JP2001095587A - Mouse epilepsy causal gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a homologous gene of human epilepsy causal gene EPM 2A in mice. SOLUTION: The homologous gene of EPM 2A in mice is isolated by amplifying a partial fragment of human EPM 2A by means of PCR using a primer prepared on the basis of the sequence of the partial fragment, screening out a mouse brain cDNA library by using the partial fragment as a probe, acquiring a cDNA encoding the amino acid sequence represented by sequence number 1 and screening out a mouse genomic library by using the resultant cDNA as a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a mouse homologous gene to human epilepsy-causing gene EPM2A.

Lafora-type progressive myoclonic epilepsy is a rare but lethal autosomal recessive disease characterized by the presence of intracellular inclusions that are positive for periodate staining. (1, 2). Lafora's disease (LD) is manifested by epileptic seizures, which usually develop in adolescence at an average age of 15 years. Often, rapid and progressive confusion follows its psychotic appearance, with the patient dying within 10 years of illness (3).

[0002] Serratosa et al. (4) and Sainz et al. (5) have already revealed by mapping that LD is located on chromosome 6p24. Also recently, Mi
(6) identified a novel gene at 6p24, named EPM2A, by positional cloning. This gene encodes a putative protein with a protein-tyrosine phosphatase region. Also, the EPM2A transcript is alternatively spliced and detected in all analyzed tissues, including the brain. EPM2A mutations have been found in many LD families, which are separated from each other by the phenotype of the disease (6, 7). Although dysfunction of protein-tyrosine phosphatase (PTP) has been linked to many neoplastic diseases (8,9), the discovery of EPM2A is the second example of their involvement in non-neoplastic diseases. (6). Although PTP is known to play many important roles in cell signaling pathways, the detailed role of EPM2A in normal brain and LD has not been elucidated.

[0003]

In recent years, the role of various genes related to neurological diseases in cells has been elucidated by using a model system. Therefore, the function of EPM2A is also similar to this. Studies may reveal this. For that purpose, a model system using animals such as mice is required, but homologous genes to human EPM2A of these animals are not known.

[0004] The present invention has been made in view of the above, and it is an object of the present invention to provide a mouse homologous gene of human EPM2A.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors have intensively studied. Then, they succeeded in isolating a mouse homologous gene to the human epilepsy-causing gene EPM2A, and found that the gene encodes a putative protein-tyrosine phosphatase, thereby completing the present invention.

That is, the present invention is as follows. (1) DNA encoding a protein shown in the following (A) or (B). (A) DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2. (B) a protein consisting of an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, or inversions in the amino acid sequence shown in SEQ ID NO: 2, and having a protein-tyrosine phosphatase activity; (2) a DNA represented by the following (a) or (b): (1)
The described DNA. (A) DNA comprising at least the nucleotide sequence consisting of nucleotide numbers 67 to 1056 in the nucleotide sequence shown in SEQ ID NO: 1. (B) of the nucleotide sequence shown in SEQ ID NO: 1, which hybridizes under stringent conditions with at least the nucleotide sequence consisting of nucleotide numbers 67 to 1056, and
DNA encoding a protein having tyrosine phosphatase activity. (3) The DNA according to (1) or (2) is modified so that it does not function properly, and the resulting modified DNA is introduced into mouse cells, and the DNA is homologous to the DNA according to (1) or (2). A method for producing a mouse deficient in an epilepsy-causing gene, comprising causing recombination between an endogenous gene having a unique sequence and the modified gene to destroy the endogenous gene.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Hereinafter, a method for obtaining the DNA of the present invention will be exemplified.
Since the nucleotide sequence of the DNA of the present invention and the amino acid sequence encoded thereby have been elucidated, the DNA of the present invention can be amplified by PCR from mouse chromosomal DNA based on those sequences or by chemical synthesis. D
The NA can be obtained.

First, a probe for screening a mouse brain cDNA library is prepared. This probe is obtained by amplifying a partial coding sequence of human EPM2A by PCR using human chromosomal DNA as a template. As primers used for PCR, SEQ ID NOs: 3 to 6
And oligonucleotides having the base sequence shown in Table 1.

