JP2004196817A - Treatment of prostate carcinoma - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a human gene enabling detection of expression in various human tissues, analysis of the structure and function of the same, gene technological preparation of human proteins, and to enable analysis of the expression, pathological analysis, diagnosis and treatment of related diseases to a gene disease, cancer, or the like. <P>SOLUTION: The subject treating method for prostate cancer comprises administration of a pharmaceutical composition comprising an antibody to human TMP (transmembrane protein)-2 protein, to a patient requiring the treatment. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a gene useful as a guide for the prevention, diagnosis and treatment of human diseases, and more specifically to a novel human gene having similarities to rats, mice, yeasts, nematodes and human genes, and the cDNA of the gene. The present invention relates to a novel human gene that can be used for gene diagnosis and development of a new therapeutic method using the gene by analysis, mapping to cDNA chromosome, and functional analysis of cDNA.

  Genetic information of organisms is accumulated as a sequence of four bases (DNA) A, C, G and T existing in the nucleus of cells, and this genetic information is used to maintain the lineage and ontogeny of individual organisms. Is stored in

In the case of humans, the number of bases is said to be about 3 billion (3 × 10 ⁹ ), and it is estimated that 50,000 to 100,000 genes are contained therein. The genetic information follows a process in which mRNA is transcribed from a gene (DNA) and then translated into a protein, whereby regulatory proteins, structural proteins or enzymes are produced to maintain a life phenomenon. Abnormalities in the flow from the above genes to protein translation cause abnormalities in life support systems such as cell proliferation and differentiation, and are thought to cause various diseases.

  From the results of the gene analysis to date, it is useful for drug development such as various receptors such as insulin receptor and LDL receptor and those involved in cell proliferation and differentiation, and metabolic enzymes such as protease, ATPase and superoxide dismutase. Many potential genes have been found.

  However, research on the analysis of human genes and the functions of the analyzed genes and the relationship between the analyzed genes and various diseases has only just begun, and there are many unclear points. There is a need in the art for the analysis of the function of, the study of the relationship between the analyzed gene and the disease, the gene diagnosis using the analyzed gene, and the study of the application of the gene to medical use.

  If the new human gene can be provided, the expression level in each cell, its structure and function can be analyzed, and the expression involved in the disease can be analyzed and diagnosed by analyzing the expression of the disease, such as genetic diseases and cancer. The present invention aims to provide such a new human gene.

  The present inventors have conducted intensive studies for the above purpose, and as a result, have obtained the following findings, and have completed the present invention.

  That is, the present inventors synthesized cDNA from mRNA extracted from various tissues such as human fetal brain, adult blood vessels, and placenta, constructed a library by incorporating the cDNA into a vector, and transformed the library. E. coli colonies were formed on an agar medium, the colonies were randomly picked and transferred to a 96-well microplate, and a number of E. coli clones containing human genes were registered.

  After culturing a small amount of each registered clone, DNA was extracted and purified, and an elongation reaction was performed to specifically stop the four bases by the deoxyterminator method using the extracted cDNA as a template, and an automatic DNA sequencer was used to extend the 5 ′ end of the gene. From about 400 base sequences. Based on the nucleotide sequence information thus obtained, novel family genes having similarity to animal, plant, and other species such as yeast, nematodes, mice, and humans were searched from known bacteria.

The cDNA analysis method is described in detail by Fujiwara et al., One of the present inventors (Fujiwara Riki, Cell Engineering, 14 , 645-654 (1995)).

  This group includes new receptors, transcription factors having DNA binding domains, signal transduction factors, metabolic enzymes, etc., and homology with the novel gene of the present invention obtained by genetic analysis and with similar genes From the above, it is possible to infer an approximate function of the gene product, that is, the protein. Furthermore, the candidate gene can be incorporated into an expression vector to produce a recombinant, and functions such as enzyme activity and binding activity can be examined.

  According to the present invention, SEQ ID NOs: 1,: 4,: 7,: 10,: 13,: 16,: 19,: 22,: 25,: 28,: 31,: 34,: 37 and: 40 A novel human gene comprising a base sequence encoding the amino acid sequence shown; SEQ ID NOS: 2, 5, 5, 8, 11, 11, 14, 14, 17, 20, 20; : 23,: 26,: 29,: 32,: 35,: 38 and: 41, a human gene comprising the nucleotide sequence represented by SEQ ID NO: 3,: 6,: 9,: 12, : 15,: 18,: 21,: 24,: 27,: 30,: 33,: 36,: 39 and: 42, and a novel human gene is provided.

  Hereinafter, the abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in the present specification are referred to in the IUPAC and IUB regulations, "Guidelines for the preparation of specifications containing base sequences or amino acid sequences" (Japan Patent Office) And conventional symbols in the field.

  As a specific example of the gene of the present invention, `` GEN-501D08 '', `` GEN-080G01 '', `` GEN-025F07 '', `` GEN-076C09 '', `` GEN-331G07 '' shown in Examples 1 to 11 described below , `` GEN-163D09 '', `` GEN-078D05TA13 '', `` GEN-423A12 '', `` GEN-092E10 '', `` GEN-428B12 '', `` GEN-073E07 '', `` GEN-093E05 '' and `` GEN-077A09 '' respectively Those deduced from the DNA sequence of each named clone can be mentioned, and their base sequences are as shown in the sequence listing.

  In addition, each of these clones had an open reading frame of a nucleotide (nucleic acid) encoded by each amino acid shown in the same sequence listing, and was calculated to have the molecular weight shown in Examples described later. . Therefore, each human gene of the present invention may be referred to as shown in Examples 1 to 11 below.

  Hereinafter, the human gene of the present invention will be described in detail.

  As described above, each of the human genes of the present invention has similarities to rats, mice, yeasts, nematodes and known human genes, and the analysis of human genes based on information on those similar genes and their analysis. The present invention can be used for research on the relationship between the function and analysis of genes and various diseases, and can be used for gene diagnosis of diseases related to genes and research on application of the genes to pharmaceutical applications.

The gene of the present invention is represented by a single-stranded DNA sequence, for example, as shown in SEQ ID NO: 2, but the present invention also includes a DNA sequence complementary to the single-stranded DNA sequence and a component containing both of them. Also encompasses. The sequence of the gene of the present invention shown in SEQ ID NO: 3n-1 (n = 1 to 14) is an example of one combination of codons indicating each amino acid residue encoded by the gene of the present invention. The present invention is not limited to this, and it is of course possible to have a DNA base sequence selected by combining any codon for each amino acid residue. The selection of the codon can be performed according to a conventional method, for example, by considering the codon usage of the host to be used [Ncl. Acids Res., 9 , 43-74 (1981)].

Further, in the gene of the present invention, a partial amino acid to amino acid sequence of the amino acid sequence shown above is modified by substitution, deletion, addition or the like, and a DNA sequence encoding the same compound having the same function is also included. Included. Production, modification (mutation) and the like of these polypeptides may occur naturally, and natural genes (the gene of the present invention) may be prepared by post-translational modification or by genetic engineering techniques, for example, cytospecific mutagenesis [ Methods in Enzymology, 154 , p350, 367-382 (1987); pp. 100 , p468 (1983); Nucleic Acids Research, 12 , p9441 (1984); eds, or modified by a method such as p105 (1986)], the chemical synthesis means such as phosphate triester method and phosphate amidite method [J. Am Chem Soc, 89, p4801 (1967);... the 91, p3350 (1969); Science, 150 , p178 (1968); Tetrahedron Lett., 22 , p1859 (1981); and DNA mutated by 24 , p245 (1983)], or a DNA obtained by a combination thereof. Can be.

  The gene of the present invention can easily and stably express the protein encoded by each of the above-mentioned genes, that is, for example, by incorporating the gene into a microorganism vector and culturing the transformed microorganism.

  Each protein obtained by using the gene of the present invention can be used to prepare a specific antibody. The component used as the antigen here can be a protein produced in large amounts according to the above-described genetic engineering technique, and the obtained antibody may be any of a polyclonal antibody and a monoclonal antibody. It can be used advantageously for measurement, identification, etc.

  According to the sequence information on the gene of the present invention disclosed by the present invention, production of the gene of the present invention can be easily performed by general genetic engineering techniques [Molecular Cloning 2nd Ed, Cold Spring Harbor Laboratory Press (1989); See "Seismic Chemistry Laboratory Course, Genetic Research Methods I, II, and III", edited by The Biochemical Society of Japan (1986)).

This involves selecting a desired clone from a human cDNA library (prepared from an appropriate source cell in which each gene is expressed in accordance with a conventional method) using an appropriate probe or antibody specific to the gene of the present invention. [Proc. Natl. Acad. Sci. USA, 78 , 6613 (1981); Science, 222 , 778 (1983) and the like].

  In the above method, the source cells include various cells and tissues expressing the target gene, cultured cells derived therefrom, and the like. From this, separation of total RNA, separation and purification of mRNA, conversion to cDNA ( Synthesis) and cloning thereof can be carried out according to a conventional method. In addition, cDNA libraries are commercially available, and in the present invention, those cDNA libraries, for example, various cDNA libraries commercially available from Clontech Lab. Inc. can also be used.

  The screening of the gene of the present invention from the cDNA library can be carried out according to the above-mentioned usual method. As the screening method, for example, a method for selecting a corresponding cDNA clone by immunological screening using a protein-specific antibody for a protein produced by a cDNA, and using a probe that selectively binds to a target DNA sequence Plaque hybridization, colony hybridization, and the like, and combinations thereof. As the probe used here, it is common to use a DNA sequence or the like chemically synthesized based on the information on the DNA sequence of the gene of the present invention. Can be used as a probe.

  Further, based on partial amino acid sequence information of a natural extract extracted, isolated and purified from each cell or tissue, a sense primer or an antisense primer can be used as a screening probe.

In addition, in obtaining the gene of the present invention, a DNA / RNA amplification method by a PCR method [Science, 230 , 1350-1354 (1985)] can be suitably used. Especially when it is difficult to obtain a full-length cDNA from the library, the lace method (RACE: Rapid amplification of cDNA ends; Experimental Medicine, 12 (6), 35-38 (1994)), especially the 5 ′ race ( The 5'RACE method (Frohman, MA, et al., Proc. Natl. Acad. Sci., USA., 8 , 8998-9002 (1988)) is preferred. Primers to be used when employing such a PCR method can be appropriately set based on the sequence information of the gene of the present invention already revealed by the present invention, and can be synthesized according to a conventional method.

  In addition, isolation and purification of the amplified DNA / RNA fragment can be performed according to a conventional method as described above, for example, by gel electrophoresis.

The determination of the nucleotide sequence of the gene of the present invention or various DNA fragments obtained above can be performed according to a conventional method, for example, the dideoxy method [Proc. Natl. Acad. Sci. USA, 74 , 5463-5467 (1977)]. And the Maxam-Gilbert method [Method in Enzymology, 65 , 499 (1980)]. Such determination of the nucleotide sequence can be easily carried out using a commercially available sequence kit or the like.

According to the use of the gene of the present invention, conventional gene recombination techniques [for example, Science, 224 , p1431 (1984); Biochem. Biophys. Res. Comm., 130 , p692 (1985); Proc. Natl. Acad. Sci. USA, 80 , p5990 (1983) and the above-cited references] can be used to obtain each recombinant protein. More specifically, the protein is produced by preparing a recombinant DNA capable of expressing the gene of the present invention in a host cell, introducing the gene into a host cell, transforming the same, and culturing the transformant. It is.

Here, both eukaryotes and prokaryotes can be used as host cells. The eukaryotic cells include cells such as vertebrates and yeasts. Examples of the vertebrate cells include COS cells (Cell, 23 , 175-182 (1981)) which are monkey cells and Chinese hamsters. Ovarian cells and their dihydrofolate reductase deficient strains [Proc. Natl. Acad. Sci. USA, 77 , 4216-4220 (1980)] and the like are often used, but are not limited thereto.

