 gene that has been artificially modified as described above.  
     [0159] Such screening can be conducted, for example, by allowing host cells to exogenously express the indicator gene and measuring the activity of the indicator protein. In this case, the indicator gene may be inserted into an expression vector, and the resulting recombinant vector is introduced into appropriate hosts such as mammalian cells. The vectors can be introduced into the host by, for example, biological, physical, or chemical methods. Examples of the biological method are a method using viral vectors, a method using a specific receptor, and cell fusion method (via HVJ (Sendai virus), polyethylene glycol (PEG) method, electric cell fusion method, and microcell-mediated chromosome transfer). Examples of the physical method are a microinjection method, electroporation method, and a method using the gene particle gun (gene gun). Examples of the chemical method are a calcium phosphate precipitation method, liposome method, DEAE-dextran method, protoplast method, red cell ghost method, red cell membrane ghost method, and microcapsule method.  
     [0160] Activities of the indicator protein measured in the screening include, for example, binding activity to GTP binding proteins or GAP activity. Furthermore, for example, the indicator protein is suggested to be related to the regulation of intracellular signal transduction in T cells, and the regulation of differentiation or proliferation of T cells. Therefore, using these activities as an index, compounds having an activity to inhibit these activities may be screened. Such compounds obtained by the method repress the function of the indicator protein. As a result, allergic immune response can be suppressed through the inhibition of the indicator protein activity that is induced in the T cells.  
     [0161] The polynucleotide, antibody, cell line, or animal model required for the various screening methods of the present invention may be previously combined into a kit. More specifically, a kit is composed of, for example, a cell (cultured cell, animal, or such) expressing the indicator gene, and a reagent for measuring the expression level of the indicator gene. Reagents used for measuring the expression level of the indicator gene include, for example, oligonucleotides comprising at least 15 continuous nucleotides of the nucleotide sequence of the indicator gene or the complementary sequence thereof. Alternatively, antibodies binding to polypeptides comprising the amino acid sequence of the indicator protein can be used as the reagent. In the kit may be packaged a substrate compound used for the detection of a label, medium and a container for cell culturing, positive and negative standard samples, and furthermore, a manual describing how to use the kit.  
     [0162] Candidate compounds used in these screening include compound preparations, such as immunosuppressors, synthesized by existing chemical methods, compound preparations synthesized by combinatorial chemistry, mixtures of multiple compounds such as extracts from animal or plant tissues, or microbial cultures, and their purified preparations.  
     [0163] Compounds selected by the screening method of the present invention are useful as a therapeutic agent for an allergic disease. Alternatively, antisense DNA capable of inhibiting the expression of the indicator gene in this invention is useful for this purpose. Such antisense DNAs are those comprising sequences complementary to the sequences containing preferably at least 20, more preferably 25, 30, 40, 50, and 100 or more continuous nucleotides of the sense strand of the indicator gene of this invention. The antisense region may be antisense to any region of transcripts (any transcripts before and after the processing, including the intermediary product) of the indicator gene. One example of such region is that containing the translation initiation codon. Furthermore, an antibody binding to the protein encoded by the indicator gene used in the present invention is useful as a therapeutic agent for an allergic disease. Preferable antibodies are those binding to the domain interacting with a GTP binding protein, such as, antibodies to the TBC domain (the amino acid region from position 965 to 1184 of SEQ ID NO: 2 or homologous region thereto), specifically antibodies that identify as an epitope a peptide comprising the Arg residue (e.g., the position 973 Arg residue of SEQ ID NO:2) necessary as the GTPase activating site, are preferred. Antibodies against a phosphotyrosine interaction domain (PID) (the amino acid region from position 167 to 237 or from 415 to 487 of SEQ ID NO: 2, or homologous region thereto) are also preferred. The therapeutic agent for an allergic disease according to the present invention contains a compound selected by the screening method, the antisense DNA, or the antibody as at least one main ingredient, and can be prepared by mixing the ingredient with a physiologically acceptable carrier, excipient, diluent, etc. The therapeutic agent for an allergic disease according to this invention can be administered orally or parenterally to ameliorate allergic symptoms.  
     [0164] For an oral drug, the dosage form can be granules, powder, tablets, capsules, solution, emulsion, suspension, etc. Examples of injections are subcutaneous, intramuscular and peritoneal injections.  
     [0165] Furthermore, in the case where a compound to be administered is a protein, the therapeutic effect can be achieved by introducing a gene encoding the protein into the living body using a gene therapy technique. A technique for treating a disease by introducing a gene into the living body that encodes a protein having a therapeutic effect is well-known in the art.  
     [0166] Alternatively, an antisense DNA can be directly administered. In that case, cell membrane permeability or stability of the DNA can be elevated by, for example, 5′ end and/or 3′ end modification. Alternatively, the antisense DNA may be incorporated downstream of an appropriate promoter sequence to be administered as an antisense RNA expression vector. When this expression vector is introduced into T cells of an allergic disease patient, the expression level of the indicator gene can be reduced due to the expression of antisense of the gene, thereby achieving a therapeutic effect on an allergic disease. For introducing the expression vector into T cells, methods performed either in vivo or ex vivo are known.  
     [0167] Although the dosage of the therapeutic agent for an allergic disease according to the invention may vary depending on the age, sex, body weight, and symptoms of a patient; therapeutic effects; methods for administration; treatment duration; types of active ingredient contained in the pharmaceutical composition or such, it can be usually administered in the range of 0.1 mg to 500 mg, preferably 0.5 mg to 20 mg per dose for an adult. However, since the dosage varies according to various conditions, an amount less than the above-described dosage may be sufficient in some cases, and a dosage exceeding the above-described range may be required in other cases.  
     [0168] The present invention provided a gene that shows a difference in expression between healthy subjects and patients with allergic diseases. Using the expression of the gene of the present invention as an indicator, it became possible to test for allergic diseases and screen for candidate therapeutic compounds. The test of the present invention can be readily conducted by measuring the expression level of the indicator gene and can quickly find the pathological state of allergic reaction. Additionally, according to this invention, the method of testing for allergies has low invasiveness towards patients since analysis of the expression level can be carried out using biological samples, like peripheral blood leukocytes. Furthermore, highly sensitive measurements in gene expression analysis are possible using small sample amounts. Year after year, high throughput methods and cost reduction are progressing in gene analysis technology. Therefore, in the near future, the method of testing for allergies of this invention is expected to be come an important bed-side diagnostic method. In this sense, the diagnostic value of these pathology-related genes is high. Furthermore, the screening methods of the present invention are expected to be applied to development of novel therapeutic agents for allergic diseases.  
     [0169] The present invention will be explained in detail below with reference to examples, but it is not to be construed as being limited thereto.  
     EXAMPLE 1  
     Identification of B1799  
     [0170] The Differential Display (DD) method (Liang and Pardee, Science, 1992, 257: 967-971; T. Ito et al., 1994, FEBS Lett. 351: 231-236) discovered a 164 bp DNA fragment (clone B1799-01) (SEQ ID NO: 3; except for primer sequence) that is expressed much stronger in T cells of atopic dermatitis patients and allergic asthma patients compared to that of healthy subjects. For the DD analysis to detect the fragment, GT15C (SEQ ID NO: 5) and AG00189 (TCTCTGGAGT; SEQ ID NO: 6) were used as anchor primer and arbitrary primer, respectively. The gene from which the fragment was derived was designated as B1799.  
     EXAMPLE 2  
     Cloning of B1799  
     [0171] PCR cloning was performed based on the 5′-RACE method using the DD sequence and as a template a human leukocyte cDNA library to obtain a DNA fragment of a full length of 486 bp (SEQ ID NO: 4). BLAST search on a public database revealed that the sequence at the position 27 to 255 of this nucleotide sequence was 100% identical to the gene KIAA0603 (GenBank Accession No. AB011175) that was identified by Kazusa DNA Research Institute. KIAA0603 is a cDNA of 5922 bp that encodes a protein consisting of 1299 amino acid residues starting with methionine in its ORF. The homologous sequence region was within the ORF region, i.e., from nt 2152 to nt 2380, of KIAA0603 gene. Furthermore, 486 bp sequence was completely identical with the nt 128071 to nt 127586 region (reverse strand) of GenBank Accession No. AL162571 that is a genomic sequence of chromosome 13.  
     EXAMPLE 3  
     Expression Quantification of B1799 by Quantitative PCR  
     [0172] The DD sequence was determined to be derived from the immature mRNA intron region of KIAA0603 gene. Assuming that B1799 is identical to KIAA0603, KIAA0603 gene was analyzed. The following primers and probes for mRNA quantification were designed within the ORF of KIAA0603 gene and used for expression quantification. TQ1799orf is a TaqMan probe used in gene expression quantification by TaqMan method, whose 5′ end and 3′ end are fluorescent labeled with FAM (6-carboxyfluorescein) and TAMRA (6-carboxy-methyl-rhodamine), respectively. Using ABI-PRISM 7700, DNA amplification was detected at real time.  
                                  1799orf-f:   AAGACAGTGGAGCAACTCCGG   (SEQ ID NO: 7)                   1799orf-r:   CAGCAACAGGTCACAATTGGC   (SEQ ID NO: 8)               TQ1799orf:   AGCTGCTGCCCGCGGATGCT   (SEQ ID NO: 9)          
 
