JPH11276173A - Gene encoding helicase, recq5 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein consisting of a protein having a specific amino acid sequence and helicase activity and useful for e.g. the elucidation of the relationship between the human homeostasis maintenance and cellular senescence, the elucidation of etiology of disorders accompanying growth and senescence and the treatment and diagnosis of the disorders. SOLUTION: This gene is such one as to encode a protein having an amino acid sequence expressed by the formula or an amino acid sequence wherein at least one amino acid(s) is (are) deleted, substituted or added in the amino acid sequence expressed by the formula and having helicase activity and to be useful for, e.g. studies on the elucidation of the relationship between human homeostasis maintenance and cellular senescence, elucidation of etiology of disorders associated with growth and senescence, and the improvement, remission and diagnosis of symptoms. This gene is obtained by preparing cDNA from mRNA derived from human testis by use of reverse transcriptase and conducting polymerase chain reaction(PCR) using the cDNA as a template and using RecQ5 primer and Taq DNA polymerase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding helicase, a protein encoded by the gene, a method for producing the protein, and uses of the protein and the gene.

[0002]

2. Description of the Related Art DNA helicase is an important enzyme that acts on various biological reactions involving DNA in a living body or in a microbial cell, and there are many kinds thereof. The existence of a wide variety of DNA helicases is apparent from the fact that reactions involving DNA are diverse, such as cell replication and proliferation, individual development and growth, and life support. Known biochemical reactions at the cellular level include DN
A replication, repairing, transcription
nscription), DNA separation (segregation) and dissociation (reco
It has been suggested that there are at least five types of mbination. Generally, DNA helicase is known to have the effect of separating double-stranded DNA into single-stranded DNA, and the energy required for this action is thought to be obtained by hydrolysis of ATP. .

[0003] Among the many DNA helicases, recently, Rec.
A Q-type DNA helicase (a helicase having a helicase domain that shows about 40% or more homology at the amino acid level with the helicase domain of the Escherichia coli recQ gene) has been discovered, and has attracted attention in terms of being related to human diseases and aging. Are gathering. For example, Bloom's syndrome is a disease in which various cancers occur frequently at a young age, and Werner's syndrome is a hereditary disease that causes premature aging and abnormal cancer. Recently, it has been revealed that the causes of these diseases are mutations in genes encoding different human RecQ-type DNA helicases (Cell, 83, pp655-666 (1995) and Science, 272, p.
p258-262 (1996)).

The RecQ type helicase was originally discovered in Escherichia coli by Nakayama et al. (Mol. Gen. Genet., 195, pp. 474-480 (1984)) and has a high homology to this helicase. Have been found in yeast and human cancer cells, and have been identified as sgs1 (Gan
gloff et al., Mol. Cell. Biol. 14, pp8391-8398 (199
4)) and RecQ1 (Seki et al., Nuc.Scida Res. 22, pp.
4566-4573 (1994)).

[0005] In single cell organisms such as E. coli and yeast, helicases belonging to this family include:
Each of Escherichia coli RecQ helicase and sgs1, which were first found, are known. On the other hand, in humans composed of multiple cells, bloom DNA helicase (Ellis et al., Cell, 83, pp6555-666 (1995)) and Werner DNA helicase (Yu et al., Science, 272, pp258
-262 (1996)) and a total of three RecQ1 helicases that have not yet been found to be associated with disease.

[0006]

SUMMARY OF THE INVENTION The present invention relates to human RecQ5
It is an object of the present invention to provide a type DNA helicase gene, a protein encoded by the gene, a method for producing the protein, and uses of the protein and the gene.

[0007]

Means for Solving the Problems The present inventors have thought that there may be many DNA helicases belonging to the RecQ family in addition to the above three types in humans.
And when those genes were mutated, as seen in the cases of Bloom syndrome and Werner syndrome,
The authors considered that it may be a causative gene for intractable diseases that are still unknown for inducing various diseases. The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, a new human RecQ5
Succeeded in cloning the type-DNA helicase gene, and completed the present invention.

[0008] That is, the present invention is a gene encoding the following protein (a) or (b). (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having helicase activity Protein Examples of the gene include those consisting of the following DNA (a) or (b). (A) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 (b) DNA which hybridizes with the DNA comprising the nucleotide sequence of (a) under stringent conditions and encodes a protein having helicase activity

Further, the present invention relates to a mouse or rat-derived protein comprising an amino acid sequence having at least 70% homology with the amino acid sequence of the following protein (a) or (b) and encoding a protein having helicase activity: Is the gene. (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having helicase activity protein

[0010] Further, the present invention is an oligonucleotide probe capable of hybridizing with at least a part of the gene. Further, the present invention is a recombinant vector containing the gene. Further, the present invention provides
A transformant containing the recombinant vector. further,
The present invention provides the method for producing a protein, comprising culturing the transformant and collecting a protein having helicase activity from the resulting culture.

Furthermore, the present invention relates to the following recombinant protein (a) or (b). (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having helicase activity Protein Here, the present invention also includes a mouse or rat protein containing an amino acid sequence having at least 70% homology with the amino acid sequence of the protein and having helicase activity.

Further, the present invention relates to a monoclonal antibody or a polyclonal antibody against the protein.
Furthermore, the present invention is the above-described monoclonal antibody-producing hybridoma, which can be obtained by fusion of an antibody-producing cell immunized with the protein with a myeloma cell. Further, the present invention is a reagent for detecting a gene encoding helicase, comprising the oligonucleotide probe.

Further, the present invention is a kit for diagnosing a disease caused by an abnormality in a gene encoding helicase, comprising the protein and the monoclonal antibody and / or the polyclonal antibody. Further, the present invention is a transgenic animal into which a gene modified to increase or decrease the expression level of the gene has been introduced. Further, the present invention is a knockout mouse which has been treated so that the function of the gene is lost. Hereinafter, the present invention will be described in detail.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The gene of the present invention is a novel human RecQ
It encodes type 5 DNA helicase, and has the sequence number 2
A protein comprising an amino acid sequence represented by, or at least one amino acid is deleted in the amino acid sequence,
It encodes a protein having a substituted or added amino acid sequence and having helicase activity.

As shown in FIG. 2, the protein of the present invention comprises, as shown in FIG. 2, seven helicases which are well-conserved at the amino acid level among Escherichia coli, yeast, and humans.・ Includes motifs. In addition, as is clear from the results of FISH (fluorescence in situ hybridization) analysis shown in FIG. 3, the human RecQ5-type DNA helicase gene of the present invention was obtained from human 17th DNA shown in FIG.
It was confirmed to be located on the long arm of the chromosome, 17q25. In addition, the gene derived from other species corresponding to the human gene of the present invention can be cloned by a known technique.

From the results of multi-tissue Northern blot analysis (FIG. 5) for examining how the gene of the present invention is expressed in various organs (FIG. 5), the gene is expressed in all tissues. In particular, it was found to be extremely strongly expressed in pancreas and testis.

[0017] The above results together strongly suggest that the gene of the present invention is one of the genes involved in the maintenance of basic homeostasis in living organisms. Therefore, the gene of the present invention is useful for research for elucidating the relationship between development and aging, and elucidation of the expression control of this gene is also useful for the creation of new drugs for maintaining homeostasis in living organisms. Useful. The gene of the present invention can be identified and obtained, for example, as follows.

1. Cloning of human RecQ5-type DNA helicase gene The gene of the present invention (human RecQ5-type gene or human RecQ5-type gene)
DNA helicase gene) is long-distance (L
D) Based on the two principles of PCR and Suppression PCR
RACE [Raped Amplification of cDNA Ends; Frohman, M.
A. et al., Methods Enzymol. Vol. 218, pp340-358 (1
993)].

The human RecQ5 type DNA helicase gene of the present invention comprises a DNA fragment having a known partial sequence and a 5 ′ terminal or 3 ′
It can be obtained by assembling (reconstructing) a DNA fragment having a terminus whose sequence is unknown. In the present invention, cloning of a full-length cDNA is performed, for example, using a commercially available kit (Marathon ^™ cDNA AmplificationKi
t; CLONETECH).

First, a DNA fragment having a known human-derived sequence is amplified. The known sequence is a po from human tissue or organ.
ly (A) + RNA, e.g., poly (A) + R from human testis or spleen
Can be obtained from NA. That is, cDNA is synthesized from the RNA using reverse transcriptase, and a partial cDNA fragment is prepared by RT-PCR (FIG. 1 (1)). Next, after determining the sequence of the obtained partial cDNA fragment, four types of gene-specific primers (GSPs) were determined based on the sequence of the partial cDNA.
(In Fig. 1, 5 'GSP1 and 5' GSP2 and 3 'GSP
SP1 and 3'GSP2). GSP is the partial cDNA
It is a primer required for amplifying a DNA fragment whose sequence is unknown, which is a DNA fragment present in the 5 ′ and 3 ′ regions from the sequence. The sequence of GSP can be arbitrarily selected from the partial cDNA sequence, and the sequence can be obtained by chemical synthesis.

In the present invention, the GSP used for amplifying an unknown sequence 5 ′ to the partial cDNA is 5′GSP1 and 5′GSP2.
GSP used to amplify the 3'-side unknown sequence
Are defined as 3′GSP1 and 3′GSP2 (frame part in FIG. 1A).
Next, a DNA fragment on the 5 ′ side and 3 ′ side of the partial cDNA is amplified (FIG. 1 (2)). As a template, commercially available human testis-derived cD
NA [eg, cDNA Ready ^{™ from} CLONETECH] can be used. Although the sequence of the DNA fragment serving as the template is unknown, an adapter sequence is added to each DNA fragment. Thus, using a primer capable of hybridizing to an adapter sequence [referred to as an adapter primer (AP)] and the GSP as primers, an amplification reaction (LD
PCR) twice (Fig. 1 (2)).

