JPH11164694A - Cell cycle regulatory proteins - Google Patents

Abstract

(57) [Summary] (Modified) [PROBLEMS] To search for cell cycle regulatory molecules in mammals, to determine the nucleotide sequence of the gene encoding the same, to produce the molecule by gene recombination techniques, and to develop and use pharmaceuticals. A nucleotide sequence encoding an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, or a nucleotide sequence encoding a protein having a cell cycle regulatory activity, wherein a part of the amino acid sequence is substituted, deleted or added, Alternatively, a DNA containing a nucleotide sequence hybridizing thereto, a recombinant vector containing the DNA, a transformant thereby,
Method for producing AIM-1 using NA, recombinant AIM-1 protein produced by the method, oligonucleotide or peptide nucleic acid capable of specifically hybridizing with a nucleotide sequence encoding the same, antibody recognizing AIM-1, AIM-1
A therapeutic agent for a disease associated with abnormal cell proliferation, which comprises an inhibitor against -1 protein, and a method for screening an AIM-1 gene or a serine-threonine kinase inhibitory active substance using the same.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel cell cycle regulatory protein, AIM-1 (aurora and IPL-1 like midb
Gene encoding ody-associated protein kinase), recombinant vector containing the gene, transformant using the vector, method for producing AIM-1 using the gene, recombinant AIM-1 protein obtained by the method About. The present invention also involves an abnormal cell proliferation comprising an oligonucleotide or a peptide nucleic acid capable of specifically hybridizing with a nucleotide sequence encoding the AIM-1 protein, an antibody recognizing AIM-1, and an inhibitor against the AIM-1 protein. A therapeutic agent for a disease, and AIM-
The present invention relates to a method for screening a substance having serine-threonine inhibitory activity using one gene or AIM-1 protein.

[0002]

BACKGROUND OF THE INVENTION Mitosis is the fundamental mode of division of the nucleus of eukaryotic cells and is a highly regulated process by which eukaryotic cells can ensure the accuracy of chromosome segregation. The basic number of chromosomes is determined depending on the species and is often an integral multiple of that number. However, if an error occurs during the mitosis stage, the number of chromosomes increases or decreases from one to several from the integral multiple ( Aneuploidy), which is thought to lead to cell death and carcinogenesis.

[0003] Drosophila (Drosophia melanogaste)
r) aurora (Glover et al, Cell 81: 95-105, 199
5) and the most related germinating yeast (Saccharomyc
es cerevisiae) IPL-1 (Francisco et al., Mol.
Cell. Biol. 14: 4731-40, 1994) is a molecule involved in the M phase of mitosis, and is considered to be a molecule necessary for maintaining the accuracy of chromosome segregation.

[0004]

However, to date, a
No molecule corresponding to urora or IPL-1 has been reported in mammals. If a gene encoding a molecule that regulates the cell cycle can be obtained in a mammal and its function can be elucidated, it can be used for pharmaceutical applications such as anticancer agents using this, Extremely interesting.

An object of the present invention is to search for a molecule that regulates the cell cycle in mammals and determine the nucleotide sequence of the gene encoding the same. In addition, a gene set using a recombinant vector containing the sequence is provided. It is to show the possibility of developing a new drug by producing such a molecule using a replacement technique and constructing a screening system or the like using the molecule.

[0006]

Means for Solving the Problems The present inventors have conserved the serine threonine kinase domain (FEBS LETT. 320: 2
46-250, 1993), a novel cell cycle regulatory protein kinase AIM-1 (aurora and I) was screened using a rat cDNA library as a probe.
We isolated the gene encoding PL-1 like midbody-associated protein kinase and succeeded in elucidating its function.

[0007] That is, the present invention relates to SEQ ID NO: 2 in the sequence listing.
A nucleotide sequence encoding an amino acid sequence shown in, or a nucleotide sequence encoding a protein having a cell cycle regulatory activity, wherein the amino acid sequence shown in SEQ ID NO: 2 is partially substituted, deleted or added, and Alternatively, a DNA comprising a base sequence hybridizing thereto is provided.

[0008] The present invention also provides a recombinant vector containing a gene encoding AIM-1 protein.

[0009] The present invention further provides a prokaryotic or eukaryotic host cell transformed with a recombinant vector containing the gene encoding the AIM-1 protein.

[0010] The present invention is further characterized in that a transformant obtained by transforming with a recombinant vector containing a gene encoding AIM-1 protein is cultured, and the produced target protein is separated and purified. AIM-
A method for producing a protein is provided.

[0011] The present invention further provides a recombinant AIM-1 protein produced by the above production method.

[0012] The present invention further provides an oligonucleotide or peptide nucleic acid capable of specifically hybridizing with a gene encoding AIM-1 protein.

[0013] The present invention further provides an antibody that recognizes a peptide having at least five consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

[0014] The present invention further provides a therapeutic agent for a disease associated with abnormal cell proliferation, which comprises an inhibitor for AIM-1 protein.

The present invention further provides the AIM-1 gene or AIM-1 gene.
Provided is a method for screening a substance having serine-threonine inhibitory activity using IM-1 protein.

Furthermore, the present inventors examined how AIM-1 is expressed at each stage of cell division, and examined the role of AIM-1 in the cell cycle.

In the present invention, a cDN encoding AIM-1 is used.
A was obtained as follows.

Oligonucleotide primer 1 for the conserved sequence MHRDVKP (SEQ ID NO: 3) of the serine threonine kinase domain and DFGVSGQ
Using antisense oligonucleotide primer 2 against (SEQ ID NO: 4) as a probe, rat cDN
PCR was performed using the A library as a template. The PCR product was separated by agarose gel electrophoresis to obtain a cDNA fragment, which was identified.

On the other hand, a rat NRK-49F fibroblast cDNA library was screened by polymerase chain reaction (PCR) to
VI-VII threonine-type protein kinases
The subdomain was isolated. Using the above cDNA fragment (SEQ ID NO: 1) as a probe,
By screening x10 ⁶ clones, identified three full-length clone. AIM-1 cD
The sequence of NA is shown in SEQ ID NO: 2, and the amino acid sequence predicted therefrom is shown in SEQ ID NO: 2.

The amino acid sequence of AIM-1 consists of 344 amino acids and has a molecular weight of 39.2 kD. Eighty amino acid residues of its N-terminal sequence correspond to the Drosophila auro
Ra and serine-threonine type protein kinase catalytic domains with homology to yeast IPL-1 (Hanks et al., Science 241: 42-52, 1988). au
FIG. 1 shows the comparison between the catalytic domains of the Rora and IPL-1 genes and the AIM-1 gene. AIM-1 shows 60% and 46% homology with aurora and IPL-1 in their catalytic domains, respectively, but the N-terminal amino acid sequences are not similar.

Using the rat AIM-1 cDNA fragment of the present invention as a probe, cDNA prepared from a human tissue that seems to be rich in AIM-1 (eg, a testis, lung, spleen, or other proliferating tissue). By screening the library, the human AIM-1 cDNA
Can be obtained. In the present invention, human cDNA was isolated by screening a cDNA library derived from human intestine and heart by this method. Human c
The human AIM-1 protein encoded by DNA had 81% amino acid identity with rat AIM-1. Further, as described in Example 10, human A
IM-1 protein showed cell cycle regulatory activity similar to rat AIM-1, indicating that it is a homolog of rat AIM-1.

