Methods of screening for modifiers of potassium channel protein activity

This is a Continuation of application Ser. No. 09/600,776, filed Jul. 21, 2000, issued as U.S. Pat. No. 6,326,168 on Dec. 4, 2001, the disclosure of which is incorporated herein by reference.

The present invention belongs to the field of genetic engineering, and particularly relates to a novel potassium channel protein expressed exclusively in the brain, or an equivalent thereof, a gene encoding the protein or an equivalent thereof, a vector containing the gene, a host cell containing the vector, and so on.

Potassium channel is a protein which is distributed in the surface membrane of cells and selectively allows potassium ions to pass through it, and it is considered that it takes an important role in controlling membrane potential of cells. Particularly, in nerve and muscle cells, it contributes to the neurotransmission of central and peripheral nerves, pace making of the heart, contraction of muscles and the like by controlling frequency, persistency and the like of action potential. In addition, it has been shown that it is also concerned in the secretion of hormones, adjustment of cell volume, proliferation of cells and the like.

As the classification based on the opening and closing mechanism of the channel, a voltage-dependent potassium channel, an inwardly rectifying potassium channel, a calcium-dependent potassium channel, a receptor coupling type potassium channel and the like have so far been identified. In addition, an ATP-dependent potassium channel, a pH-dependent potassium channel and the like have also been reported. Among them, the voltage-dependent potassium channel has a characteristic in that it opens when the membrane potential is depolarized. In general, potassium ions are present in a non-equilibrium state of about 5 mM outside the cell and about 150 mM inside the cell. Thus, when the voltage-dependent potassium channel is opened by depolarization, potassium ions flow out from intracellular part to extracellular part and, as a result, induce restoration of the membrane potential (re-polarization). Accordingly, the opening of voltage-dependent potassium channel induces reduction of excitability of nerve and muscle cells and the like. Also, it causes changes in cellular functions in non-excitatory cells too, such as increase in the driving force for Ca 2+ and subsequent increase in the flow of the same ion into the intracellular part. A compound capable of modifying opening of the voltage-dependent channel has a possibility of controlling various functions of cells, including excitability of nerve and muscle cells.

Genes of some types of the voltage-dependent potassium channel have been isolated from the brain and heart, and primary structure of the protein has been revealed. Based on the primary structure, it has been suggested that the voltage-dependent potassium channel has six transmembrane domains (S1 to S6) and one ion permeation region (H5). Also, it is assumed that the fourth transmembrane domain S4 contains basic amino acids having positive charge at intervals of 3 to 4 bases and functions as a voltage sensor.

These channels are roughly divided into Shaker type and eag type, based on the similarity of amino acid sequences. The Shaker type is a family having markedly high diversity and can be further divided into four groups of Kv1, Kv2, Kv3 and Kv4. On the other hand, the eag type is constituted by eag, eag-related gene and elk, and it related genes include hyperpolarization activation type potassium channels corresponding to KAT gene cluster and a cation channel which is activated by a cyclic nucleotide.

Regarding the importance of voltage-dependent potassium current in the brain, several findings have been obtained using these cloned voltage-dependent potassium channels. For example, relationship of Kv1.1 with memory and pain has been suggested by antisense-aided in vivo experiments (Meiri, N. et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 4430-4434; Galeotti, N. et al. (1997) J. Pharmacol. Exp. Ther., 281, 941-949). Regarding Kv3.1, its participation in the excitability of GABA activating interneuron in cerebral cortex has been shown (Massengill, J. et al. (1997) J. Neurosci., 3136-3147). On the other hand, some experiments carried out using tetraethylammonium and 4-aminopyridine as non-selective inhibitors of the voltage-dependent potassium channel have also been reported. It has been shown that tetraethylammonium suppresses voltage-dependent potassium current in cerebral cortex nerve cells and also inhibits apoptosis of the same nerve cells (Yu, S. P. et al. (1997), Science, 278, 114-117). Also, it is known that intraventricular administration of 4-aminopyridine causes epileptic attack. These results suggest a possibility that an agent capable of controlling the activity of voltage-dependent potassium channel in the brain will become a therapeutic agent for central nervous system disorders such as dementia due to disturbance of memory and so on, nerve cell death accompanied by cerebral ischemia, epilepsy and the like.

On the other hand, most of the voltage-dependent potassium channels so far cloned are distributed in a large number of tissues among organs in the whole body. Thus, even when an agent which acts selectively on a specified voltage-dependent potassium channel is found, there is a possibility that the agent acts on many tissues and thereby induces originally unexpected agent effects. In order to find an agent having less side effects by targeting a potassium channel, it is necessary to clone a potassium channel in which its expressing tissue is restricted.

An object of the present invention is to provide a novel potassium channel protein expressed exclusively in the brain, as a target of therapeutic agents for central nervous system disorders such as dementia, cerebral ischemic disorder, epilepsy and the like, and another object of the present invention is to provide a method for screening compounds and peptides capable of modifying activity of the same potassium channel protein, which are useful as therapeutic agents for central nervous system disorders, and a novel agent for use in the treatment of central nervous system disorders, which specifically acts upon the central nervous system and generates less side effects.

With the aim of solving the aforementioned problems, the present inventors have conducted intensive studies and, as a result, succeeded in isolating a gene coding for a novel potassium channel protein expressed exclusively in the brain. The inventors have also succeeded in expressing the novel potassium channel protein expressed exclusively in the brain and establishing a method for the screening of compounds and peptides capable of modifying activity of the same potassium channel protein.

The present invention relates to:

1) a potassium channel protein or an equivalent thereof, which has an amino acid sequence selected from either of Sequence Nos. 2 and 6, or an amino acid sequence resulting from said amino acid sequence by substitution, deletion or insertion of certain amino acid(s), and is expressed exclusively in the brain,

2) the potassium channel protein or an equivalent thereof according to the item 1), which is expressed exclusively in the human brain,

3) a potassium channel protein which has an amino acid sequence selected from either of Sequence Nos. 2 and 6,

4) a gene which has a gene sequence encoding the potassium channel protein or an equivalent thereof described in the items 1) to 3),

5) a gene which has a gene sequence encoding an amino acid sequence selected from either of Sequence Nos. 2 and 6,

6) a gene which has a gene sequence selected from either of the 6th to 3257th gene sequence of Sequence No. 1 or the 4th to 3057th gene sequence of Sequence No. 5, or a gene which is degenerate with respect to said gene,

7) a vector which contains the gene of the items 4) to 6),

8) a host cell which contains the vector of the item 7), or

9) a method for producing the potassium channel protein described in the items 1) to 3), which uses the host cell of the item 8).

The terms to be used in the present invention are explained in the following.

The term “substitution, deletion or insertion of amino acid” means that one or a plurality of amino acids are substituted, deleted or inserted in the amino acid sequence selected from either of the Sequence Nos. 2 and 6.

The term “expressed exclusively in the brain” means that it is expressed in the brain but not expressed in the heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestines, large intestine and peripheral blood leukocytes, illustratively, it means that when Northern blotting is carried out under the conditions of Examples, the signal is detected only in the brain and not detected in the heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestines, large intestine and peripheral blood leukocytes.

Also, the term “equivalent” means a protein having a sequence in which one or a plurality of amino acids are substituted, deleted or inserted in the amino acid sequence of the protein which is expressed in the brain but not expressed in the heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestines, large intestine and peripheral blood leukocytes, but still having the same functions when compared with the protein without alteration of its amino acid sequence.

The term “human origin” means that it is the same amino acid sequence of a potassium channel protein expressed in human.

In this connection, the potassium channel and the potassium channel protein are used as synonyms.

The novel potassium channel protein of the present invention or an equivalent thereof may be any potassium channel protein or an equivalent thereof, with the proviso that it is expressed exclusively in the brain, but is preferably a human origin. Illustratively, a potassium channel protein or an equivalent thereof, which has an amino acid sequence selected from either of Sequence Nos. 2 and 6, or has an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence selected from either of Sequence Nos. 2 and 6 are substituted, deleted or inserted, and is expressed exclusively in the brain, is included in the present invention and is preferably a human origin. More preferred is a potassium channel protein which has an amino acid sequence selected from either of Sequence Nos. 2 and 6.

The gene which has a gene sequence encoding the novel potassium channel protein of the present invention or an equivalent thereof may be any gene which has a gene sequence encoding a potassium channel protein or an equivalent thereof expressed exclusively in the brain, but is preferably a gene which epcodes a potassium channel protein of human origin. Illustratively, a gene which encodes a potassium channel protein or an equivalent thereof having an amino acid sequence selected from either of Sequence Nos. 2 and 6, or a gene which encodes a potassium channel protein or an equivalent thereof having an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence selected from either of Sequence Nos. 2 and 6 are substituted, deleted or inserted, and is expressed exclusively in the brain, is included in the present invention, and the gene is preferably a gene which encodes a human origin potassium channel protein or an equivalent thereof. More preferred is a gene which encodes an amino acid sequence selected from either of Sequence Nos. 2 and 6. Most preferred is a gene which has a gene sequence selected from either of the 6th to 3257th gene sequence of Sequence No. 1 or the 4th to 3057th gene sequence of Sequence No. 5. Also included in the present invention is a gene which hybridizes with the gene of Sequence No. 1 or 5 under a stringent condition.

Hybridization can be carried out in accordance with a known method (Maniatis, T. et al. (1982): “Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory, NY). The term “stringent condition” means a condition that a sample after hybridization is washed twice in 2×SSC containing 0.1% SDS and then subjected to the following washing step.

This washing step means that the washing (65° C.) is carried out in 0.5×SSC containing 0.1% SDS, preferably in 0.2×SSC containing 0.1% SDS, most preferably in 0.1×SSC containing 0.1% SDS. A gene which encodes the novel potassium channel protein of the present invention can be obtained by the following methods.

1) Method for Producing Novel Potassium Channel Gene

a) First Production Method

mRNA is extracted from human cells or tissue capable of producing a novel potassium channel protein. Two primers between which the channel protein mRNA or a part of the mRNA region is located are used with the thus extracted mRNA as a template. By carrying out a reverse transcriptase-polymerase chain reaction (to be referred to as RT-PCR hereinafter), the channel protein cDNA or a part thereof can be obtained. Thereafter, the channel protein can be produced by integrating the thus obtained novel potassium channel cDNA or a part of the same into an appropriate expression vector to carry out its expression in a host cell.

Firstly, from human cells or tissue capable of producing the novel potassium channel protein of the present invention, such as human cerebral cortex, mRNA containing that encoding the protein is extracted by a known method. Guanidine thiocyanate-hot phenol method, guanidine thiocyanate-guanidine hydrochloride method and the like can be exemplified as the extraction method, but guanidine thiocyanate-cesium chloride method can be cited as a preferred method. The cells or tissue capable of producing the protein can be identified, for example, by a western blotting method which uses an antibody specific for the protein.

Purification of the mRNA can be made in accordance with a usual method, for example, the mRNA can be purified by the adsorption and elution using an oligo(dT)-cellulose column. The mRNA can be further fractionated by a sucrose density gradient centrifugation or the like.

Alternatively, a commercially available mRNA extracted sample may be used without carrying out the extraction of the mRNA.

Next, single-stranded cDNA is synthesized by carrying out reverse transcriptase reaction of the thus purified mRNA in the presence of a random primer or an oligo dT primer. This synthesis can be carried out in the usual way. The thus obtained single-stranded cDNA is subjected to PCR using two primers between which a partial region of the gene of interest is located, thereby amplifying the novel potassium channel DNA of interest. The thus obtained DNA is fractionated by an agarose gel electrophoresis or the like. As occasion demands, a DNA fragment of interest can be obtained by carrying out digestion of the DNA with restriction enzymes and subsequent ligation.

b) Second Production Method

In addition to the above production method, the gene of the present invention can also be produced using conventional genetic engineering techniques. Firstly, single-stranded cDNA is synthesized using reverse transcriptase, making use of the mRNA obtained by the aforementioned method as a template, and then double-stranded cDNA is synthesized from the single-stranded cDNA. Examples of this method include S1 nuclease method (Efstratiadis, A. et al. (1976), Cell, 7, 279-288), Land method (Land, H. et al. (1981), Nucleic Acids Res., 9, 2251-2266), O. Joon Yoo method (Yoo, O. J. et al. (1983), Proc. Natl. Acad. Sci. USA, 79, 1049-1053) and Okayama-Berg method (Okayama, H. and Berg, P. (1982), Mol. Cell. Biol., 2, 161-170).

