JPH11235187A - Cyp7 promotor binding factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an isolated polypeptide CYP7 promotor binding factor which can regulate activation of transcription, and is useful for screening of pharmacological substances, diagnosis, and treatment by making the isolated polypeptide include a specific amino acid sequence. SOLUTION: This factor is obtained by including the sequence shown by formula I, II, or III, or at least ten-residue domain of formula I containing at least one of residues 1-10, 11-15, 16-21, 204-207, and 299-307, ten-residue domain of formula II containing residue 154, or ten-residue domain of formula III containing at least one of residues 3-10, 13-22, and 30-38. It is preferable that the domain binds specifically with CYP7 gene promotor, and that SPF polypeptide is produced by culturing host cells transformed by a vector containing a recombinant nucleic acid coding for the isolated polypeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The field of the invention is transcription factors that bind to the CYP7 promoter.

[0002]

BACKGROUND OF THE INVENTION In mammalian cells, cholesterol is an essential element for membrane formation and for the synthesis of sterols and non-sterols essential for normal cell function. However, excess cholesterol is not only lethal to cells, but also creates a major problem of atherosclerosis because it deposits in arteries. To maintain cholesterol homeostasis, cells, especially hepatocytes, have three main ways to regulate cholesterol levels: 1) LDL
Uptake of dietary cholesterol via the receptor;
It employs 2) endogenous cholesterol biosynthesis and 3) metabolic conversion of cholesterol to bile acids.
A key molecule that regulates these processes is cholesterol itself, which acts as a feedback signal. When intracellular cholesterol levels are increased, either by cholesterol uptake or biosynthesis, L
Transcription of DL receptors and genes containing important cholesterol biosynthetic enzymes such as HMG-CoA synthase and HMG-CoA reductase is suppressed. These feedback processes are mediated by a novel family of transcription factors called sterol regulatory element binding proteins (SREBPs). SREBP is N
It contains a terminal transcription factor domain, two hydrophobic transmembrane domains and a C-terminal regulatory domain. When intracellular cholesterol levels are low, a two-step proteolytic cascade occurs, which releases the N-terminal transcription factor domain of SREBP from the endoplasmic reticulum to the nucleus where activation of SRE-containing genes occurs.

[0003] Although the SREBP pathway is responsible for the uptake of cholesterol, such as the LDL receptor and HMG-CoA synthase, and the regulation of genes involved in cholesterol biosynthesis, the molecular basis of cholesterol catabolism is poorly understood. The main catabolic pathway for cholesterol removal is the production of bile acids that occurs only in the liver. Cholesterol 7α-hydroxylase is the first and rate-limiting enzyme in this pathway. The cholesterol 7α hydroxylase gene, also known as CYP7, belongs to the cytochrome P-450 family, which contains many microsomal enzymes involved in liver metabolism. CYP
The expression of 7 genes has been shown to be tightly regulated: it is expressed only in the liver, and its expression can be induced by dietary cholesterol and suppressed by bile acids. Cholesterol catabolism has been shown to play a central role in cholesterol homeostasis. Treatment of experimental animals with two bile acid binding resins, cholestide or cholestyramine, reduces serum cholesterol levels. Furthermore, overexpression of the CYP7 gene in hamsters reduces total and LDL cholesterol levels. Therefore, cholesterol 7α-hydroxylase is a potent therapeutic target for cholesterol-lowering drugs, and understanding the mechanisms by which CYP7 gene expression is regulated is of particular importance.

To study the molecular mechanism of liver-specific expression of the human CYP7 gene, the present inventors used He as a model system.
pG2 cells were used, which is one of the best studied hepatic cell lines, and also suitable for cell lines through the study of several liver-specific genes, including the CYP7 gene. This is because the line is indicated. We started with a DNase I hypersensitive mapping of the human CYP7 promoter and identified liver-specific elements of this promoter. As a result, the present inventors cloned a gene encoding this promoter binding protein and identified it as a human ortholog of the nuclear orphan receptor Ftz-F1 family.

Related Art Garano and Villanger (1997), unpublished, accession number U93553, describe a human α1-fetoprotein transcription factor (hFTF, SEQ ID NOs: 7 and 8); JD Tagwood, I. Isseman and S. Green (1991), unpublished, accession number M81385 is mouse liver receptor homologous protein (LRH-1) mRNA
And putative translations (mLRH, SEQ ID NOs: 9 and 10); and L. Garrano et al.
6), Mol. Cell Biol., 16: 3853-3865, discloses partial rat genes; they all have sequence similarity to the CPF polypeptides disclosed herein.

[0006]

SUMMARY OF THE INVENTION The present invention relates to isolated CPF polypeptides, related nucleic acids, their polypeptide domains having CPF-specific structure and activity, and modulators of CPF function, particularly CYP7 promoter binding. Methods and compositions that provide CPF polypeptides can regulate the activation of genes linked to the CYP7 promoter, and thus can be important regulators of cell function. The polypeptide may be produced by recombinant methods from a host cell transformed with the nucleic acid encoding the CPF polypeptide, or by purification from mammalian cells. The present invention relates to an isolated CPF capable of specifically hybridizing with the CPF gene disclosed herein.
Hybridization probes and primers, CPF-specific binding agents such as specific antibodies, and the compositions are prepared and used for diagnosis (eg, gene hybridization screening of CPF transcripts), therapy (eg,
CP for activating CYP7 promoter-dependent transcription
F activator) and methods for use in the biopharmaceutical industry (eg, immunogens, reagents for separating other transcriptional regulators, reagents for screening lead pharmacological substances from chemical libraries, etc.). I do.

[0007]

SUMMARY OF THE INVENTION The nucleotide sequences of the native cDNA encoding the human CPF polypeptide are shown in SEQ ID NOs: 1, 3 and 5, with the predicted complete translations as SEQ ID NOs: 2, 4 and 6. Shown respectively. The CPF polypeptide according to the present invention has SEQ ID NO: 2, 4 or 6.
Comprises one or more functional domains of at least 8, preferably at least 1
It comprises 6, more preferably at least 32, most preferably at least 64 contiguous SEQ ID NO: 2, 4 or 6 residues and has human CPF-specific amino acid sequence and activity. CPF domain-specific activity refers to CYP
7 promoter binding or transactivation activity and C
Includes PF-specific immunogenicity and / or antigenicity.
CPF-specific polypeptide sequences include hFTF and mLR
H (SEQ ID NOs: 8 and 10) and is easily identified by sequence comparison. See, for example, Sequences 1, 2 and 3 herein. Examples of sequences include residues 1-10, 11
-15, 16-21, 204-207 and 299-3
07, a 10-residue domain of SEQ ID NO: 4 including residues 154, and residues 3-10, 13-22 and 30
Includes a 10 residue domain of SEQ ID NO: 6 that includes at least one of -38.

[0008] CPF-specific activity or function is simple,
In vitro, cell-based assays or in vivo assays (eg, in vitro binding assays, cell culture assays, in animals (eg, gene therapy, transgenic animals, etc.)). Binding assays include any assay in which the molecular interaction between a CPF polypeptide and a binding target is evaluated. The binding target may be a naturally occurring intracellular binding target, eg, a CYP7 promoter binding site, a CPF regulatory protein or other modulator that directly regulates CPF activity or its localization; or a non-naturally occurring binding target, eg, an antibody. Or a synthetic nucleic acid binding site (see consensus sequences below), or a CPF-specific substance as identified in a screening assay as described below. The CPF binding specificity is determined by the binding equilibrium constant (typically at least about 10 ⁷ M ^-1 , preferably at least about 10 ⁸ M ^-1 , more preferably at least about 10 ⁸ M ^-1 .
⁹ M ^-1 ), the ability of the polypeptide to function as a negative mutant in CPF-expressing cells by CYP7 or synthetic binding site reporter expression is CPF-specific in heterologous hosts (eg, rodents or rabbits). It can be assayed by the ability to induce antibodies. For example, in this way, the domain defined by residues 33-123 of SEQ ID NO: 2 is found to be a functional DNA binding domain, and the domain defined by residues 242-333 and 383-405 of SEQ ID NO: 2 Is a functional ligand binding domain.

In one particular embodiment, functional CPF domains that bind to the CYP7 promoter element are defined using deletion mutagenesis (see Examples below). See, for example, Table 1.

[0010]

TABLE 1 TABLE 1: CPF functional examples of CPF deletion mutant domains defining the mutant sequence D NA binding △ N1 SEQ ID NO: 2, residues 4-495 + △ N2 SEQ ID NO: 2, residues 12-494 + ΔN3 SEQ ID NO: 2, residues 24-495 + ΔN4 SEQ ID NO: 2, residues 33-495 + ΔN5 SEQ ID NO: 2, residues 33-123 + ΔC1 SEQ ID NO: 2, residues 1-408 + Δ C2 SEQ ID NO: 2, residues 1-335 + ΔC3 SEQ ID NO: 2, residues 1-267 + ΔC4 SEQ ID NO: 2, residues 1-189 + ΔC5 SEQ ID NO: 2, residues 1-124 +

[0011] In one particular embodiment, the domain provides a CPF-specific antigen and / or immunogen, particularly when bound to a carrier protein. For example, peptides corresponding to CPF- and human CPF-specific domains are covalently linked to keyhole limpet antigen (KLH), and the conjugate is emulsified in complete Freund's adjuvant. Study rabbits are immunized and bled according to routine protocols. CP
The presence of F-specific antibodies is assayed by a solid phase immunosorbent assay using the immobilized CPF polypeptide of SEQ ID NO: 2, 4, or 6 (see, eg, Table 2).

[0012]

Table 2: Immunogenic CPF polypeptide that induces CPF-specific rabbit polyclonal antibody: CPF polypeptide-KLH conjugated CPF polypeptide sequence immunized according to the above protocol Immunogenic SEQ ID NO: 2, residues 1-10 ++ SEQ ID NO: 2, residues 4-15 ++ SEQ ID NO: 2, residues 8-20 ++++ SEQ ID NO: 2, residues 12-25 ++++ SEQ ID NO: 2, residues 15-30 ++++ SEQ ID NO: 2, residues 19-32 ++ SEQ ID NO: 2, residues 20-29 ++++ SEQ ID NO: 2, residues 200-211 ++ SEQ ID NO: 4, residues 150-159 ++++

[0013] The claimed CPF polypeptides are isolated or pure: an "isolated" polypeptide does not have at least some of its associated substances in its natural state; Preferably at least about 0.5% of the total polypeptide in a given sample,
More preferably, it comprises at least about 5% (by weight), and pure polypeptide comprises at least about 90%, preferably at least about 99%, by weight of the total polypeptide in a given sample. I do. CPF polypeptides and polypeptide domains can be synthesized, produced by recombinant technology, or purified from mammalian, preferably human, cells. A wide variety of molecular biological and biochemical methods are available for biochemical synthesis, molecular expression and purification of the composition. For example, Molecular Cloning, A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory), Current
See Protocol in Molecular Biology (Ed. Ausubel et al., Green Publ. Assoc., Willey-Interscience, NY) or other literature known in the art.

The present invention provides an agonist, an antagonist,
CPF polypeptides, including naturally occurring intracellular binding targets and the like, preferably binding agents specific for the claimed CPF polypeptides, methods of identifying and making such binding agents, and diagnostic, therapeutic and pharmaceutical products Provide their use in development. For example, a specific binding substance is
It is useful for a variety of diagnostic and therapeutic applications, especially when the disease or prognosis of the disease is associated with inappropriate use of pathways involving the protein, such as CYP7 promoter-dependent transcriptional activation. Novel CPF-specific binding substances include C
PF-specific receptor / CPF-specific binding protein, eg, a polypeptide receptor produced by somatic recombination such as a specific antibody or T cell antigen receptor (eg, Harrow and Rein (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binders identified by assays such as 1, 2 and 3 hybrid screening, non-natural cells identified by screening chemical libraries as described below. Internal binding substances and the like. Of particular interest are those that modulate CPF function, eg, CPF-dependent transcriptional activity.

Accordingly, the present invention provides a method for regulating signaling involving a CPF or CYP7 promoter in a cell, comprising the step of regulating CPF activity. The cells may be in culture or in situ, i.e., present inside a natural host. For diagnostic applications, the CPF binding agent may be conjugated directly to the binding agent, or conjugated to a probe specific for the binding agent, eg, fluorescent, radioactive, chemiluminescent, or other readily detectable. Often labeled with a molecule that can. Examples of inhibitors include nucleic acids encoding dominant / negative mutant forms of CPF as described above.

Using the amino acid sequences of the disclosed CPF polypeptides, a CP optimized for an expression system of choice.
Translation back to a nucleic acid encoding the F polypeptide (Horror et al. (1993), Gene, 136, 323-328).
Page; Martin et al. (1995), Gene, 154, 150
-Page 166) or to create probes and degenerate oligonucleotide primers for use in isolating nucleic acid sequences encoding native CPF ("GCG" software, Genetics Computer Group, In.
c., Madison, WI). The nucleic acid encoding CPF is C
Used in PF expression vectors and incorporated into recombinant host cells for expression and screening, for example, to study functional studies such as the efficacy of candidate drugs for diseases associated with cell functions regulated by CPF. For transgenic animals.

Furthermore, the present invention relates to SEQ ID NO: 1, 3 or 5
At least 12, preferably at least 24, more preferably at least 36, which hybridizes specifically with a second nucleic acid comprising the complementary strand of hFTF and mLRH cDNA (SEQ ID NOs: 7 and 9).
And most preferably, it provides a replication / amplification primer and a nucleic acid hybridization probe having a CPF cDNA specific sequence comprising at least 96 contiguous bases of SEQ ID NO: 1, 3 or 5. Such a CP
F-specific sequences can be easily identified by sequence comparison. See, for example, sequences 4 and 5 herein. Demonstrating specific hybridization generally requires stringent conditions, such as, for example,
5xSSPE (0.18 M NaCl, 0.01 M Na
PO ₄ , pH 7.7, 0.001 MEDTA) Hybridization at a temperature of 42 ° C. in a buffer containing 30% formamide in a buffer and washing at 42 ° C. with 0.2 × SSPE, the binding persists, preferably Hybridization is carried out at 42 ° C. in a buffer containing 50% formamide in 5 × SSPE buffer at 42 ° C. and the binding is sustained when subjected to washing with 0.2 × SSPE buffer at 42 ° C.

[0018]

Table 3: Examples of CPF nucleic acids that hybridize to the strands of SEQ ID NO: 1, 3 and / or 5 under conditions I and / or II CPF nucleic acid hybridization SEQ ID NO: 1, nucleotides 1-26 + sequence SEQ ID NO: 1, nucleotides 52-62 + SEQ ID NO: 1, nucleotides 815-825 + SEQ ID NO: 1, nucleotides 1120-1135 + SEQ ID NO: 1, nucleotides 1630-1650 + SEQ ID NO: 1, nucleotides 1790-1810 + SEQ ID NO: 1, nucleotide 1855 -1875 + SEQ ID NO: 1, nucleotides 1910-1925 + SEQ ID NO: 1, nucleotides 2090-2110 + SEQ ID NO: 1, nucleotides 2166-2186 + SEQ ID NO: 1, nucleotides 2266-2286 + SEQ ID NO: 1, nucleotides 2366-2386 SEQ ID NO: 1, nucleotides 2466-2486 + SEQ ID NO: 1, nucleotides 2566-2586 + SEQ ID NO: 1, nucleotides 2666-2686 + SEQ ID NO: 1, nucleotides 2766-2786 + SEQ ID NO: 1, nucleotides 2866-2886 + SEQ ID NO: 1, nucleotides 2966-2986 + SEQ ID NO: 1, nucleotides 3066-3086 +

The nucleic acid is a synthetic / unnatural sequence,
And / or is separated, ie, without at least some of the associated substances in its natural state, and is preferably at least about 0.5%, preferably at least about 0.5% of the total weight of nucleic acids present in a given fraction Constitute at least about 5% and are usually recombinant (either they contain non-natural sequences or bind to nucleotides on the native chromosome other than the nucleotides to which they are attached). Including the native sequence of the present invention). SEQ ID NOs: 1, 3
Alternatively, a recombinant nucleic acid comprising the nucleotide sequence of 5 or an essential fragment thereof may have such a sequence or fragment terminated directly adjacent (ie, connected) to a sequence other than the sequence associated on the native chromosome. ) Contains
Alternatively, it is adjacent to a natural flanking region of less than 10 kb, preferably less than 2 kb, which is directly adjacent to a sequence other than the sequence at the end or on the natural chromosome. This nucleic acid is usually RNA or DNA, but it is often advantageous to modify the stability or the like using a nucleic acid containing other base or nucleotide analogs.

The nucleic acids may be used to detect the presence of CPF genes and gene transcripts, such as translatable transcripts, hybridization probes, PCR primers, and diagnostic nucleic acids, and to provide additional CPF homologs and structural analogs. It can be widely used, for example, for detecting or amplifying an encoding nucleic acid. In diagnosis, CPF hybridization probes are:
It finds use in identifying wild-type and CPF allelic variants in clinical and experimental samples. Allelic variants can be used to generate allele-specific oligonucleotide (ASO) probes for highly efficient clinical diagnosis.
In therapy, therapeutic CPF nucleic acids are used to modulate cellular expression, intracellular concentration or utilization of active CPF.

