JPH06189770A - Expression promoter and its use - Google Patents

Abstract

PURPOSE:To provide a promoter acting at specific stages in generation and differentiation. CONSTITUTION:The objective promoter is composed of a recombinant DNA containing the promoter region of a human nerve growth factor 2. The promoter is useful as a material for analyzing the expression-controlling mechanism of a nerve-specific protein for the differentiation of function, preparation of a disease model animal or setting of a drug-screening system.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel promoter for gene expression and its use.

More specifically, the present invention relates to a recombinant DNA containing a promoter region of nerve growth factor-2 (NGF-2) gene, a recombinant vector containing the DNA and a transformant.

[0003]

2. Description of the Related Art In animals, especially higher animals, various organs are differentiated and matured from the time of their development, and various functions of living bodies can be exerted. During this process, transient or constitutive expression of various organ-specific proteins occurs,
It gives specificity to the organ. The gene of a protein that is specifically expressed in the fetal and maturation stages of development / differentiation has a transcription induction system (promoter, enhancer) that operates only during that period, and this promoter part is responsible for protein synthesis. It controls the transcription of directed mRNAs. Furthermore, promoters that are hormone-dependent or growth-factor-dependent are also known, and drug screening systems and transgenic mice using these promoters have already been created and used for drug screening and analysis of biological functions. Has been done. Proteins classified into the neurotrophic factor family are trophic factors essential for the differentiation, growth and survival of nerve cells.
Annual of New York Academy of
Science (Anu. NY Acad. Sci.) 55: 330,
1952], polypeptide (I) (hereinafter referred to as NGF-2) (European Patent Application Publication No. 386,752) and BDNF (J. Leibrock et al., Nature 341: 149, 1989). Are known. A peptide having the same amino acid sequence as NGF-2 has the name NT-3 and is named by A. Hohn et al., Nature, 34.
4, 339 (1990), and also P
It is shown in CT publication WO 91/03569. Regarding NGF-2 / NT-3, the analysis of its mRNA suggests that the synthesis starts from the embryonic stage and is most abundant at 1-2 weeks after birth [Y. Kaisho.
Et al., Biochemical and Biophysical Research Communication (Biochem. Biophy. Res. Co.
mmun.,) 174, 379, 1991].

[0004]

[Problems to be Solved by the Invention] Therefore, by using a promoter portion that operates only during the fetal or maturation stage of development / differentiation, genes encoding various proteins are ligated downstream thereof, and this is linked to animal egg cells. If you create a so-called transgenic animal (transgenic animal) by injecting
It is also possible to synthesize the protein only during the fetal or maturation stage of differentiation and study its action in the living body. Further, by linking an appropriate reporter gene to the above promoter portion and establishing a cell line capable of expressing this, it can be used as a screening system for a drug having an action of promoting or suppressing the synthesis of a protein originally regulated by the promoter. it can. However, no clear expression time specificity is detected in the promoters that direct the transcription of most genes, and promoters exhibiting such specificity are craving.

The central nervous system is composed of nerve cells (neurons) and glial cells (glial cells) differentiated from the neural tube, and can be said to be a cellular society having complex functions. The promoter that specifically functions at a specific stage of differentiation in the nervous system is useful as a means for analyzing the regulatory mechanism of the expression of nerve-specific proteins for functional differentiation. Also, by linking various genes downstream and transferring them into cells or animals, they can be specifically expressed in neural cells or brain at a specific time.

[0006]

[Means for Solving the Problems] As a promoter that operates at a specific time of development / differentiation, the present inventors
-2 / NT-3 gene human NGF-2 / NT-3 c with the goal of finding and removing the promoter
Human NGF-2 / NT-3 genomic gene was cloned using a synthetic oligonucleotide corresponding to DNA as a probe. This gene was digested with a restriction enzyme to produce NGF-2.
/4.0 kb of its upstream portion containing part of the NT-3 cDNA
DNA was obtained, and the upstream portion of 3.4 kbp was cloned again on the plasmid DNA. This 3.4 kbp DN
A plasmid DNA in which a chloramphenicol acetyltransferase (CAT) gene as a reporter gene was ligated downstream of A was constructed, and a transformant such as glial cells or other animal cells transformed with the DNA was constructed. CA
By measuring the T activity, NGF-2 / N was added to the NGF-2 / N-3 structural gene upstream portion 3.4 kbp DNA.
We were able to find the T-3 promoter.

