WO1999015558A1 - Proteine de verrues, polynucleotide codant cette proteine, polynucleotide antisens de celui-ci et anticorps reconnaissant la proteine - Google Patents

Proteine de verrues, polynucleotide codant cette proteine, polynucleotide antisens de celui-ci et anticorps reconnaissant la proteine Download PDF

Info

Publication number
WO1999015558A1
WO1999015558A1 PCT/JP1998/003739 JP9803739W WO9915558A1 WO 1999015558 A1 WO1999015558 A1 WO 1999015558A1 JP 9803739 W JP9803739 W JP 9803739W WO 9915558 A1 WO9915558 A1 WO 9915558A1
Authority
WO
WIPO (PCT)
Prior art keywords
warts
polynucleotide
protein
gene
dna
Prior art date
Application number
PCT/JP1998/003739
Other languages
English (en)
Japanese (ja)
Inventor
Toshihiko Kishimoto
Shin-Ichiro Niwa
Yasuko Nagamine
Yasuyuki Nishiyama
Hideyuki Saya
Original Assignee
Sumitomo Electric Industries, 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 Sumitomo Electric Industries, Ltd. filed Critical Sumitomo Electric Industries, Ltd.
Priority to CA002304793A priority Critical patent/CA2304793A1/fr
Publication of WO1999015558A1 publication Critical patent/WO1999015558A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to a warts protein, a polynucleotide encoding the protein, an antisense polynucleotide thereof, and an antibody recognizing the protein.
  • the present invention relates to a homologous protein of Drosophila warts protein in vertebrates, particularly mammals, and a polynucleotide encoding the protein.
  • the present invention also relates to an antisense polynucleotide of the polynucleotide.
  • the present invention also relates to an antibody that recognizes the homolog protein. Background art
  • the Drosophila war ts protein (hereinafter sometimes referred to as d-warts) encoded by the Drosophila wa rts gene (hereinafter sometimes referred to as d-warts gene) is a DM protein kinase that is a serine / sleonin kinase. It has a homology with DM-PK, suggesting its involvement in the signal transduction system.
  • d-wart s gene discloses the c invention that are known to cause hyperplastic overgrowth and imaginal disc epithelial cells by its deletion
  • Homologous genes of Drosophila genes are also found in vertebrates, especially mammals Its presence is difficult to predict immediately, but if the homologous gene of the d-warts gene is present in vertebrates, especially mammals, it will occur in vertebrates, especially in the mammalian nervous system. It can be used to elucidate the mechanism of tumor development.
  • An object of the present invention is to isolate a homolog gene of the d-warts gene from a vertebrate, determine its nucleotide sequence, and provide a homolog protein encoded by the gene.
  • Another object is to provide an antibody that recognizes the homolog protein.
  • the present invention provides h-warts, which is a homologous protein of d-warts in humans, and a polynucleotide encoding the protein.
  • Polynucleotides are typically DNA and RNA.
  • the present inventors searched for an EST database based on the base sequence of the d-warts gene, prepared primers, and converted the human brain fetal cDNA library into type III using the primer.
  • the h-warts gene which is a homologous gene of the d-warts gene in humans, was isolated and its nucleotide sequence was determined.
  • the amino acid sequence of h-warts which is a homologous protein of d-warts in humans, was determined.
  • the present invention also provides a protein (h-warts mutant) comprising an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of h-warts. is there.
  • the homology between the mutant and h-warts is preferably at least 75%. Further, it is preferable that the portion conserved between d-warts and h-warts has no mutation.
  • the portion from the 354th Tyr to the 366th His in the amino acid sequence of h—wart s (amino acid sequence described in SEQ ID NO: 1 in the sequence listing); From the Gin of the eye to the 427th Lys, from the 464th Arg to the 482th Asn, from the 488th Leu to the 509th Asp, from the 522th G1u 531st part from G1u, 538th part from Ser to 577th His, 626th part from Leu to 642th Leu, 662th G1u to 672th A Up to 1a or
  • the portion from the 752th His to the 764th Pro has no mutation.
  • the present invention also discloses an antisense polynucleotide comprising a polynucleotide encoding the h-warts or a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the h-warts mutant.
  • the present invention also discloses polynucleotides that are all or part of 12 or more nucleotides encoding polynucleotides encoding h-warts or h-warts mutants.
  • This polynucleotide for portions of the coding region, can be used to produce partial length proteins of the h_warts or h-warts mutants, respectively. It can also be used as a probe.
  • the present invention also discloses an antisense polynucleotide which is a whole or a part consisting of 12 or more bases of the antisense polynucleotide of the h-warts or mutant h-warts.
  • This antisense polynucleotide is capable of inhibiting the biosynthesis of the h-warts or h-warts mutants, respectively. It can also be used as a probe.
  • the present invention provides a polynucleotide or an antisense polynucleotide obtained by chemically modifying each of the above-mentioned polynucleotides or antisense polynucleotides. It is disclosed.
  • the present invention also relates to a part of a polynucleotide encoding the above-mentioned h-warts or h-warts mutant or an antisense polynucleotide of the above !!-warts or h-warts mutant.
  • a portion single-stranded DNA, including NA or those chemically modified