     EXAMPLE 4  
     Relevance to Pathology  
     [0173] The expression of B1799 gene was measured using RNAs prepared from T cells of atopic dermatitis patients as described in Example 3. The results are shown in Table 1. According to a t-test of the subjects divided into two groups, i.e., healthy subject group and atopic dermatitis patient group, the expression level was significantly higher (p&lt;0.05) in the patient group.  
               TABLE 1                          Expression level oif B1799 in atopic dermatitis patients                                             B1799 (copy/ng           B1799 (copy/ng           Pathology   RNA)       Pathology   RNA)                                                 1   normal   5196.77   21   moderate   3865.66       2   normal   2813.08   22   moderate   3931.79       3   normal   4443.58   23   moderate   6614.39       4   normal   4115.51   24   moderate   6352.02       5   normal   3890.60   25   moderate   15627.77       6   normal   1897.45   26   moderate   4516.00       7   normal   2163.33   27   moderate   8289.22       8   normal   154.31   28   moderate   17001.41       9   normal   3542.13   29   moderate   5502.07       10   normal   1647.13   30   moderate   21914.55       11   mild   1176.89   31   severe   2645.44       12   mild   10222.95   32   severe   3492.70       13   mild   15579.36   33   severe   5810.25       14   mild   18515.82   34   severe   13260.42       15   mild   3200.39   35   severe   736.77       16   mild   1862.32   36   severe   6562.17       17   mild   5031.60   37   severe   11227.19       18   mild   5999.48   38   severe   5974.82       19   mild   5382.48   39   severe   3249.61       20   mild   1019.33   40   severe   609.08                  
 
     [0174] Specifically, the expression level was high in patients with moderate pathology compared to healthy subjects and patients with mild pathology. More specifically, analysis of variance was performed among four groups consisting of groups of atopic dermatitis classified into groups of mild, moderate, and severe pathologies, and the healthy subject group, indicating that there are significant differences (p&lt;0.05) among these four groups. Comparison of the differences among the groups by post hoc test according to the PLSD method of Fisher, showed the significant difference and bias between the groups as follows. Expression in each group is shown in FIG. 1.  
                                  healthy subject &lt; mild pahology subject                 (bias p &lt; 0.1)                 healthy subject &lt; moderate pathology subject                 (significant difference p &lt; 0.01)                 severe pathology subject &lt; moderate pathology subject                 (bias p &lt; 0.1)                  
 
