JP2000217578A - Masl1 gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an MASL1 gene which has high homology to a nucleic acid coding for a polypeptide containing a specific amino acid sequence, has homology to ALS, is involved in proliferation, differentiation, and apoptosis of cells, and can be used for diagnosis and treatment of carcinoma and so on. SOLUTION: This new MASL1 gene contains a nucleotide selected from a polynucleotide coding for a polypeptide containing the amino acid sequence shown by formula I, a polynucleotide having homology to the polynucleotide shown by formula II at 95% or higher, or polynucleotides complementary to these nucleotides, and also has a homology to ALS, plays important roles in proliferation, differentiation, and apoptosis of cells, and can be used for elucidation of pathology, diagnosis, and treatment of diseases which are involved in differentiation of cells such as carcinoma. This gene is obtained by preparing a cDNA library derived from various kinds of cells, tissues, and so on, followed by screening the obtained library using an appropriate probe or antibody specific to the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention has isolated a novel gene containing a highly conserved leucine tandem repeat region characteristic of a protein binding molecule from the region of commonality of amplification at 8p23.1. The gene has an ATP / GTP binding site, three leucine-zipper domains, and a leucine-rich tandem repeat, all of which are important for cell cycle-related protein-protein interactions. Are functional components. By a cDNA analysis of such a gene, a novel gene (MA) capable of diagnosing and treating MFH (malignant fibrous histiocytomas), developing a new therapeutic agent for such treatment, etc.
The present invention relates to a SL1 gene), a cDNA having the gene, an expression product thereof (MASL1 protein), a pharmaceutical composition using the same, and a method for gene diagnosis.

[0002]

2. Description of the Related Art MFH is the most common soft tissue sarcoma found in adults (Enzinger, FMandWeiss, SW, malig).
nant fibrohistocyttic tumors: Soft Tissue Tumors.
CVMosby: St. louis, pp. 351-380 (1995)). Recent WH
In the O classification, MFH is defined as "Pluriform spindle cell sarcoma: ple
defined as `` morphic spindle-cell sarcoma '', a cell line that usually occurs in adults and has not yet been clearly differentiated (Weiss, SW, Histological Typing of Soft Tissue T
umors, Springer-Verlag: Berlin (1994)). Histologically, MFH is defined as a heterogeneous heterogeneous group of sarcomas of fibroblasts, histiocytic cells, and malformed cells, the stroma of which is often of varying amounts of inflammatory cells, collagen, And mucus-like substances. It is not known whether the tumors originate from fibroblasts, histiocytic cells or undifferentiated mesenchymal cells. Of the four histological subtypes of MFH, the flower-like polymorphism and the malformed subtype
Type is the most common, while giant cell or inflammation subtypes are rare. MFH karyotypic abnormalities are usually complex with structural rearrangements that exhibit complex numerical or structural abnormalities. Chromosome aberrations specific to MFH have not been identified to date. However, chromosome end fusions, unidentified circular chromosomes, and dicentric chromosomes that are often found in this type of tumor, also have double microchromosomes and homogeneous chromosomes as chromosomal alterations in gene amplification. Region appears (LeD
oussal, V., et al., Cancer, 77,1823-1830 (1992): Pe
zzi, ME, et al., Cancer, 69, 2098-2103 (1992)).
Several known proto-oncogenes that play important roles as potential etiologies for the development of tumors in soft tissues have been studied for the involvement of MFH:
Gene (SAS: the sarcoma-amplified-sequence gene),
MDM2 gene (the murine double-minute 2 gene: MD
M2) human homolog, cyclin-dependent kinase 4 (cy
cline-dependent kinase 4: CDK4), and homologous protein (CHOP) encoding C / EBP. All of the genes described above map to chromosome 12q13-15, and all but CHOP are amplified in more than one-third of the MFHs tested (Nilbert, M., et al., Cancer Genet. Cytog
enet., 83, 32-36 (1995)).

[0003] The tumor suppressor gene TP53 (17q13) or RB1 (1
Mutations affecting 3q14) have been reported in approximately one third in MFH tumors (Wadayama, B., et al., B
rJCancer, 68,1134-1139 (1993): Wunder, JS, et a
l., J. Natl. Cancer Inst., 83, 194-200 (1991)).

[0004]

We analyzed 19 MFH tumors by CGH to explore genomic mutations that might affect the development and / or progression of this type of tumor. . We combined with deletions and gains involving several regions of different chromosomes, with a distinct high level of amplification at six locus, chromosomal loci 4q12-21, 8p2.
1-pter, 8q24.1-qter, 9q12-13, 12p11.2-pter, and 15
q11.2-15. Each of these regions may contain one or more potential proto-oncogenes involved in the establishment of MFH tumors. As a first step towards the identification of such genes, we focused on the 8p amplicon to characterize the structure of the molecule in detail. From the common region of amplification at 8p23.1, we have found that MASLs contain highly conserved leucine tandem repeat regions characteristic of protein binding molecules.
A new gene, designated No. 1, was isolated. The present invention has been completed based on such knowledge.

[0005]

According to the present invention, first, a gene containing any of the following polynucleotides (a) and (b): (a) an amino acid represented by SEQ ID NO: 1 A polynucleotide encoding a polypeptide comprising the sequence or a polynucleotide having at least 95% homology to the polynucleotide having SEQ ID NO: 2, (b) to the polynucleotide of (a) above. A polynucleotide that is the complementary strand.

According to the present invention, secondly, a gene consisting of any one of the following polynucleotides (a) and (b): (a) a base sequence represented by SEQ ID NO: 2 or a complementary strand thereof; b) a polynucleotide that hybridizes under stringent conditions to the polynucleotide comprising the nucleotide sequence of (a).

According to the present invention, thirdly, a gene having the nucleotide sequence represented by SEQ ID NO: 2 or a gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, and gene expression products and genes thereof And a host cell having the recombinant expression vector, or cloned c that expresses the MASL1 protein
DNA and fragments thereof, derivatives and homologs thereof.

Further, according to the present invention, fourthly, an oligonucleotide comprising at least 15 or 30 contiguous nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 2, or

According to the present invention, fifthly, an antisense oligonucleotide corresponding to the nucleotide sequence represented by SEQ ID NO: 2, and the antisense oligonucleotide is prepared using at least 15 or 30 consecutive nucleotide sequences. And a gene therapy agent comprising these antisense oligonucleotides as active ingredients.

Furthermore, according to the present invention, sixthly, SEQ ID NO:
A DNA probe consisting of the nucleotide sequence represented by SEQ ID NO: 2, or an oligonucleotide probe thereof, comprising at least 15 or 30 consecutive nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 2; A diagnostic agent and a diagnostic kit using an oligonucleotide probe can be provided.

According to the present invention, seventhly, a MASL1 protein which is the following protein (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, (b) A) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence according to the above), and having MASL1 activity;

Further, according to the present invention, eighthly, a method for screening an interactant using the MASL1 gene or gene expression product.

[0013] Further, it is a homologue of the gene of the present invention, which is a mammalian gene homolog selected from human, dog, monkey, horse, pig, sheep and cat, and has a binding property to the expression product of the gene of the present invention. An antibody, a diagnostic method using the antibody for diagnosis of cancer, and a cancer therapeutic agent comprising the antibody as an active ingredient are provided.

[0014]

DETAILED DESCRIPTION OF THE INVENTION The present inventors have developed CGH (Comparative genomic hyb) to search for genomic mutations that may affect the development and / or progression of this type of tumor.
This was completed as a result of studying 19 MFH tumors by ridization (hybridization of comparative genome) and searching for new related genes expressed on chromosomes of MFH patients.

That is, from the study of 19 MFH tumors, together with deletions and gains involving several regions of different chromosomes, a distinct high level of amplification was achieved at six locus chromosomal loci,
4q12-21, 8p21-pter, 8q24.1-qter, 9q12-13, 12p11.2-
pter, and 15q11.2-15. We focused on the 8p amplicon to characterize the structure of the molecule in detail, and from the common region of amplification at 8p23. MAS containing certain highly conserved leucine tandem repeat regions
A new gene, designated L1, was isolated.

The MASL1 gene of the present invention is obtained by performing reverse chromosome FISH using whole genomic DNA from a patient sample in which a high level of amplification was observed on the short arm of chromosome 8 by CGH from a sample of an MFH patient. After localizing the band to 8p23.1, Southern blot analysis identified genomic fragments within the amplified region. Then, Southern blot analysis showed that ES with high amplified copy number
Using T, GEN-024H10 as a probe, a human fetal brain cDNA library was screened, a positive clone was isolated, and the nucleotide sequence was determined, whereby the sequence was determined.

The MASL1 gene of the present invention is specified as a gene having an open reading frame encoding 1052 amino acids shown in SEQ ID NO: 1.

The calculated molecular weight of the amino acid sequence encoded by the MASL1 gene of the present invention is 116890D (11
7 kD).

The amino acid sequence encoded by the MASL1 gene of the present invention has a leucine-rich tandem repeat region,
The repetitive sequence of MASL1 is very similar to the gene sequence generated in yeast adenylate cyclase, and the deduced amino acid sequence of MASL1 is ALS (Kobe, B., a
nd Deisenhofer, J., Trends Biochem.Sci., 19, 415-
421 (1994)).

As described above, the basic structure of the putative product of the novel MASL1 gene provided by the present invention is ATP / GTP
Reveals binding sites, three leucine-zipper domains, and leucine-rich tandem repeats, all of which are responsible for cell growth, differentiation, apoptosis, and DNA
MASL1 is a structural or functional component that is important for the interaction between proteins related to the cycle such as synthesis.
It is thought that the binding of the molecule predicted to bind to the protein, and further to the MASL1 protein, affects the growth and differentiation of cells and the regulation of biological functions such as the function of the complex protein molecule.

Further, as described later, it is considered that the overexpression of MASL1 belongs to the oncogene with respect to MFH.

According to the provision of the gene of the present invention, the action of regulating the proliferation, differentiation and apoptosis of each cell can be used to elucidate, diagnose, and treat diseases associated with the action, such as malignant tumors. It is possible.

The whole or a part of the MASL1 gene of the present invention can be used for producing an antibody that binds to an expression product utilizing the MASL1 gene, and for diagnosis using the antibody.

Furthermore, according to the provision of the gene of the present invention, the use of antisense fragments of the gene sequence and the expression products thereof can suppress the formation of the above-mentioned diseases.

The whole or a part of the MASL1 gene of the present invention can be used as a probe, and can be used as a part of a diagnostic kit for cancer and a diagnostic kit. Furthermore, gene amplification and enhanced expression of MASL1 gene in tumors
It can be used not only for cancer diagnosis, but also for determining the malignancy of cancer.

The present invention can also be used for screening for a MASL1 protein interacting substance using the MASL1 gene or its expression product.

[0027] One example of the MASL1 gene provided by the present invention is derived from human cancer cells, and its use can provide homologous genes of various mammals including humans. Further, by utilizing the gene of the present invention, it is also possible to identify a gene that binds to the C-terminal side of the amino acid sequence encoded by the gene of the present invention.

Hereinafter, the DNA molecule of the present invention (MASL1 gene) will be described in detail.

In the present specification, abbreviations for amino acids, peptides, base sequences, nucleic acids and the like are used in IUPAC, IUPAC,
The rules of UB, “Guidelines for preparation of specifications including base sequence or amino acid sequence” (Japan Patent Office), and commonly used symbols in the art shall be followed.

In the present invention, a gene (DNA) is
It is intended to include not only double-stranded DNA but also single-stranded DNAs such as a sense strand and an antisense strand constituting the double-stranded DNA, and the length is not limited at all.
Therefore, unless otherwise specified, the gene (DNA) of the present invention includes double-stranded DNA including human genomic DNA, and cDN
A single-stranded DNA containing A (sense strand), a single-stranded DNA having a sequence complementary to the sense strand (antisense strand), and fragments thereof are also included.

As a specific example of the MASL1 gene of the present invention,
Examples include those deduced from the DNA sequence of the clone shown in Examples described later.

The gene of this clone has an open reading frame of 3159 nucleotides which encodes a protein consisting of 1052 amino acid residues shown in SEQ ID NO: 1 in the sequence listing (showing the base sequence shown in SEQ ID NO: 2). Having. From the unidirectional sequence of the cDNA sequence from the positive clone, a single open
A 6242 bp transcript containing the reading frame was identified. Since the consensus sequence for the start of transcription ("Kozak rule") is well conserved, the start codon for transcription was considered to be nucleotides 417 to 419.

The base sequence of the full-length cDNA is 6345 nucleotides as shown in SEQ ID NO: 3.

The PSORT program for the MASL1 gene of the present invention (Nakai, K. and Kanehisa, MA, Genomics, 1
4,897-911 (1992)) showed one hydrophobic region in the middle of the amino acid sequence (residues 486-502), however, the hydrophobicity plot (Kyte, J. and Doolittle, RF,
J. Mol. Biol., 157, 105-132 (1982) did not predict hydrophobic regions. One ATP / GTP binding site
The motif "A" ([AG] -X (4) -GK- [ST]) was found at residues 416-423. Leucine residues were repeated as each of the seven amino acids capable of forming a structural motif known as the "leucine zipper" (Landschulz,
WH, et al., Science, 240, 1759-1764 (1988)).

The deduced gene product contains three leucine-
It seems to contain a zipper region (68-89 residues, 102-123
(Residues, and 962-983 residues), each with four leucine residues, were regularly spaced with seven amino acid repeats.

The DNA molecule of the present invention includes, for example, the MASL1 gene having a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, but is not particularly limited thereto. Homologs of the MASL1 gene are also included.

As used herein, the term "homolog of the MASL1 gene" means that it has sequence homology to the MASL1 gene of the present invention (or a gene product thereof), has the above-mentioned structural characteristics and commonality in gene expression patterns, and It refers to a series of related genes that are recognized as one gene family due to the similarity of their biological functions, and naturally includes alleles (alleles) of the MASL1 gene.

Specifically, a protein having a certain modification in the specific amino acid sequence represented by SEQ ID NO: 1 and having the same activity as the MASL1 protein having the specific amino acid sequence is encoded. Genes that can be used.

The homology in the amino acid sequence is as follows:
Genome Net: Measurement using the FASTA program using Genome Net (Clustal, V., Methods Mol. Bio
l., 25, 307-318 (1994)), the total amino acid sequence can usually be about 45% or more, preferably about 50% or more.

The above homology preferably satisfies at least one of about 35% or more, preferably about 45% or more of the leucine-rich repeat motif region.

That is, the MASL1 gene of the present invention has a nucleotide sequence encoding a protein consisting of an amino acid sequence in which one or several to a plurality of amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Containing DNA molecules are also included.

Here, the degree of “deletion, substitution or addition of amino acids” and their positions are the same as those of the modified protein (MASL1 protein) having the amino acid sequence represented by SEQ ID NO: 1. There is no particular limitation as long as it has the same function. In the present invention, “MASL1 activity” refers to the activity and function of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and specifically, binds to a protein that regulates cell growth, differentiation, and apoptosis. , Dissociation, or the like, which regulates the concentration or translocation of a binding protein in blood or tissue, or a function expected to regulate the activity itself.

The above amino acid sequence modification (mutation) and the like
Although it may occur in nature due to, for example, mutation or post-translational modification, it can also be artificially modified based on a naturally-derived gene (for example, the human MASL1 or human MASL1 gene of the present invention). The present invention provides such modifications
All modified genes having the above characteristics are included, regardless of the cause and means of the mutation. The gene of the present invention (human MASL
1 gene) also includes an allele (allele) of a gene encoding a protein having the amino acid sequence represented by SEQ ID NO: 1.

As the above-mentioned artificial means, for example, site-specific mutagenesis [Methods in Enzym
ology, 154, 350, 367-382 (1987);
3); Nucleic Acids Res., 12, 9441 (1984); Lectures on Sequent Chemistry Experiment 1, Genetic Research Method II, edited by The Biochemical Society of Japan, p105
(1986)) and chemical synthesis means such as the phosphate triester method and the phosphate amidite method (J. Am.
Chem. Soc., 89, 4801 (1967); id. 91, 3350 (1969); S
cience, 150, 178 (1968); Tetrahedron Lett., 22, 185
9 (1981); ibid. 24, 245 (1983)] and methods of combining them. More specifically, the synthesis of DNA can be performed by a chemical synthesis using a phosphoramidite method or a triester method, or can be performed on a commercially available automatic oligonucleotide synthesizer. The double-stranded fragment is synthesized chemically to produce a single-stranded fragment, either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or adding the complementary strand using a DNA polymerase with the appropriate primer sequences. It can also be obtained from things.

