JP2001190283A - New peptide for mammal and polynucleotide encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new peptide for mammals, in particular a peptide having the activity to inhibit cell carcinogenesis due to a proto oncogene H-ras or the proliferation of cells transformed by H-ras, and to provide a polypeptide encoding the above peptide. SOLUTION: The above-mentioned polypeptide (polynucleotide) encoding this new peptide (A) or (B) described below is isolated by performing a differential display using the whole RNA extracted from ATDC5 cells as a chondrogenic EC cell strain: (A) a peptide having a mouse- or human-derived specific amino acid sequence, or (B) a peptide having an amino acid sequence with at least one amino acid substituted, deleted, inserted or transferred in the above- mentioned amino acid sequence and also having the activity to inhibit cell carcinogenesis due to H-ras or the proliferation of cells transformed by H-ras.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a mammalian protein having a novel physiological activity, in particular, an activity of inhibiting cell carcinogenesis by H-ras, or inhibiting the growth of cells transformed with H-ras. A peptide having activity.

[0002]

BACKGROUND OF THE INVENTION Mammalian ras proto-oncogenes (proto-oncogenes) such as H-ras, K-ras and N-ras are expressed in various tissues (Oncogene, 1 (1),
47-58, 1987). For example, H-ras is highly expressed in skin and skeletal muscle, K-ras is highly expressed in intestine and thymus, and N-ras is highly expressed in testis and thymus. Ras protein is an endogenous GTPase that binds to guanine nucleotides and functions as a transducer of various physiological signals, including signals that control cell growth and differentiation.
Having activity (Annu. Rev. Biochem., 62, 851-891, 199)
3). As the biological activity of Ras protein, Krev
-1 (Cell, 56, 77-84, 1989) and H-rev107 (O
ncogene, 9, 479-490, 1994) and is known to be regulated by other guanine nucleotide-binding proteins. However, there are still many unclear points about the physiological functions of ras protein and these regulatory proteins in mammalian cells.

[0003] On the other hand, cartilage formation is important for the development of the vertebrate skeleton. The inventors of the present invention have previously described cartilage precursor cell-like (ch
Culture of ondroprogenitor-like EC cells (ATDC5) in the presence of insulin can induce a high incidence of chondrogenesis, and that ATDC5 cells leave a clear sign of chondrogenesis in vitro. Reported (J.
Bone Miner. Res., 11, 22-28, 1996; Biochem. Biophy.
s. Res. Connun., 235, 142-147, 1997; J. Cell Biol.,
133, 457-468, 1996; J. Bone and Mineral Res., 12,
1174-1188, 1997; Exp.Cell Res., 241, 1-11, 199
8). Also, embryonic fibroblast C3H10 of cloned mouse
T1 / 2 maintains the properties of pluripotent mesoderm-like ancestors and adipocytes, myoblasts, osteoblasts as well as chondrocytes (in the presence of 5-azacytidine or high-dose bone morphogenetic protein-2) Shows differentiation into cells (Cell, 17, 771-779, 1979; DNA Ce
ll Biol., 12, 871-880, 1993).

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above, and has as its object to provide a DNA encoding a peptide having a function of suppressing the proto-oncogene H-ras.

[0005]

Means for Solving the Problems In order to search for a gene that is specifically expressed in chondrogenic cells, the present inventors have investigated all Rs extracted from ATDC5 cells, a chondrogenic EC cell line.
As a result of performing differential display using NA, a novel gene that is differentially expressed was found, and the gene product had an activity of suppressing H-ras cell carcinogenesis and suppressing the growth of transformed cells. And completed the present invention.

That is, the present invention relates to the following peptide (A) or (B). (A) a peptide having the amino acid sequence of SEQ ID NO: 2; (B) in the amino acid sequence of SEQ ID NO: 2, having an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and in H-ras canceration of cells or H-ras A peptide having an activity of suppressing the growth of transformed cells.

[0007] The present invention also provides a polynucleotide encoding any of the following peptides (A) or (B). (A) a peptide having the amino acid sequence of SEQ ID NO: 2; (B) in the amino acid sequence of SEQ ID NO: 2, having an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and in H-ras canceration of cells or H-ras A peptide having an activity of suppressing the growth of transformed cells. Examples of the polynucleotide include polynucleotides shown in the following (a) or (b). (A) a polynucleotide having a base sequence represented by base numbers 160 to 660 of SEQ ID NO: 1; (B) hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence represented by nucleotide numbers 160 to 660 of SEQ ID NO: 1 and transfects with H-ras H-ras A polynucleotide encoding a peptide having an activity of suppressing the growth of a formed cell. The present invention also provides the following peptide (C) or (D). (C) a peptide having the amino acid sequence of SEQ ID NO: 14; (D) in the amino acid sequence of SEQ ID NO: 14, which has an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and is used for H-ras cell carcinogenesis or H-ras A peptide having an activity of suppressing the growth of transformed cells. The present invention also provides a polynucleotide encoding any of the following peptides (C) and (D). (C) a peptide having the amino acid sequence of SEQ ID NO: 14; (D) in the amino acid sequence of SEQ ID NO: 14, which has an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and is used for H-ras cell carcinogenesis or H-ras A peptide having an activity of suppressing the growth of transformed cells. Examples of the polynucleotide include polynucleotides shown in the following (c) or (d). (C) a polynucleotide having a base sequence represented by base numbers 402 to 905 of SEQ ID NO: 13. (D) hybridizing under stringent conditions with a polynucleotide having a nucleotide sequence represented by nucleotide numbers 402 to 905 of SEQ ID NO: 13 and transforming cells into cancerous cells with H-ras, or transforming with H-ras A polynucleotide encoding a peptide having an activity of suppressing the growth of a formed cell.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The peptide of the present invention is a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2. A peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is about 70% or more, preferably about 80% or more, of the amino acid sequence of SEQ ID NO: 2.
%, More preferably about 90% or more, most preferably about 95% or more, and has the same activity as the peptide of SEQ ID NO: 2. Another embodiment of the peptide of the present invention is a peptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 14. A peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 14 is about 70% or more, preferably about 80%
%, More preferably about 90% or more, most preferably about 95% or more, and has the same activity as the peptide of SEQ ID NO: 14. Examples of the activity as described above include an activity of suppressing canceration of cells by H-ras, an activity of suppressing proliferation of cells transformed with H-ras, or both. The activity of suppressing canceration of cells by H-ras includes
The activity of restoring the form of cancerous cells is also included. The degree of its activity can vary depending on the amino acid sequence of the peptide, but as long as such biological activity has utility, the amino acid sequence of the peptide of the present invention is SEQ ID NO: 2
Or, it may be different from the amino acid sequence represented by 14.

Specifically, the peptide of the present invention comprises one or more, preferably 1 to 10, more preferably 1 to 5, and further preferably 1, 2, 3 amino acids in the amino acid sequence of SEQ ID NO: 2 or 14. Or a peptide having an amino acid sequence in which four amino acids have been substituted, deleted, inserted or transferred, or a fragment thereof, and an analog thereof. The fragments and analogs are obtained by substituting, deleting, inserting or transferring amino acids at sites not important for the function of the peptide of the present invention. Amino acids at sites important for peptide function cannot be substituted. Others
The sites involved in preserving the overall shape of the peptide in relation to its biological activity cannot be altered by amino acid deletions, substitutions or insertions.

[0010] Substitutable amino acids are usually of similar size or polarity. Examples of such are the aliphatic amino acids Ala, Val, Leu and Ile.
Mutual substitution between Ser and Thr having a hydroxyl group, mutual substitution between Asp and Glu having an acidic residue, substitution between Asn and Gln having an amide residue, having a basic residue A substitution between Lys and Arg,
Substitution between Phe and Tyr having an aromatic residue is mentioned.

[0011] The precursor of the peptide of the present invention is also included in the peptide of the present invention as long as it has the above-mentioned physiological activity. Examples of such a precursor include those obtained by adding one or more amino acids to the N-terminal and / or C-terminal of the peptide of the present invention.

