JP3298901B2 - Method for detecting carbamyl phosphate synthase I mutant gene and DNA probe used therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a carbamyl phosphate associated with delayed onset carbamyl phosphate synthase I deficiency.
DN used detection method and that of synthase I variant gene
Regarding the A probe.

[0002]

BACKGROUND OF THE INVENTION Carbamyl phosphate synthase I deficiency is a disease caused by a deficiency of carbamyl phosphate synthase I (CPSI), which is the first stage enzyme of the urea cycle. Although the disease is not conclusive, it is known to have an autosomal recessive pattern.

[0003] CPSI is localized in the liver mitochondria of urea excreting animals including humans, and accounts for about 20% of matrix proteins. In addition, CPSI is purified from human liver, rat liver, etc., has a large subunit molecular weight of about 160,000, and is known to exist in an equilibrium state of a monomer or a monomer and a dimer. J. Biol. Chem.,
255, 7891-7895, (1980); Eur. J. Biochem., 7, 119
-127, (1968); Eur. J. Biochem., 85, 373-383, (197
8)].

A full-length cDNA clone of rat CPSI is
Complete primary structure of rat CPSI precursor spanning 1500 amino acid residues, including an extended peptide of 38 or 39 amino acid residues, isolated by Nyunoya et al.
[J. Biol. Chem., 260, 9346-9356, (1985)]. Further, a part of the chromosomal gene has been cloned, and from its structural analysis, it is estimated that there is no pseudogene in the rat CPSI gene and its size is over 100 kb.

[0005] A partial-length cDNA clone has been isolated for human CPSI, and the human CPSI gene has been mapped to the short arm of chromosome 2 from the analysis of human and Chinese hamster hybrid cell clone panels and translocation chromosomes. [J. Biol. Chem., 25
9, 13471-13476, (1984)]. That is, Adcock and O '
Brien first isolated a partial cDNA of human liver CPSI (the sequence of which is not shown) and they
Mapping was performed using one of the DNAs as a probe, and it was found that the human CPSI gene was located on the short arm of chromosome 2. Furthermore, 7 CPSs using cDNA as a probe
The Southern blot analysis of the CPSI gene of I deficiency patients has been performed, but no gene deletion or translocation has been detected. In the etiology analysis of this disease, mutations of the CPSI gene, impairment of transcription, impairment of post-transcriptional modification including mRNA splicing, or impairment of translation have been considered, but have not been clarified yet.

[0006] In the diagnosis of this disease, hyperammonemia is generally observed, no increase in specific amino acids such as citrulline is observed in body fluids, and abnormal excretion of orotic acid and various organic acids is not observed in urine. In fact, it is necessary to perform a liver biopsy to suspect the disease and confirm the diagnosis. Also,
Acetylglutamic acid is essential for the expression of CPSI activity, and since CPSI activity is controlled by fluctuations in acetylglutamic acid concentration in mitochondria, differentiation from acetylglutamic acid synthetase deficiency is currently limited to hepatic enzyme activity. The fact is that there is no other method than measurement. In recent years, DNA diagnosis for directly diagnosing genes in many diseases has become possible. However, since this disease is also one of genetic diseases, analysis at the molecular level using DNA has CPSI by gene detection
There is a demand for the development of a method for diagnosing, for the discovery of carriers and for the prenatal diagnosis, but no useful method has yet been found.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, a method for detecting a CPSI mutant gene by preparing a cDNA encoding human liver CPSI and using it as a probe. Heading,
The present invention has been reached.

That is, the gist of the present invention is as follows: (1) Detection of mutation in carbamyl phosphate synthase I gene
In (i) of nucleotide 1-1184 in SEQ ID NO: 1,
cDNA fragment and cD of nucleotides 1185-2550
A mixture with the NA fragment, or (ii) nucleotide 2551-3 in the SEQ ID NO: 1.
636 cDNA fragment and nucleotides 3637-509
Using the mixture with the cDNA fragment of No. 2 as a probe,
Hybridization with DNA of subject sample digested with restriction enzyme
Perform an extraction and check the resulting band
Band different from the normal sample
Insertion into the carbamyl phosphate synthase I gene
Or delayed onset, characterized by detecting rearrangements.
Carbamyl associated with rubamyl phosphate synthase I deficiency
Detection method phosphate synthase I mutant gene, and (2) (i) SEQ ID NO: nucleotides 1-11 in 1
84 cDNA fragments and nucleotides 1185-2550
Or (ii) nucleotide 2551-3 in SEQ ID NO: 1.
636 cDNA fragment and nucleotides 3637-509
Consisting of a mixture of 2 of cDNA fragments, associated with delayed onset type cull <br/> Bamirurin synthase I deficiency Karubamiruri
The present invention relates to a DNA probe for detecting a mutated acid synthase I gene.