As a specific embodiment of the gene of the present invention, a gene having the nucleotide sequence shown in SEQ ID NO: 2 can be exemplified. The coding region in this nucleotide sequence corresponds to SEQ ID NO:
1 shows an example of one combination of codons indicating each amino acid residue of the amino acid sequence shown in FIG. The gene of the present invention
Not only the gene having such a specific base sequence but also a base sequence selected by combining arbitrary codons for each amino acid residue is also possible. Codon selection can be performed according to a conventional method, for example, considering the codon usage frequency of a host to be used (Ncleic Acids Res.,
9, 43 (1981)].

The DNA molecule of the present invention is, as described above,
Also included are those consisting of a base sequence having a certain homology with the base sequence shown in SEQ ID NO: 2.

The above homology is at least 70% identity, preferably at least 90%, to a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 2. And more preferably at least 95%, even most preferably at least 97%, and its complementary strand polynucleotide.

As such a gene, for example, 0.1%
50 ° C. or 0.1% SD in 0.2 × SSC containing SDS
2361-3 in the base sequence shown in SEQ ID NO: 2 under stringent conditions of 60 ° C. in 1 × SSC containing S
A gene having a base sequence that hybridizes with a DNA consisting of the base sequence of 858 can also be exemplified.

The gene of the present invention can be prepared based on the sequence information of the specific example of the gene of the present invention disclosed by the present invention.
It can be easily produced and obtained by general genetic engineering techniques [Molecular Cloning 2d Ed, Cold Spring H
arbor Lab. Press (1989); see Seismic Chemistry Laboratory Course "Gene Research Methods I, II, III", edited by The Biochemical Society of Japan (1986), etc.).

Specifically, a cDNA library is prepared from an appropriate source in which the gene of the present invention is expressed according to a conventional method, and the desired library is prepared from the library using an appropriate probe or antibody specific to the gene of the present invention. Acad. Sci., USA., Proc. Natl. Acad.
8, 6613 (1981); Science, 222, 778 (1983) and the like].

In the above, the origin of cDNA is as follows:
Examples include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Separation of total RNA therefrom, separation and purification of mRNA, acquisition of cDNA, and cloning thereof can all be carried out according to conventional methods. In addition, cDNA libraries are commercially available, and in the present invention, such cDNA libraries, for example, Clontech Lab.
Various cDNA libraries commercially available from c.) can also be used.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used.

Specifically, for example, a method for selecting a corresponding cDNA clone from a protein produced by cDNA by immunoscreening using an antibody specific to the protein, and selectively binding to a target DNA sequence Examples include plaque hybridization and colony hybridization using a probe, and combinations thereof.

As the probe used herein, DNA or the like chemically synthesized based on the information on the nucleotide sequence of the gene of the present invention can be generally used. Good use. In addition, sense primers and antisense primers set based on the nucleotide sequence information of the gene of the present invention can be used as screening probes.

The nucleotide sequence used as the probe is a partial nucleotide sequence corresponding to SEQ ID NO: 2, and comprises at least 15 continuous bases, preferably 20 continuous bases, more preferably 30 continuous bases. Bases, most preferably those having 50 contiguous bases, are also included, or the positive clones themselves having the above sequences can also be used as probes.

In obtaining the gene of the present invention, PCR
DNA / RN by the method [Science, 230, 1350 (1985)]
The A amplification method can be suitably used. Particularly, when it is difficult to obtain a full-length cDNA from the library, RACE
Method [Rapid amplification of cDNA ends; Experimental Medicine, 12
(6), 35 (1994)], especially the 5′-RACE method [MA Frohma
n, et al., Proc. Natl. Acad. Sci., USA., 8, 8998
(1988)].

The primers used when employing the PCR method can be appropriately set based on the sequence information of the gene of the present invention revealed by the present invention, and can be synthesized according to a conventional method. The isolation and purification of the amplified DNA / RNA fragment can be carried out according to a conventional method as described above, for example, by gel electrophoresis.

The gene of the present invention or various DNA fragments obtained as described above can be obtained by a conventional method, for example, the dideoxy method [Proc.
Natl. Acad. Sci., USA., 74, 5463 (1977)) and the Maxam-Gilbert method (Methods in Enzymology, 65, 499).
(1980)], or simply using a commercially available sequencing kit or the like.

According to the gene of the present invention thus obtained, the presence or absence of the expression of the gene of the present invention in an individual or various tissues can be specifically determined by using, for example, a part or the entire nucleotide sequence of the gene. Can be detected.

Such detection can be carried out according to a conventional method, for example, RT-PCR [Reverse transcribed-Polyme
rase chain reaction; ES Kawasaki, et al., Amplif
ication of RNA.In PCR Protocol, A Guide to method
s and applications, AcademicPress, Inc., SanDiego, 2
RNA amplification and Northern blotting analysis [Molecular Cloning, Cold Spring Harbor La.
b. (1989)], in situ RT-PCR [Nucl.
s., 21, 3159-3166 (1993)] and cell-level measurement using in situ hybridization, NASB
Method A [Nucleic acid sequence-based amplification, N
, 350, 91-92 (1991)] and various other methods. Preferably, a detection method by RT-PCR can be mentioned.

The primers used in the case of employing the PCR method are not particularly limited as long as they are specific to the gene of the present invention and can specifically amplify only the gene of the present invention. Can be set appropriately based on Usually, primers having a partial sequence of the gene of the present invention having a length of about 10 to 35 nucleotides, preferably about 15 to 30 nucleotides can be mentioned.

As described above, the gene of the present invention also includes a DNA fragment used as a specific primer and / or a specific probe for detecting the MASL1 gene according to the present invention.

The DNA fragment can be defined as a DNA characterized in that it hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2. Here, examples of the stringent conditions include ordinary conditions used as a primer or a probe, and are not particularly limited. For example, 0.2 × S containing 0.1% SDS as described above may be used.
1 × SS containing 50% in SC or 0.1% SDS in SC
The condition of 60 ° C. in C can be exemplified.

According to the MASL1 gene of the present invention, the gene product (MA
SL1 protein) or a protein containing it can be easily and stably produced in large quantities.

Accordingly, the present invention relates to a protein such as MASL1 protein encoded by the gene of the present invention, a vector for producing the protein, for example, a vector containing the gene of the present invention, a host cell transformed with the vector, The present invention also provides a method for producing the protein of the present invention by culturing the host cell.

Specific examples of the protein of the present invention include a MASL1 protein having the amino acid sequence shown in SEQ ID NO: 1. The protein of the present invention includes not only the MASL1 protein but also homologs thereof. Is done. Examples of the homologue include a protein having an amino acid sequence in which one or several to a plurality of amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and having the MASL1 activity. be able to. Specifically, there may be mentioned a gene product of a homologue of the MASL1 gene (a MASL1 equivalent gene including an allele).

Further, homologs of the MASL1 protein of the present invention include:
Mammalian, for example, rodent proteins such as humans, horses, sheep, cows, dogs, monkeys, cats, bears, rats, rabbits and the like, which have the same activity as the MASL1 protein of the amino acid sequence shown in SEQ ID NO: 1 Included.

The protein of the present invention can be obtained by a conventional gene recombination technique (eg, Science, 224, 1431 (1984); Bioc) based on the sequence information of the MASL1 gene provided by the present invention.
hem. Biophys. Res. Comm., 130, 692 (1985); Proc. Na
Acad. Sci., USA., 80, 5990 (1983) and the like].

More specifically, the production of the protein is carried out by preparing a recombinant DNA (expression vector) capable of expressing the gene encoding the desired protein in a host cell and introducing it into a host cell for transformation. Then, the transformant is cultured, and then recovered from the resulting culture.

As the host cell, any of prokaryote and eukaryote can be used. For example, prokaryote hosts widely include commonly used hosts such as Escherichia coli and Bacillus subtilis. Escherichia coli, especially those contained in the Escherichia coli K12 strain can be exemplified. Eukaryotic host cells also include
Cells such as vertebrates and yeast are included, and the former includes, for example, COS cells [Cell, 23: 175 (198
1)] and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad. Sc.
i., USA., 77: 4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces are preferably used. Of course, it is not limited to these.

When a prokaryotic cell is used as a host, a promoter and an SD (Shine & Synthesis) are used upstream of the gene so that the gene of the present invention can be expressed in the vector using a vector that can be replicated in the host cell.・ Dargano)
An expression plasmid to which a base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis are added can be suitably used. As the above vector, generally, a plasmid derived from Escherichia coli, for example, pBR322, pBR325, pUC1
2, pUC13 and the like are often used, but are not limited thereto, and various known vectors can be used.
Commercially available products of the above-mentioned vectors used in an expression system using Escherichia coli include, for example, pGEX-4T (Amersham Pharm
acia Biotech), pMAL-C2, pMAl-P2
(New England Biolabs), pET21, pET21
/ Lacq (Invitrogen), pBAD / His (Invitrogen)
trogen).

In the case of using a vertebrate cell as a host, the expression vector usually has a promoter, an RNA splice site, a polyadenylation site and a transcription termination sequence located upstream of the gene of the present invention to be expressed. Which may optionally have a replication origin. Specific examples of the expression vector include, for example, pS having the early promoter of SV40.
V2 dhfr [Mol. Cell. Biol., 1: 854 (1981)] and the like. In addition to the above, various known commercial vectors can be used. Commercially available products of such a vector used in an expression system using animal cells include, for example, pEGF
PN, pEGFP-C (Clontrech), pIND (I
nvitrogen), pcDNA3.1 / His (Invitroge
n) and animal cells such as pFastBac
HT (GibciBRL), pAcGHLT (PharMi
ngen), pAc5 / V5-His, pMT / V5-H
is, pMT / Bip / V5-his (Invitrogen
And insect cell vectors.

Further, as a specific example of an expression vector when a yeast cell is used as a host, for example, pAM82 having a promoter for the acid phosphatase gene [Proc.
Natl. Acad. Sci., USA., 80: 1 (1983)]. Commercially available expression vectors for yeast cells include, for example, pP
ICZ (Invitrogen), pPICZα (Invitrogen
Company).

There is no particular limitation on the promoter. When Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter and the like can be preferably used. When the host is a bacterium belonging to the genus Bacillus, SP01 promoter, SP02 promoter, penP promoter and the like are preferable. When yeast is used as a host, for example, a pH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter and the like can be suitably used. When an animal cell is used as a host, preferable promoters include an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, and an SRα promoter.

[0075] As an expression vector for the gene of the present invention, an ordinary fusion protein expression vector can also be preferably used. Specific examples of the vector include glutathione-S-
Examples include pGEX (Promega) for expression as a fusion protein with transferase (GST).

The method for introducing a desired recombinant DNA (expression vector) into a host cell and the method for transforming with the same include the following.
There is no particular limitation, and various general methods can be employed.

The resulting transformant can be cultured according to a conventional method, and the target protein of the present invention encoded by a gene designed as desired by the culture can be cultured in the cell, extracellularly or on the cell membrane of the transformant. Expression, production (accumulation, secretion)
Is done.

As the medium used for the culture, various types of media commonly used depending on the host cells used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cells.

The recombinant protein of the present invention thus obtained may be subjected to various separation operations utilizing its physical properties, chemical properties and the like [“Biochemical Data Book II”,
1175-1259, 1st edition, 1st printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274 (198
6); Eur. J. Biochem., 163, 313 (1987)].

The method includes, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation,
Osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPL
Various types of liquid chromatography such as C), dialysis, and combinations thereof can be exemplified. Particularly preferred methods include affinity chromatography using a column to which a specific antibody against the protein of the present invention is bound. it can.

In designing a desired gene encoding the protein of the present invention, the base sequence of the MASL1 gene shown in SEQ ID NO: 2 can be used favorably. The gene can be used by appropriately selecting and changing codons indicating each amino acid residue, if desired.

In the amino acid sequence encoded by the MASL1 gene, when a part of the amino acid residues or the amino acid sequence is modified by substitution, deletion, addition, etc., the amino acid sequence may be modified as described above, for example, by cytospecific mutagenesis. It can be performed by various methods.

The protein of the present invention can be produced by a general chemical synthesis method according to the amino acid sequence shown in SEQ ID NO: 1. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis.

[0084] Such a peptide synthesis method is described in more detail below.
Based on the amino acid sequence information, a so-called stepwise elongation method in which each amino acid is sequentially linked one by one to extend the chain, a fragment consisting of several amino acids is synthesized in advance, and then each fragment is subjected to a coupling reaction. And the fragment condensation method, and the synthesis of the protein of the present invention may be carried out by any of them.

The condensation method adopted for the peptide synthesis described above also includes
Conventional methods can be followed, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPP
A (diphenylphosphoryl azide) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-norbornene-
2,3-dicarboximide) method and Woodward method.

The solvent that can be used in each of these methods can also be appropriately selected from well-known general solvents used in this kind of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof.

In the above peptide synthesis reaction, carboxyl groups in amino acids or peptides not involved in the reaction are generally esterified to lower alkyl esters such as methyl ester, ethyl ester and tertiary butyl ester such as benzyl ester. And aralkyl esters such as p-methoxybenzyl ester and p-nitrobenzyl ester.

An amino acid having a functional group in a side chain, for example, a hydroxyl group of a tyrosine residue may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group or the like. No need to do. Further, for example, a guanidino group of an arginine residue may be a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, an adamantyloxycarbonyl group or the like. Can be protected by various protecting groups.

The deprotection reaction of these protecting groups in the amino acids and peptides having the above protecting groups and the finally obtained protein of the present invention can also be carried out by a conventional method such as a catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride and the like. , Hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid and the like.

The protein of the present invention thus obtained is appropriately purified according to the above-mentioned various methods, for example, methods widely used in the field of peptide chemistry such as ion exchange resin, partition chromatography, gel chromatography, and countercurrent partitioning. It can be carried out.

The MASL1 protein of the present invention can be suitably used as an immunizing antigen for preparing its specific antibody, and by using this antigen, desired antisera (polyclonal antibody) and monoclonal antibody can be obtained. it can.

The method of producing the antibody itself is well understood by those skilled in the art, and the present invention can also follow these conventional methods [for example, Seijisei Kagaku Kenkyusho “Immunobiochemical Research Method”, Japan See Biochemical Society (ed., 1986).

The antibody thus obtained can be advantageously used, for example, for purification of the MASL1 protein and its measurement or identification by immunological techniques. More specifically,
Since amplification and enhanced expression of the gene of the present invention have been confirmed in cancer cells, cancer diagnosis or
It can be used to determine the malignancy of cancer.

Further, it can be used as a therapeutic agent for cancer containing an antibody that binds to MASL1 as an active ingredient.

Therefore, the antibody that binds to the MASL1 protein of the present invention is useful in the field of medicine as a drug containing it as an active ingredient. Therefore, the present invention also provides a pharmaceutical composition containing the protein of the present invention as an active ingredient.

The protein as an active ingredient in the pharmaceutical composition also includes a pharmaceutically acceptable salt thereof.
Such salts include those prepared by methods well known in the art,
For example, sodium, potassium, lithium, calcium,
Non-toxic alkali metal salts such as magnesium, barium and ammonium, alkaline earth metal salts, ammonium salts and the like are included. Further, the above salts also include non-toxic acid addition salts obtained by reacting the protein of the present invention with a suitable organic or inorganic acid. Representative non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, Borate, benzoate, lactate, phosphate, p-
Toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate and napsilate Is exemplified.

The above-mentioned pharmaceutical compositions include those containing the protein of the present invention as an active ingredient and a pharmaceutically effective amount thereof together with a suitable nontoxic pharmaceutical carrier or diluent.

Pharmaceutical carriers that can be used in the above-mentioned pharmaceutical composition (pharmaceutical preparation) include fillers, extenders, binders, humectants, disintegrants, and surfactants that are usually used depending on the use form of the preparation And diluents such as lubricants or excipients, which are appropriately selected and used according to the dosage unit form of the obtained preparation.

Particularly preferred pharmaceutical preparations of the present invention include various components which can be used in ordinary protein preparations, for example, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH adjusters, surfactants It is prepared by using such as appropriate.

Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, cellulose derivatives, and the like. These can be used alone or in combination with a surfactant. In particular, according to this combination, there are cases where the stability of the active ingredient can be further improved.

The L-amino acid is not particularly limited, and may be, for example, any of glycine, cysteine, glutamic acid and the like.

The above-mentioned sugars are not particularly limited. For example, monosaccharides such as glucose, mannose, galactose and fructose; sugar alcohols such as mannitol, inositol and xylitol; disaccharides such as sucrose, maltose and lactose; dextran; Polysaccharides such as starch, chondroitin sulfate, and hyaluronic acid and derivatives thereof can be used.

The surfactant is not particularly limited, and both ionic and nonionic surfactants can be used. For example, polyoxyethylene glycol sorbitan alkyl ester-based, polyoxyethylene alkyl ether-based,
Sorbitan monoacyl esters, fatty acid glycerides and the like can be used.

