JPH07203969A - Novel tyrosine phosphatase gene - Google Patents

Abstract

PURPOSE:To provide a novel gene relating to the production of a novel tyrosine phosphatase useful for the searches of neural function disorder-improving medicines, etc., the evaluations of medicines, the researches of drug designs, etc. CONSTITUTION:A tyrosine phosphatase gene coding a polypeptide having an amino acid sequence of the formula. This tyrosine phosphatase cDNA is obtained by screening the cDNA library of a cell originated from a rat adrenal medulla, while using the homology of a conventional tyrosine phosphatase such as STEP as an indicator.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel tyrosine phosphatase gene.

[0002]

BACKGROUND OF THE INVENTION Proteins such as growth factors produced by cells are involved in the differentiation and proliferation of cells. Stimulation of these growth factors causes intracellular phosphorylation and dephosphorylation of various proteins, which causes proliferation. It is known to regulate the signal of. There are protein kinases and protein phosphatases that regulate phosphorylation of this protein, and many genes have been cloned. As one of the protein phosphatases, there is tyrosine phosphatase (PTPase), and the tyrosine phosphatase is classified into two types, intracellular type and transmembrane type. The first isolated tyrosine phosphatase is an intracellular form of human placenta. This tyrosine phosphatase is named PTPase1B (E.
H. Fisher, J. Biol. Chem., Vol 263, No.14, p6722-67
30, 1988), the PTPase1B gene has a high homology with CD45 which is a leukocyte common antigen. Above P
By using the TPase1B gene, P using mixed primers from conserved amino acid sequences with high homology
Tyrosine phosphatase was cloned using CR reaction (Wilks. AF, Methods in Enzimolog
y 200, p533-546, 1991).

Although many tyrosine phosphatases have been cloned by the above method, the functions of most tyrosine phosphatases are unknown. It is considered that intracellular tyrosine phosphatase regulates intracellular signal transduction via phosphorylated tyrosine by tyrosine kinases, but it is a target that controls signal transduction, that is, a specific tyrosine residue that is dephosphorylated. The group has not been elucidated. In addition, transmembrane tyrosine phosphatase is expected to have a receptor function, but to date no transmembrane tyrosine phosphatase with known ligand exists.

As described above, the function of tyrosine phosphatase is currently beyond the scope of analogy. However, the biodistribution of tyrosine phosphatase,
It is becoming clear that tyrosine phosphatase has a cell- or tissue-specific function due to quantitative increase or decrease or qualitative change of tyrosine phosphatase during cell differentiation or cell proliferation. For example, STEP (striatum enriched phosphatas) obtained from bovine striatal tissue is shown to have specific expression in nerve tissue.
e) has been reported to be expressed specifically in brain tissue (M. Le.
rner, Proc. Natl. Acad. Sci. USA, vol 88, p72
42-7246, 1991). In human tissues, He PTPase (B.Zunk
e, Eur.J.Immunol, 22, p235-239,1992) and LC-PTP (M.Adach
i, Biochem.Biophys.Res.Commun.vol 186, No.3, p16
07-1615,1992) has been reported.

[0005] In addition to the above, intracellular type tyrosine phosphatase showing brain tissue-specific expression has not been reported. At present, two types of transmembrane tyrosine phosphatases have been reported (H. Saito, Proc. Natl. Aca
d. Sci. USA, vol 89, p7417-7421,1992; J. Schle
ssinger, J. Biol. Chem., vol 268, No.14, p10573-105
81, 1993). As described above, tyrosine phosphatase (eg, STEP) is considered to control the signal transduction via tyrosine kinase in nerve cells.
It is completely unknown what role tyrosine phosphatase plays in nerve cells.
To clarify this mechanism, an in vitro experimental system is required.

[0006]

The object of the present invention is to provide a novel tyrosine phosphatase that is structurally different from conventional tyrosine phosphatase and is expressed specifically in brain tissue, or a polypeptide having the same function as the phosphatase. To provide the encoding gene.

[0007]

As a result of intensive studies on tyrosine phosphatase, the present inventors have found that a cDNA library of cells derived from rat adrenal medulla using the homology of the known tyrosine phosphatase (for example, STEP) as an index. And a novel tyrosine phosphatase gene cDNA from a human brain-derived cell cDNA library
Isolated and cloned (hereinafter, the clone isolated from rat adrenal medulla-derived cells is referred to as PCPTP1 and the clone isolated from human brain-derived cells is referred to as HPCPTP1). ・ The full-length nucleotide sequence was determined. The present invention was completed by finding that it is specifically expressed in brain tissue. The novel tyrosine phosphatase gene provided by the present invention is a gene for tyrosine phosphatase containing a nucleotide sequence encoding a polypeptide consisting of the group of amino acid sequences set forth in SEQ ID NOS: 2 and 4 of the Sequence Listing, SEQ ID NO: 1 in the Sequence Listing. Or a gene encoding the tyrosine phosphatase described in 3 above.

