JP3391484B2 - DNA containing a region having promoter activity of human endothelin-2 gene - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA containing a DNA encoding a region having a promoter activity of human endothelin-2 gene which is a human vasoconstrictor peptide,
Characterized by expressing a vector containing the DNA, a transformant containing the vector, and culturing the transformant to express a structural gene encoding a protein or peptide connected downstream of the DNA having promoter activity The present invention relates to a method for producing a protein or peptide. In the present specification, the promoter refers to a region of DNA that binds RNA polymerase and serves as a signal for initiating transcription of RNA.

[0002]

2. Description of the Related Art In addition to endothelium-dependent vasodilatory responses, endothelium-dependent vasoconstrictor responses to various stimuli have been reported. Contraction by mechanical load such as blood vessel expansion and increase in internal pressure, contraction by thrombin, contraction by decrease of blood oxygen, and further neuropeptide Y [Proc.Natl.Acad.
An example is the enhancement of noradrenaline contraction by Sci., USA, 79, 5485 (1982); ibid, 81, 4577 (1984)]. American Journal of Physiology (Amer.JP
hysiol.), 248, c550 (1985) and Journal of Cell Physiology (J. Cell.Physiol), 132, 263 (1982), endothelial cell-derived coronary vasoconstrictor (molecular weights 8500 and 3000, respectively). , But the structure is unknown. Also, Journal of Pharmacology and Experimental Therapeutics (J.Ph
Armach.Exp.Ther.), 236, 339 (1985) also describes a peptide-like substance derived from endothelial cells, but the structure of this substance is also unknown.

Vasopressin is known as a peptide having a vasoconstrictor action and its amino acid sequence has been clarified. However, it was reported that vasopressin was isolated from vascular endothelial cells of mammals or birds. There is no. Furthermore, it was reported that angiotensin having vasoconstrictor effect was obtained from bovine aortic endothelial cells [Circulation Research, 60,422.
(1987)], but angiotensin has a molecular weight of about 100.
0 peptides.

[0004] Some of the inventors of the present invention have previously reported that as a peptide having a vasoconstrictor action, the
Succeeded in isolating endothelin (Japanese Patent Laid-Open No. 1-20
6997), and some of the present inventors have also succeeded in isolating human endothelin and cloning cDNAs of porcine endothelin and human endothelin (JP-A-2-72877). The amino acid sequences of this porcine and human mature endothelin polypeptide are identical and are referred to as endothelin-1. The present applicant has also applied for isolation of rat endothelin and cloning of cDNA (JP-A-2-27983), which is referred to as endothelin-3.

Further, the present applicant has filed an application for the isolation of mouse endothelin and the cloning of cDNA (Japanese Patent Laid-Open No. 2-76583), which is called endothelin B. Furthermore, the applicant has a synthetic DNA encoding a portion of endothelin-1 was used as a probe to clone DNA encoding endothelin having an amino acid sequence different from human genomic DNA library with an endothelin -1 (JP Kaihei 3-72884
News ). The present inventors have designated this endothelin as endothelin-
It was named 2. Endothelin-2 is mainly produced in the kidney and small intestine, and it has been revealed that it has actions such as smooth muscle contraction. Here, endothelin is a general term for peptides having a vasoconstrictor action consisting of 21 amino acids with a molecular weight of 2500 ± 300, and the amino acid N
1st, 3rd, 11th, 1st from the end
It has a structure in which the four cysteines located at the fifth position form two sets of disulfide bonds. The combination of disulfide bonds includes 1-15 and 3-11.
There is a combination of, and a combination of 1-11 and 3-15,
The one having the former combination has a higher production ratio and the greater activity.

[0006]

Among the above endothelins, the expression mechanism of human endothelin-2, particularly the promoter of human endothelin-2 gene, is still unknown, and the minimum region having promoter activity, promoter activity. The strength of the has not been clarified. The present invention clones DNA containing the promoter region of human endothelin-2 gene, elucidates the minimum region having promoter activity, the strength of promoter activity, etc., and produces a protein or peptide using gene recombination technology. The purpose is to help.

[0007]

[Means for Solving the Problems] The present inventors have developed a synthetic DNA encoding a part of the genomic DNA of endothelin-1.
Was used as a probe to isolate the human endothelin-2 gene from a human genomic DNA library. Then, DNA fragments of 5'-terminal regions of various lengths of human endothelin-2 gene are cloned, and the fragments are ligated upstream of structural genes such as chloramphenicol acetyltransferase and luciferase to obtain the DNA fragments. The promoter activity of was confirmed.
Then, the DNA region required to have promoter activity was determined, and the DNA containing this region was successfully used for the large-scale production of protein or peptide using gene recombination technology.

