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  • 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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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the invention concerns genomic and cDNA sequences of the human TBC-1 gene.
• the invention also concerns polypeptides encoded by the TBC-1 gene.
• the invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents.
• the invention further encompasses biallelic markers of the TBC-1 gene useful in genetic analysis.
• prostate cancer has dramatically increased over the last decades. It averages 30-50/100,000 males in Western European countries as well as within the US White male population. In these countries, it has recently become the most commonly diagnosed malignancy, being one of every four cancers diagnosed in American males. Prostate cancer's incidence is very much population specific, since it varies from 2/100,000 in China, to over 80/100,000 among African-American males. In France, the incidence of prostate cancer is 35/100,000 males and it is increasing by
• Prostate cancer is a latent disease. Many men carry prostate cancer cells without overt signs of disease. Autopsies of individuals dying of other causes show prostate cancer cells in 30 % of men at age 50 and in 60 % of men at age 80. Furthermore, prostate cancer can take up to 10 years to kill a patient after the initial diagnosis.
• the progression of the disease usually goes from a well-defined mass within the prostate to a breakdown and invasion of the lateral margins of the prostate, followed by metastasis to regional lymph nodes, and metastasis to the bone marrow. Cancer metastasis to bone is common and often associated with uncontrollable pain.
• prostate cancer is established when the disease has already metastasized to the bones.
• prostate cancers frequently grow more rapidly in sites of metastasis than within the prostate itself.
• Early-stage diagnosis of prostate cancer mainly relies today on Prostate Specific Antigen
• PSA dosage allows the detection of prostate cancer seven years before clinical symptoms become apparent.
• the effectiveness of PSA dosage diagnosis is however limited, due to its inability to discriminate between malignant and non-malignant affections of the organ and because not all prostate cancers give rise to an elevated serum PSA concentration.
• PSA dosage and other currently available approaches such as physical examination, tissue biopsy and bone scans are of limited value in predicting disease progression.
• a further consequence of the slow growth rate of prostate cancer is that few cancer cells are actively dividing at any one time, rendering prostate cancer generally resistant to radiation and chemotherapy.
• Surgery is the mainstay of treatment but it is largely ineffective and removes the ejaculatory ducts, resulting in impotence.
• Oral oestrogens and lutemizing releasing hormone analogs are also used for treatment of prostate cancer. These hormonal treatments provide marked improvement for many patients, but they only provide temporary relief. Indeed, most of these cancers soon relapse with the development of hormone-resistant tumor cells and the oestrogen treatment can lead to se ⁇ ous cardiovascular complications. Consequently, there is a strong need for preventive and curative treatment of prostate cancer.
• each of these steps compnses the deregulation of an important gene intervening m the normal metabolism of the cell and the emergence of an abnormal cellular sub-clone which overwhelms the other cell types because of a prohferative advantage.
• the genetic o ⁇ gm of this concept has found confirmation in the isolation and the characterization of genes which could be responsible. These genes, commonly called “cancer genes”, have an important role in the normal metabolism of the cell and are capable of intervening in carcmogenesis following a change.
• oncogenes genes whose products activate cell proliferation
• protooncogenes The normal non-mutant versions are called protooncogenes.
• the mutated forms are excessively or inappropriately active in promoting cell proliferation, and act in the cell in a dominant way in that a single mutant allele is enough to affect the cell phenotype.
• Activated oncogenes are rarely transmitted as germhne mutations since they may probably be lethal when expressed m all the cells. Therefore oncogenes can only be investigated in tumor tissues.
• the second group of genes which are frequently mutated in cancer called tumor suppressor genes, are genes whose products inhibit cell growth.
• Mutant versions in cancer cells have lost their normal function, and act in the cell in a recessive way m that both copies of the gene must be inactivated in order to change the cell phenotype. Most importantly, the tumor phenotype can be rescued by the wild type allele, as shown by cell fusion experiments first desc ⁇ bed by Harris and colleagues (1969). Germhne mutations of tumor suppressor genes may be transmitted and thus studied in both constitutional and tumor DNA from familial or sporadic cases.
• the current family of tumor suppressors includes DNA-binding transc ⁇ ption factors (i.e., p53, WT1), transc ⁇ ption regulators (i.e., RB, APC, probably BRCA1), protein kmase inhibitors (i.e., pl6), among others (for review, see Haber D & Harlow E, 1997).
• the third group of genes which are frequently mutated m cancer, called mutator genes, are responsible for maintaining genome integrity and/or low mutation rates. Loss of function of both alleles increases cell mutation rates, and as a consequence, proto-oncogenes and tumor suppressor genes may be mutated.
• Mutator genes can also be classified as tumor suppressor genes, except for the fact that tumo ⁇ genesis caused by this class of genes cannot be suppressed simply by restoration of a wild-type allele, as described above.
• Genes whose mactivation may lead to a mutator phenotype include mismatch repair genes (i.e., MLH1, MSH2), DNA hehcases (i.e., BLM, WRN) or other genes involved in DNA repair and genomic stability (i.e., p53, possibly BRCA1 and BRCA2) (For review see Haber D & Harlow E, 1997; Fishel R & Wilson T. 1997, Ellis NA.1997).
• the present invention pertains to nucleic acid molecules comprising the genomic sequence of a novel human gene which encodes a TBC-1 protein.
• the TBC-1 genomic sequences comprise regulatory sequence located upstream (5 '-end) and downstream (3 '-end) of the transcribed portion of said gene, these regulatory sequences being also part of the invention.
• the human TBC-1 genomic sequence is included in a previously unknown candidate region of prostate cancer located on chromosome 4.
• the invention also deals with the two complete cDNA sequences encoding the TBC-1 protein, as well as with the corresponding translation product.
• Ohgonucleotide probes or p ⁇ mers hybridizing specifically with a TBC-1 genomic or cDNA sequence are also part of the present invention, as well as DNA amplification and detection methods using said p ⁇ mers and probes.
• a further object of the invention consists of recombmant vectors comp ⁇ sing any of the nucleic acid sequences desc ⁇ bed above, and in particular of recombmant vectors comprising a TBC-1 regulatory sequence or a sequence encoding a TBC-1 protein, as well as of cell hosts and transgenic non human animals comprising said nucleic acid sequences or recombmant vectors.
• the invention also concerns a 7SC-7-related biallehc marker and the use thereof.
• the invention is directed to methods for the screening of substances or molecules that inhibit the expression of TBC-1, as well as with methods for the screening of substances or molecules that interact with a TBC-1 polypeptide.
• Figure 1 An amino acid alignment of a portion of the amino acid sequence of the TBC-1 protein of SEQ ID No 5 with other proteins sharing amino acid homology with TBC-1.
• the amino acid numbering refers to the murine TBC-1.
• SEQ ID No 1 contains a first part of the TBC-1 genomic sequence comprising the 5' regulatory sequence and the exons 1, Ibis, and 2.
• SEQ ID No 2 contains a second part of the TBC-1 genomic sequence comprising the 12 last exons of the TBC-1 gene and the 3 'regulatory sequence.
• SEQ LD No 3 contains a first cDNA sequence of the TBC-1 gene.
• SEQ ID No 4 contains a second cDNA sequence of the TBC-1 gene.
• SEQ ID No 5 contains the amino acid sequence encoded by the cDNAs of SEQ ID Nos 3 and 4.
• SEQ ID No 6 contains a primer containing the additional PU 5' sequence described further in Example 3.
• SEQ ID No 7 contains a primer containing the additional RP 5' sequence described further in Example 3.
• the following codes have been used in the Sequence Listing to indicate the locations of biallelic markers within the sequences and to identify each of the alleles present at the polymorphic base.
• the code “r” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is an adenine.
• the code “y” in the sequences indicates that one allele of the polymorphic base is a thymine, while the other allele is a cytosine.
• the code “m” in the sequences indicates that one allele of the polymorphic base is an adenine, while the other allele is an cytosine.
• the code “k” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is a thymine.
• the code “s” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is a cytosine.
• the code “w” in the sequences indicates that one allele of the polymorphic base is an adenine, while the other allele is an thymine.
• the nucleotide code of the original allele for each biallelic marker is the following: Biallelic marker Original allele
• the present invention concerns polynucleotides and polypeptides related to the human TBC-1 gene (also termed "TBC-1 gene" throughout the present specification) , which is potentially involved in the regulation of the differentiation of various cell types in mammals.
• TBC-1 gene also termed "TBC-1 gene” throughout the present specification
• a deregulation or an alteration of TBC-1 expression, or alternatively an alteration in the ammo acid sequence of the TBC-1 protein may be involved m the generation of a pathological state related to cell differentiation in a patient, more particularly to abnormal cell proliferation leading to cancer states, such as prostate cancer.
• the term "functionally active fragment" of the TBC-1 protein is intended to designate a polypeptide carrying at least one of the structural features of the TBC-1 protein involved in at least one of the biological functions and/or activity of the TBC-1 protein.
• a “heterologous” or “exogenous” polynucleotide designates a purified or isolated nucleic acid that has been placed, by genetic engmee ⁇ ng techniques, in the environment of unrelated nucleotide sequences, such as the final polynucleotide construct does not occur naturally.
• An illustrative, but not limitative, embodiment of such a polynucleotide construct may be represented by a polynucleotide comp ⁇ smg (1) a regulatory polynucleotide derived from the TBC-1 gene sequence and (2) a polynucleotide encoding a cytokme, for example GM-CSF.
• the polypeptide encoded by the heterologous polynucleotide will be termed an heterologous polypeptide for the purpose of the present invention.
• a “biologically active fragment or vanant" of a regulatory polynucleotide according to the present invention is intended a polynucleotide comprising or alternatively consisting in a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombmant polypeptide or a recombmant polynucleotide m a recombmant cell host.
• a sequence which is "operably linked" to a regulatory sequence such as a promoter means that said regulatory element is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the nucleic acid of interest.
• the promoter polynucleotide would be operably linked to a polynucleotide encoding a desired polypeptide or a desired polynucleotide if the promoter is capable of effecting transc ⁇ ption of the polynucleotide of interest.
• primer denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
• a primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transc ⁇ ptase.
• a substantially pure polypeptide typically comprises about 50%, preferably 60 to 90% weight/weight of a protein sample, more usually about 95%, and preferably is over about 99% pure.
• Polypeptide pu ⁇ ty or homogeneity is indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.
• single nucleotide polymorphism preferably refers to a single nucleotide substitution.
• the polymorphism can also involve an insertion or a deletion of at least one nucleotide, preferably between 1 and 5 nucleotides.
• heterozygosity rate of at least 0.32 even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42).
• a biallelic marker wherein the frequency of the less common allele is 30% or more is termed a "high quality biallelic marker".
• the invention also relates to va ⁇ ants and fragments of the polynucleotides described herein, particularly of a TBC-1 gene containing one or more biallelic markers according to the invention.
• Va ⁇ ants of polynucleotides are polynucleotides that differ from a reference polynucleotide.
• a variant of a polynucleotide may be a naturally occurring variant such as a naturally occur ⁇ ng allehc va ⁇ ant, or it may be a variant that is not known to occur naturally.
• Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences that are at least 95% identical to any of SEQ ID Nos 1-4 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ ID Nos 1-4 or the sequences complementary thereto, and preferably at least 98% 5 identical, more particularly at least 99.5% identical, and most preferably at least 99 9% identical to any of SEQ ID Nos 1 -4 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ ID Nos 1 -4 or the sequences complementary thereto
• a polynucleotide fragment is a polynucleotide having a sequence that entirely is the same 20 as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a TBC-1 gene, and variants thereof.
• the fragment can be a portion of an exon or of an intron of a TBC-1 gene. It can also be a portion of the regulatory sequences of the TBC-1 gene.
• such fragments comprise the polymo ⁇ hic base of a biallelic marker selected from the group consisting of the biallelic markers Al to A19 and the complements thereof. 25
• Such fragments may be "free-standing", i.e.
• polynucleotides not part of or fused to other polynucleotides, or they may be comprised withm a single larger polynucleotide of which they form a part or region. However, several fragments may be comprised with a single larger polynucleotide.
• the invention also relates to va ⁇ ants, fragments, analogs and derivatives of the polypeptides descnbed herein, including mutated TBC-1 proteins.
• the variant may be 1) one in which one or more of the ammo acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved ammo acid residue) and such substituted ammo acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the ammo acid residues includes a substituent group, or 3) one m which the mutated TBC-1 is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the mutated TBC-1, such as a leader or secretory sequence or a sequence which is employed for purification of the mutated TBC-1 or a preprotem sequence.
• Such variants are deemed to be withm
• ammo acids represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Tip, His.
• polypeptide fragments of the invention there may be mentioned those which comp ⁇ se at least about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 55 ammo acids of the TBC-1.
• the fragments contain at least one ammo acid mutation m the TBC-1 protein.
• percentage of sequence identity and “percentage homology” are used interchangeably herein to refer to compa ⁇ sons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comp ⁇ se additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comp ⁇ se additions or deletions) for optimal alignment of the two sequences.
• BLAST Basic Local Alignment Search Tool
• BLASTP and BLAST3 compare an ammo acid query sequence against a protein sequence database
• BLASTN compares a nucleotide query sequence against a nucleotide sequence database
• BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
• the BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user- specified percent homology.
• a user-specified threshold of significance such as a user- specified percent homology.
• the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karhn (see, e.g., Karhn and Altschul, 1990).
• Filters are hybridized for 48 h at 65°C, the preferred hyb ⁇ dization temperature, in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe
• the hybridization step can be performed at 65°C in the presence of SSC buffer, 1 x SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.
