JP2000228988A - Dna helicase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA helicase which has a specific amino acid sequence, has a DNA helicase activity, has homology to a part of TIP49, and is useful for diagnosing and treating genetic diseases which are caused by the abnormality of helicase and so on. SOLUTION: This is a new DNA helicase which has the amino acid sequence shown by the formula or an amino acid sequence prepared by deleting, substituting, adding, or inserting one or more amino acid(s) from, in, to, or into the amino acid sequence, has a DNA helicase activity, has homology to a part of TIP49, and is useful for diagnosing and treating genetic diseases which are caused by the abnormality of helicase and so on. This DNA helicase is obtained by designing PCR primers from the base sequence coding for the amino acid sequence having homology to the amino acid sequence encoded by TIP49 gene among sequences registered in EST database, carrying out PCR of a human liver cDNA library using the obtained primers, followed by expressing the obtained gene using an expression system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protein having DNA helicase activity, a polynucleotide encoding the protein, an antisense polynucleotide against the polynucleotide, and an antibody recognizing the protein.

[0002]

2. Description of the Related Art As a central factor in gene transcription, TBP (TATA binding protein) is used.
n) is known to be involved in the transcription of all genes of an organism. Specifically, transcription of a gene is controlled by binding of TBP to the promoter region of the DNA when translation from DNA to mRNA is performed. T
When BP forms a complex with various other proteins, depending on the structure of the partner protein or complex, the complex will have a function corresponding to the different transcriptional regulation of each gene. It is considered.

[0003] The present inventors have isolated and identified a TIP49 protein as a protein that binds to TBP and a gene encoding the same (Biochem. Biophys. Re.
s. Commun. , 235, 64-68).

[0004]

SUMMARY OF THE INVENTION The present invention provides a TIP49
An object of the present invention is to provide a novel protein having homology to a part of the protein. Another object of the present invention is to provide a polynucleotide encoding the protein. Another object of the present invention is to provide an antibody that recognizes the protein.

[0005]

The present inventor has proposed a TIP49.
CDNA encoding a protein having homology to a portion of the cDNA was isolated and its nucleotide sequence was determined. And the cDNA
The amino acid sequence encoded by was determined. Furthermore, the present inventor produced a recombinant protein encoded by this cDNA and confirmed that the molecular weight of the recombinant protein was 50 kD. The present inventors have named this protein HEL50. Furthermore, the present inventors have confirmed that HEL50 has DNA helicase activity. When the animal from which HEL50 is derived is specified, for example, human HEL50
And the animal name derived from before HEL50. When expressed as HEL50, it means HEL50 derived from an eukaryote.

[0006] The present invention also discloses an antibody that recognizes HEL50.

[0007] Specifically, the present invention provides the following proteins. 1. A protein comprising the amino acid sequence of SEQ ID NO: 1 in the sequence listing. This protein has DNA helicase activity.
This protein is human HEL50. 2. A protein comprising the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids are substituted, deleted or added, and having DNA helicase activity. Hereinafter, this protein is referred to as a human HEL50 mutant. A protein consisting of a part of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing and having DNA helicase activity is one embodiment of a human HEL50 mutant. 3. The amino acid sequence of SEQ ID NO: 1 in the sequence listing and 65%
A protein having the above homology and having DNA helicase activity. This protein is eukaryotic HEL50. 4. A protein comprising the amino acid sequence of SEQ ID NO: 12 in the sequence listing. This protein is yeast HEL50, which is one embodiment of the eukaryotic HEL50 described in the above item 4.

[0008] The present invention also provides the following polynucleotides. In the present specification, the term "polynucleotide" refers to DNA or RNA, or those in which a plurality of nucleotides consisting of bases, phosphates, and sugars are bonded thereto, including those that do not exist in nature. 5. A polynucleotide encoding the protein according to any one of the above items 1 to 4. 6. The first A of the base sequence described in SEQ ID NO: 2 in the sequence listing
DNA having a nucleotide sequence from 1 to 1389th C.
The base sequence from the 1st A to the 1389th C of the base sequence described in SEQ ID NO: 2 in the sequence listing is the base sequence of the coding region of human HEL50.
NA is hereinafter referred to as human HEL50 DNA. In addition,
Although cDNA is included in DNA, in the present specification, the D
When it is specifically indicated that NA is cDNA, c
Notated as DNA. 7. DN consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing
A. This DNA is an embodiment of the DNA described in the above item 6. 8. D consisting of the nucleotide sequence of SEQ ID NO: 12 in the sequence listing
NA. This DNA is yeast HEL50 DNA. 9. D according to any one of the above items 6 to 8,
RNA encoded by NA. Hereinafter, human HEL50 DNA
Is referred to as human HEL50 RNA. In addition, although mRNA is contained in RNA, in this specification, when it is specifically indicated that the RNA is mRNA, it is described as mRNA. 10. 10. The antisense DNA of the DNA according to any one of the above items 6 to 8, the RN according to the above item 9.
An antisense polynucleotide comprising the antisense RNA of A or a derivative thereof. The antisense polynucleotide is included in the polynucleotide, but in this specification, when it is particularly specified that the polynucleotide is an antisense strand, it is referred to as an antisense polynucleotide. 11. 11. A part of the polynucleotide according to any one of the above items 5 to 10, wherein
A polynucleotide consisting of bases or more. 12. 12. The polynucleotide according to any one of the above items 5 to 11, which is chemically modified.

[0009] The present invention also provides an antibody and an active fragment of a monoclonal antibody that recognize the protein described in any one of the above items 1 to 3. As used herein, antibodies include all antisera, polyclonal or monoclonal antibodies, as well as active fragments of the antibodies.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS HEL50 and HEL of the present invention
Fifty mutants are characterized by having DNA helicase activity, which can be confirmed using, for example, the method disclosed in Example 4. HEL50 variants can be made using known methods. For example, HEL50
A DNA encoding the mutant is prepared, the DNA is incorporated into an appropriate host, and HEL50 can be expressed in the resulting transformant. The DNA encoding the HEL50 mutant can be obtained, for example, by using a restriction enzyme to construct the HEL50 mutant.
It is obtained by deleting or substituting a base at a desired site in DNA with another base, or inserting a base at a desired site. HEL50 mutants can also be prepared by the method of site-directed mutagenesis. Also,
Random mutagenesis can also be caused by PCR with relaxed hybridization conditions.

The DNA or RNA encoding HEL50 or HEL50 mutant of the present invention comprises all base sequences that can exist due to degeneracy.

[0012] For the sense strand or RNA of DNA, there are antisense DNA and antisense RNA each having a base sequence complementary to the base sequence. In the present specification, unless otherwise specified, DNA (cD
NA includes a sense strand and an antisense strand, and RNA refers to a single strand.
Antisense DNA or antisense RNA refers to those consisting of a single strand. The present invention includes all derivatives of the antisense polynucleotide. Derivatives are, for example, those obtained by substituting or deleting at least one part of the base, sugar, or phosphate of the antisense polynucleotide with another substance bound to the 3 ′ end or 5 ′ end of the antisense polynucleotide. It is a substance having a modification such as addition, or a substance having a base, sugar, or phosphate that does not exist in nature, or a substance having a skeleton other than the sugar-phosphate skeleton. The antisense polynucleotide or derivative thereof of the present invention may hybridize to any part of the coding region of the polynucleotide encoding HEL50. Those which have a base sequence complementary to a part of mRNA and which hybridize to the mRNA are preferable.

