WO2004058815A2 - Proteine associee aux centrosomes et ses applications - Google Patents
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- WO2004058815A2 WO2004058815A2 PCT/FR2003/003895 FR0303895W WO2004058815A2 WO 2004058815 A2 WO2004058815 A2 WO 2004058815A2 FR 0303895 W FR0303895 W FR 0303895W WO 2004058815 A2 WO2004058815 A2 WO 2004058815A2
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Classifications
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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 new protein associated with centrosomes, to the polynucleotide encoding said protein as well as to the applications of said protein and said polynucleotide.
- the cell division process consists of a nuclear division (mitosis) followed by a cytoplasmic division (cytokinesis). Mitosis is dominated by the formation of a highly organized polar spindle (the mitotic spindle) made up of two families of microtubules: polar microtubules and kinetochorean microtubules.
- Microtubules are polymers composed of ⁇ - and ⁇ -tubulin subunits. Their growth is initiated in the peripheral region of the centrosome by a complex mainly containing a related protein, ⁇ -tubulin.
- the polar microtubules are composed of rows of microtubules and associated proteins which are put in place by the two mitotic centers, associated with centrioles, located at opposite poles of the spindle (asters).
- Each replicated chromosome is made up of two sister chromatids linked together by the centromere.
- the kinetochore microtubules are linked to the chromosomes replicated by specialized structures called kinetochores which are formed during prophase on each of the two faces of the centromere.
- the chromosomes condense during prophase and form the kinetochoreal microtubules which begin to interact with the polar spindle microtubules after rupture of the nuclear envelope during prometaphase.
- the chromosomes align in the equatorial zone of the spindle during the metaphase.
- the sister chromatids detach and are attracted towards the opposite poles.
- the two cellular poles move apart.
- the nuclear envelope is reformed on the surface of each group of chromosomes.
- Cell division ends when the cytoplasmic content is divided according to the cytokinesis process.
- the mitotic spindle plays an important role in the cytokinesis process, by fixing the establishment of cell segmentation.
- the division groove invariably appears in the plane of the equatorial plate, perpendicular to the axis of the mitotic spindle.
- the aurora-type kinases located at the level of the centrosomes, including the aurora-type kinases, required for the separation of the centrosomes and the assembly of the mitotic spindle, the polo-type kinases, involved in the maturation and formation of the spindle.
- bipolar and NIMA kinases that regulate the separation of centrosomes.
- Mammals have at least three aurora protein kinases. In humans, these three protein kinases are overexpressed in cancer pathologies due to chromosomal abnormalities. Thus, these proteins seem to play an important role in the control of ploidy. For example, inactivation or overexpression of two of these kinases leads to polyploidy.
- aurora A kinase activity leads to the formation of monopolar spindles. Inhibition of the activity of aurora B kinase leads to the formation of multinucleated cells due to cytokinesis defect. These chromosomal anomalies appear linked to disturbances in the formation of the mitotic spindle. Little is known about the partners and the substrates of these protein kinases. For example, in xenopus, aurora A interacts with a kinesin involved in the dynamics of microtubules. In humans, it phosphorylates the protein HsTACC-3, also overexpressed in many cancer cell lines.
- aurora A phosphorylates the protein D-TACC and is necessary for its localization at centrosomes in order to regulate the astral microtubules.
- D-TACC interacts with the microtubule associated protein (MAP: Microtubule Associated Protein) Msp, which is part of the family of proteins XMAO215 / ch-TOC / Msps, which stimulate the growth of microtubules in vitro and are concentrated at the level of centrosomes in vivo.
- MAP brings together a collection of various proteins defined on the basis of their ability to interact with microtubules. MAPs appear as partners / substrates of centrosome kinases such as aurora or polo.
- the present invention is placed. Indeed, surprisingly and unexpectedly, the inventors have highlighted a new human protein associated with centrosomes. By immunofluorescence, it is detected in collocation with the ⁇ -tubulin of the microtubules of the mitotic spindle, in particular with the aster. This protein was named ASAP for Aster Associated Protein by the Inventors.
- the overexpression of the protein according to the invention leads to disturbances in the organization of the mitotic spindle and induces aberrant and abortive mitoses (plurinucleated cells, mono- or multipolar spindles). Its overexpression blocks cell division and therefore cell proliferation.
- the subject of the invention is an isolated protein, called ASAP, characterized in that it is selected from the group consisting of: a) a protein corresponding to the sequence represented in the sequence list in the annex under the number SEQ ID NO: 1; b) a protein having, over its entire sequence, at least 80% identity or at least 90% similarity, preferably at least 90% identity or at least 95% similarity, with the protein of SEQ ID NO: 1.
- a protein according to the invention is characterized by the following properties: it has a molecular weight of between 60 and 100 kDa, preferably between 65 and 80 kDa; it is associated with centrosomes; - it is collocated by immunofluorescence with the ⁇ -tubulin of the microtubules of the mitotic spindle; it has a weak identity (23%) with the protein MAP1A (Microtubule Associated Protein 1A); it has coiled-coil domains essentially grouped in its C-terminal part between amino acids 297 and 327 on the one hand and 477 and 628 on the other hand, indicating either that the protein is oligomerized, or that it interacts with other proteins; it has a weak identity (20%), between amino acids 300 and 600 with a caldesmon-like domain (Gusev, NB, Biochemistry, 10: 1112-1121, 2000), referenced pfam00769 (NCBI, domains, http: // www.ncbi.
- the proteins caldesmon and ERM are also considered as MAP; - It also has, between positions 65 and 303, a BRCT domain (Breast cancer carboxy-terminal domain; Bork, P., et al., FASEB J., 11, 68-76 (1997)), indicating that the protein is involved in cell cycle control '; it has a great richness in ⁇ helices in its C-terminal part, in particular in the region between amino acids 420-620, almost exclusively formed of ⁇ helices.