The present invention provides an effective method for identifying substances, compounds or their lead compounds that are active at the level of cellular function that can be regulated by CPF. In general, these screening methods involve CPF binding to natural CPF binding targets.
Assaying for compounds that modulate the interaction of. A wide variety of assays for binding agents are provided, including labeled in vitro protein-protein binding assays, immunoassays, DNA binding assays, cell-based assays, and the like.
This method relies on automated, cost-effective and efficient screening of chemical libraries for lead compounds. The identified reagents find use in the pharmaceutical industry for animal and human testing. For example, the reagents can be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for drug development.

In an in vitro binding assay, another peptide or polypeptide (eg, detection or anchoring)
May be part of a fusion product with
A mixture of elements containing an F polypeptide is used.
This assay mixture contains the natural intracellular CPF binding target. Natural full-length binding targets can be used, but portions of them (eg, oligonucleotides) are used as long as they provide the binding affinity and avidity for the CPF polypeptide that can be conveniently measured in this assay. Is often preferred. The assay mixture further contains a candidate pharmacological substance. Candidate substances include numerous chemical classes, but are typically organic compounds, preferably small organic compounds, obtained from a variety of sources including libraries of synthetic or natural compounds. . Various other reagents may also be included in the mixture. These include reagents such as salts, buffers and neutral proteins, and for example, albumin, detergents, protease inhibitors, nuclease inhibitors, antibacterial agents and the like can be used.

The resulting mixture is incubated under conditions such that, in the absence of the candidate pharmacological agent, the CPF polypeptide specifically binds to a cellular binding target, a portion or analog thereof having the stated binding affinity. . The components of the mixture can be added in any order that provides for the requisite binding, and the incubation can be performed at any temperature that promotes optimal binding. Incubation times are likewise selected for optimal binding and minimized to facilitate rapid high efficiency screening.

After incubation, the substance-altered binding between the CPF polypeptide and one or more binding targets is detected by any convenient method. The difference in the binding affinity of a CPF polypeptide for a target in the absence of the substance compared to the binding affinity in the presence of the substance indicates that the substance has a binding affinity for the CPF binding target.
It shows that it modulates F polypeptide binding. Similarly, in cell-based assays described below, differences in CPF-dependent transcriptional activation in the presence and absence of a substance indicate that the substance modulates CPF function. I have. As used herein, a difference is statistically significant and preferably represents a difference of at least 50%, more preferably at least 90%.

The following experimental part and examples are provided by way of illustration and not by way of limitation.

[0026]

[Examples] 1. CPF and CYP7 promoter elements
Placement of separation and characterization cells and plasmid HepG2, human hepatocarcinoma cells lines, 293, Buy transformed embryonic kidney cell line, and Caco2, colon adenocarcinoma cell line from ATCC. SV589 is a transformed human fibroblast cell line. Cells were cultured in Dulbecco's modified Eagle's medium-Ham F12 (1: 1) supplemented with 10% fetal calf serum in a humidified incubator at 37 ° C., 5% CO ₂ . pGL3: CYP7 is the − of the human CYP7α gene.
A DNA fragment of the 716 / + 14 region was cloned into the pGL3-luciferase reporter plasmid (Promega). pGL3: SFM or pGL3: BAM are -130 and -129, respectively.
Include mutations at positions (GG to TT) or -62 and -61 (AA to TC). Excite (ExS
it) Mutagenesis kit (Stratagene) was used to introduce a two base pair substitution in pGL3: CYP7. Either the -135 to -118 wild-type three tendon repeats of the promoter, or those repeats having a G to T double base pair substitution at positions -130 and -129, were added to the HSV TK gene PGL3: 3xwt and pGL3: 3xmut were assembled by cloning into modified pGL3 with the derived TATA sequence. pfCPF is p
It contains a flag-labeled sequence at the N-terminus of the gene cloned into cDNA3 (Invitrogen).
pfCPF-AF2 has a 15 amino acid deletion of the AF-2 domain at the C-terminus of the gene. pfCPF-VP
Is the transactivation domain of HSV VP16 (aa
412-490), which are AF-2 of pfCPF.
It replaces the domain.

[0027] DNase I hypersensitive mapping. Cells (3 × 10 ⁶ ) are harvested and 50 mM Tris-HCl (pH
7.9), 100 mM KCl, 5 mM MgCl ₂ , 0.0
5% saponin, 200 mM 2-mercaptoethanol,
Lysis was performed in 1.5 mL of lysis buffer containing 50% glycerol. The nuclei were collected by centrifugation and 100 mM NaCl,
50 mM Tris-HCl (pH 7.9), 3 mM MgC
Resuspended in buffer containing l ₂ , 1 mM DTT, 1 × complete protease inhibitor cocktail (Boehringer Mannheim), and serially diluted DNase I (5, 1.7, 0.6 units / mL). 3 nuclear suspensions
Incubated at 7 ° C for 20 minutes. The reaction was stopped by adding EDTA to a final concentration of 100 mM. After treatment with RNase A and protease K, genomic DNA was prepared and subjected to Southern hybridization.

Electrophoretic mobility shift assay. Nuclear extract,
It was prepared from cultured cells using KCl instead of NaCl. In vitro transcription and translation using the TNT system (Promega)
It was carried out in. 1 μg of the protein of the nuclear extract or 0.1-1 μL of the in vitro translation product was added to 10 mM Hepes (pH
7.6), 1 μg of poly (dI-dC), 100 mM KC
1, 7% glycerol, 1 mM EDTA, 1 mM DTT,
5 mM MgCl ₂ and irrelevant single-stranded oligo DNA 4
40000 cpm in a reaction buffer containing 0 pmole
And mixed with ³² P-labeled oligonucleotide and incubated at room temperature for 20 minutes. The reaction mixture was separated on a 4% polyacrylamide-0.5 × TBE gel. The gel was dried and exposed to X-ray film. In the competition experiment, 3
A 0 or 60-fold molar excess of competitor DNA was added. In antibody supershift experiments, anti-CPF antiserum or pre-immune serum was added to the reaction mixture before the addition of probe DNA.

Transfection and reporter gene analysis. One day prior to transfection, cells were seeded into 6-well culture dishes (4 × 10 ⁵ / well). Generally, 2 ug of the luciferase reporter plasmid and 0.1 ug of the RSV LTR-driven b-galactosidase expression vector were transfected into the cultured cells by the calcium phosphate method for 48 hours. Cell extracts were prepared and assayed for luciferase activity using a luciferase assay system (Promega). Luciferase activity was normalized by b-galactosidase activity.

Molecular cloning of CPF. Ftz-F1
Using a human EST clone containing a box sequence (GenBank, accession number N59515), a human liver cDNA library purchased from Clontech was screened. The cDNA in the positive clone was recovered by converting the phage DNA into the pTriplEx plasmid and sequenced. One of several positive clones (pTriplEx-113) was selected for further analysis out of several positive clones that would have been variously spliced from the same gene.

Tissue-specific expression of CPF. Northern blot of poly A + RNA derived from human tissue was purchased from Clontech. Hybridization reactions were performed using Northern MAX hybridization buffer (Ambion).

Immunoprecipitation. Peptides derived from the CPF cDNA sequence (DRMRGGRNFKGGPMYKRD
R) was used to generate anti-CPF polyclonal antibodies. HepG2 or 293 cells (1 × 10 ⁷ )
In a medium containing 00 μCi / mL ³⁵ S-methionine,
Incubate for minutes. Harvest cells and add 50 mM Tris-HCl
(PH 7.5), 125 mM NaCl, 5 mM EDTA,
It was thawed by three freeze-thaw cycles in a buffer containing 0.1% NP-40. The cell lysate is then washed with anti-CPF
Used for immunoprecipitation with antibodies. The precipitated sample was separated by 10% SDS-PAGE and exposed to X-ray film.

[0033] DNase I hypersensitive site mapping of the human CYP7 gene. In order to study the mechanism of liver-specific expression of the human CYP7 gene, the present inventors first attempted to identify elements believed to be responsible for liver-specific expression by DNase I hypersensitive mapping of the gene. D
Nase I hypersensitivity is known to be associated with transcriptional activity. Nuclei prepared from HepG2, 293 and Caco2 cells were treated with increasing amounts of DNaseI. The DNA is then extracted and digested with appropriate restriction enzymes,
Southern blotting was performed using a labeled fragment containing nucleotides from -944 to -468 as a probe. In addition to the expected 5 kb PstI fragment, a second 2.8 kb band was observed. The increase in the intensity of the 2.8 kb band with the increased amount of DNase I treatment and the decrease in the intensity of the 5 kb band as the parent fragment at the same time indicate the presence of a DNase hypersensitive site. Importantly, this 2.8 kb band was observed only in HepG2 cells and not in the other cells examined. The size of the fragment indicates that the liver-specific DNase I hypersensitive site is between -100 bp and -300 bp from the transcription start site of the human CYP7 gene. The location of this site was further confirmed by using different restriction enzymes with probes from different regions.

Identification of liver-specific CYP7 promoter elements. To further identify the liver-specific element of the CYP7 gene, seven partially overlapping oligonucleotides (CL5, bp-368-291; CL6, bp-
311-232; CL7, bp-256-177; CL
1, bp-201-122; CL2, bp-140-6
1: CL3, bp-121-42; CL4, bp-60
− + 20) was synthesized and used for gel mobility shift experiments. When using labeled oligonucleotides CL1 and CL2, hepatocyte-specific DNA
-There was formation of a protein complex. Because unlabeled oligonucleotide CL1 competes with oligonucleotide CL2, oligonucleotides CL1 and CL2
Clearly recognized the same complex. These can compete with excess unlabeled oligonucleotides CL1 and CL2, but the oligonucleotides CL adjacent to this region
3-7, this DNA-
Protein complexes are sequence-specific. This promoter complex was not observed in 293, Caco2 or SV589 nuclear extracts, but only in HepG2 nuclear extracts, consistent with the liver-specific DNase I hypersensitive sites identified above. It is clear that sequences overlapping with these two oligonucleotides are involved in the formation of a liver-specific DNA-protein complex

Sequence analysis revealed that this region contains several 6 bp repeat elements known to be binding sites for nuclear hormone receptors.
To determine the exact sequence involved in liver-specific binding, several oligonucleotides were synthesized containing mutations in this repeat or each of its flanking sequences. Table 4
As shown in Figure 5, oligonucleotides containing mutations in repeats A and B compete for complex formation, whereas oligonucleotides containing mutations in repeat C do not compete, indicating that repeats C
Is essential for binding. To further determine the nucleotides required for complex formation, several oligonucleotides containing detailed mutations in repeat C and adjacent sequences were synthesized and used in gel shift experiments. Our results have shown that complex formation requires a common element containing 9 nucleotides. This element is known to be a binding site for a family of nuclear hormone receptors called Ftz-F1.

[0036]

Table 4 Oligonucleotide DNA binding TCTGATACCTGTGGACTTAGTTCAAGGCCAGTTA + TCTG GAGGA TGTGGACTTAGTTCAAGGCCAGTTA + TCTGATACCTGT TATA TTAGTTCAAGGCCAGTTA + TCTGGAGGATGTGGACTT CTA TCAAGGCCAGTTA + TCTGATACCTGT TATA TT CTA TCAAGGCCAGTTA + TCTGGAGGATGTGGACTTAGTTCA CACAGAG TTA + TCTGATACCTGTGGACTTAGT AG AAGGCCAGTTA - TCTGATACCTGTGGACTTAGTTC TT GGCCAGTTA - TCTGATACCTGTGGACTTAGTTCAA T GCCAGTTA - TCTGATACCTGTGGACTTAGTTCAAG T CCAGTTA - TCTGATACCTGTGGACTTAGTTCAAGG AG AGTTA - TCTGATACCTGTGGACTTAGTTCAAGGCC TA TTA - TCTGATACCTGTGGACTTAGTTCAAGGCCA A TTA + TCTGATACCTGTGGACTTAGTTCAAGGCCAG G TA + TCAAGGCCA CYP7P- binding site YCAAGGYCR FTZ-F1 consensus AAAGGTCA NGFI-B consensus TCTGATACCTGTGGACTTAGT CA AAGGCCAGTTA - TCTGATACCTGTGGACTTAGT A C C AGGCCAGTTA - TCTGATACCTGTGGACTTAGT AGG AGGCCAGTTA - TCTGATACCTGTGGACTTAGT AAG AGGCCAGTTA - TCTGATACCTGTGGACTTAGTT TC AGGCCAGTTA -TCTGATACCTGTGGACTTAGT CTC AGGCCAGTTA-

The Ftz-F1 binding site is essential for liver-specific expression of the human CYP7 gene. Human CYP7
To determine the role of the Ftz-F1 site in gene expression, this site was mutated by a two nucleotide substitution. As a control, mutations in unrelated regions were also made. The promoter sequence from +14 to -716, containing either the wild-type or mutated Ftz-F1 site, or a control, was cloned into the luciferase reporter plasmid pGL3. Next, this plasmid DNA was
The cells were transfected into pG2, 293 and Caco2 cells, and the promoter activity was measured by luciferase activity. Mutation at the Ftz-F1 site completely abolishes promoter activity in HepG2 cells, while 293
And Caco2 cells showed little or no effect. As a control, mutations in unrelated regions had no effect on promoter activity in all cells examined.

Cloning of liver-specific CYP7 promoter binding protein. Nuclear hormone receptors are DNA-specific, often ligand-dependent, transcription factors. Ft
z-F1, a Drosophila DNA binding protein, is the prototype of a subgroup of the nuclear hormone receptor family. Like most nuclear hormone receptors, Ftz-F1
Contains a zinc finger DNA binding domain and a putative ligand binding domain. The DNA binding domain of Ftz-F1 family members contains a unique 26 amino acid extension (termed the Ftz-F1 box) at the C-terminus of the two zinc finger modules. The sequence of the Ftz-F1 box is conserved from Drosophila to rodents and is deeply involved in sequence-specific binding to DNA. The ability to identify the Ftz-F1 binding site in the human CYP7 promoter indicates that human Ft-F1
The z-F1-like protein is Ftz-F1 in the human CYP7 gene.
Suggests binding to the element. Human Ft
To clone z-F1, the EST database was searched using the DNA sequence of the Ftz-F1 box,
A human EST clone was found. The EST sequence was then used as a probe to screen a human liver cDNA library. Several clones were isolated and one of them, clone # 113, was used for further analysis.

Characterization of CPF. Clone # 113 is
It encodes a 495 amino acid full-length polypeptide, including an in-frame stop codon 30 nucleotides upstream of the first ATG. We call this protein CYP
7 promoter binding factor (C YP7 P romoter-binding
It was named the CPF in honor of the F actor). Sequence analysis has revealed that CPF is a new member of the Ftz-F1 family. The closest homologs of CPF are the murine version of this family, LRH-1 (SEQ ID NOs: 7 and 8) and the human variant, hFTF (SEQ ID NOs: 9 and 10). To confirm that the cloned CPF was a factor involved in CYP7 promoter binding activity, in vitro translated CPF was used in gel shift experiments in parallel with HepG2 nuclear extract. We have found that in vitro translated CPF recognizes the same DNA sequence as the endogenous protein does, and that the gel shift patterns of the two are identical. Antibodies raised against peptides containing the Ftz-F1 box were used in gel shift experiments. We have found that DNA-protein complexes formed using either HepG2 nuclear extract or in vitro translated CPF are destroyed by specific antibodies, but not by pre-immune serum. Was. Further, the antibody
Liver-specific cellular proteins that migrated with in vitro translated CPF were recognized. Since there is no corresponding protein in 293 cells, the endogenous gene product recognized by the antibody specific for Ftz-F1 is clearly liver-specific.

Transcription activity of CPF. To measure the transcriptional activity of CPF, a flag-labeled expression plasmid pfC
PF was transfected into 293 cells with a luciferase reporter plasmid containing three copies of the wild-type Ftz-F1 binding site. The present inventors have found that pfCPF has limited transcription activity. To determine if this weak transcriptional activity is due to the weakly transcriptional domain AF2 of the gene, which activity is also likely ligand dependent, by replacing the AF2 domain of CPF with a strong viral transactivation domain, p
fCPF-VP was assembled. When fCPF-VP was transfected into 293 cells with a reporter plasmid, strong transcriptional activity was observed,
It has been suggested that transcriptional activation of F requires the help of either ligand-dependent processes or cofactors.

Tissue-specific expression of CPF. CY in rodents
It has been reported that the P7 gene is exclusively expressed in the liver. To determine tissue-specific expression of the CPF gene, a pair of RNA tissue blots was performed using either the labeled CPF cDNA or CYP7 cDNA as a probe. The present inventors have found that CPF gene expression appears to be strong in pancreas and liver, with low levels of expression in heart and lung and little or no expression in other tissues. Human CYP7 appears to be expressed only in the liver. Interestingly, low molecular weight pancreatic-specific transcripts were recognized by the human CYP7 probe.

