JPH0892291A - Prlts protein and dna coding for the same - Google Patents

Abstract

PURPOSE: To obtain a new protein, (PRLTS protein) existing in a common deficient domain of a chromosome in lung cancer, hepatoma and colon cancer, capable of diagnosing a cancer by using a monoclonal antibody to be bonded to the protein. CONSTITUTION: This new protein (PRLTS protein) is a new protein comprising the whole or a part of an amino acid sequence containing an amino acid sequence of the formula, for which a gene existing in a common deficient domain of a 8th chromosome in lung cancer, hepatoma and colon cancer codes, and is capable of diagnosing a cancer by using an antibody to be bonded to the protein. The protein is obtained by cloning a gene exising in the common deficient domain of the chromosome, incorporating the gene to a manifestation vector, transducing the vector to a host cell, transforming, culturing the transformed cell and manifesting the gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a PRLTS protein, a gene DNA encoding the same and a gene analysis method using the DNA, and is used in the field of medicine.

[0002]

2. Description of the Related Art The idea that mutations in cellular proteins play an important role in the development of cancer has long been known.
Recent advances in genetic engineering have made it possible to amplify a gene DNA encoding a specific protein and analyze gene mutations in cancer cells, which has led to dramatic progress in the field of cancer research. Up to now, analysis and identification of genes (oncogenes) encoding proteins that are considered to be involved in canceration of cells and abnormal growth of cancer cells have progressed, and the number of them has reached dozens. On the other hand, what acts in the opposite manner (tumor suppressor gene) has been in the spotlight for several years, and the Rb gene of retinoblastoma (Friend, SH et al.,
Proc. Natl. Acad. Sci. USA. 84, 9095, 1987), p53 gene of colon cancer (Lane, DP et al., Nature, 278, 2)
61, 1979) and the APC gene (Kenneth, WK, et al,
Science 253, 661, 1991) and the WT1 gene of Wilms tumor (Call, KM et al., Cell, 60, 509, 1990) have been discovered. In the case of the p53 gene, the mutated gene is transmitted through the family line. There is an example (“Li-Fraumeni syndrome” (Makin, D. et al, Science 250, 1233, 1990; S
rivastava, S. et al, Nature 348, 747, 1990)). In addition, it is gradually becoming clear that not only single gene abnormalities but also multiple gene abnormalities are involved in the development, malignant transformation, and metastasis of cancer.
It is considered that there are more unidentified oncogenes and tumor suppressor genes. Research and discovery of those
It is expected from clinical experts as well as people from all over the world.

It is estimated that there are about 4,500 genes on human chromosome 8, and Langer-
The existence of genes responsible for inherited diseases such as Gideorn syndrome, Werner syndrome, and retinitis pigmentosa has been suggested. Already, the present inventors and other researchers have found that lung cancer (Ohata H. et al., Genes Chromosome) is also present on the short arm of human chromosome 8.
s & Cancer 7, 85-88, 1993), liver cancer (Emi M. et al., Gene
s Chromosomes & Cancer 7, 152-157, 1993), colorectal cancer (F
ujiwara Y. et al., Cancer Res. 53, 1172-1174, 199
3) and prostate cancer (Bererheim U. et al., Genes Chrom
osomes & Cancer3, 215-220, 1991), bladder cancer (Knowle
s MA et al, Oncogene 8, 1357-1364, 1993), etc.
Therefore, it has been shown that the presence of important tumor suppressor genes involved in various cancers is predicted at this deletion site (Emi M.et
al., Cancer Res. 52, 5368-5372, 1992). Therefore, isolating the causative gene present at this site and identifying the protein are now expected to be important problems not only for doctors and researchers around the world but also for the general public.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel protein involved in cancers such as lung cancer, liver cancer, colon cancer and prostate cancer, a gene encoding the same, and a method for examining cancer using the same. It is to provide a diagnostic method and the like.

[0005]

[Means for Solving the Problems]
The common deletion region in lung cancer, liver cancer, and colon cancer in the short arm of chromosome number was further localized. That is, a large number of cosmid clones introduced with a DNA fragment of human chromosome 8 were prepared and subjected to the fluorescence in situ hybridization method (F
ISH method; Inazawa et al., Genomics, 10, 1075-1078,
1991) determined the location on the chromosome. Among these markers, the property that the length of the restriction enzyme fragment differs depending on the individual (RFLP: Restriction Fragment L
ength Polymorphism (restriction enzyme fragment length polymorphism), that is, RFLP marker was selected.

By using this, the RFLP marker can distinguish two homologous chromosomes inherited from parents by the difference in polymorphism (provided that both do not show the same polymorphism pattern: not informative). It has the characteristics of The difference in the polymorphic pattern of these two homologous chromosomes (heterozygosity) exists in normal tissues,
Phenomenon disappearing in cancer tissue (LOH: loss of heterozy
gosity) is detected in one chromosome due to its RF
It means that the LP marker site is deleted. In addition, it is generally considered that inactivation of the tumor suppressor gene on both chromosomes due to the deletion of one of the pair of chromosomes and the mutation of the other leads to canceration, which is common to many cancers. It is considered that the tumor suppressor gene exists in the deleted region. Using the detailed chromosomal map thus obtained and the RFLP marker, we have obtained about 1
As a result of examining LOH of the short arm of chromosome 8 in 00 cases of hepatocellular carcinoma, about 120 cases of colon cancer and about 50 cases of non-small cell lung cancer, the most localized deletion region common to these was revealed. Became. Liver cancer (HCC, Human Hepatocellula
r Carcinoma) and lung cancer (LC, Lung Cancer) partial deletion of the short arm of chromosome 8 is shown in FIG. Black circles represent loss of heterozygosity (LOH), open circles represent retention. Partial deletion of the short arm of chromosome 8 in colorectal cancer (CRC) is shown in FIG. Black circles represent loss of heterozygosity (LOH), open circles represent retention. Table 1 shows a list of clone names of each cosmid used as a probe, the determined position on the chromosome and LOH.

[0007]

[Table 1]

These regions are in agreement with each other and have a common most localized deletion region (8p21.3-8p2).
2 YAC clones that cover 2) (Y738, Y
812) was isolated (Figure 3). By subcloning this YAC DNA into a cosmid, a cosmid contig covering the common deletion region was constructed. From these cosmids, DNA fragments capable of becoming exons were selected by the exon amplification method. Then
A plurality of cDNA clones derived from the gene existing in the common deletion region were obtained by screening a cDNA library using the DNA fragment as a probe.
Using these cDNA clones as probes, whether or not tumor tissue-specific gene rearrangement was detected was examined by Southern blot analysis. As a result, when one cDNA clone was used as a probe, one lung non-small A clear gene rearrangement was observed in cell carcinoma. This clone is PDGF (Platelet Derived Growth Factor) receptor b (PDGFR-b) (Ya
rden Y. et al., Nature 323, 226-232, 1986) and
fms-like tyrosine kinase (flt) (Shibuya M. e
al., Oncogene 5, 519-524, 1990), which encoded a novel protein showing significant similarity to the N-terminal domain (extracellular domain). This protein is called PRLTS (PDGF recep
tor beta-like tumor suppressor) protein.
When the nucleotide sequence of the coding region of this gene was examined for the presence or absence of mutation in hepatocellular carcinoma, non-small cell lung cancer, and colon cancer, three clear gene mutations were identified. From these, it is clear that the deletion or abnormality of this protein and the deletion or mutation of the gene DNA encoding this protein are deeply involved in the development of many cancers including hepatocellular carcinoma and non-small cell lung cancer. became.