Next, a mouse cDNA library is screened by hybridization using the obtained PCR product as a probe. The mouse cDNA library is not particularly limited, and for example, a commercially available library such as a mouse brain cDNA library of ICR out-bred strain (Stratagene) can be used.

An example of the nucleotide sequence of the cDNA obtained as described above is shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequences predicted to be encoded by this sequence are shown in SEQ ID NOs: 1 and 2. These sequences are stored in GenBank
It is registered with the ession AF124044 number. SEQ ID NO: 2
Wherein amino acids 263 to 273 are a putative phosphatase region.

[0011] In addition, a genomic gene can be obtained by screening a mouse genomic library using the obtained cDNA as a probe.
Methods of PCR, preparation of primers used for PCR, preparation of chromosomal DNA, preparation of a chromosomal DNA library, hybridization and the like are performed by ordinary methods well-known to those skilled in the art described in literature (10) and the like. ,It can be carried out.

The DNA of the present invention includes one or several amino acid substitutions, deletions, insertions, additions, or inversions at one or more positions, as long as the activity of the encoded protein is not impaired. It may be. Where "several"
Is specifically 2 to 30, preferably 2 to 20
And more preferably 2 to 10.

The above-mentioned substitution, deletion, insertion, addition, or inversion of a base includes not only an artificial mutation but also a naturally occurring mutation (mutant or variant) caused by mouse species or individual differences. Is also included. Specifically, such a DNA hybridizes with at least nucleotides 67 to 1056 of the nucleotide sequence shown in SEQ ID NO: 1 or a probe which can be prepared from the sequence under stringent conditions, and DNA encoding a protein having tyrosine phosphatase activity is included.
As stringent conditions, specifically,
C., conditions performed at 0.1 × SSC / 0.1% SDS.

According to the present invention, the human epilepsy gene EPM2A
Mouse homologous gene of Epm2a was isolated. Mouse E
We have shown that pm2a encodes a putative protein-tyrosine phosphatase and is located in the proximal region of chromosome 10, which is the synteny and the 6p24 and EPM2A loci (6,14) in humans.

The putative protein encoded by Epm2a shows 88% identity and 93% similarity to homologous proteins in humans, and is a catalytic termed PTP (protein-tyrosine phosphatase) region. The consensus sequence in region (6) is 100% identical.

In the testis, there is an additional transcript at Epm2a, which in other tissues is:
Expression is low compared to testis. Different spliced transcripts were identified in human EPM2A (6,
7), the additional transcript may indicate an isoform of Epm2a in mice. However,
Very high sequence homology and identical expression in both human and mouse species suggest that EPM2A is functionally highly conserved in animals. The Lafora body found in patients with LD disease has the properties of an acidic mucopolysaccharide, thus suggesting that it may be polyglucosan and that enzyme defects may cause these depositions and the disease (15, 16). The fact that glycogen metabolism is known to be regulated by protein phosphatase (8, 9) predicts a putative role for EPM2A in lafora body formation (6, 7). This work on analyzing the features of Epm2a
It allows the mouse to be an attractive animal model to test the hypothesis described above. Cystatin B gene
Since it was recently revealed that they cause EPM1 (17), the generation of mutant mice in Epm2a
It will help to understand the cellular function of PM2A and the pathological role of LD.

The DNA of the present invention can be used, for example, for expression analysis of Epm2a. In addition, the present invention can be used for producing an epilepsy model animal in which Epm2a does not function normally and for constructing a targeting vector used therefor. Such a model animal is, for example, a DNA of the present invention.
Is modified so that it does not function normally, and the resulting modified DNA
Is introduced into mouse cells to cause recombination between the modified DNA and the endogenous Epm2a gene, thereby disrupting the endogenous Epm2a gene. Specifically, a method usually used for mouse genes, for example, a gene disruption method using Cre-loxP can be applied. The epilepsy model mouse can be used for the development of a therapeutic agent and a therapeutic method for epilepsy.

[0018]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

<1> Preparation of a Probe for Screening a Mouse cDNA Library In order to prepare a probe for screening a mouse brain cDNA library, a partial coding sequence of human EPM2A was amplified by PCR and purified.

