JP2000226400A - Protein having apoptosis controlling ability, its gene and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein which is a protein capable of binding to a specific caspase without having a death effector domain and capable of controlling the apoptosis. SOLUTION: This protein is capable of binding to caspase 8 without any death effctor domain, preferably a protein represented by the formula (Met is methionine; Ala is alanine; Asp is aspartic acid; Asn is asparagine; Gly is glycine; Thr is threonine; Ser is serine; Leu is leucine; Phe is phenylalanine; Val is valine; Lys is lysine; Glu is glutamic acid; Ile is isoleucine; Trp is tryptophan; Arg is arginine). The protein is obtained by culturing a transformant or a transductant containing a recombinant plasmid containing a gene (e.g. aip-1 gene) encoding the protein and integrated therein, expressing the protein and collecting the above protein from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel protein that controls apoptosis, and a gene encoding the protein. The present invention also relates to a series of uses of these proteins and genes. Furthermore, the present invention relates to an antibody against the protein and its use.

[0002]

2. Description of the Related Art Apoptosis is a form of cell death and is known to exhibit characteristic forms such as cytoplasmic condensation, nuclear fragmentation, and chromosomal DNA fragmentation. Apoptosis plays a very important role in living organisms,
It has been shown that this abnormality is involved in many pathological conditions including cancer and autoimmune diseases. In recent years, molecular mechanisms such as apoptosis control, ie, induction or inhibition, have been actively studied, and some of them have been revealed.

[0003] Apoptosis is induced in response to various stimuli, one of which is known to be Fas-dependent apoptosis. Fas is a single transmembrane receptor present on the cell membrane surface and is classified into tumor necrosis factor / nerve growth factor receptors. Fas has its ligand (Fas
The binding of an antibody to ligand (Lig) or Fas (anti-Fas antibody) conveys a stimulus to induce cell death. This cell death induction mechanism is considered as follows. As described above, when Fas binds to Fas ligand or anti-Fas antibody, Fas will form a trimer. Next, the adapter molecule in the cell is recognized by recognizing this higher-order structure.
FADD joins. Further, Caspase-8 known as a cell death-related protease binds to this FADD. Ca
Although spase-8 is usually inactive, it is converted to active form by forming a complex with Fas / FADD. Caspase-8 converted to the active form activates various Caspase family proteases in cells. Caspase family proteases destroy cytoskeletal proteins such as actin and fodrin, and cell cycle regulatory proteins such as Rb.
Complete apoptosis by activating DNase such as D. In addition, apoptosis has been found to be induced by certain oncogenes and tumor suppressor genes, and furthermore, irradiation and administration of anticancer drugs.

[0004] In recent years, two important discoveries have been made in the area of cancer research. One is that the administration of anticancer drugs to cancer cells induces apoptosis, while the administration of Fas neutralizing antibody suppresses this apoptosis (Nature Medicine Vol. 2, pp. 574-577 (1996)). . This result indicates that apoptosis by anticancer drugs is via a Fas-dependent pathway. Conversely, it can be said that enhancing the signaling of the Fas pathway can enhance the effects of anticancer drugs. Another finding is that many cancer cells express Fas ligand (Science 274, 1363-1366 (1996)). In other words, even if the immunocompetent cells approach to eliminate the cancer cells, apoptosis is induced in the immune cells by reacting the Fas ligand of the cancer cells with the Fas of the immune cells. That is, cancer cells are thought to have escaped the immune surveillance mechanism by using Fas-dependent apoptosis skillfully. However, on the other hand, cancer cells have both Fas and Fas
The ligand is also expressed. So why do cancer cells themselves not undergo apoptosis? This is in cancer cells
It is thought that signaling of Fas-dependent apoptosis is inhibited. Little is known about the molecules involved in the inhibition of Fas-dependent apoptosis. However, as data supporting this hypothesis, Fas
Dependent apoptosis has been shown to be enhanced by protein synthesis inhibitors. As a protein that inhibits apoptosis, FLAME-1 is known (J. Biol. Che.
m., Vol.272, No.30, p18542-18545 (1997)). FLAME
-1 is a protein that has a death effector domain and interacts with a Caspase family protease such as Caspase-8 to exert apoptosis inhibitory ability.

[0005]

An object of the present invention is to clarify a gene encoding a novel protein that regulates apoptosis, provide a method for cloning the gene, provide a novel protein encoded by the gene, and further provide the gene. To provide an antisense DNA and an antisense RNA, a nucleic acid probe obtained based on the gene, a method for detecting the gene using the nucleic acid probe, and a novel apoptosis controlling ability using the gene. An object of the present invention is to provide a method for producing a protein, an antibody of a novel protein having an ability to control apoptosis, and a method for detecting a novel protein having an ability to control apoptosis using the antibody.

[0006]

Means for Solving the Problems The inventor of the present invention first employs Caspas
In order to find a protein that regulates Fas-dependent apoptosis in proteins that bind to e-8, we have conducted intensive studies and completed the present invention including a novel protein that has apoptosis-regulating ability and does not have a death effector domain. . The present inventors searched and collected a protein fragment that binds to the death effector domain corresponding to the N-terminal partial amino acid sequence of Caspase-8 protein by a yeast two-hybrid screening method. Further, by using a part of the gene encoding the protein fragment as a probe, the gene encoding the full length protein was isolated from a human brain cDNA library. Further, a plasmid expressing the full length of the gene was constructed, and a transformant was introduced by introducing the plasmid into a human cell line. Furthermore, by analyzing this transformant, it was found that the novel protein encoded by the gene has apoptosis-inhibiting ability. Also, they have found that antisense DNA and antisense RNA that inhibit this gene expression may be used as therapeutic agents for diseases involving Fas-dependent apoptosis, such as anticancer agents. Further, they have found that a protein having a sequence similar to the protein having the ability to inhibit apoptosis may be involved in induction of apoptosis.

Accordingly, the present invention provides the following (1) to (3)
0). (1) A protein comprising the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. (2) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having an ability to control apoptosis. (3) A gene comprising the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. (4) A gene comprising the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and encoding a protein having apoptosis controlling ability. (5) DN containing the base sequence shown in SEQ ID NO: 1 in the sequence listing
A, which hybridizes with A under stringent conditions and encodes a protein capable of controlling apoptosis
A, a gene having the ability to control apoptosis. (6) A gene encoding a protein containing the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. (7) A gene that includes an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and encodes a protein having apoptosis controlling ability. (8) The gene according to the above (6), which has an ability to control apoptosis. (9) The gene according to any one of (4) to (8) above, wherein the ability to control apoptosis is the ability to inhibit apoptosis. (10) A protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing. (11) A protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and having apoptosis inhibitory ability. (12) A protein that binds to Caspase 8 and has no death effector domain. (13) The protein of the above (1), (2) or (10), which has apoptosis controlling ability. (14) The protein according to the above (13), wherein the ability to control apoptosis is the ability to inhibit apoptosis. (15) A recombinant plasmid comprising the gene according to any one of (3) to (9). (16) A transformant or a transductant containing the recombinant plasmid of the above (15). (17) A method for producing a protein having an apoptosis controlling ability, which comprises culturing the transformant or the transductant of the above (16) and collecting a protein having an apoptosis controlling ability from the culture. (18) The method for producing a protein according to the above (17), wherein the ability to control apoptosis is the ability to inhibit apoptosis. (19) A nucleic acid probe comprising the whole or a part of the nucleotide sequence of the gene according to any of (3) to (9). (20) A method for detecting a gene encoding a protein capable of controlling apoptosis by hybridization using the nucleic acid probe according to (19). (21) The method for detecting a gene according to (20), wherein the ability to control apoptosis is the ability to inhibit apoptosis. (22) A method for cloning a gene encoding a protein capable of controlling apoptosis, which comprises using the nucleic acid probe of (19). (23) The method for cloning the gene according to (22), wherein the ability to control apoptosis is the ability to inhibit apoptosis. (24) Antisense to the whole or a part of the nucleotide sequence of any of the above genes (3) to (9),
DNA sequence capable of inhibiting the expression of the gene. (25) Antisense to the whole or a part of the nucleotide sequence of the gene of any of (3) to (9) above,
An RNA sequence that can inhibit the expression of that gene. (26) An antibody against the protein of (1), (2) or (10). (27) A method for detecting a protein having the ability to control apoptosis using the antibody of (26). (28) The method for detecting a protein according to the above (27), wherein the ability to control apoptosis is the ability to inhibit apoptosis. (29) A method for screening a drug using the protein according to any of (1), (2) or (10) or the transformant or transductant of (16). (30) The DNA of the above (24), the RNA of the above (25)
Or a pharmaceutical preparation comprising the antibody of the above (26).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The protein having the ability to control apoptosis according to the present invention, in particular, the protein having the ability to inhibit apoptosis will be described more specifically. [I] Novel protein capable of inhibiting apoptosis of the present invention and gene encoding the same (1) To isolate a gene encoding a novel protein capable of inhibiting apoptosis of the present invention, first, Caspase-8
CDNA encoding a protein fragment that binds to the N-terminal 183 amino acids corresponding to the death effector domain of human placenta cDNA was determined using yeast two-hybrid screening.
Isolate from NA library. Next, a cDNA library of human brain is prepared, and from this library, a cDNA molecule that hybridizes with the cDNA is isolated and cloned.
A gene encoding a novel protein capable of inhibiting apoptosis can be isolated. Libraries are especially useful for the human brain
It is not limited to cDNA libraries. As a gene encoding a novel protein having the ability to inhibit apoptosis of the present invention, aip-1 (apoptosis inhibitor
ry protein) gene was found. The nucleotide sequence of this gene is as shown in SEQ ID NO: 1 in the sequence listing. In this nucleotide sequence, the starting methionine is encoded by the 25th to 27th ATG in the sequence listing. The open reading frame (ORF) is encoded by the 25th to 5817th bases in the same sequence listing. From the nucleotide sequence of this gene, it was found that the novel protein having the ability to inhibit apoptosis of the present invention had the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence shown in SEQ ID NO: 2 revealed that the novel protein having the ability to inhibit apoptosis of the present invention did not have a death effector domain. Here, the death effector domain is
Known amino acid sequence of FADD (Cell, Vol. 81, pp. 505-512 (1
995)) is defined as an amino acid sequence that binds to the 4-76 portion and shows at least 25% homology with the 4-76 portion at the amino acid level. In addition, the aip-1 gene shown in SEQ ID NO: 1 in the sequence listing, and the genes and proteins having homology with the amino acid sequence of the protein encoded by them shown in SEQ ID NO: 2 were searched in a database.
None had a homology of 30% or more.
Furthermore, for the purpose of confirming the function of aip-1, as shown in the Examples below, a plasmid containing the full length of the aip-1 gene was constructed, and a transformant expressed in a human cultured cell line was prepared. . This transformant was significantly different from the control group.
It showed resistance to Fas-dependent apoptosis. These results revealed that the protein encoded by the aip-1 gene is a protein that binds to Caspase-8 and has the ability to inhibit Fas-dependent apoptosis. (2) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and which has an ability to inhibit apoptosis is also within the scope of the present invention. . Amino acid deletions, substitutions or additions are those which do not substantially affect the structure and function of the entire protein.For example, amino acid substitutions have the same hydrophobicity, size, charge and / or
Or substitution of aromatic amino acids.
Such substitutions include, for example, glycine and proline,
Substitutions between tyrosine and tryptophan and between asparagine and serine are mentioned. The degree of deletion, substitution, or addition of these amino acids is within a range in which homology to the original amino acid sequence is 30% or more, preferably 60% or more, and more preferably 80% or more.

Further, in the present invention, in addition to the coding gene shown in SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence in which one or several amino acids have been deleted, substituted or added. Also, a gene encoding a protein capable of inhibiting apoptosis is within the scope of the present invention. Furthermore, a gene that hybridizes with the coding gene shown in SEQ ID NO: 1 in the sequence listing under stringent conditions and encodes a protein having an ability to inhibit apoptosis is also within the scope of the present invention. Such hybridizing DNA mutants are those in which the DNA sequence is partially changed due to site-specific mutagenesis, random mutation by treatment with a mutagen, mutation, deletion, ligation, etc. of DNA fragments by restriction enzyme cleavage. Is mentioned. These DNA
The degree to which the mutant hybridizes with the coding gene shown in SEQ ID NO: 1 may be determined under stringent conditions, for example, by using a hybridization solution (7% polyethylene glycol 8000) with a radioisotope-labeled probe at 1,000,000 dpm / ml. , 10% SDS) and hybridization at 65 ° C for 16 hours, followed by 2% containing 0.1% SDS.
× SSC (300 mM NaCl, 30 mM trisodium citrate)
It hybridizes under the condition of washing at 60 ° C. for 30 minutes.

[II] Production of a novel protein having the ability to inhibit apoptosis of the present invention As described below, the protein of the present invention can be produced by a DNA recombination method utilizing a gene encoding the protein of the present invention. it can. (1) Recombinant plasmid In order to produce the protein having the ability to inhibit apoptosis of the present invention by DNA recombination, first, a gene encoding the protein having the ability to inhibit apoptosis of the present invention was inserted into an appropriate vector. , Construct a recombinant plasmid. The recombinant plasmid can be stably retained in Escherichia coli or the like. In this case, pBluescript SK (-) can be used as a vector. In the examples described below, the aip-1 gene was replaced with pBluescript SK (-)
PBS / aip-1 incorporated by way of example. If necessary, cut these plasmids into appropriate regions (open reading frame, ORF) to be translated into proteins with appropriate restriction enzymes, etc., and connect them to appropriate vectors to obtain recombinant plasmids for expression. Can be. When the host is Escherichia coli, a plasmid such as pTV118 is used as an expression plasmid.
When the host is a yeast, a plasmid such as pYES2 may be used as a vector, and when the host is a mammalian cell, a plasmid such as pcDNA3 may be used as a vector.