Next, the recombinant plasmid obtained by the above method is introduced into an Escherichia coli strain, such as DH 5α, to effect its transformation, and then a transformant can be selected making use of tetracycline resistance or ampicillin resistance as a marker. When the host cell is E. coli , transformation of the host cell can be carried out, for example, by the method of Hanahan (Hanahan, D. (1983), J. Mol. Biol., 166, 557-580), namely a method in which the recombinant DNA is added to competent cells prepared in the presence of CaCl 2 and MgCl 2 or RbCl. In this connection, phage vectors such as a lambda system or the like can also be used as the vector in addition to a plasmid.

Regarding the method for selecting a strain containing DNA which encodes the novel potassium channel protein of interest from the transformants obtained above, various methods such as those shown below can be employed.

(i) A screening method which uses a synthetic oligonucleotide probe

An oligonucleotide which corresponds to the entire portion or a part of the novel potassium channel protein is synthesized (in this case, it may be either a nucleotide sequence taking the codon usage into consideration or a plurality of nucleotide sequences as a combination of possible nucleotide sequences, and in the latter case, their numbers can be reduced by including inosine) and, using this as a probe (labeled with 32 P or 33 P), hybridized with transformant DNA samples denatured and fixed on a nitrocellulose filter and the resulting positive strains are screened and selected.

(ii) A screening method which uses a probe prepared by polymerase chain reaction

Oligonucleotides of sense primer and antisense primer corresponding to a part of the novel potassium channel protein are synthesized, and a DNA fragment which encodes the entire portion or a part of the novel potassium channel protein of interest is amplified by carrying out polymerase chain reaction (Saiki, R. K. et al. (1988), Science, 239, 487-491) using these primers in combination. As the template DNA to be used herein, cDNA synthesized by reverse transcription reaction from mRNA of cells capable of producing the novel potassium channel protein, or genomic DNA, can be used. The thus prepared DNA fragment is labeled with 32 P or 33 P, and the clone of interest is selected by carrying out colony hybridization or plaque hybridization using this fragment as a probe.

(iii) A method in which screening is carried out by producing the novel potassium channel protein in other animal cells

Genes are amplified by culturing the transformants, and transfection of animal cells with the genes is carried out (in this case, either a plasmid which can replicate by itself and contains a transcription promoter region or a plasmid which can be integrated into the chromosome of animal cells may be used), thereby effecting production of proteins encoded by the genes on the cell surface. A strain containing cDNA which encodes the novel potassium channel protein of interest is selected by detecting the protein using an antibody for the novel potassium channel protein, or from the original transformants using the channel activity as a marker.

(iv) A method in which the selection is carried out using channel activity for the novel potassium channel protein as a marker

cDNA is integrated into an expression vector in advance, proteins are produced on the cell surface of transformants, and the strain of interest is selected by detecting desired novel potassium channel protein producing strains using the channel activity as a marker.

(v) A method in which the selection is carried out using an antibody for the novel potassium channel protein

cDNA is integrated into an expression vector in advance, proteins are produced on the cell surface of transformants, and the strain of interest is selected by detecting desired novel potassium channel protein producing strains using an antibody for the novel potassium channel protein and a second antibody for the first antibody.

(vi) A method which uses a selective hybridization translation system

cDNA obtained from each transformant is blotted for example on a nitrocellulose filter and hybridized with mRNA prepared from the novel potassium channel protein producing cells, and then the mRNA bonded to the cDNA is dissociated and recovered. The thus recovered mRNA is translated into protein in a protein translation system such as injection into Xenopus oocyte or a cell-free system such as a rabbit reticulocyte lysate, wheat germ or the like. A strain of interest is selected by the detection using an antibody for the novel potassium channel protein.

The method for collecting DNA which encodes the novel potassium channel protein from the thus obtained transformant of interest can be carried out in accordance with a known method (Maniatis, T. et al. (1982): “Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory, NY). For example, it can be made by separating a fraction corresponding to the plasmid DNA from cells and cutting out the cDNA region from the plasmid DNA.

The method for collecting DNA which encodes the novel potassium channel protein from the thus obtained transformant of interest can be carried out in accordance with a known method (Maniatis, T. et al. (1982): “Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory, NY). For example, it can be made by separating a fraction corresponding to the plasmid DNA from cells and cutting out the cDNA region from the plasmid DNA.

c) Third Production Method

The DNA having a nucleotide sequence which encodes the amino acid sequence selected from either of Sequence Nos. 2 and 6 can also be produced by binding a gene fragment produced by-a chemical synthesis method. Each DNA can be synthesized using a DNA synthesizer (e.g., Oligo 1000M DNA Synthesizer (Beckman) or 394 DNA/RNA Synthesizer (Applied Biosystems)).

d) Fourth Production Method

In order to effect expression of the novel potassium channel protein function expressed exclusively in the brain by the substance obtained by genetic engineering techniques making use of the DNA of the present invention, it is not always necessary to have entire portion of the amino acid sequence selected from either of Sequence Nos. 2 and 6; for example, when a part of the sequence shows functions of the novel potassium channel protein expressed exclusively in the brain, such an amino acid sequence is also included in the potassium channel protein of the present invention. Also, as is known in the interferon gene and the like, it is considered that genes of eucaryote generally show polymorphism (e.g., see Nishi, T. et al. (1985), J. Biochem., 97, 153-159), so that there will be a case in which one or a plurality of amino acids are substituted due to the polymorphism or a case in which the nucleotide sequence is changed but the amino acids are not changed. In consequence, even in the case of a protein in which one or a plurality of amino acid residues are substituted, deleted or inserted at one or a plural positions in the amino acid sequence selected from either of Sequence Nos. 2 and 6, it is possible that it has the channel activity and is expressed exclusively in the brain. In the present invention, such a protein is called an equivalent of the novel potassium channel protein.

All genes which encode such equivalents of the novel potassium channel protein and have equivalent nucleotide sequences are included in the present invention. These various types of DNA of the present invention can also be produced by nucleic acid chemical synthesis in accordance with a usual method such as phosphite triester method (Hunkapiller, M. et al. (1984), Nature, 10, 105-111), based on the information on the aforementioned novel potassium channel protein. In this connection, codons for each amino acid are well known and can be optionally selected and determined in the usual way, for example by taking codon usage of each host to be used into consideration (Crantham, R. et al. (1981), Nucleic Acids Res., 9, r43-r74). In addition, partial modification of codons of these nucleotide sequences can be carried out in accordance with a usual method such as site specific mutagenesis which uses a primer comprised of a synthetic oligonucleotide coding for a desired modification (Mark, D. F. et al. (1984), Proc. Natl. Acad. Sci. USA, 81, 5662-5666).

Determination of the DNA sequences obtained by the above methods a) to d) can be carried out by, for example, the Maxam-Gilbert chemical modification method (Maxam, A. M. and Gilbert, W. (1980): “Methods in Enzymology” 65, 499-559) or the dideoxynucleotide chain termination method which uses M13 (Messing, J. and Vieira, J (1982), Gene, 19, 269-276).

Also, the vector of the present invention, the host cell of the present invention and the potassium channel protein of the present invention can be obtained by the following methods.

2) Method for Producing Recombinant Protein of the Novel Potassium Channel

An isolated fragment containing a gene coding for the novel potassium channel protein can transform a host cell of other eucaryote by re-integrating it into an appropriate vector DNA. Also, it is possible to effect expression of the gene in respective host cell, by introducing an appropriate promoter and a sequence related to the gene expression into the vector.

Cells of vertebrates, insects, yeast and the like are included in the eucaryotic host cells, and COS cell as a simian cell (Gluzman, Y. (1981), Cell, 23, 175-182), a dihydrofolate reductase defective strain of Chinese hamster ovary cell (CHO) (Urlaub, G. and Chasin, L. A. (1980), Proc. Natl. Acad. Sci. USA, 77, 4216-4220) and the like are frequently used as the vertebral cells, though not limited thereto.

The expression vector which can be used in vertebral cells generally has a promoter positioned at the upstream of the gene to be expressed, an RNA splicing region, a polyadenylation region, a transcription termination sequence and the like, and it may further have a replication origin as occasion demands. Examples of the expression vector include pSV2dhfr which has SV40 early promoter (Subramani, S. et al. (1981), Mol. Cell. Biol., 1, 854-864), though not limited thereto.

When COS cell is used as the host cell, a vector which has the SV40 replication origin, can perform autonomous replication and has a transcription promoter, a transcription termination signal and an RNA splicing region can be used as the expression vector, and its examples include pME18S (Maruyama, K. and Takebe, Y. (1990), Med. Immunol., 20, 27-32), pEF-BOS (Mizushima, S. and Nagata, S. (1990), Nucleic Acids Res., 18, 5322), pCDM8 (Seed, B. (1987), Nature, 329, 840-842) and the like. The expression vector can be incorporated into COS cell, for example, by DEAE-dextran method (Luthman, H. and Magnusson, G. (1983), Nucleic Acids Res., 11, 1295-1308), calcium phosphate-DNA co-precipitation method (Graham, F. L. and van der Ed, A. J. (1973), Virology, 52, 456-457) or electroporation (Neumann, E. et al. (1982), EMBO J., 1, 841-845), thereby obtaining a desired transformant cell. Also, when CHO cell is used as the host cell, a transformant cell capable of stably producing the novel potassium channel protein can be obtained by carrying out co-transfection of a expression vector together with a vector capable of expressing neo gene which functions as a G418 resistance marker, such as pRSVneo (Sambrook, J. et al. (1989): “Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory, NY) or pSV2-neo (Southern, P. J. and Berg, P. (1982), J. Mol. Appl. Genet., 1, 327-341) or the like, and selecting G418 resistant colonies.

The thus obtained transformant of interest can be cultured in the usual way, and the novel potassium channel protein is produced inside the cells or on the cell surface. Regarding the medium to be used in the culturing, it can be optionally selected from various commonly used types depending on the host cell used, and in the case of the aforementioned COS cell for example, a medium such as RPMI-1640 medium, Dulbecco's modified Eagle's minimum essential medium (DMEM) or the like can be used by supplementing it with serum component such as fetal bovine serum (FBS) or the like as occasion demands.

The novel potassium channel protein thus produced in the cells or on the cell surface of the transformant can be separated and purified therefrom by various known separation techniques making use of the physical properties, chemical properties and the like of the channel protein. Illustrative examples of such techniques, to be carried out after solubilization of the channel protein-containing membrane fraction, include usual treatment with a protein precipitant, ultrafiltration, various liquid chromatography techniques such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and the like, dialysis and combinations thereof. In this connection, the membrane fraction can be obtained in the usual way. For example, it can be obtained by culturing the cells which expressed the novel potassium channel protein on the cell surface, suspending them in a buffer and then homogenizing and centrifuging them. Also, when the channel protein is solubilized using a solubilizing agent as mild as possible (CHAPS, Triton X-100, digitonin or the like), characteristics of the channel can be maintained after the solubilization.

A method for the screening of compounds and peptides capable of modifying activities of the potassium channel protein is included in the present invention. This screening method includes a means of adding an agent to be tested to the system and measuring the index, in which an index of the modification of potassium channel protein in response to a physiological characteristic of the potassium channel protein is measured making use of the thus constructed potassium channel protein which is expressed exclusively in the brain. The following screening methods can be cited as illustrative examples of this measuring system. Also, examples of the agents to be tested include compounds or peptides which are conventionally known to have potassium channel ligand activities but their ability to selectively modify activities of the potassium channel protein expressed exclusively in the brain is not clear (e.g., compounds described in JP-A-4-178375; the term “JP-A” as used herein means an “unexamined published Japanese patent application”), various known compounds and peptides registered in chemical files but their potassium channel ligand activities are unknown, compounds obtained by combinatorial chemistry techniques and random peptides prepared by employing a method such as phage display and the like. In addition, culture supernatants of microorganisms and natural components originated from plants and marine organisms are also objects of the screening. Also useful are compounds or peptides obtained by chemically or biologically modifying a compound or peptide selected by the screening method of the present invention.