The present inventors have completed the present invention as a result of further research based on these findings. That is, the present invention
(1) DNA containing a promoter region of NGF-2 / NT-3 gene, (2) Recombinant DNA containing a promoter region of NGF-2 / NT-3 gene, (3) Above (2)
Recombinant vector containing the recombinant DNA described above, (4) Recombinant vector according to (3) above, in which a structural gene is linked downstream, (5) Retaining the vector according to (3) or (4) above The present invention relates to transformants. N described in (1) above
The GF-2 / NT-3 gene promoter is preferably of human origin. Further, it is preferable that the promoter region contains the DNA represented by the following nucleotide sequence TGAGCGCGGA GCCATCTGGC CGGGTTGGCT GGTTATAACC GCGCAGATTC TGTTCACGGG ACTC (SEQ ID NO: 1) or a part thereof, and further the following sequence AAGAGGGGGGGGCGCGCGCGCGCGGGCGCGCGCGCGGTGGCGCGCGCGGGGGCGCGCGCGCGGG GGCGGGGGAG GGCGGCGCGG CGCGGAAGGG GTTAAGGCGC TGAGCGCGGA GCCATCTGGC CGGGTTGGCT GGTTATAACC GCGCAGATTC TGTTCACGGG ACTC (SEQ ID NO: 2) or a part thereof is preferable, and a part of the above nucleotide sequence has promoter activity. The part that has.

The recombinant DNA containing the NGF-2 / NT-3 promoter region of the present invention can be obtained, for example, as follows. First, Febs Letters (FE
BS Lett.) 266, 187-191 (1990), using a synthetic oligonucleotide corresponding to the most upstream region of human NGF-2 / NT-3 cDNA as a probe to screen a human gene library. , Obtain a clone of λ phage that reacts with this probe. DNA was extracted from this phage clone and a restriction enzyme map of the integrated human gene part was prepared.
A DNA fragment that reacts with the uppermost stream region probe of DNA, for example, by digestion with a restriction enzyme, is prepared by, for example, plasmid pUC11.
8 [Vieira. J. and Messing J. (1987), Methods in Enzymology]
153, 3-11]. The position of the translation initiation codon on the gene can be known by determining the nucleotide sequence of the cloned DNA and comparing it with the nucleotide sequence of the cDNA, for example. Further, using human mRNA, for example, SI mapping method [B
erk, AJ, and Sharp, PA, Cell, 12,
721, 1977], the transcription start point of the gene can be known.

NGF-2 / NT thus obtained
In a recombinant vector containing a recombinant DNA containing a promoter region of -3, NGF-2 / NT-
As a recombination vector incorporating 3 promoters,
Although not particularly limited, for example, pCD vector, cDM8
Vector [Aruffo, A. and Seed, B., Proc. Natl. Acad. Sic. USA], 84,857
3-8577 (1987)], retrovirus vector [Cone, RD and Mulligan, RC, Procedures of the National Academy of Science (Proc. Natl. Sic, USA), 81, 6349-6.
353 (1984)] for animal cells, pUC (Vie
ira, J. and Messing J. (1987), Methods in Enzy
mology 153, 3-11) and the like for E. coli.

The structural gene linked to the downstream of the promoter region in the recombinant vector encodes a polypeptide for knowing the functions of various gene products in the central nervous system during the fetal and maturation stages of development and differentiation. Structural genes are mentioned. Examples thereof include nerve cell growth factor (NGF) and basic fibroblast growth factor (basi).
c FGF), acidic fibroblast growth factor (acidic FG)
Other growth factors such as F) and other neurotrophic factors, lymphokines and the like can be mentioned. As the structural gene,
You may use the below-mentioned reporter gene. As a reporter gene, CAT (Chloramphenicol acetyl trans
ferase) gene and alkaline phosphatase gene, β-galactosidase gene is widely used, but any other structural gene can be used if there is a method for detecting its gene product.

In order to incorporate the above structural gene into a vector, it may be ligated to an appropriate restriction enzyme cleavage site located downstream of the promoter region in such a direction that the above structural gene is correctly transcribed. Animal cells, particularly glial cells and cranial nervous system cells, can be used as the host transformed with the recombinant vector. In addition, egg cells or ES cells [Evans, as a process for DNA transfer into individual animals]
MJ and Kaufman, KH, Nature, 2
92, 154 (1981)] is also used. As a method for transforming these cells, the calcium phosphate method [Grah
am et al., Virology 52,456 (197)
3)], an electroporation method [Ishizaki et al., Cell Engineering, 5,557 (1986)], a microinjection method and the like.