2. High throughput in vitro fluorescence polarization
Assay reagent: Sensor: rhodamine-labeled ILRKLLQE peptide (final concentration = 1-5 nM) Receptor: glutathione-S-transferase /
CPF ligand binding domain (SEQ ID NO: 2, residues 1-1)
23) Fusion protein (final concentration = 100-200 nM) Buffer: 10 mM HEPES, 10 mM NaCl, 6 mM
Magnesium chloride, pH 7.6

Protocol: 1. Add 90 μL of the sensor / receptor mixture to each well of a 96-well microtiter plate.

2. Add 10 μL of test compound per well.

Shake for 3.5 minutes, Fluorite FPM
-2 Measure the amount of fluorescence polarization within 5 minutes by using a fluorescence polarization micro-quantification system (Dynatech Laboratories, Inc.).

3. For cell-based reporter assays
Protocol CPF can transactivate FTZ-F1 reporter constructs when overexpressed in 293 or HeLa cells. 293 cells are transfected with a plasmid encoding the 3FTZ-F1 binding site-luciferase reporter assembly and various amounts of expression vector encoding CPF using the calcium phosphate precipitation method. After 36-48 hours, the cells are left untreated, or the candidate ligand (1
(0-50 ng / mL) for 6 hours before harvesting. Cells are lysed and luciferase activity is measured using a luciferase assay kit (Promega). Luciferase activity in each transfection was determined by pRSV-βga
Normalize by co-transfecting the control vector.

4. Sequence alignments Various alignments of the polynucleotide and polypeptide sequences are shown in Tables 5-8, representing sequence-specific fragments. For example, Table 7 shows 105, hFTF- and mLRH-specific peptides while expressing 105, hFT-
1 shows the alignment of the F and mLRH polypeptide sequences. Similarly, the alignment of their respective cDNA sequences (SEQ ID NOs: 5, 7 and 9, respectively) represents 105-, hFTF- and mLRH-specific cDNA fragments.

All publications and patent applications cited in this specification are expressly and individually indicated to state that each publication or patent application is incorporated by reference. As if incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, any changes and modifications may be made without departing from the spirit or scope of the appended claims. What can be done will be very apparent to those skilled in the art in light of the teachings of the present invention.

[0049]

TABLE 5 113PRO = SEQ ID NO: 2 hFTFpro = SEQ ID NO: 8 113PRO MSSNSDTGDL QESLKHG --- -LTP - IVSQ FKMVNYSYDE DLEELCPVCG 44 hFTFpro MLPKVETEAL GLARSHGEQG QMPENMQVSQ FKMVNYSYDE DLEELCPVCG 50 113PRO DKVSGYHYGL LTCESCKGFF KRTVQNNKRY TCIENQNCQI DKTQRKRCPY 94 hFTFpro DKVSGYHYGL LTCESCKGFF KRTVQNNKRY TCIENQNCQI DKTQRKRCPY 100 113PRO CRFQKCLSVG MKLEAVRADR MRGGRNKFGP MYKRDRALKQ QKKALIRANG 144 hFTFpro CRFQKCLSVG MKLEAVRADR MRGGRNKFGP MYKRDRALKQ QKKALIRANG 150 113PRO LKLEAMSQVI QAMPSDLTIS SAIQNIHSAS KGLPLNHAAL PPTDYDRSPF 194 hFTFpro LKLEAMSQVI QAMPSDLTIS SAIQNIHSAS KGLPLNHAAL PPTDYDRSPF 200 113PRO VTSPISMTMP PHGSLQGYQT YGHFPSRAIK SEYPDPYTSS PESIMGYSYM 244 hFTFpro VTSPISMTM- LHGSLQGYQT YGHFPSRAIK SEYPDPYTSS PESIMGYSYM 249 113PRO DSYQTSSPAS IPHLILELLK CEPDEPQVQA KIMAYLQQEQ ANRSKHEKLS 294 hFTFpro DSYQTSSPAS IPHLILELLK CEPDEPQVQA KIMAYLQQEQ ANRSKHEKLS 299 113PRO TFGLMCKMAD QTLFSIVEWA RSSIFFRELK VDDQMKLLQN CWSELLILDH 344 hFTFpro TFGLMCKMAD QTVFSIVEWA RSSIFFRELK VDDQMKLLQN CWSELLILDH349 YRQVVHGKE GSIFLVTGQQ VDYSIIASQA GATLNNLMSH AQELVAKLRS 394 hFTFpro IYRQVVHGKE GSIFLVTGQQ VDYSIIASQA GATLNNLMSH AQELVAKLRS 399 113PRO LQFDQREFVC LKFLVLFSLD VKNLENFQLV EGVQEQVNAA LLDYTMCNYP 444 hFTFpro LQFDQREFVC LKFLVLFSLD VKNLENFQLV EGVQEQVNAA LLDYTMCNYP 449 113PRO QQTEKFGQLL LRLPEIRAIS MQAEEYLYYK HLNGDVPYNN LLIEMLHAKR 494 hFTFpro QQTEKFGQLL LRLPEIRAIS MQAEEYLYYK HLNGDVPYNN LLIEMLHAKR 499 113PRO A 495 hFTFpro A 500

[0050]

[Table 6] 113PRO = SEQ ID NO: 1 36PRO = SEQ ID NO: 4 hFTFpro = SEQ ID NO: 8 mLRHpro = SEQ ID NO: 10 ---------- 17 36pro MSSNSDTGDL QESLKHG --- ---------- ---------- ---------- 17 hFTFpro MLPKVETEAL GLARSHG --- ---------- ---------- ---------- 17 mLRHpro MSASLDTGDF QEFLKHGLTA IASAPGSETR HSPKREEQLR EKRAGLPDRH 50 113PRO ------- --- ---------- ---------- --LTP--IVS QFKMVNYSYD 33 36pro ---------- -------- ----------- --LTP--IVS QFKMVNYSYD 33 hFTFpro ---------- ---------- -------- EQ GQMPENMQVS QFKMVNYSYD 39 mLRHpro RRPIPARSRL VMLPKVETEA PGLVRSHGEQ GQMPENMQVS QFKMVNYSYD 100 113PRO EDLEELCPVC GDKVSGYHYG LLTCESCKGF FKRTVQNNKR YTCIENQNCQ 83 36pro EDLEELCPVC GDKVSGYHYG LLTCESCKGF FKRTVQNNKR YTCIENQNCQ 83 hFTFpro EDLEELCPVC GDKVSGYHYG LLTCESCKGF FKRTVQNNKR YTCIENQNCQ 89 mLRHpro EDLEELCPVC GDKVSGYHYG LLTCESCKGF FKRTVQNQKR YTCIENQNCQ 150 113PRO IDKTQRKRCP YCRFQKCLSV GMKLEAVRAD RMRGGRNKFG PMYKRDRALK 133 36pro IDKTQRKRCP YCRFQKCLSV GMKL EAVRAD RMRGGRNKFG PMYKRDRALK 133 hFTFpro IDKTQRKRCP YCRFQKCLSV GMKLEAVRAD RMRGGRNKFG PMYKRDRALK 139 mLRHpro IDKTQRKRCP YCRFKKCIDV GMKLEARNAD RMRGGRNKFG PMYKRDRALK IQ 113LK -------- 154 hFTFpro QQKKALIRAN GLKLEAMSQV IQAMPSDLTI SSAIQNIHSA SKGLPLNHAA 189 mL RHpro QQKKALIRAN GLKLEAMSQV IQAMPSDLT- -SAIQNIHSA SKGLPLSHVA 248 113PRO LPPTDYDRSP FVTSPISMTM PPHGSLQGYTSY PGFP --- ---------- ---------- ---------- 154 hFTFpro LPPTDYDRSP FVTSPISMTM -LHGSLQGYQ TYGHFPSRAI KSEYPDPYTS 238 mLRHpro LPPTDYDRSP FVTSPISMTM PPHSSLHGYQ PYGHFPSRAI KSEYPDPYSS 298 113PRO SPESIMGYSY MDSYQTSSPA SIPHLILELL KCEPDEPQVQ AKIMAYLQQE 283 36pro ---------- ---------- ---------- ---------- --- ------- 154 hFTFpro SPESIMGYSY MDSYQTSSPA SIPHLILELL KCEPDEPQVQ AKIMAYLQQE 288 mLRHpro SPESMMGYSY MDGYQTNSPA SIPHLILELL KCEPDEPQVQ AKIMAYLQQE 348 113PRO QANRSKHEKL STFGLMCKMA DQTLFSIVE W ARSSIFFREL KVDDQMKLLQ 333 36pro ---------- ---------- ---------- ---------- --- DQMKLLQ 161 hFTFpro QANRSKHEKL STFGLMCKMA DQTVFSIVEW ARSSIFFREL KVDDQMKLLQ 338 mLRHpro QSNRNRQEKL SAFGLLCKMA DQTLFSIVEW ARSSIFFREL KVDDQMKLLQ 398 113PRO NCWSELLILD HIYRQVVHGK EGSIFLVTGQ QVDYSIIASQ AGATLNNLMS 383 36pro NCWSELLILD HIYRQVVHGK EGSIFLVTGQ QVDYSIIASQ AGATLNNLMS 211 hFTFpro NCWSELLILD HIYRQVVHGK EGSIFLVTGQ QVDYSIIASQ AGATLNNLMS 388 mLRHpro NCWSELLILD HIYRQVAHGK EGTIFLVTGE HVDYSTIISH TEVAFNNLLS 448 113PRO HAQELVAKLR SLQFDQREFV CLKFLVLFSL DVKNLENFQL VEGVQEQVNA 433 36pro HAQELVAKLR SLQFDQREFV CLKFLVLFSL DVKNLENFQL VEGVQEQVNA 261 hFTFpro HAQELVAKLR SLQFDQREFV CLKFLVLFSL DVKNLENFQL VEGVQEQVNA 438 mLRHpro LAQELVVRLR SLQFDQREFV CLKFLVLFSS DVKNLENLQL VEGVQEQVNA 498 113PRO ALLDYTMCNY PQQTEKFGQL LLRLPEIRAI SMQAEEYLYY KHLNGDVPYN 483 36pro ALLDYTMCNY PQQTEKFRQL LLRLPEIRAI SMQAEEYLYY KHLNGDVPYN 311 hFTFpro ALLDYTMCNY PQQTEKFGQL LLRLPEIRAI SMQAEEYLYY KHLNGDVPYN 488 mLRHpro ALLDYTVCNY PQQTEKFGQL LLRLPEIRAI SKQA EDYLYY KHVNGDVPYN 548 113PRO NLLIEMLHAK RA 495 36pro NLLIEMLHAK RA 323 hFTFpro NLLIEMLHAK RA 500 mLRHpro NLLIEMLHAK RA 560

[0051]

[Table 7] 105pro = SEQ ID NO: 6 hFTFpro = SEQ ID NO: 3 mLRHpro = SEQ ID NO: 10 105pro MSSNSDTGDL QESLKHGLTP IG ------------------ --- AGLPDRH 29 hFTFpro ------- ---------- ---------- ---------- ---------- mLRHpro MSASLDTGDF QEFLKHGLTA IASAPGSETR HSPKREEQLR EKRAGLPDRH 50 105pro GSPIPARGRL VMLPKVETEA LGLARSHGEQ GQMPENMQVS QFKMVNYSYD 79 hFTFpro ---------- -MLPKVETEA LGLARSHGEQ GQMPENMQVS QFKMVNYSYD 39 mLRHpro RRPIPARSRL VMLPKVETEA PGLVRSHGEQ GQMPENMQVS QFKMVNYSYD 100 105pro EDLEELCPVC GDKVSGYHYG LLTCESCKGF FKRTVQNNKR YTCIENQNCQ 129 hFTFpro EDLEELCPVC GDKVSGYHYG LLTCESCKGF FKRTVQNNKR YTCIENQNCQ 89 mLRHpro EDLEELCPVC GDKVSGYHYG LLTCESCKGF FKRTVQNQKR YTCIENQNCQ 150 105pro IDKTQRKRCP YCRFQKCLSV GMKLEAVRAD RMRGGRNKFG PMYKRDRALK 179 hFTFpro IDKTQRKRCP YCRFQKCLSV GMKLEAVRAD RMRGGRNKFG PMYKRDRALK 139 mLRHpro IDKTQRKRCP YCRFKKCIDV GMKLEAVRAD RMRGGRNKFG PMYKRDRALK 200 105pro QQKKALIRAN GLKLEAMSQV IQAMPSDLTI SSAIQNIHSA SKGLPLNHAA 229 hFTFpro QQKKALIRAN GLKLEAMSQV IQAMPS DLTI SSAIQNIHSA SKGLPLNHAA 189 mLRHpro QQKKALIRAN GLKLEAMSQV IQAMPSDLT- -SAIQNIHSA SKGLPLSHVA 248 105pro LPPTDYDRSP FVTSPISMTM PPHGSLQGYQ TYGHFPSRAI KSEYPDPYTS 279 hFTFpro LPPTDYDRSP FVTSPISMTM -LHGSLQGYQ TYGHFPSRAI KSEYPDPYTS 238 mLRHpro LPPTDYDRSP FVTSPISMTM PPHSSLHGYQ PYGHFPSRAI KSEYPDPYSS 298 105pro SPESIMGYSY MDSYQTSSPA SIPHLILELL KCEPDEPQVQ AKIMAYLQQE 329 hFTFpro SPESIMGYSY MDSYQTSSPA SIPHLILELL KCEPDEPQVQ AKIMAYLQQE 288 mLRHpro SPESMMGYSY MDGYQTNSPA SIPHLILELL KCEPDEPQVQ AKIMAYLQQE 348 105pro QANRSKHEKL STFGLMCKMA DQTLFSIVEW ARSSIFFREL KVDDQMKLLQ 379 hFTFpro QANRSKHEKL STFGLMCKMA DQTVFSIVEW ARSSIFFREL KVDDQMKLLQ 338 mLRHpro QSNRNRQEKL SAFGLLCKMA DQTLFSIVEW ARSSIFFREL KVDDQMKLLQ 398 105pro NCWSELLILD HIYRQVVHGK EGSIFLVTGQ QVDYSIIASQ AGATLNNLMS 429 hFTFpro NCWSELLILD HIYRQVVHGK EGSIFLVTGQ QVDYSIIASQ AGATLNNLMS 388 mLRHpro NCWSELLILD HIYRQVAHGK EGTIFLVTGE HVDYSTIISH TEVAFNNLLS 448 105pro HAQELVAKLR SLQFDQREFV CLKFLVLFSL DVKNLENFQL VEGVQEQVNA 479 hFTFpro HAQELVAKLR SLQFDQREFV CLKFLV LFSL DVKNLENFQL VEGVQEQVNA 438 mLRHpro LAQELVVRLR SLQFDQREFV CLKFLVLFSS DVKNLENLQL VEGVQEQVNA 498 105pro ALLDYTMCNY PQQTEKFGQL LLRLPEIRAI SMQAEEYLYY KHLNGDVPYN 529 hFTFpro ALLDYTMCNY PQQTEKFGQL LLRLPEIRAI SMQAEEYLYY KHLNGDVPYN 488 mLRHpro ALLDYTVCNY PQQTEKFGQL LLRLPEIRAI SKQAEDYLYY KHVNGDVPYN 548 105pro NLLIEMLHAK RA 541 hFTFpro NLLIEMLHAK RA 500 mLRHpro NLLIEMLHAK RA 560

[0052]

