JPH0965900A - Gene diagnosis using cck-a receptor gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To genetically conduct a diagnosis of a disease involved in the gene abnormality of CCK-A receptor such as type II diabetes or biliary calculus by using a deletion site in the cholecystokinin (CCK)-A receptor gene of type II diabetics. SOLUTION: In this gene diagnostic method for type II diabetes using a deletion site in the CCK-A receptor gene of type II diabetics concomitant with biliary calculus or obesity, the deletion site is a portion of normal CCK-A receptor gene of formula I, comprising exon 1 having a base sequence of formula II or exon 2 having a base sequence of formula III. Using the abnormal gene described above, a Southern blotting hybridization assay is conducted to accurately diagnose a disease involved in CCK-A receptor gene abnormality such as type II diabetes or biliary calculus by using the abnormal gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for diagnosing a gene associated with a gene abnormality of CCK-A receptor using a cholecystokinin (hereinafter abbreviated as CCK) -A receptor gene. More specifically, it relates to a method for diagnosing type II diabetes and cholelithiasis using the gene.

[0002]

2. Description of the Related Art Conventionally, it has been considered that both genetic factors and environmental factors contribute to the onset of type II diabetes, that is, adult diabetes. However, the reality of genetic factors remains unclear. In addition, experimental animals such as diabetic rats (DM rats) are actively used in the study of diabetes, but the elucidation of the genetic factors thereof has not progressed yet, and the progress of the studies is expected. Therefore, for the diagnosis of type II diabetes, blood glucose level measurement, blood insulin measurement, urinary CPR
Only the measurements were done.

[0003]

SUMMARY OF THE INVENTION The object of the present invention is to provide CC
An object of the present invention is to provide a gene diagnosis method for a disease associated with a gene abnormality of CCK-A receptor using the KA receptor gene. That is, based on the recent achievements of the present inventors regarding the type II diabetes gene that is characteristic of type II diabetic rats (DM rats) and is considered to be the cause of human type II diabetes, which is involved in the development of diabetes, type II diabetic patients The purpose of the present invention is to provide a method for diagnosing type II diabetes mellitus using a deletion site existing in the CCK-A receptor gene. The diagnostic method of the present invention is also useful for diagnosing type II diabetes associated with gallstones or obesity,
It is also useful for predicting the risk of type II diabetes. Furthermore, the present invention provides a method for diagnosing a cholelithiasis, which uses as an index the mRNA expression level of the CCK-A receptor gene in the tissues of patients with cholelithiasis is reduced or not expressed at all.

[0004]

To achieve the above object, the present inventors have established an Otsuka Long-Evans Tokushima Fatty (OLE), which has been established as an animal model for type II diabetes.
When a comparative study was carried out on TF) rats and LETO rats (normal rats), there was almost no difference in the insulin level in the pancreas of both animals, and no histological difference was observed. No difference was observed in the expression of the CCK-B receptor gene, but the CCK-A receptor gene was expressed in the normal rat but not in the type II diabetic rat (OLETF rat), and the CCK-A receptor gene was not expressed. It was discovered that there were marked differences in the expression of somatic genes.
Therefore, the present inventors conducted perfusion experiments and the like using the pancreas of both animals, and examined the effect of CCK on insulin secretion. As a result, in normal rats, a marked increase in insulin concentration was observed by the addition of CCK. In contrast, the pancreas of type II diabetic rats showed no response to the addition of CCK.

From the above findings, it was determined that the appearance of pancreatic endocrine disorders in type II diabetic rats was not due to insulin deficiency but to defects in the expression of the CCK-A receptor gene (Funakoshi, A. et al. et a
l., Biochem. and Biophys. Res. Commun. 210, 787-79
6 (1995)).

Furthermore, in order to elucidate the cause of the defect in the expression of the CCK-A receptor gene, II rats and normal rats were examined.
When the gene structure of the CCK-A receptor gene in type 2 diabetic rats was examined, it was found that in normal rats, it consisted of 5 exons, whereas in type II diabetic rats it consisted of 3 exons. It was discovered that there is a deletion of exons. Therefore, a partial deletion of the CCK-A receptor gene
It was determined to be one of the causes of type II diabetes. The present invention has been completed based on these findings.

Further, it was found that in patients with cholelithiasis, the mRNA expression level of the CCK-A receptor gene was unexpectedly decreased or not expressed even in the case of type II diabetes, and based on this finding. As a result, we have completed a method for genetic diagnosis of gallstone disease.

Cholecystokinin (CCK) is a well-known gastrointestinal hormone. Its receptors in peripheral tissues and the central nervous system are classified into two subtypes (CCK-A receptor and CCK-B receptor) based on their affinity for a group of structurally and functionally related peptides. It is classified. That is, the CCK-A receptor has affinity for the sulfate type of a pentapeptide having the same carboxyl terminus, and the CCK-B receptor has a non-sulfate (N
It has an affinity for S) type.

