JP2000224992A - New phosphodiesterase and gene coding for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new 'type-10' phosphodiesterase which has a specific amino acid sequence, has a cyclic nucleotide-hydrolyzing activity, and is useful for the study on the mechanism of intracellular propagation of information, the development of a new medicine by the screening of an inhibitor, and so on. SOLUTION: This is a new 'type-10' phosphodiesterase which has an amino acid sequence shown by formula I or II, or an amino acid sequence obtained by deleting, substituting, adding, or inserting one or more amino acid(s) from, in, to, or into the amino acid sequence, has a cyclic nucleotide-hydrolyzing activity, and is useful for the study on the complex mechanism of intracellular propagation of information, the development of a new medicine by screening an inhibitor having high specificity to the enzyme, and so on. This enzyme was obtained by screening EST database using a catalytic region of human phosphodiesterase 5A as a query sequence, followed by expressing a gene obtained by carrying out PCR using the obtained new sequences as primers.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel phosphodiesterase and its gene.

[0002]

BACKGROUND ART Cyclic nucleotides such as cAMP and cGMP are involved in the regulation of many functions in vivo as second messengers of intracellular signal transduction (Kukove).
tz et al., Naunyn Schmiedeberg's Arch. Pharmacol., 31.
0, 129-138, 1979; Schram et al., Science, 225.
Vol. 1350-1356, 1984; Ignarro et al., Annu. Rev. Ph.
armacol. Toxicol., 25, pp. 171-191, 1985; Ma
rtin et al., J. Pharmacol. Exp., 237, 539-547, 1
986).

[0003] The concentration of intracellular cAMP and cGMP, which fluctuates in response to extracellular signals, is regulated by the balance between adenyl cyclase and guanyl cyclase, which are involved in their synthesis, and phosphodiesterase (PDE), which is involved in cyclic nucleotide degradation. ing.

To date, many phosphodiesterases that degrade cyclic nucleotides have been found in mammalian tissues, and are classified into multiple types based on amino acid sequence homology, biochemical properties, and characterization by inhibitors. (Beavo, Physiol. Rev., 75, 725-748, 199
5 years).

[0005] For example, PDE1 is a Ca ²⁺ / calmodulin-dependent PDE, and hydrolyzes both cAMP and cGMP. PDE2 is activated by cGMP and c
Hydrolyzes both AMP and cGMP. PDEs classified as PDE3 are inhibited by cGMP. PDE4
Uses cAMP specifically as a substrate, and rolipram (Ro
lipram) sensitive. PDE5 specifically uses cGMP as a substrate. PDE6 is a photoreceptor cGMP
-PDE. PDE7 uses cAMP specifically as a substrate and is insensitive to rolipram.

[0006] More recently, the existence of two new types of PDEs has been reported (Soderling et al., Proc. Natl. Acad.
Sci. USA, Vol. 95, pp. 8991-8996, 1998; Fisher
Et al., Biochem. Biophys. Res.Commun., 246, 570-5.
77, 1998; Soderling et al., J. Biol. Chem., 273,
15553-15558, 1998; Fisher et al., J. Biol. Chem.,
273, 15559-15564, 1998; Hayashi et al., Bioc.
hem. Biophys. Res.Commun., 250, pp. 751-756, 19
1998). One is called PDE8 and uses cAMP specifically as a substrate. The other is called PDE9 and uses cGMP specifically as a substrate. These two PDEs are IBMX
It has been reported that it is insensitive to (3-isobutyl-1-methylxanthine).

[0007] PDE is also an important target molecule in drug development research, and inhibitor research is being vigorously pursued. Some known drugs have been found to have PDE inhibitory effects, and it has been found that specific PDE inhibitors can be useful therapeutic agents.

For example, the inotropic agent Milrinone (Milrin)
one) and Zaprinast are PDE3 and P respectively
DE5 inhibitors (Harrison et al., Mol. Pharmacol.,
29, 506-514, 1986; Gillespie et al., Mol. Pha.
rmacol., 36, 773-781, 1989). Rolipram, an antidepressant, is a PDE4 inhibitor (Schn
eider et al., Eur. J. Pharmacol., Vol. 127, pp. 105-115,
1986). PDE4 inhibitors have also been developed as anti-inflammatory agents or asthma therapeutics. Also, selective inhibitors of PDE5 have been developed as therapeutics for erectile dysfunction.

[0009] In addition, IBMX is known as a non-selective inhibitor acting on many types of PDEs. Vinpocetin is a PDE1 inhibitor, EHNA (erythro-9- (2-hydroxy-3-nonyl) adenine) is P
DE2 inhibitor, dipyridamole is PD
SCH51866, an inhibitor of E5 and PDE6, is a PDE1 and PDE
5 inhibitor, E4021, is known as a PDE5 inhibitor.

In order to develop an excellent drug having a high therapeutic effect and few side effects, it is desired to select an inhibitor which is highly selective for a certain type of PDE to be targeted.

Furthermore, finding a new type of PDE, which is a different molecular species from the conventional one, is useful for studying the complex mechanism of intracellular signal transduction and also as a target molecule for a new therapeutic agent. It was also desired from the possibility.

[0012]

It is an object of the present invention to provide a novel type of phosphodiesterase (type 10 phosphodiesterase (PDE10)) and its gene. Another object of the present invention is to provide a new method for characterizing, identifying and selecting a phosphodiesterase inhibitor. The purpose other than the above is clear from the following description.

[0013]

Means for Solving the Problems The present inventors have developed a full-length cDNA encoding a new type of phosphodiesterase (also referred to as PDE10), which is a different molecular species from the conventional one.
Was isolated from humans and rats. In addition, human phosphodiesterase (PDE10) was successfully expressed and isolated in COS cells by genetic recombination technology. Further, the present invention was characterized as an enzyme, and the present invention was completed.

That is, the present invention relates to phosphodiesterase type 10 (PDE10) and its gene. More specifically, it is a phosphodiesterase selected from the following (A) or (B).

(A) a protein having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 16, and (B) a protein having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 16. A protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown, and which has an activity of hydrolyzing a cyclic nucleotide.

Further, the present invention is a gene or nucleic acid selected from the following (a) or (b):

(A) DN having the nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 16
A gene or nucleic acid consisting of A, and (b) SEQ ID NO: 1,
A gene or nucleic acid consisting of a DNA that hybridizes under stringent conditions with a DNA having the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 16, and has an activity of hydrolyzing cyclic nucleotides A gene or nucleic acid encoding a protein.

The present invention further relates to a recombinant vector and a host cell containing the same. In addition, they can be used to characterize, identify, and identify phosphodiesterase inhibitors.
It is a way to make a selection.

The SEQ ID Nos. 1 and 2 in the sequence listing below are the nucleotide sequence of the cDNA containing the full length of the translation region of the novel human PDE gene (human PDE10 gene) isolated by the inventors (upper row) and the novel human encoded by it. PDE (human PD
E10) represents the amino acid sequence (lower).

SEQ ID NOs: 1 and 2 are sequences derived from two variants of the novel human PDE gene (human PDE10 gene).

For the base sequence or amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, the known DNA database (GenBank and EMBL) and the protein database (NBRF and SWISS-PR)
OT), a homology search resulted in EST
(Genbank / EMBL ID No: W04835 and AI300062)
Nothing considered to be derived from the same molecular species was found.

SEQ ID Nos. 15 and 16 in the Sequence Listing are a novel rat PDE gene (rat PD) isolated by the present inventors.
(E10 gene) The nucleotide sequence of the cDNA including the entire translation region (upper) and the novel rat PDE encoded thereby
(Rat PDE10) represents the amino acid sequence (lower).

SEQ ID NOS: 15 and 16 are novel rat PDEs
Sequences derived from two variants of the gene (rat PDE10 gene).

The nucleotide sequence or amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16 is a known DNA
Database (GenBank and EMBL) and protein database (NBRF and SWISS-
PROT), the result of homology search was ES
Except for T (Genbank / EMBL ID No: H32734), none of those considered to be derived from the same molecular species was found.

When the amino acid sequences of human and rat PDE10 are compared, homology as high as about 96% is observed.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION The proteins of the present invention include those having the amino acid sequence shown in SEQ ID NO: 1, 2, 15, or 16. In addition, those having an amino acid sequence represented by SEQ ID NO: 1, 2, 15, or 16 in which one or more amino acids are deleted, substituted, or added. Deletion, substitution or addition of amino acids may be performed so long as the activity of hydrolyzing cyclic nucleotides is not lost, and usually 1 to about 160 amino acids,
Preferably from 1 to about 80, more preferably from 1 to about 40. Such a protein usually has about 80% or more, preferably about 90% or more, more preferably about 95% or more amino acid level homology with the amino acid sequence shown in SEQ ID NO: 1, 2, 15 or 16. Such proteins include, in addition to mutant proteins found in nature, artificially modified mutant proteins, proteins derived from heterologous organisms, and the like.

[0027] The gene or nucleic acid of the present invention may be a D or D nucleic acid having the nucleotide sequence of SEQ ID NO:
And those containing NA. In addition, SEQ ID NOs: 1, 2,
Those containing a DNA that can hybridize under stringent conditions with a DNA having the nucleotide sequence represented by 15 or 16 are included. Such hybridizable DNA may be any that encodes a protein having an activity of hydrolyzing cyclic nucleotides. Such DNA has a homology of usually about 70% or more, preferably about 80% or more, and more preferably about 90% or more, with the nucleotide sequence shown in SEQ ID NO: 1, 2, 15 or 16. Examples of such a gene or nucleic acid include a mutant gene found in nature, an artificially modified mutant gene,
It includes homologous genes derived from heterologous organisms.

In the present invention, hybridization under stringent conditions is carried out under a normal stringent condition in a hybridization solution of 6 × SSC or a salt concentration equivalent thereto at a temperature of 50 to 70 ° C. Hybridization for about 16 hours,
Preliminary washing may be performed as necessary with a solution of × SSC or a salt concentration equivalent thereto, followed by washing in a solution of 1 × SSC or a salt concentration equivalent thereto.
Further, under conditions having higher stringency (high stringency conditions), the washing can be performed in the above-mentioned manner in a solution having a salt concentration of 0.1 × SSC or an equivalent salt concentration.

The gene or nucleic acid of the present invention can be isolated and obtained by performing screening using mammalian tissues or cells as a gene source. As mammals, dogs, cows,
Non-human animals such as horses, goats, sheep, monkeys, pigs, rabbits, rats and mice, as well as humans.

The gene or nucleic acid of the present invention can be obtained from the sequence information disclosed in the present specification (SEQ ID NO: 1, 2, 1
5 and 16). For example, primers and probes are designed based on the disclosed nucleotide sequence information, and PCR (polymerase c)
The combination can be selected and obtained from a DNA library by appropriately combining a hain reaction) method, a colony hybridization method, and a plaque hybridization method.

For example, cDNA is synthesized from mRNA prepared from mammalian cells or tissues, and using this as a template,
A cDNA fragment is obtained by the PCR method. Obtained cDNA
Was used as a probe, and cD was detected by colony hybridization or plaque hybridization.
Screen the NA library for full-length cDNA
Can be obtained. In addition, genomic genes can be isolated by screening a genomic DNA library. Further, by screening a DNA library of another mammal, a homologous gene derived from a heterologous organism can be isolated. Such mammals include, for example, dogs, cows, horses, goats, sheep,
Examples include non-human animals such as monkeys, pigs, rabbits and rats, as well as humans.

CDNA library and genomic D
DNA libraries such as NA libraries are, for example,
"Molecular Cloning" (Sambrook,
J., Fritsch, EF and Maniatis, T., Cold Spring
Harbor Laboratory Press, published in 1989). Alternatively, if there is a commercially available library, this may be used.

By determining the nucleotide sequence of the obtained cDNA, the translation region encoding the protein of the gene product can be determined, and the amino acid sequence of this protein can be obtained.

The PDE of the present invention can be produced by overexpression by a conventional gene recombination technique. It can also be expressed and produced in the form of a fusion protein with another protein or peptide.

For example, an expression vector is constructed by inserting a DNA encoding PDE into a vector in a form ligated downstream of an appropriate promoter. Next, the obtained expression vector is introduced into a host cell.

The expression system (host-vector system) includes, for example, bacterial, yeast, insect cell and mammalian cell expression systems. Of these, insect cells (Spodoptera frugiperd) are needed to obtain proteins with well-preserved functions.
a SF9, SF21, etc.) and mammalian cells (monkey COS-7)
Cells, Chinese hamster CHO cells, human HeL
a) is preferably used as a host.

As the vector, a retrovirus vector, a papilloma virus vector, a vaccinia virus vector, an SV40 vector or the like can be used for a mammalian cell system, and a baculovirus vector or the like can be used for an insect cell system.

As a promoter, an SV40 promoter, an LTR promoter, an elongation 1α promoter and the like can be used in a mammalian cell system, and a polyhedrin promoter and the like can be used in an insect cell system.

As a DNA encoding PDE, a cDNA corresponding to mRNA existing in nature (for example, a cDNA having the nucleotide sequence shown in SEQ ID NOs: 1, 2, 15, and 16) can be used. Not limited. DNA corresponding to the amino acid sequence of the target protein
Can be designed and used. In this case, codons encoding one amino acid are each known in 1 to 6 types, and the codon to be used may be selected arbitrarily. For example, in consideration of the frequency of codon usage of the host used for expression, the expression efficiency can be increased. Tall arrays can be designed. DNA having the designed base sequence can be obtained by chemical synthesis of DNA, fragmentation and binding of the cDNA, partial modification of the base sequence, and the like. Artificial partial sequence modification and mutagenesis are performed using site-specific mutagenesis (site specifi) using a primer consisting of a synthetic oligonucleotide encoding the desired modification.
c mutagenesis) (Proceedings of National Academy o
f Sciences, Vol. 81, pp. 5662-5666, 1984) and the like.