[Table 8] 113 = SEQ ID NO: 1 hFTF = SEQ ID NO: 7 113 ---------- ---------- ---------- ----- --- GA AAAAAGTACA 12 hFTF GAAACTGGAT ACATGGTTTA CAGCAGGTCA CTAATGTTGG AAAAAGTACA 50 113 GAGTCCAGGG AAAAGACTTG CTTGTAACTT TATGAATTCT GGATTTTTTT 62 hFTF GAGTCCAGGG AAA-GACTTG CTTGTAACTT TATGAATTCT GGA --- TTTT 96 113 TTTTCCTTTG CTTTTTCTTA ACTTTCACTA AGGGTTACTG TAGTCTGATG 112 hFTF TTTTCCTTTG CTTTTTCTTA ACTTTCACTA AGGGTTACTG TAGTCTGATG 146 113 TGTCCTTCCC AAGGCCACGA AATTTGACAA GCTGCACTTT TCTTTTGCTC 162 hFTF TGTCCTTCCC AAGGCCACGA AATTTGACAA GCTGCACTTT TCTTTTGCTC 196 113 AATGATTTCT GCTTTAAGCC AAAGAACTGC CTATAATTTC ACTAAGAATG 212 hFTF AATGATTTCT GCTTTAAGCC AAAGAACTGC CTATAATTTC ACTAAGAATG 246 113 TCTTCTAATT CAGATACTGG GGATTTACAA GAGTCTTTAA AGCACGGACT 262 hFTF TCTTCTAATT CAGATACTGG GGATTTACAA GAGTCTTTAA AGCACGGACT 296 113 TACACCTATT --------- ----------- ---------- ---------- 272 hFTF TACACCTATT GGTGCTGGGC TTCCGGACCG ACACGGATCC CCCATCCCGC 346 113 ---------- ---------- ---------- ---------- --------- -272 hFTF CCGCGGTCGC CTTGTCATGC TGCCCAAAGT GGAGACGGAA GCCCTGGGAC 396 113 ---------- ---------- ---------- ---------- ------- GTG 275 hFTF TGGCTCGATC GCATGGGGAA CAGGGCCAGA TGCCGGAAAA CATGCAAGTG 446 113 TCTCAATTTA AAATGGTGAA TTACTCCTAT GATGAAGATC TGGAAGAGCT 325 hFTF TCTCAATTTA AAATGGTGAA TTACTCCTAT GATGAAGATC TGGAAGAGCT 496 113 TTGTCCCGTG TGTGGAGATA AAGTGTCTGG GTACCATTAT GGGCTCCTCA 375 hFTF TTGTCCCGTG TGTGGAGATA AAGTGTCTGG GTACCATTAT GGGCTCCTCA 546 113 CCTGTGAAAG CTGCAAGGGA TTTTTTAAGC GAACAGTCCA AAATAATAAA 425 hFTF CCTGTGAAAG CTGCAAGGGA TTTTTTAAGC GAACAGTCCA AAATAATAAA 596 113 AGGTACACAT GTATAGAAAA CCAGAACTGC CAAATTGACA AAACACAGAG 475 hFTF AGGTACACAT GTATAGAAAA CCAGAACTGC CAAATTGACA AAACACAGAG 646 113 AAAGCGTTGT CCTTACTGTC GTTTTCAAAA ATGTCTAAGT GTTGGAATGA 525 hFTF AAAGCGTTGT CCTTACTGTC GTTTTCAAAA ATGTCTAAGT GTTGGAATGA 696 113 AGCTAGAAGC TGTAAGGGCC GACCGAATGC GTGGAGGAAG GAATAAGTTT 575 hFTF AGCTAGAAGC TGTAAGGGCC GACCGAATGC GTGGAGGAAG GAATAAGTTT 746 113 GGGCCAATGT ACAAGAGAGA CAGGGCCCTG AAGC AACAGA AAAAAGCCCT 625 hFTF GGGCCAATGT ACAAGAGAGA CAGGGCCCTG AAGCAACAGA AAAAAGCCCT 796 113 CATCCGAGCC AATGGACTTA AGCTAGAAGC CATGTCTCAG GTGATCCAAG 675 hFTF CATCCGAGCC AATGGACTTA AGCTAGAAGC CATGTCTCAG GTGATCCAAG 846 113 CTATGCCCTC TGACCTGACC ATTTCCTCTG CAATTCAAAA CATCCACTCT 725 hFTF CTATGCCCTC TGACCTGACC ATTTCCTCTG CAATTCAAAA CATCCACTCT 896 113 GCCTCCAAAG GCCTACCTCT GAACCATGCT GCCTTGCCTC CTACAGACTA 775 hFTF GCCTCCAAAG GCCTACCTCT GAACCATGCT GCCTTGCCTC CTACAGACTA 946 113 TGACAGAAGT CCCTTTGTAA CATCCCCCAT TAGCATGACA ATGCCCCCTC 825 hFTF TGACAGAAGT CCCTTTGTAA CATCCCCCAT TAGCATGACA ATGC --- TGC 993 113 ACGGCAGCCT GCAAGGTTAC CAAACATATG GCCACTTTCC TAGCCGGGCC 875 hFTF ACGGCAGCCT GCAAGGTTAC CAAACATATG GCCACTTTCC TAGCCGGGCC 1043 113 ATCAAGTCTG AGTACCCAGA CCCCTATACC AGCTCACCCG AGTCCATAAT 925 hFTF ATCAAGTCTG AGTACCCAGA CCCCTATACC AGCTCACCCG AGTCCATAAT 1093 113 GGGCTATTCA TATATGGATA GTTACCAGAC GAGCTCTCCA GCAAGCATCC 975 hFTF GGGCTATTCA TATATGGATA GTTACCAGAC GAGCTCTCCA GCAAGCATCC 1143 113 CACATCTGAT ACTGGAA CTT TTGAAGTGTG AGCCAGATGA GCCTCAAGTC 1025 hFTF CACATCTGAT ACTGGAACTT TTGAAGTGTG AGCCAGATGA GCCTCAAGTC 1193 113 CAGGCTAAAA TCATGGCCTA TTTGCAGCAA GAGCAGGCTA ACCGAAGCAA 1075 hFTF CAGGCTAAAA TCATGGCCTA TTTGCAGCAA GAGCAGGCTA ACCGAAGCAA 1243 113 GCACGAAAAG CTGAGCACCT TTGGGCTTAT GTGCAAAATG GCAGATCAAA 1125 hFTF GCACGAAAAG CTGAGCACCT TTGGGCTTAT GTGCAAAATG GCAGATCAAA 1293 113 CTCTCTTCTC CATTGTCGAG TGGGCCAGGA GTAGTATCTT CTTCAGAGAA 1175 hFTF CTGTCTTCTC CATTGTCGAG TGGGCCAGGA GTAGTATCTT CTTCAGAGAA 1343 113 CTTAAGGTTG ATGACCAAAT GAAGCTGCTT CAGAACTGCT GGAGTGAGCT 1225 hFTF CTTAAGGTTG ATGACCAAAT GAAGCTGCTT CAGAACTGCT GGAGTGAGCT 1393 113 CTTAATCCTC GACCACATTT ACCGACAAGT GGTACATGGA AAGGAAGGAT 1275 hFTF CTTAATCCTC GACCACATTT ACCGACAAGT GGTACATGGA AAGGAAGGAT 1443 113 CCATCTTCCT GGTTACTGGG CAACAAGTGG ACTATTCCAT AATAGCATCA 1325 hFTF CCATCTTCCT GGTTACTGGG CAACAAGTGG ACTATTCCAT AATAGCATCA 1493 113 CAAGCCGGAG CCACCCTCAA CAACCTCATG AGTCATGCAC AGGAGTTAGT 1375 hFTF CAAGCCGGAG CCACCCTCAA CAACCTCATG AGTCATGCAC AGGAGT TAGT 1543 113 GGCAAAACTT CGTTCTCTCC AGTTTGATCA ACGAGAGTTC GTATGTCTGA 1425 hFTF GGCAAAACTT CGTTCTCTCC AGTTTGATCA ACGAGAGTTC GTATGTCTGA 1593 113 AATTCTTGGT GCTCTTTAGT TTAGATGTCA AAAACCTTGA AAACTTCCAG 1475 hFTF AATTCTTGGT GCTCTTTAGT TTAGATGTCA AAAACCTTGA AAACTTCCAG 1643 113 CTGGTAGAAG GTGTCCAGGA ACAAGTCAAT GCCGCCCTGC TGGACTACAC 1525 hFTF CTGGTAGAAG GTGTCCAGGA ACAAGTCAAT GCCGCCCTGC TGGACTACAC 1693 113 AATGTGTAAC TACCCGCAGC AGACAGAGAA ATTTGGACAG CTACTTCTTC 1575 hFTF AATGTGTAAC TACCCGCAGC AGACAGAGAA ATTTGGACAG CTACTTCTTC 1743 113 GACTACCCGA AATCCGGGCC ATCAGTATGC AGGCTGAAGA ATACCTCTAC 1625 hFTF GACTACCCGA AATCCGGGCC ATCAGTATGC AGGCTGAAGA ATACCTCTAC 1793 113 TACAAGCACC TGAACGGGGA TGTGCCCTAT AATAACCTTC TCATTGAAAT 1675 hFTF TACAAGCACC TGAATGGGGA TGTGCCCTAT AATAACCTTC TCATTGAAAT 1843 113 GTTGCATGCC AAAAGAGCAT AAGTTACAAC CCCTAGGAGC TCTGCTTTCA 1725 hFTF GTTGCATGCC AAAAGAGCAT AAGTTACAAC CCCTAGGAGC TCTGCTTTCA 1893 113 AAACAAAAAG AGATTGGGGG AGTGGGGAGG GGGAAGAAGA ACAGGAAGAA 1775 hFTF AAACAAAAAG AGATTGG GGG AGTGGGGAGG GGGAAGAAGA ACAGGAAGAA 1943 113 AAAAAGTACT CTGAACTGCT CCAAGCAACG CTAATTAAAA ACTTGCTTTA 1825 hFTF AAAAAGTACT CTGAACTGCT CCAAGTAACG CTAATTAAAA ACTTGCTTTA 1993 113 AAGATATTGA ATTTAAAAAG GCATAATAAT CAAATACTTA ATAGCAAATA 1875 hFTF AAGATATTGA ATTTAAAAAG GCATAATAAT CAAATACT-A ATAGCAAATA 2042 113 AATGATGTAT CAGGGTATTT GTATTGCAAA CTGTGAATCA AAGGCTTCAC 1925 hFTF AATGATGTAT CAGGGTATTT GTATTGCAAA CTGTGAATCA AA-GCTTCAC 2091 113 AGCCCCAGAG GATTCCATAT AAAAGACATT GTAATGGAGT GGATTGAACT 1975 hFTF AGCCCCAGAG GATTCCATAT AAAAGACATT GTAATGGAGT GGATTGAACT 2141 113 CACAGATGGA TACCAACACG GTCAGAAGAA AAACGGACAG AACGGTTCTT 2025 hFTF CACAGATGGA TACCAACACG GTCAGAAGAA AAACGGACAG AACGGTTCTT 2191 113 GTATATTTAA ACTGATCTCC ACTATGAAGA AATTTAGGAA CTAATCTTAT 2075 hFTF GTATATTTAA ACTGATCTCC ACTATGAAGA AATTTAGGAA CTAATCTTAT 2241 113 TAATTAGGCT TATACAGCGG GGGATTTGAG CTTACAGGAT TCCTCCATGG 2125 hFTF TAATTAGGCT TATACAGCGG GG-ATTTGAG CTTACAGGAT TCCTCCATGG 2290 113 TAAAGCTGAA CTGAAACAAT TCTCAAGAAT GCATCAGCTG TACCTAC AAT 2175 hFTF TAAAGCTGAA CTGAAACAAT TCTCAAGAAT GCATCAGCTG ---------- 2330 113 AGCCCCTCCC TCTTCCTTTG AAGGCCCGAG CACCTCTGCC CTGTGGTCAC 2225 hFTF ---------- ---------- ---- ------ ---------- ---------- 2330 113 CGAATCTGTA CTAAGGACCT GTGTTCAGCC ACACCCAGTG GTAGCTCCAC 2275 hFTF ---------- ----- ----- ---------- ---------- ---------- 2330 113 CAAATCATGA ACAGCCTAAT TTTGAGTGTC TGTGTCTTAG ACCTGCAAAC 2325 hFTF ------ ---- ---------- ---------- ---------- ---------- 2330 113 AGCTAATAGG AAATTCTATT AATATGTTAG CTTGCCATTT TAAATATGTT 2375 hFTF ---------- ---------- ---------- ---------- ------- --- 2330 113 CTGAGGGTTG TTTTGTCTCG TGTTCATGAT GTTAAGAAAA TGCAGGCAGT 2425 hFTF ---------- ---------- ---------- -------- ----------- 2330 113 ATCCCTCATC TTATGTAAGT GTGAATTAAT ATTAAGGGAA ATGACTACAA 2475 hFTF ---------- ---------- --------- ----------- ---------- 2330 113 ACTTTCAAAG CAAATGCTCC ATAGCTAAAG CAACTTAGAC CTTATTTCTG 2525 hFTF ---------- ---------- ---------- ---------- ---------- 2330 113 CTACTGTTGC TGAAATGT GG CTTTGGCATT GTTGGATTTC ATAAAAAATT 2575 hFTF ---------- ---------- ---------- ---------- ---- ------ 2330 113 TCTGGCAGGA AGTCTTGTTA GTATACATCA GTCTTTTTCA TCATCCAAGT 2625 hFTF ---------- ---------- ---------- ----- ----- ---------- 2330 113 TTGTAGTTCA TTTAAAAATA CAACATTAAA CACATTTTGC TAGGATGTCA 2675 hFTF ---------- ---------- ------ ---- ---------- ---------- 2330 113 AATAGTCACA GTTCTAAGTA GTTGGAAACA AAATTGACGC ATGTTAATCT 2725 hFTF ---------- ------- --- ---------- ---------- ---------- 2330 113 ATGCAAAGAG AAAGGAAAGG ATGAGGTGAT GTATTGACTC AAGGTTCATT 2775 hFTF -------- ----------- ---------- ---------- ---------- 2330 113 CTTGCTGCAA TTGAACATCC TCAAGAGTTG GGATGGAAAT GGTGATTTTT 2825 hFTF ---------- ---------- ---------- ---------- --------- -2330 113 ACATGTGTCC TGGAAAGATA TTAAAGTAAT TCAAATCTTC CCCAAAGGGG 2875 hFTF ---------- ---------- ---------- ---------- ---------- 2330 113 AAAGGAAGAG AGTGATACTG ACCTTTTTAA GTCATAGACC AAAGTCTGCT 2925 hFTF ---------- ---------- ----------- --------- ------- --- 2330 113 GTAGAACAAA TATGGGAGGA CAAAGAATCG CAAATTCTTC AAATGACTAT 2975 hFTF ---------- ---------- ---------- -------- ----------- 2330 113 TATCAGTATT ATTAACATGC GATGCCACAG GTATGAAAGT CTTGCCTTAT 3025 hFTF ---------- ---------- --------- ----------- ---------- 2330 113 TTCACAATTT TAAAAGGTAG CTGTGCAGAT GTGGATCAAC ATTTGTTTAA 3075 hFTF ---------- ---------- ---------- ---------- ---------- 2330 113 AATAAAGTAT TAATACTTTA AAGTCAAAAA AAAAAAAAAA 3115 hFTF ----------- -------- ---------- ---------- 2330

[0053]