3) Method for the Screening of Compounds and Peptides Capable of Modifying Activities of the Novel Potassium Channel Protein

a) Screening Method to Which Voltage-clamp Method is Applied

Channel activities of the novel potassium channel protein can be measured by the whole-cell voltage-clamp method. Cells in which the channel protein is expressed are subjected to membrane potential fixation by the whole-cell voltage-clamp method and whole-cell current is measured. A solution containing 145 mM NaCl, 5.4 mM KCl, 2 mM CaCl 2 and 0.8 mM MgCl 2 is used as the extracellular liquid, and a solution containing 155 mM KCl is used as the intracellular liquid (patch electrode solution). Compounds and peptides capable of modifying activities of the novel potassium channel protein can be screened by comparing outward currents generated by a depolarization stimulus, namely shifting of membrane potential from the holding potential (e.g., −70 mV) to the depolarization side (e.g., −80 mV), in the presence and absence of an agent to be tested.

b) Screening Method Utilizing Rb + ion release In general, the potassium channel can pass Rb + ion similar to K + ion, so that its channel activity can be measured using radioactive isotope 86 Rb + release as an index. By carrying out incubation of novel potassium channel protein-expressed cells together with 86 RbCl (e.g., 18 hr at 37° C.), 86 Rb + can be incorporated into the cells. The cells are washed with low K + concentration physiological saline (e.g., 4.5 mM K + ) and then suspended in the same solution. When a high K + concentration solution (e.g., 100 mM in final concentration) is added to the cell suspension, membrane potential of the cells is depolarized and the potassium channel is activated. Since 86 Rb + inside the cells is thereby released into the extracellular moiety, the radioactivity in the extracellular liquid can be used as an index of the channel activity. Compounds and peptides capable of modifying activity of the novel potassium channel protein can be screened by comparing radioactivity released into the extracellular moiety when the high K + concentration solution is added, in the presence and absence of an agent to be tested.

c) Screening Method Which Uses Voltage-sensitive Dye or Intracellular K + Detecting Dye

A voltage-sensitive dye or a intracellular K + detecting dye can optically detect changes in the membrane potential or intracellular K + concentration accompanied by the opening of the potassium channel. As the voltage-sensitive dye, RH155, WW781, Di-4-ANEPPS or derivatives thereof can be used. Also, a chimeric protein prepared by inserting the amino acid sequence of green fluorescent protein into the C-terminal intracellular region of Shaker type voltage-dependent potassium channel can be used for the detection of membrane potential (Siegel, M. S. and Isacoff, E. Y. (1997), Neuron, 19, 735-741). As the intracellular K + detecting dye, K + -binding benzofuran isophthalate and the like can be used. Since channel activity of the novel potassium channel can be measured by the use of these dyes, compounds and peptides capable of modifying activity of the novel potassium channel protein can be screened by comparing the changing amounts in the presence and absence of an agent to be tested.

A medicament which contains a compound or peptide capable of significantly modifying the activity of potassium channel protein exclusively expressed in the brain, selected by the aforementioned screening as an active ingredient, is included in the present invention.

The medicament of the present invention is characterized in that it has novel pharmacological action to selectively control activities of potassium channel in the brain, and the use of the medicament is for diseases caused by abnormalities (e.g., acceleration, reduction, and denaturation) of potassium channel activities in the brain or those which express and complicate such abnormalities, wherein their illustrative examples include central nervous system disorders such as dementia, cerebral ischemic disorder, epilepsy and the like.

The pharmaceutical preparation which contains a compound or peptide capable of modifying activity of the potassium channel protein of the present invention, as an active ingredient, can be prepared using carriers, fillers and other additives generally used in the preparation of medicaments, in response to each type of the active ingredient.

Examples of its administration include oral administration by tablets, pills, capsules, granules, fine granules, powders, oral solutions and the like, and parenteral administration by injections (e.g., intravenous, intramuscular or the like), suppositories, transdermal preparations, transmucosal absorption preparations and the like. Particularly, in the case of peptides which are digested by gastric acid, parenteral administration such as intravenous injection or the like, or lower gastrointestinal delivery administration is desirable.

In the solid composition for use in the oral administration according to the present invention, one or more active substances are mixed with at least one inert diluent such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinyl pyrrolidone or aluminum magnesium silicate. In the usual way, the composition may contain other additives than the inert diluent, such as a lubricant, a disintegrating agent, a stabilizing agent and a solubilizing or solubilization assisting agent. If necessary, tablets or pills may be coated with a sugar coating or a film of a gastric or enteric substance.

The liquid composition for oral administration includes emulsions, solutions, suspensions, syrups and elixirs and contains a generally used inert diluent such as purified water or ethyl alcohol. In addition to the inert diluent, this composition may also contain other additives such as moistening agents, suspending agents, sweeteners, flavors and antiseptics.

The injections for parenteral administration includes aseptic aqueous or non-aqueous solutions, suspensions and emulsions. Examples of the diluent for use in the aqueous solutions and suspensions include distilled water for injection use and physiological saline. Examples of the diluent for use in the non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, plant oil (e.g., olive oil or the like), alcohols (e.g., ethanol or the like), polysorbate 80 and the like. Such a composition may further contain a moistening agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a solubilizing or solubilization assisting agent, an antiseptic and the like. These compositions are sterilized for example by filtration through a bacteria retaining filter, blending of a germicide or irradiation. Alternatively, they may be used by firstly making into sterile solid compositions and dissolving them in sterile water or other sterile solvent for injection use prior to their use.

The dose is optionally decided by taking into consideration strength of each active ingredient selected by the aforementioned screening method and symptoms, age, sex and the like of each patient to be administered.

FIG. 1 shows amino acid sequences of Sequence No. 2 (upper line) and Sequence No. 6 (lower line) potassium channels.

FIG. 2 shows results of Northern analysis on the novel potassium channel in each human organ. A and B represent results on the potassium channel of Sequence No. 2, and C and D on the potassium channel of Sequence No. 6.

FIG. 3 shows results of Northern analysis on the novel potassium channel in each region of the human brain. A and B represent results on the potassium channel of Sequence No. 2, and C and D on the potassium channel of Sequence No. 6.

In FIG. 4, A and B show results of in situ hybridization regarding potassium channel (Sequence No. 2) (DG, the granule cell layer of the dentate gyrus; CA1 and CA3, the pyramidal cell layer of the CA1 and CA3 fields of Ammon's horn; Cx, cerebral cortex). C and D represent enlarged images of cerebral cortex (arrows indicate signals in typical pyramidal cells). An antisense probe (in A and C) or a sense probe (in B and D) was used in the hybridization. The scale bar indicates 1.5 mm (A and B) or 50 μm (B and D).

FIG. 5 shows results of the detection of the channel activity of potassium channel (Sequence No. 2) by depolarization stimulus.

FIG. 6 shows results of the detection of the channel activity of potassium channel (Sequence No. 6) by depolarization stimulus.

FIG. 7 shows tail current of the potassium channel shown in Sequence No. 2.

FIG. 8 shows current response of the potassium channel shown in Sequence No. 6 in high potassium extracellular liquid.

In order to disclose the present invention more illustratively, examples are described in the following, but the present invention is not limited to the examples. In this connection, unless otherwise noted, they can be carried out in accordance with known methods (Maniatis, T. et al. (1982): “Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory, NY).

A novel potassium channel protein expressed exclusively in the brain, a gene encoding this protein, a vector containing this gene, a host cell containing this vector and a method for producing this potassium channel protein were provided by the present invention.

Also, there was provided a method for the screening of new medicaments, particularly new therapeutic agents for central nervous system disorders, in which compounds and peptides capable of modifying activity of the potassium channel protein are screened by allowing the potassium channel protein of the present invention to contact with agents to be tested.

For example, among tissues in which the potassium channel of the present invention shown in Sequence No. 2 was expressed as the result of Example 3, hippocampus is a region where its relationship with memory and learning is strongly suggested (Lavitan, I. B. and Kaczmarek, L. K. (1991), The Neuron: Cell and Molecular Biology , Oxford University Press, New York, N.Y.). Particularly, granule cells of the dentate gyrus and pyramidal cells of the CA1 and CA3 fields in which expression of the potassium channel was confirmed form a neural circuit, and various memory inputs are transmitted from the granule cells of the dentate gyrus to pyramidal cells of the CA3 field via pyramidal cells of the CA1 field, mediated by an excitatory synapse which uses glutamic acid as a neurotransmitter. It is considered that long-term changes in the efficiency of synaptic transmission observed in respective synapse, such as long-term increase and long-term repression, are deeply concerned in memory and learning. These long-term changes are controlled by the excitation frequency and excitation strength of nerve cells, and voltage-dependent potassium channels generally have a possibility of being able to control excitability of neurons, so that the potassium channel protein of the present invention which is a voltage-dependent potassium channel has a possibility that it is concerned in the formation of memory and learning via the excitability control of neurons.

Regarding the medicament which contains a compound or peptide capable of-specifically modifying activity of the potassium channel protein of the present invention as its active ingredient, its usefulness is expected for example as a therapeutic agent for central nervous system disorders such as dementia, cerebral ischemic disorders and epilepsy, which acts central nervous system-specifically and has less side effects.