Furthermore, the peptide of the present invention may be conjugated to polyethylene glycol to increase the half-life, conjugated to a secretory sequence or a leader sequence for secretion, or produced as a fusion peptide for purification. be able to.

Also useful are fragments characterized by the structural or functional properties of the peptides of the invention.
Physiologically acceptable acid addition salts of the peptide of the present invention or its precursor are also included in the present invention. Examples of such acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid,
Examples thereof include salts with organic acids such as acetic acid, formic acid, fumaric acid, maleic acid, succinic acid, citric acid, tartaric acid, malic acid, benzoic acid, and benzenesulfonic acid.

The peptides and polynucleotides of the present invention are in an isolated form, and preferably have been purified to a uniform degree. The polynucleotide of the present invention,
A polynucleotide encoding the peptide of the present invention. Specifically, as the polynucleotide of the present invention,
A polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 is included. As such a polynucleotide, specifically, base numbers 160 to 660 of SEQ ID NO: 1
And a codon may be replaced with another equivalent codon as long as it encodes the same amino acid sequence.

The polynucleotide of the present invention has SEQ ID NO: 2.
May encode a peptide having an amino acid sequence substantially identical to the amino acid sequence represented by As such a polynucleotide, the nucleotide sequence represented by nucleotide numbers 160 to 660 of SEQ ID NO: 1 and at least 8
Those having 0% identity and those complementary thereto are preferred. Furthermore, polynucleotides having 90% identity are preferred, and those with 95% or more identity are most preferred. A polynucleotide having a sequence having an identity of 95% or more, preferably 97% or more, can be used under the stringent conditions under the conditions of base number 160 of SEQ ID NO: 1.
Hybridizes with a polynucleotide having a base sequence represented by ６660 or a probe that can be prepared from the DNA. Another example of the polynucleotide of the present invention is a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 14. As such a polynucleotide, specifically, base number 402 of SEQ ID NO: 13
Polynucleotides having a base sequence represented by ９０905 are exemplified, but individual codons may be replaced with other equivalent codons as long as they encode the same amino acid sequence. The polynucleotide of the present invention may encode a peptide having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 14. As such a polynucleotide, a nucleotide sequence represented by nucleotide numbers 402 to 905 of SEQ ID NO: 13 and at least 8
Those having 0% identity and those complementary thereto are preferred. Furthermore, polynucleotides having 90% identity are preferred, and those with 95% or more identity are most preferred. A polynucleotide having a sequence having an identity of 95% or more, preferably 97% or more, has a nucleotide number of 40
It hybridizes with a polynucleotide having a base sequence represented by 2-905 or a probe which can be prepared from the DNA. The nucleotide sequence shown in SEQ ID NO: 1 is the nucleotide sequence of the DNA of the present invention named A-C1 derived from mouse, and the nucleotide sequence shown in SEQ ID NO: 13 is the A-C1 derived from human.
This is the nucleotide sequence of the homolog of the gene. These nucleotide sequences have 83% homology at the protein level.

The present invention further includes polynucleotides encoding fragments characterized by the structural or functional properties of the peptides of the present invention. Such a polynucleotide fragment and a polynucleotide that hybridizes to a polynucleotide encoding the fragment are also useful as a primer for PCR or as a probe for detecting DNA encoding the peptide of the present invention.

The DNA encoding the peptide of the present invention is:
For example, chondrogenic EC cell lines (eg, ATDC5 cells),
Poly (A) ⁺ R from rat skeletal muscle, heart or brain hippocampus
It can be cloned from a cDNA library obtained by reverse transcription of NA. Similarly, a homolog of the gene encoding the peptide of the present invention can be cloned from a mammal other than a human.

Specifically, using a synthetic DNA primer having a partial base sequence of the DNA encoding the peptide of the present invention, a target DNA is amplified from the above rat hippocampus cDNA library or the like by PCR, and this is amplified. Subcloning into vector. A new clone is selected and its DNA sequence is determined. A full-length cDNA is cloned using a primer synthesized based on this sequence.

In addition, DN incorporated into an appropriate vector
DNA encoding the peptide of the present invention can be cloned from A by using a DNA fragment encoding a partial or entire region of the peptide of the present invention or a labeled synthetic oligonucleotide as a probe.

The preparation of poly (A) ⁺ RNA from cells involves:
First, total RNA can be extracted and then purified from the total RNA using a carrier for purifying poly (A) ⁺ RNA such as oligo (dT) cellulose. As a method for preparing total RNA, a guanidine thiocyanate / phenol method, a guanidine thiocyanate / trifluorocesium method, an alkali sucrose density gradient centrifugation method, a method using guanidine thiocyanate and cesium chloride are preferable.

The poly (A) ⁺ R obtained as described above
Using NA as a template and using, for example, a 6-base random oligonucleotide as a primer, a single-stranded cDNA is synthesized by a reverse transcriptase, and then a double-stranded cDNA is synthesized by a DNA polymerase. About 200 bp of DN obtained
A was cut with restriction enzymes EcoRI and XhoI, and pBlues
The resulting D was subcloned into the cript IISK vector.
Transform E. coli with NA. Transformation of Escherichia coli can be carried out efficiently using the Hanahan method (J. Mol. Biol, 166, 557-580 (1983)). Plasmid DNA was purified from 100 clones by miniprep plasmid purification (Sambrook et al., MOLECULAR CLONING, A LABORATORY MANU
AL, 2nd Ed; Cold Spring Harbor Laboratory Press, C
Purification by old SpringHarbor, New York (1989) 1.25) and determination of the DNA sequence will select one new clone. Using a primer synthesized based on the sequence of this clone, a full-length cDNA is synthesized.

A polynucleotide encoding a mutant peptide having an amino acid sequence in which one or several (one to several) amino acids have been substituted, deleted, inserted or transferred in the amino acid sequence represented by SEQ ID NO: 2 or 14 Can be easily synthesized from a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or 14 by known in vitro mutagenesis. Known kits, for example, Mutan ^™ -K
(Takara Shuzo Co., Ltd.) and Mutan ^™ -G (Takara Shuzo Co., Ltd.) can be used to easily prepare a polynucleotide encoding the above mutant peptide. Further, the phosphate enzyme activity of the mutant peptide having the amino acid sequence represented by SEQ ID NO: 2 or 14 encoded by the obtained polynucleotide encoding the mutant peptide can be easily detected. The range of peptides included in the invention can be easily determined.

A recombinant vector containing the polynucleotide of the present invention is used for producing the peptide of the present invention.
As the vector of the present invention, those suitable for maintaining, propagating or expressing the polynucleotide in the host cell are used. Cloned D encoding the peptide of the invention
NA is inserted into a vector as it is, or digested with a restriction enzyme or added to a linker. The DNA
Has a translation initiation codon (ATG) at its 5 'end and a translation stop codon (TAA, TGA) at its 3' end.
Or TAG). The DNA is located downstream of the promoter in the expression vector. Such vectors include E. coli-derived plasmids (pBR322, p
Plasmids such as BR325, pUC12), plasmids derived from Bacillus subtilis (pUB110, pC194), plasmids derived from Streptomyces sp., Plasmids derived from Salmonella sp. bacteriophage such as pYAC-type plasmid), λ phage,
Vectors derived from viruses such as vaccinia virus, adenovirus, retrovirus, and baculovirus are used, and many of these vectors are commercially available.

The DNA sequence in the recombinant vector is operably linked to an appropriate expression control sequence (promoter). Such promoters include phage λ
PL promoter, T7 promoter, E. coli la
c, trp, lpp and c promoters, Bacillus spO1 promoter, penP promoter, yeast PHO5 promoter, PGK promoter, GA
P promoter, ADH1 promoter, SUC2 promoter, GAL4 promoter, Mfα promoter, insect cell polyhedron promoter, P10 promoter, SV40 early and late promoter for animal cells,
Examples include the retrovirus LTR promoter, CMV promoter, HSV-TK promoter, metallothionein promoter, 35S promoter for plant cells, rice actin gene promoter, and the like.

