JPH1189577A - Human bnip3l gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene BNIP3L having a growth-inhibiting activities against cancer cells when introduced into the various cancer cells. SOLUTION: This human BNIP3L gene is the one containing an amino acid sequence of the formula. The subject gene can be obtained by preparing cDNA library from a human placenta mRNA or the like, selecting desired clone from the prepared cDNA library by using a proper probe or antibody specific to the gene of this invention. The peptide having an amino acid sequence encoded by the gene is useful for a medicine for a treatment, etc., of various diseases and conditions caused by the regulation of apoptosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene useful as a guide for the prevention, diagnosis and treatment of human diseases, and more particularly to a novel human gene having high homology to human Nip3 gene, and cDNA analysis of the gene. The present invention relates to a gene that can be used for gene diagnosis using the gene and development of a new therapeutic method, for example, a therapeutic method for cancer, by mapping to a cDNA chromosome, analyzing the function of the cDNA, and the like.

[0002]

2. Description of the Related Art From the results of human gene analysis to date, various receptors such as insulin receptor and LDL receptor,
Related to cell growth and differentiation, protease, ATPas
e. Many genes, such as metabolic enzymes such as superoxide dismutase, have been found to be useful materials for drug development. However, studies on the analysis of human genes and the functions of the analyzed genes and the relationship between the analyzed genes and various diseases have just begun, and there are many unclear points. There is a need in the art for elucidation of the gene, research on the relationship between the gene and disease, and further, gene diagnosis using the gene, research on application of the gene and its expressed protein to pharmaceutical use, and the like.

[0003] Nip3 is adenovirus E1B19kD and human Bcl-2 protein is inhibiting molecule cell death (Boyd, JM, et al. , Cell, 7 9, 341-351 (1994)) and cytosolic mutually interact It is reported to be a protein. It has been reported that the 19 kD protein of adenovirus E1B has an effect of preventing cell death induced by viral infection or external stimuli (Rao,
L., et al., Proc. Natl. Acad. Sci. USA, 89 , 774
2-7746 (1992), Debbas, M. and White, E., GenesDe
v., 7 , 546-554 (1993)).

[0004] The activity of the proto-oncogene Bcl-2 expression protein is similar to the cell death inhibitory effect demonstrated by E1B19kD (Tarodi, B., et al., Int.
J. Oncol., 3 , 467-472 (1993); Huang, DCS, et a
l., Oncogene, 14 , 405-414 (1997)). High-level expression of each of these proteins suppresses apoptosis induced by growth factor deficiency, irradiation, glucocorticoids and various cytotoxic drugs. And the two cell death suppressor proteins are functionally equivalent and appear indistinguishable in their ability to suppress apoptosis (Huang, DCS, et al., Oncogene, 14 , 405-414 (1
997)).

[0005] Boyd et al. Used a yeast two-hybrid screening method (Boyd, JM, et al., Cell, 79 , 341-351 (1) to detect proteins that interact with E1B.
994)) using a protein that interacts with 19 kD (Nip
1, Nip2, Nip3), as well as three cDNAs (BNIP1, BNIP
2, BNIP3) was isolated. Boyd et al. Have demonstrated a direct interaction of E1B with these proteins and have
In all three, a B similar to the 19 kDa molecule of E1B
It has also been shown to interact with several motifs in the cl-2 protein. From these results, E1B19 kD and Bc
Although both 1-2 are different substances, they have been suggested to interact with certain common cellular proteins to promote cell survival. Although the physiological role of Nip proteins is still unknown, they are thought to have either an anti-apoptotic function or a function to induce apoptosis.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide Ni
A new gene encoding the p homologue, namely human Ni
to provide a p-homologous gene. The new human Ni
If a p-homologous gene can be provided, its expression level, its structure and function in each cell can be analyzed, and its expression can be analyzed to elucidate the diseases involved in such diseases, such as apoptosis including cancer. It is considered that diagnosis, treatment, and the like can be performed.

Further, the present invention has remarkable homology to the novel human gene, and has similarity in structural and gene expression patterns and similarity in biological functions thereof.
It is also an object to provide a series of novel related genes that are recognized as a family of the above genes. If such a gene can be provided, it is also possible to provide a novel peptide having an amino acid sequence corresponding to a specific region encoded by the gene, and a drug containing the peptide as an active ingredient.

The present inventors have conducted intensive studies for the above purpose and obtained the following findings, and succeeded in cloning a novel gene BNIP3L encoding the desired Nip homologue, and simultaneously cloned the gene into various cancer cells. By introducing the gene, they have found that they have a cancer cell growth inhibitory action.

That is, the present inventors synthesized cDNA from mRNA extracted from human placental tissue, incorporated it into a vector to construct a library, and transformed E. coli colonies transformed with the library on an agar medium. The colonies were randomly picked up and transferred to a 96-well microplate, and a large 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 for terminating specifically the four bases was carried out by the deoxyterminator method using the extracted cDNA as a template, and about 400 base sequences were determined from the 5 'end of the gene by an automatic DNA sequencer. Based on the nucleotide sequence information thus obtained, a known human Nip
A new gene having similarity to the gene was searched.

The above cDNA analysis method is described in detail by one of the present inventors, Fujiwara et al. (Riki Fujiwara, Cell Engineering, 14 , 645-654 (1995)). From the similarity (homology) of a candidate gene obtained by gene analysis to a known human gene, it is possible to roughly estimate the function of the gene product, that is, the function of the protein. Furthermore, incorporating the candidate gene into an expression vector,
By preparing a recombinant, functions such as enzyme activity and binding activity can be examined.

[0012]

According to the present invention, there is provided a human BNIP3L gene comprising a nucleotide sequence encoding the amino acid sequence shown by SEQ ID NO: 1, a human BNIP3L gene encoding the amino acid sequence, SEQ ID NO: A human BNIP3L gene comprising the nucleotide sequence represented by SEQ ID NO: 2; and a human BNIP3L gene comprising the nucleotide sequence represented by SEQ ID NO: 3.

In the present specification, abbreviations for amino acids, peptides, base sequences, nucleic acids, and the like are used in IUPAC and IUPAC.
Regulation B [IUPAC-IUB Communication on Biological No
menclature, Eur.J. Biochem., 138 , 9 (1984)],
The guidelines shall be in accordance with “Guidelines for Preparing Specifications and the Like Containing Base Sequence or Amino Acid Sequence” (edited by the Patent Office) and conventional symbols in the relevant field. The term “peptide” used in the present specification is not limited to the number of constituent amino acids, and includes, for example, oligopeptides, polypeptides, macropeptides and proteins.