The PDE of the present invention can be prepared from a culture of cells into which an expression vector has been introduced, by a known purification method (salting out with inorganic salts, fractional precipitation with an organic solvent, ion exchange resin column chromatography, affinity column chromatography). , Gel filtration, etc.) can be separated and purified.

A nucleic acid (oligonucleotide or polynucleotide) that hybridizes with the gene or nucleic acid of the present invention under stringent conditions can be used as a probe for detecting the gene of the present invention. In addition, in order to modulate gene expression, it can be used as, for example, an antisense oligonucleotide, a ribozyme, or a decoy. As such a nucleic acid, for example, a nucleotide having a continuous partial sequence of 14 or more bases or a sequence complementary thereto, usually in the base sequence represented by SEQ ID NO: 1, 2, 15, or 16 can be used. .

An antibody recognizing the PDE of the present invention is obtained by using the PDE of the present invention or a protein or peptide having immunological equivalence to the PDE (a synthetic peptide having a protein fragment or a partial sequence) as an antigen. be able to. Having immunological equivalence means, for example, that P
Means cross-reactive with antibodies to DE.

A polyclonal antibody can be produced by a usual method of inoculating a host animal (for example, rat or rabbit) with an antigen and collecting an immune serum. Monoclonal antibodies can be produced by conventional techniques such as the hybridoma method. In addition, a humanized monoclonal antibody or the like can be prepared by modifying the gene of the monoclonal antibody.

Using the antibody obtained above, the expression of the PDE of the present invention in cells or tissues can be detected by a usual immunochemical method (enzyme immunoassay, etc.). Alternatively, the PDE of the present invention can be purified by affinity chromatography using an antibody.

The PDE of the present invention comprises a cyclic nucleotide (c
Having the activity of hydrolyzing AMP or cGMP can be determined by a generally known method for measuring PDE activity (Th
ompson et al., Adv. Cyclic Nucleotide Res., Volume 10, 69
-92, 1979; Yanaka et al., Eur. J. Biochem., 255.
Vol., Pp. 391-399, 1998).

As substrates for the enzymatic reaction, cAMP and c
Cyclic nucleotides such as GMP and derivatives thereof can be used. The PDE of the present invention comprises cAMP and cGM.
Each of P is used as a substrate to hydrolyze them.

The PDEs of the present invention can be used for the characterization, identification or selection of phosphodiesterase inhibitors.

For example, an enzyme reaction is carried out in a system containing the PDE of the present invention, an enzyme substrate and a test substance (preferably a low-molecular compound or the like), and inhibition of the test substance on enzyme activity (activity of hydrolyzing cyclic nucleotides). Test the effect.

Alternatively, a binding reaction is performed in a system containing the PDE of the present invention and a test substance (preferably a low-molecular compound or the like), and it is determined whether the test substance has a binding ability to the PDE of the present invention. . A test substance (ligand) having binding ability is likely to be an inhibitor.

Further, the inhibitory effect (or binding ability) of the test substance (preferably a low molecular weight compound or the like) on the PDE of the present invention is examined and compared with the inhibitory action (or binding ability) on other types of PDE. The selectivity of the inhibitory action (or binding ability) can be determined. Thereby, an inhibitor having a relatively high effect on a specific type of PDE (selective inhibitor) can be selected. In addition, inhibitors can be identified and characterized.

Hereinafter, the present invention will be described in more detail by way of examples, but these examples do not limit the present invention.

In the following examples, unless otherwise specified, each operation is referred to as “molecular cloning (Molecu
lar Cloning) "(Sambrook, J., Fritsch, EF and Ma
niatis, T., Cold Spring Harbor Laboratory Press
Published in 1989), or when a commercially available reagent or kit is used, it was used according to the instructions of a commercially available product.

[0053]

EXAMPLES Example 1 Isolation of cDNA for Novel Human PDE (PDE10) (1) Human PDE5A (Genbank / EMBL Database Accessio
n No. D89094; Yanaka et al., Eur. J. Biochem., 255,
391-399, 1998) C-terminal catalytic region (500
The amino acid sequence of the region corresponding to the -875th amino acid residue) was used as a query sequence and BLAST (Basic
Local Alignment Search T
EST (Expressed se)
query tags) database. As a result, several ESTs were found to have homology to the C-terminal catalytic region of human PDE5A, and among these, one EST having a novel sequence different from the sequence of a known PDE (ID No. : W04835).

D corresponding to this EST (ID No. W04835)
The NA fragment (about 400 bp) was subjected to PCR (polymerase chai
n reaction) method.

The PCR reaction was carried out at 94 ° C. for 1 minute, 55 ° C. for 2 minutes, and 72 ° C. for 2 minutes, for a total of 60 cycles.

In the first 30 cycles, oligonucleotides having the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the below-mentioned Sequence Listing were used as primers as sense primers and antisense primers, respectively. As a template, a human fetal lung-derived cDNA library (vector: λgt11 phage) (manufactured by Clontech) was used.

The following 30 cycles were performed using 1/10 of the PCR product as a template. As the primers, oligonucleotides having the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the Sequence Listing were used as sense primers and antisense primers, respectively.

After treating the obtained PCR product with restriction enzymes, the vector plasmid pGEM-Teasy (Pro
(Mega) and its base sequence was determined. The nucleotide sequence was determined using an automatic DNA sequencer (LI-COR40).
00L; manufactured by LI-COR and ABI PRISM3
10; manufactured by PE Applied Biosystems)
It was determined by the dideoxy method (hereinafter the same). As a result, the obtained DNA fragment was used as the EST (ID No. W0483).
It was confirmed that they had almost the same sequence as in 5).

This DNA fragment (labeled with ³² P)
Was used as a probe to perform plaque hybridization. As the cDNA library, a human fetal lung-derived cDNA library as described above was used.

The plaque hybridization was performed under high stringency conditions as follows. That is, a nylon membrane (Hybond-N +) in which a plaque was alkali-treated and DNA was denatured and fixed was placed in a hybridization solution (6 × SSC, 0.5% S
DS, 5x Denhardt's solution, 100 µg / ml salmon sperm DNA) at 65 ° C for 16 hours for hybridization. (Note that the composition of 1 × SSC is 0.
15 M sodium chloride and 15 mM sodium citrate, pH 7.0, the composition of the 1x Denhardt solution is:
0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone and Ficoll 400. Less than,
Preliminary washing solution (2xSSC, 0.5% SD)
S), the membrane was washed at room temperature for 10 minutes, and then further washed with a washing solution (0.1 × SSC, 0.5% SD).
Washed twice at 65 ° C. for 30 minutes using S). Thereafter, autoradiography was performed at -70 ° C for 1 day, and positive clones were detected.

The obtained positive clone (clone human PD)
(Referred to as E10A1),
Vector plasmid pBluescriptII SK
(+) (Stratagene) was subcloned. The nucleotide sequence of the cDNA fragment was determined using the obtained plasmid.

The isolated cDNA (4576 bp) was considered to be a full-length cDNA of a novel human PDE (hereinafter referred to as PDE10) gene. The nucleotide sequence of this cDNA was analyzed to identify an open reading frame, and the amino acid sequence of the protein encoded thereby was determined.

The full length cD is shown in SEQ ID NO.
The nucleotide sequence of NA (the upper part of SEQ ID NO: 1) and the amino acid sequence of the protein encoded by it (new human PDE (PDE10)) (the lower part of SEQ ID NO: 1) are shown. The molecular weight of PDE10 deduced from the amino acid sequence (779 amino acid residues) was about 88 Kd.

When the homology of the amino acid sequence with the known PDE was examined, the PDE1
0 of the cGMP binding region and the catalytic region
It was estimated to be a region corresponding to the 445th and 493-728 amino acid residues.

When the amino acid sequence of PDE10 is compared with various known cGMP-binding human PDEs, PDE2A and PDE5A have 42% and 47%,
It showed 40% homology with DE6A and 42% with PDE6B.

In the cGMP binding region, PD
It showed 18-38% homology with E2A, PDE5A and photoreceptor phosphodiesterase, respectively.

PDE10 has one cGMP binding region containing a typical cGMP binding motif. But P
The phosphorylated region of cAMP or cGMP-dependent kinase found in DE5A was not found in this sequence.

Further, a PCR primer surrounding the translation region is designed based on the obtained cDNA sequence information of PDE10, and RT-PCR (reverse transcriber) using the primer is designed.
The presence of the same PDE10 clone in human lung-derived mRNA was confirmed as follows by pt-polymerase chain reaction).

That is, human fetal lung poly (A) + RNA
(Clontech Laboratories)
And 42 ° C using a random primer (hexamer)
After performing a reverse transcription reaction for 60 minutes, PCR was performed using the obtained cDNA as a template. As PCR primers, oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 7 to 10 in the sequence listing described below were synthesized and used. The PCR reaction was performed under the same conditions as described above.

The DNA fragment amplified by PCR (about 2.4
The nucleotide sequence of kb) was determined and compared with the cDNA sequence. As a result, the nucleotide sequence of the portion amplified by PCR was identical.

Example 2 New human PDE (PDE10)
(2) PCR primers surrounding the translation region were designed based on the cDNA base sequence information of PDE10 obtained in Example 1, and a new cDNA was obtained by RT-PCR using the primers.
An attempt was made to obtain a clone.

First, human fetal lung poly (A) + RNA (C
lowtech Laboratories) and random primers (hexamer) at 42 ° C, 60 ° C.
After performing a reverse transcription reaction for minutes, PCR was performed using the obtained cDNA as a template. As PCR primers, oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 11 to 14 of the Sequence Listing described below were synthesized and used. The reaction of PCR is
One cycle was performed at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 2 minutes, for a total of 60 cycles, and a final cycle was performed at 72 ° C. for 5 minutes.

In the first 30 cycles, oligonucleotides having the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 in the below-mentioned sequence listing were used as primers, respectively, as sense primers and antisense primers. In the subsequent 30 cycles, oligonucleotides having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 in the Sequence Listing below were used as primers as sense primers and antisense primers, respectively.

The DNA fragment amplified by PCR (about 2.4
kb) with a new cDNA clone (clone human PDE1)
0A2) and its base sequence was determined.

When this was compared with the cDNA sequence obtained in Example 1, a partial difference in the base sequence was found in the 5 ′ terminal region, but the other sequences were identical.

The newly obtained clone (human PDE10)
The nucleotide sequence of the cDNA of A2) and the amino acid sequence of the protein encoded thereby are shown in SEQ ID NO: 2 in the sequence listing below.

A newly obtained clone (human PDE10)
A2) is the PDE10 of Example 1 (human PDE10A1).
(Referred to as a splicing variant). Human PDE10A1 and human PDE10A2
Is the N-terminal side of the amino acid sequence (first to first
The 3rd amino acid residue, the 1st to 23rd amino acid residues of SEQ ID NO: 2) and their corresponding cDNA sequences are different. Also, this difference includes cAMP or cG
It was confirmed that there was an amino acid sequence motif that was supposed to be phosphorylated by MP-dependent kinase.

Example 3 Expression and Purification of Human PDE10 in COS Cells (1) Construction of Vector Plasmid for Expression of PDE10 The cDNA of human PDE10 obtained in Example 1 was digested with restriction enzymes SspI and NotI, and The obtained fragment (about 2.8 kb) was converted into a vector plasmid pBluescript.
pt II SK (+) (Stratagene)
(Blended) -NotI site.
The obtained plasmid was digested with restriction enzymes NotI (blunt-ended) and BamHI (about 2.8 kF).
b) was replaced with the vector plasmid pSVL (Amersha)
Xb of m Pharmacia Biotech)
aI (blunt-ended) -inserted at BamHI site to create PD
E10 expression vector plasmid pSVL-PDE10
I got In pSVL-PDE10, human PDE10 cDNA is operably linked downstream of the SV40 promoter.

(2) Expression in COS cells COS-7 cells (ATCC; CRL1651)
% Fetal bovine serum, 100 units / ml penicillin, and 100 μM / streptomycin (Dulbecco's modified Eagle's mediu)
m) Subculture in (Gibco) at 37 ° C. under 5% carbon dioxide.

[0086] The pSVL-PDE was added to COS-7 cells.
10 (or vector pSVL as a control) (transient transfection; transient
transfection). Transfection is performed using a polycationic liposome reagent (trade name: LipofectAMINE, G
ibco).

(3) Purification of Recombinant Human PDE10 48 hours after transfection, the cells were washed with ice-cold phosphate buffer, and then ice-cold homogenization buffer (20 mM Tris-HCl, pH 7.4). 2 mM
Magnesium acetate, 0.3 mM calcium chloride, 1 mM
Dithiothreitol, 40 μM leupeptin, 1.3 m
M benzamidine, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM sodium azide) and sonicated. The obtained homogenate is centrifuged (10
0000 g for 60 minutes), and the supernatant was collected.

Elution buffer (20 mM Tris-HC)
MonoQ Sepharose high-speed column (Amersham Pharmacia Biote) equilibrated with 1, pH 7.4, 1 mM calcium chloride, 1 mM dithiothreitol, 2 μM leupeptin, 5 mM benzamidine).
ch.) was supplied with the supernatant obtained above. After the column was washed with 20 ml of elution buffer, the protein was eluted with a sodium chloride gradient (0, 350 and 800 mM, 20 ml each), and fractionated in 2 ml portions under ice-cooling. For each fraction, c
Hydrolysis activity using AMP and cGMP as substrates (PDE
Activity) was measured.