In general, an expression vector contains a repressor binding site and an expression control region which is operated by an enhancer or the like. In addition, the expression vector contains a selection marker. Suitable markers are the dihydrofolate reductase (dhfr) gene for eukaryotic cells, the neomycin resistance gene, the tetracycline or ampicillin resistance gene for bacteria, and the like. The dhfr gene confers methotrexate resistance to transformed cells, and the neomycin resistance gene confers G418 resistance on transformed cells. d
When the hfr gene-deficient CHO cells are used as a host and the dhfr gene is used as a selection marker, transformants can be selected in a thymidine-free medium. In this case, by gradually increasing the concentration of methotrexate (MTX) and culturing, and selecting a resistant strain, the DNA encoding the peptide of the present invention is amplified in the cell simultaneously with the dhfr gene, and high expression of CHO ( dhfr ^-) cells are obtained.

The recombinant vector of the present invention is constructed so as to add a signal sequence to the N-terminal side of the peptide, if necessary. Such a signal sequence is a signal sequence of PhoA, OmpA or the like in the case of an E. coli host, an Mfα or SUC2 signal sequence in the case of a yeast host, or α- in the case of an animal cell host. Interferon signal sequence and the like.

The present invention also relates to a host cell containing the above-mentioned recombinant vector. The host cells are mammalian cells, plant cells, insect cells, yeast cells, eukaryotic cells such as Aspergillus, and prokaryotic cells such as bacterial cells. Calcium phosphate transfection, electroporation,
The recombinant vector of the present invention is introduced into host cells by transduction, infection or other methods.

Under the control of the above-mentioned promoter, the peptide of the present invention can be expressed in the above-mentioned hosts such as mammalian cells, yeasts and bacteria. Examples of prokaryotic hosts are Escherichia coli, Bacillus subtilis, Salmonella, Pseudomonas, Streptomyces, Staphylococcus and the like. Examples of yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like.

The transformed prokaryotic host is propagated and, in the case of a vector containing an inducible promoter, is induced by temperature or a chemical inducer, and the cells are converted to a carbon source (glucose, dextran, soluble starch, etc.), a nitrogen source. In a liquid medium containing (ammonium salts, nitrates, peptone, casein, meat extract, soybean meal, etc.) and inorganic substances (calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc.), an appropriate pH (pH of about 5 to 5). In step 8), culture for an appropriate time (about 3 to 24 hours). Suitable cultivation temperatures are about 14 to 43 ° C. for Escherichia coli and for Bacillus,
About 30-40 ° C. After the culture, the cells are disrupted by a physical or chemical method, and the peptide of the present invention is purified from the obtained crude extract.

The transformed yeast is cultured in a medium such as a minimal medium at a pH of about 5 to 8 and a temperature of about 20 to 35 ° C. for about 24 to 7 hours.
Incubate for 2 hours. As insect cells, AcNPV (Au
tographacalifornicaNPV)
Sf cells, MG1 cells and the like are used, and when using silkworm polynucleotic disease virus (BmPV) as a virus, silkworm larvae and silkworm cultured cells (BM-N cells) are used. Silkworm cells are made subconfluent at about 27 ° C. using a TC-10 medium containing 10% heat-inactivated fetal bovine serum and then subcultured.

Examples of mammalian cells include COS-7 cells, mouse AtT-20 cells, rat GH3 cells, rat Mt
T cells, mouse MIN6 cells, Vero cells, C127
Cells, CHO cells, dhfr gene-deficient CHO cells, H
eLa cells, L cells, BHK cells, BALB3T3 cells, 293 cells, Bowes melanoma cells and the like.

The mammalian cell expression vector comprises an origin of replication,
Promoters (the above SV40 early and late promoters, retroviral LTR promoter, CMV promoter, HSV-TK promoter, metallothionein promoter, etc.), enhancers (SV40 enhancer, adenovirus enhancer, cytomegalovirus early promoter, etc.), selectable markers (above) dh
fr gene, neomycin resistance gene, etc.), ribosome binding site, polyadenylation site (SV40 polyadenylation site, etc.), splice donor and acceptor sites (DNA sequence derived from SV40 splice site), transcription termination sequence and 5 'non-transcription sequence .

As such a vector, a plasmid vector, a single-stranded or double-stranded phage vector, a single-stranded or double-stranded RNA or DNA virus vector and the like are used. Culture of the transformed mammalian cells includes MEM medium containing about 5 to 20% fetal bovine serum, DM
An EM medium, an RPMI 1640 medium, or the like is used, and the culture is performed at a pH of about 6 to 8 and a temperature of about 30 to 40 ° C. for about 15 to 72 hours.

To introduce a gene encoding the peptide of the present invention into plant cells, a Ti plasmid present in Agrobacterium tumefaciens is used. The foreign gene is inserted into a 25 bp repeat sequence at both ends of the T-DNA of the Ti plasmid. The A. rhizogenes Ri plasmid can be used as well. When T-DNA is introduced into plant cells, a group of genes called a vir region is important. T- by activated vir gene
The DNA produces single-stranded DNA (T-strand), which integrates into the chromosome in plant cells. The target gene can be introduced into the Ti plasmid by co-integration. In addition, the Ti plasmid can be introduced into Agrobacterium by a binary method in which the T-DNA and the vir region are present on separate plasmids. Next, the plant is infected with Agrobacterium containing the target gene,
The gene is introduced into a plant.

When a mammalian cell is used as a host, the peptide of the present invention can be recovered and purified from a recombinant cell culture by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, or the like. The peptides of the present invention need not be glycosylated. Also, depending on the host, N
A peptide having a methionine terminal is obtained. See Sambrook et al., MOLECULAR CLONING, AL
ABORATORY MANUAL, 2nd Ed; Cold Spring Harbor Labor
atory Press, Cold Spring Harbor, New York (1989).

Since the peptide of the present invention is considered to be involved in information transmission in the brain, it can be used for treating cerebral nervous system diseases. Further, the substance that activates the peptide of the present invention includes:
It is considered to be useful for maintaining and activating nerve activity.
Further, the peptide of the present invention is expected to be useful for treating cancer.

[0037]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the scope of the present invention. Unless otherwise noted,
The methods of the following examples were performed by standard methods described in the experimental manual of Sambrook et al., Supra.

[0038]

Example 1 <1> Culture of Various Cells The cells used in this example were cultured as follows.
ATDC5 cells were seeded on a 6-multiwell plate at an initial density of 6 × 10 ⁴ cells / well, and
e Miner. Res., 11, 22-28, 1996 and Biochem. Biophys.
The cells were cultured according to the method described in Res. Commun., 235, 142-147, 1997.

C3H10T1 / 2 cells, NIH3T3 cells, Balb / c3T3 cells and C2C12 cells (all obtained from RIKEN cell bank) have an initial density of 6 × 1.
Seed in a 6-multiwell plate at a density of 0 ⁴ cells / well, DME containing 10% FBS (fetal bovine serum)
The cells were cultured in M medium for 3 days. MC3T3-E1 cells and ST2 cells were seeded in a 6-multiwell plate at an initial density of 6 × 10 ⁴ cells / well, and 10%
The cells were cultured in an αMEM medium containing FBS for 3 days.

Cultured adherent cells derived from bone marrow were treated with 6-week-old IC.
From R mouse (purchased from SRL), J-Cell-Physiol,
122, 81-90 issn: 0021-9541, based on the method described in 1985
Prepared. At that time, the tibia and thigh are incised and the bone ends
The ends were excised and the bone marrow was DMEM / Ha containing 10% FBS.
Washed off with m's F12 mixed medium. Medullary cells are
And disperse it in a 6-multiwell plate.
Sown (2 × 10 ⁷ cells / well). 48 hours later, medium
Replace to remove non-adherent cells and keep adherent cells humidified
5% CO_Two/ 95% air at 37 ° C for 5 days, 1 day
Then, the medium was replaced and the cells were cultured.

