Human gene RecQ4 encoding helicase

Abstract

A gene encoding: (a) a protein comprising an amino acid sequence of SEQ ID NO: 2; or (b) a protein having deletion, substitution or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2, which has a helicase activity.

Description

TECHNICAL FIELD

The present invention relates to a gene encoding a protein having a helicase activity, a protein encoded by the gene, a method for producing the protein, and use of the gene and the protein.

BACKGROUND ART

DNA helicases are important enzymes that act on various biological reactions involving DNA in living bodies or microbial cells, and there are a number of types of DNA helicases.

The occurrence of a wide variety of DNA helicases is clearly demonsrated by the fact that the reactions involving DNA include a variety of reactions, such as replication and proliferation of cells, development and growth of an individual, and sustainment of life. As the recognized biochemical reactions occurring at the cellular level, there are assumed at least five reactions including replication, repairing, transcription, segregation and recombination of DNAs. DNA helicases are generally known to act to unwind a duplex DNA into single-stranded forms, and the energy required to such an action is considered to be provided by the hydrolysis of ATPs.

Among many types of DNA helicases, RecQ-type DNA helicases have recently been found, which individually have a helicase domain with at least about 40% homology to the helicase domain of Escherichia coli ( E. coli ) recQ gene at the amino acid level. The RecQ-type helicases have been focused on due to their involvement in various diseases and aging of humans. For example, Bloom's syndrome is a disease frequently inducible various cancers in younger age, and Werner's syndrome is a genetic disease inducible premature aging and abnormal cancer. It has recently been found that these syndromes are caused by mutation of different genes respectively encoding different human RecQ-type DNA helicases ( Cell, 83, pp.655-666, 1995; and Science, 272, pp.258-262, 1996).

A RecQ-type helicase was originally found in E. coli by Nakayama et al. ( Mol. Gen. Genet., 195, pp.474-480, 1984). Two kinds of genes encoding proteins having high homology to the helicase have been found in an yeast cell and a human cancer cell, which were designated sgsl (Gangloff et at., Mol. Cell. Biol. 14, pp.8391-8398, 1994) and RecQ1 (Seki et al., Nuc. Acids Res., 22, pp.4566-4573, 1994), respectively.

Known helicases belonging to this family are, in uni-cellular organisms such as E. coli and yeast, only the above-mentioned E. coli RecQ helicase and sgs1; and in multi-cellular organisms (i.e., human), Bloom DNA helicase (Ellis et al, Cell, 83, pp.655-666, 1995) and Werner DNA helicase (Yu et al., Science, 272, pp.258-262, 1996) both responsible for the above-mentioned diseases, and RecQ1 helicase whose involvement in diseases is as yet unknown.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a human RecQ4 DNA helicase gene, a protein encoded by the gene, a method for producing the protein, and use of the protein and the gene.

The present inventors assumed if there would be many RecQ family of DNA helicases other than the above three kinds in human. The inventors also assumed that, when genes of such helicases underwent mutation, the genes might induce various diseases as observed in Bloom's syndrome and Werner's syndrome and even be causative genes for refractory diseases of which etiologies have been undetermined yet. The inventors have made intensive and extensive studies for solving the above-mentioned problems. As a result, the inventors have succeeded in the cloning of a novel human RecQ4 DNA helicase gene by so-called RACE method. This success led to the achievement of the invention.

That is, the present invention provides a gene encoding:

(a) a protein comprising an amino acid sequence of SEQ ID NO: 2; or

(b) a protein having deletion, substitution or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2, which has a helicase activity.

The present invention further provides a gene comprising:

(c) DNA comprising a nucleotide sequence of SEQ ID NO: 1; or

(d) DNA hybridizing to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, which encodes a protein having a helicase activity.

The present invention further provides an oligonuleotide probe hybridizing to at least a portion of the gene.

The present invention further provides a recombinant vector containing the gene.

The present invention further provides a transformant containing the recombinant vector.

The present invention further provides a method for producing a protein having a helicase activity, comprising culturing the transformant and then collecting the protein from the resultant culture.

The present invention further provides a recombinant protein of:

(a) a protein comprising an amino acid sequence of SEQ ID NO: 2; or

(b) a protein having deletion, substitution or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2, which has a helicase activity.

The present invention further provides a mouse- or rat-derived protein having a helicase activity, comprising an amino acid sequence having at least 70% homology to the amino acid sequence of the protein; and a mouse- or rat-derived gene encoding the protein.

The present invention further provides a method for producing a protein having a helicase activity, comprising culturing the transformant and then collecting the protein from the resultant culture.

The present invention further provides a monoclonal or polyclonal antibody specifically reacting with the protein.

The present invention further provides a hybridoma producing the monoclonal antibody, which is prepared by cell fusion of an antibody-producing cell immunized with the protein with a myeloma cell.

The present invention further provides a reagent for detecting a gene encoding helicase, comprising the oligonucleotide probe.

The present invention further provides a kit for diagnosing a disease caused by the genetic abnormality of a gene encoding a protein with a helicase activity, the kit comprising the protein and the monoclonal antibody and/or the polyclonal antibody.

The present invention further provides a transgenic animal having the gene introduced therein in the modified form, the modification being made such that the expression level of the gene is increased or decreased; and a knockout mouse in which the function of the gene has been treated to be lost.

Hereinafter, the present invention is described in detail.

The gene according to the present invention encodes a novel human RecQ4 DNA helicase, and encodes a protein which comprises an amino acid sequence of SEQ ID NO: 2 or a protein which comprises an amino acid sequence having deletion, substitution or addition of at least one amino acid residue in the amino acid sequence of SEQ ID NO: 2 and has a helicase activity.

The protein according to the present invention contains seven helicase motifs of the known RecQ-type DNA helicase, which are well-conserved between E. coli , yeast and human at the amino acid level, as shown in FIG. 2 . As is clearly demonstrated by the results of the radiation hybrid mapping shown in FIG. 3 , it is confirmed that the human RecQ-type DNA helicase gene of the present invention is located on the long arm of human chromosome 8, 8q24.3, as shown in FIG. 4 . Genes derived from other species which correspond to the human-derived gene of the present invention can also be cloned by known techniques.

From the results of the multi-tissue northern blot analysis for determining the expression level of the gene of the present invention in different organs (see FIG. 5 ), it is found that the expression of the gene is observed in all of the tissues examined, and a remarkably intensive expression is particularly observed in thymus and testis.

These results strongly suggest that the gene of the present invention is one of the genes responsible for the maintenance of the fundamental homeostasis of living bodies. Accordingly, the gene is useful for studying on the relation with development and aging of individuals. The elucidation of the expression control of the gene is also useful in the elucidation of the mechanism for maintaining the fundamental homeostasis of living bodies, as well as in the development of novel pharmaceuticals for maintaining the fundamental homeostasis for life.

The gene of the present invention can be identified and obtained by the following procedures, for example.

1. Cloning of Human RecQ-type DNA Helicase Gene

The gene of the present invention (hereinafter, also referred to as “human RecQ4 gene” or “human RecQ-type DNA helicase gene”) can be produced by performing RACE method (Rapid Amplification of cDNA Ends; Frohman, M. A. et al., Methods Enzymol. Vol. 218, pp.340-358, 1993) based on the combination of the principles of Long-distance (LD) PCR method and Suppression PCR method.

That is, a DNA fragment comprising a partial sequence of the known human RecQ-type DNA helicase gene and DNA fragments of unknown sequences respectively having the 5′ and 3′ termini are amplified separately and then linked together, thereby giving the human RecQ-type DNA helicase gene of the present invention in the form of a full-length cDNA. In the present invention, the full-length cDNA may be cloned by, for example, using a commercially available kit such as Marathon™ cDNA Amplification Kit produced by CLONTECH.

At first, a DNA fragment comprising the human-derived known sequence is amplified. The known sequence may be prepared from poly(A)+RNA derived from a human tissue or organ, such as poly(A)+RNA derived from human testis or spleen. The RNA is treated with reverse transcriptase to synthesize cDNA, which is then subjected to RT-PCR to prepare a partial cDNA fragment [ FIG. 1 (1)]. The partial cDNA fragment is sequenced. Based on the determined sequence, four kinds of gene-specific primers (GSPs) are designed, which are designated “5′GSP1” and “5′GSP2”, and “3′GSP1” and “3′GSP2”, respectively. These GSPs are required for the amplification of DNA fragments of unknown sequences to be located upstream to the 5′ region and downstream to the 3′ region of the partial cDNA sequence, respectively. The GSPs have nucleotide sequences suitably selected among the sequence of the partial cDNA, and may be synthesized chemically. In the present invention, the GSPs used for the amplification of the fragment of unknown sequence to be located upstream to the 5′ region of the partial cDNA are “5′GSP1” and “5′GSP2”, and those used for the amplification of the fragment of unknown sequence to be located downstream to the 3′ region of the partial cDNA are “3′GSP1” and “3′GSP2”, as shown in a open box of FIG. 1 (1).

Next, the DNA fragments to be located upstream to the 5′ region and downstream to the 3′ region of the partial cDNA are separately amplified [ FIG. 1 (2)]. As templates for the amplification, commercially available cDNAs derived from human testis, spleen or the like may be used, such as cDNA Ready™ made by CLONTECH. Although the sequences of the template DNA fragments are unknown, each of them has an adapter sequence attached to the terminus. Amplification reaction (LD PCR) of the cDNA fragments of unknown sequences having adapter sequences attached was separately performed two rounds using primers hybridizing to the adapter sequences (hereinafter, simply referred to as “adapter primers (APs)”) and the GSPs as primers (FIG. 1 (2)). For example, the 5′ unknown sequence may be amplified by performing the first PCR using AP1 and 5′GSP1, and then performing the second PCR using the fragment produced by the first PCR as a template and primers (AP2 and 5′GSP2) hybridizing to the inner regions relative to the regions for AP1 and 5′GSP1, respectively (i.e., nested PCR). The 3′ unknown sequence can also be amplified in the same manner as for the 5′ unknown sequence using 3′GSP1 and 3′GSP2.

In the present invention, the DNA fragment to be located upstream to the known fragment is referred to as a “5′-RACE product”, and the DNA fragment to be located downstream to the known fragment is referred to as a “3′-RACE product”. Since each AP has the same sequence as the protrusion sequence of the corresponding adapter, it cannot anneal to the adapter and the extension reaction in the first amplification only starts from the gene-specific primer (GSP) (this is called “Suppression PCR”).

The nucleotide sequence of the resultant cDNA may be determined by a PCR-based method as described by Hattori et al. (Electrophoresis 13, pp.560-565, 1992). That is, the reaction is performed using PRISM sequencing kit including a fluorescent dye deoxyterminetor produced by Perkin Elmer, the nucleotide sequence is read out using an autosequencer manufactured by Applied Biosystem (Model ABI 373) and the data is analyzed by the computer (e.g., Macintosh computer) attached to the autosequencer.

The nucleotide sequences of the known partial DNA sequence, the 5′-RACE product and the 3′-RACE product are assembled together, thereby giving a nucleotide sequence of a full-length cDNA. That is, the overlapped portions between the sequences are linked to give a nucleotide sequence having the 5′ and 3′ terminal regions (FIG. 1 ).

The nucleotide sequence of the gene of the present invention and the amino acid sequence of the protein encoded by the gene are shown in SEQ ID NOs: 1 and 2, respectively. However, the gene or protein may have mutation such as deletion, substitution, addition or insertion in the nucleotide or amino acid sequence, provided that the protein comprising the amino acid sequence of SEQ ID NO: 2 exerts a helicase activity. For example, the amino acid sequence of SEQ ID NO: 2 may have deletion of at least one, preferably 1-10, more preferably 1-5 of amino acid residues, addition of at least one, preferably 1-10, more preferably 1-5 of amino acid residues, or substitution of at least one, preferably 1-10, more preferably 1-5 of amino acid residues by other amino acid residues. Accordingly, a protein having deletion of the first methionine (Met) in the amino acid sequence of SEQ ID NO: 2 is also encompassed in the proteins having these amino acid alternations of the present invention. In addition to the nucleotide sequences encoding the amino acids contained in the protein of the present invention, the gene of the present invention also encompasses a degenerate variant of the gene encoding the same protein which is only different at a degenerated genetic codon.

In addition to the gene comprising DNA comprising a nucleotide sequence of SEQ ID NO: 1, the gene of the present invention also encompasses a gene comprising DNA hybridizing to such DNA under stringent conditions and encoding a protein with a helicase activity. As used herein, the term “stringent conditions” refer to conditions such as those at a sodium concentration of 15-60 mM, preferably 15-30 mM, and a temperature of 55-70° C., preferably 60-70° C.

The above-mentioned mutation may be introduced by any one of the known techniques. For this purpose, for example, a commercially available point mutagenesis kit (e.g., “TAKARA LA PCR in vitro Mutagenesis Kit” produced by Takara Shuzo Co., Ltd.) may be employed.

The protein of the present invention also encompasses a protein comprising an amino acid sequence with at least 70% homology to the amino acid sequence of the above-mentioned protein. The gene encoding such a protein is also encompassed in the gene of the present invention. Examples of such protein and gene include those derived from a mouse and rat.