• filter washes can be done at 37°C for 1 h in a solution containing 2 x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1 X SSC at 50°C for 45 min.
• filter washes can be performed in a solution containing 2 x SSC and 0.1% SDS, or 0.5 x SSC and 0.1% SDS, or 0.1 x SSC and 0.1% SDS at 68°C for 15 mmute intervals.
• the hybridized probes are detectable by autoradiography.
• Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989; and Ausubel et al., 1989, are inco ⁇ orated herein m their entirety. These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length.
• the cDNA of the murine tbcl gene has been desc ⁇ bed in US Patent No US 5,700,927 and it encodes a putative protein product of 1141 ammo acids.
• the N-termmus of the murine tbcl protein contains stretches of cystemes and histidmes which may form zinc finger structures m the mature polypeptides.
• the N-termmus also comprises short stretches of basic ammo acids which may be involved m a nuclear localization signal.
• the TBC domain of the murine tbcl protein contains several tyrosine residues which are conserved in BUB2 and cdcl ⁇ .
• the inventors have sequenced two portions of the TBC-1 genomic sequence.
• the first portion of the TBC-1 gene sequence contains the three first exons of the TBC-1 gene, designated as Exon 1, Exon Ibis and Exon 2, and the 5' regulatory sequence located upstream of the transcribed sequences.
• the sequence of the first portion of the genomic sequence is disclosed in SEQ ID No 1.
• the second portion contains the twelve last exons of the TBC-1 gene, designated as exons A, B, C, D, E, F, G, H, I, J, K, and L, and the 3' regulatory sequence which is located downstream of the transcribed sequences.
• Intron 1 refers to the nucleotide sequence located between Exon 1 and Exon 2; Intron Ibis refers to the nucleotide sequence located between Exon Ibis and Exon 2; Intron A refers to the nucleotide sequence located between Exon A and Exon B; and so on.
• the position of the mtrons is detailed in Table A.
• the TBC-1 mtrons defined hereinafter for the pu ⁇ ose of the present invention are not exactly what is generally understood as "introns" by the one skilled in the art and will consequently be further defined below.
• the first spliced mRNA molecule comprises Exons 1 and 2
• the genomic nucleotide sequence comprised between Exon 1 and Exon 2 is an mtronic sequence as regards to this first mRNA molecule, despite the fact that this lntronic sequence contains Exon Ibis.
• Exon ⁇ b ⁇ s is of course an exonic nucleotide sequence as regards to the second TBC-1 mRNA molecule.
• the polynucleotides contained both m any of the nucleotide sequences of SEQ ID Nos 1 or 2 and in any of the nucleotide sequences of SEQ ID Nos 3 or 4 are considered as exonic sequences.
• the polynucleotides contained any of the nucleotide sequences of SEQ ID Nos 1 or 2 but which are absent both from the nucleotide sequence of SEQ ID No 3 and from the nucleotide sequence of SEQ ID No 4 are considered as mtronic sequences
• the nucleic acids defining the TBC-1 introns described above, as well as their fragments and variants, may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the TBC-1 gene in a test sample, or alternatively in order to amplify a target nucleotide sequence with the TBC-1 mtronic sequences.
• the invention embodies pu ⁇ fied, isolated, or recombmant polynucleotides comprising a nucleotide sequence selected from the group consisting of the mtrons of the TBC-1 gene, or a sequence complementary thereto.
• the invention also encompasses a pu ⁇ fied, isolated, or recombmant polynucleotide comprising a nucleotide sequence having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a sequence selected from the group consisting of SEQ ID Nos 1 and 2 or a complementary sequence thereto or a fragment thereof.
• the nucleotide differences as regards to the nucleotide sequence of SEQ ID Nos 1 or 2 may be generally randomly distributed throughout the entire nucleic acid. Nevertheless, prefe ⁇ ed nucleic acids are those wherein the nucleotide differences as regards to the nucleotide sequence of SEQ ID Nos 1 or 2 are predominantly located outside the coding sequences contained in the exons.
• nucleic acids as well as their fragments and vanants, may be used as oligonucleotide p ⁇ mers or probes in order to detect the presence of a copy of the TBC-1 gene in a 5 test sample, or alternatively order to amplify a target nucleotide sequence withm the TBC-1 sequences.
• Another object of the invention consists of a purified, isolated, or recombmant nucleic acid that hybridizes with a sequence selected from the group consisting of SEQ ID Nos 1 and 2 or a complementary sequence thereto or a variant thereof, under the stringent hybridization conditions as
• nucleic acids of the invention include isolated, pu ⁇ fied, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID Nos 1 and 2, or the complements thereof. Additionally prefe ⁇ ed
• 15 nucleic acids of the invention include isolated, purified, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 1-1000, 1001-2000, 2001-3000, 3001-4000, 4001-5000, 5001-6000, 6001-7000, 7001-8000, 8001-9000,
• nucleic acids of the invention include isolated, purified, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the
• SEQ ID No 2 25 following nucleotide positions of SEQ ID No 2: 1-5000, 5001-10000, 10001-15000, 15001-20000, 20001-25000, 25001-30000, 30001-35000, 35001-40000, 40001-45000, 45001-50000, 50001- 55000, 55001-60000, 60001-65000, 65001-70000, 70001-75000, 75001-80000, 80001-85000, 85001-90000, 90001-95000, and 95001-99960.
• nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of TBC-1 on either side or between two or more such genomic sequences.
• the inventors have discovered that the expression of the TBC-1 gene leads to the 35 production of at least two mRNA molecules, respectively a first and a second TBC-1 transcription product, as the results of alternative splicing events. They result from two distinct first exons, namely Exon 1 and Exon Ibis.
• the first transcription product comprises Exons 1, 2, A, B, C, D, E, F, G, H, I, J, K, and L.
• This cDNA of SEQ ID No 3 includes a 5'-UTR region, spanning the whole Exon 1 and part of Exon 2. This 5'-UTR region starts from the nucleotide at position 1 and ends at the nucleotide at position 170 of the nucleotide sequence of SEQ ID No 3.
• the cDNA of SEQ ID No 3 includes a 3'- UTR region starting from the nucleotide at position 3726 and ending at the nucleotide at position 3983 of the nucleotide sequence of SEQ ID No 3.
• This first transc ⁇ ption product harbors a polyadenylation signal located between the nucleotide at position 3942 and the nucleotide at position 3947 of the nucleotide sequence of SEQ ID No 3.
• the second TBC-1 transc ⁇ ption product comprises Exons Ibis, 2, A, B, C, D, E, F, G, H, I, J, K, and L.
• This cDNA of SEQ ID No 4 includes a 5'-UTR region starting from the nucleotide at position 1 and ending at the nucleotide at position 175 of the nucleotide sequence of SEQ ID No 4.
• This second cDNA also includes a 3'-UTR region starting from the nucleotide at position 3731 and ending at the nucleotide at position 3988 of the nucleotide sequence of SEQ ID No 4.
• This second transc ⁇ ption product harbors a polyadenylation signal located between the nucleotide at position 3947 and the nucleotide at position 3952 of the nucleotide sequence of SEQ JD No 4.
• the 5 '-end sequence of this second TBC-1 mRNA corresponds to the nucleotide sequence of a 5'-EST that has been obtained from a human pancreas cDNA library and characte ⁇ zed following the teachings of the PCT Application No WO 96/34981.
• This 5'-EST is also part of the invention.
• nucleic acid fragments of the nucleotide sequences of SEQ ID Nos 3 and 4 consist in polynucleotides comprising their respective Open Reading Frames encoding the TBC-1 protein.
• Other prefe ⁇ ed nucleic acid fragments of the nucleotide sequences of SEQ ID Nos 3 and 4 consist in polynucleotides comprising at least a part of their respective 5'-UTR or 3'-UTR regions.
• the invention also pertains to a purified or isolated nucleic acid having at least a 95% of nucleotide identity with any one of the nucleotide sequences of SEQ ID Nos 3 and 4, or a fragment thereof.
• Another object of the invention consists of pu ⁇ fied, isolated or recombmant nucleic acids comprising a polynucleotide that hybridizes, under the st ⁇ ngent hybridization conditions defined herein, with any one of the nucleotide sequences of SEQ ID Nos 3 and 4, or a sequence complementary thereto or a fragment thereof.
• the invention also relates to isolated, punfied, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID Nos 3 and 4, or the complements thereof.
• nucleic acids of the invention include isolated, pu ⁇ fied, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ TD No 3- 1-500, 501-1000, 1001-1500, 1501-2000, 2001- 2500, 2501-3000, 3001-3500, and 3501-3983.
• nucleic acid is notably useful as polynucleotide probe or p ⁇ mer specific for the TBC-1 gene or the TBC-1 mRNAs and cDNAs While this section is entitled " TBC-1 cDNA Sequences," it should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section, flanking the genomic sequences of TBC-1 on either side or between two or more such genomic sequences.
• the effective TBC-1 coding sequence is comprised between the nucleotide at position 171 and the nucleotide at position 3725 of SEQ ID No 3, and between the nucleotide at position 176 and the nucleotide at position 3730 of the nucleotide sequence of SEQ ID No 4.
• the invention further provides a purified or isolated nucleic acid comprising a polynucleotide selected from the group consisting of a polynucleotide compnsmg a nucleic acid sequence located between the nucleotide at position 171 and the nucleotide at position 3725 of SEQ ID No 3, and a polynucleotide comprising a nucleic acid sequence located between the nucleotide at position 176 and the nucleotide at position 3730 of SEQ ID No 4 or a variant or fragment thereof or a sequence complementary thereto.
• the present invention concerns a punfied or isolated nucleic acid encoding a human TBC-1 protein, wherein said TBC-1 protein comprises an ammo acid sequence of SEQ ID No 5, a nucleotide sequence complementary thereto, a fragment or a va ⁇ ant thereof.
• the present invention also embodies isolated, pu ⁇ fied, and recombmant polynucleotides which encode a polypeptides comprising a contiguous span of at least 6 ammo acids, preferably at least 8 or 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No 5
• the present invention embodies isolated, purified, and recombmant polynucleotides which encode a polypeptides comprising a contiguous span of at least 6 ammo acids, preferably at least 8 or 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 ammo acids of SEQ ID No 5 wherein said contiguous span includes at least 1 , 2, 3, 5 or 10 of the following ammo acid positions in SEQ ID No 5: 1-300, 301-600, 601-900, and 901-1168.
• the above disclosed polynucleotide that contains only coding sequences de ⁇ ved from the TBC-1 ORF may be expressed in a desired host cell or a desired host organism, when said polynucleotide is placed under the control of suitable expression signals.
• Such a polynucleotide, when placed under the suitable expression signals, may be inserted in a vector for its expression
• the invention further deals with a punfied or isolated nucleic acid comprising the nucleotide sequence of a regulatory region which is located either upstream of the first exon of the TBC-1 gene and which is contained in the TBC-1 genomic sequence of SEQ ID No 1, or downstream of the last exon of the TBC-1 gene and which is contained m the TBC-1 genomic sequence of SEQ ID No 2.
• the 5 '-regulatory sequence of the TBC-1 gene is localized between the nucleotide in position 1 and the nucleotide in position 2000 of the nucleotide sequence of SEQ ID No 1.
• the 3'- regulatory sequence of the TBC-1 gene is localized between nucleotide position 97961 and nucleotide position 99960 of SEQ ID No 2.
• Polynucleotides derived from the 5 ' and 3 ' regulatory regions are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID Nos 1 or 2 or a fragment thereof in a test sample.
• the promoter activity of the 5' regulatory regions contained in TBC-1 can be assessed as described below.
• Genomic sequences lying upstream of the TBC-1 Exons are cloned into a suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer, p ⁇ gal-Basic, p ⁇ gal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech.
• a suitable promoter reporter vector such as the pSEAP-Basic, pSEAP-Enhancer, p ⁇ gal-Basic, p ⁇ gal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech.
• each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, beta galactosidase, or green fluorescent protein.
• the sequences upstream of the TBC-1 coding region are inserted into the cloning sites upstream of the reporter gene m both orientations and introduced into an approp ⁇ ate host cell.
• the level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site.
• the presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert.
• the upstream sequences can be cloned into vectors which contain an enhancer for increasing transcnption levels from weak promoter sequences A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.
• Promoter sequences withm the upstream genomic DNA may be further defined by constructing nested deletions in the upstream DNA using conventional techniques such as
• the resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites withm the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter, individually or in combination. The effects of these mutations on transcnption levels may be determined by inserting the mutations into the cloning sites m the promoter reporter vectors.
• the minimal size of the promoter of the TBC-1 gene can be determined through the measurement of TBC-1 expression levels.
• an expression vector comprising decreasing sizes from the promoter generally ranging from 2 kb to 100 bp, with a 3 ' end which is constant, operably linked to TBC-1 coding sequence or to a reporter gene is used.
• Cells which are preferably prostate cells and more preferably prostate cancer cells, are transfected with this vector and the expression level of the gene is assessed.
• the strength and the specificity of the promoter of the TBC-1 gene can be assessed through the expression levels of the gene operably linked to this promoter m different types of cells and tissues.
• the efficacy of the promoter of the TBC-1 gene is assessed in normal and cancer cells.
• the efficacy of the promoter of the TBC-1 gene is assessed in normal prostate cells and m prostate cancer cells which can present different degrees of malignancy.
• Polynucleotides carrying the regulatory elements located both at the 5' end and at the 3' end of the TBC-1 cDNAs may be advantageously used to control the transc ⁇ ptional and translational activity of an heterologous polynucleotide of interest.
