WO2001009346A1

WO2001009346A1 - Gene encoding novel adenylate kinase 3 (ak3)-like protein

Info

Publication number: WO2001009346A1
Application number: PCT/JP2000/005066
Authority: WO
Inventors: Research Institute Helix
Original assignee: Ota, Toshio; Isogai, Takao; Hayashi, Koji; Saito, Kaoru; Yamamoto, Jun-Ichi; Ishii, Shizuko; Sugiyama, Tomoyasu; Wakamatsu, Ai; Nagai, Keiichi; Otsuki, Tetsuji; Ihara, Shigeo; Nakae, Hiroki; Nishikawa, Tetsuo; Kimura, Koichi
Priority date: 1999-07-29
Filing date: 2000-07-28
Publication date: 2001-02-08
Also published as: WO2001009320A1

Abstract

By an oligocap method originally developed for isolating full-length cDNA, a plural number of full-length cDNAs are isolated from a cDNA library originating in NT-2 cells treated with retinoic acid. Among these cDNAs, a clone (C-NT2RP2000329) encoding a novel AK-like protein is isolated. It is considered that C-NT2RP2000329 has functions of converting extracellular signals into intracellular signals and changing cell morphology. Therefore, it is expected that C-NT2RP2000329 is usable as an important target in diagnosing diseases associated with abnormality in these functions and developing drugs for preventing or treating these diseases.

Description

Gene encoding a novel adenylate kinase 3 (AK 3) -like protein

The present invention relates to novel AK3-like proteins, their genes, and their production and use. Background art

Adenylate kinase (AK) is a phosphotransferase that catalyzes the reaction that produces MgATP and AMP by reversible phosphorylation of one molecule of ADP by one molecule of MgADP. Three isozymes, AK1, AK2, and AK3, have been reported for vertebrate adenylate kinase. Among them, AK3 is a GTP-binding protein, AMP phosphotransferase, encoded in the nucleus of cells, and is present in the mitochondrial matrix of liver, heart, and muscle (Shahjahan M. et al., (1991) ) Gene 107: 313-317). Recently, it has been suggested that AK3 is involved in apoptosis (Kohler C. et al., (1999) FEBS Lett 447: 10). In other literature, AK2 was released from the mitochondrial intermembrane early in apoptosis, whereas AK3 was not released into the intermembrane during apoptosis. (Noma T. et al., (1999) Biochem. Biophys. Res. Commun 264: 990). Additionally, for AK3, it was shown Rereru that force ^s involved in the maturation regeneration of nerve in rats (Inoue S. et al, (1999 ) Biochem Biophys Res Commun 2 54:.... 681). These reports suggest that AK3 is present in mitochondria and plays a role in converting extracellular information into intracellular information, changing cell morphology, apoptosis, cell motility, and transporting intracellular substances. .

The interaction between mitochondria and the nucleus has not yet been fully analyzed. Although there is not much medical or biological literature, MELAS (stroke-like symptoms or hyperlactic acidemia), central nervous disease, epilepsy, skeletal muscle pathology, and muscular disease have been identified as mitochondrial-related diseases. , Electron transmission abnormalities, Leber's disease, diabetes, Peason's disease, Perkinson's disease, metabolic abnormalities, etc. have been reported.

Because of its properties, AK3 is expected to be involved in these diseases, and is considered to be an important target for drug development for the diagnosis, prevention and treatment of these diseases. Disclosure of the invention

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a novel AK3-like protein, a gene thereof, and a method for producing the same and use thereof.

The present inventors isolated a plurality of full-length cDNAs from a cDNA library derived from retinoic acid-treated NT-2 cells by an oligocap method uniquely developed for isolating full-length cDNAs. The nucleotide sequence of one of the isolated cDNAs was determined, and its structural analysis revealed significant homology to the known AK3, indicating that the cDNA encodes a novel AK3-like protein. (This clone was named "C-NT2RP20 00329"). As described above, AK3 is present in mitochondria and plays a role in converting extracellular information into intracellular information, changing cell morphology, etc., and plays a wide variety of diseases including MELAS and central nervous system disorders. The association has been suggested. Because C-NT2RP200 0329 is a novel human AK3, it is considered to be an important target for the diagnosis of these diseases and the development of drugs to prevent or treat these diseases.

The present invention relates to a novel AK3-like protein C-NT2RP2000329 and a DNA encoding the protein, and their production and use.

(1) a DNA according to any of (a) to (d) below,

(a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2. (b) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 1. (c) having an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2,

: DNA encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of 2.

(d) a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and encodes a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2 .