[0013] The antisense polynucleotide can be prepared by any of the following steps: transcription of a gene to pre-mRNA, processing of pre-mRNA to mature mRNA, translocation of mRNA through the nuclear membrane, and translation of mRNA to protein. It is believed that it hybridizes to DNA or RNA, which carries the genetic information, and regulates the expression of the protein by affecting the normal flow of genetic information and inhibiting the biosynthesis of the protein.

In order to facilitate the hybridization of an antisense polynucleotide to RNA, generally,
It is said that an antisense polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the loop region is designed. In addition, an antisense polynucleotide having a sequence complementary to the sequence near the translation initiation codon of RNA, a ribosome binding site, a capping site or a splice site can generally be expected to have a large effect of inhibiting biosynthesis. Therefore, the antisense polynucleotide of the present invention or a derivative thereof, comprising HEL50R
Those having a nucleotide sequence complementary to the nucleotide sequence of the NA loop region, the vicinity of the translation initiation codon, the ribosome binding site, the capping site or the splice site are expected to have a large effect of inhibiting biosynthesis.

Antisense polynucleotides inhibit specific genes and RNAs in inhibiting biosynthesis of specific proteins.
Are preferred. Therefore, 12
Those composed of the above bases are preferred, and those composed of the bases of 16 or more are particularly preferred. On the other hand, those composed of 35 or less bases are preferable in terms of cell membrane permeability.

[0016] The antisense polynucleotide derivative is
It is preferred that the derivative has at least one of nuclease resistance, tissue selectivity, cell membrane permeability, or binding strength enhanced. It is particularly preferable that the derivative is a derivative having a phosphorothioate bond as a skeleton structure.
The antisense polynucleotide derivative of the present invention also includes derivatives having these functions or structures.

As a method for producing the antisense polynucleotide and its derivative of the present invention, for example, natural DNA or RNA can be synthesized using a chemical synthesizer, or PCR can be performed using HEL50 cDNA as a template. It is mentioned. Some derivatives such as a methylphosphonate type and a phosphorothioate type can be synthesized using a chemical synthesizer (for example, Model 394 manufactured by ABI). In this case, the desired antisense polynucleotide can be obtained by performing the operation according to the instructions attached to the chemical synthesizer and purifying the obtained synthetic product by HPLC using reverse phase chromatography or the like. Alternatively, a derivative thereof can be obtained.

The full length or a part of the polynucleotide or antisense polynucleotide of the present invention is HEL5
0 gene, HEL50 cDNA or HEL50mR
It can be used as a probe for detecting NA or a primer for PCR. Now, it is said that there are 3 × 10 ⁹ human proteins. 16 because bases of DNA 4 ¹⁶ types are present, can identify all proteins of human if any DNA of this length. That is, the length required as a probe or primer is theoretically 16 bases. Needless to say, it is desirable that the length be longer than this length in practical use, but in practice, it is often used with 12 bases or more. In addition, a non-coding region or a coding region can be used as a portion used as a probe or a primer. Those having a GC content of 30 to 70% are preferable because they do not easily cause a problem of the three-dimensional structure of the polynucleotide and easily hybridize.

Specific examples of the method for detecting the HEL50 gene include Northern blot hybridization and RT-PCR (“Current Protocol”).
ols in Molecular Biology "
(Greeue Publishing Associate
ates and Wiley-Juter Scie
nce) Chapter 15.1.1-15.1.9 and ibid. 15.4.1-15.4.6) or in situ hybridization (ibid.
4.3.1-14.3.14). The optimum conditions for hybridization differ depending on the length of the probe and the membrane used. That is, the hybridization conditions naturally have a certain width. Embodiments of the present invention disclose the optimum conditions for the properties of the membrane used and the length of the probe. If the length of the membrane or the probe is different, the hybridization can naturally be performed under different hybridization conditions.

When chemically synthesizing DNA or RNA, chemical modification such as labeling, biotinylation, methylation of the side chain, or substitution of O for S in the phosphate group is often performed. Are known. For example, when chemically synthesizing the DNA described in SEQ ID NO: 2 in the sequence listing, it is possible to synthesize the DNA different from the DNA itself shown in the sequence listing by performing the above chemical modification. Further, even a cDNA obtained from a cDNA library can be labeled with a radioisotope. Therefore, the DNA and RNA of the present invention include the above-mentioned chemically modified DNA, RNA or antisense polynucleotide in its range. The chemically modified DNA or RNA of the present invention can exhibit both a protein-encoding function and a probe function, and the chemically-modified antisense polynucleotide of the present invention inhibits protein biosynthesis. Function or a function as a probe.

The present invention relates to an antibody obtained by immunizing a non-human animal with HEL50, wherein the antibody recognizes HEL50 of the present invention.
ELISA method and immunostaining method (for example, measurement by FACS)
Etc. are included in the range. The animal to be immunized is preferably selected from animals other than the animal from which HEL50 to be administered as the immunogen is derived and other than human. For example, in the case of rat HEL50, it is preferable to immunize animals other than rats and other than humans (for example, rabbits and mice).

It is a commonly used method to use an immunogen obtained by binding a part of a protein to another carrier protein such as bovine serum albumin.
A portion of the protein may be synthesized, for example, using a peptide synthesizer. Preferably, a part of the protein is composed of eight or more amino acid residues. If an antiserum or polyclonal antibody can be obtained by purifying immunoglobulin from blood collected from an immunized animal, monoclonal antibodies can be obtained by hybridoma obtained by cell fusion of lymphocytes of the immunized animal with myeloma cells and the like. It is well known that is produced ("Antibod
ies A Laboratory Manual "
(Cold Spring Harbor Labor
atry Press, 1988), 141-14.
7).

The antibodies of the present invention also include active fragments. The active fragment means a fragment of an antibody having antigen-antibody reaction activity, and specifically, F (a
b ') ₂ , Fab', Fab, Fv and the like. For example, when the antibody of the present invention is digested with pepsin, F (ab ′) ₂ is obtained, and when digested with papain, Fa (ab ′) ₂ is obtained.
b is obtained. Reduction of F (ab ') ₂ with a reagent such as 2-mercaptoethanol and alkylation with monoiodoacetic acid gives Fab'. Fv is a monovalent antibody active fragment in which a heavy chain variable region and a light chain variable region are linked by a linker. A chimeric antibody can be obtained by retaining these active fragments and substituting other portions with fragments of another animal.

The detection of HEL50 includes a method using an antibody and a method using an antibody and an enzyme reaction. As a method using an antibody, specifically, HEL5
A method for detecting HEL50 using an antibody that recognizes
A method of detecting HEL50 using an antibody that recognizes HEL50 and a labeled secondary antibody of the antibody is used. As the label, for example, a radioisotope (RI), an enzyme, avidin or biotin, or a fluorescent substance (FITC, rhodamine, or the like) is used.

As a method utilizing an antibody and an enzymatic reaction, for example, ELISA is mentioned.

[0026]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. <Example 1> Isolation of HEL50 cDNA and HEL5
Determination of the amino acid sequence of I.I. EST database search Internet EST database (database in which sequences of mRNA fragments are registered, http: // w
ww. ncbi. nlm. nih. gov. / DbES
From the sequence (EST) registered in (T), a nucleotide sequence encoding an amino acid sequence having homology to the amino acid sequence encoded by the TIP49 gene was converted to TBLASTN (http
p: // www. blast. genome. ad. j
Searched from dbEST using p). as a result,
EST and AA registered with R19091 number
The EST registered with the number 374580 was extracted.