Once the nucleotide sequence is determined, the desired gene can be prepared by PCR using the primers (e.g., those as shown in SEQ ID NOs: 35 and 36) that are synthesized chemically or based on the determined nucleotide sequence or by hybridization using, as a probe, a DNA fragment having the determined nucleotide sequence.

2. Preparation of Recombinant Vector and Transformant

The recombinant vector of the present invention can be prepared by integrating the gene into a suitable vector. The transformant of the present invention can be prepared by introducing the recombinant vector DNA into a host compatible with a vector which is used for the preparation of the recombinant vector.

The gene is introduced in the purified form into a restriction site or a multi-cloning site of a suitable vector to give a recombinant vector, which recombinant vector is then used to transform a host cell.

The vector DNA into which the DNA fragment is introduced is not particularly limited, and any one may be used provided that it can be replicated in the host cell, such as plasmid DNA, phage DNA and the like. Examples of the plasmid DNA include plasmid pUC118 (Takara Shuzo Co., Ltd.), pUC119 (Takara Shuzo Co., Ltd.), pBluescript SK+(Stratagene) and pGEM-T (Promega). Examples of the phage DNA include M13mp18 and M13mp19.

The host is not particularly limited provided that it can express the gene of interest, and may be either a eukaryotic or prokaryotic cell. Examples of the host include bacteria (e.g., E. coli, Bacillus subtilis ), yeast (e.g., Saccharomyces cerevisiae ), and animal cells (e.g., COS cell, CHO cell).

When a bacterium such as E. coli is used as the host, it is preferable that the recombinant vector of the present invention be capable of autonomous replication and, at the same time, have a constitution comprising a promoter, the DNA of the present invention and a transcription termination sequence. For example, E. coli may be XL1-Blue (Stratagene) or JM10 (Takara Shuzo Co., Ltd.) and the expression vector may be pBTrp2. The promoter may be of any type provided that it can be expressed in a host (e.g., E. coli ). Examples of such a promoter include trp promoter, lac promoter, PL promoter and PR promoter which are derived from E. coli and phages.

In the present invention, the transformation can be performed by, for example, the method by Hanahan (Techniques for Transformation of E. coli In DNA Cloning, vol.1, Glover, D. M. ed., pp.109-136, IRL Press, 1985).

When yeast is used as the host, the expression vector may be YEp13 or YCp50, and the promoter may be gal 1 promoter or gal 10 promoter. The introduction of the recombinant vector into the yeast may be performed by electroporation method (Methods. Enzymol., 194, pp.182-187, 1990), spheroplast method (Proc. Natl. Acad. Sci. USA, 84, pp.1929-1933, 1978), lithium acetate method (J. Bacteriol., 153, 163-168, 1983), or the like.

When an animal cell is used as the host, the expression vector may be pcDNAI and pcDNAI/Amp (Invitrogen). The introduction of the recombinant vector into the animal cell may be performed by electroporation method, calcium phosphate precipitation method or the like.

For example, when a plasmid DNA is used as a vector DNA and an EcoRI DNA fragment is inserted thereinto, the plasmid DNA may be previously digested with a restriction enzyme EcoRI (NEB). The digested vector DNA may be mixed with the DNA fragment of interest, and then T4 DNA ligase (Takara Shuzo Co., Ltd.), for example, may be reacted with the mixture, thereby giving a recombinant vector.

The screening of the transformant strain may be performed by colony hybridization using, as a probe, a DNA fragment containing a portion of the gene of interest, or by PCR method using a 5′ primer (FP; forward primer) synthesized based on the nucleotide sequence of the gene of interest and a 3′ primer (RP; reverse primer) synthesized based on the nucleotide sequence of DNA complementary to the gene of interest, and selecting colonies containing the gene of interest.

3. Production of Protein (Polypeptide) Encoded by Human RecQ4 Gene

The transformant containing the recombinant vector prepared as mentioned above is cultured to produce the protein of the present invention. The culture method may be a conventional solid culture method. However, a liquid culture method is preferably employed for this purpose.

The culture medium used for culturing the transformant may be, for example, one comprising at least one nitrogen source (e.g., yeast extract, peptone, meat extract), which is supplemented with at least one inorganic salt (e.g., dipostassium hydrogenphosphate, magnesium sulfate, iron(II) chloride) and optionally other additive(s) (e.g., a carbohydrate source, an antibiotic, a vitamin) in an appropriate manner. If necessary, IPTG or the like may also be added to the culture medium to induce the expression of the gene. The culture medium is initially adjusted to pH 7.2-7.4, and the culture is usually performed at 36-38° C., preferably about 37° C., for 14-20 hours by aerated spinner culture method, shaking culture method or the like.

After the culture is completed, the protein of the present invention may be collected from the culture by a conventional protein purification method as follows.

The cells are disrupted by lytic treatment with an enzyme (e.g., lysozyme), ultrasonic disruption treatment, mechanical disruption treatment or the like, thereby releasing the protein encoded by the gene of the present invention from the cells. Then, insoluble matters are removed from the solution by filtration, centrifugation or the like to give a crude protein solution.

The protein may be purified from the crude protein solution by a conventional protein purification method. As such a method, for example, salting out with ammonium sulfate, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, electrophoresis or the like may be employed singly or in combination.

4. Production of Monoclonal Antibody

The monoclonal antibody specific to the protein encoded by human RecQ4 gene of the present invention may be produced as follows.

(1) Preparation of Antigen

The protein prepared as described in Section 3 above is dissolved in a buffer solution, and an adjuvant is then added thereto. As such an adjuvant, commercially available Freund's complete or incomplete adjuvant or the like may be used singly or in the mixed form.

(2) Immunization and Isolation of Antibody-producing Cells

The immunogen prepared as described above is administered to a mammalian animal (e.g., rat, mouse). A single dose of the antigen used for the immunization may be 10-500 μg per animal. The animal may be immunized by injecting the antigen intravenously, subcutaneously or intraperioneally. The interval of the immunization is not particularly limited, and the immunization may be performed at intervals of several days to several weeks, preferably 1-3 weeks, for 2-5 times, preferably 3-4 times. Two to seven days, preferably four to five days, after the final immunization, antibody-producing cells are isolated. Such antibody-producing cells may be spleen cells, lymph node cells, peripheral blood cells, and preferably spleen cells or localized lymph node cells.

(3) Cell Fusion

The myeloma cells used for cell fusion with the antibody-producing cells may be of a usually commonly available established cell line of an animal (e.g., mouse). The cell line used is preferably one having drug selectivity, and incapable of surviving in a selective medium (HAT medium; comprising hypoxantine, aminopterin and thymidine) in its non-fused form but capable of surviving in such a selective medium only in its fused form with an antibody-producing cell. Specific examples of the myeloma cell include mouse myeloma cell line such as P3U-1 (Dainippon Pharmaceutical Co., Ltd.) and P3×63Ag8.653 The myeloma cells are then fused with the antibody-producing cells. The cell fusion may be performed by mixing the antibody-producing cells with the myeloma cells at a ratio of 100-500 cells of the antibody-producing cells per 1 myeloma cell, for example, by mixing equivalent volumes of a culture medium containing 10 8 cells/ml of the antibody-producing cells and a culture medium containing 2×10 5 cells/ml of the myeloma cells, and the mixture is then subjected to fusion reaction in the presence of a fusion promoting agent. For promoting the cell fusion, polyethylene glycol with a mean molecular weight of 1,500 daltons or the like may be used. Alternatively, a commercially available cell fusion apparatus utilizing electrical stimulation (e.g., electroporation) may be employed to cause the cell fusion of the antibody-producing cells with the myeloma cells.

(4) Screening and Cloning of Hybridomas

The desired hybridomas are screened from the cells after the cell fusion treatment. The screening may be performed by appropriately diluting the cell suspension with RPMI-1640 medium containing fetal bovine serum or the like, inoculating the resultant dilution solution into each well of a microtiter plate in an amount of about 5-10 cells/well, adding a selective medium to each well, and then incubating the plate while appropriately replacing the selective medium in the wells by a fresh one. The desired hybridomas can be obtained as the cells grown about 14 days after the culture is started. The culture supernatant of the grown hybridomas is then screened for the presence of the antibodies of interest. The screening may be performed by a conventional method, and the method is not particularly limited. For example, a portion of the hybridoma-containing culture supernatant may be removed from the individual wells and subjected to screening by enzyme immunoassay (EIA), radio immunoassay (RIA) or the like.

The cloning of the fused cells is then performed by a limiting dilution method or the like to ultimately establish hybridomas which produce monoclonal antibodies.

(5) Collection of Monoclonal Antibodies

As the method for collecting monoclonal antibodies from the above-established hybridomas, a conventional cell culture method or ascites fluid production method may be employed. In the case of a cell culture method, the hybridomas are cultured in a culture medium for animal cells, such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium or a serum-free medium, under conventional culture conditions (e.g., 37° C., 5% CO 2 ) for 10-14 days, and the antibodies can be obtained from the culture supernatant. In the case of an ascites fluid production method, the hybridomas (about 5×10 6 cells) are administered intraperitoneally to an animal of the same species as that of the mammal from which the myeloma cells are derived, thereby causing to grow the hybridomas in a large scale. One to two weeks later, the ascites fluid or serum is collected from the animal. In these antibody-collecting methods, if it is required to purify the antibodies, a known method such as salting out with ammonium sulfate, ion exchange chromatography, affinity chromatography or gel chromatography may be employed singly or in combination.

5. Production of Polyclonal Antibodies

(1) Preparation of Antigen

The protein prepared as described in Section 3 above is dissolved in a buffer solution, and then an adjuvant is added thereto. Such an adjuvant may be commercially available Freund's complete or incomplete adjuvant.

(2) Immunization

The animal used for the immunization may be a rabbit, guinea pig, goat, sheep or the like. In the case of a rabbit, for example, the protein is injected subcutaneously to the foot paw usually at a dose of 100-500 μg together with Freund's complete adjuvant. Two weeks later, the same dose of the antigen mixed with Freund's incomplete adjuvant is injected intramuscularly. Additional two weeks later, the intramuscular injection is repeated. One week after the final immunization, a portion of the blood was collected from the ear and determined for the antibody titer by EIA method or the like. When the antibody titer reaches the desired value, the whole blood was collected. However, if the antibody titer is low, the immunization is repeated until the antibody titer reaches the desired value. The antibodies are then purified from the serum by ammonium sulfate fractionation as mentioned in the above-described relevant section for the purification of the monoclonal antibodies.

6. Reagent for Detecting Human RecQ4 Gene and the Protein Encoding the Gene

Human RecQ4 gene has high degree of homology to the causative genes of Bloom's syndrome and Werner's syndrome which cause chromosomal instability and induce high frequency carcinogenesis and progeria (FIG. 2 ), and the high expression of the gene is observed in various human tissues (FIG. 5 ). Therefore, it can be concluded that the gene is one of genes encoding DNA helicases responsible for the maintenance of the fundamental homeostasis of a living body. Accordingly, the elucidation of the expression control of the gene may be useful for the elucidation of the mechanism of the homeostasis maintenance of a living body, useful for the development of novel drugs for maintaining the fundamental homeostasis of a living body, and useful in the study on the elucidation of the relation with aging. The reagent of the present invention is useful for the detection and diagnosis of diseases cause by the abnormality in genes encoding proteins having helicase activity, including, but not limited to, Bloom's syndrome and Werner's syndrome.

When the gene of the present invention is used as a reagent, the hybridization may be performed using an oligonucleotide containing at least a portion of the cloned human RecQ4 gene as a probe, and the detection may be performed by Southern or Northern blotting method. As such an oligonucleotide probe, a DNA probe, an RNA probe or the like may be used.

When the polyclonal or monoclonal antibody to the protein encoded by the gene of the present invention is used as a detection reagent, the detection may be performed by EIA, RIA or Western blotting analysis.

7. Transgenic Animals Having the Gene Introduced therein in the Modified Form such that the Expression Level of the Gene Increases or Decreases

In the present invention, the gene can be artificially modified to increase or decrease the expression level thereof compared to the normal level by causing a mutagenesis (e.g., deletion, substitution, addition, insertion) at a portion of the several sites important for controlling the expression of the gene (e.g., enhancer, promoter, intron).

The mutagenesis may be performed by any one of the known methods. For example, a commercially available point mutagenesis kit (e.g., TAKARA LA PCR in vitro Mutagenesis kit produced by Takara Shuzo Co., Ltd.) may be used. The transgenic animal includes, for example, a transgenic mouse and a transgenic rat. The vector containing the mutagenized gene may be a vector capable of over-expression of RecQ4 genes from different animal species or a vector capable of suppressing the expression of such RecQ4 genes. In either case, a drug-resistance gene (e.g., a neomycin-resistance gene) is ligated to the gene for the positive selection.