(2) Transformant or transductant The above-mentioned recombinant plasmid is introduced into an appropriate host to construct a transformant or transductant. As a host, Escherichia coli, yeast, and mammalian cells can be used. A transformant that expresses a protein having the ability to inhibit apoptosis can be obtained by transforming an appropriate host with the recombinant plasmid incorporated in the expression vector as described above. For example, a transformant can be obtained by introducing a recombinant plasmid as shown in FIG. 1 into cultured mammalian cells. (3) Culture of Transformant or Transducer In order to produce the protein having the ability to inhibit apoptosis of the present invention, a transformant for expression or transduction into which a recombinant plasmid containing a gene encoding the protein has been introduced Body
The cells may be cultured in an appropriate nutrient medium to express the protein, and the protein may be recovered and purified from the cells or the medium.

[III] Nucleic acid probe, cloning and detection of gene using the same (1) Nucleic acid probe In the present invention, one of the genes encoding the protein having the ability to inhibit apoptosis of the present invention obtained as described above is provided. The whole or part thereof can be used as a nucleic acid probe to be used for cloning of a gene as described below. Examples of the nucleic acid probe comprising a part of the gene of the present invention include a nucleic acid probe comprising an oligonucleotide having 15 nucleotides or more. The nucleic acid probe is a restriction enzyme which connects the gene of the present invention or a gene fragment thereof to an appropriate vector, introduces it into a bacterium, replicates it, extracts it from crushed bacterial cells with phenol or the like, and recognizes a site connected to the vector. After cutting and electrophoresis, it can be prepared by cutting out from the gel. The nucleic acid probe can also be prepared by a chemical synthesis using a DNA synthesizer or a gene amplification technique using a PCR method based on the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. The nucleic acid probe can be labeled with a radioisotope or fluorescence to increase the detection sensitivity at the time of use. The nucleic acid probe can be used in a method for cloning a gene encoding a protein other than the above-described proteins having the ability to inhibit apoptosis. That is, if a cDNA library derived from various organs or tissues is searched by the hybridization method or the polymerase chain reaction (PCR) method using the nucleic acid probe, a protein having the same function as the protein of the present invention can be obtained. Can be isolated.
In addition, a gene encoding a protein capable of inhibiting apoptosis can be detected using this nucleic acid probe. An example is the detection of the mRNA of the gene in a cell. For example, nucleic acids are precipitated from crushed cells, and mRNA is
Is hybridized to a radioisotope-labeled nucleic acid probe on a nitrocellulose membrane, and binding can be detected by autoradiography (FIG. 2) or by measuring the amount of binding using a scintillation counter. Furthermore, the nucleic acid probe is also used for in situ hybridization, confirmation of tissues expressing a gene encoding a protein capable of inhibiting apoptosis, confirmation of the presence of genes and mRNA in various biological tissues, and the like. It is possible.

[IV] Antisense DNA and antisense
RNA The present invention also provides an antisense DNA and an antisense RNA of a gene of the protein having the ability to inhibit apoptosis. These antisense DNA or antisense
By introducing RNA into cells, it is possible to suppress the expression of a gene encoding a protein capable of inhibiting apoptosis. As antisense DNA to be introduced,
For example, the corresponding antisense DN of SEQ ID NO: 1 in the sequence listing
A or a part thereof can be used. The antisense
An example of the DNA is shown in SEQ ID NO: 3 in the sequence listing. This shows the sequence of the antisense DNA of the gene of the protein having the ability to inhibit apoptosis of SEQ ID NO: 1 in the sequence listing. As antisense DNA, for example, these antisense
A fragment obtained by appropriately cutting a part of DNA may be used, or a fragment synthesized based on these antisense DNA sequences may be used.
DNA may be used.

As the antisense RNA, for example, an antisense RNA corresponding to a gene of a protein capable of inhibiting apoptosis of SEQ ID NO: 1 in the sequence listing or a part thereof can be used. An example of the antisense RNA is shown in SEQ ID NO: 4 in the sequence listing. This is the antisense RNA of the gene of the protein having the ability to inhibit apoptosis of SEQ ID NO: 1 in the sequence listing.
FIG. As antisense RNA,
For example, a fragment obtained by appropriately cleaving a part of these antisense RNAs may be used, or these antisense RNAs may be used.
RNA synthesized based on the RNA sequence may be used. Further, for example, a gene or a part thereof of the protein having the ability to inhibit apoptosis of SEQ ID NO: 1 in the sequence listing is connected to an appropriate vector, introduced into a bacterium, replicated, and extracted from a cell lysate with phenol or the like. As a template, RNA prepared by the action of RNA polymerase in an in vitro transcription system may be used. Antisense DNA and antisense RNA can be chemically modified so that they are not easily degraded in vivo and can pass through cell membranes. A substance capable of inactivating mRNA, for example, a ribozyme may be bound thereto. The antisense DNA and antisense RNA thus prepared encode apoptosis-inhibiting proteins such as malignant tumors
It can be used for the treatment of various diseases in which the amount of mRNA is increased.

[V] Antibodies to the novel protein of the present invention having the ability to inhibit apoptosis The present invention also provides antibodies to the novel protein of the present invention having the ability to inhibit apoptosis. In order to produce this antibody, for example, a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing, a peptide fragment of a partial region of the amino acid sequence, or the like can be used as an antigen. The former can be prepared by expressing the above transformant or transductant and purifying it, and the latter can be synthesized using a commercially available amino acid synthesizer or the like. Antibodies are produced by a conventional method.For example, a monoclonal antibody is obtained by fusing antibody-producing B cells obtained by immunization with an antigen with myeloma cells, selecting a hybridoma that produces the desired antibody, and isolating the cells. It can be prepared by culturing. This antibody can be used for the treatment and diagnosis of human and animal diseases associated with the protein, including malignant tumors, as described below.

[VI] Detection of Protein Having Inhibition of Apoptosis Using Antibody The present invention provides a method of detecting a protein having the ability to inhibit apoptosis using the above antibody. Such detection is
It can be performed by measuring the binding or the amount of binding between the antibody and the protein. For example, if the cells are treated with a fluorescently labeled antibody and then observed with a fluorescence microscope, the presence of the protein or producer cells can be detected. The amount of the bound antibody can be measured by various known methods.

[VII] Drug Screening The present invention provides a method for screening a novel drug using the transformant or transductant of the present invention or the protein having the ability to inhibit apoptosis of the present invention. For example, a substance that inhibits the binding of the protein to Caspase-8
It can be found using a screening system using yeast transformants. In addition, it is possible to discover a substance that inhibits the protein or a gene encoding the same by screening for a drug showing a difference in sensitivity between the two cells, using cells into which the protein has been introduced and cells not having the protein. it can.

[VIII] Pharmaceutical preparations Antibodies against the above-mentioned antisense DNA, antisense RNA, and a novel protein capable of inhibiting apoptosis can be used as therapeutic agents for various diseases considered to be caused by abnormal execution of apoptosis such as cancer. .

[0019]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. Example 1 Gene Encoding a Novel Protein with Inhibition of Apoptosis
Isolation of the gene aip-1 1-a. Construction of a two-hybrid screening system A gene encoding a protein that binds to 183 amino acids on the N-terminal side of Caspase-8 was screened. As the method, a yeast two-hybrid screening method was used. The experimental procedure described below is based on the method of Fields et al.
Volume 340, pp. 245-246 (1989)).

1-a-1. Caspase-8 N-terminal 183 amino acid region
Region, two-hybrid screening vector
Preparation i) Caspase-8 N-terminal 183 cDNA to amino acid region code
From Preparation Human peripheral blood, using ISOGEN (Nippon Gene) to extract RNA. Using this RNA, a cDNA encoding the first to 183rd amino acids corresponding to the death effector domain of Caspase-8 (hereinafter referred to as Caspase-8 (1-183)) was subjected to RT-PCR. Amplified. A recognition site for the restriction enzyme SalI was added to the end of the PCR primer, and the reading frame was designed to match the reading frame when the two-hybrid vector pBTM116 described below was cut with the restriction enzyme SalI. The sequences of both primers are shown in SEQ ID NOs: 5 and 6 in the sequence listing. PCR conditions are:
One cycle of 95 ° C. (1 minute), 55 ° C. (1 minute), and 72 ° C. (2 minutes) was used to amplify 30 cycles. The PCR amplification product was deproteinized by phenol treatment and chloroform treatment, and then ethanol precipitated. These were washed with 70% ethanol and then dissolved in sterile water. Next, the purified DNA was combined with 10 units of restriction enzyme.
After digestion with SalI (manufactured by Toyobo), fractionation by 0.8% agarose electrophoresis, extraction and purification were performed. EASYTRAP for purification
(Manufactured by Takara Shuzo) was used. This DNA and 4 μg of E. coli vector pTZ19R were digested with 10 units of restriction enzyme SalI, and further dephosphorylated with 1 unit of bovine alkaline phosphatase (Boehringer Mannheim).
DNA was ligated with a DNA ligation kit (Takara Shuzo) (hereinafter referred to as pTZ19R / caspase-8 (1-183)).

Ii) A vehicle for two-hybrid screening
Compactors Preparation Next, a 11.1 [mu] g of pTZ19R / caspase-8 (1-183) , was digested with restriction enzyme SalI in 30 units, cut pBTM116 4.2Myug a vector for two-hybrid screen with 30 units of the restriction enzyme SalI After that, it was connected to one further dephosphorylated with one unit of bovine alkaline phosphatase (hereinafter referred to as pBTM116 / caspase-8 (1-183)). The absence of mutation in the amplified gene was confirmed by pTZ19R / casp
It was confirmed by determining the gene sequence of ase-8 (1-183). The determination of the gene sequence was performed using an automatic sequencer 370 (manufactured by Applied Biosystems). This is a two-hybrid screening vector.
pBTM116 is designed so that the target gene, that is, the gene encoding Caspase-8 (1-183), can be connected downstream of the gene encoding the DNA binding region of LexA protein. A fusion protein of the region and the target gene product can be expressed. Also, pBTM116 is
This is a vector designed to express the TRPI gene. Accordingly, the obtained two-hybrid screening vector pBTM116 / caspase-8 (1-183) can express a fusion protein of the DNA binding region of LexA protein and Caspase-8 (1-183), It is a vector that can express the TRPI gene.

1-a- 2.Extraction of pBTM116 / caspase-8 (1-183)
Introduction to the bud yeast Next, pBTM116 / caspase-8 (1-183 ) the budding yeast Saccharomy
ces cerevisiae L40 (MATa his3 △ 200 trp1-901 leu2-
_{3,112 ade2 LYS2: :( lexAop) 4} -HIS3 URA3: :( lexAop) 8 -
lacZ GAL4 gal80). Introductory experiment is Current p
The procedure was performed according to the procedure described in rotocols in molecular biology (Willy Interscience), Vol. 2, Chapter 13, 7.1 (1994). Further, since the yeast L40 strain is a tryptophan-requiring strain, the yeast L40 strain into which the plasmid was introduced was selected by culturing on a plate from which tryptophan was removed. That is, L40 was added to a 50 ml YPAD solution (1%
Yeast extract, 2% peptone, 2% glucose, 0.01% adenine) at 30 ° C. and a bacterial concentration of 2 × 10 ⁷ /
After culturing with shaking until the volume reached 100 ml, the cells were collected and washed with sterile water. The yeast was suspended in 2 ml of lithium acetate solution-1 (10 mM Tris-HCl pH 7.5, 0.1 M lithium acetate, 1 mM EDTA), and 0.2 μg of the suspension was added with 1 μg of pBTM116 / caspa.
se-8 (1-183) and 100 μg of bovine thymus DNA were added, and 1.2 ml of a polyethylene glycol / lithium acetate solution (10 mM Tris-HCl pH7.5, 40% (w / w) polyethylene glycol 4000, 0.1%) was added. M lithium acetate, 1 mM EDTA) and the mixture was kept at 30 ° C. for 30 minutes. Then, add 88 μl of dimethyl sulfoxide, mix gently and at 42 ° C.
Incubated for 15 minutes. The bacteria were precipitated by centrifugation at 12,000 rpm for 1 minute, and TE solution (10 mM Tris-HCl pH 7.5, 1 mM
After suspending in EDTA, -UT plate (0.12% yeast nitrogen base (amino acid, without ammonium sulfate), 0.5% ammonium sulfate, 1% succinic acid, 0.6%
Sodium hydroxide, 2% glucose, 0.01% adenine,
Arginine, cysteine, leucine, lysine, threonine, 0.005% aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, valine plus 1.5% agar to form an agar medium) and spray at 30 ° C. For 2 days. The resulting transformant, i.e., the yeast into which pBTM116 / caspase-8 (1-183) was introduced, was transformed into L40 / ｛pBTM116 / caspase-8 (1-183))
I named it.