The DNA probe of the present invention has SEQ ID NO: 1.
DNA consisting of all or part of the DNA according to,
Alternatively, it is a DNA that can hybridize with the DNA. The DNA described in SEQ ID NO: 1 is a cDNA encoding human liver CPSI, and a full-length cDNA insert can be prepared by connecting cDNA fragments obtained by cloning the human liver CPSI gene by a conventional method. For example, hCPSI-11, hC
Since PSI-4, hCPSI-21, and hCPSI-011 are clones that overlap the DNA of SEQ ID NO: 1, they are appropriately cleaved with restriction enzymes to obtain full length c.
By ligating into a DNA, a full-length cDNA insert can be easily prepared.

The DNA consisting of a part of the DNA of SEQ ID NO: 1 is not particularly limited as long as it constitutes a part of the human liver CPSI gene. It may be used as a mixture. In the present invention, for example, a human full-length CPSI cDNA is divided into two, and a 5 ′ half which is a half at the 5 ′ end and / or a 3 ′ half which is a half at the 3 ′ end can be used as a probe. The 5 ′ half or 3 ′ half is prepared by cutting various overlapping clones with restriction enzymes as appropriate in the same manner as in the preparation of the full-length cDNA insert described above.
It can be easily obtained by preparing a single cDNA half by tying together to form a DNA half, or by preparing a combination that becomes a cDNA half as a mixture of various cDNA fragments. For example,
Nucleotides 1-1184 as 5 'half probes
And a mixture of a cDNA fragment of nucleotides 2551-3636 and a fragment of nucleotides 3637-5092 can be used as the 3 'half probe.

Further, D which can hybridize with the DNA
NA refers to the full-length carbamyl phosphate synthase I gene or a fragment thereof as described above, even if a part of the DNA set forth in SEQ ID NO: 1 has a mutation such as base substitution, deletion, or insertion. Those that can hybridize with are within the scope of the present invention as they can be used in the method of the present invention.

In the present invention, the DN of SEQ ID NO: 1
A clone can be obtained by a known method. For example, λ prepared using an oligo (dT) primer by a plaque hybridization method
The human liver cDNA library in gt11 and the library in λgt11 prepared using random primers are screened by a conventional method. Here, a probe used for plaque hybridization was used by radiolabeling a 3 ′ fragment of rat liver CPSI cDNA,
Oligo (dT) primed human liver cDNA in λgt11
The library can be screened to isolate positive clones. Similarly, it is possible to isolate a positive clone by screening a random primer human liver cDNA library in λgt11 using radiolabeled 5 ′ fragment, intermediate fragment and 3 ′ fragment of rat cDNA as a probe. it can.

[0013] The various cDNA inserts obtained in this manner have a high homology in the coding region of human CPSI cDNA with rat CPSI cDNA, so that human CPSIc
One containing the entire DNA sequence is obtained. The obtained cDNA inserts of various clones are subcloned into pUC18 or the like by a conventional method and used. The method for detecting a CPSI mutant gene of the present invention comprises the above-described DN
A is used as a probe. Specifically, it is used when performing analysis at the molecular level in the diagnosis of CPSI abnormality. The detection method is not particularly limited as long as it is an analysis method at a molecular level that requires a probe, and is usually used in analysis by Southern blotting.

When performing Southern blotting, a sample is usually prepared by preparing a patient's genomic DNA from peripheral blood lymphocytes by a conventional method, or preparing DNA from a liver tissue obtained by biopsy by a conventional method. After digesting the sample DNA with an appropriate restriction enzyme such as EcoRI or BamHI,
Separation by agarose gel electrophoresis, transfer to a nitrocellulose filter, and hybridization using a probe labeled with ³² P, for example, 5 ′ half and / or 3 ′ half of human liver CPSI cDNA, are carried out according to a conventional method. From the band obtained, the DN of the normal sample
In case A, EcoRI digestion shows at least 19 fragments, and BamHI digestion shows at least 10 fragments. Here, a CPSI mutant gene can be detected by finding a band different from a normal sample serving as a control.

By the way, CPSI deficiency often occurs during the neonatal period, and hyperammonemia is severe and often leads to death. If you survive the neonatal period, the course is not much different from other urea cycle disorders. Therefore, it is important that a diagnosis of this disease be made immediately after birth or in the fetal period, and it is likely that it will affect the prognosis. This disease was reported by Nagata et al. In 1989 (Journal of the Japanese Society for Congenital Metabolism Disorders, 5;
In 9), 19 cases are known in Japan, but the number of cases has further increased since then. In many cases, parents are unrelated, and carriers of the mutated CPSI gene may not be uncommon. From these facts, genetic diagnosis is useful for early diagnosis of this disease and searching for carriers. For example, using the probe of the present invention, in addition to the above-described search for insertion or rearrangement mutation, a restriction enzyme cleavage polymorphism (R
Gene diagnosis using FLPs) can be considered. Fea
ron et al., using the partial-length cDNA,
P reported (Fearon et al .; Hum. Genet. 70; 207-210 198
5) However, if full-length cDNA is used, other restriction enzymes may be able to find RFLPs.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 Isolation of human CPSI cDNA clone and determination of nucleotide sequence