The gist of the present invention is (1) II using a deletion site in the CCK-A receptor gene of type II diabetic patients.
Method for genetic diagnosis of type 2 diabetes, (2) Method for genetic diagnosis according to the above (1), wherein type II diabetes is type II diabetes associated with cholelithiasis or obesity, and (3) deletion existing in CCK-A receptor gene Of the normal rat CCK-A receptor gene shown in SEQ ID NO: 1 of Sequence Listing, the site is exon 1 shown in SEQ ID NO: 2 of Sequence Listing or exon 2 shown in SEQ ID NO: 3 of Sequence Listing. The gene diagnosis method according to (1) or (2) above, which is a part including
(4) A method for diagnosing type II diabetes by comparing the expression level of CCK-A receptor gene mRNA in the tissue of type II diabetes patient with that of a healthy person, and (5)
The present invention relates to a method for diagnosing cholelithiasis, which uses as an index the mRNA expression level of the CCK-A receptor gene in the tissues of patients with cholelithiasis is reduced or not expressed at all.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The deletion site of the type II diabetes gene used in the present invention will be described in detail below. (1) The diabetes gene that is the target of the diagnostic method of the present invention is
It is a CCK-A receptor gene, which has the nucleotide sequence of SEQ ID NO: 1 in the sequence listing in normal rats, but lacks a part of the CCK-A receptor gene in normal rats in type II diabetic rats. I have lost. CCK-A in normal rat
The genomic structure of the receptor gene was found to consist of 5 exons (see Figure 1). In contrast, the type II diabetic rat CCK-A receptor gene has a normal gene of 5
Two of the exons, namely, exon 1 having the base sequence shown in SEQ ID NO: 2 and exon 2 having the base sequence shown in SEQ ID NO: 3 are deleted, and exons 3, 4 and 5 are deleted. It consists of only one (see Figure 2). The mechanism by which type II diabetic rats develop type II diabetes has not been previously clarified.
It was confirmed for the first time in the present invention that a partial deletion of the -A receptor gene results in an inherent defect in CCK-A receptor expression, which is one of the causes of the development of type II diabetes. . The facts that support this are explained below.

(2) First, it is shown that the type II diabetic rat is different from the normal rat in that the expression of the CCK-A receptor is defective.

CCK-A receptors and CCK- in the pancreas and brain of adult rats and in the pancreas of fetal rats
Northern transfer analysis of B receptor revealed that ³² P-labeled cDNA in the coding region of CCK-A receptor was detected.
The probe was 2.7 kb mR from the pancreas of a normal adult rat.
Approximately 2.7 kb of mRNA which hybridized with NA but hybridized with the probe of the coding region of CCK-A receptor was not detected in the pancreas of type II diabetic adult rat and fetal rat (Funakoshi, A. et al., B
iochem. and Biophys. Res. Commun. 210, 787-796 (19
95)).

As a primer, rat CCK-
CCK-A receptor and C from rat tissue were prepared using a sense primer and an antisense primer corresponding to the nucleotide sequence of the A receptor precursor cDNA or the CCK-B receptor precursor cDNA, respectively.
The coding region of the CK-B receptor cDNA was amplified by the polymerase chain reaction (PCR). As a result, the expected size (CCK-
Amplified DNA of 495 base pairs was obtained for the DNA amplified from A receptor precursor mRNA, but the corresponding one was not obtained from type II diabetic rats. In addition, D of CCK-A receptor in adult rat pancreas
DNA of the same size as NA was also amplified from normal rat brain RNA, but not from type II diabetic rat brain (Funakoshi, A. et al., Biochem. And Bioph.
ys. Res. Commun. 210, 787-796 (1995)).

[0014] Southern blot analysis of genomic DNA also revealed a clear difference between the type II diabetic rat genome and the normal rat genome. That is, the genomic DNAs of type II diabetic rats and normal rats were converted into Kpn I, Sma
Digestion with restriction enzymes such as I, Pvu II, or Xba I followed by Southern blot hybridization analysis with CCK-A receptor cDNA yielded different restriction fragments (Funakoshi, A. et. al., Bi
ochem. and Biophys. Res. Commun. 210, 787-796 (199
Five)). This fact indicates that CCK- in type II diabetic rats.
It shows that the structure of the A receptor gene is changed.
As is clear from the above-described facts, the type II diabetes gene is the cause of diabetes in type II diabetic rats. And it is considered that this also applies to type II diabetes in humans.

(3) Gene Diagnosis of Type II Diabetes and Cholelithiasis As is clear from the above findings, the method of gene diagnosis of type II diabetes can be exemplified by the following method. There is a method of analyzing the CCK-A receptor gene (type II diabetes gene) of a subject and detecting whether or not the CCK-A receptor gene of type II diabetes patient contains a deletion site. . Southern blot analysis is performed for analysis of genomic DNA. For humans, the PCR-Southern blot method is preferred. That is, PCR was performed using the CCK-A receptor gene of the subject as a target sequence, the PCR reaction product was subjected to electrophoresis, and then Southern blot was performed on a nylon membrane, and the obtained blot was used for CCK-A of type II diabetic patients. ³² P-labeled cDNA corresponding to the deletion site in the receptor gene
After hybridizing with A, specifically, Biochemical
and Biophysical Research Communications 194 (2), 8
Human C described in FIG. 1 of the report of 11-818 (1993).
In the nucleotide sequence of the cDNA clone of the CK-A receptor (shown in FIG. 7), positions 154 to 265 (exon 1), positions 266 to 517 (exon 2), positions 518 to 518 Up to 779th place (Exon 3),
D from 780th to 907th (exon 4) and 908th to 1440th (exon 5)
The NA fragment is ³² P-labeled, hybridized using it as a probe, and then subjected to autoradiography, and it is the normal sequence that hybridizes with all exons, and any of the above 5 exons Those that do not hybridize with this can be diagnosed as the type II diabetes gene.