The method for producing the tyrosine phosphatase gene or the polypeptide of the present invention will be described in detail below. Rat adrenal medulla-derived cells (hereinafter P
It is called C12h cell. Also, PC12h cells have been deposited as FERM P-13913 at the Institute of Biotechnology and Industrial Technology. A series of molecules for producing a cDNA required for PCPTP1 cDNA cloning or HPCTPTP1 cDNA cloning, Northern blotting expression study, hybridization screening, recombinant DNA production, DNA nucleotide sequence determination, etc. Biological experiments are based on the publicly known experimental book (Molecula
r Cloning, A laboratory Manual, 1989, Eds., Sambroo
k, J. Fritsch, EF, Maniatis, T., Cold Spring Har
bor Laboratory Press, etc.).

Method for Extracting mRNA The tyrosine phosphatase DNA of the present invention includes cells encoding the phosphatase from cells having the phosphatase-producing ability, for example, PC12h cells.
It can be extracted from RNA or commercially available mRNA by a conventional method such as the method described in the above experimental manual. As an extraction method,
Guanidine thiocyanate hot phenol method,
The guanidine-thiocyanate-cesium chloride method and the like can be mentioned, and the guanidine-thiocyanate-cesium chloride method is preferable. Purification method of mRNA Purification of mRNA may be carried out according to a conventional method, for example, it can be purified by adsorbing and eluting mRNA on an oligo (dT) cellulose column. Furthermore, mRNA can be further fractionated by a sucrose density gradient centrifugation method or the like.

Preparation method of cDNA Preparation of cDNA and preparation of cDNA library
It can be carried out by a conventional method such as the method described in the above experimental book. Single-stranded cDNA can be prepared, for example, by subjecting purified mRNA to a reverse transcriptase reaction in the presence of a random primer or an oligo dT primer. This single-stranded cDNA
A to S1 nuclease method (Efstratiadis, A. et al.,
Cell, vol 7, p279-288, 1976), etc.
NA can be created. The method of preparing a cDNA library includes two methods, that is, a method using a plasmid and a method using λ phage, and the method using λ phage is preferable. As a method of using a plasmid and increasing the efficiency, there is the method of Okayama-Berg et al. (Mol. Cell. Biol., Vol 2, p161-170, 1982).

Method for preparing tyrosine phosphatase-specific probe Tyrosine phosphatase-specific primers are synthetic D
By the NA method, a commercially available DNA synthesizer is used to prepare an oligonucleotide, and the oligonucleotide is purified by a predetermined purification method, followed by PC.
It can be used in the step of growing a specific site by R. About 380 bp of tyrosine phosphatase domain can be amplified by PCR using these primers. This PCR product is separated by electrophoresis and cut out from the gel to recover a DNA fragment. Recovered DNA
The fragment is used as a probe to search the cDNA library. By labeling a DNA fragment used as a probe with an isotope, highly sensitive screening becomes possible. As the isotope labeling, for example, a method of end-labeling using [ ³² P] γ-ATP and T4 polynucleotide kinase, or labeling by other nick translation method or primer extension method may be used, but a random primer is used. The labeling method described above can produce a probe with higher efficiency and higher specific activity.

Cloning of a novel tyrosine phosphatase By comparing the amino acid sequences of the tyrosine phosphatase family, it can be seen that the approximately 150 amino acid region upstream of its active center is a region of high homology. Based on this fact, RT-PCR was carried out using an oligonucleotide primer prepared by analogy with the base sequence of the amino acid from the conserved amino acid sequence, whereby various tyrosine phosphatases having the conserved amino acid moiety at both ends were obtained. It is known that a partial gene of ze can be obtained.
PC12h which is a cell line derived from rat using this method
Various tyrosine phosphatase partial genes were obtained from cells and further synthesized from PC12h cell mRNA.
A target tyrosine phosphatase cDNA can be obtained from a DNA library by screening with the above-mentioned partial gene of tyrosine phosphatase as a probe.

As a method for cloning the tyrosine phosphatase gene, a method of deducing the gene sequence from the amino acid sequence of the purified protein as described below and screening with a synthetic oligonucleotide as a probe is considered.
That is, the protein is purified using the activity of tyrosine phosphatase as an index, and the amino acid sequence on the amino terminal side is determined by a known method. The corresponding oligonucleotide is synthesized based on this amino acid sequence (in this case, the method of mixing multiple possible nucleotides using the codon usage is generally used, but using inosine to reduce the combination) It is also possible to use this as a probe (labeled with ³² P or ³⁵ S) to transform the transformed strain D.
NA is hybridized with a denaturation-immobilized nitrocellulose filter, and the obtained positive strain is searched and selected. The cDNA base sequences of novel tyrosine phosphatases (PCPTP1 and HPCPTP1) elucidated by the present inventors are shown in SEQ ID NOs: 1 and 3 of the sequence listing as DN.
The coding strand (RNA-like) of the double strand of A is shown. The base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 in the sequence listing are from the first ATG encoding cDNA to the termination codon TGA.