     [0175] As demonstrated above, the expression level was revealed to be higher in atopic dermatitis patients, especially in patients with moderate pathology, compared to normal healthy subjects.  
     EXAMPLE 5  
     Expression Levels in Various Immunocytes  
     [0176] Expression levels in T cells, B cells, eosinophils, monocytes, and neutrophils were measured in RNA samples derived from 5 subjects. The result revealed that the expression level was highest in T cells (FIG. 2). Among the T cells, the expression was higher in T cells with the phenotypes of CD4 +  and CD45RO + , i.e., memory T cells (FIG. 3).  
     EXAMPLE 6  
     Changes in Expression Due to T Cell Activation  
     [0177] Once mature T cells are stimulated with antigen, a further stimulation with the antigen is known to cause apoptosis which is called activated cell death of T cells. Activated cell death of T cells is suggested to be involved in the control of immunological tolerance and immune response in periphery (Yili Yang et al., J. Exp. Med. 181: 1673-1682, 1995). To examine the role of B1799 in these controls, the expression changes of B1799 due to activated cell death of T cells and cell death caused by glucocorticoid were measured. Peripheral blood-derived T cells were cultured in 5% FCS-containing RPMI1640 medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 100 u/ml penicillin, and 100 μg/ml streptomycin, further with 200 u/ml IL-2 (Imunace®, Shionogi &amp; Co., Ltd.) under 5% carbon dioxide and 95% humidity at 37° C. First, CD3+ cells isolated from periphery were cultured for 5 days on plates coated with an anti-CD3 antibody (OKT3, Janssen-Kyowa Co., LTD), followed by cultivation for 3 days without the anti-CD3 antibody. The obtained T cells were cultured again with stimulus on the anti-CD3 antibody coated plates to measure activated cell death. Cells are collected over the course of time, and RNA was extracted according to conventional methods for quantitative PCR. For control without stimulus, plates without an anti-CD3 antibody were used.  
     [0178] Further, dexamethasone was added to unstimulated control as glucocorticoid that induces apoptosis of T cells (Kofler R., Histochem. Cell Biol., 2000 July; 114(1): 1-7), and its influence was examined.  
     [0179] As depicted in FIG. 4 a , obvious cell death was induced 49 hours after the second stimulation via the T cell receptor on human periphery-derived T cells using anti-CD3 antibodies. B1799 was markedly induced 2 hours after the stimulation during the T cell activation process. In contrast, no notable cell death of peripheral T cells or changes in the expression of B1799 was observed in the presence of dexamethasone (FIG. 4 b  “DEX”). Through the stimulation of T cells by various reagents in similar experiments, the expression of B1799 was shown to be markedly induced by stimulation with 1 μg/ml of ionomycin (Sigma), calcium ionophore, alone (described as “ionomycin” in the Figure), or with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma) (described as “io+PMA” in the Figure) (FIG. 5), in addition to anti-CD3 antibodies. The above results show that the expression of B1799 was enhanced together with the activation of the T cell. Furthermore, B1799 was suggested to be involved in some way to subsequent activated cell death.  
     EXAMPLE 7  
     Analysis of Amino Acid Sequence Structure  
     [0180] The structure of the protein encoded by B1799 is shown in FIG. 6. The protein has, at its N-terminus region, two phosphotyrosine interaction domain (PID) that is involved in the inter-protein binding in the phosphorylated tyrosine binding region. Furthermore, the protein has a TBC domain at its C-terminus.  
     [0181] BLAST search revealed that the region of amino acids from the position 886 to 1237, which contains the TBC domain, shows a homology of 31% to human rab6 GTPase activating protein (Cuif M. H. et al., EMBO J. :18(7) 1772-82, 1999) (ACCESSION NP — 036329). Further, BLAST search showed that the amino acid region from the position 853 to 1107 has a homology of 21% to Gyp1p protein (ACCESSION NP — 014713), yeast GTPase activating protein (GAP). As demonstrated in FIG. 7, B1799 protein was predicted to have a very similar tertiary structure to the GTPase activating site structure of Gyp1, whose crystal structure had been determined (Rak A. et al., EMBO J. 19: 5105-5113, 2000). Arg973 residue of B1799 protein corresponds to Arg343 residue of Gyp1p, which is essential as the GTPase activating site. Therefore, there is a high possibility that the B1799 protein is a GTPase activating protein, and the Arg973 residue was predicted to play an important role in its activity.  
     [0182] The GTP binding form of the GTP binding protein, which protein has an important role in the signal transduction, is the activated form in the signal transduction and changes to the inactivated form when the bound GTP is converted to GDP. The GTP binding protein itself has the GTPase activity and GTPase activating proteins have the function to enhance the GTPase activity. GTPase activating proteins help the conversion of GTP binding proteins to the inactivated form, thereby regulating signal transduction.  
     [0183] As described above, B1799 would play an important role in signal transduction where the T cell gets activated by regulating the activity of the GTP binding protein.  
     [0184] Furthermore, throughout the whole sequence, the amino acid sequence encoded by B1799 showed a high homology to mouse Tbc1. Tbc1 was discovered as a gene whose expression changes during mast cell differentiation, and is suggested to be involved in cell differentiation and cell cycle suppression (Paul M. Richardson and Leonard I. Zon., Oncogene 11: 1139-1148, 1995). Thus, similarly to Tbc1, B1799 maybe involved in cell differentiation and cell cycle suppression.  
    
     
       
         1 
         
           
             9  
           
           
             1  
             5922  
             DNA  
             Homo sapiens  
             
               CDS  
               (348)..(4244)  
             
           
            1 

gcggccgcgg ggaccctcgg cgtggtcctc tgaccctgca aacccgcgac ggaggaaggg     60 

gaggtcctgc ccgaggcgcc agcccgagga ggaggatgcc catttaaccc gccctcgcct    120 

gccgggcgct tgcctcggtg cccgccgccg gagcctccga gccgcgcccg tggaagtgct    180 

gcatggggca gggctgctga agcgcggagt tcggggtcgc gccgctccca ggcaggcgcg    240 

ggagcccggt gcggcagttg gcacagtttc ggcggcgcct tctgcgcggg agtggggggc    300 

gcggtgcgcc cggccggcct ccgcggtgcc ctggtgaggc gagagtt atg gag ccg      356 
                                                    Met Glu Pro 
                                                    1 

ccc agc tgc att cag gat gag ccg ttc ccg cac ccc ctg gag ccc gag      404 
Pro Ser Cys Ile Gln Asp Glu Pro Phe Pro His Pro Leu Glu Pro Glu 
    5                   10                  15 

ccg ggc gtc tca gct cag ccc ggc ccc ggg aag cca agc gat aag cgg      452 
Pro Gly Val Ser Ala Gln Pro Gly Pro Gly Lys Pro Ser Asp Lys Arg 
20                  25                  30                  35 

ttc cgg ctg tgg tac gtt ggg ggg tcg tgc ctg gac cac agg acc acg      500 
Phe Arg Leu Trp Tyr Val Gly Gly Ser Cys Leu Asp His Arg Thr Thr 
                40                  45                  50 

ctg cct atg ctg ccc tgg ctc atg gcc gag atc cgc agg cgc agc cag      548 
Leu Pro Met Leu Pro Trp Leu Met Ala Glu Ile Arg Arg Arg Ser Gln 
            55                  60                  65 

aag ccc gag gcg ggc ggc tgc ggg gcg ccg gcg gcc cga gag gtg atc      596 
Lys Pro Glu Ala Gly Gly Cys Gly Ala Pro Ala Ala Arg Glu Val Ile 
        70                  75                  80 

ctg gtg ctc agc gcg ccc ttc ctg cgt tgc gtc ccc gcg ccg ggc gct      644 
Leu Val Leu Ser Ala Pro Phe Leu Arg Cys Val Pro Ala Pro Gly Ala 
    85                  90                  95 

ggg gcc tcg ggg ggc act agt ccg tcg gcc acg cag ccc aac ccg gcg      692 
Gly Ala Ser Gly Gly Thr Ser Pro Ser Ala Thr Gln Pro Asn Pro Ala 
100                 105                 110                 115 

gta ttc atc ttc gag cac aag gcg cag cat atc tcg cgc ttc atc cac      740 
Val Phe Ile Phe Glu His Lys Ala Gln His Ile Ser Arg Phe Ile His 
                120                 125                 130 

aac agc cac gac ctc acc tac ttt gcc tac ctg atc aag gcg cag ccc      788 
Asn Ser His Asp Leu Thr Tyr Phe Ala Tyr Leu Ile Lys Ala Gln Pro 
            135                 140                 145 

gac gac ccc gag tcg cag atg gcc tgc cac gtt ttc cgc gcc aca gac      836 
Asp Asp Pro Glu Ser Gln Met Ala Cys His Val Phe Arg Ala Thr Asp 
        150                 155                 160 

ccc agc cag gtt cct gat gtt att agc agc ata agg caa tta tct aaa      884 
Pro Ser Gln Val Pro Asp Val Ile Ser Ser Ile Arg Gln Leu Ser Lys 
    165                 170                 175 

gcg gcc atg aaa gag gat gcc aaa ccc agc aaa gat aat gag gac gcc      932 
Ala Ala Met Lys Glu Asp Ala Lys Pro Ser Lys Asp Asn Glu Asp Ala 
180                 185                 190                 195 