(1) Preparation of Rat CPSI cDNA Probe In this experiment, three fragments derived from the protein coding region of rat CPSI cDNA were amplified as follows and used as probes. That is, λ phage DNA having a rat liver cDNA sequence was prepared from a random primer cDNA library. Using 1 to 2 μg of the obtained λ phage DNA, amplification by PCR was performed according to a known method. Primer set (sense / antisense)
In order to obtain a 5 'fragment (597 bp, nucleotides 4-600 of rat cDNA), 5'-ACGA
GGATTTTGACAGGCATGCAAAA-3 '/
5'-CAAATTCTGCTTATTTAGGATCC
AC-3 ′, intermediate fragment (402 bp, nucleotide 1)
84'-2244) to obtain 5'-GCTATG
CCAACCAGATCCTGGGTG-3 '/ 5'-T
GGGATTCCTAGAGCGATCTTTGC-
The 3 ', 3' fragment (731 bp, nucleotide 3451)
4181) to obtain 5′-TTCCTTGAGG
AGGCCACTCGAGTC-3 '/ 5'-GTGG
CTCTGTGGGCAAAAGCTCT-3 'was used. In addition, the PCR-amplified fragment of the random primer library in λgt11 was labeled with [α- ³² P] dCTP using a random primer labeling kit (manufactured by Takara Shuzo), and pre-hybridization, hybridization, And washes.

(2) Isolation of a human CPSI cDNA clone λgt11
The oligo (dT) primed human liver cDNA library in and the random primer library in λgt11 were screened by conventional methods. These cDNAs
The libraries are respectively G. C. Ricca (Me
loy Laboratories, Inc., Springfield, VA); Ebina (Tokushima University)
And J. Courtesy of Mr. Ou (San Francisco University, California).

The probe was the rat liver CPS obtained above.
Using the 3 ′ fragment of IcDNA, about 3 × 10 ⁵ clones were screened from an oligo (dT) primed human liver cDNA library in λgt11. as a result,
One positive clone was isolated and the cDNA insert of this phage clone was designated as hCPSI011 (FIG. 1).
reference). Rat c, also obtained above as probe
Using the 5 ', intermediate and 3' fragments of DNA,
About 6 × 10 ⁵ clones were screened from the random primer human liver cDNA library in gt11.

The rat liver CPSI cDNA is about 5.6 kb.
And the human CPSI cDNA was estimated to have a similar length. Therefore, in order to obtain cDNA containing the entire human CPSI cDNA sequence, screening was carried out using the above three rat cDNA fragments as probes, and a total of six positive clones were isolated. Obtained cD
The NA inserts (hCPSI 2, 3, 4, 11, 13, and 20) are shown in FIG. One clone (hCPSI
4) was positive for both the 5 'probe and the intermediate probe. Another clone (hCPSI2) was positive for both the intermediate and 3 'probes.
The insert cDNA of the hCPSI4 clone has a length of 2.
It was 2 kb and occupied the human CPSI cDNA from the 5 'part to the middle part. On the other hand, the insert fragment cDNA of the hCPSI2 clone was 2.4 kb in length and occupied the middle to 3 ′ portions. Next, the cDNA inserts of these seven clones were subcloned into the EcoRI site of pUC18 for further detailed examination.

(3) Determination of Nucleotide Sequence The nucleotide sequence of the pUC18 subclone was directly determined by the dideoxy chain termination method using an alkali-denatured plasmid as a template and a synthetic oligonucleotide as a primer. Oligonucleotide primers were synthesized using a DNA synthesizer model 381A (manufactured by Applied Biosystems). C obtained from 7 clones
The restriction map of the DNA insert and the procedure for sequencing the insert are shown in FIG. The cDNA insert had a 118 bp 5 'untranslated sequence, 4500 bp (1500 amino acid residues).
) Of the protein coding sequence and 597 bp of 3 ′
It contained untranslated sequences. 2 to 10 show the full-length human CPSI cDNA and the deduced amino acid sequence.

The 3 'cDNA clone (hCPSI01)
1) was isolated from an oligo (dT) primed cDNA library but lacked the polyadenylation signal and the poly (A) tract. As seen in most eukaryotic genes, before the start codon ATG at nucleotide +1, a purine (A) is located at position -3,
C was present at position -4. There was an out-of-frame ATG at nucleotide position -37, which was located in the incomplete inverted repeat.

Predicted Mr of human liver CPSI precursor
Was 164828 and 160438 or 160324 in the mature matured form, which were in good agreement with the numbers reported for purified human liver CPSI (165000).