The PDE activity was measured by the radiolabeled nucleic acid method. That is, 1 μM unlabeled cGMP (or cAMP) and 22 nM [ ³ H] -cGMP (or [ ³ H] -cAMP) (Amersham Pharm)
assay buffer (50 mM Tris-HCl, pH 8.0, 5 mM)
The reaction was started by adding 10 to 30 μl of the enzyme solution to 500 μl of magnesium chloride, 4 mM 2-mercaptoethanol, 0.33 mg / ml bovine serum albumin (containing no fatty acid, manufactured by Sigma). After incubating at 37 ° C. for 30 minutes to carry out the reaction, the reaction was stopped by boiling for 1.5 minutes, and 1 mg / ml of snake venom (Crotalus atrox) was further added.
snake venom) was added, and the mixture was incubated at 37 ° C for 30 minutes. Then, 500 μl of methanol was added,
After treating the reaction solution with a Dowex column (1 × 8-400), a liquid scintillation cocktail was added to each eluate, and the radioactivity was measured.

FIG. 1 shows the PDE activity of each fraction of the cell extract fractionated by MonoQ Sepharose column chromatography.

The vector for expression of PDE10 (pSLV-P
Extract N of cells transfected with DE10)
In o.14, a strong hydrolysis activity peak for cAMP and cGMP was observed. This peak is the peak of the vector (p
SLV) was not observed in the extract of cells transfected alone, and was considered to be derived from recombinant human PDE. Also, PDE10 is cAMP and cGM.
It was found that all of P had an activity of hydrolyzing. This fraction No. 14 was used as partially purified recombinant human PDE.

An activity peak was also observed in the vicinity of fraction No. 24, but a similar peak was observed in the extract of cells transfected with the vector alone. It was thought to be from.

Example 4 Analysis of Enzymatic Properties of Human PDE10 Using the purified recombinant human PDE obtained in the same manner as in Example 3, various enzymatic properties were analyzed as follows.

(1) Kinetic Analysis of Enzyme Reaction An enzyme reaction was carried out using various substrate (cGMP or cAMP) concentrations, and PDE activity (initial rate of the reaction) was measured. The enzyme reaction and PDE activity measurement were performed in the same manner as in Example 3 (3). However, in the reaction solution, the concentration when adding unlabeled cAMP was 0.1 to 2 μM, and the concentration when adding unlabeled cGMP was 0.2 to 16 μM. In addition, the amount of the enzyme was set so that the decomposition rate of the substrate after the reaction was about 10%, and the reaction time was 30 minutes.

The results (Lineweaver-Burk plot) are shown in FIG. The results of the analysis, K _m values when the cAMP human PDE10 the substrate is 7.2 ± 0.62μM,
Vmax was 1.4 ± 0.077 pmol / min / μg. Also, K _m values when the cGMP as a substrate, 0.
26 ± 0.048 μM, and Vmax is 0.63 ±
It was 0.12 pmol / min / μg.

From these facts, PDE10 is cAMP
And cGMP, but had a stronger affinity for cAMP.

(2) cGMP for cAMP degradation activity
Influence of cAMP An enzymatic reaction was performed using cAMP as a substrate, and various concentrations of cGMP were added to the reaction solution to examine the effect on PDE activity.

The enzymatic reaction and PDE activity measurement were carried out in the same manner as in Example 3 (3). However, in the reaction solution, unlabeled cAMP was 0.3 μM ([ ³ H] -cAMP was 22 nM).
M) was added and cGMP (unlabeled) was added to 0.01 to 3
0 μM was added (or not added). In addition, the amount of the enzyme was set so that the decomposition rate of the substrate after the reaction was about 10%, and the reaction time was 30 minutes.

The relative value (%) of the activity was calculated with the cAMP degrading activity when no cGMP was added as 100%. The results are shown in FIG. As is clear from FIG. 3, a decrease in cAMP degradation activity was observed depending on the concentration of cGMP added. Unlike PDE2, PDE10 did not show an increase in PDE activity depending on the cGMP concentration.

(3) Inhibition of Activity by Various Known PDE Inhibitors Various known PDE inhibitors against the cAMP and cGMP hydrolysis activity (PDE activity) of human PDE10 (IBM
X, vinpocetine, EHNA, zaprinast, dipyridamole, rolipram, milrinone, SCH51866 and E-4021) were examined as follows.

An enzymatic reaction was carried out using cAMP or cGMP as a substrate, and various known PDE inhibitors were added to the reaction solution, and the hydrolysis activity was measured. The enzymatic reaction and PDE activity measurement were performed in the same manner as in Example 3 (3). However,
In the reaction solution, the concentration when adding unlabeled cAMP is 0.1.
3 μM, and the concentration when adding unlabeled cGMP is 7
μM, and add various PDE inhibitors to the reaction
M was added.

The amount of the enzyme was set such that the decomposition rate of the substrate after the reaction was about 10%, and the reaction time was 30 minutes.

Various PDEs for the activity of human PDE10
The results obtained by expressing the inhibitory action of the inhibitor by IC50 are shown in Table 1.

[0098]

[Table 1]

Example 5 PDEs in Various Human Tissues
Expression of 10 PDE10 in various human tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, femur, parietal bone)
The presence or absence of gene expression was examined as follows.

Various mRNAs derived from human tissues (poly (A)
+ RNA) (trade name) Human RNA Multiple Tissue Northern (MTN) Blot (Clontech L
dot blot analysis was carried out using the same method as described above. As the probe, a fragment (E) corresponding to the catalytic region in the human PDE10 cDNA obtained in Example 1 was used.
coRI digestion fragment; 1509-2308 of SEQ ID NO: 1
(Corresponding to the third base sequence) was used after being labeled with ³² P.

The amount of poly (A) + RNA was adjusted using human ubiquitin cDNA and main histocompatibility complex class Ic as a probe and adjusting the amount of the signal to be constant. Hybridization was performed under the following conditions. That is, a nylon membrane (Hy
bond-N +) was added to a hybridization solution (50% formamide, 4 × SS) containing a probe labeled with ³² P.
C, 0.5% SDS, 5x Denhardt's solution, 100 μg
/ Ml of salmon sperm DNA) at 42 ° C for 16 hours for hybridization. Then, washing was performed at 60 ° C. for 1 hour using a washing solution (0.2 × SSC, 0.1% SDS). Thereafter, autoradiography was performed at -70 ° C for 5 days.

As a result of the dot blot, as shown in FIG. 4, strong expression of PDE10 mRNA was detected in the fruit nucleus, caudate nucleus and testis. Also, weak expression was observed in the thyroid gland, pituitary gland, thalamus, and cerebellum (the order of expression intensity was as described).

[0103] Northern blot analysis of various human tissue-derived mRNAs was performed using the same probes as described above under the same conditions.

As a result of Northern blotting, about 10 k
Band b was detected in many tissues. However, in particular, in the testis, a band of about 4.0 kb was observed in addition to a band of about 10 kb, and it was considered that PDE mRNA of variant form was expressed in these tissues. Also, since the size of the mRNA band is longer than the isolated cDNA,
The DE10 mRNA was thought to have a long untranslated portion.

Example 6 Rat Novel PDE (PDE1
0) Isolation of cDNA of human PDE10cDN shown in SEQ ID NO: 1
Using BLAST (Basic Local Align) as a query sequence, the cDNA base sequence of the translation region of A
EST by the Ment Search Tool method.
(Expressed sequence tags)
Searched the database. As a result, one EST (IDNo: H32734) derived from rat PC12 cells, which has extremely high homology to the query sequence, was found.

A DNA fragment corresponding to the EST (ID No. H32734) was obtained by a PCR (polymerase chain reaction) method.

The PCR reaction was carried out at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute, for a total of 30 cycles.

As primers, oligonucleotides having the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18 in the Sequence Listing below were used. In addition, a rat brain-derived cDNA (manufactured by Clontech) was used as a template.

The obtained PCR product was used as a TA-cloning vector plasmid pGEM-Teasy (Prome
ga (manufactured by Ga, Inc.), and its base sequence was determined. The nucleotide sequence was determined using an automatic DNA sequencer (ABI PRISM3).
10; manufactured by PE Applied Biosystems)
Determined by the dideoxy method.

As a result, it was confirmed that the obtained DNA fragment had almost the same sequence as that of the EST (ID No. H32734).

This DNA fragment (labeled with ³² P)
Was used as a probe to perform plaque hybridization. The cDNA library is a rat brain-derived cDNA library (St) constructed by the λZAP system.
ratagene) was used.

The plaque hybridization was performed under the following conditions. That is, the plaques of 18 plates were transferred to a nylon membrane (Hybond-N +) so that about 5 × 10 ⁴ plaques were formed per plate. This nylon membrane is mixed with a hybridization solution containing a probe (6 × SSC, 0.5% SD).
S, 5x Denhardt's solution, 100 μg / ml salmon sperm DNA) at 65 ° C. for 16 hours for hybridization. (Note that the composition of 1 × SSC is 0.15
M sodium chloride and 15 mM sodium citrate,
The pH is 7.0 and the composition of the 1x Denhardt solution is 0.1.
02% bovine serum albumin, 0.02% polyvinylpyrrolidone and Ficoll 400. Then, the membrane is washed with a pre-washing solution (2 × SSC, 0.5% SDS) at room temperature for 10 minutes, and further with a washing solution (0.2 × SSC, 0.1% SDS) at 65 ° C. for 30 minutes. Washed twice. Then -70 ° C
For 2 days, autoradiography was performed to detect positive clones.

Of the 53 positive clones obtained, 1
The inserted cDNA fragments were isolated and recovered as plasmids for five of them. Fragment corresponding to the N-terminal part of human PDE10 (corresponding to the 68th to 448th base sequence of SEQ ID NO: 1)
And a fragment corresponding to the C-terminal portion (1990th of SEQ ID NO: 1)
~ 2304th base sequence)
Dot blot analysis was performed using the cDNA fragment of DE10 as a probe.

The hybridization was performed under the following conditions. That is, a nylon membrane (Hybo
nd-N +) and hybridization solution containing the labeled probe with ^{32 P (6xSSC, 0.5% SDS} , 5x
Denhardt's solution, salmon sperm DNA at 100 μg / ml)
The hybridization was carried out at 55 ° C. for 16 hours in a medium. Then, the washing solution (1 × SSC, 0.5% SD)
Washing was performed at 55 ° C. for 10 minutes using S). Then -7
Autoradiography was performed at 0 ° C. for 4 hours.

Five clones hybridizing with both probes (possibly holding full-length cDNA)
Was selected. Two of these clones (clone No. 8)
And 17) the entire or partial nucleotide sequence of the inserted cDNA fragment (referred to as cloned rat PDE10A2 and A3).

The isolated cDNAs (3427 and 3080)
bp) was considered to be a cDNA encoding a novel rat PDE (hereinafter referred to as PDE10). The nucleotide sequence of this cDNA was analyzed to identify an open reading frame, and the amino acid sequence of the protein encoded thereby was determined.

The nucleotide sequences of these cDNAs (upper lines of SEQ ID NOs: 15 and 16) and the proteins encoded by them (a novel human PDE) are shown in SEQ ID NOS: 15 and 16 in the Sequence Listing below.
(PDE10)) (bottom row of SEQ ID NO: 15 and bottom row of 16). Amino acid sequence (794 and 78)
The molecular weight of PDE10 estimated from 8 amino acid residues) was about 90 Kd.

Rat PDE10A2 and rat PDE10
A3 is on the N-terminal side of the amino acid sequence (1st to 23rd amino acid residues of SEQ ID NO: 15;
7th amino acid residue) and cDNAs corresponding to them
Sequence is different.

Two rat-derived PDE10s (PDE1
0A2 and PDE10A3) were converted to human-derived PDE10 (PDE10A1 and PDE10A2).
As a result, a high homology of about 96% was recognized, but differences were found in the N-terminal portion and the C-terminal portion.

Example 7 PD in Various Rat Tissues
Expression of E10 PDE10 in various rat tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, testis, femur, parietal bone)
The presence or absence of gene expression was examined as follows.

Various rat tissue-derived mRNAs (poly (A) + RNA) (trade name: rat RNA multiple
Tissue Northern (MTN) blot (Clonte)
ch Laboratories) and Northern blotting was carried out in the same manner as in Example 5. However, as the probe, rat PDE1 obtained in Example 6 was used.
0 cDNA fragment (1172-1 to 1172 of SEQ ID NO: 15)
(Corresponding to the 519th nucleotide sequence) was used after being labeled with ³² P.

As a result of Northern blotting, expression of PDE10 mRNA was observed in the brain and testis, and a band of about 9.5 kb was observed in the brain and a band of about 3.5 kb was observed in the testis.

Further, in situ hybridization was performed on rat brain. RNA probes for in situ hybridization were prepared by in vitro transcription using a digoxigenin-UTP and DIG RNA labeling kit (Boehringer Mannheim). At this time, the DNA used as a template was c used as a probe for plaque hybridization in Example 6.
The DNA fragment was converted to the vector plasmid pGEM-Teas.
After subcloning into y (Promega),
1 μg each of those linearized with the restriction enzyme SphI (for antisense probe) and the restriction enzyme SacI (for sense probe) was used. Antisense probe is T7
RNA polymerase and sense probe SP6
Synthesized using RNA polymerase.

Using a brain obtained from a 10-week-old male rat, a 10 μm-thick frozen section was prepared, fixed with 4% formaldehyde, and then 10 μg / ml proteinase K and 0.1 M triethanolamine (0.25 % Acetic anhydride). Then, it was dehydrated with alcohol. 800
μl of hybridization solution (20 mM Tri)
s-HCl, pH 8.0, 300 mM sodium chloride,
10% Sodiumdextran sulfate,
After prehybridization with 50% formamide, 0.2% N-lauroyl sarcosine, 100 μg / ml salmon sperm DNA, 1 × Denhardt's solution) for 30 minutes at 50 ° C., 800 μl of hybridization was carried out using an RNA probe labeled with digoxigenin. Hybridization was performed in a solution in a humid box at 50 ° C. for 16 hours.