The promoter of the human endothelin-2 gene of the present invention contains or is a part of the nucleotide sequence shown in SEQ ID NO: 1 or FIG. 2 and is different from known ones. It is a thing. The expression vector containing the promoter of the human endothelin-2 gene in the present invention is, for example, (i) from human cells to genomic DN.
A is isolated, this DNA is incorporated into a phage or plasmid, (ii) the host is transformed with the obtained recombinant phage or plasmid, (iii) the obtained transformant is cultured, and then a suitable transformant is selected from the transformants. Method (eg human
Target DNA by hybridization with a DNA probe encoding a part of endothelin-2)
Isolate the phage or plasmid containing
v) The desired DNA can be excised from the recombinant DNA, and (v) the DNA or a part thereof can be replaced with a promoter region in a known expression vector for animal cells to produce the DNA.

As a method for preparing genomic DNA from human cells, for example, the method of T. Maniatis et al. [Molecular Cloning, Cold Spring Harbor Laboratory
(1982), p.280-285] and the like. The genomic DNA obtained in this manner was used as a marker for J. Collins and horn.
(B. Hohn) method [Proc.Natl.Acad.Sci., USA, 71,2442
(1978)] and incorporated into a phage or a plasmid.
Examples of phage vectors that incorporate DNA include λ
gt11 (Proc. Natl. Acid. Sci., USA, 80, 1194 (198
3)] and the like, but other ones can be used as long as they can be replicated and propagated in the host. As a method for incorporating DNA into a phage vector, for example, the method of Hyunh T.V. (DNA Cloni
ng, A Practical Approuch, 1, 49 (1985)].

As a plasmid incorporating DNA, for example, pBR322 derived from Escherichia coli [Gene, 2, 95 (1977)], p
BR325 [Gene, 4,121 (1978)], pUC12 [Gene, 19,2
59 (1982)], pUC13 [Gene, 19,259 (1982)], pUB110 derived from Bacillus subtilis [Biochemical and Biophisical Resea]
rch Communication, 112, 678 (1983)] and the like, but any other one can be used as long as it is replicated and propagated in the host. As a method for incorporating DNA into a plasmid, Mesoz.
The method described in Molecular Biology [Methods in
Moleculer Biorogy, Elsevier (1986), p222-226] and the like. The phage vector or plasmid thus obtained may be used in a suitable host such as Escherichia (Es.
cherichia) Introduced into bacteria.

As an example of the bacterium of the genus Escherichia, strain K12DH of Escherichia coli is used.
1 (Proc. Natl. Acid. Sci. USA, 60, 160 (1968)],
M103 [(Nucleic Acids Research, 9,309 (1981)], J
A221 [Journal of Molecular Biology, 120,517 (197
8)], HB101 [Journal of Molecular Biology, 41, 45
9 (1969)], C600 [Genetics, 39,440 (1954)] and the like. Examples of methods for transforming a host with a phage vector include in vitro packaging method [Molecul
ar Cloning, Cold Spring Harbor Laboratory (1982), p.
256-268] and the like.

As a method for transforming a host with a plasmid, for example, the calcium chloride method or the calcium chloride / rubidium chloride method of T. Maniatis et al. [Molecular Cloning, Cold Spring
Harbor Laboratory (1982), p.249-255] and the like. The human genomic DNA library containing the human endothelin-2 gene can be obtained by the above-mentioned method or the like, but it can also be purchased as a commercial product, for example, Clonetech Lt.
aboratories Inc., USA).

Human genome DNA library
As a method for cloning the endothelin-2 gene, for example, from a DNA library obtained by inserting a human genomic DNA fragment into a phage vector-EMBL3,
Plaque hybridization method using oligonucleotide chemically synthesized based on the amino acid sequence of endothelin-2 as a probe [Molecular Cloning Cold
Spring Harbor La-boratory (1982), p. 320-328] and the like. The human endothelin-2 gene cloned in this manner can be plasmids such as pBR322, pUC12, pUC12, if necessary.
UC13, pUC18, pUC19, pUC118, p
It can be subcloned into UC119 or the like.
The nucleotide sequence of the DNA thus obtained is determined, for example, by the Maxam-Gilbert method [Proc. Natl. Ac
id. Sci., USA, 74,560 (1977)] or dideoxy method
[Nucleic Acids Research, 9,309 (1981)] and confirms the presence of the human endothelin-2 gene by comparison with known amino acid sequences. As described above, the human endothelin-2 gene is obtained.

A restriction enzyme map of the human endothelin-2 gene fragment obtained in Example 2 described below is shown in FIG. The nucleotide sequences of DNA determined by the dideoxy method are shown in FIGS. 2 to 4. The human endothelin-2 gene cloned as described above can be used as it is, or if desired, after digestion with a restriction enzyme, before use. A region used as a promoter is cut out from the cloned DNA, replaced with a promoter in a vector suitable for expression, and a structural gene to be expressed is ligated downstream of the DNA fragment containing the endothelin-2 derived promoter, An expression vector can be obtained.
Examples of the vector used as a raw material for preparing the expression vector include animal viruses such as retrovirus and vaccinia virus.

This human endothelin-2 gene promoter can be used in place of the SV-40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, etc. in the animal virus-derived expression vector. Human endothelin-2 constructed in this way
A transformant is produced using a vector containing a gene promoter. Examples of the host include Escherichia, animal cells and the like. Examples of the Escherichia bacterium include the same ones as described above.