For example, in order to amplify the 5′-side unknown sequence, PCR is first performed using AP1 and 5′GSP1, and the obtained fragment is used as a template. Primers that hybridize to the inner region (AP2 and
Perform PCR using 5'GSP2) (nested PCR). The 3′-side unknown sequence can be amplified in the same manner as when the 5′-side unknown sequence is amplified using 3′GSP1 and 3′GSP2.

In the present invention, the fragment is located upstream of the known sequence with reference to the position of the fragment of the known partial sequence.
DNA fragment is 5'-RACE product, downstream DNA fragment is 3'-RA
CE products. Since the AP has the same sequence as the protruding end of the adapter, it is not annealed to the adapter, and is extended only from the gene-specific primer (GSP) in the first amplification (this is called Suppression PCR).

The nucleotide sequences of the obtained 5'-RACE product and 3'-RACE product were determined by Hattori et al. [Electrophoreses 13, pp56.
0-565 (1992)]. That is, the reaction was performed using a PRISM sequencing kit containing a fluorescent dideoxy terminator manufactured by Perkin Elmer. Perform analysis.

From the known partial sequence obtained as described above, the base sequences of the 5'-RACE product and the 3'-RACE product, and the 5 'upstream sequence obtained by the oligocapping method, the full-length cDNA was assembled by assembly. A fragment having a base sequence can be obtained. That is, after appropriate restriction enzyme sites present in the overlapping portions between base sequences are treated with restriction enzymes, the obtained fragments are connected by a ligation reaction to obtain a full-length cDNA fragment.
(Figure 1). Confirmation of the overlapping portion and confirmation of the base sequence of the full-length cDNA can be performed using, for example, base sequence analysis software (such as DNASYS).

SEQ ID NO: 1 shows the nucleotide sequence of the gene of the present invention, and SEQ ID NO: 2 shows the amino acid sequence encoded by the gene, provided that the protein having the amino acid sequence represented by SEQ ID NO: 2 has helicase activity. In addition, mutations such as deletion, substitution, addition, and insertion may occur in the amino acid sequence contained in the protein or the base sequence of the gene. For example, at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be deleted, or the amino acid sequence represented by SEQ ID NO: 2 May be added with at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids, or at least one of the amino acid sequences represented by SEQ ID NO: 2, preferably 1 to
About 10, more preferably 1 to 5 amino acids may be substituted with another amino acid. Therefore, proteins in which the first methionine (Met) in the amino acid sequence represented by SEQ ID NO: 2 is deleted are also included in proteins resulting from the change in the amino acid sequence. In addition to the nucleotide sequence encoding the amino acid contained in the protein of the present invention, the gene of the present invention also includes degenerate isomers encoding the same protein that differs only in degenerate codons.

The above mutation can be introduced by any known method, for example, a point mutation introduction kit (for example, TAKARA LA PCR in virtro Mu manufactured by Takara Shuzo Co., Ltd.).
tagenesis Kit) or the like. In addition, at least the amino acid sequence of the protein
A protein containing an amino acid sequence having 70% homology and having helicase activity is also included in the protein of the present invention, and a gene encoding the protein is also included in the gene of the present invention. The homology is at least 70
%, More preferably 80% or more, and 90% or more.
% Or more is more preferable. Examples of the protein and gene include those derived from mouse or rat.

Further, in the present invention, the DNA which hybridizes with the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, for example, under low stringent conditions, and encodes a protein having helicase activity. Genes are also included in the gene of the present invention. Here, "low stringent conditions" means, for example, conditions in which the sodium concentration is 1 to 1000 mM, preferably 10 to 300 mM, and the temperature is 40 to 55 ° C.

Once the base sequence has been determined,
The desired gene can be obtained by chemical synthesis or by PCR using primers synthesized from the determined base sequence. For example, when expressing a recombinant protein using Escherichia coli, since the necessary gene is an open reading frame (ORF) encoding the protein, PCR is performed using a primer capable of amplifying the ORF, ORF genes can be cloned.

2. Preparation of Recombinant Vector and Transformant The recombinant vector of the present invention can be prepared by incorporating a purified gene into a restriction enzyme site or a multiple cloning site of an appropriate vector DNA. Further, the transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host compatible with the vector used in producing the recombinant vector.

The vector DNA for inserting the DNA fragment is
There is no particular limitation as long as it can be replicated in a host cell, and examples include plasmid DNA and phage DNA. Examples of plasmid DNA include plasmid pUC118 (Takara Shuzo), pBluescriptSK + (Stratagene), pGEM-T
(Manufactured by Promega). Phage DNA includes
For example, M13mp18, M13mp19 and the like can be mentioned. Vector DN
When using plasmid DNA as A, for example, EcoRI DN
When inserting the A fragment, the plasmid DNA is
(NEB). Next, the DNA fragment and the cut vector DNA are mixed, and, for example, T4D
A recombinant vector is obtained by reacting NA ligase (Takara Shuzo).

The host is not particularly limited as long as it can express the target gene, and any of eukaryotic cells and prokaryotic cells can be used. For example, Escherichia coli, Bacillus subtilis (Bacchar
omyces cerevisiae) and other yeasts,
Animal cells such as COS cells and CHO cells, and insect cells and the like are also included. When using bacteria such as E. coli as a host,
It is preferable that the recombinant vector of the present invention be capable of autonomous replication in the host and, at the same time, contain a promoter, the gene of the present invention, and a transcription termination sequence.

As Escherichia coli, for example, XL1-Blue (Stratagen
e) and JM109 (Takara Shuzo). Examples of the expression vector include pBTrp2. Also,
Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli, phage, etc., such as a trp promoter, a lac promoter, a PL promoter, and a PR promoter are used. In the present invention, the transformation is, for example, Ha
nahan's method [Techniques for Transformation of E. co
li In DNA Cloning, vol.1, Glover, DM (ed.), pp109
-136, IRLPress (1985)].

When yeast is used as a host, examples of expression vectors include Yep13 and Ycp50. Examples of the promoter include gal1 promoter, gal10
Promoters and the like. Methods for introducing a recombinant vector into yeast include, for example, electroporation [Methods. Enzymol., 194, pp. 182-187 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 84,
pp1929-1933 (1978)], Lithium acetate method [J. Bacteri
153, 163-168 (1983)].

When an animal cell is used as a host, for example, pcDNAI, pcDNAI / Amp (Invitrogen) or the like is used as an expression vector. Examples of a method for introducing a recombinant vector into animal cells include an electroporation method and a calcium phosphate precipitation method. When an insect cell is used as a host, expression vectors such as pVL1392 and pVL1393 (both from Invitrogen) and pMBac
(Stratagene), pBacPAK8 / 9 (Clontech) and the like are used.

Examples of a method for introducing a recombinant vector into insect cells include a calcium phosphate method, a lipofection method, and an electroporation method. The above-mentioned screening of the transformant contains a part of the target gene.
Colony hybridization using a DNA fragment as a probe, or 5 'based on the base sequence of the target gene
A primer (FP; forward primer) was synthesized, and then a 3 ′ primer (RP; revers
e primer), and colonies (transformants) containing the desired gene can be selected and collected by PCR using these primers.

3. Production of Protein (Polypeptide) Encoded by Human RecQ5 Type Gene The protein of the present invention can be produced by culturing a transformant having the recombinant vector obtained as described above. The culture method is exemplified as follows, but it is preferable to employ a liquid culture method. The method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host.

The medium for culturing a transformant using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the microorganism, so that the cultivation of the transformant is efficient. The medium is not particularly limited as long as it can be performed in any of the above conditions, and either a natural medium or a synthetic medium may be used.
Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, starch, maltose and dextrin; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol.

Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, and other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, casamino. Acid, NZ amine and the like. As inorganic substances, potassium monophosphate,
Potassium phosphate, calcium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, zinc sulfate, cobalt chloride and the like. Cultivation is usually performed under aerobic conditions such as shaking culture or aeration-agitation culture at 36 to 36 ° C, preferably 14 to 37 ° C.
Perform for 20 hours. During the culture period, the pH is adjusted to 7.2-7.4.

During the culture, antibiotics such as ampicillin and tetracycline, vitamins and the like may be added to the medium as needed. When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using a Lac promoter, culturing a microorganism transformed with an expression vector using a trp promoter, such as isopropyl-β-D-thiogalactopyranoside (IPTG). At times, indole acrylic acid (IAA) or the like may be added to the medium. As a medium for culturing transformants using animal cells as a host, commonly used RPMI1640 medium, DMEM
A medium or a medium obtained by adding fetal bovine serum or the like to these mediums is used.

The cultivation is usually performed at 37 ° C. in the presence of 5% CO ₂ . During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed. The medium for culturing the transformants obtained using insect cells as a host includes Grac
e's Insect Medium [Grace, TCC Nature, 195: 788,
(1962)] to which additives such as 10% fetal bovine serum are appropriately added. The pH of the medium is adjusted to 6.6 to 7.0,
Culture is usually performed at 25 ° C for 1 to 7 days, and aeration and / or agitation are added as necessary.

After completion of the cultivation, the protein of the present invention can be collected from the culture (in the cell lysate, the culture solution or the supernatant thereof) by a conventional protein purification means. After culturing, when the protein of the present invention is produced in the cells or cells, the cells are disrupted by lysis treatment using an enzyme such as lysozyme, ultrasonic crushing treatment, grinding treatment, etc. The protein is excreted outside the cells. Next, insoluble materials are removed by filtration or centrifugation to obtain a crude protein solution.

On the other hand, when the protein of the present invention is produced extracellularly or extracellularly, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like to obtain a supernatant. Then, general biochemical methods used for the isolation and purification of proteins, such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, electrophoresis, etc., alone or By combining as appropriate,
The protein of the present invention can be isolated and purified from the culture.

4. Preparation of Monoclonal Antibody A monoclonal antibody specific to the protein encoded by the human RecQ5 gene of the present invention can be obtained as follows. (1) Preparation of antigen The polypeptide obtained by the method of the above 3. is dissolved in a buffer, and then an adjuvant is added. Examples of the adjuvant include commercially available complete Freund's adjuvant, incomplete Freund's adjuvant, and the like, and any of these may be mixed.