II. Isolation of HEL50 DNA and determination of base sequence Synthesis of Primers The following primers P1 and AA374580 were obtained from the following anchor primers and the base sequence of R19091.
The following primer P2 was prepared from the base sequence using a DNA synthesizer (ABI, Model 392). Anchor primer: 5'-CTGGTTCGGC C
CA-3 ′ (base sequence described in SEQ ID NO: 3 in Sequence Listing) P1: 5′-GAGATCCGTG ATGTAACA
AG GATTGAG-3 ′ (base sequence described in SEQ ID NO: 4 in Sequence Listing) P2: 5′-CTTGGTCTGG GAGCCAT
AG CGTCG-3 ′ (base sequence described in SEQ ID NO: 5 in Sequence Listing) The synthesized primer was adjusted to 20 pmol / μl with distilled water. Using this as a PCR primer, the following PCR operation was performed.

2. PCR was performed under the following conditions using Marathon (registered trademark) human liver cDNA library (manufactured by Clontech) as the PCR cDNA library. First, use the anchor primer and the P2 primer as primers,
The test was performed under the following conditions using Takara Taq (registered trademark, manufactured by Takara Shuzo). Placed at 94 ° C. for 1 minute. Then, a cycle of “94 ° C. for 45 seconds, followed by 60 ° C. for 2 minutes, and then 72 ° C. for 2 minutes” was repeated 30 times. Thereafter, the PCR operation was completed by placing at 4 ° C. The composition of the PCR reaction solution was as follows. 0.5 ng / μl HEL50 1 μl Taq pol (Takara Shuzo) 0.1 μl 10 times concentration PCR buffer (Takara Shuzo) 2 μl dNTP mix (Takara Shuzo) 1.6 μl Anchor primer 2 μl Primer P2 2 μl Sterile distilled water 3μl Total 20μl

Thereafter, a second PCR operation was performed using the PCR product obtained above as a template and P1 and P2 primers. The second PCR operation was performed as described above.

3. Preparation of DNA Probe The DNA fragment (hereinafter, sometimes referred to as fragment A) amplified by the PCR operation is subjected to minigel electrophoresis (0.
75% agarose gel) and the fragment A band was excised from the gel. Fragment A was collected using GeneClean (manufactured by Bio 101), and bands were checked by minigel electrophoresis.

The fragment A recovered as described above was converted into pGE
Plasmids were inserted into the plasmid vector pGEM-T Easy using MT Easy (promega). Thereafter, pGEM-T Easy containing the fragment A was introduced into Escherichia coli, the Escherichia coli was cultured, and plasmid DNA, that is, fragment A was prepared from the white colonies of Escherichia coli using the Midi kit (manufactured by Qiagen). This fragment A is subjected to an auto-sequencer (ABI, 373A type) by a dye terminator method.
Was used to perform DNA sequencing, and the nucleotide sequence was confirmed.

Fragment A prepared from Escherichia coli having formed the white colony was cut out with EcoRI, and the obtained DNA fragment was mixed with a random labeling kit (Boehringer Mannheim) in a solution having the following composition.
After 30 minutes at 65 ° C, labeling was carried out at 65 ° C for 10 minutes. Fragment A 9 μl dA, G, TTP mix (Boehringer Mannheim) 3 μl Reaction solution mix (Boehringer Mannheim) 2 μl d- ³² P-dCTP (Pharmacia Amersham) 5 μl Klenow solution (Boehringer Mannheim) 1 μl Total 1 μl From this solution, labeled fragment A was purified using a G-50 micro spin column. 3 columns with swing rotor
After rotating at 000 rpm for 1 minute, packing was performed.
20 μl of the labeled fragment A solution was added to the column,
The column was rotated at 3000 rpm for 2 minutes and eluted with T50E to separate labeled fragment A.

4. Hybridization This DNA probe is hybridized to a human liver cDNA library λgt10 (manufactured by Clontech), and a cDNA hybridizing to the DNA probe is hybridized.
Was cloned. Hybridization conditions were as follows. Prehybridization 6 times SSC 0.05 times BLOTTO (2.5% nonfat dry milk, 0.01
0.1% NP40 100 µg / ml Denatured salmon sperm DNA total volume 50 ml Reaction temperature 60 ° C Reaction time 1 hour Hybridization 6 times SSC 0.05 times BLOTTO 0.1% NP40 100 µg / ml denatured salmon sperm DNA 1 × 10 ⁶ cpm / ml DNA probe Total volume 50 ml Reaction temperature 60 ° C. Reaction time 12 hours

The nitrocellulose membrane after completion of the hybridization was washed under the following conditions. Wash twice with a washing solution of 2 × SSC and 0.1% SDS for 5 minutes at room temperature. One time with a washing solution of 0.2% SSC and 0.1% SDS at room temperature.
X-ray film (manufactured by Fuji Photo Film Co., Ltd.) was exposed to the washed nitrocellulose membrane at -80 ° C. overnight to prepare an autoradiograph. From the obtained autoradiograph, the position of a positive plaque was determined. "The phage is collected from the corresponding plaque, suspended in the SM solution, then the phage is recovered from the SM solution, plaque formation is again performed on the NZY agar medium by a conventional method, and immobilized on the nitrocellulose membrane." Was repeated three times. The result was a single positive plaque. Phage was recovered from the plaque and suspended in 100 μl of SM solution to stabilize the phage.

5. Determination of Nucleotide Sequence cDNA was cut out from the phage with EcoRI, and the obtained cDNA was inserted into pBluescript SK + vector (Stratagene) and cloned. The DNA obtained as a result of the cloning was subjected to DNA sequencing by a dye terminator method using an auto sequencer (manufactured by ABI, 373A and 310), and its nucleotide sequence was determined. as a result,
The cDNA was found to contain the entire coding region.
That is, full-length HEL50 cDNA was isolated and its nucleotide sequence was determined. The nucleotide sequence of HEL50 cDNA is shown in SEQ ID NO: 2 in the sequence listing. HEL50 cDNA
Is inserted into the pBluescript SK + vector
Named EL50SK +.

6. Determination of amino acid sequence of HEL50 From the nucleotide sequence determined in the above step 5, HEL50cD
The amino acid sequence of HEL50, a protein encoded by NA, was determined. The results are shown in SEQ ID NO: 1 in the sequence listing. In addition, the portion from Gly at position 77 to Thr at position 84 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing and 29
The part from the 9th Asp to the 302nd His is
It is a site of a sequence characteristic of ATPase and DNA helicase.

<Example 2> Northern blotting Preparation of probe HEL50cDN having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing
The part of A from the 387th T to the 796th G is P
It was amplified by CR and labeled to use as a Northern blotting probe. The operation is shown below. (1) Synthesis of primers From the nucleotide sequence of HEL50, the following primers P3 and P4 were prepared using a DNA synthesizer (392, manufactured by ABI).
Mold). P3: 5'-TCGCATCAAG GAGGAGAC
-3 '(base sequence described in SEQ ID NO: 6 in Sequence Listing) P4: 5'-CTGAGAAGAG CGCCAGGG-
3 ′ (base sequence described in SEQ ID NO: 7 in Sequence Listing) The synthesized primer was prepared at 20 pmol / μl with distilled water. Using this as a PCR primer, the following PCR operation was performed.