The cellulose derivative is not particularly limited, and methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and the like can be used.

The saccharide is added in an amount of about 0.0001 mg or more, preferably about 0.01 to 1 mg / μg of the active ingredient.
Suitably, the range is about 10 mg. The amount of the surfactant added is about 0.00001 mg per 1 μg of the active ingredient.
It is appropriate that the amount is not less than about, preferably about 0.0001 to 0.01 mg. The amount of human serum albumin to be added is about 0.0001 mg or more, preferably about 0.001 to 0.1 mg per 1 μg of the active ingredient. The amino acid is suitably used in an amount of about 0.001 to 10 mg per 1 μg of the active ingredient.
The amount of the cellulose derivative to be added is about 0.00001 mg or more per 1 μg of the active ingredient, preferably about 0.00001 mg.
It is appropriate to set the range of about 01 to 0.1 mg.

The amount of the active ingredient contained in the pharmaceutical preparation of the present invention is appropriately selected from a wide range, but is usually about 0.00000.
It is appropriate that the content is in the range of about 1 to 70% by weight, preferably about 0.0001 to 5% by weight.

In the pharmaceutical preparation of the present invention, various additives,
For example, a buffer, an isotonic agent, a chelating agent and the like can be added. Here, examples of the buffer include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid, and / or a salt corresponding thereto (for example, alkali salts such as sodium salt, potassium salt, calcium salt, magnesium salt, etc.). Metal salts and alkaline earth metal salts). Examples of the tonicity agent include sodium chloride, potassium chloride, sugars, glycerin and the like. Examples of the chelating agent include sodium edetate and citric acid.

The pharmaceutical preparation of the present invention can be used as a solution preparation. Alternatively, the preparation can be freeze-dried to make it storable, and then dissolved in a buffer solution containing water, saline, or the like for use before use. It is also possible to use it after adjusting it to an appropriate concentration.

As the dosage unit form of the pharmaceutical preparation of the present invention, various forms can be selected according to the purpose of treatment. Representative examples are tablets, pills, powders, powders, granules, capsules Solid dosage forms, such as solutions, suspensions,
Includes liquid dosage forms such as emulsions, syrups and elixirs which, depending on the route of administration, may further be oral, parenteral,
They are classified into nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments and the like, and can be prepared, molded or prepared according to the usual methods.

For example, in the case of molding into a tablet form, the above-mentioned preparation carrier such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. Agent, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose,
Methylcellulose, binders such as polyvinylpyrrolidone, carboxymethylcellulose sodium, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate,
Disintegrating agents such as calcium carbonate, polyoxyethylene sorbitan fatty acid esters, surfactants such as sodium lauryl sulfate and stearic acid monoglyceride, disintegrating inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; quaternary ammonium bases; Absorption enhancers such as sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid, and lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol. Sourcing agents and the like can be used.

Further, the tablet may be a tablet coated with a usual coating, if necessary, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, or a double tablet or a multilayer tablet. Can also.

In the case of molding into the form of pills, pharmaceutical carriers such as glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin, and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran and agar can be used.

Capsules are usually prepared by mixing the active ingredient of the present invention with the above-mentioned various pharmaceutical carriers and filling the mixture into hard gelatin capsules, soft capsules and the like according to a conventional method.

Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs and the like containing conventional inert diluents, for example, water, and further include wetting agents, Auxiliaries such as emulsions and suspensions can be included, which are prepared according to conventional methods.

In preparing liquid dosage forms for parenteral administration, such as sterile aqueous to non-aqueous solutions, emulsions and suspensions, diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol,
Vegetable oils such as ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and olive oil can be used, and injectable organic esters such as ethyl oleate can be blended. These further include ordinary solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives,
A dispersant or the like can be added. Sterilization can be performed by, for example, a filtration operation of passing through a bacteria retaining filter, blending of a bactericide, irradiation treatment, heat treatment, and the like. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.

For shaping in the form of a suppository or a preparation for vaginal administration, for example, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glyceride and the like can be used as the preparation carrier.

When shaping into the form of an ointment such as a paste, cream, gel or the like, diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, propylene glycol, polyethylene glycol,
Vegetable oils such as silicon, bentonite and olive oil can be used.

A composition for nasal or sublingual administration can be prepared by a conventional method using well-known standard excipients.

The drug of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like. For example, tablets, pills,
Solutions, suspensions, emulsions, granules and capsules are administered orally, injections are administered alone or mixed with normal replenishers such as glucose and amino acids, and administered intravenously. Intradermal, subcutaneous or intraperitoneal administration, suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, sublingual agents are administered intraorally, ointments are transdermal Is administered locally.

The amount of the active ingredient to be contained in the above-mentioned pharmaceutical preparation and the dose thereof are not particularly limited, and may be determined according to the desired therapeutic effect, administration method, treatment period, patient age, sex and other conditions. It is appropriately selected from a wide range. In general,
The dose is usually about 0.0 / kg of body weight per day.
About 1 μg to 10 mg, preferably about 0.1 μg to 1 m
g, and the preparation can be administered once or several times a day.

The present invention also provides a method for producing RNA having a sequence complementary to intracellular mRNA in cells expressing the MASL1 gene of the present invention, inhibiting translation,
A gene therapy method for cancer by providing an antisense drug for suppressing the expression of one gene is provided. The method of treatment involves inhibiting the transcription or translation process by binding to the native mRNA of the cancer cell carrying the MASL1 gene, or by intercalating between DNA double helices to form a triplex, for example. This is a method for suppressing the expression of a gene that causes the gene. For this purpose, it can be considered as a method for producing an antisense oligonucleotide complementary to the mRNA of the gene and supplying the antisense oligonucleotide to the target cell. Providing such an action of suppressing the expression function of the MASL1 gene can suppress the growth of neoplasms in recipient cells / target cells. It can be maintained extrachromosomally using a vector or plasmid containing the antisense oligonucleotide and introduced into a target cell.

According to the cancer gene therapy using the antisense oligonucleotide, the antisense oligonucleotide is incorporated into a vector derived from a retrovirus, an adenovirus, or an AAV, and the resultant is infected to a cancer cell to produce the antisense oligonucleotide. By overexpressing the oligonucleotide, a desired antitumor effect can be obtained.

When the antisense oligonucleotide is introduced into cells having the MASL1 gene to suppress the expression of the MASL1 protein, the antisense oligonucleotide must correspond to the full length of the corresponding MASL1 gene. For example, as long as it retains a function substantially equivalent to the function of suppressing the expression function of the MASL1 gene, even the above-mentioned variant or a gene consisting of a partial sequence retaining a specific function can be used. Can also be used.

Vectors for introducing a desired gene for both recombination and extrachromosomal maintenance are already known in the art, and any of such known vectors can be used in the present invention. For example, a viral vector or a plasmid vector containing a copy of the MASL1 antisense oligonucleotide linked to the expression control element and capable of expressing the antisense oligonucleotide product in a target cell can be mentioned. As such a vector, the above-mentioned expression vector can be usually used, but preferably, for example, the vector disclosed in US Pat. No. 5,252,479 and PCT International Publication WO93 / 07282 (for example, as a source vector) pWP-7A, pWP-19, pWU-
1, pWP-8A, pWP-21 and / or pRSVL
And pRC / CMV (manufactured by Invitrogen) and the like. More preferred are various viral vectors described below.

As a promoter used for a vector used in gene transfer therapy, a promoter specific to an affected tissue to be treated for various diseases can be suitably used.

Specific examples thereof include, for liver, albumin, α-fetoprotein, α1-antitrypsin, transferrin, transstyrene and the like. For the colon, examples thereof include carboxylate anhydrase I and carcinoembrogen antigen. For the uterus and placenta, estrogen, aromatase, cytocytochrome P450, cholesterol side chain cleavage P450, 17 alpha-hydroxylase P4
50 and the like.

For the prostate, the prostate antigen, gp91
-Fox gene, prostate-specific kallikrein and the like. For the breast, erb-B2, erb-
Examples include B3, β-casein, β-lactoglobin, whey protein and the like. For the lung, the activator protein C uroglobulin can be exemplified. For skin, K-1
Examples thereof include 4-keratin, human keratin 1 or 6, and leucrin.

For the brain, glial fibrillary acidic protein,
Examples include mature astrocyte-specific proteins, myelin, tyrosine hydroxylase pancreatic villin, glucagon, amyloid polypeptide in the islet of Langerhans, and the like. For the thyroid gland, thyroglobulin, calcitonin and the like can be exemplified. For bone, α1 collagen, osteocalcin, bone sialoglycoprotein and the like can be exemplified.
For the kidney, renin, liver / bone / kidney alkaline phosphatase, erythropoietin and the like can be exemplified, and for the pancreas, amylase, PAP1 and the like can be exemplified.

In the production of the vector for introducing an antisense oligonucleotide, the introduced antisense oligonucleotide (all or a part of the complementary sequence corresponding to the MASL1 gene sequence) is included in the base sequence information of the MASL1 gene of the present invention. As described above, it can be easily manufactured and obtained by a general genetic engineering technique.

The introduction of such antisense oligonucleotide introduction vectors into cells is already known in the art for introducing DNA into cells, including, for example, electroporation, calcium phosphate co-precipitation, and virus transduction. Can be performed according to various methods. In addition, cells transformed with antisense oligonucleotides by MASL1 can be used as a drug for cancer suppression or cancer metastasis suppression in isolation, or as a model system for therapeutic research. It is.

In gene therapy, the above-mentioned vector for introducing an antisense oligonucleotide can be introduced into a patient's tumor cell by injecting locally or systemically into a patient's tumor site. At this time, systemic administration can reach any tumor cells that can metastasize to other sites. If the transduced gene is not permanently integrated into the chromosome of each target tumor cell, this can be achieved by repeating the administration periodically.

The gene therapy method of the present invention includes an in vivo method in which the above-described material for introducing an antisense oligonucleotide (an antisense oligonucleotide introduction vector) is directly administered to a body, Once the target cells are removed, the gene is introduced outside the body, and then the cells are returned to the body.
o) Include both methods of the method.

In addition, MASL1 can be directly introduced into cells by introducing an antisense oligonucleotide, and gene therapy using a ribozyme, which is an active molecule that cleaves an RNA strand, is also possible.

A gene transfer vector containing all or a fragment of an antisense oligonucleotide having a sequence corresponding to MASL1 of the present invention, and a cell into which human MASL1 has been introduced by the vector, as an active ingredient. The gene therapy agent of the present invention
In particular, the above-mentioned gene therapy (treatment) can be used for the treatment of hereditary diseases, viral diseases such as AIDS, and gene labeling in addition to cancer. .

The target cell into which the antisense oligonucleotide is introduced can be appropriately selected depending on the target of gene therapy (treatment). For example, as target cells, besides cancer cells and tumor tissues, lymphocytes, fibroblasts,
Examples include hepatocytes, hematopoietic stem cells, cells such as, and the like.

The method of introducing an antisense oligonucleotide in the above gene therapy includes a viral introduction method and a non-viral introduction method.

[0138] As a viral introduction method, for example, MA
In view of the fact that SL1 is a foreign substance whose antisense oligonucleotide is expressed in normal cells, a method using a retrovirus vector as a vector can be mentioned. Other viral vectors include adenovirus vectors, HIV (human immunodeficiency vir)
us) vector, adeno-associated virus vector (AAV,
adeno-associated virus), herpes virus vector, herpes simplex virus (HSV) vector, and Epstein-Barr virus (EBV).
s) Vectors and the like.

[0139] Non-viral gene transfer methods include:
Calcium phosphate coprecipitation method: Membrane-fused liposome in which DNA-encapsulated liposome is fused with inactivated Sendai virus whose gene has been disrupted in advance by ultraviolet rays to produce a membrane-fused liposome, and directly fused with a cell membrane to introduce DNA into cells Method (Kato, K., et al., J. Biol. Chem., 266, 22071-2207
4 (1991)]; a method of coating plasmid DNA with gold and physically introducing the DNA into cells by high-pressure discharge [Ya
ng, NS et al., Proc. Natl. Acad. Sci., 87, 9568-9572 (1
990)]; a naked DNA method for injecting plasmid DNA directly into organs and tumors in vivo [Wolff, JA,
et al., Science, 247, 1465-1467 (1990)]; Cationic liposome method for introducing genes embedded in multilamellar positively charged liposomes into cells [Kunio Yagi, History of Medicine, Vol.
175, No. 9, 635-637 (1995)]; In order to introduce a gene only into a specific cell and prevent the gene from entering another cell, a ligand binding to a receptor expressed in a target cell is used.
Ligand-DNA complex method for binding and administering NA (Frindeis, et al., Trends Biotechnol., 11, 202 (1
993); Miller, et al., FASEB J., 9, 190 (1995)].

The ligand-DNA complex method includes, for example, a method using asialoglycoprotein as a ligand targeting asialoglycoprotein receptor expressed by hepatocytes [Wu, et al., J. Biol. Chem., 266, 14338 (1991); Ferko
l, et al., FASEB J., 7, 1081-1091 (1993)], and a method using transferrin as a ligand targeting a transferrin receptor strongly expressed by tumor cells [Wagner et al., Proc. Natl. .Acad.Sci., USA., 87,3410
(1990)].

The gene transfer method used in the present invention is as follows.
Any of the various biological and physical gene transfer methods described above may be appropriately combined. Examples of the method by the combination include a method of combining a plasmid DNA of a certain size with a polylysine-conjugated antibody specific to an adenovirus hexon protein. According to this method, the antisense oligonucleotide of the present invention can be introduced by binding the obtained complex to the adenovirus vector and infecting the cell with the thus obtained trimolecular complex. This method allows for efficient binding, internalization and endosomal degradation before the DNA coupled to the adenovirus vector is damaged. In addition, the liposome / DNA complex can directly mediate gene transfer in vivo.

Hereinafter, a specific method for preparing a virus vector for introducing an antisense oligonucleotide of the present invention and a method for introducing an antisense oligonucleotide into a target cell or target tissue will be described.

[0143] The retroviral vector system comprises a viral vector and a helper cell (packaging cell).
Consists of Here, the helper cells previously express genes such as retroviral structural proteins gag (structural protein in virus particles), pol (reverse transcriptase), and env (envelope protein). Say not cells. On the other hand, viral vectors use packaging signals or LTRs (long terminal repeats).
s), but gag, po necessary for virus replication
It does not have structural genes such as l and env. The packaging signal is a sequence that serves as a tag during the assembly of the virus particles, and includes the selection gene (neo, hyg) and the desired introduced antisense /
An oligonucleotide (the entire antisense oligonucleotide corresponding to MASL1 or a fragment thereof) is inserted in place of the viral gene. Here, in order to obtain a high titer virus particle, it is important to shorten the insert as much as possible, widen the packaging signal including a part of the gag gene, and not leave the ATG of the gag gene. is there.

By transferring the vector DNA incorporating the desired MASL1 into the antisense oligonucleotide into the helper cells, the vector structural RNA is packaged by the viral structural proteins produced by the helper cells to form virus particles, which are secreted. You. After a virus particle as a recombinant virus infects a target cell, DNA reversely transcribed from viral genomic RNA is integrated into a cell nucleus, and an antisense gene inserted into a vector is expressed.

As a method for increasing the efficiency of introducing a desired gene, a method using a fragment containing a cell adhesion domain of fibronectin, a heparin-binding site and a junction segment [Hanenberg, H., et al., Exp. Hemat., 23,747 (1995)].

The vector used in the above retrovirus vector system includes, for example, a retrovirus [McLa
chlin, JR, et al., Proc. Natl. Acad. Res. Molec.
Biol., 38, 91-135 (1990)].

The method using an adenovirus vector will be described in detail.
Berkner, KL, Curr.Topics Microbiol.Imm
unol., 158, 39-66 (1992)), Yasuhiro Setoguchi et al. (Setoguch
i, Y., et al., Blood, 84,2946-2953 (1994)), Hiromi Kaneka et al. (Experimental Medicine, 12, 28-34 (1994)) and Kenner et al. (K
etner, G., et al., Proc. Natl. Acad. Sci., USA., 91,6186-
6190 (1994)].

For example, to construct a non-competent adenovirus vector, the E.
Remove the 1 and / or E3 gene region. Next, the desired desired foreign gene expression unit (the desired introduced antisense oligonucleotide, ie, MASL1, the antisense oligonucleotide of the present invention, a promoter for transcribing the antisense oligonucleotide, the transcribed gene) And a plasmid vector containing a part of the adenovirus genomic DNA, and a plasmid containing the adenovirus genome.
For example, 293 cells are transfected simultaneously.
By causing homologous recombination between the two and replacing the gene expression unit with E1, the non-proliferative adenovirus which is the gene therapy vector of the present invention containing the desired MASL1 antisense oligonucleotide Vectors can be created. In addition, an adenovirus genomic DNA can be incorporated into a cosmid vector to prepare a 3 'adenovirus vector to which a terminal protein has been added. Furthermore, a YAC vector can also be used for producing a recombinant adenovirus vector.

