[go: up one dir, main page]

WO2003035871A1 - Adn promoteur - Google Patents

Adn promoteur Download PDF

Info

Publication number
WO2003035871A1
WO2003035871A1 PCT/JP2002/010882 JP0210882W WO03035871A1 WO 2003035871 A1 WO2003035871 A1 WO 2003035871A1 JP 0210882 W JP0210882 W JP 0210882W WO 03035871 A1 WO03035871 A1 WO 03035871A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
cell
protein
activity
nucleotide sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2002/010882
Other languages
English (en)
Japanese (ja)
Inventor
Takafumi Kohama
Masako Sugiura
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sankyo Co Ltd
Original Assignee
Sankyo Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sankyo Co Ltd filed Critical Sankyo Co Ltd
Publication of WO2003035871A1 publication Critical patent/WO2003035871A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel DNA having one promoter activity, which is useful for searching for a medicament for treating or preventing arteriosclerosis, diabetes, thrombosis, inflammation, immune abnormality, allergy, cancer and metastasis of cancer.
  • sphingolipids have been considered one of the major components of cell membranes alongside glycemic phospholipids and cholesterol. It has been known that glycerol phospholipids not only maintain cell membrane structure, but also produce many bioactive substances as metabolites. However, little has been known about the biological activity of metabolites of sphingolipids until recently. In recent years, various metabolites of sphingolipids such as ceramide have been known to have physiological activities such as inducing apoptosis and stimulating cell proliferation, and metabolic enzymes of sphingolipids may cause physiological reactions and various pathological conditions. The possibility of a close association has been suggested (see Hannun, YA and Obeid, LM (1995) TIBS 20, 73-77).
  • the metabolic enzymes of sphingolipids include sphingomyelin, which is a major component of cell membranes, and those that act on its biosynthesis and degradation, and those that act on the synthesis and degradation of glycolipids such as gandarioside.
  • Various enzymes are known.
  • the sphingosine kinase is an enzyme that acts on the decomposition system of sphingolipids, and is an enzyme that phosphorylates sphingosine and generates sphingosine 1-phosphate.
  • Many biological activities have recently been reported for sphingosine and sphingosine 1-phosphate, which are substrates and products of this enzyme.
  • sphingosine a substrate, inhibits protein kinase C (hereinafter referred to as “PKC”), and it is apparent that the action of phorpol ester, an activator of PKC, is counteracted in many cell lines. It has become. For example, platelet aggregation, neutrophil activation, and leukemic cell differentiation induced by phorpol ester are suppressed by sphingosine (Hannun, YA and Cori, ML, (1993) Bi atmi ca ei Biophys ica Acta 1154, 223-236).
  • PKC protein kinase C
  • sphingosin acts on various other signal transduction pathways (see Hannun, YA and Corinne, ML (1993) Biochimi ca et Biophysica Acta 1154, 223-23 ⁇ 6). These signaling pathways are closely related to cancer progression, immunity, inflammation, lipid sugar metabolism, blood coagulation, and other reactions. And is known.
  • sphingosine kinase is used for various factors such as platelet-derived growth factor (hereinafter referred to as “PDGF”), nerve growth factor, vitamin D3, epidermal growth factor, and tumor necrosis factor (hereinafter “TNF- ⁇ ”). It is known to be activated by stimuli, and various physiological actions of sphingosine 11-phosphate, its product, are being revealed.
  • PDGF platelet-derived growth factor
  • nerve growth factor nerve growth factor
  • vitamin D3, epidermal growth factor epidermal growth factor
  • TNF- ⁇ tumor necrosis factor
  • PDGF is thought to play an important role in the development of vascular lesions by stimulating the proliferation and migration of vascular smooth muscle cells (Ross R. et al (1990) Science 248, 1009-1012
  • sphingosine-1-phosphate acts as a second messenger through activation of sphingosine kinase in the mechanism of action (Olivera, A. and Spiegel, S. (1993) Nature 365) , 557-560 and Spiegel, S. et al, (1994) Breast Cancer Research and Treatment 31, 337-348). Therefore, blocking the signal would be useful for preventing or treating arteriosclerosis in hyperlipidemia, diabetic vascular disorders, restenosis after percutaneous coronary angioplasty (PTCA), and so on.
  • PTCA percutaneous coronary angioplasty
  • Inflammatory cytokins such as TNF— express E-selectin, an adhesion factor, and vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells, and accumulate neutrophils and other blood cells at the site of inflammation. It is thought to play an important role in the development of inflammation. The mechanism of action is also thought to occur through activation of sphingosine kinase (see Xia, P. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 14196-14201). Therefore, if sphingosine kinase is inhibited and the expression of the adhesion factor is suppressed, it is thought that the anti-inflammatory effect is achieved.