Examples of animal cells include monkey cells COS
-7, Vero, Chinese hamster cell CHO,
Examples include mouse L cells and human FL cells. The transformation of the bacterium of the genus Escherichia, for example,
Of the National Academy of Sciences
[Proc.Natl.Acid.Sci.USA, 69,2110 (1972)] and Jean
It can be performed according to the method described in [Gene, 17, 107 (1982)]. Transformation of animal cells can be performed, for example, by using the virology [V
irology, 52, 456 (1973)]. Thus, a transformant transformed with the expression vector containing the promoter of human endothelin-2 gene is obtained.

When culturing a transformant whose host is a bacterium of the genus Escherichia, a liquid medium is suitable as a medium used for culturing, and among them, a carbon source and nitrogen necessary for producing the transformant are nitrogen. Sources, minerals, etc. are included. Examples of the carbon source include glucose, dextrin, soluble starch, sucrose, etc., and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, and an inorganic substance such as potato extract. Alternatively, examples of the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factor and the like may be added. The pH of the medium is about 5.
8 is desirable.

When culturing a transformant whose host is an animal cell, the medium is, for example, MEM medium containing 5 to 20% fetal calf serum [Science, 122,501 (1952)], DMEM medium [Virology, 8,396 ( 1959)], RPMI 1640 medium [The
Journal of the American Medical Association, 199,5
19 (1967)], 199 medium [Proceeding of the Society fo
r the Biological Medicine, 73, 1 (1950)]. The pH is preferably about 6.8. Culturing is usually carried out at about 30 to 40 ° C. for about 15 to 60 hours, and aeration and stirring are added if necessary. The product expressed from the structural gene bound to the downstream of the promoter of the human endothelin-2 gene can be separated and purified from the culture described above, for example, by the following method.

When the expressed protein or peptide is extracted from the cultured cells or cells, the cells or cells are collected by a known method after culturing, and the cells or cells are suspended in an appropriate buffer solution and subjected to ultrasonic wave, lysozyme and / or Alternatively, a method of disrupting the cells or cells by freeze-thawing or the like and then obtaining a crude extract of protein or peptide by centrifugation or filtration may be appropriately used. The buffer solution may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When the protein or mature peptide is secreted into the culture medium, after the completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.

The protein or mature peptide contained in the thus obtained culture supernatant or extract can be purified and separated by appropriately combining known separation and purification methods. Known separation and purification methods for these include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, which are mainly used for measuring molecular weight. Method utilizing difference, method utilizing difference in charge such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography , A method of utilizing a difference in isoelectric points such as an isoelectric focusing method.

The protein or peptide encoded by the structural gene linked to the promoter downstream of the human endothelin-2 gene thus obtained can be measured by enzyme immunoassay or the like. When the product has physiological activity, it can be measured using the activity as an index. In the present specification and drawings, when an abbreviation is used for a base or the like, IUPAC-IUB Commisi
It is based on the abbreviations based on on Biochemical Nomenclature or the symbols conventionally used in the field, examples of which are given below.

DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid RNA: ribonucleic acid mRNA: messenger ribonucleic acid A: adenine T: thymine G: guanine C: cytosine In addition, in the promoter DNA of human endothelin-2 of the present invention, A part of the base sequence is modified (addition,
Removal, substitution of other bases, etc.).

[0023]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.
The transformant Escherichia Coli DH10B / pHGET2 obtained in Example 3 was
It has been deposited with the deposit number FERM BP-4083 at the Research Institute for Microbial Industry and Technology (FRI) of Ministry of International Trade and Industry since November 26, 1992.

Example 1 Preparation of a DNA probe coding for a part of the human endothelin-2 gene The nucleotide sequence of SEQ ID NO: 4 which is the sequence on the 5'end side of the human endothelin-2 cDNA. Chemically synthesizes the DNA of
Using [T4 Polynucleotide] according to a conventional method [M
olecular Cloning, Cold Spring Harbor Laboratory (198
2), p.122-127] The 5'end of DNA was labeled with 32P and used for screening a human genomic DNA library.

Example 2 Isolation of human endothelin-2 gene and determination of its nucleotide sequence Escherichia coli Y1090 was infected with a genomic DNA library derived from human leukocytes (manufactured by Clontech Laboratories Inc.) and plated to obtain a phage. A zip plaque appeared.
Benton and Davis's method [Sci
ence, 196, 180 (1977)], a part of the plaque-formed phasic DNA was replicated on a nylon membrane and subjected to plaque hybridization with the 32P-labeled DNA probe described in Example 1. It was Hybridization was performed at 42 ° C. in the presence of 20% formamide and the membrane was
Washed in SSC, 0.1% SDS at 40 ° C. A clone hybridized with the probe was isolated, and a human genomic DNA insert was cut out from one clone of the clone with a restriction enzyme HindIII to obtain a plasmid pUC11.
Subcloning to 8 gave pHGEH1. Escherichia coli DH5α was transformed with this plasmid, and the transformant Escherichia Coli DH5α / pH was transformed.
GEH1 was obtained.