(2) Immunization and collection of antibody-producing cells The immunogen obtained as described above is administered to mammals such as rats and mice. The immunological amount of the antigen is 10 to 500 μg per animal at a time. Immunization sites are mainly intravenous, subcutaneous, and intraperitoneal. The interval between immunizations is not particularly limited, and may be every several days to several weeks, preferably one to several weeks.
Immunization is performed 2 to 5 times, preferably 3 to 4 times at an interval of 3 weeks. The antibody-producing cells are collected 2 to 7 days, preferably 4 to 5 days after the last immunization. Antibody-producing cells include B cells derived from spleen cells, lymph node cells, peripheral blood cells, etc., and B cells derived from spleen cells or local lymph node cells.
Cells are preferred.

(3) Cell fusion As a myeloma cell to be fused with an antibody-producing cell, a commonly available cell line of an animal such as a mouse is used.
The cell line used has drug selectivity and cannot survive in a selective medium (HAT medium: containing hypoxanthine, aminopterin and thymine) in an unfused state, but survives only in a state fused to antibody-producing cells. Those having the property which can be performed are preferred. Specific examples of myeloma cells include mouse myeloma cell lines such as P3U-1 (manufactured by Dainippon Pharmaceutical Co., Ltd.) and P3x63Ag8.653. Next, the myeloma cells are fused with the antibody-producing cells. Cell fusion is performed by mixing equal volumes of 10 ⁸ cells / ml of antibody-producing cells and 2 × 10 ⁵ cells / ml of myeloma cells in an animal cell culture medium such as serum-free DMEM or RPMI-1640 medium. The fusion reaction is performed in the presence of a fusion promoter. To promote cell fusion,
For example, polyethylene glycol having an average molecular weight of 1,500 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (eg, electroporation).

(4) Selection and Cloning of Hybridoma A desired hybridoma is selected from the cells after the cell fusion treatment. As a method, after appropriately diluting the cell suspension with, for example, RPMI-1640 medium containing fetal bovine serum, spread about 5 to 10 cells / well on a microtiter plate,
A selection medium is added to each well, and thereafter the culture is performed by appropriately replacing the selection medium. As a result, after starting cultivation in the selective medium,
Cells that grow from around 14 days can be obtained as hybridomas.

Screening is performed to determine whether the target antibody is present in the culture supernatant of the hybridoma that has grown. Hybridoma screening may be performed according to a conventional method, and is not particularly limited. For example,
A part of the culture supernatant contained in the wells grown as hybridomas was collected and subjected to enzyme immunoassay (EIA; enzyme immu
no assay), RIA (radio immunoassay) and the like. Cloning of the fused cells is performed by a limiting dilution method or the like, and finally, a hybridoma that is a monoclonal antibody-producing cell is established.

(5) Collection of Monoclonal Antibody As a method for collecting a monoclonal antibody from the established hybridoma, a usual cell culture method or ascites formation method or the like can be employed. In the cell culture method, the hybridoma was RPMI-1640 medium containing 10% fetal bovine serum, MEM
In an animal cell culture medium such as a culture medium or a serum-free medium, the cells can be cultured under ordinary culture conditions (for example, 37 ° C., 5% CO ₂ concentration) for 10 to 14 days, and the antibody can be obtained from the culture supernatant.
In the case of the ascites formation method, about 5 × 10 ⁶ hybridomas are administered intraperitoneally to mammals and allogeneic animals derived from myeloma cells, and the hybridomas are grown in large quantities. And 1
After 2 weeks, ascites or serum is collected.

When antibody purification is required in the above antibody collection method, a known method such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, or gel chromatography is appropriately selected. Alternatively, they can be purified by combining them.

5. Preparation of Polyclonal Antibody (1) Preparation of Antigen The polypeptide obtained by the method of the above 3. is dissolved in a buffer, and then an adjuvant is added. Commercially available complete Freund's adjuvant and incomplete Freund's adjuvant are used as adjuvants.

(2) Immunization Rabbits, guinea pigs, goats, sheep and the like are usually used as animals. Taking rabbits as an example, rabbits are usually injected subcutaneously in the footpad with 100 μg to 500 μg of polypeptide together with complete Freund's adjuvant. Two weeks later, the same amount of antigen is mixed with Freund's incomplete adjuvant and injected intramuscularly. Two weeks later, the intramuscular injection was repeated, and one week after the final immunization, partial blood was collected from the ear and EIA was performed.
The antibody titer is measured by a method or the like. If the antibody titer has reached the target value, the whole blood is collected. If the antibody titer is low, intramuscular injection is repeated, and immunization is repeated until the antibody titer reaches the target value.
For purification of the antibody from the serum by ammonium sulfate fractionation, the method described in the section of the monoclonal antibody can be employed.

6. Human RecQ5 gene and reagent for detecting polypeptide encoded by the gene The amino acid sequence of the protein encoded by the human RecQ5 gene has Bloom syndrome or Werner, which causes chromosome instability and causes high-frequency carcinogenesis or progeria It has high homology with the amino acid sequence of the protein encoded by the causative gene of the syndrome (Fig. 2), and also has human RecQ5
The type gene was expressed in various human tissues (FIG. 5). Therefore, it can be said that the human RecQ5-type gene is one of the genes encoding a DNA helicase involved in the maintenance of basic homeostasis in living organisms. From these facts, it is useful to elucidate the regulation of human RecQ5 gene expression to elucidate the mechanism that controls the homeostasis of living organisms, and to develop new drugs to maintain the basic homeostasis of living organisms. It is useful not only for creation but also for research to elucidate the relationship with aging. The reagent of the present invention is useful for detecting and diagnosing a disease caused by a genetic abnormality encoding a protein having helicase activity, such as, but not limited to, Bloom syndrome and Werner syndrome.

When the gene of the present invention is used as a detection reagent, hybridization is performed using an oligonucleotide containing at least a part of the cloned human RecQ5 gene as a probe, and detection is performed by Southern or Northern blotting. Note that examples of the oligonucleotide probe include a DNA probe and an RNA probe. When designing an oligonucleotide, any region of the RecQ5 gene is selected, but an open reading frame region is preferable, and the length of the oligonucleotide is not particularly limited,
Any length can be used. When a polyclonal antibody and / or a monoclonal antibody against the polypeptide encoding the gene of the present invention or any one of them is used as a detection reagent, detection is performed by EIA, RIA or Western blot analysis.

7. Transgenic Animals Introduced with a Gene Modified to Increase or Decrease the Expression Level of the Gene In the present invention, several important sites that normally regulate gene expression (enhancers) ,promoter,
Introduced mutations such as deletions, substitutions, additions, insertions, etc. can partially modify the gene so that it is artificially increased or decreased compared to the original gene expression level.

The above-mentioned mutation can be introduced by any known method, for example, a point mutation introduction kit.
(For example, Takara Shuzo's TAKARA LA PCR in vitro Mutagene
siskit) can be used. Transgenic animals include, for example, transgenic mice, transgenic rats, and the like. As a vector having the mutation-introduced gene, each animal species RecQ
There are two types of vectors: a vector that overexpresses the type 5 gene and an antisense vector that suppresses its expression. These vectors include, for example, pcDN
A, pRC / RSV (Invitrogen) and the like. In each case, a positive selection agent for gene selection
A resistance gene (eg, neomycin) is linked.

The introduction of a gene into a cell is performed by directly adding D
Although a method of injecting NA can also be used, ES cells have the advantage that they can be cultured and mice and the like can be generated from these cells, so that a method using various ES cells is more efficient. Is preferred. Examples of ES cells include TT2 cells (Shinichi Aizawa, Gene Targeting, 1995, Yodosha). And, for example, mouse RecQ
The above vector DNA containing the type 5 gene is introduced into ES cells by electroporation, and positively selected with neomycin to obtain a target mutant ES cell. The ES cells are injected into a blastocyst or an 8-cell embryo using a capillary or the like. Thereafter, the blastocyst or the 8-cell stage embryo is directly transferred to the fallopian tube of the foster mother, or the one that has been cultured for one day and has developed to the blastocyst is transferred to the foster parent's uterus. Select chimeric animals from offspring. An animal having a high chimeric contribution rate has a high possibility of a germ line, but by crossing the chimeric animal with a normal animal, it can be confirmed that the animal is a germ line chimeric animal. By mating a germ-line chimeric animal with a normal animal, a heterozygous animal can be obtained, and by mating between heterozygotes, a homozygous animal can be obtained.

8. Knockout mouse The knockout mouse of the present invention has been processed so that the function of the mouse RecQ5 gene is lost. The processing method will be described. Genomic DNA containing mouse RecQ5 gene was converted from genomic DNA prepared from mouse ES cells to PC
A vector is constructed, obtained by R or from a genomic library, with the neo resistance gene inserted into any of its exons. This operation destroys the function of this exon. At the same time, a thymidine kinase (tk) gene or a diphtheria (DT) toxin gene for negative selection is linked to this vector. The vector DNA is introduced into ES cells by electroporation. Next, the cells were treated with neomycin for positive selection and the nucleic acid analog FIAU (fluoroiodoadenosy) for negative selection.
luracil) or diphtheria toxin. By this operation, diphtheria toxin-sensitive cells that have undergone non-homologous recombination and G418-sensitive cells that do not undergo any recombination are removed, leaving only cells that have undergone homologous recombination.
In cells that have undergone this homologous recombination, the gene containing the disrupted exon is knocked out. The obtained cells are injected into mouse blastocysts or 8-cell stage embryos. Thereafter, a knockout mouse can be produced by the same method as that for producing a transgenic animal.