The nucleotide sequence of the human liver cDNA in the protein coding region is 88.9% homologous to the nucleotide sequence of the rat cDNA, and the deduced amino acid sequence is 94.4% homologous. The homology between the 5 'and 3' untranslated sequences of human and rat cDNA was much lower than the homology of the protein coding sequence.

N corresponding to the rat liver CPSI presequence
The amino acid sequence at the terminal position 38 or 39 is highly basic (consisting of 9 basic amino acid residues and 2 acidic amino acid residues) and contains many hydroxylated residues (8 residues). And did not contain a hydrophobic amino acid moiety. These features are common to the pre-sequences. The deduced amino acid sequence is similar to that found in the rat enzyme, with the N-terminal half (residues 425-813) and the C-terminal half (residues 975-975).
1349), each half containing a consensus sequence (residues 718-768 for the N-terminal half and residues 1259-1302 for the C-terminal half) at nucleotide linkages. Was out.

(4) Acquisition of a full-length cDNA insert A full-length cDNA insert can be easily obtained by a known method. For example, among the clones obtained in this example, hCPSI-11 and hCPSI-
4, hCPSI-2 and hCPSI-011 are full length cDN
A has a sequence that overlaps with A.
An NA can be constructed.

For example, first, hCPSI-11 is converted to Hinc
After digestion with II and HindIII, and hCPSI-4 similarly digested with HincII and HindIII, a combination of both is prepared (hCPSI). next,
hCPSI-2 was digested with AvaI and HindIII, and hCPSI-011 was similarly digested with AvaI and HdIII.
After digestion with indIII, a mixture of both is prepared (hCPSI). Next, a full-length cDNA can be constructed by connecting these hCPSIs.

In order to extract only the full-length cDNA insert, a linker may be synthesized and used according to a conventional method. For example, 5 'GAATTCAAGC
The 18-mer of TTGAATTC3 'is synthesized, the 18-mer is allowed to self-anneal, and then cut with EcoRI. After cutting hCPSI with EcoRI and treating with alkaline phosphatase, the above linker was bound and
Digest with indIII. This is called hCPSI
Among those obtained by digestion with dIII and binding to the plasmid after alkaline phosphatase treatment, those obtained in the forward direction are selected. The full-length cDNA insert is EcoRI
It can be taken out by cutting it out.

Example 2 Analysis of CPSI Deficiency at Molecular Level (1) Subjects In order to analyze CPSI deficiency at the molecular level, proteins, genomic DNA and mR collected from three patients were analyzed.
NA was analyzed. Three Japanese patients were diagnosed with CPSI deficiency by measuring liver urea cycle enzyme activity according to known methods. All three patients showed symptoms of hyperammonemia and decreased plasma citrulline concentration, and urinary orotate levels were normal. One girl patient (Case 1) (parents were not married) was of late onset type. Another girl patient (Case 2) had a sibling who died during the neonatal period and his parents were closely related. The other one was a boy (Case 3), had a sibling with the same illness, but his parents were not married. These two cases were of neonatal onset. Liver CPSI activity in these three patients was 9%, 11%, and 0%, respectively, relative to controls.

(2) SDS-PA of human liver homogenate
GE SDS-PAGE of the human liver homogenate was performed, followed by staining of the protein to detect the CPSI polypeptide. First, liver samples were collected from control and case 1 by liver biopsy, and samples from case 2 and case 3 were collected from autopsy livers within several hours after death. All samples were stored at -70 ° C until the start of the test. 0.5% (w /
v) containing Triton X-100, 20% (w / v) glycerin and 1 mM DTT, pH 7.4.
The liver was homogenized in 9 volumes of 0 mM potassium Hepes (pH 7.4), and the resulting homogenate was centrifuged with a microcentrifuge. The supernatant containing 70 μg of protein was then purified according to known methods using SDS-6.
% Polyacrylamide gel electrophoresis. As a result, CPSI polypeptide was detected in the control liver, but not at all or hardly detected in the liver of three patients (FIG. 11). These results suggest that the decrease in hepatic CPSI activity in the three patients was due to a decrease in the amount of CPSI enzyme protein.

(3) Analysis by Southern Blotting The rat CPSI gene is estimated to have a length of 100 kb or more. It is known to be divided into exons. Human CP
Since the SI gene is also considered to be very large and divided into many exons, hybridization using the 5 ′ half or 3 ′ half of the human CPSI cDNA as a probe was performed as described below. First, genomic DNA was prepared from peripheral blood lymphocytes from control and case 1 according to a conventional method. After digesting DNA (7-10 μg) with restriction enzymes EcoRI or BamHI,
Separated by 0.7% agarose gel electrophoresis and transferred to nitrocellulose filters. Next, hybridization was performed according to the method of Maniatis et al. Using the 5 ′ or 3 ′ half of the human liver CPSI cDNA labeled with ³² P as a probe, and the filter was washed.
An X-ray film (New RX, manufactured by Fuji Photo Film Co., Ltd.) was exposed at 0 ° C. for one day.