After the hybridization is completed, the solution is treated with a highly stringent solution (50% formamide, 300 mM sodium chloride, 30 mM sodium citrate) for 60 minutes.
And washed with 1 μg / ml RNaseA for 3 minutes.
The mixture was treated at 7 ° C. for 10 minutes, and washed again with the above-mentioned high stringency solution.

Further, the sections were subjected to anti-digoxigenin poroclonal antibody (1.5% blocking reagent (Boehringer
Buffer A (100 mM T) containing Mannheim
ris-HCl, pH 7.5, diluted 1/500 with 150 mM sodium chloride) for 5 minutes at room temperature, and then washed thoroughly with buffer A.

Further sections were taken from freshly prepared nitroblue tetrazolium and 5-brumo-4-chloro-3-
Color-Substrate solution containing indolyl phosphate (100 mM Tris-HCl, pH 9.5, 10
(0 mM sodium chloride, 50 mM magnesium chloride) and stained for 2 days at room temperature in a dark box.

As a result of the in situ hybridization,
Strong expression was observed in neurons of the striatum and olfactory tubercle.

[0129]

The novel PDE of the present invention and its gene are
It is useful for studying multiple mechanisms of intracellular signal transduction. In addition, it can be a target molecule of a therapeutic drug for a new disease.

Further, the novel PDE of the present invention and a method for characterizing, identifying and selecting an inhibitor utilizing the gene thereof are described in the following.
It is useful for developing highly selective inhibitors and excellent drugs with high therapeutic effects and few side effects.

[0131]

[Sequence list] SEQ ID NO: 1 Sequence length: 4576 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Human Sequence characteristics Characteristic symbols : CDS existing position: 251..2590 sequence: GAATTCCGGG CGGCGGCGGC CAAACTCCGC GGCGTCCCCA GGGCGCCACG TTCGCCCTCG 60 CCGCCGCGGC CGCGCTGCTC TTCGGCTCCG ACATGGAAGA TGGACCTTCT AATAATGCGA 120 GCTGCTTCCG AAGGCTGACC GAGTGCTTCC TGAGCCCCAA AGCTAATGGT ATAGATATAG 180 AAGTCATCCA CAGAGATGTT ACAGTTGAAG AGATGGGGGT AGAGAAGACT TTGAAGGAAA 240 AGAATGTAGA 250 ATG AGG ATA GAA GAG AGG AAA TCC CAA CAT TTA ACA GGT TTG ACA GAT 298 Met Arg Ile Glu Glu Arg Lys Ser Gln His Leu Thr Gly Leu Thr Asp 1 5 10 15 GAA AAA GTG AAG GCA TAT CTT TCT CTT CAC CCC CAG GTA TTA GAT GAA 346 Glu Lys Val Lys Ala Tyr Leu Ser Leu His Pro Gln Val Leu Asp Glu 20 25 30 TTT GTA TCT GAA AGT GTT AGT GCA GAG ACA GTA GAG AAA TGG CTG AAG 394 Phe Val Ser Glu Ser Val Ser Ala Glu Thr Val Glu Lys Trp Leu Lys 35 40 45 AGG AAG AAC AAC AAA TCA GAA GAT GAA TCA GCT CCT AAG GAA GTC AGC 442 Arg Lys Asn Asn Lys Ser Glu Asp Glu Ser Ala Pro Lys Glu Val Ser 50 55 60 AGG TAC CAA GAT ACG AAT ATG CAG GGA GTT GTA TAT GAA CTA AAC AGC 490 Arg Tyr Gln Asp Thr Asn Met Gln Gly Val Val Tyr Glu Leu Asn Ser 65 70 75 80 TAT ATA GAA CAA CGG TTG GAC ACA GGA GGA GAC AAC CAG CTA CTC CTC 538 Tyr Ile Glu Gln Arg Leu Asp Thr Gly Gly Asp Asn Gln Leu Leu Leu 85 90 95 TAT GAA CTG AGC AGC ATC ATT AAA ATA GCC ACA AAA GCC GAT GGA TTT 586 Tyr Glu Leu Ser Ser Ile Ile Lys Ile Ala Thr Lys Ala Asp Gly Phe 100 105 110 GCA CTG TAT TTC CTT GGA GAG TGC AAT AAT AGC CTG TGT ATA TTC ACG 634 Ala Leu Tyr Phe Leu Gly Glu Cys Asn Asn Ser Leu Cys Ile Phe Thr 115 120 125 CCA CCT GGG ATA AAG GAA GGA AAA CCC CGC CTC ATC CCT GCT GGG CCC 682 Pro Pro Gly Ile Lys Glu Gly Lys Pro Arg Leu Ile Pro Ala Gly Pro 130 135 140 ATC ACT CAG GGC ACC ACC GTC TCT GCT TAT GTG GCC AAG TCC AGG AAA 730 Ile Thr Gln Gly Thr Thr Val Ser Ala Tyr Val Ala Lys Ser Arg Lys 145 150 155 160 A CA CTG CTA GTA GAA GAC ATC CTT GGA GAT GAA CGA TTT CCA AGA GGT 778 Thr Leu Leu Val Glu Asp Ile Leu Gly Asp Glu Arg Phe Pro Arg Gly 165 170 175 ACT GGA CTG GAA TCA GGG ACT CGT ATC CAG TCT GTT CTT TGC TTA CCA 826 Thr Gly Leu Glu Ser Gly Thr Arg Ile Gln Ser Val Leu Cys Leu Pro 180 185 190 ATT GTC ACT GCA ATT GGT GAC TTG ATT GGT ATT CTC GAG CTG TAT CGG 874 Ile Val Thr Ala Ile Gly Asp Leu Ile Gly Ile Leu Glu Leu Tyr Arg 195 200 205 CAC TGG GGC AAA GAA GCC TTC TGT CTT AGT CAC CAG GAG GTT GCA ACA 922 His Trp Gly Lys Glu Ala Phe Cys Leu Ser His Gln Glu Val Ala Thr 210 215 220 220 GCA AAT CTT GCC TGG GCT TCA GTA GCA ATA CAT CAG GTG CAG GTA TGC 970 Ala Asn Leu Ala Trp Ala Ser Val Ala Ile His Gln Val Gln Val Cys 225 230 235 240 AGA GGC CTT GCC AAA CAG ACA GAA TTG AAT GAC TTC CTA CTC GAC GTA 1018 Arg Gly Leu Ala Lys Gln Thr Glu Leu Asn Asp Phe Leu Leu Asp Val 245 250 255 TCA AAA ACA TAT TTT GAT AAC ATA GTT GCA ATA GAT TCT CTA CTT GAA 1066 Ser Lys Thr Tyr Phe Asp Asn Ile Val Ala Ile Asp Ser Leu Leu Glu 260 265 270 CAC ATA ATG ATA TAT GCA AAA AAC CTG GTG AAT GCC GAT CGT TGT GCG 1114 His Ile Met Ile Tyr Ala Lys Asn Leu Val Asn Ala Asp Arg Cys Ala 275 280 285 CTT TTC CAG GTG GAC CAT AAG AAC AAG GAG TTA TAT TCA GAC CTT TTT 1162 Leu Phe Gln Val Asp His Lys Asn Lys Glu Leu Tyr Ser Asp Leu Phe 290 295 300 GAT ATT GGA GAG GAA AAG GAA GGA AAA CCT GTC TTC AAG AAG ACC AAA 1210 Asp Ile Gly Glu Glu Lys Glu Gly Lys Pro Val Phe Lys Lys Thr Lys 305 310 315 320 GAG ATA AGA TTT TCA ATT GAG AAA GGA ATT GCT GGC CAA GTA GCA AGA 1258 Glu Ile Arg Phe Ser Ile Glu Lys Gly Ile Ala Gly Gln Val Ala Arg 325 330 335 ACA GGG GAA GTC CTG AAC ATT CCA GAT GCC TAT GCA GAC CCA CGC TTT 1306 Thr Gly Glu Val Leu Asn Ile Pro Asp Ala Tyr Ala Asp Pro Arg Phe 340 345 350 AAC AGA GAA GTA GAC TTG TAC ACA GGC TAC ACC ACG CGG AAC ATC CTG 1354 Asn Arg Glu Val Asp Leu Tyr Thr Gly Tyr Thr Thr Arg Asn Ile Leu 355 360 365 TGC ATG CCC ATC GTC AGC CGA GGC AGC GTG ATA GGT GTG GTG CAG ATG 1402 Cys Met Pro Ile Val Ser Arg Gly Ser Val Ile Gly Val Val Gln Met 370 375 380 GTC AAC AAA ATC AGT GGC AGT GCC TTC TCT AAA ACA GAT GAA AAC AAC 1450 Val Asn Lys Ile Ser Gly Ser Ala Phe Ser Lys Thr Asp Glu Asn Asn 385 390 395 400 TTC AAA ATG TTT GCC GTC TTT TGT GCT TTA GCC TTA CAC TGT GCT AAT 