JPH09206080A - New gene being in domain in which causative gene of werner syndrome exists, ws-2 and protein coded by the same gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene as a causative gene of Werner diseases useful for manufacturing a medicine for detecting the cause of a disease such as sterilitas and remitting the disease. SOLUTION: Gene WS-2 codes a polypeptide substantially containing an amino acid sequence of formula I or an amino acid sequence of formula II as its variant sequence. A coding map of a gene domain (WS domain) is formed by a position cloning method and detection of cDNA and determination of a base sequence is carried out by using a cloned PI/PAC DNA constituting the map to obtain a new gene WS-2.

Description

Detailed Description of the Invention

In recent years, it has become possible to clarify a specific gene on a long chromosome by the technique of positional cloning. The present inventor makes full use of this positional cloning method to create a contig map (hereinafter, referred to as gene physical map) of a gene region that causes Werner syndrome (hereinafter, referred to as Werner region or WS region), Finally, a novel gene, WS-2, was found in this WS region, and the present invention was reached.

That is, the present invention is a gene encoding a polypeptide substantially containing the amino acid sequence represented by SEQ ID NO: 2 or 5. Examples of the gene include any of the nucleotide sequences represented by SEQ ID NOs: 1, 3, 4 or 6.

Further, the present invention is an oligonucleotide probe which hybridizes with at least a part of the above gene. Furthermore, the present invention is a recombinant DNA containing the gene. Furthermore, the present invention provides the recombinant DN
It is a transformant transformed with A.

Further, the present invention is a method for producing the above-mentioned polypeptide, which comprises culturing the above-mentioned transformant and collecting a polypeptide encoded by a human gene from the resulting culture. Furthermore, the present invention provides SEQ ID NO: 2 or 5.
It is a polypeptide encoded by a human gene, which substantially contains the amino acid sequence represented by:

Furthermore, the present invention is a monoclonal or polyclonal antibody that specifically reacts with the above-mentioned polypeptide. Furthermore, the present invention is a hybridoma that produces the monoclonal antibody, obtained by fusing antibody-producing cells immunized with the polypeptide with myeloma cells.

Furthermore, the present invention is a reagent for detecting a gene containing the above-mentioned oligonucleotide probe. further,
The present invention is a diagnostic kit containing the polypeptide and the monoclonal antibody or polyclonal antibody. Furthermore, the present invention is a mouse into which a gene modified so as to increase or decrease the expression level of the gene is introduced.

Further, the present invention is a transgenic mouse into which the gene causing Werner's disease has been introduced. Here, the term “substantially includes” or “substantially encodes” means the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 as long as it has a function as the polypeptide or gene of the present invention. It means that mutations such as substitution, insertion or deletion may occur in the represented amino acid sequence.

Hereinafter, the present invention will be described in detail. In recent years, it has become possible to clarify a specific gene on a long chromosome by the technique of positional cloning. What is positional cloning? (1) First,
A family survey is conducted on the onset of a specific disease, and a family with a high onset of the disease is sought. (2) Next, linkage analysis is performed on the DNA of not only the patient but also the relatives, and the distribution of various genetic markers on the chromosome is determined. Analyze how it differs from a healthy family, and determine from this result which region of which chromosome is present,
Then, (3) a contig map of this region (physical physical map of the gene) was created, and (4) finally, a cDNA clone with a different expression size / level between a healthy person and a patient was found in this physical map, and its cause It is a method to clarify the structural variation of the target gene.

The gene physical map can be prepared by precisely connecting human DNA fragments incorporated in a vector such as YAC (Yeast Artificial Chromosome), cosmid, and P1 and PAC phages, It facilitates subsequent cloning experiments. In addition, the gene physical map can represent a conceptual genetic map that is predicted by the mathematical probability of recombination as an accurate map that is defined by the actual length of DNA. The distance (bp) between the gene marker and the target gene can be accurately known.

The present inventor makes full use of this positional cloning method to prepare a contig map (hereinafter, referred to as physical gene map) of a gene region (hereinafter, referred to as Werner region or WS region) that causes Werner syndrome. Finally, a novel gene WS-2 was finally found in this WS region, and the present invention was achieved.

First, in searching for the location of the WS gene (gene that causes Werner syndrome), WS
Of the regions, we will create a physical map of the gene using P1 and PAC phage DNA for the region that is presumed to have the highest presence (Fig. 1). The physical map of genes is P1 and PAC phage
It can be prepared by accurately connecting human DNA fragments with an average chain length of 90 Kbp incorporated in a vector such as the following [(1) to (4) below], and facilitates subsequent cloning operations. Can be

(1) A DNA fragment (P1 / PAC clone) containing the same nucleotide sequence as the target marker DNA is selected from the library. (2) Next, the obtained clone is purified by ultracentrifugation using cesium chloride (hereinafter abbreviated as CsCl), and then the nucleotide sequences at both ends of the incorporated DNA fragment are determined. (3) Further, based on the information on the nucleotide sequence, the polymerase chain reaction method [US Patent No. 46831
95, July 28th, 1987] Hereinafter, at least one pair (that is, two) of primers for PCR will be prepared and the PCR reaction will be performed. (4) Using the PCR product obtained in (3) as a probe, select a DNA fragment (P1 / PAC clone) containing the same nucleotide sequence as the target from the library. (5) (1) ~ (4)
By repeating the above procedure, the precise operation of connecting the DNA fragments in sequence is performed to construct a gene physical map consisting of a number of P1 / PAC DNA chains in which a large region of several million base pairs has been cloned (Fig. 1 ). The physical map can be confirmed by Fluorescence in situ hybridization (hereinafter referred to as FISH) using lymphocyte cells. FISH is P
1 / PAC DNA is connected in the correct order,
It is one of the methods to know the distance of the gap that is not yet connected in P1 / PAC DNA starting from multiple starting points. The physical map of the gene thus obtained and the cloned P1 / P1 constituting it
PAC DNA is used to detect cDNA and determine the nucleotide sequence.

The WS-2 gene of the present invention substantially contains the amino acid sequence represented by SEQ ID NO: 2. Examples of genes in which the gene containing the base sequence represented by SEQ ID NO: 1 or 3 is mutated such as deletion include those containing the base sequence represented by SEQ ID NO: 4 or 6. Examples of the polypeptide having a mutation such as a deletion in the amino acid sequence represented by SEQ ID NO: 2 include those represented by SEQ ID NO: 5.

According to the present invention, the WS-2 gene has at least 8 genes.
It was found to contain individual (I, II, III, IV, V, VI, VII, VIII) exons (Fig. 2A). These exons are
Although it is within the WS region on the human genome, the transcription direction of the WS-2 gene is unknown. Further, as is clear from the result of FISH shown in FIG. 3, the WS-2 gene is located in human chromosome 8
It was confirmed that it exists near 8p11.2-p12.1.
The present inventors revealed that the WS-2 gene exists as a single copy on the human genome by Southern blot analysis detailed in [Example 5] (FIG. 4A).

It should be noted that, among the genes corresponding to the human WS-2 gene, genes of other species except monkeys do not have high homology with the nucleotide sequence of the human WS-2 gene according to the analysis result by Zoo Blot. (FIG. 4B), it is expected to be difficult to clone by known techniques.

As shown in FIG. 2A, two types of mRNA (Type
I, Type II mRNA, corresponding to the respective cDNA genes) are selected by alternative splicing [Reed and Maniatis, C
ell 46,681-690 (1986)], and can be made from the above exons. The inventor
(Sequence represented by SEQ ID NO: 1 or 3) and Type II
It was found that at least two types of mRNA (sequences represented by SEQ ID NO: 4 or 6) are produced by alternative splicing.

As a result, the polypeptide encoded by Type II mRNA (ie, cDNA gene) is the same as Type I in most of the region including the N-terminal and C-terminal regions, but has a short central region. , It was found to have an amino acid sequence lacking 83 amino acids. That is, the polypeptide encoded by the Type I gene (FIG. 2B) is represented by SEQ ID NO: 2 (270 amino acids), and the polypeptide encoded by the Type II gene is represented by SEQ ID NO: 5 (187 amino acids).

The basic factor of finding the WS-2 gene of the present invention described above is that the WS-2 gene is "STS marker showing the strongest linkage with the Werner-causing gene on the physical map of the gene,
This is due to the fact that it was in the "D8S339, region containing GSR" (Fig. 1).

The shaded boxes at the top of FIG. 1 indicate the names of typical genes transcribed from the boxes and their approximate transcription positions. These genes are tubulin pseudogene, WS gene, TFIIEβ (Transcripti) from the telomere side.
on factor, TFIIE subunit) gene, GSR (Gluta
thione S-reductase) gene, WS-2 gene (a novel gene according to the present invention) and PP2A β (Protein phosphatase 2A)
Subunit) gene. The line on the figure is a scale of 100 Kb unit, and the thick line (the thick line indicated by ↓) shows the human genomic DNA in Schematic. Numbers between the scale line and the bold line, such as D8S1055 and D8S339, are STS (Sequence Tagged Sequence) markers known so far and indicate their approximate positions. Many short solid lines underneath human genomic DNA (shown in bold) are P1 or PAC-derived DNA constructing the physical map of genes.
For convenience of the present inventors, numbers (for example, # 4370 and # 4117 derived from P1) are attached. The numbers in parentheses are
The approximate size of each DNA (eg 100 Kb) is shown. The symbols "□" and "◇" at the ends of these DNAs are used to determine the direction of the DNA.
It shows that the SP6 promoter and T7 promoter, which were placed in the 1 / PAC phage vector, are present at these ends.

Whether the WS-2 gene is the causative gene itself of WS was determined from the fibroblasts of Werner's disease patients (3 persons) and healthy persons (3 persons) using the WS-2 cDNA clone as a probe. Even if Northern blot analysis was performed using the obtained total RNA, the size and
No difference was observed in the expression level (Fig. 5 and Table 1). Therefore, it is necessary to investigate whether this gene causes Werner syndrome at the individual level.

The above-mentioned results show that mRNAs derived from other genes existing in the WS region, such as protein phosphatase mRNA (PP2Aβ) and glutathione-S-reductase (G
SR) mRNA, Transcription factor TFIIE (TFIIEβ) mRNA
This was also the case with Northern blot analysis using various probes for detecting the same, and there was no difference in the chain length and expression level of any mRNA in both Werner's disease patients and healthy subjects.

A multi-tissue northern blot (Mul) for investigating how the human WS-2 gene is expressed in various organs
The ti-Tissue Northern Blot) analysis is shown in [Example 6] (FIG. 6). From this result, WS-2 gene is strongly expressed in testis and ovary, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, small intestine,
It is moderately expressed in the large intestine, and in peripheral lymphocytes,
It was found that the expression was extremely low.