By using the promoter of the present invention, cells such as the cranial nervous system can be made to produce various polypeptides as described above. When an oncogene such as myc or ras is used as the structural gene, the obtained vector is administered to an egg cell of an animal (eg, mouse, rat, dog, cat, etc.) to cause development and differentiation in the cranial nervous system of the animal. It is possible to produce a disease model animal that causes specific lesions such as cancer in the fetal period and the maturity stage. Other model animals can be created by the same transformation method. Furthermore, when the above structural gene encodes a peptide useful for treatment of genetic diseases of the brain (eg, Alzheimer's disease, Parkinson's disease, etc.),
By directly administering the vector of the present invention into the brain of a mammal (eg, mouse, rat, dog, cat, human, etc.) or introducing the vector into cultured brain-derived cells, and then transplanting into the brain, Treatment of the disease can be performed. In addition, a brain tumor can be treated by constructing a vector using a tumor suppressor gene as a structural gene and directly introducing the vector into tumor cells. Furthermore,
If the promoter of the present invention is known to control the promoter activity of a specific compound in cells of the cranial nervous system, when the specific compound is administered so as to reach the brain of the living body, the promoter of the specific compound Since the ability to control the activity can be known, the compound activates the promoter of the NGF-2 / NT-3 gene in the brain and NGF-2 / NT- in the brain.
The production amount of 3 can be increased, whereby dementia can be treated.

The transformant can be cultured in the presence of the specific compound, and the amount of the gene product in the culture can be measured and compared to determine the ability of the compound to control the promoter activity.

Culturing of the transformant is carried out by a method known per se. Examples of the medium include MEM medium containing about 5 to 20% bovine serum [Science 122,
501 (1952)], DMEM medium [Virology (Vi
rology) 8, 396 (1959)], RPMI 1640
Medium [The Journal of the America Medical Association (The Journal of the America
n Medical Association) 199, 519 (196
7)] 199 medium [Procedure of the Society for the Biological Medicine (Pr
oceeding of the Society for the Biological Medici
ne) 73, 1 (1950)] and the like. The pH is preferably about 6-8. Culturing is usually about 30 ° C-40
It is carried out at ℃ for about 15 to 60 hours, and aeration and stirring are added if necessary.

In the present specification and drawings, when abbreviations are used for bases, amino acids, etc., IUPAC-IUB
It is based on the abbreviations by Commision on Biochemical Nomenclature or the abbreviations commonly used in this field, and examples are given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. DNA: Deoxyribonucleic acid cDNA: Complementary deoxyribonucleic acid A: Adenine T: Thymine G: Guanine C: Cytosine A, Ala: Alanine C, Cys: Cysteine D, Asp: Aspartic acid E, Glu: Glutamic acid F, Phe: Phenylalanine G, Gly: Glycine H, His: Histidine I, Ile: Isoleucine K, Lys: Lysine L, Leu: Leucine M, Met: Methionine N, Asn: Asparagine P, Pro: Proline Q, Gln: Glutamine R, Arg: Arginine S, Ser: Serine T, Thr: Threonine V, Val: Valine W, Trp: Tryptophan Y, Tyr: Tyrosine The transformant Escherichia col obtained in Example 2 described later.
i DH1 / pTB1518 and transformant Escherichi
a coli DH1 / pTB1534 has been deposited with the Fermentation Research Institute (IFO) and the Institute for Microbial Technology (FRI), Ministry of International Trade and Industry. The deposit date and deposit number are shown in Table 1 below.

[0016]

[Table 1]

Example 1 Cloning of human NGF-2 / NT-3 genomic DNA The previously reported NGF-2 / NT-3 cDNA (Y. Kaisho
FEBS Lett., 266,1
87-191 (1990)) with base numbers 1 to 22 5'-TGCCATGGTTACTTTTTGCCA
An oligonucleotide of CG-3 '(SEQ ID NO: 3) was synthesized, and using this as a probe, a human genomic DNA library (from Clontech human placenta genome.DNA)
2.3 × 10 ⁶ phages were screened. The reaction was 5 × SSPE (1 × SSPE = 180 mM NaCl,
10 mM sodium phosphate, 1 mM EDTA; pH 7.
7), 5 x Denhalt's solution (1 x Denhalt's solut
ion = 0.02% Polyvinylpyrrolidone, 0.02% Fic
oll, 0.02% bovine serum albumin), 100 μg / ml
Heat-denatured salmon sperm DNA, 0.1% in SDS 42 ° C 16
I went on time. After reaction, filter the filter 6 x SSC (1 x SS
C = 150 mM NaCl, 15 mM sodium citrate) 45
After washing at 30 ° C. for 30 minutes and 6 × SSC at 60 ° C. for 30 minutes, an autoradiogram of the filter was taken to search for a phage that reacts with the probe.