CCK in tissues of type II diabetic patients
There is a method of comparing the expression level of mRNA of the -A receptor gene with that of a healthy person. For the analysis of mRNA in this method, Northern blotting well known to those skilled in the art was carried out, and CC
Confirm that KA receptor mRNA is not expressed. Alternatively, the RT-PCR method, the RFLP method or the like may be used.

The above method can be used for diagnosing type II diabetes not only when the CCK-A receptor is not expressed at all but also when the expression is decreased. CCK-A
The above-mentioned event that the receptor is not expressed due to a gene defect is a typical example of decreased expression. The decreased expression of the CCK-A receptor is decreased due to various factors related to the expression of the CCK-A receptor. For example, a decrease in the expression of the CCK-A receptor is observed due to an abnormality in the promoter region that controls the CCK-A coding region, some processing abnormality, and the like. Therefore, the present invention also includes a diagnosis using a decrease in CCK-A receptor expression as an index.

As shown in Examples 3 and 4, the present inventors confirmed that CCK-A receptor expression was reduced in the gallbladder of human cholelithiasis patients by using the same method. From this, it was considered that poor gallbladder contraction due to a decrease in CCK-A receptor function may be one of the causes of gallstone formation. Therefore, the present invention uses the same method as described above to determine whether the expression level of CCK-A receptor gene mRNA in the gallbladder is decreased or not expressed even in cholelithiasis without type II diabetes. It can be detected by analyzing the mRNA, and the gene diagnosis of cholelithiasis can be performed.

The present invention is useful not only for diagnosing diabetes but also for diagnosing type II diabetes associated with cholelithiasis or obesity. A common event in diabetes, gallstones, and obesity is that they are caused by overeating. Therefore, the present invention is useful for predicting the risk of diseases caused by overeating. That is,
When the expression of the CCK-A receptor is decreased (including the case where it is not expressed at all) according to the present invention, the diet can prevent the disease caused by overeating.

[0020]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 (CCK-A of type II diabetic rat)
Sequencing of Receptor Genes) Preparation of Genomic DNA from Type II Diabetic Rats The general DNA technique used below is Molecular
Cloning was performed by the method described in the Laboratory Manual (T. Maniatis, 1982). Pancreatic cells of type II diabetic male rats donated by Otsuka Pharmaceutical were excised and genomic DNA was prepared by the standard phenol-chloroform method.

Southern blot analysis 10 μg of genomic DNA of type II diabetic rat was digested with a restriction enzyme, separated by 0.7% agarose gel electrophoresis, and blotted on Hybond-N nylon membrane (Amersham). This blot was labeled with ³² P-labeled CCK-A.
It was hybridized with a cDNA containing the entire region of the receptor (a gift from Dr SA Wank). This membrane was washed with SSC containing SDS by a conventional method and then autoradiographed.

Construction of genomic DNA library of type II diabetic rat Genome D of type II diabetic rat obtained as described above
NA was partially digested with Sau3AI and separated by sodium chloride density gradient ultracentrifugation. 10 kb to 20 kb
A fraction containing a long DNA fragment was collected and digested with BamHI.
It was ligated with the EMBL3 arm. Then, it was packed with GIGA Pack Gold (manufactured by Stratagene). this
Type II diabetic rat genomic DNA library is 1.8
It contained x10 ⁶ independent phage clones.

Isolation of Genomic DNA Clones A type II diabetic rat genomic DNA library was screened with the same ³² P-labeled CCK-A receptor cDNA probe used for genomic Southern blotting. About 1.0 × 10 ⁶ phage clones were screened under standard plaque hybridization conditions, 7 positive phage clones were isolated and further identified by Southern blot analysis and restriction enzyme mapping.

DNA Sequence Analysis A SalI digested DNA fragment hybridized with a CCK-A receptor cDNA probe was isolated from λN15 and λN25 phage clones, further randomly digested with DNase I for subsequencing and subcloned into M13mp19. . DNA sequencing is done by ABI Dy
e Primer UniCycle sequencing kit was used to perform with ABI model 373A DNA sequencer, and the obtained sequences were ligated using Inherit Sequence Analysis System (manufactured by Perkin Elmer). Nucleotide sequence analysis was performed using a personal computer program (Geneworks, manufactured by Intelligenetics).

As a result of the above experiment, as the base sequence of the CCK-A receptor gene of type II diabetic rat, as shown in FIG. 3, the base of the base sequence of the normal rat described in SEQ ID NO: 1 in the sequence listing. SEQ ID NOs: -2382 to 4465
A base sequence deleted was obtained. This deletion portion contained exon 1 (SEQ ID NO: 2 in the sequence listing) and exon 2 (SEQ ID NO: 3 in the sequence listing) (FIG. 3).