  • sphingosine kinase is involved in the signaling of immunoglobulin E (IgE) in mast cells. Specifically, an increase in intracellular calcium ion concentration ([Ca 2+ ]) via the receptor Yuichi (FcsRI) against the Fc fragment of IgE on mast cells, It has been reported that Ridge is deeply involved (see Choi, 0. H., et al. (1996) Nature 380, 634-636). Therefore, inhibitors of sphingosine kinase are expected to have antiallergic activity.
  • sphingosine and sphingosine 1-phosphate act on proliferation and migration of various cancer cells (Spiegel, S. et al, (1994) Breast Cancer Research and Treatment 31, 337-348). The results vary depending on the type and condition of the cancer cells. Depending on the type of cancer, regulating the amount of sphingosine-1-phosphate may be effective in preventing cancer and preventing metastasis.
  • sphingosine monophosphate include activation of phospholipase D and promotion of calcium ion release from the intracellular calcium ion pool (Spiegel, S. et al, (1994) Breast Cancer Research and Treatment 31, 337-348), the regulation of migration of vascular smooth muscle cells (see Bornieldt, KE eal, (1995) The Journal of Cell Biology 130, 193-206), and the effect of increasing the rheumatoid ion potential in cardiomyocytes (Bunemann, M. et al. (1996) The EMBO Journal 15, 5527-5534).
  • ISP-1 a microbial inhibitor of serine palmi toyl transferase
  • Sphingocin also has a down-regulating effect on serine palmitoyltransferase (see Hannun, YA and Corinne, ML (1993) Biochimica et Biophysica Acta 1154, 223-236).
  • Apoptosis-inducing effect is restored by sphingosine or sphingosine-1-phosphate (Miyake, Y. et al. (1995) Biochemical and
  • sphingosine and sphingosine monophosphate have various physiological effects, and are useful pharmacology by regulating sphingosine monophosphate production at the cellular level in many disease states. An effect is expected to be obtained.
  • an apron that regulates the production of sphingosine 1-phosphate by suppressing or promoting the expression itself of the sphingosine kinase 1 gene.
  • mouse sphingosine kinase 1 has been cloned (Kohama, T., et al.
  • An object of the present invention is to specify a promoter region involved in the transcriptional control of sphingosine kinase 1 gene and to provide a method for screening a compound that regulates the activity of the promoter. More specifically, anti-atherosclerotic agents, anti-diabetic agents, anti-thrombotic agents, anti-inflammatory agents, immunosuppressants, anticancer agents, cancer metastasis inhibitors, antiallergic agents, post-percutaneous coronary angioplasty (PTCA) It is an object of the present invention to provide a novel method for searching for a candidate substance for a preventive or therapeutic agent for restenosis and the like, and a DNA used in the method.
  • the present invention is to specify a promoter region involved in the transcriptional control of sphingosine kinase 1 gene and to provide a method for screening a compound that regulates the activity of the promoter. More specifically, anti-atherosclerotic agents, anti-diabetic agents, anti-thrombotic
  • DNA represented by any one of the following 1) to 3):
  • nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing a DNA comprising a nucleotide sequence having a nucleotide represented by nucleotide number 1 as a 5 ′ end and a nucleotide represented by nucleotide number 2016 as a 3 ′ end;
  • E. coli pGV-h spk 1 SANK 73101 (FERM BP-7780), a recombinant plasmid carried by the DNA consisting of 2016 bp linked to the 5 'end of the nucleotide sequence encoding firefly luciferase;
  • the protein is selected from the group consisting of luciferase, horseradish peroxidase, alkaline phosphatase, ⁇ 8-galactosidase and green fluorescent protein;
  • a method for screening a substance having an action of regulating a sphingosine kinase gene promoter activity comprising the following steps i) to iii):
  • iii) comparing the results of the above ii) between cells cultured in the presence of the test substance and cells cultured in the absence of the test substance, (17) One or more selected from the group consisting of arteriosclerosis, diabetes, thrombosis, inflammation, immune abnormalities, arregii, cancer and cancer metastasis, including the following steps i) to iii) Method for screening for a substance having a therapeutic or preventive effect on a disease:
  • Cell B a cell transformed with the vector according to any one of (6) to (9) and not expressing the protein encoded by the test cDNA;
  • a cell ⁇ a cell transformed with the vector according to any one of claims 6 to 9, wherein the cell does not express the protein encoded by the test cDN ⁇ ;
  • a method for separating a substance that regulates the activity of a sphingosine kinase 1 gene promoter comprising: contacting the DNA containing the substance with the DNA according to any one of (1) to (4); Next, purifying the substance bound to the DNA,
  • a sample containing a human cell nuclear extract is brought into contact with the DNA according to any one of (1) to (4), and then the protein bound to the DNA is separated.