1498 Phe Lys Met Phe Ala Val Phe Cys Ala Leu Ala Leu His Cys Ala Asn 405 410 415 ATG TAT CAT AGA ATT CGC CAC TCA GAG TGC ATT TAC CGG GTA ACG ATG 1546 Met Tyr His Arg Ile Arg His Ser Glu Cys Ile Tyr Arg Val Thr Met 420 425 430 GAA AAG CTG TCC TAC CAT AGC ATT TGT ACT TCA GAA GAG TGG CAA GGT 1594 Glu Lys Leu Ser Tyr His Ser Ile Cys Thr Ser Glu Glu Trp Gln Gly 435 440 445 CTC ATG CAA TTC ACC CTT CCC GTG CGT CTC TGC AAA GAA ATT GAA TTA 1642 Leu Met Gln Phe Thr Leu Pro Val Arg Leu Cys Lys Glu Ile Glu Leu 450 455 460 TTC CAC TTT GAC ATT GGT CCT TTT GAA AAC ATG TGG CCT GGA ATT TTT 1690 Phe His Phe Asp Ile Gly Pro Phe Glu Asn Met Trp Pro Gly Ile Phe 465 470 475 480 GTC TAC ATG GTT CAT CGG TCC TGT GGG ACA TCC TGC TTT GAG CTT GAA 1738 Val Tyr Met Val His A rg Ser Cys Gly Thr Ser Cys Phe Glu Leu Glu 485 490 495 AAG TTG TGT CGT TTT ATT ATG TCT GTG AAG AAG AAC TAT CGG CGG GTT 1786 Lys Leu Cys Arg Phe Ile Met Ser Val Lys Lys Asn Tyr Arg Arg Val 500 505 510 510 CCT TAT CAC AAC TGG AAG CAT GCG GTC ACT GTA GCA CAC TGC ATG TAT 1834 Pro Tyr His Asn Trp Lys His Ala Val Thr Val Ala His Cys Met Tyr 515 520 525 GCC ATA CTT CAG AAC AAT CAC ACG CTT TTC ACA GAC CTT GAG CGC AAA 1882 Ala Ile Leu Gln Asn Asn His Thr Leu Phe Thr Asp Leu Glu Arg Lys 530 535 540 GGA CTG CTG ATT GCG TGT CTG TGT CAT GAC CTG GAC CAC AGG GGC TTC 1930 Gly Leu Leu Ile Ala Cys Leu Cys His Asp Leu Asp His Arg Gly Phe 545 550 555 560 AGT AAC AGC TAC CTG CAG AAG TTC GAC CAC CCT CTG GCC GCT CTC TAC 1978 Ser Asn Ser Tyr Leu Gln Lys Phe Asp His Pro Leu Ala Ala Leu Tyr 565 570 575 575 TCC ACT TCC ACC ATG GAG CAG CAC CAC TTC TCC CAG ACT GTG TCC ATC 2026 Ser Thr Ser Thr Met Glu Gln His His Phe Ser Gln Thr Val Ser Ile 580 585 590 CTT CAG TTG GAA GGG CAC AAT ATC TTC TCC ACT CTG AGC TCC AGT GAA 2074 Le u Gln Leu Glu Gly His Asn Ile Phe Ser Thr Leu Ser Ser Ser Glu 595 600 605 TAT GAG CAG GTG CTT GAG ATC ATC CGC AAA GCC ATC ATT GCC ACA GAC 2122 Tyr Glu Gln Val Leu Glu Ile Ile Arg Lys Ala Ile Ile Ala Thr Asp 610 615 620 CTT GCT TTA TAC TTT GGA AAC AGG AAG CAG TTG GAA GAG ATG TAC CAG 2170 Leu Ala Leu Tyr Phe Gly Asn Arg Lys Gln Leu Glu Glu Met Tyr Gln 625 630 635 640 ACC GGA TCA CTA AAC CTT AAT CAA TCA CAT AGA GAC CGT GTA ATT 2218 Thr Gly Ser Leu Asn Leu Asn Asn Gln Ser His Arg Asp Arg Val Ile 645 650 655 GGT TTG ATG ATG ACT GCC TGT GAC CTT TGT TCT GTG ACA AAA CTG TGG 2266 Gly Leu Met Met Thr Ala Cys Asp Leu Cys Ser Val Thr Lys Leu Trp 660 665 670 CCC GTT ACA AAA TTG ACG GCA AAT GAT ATA TAT GCA GAA TTC TGG GCT 2314 Pro Val Thr Lys Leu Thr Ala Asn Asp Ile Tyr Ala Glu Phe Trp Ala 675 680 685 GAG GGT GAT GAA ATG AAG AAA TTG GGA ATA CAG CCT ATT CCT ATG ATG 2362 Glu Gly Asp Glu Met Lys Lys Leu Gly Ile Gln Pro Ile Pro Met Met 690 695 700 GAC AGA GAC AAG AAG GAT GAA GTC CCC CAA GGC CAG CTT GGG TTC TAC 2410 Asp Arg Asp Lys Lys Asp Glu Val Pro Gln Gly Gln Leu Gly Phe Tyr 705 710 715 720 AAT GCC GTG GCC ATT CCC TGC TAT ACA ACC CTT ACC CAG ATC CTC CCT 2458 Asn Ala Val Ala Ile Pro Cys Tyr Thr Thr Leu Thr Gln Ile Leu Pro 725 730 735 CCC ACG GAG CCT CTT CTG AAA GCA TGC AGG GAT AAT CTC AGT CAG TGG 2506 Pro Thr Glu Pro Leu Leu Lys Ala Cys Arg Asp Asn Leu Ser Gln Trp 740 745 750 750 GAG AAG GTG ATT CGA GGG GAG GAG ACT GCA ACC TGG ATT TCA TCC CCA 2554 Glu Lys Val Ile Arg Gly Glu Glu Thr Ala Thr Trp Ile Ser Ser Pro 755 760 765 TCC GTG GCT CAG AAG GCA GCT GCA TCT GAA GAT TGA 2590 Ser Val Ala Gln Lys Ala Ala Ala Ser Glu Asp 770 775 779 GCACTGGTCA CCCTGACACG CTGTCCCACC TACAGATCCT CATCTTGCTT CTTTGACATT 2650 CTTTTCCTTT TTTTGGGGGG GGTGGGGGGA ACCTGCACCT GGTAACTGGG GTGCAAACCT 2710 CTTCAAGAAG GTAACATCAA ATAAATAAGT CAAGCAGAGG ACTTCCTGCC AATCTCTTCT 2770 GTGAGGCATC ATAGACACTG AGCAACCAGG ACCACCCCCA CGTTCAGAAA TCAGCTGGCC 2830 AAGTGACTCC ATTTGACTTG CAAACCAGCC TTTTCTAATA GGCTAATATT GCTGAGGCCT 2890 TAAA GGAAAT GGACAAAAAT TATCCAGAAG GGGTACTTTT CCATTGTATC TTTCTAATAA 2950 GGGTTTAAAA TGGTACTATT ATGGTATTGT ACTTGGGCTT TAACATCAAT GTTGCTTTGA 3010 TGTTGTTGGA TATAAATAGG AATTTTACAC ATTACTATTG TGAATGGTGA ATGTTCATGT 3070 ATGACCTACT TGTAATTAAC TTGAGTTGTA GTCCACAGCC TCAGGACAAA TGTCGTTGAG 3130 GTTACAGAGT AAGAAATGAT GGCAAAACGT CAAACTCTTA TTTCAGAGCT TCATGAATTT 3190 AGTTAGACTA AACATAATTC TTTAAGTTCA ACCTAAAGGG CTGAGATCAA TAAATTTAAC 3250 ACTAGACGAA GTAGACTTCC TGTCTTTTTG AGAAGAGATG AGGTATATGT TACAATAAAT 3310 CTCAGAACTT CAAGTAGCAG TTCAAAAGAT GTCAGTTTTT AAAATTGTTT TTGTTGTTGT 3370 CTTGGCAGTT TTACTGAACC CTTTGCATAA AGAACAAAAT AAAAGCTCGG CATTGTAATT 3430 TTTTTAATGG ACAAGTCTTA TGGATACGAG GGGTACATTT TTCATAATGA TTCCTTTATA 3490 TTTTCACTTT GTGTCATATG CAGAATTTTA GACTCTCATT CACAATGAAA AGTTTATTTT 3550 AAACATTGTT TAATTAAAAT ACCATACAGT TCTCTTTTAA ACATCAAACC ATAAAAAGTG 3610 TATTTTGTAA TTTTACTCTG ACCTGCCGCA GTCACCTCTC ACTTATCTCT TCCACGTACT 3670 GCACGGTCGT ATTTCATGAG CTTTCTGTCC ATAGCACAGA AACAGAGCAG AAAGTAGTAC 3730 AATCATGTTG GACCTTCTTT CTGTTCTCTT TACTCTTCTC ACAGATCAGA TCACTCCATA 3790 GAAGCCTGTG GGTTTCGATG GTTTCTTCTA TACACCTTTT TGGTTGACCA GTATTACTAT 3850 ACAATGTAAG TGTTTTAAAA AATACGAAAG TAATACTCTG CACCCCTTCC TACAAAGATG 3910 ATAAAGCAGT CACTTCTGGC GCATTTTAAT AATTTAAAGA TTTTTAGTGC AATGGCACGG 3970 TAACCTCCAA ACCTGAATTA GACAGAGACT CACTCAGGAA GTGACAGGCC CATCATATCA 4030 AATAACTTAT TCACTTTTCA TGTGGCAGGA AACTGGAATA TCGCTTTTAA TAAAATGGAA 4090 AAATATGCTT CTACATATTT ACCACCATAG GCGTTTTGTT CATATGAGCC TGGTTTGTGC 4150 AAAATTAAAT CAGAGGCTTC TACAACATGG TTTATTTATG TTGTAGCAAA GTTGGCTCTA 4210 CATAAACATT GTTCTTATTT TAAAATTAAC ACTATGTGTT CAGTTTTCTT GTGGGCTTCT 4270 GAAAGTTGCC ATCTTCCCTC CGTGGAGCTC CATTTGCTAT TTTCATTATA CACTATGAGG 4330 TAAAATGTAA TAACAAAAGA GAGAGAAGTA CCACTGTGGC TAGATATATA CACACACATA 4390 TATATATGGA TGGATGTAAT ATATGTAGCA CACACACATA GATGTATATA GGATACACAC 4450 TCATGTATGT AAACGTATAC ATATGTGTAT ATATGATACA TACACATACA CACACACGAG 4510 AGACAGAAGG AAAGAGAGGA AGAGAGAAGC AAACATGTAG GAAAAAATAT AAATCAGCCG 4570 GAATTC 4576.