(2) DNA encoding a partial peptide of a protein consisting of the amino acid sequence of SEQ ID NO: 2,

(3) a protein or peptide encoded by the DNA according to (1),

(4) a vector into which the DNA of (1) has been inserted,

(5) a transformed cell carrying the DNA of (1) or the vector of (4),

(6) The protein or peptide according to (3), which comprises a step of culturing the transformed cell according to (5) and collecting a protein or a peptide expressed from the transformed cell or a culture supernatant thereof. Manufacturing method,

(7) an antibody that binds to the protein according to (3),

(8) a polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof,

(9) A method for screening a compound that binds to a protein according to (3),

(a) contacting a test sample with the protein or a partial peptide thereof,

(b) detecting the binding activity between the protein or its partial peptide and a test sample,

(c) selecting a compound having an activity of binding to the protein or a partial peptide thereof, and (10) A method for diagnosing a disease caused by a DNA abnormality according to (1),

(a) preparing a cell sample from a patient; and

(b) detecting the expression level or mutation of the DNA according to (1) in the cell,

(11) A method for screening a candidate compound for a drug for prevention or treatment of a disease caused by DNA abnormality according to (1), which comprises:

(a) contacting the test sample with cells expressing the DNA according to (1),

(b) measuring the expression level of the DNA according to (1) in the cells; and

(c) selecting a compound that increases or decreases the level of DNA expression according to (1), which is measured in step (b) and compared to the case where the test sample is not contacted, according to (1).

(1 2) The DNA according to (1) or (2), the protein or peptide according to (3), the vector according to (4), or the screening according to (9) or (11). A pharmaceutical composition containing the compound to be isolated,

(13) A DNA chip provided with the DNA according to (1) or (2) or the polynucleotide according to (8).

The present invention provides a novel protein "C-NT2RP2000329". The amino acid sequence of the human C-NT2RP2000329 protein contained in the protein of the present invention is shown in SEQ ID NO: 2, and the nucleotide sequence of the cDNA encoding the protein is shown in SEQ ID NO: 1. The C-NT2RP2000329 gene encodes a protein of 227 amino acids with significant homology to adenylate kinase 3 (AK3). AK3, which is present in mitochondria, plays a role in converting extracellular information into cytoplasm information, changing cell morphology, apoptosis, cell movement, and intracellular substance transport. Since the C-NT2RP2000329 protein is also considered to have such a function in vivo due to its structural characteristics, the C-NT2RP2000329 gene and protein are used for diagnosis of diseases caused by abnormalities in these functions. It is expected to be used for the development of drugs for preventing or treating the disease.

The C-NT2RP2000329 protein can be prepared as a recombinant protein or as a natural protein. The recombinant protein is prepared, for example, by introducing a vector into which a DNA encoding the protein of the present invention has been inserted into an appropriate host cell as described below, and purifying the protein expressed in the transformant. Is possible

. On the other hand, a natural protein can be prepared using, for example, an affinity column to which an antibody against the protein of the present invention described below is bound (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) ) Publish. John Wiley & Sons Section 16. 1-16. 19). The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. In addition, in vitro translation (for example, “0n the fidelity of raR A translation in the nuc 丄 ease-treated rabbit ret iculocyte lysate system. Dasso, MC, Jackson, RJ (1989) Nucleic Acids Res. 17: 3129 It is also possible to prepare the protein of the present invention by using, for example, -3144J.

The present invention includes a protein functionally equivalent to the human-derived C-NT2RP2000329 protein identified in this example. Such proteins include, for example, mutants, homologs, and cryptants of the C-NT2RP2000329 protein described in SEQ ID NO: 2. Here, “functionally equivalent” means that the target protein has the same biological or biochemical function as the C-NT2RP2000329 protein. Examples of such a function include a function as a GTP-binding protein AMP phosphotransferase. Whether the protein functions as a GTP-binding protein AMP phosphotransferase can be determined, for example, by mixing AMP with GTP containing ³² P at the γ-phosphate and confirming that ADP containing ³² P is purified Can be detected. GTP and ADP can be separated, for example, by thin-layer chromatography.

These proteins functionally equivalent to the proteins identified in this example are For example, a method for introducing a mutation into an amino acid sequence in a protein (eg, a site-directed mutagenesis method (Current Protocols in Molecular Biology edit. Au subel et al. (1987) Publish. John Wiley & It can be prepared using Sons Section 8. 1-8.5)). Such proteins may also be generated by mutations in amino acids in nature. According to the present invention, one or more amino acids in the amino acid sequence (SEQ ID NO: 2) may be substituted, deleted, inserted, and substituted with Z or Z as long as it has a function equivalent to the protein identified in this example. Different proteins are included by addition and the like.

The number of amino acid mutations and mutation sites in a protein are not limited as long as the function is maintained. The number of mutations is typically within 30 amino acids, preferably within 10 amino acids, and more preferably within 5 amino acids (eg, within 3 amino acids). The amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution from the viewpoint of maintaining the function of the protein. For example, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are considered to have similar properties because they are all classified as nonpolar amino acids. In addition, examples of the non-charger include Gly, Ser, Thr, Cys, Tyr, Asn, and Gin. In addition, acidic amino acids include Asp and Glu, and basic amino acids include Lys, Arg, and His.

Proteins functionally equivalent to the proteins identified in this example can be obtained by using known techniques known to those skilled in the art (Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section). 6.3.6.4) or use gene amplification technology (PCR) (Current protocols in Molecular Biologic edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1.6.4.4) It is also possible to isolate them. That is, those skilled in the art can use a DNA encoding the protein identified in this example (SEQ ID NO: 1) or a part thereof as a probe, or an oligonucleotide specifically hybridizing with the DNA as a primer. Isolating DNA that hybridizes with DNA it can. Further, based on the isolated DNA, a protein encoded by the DNA can be prepared. The present invention includes proteins encoded by DNAs that hybridize with DNAs encoding these proteins, as long as they have the same function as the proteins identified in the present example. Examples of organisms for isolating functionally equivalent proteins include, but are not limited to, vertebrate animals such as humans, mice, rats, rabbits, pigs, and rabbits.