As a result, 1 out of 2.3 × 10 ⁶ phages
Southern blot was obtained after obtaining positive clones.
Hybridization showed that a fragment of BamHI-HindIII approximately 3.0 kbp crossed the probe.
Therefore, the entire nucleotide sequence of BamHI-HindIII 3.0 kbp and its upstream region of about 1.0 kbp was determined, and this was determined [Fig. 1-1].
~ [Fig. 1-2] (SEQ ID NO: 4). That is, the nucleotide sequence of about 4.0 kbp containing the 5'upstream region of the human NGF-2 / NT-3 gene obtained here is shown in [Fig. 1-1] to [Fig. 1-2]. As shown in [Fig. 1-1] to [Fig. 1-2], +210 to +231 are human NGF-2 / NT-.
3 It was completely in agreement with the nucleotide sequence of 3 cDNA. Furthermore, cDN
TATAbox-like sequence (-32-
27), it was confirmed that human N
A promoter region of the GF-2 / NT-3 gene,
It was shown that the region downstream from +232 is an intron.

Example 2 Assay of Promoter Activity of Human NGF-2 / NT-3 Gene In order to confirm that the cloned genomic DNA fragment has promoter activity, a CAT assay method, which is one of the promoter activity test methods, was performed. It was The plasmid having CAT as a reporter gene is pCAT-Basic plasm.
id (manufactured by Promega) was used as a vector. PCAT-Control plasmid (manufactured by Promega) was used as a positive control. The 5'upstream region and 3'end of the human NGF-2 / NT-3 gene were deleted using a TAKARA deletion kit for kilosequencing, and introduced between the Hind III site and the XbaI site upstream of the CAT gene of the vector. . The calcium phosphite method (M. Wigler et al., Cell 16,777 (1979)) was used to infect the human glioma Hs683 cells with the plasmid. After infection, the cells were cultured in DMEM medium containing 10% FCS (fetal calf serum), and cells were collected 48 hours later. The CAT enzyme activity of the cell extract was measured. CAT enzyme activity was measured by adding 5 mM acetyl CoA, 0.25 μCi ¹⁴ C- to the cell extract.
After reacting for 4 hours at 37 ° C in the presence of chloramphenicol, the mixture was extracted with ethyl acetate, the ethyl acetate was evaporated to dryness, and the residue was dissolved in 15 µl ethyl acetate. ) Was spotted. After developing with a developing solvent of CHCl ₃ : methanol = 95: 1, an autoradiogram was taken.

First, the 3.4 kbp DNA fragment from −3275 to +91 in FIGS. 1-1 to 1-2 was pC.
Expression plasmid pTB inserted into AT-Basic plasmid
1518 was constructed. In order to preserve this plasmid pTB1518, it was used to transform E. coli DH1 and transformant E. coli DH1 / pTB1518 (IF
O 15283, FERM BP-3849). Human glioma Hs683 cells were transformed with the above plasmid pTB1518 to prepare transformant human glioma Hs683 / pTB1518. After this transformant was cultured in DMEM medium containing 10% FCS for 2 days, cells were collected, and the cell extract was collected using Widen.
SG et al. (Journal of Biological Chemistry, 263, 16992-1).
6998 (1988)). The results are shown in lane 1 of FIGS. 2 and 3. Note that [Fig. 2] shows human NGF- contained in the plasmid used.
The range of the 5'-upstream region of the 2 / NT-3 gene is shown, and [Fig. 3] shows the result of the autoradiogram of the result of the CAT assay. As shown in FIG. 3, a CAT activity similar to that expressed under the control of the SV40 early promoter used as a positive control (shown as lane P in FIG. 3) was observed. DN having functional promoter activity in genomic DNA fragment
It was confirmed that the A fragment was present. Next, various deletion type muteins were constructed in order to know whether the expression of activity was controlled by the presence of so-called silencer enhancers and the like, and the same CAT assay was performed.

The results are shown in FIGS. 2 and 3.
In FIGS. 2 and 3, lanes 1 to 10 are pTB1518, pTB1519, and pTB152, respectively.
0, pTB1521, pTB1522, pTB1523, p
TB1524, pTB1525, pTB1526, pTB
The results obtained by transfecting each of 1527 are shown. In addition, pTB1519 is a region from −2218 to +91, pTB1520 is a region from −1507 to +91, pTB1521 is a region from −1312 to +91, and pTB1522 is a region from −1087 to +91. pTB1523 is a region from −838 to +91, pTB1524 is a region from −988 to +91, pTB1525 is a region from −204 to +91, and pTB1526 is a region from −64 to +91.
TB1527 is a vector in which the regions from −39+ to 91 are introduced into pCAT-Basic plasmid. As shown in FIGS. 2 and 3, -39 to +
On the upstream side of 91, CAT activities were observed without any significant difference, whereas at -39 to +91, the activity was very low. From the above, human NGF
There was no clear silencer in the -2 / NT-3 gene promoter, suggesting that it must have at least the region -64 to +91 to function as a promoter.