1

ctaag atg ccg gcc atg cgg ggc ctc ctg gcg cct cag aac acc ttc ctg 50

Met Pro Ala Met Arg Gly Leu Leu Ala Pro Gln Asn Thr Phe Leu

1 5 10 15

gac acc atc gct acg cgc ttc gac ggc acg cac agt aac ttc gtg ctg 98

Asp Thr Ile Ala Thr Arg Phe Asp Gly Thr His Ser Asn Phe Val Leu

20 25 30

ggc aac gcc cag gtg gcg ggg ctc ttc ccc gtg gtc tac tgc tct gat 146

Gly Asn Ala Gln Val Ala Gly Leu Phe Pro Val Val Tyr Cys Ser Asp

35 40 45

ggc ttc tgt gac ctc acg ggc ttc tcc cgg gct gag gtc atg cag cgg 194

Gly Phe Cys Asp Leu Thr Gly Phe Ser Arg Ala Glu Val Met Gln Arg

50 55 60

ggc tgt gcc tgc tcc ttc ctt tat ggg cca gac acc agt gag ctc gtc 242

Gly Cys Ala Cys Ser Phe Leu Tyr Gly Pro Asp Thr Ser Glu Leu Val

65 70 75

cgc caa cag atc cgc aag gcc ctg gac gag cac aag gag ttc aag gct 290

Arg Gln Gln Ile Arg Lys Ala Leu Asp Glu His Lys Glu Phe Lys Ala

80 85 90 95

gag ctg atc ctg tac cgg aag agc ggg ctc ccg ttc tgg tgt ctc ctg 338

Glu Leu Ile Leu Tyr Arg Lys Ser Gly Leu Pro Phe Trp Cys Leu Leu

100 105 110

gat gtg ata ccc ata aag aat gag aaa ggg gag gtg gct ctc ttc cta 386

Asp Val Ile Pro Ile Lys Asn Glu Lys Gly Glu Val Ala Leu Phe Leu

115 120 125

gtc tct cac aag gac atc agc gaa acc aag aac cga ggg ggc ccc gac 434

Val Ser His Lys Asp Ile Ser Glu Thr Lys Asn Arg Gly Gly Pro Asp

130 135 140

aga tgg aag gag aca ggt ggt ggc cgg cgc cga tat ggc cgg gca cga 482

Arg Trp Lys Glu Thr Gly Gly Gly Arg Arg Arg Tyr Gly Arg Ala Arg

145 150 155

tcc aaa ggc ttc aat gcc aac cgg cgg cgg agc cgg gcc gtg ctc tac 530

Ser Lys Gly Phe Asn Ala Asn Arg Arg Arg Ser Arg Ala Val Leu Tyr

160 165 170 175

cac ctg tcc ggg cac ctg cag aag cag ccc aag ggc aag cac aag ctc 578

His Leu Ser Gly His Leu Gln Lys Gln Pro Lys Gly Lys His Lys Leu

180 185 190

aat aag ggg gtg ttt ggg gag aaa cca aac ttg cct gag tac aaa gta 626

Asn Lys Gly Val Phe Gly Glu Lys Pro Asn Leu Pro Glu Tyr Lys Val

195 200 205

gcc gcc atc cgg aag tcg ccc ttc atc ctg ttg cac tgt ggg gca ctg 674

Ala Ala Ile Arg Lys Ser Pro Phe Ile Leu Leu His Cys Gly Ala Leu

210 215 220

aga gcc acc tgg gat ggc ttc atc ctg ctc gcc aca ctc tat gtg gct 722

Arg Ala Thr Trp Asp Gly Phe Ile Leu Leu Ala Thr Leu Tyr Val Ala

225 230 235

gtc act gtg ccc tac agc gtg tgt gtg agc aca gca cgg gag ccc agt 770

Val Thr Val Pro Tyr Ser Val Cys Val Ser Thr Ala Arg Glu Pro Ser

240 245 250 255

gcc gcc cgc ggc ccg ccc agc gtc tgt gac ctg gcc gtg gag gtc ctc 818

Ala Ala Arg Gly Pro Pro Ser Val Cys Asp Leu Ala Val Glu Val Leu

260 265 270

ttc atc ctt gac att gtg ctg aat ttc cgt acc aca ttc gtg tcc aag 866

Phe Ile Leu Asp Ile Val Leu Asn Phe Arg Thr Thr Phe Val Ser Lys

275 280 285

tcg ggc cag gtg gtg ttt gcc cca aag tcc att tgc ctc cac tac gtc 914

Ser Gly Gln Val Val Phe Ala Pro Lys Ser Ile Cys Leu His Tyr Val

290 295 300

acc acc tgg ttc ctg ctg gat gtc atc gca gcg ctg ccc ttt gac ctg 962

Thr Thr Trp Phe Leu Leu Asp Val Ile Ala Ala Leu Pro Phe Asp Leu

305 310 315

cta cat gcc ttc aag gtc aac gtg tac ttc ggg gcc cat ctg ctg aag 1010

Leu His Ala Phe Lys Val Asn Val Tyr Phe Gly Ala His Leu Leu Lys

320 325 330 335

acg gtg cgc ctg ctg cgc ctg ctg cgc ctg ctt ccg cgg ctg gac cgg 1058

Thr Val Arg Leu Leu Arg Leu Leu Arg Leu Leu Pro Arg Leu Asp Arg

340 345 350

tac tcg cag tac agc gcc gtg gtg ctg aca ctg ctc atg gcc gtg ttc 1106

Tyr Ser Gln Tyr Ser Ala Val Val Leu Thr Leu Leu Met Ala Val Phe

355 360 365

gcc ctg ctc gcg cac tgg gtc gcc tgc gtc tgg ttt tac att ggc cag 1154

Ala Leu Leu Ala His Trp Val Ala Cys Val Trp Phe Tyr Ile Gly Gln

370 375 380

cgg gag atc gag agc agc gaa tcc gag ctg cct gag att ggc tgg ctg 1202

Arg Glu Ile Glu Ser Ser Glu Ser Glu Leu Pro Glu Ile Gly Trp Leu

385 390 395

cag gag ctg gcc cgc cga ctg gag act ccc tac tac ctg gtg ggc cgg 1250

Gln Glu Leu Ala Arg Arg Leu Glu Thr Pro Tyr Tyr Leu Val Gly Arg

400 405 410 415

agg cca gct gga ggg aac agc tcc ggc cag agt gac aac tgc agc agc 1298

Arg Pro Ala Gly Gly Asn Ser Ser Gly Gln Ser Asp Asn Cys Ser Ser

420 425 430

agc agc gag gcc aac ggg acg ggg ctg gag ctg ctg ggc ggc ccg tcg 1346

Ser Ser Glu Ala Asn Gly Thr Gly Leu Glu Leu Leu Gly Gly Pro Ser

435 440 445

ctg cgc agc gcc tac atc acc tcc ctc tac ttc gca ctc agc agc ctc 1394

Leu Arg Ser Ala Tyr Ile Thr Ser Leu Tyr Phe Ala Leu Ser Ser Leu

450 455 460

acc agc gtg ggc ttc ggc aac gtg tcc gcc aac acg gac acc gag aag 1442

Thr Ser Val Gly Phe Gly Asn Val Ser Ala Asn Thr Asp Thr Glu Lys

465 470 475

atc ttc tcc atc tgc acc atg ctc atc ggc gcc ctg atg cac gcg gtg 1490

Ile Phe Ser Ile Cys Thr Met Leu Ile Gly Ala Leu Met His Ala Val

480 485 490 495

gtg ttt ggg aac gtg acg gcc atc atc cag cgc atg tac gcc cgc cgc 1538

Val Phe Gly Asn Val Thr Ala Ile Ile Gln Arg Met Tyr Ala Arg Arg

500 505 510

ttt ctg tac cac agc cgc acg cgc gac ctg cgc gac tac atc cgc atc 1586

Phe Leu Tyr His Ser Arg Thr Arg Asp Leu Arg Asp Tyr Ile Arg Ile

515 520 525

cac cgt atc ccc aag ccc ctc aag cag cgc atg ctg gag tac ttc cag 1634

His Arg Ile Pro Lys Pro Leu Lys Gln Arg Met Leu Glu Tyr Phe Gln

530 535 540

gcc acc tgg gcg gtg aac aat ggc atc gac acc acc gag ctg ctg cag 1682

Ala Thr Trp Ala Val Asn Asn Gly Ile Asp Thr Thr Glu Leu Leu Gln

545 550 555

agc ctc cct gac gag ctg cgc gca gac atc gcc atg cac ctg cac aag 1730

Ser Leu Pro Asp Glu Leu Arg Ala Asp Ile Ala Met His Leu His Lys

560 565 570 575

gag gtc ctg cag ctg cca ctg ttt gag gcg gcc agc cgc ggc tgc ctg 1778

Glu Val Leu Gln Leu Pro Leu Phe Glu Ala Ala Ser Arg Gly Cys Leu

580 585 590

cgg gca ctg tct ctg gcc ctg cgg ccc gcc ttc tgc acg ccg ggc gag 1826

Arg Ala Leu Ser Leu Ala Leu Arg Pro Ala Phe Cys Thr Pro Gly Glu

595 600 605

tac ctc atc cac caa ggc gat gcc ctg cag gcc ctc tac ttt gtc tgc 1874

Tyr Leu Ile His Gln Gly Asp Ala Leu Gln Ala Leu Tyr Phe Val Cys

610 615 620

tct ggc tcc atg gag gtg ctc aag ggt ggc acc gtg ctc gcc atc cta 1922

Ser Gly Ser Met Glu Val Leu Lys Gly Gly Thr Val Leu Ala Ile Leu

625 630 635

ggg aag ggc gac ctg atc ggc tgt gag ctg ccc cgg cgg gag cag gtg 1970

Gly Lys Gly Asp Leu Ile Gly Cys Glu Leu Pro Arg Arg Glu Gln Val

640 645 650 655

gta aag gcc aat gcc gac gtg aag ggg ctg acg tac tgc gtc ctg cag 2018

Val Lys Ala Asn Ala Asp Val Lys Gly Leu Thr Tyr Cys Val Leu Gln

660 665 670

tgt ctg cag ctg gct ggc ctg cac gac agc ctt gcg ctg tac ccc gag 2066

Cys Leu Gln Leu Ala Gly Leu His Asp Ser Leu Ala Leu Tyr Pro Glu

675 680 685

ttt gcc ccg cgc ttc agt cgt ggc ctc cga ggg gag ctc agc tac aac 2114

Phe Ala Pro Arg Phe Ser Arg Gly Leu Arg Gly Glu Leu Ser Tyr Asn

690 695 700

ctg ggt gct ggg gga ggc tct gca gag gtg gac acc agc tcc ctg agc 2162

Leu Gly Ala Gly Gly Gly Ser Ala Glu Val Asp Thr Ser Ser Leu Ser

705 710 715

ggc gac aat acc ctt atg tcc acg ctg gag gag aag gag aca gat ggg 2210

Gly Asp Asn Thr Leu Met Ser Thr Leu Glu Glu Lys Glu Thr Asp Gly

720 725 730 735

gag cag ggc ccc acg gtc tcc cca gcc cca gct gat gag ccc tcc agc 2258

Glu Gln Gly Pro Thr Val Ser Pro Ala Pro Ala Asp Glu Pro Ser Ser

740 745 750

ccc ctg ctg tcc cct ggc tgc acc tcc tca tcc tca gct gcc aag ctg 2306

Pro Leu Leu Ser Pro Gly Cys Thr Ser Ser Ser Ser Ala Ala Lys Leu

755 760 765

cta tcc cca cgt cga aca gca ccc cgg cct cgt cta ggt ggc aga ggg 2354

Leu Ser Pro Arg Arg Thr Ala Pro Arg Pro Arg Leu Gly Gly Arg Gly

770 775 780

agg cca ggc agg gca ggg gct ttg aag gct gag gct ggc ccc tct gct 2402

Arg Pro Gly Arg Ala Gly Ala Leu Lys Ala Glu Ala Gly Pro Ser Ala

785 790 795

ccc cca cgg gcc cta gag ggg cta cgg ctg ccc ccc atg cca tgg aat 2450

Pro Pro Arg Ala Leu Glu Gly Leu Arg Leu Pro Pro Met Pro Trp Asn

800 805 810 815

gtg ccc cca gat ctg agc ccc agg gta gta gat ggc att gaa gac ggc 2498