(2) Vector HEL50S into which PCR HEL50 cDNA has been introduced
K + was used as a template for PCR, and primers P3 and P4 were used, and Takara Taqpol (registered trademark, manufactured by Takara Shuzo Co., Ltd.) was used under the following conditions.
Placed at 94 ° C. for 1 minute. Then, a cycle of “94 ° C. for 45 seconds, followed by 60 ° C. for 2 minutes, and then 72 ° C. for 2 minutes” was repeated 30 times. Thereafter, the PCR operation was completed by placing at 4 ° C. The composition of the PCR reaction solution was as follows. 0.5 ng / μl HEL50SK + 1 μl Taq pol (Takara Shuzo) 0.1 μl 10-fold concentration PCR buffer (Takara Shuzo) 2 μl dNTP mix (Takara Shuzo) 1.6 μl Primer P3 2 μl Primer P4 2 μl Sterile distilled water 3 μl total 20 μl PCR products were electrophoresed on a 0.7% agarose gel. A PCR product (hereinafter, referred to as fragment B) was cut out from the gel, the fragment B was recovered using GeneClean (manufactured by Bio 101), and bands were checked by minigel electrophoresis. As a result, D of fragment B
The concentration of the NA solution was estimated to be 25 ng / μl.

(3) Labeling 4 μl of sterile distilled water was added to 5 μl of the DNA solution of fragment B, and the mixture was kept at 98 ° C. for 10 minutes. Thereafter, the mixture was cooled on ice to denature the fragment B. Using a random labeling kit (manufactured by Boehringer Mannheim), the denatured fragment B was placed in a solution having the following composition at 37 ° C.
After leaving for 0 minutes, labeling was carried out at 65 ° C. for 10 minutes. Modified fragment B 9μl dA, G, TTP mix ( Boehringer Mannheim) 3 [mu] l reaction mix (Boehringer Mannheim) 2μl d- ³² P-dCTP (Pharmacia Amersham) 5 [mu] l Klenow solution (Boehringer Mannheim) 1 [mu] l Total 20 μl Labeled fragment B was purified from this solution using a G-50 micro spin column. 3 columns with swing rotor
After rotating at 000 rpm for 1 minute, packing was performed.
20 μl of labeled fragment B solution was added to the column,
The column was spun at 3000 rpm for 2 minutes and eluted with T50E to separate labeled fragment B.

2. Hybridization (1) Prehybridization Membrane (human MTN (registered trademark) Blot II (manufactured by Clontech), immobilized mRNA, rat MT
N (registered trademark) No. 3 (manufactured by Clontech) and a mouse fetus (manufactured by Clonetech)) were placed in 10 ml of a hybridization solution having the following composition, and incubated at 42 ° C.
Every 50 minutes, the membrane was blocked with denatured salmon sperm DNA. Hybridization solution 5-fold concentration SSPE 10-fold concentration Denhardt's solution 2% SDS, 50% formamide 100 μg / ml denatured salmon sperm DNA

(2) Hybridization 7 μl of the labeled fragment B solution was added to 20 ml of the hybridization solution and mixed well. 5 ml of the hybridization solution is added to the square Petri dish, and the above-mentioned membranes are placed one by one in the Petri dish. A vinyl sheet is placed on each of the membranes, and overnight at about 42 ° C. (about 15 hours).
Hybridized. The hybridization solution was discarded, the washing solution (double concentration SSC, 0.1% SDS) was added to the petri dish, and washing at room temperature for 5 minutes was repeated four times.

(3) Autoradiogram The membrane was exposed to radiation-sensitive paper for 16 hours, and an autoradiograph was taken. The results for the human MTN membrane are shown in FIG. HEL50mR in human tissues
Although NA was expressed, the expression in testis was the highest.
Fragment B hybridized to rat mRNA and mouse mRNA, and the result was similar to that of human for rat. HEL50 mRNA in mouse embryo
Was observed, and the highest expression was observed in the mouse fetus on the 11th day of embryo.

<Embodiment 3> Mass adjustment of HEL50 Large-scale preparation of HEL50 cDNA 1) Preparation of recombinant vector The first methionine portion of HEL50 cDNA was replaced with EcoRI
Modified by PCR so that the sites are adjacent to each other, and p
It was inserted into the EcoRI site of the ET FLAG-His vector. The vector incorporating HEL50 cDNA was introduced into JM109 competent cells (Takara Shuzo) according to the instruction manual, cultured overnight in an LB medium containing 800 ml of ampicillin, and collected from the collected JM109 competent cells using the alkaline method (Molecular method described above).
Cloning Second Edition, 1.
The vector was recovered by the method described on page 33-1.43). Ultracentrifugation for recovery of the vector was performed three times.

2. Expression of HEL50 1) Escherichia coli BL21 pLys
S (manufactured by Novagen). 2) The obtained Escherichia coli was cultured in an LB medium containing 100 μg / ml of ampicillin, and a spectrophotometer (manufactured by Beckman) was used.
When the turbidity at a wavelength of 600 nm becomes 0.6 to 0.7, IPTG is added to be 0.1 mM,
HEL50 expression was induced.

3. Purification of HEL50 1) After 2 hours, Escherichia coli was recovered and lysate 30
Suspended in ml. The obtained suspension was sonicated at 4 ° C., and ultracentrifuged at 50,000 rpm and 4 ° C. for 1 hour using an ultracentrifuge and a 45Ti rotor (both manufactured by Beckman). Lysate composition: 10 mM Tris-HCl pH 7.9 10% glycerol 0.5 M NaCl 0.1% NP40 5 mM 2-mercaptoethanol (ME) 1 mM PMSF

2) Take the obtained supernatant, add 1 M imidazole (pH 7.5) to a final concentration of 1 mM, and add N
Purification was performed using an i-NTA agarose column (Qiagen) under non-denaturing conditions according to the instruction manual. The bed volume of the column was 1 ml. The column was washed with 30 ml of a first washing solution having the following composition, and then washed with 5 ml of a second washing solution having the following composition. First wash solution composition: 20 mM Tris-HCl pH 7.9 10% glycerol 0.1 M KCl 5 mM 2-ME 0.5 mM PMSF 20 mM imidazole Second wash solution composition: 20 mM Tris-HCl pH 7.9 10% glycerol 0.1 M KCl 5 mM 2 -ME 0.5 mM PMSF 40 mM imidazole

At the time of elution, 1 ml of the eluate having the following composition was applied to the column five times. Eluent composition: 20 mM Tris-HCl pH 7.9 10% glycerol 0.1 M KCl 5 mM 2-ME 0.5 mM PMSF 200 mM imidazole

3) The eluted HEL50 fraction was dialyzed against a phosphate buffer 1 having the following composition, and then using an HAP (hydroxyapatite) column (manufactured by Bio-Rad) using an FPLC system. 0.01-0.3M with phosphate buffer 1 and phosphate buffer 2 having the following composition
Purified with a gradient of. Bed volume is 2ml
And Phosphate buffer 1 composition: 10 mM phosphate buffer pH 7.2 50 mM KCl 5 mM 2-ME 10% glycerol Phosphate buffer 2 composition: 300 mM phosphate buffer pH 7.2 50 mM KCl 5 mM 2-ME 10% glycerol

4) The HEL50 fraction eluted was dialyzed against potassium chloride buffer 1 having the following composition, and then, using an ion exchange column MonoQ (registered trademark, manufactured by Pharmacia Amersham), using an FPLC system. Potassium chloride buffer 1 and potassium chloride buffer 2 having the following composition
With a gradient of 0.05-0.5M.
The bed volume was 1 ml. Potassium chloride buffer 1 composition: 20 mM Tris-HCl pH 7.9 10% glycerol 50 mM KCl 5 mM 2-ME 0.5 mM PMSF Potassium chloride buffer 2 composition: 20 mM Tris-HCl pH 7.9 10% glycerol 0.5 M KCl 5 mM 2 -ME 0.5 mM PMSF

5) The eluted HEL50 fraction was dialyzed against BC100 having the following composition and stored at -80 ° C. 20 mM Tris-HCl pH 7.9 20% glycerol 0.1 M KCl 5 mM 2-ME 0.5 mM PMSF The results of electrophoresis (Coomassie staining) of the purified protein are shown in FIG. HEL5 at the position of 50 kD in each fraction
A band of 0 was confirmed (the band appears at a slightly larger position by the amount of the tag).