[0059]

The present invention will be described more specifically with reference to the following examples. However, the present invention does not limit the technical scope to these examples. [Example 1] Cloning of full-length human RecQ5 cDNA (1) Identification of cDNA fragment having homology to E. coli RecQ DNA helicase gene. First, in cloning the DNA of the present invention, a cDNA sequence having homology to Escherichia coli RecQ helicase was searched in the dbest database. As a result, more than a dozen ESTs (Expessed Se) including EST-DNAACC.AA155882 (FIG. 6, DNA encoding the amino acid sequence of AA155882).
quence Tag) DNA sequence was obtained. However, these ESTs
Homology in the nucleotide sequence with EST-DNA derived from a species other than human, for example, Escherichia coli, yeast, nematode, etc .; or EST and human-known Bloom's disease helicase, Werner's disease Helicase and RecQ1
Considering the homology in nucleotide sequence with each gene of helicase, EST-DNA with high homology to these genes
Was excluded.

(2) Confirmation that EST-DNA ACC.AA155882 is a gene expressed in human tissues Next, the sequence of EST-DNA ACC.AA155882 is a gene that is truly expressed in human tissues. A series of preliminary experiments to confirm this was performed. That is, a sense primer (SEQ ID NO: 3) and an antisense primer (SEQ ID NO: 4) were prepared as primers for PCR (Polymerase Chain Reaction) from the base sequence contained in the EST-DNA fragment. On the other hand, cDNA was prepared from the human testis-derived mRNA by reverse transcription, and PCR was performed using the primers prepared above.
It was confirmed whether EST-DNA was present in NA.

That is, the RT-P
By performing CR, a PCR product of about 320 base pairs predicted from the sequence of EST-DNA ACC.AA155882 could be detected. PCR
The presence or absence of the EST-DNA sequence was also confirmed by incorporating the DNA fragment amplified by PCR into Escherichia coli plasmid DNA, cloning it, and analyzing the nucleotide sequence of the obtained plasmid cloned DNA. Thus, it was confirmed that EST-DNA ACC.AA155882 was a part of the novel helicase gene (the method was described in (3-ii) and (3
-iii))).

(3) Cloning of partial cDNA fragment of human RecQ5 type gene by RT-PCR (3-i) Preparation and amplification of cDNA by reverse transcription Poly (A) + RNA derived from human testis (CLONETECH) About 1 μm
g, dithiothreitol, dNTP (dATP, dCTP, dGTP and
dTTP), random hexamer, reverse transcriptase buffer and reverse transcriptase Super Sucript II.
The reaction was carried out at 30 ° C for 30 minutes, followed by RNase treatment to prepare cDNA. PCR was performed using this cDNA as a template.

For PCR, TAKARA Taq manufactured by Takara Shuzo and a buffer attached thereto were used. Taq DNA polymerase and PCR buffer described below are used unless otherwise specified.
TAKARA Taq and the buffer attached to it. PCR was performed in 1x PCR buffer, 0.2 mM dNT
P, a solution containing 0.4 μM RecQ5 primer (SEQ ID NOs: 3 and 4) and 0.625 unit Taq DNA polymerase, and an appropriate amount of the above-mentioned human testis cDNA were added to a 25 μl mixed reaction solution. First, the above reaction solution was reacted at 94 ° C. for 5 minutes, and then reacted at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute.
This was done for 35 cycles. The obtained reaction solution was further reacted at 72 ° C. for 5 minutes.

(3-ii) Subcloning of RT-PCR product for base sequence determination RT-PCR product obtained in (3-i), T4 DNA ligase (Takara Shuzo), buffer (Takara Shuzo) And a solution containing the pGEM-T vector (Promega) was reacted at 15 ° C. for 3 hours to incorporate the RT-PCR product fragment into the pGEM-T vector. E. coli JM109 was transformed with this vector, and the E. coli was transformed with X-gal, IPTG and ampicillin.
(Final concentration: 50 μg / ml) was seeded on an LB Bacto agar plate and incubated at 37 ° C. for 12 hours. For the white E. coli colonies that appeared, select ampicillin (final concentration
(50 μg / ml) in LB medium at 37 ° C. for 16 hours or more with shaking, and a plasmid DNA was prepared using a Kurobo robot (PI-100Σ). DH ₂ O 1 containing 10 μg / ml RNase
The plasmid DNA was dissolved in 00 μl to prepare a sample for DNA sequencing.

(3-iii) Determination of base sequence Using the plasmid DNA prepared in the above (3-ii) as a template DNA, an unlabeled primer and four types of fluorescently labeled nucleotides
PCR was performed in a reaction system to which -5'-triphosphate and Taq polymerase were added. The reaction mixture is as follows.

[0066]

As a primer for determining a nucleotide sequence,
Those shown in SEQ ID NOs: 5 to 17 were used. PCR should be performed at 96 ° C for 30
Sec (denaturation), 15 sec at 55 ° C (annealing) and 4 sec at 60 ° C
This was performed for 25 cycles, with the reaction of 1 minute (elongation) as one cycle. In this reaction, DNA containing a random fluorescent dye
A fragment is synthesized, and by analyzing it with a sequencer, a continuous base sequence can be finally determined. Analysis of the PCR reaction was performed using an automatic DNA sequencer (model ABI 373) manufactured by Applied Biosystem.
SEQ ID NO: 29 shows the nucleotide sequence of the obtained human RecQ5 partial cDNA.

(4) Cloning of 5 ′ and 3 ′ Regions of Human RecQ5 Gene Using Marathon cDNA Amplification Kit 5 ′ and 3 ′ based on the nucleotide sequence of the RecQ5 partial cDNA fragment obtained in (3) above Cloning of the region is performed by Marathon c
This was performed using a DNA Amplification kit (CLONTECH). First, to amplify the 5'-RACE product, use Marathon Ready
The first PCR was performed using the testis cDNA as a template and a primer AP1 (SEQ ID NO: 18) specific to the adapter sequence and a primer 5′GSP1 (SEQ ID NO: 20) specific to the RecQ4 partial cDNA fragment. The PCR reaction mixture is as follows.

[0069]

In the PCR, denaturation was performed at 94 ° C. for 1 minute, the reaction was performed at 94 ° C. for 30 seconds (denaturation) and at 72 ° C. for 4 minutes (annealing and extension) as one cycle. One cycle of the reaction was 30 seconds (denaturation) and 4 minutes (annealing and extension) at 70 ° C, and this was repeated for 5 cycles.
The reaction was performed for 25 minutes, with a reaction of 2 seconds (denaturation) and 4 minutes at 68 ° C. (annealing and extension) as one cycle.

Using a dilution of this reaction solution as a template,
Further inner primers, AP2 (SEQ ID NO: 19) and 5'G
A second PCR using SP2 (SEQ ID NO: 21) was performed. This resulted in a 5 'RACE product of approximately 1.2 kilobase pairs. In a similar manner, a 3 'RACE product of about 1.7 kilobase pairs was obtained using the 3'GSP1 (SEQ ID NO: 22) and 3'GSP2 (SEQ ID NO: 23) primers. These were subcloned into the pGEM-T vector, and the full-length sequences of the 5 'and 3' RACE products were determined.
(Described in (3-ii) and (3-iii)]]. The nucleotide sequences of the 5 ′ and 3 ′ RACE products of the obtained human RecQ5 gene are shown in SEQ ID NOs: 30 and 31, respectively.

(5) Determination of Transcription Start Point of RecQ5 Gene The 5 ′ region containing the transcription start point of RecQ5 gene was determined by the oligocapping method. Human testis-derived mRNA was treated with calf small intestine alkaline phosphatase (CIAP) manufactured by Takara Shuzo to dephosphorylate it. The reaction mixture is as follows.

[0073]

The reaction was carried out at 37 ° C. for 30 minutes. Next, in order to remove the 5 'CAP structure contained in the dephosphorylated mRNA, it was treated with Nippon Gene tobacco acid pyrophosphatase (TAP).
The reaction mixture is as follows.

[0075]

The reaction was carried out at 37 ° C. for 60 minutes. Next, an oligo RNA consisting of 30 bases (SEQ ID NO: 24) was connected as an adapter to the 5 'end of the mRNA (TAP-RNA) from which the CAP structure had been removed using T4RNA ligase manufactured by Takara Shuzo. The reaction mixture is as follows.

[0077]

The reaction was carried out at 18 ° C. for 16 hours. This reaction product was deproteinized by phenol treatment, and oligocapping RNA was recovered by ethanol precipitation, and then dissolved in 50 μl of dH ₂ O. Next, a single-stranded cDNA was synthesized using the oligocapping RNA as a template. The reaction mixture is as follows.

[0079]

After heating at 70 ° C. for 10 minutes, the mixture was cooled in ice,
Further, the following reagents were added.

[0081]

Incubate at 37 ° C for 2 minutes, and further add Superscript
II (Gibco) (5 μl) was added and reacted at 37 ° C. for 30 minutes. After the reaction, heat at 95 ° C for 10 minutes, add dH ₂ O, and add a total volume of 500 μl
These were used as oligocapping cDNAs. Next, PCR was performed using the oligocapping cDNA as a template. PCR is 2
Nested PCR using the first PCR product as a template for the second PCR. The first reaction mixture is as follows.

[0083]

The PCR was carried out once at 95 ° C. for 5 minutes (denaturation), followed by one cycle at 94 ° C. for 30 seconds (denaturation), 60 ° C. for 30 seconds (annealing), and 72 ° C. for 1 minute (extension). This is 35
Cycled. Finally, the reaction was carried out at 72 ° C. for 5 minutes.
Next, a second PCR was performed using the PCR product as a template. The reaction mixture is as follows.

[0085]

The second PCR cycle was performed in the same manner as the first cycle. Approximately 400 base pairs of PCR product was observed by 2% agarose gel electrophoresis.
It was subcloned into a vector and the nucleotide sequence was determined [the method was described in (3-ii) and (3-iii)]. The primers described in SEQ ID NOs: 4 and 5 were used for nucleotide sequence determination. SEQ ID NO: 32 shows the nucleotide sequence of the 5 'transcription initiation site of the obtained human RecQ5 gene.