The thus obtained AIM- of the present invention
When a gene encoding one protein is used, it is possible to produce AIM-1 protein in large quantities by genetic recombination technology and use it for pharmaceutical applications.

That is, prokaryotic or eukaryotic host cells can be transformed by incorporating the gene encoding the AIM-1 protein of the present invention into an appropriate vector.

Furthermore, by introducing an appropriate promoter and a sequence related to expression into these vectors, the gene can be expressed in each host cell. In addition, by linking a gene encoding another polypeptide to the gene of interest and expressing it as a fusion protein, facilitating purification, increasing the amount of expression, or by performing an appropriate treatment in the purification step, It is also possible to cut out the target protein.

In general, eukaryotic genes are considered to exhibit polymorphism, as is known for the human interferon gene, which may result in the replacement of one or more amino acids. For example, the amino acid may not change at all even if the nucleotide sequence changes.

A polypeptide lacking or adding one or more amino acids in the amino acid sequence of SEQ ID NO: 2 in the sequence listing, or a polypeptide in which one or more amino acids have been substituted with one or more amino acids, May have cycle-regulating activity. For example, it has already been known that a polypeptide obtained by converting a nucleotide sequence corresponding to cysteine of the human interleukin 2 (IL-2) gene to a nucleotide sequence corresponding to serine retains IL-2 activity. (Wang et al., Science 224: 1431, 198
Four). Techniques for preparing these variants of the gene encoding AIM-1 protein are known to those skilled in the art.

In addition, when expressed in eukaryotic cells, most of them are added with sugar chains, and the addition of sugar chains can be regulated by converting one or more amino acids.
Even in this case, they may have cell cycle regulatory activity. Therefore, as long as the obtained polypeptide has a cell cycle regulatory activity using an artificially modified gene encoding the AIM-1 protein in the present invention, all the genes encoding those polypeptides can be used in the present invention. Included in the invention.

Furthermore, the present invention also includes a gene in which the obtained polypeptide has cell cycle regulatory activity and hybridizes with the gene shown in SEQ ID NO: 2. The hybridization conditions may be the same as those used in conventional probe hybridization (eg, Molecular Cloning: A Laboratory Manual, Sambr
ook et al., Cold Spring Harbor Laboratory Press, 1
989). Standard methods include literature (Molecular Cloni
ng: A Laboratory Mannual, Sambrook et al., Cold Sp
ring Harbor Laboratory Press, 1989), for example, 6XSSC, 0.05XBLOTTO (Bovine Lacto Tr
ansfer Technique Optimizer) under the conditions of the solution 68 ° C. in, preferably Express Hyb ^TM Hybridization Solution
(Clontech) solution at 60-68 ° C for 18 hours, or 60-68 ° C in Rapid-hyb Buffer (Amersham) solution.
2. A gene capable of hybridizing under conditions of 18 ° C. for 18 hours, a gene recognized by the probe complementary to the DNA according to claim 1, and a gene encoding a protein having a biological function of the AIM-1 protein. Included in the present invention. Further, by changing the salt concentration and / or the hybridization temperature using the above-mentioned probe, cD showing homology to the base sequence shown in SEQ ID NO: 2 can be obtained.
Many methods for isolating NA clones have already been established. Gene isolated by these methods and AIM
The present invention also encompasses a gene encoding a protein having the biological function of -1 protein (Molecular Clonin
g: A Laboratory Mannual, Sambrook et al., Cold Spr
ing Harbor Laboratory Press, 1989).

The expression vector can include an origin of replication, a selectable marker, a promoter, an RNA splice site, a polyadenylation signal, and the like.

Prokaryotic host cells among the hosts used in the expression system include, for example, Escherichia coli and Bacillus subtilis. Among eukaryotes, host cells of eukaryotic microorganisms include, for example, yeast and slime mold. Or,
Insect cells such as Sf9 may be used as host cells. Furthermore, host cells derived from animal cells include, for example, COS cells, CHO cells, and the like.

The protein produced by culturing the transformant transformed with the gene encoding the AIM-1 protein as described above can be isolated from the inside or outside of the cell and purified. In addition, not only the protein obtained from the gene containing the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 of the above-mentioned Sequence Listing, but also a part of the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing is substituted, deleted or Even if a protein is obtained using a base sequence encoding the added amino acid sequence or a gene containing a base sequence hybridizing thereto, AIM
As long as it has a biological function of the -1 protein, ie, a cell cycle regulatory activity, it is included in the present invention.

For separation and purification of the AIM-1 protein, separation and purification methods used for ordinary proteins can be used. For example, various types of chromatography, ultrafiltration, salting out, dialysis and the like can be appropriately selected and used in combination.

According to the present invention, antisense DNA can be prepared based on the nucleotide sequence of the gene encoding AIM-1 protein. Antisense DNA is mRN
By having a base sequence complementary to A and forming a base pair with mRNA, it blocks the flow of genetic information and suppresses the synthesis of the final product AIM-1 protein. The antisense DNA that can be used in the present invention is an oligonucleotide that can specifically hybridize with the base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

As used herein, the term "oligonucleotide" refers to oligonucleotides and analogs thereof formed from naturally occurring bases and sugar moieties linked by natural phosphodiester bonds. Thus, the first group included by this term is a naturally occurring species or a synthetic species produced from a naturally occurring subunit or a homolog thereof. A subunit refers to a base-sugar combination linked to an adjacent subunit by a phosphodiester bond or another bond. Also, a second group of oligonucleotides are analogs thereof, which refers to residues that function similarly to oligonucleotides, but have non-naturally occurring portions. These include oligonucleotides with chemically modified phosphate groups, sugar moieties, and 3 ', 5' ends to increase stability. For example, oligophosphorothioate in which one of the oxygen atoms of a phosphodiester group between nucleotides is substituted with sulfur, -C
Oligomethylphosphonate substituted with H ₃ is exemplified. Further, the phosphodiester bond may be substituted with another structure that is nonionic and nonchiral. In addition, modified oligonucleotide forms may be used as oligonucleotide analogs, ie, species containing purines and pyrimidines other than those normally found in nature.

The oligonucleotide according to the invention has preferably 5 to 40, more preferably 8 to 30, more preferably 12 to 30, subunits.

In the present invention, the target portion of the mRNA to which the oligonucleotide hybridizes is preferably a transcription initiation site, a translation initiation site, an intron / exon binding site or a 5 ′ cap site, but in consideration of the secondary structure of the mRNA. A site without steric hindrance should be selected.

Further, in the present invention, a peptide nucleic acid (for example, Bioconjugate Chloride) is used instead of an oligonucleotide.
em. Vol. 5, No. 1, 1994) can also be used.

A particularly preferred embodiment of the present invention is an oligonucleotide or a peptide nucleic acid that hybridizes with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and can inhibit the expression of AIM-1 protein.

