JPWO2001000818A1 - Gene encoding the promoter region of the tumor suppressor gene p51 and its uses - Google Patents

Abstract

(57) [Abstract] The present invention provides a gene encoding the promoter region of the cell death-inducing protein p51, as well as a gene encoding the 5-prime untranslated region of p51. These genes are useful for the diagnosis and treatment of diseases such as cancer caused by abnormalities in cell proliferation control. The present invention also provides antisense DNA and antisense RNA of these genes, nucleic acid probes consisting of part or all of these genes, methods for detecting the gene of the present invention or genes analogous thereto using the nucleic acid probes, transformants into which the gene of the present invention has been introduced, and drug screening methods using these. These genes are also useful for the diagnosis and treatment of diseases such as cancer.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a gene encoding the promoter region of p51, a protein having the ability to induce cell death and inhibit cell proliferation, and a gene encoding the 5'-terminal untranslated region of p51. The present invention also relates to a series of uses of these genes.
BACKGROUND ART The tumor suppressor gene p53 encodes a protein with a variety of functions, including the ability to induce apoptosis, arrest the cell cycle, and repair DNA damage. It has also been discovered that p53 is a transcription factor and controls the expression of various proteins. This protein is currently believed to play a central role in controlling cell proliferation. It has also been reported that p53 mutations are found in approximately half of cancer cells, and that this is the main cause of abnormal proliferation and resistance to anticancer drugs. Until very recently, no proteins with p53-like structure or function were known, but in recent years, two new p53-like molecules have been reported. One is p73 (Cell, Vol. 90, pp. 809-819, 1997).
The other is p51 (Nature Medicine, Vol. 4, pp. 839-843, 1998). Regarding p51, a protein encoded by the same gene has been reported under the name p63 (Molecular Cell
11, Vol. 2, pp. 305-316, 1998). p51 is structurally very similar to p53, and like p53, it has the ability to activate the transcription of the cell cycle progression inhibitor protein p21, suggesting that it is functionally homologous to p53. Furthermore, p51, like p53, has been reported to have the ability to induce cell death and inhibit cell proliferation. However, unlike p53, it has been shown that p51 rarely undergoes mutations even in cancer cells. It has also been revealed that p51 is expressed only in very limited organs, such as muscle cells. Disclosure of the Invention The objectives of the present invention are to identify the gene encoding the promoter region of the tumor suppressor gene p51 and the gene encoding the 5-prime untranslated region of p51, to provide a method for cloning these genes, to provide a method for discovering new drugs using these genes, and to provide a new method for gene therapy using these genes. The present inventors have conducted extensive research to find a gene fragment containing the p51 promoter in a human genome library, and have determined the gene sequence containing the p51 promoter region and the gene sequence encoding the 5-prime terminal untranslated region of p51, thereby completing the present invention. Specifically, a human genome fragment containing a sequence similar to the cDNA complementary to p51 mRNA was searched for and isolated by plaque hybridization screening. Furthermore, by determining the base sequence of this gene fragment, it was found that p51 was present in this fragment.
It was confirmed that the gene fragment contained a promoter region and a region estimated to be the 5-prime terminal untranslated region of p51. Furthermore, a plasmid was constructed by connecting this gene fragment to a luciferase reporter gene, and this was introduced into a human cell line to produce a transformant. Furthermore, by analyzing this transformant, it was found that the gene has promoter activity. Furthermore, it was found that a method for screening substances that act on the p51 promoter using these, and a drug that enhances this promoter activity may be used as a therapeutic agent for diseases involving p53-dependent apoptosis abnormalities, for example, an anticancer drug. Therefore, the present invention provides the following (1), (2), (3), (4), (5) or (
(1) DNA encoding a p51 promoter region having the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing; (2) DNA having a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing and having p51 promoter activity; (3) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing and has p51 promoter activity; (4) DNA encoding a p51 promoter region and a 5-prime terminal untranslated region of p51 having the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing; (5) DNA having a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing and having p51 promoter activity; (6) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing and has p51 promoter activity. Furthermore, the present invention relates to a gene encoding the 5-prime terminal untranslated region of p51 shown in (7), (8), or (9) below: (7) A gene encoding the region from 5677th to 596th in the base sequence shown in SEQ ID NO: 2 in the Sequence Listing.
(8) DNA having a base sequence of base 0; (9) DNA having a base sequence of bases 5677 to 596 in the base sequence shown in SEQ ID NO: 2 in the sequence listing;
(9) A DNA having a base sequence in which one or more bases are deleted, substituted or added in the base sequence at base 0, and having the same function as the DNA of (7) above; (9) A DNA having a base sequence from base 5677 to base 596 in the base sequence shown in SEQ ID NO: 2 in the Sequence Listing;
A DNA that hybridizes under stringent conditions with a DNA consisting of the 0th base sequence and has the same function as the DNA of (7) above. The present invention further relates to a recombinant plasmid characterized by containing the genes of (1) to (6) above. The present invention further relates to a transformant or transductant containing the recombinant plasmid. The present invention further relates to a nucleic acid probe consisting of the whole or part of the base sequence of the genes of (1) to (9) above. The present invention further relates to a pDNA hybridization product obtained by hybridization using the nucleic acid probe.
The present invention relates to a method for cloning the p51 promoter. Furthermore, the present invention relates to a DNA sequence that is antisense to the whole or part of the genes (1) to (9) above and is capable of modifying the function of the p51 promoter. Furthermore, the present invention relates to an RNA sequence that is antisense to the whole or part of the genes (1) to (9) above and is capable of modifying the function of the p51 promoter. Furthermore, the present invention relates to a method for screening for drugs that act on the p51 promoter using the transformant or transduced body. Furthermore, the present invention relates to compounds that increase or inhibit the expression of the p51 gene, selected by the screening method. Furthermore, the present invention relates to pharmaceutical preparations containing the DNA or RNA. Best Mode for Carrying Out the Invention The present invention is explained in more detail below. (1) Genes Having the p51 Promoter Region of the Present Invention (1-1) To isolate genes having the p51 promoter region of the present invention, a human genomic DNA library is constructed, and the Na
Using a portion of the sequence described in the article in the journal Therapeutic Medicine,
cDNA that hybridizes with the cDNA complementary to the 5-prime terminal untranslated region of A
The NA molecule can be isolated and cloned to isolate a gene encoding the p51 promoter region. The base sequence of the gene having the p51 promoter region of the present invention is as shown in SEQ ID NO: 1 in the Sequence Listing. In this gene sequence, TATA boxes are encoded between bases 5630 and 5636 and between bases 5659 and 5665 in the Sequence Listing. This is because the mRNA of p51A protein is encoded by the TATA box.