A restriction enzyme map of the human endothelin-2 gene contained in this plasmid is shown in FIG. The square area in the figure shows a part of the code region of the mature human endothelin-2. Further, in FIG. 1, H is HindIII,
P is PstI, B is BamHI, E is EcoRI, Sm
Represents SmaI, Sa represents SacI, Sp represents SphI, Ac represents AccI, and Hc represents HincII. The nucleotide sequence of the coding region of this matured body and its surroundings was determined by the Sanger method [Proc. Natl. Acad. Sci., USA,
74,5463 (1977)]. A part of this base sequence is shown in FIGS. 2 to 4 (SEQ ID NO: 1) and FIGS. 5 to 6. In FIG. 5 to FIG. 6, the origin (G) of the arrow indicates the transcription start point, the □ area indicated by TATAbox indicates the TATA box which is the consensus sequence of the eukaryotic promoter, and the negative numbers indicate the transcription start point. Indicates the distance.

Example 3 Determination of promoter region of human endothelin-2 gene (1) (i) Nucleotide sequence of human endothelin-2 gene subcloned into plasmid pHGEH1 in Example 2 (ii) human endothelin- The nucleotide sequence of the cDNA encoding 2 (described in Japanese Patent Application Laid-Open No. 4-281790 ) was compared, and a human endothelin-2 precursor was found in the nucleotide sequence of the human endothelin-2 gene subcloned into the plasmid pHGEH1. Exon was confirmed to exist, and the 3′-end boundary of the 5 ′ upstream region of the human endothelin-2 gene (corresponding to the 5 ′ end of exon I) was determined.

As shown in FIG. 1, the plasmid pHGEH
5'upstream of the human endothelin-2 gene subcloned into 1 has restriction enzymes HindIII and BamH.
There are I and EcoRI cleavage sites. pHGEH1 to B
After partial digestion with amHI or EcoRI, Escherichia coli DNA polymerase I treatment was performed to obtain staggered-ends.
end) converted to blant-end [Molecular
Cloning, Cold Spring Harbor Laboratory (1982), p.113-1
14 According to the method described. Then, a HindIII linker was connected to the formed blunt end by T4 DNA ligase. Subsequently, after further DNA cleavage with BssI (the BssII cleavage site is located immediately downstream of exon I of human endothelin-2), BssI was treated by the same method.
The I cleavage site was made blunt-ended and a SalI linker was connected. These operations have a HindIII cleavage site at the 5'end and a SalI cleavage site at the 3'end, and D in the 5'upstream region of the human endothelin-2 gene of different lengths.
Three types of DNA fragments consisting of NA were obtained (about 3 Kb, 2 Kb, 1 Kb, respectively).

A plasmid pCAT-basic containing DNA encoding chloramphenicol acetyltransferase (CAT) was prepared from these three DNA fragments.
(Promega) and the HindIII cleavage site and Sa in the multi-cloning site located immediately upstream of the CAT gene.
Insertion was made between the II cleavage site and 3 types of plasmids (A, D and E, respectively) were obtained (FIG. 7). This plasmid A
Is a transformant Escherichia Col.
i) DH10B / pGET2 (FERM BP-408
Held in 3). In FIG. 7, H is HindIII,
B is BamHI, E is EcoRI, Sm is SmaI, S
al represents the cleavage site of SalI. The region indicated by ■ is the DNA of the 5'upstream region of the human endothelin-2 gene, and the region indicated by □ marked CAT is the DNA encoding chloramphenicol acetyltransferase, The area indicated by □ marked SV40 is S
Each represents a DNA containing the origin of replication of V40. Plasmid A has an insert of about 3 Kb, plasmid D has an insert of about 2 Kb, and plasmid E has an insert of about 1 Kb. The plasmid A was used as a transformant Escherichia Coli DH10.
B / pHGET2 (FERM BP-4083).

After purification of these plasmids [Molecul
ar Cloning, Cold Spring Harbor Laboratory (1982), p88
-By the method described in 96],
Human kidney cells ACHN (ATCC CRL1611) cultured in a dish (Falcon) were subjected to the calcium phosphate method [Methods in Molecular Biology, Elsevier (1986).
p.286-289]. Cells were harvested 48 hours after transformation and CAT assayed [Proc. Natl. A.
cad.Sci., USA, 83, 1598 (1986)].

The autoradiogram shown in FIG. 8 shows that cells transformed with plasmid A, plasmid D and plasmid E all produced chloramphenicol. That is, it was revealed that all of the above three types of DNA fragments isolated from the human endothelin-2 gene had promoter activity. In addition, in FIG. 5, A,
D and E are CAT assay lanes for cells transformed with plasmid A, plasmid D, and plasmid E, B is a CAT assay lane for cells transformed with plasmid B as a negative control, and C is a positive control. 3 shows a CAT assay lane for cells transformed with plasmid C of FIG.