Example 2 (Diagnosis of type II diabetic rats by PCR-Southern blot analysis of CCK-A receptor gene from rat tissue) For cDNA synthesis, first-strand cD
Using an NA synthesis kit (Pharmacia Biotech), 1 μg of mRNA was incubated with reverse transcriptase of MLV at 37 ° C. for 60 minutes. In order to amplify the coding region of CCK-A receptor precursor cDNA, the above cDNA solution, 2.5 units of Taq DNA polymerase (manufactured by Seikagaku Corp.), and the nucleotide sequence of rat CCK-A receptor precursor cDNA were used. Sense primer (nucleotides 313-337, SEQ ID NO:
4) and antisense primer (nucleotide 11
29-1109, 50 pmol of each of SEQ ID NO: 5) and a sense primer (nucleotides 284-303, SEQ ID NO: corresponding to the nucleotide sequence of rat β ₂ microglobulin precursor cDNA as an internal standard).
6) and antisense primer (nucleotide 37
PC in a reaction mixture containing 3-392, SEQ ID NO: 7).
R was performed for 40 cycles.

The cycle consists of 96 ° C. for 3 minutes and 54 ° C. for 3 minutes.
1 cycle of 0 seconds at 72 ° C for 60 seconds, then 94 ° C
For 30 seconds, 54 ° C. for 3 minutes, and 72 ° C. for 3 minutes for 40 cycles. The reaction products were fractionated by NuSieve agarose (3%) gel electrophoresis and the resulting gel was then stained with ethidium bromide.

As a result, from normal rat pancreatic RNA, by PCR of 40 cycles, the expected size (495 base pairs for DNA amplified from CCK-A receptor precursor mRNA, and β ₂ -micro) was obtained. DNA amplified from globulin precursor mRNA
(109 base pairs) were obtained, but not from type II diabetic rats (Fig. 4). In addition, DNA of the same size as the CCK-A receptor DNA of the pancreas of adult rat is RNA of the brain of normal rat.
, But not from the brain of type II diabetic rats (Fig. 4).

PCR-Southern blotting was performed on the obtained DNA. The PCR-Southern blot method was performed as follows. That is, the PCR reaction product was
They were electrophoretically separated in ieve agarose gels and then blotted onto nylon membranes. The resulting blot, sequence numbers in the sequence listing of the CCK-A receptor in rats: SEQ ID NO: exon 1 and / or the sequence listing described in 2: the coding region of the portion including the exon 2 described in 3 ^[32 P] sign c
The DNA probe was hybridized at 37 ° C. for 12 hours, and the SSC solution washing solution containing 0.1% SDS was sequentially diluted to a final concentration of 0.1 × SSC at 65 ° C. and then autoradiographed. This yielded one 0.5 kb hybridizing fragment in the case of normal rats. However, in the case of type II diabetic rats, no fragment hybridizing with the probe was obtained.

Example 3 (Reduction of CCK-A Receptor Expression in Gallbladder of Human Cholelithiasis Patient) The expression of CCK-A receptor gene in the gallbladder of human gallstone disease patient was compared with that in the gallbladder of healthy subjects by the Northern transfer method. Compared. As a control, actin mR
The expression of NA was also examined. All gallbladders of human cholelithiasis patients were excised at the time of surgery. As a result, as shown in FIG. 5, in the case of the expression of actin mRNA, no difference was observed between the gallbladder of the cholelithiasis patient and the gallbladder of the healthy person.
In the case of KA receptor mRNA expression, a significant decrease in expression was observed in human cholelithiasis patients. In this example, autoradiography was performed using Biochemical and Bi
ophysicalResearch Communications 194 (2), 811-818
(1993), the human CCK-described in FIG. 1 of the report.
The full length A receptor cDNA was used as a probe.

Example 4 (Reduced expression and inability of expression of CCK-A receptor in the gallbladder of human cholelithiasis patients) As in Example 3, DNA was extracted from peripheral blood of 3 cholelithiasis patients.
Was extracted and subjected to Southern amplification and amplification of each exon site including the promoter region by PCR to search for human gene abnormalities. Furthermore, gallbladder tissues were collected by surgery to examine the expression level of CCK-A receptor mRNA. As a result, cholelithiasis patients (No. 4
6), the amount of CCK-A receptor mRNA in the gallbladder was significantly decreased in 2 cases (No. 1 to 3) as compared with the normal gallbladder (No. 1 to 3).
5 and 6), one case (No. 4) was not normally expressed. In the case of No. 4 in which the CCK-A receptor mRNA was not normally expressed, as shown in FIG.
form) was expressed, suggesting an abnormal splice.
From these, it was considered that the poor gallbladder contraction due to the decrease in CCK-A receptor function may be one of the causes of gallstone formation. In addition, the autoradiography in this example is the same as in Example 3, and the biochemical and biophysical research is performed.
ch Communications 194 (2), 811-818 (1993) reported full length c of human CCK-A receptor described in FIG.
DNA was used as a probe.

Example 5 (Use of Human CCK-A Receptor Gene in Diagnosis of Human Type II Diabetes) Biochemical and Biophysical Research Communication
s 194 (2), 811-818 (1993), the nucleotide sequence of the human CCK-A receptor cDNA shown in FIG. 1 has exons 1 to 266 at positions 154 to 265. Exons 2 to 517th, Exons 3 to 518 to 779, 780 to 9th
Exon 4 up to 07, and 908 to 14
Exon 5 is up to the 40th place. Therefore, based on the findings in the rat, even in the case of human, CCK in the pancreas of human type II diabetic patients was probed using each of exon 1 to exon 5 of the CCK-A receptor gene.
-Discovering human type II diabetic patients by examining the expression of A-receptor gene, using the fact that normal CCK-A receptor gene that hybridizes with any of the probes is not expressed or its expression is decreased Is possible.