  • the present invention also discloses a cDNA obtained by the above method for obtaining a homologous gene of the h-warts gene.
  • the homologous gene of the h-warts gene is referred to as a warts gene.
  • the present invention discloses a h-warts homolog protein (also a d-warts homolog protein) encoded by the h-warts gene homolog gene.
  • a homologous protein of h-warts is referred to as warts.
  • warts The warts described above can be produced in a transformant into which the gene has been introduced.
  • the present invention discloses an antibody that recognizes any of h-warts, h-warts mutant, or wrtts.
  • FIG. 1 is a diagram comparing the amino acid sequences of h-warts and d-warts.
  • FIG. 2 is a diagram showing the positional relationship between the cDNA fragment, EST, and each primer obtained in the process of obtaining the h-warts gene.
  • Figure 3 shows the results of Northern blot hybridization of h-warts cDNA hybridized for mRNA from human tissues. It is a photograph.
  • FIG. 4 is an electrophoretic photograph showing the results of Northern blot hybridization obtained by hybridizing h-warts cDNA with respect to mRNA of each human tissue.
  • FIG. 5 is an electrophoretic photograph showing the results of Northern plot hybridization in which mRNA of human cell lines was hybridized with cDNA of h-warts.
  • Probes used to obtain the wa rts gene in the animal from the cDNA library of each vertebrate include those with a high homology between d-wa rts and h-wa rts, that is, over the species.
  • a DNA containing a portion that codes for an amino acid sequence of a well-conserved portion can be used.
  • the amino acid sequence of d-war ts and h-war ts where the amino acid sequence is well preserved is the amino acid sequence of h-war ts (the amino acid sequence described in SEQ ID NO: 1 in the sequence listing).
  • 354th Tyr to 366th His, 407th Gin to 427th Lys, 464th A to 482th Asn, 488th Leu 509th Asp, 522th Glu to 531th G1u, 538th Ser to 577th Is, 626th Leu to 642th L The part from eu to eu, the part from Glu at 662 to A1a at 672, and the part from His at 752 to Pro at 764.
  • FIG. 1 the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.
  • the rightmost numeral indicates the number of the rightmost amino acid in the row.
  • the DNA encoding this part is composed of the base sequence of the h—warts gene (base sequence described in SEQ ID NO: 2 in the sequence listing) from the 1069th to the 1107th.
  • the warts gene of each vertebrate can be obtained from one of the vertebrate cDNA libraries, using the DNA consisting of the nucleotide sequence containing the above-described portion as a probe.
  • the warts gene of a mammal can be obtained from a cDNA library of the mammal.
  • an RNA consisting of a base sequence corresponding to the DNA can also be used as the probe.
  • a method for obtaining a wrtss gene from a cDNA library of each vertebrate is exemplified below.
  • a vertebrate cDNA library of interest is prepared from the mRNA of the animal.
  • a commercially available cDNA library may be used.
  • About 10 6 plaques were prepared on NZY agar medium, and this cDNA library was
  • the above cDNA library is obtained by the following operation
  • the full-length warts gene can be obtained from
  • ABEL kit (registered trademark, manufactured by Takara Shuzo) can be used. 2) Next, prepare a DNA reaction solution having the following composition, incubate the solution at 37 ° C for 30 minutes, and heat treat it at 70 ° C for 10 minutes to inactivate the enzyme.
  • a 5 ml polyprep column (manufactured by Bio-Rad) is packed so that the bed volume becomes 1 ml, and the DNA reaction solution heat-treated in 2) is placed on the column.
  • the entire base sequence of the warts gene is determined by the dye-one-mine-one-one method.
  • a software controller can be used.
  • a combination of DNAs was selected so that the coding region was covered.
  • primers should be included to include the entire coding region.
  • a full-length DNA can also be obtained by designing a vertebrate, preferably a mammalian vertebrate, preferably a mammalian cDNA library, and performing PCR using the primer.
  • the amino acid sequence of warts can be determined.
  • a commercially available program for example, GENETYX (registered trademark) —CD program Ver. 34 (manufactured by Software Development Corporation).
  • a transformant can be prepared by inserting the warts gene into a vector and introducing it into an appropriate host.
  • the transformant can be cultured in an appropriate medium to produce a protein.
  • natural mutation or by artificial mutation for example, the method described on pages 15.1 to 15.113) (Molecular Cloning 2nd Edition (Cold Singing Harbor Laboratory Press, 1989)).
  • the main mechanism of the polypeptide encoded by the polynucleotide The polynucleotide can be changed without changing the function.
  • the h-warts of the present invention also includes a protein comprising an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, ie, h — It is possible to create wa rts mutants.
  • the homology of the amino acid with h-warts is preferably at least 75%, more preferably at least 90%, even more preferably at least 95%.
  • the amino acid sequence of the h-warts mutant of the present invention can be determined from the nucleotide sequence of the gene encoding the mutant.
  • a commercially available program for example, GENETYX (registered trademark) —CD program Ver. 34 (manufactured by Software Development Corporation).
  • the polynucleotide encoding the wart s of the present invention includes all patterns of degeneracy.
  • DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing is a naturally occurring h-warts gene.