Briefly describing the production of the adeno-associated virus (AAV) vector, AAV was discovered as a small virus that entered the adenovirus culture system. This confirms the existence of the genus Parvovirus, which grows autonomously in host cells without requiring a helper virus for virus replication, and the genus Dependovirus, which requires a helper virus. The AAV is a common virus that has a wide host range and infects a variety of cells. The virus genome is composed of linear single-stranded DNA having a size of 4680 bases, and 145 bases at both ends are ITRs (inverted terminal).
exists) with a characteristic sequence called "repeat". This ITR portion serves as an origin of replication and serves as a primer. Furthermore, the ITR is also essential for packaging into virus particles and integration into chromosomal DNA of host cells. As for viral proteins, the left half of the genome encodes a nonstructural protein, that is, Rep, a regulatory protein that controls replication and transcription.

The production of recombinant AAV was performed by
It can be carried out using the property of being incorporated into NA, and thus a desired gene transfer vector can be prepared. More specifically, this method uses a plasmid (AAV) in which the ITRs at both ends of 5 ′ and 3 ′ of wild-type AAV are left, and a desired antisense oligonucleotide for introduction (MASL1 antisense oligonucleotide) is inserted between them. Vector plasmid). On the other hand, viral proteins required for virus replication and virus particle formation are supplied by another helper plasmid. It is necessary to prevent a common base sequence from being present between the two, and to prevent the appearance of wild-type virus due to genetic recombination. Thereafter, both plasmids are introduced, for example, by transfection into 293 cells, and further infected with an adenovirus (if 293 cells are used, a non-proliferating type) may be used as a helper virus to obtain a desired non-proliferating set. A recombinant AAV is produced. Subsequently, since the recombinant AAV is present in the nucleus, the cells are freeze-thawed and recovered, and the contaminating adenovirus is inactivated by heating at 56 ° C. Further, if necessary, the recombinant AAV is separated and concentrated by ultracentrifugation using cesium chloride. As described above, a desired recombinant AAV for gene introduction can be obtained.

The EBV vector can be produced, for example, according to the method of Shimizu et al. [Norio Shimizu, Kenzo Takada,
Cell Engineering, 14 (3), 280-287 (1995)].

The production of the EBV vector for introducing an antisense oligonucleotide of the present invention is briefly described as follows.
The virus (Epstein-Barr virus: EBV) is a virus belonging to the family Herpes isolated from cultured cells derived from Burkitt's lymphoma in 1964 by Epstein et al. [Kieff, E. and Liebowitz, D. .: Vir
ology, 2nd ed. Raven Press, New York, 1990, pp.188
9-1920]. Since the EBV has the activity of transforming cells, in order to use it as a vector for gene transfer,
Viruses lacking this transforming activity must be prepared. This can be done as follows.

That is, first, the EBV genome near the target DNA into which the desired foreign gene is to be incorporated is cloned. The DNA fragment of the foreign gene and the drug resistance gene are inserted therein, and used as a vector for producing a recombinant virus. Next, the EBV-positive Akata cells are transfected with the vector for producing a recombinant virus cut out by an appropriate restriction enzyme. Recombinant virus generated by homologous recombination can be recovered together with wild-type Akata EBV by stimulating virus production by anti-surface immunoglobulin treatment. This is EBV
By infecting negative Akata cells and selecting resistant strains in the presence of the drug, Akata cells infected with only the desired recombinant virus without wild-type EBV can be obtained. Further, by inducing the virus activity in the recombinant virus-infected Akata cells, a large amount of the desired recombinant virus vector can be produced.

The production of a non-viral vector in which a desired antisense oligonucleotide is introduced into a target cell without using a recombinant viral vector can be performed, for example, by a gene transfer method using membrane-fused liposomes. These are membrane liposomes (vesicles composed of lipid bilayers)
In this method, the content of the liposome is directly introduced into cells by giving the cell a fusion activity to the cell membrane.

The introduction of the antisense oligonucleotide using the membrane-fused liposome can be carried out, for example, by the method of Nakanishi et al. [Nakanishi, M., et al., Exp.
ll Res., 159, 399-499 (1985); Nakanishi, M., et al., Ge.
ne introduction into animal tissues.In Trends and
Future Perspectives in Peptide and Protein DrugDel
ivery (ed. by Lee, VH et al.)., Harwood Academic
Publishers Gmbh. Amsterdam, 1995, pp. 337-349].

Hereinafter, a method for introducing an antisense oligonucleotide using the membrane-fused liposome will be briefly described. That is, a Sendai virus whose gene has been inactivated by ultraviolet rays and a liposome encapsulating a high molecular substance such as a desired antisense oligonucleotide or expressed protein are fused at 37 ° C. This membrane-fused liposome has a structure called a pseudovirus in which a liposome-derived cavity is present on the inside and the same spike as the virus envelope is on the outside. After further purification by sucrose density gradient centrifugation, the membrane-fused liposome is adsorbed at 4 ° C. to the target cultured cells or tissue cells. Then, at 37 ° C., the contents of the liposome are introduced into the cells, and the desired antisense oligonucleotide can be introduced into the target cells. Here, as the lipid used as the liposome, it is preferable to prepare and use a unilamellar liposome having a diameter of 300 nm using 50% (molar ratio) cholesterol, lecithin, and a synthetic phospholipid having a negative charge.

As a method for introducing an antisense oligonucleotide into a target cell using another liposome, an antisense oligonucleotide using a cationic liposome is used.
An oligonucleotide introduction method can be mentioned. The method can be carried out according to the method of Yagi et al. [Yagi, K., et a
l., BBRC, 196, 1042-1048 (1993)]. This method focuses on the fact that both plasmids and cells are negatively charged, and gives a positive charge to both the inside and outside of the liposome membrane to increase plasmid uptake by static electricity and enhance interaction with cells. Is what you do. The liposome used here is a multilamellar large liposome (multil
Although amellar large vesicles (MLV) is useful, large unilamellar vesicles:
LUV) and small unilamellar liposomes (small unilamella)
R vesicles (SUV) can be used to form a complex with the plasmid, and the desired antisense oligonucleotide can be introduced.

The method for preparing plasmid-embedded cationic MLV is first described.
(α-trimethylammonioacetyl) -didodecyl-D-glutamate
chloride), DLPC (dilauroyl phosphatidylcholin)
e) and DOPE (dioleoyl phosphatidylethanolamin)
A chloroform solution (1 mM in lipid concentration) containing e) in a molar ratio of 1: 2: 2 is prepared. Next, a total amount of 1 μmol of lipid is put into a Spitz-type test tube, and chloroform is removed under reduced pressure using a rotary evaporator to prepare a lipid thin film. Further, chloroform is completely removed under reduced pressure and dried. Then, 0.5 ml of Dulbecco's phosphate-buffered saline containing Mg and Ca containing 20 µg of the plasmid for gene transfer was added, and after purging with nitrogen gas, the mixture was stirred with a vortex mixer for 2 minutes to give the desired antisense. A plasmid-embedded cationic MLV suspension containing the oligonucleotide can be obtained.

An example of using the plasmid-embedded cationic MLV obtained above as a gene therapy agent is as follows:
For example, an expression plasmid incorporating an antisense oligonucleotide for expression purpose is added to the above cationic MLV by D.
NA amount: 0.6 μg, liposome lipid amount: 30 n
For example, a method of embedding to a molar concentration, suspending the suspension in 2 μl of a phosphate buffered saline, and administering the cells to target cells or patient tissues extracted from a patient every other day can be exemplified.

By the way, gene therapy is defined in the Ministry of Health and Welfare guidelines as "administering a gene or a cell into which a gene is introduced into a human body for the purpose of treating a disease". However, gene therapy in the present invention means, in addition to the definition of the guideline, an antisense DNA which is characterized as a tumor-suppressing antisense DNA of a MASL1 antisense oligonucleotide in the target cell described above.
By introducing the oligonucleotide, the method includes not only the treatment of various diseases including cancer, but also the introduction of a gene as a label or cells into which a gene as a label has been introduced into a human body.

In the gene therapy of the present invention, the method of introducing a desired gene into a target cell or target tissue typically includes two types of methods.

In the first method, after collecting target cells from a patient to be treated, the cells are cultured outside the body under the addition of, for example, interleukin-2 (IL-2) and the like. This is a method (ex vivo method) of re-transplanting the obtained cells after introducing antisense oligonucleotide into the contained MASL1 of interest. The method is ADA
It is suitable for treating deficiencies, genetic diseases caused by defective genes, cancer, AIDS, and the like.

In the second method, the target antisense oligonucleotide (MASL1 antisense oligonucleotide)
Is a direct gene transfer method (direct method) in which is directly injected into a target body such as a patient's body or tumor tissue.

The first method of the gene therapy is performed in more detail, for example, as follows. That is, mononuclear cells collected from a patient are collected from monocytes using a blood separator, and the collected cells are cultured in an appropriate medium such as AIM-V medium for about 72 hours in the presence of IL-2, Add the vector containing the antisense oligonucleotide to be introduced (MASL1 antisense oligonucleotide). In order to increase the introduction efficiency of the antisense oligonucleotide, the mixture was centrifuged at 2500 rpm for 1 hour at 32 ° C. in the presence of protamine, and then centrifuged at 37 ° C. under 10% CO 2 for 24 hours.
You may culture for hours. After repeating this operation several times, the cells were further cultured for 48 hours in an AIM-V medium or the like in the presence of IL-2, the cells were washed with physiological saline, the number of viable cells was calculated, and the efficiency of introduction of the antisense oligonucleotide was increased. The in situ
u If the desired target is an enzyme activity, for example, by measuring the degree of the activity,
Confirm the oligonucleotide introduction effect.

In addition, the safety is checked by culturing bacteria and fungi in the cultured cells, detecting the presence or absence of mycoplasma infection, searching for endotoxin, etc., and confirming the safety. (MA
The cultured cells into which the SL1 antisense oligonucleotide has been introduced are returned to the patient by intravenous infusion. Gene therapy is performed by repeating such a method at intervals of several weeks to several months, for example.

Here, the dosage of the viral vector is appropriately selected depending on the target cells to be introduced. Generally, the virus titer is, for example, 1 × 1 for 1 × 10 ⁸ target cells.
It is preferable to adopt a dose in the range of 0 ³ cfu to 1 × 10 ⁸ cfu.

As an alternative to the first method, a virus-producing cell containing a retroviral vector containing a target antisense oligonucleotide (MASL1 antisense oligonucleotide) and, for example, a patient's cell are co-cultured. A method of introducing an antisense oligonucleotide (MASL1 antisense oligonucleotide) into a target cell can also be adopted.

In carrying out the second method (direct method) of gene therapy, the target antisense oligonucleotide (MASL1 antisense oligonucleotide) is actually obtained by a gene transfer method, especially by a preliminary experiment in vitro.
Whether or not is introduced is confirmed in advance by searching the vector gene cDNA by PCR or in situ PCR, or a desired therapeutic effect based on the introduction of the target antisense oligonucleotide (MASL1 antisense oligonucleotide). It is desirable to confirm the increase in specific activity, the increase in growth of target cells, and the suppression of growth. When a viral vector is used, a search for a replication-competent retrovirus or the like is performed by the PCR method, reverse transcriptase activity is measured, or the membrane protein (env) gene is
Needless to say, it is important to confirm the safety by introducing antisense oligonucleotides during gene therapy, for example, by monitoring with a method.

In the gene therapy method of the present invention, particularly when targeting cancer or malignant tumor, after collecting cancer cells from the patient,
After enzymatic treatment and the like to establish cultured cells, the cells are introduced into cancer cells targeting the desired antisense oligonucleotide using, for example, a retrovirus, and screened in G418 cells. Measure (i
n vivo measurement), followed by radiation treatment, and inoculating the tumor in or inside a patient's tumor.

The present invention also provides, as an active ingredient, a cell into which the vector for introducing an antisense oligonucleotide of the present invention or a target antisense oligonucleotide (such as MASL1 antisense oligonucleotide) has been introduced as an active ingredient. Provide a pharmaceutical composition or a pharmaceutical preparation (gene therapeutic agent) containing an effective amount together with a suitable non-toxic pharmaceutical carrier or diluent.

Pharmaceutical carriers which can be used in the pharmaceutical composition (pharmaceutical preparation) of the present invention include fillers, extenders, binders, humectants, disintegrants and the like which are usually used according to the use form of the preparation.
Diluents or excipients such as surfactants and lubricants can be exemplified, and these can be appropriately selected and used depending on the dosage unit form of the resulting preparation.

The dosage unit form of the pharmaceutical preparation of the present invention includes
Formulation examples of the MASL1 protein antibody preparation described above can be mentioned in the same manner, and can be appropriately selected from various forms according to the purpose of treatment.

For example, a pharmaceutical preparation containing the vector for introducing an antisense oligonucleotide of the present invention may be a virus containing a retrovirus vector in which the vector is embedded in a liposome or a desired antisense oligonucleotide is included. Prepared in the form of infected cells.

These are phosphate buffered saline (pH
7.4), can be prepared in a form mixed in Ringer's solution, injection for intracellular composition liquid, etc., or can be prepared in a form to be administered together with a substance such as protamine which enhances gene transfer efficiency. .

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like.

The amount of the active ingredient to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and may be selected according to the desired therapeutic effect, administration method, treatment period, patient age, gender and other conditions. It is appropriately selected from a wide range.

Generally, the dosage of the desired antisense oligonucleotide-containing retroviral vector as a pharmaceutical preparation is from about 1 × 10 ³ pfu to 1 × 10 ¹⁵ per kg body weight per day, for example, as a retrovirus titer.
It is good to be about pfu.

In the case of cells into which a desired antisense oligonucleotide for introduction has been introduced, it is appropriate to select from a range of about 1 × 10 ⁴ cells / body to about 1 × 10 ¹⁵ cells / body.

The preparation may be administered once or several times a day, or may be administered intermittently at intervals of one to several weeks. Preferably, it can be co-administered with a substance such as protamine which enhances gene transfer efficiency or a preparation containing the same.

When the gene therapy according to the present invention is applied to the treatment of cancer, the above-mentioned various gene therapies can be appropriately combined (combined gene therapy). , Radiation therapy, immunotherapy and the like. Furthermore, the gene therapy of the present invention can be carried out with reference to NIH guidelines including its safety [Recombinant DNA Advi
sory Committee, Human Gene Therapy, 4, 365-389 (19
93)].

According to the present invention, a MA that promotes tumorigenesis of cells
To detect the presence of the SL1 gene, prepare a biological sample, such as blood or serum, optionally extract nucleic acids,
It is possible to analyze whether a susceptibility gene for MASL1 is present. Also, according to the present invention, to detect neoplasms in cells or tissues, progression to a malignant progenitor disorder, or presence as a prognostic indicator, preparing a biological sample having a disorder, MASL1 It is possible to analyze whether or not neoplastic genes are present. By using this method, it is possible to detect neoplasms in cells or tissues, progression to a malignant progenitor disorder, or presence as a prognostic indicator, and diagnose them, for example, diagnosis of cancer and determination of cancer therapeutic effect, and The prognosis can be predicted.

In the detection method, for example, the DNA fragment is prepared based on information on the MASL1 gene obtained in advance from a patient sample having a tumor and is designed to be used for screening and / or amplification of the MASL1 gene. Is done. More specifically, for example, MASL1 amplified by a polymerase chain reaction (PCR) that amplifies a nucleic acid sequence with a polymerase, such as those having a property as a probe in plaque hybridization, colony hybridization, Southern blotting, Northern blotting, and the like. A probe having properties as a probe for obtaining all or part of a DNA fragment can be prepared. For this purpose, first, a primer having the same sequence as that of MASL1 is prepared and used as a screening probe to react with a biological sample (nucleic acid sample), whereby the presence of the gene having the MASL1 sequence can be confirmed. The nucleic acid sample may be prepared by various methods that facilitate detection of the target sequence, such as denaturation, restriction digestion, electrophoresis or dot blotting.

As the screening method, in particular,
From the viewpoint of sensitivity, it is preferable to use the CR method.
The method is not particularly limited as long as the method uses the MASL1 fragment as a primer, and a conventionally known method (Science, 230, 1350-13
54 (1985)) and a newly developed or future modified PCR method (Yoshiyuki Sakaki, et al., Yodosha, Experimental Medicine, extra edition,
8 (9) (1990); Protein, nucleic acid, enzyme, extra edition, Kyoritsu Publishing
Co., Ltd., 35 (17) (1990)) can be used.