[Sequence list] SEQUUE
NCE LISTING <110> Sumitomo Pharmaceuticals Compa
ny Limited <110> Tularik Inc. <120> CYP7 Promoter-Binding Factors <130> 162467 <140><141><150> 60 / 067,708 <151> 1997-12-08 <160> 10 <170> PatentIn Ver. 2.0 <210> 1 <211> 3115 <212> DNA <213> Human <220><221> CDS <222> (210). . (1694) <400> 1 gaaaaaaattagagttccag ggaaaaagact tgc
ttgtaac tttattgaatt ctggatttttt 60 tttttttcctt tgctttttttct taactttcac taa
gggttact tgtaggtctga tgtgtcccttc 120 ccaaggccac gaattttgac aagctgcact ttt
ctttttgc tcaatgattt ctgcttttag 180 ccaaaagaact gcctatatatt tcactataaga atg
tct tct aat tca ga t act ggg 233 Met
Ser Ser Asn Ser Asp Thr Gly 1
5 tat tta caa gag tct tta aag cac gga
ctt aca cct att gtg tct caa 281 Asp Leu Gln Glu Ser Leu Lys His Gly
Leu Thr Pro Ile Val Ser Gln 10 15
20 ttt aaa atg gtg aat tac tcc tat gat
gaa gat ctg gaa gag ctt tgt 329 Phe Lys Met Val Asn Tyr Ser Tyr Asp
Glu Asp Leu Glu Glu Leu Cys 25 30
35 40 ccc gtg tgt gga gaa aaa gtg tct ggg
tac cat tat ggg ctc ctc acc 377 Pro Val Cys Gly Asp Lys Val Ser Gly
Tyr His Tyr Gly Leu Leu Thr 45
50 55 tgt ga agc tgc aag gga ttt ttt aag
cga aca gtc ca aat aat aaa 425 Cys Glu Ser Cys Lys Gly Phe Phe Lys
Arg Thr Val Gln Asn Asn Lys 60 65
70 agg tac aca tgt at ga aaa aac cag aac
tgc caa att gac aaa cacag 473 Arg Tyr Thr Cys Ile Glu Asn Gln Asn
Cys Gln Ile Asp Lys Thr Gln 75 80
85 aga aag cgt tgt cct tact tgt cgt ttt
aaa aaa tgt cta agt gtt gga 521 Arg Lys Arg Cys Pro Tyr Cys Arg Phe
Gln Lys Cys Leu Ser Val Gly 90 95
100 atg aag cta gaa gct gta agg gcc gac
cga atg cgt gga gga agg aat 569 Met Lys Leu Glu Ala Val Arg Ala Asp
Arg Met Arg Gly Gly Arg Asn 105 110
115 120 aag ttt ggg cca atg tac aag aga gac
agg gcc ctg aag caag aaa 617 Lys Phe Gly Pro Met Tyr Lys Arg Asp
Arg Ala Leu Lys Gln Gln Lys 125
130 135 aaa gcc ctc atc cga gcc aat gga ctt
aag cta gaa gcc atg tct cag 665 Lys Ala Leu Ile Arg Ala Asn Gly Leu
Lys Leu Glu Ala Met Ser Gln 140 145
150 gtg atc caa gct atg ccc tct gac ctg
acc att tcc tct gca attca 713 Val Ile Gln Ala Met Pro Ser Asp Leu
Thr Ile Ser Ser Ala Ile Gln 155 160
165 aac atc cac tct gcc tcc aaa ggc cta
cct ctg aac cat gct gcc ttg 761 Asn Ile His Ser Ala Ser Lys Gly Leu
Pro Leu Asn His Ala Ala Leu 170 175
180 cct cct aca gac tat gac aga agt ccc
ttt gta aca tcc ccc att agc 809 Pro Pro Thr Asp Tyr Asp Arg Ser Pro
Phe Val Thr Ser Ser Pro Ile Ser 185 190
195 200 atg aca atg ccc cct cac ggc agc ctg
caa ggt tac caa aca tat ggc 857 Met Thr Met Pro Pro His Gly Ser Leu
Gln Gly Tyr Gln Thr Tyr Gly 205
210 215 cac ttt cct agc cgg gcc atc aag tct
gag tac cca gac ccc tat acc 905 His His Pro Ser Ser Arg Ala Ile Lys Ser
Glu Tyr Pro Asp Pro Tyr Thr 220 225
230 agc tca ccc gag tcc ata atg ggc tat
tca tat atg gat ag tac cag 953 Ser Ser Pro Glu Ser Ile Met Gly Tyr
Ser Tyr Met Asp Ser Tyr Gln 235 240
245 acg agc tct cca gca agc atc cca cat
ctg ata ctg gaa ctt ttg aag 1001 Thr Ser Ser Pro Ala Ser Ile Pro His
Leu Ile Leu Glu Leu Leu Lys 250 255
260 tgt gag cca gat gag cct caa gtc cag
gct aaa atc atg gcc tat ttg 1049 Cys Glu Pro Asp Glu Pro Gln Val Gln
Ala Lys Ile Met Ala Tyr Leu 265 270
275 280 cag caa gag cag gct aac cga agc aag
cac gaa aag ctg agc acc ttt 1097 Gln Gln Glu Gln Ala Asn Arg Ser Lys
His Glu Lys Leu Ser Thr Phe 285
290 295 ggg ctt atg tgc aaa atg gca gait caa
act ctc ttc tcc att gtc gag 1145 Gly Leu Met Cys Lys Met Ala Asp Gln
Thr Leu Phe Ser Ile Val Glu 300 305
310 tgg gcc agg agt agt atc ttc ttc aga
gaa ctt aag gtt gat gac caa 1193 Trp Ala Arg Ser Ser Ile Phe Phe Arg
Glu Leu Lys Val Asp Asp Gln 315 320
325 atg aag ctg ctt cag aac tgc tgg agt
gag ctc tta atc ctc gac cac 1241 Met Lys Leu Leu Gln Asn Cys Trp Ser
Glu Leu Leu Ile Leu Asp His 330 330 335
340 att tac cga caa gtg gta cat gga aag
gaa gga tcc atc ttc ctg gtt 1289 Ile Tyr Arg Gln Val Val His Gly Lys
Glu Gly Ser Ile Phe Leu Val 345 350
355 360 act ggg caa ca gtg gac tat tcc data
ata gca tca caa gcc gga gcc 1337 Thr Gly Gln Gln Val Asp Tyr Ser Ile
Ile Ala Ser Gln Ala Gly Ala 365
370 375 acc ctc aac aac ctc atg agt cat gca
cag gag tta gtg gca aaa ctt 1385 Thr Leu Asn Asn Leu Met Ser His Ala
Gln Glu Leu Val Ala Lys Leu 380 385
390 cgt tct ctc cag ttt gaa caa cga gag
ttc gta tgt ctg aaa ttc ttg 1433 Arg Ser Leu Gln Phe Asp Gln Arg Glu
Phe Val Cys Leu Lys Phe Leu 395 400
405 gtg ctc ttt agt tta gat gtc aaa aac
ctt ga aac ttc cag ctg gta 1481 Val Leu Phe Ser Leu Asp Val Lys Asn
Leu Glu Asn Phe Gln Leu Val 410 415
420 gaa ggt gtc cag gaa caa gtc aat gcc
gcc ctg ctg gac tac aca atg 1529 Glu Gly Val Gln Glu Gln Val Asn Ala
Ala Leu Leu Asp Tyr Thr Met 425 430
435 440 tgt aac tac ccg cag cag ca gag aaa
ttt gga cag cta ctt ctt cga 1577 Cys Asn Tyr Pro Gln Gln Thr Glu Lys
Phe Gly Gln Leu Leu Leu Arg 445
450 455 cta ccc ga atc cgg gcc atc agt atg
cag gct gaa gaa tac ctc tac 1625 Leu Pro Glu Ile Arg Ala Ile Ser Met
Gln Ala Glu Glu Tyr Leu Tyr 460 465
470 tac aag cac ctg aac ggg gat gtg ccc
tat aat aac ctt ctc att ga 1673 Tyr Lys His Leu Asn Gly Asp Val Pro
Tyr Asn Asn Leu Leu Ile Glu 475 480
485 atg ttg cat gcc aaa aga gca taatttac
aa cccctaggag ctctgctttc 1724 Met Leu His Ala Lys Arg Ala 490 495 aaaacaaaaaa gagttgggg gaggtgggggggggg
gaagaag aacaggaaga aaaaaaagtac 1784 tctgaactgc tccaaagcaac gcttaattaaa aac
ttgcttt aaaattattg aatttaaaaa 1844 gggataataa tcaaattact aatagcaaat aaa
tgagtta tcagggtatt tgtattgcaa 1904 actgtgaatc aaaggcttca cagccccaga gga
ttccata taaaagacat tgtaatggag 1964 tggattgaac tcacagatgg ataccaacac ggt
caagaaga aaaacgggaca gaacggttct 2024 tgtattatta aactgatctc cactatgaag aaa
tttagga actaattctta ttatattaggc 2084 ttatacagcg ggggattttga gcttacagga ttc
ctccatg gtaaagctga actgaaaacaa 2144 ttctcaagaa tgcatcagct gtacctacaa tag
cccctcc ctcttcctttt gaaggcccga 2204 gcaccctctgc cctgtggtca ccgaatctgt act
aaggcc tgtgttcagc caccaccagt 2264 ggtagctcca ccaatcatg aacagcctaa ttt
tgagtgt ctgttgtctta gacctgcaa 2324 cagctaatag gaatttctat taatatgtta gct
tgccatt ttaatatgt tctgagggtt 2384 gttttgtctc gtgttcatga tgttaagaaaa atg
caggcag tatccctcat cttatgtag 2444 tgtgaattta tattaagggga aatgactaca aac
tttcaaaa gcaatgctc catagctaaa 2504 gcaacttaga ccttattttct gctactgttg ctg
aaagttg gctttggcat tgttggattt 2564 cataaaaaaat ttctggcaggg aagtcttgtt agt
atacatc agtctttttttc atcatccaag 2624 tttttagttttc attaaaaaaat acaacattaaa aca
catttttg ctaggatgtc aaatagtcac 2684 agttctaagt agttggaaac aaaattgacg cat
gttaatc tatgcaagaga aaaaggaaag 2744 gatagggtga tgtattgact caaggttcat tct
tgctgca attgaacatc ctcaagagtt 2804 gggatggaaa tggtgattttt tacatggtgtc ctg
gaagat attaaagtaa ttcaaattctt 2864 ccccaaaaagg gaagaggagaga gagtgactact gac
cttttta agtcatagac caagtctgc 2924 tgttagacaa atatgggggg acaaaagaatc gca
aattcttt caatgacta tattagcatt 2984 tattaacatg cgatgccaca ggttatgaaaag tct
tgccttta tttcacaatt ttaaaaggta 3044 gctgtgcaga tgtggattaca catttgtttta aaa
taaagttattaacttt aaagtcaaaa 3104 aaaaaaaaaaaa a
3115 <210> 2 <211> 495 <212> PRT <213> Human <400> 2 Met Ser Ser Asn Ser Asp Thr Gly Asp
Leu Gln Glu Ser Leu Lys His 15
10 15 Gly Leu Thr Pro Ile Val Ser Gln Phe
Lys Met Val Asn Tyr Ser Tyr 20 25
30 Asp Glu Asp Leu Glu Glu Leu Cys Pro
Val Cys Gly Asp Lys Val Ser 35 40
45 Gly Tyr His Tyr Gly Leu Leu Thr Cys
Glu Ser Cys Lys Gly Phe Phe 50 55
60 Lys Arg Thr Val Gln Asn Asn Lys Arg
Tyr Thr Cys Ile Glu Asn Gln 65 70
7580 Asn Cys Gln Ile Asp Lys Thr Gln Arg
Lys Arg Cys Pro Tyr Cys Arg 85
90 95 Phe Gln Lys Cys Leu Ser Val Gly Met
Lys Leu Glu Ala Val Arg Ala 100 105
110 Asp Arg Met Arg Gly Gly Arg Asn Lys
Phe Gly Pro Met Tyr Lys Arg 115 120
125 Asp Arg Ala Leu Lys Gln Gln Lys Lys
Ala Leu Ile Arg Ala Asn Gly 130 135
140 Leu Lys Leu Glu Ala Met Ser Gln Val
Ile Gln Ala Met Pro Ser Asp 145 150
155 160 Leu Thr Ile Ser Ser Ala Ile Gln Asn
Ile His Ser Ala Ser Lys Gly 165
170 175 Leu Pro Leu Asn His Ala Ala Leu Pro
Pro Thr Asp Tyr Asp Arg Ser 180 185
190 Pro Phe Val Thr Thr Ser Pro Ile Ser Met
Thr Met Pro Pro His His Gly Ser 195 200
205 Leu Gln Gly Tyr Gln Thr Tyr Gly His
Phe Pro Ser Arg Ala Ile Lys 210 215
220 Ser Glu Tyr Pro Asp Pro Tyr Thr Ser
Ser Pro Glu Ser Ile Met Gly 225 230
235 240 Tyr Ser Tyr Met Asp Ser Tyr Gln Thr
Ser Ser Pro Ala Ser Ile Pro 245
250 255 His Leu Ile Leu Glu Leu Leu Lys Cys
Glu Pro Asp Glu Pro Gln Val 260 265
270 Gln Ala Lys Ile Met Ala Tyr Leu Gln
Gln Glu Gln Ala Asn Arg Ser 275 280
285 Lys His Glu Lys Leu Ser Thr Phe Gly
Leu Met Cys Lys Met Ala Asp 290 295
300 Gln Thr Leu Phe Ser Ile Val Glu Trp
Ala Arg Ser Ser Ile Phe Phe 305 310
315 320 Arg Glu Leu Lys Val Asp Asp Gln Met
Lys Leu Leu Gln Asn Cys Trp 325
330 335 Ser Glu Leu Leu Ile Leu Asp His Ile
Tyr Arg Gln Val Val His Gly 340 345
350 Lys Glu Gly Ser Ile Phe Leu Val Thr
Gly Gln Gln Val Asp Tyr Ser 355 360
365 Ile Ile Ala Ser Gln Ala Gly Ala Thr
Leu Asn Asn Leu Met Ser His 370 375
380 Ala Gln Glu Leu Val Ala Lys Leu Arg
Ser Leu Gln Phe Asp Gln Arg 385 390
395 400 Glu Phe Val Cys Leu Lys Phe Leu Val
Leu Phe Ser Leu Asp Val Lys 405
410 415 Asn Leu Glu Asn Phe Gln Leu Val Glu
Gly Val Gln Glu Gln Val Asn 420 425
430 Ala Ala Leu Leu Asp Tyr Thr Met Cys
Asn Tyr Pro Gln Gln Thr Glu 435 440
445 Lys Phe Gly Gln Leu Leu Leu Arg Leu
Pro Glu Ile Arg Ala Ile Ser 450 455
460 Met Gln Ala Glu Glu Tyr Leu Tyr Tyr
Lys His Leu Asn Gly Asp Val 465 470
475 480 Pro Tyr Asn Asn Leu Leu Ile Glu Met
Leu His Ala Lys Arg Ala 485
490 495 <210> 3 <211> 1245 <212> DNA <213> Human <220><221> CDS <222> (202). . (1170) <400> 3 cggccgcgtc gacgggaaaa ctttgctttgta act
tttatgaa ttctggattt ttttttttttcc 60 ttttgttttttt cttaacttttc actaagggtt act
gtagtct gatgtgtcct tcccaaggcc 120 acgaaaatttt acaagctgca cttttcttttt gct
caatgat tctctgcttta agccaaaagaa 180 ctgcctata tttcactaag a atg tct tct
aat tca gat act ggg gat tta 231 Met Ser Ser
Asn Ser Asp Thr Gly Asp Leu 1
5 10 caa gag tct tta aag cac gga ctt aca
cct att gtg tct ca ttt aaa 279 Gln Glu Ser Leu Lys His Gly Leu Thr
Pro Ile Val Ser Gln Phe Lys 15
20 25 atg gtg aat tac tcc tat ga t ga ga t
ctg gaa gag ctt tgt ccc gtg 327 Met Val Asn Tyr Ser Tyr Asp Glu Asp
Leu Glu Glu Leu Cys Pro Val 30 35
40 tgt gga gaa aaa gtg tct ggg tac cat
tat ggg ctc ctc acc tgt gaa 375 Cys Gly Asp Lys Val Ser Gly Tyr His
Tyr Gly Leu Leu Thr Cys Glu 45 50
55 agc tgc aag gga ttt ttt aag cga aca
gtc caa aat aat aaa agg tac 423 Ser Cys Lys Gly Phe Phe Lys Arg Thr
Val Gln Asn Asn Lys Arg Tyr 60 65
70 aca tgt ata gaa aac cag aac tgc caa
att gac aaa aca cag aga aag 471 Thr Cys Ile Glu Asn Gln Asn Cys Gln
Ile Asp Lys Thr Gln Arg Lys 75 80
85 90 cgt tgt cct tact tgt cgt ttt aaa
tgt cta agt gtt gga atg aag 519 Arg Cys Pro Tyr Cys Arg Phe Gln Lys
Cys Leu Ser Val Val Gly Met Lys 95
100 105 cta gaa gct gta agg gcc gac cga atg
cgt gga gga agg aat aag ttt 567 Leu Glu Ala Val Arg Ala Asp Arg Met
Arg Gly Gly Arg Asn Lys Phe 110 115
120 ggg cca atg tac aag ag a gac agg gcc
ctg agag caa aaa aaa gcc 615 Gly Pro Met Tyr Lys Arg Asp Arg Ala
Leu Lys Gln Gln Lys Lys Ala 125 130
135 ctc atc cga gcc aat gga ctt aag cta
gaa gcc atg tct cag gtt gat 663 Leu Ile Arg Ala Asn Gly Leu Lys Leu
Glu Ala Met Ser Gln Val Asp 140 145
150 gac caa atg aag ctg ctt cag aac tgc
tgg agt gag ctc tta atc ctc 711 Asp Gln Met Lys Leu Leu Gln Asn Cys
Trp Ser Glu Leu Leu Ile Leu 155 160
165 170 gac cac att tac cga caa gtg gta cat
gga aag gaa gga tcc atc ttc 759 Asp His Ile Tyr Arg Gln Val Val His
Gly Lys Glu Gly Ser Ile Phe 175
180 185 ctg gtt act ggg caa caa gtg gac tat
tcc ata at gca tca ca gcc 807 Leu Val Thr Gly Gln Gln Val Asp Tyr
Ser Ile Ile Ala Ser Gln Ala 190 195
200 gga gcc acc ctc aac aac ctc atg agt
cat gca cag gag tta gtg gca 855 Gly Ala Thr Leu Asn Asn Leu Met Ser
His Ala Gln Glu Leu Val Ala 205 210
215 aaa ctt cgt tct ctc cag ttt gaa caa
cga gag ttc gta tgt ctg aaa 903 Lys Leu Arg Ser Leu Gln Phe Asp Gln
Arg Glu Phe Val Cys Leu Lys 220 225
230 ttc ttg gtg ctc ttt agt tta gat gtc
aaa aac ctt gaa act tc cag 951 Phe Leu Val Leu Phe Ser Leu Asp Val
Lys Asn Leu Glu Asn Phe Gln 235 240
245 250 ctg gta ga ggt gtc cag gaa caa gtc
aat gcc gcc ctg ctg gactac 999 Leu Val Glu Gly Val Gln Glu Gln Val
Asn Ala Ala Leu Leu Asp Tyr 255
260 265 aca atg tgt aac tac ccg cag cag aca
gag aaa ttt cga cag ctactt 1047 Thr Met Cys Asn Tyr Pro Gln Gln Thr
Glu Lys Phe Arg Gln Leu Leu 270 275
280 ctt cga cta ccc ga atc cgg gcc atc
agt atg cag gct gaa gatac 1095 Leu Arg Leu Pro Glu Ile Arg Ala Ile
Ser Met Gln Ala Glu Glu Tyr 285 290
295 ctc tact tac aag cac ctg aac ggg gat
gtg ccc tat aat aac ctt ctc 1143 Leu Tyr Tyr Lys His Leu Asn Gly Asp
Val Pro Tyr Asn Asn Leu Leu 300 305
310 att ga atg ttg cat gcc aaa aga gca
tagttacaaa cccctaggag 1190 Ile Glu Met Leu His Ala Lys Arg Ala 315 320 ctctgctttc aaaacaaaaaa gagattgggggggag
tgggggag ggggaagaag aacag 1245 <210> 4 <211> 323 <212> PRT <213> Human <400> 4 Met Ser Ser Asn Ser Asp Thr Gly Asp
Leu Gln Glu Ser Leu Lys His 15
10 15 Gly Leu Thr Pro Ile Val Ser Gln Phe
Lys Met Val Asn Tyr Ser Tyr 20 25
30 Asp Glu Asp Leu Glu Glu Leu Cys Pro
Val Cys Gly Asp Lys Val Ser 35 40
45 Gly Tyr His Tyr Gly Leu Leu Thr Cys
Glu Ser Cys Lys Gly Phe Phe 50 55
60 Lys Arg Thr Val Gln Asn Asn Lys Arg
Tyr Thr Cys Ile Glu Asn Gln 65 70
7580 Asn Cys Gln Ile Asp Lys Thr Gln Arg
Lys Arg Cys Pro Tyr Cys Arg 85
90 95 Phe Gln Lys Cys Leu Ser Val Gly Met
Lys Leu Glu Ala Val Arg Ala 100 105
110 Asp Arg Met Arg Gly Gly Arg Asn Lys
Phe Gly Pro Met Tyr Lys Arg 115 120
125 Asp Arg Ala Leu Lys Gln Gln Lys Lys
Ala Leu Ile Arg Ala Asn Gly 130 135
140 Leu Lys Leu Glu Ala Met Ser Gln Val
Asp Asp Gln Met Lys Leu Leu 145 150
155 160 Gln Asn Cys Trp Ser Glu Leu Leu Ile
Leu Asp His Ile Tyr Arg Gln 165
170 175 Val Val His His Gly Lys Glu Gly Ser Ile
Phe Leu Val Thr Gly Gln Gln 180 185
190 Val Asp Tyr Ser Ile Ile Ala Ser Gln
Ala Gly Ala Thr Leu Asn Asn 195 200
205 Leu Met Ser His His Ala Gln Glu Leu Val
Ala Lys Leu Arg Ser Leu Gln 210 215
220 Phe Asp Gln Arg Glu Phe Val Cys Leu
Lys Phe Leu Val Val Leu Phe Ser 225 230
235 240 Leu Asp Val Lys Asn Leu Glu Asn Phe
Gln Leu Val Glu Gly Val Gln 245