Expression of novel tyrosine phosphatase The expression of tyrosine phosphatase is carried out by a conventional method by inserting the tyrosine phosphatase DNA nucleotide sequence of the present invention into an appropriate expression vector to obtain a microorganism or cell (for example, insect cell or This can be confirmed by obtaining a transformant using an animal cultured cell) or an individual such as a mouse as a host, and showing tyrosine phosphatase activity in the culture supernatant of the transformed cell and in the transformed animal. The obtained transformed cells and transformed animals expressing the gene of the present invention are PC
It can be used for screening studies of inhibitors, activators for PTP1 or HPCPTP1 or for drug design studies. As animal culture cells used as a host, mouse L, mouse 3T3, mouse myeloma, CHO, C, which are easily available, are available.
HL, Hela, COS, PC12h, SKNMC, human myeloma cells and the like can be mentioned. Examples of insect cells include Sf-9 cells derived from Spodoptera frugiperda. As the expression vector, a known vector developed for each host can be used. For example, when using E. coli as a host, pGEX-2T, pGEX-
Vectors of the pGEX system such as 3X are effective. Also,
When COS cells are used as a host, pCDL type vectors and pCDM8 vectors can be used. Also C
PCD when HO cells or PC12h cells are used as hosts
Vectors such as L-based vectors can be used.

The cD of PCPTP1 revealed by the present inventors
The amino acid sequence of NA is shown in SEQ ID NO: 2 in the sequence listing, and
The amino acid sequence of HPCPTP1 cDNA is shown in SEQ ID NO: 4 in the sequence listing by the three-letter code of amino acids. P of the present invention consisting of the amino acid sequence shown by SEQ ID NO: 2 in the sequence listing
The CPTP1 polypeptide or the HPCPTP1 polypeptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 4 does not impair the activity as tyrosine phosphatase, and one or more amino acids Mutant polypeptides having mutations such as deletions, additions or substitutions, that is, mutant polypeptides found in nature or artificially modified mutant polypeptides are included. Further, the DNA according to the present invention encoding the above-mentioned polypeptide includes a cDNA encoding the desired PCPTP1 or HPPCTP1, a chromosomal DNA containing an intron and an exon, a DNA in which an intron of the same chromosome is removed and an exon is linked, and Synthetic DN
DNAs and the like obtained by ligating oligonucleotides artificially produced by Method A are included.

According to the method of artificially producing oligonucleotides by the synthetic DNA method and ligating them to prepare synthetic DNA, the degeneracy of the genetic code allows the nucleotide sequence of the gene to be changed without changing the amino acid sequence. The target recombinant DNA can be prepared by changing the DNA. The DNA of the present invention includes all nucleotide sequences based on the degeneracy of the gene encoding the polypeptide of interest. Recombinant DNA encoding a mutated polypeptide, such as chromosomal DNA or cDNA, is used as chromosomal DNA or cDNA encoding mutated PCPTP1 or HPCPTP1 having an amino acid level mutation based on naturally occurring intraspecific or allelic mutations, and artificially. Mutation P that has a mutation that can be produced by the point mutation method
Chromosome DN encoding CPTP1 or HPCPTP1
A or cDNA, or mutant P prepared by the synthetic DNA method
Recombinant DN encoding CPTP1 or HPCPTP1
Obtained as A. Even if there is no change at the amino acid level,
In chromosomal DNA or cDNA separated from the natural world, there are often cases in which the DNA base sequence is mutated without changing the amino acid sequence due to the degeneracy of the genetic code. Such nucleotide sequence mutations are included in the present invention.

[0017]

INDUSTRIAL APPLICABILITY The PCPTP1 gene of the present invention is the result of Northern hybridization with rat organs.
Strong detection at 9 kb (Fig. 1) and HPPCTP
One gene is a neuron-specific tyrosine phosphatase gene, since mRNA expression was brain-specific as a result of Northern hybridization to a human MTN membrane (FIG. 2). Therefore, the present invention is
The tyrosine phosphatase is prepared by utilizing DNA encoding CPTP1 and HPCPTP1, a recombinant DNA or cDNA base sequence and a gene recombination technique,
Useful for the search for inhibitors or activators that inhibit or activate signal transduction in nerve cells related to tyrosine phosphatase, such as drugs for improving neurological dysfunction, drug evaluation, drug design research, and elucidation of signal transduction in nerve cells Is.

[0018]

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the present invention. Example 1 Determination of nucleotide sequence of PCPTP1 Extraction of total RNA From 1 × 10 ⁸ rat adrenal medulla-derived PC12h cells (FERM P-13913), guanidine thiocyanate / cesium chloride method (the above MOLECULAR CLONING,
Total RNA was extracted according to 7.19). Purification of mRNA Most of the mRNAs are known to have a poly A sequence on the 3'-terminal side, and thus mRNA was adsorbed on a column of oligo dT cellulose and eluted and purified. Synthesis of cDNA Using 1 μg of this mRNA as a template, a cDNA synthesis kit (Boehringer Mannheim) (cDNA Synthesi
Single-stranded c using random primers according to
DNA was synthesized.