• the present invention also concerns a purified or isolated nucleic acid comp ⁇ smg a polynucleotide which is selected from the group consisting of the 5' and 3' regulatory regions, or a sequence complementary thereto or a biologically active fragment or variant thereof.
• 5' regulatory region refers to the nucleotide sequence located between positions 1 and 2000 of SEQ ID No 1.
• 3' regulatory region refers to the nucleotide sequence located between positions 97961 and 99960 of SEQ ID No 2.
• the invention also pertains to a pu ⁇ fied or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of the 5' and 3' regulatory regions, advantageously 99 % nucleotide identity, preferably 99.5%> nucleotide identity and most preferably 99.8%> nucleotide identity with a polynucleotide selected from the group consisting of the 5' and 3' regulatory regions, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.
• Another object of the invention consists of purified, isolated or recombmant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide selected from the group consisting of the nucleotide sequences of the 5'- and 3' regulatory regions, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.
• the 5'UTR and 3'UTR regions of a gene are of particular importance in that they often comprise regulatory elements which can play a role in providing approp ⁇ ate expression levels, particularly through the control of mRNA stability.
• a 5' regulatory polynucleotide of the invention may include the 5'-UTR located between the nucleotide at position 1 and the nucleotide at position 170 of SEQ ID No 3, or a biologically active fragment or vanant thereof.
• a 5'-regulatory polynucleotide of the invention may include the 5'-UTR located between the nucleotide at position 1 and the nucleotide at position 175 of SEQ ID No 4, or a biologically active fragment or variant thereof.
• a 3' regulatory polynucleotide of the invention may include the 3'-UTR located between the nucleotide at position 3726 and the nucleotide at position 3983 of SEQ ID No 4, or a biologically active fragment or variant thereof.
• the invention also pertains to a purified or isolated nucleic acid which is selected from the group consisting of : a) a nucleic acid comprising the nucleotide sequence of the 5' regulatory region; b) a nucleic acid compnsmg a biologically active fragment or va ⁇ ant of the nucleic acid of the 5' regulatory region.
• Preferred fragments of the nucleic acid of the 5' regulatory region have a length of about
• the invention further includes specific elements withm this regulatory region, these elements preferably including the promoter region.
• Preferred fragments of the 3' regulatory region are at least 50, 100, 150, 200, 300 or 400 bases m length.
• a "biologically active fragment or variant" of a TBC-1 regulatory polynucleotide according to the present invention is intended a polynucleotide comprising or alternatively consisting in a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombmant polypeptide or a recombmant polynucleotide in a recombmant cell host.
• a nucleic acid or polynucleotide is "functional" as a regulatory region for expressing a recombmant polypeptide or a recombmant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and if such sequences are "operatively linked" to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.
• An operable linkage is a linkage in which the regulatory nucleic acid and the DNA sequence sought to be expressed are linked in such a way as to permit gene expression.
• Regulatory polynucleotides of the invention may be prepared from any of the nucleotide sequences of SEQ ID Nos 1 or 2 by cleavage using the suitable rest ⁇ ction enzymes, the one skill in the art being guided by the book of Sambrook et al. (1989). Regulatory polynucleotides may also be prepared by digestion of any of the nucleotide sequences of SEQ ID Nos 1 or 2 by an exonuclease enzyme, such as Bal31 (Wabiko et al., 1986). These regulatory polynucleotides can also be prepared by chemical synthesis, as desc ⁇ bed elsewhere in the specification, when the synthesis of oligonucleotide probes or pnmers is disclosed.
• the regulatory polynucleotides according to the invention may be advantageously part of a recombmant expression vector that may be used to express a coding sequence in a desired host cell or host organism.
• the recombmant expression vectors according to the invention are desc ⁇ bed elsewhere m the specification.
• the invention also encompasses a polynucleotide comprising : a) a nucleic acid comprising a regulatory nucleotide sequence of the 5' regulatory region, or a biologically active fragment or variant thereof; b) a polynucleotide encoding a desired polypeptide or nucleic acid, operably linked to the nucleic acid comprising a regulatory nucleotide sequence of the 5' regulatory region, or its biologically active fragment or variant. c) Optionally, a nucleic acid comprising a 3' regulatory polynucleotide, preferably a 3 'regulatory polynucleotide of the invention.
• the desired polypeptide encoded by the above desc ⁇ bed nucleic acid may be of various nature or o ⁇ gin, encompassing proteins of prokaryotic or eukaryotic o ⁇ gin.
• polypeptides expressed under the control of a TBC-1 regulatory region it may be cited bactenal, fungal or viral antigens.
• eukaryotic proteins such as mtracellular proteins, such as "house keeping” proteins, membrane-bound proteins, like receptors, and secreted proteins like the numerous endogenous mediators such as cytokmes.
• the desired nucleic acid encoded by the above described polynucleotide usually a RNA molecule, may be complementary to a TBC-1 coding sequence and thus useful as an antisense polynucleotide.
• Such a polynucleotide may be mcluded in a recombmant expression vector in order to express a desired polypeptide or a desired polynucleotide in host cell or in a host organism Suitable recombmant vectors that contain a polynucleotide such as descnbed hereinbefore are disclosed elsewhere in the specification.
• the present invention also concerns a method for producing one of the polypeptides described herein, and especially a polypeptide of SEQ ID No 5 or a fragment or a variant thereof, wherein said method comprises the steps of : a) cultu ⁇ ng, in an appropnate culture medium, a cell host previously transformed or transfected with the recombmant vector comprising a nucleic acid encoding a TBC-1 polypeptide, or a fragment or a va ⁇ ant thereof; b) harvesting the culture medium thus conditioned or lyse the cell host, for example by sonication or by an osmotic shock; c) separating or purifying, from the said culture medium, or from the pellet of the resultant host cell lysate the thus produced polypeptide of interest. d) Optionally characterizing the produced polypeptide of interest.
• step a) is preceded by a step wherein the nucleic acid coding for a TBC-1 polypeptide, or a fragment or a va ⁇ ant thereof, is inserted in an appropriate vector, optionally after an appropriate cleavage of this amplified nucleic acid with one or several rest ⁇ ction endonucleases.
• the nucleic acid coding for a TBC-1 polypeptide or a fragment or a va ⁇ ant thereof may be the resulting product of an amplification reaction using a pair of primers according to the invention (by SDA, TAS, 3SR NASBA, TMA etc.).
• polypeptides according to the invention may be charactenzed by binding onto an lmmunoaffmity chromatography column on which polyclonal or monoclonal antibodies directed to a polypeptide of SEQ ID No 5, or a fragment or a va ⁇ ant thereof, have previously been immobilized.
• Purification of the recombmant proteins or peptides according to the present invention may be carried out by passage onto a Nickel or Cupper affinity chromatography column.
• the Nickel chromatography column may contain the Ni-NTA resin (Porath et al., 1975).
• the polypeptides or peptides thus obtained may be purified, for example by high performance liquid chromatography, such as reverse phase and/or catio c exchange HPLC, as described by Rougeot et al. (1994).
• the reason to prefer this kind of peptide or protein punfication is the lack of byproducts found in the elution samples which renders the resultant punfied protein or peptide more suitable for a therapeutic use.
• Another object of the present invention consists in a punfied or isolated TBC-1 polypeptide or a fragment or a va ⁇ ant thereof.
• the TBC-1 polypeptide comprises an amino acid sequence of SEQ ID No 5 or a fragment or a variant thereof.
• the present invention also embodies isolated, punfied, and recombmant polypeptides comprising a contiguous span of at least 6 ammo acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200 ammo acids of SEQ ID No 5.
• the present invention also embodies isolated, purified, and recombmant polypeptides comprising a contiguous span of at least 6 ammo acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200 ammo acids of SEQ ID No 5, wherein said contiguous span includes at least 1, 2, 3, 5 or 10 of the following ammo acid positions: 1-200, 201-400, 401-600, 601-800, 801-1000, 1001-1168.
• the invention also encompasses a punfied, isolated, or recombmant polypeptides comprising an ammo acid sequence having at least 90, 95, 98 or 99%o ammo acid identity with the ammo acid sequence of SEQ ID No 5 or a fragment thereof.
• the TBC-1 polypeptide of the invention possesses ammo acid homologies as regards to the murine TBC-1 protein of 1141 ammo acids in length which is described in US Patent No US 5,700,927.
• the TBC-1 protein of the invention also possesses some homologies with two other proteins : the Pollux drosophila protein (Zhang et al., 1996) and the CDC16 protein from Caenorhabditis elegans (Wilson et al., 1994).
• Figure 1 represents an ammo acid alignment of a portion of the ammo acid sequence of the TBC-1 protein of SEQ ID No 5 with other proteins shanng ammo acid homology with TBC-1.
• the upper line shows the whole ammo acid sequence of the murine tbc-1 protein descnbed m US Patent No US 5,700,927; the second line represents part of the ammo acid sequence of the TBC-1 protein of SEQ ID No 5; the third line (Genbank access No : dmu50542) depicts the ammo acid sequence of the Pollux protein mentioned above; the fourth line (Genbank access No : cel ⁇ 5h!2) shows the ammo acid sequence of the C elegans protein mentioned above; the fifth line presents positions in which consensus ammo acids are identified, i.e. ammo acids shared by the sequences presented m the four upper lines, when present
• the TBC-1 polypeptide of the ammo acid sequence of SEQ ID No 5 has 1168 ammo acids in length.
• the TBC-1 polypeptide includes a "TBC domain" which is spanning from the amino acid in position 786 to the ammo acid in position 974 of the ammo acid sequence of SEQ ID No 5.
• This TBC domain is represented m Figure 1 as a grey area spanning from the ammo acid numbered 758 to the ammo acid numbered 949. This TBC domain is likely to regulate protem-protem interactions.
• the TBC-1 TBC domain includes the ammo acid sequence EVGYCQGL, spanning from the ammo acid in position 886 to the amino acid in position 893 of the amino acid sequence of SEQ ID No 5.
• the EVGYCQGL ammo acid sequence spans from the ammo acid numbered 861 to the amino acid numbered 868 of Figure 1. This site may interact with a kmase. Based on the structural simila ⁇ ty to cdcl ⁇ , a yeast regulator of mitosis, TBC-1 is likely to regulate mitosis and cytokinesis by interacting with other proteins which also participate with the regulation of mitosis, cytokinesis and septum formation.
• Prefe ⁇ ed polypeptides of the invention comprise the TBC domain of TBC-1, or alternatively at least the EVGYCQGL ammo acid sequence motif.
• a further object of the present invention concerns a punfied or isolated polypeptide which is encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOS 1, 2, 3, and 4 or fragments or variants thereof.
• TBC-1 protein A single variant molecule of the TBC-1 protein is explicitly excluded from the scope of the present invention, which is a polypeptide having the same ammo acid sequence than the murme tbcl protein descnbed in the US Patent No 5,700,927.
• Amino acid deletions, additions or substitutions in the TBC-1 protein are preferably located outside of the TBC domain as defined above. Most preferably, a mutated TBC-1 protein has an intact "EVGYCQGL" amino acid motif.
• Such a mutated TBC-1 protein may be the target of diagnostic tools, such as specific monoclonal or polyclonal antibodies, useful for detecting the mutated TBC-1 protein in a sample.
• the invention also encompasses a TBC-1 polypeptide or a fragment or a variant thereof in which at least one peptide bound has been modified as described in the "Definitions" section.
• Any TBC-1 polypeptide or whole protein may be used to generate antibodies capable of specifically binding to an expressed TBC-1 protein or fragments thereof as descnbed.
• One antibody composition of the invention is capable of specifically binding or specifically bind to the variant of the TBC-1 protein of SEQ ID No 5.
• an antibody composition to specifically bind to TBC-1 it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for TBC-1 protein than for another protein m an ELISA, RIA, or other antibody-based binding assay.
• the invention concerns antibody compositions, either polyclonal or monoclonal, capable of selectively binding, or selectively bind to an epitope-containmg a polypeptide comprising a contiguous span of at least 6 ammo acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, 100, 150 or 200 ammo acids of SEQ ID No 5;
• said epitope comprises at least 1, 2, 3, 5 or 10 of the following amino acid positions : 1-200, 201-400, 401-600, 601-800, 801-1000, 1001-1168.
• the invention also concerns a punfied or isolated antibody capable of specifically binding to a mutated TBC-1 protein or to a fragment or variant thereof comprising an epitope of the mutated TBC-1 protein.
• the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive ammo acids of a TBC-1 protein and including at least one of the ammo acids which can be encoded by the trait causing mutations.
• the antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known m the art.
• TBC-1 polypeptide of SEQ JJD No 5 or a fragment thereof can be used for the preparation of polyclonal or monoclonal antibodies.
• the TBC-1 polypeptide expressed from a DNA sequence comprising at least one of the nucleic acid sequences of SEQ ID Nos 1, 2, 3 and 4 may also be used to generate antibodies capable of specifically binding to the TBC-1 polypeptide of SEQ ID No 5 or a fragment thereof .
• Prefe ⁇ ed antibodies according to the invention are prepared using TBC-1 peptide fragments that do not comprise the EVGYCQGL ammo acid motif
• prefe ⁇ ed antibodies of the invention are prepared using TBC-1 peptide fragments that do not comprise the TBC domain defined elsewhere m the specification.
• the antibodies may be prepared from hyb ⁇ domas according to the technique desc ⁇ bed by Kohler and Milstem in 1975.