  • the present invention includes an antisense polynucleotide comprising a base sequence of an antisense strand of the polynucleotide encoding h-warts and a derivative thereof.
  • the antisense polynucleotide is capable of hybridizing to a polynucleotide encoding h-warts, which is used for hybridization. If the soybean polynucleotide is a polynucleotide in the coding region, it is possible to inhibit the biosynthesis of the polypeptide encoded by the polynucleotide.
  • the antisense polynucleotide for inhibiting the biosynthesis of the polypeptide preferably comprises 12 or more bases.
  • antisense polynucleotide incorporation of full-length antisense polynucleotides into cells is inappropriate even if they are too long.
  • the antisense polynucleotide is incorporated into cells to inhibit warts biosynthesis, 12 to 30 bases, preferably 15 to 25 bases, and more preferably 18 to bases 2 It is preferable to use an antisense polynucleotide consisting of 2 or less bases.
  • Antisense polynucleotides can also be used as probes.
  • the antisense polynucleotide of the present invention or a part thereof includes all of a plurality of nucleotides consisting of a base, a phosphate, and a sugar, including non-naturally occurring nucleotides. Typical examples are antisense DNA and antisense RNA.
  • the antisense polynucleotide of the present invention can be prepared by using known antisense technology to obtain various DNAs having high binding strength to target DNA or mRNA, tissue selectivity, cell permeability, nuclease resistance, and intracellular stability. Thus, a novel antisense polynucleotide derivative can be obtained.
  • an antisense polynucleotide or a derivative thereof having a base sequence complementary to the base sequence of the region forming the stem loop should be designed.
  • the antisense polynucleotide and the derivative thereof of the present invention can form a stem loop as necessary.
  • an antisense polynucleotide having a sequence complementary to the vicinity of the initiation codon, a ribosome binding site, a cabling site, or a splice site can generally be expected to have a high expression suppressing effect. Therefore, the antisense poly Nucleotides or their derivatives, which contain a complementary sequence of the gene encoding warts or the translation initiation codon of mRNA, the ribosome binding site, the cabling site, and the splice site, are expected to have a high expression suppression effect.
  • the currently generally known derivative is a derivative in which at least one of nuclease resistance, tissue selectivity, cell permeability, and binding strength is enhanced. Particularly preferred are derivatives having a phosphorothioate bond as a skeletal structure.
  • the polynucleotides and derivatives thereof of the present invention also include derivatives having these functions or structures.
  • the antisense polynucleotide is a natural antisense polynucleotide, it can be synthesized using a chemical synthesizer, or the antisense polynucleotide of the present invention can be produced by a PCR method using the gene encoding h-warts as a type II. it can. Some derivatives, such as methylphosphonate type and phosphorothioate type, can be chemically synthesized. In this case, the desired operation can also be achieved by performing the operation according to the instructions attached to the chemical synthesizer, and purifying the resulting synthetic product by HPLC using reverse phase chromatography or the like. An antisense polynucleotide or a derivative thereof can be obtained.
  • the polynucleotide encoding warts, the antisense polynucleotide thereof, or a part thereof is obtained from a cDNA library It can be used as a probe for screening the rts gene. At this time, those having a GC content of 30 to 70% can be suitably used. Further, a polynucleotide consisting of a continuous base sequence of 15 bases or more is particularly preferred.
  • the polynucleotide used as a probe may be a derivative. Usually, a sequence having the number of bases or more is recognized as a sequence having specificity.
  • a library prepared from mRNA can be preferably used as a cDNA library used in the screening using the probe.
  • these cDNA libraries A selected group of cDNAs can be used as a sample for the search. Also, commercially available products can be used.
  • the polynucleotide encoding the h-warts of the present invention, the antisense polynucleotide thereof, or a polynucleotide thereof (a polynucleotide comprising a continuous base sequence of 12 or more bases) can be used as a probe.
  • a probe By performing Northern blot hybridization on mRNA derived from each tissue using the polynucleotide as a probe, it is possible to find a tissue in which mRNA derived from the h-warts gene is expressed.
  • the DNA and RNA of the present invention include the above-mentioned chemically modified DNA, RNA or antisense polynucleotide within its scope.
  • Chemically modified DNA or RNA can exert both the function of encoding a protein and the function of a probe, and the chemically modified antisense polynucleotide can enhance the biosynthesis of proteins or proteins. Both of these functions can be inhibited.
  • a transformant by introducing the plasmid into an appropriate host such as Escherichia coli by a known method. It is possible to produce warts by culturing the obtained transformant to amplify the gene or express the protein. Similarly, it is possible to produce a warts mutant by culturing a transformant into which DNA encoding the warts mutant has been introduced.