Amplification of specific DNA fragment by PCR Mixed primers, PTPF and PTPR, were synthesized from the sequence of amino acids conserved in tyrosine phosphatase. The sequence of PTPF is 5'-TT (C / T) TG (A / G) C (A / G /
T) (A / G / C / T) ATGGT (A / G / C / T) TGGCA-3 'PTPR sequence is 5'
-CGC CC (A / G) AT (A / G / C / T) CC (A / G / C / T) GC (A / G / C / T) CT (A /
G) CAGTG-3 '. The synthetic oligonucleotide was prepared using a fully automatic DNA synthesizer based on the solid phase method.
As a fully automatic DNA synthesizer, Model 394 manufactured by Applied Biosystems was used. The reaction solution containing the nucleic acid was then incubated in a sealed vial with concentrated ammonia solution at 55 ° C. for 12 hours in order to release the protecting groups on the nucleic acid and the phosphoric acid. Purification of each oligonucleotide in which the carrier and the protecting group were released was carried out using an OPC cartridge manufactured by Applied Biosystems, and detritylated with 2% trifluoroacetic acid. Amplification by PCR was performed as follows. That is, 2μ of the obtained cDNA
10 l buffer (100 mM Tris-HCl pH 9.0,500)
mMKCl, 10% Triton-100), 25 mM MgCl ₂ 10μ
l, dNTP (2.5 mM) 10 μl, and TaqDN
1 μl of A polymerase (5 U / ul) was added, and finally 67 μl of deionized sterilized water was added to make the total volume 100 μl.
A cycle consisting of 94 ° C. for 1 minute, 37 ° C. for 2 minutes, and 72 ° C. for 2 minutes was defined as one cycle, and this step was repeated 30 times. A part of the PCR product was separated on a 1% agarose gel, and it was confirmed that a DNA of about 380 bp was amplified.

Approximately 380 bp of cDNA was obtained by the above PCR process.
After confirming that A was amplified, the DNA fragment was gel-extracted and purified according to a conventional method. This purified DNA
Using 100 ng of the fragment, pCRII vector (TA cloning
kit: Invitrogen) and subcloned into E. coli J
Among the colonies generated by transforming M109, cDNA
The one in which A was incorporated was selected. Each cDNA
Of the 45 clones, 19 of which were novel genes, were highly homologous to the tyrosine phosphatase domain, and were predicted to belong to the tyrosine phosphatase family. went.

Cloning of PCPTP1 When cloning the gene of PCPTP1,
12h cells were used as a cDNA library source, and the gene fragment of the novel clone was labeled with ³² P and used as a probe. The probe was prepared as follows. That is,
The plasmid containing the fragment of the above new clone was digested with restriction enzyme E
It was digested with coRI, cut out from the vector, and subjected to gel extraction by a conventional method for purification. The obtained DNA fragment is DNA
Labeling kit (BcaBEST DNA labeling system: Takar
a company). Add 10 μl of random primer solution and sterile distilled water to 125 ng of DNA, and add 2 to the total volume.
The volume was adjusted to 5 μl, boiled for 3 minutes, and then rapidly cooled. Then, 12.5 μl of reaction buffer, 12.5 μl of dNTP mixture, [α
25 μl of ³² P] dCTP and BcaBEST DN
After adding 5 ul of A polymerase and reacting at 53 ° C. for 30 minutes, it was boiled for 3 minutes and ice-cooled and used as a probe.

The cDNA library was prepared as follows. That is, total RNA was extracted from PC12h cells by the guanidinic acid thiocyanate and cesium chloride method, and oligo dT cellulose column (oligo dT cellulose type3: c
PolyA + RNA was purified using ollaborative biorab). cDNA synthesis system (ZAP-cDNA synthes
A cDNA library was prepared according to is kit: strategene. Escherichia coli XL1-Blue was infected with the phage containing this cDNA, 3 × 10 ⁵ plaques were made to appear on the plate, and candidate recombinant phages were screened by the plaque hybridization method shown below. Phage plaques that appeared were transferred to a nylon filter (Biodyne A BNGNG137; Nippon Pall), and the transferred nylon filter was denatured with a denaturing buffer solution (1.5M NaCl, 0.5M NaOH) for 3 minutes to neutralize. Buffer (1.5M NaCl / 0.5M Tris-HCl pH 8.0)
Neutralize for 5 minutes with 2xSSC / 0.2M Tris-
It was washed with HCl, pH 7.5 for 3 minutes and air dried.

UV-X the dried nylon membrane
Cross-linking was performed with a linker (strategene), and then prehybridization solution (6xSSC,
0.1% SDS, 0.2% Ficoll, 0.2% Polyvinylpyrrolidone, 0.2
% BSA, 50ug / ml salmon spar DNA) at 65 ℃,
After incubation for 4 hours, prehybridization was performed. To the filter after prehybridization, add 200 ml of the same composition as the prehybridization solution containing ³² P-labeled probe,
Hybridization was performed by incubating at 16 ° C. for 16 hours. Next, the filter is 2xSSC / 0.1% S
Wash with DS solution at 65 ℃ for 10 minutes, then 0.5x
The plate was washed twice with an SSC / 0.1% SDS solution at 65 ° C for 20 minutes. Further, it was washed twice with a 0.2 × SSC / 0.1% SDS solution at 65 ° C. for 30 minutes, and the filter was air-dried and then exposed to an X-ray film. The exposure was carried out at −80 ° C. for 2 days. As a result of autoradiography, 4 positive clones were obtained. Scrap the agar from this plaque S
After suspension in an M (100 mM NaCl, 8 mM MgSO ₄ .7H ₂ O) solution, this phage solution was used to perform a second screening in the same manner as in the above method to purify a single target phage.