• the polyclonal antibodies may be prepared by immunization of a mammal, especially a mouse or a rabbit, with a polypeptide according to the invention that is combined with an adjuvant of immunity, and then by punfymg of the specific antibodies contained m the semm of the immunized animal on a affinity chromatography column on which has previously been immobilized the polypeptide that has been used as the antigen.
• the present invention also includes, chime ⁇ c single chain Fv antibody fragments (Marhneau et al., 1998), antibody fragments obtained through phage display libra ⁇ es (Ridder et al., 1995; Vaughan et al., 1995) and humanized antibodies (Remmann et al., 1997; Leger et al., 1997).
• Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi -quantitatively or qualitatively to identify the presence of antigen m a biological sample.
• the antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
• the invention is also directed to a method for detecting specifically the presence of a TBC-1 polypeptide according to the invention in a biological sample, said method comprising the following steps : a) bringing into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a TBC-1 polypeptide comprising an ammo acid sequence of SEQ ID No 5, or to a peptide fragment or vanant thereof; and b) detecting the antigen-antibody complex formed.
• the invention also concerns a diagnostic kit for detecting in vitro the presence of a TBC-1 polypeptide according to the present invention m a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a TBC-1 polypeptide comprising an ammo acid sequence of SEQ ID No 5, or to a peptide fragment or variant thereof, optionally labeled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.
• the inventors have discovered nucleotide polymo ⁇ hisms located withm the genomic DNA containing the TBC-1 gene, and among them SNP that are also termed biallelic markers.
• the biallelic markers of the invention can be used for example for the generation of genetic map, the linkage analysis, the association studies.
• a second method for generating biallelic markers the DNA samples are not pooled and are therefore amplified and sequenced individually. The resulting nucleotide sequences obtained are then also analyzed to identify significant polymo ⁇ hisms. It will readily be appreciated that when this second method is used, a substantially higher number of DNA amplification reactions must be ca ⁇ ied out. It will further be appreciated that including such potentially less informative biallelic markers in association studies to identify potential genetic associations with a trait may allow m some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations. This method is usually prefe ⁇ ed when biallelic markers need to be identified in order to perform association studies withm candidate genes
• the genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals co ⁇ espondmg to a heterogeneous population of known ethnic background, or from familial cases.
• the number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, preferably from about 50 to about 200 individuals. It is usually prefe ⁇ ed to collect DNA samples from at least about 100 individuals in order to have sufficient polymo ⁇ hic diversity in a given population to generate as many markers as possible and to generate statistically significant results.
• Example 2 The techniques of DNA extraction are well-known to the skilled technician Details of a prefe ⁇ ed embodiment are provided in Example 2.
• DNA samples can be pooled or unpooled for the amplification step.
• DNA amplification techniques are well-known to those skilled in the art.
• Amplification techniques that can be used in the context of the present invention include, but are not limited to, the hgase chain reaction (LCR) descnbed in EP-A- 320 308, WO 9320227 and EP-A-439 182, the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) descnbed in Guatelh J.C., et al.(1990) and in Compton J.(1991), Q-beta amplification as descnbed in European Patent Application No 4544610, strand displacement amplification as descnbed in Walker et al.(1996) and EP A 684 315 and, target mediated amplification as desc ⁇ bed in PCT Publication WO 9322461.
• LCR hgase chain reaction
• PCR polymerase chain reaction
• RT-PCR polymerase chain reaction
• NASBA nucleic acid sequence based amplification
• NASBA nucleic acid
• LCR and Gap LCR are exponential amplification techniques, both depend on DNA hgase to join adjacent pnmers annealed to a DNA molecule.
• probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target.
• the first probe hybridizes to a first segment of the target strand and the second probe hybndizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate- 3 'hydroxyl relationship, and so that a ligase can covalently fuse or hgate the two probes into a fused product.
• a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hyb ⁇ dize to a portion of the second probe in a similar abutting fashion.
• the secondary probes also will hybridize to the target complement in the first instance.
• the third and fourth probes Once the hgated strand of pnmary probes is separated from the target strand, it will hybndize with the third and fourth probes, which can be hgated to form a complementary, secondary hgated product. It is important to realize that the hgated products are functionally equivalent to either the target or its complement.
• Gap LCR is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.
• RT-PCR polymerase chain reaction
• AGLCR is a modification of GLCR that allows the amplification of RNA.
• PCR technology is the prefe ⁇ ed amplification technique used in the present invention.
• a variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White (1997) and the publication entitled “PCR Methods and Applications” (1991 , Cold Spnng Harbor Laboratory Press).
• PCR pnmers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
• the nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample.
• the hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the pnmer sites.
• PCR has further been described in several patents including US Patents 4,683,195; 4,683,202; and 4,965,188.
• the PCR technology is the prefe ⁇ ed amplification technique used to identify new biallelic markers.
• a typical example of a PCR reaction suitable for the pu ⁇ oses of the present invention is provided in Example 3.
• One of the aspects of the present invention is a method for the amplification of a TBC-1 gene, particularly the genomic sequences of SEQ ID Nos 1 and 2 or of the cDNA sequence of SEQ ID Nos 3 or 4 or a fragment or variant thereof in a test sample, preferably using the PCR technology.
• the method comprises the steps of contacting a test sample suspected of containing the target TBC-1 sequence or portion thereof with amplification reaction reagents comprising a pair of amplification primers.
• the present invention also relates to a method for the amplification of a TBC-1 gene sequence, particularly of a fragment of the genomic sequence of SEQ ID No 1 or of the cDNA sequence of SEQ ID No 2 or 3, or a fragment or a vanant thereof in a test sample, said method comprising the steps of : a) contacting a test sample suspected of containing the targeted TBC-1 gene sequence or portion thereof with amplification reaction reagents comprising a pair of amplification primers located on either side of the TBC-1 region to be amplified, and b) optionally, detecting the amplification products
• the invention also concerns a kit for the amplification of a TBC-1 gene sequence, particularly of a portion of the genomic sequence of SEQ ID Nos 1 or 2, or of the cDNA sequence of SEQ ID Nos 3 or 4, or a variant thereof in a test sample, wherein said kit comprises: a) a pair of oligonucleotide pnmers located on either side of the TBC-1
• the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region.
• p ⁇ mers comprise a sequence which is selected from the group consisting of Bl to B15, Cl to C15, Dl to D19, and El to E19.
• biallelic markers are identified using genomic sequence information generated by the inventors
• Sequenced genomic DNA fragments are used to design pnmers for the amplification of 500 bp fragments
• These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers.
• P ⁇ mers may be designed using the OSP software (Hillier L. and Green P., 1991) All pnmers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing pnmer.
• Those skilled in the art are familiar with primer extensions, which can be used for these pu ⁇ oses.
• Prefe ⁇ ed pnmers useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes A biallelic marker presents a higher probability to be an eventual causal mutation if it is located m these functional regions of the gene.
• Prefe ⁇ ed amplification p ⁇ mers of the invention include the nucleotide sequences of Bl to B15 and Cl to C15 further detailed m Example 3.
• the amplification products generated as desc ⁇ bed above with the primers of the invention are then sequenced using methods known and available to the skilled technician.
• the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye- primer cycle sequencing protocol.
• sequence data are automatically processed with adequate software to assess sequence quality.
• a polymo ⁇ hism analysis software is used that detects the presence of biallelic sites among individual or pooled amplified fragment sequences.
• Polymo ⁇ hism search is based on the presence of supenmposed peaks in the electrophoresis pattern These peaks which present distinct colors co ⁇ espond to two different nucleotides at the same position on the sequence.
• the polymo ⁇ hism has to be detected on both strands for validation 19 biallelic markers were found in the TBC-1 gene They are detailed m the Table 2 They are located in mtronic regions.
• the polymo ⁇ hisms identified above can be further confirmed and their respective frequencies can be determined through various methods using the previously descnbed pnmers and probes These methods can also be useful for genotyping either new populations in association studies or linkage analysis or individuals in the context of detection of alleles of biallelic markers which are known to be associated with a given trait The genotyping of the biallelic markers is also important for the mapping.
• the methods described below can be equally performed on individual or pooled DNA samples
• biallelic markers described previously allows the design of appropnate oligonucleotides, which can be used as probes and pnmers, to amplify a TBC-1 gene containing the polymo ⁇ hic site of interest and for the detection of such polymo ⁇ hisms.
• the biallelic markers according to the present invention may be used in methods for the identification and characterization of an association between alleles for one or several biallelic markers of the sequence of the TBC-1 gene and a trait
• the identified polymo ⁇ hisms, and consequently the biallelic markers of the invention may be used in methods for the detection in an individual of TBC-1 alleles associated with a trait, more particularly a trait related to a cell differentiation or abnormal cell proliferation disorders, and most particularly a trait related to cancer diseases, specifically prostate cancer.
• the invention encompasses methods of genotyping compnsing determining the identity of a nucleotide at a 7BC-/-related biallelic marker or the complement thereof in a biological sample; optionally, wherein said 7BC-7-related biallelic marker is selected from the group consisting of A 1 to A 19, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said biological sample is denved from a single subject; optionally, wherein the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome; optionally, wherein said biological sample is denved from multiple subjects;
• the genotyping methods of the invention encompass methods with any further limitation descnbed in this disclosure, or those following, specified alone or m any combination;
• said method is performed in vitro, optionally, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step,
• said method is performed
• nucleic acids in purified or non-pu ⁇ fied form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired.
• DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above. While nucleic acids for use in the genotyping methods of the invention can be denved from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human. Amplification Of DNA Fragments Comprising Biallelic Markers
• Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention
• amplification of DNA fragments comprising biallelic markers may be used m various methods and for vanous pu ⁇ oses and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention. Amplification of DNA may be achieved by any method known in the art. Amplification techniques are described above in the section entitled, "Identification of IBC-7-related biallelic markers.”
• Some of these amplification methods are particularly suited for the detection of single nucleotide polymo ⁇ hisms and allow the simultaneous amplification of a target sequence and the identification of the polymo ⁇ hic nucleotide as it is further desc ⁇ bed below.
• the identification of biallelic markers as described above allows the design of appropnate oligonucleotides, which can be used as pnmers to amplify DNA fragments comprising the biallelic markers of the present invention.
• Amplification can be performed using the primers initially used to discover new biallelic markers which are descnbed herein or any set of p ⁇ mers allowing the amplification of a DNA fragment compnsing a biallelic marker of the present invention.
• the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention.
• Prefe ⁇ ed amplification primers are listed m Example 2. It will be appreciated that the p ⁇ mers listed are merely exemplary and that any other set of p ⁇ mers which produce amplification products containing one or more biallelic markers of the present invention are also of use.
• the spacing of the p ⁇ mers determines the length of the segment to be amplified
• amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp.
• Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are prefe ⁇ ed and fragments from 100-600 bp are highly prefe ⁇ ed. It will be appreciated that amplification p ⁇ mers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification p ⁇ mers may be labeled or immobilized on a solid support as described in "Oligonucleotide probes and primers".
• any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often prefe ⁇ ed at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods.
• Methods well-known to those skilled m the art that can be used to detect biallelic polymo ⁇ hisms include methods such as, conventional dot blot analyzes, single strand conformational polymo ⁇ hism analysis (SSCP) described by O ⁇ ta et al.(1989), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as descnbed in Sheffield et al.(1991), White et al.(1992), Grompe et al.(1989 and 1993).
• Another method for determining the identity of the nucleotide present at a particular polymo ⁇ hic site employs a specialized exonuclease-resistant nucleotide de ⁇ vative as descnbed in US patent 4,656,127.
• Prefe ⁇ ed methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some prefe ⁇ ed methods.
• a highly prefe ⁇ ed method is the microsequencing technique.
• the term "sequencing" is generally used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.
• the nucleotide present at a polymo ⁇ hic site can be determined by sequencing methods.
• DNA samples are subjected to PCR amplification before sequencing as described above.
• DNA sequencing methods are described in "Sequencing Of Amplified Genomic DNA And Identification Of Single Nucleotide Polymo ⁇ hisms".
• the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-pnmer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site.
• microsequencing Assays the nucleotide at a polymo ⁇ hic site m a target DNA is detected by a single nucleotide primer extension reaction
• This method involves appropriate microsequencing primers which, hybndize just upstream of the polymo ⁇ hic base of interest in the target nucleic acid.
• a polymerase is used to specifically extend the 3' end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymo ⁇ hic site.
• the identity of the inco ⁇ orated nucleotide is determined m any suitable way.
• microsequencing reactions are ca ⁇ ied out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the inco ⁇ orated nucleotide as described in EP 412 883, the disclosure of which is inco ⁇ orated herein by reference in its entirety.
• capillary electrophoresis can be used in order to process a higher number of assays simultaneously.
• An example of a typical microsequencing procedure that can be used in the context of the present invention is provided m Example 4.
• a homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (1997) and Chen et al.(1997).
• amplified genomic DNA fragments containing polymorphic sites are incubated with a 5'-fluorescein-labeled primer in the presence of allehc dye-labeled dideoxy ⁇ bonucleoside t ⁇ phosphates and a modified Taq polymerase.
• allehc dye-labeled dideoxy ⁇ bonucleoside t ⁇ phosphates and a modified Taq polymerase The dye-labeled pnmer is extended one base by the dye-termmator specific for the allele present on the template.
• the fluorescence intensities of the two dyes m the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time.
• the extended pnmer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymo ⁇ hic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, 1997).
• Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof.
• Alternative methods include several solid-phase microsequencing techniques.
