WO2000047731A1 - Protein having dna helicase activity, polynucleotide encoding the protein, antisense polynucleotide against the polynucleotide and antibody recognizing the protein - Google Patents

Protein having dna helicase activity, polynucleotide encoding the protein, antisense polynucleotide against the polynucleotide and antibody recognizing the protein Download PDF

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Publication number
WO2000047731A1
WO2000047731A1 PCT/JP1999/006519 JP9906519W WO0047731A1 WO 2000047731 A1 WO2000047731 A1 WO 2000047731A1 JP 9906519 W JP9906519 W JP 9906519W WO 0047731 A1 WO0047731 A1 WO 0047731A1
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dna
protein
polynucleotide
hel50
seq
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PCT/JP1999/006519
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French (fr)
Japanese (ja)
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Taka-Aki Tamura
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Sumitomo Electric Industries, Ltd.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)

Definitions

  • 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.
  • 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.
  • TBP TATAbindngprotein
  • TIP49 protein As a protein that binds to TBP and the gene encoding it (Biochem. Biophys. R Rs. Commun., 235, 64). — 68). Disclosure of the invention An object of the present invention is to provide a novel protein having homology to a part of TIP49.
  • 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.
  • the present inventors isolated a cDNA encoding a protein having homology to a part of TIP49 and determined the nucleotide sequence thereof. Then, the amino acid sequence encoded by the cDNA was determined. Further, the present inventors produced a recombinant protein encoded by the cDNA, and confirmed that the molecular weight of the recombinant protein was 5 OkD. The present inventors named this protein HEL50. Furthermore, the present inventors have confirmed that HE L50 has DNA helicase activity. When the animal from which HEL50 is derived is specified, an animal name derived from before HEL50, such as human HEL50, is added. The notation HEL50 means HEL50 derived from an eukaryote.
  • the present invention provides the following proteins.
  • 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 HE L50.
  • 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 which has DNA helicase activity.
  • 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 an embodiment of a human HEL50 mutant.
  • polynucleotide refers to DNA or RNA, or those in which a plurality of nucleotides consisting of a base group, a phosphate, or a sugar are bonded thereto, including those that do not exist in nature.
  • DNA having a nucleotide sequence from the 1st A to the 1389th C of the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing is the nucleotide sequence of the coding region of human HEL50.
  • human HEL50 DNA it is referred to as human HEL50 DNA.
  • cDNA is a force contained in DNA.
  • cDNA is referred to as cDNA when it is specifically indicated that the DNA is cDNA.
  • DNA comprising the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. This DNA is one embodiment of the DNA described in 6 above.
  • DNA comprising the nucleotide sequence of SEQ ID NO: 13 in the sequence listing. This DNA is yeast HE L50 DNA.
  • RNA encoded by the DNA is referred to as human HE L50 RNA.
  • mRNA is a force contained in RNA.
  • the RNA is mRNA, it is referred to as mRNA.
  • An antisense polynucleotide comprising the antisense DNA of the DNA described in any one of the above 6 to 8, the antisense RNA of the RNA described in the above 9, or a derivative thereof.
  • the antisense polynucleotide is a polynucleotide Although contained in the nucleotide, in the present specification, when it is particularly specified that the polynucleotide is an antisense strand, it is referred to as an antisense polynucleotide.
  • the present invention also provides an antibody that recognizes the protein described in any one of 1 to 3 above, and an active fragment of a monoclonal antibody.
  • antibody includes all antisera, polyclonal or monoclonal antibodies, as well as active fragments of the antibody.
  • FIG. 1 is an electrophoretic photograph showing the results of Northern blot hybridization of mRNA derived from human tissues using a part of the HEL50 gene as a probe.
  • FIG. 2 is a diagram showing the pET-3aHis vector used to introduce the HEL50 gene in Example 3.
  • FIG. 3 is a photograph showing the results of electrophoresis of HEL50 purified by MonoQ (registered trademark).
  • FIG. 4 is an electrophoresis photograph showing the result of electrophoresis of a reaction product obtained by reacting HEL50 purified by MonoQ (registered trademark) with substrate DNA.
  • FIG. 5 is a diagram showing an outline of production of one directional substrate used in the present invention.
  • FIG. 6 is an electrophoretic photograph showing the result of electrophoresis of a reaction product obtained by reacting the directional substrate shown in FIG. 5 with HEL50 or TIP49.
  • the HEL50 and HEL50 mutants of the present invention are characterized by having DNA helicase activity, which can be confirmed, for example, by using the method disclosed in Example 4.
  • the HEL50 mutant of the present invention can be prepared using a known method.
  • HE HE
  • a DNA encoding the L50 mutant is prepared, the DNA is incorporated into an appropriate host, and the resulting transformant can express the HEL50 mutant.
  • DNA encoding the HEL50 mutant can be obtained by, for example, using a restriction enzyme to delete or substitute a base at a desired site of HEL50 DNA with another base, or to insert a base at a desired site.
  • Can be The HEL50 mutant can also be produced by the method of site-directed mutagenesis.
  • random mugenesis can be caused by a PCR with relaxed hybridization conditions.
  • the DNA or RNA encoding HEL50 or HEL50 mutant of the present invention is composed of all nucleotide sequences that can exist due to degeneracy.
  • DNA refers to a double-stranded sense strand and antisense strand
  • RNA refers to a single-stranded strand
  • antisense DNA or Antisense RNA refers to one consisting of a single strand.
  • polynucleotide refers to DNA or RNA, or one or more nucleotides consisting of bases, phosphates, and sugars linked to them, and includes naturally occurring or non-naturally occurring ones. Including both. The same applies to antisense polynucleotides.
  • the antisense polynucleotides of the present invention include all derivatives whose tertiary structure and function are similar to polynucleotides.
  • Derivatives are, for example, antisense polynucleotides that have other substances bound to the 3, 5 or 5, A substance in which a modification such as substitution, deletion, or addition has occurred in at least a part of the base, sugar, or phosphate of the sense polynucleotide, or a substance having a non-naturally occurring base, sugar, or phosphate; It has a skeleton other than the sugar monophosphate 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. Particularly, those having a base sequence complementary to a part of the mRNA and hybridizing to the mRNA are preferable.
  • Antisense polynucleotides are provided.
  • an antisense polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the RNA loop region should be designed.
  • an antisense polynucleotide having a sequence complementary to the sequence near the translation initiation codon of RNA, a ribosome binding site, a cabling site or a splice site can generally be expected to have a large effect of inhibiting biosynthesis.
  • the effect of inhibiting the biosynthesis of HEL50 is expected to be large.
  • Antisense polynucleotides are unique in that they inhibit the biosynthesis of certain proteins. Those that hybridize to a certain gene or RNA are preferred. For this reason, those comprising 12 or more bases are preferred, and those comprising 16 or more bases are particularly preferred. On the other hand, those having 35 or less bases are preferable in terms of cell membrane permeability.
  • the antisense polynucleotide derivative is preferably a derivative having at least one of increased nuclease resistance, tissue selectivity, cell membrane permeability, or avidity. Particularly preferred derivatives are those having a phosphorothioate bond as a skeletal structure.
  • the antisense polynucleotide derivative of the present invention also includes derivatives having these functions or structures.
  • Examples of the method for producing the antisense polynucleotide and the derivative thereof of the present invention include, for example, natural DNA and RNA, which can be synthesized using a chemical synthesizer, and Performing the method.
  • Some derivatives such as a methylphosphonate type and a phosphorothioate type can be synthesized using a chemical synthesizer (for example, Model 394 manufactured by 881).
  • the desired antisense poly- mer is purified by performing the operation according to the instructions attached to the chemical synthesizer, and purifying the resulting synthetic product by HPLC using reverse phase chromatography or the like. Nucleotides or derivatives thereof can be obtained.
  • any DNA of this length can identify all human proteins. 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 in practical use, but practically, it is often used with 12 bases or more.
  • 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 the problem of the three-dimensional structure of the polynucleotide and easily hybridize.
  • the method for detecting the HEL50 gene include the Northern blot hybridization method and the RT-PCR method (“(urrent Pr otocolsin Molecular Biology” (Greene Pub). 1 ishing As soci at es and Wiley—Interscience) Chapterl 5. 1. 1-15. 1. 9 and ibid 15. 4. 1-15. 4. 6) or in situ hybridization method (Chapter 14.3.1-14.3.14 in the same book).
  • the optimum conditions for hybridization differ depending on the length of the probe and the membrane used. In other words, the hybridization condition naturally has 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 be performed under different hybridization conditions.
  • the DNA and RNA of the present invention include the above-mentioned chemically modified DNA, RNA or antisense polynucleotide within its scope.
  • the chemically modified DNA or RNA of the present invention can exhibit both the function of encoding a protein and the function as a probe, and the chemically modified DNA or RNA of the present invention.
  • the sense polynucleotide can exhibit both the function of inhibiting protein biosynthesis and the function as a probe.
  • the present invention relates to an antibody obtained by immunizing a non-human animal with HEL50, and it is confirmed that the HEL50 of the present invention can be recognized by Western blotting, ELISA, immunostaining, or the like.
  • the antibody confirmed by FACS is included in the range.
  • the animal to be immunized is preferably selected from animals other than the animal from which the HEL50 to be administered as the immunogen is derived and other than human.
  • rat HEL50 it is preferable to immunize an animal other than a rat and a human (eg, a heron mouse).
  • an immunogen obtained by binding a part of a protein to another carrier protein such as serum albumin is a commonly used method to use an immunogen obtained by binding a part of a protein to another carrier protein such as serum albumin.
  • a part of the protein may be synthesized using, for example, a peptide synthesizer.
  • a part of the protein is composed of eight or more amino acid residues.
  • an antiserum or a polyclonal antibody can be obtained by purifying immunoglobulin from blood collected from the immunized animal, a hybridoma obtained by cell fusion of lymphocytes of the immunized animal with myeloma cells or the like can be used. Is well known to produce monoclonal antibodies (Antibodies A Laboratory or Manual, Cold Srin Harb or Laboratory Press, 1988, 141-147).
  • the antibodies of the present invention also include active fragments thereof.
  • the active fragment means a fragment of an antibody having antigen-antibody reaction activity, and specific examples include F (ab ') 2 , Fab', Fab, Fv, and the like.
  • F (ab ') 2 when the antibody of the present invention is digested with pepsin, F (ab ') 2 is obtained, and when digested with papain, Fab is obtained.
  • F (ab ') 2 is obtained, and when digested with papain, Fab is obtained.
  • Reduction of F (ab ') with a reagent such as 2-mercaptoethanol and alkylation with monoacetic acid gives Fab
  • Fv is a monovalent antibody active fragment obtained by linking the heavy chain variable region and the light chain variable region with a linker. This A chimeric antibody can be obtained by retaining these active fragments and replacing the other parts 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.
  • Specific examples of the method using an antibody include (1) a method of detecting HEL50 using an antibody that recognizes HEL50, (2) an antibody that recognizes HEL50, and a labeled secondary antibody of the antibody. And a method for detecting HEL50.
  • the label for example, a radioisotope (RI), an enzyme, avidin or biotin, or a fluorescent substance (FITC, rhodamine, or the like) is used.
  • RI radioisotope
  • FITC avidin or biotin
  • rhodamine or the like
  • Anchor primer 5, -CTGGTT CGGC CCA-3, (base sequence described in SEQ ID NO: 3 in Sequence Listing)
  • the synthesized primer was adjusted to 20 pmo1 / 1/1 with distilled water. This is a PC
  • PCR was performed under the following conditions using Marathon (registered trademark) human liver cDNA library (Clontech).
  • an anchor primer and a P2 primer were used as primers and Takar a Taq (registered trademark, manufactured by Takara Shuzo) was used under the following conditions.
  • composition of the reaction solution of PCR was as follows.
  • Taq po 1 (Takara Shuzo) 0.1 1 0 1
  • fragment A The DNA fragment amplified by the above PCR operation (hereinafter sometimes referred to as fragment A) was subjected to minigel electrophoresis (0.75% agarose gel), and the fragment A band was cut out from the gel. Fragment A was collected using GeneClean (manufactured by Bio101) and bands were checked by minigel electrophoresis.
  • the fragment A recovered above was inserted into the plasmid vector pGEM-TEASy using pGEM-TEASy (promega). Thereafter, the pGEM-Teasy into which the fragment A was inserted was introduced into E. coli, the E. coli was cultured, and plasmid DNA, ie, a fragment, was obtained from the white colonies that appeared by using a Midi kit (Qiagen). A was prepared.
  • the DNA sequence of the fragment A was determined by the Dye-Mine-To-One method using a DNA sequencer (manufactured by ABI, type 373A), and the nucleotide sequence was confirmed.
  • Fragment A prepared from Escherichia coli that formed the white colony was excised with EcoR I, and the obtained DNA fragment was dissolved in a liquid having the following composition using a random labeling kit (manufactured by Behringaman Heim). After 30 minutes at 37 ° C, labeling was performed at 65 ° C for 10 minutes.
  • labeled fragment A was purified using a G-50 micro spin column. Packing was performed by rotating the column at 3000 rpm for 1 minute using a swing rotor. 20/1 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.
  • This DNA probe was hybridized to a human liver cDNA library gtlO (manufactured by Clontech), and a cDNA hybridizing to the DNA probe was cloned.
  • Hybridization conditions were as follows.
  • the ditrocellulose membrane after the completion of the hybridization was washed under the following conditions.
  • HEL50SK + The pB1uescript SK + vector-1 (Stratagene) and cloned.
  • the cDNA obtained as a result of the cloning was subjected to DNA sequencing using the Auto Sequencer (ABI, 373A and 310) by the Ditamine-One-One Method.
  • the nucleotide sequence was determined. As a result, it was found that the cDNA contained 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 as SEQ ID NO: 2 in the sequence listing.
  • the pB1uescripptSK + vector into which the HEL50 cDNA was inserted was named HEL50SK +.
  • HEL50 The amino acid sequence of HEL50, which is a protein encoded by HEL50 cDNA, was determined from the nucleotide sequence determined in step 5 above. The results are shown in SEQ ID NO: 1 in the sequence listing. In addition, the portion from the 77th Gly to the 84th Thr and the portion from the 299th Asp to the 302nd His in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing are ATPase and This is the site of the sequence characteristic of the DNA helicopter. (Example 2) Northern blotting
  • primer P3 and primer P4 were prepared from the nucleotide sequence of HE L50 using a DNA synthesizer (ABI, Model 392).
  • the synthesized primer was adjusted to 20 pmo1 / 1/1 with distilled water. Using this as a PCR primer, the following PCR operation was performed.
  • the vector HE L50 SK + into which the HE L50c DNA was introduced was used as the type III of PCR, and Takara Taqo 1 (registered trademark, manufactured by Takara Shuzo Co., Ltd.) was used using primer P3 and primer P4. ) was performed under the following conditions.
  • the cycle of 94 ° (: 1 minute. Then, the cycle of “45 seconds at 94, followed by 2 minutes at 60 ° C, and then 2 minutes at 72 ° C” was repeated 30 times. Put down and complete the PCR procedure.