Example 4 Determination of Promoter Region of Human Endothelin-2 Gene (2) Of the DNA fragments having promoter activity obtained in Example 3, cloned into plasmid E. Regarding the shortest fragment of about 1 Kb, in order to further limit the region having promoter activity, it was examined whether the shorter DNA fragment contained in the fragment had promoter activity. In place of the DNA encoding chloramphenicol acetyltransferase (CAT) used in Example 3, DNA encoding firefly luciferase was used as a DNA fragment for measuring promoter activity. A plasmid connected downstream was constructed. The construction method is as follows.

The plasmid E was cut with HindIII,
Exonuclease Bal3 against the cleaved DNA
1 was used to digest DNA from the 5'end [Molec
ularCloning, Cold Spring Harbor Laboratory (1982), p.
135-139 According to the method described.] After blunting the ends of the DNA using T4 DNA polymerase, the SacI linker was connected. After that, SalI (SalI cleavage site is
The DNA was cleaved with human endothelin-2 (immediately downstream of exon I), the ends of the DNA were blunted with T4 DNA polymerase, and then the BglII linker was ligated. By these operations, Sac was added to the 5'end.
DNAs of various lengths having an I cleavage site, a BglII cleavage site at the 3'end, and a portion of the DNA of the 5'upstream region of the human endothelin-2 gene of about 1 Kb cloned into the plasmid E. Fragments were obtained.

These DNA fragments were integrated into the SacI-BglII site of luciferase expression vector-pGV to obtain pGV (-1173 / + 69LUC) and pGV (-
658 / + 69LUC), pGV (-355 / + 69L)
UC) was constructed (Fig. 9). In FIG. 9, luc is a firefly.
The DNA encoding luciferase is shown. A DNA fragment that is larger than the above fragment and lacks the 5'region is PCR
It was synthesized using the method. The synthesis was carried out so that the MluI recognition sequence was present at the 5'end of the DNA and the BglII recognition sequence was present at the 3'end. Synthesized DNA fragments of various lengths were
Incorporation into the MluI-BglII site of GV, pGV
(-201 / + 69LUC), pGV (-147 / + 6)
9LUC), pGV (-117 / + 69LUC), pG
V (-45 / + 69 LUC), pGV (-25 / + 69)
LUC) and pGV (+ 1 / + 69LUC) were constructed (FIG. 9).

After purification of these plasmids [Molecul
ar Cloning, Cold Spring Harbor Laboratory (1982), p88
-By the method described in 96],
Human kidney cells AC cultured in a dish (Falcon)
Calcium phosphate method for HN [Methods in Molecular Biolog
y, Elsevier (1986) p. 286-289]. Then, the expressed luciferase activity was measured [Mol. Cel.
l.Biol., 725 (1987)]. As a result, 4 points upstream from the transcription initiation site of the human endothelin-2 gene.
PGV (-45 / + 69LU) in which a DNA fragment having a region of up to 5 bases was ligated upstream of the luciferase gene.
C) was also found to express the luciferase gene (Figure 10). That is, the region from the transcription start site of the human endothelin-2 gene up to 45 bases upstream is a promoter.
It has been shown to be the smallest unit with targeted activity.

Further, a DNA fragment having a region from the transcription initiation site of the human endothelin-2 gene to the upstream 1173 bases was ligated upstream of the luciferase gene.
GV (-1173 / + 69LUC) showed about 4 times the expression level as compared with the plasmid pGV-P in which the luciferase structural gene was linked downstream of the SV40 promoter (Fig. 10), It has been found that the DNA fragment having the following is very useful as a promoter for animal cells. The above pGV (-1173 / + 69LUC)
Is observed not only in human kidney-derived ACNH cells but also in monkey kidney-derived COS7 cells, and that the promoter of the human endothelin-2 gene has broad activity in animal-cell kidney-derived cells. There was found.

[0037]

INDUSTRIAL APPLICABILITY The cells transformed with the expression vector in which the promoter of the human endothelin-2 gene of the present invention and the structural gene coding for the protein or peptide to be produced are linked, can produce a large amount of the target protein or peptide. Since it is produced, the target protein or peptide can be efficiently produced. In addition, the vector containing the promoter of the human endothelin-2 gene produced here has the same promoter as other promoters (for example, the promoter of the human endothelin-1 gene and the promoter of the human endothelin-3 gene). It can also be used to search for substances that increase or decrease the activity. In addition, humans manufactured here
By using a vector containing the promoter of the endothelin-2 gene, it becomes possible to analyze the expression mechanism of human endothelin-2 in the living body and elucidate the regulatory substance of the promoter of the human endothelin-2 gene.