Based on the reported sequence (6) of the exon portion of human EPM2A, exon 2 of human genomic DNA was used.
The primers for amplifying the portion from to 4 were prepared.
The forward and reverse primers corresponding to exons 2 and 4, respectively, are as follows.

(Exon 2 forward primer) 5'-GCTCTTTACAAATTCCTACCGTTCTGT-3 '(SEQ ID NO: 3) (Exon 2 reverse primer) 5'-AGTGAGGCACTGCAGTTTCGA-3' (SEQ ID NO: 4) (Exon 4 forward primer) 5'-TGTGATGGGCTGGAATCTGAG- 3 '(SEQ ID NO: 5) (Exon 4 reverse primer) 5'-GAGCAGGAACTGCACGCATTA-3' (SEQ ID NO: 6)

Using the above primers, human chromosome DN
PCR was performed using A as a template. The PCR conditions are as follows.

(Reaction solution) Human genomic DNA 4 μl 2 mM dNTP 10 μl 25 μM forward primer 1 μl 25 μM reverse primer 1 μl 10 × PCR buffer (+15 mM MgCl ₂ ) 10 μl distilled water 72.5 μl enzyme mixture 1.5 μl

(Reaction conditions) Step 1 94 ° C. 5 minutes Step 2 94 ° C. 45 seconds Step 3 60 ° C. 45 seconds Step 4 72 ° C. 2 minutes Step 5 72 ° C. 10 minutes Step 6 4 ° C. Long time Steps 2 to 4 42 times repeat.

<2> Screening of mouse cDNA library Using the PCR product obtained above as a probe, ICR out-br
The mouse brain cDNA library of the ed strain (Stratagene) was screened by hybridization. The cDNA library is a mouse brain-derived poly
(A) Lambda ZAP, which is constructed from cDNA obtained from RNA using oligo (dT) and random primers
-Integrated into the II vector.

Method established by Shambrook et al. (10)
Was used to screen the mouse brain cDNA library. The phages picked up from 20 plates containing about 2 × 10 ⁶ plaques were hybridized overnight, and further subjected to alkaline phosphatase labeling and detection kit (AlkPhos direct labeling and detection kit).
A positive kit was identified using a detection kit (Amersham Life Sciences). These filters were washed at 55 ° C. in a washing solution recommended by Amersham Life Sciences, and chemifluorescence detection was performed according to the instructions. Selected phage were transferred into plasmid by in vivo excision as recommended by Stratagene. As a result, 0.6k
Clones with three insert sizes of b, 0.8 kb and 1.4 kb could be obtained. These nucleic acid sequences were determined by the dye-terminator method using ABI autosequencer type 377. By analyzing these base sequences, two inserts with a small size (0.6 kb, 0.8 kb)
Was confirmed to be part of the longest insert (1.4 kb).

The nucleotide sequence of the longest cDNA clone LDM3 (1.4 kb) is shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequences predicted to be encoded by this sequence are shown in SEQ ID NOs: 1 and 2. These sequences are stored in GenBank
Registered with AF124044 number.

The previously reported cDNA for human EPM2A has a 5 '
Partial due to lack of coding region (6, 7). However, the possible first exon can be inferred from the genomic sequence. Because the genomic sequence extends to the ORF in most 5 'sequences of the cDNA clone,
It shows all the expected features of eukaryotic genes (6). Interestingly, mouse cDNA
The 5 ′ coding sequence of clone LDM3 was found to have high homology to the predicted nucleic acid sequence of exon 1 and the amino acid sequence derived therefrom.

The start site of the coding region in the mouse clone was revealed by aligning the amino acid sequence deduced therefrom with the amino acid sequence deduced from human EPM2A. However, in 66b before the start site, a sequence adjacent to the predicted start site (ACCGCCATGC: base numbers 1 to 10 in SEQ ID NO: 1) despite no in-frame stop codon.
Are characterized by a Cossack consensus sequence (12) that suggests a eukaryotic transcription initiation site.