The gene sequence reported as the sequence of NA (p51A mRNA) (GenBa
nkABO16072) at positions 47 and 18 bases upstream. Furthermore, there are binding sites for MEF2, which is known to be a muscle cell-specific transcription factor.
It is located between bases 1211 and 1220 in the Sequence Listing. Furthermore, a database search was conducted for genes having homology to the gene encoding the p51 promoter region set forth in SEQ ID NO: 1 in the Sequence Listing, but no genes were found to have a homology of 30% or more over the entire length. Furthermore, in order to confirm the function of the gene having the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing, a plasmid was constructed in which a gene having the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing was linked to a luciferase reporter gene, and a transformant was produced by expressing the gene in a human cultured cell line, as shown in the Examples below. This transformant showed significantly higher expression of the luciferase reporter gene than the control group.
Furthermore, simple screening using transformants identified p5 in Trichostan A.
We have found that 1 μg/ml of trichostatin A (Wako Pure Chemical Industries, Ltd.) transcriptionally activates p51 in a human cultured cell line, and that under these conditions, the promoter activity of a gene containing the isolated p51 promoter region is significantly increased. These results demonstrate that the gene having the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing functions as a p51 promoter. (1-2) DNA having the nucleotide sequence set forth in SEQ ID NO: 1 in the Sequence Listing, with one or more bases deleted, substituted, or added, and having p51 promoter activity, is also within the scope of the present invention. The deletion, substitution, or addition of bases is to a degree that does not substantially affect the overall structure and function of the promoter. The degree of deletion, substitution, or addition of these bases is acceptable as long as the DNA has p51 promoter activity and has a homology of 80% or more, preferably 90% or more, and more preferably 99% or more, with the original nucleotide sequence. (1-3) A DNA that hybridizes under stringent conditions with a DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing and has p51 promoter activity.
A is also within the scope of the present invention. Such hybridizing DNA variants include those in which the DNA sequence has been partially altered by site-specific mutagenesis, random mutation by mutagen treatment, mutation of DNA fragments by restriction enzyme cleavage, deletion, ligation, etc. The degree to which these DNA variants hybridize with the coding gene shown in SEQ ID NO: 1 can be determined under stringent conditions, for example, by prehybridizing the membrane in a hybridization solution (50 mM Tris-HCl (pH 7.5), 1 M sodium chloride, 1% sodium dodecyl sulfate, 10% dextran sulfate, 0.2 mg/ml yeast RNA, 0.2 mg/ml salmon sperm DNA) at 65°C for 1 hour, adding a radioisotope-labeled cDNA fragment to a radioisotope concentration of 1 million dpm/ml, and incubating at 65°C for 16 hours to perform hybridization, and then hybridizing the membrane in 2x SS containing 0.1% sodium dodecyl sulfate.
In solution C (300 mM sodium chloride, 30 mM trisodium citrate),
After washing at 5°C for 30 minutes, hybridization is confirmed on X-ray film when analyzed by autoradiography. (1-4) DNA encoding the promoter region of p51 and the 5-prime terminal untranslated region of p51 shown in SEQ ID NO: 2 in the Sequence Listing is also within the scope of the present invention.
This corresponds to DNA encoding the dash-end untranslated region. Of these, the base sequence from base 5767 to base 5960 corresponds to the intron region. (1-5) DNA having a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 2 in the Sequence Listing and having p51 promoter activity is also within the scope of the present invention. The deletion, substitution, or addition of bases is to an extent that does not substantially affect the overall structure and function of the promoter. The degree of deletion, substitution, or addition of these bases is acceptable as long as it has p51 promoter activity and has a homology of 80% or more, preferably 90% or more, and more preferably 99% or more with the original base sequence. (1-6) DNA that hybridizes under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 2 in the Sequence Listing and has p51 promoter activity is also within the scope of the present invention. Examples of such hybridizing DNA variants include those similar to those exemplified in (1-3) above. These D
The degree to which the NA mutant hybridizes with the gene shown in SEQ ID NO: 2 is such that hybridization is confirmed under the same stringent conditions as those described in (1-3) above. (2) Gene encoding the dash-terminal untranslated region of p51 of the present invention (2-1) The base sequence from 5677 to 5678 in the base sequence shown in SEQ ID NO: 2 in the Sequence Listing
The DNA having the 960th base sequence encodes the 5-prime terminal untranslated region of p51, located downstream of the promoter region of p51, and such DNA is also within the scope of the present invention. The 5767th to 5960th base sequence corresponds to the intron region. As will be described later, the DNA encoding the 5-prime terminal untranslated region of p51 can be used, for example, in part or in its entirety, as a nucleic acid probe for gene cloning. (2-2) The 5677th to 5960th bases in the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing
DNA having a base sequence in which one or more bases are deleted, substituted or added in the 960th base sequence, and having the same function as the DNA of (2-1) above.
(2-3) The deletion, substitution or addition of bases is to such an extent that it does not substantially affect the structure and function of the DNA. The degree of deletion, substitution or addition of these bases is acceptable if it has the same function as the DNA of (2-1) above and has a homology of 80% or more, preferably 90% or more, more preferably 99% or more with the original base sequence. (2-4) The deletion, substitution or addition of bases from 5677th to 5678th in the base sequence shown in SEQ ID NO: 2 of the Sequence Listing is acceptable.
The present invention also includes DNA that hybridizes under stringent conditions with the DNA consisting of the 960th base sequence and has the same function as the DNA of (2-1) above.
Examples of the hybridization of the DNA mutants with the gene shown in SEQ ID NO: 2 include those similar to those exemplified in (1-3). The degree to which these DNA mutants hybridize with the gene shown in SEQ ID NO: 2 is such that hybridization is confirmed under the same stringent conditions as those described in (1-3) above. (3) Recombinant Plasmid Containing a Gene Having the p51 Promoter Region of the Present Invention By constructing a recombinant plasmid containing a gene having the p51 promoter region of the present invention, it is possible to stably retain the gene in E. coli or the like,
In this case, any commonly used vector can be used, for example, pBluescript II SK(-). In the examples described below, a gene having a p51 promoter region is inserted into pBluescript II SK(-).