  • a nucleic acid or a derivative thereof comprising a nucleotide having the characteristics represented by any one of the following a) to b) and having an activity of regulating the activity of the sphingosine kinase 1 gene promoter: a) SEQ ID NO: Consisting of at least 10 contiguous nucleotides in the nucleotide sequence represented by nucleotide numbers 1 to 2016 of 1;
  • the present inventors set the site predicted to be the transcription start site of the sphingosine kinase 1 gene in the human genome (nucleotide number 2026 of SEQ ID NO: 1 in the sequence listing) as “1” and set it to 2025 nucleotides upstream of its 5 ′ end.
  • ⁇ -2025 to 1-10 To the 10-nucleotide site upstream of the 5'-end (hereinafter referred to as ⁇ -2025 to 1-10.)
  • the DNA consisting of the nucleotide sequence of nucleotides 1 to 2016 in SEQ ID NO: 1 in the sequence listing
  • a reporter gene linked to DNA encoding luciferase was introduced into human HEK293 cells, and the amount of luciferase produced was measured.
  • the DNA of the present invention may contain any nucleotide sequence in the 5'-terminal upstream region of the human sphingosine kinase 1 gene as long as it has promoter activity in mammalian cells.
  • Preferable examples include a DNA consisting of the nucleotide sequences shown in nucleotide numbers 1 to 2016 of SEQ ID NO: 1 in the Sequence Listing, but the present invention is not limited thereto, and the nucleotide sequence of SEQ ID NO: 1 in the Sequence Listing is preferably used. And at least a part of the nucleotide sequence represented by C.
  • the DNA of the present invention is obtained, for example, from the gene library prepared from the human genome, for example, by the nucleotide sequence of cDNA encoding human sphingosine kinase 1 (Nava, VE, et al. (2000) FEBS 473, 81-84) can be isolated by screening using the 5 'terminal side as a probe. Alternatively, based on this sequence information or the sequence information published by the Human Genome Project, the library is screened by the polymerase chain reaction (hereinafter referred to as “PCR”) using human genomic DNA as type II. Alternatively, the DNA of the present invention can be directly amplified.
  • PCR polymerase chain reaction
  • the transformed Escherichia coli strain E.co1ipGV-hspk1pSANK73101 carrying the recombinant plasmid into which the DNA of the present invention has been inserted was established as an independent administrative authority on October 19, 2001 (Heisei 13).
  • the National Institute of Advanced Industrial Science and Technology (AIST) is deposited internationally at the Patent Organism Depositary Center (1-1-1, Higashi, Tsukuba, Ibaraki, Japan) (Central No. 6 (Zip code: 305-8566)) with accession number FERM BP-7780 Have been. Therefore, the DNA of the present invention can be obtained from the plasmid.
  • Modification of the nucleotide sequence includes, for example, introduction of a deletion using a restriction enzyme or DNA exonuclease, introduction of a mutation using a site-directed mutagenesis method, modification of a promoter sequence by a PCR method using a mutant primer, synthesis of a synthetic variant DN. It can be carried out by a method such as direct introduction of A. Preferred among these are DNAs containing the nucleotide sequences shown in nucleotide numbers 1 to 2016 of SEQ ID NO: 1 in the sequence listing.
  • the DNA of the present invention includes a nucleotide sequence that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence represented by nucleotide numbers 1 to 2016 in SEQ ID NO: 1 in the sequence listing. And DNA having a promoter activity.
  • a DNA usually has 70% or more, preferably 80% or more, more preferably 95% or more nucleotide sequence identity with the nucleotide sequence shown by nucleotide numbers 1 to 2016 of SEQ ID NO: 1 in the sequence listing.
  • Such DNAs include mutant genes found in nature, artificially modified mutant genes, and homologous genes derived from heterologous organisms.
  • the hybridization under stringent conditions is such that under the conditions having normal stringency (roast stringent conditions), eight hybridizations are performed under 5 XSSC (0.75 M sodium chloride, 0 075M sodium citrate) or an equivalent salt
  • a hybridization solution at a concentration of 37-42 ° C for about 12 hours pre-wash as needed with 5 XSSC or a solution with a salt concentration equivalent to this, This can be done by washing in 1 XSSC or a solution with a salt concentration equivalent to this.
  • the washing can be carried out in the above by performing the washing in 0.1 XSSC or a solution having a salt concentration equivalent thereto.
  • Whether or not the thus obtained DNA of the present invention has a promoter activity is determined by ligating a marker gene such as luciferase downstream thereof (hereinafter referred to as “repo-protein”) as described below. This can be confirmed by transforming a mammalian cell with the “Yuichi gene” and examining whether or not expression of the marker-1 gene in the transformed cell is detected.
