JP2001000193A - Apoptosis-related protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA encoding a polypeptide including a specific amino acid sequence and having activities for enhancing the apoptosis induction of a cell through Fas, and usable for production, or the like, of a protein useful for detection, or the like, of an inhibitor and an induction accelerator of the apoptosis. SOLUTION: This new DNA encodes a polypeptide including an amino acid sequence of amino acid number 1 to 1107 of the formula in the molecule, and having activities for enhancing the apoptosis induction of a cell through Fas, or a polypeptide including an amino acid sequence having deletion, insertion or substitution of one or more amino acids in the amino acid sequence of the formula in the molecule, and having activities for enhancing the apoptosis induction of the cell through Fas. The DNA is usable for production, or the like, of a polypeptide useful for detection, or the like, of an apoptosis inhibitor and an accelerator of apoptosis induction. The DNA is obtained by screening a cDNA library derived from a mammal cell in which the apoptosis is induced through the Fas.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide having an activity of enhancing the induction of cell apoptosis via Fas, a DNA encoding the polypeptide, a recombinant DNA vector comprising the DNA, The present invention relates to a host transformed with the recombinant DNA vector, an antibody that specifically recognizes the polypeptide, a method for producing the polypeptide, and a method for searching for an apoptosis inhibitor utilizing the activity of the polypeptide.

[0002]

2. Description of the Related Art Fas (CD95 / APO-1) is a widely expressed tumor necrosis factor.
r, a cell surface receptor molecule belonging to the receptor family (hereinafter referred to as "TNF") (Itoh, N., et al.
(1991) Cell 66,233-243 and Nagata, S., et al.
(1997) Cell 88, 355-365), an agonistic anti-Fas monoclonal antibody and F, a natural ligand.
An apoptosis signal is induced in cells expressing Fas by being cross-linked with an as ligand (hereinafter referred to as “FasL”) (Yonehara, S., et al. (1989) J. Exp. Me
d. 169, 1747-1756, Trauth, BC, et al. (1989) Sc
ience 245, 301-305 and Suda, T., et al. (1993) Ce
ll 75, 1169-1178). The mechanism of Fas-mediated signal transduction of apoptosis has been studied as a model of apoptotic cell death in mammals.
Recent studies have identified several proteins that bind to the intracellular domain of Fas (the death domain; hereinafter referred to as “DD”) that is essential for Fas-mediated apoptosis signal transduction (see above). Naga
ta, S., et al. (1997)). DD is not only Fas but also T
This is a unique motif also found in NF receptors and other apoptosis-related molecules (see Nagata, S., et al. (19).
97)). Among the Fas binding molecules, the adapter protein FADD (MORTI) having DD in the C-terminal region is Fa
has been shown to play an important role in s-mediated apoptosis (Boldin, MP, et al. (1995) J. B.
iol. Chem. 270, 7795-7798, Chinnaiyan, AM, et a
l. (1995) Cell 81, 505-512, Yeh, WC, et al. (19
98) Science 279, 1954-1958 and Zhang, J., et al.
(1998) Nature 392, 296-300). FADD associates with Fas after stimulation of Fas (Kischkel, FC, et al.
(1995) EMBO J. 14, 5579-5588), followed by caspase-8 (FLIC), a member of the mammalian caspase family.
E, MACH or Mch5) are thought to associate through a homophilic interaction between the N-terminal regions of FADD and caspase-8, called the death effector domain (hereinafter "DED"). (Boldin, MP, et al. (1996) Cell 85, 803-
815, Muzio, M., et al. (1996) Cell 85, 817-827).
A complex of Fas, FADD and caspase-8 called Death Induce Signal Complex (hereinafter "DISC") (Kischkel, FC, et al., Supra).
al. (1995)), it is believed that caspase-8 is activated by oligomerization during proteolysis (Muzio, M., et al. (1998) J. Biol. Chem. 273,
2926-2930 and Yang, X., et al. (1998) Mol. Cell
1,319-325). Next, caspase-8 is converted to caspase-
3 (Enari, M., et al. (1996) Nature 380, 723-726 and Stennicke, HR, et al. (1998) J. Biol. Chem.
273, 27084-27090), caspase-6 (Takahashi,
A., et al. (1997) Oncogene 14, 2741-2752) and caspase-7 (Fernandes-Alnemri, T., et al. (1995)
It is activated by cleaving downstream caspases including Cancer Res. 55, 2737-2742). These downstream caspases are associated with morphological changes (Rudel, T. and Bokoch, GM
(1997) Science 276, 1571-1574) and DNA fragmentation (Li
u, X. etal., (1997) Cell 89, 175-184 and Enari,
M., et al. (1998) Nature 391, 43-50) have been suggested to cleave many cell death substrates that play a central role in each apoptotic event (Vi.
lla, P., et al. (1997) Trends Biochem. Sci. 22, 38
8-393 and Lee, KK, et al. (1998) Oncogene 16,
3029-3037).

In Fas-mediated apoptosis, caspase-8 has been thought to be activated after proteolytic DED oligomerization in DISC (Muzio, M., et al. (1998) supra). Yang, X., et a
l. (1998)).

However, Bcl-2 and Bc-2 which bind to Fas and FADD but do not bind to caspase-8
anti-apoptotic proteins of the Bcl-2 family, such as _{l-X L (Medema, JP} , et al. (1998) J. Biol. Ch
em. 273, 3388-3393) has been reported to produce Fas in certain cells.
Has been reported to suppress the induction of apoptosis through MAP (Scaffidi, C., et al. (1998) EMBO J. 17, 16)
75-1687). In particular, it has been reported that the adenovirus E1B19K protein belonging to the Bcl-2 family indirectly attenuates Fas-mediated activation of caspase-8 (Pr
ez, D. and White, E. (1998) J. Cell Biol. 141, 125
5-1266), suggesting that Bcl-2 family proteins, particularly the adenovirus E1B19K protein may inhibit apoptosis via Fas through binding to an unknown protein that contributes to activation of caspase-8. ing.
In addition, there was little formation of Fas-induced DISC in certain Fas-sensitive cells expressing sufficient amounts of the known DISC-constituting proteins (Scaffidi, supra).
C., et al. (1988)), suggesting the possibility of the presence of additional constituent proteins in DISC. Such a substance that inhibits the function of the protein is considered to be an apoptosis inhibitor useful as an anti-neurodegenerative disease agent or an anti-immunodeficiency agent, but the protein has not been identified, and a novel apoptosis inhibitor has been identified. It has been desired to isolate a novel polypeptide useful for searching for an apoptosis inhibitor as a target or a gene encoding the same.

[0005]

DISCLOSURE OF THE INVENTION A first object of the present invention is to provide a D-encoding polypeptide which is useful for searching for apoptosis inhibitors and apoptosis induction promoters.
NA, polypeptide encoded by said DNA, said DNA
And a cell carrying the vector. A second object of the present invention is to provide a method for searching for an apoptosis inhibitor or an apoptosis induction promoter which acts on the above-mentioned polypeptide as a target.

[0006]

Means for Solving the Problems The present invention relates to (1) a compound having amino acids 1 to 1107 of SEQ ID NO: 2 in the molecule;
Or a polypeptide comprising an activity of enhancing Fas-mediated apoptosis induction of a cell, or one or more amino acids of the amino acid sequence being deleted from the molecule. DNA encoding a polypeptide comprising an inserted or substituted amino acid sequence and having an activity of enhancing Fas-mediated apoptosis induction of cells; (2) a molecule encoding SEQ ID NO: 2 in the molecule; Amino acids 1 to 110 of
7. A DNA encoding the polypeptide according to (1), which comprises the amino acid sequence shown in 7, and has an activity of enhancing Fas-mediated apoptosis induction of cells.
(3) Amino acid numbers 1 to 11 of SEQ ID NO: 2 in the sequence listing
DNA encoding the polypeptide consisting of the amino acid sequence represented by SEQ. coli
SANK 70599 (FERM BP-6707)
DNA inserted in the plasmid carried by (5)
DNA containing a nucleotide sequence represented by nucleotide numbers 1 to 3321 of SEQ ID NO: 1 in the molecule,
(6) a DNA which hybridizes with the DNA of (5) under stringent conditions and comprises a nucleotide sequence encoding a polypeptide having an activity of enhancing Fas-mediated cell apoptosis induction;
(7) The DN according to any one of (1) to (6)
A polypeptide having an amino acid sequence encoded by A,
(8) The DN according to any one of (1) to (6).
A. (9) The recombinant DNA vector according to (8), which is an expression vector, (10) a transformed E. coli E. coli. coli SA
(8) The recombinant DNA vector according to (9), which is held by NK 70599 (FERMBP-6707), (11) the set according to any one of (8) to (10). (12) a host cell transformed with the recombinant DNA vector; coli
SANK 70599 (FERMBP-6707), the host cell according to (11),
3) A method for testing the effect of a compound or composition sample as an apoptosis inhibitor, comprising the following steps 1) to 3): 1) Any of (1) to (6) Animal cells transformed with the DNA of any one of the above and expressing Fas (hereinafter referred to as "group A"); and animals not transformed with the DNA and expressing Fas A cell group (hereinafter referred to as "group B") is cultured in the presence of an apoptosis-inducing anti-Fas monoclonal antibody and a test sample, respectively. 3) As a result of the above 2), a test sample having a higher apoptosis-inducing inhibitory activity in Group A than that in Group B was selected. (14) A method for testing the effect of a compound or composition sample as an apoptosis induction promoter, comprising the following steps 1) to 3): 1) (1) to (1) A group of animal cells transformed with the DNA of any one of (6) and expressing Fas (hereinafter referred to as “group A”); The expressing animal cell group (hereinafter referred to as "group B") is cultured in the presence of an apoptosis-inducing anti-Fas monoclonal antibody and a test sample, respectively; 2) Then, the apoptosis-inducing activity of each of the above cell groups by the antibody 3) detect and compare the activity promoted by the test sample; 3) as a result of the above 2), promote apoptosis induction in group A rather than activity promoting apoptosis induction in group B A test sample with high activity to be selected (15)
(7) An antibody which specifically binds to the polypeptide according to (7).

That is, the present invention provides a novel DNA encoding a polypeptide having an activity of enhancing Fas-mediated apoptosis induction of cells, a polypeptide encoded by the DNA, a recombinant vector containing the DNA, and a recombinant vector comprising the DNA. It is intended to provide a transformed host cell, a use of the DNA, and an antibody that specifically recognizes the polypeptide.

The present inventors searched for a protein that directly interacts with mouse caspase-8, and as a result, cloned a cDNA encoding a novel polypeptide having an activity of enhancing Fas-mediated apoptosis induction of cells. It was confirmed that the novel polypeptide has an activity of enhancing Fas-mediated induction of cell apoptosis. Also,
The present invention has been completed by elucidating the entire primary structure of the novel polypeptide and developing a screening method for an apoptosis inhibitor and an apoptosis induction promoter using the novel polypeptide.

[0009] The phrase that the polypeptide encoded by the DNA of the present invention "enhances the induction of apoptosis of cells through Fas" refers to, for example, the anti-human Fas monoclonal antibody CH.
In an experimental system in which apoptosis is induced in cells expressing Fas by stimulation with 11 or Fas ligand, if the cells are transformed with the DNA of the present invention in advance and overexpressed, the cells are not transformed with the DNA of the present invention. Induces apoptosis in more cells as compared to a group of cells.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA encoding the polypeptide of the present invention can be obtained, for example, from a cDNA library derived from a mammalian cell in which apoptosis can be induced via Fas, from a polypeptide that directly interacts with caspase-8. Based on the cDNA fragment obtained by screening the encoding DNA, the full length of the cDNA (SEQ ID NO: 21 in the sequence listing) is obtained, and the DNA encoding the portion obtained by removing the N-terminal 855 residue from the coding region thereof Is obtained by subcloning. As a method for screening such a gene, a method for screening a gene encoding a polypeptide having an affinity for a desired protein, for example, a two-hybrid screening method using yeast as a host (Chien, CT et al. (199)
1) Proc. Natl. Acad. Sci. USA 88, 9578-9582, Field
s, S. and Song, O. (1989) Nature 340, 245-246 and
Guarente, L. (1993) Proc. Natl. Acad. Sci. USA 90,
1639-1641). More specifically,
For example, an expression vector for yeast in which the DNA binding region of yeast transcription regulator GAL4 is linked to the DED region of mouse caspase-8, and a transcription activation region of GAL4 is linked to each cDNA of a mammalian cell-derived cDNA library. The expression vector for yeast is introduced into yeast cells and expressed. In a clone into which a cDNA encoding a polypeptide having an affinity for the DED region of mouse caspase-8 (in the yeast two-hybrid screening method, such a probe molecule is generally referred to as “bait”), the DNA binding region of GAL4 is used. And the transcription activation region is coupled to enhance the production of a protein having β-galactosidase activity, which is retained by the host yeast. By detecting this β-galactosidase activity, a clone into which a cDNA encoding a polypeptide having an affinity for the DED region of mouse caspase-8 can be selected (these series of steps are performed). Kit is commercially available from Clontech (Match Maker / Two Hybrid System Kit). The nucleotide sequence of the cDNA retained by the clone thus selected is analyzed, and the full-length cDNA is obtained using a method well-known to those skilled in the art as described below.

First, an animal cell serving as a source of mRNA which is a template of a cDNA library derived from a mammalian cell capable of inducing apoptosis via Fas is a mouse T cell or a cell of a mouse T cell line in the present invention. Strains are preferred, of which the mouse T cell lines WR19L12a and F9 are preferred, but F.
Cells or tissues in which apoptosis can be induced via as, or other animal cell lines, can also be used.

For the extraction of mRNA, guanidine thiocyanate / cesium chloride ultracentrifugation method, guanidine thiocyanate / hot phenol method, guanidine hydrochloride method and the like can be employed. . and Sacchi,
N., (1987) Anal. Biochem., 162, 156-159).

It is known that many mRNAs present in the cytoplasm of eukaryotic cells have a poly (A) sequence at the 3 'end. The paramagnetic particles on which mRNA is adsorbed and streptavidin is further immobilized,
The mRNA can be purified by capturing mRNA using the bond between streptavidin and washing, and then eluting the mRNA. Alternatively, a method in which mRNA is adsorbed on an oligo (dT) cellulose column and then eluted and purified may be employed. Further, mRNA can be further fractionated by sucrose density gradient centrifugation or the like.

The mRNA obtained as described above is Fa
In order to confirm that the polypeptide encodes a polypeptide having an activity of enhancing the induction of apoptosis of cells through s, the mRNA is translated into a polypeptide and the activity is examined, or an antibody specific to the polypeptide is used. Can be used to identify the polypeptide. For example, mRNA can be injected into oocytes of Xenopus laevis and translated (Gardon, j. B. et al. (1972) Nature 233, 177-18).
2) Alternatively, cell-free translation systems such as rabbit reticulocyte and wheat germ can be used (Schleif, RF and W
ensink, PC (1981): "Practical Methods in Molecu
lar Biology ", Springer-Verlag, NY.).

Further, using the mRNA obtained by the above method as a template and synthesizing a single-stranded cDNA using a reverse transcriptase,
A double-stranded cDNA can be synthesized from the single-stranded cDNA. The method includes the S1 nuclease method (Ef
stratiadis, A. et al. (1976) Cell 7, 279-288), La
nd method (Land, H. et al. (1981) Nucleic Acids Res. 9,
2251-2266), O. Joon Yoo method (Yoo, OJ et al. (198
3) Proc. Natl. Acad. Sci. USA 79, 1049-1053), Oka
yama-Berg method (Okayama, H. and Berg, P. (1982) Mol.
Cell. Biol. 2, 161-170) and the like.

Next, the obtained cDNA fragment was inserted into a lambda phage vector and allowed to self-replicate to thereby obtain cDN.
The recombinant phage having the A fragment can be stably retained and amplified. For example, lambda phage λZAPII
(Stratagene), the host E. coli XL
Plaque was produced by 1-Blue MRF 'strain or JM109 strain, and the presence or absence of color development due to the metabolism of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (hereinafter referred to as “X-gal”). From the recombinant. As a vector, a plasmid vector can be used in addition to a lambda phage vector.

A method for selecting a clone having a cDNA encoding a polypeptide having an activity of enhancing the desired Fas-mediated apoptosis induction of cells from a gene library such as a recombinant phage obtained as described above, For example, any of the following various methods can be adopted.

(1) Screening Method Using Synthetic Oligonucleotide Probe: When the whole or part of the amino acid sequence of the target polypeptide has been elucidated (if the sequence is a plurality of continuous specific sequences, Any part of the polypeptide of interest may be synthesized, and an oligonucleotide encoding the amino acid sequence may be synthesized (for codon degenerate amino acids, frequently used codons may be used or possible codons may be replaced). A plurality of nucleotide sequences may be synthesized in combination, and in the latter case,
Inosine can be included to reduce that variety),
This was labeled with ³² P, ³⁵ S or biotin and used as a probe to hybridize with a nitrocellulose filter or nylon filter on which the recombinant phage DNA was denatured and fixed, and the resulting positive clones were searched and selected. I do.

(2) Screening method using a probe prepared by the polymerase chain reaction: When the whole or part of the amino acid sequence of the target polypeptide has been elucidated, a sense corresponding to a part of the amino acid sequence on the N-terminal side The oligonucleotide and the antisense strand oligonucleotide corresponding to a part of the C-terminal side are synthesized, and polymerase chain reaction (hereinafter referred to as “PCR”. Saiki, RK et al. (198
8) See Science 239, 487-491) to amplify a cDNA fragment encoding the desired polypeptide. The template DNA used here is cDN synthesized by reverse transcription from mRNA of a cell producing the polypeptide of the present invention.
A, or genomic DNA can be used. The DNA fragment thus prepared is labeled with ³² P, ³⁵ S, biotin, or the like, and a cDNA library or a genomic library is screened by plaque hybridization or colony hybridization using the probe as a probe. Select the desired clone.

The DNA encoding the polypeptide of the present invention can be collected from the target clone obtained as described above by a known method (Maniatis, T. et al. (1982): "Molec
ularCloning A Laboratory Manual "Cold Spring Harb
or Laboratory, NY). For example, it can be carried out by separating a fraction corresponding to plasmid DNA from cells and cutting out a cDNA region from the plasmid DNA.

The nucleotide sequence of the DNA thus obtained is determined, for example, by the Maxam-Gilbert chemical modification method (Maxam, AM and Gilbert, W. (1980): "M
ethods in Enzymology "65, 499-559) and dideoxynucleotide chain termination using M13 phage (Messing,
J. and Vieira, J. (1982) Gene 19, 269-276). Further, an automatic DNA sequence analyzer using a fluorescent dye instead of the radioisotope (for example, Model 373A manufactured by PerkinElmer Japan Applied Biosystems) can be used.