SEQ ID NO: 2 Sequence length: 2406 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Human Sequence characteristics Characteristic symbols: Location of CDS: 15..2384 Sequence: TCTTCGGCTC CGAC 14 ATG GAA GAT GGA CCT TCT AAT AAT GCG AGC TGC TTC CGA AGG CTG ACC 62 Met Glu Asp Gly Pro Ser Asn Asn Ala Ser Cys Phe Arg Arg Leu Thr 1 5 10 15 GAG TGC TTC CTG AGC CCC AGT TTG ACA GAT GAA AAA GTG AAG GCA TAT 110 Glu Cys Phe Leu Ser Pro Ser Leu Thr Asp Glu Lys Val Lys Ala Tyr 20 25 30 CTT TCT CTT CAC CCC CAG GTA TTA GAT GAA TTT GTA TCT GAA AGT GTT 158 Leu Ser Leu His Pro Gln Val Leu Asp Glu Phe Val Ser Glu Ser Val 35 40 45 AGT GCA GAG ACA GTA GAG AAA TGG CTG AAG AGG AAG AAC AAC AAC AAA TCA 206 Ser Ala Glu Thr Val Glu Lys Trp Leu Lys Arg Lys Asn Asn Lys Ser 50 55 60 GAA GAT GAA TCA GCT CCT AAG GAA GTC AGC AGG TAC CAA GAT ACG AAT 254 Glu Asp Glu Ser Ala Pro Lys Glu Val Ser Arg Tyr Gln Asp Thr Asn 65 70 75 80 ATG CAG GGA GTT GTA TAT GAA CTA AAC AGC TAT ATA GAA CAA CGG TTG 302 Met Gln Gly Val Val Tyr Glu Leu Asn Ser Tyr Ile Glu Gln Arg Leu 85 90 95 GAC ACA GGA GGA GAC AAC CAG CTA CTC CTC TAT GAA CTG AGC AGC ATC 350 Asp Thr Gly Gly Asp Asn Gln Leu Leu Leu Tyr Glu Leu Ser Ser Ile 100 105 110 ATT AAA ATA GCC ACA AAA GCC GAT GGA TTT GCA CTG TAT TTC CTT GGA 398 Ile Lys Ile Ala Thr Lys Ala Asp Gly Phe Ala Leu Tyr Phe Leu Gly 115 120 125 GAG TGC AAT AAT AGC CTG TGT ATA TTC ACG CCA CCT GGG ATA AAG GAA 446 Glu Cys Asn Asn Ser Leu Cys Ile Phe Thr Pro Pro Gly Ile Lys Glu 130 135 140 GGA AAA CCC CGC CTC ATC CCT GCT GGG CCC ATC ACT CAG GGC ACC ACC 494 Gly Lys Pro Arg Leu Ile Pro Ala Gly Pro Ile Thr Gln Gly Thr Thr 145 150 155 160 GTC TCT GCT TAT GTG GCC AAG TCC AGG AAA ACA CTG CTA GTA GAA GAC 542 Val Ser Ala Tyr Val Ala Lys Ser Arg Lys Thr Leu Leu Val Glu Asp 165 170 175 ATC CTT GGA GAT GAA CGA TTT CCA AGA GGT ACT GGA CTG GAA TCA GGG 590 Ile Leu Gly Asp Glu Arg Phe Pro Arg Gly Thr Gly Leu Glu Ser Gly 180 185 190 ACT CGT ATC CAG TCT GTT CTT TGC TTA CCA ATT GTC ACT GCA ATT GGT 638 Thr Arg Ile Gln Ser Val Leu Cys Leu Pro Ile Val Thr Ala Ile Gly 195 200 205 GAC TTG ATT GGT ATT CTC GAG CTG TAT CGG CAC TGG GGC AAA GAA GCC 686 Asp Leu Ile Gly Ile Leu Glu Leu Tyr Arg His Trp Gly Lys Glu Ala 210 215 220 TTC TGT CTT AGT CAC CAG GAG GTT GCA ACA GCA AAT CTT GCC TGG GCT 734 Phe Cys Leu Ser His Gln Glu Val Ala Thr Ala Asn Leu Ala Trp Ala 225 230 235 240 TCA GTA GCA ATA CAT CAG GTG CAG GTA TGC AGA GGC CTT GCC AAA CAG 782 Ser Val Ala Ile His Gln Val Gln Val Cys Arg Gly Leu Ala Lys Gln 245 250 255 ACA GAA TTG AAT GAC TTC CTA CTC GAC GTA TCA AAA ACA TAT TTT GAT 830 Thr Glu Leu Asn Asp Phe Leu Leu Asp Val Ser Lys Thr Tyr Phe Asp 260 265 270 AAC ATA GTT GCA ATA GAT TCT CTA CTT GAA CAC ATA ATG ATA TAT GCA 878 Asn Ile Val Ala Ile Asp Ser Leu Leu Glu His Ile Met Ile Tyr Ala 275 280 285 AAA AAC CTG GTG AAT GCC GAT CGT TGT GCG CTT TTC CAG GTG GAC CAT 926 Lys Asn Leu Val Asn Ala Asp Arg Cys Ala Leu Phe GlnVal Asp His 290 295 300 AAG AAC AAG GAG TTA TAT TCA GAC CTT TTT GAT ATT GGA GAG GAA AAG 974 Lys Asn Lys Glu Leu Tyr Ser Asp Leu Phe Asp Ile Gly Glu Glu Lys 305 310 315 320 GAA GGA AAA CCT GTC TTC AAG AAG ACC AAA GAG ATA AGA TTT TCA ATT 1022 Glu Gly Lys Pro Val Phe Lys Lys Thr Lys Glu Ile Arg Phe Ser Ile 325 330 335 GAG AAA GGA ATT GCT GGC CAA GTA GCA AGA ACA GGG GAA GTC CTG AAC 1070 Glu Lys Gly Ile Ala Gly Gln Val Ala Arg Thr Gly Glu Val Leu Asn 340 345 350 ATT CCA GAT GCC TAT GCA GAC CCA CGC TTT AAC AGA GAA GTA GAC TTG 1118 Ile Pro Asp Ala Tyr Ala Asp Pro Arg Phe Asn Arg Glu Val Asp Leu 355 360 365 TAC ACA GGC TAC ACC ACG CGG AAC ATC CTG TGC ATG CCC ATC GTC AGC 1166 Tyr Thr Gly Tyr Thr Thr Arg Asn Ile Leu Cys Met Pro Ile Val Ser 370 375 380 CGA GGC AGC GTG ATA GGT GTG GTG CAG ATG GTC AAC AAA ATC AGT GGC 1214 Arg Gly Ser Val Ile Gly Val Val Gln Met Val Asn Lys Ile Ser Gly 385 390 395 400 AGT GCC TTC TCT AAA ACA GAT GAA AAC AAC TTC AAA ATG TTT GCC GTC 1262 Ser Ala Phe Ser Lys Thr Asp Gl u Asn Asn Phe Lys Met Phe Ala Val 405 410 415 TTT TGT GCT TTA GCC TTA CAC TGT GCT AAT ATG TAT CAT AGA ATT CGC 1310 Phe Cys Ala Leu Ala Leu His Cys Ala Asn Met Tyr His Arg Ile Arg 420 425 430 CAC TCA GAG TGC ATT TAC CGG GTA ACG ATG GAA AAG CTG TCC TAC CAT 1358 His Ser Glu Cys Ile Tyr Arg Val Thr Met Glu Lys Leu Ser Tyr His 435 440 445 AGC ATT TGT ACT TCA GAA GAG TGG CAA GGT CTC ATG CAA TTC ACC CTT 1406 Ser Ile Cys Thr Ser Glu Glu Trp Gln Gly Leu Met Gln Phe Thr Leu 450 455 460 CCC GTG CGT CTC TGC AAA GAA ATT GAA TTA TTC CAC TTT GAC ATT GGT 1454 Pro Val Arg Leu Cys Lys Glu Ile Glu Leu Phe His Phe Asp Ile Gly 465 470 475 480 480 CCT TTT GAA AAC ATG TGG CCT GGA ATT TTT GTC TAC ATG GTT CAT CGG 1502 Pro Phe Glu Asn Met Trp Pro Gly Ile Phe Val Tyr Met Val His Arg 485 490 495 TCC TGT GGG ACA TCC TGC TTT GAG CTT GAA AAG TTG TGT CGT TTT ATT 1550 Ser Cys Gly Thr Ser Cys Phe Glu Leu Glu Lys Leu Cys Arg Phe Ile 500 505 510 ATG TCT GTG AAG AAG AAC TAT CGG CGG GTT CCT TAT CAC AAC TGG AAG 1598 Met Ser Val Lys Lys Asn Tyr Arg Arg Val Pro Tyr His Asn Trp Lys 515 520 525 CAT GCG GTC ACT GTA GCA CAC TGC ATG TAT GCC ATA CTT CAG AAC AAT 1646 His Ala Val Thr Val Ala His Cys Met Tyr Ala Ile Leu Gln Asn Asn 530 535 540 CAC ACG CTT TTC ACA GAC CTT GAG CGC AAA GGA CTG CTG ATT GCG TGT 1694 His Thr Leu Phe Thr Asp Leu Glu Arg Lys Gly Leu Leu Ile Ala Cys 545 550 555 560 560 CTG TGT CAT GAC CTG GAC CAC AGG GGC TTC AGT AAC AGC TAC CTG CAG 1742 Leu Cys His Asp Leu Asp His Arg Gly Phe Ser Asn Ser Tyr Leu Gln 565 570 575 AAG TTC GAC CAC CCT CTG GCC GCT CTC TAC TCC ACT TCC ACC ATG GAG 1790 Lys Phe Asp His Pro Leu Ala Ala Leu Tyr Ser Thr Ser Thr Met Glu 580 585 590 CAG CAC CAC TTC TCC CAG ACT GTG TCC ATC CTT CAG TTG GAA GGG CAC 1838 Gln His His Phe Ser Gln Thr Val Ser Ile Leu Gln Leu Glu Gly His 595 600 605 AAT ATC TTC TCC ACT CTG AGC TCC AGT GAA TAT GAG CAG GTG CTT GAG 1886 Asn Ile Phe Ser Thr Leu Ser Ser Ser Glu Tyr Glu Gln Val Leu Glu 610 615 620 ATC ATC CGC AAA GCC ATC ATT GCC ACA GAC CTT GCT TTA TAC TTT G GA 1934 Ile Ile Arg Lys Ala Ile Ile Ala Thr Asp Leu Ala Leu Tyr Phe Gly 625 630 635 640 AAC AGG AAG CAG TTG GAA GAG ATG TAC CAG ACC GGA TCA CTA AAC CTT 1982 Asn Arg Lys Gln Leu Glu Glu Met Tyr Gln Thr Gly Ser Leu Asn Leu 645 650 655 AAT AAT CAA TCA CAT AGA GAC CGT GTA ATT GGT TTG ATG ATG ACT GCC 2030 Asn Asn Gln Ser His Arg Asp Arg Val Ile Gly Leu Met Met Thr Ala 660 665 670 TGT GAC CTT TGT TCT GTG ACA AAA CTG TGG CCC GTT ACA AAA TTG ACG 2078 Cys Asp Leu Cys Ser Val Thr Lys Leu Trp Pro Val Thr Lys Leu Thr 675 680 685 GCA AAT GAT ATA TAT GCA GAA TTC TGG GCT GAG GGT GAT GAA ATG AAG 2126 Ala Asn Asp Ile Tyr Ala Glu Phe Trp Ala Glu Gly Asp Glu Met Lys 690 695 700 AAA TTG GGA ATA CAG CCT ATT CCT ATG ATG GAC AGA GAC AAG AAG GAT 2174 Lys Leu Gly Ile Gln Pro Ile Pro Met Met Asp Arg Asp Lys Lys Asp 705 710 715 720 GAA GTC CCC CAA GGC CAG CTT GGG TTC TAC AAT GCC GTG GCC ATT CCC 2222 Glu Val Pro Gln Gly Gln Leu Gly Phe Tyr Asn Ala Val Ala Ile Pro 725 730 735 TGC TAT ACA ACC CTT ACC CAG ATC CTC CTC CT CCC ACG GAG CCT CTT CTG 2270 Cys Tyr Thr Thr Leu Thr Gln Ile Leu Pro Pro Thr Glu Pro Leu Leu 740 745 750 AAA GCA TGC AGG GAT AAT CTC AGT CAG TGG GAG AAG GTG ATT CGA GGG 2318 Lys Ala Cys Arg Asp Asn Leu Ser Gln Trp Glu Lys Val Ile Arg Gly 755 760 765 GAG GAG ACT GCA ACC TGG ATT TCA TCC CCA TCC GTG GCT CAG AAG GCA 2366 Glu Glu Thr Ala Thr Trp Ile Ser Ser Pro Ser Val Ala Gln Lys Ala 770 775 780 GCT GCA TCT GAA GAT TGA 2384 Ala Ala Ser Glu Asp 785 789 GCACTGGTCA CCCTGACACG CT 2406.