An example is the pBS/p51 promoter integrated into SK(-). These plasmids can be cleaved with appropriate restriction enzymes, etc., as needed, and then ligated into an appropriate vector to produce a plasmid for measuring promoter activity. Plasmids such as pGL2 can be used as the promoter activity measurement plasmid. (4) Transformant or Transducer: A transformant or transducer can be constructed by introducing the above-mentioned recombinant plasmid into an appropriate host. E. coli, yeast, or mammalian cells can be used. A transformant harboring a promoter activity measurement plasmid can be obtained by transforming an appropriate host with the recombinant plasmid integrated into the activity measurement vector as described above. For example, a transformant can be obtained by introducing a recombinant plasmid such as that shown in Figure 1 into cultured mammalian cells. The transformant or transducer can be cultured in an appropriate nutrient medium, and promoter activity can be measured by measuring the expression level of the reporter gene in the cells. (5) Nucleic Acid Probes and Gene Cloning and Detection Using Them. In the present invention, a portion or all of the gene containing the p51 promoter region of the present invention obtained as described above, or a portion or all of the gene encoding the 5-prime untranslated region of p51 of the present invention, can be used as a nucleic acid probe for gene cloning, as described below. Examples of nucleic acid probes consisting of a portion of the gene of the present invention include nucleic acid probes consisting of oligonucleotides of 15 or more nucleotides. Such nucleic acid probes can be prepared by ligating the gene of the present invention or a gene fragment thereof to an appropriate vector, introducing it into bacteria, allowing it to replicate, extracting it from the bacterial cell lysate with phenol or the like, cleaving it with a restriction enzyme that recognizes the site of ligation with the vector, electrophoresing it, and then excising it from the gel. Alternatively, such nucleic acid probes can be prepared by chemical synthesis using a DNA synthesizer or gene amplification techniques such as polymerase chain reaction (PCR) based on the nucleotide sequence of SEQ ID NO: 1 or 2 in the Sequence Listing. The nucleic acid probes can also be labeled with radioisotopes or fluorescent labels to increase detection sensitivity during use. The nucleic acid probe can be used in a method for cloning genes other than those mentioned above that have promoter activity, as well as in a method for cloning genes that encode downstream proteins.
Alternatively, by searching genome libraries of various biological tissues using PCR, it is possible to isolate genes having the same function as the gene of the present invention or genes encoding proteins located downstream thereof. (6) Antisense DNA and Antisense RNA In the present invention, antisense DNA of the gene having the above-mentioned p51 promoter region is used.
Also provided are antisense DNA and antisense RNA for a gene having the 5-prime terminal untranslated region of p51. By introducing these antisense DNA and antisense RNA into cells, p51 can be inhibited.
It is possible to suppress or increase the expression of the gene encoding p51. The antisense DNA to be introduced can be, for example, the antisense DNA corresponding to SEQ ID NO: 1 or 2 in the sequence listing, or a part thereof. An example of the antisense DNA is shown in SEQ ID NO: 3 in the sequence listing. This is the antisense DNA corresponding to p51 in SEQ ID NO: 1 in the sequence listing.
The sequences shown in the figures are antisense DNA sequences of genes having p51 promoter activity. For example, fragments obtained by appropriately cleaving a portion of these antisense DNAs may be used as antisense DNA, or DNA synthesized based on these antisense DNA sequences may be used. For example, antisense RNAs corresponding to SEQ ID NO: 1 or 2 in the Sequence Listing, or portions thereof, may be used as antisense RNA. An example of such antisense RNA is shown in SEQ ID NO: 4 in the Sequence Listing. This shows the sequence of the antisense RNA of the gene having p51 promoter activity of SEQ ID NO: 1 in the Sequence Listing. For example, fragments obtained by appropriately cleaving a portion of these antisense RNAs may be used as antisense RNA, or RNA synthesized based on these antisense RNA sequences may be used. Alternatively, for example, a gene having p51 promoter activity of SEQ ID NO: 1 in the Sequence Listing, or a portion thereof, may be ligated to an appropriate vector, introduced into bacteria, and replicated. A lysate of the bacteria may be extracted with phenol or the like as a template, and the resulting product may be used for replication.
RNA prepared by the action of RNA polymerase in an in vitro transcription system may also be used. Antisense DNA and antisense RNA can be chemically modified to resist degradation in vivo and to allow them to pass through cell membranes. Antisense DNA and antisense RNA prepared in this manner can be used to treat various diseases, including malignant tumors. (7) Drug Screening : The present invention provides a method for screening novel drugs using the transformant or transducant of the present invention. For example, by using cultured cells transfected with a luciferase reporter gene and a recombinant plasmid containing the gene, drugs that increase luciferase activity can be screened to discover substances that have the ability to activate the p51 promoter and increase the expression of p51 protein. Cultured cells into which the recombinant plasmid is introduced can include any cultured cell line that can be subcultured, such as the colon cancer cell line HCT-116 used in the examples. Recombinant plasmids can include any vector containing a reporter gene, such as the pGL2-neo vector, which contains the neomycin gene ligated to the pGL2 plasmid used in the examples. To construct a screening system, a cultured cell line is selected and the screening vector is introduced using lipofection or other methods. By culturing these transformed cells with a selection drug, only cells containing the screening vector can be grown. These cells can be used for screening as is, or they can be cloned to prepare a single cell line and then used for screening. By adding a sample to these cells and measuring the activity of the reporter protein, substances that regulate p51 transcription can be identified. Examples of samples include microbial secondary metabolites and synthetic compounds. Substances that activate the p51 promoter increase p51 protein production and exhibit growth-inhibiting effects even in cancer cells with p53 mutations, potentially making them potential new cancer inhibitors. (8) Pharmaceutical formulations : p51 expression is observed only in very localized tissues, such as muscle cells. Therefore, by connecting a gene of interest downstream of the p51 promoter gene of the present invention and expressing the gene of interest only in specific tissues, it can be used as a gene therapy vector. Furthermore, the above-mentioned antisense DNA and antisense RNA can be used as therapeutic agents for various diseases including malignant tumors. The present invention will be specifically explained below using examples, but the present invention is not limited to these examples. Example 1 Isolation of a novel gene fragment encoding the p51 promoter region (1-1) Preparation of a screening probe A screening probe for a novel gene fragment encoding the p51 promoter region was prepared. RT-PCR was used as the method. The experimental procedure is as follows. 6 μg of human muscle-derived RNA (OriGene) was subjected to reverse transcriptase Super
cDNA was prepared using 200 units of .rscript (GIBCO BRL). Subsequently, using this as a template, PCR was performed to amplify a gene fragment derived from p51 mRNA using the primer set shown in SEQ ID NOS: 5 and 6, and the primer set shown in SEQ ID NOS: 7 and 8. The gene amplified using the former primer set contains 143 bases of the 5-primer terminal untranslated region of p51A mRNA and 5 bases of the open reading frame, which have been reported to date.