Furthermore, in the genomic sequence, an in-frame TAG precedes the 70 base 5 'side of the putative start codon of human EPM2A (6). The putative EPM2A protein in mice is the protein-
It has a region thought to have tyrosine phosphatase (PTP) function (amino acids 263 to 273 of SEQ ID NO: 2), encodes 331 amino acids, and lacks one amino acid in human EPM2A protein . Human
Compared to EPM2A, the mouse homologous gene named Epm2a showed 86% identity at the nucleic acid level, 88% identity at the protein level, and 93% similarity in the coding region. In the homology comparison, a blank was inserted at position 73 of the mouse sequence to supplement the additional amino acids deduced from human EPM2A.

<3> Northern blot analysis Northern blot of a complex tissue of a mouse was purchased from Clontech Laboratory and hybridized under the following conditions. The 1.4 kb insert of mouse EPM2A in cDNA clone LDM3 was labeled with [ ³² P] dCTP and hybridized overnight with the hybridization solution Express Hyb (Clontech Laboratories). The blots were finally purified at 55 ° C for 0.1
It was washed with × SSC / 0.1% SDS and exposed to an X-ray film at −80 ° C.

To determine the tissue specificity of the Epm2a transcript, a 1.4 kb cDNA clone, LDM3, was hybridized to Northern blots in various mouse tissues. As a control, hybridization was similarly performed using actin as a probe. Epm2a, compared to spleen, lung, testis,
Relatively high transcripts were found in brain, liver and kidney. In all tissues analyzed, a single band of 3.5 kb size was identified, while an additional 1.5 kb band was detected in testis.

<4> Screening of genomic library Using a 1.4 kb mouse Epm2a cDNA clone LDM3 as a probe, screening was performed on a mouse C57BL / 6N strain genomic library constructed with the EMBL3 SP6 / T7 vector (Clontech). Was. About 1 × 10 ⁶ plaques were screened using the alkaline phosphatase labeling / detection kit. As a result, two positive clones were obtained. These plaques were partially sequenced using primers derived from the mouse cDNA sequence. 500 n of their DNA to select inserts
To 3 g of SalI was added, and treatment with a restriction enzyme was performed for 4 hours to remove the insert from the arm of the vector. The samples were run on a 1% agarose gel (SeaKem GTG, FM
Electrophoresis was performed using C) and CHEF mapper (Bio Rad).

[0034] End-sequencing and restriction enzyme patterns indicated that the clones were clearly identical. Insert size 15 by gel electrophoresis
exon 4 of the human EPM2A gene
Hybridization with the PCR product against indicated that both clones had exons encoding a putative PTP region. Therefore, a primer was prepared for the coding region, and sequencing was performed for an adjacent region using one of the genomic clones (B6LDM8). This partial sequencing results in a 5 'intron / exon junction (tttctctagGCC
GA: SEQ ID NO: 7), and the 3 ′ UTR sequence for the last exon encoding the putative PTP region was revealed. Genomic organization for the last exon of mouse Epm2a is identical to its human gene because it has a minor consensus sequence such as AG / GC in mRNA splicing, and in both species this exon has 91 amino acids. Coded (6, 13).

<5> Fluorescence in situ hybridization (FISH) Next, in order to determine the mouse EPM2A gene locus on the chromosome, direct R band staining FISH by Mazda et al.
The method (11) was performed.

The mitogen-stimulated spleen lymphocyte culture was
Synchronized with a thymidine block and during the late replication stage, 5-bromodeoxyuridine was added. R band staining was performed by exposing to UV light after staining with Hoechst 33258. For hybridization, chromosomal slides were denatured at 70 ° C. in 70% formamide containing 2 × SSC. The DNA of the mouse 15 kb genomic clone B6LDM8 was labeled by nick translation with biotin 16-dUDP (Boehringer Mannheim). This was hybridized to slides subjected to R band staining at 37 ° C. in the presence of Cot-I DNA and salmon sperm DNA. The composition of the hybridization solution was 50% formamide, 2 x SSC, 1
0% dextran sulfate and 2 mg / ml BSA.
The slides were washed with 50% formamide containing 2 × SSC at 37 ° C., followed by 2 × SSC and 1 × SSC at room temperature. In addition, these slides were fluorescein-
Stained with streptavidin and post-stained with propidium iodide. The stained images were captured with a photometric schooled CCD camera using the Leica QFISH system (Leica).

In the metaphase spread stained with the R band, the genomic probe showed a clear signal on mouse chromosome 10A corresponding to the region of human chromosome band 6q24.