SEQ ID NO: 3 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: AAGCTGTCCT ACCATAGC 18

SEQ ID NO: 4 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: GGCTGCGGCC AGAGGGTG18.

SEQ ID NO: 5 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: ACTTTCAGAA GAGTGGCC18.

SEQ ID NO: 6 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: TGCAGGTAAC TGTTACTG 18.

SEQ ID NO: 7 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: CCGTCGCTCTT CGGCTCCG 18.

SEQ ID NO: 8 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: GGATCTGTAG GTGGGACAGC G 21.

SEQ ID NO: 9 Sequence length: 31 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: GGGAATTCAT GAGGATAGAA GAGAGGAAAT C31.

SEQ ID NO: 10 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: AGCGTGTCAG GGTGACCAGT GC22.

SEQ ID NO: 11 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: CGCTGCTCTT CGGCTCCG18.

SEQ ID NO: 12 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: GGATCTGTAG GTGGGACAGC G 21.

SEQ ID NO: 13 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: TCTTCGGCTC CGACATGG18

SEQ ID NO: 14 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: AGCGTGTCAG GGTGACCAGT GC22.

SEQ ID NO: 15 Sequence length: 3427 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA origin Organism name: rat Sequence characteristics Characteristic symbols: CDS existing position: 281..2665 sequence: GAATTCGGCA CGAGGCAGCG GCGGGCGGCG GCGGCTCTTC CTTTCGCCTG CGATCCAAGG 60 CTTGCTGCTC CCAGCCCGCT CCGGGCCCCG GCCACCTCCA CCGCCGCGGC TCCCCTTACA 120 CCCGGGCGCA CACCGCGCGG ACTCCTTGGG TTTTCCGGGT GCCGGCGGGG GCTGCCCTGG 180 CCTCGGCCCC GGCTCTGCGG CCGGTGGCCG AACTCCGTGG CGGCCCCGAG GCACCGCCCT 240 CCCCCTTGCC ACTGCCTGGC CGCTGCTCTT CGGCTCCGAC 280 ATG GAA GAT GGA CCC TCT AAC AAT GCG AGT TGC TTC CGA AGG CTG ACC 328 Met Glu Asp Gly Pro Ser Asn Asn Ala Ser Cys Phe Arg Arg Leu Thr 1 5 10 15 GAG TGT TTC CTC AGC CCC AGT TTG ACG GAT GAA AAG GTG AAG GCC TAT 376 Glu Cys Phe Leu Ser Pro Ser Leu Thr Asp Glu Lys Val Lys Ala Tyr 20 25 30 CTT TCC CTC CAT CCC CAG GTA TTA GAC GAG TTT GTT TCT GAA AGT GTT 424 Leu Ser Leu His Pro Gln Val Leu Asp Glu Phe Val Ser Glu Ser Val 35 40 45 AGT GCG GAG ACT GTG GAG AAG TGG CTG AAG AGG AAA AAC AAC AAA GCA 472 Ser Ala Glu Thr Val Glu Lys Trp Leu Lys Arg Lys Asn Asn Lys Ala 50 55 60 GAA GAT GAA CCA TCT CCT AAG GAA GTC AGC AGG TAC CAG GAC ACG AAC 520 Glu Asp Glu Pro Ser Pro Lys Glu Val Ser Arg Tyr Gln Asp Thr Asn 65 70 75 80 ATG CAG GGA GTC GTG TAC GAG CTG AAC AGC TAC ATA GAG CAG CGC CTG 568 Met Gln Gly Val Val Tyr Glu Leu Asn Ser Tyr Ile Glu Gln Arg Leu 85 90 95 GAC ACC GGC GGG GAC AAC CAC CTG CTC CTG TAC GAG CTA AGC AGT ATC 616 Asp Thr Gly Gly Asp Asn His Leu Leu Leu Tyr Glu Leu Ser Ser Ile 100 105 110 ATC AGG ATA GCC ACA AAA GCC GAC GGA TTT GCA CTG TAC TTC CTT GGA 664 Ile Arg Ile Ala Thr Lys Ala Asp Gly Phe Ala Leu Tyr Phe Leu Gly 115 120 125 GAG TGC AAT AAT AGT CTG TGT GTC TTC ACA CCA CCC GGA ATG AAG GAA 712 Glu Cys Asn Asn Ser Leu Cys Val Phe Thr Pro Pro Gly Met Lys Glu 130 135 140 GGT CAA CCC CGT CTC ATC CCC GCA GGG CCC ATC ACC CAG GGC ACC ACC 760 Gly Gln Pro Arg Leu Ile Pro Ala Gly Pro Ile Thr Gln Gly Thr Thr 145 150 155 160 ATC TCT GCC TAT GTG GCC AAG TCT AGG AAG ACC CTG CTG GTA GAG GAC 808 Ile Ser Ala Tyr Val Ala Lys Ser Arg Lys Thr Leu Leu Val Glu Asp 165 170 175 ATC CTT GGG GAT GAG CGA TTT CCC AGA GGC ACT GGT CTG GAG TCA GGA 856 Ile Leu Gly Asp Glu Arg Phe Pro Arg Gly Thr Gly Leu Glu Ser Gly 180 185 190 ACC CGA ATC CAG TCT GTC CTT TGC TTG CCT ATT GTC ACT GCC ATT GGA 904 Thr Arg Ile Gln Ser Val Leu Cys Leu Pro Ile Val Thr Ala Ile Gly 195 200 205 GAC TTG ATT GGC ATC CTT GAA CTG TAC AGG CAC TGG GGC AAA GAG GCC 952 Asp Leu Ile Gly Ile Leu Glu Leu Tyr Arg His Trp Gly Lys Glu Ala 210 215 220 TTC TGC CTC AGC CAT CAG GAG GTT GCA ACC GCC AAT CTC GCT TGG GCT 1000 Phe Cys Leu Ser His Gln Glu Val Ala Thr Ala Asn Leu Ala Trp Ala 225 230 235 240 TCC GTA GCA ATA CAC CAG GTG CAG GTG TGC AGA GGT CTC GCC AAG CAG 1048 Ser Val Ala Ile His Gln Val Gln Val Cys Arg Gly Leu Ala Lys Gln 245 250 255 ACC GAA CTG AAT GAC TTC CTG CTC GAT GTA TCA AAG ACA TAC TTT GAT 1096 Thr Glu Leu Asn Asp P he Leu Leu Asp Val Ser Lys Thr Tyr Phe Asp 260 265 270 AAC ATA GTC GCC ATA GAC TCT CTA CTT GAA CAC ATC ATG ATA TAT GCA 1144 Asn Ile Val Ala Ile Asp Ser Leu Leu Glu His Ile Met Ile Tyr Ala 275 280 285 285 AAA AAT CTA GTG AAC GCC GAC CGC TGC GCG CTC TTC CAG GTG GAC CAC 1192 Lys Asn Leu Val Asn Ala Asp Arg Cys Ala Leu Phe Gln Val Asp His 290 295 300 AAG AAC AAG GAG CTG TAC TCG GAC CTG TTT GAC ATT GGG GAG GAG AAG 1240 Lys Asn Lys Glu Leu Tyr Ser Asp Leu Phe Asp Ile Gly Glu Glu Lys 305 310 315 320 GAG GGG AAG CCC GTC TTC AAG AAG ACC AAG GAG ATC AGA TTT TCC ATT 1288 Glu Gly Lys Pro Val Phe Lys Lys Thr Lys Glu Ile Arg Phe Ser Ile 325 330 335 GAG AAA GGG ATT GCT GGT CAA GTG GCA AGA ACG GGA GAA GTC CTG AAC 1336 Glu Lys Gly Ile Ala Gly Gln Val Ala Arg Thr Gly Glu Val Leu Asn 340 345 350 ATT CCT GAT GCC TAC GCA GAC CCG CGC TTT AAC AGG GAG GTG GAC CTG 1384 Ile Pro Asp Ala Tyr Ala Asp Pro Arg Phe Asn Arg Glu Val Asp Leu 355 360 365 TAC ACA GGC TAT ACC ACG CGG AAC ATT CTG TGT ATG CCC ATA GTG AGC 1432 Ty r Thr Gly Tyr Thr Thr Arg Asn Ile Leu Cys Met Pro Ile Val Ser 370 375 380 CGC GGC AGC GTG ATC GGT GTG GTG CAA ATG GTT AAC AAG ATC AGC GGC 1480 Arg Gly Ser Val Ile Gly Val Val Gln Met Val Asn Lys Ile Ser Gly 385 390 395 400 AGC GCC TTC TCC AAG ACG GAT GAG AAC AAC TTC AAG ATG TTT GCT GTC 1528 Ser Ala Phe Ser Lys Thr Asp Glu Asn Asn Phe Lys Met Phe Ala Val 405 410 415 TTC TGC GCT CTG GCC CTG CAC TGC GCT AAC ATG TAC CAC AGG ATC CGC 1576 Phe Cys Ala Leu Ala Leu His Cys Ala Asn Met Tyr His Arg Ile Arg 420 425 430 CAC TCA GAG TGC ATC TAC AGG GTT ACC ATG GAG AAG CTG TCT TAC CAC 1624 His Ser Glu Cys Ile Tyr Arg Val Thr Met Glu Lys Leu Ser Tyr His 435 440 445 AGC ATC TGC ACC TCT GAG GAA TGG CAA GGC CTC ATG CAC TTC AAC TTG 1672 Ser Ile Cys Thr Ser Glu Glu Trp Gln Gly Leu Met His Phe Asn Leu 450 455 460 CCA GCA CGC ATC TGC CGG GAC ATC GAG CTA TTC CAC TTT GAC ATT GGT 1720 Pro Ala Arg Ile Cys Arg Asp Ile Glu Leu Phe His Phe Asp Ile Gly 465 470 470 475 480 CCT TTC GAG AAC ATG TGG CCT GGG ATC TTT GTC TAC ATG ATC CAT CGG 1768 Pro Phe Glu Asn Met Trp Pro Gly Ile Phe Val Tyr Met Ile His Arg 485 490 495 TCT TGT GGG ACA TCC TGT TTT GAA CTT GAA AAA TTG TGC CGT TTT ATC 1816 Ser Cys Gly Thr Ser Cys Phe Glu Leu Glu Lys Leu Cys Arg Phe Ile 500 505 510 ATG TCT GTG AAG AAG AAC TAT AGG CGG GTT CCT TAC CAC AAC TGG AAG 1864 Met Ser Val Lys Lys Asn Tyr Arg Arg Val Pro Tyr His Asn Trp Lys 515 520 525 CAT GCA GTC ACG GTG GCG CAC TGC ATG TAC GCC ATA CTT CAA AAC AAC 1912 His Ala Val Thr Val Ala His Cys Met Tyr Ala Ile Leu Gln Asn Asn 530 535 540 AAT GGC CTC TTC ACA GAC CTT GAG CGC AAA GGC CTG CTA ATT GCC TGT 1960 Asn Gly Leu Phe Thr Asp Leu Glu Arg Lys Gly Leu Leu Ile Ala Cys 545 550 555 560 CTG TGC CAT GAC CTG GAC CAC AGG GGC TTC AGT AAC AGC TAC CTG CAG 2008 Leu Cys His Asp Leu Asp His Arg Gly Phe Ser Asn Ser Tyr Leu Gln 565 570 575 AAA TTC GAC CAC CCC CTG GCT GCG TTG TAC TCC ACC TCC ACC ATG GAG 2056 Lys Phe Asp His Pro Leu Ala Ala Leu Tyr Ser Thr Ser Thr Met Glu 580 585 590 CAA CAC CAC TTC TCC CAG ACG G TG TCC ATC CTC CAG CTG GAA GGA CAC 2104 Gln His His Phe Ser Gln Thr Val Ser Ile Leu Gln Leu Glu Gly His 595 600 605 AAC ATC TTC TCC ACC CTG AGC TCC AGC GAG TAC GAG CAG GTG CTG GAG 2152 Asn Ile Phe Ser Thr Leu Ser Ser Ser Glu Tyr Glu Gln Val Leu Glu 610 615 620 ATC ATC CGC AAA GCC ATC ATC GCC ACT GAC CTC GCA CTG TAC TTT GGG 2200 Ile Ile Arg Lys Ala Ile Ile Ala Thr Asp Leu Ala Leu Tyr Phe Gly 625 630 635 640 AAC AGG AAG CAG TTG GAG GAG ATG TAC CAG ACA GGG TCG CTG AAC CTC 2248 Asn Arg Lys Gln Leu Glu Glu Met Tyr Gln Thr Gly Ser Leu Asn Leu 645 650 655 655 CAC AAC CAG TCC CAT CGA GAC CGC GTC ATC GTG ATG ATG ACT GCC 2296 His Asn Gln Ser His Arg Asp Arg Val Ile Gly Leu Met Met Thr Ala 660 665 670 TGC GAT CTT TGC TCT GTG ACG AAA CTA TGG CCA GTT ACA AAA TTG ACA 2344 Cys Asp Leu Cys Ser Val Thr Lys Leu Trp Pro Val Thr Lys Leu Thr 675 680 685 GCA AAT GAT ATA TAT GCA GAG TTC TGG GCT GAG GGG GAT GAG ATG AAG 2392 Ala Asn Asp Ile Tyr Ala Glu Phe Trp Ala Glu Gly Asp Glu Met Lys 690 695 700 AAG TTG GG G ATA CAG CCC ATC CCT ATG ATG GAC AGA GAC AAG CGA GAT 2440 Lys Leu Gly Ile Gln Pro Ile Pro Met Met Asp Arg Asp Lys Arg Asp 705 710 715 715 720 GAA GTC CCT CAA GGA CAG CTT GGA TTC TAC AAT GCT GTG GCC ATC CCC 2488 Glu Val Pro Gln Gly Gln Leu Gly Phe Tyr Asn Ala Val Ala Ile Pro 725 730 735 TGC TAT ACC ACC CTG ACG CAG ATC CTC CCA CCC ACA GAG CCT CTG CTG 2536 Cys Tyr Thr Thr Leu Thr Gln Ile Leu Pro Pro Thr Glu Pro Leu Leu 740 745 750 AAG GCC TGC AGG GAT AAC CTC AAT CAG TGG GAG AAG GTA ATT CGA GGG 2584 Lys Ala Cys Arg Asp Asn Leu Asn Gln Trp Glu Lys Val Ile Arg Gly 755 760 765 GAA GAG ACA GCA ATG TGG ATT TCA GGC CCA GCA ACT AGC AAA AGC ACA 2632 Glu Glu Thr Ala Met Trp Ile Ser Gly Pro Ala Thr Ser Lys Ser Thr 770 775 780 TCT GAG AAG CCG ACC AGG AAG GTC GAT GAC TGA 2665 Ser Glu Lys Pro Thr Arg Lys Val Asp Asp 785 790 794 TCCTGAGGTG ATGTCTGCCT AGCAACTGAC TCAACCTGCT TCTGTGACTT CGTTCTTTTT 2725 ATTTTTATTT TTTTAACGGG GTGAAAACCT CTCTCAGAAG GTACCGTCGC ATATCCATGT 2785 GAAGCAGATG ACTCCCTGCG CACACCTCGG ACCGT GAGCA ACCCGGGCTC CACCGTGTTC 2845 AGACGTCGGC TATTCCATGG CTCCGCCTGA CCCCCGAATG CCATTTGCTA CCAGGCCAGA 2905 ACTGCGCTGG CTGGAGGGGG CAGAGACGAC AGGAGGGGTT CTTACCTGCA TCCTTCCATG 2965 AGGGTGTGGT TCTGTGTTTC ATCTCTAACA GAGATGCTAC TGCTTGGTGG CGTTTGTTAG 3025 AAATGGGACA CATGCCCCTG TCGTGAAGTT TACATGTGAC CTTCTTGTAG GTAACTTGAG 3085 TTCGTAGCCT GGGACCCCTG TAATGAAGGT TACAGTCCAC AGGTGATAGA GAAATTCAAG 3145 CTGTAAGTTA CAGGTGCACT ACAAGTGTGT TCATTCAGTT TACCTGGGGG CATGGAGGTG 3205 AGTCAGCTCC ACGAGGAAGG AAGCATACCT CTGCCCTCAT CAAGGGGACA CAGGGTACAT 3265 CCCAGGCATC AGAGAACTGC AGCTCACCTC AAACCATGTC AAAGAATTAA AACACACCCC 3325 CATCCCCTCA CTGTAGCCTT TGGCAACTTC GCCAAACCCT TCACACAAAG AAAATAAAAG 3385 TAAGGCGTAT AAATTTCCTC CAGCAAGCCT CGTGCCGAAT TC 427