According to the use of the gene provided by the present invention, a peptide having an amino acid sequence encoded by the gene can be provided, and a drug containing the peptide as an active ingredient, in particular, for regulating apoptosis. It is also possible to provide a medicament which can be used for treating various diseases and pathological conditions caused, for example, for cancer.

[0015]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As a specific example of the gene of the present invention, "GEN-551D1"
2 "can be deduced from the DNA sequence of the clone designated as" 2 ". Its base sequence is
As set forth in SEQ ID NO: 3. The gene has an open reading frame of a nucleotide (nucleic acid) encoded by the amino acid shown in SEQ ID NO: 1, and as shown in Examples described later, the BNIP3 gene product is different from the BNIP3 gene product in N-terminal protein and size. Although it is different, it has high homology in some regions.
Well stored with IP3. Therefore, the present inventors named this newly isolated gene the BNIP3L gene. Hereinafter, the BNIP3L gene which is a human Nip homologous gene of the present invention is referred to as "human BNIP3L gene".
There is that.

The human BNIP3L gene is a Nip homologous human gene expressed in various tissues. The predicted protein consisting of a 219 amino acid peptide expressed by the gene contains a transmembrane region. According to the introduction of the gene cDNA into various cancer cells, the growth of the cell is significantly inhibited, while the antisense cDNA or vector DNA of the gene is produced.
No such effect is shown by the introduction of. In consideration of such structural characteristics, physiological activities, gene expression patterns, and the like, the human BNIP3L gene of the present invention is considered to be a gene that produces a membrane protein having a function as a tumor suppressor, which suppresses the growth of tumor cells. Is considered to play an important role.

The human BNIP3L gene family gene of the present invention refers to human BN which is an expression product of the gene of the present invention.
Expressing a gene product having sequence homology to IP3L;
It refers to a series of related genes that can be recognized as one gene family because they have common features in structural characteristics and gene expression patterns, similarities in biological functions, and the like. The above sequence homology usually means that about 50% or more of the entire amino acid sequence is the same.

The gene of the present invention includes, for example, a gene containing a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in the specific example of SEQ ID NO: 1 or all or part of the nucleotide sequence shown in SEQ ID NO: 2. It is exemplified as a gene consisting of a polynucleotide. However, the present invention is not particularly limited thereto, and may be, for example, a gene having a certain modification in the above-mentioned specific amino acid sequence or a gene having a certain homology with the above-mentioned specific base sequence.

That is, the gene of the present invention has a nucleotide sequence encoding a protein consisting of a modified amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Containing genes are also included. Here, the degree of “deletion, substitution or addition of amino acids” and the positions at which they can be performed are determined by the fact that the modified protein has the same function as the protein having the amino acid sequence represented by SEQ ID NO: 1. However, there is no particular limitation on the premise that it has a tumor cell growth inhibitory activity.

The amino acid sequence modification (mutation)
In nature, it may be caused by, for example, mutation or post-translational modification. However, it may be artificially performed based on a natural gene (for example, a specific example gene obtained according to the embodiment of the present invention). The gene of the present invention includes all modified genes having the above-mentioned properties, regardless of the cause and means of such modification.

Examples of the artificial modification means include, for example, site-specific mutagenesis [Methods in Enz
ymology, 154 , 350, 367-382 (1987); and 100 , 468 (198
3); Nucleic Acids Res., 12 , 9441 (1984); Lecture on Sequent Chemistry Experiment 1, Genetic Research Method II, edited by The Japanese Biochemical Society, p105
(1986)] and chemical synthesis means such as the phosphate triester method and the phosphate amidite method [J. Am. Che.
m. Soc., 89 , 4801 (1967); ibid., 91 , 3350 (1969); Scien
ce, 150 , 178 (1968); Tetrahedron Lett., 22 , 1859
(1981); ibid., 24 , 245 (1983)], and methods of combining them.

The nucleotide sequence of a gene comprising a polynucleotide containing all or a part of the nucleotide sequence represented by SEQ ID NO: 2, which is exemplified as an embodiment of the gene of the present invention, has the above amino acid sequence (SEQ ID NO: 1). 1) is an example of one combination of codons encoding each amino acid residue. The gene of the present invention is not limited to this combination, and it is of course possible to select various codons for each amino acid residue and have various base sequences obtained by combining these. The choice of codons above
Conventional methods can be followed, for example, the codon usage of the host to be used can be considered (Ncleic Acids Res.,
9 , 43 (1981)].

Further, the gene of the present invention is, for example, SEQ ID NO:
As in the specific example shown as 3, the nucleotide sequence is represented as the base sequence of single-stranded DNA, but the present invention naturally includes a polynucleotide comprising a base sequence complementary to such a base sequence and a component containing both of them. It is not limited to DNA such as cDNA.

In addition, the gene of the present invention also includes a gene consisting of a base sequence having sequence homology to the base sequence shown in SEQ ID NO: 3, as described above. As such a gene, for example, in 6 × SSC containing 0.1% SDS,
Under stringent conditions such as at 60 ° C., the DNA hybridized with the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 was synthesized in 2 × SSC containing 0.1% SDS.
Those which are not desorbed even when washed under conditions of ６０60 ° C. can be mentioned.

The gene of the present invention can be easily produced and obtained by general genetic engineering techniques based on the specific sequence information described in this specification [Molecular Cloning
2d Ed, Cold Spring Harbor Lab. Press (1989); see Seismic Chemistry Laboratory Course “Gene Research Methods I, II, III”, edited by The Biochemical Society of Japan (1986), etc.).

Specifically, a cDNA library is prepared from an appropriate source in which the gene of the present invention is expressed according to a conventional method, and the desired library is prepared from the library using an appropriate probe or antibody specific to the gene of the present invention. By selecting a clone, the desired gene of the present invention can be obtained (for the method, see, for example, Proc. Natl. Acad. Sci., U.
SA., 78 , 6613 (1981); Science, 222 , 778 (1983), etc.].

In the above, the origin of the cDNA is as follows:
Examples include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Isolation of total RNA, isolation and purification of mRNA therefrom, acquisition of cDNA and cloning of cDNA, etc., are all in accordance with conventional methods. be able to. In addition, cDNA libraries are commercially available, and the gene of the present invention can be obtained using various commercially available cDNA libraries, such as Clontech Lab. Inc.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used. As a specific method, for example, c
A method for selecting a corresponding cDNA clone by immunoscreening using a specific antibody against a protein produced by DNA, plaque hybridization using a probe that selectively binds to a target DNA sequence, colony hybridization, and the like. Combinations and the like can be exemplified.