The present invention provides a risk diagnosis for at least hepatocellular carcinoma, non-small cell lung cancer, and colon cancer by examining the presence or absence of a deficiency or abnormality of the present protein or the deletion or mutation of a gene encoding the protein. It is extremely important in that it provides a solution and materials for difficult problems such as early detection, follow-up, decision of treatment course, estimation of prognosis, etc., and can make a great advance in the technology in this field. That is, the present invention includes (1) a PRLTS protein containing all or part of the amino acid sequence shown in SEQ ID NO: 1, and (2) DN containing all or part of the gene DNA shown in SEQ ID NO: 2 to 9.
A, (3) A vector containing all or part of the DNA of SEQ ID NO: 2, and a transformant carrying the vector,
(4) A method for producing a PRLTS protein using a transformant, (5) an antibody which binds to the protein of SEQ ID NO: 1,
(6) A method for gene analysis, which comprises using a single-stranded DNA containing all or part of the DNAs of SEQ ID NOs: 2 to 9 or a complementary sequence thereof. The protein and DNA of the present invention also include substantially equivalent amino acids or bases with one or more amino acids or bases added, deleted or substituted.

By using a single-stranded DNA containing all or part of the DNA of the present invention or a complementary sequence thereof as a primer or a probe, it can be used for gene analysis and diagnosis. The term "part" means that the oligonucleotide used as a primer or probe consists of at least about 6 corresponding nucleotide sequences based on the DNA sequence of the present invention, preferably at least about 8 nucleotide sequences, and more preferably It means a corresponding polynucleotide consisting of about 10 to 12, more preferably about 15 to 25 nucleotide sequences. PRL coded by this DNA
The TS protein can be used as a reagent for the production of antibodies and for research and diagnostics using the antibodies by using all or part of the TS protein as an epitope. The "epitope" means an antigenic determinant of a polypeptide, and it is known that a polypeptide composed of 6 amino acids binds to an antibody (Japanese Patent Publication No. 60-500684). Part of the PRLTS protein means at least 6 amino acids in a continuous sequence, preferably at least about 8 to 10 amino acids, and more preferably at least about 11 to 20 based on the amino acid sequence of the PRLTS protein of the present invention. It means a polypeptide consisting of 4 amino acids. It goes without saying that even a polypeptide consisting of about 20 or more amino acids can be used.

The present invention will be described in detail below. (1) Isolation of cDNA Clone For the cosmid clone of human chromosome 8, for example, chromosomal DNA was extracted from human-mouse hybrid cells containing only human chromosome 8 and the method reported by Tokino et al. et
al., Am.J.Hum.Genet., 48, 258-268, 1991),
It can be prepared by incorporating the chromosomal DNA fragment into a vector such as pWEX15. From among these, colony hybridization can be performed using all human DNA as a probe to select a clone having an insertion sequence (insert) derived from a human chromosome. With respect to a large number of cosmid clones containing the human chromosome 8-derived DNA thus obtained, the position on the chromosome can be determined by the FISH method and used as a marker to create a detailed physical chromosome map. In addition, the RFLP marker can be selected from the fragment length pattern cut with a restriction enzyme (Nakamura et al., Am. J. Hum. Gen.
et. 43, 854-859, 1988). Using this map and the RFLP marker, LOH (loss of heterozygosity) was examined in the DNA of the cancer patient's cancer tissue, and the common deletion region on the chromosome in the cancer tissue was located in the vicinity of p21.3 to 22 of chromosome 8. It can be localized to a very small area. A YAC clone having a genomic DNA in this region can be selected, and then a cosmid library can be prepared based on the DNA of this YAC clone to construct a cosmid contig of this localized common deletion region. Furthermore, from the restriction enzyme fragment of the cosmid clone, the exon amplification method (Buckler et al, Proc. Natl. Acad Sc
i.USA, 88, 4005-4009, 1991), a DNA fragment having a sequence that can be an exon can be selected.
The DNA fragment thus obtained is used as a probe for c
By screening a DNA library,
It is possible to clone the cDNA of the gene existing in the localized region of human chromosome 8 around p21.3-22. Of these cDNAs, clones having a sequence contained in cancer tissue-specific gene rearrangement can be selected by Southern blot analysis. cd
The nucleotide sequence of NA can be determined according to a conventional method (Maniatis, J. et al. Molecular Cloning 2nd.ed., Co
ld Spring Harbor Laboratory Press, NY1989). The clone of the gene DNA thus obtained was obtained by the SSCP method (Orita, M. et al, Genomics,
5, 874-879, 1984) (Orita, M. et al. Cell, 60, 509-
520, 1990), RNase Protection method (Winter, E., Peru
cho, M. et al. Proc. Natl. Acad. Sci. USA, 82, 7575-
7579, 1985) (Myers, RM, et al. Science, 230, 124
2-1246, 1985), etc. for the presence or absence of abnormalities in cancer patients,
It is possible to confirm that it is a clone of the target causative gene by searching the frequency of occurrence of abnormalities.

(2) Confirmation of the entire structure of gene The cDNA obtained by the above method was confirmed to be the novel DNA sequence of SEQ ID NO: 2, and the amino acid sequence of the novel protein encoded by it was SEQ ID NO: 1. It was deduced as follows. The present inventors named the protein containing all or part of the sequence shown in SEQ ID NO: 1 as PRLTS protein, and hereinafter referred to as PRLTS protein. Genome D
By analyzing the base sequence of NA and comparing it with the base sequence of cDNA, the intron-exon junction was confirmed, and SEQ ID NOs: 3, 4, 5 containing intron / exon,
The structures of 6, 7, 8 and 9 were revealed.

(3) Recombinant expression vector and transformant thereof The gene DNA encoding the human PRLTS protein obtained by the method described above or a fragment thereof is incorporated into an appropriate vector, and the vector is transferred into an appropriate host cell. By doing so, a transformant can be obtained. By culturing this in a conventional manner, a large amount of human PRLTS protein can be produced from the culture. More specifically, human PR
A DNA encoding the LTS protein or a fragment thereof can be recombined by a known method using a restriction enzyme and DNA ligase downstream of the promoter of a vector suitable for its expression to prepare a recombinant expression vector. Vectors that can be used include, for example, plasmid p derived from E. coli.
RB322, pUC18, Bacillus subtilis derived plasmid pU
B110, plasmid pRB15 derived from yeast, bacteriophage λgt10, λgt11 or SV40
However, the vector is not particularly limited as long as it can be replicated and amplified in the host. The promoter and terminator are not particularly limited as long as they correspond to the host used for expression of the DNA base sequence encoding the human PRLTS protein, and suitable combinations are also possible depending on the host. The DNA to be used may be any DNA as long as it encodes the human PRLTS protein, and is not limited to the nucleotide sequences shown in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8 or 9 and is intentional. Regardless of whether or not it is a DNA having a base sequence in which a part of the base sequence is replaced, deleted, inserted, added, or a combination thereof. Any of a biological method, a chemical method and the like may be used to prepare these DNAs.