250 255 Glu Gln Val Asn Ala Ala Leu Leu Asp
Tyr Thr Met Cys Asn Tyr Pro 260 265
270 Gln Gln Thr Glu Lys Phe Arg Gln Leu
Leu Leu Arg Leu Pro Glu Ile 275 280
285 Arg Ala Ile Ser Met Gln Ala Glu Glu
Tyr Leu Tyr Tyr Lys His Leu 290 295
300 Asn Gly Asp Val Pro Tyr Asn Asn Leu
Leu Ile Glu Met Leu His Ala 305 310
315 320 Lys Arg Ala <210> 5 <211> 3251 <212> DNA <213> Human <220><221> CDS <222> (208). . (1830) <400> 5 cgcggccgcg tcgaccaggg aaaagacttg ctt
gtaactt tatgaattct ggatttttttt 60 tttttccttttg cttttttctta actttcacta agg
gttactg tagtctgatg tgtcctttccc 120 aaggccacga aatttgacaa gctgcacttt tct
tttgctc aatgattttct gctttaagcc 180 aaaagaactgc ctataattttc actaaga atg tc
t tct aat tca gat act ggg gat 234 Met Se
r Ser Asn Ser Asp Thr Gly Asp 1
5 tta caa gag tct tta aag cac gga ctt
aca cct att ggt gct ggg ctt 282 Leu Gln Glu Ser Leu Lys His Gly Leu
Thr Pro Ile Gly Ala Gly Leu 10 15
20 25 ccg gac cga cac gga tcc ccc atc ccc
gcc cgc ggt cgc ctt gtc atg 330 Pro Asp Arg His Gly Ser Pro Ile Pro
Ala Arg Gly Arg Leu Val Met 30
35 40 ctg ccc aaa gtg gag acg gaa gcc ctg
gga ctg gct cga tcg cat ggg 378 Leu Pro Lys Val Glu Thr Glu Ala Leu
Gly Leu Ala Arg Ser His His Gly 4550
55 gaa cag ggc cag atg ccg gaaac atg
ca ggtg tct caattt aaa atg 426 Glu Gln Gly Gln Met Pro Glu Asn Met
Gln Val Ser Ser Gln Phe Lys Met 60 65
70 gtg aat tac tcc tat gat ga ga ctg
gaa gaa ctt tgt ccc ggtg tgt 474 Val Asn Tyr Ser Tyr Asp Glu Asp Leu
Glu Glu Leu Cys Pro Val Cys 75 80
85 gga gaa aaa gtg tct ggg tac cat tat
ggg ctc ctc acc tgt ga agc 522 Gly Asp Lys Val Ser Gly Tyr His Tyr
Gly Leu Leu Thr Cys Glu Ser 90 95
100 105 tgc aag gga ttt ttt aag cga aca gtc
caa aat aat aaa agg tac aca 570 Cys Lys Gly Phe Phe Lys Arg Thr Val
Gln Asn Asn Lys Arg Tyr Thr 110
115 120 tgt at ga aaa aac cag aac tgc aaa att
gac aaa aca cag aga aag cgt 618 Cys Ile Glu Asn Gln Asn Cys Gln Ile
Asp Lys Thr Gln Arg Lys Arg 125 130
135 tgt cct tact tgt cgt ttt caaaaa tgt
cta agt gtt gga atg aag cta 666 Cys Pro Tyr Cys Arg Phe Gln Lys Cys
Leu Ser Val Gly Met Lys Leu 140 145
150 ga gct gta agg gcc gac cga atg cgt
gga gga agg aat aag ttt ggg 714 Glu Ala Val Arg Ala Asp Arg Met Arg
Gly Gly Arg Asn Lys Phe Gly 155 160
165 cca atg tac aag agga gac agg gcc ctg
aag caa cag aaa aaa gcc ctc 762 Pro Met Tyr Lys Arg Asp Arg Ala Leu
Lys Gln Gln Lys Lys Ala Leu 170 175
180 185 atc cga gcc aat gga ctt aag cta gaa
gcc atg tct cag gtg atc caa 810 Ile Arg Ala Asn Gly Leu Lys Leu Glu
Ala Met Ser Gln Val Ile Gln 190
195 200 gct atg ccc tct gac ctg acc att tcc
tct gca att caa aac atc cac 858 Ala Met Pro Ser Asp Leu Thr Ile Ser
Ser Ala Ile Gln Asn Ile His 205 205
215 tct gcc tcc aaa ggg cta cct ctg aac
cat gct gcc ttg cct cct aca 906 Ser Ala Ser Lys Gly Leu Pro Leu Asn
His Ala Ala Leu Pro Pro Thr 220 225
230 gac tat gac aga agt ccc ttt gta aca
tcc ccc att agc atg aca atg 954 Asp Tyr Asp Arg Ser Pro Pro He Val Val Thr
Ser Pro Ile Ser Met Thr Met 235 240
245 ccc cct cac ggc agc ctg caa ggt tac
caa aca tat ggg cac ttt cct 1002 Pro Pro His Gly Ser Leu Gln Gly Tyr
Gln Thr Tyr Gly His Phe Pro 250 255
260 265 agc cgg gcc atc aag tct gag tac cca
gac ccc tat acc agc tca ccc 1050 Ser Arg Ala Ile Lys Ser Glu Tyr Pro
Asp Pro Tyr Thr Ser Ser Pro 270
275 280 gag tcc atat ggc tat tca tat atg
gat agt tac cag acg agc tct 1098 Glu Ser Ile Met Gly Tyr Ser Tyr Met
Asp Ser Tyr Gln Thr Ser Ser 285 290
295 cca gca agc atc cca cat ctg ata ctg
gaa ctt ttg aag tgt gag cca 1146 Pro Ala Ser Ile Pro His Leu Ile Leu
Glu Leu Leu Lys Cys Glu Pro 300 305
310 gat gag cct caa gtc cag gct aaa atc
atg gcc tat ttg cag gag 1194 Asp Glu Pro Gln Val Gln Ala Lys Ile
Met Ala Tyr Leu Gln Gln Glu 315 320
325 cag gct aac cga agc aag cac gaa aag
ctg agc acc ttt ggg ctt atg 1242 Gln Ala Asn Arg Ser Lys His Glu Lys
Leu Ser Thr Phe Gly Leu Met 330 335
340 345 tgc aaa atg gca gat caa act ctc ttc
tcc att gtc gag tgg gcc agg 1290 Cys Lys Met Ala Asp Gln Thr Leu Phe
Ser Ile Val Glu Trp Ala Arg 350
355 360 agt agt atc ttc ttc aga gaa ctt aag
gtt gat gac caatg aag ctg 1338 Ser Ser Ile Phe Phe Arg Glu Leu Lys
Val Asp Asp Gln Met Lys Leu 365 370
375 ctt cag aac tgc tgg agt gag ctc tta
atc ctc gac cac att tac cga 1386 Leu Gln Asn Cys Trp Ser Glu Leu Leu
Ile Leu Asp His Ile Tyr Arg 380 385
390 caa gtg gta cat gga aag gaa gga tcc
atc ttc ctg gtt act ggg caa 1434 Gln Val Val His His Gly Lys Glu Gly Ser
Ile Phe Leu Val Thr Gly Gln 395 400
405 caa gtg gac tat tcc ata ata gca tca
caa gcc gga gcc acc ctc aac 1482 Gln Val Asp Tyr Ser Ile Ile Ala Ser
Gln Ala Gly Ala Thr Leu Asn 410 415
420 425 aac ctc atg agt cat gca cag gag tta
gtg gca aaa ctt cgt tct ctc 1530 Asn Leu Met Ser His Ala Gln Glu Leu
Val Ala Lys Leu Arg Ser Leu 430
435 440 cag ttt gat caa cga gag ttc gta tgt
ctg aaa ttc ttg gtg ctc ttt 1578 Gln Phe Asp Gln Arg Glu Phe Val Cys
Leu Lys Phe Leu Val Val Leu Phe 445 450
455 agt tta gat gtc aaa aac ctt gaa aac
ttc cag ctg gta gaa ggt gtc 1626 Ser Leu Asp Val Lys Asn Leu Glu Asn
Phe Gln Leu Val Glu Gly Val 460 465
470 cag gaa caa gtc aat gcc gcc ctg ctg
gac tac aca atg tgt aact tac 1674 Gln Glu Gln Val Asn Ala Ala Leu Leu
Asp Tyr Thr Met Cys Asn Tyr 475 480
485 ccg cag cag ca gag aaa ttt gga cag
cta ctt ctt cga cta ccc gaa 1722 Pro Gln Gln Thr Glu Lys Phe Gly Gln
Leu Leu Leu Arg Leu Pro Glu 490 495
500 505 atc cgg gcc atc agt atg cag gct gaa
gaa tac ctc tac tac aag cac 1770 Ile Arg Ala Ile Ser Met Gln Ala Glu
Glu Tyr Leu Tyr Tyr Lys His 510
515 520 ctg aac ggg gt gtg ccc tat aat aac
ctt ctc att gaa atg ttg cat 1818 Leu Asn Gly Asp Val Pro Tyr Asn Asn Asn
Leu Leu Ile Glu Met Leu His 525 530
535 gcc aaa aga gca taagttacaa cccctagga
g ctctgctttc aaaacaaaaaa 1870 Ala Lys Arg Ala 540 gagattggggg gaggggggg ggggggaagaag aac
agagaaga aaaaaaagtact tctgaactgc 1930 tccagagcaac gctataattaa aaactgcttt aaa
gattattg aatttaaaa gggataataa 1990 tcaaattact aatagcaaat aaatgattgta tca
gggtatt tgttattgcaa actgttgaatc 2050 aaaggcttca cagccccaga ggattccata taa
aagacat tgtaatggag tggattgaac 2110 tcacagatgg ataccacaacac ggtcagaagaaaa
acggaca gaacgggttct tgttatttatta 2170 aactgattctc actatgaag aaaatttagga act
aattctta ttaatttaggc tatataagcg 2230 ggggatttga gcttacagaga ttcctccatg gta
aagctga actgaaaaca ttctcaagaa 2290 tgcatcaggt gacctacaa tagcccctcc ctc
ttcctttt gaaggcccga gcacctctgc 2350 cctgtgtgtca ccgaatctgtt actaaggacc tgt
gttcagc caaccccaggt gtagtagccca 2410 ccaatcatg aacagcctaa tttttgaggtt ctg
tgtctta gacctgcaaaa cagctatatag 2470 gaatttct taatatgtta gctttgccatt tta
aatattgt tctgagggtt gttttgtctcc 2530 gtgtttcatga tgttaagaaaa atgcaggcag tat
cccctcat cttatgtagag tgtgaattaa 2590 tattaaggga aatgactaca aactttcaaaa gca
aatgctc catagctaaa gcaacttaga 2650 ccttattttct gctactgttg ctgaaatgtg gct
ttggcat tgttggattt cataaaaaaat 2710 ttctggcag aagctcttgttt agtatacatc agt
cttttttc atcatccaag tttttagtagtc 2770 attaaaaaaat acaactatta acacatttttg cta
ggattgtc aaatagtacac gtttctaagt 2830 aggttggaac aaaattgacg catgttaatc tat
gcaagaga gaaagggaaag gatgaggtga 2890 tgtattgact caaggttcat tcttgctgca att
gaactc ctcaagagtt gggatgggaaaa 2950 tggtgattttt tacatgtgtgtc ctggaaaagatt
aaagtaa ttcaaattctt ccccaaaaagg 3010 aaaaggaaga gagtgactact gacctttttta agt
catacac caaaagtctgc tgtagagaacaa 3070 atatgggggg acaaaagaatc gcaattttttcaa
atgacta tattagcattat ttattacatg 3130 cgatgccaca ggtatgaaaag tctttgccttta ttt
cacaatt ttaaaaggta gctgtggaga 3190 tgtggtcaa catttgtttta aaaataagta tta
attacht aaagtcaaaa aaaaaaaaaaaa 3250 a
3251 <210> 6 <211> 541 <212> PRT <213> Human <400> 6 Met Ser Ser Asn Ser Asp Thr Gly Asp
Leu Gln Glu Ser Leu Lys His 15
10 15 Gly Leu Thr Pro Ile Gly Ala Gly Leu
Pro Asp Arg His Gly Ser Pro 20 25
30 Ile Pro Ala Arg Gly Arg Leu Val Met
Leu Pro Lys Val Glu Thr Glu 35 40
45 Ala Leu Gly Leu Ala Arg Ser His Gly
Glu Gln Gly Gln Met Pro Glu 50 55
60 Asn Met Gln Val Ser Gln Phe Lys Met
Val Asn Tyr Ser Tyr Asp Glu 65 70
7580 Asp Leu Glu Glu Leu Cys Pro Val Cys
Gly Asp Lys Val Ser Gly Tyr 85
90 95 His Tyr Gly Leu Leu Thr Cys Glu Ser
Cys Lys Gly Phe Phe Lys Arg 100 105
110 Thr Val Gln Asn Asn Lys Arg Tyr Thr
Cys Ile Glu Asn Gln Asn Cys 115 120
125 Gln Ile Asp Lys Thr Gln Arg Lys Arg
Cys Pro Tyr Cys Arg Phe Gln 130 135
140 Lys Cys Leu Ser Val Val Gly Met Lys Leu
Glu Ala Val Arg Ala Asp Arg 145 150
155 160 Met Arg Gly Gly Gly Arg Asn Lys Phe Gly
Pro Met Tyr Lys Arg Asp Arg 165
170 175 Ala Leu Lys Gln Gln Lys Lys Ala Leu
Ile Arg Ala Asn Gly Leu Lys 180 185
190 Leu Glu Ala Met Ser Gln Val Ile Gln
Ala Met Pro Ser Asp Leu Thr 195 200
205 Ile Ser Ser Ala Ile Gln Asn Ile His
Ser Ala Ser Lys Gly Leu Pro 210 215
220 Leu Asn His His Ala Ala Leu Pro Pro Pro Thr
Asp Tyr Asp Arg Ser Pro Pro He 225 230
235 240 Val Thr Ser Pro Ile Ser Met Thr Met
Pro Pro His His Gly Ser Leu Gln 245
250 255 Gly Tyr Gln Thr Tyr Gly His Phe Pro
Ser Arg Ala Ile Lys Ser Glu 260 265
270 Tyr Pro Asp Pro Tyr Thr Ser Ser Pro
Glu Ser Ile Met Gly Tyr Ser 275 280
285 Tyr Met Asp Ser Tyr Gln Thr Ser Ser
Pro Ala Ser Ile Pro His Leu 290 295
300 Ile Leu Glu Leu Leu Lys Cys Glu Pro
Asp Glu Pro Gln Val Gln Ala 305 310
315 320 Lys Ile Met Ala Tyr Leu Gln Gln Glu
Gln Ala Asn Arg Ser Lys His 325
330 335 Glu Lys Leu Ser Thr Phe Gly Leu Met
Cys Lys Met Ala Asp Gln Thr 340 345
350 Leu Phe Ser Ile Val Glu Trp Ala Arg
Ser Ser Ile Phe Phe Arg Glu 355 360
365 Leu Lys Val Asp Asp Gln Met Lys Leu
Leu Gln Asn Cys Trp Ser Glu 370 375
380 Leu Leu Ile Leu Asp His Ile Tyr Arg
Gln Val Val His His Gly Lys Glu 385 390
395 400 Gly Ser Ile Phe Leu Val Thr Gly Gln
Gln Val Asp Tyr Ser Ile Ile 405
410 415 Ala Ser Gln Ala Gly Ala Thr Leu Asn
Asn Leu Met Ser His His Ala Gln 420 425
430 Glu Leu Val Ala Lys Leu Arg Ser Leu
Gln Phe Asp Gln Arg Glu Phe 435 440
445 Val Cys Leu Lys Phe Leu Val Val Leu Phe
Ser Leu Asp Val Lys Asn Leu 450 455
460 Glu Asn Phe Gln Leu Val Glu Gly Val
Gln Glu Gln Val Asn Ala Ala 465 470
475 480 Leu Leu Asp Tyr Thr Met Cys Asn Tyr
Pro Gln Gln Thr Glu Lys Phe 485
490 495 Gly Gln Leu Leu Leu Arg Leu Pro Glu
Ile Arg Ala Ile Ser Met Gln 500 505
510 Ala Glu Glu Tyr Leu Tyr Tyr Lys His
Leu Asn Gly Asp Val Pro Tyr 515 520
525 Asn Asn Leu Leu Ile Glu Met Leu His
Ala Lys Arg Ala 530 535
540 <210> 7 <211> 2330 <212> DNA <213> Human <220><221> CDS <222> (363). . (1862) <400> 7 gaactggat atatggttta cagcaggtca cta
atgttgg aaaaaagtaca gagttccaggg 60 aaagtactgc ttgttaacttt atgaatttctg ga
ttttttt ccttttgctttt tctttaactt 120 tcactaaggg ttactgtagt ctgatgtgtc ctt
cccaagg cccagaaatt tgacaagctg 180 actttttctt ttgctcaatg attctctgcttt taa
gccaaag aactgcctat aatttcacta 240 agaatgtctt ctaattcaga tactggggtat tta
caaggt ctttaaaagca cggacttca 300 cctattgggtg ctgggcttcc ggaccgacac gga
tccccca tcccgcccgc ggtcgcctttg 360 tc atg ctg ccc aaa gtg gag acg gaag
cc ctg gga ctg gct cga tcg 407 Met Leu Pro Lys Val Glu Thr Glu A
la Leu Gly Leu Ala Arg Ser 15
10 15 cat ggg gaa cag ggc cag atg ccg gaa
aac atg caa gtg tct caattt 455 His Gly Glu Gln Gly Gln Met Pro Glu
Asn Met Gln Val Ser Gln Phe 20
25 30 aaa atg gtg aat tact tcc tat ga t gaa
ga tctg gaa gag ctt tgt ccc 503 Lys Met Val Asn Tyr Ser Tyr Asp Glu
Asp Leu Glu Glu Leu Cys Pro 35 40
45 gtg tgt gga gaaa gtg tct ggg tac
cat tat ggg ctc ctc acc tgt 551 ValCys Gly Asp Lys Val Ser Gly Tyr
His Tyr Gly Leu Leu Thr Cys 50 55
60 ga agc tgc aag gga ttt ttt aag cga
aca gtc caa aat aat aaa agg 599 Glu SerCys Lys Gly Phe Phe Lys Arg
Thr Val Gln Asn Asn Lys Arg 65 70
75 tac aca tgt ata gaa aac cag aac tgc
caa att gac aaa aca cag aga 647 Tyr Thr Cys Ile Glu Asn Gln Asn Cys
Gln Ile Asp Lys Thr Gln Arg 80 85
90 aag cgt tgt cct tact tgt cgt ttt caa
aaa tgt cta agt gtt gga atg 695 Lys Arg Cys Pro Tyr Cys Arg Phe Gln
Lys Cys Leu Ser Val Gly Met 100
105 110 aag cta gaa gct gta agg gcc gac cga
atg cgt gga gga agg aat aag 743 Lys Leu Glu Ala Val Arg Ala Asp Arg
Met Arg Gly Gly Arg Asn Lys 115 120
125 ttt ggg cca atg tac aag agaga gac agg
gcc ctg aaa caag aaa aaa 791 Phe Gly Pro Met Tyr Lys Arg Asp Arg
Ala Leu Lys Gln Gln Lys Lys 130 135
140 gcc ctc atc cga gcc aat gga ctt aag
cta gaa gcc atg tct cag gtg 839 Ala Leu Ile Arg Ala Asn Gly Leu Lys
Leu Glu Ala Met Ser Gln Val 145 150
155 atc caa gct atg ccc tct gac ctg acc
att tcc tct gca att caa aac 887 Ile Gln Ala Met Pro Ser Asp Leu Thr
Ile Ser Ser Ala Ile Gln Asn 160 165
170 175 atc cac tct gcc tcc aaa ggg cta cct
ctg aac cat gct gcc ttg cct 935 Ile His Ser Ala Ser Lys Gly Leu Pro
Leu Asn His Ala Ala Leu Pro 180
185 190 cct aca gac tat gac aga agt ccc ttt
gta aca tcc ccc att agc atg 983 Pro Thr Asp Tyr Asp Arg Ser Pro Pro He
Val Thr Ser Pro Ile Ser Met 195 200
205 aca atg ctg cac ggc agc ctg caa ggt
tac caa aca tat gggc cac ttt 1031 Thr Met Leu His Gly Ser Leu Gln Gly
Tyr Gln Thr Tyr Gly His Phe 210 215
220 cct agc cgg gcc atc aag tct gag tac
cca gac ccc tat acc agc tca 1079 Pro Ser Arg Ala Ile Lys Ser Glu Tyr
Pro Asp Pro Tyr Thr Ser Ser 225 230
235 ccc gag tcc ata atg ggc tat tca tat
atg gat agt tac cag acg agc 1127 Pro Glu Ser Ile Met Gly Tyr Ser Tyr
Met Asp Ser Tyr Gln Thr Ser 240 245
250 255 tct cca gca agc atc cca cat ctg data
ctg gaa ctt ttg aag tgt gag 1175 Ser Pro Ala Ser Ile Pro His Leu Ile
Leu Glu Leu Leu Lys Cys Glu 260
265 270 cca gat gag cct caa gtc cag gct aaa
atc atg gcc tat ttg cag ca1223 Pro Asp Glu Pro Gln Val Gln Ala Lys
Ile Met Ala Tyr Leu Gln Gln 275 280
285 gag cag gct aac cga agc aag cac gaa
aag ctg agc acc ttt ggg ctt 1271 Glu Gln Ala Asn Arg Ser Lys His Glu
Lys Leu Ser Thr Phe Gly Leu 290 295
300 atg tgc aaa atg gca ga t caa act gtc
ttc tcc att gtc gag tgg gcc 1319 Met Cys Lys Met Ala Asp Gln Thr Val
Phe Ser Ile Val Glu Trp Ala 305 310
315 agg agt agt atc ttc ttc aga gaa ctt
aag gtt gat gac caat atg aag 1367 Arg Ser Ser Ile Phe Phe Arg Glu Leu
Lys Val Asp Asp Gln Met Lys 320 325
330 335 ctg ctt cag aac tgc tgg agt gag ctc
tta atc ctc gac cac att tac 1415 Leu Leu Gln Asn Cys Trp Ser Glu Leu
Leu Ile Leu Asp His Ile Tyr 340
345 350 cga caa gtg gta cat gga aag gaa gga
tcc atc ttc ctg gtt act ggg 1463 Arg Gln Val Val His Gly Lys Glu Gly Gly
Ser Ile Phe Leu Val Thr Gly 355 360