- BRCT domain Breast cancer carboxy-terminal domain
- ASAP protein is a new MAP.
- the proteins according to the invention include any protein (natural, synthetic, semi-synthetic or recombinant) of any prokaryotic or eukaryotic organism, in particular of a mammal, comprising or consisting of an ASAP protein.
- said protein is a functional ASAP protein.
- telomere a protein having normal biological activity, that is to say capable of intervening in the organization of the mitotic spindle and in cell division. This protein can include silent mutations inducing no substantial change in its activity and producing no phenotypic modification. Proteins in accordance with the invention are in particular represented by the human ASAP (SEQ ID NO: 1) and murine (SEQ ID NO: 46) proteins.
- variant proteins of the sequences SEQ ID NO: 1 and 46 are included in the proteins according to the invention defined in b), the variant proteins of the sequences SEQ ID NO: 1 and 46, in particular the proteins whose amino acid sequence has at least one mutation corresponding in particular to a truncation, a deletion , a substitution and / or an addition of at least one amino acid residue relative to to sequences SEQ ID NO: 1 and 46.
- the variant proteins have a mutation causing a dysfunction (activation or inhibition) of the protein, of other genes or proteins or of the cell in general.
- said protein is a mammalian protein, preferably a protein of human origin.
- ID NO: 1 as a reference sequence is assessed according to the percentage of amino acid residues which are identical, when the two sequences are aligned, so as to obtain the maximum correspondence between them.
- the percentage of identity can be calculated by a person skilled in the art using a computer program for comparing sequences such as, for example, that of the BLAST suite (Altschul et al., NAR, 1997, 25, 3389-3402).
- BLAST programs are implemented on the comparison window consisting of the entire SEQ ID NO: 1, indicated as the reference sequence.
- a protein having an amino acid sequence having at least X% identity with a reference sequence is defined, in the present invention as a protein whose sequence can include up to 100-X alterations per 100 amino acids of the sequence of reference, while retaining the functional properties of said reference protein.
- alteration includes deletions, substitutions or consecutive or dispersed insertions of amino acids in the reference sequence.
- the similarity of a sequence compared to a reference sequence is assessed according to the percentage of amino acid residues which are identical or which differ by conservative substitutions, when the two sequences are aligned so as to obtain the maximum correspondence between them.
- the term “conservative substitution” is intended to mean the substitution of one amino acid with another which has similar chemical or physical properties (size, charge or polarity), which generally does not modify the functional properties of the protein. .
- a protein having an amino acid sequence having at least X% similarity to a reference sequence is defined, in the present invention as a protein whose sequence can include up to 100-X non-conservative alterations per 100 amino acids of the reference sequence.
- non-conservative alterations includes deletions, non-conservative substitutions or consecutive or dispersed insertions of amino acids in the reference sequence.
- techniques or methods well known to those skilled in the art here is meant to refer to the techniques or methods conventionally used by those skilled in the art and described in numerous works, such as in particular that entitled Molecular Cloning. A Laboratory Manual (Sambrook J, Russell DW. (2000) Cold Spring Harbor Laboratory Press).
- the protein according to the invention is obtained either from a cell, or by chemical synthesis, or by genetic recombination.
- the protein can be obtained using one of the many known peptide synthesis routes, for example techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by synthesis in classic solution.
- the protein sequence can be changed to improve its solubility, especially in aqueous solvents.
- modifications are known to those skilled in the art, such as the deletion of hydrophobic domains or the substitution of hydrophobic amino acids with hydrophilic amino acids.
- the protein according to the invention consists of the chain of 13 peptides corresponding to the translation products of 13 of the 14 exons that contains the corresponding gene, the first exon not being translated (see below).
- said peptides respond to the following sequences (positions given relative to the numbering of the sequence SEQ ID NO: 1):
- - Peptide 1 it comprises 25 amino acids corresponding to positions 1 to 25 (SEQ ID NO: 2);
- - Peptide 2 it comprises 28 amino acids corresponding to positions 26 to 53 (SEQ ID NO: 3); - Peptide 3: it comprises 107 amino acids corresponding to positions 54 to 160 (SEQ ID NO: 4);
- - Peptide 4 it comprises 76 amino acids corresponding to positions 161 to 236 (SEQ ID NO: 5);
- - Peptide 5 it comprises 31 amino acids corresponding to positions 237 to 267 (SEQ ID NO: 6);
- - Peptide 6 it comprises 83 amino acids corresponding to positions 268 to 350 (SEQ ID NO: 7);
- - Peptide 7 it comprises 24 amino acids corresponding to positions 351 to 374 (SEQ ID NO: 8); - Peptide 8: it comprises 54 amino acids corresponding to positions 375 to 428 (SEQ ID NO: 9);
- - Peptide 9 it comprises 32 amino acids corresponding to positions 429 to 460 (SEQ ID NO: 10);
- - Peptide 10 it comprises 54 amino acids corresponding to positions 461 to 514 (SEQ ID NO: 11);
- - Peptide 11 it comprises 49 amino acids corresponding to positions 515 to 563 (SEQ ID NO: 12);
- - Peptide 12 it comprises 43 amino acids corresponding to positions 564 to 606 (SEQ ID NO: 13); - Peptide 13: it comprises 41 amino acids corresponding to positions 607 to 647 (SEQ ID NO: 14).