<2> Search for a gene specifically expressed in chondrogenic cells In order to search for a gene specifically expressed in chondrogenic cells, total RNA extracted from ATDC5 cells, a chondrogenic EC cell line, was used. 500b corresponding to the 3 'untranslated sequence of a novel gene differentially expressed as A-C1
A cDNA fragment of p was obtained.

Specifically, total RNA was obtained from Biochem. Bioph.
ys. Res. Commun., 235, 142-147, 1997, C3H10T1 / 2 cells and AT
It was isolated from DC5 cells and analyzed using differential display (RNAmap ^™ , manufactured by GenHunter). As a result, about 5 expressed only in undifferentiated ATDC5 cells.
A 00 bp DNA fragment was identified and its sequence was
The analysis was performed using an ALFred DNA Sequencer (Amersham Pharmacia Biotech).

Using the above cDNA fragment, AT
A mouse cDNA library prepared from DC5 cells was screened, and the full-length A-C1 gene was cloned. Poly (A) ⁺ R derived from undifferentiated ATDC5 cells
CDNA is synthesized from NA using oligo (dT),
A library was constructed by ligating to a λZAP expression vector (Stratagene). 1 × 10 ⁶ of this library
Individual plaques were screened by plaque hybridization. The probe is 5
The 00 bp fragment was amplified by PCR, and the obtained DNA fragment was labeled with [ ³² P] -label using a BcaBEST labeling kit (manufactured by Takara Shuzo Co., Ltd.), and the 6 × SSP
E, 0.2% BSA (bovine serum albumin), 0.2
% Ficoll 400 (Pharmacia), 0.2%
Polyvinylpyrrolidone, 0.1% SDS, 100 μg
/ Ml denatured salmon sperm DNA with a membrane (137 m
The hybridization was carried out at 42 ° C. for 16 hours on plaques transferred to a nylon membrane (DuPont NEN). Thereafter, the membrane was heated at 55 ° C. for 0.1
Washed off with × SSPE and 0.1% SDS.

As a result, 10 cDNA clones were obtained from 1 × 10 ⁶ independent plaques. 8 of them
Clones contained approximately 3.0 kb of cDNA. SEQ ID Nos. 1 and 2 in the sequence listing include A-C1 cDNA
And the amino acid sequence deduced therefrom are shown. A-C1 cDNA has a 501 bp open reading frame (OR) starting from the ATG codon.
F). The sequence around the ATG codon is the Kozak consensus sequence (Kozak, M. Nucleic
Acids Res., 15 (20), 8125-8148 (1987)). Therefore, the A-C1 protein was estimated to be composed of 167 amino acid residues. The calculated molecular weight of the A-C1 protein was 18,809, and the isoelectric point was 6.1. At the 3 'end of the sequence, poly (A)
It contained the sequence followed by a polyadenylation signal (AATAAA).

The Kyte-Doolittle algorithm (J. Mol. Bi
157, 105-132, 1982) showed the presence of a transmembrane domain at the C-terminus (143-159 amino acid portion). The amino acid sequence lacked the N-terminal signal peptide. These results indicate that A-C1 cD
This suggested that NA encodes an intracellular, membrane-bound protein. The amino acid sequence of the A-C1 protein was determined to be the class II tumor suppressor rat H-rev (Onco
gene, 9, 479-490, 1994) and 46% homology at the amino acid level.

<3> Confirmation that A-C1 cDNA contains ORF The 3.0 kb A-clone cloned as described above.
To confirm that the C1 cDNA contained the ORF, in vitro transcription / translation was performed using a rabbit reticulocyte lysate system in the presence of [ ³⁵ S] -methionine, and the translation product was analyzed.

The transcription / translation reaction was performed in the presence of [ ³⁵ S] -methionine (Amersham) in the presence of a rabbit reticulocyte lysate system (TnT Coupled Transcription / Translation Sy).
stems, Promega). pcDNA3.
3.0 kb A-C1 cDNA (pCMV-AC1) cloned into one vector (Invitrogen)
Was used as a template for transcription. The translation product is 10
Electrophoresed on 20% polyacrylamide gel, X-Om
Detection was performed by autoradiography using an at film (Kodak, exposed at room temperature for 8 hours).

As a result, a single protein product having the expected size was detected, whereas nothing was detected in the vector control (pcDNA3.1). This confirmed that the A-C1 cDNA contained the ORF.

<4> Analysis of cell localization of A-C1 protein To analyze the cell localization of A-C1 protein, COS
-7 cells were transformed with A- having FLAG-tag sequence at the C-terminal.
The pcDNA3.1 plasmid containing the C1 cDNA (p
(CMV / A-C1FLAG) and immunostained with anti-FLAG M2 monoclonal antibody.

A-C1 having FLAG-tag at C-terminal
The DNA fragment encoding the protein is 3.0k
It was prepared by amplifying by PCR using the A-C1 cDNA fragment of b as a template and primers having the nucleotide sequences of SEQ ID NO: 3 (sense primer) and SEQ ID NO: 4 (antisense primer) in the sequence listing. The antisense primer was FLG-t
ag (from the fourth base C to the 27th C in SEQ ID NO: 4)
Up to 24 bases) and a stop codon.

Amplification was performed after initial denaturation at 94 ° C. for 5 minutes, followed by 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 3 seconds.
The reaction of 0 seconds was repeated 25 times. The PCR product was gel purified and cloned into the pCRII vector (pC
RII / A-C1FLAG). Cloned PC
The base sequence of the R product was confirmed by ALFred DNA Sequencer. The insert in pCRII / A-C1FLAG was S
It was excised with peI and XhoI and recloned into the SpeI-XhoI site of pcDNA3.1 vector. Expression vector pCMV / A-C1 thus obtained
FLAG and control, pcDNA3.1
By Lab-Tec Chambe by lipofection using SuperFect Transfection Reagent (manufactured by Qiagen GmbH).
COS-7 cells cultured on r Slide (Nalge Nunc International) were transfected.

Two days later, the transfected cells were fixed with 4% paraformaldehyde and
n Permeabilized with X-100. Then, the cells were cultured at room temperature for 20 minutes with the diluted normal blocking serum. The cells were cultured for 30 minutes at room temperature with anti-FLAG M2 monoclonal antibody. The bound antibody was detected using Vectastain ABC Kit (Vector Laboratories). After rinsing three times with PBS, DAKO
1 using AEC Substrate System (DAKO Corp.)
Stained for 0 minutes.

As a result, A-C1 was detected by immunocytochemical staining using an anti-FLAG M2 monoclonal antibody.
The protein was found to be localized in the cytoplasm and perinuclear region, but not in the nucleus. COS-7 cells transfected with the pcDNA3.1 vector did not stain at all.

<5> AC1 mRN in various cells
Analysis of the expression of A By Northern analysis and RT-PCR,
TDC5, C3H10T1 / 2, MC3T3-E1, N
IH3T3, Balb / c3T3, ST2 and C2C1
2 was examined for A-C1 mRNA expression.

(1) Analysis by Northern Hybridization ATDC5 cells and C3H10T1 / 2 cells were seeded on a 6-multiwell plastic plate and cultured as described above. Isolate total RNA from these cells,
Biochem. Biophys. Res. Commun., 235, 142-147, 1997.
The analysis was carried out by Northern hybridization as described in (1). That is, poly (A) ⁺ RNA (5 μg)
, And separated by 1% agarose gel electrophoresis.
It was transferred onto a tran membrane (Schleicher & Schuell). A 3.0 kb cDNA fragment of A-C1 was used for hybridization as a probe.

In analyzing the tissue distribution of mature mice, the labeled cDNA was used for mouse multiple tissue
It hybridized to northern blot (made by Clontech). After the hybridization, the membrane is replaced with Cronex lightning plus intensifying screens (DuPo
using an X-Omat film (Kodak) at -80 ° C.
(Made by the company).

As a result, a major hybridization band of about 3.2 kb was detected in chondrogenic ATDC5 cells, but not in C3H10T1 / 2 cells.