365 caa caa gtg gac tat tcc ata ata gca
tca caa gcc gga gcc acc ctc 1511 Gln Gln Val Asp Tyr Ser Ile Ile Ala
Ser Gln Ala Gly Ala Thr Leu 370 375
380 aac aac ctc atg agt cat gca cag gag
tta gtg gca aaa ctt cgt tct 1559 Asn Asn Leu Met Ser His Ala Gln Glu
Leu Val Ala Lys Leu Arg Ser 385 390
395 ctc cag ttt gat caa cga gag tttc gta
tgt ctg aaa ttc ttg gtg ctc 1607 Leu Gln Phe Asp Gln Arg Glu Phe Val
Cys Leu Lys Phe Leu Val Leu 400 405
410 415 ttt agt tta gat gtc aaa aac ctt gaa
aac ttc cag ctg gta gaa ggt 1655 Phe Ser Leu Asp Val Lys Asn Leu Glu
Asn Phe Gln Leu Val Glu Gly 420
425 430 gt cag gaa caa gtc aat gcc gcc ctg
ctg gac tac aca atg tgt aac 1703 Val Gln Glu Gln Val Asn Ala Ala Leu
Leu Asp Tyr Thr Met Cys Asn 435 440
445 tac ccg cag cag ca gag aaa ttt gga
cag cta ctt ctt cga cta ccc 1751 Tyr Pro Gln Gln Thr Glu Lys Phe Gly
Gln Leu Leu Leu Arg Leu Pro 450 455
460 gaa atc cgg gcc atc agt atg cag gct
gaa gaa tac ctc tac tac aag 1799 Glu Ile Arg Ala Ile Ser Met Gln Ala
Glu Glu Tyr Leu Tyr Tyr Lys 465 470
475 cac ctg aat ggg gat gtg ccc tat aat
aac ctt ctc att ga atg ttg 1847 His Leu Asn Gly Asp Val Pro Tyr Asn
Asn Leu Leu Ile Glu Met Leu 480 485
490 495 cat gcc aaa aga gca taagttacaaa cccct
aggag ctctgcttttc aaaacaaaaaa 1902 His Ala Lys Arg Ala
500 gagattggggg gagttgggggg gggggagaagaag aac
agagaaga aaaaaaagtac tctgaactgc 1962 tccaagtaac gctataattaaa aactgcttt aaa
gattattg aatttaaaaa ggcataataa 2022 tcaaactacta atgcaaata aatgatgtat cag
ggtatttt gtattgcaa ctgtgaatca 2082 aagcttcaca gccccagaggg attccatata aaa
gactattg taatggagtg gattgaactc 2142 acagagggt accaacacgg tcagaagaaaa aac
ggacaga acgggttctttg tatttattaa 2202 ctgatctcca tattatagaagaa atttaggaac taa
tctttatt aatttaggtt atacagcgggg 2262 gattttgagct tacaggatttc ctccatgggta aag
ctgaact gaaaaaattc tcaagaatgc 2322 atcagctg
2330 <210> 8 <211> 500 <212> PRT <213> Human <400> 8 Met Leu Pro Lys Val Glu Thr Glu Ala
Leu Gly Leu Ala Arg Ser His 15
10 15 Gly Glu Gln Gly Gln Met Pro Glu Asn
Met Gln Val Ser Gln Phe Lys 20 25
30 Met Val Asn Tyr Ser Tyr Asp Glu Asp
Leu Glu Glu Leu Cys Pro Val 35 40
45 Cys Gly Asp Lys Val Ser Gly Tyr His
Tyr Gly Leu Leu Thr Cys Glu 50 55
60 Ser Cys Lys Gly Phe Phe Lys Arg Thr
Val Gln Asn Asn Lys Arg Tyr 65 70
7580 Thr Cys Ile Glu Asn Gln Asn Cys Gln
Ile Asp Lys Thr Gln Arg Lys 85
90 95 Arg Cys Pro Tyr Cys Arg Phe Gln Lys
Cys Leu Ser Val Gly Met Lys 100 105
110 Leu Glu Ala Val Arg Ala Asp Arg Met
Arg Gly Gly Arg Asn Lys Phe 115 120
125 Gly Pro Met Tyr Lys Arg Asp Arg Ala
Leu Lys Gln Gln Lys Lys Ala 130 135
140 Leu Ile Arg Ala Asn Gly Leu Lys Leu
Glu Ala Met Ser Gln Val Ile 145 150
155 160 Gln Ala Met Pro Ser Asp Leu Thr Ile
Ser Ser Ala Ile Gln Asn Ile 165
170 175 His Ser Ala Ser Lys Gly Leu Pro Leu
Asn His Ala Ala Leu Pro Pro 180 185
190 Thr Asp Tyr Asp Arg Ser Pro Pro He Val
Thr Ser Pro Ile Ser Met Thr 195 200
205 Met Leu His Gly Ser Leu Gln Gly Tyr
Gln Thr Tyr Gly His Phe Pro 210 215
220 Ser Arg Ala Ile Lys Ser Glu Tyr Pro
Asp Pro Tyr Thr Ser Ser Pro 225 230
235 240 Glu Ser Ile Met Gly Tyr Ser Tyr Met
Asp Ser Tyr Gln Thr Ser Ser 245
250 255 Pro Ala Ser Ile Pro His Leu Ile Leu
Glu Leu Leu Lys Cys Glu Pro 260 265
270 Asp Glu Pro Gln Val Gln Ala Lys Ile
Met Ala Tyr Leu Gln Gln Glu 275 280
285 Gln Ala Asn Arg Ser Lys His Glu Lys
Leu Ser Thr Phe Gly Leu Met 290 295
300 Cys Lys Met Ala Asp Gln Thr Val Phe
Ser Ile Val Glu Trp Ala Arg 305 310
315 320 Ser Ser Ile Phe Phe Arg Glu Leu Lys
Val Asp Asp Gln Met Lys Leu 325
330 335 Leu Gln Asn Cys Trp Ser Glu Leu Leu
Ile Leu Asp His Ile Tyr Arg 340 345
350 Gln Val Val His His Gly Lys Glu Gly Ser
Ile Phe Leu Val Thr Gly Gln 355 360
365 Gln Val Asp Tyr Ser Ile Ile Ala Ser
Gln Ala Gly Ala Thr Leu Asn 370 375
380 Asn Leu Met Ser His His Ala Gln Glu Leu
Val Ala Lys Leu Arg Ser Leu 385 390
395 400 Gln Phe Asp Gln Arg Glu Phe Val Cys
Leu Lys Phe Leu Val Leu Phe 405
410 415 Ser Leu Asp Val Lys Asn Leu Glu Asn
Phe Gln Leu Val Glu Gly Val 420 425
430 Gln Glu Gln Val Asn Ala Ala Leu Leu
Asp Tyr Thr Met Cys Asn Tyr 435 440
445 Pro Gln Gln Thr Glu Lys Phe Gly Gln
Leu Leu Leu Arg Leu Pro Glu 450 455
460 Ile Arg Ala Ile Ser Met Gln Ala Glu
Glu Tyr Leu Tyr Tyr Lys His 465 470
475 480 Leu Asn Gly Asp Val Pro Tyr Asn Asn
Leu Leu Ile Glu Met Leu His 485
490 495 Ala Lys Arg Ala 500 <210> 9 <211> 3027 <212> DNA <213> Mouse <220><221> CDS <222> (159). . (1838) <400> 9 tgtttttttcc cccttttttct taactttcac taa
ggaaatg agggttactg tagtctgaggg 60 tttccttccc aaagtcacaa aatatgacaa gct
gcaatct ttctcacatt caatgatttc 120 tgctgtaagc caaaggactg ccaataattt cgc
taaga atg tct gct agt ttg gat 176
Met Ser Ala Ser Leu Asp
15 act gga gatt ttt caa gaa ttt ctt aag
cat gga ctt aca gct att gcg 224 Thr Gly Asp Phe Gln Glu Phe Leu Lys
His Gly Leu Thr Ala Ile Ala 10 15
20 tct gca cca ggg tca gag act cgc cac
tcc ccc aaa cgt gag ga caa 272 Ser Ala Pro Gly Ser Glu Thr Arg His
Ser Pro Lys Arg Glu Glu Gln 25 30
35 ctc cgg aaa aaa cgt gct ggg ctt ccg
gac cga cac cga cgc ccc att 320 Leu Arg Glu Lys Arg Ala Gly Leu Pro
Asp Arg His Arg Arg Pro Ile 40 45
50 ccc gcc cgc agc cgc ctt gtc atg ctg
ccc aaa gtg gag acg gaa gcc 368 Pro Ala Arg Ser Arg Leu Val Met Leu
Pro Lys Val Glu Thr Glu Ala 55 60
65 70 cca gga ctg gtc cga tcg cat ggg gaa
cag ggg cag atg cca ga aac 416 Pro Gly Leu Val Arg Ser His Gly Glu
Gln Gly Gln Met Pro Glu Asn 75
80 85 atg caa gtg tct caattt aaa atg gtg
aat tac tcc tat gat ga gat 464 Met Gln Val Ser Gln Phe Lys Met Val
Asn Tyr Ser Tyr Asp Glu Asp 90 95
100 ctg gaa gag cta tgt cct gtg tgt gggc
gat aaa gtg tct ggg tac cat 512 Leu Glu Glu Leu Cys Pro Val Cys Gly
Asp Lys Val Ser Gly Tyr His 105 110
115 tac ggt ctc ctc acg tgc gaa agc tgc
aag ggt ttt ttt aag cga act 560 Tyr Gly Leu Leu Thr Cys Glu Ser Cys
Lys Gly Phe Phe Lys Arg Thr 120 125
130 gtc caa aac caa aa agg tac acg tgc
ata gag aac cag aat tgc caa 608 Val Gln Asn Gln Lys Arg Tyr Thr Cys
Ile Glu Asn Gln Asn Cys Gln 135 140
145 150 att gac aaa acg cag aga aaa cga tgt
ccc tact tgt cga ttc aaa aaa 656 Ile Asp Lys Thr Gln Arg Lys Arg Cys
Pro Tyr Cys Arg Phe Lys Lys 155
160 165 tgt atc gat gtt ggg atg aag ctg gaa
gcc gta aga gcc gac cgc atg 704 Cys Ile Asp Val Gly Met Lys Leu Glu
Ala Val Arg Ala Asp Arg Met 170 175
180 cga ggg gggc aga aat aag ttt ggg cca
atg tac aag aga gac agg gct 752 Arg Gly Gly Arg Asn Lys Phe Gly Pro
Met Tyr Lys Arg Asp Arg Ala 185 190
195 ttg aag cag cag aag aaa gcc ctc att
cga gcc aat gga ctt aag ctg 800 Leu Lys Gln Gln Lys Lys Ala Leu Ile
Arg Ala Asn Gly Leu Lys Leu 200 205
210 ga gcc atg tct cag gtg atc caa gca
atg ccc tca gac ctg acc tct 848 Glu Ala Met Ser Gln Val Ile Gln Ala
Met Pro Ser Asp Leu Thr Ser 215 220
225 230 gca att cag aac att cat tcc gcc tcc
aaa gc cta cct ctg agc cat 896 Ala Ile Gln Asn Ile His Ser Ala Ser
Lys Gly Leu Pro Leu Ser His His 235
240 245 gta gcc ttg cct ccg aca gac tat gac
aga agt ccc ttt gtc aca tct 944 Val Ala Leu Pro Pro Thr Asp Tyr Asp
Arg Ser Pro Phe Val Thr Ser 250 250
260 ccc att agc atg aca atg cca cct cac
agc agc ctg cat ggt tac caa 992 Pro Ile Ser Met Thr Met Pro Pro His
Ser Ser Leu His Gly Tyr Gln 265 270
275 ccc tat ggt cac ttt cct agt cgg gcc
atc aag tct gag tac cca gac 1040 Pro Tyr Gly His Phe Pro Ser Arg Ala
Ile Lys Ser Glu Tyr Pro Asp 280 285
290 ccc tac tcc agc tca cct gag tca atg
atg ggt tac tcc tac atg gat 1088 Pro Tyr Ser Ser Ser Pro Glu Ser Met
Met Gly Tyr Ser Tyr Met Asp 295 300
305 310 ggt tac cag aca aac tcc ccg gcc agc
atc cca cac ctg ata ctg gaa 1136 Gly Tyr Gln Thr Asn Ser Pro Ala Ser
Ile Pro His Leu Ile Leu Glu 315
320 325 ctt ttg aag tgt gaa cca gat gag cct
ca gtt caa gcg aag atc atg 1184 Leu Leu Lys Cys Glu Pro Asp Glu Pro
Gln Val Gln Ala Lys Ile Met 330 335
340 gct tac ctc cag caa gag cag agt aac
cga aac agg caa aaa aag ctg 1232 Ala Tyr Leu Gln Gln Glu Gln Ser Asn
Arg Asn Arg Gln Glu Lys Leu 345 350
355 agc gca ttt ggg ctt tta tgc aaa atg
gcg gac cag acc ctg ttc tcc 1280 Ser Ala Phe Gly Leu Leu Cys Lys Met
Ala Asp Gln Thr Leu Phe Ser 360 365
370 att gtt gag tgg gcc agg agt agt atc atc
ttc ttc agg gaa ctg aag gtt 1328 Ile Val Glu Trp Ala Arg Ser Ser Ile
Phe Phe Arg Glu Leu Lys Val 375 380
385 390 gat gac caaatg aag ctg ctt caa aac
tgc tgg agt gag ctc ttg att 1376 Asp Asp Gln Met Lys Leu Leu Gln Asn
Cys Trp Ser Glu Leu Leu Ile 395
400 405 ctc gat cac att tac cga caa gtg gcg
cat ggg aag gaaggg aca atc 1424 Leu Asp His Ile Tyr Arg Gln Val Ala
His Gly Lys Glu Gly Thr Ile 410 415
420 ttc ctg gtt act gga gaa cac gtg gac
tac tcc acc atc atc tca cac 1472 Phe Leu Val Thr Gly Glu His Val Asp
Tyr Ser Thr Ile Ile Ser His 425 430
435 aca gaa gtc gcg ttc aac aac ctc ctg
agt ctc gca cag gag ctg gtg 1520 Thr Glu Val Ala Phe Asn Asn Leu Leu
Ser Leu Ala Gln Glu Leu Val 440 445
450 gtg agg ctc cgt tcc ctt cag ttc gat
cag cgg gag ttt gta tgt ctc 1568 Val Arg Leu Arg Ser Leu Gln Phe Asp
Gln Arg Glu Phe Val Cys Leu 455 460
465 470 aag ttc ctg gtg ctg ttc agc tca gat
gtg aag aac ctg gag aac ctg 1616 Lys Phe Leu Val Leu Phe Ser Ser Asp
Val Lys Asn Leu Glu Asn Leu 475
480 485 cag ctg gtg gaa ggt gtc caa gag cag
gtg aat gcc gcc ctg ctg gac 1664 Gln Leu Val Glu Gly Val Gln Glu Gln
Val Asn Ala Ala Leu Leu Asp 490 495
500 tac acg gtt tgc aac tac cca caa cag
act gag aaa tc gga cag cta 1712 Tyr Thr Val Cys Asn Tyr Pro Gln Gln
Thr Glu Lys Phe Gly Gln Leu 505 510
515 ctt ctt cgg cta ccc gag atc cgg gca
atc agc aag cag gca ga gac 1760 Leu Leu Arg Leu Pro Glu Ile Arg Ala
Ile Ser Lys Gln Ala Glu Asp 520 525
530 tac ctg tac tat aag cac gtg aac ggg
gat gtg ccc tat aat aac ctc 1808 Tyr Leu Tyr Tyr Lys His Val Asn Gly
Asp Val Pro Tyr Asn Asn Leu 535 540
545 550 ctc att gag atg ctg cat gcc aaa aga
gcc tagctcccca cccctggagaag 1858 Leu Ile Glu Met Leu His Ala Lys Arg
Ala 555
560 cttgctctag gaacacagac tggaaggaga aga
ggaggac gatgacagaa acacaatact 1918 ctgaactgct ccaagcaatg ctattataa act
tggttta aagacactga attttaaaag 1978 catataattatt aaaattacctaa tagcaaaaata atg
atatatc agggtattttg tactgcaaac 2038 tgtgaatcaa aggctgtattg aatcaaagga ttc
atatgaa agacattgta atgggggtgga 2098 ttgaacttac agatgggagac caataccaca gca
gaataaa aatgggacaga acaatcctttg 2158 tatttattaa ctatctctgct attaagaaat tca
gaagttg attctctgtta ttaattggat 2218 ttgtcctgaa ttactccgtg gtgacgctga aca
actcaag aatacatggg ctgtgctttgg 2278 cagcccctcc ccatccctcc caccaccacc acc
cccaccc cccacaaggcc ctataccttc 2338 tgacctgtga gccctgaagc tattttaagg act
tctgtttc agccataccc aggtagtagct 2398 ccctaaaacc atgatttctgg gattctctgtg tct
tagacct gccacaagct aataagaacaca 2458 atgtataaat atgtcagcttt gcatttttaaa tat
gtgctga agttttgttttt gtcgtgttgtt 2518 cgtaattaaaaaagaaaaacgg gcagtaacccctct
tctatat aagcattagt taatttaag 2578 ggaatcaaaa caatctagag ccaatactcc caa
caagcaa gttagattctt actcttgctg 2638 ctgttgctga aatgtgggctt tggcatggtt ggg
tttcat aaactttttg gccaagaggc 2698 ttgttagtacatccatct gtttagtcat caa
ggtttgt agttcactta aaaaaaaaaata 2758 accactaga catctttttgc tgaatgtcaa data
gtcacag tctaagtagc caaaaaagtca 2818 aagcgtgtta aaattttgcca aatgaaggaa agg
gtgagct gcaaaaggggga tggttcgagg 2878 ttcattccag ttgttgaccccg agcgtcccc aaa
cctggga tgcaaaagaca gtgattctgc 2938 atatggcctg aaaagacagg aaaagccaggtc tcc
tacaaag gggaatggaa gatcctggcc 2998 tctaagtcat agccaaagt ctgctgtag
3027 <210> 10 <211> 560 <212> PRT <213> Mouse <400> 10 Met Ser Ala Ser Leu Asp Thr Gly Asp
Phe Gln Glu Phe Leu Lys His 15
10 15 Gly Leu Thr Ala Ile Ala Ser Ala Pro
Gly Ser Glu Thr Arg His Ser 20 25
30 Pro Lys Arg Glu Glu Gln Leu Arg Glu
Lys Arg Ala Gly Leu Pro Asp 35 40
45 Arg His Arg Arg Pro Ile Pro Ala Arg
Ser Arg Leu Val Met Leu Pro 50 55
60 Lys Val Glu Thr Glu Ala Pro Gly Leu
Val Arg Ser His His Gly Glu Gln 65 70
75 80 Gly Gln Met Pro Glu Asn Met Gln Val
Ser Gln Phe Lys Met Val Asn 85
90 95 Tyr Ser Tyr Asp Glu Asp Leu Glu Glu
Leu Cys Pro Val Cys Gly Asp 100 105
110 Lys Val Ser Gly Tyr His Tyr Gly Leu
Leu Thr Cys Glu Ser Cys Lys 115 120
125 Gly Phe Phe Lys Arg Thr Val Gln Asn
Gln Lys Arg Tyr Thr Cys Ile 130 135
140 Glu Asn Gln Asn Cys Gln Ile Asp Lys
Thr Gln Arg Lys Arg Cys Pro 145 150
155 160 Tyr Cys Arg Phe Lys Lys Cys Ile Asp
Val Gly Met Lys Leu Glu Ala 165
170 175 Val Arg Ala Asp Arg Met Arg Gly Gly
Arg Asn Lys Phe Gly Pro Met 180 185
190 Tyr Lys Arg Asp Arg Ala Leu Lys Gln
Gln Lys Lys Ala Leu Ile Arg 195 200
205 Ala Asn Gly Leu Lys Leu Glu Ala Met
Ser Gln Val Ile Gln Ala Met 210 215
220 Pro Ser Asp Leu Thr Ser Ala Ile Gln
Asn Ile His Ser Ala Ser Lys 225 230
235 240 Gly Leu Pro Leu Ser His His Val Val Ala Leu
Pro Pro Thr Asp Tyr Asp Arg 245
250 255 Ser Pro Pro Val Val Thr Ser Pro Ile Ser
Met Thr Met Pro Pro His Ser Ser 260 265
270 Ser Leu His Gly Tyr Gln Pro Tyr Gly
His Phe Pro Ser Arg Ala Ile 275 280
285 Lys Ser Glu Tyr Pro Asp Pro Tyr Ser
Ser Ser Pro Glu Ser Met Met 290 295
300 Gly Tyr Ser Tyr Met Asp Gly Tyr Gln
Thr Asn Ser Pro Ala Ser Ile 305 310
315 320 Pro His Leu Ile Leu Glu Leu Leu Lys
Cys Glu Pro Asp Glu Pro Gln 325
330 335 Val Gln Ala Lys Ile Met Ala Tyr Leu
Gln Gln Glu Gln Ser Asn Arg 340 345
350 Asn Arg Gln Glu Lys Leu Ser Ala Phe
Gly Leu Leu Cys Lys Met Ala 355 360
365 Asp Gln Thr Leu Phe Ser Ile Val Glu
Trp Ala Arg Ser Ser Ile Phe 370 375
380 Phe Arg Glu Leu Lys Val Asp Asp Gln
Met Lys Leu Leu Gln Asn Cys 385 390
395 400 Trp Ser Glu Leu Leu Ile Leu Asp His
Ile Tyr Arg Gln Val Ala His 405
410 415 Gly Lys Glu Gly Thr Ile Phe Leu Val
Thr Gly Glu His Val Asp Tyr 420 425
430 Ser Thr Ile Ile Ser His Thr Glu Val
Ala Phe Asn Asn Leu Leu Ser 435 440
445 Leu Ala Gln Glu Leu Val Val Arg Leu
Arg Ser Leu Gln Phe Asp Gln 450 455
460 Arg Glu Phe Val Cys Leu Lys Phe Leu
Val Leu Phe Ser Ser Asp Val 465 470
475 480 Lys Asn Leu Glu Asn Leu Gln Leu Val
Glu Gly Val Gln Glu Gln Val 485
490 495 Asn Ala Ala Leu Leu Asp Tyr Thr Val
Cys Asn Tyr Pro Gln Gln Thr 500 505
510 Glu Lys Phe Gly Gln Leu Leu Leu Arg
Leu Pro Glu Ile Arg Ala Ile 515 520
525 Ser Lys Gln Ala Glu Asp Tyr Leu Tyr
Tyr Lys His Val Val Asn Gly Asp 530 535
540 Val Pro Tyr Asn Asn Leu Leu Ile Glu
Met Leu His Ala Lys Arg Ala 545 550
555 560