ttt tac aac tct cag aag ttc gaa gtc ctg tac tgt gga aag gtg acc      980 
Phe Tyr Asn Ser Gln Lys Phe Glu Val Leu Tyr Cys Gly Lys Val Thr 
                200                 205                 210 

gtg acc cac aag aag gcc ccc tca agc ctc atc gat gac tgc atg gag     1028 
Val Thr His Lys Lys Ala Pro Ser Ser Leu Ile Asp Asp Cys Met Glu 
            215                 220                 225 

aag ttc agc ctg cac gaa cag cag cgc ctg aag atc caa ggc gag cag     1076 
Lys Phe Ser Leu His Glu Gln Gln Arg Leu Lys Ile Gln Gly Glu Gln 
        230                 235                 240 

cgc ggt ccg gac cca gga gag gac ctg gct gac ttg gag gtg gtg gtg     1124 
Arg Gly Pro Asp Pro Gly Glu Asp Leu Ala Asp Leu Glu Val Val Val 
    245                 250                 255 

ccc ggg tcc ccc gga gac tgc ctg ccg gag gag gct gac ggc acc gac     1172 
Pro Gly Ser Pro Gly Asp Cys Leu Pro Glu Glu Ala Asp Gly Thr Asp 
260                 265                 270                 275 

acc cac ctt ggc tta cct gcc ggg gcc agc cag cct gcc ctg acc agc     1220 
Thr His Leu Gly Leu Pro Ala Gly Ala Ser Gln Pro Ala Leu Thr Ser 
                280                 285                 290 

tct cgg gtc tgc ttc cct gag cgg att ttg gaa gat tct ggc ttt gat     1268 
Ser Arg Val Cys Phe Pro Glu Arg Ile Leu Glu Asp Ser Gly Phe Asp 
            295                 300                 305 

gag cag cag gag ttt cgg tct cgg tgc agc agt gtc acc ggc gtg caa     1316 
Glu Gln Gln Glu Phe Arg Ser Arg Cys Ser Ser Val Thr Gly Val Gln 
        310                 315                 320 

cgg aga gtt cac gag ggc agc cag aaa tcc cag ccg cga cgg aga cac     1364 
Arg Arg Val His Glu Gly Ser Gln Lys Ser Gln Pro Arg Arg Arg His 
    325                 330                 335 

gcg agc gca ccc agt cac gtc cag ccc tcg gac tcg gag aag aac agg     1412 
Ala Ser Ala Pro Ser His Val Gln Pro Ser Asp Ser Glu Lys Asn Arg 
340                 345                 350                 355 

acc atg ctc ttc cag gtt ggg cga ttt gag att aac ctt atc agt cca     1460 
Thr Met Leu Phe Gln Val Gly Arg Phe Glu Ile Asn Leu Ile Ser Pro 
                360                 365                 370 

gac act aaa tca gtt gtg cta gaa aag aat ttt aaa gat atc tcc tct     1508 
Asp Thr Lys Ser Val Val Leu Glu Lys Asn Phe Lys Asp Ile Ser Ser 
            375                 380                 385 

tgt tct cag ggt ata aag cat gtg gat cac ttt ggc ttt atc tgc cgg     1556 
Cys Ser Gln Gly Ile Lys His Val Asp His Phe Gly Phe Ile Cys Arg 
        390                 395                 400 

gag tct cca gag cct gga ctt agc cag tat att tgt tat gta ttc cag     1604 
Glu Ser Pro Glu Pro Gly Leu Ser Gln Tyr Ile Cys Tyr Val Phe Gln 
    405                 410                 415 

tgt gcc agc gaa tct ctg gtt gat gag gta atg ctg act ctg aaa cag     1652 
Cys Ala Ser Glu Ser Leu Val Asp Glu Val Met Leu Thr Leu Lys Gln 
420                 425                 430                 435 

gcc ttc agt acg gcg gct gcc ctg cag agt gcc aag acg cag att aaa     1700 
Ala Phe Ser Thr Ala Ala Ala Leu Gln Ser Ala Lys Thr Gln Ile Lys 
                440                 445                 450 

ctg tgt gag gcc tgc ccg atg cac tct ttg cat aag ctc tgt gaa agg     1748 
Leu Cys Glu Ala Cys Pro Met His Ser Leu His Lys Leu Cys Glu Arg 
            455                 460                 465 

att gaa ggt ctc tac cca cca aga gcc aag ctg gtg ata cag agg cat     1796 
Ile Glu Gly Leu Tyr Pro Pro Arg Ala Lys Leu Val Ile Gln Arg His 
        470                 475                 480 

ctc tca tca ctg aca gat aat gag caa gct gac atc ttt gaa aga gtt     1844 
Leu Ser Ser Leu Thr Asp Asn Glu Gln Ala Asp Ile Phe Glu Arg Val 
    485                 490                 495 

cag aaa atg aag cca gtc agt gac cag gaa gaa aat gaa ctt gtg att     1892 
Gln Lys Met Lys Pro Val Ser Asp Gln Glu Glu Asn Glu Leu Val Ile 
500                 505                 510                 515 

tta cac ctg agg cag ctg tgt gaa gcc aag cag aag aca cac gtg cac     1940 
Leu His Leu Arg Gln Leu Cys Glu Ala Lys Gln Lys Thr His Val His 
                520                 525                 530 

atc ggg gaa ggc cct tct act att tca aat agt aca atc cca gaa aat     1988 
Ile Gly Glu Gly Pro Ser Thr Ile Ser Asn Ser Thr Ile Pro Glu Asn 
            535                 540                 545 

gca aca agc agt gga agg ttc aaa ctt gac att ctg aaa aat aaa gct     2036 
Ala Thr Ser Ser Gly Arg Phe Lys Leu Asp Ile Leu Lys Asn Lys Ala 
        550                 555                 560 

aag aga tcc tta act agc tcc ctg gaa aat atc ttc tca agg gga gct     2084 
Lys Arg Ser Leu Thr Ser Ser Leu Glu Asn Ile Phe Ser Arg Gly Ala 
    565                 570                 575 

aac aga atg aga ggt cgg ctt gga agt gtg gac agt ttt gaa cgg tcc     2132 
Asn Arg Met Arg Gly Arg Leu Gly Ser Val Asp Ser Phe Glu Arg Ser 
580                 585                 590                 595 

aac agt ctt gct tca gag aag gac tac tca cca ggg gat tct cca cca     2180 
Asn Ser Leu Ala Ser Glu Lys Asp Tyr Ser Pro Gly Asp Ser Pro Pro 
                600                 605                 610 

ggg aca ccg cca gcg tcc cca ccg tcc tca gct tgg caa acg ttt ccc     2228 
Gly Thr Pro Pro Ala Ser Pro Pro Ser Ser Ala Trp Gln Thr Phe Pro 
            615                 620                 625 

gaa gag gat tcc gac tcc ccg cag ttt cga aga cgg gca cac acg ttc     2276 
Glu Glu Asp Ser Asp Ser Pro Gln Phe Arg Arg Arg Ala His Thr Phe 
        630                 635                 640 