This phage solution was added to E. coli XL1-BLUE
The strain was infected with the helper phage EXASSIST to excise it into a phagemid containing the cloned inserted gene. The solution containing this phagemid was heat-treated at 70 ° C. to kill E. coli, and then again infected with a new E. coli SOLR strain and plated, to obtain colonies of E. coli containing phagemid. A phagemid containing the gene of interest was isolated from these colonies, and the length of this gene was determined. The longest was 3.6 kb.
It turned out to have the gene of. This gene is PCPT
It was named P1. The whole base sequence of PCPTP1 was determined by the dideoxy method using synthetic primers. The nucleotide sequence presumed to correspond to the more mature protein is shown in SEQ ID NO: 1 in the sequence listing. Nucleotide sequence analysis The amino acid sequence encoded by the determined nucleotide sequence may have a tyrosine phosphatase domain (299 His to 306 Arg) containing the active center reported in the tyrosine phosphatase family. Is recognized.

Hybridization with mRNA Expression of mRNA was examined by Northern blotting. Rat heart, skeletal muscle, fat, spleen, kidneys, lungs,
Poly (A) ⁺ from brain, liver, and cell line PC12h
RNA was prepared and placed on 1.0% agarose at 3 μg / lane to give 50 mM MOPS, 1 mM E.
DTA, 20xSSPE (3.6M NaCl, 0.2
M sodium phosphate pH 7.7, 0.02M EDT
A), electrophoresed in 2.2M formaldehyde.
After completion of the electrophoresis, capillary blotting was carried out using 20 × SSPE, and transferred to a nylon membrane (Hybond-N membrane: Amersham). Hybridization was performed using the transferred membrane. The same procedure was performed using the above-mentioned DNA labeling kit with the 3.6 kb gene obtained by the above screening as a probe as a source.

The hybridization solution was 50% formamide, 5xSSPE, 5xdenhardt (0.
1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA),
Salmon spalm DNA denatured with 0.5% SDS and heat
(200 μg / ml). Prehybridization at 42 ° C for 2 hours, hybridization at 4
It was carried out at 2 ° C for 16 hours. Also, the membrane cleaning is 2
xSSPE / 0.1% SDS 10 minutes at room temperature, 1x
It was washed with SSPE / 0.1% SDS at 65 ° C. for 15 minutes, further washed with 0.5 × SSPE / 0.1% SDS at 65 ° C. for 15 minutes, dried in air, and autoradiography was performed using an intensifying screen. As a result, the size of cDNA was found to be about 3.9 kb in PC12h cells (Fig. 1).

Example 2 Determination of base sequence of HPCPTP1 Cloning of human homolog using PCPTP1 as a probe Human whole brain mRNA was purchased from CLONTECH.
Based on this mRNA, a cDNA synthesis system (ZAP-
cDNA synthesis kit (strategene)
A library was prepared. In cloning the full-length gene, the PCPTP1 gene described in Example 1 above was labeled with ³² P and used as a probe. The probe was prepared as follows. That is, the above PCPTP1
The gene is labeled with the DNA labeling kit (BcaBEST DNA labe
lling system: Takara). DNA 1
To 25 ng, 10 μl of the random primer solution and sterile distilled water were added to make a total volume of 25 μl, which was boiled for 3 minutes and then rapidly cooled.

Thereafter, 12.5 μl of reaction buffer and dNTP
12.5 μl of the mixed solution, 25 μl of [α ³² P] dCTP and 5 μl of BcaBEST DNA polymerase were added, reacted at 53 ° C. for 30 minutes, boiled for 3 minutes, cooled with ice,
Used as a probe. E. coli XL1-Blue was infected with this human whole brain cDNA-containing phage, and 3 × 10
^Five plaques were made to appear on the plate, and a candidate recombinant phage was screened by the plaque hybridization method shown below. Phage plaques that appeared were nylon filters (Biodyne A BN
NG137; Pall Japan Co., Ltd.) and transferred the nylon filter to a denaturing buffer solution (1.5 M NaCl, 0.
Denaturing treatment with 5M NaOH for 3 minutes, neutralization buffer (1.5M NaCl / 0.5M Tris-Cl)
Neutralization treatment for 5 minutes at pH 8.0), 2xSSC / 0.2
It was washed with M Tris-Cl pH 7.5 for 3 minutes and air dried.

UV-X the dried nylon membrane
Cross-linking was performed with a linker (strategene), and then prehybridization solution (6xSSC, 0.
1% SDS, 0.2% Ficoll, 0.2% Polyvinylpyrrolidone, 0.2% BSA,
Pre-hybridization was carried out by incubating at 50 ° C. for 4 hours in 50 ug / ml salmon spar DNA. Add 200 ml of the same composition as the prehybridization solution containing ³² P-labeled probe to the filter after prehybridization,
Hybridization was carried out by incubating for 16 hours. Then filter 2x SSC / 0.1% SDS
Wash with solution at 65 ℃ for 10 minutes, then 0.5xSSC
/0.1% SDS solution wash | cleaned twice at 65 degreeC for 20 minutes. 65 with 0.2xSSC / 0.1% SDS solution
The filter was washed twice at 30 ° C. for 30 minutes, air-dried, and then exposed to an X-ray film. The exposure was carried out at −80 ° C. for 2 days.