The gene amplified using the latter primer set encodes the full length of the p51A mRNA open reading frame. The base sequence of the former gene is shown in SEQ ID NO: 9 in the Sequence Listing. In carrying out the PCR method, EX Taq (manufactured by Takara Shuzo Co., Ltd.) was used as the DNA polymerase, and the PCR conditions were 95°C (1 minute), 55°C (1 minute), and 72°C (1 minute) for one cycle, with 30 cycles.
The PCR amplification product was subjected to cycle amplification. After deproteinization by phenol treatment and chloroform treatment, it was precipitated with ethanol. Subsequently, the DNA was washed with 70% ethanol and dissolved in sterile water. Next, these purified DNAs and 20 ng of E. coli vector pBluescript KS(+) (manufactured by Toyobo Co., Ltd.) were subjected to restriction enzyme EcoR
After digestion with pBS/EcoRV (manufactured by Takara Shuzo Co., Ltd.), a DNA with one thymine added to the digested end (hereinafter referred to as pBS/EcoRV TA) was ligated using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.) (hereinafter, the plasmids containing the amplified genes obtained with the primer sets shown in SEQ ID NOS: 5 and 6 are referred to as pBS/p51-1 and pBS/p51-2, respectively).
The plasmid containing the amplified gene obtained by the primer set shown in 8 is referred to as pBS/p51A ORF. The absence of mutations in the amplified gene was confirmed by determining the nucleotide sequences of pBS/p51-1 and pBS/p51A ORF. The nucleotide sequences were determined using an automatic sequencer, LONG READ.
The analysis was carried out using an IR4200 (Leica).
After cleavage with restriction enzymes EcoRV and PstI (manufactured by Toyobo Co., Ltd.), the DNA was fractionated by 0.8% agarose electrophoresis, extracted, and purified. EASYTRAP (manufactured by Takara Shuzo Co., Ltd.) was used for purification. This purified DNA was used as a template for a screening probe. (1-2) Screening for novel gene fragments encoding the p51 promoter region Next, screening for gene fragments encoding the p51 promoter region was attempted from a human genome library. As a human genome library, Easy-TRAP was used.
to-Handle Eukaryotic Genomic Library
The infection efficiency of this library was 3 million plaque-forming units per μl. Using this library, membranes for plaque hybridization were prepared as follows: 0.02 μl of the library solution and 0.9 ml of C6
A 00 E. coli solution (E. coli strain C600) was cultured in LB medium (0.5% yeast extract, 1% peptone, 0.2% maltose, 10 mM magnesium sulfate) containing 0.
The mixture was incubated with shaking at 37°C for 16 hours in 50 ml of 5% sodium chloride.
The cells were collected by centrifugation at 100 rpm for 5 minutes, and then suspended in 25 ml of 10 mM magnesium sulfate. The mixture was mixed and incubated at 37° C. for 15 minutes to allow infection with the phage.
To this, add 7 ml of LB soft agar (0.5% yeast extract, 1% peptone, 0.5% sodium chloride, 10 mM magnesium sulfate, and 0.7% agar) that has been kept warm at 47°C after melting, mix gently, and
The bacteria were spread onto 150 mm diameter LB-magnesium sulfate plates (agar medium prepared by adding 1.5% agar to 0.5% yeast extract, 1% peptone, 0.5% sodium chloride, and 10 mM magnesium sulfate). This procedure resulted in the appearance of 60,000 plaques per plate. A total of 23 plates were spread using the same procedure. These were cultured at 37°C for 16 hours to allow plaques to form. Next, the plates were cooled at 4°C for 1 hour, and the plaques were then attached to a nylon membrane, Colony Plaque Screen (manufactured by NEN Co., Ltd.). After drying the membrane, 500 ml of alkaline solution (0.2 N sodium hydroxide, 1.
The membrane was denatured in 500 ml of neutralizing solution (0.5 M Tris-HCl (pH 7.2), 1.5 M sodium chloride, 1 mM disodium ethylenediaminetetraacetate) at room temperature for 2 minutes. The membrane was then incubated at 55°C for 1 hour in 3xSSC solution (450 mM sodium chloride, 45 mM trisodium citrate) and then dried to prepare a membrane for plaque hybridization. pBS/p51-1 was denatured using the restriction enzyme EcoRV.
Furthermore, 50 ng of the aforementioned cDNA fragment, which had been digested with PstI and purified, was used as Prime-I.
The membrane was radioisotope-labeled using tII (Stratagene) to prepare a screening probe. Plaque hybridization was then performed using this membrane. The hybridization solution (50 mM Tris-HCl (pH 7.5), 1 M sodium chloride, 1% sodium dodecyl sulfate, 10% dextran sulfate, 0.2 mg
The membrane was incubated in 50 ml of a 500 kJ/ml yeast RNA solution (0.2 mg/ml salmon sperm DNA) at 65°C for 1 hour for prehybridization.
The radioisotope-labeled cDNA fragments were converted into radioisotope amounts of 1 million dpm/m
Hybridization was carried out by adding 1 ml of PBS to the membrane and incubating at 65°C for 16 hours. The membrane was then washed in 2xSSC solution (300 mM sodium chloride, 30 mM trisodium citrate) containing 0.1% sodium dodecyl sulfate at 65°C for 30 minutes. This washing was carried out twice. Positive plaques were then detected by autoradiography of the membranes. Next, a group of plaques containing plaques corresponding to positive signals was isolated and suspended in 1 ml of SM buffer (50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium sulfate, 0.01% gelatin). This solution was used for 4
The mixture was incubated at ℃ for 16 hours, and the phages were eluted. After centrifugation at 13,000 rpm for 10 minutes, the phages were collected in the supernatant. This phage solution was spread on an LB-magnesium sulfate plate in the same manner as above, and positive plaques were screened. As a result, positive plaques were successfully isolated as completely isolated plaques. Phages were isolated from these plaques in the same manner as above. Next, the following experiment was performed to determine the base sequence of the library DNA contained in this phage. This library was created using lambda PS phage (manufactured by MoBiTec). This phage has a loxP recombination site in its base sequence and was cloned into E. coli BNN13 carrying Cre recombinase.
By introducing the vector into strain 2, the vector portion containing the genome library sequence can be excised from the phage. This procedure was carried out according to the following method.