The oligonucleotide according to the present invention can be produced by a synthesis method known in the art, for example, a solid phase synthesis method using a synthesizer such as Applied Biosystems. Similar methods can be used to produce other oligonucleotide analogs, such as phosphorothioates and alkylated derivatives (Akira Murakami et al., "Functional Antisense DN").
Chemical synthesis of A ”, Synthetic Organic Chemistry, 48 (3): 180-193, 199
0).

The administration of an oligonucleotide or peptide nucleic acid capable of specifically hybridizing with the gene encoding AIM-1 of the present invention to an animal can suppress the production of AIM-1 protein in the animal. . In addition, since AIM-1 has a function necessary for progression of the M phase of cell division as described in detail below, the growth of cancer cells can be promoted by administering the oligonucleotide or peptide nucleic acid of the present invention to the cancer cells. Can be suppressed or stopped. The oligonucleotide of the present invention can be expected to be effective not only for treating cancer cells but also for treating other proliferative diseases.

In order to prepare an antibody that recognizes a peptide having at least five consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing of the present invention, a conventional method (for example, Shinsei Kagaku Koza Kozai 1, Protein I, p.389
-397, 1992), at least 5 of the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing as antigens
It can be obtained by immunizing an animal with a peptide having two consecutive amino acids and collecting and purifying an antibody produced in the living body. Antibodies include polyclonal and monoclonal antibodies, and methods for making them are also known to those skilled in the art. The anti-AIM-1 antibody thus obtained can be used for various immunological measurement methods such as enzyme immunoassays such as ELISA, radioimmunoassay, and immunofluorescence, or for purification of AIM-1 protein by a column.

According to the present invention, an inhibitor of AIM-1 (for example, an antagonist, an antibody such as AIM-1 (KR) or an inhibitor of protein phosphorylation activity, or an antisense chain) is used for cancer cells. Can suppress or stop diseases accompanied by abnormal cell growth such as cancer cell growth or psoriasis.

Further, the AIM- produced by the present invention
Gene therapy that suppresses or stops the growth of cancer cells by administering the gene encoding the 1 (KR) protein to the cancer cells in a site-specific manner and expressing the gene at the site of the cancer cells can also be expected.

Furthermore, in the present invention, a method for screening a substance having serine-threonine kinase inhibitory activity using the AIM-1 gene or AIM-1 protein can be performed. For example, a reaction solution containing [γ- ³² P] ATP is added to a substrate (for example, myosin light chain, histone protein,
(Synthetic substrate) and AIM-1 protein and an inhibitor candidate substance or a solvent, and after incubating at, for example, 30 ° C. for several minutes, a part of the reaction solution was soaked in Wattmann 3MM filter paper.
The reaction is stopped by immersion in ice-cooled 10% TCA-1% sodium pyrophosphate. After the filter paper is washed and dried, it is placed in a toluene scintillator, and ³² P incorporated into the protein is measured with a liquid scintillation counter, and the inhibitory activity is evaluated by decreasing the measured value of ³² P relative to a solvent control group. AIM-1 protein function The expression of the AIM-1 gene was tested by Northern blot analysis (FIG. 2a). AIM-1 cDNA labeled with ³² P-labeled poly (A) ⁺ RNA isolated from various rat tissues
Hybridized with the fragment. An AIM-1 band of about 2.0 kb was detected in all tissues tested, but particularly in testis, spleen and lung.

Since aurora and IPL-1 are known to be involved in the progression of mitosis during the M phase (chromosome segregation), it was determined whether the expression pattern of AIM-1 shows a cell cycle-dependent movement. investigated. As a result, m of AIM-1
RNA was induced in late S (late-S) and reached a maximum during the transition from G2 to M phase (FIG. 2b). Furthermore, when M phase is stopped by colcemide treatment, AIM-1 mRN
A significant accumulation of A was induced (data not shown). These results suggest that AIM-1 functions in the M phase.

To study the function of the AIM-1 protein, a peptide consisting of the C-terminal 13 amino acids of the AIM-1 sequence was synthesized, and an antibody against this peptide was prepared. Using this antibody, the NR at each time point in the cell cycle
Changes in the amount of AIM-1 protein in K-49F cells were analyzed (FIG. 2c). The results showed that the AIM-1 protein detected as a band of about 40 Kd started to accumulate at the borderline phase of S / G2, reached the highest level in M phase, and decreased dramatically in the next G1 phase. Similar results were obtained using cells whose cell cycle was synchronized by the double thymidine block and release method. These data are consistent with the Northern blot analysis. Thus, these data indicate that the AIM-1 protein is best expressed during the M phase of cell division. Further, the C-terminal of AIM-1 contains a deduced consensus sequence of the destruction box recognized by the proteosome (FIG. 1).
The protein was cyclin B (Glotzer et al., Na
349: 132-138, 1991) and cut2 (Funabiki et a
l., Nature 381: 438-441, 1996).
-Like the M phase regulatory protein, it may undergo ubiquitin proteasome-dependent proteolysis.

To determine how the AIM-1 protein is involved in mitotic machinery during the cell cycle,
Synchronously growing NRK using antibodies to IM-1
-49F cells were subjected to immunocytochemical studies. No signal of AIM-1 protein was detected in metaphase (metaphase) (FIGS. 3a-c), but in late metaphase (la
te anaphase) began to be detected as a distinct band spreading in the center of the central spindle (FIG. 3d). End (te
lophase) and with the progress of cytokinesis, AIM-1
Proteins increasingly accumulate in the midbody (Fig. 3e, f).

In a mink lung epithelial (Mv1Lu) cell, a tetracycline induction system (Gossenand Bujard, Proc.
Natl. Acad. Sci. USA 89: 5547-51, 1992) under the control of FLAG-AIM-
When one fusion protein was induced, the same results were observed (FIG. 3g). When the induced FLAG-AIM-1 was immunodetected with an anti-FLAG antibody, FLAG-AIM-1 localized in the central body was observed. This is this anti-
AIM-1 antibody specifically recognizes AIM-1 protein and shows that AIM-1 is localized in the central body.

Since to date gamma-tubulin has been identified at the organizing center of central body microtubules (Shu et al., JCS 108: 2955-62, 1995), FL
The intracellular localization of γ-tubulin in Mv1Lu cells expressing AG-AIM-1 was examined. As shown in FIG. 3d, FLAG-AIM-1 protein and γ-tubulin were co-localized in the central body. These data suggest that the appearance of AIM-1 protein in mitotic cells is consistent with the rate of protein expression by Western blot, suggesting that AIM-1 regulates the late-to-end cytokinesis process. I have.

To further confirm this possibility, the AIM
The inactivated FLAG-AIM-1 (KR) in which endogenous protein phosphorylation activity was blocked was prepared by replacing the lysine at position 109 of -1 with arginine by a point mutation. Also, a system inducible with tetracycline in Mv1Lu cells was used.