(6) Full length RecQ5 by base sequence assembly
Construction of cDNA The four nucleotide sequences of the human RecQ5 cDNA obtained as a result of the above (3), (4) and (5) are shown in SEQ ID NOs: 29 to 32. These sequences partially overlap, and their relative positional relationships can be shown as in FIG. In FIG. 7, the transcription initiation region is SEQ ID NO: 32, the 5 'RACE product is SEQ ID NO: 30, and the partial cDNA is SEQ ID NO:
The 29, 3 'RACE product is that represented by SEQ ID NO: 31.

This region was analyzed by computer using DNASYS software, and the above four base sequences were connected to form the RecQ5 cDNA.
A full-length sequence could be obtained. The cDNA obtained in this manner was represented by a total length of 3707 nucleotides excluding the 3′-end poly sequence (SEQ ID NO: 1). Of which the open reading frame (ORF) is 1230 nucleotides,
The protein encoded by the ORF consists of 410 amino acids,
The molecular weight was found to be 45,680 daltons. The amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO: 2.

Example 2 FISH (fluoresence in situ)
Identification of human RecQ5-type locus by hybridization) (1) Screening of P1 clone containing human RecQ5-type gene Primer specific to 3′-untranslated region (3′-UTR) of human RecQ5-type gene (SEQ ID NO: 33) , 34) was used to screen the P1 human genomic library, and p1 # 1503 was obtained. (2) P1 DN with digoxigenin label
Preparation of A Probe P1 # 15031 DNA was labeled with digoxigenin-dUTP by the nick translation method.

(3) Hybridization PHA-stimulated metaphase chromosomes derived from human peripheral blood lymphocytes
It was incubated with a digoxigenin-labeled probe, human COT-1 DNA, in a solution of 0% formulated, 10% dextran sulfate, 2 × SSC.
A human chromosome 17 centromere-specific probe was also added and incubated. (4) Detection of Probe Detection was performed with a biotinylated anti-digoxigenin antibody, Texas Red Avidin (FIG. 3). (5) Identification of gene locus It was found that the signal of the P1 DNA probe was present on the long arm of human chromosome 17, and was located at 17q25 which was 88% apart from the distance from the centromere to the end.

Example 3 Analysis of Human RecQ5-Type Gene by Northern Blot (1) Northern Blot Analysis of Human RecQ5-Type Gene (1-i) Human Multiple Tissue Northern
rn; MTN-1 and -11) Blot In this example, a commercially available pre-made filter (Cl
ontech) was used to perform the MTN blot. In addition,
The pre-made filter was prepared by subjecting 2 μg each of poly (A) + RNA extracted from 16 types of human tissues and organs to agarose electrophoresis and blotting it on a nylon membrane.

(1-ii) Human RecQ5 cDNA Probe An open reading frame (residues at positions 1068 to 2175 of the base sequence represented by SEQ ID NO: 1, 1108 bp) which is a part of human RecQ5 cDNA was prepared according to the method described below. It was radioactively labeled and used as a detection probe for the human RecQ5 gene.

(2) Hybridization (2-i) Prehybridization The filter was immersed in 100 ml of prehybridization buffer in a lunch box-shaped plastic container and incubated at 42 ° C. for 4 hours. Prehybridization buffer includes 50% formamide, 5xSSPE, 10xDen
Hardt's solution, 2% SDS and 100μg / ml modified salmon sperm DNA
Those containing fragments were used.

(2-ii) Radioactive Labeling of Human RecQ5 cDNA Probe As a template, 50 ng of the aforementioned human RecQ5 cDNA fragment,
Using 50 pmol of ransom hexamer as a primer,
Random Primer DNA Labeling Kit Ver.2 (manufactured by Takara ^{Shuzo) [α- 32 p] -dCTP (} NEN Corp., first manufactured by Daiichi Pure Chemicals Co., Ltd.) were radioactively labeled with.

(2-iii) Hybridization The prehybridization buffer was discarded, a new 50 ml prehybridization buffer was added, and the mixture was gently shaken so that air bubbles did not enter under the filter. To this, a probe labeled with [ ³² P] -dCTP was added so that the specific activity became about 1 × 10 ⁶ cpm / ml.
Hybridization was performed for hours.

After hybridization, the filter was washed with 100 ml of a 0.2 × SSC, 0.1% SDS solution at room temperature.
Repeat the 15 minute rinse twice, then 100 ml of 0.2xSSC, 0.
Rinsing was performed twice at room temperature for 15 minutes using a 1% SDS solution.
The water was removed until the filter became slightly moist, and autoradiography analysis was performed using a BAS 1500 system (manufactured by FUJIFILM Corporation) with Saran Wrap (manufactured by Asahi Kasei Corporation).

(3) Analysis by Fuji BAS 1500 System A sample was contact-exposed to a radiation energy memory type two-dimensional sensor (imaging plate; IP) using a stimulable phosphor like an X-ray film, and this IP was Excitation with -Ne laser light and PSL
(Photo Stimulated Luminescence) was converted to a digital quantity and quantified. The quantification used the BAS 1500 system.
The linearity of the quantitative value by this system is good, the background is reduced, the radiation intensity in the designated area is measured,
You can make comparisons.

(4) Results of tissue-specific expression of human RecQ5 mRNA MTN containing 2 μg poly (A) + RNA derived from each human tissue / organ
[ ³² P] -labeled human RecQ5 was added to the blot (CLONTECH).
Hybridization was carried out using ORF (1068 to 2175th base sequence: 1108 bp of the nucleotide sequence represented by SEQ ID NO: 1) which is a part of cDNA as a probe.

The expression of human RecQ5 mRNA was widely observed in various organs, and showed a pattern as shown in FIG. That is, the human RecQ5 gene is expressed in all tissues,
In particular, it was found that the expression was extremely strong in the pancreas and testis. In FIG. 5, the origin of poly (A) + RNA is as follows. a, heart; b, brain; c, placenta; d, lung; e, liver; f, skeletal muscle; g, kidney; h, pancreas; i, spleen; j, thymus;
Prostate; l, testis; m, ovary; n, small intestine; o, large intestine;
p, peripheral blood lymphocytes

[0100]

According to the present invention, a human RecQ5-type gene encoding helicase is provided. The human RecQ5-type gene of the present invention is useful for research to elucidate the relationship between homeostasis maintenance and cell aging, elucidating the etiology of diseases that develop with development and aging, and improving and alleviating symptoms. As a therapeutic drug aiming at aging, as a diagnostic probe for testing / prevention of diseases related to other diseases associated with aging, or as a medical, cell biology, immunology It is useful as a reagent for chemical, biochemical and molecular biological studies.