- the present invention also relates to a peptide consisting of a fragment of at least 10 consecutive amino acids of a protein defined above in a) or b), particularly a peptide selected from:
- sequences corresponding to the peptides 1 to 13 described above that is to say selected from the sequences SEQ ID NO: 2 to SEQ ID NO: 14, and
- sequences SEQ ID NO: 47 to 53 corresponding to mutants of the hASAP protein deleted from the N-terminal part containing the BRCT domain (NdeM: residues 304 -647 (SEQ ID NO: 48); Ndel2: residues 411-647 (SEQ ID NO: 49); Ndel3: residues 478-647 (SEQ ID NO: 50)) or of the C-terminal part containing the MAP domain (CdeM: residues 1 to 477 (SEQ ID NO: 51); Cdel2: residues 1 to 418 (SEQ ID NO: 52); Cdel3: residues 1 to 303 (SEQ ID NO: 53); residues 1 to 421 (SEQ ID NO: 47)).
- said peptide is useful for the production of antibodies specifically recognizing a protein as defined above, preferably recognizing the ASAP protein of sequence SEQ ID NO: 1 or SEQ ID NO: 46 .
- the invention thus also relates to monoclonal or polyclonal antibodies, characterized in that they are capable of specifically recognizing a protein according to the invention.
- the antibodies recognize, among the MAPs, only and specifically the ASAP protein of sequence SEQ ID NO: 1 or SEQ ID NO: 46.
- the antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') 2 fragments. They can also be in the form of immunoconjugates or labeled antibodies in order to obtain a delectable and or quantifiable signal.
- Said antibodies can be obtained directly from human serum or from serum from animals immunized with the proteins or peptides according to the invention.
- Specific polyclonal or monoclonal antibodies can be obtained according to techniques well known to those skilled in the art.
- the invention also relates to the use of antibodies according to the invention for the detection and / or purification of a protein according to the invention.
- the antibodies according to the invention can be advantageously used to detect the presence of a protein according to the invention, normal or mutated.
- monoclonal antibodies can be used for the detection of these proteins in a biological sample. They thus constitute a means of immunocytochemical or immunohistochemical analysis of the expression of proteins according to the invention, in particular the protein of sequence SEQ ID NO: 1, on tissue sections. Generally for such analyzes, the antibodies used are labeled in order to be detectable for example by immunofluorescent compounds, by labeling with gold or in the form of enzyme immunoconjugates.
- the subject of the invention is also a method of detection in a biological sample of the protein according to the invention, particularly of the ASAP protein, comprising a first step of suitable treatment of the cells by any appropriate means making it possible to make the intracellular medium accessible, a second step of bringing said intracellular medium thus obtained into contact with an antibody according to the invention and a third step of demonstrating by any appropriate means the ASAP protein-antibody complex formed.
- This method can also make it possible to measure the level of expression of the protein according to the invention in cells, particularly in cancer cells.
- the study of the expression of the ASAP protein is an element of evaluation of the capacity of proliferation or aggressiveness (ability to progress to cancers of poor prognosis) of cancer cells.
- the invention therefore also relates to a method for in vitro evaluation of the proliferation or aggressiveness of cancer cells contained in a biological sample, characterized in that it comprises a first step of suitable treatment of the cells by any appropriate means for making the intracellular medium accessible, a second step of bringing said intracellular medium thus obtained into contact with an antibody according to the invention, a third step of highlighting and / or measuring by any appropriate means the ASAP protein complex -antibody formed and a fourth step in evaluating the rate of transcription of the gene by comparing the rate of ASAP protein-antibody complexes formed with that of a previously chosen control biological sample.
- Said control can be constituted for example by a biological sample containing cells having a normal or altered protein level, to which said method is applied under the same conditions.
- the invention also relates to a kit making it possible to implement any of the methods described above comprising: a) at least one monoclonal or polyclonal antibody according to the invention; b) reagents for the detection of the ASAP protein-antibody complex produced during the immunological reaction.
- the kit may optionally include reagents necessary to make the intracellular medium accessible.
- the term “means of making the intracellular medium accessible” means any means known to those skilled in the art, such as, for example, cell lysis by enzymatic or chemical means, or even sonication, membrane permeation, thermal shocks.
- the present invention also relates to an isolated polynucleotide (cDNA or genomic DNA fragment), characterized in that its sequence is selected from the group consisting of: the sequences encoding a protein or a peptide as defined above, and the complementary sequences of the preceding ones, sense or antisense.
- the invention encompasses alleles of the asap gene from any mammal, as well as the polynucleotides of natural or artificial mutants of the asap gene coding for an ASAP protein, particularly for a functional ASAP protein as defined above.
- said polynucleotide coding for an ASAP protein responds to a sequence selected from the group consisting of: - the sequence SEQ ID NO: 15, corresponding to the DNA complementary to 2575 nucleotides of the MRNA encoding the human ASAP protein (hASAP); the sequence SEQ ID NO: 45, corresponding to the DNA complementary to 2767 nucleotides of the mRNA coding for the murine ASAP protein (mASAP) the genomic DNA fragment of 29750 nucleotides corresponding to the sequence represented in the list of sequences in annex under number SEQ ID NO: 16, corresponding to the human asap gene comprising 14 exons of which only 13 are translated, the first exon not being translated, contained in the contig AC097467 (length 178204 base pairs) between the bases 115117 and 143828 (version v.7.29a3 NCBI / Ensembl of July 12, 2002, http://www.ensembl.org), also located on chromosome 4q32.1 between the
- poly-dA deoxyadenosines
- ID NO: 15 is specifically expressed in the testis as a polynucleotide with a length of about 2.9 kilobases and in the brain as a polynucleotide with a length of about 9 kilobases which can correspond either to a premessager or to an isoform of high molecular weight.