Val Pro Pro Asp Leu Ser Pro Arg Val Val Asp Gly Ile Glu Asp Gly

820 825 830

tgt ggc tcg gac cag ccc aag ttc tct ttc cgc gtg ggc cag tct ggc 2546

Cys Gly Ser Asp Gln Pro Lys Phe Ser Phe Arg Val Gly Gln Ser Gly

835 840 845

ccg gaa tgt agc agc agc ccc tcc cct gga cca gag agc ggc ctg ctc 2594

Pro Glu Cys Ser Ser Ser Pro Ser Pro Gly Pro Glu Ser Gly Leu Leu

850 855 860

act gtt ccc cat ggg ccc agc gag gca agg aac aca gac aca ctg gac 2642

Thr Val Pro His Gly Pro Ser Glu Ala Arg Asn Thr Asp Thr Leu Asp

865 870 875

aag ctt cgg cag gcg gtg aca gag ctg tca gag cag gtg ctg cag atg 2690

Lys Leu Arg Gln Ala Val Thr Glu Leu Ser Glu Gln Val Leu Gln Met

880 885 890 895

cgg gaa gga ctg cag tca ctt cgc cag gct gtg cag ctt gtc ctg gcg 2738

Arg Glu Gly Leu Gln Ser Leu Arg Gln Ala Val Gln Leu Val Leu Ala

900 905 910

ccc cac agg gag ggt ccg tgc cct cgg gca tcg gga gag ggg ccg tgc 2786

Pro His Arg Glu Gly Pro Cys Pro Arg Ala Ser Gly Glu Gly Pro Cys

915 920 925

cca gcc agc acc tcc ggg ctt ctg cag cct ctg tgt gtg gac act ggg 2834

Pro Ala Ser Thr Ser Gly Leu Leu Gln Pro Leu Cys Val Asp Thr Gly

930 935 940

gca tcc tcc tac tgc ctg cag ccc cca gct ggc tct gtc ttg agt ggg 2882

Ala Ser Ser Tyr Cys Leu Gln Pro Pro Ala Gly Ser Val Leu Ser Gly

945 950 955

act tgg ccc cac cct cgt ccg ggg cct cct ccc ctc atg gca ccc tgg 2930

Thr Trp Pro His Pro Arg Pro Gly Pro Pro Pro Leu Met Ala Pro Trp

960 965 970 975

ccc tgg ggt ccc cca gcg tct cag agc tcc ccc tgg cct cga gcc aca 2978

Pro Trp Gly Pro Pro Ala Ser Gln Ser Ser Pro Trp Pro Arg Ala Thr

980 985 990

gct ttc tgg acc tcc acc tca gac tca gag ccc cct gcc tca gga gac 3026

Ala Phe Trp Thr Ser Thr Ser Asp Ser Glu Pro Pro Ala Ser Gly Asp

995 1000 1005

ctc tgc tct gag ccc agc acc cct gcc tcc cct cct cct tct gag gaa 3074

Leu Cys Ser Glu Pro Ser Thr Pro Ala Ser Pro Pro Pro Ser Glu Glu

1010 1015 1020

ggg gct agg act ggg ccc gca gag cct gtg agc cag gct gag gct acc 3122

Gly Ala Arg Thr Gly Pro Ala Glu Pro Val Ser Gln Ala Glu Ala Thr

1025 1030 1035

agc act gga gag ccc cca cca ggg tca ggg ggc ctg gcc ttg ccc tgg 3170

Ser Thr Gly Glu Pro Pro Pro Gly Ser Gly Gly Leu Ala Leu Pro Trp

1040 1045 1050 1055

gac ccc cac agc ctg gag atg gtg ctt att ggc tgc cat ggc tct ggc 3218

Asp Pro His Ser Leu Glu Met Val Leu Ile Gly Cys His Gly Ser Gly

1060 1065 1070

aca gtc cag tgg acc cag gaa gaa ggc aca ggg gtc tga gtaccagccc 3267

Thr Val Gln Trp Thr Gln Glu Glu Gly Thr Gly Val

1075 1080

tagaactcag cgttgccagg tgtgctgcca tctgctgttc ggcccaacct cagagt 3323

2

1083

Homo sapiens

2

Met Pro Ala Met Arg Gly Leu Leu Ala Pro Gln Asn Thr Phe Leu Asp

1 5 10 15

Thr Ile Ala Thr Arg Phe Asp Gly Thr His Ser Asn Phe Val Leu Gly

20 25 30

Asn Ala Gln Val Ala Gly Leu Phe Pro Val Val Tyr Cys Ser Asp Gly

35 40 45

Phe Cys Asp Leu Thr Gly Phe Ser Arg Ala Glu Val Met Gln Arg Gly

50 55 60

Cys Ala Cys Ser Phe Leu Tyr Gly Pro Asp Thr Ser Glu Leu Val Arg

65 70 75 80

Gln Gln Ile Arg Lys Ala Leu Asp Glu His Lys Glu Phe Lys Ala Glu

85 90 95

Leu Ile Leu Tyr Arg Lys Ser Gly Leu Pro Phe Trp Cys Leu Leu Asp

100 105 110

Val Ile Pro Ile Lys Asn Glu Lys Gly Glu Val Ala Leu Phe Leu Val

115 120 125

Ser His Lys Asp Ile Ser Glu Thr Lys Asn Arg Gly Gly Pro Asp Arg

130 135 140

Trp Lys Glu Thr Gly Gly Gly Arg Arg Arg Tyr Gly Arg Ala Arg Ser

145 150 155 160

Lys Gly Phe Asn Ala Asn Arg Arg Arg Ser Arg Ala Val Leu Tyr His

165 170 175

Leu Ser Gly His Leu Gln Lys Gln Pro Lys Gly Lys His Lys Leu Asn

180 185 190

Lys Gly Val Phe Gly Glu Lys Pro Asn Leu Pro Glu Tyr Lys Val Ala

195 200 205

Ala Ile Arg Lys Ser Pro Phe Ile Leu Leu His Cys Gly Ala Leu Arg

210 215 220

Ala Thr Trp Asp Gly Phe Ile Leu Leu Ala Thr Leu Tyr Val Ala Val

225 230 235 240

Thr Val Pro Tyr Ser Val Cys Val Ser Thr Ala Arg Glu Pro Ser Ala

245 250 255

Ala Arg Gly Pro Pro Ser Val Cys Asp Leu Ala Val Glu Val Leu Phe

260 265 270

Ile Leu Asp Ile Val Leu Asn Phe Arg Thr Thr Phe Val Ser Lys Ser

275 280 285

Gly Gln Val Val Phe Ala Pro Lys Ser Ile Cys Leu His Tyr Val Thr

290 295 300

Thr Trp Phe Leu Leu Asp Val Ile Ala Ala Leu Pro Phe Asp Leu Leu

305 310 315 320

His Ala Phe Lys Val Asn Val Tyr Phe Gly Ala His Leu Leu Lys Thr

325 330 335

Val Arg Leu Leu Arg Leu Leu Arg Leu Leu Pro Arg Leu Asp Arg Tyr

340 345 350

Ser Gln Tyr Ser Ala Val Val Leu Thr Leu Leu Met Ala Val Phe Ala

355 360 365

Leu Leu Ala His Trp Val Ala Cys Val Trp Phe Tyr Ile Gly Gln Arg

370 375 380

Glu Ile Glu Ser Ser Glu Ser Glu Leu Pro Glu Ile Gly Trp Leu Gln

385 390 395 400

Glu Leu Ala Arg Arg Leu Glu Thr Pro Tyr Tyr Leu Val Gly Arg Arg

405 410 415

Pro Ala Gly Gly Asn Ser Ser Gly Gln Ser Asp Asn Cys Ser Ser Ser

420 425 430

Ser Glu Ala Asn Gly Thr Gly Leu Glu Leu Leu Gly Gly Pro Ser Leu

435 440 445

Arg Ser Ala Tyr Ile Thr Ser Leu Tyr Phe Ala Leu Ser Ser Leu Thr

450 455 460

Ser Val Gly Phe Gly Asn Val Ser Ala Asn Thr Asp Thr Glu Lys Ile

465 470 475 480

Phe Ser Ile Cys Thr Met Leu Ile Gly Ala Leu Met His Ala Val Val

485 490 495

Phe Gly Asn Val Thr Ala Ile Ile Gln Arg Met Tyr Ala Arg Arg Phe

500 505 510

Leu Tyr His Ser Arg Thr Arg Asp Leu Arg Asp Tyr Ile Arg Ile His

515 520 525

Arg Ile Pro Lys Pro Leu Lys Gln Arg Met Leu Glu Tyr Phe Gln Ala

530 535 540

Thr Trp Ala Val Asn Asn Gly Ile Asp Thr Thr Glu Leu Leu Gln Ser

545 550 555 560

Leu Pro Asp Glu Leu Arg Ala Asp Ile Ala Met His Leu His Lys Glu

565 570 575

Val Leu Gln Leu Pro Leu Phe Glu Ala Ala Ser Arg Gly Cys Leu Arg

580 585 590

Ala Leu Ser Leu Ala Leu Arg Pro Ala Phe Cys Thr Pro Gly Glu Tyr

595 600 605

Leu Ile His Gln Gly Asp Ala Leu Gln Ala Leu Tyr Phe Val Cys Ser

610 615 620

Gly Ser Met Glu Val Leu Lys Gly Gly Thr Val Leu Ala Ile Leu Gly

625 630 635 640

Lys Gly Asp Leu Ile Gly Cys Glu Leu Pro Arg Arg Glu Gln Val Val

645 650 655

Lys Ala Asn Ala Asp Val Lys Gly Leu Thr Tyr Cys Val Leu Gln Cys

660 665 670

Leu Gln Leu Ala Gly Leu His Asp Ser Leu Ala Leu Tyr Pro Glu Phe

675 680 685

Ala Pro Arg Phe Ser Arg Gly Leu Arg Gly Glu Leu Ser Tyr Asn Leu

690 695 700

Gly Ala Gly Gly Gly Ser Ala Glu Val Asp Thr Ser Ser Leu Ser Gly

705 710 715 720

Asp Asn Thr Leu Met Ser Thr Leu Glu Glu Lys Glu Thr Asp Gly Glu

725 730 735

Gln Gly Pro Thr Val Ser Pro Ala Pro Ala Asp Glu Pro Ser Ser Pro

740 745 750

Leu Leu Ser Pro Gly Cys Thr Ser Ser Ser Ser Ala Ala Lys Leu Leu

755 760 765

Ser Pro Arg Arg Thr Ala Pro Arg Pro Arg Leu Gly Gly Arg Gly Arg

770 775 780

Pro Gly Arg Ala Gly Ala Leu Lys Ala Glu Ala Gly Pro Ser Ala Pro

785 790 795 800

Pro Arg Ala Leu Glu Gly Leu Arg Leu Pro Pro Met Pro Trp Asn Val

805 810 815

Pro Pro Asp Leu Ser Pro Arg Val Val Asp Gly Ile Glu Asp Gly Cys

820 825 830

Gly Ser Asp Gln Pro Lys Phe Ser Phe Arg Val Gly Gln Ser Gly Pro

835 840 845

Glu Cys Ser Ser Ser Pro Ser Pro Gly Pro Glu Ser Gly Leu Leu Thr

850 855 860

Val Pro His Gly Pro Ser Glu Ala Arg Asn Thr Asp Thr Leu Asp Lys

865 870 875 880

Leu Arg Gln Ala Val Thr Glu Leu Ser Glu Gln Val Leu Gln Met Arg

885 890 895

Glu Gly Leu Gln Ser Leu Arg Gln Ala Val Gln Leu Val Leu Ala Pro

900 905 910

His Arg Glu Gly Pro Cys Pro Arg Ala Ser Gly Glu Gly Pro Cys Pro

915 920 925

Ala Ser Thr Ser Gly Leu Leu Gln Pro Leu Cys Val Asp Thr Gly Ala

930 935 940

Ser Ser Tyr Cys Leu Gln Pro Pro Ala Gly Ser Val Leu Ser Gly Thr

945 950 955 960

Trp Pro His Pro Arg Pro Gly Pro Pro Pro Leu Met Ala Pro Trp Pro

965 970 975

Trp Gly Pro Pro Ala Ser Gln Ser Ser Pro Trp Pro Arg Ala Thr Ala

980 985 990

Phe Trp Thr Ser Thr Ser Asp Ser Glu Pro Pro Ala Ser Gly Asp Leu

995 1000 1005

Cys Ser Glu Pro Ser Thr Pro Ala Ser Pro Pro Pro Ser Glu Glu Gly

1010 1015 1020