When the above results are summed up, the WS-2 gene is
It is specifically retained only in primates, which are higher organisms,
Moreover, the expression of this gene is very strongly expressed in the reproductive system of the testis and ovary, suggesting that it plays an important role in the conservation or regeneration system of species. Therefore, the WS-2 gene is useful for understanding the gene expression regulation related to the primate regeneration system, and is also useful for detecting the cause of diseases such as infertility and creating a new drug for relieving the cause. In addition, it is also useful for research to elucidate the relationship with the onset of Werner syndrome.

The cloning of the gene of the present invention will be described below. I. Preparation of genomic DNA and RNA (1) Collection of cell samples and preparation of genomic DNA The preparation of genomic DNA from human blood lymphocytes is described in Molecular
Cloning (Sanbrook, Fritschand Maniatis, Cold Sprin
g Harbor Publishing).

First, fresh blood (10 ml) was added to 80 ml of 0.3
2M Sucrose, 10 mM Tris-HCl buffer (pH 7.5), 5 mM Mg
A solution containing Cl ₂ and 1% Triton X-100 is mixed to hemolyze, and the mixture is allowed to stand on ice for 20 minutes and then centrifuged to obtain a pellet fraction containing lymphocytes. Add this pellet to 2.25 ml of 0.075M NaCl.
Suspended in a solution containing 0.025M EDTA and 0.25ml of 2mg / m
l Proteinase K, 5% SDS and 10 mM Tris-HCl buffer (pH 8.0) are added, and the mixture is incubated at 37 ° C overnight. Then, 5 ml of 1 volume of phenol and 1 volume of a mixture of chloroform-isoamyl alcohol (24: 1) are added, and the genomic DNA is extracted while gently turning. After centrifugation (3,000 rpm, 10 minutes), the supernatant fraction is collected and the extraction with phenol / chloroform is repeated. Add 20% 3M sodium acetate solution (pH 5.2) to the supernatant obtained by centrifugation.
, And then 2.5 volumes of ethanol to add genomic DNA.
To precipitate. Filamentous DNA is wound up with a glass Pasteur pipette and dissolved in 1.25 ml buffer containing 10 mM Tris-HCl (pH 8.0) and 0.2 mM EDTA.

Purification of genomic DNA from human placental tissue
A fresh placenta obtained from a healthy pregnant woman is frozen on dry ice and sliced into very thin slices using a slicer, or finely ground pieces are used as a material. From frozen frozen placenta fragments, the method of Gross-Bellard et al. [Eur. J. Bio
chem. 36, 32-38 (1973)].
Purify.

(2) Establishment of cell line and preparation of total RNA Werner's disease patients (hereinafter referred to as WS) clinically certified
WS fibroblast cell line was established based on the skin tissue section obtained by biopsy from
Purify oly A + -containing mRNA (hereinafter referred to as poly A + mRNA). Similarly, RNA is purified with establishment of a healthy human fibroblast cell line from a healthy human skin tissue section.

The mRNA is prepared by the following method. Cultured human fibroblasts derived from WS patients or healthy individuals were detached from the Petri dish with trypsin, washed thoroughly with PBS, and homogenized in 6M guanidinium thiocyanate solution.
irgwin et al. [Biochemistry 18, 5294-9299 (197
9)] according to the cesium chloride equilibrium density gradient centrifugation method.
Separate the RNA as a precipitate. After separation, phenol extraction,
Purify total RNA by ethanol precipitation. polyA + mRN
To obtain group A, latex particles with oligo (dT) covalently bound to total RNA (oligotex (dT) -30
(Manufactured by Takara Shuzo Co., Ltd.), and added by the method of Kuribayashi-Ohta et al. [Biochem. Biophys. Acata. 1156, 204-112 (1993)].
Purify by affinity chromatography according to the method described above.

The mRNAs from these fibroblasts are used not only as a sample for preparing cDNA using reverse transcriptase, but also when certain mRNAs in WS patients are qualitative (size and existence of family) and Quantitative (expression level and copy number)
In addition, it can be used as a sample for Northern blot analysis to determine whether or not there is a change compared with a healthy person.

II. Construction of physical map of gene (1) Construction of P1 / PAC phage library P1 / PAC containing human DNA fragments for construction of P1 / PAC phage library and construction of gene physical map of P1 / PAC clone
1 / PAC phage DNA is selected from the P1 / PAC phage library by PCR using a DNA primer with a specific sequence. Here, P1 / PAC is a λ phage system (10
~ 20 kb), YAC (1-2 Mb) and cosmid (20
It refers to a phage cloning vector developed as a system capable of cloning an intermediate size DNA (~ 40 kb) (80-150 kb). The P1 / PAC phage library is available from Smoller et al. [Chromosoma 100, 487-494 (1991)] or Tashiro et al. (Experimental Medical Supplement, "Genome Mapping and Sequence Analysis", Biomanual Series 6, 1994, Yodosha).
The production method described in Genome System (8620 Pennell Dr. St. Louis, Missouri, USA) was used.

The present inventors have selected P1 / PAC phage as a cloning system for rapidly and accurately analyzing the WS region because (a) cloning is performed using E. coli as a host.
Advantages of easy DNA recovery and stable integrated DNA, (b) Sp6 and T7 promoter sequences are arranged (when contigs are created or walking, the nucleotide sequences at both ends of the insert DNA are It is necessary to read that the Sp6 and T7 promoters make this possible. (C) The phage DNA infected with E. coli as a host can be treated as a normal plasmid and can be used as a normal colony This is due to the excellent feature that the hybridization method can be used.

(2) Screening of P1 / PAC phage clones Screening of P1 / PAC phage clones was carried out by Smoller.
Et al. [Chromosoma 100, 487-494 (1991)]. That is, E. coli infected with P1 / PAC phage was spread on a nylon membrane and cultured, and then LB plate containing 1 mM isopropyl-1-thio-D-galactopranoside (hereinafter referred to as IPTG) and 25 mg / ml kanamycin. Incubate at 37 ° C. for 6 hours to amplify the copy number of P1 / PAC phage per E. coli. After that, Escherichia coli on the filter is lysed by a conventional method, and double-stranded DNA containing phage DNA is alkali-denatured into single-stranded DNA, which is fixed on a nylon membrane by UV irradiation or heating.

In order to obtain the desired P1 / PAC phage clone, a random hexamer reaction [Feinberg and Vogelstein, with a DNA probe tested with radioactive phosphate [ ³² P] was used.
Addendum.Anal. Biochem. 137, 266-267 (1984)] and PC
Prepare by R method and select and isolate the desired P1 / PAC clone by hybridization using it.

(3) Preparation of P1 / PAC DNA Recovery and purification of DNA from P1 / PAC phage clone
The procedure described by Smoller et al. [Chromosoma 100, 487-494 (1991)] was used with some modifications. Details will be described in [Example 1].

(4) Determination of nucleotide sequence of P1 / PAC DNA terminal region Inserts inserted in each P1 / PAC DNA clone in order to align P1 / PAC DNAs while overlapping them to complete a physical map of gene. Determine the nucleotide sequences at both ends of the DNA. The nucleotide sequence is determined by Hattori et al. [Electr
PCR described by Ophoresis 13,560-565 (1992)]
The method is based on. That is, using two kinds of primers [SP6 side primer (SEQ ID NO: 22) and T7 side primer (SEQ ID NO: 23)] that bind to the SP6 and T7 promoters present in the P1 / PAC vector, PRISM containing a fluorescent dideoxy terminator was used. Sequenceing Kit (Pe
rkin Elmer) to perform the reaction, and then use Applied Biosy
Read the nucleotide sequence of the gene fragment after the PCR reaction using the stem auto sequencer (model ABI 373),
Data analysis is performed using the attached Macintosh computer.

(5) Each P1 / PA by Dual-color FISH analysis
Assignment of C DNA Clone to WS Region FISH is a method in which a fluorescently labeled DNA probe is hybridized with the cell nucleus and the position of the DNA is visibly detected on the cell nucleus. As the specimen, cells in metaphase or metaphase where chromosomes are clearly visible are used.

Metaphase cells can be obtained by the following procedure. Peripheral blood cells are stimulated with phytohemagglutinin (PHA) to promote cell division and colcemid is used to arrest division at metaphase. The cells are fixed with an acetic acid / methanol solution and then spread on a slide glass. Specimens on slides are formamide and SSC [Standard SalineC
itrate; 0.15M NaCl, 0.015M Sodium Citrate (pH 7.
0)] to modify. Next, when a DNA probe labeled with digoxigenin or biotin is reacted, this label
DNA hybridizes to the specimen.

Next, when treated with a rhodamine-labeled anti-digoxigenin antibody, FITC-labeled avidin or the like and fluorescently labeled, the position where the DNA probe hybridizes can be seen as a red signal for lodamine and a green signal for FITC. .

A probe of P1 clone DNA (# 4117) containing the WS-2 gene labeled with biotin was hybridized with a sample of human peripheral blood lymphocytes in metaphase. Then, this specimen was stained with avidin labeled with a fluorescent dye (FITC). As shown by arrow 1 in FIG. 3, the FITC signal is a standard DN that specifically stains the centromere of chromosome 8.
It is present in the short arm near the A probe (labeled with rhodamine; indicated by arrow 2). Therefore, it was found that the P1 clone DNA containing the WS-2 gene is present in the short arm of chromosome 8.

III. CD by exon trapping method
Preparation of NA exon (1) Exon trapping method After creating a gene physical map covering the WS gene region, the exon portion was directly extracted from the P1 / PAC DNA constituting this gene physical map by the exon trapping method. Isolated. Specific details will be described in [Example 2]. Regarding the determined nucleotide sequence, the data bank is searched for the presence or absence of those that correspond to existing genes, or the presence or absence of those that have homologous sequences with existing genes.

(2) Exon Joining
ng) The length of exon fragments obtained by the exon trapping method is in the range of several tens to 400 bp, with an average of 200 b.
It is around p. For this size, cDNA cloning,
Since it is too small to perform various analyzes such as Southern blotting and Northern blotting, we will try to connect exons with each other using PCR.

Primers of about 20 bases are prepared at both ends of each exon by selecting one from a sense sequence (FP; forward primer) and the other from a nonsense sequence (RP; reverse primer). Exon fragments obtained by the exon trapping method have the same or neighboring P1 /
A primer for each exon of PAC DNA was selected, and a set of FPs was prepared using the cDNA prepared by RT-PCR as a template.
And PCR using RP. In this way, PCR products spanning more than one exon can be obtained.

(3) Determination of nucleotide sequence The nucleotide sequence was determined by Hattori et al. [Electrophoresis 13, 5
60-565 (1992)]. The reaction is performed using the PRISM Sequenceing Kit containing a fluorescent dideoxy terminator manufactured by Perkin Elmer, and the base sequence is read by an auto sequencer (Model ABI 373) manufactured by Applied Biosystem, and the data is analyzed by the attached Macintosh computer. Specific details will be described in [Example 3].