The introduction of the gene may be performed by directly injecting the DNA into fertilized eggs. However, it is preferable to utilize embryonic stem (ES) cells to achieve the efficient introduction of the gene, because ES cells have such an advantage that they can be cultured and they can develop mice or the like therefrom. The ES cells may be TT2 cells (Shin-ichi AIZAWA, “Gene Targeting”, 1995, Yodosha). For example, the vector DNA containing murine RecQ4 gene is introduced into ES cells by electroporation, and positive clones are selected with neomycin, thereby giving mutant ES cells of interest. The mutant ES cells are then injected to blastcysts or 8-celled embryos through a capillary or the like. The blastcysts or 8-celled embryos may be implanted direct into the oviduct of a foster parent. Alternatively, they may be developed to the blastocyst stage and then implanted to the uterus of a foster parent. Among the progenies bred from the foster parent, chimeras are selected. The animals with high contribution to the chimeras have high possibility of animals of the germ line of the chimeras. Whether an animal is a chimera of the germ line of this chimera can be determined by mating the animal with a normal (i.e., wild-type) one. The mating of the germ line of the chimera with a normal one results in the production of heterozygotes, and the mating between the heterozugotes results in the production of homozygotes.

8. Knockout Mouse

The knockout mouse of the present invention is prepared by treating a mouse so that the function of murine RecQ4 gene is lost. The method for the treatment is described below.

A genomic DNA containing murine RecQ4 gene is prepared from the genomic DNA from a murine ES cell by PCR or from a genomic library. A neo-resistance gene is introduced into any one of the exons of the genomic DNA to construct a vector. This procedure results in the disruption of the function of the exon. At the same time, a thymidine kinase (tk) gene or a diphtheria toxin (DT) gene is also introduced into the vector for the negative selection. The vector DNA is introduced into ES cells by electroporation. The cells are then cultured in the presence of neomycin for the selection of positive clones or a nucleic acid analogue FIAU (fluoroiodoadenosyluracil) or diphtheria toxin for the selection of negative clones. By this procedure, diphtheria toxin-sensitive cells in which non-homologous recombination occurs and G418-sensitive cells in which no recombination occurs are removed out, and only cells in which homologous recombination occurs are remained. In the cells in which homologous recombination occurs, the gene containing the disrupted exon undergoes knockout. The resulted cells are injected into murine blastcysts or 8-celled embryos. Thereafter, the same procedures as for the preparation of the transgenic animal are performed to produce knockout mice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the procedures for cloning the gene according to the present invention.

FIG. 2 illustrates the comparison in amino acid sequence homology between the protein encoded by human RecQ4 gene (SEQ ID NO: 37) and proteins ( E. coli RecQ (SEQ ID NO: 38), yeast SES1 (SEQ ID NO: 39), RecQ, (SEQ ID NO: 40), Bloom (SEQ ID NO: 41), and Werner (SEQ ID NO: 42) derived from other genes.

FIG. 3 illustrates the radiation mapping analysis indicating the location of human RecQ4 gene on human chromosome 8.

FIG. 4 illustrates human chromosome 8.

FIG. 5 is a photograph showing the results of the Northern blotting analysis of human RecQ4 gene.

FIG. 6 illustrates the comparison in amino acid sequence homology between the protein encoded by EST-DNA ACC. H16879 (SEQ ID NO: 43)and a protein derived from E. coli (SEQ ID NO: 44).

FIG. 7 illustrates the assembly of the gene according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is illustrated in more detail, However, the following examples are intended to illustrate but not admitted to limit the technical scope of the invention.

EXAMPLE 1

Cloning of Full-length Human RecQ4 cDNA

(1) Identification of a cDNA Fragment having Homology to E. coli RecQ4 DNA Helicase

Before the cloning of the DNA of the present invention, the dbest database was searched for a cDNA sequence having homology to E. coli RecQ helicase. As a result, there were found more than ten kinds of EST (Expressed Sequence Tag) DNAs including EST-DNA ACC. H 16879 (i.e., DNA encoding the amino acid sequence of H16879 shown in FIG. 6 ). In consideration of the nucleotide sequence homology of these ESTs to the EST-DNAs derived from organisms other than human (e.g., E. coli , yeast, threadworms) or with the genes of the known human-derived Blooms's disease helicase, Werner's disease helicase and RecQ1 helicase, those having high nucleotide sequence homology to such genes were omitted from the above-found ESTs.

(2) Identification of EST-DNA ACC.H16879 as the Gene Expressed in Human Tissues

Next, a series of preliminary experiments were performed to identify the sequence of EST-DNA ACC.H16879 as the gene truly expressed in human tissues. That is, a sense primer (SEQ ID NO: 3) and an antisense primer (SEQ ID NO: 4) for PCR (Polymerase Chain Reaction) were provided based on the nucleotide sequence contained in the EST-DNA fragment. On the other hand, cDNA was prepared from mRNA derived from human testis by reverse transcription reaction. PCR was performed using the above-prepared primers to examine for the presence of the EST-DNA in the human testis-derived cDNA.

RT-PCR was performed as described in (3-i) below. As a result, a predicted PCR product of about 320 bp could be detected from the sequence of EST-DNA ACC.H16879.

The confirmation of the presence or absence of the EST-DNA by PCR could also be performed by integrating the DNA fragment amplified by PCR into a plasmid DNA of E. coli , cloning the DNA fragment, and then analyzing the nucleotide sequence of the resultant plasmid clone DNA. In this manner, it was confirmed that EST-DNA ACC.H16879 was a portion of the novel helicase gene. [As for the procedures, see (3-ii) and (3-iii) below.]

(3) Cloning of a Partial cDNA Fragment of Human RecQ4 Gene by RT-PCR

(3-i) Preparation of cDNA by Reverse Transcription and Amplification

A reaction solution comprising human testis-derived poly(A)+RNA (CLONTECH) (about 1 μg), dithiothreitol, dNTPs (dATP, dCTP, dGTP and dTTP), a buffer for a reverse transcriptase, and a reverse transcriptase Super Sucript II, was allowed to react at 42° C. for 30 minutes, and then subjected to RNase treatment, thereby preparing cDNA. The cDNA was used as a template for the subsequent PCR.

In the PCR, TAKARA Taq (Takara Shuzo Co., Ltd.) and a buffer attached thereto were used. If not otherwise stated, the “Taq DNA polymerase” and “PCR buffer” used hereinbelow refer to TAKARA Taq and the buffer attached thereto, respectively. PCR was performed in a mixed reaction solution (25 μl) comprising a solution containing 1×PCR buffer, 0.2 mM dNTPs, 0.4 μM RecQ4 primers (SEQ ID NOs: 3 and 4) and 0.625 unit Taq DNA polymerase with an appropriate amount of the human testis-derived cDNA.

The reaction solution was allowed to react, starting with at 94° C. for 5 min., and then at a program of 94° C. for 30 sec.; 55° C. for 30 sec.; and 72° C. for 1 min. for 35 cycles. The resultant reaction solution was additionally allowed to react at 72° C. for 5 min.

(3-ii) Subcloning of PT-PCR Product for Sequencing

A solution containing the PT-PCR product obtained in (3-i) above, T4 DNA ligase (Takara Shuzo Co., Ltd.), buffer (Takara Shuzo Co., Ltd.) and pGEM-T vector (Promega) was allowed to react at 15° C. for 3 hours, thereby causing the integration of the PT-PCR product fragment into pGEM-T vector. E. coli JM109 was transformed with the vector, and the resultant E. coli transformant was plated on LB bactoagar plate containing X-gal, IPTG and ampicillin (final concentration: 50 μg/ml) and incubated at 37° C. for 12 hours. White E. coli colonies that appeared on the plate were subjected to shaking culture in LB medium containing ampicillin (final concentration: 50 μg/ml) at 37° C. for 16 hours or more, and plasmid DNA was prepared using a robot (PI-100Σ manufactured by Kurabo).

The resultant plasmid DNA was dissolved in dH 2 O (100 μl) containing 10 μg/ml of RNase for use as a sample for the subsequent DNA sequencing.

(3-iii) Sequencing

PCR was performed using the plasmid DNA prepared in (3-ii) above as a template in a reaction system containing non-labeled primers, four kinds of fluorescent labeled nucleotide-5′-triphosphate and Taq polymerase. The composition of the reaction solution is as follows.

	Thermal Ready reaction mixture	8.0	μl
	Template DNA	3.0	μl
	Primer (3.2 pmol/μl)	1.0	μl
	dH 2 O	8.0	μl
	Total	20	μl

In this PCR, primers shown in SEQ ID NOs: 5-19 were used as the primers for the sequencing.

PCR was performed at a program of 96° C. for 30 sec. (denaturation); 55° C. for 15 sec. (annealing); and 60° C. for 4 min. (elongation), for 25 cycles. By this reaction, DNA fragments each having a randomly inserted fluorescent dye were synthesized. The nucleotide sequences of these DNA fragments were individually analyzed using a sequencer. Thus, finally, a continuous nucleotide sequence could be determined. The analysis of the PCR products was performed using an automated DNA sequencer, model ABI 373 manufactured by Applied Biosystem. The nucleotide sequence of the partial human RecQ4 cDNA is shown in SEQ ID NO: 31.

(4) Cloning of the 5′ and 3′ Regions of Human RecQ4 Gene using Marathon cDNA Amplification Kit

Based on the nucleotide sequence of the partial human RecQ4 cDNA obtained in (3) above, cloning of the 5′ and 3′ regions of the human RecQ4 gene was performed using Marthon cDNA Amplification kit (CLONTECH). At first, to amplify the 5′ RACE product, the first round RCR was performed using Marathon Ready testis-derived cDNA as a template and primer AP1 (SEQ ID NO: 20) specific to the adapter sequence and primer 5′GSP1 (SEQ ID NO: 22) specific to the partial RecQ4 cDNA fragment. The composition of the reaction solution used for this PCR is as follows.

	Template	5	μl
	Primers (10 mM)	1	μl × 2
	10x Klen Taq reaction buffer	5	μl
	2.5 mM dNTPs mix	4	μl
	Klen Taq (CLONTECH)	1	μl
	dH 2 O	33	μl
	Total	50	μl

The PCR was performed, starting with denaturation step at 94° C. for 1 min., at a program of 94° C. for 30 sec. (denaturation) and 72° C. for 4 min. (annealing and elongation) for 5 cycles; then at a program of 94° C. for 30 sec. (denaturation) and 70° C. for 4 min. (annealing and elongation) for 5 cycles; and then at a program of 94° C. for 30 sec. (denaturation) and 68° C. for 4 min. (annealing and elongation) for 25 cycles.

Using the resultant reaction solution in a diluted form as a template, the second round PCR was performed using the further inner primer pairs, AP2 (SEQ ID NO: 21) and 5′GSP2 (SEQ ID NO: 23), thereby giving a 5′ RACE product of about 2 kbp. The same procedures were repeated using primer pairs, 3′GSP1 (SEQ ID NO: 24) and 3′GSP2 (SEQ ID NO: 25), thereby giving a 3′ RACE product of about 1.5 kbp. These products were separately subcloned into pGEM-T vector, and the full-length sequences of the 5′ and 3′ RACE products were determined [as for the method, see (3-ii) and (3-iii) above].

The nucleotide sequences of the obtained 5′ and 3′ RACE products of human RecQ4 gene are shown in SEQ ID NOs: 32 and 33, respectively.

(5) Determination of the Transcription Origin for RecQ4 Gene

As a result of the analysis of the nucleotide sequence of the 5′ RACE product, the first methionine of the predicted protein encoded by RecQ4 gene was not found in the 5′ RACE product, and it was assumed that the first methionine would be located further upstream to the 5′ RACE product. Then, the transcription origin of the gene was determined as follows.

A 5′ region containing the transcription origin for RecQ4 gene was determined by oligo-capping method. Human testis-derived mRNA was treated with calf small intestine-derived alkaline phosphatase (CIAP) (Takara Shuzo Co., Ltd.) to dephosphorylate the mRNA. The composition of the reaction solution used is as follows.

	Human testis-derived mRNA	10	μg
	10x CIAP buffer (Takara Shuzo Co., Ltd.)	10	μl
	RNasin (Promega)	4	μl
	CIAP (Takara Shuzo Co., Ltd.)	1	μl
	dH 2 O	to 100	μl

The reaction was performed at 37° C. for 30 min. Subsequently, the dephosphorylated mRNA was treated with tobacco acid pyrophosphatase (TAP) (Nippon Gene) to remove the 5′ CAP structure therefrom. The composition of the reaction solution used is as follows.

	Dephosphorylated mRNA	10	μg
	10x TAP buffer (Nippon Gene)	10	μl
	RNasin (Promega)	4	μl
	TAP (Nippon Gene, 300 U/μl)	1	μl
	dH 2 O	to 100	μl

The reaction was performed at 37° C. for 60 min. Subsequently, the mRNA (TAP-RNA) without CAP structure was ligated with an oligo RNA (SEQ ID NO: 26) of 30 bp as an adapter using T4 RNA ligase (Takara Shuzo Co., Ltd.). The composition of the reaction solution used is as follows.

	TAP-RNA	10	μg
	Oligo RNA (2 μg/μl)	5	μl
	10x ligase buffer (Takara Shuzo Co., Ltd.)	10	μl
	100 mM ATP	0.5	μl
	RNasin (Promega)	4	μl
	RNA ligase (Takara Shuzo Co., Ltd.)	5	μl
	60% PEG6000 (Nippon Gene)	42	μl
	dH 2 O	to 100	μl

The reaction was performed at 18° C. for 16 hours. The resultant reaction product was treated with phenol to remove proteins therefrom and then subjected to ethanol precipitation, thereby giving an oligo-capping RNA. The oligo-capping RNA was dissolved in dH 2 O(50 μl). A single stranded cDNA was synthesized using the oligo-capping RNA as a template. The composition of the reaction solution used is as follows.