- exons are distributed as follows over said genomic sequence (with respect to the numbering of the sequence SEQ ID NO: 16):
- - exon 1 it comprises 200 base pairs corresponding to positions 101 to 300 (SEQ ID NO: 17);
- - exon 2 it comprises 139 base pairs corresponding to positions 1157 to 1295 (SEQ ID NO: 18);
- - exon 3 it comprises 85 base pairs corresponding to positions 2050 to 2134 (SEQ ID NO: 19);
- - exon 4 it comprises 321 base pairs corresponding to positions 3615 to 3935 (SEQ ID NO: 20);
- - exon 5 it comprises 227 base pairs corresponding to positions 8259 to 8485 (SEQ ID NO: 21);
- - exon 6 it comprises 94 base pairs corresponding to positions 14930 to 15023 (SEQ ID NO: 22);
- - exon 7 it comprises 248 base pairs corresponding to positions 16715 to 16962 (SEQ ID NO: 23);
- - exon 8 it comprises 71 base pairs corresponding to positions 19552 to 19622 (SEQ ID NO: 24); - exon 9: it comprises 169 base pairs corresponding to positions 21187 to 21355 (SEQ ID NO: 25); - exon 10: it comprises 90 base pairs corresponding to positions 21911 to 22000 (SEQ ID NO: 26);
- - exon 11 it comprises 162 base pairs corresponding to positions 23731 to 23892 (SEQ ID NO: 27);
- - exon 12 it comprises 146 base pairs corresponding to positions 24014 to 24159 (SEQ ID NO: 28);
- - exon 13 it comprises 133 base pairs corresponding to positions 24343 to 24475 (SEQ ID NO: 29);
- - exon 14 it comprises 485 base pairs corresponding to positions 29166 to 29650 (SEQ ID NO: 30).
- the subject of the invention is also: a fragment of any one of the polynucleotides according to the invention, of at least 15 to 1500 consecutive nucleotides excluding the sequences listed under the access numbers AK024730 and AK024812 and ESTs listed under the access numbers BU198882, BM693711, AW372449, BM021380, BU928828, AL707573, AI885274, AI671785, AA805679, BU619959, BM021126, AL598336, AW976973, BU629726, AI433877, AV751613, BQ75275, AI7575213 , BF958121, BQ351941, AW194906, BG203580, BF078132, AW486134, AL600279, AA025538, AL600264, BF170676, BU759494, BB025236, BF214179, AI283076, BE6942
- polynucleotide having a percentage identity of at least 80%, preferably at least 90%, according to the invention are included in a polynucleotide having a percentage identity of at least 80%, preferably at least 90%, according to the invention, the polynucleotides variants of the sequences SEQ ID NO: 15 and 45, that is to say all the polynucleotides corresponding to allelic variants, that is to say to individual variations of the sequences SEQ ID NO: 15 and 45.
- allelic variants that is to say to individual variations of the sequences SEQ ID NO: 15 and 45.
- These natural variant sequences correspond to polymorphisms present in mammals, in particular in human being and, in particular to polymorphisms which can lead to the onset of a pathology.
- variant polynucleotide is also intended to denote any RNA or cDNA resulting from a mutation and / or from a variation of a splicing site of the genomic sequence whose mRNA has as complementary DNA the polynucleotide of sequence SEQ ID NO : 15 or SEQ ID NO: 45.
- the present invention relates to the polynucleotides or the variant fragments of the sequences SEQ ID NO: 15 and 45, particularly those in which the mutations lead to a modification of the amino acid sequence of the proteins of sequence SEQ ID NO: 1 and SEQ ID NO: 46.
- the polynucleotides according to the invention can be isolated from cells, particularly testis or brain cells or from cell DNA libraries. They can also be obtained by a polymerase chain reaction (PCR) carried out on the total DNA of the cells or also by RT-PCR carried out on the total RNA of the cells or by chemical synthesis.
- PCR polymerase chain reaction
- polynucleotides according to the invention particularly the fragments of any one of the polynucleotides according to the invention, and the sequences listed under the access numbers AK024730 and AK024812 and the ESTs listed under the access numbers BU198882, BM693711, AW372449 , BM021380, BU928828, AL707573, AI885274, AI671785, AA805679, BU619959, BM021126, AL598336, AW976973, BU629726, AI433877, AV751613, BQ372751, AI827535, B8191, BQ195, B8195, BQ19, BQ19, BQ19, B, B, B) , AL600279, AA025538, AL600264, BF170676, BU759494, BB025236, BF214179, AI283076, BE694273, AI266380, BM670854
- the transcripts of the asap gene are for example preferably detected using probes selected from the group consisting of the sequences SEQ ID NO: 15, SEQ ID NO: 45, SEQ ID NO: 17 to SEQ ID NO: 44 or using an EST as defined above or amplified by RT-PCR using primers selected from the group consisting of the sequences SEQ ID NO: 31 to 43.
- the polynucleotide according to the invention can make it possible to diagnose a pathological condition or a genetic disease involving a dysfunction of the asap gene and to screen for substances capable of modulating (activating or inhibiting) the transcription of said gene.
- the subject of the invention is also the polynucleotides capable of being obtained by amplification using the primers according to the invention.
- the probes and primers according to the invention can be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a delectable and / or quantifiable signal.
- the labeling of the probes according to the invention is carried out with radioactive elements or with non-radioactive molecules.
- radioactive isotopes used, mention may be made of 32 P, 33 P, 35 S, 3 H or 125 l.
- the non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, digoxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemo-luminescent, bioluminescent, fluorescent, phosphorescent agents.
- polynucleotides according to the invention can thus be used as a primer and / or probe in methods implementing in particular the technique of PCR (polymerase chain reaction) (US No. 4,683,202).
- PCR polymerase chain reaction
- Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR.
- the different polynucleotides according to the invention can make it possible either to determine the transcription profile of the corresponding asap gene or a possible alteration of this profile in a biological sample, or to highlight the corresponding gene in other species, allelic variants of this gene or a possible functional alteration of this gene (substantial change in the activity of the protein encoded by said gene) resulting from a mutation (insertion, deletion or substitution) of one or more nucleotides at the level of at least one exon of said gene.