  • a marker gene such as luciferase downstream thereof
  • a vector or the like having the DNA of the present invention inserted upstream of a desired gene is prepared and introduced into somatic cells. By adding stimulus, the desired gene can be inducibly expressed.
  • the present invention relates to a DNA comprising at least a part of the promoter-DNA sequence (including derivatives thereof; hereinafter, referred to as "partial sequence").
  • partial sequence By using such a partial sequence, it is possible to antagonize the binding between the promoter DNA and a protein (for example, a transcription factor) capable of binding to the promoter DNA regardless of whether the partial sequence itself has the promoter activity. It is possible to carry out the method of inhibition.
  • the partial sequence when the partial sequence corresponds to the binding site of a protein that inhibits its promoter activity on the DNA sequence of the present invention, this method can promote the promoter overnight activity, and conversely, Promoter activity can be inhibited if it corresponds to the binding site of a protein (including a transcription factor) that promotes promoter activity.
  • the partial sequence DNA used for such competitive inhibition usually has a chain length of at least 6 nucleotides, preferably at least 10 nucleotides. Examples of the type of the partial sequence selected for use in competitive inhibition include, for example, a sequence containing a binding consensus site of a transcription factor on a promoter.
  • Derivatives of such partial sequence DNAs include those in which several partial sequences are linked, those that are incorporated into an adenovirus vector or the like to enable gene transfer into animal cells, and those that contain DNA. Those in which a biotin is linked to the 3 ′ end or the 5 ′ end can be used. Also, nucleotides containing an antisense sequence of such a partial sequence, which have an activity of regulating the activity of the promoter, are included in the present invention. 3) Screening for proteins that regulate promoter activity
  • a method using a protein that regulates the promoter activity of the DNA of the present invention may be used. it can.
  • the DNA of the present invention can be used for screening a protein that regulates its promoter activity.
  • the present invention relates to a method for screening a protein that regulates the promoter activity of the DNA as described below.
  • proteins include those that directly bind to the DNA of the present invention to promote or inhibit its promoter activity, and those that act on cell membrane receptors and intracellular proteins to indirectly bind to the DNA of the present invention. Includes proteins that promote or inhibit promoter one activity.
  • This method includes a step of bringing a test protein sample into contact with the DNA of the present invention and selecting a protein that binds to the DNA of the present invention.
  • a method includes, for example, affinity purification of a protein binding thereto using the promoter-DNA of the present invention.
  • the promoter DNA of the present invention is biotinylated and bound to streptavidin-bound magnetic beads to prepare DNA affinity peas. Next, this is incubated with the nuclear extract of the cell, and the protein in the nuclear extract that specifically binds to the DNA of the present invention is purified, and its structure is determined.
  • the nuclear extract can be prepared by a conventional method using cultured cells or animal tissues.
  • an animal tissue is excised, cut into small pieces using a scalpel or scissors, suspended in a 0.25 M sucrose solution 5 to 10 times the weight of the tissue, and treated with a protease inhibitor (eg, 2 to 10 times). g / ml of leptin or capsule) and pulverize with a Potter-Elvehgem homogenizer. Dilute the obtained homogenate 10-fold with 0.25M sucrose solution, centrifuge (600 xg, 10 minutes), and collect the precipitate.
  • a nuclear extract can be prepared by suspending the obtained precipitate in a small amount of 0.25M sucrose solution and adding a 10-fold amount of 1 OmM Tris-HC1 thereto, but is not limited to this method. .
  • a protein that directly binds to the DNA of the present invention and a protein that does not have DNA binding activity but forms a complex with the protein as a subunit and binds to the DNA of the present invention can be purified (Gabrielsen 0.S. et al., Nucleic acid Research 17, 6253-6267 (1989), Savoysky E et al., Oncogene 9, 1839-1846 (1994)).
  • a method is not limited to screening for proteins, and can be used for screening for a substance that binds to the DNA of the present invention.
  • This method comprises introducing a test DNA into cells carrying a marker gene linked downstream of the DNA of the present invention [described in detail in 5-2) below, and controlling expression of the marker gene. Including the step of selecting the product.
  • Such methods include, for example, the one-hybrid method using yeast or animal cells. Specifically, a reporter gene containing the DNA of the present invention and a marker gene was stably introduced into cells, and then a gene library was introduced into the cell to promote or enhance the expression of the repo overnight gene. A clone showing inhibition is selected, and a protein that binds to the DNA of the present invention is selected.
  • a protein that acts on an endogenous protein in a cell and indirectly regulates the promoter activity of the DNA of the present invention can be obtained.
  • the yeast one-hybrid method Li JJ and Hersktiwitz L'Science 262, 1870-1873 (1993), Wang MM and Reed R, R., Nature 364, 121-126 (1993)), etc. "Matchmaker system (Clontech)" and others are sold as kits.