agc cac cca cct tca agc aca aag aga aag ctg aat ttg cag gat ggg     2324 
Ser His Pro Pro Ser Ser Thr Lys Arg Lys Leu Asn Leu Gln Asp Gly 
    645                 650                 655 

agg gct cag ggt gtg cgt tcc cct ctg ctg agg cag agc tcc agt gaa     2372 
Arg Ala Gln Gly Val Arg Ser Pro Leu Leu Arg Gln Ser Ser Ser Glu 
660                 665                 670                 675 

cag tgc agc aat ctt tcg tca gtt cga cgc atg tac aag gag agt aat     2420 
Gln Cys Ser Asn Leu Ser Ser Val Arg Arg Met Tyr Lys Glu Ser Asn 
                680                 685                 690 

tct tcc tcc agt ctt cca agt ctt cac act tcc ttc tct gcc cct tcc     2468 
Ser Ser Ser Ser Leu Pro Ser Leu His Thr Ser Phe Ser Ala Pro Ser 
            695                 700                 705 

ttc act gcc ccc tct ttc ctg aaa agc ttt tac cag aat tca ggt aga     2516 
Phe Thr Ala Pro Ser Phe Leu Lys Ser Phe Tyr Gln Asn Ser Gly Arg 
        710                 715                 720 

ctg tcc cca cag tat gaa aat gaa atc aga caa gac act gct tca gaa     2564 
Leu Ser Pro Gln Tyr Glu Asn Glu Ile Arg Gln Asp Thr Ala Ser Glu 
    725                 730                 735 

tca agt gat gga gaa ggg aga aaa agg acc tca tct acc tgc agc aat     2612 
Ser Ser Asp Gly Glu Gly Arg Lys Arg Thr Ser Ser Thr Cys Ser Asn 
740                 745                 750                 755 

gag tcc cta agt gtg gga gga acc tct gtc act cct cgc cgg atc tcc     2660 
Glu Ser Leu Ser Val Gly Gly Thr Ser Val Thr Pro Arg Arg Ile Ser 
                760                 765                 770 

tgg cgg cag cgc att ttc ctc agg gtt gct tct ccc atg aac aaa tct     2708 
Trp Arg Gln Arg Ile Phe Leu Arg Val Ala Ser Pro Met Asn Lys Ser 
            775                 780                 785 

ccc tca gca atg caa cag caa gat gga ttg gac agg aac gag ctg ctg     2756 
Pro Ser Ala Met Gln Gln Gln Asp Gly Leu Asp Arg Asn Glu Leu Leu 
        790                 795                 800 

cca ctg tcc ccc ctc tct cca acc atg gag gag gaa ccg ctg gtt ata     2804 
Pro Leu Ser Pro Leu Ser Pro Thr Met Glu Glu Glu Pro Leu Val Ile 
    805                 810                 815 

ttc ctg tct ggg gag gat gac cca gaa aag att gaa gaa aga aag aaa     2852 
Phe Leu Ser Gly Glu Asp Asp Pro Glu Lys Ile Glu Glu Arg Lys Lys 
820                 825                 830                 835 

tca aaa gaa ctg agg agc ttg tgg aga aaa gct ata cac caa caa atc     2900 
Ser Lys Glu Leu Arg Ser Leu Trp Arg Lys Ala Ile His Gln Gln Ile 
                840                 845                 850 

ttg tta ctt cga atg gaa aaa gaa aac cag aaa ctt gaa gga gca agc     2948 
Leu Leu Leu Arg Met Glu Lys Glu Asn Gln Lys Leu Glu Gly Ala Ser 
            855                 860                 865 

aga gat gaa ctc cag tcc aga aaa gtt aaa tta gac tat gaa gaa gtt     2996 
Arg Asp Glu Leu Gln Ser Arg Lys Val Lys Leu Asp Tyr Glu Glu Val 
        870                 875                 880 

ggt gca tgt cag aaa gag gtc tta ata act tgg gat aag aag ttg tta     3044 
Gly Ala Cys Gln Lys Glu Val Leu Ile Thr Trp Asp Lys Lys Leu Leu 
    885                 890                 895 

aac tgc aga gct aaa atc aga tgt gat atg gaa gat att cat act ctt     3092 
Asn Cys Arg Ala Lys Ile Arg Cys Asp Met Glu Asp Ile His Thr Leu 
900                 905                 910                 915 

ctt aaa gaa gga gtt ccc aaa agt cga cga gga gaa att tgg cag ttt     3140 
Leu Lys Glu Gly Val Pro Lys Ser Arg Arg Gly Glu Ile Trp Gln Phe 
                920                 925                 930 

ctg gct tta cag tac cga ctc aga cac aga ttg cct aat aaa caa cag     3188 
Leu Ala Leu Gln Tyr Arg Leu Arg His Arg Leu Pro Asn Lys Gln Gln 
            935                 940                 945 

cct cct gac ata tcc tat aag gaa ctt ttg aag cag ctc act gct cag     3236 
Pro Pro Asp Ile Ser Tyr Lys Glu Leu Leu Lys Gln Leu Thr Ala Gln 
        950                 955                 960 

cag cat gcg att ctc gtg gat tta gga agg acg ttt cct act cac cct     3284 
Gln His Ala Ile Leu Val Asp Leu Gly Arg Thr Phe Pro Thr His Pro 
    965                 970                 975 

tac ttt tca gta cag ctt ggg cca gga cag ctg tca ctg ttt aac ctc     3332 
Tyr Phe Ser Val Gln Leu Gly Pro Gly Gln Leu Ser Leu Phe Asn Leu 
980                 985                 990                 995 

ctg aaa gcc tat tct  ttg ctg gac aaa gaa  gtg gga tac tgt cag       3377 
Leu Lys Ala Tyr Ser  Leu Leu Asp Lys Glu  Val Gly Tyr Cys Gln 
                1000                 1005                 1010 

ggg atc agc ttt gtg  gct gga gtc ctg ctt  ctg cac atg agt gaa       3422 
Gly Ile Ser Phe Val  Ala Gly Val Leu Leu  Leu His Met Ser Glu 
                1015                 1020                 1025 

gag caa gcc ttt gaa  atg ctg aaa ttc ctc  atg tat gac ctc ggc       3467 
Glu Gln Ala Phe Glu  Met Leu Lys Phe Leu  Met Tyr Asp Leu Gly 
                1030                 1035                 1040 

ttc cgc aag cag tac  aga cct gac atg atg  tcg ctg cag att caa       3512 
Phe Arg Lys Gln Tyr  Arg Pro Asp Met Met  Ser Leu Gln Ile Gln 
                1045                 1050                 1055 

atg tac cag ctg tcc  agg ctc ctt cat gac  tat cac aga gat ctc       3557 
Met Tyr Gln Leu Ser  Arg Leu Leu His Asp  Tyr His Arg Asp Leu 
                1060                 1065                 1070 