The recombinant expression vector thus obtained was obtained by the competent cell method (J. Mol. Biol., 53, 154, 197).
0), protoplast method (Proc. Natl. Acad. Sci. USA,
75, 1929, 1978), calcium phosphate method (Science, 22
1, 551, 1983), in vitro packaging method (Proc. N
Atl, Acad. Sci. USA, 72, 581, 1975), virus vector method (Cell, 37, 1053, 1984) and the like are introduced into a host to prepare a transformant. As a host, Escherichia coli, Bacillus subtilis, yeast, animal cells and the like are used, and the obtained transformant is cultured in an appropriate medium according to the host. Cultivation is usually carried out in the range of 20 ° C to 45 ° C and pH 5 to 8,
Aeration and agitation are performed as necessary. PR from culture
Separation / purification of the LTS protein may be carried out by appropriately combining known separation / purification methods. These known methods include salting out, solvent precipitation, dialysis gel filtration, electrophoresis,
Ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography and the like can be mentioned.

(4) Preparation of antibody The antibody can be prepared by a conventional method using all or part of the PRLTS protein as an antigen. For example,
The polyclonal antibody is prepared by subcutaneously, intramuscularly, intraperitoneally, intravenously inoculating a plurality of animals such as mice, guinea pigs and rabbits a plurality of times to obtain sufficient immunity, and then collecting blood and separating serum from the animals. In addition, a commercially available adjuvant can also be used.
The monoclonal antibody may be prepared, for example, by preparing a hybridoma obtained by cell fusion of splenocytes of a mouse immunized with the PRLTS protein and commercially available mouse myeloma cells, and then preparing the hybridoma culture supernatant or the hybridoma-administered mouse ascites. it can.

The PRLTS protein used as an antigen does not necessarily have to have the entire amino acid structure, and may be a peptide having a partial structure, a variant thereof, a derivative thereof, or a fusion peptide with another peptide. Either a physical method or a chemical synthesis method may be used. These antibodies enable the identification and quantification of the PRLTS protein in human biological samples and can be used as cancer diagnostic reagents. The PRLTS protein immunoassay may be based on a known method, for example, a fluorescent antibody method, a passive agglutination method, an enzyme antibody method or the like.

(5) Gene analysis of human cancer tissue As a biological sample to be genetically analyzed, human normal tissue, various human cancer tissues, human blood, body fluid, secretory fluid, etc. can be used. For example, Sato et al. (Sa
to T., et al. Cancer Res., 50, 7184, 1990). By using a restriction enzyme fragment of the gene DNA encoding the human PRLTS protein provided by the present invention as a probe or appropriately selecting a base sequence at an appropriate position from the gene DNA and using the synthetic oligonucleotide as a primer The presence or absence of mutation of the gene can be analyzed. In addition, abnormalities such as insertion and deletion in the gene in the sample can also be detected by these analyses. The site of the base sequence to be selected may be any of the exon portion, the intron portion or the binding portion of both portions of the gene. Needless to say, it is also possible to use an artificially modified base sequence to be used, whereby the corresponding gene mutation can be detected. As a method of analysis, for example, a partial sequence is amplified by a PCR method using primers of two kinds of sequences selected,
The base sequence of the amplification product can be directly analyzed, or this amplification product can be incorporated into a plasmid in the same manner as described above, the host cell can be transformed and cultured, and the base sequence of the obtained clone can be analyzed. Alternatively, LigaseChain
Reaction method (Wu et al., Genomics, 4, 560-569, 1989)
In addition, mutant sequence-specific PCR method (Ruano and Kidd,
Nucleic Acid Research, 17, 8392, 1989), (CRNewt
on et al., Nucleic Acid Research, 17, 2503-2517, 1
989) can be used to directly detect the presence or absence of a specific mutation of the gene in the sample. Similarly,
SSCP method, RNase-Protecti using the probe containing the selected DNA sequence or RNA sequence derived from it
The point mutation can be detected by the on method. Further, by using these probes, detection of mutation of the gene in the sample by Southern hybridization,
It is possible to detect an abnormal expression level of the gene in a sample by the Northern hybridization method. The Escherichia coli 68cDNA carrying a plasmid containing the gene DNA encoding the PRLTS protein was deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, under the deposit number FERM B.
It was deposited on April 27, 1994 as P-4658.

[0018]

INDUSTRIAL APPLICABILITY The human PRLTS protein and the gene DNA encoding the protein of the present invention, which contains all or a part thereof, are expected as a research reagent for cancer, a test / diagnosis reagent and a therapeutic drug.

[0019]

The present invention will be described in detail and specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Isolation of Human Chromosome 8 Specific Cosmid Clone and Creation of Chromosome Map Method by Tokino et al. (Tokino et al., Am.J.Hum.Genet., 48, 2)
58-268, 1991), a human chromosome 8-specific cosmid clone was isolated. That is, the 8th part of the human chromosome
Human / mouse hybrid cell line A9n carrying only chromosome number
eo8 / t (Koi et al, Jpn.J.Cancer Res. 80, 413-4
Chromosomal DNA (18, 1989) was appropriately digested with a restriction enzyme Sau3AI, and the ends of the fragment were treated with a partial fill in using dATP and dGTP. 35 to 4 of these
Fractions of 2 kb in size were fractionated and the restriction enzyme XhoI was previously prepared.
Cosmid vector pWE, which had been cleaved with PEG and similarly treated at the ends by partial fill in with dCTP and dTTP
It was inserted in X15. From the cosmid clones thus obtained, a clone containing a human DNA fragment was selected by a colony hybridization method using a 32P-labeled human chromosomal DNA as a probe to obtain a human chromosome 8-specific cosmid clone. Released.
The position on the chromosome to which the DNA fragments of these cosmid clones hybridize is determined by the FISH method (Inazawa et al.,
Genomics, 10, 1075-1078, 1991).
Regarding a cosmid clone (cosmid marker) whose position on the chromosome has been determined, whether or not RFLP can be detected is determined by a known method (Nakamura et al., Am.J.Hum.Genet., 4
3, 854-859, 1988). The restriction enzyme is M
sp I, Taq I, Bgl II, Pst I, Pvu II, R
Either sa I or EcoR I was used.

(Example 2) Determination of the order of loci of cosmid markers on the short arm of human chromosome 8 (Emi M. et al., Cancer Res. 52,
5368-5372, 1992) In order to further narrow down the found common deletion region (8p21-8p23) in hepatocellular carcinoma, colon cancer, and non-small cell lung cancer on the short arm of human chromosome 8, Example 1 was used. The order of 14 polymorphic cosmid markers (RFLP markers) in this region obtained in 1. was determined (Table 1). Of these, four sitting positions (CI8-190, CI8-
245, CI8-487, MSR-32) has already been determined by our genetic linkage analysis (Emi et al, Genomics 15, 530-534, 1993) and CI8
Genetic distance between -190 and MSR-32 loci is 1
It was estimated to be 0 cM. The multi-color FISH method (In
azawa et al, Jpn.J. Cancer Res., 20, 1248-1252, 199
2; Inazawa et al, Cytogenet. Cell Genet., 65, 130-
135, 1994) to analyze the relative positional relationship of these markers. As a result, the order of these 14 RFLP markers (CI8- is abbreviated and indicated only by numbers) is Centromere-1308-101.
3-190- (1312, 2439)-(245, 48
7) -1051- (2644, MSR35, MSR3
2) -2014-2195-1344-telomere. However, the relative positions of the bracketed markers here were not separated by this method.