[0038]

[Sequence list]

SEQ ID NO: 1 Sequence length: 3367 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA Hypothetical array: No origin: Organism name: Homo sapiens Cell type: white blood cell Sequence features Characteristic symbol: promoter Location: 3323..3367 How the feature was determined: E Characteristic symbol: TATA signal Location: 3338..3344 How the features were determined: S Array: AAGCTTCCTG GTCCAGGGCC TCGATTGCAA TCCCAGGCAC CTGCTGCAGC CCAGCCCTGT 60 TGTGTCAGGC ATCACTAGGA GACTTAGTCT TCACTCCCAT GCAACCTCCA TTCAGCTCAG 120 TGTCCACAGC TGCTGGACAG CTGCTTGACG GGACCATATT TGCTCTTAGA GCTGGGACTA 180 TAGAGGGAGC TTTGGGGTGC ACAGAGAGAC AGCCTAACCT CATCCCAATG CCCCAGCCTT 240 CCTGCCAGTG GGGTCCTTCC TGACGGCCTT TCTCCAAGGG ACCCAGGATC CTGGGCTCAG 300 TTTGGACCCC TGGGAGAGCT TCACGGGTGT ACAGGAAAGG GTGAGGCAGG TTAGGAACCA 360 AGAGAACACA CCCAGCCCCA TCAGTGTCTT GAGCCAGTTG CTCCTCCTCT TGCACCTCAG 420 TTATCTCATC TACCAAATGG GAGTGGAGAC GGGGATTTCT AGGGGGAAGT TTCTAGCCCA 480 GTGTCTGACT CAGAAATACA CTCAACTGCT CTCTTCTGCC AGCCCTCCCT CCCCATACAT 540 CCCTCTTTCT TGGATTGGGC TGCTGCAAGC ATAAGGGAAA CAAATCGCTT AGCGAGTAAA 600 GCAATTCTCT TCAAATTCTG AGAGGGTGAC CTTCGGTGGC TCCCTGCCCT TCCCCCTGCA 660 GCCTCAACAA ACTCGGGCAG GTGCCAGGGC CAGAGAAGTG GGGCCAGGGG GTCGTGAAGC 720 CAGGCCACCT AGAAAGGACA GGGACAGTGA GGCCAGGACA GTTATAGGTG GCAGGGTCAT 780 GCAGTCCGAC CAGCCACGAG GGGCAGAGGC AACGGACAGA GGCCCAGGCT AAGCGACCCA 840 GGGAGATGGG GCTATTTTTA ACTCAGCTTA ATTTTCCCTA CCTGGAAACT AACTCTTGTC 900 AGTGGAGCAG CGCTGTTCCG GGAATGGCTT CATCCCCTAA GATCTGTGCT CGCTTAGGTT 960 CCTGGGCCAG TCCTCTCCAT TTCACAGGAG CCCTGCTTGT TCAGCTCAGG CAGAGGGCAG 1020 AGAAGGAGAG AAACCGGAGC CCGGCCCAGC TCGCAGCTGG CTGTGCTGAC CCTGCGGGGC 1080 TCGGGCAGCA GCCCCTGCCT TGTAGTCACC CTTCTTTCAC ACCCGGGAGG GATCCTGTCT 1140 CTGGGTTCAT TTAATGCTTC TATTATGTTT CCCCGGCTCG GAGCCTCTGG TTCCCTATTG 1200 TCTTGAGCAG GCGTTTTGCA TTTGTGTAAA GATTAGACGC TTTATAACCT TCCCTCTTCT 1260 CCCTATGGTT TCCCTCTTTT ACTTTCTCAA ATCTGAGTTC TTTCTTTCTG GCTACGCACT 1320 GCTTCCTCAT TCTCCCCCTC TCTCCCTGCC TGTCTTTATC TGCCTCCCTG TTTGTTTTCC 1380 TCCCCACCAT GACCCCCTCC GTCTCCCCGC TGACCTGCTT TTCCTTCTTT CTTCTCTTCA 1440 CGGTCCAGCG ATTTGAATTC TCTATCCACC CCCAAGCAGC CCCCAGCCCT GCTTAATTCC 1500 GTATTCATAT TCTTGCAGAG GCTGATAGAA ATGACTGTGA GTATTGCTCA CCTCATTCCA 1560 GAAATACTCT TCTAAATTCT TCGAGAGGCA CGACGTGGGA GGGCAGAGAG CACCTCACGG 1620 GGAGTCCCTT GTGCTTTGCT TGATAGACCA AGCCACCTGC CCGCTGGGTG GCCTTGACCA 1680 GGCCCTGCTC CTCTCTGAGC CTCGTGGGCA GTCCTGAGCT GACTCCCCTG TGATAATAGC 1740 TGCCTCCTGG GGTTGTGGTG AGGAAGAGTC GATAGTGCTC CAAGCACGTA GTAGGTGCTC 1800 AGGAAACATT TGTTTGTTTT TTTCACAGGA GCCAGCTCAG TCTCCAGGAA GGAAGATCCT 1860 GCTCTCACCC TCCATCAGTT CTGCCAGTTT CTCCAGGATT TCACACATGC ACGCTTGCTC 1920 TGGGCACACC CTAGGAAGAT GCAAACTTGC CTGTTCCTGG TGGTCACCTA CTGTCCCCTT 1980 CATAAATGTC CAAGGACAAA GGAGCTGCTG AATACAGGAA CTGAGCATTG GGCATCCTGG 2040 GTTCTGTAAG TGGAAAGCCA GGTGTGCATC TCTTGACCCC CCTGCCCTCA GGAAGCACTC 2100 GCAAGGCCCC TACTGTGTGC AGGGTAGGTG TCAGGCCCTG CCTCCTCTGG GGCTCTGGAC 2160 CTTGAGATTG CAGGGGAGAT AAGTGCAATG GGATGAGTCA GGCAGGCACG AGAGGCACAG 2220 GCCCTGGGAA TTCCAAGAAA CGAGAGGGCC TTTCCTGTTG TGCCTGGAGA CAGGGTCCCT 2280 GAGAAGACTC CAGCAGGAAG TGGACTCTCA GCCAGGGCAG GAACAGAGGG TGCCTGCCAG 2340 TTCCCTGAGC TGCTCAGCAC AGCTGTGGTT CATCCCTGCT TTCCCCTCCC CGGCAAGGCT 2400 TCTGGGCCAG CAGAGGGGCA GGAGCAGAAT GCCAGTCCCC AGAGAAGTCC CCTGGCTTAC 2460 TTCTTTTGCA GACAGTAGCC CCCGGTGGAA GCCAAGACAT CCTGTCATGG CCAGGACAGG 2520 AGACTGTTCA