• the basic microsequencing protocol is the same as desc ⁇ bed previously, except that the method is conducted as a heterogeneous phase assay, in which the pnmer or the target molecule is immobilized or captured onto a solid support.
• oligonucleotides are attached to solid supports or are modified m such ways that permit affinity separation as well as polymerase extension.
• the 5' ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotmylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the inco ⁇ orated terminator regent. This eliminates the need of physical or size separation More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template.
• immobilization can be ca ⁇ ied out via an interaction between biotinylated DNA and streptavidm- coated microtitration wells or avidin-coated polystyrene particles
• oligonucleotides or templates may be attached to a solid support in a high-density format.
• inco ⁇ orated ddNTPs can be radiolabeled (Syvanen, 1994) or linked to fluorescem (Livak and Hamer, 1994). The detection of radiolabeled ddNTPs can be achieved through scmtillation-based techniques.
• the detection of fluorescem-lmked ddNTPs can be based on the binding of antifluorescem antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as 7-n ⁇ trophenyl phosphate)
• a chromogenic substrate such as 7-n ⁇ trophenyl phosphate
• Other possible reporter-detection pairs include: ddNTP linked to dmitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotinylated ddNTP and horseradish peroxidase- conjugated streptavidm with o-phenylenediamme as a substrate (WO 92/15712).
• Nyren et al.(1993) desc ⁇ bed a method relying on the detection of DNA polymerase activity by an enzymatic lummometnc inorganic pyrophosphate detection assay (ELIDA).
• ELIDA enzymatic lummometnc inorganic pyrophosphate detection assay
• Pastinen et al.(1997) describe a method for multiplex detection of single nucleotide polymo ⁇ hism in which the solid phase mmisequencmg principle is applied to an oligonucleotide a ⁇ ay format.
• High-density a ⁇ ays of DNA probes attached to a solid support (DNA chips) are further desc ⁇ bed below.
• the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay.
• Prefe ⁇ ed microsequencing primers include the nucleotide sequences Dl to D15 and El to El 5. It will be appreciated that the microsequencing primers listed in Example 5 are merely exemplary and that, any primer having a 3' end immediately adjacent to the polymorphic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention.
• One aspect of the present invention is a solid support which includes one or more microsequencing p ⁇ mers listed in Example 5, or fragments compnsing at least 8, 12, 15, 20, 25, 30, 40, or 50 consecutive nucleotides thereof, to the extent that such lengths are consistent with the primer described, and having a 3' terminus immediately upstream of the co ⁇ esponding biallelic marker, for determining the identity of a nucleotide at a biallelic marker site.
• the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or hgases These assays are based on the specificity of polymerases and hgases.
• Allele Specific Amplification Primers Discnmination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele.
• allele specific amplification at least one member of the pair of primers is sufficiently complementary with a region of a TBC-1 gene comprising the polymo ⁇ hic base of a biallelic marker of the present invention to hybridize therewith and to initiate the amplification
• Such primers are able to disc ⁇ mmate between the two alleles of a biallelic marker This is accomplished by placing the polymorphic base at the 3' end of one of the amplification primers.
• OLA Oligonucleotide Ligation Assay
• OLA uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules.
• One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected.
• OLA is capable of detecting single nucleotide polymo ⁇ hisms and may be advantageously combined with PCR as described by Nickerson et al.(1990).
• PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
• Other amplification methods which are particularly suited for the detection of single nucleotide polymo ⁇ hism include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in "DNA Amplification”.
• LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase.
• LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site.
• either oligonucleotide will be designed to include the biallelic marker site
• the reaction conditions are selected such that the oligonucleotides can be hgated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide.
• the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a "gap" is created as described in WO 90/01069.
• Ligase/Polymerase-mediated Genetic Bit AnalysisTM is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271).
• This method involves the inco ⁇ oration of a nucleoside t ⁇ phosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a pnmer molecule, and their subsequent ligation to a second oligonucleotide
• the reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.
• a prefe ⁇ ed method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization.
• the hybridization probes which can be conveniently used m such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., 1989).
• Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pamng. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to disc ⁇ mmate between different alle c forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the ongmal allele and the other showing a perfect match to the target sequence containing the alternative allele.
• Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hyb ⁇ dizes to only one of the alleles.
• Stringent, sequence specific hyb ⁇ dization conditions under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., 1989).
• Stringent conditions are sequence dependent and will be different m different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
• the target DNA comprising a biallelic marker of the present invention may be amplified pnor to the hybridization reaction.
• the presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA.
• the detection of hybrid duplexes can be earned out by a number of methods.
• Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybnd duplexes
• hybridization duplexes are separated from unhyb ⁇ dized nucleic acids and the labels bound to the duplexes are then detected.
• wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
• standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.
• the TaqMan assay takes advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product.
• TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase du ⁇ ng amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.
• molecular beacons are hai ⁇ in-shaped oligonucleotide probes that report the presence of specific nucleic acids homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., 1998).
• the polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples.
• These probes are charactenzed in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide vanation.
• a particularly prefe ⁇ ed probe is 25 nucleotides in length.
• the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe.
• the biallelic marker is at the center of said polynucleotide.
• Prefe ⁇ ed probes comprise a nucleotide sequence selected from the group consisting of amphcons listed in Table 1 and the sequences complementary thereto, or a fragment thereof, said fragment compnsing at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymo ⁇ hic base.
• Prefe ⁇ ed probes comprise a nucleotide sequence selected from the group consisting of PI to P7, P9 to PI 3, PI 5 to PI 9 and the sequences complementary thereto.
• the polymo ⁇ hic base(s) are within 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.
• the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in “Oligonucleotide Probes and Primers”. The probes can be non-extendable as described in “Oligonucleotide Probes and Primers”.
• hybridization assays By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.
• High-Throughput parallel hybridization in a ⁇ ay format is specifically encompassed within "hybridization assays" and are described below.
• Hybridization assays based on oligonucleotide a ⁇ ays rely on the differences m hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence vanants. Efficient access to polymo ⁇ hism information is obtained through a basic structure comprising high-density a ⁇ ays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions.
• a solid support e.g., the chip
• Each DNA chip can contain thousands to millions of individual synthetic DNA probes a ⁇ anged in a grid-like pattern and miniaturized to the size of a dime.
• the chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken m the BRCA1 gene, in S.
• Chips of vanous formats for use in detecting biallelic polymo ⁇ hisms can be produced on a customized basis by Affymet ⁇ x (GeneChipTM), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.
• a ⁇ ays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymo ⁇ hic marker.
• EP 785280 descnbes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, a ⁇ ays may generally be "tiled" for a large number of specific polymo ⁇ hisms.
• tileing is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected vanations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of nucleotides. Tiling strategies are further descnbed m PCT application No. WO 95/11995.
• a ⁇ ays are tiled for a number of specific, identified biallelic marker sequences.
• the a ⁇ ay is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers.
• a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymo ⁇ hism. To ensure probes that are complementary to each allele, the probes are synthesized m pairs differing at the biallelic marker.
• monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymo ⁇ hism, substituted with the remaining nucleotides (selected from A, T, G, C and U).
• the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker.
• the monosubstituted probes provide internal controls for the tiled a ⁇ ay, to distinguish actual hybridization from artefactual cross-hybridization.
• the a ⁇ ay is scanned to determine the position on the a ⁇ ay to which the target sequence hybridizes.
• the hybridization data from the scanned a ⁇ ay is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample.
• Hybndization and scanning may be ca ⁇ ied out as described in PCT application No. WO 92/10092 and WO 95/11995 and US patent No. 5,424,186.
• the chips may comprise an a ⁇ ay of nucleic acid sequences of fragments of about 15 nucleotides length.
• the chip may comprise an a ⁇ ay including at least one of the sequences selected from the group consisting of amplicons listed m table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymo ⁇ hic base.
• the polymo ⁇ hic base is withm 5, 4, 3, 2, 1 , nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.
• the chip may comprise an a ⁇ ay of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention.
• Integrated Systems Another technique, which may be used to analyze polymo ⁇ hisms, includes multicomponent integrated systems, which mmiatunze and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.
• An example of such technique is disclosed in US patent 5,589,136, which descnbes the integration of PCR amplification and capillary electrophoresis in chips.
• Integrated systems can be envisaged mamly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip.
• the movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.
• the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser- induced fluorescence detection.
• TBC-1 The identification of genes involved in suspected heterogeneous, polygemc and multifacto ⁇ al traits such as cancer can be ca ⁇ ied out through two main strategies cu ⁇ ently used for genetic mapping: linkage analysis and association studies. Association studies examine the frequency of marker alleles in unrelated trait positive (T+) individuals compared with trait negative (T-) controls, and are generally employed in the detection of polygemc inheritance. Association studies as a method of mapping genetic traits rely on the phenomenon of linkage disequihbnum.
• haplotypes sets of alleles of these loci on the same chromosomal segment (called haplotypes) tend to be transmitted as a block from generation to generation
• haplotypes can be tracked not only through pedigrees but also through populations.
• the resulting phenomenon at the population level is that the occu ⁇ ence of pairs of specific alleles at different loci on the same chromosome is not random, and the deviation from random is called linkage disequihbnum (LD).
• LD linkage disequihbnum
• a specific allele in a given gene is directly involved in causing a particular trait T, its frequency will be statistically increased m a trait positive population when compared to the frequency m a trait negative population.
• the frequency of all other alleles present m the haplotype carrying the trait-causmg allele (TCA) will also be increased in trait positive individuals compared to trait negative individuals. Therefore, association between the trait and any allele in linkage disequihbnum with the trait-causmg allele will suffice to suggest the presence of a trait-related gene in that particular allele' s region.
• Linkage disequilibrium allows the relative frequencies in trait positive and trait negative populations of a limited number of genetic polymo ⁇ hisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymo ⁇ hisms m order to find trait-causmg alleles.
• the general strategy to perform association studies using biallelic markers derived from a candidate region is to scan two groups of individuals (trait positive and trait negative control individuals which are charactenzed by a well defined phenotype as desc ⁇ bed below) in order to measure and statistically compare the allele frequencies of such biallelic markers in both groups. If a statistically significant association with a trait is identified for at least one or more of the analyzed biallelic markers, one can assume that : either the associated allele is directly responsible for causing the trait (associated allele is the trait-causmg allele), or the associated allele is in linkage disequihbnum with the trait-causmg allele.
• the trait-causmg allele and by consequence the gene carrying the trait-causing allele, can be found by sequencing the vicinity of the associated marker.
• the trait under study should preferably follow a bimodal dist ⁇ bution in the population under study, presenting two clear non-overlapping phenotypes, trait positive and trait negative.
• any genetic trait may still be analyzed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups.
• the selection procedure involves to select individuals at opposite ends of the non-bimodal phenotype spectra of the trait under study, so as to include in these trait positive and trait negative populations individuals which clearly represent extreme, preferably non- overlapping phenotypes.
• the definition of the inclusion criteria for the trait positive and trait negative populations is an important aspect of the present invention.
• the selection of drastically different but relatively uniform phenotypes enables efficient comparisons m association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.
• trait positive and trait negative populations to be included in association studies consist of phenotypically homogenous populations of individuals each representing 100% of the co ⁇ espondmg trait if the trait distribution is bimodal.
• a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, can be recruited according to clinical inclusion cnte ⁇ a.
• trait negative individuals preferably more than 100 individuals
• trait positive and trait negative individuals are mcluded in such studies who are preferably both ethnically- and age-matched to the trait positive cases. They are checked for the absence of the clinical criteria defined above. Both trait positive and trait negative individuals should co ⁇ espond to unrelated cases. Genotyping of trait positive and trait negative individuals
• Allehc frequencies of the biallelic markers m each of the above descnbed population can be determined using one of the methods described above under the heading "Methods of Genotyping DNA samples for biallelic markers”. Analyses are preferably performed on amplified fragments obtained by genomic PCR performed on the DNA samples from each individual in similar conditions as those desc ⁇ bed above for the generation of biallelic markers.
• amplified DNA samples are subjected to automated microsequencing reactions using fluorescent ddNTPs (specific fluorescence for each ddNTP) and the appropnate microsequencing oligonucleotides which hybridize just upstream of the polymo ⁇ hic base.
• fluorescent ddNTPs specific fluorescence for each ddNTP
• appropnate microsequencing oligonucleotides which hybridize just upstream of the polymo ⁇ hic base.
• association studies can be ca ⁇ ied out by the skilled technician using the biallelic markers of the invention defined above, with different trait positive and trait negative populations. Suitable examples of association studies using biallelic markers of the TBC-1 gene, including the biallelic markers Al to A 19, involve studies on the following populations-
• a trait positive population suffenng from a cancer preferably prostate cancer and a healthy unaffected population
• a trait positive population suffenng from prostate cancer treated with agents acting against prostate cancer and suffering from side-effects resulting from this treatment and an trait negative population suffenng from prostate cancer treated with same agents without any substantial side- effects or
• the trait positive and trait negative individuals are selected from non- overlapping phenotypes as regards to the trait under study.
• the biallelic marker are selected from the group consisting of the biallelic markers Al to A19.
• the trait is cancer, prostate cancer, an early onset of prostate cancer, a susceptibility to prostate cancer, the level of aggressiveness of prostate cancer tumors, a modified expression of the TBC-1 gene, a modified production of the TBC-1 protein, or the production of a modified TBC-1 protein.
• the trait negative population can be replaced in the association studies by a random control population.
• the step of testing for and detecting the presence of DNA comprising specific alleles of a biallelic marker or a group of biallelic markers of the present invention can be ca ⁇ ied out as described further below.