Stringent conditions for hybridization for isolation of DNA encoding functionally equivalent proteins are usually `` lxSSC, 0.1% SDS, 37 ° C '', and are more stringent. The conditions were about 0.5 x SSC, 0.1% SDS, 42 ° C, and the more severe conditions were about 0.1 x SSC; 0.1% SDS, 65 ° C. It can be expected that DNA with higher homology to the probe sequence will be isolated as the conditions of the probe become stricter. However, the combination of the above SSC, SDS and temperature conditions is merely an example, and those skilled in the art will recognize the above or other factors that determine the stringency of the hybridization (eg, probe concentration, probe length, probe length, etc.). The same stringency as described above can be realized by appropriately combining the hybridization reaction time and the like.

The protein isolated using such a hybridization technique or gene amplification technique usually has higher homology in its amino acid sequence than the protein of the present invention described in SEQ ID NO: 2. High homology refers to sequence identity of at least 50% or more, more preferably 70% or more, and even more preferably 90% or more (eg, 93% or more, 95% or more). Amino acid sequence homology can be determined by homology search using BLAST X.

The present invention also provides a partial peptide of the protein of the present invention. The partial peptide of the protein of the present invention can be used, for example, for preparing an antibody that binds to the protein of the present invention. The partial peptide of the present invention comprises at least 7 amino acids, preferably 9 amino acids or more, more preferably 12 amino acids or more, and more preferably 15 amino acids or less. Consists of the above amino acid sequence. The partial peptide of the present invention can be produced, for example, by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase.

The present invention also provides a DNA encoding the protein of the present invention. The DNA of the present invention is not particularly limited in its form as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, and the like in addition to cDNA. Also, as long as the protein of the present invention can be encoded, it has an arbitrary base sequence based on the degeneracy of the genetic code! ) NA is included. As described above, the DNA encoding the protein of the present invention was designed based on the hybridization method using the DNA sequence of SEQ ID NO: 1 or a part thereof as a probe. It can be isolated by a conventional method such as a gene amplification method (PCR) using primers.

The present invention also provides a vector into which a DNA encoding the protein of the present invention has been inserted. The vector of the present invention is not particularly limited as long as it stably retains the inserted DNA. For example, if Escherichia coli is used as a host, a pBluescript vector (manufactured by Stratagene) or the like may be used as a cloning vector. I like it. When a vector is used for the purpose of producing the protein of the present invention, an expression vector is particularly useful. Examples of the expression vector include p BEST vector (manufactured by Promega) for in vitro expression, pET vector (manufactured by Invitrogen) for expression in E. coli, and pME18S- for expression in cultured cells. The FL3 vector (GenBank Accession No. AB009864) and the pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) can be suitably used for expression in a living organism. Insertion of a DNA encoding the protein of the present invention into a vector can be carried out by a conventional method, for example, by using a restriction enzyme Sat. 7 protocols anti fi (Current protocols in Molecular Biology edit. Ausubel et al. 1987) Publish. John Wiley & Sons. Section 11.4 to 11.11).

The present invention also relates to a DNA encoding the protein of the present invention or the DNA inserted therein. A transformant carrying the vector is provided. The host cell into which the vector of the present invention is introduced is not particularly limited, and various host cells may be used depending on the purpose. Host cells can be used, for example, for the production of the proteins of the invention. Production systems for protein production include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells. When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as hosts. The host cells of the present invention also include cells intended for use in analyzing the function of the C-NT2RP2000329 protein and screening for its function inhibitor or function promoter utilizing the C-NT2RP2000329 protein. Vector transfer into host cells can be performed, for example, by the calcium phosphate precipitation method, electric pulse mosquito L, & (Current protocols m Molecular Biology edit. Ausubel et al. (1 987) Publish. John Wiley & Sons. Section 9 1-9. 9), Lipofectamine method (manufactured by GIBCO-BRL), microinjection method, etc. Preparation of the C-NT2RP2000329 protein from the transformant can be carried out by using a protein separation / purification method known to those skilled in the art.

The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof. Here, the “complementary strand” refers to one strand of a double-stranded DNA consisting of A: T, G: C base pairs with respect to the other strand. The term "complementary" is not limited to a completely complementary sequence of at least 15 contiguous nucleotide regions, but is at least 70%, preferably at least 80%, more preferably 90%, Preferably, it should have 95% or more homology on the base sequence. The algorithm described in this specification may be used as an algorithm for determining homology.

Such a polynucleotide can be used as a probe for detecting and isolating DNA encoding the protein of the present invention, and as a primer for amplifying the DNA of the present invention. When used as a primer, usually It has a chain length of 15-100 bases, preferably 15-35 bases, more preferably 20-30 bases. When used as a probe, a DNA having at least a part or all of the sequence of the DNA of the present invention and a chain length of at least 15 bases is used. When used as a primer, the 3 'region must be complementary, but a restriction enzyme recognition sequence or a tag can be added to the 5' side. These primers are effective in diagnosing a disease associated with the DNA of the present invention.