tac aat cac ctt gaa  gaa aat gaa atc agc  ccc agt ctt tat gct       3602 
Tyr Asn His Leu Glu  Glu Asn Glu Ile Ser  Pro Ser Leu Tyr Ala 
                1075                 1080                 1085 

gcc ccc tgg ttc ctc  aca ttg ttt gcc tct  cag ttt tca tta gga       3647 
Ala Pro Trp Phe Leu  Thr Leu Phe Ala Ser  Gln Phe Ser Leu Gly 
                1090                 1095                 1100 

ttt gta gcc aga gtt  ttt gat att att ttt  ctt cag gga act gaa       3692 
Phe Val Ala Arg Val  Phe Asp Ile Ile Phe  Leu Gln Gly Thr Glu 
                1105                 1110                 1115 

gtt ata ttc aag gtt  gca ctc agc cta ctg  agc agc caa gag aca       3737 
Val Ile Phe Lys Val  Ala Leu Ser Leu Leu  Ser Ser Gln Glu Thr 
                1120                 1125                 1130 

ctt ata atg gaa tgt  gag agc ttt gaa aat  att gtt gag ttt ctt       3782 
Leu Ile Met Glu Cys  Glu Ser Phe Glu Asn  Ile Val Glu Phe Leu 
                1135                 1140                 1145 

aaa aac acg cta cct  gat atg aat acc tct  gaa atg gaa aaa att       3827 
Lys Asn Thr Leu Pro  Asp Met Asn Thr Ser  Glu Met Glu Lys Ile 
                1150                 1155                 1160 

att acc cag gtt ttt  gag atg gat att tct  aag cag ttg cat gcc       3872 
Ile Thr Gln Val Phe  Glu Met Asp Ile Ser  Lys Gln Leu His Ala 
                1165                 1170                 1175 

tat gag gtg gaa tat  cat gtg cta cag gat  gag ctt cag gaa tct       3917 
Tyr Glu Val Glu Tyr  His Val Leu Gln Asp  Glu Leu Gln Glu Ser 
                1180                 1185                 1190 

tca tat tcc tgt gag  gat agt gaa act ttg  gag aag ctg gag agg       3962 
Ser Tyr Ser Cys Glu  Asp Ser Glu Thr Leu  Glu Lys Leu Glu Arg 
                1195                 1200                 1205 

gcc aat agc caa ctg  aaa aga caa aac atg  gac ctc cta gaa aaa       4007 
Ala Asn Ser Gln Leu  Lys Arg Gln Asn Met  Asp Leu Leu Glu Lys 
                1210                 1215                 1220 

tta cag gta gct cat  act aaa atc cag gcc  ttg gaa tca aac ctg       4052 
Leu Gln Val Ala His  Thr Lys Ile Gln Ala  Leu Glu Ser Asn Leu 
                1225                 1230                 1235 

gaa aat ctt ttg acg  aga gag acc aaa atg  aag tct tta atc cgg       4097 
Glu Asn Leu Leu Thr  Arg Glu Thr Lys Met  Lys Ser Leu Ile Arg 
                1240                 1245                 1250 

acc ctg gaa caa gaa  aaa atg gct tat caa  aag aca gtg gag caa       4142 
Thr Leu Glu Gln Glu  Lys Met Ala Tyr Gln  Lys Thr Val Glu Gln 
                1255                 1260                 1265 

ctc cgg aag ctg ctg  ccc gcg gat gct cta  gcc aat tgt gac ctg       4187 
Leu Arg Lys Leu Leu  Pro Ala Asp Ala Leu  Ala Asn Cys Asp Leu 
                1270                 1275                 1280 

ttg ctg aga gac cta  aac tgc aac cct aac  aac aaa gcc aag ata       4232 
Leu Leu Arg Asp Leu  Asn Cys Asn Pro Asn  Asn Lys Ala Lys Ile 
                1285                 1290                 1295 

gga aat aag cca taattgaaga ggcacggcct cagcagaaag tgctccttag         4284 
Gly Asn Lys Pro 

aatactacag agaggaagag cctgcatgtc gctggcccaa ggctggaccc tgaagctgat   4344 

ggaaccacct aatactggtg ctgagctcct agtcacagca ggtggacctc gtgctcatca   4404 

gagcatgcca atcctaagcc attggacata tgtagactgg tttttgttgt tgctatgtac   4464 

atataaatat atatataaaa tgaacatagt tcatgctttc agataaaatg agtagatgta   4524 

tatttagatt aattttttta gtcagaactt catgaaatcc acaccaaagg aaaggtaaac   4584 

tgaaatttcc cttggacata tgtgaaatct ttttgtcttt atagtgaaac aaagccagag   4644 

catctttgta tattgcaata tacttgaaaa aaatgaatgt atttttttct ccaaagaaca   4704 

gcatgtttca ctcaatggtg aaaaggtgga aacatttatg taactttatg tgtatctgtc   4764 

ttgatatcta ctgacattgt ctatatgagg aaaatgatta ctggtcatgc tcctgtgagt   4824 

tttttgggaa ggtagggtca tttctccctg cctgctttgt gccaactagc atgttgcatc   4884 

tacatgcatt atgagtctgg ttaggcatta ctttaaacat acataaagag acagtaggac   4944 

attgtggctg agtctaccca gctcaaggta aaggagaatg ttgctaattt tttagcaaac   5004 

tagaccagca ttattactca aactaaaaat atcacacctg aaaaatttaa tttaggacct   5064 

aaaatgtcta gattagcttt ctgctttttt tatttgaata actcattcag ttgtgaatga   5124 

attcctcttt atttggtgcc acagtcacca aatgacaagg atttgccact ttcccaccaa   5184 

attgtgagtg cttgtaattt aggtctctct accttaaatt cagtataagg aaacgtaatt   5244 

atgattgatt ttttccaaag atgacaagct gtgttgaaat acattttttc ttttgaccaa   5304 

ttgacagaat ctaataagct ttaataatct tcccctttta tgtgaaaagt tttgagaact   5364 

gtgaaatgtt taggaacaaa ctgttgaaat ccattggaag ggaaaaaaga aagtggtacc   5424 

agtgttacca gctcaactaa aacctgcaat tctgcatttc aactcttcac ttcctcagcc   5484 

tacaaatagc tcattagatg acattcacgc atgctgggta taggcaagga aagtaatttt   5544 

caaagtacat ttgcagttct ctttttcaga gatgattcta tgatagtgcc tctgaaagtt   5604 

gatgcagcat ttttgccttt ccaaaaagta tttatcctca ctgctttttg cagtacttgt   5664 

attttcacag atggattatc tggggtaatt ttcttcaaag ggagtttgtt atacacagtg   5724 

aaaatgtatt atagagtaga atagtaaagc tctaggggtt tcagaaagct ttgatgaaca   5784 

gatgacaaac atctgaaacc ccctccgcac tgttacccag tgtgtatata atgacttgtt   5844 

atagctcagt gtgcccttga atccatacag tttcttaaaa gacaataaaa tcttattaat   5904 

aaagttaatg taacttct                                                 5922 

 
           