IV. Extension of exon by TAIL-PCR method From P1 / PAC DNA which constitutes the physical map of gene of WS region,
Although the exon portion can be directly isolated by the exon trapping method, the length of each exon fragment obtained by this method ranges from several tens to 400 bp and the average length is around 200 bp. Usually, this size is too small for various analyzes such as cDNA cloning and Southern blotting, and it is necessary to obtain a longer cDNA fragment. Therefore, in order to obtain a longer cDNA fragment, PCR is performed by combining the primers of each exon II
Try the exon joining method or TAIL-PCR method described in I- (2). The TAIL-PCR method is simpler than the conventional RACE method and allows various cDNA clones to be transferred to the 5'side or 3'side.
It is a method that can be extended longer and accurately,
The details will be described in [Example 3].

V. Screening of cDNA clones (1) Preparation of probe DNA Here, the DNA fragment used as the probe constitutes the physical map of the gene P1 by the exon trapping method.
It starts with an exon obtained from DNA and has a DNA sequence extended by the TAIL-PCR method. D with relatively long chain length
The NA fragment is amplified by PCR, or alternatively, inserted into a bacterial plasmid, amplified in E. coli, and then purified as a pure single DNA strand. These series of methods are described by Sambrook et al. [Molecular Cloning: A Laborator
y Manual, New York: Cold Spring Harbor Laboratory
Press (1989)]. The obtained DNA fragment is labeled with digoxigenin (hereinafter referred to as DIG) or [ ³² P] by the random hexamer method or PCR method.

(2) Screening from Zap II library A library using a λ phage as a vector has a large cDNA chain length of up to 20 Kbp, and since there is little bias in insertion efficiency depending on size, all types of libraries can be used. cDNA
Is considered to be efficiently retained. Further, in screening, many phage clones can be immobilized on a relatively small nylon membrane, so that there is an advantage that many phage clones can be examined with a small number of screenings.

The longer cDNA fragment prepared by the TAIL-PCR method is labeled with DIG or [ ³² P], and the λ phage library is screened using it. Details will be described in [Example 4].

VI. Analysis of cDNA clones (1) Analysis by Southern blot Southern blot is D cleaved by restriction enzyme or the like.
This method aims to detect a target gene from NA fragments.

First, DNA fragments are separated by electrophoresis on an agarose gel according to their size. Next, the DNA is denatured with alkali and salts and transferred to nitrocellulose filters. This filter is hybridized with a probe labeled with DIG or [ ³² P], and the filter is subjected to autoradiography. As a result, hybridized DNA fragments are detected. By using this method, the number of genes (copy number), the existence of a family, and the like can be known. As the probe, the DNA obtained by the TAIL-PCR method or the cDNA obtained by screening the λ phage library based on the DNA is used. Details will be described in [Example 5].

(2) Analysis by Northern Blot Northern blot is a specific RN from a mixture of RNA.
It is a method for detecting A, and is a method used for the purpose of detecting the size and amount of RNA of interest.

Extraction of RNA from cells or tissues was performed using high-concentration guanidine thiocyanate, deproteinized with phenol / chloroform, and then RNA precipitated by alcohol precipitation.
Is purified. RNA is fractionated by size difference by agarose gel electrophoresis and denatured with alkali and salt. Then transfer to a nitrocellulose filter and hybridize with the [ ³² P] -labeled cDNA probe. Detect by exposure to autoradiography the RNA of interest hybridized to the probe.

By this method, the mRNA corresponding to the sequence of the cDNA used as the probe can be detected, and the full-length size and relative expression level of the target gene can be known. As the probe, a cDNA fragment obtained by the TAIL-PCR method or a full-length cDNA obtained by screening a λ phage library based on the cDNA fragment is used. Details will be described in [Example 6].

VII. Preparation of transformant Recombinant D was prepared by inserting the purified gene into the restriction enzyme site or multicloning site of appropriate vector DNA.
NA is prepared, and the recombinant DNA is used to transform a host cell. Vector DNA for inserting DNA fragments
Is not particularly limited as long as it can be replicated in the host cell, and examples thereof include plasmid DNA and phage DNA. Plasmid pUC if the host cell is E. coli
118 (Takara Shuzo), pUC119 (Takara Shuzo), pBluescr
ipt SK + (manufactured by Stratagene), pGEM-T (manufactured by Promega) and the like can be used.

As the host cell, both eukaryotic cells and prokaryotic cells can be used. Examples of eukaryotic cells include cells such as animals and yeast, and examples of prokaryotic cells include Escherichia coli. For example, Escherichia coli XL1-Blue (Stratagene),
JM109 (manufactured by Takara Shuzo) or the like can be used.

When plasmid DNA is used as the vector DNA, for example, when inserting an EcoRI DNA fragment, the plasmid DNA is used as a restriction enzyme EcoRI (New England Biolab; NEB).
CIP to prevent self ligation by digesting with
(calf intestine phosphatase) and so on.
Then, the DNA fragment and the cleaved vector DNA are mixed, and, for example, T4 DNA ligase (Takara Shuzo) is allowed to act on this to obtain a recombinant DNA. By transforming the recombinant DNA thus obtained into a host cell such as Escherichia coli XL1-Blue, a colony of a transformant carrying a gene fragment containing the gene of interest can be obtained.

In the present invention, transformation can be performed, for example, by Hanahan.
Techniques for Transformation of E. coli I
n DNA Cloning, vol.1, Glover, DM (ed.) pp109-136,
IRLPress (1985)].

The above-mentioned transformants are screened by colony hybridization using a DNA fragment containing a part of the target gene as a probe, or by using a 5'primer (FP) based on the nucleotide sequence of the target gene. Synthesized, then synthesized 3'primer (RP) based on the nucleotide sequence of complementary strand DNA, and used these primers for PC
By the R method, colonies containing the target gene can be selected.

In this way, a transformant carrying the gene of the present invention can be obtained.

VIII. Production of Polypeptide Encoded by WS-2 Gene The polypeptide encoded by the gene of the present invention can be obtained by culturing a prokaryotic or eukaryotic cell carrying the recombinant DNA obtained as described above. Can be produced. The culture method may be an ordinary solid culture method, but preferably a liquid culture method.

The medium for culturing the recombinant microorganism is selected from yeast extract, peptone, meat extract, etc. 1
To one or more nitrogen sources, one or more inorganic salts such as dipotassium hydrogen phosphate, magnesium sulfate, ferric chloride are added,
Further, if necessary, a sugar raw material, an antibiotic, a vitamin and the like are appropriately added and used. In addition, if necessary, I
PTG or the like may be added to induce gene expression.
The pH of the medium at the start of the culture is adjusted to 7.2 to 7.4, and the culture is usually performed at 36 to 38 ° C., preferably around 37 ° C. for 14 to 20 hours by aeration and agitation culture, shaking culture and the like.

After the completion of the culture, the protein of the present invention can be collected from the culture by conventional protein purification means. That is, the cells are disrupted by a lysis treatment using an enzyme such as lysozyme, an ultrasonic crushing treatment, a grinding treatment, or the like, and the polypeptide encoded by the gene of the present invention is discharged outside the cells. Next, insolubles are removed by filtration or centrifugation to obtain a crude polypeptide solution.

To further purify the polypeptide from the above crude polypeptide solution, conventional protein purification methods can be used. Specifically, ammonium sulfate salting-out method, ion exchange chromatography, hydrophobic chromatography,
Gel filtration chromatography, affinity chromatography, electrophoresis, etc. may be used alone or in combination.

IX. Preparation of Monoclonal Antibody A monoclonal antibody specific to the polypeptide encoded by the WS-2 gene of the present invention can be obtained as follows.

(1) Preparation of Antigen The polypeptide obtained by the above method (VIII) is dissolved in a buffer solution, and then an adjuvant is added. Examples of the adjuvant include commercially available Freund's complete adjuvant and Freund's incomplete adjuvant, and any of these may be mixed.

(2) Collection of immunization and antibody-producing cells The immunogen obtained as described above is administered to mammals such as rats and mice. The immunological amount of the antigen is 10 to 500 μg per animal at a time. The immunization site is mainly injected intravenously, subcutaneously, or intraperitoneally. The interval of immunization is not particularly limited, and immunization is performed 2 to 5 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 1 to 3 weeks. 2 to 7 days after the last immunization day, preferably 4 to 5 days
After a day, antibody-producing cells are collected. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, and the like, with spleen cells or local lymph node cells being preferred.

(3) Cell fusion As a myeloma cell to be fused with the antibody-producing cell, a cell line which is generally available from animals such as mouse is used.
As a cell line to be used, it has drug selectivity and cannot survive in a selective medium (HAT medium: including hypoxanthine, aminopterin, and thymine) in an unfused state, but only in a state fused with antibody-producing cells. Those having the property that can be performed are preferable. Specific examples of myeloma cells include mouse myeloma cell lines such as P3U-1 (manufactured by Dainippon Pharmaceutical Co., Ltd.).

Next, the myeloma cells and antibody-producing cells are fused. Cell fusion uses serum-free DME
M, RPMI-1640 medium and other animal cell culture medium were mixed with 10 ⁸ cells / ml of antibody-producing cells and 2 × 10 ⁵ cells / ml of myeloma cells in the same volume and fused in the presence of a fusion promoter. Perform the reaction.

To promote cell fusion, polyethylene glycol having an average molecular weight of 1,500 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation (eg, electroporation).

(4) Selection and cloning of hybridomas The desired hybridomas are selected from the cells after the cell fusion treatment. As a method, a cell suspension is appropriately diluted with, for example, RPMI-1640 medium containing fetal calf serum, and then spread on a microtiter plate at about 5 to 10 cells / well,
A selective medium is added to each well, and then the selective medium is appropriately exchanged for culturing. As a result, after culturing in the selective medium,
Cells growing from around 14 days can be obtained as hybridomas.

It is screened whether the target antibody is present in the culture supernatant of the grown hybridoma. Hybridoma screening is carried out according to a conventional method and is not particularly limited. For example, a portion of the culture supernatant contained in the wells grown as hybridomas was collected and subjected to enzyme immunoassay (EIA), RI
It can be performed by A (radio immunoassay) or the like.
Cloning of the fused cells is performed by the limiting dilution method,
Finally, a hybridoma that is a monoclonal antibody-producing cell is established.

(5) Collection of Monoclonal Antibody As a method for collecting the monoclonal antibody from the established hybridoma, a usual cell culture method or ascites formation method can be adopted. In the cell culture method, the hybridoma is
Culture in animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium or serum-free medium under normal culture conditions (eg, 37 ° C., 5% CO ₂ concentration) for 10 to 14 days. Antibodies can be obtained from the culture supernatant.

In the case of the ascites formation method, about 5 × 10 ⁶ hybridomas are intraperitoneally administered to a mammal derived from a myeloma cell and an animal of the same species to proliferate the hybridomas in a large amount. Then, ascites or serum is collected 1-2 weeks later. In the above antibody collecting method, when purification of the antibody is required, a known method such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, gel chromatography, or the like is appropriately selected, or Purification can be performed by combining them.