Further, the polynucleotide of the present invention includes an antisense for suppressing the expression of the C-NT2RP2000329 protein of the present invention. The antisense has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more, and preferably has a chain length of 2000 bp or less in order to cause an antisense effect. Such an antisense can be obtained, for example, based on the DNA sequence information shown in SEQ ID NO: 1 based on the phosphorothionate method (Stem, 1988 Physicochemical properties of phosphorot hioate oligodeoxynucleotides.Nucleic Acids Res 16, 3209-21). (1988)).

The DNA of the present invention and its antisense can be applied to, for example, gene therapy. Alzheimer's disease can be considered as a target disease for gene therapy using the DNA of the present invention. When these molecules are used for gene therapy, for example, ex vivo methods such as retroviral vectors, adenoviral vectors, non-inore vectors such as ribosomes, and non-inore vectors such as ribosomes are used. It may be administered to a patient by a method such as, or in vivo.

The present invention also provides an antibody that binds to the protein of the present invention. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody, a monoclonal antibody, and a part thereof having antigen-binding properties. Also includes all classes of antibodies. Furthermore, the antibodies of the present invention also include special antibodies such as humanized antibodies.

In the case of a polyclonal antibody, the antibody of the present invention can be obtained by synthesizing an oligonucleotide corresponding to the amino acid sequence according to a conventional method and immunizing a rabbit. (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley Sons. Section 11.12-11.13), whereas for monoclonal antibodies, E. coli is routinely used. Immunize mice with the expressed and purified protein and protect them from the hybridoma cells of spleen cells and myeloma cells; protect them by T; 0 (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons.Section 11.4 to 11.11

) o

Antibodies that bind to the protein of the present invention may be used, for example, for the examination and diagnosis of abnormal expression or structural abnormality of these proteins, in addition to the purification of the protein of the present invention. Specifically, proteins are extracted from, for example, tissues, blood, or cells, and examined for abnormal expression or structure through detection of the proteins of the present invention by methods such as stamping, immunoprecipitation, and ELISA. · Can be diagnosed.

Antibodies that bind to the protein of the present invention may also be used for purposes such as treating diseases associated with the protein of the present invention. When antibodies are used for the purpose of treating patients, human antibodies or humanized antibodies are preferred because of their low immunogenicity. A human antibody is a mouse in which the immune system is replaced with that of a human (e.g., `` Functional transplant of megabase human immunoglobul in 丄 oc i recapitulates human antibody response in mice, Mendez, MJ et al. (1997) Nat. Genet. 15: 146-156 "). Moreover, a humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).

The present invention also provides a method for screening a compound that binds to the protein of the present invention. This screening method comprises: (a) a step of bringing a test sample into contact with the protein of the present invention or a partial peptide thereof; (b) a step of detecting the binding activity between the protein or its partial peptide and the test sample; (C) selecting a compound having an activity of binding to the protein or a partial peptide thereof. Specific methods include, for example, a method of contacting and purifying a test sample with an affinity column for the protein of the present invention, a method using a two-hybrid system, a West Western blotting method, a combinatorial chemistry method. Many known methods can be used, such as high-throughput screening techniques. Screening can also be performed by evaluating the binding between the protein of the present invention and a test compound using a measuring device such as BIACORE (Pharmacia). The test sample used for screening is not limited to these, but includes, for example, a cell extract, an expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, a natural compound, and the like.

The compound isolated by this screening is a candidate for a compound (agonist, antagonist) that promotes or inhibits the activity of the protein of the present invention. In addition, it is a candidate for a compound that inhibits the interaction between the protein of the present invention and a molecule that interacts with the protein in vivo.

The present invention also provides a method for diagnosing a disease caused by abnormal expression of the gene of the present invention.

This diagnosis includes, for example, MELAS (stroke-like symptoms and hyperlactemia), central nervous system, epilepsy, skeletal muscle pathology, muscular disease, abnormal electron transmission, Leber disease, diabetes, Peason disease, Perkinson disease, metabolism It is expected to be effective for diagnosis of diseases such as abnormalities.

The diagnosis of the present invention can be performed by preparing a cell sample from a patient and detecting the expression level or mutation of the gene of the present invention in the cell. The cell sample prepared from the patient may be appropriately selected according to the type of the disease to be diagnosed. For example, cells derived from muscle tissue can be suitably used.

Expression of the gene of the present invention in cells can be detected by a method known to those skilled in the art. Examples of such a method include, but are not limited to, Northern blotting and RT-PCR. In the Northern Plotting method, RNA from cells to be tested is purified and agarose gel The probe of the present invention is hybridized with a radioactive-labeled probe of the gene of the present invention, and the expression of the gene of the present invention is measured based on the presence or absence of a band that appears specifically and the density of the band. . In the RT-PCR method, RNA of a cell to be tested is purified, converted into cDNA using reverse transcriptase, and this is designated as type 铸, and a sequence characteristic of the gene of the present invention is defined as a primer, and The cDNA derived from the gene transcript is amplified by PCR. Since the amount of cDNA amplified by this is considered to be proportional to the amount of type II cDNA and, consequently, the amount of the transcript of the gene of the present invention, the amount of DNA fragment amplified by this PCR is determined by electrophoresis. By using such a method, the expression level of the gene of the present invention can be measured.