As a result of Northern analysis, A-C1 mRNA was strongly expressed in skeletal muscle in tissues of mature mice,
It was moderately expressed in heart and brain.

(2) Analysis by RT-PCR RT-PCR was performed using Biochem. Biophys. Res. Commun., 23
5, 142-147, 1997. That is, using 5 μg of total RNA extracted from various cultured cell lines as a template, SuperScript II RNase H-reverse transcriptase
(GIBCO BRL) using the first strand cDNA
Was synthesized in vitro. SEQ ID NO: 5 (sense primer) and SEQ ID NO: 6 (antisense primer) were used as primers for amplifying mouse A-C1 cDNA. Amplification was performed after initial denaturation at 94 ° C for 5 minutes, followed by 94 ° C: 30 seconds, 60 ° C: 30 seconds, and 72 ° C:
The reaction for 30 seconds was repeated 25 times.

Each PCR product (8 μl) was used together with a marker in a 3% NuSieve 3: 1 agarose gel (FMC BioProd).
ucts). The amplified PCR product is pC
Subcloned into an RII vector (manufactured by Invitrogen), and the sequences of all cDNA fragments were
Confirmed with NA Sequencer (Amersham Pharmacia Biotech).

As a result, the expression of A-C1 mRNA was
It was detected by C2C12 and ATDC5, but MC3T3
-E1, NIH3T3, Balb / c3T3 and ST2
Could not be detected. The expression of A-C1 mRNA was also detectable in cultured bone marrow-derived adherent cells.

(3) In Situ Hybridization Brains and tibias of male 5-week-old ICR mice were collected and fixed overnight at 4 ° C. with 4% paraformaldehyde in 10 mM PBS (pH 7.4). The tibia was decalcified in 10% EDTA for 4 days. These were dehydrated with a concentration series of ethanol and embedded in paraffin. 6 μm sections were subjected to in situ hybridization according to the method described in Anat Rec, 229, 462-472, 1991.

As a probe, a 3.0 kb mouse A-
C1 cDNA having a specific activity of 3.0 to 5.0 × 10 ⁸ cp
[ ³⁵ S] -dTTP (DuPont Biotechnology Syate) by nick translation so as to obtain m / μg.
(manufactured by MS Co., Ltd.). After hybridization, the slide was washed, and the dried tissue section was immersed in NTB-2 emulsion (Kodak),
Exposure was performed at 4 ° C. for 7 days. Sections were counterstained with hematoxylin. The specificity of the cDNA probe was determined by AT
This was confirmed by Northern blot analysis using total RNA extracted from DC5 cells.

As a result, A-C1 mRNA was localized in the hippocampus and bone marrow of adult mice. Prior to in situ hybridization with the cDNA probe, RN
Sections pretreated with ase showed no signal.
This indicates that the hybridization signal depends on the presence of RNA.

<6> Biological activity of A-C1 protein The amino acid sequence of A-C1 is similar to that of rat H-rev107.
Had homology. This is because A-C1 is ras
Induces reverted NIH3T3 cells
Suggest that it may have some activity. There
And H-ras ^12VOr a tiger with the vK-ras oncogene
Transformed NIH3T3 cell line (L
ras / NIH, DT) with the A-C1 gene.
By transfecting and observing the morphology, AC
The activity of one protein was examined.

Lras / NIH cells are human H-ras
A derivative of NIH3T3 containing the ^12V oncogene (unpublished), DT is NIH3T3 (HGPR) containing two copies of the Kirsten murine comba virus provirus.
T ^-...) Is a derivative strain of (Proc.Natl Acad Sci, 80, 56
02-5606, 1983).

Lras / NIH cells and DT cells were
0% FBS, 0.03% L-glutamine, 100 μg
The cells were cultured in DMEM (hereinafter, abbreviated as “growth medium”) supplemented with 100 μg / ml penicillin G and 100 μg / ml streptomycin sulfate. The transfection protocol followed the method described in Cell, 56, 77-84, 1989. pCMV-AC1, pcDNA3.1 or pKr
2.5 μg of ev-1 (Krev-1 expression vector; positive control), 1.5 μg of pSV-BSD (blasticidin S resistance vector), and 1 μg of sheared calf thymus DNA as a carrier were used.

Lras / NIH cells and DT cells (3 ×
10 ⁵ ) was seeded on a collagen-coated 35 mm diameter dish the day before co-transfection. The DNA-CaPO ₄ co-precipitate is formed in 100μl, Proc. Natl. Acad. Sci. USA, 76, 1373-1376 (1
979) and added to cells covered with 1.5 ml of growth medium. 12-1 in a CO ₂ incubator
After incubating at 37 ° C. for 6 hours, the cells were washed with 1 × DM.
Treated with 1 ml of a solution containing 25% glycerol in EM for 1 minute and rinsed with DMEM. Then, 2 ml of growth medium was added again. The next day, cells were trypsinized and
The cells were seeded on a 100 mm-diameter dish containing 15 ml of a growth medium containing g / ml blastidine-S (Funakoshi). The medium was changed the next day and after 4 days.

Four days after selection with blastidine-S, the total number of colonies was counted using a phase contrast microscope. The number of flat colonies was counted the next day. To generate a growth curve, the pCMV-AC1 vector or pcDN
Lras / transfected with A3.1 vector
NIH cells are seeded in 24-multiwell plates,
The cells were cultured in a growth medium containing 8 μg / ml of blastidine-S. Cells were rinsed twice with PBS, treated with trypsin, and counted with a hemocytometer.

As a result, Lras / NIH cells were transformed into pCM
When transfected with V-AC1, the size of colonies observed by phase contrast microscopy was relatively smaller than that of control cultures transfected with pcDNA3.1 vector. Furthermore, these colonies were composed of flat cells. On the other hand, pCMV-AC1
It did not affect T cell morphology. The total number of transfected colonies is based on the pCMV-AC1 vector or pCMV-AC1 vector.
Lr transfected with cDNA 3.1 vector
Similar between cultures of as / NIH cells. In contrast, Lras / NI transfected with pKrev-1 having a Krev-1 transform repressor gene
In both H and DT cells, an increased percentage of flat colonies was observed.

A-C1 overexpression was induced by Lras / NIH
In addition to suppressing the formation of a cancerous form (reconstructing from the cancerous form), the proliferation of cells was also suppressed. Transfection of Lras / NIH cells with the A-C1 gene inhibited cell growth. The time for Lras / NIH cells transfected with A-C1 and Lras / NIH cells transfected with the vector doubled was ２０20 hours and ３４34 hours, respectively.
However, the saturation density was similar for both (両 者 2.2 × 1).
0 ⁵ cells / cm ² ).

These facts suggest that AC1 may function as a negative regulator of H-ras-mediated signaling.

[0073]

Example 2 <1> Culture of Various Cells The cells used in this example were cultured as follows.
Human osteosarcoma-derived U ₂ OS cells have an initial density of 6 × 10 ⁴
Cells were seeded at 6 cells / well in a 6-multiwell plate, and cultured in a DMEM medium (manufactured by ICN Pharmaceuticals) containing 5% FBS (fetal calf serum) for 3 days.
RCC-K1 derived from human renal cancer (Aoki et al., Cancer 6
2, 98-104, 1988) has an initial density of 6 × 10 ⁴ cells / wel
Seed 6-multiwell plate so that l becomes 5%
DMEM / Ham's containing FBS (fetal bovine serum)
The cells were cultured in an F12 mixed medium (manufactured by ICN Pharmaceuticals, Inc) for 3 days.