Here, as the probe, DN synthesized chemically based on the information on the nucleotide sequence of the gene of the present invention is used.
A and the like can be generally used, but of course, the already obtained gene of the present invention itself or a fragment thereof can also be used. Further, a sense primer or an antisense primer set based on the nucleotide sequence information of the gene of the present invention,
It can also be used as a screening probe.

When obtaining the gene of the present invention, the PCR method
[Science,230, 1350 (1985)]
An amplification method can also be suitably used. In particular, everything from the library
If it is difficult to obtain a long cDNA, the RACE method
[Rapid amplification of cDNA ends; experimental medicine,12
(6), 35 (1994)], especially the 5'-RACE method [M.A. Frohma
n, et al., Proc. Natl. Acad. Sci., USA.,8, 8998
(1988)]. Used in the PCR method
Primers based on the sequence information of the gene of the present invention.
It can be set appropriately and can be synthesized according to the usual method. The amplified D
The isolation and purification of the NA / RNA fragment can be performed by the above-mentioned conventional method, for example,
For example, gel electrophoresis can be used.

The genes and various DNAs of the present invention obtained above
Fragments can be prepared by a conventional method, for example, the dideoxy method [Proc. Natl. Aca
d. Sci., USA., 74 , 5463 (1977)] or the Maxam-Gilbert method [Methods in Enzymology, 65 , 499 (1980)], etc., and more conveniently using a commercially available sequence kit or the like. The sequence can be determined.

The gene of the present invention obtained as described above is
For example, a part or all of the nucleotide sequence can be used for detecting the expression of the gene of the present invention in an individual or various tissues. Such detection is carried out in a conventional manner, for example RT-P
CR (Reverse transcribed-Polymerase chain reactio
n; ES Kawasaki, et al., Amplification of RNA.In
PCR Protocol, A Guide to methods and applications,
Academic Press, Inc., SanDiego, 21-27 (1991)], RNA amplification, Northern blotting analysis [Molecu
lar Cloning, Cold Spring Harbor Lab. (1989)), in
situ RT-PCR [Nucl. Acids Res., 21 , 3159-3166
(1993)], their measurement at the cell level such as in situ hybridization, and the NASBA method [Nucleic acid
sequence-based amplification, Nature, 350 , 91-92
(1991)], and can be satisfactorily carried out by other known various methods.

The primer used in the PCR method is not limited as long as it can specifically amplify only the gene of the present invention, and its sequence can be appropriately set based on the sequence information of the gene of the present invention. . Usually, the primer can have a partial sequence of about 20 to 30 nucleotides.

As described above, the gene of the present invention also includes a DNA fragment that can be used as a specific primer and / or a specific probe for detecting the BNIP3L gene according to the present invention.

In the gene therapy or treatment using the gene of the present invention, not all of the gene of the present invention, that is, a gene consisting of the entire sequence is necessarily required. As long as it retains substantially the same function, the above-mentioned modified or partially composed gene can be used.

According to the use of the gene of the present invention (BNIP3L gene), the gene product (BNIP3L protein) or a peptide containing the same (hereinafter sometimes referred to as “the peptide of the present invention”) can be easily prepared by a general genetic engineering technique. Can be manufactured stably in large quantities. According to the genetic engineering technique, a desired peptide of the present invention can be obtained by a conventional gene recombination technique [for example, Scie, using the human BNIP3L gene specifically shown in Examples and its sequence information.
nce, 224 , 1431 (1984); Biochem. Biophys. Res.
m., 130 , 692 (1985); Proc. Natl. Acad. Sci., USA.,
80 , 5990 (1983), etc.].

That is, the peptide of the present invention is a recombinant DNA capable of expressing a gene encoding a desired protein in a host cell.
Is prepared, introduced into host cells, transformed, and the transformant is cultured.

Accordingly, the present invention also includes a vector (expression vector) containing the BNIP3L gene, a host cell transformed with the vector, and a method for producing the peptide of the present invention by culturing the host cell (genetical engineering technique). To provide.

Here, the host cell includes both eukaryotes and prokaryotes. The eukaryotic cells include cells such as vertebrates and yeast, and as vertebrate cells,
For example, COS cells which are monkey cells [Cell, 23 , 175 (1
981)) and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad.Sc.
i., USA., 77 , 4216 (1980)] etc. are often used, but are not limited thereto.

As a vertebrate expression vector, a promoter usually located upstream of a gene to be expressed,
Those having an RNA splice site, polyadenylation site, transcription termination sequence, and the like can be used, and may further have an origin of replication, if necessary. Examples of the expression vector include, for example, pSV2dhfr [Mol. Cell. Biol., 1, 854 (198)
1)] and the like. As eukaryotic microorganisms, yeasts are generally used, and among them, yeasts belonging to the genus Saccharomyces can be advantageously used. As an expression vector for eukaryotic microorganisms such as the yeast, for example, pAM82 having a promoter for the acid phosphatase gene [Proc. Natl. Acad.
Sci., USA., 80 , 1 (1983)]. Preferred examples of the expression vector for the gene of the present invention include a prokaryotic gene fusion vector. Specific examples of the vector include a GST domain having a molecular weight of 26000 ( S. jap
onicum ) and pGEX-2TK and pGEX-4
T-2 and the like can be exemplified.

Escherichia coli and Bacillus subtilis are generally used as prokaryotic hosts. When these are used as a host, for example, a plasmid vector capable of replicating in the host bacterium is used, and a promoter and an SD (Shine and Dalgarno) base sequence upstream of the gene so that the desired gene can be expressed in the vector, Further, it is preferable to use an expression plasmid provided with an initiation codon (eg, ATG) necessary for initiation of protein synthesis. As the Escherichia coli as the above host, Escherichia coli K12
Strains are often used, and the vector is generally pBR3
22 and its improved vector are often used, but not limited thereto, and various known strains and vectors can also be used. As a promoter, for example, tryptophan (tr
p) Promoters, lpp promoters, lac promoters, PL / PR promoters 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 resulting transformant can be cultured according to a conventional method, and the culture produces and expresses the target protein. As a medium used for the culture,
Various conventional ones can be appropriately selected and used depending on the host cell employed, and the culture can be carried out under conditions suitable for the growth of the host cell.