Ala Arg Thr Gly Pro Ala Glu Pro Val Ser Gln Ala Glu Ala Thr Ser

1025 1030 1035 1040

Thr Gly Glu Pro Pro Pro Gly Ser Gly Gly Leu Ala Leu Pro Trp Asp

1045 1050 1055

Pro His Ser Leu Glu Met Val Leu Ile Gly Cys His Gly Ser Gly Thr

1060 1065 1070

Val Gln Trp Thr Gln Glu Glu Gly Thr Gly Val

1075 1080

3

26

Artificial Sequence

Description of Artificial SequencePrimer

3

ggaattccct aagatgccgg ccatgc 26

4

30

Artificial Sequence

Description of Artificial SequencePrimer

4

gctctagagc actctgaggt tgggccgaac 30

5

gcc atg ccg gtc atg aag ggg ttg ctg gcc ccg caa aac acc ttc ctg 48

Met Pro Val Met Lys Gly Leu Leu Ala Pro Gln Asn Thr Phe Leu

1 5 10 15

gac acc atc gcc acc cgt ttt gac gga acg cac agc aac ttc ctg ctg 96

Asp Thr Ile Ala Thr Arg Phe Asp Gly Thr His Ser Asn Phe Leu Leu

20 25 30

gcc aac gca cag ggc aca cgg ggc ttt ccc atc gtc tac tgc tcc gac 144

Ala Asn Ala Gln Gly Thr Arg Gly Phe Pro Ile Val Tyr Cys Ser Asp

35 40 45

ggc ttc tgc gag ctc aca ggc tac ggt cgc acc gag gtc atg cag aag 192

Gly Phe Cys Glu Leu Thr Gly Tyr Gly Arg Thr Glu Val Met Gln Lys

50 55 60

acc tgc agc tgc cgt ttc ctc tac ggc cca gag acc agt gag cca gcc 240

Thr Cys Ser Cys Arg Phe Leu Tyr Gly Pro Glu Thr Ser Glu Pro Ala

65 70 75

ctg cag cgt ctg cac aaa gcc ctg gag ggc cac cag gag cac cgg gct 288

Leu Gln Arg Leu His Lys Ala Leu Glu Gly His Gln Glu His Arg Ala

80 85 90 95

gaa atc tgc ttc tac cgc aag gat ggc tca gcc ttt tgg tgc ctc ctg 336

Glu Ile Cys Phe Tyr Arg Lys Asp Gly Ser Ala Phe Trp Cys Leu Leu

100 105 110

gac atg atg ccc atc aag aat gag atg ggg gag gtc gtg ctg ttc ctc 384

Asp Met Met Pro Ile Lys Asn Glu Met Gly Glu Val Val Leu Phe Leu

115 120 125

ttt tcc ttc aag gat atc act cag agt gga agc cca gga ctt ggc ccc 432

Phe Ser Phe Lys Asp Ile Thr Gln Ser Gly Ser Pro Gly Leu Gly Pro

130 135 140

caa gga ggc cgc ggg gac agt aat cac gaa aac tcc ctt ggt aga agg 480

Gln Gly Gly Arg Gly Asp Ser Asn His Glu Asn Ser Leu Gly Arg Arg

145 150 155

gga gcc acc tgg aaa ttt cgg tct gcc aga aga cgg agc cgt act gtc 528

Gly Ala Thr Trp Lys Phe Arg Ser Ala Arg Arg Arg Ser Arg Thr Val

160 165 170 175

cta cac cga ctg acc ggc cac ttt ggc cgc cgg ggc cag gga ggc atg 576

Leu His Arg Leu Thr Gly His Phe Gly Arg Arg Gly Gln Gly Gly Met

180 185 190

aag gcc aat aat aac gtg ttt gag cca aag cca tca gtg ccc gag tac 624

Lys Ala Asn Asn Asn Val Phe Glu Pro Lys Pro Ser Val Pro Glu Tyr

195 200 205

aag gtg gcc tcc gtg ggg ggg tct cgc tgc ctc ctc ctc cac tac agc 672

Lys Val Ala Ser Val Gly Gly Ser Arg Cys Leu Leu Leu His Tyr Ser

210 215 220

gtc tcc aag gcc atc tgg gac ggc ctt atc ctc ctt gcc acc ttc tac 720

Val Ser Lys Ala Ile Trp Asp Gly Leu Ile Leu Leu Ala Thr Phe Tyr

225 230 235

gtt gcg gtc acc gtc ccc tac aat gtc tgt ttc tcg ggt gac gat gac 768

Val Ala Val Thr Val Pro Tyr Asn Val Cys Phe Ser Gly Asp Asp Asp

240 245 250 255

acc ccc atc act tcg cga cac acc ctt gtc agc gac atc gcc gtg gaa 816

Thr Pro Ile Thr Ser Arg His Thr Leu Val Ser Asp Ile Ala Val Glu

260 265 270

atg ctc ttc atc cta gat atc atc ctg aac ttc cgc acc acc tat gtg 864

Met Leu Phe Ile Leu Asp Ile Ile Leu Asn Phe Arg Thr Thr Tyr Val

275 280 285

tcc cag tcc ggc cag gta atc tct gct cct cgt tcc att ggc ctc cac 912

Ser Gln Ser Gly Gln Val Ile Ser Ala Pro Arg Ser Ile Gly Leu His

290 295 300

tac ctg gcc acc tgg ttc ttc atc gac ctt att gct gct ctg ccc ttt 960

Tyr Leu Ala Thr Trp Phe Phe Ile Asp Leu Ile Ala Ala Leu Pro Phe

305 310 315

gac ctg ctt tac atc ttc aac atc acc gtg acc tcg ctg gtg cac cta 1008

Asp Leu Leu Tyr Ile Phe Asn Ile Thr Val Thr Ser Leu Val His Leu

320 325 330 335

ctg aag aca gtg cgg ctg ttg cgg ctg ctg cgg ctg ctg cag aag ctg 1056

Leu Lys Thr Val Arg Leu Leu Arg Leu Leu Arg Leu Leu Gln Lys Leu

340 345 350

gag cgg tac tct cag tgc agt gct gtg gtg ctc acg ctg ctc atg tcg 1104

Glu Arg Tyr Ser Gln Cys Ser Ala Val Val Leu Thr Leu Leu Met Ser

355 360 365

gtc ttt gcg ctc ctt gcc cac tgg atg gcc tgc atc tgg tat gtc atc 1152

Val Phe Ala Leu Leu Ala His Trp Met Ala Cys Ile Trp Tyr Val Ile

370 375 380

ggg cgc cgg gag atg gag gcc aat gac ccg ctg ctc tgg gac att ggc 1200

Gly Arg Arg Glu Met Glu Ala Asn Asp Pro Leu Leu Trp Asp Ile Gly

385 390 395

tgg ttg cat gag ttg ggc aag cgt ctg gag gtg ccc tat gtc aat ggc 1248

Trp Leu His Glu Leu Gly Lys Arg Leu Glu Val Pro Tyr Val Asn Gly

400 405 410 415

tcg gtg ggc ggc cca tca cgg cgc agc gcc tac atc gcg gca ctg tac 1296

Ser Val Gly Gly Pro Ser Arg Arg Ser Ala Tyr Ile Ala Ala Leu Tyr

420 425 430

ttc act cta agc agc ctc acc agt gtg ggc ttt ggc aac gtg tgt gcc 1344

Phe Thr Leu Ser Ser Leu Thr Ser Val Gly Phe Gly Asn Val Cys Ala

435 440 445

aac acc gac gcg gag aag atc ttc tcc atc tgc acg atg ctc ata ggc 1392

Asn Thr Asp Ala Glu Lys Ile Phe Ser Ile Cys Thr Met Leu Ile Gly

450 455 460

gcc ctg atg cac gct gtg gtg ttc ggg aac gtg aca gcc atc atc cag 1440

Ala Leu Met His Ala Val Val Phe Gly Asn Val Thr Ala Ile Ile Gln

465 470 475

cgc atg tac tcg cgc cgc tcg ctc tac cac agc cgc atg aag gac ctc 1488

Arg Met Tyr Ser Arg Arg Ser Leu Tyr His Ser Arg Met Lys Asp Leu

480 485 490 495

aag gac ttc atc cgt gtg cac cgc ctg ccg cgg ccg ctc aag cag cgc 1536

Lys Asp Phe Ile Arg Val His Arg Leu Pro Arg Pro Leu Lys Gln Arg

500 505 510

atg ctc gaa tac ttc cag acc acg tgg gcc gtc aac agc ggc atc gac 1584

Met Leu Glu Tyr Phe Gln Thr Thr Trp Ala Val Asn Ser Gly Ile Asp

515 520 525

gcc aac gag tta ctg cgt gac ttc cca gac gag ctg aga gct gac att 1632

Ala Asn Glu Leu Leu Arg Asp Phe Pro Asp Glu Leu Arg Ala Asp Ile

530 535 540

gct atg cac ctg aat cgg gag atc ctg cag ctg ccg ttg ttc ggg gca 1680

Ala Met His Leu Asn Arg Glu Ile Leu Gln Leu Pro Leu Phe Gly Ala

545 550 555

gcg agc agg ggc tgc ctg cgg gcc cta tcg ctg cac atc aag acc tcg 1728

Ala Ser Arg Gly Cys Leu Arg Ala Leu Ser Leu His Ile Lys Thr Ser

560 565 570 575

ttc tgc gct ccg ggc gag tac ctg ttg cgc cgt ggg gat gcc ctg cag 1776

Phe Cys Ala Pro Gly Glu Tyr Leu Leu Arg Arg Gly Asp Ala Leu Gln

580 585 590

gca cat tac tat gtc tgc tcc ggc tcg ctt gag gtg ctc cga gac aac 1824

Ala His Tyr Tyr Val Cys Ser Gly Ser Leu Glu Val Leu Arg Asp Asn

595 600 605

atg gtg ctg gcc atc ctg ggg aag ggg gac ctg att gga gca gat atc 1872

Met Val Leu Ala Ile Leu Gly Lys Gly Asp Leu Ile Gly Ala Asp Ile

610 615 620

cct gag ccg ggg cag gag cct ggg ttg gga gca gac cca aac ttc gtg 1920

Pro Glu Pro Gly Gln Glu Pro Gly Leu Gly Ala Asp Pro Asn Phe Val

625 630 635

cta aag acc agt gct gat gtg aaa gct ctg acc tac tgt ggc ctg cag 1968

Leu Lys Thr Ser Ala Asp Val Lys Ala Leu Thr Tyr Cys Gly Leu Gln

640 645 650 655

cag ctg agc agc cga ggg ctg gct gag gtc ctg agg ctc tat cct gag 2016

Gln Leu Ser Ser Arg Gly Leu Ala Glu Val Leu Arg Leu Tyr Pro Glu

660 665 670

tat ggg gct gcc ttc cgg gct ggc ctg ccc cgg gac ctc acc ttc aac 2064

Tyr Gly Ala Ala Phe Arg Ala Gly Leu Pro Arg Asp Leu Thr Phe Asn

675 680 685

ctg cgc cag ggc tct gac acc agt ggc ctc agc cgc ttt tcc cga tcc 2112

Leu Arg Gln Gly Ser Asp Thr Ser Gly Leu Ser Arg Phe Ser Arg Ser

690 695 700

cct cgc ctc tcc cag ccc cgc tca gaa agc ctc ggc tcc tcc tca gac 2160

Pro Arg Leu Ser Gln Pro Arg Ser Glu Ser Leu Gly Ser Ser Ser Asp

705 710 715

aag acg ctg cca tcc atc aca gag gcc gag agt ggc gcg gag cct ggg 2208

Lys Thr Leu Pro Ser Ile Thr Glu Ala Glu Ser Gly Ala Glu Pro Gly

720 725 730 735

ggt ggt ccc agg ccc cga cgg ccc ctc ctg ctg ccc aac ctc agc cca 2256

Gly Gly Pro Arg Pro Arg Arg Pro Leu Leu Leu Pro Asn Leu Ser Pro

740 745 750

gca cgg cct cgg ggc tcc ctg gtc agc ctt ttg ggc gag gag ctg ccc 2304

Ala Arg Pro Arg Gly Ser Leu Val Ser Leu Leu Gly Glu Glu Leu Pro