1 μl of the phage solution was added to 200 μl of BNN132 E. coli solution (E. coli strain BNN1
32 in 50 ml of LB medium containing 0.2% maltose and 10 mM magnesium sulfate
The cells were incubated at 37°C for 16 hours with shaking, and then centrifuged at 5000 rpm for 5 minutes to recover the cells. The cells were then mixed with 25 ml of 10 mM magnesium sulfate and incubated at 37°C.
The mixture was then plated on an LB-ampicillin plate (0.
5% yeast extract, 1% peptone, 0.5% sodium chloride, 0.1
The plasmid was prepared by spreading the plasmid onto agar medium (prepared by adding 1.5% agar to 1.5 mg/ml sodium ampicillin), and the target plasmid was prepared from the colonies that appeared. The preparation of the plasmid was carried out according to the method described in Laboratory Manual of Genetic Engineering (published by Maruzen, edited by Muramatsu Masataka, 1990) 53-
The procedure was carried out according to page 55. It was revealed that this plasmid contained a library gene of approximately 15 kilobases.
(1-3) Sequencing of gene fragment encoding p51 promoter region Next, the base sequence of a portion of the library gene in pPS/library was determined using an automatic sequencer LONG READIR 4200, and p51A m
It was confirmed that the cDNA sequence contained a portion of the RNA complementary to the cDNA sequence.
It was revealed that a 0.6 kilobase gene fragment generated when pPS/library was cleaved with the restriction enzyme EcoRV contained a portion of the cDNA sequence complementary to p51A mRNA.
The plasmid was digested with uII (Toyobo Co., Ltd.), fractionated by 0.8% agarose gel electrophoresis, and then blotted onto a nylon membrane, Hybond N+ (Amersham). Plasmid fragment blotting was performed according to the procedure in Lab Manual Human Genome Mapping (Maruzen Publishing, edited by Hori Masaaki and Nakamura Yusuke, 1991), pp. 26-36. Southern blotting was performed using this membrane as follows: pPS/library was digested with the restriction enzyme EcoRV to prepare a cDNA probe fragment, and fractionated by 0.8% agarose gel electrophoresis. A 0.6 kilobase gene fragment was excised and purified using EASYTRAP. This purified cDNA was purified using Prime-It
The membrane was radioisotope-labeled with II and used as a probe.
The mixture was incubated at 65°C for 1 hour in a 5% PEG 8000 solution for prehybridization.
Hybridization was carried out by adding 1000 kJ/ml of PBS to the membrane and incubating at 65°C for 16 hours. The membrane was then washed in 2xSSC solution containing 0.1% sodium dodecyl sulfate at 65°C for 30 minutes. This washing was carried out twice. Autoradiography of the membranes revealed that the PvuII cleavage fragment containing the region immediately upstream of p51A mRNA was 5.5 kilobases in length. Next, pPS/library was cleaved with the restriction enzyme PvuII and fractionated by 0.8% agarose electrophoresis. The 5.5 kilobase gene fragment was excised and purified by EASYTRAP. This purified c
The DNA fragment was digested with pBluescript II SK(+) using the restriction enzyme EcoR
The resulting plasmid was digested with PvuII and dephosphorylated with shrimp alkaline phosphatase, and then ligated to the resulting plasmid using a DNA ligation kit.
This base sequence is denoted as 5.5.
The base sequence was determined using the 4200.
The partial base sequences of ary were synthesized to obtain the base sequence shown in SEQ ID NO: 2 in the Sequence Listing. In addition, the base sequence obtained by removing the portion encoding the 5-prime untranslated region of p51 from the base sequence shown in SEQ ID NO: 2 in the Sequence Listing is shown in SEQ ID NO: 1 in the Sequence Listing. In addition, the 3-prime end of this PvuII cleavage fragment corresponds to the cDNA encoding the p51 mRNA.
It was revealed that the DNA sequence is located approximately 0.22 kilobases upstream of the first base of SEQ ID NO: 9 in the Sequence Listing. Therefore, the following procedure was carried out to prepare a plasmid containing up to the first base of SEQ ID NO: 9 in the Sequence Listing, which is a cDNA corresponding to a portion of the reported p51A mRNA. pPS/library was cleaved with the restriction enzyme EcoRV and fractionated by 0.8% agarose electrophoresis. A 0.6 kilobase gene fragment was excised and purified with EASYTRAP, and then purified with pBS/PvuII5.
.5 was digested with the restriction enzymes EcoRV and SmaI (Takara Shuzo Co., Ltd.), dephosphorylated with shrimp alkaline phosphatase, and ligated to the resulting plasmid using a DNA ligation kit. The resulting plasmid was analyzed, and the plasmid ligated in the correct orientation was isolated. Hereinafter, this plasmid will be referred to as pBS/p51promot
er. Furthermore, to verify the reliable presence of this base sequence in the genome, the following experiment was performed. Genomic DNA was prepared from the colon cancer cell line HCT116, and PCR was performed using this as a template. Genomic DNA was prepared according to pages 16-19 of "Cell Engineering Experimental Protocol" (published by Shujunsha, edited by the Department of Cancer Research, Institute of Medical Science, The University of Tokyo, 1993). Primers used were those shown in SEQ ID NOs: 10 and 11. These primers correspond to the sense strand of bases 3543 to 3570 and the antisense strand of bases 5458 to 5487 of the base sequence of SEQ ID NO: 1 in the Sequence Listing, respectively.
The polymerase was LA Taq (manufactured by Takara Shuzo Co., Ltd.), and PCR was performed at 94°C (1 min), 68°C
The PCR product was amplified for 30 cycles, with each cycle consisting of 3 minutes.
Agarose gel electrophoresis confirmed that a 1.9 kilobase gene fragment was specifically amplified. This fragment was then excised, purified using EASYTRAP, and ligated to pBS/EcoRV TA using a DNA ligation kit. The base sequence of this plasmid was analyzed using an automated sequencer, LONG READIR 42.