As a result of autoradiography, 4 positive clones were obtained. After suspending agar portion of this plaque scraping _{SM (100mM NaCl, 8mM MgSO 4} · 7H 2 O) was added,
Using this phage solution, the second screening was carried out in the same manner as in the above method to purify the target phage into a single one. Escherichia coli XL1-BLUE strain was infected with this phage solution in the coexistence of helper phage EXASSIST to excise a phagemid containing the cloned inserted gene. A solution containing this phagemid is heat-treated at 70 ° C. to kill E. coli, then re-infected with a new E. coli SOLR strain and plated.
E. coli colonies containing phagemid were obtained. When a phagemid containing the gene of interest was isolated from these colonies and the length of this gene was determined, it was found that the longest one had a gene of 2.8 kb. This gene was named HPCPTP1. The total nucleotide sequence of this cDNA was determined by the dideoxy method using synthetic primers. A part of the gene sequence that is considered to encode an amino acid is shown in the nucleotide sequence of SEQ ID NO: 3 in the sequence listing.

Analysis of nucleotide sequence In the amino acid sequence encoded by the determined nucleotide sequence, the tyrosine phosphatase domain containing the active center reported in the tyrosine phosphatase family contains 299th His to 306th Arg. Is allowed to do. The homology with the rat PCPTP1 gene was 89% identical in DNA and 93% identical in amino acid.

Hybridization with mRNA Expression was examined by Northern blotting. Commercially available human MTN (Lot No. 39611, CLONTEC
(Manufactured by Company H) as a source, hybridization is carried out at 42 ° C. for 16 hours, and washing is carried out with 2 × SSC, 0.05.
% SDS at room temperature for 30 minutes, 0.5xSSC, 0.1%
SDS at room temperature for 15 minutes, then 0.5% SDS, 0.1
% SDS was performed at 50 ° C. for 15 minutes. As a probe, the 2.8 kb gene obtained by the above screening was used as a source and the above DNA labeling kit was used in the same manner. Next, the membrane hybridized with the ³² P-labeled probe was subjected to autoradiography using an intensifying screen. As a result, mRNA was specifically expressed in the brain, and the size of its cDNA was about 3.9 kb (Fig. 2).

[Sequence list]

SEQ ID NO: 1 Sequence length: 1110 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Origin organism name: Rat strain name: PC12h cell sequence ATGGTCCAGC CCGACCAGGC GCCAAAGGTG CTGAATGTTG TTGTGGACCC TCAAGGCCAA 60 TGCACTCCTG AGATTCGAAA CACCGCCTCC ACTTCTGTCT GCCCTTCTCC CTTCAGGATG 120 AAGCCCATAG GACTCCAGGA GCGACGAGGT TCCAATGTAT CTCTTACACT GGACATGAGT 180 AGCCTGGGCA ACGTGGAACC CTTTGTGGCC GTCTCCACCC CCCGGGAGAA GGTAGCCATG 240 GAGTACCTGC AGTCAGCCAG CCGAGTTCTC ACAAGTCCAC AGCTGAGGGA CGTCGTGGCA 300 AGTTCCCACC TGCTTCAAAG TGAATTCATG GAAATACCAA TGAACTTTGT GGATCCCAAA 360 GAAATTGATA TTCCACGTCA TGGAACTAAA AATCGTTATA AGACCATTTT GCCAAATCCC 420 CTCAGCAGGG TGTGCTTAAG ACCAAAAAAT ATAACTGACC CATTGAGTAC TTACATCAAT 480 GCTAATTACA TTCGGGGCTA CAGTGGCAAG GAGAAAGCCT TCATTGCCAC ACAGGGCCCC 540 ATGATCAACA CTGTGAATGA CTTCTGGCAG ATGGTTTGGC AAGAAGACAG TCCTGTGATT 600 GTTATGATCA CGAAACTCAAAAAGAGAAAA TGTG TGCTCTACTG GCCAGAAAAG 660 AGAGGGATTT ACGGCAAGGT TGAGGTTCTG GTCATCGGTG TCAATGAATG TGATAACTAC 720 ACCATCCGCA ACCTCGTCTT GAAGCGAGGA AGTCACACCC AACATGTGAA GCATTACTGG 780 TACACTTCAT GGCCCGATCA CAAGACTCCA GACAGTGCCC AGCCCCTTCT GCAGCTCATG 840 CTGGACGTAG AGGAAGACAG ACTGGCCTCT GAAGGCCGAG GGCCTGTGGT TGTCCACTGC 900 AGTGCAGGGA TTGGGAGAAC CGGATGTTTC ATCGCTACAT CCATTGGCTG TCAACAGTTG 960 AAAGAAGAAG GAGTCGTAGA TGCACTAAGT ATCGTCTGCC AGCTTCGTGT AGACAGGGGT 1020 GGAATGGTCC AAACCAGTGA GCAGTATGAA TTTGTGCACC ACGCTCTGTG CCTGTTCGAG 1080 AGCAGACTTT CGCCAGAAAC GGTCCAGTGA 1110