- mutations include in particular deletions, insertions or non-conservative substitutions at the level of codons corresponding to amino acid residues located in a domain essential for the biological activity of the protein.
- the subject of the invention is a method of determining the transcription profile of the gene corresponding to the polynucleotide according to the invention or an alteration of said profile, in a biological sample, comprising a first step of obtaining by RNA any suitable means totals from the biological sample, a second step of bringing said RNAs into contact. with a probe according to the invention, previously labeled, under conventional hybridization conditions between the RNAs and the probe and a third stage of revelation by any appropriate means of the hybrids formed.
- the second step can be a step of reverse transcription and amplification of the transcripts, carried out using a pair of primers as described above and the third step, a revelation step by any suitable means of the amplified nucleic acids formed.
- Said method for determining the transcription profile of the gene may also include a step of evaluating the rate of transcription of the gene by comparison with a previously chosen control sample.
- Said control can consist, for example, of a biological sample having a normal or altered transcription of the gene corresponding to the polynucleotide according to the invention to which said method for determining the transcription profile of the gene is applied under the same conditions.
- the subject of the invention is also a method of demonstrating in other species the gene corresponding to the polynucleotide according to the invention or allelic variants of said gene or of a functional alteration of this gene, in a biological sample, comprising a first step of obtaining, by any appropriate means, DNA from the cells of a biological sample, a second step of bringing said DNA into contact with a probe according to the invention, previously labeled, under conventional hybridization conditions between the DNA and the probe and a third stage of revelation by any appropriate means of the hybrids formed.
- the second step can be an amplification step carried out using a pair of primers as described above and the third step, a revelation step by any appropriate means amplified nucleic acids formed.
- the method can possibly include a fourth step of isolation and sequencing of the nucleic acids highlighted.
- the subject of the invention is also a kit of reagents for the implementation of the methods described above comprising: a) at least one probe or a pair of primers according to the invention; b) the reagents necessary for carrying out a conventional hybridization reaction between said probe or said primers and the nucleic acid of the biological sample; c) the reagents necessary for carrying out an amplification reaction; d) the reagents necessary for the detection and / or the assay of the hybrid formed between said probe and the nucleic acid of the biological sample or of the amplified nucleic acids formed.
- kit can also contain positive or negative controls to ensure the quality of the results obtained. It can also contain the reagents necessary for the purification of nucleic acids from the biological sample.
- the polynucleotide of the invention or a fragment thereof, as well as the ESTs described above or their fragments can be used for the development of cellular or animal models not expressing the ASAP protein, by invalidating the ASAP gene by the method of If RNA (or RNAi for RNA interference; M. McManus and P. Sharp, Nature Reviews Genetics, 3, 737-747, 2002; V. Brondani, F. Kolb, E. Billy, M / S, 6-7, 665 -667, 2002) using oligonucleotides derived from their sequences.
- the invention also relates to a cloning and / or expression vector into which the polynucleotide according to the invention is inserted.
- Such a vector can contain the elements necessary for the expression and optionally for the secretion of the protein in a host cell.
- Said vectors preferably comprise: a promoter, translation initiation and termination signals, as well as suitable regions for regulating transcription. They must be able to be maintained in a stable manner in the cell and may optionally comprise sequences coding for particular signals specifying the secretion of the translated protein such as for example a strong promoter of ubiquitous nature or a selective promoter of a cell type and / or particular fabric. These different control sequences are chosen according to the cell host used.
- the polynucleotide according to the invention can be inserted into autonomously replicating vectors within the chosen host or integrative vectors of the chosen host.
- autonomously replicating systems plasmid or viral type systems are preferably used, depending on the host cell.
- the viral vectors can in particular be adenoviruses, retroviruses, lentiviruses, poxviruses or herpesviruses. Those skilled in the art know the technologies that can be used for each of these systems.
- viruses are, for example, retroviruses or viruses associated with adenoviruses (Adeno-associated virus or AAV).
- viruses are, for example, retroviruses or viruses associated with adenoviruses (Adeno-associated virus or AAV).
- naked polynucleotides such as naked DNA or RNA, artificial bacteria chromosomes (BAC), artificial yeast chromosomes (YAC, artificial yeast chromosome) are preferred. expression in yeast, mouse artificial chromosomes (MAC) for expression in murine cells and preferably human artificial chromosomes (HAC, human artificial chromosome) for expression in human cells.
- Such vectors are prepared according to methods commonly used by those skilled in the art, and the resulting recombinant vectors can be introduced into the appropriate host by standard methods, such as, for example, lipofection, electroporation, thermal shock, transformation after chemical permeabilization of the membrane, cell fusion.
- the invention also relates to transformed host cells, in particular euca ⁇ / otes and prokaryotic cells, into which at least one polynucleotide or fragment according to the invention or at least one vector according to the invention has been introduced.
- the invention also relates to non-human transgenic organisms such as transgenic animals or plants, all or part of the cells of which contain the polynucleotide according to the invention or the vector according to the invention, in a free or integrated form.
- the non-human transgenic organisms are those carrying cells containing a polynucleotide according to the invention, non-functional or carrying a mutation.
- the transgenic animals are preferably mammals, except humans, more preferably rodents, in particular mice or rats.
- the transgenic animals can be obtained by any conventional method known to a person skilled in the art, such as for example by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the chimeras affected at the level of the reproductive lines, and growth of said chimeras.
- the transformed host cells, the animals or the transgenic plants according to the invention can thus express or overexpress the gene coding for the protein according to the invention or their homologous gene or express said gene into which a mutation is introduced.
- Testis or brain cells, transformed host cells or transgenic organisms according to the invention can be used for the preparation of the protein according to the invention.