• the invention relates also to oligonucleotide molecules useful as probes or pnmers, wherein said oligonucleotide molecules hybridize specifically with a nucleotide sequence comprised in the TBC-1 gene, particularly the TBC-1 genomic sequence of SEQ ID Nos 1 and 2 or the TBC-1 cDNAs sequences of SEQ ID Nos 3 and 4. More particularly, the present invention also concerns oligonucleotides for the detection of alleles of biallelic markers of the TBC-1 gene.
• oligonucleotides are useful either as pnmers for use in vanous processes such as DNA amplification and microsequencing or as probes for DNA recognition in hybridization analyses.
• Polynucleotides derived from the TBC-1 gene are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID Nos 1 -4, or a fragment, complement, or variant thereof in a test sample.
• Particularly prefe ⁇ ed probes and primers of the invention include isolated, purified, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID Nos 1 and 2, or the complements thereof.
• prefe ⁇ ed probes and pnmers of the invention include isolated, punfied, or recombmant polynucleotides
• 20 comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 1-1000, 1001-2000, 2001-3000, 3001-4000, 4001-5000, 5001-6000, 6001-7000, 7001-8000, 8001-9000, 9001-10000, 10001-11000, 11001-12000, 12001-13000, 13001-14000, 14001-15000,
• prefe ⁇ ed probes and p ⁇ mers of the invention include isolated, pu ⁇ fied, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span compnses at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 2: 1-5000, 5001-10000, 10001-15000, 15001-
• prefe ⁇ ed probes and primers of the invention include isolated, punfied, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40,
• probes and primers of the invention include isolated, punfied, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 3 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 3.
• Additional prefe ⁇ ed probes and pnmers of the invention include isolated, punfied, or recombmant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 4: 1-500, 501-1000, 1001-1500, 1501-2000, 2001-2500, 2501-3000, 3001- 3500, and 3501-3988.
• the invention also relates to nucleic acid probes charactenzed m that they hybridize specifically, under the stnngent hybridization conditions defined above, with a nucleic acid selected from the group consisting of the nucleotide sequences of SEQ ID Nos 1-4 or a variant thereof or a sequence complementary thereto.
• the invention encompasses isolated, punfied, and recombmant polynucleotides consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos 1 and 2 and the complement thereof, wherein said span includes a TBC- 7 -related biallelic marker in said sequence, optionally, wherein said TBC- 1 -related biallelic marker is selected from the group consistmg of Al to A19, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said contiguous span is 18 to 35 nucleotides in length and said biallelic marker is withm 4 nucleotides of the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally,
• the invention encompasses isolated, purified and recombmant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID Nos 1 and 2, or the complements thereof, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide, and wherein the 3' end of said polynucleotide is located withm 20 nucleotides upstream of a 7 ?C- 7 -related biallelic marker m said sequence; optionally, wherein said !T5C-7-related biallelic marker is selected from the group consisting of Al to A19, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein the 3' end of said polynucleotide is located 1 nucleotide upstream of said 7BC-7-related biallelic marker in said sequence; and optionally, wherein said polynucleo
• the invention encompasses isolated, purified, or recombmant polynucleotides comprising, consistmg of, or consisting essentially of a sequence selected from the following sequences: Bl to B15 and Cl to C15.
• the invention encompasses polynucleotides for use in hybridization assays, sequencing assays, and enzyme -based mismatch detection assays for determining the identity of the nucleotide at a 7BC-7-related biallelic marker SEQ ED Nos 1 and 2, or the complements thereof, as well as polynucleotides for use m amplifying segments of nucleotides comprising a 7 ?C-7-related biallelic marker in SEQ ED Nos 1 and 2, or the complements thereof; optionally, wherein said 7SC-7-related biallelic marker is selected from the group consisting of Al to A19, and the complements thereof, or optionally the biallelic markers in linkage disequihbnum therewith.
• a probe or a pnmer according to the invention has between 8 and 1000 nucleotides in length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides in length More particularly, the length of these probes and pnmers can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30 nucleotides Shorter probes and pnmers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes and pnmers are expensive to produce and can sometimes self-hyb ⁇ dize to form hai ⁇ m structures.
• a prefe ⁇ ed probe or primer consists of a nucleic acid comprising a polynucleotide selected from the group of the nucleotide sequences of PI to P7, P9 to PI 3, PI 5 to P19 and the complementary sequence thereto, Bl to B15, Cl to C15, Dl to D19, El to El 9, for which the respective locations in the sequence listing are provided in Tables 2, 3 and 4.
• the formation of stable hyb ⁇ ds depends on the melting temperature (Tm) of the DNA.
• Tm depends on the length of the pnmer or probe, the ionic strength of the solution and the G+C content.
• the GC content in the probes of the invention usually ranges between 10 and 75 %, preferably between 35 and 60 %, and more preferably between 40 and 55 %.
• the pnmers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al.(1981) and the solid support method described m EP 0 707 592.
• Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, mo ⁇ hohno analogs which are desc ⁇ bed in U.S. Patents Numbered 5,185,444; 5,034,506 and 5,142,047.
• the probe may have to be rendered "non-extendable" in that additional dNTPs cannot be added to the probe.
• analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation.
• the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
• the 3' hydroxyl group simply can be cleaved, replaced or modified, U.S. Patent Application Se ⁇ al No. 07/049,061 filed Apnl 19, 1993 describes modifications, which can be used to render a probe non-extendable.
• any of the polynucleotides of the present invention can be labeled, if desired, by inco ⁇ orating any label known in the art to be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• useful labels include radioactive substances (including, P, S, H, I), fluorescent dyes (including, 5-bromodesoxyu ⁇ dm, fluorescem, acetylaminofluorene, digoxigenin) or biotin.
• polynucleotides are labeled at their 3' and 5' ends. Examples of non-radioactive labeling of nucleic acid fragments are described m the French patent No.
• the probes according to the present invention may have structural charactenstics such that they allow the signal amplification, such structural charactenstics being, for example, branched DNA probes as those descnbed by Urdea et al. in 1991 or m the European patent No. EP 0 225 807 (Chiron)
• a label can also be used to capture the pnmer, so as to facilitate the immobilization of either the pnmer or a pnmer extension product, such as amplified DNA, on a solid support.
• a capture label is attached to the pnmers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label earned by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, pnmers or probes provided herein, may, themselves, serve as the capture label.
• a solid phase reagent's binding member is a nucleic acid sequence
• it may be selected such that it binds a complementary portion of a pnmer or probe to thereby immobilize the pnmer or probe to the solid phase.
• a polynucleotide probe itself serves as the binding member
• the probe will contain a sequence or "tail" that is not complementary to the target.
• a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase.
• DNA Labeling techniques are well known to the skilled technician
• the probes of the present invention are useful for a number of pu ⁇ oses They can be notably used in Southern hybridization to genomic DNA.
• the probes can also be used to detect PCR amplification products. They may also be used to detect mismatches m the TBC-lgene or mRNA using other techniques.
• Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose stnps, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others.
• the solid support is not cntical and can be selected by one skilled in the art.
• latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples.
• a solid support refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
• the solid support can be chosen for its mtnnsic ability to attract and immobilize the capture reagent.
• the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
• the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
• the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction.
• the receptor molecule enables the indirect binding of the capture reagent to a solid support mate ⁇ al before the performance of the assay or during the performance of the assay.
• the solid phase thus can be a plastic, de ⁇ vatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill in the art.
• polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support.
• polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention.
• the invention also deals with a method for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ED Nos 1-4, a fragment or a vanant thereof and a complementary sequence thereto in a sample, said method comprising the following steps of.
• nucleic acid probe or a plurality of nucleic acid probes which can hybridize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ED Nos 1 -4, a fragment or a variant thereof and a complementary sequence thereto and the sample to be assayed; and b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample.
• the invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ED Nos 1 -4, a fragment or a variant thereof and a complementary sequence thereto in a sample, said kit comprising: a) a nucleic acid probe or a plurality of nucleic acid probes which can hybndize with a nucleotide sequence included in a nucleic acid selected form the group consisting of the nucleotide sequences of SEQ ED Nos 1-4, a fragment or a va ⁇ ant thereof and a complementary sequence thereto; and b) optionally, the reagents necessary for performing the hybridization reaction.
• said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule.
• said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate.
• the nucleic acid probe or the plurality of nucleic acid probes comprise either a sequence which is selected from the group consisting of the nucleotide sequences of PI to P7, P9 to PI 3, PI 5 to P19 and the complementary sequence thereto, Bl to B15, Cl to C15, Dl to D19, El to E19 or a biallelic marker selected from the group consisting of Al to A19 and the complements thereto.
• a substrate comprising a plurality of oligonucleotide primers or probes of the invention may be used either for detecting or amplifying targeted sequences in the TBC-1 gene and may also be used for detecting mutations in the coding or in the non-codmg sequences of the TBC-1 gene.
• Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support.
• the polynucleotides of the invention may be attached in an ordered a ⁇ ay wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide.
• such an ordered a ⁇ ay of polynucleotides is designed to be "addressable" where the distinct locations are recorded and can be accessed as part of an assay procedure.
• Addressable polynucleotide a ⁇ ays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these "addressable" a ⁇ ays particularly useful in hybridization assays. Any addressable a ⁇ ay technology known in the art can be employed with the polynucleotides of the invention.
• a ⁇ ays One particular embodiment of these polynucleotide a ⁇ ays is known as the GenechipsTM, and has been generally descnbed in US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. These a ⁇ ays may generally be produced using mechanical synthesis methods or light directed synthesis methods which mco ⁇ orate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., 1991).
• VLSIPSTM Very Large Scale Immobilized Polymer Synthesis
• an oligonucleotide probe mat ⁇ x may advantageously be used to detect mutations occurring in the TBC-1 gene and preferably in its regulatory region.
• probes are specifically designed to have a nucleotide sequence allowing their hybndization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides).
• known mutations it is meant, mutations on the TBC-1 gene that have been identified according, for example to the technique used by Huang et al.(1996) or Samson et al.(1996).
• Each oligonucleotide probe constituting a unit element of the high density DNA a ⁇ ay is designed to match a specific subsequence of the TBC-1 genomic DNA or cDNA.
• an a ⁇ ay consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the TBC-1 gene.
• 4L tiled a ⁇ ay is implemented a set of four probes (A, C, G, T), preferably 15-nucleot ⁇ de ohgomers. En each set of four probes, the perfect complement will hybridize more strongly than mismatched probes. Consequently, a nucleic acid target of length L is scanned for mutations with a tiled a ⁇ ay containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence. The hybridization signals of the 15-mer probe set tiled a ⁇ ay are perturbed by a single base change in the target sequence. As a consequence, there is a characteristic loss of signal or a "footprint" for the probes flanking a mutation position. This technique was described by Chee et al. in 1996.
• the invention concerns an a ⁇ ay of nucleic acid molecules comprising at least one polynucleotide desc ⁇ bed above as probes and primers
• the invention concerns an a ⁇ ay of nucleic acid comprising at least two polynucleotides described above as probes and primers.
• a further object of the invention consists of an a ⁇ ay of nucleic acid sequences comprising either at least one of the sequences selected from the group consisting of PI to P7, P9 to PI 3, P15 to PI 9, Bl to B15, Cl to C15, Dl to D19, El to El 9, the sequences complementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and at least one sequence comprising a biallelic marker selected from the group consisting of Al to A19 and the complements thereto.
• the invention also pertains to an a ⁇ ay of nucleic acid sequences comprising either at least two of the sequences selected from the group consisting of PI to P7, P9 to P13, PI 5 to PI 9, Bl to B15, Cl to C15, Dl to D 19, El to El 9, the sequences complementary thereto, a fragment thereof of at least 8 consecutive nucleotides thereof, and at least two sequences comprising a biallelic marker selected from the group consisting of Al to A19 and the complements thereof.
• Any of the regulatory polynucleotides or the coding polynucleotides of the invention may be inserted into recombmant vectors for expression in a recombmant host cell or a recombmant host organism.
• the present invention also encompasses a family of recombmant vectors that contains either a regulatory polynucleotide selected from the group consisting of any one of the regulatory polynucleotides denved from the TBC-1 genomic sequences of SEQ ID Nos 1 and 2, or a polynucleotide comprising the TBC-1 coding sequence, or both.
• a recombmant vector of the invention is used as an expression vector : (a) the TBC-1 regulatory sequence comprised therein drives the expression of a coding polynucleotide operably linked thereto; (b) the TBC-1 coding sequence is operably linked to regulation sequences allowing its expression in a suitable cell host and/or host organism.
• a recombmant vector of the invention is used to amplify the inserted polynucleotide denved from the TBC-1 genomic sequences of SEQ ED Nos 1 and 2 or TBC-1 cDNAs in a suitable cell host , this polynucleotide being amplified at every time that the recombmant vector replicates.
• the present invention relates to expression vectors which include nucleic acids encoding a TBC-1 protein, preferably the TBC-1 protein of the amino acid sequence of SEQ ED No 5 desc ⁇ bed therein, under the control of a regulatory sequence selected among the TBC-1 regulatory polynucleotides, or alternatively under the control of an exogenous regulatory sequence.
• a recombmant expression vector comprising a nucleic acid selected from the group consisting of 5' and 3' regulatory regions, or biologically active fragments or variants thereof, is also part of the present invention.
