JPH08217797A - Spa-1 protein and gene coding the same - Google Patents

Abstract

PURPOSE: To obtain a new cell division mechanism control protein having a specific amino acid sequence, not manifested in stationary state and manifested in a nuclei after initiating a cell cycle and controlling cell division or differentiation of lymphocyte. CONSTITUTION: This new cell division mechanism controlling protein SPA-1 protein controls cell division or differentiation of lymphocyte and is not manifested in stationary state and manifested in a nucleus after initiating a cell cycle of lymphocyte in cell cycle of cell of mammals and has an amino acid sequence from the 1st methionine to 693th alanine or the amino acid sequence in which 1 or 2 or amino acids are added, lost or replaced in an amino acid sequence containing an amino acid sequence expressed by the formula. The protein is obtained by isolating mRNA from lymph-based cell strain, preparing cDNA library by ordinary method, screening the library with anti-SPA-1 monoclonal antibody, selecting clone containing SPA-1 gene and manifesting the gene in a host cell.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to SPA-1 protein and its fragments which are proteins involved in the regulation of cell division, genes encoding them, and antibodies to these proteins.

[0002]

2. Description of the Related Art Lymphoid cells have unique characteristics in their cell proliferating ability as compared with many other somatic cells. That is, like other somatic cells, it differentiates from a certain progenitor cell through many cell divisions to become a mature cell, and once enters a quiescent state (G0 / G1). After that, when they are stimulated by an antigen or a specific growth factor, they enter the cell cycle again and undergo clonal expansion with certain redifferentiation, and then return to a quiescent state (memory cells). In the immune response of the living body, this repetitive cell proliferation (clonal amplification) constitutes an extremely large factor in addition to the unique functional differentiation and expression of lymphocytes.

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to the novel protein SPA-1 and its active fragment which are expected to be involved in the control of repetitive cell growth, genes encoding them, and antibodies to these proteins. Is to provide.

[0004]

The present invention relates to a cell division mechanism regulatory protein that is not expressed in a quiescent state in the cell cycle of mammalian cells but is expressed in the nucleus after entering the cell cycle. This protein is called SPA-1 and has the structure shown in FIG. SPA deduced from the cDNA base sequence
The amino acid sequence of -1 is M at position 1 in SEQ ID NO: 1.
It has an amino acid sequence starting from et and ending at Ala at position 693.

However, the polypeptide or protein of the present invention is not limited to the above-mentioned ones, and is included in the scope of the present invention when it has the activity of the present invention even if there is a small change in the correct amino acid sequence. . Such alterations include amino acid substitutions within the sequence, and additions or deletions of one or more amino acids, and variations thereof are also included in the definition so long as the activity of the present invention is maintained. .

[0006] Here, addition, deletion or substitution of amino acids is a well-known technique before application, such as site-directed mutagenesis (eg Nu
cleic Acid Research, Vol. 1
0, No. 20, p6487-6500, 1982), and one or more amino acids in relation to addition, deletion or substitution of amino acids,
It means a sufficient number of amino acids that can be added, deleted or substituted by the site-directed mutagenesis method.

The above-mentioned polypeptide or protein can be produced by a gene engineering method by expressing a gene encoding the amino acid sequence of the above-mentioned polypeptide or protein. The gene encoding the above polypeptide is cDNA, genomic DNA or chemically synthesized DNA.
Can be obtained as

The cDNA encoding SPA-1 is hardly expressed in the quiescent phase (G ₀ / G ₁ phase) of lymphocytes, and can be obtained by cloning a gene expressed in the proliferative phase (S phase). . For example, cDNA is prepared from G ₀ / G ₁ phase and S phase lymphocytes according to a conventional method, hybridized with these, and a G ₀ / G ₁ phase-derived cDNA that does not hybridize with the S phase-derived cDNA is selected. By doing so, a cDNA encoding SPA-1 can be obtained. One example of a specific method is described in Example 1 (1).

In order to obtain the genomic DNA, for example, a genomic DNA library is prepared from the target animal, and this is screened by using the cDNA obtained as described above, for example, the full-length cDNA as a probe. This concrete method is concretely described in Example 3. For example, a genomic DNA encoding SPA-1 is a 5.7 kbp BamHI fragment (Spa-G) of genomic DNA.
C2) and 6.6 kbp BamHI fragment (Sp
a-GC9)).

As shown in FIG. 5 and SEQ ID NOs: 2 and 3, the 5.7 kbp DNA fragment (Spa-GC2) contains four exons (exons 1 to 4), which are 5.7 kbp fragments. It is present at about 2.5 kbp site on the 3'-end side of the. Also, 12 exons (exons 5 to 16) are scattered in the 6.6 kbp fragment (Spa-GC9). These exons 1-16 contain the entire cDNA described above. The coding region of cDNA is contained in the second half of exon 5 to the first half of exon 16.

In the present invention, to obtain a DNA encoding SPA-1 or a protein fragment thereof, the above-mentioned c
After removing unnecessary parts by treating DNA or genomic DNA with exonuclease, or cleaving with an appropriate restriction enzyme, if necessary, a missing sequence is supplemented by addition of an oligonucleotide, or unnecessary parts are exonuclease, etc. It may be removed by. In addition, a gene encoding a polypeptide in which one or more amino acids are deleted, added and / or substituted with respect to the natural amino acid sequence is a cDNA or genomic DNA obtained as described above It is obtained according to the method, for example, by subjecting it to site-directed mutagenesis.

The present invention further includes DNA or RNA which hybridizes with the base sequences shown in SEQ ID NOs: 1 to 3. Such hybridizing DNA or RN
A preferably retains the biological function of SPA-1 or a protein fragment thereof, for example, 50% formamide, 5 × SSC, 10% Na-dextran, 2
0 mM Na-phosphate (pH 6.5), the above-mentioned cDNA or genome D under hybridization conditions at 42 ° C.
It hybridizes with NA.

The polypeptide or protein of the present invention can be expressed in eukaryotic cells or prokaryotic cells according to a conventional method. Eukaryotic cells include animal, eg, human or other animal base cells, eg, NIH3T3 cells, Cos −.
1 cell, CHO cell or the like is used, and further a eukaryotic microorganism such as yeast or filamentous fungus is used. The yeast Saccharomyces cerevisiae (Saccharomy
ces cerevisiae ), and the like. Examples of filamentous fungi include Aspergillus.
s ) genus, eg Aspergillus niger ( Asperg
illus niger ) and the like. Prokaryotes include bacteria. For example, Bacils
lus ), for example Bacillus subtilin ( Bacill)
us subtilis ), Escherichia coli ( Escheric
hia coli ) or the like is used.

In order to express the DNA in these hosts, the DNA containing the coding region and the DNA
An expression vector comprising an expression control region for As the expression control region used in this expression vector, a conventional one can be used. For example, for expression in animal cells, a viral promoter,
For example, LTR promoter, CMV promoter, SR
α promoter and the like can be used, T7 promoter, LacZ promoter and the like can be used for expression in E. coli, and as a yeast promoter,
For example, the α-mating factor promoter is used.

The polypeptide or protein of the present invention can be obtained by culturing a host transformed with the above expression vector and collecting the desired polypeptide from the culture. Transformation of the host with the expression vector can be performed according to a conventional method depending on the type of the host. Cultivation of the transformed host can also be performed according to a conventional method. To recover and purify the desired polypeptide from the culture, affinity chromatography, gel filtration chromatography, concentration, freeze-drying and the like can be performed by a conventional method used for protein purification.

[0016]

EXAMPLES Next, the present invention will be described more specifically by way of examples. Example 1. Cloning and Characterization of SPA-1 cDNA (1) Cloning of SPA-1 cDNA In the present invention, first, in the stationary phase (G ₀ / G _{1) of} lymphocytes.
For cloning the expressed gene in most expressed without growth phase (phase S) in the period), a conventional method from lymphoid cell lines quiescent _{(G 0 / G 1) (} LFD-14) (Auffray, C. Et al., Eur. J. Bioc.
hem. , 107 , 303, 1980).
A was prepared, and then based on this mRNA, a conventional method (Aru
ffo, A. Proc. Natl. Acad. Sc
i. USA, 84 , 8573, 1987).
An NA library was prepared.

Next, using the same cell line (LFD-14) as IL
-2 (100 U / ml) stimulated proliferation, and about 20 hours later (S phase), mRNA was prepared according to a conventional method.
Based on A, a cDNA library was prepared by a conventional method. Next, differential hybridization was carried out between these cDNA libraries to obtain a cDNA clone expressing only in S phase cells, which was designated as SPA-1 cDNA. Also, this cd
The vector containing NA was named pcSPA-1. By cutting this vector with the restriction enzyme XhoI, SP
A-1 cDNA can be excised.

(2) Structure of SPA-1 cDNA The result of determining the base sequence of SPA-1 cDNA by a conventional method is shown in SEQ ID NO: 1. This cDNA consists of a total length of about 3.5 kb and has a long (about 1.2 kb) 5'-untranslated region containing many short open reading frames (ORFs) at its 5'-terminal side. This region is a strong translational repression region commonly found in certain oncogenes.
It is shown that the gene expression is also strongly regulated at the translation level.

This cDNA further contains one open reading frame (SEQ ID NO: 1) consisting of about 2.1 kb.
From the 1200th base A (adenosine) to the 327th
Up to the 8th base C (cytosine)). A sequence consisting of 190 amino acids at the N-terminal side of the amino acid sequence encoded by this open reading frame ("S
“Pan-N”) is human Rap1 GAP (GAP).
₃ ) showed high homology. Span-N and GAP
_The homology of the amino acid sequence of ₃ is shown in FIG.

(3) Preparation of monoclonal antibody against each domain on the SPA-1 N-terminal side SPA-1 cDNA was digested with restriction enzymes BglI and Pst.
D coding for Span-N by cutting with I
D encoding a NA fragment followed by a polypeptide of about 140 amino acids (designated "Span-C")
NA fragment was obtained, and the PstI end of pGEX-1 vector (Pharmacia) was flattened with T ₄ polymerase,
By adding an EcoRI linker and inserting it into the EcoRI site of the vector, Span-N or Span-
C and GST (Glutathione-S-transferase)
The expression plasmids pGEX-SpanN and pGEX-SpanC containing the sequence encoding the fusion protein with This expression plasmid was expressed in E. coli, and the expression product was recovered and purified to obtain Span-N / GS.
A T fusion protein and a Span-C / GST fusion protein were obtained.