<Example 4> Measurement of helicase activity Preparation of substrate 30 bases (CAGTCACGAC GTTGTAAAA
A single-stranded DNA consisting of C GACGGCCAGT (base sequence described in SEQ ID NO: 8 in the sequence listing) was synthesized using a DNA synthesizer. The 5 'end is Takara ME
Using a GA LABEL (registered trademark) kit (manufactured by Takara Shuzo Co., Ltd.), 37
After 30 minutes at 70 ° C, labeling was carried out at 70 ° C for 5 minutes. 5 pmol single-stranded DNA 2 [mu] l 10 fold concentrated phosphorylation buffer (kit as shipped) 1μl γ- ³² P ATP (Pharmacia Amersham) 5 [mu] l T4 kinase (manufactured by Takara Shuzo) 1 [mu] l of sterile distilled water 1 [mu] l total 10μl

The labeled single-stranded DNA was added to M13mp
18 Add 2 μg of ssDNA (Takara Shuzo) and add
The mixture was hybridized at 10 ° C. for 10 minutes and then at 37 ° C. for 1 hour to prepare labeled DNA. The hybridized labeled DNA is applied to a micro spin column S400HR.
(Pharmacia Amersham).
First, packing was performed by rotating the column with a swing rotor at 3000 rpm for 1 minute. The entire amount (about 12 μl) of the labeled DNA solution was added to the column, and 3000
The column was rotated for 2 minutes at rpm to separate the labeled DNA. The obtained labeled DNA was used as a substrate in the following operation.

2. Measurement of helicase activity Among the eluted fractions of MonoQ obtained in Example 3, third
The following operation is performed on each of the fractions Nos. 2, 35, 38, 41, 44, 47, and 50, and HEL5 contained in each fraction is
A helicase activity of 0 was measured. 0.2 μl of the substrate prepared in the above step 1 (corresponding to about 3000 cpm) and M
onoQ elution fraction or BC100 (control) 2
μl was added to a reaction solution having the following composition, and allowed to react at 37 ° C. for 30 minutes. Reaction solution (prepared to 20 μl with sterile distilled water) 20 mM Tris-HCl pH 7.5 2 mM DTT 50 μg / μl BSA 1 mM MgCl ₂ 80 mM KCl 1 mM ATP Thereafter, 5 μl of a stop solution having the following composition was added to stop the reaction. . 60 mM EDTA 0.75% SDS 0.1% BPB 50% glycerol

The reaction solution was applied on a 10% acrylamide / 0.5-fold concentration TBE (Tris-borate / EDTA) gel in an amount of 15 μl each, and electrophoresed by applying a voltage of 100 V. After the electrophoresis, the gel was dried, and an X-ray film (manufactured by Fuji Photo Film Co., Ltd.) was exposed to the gel overnight, and an autoradiograph was taken.

FIG. 4 shows the results. Lane 1 and lane 2 are controls. Lane 1 shows the result of adding BC100 instead of the MonoQ elution fraction (negative control), and lane 2 shows the result of adding and reacting BC100 and boiling for 3 minutes (positive control). In the figure, the lanes numbered from 32 to 50 are the MonoQ elution fractions of the numbers.
In each MonoQ eluted fraction, a band was detected at the same position as the positive control (lane 2). Particularly strong bands appeared in fractions 41 and 44, and it was confirmed that HEL50 contained in these fractions had particularly large helicase activity.

<Example 5> Directionality of helicase activity Preparation of Directional Substrate The following operation was performed to prepare a directional substrate. The outline is shown in FIG. In FIG. 5, .largecircle. Indicates that the portion is labeled. 1) Preparation of 5 ′ end labeled probe 54 bases (ATGCCTGCAG GTCGACTCT
A GAGGATCCCC GGGTACCCGAG C
A single-stranded DNA consisting of TCGAATTCG TAAT (base sequence described in SEQ ID NO: 9 in the sequence listing) was synthesized using a DNA synthesizer. The 5 'end is Takara
Using a MEGA LABEL (registered trademark) kit (manufactured by Takara Shuzo Co., Ltd.), the label was placed in a solution having the following composition at 37 ° C. for 30 minutes according to the instruction manual, and then placed at 70 ° C. for 5 minutes. Thereafter, 10 μl of TE was added. 5 pmol single-stranded DNA 1 [mu] l 10 fold phosphorylation buffer (kit as shipped) 1μl γ- ³² P ATP (Pharmacia Amersham) 5 [mu] l T4 kinase (manufactured by Takara Shuzo) 1 [mu] l of sterile distilled water 2μl total 10 [mu] l 2) Preparation of 3′-end labeled probe Single-stranded D consisting of the nucleotide sequence of SEQ ID NO: 9 in the sequence listing
NA was synthesized using a DNA synthesizer, and its 3 ′ end was placed in a solution having the following composition for 30 minutes at 37 ° C. using a TdT kit (manufactured by Wako Pure Chemical Industries, Ltd.) according to the instruction manual. 5 minutes at 70 ° C. for labeling. 5 pmol / μl single-stranded DNA 1 μl 5-fold TdT buffer (supplied with the kit) 4 μl α- ³² P ddATP (Pharmacia Amersham) 5 μl TdT 1 μl sterile distilled water 9 μl total 20 μl

3) M is added to each of the labeled single-stranded DNAs.
2 μg of 13mp18 ssDNA (manufactured by Takara Shuzo Co., Ltd.) was added, and the mixture was hybridized at 75 ° C. for 5 minutes, and then at 37 ° C. for 1 to 2 hours to prepare labeled DNA. 4) The hybridized labeled DNA was purified in the same manner as in Example 4 using a micro spin column S400HR (manufactured by Pharmacia Amersham). 5) Put the purified labeled DNA in a reaction solution having the following composition.
The labeled DNA was cleaved at the SmaI site at 2 ° C. every 2 hours. Labeled DNA solution 15 μl 10-fold T buffer (supplied with SmaI enzyme) 3 μl BSA 3 μl SmaI enzyme (Takara Shuzo) 2 μl Sterile distilled water 7 μl Total 30 μl 6) Dissolve the cleaved labeled DNA in phenol / CHCl ₃ and precipitate with ethanol. I let it. Precipitate 10 μl TE
And used as a substrate for measuring Helicase activity.

2. Measurement of helicase activity In the same manner as in Example 4, 1 μl of each substrate prepared above
Each for human HEL50 or rat TIP4
The mixture was decomposed in step 9 and the reaction solution was electrophoresed. FIG. 6 shows the results. Lanes 1 and 5 are positive controls, and lanes 2 and 6 are negative controls. Lanes 3 and 7 are HEL50 and lanes 4 and 8 are T
IP49. A band appears at position 30b when Heliekes acts from the 3 'end to the 5' end, and a band appears at position 24b when a helicase acts from the 5 'end to the 3' end. is there. From FIG. 6, it can be seen that HEL50 has a helicase activity direction from the 5 ′ end to the 3 ′ end. On the other hand, the direction of the helicase activity of TIP49 was from the 3 'end to the 5' end.