  • the culture of the present invention containing the war ts or war ts mutant prepared in the transformant is collected and, if necessary, concentrated, solubilized, dialyzed, and various types of chromatography are performed to obtain the war of the present invention. It is possible to purify ts or war ts mutants.
  • any of bacteria such as Escherichia coli, yeast, and animal cells can be used, but animal cells are particularly preferable.
  • a ribosome method, an electroporation method, or the like can be used.
  • Purification methods for purifying warts or warts mutants from the resulting culture include immunoprecipitation, salting out, ultrafiltration, isoelectric point precipitation, gel filtration, electrophoresis, and ion exchange.
  • affinity chromatography such as chromatography, hydrophobic chromatography, antibody chromatography, etc., chromatography, adsorption chromatography, reverse phase chromatography, etc. Just do it.
  • the warts or warts mutant to be produced may be produced in a transformant as a fusion peptide with another polypeptide.
  • an operation of treating with a chemical substance such as bromocyan or an enzyme such as a prosthesis to cut out warts or warts mutants is required.
  • An antibody that recognizes wart s or a wart s mutant or a polypeptide consisting of an amino acid sequence specific to the wart s is immunized to animals other than humans and animals other than the animal from which the warts are derived (wart s).
  • the wa rts antibody or wa rts mutant antibody recognizes the wa rts or wa rts mutant, respectively, can be performed by Western blotting, ELISA, immunostaining (eg, FACS measurement), etc. Is possible.
  • a protein obtained by binding a part of the protein to another carrier protein such as serum albumin even if it is a part of the protein.
  • Part of the protein may be synthesized, for example, using a peptide synthesizer. It is preferable that a part of the protein is composed of eight or more amino acid residues.
  • the antibodies of the present invention also include monoclonal antibodies.
  • antibodies also include active fragments.
  • the active fragment means a fragment of an antibody having an antigen-antibody reaction activity, and specific examples thereof include F (ab ') 2 , Fab', Fab, and Fv.
  • F (ab,) 2 when the antibody of the present invention is digested with pepsin, F (ab,) 2 is obtained, and when digested with papain, F ab is obtained. Reduction of F (ab ') 2 with reagents such as 2-mercaptoethanol and alkylation with monoacetic acid gives Fab.
  • FV is a monovalent antibody active fragment in which a heavy chain variable region and a light chain variable region are linked by a linker.
  • a chimeric antibody can be obtained by retaining these active fragments and substituting other portions with fragments of another animal.
  • the antibody of the present invention can be used for elucidation of functions such as expression of warts and elucidation of a tumor formation mechanism.
  • Methods for detecting worts include a method using an antibody and a method using an enzymatic reaction.
  • the method using an antibody include (1) a method of detecting warts using a labeled warts antibody, and (2) a method of detecting warts using a warts antibody and a labeled secondary antibody of the antibody.
  • Labels include, for example, radioisotopes Elements (RIs), enzymes, avidin or biotin, or fluorescent substances (FITC or mouth-damine, etc.) are used.
  • Examples of the method using an enzyme reaction include an ELISA method, an immunoagglutination method, a Western blot method, a method for identifying an immunoreactive molecule using flow cytometry, or a method similar thereto.
  • the following primers were synthesized from a human cDNA library in order to obtain a DNA fragment consisting of a partial base sequence of the above-mentioned base sequence of Z13665.
  • PCR was performed under the following conditions using a cDNA library of human fetal brain Matahon-Ready cDNA (Clontech).
  • PCR was performed in only one direction in three directions.
  • a liquid having the following composition was placed in a test tube, and a mineral oil was layered thereon in a 15: 1 layer, and left at 94 ° C for 5 minutes.
  • cDNA library (12 ⁇
  • Taq polymerase (5uni t / l) 0.5 ⁇ 1
  • reaction was carried out at 55 ° C for 2 minutes and finally at 72 ° C for 10 minutes to carry out a fragment extension reaction to complete the PCR operation.
  • a second PCR operation was performed using the PCR product obtained as described above as a ⁇ type, using the primers warts S2 and the AP2 primer of cDNA.
  • a liquid having the following composition was placed in a test tube, and a 20/1 layer of mineral oil was layered thereon, and left at 94 ° C for 5 minutes.
  • PCR buffer 40 OmM Tris—HC1 (pH 8.3), 1 MKCl, 100 mM MgCl 2 , 100 mM DTT
  • reaction was carried out by repeating 40 times a cycle of “60 ° C. for 30 seconds, followed by 72 ° C. for 1 minute, and then 94 ° C. for 30 seconds”.
  • reaction was carried out at 55 ° C for 2 minutes and finally at 72 ° C for 10 minutes to carry out a fragment extension reaction to complete the PCR operation.
  • fragment A The DNA fragment obtained by the 3 ′ extension reaction obtained above (hereinafter sometimes referred to as fragment A) was subjected to minigel electrophoresis (0.75% agarose gel), and the fragment A band was cut out from the gel. Fragment A was collected using GeneClean (manufactured by Bio 101), and bands were checked by minigel electrophoresis.