  • the composition of the PCR reaction solution was as follows
  • fragment B (Hereinafter referred to as “fragment B”), and the fragment B was recovered using GeneClean (manufactured by Bio101), and the band was checked by minigel electrophoresis. As a result, the concentration of the DNA solution of fragment B was estimated to be 25 ng / ⁇ 1.
  • labeled fragment B was purified using a G-50 micro spin column. The column was rotated at 3000 rpm for 1 minute throughout the swinging to perform the packing. 20 ⁇ 1 of labeled fragment B solution was added to the column, the column was rotated at 3000 rpm for 2 minutes, and eluted with T50E to separate labeled fragment B. II No Predication
  • the mRNA (human MTN (registered trademark) Plot II (manufactured by Clonetech), rat MTN (registered trademark) No. 3 (manufactured by Clonetech) and mouse fetus (manufactured by Clonetech)) containing immobilized mRNA were as follows.
  • the membrane was placed in 10 ml of the hybridization solution having the composition and placed at 42 ° C for 150 minutes, and the membrane was blocked with denatured salmon sperm DNA.
  • the membrane was exposed to radiation-sensitive paper for 16 hours and autoradiographed.
  • the results for the human MTN membrane are shown in FIG. HE L in human tissues 5 OmRNA was expressed, but was most frequently expressed in testis. Fragment B hybridized to rat mRNA and mouse mRNA, and the result was similar to that of human in rat.
  • HEL5 OmRNA was also expressed in the mouse fetus, and the highest expression was observed in the mouse fetus on day 11 of embryo.
  • the first methionine portion of the HEL50c DNA was modified with PCR so that the EcoRI sites were adjacent to each other, and incorporated into the EcoRI site of the pET FLAG-His vector shown in FIG.
  • the vector incorporating HEL50c DNA was introduced into JM109 competent cells (Takara Shuzo) according to the instruction manual, cultured overnight in LB medium containing 800 ml of ampicillin, and recovered from the JM109 competent cells.
  • the vector was recovered by the alkali method (the method described above, Mo ecu a ar Cloning Se dion ion ⁇ 1.33-1.43). Ultracentrifugation for recovery of the vector was performed three times.
  • E. coli was recovered and suspended in 30 ml of a lysis solution having the following composition.
  • the resulting suspension was sonicated at 4 ° C, ultracentrifuged and 45 Ti low Ultra-centrifugation at 50,000 rpm for 1 hour at 4 ° C using Beckman.
  • the eluted HEL 50 fraction was dialyzed against phosphate buffer 1 having the following composition, and then, using a HAP (hydroxyapatite) column (manufactured by Bio-Rad), an F PLC system (trade name, Amersham) Purification was performed using a phosphate buffer solution 1 and a phosphate buffer solution 2 having the following composition in a gradient of 0.01 to 0.3 M using the following method.
  • the bed volume was 2 ml.
  • Figure 3 shows the results of electrophoresis (coomassie staining) of the purified protein.
  • a band of HEL50 was confirmed at the position of 50 kD (the band appeared slightly larger for the tag).
  • Example 4 Measurement of helicase activity
  • a single-stranded DNA consisting of 30 bases (CAGT CACGAC GTTGTAAAAC GACGGC CAGT (base sequence described in SEQ ID NO: 8 in the sequence listing)) was synthesized using a DNA synthesizer.
  • a DNA synthesizer Using the Takar MEGA LABE L (registered trademark) kit (manufactured by Takara Shuzo Co., Ltd.), place the 5 'end in a solution having the following composition at 37 ° C for 30 minutes according to the instruction manual, and then add the ⁇ 0 ° Labeled C for 5 minutes.
  • the hybridized labeled DNA was purified using a micro spin column S 400 HR (manufactured by Amersham Pharmacia). First, packing was performed by rotating the column with a swing mouth at 3000 rpm for 1 minute. The entire amount (about 12 DNA1) of the labeled DNA solution was added to the column, and the column was rotated at 3000 rpm for 2 minutes to separate the labeled DNA. The obtained labeled DNA was used as a substrate in the following procedures.
  • Example 3 Among the eluted fractions of Mo no Q obtained in Example 3, the following operations were performed on the respective fractions of Nos. 32, 35, 38, 41, 44, 47 and 50, and The helicase activity of the included HEL 50 was measured.
  • the substrate 0.2 ⁇ 1 (corresponding to about 3000 cpm) prepared in the above step I and each MonoQ-eluted fraction or BC100 (control) 2 ⁇ ⁇ 1 were added to a reaction solution having the following composition, and the mixture was added. The reaction was carried out at 30 ° C for 30 minutes.
  • Reaction solution (prepared to 201 with sterile distilled water)
  • the reaction solution was placed on a 10% acrylamide / 0.5-fold concentration TBE (Tris-borate / EDTA) gel in an amount of 15 ⁇ l, and electrophoresed by applying a voltage of 100 V. After the completion of the electrophoresis, the gel was dried, an X-ray film (manufactured by Fuji Photo Film Co., Ltd.) was exposed to the gel, and an autoradiograph was taken.
  • TBE Tris-borate / EDTA
  • Fig. 4 shows the results.
  • Lane 1 and lane 2 are controls (one control).
  • Lane 1 is a sample to which BC 100 was added instead of the Mo no Q elution fraction (negative control).
  • Lane 2 was a sample to which BC 100 was added and reacted, followed by boiling for 3 minutes (positive control) It is.
  • the lanes marked with numbers from 32 to 50 are the Mo no Q elution fractions of the numbers.
  • a band was detected at the same position as the positive control (lane 2).
  • Fractions 41 and 44 showed particularly strong bands, and HE L5 contained in these fractions. 0 was confirmed to have particularly large helicase activity.
  • Figure 5 shows the outline of the system.
  • a star indicates that the part is labeled.
  • a single-stranded DNA consisting of 54 bases was synthesized using a DNA synthesizer. 5.
  • a Takara MEGA LABEL (registered trademark) kit (Takara Shuzo Co., Ltd.) at the end, place in a solution of the following composition for 30 minutes at 37 ° C according to the instruction manual, and then Label for 5 minutes. After that, I gained 10 ⁇ calories from TE.
  • T4 kinase (Takara Shuzo) 1 ⁇ .1
  • a single-stranded DN ⁇ consisting of the nucleotide sequence of SEQ ID NO: 9 in the sequence listing was synthesized using a DN ⁇ synthesizer, and its 3 ′ end was collected using a TdT kit (Wako Pure Chemical Industries, Ltd.). After placing in a solution having the following composition at 37 ° C for 30 minutes according to the instruction manual, labeling was performed at 70 ° C for 5 minutes.
  • the purified labeled DNA was cleaved with a SmaI site every 2 hours at 37 ° C. in a reaction solution having the following composition.
  • each of the substrates prepared above was digested with human HEL50 or rat TIP49, and the reaction solution was subjected to electrophoresis.
  • 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 HEL 50 and lanes 4 and 8 are TIP 49. 3 'band appears at 30b In the case where the helix acts in the direction from the end to the 5 'end, and at the position 24b, the dod appears when the helicase acts in the direction from the 5' end to the 3 'end. From Fig. 6, it can be seen that HEL50 has a helicase activity direction of 5, 5 from the end, and 3 ends. On the other hand, the direction of the helicase activity of TIP49 was 3, 3 from the terminal, and 5 from the terminal.
  • the base sequence of yeast DNA with homology to HE L50 was obtained from TB LAS TX (http: // www. Blast, genome, ad. Jp) in Genyuichi Net's Genome Net WWW server. Used and searched. As a result, the nucleotide sequence registered under the number of YP L 235 w was extracted.
  • the following primers were prepared from this nucleotide sequence using a DNA synthesizer (ABI, 392 type).
  • Primer Y 1 (methionine side): 5 ′ — CGGAATT CAT GTCGAT TCAA ACTAGTG—3, (base sequence described in SEQ ID NO: 10)
  • Primer Y2 (3, terminal side): 5 ′ — CGGAATTCTT ATTCCGT AGT ATCCATGGC
  • SEQ ID NO: 11 in the column list The nucleotide sequence described in SEQ ID NO: 11 in the column list
  • yeast genome obtained from the National Institute of Genetics
  • KOD polymerase manufactured by Toyobo
  • composition of the PCR reaction solution was as follows.
  • the PCR product obtained above was cut at the EcoRI site using a restriction enzyme to obtain a DNA fragment.
  • the resulting DNA fragment was inserted into pBlueScriptltlSK + EcoR site and subcloned. The resulting
  • DNA sequence was carried out using a 373A (manufactured by BI Company, Inc.), and its nucleotide sequence was determined.
  • This nucleotide sequence is the nucleotide sequence of yeast HE 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.
  • SEQ ID NO: 12 in the sequence listing.
  • the determined nucleotide sequence was exactly the same as the nucleotide sequence registered in the database with the number YP L 235 w as the yeast genome.
  • the homology between human HE L50 and yeast HE L50 is 65% in total, the Walker A motif (the portion from the 77th Gly to the 84th Thr in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing). ), 69% of the war force B motif (from the 299th Asp to the 302nd
  • the homology between human TIP49 and human HEL50 is 41%, the homology between human HEL50 and yeast HEL50 in eukaryotes is higher than that between human HEL50 and human TIP49. Clearly high.
  • the eukaryotic HE L50 is considered to be more than 65% homologous to human HEL50.
  • an appropriate probe is prepared from the eukaryotic cDNA library of the target species by PCR, and the plaque hybridization method using the probe is used. It is believed that it can be isolated.
  • full-length cDNA can be obtained by PCR as exemplified in the cloning of yeast HEL50. The conditions for PCR and hybridization can be determined with reference to the conditions already shown. Industrial applicability
  • the HEL50 of the present invention has homology to TIP49 in part thereof, and thus is considered to have an interaction with TBP. Since TBP is involved in the transcription of all genes in an organism, the HEL50, the polynucleotide encoding HEL50, and the antibody recognizing HEL50 of the present invention provide a means for investigating the transcription control function of TBP. It is.
  • the HEL50 and the HEL50 mutant of the present invention can be used as a DNA helicopter. That is, it is known that certain genetic diseases are caused by abnormalities of the helicopter. Therefore, the polynucleotide of the present invention can be used as a probe or as a primer for PCR in diagnosing such a genetic disease. Furthermore, it is expected that the use of the HEL50, the polynucleotide and the antibody of the present invention will recover helicase abnormalities, that is, treat the genetic disease.

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Abstract

A protein having a homology with a part of TIP49 and cDNA encoding this protein. This protein has a DNA helicase activity.

Description

明糸田書  Akitoda
DN Aヘリケース活性を有する蛋白質、 該蛋白質をコードするポリヌクレオチ ド、 該ポリヌクレオチドに対するアンチセンスポリヌクレオチドおよび該蛋白質 を認識する抗体 技術分野  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.
本発明は、 DNAヘリケース活性を有する蛋白質、 該蛋白質をコードするポリ ヌクレオチド、 該ポリヌクレオチドに対するアンチセンスポリヌクレオチドおよ び該蛋白質を認識する抗体に関するものである。 背景技術  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. Background art
癌の発症は遺伝子の何らかの異常によって起こることが知られており、 特に、 遺伝子の転写レベルでの変動異常が癌の発症の主たる原因となると考えられてい る。  It is known that the onset of cancer is caused by some abnormality in a gene. In particular, it is thought that abnormal fluctuation in the transcription level of a gene is the main cause of the onset of cancer.
このような遺伝子転写における中心的な因子として、 TBP (TATA b i nd i ng p ro t e i n) が、 生物の全遺伝子の転写に関わっていることが 知られている。 具体的には、 DNAからmRNAへの翻訳が行ゎれるときにTB Pが該 DN Aのプロモーター領域に結合することで遺伝子の転写制御が行われて いる。 TBPが他の多様な蛋白質と複合体を形成することで、 その相手の蛋白質 や複合体の構造により、 該複合体が、 各遺伝子のそれそれ異なる転写制御に対応 するだけの機能を持つことになるからであると考えられている。  It is known that TBP (TATAbindngprotein) is involved in the transcription of all genes in an organism as a central factor in such gene transcription. Specifically, transcription of a gene is controlled by binding of TBP to the promoter region of the DNA during translation from DNA to mRNA. When TBP forms a complex with various other proteins, the complex has the function to respond to the different transcriptional regulation of each gene depending on the structure of the partner protein and complex. It is thought that it becomes.
本発明者らは、 TBPと結合する蛋白質として T I P 49蛋白質、 それをコ ードする遺伝子を既に単離同定した （B i o c h em. B i ophy s. R Θ s . C ommun. ， 235, 64— 68) 。 発明の開示 本発明は、 T I P 49の一部分との相同性を有する新規な蛋白質を提供するこ とを目的とする。 The present inventors have already isolated and identified the TIP49 protein as a protein that binds to TBP and the gene encoding it (Biochem. Biophys. R Rs. Commun., 235, 64). — 68). Disclosure of the invention An object of the present invention is to provide a novel protein having homology to a part of TIP49.
また、 本発明は、 前記蛋白質をコードするポリヌクレオチドを提供することを 目的とするものである。  Another object of the present invention is to provide a polynucleotide encoding the protein.
さらに、 本発明は、 前記蛋白質を認識する抗体を提供することを目的とするも のである。  Furthermore, another object of the present invention is to provide an antibody that recognizes the protein.
本発明者は、 T I P 49の一部と相同性を有する蛋白質をコードする cDNA を単離し、 その塩基配列を決定した。 そして、 該 c DNAがコードするアミノ酸 配列を決定した。 さらに、 本発明者は、 この cDN Aがコードする組換え蛋白質 を作製し、 該組み換え蛋白質の分子量が 5 OkDであることを確認した。 本発明 者は、 この蛋白質を HE L 50と名付けた。 さらに、 本発明者は、 HE L 50が DN Aヘリケース活性を有することを確認した。 なお、 HE L 50が由来する動 物を明示する場合は、 例えばヒト HE L 50のように、 HE L 50の前に由来す る動物名を付けて表記する。 HEL 50と表記した場合は、 真核生物である生物 に由来する HE L 50を意味する。  The present inventors isolated a cDNA encoding a protein having homology to a part of TIP49 and determined the nucleotide sequence thereof. Then, the amino acid sequence encoded by the cDNA was determined. Further, the present inventors produced a recombinant protein encoded by the cDNA, and confirmed that the molecular weight of the recombinant protein was 5 OkD. The present inventors named this protein HEL50. Furthermore, the present inventors have confirmed that HE L50 has DNA helicase activity. When the animal from which HEL50 is derived is specified, an animal name derived from before HEL50, such as human HEL50, is added. The notation HEL50 means HEL50 derived from an eukaryote.
具体的には、 本発明は、 以下の蛋白質を提供する。  Specifically, the present invention provides the following proteins.
1. 配列表の配列番号 1に記載のアミノ酸配列からなる蛋白質。 この蛋白質は DNAヘリケース活性を有する。 この蛋白質がヒト HE L 50である。  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 HE L50.