The DNA fragment used as a primer is a chemically synthesized oligo DNA.
NA can be synthesized using an automatic DNA synthesizer or the like, for example, a DNA synthesizer (Pharmacia LKB Gene Assembler Plus: manufactured by Pharmacia). The length of the synthesized primer (sense primer or antisense primer) is preferably about 10 to 30 nucleotides. The probe used for the above screening is usually a labeled probe, but may be unlabeled or may be detected by specific binding to a directly or indirectly labeled ligand. Suitable labels, and methods for labeling probes and ligands, are known in the art of the invention and include nick translation,
These techniques include radiolabels, biotin, fluorescent groups, chemiluminescent groups, enzymes, antibodies, and the like, which can be incorporated by known methods, such as random priming or kinase treatment.

As the PCR method used for the detection, for example, the RT-PCR method is exemplified, but various modified methods used in the art can be applied.

[0186] The measurement method of the present invention can be used for measuring MASL in a sample.
By using a reagent kit for detecting one gene, it can be easily carried out.

Therefore, the present invention provides a reagent kit for detecting MASL1, comprising the above-mentioned MASL1 DNA fragment.

The reagent kit comprises at least SEQ ID NO: 2
If a DNA fragment that hybridizes to a part or all of the base sequence shown in or a complementary base sequence thereof is contained as an essential component, a labeling agent, PC
Indispensable reagents for R method (for example, Taq DNA polymerase, deoxynucleotide triphosphate, primer, etc.)
May be included.

Examples of the labeling agent include a chemical modifying substance such as a radioisotope or a fluorescent substance, and the DNA fragment itself may be conjugated with the labeling agent in advance. Further, the reagent kit may contain a suitable reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., for the benefit of performing the measurement.

Furthermore, the present invention also provides a method for diagnosing cancer using the above-mentioned measuring method, a diagnostic agent and a diagnostic kit used in the method.

Further, the MASL1 sequence obtained from the test sample can be directly or indirectly sequenced by using the above-mentioned method, thereby obtaining a new MASL1 homolog having high homology with wild-type MASL1. Related genes related to genes can be found.

Accordingly, the present invention provides a method for determining human MA in a test sample by such measurement and sequencing of MASL1 DNA in the test sample.
It also provides a method of screening for a related gene related to the SL1 gene.

Also, a protein encoded by the human MASL1 gene represented by SEQ ID NO: 1 of the present invention, or an amino acid in which one or several or more amino acids are deleted, substituted or added in SEQ ID NO: 1 By synthesizing a protein from a sequence or a fragment thereof, or synthesizing an antibody against the protein, it becomes possible to measure wild-type MASL1 and / or mutant MASL1.

Accordingly, the present invention provides a method for assaying an antibody and an antigen for wild-type MASL1 and / or mutant MASL1. The degree of neoplastic condition damage or the degree of malignancy of a malignant tumor can also be detected by the assay based on a change in a wild-type MASL1 polypeptide. Such alterations can also be determined by MASL1 sequence analysis according to the conventional techniques in this field, but more preferably, using an antibody (polyclonal or monoclonal antibody),
Differences in the protein or the presence or absence of the MASL1 protein can be detected. Specific examples of the measurement method of the present invention include:
The MASL1 antibody immunoprecipitates the MASL1 protein from a solution containing a biological material sample collected from a human such as blood or serum, and can react with the MASL1 protein on a Western blot or immunoblot of a polyacrylamide gel. The MASL1 antibody can detect MASL1 protein in paraffin or frozen tissue sections using immunohistochemical techniques. Since antibody production technology and purification technology are well known in the art, these technologies can be appropriately selected.

More preferred embodiments related to the method for detecting wild-type MASL1 or a mutant thereof include an enzyme-linked immunosorbent assay including a sandwich method using a monoclonal antibody and / or a polyclonal antibody.
(ELISA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), and immunoenzymatic assay (IEMA).

The present invention also relates to a MASL1 protein for MASL1 protein.
Exists on cell membrane fraction or cell surface with binding activity
MASL1 receptors can also be provided. The MAS
To obtain L1 receptor, labeled MASL1 protein is conjugated in a biomaterial sample containing a cell membrane fraction,
It is achieved by extracting, isolating and purifying the ASL1 binding reaction product and specifying the amino acid sequence of the isolated product. The acquisition and sequencing of the MASL1 receptor protein can be easily achieved by those skilled in the art.

The present invention also provides a method for screening a compound (MASL1 receptor reactant: a compound is a low molecular weight compound, a high molecular weight compound, a protein, or a low molecular weight compound) by using the MASL1 receptor protein or a binding fragment thereof in a technique for screening any of various drugs. (Referred to as protein partial fragments, antigens, or antibodies). Preferably,
Utilizes the MASL1 receptor. The MASL1 receptor polypeptide or fragment thereof used in such a screening test may be free in solution attached to a solid support or carried to the cell surface.

As an example of drug screening, for example, a prokaryotic or eukaryotic host cell stably transformed with a recombinant protein expressing the MASL1 protein or a fragment thereof is used, preferably in a competitive binding assay. be able to. Such cells, either in free or fixed form, can also be used in standard binding assays. More specifically, the formation of a complex between the MASL1 receptor protein or a fragment thereof and the substance to be tested is measured, and the formation of a complex between the MASL1 receptor protein or a fragment thereof and the MASL1 protein or a fragment thereof is determined. Compounds can be screened by detecting the degree of inhibition by the substance being tested.

Thus, the present invention provides for contacting such a substance with a MASL1 receptor protein or a fragment thereof by methods known in the art, and then determining the presence of a complex between the substance and the MASL1 receptor protein or a fragment thereof. Or MA
It is possible to provide a drug screening method characterized by measuring the presence of a complex between a SL1 receptor protein or a fragment thereof and a ligand. further,
By measuring MASL1 receptor activity, such substances can inhibit the MASL1 receptor, and thus bind to or dissociate from the MASL1 activity defined above, e.g., proteins that regulate cell proliferation and differentiation, thereby dissociating the binding protein. It is determined whether the concentration and translocation in blood and tissue can be regulated, and whether the activity itself can be regulated. In such a competitive binding assay, more specifically, the MASL1 receptor protein or a fragment thereof is labeled. Free MASL1 receptor protein or a fragment thereof is separated from that present in the protein: protein complex, and the amount of free (uncomplexed) label is determined by the amount of binding of the agent to be tested to the MASL1 receptor or MASL1 receptor: It is a measure of inhibition of MASL1 protein binding. By analyzing a small peptide (peptide mimetic) of the MASL1 protein in this way, one having MASL1 receptor inhibitory activity can be measured.

In the present invention, another method for drug screening is a screening method for a compound having an appropriate binding affinity for the MASL1 receptor protein. Synthesis on a solid support, such as the surface of a plastic pin or other material, followed by peptide test compound
React with MASL1 receptor protein and wash. Subsequently, a method for detecting the reaction-bound MASL1 receptor protein using a known method can also be exemplified (PCT Patent Publication No. WO
84-03564). Purified MASL1 receptor can be coated directly onto plates used in the drug screening techniques described above. However, supplementing the antibody with a non-neutralizing antibody against the polypeptide,
The MASL1 receptor protein can be immobilized on a solid phase. The present invention is also directed to the use of a competitive drug screening assay in which a test compound competes with a neutralizing antibody capable of specifically binding to the MASL1 receptor protein for binding to the MASL1 receptor protein or a fragment thereof. The competition by the antibody makes it possible to detect the presence of any peptide having one or more antigenic determinants of the MASL1 receptor protein.

Further, with respect to drug screening, additional methods include the use of host eukaryotic cell lines or cells that contain a non-functional MASL1 gene. After growing the host cell line or cells in the presence of the drug compound for a period of time, the rate of growth of the host cell is measured to allow the compound to bind or dissociate, for example, from proteins that regulate cell growth or differentiation. To control the concentration and translocation of the binding protein in blood and tissues, and whether the activity itself can be regulated. As one means of measuring the growth rate, the biological activity of the MASL1 receptor can be measured.

Further, according to the present invention, a more active or stable form of the MASL1 protein derivative or, for example, in vivo
In order to develop agents that enhance or interfere with the function of the MASL1 protein (in vivo), biologically active proteins or structural analogs for which they interact, such as MASL
1 Agonists, MASL1 antagonists, MASL1 inhibitors and the like can be produced. The structural analog may be, for example, a three-dimensional structure of a complex of MASL1 and another protein.
It can be determined by line crystallography, computer modeling, or a combination of these. Information on the structure of a structural analog can also be obtained by protein modeling based on the structure of a homologous protein.

The MASL having an active or stable form as described above
1 As a method for obtaining a protein derivative, for example, alanine
It is possible to analyze by scanning. The method involves substituting Ala for an amino acid residue and measuring each of the amino acid residues of the peptide in a manner that measures its effect on the activity of the peptide, thus determining regions important for the activity and stability of the peptide. Is the way. By this method, more active or stable MASL1 derivatives can be designed.

It is also possible to isolate a target-specific antibody selected by a functional assay and then analyze its crystal structure. In principle, this approach allows the pharmacoma (pharmac
ore). By generating anti-idiotypic antibodies to functional pharmacologically active antibodies, it is possible to identify and isolate peptides from a bank of chemically or biologically generated peptides. Therefore, the selected peptide is also expected to act as a pharmacore.

Thus, a drug having improved MASL1 activity or stability or an action as an inhibitor, agonist, antagonist or the like of MASL1 activity can be designed and developed.

With the cloned MASL1 sequence, a sufficient amount of MASL1 protein can be obtained for analytical studies such as X-ray crystallography. Further, SEQ ID NO:
By providing the MASL1 protein consisting of the amino acid sequence shown in No. 1, it is possible to adapt to computer modeling technology instead of or in addition to X-ray crystallography.

According to the present invention, which parts of the MASL1 gene sequence affect the various MASL1 activities in vivo as described above by preparing MASL1 gene-containing knockout mice (mutant mice). That is, MASL1
The function of the gene product and the modified MASL1 gene product in vivo can be confirmed.

This method is a technique for intentionally modifying the genetic information of an organism using homologous recombination of a gene, and includes a method using mouse embryonic stem cells (ES cells) (Capeccchi, MR, Science, 244, 1288-1292 (198
9)).

[0209] The method for producing the above mutant mouse is already a common technique for those skilled in the art,
(Edited by Tetsuo Noda, Experimental Medicine, extra edition, 14 (20) (1996), Youtosha), the human wild-type MASL1 gene and mutant MASL1 of the present invention.
Mutated mice can be easily produced by adapting the gene. Therefore, by the adaptation of the technique, improved MASL1 activity or stability or inhibitors of MASL1 activity, agonists,
Designing drugs that act as antagonists, etc.
Can be developed.

[0210]

According to the present invention, the binding or dissociation of a protein that regulates cell growth, differentiation and apoptosis, or the like, regulates the concentration or translocation of a bound protein in blood or tissue. Thus, a novel MASL1 gene having an action of regulating the activity itself is provided. The gene of the present invention is AL
Has similarity to S, and therefore can be used for research on the relationship between the functions of these related genes analyzed and various diseases, and can be used for gene diagnosis for various diseases and antibodies and antisense against expression products of the genes. Can be used for research on application to medical use. Further, according to the use of the gene of the present invention, the expression status of the gene in various tissues can be examined, and its function in a living body can be analyzed.

According to the gene, the MASL1 protein encoded by the gene can be produced in large quantities by genetic engineering. According to the protein, the MASL1 protein activity and the binding activity of the MASL1 protein can be improved. You can also check the function.

[0212] MASL1 protein is also involved in diseases involving the MASL1 gene and its product (eg, by binding to or dissociating from proteins that regulate cell growth, differentiation, and apoptosis, thereby causing the binding protein to be expressed in blood and tissues). It is useful for elucidation of pathological conditions, diagnosis, treatment, etc. of various diseases related to the action of regulating the concentration and translocation of, and the activity itself, particularly cancer.

According to the present invention, there is further provided a gene transfer vector containing a MASL1 antisense oligonucleotide, which is useful for gene therapy, a cell into which the MASL1 has been introduced an antisense oligonucleotide, and the vector or cell as an active ingredient. And a gene therapy method utilizing the same.

According to the present invention, MASL1 has an inhibitory effect on the growth of various cancer cells, and binds MASL1 used for treatment of various cancer diseases and conditions to the antisense oligonucleotide or MASL1. A drug containing the antibody as an active ingredient can also be provided.

[0215]

The present invention will now be described in further detail with reference to examples. Example 1 Materials and Methods 1) Early MFH Tumors In all 19 cases, early tumor tissue from newly diagnosed untreated patients was collected at surgical resection. DNA from frozen samples was extracted according to standard methods.

For CGH (Kallioniemi, OP, et a
l., Science, 258, 818-821 (1992)), DNA samples isolated from tumor specimens were analyzed using Spectrum-Green-
Directly labeled with dUTP (Spectrum-green-dUTP: Vysis), and normal DNA was labeled with Texas red-dUTP (DuPont) using nick translation.

Labeled tumor and normal DNA (180 ng each)
Was combined with 15 μg of normal Cot-1 DNA (Gibco-BRL) in 10 μl of hybridization solution (50% formamide, 10% dextran sulfate, 2 × SSC).
Denatured at 5 ° C. for 5 minutes, then adapted to normal (lymphocyte) metaphase extension.

Hybridization conditions and detection of signals were performed according to the cited literature (supervised by Toshiki Mori, Yoji Ariyama, Tatsuo Abe, edited by Joji Inazawa, clinical FISH protocol, extracellular cell engineering, 179-184 (1997)). ).

Three-color fluorescent image (DAPI, Spectrum-greeen)
and Texas-red fluoresence) are epifluoresence (surface fluorescence) and microscope (epifluoresence micr)
oscope: Nikon Corporation), cooling / charging coupling device (cooled charg
e-coupled device: CCD) Camera (Photometrics: Ph)
otometrics) were collected from each metaphase extension. The relative change in the copy number of the DNA sequence was analyzed using a digital image analysis system (Quips-XL software; manufactured by Vysis, Inc.). The fluorescence ratio is 1.
Above 5, the chromosomal region was considered to have a high level of amplification. Heterochromatin regions near the centromere and near all Y chromosomes were excluded from analysis.

CGH analysis revealed that 16 of the 19 tumors tested exhibited genomic imbalance. The lower representation of the minimal region of overlap was 3p25-pter (2 cases; 11%), 10p12-pter (2 cases; 11%), 13q21 (2 cases; 11%), and 18q22-qter (2
Above 4q12-13 (7 cases; 39%), 5p15.3-pter (5 cases; 28)
%), 8q24.1-qter (4 cases; 22%), 6p12-21 (3 cases; 1
(8%) and 12p12-pter (3 cases; 18%). High level amplification of DNA was confirmed at six different loci in each of one tumor: 4q12-21,
8p21-pter, 8q24.1-qter, 9q12-13, 12p11.2-pter, and 15q11.2-15. Also, a high level of amplification on the short arm of chromosome 8 was found in Tumor No. 1. 2) FISH (Reverse-painting)
(FISH) To further narrow the region in the high level of amplified 8p21-23 in tumor No. 1 in CGH studies, we used the whole genomic DNA from tumor No. 1 as a probe to Was performed to elongate the cells (RevFISH: Joos, S., et al., Hum. Genet., 9).
0, 584-589 (1993)). Extended chromosomes were prepared from lymphocytes cultured according to the method of Inazawa et al. (Inazawa,
J., et al., Cytogenet.Cell Genet., 65, 130-135 (1
994)).

As a result, the FISH signal of the amplicon for this MFH tumor was cytogenically band 8p2
Limited the amplicon to 3.1. 3) Southern blot analysis and determination of the size of the 8p amplicon Southern hybridization was performed according to the standard protocol of Sunblock et al. To identify genomic fragments within the region amplified by 8p21-23 ( Sambrook, J., et
al., Molecular Cloning a Laboratory Manual. Cold
Spring Harbor Laboratory Press: NY. (1989)).

The probe used in this test was the following EST
s (expressed-sequence tags) or STSs (sequence-ta
gged sites) including: 757C03 (G
EN-131E06), 0600H06 (GEN-003G09), cos.024H10 (GEN-02
4H10), cos.505G01 (GEN-505G01), 0155D08 (GEN-001B1
2), cos.D8S1819 (D8S1819), and cos.D8S550 (D8S550).
ESTs and data on the localization of those chromosomes are
ENOTK human cDNA database (http: //genotk.g
enome.ad.jp).

Next, the tumor DNA and human placenta DNA digested with EcoRI were electrophoresed on a 0.8% agarose gel, and transferred to a nylon membrane (manufactured by BIODYNE). Each probe was prepared using random primers [α- ³² PdCT
P] and hybridized to the pre-hybridized filter. The present inventors have determined that BAS1000
The signal was analyzed with an image analyzer (Fuji) to calculate the degree of amplification. The filters were then exposed for autoradiography at -80 ° C overnight.