	Oligo-capping RNA	50	μl
	Random hexamer (20 μM)	5	μl
		55	μl

The reaction solution was heated at 70° C. for 10 min. and then chilled on ice, and the following reagents were added thereto.

	5x First strand buffer (Gibco)	20	μl
	0.1 M DTT (Gibco)	10	μl
	20 μM dNTPs	5	μl
	RNasin (Promega)	5	μl
		95	μl

The reaction solution was incubated at 37° C. for 2 min., then Superscript II (Gibco) (5 μl) was added thereto, and the reaction mixture was allowed to react at 37° C. for 30 min. After the reaction was completed, the resultant solution was heated at 95° C. for 10 min, and then dH 2 O was added thereto to the total volume of 500 μl, which was served as an oligo-capping cDNA. PCR was performed using the oligo-capping cDNA as a template. The PCR was performed two rounds. Using the first round PCR product as a template, the second round PCR was then performed (i.e., nested PCR). The composition of the reaction solution used for the first round PCR is as follows.

	Oligo-capping cDNA	1	μl
	Sense primer (SEQ ID NO: 27)(20 μM)	0.5	μl
	Antisense primer (SEQ ID NO: 29)(20 μM)	0.5	μl
	10x PCR buffer	2.5	μl
	2.5 mM dNTPs	2.0	μl
	50% Glycerol	2.5	μl
	Taq DNA polymerase	0.5	μl
	dH 2 O	15.5	μl
	Total	25	μl

The PCR was performed, starting with a denaturation step at 95° C. for 5 min., and then at a program of 94° C. for 30 sec. (denaturation); 60° C. for 30 sec. (annealing); and 72° C. for 1 min. (elongation) for 35 cycles, and ending with 72° C. for 5 min. Using the PCR product as a template, the second round PCR was performed. The reaction solution used for the second PCR was as follows.

	PCR product	1	μl
	Sense primer (SEQ ID NO: 28)(20 μM)	0.5	μl
	Antisense primer (SEQ ID NO: 30)(20 μM)	0.5	μl
	10x PCR buffer	2.5	μl
	2.5 mM dNTPs	2.0	μl
	50% Glycerol	2.5	μl
	Taq DNA polymerase	0.5	μl
	dH 2 O	15.5	μl
	Total	25	μl

The second round PCR was performed in the same manner as the first round PCR. Upon the 2% agarose gel electrophoresis, a PCR product of about 400 bp was observed. The PCR product was subcloned into pGEM-T vector and sequenced [as for the methods, see (3-ii) and (3-iii) above]. In the sequencing, a primer pair of SEQ ID NOs: 5 and 6 was used.

The nucleotide sequence of the obtained 5′ transcription origin region of human RecQ4 gene is shown in SEQ ID NO: 34.

(6) Construction of Full-length RecQ4 cDNA by Nucleotide Sequence Assembly

The four nucleotide sequences of human RecQ4 cDNA obtained in (3), (4) and (5) above are shown in SEQ ID NOs: 31-34, respectively. These sequences are overlapped in part one another, and the relative positions of the sequences are as shown in FIG. 7 . The transcription origin region, the 5′ RACE product, the partial cDNA and the 3′ RACE product in FIG. 7 are shown in SEQ ID NOs: 34, 32, 31 and 33, respectively.

These regions were analyzed by computer using DNASYS soft wear. The four nucleotide sequences were then ligated together to give a full-length RecQ4 cDNA sequence. The obtained cDNA was composed of 3850 nucleotides in full-length without the 3′ poly(A) sequence (see SEQ ID NO: 1). In the cDNA sequence, it was found that the open reading frame (ORF) was composed of 3624 nucleotides and a protein encoded by this sequence consisted of 1208 amino acid residues and had a molecular weight of 133070 daltons. The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

EXAMPLE 2

Analysis of Human RecQ4 Gene by Northern Blotting

(1) Northern Blotting Analysis of Human RecQ4 Gene

(1-i) Human Multiple Tissue Northern (MTN-I and -II) Blot

In this example, MTN blotting was performed using a commercially available pre-made filter (CLONTECH). The pre-made filter was prepared by electrophoresing poly(A)+RNAs extracted from 16 kinds of human tissues and organs (2 μg each) on an agarose gel and then blotting the gel on a nylon membrane.

(1-ii) Human RecQ4 cDNA Probe

The open reading frame (a portion of human RecQ4 cDNA; residues 2097-2417 in the nucleotide sequence of SEQ ID NO: 1; 321 bp) was radiolabeled by the method as described in below, which was used as a detection probe for human RecQ4 gene.

(2) Hybridization

(2-i) Pre-hybridization

A filter was immersed in a pre-hybridization buffer (100 ml) in a lunch box-type plastic container, and incubated at 42° C. for 4 hours. This pre-hybridization buffer contained 50% formamide, 5× SSPE, 10× Denhardt's solution, 2% SDS and 100 μg/ml of a denatured salmon spermatic DNA fragment.

(2-ii) Radiolabeling of Human RecQ4 cDNA Probe

Radiolabaling with [α 32 P]-dCTP (NEN, Daiichi Pure Chemicals) was performed using the above-mentioned human RecQ4 cDNA fragment (50 ng) as a template, random hexamer (50 pmol) as a primer and a random primer DNA labeling kit Ver.2 (Takara Shuzo Co., Ltd.).

(2-iii) Hybridization

The pre-hybridization buffer was discarded from the container and a fresh pre-hybridization buffer was added thereto. The container was gently shaken so that no air bubble was formed beneath the filter. The probe radiolabeled with [ 32 p]-dCTP was added to the pre-hybridization solution to a specific activity of about 1×10 6 cpm/ml, and hybridization was performed at 42° C. for 16 hours.

After the hybridization, the filter was rinsed two times with 2× SSC/0.1% SDS solution (100 ml) at room temperature for 15 min. each, and additionally rinsed two times with 0.2× SSC/0.1% SDS solution (100 ml) at room temperature for 15 min. each.

The filter was dried to such an extent that the filter became slightly moist, and then wrapped in a plastic wrap (“Saran wrap” made by Asahi Chemical Industries, Ltd.) for use in the subsequent autoradiographic analysis using BAS 1500 system (Fujifilm).

(3) Analysis by Fuji BAS 1500 System

The sample was close-exposed to a radiation energy memory type two-dimensional sensor (Imaging Plate; IP) using photostimulable phosphor, like a X-ray film. The IP was excited with a He—Ne laser beam. The luminescence emitted depending on the amount of the exposure light was determined in terms of a digital amount called PSL (Photo Stimulated Luminescence). The determination was performed using BAS 1500 system. The values determined by this system showed a good linearity, and therefore it was found this system could provide for subtraction of background, calculation and comparison of the intensities of radiation in the predetermined regions.

(4) Results of the Tissue-specific Expression of Human RecQ4 mRNA

The [ 32 P]-labeled ORF of human RecQ4 cDNA (a portion of human RecQ4 cDNA; nucleotides 2097-2417 in SEQ ID NO: 1; 321 bp) as a probe was hybridized to MTN blot (CLONTECH) containing poly(A)+RNAs derived from various human tissues and organs (2 μg each).

The expression of human RecQ4 mRNA was observed in various organs, and the expression pattern thereof is shown in FIG. 5 . That is, human RecQ4 gene was expressed in all tissues, and a remarkably strong expression was particularly observed in thymus and testis. In FIG. 5 , the sources from which the poly(A)+RNAs were derived are as follows.

a, heart; b, brain; c, placenta; d, lung; e, liver; f, skeletal muscle; g, kidney; h, pancreas; i, spleen; j, thymus; k, prostate; 1, testis; m, ovary; n, small intestine; o, colon; p, peripheral blood lymphocyte.

INDUSTRIAL APPLICABILITY

According to the present invention, human RecQ4 gene is provided.

The human RecQ4 gene of the present invention is useful for the study on the relation with the maintenance of homeostasis and cell aging in human and the elucidation of the causes of diseases associated with growth and aging, and also useful as a therapeutic for improving and alleviating the conditions of such diseases, a diagnostic probe for detecting and preventing the diseases relating to the other diseases associated with aging, and a reagent for medical, cell biological, immunological, biochemical and molecular biological studies on the development of human individuals.