2. 配列表の配列番号 1に記載のアミノ酸配列において一または複数のァミノ 酸が置換、 欠失または付加されたアミノ酸配列からなり、 かつ DNAヘリケース 活性を有する蛋白質。 以下、 この蛋白質をヒト HEL 50変異体という。 配列表 の配列番号 1に記載のァミノ酸配列の一部からなり、 かつ D N Aヘリケース活性 を有する蛋白質は、 ヒト HE L 50変異体の一態様である。  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 which has 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 an embodiment of a human HEL50 mutant.
3. 配列表の配列番号 1に記載のアミノ酸配列と 65%以上のホモロジ一を有 し、 かつ DNAヘリケース活性を有する蛋白質。 この蛋白質は、 真核生物の HE L 50である。 4. 配列表の配列番号 12に記載のアミノ酸配列からなる蛋白質。 この蛋白質 は、 酵母 HE L 50であり、 前記 3に記載の真核生物の HE L 50の一態様であ る。 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. This protein is the eukaryotic HE L50. 4. A protein comprising the amino acid sequence of SEQ ID NO: 12 in the sequence listing. This protein is yeast HEL50, and is one embodiment of the eukaryotic HEL50 described in 3 above.
また、 本発明は、 以下のポリヌクレオチドを提供するものである。 なお、 本 明細書においては、 ポリヌクレオチドとは、 DNAや RNA、 またはそれらに塩 基、 リン酸、 糖からなるヌクレオチドが複数結合したものを、 天然には存在しな いものを含めていう。  Further, the present invention 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 a base group, a phosphate, or a sugar are bonded thereto, including those that do not exist in nature.
5. 前記 1ないし 4のいずれか一つに記載の蛋白質をコードするポリヌクレオ チド。  5. A polynucleotide encoding the protein according to any one of 1 to 4 above.
6. 配列表の配列番号 2に記載の塩基配列の 1番目の Aから 1389番目の C までの塩基配列を有する DNA。 配列表の配列番号 2に記載の塩基配列の 1番目 の Aから 1389番目の Cまでの塩基配列は、 ヒト HE L 50のコード領域の塩 基配列であり、 この塩基配列を有する DN Aを、 以下ではヒト HEL 50DNA という。 なお、 c DNAは D NAに含まれる力 本明細書では、 当該 DNAが c DNAであることを特に明示する場合には、 cDNAと表記する。  6. DNA having a nucleotide sequence from the 1st A to the 1389th C of the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing. The nucleotide sequence from the 1st A to the 1389th C of the nucleotide sequence described in SEQ ID NO: 2 in the sequence listing is the nucleotide sequence of the coding region of human HEL50. Hereinafter, it is referred to as human HEL50 DNA. Note that cDNA is a force contained in DNA. In this specification, cDNA is referred to as cDNA when it is specifically indicated that the DNA is cDNA.
7. 配列表の配列番号 2に記載の塩基配列からなる DNA。 この DNAは前記 6に記載の D N Aの一態様である。  7. DNA comprising the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. This DNA is one embodiment of the DNA described in 6 above.
8. 配列表の配列番号 1 3に記載の塩基配列からなる DNA。 この DNAは、 酵母 HE L 50 DNAである。  8. DNA comprising the nucleotide sequence of SEQ ID NO: 13 in the sequence listing. This DNA is yeast HE L50 DNA.
9.前記 6ないし 8のいずれか一つに記載の DNAがコ一ドする RNA。以下、 ヒト HE L 50 DNAがコードする RNAをヒト HE L 50 RNAという。なお、 mRNAは RNAに含まれる力 本明細書では、 当該 R N Aが mR N Aであるこ とを特に明示する場合には、 mRNAと表記する。  9. An RNA encoded by the DNA according to any one of 6 to 8 above. Hereinafter, the RNA encoded by the human HE L50 DNA is referred to as human HE L50 RNA. Note that mRNA is a force contained in RNA. In this specification, when it is particularly specified that the RNA is mRNA, it is referred to as mRNA.
10. 前記 6ないし 8のいずれか一つに記載の DNAのアンチセンス DNA、 前記 9に記載の RNAのアンチセンス RNA、 またはそれらの誘導体からなるァ ンチセンスポリヌクレオチド。 なお、 アンチセンスポリヌクレオチドは、 ポリヌ クレオチドに含まれるが、 本明細書では、 当該ポリヌクレオチドがアンチセンス 鎖であることを特に明示するときには、 アンチセンスポリヌクレオチドと表記す る。 10. An antisense polynucleotide comprising the antisense DNA of the DNA described in any one of the above 6 to 8, the antisense RNA of the RNA described in the above 9, or a derivative thereof. The antisense polynucleotide is a polynucleotide Although contained in the nucleotide, in the present specification, when it is particularly specified that the polynucleotide is an antisense strand, it is referred to as an antisense polynucleotide.
1 1. 前記 5ないし 10のいずれか一つに記載のポリヌクレオチドのうちの一 部であって、 連続する 12塩基以上からなるポリヌクレオチド。  1 1. A part of the polynucleotide according to any one of the above 5 to 10, which is a polynucleotide comprising 12 or more consecutive bases.
12. 化学修飾された前記 5ないし 1 1項のいずれか一つに記載のポリヌクレ ォチド。  12. The polynucleotide according to any one of the above items 5 to 11, which is chemically modified.
また、 本発明は、 前記 1ないし 3のいずれか一つに記載の蛋白質を認識する抗 体、 およびモノクローナル抗体の活性フラグメントを提供するものである。 本明 細書では、 抗体とは、 抗血清、 ポリクローナル抗体またはモノクローナル抗体の 全てを含み、 さらに抗体の活性フラグメントも含む。 図面の簡単な説明  The present invention also provides an antibody that recognizes the protein described in any one of 1 to 3 above, and an active fragment of a monoclonal antibody. As used herein, antibody includes all antisera, polyclonal or monoclonal antibodies, as well as active fragments of the antibody. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 ヒト各組織由来の mRNAについて HE L 50遺伝子の一部をプロ一 ブとしてノ一ザンブロットハイプリダイゼーションした結果を示す電気泳動写真 である。  FIG. 1 is an electrophoretic photograph showing the results of Northern blot hybridization of mRNA derived from human tissues using a part of the HEL50 gene as a probe.
図 2は、 実施例 3で HE L 50遺伝子を導入するのに使用した pET— 3 aH i sベクターを示す図である。  FIG. 2 is a diagram showing the pET-3aHis vector used to introduce the HEL50 gene in Example 3.
図 3は、 MonoQ (登録商標） で精製した HE L 50の電気泳動の結果を示 す写真である。  FIG. 3 is a photograph showing the results of electrophoresis of HEL50 purified by MonoQ (registered trademark).
図 4は、 MonoQ (登録商標） で精製した HE L 50と基質 DNAとを反応 させた反応物を電気泳動した結果を示す電気泳動写真である。  FIG. 4 is an electrophoresis photograph showing the result of electrophoresis of a reaction product obtained by reacting HEL50 purified by MonoQ (registered trademark) with substrate DNA.
図 5は、 本発明で用いる一つの方向性基質の作製の概要を示す図である。 図 6は、 図 5に示した方向性基質と HE L 50または T I P 49とを反応させ た反応物を電気泳動した結果を示す電気泳動写真である。 発明を実施するための最良の形態 FIG. 5 is a diagram showing an outline of production of one directional substrate used in the present invention. FIG. 6 is an electrophoretic photograph showing the result of electrophoresis of a reaction product obtained by reacting the directional substrate shown in FIG. 5 with HEL50 or TIP49. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の HE L 50および HE L 50変異体は、 D N Aヘリケース活性を有す ることを特徴とするが、 これらについては、 例えば実施例 4で開示した方法を用 いて確認できる。  The HEL50 and HEL50 mutants of the present invention are characterized by having DNA helicase activity, which can be confirmed, for example, by using the method disclosed in Example 4.
本発明の HEL 50変異体は公知の方法を使用して作製できる。 例えば、 HE The HEL50 mutant of the present invention can be prepared using a known method. For example, HE
L 50変異体をコードする DNAを作製して、該 DNAを適当な宿主に組み込み、 得られる形質転換体に HE L 50変異体を発現させることができる。 HEL 50 変異体をコードする DN Aは、 例えば、 制限酵素を利用して、 HEL 50DNA の所望の部位の塩基を欠失若しくは他の塩基に置換し、 または所望の部位に塩基 を挿入して得られる。 また、 サイ トディレクトミュー夕ジヱネシスの方法によつ ても HE L 50変異体を作製することができる。 また、 ハイブリダィズの条件を 緩くした P CRによりランダムミュー夕ジエネシスを起こすこともできる。 A DNA encoding the L50 mutant is prepared, the DNA is incorporated into an appropriate host, and the resulting transformant can express the HEL50 mutant. DNA encoding the HEL50 mutant can be obtained by, for example, using a restriction enzyme to delete or substitute a base at a desired site of HEL50 DNA with another base, or to insert a base at a desired site. Can be The HEL50 mutant can also be produced by the method of site-directed mutagenesis. In addition, random mugenesis can be caused by a PCR with relaxed hybridization conditions.
本発明の HE L 50または HE L 50変異体をコードする DNAまたは RNA は、 縮重により存在しうる全ての塩基配列からなるものである。  The DNA or RNA encoding HEL50 or HEL50 mutant of the present invention is composed of all nucleotide sequences that can exist due to degeneracy.
D N Aのセンス鎖または RN Aについては、 その塩基配列と相補的な塩基配列 からなるアンチセンス DN Aまたはアンチセンス RN Aがそれぞれ存在する。 本 明細書では、 特に断りがない限り、 DNA (cDNAを含む） はセンス鎖とアン チセンス鎖の二本鎖からなるものを指し、 RNAは一本鎖からなるものを指し、 アンチセンス DN Aまたはアンチセンス RN Aは一本鎖からなるものを指す。 本明細書において、 ポリヌクレオチドとは、 DNAまたはRNA、 或いはそれ らにさらに塩基、 リン酸、 糖からなるヌクレオチドが 1または複数結合したもの をいい、 天然に存在するもの、 または天然に存在しないもののいずれも含む。 ァ ンチセンスポリヌクレオチドについても同様である。  For the sense strand or RNA of DNA, an antisense DNA or antisense RNA having a nucleotide sequence complementary to the nucleotide sequence exists, respectively. In this specification, unless otherwise specified, DNA (including cDNA) refers to a double-stranded sense strand and antisense strand, RNA refers to a single-stranded strand, and antisense DNA or Antisense RNA refers to one consisting of a single strand. As used herein, the term "polynucleotide" refers to DNA or RNA, or one or more nucleotides consisting of bases, phosphates, and sugars linked to them, and includes naturally occurring or non-naturally occurring ones. Including both. The same applies to antisense polynucleotides.
本発明のアンチセンスポリヌクレオチドは、 その立体構造および機能がポリヌ クレオチドと類似する誘導体を全て含む。 誘導体とは、 例えば、 アンチセンスポ リヌクレオチドの 3， 末端若しくは 5， 末端に他の物質が結合したものやアンチ センスポリヌクレオチドの塩基、糖、 リン酸の少なくともいずれか一部において、 置換や欠失や付加といった修飾が生じた物質、 或いは天然に存在しないような塩 基、 糖、 リン酸を有するものや、 糖一リン酸骨格以外の骨格を有するものである。 本発明のアンチセンスポリヌクレオチドまたはその誘導体は、 HE L 50をコ 一ドするポリヌクレオチドのコ一ド領域のいかなる部分にハイブリダィズするも のであってもよい。 特に、 mRNAの一部に対して相補的な塩基配列を有し、 該 m R N Aにハイブリダィズするものが好ましい。 The antisense polynucleotides of the present invention include all derivatives whose tertiary structure and function are similar to polynucleotides. Derivatives are, for example, antisense polynucleotides that have other substances bound to the 3, 5 or 5, A substance in which a modification such as substitution, deletion, or addition has occurred in at least a part of the base, sugar, or phosphate of the sense polynucleotide, or a substance having a non-naturally occurring base, sugar, or phosphate; It has a skeleton other than the sugar monophosphate 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. Particularly, those having a base sequence complementary to a part of the mRNA and hybridizing to the mRNA are preferable.
アンチセンスポリヌクレオチドは、  Antisense polynucleotides are
1. 遺伝子から p r e _ m R N Aへの転写段階、  1. the transcription stage from the gene to pre_mRNA,
2. p r e—mRNAから成熟 mRNAへのプロセッシング段階、 2. The processing stage from p r e—mRNA to mature mRNA,
3. mRNAが核膜通過する段階、  3. mRNA passes through the nuclear membrane,
4. mRNAから蛋白質への翻訳段階  4. Translation stage from mRNA to protein
のいずれかで、 遺伝情報を担う DN Aまたは RN Aにハイブリダィズし、 遺伝情 報の伝達の正常な流れに影響を与えて蛋白質の生合成を阻害することにより該蛋 白質の発現を調節すると考えられている。 In one of the two ways, it is thought to hybridize to DNA or RNA, which carries the genetic information, and to regulate the expression of the protein by affecting the normal flow of genetic information and inhibiting protein biosynthesis. Have been.
アンチセンスポリヌクレオチドが RN Aにハイブリダィズし易くするために、 一般的には、 R N Aのループ領域の塩基配列に相補的な塩基配列を有するアンチ センスポリヌクレオチドを設計するとよいとされている。 また、 RNAの翻訳開 始コドン付近、 リボソーム結合部位、 キヤッビング部位またはスプライス部位の 配列に相補的な配列を有するようなアンチセンスポリヌクレオチドは、 一般に生 合成を阻害する効果が大きいことが期待できる。  In order to facilitate the hybridization of the antisense polynucleotide to RNA, it is generally said that an antisense polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the RNA loop region should be 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 cabling site or a splice site can generally be expected to have a large effect of inhibiting biosynthesis.
したがって、 本発明のアンチセンスポリヌクレオチドまたはその誘導体であつ て、 HEL 50RNAのループ領域、 翻訳開始コドン付近、 リボソーム結合部位、 キヤッビング部位またはスプライス部位の塩基配列に相補的な塩基配列を持つも のは、 HEL 50の生合成を阻害する効果が大きいことが期待される。  Therefore, the antisense polynucleotide of the present invention or a derivative thereof having a nucleotide sequence complementary to the nucleotide sequence of the loop region of the HEL50 RNA, the vicinity of the translation initiation codon, the ribosome binding site, the cabling site or the splice site, The effect of inhibiting the biosynthesis of HEL50 is expected to be large.
アンチセンスポリヌクレオチドは、 特定の蛋白質の生合成を阻害する点で、 特 定の遺伝子や RNAにハイブリダィズするものが好ましい。 このため、 12以上 の塩基からなるものが好ましく、 1 6以上の塩基からなるものが特に好ましい。 一方、 細胞膜透過性の点では 35以下の塩基からなるものが好ましい。 Antisense polynucleotides are unique in that they inhibit the biosynthesis of certain proteins. Those that hybridize to a certain gene or RNA are preferred. For this reason, those comprising 12 or more bases are preferred, and those comprising 16 or more bases are particularly preferred. On the other hand, those having 35 or less bases are preferable in terms of cell membrane permeability.