200 μg of each of these fusion proteins was mixed with Freund's complete adjuvant, and an Armenian hamster (♂, 5w) was subcutaneously immunized. Then, every 2 weeks, 200 μg of the fusion protein was mixed with Freund's incomplete adjuvant 3 times and administered intraperitoneally to the hamster. Three days after the final immunization, the hamster spleen was excised and cut into small pieces to prepare splenocyte solution. This is the method of Leo, O et al. (PNAS)
84 : 1374, 1987) and cell fusion with mouse myeloma strain P3U1 was performed to obtain a hybridoma.

Among these hybridomas, clones producing the desired antibody were selected by the ELISA method using each fusion protein used for immunization of hamster as an antigen. That is, each fusion protein (GST-SpanN, G
A 96-well plate to which 1 μg / well of ST-SpanC) or 1 μg / well of GST protein alone was bound was prepared, and 100 μl of each hybridoma supernatant was reacted with this.

Next, this was reacted with anti-hamster IgG-peroxidase, and ABTS (2-2'-) was used as a substrate.
A color reaction was carried out using (azino-di-3-ethyl-benzothianozino-6-sulfate), and those which did not react with GST but reacted only with each fusion protein were regarded as positive. The cells in the wells showing positive were cloned by the limiting dilution method to obtain clones from single cells. The monoclonal antibody against Span-N is called F6, and the monoclonal antibody against Span-C is called H10. FIG. 7 shows the results of analysis of the reactivity between each monoclonal antibody and the fusion protein by Western blotting.

That is, FIG. 7 shows that each 10 μg of GST
-SpanN and GST-SpanC fusion protein or GST alone was developed by SDS-PAGE, blotted on a membrane, and reacted with F6 or H10 antibody solution (10 μg / ml), and ¹²⁵ I-Prot
The result detected by einA (Amersham) is shown.

The hybridoma producing the monoclonal antibody F6 is named F6 and is deposited under the deposit number FERM B.
On October 18, 1994 as P-4839, and the hybridoma producing the monoclonal antibody H10 was H
It was named 10 and was deposited with the deposit number FERM BP-4840 on October 18, 1994, at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology.

(4) SPA with monoclonal antibody
Detection of 1 protein Lymphocyte cell line LFD14 cells (Kubota, He. E.
t al. , J. et al. Immunol. 145 : 3924,
1990) from Harlow, E .; , Et
al. Mol. & Cellular Biology
6 : 1579, 1986, the protein was extracted, and the protein was identified by the immunoblotting method using the above-mentioned monoclonal antibody, the immunoprecipitation method, the immunostaining method and the like.

As a result, for example, the monoclonal antibody F6
In Western blotting using
The protein from FD14 was detected as a band with a molecular weight of about 68 kDa. From this, the SPA-1 gene has about 6
It is expected to encode an 8 kDa nuclear protein. That is, the SPA-1 protein of the present invention has SEQ ID NO:
It is presumed to have an amino acid sequence from the 1st amino acid methionine of 1 to the 693th amino acid alanine.

[0028]Example 2. Expression of SPA-1 cDNA (1) Expression of SPA-1 proteinExpression by in vitro transcription / translation method Fig. 8 shows various lengths of SPA-1 cDNA as templates
SPA-expressed by the in vitro transcription / translation method used
The analysis result of 1 protein is shown. As shown in Fig. 8 as a template
Contains pBluesc containing the full-length SPA-1 cDNA.
ript KS ⁺-Spa1 plasmid, this 5'side
Clones lacking various lengths (# 52, # 35, # 33,
# 92) Alternatively, the ORF is completely included and works negatively during translation.
The # 35 plasmid lacking the 5'untranslated region
Various positions (NcoI (1729), BalI (223
1), EcoRI (2881), DraI (303
The plasmid cleaved in 8)) was used.

Using 10 μg of these template DNAs, RN
Complementary mRN with A transcription kit (Stratagene)
A (cRNA) was synthesized. This cRNA is Tagaw
a et al. (J. Biol. Chem., 256 : 20.
021, 1990), an in vitro expression translation kit (Stratagene) was used to translate in vitro using a rabbit reticulocyte lysate (Promega) in the presence of ³⁵ S-methionine (Amersham). The above H10 antibody and protein A beads (Pharmacia)
Those immunoprecipitated by and were developed by SDS-PAGE.

As a result, full-length pBluescript-KS ⁺ -S containing the ORF and the 3'untranslated region completely.
A specific band of about 85 kDa was detected when the PA-1, # 52 and # 35 plasmids were used as templates.
In the plasmid lacking part of F (# 33), a translation product (about 50 kDa) was detected depending on the lacking length. Furthermore, a plasmid lacking the 3'untranslated region below the ORF (# 35
/ BalI, # 35 / EcoRI and # 35 / Dra
In I), a translation product smaller than 85 kDa was detected depending on its length. These results show that SPA-1 protein is 120
It shows that it is translated as a polypeptide consisting of 693 amino acids from methionine at position 0 to alanine at position 3278.

By stable animal cell transfectants
The expressed SPA-1 cDNA was excised with the restriction enzymes BglI-DraI, and the pSRα expression vector (Takebe,
Y. Et al., Mol. Cell Biol. , 8 , 466-
472, 1988) to create an expression vector SRα-SPA-1, which is combined with the plasmid pSV ₂ NeO containing the Neo gene into NIH3T3.
After introduction into cells (ATCC CRL-1658) G
Stable transfectants were obtained by selection with 418 (NIH / SPA-1 cells).

Under normal culture conditions (5% serum added), NIH / SPA-1 cells were transfected with NIH3T3 into which only the control SRα vector was introduced.
The cells show the same growth as the cells (NIH / SRα cells), but the cells are synchronized with G1 phase by serum removal (exten).
ted G1), it was found that, after adding a serum after a certain period of time and rotating the cell cycle, rapid cell death occurred in the middle to the late S phase (A in FIG. 10). Morphologically, cells became round and marked nuclear condensation was observed, which was presumed to be due to so-called mitotic catastrophes (FIG. 9B). In addition, SPA-1 shows a characteristic change in expression with such cell cycle synchronization,
It was also suggested that, like the cyclin group, its expression is regulated by the cell cycle (FIGS. 10B and 10C).

FIG. 9 shows the induction of cell death by proliferation stimulation after G ₁ phase cell cycle block in NIH3T3 cells (NIH / Spa-1) into which the SPA-1 cDNA had been gene-transfected. , NIH / SPA-1
After culturing cells (●) and NIH / SRα cells in which only pSRα vector was introduced into NIH3T3 cells (○) in the presence of 5% serum until almost saturated,
The medium was transferred to a medium containing 0.5% serum and cultured for 0, 24 or 48 hours. Then, the results are shown in which the cells were transferred to a medium containing 20% serum and the number of cells was measured over time.

FIG. 9B shows NIH / SRα cells and NIH / SPA-cultured in the presence of 0.5% serum for 48 hours.
20% serum is added to one cell and a microscopic image of the cell is obtained after 18 hours of culture. The right side shows the nuclear state of the cell at that time with Hoechst 33427 (Sigma). NIH / SP
Atrophy of the nucleus is recognized in A-1. In FIG. 10, A shows the analysis result of the cell cycle in NIH / SPA-1 cells, the upper row is NIH / SRα, and the lower row is NIH / SPA-.
1 cell. 16 hours after re-addition of serum, NIH
/ SPA-1 cells are dead (control cells are in S phase). B is a culture in the absence of serum (G ₁ a
rrest) shows the accumulation of SPA-1 protein, NIH
/ SPA-transfected SPA-1 cells
Despite detection of 1 mRNA, under normal culture (lane of oh), SPA-1 protein is hardly detected by Western blotting (probably always degraded). However, once the serum concentration was lowered to 0.5% and the cell cycle was stopped at G ₁ (G ₁ arrest)
When set to 1, accumulation of SPA-1 protein is observed.

C of FIG. 10 shows the kinetics of SPA-1 protein after the addition of serum. The cell cycle was restarted by the re-addition of serum after 48 hours of G ₁ arrest, and only the surviving cells were collected at each time. Then, the expression of SPA-1 protein was examined. Even in NIH / SPA-1 cells and some cells (about 10%) survived 24 hours after re-addition of serum, activation of cαc2 was observed in these cells. In contrast,
The SPA-1 protein is already reduced at this point.

Expression of recombinant SPA-1 in E. coli The SPA-1 cDNA was digested with the restriction enzyme BglI (base 117).
Cleavage at position 1) and DraI (at base 3038 position) to obtain BglI-DraI cleavage, which is T4.
The ends were made blunt by polymerase. This DNA fragment was used as a plasmid BS-SK digested with EcoRV.
(Stratagene) and the plasmid SK ⁺ -S
PA-1 was obtained. Next, HindIII of this plasmid
BamHI linker was added to the site, and this was added to BamHI
The resulting BamHI fragment was digested with BglII and the resulting expression plasmid pET-16b (Nova) was digested with BglII.
gen, USA) and used as an expression plasmid pET-S.
PA1 was obtained. E. coli was transformed with this expression plasmid.

This Escherichia coli was cultured, and the expression product from the culture was electrophoresed and detected by the above-mentioned monoclonal antibody F6. As a result, a band was detected at a site having a molecular weight of 85 kDa, and recombinant SPA-1 (rSPA-1) Was confirmed to have been expressed. Figure 3 shows the process of constructing the above expression plasmid.
Shown in

(2) Bioactivity of Span-N Since Span-N has homology with GAP3, GAP activity was examined using the GST-SpanN fusion protein described above. As a control, human GAP3 (75 to 663 amino acid residues) GST fusion protein was used, and yeast Rsr1 (1-
272 residues), human Rap1A (Glu ⁶³ ) (1-18
4 residues), human Ha-Ras (1-189 residues), and human RhoA (1-193 residues) GST fusion protein (reference: Nur-E-Kamal et al., Mol. Bio).
l. Cell 31 : 1437-1442, 199.
2. , Nur-E-Kamal et al., J. Am. Biol. Ch
em 267 : 1415-1418, 1992) GT
Regarding the effect on Pase activity, the method of Maruta et al. (J. Biol. Chem. 266 : 11661-
11668, 1991). As a result Sp
an-N is ineffective for Ha-Ras, Rac1, Rho1, etc. but selective GAP for Rap1 and Rsr1
It was shown to have activity. Table 1 shows the results.