Wild-type FLAG-AIM-1 (WT) or FLAG-
AIM-1 (KR) -introduced Mv1Lu cells were grown for 24 hours in the presence or absence of doxycycline (tetracycline analog), and then subjected to Western blot using an anti-FLAG antibody (FIG. 4A). FLAG-A
When IM-1 (KR) was induced for 72 hours, about 68
% Of cells failed to undergo normal cytokinesis and had more than one nucleus (FIG. 4Bf, Table 1). Furthermore, the size of these cells was much larger than normal cells (FIG. 4Bf, FIG. 5D). However, in these abnormal cells, spindle function, including chromosome segregation and late late extension, and nuclear division did not appear to be affected (data not shown). This is called flow cytometry (FA
CS) analysis further confirmed (FIG. 4C). AIM
4N or 8N DNA in 72 hours of induction of -1 (KR)
The number of cells with 劇 的 increases dramatically, suggesting the appearance of binuclear or tetranuclear cells. That is, multinucleated bodies appeared because nuclear division progressed but cytokinesis was inhibited.

In contrast, wild-type FLAG-AI
Cells expressing M-1 (WT) or cells transformed with the vector alone showed a normal cell cycle division pattern (FIG. 4C).

In order to confirm the generality of this observation, AI
M-1 (WT) and AIM-1 (KR) plasmids were transiently expressed in NRK-49F cells and human diploid fibroblast KD cells, and their effects were transformed with the vector alone. Compared. These constructs were transformed into cells with the reporter construct RSV-β-galactosidase and β-cells were used as positive internal controls for transformation.
-A galactosidase expression plasmid was prepared (Table 1). Transformation with the vector alone did not affect normal cell cycle progression, but AIM-1
Transformation with the (KR) construct increased the number of cells having two or more nuclei (NRK-49F cells: 62%, KD
Cells: 38%).

The nucleus of these AIM-1 (KR) expressing cells divides 3 to 4 times in 4 to 5 days to have 10 or more nuclei per cell (FIG. 5D). Of polynuclei will not attach to the plate without further splitting. In order to examine the effect of AIM-1 (KR) on proliferation, the cells were cultured for 2 weeks in the presence or absence of doxycycline (FIG. 5E). Mv1Lu cells expressing AIM-1 (KR) In cells, mitotic inhibition was observed, but in cells expressing AIM-1 (WT), mitotic inhibition was not observed.

From the above data, it can be seen that the overexpression of the loss-of-function AIM-1 inhibits cytokinesis during mitosis,
This led to the appearance of multinucleated cells, namely, AIM
-1 was strongly suggested to be required for cytokinesis.

However, these data for AIM-1 are in contrast to mutations in the aurora gene. That is, the mutant aurora gene shows an abnormal mutant spindle because the centrosome cannot be separated to form a bipolar spindle. This means that A
This suggests that the IM-1 gene is not a fully functional homolog of the aurora gene, but rather a functionally related molecule. Spindle functions, such as centrosome separation and cytokinesis, are regulated by kinesin-like proteins (KLP).
(Goldstein, Trends Cell Biol. 1: 93-98, 1991; Moo
re & Endow, BioEssays 18: 207-219, 1996). In Drosophila, it has been reported that KLP-61F is required for centrosome duplication, while KLP-3A is a component of the centrosome and is required for central spindle structure as well as cytokinesis (Williams et al.) ., J. Cell Biol. 129: 709
-723, 1995). The phenotypes in both KLP-61F and the aurora mutant show abnormalities that are surprisingly similar to centrosomes (Heck et al., J. Cell Biol. 123: 665-6).
79, 1993), suggesting that they are involved in a common process. Aurora kinases are thought to be involved in the function of KLP-61F that regulates kinesin motility activity. In contrast, the phenotype in the KLP3A mutation is similar to that in the loss-of-function mutant of AIM-1. Therefore, AIM-1 may be involved in the control of KLP-3A in cytokinesis.

Drosophila (Drosophia melanogast)
er) Mutations in polo cause abnormal mitosis and meiosis due to abnormal spindle formation (Fenton et al.
al., Nature 363: 637-640, 1993). This is likely to result in polyploid cells. The mammalian M-phase specific protein kinase Plk associated with polo is localized in the central body during anaphase and cytokinesis (Clay et al., Proc. Natl.
Acad. Sci. USA 90: 4882-4886, 1993; Lee et al., Mo
l. Cell. Biol. 15: 7143-7151, 1995; Golsteyn et a
l., J. Cell Biol. 129: 1617-1628, 1995). polo
And the properties of PLK are similar to those of AIM-1, but are not structurally homologous to AIM-1.

It has been observed that AIM-1 is localized in the central body together with γ-tubulin during anaphase and cytokinesis. Recently, it has been reported that suppression of γ-tubulin using the antisense RNA method causes damage to the morphogenesis of the midbody structure, leading to premature cytokinesis (Shu et al., JCS 108: 2955-62,
1995). These data show some relationship between AIM-1 and γ-tubulin. Therefore,
γ-tubulin may be involved in the biogenesis of the midbody structure in the completion of cytokinesis in cooperation with AIM-1 kinase.

The present invention is described in more detail by the following examples, which do not limit the invention.

[0060]

EXAMPLES In the following Examples, cell extracts were prepared by the following method.

The cells were placed in a RIPA buffer (10 mM Tris-HC).
l (pH7.5), 1% NP040, 150mM NaCl, 1mM EDTA, 1mM PMSF,
(4 mg / ml leupeptin) and centrifuged at 100,000 × g for 1 hour. The resulting pellet was suspended in SDS-PAGE sample buffer, boiled, and sonicated. 10,000
The supernatant obtained by centrifugation at rpm was treated with SpinBind (FMC) to remove mixed DNA fragments. Example 1: cDN encoding part of AIM-1 gene
Identification of fragment A Conserved sequence MHRD of serine threonine kinase domain
Oligonucleotide primer 1 for VKP (SEQ ID NO: 3) and DFGVSGQ (SEQ ID NO: 4)
An antisense oligonucleotide primer 2 was prepared. The sequences of primer 1 and primer 2 are as follows. Primer 1: 5'-ATGCA (T / C) (C / A) G (T / C / A / G) GA (T / C) G
T (T / C / A / G) AA (A / G) CC-3 ′ (SEQ ID NO: 5) Primer 2: 5′-TG (T / C / A / G) CC (T / C / A / G ) GA (T / C / A / G)
AC (T / C / A / G) CC (A / G) AA (A / G) TC-3 '(SEQ ID NO: 6) Rat cDNA library (FEBS LETT. 320: 246-25
0, 1993) as a template and the above primers 1 and 2 as primers, using vent DNA polymerase,
(94 ° C .: 1 minute, 55 ° C .: 1 minute, 72 ° C .: 2 minutes) and amplified by performing 40 cycles of PCR. Obtained PC
The R product was separated by agarose gel electrophoresis to obtain a fragment.