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, a gene diagnosis method for diseases associated with CCK-A receptor gene abnormality such as type II diabetes and cholelithiasis using the CCK-A receptor gene has become possible.

[0035]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 10914 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Origin of organism: Normal rat Characterization method: E Sequence AGCATTCGGT TCCCTCAGTG GGAAACTTGT ACACAATGGG TGCGAGGGAT ACCTAGCAAA 60 TAGATCAGGT CATAATACAC TTGGACAAAC CAGTCAAAAA CACAGATGTT CAGACTAAGG 120 CCAGTTGTAT AGTGAACAGT GAGGGACTTT TAAATATTGT GTAACTTCCT AAAATAAATC 180 CAGGCCCCTG AACATGTCTC TCACACTCCT CCCCGCTCCC GACATTCCCT AACTTATTCC 240 TACTTCTGTT GTTCAAACAG GCCAATCTCA TTACTGCCTA AGACCCAGAG GTAACCTCTG 300 CAAAAAATGT TGTGTCTCCT GTTGGAAGCT TTCTTATTTA CCCCTCAGAT CTTACACCAC 360 CAGTACCCCA GACCCCTACC AGGGAGGCTG CAATCCTTTC CTTCTCATCC TGGAAGGCTT 420 GAAAAGCACT CAGCAAATGG TTGACTCTTG GTGAACAGGT CTGAGTGGAC AAAGCAAAAA 480 ACAAACAAAA TAAACAAACG TGAATAAAAA CATTGCTGAT TTCTATAGTA TTAAGGACCT 540 AGGAATCTGT ATTCAGAAGA GTCCCCATTT CTGGTTAACA AGGGCAGAGA GAGACCACCCAA 600 GCCACAGAAC CGTGCACACTCACACTAGCAACTT TTGCT 660 GGGCAAGGCT CCAAAGCTAA AAGGCACTAA TTGCTGTATA TAAGGAGATA GGAATGCAGT 720 CCTTCTGTGT CCCTTAGTCC TGATGGTCTG CATTATCGGA TCTGTTACCT TGTTAATTGA 780 TGCTGTCTCT CACAGTTCAG GTCTCACTCT GAATATGTCT GGGACAATGA CAGGCAGAGG 840 GGAGAGGTAA TAATTGGAGT TCTCTCTCTG TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC 900 TCTCTCTCTC CACACTCGTG TGTGTGTGTG TGTGTGTGTC GCGCGCGCGC ACGCGAGCAC 960 ACATGCACGC ATGCAGATGT GTCCCCATTC TGGGAAGGGT TAATATGCAG CCAGCTCTGC 1020 CTGTGTTCAG GGCCTCAGCC TGGCCCAGGA GTGGGAGGGG TGGGCCTGGA AGGTGGAGTG 1080 GAGCAGGCTT GACTTGCACA GGCTGCCTTC TCCCCACAGG AGTGCAAATG CTTGCCCAGA 1140 TGCTCTGAGA ATGGCGAACT CAAGTTGCCT TTAGGAATGG CTGCAAAGCC CACACCTGGA 1200 AATCTCCCCC TCCCTGCTCC TCCACGGCAG GTTGCATTTG GGAGACTCTG TGATCATTAG 1260 AGGAGAGAGA CAGGA ATG AGC CAT TCA CCA GCT CGC CAG CAC TTG GTA GAA 1311 Met Ser His Ser Pro Ala Arg Gln His Leu Val Glu 5 10 AGC AGC AGG ATG GAC GTG GTC GAC AGC CTT CTT ATG AAT GGG AGC AAC 1359 Ser Ser Arg Met Asp Val Val Asp Ser Leu Leu Met Asn Gly Ser Asn 15 20 25 ATC ACT CCC CCC TGT GAA CTC GGA CTG GAA AAT GAG ACG CTT TTC TGC 1407 Ile Thr Pro Pro Cys Glu Leu Gly Leu Glu Asn Glu Thr Leu Phe Cys 30 35 40 TTG GAT CAA CCT CAA CCT TCA AAA GGTAGGTGCC AGACACTATG ATTAGAATTC 1461 Leu Asp Gln Pro Gln Pro Gln Lys G 45 50 AGAAAACTAC CGAGTTAAGG GAGATATAGC AACAGCAAGG CTAATTGAAA TATACATGC 1521 ATGAAATATA CA ------- 527 bp ------- GGACATCA TCAGGAACTA AGCACTCCC 2088 TTGGGGGCTT AAGGTTTTTA ACATTCTGCC CTCTAAACCC CCCATCCCCCTCATCCCCTCCT CTC CTG TAC TCC 2197 Glu Trp Gln Ser Ala Leu Gln Ile Leu Leu Tyr Ser 55 60 ATC ATA TTC CTT CTC AGT GTG CTG GGG AAC ACG CTG GTT ATA ACG GTG 2245 Ile Ile Phe Leu Leu Ser Val Leu Gly Asn Thr Leu Val Ile Thr