The mutation of the gene of the present invention can be detected by a method of directly determining the nucleotide sequence by PCR or the like. In the PCR method, DNA of a cell to be tested is prepared, and the DNA is amplified into a 铸 type, and a sequence characteristic of the gene of the present invention is used as a primer to amplify the gene portion of the present invention on the chromosome of the cell. However, the presence or absence of a mutation can be measured by confirming the nucleotide sequence. In addition, when there is a large amount of sample, it is preferable to use the SSCP method.

The present invention also provides a method for screening a candidate compound for a drug for preventing or treating a disease caused by DNA abnormality of the present invention. The screening method of the present invention is considered to be suitable for screening candidate compounds for drugs for preventing or treating the above-mentioned diseases.

The screening according to the present invention provides a method in which a test sample is brought into contact with a cell in which the gene of the present invention is expressed, the expression level of the gene of the present invention in the cell is measured, and the test sample is not contacted. This can be carried out by selecting a compound that increases or decreases the expression level of the gene of the present invention in the cells that have been brought into contact with the test sample.

The test sample is not particularly limited, and examples thereof include compounds obtained by combinatorial chemistry technology (Tetrahedron (1995) 51, 8135-8137), Alternatively, a random peptide group created by applying a phage display method (J. Mol. Biol. (1991) 222, 301-310) can be used. In addition, culture supernatants of microorganisms and natural components derived from plants and marine organisms are also subject to screening. Other examples include, but are not limited to, biological tissue extracts, cell extracts, expression products of gene libraries, synthetic low-molecular compounds, synthetic peptides, and natural compounds.

Examples of cells used for screening include, but are not limited to, COS7 cells. The expression of the gene of the present invention in the cells can be measured by a method known to those skilled in the art, for example, the Northern blotting method, the RT-PCR method, and the like, as in the above-described diagnosis.

As a result of this measurement, a compound capable of altering the expression of the gene of the present invention is expected to act, for example, to regulate the expression level of the gene of the present invention in patients with the above-mentioned diseases. Candidate for therapeutic drug.

When the gene of the present invention, its protein, a compound that regulates the expression of the gene, or a compound that regulates the activity of the protein is used as a drug, the drug itself can be used as a drug, but a known preparation can be used. It is also possible to formulate and use it by a chemical method. For example, it may be used in the form of a formulation by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent and the like. Administration to a patient can be performed by a method known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. When DNA is used as a therapeutic agent, the DNA may be incorporated into a vector for gene therapy and administered to a patient. The dose and the administration method vary depending on the patient's body weight, age, symptoms and the like, but those skilled in the art will be able to select as appropriate.

The present invention also provides a DNA chip on which the DNA or polynucleotide of the present invention is arranged. Offer In the present invention, the term "DNA chip" refers to a DNA chip having a size of 1 to 10 cm ² , such as glass or silicon, on which a large number of DNAs are precisely arranged in a predetermined order. As a result, analysis of DNA and RA can be performed in a large amount in a short time. The DNA chip can be manufactured as follows. First, a partial sequence specific to the gene of the present invention is selected as a probe. This is spotted, for example, on a commonly supplied DNA chip substrate made of glass or silicon having a size of l to 10 cm ² using a microarrayer. As a method for immobilizing the probe on the substrate, a method known in the art can be used. For example, an amino group for covalent bonding can be introduced into the end of the probe in advance, and immobilized on a silanized substrate surface by silane coupling. After immobilization of the probe, regions other than the region where the probe is immobilized are coated with, for example, polylysine to avoid nonspecific binding of sample DNA and RNA to the silane-coated surface. One or more probes can be fixed to a single chip base. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a comparison between the amino acid sequence of the AMP phosphotransferase, which is homologous to the amino acid sequence of the protein of the present invention, and the amino acid sequence of the protein of the present invention.

FIG. 2 is a diagram showing a comparison of the amino acid sequence of the rat amino acid sequence of rat mitochondrial GTP-binding protein AMP phosphotransferase showing homology with the amino acid sequence of the protein of the present invention and the protein of the present invention.

FIG. 3 is a diagram showing a comparison between the amino acid sequence of a human mitochondrial GTP-binding protein AMP phosphotransferase showing homology with the amino acid sequence of the protein of the present invention and the amino acid sequence of the protein of the present invention. . BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. Unless otherwise specified, the method can be carried out according to a known method (Mamatis, T. at al. (1982): Molecular Cloning-A Laboratory Manual, Old Spring Harbor Laboratory, NY).

[Example 1] Production of a cDNA library from NT-2 cells by the oligocap method

NT-2 neural progenitor cells (purchased from Stratagene) that can be differentiated into neural cells by retinoic acid treatment in teratocarcinoma cells derived from human fetal testis are cultured, and induced to differentiate by adding retinoic acid. Cultured. From the cells, mRNA was extracted by the method described in Molecular Coating, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989). Further, according to the method described in Molecular Cloning, A Laboratory Manual, Second Edition, and Old Spring Harbor Laboratory Press (1989), an oligo (dT) cellulose column (Col laborative labs) was used to obtain poly (A) + RNA. Was purified.