アンチセンスポリヌクレオチド誘導体は、 ヌクレア一ゼ耐性、 組織選択性、 細 胞膜透過性または結合力の少なくとも一つが高められた誘導体であることが好ま しい。 特に好ましい該誘導体は、 フォスフォロチォェ一ト結合を骨格構造として 有する誘導体である。本発明のアンチセンスポリヌクレオチド誘導体についても、 これらの機能または構造を有する誘導体が含まれる。  The antisense polynucleotide derivative is preferably a derivative having at least one of increased nuclease resistance, tissue selectivity, cell membrane permeability, or avidity. Particularly preferred derivatives are those having a phosphorothioate bond as a skeletal structure. The antisense polynucleotide derivative of the present invention also includes derivatives having these functions or structures.
本発明のアンチセンスポリヌクレオチドおよびその誘導体の製造方法としては、 例えば、 天然型の DNAや RNAであれば、 化学合成機を使用して合成したり、 HE L 50 c DNAを铸型として P CR法を行うことが挙げられる。 また、 メチ ルフォスフォネート型やフォスフォロチォェ一ト型等、 誘導体の中には、 化学合 成機 （例えば、 八81社製394型） を使用して合成できるものもある。 この場 合には、 化学合成機に添付されている説明書にしたがって操作を行い、 得られた 合成産物を、 逆相クロマトグラフィー等を用いた HPLC法により精製すること により、 目的のアンチセンスポリヌクレオチドまたはその誘導体を得ることがで きる。  Examples of the method for producing the antisense polynucleotide and the derivative thereof of the present invention include, for example, natural DNA and RNA, which can be synthesized using a chemical synthesizer, and Performing the method. Some derivatives such as a methylphosphonate type and a phosphorothioate type can be synthesized using a chemical synthesizer (for example, Model 394 manufactured by 881). In this case, the desired antisense poly- mer is purified by performing the operation according to the instructions attached to the chemical synthesizer, and purifying the resulting synthetic product by HPLC using reverse phase chromatography or the like. Nucleotides or derivatives thereof can be obtained.
本発明のポリヌクレオチドまたはアンチセンスポリヌクレオチドの全長または その一部を、 HE L 50遺伝子や HE L 50 c DNA、 或いは HEL 50mRN Aを検出するためのプロ一ブまたは P CRのプライマ一として用いることができ る。  Use of the full length or a part of the polynucleotide or antisense polynucleotide of the present invention as a probe or PCR primer for detecting HEL50 gene, HEL50 cDNA, or HEL50 mRNA. Can be done.
さて、 ヒトの蛋白質の種類は 3 X 1 09個といわれている。 1 6塩基の DNA は 416種類存在するので、 この長さの D N Aがあればヒトの蛋白質を全て識別 できる。 すなわち、 プロ一ブまたはプライマ一として必要な長さは、 理.論的には 16塩基である。 実用上もこの長さ以上であることが望ましいことは言うまでも ないが、 実用的には 12塩基以上で用いられることが多い。 また、 プローブまたはプライマ一として用いる箇所は、 非コード領域、 コード 領域のいずれも使用可能である。 GC含有率が 30ないし 70%であるものは、 ポリヌクレオチドの立体構造の問題が生じにく くハイプリダイズし易いので、 好 ましい。 Well, kind of protein in humans is said to be 3 X 1 0 9 pieces. Since there are 416 types of 16- base DNA, any DNA of this length can identify all human proteins. 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 in practical use, but practically, 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 the problem of the three-dimensional structure of the polynucleotide and easily hybridize.
HE L 50遺伝子を検出する方法としては具体的には、 ノーザンプロットハイ ブリダィゼーシヨン法や R T— P C R法 （ 『（ u r r e n t Pr o t o c o l s i n Mo l e cu l ar B i o l o gy』 (Gr e ene P u b 1 i s h i n g As s o c i at e s and Wi l e y— I nt e r s c i e n c e ) Chap t e r l 5. 1. 1 - 15. 1. 9および同書 15. 4. 1 - 15. 4. 6) またはインサイチュハイブリダィゼ一シヨン法 （同書 Chap t e r 14. 3. 1 - 14. 3. 14) が挙げられる。  Specific examples of the method for detecting the HEL50 gene include the Northern blot hybridization method and the RT-PCR method (“(urrent Pr otocolsin Molecular Biology” (Greene Pub). 1 ishing As soci at es and Wiley—Interscience) Chapterl 5. 1. 1-15. 1. 9 and ibid 15. 4. 1-15. 4. 6) or in situ hybridization method (Chapter 14.3.1-14.3.14 in the same book).
なお、 ハイブリダィズの条件は、 プローブの長さや使用するメンブランにより 最適な条件が異なる。 つまり、 ハイブリダィズ条件は自ずから或る幅をもつもの である。 本発明の実施例では、 使用したメンブランの性質とプローブの長さにお ける最適な条件を開示するものであり、 メンブランやプローブの長さが異なれば 当然異なるハイブリダイズ条件でもハイプリダイズし得る。  The optimum conditions for hybridization differ depending on the length of the probe and the membrane used. In other words, the hybridization condition naturally has 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 be performed under different hybridization conditions.
DN Aまたは RN Aを化学合成するときに、 標識すること、 ピオチン化するこ と、 側鎖をメチル化すること、 またはリン酸基部分の 0を Sに置換すること等の 化学修飾することはよく知られている。 例えば、 配列表の配列番号 2に記載の D N Aを化学合成するときに、 前記の化学修飾をして、 配列表に示された DN Aそ のものと異なるものを合成することが可能である。 また、 cDNAライブラリー から取得された c D N Aであっても放射性同位体で標識することが可能である。 従って、 本発明の DNAおよび RNAは、 上記の化学修飾された DNA、 RN Aまたはアンチセンスポリヌクレオチドをその範囲に含むものである。 本発明の 化学修飾された DNAまたは RNAは、 蛋白質をコードする機能またはプローブ としての機能をいずれも発揮可能なものであり、 本発明の化学修飾されたアンチ センスポリヌクレオチドは、 蛋白質の生合成を阻害する機能またはプローブとし ての機能をいずれも発揮可能なものである。 When chemically synthesizing DNA or RNA, labeling, biotinylation, methylation of the side chain, or chemical modification such as substitution of S in the phosphate group with 0 is not possible. well known. For example, when chemically synthesizing the DNA described in SEQ ID NO: 2 in the sequence listing, it is possible to synthesize a DNA different from the DNA itself shown in the sequence listing by performing the above chemical modification. In addition, 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 within its scope. The chemically modified DNA or RNA of the present invention can exhibit both the function of encoding a protein and the function as a probe, and the chemically modified DNA or RNA of the present invention. The sense polynucleotide can exhibit both the function of inhibiting protein biosynthesis and the function as a probe.
本発明は、 HEL 50をヒト以外の動物に免疫感作することにより得られる抗 体であって、 本発明の HE L 50を認識することがウエスタンプロッ ト法、 EL I S A法や免疫染色法 （例えば F ACSでの測定） 等により確認される抗体をそ の範囲内に含む。 なお、 免疫感作する動物は、 免疫原として投与する HEL 50 が由来する動物以外かつヒト以外の動物から選択することが好ましい。 例えば、 ラット HEL 50であれば、 ラッ ト以外かつヒト以外の動物 （例を挙げれば、 ゥ サギゃマウス等） に免疫感作することが好ましい。  The present invention relates to an antibody obtained by immunizing a non-human animal with HEL50, and it is confirmed that the HEL50 of the present invention can be recognized by Western blotting, ELISA, immunostaining, or the like. For example, the antibody confirmed by FACS) is included in the range. The animal to be immunized is preferably selected from animals other than the animal from which the 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 an animal other than a rat and a human (eg, a heron mouse).
また、 免疫原として、 蛋白質の一部分をゥシ血清アルブミン等の他のキャリア 一蛋白質に結合させたものを用いることは、 よく用いられる方法である。 蛋白質 の一部分は、 例えばペプチド合成機を用いて合成してもよい。 なお、 蛋白質の一 部分は、 8以上のアミノ酸残基からなるものであることが好ましい。  It is a commonly used method to use an immunogen obtained by binding a part of a protein to another carrier protein such as serum albumin. A part of the protein may be synthesized using, for example, a peptide synthesizer. Preferably, a part of the protein is composed of eight or more amino acid residues.
免疫感作した動物から採血した血液から免疫グロブリンを精製して抗血清やポ リクローナル抗体が得られるならば、 該免疫した動物のリンパ球をミエローマ細 胞等と細胞融合させて得られたハイプリ ドーマによりモノクローナル抗体が産生 されることはよく知られている （ 『An t i b o d i e s A Lab o r at o r y Manua l』 (Co l d S r i n Harb o r Lab o r a t o r y P r e s s, 1988 ) 、 14 1— 147) 。  If an antiserum or a polyclonal antibody can be obtained by purifying immunoglobulin from blood collected from the immunized animal, a hybridoma obtained by cell fusion of lymphocytes of the immunized animal with myeloma cells or the like can be used. Is well known to produce monoclonal antibodies (Antibodies A Laboratory or Manual, Cold Srin Harb or Laboratory Press, 1988, 141-147).
本発明の抗体はその活性フラグメントをも含む。 活性フラグメントとは、 抗原 抗体反応活性を有する抗体のフラグメントを意味し、 具体的には、 F (ab' ) 2 、 Fab' 、 Fab, F v等を挙げることができる。 例えば、 本発明の抗体 をペプシンで分解すると F (ab' ) 2 が得られ、 パパインで分解すると F a b が得られる。 F (ab' ) , を 2—メルカプトエタノール等の試薬で還元して、 モノョード酢酸でアルキル化すると Fab， が得られる。 Fvは重鎖可変領域と 軽鎖可変領域とをリンカ一で結合させた一価の抗体活性フラグメントである。 こ れらの活性フラグメントを保持し、 その他の部分を他の動物のフラグメン卜に置 換することでキメラ抗体が得られる。 The antibodies of the present invention also include active fragments thereof. The active fragment means a fragment of an antibody having antigen-antibody reaction activity, and specific examples include F (ab ') 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, Fab is obtained. Reduction of F (ab '), with a reagent such as 2-mercaptoethanol and alkylation with monoacetic acid gives Fab ,. Fv is a monovalent antibody active fragment obtained by linking the heavy chain variable region and the light chain variable region with a linker. This A chimeric antibody can be obtained by retaining these active fragments and replacing the other parts with fragments of another animal.
HE L 50の検出については、 抗体を用いる方法や抗体と酵素反応とを利用す る方法が挙げられる。 抗体を用いる方法としては具体的には、 （ 1) HEL 50 を認識する抗体を用いて HE L 50を検出する方法、 （2) HEL 50を認識す る抗体および該抗体の標識二次抗体を用いて HE L 50を検出する方法が挙げら れる。 標識としては、 例えば放射性同位元素 （R I ) 、 酵素、 アビジンまたはビ ォチン、 若しくは蛍光物質 （F I TCやローダミン等） が利用される。 抗体と酵 素反応とを利用する方法としては、 例えば、 EL I SAが挙げられる。 実施例  The detection of HEL50 includes a method using an antibody and a method using an antibody and an enzyme reaction. Specific examples of the method using an antibody include (1) a method of detecting HEL50 using an antibody that recognizes HEL50, (2) an antibody that recognizes HEL50, and a labeled secondary antibody of the antibody. And a method for detecting HEL50. 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 enzyme reaction, for example, ELISA is mentioned. Example
以下に実施例を示し、 本発明をさらに詳述するが、 本発明はこれらの実施例に 限定されるものではない。  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.
(実施例 1) HEL 50 cDNAの単離および HE L 50のアミノ酸配列の決 定  (Example 1) Isolation of HEL50 cDNA and determination of amino acid sequence of HEL50
I E STデータベースの検索  Search the I E ST database
ィン夕ーネッ卜の E S Tデータベース （mRN Aの断片の配列を登録してある デ一夕べ一ス、 h t t p ： / / www. nc b i . n 1 m. n i h . gov. Z dbEST) に登録された配列 （E ST) から、 T I P 49遺伝子がコードする アミノ酸配列とホモロジ一のあるアミノ酸配列をコードする塩基配列を、 ィン夕 —ネットのゲノムネット WWWサーバ一中の T B L A S TN (h t t p ： //w ww. b l as t, genome, ad. j p) を使用して、 dbE STから検 索した。 その結果、 R 1 909 1の番号で登録されている E S Tおよび AA 37 4580の番号で登録されている E STを抽出した。  Registered in the EST database (http: //www.ncbi.n1m.nih.gov.ZdbEST) where the sequence of the mRNA fragment is registered. From the sequence (EST), the nucleotide sequence encoding an amino acid sequence homologous to the amino acid sequence encoded by the TIP 49 gene was converted to the TBLAS TN (http: // w ww. blast, genome, ad. jp) and searched from dbEST. As a result, EST registered under the number of R 19091 and EST registered under the number of AA 374580 were extracted.
I I HE L 5 ODN Aの単離および塩基配列の決定  Isolation and nucleotide sequence determination of I I HE L5 ODN A
1. プライマ一の合成 以下のアンカ一プライマ一、 R 1 909 1の塩基配列から以下のプライマー P 1、 および A A 374580の塩基配列から以下のプライマ一 P 2をそれそれ D NA合成機 （AB I社製、 392型） を使用して作製した。 1. Primer synthesis The following primer P1, the following primer P1 from the base sequence of R19091 and the following primer P2 from the base sequence of AA 374580: DNA synthesizer (ABI, Model 392) It was prepared using.
アンカ一プライマー： 5， —CTGGTT CGGC CCA— 3， （配列表の配 列番号 3に記載の塩基配列） Anchor primer: 5, -CTGGTT CGGC CCA-3, (base sequence described in SEQ ID NO: 3 in Sequence Listing)
P 1 ： 5 ' -GAGAT C CGTG ATGT AACAAG GATTGAG - 3， （配列表の配列番号 4に記載の塩基配列）  P 1: 5'-GAGAT C CGTG ATGT AACAAG GATTGAG-3, (base sequence described in SEQ ID NO: 4 in Sequence Listing)
P 2 ： 5 ' -CTTGGTCTGG GAGC C CATAG CGT CG- 3 ' (配列表の配列番号 5に記載の塩基配列）  P 2: 5'-CTTGGTCTGG GAGC C CATAG CGT CG-3 '(base sequence described in SEQ ID NO: 5 in Sequence Listing)
合成したプライマーは、 蒸留水で 20 pmo 1/〃 1に調製した。 これを PC The synthesized primer was adjusted to 20 pmo1 / 1/1 with distilled water. This is a PC
Rプライマ一として用いて、 以下の P CR操作を行った。 The following PCR operation was performed using the R primer.