Example 3 Assay of Human NGF-2 / NT-3 Gene Promoter Activity in Human Plasma-Derived ARH77 Cells To examine the specificity of the human NGF-2 / NT-3 gene promoter, NGF-2 / NT-3. The promoter activity was measured using cells in which the gene was not expressed.
The following experiments were carried out using human plasma-derived ARH77 cells in which it was confirmed that NGF-2 / NT-3 mRNA was not detected by Northern blot hybridization. Infection of ARH77 cells with the plasmid was carried out by electroporation (Ishizaki et al., Cell Engineering,
5, 557 (1986)) and 0.3 kV 500 μF. RPMI-1 containing 10% FCS after infection
The cells were cultured in 640 medium and the cells were collected after 48 hours. The CAT enzyme activity of the cell extract was measured. CAT
The plasmid used in Example 2 was used as the reporter gene, and the results of the CAT assay at the time of transfection are shown in FIG. As shown in FIG. 2 and FIG. 4, N including the upstream side from -65
With the GF-2 / NT-3 promoter, the CAT activity was very weak, and only the CAT activity of the cell extract of pTB1526 (-64 to +91) -transfected cells showed a clear CAT activity. From the above results, it was suggested that the region upstream of -65 contains a region that controls the expression of NGF-2 / NT-3 gene.

Example 4 Human NGF-2 promoter
Establishment of human glioma Hs683 stable strain into which alkaline phosphatase fusion plasmid was introduced Construction of a plasmid using alkaline phosphatase as a reporter gene is shown in [Fig. 5-1] and [Fig. 5-2]. First, pGem-4Z / PLAP * 489 (SEA
P) (J. Berger et al., Gene 66 , 1-10)
(1988)) EcoRI-KpnI 2.0 kbp fragment was isolated and blunted with T4 DNA polymerase.
The fragment added with the coRI linker (pGGAATTCC manufactured by Takara Shuzo) was used as a vector pTB389 (Y. Ono et al., Science 236 , 1116-1120 (198).
7)) was introduced into the EcoRI site to construct pTB1330. After cutting pTB1330 with XhoI, T4DNA
After blunting with a polymerase, XbaI linker (p
(CTCTGAGA) was added to construct pTB1331 bound by T4 DNA ligase. Next, the NGF-2 promoter HindIII-XbaI 2.3 kbp fragment was isolated from pTB1519 obtained in Example 2, and introduced into the upstream HindIII-XbaI site of the alkaline phosphatase gene of pTB1331 constructed above to introduce pTB153.
4 was built. This was introduced into E. coli DH1 and Escher
ichia coli DH1 / pTB1534 (IFO 153
83, FERM BP-4038).

The drug resistance marker is p, which has the neo gene.
TB6 (R. Sasada et al., Cell Structure and Function) 12,
205-217 (1987)). pTB15
Human glioma Hs683 cells were simultaneously infected with 34 and pTB6, and 500 μg / mlG was obtained 48 hours after the infection.
418 (O-2-amino-2,7-dioxy-D-glycero-α-D-glucoheptopyranosyl [1 → 4] -O-3-deoxy-4C-methyl-3- [methylamine]- It was cultured in DMEM medium containing β-L-arabinosyl-D-streptamine, Sigma) and 10% FCS to obtain a neomycin resistant strain. From the obtained neomycin resistant strain, one clone stably expressing alkaline phosphatase was obtained. Alkaline phosphatase activity was measured as an increase in A ₄₀₅ absorbance using p-nitrophenyl phosphate as a substrate. The culture supernatant was treated at 65 ° C. for 5 minutes and then centrifuged at 12,000 × g for 5 minutes, and 100 μl of the supernatant was added to 1 × SEAP assay buffer (1.
0M diethanolamine, pH 9.8, 0.5 mM Mg
Cl ₂ , 10 mM L-homoarginine) and the final volume was 200 μl. Place in a 96-well microtiter plate, add 20 μl of 120 mM p-nitrophenyl phosphate (in 1 × SEAP assay Buffer), and add A
₄₀₅ was measured. 1 mU of alkaline phosphatase is 1
The amount of alkaline phosphatase that hydrolyzes the pmole substrate in 1 minute is defined as 1 mU = 0.4ΔA ₄₀₅ / min. Obtained clone (Hs683 / pTB1534)
The cell growth curve [Fig. 6] and alkaline phosphatase activity [Fig. 7] are shown.