<2> Extraction of RNA and RT-PCR Total RNA was extracted by a single-step method,
l., Biochem. Biophys. Res. Commun., 235, 142-147,
Analysis was performed by RT-PCR according to the method described in 1997. That is, using 5 μg of total RNA extracted from U ₂ OS cells, ROC-K1 cells and human bone cells as a template, Su
perScript II RNase H-reverse transcriptase (GIBCO
The first strand cDNA was synthesized using BRL). In the PCR, primers based on mouse A-C1 SEQ ID NO: 7 (sense primer) and SEQ ID NO: 8
(Antisense primer) was used. Amplification at 94 ° C
After initial denaturation for 5 minutes at 94 ° C. for 30 seconds, 60
The reaction of 30 ° C for 30 seconds and 30 seconds for 72 ° C was repeated 25 times. Each PCR product (8 μl) was analyzed on a 3% NuSieve 3: 1 agarose gel (FMC BioProducts). The single 128 bp fragment was transferred to the pCRII vector (Inv
subcloned into ALFred DNA Sequ
The nucleotide sequence was determined using encer (Amersham Pharmacia Biotech).

<3> Isolation of human A-C1 cDNA The entire coding sequence of human A-C1 cDNA was obtained from Marathon cD
NA Amplification kit (Clontech) and synthetic oligonucleotide primers as primers; SEQ ID NO: 9
RACE (rapid amplification of cDNA ends) using (gene specific primer 1), SEQ ID NO: 10 (gene specific primer 2), SEQ ID NO: 11 (gene specific primer 3) and SEQ ID NO: 12 (gene specific primer 4)
Method (Frohman et al., Proc. Natl. Acad. Sci., 85, 89
98-9002, 1988). Human A-C1 cDN
To amplify the 5'- and 3'-ends of A, human RC
An uncloned library of cDNA prepared from C-K1 and with an added adapter was used. 5'-
And 3′-RACE cDNA products were subcloned into a pCRII vector (Invitrogen), and ALFred
The nucleotide sequence was determined using a DNA Sequencer (Amersham Pharmacia Biotech).

The nucleotide sequence of human A-C1 cDNA and the deduced amino acid sequence are shown in SEQ ID NOS: 13 and 14, respectively.
Shown in The human A-C1 cDNA contains an open reading frame of 555 nucleotides encoding 168 amino acids. The sequence near the first ATG is
Kozak reports the start methionine codon (Koza
k, Nucleic Acids Res., 15, 8125-8148, 1987). The amino acid sequence of human A-C1 protein shows 83% homology with mouse A-C1 (FIG. 1).
In addition, human A-C1 has two consensus sequences, DXXG (SEQ ID NO: 15) and NKXD (SEQ ID NO: 16). As already reported, DXXG is a GT
NKXD is important for the binding of guanine rings, involved in binding to MG ²⁺ and γ-phosphate with P attached (Bourne et al, Nature, 346, 117-127, 1991). These data suggest that human A-C1 may be involved in the ras transduction pathway.

<4> Northern Analysis Northern analysis was performed to analyze the expression and distribution of human A-C1 in normal mature tissues. Various human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, stomach, thyroid, spinal cord, lymphoma, trachea, A membrane (Human multiple tissue n) on which RNA derived from the adrenal gland and bone marrow
The labeled 555 bp cDNA fragment of human A-C1 was hybridized to orthern blot (manufactured by Clontech). After hybridization, the membrane
Cronex lightning plus intensifying screens (DuPont
Using an X-Omat film (Kodak) at -80 ° C.

A 1.1 kb transcript specific for A-C1 was detected in skeletal muscle, testis, heart, brain and thyroid.
Furthermore, the expression level of human A-C1 mRNA was low in normal human bone cells, and high in U ₂ OS cells derived from osteosarcoma.

<5> Chromosome extraction and fluorescence in situ
u hybridization (FISH) To determine the chromosomal location of the human AC1 gene,
ISH analysis identified 150 representative R-band metaphase chromosomes. R band chromosome extraction and FIS
H analysis was performed by Takahashi et al., Hum. Genet., 86, 14-1.
6, 1990. Chromosome slide is 6
Fix for 2 hours at 5 ° C, 7 hours in 2xSSC at 70 ° C for 2 hours.
Denatured with 0% formamide and dehydrated with concentration series of ethanol (70%, 85% and 100% for 5 minutes each).
The denatured probe was treated with a final concentration of 50% formamide, 2 × SSC, 10% dextran sulfate and 1 mg / ml.
An equal volume was mixed with a hybridization solution to give bovine serum albumin (Sigma). A human chromosomal DNA library (lambda EMBL3ge) was prepared by using a human A-C1 cDNA of 0.9 kb.
nomic library of human placenta (clontech)
From. This clone was cloned into biotin 16-dUTP (Boehringer Mannheim) by an industrial method.
Labeled with Nick Translation. Labeled DNA is salmon sperm DNA and Escherichia coli
Ethanol precipitated with RNA, 75 ° C for 10 minutes, 1
Denatured in 00% formamide. The slide is 50
% Formamide in 2 × SSC at 37 ° C. for 20 minutes, followed by a wash in 2 × SSC and 1 × SSC at room temperature for 20 minutes each.

After rinsing in 4 × SSC, the plate was reacted with an anti-biotin antibody (Vector Laboratories) diluted 1: 500 with 4 × SSC at 37 ° C. for 1 hour under coverslip. Next, the slides were washed in 4 × SSC for 10 minutes, washed in 4 × SSC containing 0.1% Nonidet P-40, further washed in 4 × SSC for 10 minutes, and fluorescent anti-goat for 1 hour at 37 ° C. IgG (1: 500 dilution; Nordic Immunolog
y company). After staining, each was washed for 10 minutes with 4 × SSC on a shaker, and 0.1% Nonidet P
Washed in 4 × SSC containing -40. Furthermore, 2 × SS
Rinse with C, remove excess liquid and stain with 0.75 μg / ml propidium iodide. The slide was 450-490 nm (Nikon filter set B-2) under UV light source.
A) and near 365 nm (UV-2A) and photographed with Kodak Ectachrome ASA100 film.

The fluorescent signal of the double-stranded chromatid was detected on chromosome band 3q28-29. The probe was highly specific at this site and did not show symmetrical signals on other chromosomes.

Several studies have shown that this area and hematological malignancies (Slavutsky et al., Cancer Genet. Cytogenet.,
21, 335-342, 1986), epithelial tumors (Grati et al., Cance
r Genet. Cytogenet., 118, 57-61, 2000) and adrenocortical tumors (Dohna et al., Genes Chromosomes Cancer, 2
8, 145-152, 2000). So far, there has been accumulating evidence that ras and genes related to ras are involved in canceration and tumors (Bohle et al., Am. J. Pathol. 148, 731-
738, 1996; Bos, Cancer Res. 49, 4682-4689, 1989;
Stratton et al., Cancer Res. 49, 6324-6327, 1989; Y
oo et al., Mod. Pathol. 12, 775-780, 1999).

These facts indicate that A-C1 is H-ras
It is involved in the suppression of cell transformation by H-ras or the suppression of proliferation of cells transformed with H-ras.

[0084]

Industrial Applicability According to the present invention, a novel peptide of a mammal, in particular, an activity of inhibiting cell carcinogenesis by H-ras,
Alternatively, a peptide having an activity of suppressing the growth of cells transformed with H-ras, and a polypeptide encoding the same are provided.