<Example 6> Cloning of yeast HEL50 Database Search The base sequence of yeast DNA having homology with HEL50 was obtained from TBLASTX (http://www.blas.com) in the GenomeNet WWW server on the Internet.
t. genome. ad. jp). As a result, a base sequence registered with the number of YPL235w was extracted. From the nucleotide sequence, the following primers were prepared using a DNA synthesizer (ABI, Model 392). Primer Y1 (methionine side): 5'-CGGAAT
TCAT GTCGATTCAA ACTAGTG-
3 '(base sequence described in SEQ ID NO: 10 in the sequence listing) Primer Y2 (3' end): 5'-CGGAATT
CTT ATTCCGTAGT ATCCATGGC
(Base sequence described in SEQ ID NO: 11 in Sequence Listing) Using this as a PCR primer, the following PCR operation was performed.

2. PCR Using the yeast genome (obtained from the National Institute of Genetics) as a template, and using KOD polymerase (manufactured by Toyobo), PCR was performed under the following conditions. Placed at 95 ° C. for 5 minutes. Next, a cycle of “94 ° C. for 30 seconds, followed by 55 ° C. for 30 seconds, and then 72 ° C. for 1 minute” was repeated 45 times. Then, after 5 minutes at 72 ° C, the PCR operation was completed at 4 ° C. The composition of the PCR reaction solution was as follows. Yeast genome 500 ng Primer Y1 10 pmol Primer Y2 10 pmol KOD pol (manufactured by Toyobo) 2 μl 10-fold concentration PCR buffer (manufactured by Toyobo) 5 μl 2 mM dNTP mix (manufactured by Toyobo) 5 μl 25 mM MgCl ₂ 2 μl prepared in sterile distilled water .

Thereafter, the PCR product obtained above was cut at the EcoRI site using a restriction enzyme to obtain a DNA fragment. The obtained DNA fragment was ligated with pBluescriptI.
It was inserted into the EcoRI site of ISK + and subcloned. The resulting DNA fragment was subjected to an autosequencer (ABI) by the dye terminator method.
(Model 373A), and the nucleotide sequence was determined. This base sequence is the yeast HE
It is a base sequence of L50 cDNA. This nucleotide sequence is shown as SEQ ID NO: 13 in the sequence listing. The amino acid sequence determined from this nucleotide sequence is the amino acid sequence of yeast HEL50. This amino acid sequence is shown as SEQ ID NO: 12 in the sequence listing. The determined nucleotide sequence is represented by YPL as the yeast genome.
The nucleotide sequences registered in the database with the numbers of 235w were exactly the same.

The homology between human HEL50 and yeast HEL50 is 65% in total, and 69% in Walker A motif (portion from Gly at position 77 to Thr at position 84 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing).
The Walker B motif (the portion from Asp at position 299 to His at position 302 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) was 71%. Human TIP4
Since the homology between human 9 and human HEL50 is 41%,
The homology between human HEL50 and yeast HEL50 in eukaryotes is clearly higher than the homology between human HEL50 and human TIP49. Eukaryotic HEL50 is considered to have a homology of 65% or more with human HEL50. Other eukaryotic HEL50 cDNAs can also be prepared by PCR from eukaryotic cDNA libraries of the species of interest.
Thus, it is considered that an appropriate probe can be prepared and isolated by a plaque hybridization method using the probe. Further, as exemplified in the cloning of yeast HEL50, full-length cDN was
A can also be obtained. PCR and hybridization conditions can be determined with reference to the conditions already shown.

[0063]

The HEL50 and HEL50 mutant of the present invention can be used as a helicase.

HEL50 DNA or HEL5 of the present invention
0 mRNA can be used to produce HEL50 by introducing it into an appropriate host and culturing the resulting transformant. DNA encoding HEL50 mutant
Alternatively, mRNA can be similarly used to produce a HEL50 mutant.

HEL50 consisting of 12 or more consecutive bases
(Including antisense polynucleotides) can be used as a probe to examine HEL50 expression. In addition, it can be used as a primer for PCR. The antisense polynucleotide is H
It can also be used to inhibit the biosynthesis of EL50. HE
The same applies to polynucleotides (including antisense polynucleotides) encoding L50 variants.

The antibody that recognizes HEL50 of the present invention is H
It can be used for detection and purification of EL50. For purification,
Affinity chromatography using an antibody-bound column or immunoprecipitation can be used

Since HEL50 of the present invention has homology with TIP49 in a part thereof, it is considered that HEL50 has an interaction with TBP. Since TBP is involved in the transcription of all genes in an organism, it is strongly expected that the HEL50 of the present invention, the polynucleotide encoding HEL50, and the antibody recognizing HEL50 will provide a means of investigating the transcription control function of TBP. it can.

It is known that certain genetic diseases are caused by abnormal helicopter cases. Therefore, it can be expected that the polynucleotide of the present invention can be used as a probe or a primer for PCR in diagnosing such a genetic disease. Further, it can be expected that the antibody of the present invention can be used for diagnosis of genetic diseases. In addition, recovery of helicase abnormalities, that is, treatment of the above-mentioned genetic diseases can be expected using the HEL50, polynucleotide or antibody of the present invention.

[0069]