The sequence of the obtained cDNA fragment was determined, and the sequence described in SEQ ID NO: 1 (attcacagagacataaagcccgagaa
cctgctgttaggtctacagggagagctgaagattgcggactttggctggt
ctgtgcat). Example 2: Library screening (1) Preparation of rat NRK-49F cDNA library NRK-49F RN in logarithmic growth phase by guanidine method
A and take mRNA on oligo-dT cellulose column
Was purified. After oligo-dT / Not1 was used as a primer, cDNA was produced using reverse transcriptase, and an EcoRI adapter was bound to the cDNA. Then, the expression vector pcTerraIII (FEBS L
ETT. 320: 246-250, 1993). (2) Screening The sequence of the cDNA fragment obtained in Example 1 (SEQ ID NO: 1) was used as a probe for gene screening. The probe was labeled with ^32- P and NRK-49 bound to the filter.
It hybridized with the F cDNA library under the following conditions, and a positive clone was isolated. Hybridization solution: 6x SSPE, 0.5% SDS, 10x Denhardt sol., 10
0u / ml denatured herring sperm DNA. The filter is (2x SSC,
(15 minutes with 0.1% SDS, 15 minutes with 0.2 × SSC, 0.1% SDS).

Thus, three full-length cDNAs were isolated and sequenced. The cDNA sequence of AIM-1 and the amino acid sequence predicted therefrom are shown in SEQ ID NO: 2.

In addition, the amino acid sequence of AIM-1 was determined by comparing the Drosophila-derived aurora gene and the yeast-derived Iurora gene.
FIG. 1 shows a comparison with the amino acid sequence of the PL-1 gene. In the figure, amino acid numbers are shown on the left. Roman numbers above the amino acid sequence are described in Hanks et al. (Science 241: 42-52,
1988). The same amino acids conserved in more than one sequence are shown in black. Also, the underlined amino acid sequence in AIM-1 subdomain VIb to VII indicates the sequence first identified by PCR. The asterisks are the portions used in the preparation of the anti-peptide antibody N12 described in the following examples. AIM-1
The underlined short portion in the amino acid sequence at the C-terminus is a putative proteolytic box site. Example 3 Expression of AIM-1 Gene in Rat Tissue A membrane filter containing mRNA (2 μg) prepared from various rat tissues was used as a probe with the ³² P-labeled AIM-1 cDNA fragment described in Example 2 as a probe. Blot analysis was performed.

The result obtained is shown in FIG. 2a. An AIM-1 band of about 2.0 kb was detected in all tissues tested, but particularly in testis, spleen and lung. Example 4: Relationship between AIM-1 mRNA and cell cycle Cells were obtained by placing confluent rat NRK-49F cells in serum-free medium for 2 days and then inoculating 1: 3 in serum-containing DMEM medium. The cycle was run synchronously. Each point in the cell cycle (0, 4, 8, 1
2, 16, 20, 24 hours) and 2 μg of poly (A) ⁺
RNA was added. G3PDH gene probe (Nippon Gene) was used as a control. The results obtained are shown at the top of FIG. 2b.

Further, FACS analysis by cycle test (Becton Dickinson) and FACScan (Becton Dicki
nson), the percentage of cell number at each cell cycle stage relative to the total cell number was calculated and is shown at the bottom of FIG. 2b.

As is clear from the figure, AIM-1 mR
NA was induced in late S (late-S) and reached a maximum during the transition from G2 to M phase. Example 5: Preparation of Antibody to AIM-1 In order to prepare a polyclonal antibody to AIM-1, a peptide (A to L at the C-terminal to V-C-terminal region) indicated by an asterisk in the amino acid sequence of AIM-1 shown in FIG. Up to)
This was conjugated to KLH (keyhole limpet hemocyanin) and injected into rabbits.

Serum was obtained from rabbits, from which peptide-immobilized FMP-activated cellulose columns (Seikagaku)
Was used for affinity purification to obtain a polyclonal antibody. Example 6: Relationship between AIM-1 protein expression and cell cycle The cell cycle was synchronized and advanced as in Example 4 described above. Each point in the cell cycle (0, 4, 8, 12, 16, 2
At 0, 24, and 28 hours), insoluble proteins corresponding to 2 × 10 ⁵ cells were removed and subjected to Western blot analysis using the anti-AIM-1 antibody (N12) prepared in Example 5 as a probe. AIM-1 in vit
The ro transcription / translation product (Promega) was used as a control.

The results obtained are shown in FIG. 2c. About 40Kd
AIM-1 protein detected as a band of
It was observed that it started to accumulate at the borderline phase of G2, reached the highest level in M phase, and decreased dramatically in the next G1 phase.

Similar results were obtained using cellular proteins prepared by synchronizing the cell cycle by the double thymidine block and release method. Example 7: Expression plasmid Construction (1) Preparation of FLAG-AIM-1 (WT) The full length coding sequence of AIM-1 cDNA obtained in Example 2 was converted to pUHD10-3 (Hermann Bujard, Zentru
m fur Molekulare Biologie der UniversitatHeidelber
g, Im Neuenheimer Feld 282, W-6900, Heidelberg, Fe
deral Republic of Germany) or EF1a
Promoter (Mizushima et al., Nucleic Acids Res.
18: 5322-5326, 1990) to produce FLAG-AIM-1 (WT), which was subcloned into the pEF / hygI plasmid and labeled at the N-terminus with the FLAG protein. (2) Preparation of FLAG-AIM-1 (KR) FLAG was obtained by two-step PCR using the following primers.
-AIM-1 (KR) was produced. Primer 3: 5'-AGA GAA TTC ATG GAC TAC AAG GAC G
AT GAC GAC AAG ATG GCTCAG AAA GAG AAC-3 '(SEQ ID NO: 7) Primer 4: 5'-CTT GAA GAG GAT CCT TAG CGC CAC G
AT-3 '(SEQ ID NO: 8) Primer 5: 5'-ATC GTG GCG CTA AGG ATC CTC TTC A
AG-3 '(SEQ ID NO: 9) Primer 6: 5'-GA CTC AGA CTA AAG GGC AGA GGG AG
G CAG ACG GCG CGC-3 ′ (SEQ ID NO: 10) First PCR (A) The combination of primer 3 and primer 4
A 330 bp fragment was amplified using the M-1 gene as a template, and purified by agarose gel electrophoresis. (B) Separately, a 700 bp fragment was amplified using the AIM-1 gene as a template with a combination of primers 5 and 6, and purified by agarose gel electrophoresis. Second PCR The above fragments (A) and (B) were mixed in equal amounts, and PCR was performed using a combination of primer 3 and primer 6.
The fragment of b was purified, cut at EcoRI and XbaI sites, and inserted into the multiple cloning site of pUHD10-3. (3) Expression of plasmid FLAG-AIM-1 (WT) prepared in (1) above and FLAG-AIM-1 (KR) prepared in (2) were expressed. These were then purified using the lipofectin method according to the manufacturer's (GIBCO, BRL) protocol.
M with F / hygI hygromycin resistance plasmid
Cotransformed into v1Lu cells (ATTC CRL-6584). Hygromycin (Calbiochem) 0.2m
Clones were selected in g / ml and deoxycycline (Sigma). After 18 hours in the absence of deoxycycline, FLAG-AIM-1 (W) by Western blot using an anti-FLAG antibody (M2) as a probe.
Clones that induced T) or (KR) were selected. A clone with an empty vector was used as a control. Example 8: Involvement of AIM-1 protein in mitosis (1) Immunocytochemical test using antibody To examine how AIM-1 protein is involved in mitosis during the cell cycle , NRK-49F at various stages of development using antibodies against AIM-1
Cells were subjected to immunocytochemical studies.