As a result of the Southern blot, from the state of amplification of each of these localities at 15 MFHs, the region where the amplified DNA overlaps smaller was D8S1819 and D8S1819.
It was between 8S550. This area consists of two tumors, No. 1 and N
GEN-024H10 and GEN-505G01 amplified in o.4 (13%)
2 ESTs. However, only tumor No. 1 with high copy number amplification was detected by CGH at 8p21-pter. This was based on a comparison of the hybridization signal of the amplified DNA to the normal DNA.

The copy number was determined by
Amplification of N-024H10 and GEN-505G01 was about 8-fold and 4-fold, and in Tumor No. 4 was about 6-fold and 3-fold DNA amplification, respectively. 4) Screening of cDNA library and DNA sequencing The present inventors followed the protocol of Ishikawa et al.
ikawa, S., et al., DNA Res., 4, 35-43 (1997)), c
DNA library screening and DNA sequencing were performed.

That is, a human fetal brain cDNA library (10 × 10 6 plaque: manufactured by Stratagene) was screened using GEN-024H10 as a probe.
The DNA sequence of the positive clone was determined using an ABI automatic sequencer (Perkin-Elmer).

As a result, cD was determined from 10 positive clones.
A 6242 bp transcript was identified that contained a single open reading frame with a 3159 bp unidirectional sequence of NA sequence. Since the consensus sequence for the start of transcription ("Kozak rule") is well conserved, the start codon for transcription is the nucleotide number 417-419.
It was thought to be th.

SEQ ID NO: 1 shows the estimated 10
Shows the 52 amino acid sequence.

The structure analysis of the novel protein was performed by using the PSORT program (Nakai, K. and K) for predicting the protein localization site.
anehisa, MA, Genomics, 14,897-911 (1992)) showed one hydrophobic region in the middle of the amino acid sequence (residues 486-502), however, the hydrophobicity plot (Kyte,
J. and Doolittle, RF, J. Mol. Biol., 157, 105-13
2 (1982)) did not predict hydrophobic regions. One A
TP / GTP binding site motif “A” ([AG] -X (4) -G
-K- [ST]) was found at residues 416-423. Leucine residues were repeated as each of the seven amino acids capable of forming a structural motif known as the "leucine zipper" (Landschulz, WH, et al., Science, 240, 1
759-1764 (1988)).

[0230] The putative gene product contains three leucine-
It seems to contain a zipper region (68-89 residues, 102-123
(Residues, and 962-983 residues), each with four leucine residues, were regularly spaced with seven amino acid repeats.

According to a homology search using the FASTA program (Genome Net), the estimated protein was found to be ALS (acid-labile subunit protein) and IG of insulin growth factor.
It has been shown that they are similar to the components of a three-component complex of FI, IGF-II and IGF-binding protein-3 (IGFBP3) (Baxter, RC, et al., J. Biol. Che
m., 264, 11843-11848 (1989)). ALS belongs to a family of proteins with conserved leucine-rich repeat motifs (Kobe, B., and Deisenhofer, J., Tren
ds Biochem. Sci., 19, 415-421 (1994)).

When aligning the 11 leucine-rich tandem repeats (23 residues each) of the new protein, it is clear that some residues are highly conserved and the following consensus sequence can be deduced. : PX-α-XXL-X
-X-L-X-X-L-X-L-X-X-N-X-α-
XX-α (X is all amino acids, α is an aliphatic amino acid: L, I or V).

The basic structure of the putative MASL1 product is
It showed some features and unexpected sequence homology suggesting important functions for this protein. A striking feature was the presence of 11 leucine-rich tandem repeats of 23 amino acids. This motif usually contains proline as the first residue, with leucine repeated at some positions,
The second residue contains asparagine.

The highly conserved features of this pattern indicate that the leucine-rich repeat regions are important for maintaining structural integrity and / or function of the proteins containing them.

Since the function of known proteins containing this motif is related to protein-protein interactions (Tan, F.,
et al., J. Biol. Chem., 265, 13-19 (1990): Delhant
y, Pand Baxter, RC, Biochem. Biophys. Res. Comm
un., 227, 897-902 (1996)), this motif may be involved in binding.

The repeat sequence of MASL1 is very similar to the gene occurring in yeast adenylate cyclase
(Kataoka, T. et al., Cell, 43, 493-505 (1985)). The yeast adenylate cyclase enzyme is activated by RAS and regulates the transition of the yeast cell cycle to the G1 phase
(Thevelein, JM, Mol.Microbiol., 5, 1301-1307 (19
91)). The possession of this conserved motif is presumed that the MASL1 protein plays an important role in signal transduction and regulation of cell proliferation, differentiation and apoptosis in humans.

In addition, the deduced amino acid sequence of MASL1 has 35.9% homology with ALS. In a complex of IGFs and IGFBP, ALS
Has a central role in regulating the bioavailability of circulating IGFs (Delhanty, PJ and
Baxter, RC, Prog.Growth Factor Res., 6,141-1
49 (1995)). ALS is IGFBP-3 only in serum
In these three complexes, growth factors apparently cannot cross the capillary barrier (Binou
x, M. and Hossenlopp, P., Endocrinol. Metab., 67,
509-514 (1988)).

However, it can act as a receptor for other circulating molecules (Hook, M .;
et al., Annu. Rev. Biochem., 53, 847-869 (1984).
), When the cell associated glycosaminoglycan interacts with the ALS, the growth factor in the binary complex is easily separated from the three complexes so that the growth factor can cross the capillary barrier. IGFs from circulation in tissues
May depend on this specific separation mechanism (Baxter, RC, Biochem. J., 271, 773-777 (1992).
0)). Therefore, if MASL1 is a structural analog to ALS and binds to a protein similar to IGFBP-3, it may regulate cell proliferation, differentiation, and apoptosis.

We have identified a novel gene, MASL1
(MFH-amplified sequences with leucine-rich tandem
repeats). 5) Expression of MASL1 by Northern blot analysis From one of 16 different normal human tissues (manufactured by Clontech), a Northern blot containing 2 μg of poly (A) + RNA in each lane was prepared according to the manufacturer's instructions. Three
Hybridization was performed with a random primed ³² P-labeled probe corresponding to nucleotide 858. The blot was washed with 0.1 × SSC / 0.1% SDS at 50 ° C.
The signal was analyzed with a BAS1000 image analyzer (Fuji).

Northern blot analysis was performed to determine the level of MASL1 expression in various human tissues. As a result, a 6.6 kb transcript was universally expressed. 6) RT-PCR analysis A quantitative expression test was performed by RT-PCR to compare the expression levels of MASL1 transcripts in MFH tumors and normal tissues.

The total RNA was measured according to the manufacturer's instructions.
It was obtained from a frozen sample of MFH tumor using zol (manufactured by Gibco-BRL). Superscript II (manufactured by Gibco-BRL)
CDNA produced from 0.2 μg of total RNA with
Used as template for R. All reactions were performed on GeneAmp PC
R system 9600 (Perkin-Elmer) at 94 ° C
30 minutes at 94 ° C, 30 seconds at 58 ° C,
The reaction was carried out for 25 cycles at 72 ° C. for 30 seconds as one cycle.

The integrity of each RNA template was prepared through amplification of β-actin, which showed a similar signal in all samples.

The sequences of the primers used for RT-PCR are shown below: MASL1: 5'-GCATTTCCAGCCACC
TCAG-3 'and 5'-ACAGAAAGCCCTCC
CACATAC-3 ′ β-actin: 5′-CCAGAGATGGCCACG
GCTGCT-3'5'-TCCTTCTGCATCC
The TGTCGGCT-3 'RT-PCR product was electrophoresed through a 3.0% GTG agarose gel, Southern-blotted onto a nylon membrane, and then the 5'-ATCCTCAATGTCTTCTCTCAGAG of GEN-024H10 internal oligonucleotide.
3 ′ was labeled with [γ- ³² PdATP] and hybridized.

As a result, this gene was overexpressed not only in tumor No. 1 but also in tumors No. 3, 4, 5, 6, 7, 9 and 11.

The expression of MASL1 was determined by CGH analysis.
Only tumors that showed a high level of amplification at 8p21-23 were increased about 10-fold in tumor No. 1.

According to our quantitative gene expression test by RT-PCR, the expression of MASL1 was
o.4 increased significantly, and both of these
Clear amplification of DNA was seen at this locus. The results indicate that overexpression of MASL1 was 57% of the tumors tested by RT-PCR (8/14), even though only one of them showed a high level of amplification at 8p23.1 by CGH.
In the fact that DNA amplification was
It can be strongly suggested that this is one of the mechanisms that can lead to up regulation of 1. Together with the deduced amino acid structure of MASL1, the data may indicate that dysregulation of this gene is an important virulence factor for MFH.

[0247]