X. Preparation of Polyclonal Antibody (1) Preparation of Antigen The polypeptide obtained by the above method (VIII) is dissolved in a buffer solution, and then an adjuvant is added. As the adjuvant, commercially available Freund's complete adjuvant and Freund's incomplete adjuvant are used.

(2) Rabbits, guinea pigs, goats, sheep and the like are usually used as immunized animals. Taking a rabbit as an example, the rabbit is usually injected subcutaneously in the footpad with 100 μg to 500 μg of the polypeptide together with complete Freund's adjuvant. Two weeks later, the same amount of antigen is mixed with Freund's incomplete adjuvant for intramuscular injection. Two weeks later, intramuscular injection is repeated, and one week after the final immunization, blood is partially collected from the ear, and the antibody titer is measured by EIA or the like. If the antibody titer has reached the target value, the whole blood is collected. If the antibody titer is low, intramuscular injection is repeated, and immunization is repeated until the antibody titer reaches the target value. For purification of the antibody from serum by the ammonium sulfate fractionation, the method described in the section of the monoclonal antibody (IX) can be adopted.

XI. WS-2 gene existing in WS region and reagent for detecting polypeptide encoded by the gene WS-2 gene is specifically retained only in primate which is a higher organism, and this gene Expression is very strongly expressed in the testicular and ovarian reproductive system,
It is suggested that it plays an important role in the conservation or regeneration system of species, and is useful for understanding the gene expression regulation related to the regeneration system of primates, and also for detecting the cause of disease such as infertility and relieving it. It is useful not only for the creation of a new drug for the treatment, but also for the research for elucidating the relationship with the onset of Werner syndrome.

When the gene of the present invention is used as a detection reagent related to the above, cloned WS-2
Hybridization can be carried out using an oligonucleotide containing at least a part of the gene as a probe, and detection can be carried out by Southern or Northern blotting.
Note that examples of the oligonucleotide probe include a DNA probe and an RNA probe. When a polyclonal antibody or a monoclonal antibody against the polypeptide encoding the gene of the present invention is used as a detection reagent, it can be detected by EIA, RIA or Western blot analysis.

XII. Transgenic Mouse (1) Construction of Gene to be Introduced Regarding the gene to be introduced into the mouse, genomic DNA containing an upstream promoter and a downstream poly-A signal is used, or a cDNA sandwiched between the two is used. Either use. If a transgenic mouse capable of overexpressing the WS-2 gene is produced, it is highly likely that it can be used as a model animal (experimental animal) for analyzing the biological action of the WS-2 gene.

As the gene of the transgenic mouse, there can be considered two vectors, a vector which overexpresses the WS-2 gene and an antisense vector which suppresses its expression. In either case, a drug for positive selection (for example, neomycin) resistant gene for gene selection is ligated.

(2) Insertion of Gene into Cell To insert a gene into a cell, a method of directly injecting DNA into a fertilized egg, which has been conventionally used, can be used. embryonic stem cell: ES cell)
Have the advantage that they can be cultured and that mice can be generated from these cells. Therefore, the method using various ES cells is preferable because it is more efficient. Examples of ES cells include TT2 cells (Shinichi Aizawa, Gene Targeting, 1995, Yodosha).
The above-mentioned vector DNA containing the WS-2 gene is introduced into ES cells by electroporation and positively selected with neomycin to obtain the target mutant ES cells.

(3) Injection into embryo placenta or 8-cell stage embryo The above ES cells are injected into embryo placenta or 8-cell stage embryo using a capillary tube.

(4) Transplantation into foster mother ES cell-injected 8-cell stage embryos are directly transplanted into the oviduct of a foster mother, or cultured for one day, and embryos that have developed to blastocysts are transplanted into the uterus. Blastocysts injected with ES cells are directly transplanted into the uterus.

(5) Analysis of chimeric mice Pregnant mice are allowed to give birth. The degree of chimera is judged based on the coat color of the offspring, and those with a high degree are crossed.
Cut off the tail of the newborn mouse and examine the transmission of mutant allele by PCR. Mating hetero mice,
Create a homozygote and analyze it.

[0083]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to these examples.

[Example 1] Purification of P1 and PAC DNA Highly pure P1 and PAC DNA were separated and purified by CsCl density gradient centrifugation, and a physical map of the gene of the WS region was prepared, a gene sequence was decoded, and analysis by FISH and It was used as a material for the exon trapping method.

(1) Method for increasing / inducing P1 / PAC copy number in Escherichia coli by IPTG stimulation Recovery / purification of DNA from P1 / PAC phage clone was performed using Sm.
A modification of the method reported by oller et al. [Chromosoma 100, 487-494 (1991)]. E. coli NS3529 colonies containing a single P1 / PAC phage clone were suspended in 33 ml of LB culture medium containing a final concentration of 25 μg / ml of kanamycin, and cultured at 37 ° C overnight with shaking. On the next day, the cells were transferred to 1 L of LB culture medium containing the same concentration of kanamycin, shake-cultured for 1.5 hours, IPTG was added to a final concentration of 0.5 mM, and the culture was continuously shaken for 5 hours.

(2) P1 / PAC DN by CsCl density gradient centrifugation
Purified P1 / PAC DNA of A was prepared from the cells obtained by the method described in (1) above using the conventional alkali-SDS method [Birnboim and Doly,
Nucleic Acids Res. 7, 1513-1523 (1979)] and purified by CsCl density gradient centrifugation.

The cells recovered by the cooling centrifugation (3,500 rpm × 15 minutes) were added to 50 ml of 50 mM Sucrose-10 mM EDTA-25 mM Tr.
1.5 ml of is-HCl (pH 8.0) and 50 mg / ml lysozyme solution
Add gently and incubate at room temperature for 5 minutes. Subsequently, 100 ml of 0.2 M NaOH-1% SDS solution was added, and the mixture was gently incubated on ice for 5 minutes. In addition, 75 ml of 3M KAc-11.
5% glacial acetic acid was added, and the mixture was gently incubated on ice for 5 minutes. The supernatant was collected by cold centrifugation (4,000 rpm × 15 minutes), RNase solution was added to the final concentration of 50 μg / ml, and the mixture was incubated at 37 ° C for 1 hour and then the same amount of 2-propan was added.
ol was added and the mixture was allowed to stand at -20 ° C for 1 hour. Cold centrifugation (6,00
After that, the supernatant was removed by decantation, and the precipitate containing P1 / PAC DNA was washed with 70% EtOH and then air-dried completely. 6 ml of 10 mM Tris-HCl-1 mM EDTA (pH 8.0) was added to dissolve the precipitate, and then 6 g of CsCl was added to dissolve it sufficiently, and further 100 mg of 10 mg / ml ethidium bromide was added.
The mixture was left at -20 ° C for 20 minutes. Cooling centrifugation (6,000rpm
After 10 minutes), the supernatant was recovered, and P1 / PAC DNA was separated from the bacterial genome by ultrahigh speed centrifugation at 100,000 rpm for 4 hours or more at 22 ° C. Under UV irradiation, collect the band containing P1 / PAC DNA, remove ethidium bromide with isoamyl alcohol, and remove CsCl by dialysis twice.
Was purified. 10 mM Tris-HCl-1.25 as dialysis buffer
mM EDTA (pH 8.0) was used. These P1 / PAC DNAs are
It was used for preparing ntig (Fig. 1) and for the exon trapping method shown in [Example 2].

Example 2 Exon Trapping Method (1) Preparation of DNA Fragments Exons were recovered from P1 / PAC DNA fragments by the following series of operations. First, 100 μg of each P1 / PAC DNA fragment was added.
Cleavage was carried out by incubating the restriction enzymes BamHI and BglII of unit with a predetermined buffer manufactured by Takara Shuzo at 37 ° C. for 3 hours. The reaction solution was treated with phenol / chloroform to extract DNA and then precipitated with ethanol.

(2) Vector for exon trapping
Insertion of DNA fragment into pSPL3 After cutting with restriction enzyme Bam HI, phosphatase (Boehringer Ma
40 ng exon dephosphorylated by nnhaim treatment)
Vector pSPL3 for trapping (manufactured by Gibco BRL) and 100 ng of P1 / PAC DNA fragment digested with restriction enzymes Bam HI and Bgl II were reacted with T4 DNA ligase at a predetermined buffer at 15 ° C. for 16 hours, To pSPL3 vector P1 DN
The A fragment was inserted.

After treatment at 72 ° C. for 10 minutes to stop the reaction, E. coli HB101 was transformed. The grown E. coli was collected and the plasmid was transferred to the Qiagen column (Qiagen
(Manufactured by the company). The pSPL3 vector contains
The donor and acceptor structures for splicing of the HIV tat gene have been incorporated and are designed to be used to splice exons from inserted genes. The various primers (SEQ ID NOS: 7, 8 and 9) described below were set on this vector.

(3) Transfection of Cos-7 Cells by Electroporation Cos-7 cells were cultured in a petri dish having a diameter of 10 cm using DMEM medium containing 10% fetal bovine serum. The cells were peeled off using a 0.5% trypsin solution to obtain a cell suspension, and the cells were washed with cold PBS (Phosphate buffer-Saline pH 7.2).
After adjusting the cell number to a concentration of 0.5 × 10 ⁶ / ml, 0.8 ml of the cell suspension and 5 μg of plasmid DNA were added to a 0.4 cm chamber and ice-cooled for 10 minutes. Gene Pulser II (manufactured by BioRAD)
Was used for electroporation under the conditions of voltage 1.2 kV and capacitance 25 μFD. After ice-cooling for 10 minutes again, the cells were resuspended in a medium preheated to 37 ° C., and then cultured for 60 to 72 hours.

(4) Extraction of total RNA The transfected cells were washed with PBS warmed to 37 ° C. and treated with 0.1% trypsin solution to remove the cells to give a suspension. After suspending the cells in DMEM medium containing 10% fetal bovine serum to inactivate trypsin, 300 μl of TKM solution (10 mM Tris-HCl) containing no RNase was used.
pH 7.5, 10 mM KCl, 1 mM MgCl ₂ ) and 15 μl of 10% Triton
Add X-100 solution every 5 minutes under ice cooling and add 1,500 r at 4 ℃.
The nucleus was removed by centrifugation at pm x 5 minutes. Remove the supernatant and use RNase
After adding 20 μl of 5% SDS containing no RNA, RNA was extracted with phenol / chloroform. Usually, 30 to 50 μg of total RNA could be recovered by these series of operations.
RNA was precipitated with ethanol, treated with diethylpyrocarbonate, and dissolved in 50 μl of RNase-free MilliQ water.