A) + RNA from the pol ba! A cDNA library was prepared by the gocap method [M. Maruyama and S. Sugano, Gene, 138: 171-174 (1994)]. Using an oligocaplinker (synthetic RNA) consisting of the sequence represented by SEQ ID NO: 3 and an oligo (dT) adapter consisting of the sequence represented by SEQ ID NO: 4, the literature [Suzuki * Sugano, Protein Nucleic Acid Enzyme, 41: 197-201 (1996), Y. Suzuki et al., Gene, 200: 149-156 (1997)], BAP (Bacterial Alkaline Phosphatase) treatment, TAP (Tobacco Acid Pyrophosphatase) Treatment, RNA ligation, synthesis of first-strand cDNA and removal of RNA were performed. Then, using a 5′-end PCR primer represented by SEQ ID NO: 5 and a 3′-end PCR primer represented by SEQ ID NO: 6, it was converted into double-stranded cDNA by PCR (polymerase chain reaction). The obtained DNA fragment was cut with Sfil. Next, the direction of the cDNA was determined and cloned into the vector pME18SFL3 (GenBank AB009864) cut with Dralll to prepare a cDNA library. pME18S The cloned site of FL3 is an asymmetric Dralll site, and a complementary Sfil site is added to the end of the cDNA fragment. Is introduced in a one-way direction downstream of.

[Example 2] Analysis of a cDNA clone derived from a cDNA library prepared from NT-2 cells

(1) Isolation of cDNA clone

A part of the cDNA library prepared in Example 1 was introduced into E. coli DH10B by electroporation using Gene Pulser (manufactured by Biorad). Transformants were selected by culturing on LB agar medium containing 50 g / ml ampicillin. These transformants were cultured overnight in an LB medium containing 50 g / nLL of ampicillin, and a plasmid was extracted using an automatic plasmid extractor PI100 (manufactured by Kurabo Industries, Ltd.).

(2) Analysis of the nucleotide sequence of the isolated cDNA clone

Plasmid DNA from clones obtained from these transformants was subjected to DNA sequencing using a DNA sequencing kit (BigDye rermmator and Ycle Sequencing FII Ready Reaction Kit, manufactured by PE Biosystems) according to the manual. The nucleotide sequence from the 5 'end or 3' end of each cDNA clone was analyzed using an NA sequencer (ABI PRISM 377, manufactured by PE Biosystems).

For determination of the base sequence from the 5 'end, use ME761FW represented by SEQ ID NO: 7, and for determination of the base sequence from the 3' end, use ME1250RV represented by SEQ ID NO: 8. Used as primer.

(3) Clustering of 5 'and 3' end sequences of cDNA clones

The 5′-end sequence and the 3′-end sequence of the cDNA clone determined in (2) were separately clustered. That is, the determined 5 'end and 3' end of the cDNA clone The BLAST analysis was performed on the single-pass sequence data from each sequence data with each sequence data, and clones considered to be derived from the same gene were grouped. If the consensus sequence with a homology of 95% or more is 300 base pairs or more in the 5 'end sequence and the consensus sequence with a homology of 90% or more is 200 base pairs or more in the 3' end sequence The 'terminal sequence group' was further processed into a group (cluster) so that the 5 'terminal sequence and the 3' terminal sequence of the same clone belonged to the same group (cluster 1).

(4) Characterization of cDNA clone sequence

5 ′ terminal sequence data of the clone sequence was characterized based on the following method.

(1) By homology search using BlastN for GenBank, it is confirmed whether the sequence is identical to the human {other mRNA sequence (including the licensed sequence)} / human EST sequence.

(2) Confirm that the human mRNA sequence is longer at the 5 'end than the human EST sequence.

(3) The ATGpr program [A. Salamov, T. Nishikawa, MB Sw indells. Assessing protein coding region integrity in cDNA sequencing projects. Bioinf ormatics 14: 384-390 (1998)] Determine ATGprl and ATGpr2 values from all start codons.

(4) Determine the number of identical human EST sequences by homology search using BlastN for GenBank.

Characterization of the 3'-terminal sequence data of the clone sequence was performed for (1) and (4) above.

Based on the data of the clone sequences thus characterized, a new and potentially full-length cDNA clone was selected.

(5) Human mRNA sequence ゃ Identity to human EST sequence Comparison of 5'-end length The identity of the 5'-end and 3'-end sequences of the clone sequence to the human or other organism's mRNA sequence is at least 94% identical to the sequence compared to each sequence when the sequence length is 200 bases or more. Were considered identical. The identity to the human EST sequence was considered to be identical when the length of the comparison sequence with the 5'-terminal sequence was 200 bases or more and 90% or more matches.

When comparing the length of the 5 'end with the human mRNA sequence as a comparison sequence, when the length of the 5' end sequence is longer than the human mRNA sequence, or when the 5 'end sequence contains a translation initiation codon , And the total length. When the comparison target sequence is EST, it is convenient if the 5 'end is longer than the human EST sequence in the database, or if the difference between the two is within 50 bases even for a clone with a shorter 5' end. The total length is defined as the total length, and the shorter length is defined as the non-full length.