The base sequence of the p51 promoter was determined using pGL2 (Promega) and confirmed to be completely identical to the base sequence in pBS/p51 promoter. (1-4) Construction of a luciferase vector containing the p51 promoter region : To confirm the presence of promoter activity in the isolated region, a vector in which this gene sequence was ligated upstream of a luciferase reporter gene was prepared according to the following method. pGL2 (Promega) was used as the vector containing the luciferase reporter gene. First, a neomycin resistance gene was introduced into this vector to enable integration and selection of pGL2 in mammalian cells. pGL2 was cleaved with the restriction enzyme BamHI (Toyobo Co., Ltd.) and pMAM neo (Toyobo Co., Ltd.) was cleaved with BamHI, fractionated by 0.8% agarose gel electrophoresis, and the 2617-base neomycin resistance gene excised and purified using EASYTRAP. These were then ligated using a DNA ligation kit. This plasmid will be referred to as pGL2-neo hereinafter.
pGL2-neo was cleaved with the restriction enzyme XhoI (manufactured by Toyobo Co., Ltd.), precipitated with ethanol, washed with 70% ethanol, and then dissolved in purified water.
The cut ends were blunted using a blunting kit (manufactured by Takara Shuzo Co., Ltd.), then precipitated with ethanol, washed with 70% ethanol, and dissolved in purified water.
The DNA was cleaved with the restriction enzyme KpnI (Boehringer Mannheim), treated with phenol and chloroform to remove proteins, precipitated with ethanol, washed with 70% ethanol, and then dissolved in sterilized water.
The fragments are represented as I (blunting), KpnI. Next, pBS/p51promoter was cleaved with the restriction enzyme NotI (Boehringer Mannheim), precipitated with ethanol, washed with 70% ethanol, and dissolved in purified water. The cleaved ends of this DNA were blunted using a DNA blunting kit (Takara Shuzo), precipitated with ethanol, washed with 70% ethanol, and dissolved in purified water. Next, this DNA was cleaved with the restriction enzyme KpnI (Boehringer Mannheim), fractionated by 0.8% agarose electrophoresis, and a 5.7 kilobase gene fragment was excised and purified by EASYTRAP. This gene fragment was cleaved with pBS/p51promoter/NotI (blunting), KpnI.
pGL2-neo/XhoI (blunt
ng), KpnI and pBS/p51promoter/NotI (blunti
ng) and KpnI5.7 were ligated using a DNA ligation kit. This plasmid is designated as pGL2-neo/p51promoter. The gene sequence of this plasmid is shown in SEQ ID NO: 12 in the sequence listing. Furthermore, the restriction enzyme map of this plasmid is shown in Figure 1. (1-5) Effects of various drugs on the transcription level of p51 1) Northern hybridization Various stimuli, including drug stimulation, were applied to the colon cancer-derived cell line HCT116 to detect the transcription level of p51.
The change in the expression level of 51 was examined by Northern blotting.
The CT116 cell line was seeded onto a 60 mm diameter culture dish and cultured for 2 days at 37°C in the presence of 5% carbon dioxide. The medium was McCoy's 5A (manufactured by Nissayken Co., Ltd.) supplemented with fetal bovine serum (FBS) to a final concentration of 10%.
ovine serum) (hereinafter referred to as McCoy's 5A/10%
Subsequently, A23187 (0.1 μM, 1 μM),
Cisplatin (1 μg/ml, 10 μg/ml), CRTcAMP (0.1 μM
, 1 μM), etoposide (10 ng/ml, 0.1 μg/ml), genistein (1 μM, 10 μM), ML-236B (1 μg/ml, 10 μg/ml),
Milrinone (1 μM, 10 μM), mitomycin C (5 μg/ml, 50 μg
/ml), NKH477 (0.1 μM, 1 μM), okadaic acid (1 nM, 10 nM
), radicicol (0.1 μg/ml, 1 μg/ml), staurosporine (
1nM, 10nM), taxol (1μg/ml, 10μg/ml), trichostatin A (0.1μg/ml, 1μg/ml), radiation (50J, 100J per square meter), vitamin D3 (10nM, 100nM), vincristine (1μg/ml, 10μg/ml), wortmannin (0.1μM, 1μM)
The cells were treated with 5% CO2 at 37°C for an additional 24 hours. Subsequently, RNA was prepared from these cells using ISOGEN (Nippon Gene). 20 μg of each RNA was fractionated by electrophoresis on 1% agarose gel containing formaldehyde and blotted onto a nylon membrane Hybond N+ according to the method described in the New Cell Engineering Experimental Protocol, pages 194-197. As a probe, pBS/p51AORF was digested with the restriction enzyme EcoRI (Toyobo) and 0.
After fractionation by 8% agarose electrophoresis, a 0.6 kilobase gene fragment was excised.
The membrane was purified by EASYTRAP and radioisotope-labeled with Prime-It II. The membrane was placed in 10 ml of prehybridization solution (25 mM phosphate buffer (pH 7.0), 6×SSC, 50% formamide, 5×Denhardt's solution, 0.1% sodium dodecyl sulfate, 0.2 mg/ml
The samples were prehybridized by incubating in a 10 ml hybridization solution (20 mM phosphate buffer (pH 7.0), 6×SSC, 50% formamide, 1× Denhardt's solution, 0.1 ml of PBS containing 1000 kJ/ml ...
1% sodium dodecyl sulfate, 0.1 mg/ml salmon sperm DNA, 4% dextran sulfate), and then the radiolabeled probe was added to 1 million dp
Hybridization was performed by adding 1 μg/ml of trichostatin A to the cells and incubating at 42°C for 16 hours. The cells were then washed twice in 2x SSC containing 0.1% sodium dodecyl sulfate at 65°C for 30 minutes. The membrane was analyzed by autoradiography. 2) Results: As a result, only the cells treated with 1 μg/ml of trichostatin A contained p51A-derived cDNA.
A specific band reacting with NA was observed. No increase in p51 mRNA level was observed with any other stimuli. Figure 2 shows the increase in p51 mRNA due to trichostatin treatment. In Figure 2, lanes 1 and 2 are taxol (1 μg/ml, 10 μg/ml) treated cells, lanes 3 and 4 are trichostatin A (0.1 μg/ml, 1 μg/ml) treated cells, lanes 5 and 6 are radiation (50 J, 100 J per square meter) treated cells, lanes 7 and 8 are vitamin D3 (10 nM, 100 nM) treated cells, and lanes 9 and 10 are vincristine (1
10 μg/ml, 10 μg/ml) treated cells, lanes 11 and 12: wortmannin (0
The figure shows a blot of 20 μg of RNA from cells treated with 1 μM or 1 μM of p51. (1-6) Functional analysis of the p51 promoter region Next, the following experiment was carried out to perform functional analysis of the p51 promoter region. 200,000 HCT116 cells were seeded onto a 60 mm diameter culture dish. The medium used was McCoy's 5A/10% FBS. These were then diluted with 5%
After culturing for 48 hours at 37°C in the presence of carbon dioxide, the medium was replaced with serum-free McCoy's 5A.