- the protein according to the invention particularly the protein
- Native ASAP can be purified according to techniques known to those skilled in the art.
- the protein can be purified from lysates and cell extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, particularly affinity chromatography, techniques immunoaffinity using specific monoclonal or polyclonal antibodies, etc.
- a subject of the invention is also a method for preparing the ASAP protein, characterized in that cells expressing the protein or cells transformed according to the present invention are cultivated, in particular mammalian cells or cells of organisms. transgenic according to the invention, under conditions allowing the expression of said protein, and that said protein is purified.
- the invention also relates to a protein, characterized in that it is capable of being obtained by any of the preparation methods described above.
- the subject of the invention is also a method of screening a substance capable of interacting in vitro, directly or indirectly, with the polynucleotide or the protein according to the invention, characterized in that: in a first step, the test substance and the polynucleotide or protein according to the invention and in a second step, the complex formed between said substance and the polynucleotide or protein according to the invention is detected by any appropriate means.
- the present invention also relates to a method of screening a substance capable of modulating (activating or inhibiting) the activity of the ASAP protein, characterized in that: - in a first step, cells of a biological sample expressing the ASAP protein with a test substance,
- the effect of said substance on the activity of said ASAP protein is measured by any suitable means, and - in a third step, substances capable of modulating said activity are selected.
- the term “activity of the ASAP protein” is intended to mean both the expression of the ASAP protein or of the transcripts
- the detection of the complex formed between said substance and the polynucleotide or the protein or the measurement of the effect of said substance on the activity of said ASAP protein can be carried out by conventional techniques of analysis of mRNA or proteins which are known in themselves; by way of nonlimiting example, the following techniques can be cited: RT-PCR, Northern-blot, Westem-blot, RIA, ELISA, immunoprecipitation, immunocytochemical or immunohistochemical analysis techniques.
- said measurement is carried out using probes, primers or antibodies, as defined above.
- Such substances can be biological macromolecules such as for example a nucleic acid, a lipid, a sugar, a protein, a peptide, a hybrid protein-lipid compound, protein-sugar, peptide-iipid or peptide-sugar, a protein or a peptide to which chemical ramifications have been added or chemical molecules.
- the invention also relates to the polynucleotide, the protein, the antibodies, the vectors or the cells transformed according to the invention, used as medicaments.
- the overexpression of the protein according to the invention blocks cell division and therefore cell proliferation. This makes it an excellent candidate for use as an anti-mitotic agent, usable for example in the treatment of cancer pathologies.
- the subject of the invention is also the use of the polynucleotide, of a vector or of the protein according to the invention, in the preparation of an anti-mitotic medicament.
- the overexpression of the protein according to the invention leads to disturbances in the organization of the mitotic spindle and induces aberrant and abrupt mitoses (plurinucleated cells, mono- or multipolar spindles).
- the subject of the invention is also the use of an antisense polynucleotide or an antisense fragment, of an antibody, of a vector containing an antisense oligonucleotide according to the invention, capable of '' inhibit the expression of the polynucleotide or protein according to the invention, in the preparation of a medicament intended for the treatment of pathologies linked to disturbances in the organization of the mitotic spindle and / or to an induction of aberrant and abortive mitoses ( plurinucleated cells, mono- or multipolar spindles) linked to the overexpression of the protein according to the invention.
- Figure 1 shows the chromosomal location and structure of the human asap gene.
- FIG. 2 shows the signals obtained by Northern blots on different human tissues after hybridization with a hASAP probe.
- RT-PCR obtained with primers corresponding to the mouse polynucleotide, orthologue of the polynucleotide SEQ ID NO: 15, from different mouse tissues.
- FIG. 4 represents the cellular localization of the hASAP protein coupled to the Green Fluorescent Protein (GFP) in 3 'or the Yellow Fluorescent Protein (YFP) in 5' or to a MYC tag on the N-terminal side (fusion column).
- GFP Green Fluorescent Protein
- YFP Yellow Fluorescent Protein
- MYC tag on the N-terminal side (fusion column).
- the nuclei are stained with propidium iodide or
- Hoechst 33286 (4A: 63x objective; 4B, 4C, 4D: I00X objective).
- FIG. 5 shows the co-location of the ASAP protein human with alpha-tubulin.
- Figure 5 A cellular localization of alpha-tubulin
- Figure 5 B localization of the ASAP protein
- Figure 5 C superposition of the 2 images showing the collocation of the 2 proteins.
- constFIS-1 R (5'-AAACACTTTTGCGAACACAGTTC-3 ') (SEQ ID NO: 34).
- Figure 1 shows the chromosomal location and structure of the human asap gene.
- the human asap gene consists of 29,750 nucleotides comprising 14 exons only 13 of which are translated, the first exon not being translated. The size of the exons ranges from 71 to 321 base pairs.
- the gene sequence is contained in the contig AC097467 (length 178204 base pairs) between the bases.
- the protein sequence was compared with the sequences of the databases using the PSI-BLAST and PHI-BLAST programs of the NCBI (http://www.ncbi.nIm.nih.gov/Sitemap/). Consensus protein motifs were sought using the DART programs of the NCBI and SMART of ExPASy-Tools (http://www.expasy.ch/tools/#similariw), whose parameters make it possible to detect patterns of low homology.
- the ASAP protein has a 23% sequence identity on the C-terminal third with a protein associated with microtubules (MAP 1A for Microtubule-Associated-Protein 1A).
- the ASAP protein has coiled-coil domains essentially grouped in its C-terminal part between amino acids 297 and 327 on the one hand and 477 and 628 on the other hand, indicating either that the protein oligomerizes, or that it interacts with other proteins.