[Literature] 1. Lafora, GR (1955) Proceedings of the 2nd Int
ernational Congress of Neuropathology, Excerpta Me
dica, Part I, p.5-9, Amsterdam. 2.Schwarz, GA Yanoff, M. (1965) Arch. Neurol. 1
2, 172-188. 3. Van Heycop Ten Ham MW (1974) in Handbook of C
linical Neurology (Vinken, Pj and Bruyn GW, Ed
s.) p.171-189, North-Holland, New York. 4. Serratosa, JM et al. (1995) Hum. Mol. Genet.
4, 1657-1663. 5. Sainz, J. et al. (1997) Am. J. Hum. Genet. 61,
1205-1209. 6. Minassian, BA (1998) Nature Genet. 20, 171-17
4. 7. Serratosa, JM et al. (1999) Hum. Mol. Gent.
8.345-352. 8. Tonks, NK and Neel, BG (1996) Cell 87, 365-
368. 9. Zhang. ZY (1998) Crit. Rev. Mol. Biol. 33, 1-
52. 10. Sambook, J. et al. (1989) Molecular Cloning. A
Laboratory Manual, 2nd ed. Cold Spring Harbor Lab
oratory Press, Cold Spring Harbor, NY. 11.Matsuda, Y. et al. (1992) Cytogenet. Cell Gene
t. 61, 282-285. 12. Kozak, M. (1996) Mamm. Genome 7, 563-574. 13. Jackson, IJ (1991) Nucleic Acids Res. 19, 37
95-3798. 14. Debry, RW et al. (1996) Genomics 33, 337-35
1. 15. Yokoi, S. et al. (1968) Arch. Neurol. 19, 15-3
3. 16. Sakai, M. et al. (1970) Neurol. 20, 160-176. 17. Pennacchio, LA et al. (1998) Nature Genet. 2
0, 251-258.

[0039]

Industrial Applicability According to the present invention, a novel mouse epilepsy-causing gene Epm2a against human EPM2A is provided. The DNA of the present invention can be used for expression analysis of Epm2a, production of epilepsy model animals, and the like.

[0040]

[Sequence list] SEQUENCE LISTING <110> RIKEN <120> Mouse epilepsy-causing gene <130> P-6824 <140> <141> 1999-10-01 <160> 7 <170> PatentIn Ver. 2.0