[0039]

[Table 1]

FIG. 4 shows the Span-N concentration and Rsr.
The relationship of 1 GTPase activation is shown. This figure is Span
It is shown that the RsrGTPase activity of N is concentration-dependent. GAP activity was measured by the method of Maruta et al. (J. Biol. Chem. 266 : 11661).
-11668, 1991). SPA-1 is a nuclear protein, while there is no report that Rap1 exists in the nucleus. Therefore, when the activity against the small G protein Ran, which is known to exist only in the nucleus, was examined, Spa
It was found that n-N shows clear GAP activity against Ran. The results are shown in Table 2.

[0041]

[Table 2]

FIG. 5 shows the Span-N concentration and Ran.
The relationship with GTPase activation is shown.

Example 3. Cloning of genomic gene (1) Mouse genomic library (EMBL3-Adu
lt DBA / 2J liver DNA; CLONTECH, ML
1009d) 1.0 × 10 ⁶ was blotted on a Hybond-N ⁺ membrane (Amersham, RPN303B). Vector SPA-in which SPA-1 cDNA is inserted
1 cDNA / pBluescript to XhoI (T
The full-length SPA-1 cDNA was excised by cutting with oYoBo, XHO-101), and this DNA fragment was labeled with α ³² P-dCTP (Amersham PB (0205)) by Nick Translation Kit (Amersham, N5000).

This probe was used for Rapid Hybrid
ization Buffer (Amersham RPN1
636) and reacted with the genomic library. As a result of the primary screening, 15 signals were obtained, including positive and false positives. As a result of the secondary screening, 9 positive clones were obtained. By subjecting these to a third screening, it was confirmed that the obtained 9 strains were cloned. The genomic DNAs in these clones are referred to as GC1 to GC9, respectively.

(2) Preparation of total mouse DNA A 4 cm-old Balb / c mouse was cut into 2 cm tails.
Place in a 5 ml Eppendorf tube. Then this was chopped with scissors. To this, 500 μl of the mixed solution (439 μl of 1 × SSC, 5 μl of 1M Tris-
HCl (pH 7.5), 1 μl 0.5M EDTA (pH
8.0), 50 μl of 10% SDS, and 5 μl of 20
mg / ml proteinase K) was added and incubated at 37 ° C for 12 hours.

Next, 500 μl of buffered phenol was added to this and mixed gently for 5 minutes. Add this mixture to 10,
Centrifuge for 5 minutes at room temperature at 000 rpm. The liquid phase was transferred to a new tube, to which 700 μl of isopropanol was added, and the tube was inverted several times, causing a linear precipitation. The precipitate was transferred to a new tube containing 500 μl of 70% ethanol, then 70% ethanol was removed and the precipitate was washed with 100% ethanol. The precipitate was air dried and then 100 μl TE buffer was added to prepare total DNA.

(3) Genome DN encoding SPA-1
Screening of A The total DNA prepared in (2) above was treated with BamHI (T
oYoBo, BHA-102) or EcoRI (ToY
oBo, ECO-101) and then H
Screening was performed by blotting on a ybond-N + membrane and hybridization with the SPA-1 cDNA full-length probe prepared in (1) above.
Hybridization is performed in the same manner as in (1) above using Rapi.
dHybridization Buffer.

As a result, Bam of 5.7 kb and 6.6 kb
HI fragment and E of 9.2 kb, 5.2 kb and 1.4 kb
The coRI fragment was positive. The 5.7 kb and 6.6 kb BamHI fragments contained the full length SPA-1 cDNA. In addition, these fragments corresponded to the above-mentioned genomic fragments Spa-GC2 and Spa-GC9, respectively. The phage vectors into which these were inserted are each Spa-
GC2 / EMBL-3 and Spa-GC9 / EMBL-
It was named 3.

(4) Sequencing These phage vectors were prepared, cleaved with BamHI and 5.7 kb B from Spa-GC2 / EMBL-3 with the Gene Clean Kit (Funakoshi).
The amHI fragment and the 6.6 kb BamHI fragment from Spa-GC9 / EMBL-3 were prepared.

Next, these are added to pBluescript.
II SK (+) (ToYoBo, SC212205)
DNA Ligation Ki at the BamHI site of
T (Takara 6021) was inserted and subcloned. Next, Kilo-Sequence D
The deletion mutant was prepared by the deletion kit (Takara, 6030), and the 7-deaza Sequence was prepared.
Sequencing was performed by Nase (ToYoBo, US70777). As a result, Spa-GC2 has its 3 '
It was found that exons 1 to 4 were scattered in the side half and exons 5 to 16 were scattered in Spa-GC9.

The base sequence of Spa-GC2 is SEQ ID NO: 2.
And the base sequence of Spa-GC9 is shown in SEQ ID NO:
3 shows. In Spa-GC9, the second half of exon 5 to the first half of exon 16 contains the amino acid coding region in cDNA. In addition, Spa-GC2 and Spa-G
The relative positions of the genomic fragments, including C9, are shown in FIG.

[0052]

The SPA-1 protein is strongly expressed after the S phase of the normal lymphocyte cell cycle, suggesting that it may be involved in DNA replication and cell division. On the other hand, it was shown that the RanGAP activation domain exists at the N-terminus of the protein. Ran is a small molecule G that exists only in the nucleus
It is a protein and is associated with the RCC-1 gene. RCC-1
Is a nuclear protein that is well conserved in all cells from yeast to mammals, and is well known as a gene involved in the check mechanism of G ₂ / M transition (ie, prevention of cell division before completion of DNA replication). However, it has recently been known that it is involved in many other aspects of the function of the cell nucleus, such as initiation of DNA replication and RNA nuclear export.

In addition, RCC-1 is
Acts as P exchanger. This RCC-
The 1 / Ran system is, however, both constitutively expressed independent of the cell cycle, and therefore, cycle and RCC-1 / Ran
As a factor that connects the two, a cycle-dependent factor, especially GAP
It has long been presumed that the intervening molecules are present, but its main body was unknown. This series of results strongly suggests that SPA-1 is the correct intervening molecule. Furthermore, SPA-1 overexpression causes mitotic disruption (mit
This time, it was found that SPA-1 is cycle-dependent on this RCC.
Let us suggest that it is a central molecule that regulates the function of the −1 / Ran system.

For the cyclin / cdc2 system, which corresponds to the engine that promotes DNA synthesis and cell division, the details of how the RCC1 / Ran system, which is a brake, plays a role of fine adjustment via SPA-1 will be important in the future. It is a serious study subject. Above all, the fact that SPA-1 is highly expressed in lymphoid cells having unique cell growth characteristics suggests that it plays an important role in the growth regulation of the cell line and its check mechanism. is there.
Therefore, the protein of the present invention is expected to be useful as a regulator of lymphocyte differentiation. Furthermore, when the protein of the present invention is expressed in tumor cells, cell death can be induced in the S phase of the cell cycle, and therefore it is also useful as an antitumor agent.

[0055]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3519 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA Origin: Sequence characteristics: DNA encoding SPA-1 Sequence: GTCCTGCATG CAGCTGCCCC AGGAGCTCCT GTGTCCTTGA GGCCCATCTG 50 AACAGCCCCC TCCTCTGCAG TGCAGAAACC ACTGAAGCCT CAGCCTTCTG 100 GGTGGGCACC AAGGACCCGT GCCCACCAAT GCGGCCCGGC CCCCAGAGAG 150 TCAGGCCCAC AGGAGCACGC CCATGTGGGC CGGAGGTGTG GGGAGCCCTC 200 GGCGGGCATG GCCCCTGCAC CTACCGATGA CCTCTTTGCC CGTAAGCTTC 250 GCCAACCTGC CCGGCCCCCA CTGACACCAC AATACCTTTG AGCCGAGGCC 300 AGCTCGGGGC CCACTCTTGC GCAGTGGCAG TGATGCTGGT GAAGTCCGGC 350 CCCCTACACC AGCCAGCCCC CGTGCCCGTG CCCACAGCCA CGAGGATGCC 400 AGCCGCCCTG CTGCAACCCC TACTCGGCTC TTCACTGACC CACTGGCACT 450 GCTAGGGTTG CCAGCAGAAG AGCCAGAGCC CACCTTCCCG CCAGTGCTGG 500 AACCCCGGTG GTTTGCTCAC TATGATGTGC AGAGCTTGCT CTTTGACTGG 550 GCTCCACGAC CTCGGGGGAC AGGCAGCCAT ACAGAGGCAA ACTCTGGGAC 600 CTTAGCTGAG GGTCAGGA TGG GCTTTGTGAG CGAGCTTGGT GGTGAGGGTG AGCTAGGGCT GGGTGGGCCA 700 ATATCCCCAC CTGTGCCCCC TGCACTGCCT AATGCGGCTG TGTCTGTCCGTC CCGACGCGCCACACTTACAGTGCCGATACGACTGACCGATACTACCTGCTGACCTACTACAGTCAGCAG

[0056] GAGGAGAAAG AGGGCAGCGG AGGGGGCACC TACACAGCTA CCGGGTCATC 950 GTGCGGACCA CGCAGCTCCG GACCCTCCGT GGCACCATCT CGGAGGACGC 1000 ACTGCCTCCC GGCCCCCCGA GCGTATCTCC GAGGAAGCTT CTGGAACATG 1050 TGCTCCACGG CTGAGCCCAC CTGCCTGCGC CTGGGTTCAG CCTCTCCCAA 1100 GGTGCCCCGC AGCTGCTTAC TCTGGATGAG CAAGTGCTGA GCTTCCAACG 1150 CAAGGTGGGC ATCCTGTACT GCCGCGCAGG CCAGGGCTCT GAGGAAGAG ATG 1202 Met TAC AAC AAC CAG GAG GCC GGA GCA GCC TTT ATG CAG TTC CTT ACT 1247 Tyr Asn Asn Gln Glu Ala Gly Ala Ala Phe Met Gln Phe Leu Thr 5 10 15 TTG CTG GGT GAT GTG GTG CGA CTC AAA GGC TTT GAA AGT TAC CGG 1292 Leu Leu Gly Asp Val Val Arg Leu Lys Gly Phe Glu Ser Tyr Arg 20 25 30 GCC CAG CTG GAT ACC AAA ACG GAT TCC ACG GGC ACA CAC TCA CTC 1337 Ala Gln Leu Asp Thr Lys Thr Asp Ser Thr Gly Thr His Ser Leu 35 40 45 TAC ACC ACC TAC CAA GAC CAT GAG ATC ATG TTT CAC GTG TCC ACG 1382 Tyr Thr Thr Tyr Gln Asp His Glu Ile Met Phe His Val Ser Thr 50 55 60 ATG CTG CCT TAC ACG CCT AAT AAC CAG CAA CAG CTC CTG AGG AAG 1427 Met Leu Pro Tyr Thr Pro Asn Asn Gln Gln Gln Leu Leu Arg Lys 65 70 75 CGT CAT ATC GGC AAC GAT ATT GTG ACC ATC GTG TTC CAG GAG CCC 1472 Arg His Ile Gly Asn Asp Ile Val Thr Ile Val Phe Gln Glu Pro 80 85 90 GGT AGC AAG CCC TTC TGC CCT ACA ACA ATC CGC TCT CAC TTC CAG 1517 Gly Ser Lys Pro Phe Cys Pro Thr Thr Ile Arg Ser His Phe Gln 95 100 105