Finally, PCR is performed to obtain a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

A cDNA encoding the most preferred polypeptide of the present invention (SEQ ID NO: 2 in the sequence listing)
Transformed E. coli strain E. coli harboring the inserted plasmid. coli SANK 70599 was created in April 1999.
It has been deposited internationally with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology on March 20, and given the accession number FERM BP-6707. Therefore, the gene encoding the polypeptide of the present invention (SEQ ID NO: 1 in the sequence listing) can also be obtained from the strain.

The fragment containing the gene encoding the polypeptide of the present invention, cloned as described above, is integrated into an appropriate vector DNA to transform other prokaryotic or eukaryotic host cells. Can be switched. Furthermore, the gene can be expressed in each host by introducing an appropriate promoter and a sequence involved in expression into these vectors.

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
And the like. To transform the gene of interest into these host cells, the host cell is transformed with a plasmid vector containing replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.

For example, K12 strain and the like are often used as Escherichia coli, and pBR322 and pUC-type plasmids are generally used as vectors, but are not limited thereto, and any known various strains and vectors can be used. .

As E. coli, promoters include tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (t
ac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter and the like, and any promoter can be used for production of the polypeptide of the present invention.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura,
K. et al. (1984) J. Biochem. 95, 87-93) and the like, but are not limited thereto. Bacillus subtilis α
DN encoding the signal peptide sequence of amylase
By linking the A sequence, secretory expression outside the cells is also possible.

Eukaryotic host cells include cells of vertebrates, insects, yeasts, and the like.
For example, COS cells (Gluzman, Y. (1.
981) Cell 23, 175-182, ATCC CRL-165
0) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) deficient in dihydrofolate reductase (Urlaub, G. and Chasin, LA (1980) Proc.
Natl. Acad. Sci. USA 77, 4126-4220) and the like are often used, but are not limited thereto.

As a vertebrate cell expression promoter, those having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc. which are usually located upstream of the gene to be expressed can be used. May have a replication origin. An example of such an expression vector is pSV2dhfr having the early promoter of SV40 (Subramani, S. et al. (198
1) Mol. Cell. Biol. 1,854-864).
It is not limited to this.

As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription termination signal, Those having an RNA splice site can be used. The expression vector is diethylaminoethyl (D
EAE)-Dextran method (Luthman, H. and Magnusso)
n, G. (1983) Nucleic Acids Res, 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and
van der Eb, AJ (1973) Virology 52, 456-457),
And electric pulse drilling (Neumann, E. et al. (1982)
EMBO J. 1, 841-845) can be incorporated into COS cells, and thus desired transformed cells can be obtained. When CHO cells are used as host cells, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, J. et al. (1)
989): "Molecular CloningA Laboratory Manual" Cold
Spring Harbor Laboratory, NY) and pSV2-neo
(Southern, PJ and Berg, P. (1982) J. Mol. App
l. Genet. 1, 327-341) and the like, and by selecting G418-resistant colonies, transformed cells stably producing the polypeptide of the present invention can be obtained.

When insect cells are used as host cells, cell lines derived from ovarian cells of Spodoptera frugiperda (Sf-9 or Sf-21) of the family Lepidoptera, Noctuidae, and Trichopl
High Five cells derived from usia ni egg cells (Wickham, TJ e
tal, (1992) Biotechnol. Prog. I: 391-396) is often used as a host cell, and the baculovirus transfer vector is pVL1392 / 1393 is frequently used (Kidd, IM and VC Emery (1993) The use of b
aculoviruses as expression vectors.Applied Bioche
mistry and Biotechnology 42, 137-159). In addition to these, vectors using baculovirus P10 or a promoter of the same basic protein can also be used. Furthermore, A
By connecting the secretion signal sequence of the envelope surface protein GP67 of cNPV to the N-terminal side of the target protein,
It is also possible to express the recombinant protein as a secreted protein (Zhe-mei Wang, et al. (1998) Biol. Chem., 3
79, 167-174).

As an expression system using eukaryotic microorganisms as host cells, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevi
siae and petroleum yeast Pichia pastoris are preferred. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennetzen,
L. and Hall, BD (1982) J. Biol. Chem. 257, 301
8-3025) and the promoter of the acid phosphatase gene (Miyanohara, A. et al. (1983) Proc. Natl. Acad. S
ci. USA 80, 1-5) can be preferably used. Also,
When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site for an endogenous protease or a known protease possessed by the host cell at the N-terminal side. For example, in a system in which human mast cell tryptase of a trypsin-type serine protease is expressed in petroleum yeast, the activity is achieved by connecting the secretion signal sequence of α-factor of yeast and the cleavage site of KEX2 protease possessed by petroleum yeast to the N-terminal side and expressing it. Type tryptase is known to be secreted into the medium (Andrew, L. Niles, et al. (1998) Biotechnol. Ap
pl. Biochem. 28,125-131).

The transformant obtained as described above is
It can be cultured according to a conventional method.
Alternatively, the polypeptide of the present invention is produced extracellularly. The medium used for the culture can be appropriately selected from various ones commonly used depending on the host cell used. For example, in the case of the above COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) A medium obtained by adding a serum component such as fetal bovine serum to a medium such as the above can be used.

The recombinant protein produced in the cells of the transformant or extracellularly by the above culture can be separated and purified by various known separation procedures utilizing the physical and chemical properties of the protein. can do. As the method, specifically, for example, treatment with a normal protein precipitant,
Examples include various liquid chromatography such as ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof. . In addition, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, the protein can be efficiently purified by a nickel affinity column. By combining the above methods, the polypeptide of the present invention can be easily produced in a large amount with high yield and high purity.

The DNA of the present invention obtained as described above
Whether the polypeptide encoded by has the activity of enhancing Fas-mediated apoptosis induction of cells is determined, for example, by cells capable of inducing apoptosis via Fas.
When the DNA of the present invention is introduced as a foreign gene and transiently overexpressed to induce apoptosis via Fas in the cells, the overexpression of the DNA of the present invention increases the number of cells in which apoptosis is induced It can be detected by checking whether or not it is.

Specifically, for example, HF which stably expresses human Fas which has been transduced from human HeLa cells
One cell is co-transfected with an expression vector for animal cells containing the DNA of the present invention and an expression vector for β-galactosidase by electroporation or the like, and then the cell is stimulated with an anti-human Fas monoclonal antibody CH-11. . After culturing for a certain period of time, the cells were fixed and X-G
The cells are stained with al, and among the β-galactosidase-positive blue cells, cells having a flat and adherent shape and cells having a round atrophy are counted as viable cells and apoptotic cells, respectively. In this way, the percentage of apoptotic blue cells is calculated. D of the present invention
Overexpression of NA has no significant effect in the absence of CH11-stimulated Fas signal, but can increase the number of cells in which apoptosis is induced after stimulation with CH11.

However, the method for detecting the activity of the polypeptide of the present invention to enhance the induction of cell apoptosis via Fas is not limited to these methods, and other known methods for detecting apoptosis can be used, for example. .

DNA encoding the polypeptide of the present invention
Can be used for the following applications, for example.

(1) Search for an apoptosis inhibitor: As described above, a DNA encoding the polypeptide of the present invention is introduced into cultured cells and expressed, and a test sample (low-molecular compound, microorganism culture) An extract sample of a substance is added to the medium to detect whether the number of cells in which apoptosis is induced decreases. The apoptosis induction inhibitory activity is low in wild-type cultured cells that do not express the DNA encoding the polypeptide of the present invention, and the apoptosis induction inhibitory activity is high in cells expressing the DNA encoding the polypeptide of the present invention. Select the test sample. The test sample thus selected is considered to be effective as an apoptosis inhibitor, particularly as a therapeutic agent for hepatitis and Alzheimer's disease. In addition, among autoimmune diseases such as type I diabetes, it has been shown that tissues are destroyed through Fas ligands presented by activated T cells and host tissues. It is useful for preventing severe tissue destruction. In addition, certain cancer tissues include Fa
Expression of s-ligands has been shown to kill infiltrating T cells and escape immune surveillance, and the apoptosis inhibitors are also useful as therapeutics for such tumors (O'Connel, J. et al. (1998) J.
Pathol. 186 (3) 240-246 and Suarez-Pinzon, W. et a
l. (1999) Diabetes 48 (1) 21-28).

(2) Search for an apoptosis induction promoter:
A DNA encoding the polypeptide of the present invention is introduced and expressed in cultured cells, and a test sample is added to the cells to detect whether apoptosis-induced cells increase. . A test in which wild-type cultured cells not expressing the DNA encoding the polypeptide of the present invention have a weak apoptosis induction promoting activity, and cells expressing the DNA encoding the polypeptide of the present invention have a high apoptosis induction promoting activity. Select a sample. The test sample selected in this way is considered to be effective as an apoptosis induction promoter, particularly as a therapeutic agent for various cancers and as a therapeutic agent for autoimmune diseases such as rheumatism.

(3) Gene therapy using an adenovirus vector: The polypeptide of the present invention is incorporated into an adenovirus vector and administered to an affected part of an autoimmune disease patient such as various cancers, chronic hepatitis, rheumatism, etc.
Increases sensitivity and improves symptoms.

(4) Treatment using antisense DNA:
By administering antisense DNA of the DNA encoding the polypeptide of the present invention to patients with acute hepatitis or Alzheimer's disease, rapid death of cells is suppressed and progress of symptoms is prevented.

(5) Preparation of model animal: Since the polypeptide of the present invention is thought to play a part in the Fas-mediated apoptosis induction pathway, the gene must be so constructed that it cannot express the DNA encoding the polypeptide of the present invention. The engineered knockout mouse is considered to exhibit autoimmune disease-like symptoms because the cells producing autoantibodies survive without undergoing selection by Fas-mediated apoptosis. In addition, even when the knockout mouse is lethal, it can be a useful model for autoimmune diseases by using a method utilizing CRE-LoxP, for example, by specifically inhibiting expression in immune system cells. . Transgenic mice genetically engineered to overexpress the DNA encoding the polypeptide of the present invention are useful, for example, in acute hepatitis models.

The N-terminal amino acid sequence of the polypeptide of the present invention purified as described above is determined using an automatic protein amino acid sequencer (for example, model PPSQ-10 manufactured by Shimadzu Corporation). be able to.

In order for the substance obtained by the genetic engineering technique using the DNA of the present invention to exhibit the activity of enhancing Fas-mediated apoptosis induction of cells, the sequence in the sequence listing is not necessarily required. It is not necessary to have all of the amino acid sequences of amino acid Nos. 1 to 1107 of No. 2, for example, even if a partial sequence thereof has an activity of enhancing Fas-mediated induction of cell apoptosis, Is also encompassed by the polypeptides of the present invention. In addition, a DNA encoding the polypeptide is also included in the present invention.

The polypeptide having the activity of enhancing the induction of cell apoptosis via Fas is 1107 having a methionine residue of amino acid No. 1 in the amino acid sequence shown in SEQ ID No. 2 in the sequence listing as an N-terminal. A polypeptide consisting of amino acids can be exemplified. Also,
Preferred examples of the DNA encoding the polypeptide include a DNA consisting of the nucleotide sequence represented by nucleotide numbers 1 to 3321 in SEQ ID NO: 1 in the sequence listing.

In general, eukaryotic genes are known to be polymorphism (poly
morphism) (eg Nishi, T. et a
l. (1985) J. Biochem. 97, 153-159), one or more amino acids may be replaced by this polymorphism, or no It may not change. One or more amino acid residues are deleted at one or more sites in the polypeptide of the present invention consisting of the amino acid sequence shown by amino acid numbers 1 to 1107 of SEQ ID NO: 2 in the sequence listing , An added, inserted or substituted polypeptide often has an activity of enhancing Fas-mediated cell apoptosis induction (a protein having an amino acid sequence in which a natural amino acid sequence is substituted is a natural protein). As an example having the same activity, for example, a protein obtained by converting a nucleotide sequence corresponding to cysteine of the interleukin 2 (IL-2) gene to a nucleotide sequence corresponding to serine retains IL-2 activity. (Wang, A. et al. (1984) S
cience 224, 1431-1433)). All such polypeptides are included in the present invention as long as they have an activity of enhancing Fas-mediated apoptosis induction of cells. In addition, all DNAs having the same nucleotide sequence encoding these polypeptides are also included in the present invention.

The various DNAs of the present invention can be prepared, for example, by the phosphite-triester method (Hunkapiller, M. et al.) Based on the information on the polypeptide having the activity of enhancing the apoptosis induction of cells through Fas. al. (19
84) It can also be produced by chemical synthesis according to a conventional method such as Nature 310, 105-111).

The codon corresponding to the desired amino acid may be selected arbitrarily. For example, it can be determined according to a conventional method in consideration of the codon usage of the host to be used. (Granth
am, R. et al. (1981) Nucleic Acids Res. 9, 143-17
Four). Furthermore, the codons of these nucleotide sequences can be partially modified by site-specific mutagenesis (Mark, Mark) using a primer consisting of a synthetic oligonucleotide encoding the desired modification in a conventional manner.
DF et al. (1984) Proc. Natl. Acad. Sci. USA 8
1, 5662-5666). In order to prepare a variant in which one or two or more amino acid residues are deleted, exonuclease Bal31
Method of removing DNA from the end using the method described above (Toshimitsu Kishimoto et al., “Seismological Chemistry Laboratory Course 1: Genetic Research Method II” 335-354),
Cassette Mutation Method (Toshimitsu Kishimoto, “New Chemistry Experiment Course 2 ・
Nucleic acid III Recombinant DNA technology "242-251) and the like.

Whether or not a certain DNA hybridizes with a DNA consisting of the nucleotide sequence represented by nucleotide numbers 1 to 3321 of SEQ ID NO: 1 in the sequence listing is determined.
For example, the target DNA is subjected to the random primer method (Ana
l. Biochem., 132: 6013 (1983)) and the nick translation method (Maniatis, T. et al. (1982) "Molecular C
loning A Laboratory Mannual "Cold Spring Harbor La
boratory, NY.), etc., and hybridization can be performed using a probe labeled with [α- ³² P] dCTP or the like. The DNA used for hybridization is adsorbed on a known method, for example, a nitrocellulose membrane or a nylon membrane, and solidified by heating or irradiation with ultraviolet rays. Next, the film is, for example, 6 × SSC
(0.9M sodium chloride, 0.09M sodium citrate, pH 7.5), 5% Denhardt
The solution is immersed in a prehybridization solution containing the solution and 0.1% sodium dodecyl sulfate (hereinafter referred to as “SDS”), and kept at 55 ° C. for 4 hours or more. Thereafter, the previously prepared labeled probe is added to the same prehybridization solution so as to have a final specific activity of 1 × 10 ⁶ cpm / ml, and the mixture is kept at 60 ° C. overnight. The operation of washing the membrane at 57 ° C. for 5 minutes was repeated 5 times, and after further washing at 57 ° C. for 20 minutes, autoradiography was performed.
It can be determined whether or not hybridization has occurred. Using this method, nucleotide numbers 9 to 9 in SEQ ID NO: 1 can be obtained from cDNA libraries derived from various animal cells.
DN consisting of the nucleotide sequence shown from 1 to 831
CDNA hybridizing to A can be isolated (Maniatis, T. et al. (1982) "Molecular Cloning
A Laboratory Mannual "Cold Spring Harbor Laborator
y, NY.).

A monoclonal antibody that specifically binds to the polypeptide of the present invention can be obtained by the method described below.

In producing a monoclonal antibody,
Generally, the following work steps are required. That is,
(A) Purification of a biopolymer used as an antigen, (b) Immunization by injecting the antigen into an animal, and then collecting blood and assaying the antibody titer to determine the timing of spleen removal, and then determine the antibody. (C) preparation of myeloma cells (hereinafter referred to as "myeloma"); (d) cell fusion of antibody-producing cells with myeloma; and (e) selection of a hybridoma group producing the desired antibody. (F) splitting (cloning) into single cell clones, (g) culturing hybridomas to produce monoclonal antibodies in large quantities, or breeding hybridoma-transplanted animals, (h) Investigation of the biological activity of the monoclonal antibody produced and its recognition specificity, or assay of its properties as a labeling reagent.

Hereinafter, a method for preparing a monoclonal antibody will be described in detail along the above steps, but the method for preparing the antibody is not limited thereto. For example, antibody-producing cells other than spleen cells and myeloma can be used.

(A) Purification of antigen As the antigen, the polypeptide of the present invention or a part thereof prepared by the method described above can be used.
Furthermore, since the present invention has revealed the entire primary structure of the polypeptide of the present invention, it can be obtained by a method well known to those skilled in the art.
It is also possible to chemically synthesize a partial peptide having the amino acid sequence represented by amino acid numbers 1 to 1107 of SEQ ID NO: 2 in the sequence listing and use this as an antigen.

(B) Preparation of antibody-producing cells The antigen obtained in the step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or alum, to immunize a laboratory animal as an immunogen. I do. As an experimental animal, a mouse is most preferably used, but is not limited thereto.

The method of immunogen administration for mouse immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.

The immunization can be performed once or multiple times at appropriate intervals (preferably at intervals of one to five weeks). Thereafter, the antibody titer against the antigen in the serum of the immunized animal is measured, and the effect of the subsequent operations can be enhanced by using the animal whose antibody titer is sufficiently high as a source of the antibody-producing cells. Generally, 3 to 3 days after the last immunization
It is preferable to use antibody-producing cells derived from an animal 5 days later for cell fusion later.

The antibody titers used herein include radioisotope immunoassay (hereinafter referred to as "RIA"), enzyme-linked immunosorbent assay (hereinafter referred to as "ELISA"), fluorescent antibody assay, and passive immunoassay. Various known techniques such as a hemagglutination method can be mentioned, but from the viewpoints of detection sensitivity, speed, accuracy, and possibility of automation of the operation, the RIA method or the ELISA method is more preferable.

In the present invention, the measurement of the antibody titer
According to the LISA method, it can be performed according to the procedure described below. First, the purified or partially purified antigen is adsorbed on a solid surface such as a 96-well plate for ELISA, and the solid surface on which the antigen is not adsorbed is exposed to a protein unrelated to the antigen, for example, bovine serum albumin (hereinafter referred to as “B
After washing the surface, the sample is contacted with a serially diluted sample (eg, mouse serum) as a primary antibody, and the monoclonal antibody in the sample is bound to the antigen. Further, an antibody against an enzyme-labeled mouse antibody is added as a secondary antibody, and the antibody is bound to the mouse antibody.After washing, a substrate of the enzyme is added, and a change in absorbance due to color development based on the decomposition of the substrate is measured. calculate.