<210> 1 <211> 1419 <212> DNA <213> Mus musculus <220> <221> CDS <222> (67) .. (1056) <400> 1 gcgccgcccc ggactcgcgc cgccgccgct gccgctcgcc aggctcccag gactcgggcc 60 accgcc atg ctc ttc cgc ttc ggc gtg gtg gtg ccg ccc gcg gtg gcc 108 Met Leu Phe Arg Phe Gly Val Val Val Pro Pro Ala Val Ala 1 5 10 ggc gct cgg cag gag ctg ctg ctc gcc ggc gc ggg ccc Ala Arg Gln Glu Leu Leu Leu Ala Gly Ser Arg Pro Glu Leu Gly 15 20 25 30 cgc tgg gag ccg cac ggc gcc gtg cgt ctc agg ccc gcg ggt acc gcg 204 Arg Trp Glu Pro His Gly Ala Val Arg Leu Arg Pro Ala Gly Thr Ala 35 40 45 gcg ggc gcc gct gcg ctg gcg ctg cag gag ccc ggc ctg tgg ctc gcc 252 Ala Gly Ala Ala Ala Leu Ala Leu Gln Glu Pro Gly Leu Trp Leu Ala 50 55 60 gag gtg gag ctg gag gg gcc ggc ggg gcg gag ccg ggc 300 Glu Val Glu Leu Glu Ala Tyr Glu Glu Ala Gly Gly Ala Glu Pro Gly 65 70 75 cgc gtt gac acg ttc tgg tac aag ttc ctg cag cgc gag cct gga ggc 348 Arg Val Asp Thr Tyr Lys Phe Leu Gln Arg Glu Pro Gly Gly 80 85 90 gag ctg cac tgg gaa gga aat gga cct cac cat gac cgt tgc tgc aca 396 Glu Leu His Trp Glu Gly Asn Gly Pro His His Asp Arg Cys Cys Thr 95 100 105 110 tat aat gag gac aac ttg gtg gat ggt gtt tat tgt ctc cca gta gga 444 Tyr Asn Glu Asp Asn Leu Val Asp Gly Val Tyr Cys Leu Pro Val Gly 115 120 125 cac tgg att gag gcc act ggg cac acc aat gaa atg aag cgc aca aca 492 His Trp Ile Glu Ala Thr Gly His Thr Asn Glu Met Lys Arg Thr Thr 130 135 140 gac ttc tat ttt aat att gct ggc cac caa gcc atg cac tat tca aga 540 Asp Phe Tyr Phe Asn Ile Ala Gly His Gln Ala Met His Tyr Ser Arg 145 150 155 att cta cca aat atc tgg ctg ggt agc tgc cct cgc caa ctg gaa cat 588 Ile Leu Pro Asn Ile Trp Leu Gly Ser Cys Pro Arg Gln Leu Glu His 160 165 170 170 gtg acc atc aaa ctg aag cat gaa ctg gga gtt g atg aat 636 Val Thr Ile Lys Leu Lys His Glu Leu Gly Val Thr Ala Val Met Asn 175 180 185 190 ttc cag act gaa tgg gat atc atc cag aat tct tca ggc tgc aac cgc 684 Phe Gln Thr Glu Trp Asp Ile Ile Gln Asn Ser Ser Gly Cys Asn Arg 195 200 205 tac cct gaa ccc atg act cca gac acc atg atg aag ctg tat aag gaa 732 Tyr Pro Glu Pro Met Thr Pro Asp Thr Met Met Lys Leu Tyr Lys Glu 210 215 220 gaa ggc ttg tcc tac atc tgg atg ccc act cca gac atg agc act gag 780 Glu Gly Leu Ser Tyr Ile Trp Met Pro Thr Pro Asp Met Ser Thr Glu 225 230 235 ggc cga gtg cag atg ctg cca cag gct gtg tgt ctc ctg cac gcg ctt 828 Gly Arg Gln Met Leu Pro Gln Ala Val Cys Leu Leu His Ala Leu 240 245 250 ctg gag aat gga cac acg gtg tat gtc cac tgc aac gct ggc gtg ggt 876 Leu Glu Asn Gly His Thr Val Tyr Val His Cys Asn Ala Gly Val Gly 255 260 265 270 cgc tcc aca gct gca gtg tgc ggc tgg ctc cac tat gtg att ggc tgg 924 Arg Ser Thr Ala Ala Val Cys Gly Trp Leu His Tyr Val Ile Gly Trp 275 280 285 aat ctg cgc aag gtc atg gc gac gac tc aaa agg cct gcg gtc 972 Asn Leu Arg Lys Val Gln Tyr Phe Ile Met Ala Lys Arg Pro Ala Val 290 295 300 tac att gac gag gac gct ttg gct caa gca caa caa gac ttt tct cag 1020 Tyr Ile Asp Glu Asp Ala LeA Gln Ala Gln Gln Asp Phe Ser Gln 305 310 315 aag ttc ggg aag gtt cac tct tcc ata tgc gct ttg taggtgatca 1066 Lys Phe Gly Lys Val His Ser Ser Ile Cys Ala Leu 320 325 330 gccctccatt ccgtctagct gccttaggaa ggagtgag gaggtagaggg gaggt gaggt gggt gcc cctggagatt gccctctatt gtggcttggt 1186 tgggcttgtg tttgaaatgc tttacaaagg aaaactgcat gccacatgag aaaacttcaa 1246 agtacacttg caatgcaagt gtatgcctgt agttgctgct gccaccggca gggggccttt 1306 gcttacaggg tctgcatagg ttgttatgga tttgtgactt tggcatgcag aagaccagag 1366 tcctctcttg ggggttgctg ggaggacttg gtgtgatgat aaagatgaaa gct 1419