AGGGGCGCTT TGTGTGTGTG TCCTCAGGGA CATCTTTCTG GAAAGGGGTC 2580 TGAAGCCACC TTCTTAGTCT CAAAGGAGCC TGTAAGCCAA GCAGGTGAAG TACCTTCTTG 2640 TTTTGGCCTT GTTTGCAAAC AAGGAAACAG GCCCAGGAAG GATGCCCTTG GCCATGGCTG 2700 CATAGCTGAA CAGCTTCTGG AAGGGACTAG AACTCCTGCC CCAGGCCCTG GGGTGACAGG 2760 GCAGGGGTTG AGGGGGGCAT TTCCTGGGTG AGGTGGGGAT GAAGCAGTTC CAGGCTTGTC 2820 AGAAGTCAGG CATGAGCTGT GTCCTTCACA GAGGGAGGCA GGCCCAGAGA GGGCTCTGAC 2880 TCGCCCAAGG CCACACAGCC TTGCGTGGGC CTTTCACATC CCACACAACA GAGGGGCATC 2940 CTCAGCCTGG TTGGCAGAGG GCAGGCAGGA TAGATGGCAG AGTCTTCTCC GAGGAGAGGG 3000 GTTTTGCTCA TGGAACCTCC TCCTCCACAC TCACAGCCCT GGGCGAGACC TGTGGAGCAG 3060 CCGCCAACAG AGTGAGGGAG GGGGCTCGGG GCAGCTGGGG GTGACTTGAG GAAGTCCAGC 3120 TGGACTGCGA GGGGCCCCTG GGGACTGCCA GGGAGCCTCA GGACTCCCAG AGGTGCTCCA 3180 GGCACAGAGG GAGGAATGGG CCTTCCATCT CCCTCCCCCC TTCACTGCAG AGGCTGGGTC 3240 GGGCCAGGTG CCCGGGGAGG AGGCGGTGTC CCTGGCTCCC AGCCCGCCGG TGCAGCGGGG 3300 CAGGGCTGGA CCAGAAGGGG TGGGGCACCG TGCCTGGTAT AAGAGGCAGC CAGGGCACCG 3360 AGGCAAT 3367 SEQ ID NO: 2 Sequence length: 1173 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA Hypothetical array: No origin: Organism name: Homo sapiens Cell type: white blood cell Sequence features Characteristic symbol: promoter Location: 1129..1173 How the feature was determined: E Characteristic symbol: TATA signal Location: 1144..1150 How the features were determined: S   Array: GAGTCAGGCA GGCACGAGAG GCACAGGCCC TGGGAATTCC AAGAAACGAG AGGGCCTTTC 60 CTGTTGTGCC TGGAGACAGG GTCCCTGAGA AGACTCCAGC AGGAAGTGGA CTCTCAGCCA 120 GGGCAGGAAC AGAGGGTGCC TGCCAGTTCC CTGAGCTGCT CAGCACAGCT GTGGTTCATC 180 CCTGCTTTCC CCTCCCCGGC AAGGCTTCTG GGCCAGCAGA GGGGCAGGAG CAGAATGCCA 240 GTCCCCAGAG AAGTCCCCTG GCTTACTTCT TTTGCAGACA GTAGCCCCCG GTGGAAGCCA 300 AGACATCCTG TCATGGCCAG GACAGGAGAC TGTTCAAGGG GCGCTTTGTG TGTGTGTCCT 360 CAGGGACATC TTTCTGGAAA GGGGTCTGAA GCCACCTTCT TAGTCTCAAA GGAGCCTGTA 420 AGCCAAGCAG GTGAAGTACC TTCTTGTTTT GGCCTTGTTT GCAAACAAGG AAACAGGCCC 480 AGGAAGGATG CCCTTGGCCA TGGCTGCATA GCTGAACAGC TTCTGGAAGG GACTAGAACT 540 CCTGCCCCAG GCCCTGGGGT GACAGGGCAG GGGTTGAGGG GGGCATTTCC TGGGTGAGGT 600 GGGGATGAAG CAGTTCCAGG CTTGTCAGAA GTCAGGCATG AGCTGTGTCC TTCACAGAGG 660 GAGGCAGGCC CAGAGAGGGC TCTGACTCGC CCAAGGCCAC ACAGCCTTGC GTGGGCCTTT 720 CACATCCCAC ACAACAGAGG GGCATCCTCA GCCTGGTTGG CAGAGGGCAG GCAGGATAGA 780 TGGCAGAGTC TTCTCCGAGG AGAGGGGTTT TGCTCATGGA ACCTCCTCCT CCACACTCAC 840 AGCCCTGGGC GAGACCTGTG GAGCAGCCGC CAACAGAGTG AGGGAGGGGG CTCGGGGCAG 900 CTGGGGGTGA CTTGAGGAAG TCCAGCTGGA CTGCGAGGGG CCCCTGGGGA CTGCCAGGGA 960 GCCTCAGGAC TCCCAGAGGT GCTCCAGGCA CAGAGGGAGG AATGGGCCTT CCATCTCCCT 1020 CCCCCCTTCA CTGCAGAGGC TGGGTCGGGC CAGGTGCCCG GGGAGGAGGC GGTGTCCCTG 1080 GCTCCCAGCC CGCCGGTGCA GCGGGGCAGG GCTGGACCAG AAGGGGTGGG GCACCGTGCC 1140 TGGTATAAGA GGCAGCCAGG GCACCGAGGC AAT 1173 SEQ ID NO: 3 Sequence length: 45 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA Hypothetical array: No origin: Organism name: Homo sapiens Cell type: white blood cell Sequence features Characteristic symbol: promoter Location: 1..45 How the feature was determined: E Characteristic symbol: TATA signal Location: 16..22 How the features were determined: S   Array: GGGCACCGTG CCTGGTATAA GAGGCAGCCA GGGCACCGAG GCAAT 45 SEQ ID NO: 4 Sequence length: 45 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence type: Other nucleic acids Synthetic DNA Hypothetical array: No Array: AAAGAGTGTG TCTACTTCTG CCACCTGGAC ATCATCTGGG TCAAC 45