Example 3 Localization of Common Deletion Region of Human Chromosome 8 Short Arm in Hepatocellular Carcinoma, Non-Small Cell Lung Cancer and Colorectal Cancer 102 cases of Hepatocellular carcinoma, 53 cases of non-small cell lung cancer and Tumor tissue was obtained from surgical materials of 123 cases of colorectal cancer. Corresponding normal tissue or peripheral blood samples were obtained from each patient. A known method (S
ato et al., Cancer Res., 50, 7184-7189, 1990). The DNA was cleaved with an appropriate restriction enzyme, subjected to 1.0% agarose gel electrophoresis, and Southern-transferred to a nylon membrane with 0.1N-NaOH / 0.1M-NaCl (Sato et al., Cancer Res., 50, 7184-7
189, 1990). Example 2 for these membranes
Southern hybridization (Sato et al., With 14 RFLP markers sequenced by Sato et al.
Cancer Res., 50, 7184-7189, 1990) was performed to search for LOH (loss of heterozygosity) (Table 1). In hepatocellular carcinoma, heterozygosity was informative in 93 cases by at least one marker, and LOH was detected by at least one marker in 34 cases (37%). In non-small cell lung cancer, 56 cases were informative for at least one marker, of which 26 (46%) detected LOH for at least one marker. In colorectal cancer, 115 cases were informative for at least one marker, and in 64 cases (54%), at least one marker detected LOH.

From the above results, the short arm of human chromosome 8 (8
p) that can be determined to have a partial deletion, that is, heterozygosity can be distinguished by two or more markers (informativ
e), and an example showing a deletion part (LOH detection) and a retention part (LOH non-detection) was collected and analyzed. As a result, 11 cases of hepatocellular carcinoma (HCC) showed CI8-245.
And the common deletion region was localized between the two markers CI8-2644 (FIG. 1). Similarly, non-small cell lung cancer (L
In C), there are 12 cases of CI8-1051 and CI8-2644.
A common deletion region was localized between the two markers in (Fig. 1). In 25 cases of colorectal cancer (CRC), a common deletion region was localized between two markers CI8-245 and CI8-2644 (Fig. 2). The deletion region was common in cancers of these three tissues, and was localized particularly in a narrow region in non-small cell lung cancer.

Example 4 Preparation of Physical Map by Pulse Field Gel Electrophoresis (PFGE) Regarding the common deletion region localized in Example 3,
PFGE using 12 DNA markers in this region
A physical map was created by analysis. Eight of the used DNA markers are polymorphic cosmid clones used in the study of LOH. Other four non-polymorphic markers (CI8-1195, CI8-2429, C
I8-2003, CI8-1372) is a multi-color F
CI8-1312 and cMSR-3 by ISH method
The relative position to 2 was determined. Genomic DNA was digested with restriction enzymes having 7 rare cleavage points, subjected to PFGE, and Southern blot analysis was performed using each DNA marker as a probe. The sizes of the genomic DNA fragments identified by each of the 12 DNA markers are summarized in Figure 4. There are no gaps in this PFGE map,
The order of all markers except cMSR-32 and CI8-2429 was clarified. The narrowest localized common deletion region (CI8-1051) found in non-small cell lung cancer
And CI8-2644) was estimated to be approximately 600 kb.

Example 5 Preparation of Cosmid Contig of Common Deletion Region In order to isolate the gene existing in this 600 kb region,
We have cosmid markers CI8-48 to CI8.
Y812 and Y, including the genomic region spanning -2003
Two 738 YAC clones were isolated (Figure 3). A cosmid library was prepared based on the DNAs of these two YAC clones, and clones containing human DNA were prepared.
I got four. Cosmid contig maps of these 74 cosmid clones were constructed by Southern hybridization.

Example 6 Isolation of Gene from Common Deletion Region From the cosmid contig obtained in Example 5, 34 cosmid clones covering the entire common deletion region were selected and exon amplification was performed. Law (Buckler et
al, Proc.Natl.Acad Sci.USA, 88, 4005-4009, 1991)
Was searched for sequences that could be exons. as a result,
54 exon-like DNA fragments were obtained. These D
Using the NA fragment as a probe, a fetal lung-derived or fetal brain-derived cDNA library was screened. This resulted in 6 different cDNA clones.

Example 7 Detection of Gene Rearrangement in Cancer Tissue Whether or not tumor tissue-specific gene rearrangement was detected when each of the 6 types of cDNA clones was used as a probe was examined. That is, DNA of cancer tissue and corresponding normal tissue
Of the restriction enzyme (EcoRI + HindIII, PvuII or PstI) of Escherichia coli using the DNA of these cDNA clones as a probe was analyzed by Southern blotting, and large structural defects such as deletions, duplications, amplifications and translocations occurring in cancer cells were observed. A gene abnormality, so-called gene rearrangement, was detected. A DNA panel of 295 cases consisting of 102 cases of hepatocellular carcinoma, 70 cases of non-small cell lung cancer and 123 cases of colon cancer was examined. As a result, gene rearrangement was detected in one case of non-small cell lung cancer when one cDNA clone was used as a probe (FIG. 5). Comparison of the Southern blot patterns of DNA in tumor tissue and normal tissue showed that this gene rearrangement occurred in tumor tissue-specific manner. This clone was cosmid cCI8-3068
It contains a sequence derived from cCI8-3068, and cCI8-3068 is cCI8-1051 and cCI-2 by multicolor FISH.
Since it was located in the middle of 644, it was confirmed that it was derived from the above-mentioned common deletion region. In this case both lateral markers cCI8-245 (D8S335) and cMS
Since LOH is recognized for R-32 (MSR), it is clear that a somatic mutation of "two hit" occurs at the locus containing this cDNA.

EXAMPLE 8 cDNA Structure This cDNA clone was isolated from a fetal lung cDNA library and consists of 1502 bp, 61 bp of 5'untranslated region, 1128 bp coding region, 313b.
It was confirmed to be a novel DNA base sequence containing the 3'untranslated region of p (SEQ ID NO: 2). The open reading frame contained in this cDNA sequence encoded a novel protein consisting of 375 amino acids (PRLTS protein: SEQ ID NO: 1).

(Example 9) Structural determination of chromosomal DNA The sequence of the genomic DNA around the exon was determined by comparing the cDNA obtained in Example 8 with the sequence of the genomic DNA of the cosmid clone. The existence was confirmed. Sequences of genomic DNAs containing each exon are shown in SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9. The amino acid numbers in these sequence listings should be the same as those in SEQ ID NO: 1,
The position number as the PRLTS protein is shown.