The 5 'half probe used was a cDNA fragment of nucleotides 1-1184 and nucleotide 1185-
The 3550 half probe is a mixture of a cDNA fragment of nucleotides 2551-1636 and a fragment of nucleotides 3637-5092. This 5 'half probe converts hCPSI-11 to EcoRI.
Fragment and pCPSI digested with HincII and HincII
-4 digested with HindII and HindIII
Due to the mixture of A fragments and the 3 'half probe
PSI-2 digested with AvaI and HindIII c
The DNA fragment and pCPSI-011 were converted to AvaI and Ec
Prepared by a mixture of cDNA fragments digested with oRI.

As a result of the analysis by Southern blotting, a clear band was obtained.
For NA, EcoRI digestion revealed at least 19 fragments, with a total length of 92 kb for these fragments (FIG. 12). Similarly, BamHI digestion revealed at least 10 fragments, with a total length of 77 kb for these fragments. From these data, human liver CPS
The length of the I gene is greater than 92 kb and
The absence of an EcoRI site in A indicates that this gene is divided into at least 19 exons. On the other hand, in case 1, an extra band was observed in the autoradiogram pattern (FIG. 12, A in the figure).
See lanes 2 and 4). That is, using a 5 ′ half probe, one band of 3.9 kb by EcoRI digestion and two bands by BamHI digestion (4.1 kB)
b and 2.9 kb). Therefore, in case 1, it was determined that mutation such as insertion or rearrangement of the CPSI gene had occurred. Whether this insertion or rearrangement is responsible for the disease requires further investigation, but it is certain that the pathogenic mutant CPSI gene is linked to this insertion or rearrangement.

[0034]

According to the present invention, the full length c of human CPSI
By using an overlapping cDNA containing a DNA sequence as a probe, it is possible to detect a CPSI deficiency that may be caused by insertion or rearrangement of the CPSI gene. Thus, the family of the patient detected in this way also enables carrier discovery and prenatal diagnosis.

[0035]