(C) Step of Preparing Myeloma As a myeloma, a cell line generally obtained from a mouse, for example, an 8-azaguanine-resistant mouse (BALB)
/ C derived) myeloma strain P3X63Ag8U.1 (P3-U1) (Yelton,
DE et al. Current Topics in Microbiology and Imm
unology, 81, 1-7 (1978)), P3 / NSI / 1-Ag4-1 (NS-1)
(Kohler, G. et al. European J. Immunology, 6, 511
-519 (1976)), Sp2 / O-Ag14 (SP-2) (Shulman, M. et.
al. Nature, 276, 269-270 (1978)), P3X63Ag8.653
(653) (Kearney, JF et al. J. Immunology, 123, 15
48-1550 (1979)), P3X63Ag8 (X63) (Horibata, K. and
Harris, AW Nature, 256, 495-497 (1975)) and the like are preferably used. These cell lines are prepared in an appropriate medium, for example, 8-azaguanine medium [glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as “FCS”) in RPMI1640 medium.
Medium supplemented with 8-azaguanine in a medium supplemented with Iscove's Modified Dulbecc Medium
o's Medium; hereinafter referred to as "IMDM") or DM
The cells were subcultured in EM, but were subcultured in a normal medium (for example, ASF104 medium containing 10% FCS (manufactured by Ajinomoto Co., Inc.)) 3 to 4 days before cell fusion.
Keep a cell count of 10 ⁷ or more.

(D) Cell fusion Antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, and may be obtained from any site of an individual. Alternatively, it can be obtained from a combination of these as appropriate, and spleen cells are most commonly used.

After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. Currently, the most commonly used means for fusing the spleen cells with the myeloma obtained in step (c) is a method using polyethylene glycol, which has relatively low cytotoxicity and simple fusion operation. This method includes, for example, the following procedure.

The spleen cells and myeloma are thoroughly washed with a serum-free medium (for example, RPMI 1640) or PBS, mixed so that the ratio of spleen cells to myeloma is about 5: 1 to 10: 1, and centrifuged. To separate. The supernatant was removed, and the precipitated cell group was thoroughly loosened. Then, 1 ml of 50% (w / v) polyethylene glycol (molecular weight: 10
00-4000) is added dropwise. Thereafter, 10 ml of serum-free medium is slowly added, followed by centrifugation. The supernatant was discarded again, and the precipitated cells were placed in an appropriate amount of hypoxanthine / aminopterin / thymidine (hereinafter referred to as “HAT”).
HAT medium) and mouse interleukin-2 (hereinafter, referred to as "IL-2"), suspended in a normal medium (hereinafter, referred to as "HAT medium"), and distributed to each well of a culture plate (hereinafter, referred to as "plate"). Inject and incubate at 37 ° C for about 2 weeks in the presence of 5% carbon dioxide. HAT medium is supplemented as needed on the way.

(E) Selection of Hybridoma Group When the myeloma cell is an 8-azaguanine resistant strain, that is, a hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) -deficient strain, the unfused myeloma cell , And fusion cells of myeloma cells cannot survive in a HAT-containing medium. On the other hand, a fusion cell between antibody-producing cells or a hybridoma of an antibody-producing cell and a myeloma cell can survive, but a fusion cell between antibody-producing cells has a life span. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma between the antibody-producing cell and the myeloma cell survives, and as a result, the hybridoma can be selected.

For the hybridomas that have grown in a colony, the medium is replaced with a medium obtained by removing aminopterin from the HAT medium (hereinafter referred to as “HT medium”). Thereafter, a part of the culture supernatant is collected, and the antibody titer is measured, for example, by ELISA.

The method using an 8-azaguanine-resistant cell line has been described above, but other cell lines can also be used according to the selection method of the hybridoma, and in that case, the composition of the medium used also changes.

(F) Cloning A hybridoma that has been found to produce a specific antibody by measuring the antibody titer in the same manner as described in step (b) is transferred to another plate and cloned. As the cloning method, a limiting dilution method in which one hybridoma is diluted and cultured so that one hybridoma is contained in one well of the plate, a soft agar method in which the colony is collected by culturing in a soft agar medium, and a micromanipulator one by one. Method of removing and culturing cells from a cell sorter
A "sort clone" for separating individual cells may be mentioned, but the limiting dilution method is simple and often used.

For the wells in which the antibody titer has been recognized, for example, cloning by the limiting dilution method is repeated 2 to 4 times, and those having a stable antibody titer are selected as the monoclonal antibody-producing hybridoma strain of the present invention.

(G) Preparation of Monoclonal Antibody by Hybridoma Culture After completion of cloning, the culture medium was HT
The medium is replaced with a normal medium and cultured. Large-scale culture is performed by rotary culture using a large culture bottle or spinner culture. The monoclonal antibody that specifically binds to the polypeptide of the present invention can be obtained by purifying the supernatant in this large-scale culture using a method known to those skilled in the art such as gel filtration. In addition, mice of the same strain (for example,
By growing the hybridoma in the abdominal cavity of BALB / c) or Nu / Nu mouse, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained. As a simple method of purification, a commercially available monoclonal antibody purification kit (for example, MAbTrap GII)
Kit; manufactured by Pharmacia) or the like can also be used.

The monoclonal antibody thus obtained is
It has high antigen specificity for the polypeptide of the present invention.

(H) Assay of Monoclonal Antibody The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. First, as an identification method, Ouchterlony (Ouchterlon
y) method, ELISA method, or RIA method.
The Otterlony method is simple, but requires a concentration operation when the concentration of the monoclonal antibody is low. on the other hand,
When the ELISA method or the RIA method is used, the culture supernatant is reacted with the antigen-adsorbed solid phase as it is, and an antibody corresponding to various immunoglobulin isotypes and subclasses is used as a secondary antibody. Can be identified. Further, as a simpler method, a commercially available identification kit (for example, mouse typer kit; manufactured by Bio-Rad) can be used.

Further, the protein was quantified by the Foreign Lowry method and the absorbance at 280 nm [1.4.
(OD280) = immunoglobulin 1 mg / ml].

The monoclonal antibody of the present invention thus obtained can be used for detecting, separating and purifying the polypeptide of the present invention by utilizing its specificity.

[0075]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

Reference Example 1 D of mouse caspase-8
Cloning of molecules interacting with the ED region 1. Preparation of cDNA Library 1.1 Construction of cDNA Library Derived from Mouse WR19L12a Cell and Fusion Protein Expression Vector Consisting of GAL4 Transcriptional Activating Region After culturing until the cells are collected,
Total RNA was prepared using (Nippon Gene Co., Ltd.). From this total RNA, poly (A) ⁺ RN was purified using an mRNA purification kit (Amersham Pharmacia) ^.
A was purified. Using 5 μg of the poly (A) ⁺ RNA and oligo dT primer or random primer, cDNA was synthesized by a reverse transcriptase reaction. A cDNA library was prepared by adding a restriction enzyme EcoRI recognition site and a NotI recognition site to those obtained by priming both ends of the obtained cDNA with oligo dT primers. Primed with random primer, cDNA with restriction enzyme EcoRI recognition site added
A library was prepared. This series of steps involves cDNA
A synthesis kit (Timesaver cDNA synthesis kit: manufactured by Amersham Pharmacia) was used.

The obtained cDNA fragment was inserted into the multi-cloning site of the yeast two-hybrid system vector pGAG424 and pGAG424 containing the gene for the GAL4 transcriptional activation region (amino acids 768 to 881 of GAL4). Was inserted into the vector pGADinex (manufactured by Clontech) into which was introduced. Thus, a plasmid library for expressing a fusion protein of the mouse cDNA library and the transcriptional activation region of GAL4 was obtained.

1.2 cDNA library derived from F9 cells (for colony hybridization) Mouse testis teratoma-derived cell line F9 was cultured in (Dulbecco's modified Eagle's medium containing 10% fetal calf serum) until it became 70% confluent. After that, Isogen
(Nippon Gene) to prepare total RNA. From this total RNA, poly (A) ⁺ RNA was purified using an mRNA purification kit (Amersham Pharmacia). CDNA was synthesized by reverse transcriptase reaction using 5 μg of poly (A) ⁺ RNA and oligo dT primer or random primer, and the resulting cDN
A cDNA library was prepared in which the both ends of A were primed with an oligo dT primer and a restriction enzyme EcoRI recognition site and a NotI recognition site were primed with a random primer, and a restriction enzyme EcoRI recognition site was added to those primed with a random primer. In this series of steps, a cDNA synthesis kit (Timesaver cDNA synthesis kit: manufactured by Amersham Pharmacia) was used.

The obtained cDNA fragment was converted into a vector pGAG424 and pGAG for the yeast two-hybrid system.
424 was inserted into a vector pGADinex (Clontech) having a restriction enzyme NotI recognition sequence introduced into the multicloning site. Thus, the mouse cDN
A plasmid library for expressing a fusion protein of the A library and the transcriptional activation region of GAL4 was obtained.

2. Yeast two-hybrid screening 2.1 Preparation of probe (bait) (1) Isolation of mouse caspase-8 DED region gene cDN encoding mouse Caspase-8 DED region
A was cloned by colony hybridization from cDNA obtained by reverse transcription reaction from RNA prepared from mouse proB cell-derived cell line BaF3, and mammalian cell expression vector pME18S (Sakamaki, K. et al. (1992) E
MBO J. 11, 3541-3549) to obtain plasmid pME18S-mCaspase-8. Using this pME18S-mCaspase-8 as a template, PC
The DED region of mouse caspase-8 was obtained by the R method. Oligonucleotide primers having the following nucleotide sequence for PCR: 5'-catggaattc atggatttcc agagttgtat tgtatttat -3 '
(SEQ ID NO: 3 in the Sequence Listing); and 5′-catggtcgac ctcatccaaa actgaaggtg-3 ′ (SEQ ID NO: 4 in the Sequence Listing) were synthesized and used. These primers are based on the DNA sequence encoding mouse caspase-8 (gene sequence database Genbank accession number af067834),
Restriction enzyme recognition sites for insertion into yeast expression vectors are added to the ends. 626 bp obtained
DNA fragment of the EcoRI recognition site before the start codon,
It has a SalI recognition site after the stop codon and encodes amino acids 1 to 202 of mouse caspase-8.

(2) Construction of Fusion Protein Expression Vector Consisting of Mouse Caspase-8 DED Region and GAL4 DNA Binding Region Restriction enzyme Eco
Digested with RI and Sail. The obtained fragment was ligated to the DNA binding region of GAL4 (1 to 14 of GAL4).
It was inserted into the EcoRI-SalI site of an expression vector pGBT9 (manufactured by Clontech) containing the gene for the seventh amino acid residue) for the yeast two-hybrid system. Thus, the plasmid pGBT9- for expressing a fusion protein of amino acid residues 1 to 202 (DED) of mouse caspase-8 and the DNA binding region of GAL4 was obtained.
mCaspase-8DED was obtained.

2.2 Yeast Two-Hybrid Screening Yeast two-hybrid screening of the WR19L12a-derived cDNA library was carried out by using a Matchmaker Two-Hybrid System Kit (Clontech) according to its protocol.

Yeast cell line HF7c (Clontech)
Then, the fusion protein expression plasmid obtained in the above step was introduced. The cell line SFY526 is a cell line in which a fusion gene of GAL1 and lacZ is integrated into a chromosome and has a deletion mutation of the GAL4 gene. (Bartel, et a
l. (1993) Bio Techniques 14, 920-924). 0.1 μg
Using pGBT9-mCaspase-8 DED of
HF7c was transformed on a small scale. Transformation was performed using the lithium acetate method (Itoh, H. et al. (1983) J. Bacteriol.
153, 163-168, Hill, J. et al. (1991) Nucleic Acid
s Res. 19, 5791, Schiestl, RH and Gietz, RD
(1989) Curr.Genet. 16, 339-346 and Gietz, D. et.
al. (1992) Nucleic Acids Res. 20, 1425). In pGBT9-mCaspase-8DED,
Since the tryptophan deficiency resistance gene has been incorporated as a selection marker, the tryptophan-deficient SD
By culturing in a synthetic medium, plasmid pGBT9
-Transformant HF7c into which mCaspase-8DED has been introduced and survived [pGBT9-mCaspase-8
DED] was sorted out. Subsequently, HF7c [pGBT
9-mCaspase-8DED] with cDNA and GAL
200 μg of the plasmid into which the fusion protein of the transcription activation region No. 4 was incorporated was transfected on a large scale. cDN
Since the leucine deficiency resistance gene is incorporated as a selection marker in the plasmid into which the fusion protein of the transcription activation region of A and GAL4 has been incorporated, the plasmid is resistant to tryptophan and leucine deficiency. Furthermore, in the clones in which the polypeptide corresponding to the DED region of mouse caspase-8 derived from pGBT9-mCaspase-8DED interacts with the expression product polypeptide of the cDNA derived from WR19L12a, the clone is linked to each polypeptide. The DNA-binding domain of GAL4 and the transcriptional activation domain are coupled to GAL1 in host HF7c.
Expresses reporter genes HIS3 and lacZ, which are connected downstream of E. coli. The expression of HIS3 makes the cells resistant to histidine deficiency, and the color of the lacZ gene product changes to blue upon addition of X-Gal due to the activity of β-galactosidase. The growing colonies were detected for the activity of β-galactosidase by a filter method in accordance with the protocol of the Matchmaker Two Hybrid System Kit. Plasmids derived from the library of tryptophan, leucine and histidine-resistant colonies positive for β-galactosidase activity were purified according to the protocol of the matchmaker two-hybrid system kit. Next, yeast strain SFY526 having a reporter gene in which GAL1 was connected upstream of another promoter and lacZ was connected downstream of the promoter, and a plasmid derived from a clone selected using HF7c and pGBT9-mCaspa were used.
The cells were transformed with se-8DED, cultured in an SD synthetic medium depleted of tryptophan and leucine, and the β-galactosidase activity of the growing colonies was detected by a filter method to detect the D of mouse caspase-8.
CD encoding a polypeptide that interacts with the ED region
A clone retaining NA was selected.

As described above, 76 positive clones were selected from the 2 × 10 ⁷ transformant library.
Positive clones were classified by restriction enzyme digestion fragment analysis and nucleotide sequence analysis, and they contained two unknown clones (# 68 and # 149). Among them, # 68 was suggested to be a mouse homolog of Gal4 as a result of the search. Another clone, namely
# 149 which did not show any significant identity to any known protein in the GenBank database was selected, and an operation for obtaining the full-length cDNA containing this clone # 149 was performed.

3. To obtain the full length of PCR clone # 149, WR19L1
Using the 2a-derived cDNA library as a template,
Nested PCR (DNA amplified in the first PCR
Rapid Amplification of cDNA Ends (PCR) PCR (hereinafter referred to as "5'-RACE PCR") was performed in combination with the second PCR using the internal sequence of the fragment as a primer.

3.1 Primers A cDNA library derived from WR19L12a was prepared by using the following synthetic oligonucleotide primers, and using KOD dashboard polymerase and γ as DNA polymerases.
Directly amplified using Taq polymerase (all manufactured by Toyobo Co., Ltd.): 5'-taccactaca atggatg-3 '(F0: SEQ ID NO: 5 in the sequence listing) 5'-tgaagatacc ccaccaaacc c-3' (NF0: SEQ ID NO: 6 in the Sequence Listing 5'-gtataaatga aagaaattga gatgg-3 '(R0: SEQ ID NO: 7 in the Sequence Listing) 5'-cacgatgcac agttgaagtg-3' (NR0: SEQ ID NO: 8 in the Sequence Listing) 5'-accttatttg aggcatcttt aag -3 '(R1: SEQ ID NO: 9 in the sequence listing) 5'-catatatatc tggaacctgt gtttgtac -3' (NR1:
5′-ttagcacttg catgctgtg-3 ′ (R2: SEQ ID NO: 11 in the sequence listing) 5′-tcctcctcag tatgtccttc atc-3 ′ (NR2: SEQ ID NO: 12 in the sequence listing) 5′-catgctaaca actttgcata − 3 '(R3: SEQ ID NO: 13 in the Sequence Listing) 5'-gggtctggaa taggtctcag-3' (NR3: SEQ ID NO: 14 in the Sequence Listing) 5'-aggagaaaga gttaagttcag-3 '(F4: SEQ ID NO: 15 in the Sequence Listing) 5 '-gcttcatttg gcccactctc c-3' (NF4: SEQ ID NO: 16 in the sequence listing) 5'-ttcagaatgc gattttcctt-3 '(R5: SEQ ID NO: 17 in the sequence listing) 5'-ttccaagttt ggctgtgcag tg-3' (NR5: sequence listing SEQ ID NO: 18) 5'-gtactgtcag aaacagca-3 '(R6: SEQ ID NO: 19 in the sequence listing) and 5'-acagcactgt ccaacccagca-3' (NR6: SEQ ID NO: 20 in the sequence listing).

3.2 Cloning by PCR PCR1 to PCR7 were sequentially performed using the following combinations of templates and primers: PCR1 (Amplification of inserted fragment with sequence primer in vector)
PCR and nested PCR) Template: WR19L12a-derived cDNA library Primers: F0 and R0, then NF0 and NR0 (for nested PCR) PCR2 (first 5'-RACE and nested P
CR) Template: product of PCR1 Primers: F0 and R1 (5′-RACE PCR
FN0 and NR1 (for nested PCR) PCR3 (second 5'-RACE and nested P
CR) Template: product of PCR2 Primers: F0 and R2 (5'-RACE PCR
FN0 and NR2 (for nested PCR) PCR4 (third 5'-RACE and nested P
CR) Template: product of PCR3 Primers: R0 and R3 (5'-RACE PCR
RN0 and NR3 (for nested PCR) PCR5 (4th 5'-RACE and nested P
CR) Template: product of PCR4 Primer: F4 and F0 (5'-RACE PCR
NF4 and RN0 (for nested PCR) PCR6 (5th 5'-RACE and nested P
CR) Template: product of PCR5 Primers: R0 and R5 (5'-RACE PCR
RN0 and NR5 (for nested PCR) PCR7 (6th 5'-RACE and nested P
CR) Template: product of PCR6 Primers: R0 and R6 (5'-RACE PCR
RN0 and NR6 (for nested PCR) PCR from the first PCR of above PCR1 and PCR2
The 5'-RACE of No. 7 was prepared using a buffer solution for 1 × KOD dish (manufactured by Toyobo Co., Ltd.), 50 pmol of each primer, 1 μg of template DNA, 0.2 mM dNTPs and KO.
The reaction was performed with a reaction solution composition of 2.5 units (D reaction polymerase: 50 μl). The reaction was first heated at 95 ° C. for 1 minute, then at 95 ° C. for 30 seconds, 60- (X-1)
15 seconds at 72 ° C and 1 minute 30 seconds at 72 ° C
Cycle, then 95 ° C. for 30 seconds, 48 ° C. for 15 seconds, 72
25 cycles of a temperature cycle of 1 minute 30 seconds at ° C and finally a temperature of 72 ° C for 5 minutes (X represents the number of cycles).

After completion of the PCR, nested PCR was subsequently performed using the reaction product as a template. That is, 10
0 mM Tris-HCl (pH 8.3), 0.1% Triton X-100, 50 mM potassium chloride, 1.5 mM
A reaction solution (50 μl of reaction solution) having a composition consisting of magnesium chloride, 0.2 mM dNTPs, 50 pmol of each primer, 1 μl of the above 5′-RACE PCR reaction product and 2.5 units of γTaq DNA polymerase was prepared. After heating for 1 minute, 95 ° C for 30 seconds,
10 temperature cycles of 67- (Y-1) ° C. for 15 seconds, 72 ° C. for 1 minute 30 seconds, and then 95 ° C. for 30 seconds, 57
Temperature cycle for 10 seconds at 72 ° C and 1 minute 30 seconds at 72 ° C.
The cycle was lastly kept at 72 ° C. for 5 minutes (Y represents the number of cycles). Some of the reaction products after each nested PCR in PCR1 to PCR6 are
2 to 5 were used as templates for 5'-RACE PCR in PCR7.

From the PCR1 thus obtained, a PC
1 μl of each nested PCR product of R7 is TOP
OTA Cloning Kit (Invitrogen)
Was subcloned into the plasmid vector pCR2.1. The obtained cDNA fragment was ligated using a DNA ligation kit (version 2, manufactured by Takara Shuzo Co., Ltd.), and finally a 6779 bp DNA fragment was obtained.

4. Screening by Colony Hybridization In order to prepare a probe for colony hybridization, using the 6779 bp DNA fragment obtained in 3 above as a template, a synthetic oligonucleotide primer having the following nucleotide sequence: 5′-atggcagcag atgatgacaa tg -3 ′ ( Primer A:
5'-tgcttcattt ggcccactct c-3 '(Primer B:
5'-gactcacggt ccttggagac g-3 '(primer C:
Using two types of PCR (primers A and B and C) using 5′-aaggataagc ggaagtattt tttccaa-3 ′ (primer D: SEQ ID NO: 29 in the sequence listing).
And two types of reaction using D) [PCR conditions:
100 mM Tris-HCl (pH 8.3), 0.1%
Triton X-100, 50 mM potassium chloride, 1.5
mM magnesium chloride, 0.2 mM dNTPs, 50 pmol of each primer, 1 μl of template DNA and γT
A reaction solution (reaction volume: 50 μl) having a composition consisting of 2.5 units of aq DNA polymerase was prepared and heated at 95 ° C. for 1 minute, then at 95 ° C. for 30 seconds, at 57 ° C. for 10 seconds, and at 72 ° C.
30 cycles of a temperature cycle of 1 minute 30 seconds at ℃
Finally, it was kept at 72 ° C. for 5 minutes]. PCR product 2
DNAs (nucleotide numbers 256-1881 and 2011 of SEQ ID NO: 21 in the sequence listing)
-3291) with a DNA labeling kit (Lady to Go D
Radiolabeled with ³² P using a NA labeling kit: Amersham Pharmacia. Using these as probes, a cDNA library prepared from F9 cells (using Escherichia coli strain DH5α (manufactured by Gibco) as a host) was screened by colony hybridization.

As a result, five independent clones were selected. The nucleotide sequence analysis of these clones revealed that they covered the entire length of the coding region while overlapping each other. The fragments were ligated by ligation such that the overlapping portions did not overlap, thereby obtaining a full-length cDNA shown in SEQ ID NO: 21 in the sequence listing. In particular:
Fragment (hereinafter, referred to as "fragment (1)") from the 5 'end to the restriction enzyme AtlII recognition site from a clone having the 5' end to nucleotide number 1131 of SEQ ID NO: 21 in the sequence listing; SEQ ID NO: 21 in the sequence listing From the clone having nucleotide numbers 318 to 1422 of
A restriction enzyme AtlII-XbaI digested fragment (hereinafter, referred to as "fragment (2)"); a clone having nucleotide numbers 1112 to 2748 of SEQ ID NO: 21 in the sequence listing, a restriction enzyme XbaI-PstI digested fragment (hereinafter, referred to as "fragment (2)"); 3) "); a fragment digested with a restriction enzyme PstI-AvrII (hereinafter, referred to as" fragment (4) ") from a clone having nucleotide numbers 2087 to 5712 of SEQ ID NO: 21 in the sequence listing; From the clone having nucleotide numbers 2835 to the 3 ′ end, fragments (hereinafter referred to as “fragment (5)”) from the restriction enzyme AvrII recognition site to the 3 ′ end were prepared, and first, the fragment (3) was converted into a plasmid vector. pBlu
After incorporation into the script, fragment (2) was ligated to the 5 ′ end of the fragment (3), and then fragment (1) was ligated to the 5 ′ end. Furthermore, 3 ′ of fragment (3)
Fragment (4) was ligated to the terminal side, and then fragment (5) was ligated to the 3 ′ terminal side. In this way, pBlues
A full-length cDNA in which each of the above DNA fragments was ligated to the script in the order of (1)-(2)-(3)-(4)-(5) from the 5 'end side was obtained.

The obtained cDNA was obtained from a Kozak consensus sequence (Kozak, M. (1989) J. Cell Biol. 108, 229-).
241) had a long open reading frame (5886 bp) starting at the initiation methionine codon. 18 bp upstream of its initiation methionine codon,
Since a stop codon in the same frame as the open reading frame was present in the guanine- and cytosine-rich region at the 5 'end of the cDNA, the methionine codon was determined as the translation initiation site. This DNA consists of 1962 amino acid residues (SEQ ID NO: 22), the estimated molecular weight 219.1kDa, FLASH (FL ICE- as
sociated h uge protein) encoded a novel polypeptide, designated.

[Example 1] Preparation of 5 'terminal deletion mutant (1) 5' terminal deletion mutant of DNA obtained in Reference Example 1 (from amino acid number 1 of SEQ ID NO: 2 in the sequence listing) 110
7), the following two nucleotide sequences: 5'-ggaattcatg gagagctcat gtgcaatt-3 '(SEQ ID NO: 23 in the sequence listing); and 5'-accggtcgac cctgcatttt gacttctcaa agag-3' An oligonucleotide primer having SEQ ID NO: 24 in the column list was synthesized. Using these as primers and the DNA obtained in Reference Example 1 as a template, PCR was performed using Pfu polymerase (Pfu turbo polymerase: manufactured by Stratagene).

(2) Preparation of Vector 1) The pME18S vector was digested with EcoRI and blunt-ended, and the following nucleotide sequence was obtained: 5′-atggctgact acaaggacga cgatgacaag gaattccgg -3 ′
The plasmid vector pME18S-Flag2 was obtained by inserting the synthetic oligonucleotide having (SEQ ID NO: 25 in the sequence listing) using the above ligation kit. The 3 ′ end of the above oligonucleotide was the pME18S-F
It is designed so that a recognition site for the restriction enzyme EcoRI is generated in lag2.

2) The DNA fragment amplified in the above (1) was recovered, digested with restriction enzymes EcoRI and NotI, and inserted into the plasmid vector pME18S-Flag2 using the ligation kit described above. Competent Escherichia coli pBluesc
rint strain (Stratagene) was transformed and the plasmid vector pME18S-Flag2mFlash was transformed.
E. coli strain E. coli that retains ΔC coli SANK 70
599. This plasmid is extracted and the inserted DNA
The nucleotide sequence of the fragment (SEQ.
A sequencer (Applied Biosystems 310
(Type). The thus obtained transformed E. coli strain E. coli E. coli SANK 70599 was internationally deposited with the Institute of Biotechnology, Institute of Biotechnology on April 20, 1999, and had accession number FERM.
BP-6707 was attached.

E. Plasmid pME18S-F purified from a culture obtained by culturing E. coli SANK 70599 on a large scale using a plasmid purification kit (Qiagen)
lag2mFlashΔC was used for the following transformations. In addition, pME18S-Flag2 was also used as a negative control.

Example 2 Activity Evaluation HF1 cells stably expressing human Fas derived from human HeLa cells and stably expressing human Fas were placed in a 10 mm culture dish for 10% fetal calf serum (hereinafter referred to as "FCS"). Were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5 × 10 ⁶ cells / dish. The cells were collected, transferred to an electrode chamber for electroporation (manufactured by Bio-Rad), and the plasmid pME18S obtained in 1 above was obtained.
-Flag2mFlashΔC (30 μg) and β-
The galactosidase expression vector pJ7-LacZ (1
5 μg), and the gene was transferred by electroporation (300 V, 960 μF) using an electroporation gene transfer apparatus (Gene Pulser: manufactured by Bio-Rad).

Next, the cells were transferred to a φ10 mm culture dish, cultured for 20 hours, dispensed into a 96-well culture plate, and further cultured for 4 hours. In the presence of 10 μM cycloheximide, the anti-human Fas monoclonal antibody CH- 11 (manufactured by Medical Biology Research Institute: final concentration 100)
ng / ml). After culturing for 6 hours, cells were fixed with 0.2% glutaraldehyde and 2% formaldehyde and stained with X-Gal for 0.5-1 hour. Among the β-galactosidase-positive blue cells, the cells having a flat and adherent shape and the cells having a round atrophy were counted as viable cells and apoptotic cells, respectively. This experiment was performed in triplicate for each transfection, in each case at least 300 β-galactosidase positive cells were counted, and the mean and standard error of the percentage of apoptotic blue cells were calculated.

As a result, the plasmid p
Overexpression of ME18S-Flag2mFlashΔC was induced by stimulation of anti-Fas monoclonal antibody CH11 with F11.
In the absence of the as signal, there is no significant effect, but C
It was shown to increase the number of cells in which apoptosis was induced after stimulation with H11 (see FIG. 1, where “Δ” in the figure).
“C” indicates a group in which pME18S-Flag2mFlashΔC was overexpressed, and “control” indicates pME18S-Flag2mFlashΔC.
Flag2 is introduced).

In this experimental system, when stimulating with CH11, a test sample is added to the medium at the same time, and an apoptosis inhibitor is screened by selecting one that suppresses apoptosis. Plasmid pME18S-F
A test sample in which the amount required to inhibit apoptosis when lag2mFlashΔC is overexpressed is greater than that when no overexpression is performed, is a DNA of the present invention.
Can be a novel apoptosis inhibitor that selectively inhibits the function of the polypeptide encoded by Conversely, pME
A test sample that promotes apoptosis induction when 18S-Flag2mFlashΔC is overexpressed and does not promote apoptosis induction when not overexpressed,
It can be a novel apoptosis induction promoter that selectively enhances the function of the polypeptide encoded by the DNA of the present invention.

[Example 3] Antibody The following amino acid sequence: Leu-Ser-Pro-Asn-Ser-Asp-Arg-Asn-Gly-Asp-Ala-His-Ar
g (amino acids 988 to 100 of SEQ ID NO: 2 in the sequence listing)
0), a rabbit was immunized with this peptide according to a conventional method, and an antiserum was obtained to prepare a polyclonal antibody. W
After performing SDS-polyacrylamide electrophoresis of the R19L12a cell extract and performing Western blot using the above-mentioned polyclonal antibody according to a conventional method, it was found that the total molecular weight of FLASH described in Reference Example 1 was slightly over 200 kDa, which is close to the calculated molecular weight. A band was detected at the corresponding position.

[0102]

As described above, the present invention provides a novel DNA useful for searching for an apoptosis inhibitor and an apoptosis induction promoter, and a method for searching for an apoptosis inhibitor or an apoptosis induction promoter using the DNA. . INDUSTRIAL APPLICABILITY The present invention is useful for searching for a therapeutic agent for various diseases caused by abnormal Fas-mediated apoptosis.

[Brief description of the drawings]

FIG. 1. Plasmid pME18S- in HF1 cells.
FIG. 4 is a view showing the effect of overexpression of Flag2mFlashΔC on apoptosis induction.

[Sequence List Free Text] SEQ ID NO: 3: PCR primer for amplifying DNA fragment encoding mouse Caspase-8 DED region SEQ ID NO: 4: Amplifying DNA fragment encoding mouse Caspase-8 DED region Primer for PCR SEQ ID NO: 5: Expression vector plasmid pGAD for yeast
PCR for amplifying the DNA fragment inserted into 424
Primer: SEQ ID NO: 6: yeast expression vector plasmid pGAD
PCR for amplifying the DNA fragment inserted into 424
Primer SEQ ID NO: 7: Expression vector plasmid for yeast pGAD
PCR for amplifying the DNA fragment inserted into 424
Primer: SEQ ID NO: 8: pGAD, an expression vector plasmid for yeast
PCR for amplifying the DNA fragment inserted into 424
SEQ ID NO: 9: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 10: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 11: PCR primer for amplifying a DNA fragment encoding a partial sequence of a mouse apoptosis-related protein SEQ ID NO: 12: PCR primer for amplifying a DNA fragment encoding a partial sequence of a mouse apoptosis-related protein SEQ ID NO: 13: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 14: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 15: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 16: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 17: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 18: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 19 PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 20: PCR primer for amplifying a DNA fragment encoding a partial sequence of mouse apoptosis-related protein SEQ ID NO: 2 : PCR primer for amplifying cDNA encoding a deletion mutant of mouse apoptosis-related protein SEQ ID NO: 24: PCR primer for amplifying cDNA encoding a deletion mutant of mouse apoptosis-related protein SEQ ID NO: 25 : Expression vector pME18S for mammals
SEQ ID NO: 26: PCR primer for amplifying probe DNA fragment for colony hybridization SEQ ID NO: 27: PCR primer for amplifying probe DNA fragment for colony hybridization SEQ ID NO: 28: Colony hybridization No. 29: PCR primer for amplifying probe DNA fragment for colony hybridization

[Sequence List] SEQUENCE LISTING <110> Sankyo Company, Limited <120> Apoptosis-Related Protein <130> 2000047SS <140> <141> <150> JP H11-117103 <151> 1999-04-23 <160> 29 <170> PatentIn Ver. 2.0 <210> 1 <211> 3321 <212> DNA <213> Mus musculus <220> <221> CDS <222> (1) .. (3321) <400> 1 atg gag agc tca tgt gca att cca ctg tat gat agc agt cat aaa gat 48 Met Glu Ser Ser Cys Ala Ile Pro Leu Tyr Asp Ser Ser His Lys Asp 1 5 10 15 gag ttt cca tcc aat tca act ctt tct acc ttc aag agt cag tct gat 96 Glu Phe Pro Ser Asn Ser Thr Leu Ser Thr Phe Lys Ser Gln Ser Asp 20 25 30 cta aat aag gaa aat gaa aaa cca gtt ccc aaa ttt gac aaa tgt tca 144 Leu Asn Lys Glu Asn Glu Lys Pro Val Pro Lys Phe Asp Lys Cys Ser 35 40 45 gaa gca gat tct tgt aag cat ttg tct tta gat gaa tta gaa gaa ggc 192 Glu Ala Asp Ser Cys Lys His Leu Ser Leu Asp Glu Leu Glu Glu Gly 50 55 60 gaa att aga agt gat gat gaa gaa tct gta gca caa aaa cgc ttg gaa 240 Glu Ile Arg Ser Asp Asp Glu Glu Ser Val Ala Gln Lys Arg Leu Glu 65 70 75 80 aag agt gca aga cct aga gtt tct gct gaa gtg cagca g aaa agc 288 Lys Ser Ala Arg Pro Arg Val Ser Ala Glu Val Gln Pro Gly Lys Ser 85 90 95 agc cca gga agc agg agg agc act gtg cat gtg cat aag gac aac ggg 336 Ser Pro Gly Ser Arg Arg Ser Thr Val His Val His Lys Asp Asn Gly 100 105 110 agg act gct gta aaa ctc cct cgg gac aga ctc aca tgg agc aaa aga 384 Arg Thr Ala Val Lys Leu Pro Arg Asp Arg Leu Thr Trp Ser Lys Arg 115 120 125 tca agt gaa tca aga ccc tcc aac acc gaa agg aaa agt aaa aca atg 432 Ser Ser Glu Ser Arg Pro Ser Asn Thr Glu Arg Lys Ser Lys Thr Met 130 135 140 agc atc tcc agc ttg gaa aaa ata ctt ccg ctt atc ctt gta ccc tct 480 Ser Ile Ser Ser Leu Glu Lys Ile Leu Pro Leu Ile Leu Val Pro Ser 145 150 155 160 tcc ctg tgg gag gtt atg cat atg tta cgg ttg cta ggg aaa cat gta 528 Ser Leu Trp Glu Val Met His Met Leu Arg Leu Leu Gly Lys His Val 165 170 175 aga aaa aat tat atg aaa ttc aag ata aaa ttt tca ttg aca caa ttt 576 Arg Lys Asn Tyr Met Lys Phe Lys Ile Lys Phe Ser Leu Thr Gln Phe 180 185 190 cat aga att att gaa tct gca att ttg agt ttc aca tca cta att aaa 624 His Ile Ile Glu Ser Ala Ile Leu Ser Phe Thr Ser Leu Ile Lys 195 200 205 tgc ctt gat ttg tct aag atc tgt aag tca gta agt act tta caa aag 672 Cys Leu Asp Leu Ser Lys Ile Cys Lys Ser Val Ser Thr Leu Gln Lys210 215 220 agt ctt tgt gaa gtt ata gaa tct aac ctt aaa caa gtg aag aag aat 720 Ser Leu Cys Glu Val Ile Glu Ser Asn Leu Lys Gln Val Lys Lys Asn 225 230 235 240 ggc ata gtt gac cgt tta ttt gaa cag caa caa aca gat atg aaa aaa 768 Gly Ile Val Asp Arg Leu Phe Glu Gln Gln Gln Thr Asp Met Lys Lys 245 250 255 aaa ttg tgg aag ttt gta gat gaa caa ctt gat tat ttg ttt gaa aag 816 Lys Leu Trp Lys Phe Val Glu Gln Leu Asp Tyr Leu Phe Glu Lys 260 265 270 ctt aaa aaa atc cta cta aag ttt tgt gat tct gta aac ttt gaa aat 864 Leu Lys Lys Ile Leu Leu Lys Phe Cys Asp Ser Val Asn Phe Glu Asn 275 280 285 gag a agt gaa gga aaa ctt gga aaa aaa tat aaa gag aga acc caa 912 Glu Asn Ser Glu Gly Lys Leu Gly Lys Lys Tyr Lys Glu Arg Thr Gln 290 295 300 cat tca aat tgt cag aag aaa aaa atg gac aac aaa gaa ata cgg aga 960 His Ser Asn Cys Gln Lys Lys Lys Met Asp Asn Lys Glu Ile Arg Arg 305 310 315 320 gaa aaa gtg cta aaa tca gaa aat act gtg aat ttt aaa tct tca ctg 1008 Glu Lys Val Leu Lys Ser Glu Asn Thr Val Asn Ph e Lys Ser Ser Leu 325 330 335 gga tgt gaa aaa tct gaa gaa aaa cat cag gac caa aat aaa acc aat 1056 Gly Cys Glu Lys Ser Glu Glu Lys His Gln Asp Gln Asn Lys Thr Asn 340 345 350 gct agt ata gta aag cat gat gta aaa aga act ttt agc act tgc agt 1104 Ala Ser Ile Val Lys His Asp Val Lys Arg Thr Phe Ser Thr Cys Ser 355 360 365 gat aat aca aag aac gct gaa tgt aaa gag cag ttt ctg gaa aag agc 1152 Asp Asn Thr Lys Asn Ala Glu Cys Lys Glu Gln Phe Leu Glu Lys Ser 370 375 380 tgc ccg agt acc cct agg cca gga aaa gat gaa gga cat act gag gag 1200 Cys Pro Ser Thr Pro Arg Pro Gly Lys Asp Glu Gly His Thr Glu Glu 385 390 395 400 gag gca cag gcg gca cag cat gca agt gct aag tcc gaa cgg agc ttt 1248 Glu Ala Gln Ala Ala Gln His Ala Ser Ala Lys Ser Glu Arg Ser Phe 405 410 415 gag atc ctt act gag cag cag gca tcc agc ctt act ttt aac tta gtg 1296 Glu Ile Leu Thr Glu Gln Gln Ala Ser Ser Leu Thr Phe Asn Leu Val 420 425 430 agt gat gca cag atg ggt gag ata ttt aaa agc ttg cta caa ggt tct 1344 Ser Asp Ala Gln Met Gly Glu Ile Phe Lys Ser Leu Leu Gln Gly Ser 435 440 445 gat ctg ttg gac aca agt ggc act gaa aag gca gag tgg gaa tta aag 1392 Asp Leu Leu Asp Thr Ser Gly Thr Glu Lys Ala Glu Trp Glu Leu Lys 450 455 460 460 cca gag aaa cag ctg ctg gaa agc ctc aag tgt gaa tct gca cca 1440 Thr Pro Glu Lys Gln Leu Leu Glu Ser Leu Lys Cys Glu Ser Ala Pro 465 470 475 475 480 gct tgt gca aca gaa gag ctg gtt gca tgt cca 1488 Ala Cys Ala Thr Glu Glu Leu Val Ser Glu Gly Ala Ser Leu Cys Pro 485 490 495 aaa gtg atc agt gat gat aat tgg tct tta tta tca tct gaa aag ggt 1536 Lys Val Ile Ser Asp Asp Asn Trp Ser Leu Leu Ser Ser Glu Lys Gly 500 505 510 cca tct tta tct tca ggg ctt tca ctg cca gtt cat cct gat gtg tta 1584 Pro Ser Leu Ser Ser Gly Leu Ser Leu Pro Val His Pro Asp Val Leu 515 520 525 gat gaa aat tgt atg ttt gaa gta tct tct aac act gct tta ggt aaa 1632 Asp Glu Asn Cys Met Phe Glu Val Ser Ser Asn Thr Ala Leu Gly Lys 530 535 540 gat aat gta tac agc tca gaa aag agt aag ccc tgc atc tct tcc ata 1680 Asp Asn Val Tyr Ser Ser Glu Lys Ser Lys Pro Cys Ile Ser Ser Ile 545 550 555 560 ctc tta gaa gat ctt gcg gtc tct tta aca gta ccg tca cct ctg aaa 1728 Leu Leu Glu Asp Leu Ala Val Ser Leu Thr Val Pro Ser Pro Leu Lys 565 570 575 tca gat ggc cat ttg agt ttc tta aag cca gaa gtt ttg tca act tca 1776 Ser Asp Gly His Leu Ser Phe Leu Lys Pro Glu Val Leu Ser Thr Ser 580 585 590 act cct gaa gaa gtt att agt gca cat ttt agt gag gat gct ttg ctt 1824 Thr Pro Glu Glu Val Ile Ser Ala His Phe Ser Glu Asp Ala Leu Leu 595 600 605 gag gaa gag gat gca tct gaa cag gac att cat cta gct ctg gag tct 1872 Glu Glu Glu Asp Ala Ser Glu Gln Asp Ile His Leu Ala Leu Glu Ser 610 615 620 gat aac tca agc agt aag tca agc tgt tca tca tgg aca agc cgg tct 1920 Asp Asn Ser Ser Ser Lys Ser Ser Cys Ser Ser Trp Thr Ser Arg Ser 625 630 635 640 640 gtt gct tca ggc ttt cag tac cac cct aat ctt ccc atg cat gct gtc 1968 Val Ala Ser Gly Phe Gln Tyr His Pro Asn Leu Pro Met His Ala Val 645 650 655 ata atg gaa aag tcc aat gat cat ttc att gtg aaa ata c gg cgt gca 2016 Ile Met Glu Lys Ser Asn Asp His Phe Ile Val Lys Ile Arg Arg Ala 660 665 670 aca cca tct acc tcc cct ggc ctt aaa cat ggt gtg gta gct gag gag 2064 Thr Pro Ser Thr Ser Pro Gly Leu Lys His Gly Val Val Ala Glu Glu 675 680 685 tca ttg aca tct ttg cct aga act gga aaa gaa gct ggt gta gca aca 2112 Ser Leu Thr Ser Leu Pro Arg Thr Gly Lys Glu Ala Gly Val Ala Thr 690 695 700 gag aaa gaa cct aac ctg ttt cag agt aca gtt tta aaa cct gtc aag 2160 Glu Lys Glu Pro Asn Leu Phe Gln Ser Thr Val Leu Lys Pro Val Lys 705 710 715 720 gac ttg gaa aat act gat aaa aat att gat aag agt aaa cta act cat 2208 Asp Leu Glu Asn Thr Asp Lys Asn Ile Asp Lys Ser Lys Leu Thr His 725 730 735 gaa gaa cag aac tct ata gta caa aca cag gtt cca gat ata tat gaa 2256 Glu Glu Gln Asn Ser Ile Val Gln Thr Gln Val Pro Asp Ile Tyr Glu 740 745 750 ttt ctt aaa gat gcc tca aat aag gtg gtt cat tgt gat caa gtg gtt 2304 Phe Leu Lys Asp Ala Ser Asn Lys Val Val His Cys Asp Gln Val Val 755 760 765 765 gat gat tgc ttc aag ttg cat caa g ta tgg gaa cca aaa gtt tct gag 2352 Asp Asp Cys Phe Lys Leu His Gln Val Trp Glu Pro Lys Val Ser Glu 770 775 780 aac ctt caa gag ttg cct tca atg gag aaa atc cca cac tct ctt gat 2400 Asn Leu Gln Glu Leu Pro Ser Met Glu Lys Ile Pro His Ser Leu Asp 785 790 795 800 aat cat ctt cct gat aca cac ata gat cta acg aaa gat tca gcc act 2448 Asn His Leu Pro Asp Thr His Ile Asp Leu Thr Lys Asp Ser Ala Thr 805 810 815 gag acc aaa agc ttg ggg gag cta atg gaa gta aca gtt tta aat gtt 2496 Glu Thr Lys Ser Leu Gly Glu Leu Met Glu Val Thr Val Val Leu Asn Val 820 825 830 gat cac ttg gaa tgt tct caa acc aac tta gat caa gat gca gag ata 2544 Asp His Leu Glu Cys Ser Gln Thr Asn Leu Asp Gln Asp Ala Glu Ile 835 840 845 aca tgt agt tct tta cag cct gat act ata gat gct ttt att gat ttg 2592 Thr Cys Ser Ser Leu Gln Pro Asp Thr Ile Asp Ala Phe Ile Asp Leu 850 855 860 aca cat gat gct tca agt gag agt aaa aat gaa ggt agt gaa cct gtg 2640 Thr His Asp Ala Ser Ser Glu Ser Lys Asn Glu Gly Ser Glu Pro Val 865 870 875 880 880 tta gct gt t gaa ggt atg gga tgc cag gta ata tgt ata gat gag gat 2688 Leu Ala Val Glu Gly Met Gly Cys Gln Val Ile Cys Ile Asp Glu Asp 885 890 895 acc aac aaa gaa gga aag atg gga agg gca aac agt cct ttaga 2736 Thr Asn Lys Glu Gly Lys Met Gly Arg Ala Asn Ser Pro Leu Glu Ser 900 905 910 att gtt gaa gaa act tgt att gat ttg acc tca gag tct cct ggc tcc 2784 Ile Val Glu Glu Thr Cys Ile Asp Leu Thr Ser Glu Ser Pro Gly Ser 915 920 925 tgt gaa atc aag aga cat aat tta aag tcg gag cct cca tca aag ttg 2832 Cys Glu Ile Lys Arg His Asn Leu Lys Ser Glu Pro Pro Ser Lys Leu 930 935 940 gat tgt tta gag ttg cct gaa act ctg ggt aat ggt cac aag aag agg 2880 Asp Cys Leu Glu Leu Pro Glu Thr Leu Gly Asn Gly His Lys Lys Arg 945 950 955 960 aaa aac agt cct ggt gtt agt cac tct tct cag aaa aaa caa aga aag 2928 Lys Asn Ser Pro Gly Val Ser His Ser Ser Gln Lys Lys Gln Arg Lys 965 970 975 gac ata gac tta agt agt gaa aag acc cag aga ctt agt ccc aat tct 2976 Asp Ile Asp Leu Ser Ser Glu Lys Thr Gln Arg Leu Ser Pro Asn Ser 980 985 990 gat aga aat ggt gat gct cac aga aag caa gcg agc aag aaa aga gaa 3024 Asp Arg Asn Gly Asp Ala His Arg Lys Gln Ala Ser Lys Lys Arg Glu 995 1000 1005 cct gca gta aat gaa acg tcc ttg gca tca agc cca gag gtg 3072 Pro Ala Val Asn Glu Thr Ser Leu Ser Ser Glu Ala Ser Pro Glu Val 1010 1015 1020 aag ggt tca aca gca gta ctt gct gct tcc cca gca agc ctt tct gca 3120 Lys Gly Ser Thr Ala Val Leu Ala Ala Ser Pro Ala Ser Leu Ser Ala 1025 1030 1035 1040 aaa aat gtt atc aaa aag aag gga gaa att ata gtt tca tgg aca aga 3168 Lys Asn Val Ile Lys Lys Lys Gly Glu Ile Ile Val Ser Trp Thr Arg 1045 1050 1055 aat gat gac cgg gaa att tta ctg gaa tgt cag aaa aga atg ccg tcc 3216 Asn Asp Asp Arg Glu Ile Leu Leu Glu Cys Gln Lys Arg Met Pro Ser 1060 1065 1070 ctg aaa aca ttt act tat tta gct gtc aag ctg aat aaa a Leu Lys Thr Phe Thr Tyr Leu Ala Val Lys Leu Asn Lys Asn Pro Asn 1075 1080 1085 cag gtt tca gag agg ttc cag cag ctg aaa aag ctc ttt gag aag tca 3312 Gln Val Ser Glu Arg Phe Gln G ln Leu Lys Lys Leu Phe Glu Lys Ser 1090 1095 1100 aaa tgc agg 3321 Lys Cys Arg 1105 <210> 2 <211> 1107 <212> PRT <213> Mus musculus <400> 2 Met Glu Ser Ser Cys Ala Ile Pro Leu Tyr Asp Ser Ser His Lys Asp 1 5 10 15 Glu Phe Pro Ser Asn Ser Thr Leu Ser Thr Phe Lys Ser Gln Ser Asp 20 25 30 Leu Asn Lys Glu Asn Glu Lys Pro Val Pro Lys Phe Asp Lys Cys Ser 35 40 45 Glu Ala Asp Ser Cys Lys His Leu Ser Leu Asp Glu Leu Glu Glu Gly 50 55 60 Glu Ile Arg Ser Asp Asp Glu Glu Ser Val Ala Gln Lys Arg Leu Glu 65 70 75 80 Lys Ser Ala Arg Pro Arg Val Ser Ala Glu Val Gln Pro Gly Lys Ser 85 90 95 Ser Pro Gly Ser Arg Arg Ser Thr Val His Val His Lys Asp Asn Gly 100 105 110 Arg Thr Ala Val Lys Leu Pro Arg Asp Arg Leu Thr Trp Ser Lys Arg 115 120 125 Ser Ser Glu Ser Arg Pro Ser Asn Thr Glu Arg Lys Ser Lys Thr Met 130 135 140 Ser Ile Ser Ser Leu Glu Lys Ile Leu Pro Leu Ile Leu Val Pro Ser 145 150 155 160 Ser Leu Trp Glu Val Met His Met Leu Arg Leu Leu Gly Lys His Val 165 170 175 Arg Lys Asn Tyr Met Lys Phe Lys Ile Lys Phe Ser Leu Thr Gln Phe 180 185 190 His Arg Ile Ile Glu Ser Ala Ile Leu Ser Phe Thr Ser Leu Ile Lys 195 200 205 Cys Leu Asp L eu Ser Lys Ile Cys Lys Ser Val Ser Thr Leu Gln Lys 210 215 220 Ser Leu Cys Glu Val Ile Glu Ser Asn Leu Lys Gln Val Lys Lys Asn 225 230 235 240 Gly Ile Val Asp Arg Leu Phe Glu Gln Gln Gln Thr Asp Met Lys Lys 245 250 255 Lys Leu Trp Lys Phe Val Asp Glu Gln Leu Asp Tyr Leu Phe Glu Lys 260 265 270 Leu Lys Lys Ile Leu Leu Lys Phe Cys Asp Ser Val Asn Phe Glu Asn 275 280 285 Glu Asn Ser Glu Gly Lys Leu Gly Lys Lys Tyr Lys Glu Arg Thr Gln 290 295 300 His Ser Asn Cys Gln Lys Lys Lys Met Asp Asn Lys Glu Ile Arg Arg 305 310 315 320 Glu Lys Val Leu Lys Ser Glu Asn Thr Val Asn Phe Lys Ser Ser Leu 325 330 335 Gly Cys Glu Lys Ser Glu Glu Lys His Gln Asp Gln Asn Lys Thr Asn 340 345 350 Ala Ser Ile Val Lys His Asp Val Lys Arg Thr Phe Ser Thr Cys Ser 355 360 365 Asp Asn Thr Lys Asn Ala Glu Cys Lys Glu Gln Phe Leu Glu Lys Ser 370 375 380 Cys Pro Ser Thr Pro Arg Pro Gly Lys Asp Glu Gly His Thr Glu Glu 385 390 395 400 Glu Ala Gln Ala Ala Gln His Ala Ser Ala Lys Ser Glu Arg Ser Phe 405 410 415 Glu Ile Leu T hr Glu Gln Gln Ala Ser Ser Leu Thr Phe Asn Leu Val 420 425 430 Ser Asp Ala Gln Met Gly Glu Ile Phe Lys Ser Leu Leu Gln Gly Ser 435 440 445 Asp Leu Leu Asp Thr Ser Gly Thr Glu Lys Ala Glu Trp Glu Leu Lys 450 455 460 Thr Pro Glu Lys Gln Leu Leu Glu Ser Leu Lys Cys Glu Ser Ala Pro 465 470 475 480 Ala Cys Ala Thr Glu Glu Leu Val Ser Glu Gly Ala Ser Leu Cys Pro 485 490 495 Lys Val Ile Ser Asp Asp Asn Trp Ser Leu Leu Ser Ser Glu Lys Gly 500 505 510 Pro Ser Leu Ser Ser Gly Leu Ser Leu Pro Val His Pro Asp Val Leu 515 520 525 Asp Glu Asn Cys Met Phe Glu Val Ser Ser Asn Thr Ala Leu Gly Lys 530 535 540 Asp Asn Val Tyr Ser Ser Glu Lys Ser Lys Pro Cys Ile Ser Ser Ile 545 550 555 560 Leu Leu Glu Asp Leu Ala Val Ser Leu Thr Val Pro Ser Pro Leu Lys 565 570 575 Ser Asp Gly His Leu Ser Phe Leu Lys Pro Glu Val Leu Ser Thr Ser 580 585 590 Thr Pro Glu Glu Val Ile Ser Ala His Phe Ser Glu Asp Ala Leu Leu 595 600 605 Glu Glu Glu Asp Ala Ser Glu Gln Asp Ile His Leu Ala Leu Glu Ser 610 615 620 Asp Asn Ser SerSer Lys Ser Ser Cys Ser Ser Trp Thr Ser Arg Ser 625 630 635 640 Val Ala Ser Gly Phe Gln Tyr His Pro Asn Leu Pro Met His Ala Val 645 650 655 Ile Met Glu Lys Ser Asn Asp His Phe Ile Val Lys Ile Arg Arg Ala 660 665 670 Thr Pro Ser Thr Ser Pro Gly Leu Lys His Gly Val Val Ala Glu Glu 675 680 685 Ser Leu Thr Ser Leu Pro Arg Thr Gly Lys Glu Ala Gly Val Ala Thr 690 695 700 Glu Lys Glu Pro Asn Leu Phe Gln Ser Thr Val Leu Lys Pro Val Lys 705 710 715 720 Asp Leu Glu Asn Thr Asp Lys Asn Ile Asp Lys Ser Lys Leu Thr His 725 730 735 Glu Glu Gln Asn Ser Ile Val Gln Thr Gln Val Pro Asp Ile Tyr Glu 740 745 750 Phe Leu Lys Asp Ala Ser Asn Lys Val Val His Cys Asp Gln Val Val 755 760 765 Asp Asp Cys Phe Lys Leu His Gln Val Trp Glu Pro Lys Val Ser Glu 770 775 780 Asn Leu Gln Glu Leu Pro Ser Met Glu Lys Ile Pro His Ser Leu Asp 785 790 795 800 Asn His Leu Pro Asp Thr His Ile Asp Leu Thr Lys Asp Ser Ala Thr 805 810 815 Glu Thr Lys Ser Leu Gly Glu Leu Met Glu Val Thr Val Leu Asn Val 820 825 830 Asp His Leu GluCys Ser Gln Thr Asn Leu Asp Gln Asp Ala Glu Ile 835 840 845 Thr Cys Ser Ser Leu Gln Pro Asp Thr Ile Asp Ala Phe Ile Asp Leu 850 855 860 Thr His Asp Ala Ser Ser Glu Ser Lys Asn Glu Gly Ser Glu Pro Val 865 870 875 875 880 Leu Ala Val Glu Gly Met Gly Cys Gln Val Ile Cys Ile Asp Glu Asp 885 890 895 Thr Asn Lys Glu Gly Lys Met Gly Arg Ala Asn Ser Pro Leu Glu Ser 900 905 910 Ile Val Glu Glu Thr Cys Ile Asp Leu Thr Ser Glu Ser Pro Gly Ser 915 920 925 Cys Glu Ile Lys Arg His Asn Leu Lys Ser Glu Pro Pro Ser Lys Leu 930 935 940 Asp Cys Leu Glu Leu Pro Glu Thr Leu Gly Asn Gly His Lys Lys Arg 945 950 955 960 Lys Asn Ser Pro Gly Val Ser His Ser Ser Gln Lys Lys Gln Arg Lys 965 970 975 Asp Ile Asp Leu Ser Ser Glu Lys Thr Gln Arg Leu Ser Pro Asn Ser 980 985 990 Asp Arg Asn Gly Asp Ala His Arg Lys Gln Ala Ser Lys Lys Arg Glu 995 1000 1005 Pro Ala Val Asn Glu Thr Ser Leu Ser Ser Glu Ala Ser Pro Glu Val 1010 1015 1020 Lys Gly Ser Thr Ala Val Leu Ala Ala Ser Pro Ala Ser Leu Ser Ala 1025 1030 1035 1040 Lys As n Val Ile Lys Lys Lys Gly Glu Ile Ile Val Ser Trp Thr Arg 1045 1050 1055 Asn Asp Asp Arg Glu Ile Leu Leu Glu Cys Gln Lys Arg Met Pro Ser 1060 1065 1070 Leu Lys Thr Phe Thr Tyr Leu Ala Val Lys Leu Asn Lys Asn Pro Asn 1075 1080 1085 Gln Val Ser Glu Arg Phe Gln Gln Leu Lys Lys Leu Phe Glu Lys Ser 1090 1095 1100 Lys Cys Arg 1105 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the DED of mouse caspase-8 <400> 3 catggaattc atggatttcc agagttgtat tgtatttat 39 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the DED of mouse caspase-8 <400> 4 catggtcgac ctcatccaaa actgaaggtg 30 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment inserted in a yeast expression vector plasmid pGAD424 <400> 5 taccactaca atggatg 17 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment inserted in a yeast expression vector plasmid pGAD424 <400> 6 tgaagatacc ccaccaaacc c 21 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment inserted in a yeast expression vector plasmid pGAD424 <400> 7 gtataaatga aagaaattga gatgg 25 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment inserted in a yeast expression vector plasmid pGAD424 <400> 8 cacgatgcac agttgaagtg 20 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 9 accttatttg aggcatcttt aag 23 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 10 catatatatc tggaacctgt gtttgtac 28 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 11 ttagcacttg catgctgtg 19 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 12 tcctcctcag tatgtccttc atc 23 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 13 catgctaaca actttgcata 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 14 gggtctggaa taggtctcag 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 15 aggagaaaga gttaagttca g 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 16 gcttcatttg gcccactctc c 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 17 ttcagaatgc gattttcctt 20 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 18 ttccaagttt ggctgtgcag tg 22 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 19 gtactgtcag aaacagca 18 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment encoding the partial sequence of a mouse apoptosis-related protein <400> 20 acagcactgt ccaacccagc a 21 <210> 21 <211> 6763 <212> DNA <213> Mus musculus <220> <221> CDS <222> (256) .. (6141) <400> 21 cgcggggctg ctggggagag gcagcggggt gccgcctggg atggcttacg ctctgggcga 60 agggttgtgc ctggctgggc ctcgaggagc cagctgccgt cctcggggag cgcccctcgg 120 agcgtctcgg ggggcgctgg ggcgagcacc cggagcatcg ccgggtctac agcgcgcccc 180 tcccctcccc cacccgtgtc tcccaagtcc ctactgccga gctctcgatg gcacctatga 240 gaaggatata ggacc atg gca gca gat gat gac aat ggt gat gga acg ggt 291 Met Ala Ala Asp Asp Asp Asn Gly Asp Gly Thr Gly 1 5 10 ttg ttt gat gtc tgt cct gcc tct cca ctt aaa aat aat gat gaa ggc 339 Leu Phe Asp Val Cys Pro Ala Ser Pro Leu Lys Asn Asn Asp Glu Gly 15 20 25 tca ttg gac ata tat gct ggg ttg gac agt gct gtt tct gac agt act 387 Ser Leu Asp Ile Tyr Ala Gly Leu Asp Ser Ala Val Ser Asp Ser Thr 30 35 40 gct aga tcc tgt gtg tca ttc aga aac tgt tta gat ttg tat gaa gag 435 Ala Arg Ser Cys Val Ser Phe Arg Asn Cys Leu Asp Leu Tyr Glu Glu 45 50 55 60 atc ctg act gaa gaa gga act gcg aag gag gca acg tac aat gac ttg 483 Ile Leu Thr Glu Glu Gly Thr Ala Lys Glu Ala Thr Tyr Asn Asp Leu 65 70 75 cag ata gaa tat ggg a aa tgt cag cag caa atg aaa gac ctg atg aaa 531 Gln Ile Glu Tyr Gly Lys Cys Gln Gln Gln Met Lys Asp Leu Met Lys 80 85 90 agg ttt aag gaa ata cag aca cag aac ttg aac tta aaa aat gaa aac 579 Lys Glu Ile Gln Thr Gln Asn Leu Asn Leu Lys Asn Glu Asn 95 100 105 cag tct ctt aag aag aat atc tca gca ctt atc aaa act gcc aga gtg 627 Gln Ser Leu Lys Lys Asn Ile Ser Ala Leu Ile Lys Thr Ala Arg Val 110 115 120 gag ata aac cgt aag gat gaa gag ata aat cat ctt cac caa aga ttg 675 Glu Ile Asn Arg Lys Asp Glu Glu Ile Asn His Leu His Gln Arg Leu 125 130 135 140 tct gag ttt cca cat ttt cga aat aac cat aaa gct gca aga aca aaa 723 Ser Glu Phe Pro His Phe Arg Asn Asn His Lys Ala Ala Arg Thr Lys 145 150 155 gat tca cag tcc aca tct ccc cat ttg gat gat tgt tca aag act gat 771 Asp Ser Gln Ser Thr Ser Pro His Leu Asp Asp Cys Ser Lys Thr Asp 160 165 170 cac gga gtt aaa agt gat gtt cag aaa gat gta cat cct aac act gcg 819 His Gly Val Lys Ser Asp Val Gln Lys Asp Val His Pro Asn Thr Ala 175 180 185 cag cca aac ttg gaa aag gaa gga aaa tcg cat tct gaa gca cag aat 867 Gln Pro Asn Leu Glu Lys Glu Gly Lys Ser His Ser Glu Ala Gln Asn 190 195 200 cct ttg cac ttg tct acg ggt gtt gag aaa cat tgt gcc aat aatgt Pro Leu His Leu Ser Thr Gly Val Glu Lys His Cys Ala Asn Asn Val 205 210 215 220 tgg tca cgt tct cct tac cag gtt gga gaa ggt aac tca aat gag gat 963 Trp Ser Arg Ser Pro Tyr Gln Val Gly Glu Gly Asn Ser Asn Glu Asp 225 230 235 aat agg aga gga agg agt gga act aga cat agc caa tgt agc aga gga 1011 Asn Arg Arg Gly Arg Ser Gly Thr Arg His Ser Gln Cys Ser Arg Gly 240 245 250 act gat aga aca cag aaa gac tta cat agc agc tgt aat gac agt gag 1059 Thr Asp Arg Thr Gln Lys Asp Leu His Ser Ser Cys Asn Asp Ser Glu 255 260 265 cca agg gac aag gag gct aac tcc aga cta caa gga cac cct gag aaa 1107 Pro Arg Asp Lys Glu Ala Asn Ser Arg Leu Gln Gly His Pro Glu Lys 270 275 280 cat ggc aac agt gaa gca agg acg gag agc aaa att tca gag agt aaa 1155 His Gly Asn Ser Glu Ala Arg Thr Glu Ser Lys Ile Ser Glu Ser Lys 285 290 295 300 agc agc act ggt atg gga tat aaa agt gag cgc agt gcc tct tct tgg 1203 Ser Ser Thr Gly Met Gly Tyr Lys Ser Glu Arg Ser Ala Ser Ser Trp 305 310 315 gaa aaa gag act tcc aga ga gaa agg cca cac act cga gtg gaa tct caa 1251 Glu Lys Glu Thr Ser Arg Glu Arg Pro His Thr Arg Val Glu Ser Gln 320 325 330 cat gac aaa aat cta gag aaa caa aat gaa aga tta caa aat atg cac 1299 His Asp Lys Asn Leu Glu Lys Gln Asn Glu Arg Leu Gln Asn Met His 335 340 345 aga aaa gag ctt ccg tct cag gac aaa aca gaa aga aaa gtt gat gtg 1347 Arg Lys Glu Leu Pro Ser Gln Asp Lys Thr Glu Arg Lys Val Asp Val 350 355 360 aag ttt aaa cca gca gga gag gag cag ggg cat cgg gga aga gtg gac 1395 Lys Phe Lys Pro Ala Gly Glu Glu Gln Gly His Arg Gly Arg Val Asp 365 370 375 380 cgg gcg tta cct cct cat ccc aag aat gac gtg aaa cat tac ggc ttc 1443 Arg Ala Leu Pro Pro His Pro Lys Asn Asp Val Lys His Tyr Gly Phe 385 390 395 aat aag tat cat cca gaa gag aga agg gga agg gaa gat tgt aaa aga 1491 Asn Lys Tyr His Pro Glu Glu Arg Arg Gly Arg Glu A sp Cys Lys Arg 400 405 410 gac aga ggg atg aac agt cat ggc ttt caa gat aga aga tgt tca tct 1539 Asp Arg Gly Met Asn Ser His Gly Phe Gln Asp Arg Arg Cys Ser Ser 415 420 425 ttt ctt tca agc aac aga aat agc aaa tac ccg cac tcc aag gaa gtt 1587 Phe Leu Ser Ser Asn Arg Asn Ser Lys Tyr Pro His Ser Lys Glu Val 430 435 440 agt gtt gca cac cag tgg gaa aat act cct ttc aaa gca gaa aga cat 1635 Ser Val Ala His Gln Trp Glu Asn Thr Pro Phe Lys Ala Glu Arg His 445 450 455 460 aga act gag gac agg agg aaa aga gaa cga gaa aac aaa gaa gaa agt 1683 Arg Thr Glu Asp Arg Arg Lys Arg Glu Arg Glu Asn Lys Glu Glu Ser465 470 475 aga cat gtg aaa agt gac aaa aaa tca cct cca gaa cac tta caa agg 1731 Arg His Val Lys Ser Asp Lys Lys Ser Pro Pro Glu His Leu Gln Arg 480 485 490 act cat aaa gac act aag aaa agc act gct gat gga aag aga cag act 1779 Thr His Lys Asp Thr Lys Lys Ser Thr Ala Asp Gly Lys Arg Gln Thr 495 500 505 gag ccc aaa cat ggt aaa ggt gca gtc tct aac agt gag ctt tct aaa 1827 Glu Pro Lys His Gly Lys Gly Al a Val Ser Asn Ser Glu Leu Ser Lys 510 515 520 ggg aca gac agt aaa gaa ggt gca aca aag gtg gag agt ggg cca aat 1875 Gly Thr Asp Ser Lys Glu Gly Ala Thr Lys Val Glu Ser Gly Pro Asn 525 530 535 535 540 gaa gca aaa ggc aag gac tta aag tta agc ttc atg gaa aaa ctg aac 1923 Glu Ala Lys Gly Lys Asp Leu Lys Leu Ser Phe Met Glu Lys Leu Asn 545 550 555 tta act ctt tct cct gct aaa aag cag cct gct tgt ca cca 1971 Leu Thr Leu Ser Pro Ala Lys Lys Gln Pro Ala Cys Gln Asp Asn Pro 560 565 570 cat caa ata act ggt gtt ccc gag ccc agt ggc acg tgt gac tca cgg 2019 His Gln Ile Thr Gly Val Pro Glu Pro Ser Gly Thr Cys Asp Ser Arg 575 580 585 tcc ttg gag acg act gga acg gtg gca tgc ctt cct tct ggc agt gaa 2067 Ser Leu Glu Thr Thr Gly Thr Val Ala Cys Leu Pro Ser Gly Ser Glu 590 595 600 cat aat aga gag gaa acc aaa tca gag tta cca gag cca aag gag gct 2115 His Asn Arg Glu Glu Thr Lys Ser Glu Leu Pro Glu Pro Lys Glu Ala 605 610 615 620 620 ctt ttg gca aca tct caa ctc agg atc agc att cca gaa aac aaa atg 2163 Leu Leu Ala Thr Ser Gln Leu Arg Ile Ser Ile Pro Glu Asn Lys Met 625 630 635 aag gaa gaa aag agg ttg tta ttt aaa tct gtt gag aat act gtg cct 2211 Lys Glu Glu Lys Arg Leu Leu Phe Lys Ser Val Glu Asn Thr Val Pro 640 645 650 tgt gaa ctg ctt gct tgt ggc acg gaa att tcc ctc cca gca cct gta 2259 Cys Glu Leu Leu Ala Cys Gly Thr Glu Ile Ser Leu Pro Ala Pro Val 655 660 665 gaa att gaa caa gca aga tgcca g gtg gaa gtg gaa gag 2307 Glu Ile Glu Gln Ala Arg Cys Leu Leu Gly Ser Val Glu Val Glu Glu 670 675 680 acc tgt ggt ggt gca agg aca gcc gct tct gtg gtg atg cat gtg tta 2355 Thr Cys Gly Ala Arg Thr Ala Ser Val Val Met His Val Leu 685 690 695 700 cca gaa cat gct tct gaa gat gcc agc caa gaa ttg gac acc aaa aga 2403 Pro Glu His Ala Ser Glu Asp Ala Ser Gln Glu Leu Asp Thr Lys Arg 705 710 715 cac gat ggt ata aat gct tgt gct att tct gaa ggt gtg aaa aca aag 2451 His Asp Gly Ile Asn Ala Cys Ala Ile Ser Glu Gly Val Lys Thr Lys 720 725 730 gtg att ctt tca cca aaa gca gct gca gcc agt gag agc cat c tt gca 2499 Val Ile Leu Ser Pro Lys Ala Ala Ala Ala Ser Glu Ser His Leu Ala 735 740 cct ttg gtt gaa gaa cct agc att tca cta gta aac tgt tcg gga gac 2547 Pro Leu Val Glu Glu Pro Ser Ile Ser Leu Val Asn Cys Ser Gly Asp 750 755 760 aat aat cct aaa ctt gaa cct tct ctt gaa gag agg cct ata gtt gag 2595 Asn Asn Pro Lys Leu Glu Pro Ser Leu Glu Glu Arg Pro Ile Val Glu 765 770 775 775 780 act aaa tcc tgt cct ttg gag tct tgt tta cct aaa gag act ttt gta 2643 Thr Lys Ser Cys Pro Leu Glu Ser Cys Leu Pro Lys Glu Thr Phe Val 785 790 795 cct tca cca cag aag act gag ttg att gac cac aaa ata gaa act gga 2691 Pro Ser Pro Gln Lys Thr Glu Leu Ile Asp His Lys Ile Glu Thr Gly 800 805 810 gaa tca aac tca gta tat caa gat gat gat aac tca gtt ttg agt att 2739 Glu Ser Asn Ser Val Tyr Gln Asp Asp Asp Asn Ser Val Leu Ser Ile 815 820 825 gac ttt aat aat ctg aga cct att cca gac ccc atc agc cct ctg aat 2787 Asp Phe Asn Asn Leu Arg Pro Ile Pro Asp Pro Ile Ser Pro Leu Asn 830 835 840 agt cca gtg aga ccg gta gtc aaa gtt t agc atg gag agc tca tgt 2835 Ser Pro Val Arg Pro Val Cys Lys Val Val Ser Met Glu Ser Ser Cys 845 850 855 860 gca att cca ctg tat gat agc agt cat aaa gat gag ttt cca tcc aat 2883 Ala Ile Pro Leu Tyr Asp Ser Ser His Lys Asp Glu Phe Pro Ser Asn 865 870 875 tca act ctt tct acc ttc aag agt cag tct gat cta aat aag gaa aat 2931 Ser Thr Leu Ser Thr Phe Lys Ser Gln Ser Asp Leu Asn Lys Glu Asn 880 885 890 gaa aaa cca gtt ccc aaa ttt gac aaa tgt tca gaa gca gat tct tgt 2979 Glu Lys Pro Val Pro Lys Phe Asp Lys Cys Ser Glu Ala Asp Ser Cys 895 900 905 aag cat ttg tct tta gat gaa tta gaa gaa atc gaa ata agt gat 3027 Lys His Leu Ser Leu Asp Glu Leu Glu Glu Gly Glu Ile Arg Ser Asp 910 915 920 gat gaa gaa tct gta gca caa aaa cgc ttg gaa aag agt gca aga cct 3075 Asp Glu Glu Ser Val Ala Gln Lylu Arg Leu Lys Ser Ala Arg Pro 925 930 935 940 aga gtt tct gct gaa gtg cag cca ggg aaa agc agc cca gga agc agg 3123 Arg Val Ser Ala Glu Val Gln Pro Gly Lys Ser Ser Pro Gly Ser Arg 945 950 955 agg agc act gtg cat gtg cat aag gac aac ggg agg act gct gta aaa 3171 Arg Ser Thr Val His Val His Lys Asp Asn Gly Arg Thr Ala Val Lys 960 965 970 ctc cct cgg gac aga ctc aca tgg agc aaa aga tca agt gaa tca aga 3219 Leu Pro Arg Asp Arg Leu Thr Trp Ser Lys Arg Ser Ser Glu Ser Arg 975 980 985 ccc tcc aac acc gaa agg aaa agt aaa aca atg agc atc tcc agc ttg 3267 Pro Ser Asn Thr Glu Arg Lys Ser Lys Thr Met Ser Ile Ser Ser Leu 990 995 1000 gaa aaa ata ctt ccg ctt atc ctt gta ccc tct tcc ctg tgg gag gtt 3315 Glu Lys Ile Leu Pro Leu Ile Leu Val Pro Ser Ser Leu Trp Glu Val 1005 1010 1015 1020 atg cat atg tta cgg ttg cta ggg aaa cat gta aga aaa aat tat atg 3363 Met His Met Leu Arg Leu Leu Gly Lys His Val Arg Lys Asn Tyr Met 1025 1030 1035 aaa ttc aag ata aaa ttt tca ttg aca caa ttt cat aga att att gaa 3411 Lys Phe Lys Phe Lys Ple Ser Leu Thr Gln Phe His Arg Ile Ile Glu 1040 1045 1050 tct gca att ttg agt ttc aca tca cta att aaa tgc ctt gat ttg tct 3459 Ser Ala Ile Leu Ser Phe Thr Ser Leu Ile Lys Cys Leu Asp Leu Ser 1055 1060 1065 aag atc tgt aag tca gta agt act tta caa aag agt ctt tgt gaa gtt 3507 Lys Ile Cys Lys Ser Val Ser Thr Leu Gln Lys Ser Leu Cys Glu Val 1070 1075 1080 ata gaa tct aac ctt aaa caa gtg aag aag aat ggc ata gtt gac cgt 3555 Ile Glu Ser Asn Leu Lys Gln Val Lys Lys Asn Gly Ile Val Asp Arg 1085 1090 1095 1100 tta ttt gaa cag caa caa aca gat atg aaa aaa aaa ttg tgg aag ttt 3603 Leu Phe Glu Gln Gln Thr Asp Met Lys Lys Lys Leu Trp Lys Phe 1105 1110 1115 gta gat gaa caa ctt gat tat ttg ttt gaa aag ctt aaa aaa atc cta 3651 Val Asp Glu Gln Leu Asp Tyr Leu Phe Glu Lys Leu Lys Lys Ile Leu 1120 1125 aag ttt tgt gat tct gta aac ttt gaa aat gag aat agt gaa gga 3699 Leu Lys Phe Cys Asp Ser Val Asn Phe Glu Asn Glu Asn Ser Glu Gly 1135 1140 1145 aaa ctt gga aaa aaa tat aaa gag aga acc caa cat tca aatgt cag 3747 Lys Leu Gly Lys Lys Tyr Lys Glu Arg Thr Gln His Ser Asn Cys Gln 1150 1155 1160 aag aaa aaa atg gac aac aaa gaa ata cgg aga gaa aaa gtg cta aaa 3795 Lys Lys Lys Met Asp Asn Lys Glu Ile Arg Arg Glu Lys Val Leu Lys 1165 1170 1175 1180 tca gaa aat act gtg aat ttt aaa tct tca ctg gga tgt gaa aaa tct 3843 Ser Glu Asn Thr Val Asn Phe Lys Ser Ser Leu Gly Cys Glu Lys Ser 1185 1190 1195 gaa gaa aaa cat cag gac caa aat aaa acc aat gct agt ata gta aag 3891 Glu Glu Lys His Gln Asp Gln Asn Lys Thr Asn Ala Ser Ile Val Lys 1200 1205 1210 cat gat gta aaa aga act ttt agc act tgc agt gat aat aca aag aac 3939 His Asp Val Lys Arg Thr Phe Ser Thr Cys Ser Asp Asn Thr Lys Asn 1215 1220 1225 gct gaa tgt aaa gag cag ttt ctg gaa aag agc tgc ccg agt acc cct 3987 Ala Glu Cys Lys Glu Gln Phe Leu Glu Ser Cys Pro Ser Thr Pro 1230 1235 1240 agg cca gga aaa gat gaa gga cat act gag gag gag gca cag gcg gca 4035 Arg Pro Gly Lys Asp Glu Gly His Thr Glu Glu Glu Ala Gln Ala Ala 1245 1250 1255 1260 cag cat gca agt gct aag tcc gaa cgg agc ttt gag atc ctt act gag 4083 Gln His Ala Ser Ala Lys Ser Glu Arg Ser Phe Glu Ile Leu Thr Glu 1265 1270 1275 cag cag gca tcc agc ctt act ttt aac tta g tg agt gat gca cag atg 4131 Gln Gln Ala Ser Ser Leu Thr Phe Asn Leu Val Ser Asp Ala Gln Met 1280 1285 1290 ggt gag ata ttt aaa agc ttg cta caa ggt tct gat ctg ttg gac aca 4179 Gly Glu Ile Phe Lys Seru Leu Gln Gly Ser Asp Leu Leu Asp Thr 1295 1300 1305 agt ggc act gaa aag gca gag tgg gaa tta aag act cca gag aaa cag 4227 Ser Gly Thr Glu Lys Ala Glu Trp Glu Leu Lys Thr Pro Glu Lys Gln 1310 1315 1320 ctg ctg gaa agc ctc aag tgt gaa tct gca cca gct tgt gca aca gaa 4275 Leu Leu Glu Ser Leu Lys Cys Glu Ser Ala Pro Ala Cys Ala Thr Glu 1325 1330 1335 1340 gag ctg gtt tca gag ggg gct tct ctt tgt cca gt gat 4323 Glu Leu Val Ser Glu Gly Ala Ser Leu Cys Pro Lys Val Ile Ser Asp 1345 1350 1355 gat aat tgg tct tta tta tca tct gaa aag ggt cca tct tta tct tca 4371 Asp Asn Trp Ser Leu Leu Ser Ser Glu Lys Gly Pro Ser Leu Ser Ser 1360 1365 1370 ggg ctt tca ctg cca gtt cat cct gat gtg tta gat gaa aat tgt atg 4419 Gly Leu Ser Leu Pro Val His Pro Asp Val Leu Asp Glu Asn Cys Met 1375 1380 1385 ttt gaa gta tct tct aac act gct tta ggt aaa gat aat gta tac agc 4467 Phe Glu Val Ser Ser Asn Thr Ala Leu Gly Lys Asp Asn Val Tyr Ser 1390 1395 1400 tca gaa aag agt aag ccc tgc atc tct tcc ata ctc tta gat ctt 4515 Ser Glu Lys Ser Lys Pro Cys Ile Ser Ser Ile Leu Leu Glu Asp Leu 1405 1410 1415 1420 gcg gtc tct tta aca gta ccg tca cct ctg aaa tca gat ggc cat ttg 4563 Ala Val Ser Leu Thr Val Pro Ser Pro Leu Lys Ser Asp Gly His Leu 1425 1430 1435 agt ttc tta aag cca gaa gtt ttg tca act tca act cct gaa gaa gtt 4611 Ser Phe Leu Lys Pro Glu Val Leu Ser Thr Ser Thr Pro Glu Glu Val 1440 1445 1450 att agt gca cat ttt agt gag gat gct ttg ctt gag gaa gag gat gca 4659 Ile Ser Ala His Phe Ser Glu Asp Ala Leu Leu Glu Glu Glu Asp Ala 1455 1460 1465 tct gaa cag gac att cat cta gct ctg gag tct gat aac tca agc agt 4 Ser Gln Asp Ile His Leu Ala Leu Glu Ser Asp Asn Ser Ser Ser 1470 1475 1480 aag tca agc tgt tca tca tgg aca agc cgg tct gtt gct tca ggc ttt 4755 Lys Ser Ser Cys Ser Ser Trp Thr Ser Ar g Ser Val Ala Ser Gly Phe 1485 1490 1495 1500 cag tac cac cct aat ctt ccc atg cat gct gtc ata atg gaa aag tcc 4803 Gln Tyr His Pro Asn Leu Pro Met His Ala Val Ile Met Glu Lys Ser 1505 1510 1515 aat gat cat ttc att gtg aaa ata cgg cgt gca aca cca tct acc tcc 4851 Asn Asp His Phe Ile Val Lys Ile Arg Arg Ala Thr Pro Ser Thr Ser 1520 1525 1530 cct ggc ctt aaa cat ggt gtg gta gct gag gag tca ttg aca tct ttg 4899 Pro Gly Leu Lys His Gly Val Val Ala Glu Glu Ser Leu Thr Ser Leu 1535 1540 1545 cct aga act gga aaa gaa gct ggt gta gca aca gag aaa gaa cct aac 4947 Pro Arg Thr Gly Lys Glu Ala Gly Val Ala Thr Glu Lys Glu Pro Asn 1550 1555 1560 ctg ttt cag agt aca gtt tta aaa cct gtc aag gac ttg gaa aat act 4995 Leu Phe Gln Ser Thr Val Leu Lys Pro Val Lys Asp Leu Glu Asn Thr 1565 1570 1575 1580 gat aaa aat att gat aag agt aaa cta act cat gaa gaa cag aac tct 5043 Asp Lys Asn Ile Asp Lys Ser Lys Leu Thr His Glu Glu Gln Asn Ser 1585 1590 1595 ata gta caa aca cag gtt cca gat ata tat gaa ttt ctt aaa gat g cc 5091 Ile Val Gln Thr Gln Val Pro Asp Ile Tyr Glu Phe Leu Lys Asp Ala 1600 1605 1610 tca aat aag gtg gtt cat tgt gat caa gtg gtt gat gat tgc ttc aag 5139 Ser Asn Lys Val Val His Cys Asp Gln Val Val Asp Asp Cys Phe Lys 1615 1620 1625 ttg cat caa gta tgg gaa cca aaa gtt tct gag aac ctt caa gag ttg 5187 Leu His Gln Val Trp Glu Pro Lys Val Ser Glu Asn Leu Gln Glu Leu 1630 1635 1640 cct tca atg gag aaa atcca cac tct ctt gat aat cat ctt cct gat 5235 Pro Ser Met Glu Lys Ile Pro His Ser Leu Asp Asn His Leu Pro Asp 1645 1650 1655 1660 aca cac ata gat cta acg aaa gat tca gcc act gag acc aaa agc ttg 5283 Thr His Ile Asp Leu Thr Lys Asp Ser Ala Thr Glu Thr Lys Ser Leu 1665 1670 1675 ggg gag cta atg gaa gta aca gtt tta aat gtt gat cac ttg gaa tgt 5331 Gly Glu Leu Met Glu Val Thr Val Leu Asn Val Asp His Leu Glu Cys 1680 1685 1690 tct caa acc aac tta gat caa gat gca gag ata aca tgt agt tct tta 5379 Ser Gln Thr Asn Leu Asp Gln Asp Ala Glu Ile Thr Cys Ser Ser Leu 1695 1700 1705 cag cct gat act ata gat gct ttt att gat ttg aca cat gat gct tca 5427 Gln Pro Asp Thr Ile Asp Ala Phe Ile Asp Leu Thr His Asp Ala Ser 1710 1715 1720 agt gag agt aaa aat gaa ggt agt gaa cct gtg tta gct gtt gaa ggt 5475 Ser Glu Ser Lys Asn Glu Gly Ser Glu Pro Val Leu Ala Val Glu Gly 1725 1730 1735 1740 atg gga tgc cag gta ata tgt ata gat gag gat acc aac aaa gaa gga 5523 Met Gly Cys Gln Val Ile Cys Ile Asp Glu Asp Thr Asn Lys Glu Gly 1745 1750 1755 aag atg gga agg gca aac agt cct tta gaa agt att gtt gaa gaa act 5571 Lys Met Gly Arg Ala Asn Ser Pro Leu Glu Ser Ile Val Glu Glu Thr 1760 1765 1770 tgt att gat ttg acc tca gag tct cct gg tcc tgt gaa atc aag aga 5619 Cys Ile Asp Leu Thr Ser Glu Ser Pro Gly Ser Cys Glu Ile Lys Arg 1775 1780 1785 cat aat tta aag tcg gag cct cca tca aag ttg gat tgt tta gag ttg 5667 His Asn Leu Lys Ser Glu Pro Pro Ser Lys Leu Asp Cys Leu Glu Leu 1790 1795 1800 cct gaa act ctg ggt aat ggt cac aag aag agg aaa aac agt cct ggt 5715 Pro Glu Thr Leu Gly Asn Gly His Lys Lys Arg Lys Asn Ser Pr o Gly 1805 1810 1815 1820 gtt agt cac tct tct cag aaa aaa caa aga aag gac ata gac tta agt 5763 Val Ser His Ser Ser Gln Lys Lys Gln Arg Lys Asp Ile Asp Leu Ser 1825 1830 1835 agt gaa aag acc cag aga ctt agt ccc aat tct gat aga aat ggt gat 5811 Ser Glu Lys Thr Gln Arg Leu Ser Pro Asn Ser Asp Arg Asn Gly Asp 1840 1845 1850 gct cac aga aag caa gcg agc aag aaa aga gaa cct gca gta aat gaa 5859 Ala His Arg Lys G Ala Ser Lys Lys Arg Glu Pro Ala Val Asn Glu 1855 1860 1865 acg tcc ttg tca tca gag gcc agc cca gag gtg aag ggt tca aca gca 5907 Thr Ser Leu Ser Ser Glu Ala Ser Pro Glu Val Lys Gly Ser Thr Ala 1870 1875 1880 gta ctt gct gct tcc cca gca agc ctt tct gca aaa aat gtt atc aaa 5955 Val Leu Ala Ala Ser Pro Ala Ser Leu Ser Ala Lys Asn Val Ile Lys 1885 1890 1895 1900 aag aag gga gaa att ata gtt tca tgg aca aga aat gat gac cgg gaa 6003 Lys Lys Gly Glu Ile Ile Val Ser Trp Thr Arg Asn Asp Asp Arg Glu 1905 1910 1915 att tta ctg gaa tgt cag aaa aga atg ccg tcc ctg aaa aca ttt act 6051 Ile Leu Leu Glu Cys Gln Lys Arg Met Pro Ser Leu Lys Thr Phe Thr 1920 1925 1930 tat tta gct gtc aag ctg aat aaa aat cca aat cag gtt tca gag agg 6099 Tyr Leu Ala Val Lys Leu Asn Lys Asn Pro Asn Gln Val Ser Glu Arg 1935 1940 1945 ttc cag cag ctg aaa aag ctc ttt gag aag tca aaa tgc agg 6141 Phe Gln Gln Leu Lys Lys Leu Phe Glu Lys Ser Lys Cys Arg 1950 1955 1960 tagtagtgga tccatcaagg aggctaatgc tatgagttgc tggatttcat ggtgcctcac 6201 tgataattct tctgcacaga agcttcgatt cccgtgtttt aaatggggaa ctaggtggag 6261 ggtttgtctc cgtttgacag ctagatgatc atctaccttt ttttttttac tttcctggtc 6321 attgtgcttg gcgtattcag atcaacagtc tgattcactt acctgttatt ggtcagaatg 6381 tttttaatca ttaaaagttg ttatcaaatg aaagactaaa agcaaccttg atgtaatact 6441 gaaaccttag ttttgatatt ggatgaaata aattctttaa aaaacaattt tgtagcattt 6501 taaatgtaaa aactatttag ttcacctttc tattcatttt tgtttttata attgcctcat 6561 tttggaaaat tgtgaaataa tttaaatagt atatttgtat atatgtacaa ttattgtgat 6621 gtaaaatact gtaagtgtat gtgtctacgt ctatgtatgc acaccaatgg ttttgctagc 6681 caaggttttt tt tatctata ttaaatattt tgtataaact ttcctttcag ttagtcatgt 6741 ataataaaaa catgaagtcc tg 6763 <210> 22 <211> 1962 <212> PRT <213> Mus musculus <400> 22 Met Ala Ala Asp Asp Asp Asp Asn Gly Asp Gly Thr Gly Leu Phe Asp Val 1 5 10 15 Cys Pro Ala Ser Pro Leu Lys Asn Asn Asp Glu Gly Ser Leu Asp Ile 20 25 30 Tyr Ala Gly Leu Asp Ser Ala Val Ser Asp Ser Thr Ala Arg Ser Cys 35 40 45 Val Ser Phe Arg Asn Cys Leu Asp Leu Tyr Glu Glu Ile Leu Thr Glu 50 55 60 Glu Gly Thr Ala Lys Glu Ala Thr Tyr Asn Asp Leu Gln Ile Glu Tyr 65 70 75 80 Gly Lys Cys Gln Gln Gln Met Lys Asp Leu Met Lys Arg Phe Lys Glu 85 90 95 Ile Gln Thr Gln Asn Leu Asn Leu Lys Asn Glu Asn Gln Ser Leu Lys 100 105 110 Lys Asn Ile Ser Ala Leu Ile Lys Thr Ala Arg Val Glu Ile Asn Arg 115 120 125 Lys Asp Glu Glu Ile Asn His Leu His Gln Arg Leu Ser Glu Phe Pro 130 135 140 His Phe Arg Asn Asn His Lys Ala Ala Arg Thr Lys Asp Ser Gln Ser 145 150 155 160 Thr Ser Pro His Leu Asp Asp Cys Ser Lys Thr Asp His Gly Val Lys 165 170 175 Ser Asp Val Gln Lys Asp Val His Pro Asn Thr Ala Gln Pro Asn Leu 180 185 190 Glu Lys Glu Gly Gly Lys Ser His Ser Glu Ala Gln Asn Pro Leu His Leu 195 200 205 Ser Thr Gly V al Glu Lys His Cys Ala Asn Asn Val Trp Ser Arg Ser 210 215 220 Pro Tyr Gln Val Gly Glu Gly Asn Ser Asn Glu Asp Asn Arg Arg Gly 225 230 235 240 Arg Ser Gly Thr Arg His Ser Gln Cys Ser Arg Gly Thr Asp Arg Thr 245 250 255 Gln Lys Asp Leu His Ser Ser Cys Asn Asp Ser Glu Pro Arg Asp Lys 260 265 270 Glu Ala Asn Ser Arg Leu Gln Gly His Pro Glu Lys His Gly Asn Ser 275 280 285 Glu Ala Arg Thr Glu Ser Lys Ile Ser Glu Ser Lys Ser Ser Thr Gly 290 295 300 Met Gly Tyr Lys Ser Glu Arg Ser Ala Ser Ser Trp Glu Lys Glu Thr 305 310 315 320 Ser Arg Glu Arg Pro His Thr Arg Val Glu Ser Gln His Asp Lys Asn 325 330 335 Leu Glu Lys Gln Asn Glu Arg Leu Gln Asn Met His Arg Lys Glu Leu 340 345 350 Pro Ser Gln Asp Lys Thr Glu Arg Lys Val Asp Val Lys Phe Lys Pro 355 360 365 Ala Gly Glu Glu Gln Gly His Arg Gly Arg Val Asp Arg Ala Leu Pro 370 375 380 Pro His Pro Lys Asn Asp Val Lys His Tyr Gly Phe Asn Lys Tyr His 385 390 395 400 400 Pro Glu Glu Arg Arg Gly Arg Glu Asp Cys Lys Arg Asp Arg Gly Met 405 410 415 Asn Ser HisGly Phe Gln Asp Arg Arg Cys Ser Ser Phe Leu Ser Ser 420 425 430 Asn Arg Asn Ser Lys Tyr Pro His Ser Lys Glu Val Ser Val Ala His 435 440 445 Gln Trp Glu Asn Thr Pro Phe Lys Ala Glu Arg His Arg Thr Glu Asp 450 455 460 Arg Arg Lys Arg Glu Arg Glu Asn Lys Glu Glu Ser Arg His Val Lys 465 470 475 480 Ser Asp Lys Lys Ser Pro Pro Glu His Leu Gln Arg Thr His Lys Asp 485 490 495 Thr Lys Lys Ser Thr Ala Asp Gly Lys Arg Gln Thr Glu Pro Lys His 500 505 510 Gly Lys Gly Ala Val Ser Asn Ser Glu Leu Ser Lys Gly Thr Asp Ser 515 520 525 Lys Glu Gly Ala Thr Lys Val Glu Ser Gly Pro Asn Glu Ala Lys Gly 530 535 540 Lys Asp Leu Lys Leu Ser Phe Met Glu Lys Leu Asn Leu Thr Leu Ser 545 550 555 560 Pro Ala Lys Lys Gln Pro Ala Cys Gln Asp Asn Pro His Gln Ile Thr 565 570 575 Gly Val Pro Glu Pro Ser Gly Thr Cys Asp Ser Arg Ser Leu Glu Thr 580 585 590 Thr Gly Thr Val Ala Cys Leu Pro Ser Gly Ser Glu His Asn Arg Glu 595 600 605 Glu Thr Lys Ser Glu Leu Pro Glu Pro Lys Glu Ala Leu Leu Ala Thr 610 615 620 Ser Gln Leu ArgIle Ser Ile Pro Glu Asn Lys Met Lys Glu Glu Lys 625 630 635 640 Arg Leu Leu Phe Lys Ser Val Glu Asn Thr Val Pro Cys Glu Leu Leu 645 650 655 Ala Cys Gly Thr Glu Ile Ser Leu Pro Ala Pro Val Glu Ile Glu Gln 660 665 670 Ala Arg Cys Leu Leu Gly Ser Val Glu Val Glu Glu Thr Cys Gly Gly 675 680 685 Ala Arg Thr Ala Ala Ser Val Val Met His Val Leu Pro Glu His Ala 690 695 700 Ser Glu Asp Ala Ser Gln Glu Leu Asp Thr Lys Arg His Asp Gly Ile 705 710 715 715 720 Asn Ala Cys Ala Ile Ser Glu Gly Val Lys Thr Lys Val Ile Leu Ser 725 730 735 Pro Lys Ala Ala Ala Ala Ala Ser Glu Ser His Leu Ala Pro Leu Val Glu 740 745 750 Glu Pro Ser Ile Ser Leu Val Asn Cys Ser Gly Asp Asn Asn Pro Lys 755 760 765 Leu Glu Pro Ser Leu Glu Glu Arg Pro Ile Val Glu Thr Lys Ser Cys 770 775 780 780 Pro Leu Glu Ser Cys Leu Pro Lys Glu Thr Phe Val Pro Ser Pro Gln 785 790 795 800 Lys Thr Glu Leu Ile Asp His Lys Ile Glu Thr Gly Glu Ser Asn Ser 805 810 815 Val Tyr Gln Asp Asp Asp Asn Ser Val Leu Ser Ile Asp Phe Asn Asn 820 825 830 Leu Arg Pro Ile Pro Asp Pro Ile Ser Pro Leu Asn Ser Pro Val Arg 835 840 845 Pro Val Cys Lys Val Val Ser Met Glu Ser Ser Cys Ala Ile Pro Leu 850 855 860 Tyr Asp Ser Ser His Lys Asp Glu Phe Pro Ser Asn Ser Thr Leu Ser 865 870 875 880 Thr Phe Lys Ser Gln Ser Asp Leu Asn Lys Glu Asn Glu Lys Pro Val 885 890 895 Pro Lys Phe Asp Lys Cys Ser Glu Ala Asp Ser Cys Lys His Leu Ser 900 905 910 Leu Asp Glu Leu Glu Glu Gly Glu Ile Arg Ser Asp Asp Glu Glu Ser 915 920 925 925 Val Ala Gln Lys Arg Leu Glu Lys Ser Ala Arg Pro Arg Val Ser Ala 930 935 940 Glu Val Gln Pro Gly Lys Ser Ser Pro Gly Ser Arg Arg Ser Thr Val 945 950 955 960 His Val His Lys Asp Asn Gly Arg Thr Ala Val Lys Leu Pro Arg Asp 965 970 975 Arg Leu Thr Trp Ser Lys Arg Ser Ser Glu Ser Arg Pro Ser Asn Thr 980 985 990 Glu Arg Lys Ser Lys Thr Met Ser Ile Ser Ser Leu Glu Lys Ile Leu 995 1000 1005 Pro Leu Ile Leu Val Pro Ser Ser Leu Trp Glu Val Met His Met Leu 1010 1015 1020 Arg Leu Leu Gly Lys His Val Arg Lys Asn Tyr Met Lys Phe Lys Ile 025 1030 1035 1040 Lys Phe Ser Leu Thr Gln Phe His Arg Ile Ile Glu Ser Ala Ile Leu 1045 1050 1055 Ser Phe Thr Ser Leu Ile Lys Cys Leu Asp Leu Ser Lys Ile Cys Lys 1060 1065 1070 Ser Val Ser Thr Leu Gln Lys Ser Leu Cys Glu Val Ile Glu Ser Asn 1075 1080 1085 Leu Lys Gln Val Lys Lys Asn Gly Ile Val Asp Arg Leu Phe Glu Gln 1090 1095 1100 Gln Gln Thr Asp Met Lys Lys Lys Leu Trp Lys Phe Val Asp Glu Gln 105 1110 1115 1120 Leu Asp Tyr Leu Phe Glu Lys Leu Lys Lys Ile Leu Leu Lys Phe Cys 1125 1130 1135 Asp Ser Val Asn Phe Glu Asn Glu Asn Ser Glu Gly Lys Leu Gly Lys 1140 1145 1150 Lys Tyr Lys Glu Arg Thr Gln His Ser Asn Cys Gln Lys Lys Lys Met 1155 1160 1165 Asp Asn Lys Glu Ile Arg Arg Glu Lys Val Leu Lys Ser Glu Asn Thr 1170 1175 1180 Val Asn Phe Lys Ser Ser Leu Gly Cys Glu Lys Ser Glu Glu Lys His 185 1190 1195 1200 Gln Asp Gln Asn Lys Thr Asn Ala Ser Ile Val Lys His Asp Val Lys 1205 1210 1215 Arg Thr Phe Ser Thr Cys Ser Asp Asn Thr Lys Asn Ala Glu Cys Lys 1220 1225 1230 Glu Gln Phe Leu Glu Lys Ser Cys Pro Ser Thr Pro Arg Pro Gly Lys 1235 1240 1245 Asp Glu Gly His Thr Glu Glu Glu Ala Gln Ala Ala Gln His Ala Ser 1250 1255 1260 Ala Lys Ser Glu Arg Ser Phe Glu Ile Leu Thr Glu Gln Gln Ala Ser 265 1270 1275 1280 Ser Leu Thr Phe Asn Leu Val Ser Asp Ala Gln Met Gly Glu Ile Phe 1285 1290 1295 Lys Ser Leu Leu Gln Gly Ser Asp Leu Leu Asp Thr Ser Gly Thr Glu 1300 1305 1310 Lys Ala Glu Trp Glu Leu Lys Thr Pro Glu Lys Gln Leu Leu Glu Ser 1315 1320 1325 Leu Lys Cys Glu Ser Ala Pro Ala Cys Ala Thr Glu Glu Leu Val Ser 1330 1335 1340 Glu Gly Ala Ser Leu Cys Pro Lys Val Ile Ser Asp Asp Asn Trp Ser 345 1350 1355 1360 Leu Leu Seru Ser Glu Lys Gly Pro Ser Leu Ser Ser Gly Leu Ser Leu 1365 1370 1375 Pro Val His Pro Asp Val Leu Asp Glu Asn Cys Met Phe Glu Val Ser 1380 1385 1390 Ser Asn Thr Ala Leu Gly Lys Asp Asn Val Tyr Ser Ser Glu Lys Ser 1395 1400 1405 Lys Pro Cys Ile Ser Ser Ile Leu Leu Glu Asp Leu Ala Val Ser Leu 1410 1415 1420 Thr Val Pro Ser Pro Leu Lys Ser Asp Gly His Leu Ser Phe Leu Lys 425 1430 1435 1440 Pro Glu Val Leu Ser Thr Ser Thr Pro Glu Glu Val Ile Ser Ala His 1445 1450 1455 Phe Ser Glu Asp Ala Leu Leu Glu Glu Glu Asp Ala Ser Glu Gln Asp 1460 1465 1470 Ile His Leu Ala Leu Glu Ser Asp Asn Ser Ser Ser Lys Ser Ser Cys 1475 1480 1485 Ser Ser Trp Thr Ser Arg Ser Val Ala Ser Gly Phe Gln Tyr His Pro 1490 1495 1500 Asn Leu Pro Met His Ala Val Ile Met Glu Lys Ser Asn Asp His Phe 505 1510 1515 1520 Ile Val Lys Ile Arg Arg Ala Thr Pro Ser Thr Ser Pro Gly Leu Lys 1525 1530 1535 His Gly Val Val Ala Glu Glu Ser Leu Thr Ser Leu Pro Arg Thr Gly 1540 1545 1550 Lys Glu Ala Gly Val Ala Thr Glu Lys Glu Pro Asn Leu Phe Gln Ser 1555 1560 1565 Thr Val Leu Lys Pro Val Lys Asp Leu Glu Asn Thr Asp Lys Asn Ile 1570 1575 1580 Asp Lys Ser Lys Leu Thr His Glu Glu Gln Asn Ser Ile Val Gln Thr 585 1590 1595 1600 Gln Val Pro Asp Ile Tyr Glu Phe Leu Lys Asp Ala Ser Asn Lys Val 1605 1610 1615 Val His Cys Asp Gln Val Val Asp Asp Cys Phe Lys Leu His Gln Val 1620 1625 1630 Trp Glu Pro Lys Val Ser Glu Asn Leu Gln Glu Leu Pro Ser Met Glu 1635 1640 1645 Lys Ile Pro His Ser Leu Asp Asn His Leu Pro Asp Thr His Ile Asp 1650 1655 1660 Leu Thr Lys Asp Ser Ala Thr Glu Thr Lys Ser Leu Gly Glu Leu Met 665 1670 1675 1680 Glu Val Thr Val Le Le Asn Val Asp His Leu Glu Cys Ser Gln Thr Asn 1685 1690 1695 Leu Asp Gln Asp Ala Glu Ile Thr Cys Ser Ser Leu Gln Pro Asp Thr 1700 1705 1710 Ile Asp Ala Phe Ile Asp Leu Thr His Asp Ala Ser Ser Glu Ser Lys 1715 1720 1725 Asn Glu Gly Ser Glu Pro Val Leu Ala Val Glu Gly Met Gly Cys Gln 1730 1735 1740 Val Ile Cys Ile Asp Glu Asp Thr Asn Lys Glu Gly Lys Met Gly Arg 745 1750 1755 1760 Ala Asn Ser Pro Leu Glu Ser Ile Val Glu Glu Thr Cys Ile Asp Leu 1765 1770 1775 Thr Ser Glu Ser Pro Gly Ser Cys Glu Ile Lys Arg His Asn Leu Lys 1780 1785 1790 Ser Glu Pro Pro Ser Lys Leu Asp Cys Leu Glu Leu Pro Glu Thr Leu 1795 1800 1805 Gly Asn Gly His Lys Lys Arg Lys Asn Ser Pro Gly Val Ser His Ser 1810 1815 1820 Ser Gln Lys Lys Gln Arg Lys Asp Ile Asp Leu Ser Ser Glu Lys Thr 825 1830 1835 1840 Gln Arg Leu Ser Pro Asn Ser Asp Arg Asn Gly Asp Ala His Arg Lys 1845 1850 1855 Gln Ala Ser Lys Lys Arg Glu Pro Ala Val Asn Glu Thr Ser Leu Ser 1860 1865 1870 Ser Glu Ala Ser Pro Glu Val Lys Gly Ser Thr Ala Val Leu Ala Ala 1875 1880 1885 Ser Pro Ala Ser Leu Ser Ala Lys Asn Val Ile Lys Lys Lys Gly Glu 1890 1895 1900 Ile Ile Val Ser Trp Thr Arg Asn Asp Asp Arg Glu Ile Leu Leu Glu 905 1910 1915 1920 Cys Gln Lys Arg Met Pro Ser Leu Lys Thr Phe Thr Tyr Leu Ala Val 1925 1930 1935 Lys Leu Asn Lys Asn Pro Asn Gln Val Ser Glu Arg Phe Gln Gln Leu 1940 1945 1950 Lys Lys Leu Phe Glu Lys Ser Lys Cys Arg 1955 1960 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a cDNA encoding the deletion-mutant of a mouse apoptosis-related protein <400> 23 ggaattcatg gagagctcat gtgcaatt 28 <210> 24 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a cDNA encoding the deletion-mutant of a mouse apoptosis-related protein <400> 24 accggtcgac cctgcatttt gacttctcaa agag 34 <210> 25 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Oligonucleotide linker which is inserted in a mammarian expression vector pME18S <400> 25 atggctgact acaaggacga cgatgacaag gaattccgg 39 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment used as a probe for colony-hybridization <400> 26 atggcagcag atgatgacaa tg 22 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment used as a probe for colony-hybridization <400> 27 tgcttcattt ggcccactct c 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment used as a probe for colony-hybridization <400> 28 gactcacggt ccttggagac g 21 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer to amplify a DNA fragment used as a probe for colony-hybridization <400> 29 aaggataagc ggaagtattt tttccaa 27