2. PCR  2. PCR
c DNAライブラリ一には、 マラソン （登録商標） ヒト肝臓 cDNAライブラ リー （クロ一ンテック社製） を用いて、 以下の条件で P CRを行った。  For the cDNA library, PCR was performed under the following conditions using Marathon (registered trademark) human liver cDNA library (Clontech).
まず、 プライマーとしてアンカープライマーと P 2プライマ一を使用し、 Ta kar a Taq (登録商標、 宝酒造社製） を使用して以下の条件で行った。  First, an anchor primer and a P2 primer were used as primers and Takar a Taq (registered trademark, manufactured by Takara Shuzo) was used under the following conditions.
94°Cに 1分間おいた。 次いで 「94 で45秒間、 続いて 60°Cで 2分間、 続いて 72 °Cで 2分間」 のサイクルを 30回繰り返した。 その後、 4°Cに置いて PCR操作を完了した。  Placed at 94 ° C for 1 minute. Then, a cycle of “45 seconds at 94, followed by 2 minutes at 60 ° C., and then 2 minutes at 72 ° C.” was repeated 30 times. Thereafter, the temperature was kept at 4 ° C. to complete the PCR operation.
P C Rの反応液の組成は下記の通りとした。  The composition of the reaction solution of PCR was as follows.
0. 5 ng/u 1 c DNAライブラリー 1〃 1  0.5 ng / u 1 c DNA library 1〃 1
Taq po 1 (宝酒造社製） 0. 1〃 1 Taq po 1 (Takara Shuzo) 0.1 1 0 1
10倍濃度 P CR緩衝液 （宝酒造社製） 2μΛ  10 times concentration PCR buffer (Takara Shuzo) 2μΛ
dNTP mix (宝酒造社製） 1. 6 1  dNTP mix (Takara Shuzo) 1. 6 1
アンカープライマー 2 1 Anchor primer 2 1
プライマー P 2 2 μΛ 滅菌蒸留水 1 1. 3〃 1 合計 20〃 1 Primer P 2 2 μΛ Sterile distilled water 1 1. 3〃 1 Total 20〃 1
その後、 前記で得た PCR産物を铸型として、 P 1プライマーと P 2プライマ 一を用いて 2度目の PCR操作を行った。 2度目の P CR操作は、 前記と同様に 行った。  Thereafter, a second PCR operation was carried out using the P1 primer and the P2 primer as the 铸 -type PCR product obtained above. The second PCR operation was performed as described above.
3. DN Aプローブの作製  3. Preparation of DNA probe
前記の PCR操作で増幅した DNA断片 （以降、 フラグメント Aということが ある） をミニゲル電気泳動 （0. 75%ァガロースゲル） させて、 フラグメント Aのバンドをゲルから切り出した。 Gene C l e an (バイオ 10 1社製） で フラグメント Aを回収して、 ミニゲル電気泳動でバンドをチェックした。  The DNA fragment amplified by the above PCR operation (hereinafter sometimes referred to as fragment A) was subjected to minigel electrophoresis (0.75% agarose gel), and the fragment A band was cut out from the gel. Fragment A was collected using GeneClean (manufactured by Bio101) and bands were checked by minigel electrophoresis.
前記で回収したフラグメント Aを、 pGEM—T Ea s y (プロメガ社製） を用いてプラスミ ドベクター p GEM— T E a s yに挿入した。 その後、 フラ グメント Aを挿入した pGEM— T Ea syを大腸菌に導入し、 該大腸菌を培 養し、 現れた白色コロニーの大腸菌から Mi d iキッ ト （キアゲン社製） を用い てプラスミ ド DNAすなわちフラグメント Aを調製した。 このフラグメント Aに ついて、 ダイターミネ一夕一法により、 ォ一トシ一クェンサ一 （AB I社製、 3 73 A型） を用いて、 DNAシークェンスを行い、 その塩基配列を確認した。 前記白色コロニーを形成した大腸菌から調製したフラグメント Aを E c oR I で切り出し、 得られた DN A断片をランダムラベリングキット （ベ一リンガマン ハイム社製）を使用して、下記の組成の液中に 37°Cに 30分間おいた後、 65°C に 10分間おいて標識した。  The fragment A recovered above was inserted into the plasmid vector pGEM-TEASy using pGEM-TEASy (promega). Thereafter, the pGEM-Teasy into which the fragment A was inserted was introduced into E. coli, the E. coli was cultured, and plasmid DNA, ie, a fragment, was obtained from the white colonies that appeared by using a Midi kit (Qiagen). A was prepared. The DNA sequence of the fragment A was determined by the Dye-Mine-To-One method using a DNA sequencer (manufactured by ABI, type 373A), and the nucleotide sequence was confirmed. Fragment A prepared from Escherichia coli that formed the white colony was excised with EcoR I, and the obtained DNA fragment was dissolved in a liquid having the following composition using a random labeling kit (manufactured by Behringaman Heim). After 30 minutes at 37 ° C, labeling was performed at 65 ° C for 10 minutes.
フラグメント A 9 1 Fragment A 9 1
d A, G, TTP mix (ベーリンガーマンハイム社製） 3〃1 d A, G, TTP mix (Boehringer Mannheim) 3〃1
反応液 m i X (ベーリンガーマンハイム社製） 2〃1 Reaction liquid miX (Boehringer Mannheim) 2〃1
d-32P-d CTP (アマシャムフアルマシア社製） 5 1 d- 32 Pd CTP (manufactured by Amersham Fulmasia) 5 1
クレノウ液 （ベーリンガーマンハイム社製） 1〃1 合計 20 1 Klenow liquid (Boehringer Mannheim) 1〃1 Total 20 1
この液から標識フラグメント Aを G— 50マイクロスピンカラムで精製した。 スウィングローターでカラムを 3000 r pmで 1分間回転させ、 パッキング を行った。 そのカラムに標識フラグメント A液を 20 /1添加し、 3000 r p mで 2分間カラムを回転させ、 T 50 Eで溶出して標識フラグメント Aを分離し た。  From this solution, labeled fragment A was purified using a G-50 micro spin column. Packing was performed by rotating the column at 3000 rpm for 1 minute using a swing rotor. 20/1 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. ハイブリダィゼ一シヨン  4. Hybridization
この DNAプロ一ブを、 ヒト肝臓 c D N Aライブラリー え gt l O (クロ一 ンテック社製） にハイブリダィズさせ、 該 DN Aプローブにハイブリダィズする cDNAをスクローニングした。  This DNA probe was hybridized to a human liver cDNA library gtlO (manufactured by Clontech), and a cDNA hybridizing to the DNA probe was cloned.
ハイブリダイズ条件は以下のようにした。  Hybridization conditions were as follows.
( 1) プレハイブリダィゼーシヨン  (1) Pre-hybridization
6倍 S S C 6 times S S C
0. 05倍 BLOTTO (2. 5 %脱脂粉乳、 0. 0 1%アジ化ナトリウム） 0. 1 %NP 40  0.05 times BLOTTO (2.5% skim milk powder, 0.0 1% sodium azide) 0.1% NP 40
100 g/ml 変性サケ精子 DNA  100 g / ml denatured salmon sperm DNA
総液量 50ml Total liquid volume 50ml
反応温度 60 °C Reaction temperature 60 ° C
反応時間 1時間 Reaction time 1 hour
(2) ハイブリダィゼーシヨン  (2) Hybridization
6倍 S S C  6 times S S C
0. 05倍 BLOTTO  0.05x BLOTTO
0. 1 %NP 40  0.1% NP 40
10 O g/ml 変性サケ精子 DNA  10 O g / ml denatured salmon sperm DNA
1 X 106 c pm/m 1 DNAプローブ 1 X 10 6 cpm / m 1 DNA probe
総液量 50ml 反応温度 60 °C Total liquid volume 50ml Reaction temperature 60 ° C
反応時間 12時間 Reaction time 12 hours
ハイプリダイズの終了した二トロセルロース膜を以下の条件で洗浄した。  The ditrocellulose membrane after the completion of the hybridization was washed under the following conditions.
(a) 2倍 SSC；、 0. 1%SDSの洗浄液で室温で 5分間洗浄することを 2 回  (a) 2x SSC ;, washing twice with 0.1% SDS washing solution at room temperature for 5 minutes
(b) 0. 2%SSC, 0. 1%SDSの洗浄液で室音で 10分間洗浄するこ とを 3回  (b) 3 times washing with 0.2% SSC, 0.1% SDS washing solution with room sound for 10 minutes
X線フィルム （富士写真フィルム社製） を洗浄したニトロセルロース膜に一 8 0°Cでー晚曝し、オートラジオグラフとした。得られたオートラジオグラフから、 陽性のプラークの位置を決定した。 「対応するプラークからファージを回収して SM溶液に懸濁し、 続いて SM溶液からファージを回収し、 常法により再度 NZ Y寒天培地上にプラーク形成を行わせ、 ニトロセルロース膜上に固定すること」 を 3回繰り返した。 その結果、 陽性のプラークは単一なものとなった。 該プラー クからファージを回収し、 100 1の SM溶液に懸濁し、 ファージを安定化さ せた。  An X-ray film (manufactured by Fuji Photo Film Co., Ltd.) was exposed to the washed nitrocellulose membrane at 180 ° C. for autoradiography. From the obtained autoradiograph, the position of the positive plaque was determined. `` Recover the phage from the corresponding plaque, suspend it in the SM solution, then recover the phage from the SM solution, form the plaque on the NZY agar medium again by the usual method, and fix it on the nitrocellulose membrane. Was repeated three times. The result was a single positive plaque. The phage was recovered from the plaque and suspended in a 100 1 SM solution to stabilize the phage.
5. 塩基配列の決定  5. Determination of nucleotide sequence
該ファージから E c oR Iで cDNAを切り出し、 得られた cDNAを pB 1 ue s cr ipt SK+ベクタ一 （ストラタジーン社製） に挿入して、 クロー ニングした。 クロ一ニングの結果得られた c DNAをダイタ一ミネ一夕一法によ り、 オートシークェンサ一 （AB I社製、 373 A型および 310型） を用いて、 DNAシークェンスを行い、 その塩基配列を決定した。 その結果、 コード領域を 全て含む c DNAであることが分かった。 すなわち全長の HEL50 cDNAが 単離され、 その塩基配列が決定された。 HE L 50 c DNAの塩基配列を配列表 の配列番号 2に示す。 なお、 HEL 50 cDNAを挿入した pB 1 u e s c r i p t SK+ベクタ一を HE L 50 SK +と名付けた。  CDNA was excised from the phage with EcoRI and the obtained cDNA was inserted into pB1uescript SK + vector-1 (Stratagene) and cloned. The cDNA obtained as a result of the cloning was subjected to DNA sequencing using the Auto Sequencer (ABI, 373A and 310) by the Ditamine-One-One Method. The nucleotide sequence was determined. As a result, it was found that the cDNA contained 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 as SEQ ID NO: 2 in the sequence listing. The pB1uescripptSK + vector into which the HEL50 cDNA was inserted was named HEL50SK +.
6. HEL50のァミノ酸配列の決定 前記 5の工程で決定した塩基配列から、 HEL 50 cDNAがコードする蛋白 質である HEL 50のァミノ酸配列を決定した。 結果を配列表の配列番号 1に示 す。 また、 配列表の配列番号 1に記載のアミノ酸配列の 77番目の G l yから 8 4番目の Thrまでの部分および 299番目の As pから 302番目の H i sま での部分が、 AT P a s eおよび DN Aヘリケースに特徴的な配列の部位である。 (実施例 2 ) ノーザンブロッテイング 6. Determination of amino acid sequence of HEL50 The amino acid sequence of HEL50, which is a protein encoded by HEL50 cDNA, was determined from the nucleotide sequence determined in step 5 above. The results are shown in SEQ ID NO: 1 in the sequence listing. In addition, the portion from the 77th Gly to the 84th Thr and the portion from the 299th Asp to the 302nd His in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing are ATPase and This is the site of the sequence characteristic of the DNA helicopter. (Example 2) Northern blotting
I プローブの作製  Preparation of I probe
配列表の配列番号 2に塩基配列を示す HE L 50 cDNAの 387番目の Tか ら 796番目の Gまでの部分を P CRにより増幅し、 標識してノーザンプロッテ イングのプローブとした。 以下に操作を示す。  The portion from the 387th T to the 796th G of the HEL50 cDNA whose nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing was amplified by PCR and labeled to obtain a Northern blotting probe. The operation is shown below.
1 · プライマーの合成  1 · Synthesis of primer
HE L 50の塩基配列から以下のプライマ一 P 3およびプライマー P 4を DN A合成機 （AB I社製、 392型） を使用してそれそれ作製した。  The following primer P3 and primer P4 were prepared from the nucleotide sequence of HE L50 using a DNA synthesizer (ABI, Model 392).
P 3 ： 5 ' -T CGCAT CAAG GAGGAGAC- 3 ' (配列表の配列番 号 6に記載の塩基配列） P 3: 5'-T CGCAT CAAG GAGGAGAC- 3 '(base sequence described in SEQ ID NO: 6 in Sequence Listing)
P 4 ： 5 ' -CTGAGAAGAG CGCCAGG— 3， （配列表の配列番号 7に記載の塩基配列）  P 4: 5'-CTGAGAAGAG CGCCAGG-3, (base sequence described in SEQ ID NO: 7 in Sequence Listing)
合成したプライマ一は、 蒸留水で 20 pmo 1/〃 1に調製した。 これを PC Rプライマーとして用いて、 以下の P CR操作を行った。  The synthesized primer was adjusted to 20 pmo1 / 1/1 with distilled water. Using this as a PCR primer, the following PCR operation was performed.
2. P CR  2. PCR
HE L 50 c DN Aが導入されたベクター HE L 50 S K +を P CRの铸型と し、 プライマー P 3およびプライマ一 P 4を使用して、 T akara Taq o 1 (登録商標、 宝酒造社製） を使用して以下の条件で行った。  The vector HE L50 SK + into which the HE L50c DNA was introduced was used as the type III of PCR, and Takara Taqo 1 (registered trademark, manufactured by Takara Shuzo Co., Ltd.) was used using primer P3 and primer P4. ) Was performed under the following conditions.