NRK-49F cells were stained with the anti-AIM-1 polyclonal antibody prepared in Example 5 (left column in FIG. 3) and the anti-α-tubulin monoclonal antibody (middle column in FIG. 3), and the dye Hoechst 33258 was used. With D
NA staining (right column in FIG. 3) was performed.

The cells grown on the Labtec chamber slide (Nunc) were transferred to a microtubule stabilizing buffer (Boleti) containing 0.5% Triton X-100 (MSB: 80 mM K-PIPES (pHH6.8), 5 mM EGT).
A, 1 mM MgCl ₂ ) and fixed with methanol (−
10 minutes at 20 ° C). Fix the fixed cells with 0.1M PIPES (pH7.2), 1m
M MgSO4, 1mM EGTA, 1.83% L-lysine, 1% BSA and 0.1%
Wash in a solution consisting of sodium azide and then monoclonal mouse anti-α-tubulin antibody # 7-516
8 (Sigma) and affinity purified rabbit AIM
-1 antibody N12 or anti-FLAG monoclonal antibody (M2) and rabbit anti-γ-tubulin antibody (Masuda)
For 1 hour. Next, FITC-
Incubated with conjugated goat anti-rabbit IgG antibody and rhodamine-conjugated goat anti-mouse IgG antibody. The signal was observed using a microscope (Olympus).

According to the progress of cell division, a in FIG. 3 shows the interphase, b shows the prophase, c shows the metaphase, d shows the late anaphase, and e shows the late phase.
Represents terminal phase (telophase), and f represents cytokinesis.
s).

AIM-1 is not detected in metaphase, but is detected late in the center of the spindle. This staining is shown in the midbody (midbod) with the progression of anaphase and cytokinesis.
It was observed that it increased in y). (2) Test using expression plasmid pUHD10-3 / FLAG-AI prepared in Example 7
Mink lung epithelial (Mv1Lu) cells with M-1 (WT) were cultured in doxycycline-free medium for 24 hours, then anti-FLAG monoclonal antibody M2 (KODAK) and rabbit anti-γ-tubulin antibody (Masuda et al., J. Cell
Sci. 109: 165-177, 1996) and Hoechst 33258.

As shown in FIG. 3g, during cytokinesis FL
AG-AIM-1 protein was localized in the central body along with γ-tubulin. This is consistent with the protein expression pattern by Western blot,
-1 implicates in the regulation of the cytokinesis process from late to late. Example 9: Appearance of AIM-1 and polynuclear body (1) AIM-1 expression and cell abnormality Wild-type FLAG-AIM-1 (W
T) or kinase-inactivated FLAG-AIM-1 (K-
R) (dominant negative type) was induced in Mv1Lu cells. After removing doxycycline (DOX),
Cells were cultured for 18 hours. Cells were harvested and cell lysates were subjected to immunoblot analysis using an anti-FLAG monoclonal antibody. The results obtained are shown in FIG. 4A.

Next, only the vector (a, d), FLAG
-AIM-1 (WT) (b, e) or FLAG-AI
Asynchronous cells transformed with M-1 (KR) (c, f) were cultured for 72 hours in the presence (ac) or absence (df) of DOX, respectively, and stained with Giemsa solution. did.
The obtained results are shown in FIG. 4B (note that in FIG. 4B, a, b, c are shown from left to right in the upper column, and d, d are shown from left to right in the lower column.
e, f). Kinase-inactivated FLAG-AIM
It was observed that the expression of -1 (KR) resulted in the appearance of cells having two or more nuclei (FIG. 4f).

Further, a cell sample was collected, the cells were fixed with ethanol, stained with propidium iodide, and subjected to FACS analysis. The obtained result is shown in FIG. 4C. Induction of AIM-1 (KR) 4N or 8 at 72 hours
The number of cells with N DNA increased dramatically. This indicates the appearance of dinuclear or tetranuclear cells.

Further, the FLAG-AIM shown in FIG.
Superimposing the respective images of Mv1Lu cells expressing -1 (KR) showed double staining of α-tubulin (green) and DNA (red) (propidium iodide). It is FIG. 5D. After removal of doxycycline, binuclear (a), quadrinuclear (b), 8
Nuclei (c) or abnormal cells having 10 or more nuclei appeared.
α-tubulin is anti-α-tubulin antibody and FITC
-Detected with conjugated goat anti-mouse IgG (Cappel) and the signal was observed with a confocal laser microscope (Fluoview, Olympus).

FLAG-AIM-1 (WT) or FLA
Mv transfected with G-AIM-1 (KR)
Each of the 1 Lu cells was cultured for 2 weeks in the presence or absence of DOX and stained with Giemsa solution. The results obtained are shown in FIG. 5E. As is clear from the figure, AIM-1 (W
Division inhibition was observed in Mv1Lu cells expressing T), but division inhibition was not observed in cells expressing AIM-1 (KR). (2) Abnormal cytokinesis in cells expressing AIM-1 (KR) FLAG-AIM-1 (WT), FLAG-AIM-1
(KR) or the control plasmid pEF / hygI
Plasmids were combined with the reporter construct RSV-β-galactosidase at a ratio of 10: 1 into NRK-49F cells and human diploid fibroblast KD cells (gifted by the late Dr. Takeo Kakunaga) using the lipofectin method. Co-transfection. Twenty-four hours later, cells were seeded at 1 × 10 ³ cells per 60 mm dish, fixed in 1.25% glutaraldehyde / PBS 72 hours post-transfection, and described in the literature (Shu et al., J. Biol.
CS 108: 2955-62, 1995), and the expression of β-galactosidase was observed. Mv1Lu cells were performed under the conditions described in (1) B above. Table 1 shows the obtained results. Numbers in the table indicate NRK-49F cells or K
For D cells, it means the percentage of cells having two or more nuclei to β-galactosidase positive cells, and Mv1L
For u cells, means the percentage of cells with two or more nuclei relative to total cells. More than 160 cells were tested for each cell line.

[0080]

[Table 1]Table 1 Mv1LuPlasmid NRK-49F KD DOX (+) DOX (-)  Vector only 0.5 0.2 0.4 0.7 AIM-1 (WT) 7.8 4.8 0.9 2.8AIM-1 (K-R) 62.0 38.4 7.7 68.4 Example 10: Isolation of human cDNA, AIM-1 protein
Expression and activity thereof (1) Isolation of human AIM-1 cDNA Conserved sequence IHRD of serine threonine kinase domain
Oligonucleotide primers for sense to IKP
7, and antisense oligos against DFGWSVH
Nucleotide primer 8 was prepared. Primer 7: 5'-AT (A / T / C) CA (T / C) (A / C) G (A / T / C / G) GA
(T / C) AT (A / T / C) AA (A / G) CC (A / T / C / G) -3 ′ (SEQ ID NO: 1
1) Primer 8: 5 '-(A / G) TG (A / T / C / G) AC (A / T / C / G) GACCA
(A / T / C / G) CC (A / G) AA (A / G) T-3 ′ (SEQ ID NO: 12) “lone linker” method (Abe, Mamm. Genome 2: 252-259, 199)
C from human cells (gut and heart) prepared according to 2)
Using the DNA library as a template, the primers 7 and
ExTaq DNA Polymerase
Amplified (Takara, Tokyo). The resulting PCR product
Were subcloned and about 5 for each library.
Zero fragments were obtained and sequenced.