As a vertebrate expression vector, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like, which is usually located upstream of a gene to be expressed, can be used. You may have. Examples of the expression vector include, for example, pSV2dhfr [Mol. Cell. Biol., 1 , 854 (1981)] which has an early promoter of SV40. As eukaryotic microorganisms, yeasts are generally used, and among them, yeast of the genus Saccharomyces can be advantageously used. As an expression vector for eukaryotic microorganisms such as the yeast, for example, pAM82 (Proc. Natl. Acad. Sci. USA, 80 , 1-5 (1983)) having a promoter for the acid phosphatase gene can be used.

  As the expression vector of the gene of the present invention, a prokaryotic gene fusion vector can be preferably used. Specific examples of the vector include, for example, pGEX-2TK having a GST domain having a molecular weight of 26000 (derived from S. japonicum) and pGEX-4T-2 and the like can be exemplified.

  Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. When these are used as a host, the present invention uses, for example, a plasmid vector that can be replicated in the host bacterium, and a promoter and an SD (Shine and & Co.) upstream of the gene so that the gene of the present invention can be expressed in the vector. (Dalgarno) It is preferable to use an expression plasmid to which a base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis have been added. As the Escherichia coli as the host, Escherichia coli K12 strain or the like is often used, and as the vector, pBR322 and its improved vector are generally used, but not limited thereto, and various known strains and vectors are generally used. Is also available. As the promoter, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, PL / PR promoter and the like can be used.

  As a method for introducing the desired recombinant DNA thus obtained into a host cell and a transformation method using the same, various general methods can be employed. The obtained transformant can be cultured according to a conventional method, and the target protein encoded by the gene of the present invention is produced and expressed by the culture. As the medium used for the culture, various types of media commonly used depending on the host cells used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cells.

  As described above, the desired recombinant protein is expressed, produced, accumulated or secreted inside, outside, or on the cell membrane of the transformant.

Each recombinant protein, if desired, may be subjected to various separation procedures utilizing its physical properties, chemical properties, etc. (“Biochemical Data Book II”, pp. 1175-1259, 1st edition, 1st printing, June 1980 23, Tokyo Chemical Doujinsha Co., Ltd .; see Biochemistry, 25 (25), 8274-8277 (1986); Eur. J. Biochem., 163 , 313-321 (1987), etc.]. Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration) And various types of liquid chromatography such as adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC), dialysis, and combinations thereof. Particularly preferred methods include binding a desired protein. An example is affinity chromatography using a column that has been subjected to aging.

  Further, based on the sequence information of the gene of the present invention revealed by the present invention, for example, by using a part or all of the nucleotide sequence of the gene, detection of the expression of the gene of the present invention in various human tissues can be detected. Can do it. This can be performed according to a conventional method, for example, RT-PCR (Reverse transcribed-Polymerase chain reaction) (Kawasaki, ES, et al., Amplification of RNA. In PCR Protocol, A Guide to methods and applications, Academic Press, Inc. , SanDiego, 21-27 (1991)) and Northern blotting analysis (Molecular Cloning, Cold Spring Harbor Laboratory (1989)).

  In the case of employing the PCR method, the primers used are not particularly limited as long as they are specific to the gene of the present invention capable of specifically amplifying only the gene of the present invention. Can be set as appropriate. Usually, it can have a partial sequence of about 20 to 30 nucleotides according to a conventional method. Preferred examples thereof are as shown in Examples 1-11 described below.

  Thus, the present invention also provides primers and / or probes useful for detection unique to such a novel human gene.

  According to the present invention, a novel human gene is provided, and by using the gene, expression of the gene in various tissues can be detected, its structure and function can be analyzed, and the human protein encoded by the gene can be analyzed. It is possible to produce genetically engineered products, thereby enabling analysis of the expression of the products, and elucidation, diagnosis, treatment, and the like of diseases involving them, such as genetic diseases and cancer.

  Hereinafter, examples will be given to describe the present invention in more detail.

GDP dissociation-promoting protein gene (1) Cloning and DNA sequencing of GDP dissociation-promoting protein gene mRNA extracted from human fetal brain, adult blood vessels, and placenta was purchased from Clonetech and used as a starting material.

  CDNA was synthesized from each of the above mRNAs and inserted into the vector λZAPII (Stratagene) to construct a cDNA library (Otsuka GEN Research Institute, Otsuka Pharmaceutical Co., Ltd.).

Escherichia coli colonies containing human genes were formed on an agar medium at random by the in vivo excision method (In vivo excision: Short, JM, et al., Nucleic Acids Res., 16 , 7583-7600 (1988)). A colony was picked up and an E. coli clone containing a human gene was registered in a 96-well microplate. The registered clone was stored at -80 ° C.

  Next, each registered clone was cultured overnight in 1.5 ml of LB medium, and DNA was extracted and purified using a plasmid automatic extractor PI-100 (manufactured by Kurabo Industries, Ltd.). The contaminated RNA of Escherichia coli was decomposed and removed by RNase treatment. Finally, the DNA was dissolved in 30 μl, 2 μl was roughly checked for the size and amount of DNA using a minigel, 7 μl was used for a sequencing reaction, and the remaining 21 μl was stored at 4 ° C. as plasmid DNA.

  According to this method, a cosmid that can be used also as a probe for FISH (fluoresence in situ hybridization) shown in each of the following examples can be extracted by slightly changing the program.

Subsequently, the dideoxy terminator method of Sanger et al. Using T3, T7 or synthetic oligonucleotide primers (Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74 , 5463-5467 (1977)). Alternatively, a cycle sequence method (Carothers, AM, et al., Bio. Techniques, 7 , 494-499 (1989)), which is a method in which the PCR method is added to the dideoxy terminator method, was performed. In these methods, a small amount of plasmid DNA (approximately 0.1-0.5 μg) is used as a template (template) to perform an elongation reaction in which four kinds of bases are specifically stopped.

As a sequencing primer, a fluorescently labeled FITC (fluorescein isothiocyanate) was used, and the reaction was generally performed for about 25 cycles with Taq polymerase. The PCR product was separated on a polyacrylamide-urea gel, and the sequence of about 400 bases from the 5 ′ end of the cDNA was determined for the fluorescently labeled DNA fragment using an automatic DNA sequencer, ALF ^™ DNA sequencer (Pharmacia).

  In addition, since the 3 'untranslated region has a high heterogeneity of each gene and is suitable for distinguishing individual genes, the 3' side was also sequenced in some cases.

The vast amount of nucleotide sequence information obtained by the DNA sequencer was transferred to a 64-bit computer DEC3400 and used for homology analysis by computer. Homology analysis was performed by searching the database (GenBank, EMBL) using the UWGCG FASTA program (Pearson, WR and Lipman, DJ, Proc. Natl. Acad. Sci., USA, 85 , 2444-2448 (1988)). Was.

  By arbitrarily selecting according to the above method and analyzing the cDNA sequence, one clone having a 0.8-kb insertion named GEN-501D08 was found to have the C-terminal of the human RalGDP dissociation stimulator (RalGDS) gene. It showed high homology to the region. Since RalGDS appears to play a role in the signal transduction pathway, the complete nucleotide sequence of the cDNA insert providing its human homolog was further determined.

Small GTPases play a critical role in signaling many cell functions, including cell proliferation, differentiation, and transcription (Bourne, HR, et al., Nature, 348 , 125- 132 (1990); Bourne et al., Nature, 349 , 117-127 (1991)).

Among them, the protein encoded by the ras gene family functions as a molecular switch, that is, the function of the ras gene family is a binding protein such as a biologically inactive GDP binding protein or an active GDP binding protein. It is well known that these two conditions are induced by GTPase activating proteins (GAPs) or GDS. The former induces GDP binding by stimulating the hydrolysis of bound GTP, and the latter induces defined GTP binding by releasing bound GDP (Bogusuki, MS and McCormick, F., Nature, 366 , 643-654 (1993)).

RalGDS was first discovered as a member of the ras gene family without transforming activity as a specific GDP dissociation promoting protein for RAS (Chardin, P. and Tavitian, A., EMBO J., 5 , 2203-2208 (1986); Albright, CF, et al., EMBO J., 12 , 339-347 (1993)).

In addition to Ral, RalGDS was found to function as an effector molecule for N-ras, H-ras, K-ras, and Rap by interacting with these proteins (Spaargaren, M. and Bischoff, JR Natl. Acad. Sci. USA, 91 , 12609-12613 (1994)).

  SEQ ID NO: 3 shows the nucleic acid sequence of a cDNA clone named GEN-501D08, SEQ ID NO: 2 shows the nucleic acid sequence of the coding region of the clone, and SEQ ID NO: 1 shows the amino acid sequence encoded by the nucleic acid sequence.

  This cDNA was composed of 842 bases and contained an open reading frame of 366 bases encoding 122 amino acids. The transcription initiation codon was located at the 28th nucleic acid residue.

  Comparison of the amino acid sequence deduced from this cDNA with the RalGDS protein known in the general database revealed that the protein encoding this cDNA is a homologue to the C-terminal region of human RalGDS. It became. The amino acid sequence encoded by this novel gene had 39.5% identity to the C-terminal region of RalGDS, which is considered necessary for binding to ras.

Thus, as noted above, it is believed that this gene product may interact with ras family proteins or affect the ras-mediated signal transduction pathway. However, this novel gene does not contain a region encoding the GDS active region and appears to have a different function than the GDS protein. Thus, we named this gene human RalGDS.
(2) Northern blot analysis The expression of mRNA of RalGDS protein in normal human tissues was evaluated by Northern blot using a human cDNA clone labeled by a random oligonucleotide priming method as a probe.

  Northern blot analysis was performed using a human MTN blot (Human Multiple Tissue Northern blot; Clontech, Palo Alto, California, USA) according to the product usage.

That is, the PCR amplification product of the GEN-501D08 clone was labeled with [ ³² P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim) to obtain a probe.

  Blotting was carried out at 42 ° C. overnight in a solution of 50% formamide / 5 × SSC / 50 × Denhardz solution / 0.1% SDS solution (containing 100 μg / ml denatured salmon sperm DNA). After washing twice with 2 × SSC / 0.01% SDS at room temperature, then, washing with 0.1 × SSC / 0.05% SDS three times at 50 ° C. for 40 minutes. The filter was exposed to an X-ray film (manufactured by Kodak) at -70 ° C for 18 hours.

As a result, it was clarified that a 900 base transcript was expressed in all of the tested human tissues. In addition, a 3.2 kilo transcript was specifically observed in heart and skeletal muscle. Expression of transcripts of these different sizes may be owed to alternative splicing or cross-hybridization to homologous genes.
(3) Cosmid clone and chromosome localization by FISH
FISH was performed by screening a library of human chromosomes cloned into the cosmid vector pWE15 using the 0.8 kilobase insert of the cDNA clone as a probe (Sambrook, J., et al., Molecular Cloning, 2nd Ed., Pp.3.1-3.58, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York (1989)).

FISH for chromosomal locus localization was performed in the same manner as the method of Inazawa et al., Which confirmed by comparing G band patterns (Inazawa, J .. et al., Genomics, 17 , 153-162 (1993)). .

  0.5 μg of cosmid DNA corresponding to GEN-501D08 obtained from chromosome screening was labeled with biotin 16dUTP by nick translation to obtain a probe.

  In addition, 0.5 μl of sonicated human placental DNA (10 mg / ml) was added to 9.5 μl of the probe solution to remove background noise caused by the repetitive sequence. The mixture was denatured at 80 ° C. for 5 minutes, and an equal volume of 4 × SSC containing 20% dextran sulfate was mixed. The hybridization mixture was then seeded on denatured slides, covered with paraffin, and incubated at 37 ° C. for 16-18 hours in a wet box. After washing with 50% formamide / 2 × SSC at 37 ° C. for 15 minutes, washing was performed with 2 × SSC for 15 minutes and further with 1 × SSC for 15 minutes.