  • a step of contacting a test sample with a cell holding a chimera gene linked downstream of the DNA of the present invention and selecting a protein that regulates the expression of the chimera gene including.
  • a cell into which a DNA and a marker gene of the present invention have been stably introduced and a test sample may be used. Incubate with the culture supernatant) and select a protein that shows promotion or inhibition of marker-1 gene expression.
  • a protein which indirectly affects the promoter activity of the DNA of the present invention via a cell membrane receptor or the like is obtained.
  • the present invention also relates to a method for screening for a cDNA encoding a protein that regulates the promoter activity of the DNA of the present invention.
  • This screening method includes a step of contacting the expression product of a test DNA with the DNA of the present invention and selecting cDNA encoding a protein that binds to the DNA of the present invention. Such methods include, for example, the Southwestern method.
  • each protein is expressed in Escherichia coli into which the gene library has been introduced, and these proteins are transferred to a filter membrane. Then, the DNA of the present invention is directly plotted as a probe, and the DNA is bound to the DNA probe.
  • a gene encoding a protein having the activity of binding to the DNA of the present invention can be obtained by this method (Experimental Medicine Separate Volume Bio Manual Series) “Transcription Factor Research Method”, published by Yodosha Co., Ltd., pp.177-188).
  • the test cDNA is introduced into cells containing a marker gene linked downstream of the DNA of the present invention, and a cDNA encoding an expression product that regulates the expression of a marker gene is selected.
  • a cDNA encoding an expression product that regulates the expression of a marker gene is selected.
  • Such methods include, for example, the above-described one-hybrid method using yeast or animal cells. That is, a reporter gene into which a DNA of the present invention and a human gene have been introduced is stably introduced into cultured cells derived from mammals (hereinafter referred to as “cell A”). Culture the cells (hereinafter referred to as “cell B”) into which the recombinant plasmid containing the test cDNA has been inserted into a cell expression vector.
  • cell A is cultured as it is, or an expression vector for mammalian cells that does not contain the test cDNA in cell A, or a recombinant plasmid that does not express the test cDNA even if it contains the test cDNA.
  • the cells into which the cells have been introduced (hereinafter referred to as “cell C”) are cultured. Compare the marker gene expression levels between cell B and cell A or cell C, and select a cDNA clone that promotes or inhibits the expression of the marker gene.
  • cDNA which regulates the protein can also be obtained.
  • the expression product of the test cDNA is brought into contact with cells carrying the chimera gene linked downstream of the DNA of the present invention to regulate the expression of the chimera gene Selecting a cDNA encoding the expression product.
  • An example of a specific method is as follows. A cell into which a reporter gene into which the DNA of the present invention and a marker gene have been inserted is stably introduced, and an expression product of a test cDNA (for example, a cell into which a cDNA library is introduced). To isolate a protein or a cDNA encoding the protein that promotes or inhibits expression of the marker gene. By this method, it is possible to obtain cDNA which encodes a protein which acts on the promoter activity of the DNA of the present invention indirectly via a cell membrane receptor or the like.
  • the present invention relates to a method for screening a compound that regulates the promoter activity of the DNA as described below.
  • a DNA of the present invention is brought into contact with a test sample in the presence of a test compound, and a compound that promotes or inhibits the binding of the DNA of the present invention to a protein in the test sample is selected.
  • a nuclear extract of a cell is bound to a probe obtained by labeling the DNA of the present invention with a radioisotope or the like, and the proteins in the nuclear extract and the DNA of the present invention are subjected to polyacrylamide gel electrophoresis.
  • Compound of The body pan is detected by the gel shift method (Experimental Medicine Separate Volume, Bio Manual Series, “Transcription Factor Research Method”, published by Yodosha Co., Ltd., pages 107-112).
  • a compound that inhibits the binding of the protein to the DNA of the present invention is as follows: It is believed that the promoter activity of the DNA of the invention can be promoted.
  • the protein that binds to the DNA of the present invention has already been isolated, it is possible to use a recombinant protein of the protein instead of the cell nuclear extract.
  • a test compound is brought into contact with a cell holding a marker gene linked to the 3 ′ end of the DNA of the present invention, and a compound that regulates the expression of the marker gene is selected. Process. In this way, compounds that directly or indirectly modulate the promoter activity of the DNA of the present invention are obtained.
  • the chimeric protein encoded by the chimeric gene linked to the 3 ′ end of the DNA of the present invention is produced by the host cell in a series of steps of the method of the present invention. If it can be distinguished from any other possible proteins (preferably, the cells before transformation do not have a gene encoding the same or similar protein as the marker protein). Good. For example, even when the marker protein is toxic to the cell or confers resistance to a susceptible antibiotic, the presence or absence of the marker gene is determined by the cell. It can be determined by the survival rate. However, a more preferable marker gene used in the present invention can specifically and quantitatively detect the expression level (for example, when a specific antibody against the protein encoded by the marker gene is obtained). It is a structural gene.