94°(：に 1分間ぉぃた。 次いで 「94 で45秒間、 続いて 60 °Cで 2分間、 続いて 72°Cで 2分間」 のサイクルを 30回繰り返した。 その後、 4°Cに置いて PCR操作を完了した。 PCRの反応液の組成は下記の通りとした The cycle of 94 ° (: 1 minute. Then, the cycle of “45 seconds at 94, followed by 2 minutes at 60 ° C, and then 2 minutes at 72 ° C” was repeated 30 times. Put down and complete the PCR procedure. The composition of the PCR reaction solution was as follows
0. 5 ng/u 1 HE L 50 SK + 1 j 1 0.5 ng / u 1 HE L 50 SK + 1 j 1
Taq p o 1 (宝酒造社製） 0. 1 j 1 Taq p o 1 (Takara Shuzo) 0.1 j 1
10倍濃度 P CR緩衝液 （宝酒造社製） 2j l 10x concentration PCR buffer (Takara Shuzo) 2jl
dNTP mix (宝酒造社製） . Q JLL I dNTP mix (Takara Shuzo). Q JLL I
プライマー P 3 2 /JL I Primer P32 / JL I
プライマー P 4 2μ.1 Primer P 4 2μ.1
滅菌蒸留水 1 1. 3 1 Sterile distilled water 1 1. 3 1
PI口 Γ 20 fi l  PI mouth Γ 20 fi l
。11産物を0. 7%ァガロースゲル上で電気泳動した。 ゲルから P C R産物 . The 11 products were electrophoresed on a 0.7% agarose gel. PCR product from gel
(以下、 フラグメント Bという） を切り出し、 Ge ne C l e an (バイオ 1 0 1社製） でフラグメント Bを回収して、 ミニゲル電気泳動でバンドをチェックし た。 その結果、 フラグメント Bの DNA液の濃度は、 25 ng/〃 1と推定され ノ 0 (Hereinafter referred to as “fragment B”), and the fragment B was recovered using GeneClean (manufactured by Bio101), and the band was checked by minigel electrophoresis. As a result, the concentration of the DNA solution of fragment B was estimated to be 25 ng / 〃1.
O . ネ示  O.
フラグメント Bの DNA液 5〃 1に滅菌蒸留水 4 / 1を加えて 98°Cに 1 0分 間おいた。 その後、 氷上で冷却し、 フラグメント Bを変性させた。 この変性フラ グメント Bをランダムラベリングキッ ト （ベ一リンガーマンハイム社製） を使用 して、 下記の組成の液中に 37°Cに 30分間おいた後、 65°Cに 1 0分間おいて 標識した。  5/1 of the DNA solution of fragment B was added with 4/1 of sterile distilled water and kept at 98 ° C for 10 minutes. Then, it was cooled on ice to denature the fragment B. Using a random labeling kit (manufactured by Behringer Mannheim), place this denatured fragment B in a solution of the following composition at 37 ° C for 30 minutes, and then at 65 ° C for 10 minutes to label it. did.
変性フラグメント B μ.1 Denatured fragment B μ.1
dA， G, TTP mix (ベ一リンガーマンハイム社製） 3 1 dA, G, TTP mix (Beringermannheim) 3 1
反応液 m i X (ベ一リンガーマンハイム社製） 2 μ ΐ Reaction mixture miX (Beringermannheim) 2 μΐ
d-32P-d CTP (アマシャムフアルマシア社製） 5 丄 d- 32 Pd CTP (manufactured by Amersham Fulmasia) 5 丄
クレノウ液 （ベ一リンガーマンハイム社製） 1 jiL 1 Klenow liquid (from Behringer Mannheim) 1 jiL 1
20 i 1 この液から標識フラグメント Bを G— 50マイクロスピンカラムで精製した。 スウイングロ一夕一でカラムを 3000 r pmで 1分間回転させ、 パヅキングを 行った。 そのカラムに標識フラグメント B液を 20〃 1添加し、 3000 r pm で 2分間カラムを回転させ、 T 50 Eで溶出して標識フラグメント Bを分離した。 I I ノヽィプリダイゼーシヨン 20 i 1 From this solution, labeled fragment B was purified using a G-50 micro spin column. The column was rotated at 3000 rpm for 1 minute throughout the swinging to perform the packing. 20〃1 of labeled fragment B solution was added to the column, the column was rotated at 3000 rpm for 2 minutes, and eluted with T50E to separate labeled fragment B. II No Predication
1. プレハイブリダィゼーシヨン  1. Prehybridization
mRN Aを固定したメンブラン （ヒト MTN (登録商標） プロット I I (クロ ーンテック社製） 、 ラット MTN (登録商標） No. 3 (クローンテック社製） およびマウス胎児 （クローンテック社製） ） を、 下記組成のハイブリダィゼーシ ヨン液 10mlに入れて、 42°Cで 150分おき、 変性サケ精子 DNAでメンブ ラン上をブロッキングした。  The mRNA (human MTN (registered trademark) Plot II (manufactured by Clonetech), rat MTN (registered trademark) No. 3 (manufactured by Clonetech) and mouse fetus (manufactured by Clonetech)) containing immobilized mRNA were as follows. The membrane was placed in 10 ml of the hybridization solution having the composition and placed at 42 ° C for 150 minutes, and the membrane was blocked with denatured salmon sperm DNA.
ハイブリダーゼーション液  Hybridization solution
5倍濃度 SSPE 5-fold concentration SSPE
10倍濃度 Denhar t d' s液  10 times concentration Denhar t d 's solution
2%SD S、 50%ホルムアミ ド 2% SD S, 50% formamide
100〃g/ml変性サケ精子 DNA  100〃g / ml denatured salmon sperm DNA
2. ノヽィブリダイゼ一ション  2. Noidization
前記ハイブリダイゼ一シヨン液 20 m 1に標識フラグメント B液を 7〃 1加え、 よくかき混ぜた。 角形シャーレにハイプリダイゼ一シヨン液を 5 mlずつ加え、 上記メンブランをシャーレに 1枚ずつ入れ、 各メンブランの上にビニールシ一ト を置き、 42°Cでー晚 （約 15時間） ハイブリダィズさせた。 ハイブリダィゼー シヨン液を捨て、 洗浄液 （2倍濃度 S SC、 0. 1 % SDS) をシャーレに加 え、 室温で 5分間洗浄することを 4回繰り返した。  To 20 ml of the above hybridization solution, 7〃1 of the labeled fragment B solution was added and mixed well. 5 ml of Hydride Solution was added to the square petri dish, and the above-mentioned membranes were placed one by one in the Petri dish. A vinyl sheet was placed on each membrane, and hybridized at 42 ° C for about 15 hours. The hybridization solution was discarded, and a washing solution (double concentration SSC, 0.1% SDS) was added to the petri dish, and washing for 5 minutes at room temperature was repeated four times.
3. ォートラジオグラム  3. Autoradiogram
このメンブランを放射線感光紙に 1 6時間さらして、 オートラジオグラフを取 つた。 ヒト MTNメンブランについての結果を図 1に示す。 ヒト各組織で HE L 5 OmRNAが発現していたが、 精巣での発現が最も多かった。 ラッ トの mRN Aおよびマウスの mRNAにもフラグメント Bがハイブリダィズし、 ラットにつ いてもヒトと同様の結果であった。 マウス胎児でも HE L 5 OmRNAの発現が みられ、 一番多く発現しているのは胎生 1 1日目のマウス胎児であることが観察 された。 The membrane was exposed to radiation-sensitive paper for 16 hours and autoradiographed. The results for the human MTN membrane are shown in FIG. HE L in human tissues 5 OmRNA was expressed, but was most frequently expressed in testis. Fragment B hybridized to rat mRNA and mouse mRNA, and the result was similar to that of human in rat. HEL5 OmRNA was also expressed in the mouse fetus, and the highest expression was observed in the mouse fetus on day 11 of embryo.
(実施例 3 ) HEL 50の大量調整  (Example 3) Mass adjustment of HEL 50
I HE L 50 c DNAの大量調整  Mass preparation of I HE L 50 c DNA
1. 組換えベクターの作製  1. Construction of recombinant vector
HE L 50 c DNAの第一メチォニン部分を E c o R Iサイ 卜が隣接するよう に P CRで改変し、 図 2に示す pET F LAG— H i sベクターの E c oR I サイ 卜に組み込んだ。 HE L 50 c DNAを組み込んだベクターを JM 109コ ンピテントセル （宝酒造社製） に、 取扱説明書に従って導入し、 800mlのァ ンピシリンを含む LB培地で一晩培養し、 回収した JM 109コンビテントセル からアルカリ法 （前掲の Mo 1 e c u 1 a r Cl oning Se cond Edi t ion^ 1. 33- 1. 43ページに記載の方法） によりベクターを回 収した。 ベクターの回収のための超遠心分離は 3回行った。  The first methionine portion of the HEL50c DNA was modified with PCR so that the EcoRI sites were adjacent to each other, and incorporated into the EcoRI site of the pET FLAG-His vector shown in FIG. The vector incorporating HEL50c DNA was introduced into JM109 competent cells (Takara Shuzo) according to the instruction manual, cultured overnight in LB medium containing 800 ml of ampicillin, and recovered from the JM109 competent cells. The vector was recovered by the alkali method (the method described above, Mo ecu a ar Cloning Se dion ion ^ 1.33-1.43). Ultracentrifugation for recovery of the vector was performed three times.
I I HE L 50の発現  Expression of I I HE L50
(1) 大量調製したベクタ一を大腸菌 BL21 pLy s S (ノバジェン社 製） に導入した。  (1) The vector prepared in a large amount was introduced into E. coli BL21 pLysSS (manufactured by Novagen).
(2) 得られた大腸菌をアンピシリン 100〃g/mlを含む LB培地で培養 し、 分光光度計 （ベックマン社製） で 60 Onmの波長における濁度が 0. 6か ら 0. 7になった時点で I P T Gを 0. ImMになるように加え、 HE L 50の 発現誘導を行った。  (2) The obtained Escherichia coli was cultured in an LB medium containing 100 μg / ml of ampicillin, and the turbidity at a wavelength of 60 Onm was changed from 0.6 to 0.7 using a spectrophotometer (Beckman). At this time, IPTG was added so as to be 0.1 ImM, and the expression of HEL50 was induced.
I I I HE L 50の精製  Purification of I I I HE L50
(1) 2時間後、 大腸菌を回収し、 下記組成の溶解液 30mlで懸濁した。 得 られた懸濁液を 4°Cで超音波処理し、 超遠心分離器および 45 T iロー夕 （いず れもベックマン社製） を使用して 50000 r pm、 4 °Cで 1時間超遠心分離し た。 (1) Two hours later, E. coli was recovered and suspended in 30 ml of a lysis solution having the following composition. The resulting suspension was sonicated at 4 ° C, ultracentrifuged and 45 Ti low Ultra-centrifugation at 50,000 rpm for 1 hour at 4 ° C using Beckman.
溶解液組成： Solution composition:
10mM Tr i s— HC1 pH 7. 9  10mM Tris—HC1 pH 7.9
10%グリセロール 10% glycerol
0. 5M NaC 1 0.5 M NaC 1
0. 1 %NP 40 0.1% NP 40
5mM 2—メルカプトエタノール （ME)  5mM 2-mercaptoethanol (ME)
1 mM PMS F  1 mM PMS F
(2) 得られた上清を取り、 1Mイ ミダゾ一ル （pH7. 5) を終濃度 ImM になるように加え、 N i—NT Aァガロースカラム （キアゲン社製） を用いて非 変性条件で取扱説明書に従って精製した。 カラムのベッドボリュームは、 1ml とした。 カラムは、 下記組成の第一洗浄液 3 Omlで洗浄した後、 下記組成の第 二洗浄液 5 mlで洗浄した。  (2) Take the resulting supernatant, add 1M imidazole (pH 7.5) to a final concentration of ImM, and use a Ni-NT A agarose column (Qiagen) under non-denaturing conditions. Purified according to the instructions. The bed volume of the column was 1 ml. The column was washed with 3 Oml of a first washing solution having the following composition, and then washed with 5 ml of a second washing solution having the following composition.
第一洗浄液組成： First cleaning liquid composition:
2 OmM Tr i s—HCl pH 7. 9  2 OmM Tris-HCl pH 7.9
10 %グリセロール  10% glycerol
0. 1 M KC 1  0.1 M KC 1
5 mM 2 -ME  5 mM 2 -ME
0. 5 mM PMS F  0.5 mM PMS F
20 mM ィミダゾール  20 mM imidazole
第二洗浄液組成： Second cleaning liquid composition:
2 OmM T r i s— HC 1 pH 7. 9  2 OmM T ris — HC 1 pH 7.9
10 %グリセ口ール  10% glycerol
0. 1 M K C 1  0.1 M K C 1
5 mM 2 -ME 0. 5 mM PMS F 5 mM 2 -ME 0.5 mM PMS F
40 mM ィミダゾール  40 mM imidazole
溶出時には、 下記組成の溶出液を 1mlずつ 5回カラムに流した。  At the time of elution, 1 ml of the eluate having the following composition was applied to the column five times.
溶出液組成： Eluate composition:
2 OmM Tr i s— HC 1 H 7. 9 2 OmM Tr i s—HC 1 H 7. 9
10%グリセロール 10% glycerol
0. 1 M K C 1 0.1 M K C 1
5mM 2 - ME 5mM 2-ME
0. 5 mM PMS F 0.5 mM PMS F
200 mM イミダゾ'一ル 200 mM imidazo
(3) 溶出された H E L 50画分を下記組成のリン酸緩衝液 1で透析した後、 HAP (ハイ ドロキシアパタイ ト） カラム （バイオラッド社製） を使用して、 F PLCシステム （商標、 アマシャムフアルマシア社製） を使用して、 リン酸緩衝 液 1および下記組成のリン酸緩衝液 2により 0. 0 1—0. 3 Mのグラジェント で精製した。 ベッドボリュームは 2 mlとした。  (3) The eluted HEL 50 fraction was dialyzed against phosphate buffer 1 having the following composition, and then, using a HAP (hydroxyapatite) column (manufactured by Bio-Rad), an F PLC system (trade name, Amersham) Purification was performed using a phosphate buffer solution 1 and a phosphate buffer solution 2 having the following composition in a gradient of 0.01 to 0.3 M using the following method. The bed volume was 2 ml.
リン酸緩衝液 1組成： Phosphate buffer 1 composition:
1 OmMリン酸緩衝液 pH 7. 2  1 OmM phosphate buffer pH 7.2
50 mM K C 1  50 mM K C 1
5mM 2 -ME  5mM 2 -ME
10%グリセロール  10% glycerol
リン酸緩衝液 2組成：  Phosphate buffer 2 composition:
300 mMリン酸緩衝液 p H 7. 2  300 mM phosphate buffer pH 7.2
5 OmM K C 1  5 OmM K C 1
5mM 2 -ME  5mM 2 -ME
10 %グリセ口ール  10% glycerol
(4) 溶出された HE L 50画分を下記組成の塩化力リゥム緩衝液 1で透析し た後、 イオン交換カラム Mo no Q (登録商標、 アマシャムフアルマシア社製） を使用して、 FPLCシステム （商標、 アマシャムフアルマシア社製） を使用し て、 塩化カリウム緩衝液 1および下記組成の塩化カリウム緩衝液 2により 0. 0(4) The eluted HE L50 fraction was dialyzed against chlorinated REAME buffer 1 having the following composition. After that, using an ion exchange column Mo no Q (registered trademark, manufactured by Amersham Pharmacia), using an FPLC system (trademark, manufactured by Amersham Pharmacia), potassium chloride buffer 1 and the following composition With potassium chloride buffer 2 at 0.0
5— 0. 5Mのグラジェン卜で精製した。 べヅドボリュームは lmlとした。 塩化カリウム緩衝液 1組成： Purification was performed using a 5-0.5 M gradient. The bed volume was lml. Potassium chloride buffer 1 Composition:
2 OmM T r i s— HC 1 pH 7. 9  2 OmM T ris — HC 1 pH 7.9
10%グリセロール  10% glycerol
5 OmM K C 1  5 OmM K C 1
5mM 2 -ME  5mM 2 -ME
0. 5 mM PMS F 0.5 mM PMS F
塩化カリウム緩衝液 2組成： Potassium chloride buffer 2 composition:
2 OmM T r i s -H C 1 pH 7. 9  2 OmM T ris -H C 1 pH 7.9
10 %グリセロール  10% glycerol
0. 5M K C 1  0.5 M K C 1
5mM 2 -ME 5mM 2 -ME
0. 5 mM PMS F  0.5 mM PMS F
(5 )溶出された HE L 50画分を下記組成の B C 1 00で透析して、— 80°C で保存した。  (5) The eluted HEL50 fraction was dialyzed against BC100 having the following composition and stored at -80 ° C.