Then, the slide was incubated at 37 ° C. for 40 minutes in 4 × SSC to which “1% BlockAce” (trade name, manufactured by Dainippon Pharmaceutical) containing avidin-FITC (5 μg / ml) was added. Thereafter, the slides were washed with 4xSSC and 4xSSC containing 0.05% Triton X-100 for 10 minutes each, and 1% DABCO (PBS (-): glycerol in 1: 9 (v: v) 1% DABCO, Sigma) 100 mg of PPD solution (PPD (Wako Catalog No.164-015321) and 10 ml of PBS (-) (pH 7.4) containing 0.5 M Na ₂ CO ₃ /0.5 M NaHCO ₃ (9: 1, v / v) After adjusting the pH to 8.0 with the buffer solution (pH 9.0), glycerin was added thereto, and the mixture was immersed in a total volume of 100 ml), and DAPI (4,6-diamino-2-phenylindole: (Sigma).

  From more than 100 metaphase cells tested, specific hybridization signals without signals on other chromosomes were observed on chromosome band 6p21.3, confirming that the RalGDS gene is located on chromosome 6p21.3. confirmed.

  According to the use of the novel human RalGDS-related gene of the present invention obtained in this example, detection of the expression of the gene in various tissues and the production of human RalGDS protein by genetic engineering become possible. In addition to studying the role of proteins and ras-mediated signals in transduction pathways, it is thought that it will be possible to elucidate the pathology, diagnose, and treat diseases associated with these diseases, such as cancer. It is possible to study the relationship between the occurrence and progression of various diseases such as ankylosing spondylitis, atrial septal defect, pigmentary retinopathy, and dyslexia.

Cytoskeleton-related protein 2 gene (CKAP2 gene)
(1) Cloning and DNA Sequencing of Cytoskeleton-Related Protein 2 Gene The sequence analysis of a cDNA clone arbitrarily selected from a human fetal brain cDNA library in the same manner as in Example 1- (1) revealed A clone having a base sequence highly homologous to several CAP family genes containing the CAP-glycine region of the gene (cytoskeleton-associated protein: CAP) was found and named GEN-080G01.

  By the way, the cytoskeleton is located just inside the cytoplasm or cell membrane of eukaryotic cells, and is a network-like structure in which fibrous substances are intricately intertwined. The cytoskeleton is composed of microparticles made of tubulin, microfibrils made of actin, and intermediate fibers made of desmin and vimentin. The cytoskeleton not only plays a role as a skeleton of the cell, but also performs a dynamic function such as causing a morphological change of the cell by polymerization and depolymerization of the fibrous system. The cytoskeleton binds to organelles and receptors on cell membranes and ion channels, plays an important role in their movement and localization in cells, and regulates activities and mutual information. It is considered to have a function of transmission, and is supposed to occupy a very important position in regulating the physiological activity of whole cells. In particular, since cancer cells show morphology and recognition response different from normal cells, attention is paid to the relationship between canceration of cells and qualitative changes in cytoskeleton.

This cytoskeletal activity is regulated by a number of cytoskeletal-related proteins (CAPs). One group of CAPs has been characterized by a highly conserved glycine motif that is presumed to contribute to its association with microcontrollers (CAP-GLY region: Riehemann K. and Song C., Trends Biochem. Sci., 18 , 82-83 (1993)).

Members of this group of CAPs are CLIP-170, 150 kDa DAP (dynein-associated protein, or dynactin), D. melanogaster GLUED, S. cerevisiae BIK1, restin (restin: Bilbe G., et al., EMBO J., 11 , 2103-2113 (1992); Hilliker C., et al., Cytogenet. Cell Genet., 65 , 172-176 (1994)) and the 113.5 kDa protein of C. elegans (Wilson R., et al., Nature 368 , 32-38 (1994)), except for the latter two proteins, which have been suggested by direct or indirect evidence that they can interact with microcontrollers. .

CLIP-170 is important for the binding of endocrine vesicles to microgranules in vitro and co-localizes with endocrine organelles (Rickard JE and Kreis TE, J. Biol. Chem., 18 , 82-83 ( 1990); Pierre P., et al., Cell, 70 , 887-900 (1992)).

The dynactin acts in retrograde vesicle transport (Schroer TA and Sheetz MP, J. Cell Biol., 115 , 1309-1318 (1991)) or, possibly, in chromosome movement during mitosis. Allegedly work (Pfarr CM, et al., Nature, 345 , 263-265 (1990); Steuer ER, et al., Nature, 345 , 266-268 (1990); Wordeman L., et al., J Cell Biol., 114 , 285-294 (1991)) is one of the factors that make up the cytoplasmic dynein movement.

The mammalian homolog of dynactin, Drosophila GLUED, is important for the survival of almost all cells and for the formation of some neurons (Swaroop A., et al., Proc. Natl. Acad. Sci. USA, 84 , 6501-6505 (1987); Holzbaur ELF, et al., Nature, 351 , 579-583 (1991)).

BIK1 interacts with microcontrollers and plays an important role in spindle formation during mitosis in yeast (Trueheart J., et al., Mol. Cell. Biol., 7 , 2316-2326 ( 1987); Berlin V., et al., J. Cell. Biol., 111 , 2573-2586 (1990)).

  Currently, these genes have been classified as CAP families (CAPs).

Based on a database search, the cDNA clone of 463 bases (excluding the poly A signal) showed significant nucleic acid sequence homology to the genes encoding restin and CLIP-170. However, when compared to the restin gene, this clone lacked the 5 'portion, so the 5' race technique was used to isolate this missing segment (Frohman MA, et al., Proc. Natl. Acad. Sci. USA, 8 , 8998-9002 (1988)).
(2) 5'RACE: 5'rapid amplification of cDNA ends
The cDNA clone containing the 5 'portion of the gene of the present invention is isolated and analyzed by partially modifying the protocol for using the product, and performing the 5' race method using a commercially available kit (5'-Rapid AmpliFinder RACE Kit, Clontech). Was isolated as follows.

  The gene-specific primers P1 and P2 used here were synthesized according to a conventional method, and their nucleotide sequences are as shown in SEQ ID NOs: 43 (primer P1) and 44 (primer P2), respectively. The anchor and primer used in the commercial kit were used.

  0.1 μg of human fetal brain poly (A) + RNA was reverse transcribed by random hexamer using reverse transcriptase (Superscript TMII RNase H Reverse Transcriptase, Life Technologies) to obtain cDNA, which was used for P1 primer and anchor primer. Amplification was carried out by the method of Watanabe et al. (Watanabe T., et al., Cell Genet.,) Using the first PCR.

  That is, 0.1 μg of the poly (A) + RNA was combined with 2.5 mM dNTP / 1 × Taq buffer (Takara Shuzo) /0.2 μM P1 primer, 0.2 μM adapter / primer / ExTaq enzyme (Takara Shuzo) 0.25 units, and the total amount was 50 μl. After adding an anchor primer thereto and reacting at room temperature, it was subjected to PCR. That is, 35 cycles of 94 ° C. for 1 minute, then 94 ° C. for 45 seconds, 60 ° C. for 45 seconds, and 72 ° C. for 2 minutes were performed and reacted at 72 ° C. for 5 minutes.

  Thereafter, 1 μl in 50 μl of the first PCR reaction was amplified by a second PCR reaction using specific nested P2 primers and anchor primers. The second PCR product was analyzed by 1.5% agarose gel electrophoresis.

  A single band having a size of about 650 bases was detected by agarose gel electrophoresis, and the product of this band was inserted into a vector (pT7Blue (R) T-Vector, Novagen), and an appropriate Multiple clones with size insertions were selected.

  Six of the 5 'race clones obtained from the PCR reaction had the same sequence but different lengths. By sequencing a cDNA clone with two overlaps of GEN-080G01 and GEN-080G0149, the sequence encoding the protein, the 5 'and 3' flanking sequences and the sequence of 1015 bases in total length were determined, The gene was designated as cytoskeleton-related protein 2 gene (CKAP2 gene).

  SEQ ID NO: 6 shows the nucleic acid sequence obtained from the two overlapping cDNA clones of GEN-080G01 and GEN-080G0149, SEQ ID NO: 5 shows the nucleic acid sequence of the coding region of the clone, and SEQ ID NO: 4 shows the nucleic acid sequence Each shows the amino acid sequence encoded by the sequence.

  As shown in SEQ ID NO: 6, the CKAP2 gene has a relatively GC-rich 5 ′ non-coding region and an incomplete triplet of (CAG) 4 (CGG) 4 (CTG) (CGG). It was present at positions 40-69 bases.

  The ATG at positions 274-276 bases is a putative start codon. The stop codon (TGA) was located at bases 853-855. The polyadenylation signal (ATTAAA) was at 16 bases before the poly (A) start. The predicted open reading frame consists of 579 bases encoding 193 amino acids with a calculated molecular weight of 21,800.

Furthermore, the coding region was amplified by RT-PCR to exclude the possibility that the obtained synthetic sequence was a cDNA chimera.
(2) Similarity between other CRPs and CKAP2
The sequence of CKAP2 revealed homology between the sequences of restin and CLIP-170, but the homology region was restricted to the short sequence of the CAP-GLY domain. At the amino acid level, the deduced CKAP2 also showed high homology with the other five CAPs.

CKAP2 did not possess other motifs characteristic of some CAPs, such as the alpha-helix rod, zinc finger motifs. Alpha-helical rods appeared to contribute to dimer formation and increase microtubule binding (Pierre P., et al., Cell, 70 , 887-900, (1992)). . Deletion of the alpha-helical region may mean that CKAP2 cannot form homo- or hetero-dimers.

  Alignment of the CAP-GLY regions of these proteins revealed that in addition to glycine, conserved residues are also present in CKAP2. CAPs with CAP-GLY domains were also thought to be involved in organelle activity and in their interaction with microcontrollers. CKAP2 is located within these families because it contains the CAP-GLY region as described above.

Studies using mutants of Glued show that Glued products play a critical role in almost all cells (Swaroop A., et al., Proc. Natl. Acad. Sci. USA, 84 , 6501- 6505 (1987)) and performing other neuro-specific functions in nerve cells (Meyerowizt EM and Kankel DR, Dev. Biol., 62 , 112-142 (1978)). These microgovernment-related proteins are thought to act on vesicle trafficking and mitosis. Thus, the importance of the vesicle transport system in neurons, the deficiency of these components may lead to abnormal neuronal cell lines.

As such, CKAP2 may be involved in specific neuronal functions as well as essential cellular functions.
(3) Northern blot analysis The expression of human CKAP2 mRNA in normal human tissues was evaluated by Northern blot using the GEN-080G01 clone (corresponding to 553-1015 bases) as a probe in the same manner as in Example 1- (2).

  As a result, a transcript of 1.0 kb corresponding to the size of CKAP2 cDNA was detected in all eight tissues of the tested human heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. The 1.0 kb transcript was expressed at significantly higher levels in heart and brain than in other tissues tested. Two weak bands of 3.4 kb and 4.6 kb were detected in all tissues, respectively.

According to Northern blot analysis, the 3.4 kb and 4.6 kb transcripts were either derived from the same gene encoding the 1.0 kb transcript of CKAP2 by alternative splicing, or were transcribed from another related gene. It is also possible that The characteristics of these transcripts may indicate that CKAP2 also encodes a protein having a CAP-GLY region and also an α-helix.
(4) Cosmid clones and chromosomal localization by direct R-binding FISH
Two cosmids corresponding to CKAP2 cDNA were obtained. For these two cosmid clones, CKAP2 chromosomal loci were mapped by direct R-binding FISH in the same manner as in Example 1- (3).

Also, due to the suppression of background by repetitive sequences, a 20-fold excess of human as described by Richter (Lichter P. et al., Proc. Natl. Sci. USA, 87 , 6634-6638 (1990)). Cot-I DNA (BRL) was added. Provia 100 film (Fuji ISO100; manufactured by Fuji Film Co., Ltd.) was used for microphotography.

 As a result, CKAP2 was mapped on chromosome band 19q13.11-q13.12.