- the tissues tested are: the spleen, thymus, prostate, testis, ovary, small intestine, colon, blood leukocytes, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. a.2) Results
- RNAs from different tissues of mice namely the brain, the heart, the colon, the liver, the small intestine, the skeletal muscle, the pancreas, the lung, the kidney, the spleen and the testicle.
- b.1 Obtaining the AD Ne .Qrth jpgue.de sgurjs
- RNAs of cells from different mouse tissues are extracted with the "mammalian total RNA kit" from the company Sigma.
- the RNAs are retro-transcribed with the Superscript II kit from the company Invitrogen according to the conditions prescribed by the supplier and using oligodT primers.
- the products obtained are checked by electrophoresis on 1% agarose gel.
- 1 ⁇ l of each sample thus obtained is in turn amplified by PCR (25 ⁇ l of reaction medium, 30 cycles (94 ° C for 15 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds)) with primers specific to the mouse asap gene (mFIS-1F, 5'-ACA ACG AAT AAC AGA GTG TCC-3 '(SEQ ID NO: 35) and mFIS-2R, 5'-ACT CCT GAT AAA CAG CTG CC-3' ( SEQ ID NO: 36).
- the amplified products obtained are analyzed by electrophoresis on a 1% agarose gel, stained with ethydium bromide and their size compared to a size marker deposited on the gel in parallel.
- the amplified products obtained are transferred by capillarity onto a charged nylon membrane in the buffer. 1.5M NaCl / 0.5M NaOH, according to the Southern technique (alkaline transfer).
- the membrane is then hybridized with a radiolabeled mASAP probe, (SEQ ID NO: 44), generated by amplification of the sequence contained in the mouse clone AW06131 selected after comparison of the human ASAP sequence in the databases (GenBank) (http : //expression.gnf.org/promoter/tissue/images/41739_s_at.png).
- the amplification was carried out by PCR (conditions as described above in which the reaction volume is 50 ⁇ l and the cold dCTP is at the concentration of 10 ⁇ M supplemented with 50 ⁇ Ci of ⁇ -P 32 - dCTP at 3000Ci / mmol), using the primers mFIS-1F (SEQ ID NO: 35) and mFis-2R (SEQ ID NO: 36). Hybridizations are carried out at 65 ° C. (in 6X SSC / 0.5% SDS / 5X Denhardt buffer). The membrane is rinsed with high stringency (0, IX SSC / 0.1% SDS), then analyzed by autoradiography or with the Phosphorlmager. b.2) .Results
- the hASAP cDNA obtained in Example 1 is inserted into three expression vectors:
- pEAK10-EGFP in phase with the Green Fluorescent Protein (GFP) fused in C-terminal (vector 1) (pEAK10, Edge vector Biosystems (distributed by Q.BIOgene, Illkirch in France) into which the EGFP protein (enhanced Green Fluorescent Protein) was introduced according to the reference Gaillard, I., et al., Eur. J. Neurosci., 15, 409-418, 2002);
- GLOMYC3-1 comprising a MYC tag on the N-terminal side (vector 3), vector derived from the vector pcDNA3.1 (Invitrogen), into which a 5 'untranslated region (5' UTR) and a MYC tag at Hindlll-BamHI sites, and the 3'UTR region of globin (Spel-Xbal fragment in the Xbal site).
- the hASAP cDNA is amplified from its initial cloning vector (pCR4-TOPO) by PCR using the high-fidelity polymerase pfu
- hFIS-Exp1 R (5'-GAAACACTTTTGCGAACACAGTTC-3 ') (SEQ ID NO: 38).
- the vector is cut with EcoRV and dephosphorylated: 10 ng of vector are used for ligation with the DNA insert.
- the PCR product is phosphorylated then purified on high PURE PCR kit (Roche): 100 ng of insert are used for ligation [12 h at 16 ° C in 10 ⁇ l final (Biolabs ligase), according to standard conditions (Sambrook and Russell) ].
- Glomyc-FIS1 F (5'-TAATGTCTGATGAAGTTTTTAGCACC-3 ') (SEQ ID NO:
- YFP-FIS1 F (5'-AATGTCTGATGAAG I I I I IAGCACC-3 ') (SEQ ID NO: 41)
- the recombinants are analyzed by PCR using a vector primer and an internal primer.
- PEAK-GFP annealing at 58 ° C, extension 45 sec. at 72 ° C. and standard conditions for the rest.
- YFP annealing 55 ° C for 1 min. ; Primers: YFP-2F (5'-
- the hASAP cDNA was cloned into the vector pGEX-4T2 (AMERSHAM), so as to produce a fusion protein with GST, purifiable according to the standard protocols.
- pGEX-4T2 AERSHAM
- Subcloning of the mASAP cDNA into a procarvote or eukaryotic expression vector Using a strategy similar to that used in paragraph a) above, the mASAP cDNA was cloned into the following vectors: - pGEX-4T2, (AMERSHAM), so as to produce a fusion protein with GST, purifiable according to standard protocols.
- the vectors obtained are transfected according to the calcium phosphate technique or more routinely using the jetPE1 process (GDSP10101, Qbiogene) according to the manufacturer's recommendations, in the following cell lines:
- transfected cells have division defects because the nuclei are always larger than in the non-transfected cells ( Figure 4A and 4B).
- some of the transfected cells appear to be plurinucleated ( Figure 4B). This suggests an abnormal division of the transfected cells.
- the localization profile of the hASAP and mASAP proteins, in a star, is characteristic of the nucleation of the microtubules in an aster around the centrosome ( Figures 4C and 4D).
- a similar localization profile for the ASAP protein is detected in the U-2 OS line (p53 +/-) overexpressing hASAP and in the non-transformed NI H 3T3 line overexpressing mASAP; an accumulation of monopolar cells in mitosis is observed.