44 1 3850 DNA Homo sapiens CDS (85)..(3708) 1gcattggctg tcggcccccg cgacggctgc gcgggagatt cgctggacga tcgcaagcgc 60ggaggccggg cgggcgcgcg cgcc atg gag cgg ctg cgg gac gtg cgg gag 111 Met Glu Arg Leu Arg Asp Val Arg Glu 1 5cgg ctg cag gcg tgg gag cgc gcg ttc cga cgg cag cgc ggg cgg cga 159Arg Leu Gln Ala Trp Glu Arg Ala Phe Arg Arg Gln Arg Gly Arg Arg10 15 20 25ccg agc cag gac gac gtg gag gcg gcg ccg gag gag acc cgc gcg ctc 207Pro Ser Gln Asp Asp Val Glu Ala Ala Pro Glu Glu Thr Arg Ala Leu 30 35 40tac cgg gag tac cgc act ctg aag cgt acc acg ggc cag gcc ggc ggc 255Tyr Arg Glu Tyr Arg Thr Leu Lys Arg Thr Thr Gly Gln Ala Gly Gly 45 50 55ggg ctc cgc agc tcc gag tcg ctc ccc gcg gcg gcc gaa gag gcg cca 303Gly Leu Arg Ser Ser Glu Ser Leu Pro Ala Ala Ala Glu Glu Ala Pro 60 65 70gag ccc cgc tgc tgg ggg ccc cat ctg aat cgg gct gcg acc aag agt 351Glu Pro Arg Cys Trp Gly Pro His Leu Asn Arg Ala Ala Thr Lys Ser 75 80 85cca cag cct acg cca ggg cgg agc cgc cag ggc tcg gtg ccg gac tac 399Pro Gln Pro Thr Pro Gly Arg Ser Arg Gln Gly Ser Val Pro Asp Tyr90 95 100 105ggg cag cgg ctc aag gcc aat ctg aaa ggc acc ctg cag gcc gga cca 447Gly Gln Arg Leu Lys Ala Asn Leu Lys Gly Thr Leu Gln Ala Gly Pro 110 115 120gcc ctg ggc cgc aga ccg tgg cct cta gga aga gcc tca tct aag gca 495Ala Leu Gly Arg Arg Pro Trp Pro Leu Gly Arg Ala Ser Ser Lys Ala 125 130 135tcc acc cca aag ccc cca ggt aca ggg cct gtc ccc tcc ttt gca gaa 543Ser Thr Pro Lys Pro Pro Gly Thr Gly Pro Val Pro Ser Phe Ala Glu 140 145 150aaa gtc agt gat gag cct cca cag ctc cct gag ccc cag cca agg cca 591Lys Val Ser Asp Glu Pro Pro Gln Leu Pro Glu Pro Gln Pro Arg Pro 155 160 165ggc cgg ctc cag cat ctg cag gca tcc ctg agc cag cgg ctg ggc tcc 639Gly Arg Leu Gln His Leu Gln Ala Ser Leu Ser Gln Arg Leu Gly Ser170 175 180 185cta gat cct ggc tgg tta cag cga tgt cac agt gag gtc cca gat ttt 687Leu Asp Pro Gly Trp Leu Gln Arg Cys His Ser Glu Val Pro Asp Phe 190 195 200ctg ggg gcc ccc aaa gcc tgc agg cct gat cta ggc tca gag gaa tca 735Leu Gly Ala Pro Lys Ala Cys Arg Pro Asp Leu Gly Ser Glu Glu Ser 205 210 215caa ctt ctg atc cct ggt gag tcg gct gtc ctt ggt cct ggt gct ggc 783Gln Leu Leu Ile Pro Gly Glu Ser Ala Val Leu Gly Pro Gly Ala Gly 220 225 230tcc cag ggc cca gag gct tca gcc ttc caa gaa gtc agc atc cgt gtg 831Ser Gln Gly Pro Glu Ala Ser Ala Phe Gln Glu Val Ser Ile Arg Val 235 240 245ggg agc ccc cag ccc agc agc agt gga ggc gag aag cgg aga tgg aac 879Gly Ser Pro Gln Pro Ser Ser Ser Gly Gly Glu Lys Arg Arg Trp Asn250 255 260 265gag gag ccc tgg gag agc ccc gca cag gtc cag cag gag agc agc caa 927Glu Glu Pro Trp Glu Ser Pro Ala Gln Val Gln Gln Glu Ser Ser Gln 270 275 280gct gga ccc cca tcg gag ggg gct ggg gct gta gca gtt gag gaa gac 975Ala Gly Pro Pro Ser Glu Gly Ala Gly Ala Val Ala Val Glu Glu Asp 285 290 295cct cca ggg gaa cct gta cag gca cag cca cct cag ccc tgc agc agc 1023Pro Pro Gly Glu Pro Val Gln Ala Gln Pro Pro Gln Pro Cys Ser Ser 300 305 310cca tcg aac ccc agg tac cac gga ctc agc ccc tcc agt caa gct agg 1071Pro Ser Asn Pro Arg Tyr His Gly Leu Ser Pro Ser Ser Gln Ala Arg 315 320 325gct ggg aag gct gag ggc aca gcc ccc ctg cac atc ttc cct cgg ctg 1119Ala Gly Lys Ala Glu Gly Thr Ala Pro Leu His Ile Phe Pro Arg Leu330 335 340 345gcc cgc cat gac agg ggc aat tac gta cgg ctc aac atg aag cag aaa 1167Ala Arg His Asp Arg Gly Asn Tyr Val Arg Leu Asn Met Lys Gln Lys 350 355 360cac tac gtg cgg ggc cgg gca ctc cgt agc agg ctc ctc cgc aag cag 1215His Tyr Val Arg Gly Arg Ala Leu Arg Ser Arg Leu Leu Arg Lys Gln 365 370 375gca tgg aag cag aag tgg cgg aag aaa ggg gag tgt ttt ggg ggt ggt 1263Ala Trp Lys Gln Lys Trp Arg Lys Lys Gly Glu Cys Phe Gly Gly Gly 380 385 390ggt gcc aca gtc aca acc aag gag tct tgt ttc ctg aac gag cag ttc 1311Gly Ala Thr Val Thr Thr Lys Glu Ser Cys Phe Leu Asn Glu Gln Phe 395 400 405gat cac tgg gca gcc cag tgt ccc cgg cca gca agt gag gaa gac aca 1359Asp His Trp Ala Ala Gln Cys Pro Arg Pro Ala Ser Glu Glu Asp Thr410 415 420 425gat gct gtt ggg cct gag cca ctg gtt cct tca cca caa cct gta cct 1407Asp Ala Val Gly Pro Glu Pro Leu Val Pro Ser Pro Gln Pro Val Pro 430 435 440gag gtg ccc agc ctg gac ccc acc gtg ctg cca ctc tac tcc ctg ggg 1455Glu Val Pro Ser Leu Asp Pro Thr Val Leu Pro Leu Tyr Ser Leu Gly 445 450 455ccc tca ggg cag ttg gca gag acg ccg gct gag gtg ttc cag gcc ctg 1503Pro Ser Gly Gln Leu Ala Glu Thr Pro Ala Glu Val Phe Gln Ala Leu 460 465 470gag cag ctg ggg cac caa gcc ttt cgc cct ggg cag gag cgt gca gtc 1551Glu Gln Leu Gly His Gln Ala Phe Arg Pro Gly Gln Glu Arg Ala Val 475 480 485atg cgg atc ctg tct ggc atc tcc acg ctg ctg gtg ctg cct aca ggt 1599Met Arg Ile Leu Ser Gly Ile Ser Thr Leu Leu Val Leu Pro Thr Gly490 495 500 505gcc ggc aag tcc ctg tgc tac cag ctc cca gcg ctg ctc tac agc cgg 1647Ala Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Leu Leu Tyr Ser Arg 510 515 520cgc agc ccc tgc ctc acg ttg gtc gtc tct ccc ctg ctg tca ctc atg 1695Arg Ser Pro Cys Leu Thr Leu Val Val Ser Pro Leu Leu Ser Leu Met 525 530 535gat gac cag gtg tct ggc ctg cca ccg tgt ctc aag gcg gcc tgc ata 1743Asp Asp Gln Val Ser Gly Leu Pro Pro Cys Leu Lys Ala Ala Cys Ile 540 545 550cac tcg ggc atg acc agg aag caa cgg gaa tct gtc ctg cag aag att 1791His Ser Gly Met Thr Arg Lys Gln Arg Glu Ser Val Leu Gln Lys Ile 555 560 565cgg gca gcc cag gta cac gtg ctg atg ctg aca cct gag gca ctg gtg 1839Arg Ala Ala Gln Val His Val Leu Met Leu Thr Pro Glu Ala Leu Val570 575 580 585ggg gcg gga ggc ctc cct cca gcc gca cag ctg cct cca gtt gct ttt 1887Gly Ala Gly Gly Leu Pro Pro Ala Ala Gln Leu Pro Pro Val Ala Phe 590 595 600gcc tgc att gat gag gcc cac tgc ctc tcc cag tgg tcc cac aac ttc 1935Ala Cys Ile Asp Glu Ala His Cys Leu Ser Gln Trp Ser His Asn Phe 605 610 615cgg ccc tgc tac ctg cgc gtc tgc aag gtg ctt cgg gag cgc atg ggc 1983Arg Pro Cys Tyr Leu Arg Val Cys Lys Val Leu Arg Glu Arg Met Gly 620 625 630gtg cac tgc ttc ctg ggc ctc aca gcc aca gcc aca cgc cgc act gcc 2031Val His Cys Phe Leu Gly Leu Thr Ala Thr Ala Thr Arg Arg Thr Ala 635 640 645agt gac gtg gca cag cac ctg gct gtg gct gaa gag cct gac ctc cac 2079Ser Asp Val Ala Gln His Leu Ala Val Ala Glu Glu Pro Asp Leu His650 655 660 665ggg cca gcc cca gtt ccc acc aac ctg cac ctt tcc gtg tcc atg gac 2127Gly Pro Ala Pro Val Pro Thr Asn Leu His Leu Ser Val Ser Met Asp 670 675 680agg gac aca gac cag gca ctg ttg acg ctg ctg caa ggc aaa cgt ttt 2175Arg Asp Thr Asp Gln Ala Leu Leu Thr Leu Leu Gln Gly Lys Arg Phe 685 690 695caa aac ctc gat tcc att atc att tac tgc aac cgg cgc gag gac aca 2223Gln Asn Leu Asp Ser Ile Ile Ile Tyr Cys Asn Arg Arg Glu Asp Thr 700 705 710gag cgg atc gct gcg ctc ctc cga acc tgc ctg cac gca gcc tgg gtc 2271Glu Arg Ile Ala Ala Leu Leu Arg Thr Cys Leu His Ala Ala Trp Val 715 720 725cca ggg tct gga ggt cgt gcc ccc aaa acc aca gcc gag gcc tac cac 2319Pro Gly Ser Gly Gly Arg Ala Pro Lys Thr Thr Ala Glu Ala Tyr His730 735 740 745gcg ggc atg tgc agc cgg gaa cgg cgg cgg gta cag cga gcc ttc atg 2367Ala Gly Met Cys Ser Arg Glu Arg Arg Arg Val Gln Arg Ala Phe Met 750 755 760cag ggc cag ttg cgg gtg gtg gtg gcc acg gtg gcc ttt ggg atg ggg 2415Gln Gly Gln Leu Arg Val Val Val Ala Thr Val Ala Phe Gly Met Gly 765 770 775ctg gac cgg cca gat gtg cgg gct gtg ctg cat ctg ggg ctg ccc cca 2463Leu Asp Arg Pro Asp Val Arg Ala Val Leu His Leu Gly Leu Pro Pro 780 785 790agc ttc gag agc tac gtg cag gcc gtg ggc cgg gcc ggg cgt gac ggg 2511Ser Phe Glu Ser Tyr Val Gln Ala Val Gly Arg Ala Gly Arg Asp Gly 795 800 805cag cct gcc cac tgc cac ctc ttc ctg cag ccc cag ggc gaa gac ctg 2559Gln Pro Ala His Cys His Leu Phe Leu Gln Pro Gln Gly Glu Asp Leu810 815 820 825cga gag ctg cgc aga cat gtg cac gcc gac agc acg gac ttc ctg gct 2607Arg Glu Leu Arg Arg His Val His Ala Asp Ser Thr Asp Phe Leu Ala 830 835 840gtg aag agg ctg gta cag cgc gtg ttc cca gcc tgc acc tgc acc tgc 2655Val Lys Arg Leu Val Gln Arg Val Phe Pro Ala Cys Thr Cys Thr Cys 845 850 855acc agg ccg ccc tcg gag cag gaa ggg gcc gtg ggt ggg gag agg cct 2703Thr Arg Pro Pro Ser Glu Gln Glu Gly Ala Val Gly Gly Glu Arg Pro 860 865 870gtg ccc aag tac ccc cct caa gag gct gag cag ctt agc cac caa gca 2751Val Pro Lys Tyr Pro Pro Gln Glu Ala Glu Gln Leu Ser His Gln Ala 875 880 885gcc cca gga ccc aga agg gtc tgc atg ggc cat gag cgg gca ctc cca 2799Ala Pro Gly Pro Arg Arg Val Cys Met Gly His Glu Arg Ala Leu Pro890 895 900 905ata cag ctt acc gta cag gct ttg gac atg ccg gag gag gcc atc gag 2847Ile Gln Leu Thr Val Gln Ala Leu Asp Met Pro Glu Glu Ala Ile Glu 910 915 920act ttg ctg tgc tac ctg gag ctg cac cca cac cac tgg ctg gag ctg 2895Thr Leu Leu Cys Tyr Leu Glu Leu His Pro His His Trp Leu Glu Leu 925 930 935ctg gcg acc acc tat acc cat tgc cgt ctg aac tgc cct ggg ggc cct 2943Leu Ala Thr Thr Tyr Thr His Cys Arg Leu Asn Cys Pro Gly Gly Pro 940 945 950gcc cag ctc cag gcc ctg gcc cac agg tgt ccc cct ttg gct gtg tgc 2991Ala Gln Leu Gln Ala Leu Ala His Arg Cys Pro Pro Leu Ala Val Cys 955 960 965ttg gcc