CAC GTT TTC TTG GTG GTG CGT GCG CAT GCT CCC TGC ACC CCA CAC 1562 His Val Phe Leu Val Val Arg Ala His Ala Pro Cys Thr Pro His 110 115 120 ACC TCA TAC AGG GTG GCA GTG AGC CGC ACC CAG GAC ACT CCT GCC 1607 Thr Ser Tyr Arg Val Ala Val Ser Arg Thr Gln Asp Thr Pro Ala 125 130 135 TTC GGG CCT GCG CTG CCA GAA GGC GGA GGC CCC TTT GCA GCC AAT 1652 Phe Gly Pro Ala Leu Pro Glu Gly Gly Gly Pro Phe Ala Ala Asn 140 145 150 GCC GAT TTC CGG GCC TTT CTG TTG GCT AAG GCA CTC AAT GGT GAG 1697 Ala Asp Phe Arg Ala Phe Leu Leu Ala Lys Ala Leu Asn Gly Glu 155 160 165 CAA GCG GCT GGT CAT GCA CGC CAG TTC CAC GCC ATG GCT ACA CGC 1742 Gln Ala Ala Gly His Ala Arg Gln Phe His Ala Met Ala Thr Arg 170 175 180 ACA CGC CAA CAG TAC CTG CAG GAC CTG GCT ACT AAT GAA GTG ACC 1787 Thr Arg Gln Gln Tyr Leu Gln Asp Leu Ala Thr Asn Glu Val Thr 185 190 195 ACT ACT TCG CTG GAC TCG GCT TCG CGG TTT GGC CTG CCA TCT CTG 1832 Thr Thr Ser Leu Asp Ser Ala Ser Arg Phe Gly Leu Pro Ser Leu 200 205 210 GGG GGT AGG CGC CGG GC A ACC CCT CGG AGC CCA GGC GCG GAC GTA 1877 Gly Gly Arg Arg Arg Ala Thr Pro Arg Ser Pro Gly Ala Asp Val 215 220 225 CAG GCG GCG GGT GCG CTG ATG TGG GGC GTA CGC GCG GCT CCA GGG 1922 Gln Ala Ala Gly Ala Leu Met Trp Gly Val Arg Ala Ala Pro Gly 230 235 240

GCG CGG GTC GCA GCG GGA GCT GAA ACG AGC GGT CCG GAC GAC GCC 1967 Ala Arg Val Ala Ala Gly Ala Glu Thr Ser Gly Pro Asp Asp Ala 245 250 255 GAG GTG CCC TGC TTG TTG GGC ATC TCA GCA GAG ACA CTG GTG CTG 2012 Glu Val Pro Cys Leu Leu Gly Ile Ser Ala Glu Thr Leu Val Leu 260 265 270 GTG GCA CCT CGC GAC GGC CGC GTG GTC TTC AAT TGT GCC TGT CGC 2057 Val Ala Pro Arg Asp Gly Arg Val Val Phe Asn Cys Ala Cys Arg 275 280 285 GAC GTA TTG GCC TGG ACC TTC TCA GAG CAC CAA CTC GAT CTG TAC 2102 Asp Val Leu Ala Trp Thr Phe Ser Glu His Gln Leu Asp Leu Tyr 290 295 300 CAC GGG CGC GGG GAG GCG ATC ACG CTG CGG CTC GAC GGG GCC CCA 2147 His Gly Arg Gly Glu Ala Ile Thr Leu Arg Leu Asp Gly Ala Pro 305 310 315 GGG CAA GCC GTG GGC GAA GTC GTG GCA CGT CTG CAG CTG GTG AGC 2192 Gly Gln Ala Val Gly Glu Val Val Ala Arg Leu Gln Leu Val Ser 320 325 330 CGC GGG TGT GAG ACC AGA GAA CTA GCG CTG CCC AGA GAT GGC CAA 2237 Arg Gly Cys Glu Thr Arg Glu Leu Ala Leu Pro Arg Asp Gly Gln 335 340 345 GGT CGC CTG GGC TTC GA G GTG GAT GCA GAA GGC TTC ATC ACG CAC 2282 Gly Arg Leu Gly Phe Glu Val Asp Ala Glu Gly Phe Ile Thr His 350 355 360 GTG GAG CGC TTC ACG TTT GCG GAG ACC ACG GGG CTT CGG CCT GGA 2327 Val Glu Arg Phe Thr Phe Ala Glu Thr Thr Gly Leu Arg Pro Gly 365 370 375

GCT CGT TTG CTG CGA GTC TGC GGC CAG ACG CTG CCC AAG CTG GGT 2372 Ala Arg Leu Leu Arg Val Cys Gly Gln Thr Leu Pro Lys Leu Gly 380 385 390 CCC GAA GCT GCT GCC CAG ATG CTG CGC TCT GCG CCG AAG GTC TGC 2417 Pro Glu Ala Ala Ala Gln Met Leu Arg Ser Ala Pro Lys Val Cys 395 400 405 GTC ACG GTC CTA CCC CCA GAC GAG AGC GGC CGG CCG CAG AGG AGC 2462 Val Thr Val Leu Pro Pro Asp Glu Ser Gly Arg Pro Gln Arg Ser 410 415 420 TTT TCG GAG CTC TAT ATG CTC TCT CTG AAG GAA CCC AGC CGG CGG 2507 Phe Ser Glu Leu Tyr Met Leu Ser Leu Lys Glu Pro Ser Arg Arg 425 430 435 GGG GGC CCA GAG CCA GTA CAG GAT GAA ACT GGG AAG TTG GTC ATA 2552 Gly Gly Pro Glu Pro Val Gln Asp Glu Thr Gly Lys Leu Val Ile 440 445 450 TTG CCT CCC ACC AAG CAG CTG CTA CAT TTT TGC CTG AAA GAC AGC 2597 Leu Pro Pro Thr Lys Gln Leu Leu His Phe Cys Leu Lys Asp Ser 455 460 465 AGC AGT CCT CCG GGG CCT GGG GAT CTG ACT GAG GAG AGG ACA GAG 2642 Ser Ser Pro Pro Gly Pro Gly Asp Leu Thr Glu Glu Arg Thr Glu 470 475 480 TTC CTG CGC AGC CAC A AC TCC CTG TCA TCT GGA AGC TCC CTG TCC 2687 Phe Leu Arg Ser His Asn Ser Leu Ser Ser Gly Ser Ser Leu Ser 485 490 495 GAT GAG GCT CCA GTC CTG CCC AAC ACC ACT CCA GAC CTC CTC CTT 2732 Asp Glu Ala Pro Val Leu Pro Asn Thr Thr Pro Asp Leu Leu Leu 500 505 510

GTC ACC ACT GCC AAC CCA TCT GCA CCT GGT ACT GAC AGA GAA ACA 2777 Val Thr Thr Ala Asn Pro Ser Ala Pro Gly Thr Asp Arg Glu Thr 515 520 525 CCC CCT TCC CAG GAC CAG TCA GGA AGC CCC AGT AGC CAT GAA GAC 2822 Pro Pro Ser Gln Asp Gln Ser Gly Ser Pro Ser Ser His Glu Asp 530 535 540 ACC AGT GAC TCA GGC CCA GAA CTG AGG GCC TCC ATC CTG CCC AGA 2867 Thr Ser Asp Ser Gly Pro Glu Leu Arg Ala Ser Ile Leu Pro Arg 545 550 555 ACC TTG TCT CTG CGG AAT TCC ATC AGT AAG ATT ATG TCG GAA GCT 2912 Thr Leu Ser Leu Arg Asn Ser Ile Ser Lys Ile Met Ser Glu Ala 560 565 570 GGC AGT GAG ACC CTG GAG GAT GAG TGG CAG TCC ATC TCA GAG ATC 2957 Gly Ser Glu Thr Leu Glu Asp Glu Trp Gln Ser Ile Ser Glu Ile 575 580 585 GCC TCC ACT TGC AAC ACA ATT CTG GAG TCA CTG TCC CGG GAG GGA 3002 Ala Ser Thr Cys Asn Thr Ile Leu Glu Ser Leu Ser Arg Glu Gly 590 595 600 CAA CCC ATC TCA GAG AGC GGA GAC CCC AAG GAA GCT TTA AAG TGT 3047 Gln Pro Ile Ser Glu Ser Gly Asp Pro Lys Glu Ala Leu Lys Cys 605 610 615 GAT TCT GAG CCA GAA CC C GGG AGC CTG TCA GAA AAG GTC TCT CAC 3092 Asp Ser Glu Pro Glu Pro Gly Ser Leu Ser Glu Lys Val Ser His 620 625 630 CTA GAG TCC ATG CTC TGG AAG CTC CAG GAG GAC CTG CAG AGG GAG 3137 Leu Glu Ser Met Leu Trp Lys Leu Gln Glu Asp Leu Gln Arg Glu 635 640 645