(Example 10) Detection of gene mutation in tumor tissue DNA of 48 cases of hepatocellular carcinoma, 31 cases of non-small cell lung cancer and 28 cases of colon cancer single strand conformatio
n polymorphism (SSCP) analysis (Orita, M. et al, Genomi
cs, 5, 874-879, 1984) (Orita, M. et al. Cell, 60,
509-520, 1990) to search for mutations in this gene. PCR primers designed from the genomic DNA sequence obtained in Example 9 (SEQ ID NOS: 10/11, 12/13,
14/15, 16/17, 18/19, 20/21), each exon was amplified and SSCP analysis was performed. The base sequence of SEQ ID NO: 10-21 is SEQ ID NO: 3-9.
It has been synthesized based on the following relationship. Sequence number 10 = Sequence number 4, No. 3-24 Sequence number 11 = Sequence number 4, No. 281-300 Antisense Sequence number 12 = Sequence number 5, No. 49-71 Sequence number 13 = Sequence number 5, No .393-415 Antisense SEQ ID NO: 14 = SEQ ID NO: 6, No. 5-24 SEQ ID NO: 15 = SEQ ID NO: 6, No.207-231 Antisense SEQ ID NO: 16 = SEQ ID NO: 7, No. 34-53 SEQ ID NO: 17 = SEQ ID NO: 7, No. 372-393 Antisense SEQ ID NO: 18 = SEQ ID NO: 8, No. 5-24 SEQ ID NO: 19 = SEQ ID NO: 8, No. 191-211 Antisense SEQ ID NO: 20 = SEQ ID NO: 9, No. 21-41 SEQ ID NO: 21 = SEQ ID NO: 9, No.263-285 As a result of antisense SSCP analysis, a PCR product in which a change in electrophoretic pattern was observed was cloned and the nucleotide sequence was determined. As a result, colon cancer (CRC) 1 case and hepatocellular carcinoma (HCC) 1 case
A point mutation accompanied by amino acid substitution was found in an example, and a deletion mutation of 2 bases was found in one case of hepatocellular carcinoma (HCC) (Table 2). By comparing the nucleotide sequence of the tumor with the sequence of the corresponding normal tissue, it was confirmed that these mutations were present only in the tumor tissue.

[0031]

[Table 2]

One case of colon cancer, C to T in CRC20
A point mutation to 6 results in a change from histidine to tyrosine at codon 23. In this case, a sequence containing normal C was not detected in the nucleotide sequence of tumor DNA,
It has been shown that a deletion occurs in the allele. In hepatocellular carcinoma HCC74, a point mutation from C to T resulted in a change of codon 302 from valine to alanine. In another example, hepatocellular carcinoma HCC107, codon 175
A frame-shift occurred due to a deletion mutation of 2 bases in the gene, and a stop codon was generated downstream. In these cases also, allelic loss of tumor cells occurred.

[0033]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 375 Sequence type: Amino acid Sequence type: Protein Topology: Linear Origin Biological name: Homo sapiens Direct origin Library name: Human fetal lung cDNA library Sequence Met Lys Val Trp Leu Leu Leu Gly Leu Leu Leu Val His Glu Ala Leu 1 5 10 15 Glu Asp Val Thr Gly Gln His Leu Pro Lys Asn Lys Arg Pro Lys Glu 20 25 30 Pro Gly Glu Asn Arg Ile Lys Pro Thr Asn Lys Lys Val Lys Pro Lys 35 40 45 Ile Pro Lys Met Lys Asp Arg Asp Ser Ala Asn Ser Ala Pro Lys Thr 50 55 60 Gln Ser Ile Met Met Gln Val Leu Asp Lys Gly Arg Phe Gln Lys Pro 65 70 75 80 Ala Ala Thr Leu Ser Leu Leu Ala Gly Gln Thr Val Glu Leu Arg Cys 85 90 95 Lys Gly Ser Arg Ile Gly Trp Ser Tyr Pro Ala Tyr Leu Asp Thr Phe 100 105 110 Lys Asp Ser Arg Leu Ser Val Lys Gln Asn Glu Arg Tyr Gly Gln Leu 115 120 125 Thr Leu Val Asn Ser Thr Ser Ala Asp Thr Gly Glu Phe Ser Cys Trp 130 135 140 Val Gln Leu Cys Ser Gly Tyr Ile Cys Arg Lys Asp Glu Ala Ly s Thr 145 150 155 160 Gly Ser Thr Tyr Ile Phe Phe Thr Glu Lys Gly Glu Leu Phe Val Pro 165 170 175 Ser Pro Ser Tyr Phe Asp Val Val Tyr Leu Asn Pro Asp Arg Gln Ala 180 185 190 Val Val Pro Cys Arg Val Thr Val Leu Ser Ala Lys Val Thr Leu His 195 200 205 Arg Glu Phe Pro Ala Lys Glu Ile Pro Ala Asn Gly Thr Asp Ile Val 210 215 220 Tyr Asp Met Lys Arg Gly Phe Val Tyr Leu Gln Pro His Ser Glu His 225 230 235 240 Gln Gly Val Val Tyr Cys Arg Ala Glu Ala Gly Gly Arg Ser Gln Ile 245 250 255 Ser Val Lys Tyr Gln Leu Leu Tyr Val Ala Val Pro Ser Gly Pro Pro 260 265 270 Ser Thr Thr Ile Leu Ala Ser Ser Asn Lys Val Lys Ser Gly Asp Asp 275 280 285 Ile Ser Val Leu Cys Thr Val Leu Gly Glu Pro Asp Val Glu Val Glu 290 295 300 Phe Thr Trp Ile Phe Pro Gly Gln Lys Asp Glu Arg Pro Val Thr Ile 305 310 315 320 Gln Asp Thr Trp Arg Leu Ile His Arg Gly Leu Gly His Thr Thr Arg 325 330 335 Ile Ser Gln Ser Val Ile Thr Val Glu Asp Phe Glu Thr Ile Asp Ala 340 345 350 Gly Tyr Tyr Ile Cys Thr Ala Gln Asn Leu Gln Gly Gln Thr Th r Val 355 360 365 Ala Thr Thr Val Glu Phe Ser 370 375.

SEQ ID NO: 2 Sequence length: 1502 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Homo sapiens Direct origin Library name: Human fetal lung cDNA library Sequence features Characteristic symbol: CDS Location: 62. ． 1189 Methods of characterizing: E sequence CCTGCGTCCC CGCCCCGCNC AGCCGCCGCG CTCCTGCNCT CCGAGGTCCG AGGTTCCCGA 60 G ATG AAG GTC TGG CTG CTG CTT GGT CTT CTG CTG GTG CAC GAA GCG 106 Met Lys Val Trp Leu Leu Leu Gly Leu Glu Leu Glu Leu Glu 10 15 CTG GAG GAT GTT ACT GGC CAA CAC CTT CCC AAG AAC AAG CGT CCA AAA 154 Leu Glu Asp Val Thr Gly Gln His Leu Pro Lys Asn Lys Arg Pro Lys 20 25 30 GAA CCA GGA GAG AAT AGA ATC AAA CCT ACC AAC AAG AAG GTG AAG CCC 202 Glu Pro Gly Glu Asn Arg Ile Lys Pro Thr Asn Lys Lys Val Lys Pro 35 40 45 AAA ATT CCT AAA ATG AAG GAC AGG GAC TCA GCC AAT TCA GCA CCA AAG 250 Lys IlePro Lys Met Lys Asp Arg Asp Ser Ala Asn Ser Ala Pro Lys 50 55 60 ACG CAG TCT ATC ATG ATG CAA GTG CTG GAT AAA GGT CGC TTC CAG AAA 298 Thr Gln Ser Ile Met Met Gln Val Leu Asp Lys Gly Arg Phe Gln Lys 65 70 75 CCC GCC GCT ACC CTG AGT CTG CTG GCG GGG CAA ACT GTA GAG CTT CGA 346 Pro Ala Ala Thr Leu Ser Leu Leu Ala Gly Gln Thr Val Glu Leu Arg 80 85 90 95 TGT AAA GGG AGT AGA ATT GGG TGG AG C TAC CCT GCG TAT CTG GAC 394 Cys Lys Gly Ser Arg Ile Gly Trp Ser Tyr Pro Ala Tyr Leu Asp Thr 100 105 110 TTT AAG GAT TCT CGC CTC AGC GTC AAG CAG AAT GAG CGC TAC GGC CAG 442 Phe Lys Asp Ser Arg Leu Ser Val Lys Gln Asn Glu Arg Tyr Gly Gln 115 120 125 TTG ACT CTG GTC AAC TCC ACC TCG GCA GAC ACA GGT GAA TTC AGC TGC 490 Leu Thr Leu Val Asn Ser Thr Ser Ala Asp Thr Gly Glu Phe Ser Cys 130 135 140 TGG GTG CAG CTC TGC AGC GGC TAC ATC TGC AGG AAG GAC GAG GCC AAA 538 Trp Val Gln Leu Cys Ser Gly Tyr Ile Cys Arg Lys Asp Glu Ala Lys 145 150 155 ACG GGC TCC ACC TAC ATC TTT TTT ACA GAG AAA GGA GAA CTC TTT GTA 586 Thr Gly Ser Thr Tyr Ile Phe Phe Thr Glu Lys Gly Glu Leu Phe Val 160 165 170 175 CCT TCT CCC AGC TAC TTC GAT GTT GTC TAC TTG AAC CCG GAC AGA CAG 634 Pro Ser Pro Ser Tyr Phe Asp Val Val Tyr Leu Asn Pro Asp Arg Gln 180 185 190 GCT GTG GTT CCT TGT CGG GTG ACC GTG CTG TCG GCC AAA GTC ACG CTC 682 Ala Val Val Pro Cys Arg Val Thr Val Leu Ser Ala Lys Val Thr Leu 195 200 205 CAC AGG GAA TTC CCA GC C AAG GAG ATC CCA GCC AAT GGA ACG GAC ATT 730 His Arg Glu Phe Pro Ala Lys Glu Ile Pro Ala Asn Gly Thr Asp Ile 210 215 220 GTT TAT GAC ATG AAG CGG GGC TTT GTG TAT CTG CAA CCT CAT TCC GAG 778 Val Tyr Asp Met Lys Arg Gly Phe Val Tyr Leu Gln Pro His Ser Glu 225 230 235 CAC CAG GGT GTG GTT TAC TGC AGG GCG GAG GCC GGG GGC AGA TCT CAG 826 His Gln Gly Val Val Tyr Cys Arg Ala Glu Ala Gly Gly Arg Ser Gln 240 245 250 255 ATC TCC GTC AAG TAC CAG CTG CTC TAC GTG GCG GTT CCC AGT GGC CCT 874 Ile Ser Val Lys Tyr Gln Leu Leu Tyr Val Ala Val Pro Ser Gly Pro 260 265 270 CCC TCA ACA ACC ATC TTG GCT TCT TCA AAC AAA GTG AAA AGT GGG GAC 922 Pro Ser Thr Thr Ile Leu Ala Ser Ser Asn Lys Val Lys Ser Gly Asp 275 280 285 GAC ATC AGT GTG CTC TGC ACT GTC CTG GGG GAG CCC GAT GTG GAG GTG 970 Asp Ile Ser Val Leu Cys Thr Val Leu Gly Glu Pro Asp Val Glu Val 290 295 300 GAG TTC ACC TGG ATC TTC CCA GGG CAG AAG GAT GAA AGG CCT GTG ACG 1018 Glu Phe Thr Trp Ile Phe Pro Gly Gln Lys Asp Glu Arg Pro Val Thr 305 310 315 ATC CAAGAC ACT TGG AGG TTG ATC CAC AGA GGA CTG GGA CAC ACC ACG 1066 Ile Gln Asp Thr Trp Arg Leu Ile His Arg Gly Leu Gly His Thr Thr 320 325 330 335 AGA ATC TCC CAG AGT GTC ATT ACA GTG GAA GAC TTC GAG ACG ATT GAT 1114 Arg Ile Ser Gln Ser Val Ile Thr Val Glu Asp Phe Glu Thr Ile Asp 340 345 350 GCA GGA TAT TAC ATT TGC ACT GCT CAG AAT CTT CAA GGA CAG ACC ACA 1162 Ala Gly Tyr Tyr Ile Cys Thr Ala Gln Asn Leu Gln Gly Gln Thr Thr 355 360 365 GTA GCT ACC ACT GTT GAG TTT TCC TGACTTGGAA AAGGAAATGT AATGAACTTA 1216 Val Ala Thr Thr Val Glu Phe Ser 370 375 TGGAAAGCCC ATTTGTGTAC ACAGTCAGCT TTGGGGTTCC TTTTATTAGT GCTTTGCCAG 1276 AGGCTGATGT CAAGCACCAC ACCCCAACCC CAGCGTCTCG TGAGTCCGAC CCAGACATCC 1336 AAACTAAAAG GAAGTCATCC AGTCTATTCA CAGAAGTGTT AACTTTTCTA ACAGAAAGCA 1396 TGATTTTGAT TGCTTACCTA CATACGTGTT CCTAGTTTTT ATACATGTGT AAACAATTTT 1456 ATATAATCAA TCATTTCTAT TAAATGAGCA CGTTTTTGTA AAAAAT 1502.

SEQ ID NO: 3 Sequence length: 281 Sequence type: Nucleic acid Number of strands: Double strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Homo sapiens Direct origin Library name: Human DNA cosmid library Sequence features Characteristic symbols: exon1 Location: 99. ． How to determine the 150 features: E SEQ AAATTCCCCA ACTTTTTCCC CCAAACCTTG TTCCTCCTGA AGAAACCGAA TCCTCCCGCT 60 TCGGCGTCCC AGGAGCCCGC CCCTCGCCCG CCGCCTCCCC TGCGTCCCCG CCCCGCNCAG 120 CCGCCGCGCT CCTGCNCTCC GAGGTCCGAG GTTCCCGAGA TNAAGGTCTG GCTGCTGCTT 180 GGTCTTCTGC TGGTGCNCCA AGCGATGGAG GATGGTGAGT GACTCTGGGC GCGGGGCCAC 240 CTAGCTTGGT GCCCTGACTT TAGCCGGGAC CCGAAGTTTT T 281.

SEQ ID NO: 4 Sequence length: 323 Sequence type: Nucleic acid Number of strands: Double strand Topology-: Linear Sequence type: Genomic DNA Origin organism name: Homo sapiens Direct origin Library name: Human DNA cosmid library Sequence features Characteristic symbol: exon2 Location: 128. ． 191 Method for determining the characteristics: E sequence AAACCTTGTT CCTCCTGAAG AAACCGAATC CTCCCGCTTC GGCGTCCCAG GAGCCCGCCC 60 CTCGCCCGCC GCCTCCCCTG CGTCCCCGCC CCGCGCAGCC GCCGGCTCCT GCLCTCu Gru CTG Leu Ctu Glu CTG Leu CTG Leu Ctu Glu 1 5 10 CAC GAA GCG CTG GAG GAT G GTGAGTGACT CTGGGCGCGG GGCCACCTAG 221 His Glu Ala Leu Glu Asp Val 15 CTTGTGCCCT GACTTTAGCC GGGACCCGAA GCCCCCGCCG CCCTCCTGCC AGCTCTTGGT 281 CTAACGTTCG GCCCTCGGTG GCTCAGCC CC CGCNCC.

SEQ ID NO: 5 Sequence length: 486 Sequence type: Nucleic acid Number of strands: Double strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Homo sapiens Direct origin Library name: Human DNA cosmid library Sequence features Characteristic symbols: exon 3 Location: 74. ． 371 Method of Characterizing: E Sequence AAAAAGTTAT TGGCCTCAAA TATTCCAAAA ATGTCATTAC TACAGNGNAT TTCTCTCTCCC 60 TTACGTTTTG CAG TT ACT GGC CAA CAC CTT CCC AAG AAC AAG CGT CCA AAA 111 Val Thr Gly Gln His Leu Pro Lys Asn Lys Ars 20n Lys Ars 20n GGA GAG AAT AGA ATC AAA CCT ACC AAC AAG AAG GTG AAG CCC 159 Glu Pro Gly Glu Asn Arg Ile Lys Pro Thr Asn Lys Lys Val Lys Pro 35 40 45 AAA ATT CCT AAA ATG AAG GAC AGG GAC TCA GCC AAT TCA GCA CCA AAG 207 Lys Ile Pro Lys Met Lys Asp Arg Asp Ser Ala Asn Ser Ala Pro Lys 50 55 60 ACG CAG TCT ATC ATG ATG CAA GTG CTG GAT AAA GGT CGC TTC CAG AAA 255 Thr Gln Ser Ile Met Met Gln Val Leu Asp Lys Gly Arg Phe Gln Lys 65 70 75 CCC GCC GCT ACC CTG AGT CTG CTG GCG GGG CAA ACT GTA GAG CTT CGA 303 Pro Ala Ala Thr Leu Ser Leu Leu Ala Gly Gln Thr Val Glu Leu Arg 80 85 90 95 TGT AAA GGG AGT AGA ATT GGG TGG AGC TAC CCT GCG TAT CTG GAC ACC 351 Cys Lys Gly Ser Arg Ile Gly Trp Ser Tyr Pro Ala Tyr Leu Asp Thr 100 105 110 TTT AAG GAT TCT CGC CTC AG GTAANCATTT TTTTTTAAAN CTGTGTAGGG 401 Phe Lys Asp Ser Arg Leu Ser 115 TTGAGGATTT GTAATAGTTC AAAATTCCTT CTTAACTATT ATTACACATT GTTTCTGACA 461 TGTCCCATTT TCCCCTAATA GATCA 486.

SEQ ID NO: 6 Sequence length: 231 Sequence type: Nucleic acid Number of strands: Double strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Homo sapiens Direct origin Library name: Human DNA cosmid library Sequence features Characteristic symbols: exon 4 Location: 53. ． 204 Method of characterizing: E sequence GGGCCCCAGC CAGGTCTGAT TTGCTTTGAG TTCATGTGTC TNTTATTCCT NG C GTC 56 Ser Val AAG CAG AAT GAG CGC TAC GGC CAG TTG ACT CTG GTC AAC TCC ACC TCG 104 Lys Gln Asn Glu Arn Leu Leu Gru Arn Tru Gln Ser Thr Ser 120 125 130 135 GCA GAC ACA GGT GAA TTC AGC TGC TGG GTG CAG CTC TGC AGC GGC TAC 152 Ala Asp Thr Gly Glu Phe Ser Cys Trp Val Gln Leu Cys Ser Gly Tyr 140 145 150 ATC TGC AGG AAG GAC GAG GCC AAA ACG GGC TCC ACC TAC ATC TTT TTT 200 Ile Cys Arg Lys Asp Glu Ala Lys Thr Gly Ser Thr Tyr Ile Phe Phe 155 160 165 ACA G GTAAAATACT TGGTGCATTA ATGGAAC 231 Thr Glu.

SEQ ID NO: 7 Sequence length: 423 Sequence type: Nucleic acid Number of strands: Double strand Topology-: Linear Sequence type: Genomic DNA Origin organism name: Homo sapiens Direct origin Library name: Human DNA cosmid library Sequence features Characteristic symbols: exon 5 Location: 61. ． 354 Method of Characterizing: E Sequence GNGTGCTNTN ACTTTGCGTC TCCGGAGTGN ANAAGCCTGT GCTCTTCCTT CCCTTNGCAG 60 AG AAA GGA GAA CTC TTT GTA CCT TCT CCC AGC TAC TTC GAT GTT GTC 107 GluLys Gly Glu Leu Phe Val Pro Ser Pro Val Val 170 180 TAC TTG AAC CCG GAC AGA CAG GCT GTG GTT CCT TGT CGG GTG ACC GTG 155 Tyr Leu Asn Pro Asp Arg Gln Ala Val Val Pro Cys Arg Val Thr Val 185 190 195 200 CTG TCG GCC AAA GTC ACG CTC CAC AGG GAA TTC CCA GCC AAG GAG ATC 203 Leu Ser Ala Lys Val Thr Leu His Arg Glu Phe Pro Ala Lys Glu Ile 205 210 215 CCA GCC AAT GGA ACG GAC ATT GTT TAT GAC ATG AAG CGG GGC TTT GTG 251 Pro Ala Asn Gly Thr Asp Ile Val Tyr Asp Met Lys Arg Gly Phe Val 220 225 230 TAT CTG CAA CCT CAT TCC GAG CAC CAG GGT GTG GTT TAC TGC AGG GCG 299 Tyr Leu Gln Pro His Ser Glu His Gln Gly Val Val Tyr Cys Arg Ala 235 240 245 GAG GCC GGG GGC AGA TCT CAG ATC TCC GTC AAG TAC CAG CTG CTC TAC 347 Glu Ala Gly Gly Arg Ser Gln Ile Ser Val Lys Tyr Gln Leu Leu Tyr 250 255 260 GTG GCG G GTAA GCCTGG CCACCCCTGC CTAGATTCTA GTTAGTCCCC TGGTCAGTTT 404 Val Ala Val 265 CAGGTACTGC TGTTCCCTG 423.