(5) RT-PCR About 3 μg of cytoplasmic RNA prepared by the above-mentioned procedure, primer SA2 (SEQ ID NO: 7) set in pSPL3 vector, dithiothreitol, dNTP (dATP, dCTP, dGTP and dTTP), reversal Transcriptase buffer and reverse transcriptase Super Sucript II
The reaction was carried out at 42 ° C for 30 minutes in the reaction solution containing RNase, followed by RNase treatment to prepare cDNA. Using this cDNA as a template, the two primers SD2 set in the pSPL3 vector
(SEQ ID NO: 8) and SA4 (SEQ ID NO: 9) were subjected to PCR reaction using Taq DNA polymerase. The reaction was carried out for 30 cycles, each cycle consisting of 1 minute at 94 ° C, 1 minute at 60 ° C and 1 minute at 72 ° C.

The reaction product, DNA, was precipitated with ethanol and separated into high molecular weight and low molecular weight fractions by 4% low melting point agarose electrophoresis. Cut out the gel from each fraction, dissolve at 70 ℃, and then use Resin column (Promega)
DNA was purified by. Normally, 50 to 50
200 ng of DNA could be recovered.

(6) Preparation of exon DNA for nucleotide sequence determination A solution containing the above DNA solution, T4 DNA ligase (Takara Shuzo), buffer (Takara Shuzo), and pGEM-T vector (Promega) was prepared. Exon DN is reacted at 15 ℃ for 3 hours.
The A fragment was incorporated into the pGEM-T vector. E. coli JM109 was transformed with this vector and plated on LB bacto agar plates containing X-gal, IPTG and ampicillin (final concentration 50 μg / ml). For the white E. coli colonies,
PCR was carried out using two primers, SA4 (SEQ ID NO: 9) and SD2 (SEQ ID NO: 8) set in the pSPL3 vector.
The obtained colonies of the DNA fragment were cultivated in LB medium containing ampicillin (final concentration 50 μg / ml) at 37 ° C for 16 hours or more with shaking, and the Kurabo robot (PI-100Σ) was used.
Was used to collect and purify the plasmid DNA.

After degrading RNA by RNase treatment, 20%
Small molecule compounds were removed with polyethylene glycol / 2.5M NaCl solution and ethanol precipitation was performed to obtain a sample for DNA sequencing. Finally, the exons whose nucleotide sequences were confirmed were searched in the data bank and roughly classified into known and unknown genes. The unknown exon was used as a probe when screening the library as described in [Example 4].

[Example 3] Extension of exon by TAIL-PCR method TAIL-PCR method is a temperature asymmetric alternating PCR method capable of efficiently amplifying an unknown nucleotide sequence adjacent to an existing sequence [Liu, Y.-G.
and Whittier, RL Genomics 25, pp674-681 (199
5)], using a long primer specific to a known sequence and an arbitrary primer shorter than that, alternately perform high temperature and medium temperature annealing. Furthermore, by changing this cycle to the primer site and repeating it three times, the target sequence can be preferentially and specifically amplified. Specifically, human HeLa was used as the template cDNA in the following cycle.
PCR was performed using the cell cDNA library and the human lip cell cDNA library to detect and isolate the cDNA fragment.

(1) Primary PCR reaction; 16.5 μL pre-mixture-1 * 2.5 μL template 1.0 μL AD 1-4 primer (100 pmol / μL) Total amount 20 μL * pre-mixture-1 (165 μL, 10 samples) 20.0 μL 10 × buffer 16.0 μL dNTPs 30.0 μL SP-1 primer (1 pmol / μL) 1.6 μL Taq (5 U / μL) 97.4 μL dH ₂ O 1 cycle 92 ℃ 2 minutes, 95 ℃ 1 minute, 1 cycle 94 ℃ 15 seconds, 65 ℃ 1 minute, 72 ℃ 2 minutes, 5 cycles 94 ℃ 15 seconds, 30 ℃ 3 minutes, (over 3 minutes) 72 ℃ 2 minutes, 1 cycle 94 ℃ 15 seconds, 44 ℃ 1 minute, 72 ℃ 2 minutes, 10 cycles 12 cycles 94 ℃ 5 seconds, 65 ℃ 1 minute, 72 ℃ 2 minutes, 2 cycles 94 ℃ 5 seconds, 44 ℃ 1 minute, 72 ℃ 2 minutes, 1 cycle 1 cycle 72 ℃ 5 minutes, 1 cycle 4 ℃ Hold.

(2) Secondary PCR reaction; 17.4 μL pre-mixture-2 * 2.0 μL primary PCR product (× 100 dilution) 0.6 μL AD 1-4 primer (100 pmol / μL) Total amount 20 μL * pre-mixture-2 ( 174 μL, 10 samples) 20.0 μL 10 × buffer 2.0 μL dNTPs 40.0 μL SP-2 primer (1 pmol / μL) 1.6 μL Taq (5 U / μL) 110.4 μL dH ₂ O 1 cycle 94 ° C 5 minutes 1 cycle 10 cycles 94 ° C 5 seconds, 65 ℃ 1 minute, 72 ℃ 2 minutes, 2 cycles 94 ℃ 5 seconds, 44 ℃ 1 minute, 72 ℃ 2 minutes, 1 cycle 1 cycle 72 ℃ 5 minutes Hold at 4 ℃.

(3) Tertiary PCR reaction; 42.0 μL pre-mixture-3 * 5.0 μL secondary PCR product (× 100 dilution) 3.0 μL AD 1-4 primer (100 pmol / μL) Total amount 50 μL * pre-mixture-2 ( 420 μL, 10 samples) 50.0 μL 10 × buffer 5.0 μL dNTPs 20.0 μL SP-3 primer (1 pmol / μL) 3.0 μL Taq (5 U / μL) 342.0 μL dH ₂ O 1 cycle 94 ° C 5 minutes 1 cycle 20 cycles 94 ° C 10 seconds, 44 ℃ 1 minute, 72 ℃ 2 minutes 1 cycle 72 ℃ 5 minutes Hold at 4 ℃.

(4) Primers used AD1 SEQ ID NO: 10 AD2 SEQ ID NO: 11 AD3 SEQ ID NO: 12 AD4 SEQ ID NO: 13 3 ′ extension 3 ′ SP-1 SEQ ID NO: 14 3 ′ SP-2 SEQ ID NO: 15 3 ′ SP -3 SEQ ID NO: 16 Extension to 5'side 5'SP-1 SEQ ID NO: 17 5'SP-2 SEQ ID NO: 18 5'SP-3 SEQ ID NO: 19

Usually, 7 μL was analyzed by 1.5% agarose gel, and when specific amplification was observed, 5 μL was directly used for the base sequence analysis. The DNA sequence thus obtained was cloned using the pGEM-T vector (Promega) in order to confirm that it was a gene fragment derived from the WS region, and the nucleotide sequence was determined by the method described below. The sequence was determined.

Determination of Nucleotide Sequence The target DNA fragment was pGEM-T vector (Promega).
After being inserted into Escherichia coli and propagated in E. coli, the plasmid is purified by the alkali method using a robot (PI-100Σ manufactured by Kurabo). After treating the resulting purified plasmid with RNase, treat it with a solution of polyethylene glycol / saline to remove small molecules and purify the DNA.

Using the purified DNA as a template DNA, an unlabeled primer, four types of fluorescently labeled nucleotide-5'-triphosphate, and Taq polymerase were added in a reaction system.
Perform PCR. In this reaction, a fluorescent dye was randomly added.
By synthesizing a DNA fragment and analyzing it with a sequencer, a continuous base sequence can be finally determined. The nucleotide sequence of DNA can be determined using an automated DNA sequencer (model ABI 373) manufactured by Applied Biosystem.

The position of the obtained cDNA clone was finally confirmed accurately by PCR and FISH, and the nucleotide sequence was determined using the following primers. M13F side sequence number 20 M13R side sequence number 21 SP6 side sequence number 22 T7 side sequence number 23

[Example 4] Screening of NEC 14λ Zap II cDNA library Composition in WS region owned by AGEN Institute Inc. P1
Probe a partial fragment of WS-2 gene (600bp containing a part of VIII of exon IV-V-VI-VII-VIII in Fig. 2A) isolated from clones (# 4117, # 4370) by exon trapping method. Used for. Human NEC 14 (embryonal carcinoma) λZa
The pII cDNA library was constructed by the method of Nojima et al.
ori, M. et al., Nuc. Acids. Res. 21: p2782, 1993),
This was screened with the above probe, and 3 types of cDNA
I got a clone.

(1) Preparation of Infected Host Cell Escherichia coli XL1 Blue containing 12.5 μg / ml tetracycline
The cells were cultured on an LB plate at 37 ° C overnight to obtain a single colony.
This single colony was suspended in 75 ml of an LB culture medium containing 0.2% maltose and 10 mM MgSO ₄ , shake-cultured overnight at 37 ° C., and the next day, Escherichia coli was recovered by cold centrifugation (3,500 rpm × 15 minutes). Finally, pre-cooled to 4 ° C
150 ml sterile Mi containing 0.2% maltose and 10 mM MgSO _4.
The cells were resuspended in lli-Q water and used as infected host cells.

(2) Infection and Plating With SM solution (see “(7) Reagents / Solutions” below) 1.5-3
A λZap II cDNA library phage solution diluted to × 10 ⁴ pfu was mixed well with 600 µl of XL1 Blue prepared by the above method, incubated at 37 ° C for 20 minutes, and then 9 ml
Add 0.7% top agarose-NZYM (preheated to 50 ° C) to 1.5% bottom agar-NZYM plate (15 cm diameter dish) so that the mixture is evenly distributed at 37 ° C.
I incubated for 6-8 hours.

(3) Immobilization of phage DNA on nitrocellulose filter After cooling the plaque-formed plate at 4 ° C (30 minutes or more), a nitrocellulose filter (Schleicher & Schleicher & S
chuell, Millipore, etc.) should be gently placed between the plate and the nitrocellulose filter to prevent air from entering. Let stand for about 3 minutes (load the filter while the plate is warm and agarose will stick to the filter when peeled off) while puncturing the nitrocellulose filter and plate with a needle and mark. Peel off the filter, and place the filter on the filter paper soaked with the following solutions (I to III) with the surface with the phage facing up.

I. 0.5 M NaOH, 1.5 M NaCl 1 min 30 sec II. 0.5 M Tris-HCl (pH 7.5), 1.5 M NaCl 5 min III. 3 x SSC 3 min The resulting nitrocellulose filter was air dried. rear,
The phage DNA was fixed to the filter by exposing it to 312 nm UV light for 15 seconds.

(4) Labeling of probe As the probe DNA, the P1 clone (# 4117, which constitutes the physical map of the gene by the exon trapping method) (# 4117,
# 4370) started from exon, TAIL-PCR
Partial fragment of the WS-2 gene extended in Fig. 2 (exon I in Fig. 2A
600 bp containing part of VIII of VV-VI-VII-VIII was used.
DIG-DNA Labeli using 50 ng of WS-2 cDNA fragment as template and 50 pmol of random hexamer as primer
It was labeled with DIG using ng Kit (manufactured by BMB).