AAG GCG GAC AGG GCA GCC TTG GAG GAG GAG GTT CGG AGC CTC AGA 3182 Lys Ala Asp Arg Ala Ala Leu Glu Glu Glu Val Arg Ser Leu Arg 650 655 660 CAC AAC AAC CAG AGG CTG CTG GCA GAG TCC GAG AGT GCC GCC ACC 3227 His Asn Asn Gln Arg Leu Leu Ala Glu Ser Glu Ser Ala Ala Thr 665 670 675 CGC CTG CTC CTG GCC TCT AAG CAT CTG GGT GCA CCC ACT ACT GAC 3272 Arg Leu Leu Leu Ala Ser Lys His Leu Gly Ala Pro Thr Thr Asp 680 685 690 CTG GCC TGAGTTCCAA TCTGAATCTG GACCTGCTTG GAACTGCCTG 3318 Leu Ala GCCCCTCAGA GCAACTGGGT CATACTAGTG CCCTTCCTCA GGACTTCTTC 3368 CCTGCGCTGA GGCGCGTCTT AGCACTGCCC CCTCTTCCCA GCCCATTTGG 3418 TGGCTAATGC CTGTCCCTGT TTGTAAATAT CCTGTAAAGA AAAGGAGACA 3468 TCAGAGTTTA AAAAAAAGAA ACAACAAGAA GAAGCAAAAA AAAAAAAAAA 3518 A 3519

SEQ ID NO: 2 Sequence length: 5687 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Mouse strain: Spa-GC2 Sequence characteristics Exon 1: 3109-3284 Exon 2: 3764-4555 Exon 3: 5147-5273 Exon 4: 5383-5524 Sequence:

[0063] GGATCCCAAA CTGAGGCAGC AGCCTCCTAG CCAGGCCCTA AGAGCCAAAC 50 CCATGGGCTG GTCCCTCATT GGAGCCCATG GCCAGGACTG ACTTTGCCTC 100 TGGGGCCTGC ACTGCCCCCA AGGCTGGCCT CCTTAGCCTG GACCTGGGGC 150 CCGATATGTG GCAAGGGTGG GTTCATTCGT TCTTTTGTCA TTTTTCTTTC 200 TTTTTTTTTC TGTGCTTCAG AGACACCAAA TTAATAACAC TATTTTTGAT 250 TTTGGTTGGC AGTTTTATTT TCTGTGGGAC GAGGTGAGGT TGGTAGAGGT 300 GCCGGAGGGA GGCTGAAGTC AGAAGAGTGT GAGGGATAAG GGGTCAGACT 350 GCTGGGCTCC AGGCAGACAT GAGGTGGGAT GGGCTGCCTT CCTCACCTGC 400 CTCTGCCTTT CTTTTTTTTT TTTTTTTTTT AATGGTTTAA GAGCTTATTA 450 TAGAAATGCC AGTCGAGGGA AGAGAAAAGG TAGAGAGAGA GAGTGTGTGG 500 GACGGGGAAG GCTAGAGAAG AAGAAAGAGA AAGGAGAGAA AAGACAAAGG 550 GAAGAGGAGA AAGAAGTGAG AGGATAAAGG AGAGAGCTGA GCCTCTGCTT 600 TTCAAGCAGT CCTCTATCCC CAGGTGGCTC ACACATCCAT CAGCCTTGAT 650 CTTATCAAAG ACTGCTCAAC CTCATTTGTC CAAGCTTAAG AAAATAACAG 700 GTGAATAGAA AGGATTCTAT TCGTTTTTGA GACAGGGGCT TACCATGTAC 750 TCCCTATGTA CATCAGGCTG GCCTTGACCT CAGATCCACC TGCCTCTGCC 800 TTCAGAGTGC TGGATTTAAT GGTGTTCGCC ATTGC ATAAA TGGTAGCCTG 850 TGTAGATCAG TTATCTAGCT TGGCCCAGCC CTTAAAGAGT GAAATAGTTT 900 CTGGCCCTAA TACCTGCTGT CTGCTGAGCC ACAGCAGGAC ACTAAGTGGC 950 CTCTAGCGCT CCAATTGGTC TGGAAGGCAG GTACATTTGT TCCCATTTCT 1000 CGGTGAAGTC ACTGACTGGG CTAGGACCGG GAGTTAAAAG AGCAGCTGAA 1050 GGCTGGGACA GAGAACTTCA GGCTGTCCGG GGCTGCCTAG GTTCCTGTCG 1100 GAGGTCCCCA CCCACTGTGC TTCCGCCTTA GACAGCTCCC GGGTAGTCCC 1150 GCCCCTCCAC TACGTACCGC CTCCATCCTG GCCCCGCCCC CAGGGAGGGA 1200

[0064] GGCGCCGGGA GCGGTGTGAG CAGGCAGCGG GACCTTGGTG CGGAAGGCAG 1250 CGGTGGCCAG CTTGAGCCCG AGAGGTACTG GCGGGATCAG GGATCGGGAG 1300 GCACCAGGTT CGGGCTGGAT ACCCAACAAA GTAGCCTGGA CGTGAACCCT 1350 GTAGTGTGGG GAGGAACGGG ACTATTGGCT GCTTTCGCTA CACGCACCCC 1400 ACCCAACCTC CTGCCCCAGT CCAGCCCCGA GTCAGCACGT CCAGTGTTCT 1450 GCTCCTGCTG GCAGCTCCCA CTCCCTCCTC TGCATGAGCA GATTCAGAGC 1500 TCACTGAGTG GATTCATTGG TTCTGGACTT TTCTCAGCAA TGCTGGCGCA 1550 GCTGCTCCTG CTGCTGTTGT TGTTGTGGGG CTCCCCCTAT TCTGGGGCTC 1600 CCCTGTTCCC AGTGTGACCT CTTTCCCAGC CTTTGCAATC CTGAGTCTGG 1650 CCTGGGAGGA AACATCTGCA GCACTCCCTG GCAACAGAAA TAGGGTCACG 1700 ACCTCCAGAT GTGCTGGGAA GCATCCAGCG CCTCCTCCTG GGGCAGCCAG 1750 GCCTTCCGGA TCTGTGGGGG CGGGCCCCCC CTTTCCCCCC CTCAGTGACA 1800 CAGGCTGCAA GGAATGTCTG GGCCTCAATG GACCTTGTGT AAGATGAGGG 1850 GTGGGGGGCA GAGCAAGTAC ACACCTTAAG GCAGGGCCAG AACAAGAGGG 1900 AGCTCCTGGA CTGGGCTGCA CACATTCCCA GGGCTCCTCC CGGCACTGCG 1950 GCCTCAGTCT GTGCCCACGC TTGGTCTATG GACCTGGGCG CCTGCACAGT 2000 TCACACACGG ACATAGTTGG CCTTCACCTT TCAGTTTCCA AGGAGTCTTC 2050 AAAGAACTCA TGAAGAGTTC CAGACTCAGA GAGCTTATCC TAGAAGACAG 2100 ACAGACAGAC AGGAAGACCC TGAGGAGGTC TGCTCTTATT TAATTCTGGA 2150 GACCCAGCTG AGGGGCACCG TGGAGCTGCT CCCTGTCCCC TCCCAGCCTG 2200 GCCCCCTTGA TGCCACTGGA TGATGCAAAA AAAAGTACTA ATGGAGGCCT 2250 GCCCCTGCCC CAGCTGTTGG CTCCATTCCT ACGTCACGCC GAGGTAGGCT 2300 CGGCCTTCTC ACACCTTTTG CACCTGCCTA GTGTAGCTTC ACCACATTTC 2350 CGCACTTAGT AGGTCCCTGG GGCCTTGGGT GTTTCAGCCT TACATCCTGT 2400 GAGACCTTGA GCCTCTCCCA TCTCCCCTCA CAAGGCTGCC TTACTCCTAC 2450 GCACACGGGC AGAGTAGGCA GGTGCAGCTC TGACAAGTCC AGAAGCAGCA 2500 GTCTCAACCT GTGTGCTGGG ACCCCTTTGA GGGTCGAGCA GTCCTTCACA 2550 GGGGTCACAC TTGAGATATT TATCTTCTTC TTCTTCTTCT TTTTGTTTTT 2600

[0065] CAAGACAGGG TTTCTCTGTG TAGCCCTGGC TATCCTGGAA CTCACTCTGT 2650 AGACCAGGCT GGCCTTGAAC TCGAAATCTG CCTGCCTCTG ATTCCCCAGT 2700 GCTAGGATTA AAGGAGTGTG CCAACACTGC CCGGCTCATG TTATGATCTT 2750 AAGGGCAGCA AAATTACAGT GGTGAAGTAG CAATGAAAAT AATTTTCTGG 2800 CTGTGAGGTC ACCACCGCAT TAGGGAAACT GTATTAAAGC GTCACAGAGT 2850 TAAGAAGGTT GAGAACTACT GCCTTCGAGA TTCAGAGACA AGGTTCAAAT 2900 TCTAGTTTGA ACATGGAACT AATTCAGGCA AGCTCATCTT CTTAACTGGG 2950 CCTCACTGTG ACCTGTCTCA CTGGGTTCAG ACCTCCCTGT CCATGCATGT 3000 GAGGCCAGGT AAACAGACAT CCACAGGGTC CTGATTGGGA TTAGCCTCTC 3050 TCACCCCTGG GAGTGGGCAT CGTGACCTGC AAGAGATTAG TATTAGTCTT 3100 GTCCTTTAGA CTTAGGTGTC TTGGGTCCCA TGACTGAGCT GTTGTGACCC 3150 TAGCACCTTC CTCAGGATAT AGGAGCCAAG CAGGGGGCTG GGCTGAGTTG 3200 GGGCCACTTC CTGTGTTATA GGAAGTCCTC TCACCACTGC TTCTGTCCTG 3250 CATGCAGCTG CCCCAGGAGC TCCTGTGTCC TTGAGGTATT GAGACTGCGG 3300 GAATTGAGGG CACTGAGTCT AGGCCTTGGG TGCTCAGTCT CTTTGGGGAC 3350 TCTGGAGGAA GTGGGAGGTA CCAGGGAGGA AGGTCTCTGG GGACGGACGT 3400 CTCCCTTTGT ACAAGTGGGC AAGACTCAGA CACCAGTGAC TGCTTTGATT 3450 TCCGTTCTGG TGAAAACTGT TCAGAATTTG GTGGCAACCC TCACTTTGAG 3500 CCTAGTTCCA CAGCCAAGGT GTACAGGGGA GAACTGGGAG GGGCCGGTGC 3550 CACTAGACCC AGTCACTAGC ACCCCGAGAG CAAAGCATCC CAGTTCAGCT 3600 CCCAGCCTTG ACCTAAGCCT GGGATGGGGC TGGAAACTTC AGCCCAGGCA 3650 GACAAGGAAG TGGCCAGGAA AGCGGAAGCA GCTTTGATGG TCCGGAGGGG 3700 GCCGGAAGCT AAATGGGGTG GTGGAAGACT GGGCTGGGGG CCTGAGTTCC 3750 TGTTTTCTCC CCAGGCCCAT CTGAACAGCC CCCTCCTCTG CAGTGCAGGA 3800 ACCTCTGAAC GCTCAGCCTT CTGGCTGGGC ACCAAGGACC CGTGCCCACC 3850 AATGCGGCCC GGCCCCCAGA GAGTCAGGCC CACAGGAGCA CGCCCATGTG 3900 GGCCGGCGGT GTGGGGAGCC CTCGGCGGGT GCATGGCCTG CACCTACCGA 3950 TGACCTCTTT GCCCGTAGCT TCGCCAACCT GCCCGGCCCC CACTGACACC 4000