  • Fragment A was taken in 1 ⁇ 1 and diluted with 991 TE. The absorbance at 260 nm (A260) was measured and the DNA concentration was calculated (the DNA concentration when A260 was 1.0 was 50 / l / ml). Fragment A was diluted with TE so that the DNA concentration was 1 / g / ⁇ 1.
  • the DNA sequence of this diluent is determined by using the warts S2 primer and AP2 primer, and using the dye sequencer (881 model 373A) according to the Dye-Mine-Mine-Eye method.
  • the nucleotide sequence of fragment A was determined. This nucleotide sequence is shown as SEQ ID NO: 3 in the sequence listing. 5.
  • AGTAATAGG—3 ′ (base sequence described in SEQ ID NO: 9 in the sequence listing) was designed and synthesized using a DNA synthesizer (ABI, model 392). The synthesized primer was adjusted to 2 Opmo 1 / ⁇ 1 with distilled water. Using this as a PCR primer, PCR was performed.
  • One of the cDNA libraries contains the mat of human fetal brain
  • PCR was performed using a DNA library (Clontech) under the following conditions.
  • PCR was performed in only one direction in the 5, direction.
  • a liquid having the following composition was placed in a test tube, and mineral oil was layered thereon in a ratio of 15/1, and left at 94 ° C for 5 minutes.
  • c DNA library (1 ⁇ g / ⁇ 1) 2/1 dNTP mixture each NTP is dissolved in distilled water at 20 pmo 1/11) 1/1 war ts AS 2 primer (20 pmo l / l ) 1 ⁇ .1
  • the PCR product obtained above was used as a type II, and a second PCR operation was performed using the warts AS1 primer and the AP2 primer of cDNA.
  • a solution having the following composition was placed in a test tube, and a 20-1 layer of mineral oil was layered thereon, and left at 94 ° C for 5 minutes.
  • AP 2 primer (20 pmo 1 / ⁇ .1) 1 ⁇ 1 10-fold concentration of PCR buffer (40 OmM Tris-HC 1 (pH 8.3), 1 M KC1, 10 OmM MgCl 2 , 10 OmM DTT ) 2 ⁇ 1 distilled water 8.5 1 & polymerase (5111; 17 3 / ⁇ 1) 0.5 ⁇ 1 total 20 1 Then, "60 ° C for 30 seconds, followed by 72 ° C for 1 minute, Subsequently, a cycle of 30 seconds at 94 ° C ”was repeated 40 times to carry out the reaction.
  • reaction was carried out at 55 ° C for 2 minutes and finally at 72 ° C for 10 minutes to carry out a fragment extension reaction to complete the PCR operation.
  • fragment B The DNA fragment obtained from the 5, extension reaction obtained above (hereinafter sometimes referred to as fragment B) was subjected to minigel electrophoresis (0.75% agarose gel), and the fragment B band was cut out from the gel. Fragment B was recovered using GeneClean (manufactured by Bio 101), and bands were checked by minigel electrophoresis. Fragment B was taken from 11 and diluted with 99 ⁇ 1 of TE. The absorbance at 260 nm (A 260) was measured, and the DNA concentration was calculated (the DNA concentration when A 260 was 1.0 was 50 ⁇ 1 / ml). DNA concentration should be 1 ⁇ g / 1 Fragment B was diluted with TE.
  • the DNA sequence of this diluted solution was performed using the wrts AS1 primer and the AP2 primer by the dye sequencer (Y81 model 373A) according to the Dye-Mine-Mine-Eye method.
  • the nucleotide sequence of fragment B was determined. This nucleotide sequence is shown as SEQ ID NO: 4 in the sequence listing.
  • GCT CGGGGATGACT-3 ′ base sequence described in SEQ ID NO: 11 in the sequence listing
  • the synthesized primer was adjusted to 20 pmo1 / 1/1 with distilled water. Using this as a PCR primer, PCR was performed.
  • One of the cDNA libraries contains the human fetal brain matathon—Readyc
  • PCR was performed using the DNA library 1 (Clontech) under the following conditions.
  • PCR was performed in only one direction in the 5, direction.
  • a liquid having the following composition was placed in a test tube, and a mineral oil was layered thereon in a ratio of 15/1, and left at 94 ° C for 5 minutes.
  • cDNA library (l ⁇ g / ⁇ l) 2 z 1 dNTP mixture (each NTP dissolved in distilled water at 20 pmo 1) 1 ⁇ 1 warts AS 12 primer (20 pmol / zl) 1 ju 1
  • reaction was carried out at 55 ° C for 2 minutes and finally at 72 ° C for 10 minutes to carry out a fragment extension reaction to complete the PCR operation.
  • a second PCR operation was performed using the warts AS11 primer and the cDNA AP2 primer.
  • a liquid having the following composition was placed in a test tube, and a 20/1 layer of mineral oil was layered thereon, and left at 94 ° C for 5 minutes.
  • Taq polymerase (5un i t s / / 1) 0.5 jl
  • reaction was repeated 40 times by repeating a cycle of “60 ° C. for 30 seconds, followed by 72 ° C. for 1 minute, followed by 94 ° C. for 30 seconds”.
  • reaction was carried out at 55 ° C for 2 minutes and finally at 72 ° C for 10 minutes to carry out a fragment extension reaction to complete the PCR operation.
  • fragment C The DNA fragment obtained from the 5 'extension reaction obtained above (hereinafter sometimes referred to as fragment C) was subjected to mini-gel electrophoresis (0.75% agarose gel), and fragmentation was performed. G band was excised from the gel. Fragment C was recovered using GeneClean (manufactured by Bio 101), and bands were checked by minigel electrophoresis.
  • Fragment C was removed and diluted with 991 TE.
  • the absorbance at 260 nm (A260) was measured and the DNA concentration was calculated (the DNA concentration when A260 was 1.0 was 50 / l / ml).
  • Fragment C was diluted with TE so that the DNA concentration was 1 ⁇ g / ⁇ 1.
  • the DNA sequence of this diluent is determined using the wart s AS 11 primer and the AP 2 primer, and using the auto sequencer (ABI model 373 A) by the Dye-Mine-Mine-Eye method.
  • the nucleotide sequence of fragment C was determined. This nucleotide sequence is shown as SEQ ID NO: 5 in the sequence listing.