SEQ ID NO: 2 Sequence length: 369 Sequence type: Amino acid Topology: Linear sequence type: Protein Origin organism name: Rat strain name: PC12h cell sequence Met Val Gln Pro Asp Gln Ala Pro Lys Val Leu Asn Val Val Val Asp 1 5 10 15 Pro Gln Gly Gln Cys Thr Pro Glu Ile Arg Asn Thr Ala Ser Thr Ser 20 25 30 Val Cys Pro Ser Pro Phe Arg Met Lys Pro Ile Gly Leu Gln Glu Arg 35 40 45 Arg Gly Ser Asn Val Ser Leu Thr Leu Asp Met Ser Ser Leu Gly Asn 50 55 60 Val Glu Pro Phe Val Ala Val Ser Thr Pro Arg Glu Lys Val Ala Met 65 70 75 80 Glu Tyr Leu Gln Ser Ala Ser Arg Val Leu Thr Ser Pro Gln Leu Arg 85 90 95 Asp Val Val Ala Ser Ser His Leu Leu Gln Ser Glu Phe Met Glu Ile 100 105 110 Pro Met Asn Phe Val Asp Pro Lys Glu Ile Asp Ile Pro Arg His Gly 115 120 125 Thr Lys Asn Arg Tyr Lys Thr Ile Leu Pro Asn Pro Leu Ser Arg Val 130 135 140 Cys Leu Arg Pro Lys Asn Ile Thr Asp Pro Leu Ser Thr Tyr Ile Asn 145 150 155 160 Ala Asn Tyr Ile Arg G ly Tyr Ser Gly Lys Glu Lys Ala Phe Ile Ala 165 170 175 Thr Gln Gly Pro Met Ile Asn Thr Val Asn Asp Phe Trp Gln Met Val 180 185 190 Trp Gln Glu Asp Ser Pro Val Ile Val Met Ile Thr Lys Leu Lys Glu 195 200 205 Lys Asn Glu Lys Cys Val Leu Tyr Trp Pro Glu Lys Arg Gly Ile Tyr 210 215 220 Gly Lys Val Glu Val Leu Val Ile Gly Val Asn Glu Cys Asp Asn Tyr 225 230 235 240 Thr Ile Arg Asn Leu Val Leu Lys Arg Gly Ser His Thr Gln His Val 245 250 255 Lys His Tyr Trp Tyr Thr Ser Trp Pro Asp His Lys Thr Pro Asp Ser 260 265 270 Ala Gln Pro Leu Leu Gln Leu Met Leu Asp Val Glu Glu Asp Arg Leu 275 280 285 Ala Ser Glu Gly Arg Gly Pro Val Val Val His Cys Ser Ala Gly Ile 290 295 300 Gly Arg Thr Gly Cys Phe Ile Ala Thr Ser Ile Gly Cys Gln Gln Leu 305 310 315 320 Lys Glu Glu Gly Val Val Asp Ala Leu Ser Ile Val Cys Gln Leu Arg 325 330 335 Val Asp Arg Gly Gly Met Val Gln Thr Ser Glu Gln Tyr Glu Phe Val 340 345 350 His His Ala Leu Cys Leu Phe Glu Ser Arg Leu Ser Pro Glu Thr Val 355 360 365 Gln 369

SEQ ID NO: 3 Sequence length: 1110 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Origin organism name: Human sequence ATGGTCCAAC CTGAGCAGGC CCCAAAGGTA CTGAATGTTG TCGTGGACCC TCAAGGCCGA 60 GGTGCTCCTG AGATCAGAGC TACCACCGCT ACCTCTGTTT GCCCTTCTCC TTTCAAAATG 120 AAGCCCATAG GACTTCAAGA GAGAAGAGGG TCCAACGTAT CTCTTACATT GGACATGAGT 180 AGCTTGGGGA ACATTGAACC CTTTGTGTCT ATACCAACAC CACGGGAGAA GGTAGCAATG 240 GAGTATCTGC AGTCAGCCAG CCGAATTCTC ACAAGGTCTC AGCTGAGGGA CGTCGTGGCA 300 AGTTCACATT TACTCCAAAG TGAATTCATG GAAATACCGA TGAACTTTGT GGATCCCAAA 360 GAAATTGATA TTCCGCGTCA TGGAACTAAA AATCGCTATA AGACCATTTT ACCAAATCCC 420 CTCAGCAGAG TGTGTTTAAG ACCAAAAAAT GTAACCGATT CATTGAGCAC CTACATTAAT 480 GCTAATTATA TTAGGGGCTA CAGTGGCAAG GAGAAAGCCT TCATTGCCAC GCAGGGCCCC 540 ATGATCAACA CCGTGGATGA TTTCTGGCAG ATGGTTTGGC AGGAAGACAG CCCTGTGATT 600 GTTATGATCA CAAAACTCAA AGAAAAAAAT GAGAAATGTG TGCTATACTG GCCGGAAAAG 660 AGAG GGATAT ATGGAAAAGT TGAGGTTCTG GTTATCAGTG TAAATGAATG TGATAACTAC 720 ACCATTCGAA ACCTTGTCTT AAAGCAAGGA AGCCACACCC AACATGTGAA GCATTACTGG 780 TACACCTCAT GGCCTGATCA CAAGACTCCA GACAGTGCCC AGCCCCTCCT ACAGCTCATG 840 CTGGATGTAG AAGAAGACAG ACTTGCTTCC CAGGGCCGAG GGCCTGTGGT TGTCCACTGC 900 AGTGCAGGAA TAGGTAGAAC AGGGTGTTTT ATTGCTACAT CCATTGGCTG TCAACAGCTG 960 AAAGAAGAAG GAGTTGTGGA TGCACTAAGC ATTGTCTGCC AGCTTCGTAT GGATAGAGGT 1020 GGAATGGTCC AAACCAGTGA GCAGTATGAA TTTGTGCACC ATGCTCTGTG CCTGTATGAG 1080 AGCAGACTTT CAGCAGAGAC TGTCCAGTGA 1110