[0066] ACATACCTTT GAGCCGAGGC CAGCTCGGGC CACTCTTGCG CAGTGGCAGT 4050 GATGCTGGTG AAGTCGGCCC CCTACACCAG CCAGCCCCCG TGCCCGTGCC 4100 CACAGCCACG AGGATGCCAG CCGCCCTGCT GCAACCCCTA CTCGGCTCTT 4150 CACTGACCCA CTGGCACTGC TAGGGTTGCC AGCAGAAGAG CCAGAGCCCA 4200 GGTTCCCGCC AGTGCTGGAA CCCCGGTGGT TTGCTCACTA TGATGTGCAG 4250 AGCTTGCTCT TTGACTGGGC TCCACGACCT CGGGGGACAG GCAGCCATAC 4300 AGAGGCAAAC TCTGGGACCT TAGCTGAGGG CCAGACTACC ACCTCAGATC 4350 TACTGCTCGG GGCACCTGGC TTTGTGAGCG AGCTTGGTGG TGAGGGTGAG 4400 CTAGGGCTGG GTGGGCCAAT ATCCCCACCT GTGCCCCCTG CACTGCCTAA 4450 TGCGGCTGTG TCCGTCCTGG AGGAGCCACA GACCCGGACC ACACTTACAG 4500 CCTGGAGCAC GCAGATCTGG GTGCAGGCTA CTACCGCAAG TACTTCTATG 4550 GCAAAGGTAA GGGGCAGGCG AGCCTGGGAG AGGCAGGAGA GGATCTGGGT 4600 CGGAGGTCCC TGTGGTCTTC TACATTCTAT CAGTGGGAGG CTCATGGGCT 4650 GGCCTTCCCT GTAAAAAAGG GGCAGGAGCT GAATTGGGCT CTGTTGGCTC 4700 AACTCTGACC ACCTCTTTAA GGCCAAGAAT GGTGTCACAC CTGAAGTCAG 4750 GGAGTGCACT TACCTCTGAG GCTCATCTTC ATAACCTCCA GGAGGCCAGT 4800 GAGCGATTTC CTATTTCCAT ATCTGTGTGA TGAAACCCTG TTCTCATCAT 4850 TAGCAGGAAA AGCAGCTTCC GTGTCTTGAA TGGGAGAACC TAAGCTTTGG 4900 TGGAGCCAGG GCAGCATTTA ACTAGGAGGA CTTAGGCATT TGTTCCCCGG 4950 TCCTGGGAAC AAGGTGTAAC CGTGGGTGGG ACTGCAAACT GGGGTGGAGT 5000 GAACTCCCAG GTTCAGCGCT TGGTGAGAGA ATACCTAGGG TGGTACTTCT 5050 GTGGTGGGAG TAGTCAAGAA GGGATAGGGT GGTCTGTGGG TTTGACTGAA 5100 AGGCCACCGA CCGACCAACC AACGACCCTC CACCCCCACC CCCACAGAAC 5150 ACCAGAACTT CTTTGGGTTG GATGAGGCGC TGGGTCCGGT GGCCGTGAGC 5200 CTGCGACGGG AGGAGAAAGA GGGCAGCGGA GGGGGCACCT ACACAGCTAC 5250 CGGGTCATCG TGCGGACCAC GCAGGTGGGC TGGGATTACA GGCTCAGGAG 5300 GCAGGTTTCC TCCACCACAG CCTATACAAA AACTGAATGT CTCTACATCC 5350 TTAGCTCCGG ACCCTCCGTG GCACCATCTC GGAGGACGCA CTGCCTCCCG 5400 GCCCCCCGAG CGTATCTCCG AGGAAGCTTC TGGAACATGT GCTCCACGGC 5450 TGAGCCCACC TGCCTGCGCC TGGGTTCAGC CTCTCCCAAG GTGCCCCGCA 5500 GCTGCTTACT CTGGATGAGC AAGTGGTGAG TGGCTGGGAG GTAAGGAGGG 5550 AGTGCAGCAT CCCGGGGAAG ATGGGGCTGA CCTTCATCTC CCTAACTAGC 5600 TAGCTTCCCG CTCCCTAACC CTGACCTGAT CTGACGGACC TCAAGGTACA 5650 G CTGATCCAC CTCCAAGCCT TTCCGAGAGA AGGATCC 5687

SEQ ID NO: 3 Sequence length: 6645 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin: Mouse strain: Spa-GC9 Sequence characteristics Exon 5: 891-1015 Exon 6: 1356-1459 Exon 7: 1726-1933 Exon 8: 2009-2618 Exon 9: 2890-3164 Exon10: 4291-4509 Exon11: 4598-4709 Exon12: 17x473-174714-174714-4795-4790. : 5200-5305 Exon15: 5497-5525 Exon16: 5598-5969 Open Reading Frame: 904-57.
41 Sequence:

[0068] GGATCCCCAT TACAGATGGA TGTGAGCCAC CATGTGTTGC TGGGAATTGA 50 ACTCAGGAAC CTCTGGAAGA GCAGTCAATG CTCTTAACCA CTGAGCCATC 100 TCTCCAGCTA ACCTTGTTTC AAACAAACAA AAAATTGCAG GTACGTGTCT 150 AGATTCCAAT ATTTGGGAGA TACAGGCAAA TGATCAGGAT CAGGCAGTCT 200 TAGCTATATA TGAGTTTAAG TACAGCCTGG CCTATGTACT ATAGCCTATC 250 AAAAAGACAA ACAGGAAGGG GACAGAAATG ACTCCAAACC TCAGAGGGCT 300 GGGGTGCCAG CACACTGGAG CCTTGAGCTG AGGGGGACGG GAACATGGGC 350 ACCAGTCTTG GCAGGGGATC TCAGCCTTCC TAGTGCCCTT TCCCACAGCT 400 CCACATGGTG TGGTGACCCT GATCAAAAAT TATTTTCATT GCCACTTCAT 450 AACTGTAATT CTGCTACCCT TAGGAATTGT ACCATAAACA CCTGACACAC 500 AGGGTCTCTG CTGCATGCGA CCCCTGTGAA TGGGCCGTTA GACCCTCAGA 550 AGGGTCACAA CTATAGGCTG AGAACCCACT GGTGTATAGG GTCCTTTCTG 600 GGAGTTATCT CTTTTGTTGC TGGAGAAGTC ATTAAATCCT CTGCCTCTTC 650 CCTGTGACCT CCCTGCTCTC ACGAGCACAG GAGAGGGCAG GTAGAACGCA 700 CTTGATGGGC AAAGATGCCC AAATGGCTCA GAGTTCCTAC CAGGGCAGCC 750 CAGCCCCAAA GGCCAGCTCT TCCCATTCTC TACAGGGTGG GCTGCCAGGG 800 TACTGAAGCC TTTGTCTTCT GTTGTCCATG ACCCC CTCAG CTGAGCTTCC 850 AACGCAAGGT GGGCATCCTG TACTGCCGCG CAGGCCAGGG CTCTGAGGAA 900 GAG ATG TAC AAC AAC CAG GAG GCC GGA GCA GCC TTT ATG CAG TTC 945 Met Tyr Asn Asn Gln Glu Ala Gly Ala Ala Phe Met GTT Gln Phe 10 GTT GTT CTC AAA GGC TTT GAA AGT 990 Leu Thr Leu Leu Gly Asp Val Val Arg Leu Lys Gly Phe Glu Ser 15 20 25

[0069] TAC CGG GCC CAG CTG GAT ACC AAA A GTGAGCGTCC CCCGCCCCTA 1035 Tyr Arg Ala Gln Leu Asp Thr Lys 30 35 AGGGACTGGA GATGCAGGGC AGAACTTTAT CAGTGTTCCT TAGTCTGTGG 1085 TGGCTGGGGC TGAGAATGGG GGAGTGCCCT GCTCCCTCTA AGTCTTATTT 1135 CTGGATTCGT TCTATCTCAG CACCCCTATA CTGATTCCCC TTCACCCTGG 1185 TGTGGGGCCG TAGTCTATAG GAGAGGGGAG GGAATTTACC AAGGATGGGG 1235 CTCTTGGTCT TCGTGGCCTA AGCAATAGCT GGTGGCTGGG ACATAGAAGT 1285 AAATTTAAGA CTCATTGAAG TCACCCACAC CCCCCATGTT CTCTTTGTGT 1335 CCCCAATTGT CTGGCTACAG CG GAT TCC ACG GGC ACA CAC TCA CTC TAC ATC GTC CATC ATC TCA CTC TAC 1384 Thr Asp Ser Thr Gly Thr His Ser Leu Tyr ATG 1429 Thr Thr Tyr Gln Asp His Glu Ile Met Phe His Val Ser Thr Met 50 55 60 CTG CCT TAC ACG CCT AAT AAC CAG CAA CAG GTGTGTGAGG AGCTGGGCCA 1479 Leu Pro Tyr Thr Pro Asn Asn Gln Gln Gln 65 70 GGCCAAAGAC TTTCGG GGT AGT CATTGGCG 1529 TCAGAACGGT GGTCTGAGCT CTGCTGAACC TAACAACACC CACCCCCCCA 1579 CCCCTTGGCT GTACCACCTT CGCAAATACC CTCCTCGGGC CTTTATAAGG 1629 TGCAGGTGGG GAACCACTGA CACCTTTGCC ATGCCTAAAT GAGGGACTGG 1679 GGGGGGCACA AAGCTCACCT CTCATTTGCC TACCTTTAAC CCCCAG CTC CTG 1731 Leu Leu AGG AAG CGT CAT ATC GTC CAT ATC GTC ATC GAT ATT GTG ACC GTC ATC GTC ATC GTC ATT GTG ACC 80 85