- FIG. 5 illustrates the co-location of the human ASAP protein with alpha-tubulin; similarly the murine ASAP protein co-locates with alpha-tubulin.
- Figure 5A illustrates the cellular localization of alpha-tubulin detected by immunofluorescence using an anti-tubulin antibody (Alexa-594, Molecular Probe).
- Figure 5B illustrates the location of the yellow fluorescent protein (YFP) labeled ASAP protein.
- YFP yellow fluorescent protein
- Figure 5C represents the superposition of the 2 images demonstrating the colocation of the 2 proteins.
- the proteins were expressed in E. coli and purified according to standard protocols. Rabbits were then immunized with the purified ASAP proteins according to a standard protocol, and the immune sera were harvested. b) Analysis of the reactivity of polyclonal sera vis-à-vis the endogenous ASAP protein.
- the monospecific polyclonal serum directed against the whole hASAP protein detects a protein with an apparent molecular weight of approximately 110 kDa corresponding to the endogenous ASAP protein, both in HEK-293 cells and U-2 cells BONE.
- an anti-FLAG antibody detects a molecular weight protein equivalent, in HEK-293 or U-2 OS control cells, transfected with an expression vector of the hASAP protein fused with a label
- polyclonal monospecific serum directed against the whole hASAP protein marks the microtubules of cells
- HEK-293 in interphase, the asters of the cells in mitosis and the microtubules of the residual body at the end of telophase.
- the monospecific polyclonal serum directed against the hASAP protein deleted from its C-terminal part containing the potential MAP domain has the same profile in immunofluorescence and detects a protein of approximately 110 kDa, in Western blot.
- Monospecific polyclonal serum directed against the mASAP protein is used to detect which cell types express
- Nde1 residues 478-647 (SEQ ID NO: 50)) or of the C-terminal part containing the MAP domain (CdeM: residues 1 to 477 (SEQ ID NO: 51);
- Cdel2 residues 1 to 418 (SEQ ID NO: 52); Cdel3: residues 1 to 303 (SEQ ID NO: 53)) were amplified by PCR using appropriate primers and then cloned into the expression vectors pEAK10-EGFP (C-terminal fusion with GFP) and pEYFP- C1 (N-terminal fusion with YFP) according to a protocol similar to that described in Example 4.
- the 3 deleted constructs of the C-terminal part no longer colocalize in interphase with tubulin and no longer have a fibrous appearance; these results indicate that the deletion concerns a MAP domain.
- no monopolar cell blocked in mitosis is observed in the cells overexpressing the deleted mutants of the C-terminal part containing the MAP domain.
- the 3 deleted constructs of the N-terminal part containing the BRCT domain exhibit nuclear localization in the form of foci, but there remain in the cytoplasm some fibers co-localizing with tubulin.
- RNAi interfering RNA
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US10/540,493 US7402394B2 (en) | 2002-12-24 | 2003-12-24 | Centrosome-associated protein and applications thereof |
AU2003303464A AU2003303464A1 (en) | 2002-12-24 | 2003-12-24 | Centrosome-associated protein and applications thereof |
JP2004563311A JP4660200B2 (ja) | 2002-12-24 | 2003-12-24 | 中心体結合タンパク質およびその適用 |
CA002511436A CA2511436A1 (fr) | 2002-12-24 | 2003-12-24 | Proteine associee aux centrosomes et ses applications |
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WO2000073801A2 (fr) * | 1999-05-28 | 2000-12-07 | Ludwig Institute For Cancer Research | Antigenes associes au cancer du sein, de la prostate et de l'estomac et leur utilisation |
WO2002070539A2 (fr) * | 2001-03-05 | 2002-09-12 | Hyseq, Inc. | Nouveaux acides nucleiques et polypeptides |
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WO2002070539A2 (fr) * | 2001-03-05 | 2002-09-12 | Hyseq, Inc. | Nouveaux acides nucleiques et polypeptides |
Non-Patent Citations (6)
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DATABASE EM_HTG 18 décembre 2002 (2002-12-18), ADACHI, J. ET AL.: "Mus musculus adult male pituitary gland cDNA, RIKEN full-length enriched library, clone:5330427D05 product:weakly similar to CDNA: FLJ21159 FIS, CLONE CAS09969 [Homo sapiens], full insert sequence." XP002307713 Database accession no. AK030527 * |
DATABASE EM_HUM [Online] 25 octobre 2001 (2001-10-25), WATERSTONE R.H.: "Homo sapiens BAC clone RP11-27G13 from 4, complete sequence" XP002252293 Database accession no. AC097467 * |
DATABASE EM_HUM 29 septembre 2000 (2000-09-29), SUGANO S. ET AL.: "Homo sapiens cDNA: FLJ21159 fis, clone CAS09969" XP002288171 Database accession no. AK024812 cité dans la demande * |
DATABASE EMBL 5 novembre 2003 (2003-11-05), STRAUSBERG R.L. ET AL.: "Mus musculus RIKEN cDNA 5330427D05 gene, mRNA (cDNA clone IMAGE:30254438), partial cds." XP002307714 Database accession no. BC061216 * |
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FR2849039B1 (fr) | 2010-10-01 |
US20060216710A1 (en) | 2006-09-28 |
AU2003303464A8 (en) | 2004-07-22 |
US7402394B2 (en) | 2008-07-22 |
AU2003303464A1 (en) | 2004-07-22 |
FR2849039A1 (fr) | 2004-06-25 |
CA2511436A1 (fr) | 2004-07-15 |
WO2004058815A3 (fr) | 2005-03-17 |
JP2006526387A (ja) | 2006-11-24 |
EP1578792A2 (fr) | 2005-09-28 |
JP4660200B2 (ja) | 2011-03-30 |
WO2004058815B1 (fr) | 2005-05-06 |
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