1-a-3. Caspase-8 N-terminal 183 amino acid region
Screening of Factors that Bind to Regions i) A human cDNA library was subsequently screened for a protein that binds to Caspase-8 (1-183) from the human cDNA library. This human cD
The human placenta MATCHMAKER cDNA (pACT
2 / Human placenta library, Clonetech, product number
CLHL4025A) was used. This pACT2 / human placenta library is prepared using a two-hybrid screening vector pACT2. pACT2 is designed so that the target gene can be connected downstream of the gene encoding the GAL4 transcription activation region, and can express a fusion protein of the GAL4 transcription activation region and the target gene product in yeast cells. . PACT2 is a vector designed to express the LEU2 gene.

Ii) pACT2 / L40 / ｛pB of human placenta library
TM116 / caspase-8 (1-183) electrically to the input beginning to}, were prepared of the library plasmid. Each of 230,000 E. coli BNN132 strains carrying this pACT2 / human placental library was 140 cm ²
LB-Amp plate (0.5% yeast extract, 1
% Peptone, 0.5% sodium chloride, and 100 μg / ml sodium ampicillin plus 1.5% agar to form an agar medium). This was cultured at 30 ° C. for 18 hours, and after collecting the cells, the cells were cultured with shaking in 2000 ml of LB-Amp solution at 30 ° C. for 3 hours to prepare a plasmid. Plasmid preparation
Laboratory Manual Genetic Engineering (Maruzen Edition, Muramatsu Masataka Edition) 53-5
Performed according to the procedure on page 5 (1990). Next, these library plasmids were transformed into L40 pBTM116 / caspase-8 (1-183)｝
Was introduced. Since the yeast L40 strain is a leucine-requiring strain, the yeast L40 strain into which the plasmid was introduced was selected by culturing under conditions excluding leucine. That is,
L40 ｛pBTM116 / caspase-8 in 1000 ml YPAD solution
(1-183)｝ was inoculated at a bacterial concentration of 1 × 10 ⁷ / ml, and cultured with shaking at 30 ° C. for 3 hours. The bacteria were precipitated by centrifugation at 3,000 rpm for 5 minutes and washed with 400 ml of a TE solution. Subsequently, this was added to 20 ml of lithium acetate solution-2 (5 mM
Tris-hydrochloric acid pH 7.5, 0.1 M lithium acetate, 0.5 mM EDT
A), 500 µg of library plasmid and 2 mg
Was added, and 140 ml of a polyethylene glycol / lithium acetate solution was further added, and the mixture was incubated at 30 ° C. for 30 minutes. Further, 3.5 ml of dimethyl sulfoxide was added thereto, mixed gently, and kept at 42 ° C. for 6 minutes. Next, this was washed twice with 400 ml of YPA solution (1% yeast extract, 2% peptone, 0.01% adenine), suspended in 1000 ml of YPAD solution, and cultured with shaking at 30 ° C. for 1 hour. These are centrifuged to collect the cells, and 500 ml
After washing with -UTL solution (leucine was removed from -UT solution), the cells were suspended in 1000 ml of -UTL solution and cultured with shaking at 30 ° C for 4 hours. By culturing under the conditions excluding leucine, L40 (pBTM116 / caspase-8 (1-183)) and pACT
2 / Transformant into which the human placenta library has been introduced is obtained.

Iii) Factors binding to Caspase-8 (1-183)
Selection of yeast L40 strain expressing offspring Screening of transformants obtained by introducing pBTM116 / caspase-8 (1-183) and pACT2 / human placenta library into L40 thus obtained, and further using histidine sensitivity as an indicator The yeast L40 strain that expresses a factor that binds to Caspase-8 (1-183) can be selected. That is, the yeast L40 strain for two-hybrid screening is a histidine-requiring strain, and has been modified to express the HIS3 gene under the control of LexA. In the transformant, a fusion protein of the DNA binding region of LexA protein expressed from pBTM116 / caspase-8 (1-183) and Caspase-8 (1-183) and pACT2 / human placenta library were expressed. Fusion proteins between GAL4 transcription activation region and various gene products
The fusion protein of the transcriptional activation region of GAL4 and a factor that binds to Caspase-8 (1-183) binds to each other via the factor. After binding, the conjugate binds to the upstream portion of the HIS3 gene of the yeast L40 strain via the DNA binding region of the LexA protein, and then, by the transcriptional activation region of GAL4 in the conjugate, yeast L4
The HIS3 gene of 0 strain changes from off to on, and histidine is synthesized. Therefore, by screening transformants further using histidine sensitivity as an index, Ca
yeast L40 expressing a factor that binds to spase-8 (1-183)
You can select the strain. Based on this theory, L40 obtained in the above ii) {pBTM116 / caspase-8 (1-183)}
The culture of the transformant into which the pACT2 / human placenta library has been introduced is centrifuged to collect the cells, washed twice with a -THULL solution (a solution obtained by removing leucine, lysine and histidine from the -UT solution), and then washed with 100 ml. Suspended in -THULL solution, and add 1 ml each.
The solution was spread on 100 140 cm ² -THULL plates (1.5% agar was added to the -THULL solution to form an agar medium). This was cultured at 30 ° C. for 3 days, and the appearing colony was isolated. Thus, the yeast L40 strain expressing a factor that binds to Caspase-8 (1-183) was selected.

1-b. Cloning of aip-1 gene i) Encoding factor binding to Caspase-8 (1-183)
From yeast colonies that appeared plasmid preparation -THULL plates containing gene, the plasmid was prepared containing the library gene. The experiment is Curren
The procedure was performed according to the procedure described in t protocols in molecular biology (published by Wiley Interscience), Vol. 2, Chapter 13, 11.1 (1994). That is, a colony was inoculated into 5 ml of a YPAD solution and cultured with shaking at 30 ° C. for 2 days. Then, 3000
The cells were collected by spinning and centrifuging for 5 minutes, and 0.5 ml of a sorbitol solution (1 M sorbitol, 100 mM EDTA pH7.
5) Suspension, 50 μl of 0.3 mg / ml zymolyase 100
T (manufactured by Seikagaku Corporation) was added, and the mixture was kept at 37 ° C. for 1 hour. Subsequently, the cells were collected by centrifugation at 2500 rpm for 5 minutes, suspended in 0.5 ml of a cell suspension buffer (50 mM Tris-HCl (pH 7.5), 20 mM EDTA), and then 25 μl of 20% SDS
Was added and kept at 65 ° C. for 30 minutes. In addition, 250 μl
Was added and the mixture was allowed to stand on ice for 1 hour. After centrifugation of this sample at 12,000 rpm for 10 minutes, the supernatant was recovered, and 0.5 ml of 2-propanol was added, followed by centrifugation at 12,000 rpm for 30 seconds to collect the plasmid in the precipitate. After washing the plasmid with 70% ethanol, TE
Dissolved in the solution. This plasmid was designated as pACT2 / library-1.

Ii) Specific binding to Caspase-8 (1-183)
The protein encoded by this library gene is Caspase-8
The following experiment was carried out for the purpose of confirming the binding to the N-terminal 183 amino acids of. First, a gene encoding the cytoplasmic region of Fas (Fas amino acids 192 to 335, hereinafter referred to as Fas (192-335)) was amplified by RT-PCR using human peripheral blood RNA as a template. Similarly, a gene encoding FADD was amplified using RNA of Paca-2, a cultured cell line, as a template. A recognition site for the restriction enzyme EcoRI was added to the end of the PCR primer, and the two-hybrid vector pBTM11
Designed to match the reading frame when connected to 6. The sequences of the primers are shown in SEQ ID NOs: 7 to 10 in the sequence listing. fas (19
2-335) In amplification of the gene, the primers shown in SEQ ID NOs: 7 and 8 were used, and the PCR conditions were 95 ° C (1 minute), 55 ° C
C. (1 minute) and 72.degree. C. (1 minute) were defined as one cycle, and amplification was performed for 30 cycles. SEQ ID NO: 9 for amplification of the fadd gene
PCR conditions are 96 ° C, using the primers described in and 10.
(2 minutes), 57 ° C. (30 seconds) and 72 ° C. (1 minute 30 seconds) were defined as one cycle, and amplification was performed for 35 cycles. These genes were cut with 10 units of EcoRI restriction enzyme, fractionated by 0.8% agarose electrophoresis, extracted and purified. EASYTRAP was used for purification. These DNAs were connected in the forward direction to the restriction site EcoRI recognition site of the E. coli vector pTZ19R. The experiment was performed in the same procedure as the method described above. These are referred to as pTZ19R / fas (192
-335) and named pTZ19R / fadd.

Next, 10 μg of each of pTZ19R / fas (192-335),
Cut pTZ19R / fadd with 30 units of restriction enzyme EcoRI,
After fractionation by 8% agarose electrophoresis, the DNA extracted and purified in the same manner was combined with the two-hybrid vector pBTM116
Restriction enzyme EcoRI recognition site. These are called pBTM
116 / fas (192-335) and pBTM116 / fadd. In addition,
PTZ19R / fas (192-
335), and confirmed by determining the gene sequence of pTZ19R / fadd. Automatic sequencer for gene sequence determination
370 (manufactured by Applied Biosystems). In addition, pTZ19R / fas (192-335) was digested with the restriction enzyme EcoRI, and the purified gene fragment was also connected to the restriction enzyme EcoRI recognition site of the two-hybrid screening vector pGAD424 in the forward direction, and pGAD424 / fas (192-335). 335). pGAD424, like the two-hybrid screening vector pACT2 described above, is designed so that the gene of interest can be connected downstream of the gene encoding the transcriptional activation region of GAL4, and the transcriptional activation region of GAL4 in yeast cells. And a fusion protein of the desired gene product can be expressed.

Using these, the following experiment was conducted. Plasmids were introduced into S. cerevisiae L40 in the following six combinations. 1) 1 μg of pACT2 / library-1 and 1 μg of pBTM116 / fas (192
-335); 2) 1 μg of pACT2 / library-1 and 1 μg of pBTM116 / fadd
3) 1 μg of pACT2 / library-1 and pBTM116 / caspase-8 (1-1
4) 1 μg of pGAD424 / fas (192-335) and 1 μg of pBTM116 / f
Mixture of as (192-335); 5) 1 μg of pGAD424 / fas (192-335) and 1 μg of pBTM116 / f
mixture of add; 6) 1 μg of pGAD424 / fas (192-335) and 1 μg of pBTM116 /
A mixture of caspase-8 (1-183). The introduction of the gene into the yeast cells was performed according to the above-mentioned procedure. Each of these yeasts is on a -UTL plate (
1.5% agar was added to make agar medium)
The cells were cultured at 30 ° C for 2 days. pBTM116 complements L40 for tryptophan auxotrophy, and pACT2 and pGAD424 complement leucine auxotrophy. Therefore, yeasts grown on a medium lacking tryptophan and leucine have both plasmids introduced. Subsequently, these yeasts were spread on a -THULL plate, and their growth was examined. As a result, 3), 4),
It was found that only the yeast into which the plasmid mixture of 5) was introduced proliferated on the -THULL plate. That is, library-
1 Product binds to Caspase-8 (1-183) but Fas (192-33
5) did not bind to FADD. Fas (192-335) used as a control bound Fas (192-335) itself to FADD, but did not bind to Caspase-8 (1-183). These results indicate that the pACT2 / library-1 product is a factor that specifically binds to Caspase-8 (1-183).

Iii) Specific binding to Caspase-8 (1-183)
Sequencing Subsequently vector for sequencing the library-1 gene of the gene fragment encoding that factor
Connected to pBluescript SK (-). That is, 5 μg of pA
CT2 / library-1 was digested with 10 units of restriction enzyme EcoRI, fractionated by 0.8% agarose gel electrophoresis, and the library gene was extracted and purified. EASYTRAP was used for purification. This DNA is used as the restriction enzyme EcoRI of pBluescript SK (-).
Connect to the recognition site and insert the resulting plasmid into pBS / library-
Named 1. Then, using this plasmid, librar
The nucleotide sequence of the y-1 gene was determined. The determination of the gene sequence was performed using an automatic sequencer LONG READIR4200 (manufactured by Leica). The library gene sequence was determined by the above method and compared with the known gene library. BLAST search was used as software. This is the homepage of the US NIH on the Internet (address http: // www.
ncbi.nlm.nih.gov/BLAST/) is a gene search software. As a result, it was confirmed that a gene showing no homology with the known gene was included. The sequence of this gene fragment is shown in SEQ ID NO: 11 in the sequence listing. Also, pBS / libr
FIG. 3 shows a restriction map of ary-1.

Iv) Specific binding to Caspase-8 (1-183)
Isolation of full-length cDNA encoding the factor Next, an attempt was made to isolate the full-length cDNA of this gene. The gene library is a human brain cDNA library (infection efficiency: 5 × 10 ⁶
pfu (plaque forming unit) / ml). It is prepared using a NotI-oligo dT primer (Promega) as a primer, an EcoRI adapter (Promega) as an adapter, and a λZAPII phage vector (Stratagene) cut with a restriction enzyme EcoRI as a vector. The probe was prepared as follows. 5 μg of pBS / library-1
Was digested with 20 units each of restriction enzymes NotI and XhoI (both manufactured by Toyobo), fractionated by 0.8% agarose electrophoresis, extracted and purified. EASYTRAP was used for purification. 70 ng of this library gene fragment was radioisotope-labeled using Prime-It II (manufactured by Stratagene).