cag cag ctg cct gag gac cca ggg caa ggc agc agc tcc gtg 3039Leu Ala Gln Gln Leu Pro Glu Asp Pro Gly Gln Gly Ser Ser Ser Val970 975 980 985gag ttt gac atg gtc aag ctg gtg gac tcc atg ggc tgg gag ctg gcc 3087Glu Phe Asp Met Val Lys Leu Val Asp Ser Met Gly Trp Glu Leu Ala 990 995 1000tct gtg cgg cgg gct ctc tgc cag ctg cag tgg gac cac gag ccc 3132Ser Val Arg Arg Ala Leu Cys Gln Leu Gln Trp Asp His Glu Pro 1005 1010 1015agg aca ggt gtg cgg cgt ggg aca ggg gtg ctt gtg gag ttc agt 3177Arg Thr Gly Val Arg Arg Gly Thr Gly Val Leu Val Glu Phe Ser 1020 1025 1030gag ctg gcc ttc cac ctt cgc agc ccg ggg gac ctg acc gct gag 3222Glu Leu Ala Phe His Leu Arg Ser Pro Gly Asp Leu Thr Ala Glu 1035 1040 1045gag aag gac cag ata tgt gac ttc ctc tat ggc cgt gtg cag gcc 3267Glu Lys Asp Gln Ile Cys Asp Phe Leu Tyr Gly Arg Val Gln Ala 1050 1055 1060cgg gag cgc cag gcc ctg gcc cgt ctg cgc aga acc ttc cag gcc 3312Arg Glu Arg Gln Ala Leu Ala Arg Leu Arg Arg Thr Phe Gln Ala 1065 1070 1075ttt cac agc gta gcc ttc ccc agc tgc ggg ccc tgc ctg gag cag 3357Phe His Ser Val Ala Phe Pro Ser Cys Gly Pro Cys Leu Glu Gln 1080 1085 1090cag gat gag gag cgc agc acc agg ctc aag gac ctg ctc ggc cgc 3402Gln Asp Glu Glu Arg Ser Thr Arg Leu Lys Asp Leu Leu Gly Arg 1095 1100 1105tac ttt gag gaa gag gaa ggg cag gag ccg gga ggc atg gag gac 3447Tyr Phe Glu Glu Glu Glu Gly Gln Glu Pro Gly Gly Met Glu Asp 1110 1115 1120gca cag ggc ccc gag cca ggg cag gcc aga ctc cag gat tgg gag 3492Ala Gln Gly Pro Glu Pro Gly Gln Ala Arg Leu Gln Asp Trp Glu 1125 1130 1135gac cag gtc cgc tgc gac atc cgc cag ttc ctg tcc ctg agg cca 3537Asp Gln Val Arg Cys Asp Ile Arg Gln Phe Leu Ser Leu Arg Pro 1140 1145 1150gag gag aag ttc tcc agc agg gct gtg gcc cgc atc ttc cac ggc 3582Glu Glu Lys Phe Ser Ser Arg Ala Val Ala Arg Ile Phe His Gly 1155 1160 1165atc gga agc ccc tgc tac ccg gcc cag gtg tac ggg cag gac cga 3627Ile Gly Ser Pro Cys Tyr Pro Ala Gln Val Tyr Gly Gln Asp Arg 1170 1175 1180cgc ttc tgg aga aaa tac ctg cac ctg agc ttc cat gcc ctg gtg 3672Arg Phe Trp Arg Lys Tyr Leu His Leu Ser Phe His Ala Leu Val 1185 1190 1195ggc ctg gcc acg gaa gag ctc ctg cag gtg gcc cgc tgactgcact 3718Gly Leu Ala Thr Glu Glu Leu Leu Gln Val Ala Arg 1200 1205gcattggggg atgtcgggta gagctggggt tgtcagaggc tagggcagtg actgaggacc 3778tgggcaaaac ctgccacagg gtgtgggaac gaggaggctc caaaatgcag aataaaaaat 3838gctcactttg tt 3850 2 1208 PRT Homo sapiens 2Met Glu Arg Leu Arg Asp Val Arg Glu Arg Leu Gln Ala Trp Glu Arg1 5 10 15Ala Phe Arg Arg Gln Arg Gly Arg Arg Pro Ser Gln Asp Asp Val Glu 20 25 30Ala Ala Pro Glu Glu Thr Arg Ala Leu Tyr Arg Glu Tyr Arg Thr Leu 35 40 45Lys Arg Thr Thr Gly Gln Ala Gly Gly Gly Leu Arg Ser Ser Glu Ser 50 55 60Leu Pro Ala Ala Ala Glu Glu Ala Pro Glu Pro Arg Cys Trp Gly Pro65 70 75 80His Leu Asn Arg Ala Ala Thr Lys Ser Pro Gln Pro Thr Pro Gly Arg 85 90 95Ser Arg Gln Gly Ser Val Pro Asp Tyr Gly Gln Arg Leu Lys Ala Asn 100 105 110Leu Lys Gly Thr Leu Gln Ala Gly Pro Ala Leu Gly Arg Arg Pro Trp 115 120 125Pro Leu Gly Arg Ala Ser Ser Lys Ala Ser Thr Pro Lys Pro Pro Gly 130 135 140Thr Gly Pro Val Pro Ser Phe Ala Glu Lys Val Ser Asp Glu Pro Pro145 150 155 160Gln Leu Pro Glu Pro Gln Pro Arg Pro Gly Arg Leu Gln His Leu Gln 165 170 175Ala Ser Leu Ser Gln Arg Leu Gly Ser Leu Asp Pro Gly Trp Leu Gln 180 185 190Arg Cys His Ser Glu Val Pro Asp Phe Leu Gly Ala Pro Lys Ala Cys 195 200 205Arg Pro Asp Leu Gly Ser Glu Glu Ser Gln Leu Leu Ile Pro Gly Glu 210 215 220Ser Ala Val Leu Gly Pro Gly Ala Gly Ser Gln Gly Pro Glu Ala Ser225 230 235 240Ala Phe Gln Glu Val Ser Ile Arg Val Gly Ser Pro Gln Pro Ser Ser 245 250 255Ser Gly Gly Glu Lys Arg Arg Trp Asn Glu Glu Pro Trp Glu Ser Pro 260 265 270Ala Gln Val Gln Gln Glu Ser Ser Gln Ala Gly Pro Pro Ser Glu Gly 275 280 285Ala Gly Ala Val Ala Val Glu Glu Asp Pro Pro Gly Glu Pro Val Gln 290 295 300Ala Gln Pro Pro Gln Pro Cys Ser Ser Pro Ser Asn Pro Arg Tyr His305 310 315 320Gly Leu Ser Pro Ser Ser Gln Ala Arg Ala Gly Lys Ala Glu Gly Thr 325 330 335Ala Pro Leu His Ile Phe Pro Arg Leu Ala Arg His Asp Arg Gly Asn 340 345 350Tyr Val Arg Leu Asn Met Lys Gln Lys His Tyr Val Arg Gly Arg Ala 355 360 365Leu Arg Ser Arg Leu Leu Arg Lys Gln Ala Trp Lys Gln Lys Trp Arg 370 375 380Lys Lys Gly Glu Cys Phe Gly Gly Gly Gly Ala Thr Val Thr Thr Lys385 390 395 400Glu Ser Cys Phe Leu Asn Glu Gln Phe Asp His Trp Ala Ala Gln Cys 405 410 415Pro Arg Pro Ala Ser Glu Glu Asp Thr Asp Ala Val Gly Pro Glu Pro 420 425 430Leu Val Pro Ser Pro Gln Pro Val Pro Glu Val Pro Ser Leu Asp Pro 435 440 445Thr Val Leu Pro Leu Tyr Ser Leu Gly Pro Ser Gly Gln Leu Ala Glu 450 455 460Thr Pro Ala Glu Val Phe Gln Ala Leu Glu Gln Leu Gly His Gln Ala465 470 475 480Phe Arg Pro Gly Gln Glu Arg Ala Val Met Arg Ile Leu Ser Gly Ile 485 490 495Ser Thr Leu Leu Val Leu Pro Thr Gly Ala Gly Lys Ser Leu Cys Tyr 500 505 510Gln Leu Pro Ala Leu Leu Tyr Ser Arg Arg Ser Pro Cys Leu Thr Leu 515 520 525Val Val Ser Pro Leu Leu Ser Leu Met Asp Asp Gln Val Ser Gly Leu 530 535 540Pro Pro Cys Leu Lys Ala Ala Cys Ile His Ser Gly Met Thr Arg Lys545 550 555 560Gln Arg Glu Ser Val Leu Gln Lys Ile Arg Ala Ala Gln Val His Val 565 570 575Leu Met Leu Thr Pro Glu Ala Leu Val Gly Ala Gly Gly Leu Pro Pro 580 585 590Ala Ala Gln Leu Pro Pro Val Ala Phe Ala Cys Ile Asp Glu Ala His 595 600 605Cys Leu Ser Gln Trp Ser His Asn Phe Arg Pro Cys Tyr Leu Arg Val 610 615 620Cys Lys Val Leu Arg Glu Arg Met Gly Val His Cys Phe Leu Gly Leu625 630 635 640Thr Ala Thr Ala Thr Arg Arg Thr Ala Ser Asp Val Ala Gln His Leu 645 650 655Ala Val Ala Glu Glu Pro Asp Leu His Gly Pro Ala Pro Val Pro Thr 660 665 670Asn Leu His Leu Ser Val Ser Met Asp Arg Asp Thr Asp Gln Ala Leu 675 680 685Leu Thr Leu Leu Gln Gly Lys Arg Phe Gln Asn Leu Asp Ser Ile Ile 690 695 700Ile Tyr Cys Asn Arg Arg Glu Asp Thr Glu Arg Ile Ala Ala Leu Leu705 710 715 720Arg Thr Cys Leu His Ala Ala Trp Val Pro Gly Ser Gly Gly Arg Ala 725 730 735Pro Lys Thr Thr Ala Glu Ala Tyr His Ala Gly Met Cys Ser Arg Glu 740 745 750Arg Arg Arg Val Gln Arg Ala Phe Met Gln Gly Gln Leu Arg Val Val 755 760 765Val Ala Thr Val Ala Phe Gly Met Gly Leu Asp Arg Pro Asp Val Arg 770 775 780Ala Val Leu His Leu Gly Leu Pro Pro Ser Phe Glu Ser Tyr Val Gln785 790 795 800Ala Val Gly Arg Ala Gly Arg Asp Gly Gln Pro Ala His Cys His Leu 805 810 815Phe Leu Gln Pro Gln Gly Glu Asp Leu Arg Glu Leu Arg Arg His Val 820 825 830His Ala Asp Ser Thr Asp Phe Leu Ala Val Lys Arg Leu Val Gln Arg 835 840 845Val Phe Pro Ala Cys Thr Cys Thr Cys Thr Arg Pro Pro Ser Glu Gln 850 855 860Glu Gly Ala Val Gly Gly Glu Arg Pro Val Pro Lys Tyr Pro Pro Gln865 870 875 880Glu Ala Glu Gln Leu Ser His Gln Ala Ala Pro Gly Pro Arg Arg Val 885 890 895Cys Met Gly His Glu Arg Ala Leu Pro Ile Gln Leu Thr Val Gln Ala 900 905 910Leu Asp Met Pro Glu Glu Ala Ile Glu Thr Leu Leu Cys Tyr Leu Glu 915 920 925Leu His Pro His His Trp Leu Glu Leu Leu Ala Thr Thr Tyr Thr His 930 935 940Cys Arg Leu Asn Cys Pro Gly Gly Pro Ala Gln Leu Gln Ala Leu Ala945 950 955 960His Arg Cys Pro Pro Leu Ala Val Cys Leu Ala Gln Gln Leu Pro Glu 965 970 975Asp Pro Gly Gln Gly Ser Ser Ser Val Glu Phe Asp Met Val Lys Leu 980 985 990Val Asp Ser Met Gly Trp Glu Leu Ala Ser Val Arg Arg Ala Leu Cys 995 1000 1005Gln Leu Gln Trp Asp His Glu Pro Arg Thr Gly Val Arg Arg Gly 1010 1015 1020Thr Gly Val Leu Val Glu Phe Ser Glu Leu Ala Phe His Leu Arg 1025 1030 1035Ser Pro Gly Asp Leu Thr Ala Glu Glu Lys Asp Gln Ile Cys Asp 1040 1045 1050Phe Leu Tyr Gly Arg Val Gln Ala Arg Glu Arg Gln Ala Leu Ala 1055 1060 1065Arg Leu Arg Arg Thr Phe Gln Ala Phe His Ser Val Ala Phe Pro 1070 1075 1080Ser Cys Gly Pro Cys Leu Glu Gln Gln Asp Glu Glu Arg Ser Thr 1085 1090 1095Arg Leu Lys Asp Leu Leu Gly Arg Tyr Phe Glu Glu Glu Glu Gly 1100 1105 1110Gln Glu Pro Gly Gly Met Glu Asp Ala Gln Gly Pro Glu Pro Gly 1115 1120 1125Gln Ala Arg Leu Gln Asp Trp Glu Asp Gln Val Arg Cys Asp Ile 1130 1135 1140Arg Gln Phe Leu Ser Leu Arg Pro Glu Glu Lys Phe Ser Ser Arg 1145 1150 1155Ala Val Ala Arg Ile Phe His Gly Ile Gly Ser Pro Cys Tyr Pro 1160 1165 1170Ala Gln Val Tyr Gly Gln Asp Arg Arg Phe Trp Arg Lys Tyr Leu 1175 1180 1185His Leu Ser Phe His Ala Leu Val Gly Leu Ala Thr Glu Glu Leu 1190 1195 1200Leu Gln Val Ala Arg 1205 3 24 DNA Artificial Sequence SENSE PRIMER 3caccaacctg cacctttccg tgtc 24 4 24 DNA Artificial Sequence ANTISENSE PRIMER 4agccccatcc caaaagccac cgtg 24 5 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 5cgccagggtt ttcccagtca cgac 24 6 22 DNA Artificial Sequence PRIMERS FOR SEQUENCING 6tcacacagga aacagctatg ac 22 7 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 7aatctgggac ctcactgtga catc 24 8 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 8tcatctaagg catccacccc aaag 24 9 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 9tcacaacttc tgatccctgg tgag 24 10 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 10ctcagcccct ccagtcaagc tagg 24 11 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 11gtttcctgaa cgagcagttc gatc 24 12 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 12ctgggcagga gcgtgcagtc atgc 24 13 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 13gctgcctcca gttgcttttg cctg 24 14 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 14ggacacagac caggcactgt tgac 24 15 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 15gggtacagcg agccttcatg cagg 24 16 24 DNA Artificial Sequence PRIMERS FOR SEQUENCING 16tcctggctgt gaagaggctg gtac 24 17 22 DNA Artificial Sequence PRIMERS FOR SEQUENCING 17cagcttaccg tacaggcttt gg 22 18 22 DNA Artificial Sequence PRIMERS FOR SEQUENCING 18ggggtgcttg tggagttcag tg 22 19 22 DNA Artificial Sequence PRIMERS FOR SEQUENCING 19caggccagac tccaggattg gg 22 20 27 DNA Artificial Sequence PRIMER AP1 20ccatcctaat acgactcact atagggc 27 21 23 DNA Artificial Sequence AP2 21actcactata gggctcgagc ggc 23 22 24 DNA Artificial Sequence PRIMER 5′ GSP1 22gccttgcagc agcgtcaaca gtgc 24 23 24 DNA Artificial Sequence PRIMER 5′ GSP2 23tggacacgga aaggtgcagg ttgg 24 24 24 DNA Artificial Sequence PRIMER 3′GSP1 24gttgacgctg ctgcaaggca aacg 24 25 24 DNA Artificial Sequence PRIMER 3′ GSP2 25catctcatgg aatgatcctg tcgg 24 26 30 RNA Artificial Sequence OLIGO RNA ADAPTER 26cgaaucguaa ccguucguac gagaaucgcu 30 27 23 DNA Artificial Sequence SENSE PRIMER FIRST ROUND 27cgaatcgtaa ccgttcgtac gag 23 28 26 DNA Artificial Sequence SENSE PRIMER NESTED 28atcgtaaccg ttcgtacgag aatcgc 26 29 24 DNA Artificial Sequence ANTISENSE PRIMER FIRST ROUND 29taggctgtgg actcttggtc gcag 24 30 24 DNA Artificial Sequence ANTISENSE PRIMER NESTED 30ttggtcgcag cccgattcag atgg 24 31 321 DNA Artificial Sequence RECQ4 PARTIAL cDNA 31caccaacctg cacctttccg tgtccatgga cagggacaca gaccaggcac tgttgacgct 60gctgcaaggc aaacgttttc aaaacctcga ttccattatc atttactgca accggcgcga 120ggacacagag cggatcgctg cgctcctccg aacctgcctg cacgcagcct gggtcccagg 180gtctggaggt cgtgccccca aaaccacagc cgaggcctac cacgcgggca tgtgcagccg 240ggaacggcgg cgggtacagc gagccttcat gcagggccag ttgcgggtgg tggtggccac 300ggtggccttt gggatggggc t 321 32 1954 DNA Artificial Sequence RECQ4 5′ RACE PRODUCT 32gacgacgtgg aggcggcgcc ggaggagacc cgcgcgctct accgggagta ccgcactctg 60aagcgtacca cgggccaggc cggcggcggg ctccgcagct ccgagtcgct ccccgcggcg 120gccgaagagg cgccagagcc ccgctgctgg gggccccatc tgaatcgggc tgcgaccaag 180agtccacagc ctacgccagg gcggagccgc cagggctcgg tgccggacta cgggcagcgg 240ctcaaggcca atctgaaagg caccctgcag gccggaccag ccctgggccg cagaccgtgg 300cctctaggaa gagcctcatc taaggcatcc accccaaagc ccccaggtac agggcctgtc 360ccctcctttg cagaaaaagt cagtgatgag cctccacagc tccctgagcc ccagccaagg 420ccaggccggc tccagcatct gcaggcatcc ctgagccagc ggctgggctc cctagatcct 480ggctggttac agcgatgtca cagtgaggtc ccagattttc tgggggcccc caaagcctgc 540aggcctgatc taggctcaga ggaatcacaa cttctgatcc ctggtgagtc ggctgtcctt 600ggtcctggtg ctggctccca gggcccagag gcttcagcct tccaagaagt cagcatccgt 660gtggggagcc cccagcccag cagcagtgga ggcgagaagc ggagatggaa cgaggagccc 720tgggagagcc ccgcacaggt ccagcaggag agcagccaag ctggaccccc atcggagggg 780gctggggctg tagcagttga ggaagaccct ccaggggaac ctgtacaggc acagccacct 840cagccctgca gcagcccatc gaaccccagg taccacggac tcagcccctc cagtcaagct 900agggctggga aggctgaggg cacagccccc ctgcacatct tccctcggct ggcccgccat 960gacaggggca attacgtacg gctcaacatg aagcagaaac actacgtgcg gggccgggca 1020ctccgtagca ggctcctccg caagcaggca tggaagcaga agtggcggaa gaaaggggag 1080tgttttgggg gtggtggtgc cacagtcaca accaaggagt cttgtttcct gaacgagcag 1140ttcgatcact gggcagccca gtgtccccgg ccagcaagtg aggaagacac agatgctgtt 1200gggcctgagc cactggttcc ttcaccacaa cctgtacctg aggtgcccag cctggacccc 1260accgtgctgc cactctactc cctggggccc tcagggcagt tggcagagac gccggctgag 1320gtgttccagg ccctggagca gctggggcac caagcctttc gccctgggca ggagcgtgca 1380gtcatgcgga tcctgtctgg catctccacg ctgctggtgc tgcctacagg tgccggcaag 1440tccctgtgct accagctccc agcgctgctc tacagccggc gcagcccctg cctcacgttg 1500gtcgtctctc ccctgctgtc actcatggat gaccaggtgt ctggcctgcc accgtgtctc 1560aaggcggcct gcatacactc gggcatgacc aggaagcaac gggaatctgt cctgcagaag 1620attcgggcag cccaggtaca cgtgctgatg ctgacacctg aggcactggt gggggcggga 1680ggcctccctc cagccgcaca gctgcctcca gttgcttttg cctgcattga tgaggcccac 1740tgcctctccc agtggtccca caacttccgg ccctgctacc tgcgcgtctg caaggtgctt 1800cgggagcgca tgggcgtgca ctgcttcctg ggcctcacag ccacagccac acgccgcact 1860gccagtgacg tggcacagca cctggctgtg gctgaagagc ctgacctcca cgggccagcc 1920ccagttccca ccaacctgca cctttccgtg tcca 1954 33 1503 DNA Artificial Sequence RECQ4 3′ RACE PRODUCT 33gggtacagcg agccttcatg cagggccagt tgcgggtggt ggtggccacg gtggcctttg 60ggatggggct ggaccggcca gatgtgcggg ctgtgctgca tctggggctg cccccaagct 120tcgagagcta cgtgcaggcc gtgggccggg ccgggcgtga cgggcagcct gcccactgcc 180acctcttcct gcagccccag ggcgaagacc tgcgagagct gcgcagacat gtgcacgccg 240acagcacgga cttcctggct gtgaagaggc tggtacagcg cgtgttccca gcctgcacct 300gcacctgcac caggccgccc tcggagcagg aaggggccgt gggtggggag aggcctgtgc 360ccaagtaccc ccctcaagag gctgagcagc ttagccacca agcagcccca ggacccagaa 420gggtctgcat gggccatgag cgggcactcc caatacagct taccgtacag gctttggaca 480tgccggagga ggccatcgag actttgctgt gctacctgga gctgcaccca caccactggc 540tggagctgct ggcgaccacc tatacccatt gccgtctgaa ctgccctggg ggccctgccc 600agctccaggc cctggcccac aggtgtcccc ctttggctgt gtgcttggcc cagcagctgc 660ctgaggaccc agggcaaggc agcagctccg tggagtttga catggtcaag ctggtggact 720ccatgggctg ggagctggcc tctgtgcggc gggctctctg ccagctgcag tgggaccacg 780agcccaggac aggtgtgcgg cgtgggacag gggtgcttgt ggagttcagt gagctggcct 840tccaccttcg cagcccgggg gacctgaccg ctgaggagaa ggaccagata tgtgacttcc 900tctatggccg tgtgcaggcc cgggagcgcc aggccctggc ccgtctgcgc agaaccttcc 960aggcctttca cagcgtagcc ttccccagct gcgggccctg cctggagcag caggatgagg 1020agcgcagcac caggctcaag gacctgctcg gccgctactt tgaggaagag gaagggcagg 1080agccgggagg catggaggac gcacagggcc ccgagccagg gcaggccaga ctccaggatt 1140gggaggacca ggtccgctgc gacatccgcc agttcctgtc cctgaggcca gaggagaagt 1200tctccagcag ggctgtggcc cgcatcttcc acggcatcgg aagcccctgc tacccggccc 1260aggtgtacgg gcaggaccga cgcttctgga gaaaatacct gcacctgagc ttccatgccc 1320tggtgggcct ggccacggaa gagctcctgc aggtggcccg ctgactgcac tgcattgggg 1380gatgtcgggt agagctgggg ttgtcagagg ctagggcagt gactgaggac ctgggcaaaa 1440cctgccacag ggtgtgggaa cgaggaggct ccaaaatgca gaataaaaaa tgctcacttt 1500gtt 1503 34 347 DNA Artificial Sequence RECQ4 TRANSCRIPTION ORIGIN REGION 34gcattggctg tcggcccccg cgacggctgc gcgggagatt cgctggacga tcgcaagcgc 60ggaggccggg cgggcgcgcg cgccatggag cggctgcggg acgtgcggga gcggctgcag 120gcgtgggagc gcgcgttccg acggcagcgc gggcggcgac cgagccagga cgacgtggag 180gcggcgccgg aggagacccg cgcgctctac cgggagtacc gcactctgaa gcgtaccacg 240ggccaggccg gcggcgggct ccgcagctcc gagtcgctcc ccgcggcggc cgaagaggcg 300ccagagcccc gctgctgggg gccccatctg aatcgggctg cgaccaa 347 35 24 DNA Artificial Sequence PRIMERS FOR THE RECQ4 GENE 35gacggctgcg cgggagattc gctg 24 36 24 DNA Artificial Sequence PRIMERS FOR THE RECQ4 GENE 36caggttttgc ccaggtcctc agtc 24 37 361 PRT Homo sapiens 37Gln Ala Leu Glu Gln Leu Gly His Gln Ala Phe Arg Pro Gly Gln Glu1 5 10 15Arg Ala Val Met Arg Ile Leu Ser Gly Ile Ser Thr Leu Leu Val Leu 20 25 30Pro Thr Gly Ala Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Leu Leu 35 40 45Tyr Ser Arg Arg Ser Pro Cys Leu Thr Leu Val Val Ser Pro Leu Leu 50 55 60Ser Leu Met Asp Asp Gln Val Ser Gly Leu Pro Pro Cys Leu Lys Ala65 70 75 80Ala Cys Ile His Ser Gly Met Thr Arg Lys Gln Arg Glu Ser Val Leu 85 90 95Gln Lys Ile Arg Ala Ala Gln Val His Val Leu Met Leu Thr Pro Glu 100 105 110Ala Leu Val Gly Ala Gly Gly Leu Pro Pro Ala Ala Gln Leu Pro Pro 115 120 125Val Ala Phe Ala Cys Ile Asp Glu Ala His Cys Leu Ser Gln Trp Ser 130 135 140His Asn Phe Arg Pro Cys Tyr Leu Arg Val Cys Lys Val Leu Arg Glu145 150 155 160Arg Met Gly Val His Cys Phe Leu Gly Leu Thr Ala Thr Ala Thr Arg 165 170 175Arg Thr Ala Ser Asp Val Ala Gln His Leu Ala Val Ala Glu Glu Pro 180 185 190Asp Leu His Gly Pro Ala Pro Val Pro Thr Asn Leu His Leu Ser Val 195 200 205Ser Met Asp Arg Asp Thr Asp Gln Ala Leu Leu Thr Leu Leu Gln Gly 210 215 220Lys Arg Phe Gln Asn Leu Asp Ser Ile Ile Ile Tyr Cys Asn Arg Arg225 230 235 240Glu Asp Thr Glu Arg Ile Ala Ala Leu Leu Arg Thr Cys Leu His Ala 245 250 255Ala Trp Val Pro Gly Ser Gly Gly Arg Ala Pro Lys Thr Thr Ala Glu 260 265 270Ala Tyr His Ala Gly Met Cys Ser Arg Glu Arg Arg Arg Val Gln Arg 275 280 285Ala Phe Met Gln Gly Gln Leu Arg Val Val Val Ala Thr Val Ala Phe 290 295 300Gly Met Gly Leu Asp Arg Pro Asp Val Arg Ala Val Leu His Leu Gly305 310 315 320Leu Pro Pro Ser Phe Glu Ser Tyr Val Gln Ala Val Gly Arg Ala Gly 325 330 335Arg Asp Gly Gln Pro Ala His Cys His Leu Phe Leu Gln Pro Gln Gly 340 345 350Glu Asp Leu Arg Glu Leu Arg Arg His 355 360 38 334 PRT Escherichia coli 38Phe Gly Tyr Gln Gln Phe Arg Pro Gly Gln Glu Glu Ile Ile Asp Thr1 5 10 15Val Leu Ser Gly Arg Asp Cys Leu Val Val Met Pro Thr Gly Gly Gly 20 25 30Lys Ser Leu Cys Tyr Gln Ile Pro Ala Leu Leu Leu Asn Gly Leu Thr 35 40 45Val Val Val Ser Pro Leu Ile Ser Leu