Based on the sequence information of this cDNA fragment, N
Oligonucleotide primer 9 for sense against LLLGLKGELKI (SEQ ID NO: 13) and "lone li
A universal adapter primer 10 was prepared for an oligo (dT) -containing adapter primer for the construction of an nker "prepared cDNA library. Primer 9: 5′-AATCTGCTCTTAGGGCTCAAGGGAGAGCTGAAG
ATT-3 '(SEQ ID NO: 14) Primer 10: 5'-TCCACTAATATCGGCCACGCGTCGACTAGTA
C-3 ′ (SEQ ID NO: 15) The 3 ′ end of AIM-1 cDNA was amplified using these primers, and the amplified product (550 bp) was sequenced. Then, using this amplification product as a probe, HeLa
cDNA library (Otsu et al., FEBS Lett. 320: 246
-250, 1993) to screen for full-length human AIM-1
Clones containing the cDNA were isolated and sequenced. (2) Expression and activity of AIM-1 protein Blots containing polyadenylated RNA derived from various tissues were subjected to Northern blot analysis using human AIM-1 cDNA as a probe. Expression was also found in placenta, lung, small intestine, large intestine and spleen. Several types of colorectal tumors (colorectal tum
or) The expression level of human AIM-1 in the cell line was examined by Northern blot analysis. As a result, the expression level was high in all cell lines, and multinucleated cells and polyploid cells were frequently observed (10-20%). AIM
-1 was suggested to be associated with the formation of multinucleated cells and an increase in multiploid cells. Furthermore, in order to confirm that the expression of human AIM-1 was cell cycle-dependent, the expression of AIM-1 was examined using colcemid-treated cells. Was observed to be maximal.

[0082]

[Sequence List] SEQUENCE LISTING <110> Chugai Seiyaku Kabushiki Kaisha <120> Cell cycle regulatory protein <130> 980991 <160> 14 <210> 1 <211> 84 <212> DNA <213> Rat <440> 1 attcacagagacataaagcccgagaacctgctgttaggtctacagggagagctgaagat IHRDIPEKI tgcggactttggctggtctgtgcat ADFGWSVH <210> 2 <211> 1815 <212> DNA <220> <221> CDS <222> (185) ... (1216) <213> Rat <440> 2 10 20 30 40 50 60 attcgaaagtgttcgggtcgcggggtaggttctccggtgtacgagcgcc 80 90 100 110 120 gtttcggcttttccggggactccggaggccgcagtgatcctcggggtcgggttccgttgg 130 140 150 160 170 180 gcgggtccacgtgcccgccgatccgcctagaaggagagctcccctcccgcttttgccctg 190 200 210 220 230 240 gagaatggctcagaaagagaacgtctacccgtggccctacggctcaaagacgtctcaatc MAQKENVYPWPYGSKTSQS 250 260 270 280 290 300 tggcctgaacaccttgccccagagagtcctacggaaggagcctgccgtgacacctgcaca GLNTLPQRVLRKEPAVTPAQ 310 320 330 340 350 360 ggccctcatgaaccggtccaacagccagtccacagctgtccctggtcagaagttgactga ALMNRSNSQ STAVPGQKLTE 370 380 390 400 410 420 gaacaagggtgccactgccttgcaaggatcccagagcaggcagcctttcaccattgacaa NKGATALQGSQSRQPFTIDN 430 440 450 460 470 480 ctttgagattgggcgtcctctgggcaaaggcaaatttggaaatgtgtacttggctcggga FEIGRPLGKGKFGNVYLARE 490 500 510 520 530 540 gaagaaaagccgtttcatcgtggcgctaaagatcctcttcaagtctcagattgaaaagga KKSRFIVALKILFKSQIEKE 550 560 570 580 590 600 gggggtggagcaccagctccgccgagagatcgaaatccaggcgcacctgaaacatcccaa GVEHQLRREIEIQAHLKHPN 610 620 630 640 650 660 tattcttcagctgtacaactacttctatgaccagcagaggatctacttgatactcgaata ILQLYNYFYDQQRIYLILEY 670 680 690 700 710 720 cgccccccgcggagagctctacaaggaactacagaagagcggaaccttcgatgagcagcg APRGELYKELQKSGTFDEQR 730 740 750 760 770 780 gactgccacgatcatggaggaactgtcagacgcgctgatgtactgccacaagaagaaggt TATIMEELSDALMYCHKKKV 790 800 810 820 830 840 gattcacagagacataaagcccgagaacctgctgttaggtctacagggagagctgaagat IHRDIP LLLGLQGELKI 850 860 870 880 890 900 tgcggactttggctggtctgtgcatgccccatccctgaggaggaagaccatgtgcggcac ADFGWSVHAPSLRRKTMCGT 910 920 930 940 950 960 cctggactatctgcccccagagatgattgaagggcggatgcataatgagatggtagatct LDYLPPEMIEGRMHNEMVDL 970 980 990 1000 1010 1020 gtggtgcattggagtgctctgctatgaactgatggtggggaacccaccctttgagagccc WCIGVLCYELMVGNPPFESP 1030 1040 1050 1060 1070 1080 tagccacagtgagacatatcgtcggattgtcaaggtagacctgaagtttccctcttctat SHSETYRRIVKVDLKFPSSM 1090 1100 1110 1120 1130 1140 gcctttgggggccaaggacctcatctccaagctgctcaaacataacccctcacaacgact PLGAKDLISKLLKHNPSQRL 1150 1160 1170 1180 1190 1200 gcctctggagcaggtctcggctcacccttgggtccgggccaactcacggagggttctgcc PLEQVSAHPWVRANSRRVLP 1210 1220 1230 1240 1250 1260 tccctctgccctttagcctgcttcttgatttttgttcctgtcatttctcagttttctttg PSAL * 1270 1280 1290 1300 1310 1320 tatgtctgtgtatgtgtcgtgagaaggggattagtgattggaaactatccctaaccccag 1330 1340 1350 1360 1370 1380 ttctagggaatcttattccttggggatcttattcctcttctgacctctacaggcaaaatt 1390 1400 1410 1420 1430 1440 gggcatacatgtcgtacacatatatgcaagccaaacatataaagttaaaaaacaaacagt 1450 1460 1470 1480 1490 1500 gctagagagatggcttggcagttaaaagcactggctactcttcccaagaaccaggagttc 1510 1520 1530 1540 1550 1560 aatttgagcactacaatggtgctcacaaccactgtctgtaacacctaattctggggtatc 1570 1580 1590 1600 1610 1620 gaggccttcaagcctctgcaggctagaggctaggatgtggtatcatcctatgcagggcaa 1630 1640 1650 1660 1670 1680 gacacccatgcacagaattttaaatcctctaaatgaaagaatttgtcaaatgttgaacgt 1690 1700 1710 1720 1730 1740 cattttaaaaatactataagccaaaaatgcatttaatacaatttttctctgaaacatggt 1750 1760 1770 1780 1790 1800 ttagcctactctgttttaaattcaggaaaattatgaagaataaagcatattttataataa 1810 1820 actcttaaatatttc <210> 3 <211> 7 <212> PRT <213> Unknown <440> 3 MHRDVKP <210> 4 <211> 7 <212> PRT <213> Unknown <440> 4 DFGVSQ > 5 <211> 20 <212> DNA <213> Artificial Sequence <440> 5 atgcaymgngaygtnaarcc <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <440> 6 tgnccn ganacnccraartc <210> 7 <211> 54 <212> DNA <213> Artificial Sequence <440> 7 aga gaa ttc atg gac tac aag gac gat gac gac aag atg gct cag aaa gag aac <210> 8 <211> 27 <212 > DNA <213> Artificial Sequence <440> ctt gaa gag gat cct tag cgc cac gat <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <440> atc gtg gcg cta agg atc ctc ttc aag <210 > 10 <211> 38 <212> DNA <213> Artificial Sequence <440> 10 ga ctc aga cta aag ggc aga ggg agg cag acg gcg cgc <210> 11 <211> 21 <212> DNA <213> Artificial Sequence < 440> 11 athcaymgngayathaarccn <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <440> 12 rtgnacngaccanccraart <210> 13 <211> 12 <212> PRT <213> Unknown <440> 13 NLLLGLKGELKI <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <440> 14 aatctgctcttagggctcaagggagagctgaagatt <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <440> 15 tccactaatatcggccacgcgtcgactagtac