  • genes encoding proteins such as, but not limited to, the following:
  • Chloramphenicol acetyl transferase Adds an acetyl group to chloramphenicol. Detectable by so-called CAT Atsushi etc.
  • PCAT3 Basic vector (Promega) is a vector that can be used to prepare a reporter at vector simply by incorporating a promoter. Commercially available;
  • Phosphorus luciferase Quantified by measuring the bioluminescence generated when luciferin is metabolized.
  • PGB-B2 Vector-1 manufactured by Toyo Ink Manufacturing Co., Ltd.
  • PGB-B2 Vector-1 is also commercially available as a vector for the repo one at a time;
  • 3-Galactosidase There are substrates that can be measured by color reaction, fluorescence or chemiluminescence, respectively.
  • Repo - data - p ⁇ ga (Promega) 1 -B asic as a vector for Atsusi is that are commercially available;
  • Secreted alfa phosphatase There are substances that can be measured by color reaction, bioluminescence, or chemiluminescence, respectively.
  • PSEAP2-Basic (Clontech) is commercially available as a vector for reporter access;
  • Green-fluorescent protein Although it is not an enzyme, it fluoresces itself and can be directly quantified. Similarly, pEGFP-1 (manufactured by Clontech) is commercially available as a vector for reporter attestation.
  • Methods for introducing expression plasmids into cultured cell lines include the DEAE-dextran method (Luthman, H. and Magnus son, G. (1983) Nucleic Acids Res. 11, 1295-1308), calcium phosphate-DNA coprecipitation method ( Graham, FL and van der Eb, AJ (1973) Virology 52, 456-457), electric pulse drilling
  • the transcription of the marker gene is promoted.
  • the culture was performed under conditions with and without the addition of any test substance in the medium, and then the expression level of the marker gene was measured. Then, it is examined whether or not the expression level of the marker gene is changed by the addition of the test substance.
  • the “conditions under which the marker gene can be expressed” may be any conditions under which the cells can survive and produce proteins.
  • the medium is suitable for the cell line used (such as fetal serum or the like). Serum components may be added) in the presence of air containing 4 to 6% (optimally 5%) carbon dioxide at 36 to 38 (most preferably at 37 ° C) 3 days (optimally Incubate for 2 days).
  • test substance that suppresses the induction of reporter gene expression when an expression vector having a marker gene linked to the 3 ′ end of the DNA of the present invention is introduced, It is selected as a candidate substance useful as a therapeutic or prophylactic agent for a disease selected from the group consisting of arteriosclerosis, diabetes, thrombus, inflammation, immune abnormality, allergy, cancer and cancer metastasis.
  • the protein of the present invention that regulates the promoter activity of DNA has already been obtained, the protein (or a derivative thereof) is brought into contact with the DNA of the present invention in the presence of a test compound, and By selecting a compound that promotes or inhibits the binding between the protein (or a derivative thereof) and the DNA of the present invention, it is possible to screen for a compound that regulates the promoter activity of the DNA of the present invention.
  • the protein (or only the DNA binding domain) that binds to the DNA of the present invention fused with daltathione S-transferase is purified, and the anti-glutathione S-transferase is purified.
  • the DNA of the present invention After binding to the microplate covered with the antibody, the DNA of the present invention, which has been lyophilized, is brought into contact with this protein, and the binding between the protein and the DNA of the present invention is examined using streptavidinated alkaline phosphatase. To detect.
  • a test compound is also added, and a compound that promotes or inhibits the binding between the protein and the DNA of the present invention is selected. In this way, compounds that act directly on the DNA of the present invention and compounds that act on proteins that bind to the present invention are obtained.
  • a protein that binds to the DNA of the present invention inhibits the promoter activity of the DNA of the present invention in vivo
  • a compound that inhibits the binding between the protein and the DA of the present invention is It is considered that the promoter activity of the DNA of the present invention can be promoted.
  • DNA containing the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing was selected as the target promoter DNA.
  • the transcription start site of the gene encoding human sphingosine kinase 1 was determined with reference to the sequence of human sphingosine kinase 1 (XM036215) registered on the GenBank Data Base. did. That is, in the nucleotide sequence of XM036215, the first nucleotide excluding a part of the vector was determined to be the transcription start site, and the position was defined as “1”.
  • Example 2 On the basis of the results of Example 1, a reporter plasmid having a cDNA in the 5'-terminal upstream region of the gene encoding human sphingosine kinase 1 was constructed.