GAG CCC GGT AGC AAG CCC TTC TGC CCT ACA ACA ATC CGC TCT CAC 1821 Glu Pro Gly Ser Lys Pro Phe Cys Pro Thr Thr Ile Arg Ser His 90 95 100 TTC CAG CAC GTT TTC TTG GTG GTG CGT GCG CAT GCT CCC TGC ACC 1866 Phe Gln His Val Phe Leu Val Val Arg Ala His Ala Pro Cys Thr 105 110 115 CCA CAC ACC TCA TAC AG GTGGGTGCTA GGGTGAACTC AGGTCATGGG 1913 Pro His Thr Ser Tyr Arg 120 125 CACCGATGAT TGACACATCCCC 1 CCCACACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCCCCCCCCCCCCCCCCCCCCCTTCCGA G GTG GCA 2015 Val Ala GTG AGC CGC ACC CAG GAC ACT CCT GCC TTC GGG CCT GCG CTG CCA 2060 Val Ser Arg Thr Gln Asp Thr Pro Ala Phe Gly Pro Ala Leu Pro 130 135 140 GAA GGC GGA GGC CCC TTT GCA GCC AAT GCC GAT TTC CGG GCC TTT 2105 Glu Gly Gly Gly Pro Phe Ala Ala Asn Ala Asp Phe Arg Ala Phe 145 150 155 CTG TTG GCT AAG GCA CTC AAT GGT GAG CAA GCG GCT GGT CAT GCA 2150 Leu Leu Ala Lys Ala Leu Asn Gly Glu Glu Ala Ala Gly His Ala 160 165 170 CGC CAG TTC CAC GCC ATG GCT ACA CGC ACA CGC CAA CAG TAC CTG 2195 Arg Gln Phe His Ala Met Ala Thr Arg Thr Arg Gln Gln Tyr Leu 175 180 185 CAG GAC CTG GCT ACT AAT GAA GTG ACC ACT ACT TCG CTG GAC TCG 2240 Gln Asp Leu Ala Thr Asn Glu Val Thr Thr Thr Ser Leu Asp Ser 190 195 200

GCT TCG CGG TTT GGC CTG CCA TCT CTG GGG GGT AGG CGC CGG GCA 2285 Ala Ser Arg Phe Gly Leu Pro Ser Leu Gly Gly Arg Arg Arg Ala 205 210 215 ACC CCT CGG AGC CCA GGC GCG GAC GTA CAG GCG GCG GGT GCG CTG 2330 Thr Pro Arg Ser Pro Gly Ala Asp Val Gln Ala Ala Gly Ala Leu 220 225 230 ATG TGG GGC GTA CGC GCG GCT CCA GGG GCG CGG GTC GCA GCG GGA 2375 Met Trp Gly Val Arg Ala Ala Pro Gly Ala Arg Val Ala Ala Gly 235 240 245 GCT GAA ACG AGC GGT CCG GAC GAC GCC GAG GTG CCC TGC TTG TTG 2420 Ala Glu Thr Ser Gly Pro Asp Asp Ala Glu Val Pro Cys Leu Leu 250 255 260 GGC ATC TCA GCA GAG ACA CTG GTG CTG GTG GCA CCT CGC GAC GGC 2465 Gly Ile Ser Ala Glu Thr Leu Val Leu Val Ala Pro Arg Asp Gly 265 270 275 CGC GTG GTC TTC AAT TGT GCC TGT CGC GAC GTA TTG GCC TGG ACC 2510 Arg Val Val Phe Asn Cys Ala Cys Arg Asp Val Leu Ala Trp Thr 280 285 290 TTC TCA GAG CAC CAA CTC GAT CTG TAC CAC GGG CGC GGG GAG GCG 2555 Phe Ser Glu His Gln Leu Asp Leu Tyr His Gly Arg Gly Glu Ala 295 300 305 ATC ACG CTG CGG CTC GA C GGG GCC CCA GGG CAA GCC GTG GGC GAA 2600 Ile Thr Leu Arg Leu Asp Gly Ala Pro Gly Gln Ala Val Gly Glu 310 315 320 GTC GTG GCA CGT CTG CAG GTGAGGCAGT GTCAAAAACT AAGGTCCCCT 2648 Val Val Ala Arg Leu Gln GT GG GG GG 325 GTGG GTTAGCTGCT 2698

[0072] GTGGTGGGGC GGGGAAAAGG TACTTGCACA GGTGACTCTC AGAGTCTCCA 2748 ATTCGAATAC ACAACTATCA GGTAGGTCGC TAGGGCTCCT GGGGCATGCC 2798 GGGTTAAATC GATCGAGGCA GGGGCGGGAC CAGGGCGGGG CCTCTGTGAA 2848 GCCACGCCCC AAGGCCACTC TCACCCAGCC TTTCCTTGCA G CTG GTG 2895 Leu Val 330 AGC CGC GGG TGT GAG ACC AGA GAA CTA GCG CTG CCC AGA GAT GGC 2940 Ser Arg Gly Cys Glu Thr Arg Glu Leu Ala Leu Pro Arg Asp Gly 335 340 345 CAA GGT CGC CTG GGC TTC GAG GTG GAT GCA GAA GGC TTC ATC ACG 2985 Gln Gly Arg Leu Gly Phe Glu Val Asp Ala Glu Gly Phe Ile Thr 350 355 360 CAC GTG GAG CGC TTC ACG TTT GCG GAG ACC ACG GGG CTT CGG CCT 3030 His Val Glu Arg Phe Thr Phe Ala Glu Thr Thr Gly Leu Arg Pro 365 370 375 GGA GCT CGT TTG CTG CGA GTC TGC GGC CAG ACG CTG CCC AAG CTG 3075 Gly Ala Arg Leu Leu Arg Val Cys Gly Gln Thr Leu Pro Lys Leu 380 385 390 GGT CCC GAA GCT GCT GCC CAG ATG CTG CGC TCT GCG CCG AAG GTC 3120 Gly Pro Glu Ala Ala Ala Gln Met Leu Arg Ser Ala Pro Lys Val 395 400 405 TGC GTC ACG GTC CTA CCC CCA GAC GAG AGC GGC C GG CCG CAG AG 3164 Cys Val Thr Val Leu Pro Pro Asp Glu Ser Gly Arg Pro Gln Arg 410 415 420 GTCAGGGCAC CGGGTGGGGG TTGTGGGGGG TGGGTAGGATC ATC

[0073] AAGTACACAC TAATTGACCA CTTCCTATTC TAAACCCAAT ATAGGCAAAC 3414 TTTCCCCATA GAACCCCTAA TAGTAAATAC TTTTAGACTT TTGGAGCCAC 3464 TATTCTTGCC ACAGTCACTC GACTCTTTTG TAGGAAAATG AATGTGTGCC 3514 AGCATCTACT AAAACTATGA CTGGAATTTT AGGATTTGAT TTGGAGCTCC 3564 TTGTCTTGTG AAGGGGTAAA CCCAATGTAA GGTCAAAAAA AAAAAAAAAT 3614 CCAAGTAGAA ACACATTATG CTCAGACTGT GTAATTTTTA CATGCCATGA 3664 AGTACTCTAT TAATACCTTT TAAATTATTT AAACATCTAA GAACTAAGGC 3714 CAGAGAAGTG GCTCAGCCAT TAAGAGCATT TGCTGCTCTT GCAGAGGACC 3764 TGAGTTGGAT TCCTAGCACC CACACAGTGC TCTGTGACAG CCTGTATGTA 3814 ACTTCAGATC CAGGGGTCTC ACACCCTCTT CTGGTCTCCA CAGGTGTTGC 3864 ATTCACATGT GCCTGCTCCC TCCCCACGTA AATACACATA TACACATCAA 3914 TAAATAGTTC AAGATCTCTA AAAACTATTC TTAGCAGATA GGAGTTTCAA 3964 AGACTGGCAT GTGTGCTAAT AAAAAACAAA GAGAAGCATG GGCTGGATGG 4014 CTCCAGGCAG TGCACTGTGG ATGCTAAGCG ATTTATATTA CATTGTTTCC 4064 ACTGTAAATA CTCTTATGTA TGTTTGACAG AAAACAGAGA GAGTGGCCTG 4114 CTTAGGAGAC ATGGGCAGCC ATGGATACAA AGTTAACAGT GATATTTGTC 4164 TGCTGTAGAG TCAGGATGCC TGGAGCTCTC TTCCTTTTGG ATGTCTCTGG 4214 CAGTGGCTGG GATGGGGTGG ATGCTGTGGA GGGGATGGAG GGTCCTACCT 4264 GATGCTGCCC CACCCCCACC CTCCAG G AGC TTT TCG GAG CTC TAT ATG 43GC Seruhe CTG AGC 43G Serpent Glu Pro Ser Arg Arg Gly Gly Pro Glu Pro Val 430 435 440 CAG GAT GAA ACT GGG AAG TTG GTC ATA TTG CCT CCC ACC AAG CAG 4402 Gln Asp Glu Thr Gly Lys Leu Val Ile Leu Pro Pro Thr Lys Gln 445 450 455