(5) Hybridization Filter was placed in a plastic container with 100 ml of hybridization buffer (50% formamide, 5 × SSPE,
× Denhardt's solution, 2% SDS, 100 μg / ml denatured salmon sperm D
NA fragment) and incubated at 42 ° C. for 4 hours or more. Remove the hybridization buffer, add a new 50 ml hybridization buffer,
The mixture was gently shaken so that air bubbles did not enter under the filter. Add DIG-labeled DNA probe to this,
Hybridized at 42 ° C for 16 hours or more.

(6) Washing and autoradiography After hybridization, the filter was washed with 100 ml of 2 ml.
× SSC, 0.1% SDS solution at room temperature, 10 minutes rinsing 3 times, then 100 ml of 0.1 × SSC, 0.1% SDS solution at room temperature,
Four 10 minute rinses were performed. Excessive moisture was removed so that the filter kept a slightly wet state, and autoradiographic analysis was performed by Kodak or Fuji Film sandwiched between Saran Wrap (Asahi Kasei).

[0114] (7) Reagents solution (i): SM solution; A): 5.8 (g) NaCl B): 2.0 (g) MgSO 4 · 7H 2 OC): 25.0 (ml) 2M Tris-HCl (pH 7.5 ) D): 5.0 (ml) 2% Gelatin Milli-Q Adjust to 1,000 ml with water, and sterilize by autoclaving.

(Ii): Solution I; Denature solution (1,000 ml); A): 100.00 (ml) 5N NaOH (final concentration 0.5M) B): 300.00 (ml) 5M NaCl (final concentration 1.5M) Milli-Q Adjust to 1,000 ml with water and sterilize by autoclaving.

(Iii): Solution II; Neutrize solution (1,000 ml); A): 250.00 (ml) 2M Tris-HCl (pH 7.4) (final concentration 0.5M) B): 300.00 (ml) 5M NaCl (final concentration) 1.5M) Milli-Q Adjust to 1,000 ml with water, and sterilize by autoclaving.

(Iv): Solution III; 20 x SSC (1,000 ml); A): 175.32 (g) NaCl (final concentration 3.0M) B): 88.23 (g) Na citrate (final concentration 0.3M) with 6N hydrochloric acid After adjusting the pH to 7.0, make up to 1,000 ml with Milli-Q water and sterilize by autoclaving.

(V): Hybridization solution A): 50% formamide B): 5 × SSPE C): 5 × Denhardt's Solution D): 0.1% SDS E): 100 μg / ml salmon sperm DNA

(Vi): Storage solution a): 20 x SSPE 1): 175.32 (g) NaCl 2): 27.60 (g) NaH ₂ PO ₄ 3): 7.49 (g) EDTA ・ 2Na 4): 750.00 (ml ) Milli-Q water Adjust the pH to 7.4 with 10N NaOH, make up to 1,000 ml with Milli-Q water, and sterilize by autoclaving.

B): 50 × Denhardt's Solution 1): 10.00 (g) Ficoll 400 2): 10.00 (g) BSA fraction V 3): 10.00 (g) Polyvinylpyrrolidone MilliQ Water was added to make 1000 ml, and then filtered. Store at -20 ° C.

C): 100 mg / ml salmon sperm DNA 1): Weigh 2.0 (g) salmon sperm DNA into a 50 ml tube.

2): Add 20 ml MilliQ water and incubate for 10 minutes. 3): Pass the solution above through the needle 100 times or more, or
Short DNA fragments by sonication.

4): After boiling for 10 minutes, immediately cool with ice and leave for 10 minutes.

(8) Results (A): Exon-intron structure The WS-2 gene is composed of at least 8 exons, and is composed of a population of different combinations by an alternative mechanism called gene expression due to gene expression. The present invention has been found to produce at least two types of mRNA. Eight exons are P1- # 4117
And-scattered in the common part of # 4370. alternativesplic
As a result of ing, two types of mRNA of Type I and II shown in FIG. 2A
Are produced, and these mRNAs are used in Lip fibroblast cells and NEC 1
It is known from the present invention that it is expressed in 4 (embryonal carcinoma) cells, and their cDNAs were cloned by the method shown in [Example 4]. In the figure, ATG (w
(ith an arrow) indicates the position of the initiator codon for protein synthesis, and TGA (with an arrow) indicates the position of the stop codon. (B): Computer analysis of amino acids (Fig. 2B)

Example 5 Analysis of WS-2 by Southern Blot WS-2 having a nucleotide sequence complementary to the labeled nucleic acid probe
Southern hybridization was performed as follows to identify the region of the gene DNA.

(1) Human placental genomic DNA 10 μg of human placental genomic DNA digested with restriction enzymes (EcoRI, HindIII and PstI) was separated by 0.8% agarose gel electrophoresis. The gel was immersed in 150 ml of 0.5N NaOH-1.5M NaCl solution and gently shaken for 30 minutes to denature it with alkali. Gently wash the gel with deionized water, 0.5M Tris-HCl (pH 7.5),
It was immersed in 150 ml of a 1.5 M NaCl solution and gently shaken for 30 minutes to cause a neutralization reaction.

On the other hand, a nitrocellulose membrane (manufactured by Amersham) cut into the same size as the gel was moistened with distilled water.
2 x SSC (0.3M NaCl, 0.03M sodium citrate (pH
7.0) soaked. Add 200 ml of 20 × SSC solution to the tray,
Soak the sponge, then filter paper of appropriate size (Whatmann
3 MM) was dipped in 2 × SSC and then placed on the sponge.

Next, on this filter paper, an agarose gel treated as described above and a nitrocellulose membrane thereon were placed, and a filter paper of the same size, a bundle of paper towels, and a weight were placed and transferred overnight.

After the transfer is completed, ultraviolet irradiation (wavelength
Genomic DNA was immobilized on a nitrocellulose membrane at 260 nm and 120 mJ / cm ² ) and then lightly washed with ultrapure water. After placing the nitrocellulose membrane in a plastic bag, prehybridization solution [7% SDS, 50% Formamide,
5 × SSC, 2% Blocking Reagent (Boehringer Mannheim
Co., Cat. No. 1096176), 50 mM sodium phosphate (pH 7.
0), 0.1% (w / v) N-Lauroylsarcosine, 50 μg / ml containing denatured herring sperm DNA] The sample was immersed in 5 ml and treated at 42 ° C. for 6 hours. Change the solution, hybridization solution (composition of pre-hybridization solution plus digoxigenin labeled full length WS-2 cDNA probe 5-20 ng / ml)
After pouring 5 ml, the bag was sealed and incubated overnight at 42 ° C. Take out the nitrocellulose membrane and remove 100 ml.
2 × SSC, 0.1% SDS solution, and shaken at room temperature for 5 minutes.

After repeating this operation twice,
× SSC-0.1% SDS solution was changed and shaken at 42 ° C for 15 minutes.
After changing the solution and continuing shaking at 42 ° C for 15 minutes, the nitrocellulose membrane was rinsed lightly with a maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5), and a 1% blocking solution (Blocking Reagent was added to maleic acid). (Dissolved in buffer) for 30 minutes at room temperature. After removing the solution, the alkaline phosphatase-labeled anti-digoxigenin antibody (Boehring
er Mannheim, Cat. No. 1093274) was used to react the nitrocellulose membrane at room temperature for 30 minutes.

The nitrocellulose membrane was washed with a maleic acid buffer containing 0.3% Tween 20 for 5 minutes at room temperature with shaking. After repeating this washing operation twice, the nitrocellulose membrane was buffered with alkaline phosphatase (0.1M).
Tris-HCl, pH 9.5), 0.1 M NaCl, 50 mM MgCl ₂ ) lightly rinsed, 0.25 mM chemiluminescent substrate CSPDTM (Boehringer Mannh)
It was immersed in a buffer for alkaline phosphatase reaction containing Eim's Cat. No. CD010). After accelerating the enzymatic reaction at 37 ° C for 10 minutes, the generated chemiluminescence was analyzed by Kodack X
It was detected by autoradiography with line film (Fig. 4A).

(2) Zoo Blot 9 types of genomic DNA (4 μg each) were subjected to agarose electrophoresis and a premade filter (manufactured by Clontech) blotted on a nylon membrane was used (FIG. 4B).

Composition of human chromosome 8p11.2-p12.1 contig owned by AGEN Institute Inc. P1 clone (# 411
7, # 4370), the partial fragment of WS-2 gene (600 bp including a part of VIII of exon IV-V-VI-VII-VIII in Fig. 2A) isolated by TAIL-PCR method was used as a probe. NEC14 (embryon
screening of a λZapII cDNA library, a full-length cDNA clone of WS-2 gene, pBS N9-1.1
I got By digesting this pBS N9-1.1 with restriction enzymes EcoRI and HindIII, a 1263 bp cDNA fragment containing the open reading frame (ORF) of the WS-2 gene can be obtained. This 126
The 3 bp DNA fragment was radioactively labeled according to the method described below and WS-2
It was used as a cDNA detection probe. Hybridization and washing conditions followed Clontech's protocol.

(3) Results The results of Southern hybridization are shown in FIGS. 4A and 4B. (A): Human placental genomic DNA Human placental genomic DNA was digested with a restriction enzyme, separated by agarose electrophoresis, and then blotted on a nitrocellulose membrane.
This was hybridized with DIG-labeled WS-2 cDNA as a probe and detected by an alkaline phosphatase-labeled anti-digoxigenin antibody. Lane 1 digested with PstI, lane 2 digested with HindIII, lane 3 Ec.
It was digested with oRI.

(B): Zoo Blot analysis Zoo Blot (CLONTE containing 4 μg of genomic DNA from various sources)
The CH-2) was hybridized with [ ³² P] -labeled WS-2 cDNA as a probe. Origin of Genomic DNA: a, human; b, monkey; c, rat; d,
Mouse; e, dog; f, cow; g, rabbit; h, chicken; i, yeast WS-2 mRNA expression was species-specific.

Example 6 Analysis of WS-2 by Northern Blot (1) Analysis of WS-2 Northern Blot In the present invention, after total RNA was subjected to agarose electrophoresis by the glyoxal method and transferred to a nylon membrane, The cDNA probe was labeled with [ ³² P] -dCTP and hybridized. Here, we show the analysis by the WS-2 cDNA probe using the following RNA samples (Fig. 5).
And FIG. 6).

(I) Total RNA sample (Fig. 5) Derived from healthy human fibroblasts-CS-3F0-250 (newborn skin marketed by Dainippon Pharmaceutical Co., Ltd.)-YH (male, 35 years old, skin) -HN (Male, 20 years old, skin) WS patient fibroblast origin ・ WS6201 (Female, skin) ・ WS11901 (Male, skin) ・ WS12301 (Male, 42 years old, skin)

(Ii) Human multi-tissue northern blot [Human
Multiple Tissue Northern (MTN) Blot] (Fig. 6) MTN Blot I ⅈ 2 μg each of polyA + RNA extracted from 16 types of human tissues / organs was subjected to agarose gel electrophoresis and blotted on a nylon membrane ( Clonte
ch company) was used.