A membrane for plaque hybridization was prepared as follows. 20 μl of library solution and 80 μl of SM buffer (50 mM Tris-HCl (pH 7.5),
100 mM NaCl, 10 mM MgSO ₄ , 0.01% gelatin), 200 μ
l of E. coli solution (E. coli strain XL-1 Blue is cultured with shaking at 37 ° C for 16 hours, and collected by centrifugation at 5,000 rpm for 5 minutes.
(Suspended in MgSO ₄ ) and incubated at 37 ° C. for 15 minutes to infect the phage. To this, add 7 ml of LB-soft agar (0.5% yeast extract, 1% peptone, 0.5% sodium chloride, 0.7% agar) kept at 47 ° C after melting and mix gently. MgSO ₄
Sprayed on 12 plates (0.5% yeast extract, 1% peptone, 0.5% sodium chloride, 10 mM MgSO ₄ and 1.5% agar to form an agar medium). 37 ℃
For 6 hours to form plaques. Next, the plate was kept at 4 ° C. for 1 hour, and then plaque was adhered to the nylon membrane. Nylon membrane
A colony plaque screen manufactured by NEN was used. After drying the membrane, 500 ml of alkaline solution (0.5N NaOH
, 1.5 M NaCl) at room temperature for 2 minutes, followed by 5 minutes
00ml neutralization solution (0.5M Tris-HCl (pH 7.2), 1.5M NaCl
, 1 mM EDTA) at room temperature for 2 minutes. next,
After keeping the membrane in a 3 × SSC solution (450 mM NaCl, 45 mM trisodium citrate) at 55 ° C. for 1 hour,
It was dried to obtain a plaque hybridization membrane. Next, plaque hybridization was performed using this membrane. The above membrane was kept at 65 ° C. for 1 hour in 50 ml of a hybridization solution (7% polyethylene glycol 8000, 10% SDS) for prehybridization. Next, radioisotope labeled
1,000,000 dpm / ml radioactive amount of library-1 gene fragment
, And incubated at 65 ° C. for 16 hours to perform hybridization. Subsequently, the membrane was washed with 2 × SSC (300 mM NaCl, 30%) containing 0.1% SDS.
(mM trisodium citrate) at 65 ° C. for 30 minutes. This washing was performed twice. Subsequently, positive plaques were detected by performing autoradiography on these membranes.

Next, a plaque group containing plaques corresponding to the positive signal was isolated and suspended in 1 ml of SM buffer.
This solution was kept at 4 ° C. for 16 hours to elute the phage, and then centrifuged at 13,000 rpm for 10 minutes to collect the phage in the supernatant. When this phage solution emerges,
The plaques were spread on LB-MgSO ₄ plates so that the number was 0, 1000, and positive plaques were screened in the same manner as described above. As a result, a positive plaque was successfully isolated as a completely isolated plaque. Phage was isolated from this plaque. Next, the library contained in this phage
The following experiment was performed for the purpose of determining the base sequence of DNA.
Since this library is prepared using λZAPII, pBluescript SK (−) containing the library gene can be cut out from the vector by infecting a helper phage. This operation was performed according to the following method. 50 μl of the phage solution was added to 10 μl of the R408 helper phage solution (infection efficiency 1 × 10 ¹¹ pfu (plaque formi
ng unit) / ml), mix with 40 μl of E. coli solution, and
Incubated for 15 minutes. Subsequently, 1 ml of 2 × YT solution (1.6% polypeptone, 1% yeast extract, 0.5% NaC
l) was added and the cells were cultured with shaking at 37 ° C. for 3 hours. Then 70
After heat treatment at 20 ° C for 20 minutes, 2 µl of this solution was placed in LB medium for 5 minutes.
Mix with 100 μl of E. coli strain XL-1Blue
For 15 minutes. This E. coli solution was spread on an LB-Amp plate, and the target plasmid was prepared from the colonies that appeared. This plasmid was designated as pBS / aip-1. It was confirmed that this plasmid contained a 6.3 kb library gene. The determination of the library gene sequence was performed using an automatic sequencer LONG READIR4200 (manufactured by Leica). This gene sequence is shown as SEQ ID NO: 1 in the sequence listing. The predicted starting methionine is encoded by the 25th to 27th ATG in the sequence listing. Also, the expected OR
F is encoded by the 25th to 5817th bases in the same sequence listing. In addition, an in-frame stop codon is found in this gene upstream of the predicted start methionine codon. This exists from the first to the third in the sequence listing. From this, the isolated genes were all ORF
It was shown to be a cDNA containing The predicted amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing.

1-c. Various tissues of aip-1 gene, in cancer cells
I) Northern hybridization Next, the expression of this gene in various tissues and cancer cells was examined by Northern hybridization. As a membrane, a human MTN blot of Clonetech was used. These membranes were pre-incubated in 5 ml of ExpressHyb solution (Clontech) at 68 ° C for 30 minutes. Subsequently, the radioisotope-labeled gene probe (used in the above 1-b experiment) was used.
Add 1 million dpm / ml, and further add 1
Hybridization was performed by keeping the temperature for a period of time. Then, these were placed in 2 × SSC containing 0.1% SDS,
Washed at room temperature for 10 minutes. This washing was performed three times. This was followed by 0.1 x SSC containing 0.1% SDS (15 mM NaCl
, 1.5 mM trisodium citrate) at 50 ° C. for 20 minutes. These membranes were analyzed using a Bio-Rad molecular imager system. ii) Results The results are shown in FIG. A band was observed at about 6.5 kb, strongly suggesting that the isolated gene fragment was full-length cDNA. In FIG. 2, lane 1: pancreas, lane 2: kidney, lane 3: skeletal muscle, lane 4: liver, lane 5: lung, lane 6: placenta, lane 7: brain, lane 8: heart-derived poly (A) 2 μg of each RNA is blotted.

1-d. Functional Analysis of aip-1 Gene i) Construction of aip Expression Vector Subsequently, functional analysis of the aip-1 gene was performed. First,
A vector for expressing the aip-1 gene in mammalian cells was prepared. After cutting 5 μg of pBS / aip-1 with 20 units of restriction enzymes BamHI and NotI (both manufactured by Toyobo),
Fractionation was performed by 0.8% agarose electrophoresis, and a 6.3 kb fragment corresponding to the aip-1 gene was extracted and purified. EASY for purification
TRAP was used. This purified DNA was connected to the restriction sites BamHI and NotI of the mammalian cell expression vector pcDNA3. This plasmid was named pcDNA3 / aip-1. The restriction map of this plasmid is shown in FIG. Culture 1,000 ml of Escherichia coli strain XL-1Blue carrying this plasmid and cultivate it in Lab Manual Genetic Engineering (Maruzen, Masataka Muramatsu), pp. 53-55
(1990). This plasmid was purified twice by cesium chloride density gradient centrifugation. Similarly, a vector in which a β-galactosidase gene was connected to pcDNA3 was prepared and named pcDNA3 / lacZ.

Ii) Apoptosis-suppressing ability of aip-1 Next, the method of Chinnaiyan et al. (Cell, 81, 505-512)
(1995)), the ability of aip-1 to suppress apoptosis was measured. 500,000 human embryonic kidney cell lines, 293 cells,
The cells were sprayed on a cell culture dish having a diameter of 60 mm. As the medium, D-MEM (manufactured by Nissei Laboratories) supplemented with bovine serum (FBS: fetal bovine serum) so as to have a final concentration of 10% (hereinafter referred to as D-MEM / 10% FBS) was used. After culturing them at 37 ° C. for 48 hours in the presence of 5% CO ₂ , the medium was replaced with D-MEM containing no bovine serum. Subsequently, these cells were divided into two groups, and the following plasmids were introduced. One group contains 0.2 μg pcDNA3 / lacZ and 1.8 μg pcDNA3 / aip
-1 vector mixture, 0.2 μg pcDNA3 / l for another
A mixture of acZ and 1.8 μg of pcDNA3 vector was introduced.
For the introduction of the plasmid, Lipofectamine Plus reagent from Gibco was used. The experiment followed the following procedure. That is, 125 μl of D-MEM and 8 μl of a plus solution were added to the plasmid solution, mixed well, and then kept at room temperature for 15 minutes. Next, add 12 μl Lipofectamine solution and 125 μl
l-D-MEM, mix well, then add
Incubated for a minute. This was administered to the 293 cells cultured according to the above procedure, mixed gently, and then in the presence of 5% CO ₂ at 37 ° C.
After culturing for 3 hours, the plasmid was incorporated. Subsequently, the medium was replaced with D-MEM / 10% FBS, and the cells were further cultured at 37 ° C. for 21 hours in the presence of 5% CO ₂ . Subsequently, the two groups of cells were treated with an anti-Fas antibody CH11 (Pharmingen).
Was administered at final concentrations of 0, 50, 100, and 200 ng / ml, and further cultured at 37 ° C. for 24 hours in the presence of 5% CO ₂ .
These cells were evaluated for apoptosis-inhibiting activity using the following two parameters as indices. The first index is an evaluation based on the number of cells that have undergone apoptosis and have detached from the dish. This was performed by collecting the medium in the cell culture dish and measuring the number of cells contained therein.
Another indicator is that the adherent cells are stained with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), indicating the apoptotic morphology of the stained cells. The evaluation is based on the percentage of cells. This was performed according to the following method. After removing the medium in the cell culture dish and washing with PBS (phosphate buffered saline), the cells were fixed in 2% formaldehyde and 2 ml of 0.2% glutaraldehyde for 5 minutes at room temperature. Subsequently, after removing the fixative, the plate is washed twice with PBS and stained (PBS
20 mM potassium ferricyanide, 20 mM potassium ferrocyanide, 1 mM MgCl ₂ , 0.1% X-gal)
Then, the mixture was kept at 37 ° C. for 16 hours. The morphology of the stained cells was determined under a light microscope. These results are shown in FIGS. From these results, at all concentrations of the anti-Fas antibody, apoptosis occurred in the pcDNA3 / aip-1 vector-introduced group compared to the pcDNA3-introduced group, and the number of cells detached from the dish was significantly reduced (FIG. 4). At all anti-Fas antibody concentrations, pcDNA3 / a
In the group into which the ip-1 vector had been introduced, the number of stained cells showing an apoptotic morphology was significantly reduced as compared with the group into which pcDNA3 had been introduced (FIG. 5). These results indicate that the aip-1 gene product has the ability to inhibit Fas-dependent apoptosis. From the above experimental data, aip-
1 The gene product was found to bind to the N-terminal region of Caspase-8 and to inhibit Fas-dependent apoptosis.

[0037]

Industrial Applicability According to the present invention, a novel protein having an ability to control apoptosis and a gene encoding the protein have been provided. These are useful for diagnosis and treatment of diseases such as cancer caused by abnormalities in the apoptosis control mechanism. Further, according to the present invention, antisense DNA and antisense RNA of the gene, a nucleic acid probe capable of hybridizing to the gene, a method for detecting the gene using the nucleic acid probe, and a transformant into which the gene has been introduced are used. A method for producing a novel protein having the ability to control apoptosis, an antibody to the protein, a method for detecting the protein using the antibody, and the like. These are also useful for diagnosis and treatment of diseases such as cancer.

[0038]

[Sequence List Free Text] The nucleotide sequences of SEQ ID NOS: 5 to 10 indicate PCR primers.

[0039]