• the invention also encompasses a recombmant expression vector comprising a) a nucleic acid comprising the 5' regulatory polynucleotide of the nucleotide sequence SEQ ED No 1 , or a biologically active fragment or vanant thereof; b) a polynucleotide encoding a polypeptide or a polynucleotide of interest operably linked with said nucleic acid. c) optionally, a nucleic acid comprising a 3 '-regulatory polynucleotide, preferably a 3'- regulatory polynucleotide of the invention, or a biologically active fragment or variant thereof.
• the nucleic acid comprising the 5' regulatory polynucleotide or a biologically active fragment or variant thereof may also comprises the 5'-UTR sequence from any of the two cDNA of the invention or a biologically active fragment or variant thereof.
• the invention also pertains to a recombmant expression vector useful for the expression of the TBC-1 coding sequence, wherein said vector comprises a nucleic acid selected from the group consisting of SEQ ED Nos 3 and 4 or a nucleic acid having at least 95% nucleotide identity with a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ ED Nos 3 and 4.
• Another recombmant expression vector of the invention consists in a recombmant vector comprising a nucleic acid comprising the nucleotide sequence beginning at the nucleotide in position 176 and ending in position 3730 of the polynucleotide of SEQ ED No 4.
• a recombmant vector of the invention may comprise any of the polynucleotides described herein, including regulatory sequences, and coding sequences, as well as any TBC-1 primer or probe as defined above. More particularly, the recombmant vectors of the present invention can comprise any of the polynucleotides described in the "TBC-1 cDNA Sequences” section, the “Coding Regions” section, “Genomic sequence of TBC-1" section and the "Oligonucleotide Probes And Primers” section. Some of the elements which can be found in the vectors of the present invention are described m further detail in the following sections. a) Vectors
• a recombmant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non- chromosomal and synthetic DNA.
• a recombmant vector can comprise a transcriptional unit comprising an assembly of :
• Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.
• Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
• a recombmant protein may include an N-terminal residue This residue may or may not be subsequently cleaved from the expressed recombmant protein to provide a final product.
• recombmant expression vectors will include ongins of replication, selectable markers permitting transformation of the host cell, and a promoter denved from a highly expressed gene to direct transcription of a downstream structural sequence.
• the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the pe ⁇ plasmic space or the extracellular medium.
• the selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycm resistance for eukaryotic cell culture, TRPl for S. cerevisiae or tetracyclme, ⁇ fampicm or ampicill resistance in E coli, or levan saccharase for mycobacte ⁇ a.
• useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication denved from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017).
• Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1 (Promega Biotec, Madison, Wl, USA).
• bacte ⁇ al vectors pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescnpt, ps ⁇ X174, pbluesc ⁇ pt SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRET5 (Pharmacia); or eukaryotic vectors : pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); baculovirus transfer vector pVL1392/1393 (Pharmingen);
• a suitable vector for the expression of the TBC-1 polypeptide of SEQ ED No 5 is a baculovirus vector that can be propagated in insect cells and m insect cell lines.
• a specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL 171 1) which is derived from Spodoptera frug ⁇ erda.
• Other suitable vectors for the expression of the TBC-1 polypeptide of SEQ ED No 5 m a baculovirus expression system include those descnbed by Chai et al. (1993), Vlasak et al. (1983) and Lenhard et al. (1996).
• Mammalian expression vectors will comprise an ongm of replication, a suitable promoter and enhancer, and also any necessary nbosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontransc ⁇ bed sequences.
• DNA sequences derived from the SV40 viral genome for example SV40 o ⁇ gin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required nontranscnbed genetic elements.
• the suitable promoter regions used m the expression vectors according to the present invention are chosen taking into account the cell host in which the heterologous gene has to be expressed.
• a suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombmant vector sequences withm which the construct promoter/coding sequence has been inserted.
• Prefe ⁇ ed bactenal promoters are the Lad, LacZ, the T3 or T7 bacte ⁇ ophage RNA polymerase promoters, the polyhed ⁇ n promoter, or the plO protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 1992), the lambda P R promoter or also the trc promoter.
• Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.
• Particularly prefe ⁇ ed bactenal promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp.
• Eukaryotic promoters include CMV immediate early, HSV thymidine kmase, early and late SV40, LTRs from retrovirus, and mouse metallothionem-L. Selection of a convenient vector and promoter is well withm the level of ordinary skill in the art.
• a promoter is well with the ability of a person skilled in the field of genetic egmeenng. For example, one may refer to the book of Sambrook et al. (1989) or also to the procedures described by Fuller et al. (1996).
• TBC-1 gene regulatory polynucleotide a polynucleotide encoding the TBC-1 polypeptide of SEQ ID No 5 or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.
• Other types of vectors The in vivo expression of a TBC-1 polypeptide of SEQ ED No 5 may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive TBC-1 protein.
• the present invention also deals with recombmant expression vectors mamly designed for the in vivo production of the TBC-1 polypeptide of SEQ ED No 5 by the introduction of the appropriate genetic material in the organism of the patient to be treated.
• This genetic matenal may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced m the said organism, directly in vivo into the appropriate tissue.
• One specific embodiment for a method for delivering a protein or peptide to the mtenor of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable ca ⁇ ier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.
• the invention provides a composition for the in vivo production of the TBC-1 protein or polypeptide descnbed herein.
• compositions comprising a polynucleotide are described in PCT application N° WO 90/11092 (Vical Inc.) and also in PCT application N° WO 95/11307 (Enstitut Pasteur, INSERM, Universite d'Ottawa) as well as in the articles of Tacson et al. (1996) and of Huygen et al. (1996).
• the amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0,1 and 100 ⁇ g of the vector in an animal body, preferably a mammal body, for example a mouse body.
• it may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. En a subsequent step, the cell that has been transformed with the vector coding for the desired TBC-1 polypeptide or the desired fragment thereof is reintroduced into the animal body in order to deliver the recombmant protein withm the body either locally or systemically.
• the vector is denved from an adenovirus.
• Prefe ⁇ ed adenovirus vectors according to the invention are those desc ⁇ bed by Feldman and Steg (1996) or Ohno et al. (1994).
• Another prefe ⁇ ed recombmant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin ( French patent application N° FR-93.05954).
• Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).
• Other prefe ⁇ ed retroviral vectors are those described in Roth et al. (Roth J.A. et al., 1996), PCT Application No WO 93/25234, PCT Application No WO 94/ 06920, Roux et al , 1989, Julan et al., 1992 and Neda et al., 1991.
• AAV adeno-associated virus
• the adeno-associated virus is a naturally occurnng defective virus that requires another virus, such as an adenovirus or a he ⁇ es virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989).
• AAV adeno-associated virus
• compositions containing a vector of the invention advantageously comprise an oligonucleotide fragment of a nucleic sequence selected from the group consisting of SEQ ED Nos 3 or 4 as an antisense tool that inhibits the expression of the co ⁇ esponding TBC-1 gene.
• Prefe ⁇ ed methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al. (1995) or those descnbed in PCT Application No WO 95/24223.
• Another object of the invention consists m host cell that have been transformed or transfected with one of the polynucleotides described therein, and more precisely a polynucleotide either comprising a TBC-1 regulatory polynucleotide or the coding sequence of the TBC-1 polypeptide having the ammo acid sequence of SEQ ED No 5.
• mcluded host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombmant vector such as one of those desc ⁇ bed above.
• a recombmant host cell of the invention comprises any one of the polynucleotides or the recombmant vectors described therein. More particularly, the cell hosts of the present invention can comprise any of the polynucleotides described in "TBC-1 cDNA Sequences” section, the “Coding Regions” section, “Genomic sequence of TBC-1 " section and the "Oligonucleotide Probes And Primers” section.
• Another prefe ⁇ ed recombmant cell host according to the present invention is characterized in that its genome or genetic background (including chromosome, plasmids) is modified by the nucleic acid coding for the TBC-1 polypeptide of SEQ ED No 5.
• Prefe ⁇ ed host cells used as recipients for the expression vectors of the invention are the following : a) Prokaryotic host cells : Escherichia coli strains (I.E. DH5- ⁇ strain) or Bacillus subtilis.
• Eukaryotic host cells HeLa cells (ATCC N°CCL2; N°CCL2.1; N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL1650; N°CRL1651), Sf-9 cells (ATCC N°CRL1711).
• the constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
• the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.
• Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
• Microbial cells employed in the expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by the skill artisan.
• transgenic animals or "host animals” are used herein to designate animals that have their genome genetically and artificially manipulated so as to include one of the nucleic acids according to the invention.
• Prefe ⁇ ed animals are non-human mammals and include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have their genome artificially and genetically altered by the insertion of a nucleic acid according to the invention.
• the transgenic animals of the invention all include within a plurality of their cells a cloned recombinant or synthetic DNA sequence, more specifically one of the purified or isolated nucleic acids comprising a TBC-1 coding sequence, a TBC-1 regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification.
• transgenic animals according to the invention contain in their somatic cells and/or in their germ line cells any of the polynucleotides described in "TBC-1 cDNA Sequences” section, the “Coding Regions” section, “Genomic sequence of TBC-1 " section, the “Oligonucleotide Probes And Primers” section and the “Vectors for the expression of a regulatory or coding polynucleotide of TBC-1" section.
• transgenic animals of the invention thus contain specific sequences of exogenous genetic material such as the nucleotide sequences described above in detail.
• these transgenic animals may be good experimental models in order to study the diverse pathologies related to cell differentiation, in particular concerning the transgenic animals within the genome of which has been inserted one or several copies of a polynucleotide encoding a native TBC-1 protein, or alternatively a mutant TBC-1 protein.
• these transgenic animals may express a desired polypeptide of interest under the control of the regulatory polynucleotides of the TBC-1 gene, leading to good yields in the synthesis of this protein of interest, and eventually a tissue specific expression of this protein of interest.
• transgenic animals of the invention Since it is possible to produce transgenic animals of the invention using a variety of different sequences, a general description will be given of the production of transgenic animals by refe ⁇ mg generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to mco ⁇ orate the DNA sequences into animals.
• This general description can be adapted by those skilled in the art in order to mco ⁇ orate the DNA sequences into animals.
• it may be refe ⁇ ed to Sandou et al. (1994) and also to US Patents Nos 4,873,191, issued Oct.lO, 1989, 5,968,766, issued Dec. 16, 1997 and 5,387,742, issued Feb. 28, 1995, these documents being herein inco ⁇ orated by reference to disclose methods for producing transgenic mice.
• Transgenic animals of the present invention are produced by the application of procedures which result m an animal with a genome that co ⁇ orates exogenous genetic material which is integrated into the genome.
• the procedure involves obtaining the genetic matenal, or a portion thereof, which encodes either a TBC-1 coding sequence, a TBC-1 regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described m the present specification.
• a recombinant polynucleotide of the invention is inserted into an embryonic or ES stem cell line. The insertion is made using electroporation. The cells subjected to electroporation are screened (e.g.
• Southern blot analysis to find positive cells which have integrated the exogenous recombinant polynucleotide into their genome.
• An illustrative positive-negative selection procedure that may be used according to the invention is described by Mansour et al. (1988). Then, the positive cells are isolated, cloned and injected into 3.5 days old blastocysts from mice. The blastocysts are then inserted into a female host animal and allowed to grow to term. The offsp ⁇ ngs of the female host are tested to determine which animals are transgenic e.g. include the inserted exogenous DNA sequence and which are wild-type.
• the present invention also concerns a method for the screening of new agents, or candidate substances interacting with TBC-1. These new agents could be useful against cancer.
• the invention relates to a method for the screening of candidate substances comprising the following steps: - providing a cell line, an organ, or a mammal expressing a TBC-1 gene or a fragment thereof, preferably the regulatory region or the promoter region of the TBC-1 gene. - obtaining a candidate substance preferably a candidate substance capable of inhibiting the binding of a transcription factor to the TBC-1 regulatory region,
• the cell line, organ or mammal expresses a heterologous protein, the coding sequence of which is operably linked to the TBC-1 regulatory or promoter sequence.
• they express a TBC-1 gene comprising alleles of one or more r5C-7-related biallelic markers.
• a candidate substance is a substance which can interact with or modulate, by binding or other intramolecular interactions, expression, stability, and function of TBC-1. Such substances may be potentially interesting for patients who are not responsive to existing drugs or develop side effects to them. Screening may be effected using either in vitro methods or in vivo methods.
• Such methods can be carried out in numerous ways such as on transformed cells which express the considered alleles of the TBC-1 gene, on tumors induced by said transformed cells, for example in mice, or on a TBC-1 protein encoded by the considered allelic variant of TBC-1.
• Screening assays of the present invention generally involve determining the ability of a candidate substance to present a cytotoxic effect, to change the characteristics of transformed cells such as proliferative and invasive capacity, to affect the tumor growth, or to modify the expression level of TBC-1.
• this method includes preparing transformed cells with different forms of TBC-1 sequences containing particular alleles of one or more biallelic markers and/or trait causing mutations described above. This is followed by testing the cells expressing the TBC-1 with a candidate substance to determine the ability of the substance to present cytotoxic effect, to affect the characteristics of transformed cells, the tumor growth, or to modify the expression level of TBC-1.
• Typical examples of such drug screening assays are provided below. It is to be understood that the parameters set forth in these examples can be modified by the skilled person without undue experimentation.
• a method for the screening of a candidate substance according to the invention comprises the following steps : a)providing a polypeptide comprising the amino acid sequence SEQ ID No 5, or a peptide fragment or a variant thereof; b) obtaining a candidate substance; c) bringing into contact said polypeptide with said candidate substance; d) detecting the complexes formed between said polypeptide and said candidate substance.