755 760 765

cca ttc tca gcc ctt gtc tcc tct cct tcc tta tcc cca tcc ctg tcc 2352

Pro Phe Ser Ala Leu Val Ser Ser Pro Ser Leu Ser Pro Ser Leu Ser

770 775 780

cct gcc ctg gct ggc cag ggc cac agt gcc tcc cct cac ggc ccc ccc 2400

Pro Ala Leu Ala Gly Gln Gly His Ser Ala Ser Pro His Gly Pro Pro

785 790 795

agg tgc tct gct gcc tgg aag ccc cct cag ctt ctc att ccc cca ctg 2448

Arg Cys Ser Ala Ala Trp Lys Pro Pro Gln Leu Leu Ile Pro Pro Leu

800 805 810 815

gga acc ttt gga cct ccg gac ctc agt ccc cgg ata gtg gat ggc att 2496

Gly Thr Phe Gly Pro Pro Asp Leu Ser Pro Arg Ile Val Asp Gly Ile

820 825 830

gag gac tct ggc agc aca gct gag gcc cct tca ttc cga ttc agc agg 2544

Glu Asp Ser Gly Ser Thr Ala Glu Ala Pro Ser Phe Arg Phe Ser Arg

835 840 845

agg cct gaa ctg cca agg ccc cgc tcc cag gcg ccc cct aca ggg acc 2592

Arg Pro Glu Leu Pro Arg Pro Arg Ser Gln Ala Pro Pro Thr Gly Thr

850 855 860

agg ccc agc cca gaa ttg gcc agt gag gct gag gag gtg aag gaa aag 2640

Arg Pro Ser Pro Glu Leu Ala Ser Glu Ala Glu Glu Val Lys Glu Lys

865 870 875

gtt tgc cgg ctg aac cag gag atc tct cgt ctc aat cag gag gtg tct 2688

Val Cys Arg Leu Asn Gln Glu Ile Ser Arg Leu Asn Gln Glu Val Ser

880 885 890 895

cag ctt agc cgg gag ctg cgg cac atc atg ggc ctg ctg cag gcc agg 2736

Gln Leu Ser Arg Glu Leu Arg His Ile Met Gly Leu Leu Gln Ala Arg

900 905 910

ctg ggt ccc cca ggc cac cca gca ggc tcc gct tgg acc cca gac cct 2784

Leu Gly Pro Pro Gly His Pro Ala Gly Ser Ala Trp Thr Pro Asp Pro

915 920 925

cct tgt cca cag ctg agg cca cca tgc ctc tct cct tgt gcg tcc aga 2832

Pro Cys Pro Gln Leu Arg Pro Pro Cys Leu Ser Pro Cys Ala Ser Arg

930 935 940

cca cca ccc agc ctc cag gat act acg ctt gct gaa gtt cac tgc cca 2880

Pro Pro Pro Ser Leu Gln Asp Thr Thr Leu Ala Glu Val His Cys Pro

945 950 955

gcc agt gtg ggg acc atg gag aca ggg act gcg ctc ctg gac ttg aga 2928

Ala Ser Val Gly Thr Met Glu Thr Gly Thr Ala Leu Leu Asp Leu Arg

960 965 970 975

cct tcc ata ttg ccc ccc tac ccc tca gag cct gac cct ctg gga ccc 2976

Pro Ser Ile Leu Pro Pro Tyr Pro Ser Glu Pro Asp Pro Leu Gly Pro

980 985 990

tct cca gtg cca gag gcc tca ccc cca acc cca agc ctc ttg agg cac 3024

Ser Pro Val Pro Glu Ala Ser Pro Pro Thr Pro Ser Leu Leu Arg His

995 1000 1005

agt ttc cag tcc agg tca gac aca ttc cac tga ccctggc 3064

Ser Phe Gln Ser Arg Ser Asp Thr Phe His

1010 1015

6

1017

Homo sapiens

6

Met Pro Val Met Lys Gly Leu Leu Ala Pro Gln Asn Thr Phe Leu Asp

1 5 10 15

Thr Ile Ala Thr Arg Phe Asp Gly Thr His Ser Asn Phe Leu Leu Ala

20 25 30

Asn Ala Gln Gly Thr Arg Gly Phe Pro Ile Val Tyr Cys Ser Asp Gly

35 40 45

Phe Cys Glu Leu Thr Gly Tyr Gly Arg Thr Glu Val Met Gln Lys Thr

50 55 60

Cys Ser Cys Arg Phe Leu Tyr Gly Pro Glu Thr Ser Glu Pro Ala Leu

65 70 75 80

Gln Arg Leu His Lys Ala Leu Glu Gly His Gln Glu His Arg Ala Glu

85 90 95

Ile Cys Phe Tyr Arg Lys Asp Gly Ser Ala Phe Trp Cys Leu Leu Asp

100 105 110

Met Met Pro Ile Lys Asn Glu Met Gly Glu Val Val Leu Phe Leu Phe

115 120 125

Ser Phe Lys Asp Ile Thr Gln Ser Gly Ser Pro Gly Leu Gly Pro Gln

130 135 140

Gly Gly Arg Gly Asp Ser Asn His Glu Asn Ser Leu Gly Arg Arg Gly

145 150 155 160

Ala Thr Trp Lys Phe Arg Ser Ala Arg Arg Arg Ser Arg Thr Val Leu

165 170 175

His Arg Leu Thr Gly His Phe Gly Arg Arg Gly Gln Gly Gly Met Lys

180 185 190

Ala Asn Asn Asn Val Phe Glu Pro Lys Pro Ser Val Pro Glu Tyr Lys

195 200 205

Val Ala Ser Val Gly Gly Ser Arg Cys Leu Leu Leu His Tyr Ser Val

210 215 220

Ser Lys Ala Ile Trp Asp Gly Leu Ile Leu Leu Ala Thr Phe Tyr Val

225 230 235 240

Ala Val Thr Val Pro Tyr Asn Val Cys Phe Ser Gly Asp Asp Asp Thr

245 250 255

Pro Ile Thr Ser Arg His Thr Leu Val Ser Asp Ile Ala Val Glu Met

260 265 270

Leu Phe Ile Leu Asp Ile Ile Leu Asn Phe Arg Thr Thr Tyr Val Ser

275 280 285

Gln Ser Gly Gln Val Ile Ser Ala Pro Arg Ser Ile Gly Leu His Tyr

290 295 300

Leu Ala Thr Trp Phe Phe Ile Asp Leu Ile Ala Ala Leu Pro Phe Asp

305 310 315 320

Leu Leu Tyr Ile Phe Asn Ile Thr Val Thr Ser Leu Val His Leu Leu

325 330 335

Lys Thr Val Arg Leu Leu Arg Leu Leu Arg Leu Leu Gln Lys Leu Glu

340 345 350

Arg Tyr Ser Gln Cys Ser Ala Val Val Leu Thr Leu Leu Met Ser Val

355 360 365

Phe Ala Leu Leu Ala His Trp Met Ala Cys Ile Trp Tyr Val Ile Gly

370 375 380

Arg Arg Glu Met Glu Ala Asn Asp Pro Leu Leu Trp Asp Ile Gly Trp

385 390 395 400

Leu His Glu Leu Gly Lys Arg Leu Glu Val Pro Tyr Val Asn Gly Ser

405 410 415

Val Gly Gly Pro Ser Arg Arg Ser Ala Tyr Ile Ala Ala Leu Tyr Phe

420 425 430

Thr Leu Ser Ser Leu Thr Ser Val Gly Phe Gly Asn Val Cys Ala Asn

435 440 445

Thr Asp Ala Glu Lys Ile Phe Ser Ile Cys Thr Met Leu Ile Gly Ala

450 455 460

Leu Met His Ala Val Val Phe Gly Asn Val Thr Ala Ile Ile Gln Arg

465 470 475 480

Met Tyr Ser Arg Arg Ser Leu Tyr His Ser Arg Met Lys Asp Leu Lys

485 490 495

Asp Phe Ile Arg Val His Arg Leu Pro Arg Pro Leu Lys Gln Arg Met

500 505 510

Leu Glu Tyr Phe Gln Thr Thr Trp Ala Val Asn Ser Gly Ile Asp Ala

515 520 525

Asn Glu Leu Leu Arg Asp Phe Pro Asp Glu Leu Arg Ala Asp Ile Ala

530 535 540

Met His Leu Asn Arg Glu Ile Leu Gln Leu Pro Leu Phe Gly Ala Ala

545 550 555 560

Ser Arg Gly Cys Leu Arg Ala Leu Ser Leu His Ile Lys Thr Ser Phe

565 570 575

Cys Ala Pro Gly Glu Tyr Leu Leu Arg Arg Gly Asp Ala Leu Gln Ala

580 585 590

His Tyr Tyr Val Cys Ser Gly Ser Leu Glu Val Leu Arg Asp Asn Met

595 600 605

Val Leu Ala Ile Leu Gly Lys Gly Asp Leu Ile Gly Ala Asp Ile Pro

610 615 620

Glu Pro Gly Gln Glu Pro Gly Leu Gly Ala Asp Pro Asn Phe Val Leu

625 630 635 640

Lys Thr Ser Ala Asp Val Lys Ala Leu Thr Tyr Cys Gly Leu Gln Gln

645 650 655

Leu Ser Ser Arg Gly Leu Ala Glu Val Leu Arg Leu Tyr Pro Glu Tyr

660 665 670

Gly Ala Ala Phe Arg Ala Gly Leu Pro Arg Asp Leu Thr Phe Asn Leu

675 680 685

Arg Gln Gly Ser Asp Thr Ser Gly Leu Ser Arg Phe Ser Arg Ser Pro

690 695 700

Arg Leu Ser Gln Pro Arg Ser Glu Ser Leu Gly Ser Ser Ser Asp Lys

705 710 715 720

Thr Leu Pro Ser Ile Thr Glu Ala Glu Ser Gly Ala Glu Pro Gly Gly

725 730 735

Gly Pro Arg Pro Arg Arg Pro Leu Leu Leu Pro Asn Leu Ser Pro Ala

740 745 750

Arg Pro Arg Gly Ser Leu Val Ser Leu Leu Gly Glu Glu Leu Pro Pro

755 760 765

Phe Ser Ala Leu Val Ser Ser Pro Ser Leu Ser Pro Ser Leu Ser Pro

770 775 780

Ala Leu Ala Gly Gln Gly His Ser Ala Ser Pro His Gly Pro Pro Arg

785 790 795 800

Cys Ser Ala Ala Trp Lys Pro Pro Gln Leu Leu Ile Pro Pro Leu Gly

805 810 815

Thr Phe Gly Pro Pro Asp Leu Ser Pro Arg Ile Val Asp Gly Ile Glu

820 825 830

Asp Ser Gly Ser Thr Ala Glu Ala Pro Ser Phe Arg Phe Ser Arg Arg

835 840 845

Pro Glu Leu Pro Arg Pro Arg Ser Gln Ala Pro Pro Thr Gly Thr Arg

850 855 860

Pro Ser Pro Glu Leu Ala Ser Glu Ala Glu Glu Val Lys Glu Lys Val

865 870 875 880

Cys Arg Leu Asn Gln Glu Ile Ser Arg Leu Asn Gln Glu Val Ser Gln

885 890 895

Leu Ser Arg Glu Leu Arg His Ile Met Gly Leu Leu Gln Ala Arg Leu

900 905 910

Gly Pro Pro Gly His Pro Ala Gly Ser Ala Trp Thr Pro Asp Pro Pro

915 920 925

Cys Pro Gln Leu Arg Pro Pro Cys Leu Ser Pro Cys Ala Ser Arg Pro

930 935 940

Pro Pro Ser Leu Gln Asp Thr Thr Leu Ala Glu Val His Cys Pro Ala

945 950 955 960

Ser Val Gly Thr Met Glu Thr Gly Thr Ala Leu Leu Asp Leu Arg Pro

965 970 975

Ser Ile Leu Pro Pro Tyr Pro Ser Glu Pro Asp Pro Leu Gly Pro Ser

980 985 990

Pro Val Pro Glu Ala Ser Pro Pro Thr Pro Ser Leu Leu Arg His Ser

995 1000 1005

Phe Gln Ser Arg Ser Asp Thr Phe His

1010 1015

7

19

Artificial Sequence

Description of Artificial SequencePrimer

7

gccatgccgg tcatgaagg 19

8

20

Artificial Sequence

Description of Artificial SequencePrimer

8

gccagggtca gtggaatgtg 20

9

3715

Rattus sp.