SEQ ID NO: 16 Sequence length: 3080 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA origin Organism name: rat Sequence characteristics Characteristic symbols: CDS existing position: 634..3000 sequence: GAATTCGGCA CGAGCTTCAG AGCACACGCA GCCCCCCAAC TCCGCCCCTA TGTCAAGTGC 60 ACGCGGGCAC ACGGGTTGGA CACTCGCCCA CGCACGCACA CACACGCGCG TGCACATACA 120 TGCACACTCG TGCACACACA CTACTTCTAG GTGTGTGGGT CTAAAGTTGC TTCCGTCGAG 180 AGGCTGAGCT GAGATGGACC AGTCTTCATT GGTGCCTAGA AAGCTCCTTA CAGCTTTCCT 240 GAGGATCCCA GAAAGGAAGG CGGGAGGGAG TAGCATCGCT CCTTCTTAGA GTTGTCTCCC 300 CCTTTTGCGA GATGGCCAGC GCAGAAACTC CAAGTACCCG AAGCCCTTGG AATACTGCTG 360 CATGGAGCAT ATTCGAGCAT CTTGGAGAGG TCCTGGACTT GGGCCACCCG GGTTTGGCAA 420 CTTGTTTTGA GCAGCCCTGG ATGGAGCCCG GATCCACCTT TCCCAGAGAC TTGTCCCTAC 480 GGCCCCACTT TGAATTTGTG AACTGGCAAT GAAGCATC GCTC TGAGTCGATC AGC TGATCGATC AGC TGATCGATC ATCGGTCGATCACTGAGTCGATCACTGAGGATCACTGAGGATCACTGAGGATCACTGAGTCGATCAAGG AGA CTCTCGGGGG CAG 633 ATG AGC AAT GAC TCC CCA GAA GGT GCC GTG GGC TCC TGC AAC GCA ACA 681 Met Ser Asn Asp Ser Pro Glu Gly Ala Val Gly Ser Cys Asn Ala Thr 1 5 10 15 GGT TTG ACG GAT GAA AAG GTG AAG GCC TAT CTT TCC CTC CAT CCC CAG 729 Gly Leu Thr Asp Glu Lys Val Lys Ala Tyr Leu Ser Leu His Pro Gln 20 25 30 GTA TTA GAC GAG TTT GTT TCT GAA AGT GTT AGT GCG GAG ACT GTG GAG 777 Val Leu Asp Glu Phe Val Ser Glu Ser Val Ser Ala Glu Thr Val Glu 35 40 45 AAG TGG CTG AAG AGG AAA AAC AAC AAA GCA GAA GAT GAA CCA TCT CCT 825 Lys Trp Leu Lys Arg Lys Asn Asn Lys Ala Glu Asp Glu Pro Ser Pro 50 55 60 AAG GAA GTC AGC AGG TAC CAG GAC ACG AAC ATG CAG GGA GTC GTG TAC 873 Lys Glu Val Ser Arg Tyr Gln Asp Thr Asn Met Gln Gly Val Val Tyr 65 70 75 80 GAG CTG AAC AGC TAC ATA GAG CAG CGC CTG GAC ACC GGC GGG GAC AAC 921 Glu Leu Asn Ser Tyr Ile Glu Gln Arg Leu Asp Thr Gly Gly Asp Asn 85 90 95 CAC CTG CTC CTG TAC GAG CTA AGC AGT ATC ATC AGG ATA GCC ACA AAA 969 His Leu Leu Leu Leu Tyr Glu Leu Ser Ser Ile Ile Arg Ile Ala Thr Lys 100 105 110 GCC GAC GGA TTT GCA CTG TAC TTC CTT GGA GAG TGC AAT AAT AGT CTG 1017 Ala Asp Gly Phe Ala Leu Tyr Phe Leu Gly Glu Cys Asn Asn Ser Leu 115 120 125 TGT GTC TTC ACA CCA CCC GGA ATG AAG GAA GGT CAA CCC CGT CTC ATC 1065 Cys Val Phe Thr Pro Pro Gly Met Lys Glu Gly Gln Pro Arg Leu Ile 130 135 140 CCC GCA GGG CCC ATC ACC CAG GGC ACC ACC ATC TCT GCC TAT GTG GCC 1113 Pro Ala Gly Pro Ile Thr Gln Gly Thr Thr Ile Ser Ala Tyr Val Ala 145 150 155 160 AAG TCT AGG AAG ACC CTG CTG GTA GAG GAC ATC CTT GGG GAT GAG CGA 1161 Lys Ser Arg Lys Thr Leu Leu Val Glu Asp Ile Leu Gly Asp Glu Arg 165 170 175 TTT CCC AGA GGC ACT GGT CTG GAG TCA GGA ACC CGA ATC CAG TCT GTC 1209 Phe Pro Arg Gly Thr Gly Leu Glu Ser Gly Thr Arg Ile Gln Ser Val 180 185 190 CTT TGC TTG CCT ATT GTC ACT GCC ATT GGA GAC TTG ATT GGC ATC CTT 1257 Leu Cys Leu Pro Ile Val Thr Ala Ile Gly Asp Leu Ile Gly Ile Leu 195 200 205 GAA CTG TAC AGG CAC TGG GGC AAA GAG GCC TTC TGC CTC AGC CAT CAG 1305 Glu Leu Tyr Arg His Trp Gly Lys Glu Ala Phe Cys Leu Ser His Gln 210 215 220 GAG GTT GCA ACC GCC AAT CTC GCT TGG GCT TCC GTA GCA ATA CAC CAG 1353 Glu Val Ala Thr Ala Asn Leu Ala Trp Ala Ser Val Ala Ile His Gln 225 230 235 240 GTG CAG GTG TGC AGA GGT CTC GCC AAG CAG ACC GAA CTG AAT GAC TTC 1401 Val Gln Val Cys Arg Gly Leu Ala Lys Gln Thr Glu Leu Asn Asp Phe 245 250 255 CTG CTC GAT GTA TCA AAG ACA TAC TTT GAT AAC ATA GTC GCC ATA GAC 1449 Leu Leu Asp Val Ser Lys Thr Tyr Phe Asp Asn Ile Val Ala Ile Asp 260 265 270 TCT CTA CTT GAA CAC ATC ATG ATA TAT GCA AAA AAT CTA GTG AAC GCC 1497 Ser Leu Leu Glu His Ile Met Ile Tyr Ala Lys Asn Leu Val Asn Ala 275 280 285 GAC CGC TGC GCG CTC TTC CAG GTG GAC CAC AAG AAC AAG GAG CTG TAC 1545 Asp Arg Cys Ala Leu Phe Gln Val Asp His Lys Asn Lys Glu Leu Tyr 290 295 300 TCG GAC CTG TTT GAC ATT GGG GAG GAG AAG GAG GGG AAG CCC GTC TTC 1593 Ser Asp Leu Phe Asp Ile Gly Glu Glu Lys Glu Gly Lys Pro Val Phe 305 310 315 320 AAG AAG ACC AAG GAG ATC AGA TTT TCC ATT GAG AAA GGG ATT GCT GGT 1641 Lys Lys Thr Lys G lu Ile Arg Phe Ser Ile Glu Lys Gly Ile Ala Gly 325 330 335 CAA GTG GCA AGA ACG GGA GAA GTC CTG AAC ATT CCT GAT GCC TAC GCA 1689 Gln Val Ala Arg Thr Gly Glu Val Leu Asn Ile Pro Asp Ala Tyr Ala 340 345 350 GAC CCG CGC TTT AAC AGG GAG GTG GAC CTG TAC ACA GGC TAT ACC ACG 1737 Asp Pro Arg Phe Asn Arg Glu Val Asp Leu Tyr Thr Gly Tyr Thr Thr 355 360 365 CGG AAC ATT CTG TGT ATG CCC ATA GTG AGC CGC GGC AGC GTG ATC GGT 1785 Arg Asn Ile Leu Cys Met Pro Ile Val Ser Arg Gly Ser Val Ile Gly 370 375 380 GTG GTG CAA ATG GTT AAC AAG ATC AGC GGC AGC GCC TTC TCC AAG ACG 1833 Val Val Gln Met Val Asn Lys Ile Ser Gly Ser Ala Phe Ser Lys Thr 385 390 395 400 GAT GAG AAC AAC TTC AAG ATG TTT GCT GTC TTC TGC GCT CTG GCC CTG 1881 Asp Glu Asn Asn Phe Lys Met Phe Ala Val Phe Cys Ala Leu Ala Leu 405 410 415 CAC TGC GCT AAC ATG TAC CAC AGG ATC CGC CAC TCA GAG TGC ATC TAC 1929 His Cys Ala Asn Met Tyr His Arg Ile Arg His Ser Glu Cys Ile Tyr 420 425 430 AGG GTT ACC ATG GAG AAG CTG TCT TAC CAC AGC ATC TGC ACC TCT GAG 197 7 Arg Val Thr Met Glu Lys Leu Ser Tyr His Ser Ile Cys Thr Ser Glu 435 440 445 GAA TGG CAA GGC CTC ATG CAC TTC AAC TTG CCA GCA CGC ATC TGC CGG 2025 Glu Trp Gln Gly Leu Met His Phe Asn Leu Pro Ala Arg Ile Cys Arg 450 455 460 GAC ATC GAG CTA TTC CAC TTT GAC ATT GGT CCT TTC GAG AAC ATG TGG 2073 Asp Ile Glu Leu Phe His Phe Asp Ile Gly Pro Phe Glu Asn Met Trp 465 470 475 480 480 CCT GGG ATC TTT GTC TAC ATG ATC CAT CGG TCT TGT GGG ACA TCC TGT 2121 Pro Gly Ile Phe Val Tyr Met Ile His Arg Ser Cys Gly Thr Ser Cys 485 490 495 TTT GAA CTT GAA AAA TTG TGC CGT TTT ATC ATG TCT GTG AAG AAG AAC 2169 Phe Glu Leu Glu Lys Leu Cys Arg Phe Ile Met Ser Val Lys Lys Asn 500 505 510 TAT AGG CGG GTT CCT TAC CAC AAC TGG AAG CAT GCA GTC ACG GTG GCG 2217 Tyr Arg Arg Val Pro Tyr His Asn Trp Lys His Ala Val Thr Val Ala 515 520 525 CAC TGC ATG TAC GCC ATA CTT CAA AAC AAC AAT GGC CTC TTC ACA GAC 2265 His Cys Met Tyr Ala Ile Leu Gln Asn Asn Asn Gly Leu Phe Thr Asp 530 535 540 CTT GAG CGC AAA GGC CTG CTA ATT GCC TGT CTG TGC CAT GAC CTG GAC 2313 Leu Glu Arg Lys Gly Leu Leu Ile Ala Cys Leu Cys His Asp Leu Asp 545 550 555 560 CAC AGG GGC TTC AGT AAC AGC TAC CTG CAG AAA TTC GAC CAC CCC CTG 2361 His Arg Gly Phe Ser Asn Ser Tyr Leu Gln Lys Phe Asp His Pro Leu 565 570 575 GCT GCG TTG TAC TCC ACC TCC ACC ATG GAG CAA CAC CAC TTC TCC CAG 2409 Ala Ala Leu Tyr Ser Thr Ser Thr Met Glu Gln His His Phe Ser Gln 580 585 590 590 ACG GTG TCC ATC CTC CAG CTG GAG GGA CAC AAC ATC TTC TCC ACC CTG 2457 Thr Val Ser Ile Leu Gln Leu Glu Gly His Asn Ile Phe Ser Thr Leu 595 600 605 AGC TCC AGC GAG TAC GAG CAG GTG CTG GAG ATC ATC CGC AAA GCC ATC 2505 Ser Ser Ser Glu Tyr Glu Gln Val Leu Glu Ile Ile Arg Lys Ala Ile 610 615 620 ATC GCC ACT GAC CTC GCA CTG TAC TTT GGG AAC AGG AAG CAG TTG GAG 2553 Ile Ala Thr Asp Leu Ala Leu Tyr Phe Gly Asn Arg Lys Gln Leu Glu 625 630 635 640 GAG ATG TAC CAG ACA GGG TCG CTG AAC CTC CAC AAC CAG TCC CAT CGA 2601 Glu Met Tyr Gln Thr Gly Ser Leu Asn Leu His Asn Gln Ser His Arg 645 650 655 GAC CGC GTC ATC GGC TTG A TG ATG ACT GCC TGC GAT CTT TGC TCT GTG 2649 Asp Arg Val Ile Gly Leu Met Met Thr Ala Cys Asp Leu Cys Ser Val 660 665 670 ACG AAA CTA TGG CCA GTT ACA AAA TTG ACA GCA AAT GAT ATA TAT GCA 2697 Thr Lys Leu Trp Pro Val Thr Lys Leu Thr Ala Asn Asp Ile Tyr Ala 675 680 685 GAG TTC TGG GCT GAG GGG GAT GAG ATG AAG AAG TTG GGG ATA CAG CCC 2745 Glu Phe Trp Ala Glu Gly Asp Glu Met Lys Lys Leu Gly Ile Gln Pro 690 695 700 ATC CCT ATG ATG GAC AGA GAC AAG CGA GAT GAA GTC CCT CAA GGA CAG 2793 Ile Pro Met Met Asp Arg Asp Lys Arg Asp Glu Val Pro Gln Gly Gln 705 710 710 715 720 CTT GGA TTC TAC AAT GCT GTG GCC ATC CCC TGC TAT ACC ACC CTG ACG 2841 Leu Gly Phe Tyr Asn Ala Val Ala Ile Pro Cys Tyr Thr Thr Leu Thr 725 730 735 CAG ATC CTC CCA CCC ACA GAG CCT CTG CTG AAG GCC TGC AGG GAT AAC 2889 Gln Ile Leu Pro Pro Thr Glu Pro Leu Leu Lys Ala Cys Arg Asp Asn 740 745 750 CTC AAT CAG TGG GAG AAG GTA ATT CGA GGG GAA GAG ACA GCA ATG TGG 2937 Leu Asn Gln Trp Glu Lys Val Ile Arg Gly Glu Glu Thr Ala Met Trp 755 760 765 ATT TC A GGC CCA GCA ACT AGC AAA AGC ACA TCT GAG AAG CCG ACC AGG 2985 Ile Ser Gly Pro Ala Thr Ser Lys Ser Thr Ser Glu Lys Pro Thr Arg 770 775 780 AAG GTC GAT GAC TGA 3000 Lys Val Asp Asp 785 788 TCCTGAGGTG ATGTCTGCCT AGCAACTGAC TCAACCTGCT TCTGTGACTT CGTTCTTTTT 3060 ATTTTTATTT TTTTAACGGG 3080.

SEQ ID NO: 17 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic primer) Sequence: GCGCTCTTCC AGGTGGACC19.

SEQ ID NO: 18 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic primer) Sequence: CATCTTGAAG TTGTTCTC18

[Brief description of the drawings]

FIG. 1 shows the results of fractionating a cell extract of COS cells expressing recombinant human PDE10 by MonoQ Sepharose column chromatography (cAMP or cGM of each fraction)
FIG.

FIG. 2 cAMP or cGMP by human PDE10
Kinetic analysis of hydrolysis reaction (Lineweaver-Burk pl
ot).

FIG. 3 is a view showing the results of examining the effect of cGMP on cAMP degradation activity of human PDE10. The relative value (%) of the activity was shown assuming that the cAMP degradation activity in the case where cGMP was not added was 100%.

FIG. 4 is a view showing the expression of PDE10 gene (result of dot blot) in each human tissue.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/16 C 9/16 C12P 21/08 15/02 C12Q 1/44 C12P 21/08 1/68 A C12Q 1/44 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/573 A 33/50 C12N 5/00 B 33/573 A // (C12N 9/16 15/00 (C12R 1:91) (72) Inventor Hideo Michibata 2-3-8 Kawagishi, Toda City, Saitama Prefecture Tanabe Pharmaceutical Toda Ryo (72) Inventor Keizo Yuasa 2-3-8 Kawagishi, Toda City, Saitama Prefecture Tanabe Pharmaceutical Toda Ryo

Claims (22)

[Claims] 1. A type 10 phosphodiesterase. 2. The phosphodiesterase according to claim 1, which is a protein selected from the following (A) and (B): (A) a protein having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 16; (B) a protein having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 16 in which one or more amino acids have been deleted, substituted or added, and A protein having an activity of hydrolyzing cyclic nucleotides. 3. The phosphodiesterase according to claim 1, which is a protein selected from the following (A) and (B): (A) a protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (B) a protein having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and an activity of hydrolyzing cyclic nucleotides A protein having 4. The phosphodiesterase according to claim 1, which is derived from human. 5. The phosphodiesterase according to claim 1, which is derived from a rat. 6. A gene or nucleic acid encoding the phosphodiesterase according to claim 1, 2 or 3. 7. A gene or nucleic acid selected from the following (a) and (b): (A) A gene or nucleic acid comprising a DNA having the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 16. (B) a gene or nucleic acid consisting of a DNA that hybridizes under stringent conditions with a DNA having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 16, and a cyclic nucleotide A gene or a nucleic acid encoding a protein having an activity of hydrolyzing a protein. 8. A gene or nucleic acid selected from the following (a) and (b): (A) a gene or nucleic acid consisting of a DNA having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 (B) a gene or nucleic acid consisting of a DNA that hybridizes under stringent conditions with a DNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and a protein having an activity of hydrolyzing cyclic nucleotides A gene or nucleic acid encoding 9. The gene or nucleic acid according to claim 7, which is derived from human. 10. The gene or nucleic acid according to claim 7, which is derived from a rat. 11. A recombinant vector containing the gene or nucleic acid according to claim 6, 7, or 8. 12. The recombinant vector according to claim 11, which is an expression vector. A host cell into which the recombinant vector according to claim 12 has been introduced. 14. A step of performing an enzymatic reaction in a system containing the phosphodiesterase according to claim 1, an enzyme substrate and a test substance, and a step of assaying the inhibitory effect of the test substance on the enzyme activity of the phosphodiesterase according to claim 1. A method for characterizing, identifying or selecting a phosphodiesterase inhibitor, including: 15. The enzyme substrate is cAMP and cGMP.
The method according to claim 14, which is a cyclic nucleotide selected from the group consisting of: 16. The enzyme activity of a phosphodiesterase,
15. The method of claim 14, which is a cyclic nucleotide hydrolytic activity. 17. A step of performing a binding reaction in a system containing the phosphodiesterase according to claim 1 and a test substance, and a step of testing whether the test substance has a binding ability to the phosphodiesterase according to claim 1. For characterizing, identifying or selecting phosphodiesterase inhibitors. 18. The method according to claim 14 or 17, which is used to characterize, identify or select by the selectivity of an inhibitory effect on a plurality of types of phosphodiesterases. 19. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 1
DNA having the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 16
Claims 6 and 7 using a nucleic acid that hybridizes under stringent conditions with a probe or primer.
Or a method for detecting the gene or nucleic acid according to 8. 20. A nucleotide sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15 or SEQ ID NO: 16, which is used for suppressing the expression of the gene according to claim 6, 7 or 8 in a cell. A nucleic acid that hybridizes under stringent conditions with a DNA having 21. An antibody that recognizes the phosphodiesterase according to claim 1. 22. A method for detecting the expression of the phosphodiesterase of claim 1 in a cell or tissue using an antibody that recognizes the phosphodiesterase of claim 1.