2 μg of either pGL2-promoter or pGL2-neo vector was introduced. Plasmid introduction was performed using Lipofectamine Plus reagent (GIBCO BRL). Specifically, 125 μl of McCoy's 5A and 8 μg of PEG-1000 were added to the plasmid solution.
1 μl of PLAS solution was added, stirred thoroughly, and then incubated at room temperature for 15 minutes. 125 μl of McCoy's 5A and 12 μl of Lipofectamine solution were added, stirred thoroughly, and then incubated at room temperature for another 15 minutes. This was administered to HCT116 cells cultured as described above, mixed gently, and then incubated in the presence of 5% carbon dioxide.
The cells were cultured at 37°C for 3 hours to allow the plasmid to be incorporated.
The medium was replaced with COY's 5A/10% FBS and the cells were cultured in the presence of 5% carbon dioxide at 37°C for 21 hours. Subsequently, trichostatin A was administered to the two groups of cells to a final concentration of 0 or 1 μg/ml, and the cells were cultured in the presence of 5% carbon dioxide at 37°C for 21 hours.
The cells were cultured for 24 hours at 4°C for 24 hours. The luciferase activity of these cells was measured to examine the promoter activity. In addition, pcDNA3/lacZ cells were used as negative control cells that did not contain the luciferase gene.
The luciferase activity was measured as follows: The medium in the cell culture dish was removed, and 5 ml of cell lysis reagent (25 ml) was added.
M Tris-HCl (pH 7.8), 2 mM dithiothreitol, 2 mM CDTA
, 0.2% Triton X-100, 10% glycerol) was added, and the mixture was incubated at room temperature for 15 minutes. 10, 20, 30, 40, and 50 μl of the cell lysate were taken and diluted with a substrate solution (20 mM Tricine-sodium hydroxide (pH 7.8), 1.0
7 mM basic magnesium carbonate, 2.67 mM magnesium sulfate, 0.1 mM disodium ethylenediaminetetraacetate, 33.3 mM dithiothreitol, 270
After incubation at room temperature for 15 seconds, the mixture was measured using a luminometer.
The chemiluminescence intensity was measured using SKAN (manufactured by Dainippon Pharmaceutical Co., Ltd.). The results are shown in Figure 3. These results demonstrate that the cell lysate containing pGL2-neo/p51 promoter exhibits significantly higher fluorescence intensity than pGL2-neo. Furthermore, administration of trichostatin A significantly reduced the fluorescence intensity of pGL2-neo/p51 promoter.
The chemiluminescence intensity in the culture medium was further significantly enhanced. This result is consistent with the results shown in Figure 2 above. The above experimental data revealed that the isolated gene fragment is a functional p51 promoter region. Example 2 Screening for drugs that modify p51 promoter function (2-1) Preparation of cultured cell lines for screening Cultured cell lines carrying the pGL2-neo/p51promoter plasmid can be used as cells for screening drugs that modify p51 promoter function. Such drugs are effective in treating various diseases including cancer. The cells were prepared using the following procedure. The HCT116 cell line was used as the plasmid-introduced cells. 500,000 HCT
116 cells were seeded onto a 60 mm diameter culture dish.
Y's5A/10% FBS was used. These were incubated for 37 min in the presence of 5% carbon dioxide.
After culturing for 48 hours at 4°C, the medium was replaced with serum-free McCoy's 5A.
The plasmid used for introduction was pGL2-neo/p51promot
2 μg of er was dissolved in 4 μl of TE solution to prepare a plasmid solution. The plasmid was introduced using Lipofectamine Plus reagent (GIBCO BRL). That is, 125 μl of McCoy's 5A and 8 μl of
The solution was added, stirred thoroughly, and then kept at room temperature for 15 minutes.
25 μl of McCoy's 5A and 12 μl of Lipofectamine solution were added, mixed thoroughly, and then incubated at room temperature for another 15 minutes. This was then administered to the HCT116 cells cultured as described above, gently mixed, and then incubated at 37°C in the presence of 5% carbon dioxide.
The cells were cultured at 4°C for 3 hours to allow the plasmid to be incorporated.
Y's 5A/10% FBS, and incubated at 37°C in the presence of 5% carbon dioxide.
The cells were cultured for 21 hours. The cells were harvested from the culture dish using PBS containing 0.25% trypsin and 0.02% EDTA, and 1% of the cells were seeded onto a 100 mm diameter culture dish.
After further culturing for 72 hours at 37°C in the presence of 5% carbon dioxide, 1.2 mg/ml of Geneticin (Nacalai Tesque) was added as a selective agent.
The medium was changed every three days, and the cells were cultured under the above conditions for another two weeks. Cells in which the plasmid gene had been introduced into the cells and had become geneticin-resistant were selected. Subsequently, 50 clones were isolated from the cell colonies that formed on the dish and plated onto 24-well culture dishes for further culture. These clones were expected to exhibit increased luciferase activity in response to stimuli that activate p51 promoter function. Therefore, the responsiveness of these clones to trichostatin A was examined using the following procedure. Five hundred thousand cells from each clone were plated onto a 60 mm culture dish in McCoy's 5A/10% FBS medium. After culturing these cells at 37°C in the presence of 5% carbon dioxide for 48 hours, the two groups of cells were treated with trichostatin A at a final concentration of 0 or 0.1 μg/ml and cultured for 24 hours at 37°C in the presence of 5% carbon dioxide. The effect of trichostatin A on p51 promoter activity was examined by measuring the luciferase activity of these cells. Luciferase activity was measured according to the method described above.
Several clones showing a significant increase in luciferase activity in response to /ml trichostatin A were obtained, and from these, HCT116/p
The 51 promoter clone #9 was selected. The results of the luciferase assay are shown in Table 1. In the table, TsA represents trichostatin A. The trichostatin A administration group received 0.1 μg
The values in the figure show the measured values of luciferase activity. (2-2) Screening of drugs that modify p51 promoter function The HCT116/p51 promoter clone #9 cell line was administered at a concentration of 1000 μg/ml.
This is useful for screening drugs that induce activation of the V51 promoter function.