[Sequence List] SEQUENCE LISTING <110> NIPPON KAYAKU KABUSHIKI KAISYA <120> Protein having inhibition activity on appotosis, gene there and use there <130> DA-02858 <150> JP 10-345868 <151> 1998-12-4 <160> 11 <210> 1 <211> 6309 <212> DNA <213> Homosapiens <400> 1 tgactctcaa aaggaaatag gatcatggca gcagatgatg acaatggtga tggaacaagt 60 ttatttgatg tcttttctgc ttctcctctt aagaacaatg atgaaggctc actggacata 120 tacgctgggt tggacagtgc tgtttctgac agcgcttcca aatcctgtgt accatcaaga 180 aattgtttgg acttatatga agagatcctg actgaagaag gaactgcaaa ggaggcaaca 240 tataatgatt tgcaagtaga atatggaaaa tgtcaactac aaatgaaaga gctgatgaaa 300 aaatttaaag aaatacagac acagaatttc agcttaataa acgaaaacca gtctcttaag 360 aagaatattt cagcacttat caaaactgcc agagtggaaa taaaccgcaa ggatgaagaa 420 ataagtaatc ttcaccaaag attgtctgag tttccacatt ttcgaaataa tcataaaact 480 gcaaggacat ttgatacagt taaaacaaaa gatcttaaat ctagatctcc acatttggat 540 gattgttcaa agactgatca cagagctaaa agtgatgttt ctaaagatgt acatcatagc 600 acttcactgc caaatctgga aaaggaagga aaaccacatt ctgataaaag gagtacttca 660 catttaccta catctgttga gaaacactgc actaatggtg tttggtcacg ttctcattat 720 caggttggcg agggtagctc aaatgaggat agtagaagag gaagaaaaga tattagacat 780 agccagttta acagaggaac tgaaagagta cgaaaagact taagtactgg ctgtggtgat 840 ggtgaaccaa gg atattgga ggctagtcaa aggctacaag gacatcctga gaaatatggt 900 aaaggtgaac caaagactga aagcaaaagt tcgaagttta aaagtaactc agattctgac 960 tataaaggtg aacgcattaa ctcttcttgg gagaaagaga cccctggaga aaggtcacac 1020 agtcgagtag actctcaaag tgacaaaaaa ctagaaagac aaagtgaaag atcacaaaat 1080 ataaatagga aagaagttaa atcacaagac aaagaagaaa gaaaagttga tcaaaaacct 1140 aaatcagtag taaaggacca agatcactgg agaagatctg aacgagcatc acttcctcat 1200 tccaagaatg aaataacatt ttctcataat tcaagtaaat accatctaga agagagaaga 1260 ggatgggaag attgtaaaag agacaagagt gtaaacagtc atagttttca agatggaaga 1320 tgtccatctt ctctttcaaa cagtagaact cacaaaaaca ttgactctaa ggaagttgat 1380 gccatgcatc agtgggaaaa tacaccttta aaagcagaaa gacatagaac tgaagataag 1440 aggaaaagag aacaagaaag caaagaagaa aataggcata ttagaaatga aaaaagagta 1500 cctacagaac atttgcagaa gactaataag gaaactaaga aaaccactac tgatttaaag 1560 aaacagaatg aaccaaagac tgataaggga gaagtccttg ataatggtgt ttctgaagga 1620 gcagataata aagagcttgc aatgaaagct gagagtggtc caaatgaaac aaaaaacaag 1680 gacctaaaat tgagttttat gaaaaaattg aacttaactc tttctcctgc taaaaagcaa 1740 cctgtttccc aggataatca gcataaaata actgatattc ccaagtccag tggtgtatgt 1800 gattcagagt cttcaatgca agttaaaaca gtggcatatg ttccctccat aagtgaacat 1860 atcttggggg aagcagctgt cagtgaacat accatggggg aaaccaagtc aacgttattg 1920 gaaccaaagg ttgctcttct agcagtgact gaacccagga tcggtatctc agaaaccaac 1980 aaggaagacg aaaatagttt gttagttagg tctgttgaca atactatgca ttgtgaagag 2040 cccatttgtg gtacagagac ttccttccca tctcctatgg aaatacaaca gacagaatcc 2100 ttgtttccat caacaggaat gaaacaaacc attaataatg gaagggcagc agctcctgtg 2160 gtaatggatg tattacaaac agatgtgtct caaaactttg gcttggaatt ggataccaaa 2220 agaaatgata attcagatta ttgtggtatt tctgaaggta tggagatgaa ggtggcactt 2280 tcaacaacag tgagtgaaac cactgaaagc attttgcagc cttcaattga ggaagctgat 2340 attttgccaa taatgctttc agaagataat aacccaaaat ttgagccttc tgttatagtt 2400 acaccactgg ttgagagtaa gtcgtgtcat ctggagcctt gcttacctaa agagactcta 2460 gattcttcac ttcagcagac tgagttaatg gaccacagaa tggcaactgg tgaaacaaac 2520 tcagtatatc atgatgatga taact cggtt ttgagcattg accttaatca cctgagacct 2580 attccagaag ccatcagtcc tctgaatagt ccagtgagac ctgtagcaaa agttcttaga 2640 aatgaaagcc cacctcaagt tccagtgtat aataacagtc ataaagatgt gtttttacca 2700 aattcagctc attctacctc taagagtcag tctgatctca ataaggaaaa tcaaaagcca 2760 atttacaaat ctgacaaatg tacagaagca gacacatgta agaattcacc attagatgaa 2820 ttagaagaag gagaaattag aagtgatagt gaaacatcta aaccacaaga aagttttgaa 2880 aaaaattcca aacgtagagt gtcagctgat gtgcggaagt caaagactat cccacgacgt 2940 gggaaaagta ctgtgtgttt agataaagac agtaggaaaa cacatgtaag aatccatcag 3000 accaataaca aatggaataa aagacctgat aaatctagca gatcttcaaa aacagagaag 3060 aaagataaag tgatgagcac ttccagcttg gaaaaaatag ttccaattat tgctgtaccc 3120 tcttctgaac aagagatcat gcacatgtta cgaatgataa gaaaacatgt aagaaaaaat 3180 tatatgaaat tcaaggcaaa attttcatta atacaatttc acagaattat tgagtcagca 3240 attttgagtt ttacatcttt aattaaacat ctcaacttac acaaaatctc taagtcagtg 3300 actaccttac agaagaatct ctgtgatatt atagagtcta aacttaagca agttaaaaag 3360 aatggcatag ttgatcgttt atttgaacag caactaccag atatgaaaaa aaaattgtgg 3420 aagtttgtag atgaccaact tgattatttg tttgcaaagc ttaagaaaat cttagtatgt 3480 gattccaaaa gctttggaag agatagtgat gaaggcaaac ttgaaaaaac aagtaaacag 3540 aatgcacagt attcaaatag tcagaaaagg agtgtggaca actccaacag agaattgctg 3600 aaagaaaaat tatcaaaatc agaagaccct gttcattata agtctttagt gggatgtaaa 3660 aaatctgagg aaaattatca agaccaaaat aactccagta ttaacactgt aaagcatgac 3720 attaaaaaaa attttaacat ctgctttgat aatataaaga actctcaatc cgaagagcgc 3780 tccttggaag tacactgtcc aagcacccca aagtcagaaa aaaacgaagg aagcagcata 3840 gaggatgcac agacatccca gcatgcaact ttgaagccag aacgaagttt cgagattctt 3900 accgaacagc aagcatcgag ccttactttt aatttagtga gtgatgcaca aatgggtgaa 3960 atatttaaaa gtttgttgca aggttctgat cttttagaca gcagtgttaa ctgtactgaa 4020 aaaagtgagt gggagttaaa gactccagag aagcagctgc tagagactct taagtgcgag 4080 tctataccag cttgtacaac agaagagcta gtttcagggg tggcttctcc atgtcctaaa 4140 atgattagtg atgataattg gtcattatta tcatctgaaa aaggtccatc tctgtcttca 4200 gggctttcat tgccggttca tcctgatgtg ttggat gaaa gttgtatgtt tgaagtgtct 4260 actaacctac ctttaagtaa agataatgtg tgtagtgtag aaaagagcaa gccctgcgtt 4320 tcttccatac ttcttgaaga tctagcagtc tctttaacag taccatcgcc tctgaagtca 4380 gatggtcatc tcagtttttt aaagcctgat atgtcgtcca gttcaactcc tgaagaagtc 4440 attagtgctc attttagtga agatgcctta cttgaggaag aggatgcatc tgagcaagat 4500 attcatttag ctctggagtc tgataattca agcagtaaat caagttgttc ttcttcctgg 4560 acaagccgat ctgttgctcc aggctttcag taccacccta atctacctat gcatgccgtc 4620 ataatggaaa agtccaatga tcatttcatt gtgaaaatac gacgtgcaac accatctacc 4680 tcttctggcc ttaaacagag tatgatgcct gatgaattat tgacatcttt gcccagacat 5740 ggaaaggaag ctgatgaagg accagagaaa gaatatattt catgtcagaa cacagttttt 4800 aaatctgtgg aggaattgga aaactccaac aaaaatgttg atggcagcaa gtcaactcat 4860 gaagaacaga gctctatgat acaaacacag gttcctgata tatatgaatt tcttaaagat 4920 gcttcagata agatgggtca tagtgatgaa gtggctgatg aatgtttcaa attgcatcaa 4980 gtatgggaaa caaaagtgcc tgaaagcatt gaagaattgc cttcaatgga agaaatctca 5040 cactctgttg gggaacatct tccaaacaca tacgtagatc t aacgaaaga tccagtcact 5100 gaaaccaaaa acttggggga attcatagaa gtaacagttt tacatattga tcagttggga 5160 tgttctggag gcaatttaaa tcagagtgct caaatattag acaattcttt gcaggctgat 5220 actgtaggtg cttttattga tttgacacaa gatgcttcaa gtgaggctaa aagtgaaggt 5280 aatcatcctg cattagctgt ggaagacttg ggatgtgggg tgatacaggt agatgaagat 5340 aattgtaagg aagaaaaggc acaagtggca aacaggcctt taaaatgcat tgttgaggaa 5400 acctatatcg acttgaccac agaatctccc agttcatgtg aagtaaaaaa agatgagtta 5460 aaatcagagc caggatcaaa ttgtgataac tcggagttgc ctgggacttt gcataattct 5520 cacaaaaaga gaagaaacat ttctgatcta aatcatcctc ataaaaaaca aagaaaggaa 5580 acagacttaa ctaataagga aaagaccaag aaacctaccc aagattcttg tgagaatact 5640 gaagctcacc aaaagaaagc cagtaagaag aaggcccctc ctgtgactaa agatccctca 5700 tcattaaagg caaccccagg gattaaggat tcatcagcag cacttgccac ttctacaagt 5760 ctttctgcaa aaaatgttat taaaaagaag ggagaaatta tcattttatg gacaaggtaa 5820 gaatcttgtg agacattgaa atcaccagga aattttgcac tcagaaatct aatctgtctg 5880 gctgggcgca gtggctcatg cctgtaatcc caatactttg ggacgct gag gcaggtggat 5940 cacttgagcc caggagtttg ggaccagctt gagcaacatg gcgaaattct gtctctacaa 6000 aaaaatacaa aaattagggc cgggcgcagt gctcacgcct gtaatcccag cactttggga 6060 ggctgaagcg ggcggatcat gaggtcagga gattgagacc atcctggcta acatggtgaa 6120 acccaatctc taccaaaaat acaaaactta gccagacgtg gtggcatgca cctgaaagtt 6180 ccggctggtt gggaggctga ggcaggagaa tggcatgaac ccggggggca gagcttgcag 6240 tgagccgaga tggcgccact gcattctagc ctgggcaaca gagcgagact gtcttaaaaa 6300 aaaaaaaaa 6309 <210> 2 <211> 1931 <212> PRT <213> Homosapiens <400> 2 Met Ala Ala Asp Asp Asp Asn Gly Asp Gly Thr Ser Leu Phe Asp Val 1 5 10 15 Phe Ser 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 Ala Ser Lys Ser Cys 35 40 45 Val Pro Ser 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 Val Glu Tyr 65 70 75 80 Gly Lys Cys Gln Leu Gln Met Lys Glu Leu Met Lys Lys Phe Lys Glu 85 90 95 Ile Gln Thr Gln Asn Phe Ser Leu Ile 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 Ser Asn Leu His Gln Arg Leu Ser Glu Phe Pro 130 135 140 His Phe Arg Asn Asn His Lys Thr Ala Arg Thr Phe Asp Thr Val Lys 145 150 155 160 Thr Lys Asp Leu Lys Ser Arg Ser Pro His Leu Asp Asp Cys Ser Lys 165 170 175 Thr Asp His Arg Ala Lys Ser Asp Val Ser Lys Asp Val His His Ser 180 185 190 Thr Ser Leu Pro Asn Leu Glu Lys Glu Gly Lys Pro His Ser Asp Lys 195 200 205 Arg Ser Thr Se r His Leu Pro Thr Ser Val Glu Lys His Cys Thr Asn 210 215 220 Gly Val Trp Ser Arg Ser His Tyr Gln Val Gly Glu Gly Ser Ser Asn 225 230 235 240 Glu Asp Ser Arg Arg Gly Arg Lys Asp Ile Arg His Ser Gln Phe Asn 245 250 255 Arg Gly Thr Glu Arg Val Arg Lys Asp Leu Ser Thr Gly Cys Gly Asp 260 265 270 Gly Glu Pro Arg Ile Leu Glu Ala Ser Gln Arg Leu Gln Gly His Pro 275 280 285 Glu Lys Tyr Gly Lys Gly Glu Pro Lys Thr Glu Ser Lys Ser Ser Lys 290 295 300 Phe Lys Ser Asn Ser Asp Ser Asp Tyr Lys Gly Glu Arg Ile Asn Ser 305 310 315 320 Ser Trp Glu Lys Glu Thr Pro Gly Glu Arg Ser His Ser Arg Val Asp 325 330 335 Ser Gln Ser Asp Lys Lys Leu Glu Arg Gln Ser Glu Arg Ser Gln Asn 340 345 350 Ile Asn Arg Lys Glu Val Lys Ser Gln Asp Lys Glu Glu Arg Lys Val 355 360 365 Asp Gln Lys Pro Lys Ser Val Val Lys Asp Gln Asp His Trp Arg Arg 370 375 380 Ser Glu Arg Ala Ser Leu Pro His Ser Lys Asn Glu Ile Thr Phe Ser 385 390 395 400 400 His Asn Ser Ser Lys Tyr His Leu Glu Glu Arg Arg Gly Trp Glu Asp 405 410 415 Cys Lys Arg As p Lys Ser Val Asn Ser His Ser Phe Gln Asp Gly Arg 420 425 430 Cys Pro Ser Ser Leu Ser Asn Ser Arg Thr His Lys Asn Ile Asp Ser 435 440 445 Lys Glu Val Asp Ala Met His Gln Trp Glu Asn Thr Pro Leu Lys Ala 450 455 460 Glu Arg His Arg Thr Glu Asp Lys Arg Lys Arg Glu Gln Glu Ser Lys 465 470 475 480 Glu Glu Asn Arg His Ile Arg Asn Glu Lys Arg Val Pro Thr Glu His 485 490 495 495 Leu Gln Lys Thr Asn Lys Glu Thr Lys Lys Thr Thr Thr Asp Leu Lys 500 505 510 510 Lys Gln Asn Glu Pro Lys Thr Asp Lys Gly Glu Val Leu Asp Asn Gly 515 520 525 Val Ser Glu Gly Ala Asp Asn Lys Glu Leu Ala Met Lys Ala Glu Ser 530 535 540 Gly Pro Asn Glu Thr Lys Asn Lys Asp Leu Lys Leu Ser Phe Met Lys 545 550 555 560 Lys Leu Asn Leu Thr Leu Ser Pro Ala Lys Lys Gln Pro Val Ser Gln 565 570 575 Asp Asn Gln His Lys Ile Thr Asp Ile Pro Lys Ser Ser Gly Val Cys 580 585 590 Asp Ser Glu Ser Ser Met Gln Val Lys Thr Val Ala Tyr Val Pro Ser 595 600 605 Ile Ser Glu His Ile Leu Gly Glu Ala Ala Val Ser Glu His Thr Met 610 615 620 620 Gly Glu Thr Lys Se r Thr Leu Leu Glu Pro Lys Val Ala Leu Leu Ala 625 630 635 640 Val Thr Glu Pro Arg Ile Gly Ile Ser Glu Thr Asn Lys Glu Asp Glu 645 650 655 Asn Ser Leu Leu Val Arg Ser Val Asp Asn Thr Met His Cys Glu Glu 660 665 670 Pro Ile Cys Gly Thr Glu Thr Ser Phe Pro Ser Pro Met Glu Ile Gln 675 680 685 Gln Thr Glu Ser Leu Phe Pro Ser Thr Gly Met Lys Gln Thr Ile Asn 690 695 700 Asn Gly Arg Ala Ala Ala Pro Val Val Met Asp Val Leu Gln Thr Asp 705 710 715 715 720 Val Ser Gln Asn Phe Gly Leu Glu Leu Asp Thr Lys Arg Asn Asp Asn 725 730 735 Ser Asp Tyr Cys Gly Ile Ser Glu Gly Met Glu Met Lys Val Ala Leu 740 745 750 Ser Thr Thr Val Val Ser Glu Thr Glu Ser Ile Leu Gln Pro Ser Ile 755 760 765 Glu