[Sequence list] SEQ ID NO: 1 Sequence length: 5215 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Origin Organism name: Human Tissue type: Liver Sequence AAGCAACCTT AAAATGACTG CACCCTCCCA GATTTCTTTT ACATTAACTA AAAAGTCTTA 60 TCACACAATC TCATAAAATT TATGTAATTT CATTTAATTT TAGCCACAAA TCATCAAAAT 120 GACGAGGATT TTGACAGCTT TCAAAGTGGT GAGGACACTG AAGACTGGTT TTGGCTTTAC 180 CAATGTGACT GCACACCAAA AATGGAAATT TTCAAGACCT GGCATCAGGC TCCTTTCTGT 240 CAAGGCACAG ACAGCACACA TTGTCCTGGA AGATGGAACT AAGATGAAAG GTTACTCCTT 300 TGGCCATCCA TCCTCTGTTG CTGGTGAAGT GGTTTTTAAT ACTGGCCTGG GAGGGTACCC 360 AGAAGCTATT ACTGACCCTG CCTACAAAGG ACAGATTCTC ACAATGGCCA ACCCTATTAT 420 TGGGAATGGT GGAGCTCCTG ATACTACTTC TCTGGATGAA CTGGGACTTA GCAAATATTT 480 GGAGTCTAAT GGAATCAAGG TTTCAGGTTT GCTGGTGCTG GATTATAGTA AAGACTACAA 540 CCACTGGCTG GCTACCAAGA GTTTAGGGCA ATGGCTACAG GAAGAAAAGG TTCCTGCAAT 600 TTATGGAGTG GACACAAGAA TGCTGACTAA AATAATTCGG GATAAGGGTA CCATGCTTGG 660 GAAGATTGATTGTGAGG CCAAATAAAC AGAATTTGAT 720 TGCTGAGGTT TCAACCAAGG ATGTCAAAGT GTACGGCAAA GGAAACCCCA CAAAAGTGGT 780 AGCTGTAGAC TGTGGGATTA AAAACAATGT AATCCGCCTG CTAGTAAAGC GAGGAGCTGA 840 AGTGCACTTA GTTCCCTGGA ACCATGATTT CACCAAGATG GAGTATGATG GGATTTTGAT 900 CGCGGGAGGA CCGGGGAACC CAGCTCTTGC AGAACCACTA ATTCAGAATG TTCAGAAGAT 960 TTTGGAGAGT GATCGCAAGG AGCCATTGTT TGGAATCAGT ACAGGAAACT TAATAACAGG 1020 ATTGGCTGCT GGTGCCAAAA CCTACAAGAT GTCCATGGCC AACAGAGGGC AGAATCAGCC 1080 TGTTTTGAAT ATCACAAACA AACAGGCTTT CATTACTGCT CAGAATCATT GCTATGCCTT 1140 GGACAACACC CTCCCTGCTG GCTGGAAACC ACTTTTTGTG AATGTCAACG ATCAAACAAA 1200 TGAGGGGATT ATGCATGAGA GCAAACCCTT CTTCGCTGTG CAGTTCCACC CAGAGGTCAC 1260 CCCGGGGCCA ATAGACACTG AGTACCTGTT TGATTCCTTT TTCTCACTGA TAAAGAAAGG 1320 AAAAGCTACC ACCATTACAT CAGTCTTACC GAAGCCAGCA CTAGTTGCAT CTCGGGTTGA 1380 GGTTTCCAAA GTCCTTATTC TAGGATCAGG AGGTCTGTCC ATTGGTCAGG CTGGAGAATT 1440 TGATTACTCA GGATCTCAAG CTGTAAAAGC CATGAAGGAA GAAAATGTCA AAACTGTTCT 1500 GATGAACCCA AACATTGCAT CAGTCCAGAC CAATGAGGTG GGCTTAAAGC AAGCGGATAC 1560 TGTCTACTTT CTTCCCATCA CCCCTCAGTT TGTCACAGAG GTCATCAAGG CAGAACAGCC 1620 AGATGGGTTA ATTCTGGGCA TGGGTGGCCA GACAGCTCTG AACTGTGGAG TAGAACTATT 1680 CAAGAGAGGT GTGCTCAAGG AATATGGTGT GAAAGTCCTG GGAACTTCAG TTGAGTCCAT 1740 TATGGCTACG GAAGACAGGC AGCTGTTTTC AGATAAACTA AATGAGATCA ATGAAAAGAT 1800 TGCTCCAAGT TTTGCAGTGG AATCGATTGA GGATGCACTG AAGGCAGCAG ACACCATTGG 1860 CTACCCAGTG ATGATCCGTT CCGCCTATGC ACTGGGTGGG TTAGGCTCAG GCATCTGTCC 1920 CAACAGAGAG ACTTTGATGG ACCTCAGCAC AAAGGCCTTT GCTATGACCA ACCAAATTCT 1980 GGTGGAGAAG TCAGTGACAG GTTGGAAAGA AATAGAATAT GAAGTGGTTC GAGATGCTGA 2040 TGACAATTGT GTCACTGTCT GTAACATGGA AAATGTTGAT GCCATGGGTG TTCACACAGG 2100 TGACTCAGTT GTTGTGGCTC CTGCCCAGAC ACTCTCCAAT GCCGAGTTTC AGATGTTGAG 2160 ACGTACTTCA ATCAATGTTG TTCGCCACTT GGGCATTGTG GGTGAATGCA ACATTCAGTT 2220 TGCCCTTCAT CCTACCTCAA TGGAATACTG CATCATTGAA GTGAATGCCA AGATGTCCCC 2280 GAACTCTGCT CTGGCCTCCA AAACGACTGG CTACCCATTG GCATTCATTG CTGCAAAGAT 2340 TGCCCTAGGA ATCCCACTTC CAGGAATTAA GAACGTCGTA TCCGGGAAGA CATCAG CCTG 2400 TTTTGAACCT AGCCTGGATT ACATGGTCAC CAAGATTCCC CGCTGGGATC TTGACCGTTT 2460 TCATGGAACA TCTAGCCGAA TTGGTAGCTC TATGAAAAGT GTAGGAGAGG TCATGGCTAT 2520 TGGTCGTACC TTTGAGGAGA GTTTCCAGAA AGCTTTACGG