(Iii) WS-2 cDNA probe Human chromosome 8p11.2 owned by AGEN Institute Inc.
-p12.1 Contig configuration Partial fragment of WS-2 gene isolated by TAIL-PCR from P1 clones (# 4117, # 4370) (Fig. 2
A part of exons IV-V-VI-VII-VIII VIII 600
bp) as a probe for human NEC14 (embryonal carcino
ma) λZapII cDNA library was screened to obtain a full-length WS-2 gene cDNA clone, pBS N9-1.1.
By digesting this pBS N9-1.1 with restriction enzymes EcoRI and HindIII, a 1263 bp cDNA fragment containing the ORF of WS-2 gene can be obtained. This 1263 bp DNA fragment was radiolabeled according to the method described below and used as a WS-2 cDNA detection probe.

(Iv) Human β-actin cDNA probe Human β-actin cDNA (206 to 1109 nucleotides, 904 b
p) portion is amplified and purified by RT-PCR using the primers represented by SEQ ID NO: 24 and SEQ ID NO: 25, and this is
It was subcloned into the EM-T vector (Promega). This cDNA clone was cloned into Nc
A 935 bp fragment obtained by digesting at the oI and NotI sites was used as a probe.

(2) Preparation of total RNA blotting filter (i) Tools and reagents Tools such as electrophoresis tank, comb, stirrer bar are RNase
For removal, soak in 2% Absorb (Dupont) for 1 day,
It was used after rinsing with Milli-Q water treated with diethylpyrocarbonate (DEPC). All reagents were prepared with DEPC-MilliQ water and autoclaved and used.

(Ii) Deionization treatment of glyoxal and dimethyl sulfoxide (DMSO) Saturated glyoxal (manufactured by Nakarai Co., Ltd.) and DMSO (manufactured by Nakarai Co., Ltd.) were each added to 20 ml of ion exchange resin AG501-X8.
Add (BioRad) 20g and stir for 30 minutes. The same amount of resin is added to the filtered filtrate again, and this operation is repeated until the pH becomes 6.0 or less. Finally, 1 ml each was dispensed into an Eppendorf tube and stored at -20 ° C.

(Iii) Preparation of agarose gel 1 g agarose ME (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 100 ml of 10 mM sodium phosphate (pH 6.5) to prepare a 1% agarose gel. The size of the gel is 7 cm (W) x 10 cm (L).

(Iv) Glyoxal treatment of RNA In the denatured state due to hydrogen bonding between RNA and glyoxal, RN
The following pretreatment was performed because it was not decomposed by ase. 20 μg of the above total RNA sample was ethanol precipitated and the pellet was air dried. Dissolve this with 5 μl of DEPC-MilliQ water and add 11 μl of glyoxal mix [58
μl deionized glyoxal, 200 μl deionized DMS
O, 8 μl 10% SDS, 8 μl 0.5 M sodium phosphate (pH 6.
5), Preparation before use] was added and incubated at 50 ° C. for 20 minutes. At this time, RNA ladder (Gibco
5 μg (manufactured by BRL) was similarly pretreated.

(V) Electrophoresis Add 3 μl of gel to the glyoxal-treated total RNA sample.
Loading buffer [50% glycerol, 10 mM sodium phosphate (pH 7.0), 0.25% bromophenol blue, 0.25% xylene cyanol FF] was added, and the mixture was gently centrifuged.

This was loaded onto a 1% agarose gel and 8% in 10 mM sodium phosphate buffer (pH 6.5).
Electrophoresis was carried out at 0 V for about 2 hours until just before the bromophenol blue flowed out of the gel. During migration, in order to prevent glyoxal from being dissociated from RNA due to an increase in pH, the migration buffer was stirred with a magnetic stirrer, and the + and-polar buffers were circulated by a peristaltic pump.

After the electrophoresis, the gel was immersed in a 50 mM sodium hydroxide solution for 20 minutes to dissociate the glyoxal (the next staining would not be successful unless this operation was performed). RNA was stained by adding 1 µg / ml ethidium bromide while neutralizing with 50 mM sodium phosphate (pH 6.5) for 20 minutes. The stained size markers and ribosomal RNA were photographed along with the scale.

(Vi) Blotting on Nylon Membrane RNA was transferred to a nylon membrane, Hybond N + (manufactured by Amersham) by a capillary method overnight in 20 × SSC buffer. When the nylon membrane (hereinafter referred to as a filter) was peeled from the gel, a marker was attached at the position of the comb, and the upper left corner of the RNA-transferred surface was cut off so that the inside and outside could be seen. 1 filter with 2 x SSC buffer
Rinse for 0 minutes and air dry. When the filter dries 312 n
The RNA was fixed to the filter by exposing to m UV light for 15 seconds.

(3) Hybridization (i) Prehybridization The filter is immersed in 100 ml of prehybridization buffer (WS-2 or β-actin prehybridization buffer) in a lunch box type poly container.
Incubated at 4 ° C for 4 hours.

The WS-2 prehybridization buffer contains 50% formamide, 5 × SSPE, 10 × Denhar.
A solution containing dt's solution, 2% SDS, 100 µg / ml denatured salmon sperm DNA fragment was added to β-actin prehybridization buffer with 40% formamide, 6 x SSC, 5 x Denha.
An rdt's solution containing 0.5% SDS and 100 μg / ml denatured salmon sperm DNA fragment was used.

(Ii) WS-2 and β-actin cDNA probe radioactive labeling Using the above-mentioned WS-2 cDNA fragment 50 ng as a template and a random hexamer 50 pmol as a primer, a random primer DNA labeling kit Ver.2 (Takara Shuzo) Was labeled with [α- ³² P] -dCTP (manufactured by NEN, manufactured by Daiichi Pure Chemicals). The β-actin probe was labeled in the same way.

(Iii) Hybridization Discard the pre-hybridization buffer and freshly
Add 50 ml prehybridization buffer and shake gently to prevent air bubbles from getting under the filter. WS-2 radiolabeled with [ ³² P] -dCTP
The cDNA probe was added so that the specific activity was about 1 × 10 ⁶ cpm / ml, and hybridized at 42 ° C. for 16 hours.

After hybridization, the filter was rinsed twice with 100 ml of 2 × SSC, 0.1% SDS solution at room temperature for 15 minutes, then 100 ml of 0.2 × SSC, 0.1% SDS.
The DS solution was rinsed twice at room temperature for 15 minutes. The filter was dehumidified to a slightly damp state, and autoradiography analysis was performed using a BAS1500 system (manufactured by Fuji Film Co., Ltd.) with a Saran wrap (manufactured by Asahi Kasei Corporation).

(Iv) Removal of probe from filter Be careful not to dry the filter completely,
Boiled for 20 minutes with 0.1 × SSC, 5% SDS solution. After boiling, the mixture was allowed to stand until it returned to room temperature, then the filter was taken out on filter paper and air-dried. It was confirmed that the signal disappeared by performing autoradiography before hybridizing the next probe or exposing to the Fuji BAS1500 system (manufactured by Fuji Film).

(4) Analysis by Fuji BAS1500 system The BAS1500 system is a radiation energy memory type two-dimensional sensor (imaging plate;
The sample was contact-exposed to IP) similarly to the X-ray film, and this IP was excited by He-Ne laser light and the fluorescence emitted according to the exposure amount was converted into a digital amount called PSL (Photo Stimulated Luminescence) and quantified. . The linearity of the quantitative value is good and the background can be subtracted, and the radiation intensity in the designated area can be measured and compared.

(I) Quantification of WS-2 mRNA expression The sensitivity of IP was set to about 20 times that of X-ray film, and the filter hybridized with WS-2 cDNA probe was contact-exposed to IP for 3 hours in the dark. . As a result, a clear band of about 1.5 kbp could be detected. WS-2 mRNA in healthy subjects and WS patients
There was no significant difference in size and expression level.

The quantification was performed as follows. First, an area of each band of a healthy person and a WS patient and an arbitrary background area were designated, and the background was subtracted from the quantified value of the radiation intensity of each band for comparison (Table 1).

(Ii) Normalization Between Samples The WS-2 probe was removed from the filter according to the method described above. Next, similar hybridization and quantification were performed using a β-actin probe, and the signal intensity of each sample was measured. Based on the assumption that all samples were derived from fibroblasts and the expression level of β-actin was the same,
The ratio between the expression level of WS mRNA and the expression level of β-actin mRNA was calculated (Table 1).

(5) Results (i) Tissue-specific expression of WS-2 mRNA WS-2 mRNA showed tissue-specific expression, and had a pattern as shown in FIG. That is, the WS-2 gene is strongly expressed in testis and ovary, and moderately expressed in heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, small intestine, and large intestine. It was found that the expression was extremely low in peripheral lymphocytes.

FIG. 6: Distribution of WS-2 mRNA expression in tissues and organs Multi containing 2 poly (A) + RNAs derived from human tissues and organs
ple Tissue Northern Blot I, II (CLONTECH)
WS-2 cDNA labeled with [ ³² P] was hybridized with the probe.

Origin of Poly A + RNA: a, heart: b, brain; c, placenta; d, lung; e, liver; f, skeletal muscle; g, kidney; h, pancreas; i, spleen; k, prostate l, testis; m, ovary; n, small intestine; o, large intestine, p, peripheral blood lymphocytes WS-2 mRNA expression was organ-specific.

(Ii) Comparison between WS-2 mRNA expression in healthy subjects and WS patients (FIGS. 5A, 5B and Table 1) In addition, healthy subjects and WS standardized by β-actin mRNA expression level
Regarding the appearance of about 1.5 kb WS-2 mRNA in the patient, there was no difference in size and expression level between the two.

FIG. 5A: Comparison of the amount of WS-2 mRNA using 20 μg of total RNA from fibroblasts of healthy subjects and WS patients. A band of about 1.5 kb was detected, and there was no significant difference in the size and expression level of mRNA between healthy subjects (lanes 1 to 3) and WS patients (lanes 4 to 6).

Origin of total RNA: lane 1, total RNA derived from CS-3FO-250 cells; lane 2, total RNA derived from healthy human YH cells; lane 3,
Healthy human HN cell-derived total RNA; lane 4, WS6201 patient cell-derived total RNA; lane 5, WS11901 patient cell-derived total RNA; lane
6, WS12301 Patient cell total RNA. 28S and 18S are ribosomal R
The position of the size of the NA subunit is shown.

FIG. 5B: The same filter as in (B) was hybridized with β-actin probe. Β-actin mRNA, which is a positive control, was used in WS patients (lane 4
In ~ 6), the same level of expression as in healthy subjects (lanes 1 to 3) was observed.

[0166]

[Table 1]

Table 1 shows the results of quantification of the northern blot analysis results shown in FIGS. 5A and 5B by the Fuji BAS 1500 system. Actin mRN expression of WS-2 mRNA
After normalizing with A, healthy subjects and WS patients were compared by% display. This data compares the values of 3 WS patients and 3 healthy individuals. Lanes 1 to 6 in Table 1 correspond to lanes 1 to 6 in FIGS. 5A and 5B. Lanes 1 to 3 show the values of healthy persons, and lanes 4 to 6 show the values of WS patients.