CTG CTA CAT TTT TGC CTG AAA GAC AGC AGC AGT CCT CCG GGG CCT 4447 Leu Leu His Phe Cys Leu Lys Asp Ser Ser Ser Pro Pro Gly Pro 460 465 470 GGG GAT CTG ACT GAG GAG AGG ACA GAG TTC CTG CGC AGC CAC AAC 4492 Gly Asp Leu Thr Glu Glu Arg Thr Glu Phe Leu Arg Ser His Asn 475 480 485 TCC CTG TCA TCT GGA AG GTACACTCAC TGGGCCAGCC TTTTAGGACC 4539 Ser Leu Ser Ser Gly Ser 490 TGAAAGCACA GCTCTGGAAA AGCAGCTCTCCCCCCCTC CTCTCCGCCCT GAG GCT CCA GTC CTG CCC AAC ACC 4634 Ser Leu Ser Asp Glu Ala Pro Val Leu Pro Asn Thr 495 500 505 ACT CCA GAC CTC CTC CTT GTC ACC ACT GCC AAC CCA TCT GCA CCT 4679 Thr Pro Asp Leu Leu Leu Val Thr Thr Ala Asn Pro Ser Ala Pro 510 515 520 GGT ACT GAC AGA GAA ACA CCC CCT TCC CAG GTAAGCAGAA 4719 Gly Thr Asp Arg Glu Thr Pro Pro Ser Gln 525 530 ACAAACAGAG CTCTGGAGAT TCATTGCAGA GGTGACATTG GATGCTACAG 4769 CCTTGCTGTT CACTGA CAGCCCCC Asp Gln Ser Gly Ser Pro Ser Ser 535 CAT GAA GAC ACC AGT GAC TCA GGC CCA GAA CTG AGG GCC TCC ATC 4863 His Glu Asp Thr Ser Asp Ser Gly Pro Glu Leu Arg Ala Ser Ile 540 545 550

[0075] CTG CCC AGA ACC TTG TCT CTG CGG AAT TCC ATC AGT AAG A 4903 Leu Pro Arg Thr Leu Ser Leu Arg Asn Ser Ile Ser Lys 555 560 565 GTGAGTCTGG AGCCAGGGAA TAGGGCAGGA GGAGAAGACA GCCCCTCCCC 4953 CCCATTCCAG CCCCTCCCTC CCCCCAGCCC CACCCTCCCT AAGCCTTCTC 5003 CTTTGACCTG CAG TT ATG TCG GAA GCT GGC AGT GAG ACC CTG GAG 5048 Ile Met Ser Glu Ala Gly Ser Glu Thr Leu Glu 570 575 GAT GAG TGG CAG TCC ATC TCA GAG ATC GCC TCC ACT TGC AAC ACA 5093 Asp Glu Trp Gln Ser Ile Ser Glu Ile Ala Ser Thr Cys Asn Thr 580 585 590 ATT CTG GAG TCA CTG TCC CGG GAG GTGAGGCCGC AAGGCCCAGA 5137 Ile Leu Glu Ser Leu Ser Arg Glu 595 600 GGGAGGAGCC AGGAGGATGT TTATCCCTTC AGACCTGCGC GCC GAC GAC CAG GCA CAG Pro Ile Ser Glu Ser Gly Asp Pro Lys 605 610 GAA GCT TTA AAG TGT GAT TCT GA GTAAGTTTTC TGCCCTCACA 5275 Glu Ala Leu Lys Cys Asp Ser Glu 615 TACCCACTCT TGTGTGTGTG TCCTTCCCTG CCTGCCTGTCAGCTAG ACTC 75 GGGTTCATTA TCAGTCAGGA GTGTCTGGGA GCCTGCACTG CTTCCGCTGA 5425 GTTCTGACCC CATGTCCTCA G G CCA GAA CCC GGG AGC CTG TCA GAA 5471 Pro Glu Pro Gly Ser Leu Ser Glu 620 625

AAG GTC TCT CAC CTA GAG TCC ATG CTC TGG AAG CTC CAG GAG GAC 5516 Lys Val Ser His Leu Glu Ser Met Leu Trp Lys Leu Gln Glu Asp 630 635 640 CTG CAG AGG GTGAGGAGAGAGCCTGACGG GGGCGCACAG GGCTGCCCCT 5565 TGACTACCAT TCTTCAACCT AG GAG AAG GCG GAC AGG GCA 5615 Glu Lys Ala Asp Arg Ala 650 GCC TTG GAG GAG GAG GTT CGG AGC CTC AGA CAC AAC AAC CAG AGG 5660 Ala Leu Glu Glu Glu Val Arg Ser Leu Arg His Asn 655 Glu Glu 665 CTG CTG GCA GAG TCC GAG AGT GCC GCC ACC CGC CTG CTC CTG GCC 5705 Leu Leu Ala Glu Ser Glu Ser Ala Ala Thr Arg Leu Leu Leu Ala 670 675 680 TCT AAG CAT CTG GGT GCA CCC ACT ACT GAC CTG GCC 5741 Ser Lys His Leu Gly Ala Pro Thr Thr Asp Leu Ala 685 690 TGAGTTCCAA TCTGAATCTG GACCTGCTTG GAACTGCCTG GCCCCTCAGA 5791 GCAACTGGGT CATACTAGTG CCCTTCCTCA GGACTTCTTC CCTGCGCTGA 5841 GGCGCGTCTT AGCACTGCCC CCTCTTCCCA GCCCATTTGG TGGCTAATGC 5891 CTGTCCCTGT TTGTAAATAT CCTGTAAAGA AAAGGAGACA TCAGAGTTTA 5941 AAAAAAAGAA ACAACAAGAA GAAGCAAACA ACTCTATTTG TGTTTGT GTG 5991 TCAAGATACA GAGGAGGGGG AGTCATCCCC TTTCCAAGGT CATATCAAGC 6041 TCCTAGGAGC AGTAGGACAG GTCCCAGGGG GGACATTGAC TTAGTGTTAA 6091 TCTGGCACCA AGCAGAGGCT CTGAGGATAG AACACCCCCT TGGCTCCCCT 6141 TCATTTATTG GGTTCTCTTG GAAAGCAGGT GGCCACGCTT CATGCCTGTC 6191 TGTTTGGAGC AGGAGAGGGA ACACTTCGAG CCTGCAGAGC GAACAACCAG 6241 GGGTGGGCTC TGGCCATGCA GTATGGAATT CCCGAATAGG CCCTGCTAAG 6291 CTGAGCTTCA GAGCATCATT GACTACCACT GGATGGATCA CCTGTTGCAG 6341 GCCCCAGCCA GTGCCTCATC AGCCTCTCCC CAGGGCTGCC TCTGCCTCGA 6391 GAAGCCCAGA CCCTGAGAGA GGACAGGATA AACATGGCTG AGTAACAGTG 6441 GGGCCATGAG CACAAGGAAG CCTTCTCTGA GGAGGCTAAT AAAAGGACTG 6491 AGTTTTGAAA GTTGAGTTCA CCAGCAGATCAGCCACAGAGACACTAGTCAGAGCCATAGAGCCAGAGCCATAGGTACCGAGCCACTAGGCAGGACCCCAGGACGAGACC

[Brief description of drawings]

FIG. 1 is an amino acid sequence of Span-N and GAP3.
It is the figure which compared with the amino acid sequence of m protein.

FIG. 2 is a schematic diagram of the structure of SPA-1 protein.

FIG. 3 is a diagram showing a process for producing a plasmid for recombinant expression of SPA-1 protein.

FIG. 4 shows that Span-N is Rsr1G in a concentration-dependent manner.
It is a graph which shows activating TPase.

FIG. 5 shows that Span-N is Ran1G concentration-dependently.
It is a graph which shows activating TPase.

FIG. 6 shows a restriction map of genomic DNA encoding SPA-1 of the present invention.

FIG. 7 is an electrophoretogram showing the reactivity of monoclonal antibodies F6 and H10 with GST and GST / SpanN fusion protein, and is a photograph as a drawing.

FIG. 8 is an electrophoretogram showing the appearance of the expression product from the SPA gene in which various regions have been deleted, and is a photograph as a drawing.

FIG. 9 shows S introduced into animal cells when the animal cells were subjected to synchronized culture by serum starvation and re-addition of serum.
Fig. 3 shows the effect of PA-1 gene on the cells, and is a photograph instead of a drawing showing the morphology of an organism.

FIG. 10 is an electrophoretogram showing the effect of SPA-1 gene introduced into animal cells on the cells when the animal cells were subjected to synchronized culture by serum starvation and re-addition of serum. It is a photograph instead of a drawing.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/08 9281-4B C12N 5/00 B C12Q 1/68 9162-4B 15/00 ZNAC // (C12P 21/02 C12R 1:19) (C12P 21/08 C12R 1:91) Application for application of Article 30, Paragraph 1 of the Patent Act November 28, 1994 “The 17th Japanese Molecule Biology Society Annual Meeting Program / Lecture Summary "

Claims (17)

[Claims] 1. A cell division mechanism regulating protein which is not expressed in a quiescent state in the cell cycle of mammalian cells but is expressed in the nucleus after entry into the cell cycle. 2. The cell division regulatory protein according to claim 1, which has the structure shown in FIG. 3. The protein according to claim 1, which has an amino acid sequence from the 1st methionine to the 693th alanine in the amino acid sequence shown in SEQ ID NO: 1, wherein 1 or 2 or more amino acid sequences are contained in the amino acid sequence. A cell division regulatory protein in which amino acids are added, deleted, or substituted. 4. The SPA-1 according to claim 1, which has an amino acid sequence from methionine at position 1 to alanine at position 693 in the amino acid sequence shown in SEQ ID NO: 1.
protein. 5. A fragment of the SPA-1 protein according to claim 4, which is a Span-N or Span-C protein fragment having the structure shown in FIG. 6. The Span- according to claim 5, which has an amino acid sequence from methionine at position 1 to leucine at position 190 in the amino acid sequence shown in SEQ ID NO: 1.
N protein fragment. 7. The span according to claim 5, which has an amino acid sequence from alanine at position 191 to leucine at position 327 in the amino acid sequence shown in SEQ ID NO: 1.
-C protein fragment. 8. A gene encoding the protein for regulating cell division machinery or a fragment thereof according to any one of claims 1 to 7, and a gene that hybridizes to these genes. 9. In the base sequence shown in SEQ ID NO: 1,
1200th A (adenosine) to 3278th C
The gene according to claim 8, which has a base sequence up to (cytosine). 10. The gene according to claim 8, wherein the gene is cDNA. 11. An antibody against the protein or fragment thereof according to any one of claims 1 to 7. 12. The method according to claim 1, which is a monoclonal antibody.
The antibody according to 1. 13. Genomic DNA comprising exons 1 to 4 in the sequence shown in SEQ ID NO: 2. 14. Exon 5 in the sequence shown in SEQ ID NO: 3.
Genomic DNA comprising ~ 16. 15. A genomic DNA encoding the amino acid sequence encoded by the open reading frame in the sequence shown in SEQ ID NO: 3. 16. Genomic DNA comprising the open reading frame set forth in SEQ ID NO: 3. 17. A polypeptide obtained by expression of the coding region in SEQ ID NO: 1 or 3.