[0025]

INDUSTRIAL APPLICABILITY The NGF-2 / NT-3 promoter of the present invention is useful as a material for analyzing the mechanism of controlling the expression of nerve-specific proteins for functional differentiation. In addition, by linking various genes downstream of this promoter and transferring them into cells or animals, they are expressed specifically in the nervous system of the cells or animals, at a specific time in the brain, and the setting of a drug screening system, It can be used as a promoter to be incorporated into a vector used in the preparation of disease model animals and treatment of biological diseases.

[0026]

[0027]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 64 Sequence type: Nucleic acid Sequence: TGAGCGCGGA GCCATCTGGC CGGGTTGGCT GGTTATAACC GCGCAGATTC TGTTCACGGG 60 ACTC 64.

SEQ ID NO: 2 Sequence length: 204 Sequence type: Nucleic acid Sequence: AAGAGGGGCC AGGAGAAATG ACCCCTTCCC CGCCACGGGT CCCGAAGTGA GGGCGGGGGGGGGGGGGGGGGGGGCTGG GGCGCGGGCG CGCGGGCGGC GGCGCGGTCG

SEQ ID NO: 3 Sequence Length: 22 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence: TGCCATGGTT ACTTTTGCCA CG 22.

SEQ ID NO: 4 Sequence length: 3821 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Human Sequence features Characteristic symbol: CDS Location: 3276..3821 Characteristic determination method: S Characteristic symbol: intron Location: 3507..3821 Characteristic determination method: S Sequence: GAGCTCAAAC ATAGGGAGAT AAGTGCTGTT TTCACAAGAT AAAGGCAAAA TTCAATCCCATG 60AG AGGGTAACCA GAAGGCACGC TCTGGAAATT TGCTTTAGGA GAGAGTTTTA AAGGGGGTTT 180 TCAAAAACAA GATCTGATTC CTGCTCTCAG AAATCACTTC CAGGAGTCAG GGCCTTACTC 240 TCAGATGCAG CAGGGAGAAG AAGAAAGTTC AGCAACCTAA AAATACAGTC GACAGATGGG 300 CAGCCAAAGT CATGGCCACG AAGTCAACTT GGAGAGGAGC ACCTACCTAG TGAATCCTAA 360 AAGATCTCAT CCTGGATGCT TCCTTAACCA GGCCTATGTA CAGGGCACAA GCTCGCAGCC 420 AGCTTACTTT CCAGTCCTGA TCTTTGCTTT TGCTATCCAT ACCAATGGTA TTTCTATAGA 480 AAAGAAAAAT CTCTATTTA G AAACACGGAT TTACTTAGAA GTCACAATAT TCTAGTTTAA 540 AAATGGCTCT ACATAGTAGA GAATGATCTT TTTATTCTGT CTTCTTAAAA ATACACCTTT 600 CTAATTCTTT TTTTCTTCCC ACCTTCTTCA TTCAGCACCT TGCCACTCCC TTGGAAGCCA 660 CAACAGCGAG CTGGGGGGTC AGTCCCTAGT CTTAGAGGGA AGAAATCTTT AGGTCTGAAG 720 TCTAAAGAAA AACAGTAAAG GAAAAGGCAG TTGGCGGTGC TCAAGGTAGA CTGTCTGAAA 780 GAGGTCTTCT ACTCAGAAAA GGGCTAAGGC TCTCCCTTTG GGAAACCAAT CCTTCTGAGA 840 AAAAGTGCAT CTTTCACCCT CTGCTCCTGT CTGGGTCTCT CCCTCTTCCT CCCTCCTTCC 900 CTCAGTCCCT CCTCCCCTCT CTCCACAAAG ACACAGCACA TATTTGGCAA GATTAAGGTG 960 TCACCTCTCA TATTACAAGG CCTGTTGATT GCAAGCAAAG ACAGACCCAC CAGCTTAGGA 1020 CAAAACCCCT TGGAGTTGGA AATAAGACAA ACTCTGGGAT CCCCGAAAGT CCCGGCAAAA 1080 TGACGCGGCC AGCCAGTGCA AGGCATCTGC AGAACAAATC CAAGTCCTAA ACGCACTGCT 1140 TGCTGCCTTT TCTTCTCCTT CCTTTCTTCT GATTTTTCAA GTTTGTTTGC CCCCCTTCCC 1200 CTCCTCCCTC CAGACTGCCA GGGACCTGGG AGCTGCCTGC AGATCAGCCC GCACATGTAT 1260 TTAACCCCTT CCCTGCTGCA GCAGGAGCCA ACCACCTCTT TCCTTGCAAT CTTCAGGTTC 1320 CCAGAGGACC TGGAGCTTGA