2 OmM T r i s— HC 1 pH 7. 9  2 OmM T ris — HC 1 pH 7.9
20 %グリセロール  20% glycerol
0. 1M KC 1  0.1M KC 1
5mM 2 -ME  5mM 2 -ME
0. 5mM PMS F  0.5mM PMS F
精製した蛋白質の電気泳動 （クマシ一染色） の結果を図 3に示す。 いずれの画 分にも 50 kDの位置に HE L 50のバンドが確認された （タグの分だけやや大 きめの位置にバンドが現れている） 。 (実施例 4 ) ヘリケース活性の測定 Figure 3 shows the results of electrophoresis (coomassie staining) of the purified protein. In each of the fractions, a band of HEL50 was confirmed at the position of 50 kD (the band appeared slightly larger for the tag). (Example 4) Measurement of helicase activity
I 基質の作製  Preparation of I substrate
30塩基 （C AGT CACGAC GTTGTAAAAC GACGGC CA GT (配列表の配列番号 8に記載の塩基配列） ） からなる一本鎖 DNAを DNA 合成機を使用して合成した。 その 5 ' 末端を Takar a MEGA LABE L (登録商標） キット （宝酒造社製） を使用して、 取扱説明書に従って下記の組 成の液中に 37°Cで 30分間おいた後、 Ί 0°Cに 5分間おいて標識した。  A single-stranded DNA consisting of 30 bases (CAGT CACGAC GTTGTAAAAC GACGGC CAGT (base sequence described in SEQ ID NO: 8 in the sequence listing)) was synthesized using a DNA synthesizer. Using the Takar MEGA LABE L (registered trademark) kit (manufactured by Takara Shuzo Co., Ltd.), place the 5 'end in a solution having the following composition at 37 ° C for 30 minutes according to the instruction manual, and then add the Ί0 ° Labeled C for 5 minutes.
5 pmo 1/〃 1一本鎖 DNA 2^ 1 5 pmo 1 / 〃 1 Single-stranded DNA 2 ^ 1
10倍濃度リン酸化緩衝液 （キッ トに付属のもの） 1〃 1  10-fold concentration phosphorylation buffer (supplied with kit) 1〃 1
ァ一" p ATP (アマシャムフアルマシア社製） 5〃 1 A 1 "p ATP (manufactured by Amersham Fulmasia) 5〃 1
T 4キナーゼ （宝酒造社製） 1〃 1 T4 kinase (Takara Shuzo) 1〃 1
滅菌蒸留水 1 1 Sterile distilled water 1 1
合計 1 0^ 1 Total 1 0 ^ 1
この標識した一本鎖 DN Aに、 M 1 3mp l 8 s sDNA (宝酒造社製） を 2〃g加えて、 75°Cに 1 0分間、 続いて 37°Cに 1時間おいて両者をハイプリ ダイズさせ、 標識 DN Aを作製した。  To this labeled single-stranded DNA, add 2 μg of M13mp18ssDNA (Takara Shuzo), and hybridize both at 75 ° C for 10 minutes, then at 37 ° C for 1 hour. It was soyed to produce labeled DNA.
ハイブリダィズした標識 DN Aをマイクロスピンカラム S 400 HR (アマシ ャムフアルマシア社製） を使用して精製した。 まず、 スウィング口一ターでカラ ムを 3000 r pmで 1分間回転させ、 パッキングを行った。 そのカラムに前記 標識 DNA液を全量 （約 1 2〃 1) 添加し、 3000 r pmで 2分間カラムを回 転させ、 標識 DNAを分離した。 得られた標識 DNAを基質として以下の操作に 用いた。  The hybridized labeled DNA was purified using a micro spin column S 400 HR (manufactured by Amersham Pharmacia). First, packing was performed by rotating the column with a swing mouth at 3000 rpm for 1 minute. The entire amount (about 12 DNA1) of the labeled DNA solution was added to the column, and the column was rotated at 3000 rpm for 2 minutes to separate the labeled DNA. The obtained labeled DNA was used as a substrate in the following procedures.
I I ヘリケース活性の測定  I I Measurement of helicase activity
実施例 3で得られた Mo no Qの溶出画分のうち、 第 3 2、 35、 38、 4 1、 44、 47、 50番の各画分について、 以下の操作を行い、 各画分に含まれる H EL 50のヘリケース活性を測定した。 前記 Iの工程で作製した基質 0. 2〃 1 (約 3000 c pmに相当） と、 各 M onoQ溶出画分または BC 100 (コン トロール） 2〃1とを下記組成の反応 液に加え、 37°Cに 30分間おいて反応させた。 Among the eluted fractions of Mo no Q obtained in Example 3, the following operations were performed on the respective fractions of Nos. 32, 35, 38, 41, 44, 47 and 50, and The helicase activity of the included HEL 50 was measured. The substrate 0.2〃1 (corresponding to about 3000 cpm) prepared in the above step I and each MonoQ-eluted fraction or BC100 (control) 2 加 え 1 were added to a reaction solution having the following composition, and the mixture was added. The reaction was carried out at 30 ° C for 30 minutes.
反応液 （滅菌蒸留水で 20 1に調製した。 ） Reaction solution (prepared to 201 with sterile distilled water)
2 OmM T r i s -HC 1 pH 7. 5 2 OmM Tris -HC 1 pH 7.5
2mM DTT 2mM DTT
50 / g/ 1 B S A  50 / g / 1 B S A
ImM Mg C 1  ImM Mg C 1
8 OmM K C 1  8 OmM K C 1
ImM ATP  ImM ATP
その後、 5〃 1の下記組成の停止液を加え、 反応を停止させた。  Thereafter, a stop solution having the following composition in 5-1 was added to stop the reaction.
6 OmM EDTA  6 OmM EDTA
0. 75 %S D S 0.75% S DS
0. 1 %BPB 0.1% BPB
50 %グリセロール  50% glycerol
反応液を 10%アクリルアミ ド /0. 5倍濃度 T BE (Tr i s-b o rat e/ED T A) ゲル上に 15〃 1ずつのせ、 100 Vの電圧をかけて電気泳動し た。 電気泳動終了後、 ゲルを乾燥させ、 X線フィルム （富士写真フィルム社製） を前記ゲルにー晚曝し、 オートラジオグラフを取った。  The reaction solution was placed on a 10% acrylamide / 0.5-fold concentration TBE (Tris-borate / EDTA) gel in an amount of 15 μl, and electrophoresed by applying a voltage of 100 V. After the completion of the electrophoresis, the gel was dried, an X-ray film (manufactured by Fuji Photo Film Co., Ltd.) was exposed to the gel, and an autoradiograph was taken.
結果を図 4に示す。 レーン 1とレーン 2は対照 （コント口一ル） である。 レー ン 1は Mo n o Q溶出画分の代わりに B C 100を添加したもの （ネガティブコ ントロール） であり、 レーン 2は BC 100を添加して反応させた後 3分間沸騰 させたもの （ポジティブコントロール） である。 図中、 32ないし 50の数字が ふられたレーンは当該数字の Mo no Q溶出画分である。 各 Mo no Q溶出画分 はポジティブコントロール （レーン 2) と同じ位置にバンドが検出された。 41 画分および 44画分は特に強くバンドが現れ、 これらの画分に含まれる HE L 5 0は特にヘリケース活性が大きいことが確認された， Fig. 4 shows the results. Lane 1 and lane 2 are controls (one control). Lane 1 is a sample to which BC 100 was added instead of the Mo no Q elution fraction (negative control). Lane 2 was a sample to which BC 100 was added and reacted, followed by boiling for 3 minutes (positive control) It is. In the figure, the lanes marked with numbers from 32 to 50 are the Mo no Q elution fractions of the numbers. In each of the Mo no Q elution fractions, a band was detected at the same position as the positive control (lane 2). Fractions 41 and 44 showed particularly strong bands, and HE L5 contained in these fractions. 0 was confirmed to have particularly large helicase activity.
(実施例 5 ) ヘリケース活性の方向性  (Example 5) Directionality of helicase activity
I 方向性基質の調製  Preparation of I-directional substrate
以下の操作を行い方向性基質を作製した。 の概要を図 5に示す。 図 5中、 ☆ は該部分が標識されていることを示す。  The following operation was performed to prepare a directional substrate. Figure 5 shows the outline of the system. In FIG. 5, a star indicates that the part is labeled.
( 1) 5， 末端標識プローブの作製  (1) 5, Preparation of end-labeled probe
54塩基 （ATGCCTGCAG GT CGACT CTA GAGGAT C C C C GGGTACCGAG CTCGAATTCG TAAT (配列表の配列 番号 9に記載の塩基配列） ） からなる一本鎖 DN Aを DN A合成機を使用して合 成した。 その 5， 末端を T ak a r a MEGA L A B E L (登録商標） キッ ト （宝酒造社製） を使用して、 取扱説明書に従って下記の組成の液中に 37°Cで 30分間おいた後、 70°Cに 5分間おいて標識した。 その後、 TEを 10〃1カロ えた。  A single-stranded DNA consisting of 54 bases (ATGCCTGCAG GT CGACT CTA GAGGAT CCCC CCGGGGTACCGAG CTCGAATTCG TAAT (base sequence described in SEQ ID NO: 9 in the Sequence Listing)) was synthesized using a DNA synthesizer. 5. Using a Takara MEGA LABEL (registered trademark) kit (Takara Shuzo Co., Ltd.) at the end, place in a solution of the following composition for 30 minutes at 37 ° C according to the instruction manual, and then Label for 5 minutes. After that, I gained 10〃 calories from TE.
5 pmo 1/〃 1一本鎖 DNA 1 1  5 pmo 1 / 〃 1 Single-stranded DNA 1 1
10倍リン酸化緩衝液 （キットに付属のもの） 1 PL 1  10-fold phosphorylation buffer (supplied with kit) 1 PL 1
ァ— 32P ATP (アマシャムフアルマシア社製） 5 // 1 32 32 P ATP (Amersham Armasiaia) 5 // 1
T 4キナーゼ （宝酒造社製） 1 μ.1 T4 kinase (Takara Shuzo) 1 μ.1
滅菌蒸留水 2 μΛ Sterile distilled water 2 μΛ
σ S十 10 / 1 σ S10 10/1
(2) 3， 末端標識プローブの作製  (2) 3. Preparation of end-labeled probe
配列表の配列番号 9に記載の塩基配列からなる一本鎖 DN Αを DN Α合成機を 使用して合成し、 その 3 ' 末端を TdTキット （和光純薬社製） を使用して、 取 扱説明書に従って下記の組成の液中に 37°Cで 30分間おいた後、 70°Cに 5分 間おいて標識した。  A single-stranded DNΑ consisting of the nucleotide sequence of SEQ ID NO: 9 in the sequence listing was synthesized using a DNΑ synthesizer, and its 3 ′ end was collected using a TdT kit (Wako Pure Chemical Industries, Ltd.). After placing in a solution having the following composition at 37 ° C for 30 minutes according to the instruction manual, labeling was performed at 70 ° C for 5 minutes.
5 pmo 1/〃 1一本鎖 D N A 1 1  5 pmo 1 / 〃 1 Single-stranded DNA 1 1
5倍 TdT緩衝液 （キッ 卜に ί寸属のもの） 4 / 1 ひ一32 P d d ATP (アマシャムフアルマシア社製） 5〃 15x TdT buffer solution (small size to kit) 4/1 Hiichi 32 P dd ATP (manufactured by Amersham Fulmasia) 5〃 1
TdT 1 μ.1 TdT 1 μ.1
滅菌蒸留水 9 1 Sterile distilled water 9 1
合計 20〃 1 Total 20〃 1
(3) 標識した一本鎖 DNAそれぞれに、 Μ 1 3mp 1 8 s sDNA (宝酒 造社製） を 2〃 g加えて、 75 °Cに 5分間、 続いて 37 °Cに 1時間から 2時間お いて両者をハイブリダィズさせ、 標識 D N Aを作製した。  (3) To each of the labeled single-stranded DNAs, add 2Μ g of 313mp18ssDNA (Takara Shuzo), and add them at 75 ° C for 5 minutes, then at 37 ° C for 1 hour. Both were hybridized with time to prepare labeled DNA.
(4)ハイブリダィズした標識 DN Aをマイクロスピンカラム S 400 HR (ァ マシャムフアルマシア社製） を使用して、 実施例 4の場合と同様に精製した。  (4) The hybridized labeled DNA was purified in the same manner as in Example 4 using a micro spin column S400HR (manufactured by Amersham Pharmacia).
(5) 精製した標識 DN Aを以下の組成の反応液中に 37 °Cで 2時間おき Sm a Iサイ 卜で標識 DNA開裂した。  (5) The purified labeled DNA was cleaved with a SmaI site every 2 hours at 37 ° C. in a reaction solution having the following composition.
標識 DNA溶液 1 5 1 Labeled DNA solution 1 5 1
10倍 T緩衝液 （Sma I酵素に付属のもの） 3〃 1  10x T buffer (supplied with Sma I enzyme) 3〃 1
B S A 3 21  B S A 3 21
Sma I酵素 （宝酒造社製） 2〃 1 Sma I Enzyme (Takara Shuzo) 2〃 1
滅菌蒸留水 7 1 Sterile distilled water 7 1
合計 30〃 1 Total 30〃 1
(6) 開裂した標識 DNAをフエノール/ CHC 13 に溶かし、 エタノール沈 殿させた。 沈殿物を 10〃 1の TEに溶かし、 ヘリケース活性測定の基質として 用いた。 (6) was dissolved cleaved labeled DNA to phenol / CHC 1 3, and ethanol-precipitated gluteal. The precipitate was dissolved in 10 1 TE and used as a substrate for measuring Helicase activity.
I ェ ヘリケース活性測定  I. Helicase activity measurement
実施例 4と同様の操作で、 上記で作製した各基質 1〃 1ずつを、 それそれヒト HE L 50またはラッ ト T I P 49で分解し、 反応液を電気泳動した。  In the same manner as in Example 4, each of the substrates prepared above was digested with human HEL50 or rat TIP49, and the reaction solution was subjected to electrophoresis.