Glu Ala Asp Ile Leu Pro Ile Met Leu Ser Glu Asp Asn Asn Pro 770 775 780 Lys Phe Glu Pro Ser Val Ile Val Thr Pro Leu Val Glu Ser Lys Ser 785 790 795 800 Cys His Leu Glu Pro Cys Leu Pro Lys Glu Thr Leu Asp Ser Ser Leu 805 810 815 Gln Gln Thr Glu Leu Met Asp His Arg Met Ala Thr Gly Glu Thr Asn 820 825 830 Ser Val Tyr His As p Asp Asp Asn Ser Val Leu Ser Ile Asp Leu Asn 835 840 845 His Leu Arg Pro Ile Pro Glu Ala Ile Ser Pro Leu Asn Ser Pro Val 850 855 860 Arg Pro Val Ala Lys Val Leu Arg Asn Glu Ser Pro Pro Gln Val Pro 865 870 875 880 Val Tyr Asn Asn Ser His Lys Asp Val Phe Leu Pro Asn Ser Ala His 885 890 895 Ser Thr Ser Lys Ser Gln Ser Asp Leu Asn Lys Glu Asn Gln Lys Pro 900 905 910 Ile Tyr Lys Ser Asp Lys Cys Thr Glu Ala Asp Thr Cys Lys Asn Ser 915 920 925 925 Pro Leu Asp Glu Leu Glu Glu Gly Glu Ile Arg Ser Asp Ser Glu Thr 930 935 940 Ser Lys Pro Gln Glu Glu Ser Phe Glu Lys Asn Ser Lys Arg Arg Val Ser 945 950 955 960 Ala Asp Val Arg Lys Ser Lys Thr Ile Pro Arg Arg Gly Lys Ser Thr 965 970 975 Val Cys Leu Asp Lys Asp Ser Arg Lys Thr His Val Arg Ile His Gln 980 985 990 Thr Asn Asn Lys Trp Asn Lys Arg Pro Asp Lys Ser Ser Arg Ser Ser 995 1000 1005 Lys Thr Glu Lys Lys Asp Lys Val Met Ser Thr Ser Ser Leu Glu Lys 1010 1015 1020 Ile Val Pro Ile Ile Ala Val Pro Ser Ser Glu Gln Glu Ile Met His 1025 1030 1035 1040 Met Leu A rg Met Ile Arg Lys His Val Arg Lys Asn Tyr Met Lys Phe 1045 1050 1055 Lys Ala Lys Phe Ser Leu Ile Gln Phe His Arg Ile Ile Glu Ser Ala 1060 1065 1070 Ile Leu Ser Phe Thr Ser Leu Ile Lys His Leu Asn Leu His Lys Ile 1075 1080 1085 Ser Lys Ser Val Thr Thr Leu Gln Lys Asn Leu Cys Asp Ile Ile Glu 1090 1095 1100 Ser Lys Leu Lys Gln Val Lys Lys Asn Gly Ile Val Asp Arg Leu Phe 1105 1110 1115 1120 Glu Gln Gln Leu Pro Asp Met Lys Lys Lys Leu Trp Lys Phe Val Asp 1125 1130 1135 Asp Gln Leu Asp Tyr Leu Phe Ala Lys Leu Lys Lys Ile Leu Val Cys 1140 1145 1150 Asp Ser Lys Ser Phe Gly Arg Asp Ser Asp Glu Gly Lys Leu Glu Lys 1155 1160 1165 Thr Ser Lys Gln Asn Ala Gln Tyr Ser Asn Ser Gln Lys Arg Ser Val 1170 1175 1180 Asp Asn Ser Asn Arg Glu Leu Leu Lys Glu Lys Leu Ser Lys Ser Glu 1185 1190 1195 1200 Asp Pro Val His Tyr Lys Ser Leu Val Gly Cys Lys Lys Ser Glu Glu 1205 1210 1215 Asn Tyr Gln Asp Gln Asn Asn Ser Ser Ile Asn Thr Val Lys His Asp 1220 1225 1230 Ile Lys Lys Asn Phe Asn Ile Cys Phe Asp Asn Ile Lys Asn Ser Gln 1235 1240 1245 Ser Glu Glu Arg Ser Leu Glu Val His Cys Pro Ser Thr Pro Lys Ser 1250 1255 1260 Glu Lys Asn Glu Gly Ser Ser Ile Glu Asp Ala Gln Thr Ser Gln His 1265 1270 1275 1280 Ala Thr Leu Lys Pro Glu Arg Ser Phe Glu Ile Leu Thr Glu Gln Gln 1285 1290 1295 Ala Ser Ser Leu Thr Phe Asn Leu Val Ser Asp Ala Gln Met Gly Glu 1300 1305 1310 Ile Phe Lys Ser Leu Leu Gln Gly Ser Asp Leu Leu Asp Ser Ser Val 1315 1320 1325 Asn Cys Thr Glu Lys Ser Glu Trp Glu Leu Lys Thr Pro Glu Lys Gln 1330 1335 1340 Leu Leu Glu Thr Leu Lys Cys Glu Ser Ile Pro Ala Cys Thr Thr Glu 1345 1350 1355 1360 Glu Leu Val Ser Gly Val Ala Ser Pro Cys Pro Lys Met Ile Ser Asp 1365 1370 1375 Asp Asn Trp Ser Leu Leu Ser Ser Glu Lys Gly Pro Ser Leu Ser Ser 1380 1385 1390 Gly Leu Ser Leu Pro Val His Pro Asp Val Leu Asp Glu Ser Cys Met 1395 1400 1405 Phe Glu Val Ser Thr Asn Leu Pro Leu Ser Lys Asp Asn Val Cys Ser 1410 1415 1420 Val Glu Lys Ser Lys Pro Cys Val Ser Ser Ile Leu Leu Glu Asp Leu 1425 1430 1435 1440 Ala Val S er Leu Thr Val Pro Ser Pro Leu Lys Ser Asp Gly His Leu 1445 1450 1455 Ser Phe Leu Lys Pro Asp Met Ser Ser Ser Ser Thr Thr Glu Glu Val 1460 1465 1470 Ile Ser Ala His Phe Ser Glu Asp Ala Leu Leu Glu Glu Glu Asp Ala 1475 1480 1485 Ser Glu Gln Asp Ile His Leu Ala Leu Glu Ser Asp Asn Ser Ser Ser 1490 1495 1500 Lys Ser Ser Cys Ser Ser Ser Trp Thr Ser Arg Ser Val Ala Pro Gly 1505 1510 1515 1520 Phe Gln Tyr His Pro Asn Leu Pro Met His Ala Val Ile Met Glu Lys 1525 1530 1535 Ser Asn Asp His Phe Ile Val Lys Ile Arg Arg Ala Thr Pro Ser Thr 1540 1545 1550 Ser Ser Gly Leu Lys Gln Ser Met Met Pro Asp Glu Leu Leu Thr Ser 1555 1560 1565 Leu Pro Arg His Gly Lys Glu Ala Asp Glu Gly Pro Glu Lys Glu Tyr 1570 1575 1580 Ile Ser Cys Gln Asn Thr Val Phe Lys Ser Val Glu Glu Leu Glu Asn 1585 1590 1595 1600 Ser Asn Lys Asn Val Asp Gly Ser Lys Ser Thr His Glu Glu Gln Ser 1605 1610 1615 Ser Met Ile Gln Thr Gln Val Pro Asp Ile Tyr Glu Phe Leu Lys Asp 1620 1625 1630 Ala Ser Asp Lys Met Gly His Ser Asp Glu Val Ala Asp Glu Cys Phe 1635 1640 1645 Lys Leu His Gln Val Trp Glu Thr Lys Val Pro Glu Ser Ile Glu Glu 1650 1655 1660 Leu Pro Ser Met Glu Glu Ile Ser His Ser Val Gly Glu His Leu Pro 1665 1670 1675 1680 Asn Thr Tyr Val Asp Leu Thr Lys Asp Pro Val Thr Glu Thr Lys Asn 1685 1690 1695 Leu Gly Glu Phe Ile Glu Val Thr Val Leu His Ile Asp Gln Leu Gly 1700 1705 1710 Cys Ser Gly Gly Asly Leu Asn Gln Ser Ala Gln Ile Leu Asp Asn Ser 1715 1720 1725 Leu Gln Ala Asp Thr Val Gly Ala Phe Ile Asp Leu Thr Gln Asp Ala 1730 1735 1740 Ser Ser Glu Ala Lys Ser Glu Gly Asn His Pro Ala Leu Ala Val Glu 1745 1750 1755 1760 Asp Leu Gly Cys Gly Val Ile Gln Val Asp Glu Asp Asn Cys Lys Glu 1765 1770 1775 Glu Lys Ala Gln Val Ala Asn Arg Pro Leu Lys Cys Ile Val Glu Glu 1780 1785 1790 Thr Tyr Ile Asp Leu Thr Thr Glu Ser Pro Ser Ser Cys Glu Val Lys 1795 1800 1805 Lys Asp Glu Leu Lys Ser Glu Pro Gly Ser Asn Cys Asp Asn Ser Glu 1810 1815 1820 Leu Pro Gly Thr Leu His Asn Ser His Lys Lys Arg Arg Asn Ile Ser 1825 1830 1835 1840 Asp Leu A sn His Pro His Lys Lys Gln Arg Lys Glu Thr Asp Leu Thr 1845 1850 1855 Asn Lys Glu Lys Thr Lys Lys Pro Thr Gln Asp Ser Cys Glu Asn Thr 1860 1865 1870 Glu Ala His Gln Lys Lys Ala Ser Lys Lys Lys Ala Pro Pro Val Thr 1875 1880 1885 Lys Asp Pro Ser Ser Leu Lys Ala Thr Pro Gly Ile Lys Asp Ser Ser 1890 1895 1900 Ala Ala Leu Ala Thr Ser Thr Ser Leu Ser Ala Lys Asn Val Ile Lys 1905 1910 1915 1920 Lys Lys Gly Glu Ile Ile Ile Leu Trp Thr Arg 1925 1930 <210> 3 <211> 5817 <212> DNA <213> Homosapiens <400> 3 ccttgtccat aaaatgataa tttctccctt ctttttaata acattttttg cagaaagact 60 tgtagaagtg gcaagtgctg ctgatgaatc cttaatccct ggggttgcct ttaatgatga 120 gggatcttta gtcacaggag gggccttctt cttactggct ttcttttggt gagcttcagt 180 attctcacaa gaatcttggg taggtttctt ggtcttttcc ttattagtta agtctgtttc 240 ctttctttgt tttttatgag gatgatttag atcagaaatg tttcttctct ttttgtgaga 300 attatgcaaa gtcccaggca actccgagtt atcacaattt gatcctggct ctgattttaa 360 ctcatctttt tttacttcac atgaactggg agattctgtg gtcaagtcga tataggtttc 420 ctcaacaatg cattttaaag gcctgtttgc cacttgtgcc ttttcttcct tacaattatc 480 ttcatctacc tgtatcaccc cacatcccaa gtcttccaca gctaatgcag gatgattacc 540 ttcactttta gcctcacttg aagcatcttg tgtcaaatca ataaaagcac ctacagtatc 600 agcctgcaaa gaattgtcta atatttgagc actctgattt aaattgcctc cagaacatcc 660 caactgatca atatgtaaaa ctgttacttc tatgaattcc cccaagtttt tggtttcagt 720 gactggatct ttcgttagat ctacgtatgt gtttggaaga tgttccccaa cagagtgtga 800 gatttcttcc attgaaggca attcttcaat gctttcaggc acttttgttt cccatacttg 860 atgcaatttg aa acattcat cagccacttc atcactatga cccatcttat ctgaagcatc 900 tttaagaaat tcatatatat caggaacctg tgtttgtatc atagagctct gttcttcatg 960 agttgacttg ctgccatcaa catttttgtt ggagttttcc aattcctcca cagatttaaa 1020 aactgtgttc tgacatgaaa tatattcttt ctctggtcct tcatcagctt cctttccatg 1080 tctgggcaaa gatgtcaata attcatcagg catcatactc tgtttaaggc cagaagaggt 1140 agatggtgtt gcacgtcgta ttttcacaat gaaatgatca ttggactttt ccattatgac 1200 ggcatgcata ggtagattag ggtggtactg aaagcctgga gcaacagatc ggcttgtcca 1260 ggaagaagaa caacttgatt tactgcttga attatcagac tccagagcta aatgaatatc 1320 ttgctcagat gcatcctctt cctcaagtaa ggcatcttca ctaaaatgag cactaatgac 1380 ttcttcagga gttgaactgg acgacatatc aggctttaaa aaactgagat gaccatctga 1440 cttcagaggc gatggtactg ttaaagagac tgctagatct tcaagaagta tggaagaaac 1500 gcagggcttg ctcttttcta cactacacac attatcttta cttaaaggta ggttagtaga 1560 cacttcaaac atacaacttt catccaacac atcaggatga accggcaatg aaagccctga 1620 agacagagat ggaccttttt cagatgataa taatgaccaa ttatcatcac taatcatttt 1680 aggacatgga gaagccaccc ctgaaactag ctcttctgtt gtacaagctg gtatagactc 1740 gcacttaaga gtctctagca gctgcttctc tggagtcttt aactcccact cacttttttc 1800 agtacagtta acactgctgt ctaaaagatc agaaccttgc aacaaacttt taaatatttc 1860 acccatttgt gcatcactca ctaaattaaa agtaaggctc gatgcttgct gttcggtaag 1920 aatctcgaaa cttcgttctg gcttcaaagt tgcatgctgg gatgtctgtg catcctctat 1980 gctgcttcct tcgttttttt ctgactttgg ggtgcttgga cagtgtactt ccaaggagcg 2040 ctcttcggat tgagagttct ttatattatc aaagcagatg ttaaaatttt ttttaatgtc 2100 atgctttaca gtgttaatac tggagttatt ttggtcttga taattttcct cagatttttt 2160 acatcccact aaagacttat aatgaacagg gtcttctgat tttgataatt tttctttcag 2220 caattctctg ttggagttgt ccacactcct tttctgacta tttgaatact gtgcattctg 2280 tttacttgtt ttttcaagtt tgccttcatc actatctctt ccaaagcttt tggaatcaca 2340 tactaagatt ttcttaagct ttgcaaacaa ataatcaagt tggtcatcta caaacttcca 2400 caattttttt ttcatatctg gtagttgctg ttcaaataaa cgatcaacta tgccattctt 2460 tttaacttgc ttaagtttag actctataat atcacagaga ttcttctgta aggtagtcac 2520 tgacttagag attttgtgta agttg agatg tttaattaaa gatgtaaaac tcaaaattgc 2580 tgactcaata attctgtgaa attgtattaa tgaaaatttt gccttgaatt tcatataatt 2640 ttttcttaca tgttttctta tcattcgtaa catgtgcatg atctcttgtt cagaagaggg 2700 tacagcaata attggaacta ttttttccaa gctggaagtg ctcatcactt tatctttctt 2760 ctctgttttt gaagatctgc tagatttatc aggtctttta ttccatttgt tattggtctg 2820 atggattctt acatgtgttt tcctactgtc tttatctaaa cacacagtac ttttcccacg 2880 tcgtgggata gtctttgact tccgcacatc agctgacact ctacgtttgg aatttttttc 2940 aaaactttct tgtggtttag atgtttcact atcacttcta atttctcctt cttctaattc 3000 atctaatggt gaattcttac atgtgtctgc ttctgtacat ttgtcagatt tgtaaattgg 3060 cttttgattt tccttattga gatcagactg actcttagag gtagaatgag ctgaatttgg 3120 taaaaacaca tctttatgac tgttattata cactggaact tgaggtgggc tttcatttct 3180 aagaactttt gctacaggtc tcactggact attcagagga ctgatggctt ctggaatagg 3240 tctcaggtga ttaaggtcaa tgctcaaaac cgagttatca tcatcatgat atactgagtt 3300 tgtttcacca gttgccattc tgtggtccat taactcagtc tgctgaagtg aagaatctag 3360 agtctcttta ggtaagcaag gctccagatg acacgactta ctctcaacca gtggtgtaac 3420 tataacagaa ggctcaaatt ttgggttatt atcttctgaa agcattattg gcaaaatatc 3480 agcttcctca attgaaggct gcaaaatgct ttcagtggtt tcactcactg ttgttgaaag 3540 tgccaccttc atctccatac cttcagaaat accacaataa tctgaattat catttctttt 3600 ggtatccaat tccaagccaa agttttgaga cacatctgtt tgtaatacat ccattaccac 3660 aggagctgct gcccttccat tattaatggt ttgtttcatt cctgttgatg gaaacaagga 3720 ttctgtctgt tgtatttcca taggagatgg gaaggaagtc tctgtaccac aaatgggctc 3780 ttcacaatgc atagtattgt caacagacct aactaacaaa ctattttcgt cttccttgtt 3840 ggtttctgag ataccgatcc tgggttcagt cactgctaga agagcaacct ttggttccaa 3900 taacgttgac ttggtttccc ccatggtatg ttcactgaca gctgcttccc ccaagatatg 3960 ttcacttatg gagggaacat atgccactgt tttaacttgc attgaagact ctgaatcaca 4020 tacaccactg gacttgggaa tatcagttat tttatgctga ttatcctggg aaacaggttg 4080 ctttttagca ggagaaagag ttaagttcaa ttttttcata aaactcaatt ttaggtcctt 4140 gttttttgtt tcatttggac cactctcagc tttcattgca agctctttat tatctgctcc 4200 ttcagaaaca ccattatcaa ggacttctcc cttatc agtc tttggttcat tctgtttctt 4260 taaatcagta gtggttttct tagtttcctt attagtcttc tgcaaatgtt ctgtaggtac 4320 tcttttttca tttctaatat gcctattttc ttctttgctt tcttgttctc ttttcctctt 4380 atcttcagtt ctatgtcttt ctgcttttaa aggtgtattt tcccactgat gcatggcatc 4440 aacttcctta gagtcaatgt ttttgtgagt tctactgttt gaaagagaag atggacatct 4500 tccatcttga aaactatgac tgtttacact cttgtctctt ttacaatctt cccatcctct 4560 tctctcttct agatggtatt tacttgaatt atgagaaaat gttatttcat