Val 65 70 75 80 CTG ATT CGA AAC AAG AGG ATG CGG ACG GTC ACC AAC ATC TTC CTG CTG 2293 Leu Ile Arg Asn Lys Arg Met Arg Thr Val Thr Asn Ile Phe Leu Leu 85 90 95 TCC CTG GCT GTC AGT GAC CTC ATG CTC TGC CTC TTC TGC ATG CCG TTC 2341 Ser Leu Ala Val Ser Asp Leu Met Leu Cys Leu Phe Cys Met Pro Phe 100 105 110 AAC CTC ATC CCC AAC CTG CTC AAG GAT TTC ATC TTC GGA AGT GCC GTG 2389 Asn Leu Ile Pro Asn Leu Leu Lys Asp Phe Ile Phe Gly Ser Ala Val 115 120 125 TGC AAG ACT ACC ACC TAC TTC ATG GGTGAGTCCT GCAGGTGTCT CyGG Lys Thr Thr Tyr Phe Met G 130 135 CTTATCCCCC CACACACCCC TTTGTCAGCC CCTTGATGGT CACAGCAGAC AGACCCTGTT 2503 GGGTTCCAAA A ----- 2910 bp ------ TCTCAGGCA ATTCTAAACT GCAGTTCTGA 5453 TACATTTAAG CTAATGTACA AATAAACTTT CGAGTCAACCACTTTCTGAGATTCAACA TTC AAC CTG GTA GCC 5562 ly Thr Ser Val Ser Val Ser Thr Phe Asn Leu Val Ala 140 145 ATC TCT CTG GAG AGA TAT GGC GCC ATC TGC AGA CCC CTA CAG TCC CGC 5610 Ile Ser Leu Glu Arg Tyr Gly Ala Ile Cys Arg Pro Leu Gln Ser Arg 150 155 160 165 GTC TGG CAA ACA AAG TCC CAT GCT TTG AAG GTC ATC GCT GCC ACC TGG 5658 Val Trp Gln Thr Lys Ser His Ala Leu Lys Val Ile Ala Ala Thr Trp 170 175 180 TGC CTC TCC TTT ACC ATC ATG ACT CCG TAC CCC ATT TAC AGC AAC TTG 5706 Cys Leu Ser Phe Thr Ile Met Thr Pro Tyr Pro Ile Tyr Ser Asn Leu 185 190 195 GTG CCT TTT ACT AAA AAT AAT AAC CAG ACG GCG AAC ATG TGC CGC TTC 5754 Val Pro Phe Thr Lys Asn Asn Asn Gln Thr Ala Asn Met Cys Arg Phe 200 205 210 CTG TTG CCA AGT GAC GCT ATG CAG CAG TCC TGG TAAGGCAGTG CACTTCGCTC 5914 Leu Leu Pro Ser Asp Ala Met Gln Gln Ser Tr 215 220 CTCCTTTACA ATAAAGCTAG TCTAAGTTAG CAGTGTGAGG GACCGGAAAG CCTTACCAGA 5974 TGACTATGAG TCAGATATC ------ 1800 CAT13ACTC CCCGCCCATC AGAACATGAA GGTCCAGAGT CACTAATTAG 7873 ATTTCTTTTA ATTGTATTAG G CAA ACA TTC CTG CTA CTC ATC CTC TTT CTT 7924 p Gln Thr Phe Leu Leu Leu Ile Leu Phe Leu 225 230 CTC CCT GGG ATT GTG ATG GTG GTG TTG ATG GTG GTG TTG Pro Gly Ile Val Met Val Val Ala Tyr Gly Leu Ile Ser Leu Glu 235 240 245 250 CTC TAC CAA GGA ATC AAA TTT GAT GCC AGC CAG AAG AAA TCT GCT AAA 8020 Leu Tyr Gln Gly Ile Lys Phe Asp Ala Ser Gln Lys Lys Ser Ala Lys 255 260 265 GGT ACTGATCTTT GGGCTGGATA TTTATTCCAA AAGAGCTCAA GAGCTTTCTT 8073 G TGTAAGTTAG CTAGAAGGAC TCAGGATATA TGGAGCACGT GCTGGAAA ---------- 8121 --734 bp ----- TCCAGCTCAA GTTCCTGGAT TGGGGGTAGT CAGGGGCTTT 8895 ACGTCTTCTC TCTGGCATGA CTTGTCCTGA ACACTAAAAT GATGTCCCTT TCCCACCCAG 8955 AG AAG AAG CCG AGC CGC AGC AGC CGC AGC AGT GAT 9002 lu Lys Lys Pro Ser Thr Gly Ser Ser Thr Arg Tyr Glu Asp Ser Asp 270 275 280 GGC TGT TAC TTG CAG AAG TCC CGG CCC CCG AGG AAG CTG GAG CTT CAG 9050 Gly Cys Tyr Leu Gln Lys Ser Arg Pro Pro Arg Lys Leu Glu Leu Gln 285 290 295 CAG CTG TCT AGC GGC AGC GGT GGC AGC AGA CTC AAC CGG ATC AGG AGC 9098 Gln Leu Ser Ser Gly Ser Gly Gly Ser Arg Leu Asn Arg Ile Arg Ser 300 305 310 AGC AGT TCA GCT GCC AAC CTG ATA GCC AAG AAG CGC GTG