Two autosomal forms of CADASIL (cerebral autosomal dominant artery disease with cerebral cortical infarction and cerebral white matter atrophy) between DNA markers D19S221 and D19S222 and FHM (familial hemiplegic migraine) between D19S215 and D19S216 Dominant neurological disorders have been localized in this area by linkage analysis. These two diseases may be diseases caused by alleles of the same gene (Tournier-Lasserve E. et al., Nature Genet., 3 , 256-259 (1993); Joutel A. et al., Nature Genet ., 5 , 40-45 (1993)).

  There is no evidence to support CKAP2 as FHM or as a CADASIL candidate gene, but it is speculated that the variant may lead to some neuronal disease.

  By using the novel human CKAP2 gene of the present invention obtained by this example, it is possible to detect the expression of the gene in various tissues, and to produce human CKAP2 by genetic engineering. In addition, analysis of the functions of the human CKAP2 system or human CKAP2, which is deeply involved in the essential activities of cells, and various neurological diseases involving the CKAP2, such as familial migraine, can be performed. Screening and evaluation of therapeutic and prophylactic agents can also be performed.

OTK27 gene (1) Cloning and DNA sequencing of OTK27 gene As a result of sequence analysis and database search of cDNA clone arbitrarily selected from human fetal brain cDNA library in the same manner as in Example 1- (1), GEN-025F07, a cDNA clone encoding a yeast nucleoprotein (Saccharomyces cerevisiae: Kolodrubetz, D. and Burgum, A., YEAST, 7 , 79-90 (1991)), a protein with high homology to NHP2 , Named OTK27.

Nuclear proteins are basic components of cells such as chromosomes and ribosomes, and this nuclear protein is thought to play an essential role in cell growth and viability. NHP2, a yeast nuclear protein, is a high-mobility group (HMG) -like protein and has been reported to have an essential function for cell viability like HMG (Kolodrubetz, D. et al. and Burgum, A., YEAST, 7 , 79-90 (1991)).

  It is presumed that the OTK27 gene, a novel human gene of the present invention having high homology to the above-mentioned yeast NHP2, also has a similar function.

  The nucleotide sequence of the GEN-025F07 clone is composed of 1493 bases as shown in SEQ ID NO: 9 and contains an open reading frame of 384 bases shown in SEQ ID NO: 8, and is encoded by this. The amino acid sequence consisted of 128 amino acids as shown in SEQ ID NO: 7. The start codon started at bases 95-97 of the nucleotide sequence number of SEQ ID NO: 9, and bases 479-481 indicated a stop codon.

The OTK27 peptide showed a high homology of 38% at the amino acid level with NHP2. It was 83% identical to the protein deduced from the nucleic acid sequence of the cDNA from Arabidopsis thaliana (Newman, T., unpublished; GENEMBL Accession No. T14197).
(2) Northern blot analysis The expression of human OTK27 mRNA in normal human tissues was amplified by PCR of the insert portion of the OTK27 cDNA clone, the PCR product was purified, and [ ³² P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim) And labeled with a probe, and subjected to Northern blotting according to Example 1- (2).

  Northern blot analysis detected two bands at approximately 1.6 kb and 0.7 kb indicating two potential transcripts from this gene. Although both sizes of transcript were expressed in all normal adult tissues tested, the expression of the 0.7 kb transcript was significantly reduced in the brain and compared to other tissues tested in heart, skeletal muscle, and testicles. Was more highly expressed.

  For further testing of these two transcripts, 11 cDNA clones were isolated from the testis cDNA library and their DNA sequences were determined as in Example 1- (1).

  As a result, in the six clones, they corresponded to the 0.7-kilobase transcript, the poly-A sequence started at the base around position 600, and in two of these clones, the poly-A sequence was at position 598. Bases, three clones started at base 606, and one clone started at base 613.

  In these six clones, the "TATAAA" sequence at bases 583-588 was probably found as a poly A signal. In addition, the upstream poly-A signal “TATAAA” of this gene has little effect in the brain, is recognized to be more effective in the above three tissues than in other tissues, and the stability of each transcript is different in various tissues. It was also possible that they differed.

Furthermore, the results of Zoo-blot analysis indicated that this gene was well conserved in other vertebrates. Because this gene is expressed throughout normal adult tissues and is widely conserved among species, its gene product is thought to play an important physiological role. Since yeast lacking NHP2 cannot survive, it can be suggested that human homologs may also be essential to cell viability.
(3) Localization of the OKT27 chromosome by direct R-binding FISH One cosmid clone corresponding to cDNA OTK27 was isolated from the entire human chromosome cosmid library (equivalent to 5 genomes) using the OTK27 cDNA insert as a probe. Then, direct R-binding FISH was performed according to Example 1- (3) to determine the chromosome localization of OTK27.

  As a result, two distinct spots were observed on the chromosome 12q24.3 band.

  The OTK27 gene of the present invention can be used for expression and detection of human nucleoprotein and OTK27 protein, and thus diagnoses and elucidates the pathology of various diseases in which the protein is involved, for example, because of its involvement in cell proliferation and differentiation, treatment of certain cancers It can be used for screening and evaluation of prophylactic drugs.

OTK18 gene (1) Cloning and DNA sequencing of the OTK18 gene Zinc-finger proteins have been defined as a large family of proteins that regulate eukaryotic transcription and contain evolutionarily conserved structural motifs ( Kadonaga, JT, et al., Cell, 51 , 1079-1090 (1987); Klung, A. and Rhodes, D., Trends Biol. Sci., 12 , 464-469 (1987); Evans, RM and Hollenberg, SM, Cell, 52 , 1-3 (1988)).

Zinc fingers, a loop-like motif formed by the interaction of two ions, cysteine and histidine, are involved in the specific binding of proteins to RNA or DNA. The zinc finger motif was originally identified in the amino acid sequence of the frog ( Xenopus ) transcription factor IIIA (Miller, J., et al., EMBO J., 4 , 1609-1614 (1986)).

C ₂ H ₂ finger motif is usually repeated several, intervening sequences 7 or 8 amino acids are conserved evolutionarily in between. This intervening spread was initially identified in the Drosophila kruppel segmentation gene (Rosenberg, UB, et al., Nature, 319 , 336-339 (1986)). Since then, vertebrate genomes, genes encoding C ₂ H ₂ zinc finger proteins hundreds have been found.

  Results of sequence analysis and database search of cDNA clones arbitrarily selected from human fetal brain cDNA library in the same manner as in Example 1- (1), confirming several clones containing a zinc finger structure, Here, a clone having a Kruppel-type zinc finger structure was found.

  This clone lacked the 5 'portion of the transcript, so using the approximately 1.8 kilobase insert of the cDNA clone as a probe, plaque hybridizing a fetal brain cDNA library to isolate three clones, Their base sequences were determined in the same manner as in Example 1- (1).

  The clone with the largest insert of the three clones spanned 3754 bases with an open reading frame of 2133 bases encoding 711 amino acids, and was cloned against human ZNF41 and the Drosophila kruppel gene. , A novel human gene encoding a peptide with high similarity in the zinc finger region. This gene was designated as OTK18 gene (derived from the GEN-076C09 clone).

  The nucleic acid sequence of the cDNA clone of the OTK18 gene is shown in SEQ ID NO: 12, the base sequence including the coding region is shown in SEQ ID NO: 11, and the deduced amino acid sequence encoded by the OTK18 gene is SEQ ID NO: As indicated by 10.

From SEQ ID NO: 12, it was found that the deduced peptide sequence of OTK18 contained 13 finger motifs on the carboxy side of the protein.
(2) Comparison with genes having other zinc finger motifs
OTK18, human ZNF41, by comparing the Kruppel gene in Drosophila, each finger motif had been largely conserved with respect to a common sequence CXECGKAFXQKSXLX ₂ HQRXH.

  Comparison of the consensus sequence of the zinc finger motif of OTK18 with human ZNF41 and the Drosophila kruppel gene showed that the Kruppel-type motif was well conserved in the protein encoded by the OTK18 gene. However, sequence similarity was limited to the zinc finger region, with no significant homology in other regions.

The zinc finger region specifically interacts with the target DNA, recognizes a sequence of about 5 bases, and specifically binds to the DNA helix (Rhodes, D. and Klug, A., Cell, 46 , 123-132 (1986)).

This suggests that the multi-module (the zinc finger in tandem) could target this gene product containing 13 repeat units based on the idea that it could interact with a wide range of DNA. It is assumed that the DNA is a single DNA fragment of about 65 bases in length.
(3) Northern Blot Analysis Northern blot analysis was performed according to Example 1- (2) to amplify the expression of human OTK18 mRNA in normal human tissues by PCR on the insert of the OTK18 cDNA clone, and purify the PCR product. Labeling was performed with ³² P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim), and MTN blot was used as a probe.

Northern blot analysis revealed that the transcript of OTK18 was approximately 4.3 kilobases long, which was expressed throughout various normal adult tissues. However, the level of expression in liver and peripheral blood lymphocytes appeared to be lower in other organs tested.
(4) Localization of cosmid clones and chromosomes by direct R-binding FISH
OTK18 chromosome localization was performed according to Example 1- (3).

  As a result, two perfect spots were confirmed in eight samples, and 23 samples exhibited an incomplete signal or two spots in one or both. All signals appeared in the q13.4 band on chromosome 19. No two spots were observed on other chromosomes.

As a result of FISH, this gene was found to be localized on chromosome band 19q13.4. This region is known to contain many DNA segments that hybridize to the zinc finger region oligonucleotide (Hoovers, JMN, et al., Genomics, 12 , 254-263 (1992)). Moreover, at least one other gene encoding a zinc finger protein has been found in this region (Marine, J.-C., et al., Genomics, 21 , 285-286 (1994)).

  This suggests that chromosome 19q13 is a site where many multigenes encoding proteins that regulate transcription are gathered.

  By using the novel human OTK18 gene provided by this example, it is possible to detect the expression of the gene in various tissues, and to produce human OTK18 protein by genetic engineering. Analysis of the function of the human transcription regulatory protein gene system or human transcription regulatory protein that is deeply involved in the basic activities of cells, and various diseases in which this is involved, for example, abnormal development and differentiation, cancer, or abnormal nervous system development Diagnosis, psychiatric and neurological disorders, and screening and evaluation of therapeutic and prophylactic drugs.

Human 26S proteasome component P42 protein and P27 protein gene (1) Cloning and DNA sequencing of each gene of human 26S proteasome component P42 protein and P27 protein Proteasome, a multifunctional protease, is used in eukaryotes from yeast to human. It is an enzyme that widely exists and degrades ubiquitin-binding proteins in cells in an energy-dependent manner. The proteasome is structurally composed of a 20S proteasome composed of various components having a molecular weight of 21 to 31 kDa, and a PA700 control protein group having a sedimentation coefficient of 22S composed of various components of 30 to 112 kDa, As a whole, a macromolecule having a sedimentation coefficient of 26 S and a molecular weight of about 2 million daltons is formed (Rechsteiner, M., et al., J. Biol. Chem., 268 , 6065-6068 (1993), Yoshimura, T., et al., J. Struct. Biol., 111 , 200-211 (1993), Tanaka, K., et al., New Biologist, 4 , 173-187 (1992)].

  Although the entire structure of the proteasome has not yet been clarified from its structural and mechanical analysis, the following functions have been reported mainly from studies using yeast and mice, and the functions are gradually being clarified .

That is, the energy-dependent proteolytic mechanism in cells starts with protein selection by ubiquitin binding, but the ATP-dependent activity of degrading ubiquitinated proteins is not present in the 20S proteasome but in the 26S proteasome (Chu-Ping et al., J. Biol. Chem., 269 , 3539-3547 (1994)]. This suggests that the human 26S proteasome is useful for elucidating the mechanism of energy-dependent proteolysis.