[0101]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3707 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA origin Organism name: Homo sapiens Sequence characteristics Characteristic symbol: CDS Location : 161.1393 Other information: human RecQ5 DNA helicase Sequence: ACGGATATAA GATTGCGTGG GTTCTGCCTA AAGCTGAATT CCCAGCGCTT TGGCTTCTCT 60 GAGTTGGGGT TGTGTATAGG GGTCTTCGAA CAGTTCCGGA ACCAGCCAGC AGCCTTTCAT GCTCTCGCTCGCTC ACTCTGGCTC GCTC ACTGCGTCCGTC ACTCTGGCTC ATC TTT GAC CCT GAG CGG CGA GTC CGG AGT ACG CTG AAG 223 Thr Thr Phe Pro Phe Asp Pro Glu Arg Arg Val Arg Ser Thr Leu Lys 10 15 20 AAG GTC TTT GGG TTT GAC TCT TTT AAG ACG CCT TTA CAG GAG AGT GCG 271 Lys Val Phe Gly Phe Asp Ser Phe Lys Thr Pro Leu Gln Glu Ser Ala 25 30 35 ACC ATG GCT GTA GTA AAA GGT AAC AAG GAC GTC TTT GTG TGC ATG CCC 319 Thr Met Ala Val Val Lys Gly Asn Lys Asp Val Phe Val Cys Met Pro 40 45 50 ACA GGG GCA GGA AAA TCC CTA TGC TAT CAG CTC CCT GCT CTG TTG GCC 367 Thr Gly Ala Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Leu Leu Ala 55 60 65 AAA GGC ATC ACC ATT GTA GTC TCT CCT CTC ATT GCT TTG ATT CAG GAC 415 Lys Gly Ile Thr Ile Val Val Ser Pro Leu Ile Ala Leu Ile Gln Asp 70 75 80 85 CAA GTG GAC CAC TTG CTA ACC CTA AAG GTA CGA GTA AGT TCC CTG AAC 463 Gln Val Asp His Leu Leu Thr Leu Lys Val Arg Val Ser Ser Leu Asn 90 95 100 TCG AAG CTC TCT GCA CAG GAA AGG AAG GAG CTG CTT GCT GAC CTG GAG 511 Ser Lys Leu Ser Ala Gln Glu Arg Lys Glu Leu Leu Ala Asp Leu Glu 105 110 115 CGA GAA AAG CCC CAG ACC AAG ATT CTG TAC ATC ACC CCA GAG ATG GCA 559 Arg Glu Lys Pro Gln Thr Lys Ile Leu Tyr Ile Thr Pro Glu Met Ala 120 125 130 GCT TCA TCC TCC TTC CAG CCC ACC CTG AAC TCC CTG GTG TCC CGC CAC 607 Ala Ser Ser Ser Phe Gln Pro Thr Leu Asn Ser Leu Val Ser Arg His 135 140 145 CTG CTG TCT TAC TTG GTG GTG GAT GAA GCT CAT TGT GTT TCC CAA TGG 655 Leu Leu Ser Tyr Leu Val Val Asp Glu Ala His Cys Val Ser Gln Trp 150 155 160 165 GGG CAT GAC TTT CGT CCT GAC TAC TTG CGT CTG GGT GCC CTG CGC TCC 703 Gly His Asp Phe Arg Pro Asp Tyr Leu Arg Leu Gly Ala Leu Arg Ser 170 175 180 CGC CTG GGA CAT GCC CCT TGT GTG GCT CTG ACC GCC ACA GCC ACC CCA 751 Arg Leu Gly His Ala Pro Cys Val Ala Leu Thr Ala Thr Ala Thr Pro 185 190 195 CAG GTC CAA GAG GAC GTG TTT GCT GCC CTG CAC CTG AAG AAA CCA GTT 799 Gln Val Gln Glu Asp Val Phe Ala Ala Leu His Leu Lys Lys Pro Val 200 205 210 GCC ATC TTC AAG ACT CCC TGC TTC CGG GCC AAC CTC TTC TAT GAT GTG 847 Ala Ile Phe Lys Thr Pro Cys Phe Arg Ala Asn Leu Phe Tyr Asp Val 215 220 225 CAA TTC AAG GAA CTG ATT TCT GAT CCC TAT GGG AAC CTG AAG GAC TTC 895 Gln Phe Lys Glu Leu Ile Ser Asp Pro Tyr Gly Asn Leu Lys Asp Phe 230 235 240 245 TGC CTT AAG GCT CTT GGA CAG GAG GCT GAT AAA GGG TTA TCT GGC TGC 943 Cys Leu Lys Ala Leu Gly Gln Glu Ala Asp Lys Gly Leu Ser Gly Cys 250 255 260 GGC ATT GTG TAC TGC AGG ACT AGA GAG GCT TGT GAA CAG CTG GCC ATA 991 Gly Ile Val Tyr Cys Arg Thr Arg Glu Ala Cys Glu Gln Leu Ala Ile 265 270 275 GAG CTC AGC TGC AGG GGT GTGAAC GCC AAG GCT TAC CAT GCA GGG CTG 1039 Glu Leu Ser Cys Arg Gly Val Asn Ala Lys Ala Tyr His Ala Gly Leu 280 285 290 290 AAG GCC TCT GAA AGA ACG CTG GTG CAG AAC GAC TGG ATG GAG GAG AAG 1087 Lys Ala Ser Glu Arg Thr Leu Val Gln Asn Asp Trp Met Glu Glu Lys 295 300 305 GTC CCT GTA ATT GTT GCA ACC ATT AGT TTT GGG ATG GGA GTG GAT AAA 1135 Val Pro Val Ile Val Ala Thr Ile Ser Phe Gly Met Gly Val Asp Lys 310 315 320 325 GCC AAT GTC AGG TTT GTC GCC CAT TGG AAT ATT GCC AAG TCT ATG GCT 1183 Ala Asn Val Arg Phe Val Ala His Trp Asn Ile Ala Lys Ser Met Ala 330 335 340 340 GGG TAC TAC TAC CAG GAG TCT GGC CGG GCT GGC AGG GAT GGG AAG CCT TCC 1231 Gly Tyr Tyr Gln Glu Ser Gly Arg Ala Gly Arg Asp Gly Lys Pro Ser 345 350 355 TGG TGC CGT CTC TAT TAC TCC AGG AAT GAC CGG GAC CAA GTC AGC TTC 1279 Trp Cys Arg Leu Tyr Tyr Ser Arg Asn Asp Arg Asp Gln Val Ser Phe 360 365 370 CTG ATC AGG AAG GAA GTA GCA AAA CTC CAG GAA AAG AGA GGA AAC AAA 1327 Leu Ile Arg Lys Glu Val Ala Lys Leu Gln Glu Lys Arg Gly Asn Lys 375 380 385 GCA TCT G AT AAA GCC ACT ATC ATG GCC TTT GAT GCC CTG GTG ACC TTC 1375 Ala Ser Asp Lys Ala Thr Ile Met Ala Phe Asp Ala Leu Val Thr Phe 390 395 400 405 TGT GAA GAA CTG GGG TAAGTGACTT ATTTTATATG TGGAGCAAAG TGTCAGlugAGlu30 410 ATCATTTACT TCCCCGGCAC GCCCTAGTTA AGCAGCTGAC ATAAGACAGC CCGTAGGCTA 1490 CCAAGGGACA CCTGCCTGCA AAGGCCATTG TGTCGGGCAG TGTGGAAGTC AGGACCTTGC 1550 TCTTCTCTAT TGGAGGCCCC GGGATCTCTC GCAGTGTGGG GATTTGCTCA GTATTCTGAG 1610 TGGTGTCCTT CCCTCCACTC CACCCTCTTC TGGGATGGCT GGCCCCACAA GCCACTCAGT 1670 TCCAAGGGAT GTGACCAGCT CTGAGCCATC GGCTTTGTGG TCATCATGTT CCACCCGAGC 1730 TGGGACTTCT GGCATCACTT GAGTTATCCT CACCCTTATC CAGGACCCAA CAACAGAAGC 1790 CTGTGGCCCC AACTTACCTC GGGGAGTCAT GTGCTTTGAA CTCAACGCAT CTGTTTTACC 1850 TGCATCTGCA GGCGATGGGG CAGGGGCCAC GGGAAGAGTC TGAGGGCTGC TTGGTGTAGT 1910 CAGGTTGTGT CCAGGCATGC GGAGCTGTGA GTGCCTGCAG GAGAGACACC CAGGAGGAGT 1970 TTTTACATTT TGGTCTAAAAAGCTCTTGGA TTCATCTGATCGATCGATCGATCTGATGACTGATCTCGATCGATCGATCTCGATCAGGATCAG AGAGAGGTC 2090 ACAGTCACGA AGCTCATGGT AGACTGAGGC CACTAAACAC CCGTCTCCTG ATTTTCAGTG 2150 GCGTCCTCAT TTGCATACAC CTGGGCCATC TTGGTTTTTG CAAGAAAAAA GCGAGGAAAT 2210 CAGAGAAACT CTTTGGGCTG TGTGTTTTTA TTTCCACCTC CTCCTGTTGA CCAGTGAGTC 2270 GGTGGCTTCT GGACTGAAGC TATTTTCTTC TCCAAACTGC TGTCTCTAAT TTGGCCTCTG 2330 TGGCAAGACC ACGCCCAGCA AAAGTGGTCG GCTGCAGTGC CAGGACCACC CTTGCCTGCA 2390 GCCTTTGCGA CCAGAGCCCT TTTCAAGCAC AATTAATATG GGAGTTGCTA AATTTGGCCT 2450 TCTTAGGCTT CTTGAGCAGA GCTCTTATTT CTGGTGAGGA ATGAAAGGGC CAAGTGTTCC 2510 TGTTCATTGT GAGTCCTGTT TGCTGAAAAA AGCTCTGGGG CTGTCTGAGG CTCTGGAATG 2570 CTCTCTGGAG GGTTAACTCT GCCTGTCCCC AGGGTCTGCT CCGCCTGCCA GGCAAGGGAA 2630 CAGGTCCTCC CAAGGGCCTC TGCCTTCATT TCTCCTGTCA CCTTCCGGGG AGCTGGGACT 2690 TCTTAGTGCA TCAGTGGCCT GGTTGCCAGG GGCTGGGGTC CAGGCATCTG TGGACTCAAG 2750 GAGTCCAGGT GACGGTGTTG CGCTGCCTCT TGAGCAGCCT CGTGCCTGTC TCCTTTGCTA 2810 CATGTGATGA TTCTGAAATC CAGGCAGCAG GCTGGAATAA ACGCTGCTGG TATGTCTGTA 2870 TTGGCTACTG TTCTTTGAAT AAGTGAATGT TTCTTTATTG CATAACTGGC GTGTAGC ACA 2930 AAGAAAAAGC CCCTTCTCCA CCAGATATTT GCTTCCTAAG AATAGTTCTC ATTGGAGGCT 2990 GAGGTCGAAG CAGCCTCCCA CCAGCGATCC CTTGGGGCGG GCACCTGGCA GTGAGGGACA 3050 GAGCTAGTGG GGTGGGCTCA GCAGAAGTGG GTGCATTTCT GCAAAGGAAG CTGTAGGTTT 3110 CTTGTCAGTT TGTTGTGGGG TCTGAGGCAC ATGGGAGTGG GACCAGCAGA AAAGATACTC 3170 AGGTCAGTTA ACTAATGAGG CGGGGCTGTG GCAGCCAGCG GTTTTCCTCT CTTCTCTATC 3230 TGTAGCTACT TTCTCTGGGC TGCAGCCTTG AAAAAGCAGT GTTTGATTAT TCATAACAAT 3290 GAGTGTCTTT TAGTGTCACA GGGAACAGTT ATTAGGTTTG AAGCAACCTC CAGCTTGAGG 3350 CCTCGGCATC TTGAGATGGA AGAGCAGCCT TGTGTTTAGA CCTGGATTGA ATTGGGCCCT 3410 GTTGTTTCCT GGGATCTTGG GGCTGGGGGT GCCCAACAGC CTGGCATCTT CTTGTTTCTC 3470 TGTGTGTTTA CTCATTCATT CATTTATTCA GCTGATAAAT ATTGACTGAT GCCAGCTCTG 3530 TGCCAGACAC ATTGGACATG GGGGATACAC TAGTGGGCAG AAATAGCCCC GTCCCAGAGC 3590 CGGCCATGGT GGCTCATACC TGTTATACCA GCACTTTGGG AGGCTGAAGT GGGAGGATCG 3650 CTTGAGCCTG GGAGTTCAAG ACCAGCCTGG GCAACATGGC AAGACCCTGT CTCTACT 3707