SEQ ID NO: 4 Sequence length: 369 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Human sequence Met Val Gln Pro Glu Gln Ala Pro Lys Val Leu Asn Val Val Val Asp 1 5 10 15 Pro Gln Gly Arg Gly Ala Pro Glu Ile Arg Ala Thr Thr Ala Thr Ser 20 25 30 Val Cys Pro Ser Pro Phe Lys Met Lys Pro Ile Gly Leu Gln Glu Arg 35 40 45 Arg Gly Ser Asn Val Ser Leu Thr Leu Asp Met Ser Ser Leu Gly Asn 50 55 60 Ile Glu Pro Phe Val Ser Ile Pro Thr Pro Arg Glu Lys Val Ala Met 65 70 75 80 Glu Tyr Leu Gln Ser Ala Ser Arg Ile Leu Thr Arg Ser Gln Leu Arg 85 90 95 Asp Val Val Ala Ser Ser His Leu Leu Gln Ser Glu Phe Met Glu Ile 100 105 110 Pro Met Asn Phe Val Asp Pro Lys Glu Ile Asp Ile Pro Arg His Gly 115 120 125 Thr Lys Asn Arg Tyr Lys Thr Ile Leu Pro Asn Pro Leu Ser Arg Val 130 135 140 Cys Leu Arg Pro Lys Asn Val Thr Asp Ser Leu Ser Thr Tyr Ile Asn 145 150 155 160 Ala Asn Tyr Ile Arg Gly Tyr Ser Gly Lys Glu Lys Ala Phe Ile Ala 165 170 175 Thr Gln Gly Pro Met Ile Asn Thr Val Asp Asp Phe Trp Gln Met Val 180 185 190 Trp Gln Glu Asp Ser Pro Val Ile Val Met Ile Thr Lys Leu Lys Glu 195 200 205 Lys Asn Glu Lys Cys Val Leu Tyr Trp Pro Glu Lys Arg Gly Ile Tyr 210 215 220 Gly Lys Val Glu Val Leu Val Ile Ser Val Asn Glu Cys Asp Asn Tyr 225 230 235 240 Thr Ile Arg Asn Leu Val Leu Lys Gln Gly Ser His Thr Gln His Val 245 250 255 Lys His Tyr Trp Tyr Thr Ser Trp Pro Asp His Lys Thr Pro Asp Ser 260 265 270 Ala Gln Pro Leu Leu Gln Leu Met Leu Asp Val Glu Glu Asp Arg Leu 275 280 285 Ala Ser Gln Gly Arg Gly Pro Val Val Val His Cys Ser Ala Gly Ile 290 295 300 Gly Arg Thr Gly Cys Phe Ile Ala Thr Ser Ile Gly Cys Gln Gln Leu 305 310 315 320 Lys Glu Glu Gly Val Val Asp Ala Leu Ser Ile Val Cys Gln Leu Arg 325 330 335 Met Asp Arg Gly Gly Met Val Gln Thr Ser Glu Gln Tyr Glu Phe Val 340 345 350 His His Ala Leu Cys Leu Tyr Glu Ser Arg Leu Ser Ala Glu Thr Val 355 360 365 Gln 369

[Brief description of drawings]

FIG. 1 shows the results of detection of PCPTP1 mRNA by Northern blotting.

FIG. 2 shows the results of detection of HPCPTP1 mRNA by Northern blotting.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12R 1:91) (C12N 9/12 C12R 1:91) C12R 1:91)

Claims (5)

[Claims] 1. A tyrosine phosphatase gene containing a nucleotide sequence encoding a polypeptide consisting of the group of amino acid sequences set forth in SEQ ID NOS: 2 and 4 of the Sequence Listing. 2. A gene for tyrosine phosphatase, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing. 3. A tyrosine phosphatase gene containing a nucleotide sequence encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing. 4. A gene encoding the tyrosine phosphatase described in SEQ ID NO: 1 in the sequence listing. 5. A gene encoding a tyrosine phosphatase set forth in SEQ ID NO: 3 in the sequence listing.