  • S17 sense primer corresponding to a part of fragment C as a primer-5 one TCTGGAAACATGGAATACGTAATC-3, (base sequence described in SEQ ID NO: 12 in the sequence listing); 3, a part of fragment A as a primer AS 22 antisense primer equivalent to 5, 1 TATGGGTAGC
  • CATTGTCATCAAAA-3 ′ base sequence described in SEQ ID NO: 13 in the sequence listing
  • a PCR reaction was performed using the above primers.
  • a liquid having the following composition was placed in a test tube, and a 20: 1 layer of mineral oil was layered thereon, and left at 94 ° C for 5 minutes.
  • Human fetal brain cDNA library 1 (l zg / ⁇ l) 5 l
  • reaction was repeated 40 times by repeating a cycle of “60 ° C. for 30 seconds, followed by 72 ° C. for 2 minutes, followed by 94 ° C. for 30 seconds”. Subsequently, the reaction was carried out at 55 ° C for 2 minutes and finally at 72 ° C for 10 minutes to complete the PCR operation.
  • the PCR product was subjected to mini gel electrophoresis (0.75% agarose gel). As a result, a band of about 2440 kbp was observed.
  • the h-warts gene was isolated.
  • the nucleotide sequence of the h-warts gene is shown in SEQ ID NO: 2 in the sequence listing.
  • Figure 2 shows the positional relationship between the full length gene of h-warts, fragment A, fragment B, fragment C and Z 13665.
  • amino acid sequence of h-warts was determined. This amino acid sequence is shown as SEQ ID NO: 1 in the sequence listing.
  • the homology between h-wart s protein and d-wart s protein is about 70%, and is a protein that is well conserved among species.
  • Figure 1 shows the comparison between the two.
  • h—warts is represented by h—wts
  • d—warts is represented by d—wts.
  • the DM-PK domain of h-warts is a portion from Leu at position 405 to Trp at position 768 of the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
  • the homology between h-warts and DM-PK was about 50%.
  • the primary structure of h--warts indicates signal transduction and differentiation or proliferation, particularly in relation to malignancies.
  • the full-length h-warts gene comprising the above nucleotide sequence was inserted into pBj-Myc vector, and the vector was introduced into Escherichia coli DH5 to prepare a transformant.
  • This was named my c—wart s, and on September 24, 1997 (1-1-3 Tsukuba East, Ibaraki, Japan (zip code: 305-8566)), Ministry of International Trade and Industry Deposited with the Biotechnology Research Institute (Accession No. FERM P-1644 1). Further, on August 24, 1998, the my c—warts were transferred to the International Deposit under the Budapest Treaty at the Institute (Accession No. FERM BP—
  • Membrane (Humn Mult iple Ti ssue Northern Blot I, II) prepared by blotting 2 ⁇ g each of poly A + RNA (mRNA) from human tissues and poly A + RNA (mRNA) from various human cells And Human Cancer Cell Line Mult ip 1 e Tissue Northern Blot (manufactured by Clontech)) with fragment A labeled by the following procedures 1) to 4) under the conditions of high stringency. , Mo lecular Cl onin AL aboratory Manual Second Edition 7.
  • Hybridization was performed as described on page 52, and analysis was performed by Northern plot hybridization method.
  • T50E was passed through the column four times with 200 ⁇ l each, and a labeled DNA probe was obtained from the fraction eluted with the second 200 ⁇ l.
  • Figures 3 and 4 show photographs of the results for each tissue. From the photograph, it was found that the mRNA of h-warts (band of 9.5 kb) was expressed in tissues other than lung and kidney.
  • FIG. 5 shows photographs of the results for various cells. From the photograph, expression was observed in cells other than lung cancer-derived cells A549.
  • a full-length h-warts gene was inserted into pBj-Myc vector, and the vector was introduced into COS cells (ATCC CRL-1651) to prepare a transformant. Transfer this transformant to 1 x 10 6 cells with 50 mM Tris—HC1, 150 mM NaCl, 1 Triton X_100, 50 mM odoacetamide, 2 m
  • the chromosomal mating of the full-length h-warts gene was determined by PCR using ligation hybrid cells.
  • the nucleotide sequence described in No. 15 was designed, and 93 radiation hybrid clones (radiation hybridc) of the human who lege nome of Reseach Genetics Inc. were designed. 1 One) was subjected to PCR.
  • the section number of the panel to which the primer (wt s S 6 or wt s AS 7) was hybridized was changed to “Whitehead Ad Institute / MITT Center for GenomeResearch (httptp). : www—geno me.wi.mit.e du / cgi—bincontig / rhmapperi.pi.) to obtain information on the chromosome position and nucleotide sequence of the panel with the number. .
  • the EST database was searched for that base sequence, multiple base sequences registered in the EST were found to be part of the h-warts gene. It overlapped with the array.
  • h-wart s was located between D 6 S 311 and WI 5215 registered in the EST database, and was located on chromosome 6 long arm 6q25.
  • a Drosophila warts homolog protein is also present in vertebrates.
  • the war ts of the present invention are expected to be involved in signal transduction, epithelial cell differentiation or abnormal proliferation. Therefore, the antibody of the present invention that recognizes wart s, its gene and warts is effective for analyzing the mechanism of tumor formation.
  • the h-warts gene of the present invention has a homology with the wa rts gene in other vertebrates, particularly mammalian cDNA libraries, which is higher than the d_warts gene in the animal.
  • Polynucleotides encoding warts, their antisense polynucleotides or portions thereof are suitable as probes for obtaining warts genes in other vertebrates, particularly mammals, from such animals.