Factors involved in cell cycle regulation generally have short half-lives and are often subject to strict quantitative control. In fact, it has been revealed that the oncogene products Mos, Myc, Fos, etc. are degraded by the 26S proteasome in an energy- and ubiquitin-dependent manner (Ishida, N., et al., FEBS Lett., 324 , 345-348). (1993), Hershko, A., and Ciechanover, A., Annu. Rev. Biochem., 61 , 761-807 (1992)], and the importance of the proteasome in cell cycle control is being recognized.

Its importance in the immune system has also been pointed out, suggesting that the proteasome is actively involved in class I major histocompatibility complex antigen presentation (Michalek MT., Et al., Nature, 363 , 552). -554 (1993)) and the phenomenon that ubiquitinated proteins are abnormally accumulated in the brain of Alzheimer patients (Kitaguchi, N., et al., Nature, 361 , 530-532 (1988)). Proteasomes have various functions, such as suggesting that proteasomes are involved in diseases, and their usefulness in diagnosis and treatment of various disease states has been attracting attention.

The primary function of the 26S proteasome is its activity to degrade ubiquitinated proteins. In particular, it has been found that degradation of oncogene products such as c-Myc and cell cycle-related gene products such as cyclin is degraded by a ubiquitin-dependent pathway. In addition, the proteasome gene is abnormally expressed in liver cancer cells, kidney cancer cells, leukemia cells, etc. as compared to normal cells (Kanayama, H., et al., Cancer Res., 51 , 6677-6685 (1991)). It has been observed that proteasome is abnormally accumulated in tumor cell nuclei. From this, it is expected that the components of the human proteasome will be useful for elucidating the mechanism of these canceration and diagnosing or treating cancer.

Furthermore, it is known that the expression of proteasome is induced by interferon γ and the like, and is deeply involved in antigen presentation in cells (Aki, M., et al., J. Biochem., 115 , 257-269 ( 1994)]. From this, the components of the human proteasome can be expected to elucidate the mechanism of antigen presentation by the immune system and to develop immunoregulators.

  In addition, the proteasome is also thought to be closely related to ubiquitin, which is abnormally accumulated in the brain of Alzheimer's patients, indicating that the components of the human proteasome are useful for elucidating the cause of Alzheimer's disease and treating it. Is suggested.

  In this way, the proteasome, which can be expected to have multiple functions, is thought to be able to be used for diagnosis of various disease states by immunoassay if an antibody is produced using each component as an antigen in addition to the use of the enzyme itself. Therefore, its usefulness in the diagnostic field is also attracting attention.

By the way, a protein having the properties of human 26S proteasome is disclosed in, for example, JP-A-5-292964, and a rat proteasome-constituting protein is disclosed in JP-A-5-28957 and JP-A-5-317059. However, the components of the human 26S proteasome are not known. Therefore, the present inventors obtained a further search for the components of the human 26S proteasome, and obtained two new human 26S proteasome components P42 protein and a human 26S proteasome component P27 protein, which are the following proteins. The gene was cloned and DNA sequenced by the method shown in Table 1.
(1) Purification of human 42S proteasome constituents P42 protein and P27 protein Using about 100 g of fresh human kidney, the biogel A-1.5 m (5 Human proteasome was purified by column chromatography using hydroxyapatite (1.5 x 15 cm, Biorad), Q-Sepharose (1.5 x 15 cm, Pharmacia) and glycerol density gradient centrifugation. .

The obtained human proteasome was subjected to reverse-phase HPLC using a Hitachi L6200 model high performance liquid chromatography (HPLC) system. The column was Shodex RS Pak D4-613 (0.6 × 15 cm, manufactured by Showa Denko), and the following two gradients were used for gradient elution.
First liquid: 0.06% trifluoroacetic acid Second liquid: 0.05% trifluoroacetic acid, 70% acetonitrile A part of each eluted fraction was subjected to 8.5% SDS-polyacrylamide electrophoresis under dithiothreitol reduction and detected. , P42 protein and P27 protein were isolated and purified, respectively.

  The purified P42 and P27 proteins were each digested with 1 μg of trypsin in 0.1 M Tris buffer (pH 7.8) and 2 M urea at 37 ° C. for 8 hours, and the obtained partial peptide fragments were separated by reverse phase HPLC. The sequence was determined by Edman degradation. The results were as shown in Table 1 below.

（2）cDNAライブラリーの検索、クローンの単離及びcDNA塩基配列の決定
本発明者らは、実施例1-（1）に示すように、ヒト胎児脳、血管動脈、胎盤cDNAライブラリーの大量DNA配列の決定から、約3万個のcDNAデーターを含むデーターベースを構築している。

(2) Search of cDNA library, isolation of clone, and determination of cDNA base sequence As shown in Example 1- (1), the present inventors have determined that a large amount of human fetal brain, vascular artery, From the determination of the DNA sequence, a database containing about 30,000 cDNA data has been constructed.

  A FASTA search (a homology search with the amino acid sequence deduced from the DNA database for the amino acid sequence) was performed by computer based on each amino acid sequence obtained in (1) above. A clone (GEN-331G07) having two identical amino acid sequences ((2) and (7) shown in Table 2) and a two amino acid sequence ((1) and (8) shown in Table 2) of P27 A matching clone (GEN-163D09) was found.

  For each of these clones, the sequence on the 5 'side was determined by the 5' RACE method in the same manner as in Example 2- (2), and the entire sequence of each was determined together.

  As a result, the P42 clone, GEN-331G07, was composed of the 1566 base sequence shown in SEQ ID NO: 15, and contained therein a 1167 base translation region shown in SEQ ID NO: 14, and was encoded by this. The amino acid sequence was found to be the 389 amino acid sequence shown in SEQ ID NO: 13.

  As a result of homology search using a computer, the P42 protein was found to have significant homology with the AAA (ATPases associated with a variety of cellular activities) protein family (eg, P45, TBP1, TBP7, S4, MSS1, etc.). This suggests that it is a new member of the AAA protein family.

  GEN-163D09, which is a P27 clone, is composed of the 1128 nucleotide sequence shown in SEQ ID NO: 18, and contains a 669 nucleotide translation region shown in SEQ ID NO: 17, which encodes the amino acid sequence encoded thereby. Was found to be the 223 amino acid sequence shown in SEQ ID NO: 16.

  As a result of homology search using a computer, no gene showing homology was found in the public data bank, and the P27 protein was considered to be a novel gene with an unknown function.

  Thus, both the P42 and P27 gene products were originally purified as regulatory subunit components of the proteasome complex, and they all play important roles for various biological functions through proteolysis, for example, It is thought to play a role for energy supply by ATP degradation, and these points are useful not only for elucidating the function of human 26S proteasome, but also for diagnosing and treating various pathological conditions caused by the above-mentioned decline in biological functions. It is believed that there is.

BNAP gene (1) Cloning and DNA sequencing of BNAP gene
Nucleosomes, composed of DNA and histones, are the basic structure of chromosomes in eukaryotic cells and are well conserved across species. This structure is strongly related to DNA replication and transcription. However, the formation of nucleosomes is not completely understood, and only a few specific factors relating to nucleosome assembly (NAPs) have been identified. That is, it has already been confirmed that two acidic proteins, nucleoplasmin and N1, facilitate nucleosome assembly (Kleinschmidt, JA, et al., J. Biol. Chem., 260 , 1166- 1176 (1985): Dilworth, SM, et al., Cell, 51 , 1009-1018 (1987)).

  Using a monoclonal antibody, the gene for yeast NAP-I was isolated, and it was examined whether the recombinant protein possessed nucleosome assembly activity in vitro.

More recently, the mouse NAP-I gene (Okuda, A .: registered in database as an accession number D12618), which is a mammalian homolog of yeast NAP-I, and the mouse DN38 gene (Kato, K., Eur. Neurosci., 2 , 704-711 (1990)) and human nucleosome assembly protein (hNRP) were cloned respectively (Simon, HU, et al., Biochem. J., 297 , 389-397 (1994). )), The hNRP gene was expressed in many tissues and was shown to be associated with T-lymphocyte proliferation.

  The present inventors, sequence analysis of a cDNA clone arbitrarily selected from a human fetal brain cDNA library in the same manner as in Example 1- (1), and the results of a database search, the 1125 base cDNA clone GEN- 078D05 (not containing polyA) has significantly higher homology with mouse NAP-I, a gene for nucleosome assembly proteins (NAPs) related to nucleosome assembly, partial mouse cDNA clones DN38 and hNRP Clarified to have.

  Since GEN-078D05 lacked the 5 'region, the 5' race method was performed in the same manner as in Example 2- (2) to obtain the entire coding region. For the 5 ′ race method, primers P1 and P2 having the respective nucleotide sequences shown in SEQ ID NOs: 45 and 46 were used.

  After the first 5 'race, the result was a single band with a sequence length of 1300 bases. This product was inserted into pT7Blue (R) T-Vecter, and several clones having appropriate size inserts were selected.

  Ten 5 'race clones, obtained from two independent PCR reactions, were sequenced and the longest clone, GEN-078D05TA13 (approximately 1300 bases long), was further analyzed.

  By sequencing both strands of two overlapping cDNA clones GEN-078D05 and GEN-078D05TA13, it was confirmed that the two clones did not yet cover the entire coding region, so a second The 5 'race method was implemented. In the second 5 ′ race method, primers P3 and P4 having the nucleotide sequences shown in SEQ ID NOs: 47 and 48 were used.

  Clone GEN-078D0508 obtained by the second 5 'race method was 300 bases in length. This clone contained a putative start codon and three preceding in-frame stop codons. From these three overlapping clones, the entire coding sequence consisting of 2636 bases was revealed, and this gene was designated as a brain-specific nucleosome assembly protein (BNAP) gene.

  The BNAP gene contains an open reading frame of 1518 bases shown in SEQ ID NO: 20, and the amino acid encoded thereby has 506 amino acid residues as shown in SEQ ID NO: 19; Is as shown in SEQ ID NO: 21.

As shown in SEQ ID NO: 21, the 5 'non-coding region of the gene was overall GC rich. Candidate sequences for the initiation codon were found at nucleotide positions 266-268, 287-289 and 329-331. All three sequences had well conserved sequences around the start codon (Kozak, M., J. Biol. Chem., 266 , 19867-19870 (1991)).

  According to the scanning model, the first ATG of the cDNA clone (SEQ ID NOs: 266-268) may be the start codon. The stop codon was located at nucleotide positions 1784-1786.

  The 3 ′ non-coding region was AT-rich as a whole, and the two polyadenylation signals (AATAAA) were located at nucleotide positions 2606-2611 and 2610-2615, respectively.

  The longest open reading frame consisted of 1518 bases encoding 506 amino acids, and the calculated molecular weight of the BNAP gene product was 57,600 daltons.

  Hydrophilicity plots showed that BNAP was very hydrophilic like other NAPs.

  To eliminate the possibility that recombinant BNAP expression and purification, and the possibility that the BNAP gene sequence might constitute three chimeric clones at the 5 'race stage, SEQ ID NO: RT-PCR was performed using the 326th to 356th sequences and the sequences of SEQ ID NOS: 1758 to 1786 as antisense primers.

As a result, a single product of about 1500 base pairs was obtained, and it was confirmed that the sequence was not a chimera but a single transcript.
(2) Comparison between BNAP and NAPs
The deduced amino acid sequence of BNAP showed 46% identity and 65% similarity with hNRP.

  The putative BNAP gene product had motifs characteristic of BNAP and those found in previously reported NAPs. Generally, the C-terminal half was strong and well conserved between human and yeast.

  The first motif (domain I) was KGIPDYWLI (corresponding to amino acid residues 309-317), which was also found in hNRP (KGIPSFWLT) and yeast NAP-I (KGIPEFWLT).

  The second motif (domain II) was ASFFNFFSPP (corresponding to amino acid residues 437-446), which was designated as DSFFNFFAPP in hNRP and ESFFNFFSP in yeast NAP-I.

  These two motifs were also conserved in the putative peptides of mouse NAP-I and DN38. Both conserved motifs were hydrophilic clusters, with Cys at position 402 also conserved.

  The N-terminal half has no strictly conserved motifs from yeast to mammalian species, while motifs have been found that are conserved among mammalian species.