Continuing on the front page (72) Inventor Bay Shan U.S.A. 94080 In San Francisco, California, To Corporate Drive, Tularik Inc. (72) Inventor Masahiro Nita 2 Doshomachi, Chuo-ku, Osaka, Osaka Chome 2-8 Sumitomo Pharmaceutical Co., Ltd.

Claims (9)

[Claims] 1. SEQ ID NO: 2, 4 or 6, or residues 1-10, 11-15, 16-21, 204-207
And at least one of residues 299-307, SEQ ID NO: 4 including residues 154, or residue 3-1.
An isolated polypeptide comprising a 10 residue domain of SEQ ID NO: 6 comprising at least one of 0, 13-22 and 30-38. 2. The isolated polypeptide according to claim 1, wherein said domain specifically binds to a CYP7 gene promoter. 3. In the presence of an isolated or recombinant first nucleic acid comprising the strand of SEQ ID NO: 1, 3 or 5, or a third and fourth nucleic acid comprising SEQ ID NO: 7 and 9, respectively. A sequence having at least 24 contiguous bases of the corresponding SEQ ID No. 1, 3 or 5 sufficient to specifically hybridize with a second nucleic acid comprising the complementary strand of the corresponding SEQ ID No. 1, 3 or 5 Part of the chain numbered 1, 3 or 5. 4. A recombinant nucleic acid encoding the polypeptide according to claim 1. 5. A cell containing the nucleic acid according to claim 4. 6. A method for producing a CPF polypeptide, which comprises introducing a nucleic acid according to claim 4 into a host cell or cell extract, wherein said host cell or extract comprises:
Incubating under conditions such that the nucleic acid is expressed as a transcript and the transcript is expressed as a translation product comprising the polypeptide, and separating the translation product. 7. A method for screening a substance that modulates the interaction of a CPF polypeptide with a binding target, the method comprising the steps of: SEQ ID NO: 2, 4 or 6, or
Residues 1-10, 11-15, 16-21, 204-20
7 and 299-307, at least a 10-residue domain of SEQ ID NO: 2 comprising residues 154, a 10-residue domain of SEQ ID NO: 4 comprising residues 154, or residues 3-1
An isolated polypeptide comprising a 10 residue domain of SEQ ID NO: 6 comprising at least one of 0, 13-22 and 30-38; a binding target of said polypeptide; and
A mixture comprising a candidate substance, in vitro or in culture, under conditions such that the polypeptide specifically binds to the binding target with a control affinity in the absence of the substance. By detecting the binding affinity of the polypeptide, the affinity biased by the substance (here,
The difference between the affinity biased by the substance and the affinity of the control is
Indicating that the substance modulates binding of the polypeptide to a binding target). 8. The method of claim 7, wherein the binding target is a nucleic acid comprising sufficient CYP7 promoter sequence to specifically bind to a CPF polypeptide. 9. A method for screening for a substance that specifically binds to a CPF polypeptide, the method comprising:
SEQ ID NO: 2, 4 or 6, or residues 1-10, 1
1-15, 16-21, 204-207 and 299-
At least one of SEQ ID NO: 2 comprising at least one of 307, 10 residues of SEQ ID NO: 4 comprising residue 154, or at least one of residues 3-10, 13-22 and 30-38 SEQ ID NO: 6
An isolated polypeptide comprising a 10 residue domain of
And incubating in vitro or in culture under conditions such that the substance specifically binds to the polypeptide, and detecting the specifically bound substance. .