(6) Prediction of full length by ATGpr

The results of ATGpr [A. Salamov, T. Nishikawa, Μ. Β. Swindells. As sessing protein coding region integrity in cDNA sequencing projects. Bioinformatics 14: 384-390 (1998)] were used for prediction of full length. . The ATGprl value predicts the possibility of the full length from the calculated value, and the higher the ATGpr1 value, the higher the possibility of the full length. The maximum ATGprl value and the maximum ATGpr2 value indicate the maximum ATGpr1 and ATGpr2 values predicted from all the start codons contained in the 5 'terminal sequence of the clone sequence. Using.

(7) Prediction of novelty from the number of identical EST sequences by homology search

5 'end sequence Each 3' end sequence was determined by homology search using GenBank. The human EST sequence was determined to be the same when the length of the comparison sequence portion with the 5'-terminal sequence was 90% or more over 200 bases or more. The number of EST sequences was used directly for the characterization and used as an index of novelty. Clones with 5 'and 3' end sequences that are not identical to mRNA sequences but also to EST sequences —The gene that is Similarly, clones having a small number of 5'-terminal sequences or 3'-terminal sequences having a small number of identical EST sequences were also determined to be cDNA clones encoding the novel sequences.

(8) Cluster characterization

5'-end sequence A cluster that groups the 3'-end sequences was characterized based on the following viewpoints.

(1) By homology search using BlastN for GenBank, the human mRNA sequence (including the licensed sequence) of other organisms and the human EST sequence are identical.

When at least one of the 5′-terminal sequences contained in the cluster was identical to the mRNA sequence, the cluster was regarded as the same cluster as the mRNA sequence.

(2) Is the human mRNA sequence longer at the 5 'end than the human EST sequence?

If all the 5 'terminal sequences included in the cluster were not full-length with respect to the mRNA sequence or the human EST sequence, the cluster was classified as a cluster that was non-full-length with respect to the mRNA sequence or the human EST sequence. did.

(3) ATGpr 1 value and ATGpr 2 value derived from all start codons in the 5 ′ terminal sequence by the ATGpr program for predicting full length.

All in the 5 'end sequence by the ATGpr program [A. Salamov, T. Nishikawa, MB Swindells. Assessing protein coding region integrity in cDNA sequencing projects. Bioinformatics 14: 384-390 (1998)] For the ATGprl value derived from the start codon of the above, the maximum value of the ATGprl value for all the 5 'terminal sequences included in the cluster was defined as the ATGprl value in the cluster. ATGpr2 values were also the same.

(4) Number of human EST sequences determined to be identical by homology search using BlastN for GenBank. The maximum value of the number of EST sequences was determined for each of the 5'-terminal sequences and the 3'-terminal sequences included in the cluster, and the number of the same EST sequences of the 5'-terminal sequence in the cluster and the same number of EST sequences of the 3'-terminal sequence were determined.

(9) Selection method of cluster best from characterization

First, from the data obtained by the characterization, clusters identical to the mRNA sequences of humans and other organisms (including the licensed sequences) and non-full-length clusters were excluded. From those clusters, those that met any of the following conditions were selected.

(a) A cluster in which the number of identical EST sequences in the 5'-terminal sequence in the cluster is 20 or less and the ATGprl value in the cluster exceeds 0.3.

(b) Even if the ATGprl value in the cluster is 0.3 or less, the number of identical EST sequences in the 5'-terminal sequence in the cluster is not more than ^^, and the number of identical EST sequences in the 3'-terminal sequence in the cluster is also A cluster with 5 or less and multiple clones in the cluster.

(c) Even if the cluster has an ATGprl value of 0.3 or less, the number of identical EST sequences in the 5 'terminal sequence in the cluster is 0, and the number of identical EST sequences in the 3' terminal sequence in the cluster is 0. Cluster one that is 1 or more.

(d) Even if the ATGprl value in cluster 1 is 0.3 or less, the number of identical EST sequences in the 5 'end sequence in cluster 1 is 1 or more and 5 or less, and the 3' end sequence in cluster 1 The same EST sequence number of clusters that are SO.

In the cluster selected in (a), at least one clone contains a clone with high novelty and full length. The clusters selected in (b), (c), and (d) have a lower overall length, but are still full-length and contain new clones.

(10) How to select clones from cluster 1

If only one clone is included in the same cluster, Selected. When multiple clones were included in the same cluster and there were multiple clones with an ATGprl value greater than 0.3, the clone with the higher ATGprl value was selected. If the same cluster contains multiple clones and there are multiple clones with an ATGprl value of 0.3 or less, if the ATGpr2 value is greater than 0.3, the clone with the larger ATGpr2 value was selected. . In addition, when a plurality of clones are included in the same cluster, and the ATGprl value and ATGpr2 value are both 0.3 or less, if there is a clone having the maximum ATGprl value and ATGpr2 value in the cluster, the clone is replaced. Selected . If multiple clones are included in the same cluster and selection with the ATGpr value as described above is not possible, assemble using the 5 'end sequence and the 3' end sequence and the human EST sequence. A longer clone was selected on the 5 'end side. For assembly, Sequencher (manufactured by Gene Codes) was used, and if some parts could not be determined by assembling, all target clones were judged to be full length.