  • the following oligonucleotide primers in order to specifically amplify the 5,5 terminal upstream region of the gene encoding human sphingosine kinase 1, the following oligonucleotide primers:
  • the first PCR reaction was performed using commercially available human genomic DNA as type II and LA PCR Kit 'version 2.1 (Takara Shuzo Co., Ltd.). That is, human genomic DNA (manufactured by Clontech) 51, primers SKlp- ⁇ and SKlp-2 0.4 M each, and dATP, dGTP, dCTP, dTTP 400 / M each, including 2.5 mM magnesium chloride 1
  • a 501 reaction mixture consisting of XLA PCR GC I buffer and 0.05 units of LA Taq DNA polymerase (supplied with the kit) was prepared.
  • the PCR product was purified using a micro piospin column 30 chromatography Ichiram (manufactured by Bio-Irad), and the T "A cloning method (Clark, JM et al. (1988) Nucleic Acid Res. 16: 9677-9686) and ligated into pCR2.1 vector-1 (attached to the original TA Cloning Kit (Invitrogen)) and introduced into E. coli I NVaF 'strain (attached to the kit).
  • the entire nucleotide sequence of the inserted cDNA was analyzed by the dideoxy nucleotide chain termination method, and the plasmid into which the DNA having the nucleotide sequence shown in nucleotide numbers 1 to 20 of SEQ ID NO: 1 in the sequence listing was inserted into pCR2. 1-Named hspklp.
  • This plasmid PCR2.1-hspk1p was digested with restriction enzymes EcoRV and HindIII to isolate a fragment of about 2.0 kbp containing the inserted cDNA.
  • luciferase expression vector pGV-B2 (manufactured by Toyo Ink Manufacturing Co., Ltd.) was digested with restriction enzymes SmaI and HindIII and dephosphorylated with alkaline phosphatase.
  • the 2.0 kb p fragment pGV-B2 vector was ligated by a reaction using T4DN A ligase (Takara Shuzo Co., Ltd.) and introduced into E. coli JMl09. Plasmid DNA pGV-hspk1p was extracted from the obtained transformant, and the entire nucleotide sequence of inserted cDNA was confirmed by dideoxynucleotide chain termination.
  • the transformed E. coli pGV-hspk1 SANK731101 harboring the luciferase expression vector pGV—hspk1p was obtained on October 19, 2001 (Heisei 13). It was internationally deposited at the National Institute of Advanced Industrial Science and Technology ⁇ Patent Organism Depositary, and was assigned the accession number FE RM BP-7780.
  • the luciferase expression vector pGV-B2 (manufactured by Toyo Ink Manufacturing Co., Ltd.) DNA is used to transform E. coli J Ml09 competent cells (manufactured by Takara Shuzo Co., Ltd.), and the ampicillin-resistant colonies are cultured. DNA was recovered and purified. This plasmid DNA was used as a negative control in the examples below.
  • Example 3 Promoter activity
  • plasmid for expression of the plasmid for the expression of plasmid L-luciferase (pGV-hspk 1p) was transfected into a human fetal kidney-derived cell line HEK293, and the change in the luciferase activity was examined.
  • ⁇ luciferase expression plasmid pRL-TK (manufactured by Toyo Ink Mfg. Co., Ltd.) was used.
  • HEK293 human embryonic kidney cells HEK293 (American Type Culture Collection CRL-1573) are seeded on a polylysine-coated 12 ⁇ L plate for tissue culture (Iwaki Glass) for 24 hours. After culturing, a mixture of 0.63 g of pGV-B2 or pGV-hspk 1 p and 0.07 ng of pRL-TK per well is added to the transfection reagent. (Lipofectamine 'plus, manufactured by Gibco BIRL) was used for transfection according to the attached protocol.
  • the medium was removed, and the cells were lysed in 250 ⁇ 1 / 1L cell lysing agent (PLD-30 (manufactured by Toyo Ink Mfg. Co., Ltd.)).
  • a cell extract was prepared.
  • the firefly and the firefly and the firefly were prepared by using the dual-luciferase-repo-one-night-atsushi 'system (manufactured by Toyo Ink Mfg. Co., Ltd.) according to the attached protocol.
  • the Mycobacterium luciferase activity was measured (Luminas CT-9000D (Diatron) was used as a measuring device). The experimental results were expressed as the activity of Luciferase activity corrected for the activity of the internal standard.
  • ⁇ 293 cells transfected with pGV-hspk 1 ⁇ showed about 7-fold higher luciferase activity than cells transfected with pGV-2, indicating that they were incorporated into pGV-hspk 1 p.
  • Promote activity must be present in the DNA shown in nucleotide numbers 1 to 2016 of SEQ ID NO: 1 in the sequence listing, i.e., in the region corresponding to 2025 to 110 of the 5'-end upstream of human sphingosine kinase 1 gene. Became clear.
  • the promoter activity of the DNA of the present invention was measured by measuring the luciferase activity when, for example, HEK293 cells transformed with pGV-hspk1p were cultured in the presence of a test substance. Testing for inhibitors can be performed.