[Sequence List] SEQUENCE LISTING <110> Otsuka Pharmaceutical Co., Ltd. <120> MASL1 gene <130> 4C9JP <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 1052 <212> PRT <213 > Human embryonic brain <400> 1 Met Ala Gly Met Asp Ser Gly Asn Leu Lys Thr Ala Arg Leu Trp Arg 1 5 10 15 Asp Ala Ala Leu Arg Ala Arg Lys Leu Arg Ser Asn Leu Arg Gln Leu 20 25 30 Thr Leu Thr Ala Ala Gly Ala Cys Pro Gly Ala Gly Ala Asp Ala Leu 35 40 45 Glu Ser Pro Ala Ser Pro Gln Leu Val Leu Pro Ala Asn Leu Gly Asp 50 55 60 Ile Glu Ala Leu Asn Leu Gly Asn Asn Gly Leu Glu Glu Val Pro Glu 65 70 75 80 Gly Leu Gly Ser Ala Leu Gly Ser Leu Arg Val Leu Val Leu Arg Arg 85 90 95 Asn Arg Phe Ala Arg Leu Pro Pro Ala Val Ala Glu Leu Gly His His 100 105 110 Leu Thr Glu Leu Asp Val Ser His Asn Arg Leu Thr Ala Leu Gly Ala 115 120 125 Glu Val Val Ser Ala Leu Arg Glu Leu Arg Lys Leu Asn Leu Ser His 130 135 140 Asn Gln Leu Pro Ala Leu Pro Ala Gln Leu Gly Ala Leu Ala His Leu 145 150 155 160 Glu Glu Leu Asp Val Ser Phe Asn Arg Leu Ala His Leu P ro Asp Ser 165 170 175 Leu Ser Cys Leu Ser Arg Leu Arg Thr Leu Asp Val Asp His Asn Gln 180 185 190 Leu Thr Ala Phe Pro Arg Gln Leu Leu Gln Leu Val Ala Leu Glu Glu 195 200 205 Leu Asp Val Ser Ser Asn Arg Leu Arg Gly Leu Pro Glu Asp Ile Ser 210 215 220 Ala Leu Arg Ala Leu Lys Ile Leu Trp Leu Ser Gly Ala Asp Leu Gly 225 230 235 240 Thr Leu Pro Ala Gly Phe Cys Glu Leu Ala Ser Leu Glu Ser Leu Met 245 250 255 Leu Asp Asn Asn Gly Leu Gln Ala Leu Pro Ala Gln Phe Ser Cys Leu 260 265 270 Gln Arg Leu Lys Met Leu Asn Leu Ser Ser Asn Leu Phe Glu Glu Phe 275 280 285 Pro Ala Ala Leu Leu Pro Leu Ala Gly Leu Glu Glu Leu Tyr Leu Ser 290 295 300 Arg Asn Gln Leu Thr Ser Val Pro Ser Leu Ile Ser Gly Leu Gly Arg 305 310 315 320 Leu Leu Thr Leu Trp Leu Asp Asn Asn Arg Ile Arg Tyr Leu Pro Asp 325 330 335 Ser Ile Val Glu Leu Thr Gly Leu Glu Glu Leu Val Leu Gln Gly Asn 340 345 350 Gln Ile Ala Val Leu Pro Asp His Phe Gly Gln Leu Ser Arg Val Gly 355 360 365 Leu Trp Lys Ile Lys Asp Asn Pro Leu Ile Gln Pro Pro Tyr G lu Val 370 375 380 Cys Met Lys Gly Ile Pro Tyr Ile Ala Ala Tyr Gln Lys Glu Leu Ala 385 390 395 400 His Ser Gln Pro Ala Val Gln Pro Arg Leu Lys Leu Leu Leu Met Gly 405 410 415 His Lys Ala Ala Gly Lys Thr Leu Leu Arg His Cys Leu Thr Glu Glu 420 425 430 Arg Val Glu Gly Cys Pro Gly Gly Gly Asp Lys Glu Lys Cys Tyr Pro 435 440 445 Pro Ser Pro Pro Pro Val Ser Lys Gly Ile Glu Val Thr Ser Trp Thr 450 455 460 Ala Asp Ala Ser Arg Gly Leu Arg Phe Ile Val Tyr Asp Leu Ala Gly 465 470 475 480 Asp Glu Ser Tyr Glu Val Ile Gln Pro Phe Phe Leu Ser Pro Gly Ala 485 490 495 Leu Tyr Val Leu Val Val Asn Leu Ala Thr Tyr Glu Pro Arg His Phe 500 505 510 Pro Thr Thr Val Gly Ser Phe Leu His Arg Val Gly Ala Arg Val Pro 515 520 525 Asn Ala Val Val Cys Ile Val Gly Thr His Ala Asp Leu Cys Gly Glu 530 535 540 Arg Glu Leu Glu Glu Lys Cys Leu Asp Ile His Arg Gln Ile Ala Leu 545 550 555 560 Gln Glu Lys His Asp Ala Glu Gly Leu Ser Arg Leu Ala Lys Val Val 565 570 575 Asp Glu Ala Leu Ala Arg Asp Phe Glu Leu Arg Ser Ala Ser P ro His 580 585 590 Ala Ala Tyr Tyr Gly Val Ser Asp Lys Asn Leu Arg Arg Arg Lys Ala 595 600 605 His Phe Gln Tyr Leu Leu Asn His Arg Leu Gln Ile Leu Ser Pro Val 610 615 620 620 Leu Pro Val Ser Cys Arg Asp Pro Arg His Leu Arg Arg Leu Arg Asp 625 630 635 640 Lys Leu Leu Ser Val Ala Glu His Arg Glu Ile Phe Pro Asn Leu His 645 650 655 655 Arg Val Leu Pro Arg Ser Trp Gln Val Leu Glu Glu Leu His Phe Gln 660 665 670 Pro Pro Gln Ala Gln Arg Leu Trp Leu Ser Trp Trp Asp Ser Ala Arg 675 680 685 Leu Gly Leu Gln Ala Gly Leu Thr Glu Asp Arg Leu Gln Ser Ala Leu 690 695 700 Ser Tyr Leu His Glu Ser Gly Lys Leu Leu Tyr Phe Glu Asp Ser Pro 705 710 715 715 720 Ala Leu Lys Glu His Val Phe His Asn Leu Thr Arg Leu Ile Asp Ile 725 730 735 Leu Asn Val Phe Phe Gln Arg Asp Pro Ser Leu Leu Leu His Lys Leu 740 745 750 Leu Leu Gly Thr Ser Gly Glu Gly Lys Ala Glu Gly Glu Ser Ser Pro 755 760 765 Pro Met Ala Arg Ser Thr Pro Ser Gln Glu Leu Leu Arg Ala Thr Gln 770 775 780 Leu His Gln Tyr Val Glu Gly Phe Leu Leu His Gly Leu Leu Pro A la 785 790 795 800 His Val Ile Arg Leu Leu Leu Lys Pro His Val Gln Ala Gln Gln Asp 805 810 815 Leu Gln Leu Leu Leu Glu Leu Leu Glu Lys Met Gly Leu Cys Tyr Cys 820 825 830 Leu Asn Lys Pro Lys Gly Lys Pro Leu Asn Gly Ser Thr Ala Trp Tyr 835 840 845 Lys Phe Pro Cys Tyr Val Gln Asn Glu Val Pro His Ala Glu Ala Trp 850 855 860 Ile Asn Gly Thr Asn Leu Ala Gly Gln Ser Phe Val Ala Glu Gln Leu 865 870 875 880 Gln Ile Glu Tyr Ser Phe Pro Phe Thr Phe Pro Pro Gly Leu Phe Ala 885 890 895 Arg Tyr Ser Val Gln Ile Asn Ser His Val Val His Arg Ser Asp Gly 900 905 910 Lys Phe Gln Ile Phe Ala Tyr Arg Gly Lys Val Pro Val Val Val Ser 915 920 925 Tyr Arg Pro Ala Arg Gly Val Leu Gln Pro Asp Thr Leu Ser Ile Ala 930 935 940 Ser His Ala Ser Leu Pro Asn Ile Trp Thr Ala Trp Gln Ala Ile Thr 945 950 955 960 Pro Leu Val Glu Glu Leu Asn Val Leu Leu Gln Glu Trp Pro Gly Leu 965 970 975 His Tyr Thr Val His Ile Leu Cys Ser Lys Cys Leu Lys Arg Gly Ser 980 985 990 Pro Asn Pro His Ala Phe Pro Gly Glu Leu Leu Ser Gln Pro Arg P ro 995 1000 1005 Glu Gly Val Ala Glu Ile Ile Cys Pro Lys Asn Gly Ser Glu Arg Val 1010 1015 1020 Asn Val Ala Leu Val Tyr Pro Pro Thr Pro Thr Val Ile Ser Pro Cys 1025 1030 1035 1040 Ser Lys Lys Asn Val Gly Glu Lys His Arg Asn Gln 1045 1050 1052 <210> 2 <211> 3159 <212> DNA <213> Human embryonic brain <400> 2 atggctggga tggacagtgg caacctgaag accgcgaggc tgtggcggga cgccgccctg 60 cgtgccagga agctgcggag caacctgcgc cagctcacgc tcaccgccgc cggggcctgc 120 cccggggccg gggccgacgc gctcgagtcc cccgcctccc cccagctcgt gctgccggcc 180 aacctcgggg acattgaggc actgaacctg gggaacaacg gcctggagga ggtacccgag 240 gggctggggt cggcgctggg cagcctgcgc gtcctggtcc tgcgcaggaa ccgcttcgcc 300 cggctgcccc cggcggtggc cgagctcggc caccacctca ccgagctgga cgtgagccac 360 aaccggctga ccgccctggg cgcggaggtg gtgagtgctc tgagggagct gcggaagctc 420 aacctcagcc acaaccagct gcccgccctg cccgcccagc tgggcgctct cgctcacctg 480 gaggagctgg atgtcagctt taaccggctg gcgcacctgc ctgactccct ctcctgcctc 540 tcccgcctgc gcaccctgga cgtggatcac aaccagctca ctgccttccc ccggcagc tg 600 ctgcagctgg tggccctgga ggagctggac gtgtccagca accggctgcg gggcctgcct 660 gaggatatca gtgccctgcg tgccctcaag atcctctggc tgagtggggc cgagcttggc 720 acgctgcccg ccggcttctg cgagctggcc agtttggaga gcctcatgct agacaacaac 780 gggctgcagg ctctgcccgc ccagttcagc tgcctgcagc ggctcaaaat gctcaacctg 840 tcctccaacc tcttcgagga gttccctgcc gcgctgctgc ccctggctgg tctggaggag 900 ctctacctta gtcgcaacca gctcacctcg gtgccatccc ttatctcggg cctgggccgg 960 cttctcacct tgtggctgga taataaccgc atccgctacc tgccggactc catcgtggag 1020 ctgaccggcc tggaggagct cgtgctgcag gggaaccaga tcgcggtgct gcccgaccac 1080 tttggccagc tctcccgggt gggtttgtgg aagatcaaag acaacccact gatccagccc 1140 ccctacgagg tctgcatgaa ggggatcccc tacatcgcag cctaccagaa ggaactggct 1200 cattcccagc cggcggtgca gccccggctc aagctgctcc tgatggggca taaggctgca 1260 ggaaagactt tgctgcgcca ctgcctcacc gaggagagag tggagggatg cccaggagga 1320 ggggacaagg agaagtgcta cccaccgtca cctccccctg tgagcaaggg catcgaggtg 1380 accagctgga cggccgatgc ctcccggggc ctgcggttca tcgtgtatga cttagctggg 1440 gatga aagtt atgaggtgat ccagcccttc ttcctgtccc caggggccct atacgtgctg 1500 gtggtcaact tggccaccta tgagcctcgc cactttccta ccaccgtggg ctccttcttg 1560 catcgggtcg gggcgagagt gcccaacgcg gtggtgtgca tcgtgggcac ccacgcagac 1620 ctgtgcggag agcgtgagct ggaggagaaa tgtctggaca ttcaccgcca gatcgccctg 1680 caggagaagc acgacgcgga gggactgagc cgcttggcca aggtggtgga cgaggcactg 1740 gcccgggact tcgagctgcg ctctgccagc ccccacgcag cctactatgg cgtttcggac 1800 aagaaccttc gacggcgcaa ggcccatttt caatacctgc tcaaccaccg gctgcagatc 1860 ctctcccccg tgttgcctgt tagctgcagg gacccgcgcc acttacgacg ccttcgggac 1920 aagttgctgt cagttgctga gcaccgagag atcttcccca acttacacag agtactgcct 1980 cgatcctggc aggtgctgga ggaactgcat ttccagccac ctcaggccca gcgactgtgg 2040 ctaagctggt gggactcggc gcgcttgggc ctgcaggcgg gtctgaccga ggaccgactg 2100 cagagtgccc tctcctacct gcatgagagc ggcaagctac tctactttga ggacagtccg 2160 gctctcaagg agcacgtctt ccacaacctc acccgcctca tcgacatcct caatgtcttc 2220 ttccagaggg atccctcttt gctgctgcat aagctgctcc tagggaccag tggagagggc 2280 aaggcggagg gggaaagctc cccgcccatg gcgcggtcca cccccagcca ggaactgctc 2340 cgggccaccc agctccatca gtatgtggag ggctttctgt tgcatgggct cttgccagct 2400 catgtcattc ggttgctgct taagcctcat gtccaggccc agcaggactt gcagctgttg 2460 ctggagctgc tggagaagat gggactctgt tactgcctca ataaacccaa gggcaagcct 2520 ttgaatgggt ccacagcttg gtacaagttc ccatgctatg tgcagaacga ggtgccccat 2580 gcagaagcct ggattaatgg gaccaaccta gctgggcagt cttttgtggc tgagcagttg 2640 cagattgaat atagctttcc ttttactttt ccacctgggt tgtttgcacg ctacagtgtc 2700 cagatcaaca gccatgtggt gcacaggtcg gatggtaaat ttcagatctt tgcctataga 2760 gggaaagttc ctgtggttgt gagttacaga cctgccaggg gagtcctgca gccagacacc 2820 ctgtccattg ctagccatgc atcattacca aatatatgga ccgcatggca agccataacc 2880 cccttggtgg aggaactgaa tgtcctactt caggaatggc ctggactgca ctacaccgtg 2940 cacattctct gttctaagtg ccttaagaga ggatcgccca atccacatgc ttttccaggg 3000 gagttgctga gtcagcccag accggaagga gtggcagaga tcatttgccc caagaacggc 3060 agcgagcgag taaatgttgc cttggtttac ccacccacgc cgactgtgat cagcccctgt 3120 tccaagaaga atgttg gtga aaagcacaga aaccagtga 3159 <210> 3 <211> 6345 <212> DNA <213> MASL1cDNA full length sequence <400> 3 cccctccccc aggagctcga ggaggcgacg ccgctgccgg ccgcgggacc gcggctgcga 60 gggtagcggg gccggagccg ggcaccccga ggcgccgcgc ggcgggcgga gagggaggag 120 gagcaggcat cctccgcggc gcgcgggcgg cggccctgct gcccggcgcg cccatggccc 180 ggccgcaggg agaatgcaag ctgctctcca cgcgctcggc gtcccccgga gccgctcgcg 240 tcgccgccgc cccctagccg ccagcgctcg gccgctggct gcgctaggac ccgcggccgc 300 cggccgccga gcgcgggagg gcaggcaggg gcaggagccg gagggcccgg ggcgcggcgc 360 ggcatgtagc tgcgggctcc cgcgtccgcg tgaggctgtc ggcccggggc cccgcc atg 419 Met 1 gct ggg atg gac agt ggc aac ctg aag acc gcg agg ctg tgg cgg gac 467 Ala Gly Met Asp Ser Gly Asn Leu Lys Thr Ala Arg Leu Trp Arg Asp 5 10 15 gcc gcc ctg cgt gcc agg aag ctg cgg agc aac ctg cgc cag ctc acg 515 Ala Ala Leu Arg Ala Arg Lys Leu Arg Ser Asn Leu Arg Gln Leu Thr 20 25 ctc acc gcc gcc ggg gcc tgc ccc ggg gcc ggg gcc gac gcg ctc gag 563 Leu Thr Ala Ala Gly Ala Cys Pro Gly Ala Gl y Ala Asp Ala Leu Glu 35 40 45 tcc ccc gcc tcc ccc cag ctc gtg ctg ccg gcc aac ctc ggg gac att 611 Ser Pro Ala Ser Pro Gln Leu Val Leu Pro Ala Asn Leu Gly Asp Ile 50 55 60 65 gag gca ctg aac ctg ggg aac aac ggc ctg gag gag gta ccc gag ggg 659 Glu Ala Leu Asn Leu Gly Asn Asn Gly Leu Glu Glu Val Pro Glu Gly 70 75 80 ctg ggg tcg gcg ctg ggc ag c gc ag c gc ag Gly Ser Ala Leu Gly Ser Leu Arg Val Leu Val Leu Arg Arg Asn 85 90 95 cgc ttc gcc cgg ctg ccc ccg gcg gtg gcc gag ctc ggc cac cac ctc 755 Arg Phe Ala Arg Leu Pro Pro Ala Val Ala Glu Leu Gly His Leu 100 105 110 acc gag ctg gac gtg agc cac aac cgg ctg acc gcc ctg ggc gcg gag 803 Thr Glu Leu Asp Val Ser His Asn Arg Leu Thr Ala Leu Gly Ala Glu 115 120 125 gtg gtg agt gct ctg agg gag ctg cgg ctc aac ctc agc cac aac 851 Val Val Ser Ala Leu Arg Glu Leu Arg Lys Leu Asn Leu Ser His Asn 130 135 140 145 cag ctg ccc gcc ctg ccc gcc cag ctg ggc gct ctc gct cac ctg gag 899 Gln Leu Pro Ala Le Ala Gln Leu Gly Ala Leu Ala His Leu Glu 150 155 160 gag ctg gat gtc agc ttt aac cgg ctg gcg cac ctg cct gac tcc ctc 947 Glu Leu Asp Val Ser Phe Asn Arg Leu Ala His Leu Pro Asp Ser Leu 165 170 175 tcc tgc cc tcc ctg cgc acc ctg gac gtg gat cac aac cag ctc 995 Ser Cys Leu Ser Arg Leu Arg Thr Leu Asp Val Asp His Asn Gln Leu 180 185 190 act gcc ttc ccc cgg cag ctg ctg cag ctg gtg gcc ctg gag Agg ctg 104 Phe Pro Arg Gln Leu Leu Gln Leu Val Ala Leu Glu Glu Leu 195 200 205 gac gtg tcc agc aac cgg ctg cgg ggc ctg cct gag gat atc agt gcc 1091 Asp Val Ser Ser Asn Arg Leu Arg Gly Leu Pro Glu Asp Ile Ser Ala 210 215 220 225 ctg cgt gcc ctc aag atc ctc tgg ctg agt ggg gcc gat ctt ggc acg 1139 Leu Arg Ala Leu Lys Ile Leu Trp Leu Ser Gly Ala Asp Leu Gly Thr 230 235 240 ctg ccc gcc ggc ttc tgc gag gag ttg gag agc ctc atg cta 1187 Leu Pro Ala Gly Phe Cys Glu Leu Ala Ser Leu Glu Ser Leu Met Leu 245 250 255 gac aac aac ggg ctg cag gct ctg ccc gcc cag ttc agc tgc ctg cag 1235 Asp Asn Asn Gly n Ala Leu Pro Ala Gln Phe Ser Cys Leu Gln 260 265 270 cgg ctc aaa atg ctc aac ctg tcc tcc aac ctc ttc gag gag ttc cct 1283 Arg Leu Lys Met Leu Asn Leu Ser Ser Asn Leu Phe Glu Glu Phe Pro 275 280 gcc gcg ctg ctg ccc ctg gct ggt ctg gag gag ctc tac ctt agt cgc 1331 Ala Ala Leu Leu Pro Leu Ala Gly Leu Glu Glu Leu Tyr Leu Ser Arg 290 295 300 305 aac cag ctc acc tcg gtg ccatc ttg ccaccc gc ggc cgg ctt 1379 Asn Gln Leu Thr Ser Val Pro Ser Leu Ile Ser Gly Leu Gly Arg Leu 310 315 320 ctc acc ttg tgg ctg gat aat aac cgc atc cgc tac ctg ccg gac tcc 1427 Leu Thr Leu Trp Leu Asp Asn Asn Arg Ic Arg Tyr Leu Pro Asp Ser 325 330 335 atc gtg gag ctg acc ggc ctg gag gag ctc gtg ctg cag ggg aac cag 1475 Ile Val Glu Leu Thr Gly Leu Glu Glu Leu Val Leu Gln Gly Asn Gln 340 345 350 atc gcg gtg gac cac ttt ggc cag ctc tcc cgg gtg ggt ttg 1523 Ile Ala Val Leu Pro Asp His Phe Gly Gln Leu Ser Arg Val Gly Leu 355 360 365 tgg aag atc aaa gac aac cca ctg atc cag ccc ccc tac gag gtc tgc 1571 Lys Ile Lys Asp Asn Pro Leu Ile Gln Pro Pro Tyr Glu Val Cys 370 375 380 385 atg aag ggg atc ccc tac atc gca gcc tac cag aag gaa ctg gct cat 1619 Met Lys Gly Ile Pro Tyr Ile Ala Ala Tyr Gln Lys Glu Leu Ala His 390 395 400 tcc cag ccg gcg gtg cag ccc cgg ctc aag ctg ctc ctg atg ggg cat 1667 Ser Gln Pro Ala Val Gln Pro Arg Leu Lys Leu Leu Leu Met Gly His 405 410 415 aag gct gca gga aag c ttg ctg cac tgc ctc acc gag gag aga 1715 Lys Ala Ala Gly Lys Thr Leu Leu Arg His Cys Leu Thr Glu Glu Arg 420 425 430 gtg gag gga tgc cca gga gga ggg gac aag gag aag tgc tac cca ccg 1763 Val Glu Gly Cys Pro Gly Gly Asp Lys Glu Lys Cys Tyr Pro Pro 435 440 445 tca cct ccc cct gtg agc aag ggc atc gag gtg acc agc tgg acg gcc 1811 Ser Pro Pro Pro Val Ser Lys Gly Ile Glu Val Thr Ser Trp Thr Ala 450 455 460 460 465 gat gcc tcc cgg ggc ctg cgg ttc atc gtg tat gac tta gct ggg gat 1859 Asp Ala Ser Arg Gly Leu Arg Phe Ile Val Tyr Asp Leu Ala Gly Asp 470 475 480 gaa agt tat gag gtg atc cag ccc ttc ttctt ctt ca ggg gcc cta 1907 Glu Ser Tyr Glu Val Ile Gln Pro Phe Phe Leu Ser Pro Gly Ala Leu 485 490 495 tac gtg ctg gtg gtc aac ttg gcc acc tat gag cct cgc cac ttt cct 1955 Tyr Val Leu Val Val Asn Leu Ala Thr Tyr Glu Pro Arg His Phe Pro 500 505 510 acc acc gtg ggc tcc ttc ttg cat cgg gtc ggg gcg aga gtg ccc aac 2003 Thr Thr Val Gly Ser Phe Leu His Arg Val Gly Ala Arg Val Pro Asn 515 520 525 gcg gtg gtg tgc atc gtg ggc acc cac gca gac ctg tgc gga gag cgt 2051 Ala Val Val Cys Ile Val Gly Thr His Ala Asp Leu Cys Gly Glu Arg 530 535 540 540 545 gag ctg gag gag aaa tgt ctg gac att cac cgc cag atc gcc ccg Glu Leu Glu Glu Lys Cys Leu Asp Ile His Arg Gln Ile Ala Leu Gln 550 555 560 gag aag cac gac gcg gag gga ctg agc cgc ttg gcc aag gtg gtg gac 2147 Glu Lys His Asp Ala Glu Gly Leu Ser Arg Le Val Asp 565 570 575 gag gca ctg gcc cgg gac ttc gag ctg cgc tct gcc agc ccc cac gca 2195 Glu Ala Leu Ala Arg Asp Phe Glu Leu Arg Ser Ala Ser Pro His Ala 580 585 590 gcc tac tat ggc gtt tcg g aac ctt cga cgg cgc aag gcc cat 2243 Ala Tyr Tyr Gly Val Ser Asp Lys Asn Leu Arg Arg Arg Lys Ala His 595 600 605 ttt caa tac ctg ctc aac cac cgg ctg cag atc ctc tcc ccc gtg ttg Tyr The Gln Leu Asn His Arg Leu Gln Ile Leu Ser Pro Val Leu 610 615 620 625 cct gtt agc tgc agg gac ccg cgc cac tta cga cgc ctt cgg gac aag 2339 Pro Val Ser Cys arg asp Pro arg His Leu arg arg Leu arg asp Lys 630 635 640 ttg ctg tca gtt gct gag cac cga gag atc ttc ccc aac tta cac aga 2387 Leu Leu Ser Val Ala Glu His Arg Glu Ile Phe Pro Asn Leu His Arg 645 650 655 gta ctg cct cga tcc tgg cag gtg ctggg cat ttc cag cca 2435 Val Leu Pro Arg Ser Trp Gln Val Leu Glu Glu Leu His Phe Gln Pro 660 665 670 cct cag gcc cag cga ctg tgg cta agc tgg tgg gac tcg gcg cgc ttg 2483 Pro Gln Ala Gln Arg Leu Trp Leu Ser Trp Trp Asp Ser Ala Arg Leu 675 680 685 ggc ctg cag gcg ggt ctg acc gag gac cga ctg cag agt gcc ctc tcc 2531 Gly Leu Gln Ala Gly Leu Thr Glu Asp Arg Leu Gln Ser Ala Leu Ser 690 695 700 705 tac cat gag agc ggc aag cta ctc tac ttt gag gac agt ccg gct 2579 Tyr Leu His Glu Ser Gly Lys Leu Leu Tyr Phe Glu Asp Ser Pro Ala 710 715 720 ctc aag gag cac gtc ttc cac aac ctc acc cgc atc atc gc 2627 Leu Lys Glu His Val Phe His Asn Leu Thr Arg Leu Ile Asp Ile Leu 725 730 735 aat gtc ttc ttc cag agg gat ccc tct ttg ctg ctg cat aag ctg ctc 2675 Asn Val Phe Phe Gln Arg Asp Pro Ser Leu Leu Leu Lys Leu Leu 740 745 750 cta ggg acc agt gga gag ggc aag gcg gag ggg gaa agc tcc ccg ccc 2723 Leu Gly Thr Ser Gly Glu Gly Lys Ala Glu Gly Glu Ser Ser Pro Pro 755 760 765 atg gcg cgg tcc accgcc cac gaa ctg ctc cgg gcc acc cag ctc 2771 Met Ala Arg Ser Thr Pro Ser Gln Glu Leu Leu Arg Ala Thr Gln Leu 770 775 780 785 cat cag tat gtg gag ggc ttt ctg ttg cat ggg ctc ttg cca gct cat 2819 His Gln Tyr Glu Gly Phe Leu Leu His Gly Leu Leu Pro Ala His 790 795 800 gtc att cgg ttg ctg ctt aag cct cat gtc cag gcc cag cag gac ttg 2867 Val Ile Arg Leu Leu Leu Lys Pro His Val Gln Ala Gln Gln Asp Leu 805 810 815 cag ctg ttg ctg gag ctg ctg gag aag atg gga ctc tgt tac tgc ctc 2915 Gln Leu Leu Leu Glu Leu Leu Glu Lys Met Gly Leu Cys Tyr Cys Leu 820 825 830 aat aaa ccc aag gg ccc aag gg ag ccc aag gg ag ccc aag gg ag ccc aag gg agg aca gct tgg tac aag 2963 Asn Lys Pro Lys Gly Lys Pro Leu Asn Gly Ser Thr Ala Trp Tyr Lys 835 840 845 ttc cca tgc tat gtg cag aac gag gtg ccc cat gca gaa gcc tgg att 3011 Phe Pro Cys Tyr Val Gln Aslu Val Pro His Ala Glu Ala Trp Ile 850 855 860 865 aat ggg acc aac cta gct ggg cag tct ttt gtg gct gag cag ttg cag 3059 Asn Gly Thr Asn Leu Ala Gly Gln Ser Phe Val Ala Glu Gln Leu Gln 870 875 880 880 att gaa tat agc ttt cct ttt act ttt cca cct ggg ttg ttt gca cgc 3107 Ile Glu Tyr Ser Phe Pro Phe Thr Phe Pro Pro Gly Leu Phe Ala Arg 885 890 895 tac agt gtc cag atc aac agc cat gtg gtg cac agg tcg gat 3155 Tyr Ser Val Gln Ile Asn Ser His Val Val His Arg Ser Asp Gly Lys 900 905 910 ttt cag atc ttt gcc tat aga ggg aaa gtt cct gtg gtt gtg agt tac 3203 Phe Gln Ile Phe Ala Tyr Arg Gly Lys Val Pro V al Val Val Ser Tyr 915 920 925 aga cct gcc agg gga gtc ctg cag cca gac acc ctg tcc att gct agc 3251 Arg Pro Ala Arg Gly Val Leu Gln Pro Asp Throne Leu Ser Ile Ala Ser 930 935 940 945 cat gca tca tta cca aat ata tgg acc gca tgg caa gcc ata acc ccc 3299 His Ala Ser Leu Pro Asn Ile Trp Thr Ala Trp Gln Ala Ile Thr Pro 950 955 960 ttg gtg gag gaa ctg aat gtc cta ctt cag gaa tgg cct gga ctg cac 3347u Glu Glu Leu Asn Val Leu Leu Gln Glu Trp Pro Gly Leu His 965 970 975 tac acc gtg cac att ctc tgt tct aag tgc ctt aag aga gga tcg ccc 3395 Tyr Thr Val His Ile Leu Cys Ser Lys Cys Leu Lys Arg Gly Ser Pro 980 985 990 aat cca cat gct ttt cca ggg gag ttg ctg agt cag ccc aga ccg gaa 3443 Asn Pro His Ala Phe Pro Gly Glu Leu Leu Ser Gln Pro Arg Pro Glu 995 1000 1005 gga gtg gca gag atc att tgc ccc aag aac agc gag cga gta aat 3491 Gly Val Ala Glu Ile Ile Cys Pro Lys Asn Gly Ser Glu Arg Val Asn 1010 1015 1020 1025 gtt gcc ttg gtt tac cca ccc acg ccg act gtg atc agc ccc tgt tcc 3539 Val Ala Leu Val Tyr Pro Pro Thr Pro Thr Val Ile Ser Pro Cys Ser 1030 1035 1040 aag aag aat gtt ggt gaa aag cac aga aac cag tga cgtttgtggc 3588 Lys Lys Asn Val Gly Glu Lys His Arg Asn Gln 1045 1050 1052 tgtggaattt ccatggaga gagacctg gg gg gg cagaccctct gcactcaccc cagcgtgttc tgtgaacttg agtgacaacg cgtgcttgca 3708 gggtgctttt tggatgactg gggaagaggt ggggaggggg tggtgggggg aagcatggac 3768 gagaacatgg agcaaatgtt ttacaacctg aacctcagaa ctgtgatcct ccaaggagcg 3828 cgctacttga agaaaagaaa aaaaaaaaaa aaagtcgaat ggcttctcag ggattttgtt 3888 ttccgtgcac atataagcca tagttacagt acaggtggca gtatttagag cactcagatt 3948 tcagttctgt taaatgtgaa agagggatcg actgacgttc attgctgttc cataactagt 4008 gtaaaatatg tatatgttta tctttatttt tataatatgc aaatacattt aaatttatac 4068 agtttgaagc ttctagcgtt agttcacact gggtgcaata ttctgcatga gaactgtgcc 4128 aagatggggc tgatttctta ttttagtata agactttttt gttttttctt tattctttaa 4188 tcctcttcac attaaaaaaa aaaaaatctc tctgttag gt catggattgac gtagt atcatggattca gtgatcagtattg taaatgtgca tttaagacag tgtcccttcc 4308 cttcttttca atgaaggtcc ctgcctatat aaatcatctg gcacgctggt gggaaatcct 4368 ttgctcttcc aacgtgttat tagtgctggg cagagatggg gcacactcag gggccaaaga 4428 ggacaaaaag tccatgcaaa acttgagtct tttaatggct taagataatc aggagtcagt 4488 tctgaatctt acaaagtgct ctgcttaata agtaccttac ttagcagagc actttgcaaa 4548 catattactt attagcagag ctctttgtag accttccaca tctggctgtc agatcttaag 4608 gttgtgaatt taggctccag ttatattcac tggagagcat aatcccacac gggttattta 4668 taaatacaga gcctctgatt ggacggtctc ctgccaagaa ctagtaatac ccttgtttta 4728 aaatcttcac aaggtaaaac ttaaaaagcc aaccaaacaa attgctctcc attctacttt 4788 taattgggcc aaacagcata tgctacagta gtaacatgtt tttcggagag tgtaaaaaac 4848 tctgtttaca tttgcctcct ccgtgggttg atcgaaaatg tataaaactg actgcttctc 4908 gccagcctca gacaagaaga gtgagttgct ggtactcgct actcttttac ttcttttgta 4968 aagtattgac tcttggaagg ctacagtata caaagtctca acatgttttt taaaagaaat 5028 aaggagcaag cgactgccct gctagaaatc acacaccgat ttttgtagaa tattttgtgc 5088 cccaggcatt aatttcactg actccagaac ctgcag ttca gagaatgatt tcttatgatg 5148 ataaaaatcg aatgggatca gacgatgttt gcattttttt aatacttgaa taggacacct 5208 caagtttgag atttcatttt cttttagaac acagtcacaa gattaatctg gtgaatcctt 5268 ttgtcacagt tctcgtgtgt gtgtgcgcgt ctccgtgtgt gtgtgtgtgc atgtgtgtaa 5328 aactgaatgg tcacatttaa ttgctttttg gaccattgaa tagttgggaa gtaagaattt 5388 tttaattggc atgagacggt tcctcaactg ttaaattaac caactttgac ctgtctttag 5448 aaaaaggctt atttgtatga ttttgggcta actccccggg gaccatatta aatgacaaaa 5508 atgctccttt gggtgacaca ccctacaaag tatttgctgt tacgaacata aacgcccaca 5568 ttcttaatat ctaatatttt tgaccagtga tgttttatgc tgtcatctga accctagaga 5728 agcagtgtca gaggaaacct tggtgtcaca tgtgtcttag caaaagggtt accatgatcg 5788 agggtcatgt gaccaaaaga tgctccagag aagcttgaga atttgtttca agttgggagg 5848 agggttggag atacaaaaat cactctgctc tacaggactc ttcagctgtc tatgcaagaa 5908 attccgtttt ctctttcagc acctggaaag acacagcagc ccaccgaggc gataggtgat 5968 tcactaagca caagaggaat gttttctaag caaggcgtcc cttgcctctc aaacaaatgc 6028 cctccaagtt tgttagggtt tctattcctg caacttgtag t atcaaaacc acttcctgga 6088 attgtcaaag cactgccaaa ataaatgttt ttcccccttc taagaaaaaa aaaatgacag 6148 tgctcatatt tgacacttgt gtattggact ctcttttgaa tgaataaaaa ggaaaagggg 6208 tttggtgtaa ttcctgatgg ggtgcgtgtt gtttttcatg ccatggtttg tgaattttaa 6268 ttgtggtttc ccatttcgtt gttgtaactg ggcagaaatt aaaaaagaaa aatcaataaa 6328 aatacaaaga aatggtt 6345 <210> 4 <211> 154 <212> DNA <223> Probe (GEN-505G01) <400> 4 cttaaaagga tatagactta atttatctgt gatatgcagt taagtggaat gttgcagtgt 60 atccggtaag agtgtctaac ttttgaagtt caattattct tttgtttttgg ttttgttttg 120 ttttttga gatggagtct <tt> tt <tt> tttttttttttttt120t <tt> tttttttttttttt120 5 agctccattt cactaataag gagacagatg tggaggttgg ggagttggtc ccaggtcacc 60 caactgggga gggcagaggt tggggaggga caggagtcaa taacccaaag tcatgaaatg 120 agaaaggaag taaacacttg gNtggagaNt cacacacaca cacacacaca cacacacaca 180 cacacacctc ctaacaggta tgttgtctgc aacaaggcaa aaataattca ttaatatctc 240 atttaaactt gagggcgagg gaattcctga accacctctc tggagcaaat aatggaaatt 300 ggaaattgat tgtcattta c ctttgaggaa gacttcggga tgtgccatgt ctttggtata 360 gggctgcgtg gtgttgtgac gcatgt 386 <210> 6 <211> 19 <212> DNA <400> 6 gcatttccag ccacctcag 19 <210> 7 <211> 20 <212> DNA <400> 7 acagaaagcc cc > 8 <211> 21 <212> DNA <400> 8 ccagagatgg ccacggctgc t 21 <210> 9 <211> 21 <212> DNA <400> 9 tccttctgca tcctgtcggc t 21 <210> 10 <211> 21 <212> DNA <400> 10 atcctcaatg tcttcttcca g 21