             2  
             1299  
             PRT  
             Homo sapiens  
           
            2 

Met Glu Pro Pro Ser Cys Ile Gln Asp Glu Pro Phe Pro His Pro Leu 
1               5                   10                  15 

Glu Pro Glu Pro Gly Val Ser Ala Gln Pro Gly Pro Gly Lys Pro Ser 
            20                  25                  30 

Asp Lys Arg Phe Arg Leu Trp Tyr Val Gly Gly Ser Cys Leu Asp His 
        35                  40                  45 

Arg Thr Thr Leu Pro Met Leu Pro Trp Leu Met Ala Glu Ile Arg Arg 
    50                  55                  60 

Arg Ser Gln Lys Pro Glu Ala Gly Gly Cys Gly Ala Pro Ala Ala Arg 
65                  70                  75                  80 

Glu Val Ile Leu Val Leu Ser Ala Pro Phe Leu Arg Cys Val Pro Ala 
                85                  90                  95 

Pro Gly Ala Gly Ala Ser Gly Gly Thr Ser Pro Ser Ala Thr Gln Pro 
            100                 105                 110 

Asn Pro Ala Val Phe Ile Phe Glu His Lys Ala Gln His Ile Ser Arg 
        115                 120                 125 

Phe Ile His Asn Ser His Asp Leu Thr Tyr Phe Ala Tyr Leu Ile Lys 
    130                 135                 140 

Ala Gln Pro Asp Asp Pro Glu Ser Gln Met Ala Cys His Val Phe Arg 
145                 150                 155                 160 

Ala Thr Asp Pro Ser Gln Val Pro Asp Val Ile Ser Ser Ile Arg Gln 
                165                 170                 175 

Leu Ser Lys Ala Ala Met Lys Glu Asp Ala Lys Pro Ser Lys Asp Asn 
            180                 185                 190 

Glu Asp Ala Phe Tyr Asn Ser Gln Lys Phe Glu Val Leu Tyr Cys Gly 
        195                 200                 205 

Lys Val Thr Val Thr His Lys Lys Ala Pro Ser Ser Leu Ile Asp Asp 
    210                 215                 220 

Cys Met Glu Lys Phe Ser Leu His Glu Gln Gln Arg Leu Lys Ile Gln 
225                 230                 235                 240 

Gly Glu Gln Arg Gly Pro Asp Pro Gly Glu Asp Leu Ala Asp Leu Glu 
                245                 250                 255 

Val Val Val Pro Gly Ser Pro Gly Asp Cys Leu Pro Glu Glu Ala Asp 
            260                 265                 270 

Gly Thr Asp Thr His Leu Gly Leu Pro Ala Gly Ala Ser Gln Pro Ala 
        275                 280                 285 

Leu Thr Ser Ser Arg Val Cys Phe Pro Glu Arg Ile Leu Glu Asp Ser 
    290                 295                 300 

Gly Phe Asp Glu Gln Gln Glu Phe Arg Ser Arg Cys Ser Ser Val Thr 
305                 310                 315                 320 

Gly Val Gln Arg Arg Val His Glu Gly Ser Gln Lys Ser Gln Pro Arg 
                325                 330                 335 

Arg Arg His Ala Ser Ala Pro Ser His Val Gln Pro Ser Asp Ser Glu 
            340                 345                 350 

Lys Asn Arg Thr Met Leu Phe Gln Val Gly Arg Phe Glu Ile Asn Leu 
        355                 360                 365 

Ile Ser Pro Asp Thr Lys Ser Val Val Leu Glu Lys Asn Phe Lys Asp 
    370                 375                 380 

Ile Ser Ser Cys Ser Gln Gly Ile Lys His Val Asp His Phe Gly Phe 
385                 390                 395                 400 

Ile Cys Arg Glu Ser Pro Glu Pro Gly Leu Ser Gln Tyr Ile Cys Tyr 
                405                 410                 415 

Val Phe Gln Cys Ala Ser Glu Ser Leu Val Asp Glu Val Met Leu Thr 
            420                 425                 430 

Leu Lys Gln Ala Phe Ser Thr Ala Ala Ala Leu Gln Ser Ala Lys Thr 
        435                 440                 445 

Gln Ile Lys Leu Cys Glu Ala Cys Pro Met His Ser Leu His Lys Leu 
    450                 455                 460 

Cys Glu Arg Ile Glu Gly Leu Tyr Pro Pro Arg Ala Lys Leu Val Ile 
465                 470                 475                 480 

Gln Arg His Leu Ser Ser Leu Thr Asp Asn Glu Gln Ala Asp Ile Phe 
                485                 490                 495 

Glu Arg Val Gln Lys Met Lys Pro Val Ser Asp Gln Glu Glu Asn Glu 
            500                 505                 510 

Leu Val Ile Leu His Leu Arg Gln Leu Cys Glu Ala Lys Gln Lys Thr 
        515                 520                 525 

His Val His Ile Gly Glu Gly Pro Ser Thr Ile Ser Asn Ser Thr Ile 
    530                 535                 540 

Pro Glu Asn Ala Thr Ser Ser Gly Arg Phe Lys Leu Asp Ile Leu Lys 
545                 550                 555                 560 

Asn Lys Ala Lys Arg Ser Leu Thr Ser Ser Leu Glu Asn Ile Phe Ser 
                565                 570                 575 

Arg Gly Ala Asn Arg Met Arg Gly Arg Leu Gly Ser Val Asp Ser Phe 
            580                 585                 590 

Glu Arg Ser Asn Ser Leu Ala Ser Glu Lys Asp Tyr Ser Pro Gly Asp 
        595                 600                 605 

Ser Pro Pro Gly Thr Pro Pro Ala Ser Pro Pro Ser Ser Ala Trp Gln 
    610                 615                 620 

Thr Phe Pro Glu Glu Asp Ser Asp Ser Pro Gln Phe Arg Arg Arg Ala 
625                 630                 635                 640 

His Thr Phe Ser His Pro Pro Ser Ser Thr Lys Arg Lys Leu Asn Leu 
                645                 650                 655 

Gln Asp Gly Arg Ala Gln Gly Val Arg Ser Pro Leu Leu Arg Gln Ser 
            660                 665                 670 

Ser Ser Glu Gln Cys Ser Asn Leu Ser Ser Val Arg Arg Met Tyr Lys 
        675                 680                 685 

Glu Ser Asn Ser Ser Ser Ser Leu Pro Ser Leu His Thr Ser Phe Ser 
    690                 695                 700 

Ala Pro Ser Phe Thr Ala Pro Ser Phe Leu Lys Ser Phe Tyr Gln Asn 
705                 710                 715                 720 

Ser Gly Arg Leu Ser Pro Gln Tyr Glu Asn Glu Ile Arg Gln Asp Thr 
                725                 730                 735 

Ala Ser Glu Ser Ser Asp Gly Glu Gly Arg Lys Arg Thr Ser Ser Thr 
            740                 745                 750 

Cys Ser Asn Glu Ser Leu Ser Val Gly Gly Thr Ser Val Thr Pro Arg 
        755                 760                 765 