  • a substance that inhibits this promoter overnight activity can be a therapeutic or prophylactic agent for a disease selected from the group consisting of arteriosclerosis, diabetes, thrombosis, inflammation, immune disorders, allergy, cancer and cancer metastasis.
  • the present invention provides a novel DNA having one promoter activity.
  • D of the present invention NA can be used in methods to test substances that suppress sphingosine kinase 1 expression, so it has a new mechanism of action for arteriosclerosis, diabetes, thrombosis, inflammation, immune disorders, allergy, cancer and cancer. It is useful for developing an agent for treating or preventing a disease selected from the group consisting of metastases.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un nouvel ADN présentant une activité de promoteur. Cet ADN peut être utilisé dans un procédé permettant de tester les produits qui régulent le niveau d'expression de la sphingosine kinase 1. Il est par conséquent utile pour la mise au point de médicaments curatifs et préventifs présentant un nouveau mode d'action pour les maladies du groupe suivant : artériosclérose, diabète, thrombose, inflammation, immunopathies, allergies, cancers, et métastases cancéreuses.
PCT/JP2002/010882 2001-10-23 2002-10-21 Adn promoteur Ceased WO2003035871A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001/325402 2001-10-23
JP2001325402 2001-10-23

Publications (1)

Publication Number Publication Date
WO2003035871A1 true WO2003035871A1 (fr) 2003-05-01

Family

ID=19141969

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/010882 Ceased WO2003035871A1 (fr) 2001-10-23 2002-10-21 Adn promoteur

Country Status (1)

Country Link
WO (1) WO2003035871A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1845971A4 (fr) * 2005-01-21 2009-12-30 Medvet Science Pty Ltd Procede de traitement d'un dommage cellulaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAVA V.E. ET AL.: "Functional characterization of human sphingosine kinase-1", FEBS LETTERS, vol. 473, 2000, pages 81 - 84, XP004337110 *
WILLIAMS T.M. ET AL.: "Advantages of firefly luciferase as a reporter gene: application to the interleukin-2 gene promoter", ANAL. BIOCHEM., vol. 176, 1989, pages 28 - 32, XP000601607 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1845971A4 (fr) * 2005-01-21 2009-12-30 Medvet Science Pty Ltd Procede de traitement d'un dommage cellulaire

Similar Documents

Publication Publication Date Title
CA2665489C (fr) Compositions associees a prrg4 et procedes d'utilisation de celles-ci dans des procedes de diagnostic de tumeur
Giudici et al. A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma
JP2007525956A (ja) 新規β−アクチンおよびＲＰＳ２１プロモーター、ならびにこれらの使用方法
Springhorn et al. Posttranscriptional regulation of Id1 activity in cardiac muscle. Alternative splicing of novel Id1 transcript permits homodimerization.
KR20010022741A (ko) 신규 노화-인자 유전자, ｐ23의 단리
Dehbi et al. Transcriptional activation of the CEF-4/9E3 cytokine gene by pp60v-src
US20020193289A1 (en) Methods and compositions for modulating telomerase reverse transcriptase (TERT) expression
WO2002072787A2 (fr) Répresseurs d'expression de la télomérase et leurs procédés d'utilisation
WO2003035871A1 (fr) Adn promoteur
Wu et al. Molecular characterization of the constitutive expression of the plasma platelet-activating factor acetylhydrolase gene in macrophages
US20030096732A1 (en) Telomerase expression repressor proteins and methods of using the same
WO1995032284A1 (fr) Facteur agissant en interaction avec des proteines nucleaires
WO1995032284A9 (fr) Facteur agissant en interaction avec des proteines nucleaires
US6127176A (en) Mutant cell lines unresponsive to interleukin 1
EP1027424A1 (fr) Regulateurs de l'expression du gene ucp3
AU3103899A (en) Identification of factors which mediate the interaction of heterotrimeric g proteins and monomeric g proteins
JP2003199590A (ja) プロモーターｄｎａ
US6803204B2 (en) Biologically active alternative form of the IKKα IκB kinase
WO2003025579A2 (fr) Utilisation d'une proteine de liaison des acides gras du coeur
JP4516747B2 (ja) インスリン作用促進能力の検定方法
WO2001096575A1 (fr) Ceramide kinase et adn la codant
US20100196957A1 (en) Cloning and characterization of 5' flanking regions of a human aggrecanase-1 gene
JP2004081147A (ja) セラミドキナーゼ遺伝子プロモーターｄｎａ
JP4516746B2 (ja) コレステロールエステル分泌抑制能力の検定方法
JP3942212B2 (ja) ＴＧＦ−βファミリーの情報伝達系を担う新規キナーゼ

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP