Human gene RecQ4 encoding helicase

This application is a divisional of U.S. application Ser. No. 09/463,702, filed Jan. 24, 2000, now U.S. Pat. No. 6,335,435, which is a 371 of PCT/JP98/0344 filed July 10, 1998 the disclosure of which is incorporated herein by reference in its entirety, and Japanese Application Serial No. 200387/1997, filed Jul. 25, 1997, the disclosure of which is incorporated herein by reference in its entirety.

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.

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.

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.

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.

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.

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.

1

gcattggctg tcggcccccg cgacggctgc gcgggagatt cgctggacga tcgcaagcgc 60

ggaggccggg cgggcgcgcg cgcc atg gag cgg ctg cgg gac gtg cgg gag 111

Met Glu Arg Leu Arg Asp Val Arg Glu

1 5

cgg ctg cag gcg tgg gag cgc gcg ttc cga cgg cag cgc ggg cgg cga 159

Arg Leu Gln Ala Trp Glu Arg Ala Phe Arg Arg Gln Arg Gly Arg Arg

10 15 20 25

ccg agc cag gac gac gtg gag gcg gcg ccg gag gag acc cgc gcg ctc 207

Pro Ser Gln Asp Asp Val Glu Ala Ala Pro Glu Glu Thr Arg Ala Leu

30 35 40

tac cgg gag tac cgc act ctg aag cgt acc acg ggc cag gcc ggc ggc 255

Tyr Arg Glu Tyr Arg Thr Leu Lys Arg Thr Thr Gly Gln Ala Gly Gly

45 50 55

ggg ctc cgc agc tcc gag tcg ctc ccc gcg gcg gcc gaa gag gcg cca 303

Gly Leu Arg Ser Ser Glu Ser Leu Pro Ala Ala Ala Glu Glu Ala Pro

60 65 70

gag ccc cgc tgc tgg ggg ccc cat ctg aat cgg gct gcg acc aag agt 351

Glu Pro Arg Cys Trp Gly Pro His Leu Asn Arg Ala Ala Thr Lys Ser

75 80 85

cca cag cct acg cca ggg cgg agc cgc cag ggc tcg gtg ccg gac tac 399

Pro Gln Pro Thr Pro Gly Arg Ser Arg Gln Gly Ser Val Pro Asp Tyr

90 95 100 105

ggg cag cgg ctc aag gcc aat ctg aaa ggc acc ctg cag gcc gga cca 447

Gly Gln Arg Leu Lys Ala Asn Leu Lys Gly Thr Leu Gln Ala Gly Pro

110 115 120

gcc ctg ggc cgc aga ccg tgg cct cta gga aga gcc tca tct aag gca 495

Ala Leu Gly Arg Arg Pro Trp Pro Leu Gly Arg Ala Ser Ser Lys Ala

125 130 135

tcc acc cca aag ccc cca ggt aca ggg cct gtc ccc tcc ttt gca gaa 543

Ser Thr Pro Lys Pro Pro Gly Thr Gly Pro Val Pro Ser Phe Ala Glu

140 145 150

aaa gtc agt gat gag cct cca cag ctc cct gag ccc cag cca agg cca 591

Lys Val Ser Asp Glu Pro Pro Gln Leu Pro Glu Pro Gln Pro Arg Pro

155 160 165

ggc cgg ctc cag cat ctg cag gca tcc ctg agc cag cgg ctg ggc tcc 639

Gly Arg Leu Gln His Leu Gln Ala Ser Leu Ser Gln Arg Leu Gly Ser

170 175 180 185

cta gat cct ggc tgg tta cag cga tgt cac agt gag gtc cca gat ttt 687

Leu Asp Pro Gly Trp Leu Gln Arg Cys His Ser Glu Val Pro Asp Phe

190 195 200

ctg ggg gcc ccc aaa gcc tgc agg cct gat cta ggc tca gag gaa tca 735

Leu Gly Ala Pro Lys Ala Cys Arg Pro Asp Leu Gly Ser Glu Glu Ser

205 210 215

caa ctt ctg atc cct ggt gag tcg gct gtc ctt ggt cct ggt gct ggc 783

Gln Leu Leu Ile Pro Gly Glu Ser Ala Val Leu Gly Pro Gly Ala Gly

220 225 230

tcc cag ggc cca gag gct tca gcc ttc caa gaa gtc agc atc cgt gtg 831

Ser Gln Gly Pro Glu Ala Ser Ala Phe Gln Glu Val Ser Ile Arg Val

235 240 245

ggg agc ccc cag ccc agc agc agt gga ggc gag aag cgg aga tgg aac 879

Gly Ser Pro Gln Pro Ser Ser Ser Gly Gly Glu Lys Arg Arg Trp Asn

250 255 260 265

gag gag ccc tgg gag agc ccc gca cag gtc cag cag gag agc agc caa 927

Glu Glu Pro Trp Glu Ser Pro Ala Gln Val Gln Gln Glu Ser Ser Gln

270 275 280

gct gga ccc cca tcg gag ggg gct ggg gct gta gca gtt gag gaa gac 975

Ala Gly Pro Pro Ser Glu Gly Ala Gly Ala Val Ala Val Glu Glu Asp

285 290 295

cct cca ggg gaa cct gta cag gca cag cca cct cag ccc tgc agc agc 1023

Pro Pro Gly Glu Pro Val Gln Ala Gln Pro Pro Gln Pro Cys Ser Ser

300 305 310

cca tcg aac ccc agg tac cac gga ctc agc ccc tcc agt caa gct agg 1071

Pro Ser Asn Pro Arg Tyr His Gly Leu Ser Pro Ser Ser Gln Ala Arg

315 320 325

gct ggg aag gct gag ggc aca gcc ccc ctg cac atc ttc cct cgg ctg 1119

Ala Gly Lys Ala Glu Gly Thr Ala Pro Leu His Ile Phe Pro Arg Leu

330 335 340 345

gcc cgc cat gac agg ggc aat tac gta cgg ctc aac atg aag cag aaa 1167

Ala Arg His Asp Arg Gly Asn Tyr Val Arg Leu Asn Met Lys Gln Lys

350 355 360

cac tac gtg cgg ggc cgg gca ctc cgt agc agg ctc ctc cgc aag cag 1215

His Tyr Val Arg Gly Arg Ala Leu Arg Ser Arg Leu Leu Arg Lys Gln

365 370 375

gca tgg aag cag aag tgg cgg aag aaa ggg gag tgt ttt ggg ggt ggt 1263

Ala Trp Lys Gln Lys Trp Arg Lys Lys Gly Glu Cys Phe Gly Gly Gly

380 385 390

ggt gcc aca gtc aca acc aag gag tct tgt ttc ctg aac gag cag ttc 1311

Gly Ala Thr Val Thr Thr Lys Glu Ser Cys Phe Leu Asn Glu Gln Phe

395 400 405

gat cac tgg gca gcc cag tgt ccc cgg cca gca agt gag gaa gac aca 1359

Asp His Trp Ala Ala Gln Cys Pro Arg Pro Ala Ser Glu Glu Asp Thr

410 415 420 425

gat gct gtt ggg cct gag cca ctg gtt cct tca cca caa cct gta cct 1407

Asp Ala Val Gly Pro Glu Pro Leu Val Pro Ser Pro Gln Pro Val Pro

430 435 440

gag gtg ccc agc ctg gac ccc acc gtg ctg cca ctc tac tcc ctg ggg 1455

Glu Val Pro Ser Leu Asp Pro Thr Val Leu Pro Leu Tyr Ser Leu Gly

445 450 455

ccc tca ggg cag ttg gca gag acg ccg gct gag gtg ttc cag gcc ctg 1503

Pro Ser Gly Gln Leu Ala Glu Thr Pro Ala Glu Val Phe Gln Ala Leu

460 465 470

gag cag ctg ggg cac caa gcc ttt cgc cct ggg cag gag cgt gca gtc 1551

Glu Gln Leu Gly His Gln Ala Phe Arg Pro Gly Gln Glu Arg Ala Val

475 480 485

atg cgg atc ctg tct ggc atc tcc acg ctg ctg gtg ctg cct aca ggt 1599

Met Arg Ile Leu Ser Gly Ile Ser Thr Leu Leu Val Leu Pro Thr Gly

490 495 500 505

gcc ggc aag tcc ctg tgc tac cag ctc cca gcg ctg ctc tac agc cgg 1647

Ala Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Leu Leu Tyr Ser Arg

510 515 520

cgc agc ccc tgc ctc acg ttg gtc gtc tct ccc ctg ctg tca ctc atg 1695

Arg Ser Pro Cys Leu Thr Leu Val Val Ser Pro Leu Leu Ser Leu Met

525 530 535

gat gac cag gtg tct ggc ctg cca ccg tgt ctc aag gcg gcc tgc ata 1743

Asp Asp Gln Val Ser Gly Leu Pro Pro Cys Leu Lys Ala Ala Cys Ile

540 545 550

cac tcg ggc atg acc agg aag caa cgg gaa tct gtc ctg cag aag att 1791

His Ser Gly Met Thr Arg Lys Gln Arg Glu Ser Val Leu Gln Lys Ile

555 560 565

cgg gca gcc cag gta cac gtg ctg atg ctg aca cct gag gca ctg gtg 1839

Arg Ala Ala Gln Val His Val Leu Met Leu Thr Pro Glu Ala Leu Val

570 575 580 585

ggg gcg gga ggc ctc cct cca gcc gca cag ctg cct cca gtt gct ttt 1887

Gly Ala Gly Gly Leu Pro Pro Ala Ala Gln Leu Pro Pro Val Ala Phe

590 595 600

gcc tgc att gat gag gcc cac tgc ctc tcc cag tgg tcc cac aac ttc 1935

Ala Cys Ile Asp Glu Ala His Cys Leu Ser Gln Trp Ser His Asn Phe

605 610 615

cgg ccc tgc tac ctg cgc gtc tgc aag gtg ctt cgg gag cgc atg ggc 1983

Arg Pro Cys Tyr Leu Arg Val Cys Lys Val Leu Arg Glu Arg Met Gly

620 625 630

gtg cac tgc ttc ctg ggc ctc aca gcc aca gcc aca cgc cgc act gcc 2031

Val His Cys Phe Leu Gly Leu Thr Ala Thr Ala Thr Arg Arg Thr Ala

635 640 645

agt gac gtg gca cag cac ctg gct gtg gct gaa gag cct gac ctc cac 2079

Ser Asp Val Ala Gln His Leu Ala Val Ala Glu Glu Pro Asp Leu His

650 655 660 665

ggg cca gcc cca gtt ccc acc aac ctg cac ctt tcc gtg tcc atg gac 2127

Gly Pro Ala Pro Val Pro Thr Asn Leu His Leu Ser Val Ser Met Asp

670 675 680

agg gac aca gac cag gca ctg ttg acg ctg ctg caa ggc aaa cgt ttt 2175

Arg Asp Thr Asp Gln Ala Leu Leu Thr Leu Leu Gln Gly Lys Arg Phe

685 690 695

caa aac ctc gat tcc att atc att tac tgc aac cgg cgc gag gac aca 2223

Gln Asn Leu Asp Ser Ile Ile Ile Tyr Cys Asn Arg Arg Glu Asp Thr

700 705 710

gag cgg atc gct gcg ctc ctc cga acc tgc ctg cac gca gcc tgg gtc 2271

Glu Arg Ile Ala Ala Leu Leu Arg Thr Cys Leu His Ala Ala Trp Val

715 720 725

cca ggg tct gga ggt cgt gcc ccc aaa acc aca gcc gag gcc tac cac 2319

Pro Gly Ser Gly Gly Arg Ala Pro Lys Thr Thr Ala Glu Ala Tyr His

730 735 740 745

gcg ggc atg tgc agc cgg gaa cgg cgg cgg gta cag cga gcc ttc atg 2367

Ala Gly Met Cys Ser Arg Glu Arg Arg Arg Val Gln Arg Ala Phe Met

750 755 760

cag ggc cag ttg cgg gtg gtg gtg gcc acg gtg gcc ttt ggg atg ggg 2415

Gln Gly Gln Leu Arg Val Val Val Ala Thr Val Ala Phe Gly Met Gly

765 770 775

ctg gac cgg cca gat gtg cgg gct gtg ctg cat ctg ggg ctg ccc cca 2463

Leu Asp Arg Pro Asp Val Arg Ala Val Leu His Leu Gly Leu Pro Pro

780 785 790

agc ttc gag agc tac gtg cag gcc gtg ggc cgg gcc ggg cgt gac ggg 2511

Ser Phe Glu Ser Tyr Val Gln Ala Val Gly Arg Ala Gly Arg Asp Gly

795 800 805

cag cct gcc cac tgc cac ctc ttc ctg cag ccc cag ggc gaa gac ctg 2559

Gln Pro Ala His Cys His Leu Phe Leu Gln Pro Gln Gly Glu Asp Leu

810 815 820 825

cga gag ctg cgc aga cat gtg cac gcc gac agc acg gac ttc ctg gct 2607

Arg Glu Leu Arg Arg His Val His Ala Asp Ser Thr Asp Phe Leu Ala

830 835 840

gtg aag agg ctg gta cag cgc gtg ttc cca gcc tgc acc tgc acc tgc 2655

Val Lys Arg Leu Val Gln Arg Val Phe Pro Ala Cys Thr Cys Thr Cys

845 850 855

acc agg ccg ccc tcg gag cag gaa ggg gcc gtg ggt ggg gag agg cct 2703

Thr Arg Pro Pro Ser Glu Gln Glu Gly Ala Val Gly Gly Glu Arg Pro

860 865 870

gtg ccc aag tac ccc cct caa gag gct gag cag ctt agc cac caa gca 2751

Val Pro Lys Tyr Pro Pro Gln Glu Ala Glu Gln Leu Ser His Gln Ala

875 880 885

gcc cca gga ccc aga agg gtc tgc atg ggc cat gag cgg gca ctc cca 2799

Ala Pro Gly Pro Arg Arg Val Cys Met Gly His Glu Arg Ala Leu Pro

890 895 900 905

ata cag ctt acc gta cag gct ttg gac atg ccg gag gag gcc atc gag 2847

Ile Gln Leu Thr Val Gln Ala Leu Asp Met Pro Glu Glu Ala Ile Glu

910 915 920

act ttg ctg tgc tac ctg gag ctg cac cca cac cac tgg ctg gag ctg 2895

Thr Leu Leu Cys Tyr Leu Glu Leu His Pro His His Trp Leu Glu Leu

925 930 935

ctg gcg acc acc tat acc cat tgc cgt ctg aac tgc cct ggg ggc cct 2943

Leu Ala Thr Thr Tyr Thr His Cys Arg Leu Asn Cys Pro Gly Gly Pro

940 945 950

gcc cag ctc cag gcc ctg gcc cac agg tgt ccc cct ttg gct gtg tgc 2991

Ala Gln Leu Gln Ala Leu Ala His Arg Cys Pro Pro Leu Ala Val Cys

955 960 965

ttg gcc cag cag ctg cct gag gac cca ggg caa ggc agc agc tcc gtg 3039

Leu Ala Gln Gln Leu Pro Glu Asp Pro Gly Gln Gly Ser Ser Ser Val

970 975 980 985

gag ttt gac atg gtc aag ctg gtg gac tcc atg ggc tgg gag ctg gcc 3087

Glu Phe Asp Met Val Lys Leu Val Asp Ser Met Gly Trp Glu Leu Ala

990 995 1000

tct gtg cgg cgg gct ctc tgc cag ctg cag tgg gac cac gag ccc 3132

Ser Val Arg Arg Ala Leu Cys Gln Leu Gln Trp Asp His Glu Pro

1005 1010 1015

agg aca ggt gtg cgg cgt ggg aca ggg gtg ctt gtg gag ttc agt 3177

Arg Thr Gly Val Arg Arg Gly Thr Gly Val Leu Val Glu Phe Ser

1020 1025 1030

gag ctg gcc ttc cac ctt cgc agc ccg ggg gac ctg acc gct gag 3222

Glu Leu Ala Phe His Leu Arg Ser Pro Gly Asp Leu Thr Ala Glu

1035 1040 1045

gag aag gac cag ata tgt gac ttc ctc tat ggc cgt gtg cag gcc 3267

Glu Lys Asp Gln Ile Cys Asp Phe Leu Tyr Gly Arg Val Gln Ala

1050 1055 1060

cgg gag cgc cag gcc ctg gcc cgt ctg cgc aga acc ttc cag gcc 3312

Arg Glu Arg Gln Ala Leu Ala Arg Leu Arg Arg Thr Phe Gln Ala

1065 1070 1075

ttt cac agc gta gcc ttc ccc agc tgc ggg ccc tgc ctg gag cag 3357

Phe His Ser Val Ala Phe Pro Ser Cys Gly Pro Cys Leu Glu Gln

1080 1085 1090

cag gat gag gag cgc agc acc agg ctc aag gac ctg ctc ggc cgc 3402

Gln Asp Glu Glu Arg Ser Thr Arg Leu Lys Asp Leu Leu Gly Arg

1095 1100 1105

tac ttt gag gaa gag gaa ggg cag gag ccg gga ggc atg gag gac 3447

Tyr Phe Glu Glu Glu Glu Gly Gln Glu Pro Gly Gly Met Glu Asp

1110 1115 1120

gca cag ggc ccc gag cca ggg cag gcc aga ctc cag gat tgg gag 3492

Ala Gln Gly Pro Glu Pro Gly Gln Ala Arg Leu Gln Asp Trp Glu

1125 1130 1135

gac cag gtc cgc tgc gac atc cgc cag ttc ctg tcc ctg agg cca 3537

Asp Gln Val Arg Cys Asp Ile Arg Gln Phe Leu Ser Leu Arg Pro

1140 1145 1150

gag gag aag ttc tcc agc agg gct gtg gcc cgc atc ttc cac ggc 3582

Glu Glu Lys Phe Ser Ser Arg Ala Val Ala Arg Ile Phe His Gly

1155 1160 1165

atc gga agc ccc tgc tac ccg gcc cag gtg tac ggg cag gac cga 3627

Ile Gly Ser Pro Cys Tyr Pro Ala Gln Val Tyr Gly Gln Asp Arg

1170 1175 1180

cgc ttc tgg aga aaa tac ctg cac ctg agc ttc cat gcc ctg gtg 3672

Arg Phe Trp Arg Lys Tyr Leu His Leu Ser Phe His Ala Leu Val

1185 1190 1195

ggc ctg gcc acg gaa gag ctc ctg cag gtg gcc cgc tgactgcact 3718

Gly Leu Ala Thr Glu Glu Leu Leu Gln Val Ala Arg

1200 1205

gcattggggg atgtcgggta gagctggggt tgtcagaggc tagggcagtg actgaggacc 3778

tgggcaaaac ctgccacagg gtgtgggaac gaggaggctc caaaatgcag aataaaaaat 3838

gctcactttg tt 3850

2

1208

Homo sapiens

2

Met Glu Arg Leu Arg Asp Val Arg Glu Arg Leu Gln Ala Trp Glu Arg

1 5 10 15

Ala Phe Arg Arg Gln Arg Gly Arg Arg Pro Ser Gln Asp Asp Val Glu

20 25 30

Ala Ala Pro Glu Glu Thr Arg Ala Leu Tyr Arg Glu Tyr Arg Thr Leu

35 40 45

Lys Arg Thr Thr Gly Gln Ala Gly Gly Gly Leu Arg Ser Ser Glu Ser

50 55 60

Leu Pro Ala Ala Ala Glu Glu Ala Pro Glu Pro Arg Cys Trp Gly Pro

65 70 75 80

His Leu Asn Arg Ala Ala Thr Lys Ser Pro Gln Pro Thr Pro Gly Arg

85 90 95

Ser Arg Gln Gly Ser Val Pro Asp Tyr Gly Gln Arg Leu Lys Ala Asn

100 105 110

Leu Lys Gly Thr Leu Gln Ala Gly Pro Ala Leu Gly Arg Arg Pro Trp

115 120 125

Pro Leu Gly Arg Ala Ser Ser Lys Ala Ser Thr Pro Lys Pro Pro Gly

130 135 140

Thr Gly Pro Val Pro Ser Phe Ala Glu Lys Val Ser Asp Glu Pro Pro

145 150 155 160

Gln Leu Pro Glu Pro Gln Pro Arg Pro Gly Arg Leu Gln His Leu Gln

165 170 175

Ala Ser Leu Ser Gln Arg Leu Gly Ser Leu Asp Pro Gly Trp Leu Gln

180 185 190

Arg Cys His Ser Glu Val Pro Asp Phe Leu Gly Ala Pro Lys Ala Cys

195 200 205

Arg Pro Asp Leu Gly Ser Glu Glu Ser Gln Leu Leu Ile Pro Gly Glu

210 215 220

Ser Ala Val Leu Gly Pro Gly Ala Gly Ser Gln Gly Pro Glu Ala Ser

225 230 235 240

Ala Phe Gln Glu Val Ser Ile Arg Val Gly Ser Pro Gln Pro Ser Ser

245 250 255

Ser Gly Gly Glu Lys Arg Arg Trp Asn Glu Glu Pro Trp Glu Ser Pro

260 265 270

Ala Gln Val Gln Gln Glu Ser Ser Gln Ala Gly Pro Pro Ser Glu Gly

275 280 285

Ala Gly Ala Val Ala Val Glu Glu Asp Pro Pro Gly Glu Pro Val Gln

290 295 300

Ala Gln Pro Pro Gln Pro Cys Ser Ser Pro Ser Asn Pro Arg Tyr His

305 310 315 320

Gly Leu Ser Pro Ser Ser Gln Ala Arg Ala Gly Lys Ala Glu Gly Thr

325 330 335

Ala Pro Leu His Ile Phe Pro Arg Leu Ala Arg His Asp Arg Gly Asn

340 345 350

Tyr Val Arg Leu Asn Met Lys Gln Lys His Tyr Val Arg Gly Arg Ala

355 360 365

Leu Arg Ser Arg Leu Leu Arg Lys Gln Ala Trp Lys Gln Lys Trp Arg

370 375 380

Lys Lys Gly Glu Cys Phe Gly Gly Gly Gly Ala Thr Val Thr Thr Lys

385 390 395 400

Glu Ser Cys Phe Leu Asn Glu Gln Phe Asp His Trp Ala Ala Gln Cys

405 410 415

Pro Arg Pro Ala Ser Glu Glu Asp Thr Asp Ala Val Gly Pro Glu Pro

420 425 430

Leu Val Pro Ser Pro Gln Pro Val Pro Glu Val Pro Ser Leu Asp Pro

435 440 445

Thr Val Leu Pro Leu Tyr Ser Leu Gly Pro Ser Gly Gln Leu Ala Glu

450 455 460

Thr Pro Ala Glu Val Phe Gln Ala Leu Glu Gln Leu Gly His Gln Ala

465 470 475 480

Phe Arg Pro Gly Gln Glu Arg Ala Val Met Arg Ile Leu Ser Gly Ile

485 490 495

Ser Thr Leu Leu Val Leu Pro Thr Gly Ala Gly Lys Ser Leu Cys Tyr

500 505 510

Gln Leu Pro Ala Leu Leu Tyr Ser Arg Arg Ser Pro Cys Leu Thr Leu

515 520 525

Val Val Ser Pro Leu Leu Ser Leu Met Asp Asp Gln Val Ser Gly Leu

530 535 540

Pro Pro Cys Leu Lys Ala Ala Cys Ile His Ser Gly Met Thr Arg Lys

545 550 555 560

Gln Arg Glu Ser Val Leu Gln Lys Ile Arg Ala Ala Gln Val His Val

565 570 575

Leu Met Leu Thr Pro Glu Ala Leu Val Gly Ala Gly Gly Leu Pro Pro

580 585 590

Ala Ala Gln Leu Pro Pro Val Ala Phe Ala Cys Ile Asp Glu Ala His

595 600 605

Cys Leu Ser Gln Trp Ser His Asn Phe Arg Pro Cys Tyr Leu Arg Val

610 615 620

Cys Lys Val Leu Arg Glu Arg Met Gly Val His Cys Phe Leu Gly Leu

625 630 635 640

Thr Ala Thr Ala Thr Arg Arg Thr Ala Ser Asp Val Ala Gln His Leu

645 650 655

Ala Val Ala Glu Glu Pro Asp Leu His Gly Pro Ala Pro Val Pro Thr

660 665 670

Asn Leu His Leu Ser Val Ser Met Asp Arg Asp Thr Asp Gln Ala Leu

675 680 685

Leu Thr Leu Leu Gln Gly Lys Arg Phe Gln Asn Leu Asp Ser Ile Ile

690 695 700

Ile Tyr Cys Asn Arg Arg Glu Asp Thr Glu Arg Ile Ala Ala Leu Leu

705 710 715 720

Arg Thr Cys Leu His Ala Ala Trp Val Pro Gly Ser Gly Gly Arg Ala

725 730 735

Pro Lys Thr Thr Ala Glu Ala Tyr His Ala Gly Met Cys Ser Arg Glu

740 745 750

Arg Arg Arg Val Gln Arg Ala Phe Met Gln Gly Gln Leu Arg Val Val

755 760 765

Val Ala Thr Val Ala Phe Gly Met Gly Leu Asp Arg Pro Asp Val Arg

770 775 780

Ala Val Leu His Leu Gly Leu Pro Pro Ser Phe Glu Ser Tyr Val Gln

785 790 795 800

Ala Val Gly Arg Ala Gly Arg Asp Gly Gln Pro Ala His Cys His Leu

805 810 815

Phe Leu Gln Pro Gln Gly Glu Asp Leu Arg Glu Leu Arg Arg His Val

820 825 830

His Ala Asp Ser Thr Asp Phe Leu Ala Val Lys Arg Leu Val Gln Arg

835 840 845

Val Phe Pro Ala Cys Thr Cys Thr Cys Thr Arg Pro Pro Ser Glu Gln

850 855 860

Glu Gly Ala Val Gly Gly Glu Arg Pro Val Pro Lys Tyr Pro Pro Gln

865 870 875 880

Glu Ala Glu Gln Leu Ser His Gln Ala Ala Pro Gly Pro Arg Arg Val

885 890 895

Cys Met Gly His Glu Arg Ala Leu Pro Ile Gln Leu Thr Val Gln Ala

900 905 910

Leu Asp Met Pro Glu Glu Ala Ile Glu Thr Leu Leu Cys Tyr Leu Glu

915 920 925

Leu His Pro His His Trp Leu Glu Leu Leu Ala Thr Thr Tyr Thr His

930 935 940

Cys Arg Leu Asn Cys Pro Gly Gly Pro Ala Gln Leu Gln Ala Leu Ala

945 950 955 960

His Arg Cys Pro Pro Leu Ala Val Cys Leu Ala Gln Gln Leu Pro Glu

965 970 975

Asp Pro Gly Gln Gly Ser Ser Ser Val Glu Phe Asp Met Val Lys Leu

980 985 990

Val Asp Ser Met Gly Trp Glu Leu Ala Ser Val Arg Arg Ala Leu Cys

995 1000 1005

Gln Leu Gln Trp Asp His Glu Pro Arg Thr Gly Val Arg Arg Gly

1010 1015 1020

Thr Gly Val Leu Val Glu Phe Ser Glu Leu Ala Phe His Leu Arg

1025 1030 1035

Ser Pro Gly Asp Leu Thr Ala Glu Glu Lys Asp Gln Ile Cys Asp

1040 1045 1050

Phe Leu Tyr Gly Arg Val Gln Ala Arg Glu Arg Gln Ala Leu Ala

1055 1060 1065

Arg Leu Arg Arg Thr Phe Gln Ala Phe His Ser Val Ala Phe Pro

1070 1075 1080

Ser Cys Gly Pro Cys Leu Glu Gln Gln Asp Glu Glu Arg Ser Thr

1085 1090 1095

Arg Leu Lys Asp Leu Leu Gly Arg Tyr Phe Glu Glu Glu Glu Gly

1100 1105 1110

Gln Glu Pro Gly Gly Met Glu Asp Ala Gln Gly Pro Glu Pro Gly

1115 1120 1125

Gln Ala Arg Leu Gln Asp Trp Glu Asp Gln Val Arg Cys Asp Ile

1130 1135 1140

Arg Gln Phe Leu Ser Leu Arg Pro Glu Glu Lys Phe Ser Ser Arg

1145 1150 1155

Ala Val Ala Arg Ile Phe His Gly Ile Gly Ser Pro Cys Tyr Pro

1160 1165 1170

Ala Gln Val Tyr Gly Gln Asp Arg Arg Phe Trp Arg Lys Tyr Leu

1175 1180 1185

His Leu Ser Phe His Ala Leu Val Gly Leu Ala Thr Glu Glu Leu

1190 1195 1200

Leu Gln Val Ala Arg

1205

3

24

Artificial Sequence

3

caccaacctg cacctttccg tgtc 24

4

24

Artificial Sequence

4

agccccatcc caaaagccac cgtg 24

5

24

Artificial Sequence

5

cgccagggtt ttcccagtca cgac 24

6

22

Artificial Sequence

6

tcacacagga aacagctatg ac 22

7

24

Artificial Sequence

7

aatctgggac ctcactgtga catc 24

8

24

Artificial Sequence

8

tcatctaagg catccacccc aaag 24

9

24

Artificial Sequence

9

tcacaacttc tgatccctgg tgag 24

10

24

Artificial Sequence

10

ctcagcccct ccagtcaagc tagg 24

11

24

Artificial Sequence

11

gtttcctgaa cgagcagttc gatc 24

12

24

Artificial Sequence

12

ctgggcagga gcgtgcagtc atgc 24

13

24

Artificial Sequence

13

gctgcctcca gttgcttttg cctg 24

14

24

Artificial Sequence

14

ggacacagac caggcactgt tgac 24

15

24

Artificial Sequence

15

gggtacagcg agccttcatg cagg 24

16

24

Artificial Sequence

16

tcctggctgt gaagaggctg gtac 24

17

22

Artificial Sequence

17

cagcttaccg tacaggcttt gg 22

18

22

Artificial Sequence

18

ggggtgcttg tggagttcag tg 22

19

22

Artificial Sequence

19

caggccagac tccaggattg gg 22

20

27

Artificial Sequence

PRIMER AP1

20

ccatcctaat acgactcact atagggc 27

21

23

Artificial Sequence

AP2

21

actcactata gggctcgagc ggc 23

22

24

Artificial Sequence

PRIMER 5′ GSP1

22

gccttgcagc agcgtcaaca gtgc 24

23

24

Artificial Sequence

PRIMER 5′ GSP2

23

tggacacgga aaggtgcagg ttgg 24

24

24

Artificial Sequence

PRIMER 3′GSP1

24

gttgacgctg ctgcaaggca aacg 24

25

24

Artificial Sequence

PRIMER 3′ GSP2

25

catctcatgg aatgatcctg tcgg 24

26

30

Artificial Sequence

26

cgaaucguaa ccguucguac gagaaucgcu 30

27

23

Artificial Sequence

27

cgaatcgtaa ccgttcgtac gag 23

28

26

Artificial Sequence

28

atcgtaaccg ttcgtacgag aatcgc 26

29

24

Artificial Sequence

29

taggctgtgg actcttggtc gcag 24

30

24

Artificial Sequence

30

ttggtcgcag cccgattcag atgg 24

31

321

Artificial Sequence

RECQ4 PARTIAL cDNA

31

caccaacctg cacctttccg tgtccatgga cagggacaca gaccaggcac tgttgacgct 60

gctgcaaggc aaacgttttc aaaacctcga ttccattatc atttactgca accggcgcga 120

ggacacagag cggatcgctg cgctcctccg aacctgcctg cacgcagcct gggtcccagg 180

gtctggaggt cgtgccccca aaaccacagc cgaggcctac cacgcgggca tgtgcagccg 240

ggaacggcgg cgggtacagc gagccttcat gcagggccag ttgcgggtgg tggtggccac 300

ggtggccttt gggatggggc t 321

32

1954

Artificial Sequence

RECQ4 5′ RACE PRODUCT

32

gacgacgtgg aggcggcgcc ggaggagacc cgcgcgctct accgggagta ccgcactctg 60

aagcgtacca cgggccaggc cggcggcggg ctccgcagct ccgagtcgct ccccgcggcg 120

gccgaagagg cgccagagcc ccgctgctgg gggccccatc tgaatcgggc tgcgaccaag 180

agtccacagc ctacgccagg gcggagccgc cagggctcgg tgccggacta cgggcagcgg 240

ctcaaggcca atctgaaagg caccctgcag gccggaccag ccctgggccg cagaccgtgg 300

cctctaggaa gagcctcatc taaggcatcc accccaaagc ccccaggtac agggcctgtc 360

ccctcctttg cagaaaaagt cagtgatgag cctccacagc tccctgagcc ccagccaagg 420

ccaggccggc tccagcatct gcaggcatcc ctgagccagc ggctgggctc cctagatcct 480

ggctggttac agcgatgtca cagtgaggtc ccagattttc tgggggcccc caaagcctgc 540

aggcctgatc taggctcaga ggaatcacaa cttctgatcc ctggtgagtc ggctgtcctt 600

ggtcctggtg ctggctccca gggcccagag gcttcagcct tccaagaagt cagcatccgt 660

gtggggagcc cccagcccag cagcagtgga ggcgagaagc ggagatggaa cgaggagccc 720

tgggagagcc ccgcacaggt ccagcaggag agcagccaag ctggaccccc atcggagggg 780

gctggggctg tagcagttga ggaagaccct ccaggggaac ctgtacaggc acagccacct 840

cagccctgca gcagcccatc gaaccccagg taccacggac tcagcccctc cagtcaagct 900

agggctggga aggctgaggg cacagccccc ctgcacatct tccctcggct ggcccgccat 960

gacaggggca attacgtacg gctcaacatg aagcagaaac actacgtgcg gggccgggca 1020

ctccgtagca ggctcctccg caagcaggca tggaagcaga agtggcggaa gaaaggggag 1080

tgttttgggg gtggtggtgc cacagtcaca accaaggagt cttgtttcct gaacgagcag 1140

ttcgatcact gggcagccca gtgtccccgg ccagcaagtg aggaagacac agatgctgtt 1200

gggcctgagc cactggttcc ttcaccacaa cctgtacctg aggtgcccag cctggacccc 1260

accgtgctgc cactctactc cctggggccc tcagggcagt tggcagagac gccggctgag 1320

gtgttccagg ccctggagca gctggggcac caagcctttc gccctgggca ggagcgtgca 1380

gtcatgcgga tcctgtctgg catctccacg ctgctggtgc tgcctacagg tgccggcaag 1440

tccctgtgct accagctccc agcgctgctc tacagccggc gcagcccctg cctcacgttg 1500

gtcgtctctc ccctgctgtc actcatggat gaccaggtgt ctggcctgcc accgtgtctc 1560

aaggcggcct gcatacactc gggcatgacc aggaagcaac gggaatctgt cctgcagaag 1620

attcgggcag cccaggtaca cgtgctgatg ctgacacctg aggcactggt gggggcggga 1680

ggcctccctc cagccgcaca gctgcctcca gttgcttttg cctgcattga tgaggcccac 1740

tgcctctccc agtggtccca caacttccgg ccctgctacc tgcgcgtctg caaggtgctt 1800

cgggagcgca tgggcgtgca ctgcttcctg ggcctcacag ccacagccac acgccgcact 1860

gccagtgacg tggcacagca cctggctgtg gctgaagagc ctgacctcca cgggccagcc 1920

ccagttccca ccaacctgca cctttccgtg tcca 1954

33

1503

Artificial Sequence

RECQ4 3′ RACE PRODUCT

33

gggtacagcg agccttcatg cagggccagt tgcgggtggt ggtggccacg gtggcctttg 60

ggatggggct ggaccggcca gatgtgcggg ctgtgctgca tctggggctg cccccaagct 120

tcgagagcta cgtgcaggcc gtgggccggg ccgggcgtga cgggcagcct gcccactgcc 180

acctcttcct gcagccccag ggcgaagacc tgcgagagct gcgcagacat gtgcacgccg 240

acagcacgga cttcctggct gtgaagaggc tggtacagcg cgtgttccca gcctgcacct 300

gcacctgcac caggccgccc tcggagcagg aaggggccgt gggtggggag aggcctgtgc 360

ccaagtaccc ccctcaagag gctgagcagc ttagccacca agcagcccca ggacccagaa 420

gggtctgcat gggccatgag cgggcactcc caatacagct taccgtacag gctttggaca 480

tgccggagga ggccatcgag actttgctgt gctacctgga gctgcaccca caccactggc 540

tggagctgct ggcgaccacc tatacccatt gccgtctgaa ctgccctggg ggccctgccc 600

agctccaggc cctggcccac aggtgtcccc ctttggctgt gtgcttggcc cagcagctgc 660

ctgaggaccc agggcaaggc agcagctccg tggagtttga catggtcaag ctggtggact 720

ccatgggctg ggagctggcc tctgtgcggc gggctctctg ccagctgcag tgggaccacg 780

agcccaggac aggtgtgcgg cgtgggacag gggtgcttgt ggagttcagt gagctggcct 840

tccaccttcg cagcccgggg gacctgaccg ctgaggagaa ggaccagata tgtgacttcc 900

tctatggccg tgtgcaggcc cgggagcgcc aggccctggc ccgtctgcgc agaaccttcc 960

aggcctttca cagcgtagcc ttccccagct gcgggccctg cctggagcag caggatgagg 1020

agcgcagcac caggctcaag gacctgctcg gccgctactt tgaggaagag gaagggcagg 1080

agccgggagg catggaggac gcacagggcc ccgagccagg gcaggccaga ctccaggatt 1140

gggaggacca ggtccgctgc gacatccgcc agttcctgtc cctgaggcca gaggagaagt 1200

tctccagcag ggctgtggcc cgcatcttcc acggcatcgg aagcccctgc tacccggccc 1260

aggtgtacgg gcaggaccga cgcttctgga gaaaatacct gcacctgagc ttccatgccc 1320

tggtgggcct ggccacggaa gagctcctgc aggtggcccg ctgactgcac tgcattgggg 1380

gatgtcgggt agagctgggg ttgtcagagg ctagggcagt gactgaggac ctgggcaaaa 1440

cctgccacag ggtgtgggaa cgaggaggct ccaaaatgca gaataaaaaa tgctcacttt 1500

gtt 1503

34

347

Artificial Sequence

RECQ4 TRANSCRIPTION ORIGIN REGION

34

gcattggctg tcggcccccg cgacggctgc gcgggagatt cgctggacga tcgcaagcgc 60

ggaggccggg cgggcgcgcg cgccatggag cggctgcggg acgtgcggga gcggctgcag 120

gcgtgggagc gcgcgttccg acggcagcgc gggcggcgac cgagccagga cgacgtggag 180

gcggcgccgg aggagacccg cgcgctctac cgggagtacc gcactctgaa gcgtaccacg 240

ggccaggccg gcggcgggct ccgcagctcc gagtcgctcc ccgcggcggc cgaagaggcg 300

ccagagcccc gctgctgggg gccccatctg aatcgggctg cgaccaa 347

35

24

Artificial Sequence

PRIMERS FOR THE RECQ4 GENE

35

gacggctgcg cgggagattc gctg 24

36

24

Artificial Sequence

PRIMERS FOR THE RECQ4 GENE

36

caggttttgc ccaggtcctc agtc 24

37

361

Homo sapiens

37

Gln Ala Leu Glu Gln Leu Gly His Gln Ala Phe Arg Pro Gly Gln Glu

1 5 10 15

Arg Ala Val Met Arg Ile Leu Ser Gly Ile Ser Thr Leu Leu Val Leu

20 25 30

Pro Thr Gly Ala Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Leu Leu

35 40 45

Tyr Ser Arg Arg Ser Pro Cys Leu Thr Leu Val Val Ser Pro Leu Leu

50 55 60

Ser Leu Met Asp Asp Gln Val Ser Gly Leu Pro Pro Cys Leu Lys Ala

65 70 75 80

Ala Cys Ile His Ser Gly Met Thr Arg Lys Gln Arg Glu Ser Val Leu

85 90 95

Gln Lys Ile Arg Ala Ala Gln Val His Val Leu Met Leu Thr Pro Glu

100 105 110

Ala Leu Val Gly Ala Gly Gly Leu Pro Pro Ala Ala Gln Leu Pro Pro

115 120 125

Val Ala Phe Ala Cys Ile Asp Glu Ala His Cys Leu Ser Gln Trp Ser

130 135 140

His Asn Phe Arg Pro Cys Tyr Leu Arg Val Cys Lys Val Leu Arg Glu

145 150 155 160

Arg Met Gly Val His Cys Phe Leu Gly Leu Thr Ala Thr Ala Thr Arg

165 170 175

Arg Thr Ala Ser Asp Val Ala Gln His Leu Ala Val Ala Glu Glu Pro

180 185 190

Asp Leu His Gly Pro Ala Pro Val Pro Thr Asn Leu His Leu Ser Val

195 200 205

Ser Met Asp Arg Asp Thr Asp Gln Ala Leu Leu Thr Leu Leu Gln Gly

210 215 220

Lys Arg Phe Gln Asn Leu Asp Ser Ile Ile Ile Tyr Cys Asn Arg Arg

225 230 235 240

Glu Asp Thr Glu Arg Ile Ala Ala Leu Leu Arg Thr Cys Leu His Ala

245 250 255

Ala Trp Val Pro Gly Ser Gly Gly Arg Ala Pro Lys Thr Thr Ala Glu

260 265 270

Ala Tyr His Ala Gly Met Cys Ser Arg Glu Arg Arg Arg Val Gln Arg

275 280 285

Ala Phe Met Gln Gly Gln Leu Arg Val Val Val Ala Thr Val Ala Phe

290 295 300

Gly Met Gly Leu Asp Arg Pro Asp Val Arg Ala Val Leu His Leu Gly

305 310 315 320

Leu Pro Pro Ser Phe Glu Ser Tyr Val Gln Ala Val Gly Arg Ala Gly

325 330 335

Arg Asp Gly Gln Pro Ala His Cys His Leu Phe Leu Gln Pro Gln Gly

340 345 350

Glu Asp Leu Arg Glu Leu Arg Arg His

355 360

38

334

Escherichia coli

38

Phe Gly Tyr Gln Gln Phe Arg Pro Gly Gln Glu Glu Ile Ile Asp Thr

1 5 10 15

Val Leu Ser Gly Arg Asp Cys Leu Val Val Met Pro Thr Gly Gly Gly

20 25 30

Lys Ser Leu Cys Tyr Gln Ile Pro Ala Leu Leu Leu Asn Gly Leu Thr

35 40 45

Val Val Val Ser Pro Leu Ile Ser Leu Met Lys Asp Gln Val Asp Gln

50 55 60

Leu Gln Ala Asn Gly Val Ala Ala Ala Cys Leu Asn Ser Thr Gln Thr

65 70 75 80

Arg Glu Gln Gln Leu Glu Val Met Thr Gly Cys Arg Thr Gly Gln Ile

85 90 95

Arg Leu Leu Tyr Ile Ala Pro Glu Arg Leu Met Leu Asp Asn Phe Leu

100 105 110

Glu His Leu Ala His Trp Asn Pro Val Leu Leu Ala Val Asp Glu Ala

115 120 125

His Cys Ile Ser Gln Trp Gly His Asp Phe Arg Pro Glu Tyr Ala Ala

130 135 140

Leu Gly Gln Leu Arg Gln Arg Phe Pro Thr Leu Pro Phe Met Ala Leu

145 150 155 160

Thr Ala Thr Ala Asp Asp Thr Thr Arg Gln Asp Ile Val Arg Leu Leu

165 170 175

Gly Leu Asn Asp Pro Leu Ile Gln Ile Ser Ser Phe Asp Arg Pro Asn

180 185 190

Ile Arg Tyr Met Leu Met Glu Lys Phe Lys Pro Leu Asp Gln Leu Met

195 200 205

Arg Tyr Val Gln Glu Gln Arg Gly Lys Ser Gly Ile Ile Tyr Cys Asn

210 215 220

Ser Arg Ala Lys Val Glu Asp Thr Ala Ala Arg Leu Gln Ser Lys Gly

225 230 235 240

Ile Ser Ala Ala Ala Tyr His Ala Gly Leu Glu Asn Asn Val Arg Ala

245 250 255

Asp Val Gln Glu Lys Phe Gln Arg Asp Asp Leu Gln Ile Val Val Ala

260 265 270

Thr Val Ala Phe Gly Met Gly Ile Asn Lys Pro Asn Val Arg Phe Val

275 280 285

Val His Phe Asp Ile Pro Arg Asn Ile Glu Ser Tyr Tyr Gln Glu Thr

290 295 300

Gly Arg Ala Gly Arg Asp Gly Leu Pro Ala Glu Ala Met Leu Phe Tyr

305 310 315 320

Asp Pro Ala Asp Met Ala Trp Leu Arg Arg Cys Leu Glu Glu

325 330

39

333

Saccharomyces cerevisiae

39

Phe Arg Pro Asn Gln Leu Glu Ala Val Asn Ala Thr Leu Gln Gly Lys

1 5 10 15

Asp Val Phe Val Leu Met Pro Thr Gly Gly Gly Lys Ser Leu Cys Tyr

20 25 30

Gln Leu Pro Ala Val Val Lys Ser Gly Lys Thr His Gly Thr Thr Ile

35 40 45

Val Ile Ser Pro Leu Ile Ser Leu Met Gln Asp Gln Val Glu His Leu

50 55 60

Leu Asn Lys Asn Ile Lys Ala Ser Met Phe Ser Ser Arg Gly Thr Ala

65 70 75 80

Glu Gln Arg Arg Gln Thr Phe Asn Leu Phe Ile Asn Gly Leu Leu Asp

85 90 95

Leu Val Tyr Ile Ser Pro Glu Met Ile Ser Ala Ser Glu Gln Cys Lys

100 105 110

Arg Ala Ile Ser Arg Leu Tyr Ala Asp Gly Lys Leu Ala Arg Ile Val

115 120 125

Val Asp Glu Ala His Cys Val Ser Asn Trp Gly His Asp Phe Arg Pro

130 135 140

Asp Tyr Lys Glu Leu Lys Phe Phe Lys Arg Glu Tyr Pro Asp Ile Pro

145 150 155 160

Met Ile Ala Leu Thr Ala Thr Ala Ser Glu Gln Val Arg Met Asp Ile

165 170 175

Ile His Asn Leu Glu Leu Lys Glu Pro Val Phe Leu Lys Gln Ser Phe

180 185 190

Asn Arg Thr Asn Leu Tyr Tyr Glu Val Asn Lys Lys Thr Lys Asn Thr

195 200 205

Ile Phe Glu Ile Cys Asp Ala Val Lys Ser Arg Phe Lys Asn Gln Thr

210 215 220

Gly Ile Ile Tyr Cys His Ser Lys Lys Ser Cys Glu Gln Thr Ser Ala

225 230 235 240

Gln Met Gln Arg Asn Gly Ile Lys Cys Ala Tyr Tyr His Ala Gly Met

245 250 255

Glu Pro Asp Glu Arg Leu Ser Val Gln Lys Ala Trp Gln Ala Asp Glu

260 265 270

Ile Gln Val Ile Cys Ala Thr Val Ala Phe Gly Met Gly Ile Asp Lys

275 280 285

Pro Asp Val Arg Phe Val Tyr His Phe Thr Val Pro Arg Thr Leu Glu

290 295 300

Gly Tyr Tyr Gln Glu Thr Gly Arg Ala Gly Arg Asp Gly Asn Tyr Ser

305 310 315 320

Tyr Cys Ile Thr Tyr Phe Ser Phe Arg Asp Ile Arg Thr

325 330

40

332

Homo sapiens

40

Phe Arg Pro Leu Gln Leu Glu Thr Ile Asn Val Thr Met Ala Gly Lys

1 5 10 15

Glu Val Phe Leu Val Met Pro Thr Gly Gly Gly Lys Ser Leu Cys Tyr

20 25 30

Gln Leu Pro Ala Leu Cys Ser Asp Gly Phe Thr Leu Val Ile Cys Pro

35 40 45

Leu Ile Ser Leu Met Glu Asp Gln Leu Met Val Leu Lys Gln Leu Gly

50 55 60

Ile Ser Ala Thr Met Leu Asn Ala Ser Ser Ser Lys Glu His Val Lys

65 70 75 80

Trp Val His Ala Glu Met Val Asn Lys Asn Ser Glu Leu Lys Leu Ile

85 90 95

Tyr Val Thr Pro Glu Lys Ile Ala Lys Ser Lys Met Phe Met Ser Arg

100 105 110

Leu Glu Lys Ala Tyr Glu Ala Arg Arg Phe Thr Arg Ile Ala Val Asp

115 120 125

Glu Val His Cys Cys Ser Gln Trp Gly His Asp Phe Arg Pro Asp Tyr

130 135 140

Lys Ala Leu Gly Ile Leu Lys Arg Gln Phe Pro Asn Ala Ser Leu Ile

145 150 155 160

Gly Leu Thr Ala Thr Ala Thr Asn His Val Leu Thr Asp Ala Gln Lys

165 170 175

Ile Leu Cys Ile Glu Lys Cys Phe Thr Phe Thr Ala Ser Phe Asn Arg

180 185 190

Pro Asn Leu Tyr Tyr Glu Val Arg Gln Lys Pro Ser Asn Thr Glu Asp

195 200 205

Phe Ile Glu Asp Ile Val Lys Leu Ile Asn Gly Arg Tyr Lys Gly Gln

210 215 220

Ser Gly Ile Ile Tyr Cys Phe Ser Gln Lys Asp Ser Glu Gln Val Thr

225 230 235 240

Val Ser Leu Gln Asn Leu Gly Ile His Ala Gly Ala Tyr His Ala Asn

245 250 255

Leu Glu Pro Glu Asp Lys Thr Thr Val His Arg Lys Trp Ser Ala Asn

260 265 270

Glu Ile Gln Val Val Val Ala Thr Val Ala Phe Gly Met Gly Ile Asp

275 280 285

Lys Pro Asp Val Arg Phe Val Ile His His Ser Met Ser Lys Ser Met

290 295 300

Glu Asn Tyr Tyr Gln Glu Ser Gly Arg Ala Gly Arg Asp Asp Met Lys

305 310 315 320

Ala Asp Cys Ile Leu Tyr Tyr Gly Phe Gly Asp Ile

325 330

41

320

Homo sapiens

41

Gln Leu Glu Ala Ile Asn Ala Ala Leu Leu Gly Glu Asp Cys Phe Ile

1 5 10 15

Leu Met Pro Thr Gly Gly Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala

20 25 30

Cys Val Ser Pro Gly Val Thr Val Val Ile Ser Pro Leu Arg Ser Leu

35 40 45

Ile Val Asp Gln Val Gln Lys Leu Thr Ser Leu Asp Ile Pro Ala Thr

50 55 60

Tyr Leu Thr Gly Asp Lys Thr Asp Ser Glu Ala Thr Asn Ile Tyr Leu

65 70 75 80

Gln Leu Ser Lys Lys Asp Pro Ile Ile Lys Leu Leu Tyr Val Thr Pro

85 90 95

Glu Lys Ile Cys Ala Ser Asn Arg Leu Ile Ser Thr Leu Glu Asn Leu

100 105 110

Tyr Glu Arg Lys Leu Leu Ala Arg Phe Val Ile Asp Glu Ala His Cys

115 120 125

Val Ser Gln Trp Gly His Asp Phe Arg Gln Asp Tyr Lys Arg Met Asn

130 135 140

Met Leu Arg Gln Lys Phe Pro Ser Val Pro Val Met Ala Leu Thr Ala

145 150 155 160

Thr Ala Asn Pro Arg Val Gln Lys Asp Ile Leu Thr Gln Leu Lys Ile

165 170 175

Leu Arg Pro Gln Val Phe Ser Met Ser Phe Asn Arg His Asn Leu Lys

180 185 190

Tyr Tyr Val Leu Pro Lys Lys Pro Lys Lys Val Ala Phe Asp Cys Leu

195 200 205

Glu Trp Ile Arg Lys His His Pro Tyr Asp Ser Gly Ile Ile Tyr Cys

210 215 220

Leu Ser Arg Arg Glu Cys Asp Thr Met Ala Asp Thr Leu Gln Arg Asp

225 230 235 240

Gly Leu Ala Ala Leu Ala Tyr His Ala Gly Leu Ser Asp Ser Ala Arg

245 250 255

Asp Glu Val Gln Gln Lys Trp Ile Asn Gln Asp Gly Cys Gln Val Ile

260 265 270

Cys Ala Thr Ile Ala Phe Gly Met Gly Ile Asp Lys Pro Asp Val Arg

275 280 285

Phe Val Ile His Ala Ser Leu Pro Lys Ser Val Glu Gly Tyr Tyr Gln

290 295 300

Glu Ser Gly Arg Ala Gly Arg Asp Gly Glu Ile Ser His Cys Leu Leu

305 310 315 320

42

344

Homo sapiens

42

Cys Leu Lys Met Tyr Phe Gly His Ser Ser Phe Lys Pro Val Gln Trp

1 5 10 15

Lys Val Ile His Ser Val Leu Glu Glu Arg Arg Asp Asn Val Ala Val

20 25 30

Met Ala Thr Gly Tyr Gly Lys Ser Leu Cys Phe Gln Tyr Pro Pro Val

35 40 45

Tyr Val Gly Lys Ile Gly Leu Val Ile Ser Pro Leu Ile Ser Leu Met

50 55 60

Glu Asp Gln Val Leu Gln Leu Lys Met Ser Asn Ile Pro Ala Cys Phe

65 70 75 80

Leu Gly Ser Ala Gln Ser Glu Asn Val Leu Thr Asp Ile Lys Leu Gly

85 90 95

Lys Tyr Arg Ile Val Tyr Val Thr Pro Glu Tyr Cys Ser Gly Asn Met

100 105 110

Gly Leu Leu Gln Gln Leu Glu Ala Asp Ile Gly Ile Thr Leu Ile Ala

115 120 125

Val Asp Glu Ala His Cys Ile Ser Glu Trp Gly His Asp Phe Arg Asp

130 135 140

Ser Phe Arg Lys Leu Gly Ser Leu Lys Thr Ala Leu Pro Met Val Pro

145 150 155 160

Ile Val Ala Leu Thr Ala Thr Ala Ser Ser Ser Ile Arg Glu Asp Ile

165 170 175

Val Arg Cys Leu Asn Leu Arg Asn Pro Gln Ile Thr Cys Thr Gly Phe

180 185 190

Asp Arg Pro Asn Leu Tyr Leu Glu Val Arg Arg Lys Thr Gly Asn Ile

195 200 205

Leu Gln Asp Leu Gln Pro Phe Leu Val Lys Thr Ser Ser His Trp Glu

210 215 220

Phe Glu Gly Pro Thr Ile Ile Tyr Cys Pro Ser Arg Lys Met Thr Gln

225 230 235 240

Gln Val Thr Gly Glu Leu Arg Lys Leu Asn Leu Ser Cys Gly Thr Tyr

245 250 255

His Ala Gly Met Ser Phe Ser Thr Arg Lys Asp Ile His His Arg Phe

260 265 270

Val Arg Asp Glu Ile Gln Cys Val Ile Ala Thr Ile Ala Phe Gly Met

275 280 285

Gly Ile Asn Lys Ala Asp Ile Arg Gln Val Ile His Tyr Gly Ala Pro

290 295 300

Lys Asp Met Glu Ser Tyr Tyr Gln Glu Ile Gly Arg Ala Gly Arg Asp

305 310 315 320

Gly Leu Gln Ser Ser Cys His Val Leu Trp Ala Pro Ala Asp Ile Asn

325 330 335

Leu Asn Arg His Leu Leu Thr Glu

340

43

106

Homo sapiens

43

Thr Asn Leu His Leu Ser Val Ser Met Asp Arg Asp Thr Asp Gln Ala

1 5 10 15

Leu Leu Thr Leu Leu Gln Gly Lys Arg Phe Gln Asn Leu Asp Ser Ile

20 25 30

Ile Ile Tyr Cys Asn Arg Arg Glu Asp Thr Glu Arg Ile Ala Ala Leu

35 40 45

Leu Arg Thr Cys Leu His Ala Ala Trp Val Pro Gly Ser Gly Gly Arg

50 55 60

Ala Pro Lys Thr Thr Ala Glu Ala Tyr His Ala Gly Met Cys Ser Arg

65 70 75 80

Glu Arg Arg Arg Val Gln Arg Ala Phe Met Gln Gly Gln Leu Arg Val

85 90 95

Val Val Ala Thr Val Ala Phe Gly Met Gly

100 105

44

88

Escherichia coli

44

Asn Ile Arg Tyr Met Leu Met Glu Lys Phe Lys Pro Leu Asp Gln Leu

1 5 10 15

Met Arg Tyr Val Gln Glu Gln Arg Gly Lys Ser Gly Ile Ile Tyr Cys

20 25 30

Asn Ser Arg Ala Lys Val Glu Asp Thr Ala Ala Arg Leu Gln Ser Lys

35 40 45

Gly Ile Ser Ala Ala Ala Tyr His Ala Gly Leu Glu Asn Asn Val Arg

50 55 60

Ala Asp Val Gln Glu Lys Phe Gln Arg Asp Asp Leu Gln Ile Val Val

65 70 75 80

Ala Thr Val Ala Phe Gly Met Gly

85