Arg Ile Ser Trp Arg Gln Arg Ile Phe Leu Arg Val Ala Ser Pro Met 
    770                 775                 780 

Asn Lys Ser Pro Ser Ala Met Gln Gln Gln Asp Gly Leu Asp Arg Asn 
785                 790                 795                 800 

Glu Leu Leu Pro Leu Ser Pro Leu Ser Pro Thr Met Glu Glu Glu Pro 
                805                 810                 815 

Leu Val Ile Phe Leu Ser Gly Glu Asp Asp Pro Glu Lys Ile Glu Glu 
            820                 825                 830 

Arg Lys Lys Ser Lys Glu Leu Arg Ser Leu Trp Arg Lys Ala Ile His 
        835                 840                 845 

Gln Gln Ile Leu Leu Leu Arg Met Glu Lys Glu Asn Gln Lys Leu Glu 
    850                 855                 860 

Gly Ala Ser Arg Asp Glu Leu Gln Ser Arg Lys Val Lys Leu Asp Tyr 
865                 870                 875                 880 

Glu Glu Val Gly Ala Cys Gln Lys Glu Val Leu Ile Thr Trp Asp Lys 
                885                 890                 895 

Lys Leu Leu Asn Cys Arg Ala Lys Ile Arg Cys Asp Met Glu Asp Ile 
            900                 905                 910 

His Thr Leu Leu Lys Glu Gly Val Pro Lys Ser Arg Arg Gly Glu Ile 
        915                 920                 925 

Trp Gln Phe Leu Ala Leu Gln Tyr Arg Leu Arg His Arg Leu Pro Asn 
    930                 935                 940 

Lys Gln Gln Pro Pro Asp Ile Ser Tyr Lys Glu Leu Leu Lys Gln Leu 
945                 950                 955                 960 

Thr Ala Gln Gln His Ala Ile Leu Val Asp Leu Gly Arg Thr Phe Pro 
                965                 970                 975 

Thr His Pro Tyr Phe Ser Val Gln Leu Gly Pro Gly Gln Leu Ser Leu 
            980                 985                 990 

Phe Asn Leu Leu Lys Ala Tyr Ser  Leu Leu Asp Lys Glu  Val Gly Tyr 
        995                 1000                 1005 

Cys Gln  Gly Ile Ser Phe Val  Ala Gly Val Leu Leu  Leu His Met 
    1010                 1015                 1020 

Ser Glu  Glu Gln Ala Phe Glu  Met Leu Lys Phe Leu  Met Tyr Asp 
    1025                 1030                 1035 

Leu Gly  Phe Arg Lys Gln Tyr  Arg Pro Asp Met Met  Ser Leu Gln 
    1040                 1045                 1050 

Ile Gln  Met Tyr Gln Leu Ser  Arg Leu Leu His Asp  Tyr His Arg 
    1055                 1060                 1065 

Asp Leu  Tyr Asn His Leu Glu  Glu Asn Glu Ile Ser  Pro Ser Leu 
    1070                 1075                 1080 

Tyr Ala  Ala Pro Trp Phe Leu  Thr Leu Phe Ala Ser  Gln Phe Ser 
    1085                 1090                 1095 

Leu Gly  Phe Val Ala Arg Val  Phe Asp Ile Ile Phe  Leu Gln Gly 
    1100                 1105                 1110 

Thr Glu  Val Ile Phe Lys Val  Ala Leu Ser Leu Leu  Ser Ser Gln 
    1115                 1120                 1125 

Glu Thr  Leu Ile Met Glu Cys  Glu Ser Phe Glu Asn  Ile Val Glu 
    1130                 1135                 1140 

Phe Leu  Lys Asn Thr Leu Pro  Asp Met Asn Thr Ser  Glu Met Glu 
    1145                 1150                 1155 

Lys Ile  Ile Thr Gln Val Phe  Glu Met Asp Ile Ser  Lys Gln Leu 
    1160                 1165                 1170 

His Ala  Tyr Glu Val Glu Tyr  His Val Leu Gln Asp  Glu Leu Gln 
    1175                 1180                 1185 

Glu Ser  Ser Tyr Ser Cys Glu  Asp Ser Glu Thr Leu  Glu Lys Leu 
    1190                 1195                 1200 

Glu Arg  Ala Asn Ser Gln Leu  Lys Arg Gln Asn Met  Asp Leu Leu 
    1205                 1210                 1215 

Glu Lys  Leu Gln Val Ala His  Thr Lys Ile Gln Ala  Leu Glu Ser 
    1220                 1225                 1230 

Asn Leu  Glu Asn Leu Leu Thr  Arg Glu Thr Lys Met  Lys Ser Leu 
    1235                 1240                 1245 

Ile Arg  Thr Leu Glu Gln Glu  Lys Met Ala Tyr Gln  Lys Thr Val 
    1250                 1255                 1260 

Glu Gln  Leu Arg Lys Leu Leu  Pro Ala Asp Ala Leu  Ala Asn Cys 
    1265                 1270                 1275 

Asp Leu  Leu Leu Arg Asp Leu  Asn Cys Asn Pro Asn  Asn Lys Ala 
    1280                 1285                 1290 

Lys Ile  Gly Asn Lys Pro 
    1295 

 
           
             3  
             164  
             DNA  
             Homo sapiens  
           
            3 

caaggtggat tgtgaattta tgctgtagcc aacttttagt tttgagaaac accataaaaa     60 

caaattaagt tacctcattt actaggcgaa acaggcaagg ttaaggcata cacaaaaaga    120 

agagttaatt cgtttgggtg gaaactcttt tgtttttcct ttca                     164 

 
           
             4  
             486  
             DNA  
             Homo sapiens  
           
            4 

aaagggctcc cttccgcttg tgtttcagga ctactcacca ggggattctc caccagggac     60 

accgccagcg tccccaccgt cctcagcttg gcaaacgttt cccgaagagg attccgactc    120 

cccgcagttt cgaagacggg cacacacgtt cagccaccca ccttcaagca caaagagaaa    180 

gctgaatttg caggatggga gggctcaggg tgtgcgttcc cctctgctga ggcagagctc    240 

cagtgaacag tgcaggtgag tctgaccctc tccagaatga gacctagatt ctcaaggcac    300 

acagttttga ttactctgga gtcaaggtgg attgtgaatt tatgctgtag ccaactttta    360 

gttttgagaa acaccataaa aacaaattaa gttacctcat ttactaggcg aaacaggcaa    420 

ggttaaggca tacacaaaaa gaagagttaa ttcgtttggg tggaaactct tttgtttttc    480 

ctttca                                                               486 

 
           
             5  
             17  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            5 

gttttttttt ttttttc                                                    17 

 
           
             6  
             10  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            6 

tctctggagt                                                            10 

 
           
             7  
             21  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            7 

aagacagtgg agcaactccg g                                               21 

 
           
             8  
             21  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            8 

cagcaacagg tcacaattgg c                                               21 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               Synthetic  
             
           
            9 

agctgctgcc cgcggatgct                                                 20