ATGTGCCACC CATCTATAGA 2580 GGGTTTCACT CCCCGTCTCC CAATGAACAA AGAATGGCCA TCGAATTTAG ATCTTAGAAA 2640 AGAGTTGTCT GAACCAAGCA GCACGCGTAT CTATGCCATT GCCAAGGCCA TTGATGACAA 2700 CATGTCCCTT GATGAGATTG AGAAGCTCAC ATACATTGAC AAGTGGTTTT TGTATAAGAT 2760 GCGTGATATT TTAAACATGG AAAAGACACT GAAAGGCCTC AACAGTGAGT CCATGACAGA 2820 AGAAACCCTG AAAAGGGCAA AGGAGATTGG GTTCTCAGAT AAGCAGATTT CAAAATGCCT 2880 TGGGCTCACT GAGGCCCAGA CAAGGGAGCT GAGGTTAAAG AAAAACATCC ACCCTTGGGT 2940 TAAACAGATT GATACACTGG CTGCAGAATA CCCATCAGTA ACAAACTATC TCTATGTTAC 3000 CTACAATGGT CAGGAGCATG ATGTCAATTT TGATGACCAT GGAATGATGG TGCTAGGCTG 3060 TGGTCCATAT CACATTGGCA GCAGTGTGGA ATTTGATTGG TGTGCTGTCT CTAGTATCCG 3120 CACACTGCGT CAACTTGGCA AGAAGACGGT GGTGGTGAAT TGCAATCCTG AGACTGTGAG 3180 CACAGACTTT GATGAGTGTG ACAAACTGTA CTTTGAAGAG TTGTCCTTGG AGAGAATCCT 3 240 AGACATCTAC CATCAGGAGG CATGTGGTGG CTGCATCATA TCAGTTGGAG GCCAGATTCC 3300 AAACAACCTG GCAGTTCCTC TATACAAGAA TGGTGTCAAG ATCATGGGCA CAAGCCCCCT 3360 GCAGATCGAC AGGGCTGAGG ATCGCTCCAT CTTCTCAGCT GTCTTGGATG AGCTGAAGGT 3420 GGCTCAGGCA CCTTGGAAAG CTGTTAATAC TTTGAATGAA GCACTGGAAT TTGCAAAGTC 3480 TGTGGACTAC CCCTGCTTGT TGAGGCCTTC CTATGTTTTG AGTGGGTCTG CTATGAATGT 3540 GGTATTCTCT GAGGATGAGA TGAAAAAATT CCTAGAAGAG GCGACTAGAG TTTCTCAGGC 3600 CACGCCAGTG GTGCTGACAA AATTTGTTGA AGGGGCCCGA GAAGTAGAAA TGGACGCTGT 3660 TGGCAAAGAT GGAAGGGTTA TCTCTCATGC CATCTCTGAA CATGTTGAAG ATGCAGGTGT 3720 CCACTCGGAG AATGCCACTC TGATGCTGCC CACACAAACC ATCAGCCAAG GGGCCATTGA 3780 AAAGGTGAAG GATGCTACCC GGAAGATTGC AAAGGCTTTT GCCATCTCTG GTCCATTCAA 3840 CGTCCAATTT CTTGTCAAAG GAAATGATGT CTTGGTGAAT GAGTGTAACT TGAGAGCTTC 3900 TCGATCCTTC CCCTCTGTTT CCAAGACTCT TGGGGTTGAC TTCATTGATG TGGCCACCAA 3960 GGTGTTGATT GGAGAGAATG TTGATGAGAA ACATCTTCCA ACATTGGACC ATCCCATAAT 4020 TCCTGTTGAC TATGTTGCAA TTAAGGCTCC CATGTTTTCC TGGCCCCGGT TGAGGGATGC 4080 TG ACCCCATT CTGAGATGTG AGATGGCTTC CACTGGAGAG GTGGCTTGCT TTGGTGAAGG 4140 TATTCATACA GCCTTCCTAA AGGCAATGCT TTCCACAGGA TTTAAGATAC CCCAGAAAGG 4200 CATCCTGATA GGCATCCAGC AATCATTCCG GCCAAGATTC CTTGGTGTGG CTGAACAATT 4260 ACACAATGAA GGTTTCAAGC TGTTTGCCAC GGAAGCCACA TCAGACTGGC TCAACGCCAA 4320 CAATGTCCCT GCCAACCCAG TGGCATGGCC GTCTCAAGAA GGACAGAATC CCAGCCTCTC 4380 TTCCATCAGA AAATTGATTA GAGATGGCAG CATTGACCTA GTGATTAACC TTCCCAACAA 4440 CAACACTAAA TTTGTCCATG ATAATTATGT GATTCGGAGG ACAGCTGTTG ATAGTGGAAT 4500 CCCTCTCCTC ACTAATTTTC AGGTGACCAA ACTTTTTGCT GAAGCTGTGC AGAAATCTCG 4560 CAAGGTGGAC TCCAAGAGTC TTTTCCACTA CAGGCAGTAC AGTGCTGGAA AAGCAGCATA 4620 GAGATGCAGA CACCCCAGCC CCATTATTAA ATCAACCTGA GCCACATGTT ATATAAAGGA 4680 ACTGATTCAC AACTTTCTCA GAGATGAATA TTGATAACTA AACTTCATTT CAGTTTACTT 4740 TGTTATGCCT TAATATTCTG TGTCTTTTGC AATTAAATTG TCAGTCACTT CTTCAAAACC 4800 TTACAGTCCT TCCTAAGGTT ACTCTTCATG AGATTCATCC ATTTACTAAT ACTGTATTTT 4860 TGGTGGACTA GGCTTGCCTA TGTGCTTATG TGTAGCTTTT TACTTTTTAT GGTGTGATTA 4920 ATGGTGAT CA AGGTAGGAAA AGTTGTGTTC TATTTTCTTG AACTCCTTCT ATACTTTAAG 4980 ATACTCTATT TTTAAAACAC TATCTGCAAA CTCAGGACAC TTTAACAGGG CAGAATACTC 5040 TAAAAACTTG ATAAAATTAA ATATAGATTT AATTTATGAA CCTTCCATCA TGTGTTTGTG 5100 TATTGCTTCT TTTTGGATCC TCATTCTCAC CCATTTGGCT AATCCAGGAA TATTGTTATC 5160 CCTTCCCATT ATATTGAAGT TGAGAAATGT GACAGAGCAT TTAGAGTATG AATTC 5215