ATC CGC ATG 9146 Ser Ser Ser Ala Ala Asn Leu Ile Ala Lys Lys Arg Val Ile Arg Met 315 320 325 330 CTC ATT GTC ATC GTG GTC CTC TTC TTC CTG TGC TGG ATG CCC ATC TTC 9194 Leu Ile Val Ile Val Val Leu Phe Phe Leu Cys Trp Met Pro Ile Phe 335 340 345 AGC GCC AAC GCC TGG CGG GCA TAT GAC ACG GTT TCT GCC GAG AAG CAC 9242 Ser Ala Asn Ala Trp Arg Ala Tyr Asp Thr Val Ser Ala Glu Lys His 350 355 360 CTC TCA GGG ACT CCC ATC TCC TTC ATC CTC CTC CTC TCC TAC ACC TCC 9290 Leu Ser Gly Thr Pro Ile Ser Phe Ile Leu Leu Leu Ser Tyr Thr Ser 365 370 375 TCC TGT GTT AAC CCC ATC ATC TAT TGC TTC ATG AAC AAA CGC TTT CGC 9338 Ser Cys Val Asn Pro Ile Ile Tyr Cys Phe Met Asn Lys Arg Phe Arg 380 385 390 CTG GGC TTC ATG GCC ACC TTC CCT TGT TGC CCG AAT CCC GGT CCC CCA 9386 Leu Gly Phe Met Ala Thr Phe Pro Cys Cys Pro Asn Pro Gly Pro Pro 395 400 405 410 GGG GTG AGA GGA GAG GTG GGA GAG GAG GAG GAT GGG AGG ACC ATA AGG 9434 Gly Val Arg Gly Glu Val Gly Glu Glu Glu Asp Gly Arg Thr Ile Arg 415 420 425 GCA TTG CTG TCC AGG TAT TCC TAC AGC CAC ATG AGC ACC TCT GCT CCA 9482 Ala Leu Leu Ser Arg Tyr Ser Tyr Ser His Met Ser Thr Ser Ala Pro 430 435 440 CCC CCC TGAACTCCAC CTGGTCCACT GCTGCAGCAG GAAGGAGGGG ACCGGGGAGC 9538 Pro Pro ### AAGGAGGAAG AGGAAGTGGG AGGAAAGGAG AGGGAAGACC CATTTCTCAC GGCGATTCTT 9598 CAATGTCTGC TTTTCATCCT TCACAAAGTT CCAGAAGGAA CCTAAGGTGG GGGG TCATGAAA 9658 ACGCCAAGAC ATTTTCCATA ATGTTTACCA TGGGAGAGAC CTCAGCAGGC CGGTGATAAG 9718 AGCCCCACTG GGCTCATGCG ACAAATGTCA CTGACAAGTC CATGTTCAAA ATGGAGGTGT 9778 AGACTTTGAT GAGCTTTAGA ATTTTCTAGA ACAGCCCAGG TGAAGTGTCT CTATAGTGAC 9838 ACACTGCTGT TCTCTGCTTC TGCTCCTGTA GAATTATGGC CCTCCCCAGG AAACCCATGC 9898 CTCCTTCCTA TGTACTCACT ACAACTACAC ACCTTCTCAT GGCCACCAAG GACACATGGG 9958 ACACACACAC CTCCTCTGGC CACGTCTGAA TATGGGAATA GTCTGACAAT TTTTAAAGCT10018 TGAGGTTGGC CAAGTACACT CATGGGTGTA AGCTGGGGTC ATCATCAGAA AACCAAGAAT10078 GTGTCTAAAA GACAGGTTTT AGAATTTTTA TTTCTACAAA GGAAAGCGTG TGCAAGGCCA10138 GTAGCCAGAA GGACTGTTTT CTGTCAGCAA TGTCAAGCTG CAGAAGGGAG GGTGATCTAT10198 ACGGTGTAAT CTCTCCTCTG TATCTAACCC CACAACATTT TTTTTTACCT CAACTTAATT10258 TCGTGTGTTC ACCTGTATTT CCTCCTTTCC AGAAACACCT CCAGGTAAGA AGGGATATGT10318 ACATACCAGG GCAAGGGTGG TAACATGTGT AGTCAGTCCC CAAGGAACAC TGGCATGCTG10378 GGACTCAGTT AATAAGTAGC TTTGAATGCC AAAACTATGT ATGCTTTTAA GTACTGTTGA10438 TGTAGCTCTT GGTAATTTGG CTCAGTGTGT GTGTGTGTGT GTATGTGTGT GTGTGTGA TC10498 TGAACATATG GCTACATTAT GTGTGTGTTA ATAACTTGTA AGTGGATATT GGAATTCAAC10558 AAATGTCATC ATTCTTCCAT AAAAGTTGGA GTATCGTGTG AGCTTCATTT CTTTGAAAAA10618 TTAGTGTTGA AAATACTGAA CTCTGAGAAC TCTGTTTAAA AACTGAAATA AATTACACAA10678 TGAGGGAGAC CATTTCTAGA TACCATGTAC GATCCAGTGT ACCAAGCACA CACTTCGGCT10738 CCCTTCCTCC CTCCCTCCCT TCATTCTTCT TCCTTGCACG TTATTGTCTT GTTTACTAAA10798 AAGAGTTAGT ATGAACCTTT CCAACAGAAA CAAGTTTGTA CGCGCATGTG GAAACTAAAA10858 AATATGCAAA GAAAAGGATC CAAGGCAAGT AAGTTCATTG CTGCGAAGCT GTCGAC 10914