<210> 2 <211> 330 <212> PRT <213> Mus musculus <400> 2 Met Leu Phe Arg Phe Gly Val Val Val Pro Pro Ala Val Ala Gly Ala 1 5 10 15 Arg Gln Glu Leu Leu Leu Ala Gly Ser Arg Pro Glu Leu Gly Arg Trp 20 25 30 Glu Pro His Gly Ala Val Arg Leu Arg Pro Ala Gly Thr Ala Ala Gly 35 40 45 Ala Ala Ala Ala Leu Ala Leu Gln Glu Pro Gly Leu Trp Leu Ala Glu Val 50 55 60 Glu Leu Glu Ala Tyr Glu Glu Ala Gly Gly Ala Glu Pro Gly Arg Val 65 70 75 80 Asp Thr Phe Trp Tyr Lys Phe Leu Gln Arg Glu Pro Gly Gly Glu Lelu 85 90 95 His Trp Glu Gly Asn Gly Pro His His Asp Arg Cys Cys Thr Tyr Asn 100 105 110 Glu Asp Asn Leu Val Asp Gly Val Tyr Cys Leu Pro Val Gly His Trp 115 120 125 Ile Glu Ala Thr Gly His Thr Asn Glu Met Lys Arg Thr Thr Asp Phe 130 135 140 Tyr Phe Asn Ile Ala Gly His Gln Ala Met His Tyr Ser Arg Ile Leu 145 150 155 160 Pro Asn Ile Trp Leu Gly Ser Cys Pro Arg Gln Leu Glu His Val Thr 165 170 175 Ile Lys Leu Lys His Glu Leu Gly Val Thr Ala Val Met Asn Phe Gln 180 185 190 Thr Glu Trp Asp Ile Ile Gln Asn Ser Ser Gly Cys Asn Arg Tyr Pro 195 200 205 Glu Pro Met Thr Pro Asp Thr Met Met Lys Leu Tyr Lys Glu Glu Gly 210 215 220 Leu Ser Tyr Ile Trp Met Pro Thr Pro Asp Met Ser Thr Glu Gly Arg 225 230 235 240 Val Gln Met Leu Pro Gln Ala Val Cys Leu Leu His Ala Leu Leu Glu 245 250 255 Asn Gly His Thr Val Tyr Val His Cys Asn Ala Gly Val Gly Arg Ser 260 265 270 Thr Ala Ala Val Cys Gly Trp Leu His Tyr Val Ile Gly Trp Asn Leu 275 280 285 Arg Lys Val Gln Tyr Phe Ile Met Ala Lys Arg Pro Ala Val Tyr Ile 290 295 300 Asp Glu Asp Ala Leu Ala Gln Ala Gln Gln Asp Phe Ser Gln Lys Phe 305 310 315 320 Gly Lys Val His Ser Ser Ile Cys Ala Leu 325 330

<210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for human EPM2A <400> 3 gctctttaca aattcctacc gttctgt 27

<210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for human EPM2A <400> 4 agtgaggcac tgcagtttcg a 21

<210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for human EPM2A <400> 5 tgtgatgggc tggaatctga g 21

<210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for human EPM2A <400> 6 gagcaggaac tgcacgcatt a 21

<210> 7 <211> 14 <212> DNA <213> Mus musculus <400> 7 tttctctagg ccga 14

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1:91) C12R 1:91)

Claims (3)

[Claims] 1. A DNA encoding a protein represented by the following (A) or (B): (A) DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2. (B) a protein consisting of an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, or inversions in the amino acid sequence shown in SEQ ID NO: 2, and having a protein-tyrosine phosphatase activity; 2. The DNA according to claim 1, which is a DNA shown in (a) or (b) below. (A) DNA comprising at least the nucleotide sequence consisting of nucleotide numbers 67 to 1056 in the nucleotide sequence shown in SEQ ID NO: 1. (B) of the nucleotide sequence shown in SEQ ID NO: 1, which hybridizes under stringent conditions with at least the nucleotide sequence consisting of nucleotide numbers 67 to 1056, and
DNA encoding a protein having tyrosine phosphatase activity. 3. The DNA according to claim 1 or 2 is modified so as not to function normally, and the obtained modified DNA is introduced into mouse cells, and a sequence homologous to the DNA according to claim 1 or 2 is modified. A method for producing an epilepsy-causing gene-deficient mouse, comprising recombining an endogenous gene having the modified gene with the modified gene to destroy the endogenous gene.