[0085]

[Sequence list] SEQUENCE LISTING <110> Mitsubishi Tokyo Pharmaceutical Co., Ltd. <120> Novel mammalian peptide and polynucleotide encoding it <130> P-8050 <140> <141> 2000-11-02 <150> JP 11-313955 <151> 1999-11-04 <160> 8 <170> PatentIn Ver. 2.0

<210> 1 <211> 2976 <212> DNA <213> Mus musculus <220> <221> CDS <222> (160) .. (660) <400> 1 cgagggaggc tgtggtggcc ttggctgtgg ccttggcctc agggcgatgc ggatggagtc 60 acccagggtc tgctagcggc accgcagcga gcccgtggag aacccgggaa gcccgtcact 120 gcctccaggg cactaagaca acccagcttg agcagggag atg gcg gta aat gat 174 Met Ala Val Asn Asp 15 tgc ttc agt ctg Acc tat cga c cac c tat cca c cac c tat cca c cac c tat cca c ac c tat cca c ac c tat cca c ac c tat cca c ac c His Pro Gly Asp Leu Ile 10 15 20 gaa gtg ttc cgt cct tgc tat cag cac tgg gca ctg tac ttg ggt gat 270 Glu Val Phe Arg Pro Cys Tyr Gln His Trp Ala Leu Tyr Leu Gly Asp 25 30 35 ggc tac gtg atc aac att gca cct ata gat ggc att cgc tca tca ttt 318 Gly Tyr Val Ile Asn Ile Ala Pro Ile Asp Gly Ile Arg Ser Ser Phe 40 45 50 aca agt gct aag tcc gtg ttc agc aca aag gcc ttg gtg aaa atg cag 366 Ser Ala Lys Ser Val Phe Ser Thr Lys Ala Leu Val Lys Met Gln 55 60 65 ctt ttg aag gat gtt gtg gga aat gac aca tac aga ata aat aac aag 414 Leu Leu Lys Asp Val Val Gly Asn Asp Thr Tyr Arg Ile Asn Asn Lys 70 75 80 85 tac gac aca aca tac cct cct ctt cct gtg gag gag gtg ata caa cgg 462 Tyr Asp Thr Thr Tyr Pro Pro Leu Pro Val Glu Glu Val Ile Gln Arg 90 95 100 tca gag ttc cct att ggg cag gaa gta gcc tat gac ttg ctg gtc aac 510 Ser Glu Phe Pro Ile Gly Gln Glu Val Ala Tyr Asp Leu Leu Val Asn 105 110 115 aac tgt gag cat ttt gta acc ttg ctg cgc tat gga gaga g tca 558 Asn Cys Glu His Phe Val Thr Leu Leu Arg Tyr Gly Glu Gly Val Ser 120 125 130 gag cag gcc aac cga gca atc ggc acc atc gga ttg gtg gca gct ggt 606 Glu Gln Ala Asn Arg Ala Ile Gly Thr Ile Gly Leu Val Ala Ala Gly 135 140 145 att gat atc ttc aca ttc ctc ggc ttg ttt ccc aaa aga caa aga acg 654 Ile Asp Ile Phe Thr Phe Leu Gly Leu Phe Pro Lys Arg Gln Arg Thr 150 155 160 165 aaa tat tatga t tagagat ttgaat ggagaaaaaa 710 Lys Tyr aatcctaggg tgaatactta ttttgaatgc atcattattg ctcatggtcc ccatgatgga 770 tggcagactc tgtaataaat tgcttgctga ttttaatctt atcattgagc caagaaaagt 830 ttttcccaac tagc agagat ttgccgtggc agcttgaaca aaatgcaatt gccttttgat 890 cgagccagct gaggatctta acagaaccaa gaccacattt tatcttctgc tgtaaatatt 950 gttttccttt tccctaagga cagctgtttt gccagaggtg tggagaacca tttgcataca 1010 ctgctgagag acagttgtta gggccaacat ctaaattcct tttgctttcc ttgtcagaaa 1070 aaggagcgtg aacatatcca gtagtttgga tacatggata tatatccatg atatatccaa 1130 gtacttaagg atctctgcat gacaaagcat ttgaggtagt acccccatat tgctgcacct 1190 gtgtgggtgg agcatgtaag aaggagtggg ggacttttgt gtgtgcatat gtatggaatt 1250 cttatgaatg gtgtgcatgt atagggacgg tcttggtctc acctttttta ttcttatttt 1310 attcttattc ttattttatt ttataattta taaaaggaga cagggtatgc tgtcctttat 1370 agcttatggc aaactagcta gcctgtgggc atccagagag tctcctgtct ctgcctccat 1430 ttcacggtag gagttctgga gttaccgaag tgaactaaca acccaagctt tctgtggact 1490 ctggggatct gagctcaaga catcatgctt atgtttaaga tgctttaccc actgagcccc 1550 tctccaatct ctctctctct ctttctttct ctctctctct cccttgtacg ttcagaccca 1610 gtgttcaaga ctcagtgatg aacaatgggc tgtcgtttta ttgatttaaa tggatccctc 1670 cacattgtgt agtagctcag t ggtttgacc tttggggctt ggcaagcaat cattaatatt 1730 aatgcaaaga gctatattta aaacacaaaa ttcctcattc ctttaaagat gtgaactgta 1790 aatcaaggaa tccgcttatt ttattctttt attttactga ttatccgttg aattaaactc 1850 cttcttaatg ccatttctgc agaattcttg atagcaggtc aggagaggtg ggacgttagt 1910 ctttctacat gaaggtagta taagggcatc atgacattac ctcataaacc acacagctag 1970 cccggcatcc ttttgtgcca ttatcaaaag catgtcaatc aataaggaaa gagtgccagt 2030 gaccatgggg atagaacgtg atggagtatg gtaaagtcaa gtgcacaaga ctcggtggaa 2090 agctcagacc tacttaactg atttgacctt ggtgctggaa acactcacac tgatgagggt 2150 aacagtaatg tgaacagaac tatgtgctgc cactcccatc cagacagagt cccctctgtc 2210 ccttctttac ttttctaaca ggcccttctt gtccgaacct ccagactcat tctttagttg 2270 tctcagaagg cacgaggtta cttttatcct gaatgcaggt ctctgttccc aaatagtaaa 2330 gaatattcag ttttctagaa gtttcctatt aaggtaaggg cagctgcagc ctctgggcta 2390 ctttcccctg gtgtgccacc ttctcaataa actatatttg cagggcattt gtctctggca 2450 gaattacacc gcctgtcagg aaagggacta ggactgagaa ccaaatgcct cttagggatt 2510 caaatgaaac ctttctagag gactctg aca tctccactcc agaagaaaag caaaatgtta 2570 aaaggaaaac gtcagaaatg cactttttaa tccatgggtt tatttcctta tcagttatgt 2630 gaagctttgg cagccttggt ttgctgtaaa catggccaac cttaaaatgg gcagccctaa 2690 atagctgtgg ttgttttcct gcaggtactc aatacccaca cgttcaatgc aacttatact 2750 tggatcagct atttccccaa cactatgaaa tattgtctgg gcatattttc agcagtcagg 2810 aatggagtcc acttctatca tgtttcaaaa aatggaatgt aaagatcacc tgcatcaagt 2870 gataggtttt tggtaataca aggaaagaaa tgtctgtatt tttattgaat atgtacatat 2930 aacaaataaa atatgctatc taaatgtcaa aaaaaaaaaa aaaaaa 2976

<210> 2 <211> 167 <212> PRT <213> Mus musculus <400> 2 Met Ala Val Asn Asp Cys Phe Ser Leu Thr Tyr Pro His Asn Pro His 1 5 10 15 Pro Gly Asp Leu Ile Glu Val Phe Arg Pro Cys Tyr Gln His Trp Ala 20 25 30 Leu Tyr Leu Gly Asp Gly Tyr Val Ile Asn Ile Ala Pro Ile Asp Gly 35 40 45 Ile Arg Ser Ser Phe Thr Ser Ala Lys Ser Val Phe Ser Thr Lys Ala 50 55 60 Leu Val Lys Met Gln Leu Leu Lys Asp Val Val Gly Asn Asp Thr Tyr 65 70 75 80 Arg Ile Asn Asn Lys Tyr Asp Thr Thr Tyr Pro Pro Leu Pro Val Glu 85 90 95 Glu Val Ile Gln Arg Ser Glu Phe Pro Ile Gly Gln Glu Val Ala Tyr 100 105 110 Asp Leu Leu Val Asn Asn Cys Glu His Phe Val Thr Leu Leu Arg Tyr 115 120 125 Gly Glu Gly Val Ser Glu Gln Ala Asn Arg Ala Ile Gly Thr Ile Gly 130 135 140 Leu Val Ala Ala Gly Ile Asp Ile Phe Thr Phe Leu Gly Leu Phe Pro 145 150 155 160 Lys Arg Gln Arg Thr Lys Tyr 165

<210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 gccgccacca tggacccgac acggtccc 28

<210> 4 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 ctacttgtca tcgtcgtcct tgtaatcata tttcgttctt tgtcttttgg gaaac 55