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

The peptide (recombinant protein) of the present invention can be subjected to various separation operations utilizing its physical properties, chemical properties and the like [Biochemical Data Book II, 1175-1259], if desired.
Page, 1st edition, 1st print, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274 (1986); Eur. J.
Biochem., 163 , 313 (1987)]. 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) , Adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HP
Examples thereof include various liquid chromatography such as LC), dialysis, and combinations thereof. As a particularly preferable method, affinity chromatography using a column to which a specific antibody of the peptide of the present invention is bound can be exemplified.

In designing a desired gene encoding the peptide of the present invention, the human BN represented by SEQ ID NO: 2 was designed.
The IP3L gene can be used. The gene is
If desired, codons indicating each amino acid residue can be of course appropriately selected and changed as described above.
Further, in the amino acid sequence encoded by the human BNIP3L gene contained in SEQ ID NO: 1, when some amino acids or amino acid sequences are modified by substitution, deletion, addition, etc., for example, cytospecific mutagenesis, etc. Various methods described above can be adopted.

The peptide of the present invention can also be produced by a general chemical synthesis method according to the amino acid sequence information. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis. More specifically, such a peptide synthesis method is a so-called stepwise elongation method in which each amino acid is successively linked one by one to extend the chain based on amino acid sequence information, and a fragment consisting of several amino acids is previously synthesized. And then a fragment condensation method in which each fragment is subjected to a coupling reaction.

The condensation reaction that can be employed in the above peptide synthesis method can be in accordance with a conventional method. Specifically, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide)
Method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-
5-norbornene-2,3-dicarboximide etc.)
Law, the Woodward Act, etc.

The solvent that can be used in each of these methods can be appropriately selected from well-known general solvents that are used in this kind of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof.

In the above peptide synthesis reaction, carboxyl groups in amino acids or peptides not involved in the reaction are generally esterified to form lower alkyl esters such as methyl ester, ethyl ester and tertiary butyl ester such as benzyl ester. And aralkyl esters such as p-methoxybenzyl ester and p-nitrobenzyl ester.

An amino acid having a functional group in a side chain, for example, a hydroxyl group of a tyrosine residue may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, etc., but such protection is not always required. You do not need to do it.

Further, for example, a guanidino group of an arginine residue includes a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, an adamantyloxycarbonyl group. And other suitable protecting groups.

The deprotection reaction of these protecting groups in the amino acids and peptides having the above protecting groups and the finally obtained peptide of the present invention can also be carried out by a conventional method such as a catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride or the like. , Hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid and the like.

The peptide of the present invention thus obtained can be appropriately prepared by the above-mentioned various methods, for example, according to methods widely used in the field of peptide chemistry, such as ion exchange resin, partition chromatography, gel chromatography, and countercurrent partitioning. The purification can be performed.

The peptide of the present invention can also be used as an immunizing antigen for preparing a specific antibody for the BNIP3L protein (included in the peptide of the present invention). By utilizing the antigen,
Desired antiserum (polyclonal antibody) and monoclonal antibody can be obtained. The method for producing the antibody,
It is well understood by those skilled in the art, and these methods can be used in accordance with the conventional methods [see Sequel Chemistry Laboratory Course “Immunobiochemical Research Method”, edited by The Japanese Biochemical Society (1986)). The antibody thus obtained can be advantageously used, for example, for purification of BNIP3L protein, measurement or identification of the protein by immunological techniques, and the like.

The peptide of the present invention is useful in the field of medicine as a drug containing the peptide as an active ingredient.

The peptide of the present invention useful as a pharmaceutical also includes a pharmaceutically acceptable salt thereof. Such salts include non-toxic alkali metal, alkaline earth metal and ammonium salts such as, for example, sodium, potassium, lithium, calcium, magnesium, barium, ammonium and the like, which are prepared by known methods. Further, the above salts also include non-toxic acid addition salts obtained by reacting the peptide of the present invention with a suitable organic or inorganic acid.
Representative non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, Borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate,
Examples include fumarate, succinate, tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate and napsylate.

The scope of the present invention also includes pharmaceutical compositions and preparations containing a pharmaceutically effective amount of the peptide of the present invention as an active ingredient, together with a suitable non-toxic pharmaceutical carrier or diluent.

Pharmaceutical carriers that can be used in the above-mentioned pharmaceutical composition (pharmaceutical preparation) include fillers, extenders, binders, humectants, disintegrants, and surfactants that are usually used depending on the use form of the preparation. And diluents and excipients such as lubricants. These can be appropriately selected and used according to the dosage unit form of the obtained preparation.

The dosage unit form of the pharmaceutical preparation of the present invention includes
Various forms can be selected according to the purpose of treatment. Typical examples include solid dosage forms such as tablets, pills, powders, powders, granules and capsules, and liquid dosage forms such as solutions, suspensions, emulsions, syrups and elixirs. These are further classified into oral preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments and the like according to the administration route, and can be prepared, molded or prepared according to the usual methods. it can.

In the case of molding into a tablet form, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, water ,
Ethanol, propanol, simple syrup, glucose solution,
Starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, binders such as polyvinylpyrrolidone, sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropylcellulose,
Disintegrators such as dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, etc., surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, sucrose, stearin, cocoa butter Disintegration inhibitors such as hydrogenated oils, quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, starch, lactose, kaolin,
Adsorbents such as bentonite and colloidal silicic acid, and lubricating agents such as purified talc, stearate, powdered boric acid, and polyethylene glycol can be used.

Further, the tablets can be made into tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double or multilayer tablets.

In the case of molding into a pill form, as a pharmaceutical carrier, for example, glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran and agar can be used.

Capsules are prepared according to a conventional method, usually by mixing an active ingredient with the above-mentioned various pharmaceutical carriers and filling the mixture into hard gelatin capsules, soft capsules and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs and the like containing conventional inert diluents such as water, and further include wetting agents, Auxiliaries such as emulsions and suspensions can be included, and these are prepared according to a conventional method.

In preparing liquid dosage forms for parenteral administration, such as sterile aqueous to non-aqueous solutions, emulsions and suspensions, diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol, ethoxylated Vegetable oils such as isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and olive oil can be used, and injectable esters such as ethyl oleate can be blended. These may further contain ordinary solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives, dispersants and the like. Sterilization can be performed, for example, by filtration through a bacteria-retaining filter, blending of a bactericide,
It can be performed by irradiation treatment, heat treatment, or the like. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.

For shaping in the form of suppositories or vaginal preparations, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like can be used as the preparation carriers.

When shaping into the form of an ointment such as paste, cream, gel, etc., diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives,
Vegetable oils such as propylene glycol, polyethylene glycol, silicon, bentonite and olive oil can be used.

The composition for nasal or sublingual administration can be prepared according to a conventional method using well-known standard excipients.

It should be noted that the drug prepared as described above may contain a coloring agent, a preservative, a fragrance, a flavor, a sweetener, and other pharmaceuticals, if necessary.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are orally administered, and injections are administered intravenously, alone or as a mixture with ordinary replenishers such as glucose and amino acids. Independently intramuscularly, intradermally, subcutaneously or intraperitoneally, suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, sublingual agents are administered orally, Ointments are topically administered transdermally.

The amount of the active ingredient to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and may be selected according to the desired therapeutic effect, administration method, treatment period, patient age, sex, other conditions, and the like. It is selected appropriately from a wide range, but in general,
The dose is usually about 1 to 10 per kg of body weight per day.
The dosage is preferably about 1 mg, and the preparation can be administered once or several times a day.

[0072]

According to the present invention, a novel human BNIP3
An L gene is provided. By using the gene, it is possible to detect the expression of the gene in various tissues and analyze its structure and function, and it is also possible to produce a human protein encoded by the gene by genetic engineering. By analyzing the expression of the gene, it becomes possible to elucidate the pathological condition, diagnose, treat, etc. of diseases associated with them, for example, various diseases related to apoptosis, particularly cancer.

Further, according to the present invention, there are also provided a peptide having an inhibitory effect on the growth of various cancer cells, useful for treating various cancer diseases and conditions, and a drug containing the peptide as an active ingredient.

[0074]

The present invention will now be described in further detail with reference to examples.

[0075]

Example 1 Human BNIP3L gene (GEN-551
D1 gene) (1) Cloning and DN of human BNIP3L gene
A sequencing Using a ZAP-cDNA synthesis kit (manufactured by Stratagene), according to the instruction for use of the product, human placental mRNA was used.
A cDNA library was constructed from. Sequencing of selected clones was performed using an automated DNA sequencer (ABI).
PRISM ^™ 377, manufactured by PerkinElmer) and A
The measurement was performed using an LF DNA sequencer (Pharmacia). To search for homology to known sequences, the FASTA program was used (Person, Wra.
nd Lipman, DJ, Proc. Natl. Acad. Sci. USA,
85 , 1435-1441 (1988)).

As a result, one clone GEN-551D12 having high homology with the human BNIP3 gene was selected from the above cDNA library. The 1265 base cDNA insert of this clone contained an open reading frame of 657 nucleotides encoding 219 amino acids (GenBank accession No. A
B004788).

When the gene sequence of the GEN-551D12 clone was compared with that of BNIP3, the sequence corresponding to codons 35-219 of the gene product encoded by the sequence of the open reading frame of the clone (SEQ ID NO: 1) 35-219 amino acid sequence shown)
And the sequence of codons 17 to 196 of BNIP3 are as follows:
Although 65% identical and well conserved, the N-terminal portion of the gene product (1-34 amino acid sequence shown in SEQ ID NO: 1) was different in size and amino acid sequence from that of BNIP3. Due to the high homology in the partial region (codons 35-219), the present inventors have determined that the gene (the clone, namely GEN-55).
1D12) was named "BNIP3L gene" and its expressed protein was named "BNIP3L protein".

According to the PSORT program, BNIP
The amino acid residues 188 to 208 encoded by the 3L gene are located at the transmembrane region (Nakai, K. and Kanehisa,
M., Genomics, 14 , 897-911 (1992)). This putative transmembrane region consists of BNIP3 and BNI
It was well conserved with the P3L protein.

(2) Northern Blot Analysis The expression of the gene of the present invention in normal human tissues was evaluated by Northern blot analysis using a human cDNA clone labeled by the random oligonucleotide priming method as a probe.

The Northern blot analysis was performed according to the product specification by using a human MTN blot (Human Multiple Tissue Not
hern blot; Clonetech, Palo Alto, California, USA).

That is, GEN-551D12 clone cD
The DNA of the insert portion obtained by digesting the NA clone with restriction enzymes EcoRI and XhoI was replaced with [ ³² P] -dC
The probe was labeled with TP (random primed DNA labeling kit, Boehringer Mannheim).

The blotting was performed after pre-hybridization for 6 hours, then at 42 ° C. for 12 hours, 50% formamide / 5 ×.
SSPE / 10 × Denhartz solution / 2% SDS solution (1
(Containing 00 μg / ml denatured salmon sperm DNA).

After washing with 2 × SSC / 0.05% SDS at room temperature for 40 minutes, the plate was washed twice with 0.1 × SSC / 0.1% SDS at 50 ° C. for 20 minutes. The filter is-
It was exposed to an X-ray film (manufactured by Kodak) at 70 ° C. for 12 hours.

As a result, the gene of the present invention named BNIP3L gene was tested on 16 normal human tissues (heart (h
eart), brain (placenta), lung (lung), liver (l
iver), skeletal muscle, kidney (kidney), pancreas
(pancreas), spleen (spleen), thymus (thymus), prostate (pro
state), testis (testis), ovary (ovary), small intestine (small inte
stine), colon and peripheral blood leukocytes (peripheral blo)
od leukocyte)).

(3) Localization of cosmid clone and chromosome by FISH method Using a chromosome clone containing the BNIP3L gene,
Known methods (Inazawa, J., et al., Genomics, 17 , 153-
162 (1993)).

That is, GEN-511D12 synthesized separately was used.
The cDNA clone was digested with the restriction enzymes EcoRI and XhoI.
A cosmid library of human chromosomes constructed in the cosmid vector pWEX15 (obtained from Stratagene) is used as a probe by using the DNA of the insert portion obtained by digestion with E. coli as a probe, according to a conventional method (Sambrook, J., et al. , Mole
cular Cloning, 2nd Ed., pp.3.1-3.58, Cold Spring H
arbor Laboratory Press. Cold Spring Harbor, New Yo
rk (1989)).

As a result, two independent cosmid clones were isolated. Each cosmid clone was used as a probe for the FISH test, and chromosomal locus determination was confirmed by comparing the pattern of the standard G band of each chromosome. In 100 metaphase cell specimens tested by each cosmid, a specific signal was displayed on the short arm on chromosome band 8. The signal position was determined as 8p21 by G band pattern. No specific signals on other chromosomes could be detected.

(4) Colony formation assay The gene of the present invention can be used for (1) LOH (loss of heterozygosity, loss of heterogeneity) in various cancers including colon, lung, prostate and liver.
rozygosity is mapped to the detected chromosome 8p21, and (2) the gene product clearly shows significant similarity to Nip3, which interacts with Bcl-2, a protein that suppresses apoptosis. Has become
Hereinafter, a colony formation assay was performed to examine whether the BNIP3L gene of the present invention plays an important role in either cell growth or cell death.

A) Cancer cell lines used The following cancer cell lines were used for the colony formation assay.

D98HB2-4, D98HB2-9, D98 derived from human cervical cancer cell line D98 / AH2
V1 and D98V2 (Benham, FJ and Harris, H., P
roc.Natl.Acad.Sci. USA, 76 , 4016-4019 (1979)
, Colon cancer cell line SW480 (ATCC No. CCL22)
8: Leibovitz, A., et al., Cancer Res., 36, 4562-4.
569 (1976)) and the glioblastoma cell line T98G (ATC
C No. CRL1690: Stein, GH, J. Cell Physio
l., 99 , 43-54 (1979)).

D98HB2-4 and D98HB2-9
Was transformed with a plasmid prepared to express high levels of Bcl-2. D98V1 and D98V
2 is a vector plasmid (pUC-CAGGS, Niwa, H., Yamamu) constructed to express low levels of Bcl-2.
ra, K. and Miyazaki, J., Gene, 108 , 193-200 (199
1)).

The above-mentioned D98HB2-4, D98HB2
-9, D98V1 and D98V2 cells were distributed by Yusuke Eguchi, a professor of genetic medicine at the Biomedical Education and Research Center, Osaka University School of Medicine.

B) Construction of Expression Vector The plasmid prepared for expressing the BNIP3L protein was a pCEP4 vector (manufactured by Invitrogen, San Diego, USA) having a CMV promoter and a gene conferring hygromycin (manufactured by Sigma) resistance. (California) was constructed by cloning the entire coding region of the BNIP3L cDNA (657 bases).

The sense strand (pCEP4 / BNIP3LS
Two plasmids were constructed: a plasmid prepared to express S) and an antisense plasmid prepared to express the antisense strand (pCEP4 / BNIP3LAS). The antisense plasmid is the same 657 in the opposite direction.
It was constructed by inserting the base BNIP3L cDNA.

C) Colony formation assay Each of the above six cancer cell lines was seeded in a 25 cm ² flask (2 × 10 ⁵ cells / flask), and after 24 hours, BNIP
Transformation was performed with a BNIP3L expression vector (pCEP4 / BNIP3LSS) containing the 3L cDNA sense strand.

The method is as follows. That is, the day before transfection, each cell line was transformed into 2 × 10 ⁵ cells / 2
Seeding was performed so as to form a 5 cm ² flask. As the medium, D98HB2-4, D98HB2-9, D98V1
For each cell of D98V2 and D98V2, RPMI-1640 medium (Nikken Institute of Biomedical Research) supplemented with 10% fetal bovine serum (FBS) was used. For SW480 cells, Leibovitz'L-15 medium (Gibco BRL) supplemented with 10% FBS was used. For T98G cells, a minimal essential medium (MEM, Gibco BRL) supplemented with 10% FBS was used.

Next, (1) OPTI-MEN (Gibco B
RL) DNA5 transfected into 250 μl
μg and (2) 250 μl of OPTI-MEN
Were mixed with 25 μg of TransIt ^™ -LT1, and the two were mixed and allowed to stand for 20 minutes. Each cell (1.5 ml of each medium, containing serum) was treated with the above (1) and (2)
Was added and cultured at 37 ° C. for 6 hours. After 6 hours, the medium containing the mixture was replaced with a fresh medium, and the culture was continued at 37 ° C. for 24 hours to wait for the expression of the introduced gene. Then, the cells were diluted 1: 8 and cultured in the presence of 600 μg / ml hygromycin for 14 days.

Two weeks later, after fixation with methanol, the number of colonies was counted using Giemsa staining solution.

As a control, all of the six cell lines were transformed with the pCEP4 vector alone or with the plasmid (pCEP4 / BNIP3LAS) with the BNIP3L cDNA reversed.

5 μg of plasmid DNA and 25 μg of Tr
ansl ^™ -LT1 (Pan Bella Corporation) was used for each transformation, and each transformation was performed twice.

Table 1 shows the fraction (%) of colonies (80 and 200 colonies for each test) in each flask in which the transformant of the present invention and the control (vector only) were compared. In addition, the number (%) of colonies in Table 1 indicates an average value of two independent tests.

[0102]

[Table 1]

As a result of the above test, as shown in Table 1,
The number of hygromycin-resistant clones depends on the number of cells transformed with the control vector and the amount of antisense cDNA.
Cells transformed with the sense strand cDNA from cells transformed with the plasmid prepared to express
Significantly less in dishes containing HB2-9 or D98V2 cells. The results of the colony formation assay in six cancer cell lines, including these two cell lines, are shown in Table 1.
As shown in Table 2, in all the tested cell lines, those in which the sense cDNA was induced showed a dramatic decrease in the number of colonies as compared to those in which only the antisense cDNA or vector was introduced. That is,
The number of colonies in the transformant transformed with the sense strand plasmid was reduced to 7.0 to 26.9% as compared with the control.

Boyd, JM, et al., Cancer R
es., 52 , 5368-5372 (1992)) is adenovirus 19
Three proteins (designated Nip1, Nip2, Nip3) were isolated using the yeast two-hybrid screening system, with kDaE1B as the “Otori”, but there was no significant sequence homology between them. Was. However, according to in vitro and in vivo binding assays, each Nip protein was found to be E1B19kDa protein and B
Reported to bind to cl-2 protein. These Ni
p protein is defective in suppressing cell death E
Since it did not bind to the mutant 1B protein, it was thought that the Nip protein plays an important role in the pathway that suppresses cell death. Boyd et al.'S results suggested that E1B19 kDa or Bcl-2 might work synergistically in anti-apoptotic activity.

However, because their physiological functions have not been elucidated, it is possible that they act in an inhibitory manner on anti-apoptic activity and may act as a substitute for certain growth inhibitors. It cannot be ruled out.

Bcl-2 is a member that suppresses cell death (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, A1) and a member that induces cell death (Bax, Bak, Bcl-XS, Bad). , Bid), which belong to a member of the apoptosis-regulating gene product, which form dimers with each other, and determine the relative amount of pro-apoptic and anti-apoptic proteins in the cell, Determine its susceptibility to (Kroemer,
G., Nature Med., 3 , 614-620 (1997)). Other Bcl-2 binding proteins not belonging to the family have also been reported (Fernandez-Sarabia, MJ and Bisc
hoff, JR, Nature, 366 , 274-275 (1993); Wang, H.
G., et al., Oncogene, 9 , 2751-2756 (1994); Takayam
a, S., et al., Cell, 80 , 279-284 (1995); Wang, H.
G., etal., Proc. Natl. Acad. Sci. USA, 93 , 706.
3-7068 (1996)).

The physiological role of the Nip protein is still uncertain, and whether it can have an anti-apoptotic effect or an apoptosis-inducing effect, and
How the ip protein functions when it interacts with Bcl-2 has not yet been renamed. Moreover, the human Nip homologous gene has not yet been isolated and analyzed, and elucidation of the gene has been awaited in the art.

The present inventors have developed a novel gene BNIP encoding one protein having sequence homology to Nip3, one of E1B-related proteins, as shown in the above Examples.
3L was cloned, and the BNIP3L gene c
DNA identification was performed. As a result, it was found that both the BNIP3L protein and the Nip3 protein having the deduced amino acid sequence encoded by the gene of the present invention contain a highly conserved putative transmembrane region. All Nip proteins are located in the nuclear envelope / ER, and Nip3 is also located on the mitochondrial membrane (Boyd, JM, et a).
l., Cell, 79 , 341-351 (1994)). Similarly, Bcl-2
The protein is localized at the mitochondrial and nuclear membranes and the endoplasmic reticulum (Akao, Y., et al., Cancer Res., 54 , 2468-2471).
(1994)). The E1B19 kD protein has been found to be expressed in the nuclear envelope / packet region by a transformation test (Boyd, JM, et al., Cell, 79 , 341-341).
351 (1994)). Although the location of the BNIP3L protein in cells has not been determined, if this protein is Bcl-
If it is related to 2, it is considered to be present in the membrane.

[0109] FISH revealed that the BNIP3L gene of the present invention was localized on chromosome 8p21. On the other hand, allelic loss has been detected in carcinomas of various tissues, including colon, lung, prostate and liver (Emi, M., e.
t al., Cancer Res., 52 , 5368-5372 (1992); Emi, M.,
et al., Genes Chrom. Cancer, 7 , 152-157 (1993); Bo
va, GS, Cancer Res., 53 , 3869-3873 (1993); Ohat
a, H., et al., Genes Chrom. Cancer, 7 , 85-88 (199
3); Fujiwara, Y., et al., Cancer Res., 53 , 1172-117.
4 (1993); Fujiwara, Y., et al., Genes Chrom. Cance
r, 10 , 7-14 (1994); Macoska, JA, et al., Cancer.
Res., 54 , 3824-3830 (1994)).

Further, from the results of the colony formation assay performed by introducing the BNIP3L gene into cancer cells as shown in the above Examples, it was found that temporary overexpression of the BNIP3L gene product caused significant suppression of cancer cell growth. There was found.
That is, as a feature of the BNIP3L gene expression protein of the present invention, a cell growth inhibitory effect on cancer cells was proved.

According to the present invention, the human Nip3 gene (B
Novel human BN having high homology with NIP3 gene)
By providing the IP3L gene, the use of the gene makes it possible to detect the expression of the gene in various tissues and to produce the BNIP3L protein by genetic engineering, thereby enabling the function of various diseases associated with abnormal preparation of apoptosis. Analysis,
Diagnosis and the like of these related diseases can be performed, and treatment and prevention of these diseases, particularly cancer, can also be performed.

[0112]

[Sequence list]

 (1) GENERAL INFORMATION: (i) APPLICANT: Ostuka Pharmaceutical Co., Ltd. (ii) TITLE OF INVENTION: human BNIP3L gene (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B ) FILING REFERENCE: 1B07JP (C) FILING DATE: 24-September-1997

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 219 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Ser Ser His Leu Val Glu Pro Pro Pro Pro Leu His Asn Asn Asn 1 5 10 15 Asn Asn Cys Glu Glu Asn Glu Gln Ser Leu Pro Pro Pro Ala Gly Leu 20 25 30 Asn Ser Ser Trp Val Glu Leu Pro Met Asn Ser Ser Asn Gly Asn Asp 35 40 45 Asn Gly Asn Gly Lys Asn Gly Gly Leu Glu His Val Pro Ser Ser Ser 50 55 60 Ser Ile His Asn Gly Asp Met Glu Lys Ile Leu Leu Asp Ala Gln His 65 70 75 80 Glu Ser Gly Gln Ser Ser Ser Arg Gly Ser Ser His Cys Asp Ser Pro 85 90 95 Ser Pro Gln Glu Asp Gly Gln Ile Met Phe Asp Val Glu Met His Thr 100 105 110 Ser Arg Asp His Ser Ser Gln Ser Glu Glu Glu Val Val Glu Gly Glu 115 120 125 Lys Glu Val Glu Ala Leu Lys Lys Ser Ala Asp Trp Val Ser Asp Trp 130 135 140 Ser Ser Arg Pro Glu Asn Ile Pro Pro Lys Glu Phe His Phe Arg His 145 150 155 160 Pro Lys Arg Ser Val Ser Leu Se r Met Arg Lys Ser Gly Ala Met Lys 165 170 175 Lys Gly Gly Ile Phe Ser Ala Glu Phe Leu Lys Val Phe Ile Pro Ser 180 185 190 Leu Phe Leu Ser His Val Leu Ala Leu Gly Leu Gly Ile Tyr Ile Gly 195 200 205 Lys Arg Leu Ser Thr Pro Ser Ala Ser Thr Tyr 210 215

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 657 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGTCGTCCC ACCTAGTCGA GCCGCCGCCG CCCCTGCACA ACAACAACAA CAACTGCGAG 60 GAAAATGAGC AGTCTCTGCC CCCGCCGGCC GGCCTCAACA GTTCCTGGGT GGAGCTACCC 120 ATGAACAGCA GCAATGGCAA TGATAATGGC AATGGGAAAA ATGGGGGGCT GGAACACGTA 180 CCATCCTCAT CCTCCATCCA CAATGGAGAC ATGGAGAAGA TTCTTTTGGA TGCACAACAT 240 GAATCAGGAC AGAGTAGTTC CAGAGGCAGT TCTCACTGTG ACAGCCCTTC GCCACAAGAA 300 GATGGGCAGA TCATGTTTGA TGTGGAAATG CACACCAGCA GGGACCATAG CTCTCAGTCA 360 GAAGAAGAAG TTGTAGAAGG AGAGAAGGAA GTCGAGGCTT TGAAGAAAAG TGCGGACTGG 420 GTATCAGACT GGTCCAGTAG ACCCGAAAAC ATTCCACCCA AGGAGTTCCA CTTCAGACAC 480 CCTAAACGTT CTGTGTCTTT AAGCATGAGG AAAAGTGGAG CCATGAAGAA AGGGGGTATT 540 TTCTCCGCAG AATTTCTGAA GGTGTTCATT CCATCTCTCT TCCTTTCTCA TGTTTTGGCT 600 TTGGGGCTAG GCATCTATAT TGGAAAGCGA CTGAGCACAC CCTCTGCCAG CACCTAC 657

(2) INFORMATION FOR
SEQ ID NO: 3: (i) SEQUENCE CHARACTERIST
ICS: (A) LENGTH: 1265 base pai
rs (B) TYPE: nucleic acid (C) STRANDENDESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (c)
DNA) (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 46. . 702 (xi) SEQUENCE DESCRIPTIO
N: SEQ ID NO: 3: CGGAGACGGT CCTGCTGCTG CCGCAGTCCT GCC
AGCTGTC CGACA ATG TCG TCC 54
Met Ser Ser
1 CAC CTA GTC GAG CCG CCG CCG CCC CTG
CAC AAC AAC AAC AAC AAC TGC 102 His Leu Val Glu Pro Pro Pro Pro Leu
His Asn Asn Asn Asn Asn Asn Cys 5 10
15 GAG GAA AAT GAG CAG TCT CTG CCC CCG
CCG GCC GGC CTC AAC AGT TCC 150 Glu Glu Asn Glu Gln Ser Leu Pro Pro
Pro Ala Gly Leu Asn Ser Ser 20 25
30 35 TGG GTG GAG CTA CCC ATG AAC AGC AGC
AAT GGC AAT GAT AAT GGC AAT 198 Trp Val Glu Leu Pro Met Asn Ser Ser
Asn Gly Asn Asp Asn Gly Asn 40
45 50 GGG AAA AAT GGG GGG CTG GAA CAC GTA
CCA TCC TCA TCC TCC ATC CAC 246 Gly Lys Asn Gly Gly Leu Glu His Val
Pro Ser Ser Ser Ser Ile His 55 60
65 AAT GGA GAC ATG GAG AAG ATT CTT TTG
GAT GCA CAA CAT GAA TCA GGA 294 Asn Gly Asp Met Glu Lys Ile Leu Leu
Asp Ala Gln His Glu Ser Gly 70 75
80 CAG AGT AGT TCC AGA GGC AGT TCT CAC
TGT GAC AGC CCT TCG CCA CAA 342 Gln Ser Ser Ser Arg Gly Ser Ser His
Cys Asp Ser Pro Ser Pro Gln 85 90
95 GAA GAT GGG CAG ATC ATG TTT GAT GTG
GAA ATG CAC ACC AGC AGG GAC 390 Glu Asp Gly Gln Ile Met Phe Asp Val
Glu Met His Thr Ser Arg Asp 100 105
110 115 CAT AGC TCT CAG TCA GAA GAA GAA GTT
GTA GAA GGA GAG AAG GAA GTC 438 His Ser Ser Gln Ser Glu Glu Glu Val
Val Glu Gly Glu Lys Glu Val 120
125 130 GAG GCT TTG AAG AAA AGT GCG GAC TGG
GTA TCA GAC TGG TCC AGT AGA 486 Glu Ala Leu Lys Lys Ser Ala Asp Trp
Val Ser Asp Trp Ser Ser Arg 135 140
145 CCC GAA AAC ATT CCA CCC AAG GAG TTC
CAC TTC AGA CAC CCT AAA CGT 534 Pro Glu Asn Ile Pro Pro Lys Glu Phe
His Phe Arg His Pro Lys Arg 150 155
160 TCT GTG TCT TTA AGC ATG AGG AAA AGT
GGA GCC ATG AAG AAA GGG GGT 582 Ser Val Ser Leu Ser Met Arg Lys Ser
Gly Ala Met Lys Lys Gly Gly 165 170
175 ATT TTC TCC GCA GAA TTT CTG AAG GTG
TTC ATT CCA TCT CTC TTC CTT 630 Ile Phe Ser Ala Glu Phe Leu Lys Val
Phe Ile Pro Ser Leu Phe Leu 180 185
190 195 TCT CAT GTT TTG GCT TTG GGG CTA GGC
ATC TAT ATT GGA AAG CGA CTG 678 Ser His Val Leu Ala Leu Gly Leu Gly
Ile Tyr Ile Gly Lys Arg Leu 200
205 210 AGC ACA CCC TCT GCC AGC ACC TAC TGAG
GGAAAG GAAAAGCCCC TGGAAATGCG 732 Ser Thr Pro Ser Ala Ser Thr Thr Tyr
215
TGTGACCTGT GAAGTGGTGT ATTGTCACAG TAG
CTTATTT GAACTTGGAGA CCATTGTAAG 792 CATGACCCAA CCTACACCACCC TGTTTTTTACA TAT
CCAATTC CAGTAACTCT CAAATTCAAT 852 ATTTATTCA AACTCTGTTG AGGCATTTTA CTA
ACCTTTAT ACCCTTTTGG GCCTGAAGAC 912 ATTTTAGAT ATTCTCTAACAG AGTTTACTGT TGT
TTAGAAAA TTTGCAAGGG CTTCTTTTCC 972 GCAAATTGCCA CCAGCAGATT ATAATTTTGTGT CAG
CAATGCT ATTATCTCTA ATTAGTGCCA 1032 CCAGACTAGA CCTGATCAT TCATGGGTATA AAT
TTTACTTC TTGCAACATA ACTACCATCT 1092 CTCTCTTAAA ACGAGATCAG GTTAGCAAAT GAT
GTAAAAG AAGCTTTATT GTCTAGTTGT 1152 TTTTTTTCCC CCAAGACAAA GGCAAGTTTC CCT
AAGTTTG AGTTGATAGT ATTAAAAAG 1212 AAAACAAAAACAAAAAAAAAA GGCAAGGCAC AAC
AAAAAAAA TATCCTGGGC AAT 1265

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 21/02 C07K 14/52 C12Q 1/68 A61K 37/02 // C07K 14/52 ADU (C12N 15/09 ZNA C12R 1:91) ( C12P 21/02 C12R 1:19)

Claims (3)

[Claims] 1. A human BNIP comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1.
3L gene. 2. A human BNIP3L gene comprising the nucleotide sequence of SEQ ID NO: 2. 3. The human BNIP3L gene according to claim 2, which is a nucleotide sequence represented by SEQ ID NO: 3.