GAAAAGAACT C TGCCAGTGG ATCTGAAACT GGGGCCTGAA 1380 TCCCTCCTTT GACCAGGGCG AGAAGCTGGA GGAGGGGGGC AAGTGCGGGA AGTGGGGGAG 1440 GGCAGGGAGG CGGGCCAGAT GAGAGGGAGA AAAGCAGAAC CCGACAGAGC ACGCCCAATC 1500 CAAACCTTCA TGGTGCTGTG TGGCTGGGTG GAGGGAACGA CTCGGCAGCC TCTTCTGGCC 1560 CTGAGGAAGA CGTCGATATT TTGGCACGAG GGGAGCCACT GAAGGACTAC CCTACCCTTG 1620 CGAGGGACCG CAGGAGGTGA CGCCCCTGGG CCTCGGTGGG CGCTTCTGGC GGTTTTCGAT 1680 GTGGCAACCC CCATCAGCCA GGATAATGAT GAGGCAGGTA CAATCTATCT AGTACGCAGC 1740 CCCCAGACTC CCCCCTCCCT TCCCACCTCC CCATCCAACC CCCCAGCTAC TCTCTGCGGC 1800 CGGTTGGTCC TGAACTGGTG GGTGCAGTTC CGATGTTTAA CCAAATTCTC AAGCAATTTC 1860 AAGGTATTTG GATTTTTTGA ACCTGGGCCC TAACCGAAAC GCGGAACGGC TTGCTTATTA 1920 GACACCTCGA ACGACAGCGC AGGGAGGAAA CGGGATACTC GCTGCCCTTC CCAGTCGCGC 1980 GTGAGTCAAA AGGTCCTGGC AGGAGATGAT GTGAGGAGCG GCTGAAGTGG CAGGGAGCAA 2040 GGGATGAGGG GCTTGGAGCG GAGGTCCACC ACGCAAGGAC TCGGGAAGCG GGCAAGTGGG 2100 CAAAACTCTG CTTCCGGGCT CTCGATTTCT CGTTGATCAC TAAGTGGTAT TTTTCCCCCT 2160 TCTCTCGATG GCAAATGGGC GAAATCAAGA TGACTTA ACT TGGTAAATTT AGAGAGAACG 2220 GCTCGGAGCA AGTGAGGTCT AACGGGCAGC TAAAATTATC TCCAAATAAG AGATTTTGAC 2280 CCCCTCCCCC TATCCTCTCC TCGAATGTAT CCACCGGTGG GGAAGTGAGC GTCATTACTT 2340 TCGGGGCGCC ACGACAGGTT TGTTTGTTGC TCGCCTTTCC TGCTTCTCGC GCTGTCCCCG 2400 CGTGCAGACT GGTGGGTGCT GGGCGAGTGA TTAGCTGCAG GGCCCCATCC TAGTTTGGAA 2460 GGAAGGGGTT TAGAAGTTGG AGGATGGGTG AAATGGGAGG CTGCGATCCA TCTCCCTCTC 2520 CCTTCCACAC TCAAGCTCCC GCAAACACGC GCGCGCACAC ACAGCCCCTC CCTAGTCCCT 2580 CGGACCACCC GCCCCCACGC CCCTCTACCT TGACCTCCCT TGACCGCCGA CACAGCGTCC 2640 TGGGTGCGGG TCCCCGGGAG CGGGGAGTTC GCCGGGGAGC GATTGTCCTT GGGCGTGTTC 2700 GTGCTGTGGG GTGGGGGGAG GAGTGGCGGG TGGGCTTGGT AGGGGGTGGG GAGAGATCTG 2760 GAGCTGGAAG GGTCTAAGGT TTGGAGGAGG AGTTTACCCC TCAGACCTGA TCCTCCTGAC 2820 CAAAAAGGCA GGAAAAGGCC CTGATGCCTT GTAAAGAAAA TCTTGAAAGA AAAAAGATCA 2880 AAAAGAAAAA TTTCAAGAAA AAGAACCACT AAGAAAGGCT GAAGACACTA ACATGTAACC 2940 TGTTACGATA CATTTAACGT TTCGTTTTTT CCTGGATCTC TAAAAGGGAA CTCAAGGGTG 3000 GGGGTTACTG AAGAATACTA CAGATTTGGA AGTTTTTGTT GC TGTTGTTG TTTGGTTTGG 3060 TTTTGTTTTT CAAGAGGGGC CAGGAGAAAT GACCCCTTCC CCGCCACGGG TCCCGAAGTG 3120 AGGGCGGGGG GGGGGCTCTG GGGCGCGGGC GCGCGCGGCG CGGCGCGGGC CGGCGGGGGA 3180 GGGCGGCGCG GCGCGGAAGG GGTTAAGGCG CTGAGCGCGG AGCCATCTGG CCGGGTTGGC 3240 TGGTTATAAC CGCGCAGATT CTGTTCACGG GACTCAGAGT TGAAGCTCCT CTCCCTTCCG 3300 AACACGTCCG CGCACCGCCC CGCGACGCAG CCCGGCGCAA CTACTTTCTT CTCTCTCCTT 3360 TCTTTCTTCC TCTCCTTTTT CCCCTGCTGG GTAGTGGCTG CGGCGGGGTG GGGGAGACTT 3420 TGAATGACCG AGCTCGCGTC CACCTTTCTC TTCATGTCGA CGTCCCTGGA AACGGCCACA 3480 CGGATGCCAT GGTTACTTTT GCCACGgtaa ggggaggcgg cgggcacctt gggtgggcag 3540 gtttggggat gggggtccac gtggggaggg attttccagt ggactggtgc ggggggcccc 3600 agatccgcat cccgccccac ccccatcgcg ccgcgctcac tcactttccc gggcttgtgt 3660 cttccccaaa gtttgcgctg ggatctgctc aggccgaagc gcaaccgcag ccaccccgct 3720 acacacacac acacacacac acacacacac acacacacac acagacacgg acacccttct 3780 ccacctcctc ccctcttgtc cctcggctgc ccaagaagct t 3821.