  For example, HDLERKYA (corresponding to amino acids 130-137) and IINAEYEPTEEECEW (corresponding to amino acids 150-164), which may be related to mammalian-specific functions, were strictly conserved. .

  NAPs had acidic stretches that are believed to be likely to bind to histones or other basic proteins. All NAPs had three acidic stretches, but the position of those stretches was not preserved.

  BNAP does not have such three acidic stretches, but instead has three repeats with one long acidic cluster containing 41 acidic amino acid residues out of 98 amino acid residues (Corresponding to amino acid residues 194-207, 208-221 and 222-235), the consensus of which is ExxKExPEVKxEEK (x is not conserved and is often a hydrophobic residue). Was.

  Further, it was revealed that the BNAP sequence has several BNAP-specific motifs. That is, an extremely serine-rich region (corresponding to the 24-72nd amino acid residue) in which 33 residues (67%) of 49 amino acid residues are serine was found in the N-terminal portion. It was found as an incomplete AGC repeat at the nucleic acid level.

  Subsequent to this serine-rich region, a basic region containing 10 of 19 basic amino acid residues (corresponding to amino acid residues 71 to 89) appeared.

  BNAP was thought to be localized in the nucleus, with two possible nuclear localization signals (NLSs). The first signal was found in the basic domain of BNAP, whose sequence YRKKR (corresponding to amino acids 75-79) was similar to the HIV-1 Tat NLS (GRKKR). The second signal was located at the C-terminus and its sequence KKYRK (corresponding to amino acid residues 502-506) resembled the SV40 large T antigen NLS (KKKRK). Given these two putative NLSs, BNAP appeared to be localized in the nucleus, but could not rule out the possibility that other basic clusters could serve as NLSs.

BNAP has several phosphorylation sites and phosphorylation may regulate BNAP activity.
(3) Northern Blot Analysis Northern blot analysis was performed to determine the expression of BNAP mRNA in normal human tissues by using clone GEN-078D05TA13 (corresponding to positions 1323 to 1558 from SEQ ID NO: 323 of the BNAP gene) according to Example 1- (2). Amplified by PCR, the PCR product was purified, labeled with [ ³² P] -dCTP (Random Primed DNA Labeling Kit, Boehringer Mannheim), and tested using MTN blot as a probe.

  Northern blot analysis detected a 3.0 kilogram transcript of BNAP in the brain of the eight human adult tissues tested, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (8-hour exposure). ), And a longer exposure (24 hour exposure) detected a hazy band of the same size in the heart.

BNAP was equally expressed in some parts of the brain tested, but in other tissues no signal was detected by 72 h exposure. Although hNRP mRNA was expressed everywhere in the human tissues tested, BNAP mRNA was expressed in a tissue-specific manner.
(4) Radiation Hybrid Mapping
Chromosomal mapping of BNAP clones was performed by radiation hybrid mapping (Cox, DR, et al., Science, 250 , 245-250 (1990)).

  That is, a panel of radiation hybrid clones (G3RH) of the whole human genome was purchased from Research Genetics, Inc., AL, USA, and according to the instructions for use of the product, SEQ ID NO: 49 PCR reaction for chromosome mapping analysis was performed using the A1 primer and A2 primer of each base sequence shown in FIGS.

The results obtained were analyzed by software available on the Internet (Boehnke, M., et al., Am. J. Hum. Genet., 46 , 581-586 (1991)).

  As a result, the BNAP gene was strongly linked to the marker DXS990 (LOD = 1000, cR8000 = -0.00). Since marker DXS990 is a marker located at Xq21.3-q22 on the chromosome, BNAP is located at chromosome position Xq21.3-q22, where mental retardation associated with several X chromosomes is located. It was confirmed that.

Nucleosome structure Not only the basic chromosomal structural units characteristic of eukaryotes, but also regulatory units of gene expression. Several results suggest that genes with high transcriptional activity are sensitive to nuclease treatment, and that transcriptional activity alters the structure of chromosomes (Elgin, SCR, J. Biol. Chem., 263 , 19259). -19262 (1988)).

NAP-I has been cloned in yeast, mouse, and human, and is one of the factors capable of promoting nucleosome assembly in vitro. In a sequence study performed, NAPs containing the epitope for the specific antibody 4A8 were detected in humans, mice, frogs, Drosophila, and yeast ( S. cerevisiae ) (Ishimi, Y., et al. , Eur. J. Biochem., 162 , 19-24 (1987)).

In these experiments, NAPs were electrophoresed between 50-60 kDa by SDS-PAGE analysis, while recombinant BNAP was electrophoresed slowly at approximately 80 kDa. The 4A8 epitope was shown to be localized within a second, well conserved, hydrophobic motif. It was also shown that the triplet of FNF was important as a part of the epitope (Fujii-Nakata T., et al., J. Biol. Chem., 267 , 20980-20986 (1992)).

  BNAP also contained this matching motif in domain II. The fact that domain II is significantly hydrophobic and the fact that domain II can be recognized by the immune system suggests that it is likely presented on the BNAP surface and has the potential to participate in protein-protein interactions Is done.

  Domain I may also be involved in protein-protein interactions. The functional significance of the conserved regions is still unclear, but given the relatively low but collectively conserved among these NAPs, they must be essential for nucleosome assembly. It is considered not to be.

The hNRP gene is expressed in thyroid, stomach, kidney, intestine, leukemia, lung cancer, breast cancer and the like (Simon, HU, et al., Biochem. J., 297 , 389-397 (1994)). On the other hand, NAPs are expressed everywhere and are thought to play an important role in basic nucleosome formation.

  BNAP may be involved in brain-specific nucleosome formation, and its deficiency may cause neurological illness or mental retardation as a result of deviated neuronal function.

BNAP was linked to a marker on X chromosome q21.3-q22 where the mental retardation linked to several X chromosomes was localized. The region around the center of the X chromosome was rich in genes responsible for mental retardation, such as α-thalassemia and mental retardation (ATR-X) (Gibbons, RJ, et al., Cell, 80 , 837-845 (1995)). Similar to the analysis of the ATR-X gene, which appears to regulate nucleosome structure, we believe that BNAP may be involved in some type of mental retardation linked to the X chromosome.

  According to the present example, a novel BNAP gene is provided, and the use of the gene enables detection of expression of the gene in various tissues and production of BNAP protein by genetic engineering. Research on the formation of brain nucleosomes, which are deeply involved in basic activities of cells, and research on the function of brain nerve cells, and diagnose various neurological diseases and mental retardation involving these. In addition, screening and evaluation of these therapeutic and preventive drugs can be performed.

Human skeletal muscle-specific ubiquitin conjugating enzyme gene (UBE2G gene)
The ubiquitin system is one of a group of enzymes essential for cellular processes, and is conserved from yeast to humans. The system is composed of ubiquitin-activating enzymes (UBAs), ubiquitin-conjugating enzymes (UBCs), ubiquitin protein ligases (UBRs) and 26S proteasome particles.

Ubiquitin is transferred from the UBA to several UBCs and activated. UBC transfers ubiquitin to target proteins with or without UBR involvement. These ubiquitinated target proteins are said to trigger a number of cellular responses, including proteolysis, protein modification, protein transport, DNA repair, cell cycle control, replication control, stress response, and immune responses (Jentsch, S ., et al., Biochim. Biophys. Acta, 1089 , 127-139 (1991): Hershko, A. and Ciechanover, A., Annu. Rev. Biochem., 61 , 761-807 (1992): Jentsch, S. ., Annu. Rev. Genet., 26 , 179-207 (1992): Ciechanover, A., Cell, 79 , 13-21 (1994)).

UBCs are important components of this system, have different substrate specificities, and appear to regulate different functions. For example, UBC7 of Saccharomyces cerevisiae (S. Cerevisiae) is induced by cadmium, it is believed to contribute to the resistance to cadmium poisoning (Jungmann, J., et al. , Nature, 361, 369-371 ( 1993)). Degradation of MAT-α2 is also performed by UBC7 and UBC6 (Chen, P., et al., Cell, 74 , 357-369 (1993)).

The novel gene obtained in this example has a high homology to yeast cell cycle genes UBC9 and Hus5, and is a human UBC7-like gene that is strongly expressed in human skeletal muscle. The following describes sequencing.
(1) Cloning and DNA sequencing of human skeletal muscle-specific ubiquitin-conjugating enzyme gene (UBE2G gene) Sequence analysis of cDNA clone arbitrarily selected from human fetal brain cDNA library in the same manner as in Example 1- (1) And a database search revealed that the GEN-423A12 cDNA clone had significantly higher homology to genes encoding ubiquitin-conjugating enzymes (UBCs) of various species.

  Since the GEN-423A12 lacked the 5 ′ side, the 5 ′ race method was performed in the same manner as in Example 2- (2) to obtain the entire coding region.

  For the 5 'race method, P1 and P2 primers having the nucleotide sequences shown in SEQ ID NOS: 51 and 52 were used.

  The 5 'race product was inserted into pT7Blue (R) T-Vecter and several clones with the appropriate size insert were selected.

  Obtained from two independent PCR reactions, four of the 5 'race clones had the same sequence but different lengths.

  By the above clone sequencing, the 5'- and 3'-flanking sequences adjacent to the translated sequence of the new gene were determined.

  As a result, a new gene having a total sequence of 617 bases was revealed, and this gene was named a human skeletal muscle-specific ubiquitin-conjugating enzyme gene (UBE2G gene).

  In addition, in order to exclude the possibility that this sequence is a chimeric clone, in order to amplify from a human fetal brain cDNA library, RT-PCR using sense primers was performed according to Example 6- (1) above. Carried out. As a result, a single PCR product was obtained, and it was confirmed that the sequence was not chimeric.

  The UBE2G gene contains an open reading frame of 510 bases shown in SEQ ID NO: 23, and the amino acid encoded thereby has 170 amino acid residues as shown in SEQ ID NO: 22; Is as shown in SEQ ID NO: 24.

  The start codon deduced as shown in SEQ ID NO: 24 was located at nucleotide positions 19-21, which is the first ATG triplet of the cDNA clone. Since there was no in-frame stop codon preceding this ATG, this clone was deduced to contain the entire open reading frame for the following reasons.

That supported this ATG agrees with the (a) Saccharomyces cerevisiae (S. Cerevisiae) UBC7 and higher homologous amino acid sequence, UBC7 initiation codon of the yeast.
(B) The sequence of AGGATGA was similar to the consensus sequence (A / G) CCATGG around the start codon (Kozak, M., J. Biol. Chem., 266 , 19867-19870 (1991)).
(2) Comparison of amino acid sequences between UBE2G and UBCs
Comparison of the amino acid sequences of UBE2G and UBCs appeared to indicate that the active site cysteine capable of binding to ubiquitin is the 90th cysteine residue. The peptides encoded by these genes appear to belong to the same family.
(3) Northern blot analysis According to Example 1- (2), Northern blot analysis was performed to amplify the expression of UBE2G mRNA in normal human tissues by amplifying the entire sequence of UBE2G by PCR, purify the PCR product, and perform [ ^32] P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim) was used as a probe, and the membrane was performed using an MTN blot.

As a result of Northern blot analysis, 16 human adult tissues tested, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thyroid, bladder, testicle, ovary, small intestine, large intestine and peripheral blood leukocytes, After 18 hours of exposure, 4.4 kb, 2.4 kb, and 1.6 kb transcripts were detectable, and strong expression of these transcripts was observed in skeletal muscle.
(4) Radiation Hybrid Mapping
Chromosome mapping of the UBE2G clone was performed by radiation hybrid mapping in the same manner as in Example 6- (4).

  The C1 primer and C4 primer used for the PCR reaction for chromosome mapping analysis correspond to SEQ ID NO: 415-435 and SEQ ID NO: 509-528 of SEQ ID NO: 24, respectively. The nucleotide sequence is as shown in SEQ ID NOs: 53 and 54.