<210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 cacactggta agtggggcaa gaccg 25

<210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 ggattgtgtt gtttcagggt tcggg 25

<210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 cagcactggg cactgtactt gg 22

<210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 aaaagctgca ttttcaccaa ggc 23

<210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 ggtgagacca agaccgtccc 20

<210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 10 atatttcgtt ctttgtcttt tgggaaac 28

<210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 acctgttaga tggcattcct gcgtcc 26

<210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 12 gcattcctgc gtcctttaca agcgc 25

<210> 13 <211> 1089 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (402) .. (905) <400> 13 ctcactatag ggctcgagcg gccgcccggg caggtatttt ttaatattta atccttgata 60 tttttaaaat ctgtgtaagt tatattatcc agggatttct agatttaaag agaaaattac 120 agaatgcttg ttattataaa ggggctctga aattgatcgg gatgagaagc attactatag 180 aaaattaagt aatacaagac aaattatttc tctacttgtc cccttgtgca cacatcagtg 240 ttggaatcat tcaaagaaaa ggacagggtc gtgattctaa gtctttaata tagaatgcag 300 tctcaataaa ttctgtatca ttctgctttt atggatgtct taaactgatt ttcttttggc 360 aagttgtatt agctagctct tctttgtatt tgtcattgca g atg gcg ttt aat gat 416 Met Ala Phe Asn Asp 1 5 tgc ttc agt ttg aac tac cct ggc aac ccc tgc cca ggg gac ttg atc 464 Cys Phe Ser Leu Asn Tyr Pro Gly Asn Pro Cys Pro Gly Asp Leu Ile 10 15 20 gaa gtg ttc cgt cct ggc tat cag cg tgg tac ttg ggt gat 512 Glu Val Phe Arg Pro Gly Tyr Gln His Trp Ala Leu Tyr Leu Gly Asp 25 30 35 ggt tac gtt atc aac ata gca cct gta gat ggc att cct gcg tcc ttt 560 Gly Tyr Val Ile Asn Ile A la Pro Val Asp Gly Ile Pro Ala Ser Phe 40 45 50 aca agc gcc aag tct gta ttc agc agt aag gcc ctg gtg aaa atg cag 608 Thr Ser Ala Lys Ser Val Phe Ser Ser Lys Ala Leu Val Lys Met Gln 55 60 65 ctc ttg aag gat gtt gtg gga aat gac aca tac aga ata aac aat aaa 656 Leu Leu Lys Asp Val Val Gly Asn Asp Thr Tyr Arg Ile Asn Asn Lys 70 75 80 85 tac gat gaa acg tac ccc cct ctc cct gtg ata gaa atc aag cgg 704 Tyr Asp Glu Thr Tyr Pro Pro Leu Pro Val Glu Glu Ile Ile Lys Arg 90 95 100 tca gag ttt gta att gga cag gag gtg gcc tat aac tta ctt gtc aac 752 Ser Glu Phe Val Ile Gly Gln Glu Val Ala Tyr Asn Leu Leu Val Asn 105 110 115 aac tgt gaa cat ttt gtg aca ttg ctt cgc tat gga gaa gga gtt tca 800 Asn Cys Glu His Phe Val Thr Leu Leu Arg Tyr Gly Glu Gly Val Ser 120 125 130 gag cag gcc aac cga gcg ata agt acc gtt gag ttt gtg aca gct gct 848 Glu Gln Ala Asn Arg Ala Ile Ser Thr Val Glu Phe Val Thr Ala Ala 135 140 145 gtt ggt gtc ttc tca ttc ctg ggc ttg ttt cca aaa gga caa aga gca 896 Val Gly Phe Ser Phe L eu Gly Leu Phe Pro Lys Gly Gln Arg Ala 150 155 160 165 aaa tac tat taacaattta ccaaagagat attgatattg aaggaatttg 945 Lys Tyr Tyr ggaggaggaa aagaaacctg ggtta aaa ttagtagtaaaa ggttagattac aaa

<210> 14 <211> 168 <212> PRT <213> Homo sapiens <400> 14 Met Ala Phe Asn Asp Cys Phe Ser Leu Asn Tyr Pro Gly Asn Pro Cys 1 5 10 15 Pro Gly Asp Leu Ile Glu Val Phe Arg Pro Gly Tyr Gln His Trp Ala 20 25 30 Leu Tyr Leu Gly Asp Gly Tyr Val Ile Asn Ile Ala Pro Val Asp Gly 35 40 45 Ile Pro Ala Ser Phe Thr Ser Ala Lys Ser Val Phe Ser Ser Lys Ala 50 55 60 Leu Val Lys Met Gln Leu Leu Lys Asp Val Val Gly Asn Asp Thr Tyr 65 70 75 80 Arg Ile Asn Asn Lys Tyr Asp Glu Thr Tyr Pro Pro Leu Pro Val Glu 85 90 95 Glu Ile Ile Lys Arg Ser Glu Phe Val Ile Gly Gln Glu Val Ala Tyr 100 105 110 Asn Leu Leu Val Asn Asn Cys Glu His Phe Val Thr Leu Leu Arg Tyr 115 120 125 Gly Glu Gly Val Ser Glu Gln Ala Asn Arg Ala Ile Ser Thr Val Glu 130 135 140 Phe Val Thr Ala Ala Val Gly Val Phe Ser Phe Leu Gly Leu Phe Pro 145 150 155 160 Lys Gly Gln Arg Ala Lys Tyr Tyr 165

[Brief description of the drawings]

FIG. 1. Comparison of the amino acid sequences of human and mouse AC1 (SEQ ID NO: 2) and rat Ha-rev107 (SEQ ID NO: 14). Asterisks indicate homologous residues. Sequences capable of base interaction are indicated by boxes.

Claims (6)

[Claims] 1. A peptide of the following (A) or (B): (A) a peptide having the amino acid sequence of SEQ ID NO: 2; (B) in the amino acid sequence of SEQ ID NO: 2, having an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and in H-ras canceration of cells or H-ras A peptide having an activity of suppressing the growth of transformed cells. 2. A polynucleotide encoding any one of the following peptides (A) and (B): (A) a peptide having the amino acid sequence of SEQ ID NO: 2; (B) in the amino acid sequence of SEQ ID NO: 2, having an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and in H-ras canceration of cells or H-ras A peptide having an activity of suppressing the growth of transformed cells. 3. The polynucleotide according to claim 2, which is a polynucleotide represented by the following (a) or (b): (A) a polynucleotide having a base sequence represented by base numbers 160 to 660 of SEQ ID NO: 1; (B) hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence represented by nucleotide numbers 160 to 660 of SEQ ID NO: 1 and transfects with H-ras H-ras A polynucleotide encoding a peptide having an activity of suppressing the growth of a formed cell. 4. A peptide of the following (C) or (D): (C) a peptide having the amino acid sequence of SEQ ID NO: 14; (D) in the amino acid sequence of SEQ ID NO: 14, which has an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and is used for H-ras cell carcinogenesis or H-ras A peptide having an activity of suppressing the growth of transformed cells. 5. A polynucleotide encoding the following peptide (C) or (D): (C) a peptide having the amino acid sequence of SEQ ID NO: 14; (D) in the amino acid sequence of SEQ ID NO: 14, which has an amino acid sequence having one or more amino acid substitutions, deletions, insertions, or metastases, and is used for H-ras cell carcinogenesis or H-ras A peptide having an activity of suppressing the growth of transformed cells. 6. The polynucleotide according to claim 5, which is a polynucleotide shown in the following (c) or (d). (C) a polynucleotide having a base sequence represented by base numbers 402 to 905 of SEQ ID NO: 13. (D) hybridizing under stringent conditions with a polynucleotide having a nucleotide sequence represented by nucleotide numbers 402 to 905 of SEQ ID NO: 13 and transforming cells into cancerous cells with H-ras, or transforming with H-ras A polynucleotide encoding a peptide having an activity of suppressing the growth of a formed cell.