結果を図 6に示す。 レーン 1および 5はポジティブコントロール、 レーン 2お よび 6はネガティブコントロールである。レーン 3および 7が HE L 50であり、 レーン 4および 8が T I P 49である。 30 bの位置にバンドが現れるのは 3 ' 末端から 5' 末端方向にヘリ一ケスが作用する場合であり、 24 bの位置に， ドが現れるのは 5 ' 末端から 3 ' 末端方向にヘリケースが作用する場合である。 図 6から HE L 50はヘリケース活性の方向性が 5， 末端から 3， 末端であるこ とが分かる。 一方、 T I P 49のヘリケース活性の方向性は 3， 末端から 5， 末 端であった。 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 HEL 50 and lanes 4 and 8 are TIP 49. 3 'band appears at 30b In the case where the helix acts in the direction from the end to the 5 'end, and at the position 24b, the dod appears when the helicase acts in the direction from the 5' end to the 3 'end. From Fig. 6, it can be seen that HEL50 has a helicase activity direction of 5, 5 from the end, and 3 ends. On the other hand, the direction of the helicase activity of TIP49 was 3, 3 from the terminal, and 5 from the terminal.
(実施例 6) 酵母 HE L 50のクローニング  (Example 6) Cloning of yeast HE L50
I データベースの検索 I Searching the database
HE L 50とホモロジ一を有する酵母の DNAの塩基配列を、 ィン夕一ネット のゲノムネット WWWサーバ一中の TB LAS TX (h t t p ： //www. b l as t , genome, ad. j p) を使用して、 検索した。 その結果、 YP L 235 wの番号で登録されている塩基配列が抽出された。  The base sequence of yeast DNA with homology to HE L50 was obtained from TB LAS TX (http: // www. Blast, genome, ad. Jp) in Genyuichi Net's Genome Net WWW server. Used and searched. As a result, the nucleotide sequence registered under the number of YP L 235 w was extracted.
この塩基配列から以下のプライマ一それぞれ DN A合成機 （AB I社製、 39 2型） を使用して作製した。  The following primers were prepared from this nucleotide sequence using a DNA synthesizer (ABI, 392 type).
プライマー Y 1 (メチォニン側） ： 5 ' — CGGAATT CAT GTCGAT TCAA ACTAGTG— 3， （配列表の配列番号 10に記載の塩基配列） プライマー Y2 (3， 末端側） ： 5' — CGGAATTCTT ATTCCGT AGT ATCCATGGC (配列表の配列番号 1 1に記載の塩基配列） これを PC Rプライマ一として用いて、 以下の P CR操作を行った。 Primer Y 1 (methionine side): 5 ′ — CGGAATT CAT GTCGAT TCAA ACTAGTG—3, (base sequence described in SEQ ID NO: 10) Primer Y2 (3, terminal side): 5 ′ — CGGAATTCTT ATTCCGT AGT ATCCATGGC The nucleotide sequence described in SEQ ID NO: 11 in the column list) Using this as a PCR primer, the following PCR operation was performed.
I I P CR  I I P CR
酵母ゲノム （国立遺伝学研究所から入手） を鐯型として用いて、 KODポリメ ラーゼ （東洋紡社製） を使用して、 以下の条件で PC Rを行った。  Using yeast genome (obtained from the National Institute of Genetics) as type I, PCR was performed under the following conditions using KOD polymerase (manufactured by Toyobo).
95 °Cに 5分間おいた。次いで「94°Cで 30秒間、 続いて 55°Cで 30秒間、 続いて 72°Cで 1分間」 のサイクルを 45回繰り返した。 その後、 72°Cに 5分 間おいた後、 4°Cにおいて P CR操作を完了した。  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.
P CRの反応液の組成は下記の通りとした。  The composition of the PCR reaction solution was as follows.
酵母ゲノム 500 ng プライマー Y 1 10 pmo 1 500 ng yeast genome Primer Y 1 10 pmo 1
プライマー Y 2 10 pmo 1 Primer Y 2 10 pmo 1
KOD po 1 (東洋紡社製） 2〃 1  KOD po 1 (Toyobo) 2〃 1
10倍濃度 P CR緩衝液 （東洋紡社製） 5^ 1  10x concentration PCR buffer (Toyobo) 5 ^ 1
2mM dNTP mix (東洋紡社製） 5 // 1 2mM dNTP mix (Toyobo) 5 // 1
25 mM Mg C 1 2 μΛ  25 mM Mg C 1 2 μΛ
滅菌蒸留水で 50 1に調製した。 It was adjusted to 501 with sterile distilled water.
その後、 前記で得た PCR産物を、 制限酵素を使用して E c oR Iサイ トで切 断して DN A断片を得た。 得られた DNA断片を pB lue s c r i p t l l SK +の E c oRエサイ トに挿入してサブクローニングした。 その結果得られた Thereafter, the PCR product obtained above was cut at the EcoRI site using a restriction enzyme to obtain a DNA fragment. The resulting DNA fragment was inserted into pBlueScriptltlSK + EcoR site and subcloned. The resulting
DNA断片について、 ダイ夕一ミネ一夕一法により、 オートシークェンサ一 （AFor DNA fragments, the auto sequencer (A
B I社製、 373 A型） を用いて、 DNAシークェンスを行い、 その塩基配列を 決定した。 この塩基配列が酵母 HE L 50 c DNAの塩基配列である。 この塩基 配列を配列表の配列番号 13に示す。 また、 この塩基配列から決定されたァミノ 酸配列が酵母 HE L 50のアミノ酸配列である。 このアミノ酸配列を配列表の配 列番号 1 2に示す。 決定された塩基配列は、 酵母のゲノムとして YP L 235 w の番号でデータベースに登録されている塩基配列全く同じであった。 DNA sequence was carried out using a 373A (manufactured by BI Company, Inc.), and its nucleotide sequence was determined. This nucleotide sequence is the nucleotide sequence of yeast HE 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 was exactly the same as the nucleotide sequence registered in the database with the number YP L 235 w as the yeast genome.
ヒト HE L 50と酵母 HE L 50とのホモロジ一は、 全体を通じては 65%、 ウォーカー Aモチーフ （配列表の配列番号 1に記載のアミノ酸配列の 77番目の Gl yから 84番目の Thrまでの部分）では 69 %、ウォー力一 Bモチーフ（配 列表の配列番号 1に記載のアミノ酸配列の 299番目の As pから 302番目の The homology between human HE L50 and yeast HE L50 is 65% in total, the Walker A motif (the portion from the 77th Gly to the 84th Thr in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing). ), 69% of the war force B motif (from the 299th Asp to the 302nd
Hi sまでの部分） では 7 1 %であった。 In the part up to His), it was 71%.
ヒト T I P 49とヒト HEL 50のホモロジ一が 4 1 %であるので、 真核生物 におけるヒト HEL 50と酵母 HE L 50のホモロジ一は、 ヒト HE L 50とヒ ト T I P 49とのホモロジ一よりもはっきりと高い。 真核生物の HE L 50はヒ ト HEL 50とのホモロジ一が 65 %以上であると考えられる。 他の真核生物の HE L 50 cDNAも、 目的とする種の真核生物の c D N Aラ イブラリーから P C Rにより適当なプロ一ブを作製し、 そのプローブを使用した プラークハイブリダイゼ一シヨン法により単離することができると考えられる。 また、 酵母 HE L 50のクローニングで例示したように P CRにより完全長の c DNAを得ることもできる。 P CRやハイブリダィゼ一シヨンの条件は、 すでに 示した条件を参考に決定できる。 産業上の利用可能性 Since the homology between human TIP49 and human HEL50 is 41%, the homology between human HEL50 and yeast HEL50 in eukaryotes is higher than that between human HEL50 and human TIP49. Clearly high. The eukaryotic HE L50 is considered to be more than 65% homologous to human HEL50. For other eukaryotic HEL50 cDNAs, an appropriate probe is prepared from the eukaryotic cDNA library of the target species by PCR, and the plaque hybridization method using the probe is used. It is believed that it can be isolated. Alternatively, full-length cDNA can be obtained by PCR as exemplified in the cloning of yeast HEL50. The conditions for PCR and hybridization can be determined with reference to the conditions already shown. Industrial applicability
本発明の HE L 50は、 その一部において T I P 49との相同性があるので、 TBPとの相互作用があると考えられる。 T BPは生物の全遺伝子の転写に関わ つているので、 本発明の HE L 50、 HEL 50をコードするポリヌクレオチド、 HEL 50を認識する抗体により TBPの転写制御機能の究明の一手段が供せら れる。  The HEL50 of the present invention has homology to TIP49 in part thereof, and thus is considered to have an interaction with TBP. Since TBP is involved in the transcription of all genes in an organism, the HEL50, the polynucleotide encoding HEL50, and the antibody recognizing HEL50 of the present invention provide a means for investigating the transcription control function of TBP. It is.
また、 本発明の HE L 50および HE L 50変異体は、 DN Aヘリケースとし て使用可能である。 すなわち、 ある種の遺伝病はヘリケースの異常が原因するこ とが知られている。 したがって、 本発明のポリヌクレオチドはプローブとして、 或いは P CRのプライマーとしてそのような遺伝病の診断に利用できる。 さら に、 本発明の HE L 50、 ポリヌクレオチドおよび抗体を利用して、 ヘリケース の異常を回復すること、 すなわち前記遺伝病を治療することが期待できる。  In addition, the HEL50 and the HEL50 mutant of the present invention can be used as a DNA helicopter. That is, it is known that certain genetic diseases are caused by abnormalities of the helicopter. Therefore, the polynucleotide of the present invention can be used as a probe or as a primer for PCR in diagnosing such a genetic disease. Furthermore, it is expected that the use of the HEL50, the polynucleotide and the antibody of the present invention will recover helicase abnormalities, that is, treat the genetic disease.

Claims

言青求の範囲 Scope of word blue
1. 配列表の配列番号 1に記載のァミノ酸配列からなる蛋白質。  1. A protein comprising the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
2. 配列表の配列番号 1に記載のァミノ酸配列において一または複数のァミ ノ酸が置換、 欠失または付加されたアミノ酸配列からなり、 かつ DNAヘリケー ス活性を有する蛋白質。  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 which has DNA helicase activity.
3. 配列表の配列番号 1に記載のァミノ酸配列と 65 %以上のホモロジ一を 有し、 かつ DN Aヘリケース活性を有する蛋白質。  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. 配列表の配列番号 1 2に記載のアミノ酸配列からなる蛋白質。  4. A protein comprising the amino acid sequence of SEQ ID NO: 12 in the sequence listing.
5. 請求項 1ないし 4のいずれか一項に記載の蛋白質をコードするポリヌク レオチド。  5. A polynucleotide encoding the protein according to any one of claims 1 to 4.
6. 配列表の配列番号 2に記載の塩基配列の 1番目の Aから 1389番目の Cまでの塩基配列を有する D NA。  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. 配列表の配列番号 2に記載の塩基配列からなる請求項 6に記載の D N A。  7. The DNA of claim 6, comprising the nucleotide sequence of SEQ ID NO: 2 in the sequence listing.
8. 配列表の配列番号 13に記載の塩基配列からなる DNA。 8. DNA comprising the nucleotide sequence of SEQ ID NO: 13 in the sequence listing.
9. 請求項 6ないし 8のいずれか一項に記載の DNAがコードする RNA。 9. An RNA encoded by the DNA according to any one of claims 6 to 8.
10. 請求項 6ないし 8のいずれか一項に記載の DNAのアンチセンス DN Aまたはその誘導体からなるアンチセンスポリヌクレオチド。 10. An antisense polynucleotide comprising the antisense DNA of the DNA according to any one of claims 6 to 8 or a derivative thereof.
1 1. 請求項 9に記載の RNAのアンチセンス RNAまたはその誘導体から なるアンチセンスポリヌクレオチド。  1 1. An antisense polynucleotide comprising the antisense RNA of the RNA according to claim 9 or a derivative thereof.
12. 請求項 5に記載のポリヌクレオチドの連続する 12以上の塩基からな る部分からなるポリヌクレオチド。  12. A polynucleotide comprising a portion consisting of 12 or more consecutive bases of the polynucleotide according to claim 5.
13. 請求項 6ないし 8のいずれか一項に記載の DN Aの連続する 12以上 の塩基からなる部分からなる D N A。  13. A DNA comprising a portion comprising 12 or more consecutive bases of the DNA according to any one of claims 6 to 8.
14. 請求項 9に記載の RNAの連続する 1 2以上の塩基からなる部分から なる RNA。  14. An RNA comprising a portion consisting of at least 12 consecutive bases of the RNA according to claim 9.
15. 化学修飾された請求項 5に記載のポリヌクレオチド。 15. The polynucleotide according to claim 5, which has been chemically modified.
16. 化学修飾された請求項 6ないし 8のいずれか 1項に記載の DNA。16. The DNA according to any one of claims 6 to 8, which is chemically modified.
17. 化学修飾された請求項 9に記載の RNA。 17. The RNA according to claim 9, which has been chemically modified.
18. 請求項 1ないし 4のいずれか一項に記載の蛋白質を認識する抗体。  18. An antibody that recognizes the protein according to any one of claims 1 to 4.
PCT/JP1999/006519 1999-02-10 1999-11-22 Protein having dna helicase activity, polynucleotide encoding the protein, antisense polynucleotide against the polynucleotide and antibody recognizing the protein WO2000047731A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042479A2 (en) * 1999-12-08 2001-06-14 Genoptera, Llc Insecticide targets and methods of use
WO2003028537A3 (en) * 2001-10-01 2003-12-04 Gen Hospital Corp Methods for diagnosing and treating diseases and conditions of the heart or digestive system, and cancer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Gohsi T. et al., "Molecular cloning of mouse p47, a second group mammalian RuvB DNA helicase-like protein: homology with those from human and Saccharomyces cerevisiae", The Journal of Biochemistry, Vol. 125, No. 5, pages 939-946 (May 1999) *
Kanemaki M. et al., "Molecular cloning of a rat 49-kDa TBP-interacting protein (TIP49) that is highly homologous to the bacterial RuvB", Biochem. Biophys. Res. Commun., Vol. 235, No. 1, pages 64-68 (1997) *
Kanemaki M. et al., "TIP49b, a new RuvB-like DNA helicase, is included in a complex together with another RuvB-like DNA helicase, TIP49a", The Journal of Biological Chemistry, Vol. 274, No. 32, pages 22437-22444 (August 1999) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042479A2 (en) * 1999-12-08 2001-06-14 Genoptera, Llc Insecticide targets and methods of use
WO2001042479A3 (en) * 1999-12-08 2001-12-13 Genoptera Llc Insecticide targets and methods of use
WO2003028537A3 (en) * 2001-10-01 2003-12-04 Gen Hospital Corp Methods for diagnosing and treating diseases and conditions of the heart or digestive system, and cancer
AU2002330177B2 (en) * 2001-10-01 2008-06-26 The General Hospital Corporation Methods for diagnosing and treating diseases and conditions of the heart or digestive system, and cancer

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