(11) Analysis of full-length sequence of cDNA clone

The nucleotide sequence of the full-length cDNA was determined for the NT-2 cell-derived cDNA clones selected as described in (1) to (10) and determined to be highly likely to be novel. The nucleotide sequence is mainly based on primer walking by the dideoxy terminator method using a custom synthesized DNA primer (sequencing is performed according to the manual using a DNA sequencing reagent manufactured by PE Biosystem, using a custom synthesized DNA primer). After the reaction, the DNA sequence was analyzed using the company's sequencer). The full-length nucleotide sequence was finally determined by completely overlapping the partial nucleotide sequence determined by the above method. Next, a deduced amino acid sequence was determined from the determined nucleotide sequence of the full-length cDNA.

As an example of a cDNA clone derived from NT-2 cells selected as described in (1) to (10) and determined to be highly likely to be new and full-length, cDNA clone C- The nucleotide sequence of NT2RP2000329 is shown in SEQ ID NO: 1. SEQ ID NO: 2 shows the amino acid sequence of the gene product encoded by cDNA clone C-NT2RP2000329 estimated from the full-length nucleotide sequence.

[Example 3] Function analysis of C-NT2RP2000329

C-NT2RP2000329 (SEQ ID NO: 1) was searched using the in-house GenBank database BLAST program. As a result, as shown in Fig. 1, C_NT2RP2000329 was produced in the nucleus of the cell of the moss as reported in the literature (Shahjahan M. et al., (1991) Gene 107: 313'-317). It showed 92% similarity to the amino acid sequence of the AMP phosphotransferase (AK3), a GTP-binding protein from tochondria. In addition, as shown in Figure 2, mitochondria produced in the nuclei of rat cells reported in the literature (Tanabe T. et al., (1993) J. Biochem. 113: 200-207). The amino acid sequence of GTP-binding protein AMP phosphotransferase (AK3) showed 90% similarity. In addition, as shown in Figure 3, GTP binding of mitochondria produced in the nucleus of human cells reported in the literature (Xu G. et al., (1992) Genetics 13: 537-542). The amino acid sequence of protein AMP phosphotransferase (AK3) showed 60% similarity. Thus, C-NT2RP2000329 had a similarity of 90% or more with AK3 of human and rat, but only 60% of similarity with human AK3. Therefore, C-NT2R P2000329 is a new and different human gene. Industrial applicability

The present invention provides a novel AK3-like protein (C-NT2RP2000329), a gene encoding the protein, a vector containing the gene, a transformant containing the vector, and a method for producing the protein. It is suggested that the cDNA of the present invention is associated with abnormally caused diseases such as conversion of extracellular information into intracellular information, change in cell morphology, apoptosis, cell motility, and intracellular substance transport. Therefore, the genetics of the present invention Children or proteins are effective for the development of diagnostic markers and pharmaceuticals for these diseases.

Claims

The scope of the claims

1. The DNA according to any one of (a) to (d) below.

(a) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2.

(b) DNA containing the coding region of the nucleotide sequence of SEQ ID NO: 1.

(c) one or more amino acids in the amino acid sequence of SEQ ID NO: 2 having substitution, deletion, insertion, and / or addition of an amino acid sequence;

(d) hybridizing with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions, and encoding a protein functionally equivalent to the protein consisting of the amino acid sequence of SEQ ID NO: 2; DNA.

2. A DNA encoding a partial peptide of a protein consisting of the amino acid sequence of SEQ ID NO: 2.

3. A protein or peptide encoded by the DNA of claim 1.

4. A vector into which the DNA according to claim 1 has been inserted.

5. A transformed cell carrying the DNA according to claim 1 or the vector according to claim 4.

6. The protein or peptide according to claim 3, comprising a step of culturing the transformed cell according to claim 5, and recovering the expressed protein or peptide from the transformed cell or a culture supernatant thereof. Manufacturing method.

7. An antibody that binds to the protein of claim 3.

8. A polynucleotide comprising at least 15 nucleotides complementary to DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof.

9. A method for screening a compound that binds to a protein according to claim 3, wherein (a) contacting a test sample with the protein or a partial peptide thereof,

(c) selecting a compound having an activity of binding to the protein or a partial peptide thereof.

10. A method for diagnosing a disease caused by a DNA abnormality according to claim 1,

(a) preparing a cell sample from a patient; and

(b) detecting the expression level or mutation of the DNA according to claim 1 in the cell.

1 1. A method for screening a candidate compound for a drug for preventing or treating a disease caused by DNA abnormality according to claim 1,

(a) contacting a test sample with cells expressing the DNA of claim 1

(b) measuring the expression level of the DNA according to claim 1 in the cells, and

(c) selecting a compound that increases or decreases the DNA expression level according to claim 1, which is measured in step (b), as compared with a case where the test sample is not contacted.

1 2. The DNA according to claim 1 or 2, the protein or peptide according to claim 3, the vector according to claim 4, or the compound isolated by the screening according to claim 9 or 11. A pharmaceutical composition comprising

1 3. A DNA chip on which the DNA according to claim 1 or 2 or the polynucleotide according to claim 8 is arranged.