  As a result, the UBE2G gene was linked to markers D1S446 (LOD = 12.52, cR8000 = 8.60) and D1S235 (LOD = 9.14, cR8000 = 22.46). These markers were localized to the chromosomal band lq42.13-q42.3.

  UBE2G was strongly expressed in skeletal muscle and very weakly expressed in all tissues tested. All other UBCs are involved in essential cellular functions, such as regulating the cell cycle, and these UBCs are expressed throughout tissues. However, UBE2G expression patterns may suggest a muscle-specific role.

  Three different sized transcripts were detected, but it was not possible to confirm which of them corresponded to the cDNA clone. The major structure of the UBE2G product showed strong homology to yeast UBC7. On the other hand, the nematode UBC7 showed strong homology to the yeast UBC7. It is involved in the degradation of the repressor and also confers resistance to cadmium in yeast. The similarities between these proteins suggest that they belong to the same family.

UBE2G is involved in the degradation of muscle-specific proteins, and deficiency of the gene is thought to result in diseases such as muscular dystrophy. Recently, another proteolytic enzyme, calpain 3 (Calpain 3), has been demonstrated to be the cause of limb muscular dystrophy type 2A (Richard, I., et al., Cell, 81 , 27-40 (1995)). Although the gene is currently not significantly associated with any hereditary muscular disease from the UBE2G chromosomal location, future linkage analysis may reveal a relationship with the gene.

  According to this example, a novel UBE2G gene is provided, and the use of the gene enables detection of the expression of the gene in various tissues and production of the UBE2G protein by genetic engineering. It is possible to carry out research on muscle-specific protein degradation and skeletal muscle function that are deeply involved in the basic activities of cells in various ways, and various muscle diseases involving these, such as diagnosis of muscular dystrophy. Screening and evaluation of these therapeutic and prophylactic drugs can also be performed.

TMP-2 gene (1) Cloning and DNA sequencing of TMP-2 gene Sequence analysis of cDNA clone arbitrarily selected from human fetal brain cDNA library in the same manner as in Example 1- (1) and database analysis As a result of the search, a clone (GEN-092E10) having a cDNA sequence highly homologous to the membrane protein gene (transmembrane protein: Accession No. U19878) was found.

Thus, conventionally, the membrane protein gene has been cloned in frogs ( Xenopus laevis ) and humans, and they are transmembrane proteins having a follistatin module and an EGF region (epidermal growth factor domain). It is considered to be a gene (Accession No .; U19878).

  From the sequence information of the above protein gene, since the GEN-092E10 clone lacked the 5 'region, a λgt10 cDNA library (Human Fetal Brain 5'-STRETCH PLUS cDNA; manufactured by Clontech) was probed with the GEN-092E10 clone. As a result, a cDNA clone containing an additional 5 'upstream was isolated.

  Sequencing of both strands of this cDNA clone revealed a sequence covering the entire coding region and was named the TMP-2 gene.

  The TMP-2 gene contains an open reading frame of 1122 bases shown in SEQ ID NO: 26, and the amino acid encoded thereby has 374 amino acid residues as shown in SEQ ID NO: 25 The nucleic acid sequence of all the cDNA clones of TMP-2 consisted of 1721 bases as shown in SEQ ID NO: 27.

  The 5 'non-coding region as shown in SEQ ID NO: 27 was overall GC rich. There were several candidate start codon sequences, but according to the scanning model, the fifth ATG (base sequence Nos. 368-370) of the cDNA clone was estimated as the start codon. The stop codon was located at nucleotide positions 1490-1492. The polyadenylation signal (AATAAA) was located at nucleotide positions 1703-1708. The calculated molecular weight of the TMP-2 gene product was 41,400 daltons.

  As described above, the membrane protein gene has a follistatin module and an EGF region, but these motifs are also conserved in the novel human gene of the present invention.

In addition, the TMP-2 gene of the present invention includes TGF-α (transforming growth factor-α; Derynck, R., et al., Cell, 38 , 287-297 (1984)) and beta-cellulin (Igarashi, K. and Folkman, J., Science, 259 , 1604-1607 (1993)), heparin-binding EGF-like growth factor; Higashiyama, S., et al., Science, 251 , 936-939. (1991)) and Schwannoma-derived growth factor (Kimura, H., et al., Nature, 348 , 257-260 (1990)) at the amino acid level beyond the EGF region. Therefore, it is considered that they play an important role in cell proliferation and communication between cells.
(2) Northern blot analysis Northern blot analysis was performed according to Example 1- (2) by amplifying the expression of TMP-2 mRNA in normal human tissues by PCR of clone GEN-092E10, purifying the PCR product, Labeling was performed using [ ³² P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim), and MTN blot was used as a probe.

  Northern analysis revealed high expression in brain and prostate. The size of the TMP-2 gene mRNA was about 2 kb.

  According to the present invention, a novel human TMP-2 gene is provided, and the use of the gene enables detection of the expression of the gene in various tissues and production of human TMP-2 protein by genetic engineering. As described above, brain tumors and prostate cancer that are deeply involved in cell proliferation and cell-to-cell communication can be studied, and these diseases can be diagnosed. Screening and evaluation of these therapeutic and preventive drugs can also be performed. Can do it.

Human NPIK gene (1) Cloning and DNA sequencing of human NPIK gene In the same manner as in Examples 1 and 2, the results of sequence analysis of cDNA clones arbitrarily selected from a human fetal brain cDNA library and database search Has high homology to the gene encoding the amino acid sequence conserved in phosphatidylinositol 3 and 4 kinases (Kunz, J., et al., Cell, 73 , 585-596 (1993)) Two cDNA clones were obtained, these were named GEN-428B12c1 and GEN-428B12c2, and their entire sequences were determined according to each of the above Examples.

  As a result, it was found that the GEN-428B12c1 cDNA clone and the GEN-428B12c2 clone had different coding sequences only in the 12 amino acid residues at the 5 ′ end, and the GEN-428B12c1 cDNA clone was 12 amino acid residues longer.

  The GEN-428B12c1 cDNA sequence of the human NPIK gene contains an open reading frame of 2487 bases shown in SEQ ID NO: 32, and the amino acid encoded thereby has 829 amino acid residues as shown in SEQ ID NO: 31. The nucleic acid sequence of the full-length cDNA clone, which has a group, consisted of 3324 bases as shown in SEQ ID NO: 33.

  The start codon predicted as shown in SEQ ID NO: 33 was located at nucleotide positions 115 to 117, which is the second ATG triplet of the cDNA clone. In addition, the stop codon was located at positions 2602-2604 of the nucleotide sequence number. The polyadenylation signal (AATAAA) was located at nucleotide positions 3305-3310.

  On the other hand, the GEN-428B12c2 cDNA sequence of the human NPIK gene contains an open reading frame of 2451 bases shown in SEQ ID NO: 29, and the amino acid encoded thereby is 817 as shown in SEQ ID NO: 28. The nucleic acid sequence of the full-length cDNA clone having amino acid residues was composed of 3602 bases as shown in SEQ ID NO: 30.

The start codon predicted as shown in SEQ ID NO: 30 was located at nucleotide positions 429-431, which is the seventh ATG triplet of the cDNA clone. Also, the stop codon was located at positions 2880-2882 of the nucleotide sequence number. The polyadenylation signal (AATAAA) was located at nucleotide positions 3583-3588.
(2) Northern blot analysis Northern blot analysis was performed according to Example 1- (2) by amplifying the expression of human NPIK mRNA in normal human tissues by PCR of the entire sequence of human NPIK, purifying the PCR product, Labeling was performed with [ ³² P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim), used as a probe, and the membrane was performed using an MTN blot.

  As a result of Northern analysis, the expression of the human NPIK gene was observed in various organs of the 16 human adult tissues tested, and transcripts of about 3.8 kb and about 5 kb were detectable.

  Using a primer A having a base sequence shown in SEQ ID NO: 55 and 56 containing a start codon of the GEN-428B12c2 cDNA sequence and a primer B containing a stop codon, Human Fetal Brain Marathon-Ready cDNA (Clontech) as a template PCR was performed, and the nucleotide sequence of the PCR product was determined.

  As a result, the expressed human NPIK mRNA lacked the nucleotide sequence Nos. 1060-1104 of SEQ ID NO: 33 of the GEN-428B12c1 cDNA sequence (the amino acid sequence Nos. 316-330 of the amino acid residue SEQ ID NO: 31) and It was found that there was a nucleotide sequence lacking the nucleotide sequence No. 1897-1911 of SEQ ID NO: 33 (the amino acid sequence No. 595-599 of the amino acid residue SEQ ID NO: 31).

  Furthermore, this gene has a polymorphism shown in Table 2 below (428B12c1.fasta) in the region of nucleotide sequence Nos. 1941-1966 of GEN-428B12c1 cDNA sequence of SEQ ID NO: 33 (428B12c1.fasta), whereby the GEN-428B12c1 amino acid residue was found. It was found that a mutant protein in which IQDSCEITT of amino acid sequence number 610-618 of group SEQ ID NO: 31 was mutated to YKILVISA was encoded.

（3）FISHによるヒトNPIK遺伝子の染色体のマッピング
実施例1-(3)に準じてFISHによるヒトNPIK遺伝子の染色体のマッピングを行なった。

(3) Chromosome mapping of human NPIK gene by FISH Chromosome mapping of human NPIK gene by FISH was performed according to Example 1- (3).

  As a result, the human NPIK gene was found to have a chromosomal locus at 1q21.1-q21.3.

  As described above, the human NPIK gene, which is a novel human gene of the present invention, has two cDNAs having different 5 'regions, and they contain regions that can encode 829 and 817 amino acids, respectively. In addition, the presence of two sites that can be deleted in the mRNA of this gene, and the presence of polymorphism in the specific region corresponding to amino acid sequence Nos. 610-618 of SEQ ID NO: 31 of GEN-428B12c1 amino acid residue, Since the protein is encoded, human NPIK may include human NPIK, human NPIK deletion, human NPIK mutant and / or human NPIK mutation / deletion consisting of several combinations. .

Recently, several proteins belonging to the PI3 and 4 kinase families have been shown to have protein kinase activity (Dhand, R., et al., EMBO J., 13 , 522-533 (1994): Stack). , JH and Emr, SD, J. Biol. Chem., 269 , 31552-31562 (1994): Hartley, KO, et al., Cell, 82 , 849-856 (1995)).

Furthermore, proteins belonging to this family are involved in repair of DNA damage (Hartley, K.o., et al., Cell, 82 , 849-856 (1995)), and that of ataxia (ataxia). It was found to be the causative gene (Savitsky, K., et al., Science, 268 , 1749-1753 (1995)).

  The protein encoded by the human NPIK gene having high homology to these PI kinase families can be expected to be a novel enzyme that phosphorylates lipids or proteins.

  According to this example, a novel human NPIK gene is provided, and the use of the gene enables detection of the expression of the gene in various tissues and production of human NPIK protein by genetic engineering. Thus, studies on lipid or protein kinases, studies on DNA repair, and diseases related to them, such as cancer research and diagnosis, can be performed, and screening and evaluation of therapeutic and preventive drugs for these can be performed. Can also be performed.

nel-related protein type 1 (NRP1) gene and nel-related protein type 2 (NRP2) gene (1) Cloning and DNA sequencing of NRP1 and NRP2 genes
EGF-like repeats have been identified in many membrane proteins and in proteins involved in growth regulation and differentiation. This motif appears to be involved in protein-protein interactions.

In recent years, a gene encoding nel, a novel peptide containing five EGF-like repeats, has been cloned from a chicken embryo cDNA library (Matsuhashi, S., et al., Dev. Dynamics., 203 , 212- 222 (1995)). This product is thought to be a membrane molecule with its EGF-like repeat in the extracellular domain. A 4.5 kb transcript of nelmRNA is expressed in various tissues during embryonic life, and after birth is exclusively expressed in the brain and retina.