Met Lys Asp Gln Val Asp Gln 50 55 60Leu Gln Ala Asn Gly Val Ala Ala Ala Cys Leu Asn Ser Thr Gln Thr65 70 75 80Arg Glu Gln Gln Leu Glu Val Met Thr Gly Cys Arg Thr Gly Gln Ile 85 90 95Arg Leu Leu Tyr Ile Ala Pro Glu Arg Leu Met Leu Asp Asn Phe Leu 100 105 110Glu His Leu Ala His Trp Asn Pro Val Leu Leu Ala Val Asp Glu Ala 115 120 125His Cys Ile Ser Gln Trp Gly His Asp Phe Arg Pro Glu Tyr Ala Ala 130 135 140Leu Gly Gln Leu Arg Gln Arg Phe Pro Thr Leu Pro Phe Met Ala Leu145 150 155 160Thr Ala Thr Ala Asp Asp Thr Thr Arg Gln Asp Ile Val Arg Leu Leu 165 170 175Gly Leu Asn Asp Pro Leu Ile Gln Ile Ser Ser Phe Asp Arg Pro Asn 180 185 190Ile Arg Tyr Met Leu Met Glu Lys Phe Lys Pro Leu Asp Gln Leu Met 195 200 205Arg Tyr Val Gln Glu Gln Arg Gly Lys Ser Gly Ile Ile Tyr Cys Asn 210 215 220Ser Arg Ala Lys Val Glu Asp Thr Ala Ala Arg Leu Gln Ser Lys Gly225 230 235 240Ile Ser Ala Ala Ala Tyr His Ala Gly Leu Glu Asn Asn Val Arg Ala 245 250 255Asp Val Gln Glu Lys Phe Gln Arg Asp Asp Leu Gln Ile Val Val Ala 260 265 270Thr Val Ala Phe Gly Met Gly Ile Asn Lys Pro Asn Val Arg Phe Val 275 280 285Val His Phe Asp Ile Pro Arg Asn Ile Glu Ser Tyr Tyr Gln Glu Thr 290 295 300Gly Arg Ala Gly Arg Asp Gly Leu Pro Ala Glu Ala Met Leu Phe Tyr305 310 315 320Asp Pro Ala Asp Met Ala Trp Leu Arg Arg Cys Leu Glu Glu 325 330 39 333 PRT Saccharomyces cerevisiae 39Phe Arg Pro Asn Gln Leu Glu Ala Val Asn Ala Thr Leu Gln Gly Lys1 5 10 15Asp Val Phe Val Leu Met Pro Thr Gly Gly Gly Lys Ser Leu Cys Tyr 20 25 30Gln Leu Pro Ala Val Val Lys Ser Gly Lys Thr His Gly Thr Thr Ile 35 40 45Val Ile Ser Pro Leu Ile Ser Leu Met Gln Asp Gln Val Glu His Leu 50 55 60Leu Asn Lys Asn Ile Lys Ala Ser Met Phe Ser Ser Arg Gly Thr Ala65 70 75 80Glu Gln Arg Arg Gln Thr Phe Asn Leu Phe Ile Asn Gly Leu Leu Asp 85 90 95Leu Val Tyr Ile Ser Pro Glu Met Ile Ser Ala Ser Glu Gln Cys Lys 100 105 110Arg Ala Ile Ser Arg Leu Tyr Ala Asp Gly Lys Leu Ala Arg Ile Val 115 120 125Val Asp Glu Ala His Cys Val Ser Asn Trp Gly His Asp Phe Arg Pro 130 135 140Asp Tyr Lys Glu Leu Lys Phe Phe Lys Arg Glu Tyr Pro Asp Ile Pro145 150 155 160Met Ile Ala Leu Thr Ala Thr Ala Ser Glu Gln Val Arg Met Asp Ile 165 170 175Ile His Asn Leu Glu Leu Lys Glu Pro Val Phe Leu Lys Gln Ser Phe 180 185 190Asn Arg Thr Asn Leu Tyr Tyr Glu Val Asn Lys Lys Thr Lys Asn Thr 195 200 205Ile Phe Glu Ile Cys Asp Ala Val Lys Ser Arg Phe Lys Asn Gln Thr 210 215 220Gly Ile Ile Tyr Cys His Ser Lys Lys Ser Cys Glu Gln Thr Ser Ala225 230 235 240Gln Met Gln Arg Asn Gly Ile Lys Cys Ala Tyr Tyr His Ala Gly Met 245 250 255Glu Pro Asp Glu Arg Leu Ser Val Gln Lys Ala Trp Gln Ala Asp Glu 260 265 270Ile Gln Val Ile Cys Ala Thr Val Ala Phe Gly Met Gly Ile Asp Lys 275 280 285Pro Asp Val Arg Phe Val Tyr His Phe Thr Val Pro Arg Thr Leu Glu 290 295 300Gly Tyr Tyr Gln Glu Thr Gly Arg Ala Gly Arg Asp Gly Asn Tyr Ser305 310 315 320Tyr Cys Ile Thr Tyr Phe Ser Phe Arg Asp Ile Arg Thr 325 330 40 332 PRT Homo sapiens 40Phe Arg Pro Leu Gln Leu Glu Thr Ile Asn Val Thr Met Ala Gly Lys1 5 10 15Glu Val Phe Leu Val Met Pro Thr Gly Gly Gly Lys Ser Leu Cys Tyr 20 25 30Gln Leu Pro Ala Leu Cys Ser Asp Gly Phe Thr Leu Val Ile Cys Pro 35 40 45Leu Ile Ser Leu Met Glu Asp Gln Leu Met Val Leu Lys Gln Leu Gly 50 55 60Ile Ser Ala Thr Met Leu Asn Ala Ser Ser Ser Lys Glu His Val Lys65 70 75 80Trp Val His Ala Glu Met Val Asn Lys Asn Ser Glu Leu Lys Leu Ile 85 90 95Tyr Val Thr Pro Glu Lys Ile Ala Lys Ser Lys Met Phe Met Ser Arg 100 105 110Leu Glu Lys Ala Tyr Glu Ala Arg Arg Phe Thr Arg Ile Ala Val Asp 115 120 125Glu Val His Cys Cys Ser Gln Trp Gly His Asp Phe Arg Pro Asp Tyr 130 135 140Lys Ala Leu Gly Ile Leu Lys Arg Gln Phe Pro Asn Ala Ser Leu Ile145 150 155 160Gly Leu Thr Ala Thr Ala Thr Asn His Val Leu Thr Asp Ala Gln Lys 165 170 175Ile Leu Cys Ile Glu Lys Cys Phe Thr Phe Thr Ala Ser Phe Asn Arg 180 185 190Pro Asn Leu Tyr Tyr Glu Val Arg Gln Lys Pro Ser Asn Thr Glu Asp 195 200 205Phe Ile Glu Asp Ile Val Lys Leu Ile Asn Gly Arg Tyr Lys Gly Gln 210 215 220Ser Gly Ile Ile Tyr Cys Phe Ser Gln Lys Asp Ser Glu Gln Val Thr225 230 235 240Val Ser Leu Gln Asn Leu Gly Ile His Ala Gly Ala Tyr His Ala Asn 245 250 255Leu Glu Pro Glu Asp Lys Thr Thr Val His Arg Lys Trp Ser Ala Asn 260 265 270Glu Ile Gln Val Val Val Ala Thr Val Ala Phe Gly Met Gly Ile Asp 275 280 285Lys Pro Asp Val Arg Phe Val Ile His His Ser Met Ser Lys Ser Met 290 295 300Glu Asn Tyr Tyr Gln Glu Ser Gly Arg Ala Gly Arg Asp Asp Met Lys305 310 315 320Ala Asp Cys Ile Leu Tyr Tyr Gly Phe Gly Asp Ile 325 330 41 320 PRT Homo sapiens 41Gln Leu Glu Ala Ile Asn Ala Ala Leu Leu Gly Glu Asp Cys Phe Ile1 5 10 15Leu Met Pro Thr Gly Gly Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala 20 25 30Cys Val Ser Pro Gly Val Thr Val Val Ile Ser Pro Leu Arg Ser Leu 35 40 45Ile Val Asp Gln Val Gln Lys Leu Thr Ser Leu Asp Ile Pro Ala Thr 50 55 60Tyr Leu Thr Gly Asp Lys Thr Asp Ser Glu Ala Thr Asn Ile Tyr Leu65 70 75 80Gln Leu Ser Lys Lys Asp Pro Ile Ile Lys Leu Leu Tyr Val Thr Pro 85 90 95Glu Lys Ile Cys Ala Ser Asn Arg Leu Ile Ser Thr Leu Glu Asn Leu 100 105 110Tyr Glu Arg Lys Leu Leu Ala Arg Phe Val Ile Asp Glu Ala His Cys 115 120 125Val Ser Gln Trp Gly His Asp Phe Arg Gln Asp Tyr Lys Arg Met Asn 130 135 140Met Leu Arg Gln Lys Phe Pro Ser Val Pro Val Met Ala Leu Thr Ala145 150 155 160Thr Ala Asn Pro Arg Val Gln Lys Asp Ile Leu Thr Gln Leu Lys Ile 165 170 175Leu Arg Pro Gln Val Phe Ser Met Ser Phe Asn Arg His Asn Leu Lys 180 185 190Tyr Tyr Val Leu Pro Lys Lys Pro Lys Lys Val Ala Phe Asp Cys Leu 195 200 205Glu Trp Ile Arg Lys His His Pro Tyr Asp Ser Gly Ile Ile Tyr Cys 210 215 220Leu Ser Arg Arg Glu Cys Asp Thr Met Ala Asp Thr Leu Gln Arg Asp225 230 235 240Gly Leu Ala Ala Leu Ala Tyr His Ala Gly Leu Ser Asp Ser Ala Arg 245 250 255Asp Glu Val Gln Gln Lys Trp Ile Asn Gln Asp Gly Cys Gln Val Ile 260 265 270Cys Ala Thr Ile Ala Phe Gly Met Gly Ile Asp Lys Pro Asp Val Arg 275 280 285Phe Val Ile His Ala Ser Leu Pro Lys Ser Val Glu Gly Tyr Tyr Gln 290 295 300Glu Ser Gly Arg Ala Gly Arg Asp Gly Glu Ile Ser His Cys Leu Leu305 310 315 320 42 344 PRT Homo sapiens 42Cys Leu Lys Met Tyr Phe Gly His Ser Ser Phe Lys Pro Val Gln Trp1 5 10 15Lys Val Ile His Ser Val Leu Glu Glu Arg Arg Asp Asn Val Ala Val 20 25 30Met Ala Thr Gly Tyr Gly Lys Ser Leu Cys Phe Gln Tyr Pro Pro Val 35 40 45Tyr Val Gly Lys Ile Gly Leu Val Ile Ser Pro Leu Ile Ser Leu Met 50 55 60Glu Asp Gln Val Leu Gln Leu Lys Met Ser Asn Ile Pro Ala Cys Phe65 70 75 80Leu Gly Ser Ala Gln Ser Glu Asn Val Leu Thr Asp Ile Lys Leu Gly 85 90 95Lys Tyr Arg Ile Val Tyr Val Thr Pro Glu Tyr Cys Ser Gly Asn Met 100 105 110Gly Leu Leu Gln Gln Leu Glu Ala Asp Ile Gly Ile Thr Leu Ile Ala 115 120 125Val Asp Glu Ala His Cys Ile Ser Glu Trp Gly His Asp Phe Arg Asp 130 135 140Ser Phe Arg Lys Leu Gly Ser Leu Lys Thr Ala Leu Pro Met Val Pro145 150 155 160Ile Val Ala Leu Thr Ala Thr Ala Ser Ser Ser Ile Arg Glu Asp Ile 165 170 175Val Arg Cys Leu Asn Leu Arg Asn Pro Gln Ile Thr Cys Thr Gly Phe 180 185 190Asp Arg Pro Asn Leu Tyr Leu Glu Val Arg Arg Lys Thr Gly Asn Ile 195 200 205Leu Gln Asp Leu Gln Pro Phe Leu Val Lys Thr Ser Ser His Trp Glu 210 215 220Phe Glu Gly Pro Thr Ile Ile Tyr Cys Pro Ser Arg Lys Met Thr Gln225 230 235 240Gln Val Thr Gly Glu Leu Arg Lys Leu Asn Leu Ser Cys Gly Thr Tyr 245 250 255His Ala Gly Met Ser Phe Ser Thr Arg Lys Asp Ile His His Arg Phe 260 265 270Val Arg Asp Glu Ile Gln Cys Val Ile Ala Thr Ile Ala Phe Gly Met 275 280 285Gly Ile Asn Lys Ala Asp Ile Arg Gln Val Ile His Tyr Gly Ala Pro 290 295 300Lys Asp Met Glu Ser Tyr Tyr Gln Glu Ile Gly Arg Ala Gly Arg Asp305 310 315 320Gly Leu Gln Ser Ser Cys His Val Leu Trp Ala Pro Ala Asp Ile Asn 325 330 335Leu Asn Arg His Leu Leu Thr Glu 340 43 106 PRT Homo sapiens 43Thr Asn Leu His Leu Ser Val Ser Met Asp Arg Asp Thr Asp Gln Ala1 5 10 15Leu Leu Thr Leu Leu Gln Gly Lys Arg Phe Gln Asn Leu Asp Ser Ile 20 25 30Ile Ile Tyr Cys Asn Arg Arg Glu Asp Thr Glu Arg Ile Ala Ala Leu 35 40 45Leu Arg Thr Cys Leu His Ala Ala Trp Val Pro Gly Ser Gly Gly Arg 50 55 60Ala Pro Lys Thr Thr Ala Glu Ala Tyr His Ala Gly Met Cys Ser Arg65 70 75 80Glu Arg Arg Arg Val Gln Arg Ala Phe Met Gln Gly Gln Leu Arg Val 85 90 95Val Val Ala Thr Val Ala Phe Gly Met Gly 100 105 44 88 PRT Escherichia coli 44Asn Ile Arg Tyr Met Leu Met Glu Lys Phe Lys Pro Leu Asp Gln Leu1 5 10 15Met Arg Tyr Val Gln Glu Gln Arg Gly Lys Ser Gly Ile Ile Tyr Cys 20 25 30Asn Ser Arg Ala Lys Val Glu Asp Thr Ala Ala Arg Leu Gln Ser Lys 35 40 45Gly Ile Ser Ala Ala Ala Tyr His Ala Gly Leu Glu Asn Asn Val Arg 50 55 60Ala Asp Val Gln Glu Lys Phe Gln Arg Asp Asp Leu Gln Ile Val Val65 70 75 80Ala Thr Val Ala Phe Gly Met Gly 85

Claims

1. An antibody that specifically reacts with a protein consisting of the amino acid sequence of SEQ ID NO:2.

2. The antibody of claim 1, wherein the antibody is monoclonal.

3. The antibody of claim 1, wherein the antibody is polyclonal.