tcttggaatg 4620 aggaagtgat gctcgttcag atcttctcca gtgatcttgg tcctttacta ctgatttagg 4680 tttttgatca acttttcttt cttctttgtc ttgtgattta acttctttcc tatttatatt 4740 ttgtgatctt tcactttgtc tttctagttt tttgtcactt tgagagtcta ctcgactgtg 4800 tgacctttct ccaggggtct ctttctccca agaagagtta atgcgttcac ctttatagtc 4860 agaatctgag ttacttttaa acttcgaact tttgctttca gtctttggtt cacctttacc 4920 atatttctca ggatgtcctt gtagcctttg actagcctcc aatatccttg gttcaccatc 4980 accacagcca gtacttaagt cttttcgtac tctttcagtt cctctgttaa actggctatg 5040 tctaatatct tttcttcctc ttctactatc ctcatttgag c taccctcgc caacctgata 5100 atgagaacgt gaccaaacac cattagtgca gtgtttctca acagatgtag gtaaatgtga 5160 agtactcctt ttatcagaat gtggttttcc ttccttttcc agatttggca gtgaagtgct 5220 atgatgtaca tctttagaaa catcactttt agctctgtga tcagtctttg aacaatcatc 5280 caaatgtgga gatctagatt taagatcttt tgttttaact gtatcaaatg tccttgcagt 5340 tttatgatta tttcgaaaat gtggaaactc agacaatctt tggtgaagat tacttatttc 5400 ttcatccttg cggtttattt ccactctggc agttttgata agtgctgaaa tattcttctt 5460 aagagactgg ttttcgttta ttaagctgaa attctgtgtc tgtatttctt taaatttttt 5520 catcagctct ttcatttgta gttgacattt tccatattct acttgcaaat cattatatgt 5580 tgcctccttt gcagttcctt cttcagtcag gatctcttca tataagtcca aacaatttct 5640 tgatggtaca caggatttgg aagcgctgtc agaaacagca ctgtccaacc cagcgtatat 5700 gtccagtgag ccttcatcat tgttcttaag aggagaagca gaaaagacat caaataaact 5760 tgttccatca ccattgtcat catctgctgc catgatccta tttccttttg agagtca 5817 <210> 4 <211> 5817 <212> RNA <213> Homosapiens <400> 4 ccuuguccau aaaaugauaa uuucucccuu cuuuuuaaua acauuuuuug cagaaagacu 60 uguagaagug gcaagugcug cugaugaauc cuuaaucccu gggguugccu uuaaugauga 120 gggaucuuua gucacaggag gggccuucuu cuuacuggcu uucuuuuggu gagcuucagu 180 auucucacaa gaaucuuggg uagguuucuu ggucuuuucc uuauuaguua agucuguuuc 240 cuuucuuugu uuuuuaugag gaugauuuag aucagaaaug uuucuucucu uuuugugaga 300 auuaugcaaa gucccaggca acuccgaguu aucacaauuu gauccuggcu cugauuuuaa 360 cucaucuuuu uuuacuucac augaacuggg agauucugug gucaagucga uauagguuuc 420 cucaacaaug cauuuuaaag gccuguuugc cacuugugcc uuuucuuccu uacaauuauc 480 uucaucuacc uguaucaccc cacaucccaa gucuuccaca gcuaaugcag gaugauuacc 540 uucacuuuua gccucacuug aagcaucuug ugucaaauca auaaaagcac cuacaguauc 600 agccugcaaa gaauugucua auauuugagc acucugauuu aaauugccuc cagaacaucc 660 caacugauca auauguaaaa cuguuacuuc uaugaauucc cccaaguuuu ugguuucagu 720 gacuggaucu uucguuagau cuacguaugu guuuggaaga uguuccccaa cagaguguga 780 gauuucuucc auugaaggca auucuucaau gcuuucaggc acuuuuguuu cccauacuug 840 augcaauuug aa acauucau cagccacuuc aucacuauga cccaucuuau cugaagcauc 900 uuuaagaaau ucauauauau caggaaccug uguuuguauc auagagcucu guucuucaug 960 aguugacuug cugccaucaa cauuuuuguu ggaguuuucc aauuccucca cagauuuaaa 1020 aacuguguuc ugacaugaaa uauauucuuu cucugguccu ucaucagcuu ccuuuccaug 1080 ucugggcaaa gaugucaaua auucaucagg caucauacuc uguuuaaggc cagaagaggu 1140 agaugguguu gcacgucgua uuuucacaau gaaaugauca uuggacuuuu ccauuaugac 1200 ggcaugcaua gguagauuag ggugguacug aaagccugga gcaacagauc ggcuugucca 1260 ggaagaagaa caacuugauu uacugcuuga auuaucagac uccagagcua aaugaauauc 1320 uugcucagau gcauccucuu ccucaaguaa ggcaucuuca cuaaaaugag cacuaaugac 1380 uucuucagga guugaacugg acgacauauc aggcuuuaaa aaacugagau gaccaucuga 1440 cuucagaggc gaugguacug uuaaagagac ugcuagaucu ucaagaagua uggaagaaac 1500 gcagggcuug cucuuuucua cacuacacac auuaucuuua cuuaaaggua gguuaguaga 1560 cacuucaaac auacaacuuu cauccaacac aucaggauga accggcaaug aaagcccuga 1620 agacagagau ggaccuuuuu cagaugauaa uaaugaccaa uuaucaucac uaaucauuuu 1680 aggacaugga gaagccaccc cugaaacuag cucuucuguu guacaagcug guauagacuc 1740 gcacuuaaga gucucuagca gcugcuucuc uggagucuuu aacucccacu cacuuuuuuc 1800 aguacaguua acacugcugu cuaaaagauc agaaccuugc aacaaacuuu uaaauauuuc 1860 acccauuugu gcaucacuca cuaaauuaaa aguaaggcuc gaugcuugcu guucgguaag 1920 aaucucgaaa cuucguucug gcuucaaagu ugcaugcugg gaugucugug cauccucuau 1980 gcugcuuccu ucguuuuuuu cugacuuugg ggugcuugga caguguacuu ccaaggagcg 2040 cucuucggau ugagaguucu uuauauuauc aaagcagaug uuaaaauuuu uuuuaauguc 2100 augcuuuaca guguuaauac uggaguuauu uuggucuuga uaauuuuccu cagauuuuuu 2160 acaucccacu aaagacuuau aaugaacagg gucuucugau uuugauaauu uuucuuucag 2220 caauucucug uuggaguugu ccacacuccu uuucugacua uuugaauacu gugcauucug 2280 uuuacuuguu uuuucaaguu ugccuucauc acuaucucuu ccaaagcuuu uggaaucaca 2340 uacuaagauu uucuuaagcu uugcaaacaa auaaucaagu uggucaucua caaacuucca 2400 caauuuuuuu uucauaucug guaguugcug uucaaauaaa cgaucaacua ugccauucuu 2460 uuuaacuugc uuaaguuuag acucuauaau aucacagaga uucuucugua agguagucac 2320 ugacuuagag auuuugugua aguug agaug uuuaauuaaa gauguaaaac ucaaaauugc 2380 ugacucaaua auucugugaa auuguauuaa ugaaaauuuu gccuugaauu ucauauaauu 2440 uuuucuuaca uguuuucuua ucauucguaa caugugcaug aucucuuguu cagaagaggg 2700 uacagcaaua auuggaacua uuuuuuccaa gcuggaagug cucaucacuu uaucuuucuu 2760 cucuguuuuu gaagaucugc uagauuuauc aggucuuuua uuccauuugu uauuggucug 2820 auggauucuu acauguguuu uccuacuguc uuuaucuaaa cacacaguac uuuucccacg 2880 ucgugggaua gucuuugacu uccgcacauc agcugacacu cuacguuugg aauuuuuuuc 2940 aaaacuuucu ugugguuuag auguuucacu aucacuucua auuucuccuu cuucuaauuc 3000 aucuaauggu gaauucuuac augugucugc uucuguacau uugucagauu uguaaauugg 3060 cuuuugauuu uccuuauuga gaucagacug acucuuagag guagaaugag cugaauuugg 3120 uaaaaacaca ucuuuaugac uguuauuaua cacuggaacu ugaggugggc uuucauuucu 3180 aagaacuuuu gcuacagguc ucacuggacu auucagagga cugauggcuu cuggaauagg 3240 ucucagguga uuaaggucaa ugcucaaaac cgaguuauca ucaucaugau auacugaguu 3300 uguuucacca guugccauuc ugugguccau uaacucaguc ugcugaagug aagaaucuag 3360 agucucuuua gguaagcaag gcuccagaug acacgacuua cucucaacca gugguguaac 3420 uauaacagaa ggcucaaauu uuggguuauu aucuucugaa agcauuauug gcaaaauauc 3480 agcuuccuca auugaaggcu gcaaaaugcu uucagugguu ucacucacug uuguugaaag 3540 ugccaccuuc aucuccauac cuucagaaau accacaauaa ucugaauuau cauuucuuuu 3600 gguauccaau uccaagccaa aguuuugaga cacaucuguu uguaauacau ccauuaccac 3660 aggagcugcu gcccuuccau uauuaauggu uuguuucauu ccuguugaug gaaacaagga 3720 uucugucugu uguauuucca uaggagaugg gaaggaaguc ucuguaccac aaaugggcuc 3780 uucacaaugc auaguauugu caacagaccu aacuaacaaa cuauuuucgu cuuccuuguu 3840 gguuucugag auaccgaucc uggguucagu cacugcuaga agagcaaccu uugguuccaa 3900 uaacguugac uugguuuccc ccaugguaug uucacugaca gcugcuuccc ccaagauaug 3960 uucacuuaug gagggaacau augccacugu uuuaacuugc auugaagacu cugaaucaca 4020 uacaccacug gacuugggaa uaucaguuau uuuaugcuga uuauccuggg aaacagguug 4080 cuuuuuagca ggagaaagag uuaaguucaa uuuuuucaua aaacucaauu uuagguccuu 4140 guuuuuuguu ucauuuggac cacucucagc uuucauugca agcucuuuau uaucugcucc 4200 uucagaaaca ccauuaucaa ggacuucucc cuuauc aguc uuugguucau ucuguuucuu 4260 uaaaucagua gugguuuucu uaguuuccuu auuagucuuc ugcaaauguu cuguagguac 4320 ucuuuuuuca uuucuaauau gccuauuuuc uucuuugcuu ucuuguucuc uuuuccucuu 4380 aucuucaguu cuaugucuuu cugcuuuuaa agguguauuu ucccacugau gcauggcauc 4440 aacuuccuua gagucaaugu uuuugugagu ucuacuguuu gaaagagaag auggacaucu 4500 uccaucuuga aaacuaugac uguuuacacu cuugucucuu uuacaaucuu cccauccucu 4560 ucucucuucu agaugguauu uacuugaauu augagaaaau guuauuucau ucuuggaaug 4620 aggaagugau gcucguucag aucuucucca gugaucuugg uccuuuacua cugauuuagg 4680 uuuuugauca acuuuucuuu cuucuuuguc uugugauuua acuucuuucc uauuuauauu 4740 uugugaucuu ucacuuuguc uuucuaguuu uuugucacuu ugagagucua cucgacugug 4800 ugaccuuucu ccaggggucu cuuucuccca agaagaguua augcguucac cuuuauaguc 4860 agaaucugag uuacuuuuaa acuucgaacu uuugcuuuca gucuuugguu caccuuuacc 4920 auauuucuca ggauguccuu guagccuuug acuagccucc aauauccuug guucaccauc 4980 accacagcca guacuuaagu cuuuucguac ucuuucaguu ccucuguuaa acuggcuaug 5040 ucuaauaucu uuucuuccuc uucuacuauc cucauuugag c uacccucgc caaccugaua 5100 augagaacgu gaccaaacac cauuagugca guguuucuca acagauguag guaaauguga 5160 aguacuccuu uuaucagaau gugguuuucc uuccuuuucc agauuuggca gugaagugcu 5220 augauguaca ucuuuagaaa caucacuuuu agcucuguga ucagucuuug aacaaucauc 5280 caaaugugga gaucuagauu uaagaucuuu uguuuuaacu guaucaaaug uccuugcagu 5340 uuuaugauua uuucgaaaau guggaaacuc agacaaucuu uggugaagau uacuuauuuc 5400 uucauccuug cgguuuauuu ccacucuggc aguuuugaua agugcugaaa uauucuucuu 5460 aagagacugg uuuucguuua uuaagcugaa auucuguguc uguauuucuu uaaauuuuuu 5520 caucagcucu uucauuugua guugacauuu uccauauucu acuugcaaau cauuauaugu 5580 ugccuccuuu gcaguuccuu cuucagucag gaucucuuca uauaagucca aacaauuucu 5640 ugaugguaca caggauuugg aagcgcuguc agaaacagca cuguccaacc cagcguauau 5700 guccagugag ccuucaucau uguucuuaag aggagaagca gaaaagacau caaauaaacu 5760 uguuccauca ccauugucau caucugcugc caugauccua uuuccuuuug agaguca 5817 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 ccgtcgacag atggacttca gcagaaatc 29 <210> 6 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 gggtcgactc atttgctgaa ttcttcatag tcg 33 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 7 gggaattcag aaaggaagta cagaaaacat 30 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 8 gggaattcca ctctagacca agctttgg 28 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 9 cggaattcat ggacccgttc ctggtgc 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 10 gggaattcca tcaggacgct tcggagg 27 <210> 11 <211> 1961 <212> DNA <213> Homosapiens <400> 11 gaattcgcgg ccgcgtcgac atgaaattca aggcaaaatt ttcattaata caatttcaca 60 gaattattga gtcagcaatt ttgagtttta catctttaat taaacatctc aacttacaca 120 aaatctctaa gtcagtgact accttacaga agaatctctg tgatattata gagtctaaac 180 ttaagcaagt taaaaagaat ggcatagttg atcgtttatt tgaacagcaa ctaccagata 240 tgaaaaaaaa attgtggaag tttgtagatg accaacttga ttatttgttt gcaaagctta 300 agaaaatctt agtatgtgat tccaaaagct ttggaagaga tagtgatgaa ggcaaacttg 360 aaaaaacaag taaacagaat gcacagtatt caaatagtca gaaaaggagt gtggacaact 420 ccaacagaga attgctgaaa gaaaaattat caaaatcaga agaccctgtt cattataagt 480 ctttagtggg atgtaaaaaa tctgaggaaa attatcaaga ccaaaataac tccagtatta 540 acactgtaaa gcatgacatt aaaaaaaatt ttaacatctg ctttgataat ataaagaact 600 ctcaatccga agagcgctcc ttggaagtac actgtccaag caccccaaag tcagaaaaaa 660 acgaaggaag cagcatagag gatgcacaga catcccagca tgcaactttg aagccagaac 720 gaagtttcga gattcttacc gaacagcaag catcgagcct tacttttaat ttagtgagtg 780 atgcacaaat gggtgaaata tttaaaagtt tgttgcaagg ttctgatctt ttagacagca 840 gtgttaactg t actgaaaaa agtgagtggg agttaaagac tccagagaag cagctgctag 900 agactcttaa gtgcgagtct ataccagctt gtacaacaga agagctagtt tcaggggtgg 960 cttctccatg tcctaaaatg attagtgatg ataattggtc attattatca tctgaaaaag 1020 gtccatctct gtcttcaggg ctttcattgc cggttcatcc tgatgtgttg gatgaaagtt 1080 gtatgtttga agtgtctact aacctacctt taagtaaaga taatgtgtgt agtgtagaaa 1140 agagcaagcc ctgcgtttct tccatacttc ttgaagatct agcagtctct ttaacagtac 1200 catcgcctct gaagtcagat ggtcatctca gttttttaaa gcctgatatg tcgtccagtt 1260 caactcctga agaagtcatt agtgctcatt ttagtgaaga tgccttactt gaggaagagg 1320 atgcatctga gcaagatatt catttagctc tggagtctga taattcaagc agtaaatcaa 1380 gttgttcttc ttcctggaca agccgatctg ttgctccagg ctttcagtac caccctaatc 1440 tacctatgca tgccgtcata atggaaaagt ccaatgatca tttcattgtg aaaatacgac 1500 gtgcaacacc atctacctct tctggcctta aacagagtat gatgcctgat gaattattga 1560 catctttgcc cagacatgga aaggaagctg atgaaggacc agagaaagaa tatatttcat 1620 gtcagaacac agtttttaaa tctgtggagg aattggaaaa ctccaacaaa aatgttgatg 1680 gcagcaagtc aactcatga a gaacagagct ctatgataca aacacaggtt cctgatatat 1740 atgaatttct taaagatgct tcagataaga tgggtcatag tgatgaagtg gctgatgaat 1800 gtttcaaatt gcatcaagta tgggaaacaa aagtgcctga aagcattgaa gaattgcctt 1860 caatggaaga aatctcacac tctgttgggg aacatcttcc aaacacatac gtagatctaa 1940 cgaaagatcc agtcactgaa accaaaaact tgggggaatt c 1961