  As a result of sequence analysis and database search of a cDNA clone arbitrarily selected from a human fetal brain cDNA library in the same manner as in Example 1- (1), two cDNA clones having significantly higher homology to the above nel And named GEN-073E07 and GEN-093E05, respectively.

  Since both clones lacked the 5 'region, the 5' race method was performed in the same manner as in Example 2- (2) to obtain the entire coding region.

  As a primer for the 5 ′ race method, a primer having an arbitrary sequence obtained from the cDNA sequence of the above clone was synthesized, and an anchor primer provided with a commercially available kit was used as a primer.

  Some of the 5 'race clones obtained from the PCR reaction were sequenced to identify sequences that appeared to cover the entire coding region, and each of these genes was nel-related protein type 1 (nel-related protein type). 1: NRP1) gene and nel-related protein type 2 (NRP2) gene.

  The NRP1 gene contains an open reading frame of 2430 bases shown in SEQ ID NO: 35, and the deduced amino acid encoded by the NRP1 gene has 810 amino acid residues as shown in SEQ ID NO: 34. The nucleic acid sequence of the entire NRP1 gene cDNA clone consists of 2977 bases as shown in SEQ ID NO: 36.

  On the other hand, the NRP2 gene contains an open reading frame of 2448 bases shown in SEQ ID NO: 38, and the deduced amino acid encoded thereby has 816 amino acid residues as shown in SEQ ID NO: 37. The nucleic acid sequence of the entire cDNA clone of the NRP2 gene consists of 3198 bases as shown in SEQ ID NO: 39.

  Furthermore, the coding region was amplified by RT-PCR to eliminate the possibility that the obtained sequence was a cDNA chimera.

  Both the putative NRP1 and NRP2 gene products had a highly hydrophobic N-terminus that could function as a signal peptide for membrane insertion. Neither appeared to possess a hydrophobic transmembrane domain when compared to the chick embryo nel. Comparing the deduced peptide sequences between NRP1, NRP2 and nel, NRP2 has 80% homology at the amino acid level and is more closely related to nel than 50% of that of NRP1. Cysteine-rich domains and cysteine residues within the EGF-like repeat were completely conserved.

  The most striking difference between NRPs and chicken proteins was that the human homolog lacked the putative membrane domain of nel, but the nucleic acid sequences of NRPs and nel were also very similar in this missing region. Two more NRPs carried six EGF-like repeats, while only five nels.

Other unique motifs of nel reported by Matsuhashi et al. (Matsuhashi, S., et al., Dev. Dynamics., 203 , 212-222 (1995)) were also found in NRPs at the same location. As described above, the putative peptides of the two NRPs indicate that they are not transmembrane proteins, and the NRPs may be secreted proteins or proteins anchored to the membrane by post-translational modifications .

  We believe that NRPs may function as ligands by stimulating other molecules such as the EGF receptor. The present inventors have also found that an extra EGF-like repeat is encoded in the nel by frame-shifting the membrane domain region of the nel.

When NRP2 and nel were aligned and compared, this frame-shifted three amino acid sequence showed similarity in all regions between NRP2 and nel. This suggests that NRP2 may be the human counterpart of nel. In contrast, NRP1 is considered to be a similar gene rather than a human nel counterpart.
(2) Northern blot analysis Northern blot analysis was performed according to Example 1- (2) by amplifying NRPs mRNA expression in normal human tissues by PCR of the entire sequence of both cloned cDNAs and purifying the PCR product. and, ^[32 P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim) labeled with, was performed using the MTN blot as two probes.

  The expression patterns of two NRPs were evaluated in 16 adult tissues and 4 human fetal tissues.

  Northern analysis revealed that the 3.5 kb transcript of NRP1 was weakly expressed in fetal and adult brain and kidney. The 3.6 kb transcript of NRP2 was strongly expressed only in adult and fetal brain, but only slightly in fetal kidney.

This suggests that NRPs play a brain-specific role, such as signal molecules for growth regulation. In addition, these genes may have special functions in the kidney.
(3) Chromosome mapping of NRP1 gene and NRP2 gene by FISH Chromosome mapping of NRP1 gene and NRP2 gene by FISH was performed according to Example 1- (3).

  As a result, it was found that the NRP1 gene had a chromosomal locus at 11p15.1-p15.2, and the NRP2 gene had a chromosomal locus at 12q13.11-q13.12.

  According to the present invention, a novel human NRP1 gene and NRP2 gene are provided, and the use of the gene enables the detection of expression of the gene in various tissues, and the production of human NRP1 and NRP2 proteins by genetic engineering. It can be used for research on brain neurotransmission pathways, can also diagnose various diseases related to brain neurotransmission pathways, and can screen and evaluate therapeutic and preventive drugs for these. In addition, it is suggested that these NRPs having an EGF domain may act as growth factors in the brain, and are considered to be effective in diagnosing and treating various brain tumors and in regenerating nerves in degenerative neurological diseases.

GSPT1-related protein (GSPT1-TK) gene (1) Cloning and DNA sequencing of the GSPT1-TK gene It is one of the human homologous genes. The yeast GST1 gene, originally identified as a protein that can complement and complete the temperature-sensitive gst1 (G1-to-S transposition) mutant of Saccharomyces cerevisiae, is derived from a yeast chromosome library. Isolated (Kikuchi, Y., Shimatake, H. and Kikuchi, A., EMBO J. 7 , 1175-1182 (1988)), the gene has a target site for cAMP-dependent protein kinase and a GTPase domain. Coded for protein.

The human GSPT1 gene was isolated from a cDNA library of KB cells by hybridization using the yeast GST1 gene as a probe (Hoshino, S., Miyazawa, H., Enomoto, T., Hanaoka, F., Kikucho, Y , Kikuchi, a. And Ui, m., EMBO J. 8 , 3807-3814 (1989)). Like the yeast GST1, the putative protein of the GSPT1 gene also has a GTP-binding region and a GTPase activity center site, and plays an important role in cell proliferation.

  Furthermore, a breakpoint for chromosomal rearrangement is that the GSPT1 gene in patients with acute nonlymphocytic leukemia (ANLL) is observed at chromosomal locus 16p13.3 (Ozawa, K., Murakami, Y., Eki, T. et al. , Yokoyama, K., Soeda, E., Hoshino, S., Ui, M. and Hanaoka, F., Somatic Cell and Molecular Genet., Vol. 18, 189-194 (1992)).

  As a result of sequence analysis and database search of a cDNA clone arbitrarily selected from a human fetal brain cDNA library in the same manner as in Example 1- (1), a 0.3 kb cDNA sequence highly homologous to the GSPT1 was obtained. A clone having the same was found and named GEN-077A09. Since the GEN-077A09 clone was considered to lack the 5 'region, the 5' RACE method was performed in the same manner as in Example 2- (2) to obtain the entire coding region.

  For the 5 ′ RACER method, P1 primers and P2 primers having the nucleotide sequences shown in SEQ ID NOS: 59 and 60 obtained from the known cDNA sequences of the above cDNA clones were used, and commercially available kits were used as anchor primers. The accessory was used as a primer. The PCR reaction was carried out for 35 cycles of 94 ° C. for 45 seconds, 58 ° C. for 45 seconds, 72 ° C. for 2 minutes, and finally extended at 72 ° C. for 7 minutes.

  By sequencing some of the 5'RACE clones obtained from the PCR reaction and sequencing the two overlapping cDNA clones, this 5'RACE clone and the GEN-077A09 clone, The sequence considered to cover the coding region was clarified and named GSPT1-related protein "GSPT1-TK gene".

  The GSPT1-TK gene contains an open reading frame of 1497 bases shown in SEQ ID NO: 41, and the deduced amino acid encoded thereby has 499 amino acid residues as shown in SEQ ID NO: 40 I was

  The nucleic acid sequence of all cDNA clones of the GSPT1-TK gene consisted of 2057 bases as shown in SEQ ID NO: 42, and the calculated molecular weight was 55,740 daltons.

  The first methionine (ATG) in the open reading frame had no stop codon in-frame, but because this ATG was surrounded by a sequence similar to Kozak's consensus sequence for the start of transcription, It was concluded that this ATG triplet located at positions 144-146 of SEQ ID NO: was the start codon.

  In addition, one polyadenyl signal AATAAA was found 13 bases upstream from the polyadenylation site.

  Human GSPT1-TK contains a glutamate-rich region near the N-terminus, and 18 of the 20 glutamic acids present in this region of GSPT1-TK are conserved and perfectly aligned with those of the human GSPT1 protein. I have. In the GSPT1-TK protein, several regions (G1, G2, G3, G4, and G5) of the GTP-binding protein responsible for guanine nucleic acid binding and hydrolysis, as well as those regions of the human GSPT1 protein, were conserved.

Thus, human GSPT1-TK revealed 89.4% identity in the DNA sequence and 92.4% identity in the deduced amino acid sequence with human GSPT1, which appears to play an important role in the G1 to S phase transition of the cell cycle. . The amino acid sequence was 50.8% identical to that of yeast GST1.
(2) Northern blot analysis Northern blot analysis was performed according to Example 1- (2) by amplifying GSPT1-TK mRNA expression in normal human tissues by PCR of a GEN-077A09 cDNA clone, purifying the PCR product, Labeling was performed with [ ³² P] -dCTP (random primed DNA labeling kit, Boehringer Mannheim), and the test was performed using MTN blot as a probe.

As a result of Northern analysis, a 2.7 kb major transcript was detected in various tissues. Human GSPT1-TK expression levels appeared to be most highly expressed in brain and testes.
(3) Chromosome mapping of GSPT1-TK gene by FISH Chromosome mapping of the GSPT1-TK gene by FISH was performed according to Example 1- (3).

  As a result, it was found that the GSPT1-TK gene had a chromosomal locus at 19p13.3. In this chromosome localization site, the mutual locus from acute lymphocytic leukemia (Acute lymphocytic leukemia: ALL) and acute myeloid leukemia (AML) is frequently observed. In addition, it has been reported that acute nonlymphocytic leukemia (ANLL) is associated with a rearrangement involving a region of human GSPT1 (Ozawa, K., Murakami, Y., Eki, T , Yokoyama, K., Soeda, E., Hoshino, S., Ui, M. and Hanaoka, F., Somatic Cell and Molecular Genet., Vol. 18, 189-194 (1992)).

  These suggest that ALL and AML are the most candidate genes related to this gene.

  According to the present invention, a novel human GSPT1-TK gene is provided, and the use of the gene enables detection of expression of the gene in various tissues and production of human GSPT1-TK protein by genetic engineering. Can be used for research on cell proliferation as described above, and can diagnose various diseases related to the chromosomal locus of this gene, such as acute myeloid leukemia. That is, this is because the translocation of this gene disrupts the GSPT1-TK gene, and the fusion protein expressed by this translocation is considered to be the cause of these diseases.

  Furthermore, by preparing an antibody against the fusion protein, elucidating the intracellular localization, and examining the expression specific to the disease, it is expected that diagnosis and treatment thereof will be possible. Therefore, it is considered that the use of the gene of the present invention enables screening and evaluation of these therapeutic and prophylactic agents.

  According to the present invention, a novel human gene is provided, and by using the gene, expression of the gene in various tissues can be detected, and a protein encoded by the gene and an antibody thereto can be produced. These genes and proteins are useful for studies on cell proliferation, diagnosis of various diseases related to them, and the like. In addition, antibodies against the above proteins can be used for diagnosis and treatment of related diseases. Furthermore, the use of the gene of the present invention allows screening and evaluation of therapeutic and prophylactic agents for related diseases.

SEQ ID NOs: 43 to 60 are nucleotide sequences of primers for the 5 ′ race method.

Claims (4)

A method for treating prostate cancer, which comprises administering a pharmaceutical composition containing an antibody against human TMP-2 protein to a patient in need of treatment. 2. The method of claim 1, wherein an antibody against the human TMP-2 protein is conjugated to the therapeutic moiety. 3. The method of claim 2, wherein the therapeutic agent moiety is a cytotoxic agent. 3. The method of claim 2, wherein the therapeutic moiety is a radioisotope.