[Sequence List] SEQUENCE LISTING <110> Sumitomo Electric Industries, Ltd. <120> DNA helicase <130> 098Y0518 <160> 13 <210> 1 <211> 463 <212> PRT <213> Homo sapience <400> 1 Met Ala Thr Val Thr Ala Thr Thr Lys Val Pro Glu Ile Arg Asp Val 1 5 10 15 Thr Arg Ile Glu Arg Ile Gly Ala His Ser His Ile Arg Gly Leu Gly 20 25 30 Leu Asp Asp Ala Leu Glu Pro Arg Gln Ala Ser Gln Gly Met Val Gly 35 40 45 Gln Leu Ala Ala Arg Arg Ala Ala Gly Val Val Leu Glu Met Ile Arg 50 55 60 Glu Gly Lys Ile Ala Gly Arg Ala Val Leu Ile Ala Gly Gln Pro Gly 65 70 75 80 Thr Gly Lys Thr Ala Ile Ala Met Gly Met Ala Gln Ala Leu Gly Pro 85 90 95 Asp Thr Pro Phe Thr Ala Ile Ala Gly Ser Glu Ile Phe Ser Leu Glu 100 105 110 Met Ser Lys Thr Glu Ala Leu Thr Gln Ala Phe Arg Arg Ser Ile Gly 115 120 125 Val Arg Ile Lys Glu Glu Thr Glu Ile Ile Glu Gly Glu Val Val Glu 130 135 140 Ile Gln Ile Asp Arg Pro Ala Thr Gly Thr Gly Ser Lys Val Gly Lys 145 150 155 160 Leu Thr Leu Lys Thr Thr Glu Met Glu Thr Ile Tyr Asp Leu Gly Thr 165 170 175 Ly s Met Ile Glu Ser Leu Thr Lys Asp Lys Val Gln Ala Gly Asp Val 180 185 190 Ile Thr Ile Asp Lys Ala Thr Gly Lys Ile Ser Lys Leu Gly Arg Ser 195 200 205 Phe Thr Arg Ala Arg Asp Tyr Asp Ala Met Gly Ser Gln Thr Lys Phe 210 215 220 Val Gln Cys Pro Asp Gly Glu Leu Gln Lys Arg Lys Glu Val Val His 225 230 235 240 Thr Val Ser Leu His Glu Ile Asp Val Ile Asn Ser Arg Thr Gln Gly 245 250 255 Phe Leu Ala Leu Phe Ser Gly Asp Thr Gly Glu Ile Lys Ser Glu Val 260 265 270 Arg Glu Gln Ile Asn Ala Lys Val Ala Glu Trp Arg Glu Glu Gly Lys 275 280 285 Ala Glu Ile Ile Pro Gly Val Leu Phe Ile Asp Glu Val His Met Leu 290 295 300 Asp Ile Glu Ser Phe Ser Phe Leu Asn Arg Ala Leu Glu Ser Asp Met 305 310 315 320 Ala Pro Val Leu Ile Met Ala Thr Asn Arg Gly Ile Thr Arg Ile Arg 325 330 335 Gly Thr Ser Tyr Gln Ser Pro His Gly Ile Pro Ile Asp Leu Leu Asp 340 345 350 350 Arg Leu Leu Ile Val Ser Thr Thr Pro Tyr Ser Glu Lys Asp Thr Lys 355 360 365 Gln Ile Leu Arg Ile Arg Cys Glu Glu Glu Asp Val Glu Met Ser Glu 370 375 380 Asp Al a Tyr Thr Val Leu Thr Arg Ile Gly Leu Glu Thr Ser Leu Arg 385 390 395 400 Tyr Ala Ile Gln Leu Ile Thr Ala Ala Ser Leu Val Cys Arg Lys Arg 405 410 415 Lys Gly Thr Glu Val Gln Val Asp Asp Ile Lys Arg Val Tyr Ser Leu 420 425 430 Phe Leu Asp Glu Ser Arg Ser Thr Gln Tyr Met Lys Glu Tyr Gln Asp 435 440 445 Ala Phe Leu Phe Asn Glu Leu Lys Gly Glu Thr Met Asp Thr Ser 450 455 460 <210> 2 <211 > 1512 <212> DNA <213> Homo sapience <220> <221> CDS <222> (1) ... (1389) <400> 2 atg gca acc gtt aca gcc aca acc aaa gtc ccg gag atc cgt gat gta 48 aca agg att gag cga atc ggt gcc cac tcc cac atc cgg gga ctg ggg 96 ctg gac gat gcc ttg gag cct cgg cag gct tcg caa ggc atg gtg ggt 144 cag ctg gcg gca cgg cgg gcg gct ggc gt cgg 192 gaa ggg aag att gcc ggt cgg gca gtc ctt att gct ggc cag ccg ggc 240 acg ggg aag acg gcc atc gcc atg ggc atg gcg cag gcc ctg ggc cct 288 gac acg cca ttc aca gcc atc gcc atc gcc atc gcc atc gcc atc gcc atc gcc ctg gag 336 atg agc aag acc gag gcg ctg acg cag gcc ttc cgg cgg tcc atc ggc 384 gtt cgc atc aag gag gag acg gag atc atc gaa ggg gag gtg gtg gag 432 atc cag att gat cga cca gca aca ggg acg ggc tcc aag gtg ggc aaa 480 ctg acc ctc aag acc aca gc atgag gag gac ggc acc 528 aag atg att gag tcc ctg acc aag gac aag gtc cag gcc ggg gac gtg 576 atc acc atc gac aag gcg acg ggc aag atc tcc aag ctg ggc cgc tcc 624 ttc aca cgc gcc cgc gac tac acc aag ttc 672 gtg cag tgc cca gat ggg gag ctc cag aaa cgc aag gag gtg gtg cac 720 acc gtg tcc ctg cac gag atc gac gtc atc aac tct cgc acc cag ggc 768 ttc ctg gc g cg gc gc gc gc gc g gc gc g gc gc g gc gc gc g cg gc g cg gc g gc gc g cg gc gc gc gc gc gc g gc aag tca gaa gtc 816 cgt gag cag atc aat gcc aag gtg gct gag tgg cgc gag gag ggc aag 864 gcg gag atc atc cct gga gtg ctg ttc atc gac gag gtc cac atg ctg 912 gac atc gc ag cc gc atc gc gc atc gc ag cc gc atc gc gc atc gc ag ctg gag agt gac atg 960 gcg cct gtc ctg atc atg gcc acc aac cgt ggc atc acg cga atc cgg 1008 ggc acc agc tac cag agc cct cac ggc atc ccc ata gac ctg ctg gac 1056 cgg ctg ctt acct c agc gag aaa gac acg aag 1104 cag atc ctc cgc atc cgg tgc gag gaa gaa gat gtg gag atg agt gag 1152 gac gcc tac acg gtg ctg acc cgc atc ggg ctg gag acg tca ctg cgc ata gc atc gc tacc tacc tacc gcc agc ttg gtg tgc cgg aaa cgc 1248 aag ggt aca gaa gtg cag gtg gat gac atc aag cgg gtc tac tca ctc 1296 ttc ctg gac gag tcc cgc tcc acg cag tac atg aag gag tac gc tt gac tac cac gc tac cac gc tac cac gc tac cac gc tac cac gc tac cac gc tac cac gc tac cac gc aaa ggc gag acc atg gac acc tcc tga 1392 gttggatgtc atcccccgac cccaccctgt tttccaccag agttctgaca ctgtgactct 1452 gtataaaatg gttgggaagc tgcaaaaaaa aaaaaaaaaa 1512 <210> 3 <211> 13 <212> DNA <213> Artificial Sequence ctggttcggc cca 13 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 gagatccgtg atgtaacaag gattgag 27 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 5 cttggtctgg gagcccatag cgtcg 25 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 tcgcatcaag gaggagac 18 <210 > 7 <2 11> 17 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 ctgagaagag cgccagg 17 <210> 8 <211> 30 <212> DNA <213> Artificial Sequemce <220> <223> Synthesized DNA <400> 8 cagtcacgac gttgtaaaac gacggccagt 30 <210> 9 <211> 54 <212> DNA <213> Artificial Sequemce <220> <223> Synthesized DNA <400> 9 ATGCCTGCAG GTCGACTCTA GAGGATCCCC GGGTACCGAG CTCGAATTCG TAAT 54 <210> 10 < 211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 10 CGGAATTCAT GTCGATTCAA ACTAGTG 27 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 CGGAATTCTT ATTCCGTAGT ATCCATGGC 29 <210> 12 <211> 471 <212> PRT <213> Yeast <400> 12 Met Ser Ile Gln Thr Ser Asp Pro Asn Glu Thr Ser Asp Leu Lys Ser 5 10 15 Leu Ser Leu Ile Ala Ala His Ser His Ile Thr Gly Leu Gly Leu Asp 20 25 30 Glu Asn Leu Gln Pro Arg Pro Thr Ser Glu Gly Met Val Gly Gln Leu 35 40 45 Gln Ala Arg Arg Arg Ala Ala Gly Val Ile Leu Lys Met Val Gln Asn Gly 50 55 60 Thr Ile Ala Gly Arg Ala Val Leu Val Ala Gly Pro Pro Ser Th r Gly 65 70 75 80 Lys Thr Ala Leu Ala Met Gly Val Ser Gln Ser Leu Gly Lys Asp Val 85 90 95 Pro Phe Thr Ala Ile Ala Gly Ser Glu Ile Phe Ser Leu Glu Leu Ser 100 105 110 Lys Thr Glu Ala Leu Thr Gln Ala Phe Arg Lys Ser Ile Gly Ile Lys 115 120 125 Ile Lys Glu Glu Thr Glu Leu Ile Glu Gly Glu Val Val Glu Ile Gln 130 135 140 Ile Asp Arg Ser Ile Thr Gly Gly His Lys Gln Gly Lys Leu Thr Ile 145 150 155 160 Lys Thr Thr Asp Met Glu Thr Ile Tyr Glu Leu Gly Asn Lys Met Ile 165 170 175 Asp Gly Leu Thr Lys Glu Lys Val Leu Ala Gly Asp Val Ile Ser Ile 180 185 190 Asp Lys Ala Ser Gly Lys Ile Thr Lys Leu Gly Arg Ser Phe Ala Arg 195 200 205 Ser Arg Asp Tyr Asp Ala Met Gly Ala Asp Thr Arg Phe Val Gln Cys 210 215 220 Pro Glu Gly Glu Leu Gln Lys Arg Lys Thr Val Val His Thr Val Ser 225 230 235 240 Leu His Glu Ile Asp Val Ile Asn Ser Arg Thr Gln Gly Phe Leu Ala 245 250 255 Leu Phe Thr Gly Asp Thr Gly Glu Ile Arg Ser Glu Val Arg Asp Gln 260 265 270 Ile Asn Thr Lys Val Ala Glu Trp Lys Glu Glu Gly Lys Ala Glu Ile 2 75 280 285 Val Pro Gly Val Leu Phe Ile Asp Glu Val His Met Leu Asp Ile Glu 290 295 300 Cys Phe Ser Phe Ile Asn Arg Ala Leu Glu Asp Glu Phe Ala Pro Ile 305 310 315 320 Val Met Met Ala Thr Asn Arg Gly Val Ser Lys Thr Arg Gly Thr Asn 325 330 335 Tyr Lys Ser Pro His Gly Leu Pro Leu Asp Leu Leu Asp Arg Ser Ile 340 345 350 Ile Ile Thr Thr Lys Ser Tyr Asn Glu Gln Glu Ile Lys Thr Ile Leu 355 360 365 Ser Ile Arg Ala Gln Glu Glu Glu Val Glu Leu Ser Ser Asp Ala Leu 370 375 380 Asp Leu Leu Thr Lys Thr Gly Val Glu Thr Ser Leu Arg Tyr Ser Ser 385 390 395 400 Asn Leu Ile Ser Val Ala Gln Gln Ile Ala Met Lys Arg Lys Asn Asn 405 410 415 Thr Val Glu Val Glu Asp Val Lys Arg Ala Tyr Leu Leu Phe Leu Asp 420 425 430 Ser Ala Arg Ser Val Lys Tyr Val Gln Glu Asn Glu Ser Gln Tyr Ile 435 440 445 Asp Asp Gln Gly Asn Val Gln Ile Ser Ile Ala Lys Ser Ala Asp Pro 450 455 460 Asp Ala Met Asp Thr Thr Glu 465 470 <210> 13 <211> 1416 <212> DNA <213> Yeast <220> <221> CDS <222> ( 1) ... (1413) <400> 13 atg tcg att caa act agt gat cca aat gaa aca tca gat tta aag tcg 48 tta tct tta att gct gcc cac tcc cat att aca ggt tta ggt cta gat 96 gaa aac ttg caa cca cgt ccc aca tcc gaa ggt atg gtt ggg caa ttg 144 cag gcc gt gct ggt gtg ata ttg aaa atg gtt caa aat ggc 192 acc ata gca ggt agg gct gtt ttg gta gcg ggc ccc cct tca act ggt 240 aag acc gct ctt gcc atg ggt gtt tcc cag tct ctg ggt aaa gat gta ctcca att gcg ggc tca gaa atc ttt tct tta gaa ttg agt 336 aaa act gaa gca cta act caa gct ttt agg aaa tcc atc ggt atc aaa 384 atc aag gag gag aca gaa ttg att gaa ggt gaa gtc gtg gat gtc gtg gat gtg tct att act ggt gga cac aaa caa gga aaa ttg act att 480 aaa act acc gat atg gaa aca att tat gaa tta ggc aat aaa atg att 528 gat ggc cta act aaa gaa aag gta ttg gct ggc gat gtt att tct att gct agt ggg aag att acc aag cta ggc aga tcc ttt gct aga 624 tct agg gat tat gat gcc atg ggt gct gat acc aga ttt gtt caa tgt 672 ccg gaa ggc gaa ctg caa aaa agg aaa aca gtg gttg cac g c at gaa att gat gtt att aat tca aga aca caa gga ttt ttg gca 768 tta ttt act ggt gac acc ggt gaa att agg tca gag gta aga gac cag 816 ata aac aca aaa gtg gca gaa tgg aaa gaa gaa ggt aaa g gtt cct ggt gta tta ttt atc gac gaa gtc cac atg ttg gat ata gaa 912 tgt ttt tcc ttt ata aat agg gct ttg gaa gat gag ttt gcc cca atc 960 gtc atg atg gct aca aat aga gga gtt tcc acc acc 1008 tac aaa tct cca cat ggg tta cct ctc gat ctt ttg gat agg tca att 1056 att att aca act aaa agt tat aat gag cag gag att aag aca att tta 1104 tct ata aga gca caa gag gag gaa gtt gca ccg tcc tta 1152 gat cta ttg acc aaa aca ggt gtg gaa act agt ttg cgt tac agc agt 1200 aat tta atc tct gtt gct cag caa att gca atg aag aga aaa aac aac 1248 act gtt gaa gtg gaa gat gtc tt att gct ttg gac 1296 agc gct aga tct gtt aag tat gtt caa gag aac gag tca caa tat att 1344 gat gat cag ggc aac gtt caa ata tcc att gct aaa tca gca gac cct 1392 gat gcc atg gat act acg gaa taa 1416