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of AIM-1 obtained from the Drosophila-derived aurora gene and the yeast-derived IPL-1.
It is a figure in comparison with the amino acid sequence of the gene.

FIG. 2a shows AIM-1 cDNA obtained by ³² P-labeling poly (A) ⁺ RNA isolated from various rat tissues.
It is a figure (photograph of electrophoresis) of Northern blot analysis obtained by hybridizing with a fragment. FIG. 2b shows the AIM-
FIG. 2c shows the relationship between the expression pattern of 1 mRNA and the cell cycle (photograph of electrophoresis). FIG. 2c shows each time point of the cell cycle using an antibody against a peptide consisting of the C-terminal 13 amino acids of AIM-1 sequence. FIG. 2 is a diagram (photograph of electrophoresis) for analyzing changes in the amount of AIM-1 protein in NRK-49F cells in Example 1.

[FIG. 3] NRK-49F cells were prepared using the anti-AIM-1 polyclonal antibody prepared in Example 5 (left column in FIG. 3) and anti-α.
FIG. 4 is a diagram (photograph showing the form of an organism) obtained by staining with a tubulin monoclonal antibody (center column in FIG. 3) and DNA staining using the dye Hoechst33258 (right column in FIG. 3). 3a shows the interphase, b shows the early phase (prophase), c shows the middle phase (metaphase), d shows the late anaphase, and e shows the final phase (telophase).
f indicates cytokinesis (cytokinesis). FIG. 3g shows pUHD10-3 / FLAG-AIM prepared in Example 7.
-1 (WT) mink lung epithelial (Mv1Lu) cells were cultured in doxycycline-free medium for 24 hours, then anti-FLAG monoclonal antibody M2 (KODAK) (left column), rabbit anti-γ-tubulin antibody (middle column) ) And Ho
echst33258 (right column).

FIG. 4A shows wild-type FLAG-AIM-1 (W
T) or inactivated FLAG-AIM-1 (KR) with Mv
After induction in 1 Lu cells and removal of doxycycline (DOX), the cells were cultured for 18 hours, the resulting cells were collected, and the cell lysate was subjected to immunoblot analysis using an anti-FLAG monoclonal antibody (Electroblot analysis). Photo of electrophoresis)
It is. FIG. 4B shows only vectors (a, d), FLAG
-AIM-1 (WT) (b, e) or FLAG-AI
Asynchronous cells transformed with M-1 (KR) (c, f) were cultured for 72 hours in the presence (ac) or absence (df) of DOX, respectively, and stained with Giemsa solution. It is a figure (photograph showing the form of an organism). In FIG. 4B,
FIG. 4C, which shows a, b, c from left to right in the upper column and d, e, f from left to right in the lower column, collects the cell sample,
FIG. 2 shows cells fixed with ethanol, stained with propidium iodide, and subjected to FACS analysis.

FIG. 5D is a view showing the FLAG-AIM- shown in FIG. 4B.
Α obtained by superimposing each image of Mv1Lu cells expressing 1 (KR), α
-Shows double staining of tubulin (green) and DNA (red) (propidium iodide) (photograph representing the morphology of the organism). After removing doxycycline, dinuclear (a), 4
Abnormal cells having nuclei (b), eight nuclei (c) or ten or more nuclei appeared. FIG. 5E shows FLAG-AIM-1 (WT).
Alternatively, Mv1Lu cells transfected with FLAG-AIM-1 (KR) were cultured for 2 weeks in the presence or absence of DOX, and stained with Giemsa solution (photograph showing the form of an organism). .

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI C07H 21/04 C07K 14/47 C07K 14/47 16/18 16/18 C12N 1/19 C12N 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/68 A C12P 21/02 G01N 33/53 D C12Q 1/68 33/531 A G01N 33/53 A61K 37/48 ADU 33/531 C12N 5/00 B // ( (C12N 1/19 C12R 1: 865) (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1: 125) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:19) ( C12P 21/02 C12R 1: 865)

Claims (13)

[Claims] 1. A base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, or an amino acid sequence obtained by substituting, deleting or adding a part of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, A DNA comprising a nucleotide sequence encoding a protein having a regulatory activity, or a nucleotide sequence hybridizing thereto. 2. A recombinant vector containing the DNA according to claim 1. [3] A prokaryotic or eukaryotic host cell transformed with the recombinant vector according to [2]. 4. A method for producing a recombinant protein, comprising culturing the host cell according to claim 3, and isolating and purifying the produced protein. 5. The method for producing a recombinant protein according to claim 4, wherein the recombinant protein is a protein having a cell cycle regulating activity. 6. A recombinant AIM-1 obtained by separating and purifying a culture supernatant obtained by culturing the host cell according to claim 3.
protein. 7. An oligonucleotide or peptide nucleic acid capable of specifically hybridizing with a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 8. An oligonucleotide or peptide nucleic acid capable of hybridizing with a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing and inhibiting the expression of AIM-1 protein. 9. An antibody that recognizes a peptide having at least five consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 10. A therapeutic agent for a disease associated with abnormal cell proliferation, which comprises an inhibitor against AIM-1 protein. (11) the inhibitor of AIM-1 protein is AIM-1 (KR), an inhibitor of AIM-1 protein phosphorylation activity, the oligonucleotide or the peptide nucleic acid of (7), or the (8) of claim 8; The therapeutic agent according to claim 10, which is an antibody. 12. The therapeutic agent according to claim 11, wherein the disease associated with abnormal cell proliferation is cancer. 13. A method for screening a substance having serine-threonine inhibitory activity using the AIM-1 gene or AIM-1 protein.