[Brief description of drawings]

FIG. 1-1 Human NGF-2 / NT obtained in Example 1
-3 shows the nucleotide sequence from -3275 to +546 including the 5'upstream region of gene.

1-2: Human NGF-2 / NT obtained in Example 1
-3 shows the nucleotide sequence from -3275 to +546 including the 5'upstream region of gene.

FIG. 2 shows the range of 5 ′ upstream region of human NGF-2 / NT-3 gene contained in the plasmid used in Example 2.

FIG. 3 shows an autoradiogram of the CAT assay results obtained in Example 2.

FIG. 4 shows an autoradiogram of the results of the CAT assay obtained in Example 3.

FIG. 5-1: Plasmid pTB1 obtained in Example 4
A constructional diagram of 534 is shown.

FIG. 5-2: Plasmid pTB1 obtained in Example 4
A constructional diagram of 534 is shown.

FIG. 6 shows a cell growth curve obtained in Example 4.

FIG. 7 is a graph showing the alkaline phosphatase activity obtained in Example 4.

Claims (10)

[Claims] 1. A DNA containing a human nerve growth factor 2 promoter region. 2. A recombinant DNA containing a human nerve growth factor 2 promoter region. 3. The recombinant DNA according to claim 2, wherein the promoter region contains the base sequence TGAGCGCGGA GCCATCTGGC CGGGTTGGCT GGTTATAACC GCGCAGATTC TGTTCACGGG ACTC or a part thereof. 4. A promoter region, the nucleotide sequence AAGAGGGGCC AGGAGAAATG ACCCCTTCCC CGCCACGGGT CCCGAAGTGA GGGCGGGGGG GGGGCTCTGG GGCGCGGGCG CGCGCGGCGC GGCGCGGGCC GGCGGGGGAG GGCGGCGCGG CGCGGAAGGG GTTAAGGCGC TGAGCGCGGA GCCATCTGGC CGGGTTGGCT GGTTATAACC GCGCAGATTC TGTTCACGGG ACTC the content or recombinant DNA according to claim 2, wherein the containing portion thereof. 5. A recombinant vector containing the DNA according to claim 1 or 2. 6. A structural gene is linked downstream.
The recombinant vector described. 7. The structural gene is a neurotrophic factor.
The recombinant vector described. 8. The recombinant vector according to claim 6, wherein the structural gene is a reporter gene. 9. A transformant carrying the recombinant vector according to claim 6. 10. The transformant according to claim 9, wherein the host is an animal cell.