Abstract

Cette invention concerne une protéine homologue qui est codée par un gène homologue à un gène de verrues de type d, et qui est isolée à partir d'un vertébré puis séquencée. Cette invention concerne également un polynucléotide codant cette protéine homologue, lequel contient un ARN, etc., qui comprend la séquence de base correspondant au gène homologue, ainsi que le polynucléotide antisens de celui-ci. Cette invention concerne enfin un anticorps capable de reconnaître la protéine homologue susmentionnée.
PCT/JP1998/003739 1997-09-24 1998-08-24 Proteine de verrues, polynucleotide codant cette proteine, polynucleotide antisens de celui-ci et anticorps reconnaissant la proteine WO1999015558A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA002304793A CA2304793A1 (fr) 1997-09-24 1998-08-24 Proteine de verrues, polynucleotide codant cette proteine, polynucleotide antisens de celui-ci et anticorps reconnaissant la proteine

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP9258689A JPH1189580A (ja) 1997-09-24 1997-09-24 ｗａｒｔｓ蛋白質、該蛋白質をコードするポリヌクレオチド、そのアンチセンスポリヌクレオチドおよび該蛋白質を認識する抗体
JP9/258689 1997-09-24

Publications (1)

Publication Number Publication Date
WO1999015558A1 true WO1999015558A1 (fr) 1999-04-01

Family

ID=17323737

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1998/003739 WO1999015558A1 (fr) 1997-09-24 1998-08-24 Proteine de verrues, polynucleotide codant cette proteine, polynucleotide antisens de celui-ci et anticorps reconnaissant la proteine

Country Status (3)