SEQ ID NO: 8 Sequence length: 239 Sequence type: Nucleic acid Number of strands: Double strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Homo sapiens Direct origin Library name: Human DNA cosmid library Sequence features Characteristic symbols: exon 6 Location: 46. ． 185 Method of Characterizing: E Sequence TTACACTCGG GGTACTCACT CTGCCTGTTT CGTGCTTGCT TCCAG TT CCC AGT 53 Val Pro Ser GGC CCT CCC TCA ACA ACC ATC TTG GCT TCT TCA AAC AAA GTG AAA AGT 101 Gly Pro Pro Ser Thr Thr Ile Leu Ala Ser Ser Asn Lys Val Lys Ser 270 275 280 285 GGG GAC GAC ATC AGT GTG CTC TGC ACT GTC CTG GGG GAG CCC GAT GTG 149 Gly Asp Asp Ile Ser Val Leu Cys Thr Val Leu Gly Glu Pro Asp Val 290 295 300 GAG GTG GAG TTC ACC TGG ATC TTC CCA GGG CAG AAG gtaagtgttg 195 Glu Val Glu Phe Thr Trp Ile Phe Pro Gly Gln Lys 305 310 TACCTGCATC TCAGCCCCTG CGTCTCAGCC TCTGCATCTC AGCC 239.

SEQ ID NO: 9 Sequence length: 551 Sequence type: Nucleic acid Number of strands: Double strand Topology :: Linear Sequence type: Genomic DNA Origin organism name: Homo sapiens Direct origin Library name: Human DNA cosmid library Sequence features Characteristic symbols: exon 7 Location: 50. ． 551 Method of Characterizing: E Sequence ATAGCAGCTT GTCCCTCTTG CTTCAGTCTT TGTGGGTGTC GTTAAACAG GAT GAA 55 Asp Glu 315 AGG CCT GTG ACG ATC CAA GAC ACT TGG AGG TTG ATC CAC AGA GGA CTG 103 Arg Pro Val Thr Ile Gln Asu Iru Arg Gly Leu 320 325 330 GGA CAC ACC ACG AGA ATC TCC CAG AGT GTC ATT ACA GTG GAA GAC TTC 151 Gly His Thr Thr Arg Ile Ser Gln Ser Val Ile Thr Val Glu Asp Phe 335 340 345 GAG ACG ATT GAT GCA GGA TAT TAC ATT TGC ACT GCT CAG AAT CTT CAA 199 Glu Thr Ile Asp Ala Gly Tyr Tyr Ile Cys Thr Ala Gln Asn Leu Gln 350 355 360 GGA CAG ACC ACA GTA GCT ACC ACT GTT GAG TTT TCC TGACTTGGAA 245 Gly Gln Thr Thr Val Ala Thr Thr Val Glu Phe Ser 365 370 375 AAGGAAATGT AATGAACTTA TGGAAAGCCC ATTTGTGTAC ACAGTCAGCT TTGGGGTTCC 305 TTTTATTAGT GCTTTGCCAG AGGCTGATGT CAAGCACCAC ACCCCAACCC CAGCGTCTCG 365 TGAGTCCGAC CCAGACATCC AAACTAAAAG GAAGTCATCC AGTCTATTCA CAGAAGTGTT 425 AACTTTTCTA ACAGAAAGCA TGATTTTGAT TGCTTACCTA CATACGTGTT CCTAGTTTTT 485 ATACATGTGT AAACAATTTT ATATAATCAA TCAT TTCTAT TAAATGAGCA CGTTTTTGTA 545 AAAAAT 551.

SEQ ID NO: 10 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence ACCTTGTTCC TCCTGAAGAA AC 22.

SEQ ID NO: 11 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence ACCGAGGGCC GAACGTTAGA 20.

SEQ ID NO: 12 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence ATTTCTCTCT CCTTACGTTT TGC 23.

SEQ ID NO: 13 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TTACAAATCC TCAACCCTAC ACA 23.

SEQ ID NO: 14 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence CCCAGCCAGG TCTGATTTGC 20.

SEQ ID NO: 15 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GTTCCATTAA TGCACCAAGT ATTTT 25.

SEQ ID NO: 16 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGCCTGTGCT CTTCCTTCCC 20.

SEQ ID NO: 17 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence GGGACTAACT AGAATCTAGG CA 22.

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

SEQ ID NO: 19 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence GGCTGAGATG CAGGTACAAC A 21.

SEQ ID NO: 20 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CTTCAGTCTT TGTGGGTGTC G 21.

SEQ ID NO: 21 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid (synthetic DNA) sequence GTACACAAAT GGGCTTTCCA TAA 23

[Brief description of drawings]

FIG. 1 shows partial deletion of the short arm of chromosome 8 in hepatocellular carcinoma (HCC) and lung cancer (LC). Black circles represent loss of heterozygosity (LOH), open circles represent retention. The common deletion region is indicated by a black frame. The clone name is shown only by the clone number.

FIG. 2 is a diagram showing partial deletion of the short arm of chromosome 8 in colorectal cancer (CRC). Black circles represent loss of heterozygosity (LOH), open circles represent retention. The common deletion region is indicated by a black frame. The clone name is shown only by the clone number.

FIG. 3 is a diagram showing the markers located in the common deletion region of the short arm of chromosome 8 and the positions of two YAC clones existing in this region. Marker names are shown by clone number only.

FIG. 4 is a diagram showing the size of a restriction enzyme fragment identified by using each DNA marker around a localized common deletion region as a probe. The size is shown in Kb. The clone name is shown only by the clone number.

FIG. 5 shows detection of gene rearrangement in lung cancer by Southern blot. N and T represent DNA of normal tissue and cancer tissue, respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location C12N 15/09 ZNA C12P 21/02 C 9282-4B 21/08 9358-4B C12Q 1/68 A 9453 -4B G01N 33/566 33/574 ZA

Claims (13)

[Claims] 1. A PRLTS protein containing all or part of the amino acid sequence of SEQ ID NO: 1. 2. A PRLTS protein containing all or part of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 1 has been added, deleted or substituted. 3. A DN encoding a PRLTS protein containing all or part of the amino acid sequence of SEQ ID NO: 1.
A. 4. A DNA encoding a PRLTS protein containing all or part of an amino acid sequence in which one or more amino acids of the amino acid sequence of SEQ ID NO: 1 has been added, deleted or substituted. 5. A vector containing the DNA according to claim 3 or 4. 6. A transformant carrying the vector according to claim 5. 7. The method for producing a protein according to claim 1, which comprises culturing the transformant according to claim 6 and recovering the expression product. 8. A DN containing all or part of the intron DNAs of SEQ ID NOS: 3 to 9 or a complementary sequence thereof.
A. 9. A DNA probe comprising a single-stranded DNA containing all or a part of the nucleotide sequence of SEQ ID NO: 2 or a complementary sequence thereof. 10. A DNA primer comprising a single-stranded DNA containing all or a part of the nucleotide sequence of SEQ ID NO: 2 or a complementary sequence thereof. 11. An antiserum or a monoclonal antibody that binds to the protein according to claim 1 or 2. 12. A method for identifying a cancer cell, which comprises detecting a mutation in the genome of the DNA according to claim 3. 13. The identification method according to claim 12, wherein the DNA according to claim 8 or 9 is used.