SEQ ID NO: 2 Sequence length: 410 Sequence type: amino acid Topology: linear Sequence type: protein Origin Organism name: Homo sapiens Sequence characteristics Other information: human RecQ5 DNA helicase Sequence: Met Ser Ser His His Thr Thr Phe Pro Phe Asp Pro Glu Arg Arg Val 1 5 10 15 Arg Ser Thr Leu Lys Lys Val Phe Gly Phe Asp Ser Phe Lys Thr Pro 20 25 30 Leu Gln Glu Ser Ala Thr Met Ala Val Val Lys Gly Asn Lys Asp Val 35 40 45 Phe Val Cys Met Pro Thr Gly Ala Gly Lys Ser Leu Cys Tyr Gln Leu 50 55 60 Pro Ala Leu Leu Ala Lys Gly Ile Thr Ile Val Val Ser Pro Leu Ile 65 70 75 80 Ala Leu Ile Gln Asp Gln Val Asp His Leu Leu Thr Leu Lys Val Arg 85 90 95 Val Ser Ser Leu Asn Ser Lys Leu Ser Ala Gln Glu Arg Lys Glu Leu 100 105 110 Leu Ala Asp Leu Glu Arg Glu Lys Pro Gln Thr Lys Ile Leu Tyr Ile 115 120 125 Thr Pro Glu Met Ala Ala Ser Ser Ser Phe Gln Pro Thr Leu Asn Ser 130 135 140 Leu Val Ser Arg His Leu Leu Ser Tyr Leu Val Val Asp Glu Ala His 145 150 1 55 160 Cys Val Ser Gln Trp Gly His Asp Phe Arg Pro Asp Tyr Leu Arg Leu 165 170 175 Gly Ala Leu Arg Ser Arg Leu Gly His Ala Pro Cys Val Ala Leu Thr 180 185 190 Ala Thr Ala Thr Pro Gln Val Gln Glu Asp Val Phe Ala Ala Leu His 195 200 205 Leu Lys Lys Pro Val Ala Ile Phe Lys Thr Pro Cys Phe Arg Ala Asn 210 215 220 Leu Phe Tyr Asp Val Gln Phe Lys Glu Leu Ile Ser Asp Pro Tyr Gly 225 230 235 240 Asn Leu Lys Asp Phe Cys Leu Lys Ala Leu Gly Gln Glu Ala Asp Lys 245 250 255 Gly Leu Ser Gly Cys Gly Ile Val Tyr Cys Arg Thr Arg Glu Ala Cys 260 265 270 Glu Gln Leu Ala Ile Glu Leu Ser Cys Arg Gly Val Asn Ala Lys Ala 275 280 285 Tyr His Ala Gly Leu Lys Ala Ser Glu Arg Thr Leu Val Gln Asn Asp 290 295 300 Trp Met Glu Glu Lys Val Pro Val Ile Val Ala Thr Ile Ser Phe Gly 305 310 315 320 Met Gly Val Asp Lys Ala Asn Val Arg Phe Val Ala His Trp Asn Ile 325 330 335 Ala Lys Ser Met Ala Gly Tyr Tyr Gln Glu Ser Gly Arg Ala Gly Arg 340 345 350 Asp Gly Lys Pro Ser Trp Cys Arg Leu Tyr Tyr Ser Arg Asn Asp Arg 355 360 Three 65 Asp Gln Val Ser Phe Leu Ile Arg Lys Glu Val Ala Lys Leu Gln Glu 370 375 380 380 Lys Arg Gly Asn Lys Ala Ser Asp Lys Ala Thr Ile Met Ala Phe Asp 385 390 395 400 Ala Leu Val Thr Phe Cys Glu Glu Leu Gly 405 410

SEQ ID NO: 3 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ACGACTGGAT GGAGGAGAAG GTCC 24

SEQ ID NO: 4 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CCCAGGTGTA TGCAAATGAG GACG 24

SEQ ID NO: 5 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CGCCAGGGTT TTCCCAGTCA CGAC 24

SEQ ID NO: 6 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TCACACAGGA AACAGCTATG AC22

SEQ ID NO: 7 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TTATCAGCCT CCTGTCCAAG AGCC 24

SEQ ID NO: 8 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CAAGACCACG CCCAGCAAAA GTGG 24

SEQ ID NO: 9 Sequence length: 24 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ATCCC CCATG TCCAATGTGC CTGG 24

SEQ ID NO: 10 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TAGAGAAGAG AGGAAAACCG CTGG 24

SEQ ID NO: 11 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AGGCTCTGGA ATGCTCTCTG GAGG 24

SEQ ID NO: 12 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GATATAAGAT TGCGTGGGTT CTGC 24

SEQ ID NO: 13 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TCTGTGTTCT TGGCTAAGAT GAGC 24

SEQ ID NO: 14 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTCATCCTC CTTCCAGCCC ACCC 24

SEQ ID NO: 15 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: AGGAACTGAT TTCTGATCCC TATG 24

SEQ ID NO: 16 Sequence length: 24 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TCCTGGTGCC GTCTCTATTA CTCC 24

SEQ ID NO: 17 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CCAGAGAGGT CACAGTCACG AAGC 24

SEQ ID NO: 18 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CCATCCTAAT ACGACTCACT ATAGGGC 27

SEQ ID NO: 19 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of sequence: Other nucleic acid (synthetic DNA) Sequence: ACTCACTATA GGGCTCGAGC GGC
23

SEQ ID NO: 20 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GAAGGTCACC AGGGCATCAA AGGC 24

SEQ ID NO: 21 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCATTCCTG GAGTAATAGA GACG 24

SEQ ID NO: 22 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Sequence: GGTGTAGTCA GGTTGTGTCC AGGC 24

SEQ ID NO: 23 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CATCTCATGG AATGATCCTG TCGG 24

SEQ ID NO: 24 Sequence length: 30 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic RNA) Sequence: CGAAUCGUAA CCGUUCGUAC GAGAAUCGCU 30

SEQ ID NO: 25 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CGAATCGTAA CCGTTCGTAC GAG 23

SEQ ID NO: 26 Sequence length: 26 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: ATCGTAACCG TTCGTACGAG AATCGC 26

SEQ ID NO: 27 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CTTACTCGTA CCTTTAGGGT TAGC 24

SEQ ID NO: 28 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTCCACTTGG TCCTGAGTCA AAGC 24

SEQ ID NO: 29 Sequence length: 1108 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Homo sapiens Sequence characteristics Other information : human RecQ5 partial cDNA sequence: ACGACTGGAT GGAGGAGAAG GTCCCTGTAA TTGTTGCAAC CATTAGTTTT GGGATGGGAG 60 TGGATAAAGC CAATGTCAGG TTTGTCGCCC ATTGGAATAT TGCCAAGTCT ATGGCTGGGT 120 ACTACCAGGA GTCTGGCCGG GCTGGCAGGG ATGGGAAGCC TTCCTGGTGC CGTCTCTATT 180 ACTCCAGGAA TGACCGGGAC CAAGTCAGCT TCCTGATCAG GAAGGAAGTA GCAAAACTCC 240 AGGAAAAGAG AGGAAACAAA GCATCTGATA AAGCCACTAT CATGGCCTTT GATGCCCTGG 300 TGACCTTCTG TGAAGAACTG GGGTAAGTGA CTTATTTTAT ATGTGGAGCA AAGTGTCAGT 360 GAGATCATTT ACTTCCCCGG CACGCCCTAG TTAAGCAGCT GACATAAGAC AGCCCGTAGG 420 CTACCAAGGG ACACCTGCCT GCAAAGGCCA TTGTGTCGGG CAGTGTGGAA GTCAGGACCT 480 TGCTCTTCTC TATTGGAGGC CCCGGGATCT CTCGCAGTGT GGGGATTTGC TCAGTATTCT 540 GAGTGGTGTC CTTCCCTCCA CTCCACCCTC TTCTGGGATG GCTGGCCCCA CAAGCCACTC 600 AGTTCCAAGG GATGTGACCA GCTCTGAGC C ATCGGCTTTG TGGTCATCAT GTTCCACCCG 660 AGCTGGGACT TCTGGCATCA CTTGAGTTAT CCTCACCCTT ATCCAGGACC CAACAACAGA 720 AGCCTGTGGC CCCAACTTAC CTCGGGGAGT CATGTGCTTT GAACTCAACG CATCTGTTTT 780 ACCTGCATCT GCAGGCGATG GGGCAGGGGC CACGGGAAGA GTCTGAGGGC TGCTTGGTGT 840 AGTCAGGTTG TGTCCAGGCA TGCGGAGCTG TGAGTGCCTG CAGGAGAGAC ACCCAGGAGG 900 AGTTTTTACA TTTTGGTCTA AAAAGCTCTT GGATTCATCT CATCTCATGG AATGATCCTG 960 TCGGATGACG CTGACGTGAT TGCTTCAGAC TTAGAGGTGA ATAAATTGAG GTCCAGAGAG 1020 GTCACAGTCA CGAAGCTCAT GGTAGACTGA GGCCACTAAA CACCCGTCTC CTGATTTTCA 1080 GTGGCGTCCT CATTTGCATA CACCTGGG 1108

SEQ ID NO: 30 Sequence length: 1183 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Homo sapiens Sequence characteristics Other information : human RecQ5 partial cDNA sequence: GGGGTCTTCG AACAGTTCCG GAACCAGCCA GCAGCCTTTA ATTCTTGGGC GGACCACGGC 60 CGGTTCTGTG TTCTTGGCTA AGATGAGCAG CCACCATACC ACCTTTCCTT TTGACCCTGA 120 GCGGCGAGTC CGGAGTACGC TGAAGAAGGT CTTTGGGTTT GACTCTTTTA AGACGCCTTT 180 ACAGGAGAGT GCGACCATGG CTGTAGTAAA AGGTAACAAG GACGTCTTTG TGTGCATGCC 240 CACAGGGGCA GGAAAATCCC TATGCTATCA GCTCCCTGCT CTGTTGGCCA AAGGCATCAC 300 CATTGTAGTC TCTCCTCTCA TTGCTTTGAT TCAGGACCAA GTGGACCACT TGCTAACCCT 360 AAAGGTACGA GTAAGTTCCC TGAACTCGAA GCTCTCTGCA CAGGAAAGGA AGGAGCTGCT 420 TGCTGACCTG GAGCGAGAAA AGCCCCAGAC CAAGATTCTG TACATCACCC CAGAGATGGC 480 AGCTTCATCC TCCTTCCAGC CCACCCTGAA CTCCCTGGTG TCCCGCCCT TGCTGTTCTGCCTG TGCTGTTCTGCCTG TGCTGTTCTGCCTG TGCTTCTGCCTG T GGGACATGCC CCTTGTGTGG CTCTGACCGC 660 CACAGCCACC CCACAGGTCC AAGAGGACGT GTTTGCTGCC CTGCACCTGA AGAAACCAGT 720 TGCCATCTTC AAGACTCCCT GCTTCCGGGC CAACCTCTTC TATGATGTGC AATTCAAGGA 780 ACTGATTTCT GATCCCTATG GGAACCTGAA GGACTTCTGC CTTAAGGCTC TTGGACAGGA 840 GGCTGATAAA GGGTTATCTG GCTGCGGCAT TGTGTACTGC AGGACTAGAG AGGCTTGTGA 900 ACAGCTGGCC ATAGAGCTCA GCTGCAGGGG TGTGAACGCC AAGGCTTACC ATGCAGGGCT 960 GAAGGCCTCT GAAAGAACGC TGGTGCAGAA CGACTGGATG GAGGAGAAGG TCCCTGTAAT 1020 TGTTGCAACC ATTAGTTTTG GGATGGGAGT GGATAAAGCC AATGTCAGGT TTGTCGCCCA 1080 TTGGAATATT GCCAAGTCTA TGGCTGGGTA CTACCAGGAG TCTGGCCGGG CTGGCAGGGA 1140 TGGGAAGCCT TCCTGGTGCC GTCTCTATTA CTCCAGGAAT GAC 1183