[Brief description of drawings]

FIG. 1 is a restriction map of genomic DNA containing DNA encoding a part of the precursor of human endothelin-2.

FIG. 2 is a diagram showing the nucleotide sequence of the 5 ′ upstream region of human endothelin-2 gene.

FIG. 3 is a diagram showing the nucleotide sequence of the 5 ′ upstream region of human endothelin-2 gene.

FIG. 4 is a diagram showing the nucleotide sequence of the 5 ′ upstream region of the human endothelin-2 gene.

FIG. 5 is a diagram showing a position of a transcription signal such as a TATAA box in a part of the base sequence of FIG.

FIG. 6 is a diagram showing a position of a transcription signal such as a TATAA box in a part of the base sequence shown in FIG.

FIG. 7 is a diagram showing the structures of plasmid A, plasmid D, and plasmid E, which are plasmids containing a DNA fragment containing a promoter of human endothelin-2 gene and a CAT gene connected downstream of the DNA fragment. .

FIG. 8 shows an autoradiogram of a CAT assay of human kidney cells transformed with plasmid A, plasmid D, plasmid E.

FIG. 9 is a diagram showing various DNA fragments containing a human endothelin-2 promoter contained in an expression vector and a luciferase gene linked downstream thereof.

FIG. 10 shows the activity of luciferase expressed after transforming ACNH cells with the plasmid containing the DNA fragment shown in FIG.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-3-72884 (JP, A) Chiharu Kimura, 4 others, Cloning of human endothelin-2 gene and analysis of 5'upstream region, 15th Japanese Molecule Biology Annual Meeting Program / Abstracts, Japan, 15th Annual Meeting of the Molecular Biology Society of Japan Preparatory Committee, November 20, 1992, p. 279 J. Biol. Chem. , Vol. 265, No18, p. 10446-10450 (1990) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 GenBank / EMBL / DDBJ / GeneSeq JISST file (JOIS) MEDLINE (STN) BIOSIS / WPI (DIALOG)

Claims (9)

(57) [Claims] 1. A DNA having the promoter sequence of the human endothelin-2 gene, which comprises the nucleotide sequence of SEQ ID NO: 3. 2. The DNA according to claim 1, which contains the nucleotide sequence of SEQ ID NO: 2. 3. The DNA according to claim 1, which is derived from human genomic DNA. 4. The DNA according to claim 3, wherein the human genomic DNA is a DNA having the nucleotide sequence of SEQ ID NO: 1. 5. A vector containing the DNA according to claim 1. 6. A transformant carrying the vector according to claim 5. 7. The transformant according to claim 6, which is a bacterium. 8. The transformant according to claim 6, which is an animal cell. 9. A method for producing a protein or peptide, which comprises culturing the transformant according to claim 6 and expressing a structural gene encoding a protein or peptide connected downstream of a DNA having a promoter activity. .