[Brief description of the drawings]

FIG. 1. HEL50 for mRNA from human tissues
4 is an electrophoretic photograph showing the result of Northern blot hybridization using a part of a gene as a probe.

FIG. 2 is a diagram showing a pET-3a His vector.

FIG. 3 is a photograph showing the results of electrophoresis of HEL50 purified by MonoQ.

FIG. 4 is an electrophoretic photograph showing the result of electrophoresis of a reaction product obtained by reacting HEL50 purified with MonoQ with DNA.

FIG. 5 is a diagram showing an outline of preparation of a directional substrate.

FIG. 6 is an electrophoretic photograph showing the result of electrophoresis of a reaction product obtained by reacting a directional substrate with HEL50 or TIP49.

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[Submission date] February 15, 1999 (1999.2.1
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──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) // A61K 48/00 A61K 48/00 4H045 F term (reference) 4B024 AA20 BA80 CA04 DA06 EA04 GA11 GA19 HA01 HA12 4B050 CC03 DD11 LL03 4B063 QA12 QA18 QQ21 QR32 QR55 QR62 QS03 QS34 4B064 AG27 CA10 CA20 DA13 4C084 AA13 ZC192 4H045 AA11 CA40 DA76 DA86 DA89 FA72

Claims (13)

[Claims] 1. A protein comprising the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. A protein comprising an amino acid sequence in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and having a DNA helicase activity. 3. A protein having a homology of at least 65% with the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having DNA helicase activity. 4. A protein comprising the amino acid sequence of SEQ ID NO: 12 in the sequence listing. A polynucleotide encoding the protein according to any one of claims 1 to 4. 6. A DNA having a nucleotide sequence from the 1st A to the 1389th C of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. 7. The DNA according to claim 6, which comprises the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. 8. A DNA comprising the nucleotide sequence of SEQ ID NO: 13 in the sequence listing. 9. An RNA encoded by the DNA according to any one of claims 6 to 8. 10. The antisense DNA of the DNA according to any one of claims 6 to 8, and the R antisense DNA according to claim 9,
An antisense polynucleotide comprising an antisense RNA of NA or a derivative thereof. 11. A polynucleotide comprising a portion consisting of 12 or more consecutive bases of the polynucleotide according to any one of claims 5 to 10. 12. The polynucleotide according to claim 5, which is chemically modified. An antibody that recognizes the protein according to any one of claims 1 to 4.