Country Link
JP (1) JPH1189580A (fr)
CA (1) CA2304793A1 (fr)
WO (1) WO1999015558A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1022333A1 (fr) * 1999-01-25 2000-07-26 Jcr Pharmaceuticals Co., Ltd. Gène suppresseur de tumeur d'origine humaine
WO2004030688A1 (fr) * 2002-10-01 2004-04-15 Link Genomics, Inc. Nouvelle methode de regulation de l'apoptose par gestion de l'interaction entre des proteines et un medicament utilisant ces proteines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996030402A1 (fr) * 1995-03-27 1996-10-03 Yale University Sequences nucleotidiques et proteiques de genes lats et procedes les utilisant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996030402A1 (fr) * 1995-03-27 1996-10-03 Yale University Sequences nucleotidiques et proteiques de genes lats et procedes les utilisant

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENES & DEVELOPMENT, Vol. 9, No. 5, (1995), ROBIN W. JUSTICE et al., "The Drosophila Tumor Suppressor Gene Warts Encodes a Homolog of Human Myotonic Dystrophy Kinase and is Required for the Control of Cell Shape and Proliferation", p. 534-546. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1022333A1 (fr) * 1999-01-25 2000-07-26 Jcr Pharmaceuticals Co., Ltd. Gène suppresseur de tumeur d'origine humaine
WO2004030688A1 (fr) * 2002-10-01 2004-04-15 Link Genomics, Inc. Nouvelle methode de regulation de l'apoptose par gestion de l'interaction entre des proteines et un medicament utilisant ces proteines

Also Published As

Publication number Publication date
CA2304793A1 (fr) 1999-04-01
JPH1189580A (ja) 1999-04-06

Similar Documents

Publication Publication Date Title
Asano et al. Conservation and diversity of eukaryotic translation initiation factor eIF3
CA2479498A1 (fr) Compositions et procedes destines au traitement de tumeur
JPH07133298A (ja) ヒトＩＬ−２レセプターγ鎖分子
AU713937B2 (en) Peptides capable of binding to the gap protein sh3 domain, nucleotide sequences coding therefor, and preparation and use thereof
AU673141B2 (en) Purification and cloning of P62
WO1999015558A1 (fr) Proteine de verrues, polynucleotide codant cette proteine, polynucleotide antisens de celui-ci et anticorps reconnaissant la proteine
JP4127861B2 (ja) シナプス活性化タンパク質組成物および方法
US5846822A (en) Nucleic acid molecules encoding PP32: a newly identified CD45-associated protein
EP1125947A1 (fr) Proteines humaines h37 et adnc les codant
US7427666B2 (en) Antibody directed against a ubiquitin-specific protease occurring in the brain
WO1997031110A1 (fr) Molecule de la famille des genes de transfert f, polynucleotide codant pour cette molecule et anticorps actif contre cette molecule
EP0905239A2 (fr) Gène codant Afadin-1
EP1148125B1 (fr) Nucleoproteine humaine possedant un domaine ww et polynucleotide codant cette nucleoproteine
WO1999057269A1 (fr) Gene hrpi humain
CA2322070A1 (fr) Proteine se liant a ne-dlg
EP1369478A1 (fr) Nouvelle proteine de classe a du type recepteur eboueur
WO1999009060A1 (fr) PROTEINE 1(3)mbt, POLYNUCLEOTIDE CODANT POUR LADITE PROTEINE, SON POLYNUCLEOTIDE ANTISENSE ET ANTICORPS RECONNAISSANT LADITE PROTEINE
JPH11146786A (ja) ｈ−Ｈｙｄ蛋白質、該蛋白質をコードするポリヌクレオチド、そのアンチセンスポリヌクレオチドおよび該蛋白質を認識する抗体
WO1999023218A1 (fr) PROTEINES DE FIXATION DE p16, GENE DE CES PROTEINES ET ANTICORPS CONTRE CES PROTEINES
CA2309336A1 (fr) Proteine neuralisee, polynucleotide codant cette derniere, et anticorps reconnaissant cette proteine
WO1998046745A1 (fr) NOUVELLE MOLECULE DE LA FAMILLE dlg, POLYNUCLEOTIDE CODANT CETTE MOLECULE, ANTICORPS DIRIGE CONTRE ELLE ET PROCEDE DE DETECTION DE GENE dlg
CA2330168A1 (fr) Proteine pour la regulation de l'apoptose
WO2000047731A1 (fr) Proteine a activite helicase d'adn, polynucleotide codant cette proteine, polynucleotide antisens et anticorps associes
WO1999016784A1 (fr) Proteine fixatrice de ras (pre1)
JPH0977792A (ja) Ｔｂｐと複合体を形成する蛋白質、その遺伝子及び抗体ならびにそれらを用いた癌の診断法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2304793

Country of ref document: CA

Ref country code: CA

Ref document number: 2304793

Kind code of ref document: A

Format of ref document f/p: F