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12N 1/21 C12N 1/21 5/10 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33 / 50 Z 33/50 33/53 D 33/53 C12P 21/02 C // C12P 21/02 21/08 21/08 C12N 5/00 A (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (15)

[Claims] 1. A molecule comprising an amino acid sequence represented by amino acid Nos. 1 to 1107 of SEQ ID No. 2 in the sequence listing,
a polypeptide characterized by having an activity of enhancing cell-mediated apoptosis induction through as, or an amino acid in which one or more amino acids of the amino acid sequence has been deleted, inserted or substituted in the molecule A DNA encoding a polypeptide, comprising a sequence, and having an activity of enhancing Fas-mediated induction of cell apoptosis. 2. A molecule comprising an amino acid sequence represented by amino acid Nos. 1 to 1107 of SEQ ID No. 2 in the sequence listing,
The DNA encoding the polypeptide according to claim 1, which has an activity of enhancing apoptosis induction of cells through as. 3. A DNA encoding a polypeptide having an amino acid sequence represented by amino acid numbers 1 to 1107 of SEQ ID NO: 2 in the sequence listing. 4. The transformed E. coli E. coli. coli SANK
DNA inserted into a plasmid carried by S. 70599 (FERM BP-6707). 5. A DNA comprising, in a molecule, a nucleotide sequence represented by nucleotide numbers 1 to 3321 of SEQ ID NO: 1 in the sequence listing. 6. A nucleic acid comprising a nucleotide sequence encoding a polypeptide that hybridizes with the DNA of claim 5 under stringent conditions and has an activity to enhance Fas-mediated apoptosis induction of cells. DNA. 7. A polypeptide having an amino acid sequence encoded by the DNA according to any one of claims 1 to 6. 8. A recombinant DNA vector comprising the DNA according to any one of claims 1 to 6. 9. An expression vector,
The recombinant DNA vector according to claim 8. 10. The transformed E. coli E. coli. coli SAN
The recombinant DNA vector according to claim 8 or 9, wherein the recombinant DNA vector is held by K70599 (FERM BP-6707). A host cell transformed with the recombinant DNA vector according to any one of claims 8 to 10. 12. A transformed E. coli E. coli. coli SAN
The host cell according to claim 11, which is K70599 (FERM BP-6707). 13. A method for testing the effect of a sample of a compound or composition as an apoptosis inhibitor, comprising the following steps 1) to 3): 1) The method of claim 1 to 6 An animal cell group transformed with any one of the DNAs and expressing Fas (hereinafter referred to as "group A"), and not transformed with the DNA and expressing Fas Each of the animal cell groups (hereinafter referred to as “Group B”) was apoptosis-inducing anti-F
culturing in the presence of a monoclonal antibody and a test sample; 2) detecting and comparing the activity of the test sample to inhibit apoptosis-inducing activity of the antibody in each of the above cell groups; 3) the result of 2) above , A test sample having a higher apoptosis induction inhibitory activity in Group A than an apoptosis induction inhibitory activity in Group B is selected. 14. A method for testing the effect of a compound or composition sample as an apoptosis induction promoter, comprising the following steps 1) to 3): 1) 1) to 1). A group of animal cells transformed with the DNA of any one of the above and expressing Fas (hereinafter referred to as “group A”); and a group of animals not transformed with the DNA and expressing Fas Animal cell group (hereinafter, referred to as “group B”) was apoptosis-inducing anti-F
culturing in the presence of a monoclonal antibody and a test sample; 2) detecting and comparing the activity of the test sample to promote apoptosis-inducing activity in each of the cell groups by the antibody; 3) the result of 2) above In addition, a test sample having a higher activity for promoting apoptosis induction in Group A than a activity for promoting apoptosis induction in Group B is selected. 15. An antibody that specifically binds to the polypeptide according to claim 7.