SEQ ID NO: 31 Sequence length: 1700 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Homo sapiens Sequence characteristics Other information : human RecQ5 partial cDNA sequence: CATCTCATGG AATGATCCTG TCGGATGACG CTGACGTGAT TGCTTCAGAC TTAGAGGTGA 60 ATAAATTGAG GTCCAGAGAG GTCACAGTCA CGAAGCTCAT GGTAGACTGA GGCCACTAAA 120 CACCCGTCTC CTGATTTTCA GTGGCGTCCT CATTTGCATA CACCTGGGCC ATCTTGGTTT 180 TTGCAAGAAA AAAGCGAGGA AATCAGAGAA ACTCTTTGGG CTGTGTGTTT TTATTTCCAC 240 CTCCTCCTGT TGACCAGTGA GTCGGTGGCT TCTGGACTGA AGCTATTTTC TTCTCCAAAC 300 TGCTGTCTCT AATTTGGCCT CTGTGGCAAG ACCACGCCCA GCAAAAGTGG TCGGCTGCAG 360 TGCCAGGACC ACCCTTGCCT GCAGCCTTTG CGACCAGAGC CCTTTTCAAG CACAATTAAT 420 ATGGGAGTTG CTAAATTTGG CCTTCTTAGG CTTCTTGAGC AGAGCTCTTA TTTCTGGTGA 480 GGAATGAAAG GGCCAAGTGT TCCTGTTCAT TGTGAGTCCT GTTTGCTGAA AAAAGCTCTG 540 GGGCTGTCTG AGGCTCTGGA ATGCTCTCTG GAGGGTTAAC TCTGCCTGTC CCCAGGGTCT 600 GCTCCGCCTG CCAGGCAAGG GAACAGGTC C TCCCAAGGGC CTCTGCCTTC ATTTCTCCTG 660 TCACCTTCCG GGGAGCTGGG ACTTCTTAGT GCATCAGTGG CCTGGTTGCC AGGGGCTGGG 720 GTCCAGGCAT CTGTGGACTC AAGGAGTCCA GGTGACGGTG TTGCGCTGCC TCTTGAGCAG 780 CCTCGTGCCT GTCTCCTTTG CTACATGTGA TGATTCTGAA ATCCAGGCAG CAGGCTGGAA 840 TAAACGCTGC TGGTATGTCT GTATTGGCTA CTGTTCTTTG AATAAGTGAA TGTTTCTTTA 900 TTGCATAACT GGCGTGTAGC ACAAAGAAAA AGCCCCTTCT CCACCAGATA TTTGCTTCCT 960 AAGAATAGTT CTCATTGGAG GCTGAGGTCG AAGCAGCCTC CCACCAGCGA TCCCTTGGGG 1020 CGGGCACCTG GCAGTGAGGG ACAGAGCTAG TGGGGTGGGC TCAGCAGAAG TGGGTGCATT 1080 TCTGCAAAGG AAGCTGTAGG TTTCTTGTCA GTTTGTTGTG GGGTCTGAGG CACATGGGAG 1140 TGGGACCAGC AGAAAAGATA CTCAGGTCAG TTAACTAATG AGGCGGGGCT GTGGCAGCCA 1200 GCGGTTTTCC TCTCTTCTCT ATCTGTAGCT ACTTTCTCTG GGCTGCAGCC TTGAAAAAGC 1260 AGTGTTTGAT TATTCATAAC AATGAGTGTC TTTTAGTGTC ACAGGGAACA GTTATTAGGT 1320 TTGAAGCAAC CTCCAGCTTG AGGCCTCGGC ATCTTGAGAT GGAAGAGCAG CCTTGTGTTT 1380 AGACCTGGAT TGAATTGGGC CCTGTTGTTT CCTGGGATCT TGGGGCTGGG GGTGCCCAAC 1440 AGCCTGGCAT CTTCTTGTTT CTCTGTGTGT TTACTCATTC ATTCATTTAT TCAGCTGATA 1500 AATATTGACT GATGCCAGCT CTGTGCCAGA CACATTGGAC ATGGGGGATA CACTAGTGGG 1560 CAGAAATAGC CCCGTCCCAG AGCCGGCCAT GGTGGCTCAT ACCTGTTATA CCAGCACTTT 1620 GGGAGGCTGA AGTGGGAGGA TCGCTTGAGC CTGGGAGTTCAGAGCTGCAGCTAGAGCTGCAGCTGCAGCTGCAGCTGCAGCTGCAGCTGCAGGG

SEQ ID NO: 32 Sequence length: 424 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Homo sapiens Sequence characteristics Other information : human RecQ5 partial cDNA sequence: ACGGATATAA GATTGCGTGG GTTCTGCCTA AAGCTGAATT CCCAGCGCTT TGGCTTCTCT 60 GAGTTGGGGT TGTGTATAGG GGTCTTCGAA CAGTTCCGGA ACCAGCCAGC AGCCTTTAAT 120 TCTTGGGCGG ACCACGGCCG GTTCTGTGTT CTTGGCTAAG ATGAGCAGCC ACCATACCAC 180 CTTTCCTTTT GACCCTGAGC GGCGAGTCCG GAGTACGCTG AAGAAGGTCT TTGGGTTTGA 240 CTCTTTTAAG ACGCCTTTAC AGGAGAGTGC GACCATGGCT GTAGTAAAAG GTAACAAGGA 300 CGTCTTTGTG TGCATGCCCA CAGGGGCAGG AAAATCCCTA TGCTATCAGC TCCCTGCTCT 360 GTTGGCCAAA GGCATCACCA TTGTAGTCTC TCCTCTCATT GCTTTGATTC AGGACCAAGT 420 GGAC 424

SEQ ID NO: 33 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TTGGTGTAGT CAGGTTGTGT CCAG 24

SEQ ID NO: 34 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GACGCCACTG AAAATCAGGA GACG 24

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for cloning a gene of the present invention.

FIG. 2 is a diagram comparing amino acid sequences of proteins encoded by the gene of the present invention with those derived from other genes.

FIG. 3 is a photograph showing the result of fluorescence in situ hybridization (organism form).

FIG. 4 is a diagram showing human chromosome 17;

FIG. 5 is an electrophoretic photograph showing the results of Northern blot analysis of the gene of the present invention.

FIG. 6 is a diagram comparing the amino acid sequence encoded by EST-DNA ACC.AA155882 with that derived from Escherichia coli.

FIG. 7 shows the assembly of the gene of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 21/02 C12P 21/08 21/08 C12Q 1/68 A C12Q 1/68 G01N 33/50 T G01N 33/50 33/53 D 33/53 33/577 B 33/577 C12N 5/00 B // (C12N 15/09 ZNA C12R 1:91) (C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (16)

[Claims] 1. A gene encoding the following protein (a) or (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having helicase activity protein 2. A gene comprising the following DNA (a) or (b): (A) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1 (b) DNA which hybridizes with the DNA comprising the nucleotide sequence of (a) under stringent conditions and encodes a protein having helicase activity 3. A mouse or rat-derived gene comprising an amino acid sequence having at least 70% homology with the amino acid sequence of the following protein (a) or (b), and encoding a protein having helicase activity: (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having helicase activity protein An oligonucleotide probe capable of hybridizing with at least a part of the gene according to any one of claims 1 to 3. A recombinant vector containing the gene according to any one of claims 1 to 3. A transformant comprising the recombinant vector according to claim 5. 7. A method for producing a protein, comprising culturing the transformant according to claim 6, and collecting a protein having helicase activity from the resulting culture. 8. A recombinant protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid is deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and having helicase activity protein 9. A mouse or rat protein comprising an amino acid sequence having at least 70% homology with the amino acid sequence of the protein according to claim 8, and having helicase activity. 10. A monoclonal antibody against the protein according to claim 8 or 9. 11. A polyclonal antibody against the protein according to claim 8 or 9. 12. The monoclonal antibody-producing hybridoma according to claim 10, which can be obtained by fusion of an antibody-producing cell immunized with the protein according to claim 8 or 9 with a myeloma cell. A reagent for detecting a gene encoding helicase, comprising the oligonucleotide probe according to claim 4. 14. A method for diagnosing a disease caused by an abnormality in a gene encoding helicase, comprising the protein according to claim 8 or 9 and the monoclonal antibody according to claim 10 and / or the polyclonal antibody according to claim 11. kit. 15. A transgenic animal into which a gene modified to increase or decrease the expression level of the gene according to any one of claims 1 to 3 has been introduced. 16. A knockout mouse treated so that the function of the gene according to any one of claims 1 to 3 is lost.