Example 7 Expression of WS-2 Protein (1) Expression Vector, pGEX-3X For the purpose of obtaining a protein encoded by the WS-2 gene, expression was performed using Escherichia coli. As a vector used for expression, pGEX-3X (Pharmacia Biotech) was used. This vector employs a promoter that can induce expression with high efficiency, and
It expresses a fusion protein with 6,000 GST (glutathione-S-transferase) domains (from S. japonicum) (Smith, DB and Johnson, KS, Gene 6
7. 31, 1998). Also, the expressed fusion protein is Gl
It can be easily purified using affinity chromatography on utatione Sepharose 4B. To cleave the protein of interest from the fusion protein, use the recognition sequence site of the site-specific protease (blood coagulation factor Xa) located immediately upstream of the multicloning site.
(Fikrig, E. et al., Science 250. 553, 1990).

(2) Cloning of WS-2 gene A plasmid containing the full-length WS-2 gene, pBS N9-1.1, was Ec.
After digesting with oRI and HindIII and purifying the fragment containing the ORF of WS-2 gene, fill in with Klenow enzyme to form blunt ends. After digestion with Sma I, the CIP-treated pGEX-3X vector and the purified and modified fragment containing the ORF of the WS-2 gene were reacted with T4 ligase to prepare a recombinant, which was then transduced into XL1-Blue cells. To form. Finally, a clone containing the correct copy and containing a single copy is found, and a sequence reaction is performed to reconfirm that the amino acid frame is correct.

(3) Production of protein The above vector was transformed into Escherichia coli BL21 (DE3) and then grown according to the protocol. E. coli was collected as a pellet, and 20 ml of lysis buffer [10 mM Hepes (pH
7.5), 3.4 mM EDTA-2Na (pH 8.0), 150 mM NaCl, 1% Trito
n X-100, 10mMDTT, 1mg / ml lysozyme] and then mild sonication is repeated. The supernatant was salted out with ammonium sulfate at a final concentration of 20% and then dialyzed thoroughly (50 mM Tris-HCl (pH 7.0), 50 mM N
aCl, 5mMDTT) and Glutatione Sephar according to the protocol.
Purify the protein of interest on an ose 4B column and add 10.0% S
Purity was examined by DS-PAGE (Fig. 7).

(4) Results The desired WS-2 gene product having a molecular weight of 30,000 and a molecular weight of 26,0
A polypeptide with a molecular weight of 56,000 expected as a fusion protein with the GST (glutathione-S-transferase) domain of 00 (derived from S. japonicum) was detected as a single band by Coomassie staining after SDS-polyacrylamide gel electrophoresis. It turned out that it was purified.

The protein encoded by the WS-2 gene was expressed in E. coli by the method described in [Example 7] and purified by affinity chromatography using Glutation Sepharose 4B. Purified WS-2 and GST
The fusion protein with a molecular weight of about 56,000 was analyzed for its purity by 10.0% polyacrylamide gel electrophoresis. After electrophoresis, the proteins in the gel are Coomasie Bri
Stained with lliant Blue.

Lane 1: Molecular weight marker (Bio-Rad, Low range) Lane 2: BL21 (D transformed with pGEX-3X plasmid
E3) Total protein of cells Lane 3: BL21 (D transformed with pGEX-3X plasmid
E3) IPTG-induced whole cell protein lane 4: total protein of BL21 (DE3) cell transformed with pGEX-3X plasmid containing WS-2 Lane 5: pGEX- containing WS-2 Total protein in which BL21 (DE3) cells transformed with 3X plasmid were induced with IPTG Lane 6: Fraction I recovered from Glutation Sepharose 4B Lane 7: Fraction II recovered from Glutation Sepharose 4B Lane 8: Glutation Sepharose Fraction III recovered from 4B

This protein can be used as an antigen for producing a polyclonal antibody or a monoclonal antibody. In addition, this gene is very strongly expressed in the reproductive system of testis and ovary, suggesting that it plays an important role in the conservation or regeneration system of species. Therefore, this gene is useful for elucidating the gene expression regulation related to the primate regeneration system, and is also useful for the detection of the cause of diseases such as infertility and the creation of new drugs for relieving it. , It can be used as a research material for elucidating the relationship with the onset of Werner syndrome disease. Furthermore, polyclonal antibodies and monoclonal antibodies against the WS-2 gene product may be usable as the above diagnostic reagents.

[0175]

INDUSTRIAL APPLICABILITY The present invention provides a gene (WS-2 gene) that causes Werner's disease. The WS-2 gene is
It is specifically retained only in primates, which are higher organisms,
Moreover, since it is very strongly expressed in the reproductive system of the testis and ovary, it is suggested that it plays an important role in the conservation or regeneration system of the species, and the elucidation of the gene expression regulation related to the primate regeneration system. It is also useful for detection of disease causes such as infertility and the creation of new drugs for relieving this, and also useful for research to elucidate the relationship with Werner syndrome onset. is there.

In addition, the gene of the present invention (WS-2 gene) is
It is useful not only for detecting the cause of diseases such as infertility and for gene therapy for relieving the cause, but also as a diagnostic probe for inspecting and preventing diseases related to the development of Werner syndrome.

In addition, medical and scientific studies on the development of human individuals,
As a reagent for cell biological, immunological, biochemical or molecular biological research, the WS-2 gene and the protein encoded by the WS-2 gene and antibodies to the protein are useful. In addition, the WS-2 gene is extremely useful in creating transgenic mice and creating model animals.

[0178]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1482 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence:

SEQ ID NO: 2 Sequence length: 270 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence:

SEQ ID NO: 3 Sequence Length: 1482 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA to mRNA Sequence:

SEQ ID NO: 4 Sequence length: 1077 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence:

SEQ ID NO: 5 Sequence Length: 187 Sequence Type: Amino Acid Topology: Linear Sequence Type: Protein Sequence:

SEQ ID NO: 6 Sequence length: 1077 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to mRNA Sequence:

SEQ ID NO: 7 Sequence Length: 21 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: ATCTCAGTGG TATTTGTGAG C 21

SEQ ID NO: 8 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: GTGAACTGCA CTGTGACAAG CTGC 24

SEQ ID NO: 9 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CACCTGAGGA GTGAATTGGT CG 22

SEQ ID NO: 10 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TGWGNAGWAN CASAGA 16

SEQ ID NO: 11 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: AGWGNAGWAN CAWAGG 16

SEQ ID NO: 12 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence features: Characteristic symbols: modified base Location: 6 Method of determining features: E Other information: Inosine Symbol representing characteristics: modified base Location: 11 Method of determining features: E Other information: Inosine Sequence: CAWCGICNGA IASGAA 16

SEQ ID NO: 13 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence features: Characteristic symbols: modified base Location: 5 Method of determining feature: E Other information: Inosine Symbol representing characteristic: modified base Location: 11 Method of determining feature: E Other information: Inosine Sequence: TCSTICGNAC ITWGGA 16

SEQ ID NO: 14 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: ATTGAGAAAA CTGGAGGAGC G 21

SEQ ID NO: 15 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CAAATGACGG GTGAAGCCTG G 21

SEQ ID NO: 16 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: AAGCAGTGGT CACAAACTTG G 21

SEQ ID NO: 17 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CCCAAGTTTG TGACCACTGC 20

SEQ ID NO: 18 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: ATTTCCAGGC TTCACCCGTC 20

SEQ ID NO: 19 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CAGGCTTCAC CCGTCATTTG 20

SEQ ID NO: 20 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CGCCAGGGTT TTCCCAGTCA CGAC 24

SEQ ID NO: 21 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TCACACAGGA AACAGCTATG AC 22

SEQ ID NO: 22 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: GATTTAGGTG ACACTATAG 19

SEQ ID NO: 23 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: TAATACGACT CACTATAGGG 20

SEQ ID NO: 24 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Sequence: CGACGAGGCC CAGAGCAAGA GAGG 24

SEQ ID NO: 25 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CCACATCTGC TGGAAGGTGG ACAG 24

[Brief description of drawings]

FIG. 1 is a diagram showing a gene physical map of a P1 / PAC clone in the WS gene region and the positions of the WS-2 gene and a known gene.

FIG. 2 is a schematic diagram showing the WS-2 gene and the protein encoded by the gene.

FIG. 3 is a photograph showing the results of FISH analysis using a P1 clone containing the WS-2 gene (morphology of organism).

FIG. 4 is a photograph of electrophoresis showing the results of analysis of WS-2 gene by Southern blotting.

FIG. 5 is a photograph of electrophoresis showing the results of analysis of WS-2 gene by Northern blot.

FIG. 6 is a photograph of electrophoresis showing the results of analysis of WS-2 gene by Northern blot.

FIG. 7 is a photograph of electrophoresis showing production of a protein encoded by WS-2 gene using Escherichia coli.

Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C07K 16/18 C07K 16/18 C12N 1/21 C12N 1/21 5/10 C12P 21/02 C C12P 21/02 21/08 21/08 7823-4B C12Q 1/68 A C12Q 1/68 G01N 33/53 D G01N 33/53 33/577 B 33/577 A61K 39/395 D // A61K 39/395 N 48/00 48 / 00 C12N 5/00 B (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:19) (C12P 21/08 C12R 1:91)

Claims (14)

[Claims] 1. A gene encoding a polypeptide substantially containing the amino acid sequence represented by SEQ ID NO: 2 or 5. 2. The gene according to any one of the nucleotide sequences represented by SEQ ID NO: 1, 3, 4 or 6.
The described gene. 3. An oligonucleotide probe which hybridizes with at least a part of the gene according to claim 1. 4. A recombinant DNA containing the gene according to any one of claims 1 to 2. A transformant transformed by the recombinant DNA according to claim 4. 6. A method for producing the above-mentioned polypeptide, which comprises culturing the transformant according to claim 5 and collecting a polypeptide encoded by a human gene from the resulting culture. 7. A polypeptide encoded by a human gene, which substantially comprises the amino acid sequence represented by SEQ ID NO: 2 or 5. 8. A monoclonal antibody which specifically reacts with the polypeptide according to claim 7. 9. A polyclonal antibody which specifically reacts with the polypeptide according to claim 7. 10. A hybridoma producing the monoclonal antibody according to claim 8, which is obtained by fusing antibody-producing cells immunized with the polypeptide according to claim 7 and myeloma cells. 11. A reagent for detecting a gene, which comprises the oligonucleotide probe according to claim 3. 12. The polypeptide according to claim 7, and the monoclonal antibody according to claim 8 and / or claim 9.
A diagnostic kit containing the described polyclonal antibody. 13. A mouse into which a gene modified so as to increase or decrease the expression level of the gene according to claim 1 or 2 is introduced. 14. A transgenic mouse into which the gene according to claim 1 or 2 has been introduced.