[Brief description of the drawings]

FIG. 1 is a diagram showing a restriction map of human liver CPSI cDNA and the positions of various clones obtained. An open box in the figure indicates a code area, and a line indicates a non-code area. Arrows indicate the direction and region of sequencing.

FIG. 2 shows the full-length human CPSI together with FIG. 3 to FIG.
1 shows the cDNA and deduced amino acid sequence.

FIG. 3 shows the full-length human CPSI cDNA and deduced amino acid sequence together with FIG. 2 and FIGS.

FIG. 4 shows the full-length human CPSI cDNA and the deduced amino acid sequence together with FIG. 2, FIG. 3 and FIG. 5 to FIG.

FIG. 5 shows the full-length human CPSI cDNA and the deduced amino acid sequence together with FIG. 2 to FIG. 4 and FIG. 6 to FIG.

FIG. 6 shows the full-length human CPSI cDNA and the deduced amino acid sequence together with FIG. 2 to FIG. 5 and FIG. 7 to FIG.

FIG. 7 shows the full-length human CPSI cDNA and the deduced amino acid sequence together with FIG. 2 to FIG. 6 and FIG. 8 to FIG.

FIG. 8 shows the full-length human CPSI cDNA and the deduced amino acid sequence together with FIG. 2 to FIG. 7 and FIG. 9 and FIG.

FIG. 9 shows the full-length human CPSI cDNA and the deduced amino acid sequence together with FIG. 2 to FIG. 8 and FIG.

FIG. 10 shows the full-length human CPS together with FIGS.
1 shows the IcDNA and deduced amino acid sequence.

FIG. 11 shows SDS-PAGE of liver homogenate.
FIG. 6 is a diagram showing the results of the above. Lanes 1 and 4 show the results of control, lane 2 shows the results of case 1, lane 3 shows the results of case 2, and lane 5 shows the results of case 3. The molecular weight size markers were rabbit myosin (200 kD), rabbit phosphorylase b (97.4 kD), bovine serum albumin (69 kD).
D) and chicken ovalbumin (46 kD).

FIG. 12 is a diagram showing the results of analysis by Southern blotting. A is the result when the 5 ′ half probe is used, and B is the result when the 3 ′ half probe is used. Lanes 1 and 2 are D for both A and B in the figure.
NA was digested with EcoRI, and lanes 3 and 4 were BamHI
It is a figure of the result of having digested with. Lanes 1 and 3 are controls,
Lanes 2 and 4 are case 1.

────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Ichiro Matsuda 1-1-16-35 Watashika, Kumamoto-shi, Kumamoto (56) References The Journal of Biological Chemistry, Vol. 260, no. 1 (58) Field surveyed (Int.Cl. ⁷ , DB name) C12Q 1/68 C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ / GeneSeq

Claims (4)

(57) [Claims] (1) a modification of a carbamyl phosphate synthase I gene;
In the detection of the differences : (i) nucleotides 1-1184 of SEQ ID NO: 1
cDNA fragment and cD of nucleotides 1185-2550
A mixture with the NA fragment, or (ii) nucleotide 2551-3 in the SEQ ID NO: 1.
636 cDNA fragment and nucleotides 3637-509
Using the mixture with the cDNA fragment of No. 2 as a probe,
Hybridization with DNA of subject sample digested with restriction enzyme
Perform an extraction and check the resulting band
Band different from the normal sample
Insertion into the carbamyl phosphate synthase I gene
Or delayed onset, characterized by detecting rearrangements.
Carbamyl associated with rubamyl phosphate synthase I deficiency
A method for detecting a phosphate synthase I mutant gene. 2. The restriction enzyme is EcoRI, which serves as a control.
Bands of at least 19 fragments in normal samples
The detection method according to claim 1, wherein a band different from the band is detected.
Law. 3. The restriction enzyme is BamHI, which serves as a control.
Bands of at least 10 fragments in normal samples
The band according to claim 1, wherein a band different from the band is detected.
Detection method. (I) a nucleotide in SEQ ID NO: 1
The cDNA fragment of 1-1184 and nucleotide 1185-
A mixture with the cDNA fragment of 2550, or (ii) nucleotide 2551-3 in the SEQ ID NO: 1.
636 cDNA fragment and nucleotides 3637-509
2. A late onset type calf comprising a mixture with the cDNA fragment
Carbamyluri associated with bamil phosphate synthase I deficiency
DNA probe for detection of mutated acid synthase I gene
Bu .