SEQ ID NO: 2 Sequence length: 157 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Normal rat Sequence features Characteristic symbols : Exon characterization method: E sequence ATG AGC CAT TCA CCA GCT CGC CAG CAC TTG GTA GAA AGC AGC AGG ATG 48 Met Ser His Ser Pro Ala Arg Gln His Leu Val Glu Ser Ser Arg Met 5 10 15 GAC GTG GTC GAC AGC CTT CTT ATG AAT GGG AGC AAC ATC ACT CCC CCC 96 Asp Val Val Asp Ser Leu Leu Met Asn Gly Ser Asn Ile Thr Pro Pro 20 25 30 TGT GAA CTC GGA CTG GAA AAT GAG ACG CTT TTC TGC TTG GAT CAA CCT 144 Cys Glu Leu Gly Leu Glu Asn Glu Thr Leu Phe Cys Leu Asp Gln Pro 35 40 45 CAA CCT TCA AAA G 157 Gln Pro Ser Lys G 50

SEQ ID NO: 3 Sequence length: 291 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Normal rat Sequence features Characteristic symbols : Exon characterization method: E sequence AG TGG CAG TCT GCA CTG CAG ATT CTC CTG TAC TCC ATC ATA TTC CTT 47 Glu Trp Gln Ser Ala Leu Gln Ile Leu Leu Tyr Ser Ile Ile Phe Leu 5 10 15 CTC AGT GTG CTG GGG AAC ACG CTG GTT ATA ACG GTG CTG ATT CGA AAC 95 Leu Ser Val Leu Gly Asn Thr Leu Val Ile Thr Val Leu Ile Arg Asn 20 25 30 AAG AGG ATG CGG ACG GTC ACC AAC ATC TTC CTG CTG TCC CTG GCT GTC 143 Lys Arg Met Arg Thr Val Thr Asn Ile Phe Leu Leu Ser Leu Ala Val 30 35 40 AGT GAC CTC ATG CTC TGC CTC TTC TGC ATG CCG TTC AAC CTC ATC CCC 191 Ser Asp Leu Met Leu Cys Leu Phe Cys Met Pro Phe Asn Leu Ile Pro 45 50 55 60 AAC CTG CTC AAG GAT TTC ATC TTC GGA AGT GCC GTG TGC AAG ACT ACC 239 Asn Leu Leu Lys Asp Phe Ile Phe Gly Ser Ala Val Cys Lys Thr Thr 65 70 75 ACC TAC TTC ATG GGTGAGTCCT GCAGGTGTCT GTGGACAGGG 291 Thr Tyr Phe Met G 80

SEQ ID NO: 4 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Sequence AGTCTGCACT GCAGATTCTC C 21

SEQ ID NO: 5 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Sequence GCTTCTTGGC TATCAGGTTG G 21

SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Sequence ACCGAGACCG ATGTATATGC 20

SEQ ID NO: 7 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: cDNA to mRNA Sequence TGATTCAGAG CTCCATAGAG 20

[Brief description of drawings]

FIG. 1 shows the genomic structure of the CCK-A receptor gene in normal rat.

FIG. 2 shows the genomic structure of the CCK-A receptor gene in diabetic rats (type II diabetic rats).

FIG. 3 is CCK of type II diabetic rats as compared to CCK-A receptor precursor gene of normal rats.
It is a figure which shows the deleted nucleotide sequence part of a -A receptor precursor gene.

FIG. 4 is an electrophoretogram showing the expression of CCK-A receptor precursor gene in rat tissues. M
(Marker) indicates pUC19 digested with Hap II. Lane 1 shows the pancreas of a normal rat, 2 the pancreas of a type II diabetic rat, 3 the brain of a normal rat, and 4 the brain of a type II diabetic rat.

FIG. 5 is CCK-A in the gallbladder of a patient with cholelithiasis by Northern transfer analysis in Example 3.
It is an electropherogram which shows the expression of a receptor gene. 1 is a gallbladder of a healthy person, 2 is a gallbladder of a cholelithiasis patient A, 3 is a gallstone patient B
The result about the gallbladder of is shown.

FIG. 6 is CCK-A in the gallbladder of a patient with cholelithiasis by Northern transfer analysis in Example 4.
It is an electropherogram which shows the expression of a receptor gene. 1 to 3 show the results for the gallbladder of a healthy person, and 4 to 6 show the results for the gallbladder of a cholelithiasis patient.

FIG. 7: Human CCK-A receptor full-length cDNA
FIG.

Claims (5)

[Claims] 1. A cholecystokinin (C
A method for diagnosing type II diabetes using a deletion site present in the CK) -A receptor gene. 2. Type II diabetes is associated with cholelithiasis or obesity
The method of gene diagnosis according to claim 1, wherein the method is type II diabetes. 3. The normal CCK-A according to SEQ ID NO: 1 in the sequence listing, wherein the deletion site in the CCK-A receptor gene is present.
3. The part of the receptor gene containing exon 1 described in SEQ ID NO: 2 of the sequence listing or exon 2 described in SEQ ID NO: 3 of the sequence listing, characterized in that it is a portion. Gene diagnosis method. 4. CCK- in tissues of type II diabetic patients
A method for diagnosing type II diabetes by comparing the expression level of A receptor gene mRNA with that of a healthy person. 5. A method for diagnosing cholelithiasis, which uses as an index the decrease in the mRNA expression level of the CCK-A receptor gene in the tissues of patients with cholelithiasis or no expression at all.