[Brief description of the drawings]

FIG. 1 shows a restriction map of pBS / library-1. The library-1 gene uses this vector as the restriction enzyme EcoRI
And can be isolated by cleavage. In the figure
Amp indicates the ampicillin resistance gene, and ColEI indicates the origin of replication of colicin EI.

FIG. 2 is a diagram showing the expression of aip-1 in various human organs analyzed using a Northern blotting technique. Membrane uses Clonetech human MTN blot,
In each lane, lane 1: pancreas, lane 2: kidney, lane 3: skeletal muscle, lane 4: liver, lane 5: lung, lane 6: placenta, lane 7: brain, lane 8: poly from heart
(A) 2 μg of each RNA is blotted. A band reacting with the aip-1 probe is observed at the position indicated by the arrow.

FIG. 3 is a diagram showing a restriction map of pcDNA3 / aip-1.
The aip-1 gene ligates this vector with the restriction enzymes BamHI and NotI.
And can be isolated by cleavage. In the figure
Amp is the ampicillin resistance gene, ColEI is the replication origin of colicin EI, Neo is the neomycin resistance gene, and PCV is the cytomegalovirus-derived promoter.

[FIG. 4] The inhibitory effect of the protein encoded by aip-1 on anti-Fas antibody-dependent apoptosis was analyzed. 29
3 Add 0.2μg pcDNA3 / lacZ and pcDNA3 / aip-1 1.8 to cells.
μg mixture (lane aip-1) or pcDNA3 / la
A mixture of 0.2 μg cZ and 31.8 μg pcDNA (lane empty
) Was introduced and cultured for 24 hours. Then 50, 100, 200ng /
After administration of ml of anti-Fas antibody CH11 and further culturing for 24 hours, the cells were fixed and stained. The figure shows apoptosis
It shows the number of cells detached from the culture dish.

[FIG. 5] The inhibitory effect of the protein encoded by aip-1 on anti-Fas antibody-dependent apoptosis was analyzed. 29
3 Add 0.2μg pcDNA3 / lacZ and pcDNA3 / aip-1 1.8 to cells.
μg mixture (lane aip-1) or pcDNA3 / la
A mixture of 0.2 μg cZ and 31.8 μg pcDNA (lane empty
) Was introduced and cultured for 24 hours. Then 50, 100, 200ng /
After administration of ml of anti-Fas antibody CH11 and further culturing for 24 hours, the cells were fixed and stained. The figure shows the percentage of cells on the culture dish that stained blue and showed an apoptotic morphology.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 ZNA C12Q 1/68 A C12P 21/02 A61K 37/02 21/08 C12N 5/00 B C12Q 1/68 15/00 ZNAA

Claims (2)

[Claims] 1. A death effector domain which binds to caspase-8 and binds to caspase-8.
Protein without. 2. A protein represented by the following (a) or (b): (a) a protein having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing; (b) an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having apoptosis controlling ability.