9

ctgctggggc ctacgaacct gggccgggca tagccccccg acggctactc tagggggcgc 60

ggggcccggc ggggggcggc cgagccaggc gccctccccc ggcgctgagt ccccgcgccc 120

cggagggatg gggcgggcgg tccccgccgc ctaagatgcc ggccatgcgg gggctccttg 180

cgccgcagaa caccttcctg gacaccatcg ccacccgctt cgacgggacg cacagtaact 240

tcgtcctggg caacgcccag gtggcagggc tcttccctgt ggtctactgc tccgatggct 300

tctgtgacct cacgggtttc tccagagctg aggtcatgca gcgaggctgt gcctgctcct 360

tcctctatgg gccagacacc agtgagttgg tccgccaaca gatccgaaaa gccctggatg 420

agcacaaaga attcaaggct gaactgatcc tgtaccggaa gagcgggctt ccattctggt 480

gtctcctgga tgtgatacct ataaaaaacg agaaggggga ggtggccctc ttcctggtct 540

ctcacaagga catcagtgag accaagaacc gaggaggccc tgacaactgg aaggagagag 600

gtggtggccg acgcagatat ggtcgggcag gatccaaagg ctttaatgcc aatcggaggc 660

gcagccgggc ggttctctac cacctctctg gtcacctgca gaaacaaccc aagggcaagc 720

acaaactcaa taagggtgtg tttggagaga agccaaattt gcccgaatat aaagtcgctg 780

ctatccggaa gtcacccttt atcctgctgc actgtggggc tctgagagcc acctgggatg 840

gcttcatcct gctcgccacg ctctacgtgg ctgtcactgt gccatacagc gtgtgtgtga 900

gcacagcacg ggagcccagt gctgcccgtg gcccacctag tgtctgtgac ctggccgtgg 960

aagtcctctt catcttagat attgtgctga attttcgtac tacatttgtg tccaagtcag 1020

gccaggtggt attcgcccca aagtccattt gcctccacta cgtcaccacc tggttcctgc 1080

tggatgtcat agcagcactg ccctttgacc tactacatgc cttcaaggtc aatgtgtacg 1140

ttggggctca cctactgaag accgtgcggc tgcttcggct gctgcgccta ctaccaagac 1200

tggaccggta ctctcagtat agcgctgttg tgctcacctt gctcatggct gtgtttgccc 1260

tgctcgccca ctgggtggcc tgcgtttggt tctacatcgg ccagcaagag attgagaaca 1320

gcgagtcaga gctgcctgag atcggctggc tgcaggagct ggcacgcagg ctggagacgc 1380

cctattacct ggtgagccgg agtccagatg gagggaacag ctctggccag agtgaaaact 1440

gcagtagcag tggcggcggc agcgaagcca acgggactgg gctggagctg ctgggtggcc 1500

catccctacg cagcgcctac atcacctcct tgtacttcgc gctcagcagt ctcaccagtg 1560

tgggcttcgg caatgtgtcc gctaacacag acactgagaa gattttctcc atctgcacca 1620

tgcttattgg agctctgatg catgcagtgg tgtttgggaa tgtgacagcc atcatccagc 1680

gcatgtacgc tcgccgcttt ctgtaccaca gccgcacccg tgacctgcga gactacattc 1740

gcatccaccg catccccaag cccctcaagc agcgcatgct cgagtacttc caagccacct 1800

gggccgtgaa caacggcatc gataccactg agctgctgca gagccttccg gatgagcttc 1860

gagcagacat cgccatgcac ctgcacaagg aggtcctgca gctgccattg ttcgaggcag 1920

cgagccgtgg ctgcctgcgg gcactgtctc tggccctgag gcccgccttc tgcacgccgg 1980

gcgagtacct cattcaccaa ggcgatgctc tccaggctct ctactttgtg tgctcaggtt 2040

ccatggaggt cctcaaaggt ggcaccgtcc tcgccattct agggaagggt gacctgatcg 2100

gctgtgagct gccccagcga gagcaagtag tgaaggccaa tgccgacgta aaggggctga 2160

catactgcgt cctacagtgc ctgcagctgg ctgggctgca cgagagcctc gcactgtacc 2220

ctgagtttgc cccacgcttt agccgtggcc tccgagggga gctcagctac aacctgggag 2280

ctggaggagt gtctgcagag gtggatacca gctcactgag tggtgacaac accctcatgt 2340

ccacactgga ggagaaggag acagatgggg agcaaggaca cacgatctca ccagccccag 2400

cagatgagcc ctccagcccc ctgctgtcac ctggctgtac ctcctcctcc tcagcggcca 2460

aactactctc cccacgtcga actgcacccc ggccgaggct gggtggcaga gggcggccaa 2520

gtagggcagg ggttttgaag cctgaggctg gtccttctgc tcatccacgg acacttgatg 2580

ggttgcagct gccccccatg ccatggaatg tacctccaga cctgagcccc agggtcgtag 2640

atggcattga ggatggctgc ggctctgacc agcacaagtt ctctttccgg gtgggtcagt 2700

ctggcccaga atgtagcagc agcccctccc caggaacaga gagtggcctg ctcactgtcc 2760

ccttggtgcc cagtgaggca agaaacacag acacactgga caagctacgg caggcggtga 2820

cggagctgtc tgaacaggtg ctgcagatgc gagagggact gcagtcactt cgccaggctg 2880

tgcagctcat cctggtgccc caaggggaag gccagtgtcc ccgggtatca ggagaggggc 2940

catgcccagc cactgcctct gggctcctac aacccctgcg tgtggacact ggggcatcat 3000

cctactgcct gcagccccca gcaggttcag tcttgagtgg gacctggcct cacccccgtc 3060

cagggcatcc ccctcccctc atggcaccct ggccctgggg ccccccagca tctcagagct 3120

ccccctggcc tcgagccaca gctttatgga cctccacctc agactcagag ccccctggct 3180

ctggagacct ctgctctgag cccagcaccc cagcctcacc ccctcctcct gaggaaggag 3240

ctaggactgg gactcctgca cctgtgagcc aggctgaggc taccagtact ggagagcccc 3300

ctccggggtc agggggccga gccttgccct gggatcccca cagcctagag atggtgctca 3360

tcggctgcca tggccctggc tcggtccagt ggacccagga ggagggcaca ggagtctgac 3420

cacaggcagg gagaggggtt ctgcaaacac ccgccacctg ctgaccgccc agcctcacag 3480

ggctgccctc tggctcaggg cagggaacct aaggaaggag gagggtgagc tggagcctca 3540

ggccccaggc cagggatcca cgggttctgc tccactgacc tgacccaatg ggggcagagg 3600

cctgaggacg aggaggggtt ctgccattcc ttgcatgtgc ccatctccac tgtcctctgt 3660

cctcatgttt tttatattaa aaaacataaa aaaaaaccaa taaagaaact acttt 3715

10

3736

Rattus sp.

10

caggcagcgg cggcgagagg aggggaggag gcaggccggc gcatggggcg ccccggcccc 60

gccggtagcg cgccccctcc ggccaggccg cgctgaacgc agcccgcgca acgcctcgaa 120

ttcgtacccg gggccatgcc ggtcatgaag gggttgctgg ccccgcaaaa cacctttctg 180

gacaccatcg ccactcgctt tgacggcacg cacagcaact ttcttctggc caatgcccag 240

ggcccacggg gttttcccat cgtctactgc tctgacggct tctgtgagct cacaggctac 300

ggccgcaccg aggtcatgca gaaaacctgt agctgccggt tcctctatgg cccagagacc 360

agtgagccgg ccttgcaacg gttacaaaaa gccctggagg gccaccaaga acacagagct 420

gaaatctgct tttaccgaaa ggatggctcg gccttttggt gtcttctgga catgatgccc 480

atcaaaaatg agctggggga ggtggtgctt ttcctatttt cctttaagga catctctcag 540

agtggaggcc caggacttgg ctcaccaggg atccatgggg acaataataa tcatgaaaac 600

tcccttggga ggagaggagc tagctcaaga cttaggtcca cgaggaggca gaaccggaca 660

gttctacacc ggttgactgg ccactttggt cgccgggacc agggaagcgt gaaagccaat 720

agtaacgtgt ttgagccaaa gccatcagtg cctgagtaca aagtggcctc cgtggggggc 780

tcccgctgcc tgctcctcca ctacagcatc cccaaggctg tctgggacgg tctcatcctt 840

ctcgctacgt tctacgtcgc ggtcaccgtc ccttacaacg tctgcttcgc tggtgatgac 900

gacaccccca tcacgtcccg acacaccctt gtcagtgaca tcgctgtgga gatgctcttc 960

atcctggaca tcatcttgaa cttccgcacc acctacgtgt cccagtcggg ccaggtggtt 1020

tctgctcctc ggtccattgg cctccactac ctggccacct ggttcttcgt ggacctcatt 1080

gctgctttgc cctttgacct gctgtatgtc ttcaacatca ctgtgacctc gctggtacat 1140

ctgctgaaaa ccgtgcggct cctgcggttg ctgaggctgc tgcagaagct agagcggtac 1200

tctcagtgca gcgcggtggt gctcacgctg ctcatgtccg tctttgcact ccttgcccac 1260

tggatggcct gcgtctggta tgtcatcggg cgccgggaga tggaggccaa tgacccgctg 1320

ctctgggaca ttggttggtt gcatgagctg ggtaagcggc tggaggagcc ttatgtcaat 1380

ggctcggccg gtggaccatc tcggcgcagt gcctacatcg ccgcgctgta cttcacgctg 1440

agcagcctca ccagtgtagg cttcggcaac gtttgtgcca acactgacgc tgagaagatc 1500

ttctccatct gcacgatgct cataggcgcg ctgatgcacg cggtggtgtt tgggaatgtc 1560

acagccatca tccagcgcat gtactcccga cgctcgctct accacagccg catgaaggat 1620

ctcaaggact tcatccgagt gcatcgtctg ccccgcccac tcaagcagcg catgctcgag 1680

tacttccaga ctacatgggc cgtcaacagc ggcatcgatg ccaacgagtt actgcgtgac 1740

ttcccggatg agctgcgagc tgacatcgcc atgcacctga atcgggagat cctgcagctg 1800

cctttgtttg gagcagcaag caggggctgc cttcgtgccc tctccctgca catcaagacc 1860

tcattttgtg ctcctgggga gttcctgcta cgccgtgggg atgccctgca ggcacactac 1920

tatgtctgct ctggctctct tgaggtgctc cgagacaaca cggtgctggc catccttgga 1980

aagggggact tgattggggc agacatccct gagttggggc aggagcctgg ggcaggggca 2040

ggctgcgtgc tgaagaccag cgctgatgtg aaagcactga cttactgcgg cctgcagcag 2100

ctgagcagcc gagggctggc cgaggtcctt cggttgtatc cggaatatgt ggctgccttc 2160

agggctggcc taccccggga cctaaccttc aacctgcgcc aaggctctga aaacaatggc 2220

ctcggccgct tctcacgttc tcctcgactc tcccaggcac gctccgacac tcttggttcc 2280

tcctcagaca agactctgcc atccatcaca gaaaccgagg gtggcatgga gcctggggct 2340

ggttccaagc cccgtcggcc cctcctgctg cccaacctca gtccagcacg acctcggggg 2400

tccctggtca gccttttggg cgaagagctg cccccattct cagcccttgt ctcctctcct 2460

tccctgtccc caactccttc ccctgccctg gctggccggg gttcaagtcc ctccctgcac 2520

gggcccccca ggggctctgc tgcctggaag cccccccagc tcctcacccc cccactggga 2580

acatttggac ctccggacct cagtccccgg atcgtggatg gcattgagga ctccagtaac 2640

acagctgagg ctcctacatt ccggttcagc aagaggccgg agcccaccag aacccgttca 2700

caggctcccc tttcaggccc taggctcagc cgggaactgg ccacagaggc agcagaggag 2760

gtgaaggaaa aggtctgcag gctgaaccaa gagatttcca gactcaacca ggaagtgtct 2820

cagctgagcc gggagcttcg ccaagtgatg ggcctcttac aggccaggct gggtccccca 2880

agtcacccac ctgactccac ttggctccca gaccttcctt gtccccatca gagaccgcca 2940

tgcatctctc ctcatatgtc tggacctcca cctggtctcc agaatactac acttgctgta 3000

gtccactgtc cagccagtgt tgggacagtg gagatagggg ccaccccctc agagctgagg 3060

tcttccatgg tgccaccctt tccctcagaa cctgatcctc ttggaccctc tccagtgcca 3120

gaggcttctc ctctgacccc aagcctcctg aagcacagct tccagtctgg gtcagacaca 3180

ttccactgac cctggccttg ggcccaggcc tgtctggggt gggcttaatt acctgccatc 3240

cagggaggag ctgggctcct tggcctcttg ccttggggtc agcagctgcc agctggtctg 3300

gttggctctg gattctctgg actttttaac aatgagtggc cacatctaac cccgttccat 3360

tttctaaacc atccccccca cccgtcctac tcttgtggga ggggagtcac ctgaagccgt 3420

ggaatccagc aggcatgaaa tggaccatgg tgccctgttg ggctacgcag aggagatgtc 3480

ttactttctc cccacgactt ggaggctgtc atgcaaggtg gtcccttctg ctgccagcag 3540

ctcaaagcat tctagcttta cccttctgca gttcccacct ccaaacatca gtcatatctg 3600

cccccctccc tcccaacatg gtctcgacct caatgaggat ctgggcaact cacaaaccct 3660

ctctggtttc cagctcccct ttctcactaa acaccccagg ctcacttttg gatagaaaat 3720

aaatccatat attttt 3736

11

20

Artificial Sequence

Description of Artificial Sequenceprobe

11

accttcctgg acaccatcgc 20

12

20

Artificial Sequence

Description of Artificial Sequenceprobe

12

ccaaacacca ccgcgtgcat 20