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/18 C07K 16/18 4C085 C12N 1/21 C12N 1/21 4H045 C12P 21/02 C12P 21/02 C 21/08 21/08 C12Q 1/68 C12Q 1/68 A G01N 33/50 G01N 33/50 TP 33/574 33/574 AZ 33/577 33/577 B // (C12N 1/21 C12R 1: 19) (72) Inventor Joji Inazawa 2-13-38 Kamikisaki, Shinagawa-ku, Tokyo Miki Building 501 (72) Inventor Yusuke Nakamura 1-17-33 Azamino, Aoba-ku, Yokohama-shi, Kanagawa F term (reference) 2G045 AA26 AA34 AA35 BB20 CB01 CB21 DA12 DA13 DA14 DA36 FB02 FB03 4B024 AA01 AA12 AA20 BA43 CA04 CA09 GA27 HA11 HA17 4B063 QA01 QA07 QA18 QA19 QQ03 QQ43 QQ58 QQ99 QR40 QR55 QS24 QS34 4B014 AG01 DA26 AG19 4B065 AA90Y AA91Y AA93Y CA23 CA44 CA46 4C085 AA13 BB11 GG01 GG08 GG10 4H045 AA10 AA30 BA10 CA40 DA86 EA28 EA31 EA51 FA74

Claims (26)

[Claims] 1. A gene containing any one of the following polynucleotides (a) and (b): (a) a polynucleotide encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: : At least 95 for 2 polynucleotides
(B) a polynucleotide which is a complementary strand to the polynucleotide of the above (a). 2. A gene comprising any one of the following polynucleotides (a) and (b): (a) a base sequence represented by SEQ ID NO: 2 or a complementary strand thereof; A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a base sequence. 3. The gene according to claim 2, which is a nucleotide sequence represented by SEQ ID NO: 2. 4. A gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 1. 5. An MA having the amino acid sequence according to claim 1.
SL1 gene expression product. 6. A recombinant expression vector having the gene according to claim 1 or 2. 7. A host cell having the recombinant expression vector according to claim 6. 8. A cloned cDN expressing the MASL1 protein.
A and its fragments, derivatives and homologs thereof. 9. An oligonucleotide comprising at least 15 contiguous nucleotides of the nucleotide sequence represented by SEQ ID NO: 2. 10. The oligonucleotide according to claim 9, which comprises at least 30 consecutive nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 2. (11) an antisense oligonucleotide against the nucleotide sequence according to (3); 12. An antisense oligonucleotide according to claim 11, comprising at least 15 contiguous nucleotide sequences. 13. The antisense oligonucleotide according to claim 12, comprising at least 30 contiguous nucleotide sequences. [14] An antisense oligonucleotide prepared using the sequence according to [11] to [13]. (15) A gene therapy agent comprising the antisense oligonucleotide according to (14) as an active ingredient. 16. An oligonucleotide probe comprising the nucleotide sequence according to claim 3. 17. An oligonucleotide probe comprising at least 15 consecutive nucleotide sequences of the nucleotide sequence shown by SEQ ID NO: 2. 18. The oligonucleotide probe according to claim 17, which comprises at least 30 consecutive nucleotide sequences of the nucleotide sequence represented by SEQ ID NO: 2. (19) A cancer diagnostic agent using the oligonucleotide probe according to any one of (16) to (18). (20) A kit for cancer diagnosis using the oligonucleotide probe according to any one of (16) to (18). 21. A protein of the following (a) or (b):
MASL1 protein: (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; An active protein. A method for screening an interactant using the MASL1 gene or gene expression product according to claim 1 or 2. 23. A homologue of the gene according to claim 1, which is a mammalian homologue selected from human, dog, monkey, horse, pig, sheep and cat. 24. An antibody having a binding property to the expression product of the gene according to claim 1 or 2. 25. A diagnostic method using the antibody according to claim 24 for diagnosing cancer. 26. A therapeutic agent for cancer comprising the antibody according to claim 24 as an active ingredient.