The screening was carried out as follows: 10,000 HCT116/p51 promoter clone #9 cultured cells were seeded into a 96-well culture dish and then incubated with 200 μl of RPMI1640/10%
The cells were cultured in FBS in the presence of 5% carbon dioxide at 37°C for 24 hours. Subsequently, a screening agent was added, and the culture was continued for 24 hours, after which luciferase activity was measured. Synthetic compounds or microbial secondary metabolites were used as the agents. 1,200 compounds and 9,600 microbial secondary metabolites were screened, and 71 samples were found to exhibit p51 promoter activation activity equal to or greater than that of 0.1 μg/ml trichostatin A. Substances that activate the p51 promoter include:
These genes are expected to be therapeutic agents for various diseases, including cancer. Industrial Applicability: The present invention provides a gene encoding the promoter region of the cell death-inducing protein p51, as well as a gene encoding the 5-prime untranslated region of p51. These genes are useful for the diagnosis and treatment of diseases, including cancer, caused by abnormalities in cell proliferation control. Furthermore, the present invention provides antisense DNA and antisense RNA of these genes, nucleic acid probes consisting of part or all of these genes, methods for detecting the gene of the present invention or its analogous genes using such nucleic acid probes, transformants containing the gene of the present invention, and drug screening methods using these. These genes are also useful for the diagnosis and treatment of diseases, including cancer. Sequence Listing Free Text: The nucleotide sequences of SEQ ID NOs: 5 to 8, 10, and 11 in the Sequence Listing represent PCR primers. The nucleotide sequence of SEQ ID NO: 9 encodes the 5-prime untranslated region of p51A mRNA and the 5-prime terminal 165 of the open reading frame of p51A mRNA.
The base sequence of SEQ ID NO: 12 shows the DNA sequence of a plasmid containing the p51 promoter and neomycin resistance gene.

[Sequence List]

[Brief explanation of the drawings]

Figure 1 shows the restriction enzyme map of pGL2-neo/p51 promoter. In the figure, amp represents the ampicillin resistance gene, neo represents the neomycin resistance gene, and luc represents the luciferase gene. Restriction enzymes marked with an asterisk (*) to the left of their names are enzymes that cleave the vector at a single point. Figure 2 shows the analysis of p51 expression levels in colon cancer-derived cell line HCT116 using Northern blotting techniques when various stimuli were applied. Lanes 1 and 2 show taxol (1 μg/ml, 10 μg/ml)-treated cells, lanes 3 and 4 show trichostatin A (0.1 μg/ml, 1 μg/ml)-treated cells, and
Lanes 5 and 6: cells treated with radiation (50 J and 100 J per square meter), lanes 7 and 8: cells treated with vitamin D3 (10 nM and 100 nM), lane 9
10: Vincristine (1 μg/ml, 10 μg/ml) treated cells, lane 11
12: 20 μg of RNA from cells treated with wortmannin (0.1 μM, 1 μM) was blotted. A band reacting with the p51 probe was observed at the position indicated by the arrow.
Twenty-four hours after transfection with 2 μg of either pGL2-neo or pGL2-neo vector, 0 or 1 μg/ml trichostatin A (TsA) was added and the cells were cultured for another 24 hours. The promoter activity of the isolated gene fragment was examined by measuring the luciferase activity in these cells. Cells transfected with 2 μg of pcDNA3/lacZ were used as negative control cells. As a result, pGL2-neo vector was
It was found that the extract from cells transfected with eo/p51 promoter showed significantly higher fluorescence intensity than the extract from cells transfected with pGL2-neo.
The chemiluminescence intensity in the extract of pGL2-neo/p51 promoter-transfected cells was further enhanced by administration of

─────────────────────────────────────────────────────── Continued from the front page (51) Int.Cl. ⁷ Identification Symbol FI C12N 5/10 C12Q 1/68 A C12Q 1/68 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 C12N 5/00 A (72) Inventor Fujii Shuji 3-48-21-1201 Honmachi, Shibuya-ku, Tokyo (Note) This publication is based on the gazette published internationally by the International Bureau of Patents (WIPO). The effect of the international publication of the Japanese patent application (Japanese utility model registration application) related to this publication arises pursuant to Article 184-10, Paragraph 1 of the Patent Act (Article 48-13, Paragraph 2 of the Utility Model Act), and is unrelated to this publication.

Claims (11)

[Claims] [Claim 1] A gene encoding a p51 promoter region shown in (1), (2), (3), (4), (5), or (6) below: (1) DNA encoding a p51 promoter region having the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing; (2) DNA having a nucleotide sequence in which one or more bases have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing, and having p51 promoter activity; (3) DNA that hybridizes under stringent conditions with DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing, and has p51 promoter activity; (4) DNA encoding a p51 promoter region and a 5-prime terminal untranslated region of p51, having the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing; (5) DNA having a nucleotide sequence in which one or more bases have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing, and having p51 promoter activity; (6) A DNA which hybridizes under stringent conditions with a DNA consisting of the base sequence shown in SEQ ID NO: 2 in the sequence listing and has p51 promoter activity. Claim 2: A gene encoding the 5-prime terminal untranslated region of p51 shown in (7), (8) or (9) below: (7) The base sequence from 5677th to 596th in the base sequence shown in SEQ ID NO: 2 in the Sequence Listing
(8) DNA having a base sequence of base 0; (9) DNA having a base sequence of bases 5677 to 596 in the base sequence shown in SEQ ID NO: 2 in the sequence listing;
(9) A DNA having a base sequence in which one or more bases are deleted, substituted or added in the base sequence at base 0, and having the same function as the DNA of (7) above; (9) A DNA having a base sequence from base 5677 to base 596 in the base sequence shown in SEQ ID NO: 2 in the Sequence Listing;
A DNA that hybridizes under stringent conditions with a DNA consisting of the 0th base sequence and has the same function as the DNA of (7) above. 3. A recombinant plasmid comprising the gene of claim 1. 4. A transformant or transductant containing the recombinant plasmid of claim 3. 5. A nucleic acid probe consisting of all or part of the gene of claim 1 or 2. 6. A method for cloning the p51 promoter region or a gene analogous thereto, which comprises using the nucleic acid probe of claim 5. 7. A DNA sequence which is antisense to the whole or part of the gene of claim 1 or 2 and which increases or inhibits the expression of the p51 gene. 8. An RNA sequence which is antisense to the whole or part of the gene of claim 1 or 2 and which increases or inhibits the expression of the p51 gene. 9. A method for screening for a drug acting on the p51 promoter, using the transformant or transducant of claim 4. 10. A compound that increases or inhibits the expression of the p51 gene, selected by the screening method of claim 9. 11. A pharmaceutical preparation comprising the DNA sequence of claim 1, the DNA sequence of claim 7, or the RNA sequence of claim 8.