• a ligand means a molecule, such as a protein, a peptide, an antibody or any synthetic chemical compound capable of binding to the TBC-1 protein or one of its fragments or variants or to modulate the expression of the polynucleotide coding for TBC-1 or a fragment or variant thereof.
• a biological sample or a defined molecule to be tested as a putative ligand of the TBC-1 protein is brought into contact with a punfied TBC-1 protein, for example a punfied recombinant TBC-1 protein produced by a recombinant cell host as descnbed hereinbefore, in order to form a complex between the TBC-1 protein and the putative hgand molecule to be tested.
• a punfied TBC-1 protein for example a punfied recombinant TBC-1 protein produced by a recombinant cell host as descnbed hereinbefore
• the putative ligand is the expression product of a DNA insert contained in a phage vector (Parmley and Smith, 1988). Specifically, random peptide phages hbranes are used. The random DNA inserts encode peptides of 8 to 20 aminoacids in length (Oldenburg K.R. et al., 1992,.; Valadon P., et al., 1996; Lucas A.H., 1994; Westennk M.A.J., 1995; Castagnoh L. et al., 1991).
• the recombinant phages expressing a protein that binds to the immobilized TBC-1 protein are retained and the complex formed between the TBC-1 protein and the recombinant phage may be subsequently immunoprecipitated by a polyclonal or a monoclonal antibody directed against the TBC-1 protein.
• the phage population is brought into contact with the immobilized TBC-1 protein. Then the preparation of complexes is washed in order to remove the non-specifically bound recombinant phages.
• the phages that bind specifically to the TBC-1 protein are then eluted by a buffer (acid pH) or immunoprecipitated by the ant ⁇ -TBC-1 monoclonal antibody produced by a hyb ⁇ doma, and this phage population is subsequently amplified by an over-mfection of bactena (for example E. cob).
• the selection step may be repeated several times, preferably 2-4 times, in order to select the more specific recombinant phage clones.
• the last step consists in characterizing the peptide produced by the selected recombinant phage clones either by expression m infected bactena and isolation, expressing the phage insert m another host- vector system, or sequencing the insert contained in the selected recombinant phages.
• Candidate ligands obtained through a two-hybrid screening assay The yeast two-hybrid system is designed to study protem-protem interactions in vivo (Fields and Song, 1989), and relies upon the fusion of a bait protein to the DNA binding domain of the yeast Gal4 protein. This technique is also described in US Patent N° US 5,667,973 and US Patent N° 5,283,173 (Fields et al.) the technical teachings of both patents being herein inco ⁇ orated by reference.
• the general procedure of library screening by the two-hybnd assay may be performed as described by Ha ⁇ er et al. (Ha ⁇ er JW et al., 1993) or as described by Cho et al. (1998) or also Fromont-Racine et al. (1997).
• the bait protein or polypeptide consists of a TBC-1 polypeptide or a fragment or va ⁇ ant thereof.
• nucleotide sequence encoding the TBC-1 polypeptide or a fragment or variant thereof is fused to a polynucleotide encoding the DNA binding domain of the GAL4 protein, the fused nucleotide sequence being inserted in a suitable expression vector, for example pAS2 or pM3.
• a human cDNA library is constructed in a specially designed vector, such that the human cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcriptional domain of the GAL4 protein.
• the vector used is the pACT vector.
• the polypeptides encoded by the nucleotide inserts of the human cDNA library are termed "pray" polypeptides.
• a third vector contains a detectable marker gene, such as beta galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcnptional activation domain and the DNA binding domain.
• a detectable marker gene such as beta galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcnptional activation domain and the DNA binding domain.
• the vector pG5EC may be used.
• Two different yeast strains are also used. As an illustrative but non limiting example the two different yeast strains may be the following :
• Y190 the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trpl-901, h ⁇ s3-D200, ade2-101, gal4Dgall80D URA3 GAL-LacZ, LYS GAL-HIS3, cyh r );
• Y187 the phenotype of which is (MATa gal4 gal80 h ⁇ s3 trpl-901 ade2-101 ura3-52 leu2-3, - 772 URA3 GAL-lacZmet ), which is the opposite mating type of Y190.
• pAS2/TBC-l and 20 ⁇ g of pACT-cDNA library are co-transformed into yeast strain Y190.
• the transformants are selected for growth on minimal media lacking histidine, leucme and tryptophan, but containing the histidine synthesis inhibitor 3-AT (50 mM).
• Positive colonies are screened for beta galactosidase by filter lift assay.
• the double positive colonies (H ⁇ s + , beta-gat) are then grown on plates lacking histidine, leucme, but containing tryptophan and cycloheximide (10 mg/ml) to select for loss of pAS2/TBC-l plasmids but retention of pACT-cDNA library plasmids.
• the resulting Y190 strains are mated with Y187 strains expressing TBC-1 or non-related control proteins; such as cyclophilm B, lamm, or SNF1, as Gal4 fusions as descnbed by Ha ⁇ er et al. (1993) and by Bram et al. (1993), and screened for beta galactosidase by filter lift assay.
• Yeast clones that are beta gal- after mating with the control Gal4 fusions are considered false positives.
• the interaction between TBC-1 or a fragment or va ⁇ ant thereof with cellular proteins may be assessed using the Matchmaker Two Hybrid System 2 (Catalog No. K 1604-1, Clontech). ). As desc ⁇ bed in the manual accompanying the Matchmaker Two Hybnd System 2 (Catalog No.
• nucleic acids encoding the TBC-1 protein or a portion thereof are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcnptional activator GAL4
• a desired cDNA preferably human cDNA, is inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4.
• the two expression plasmids are transformed into the yeast cells and the yeast cells are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GAL4 dependent expression of the HIS3 gene.
• Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay are those in which an interaction between TBC-1 and the protein or peptide encoded by the initially selected cDNA insert has taken place. Method for screening ligands that modulate the expression of the TBC-1 gene.
• Another subject of the present invention is a method for screening molecules that modulate the expression of the TBC-1 protein.
• Such a screening method comprises the steps of : a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the TBC-1 protein, operably linked to a TBC-1 5'-regulatory sequence; b) bnngmg into contact the cultivated cell with a molecule to be tested; c) quantifying the expression of the TBC-1 protein.
• the TBC-1 protein encoding DNA sequence is inserted into an expression vector, downstream from a TBC-1 5 '-regulatory sequence that contains a TBC-1 promoter sequence.
• the quantification of the expression of the TBC-1 protein may be realized either at the mRNA level or at the protein level. En the latter case, polyclonal or monoclonal antibodies may be used to quantify the amounts of the TBC-1 protein that have been produced, for example in an ELESA or a REA assay.
• the quantification of the TBC-1 mRNAs is realized by a quantitative PCR amplification of the cDNAs obtained by a reverse transcription of the total mRNA of the cultivated TBC- 1 -transfected host cell, using a pair of pnmers specific for TBC-1.
• TBC-1 Expression levels and patterns of TBC-1 may be analyzed by solution hyb ⁇ dization with long probes as descnbed m international Patent Application No. WO 97/05277, the entire contents of which are inco ⁇ orated herein by reference.
• the TBC-1 cDNA or the TBC-1 genomic DNA desc ⁇ bed above, or fragments thereof is inserted at a cloning site immediately downstream of a bacte ⁇ ophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA.
• the TBC-1 insert compnses at least 100 or more consecutive nucleotides of the genomic DNA sequence or the cDNA sequences, particularly those compnsing one of the nuceotide sequences of SEQ ID Nos 3, 4 and 6-8 or those encoding a mutated TBC-1.
• the plasmid is hneanzed and transc ⁇ bed in the presence of ⁇ bonucleotides compnsing modified nbonucleotides (i.e. biotm-UTP and DIG-UTP). An excess of this doubly labeled RNA is hybndized in solution with mRNA isolated from cells or tissues of interest.
• the hybridizations are performed under standard stringent conditions (40-50°C for 16 hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8).
• the unhybridized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases CL3, Tl, Phy M, U2 or A).
• RNases CL3, Tl, Phy M, U2 or A ribonucleases specific for single-stranded RNA
• the presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidin.
• the presence of the DIG modification enables the hybrid to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline phosphatase.
• a ⁇ ay means a one dimensional, two dimensional, or multidimensional a ⁇ angement of a plurality of nucleic acids of sufficient length to permit specific detection of expression of mRNAs capable of hybridizing thereto.
• the a ⁇ ays may contain a plurality of nucleic acids derived from genes whose expression levels are to be assessed.
• the a ⁇ ays may include the TBC-1 genomic DNA, the TBC-1 cDNA sequences or the sequences complementary thereto or fragments thereof, particularly those comprising at least one of the biallelic markers according the present invention.
• the fragments are at least 15 nucleotides in length.
• the fragments are at least 25 nucleotides in length. In some embodiments, the fragments are at least 50 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. In another prefe ⁇ ed embodiment, the fragments are more than 100 nucleotides in length. In some embodiments the fragments may be more than 500 nucleotides in length.
• TBC-1 gene expression may be performed with a complementary DNA microa ⁇ ay as described by Schena et al. (1995).
• Full length TBC-1 cDNAs or fragments thereof are amplified by PCR and a ⁇ ayed from a 96-well microtiter plate onto silylated microscope slides using high-speed robotics.
• Printed a ⁇ ays are incubated in a humid chamber to allow rehydration of the a ⁇ ay elements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1 min and once for 5 min in sodium borohydride solution.
• the a ⁇ ays are submerged in water for 2 min at 95°C, transfe ⁇ ed into 0.2% SDS for 1 min, rinsed twice with water, air dried and stored in the dark at 25°C.
• Probes are hybridized to 1 cm 2 microa ⁇ ays under a 14 x 14 mm glass coverslip for 6-12 hours at 60°C. A ⁇ ays are washed for 5 min at 25 °C in low stringency wash buffer (1 x SSC/0.2% SDS), then for 10 min at room temperature in high stringency wash buffer (0.1 x SSC/0.2% SDS). Arrays are scanned in 0.1 x SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations.
• TBC-1 gene expression may also be performed with full length TBC-1 cDNAs or fragments thereof in complementary DNA a ⁇ ays as described by Pietu et al. (1996).
• the full length TBC-1 cDNA or fragments thereof is PCR amplified and spotted on membranes. Then, mRNAs originating from various tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybridized mRNAs are detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.
• expression analysis using the TBC-1 genomic DNA, the TBC-1 cDNAs, or fragments thereof can be done through high density nucleotide a ⁇ ays or chips as described by Lockhart et al. (1996) and Sosnowsky et al. (1997).
• Oligonucleotides of 15-50 nucleotides from the sequences of the TBC-1 genomic DNA, the TBC-1 cDNA sequences particularly those comprising at least one of biallelic markers according the present invention, preferably at least one of SEQ TD No 7-8 or those comprising the trait causing mutation, or the sequences complementary thereto, are synthesized directly on the chip (Lockhart et al., supra) or synthesized and then addressed to the chip (Sosnowski et al., supra).
• the oligonucleotides are about 20 nucleotides in length.
• TBC-1 cDNA probes labeled with an appropriate compound such as biotin, digoxigenin or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hybridized to the chip. After washing as described in Lockhart et al., supra and application of different electric fields (Sosnowsky et al., 1997)., the dyes or labeling compounds are detected and quantified. Duplicate hybridizations are performed. Comparative analysis of the intensity of the signal originating from cDNA probes on the same target oligonucleotide in different cDNA samples indicates a differential expression of TBC-1 mRNAs.
• an appropriate compound such as biotin, digoxigenin or fluorescent dye
• a method for screening of a candidate substance or molecule that modulates the expression of the TBC-1 gene comprises the following steps : a) providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises the 5' regulatory region sequence or a biologically active fragment or variant thereof, the 5' regulatory region or its biologically active fragment or variant being operably linked to a polynucleotide encoding a detectable protein; b) obtaining a candidate substance, and c) determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.
• the nucleic acid comprising the
• 5' regulatory region sequence or a biologically active fragment or variant thereof also includes a 5 'UTR region of one of the TBC-1 cDNAs of SEQ ED Nos 3 and 4, or one of their biologically active fragments or variants thereof.
• a second method for the screening of a candidate substance or molecule that modulates the expression of the TBC-1 gene comprises the following steps : a) providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises a 5 'UTR sequence of one of the TBC-1 cDNAs of SEQ ED Nos 3 and 4, or one of their biologically active fragments or variants, the 5 'UTR sequence or its biologically active fragment or variant being operably linked to a polynucleotide encoding a detectable protein; b) obtaining a candidate substance, and c) determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.
• the nucleic acid that comprises a nucleotide sequence selected from the group consisting of the 5'UTR sequence of one of the TBC-1 cDNAs of SEQ ED Nos 3 and 4 or one of their biologically active fragments or variants includes a promoter sequence, wherein said promoter sequence can be either endogenous, or in contrast exogenous with respect to the TBC-1 5'UTR sequences defined therein.
• polynucleotides encoding a detectable protein there may be cited polynucleotides encoding beta galactosidase, green fluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT).
• GFP green fluorescent protein
• CAT chloramphenicol acetyl transferase
• Nucleic acids including at least one of the biallelic polymo ⁇ hisms of interest can be used to generate genetically modified non-human animals or to generate site specific gene modifications in cell lines.
• transgenic is intended to encompass genetically modified animals having a deletion or other knock-out of TBC-1 gene activity, having an exogenous TBC-1 gene that is stably transmitted in the host cells, or having an exogenous TBC-1 promoter operably linked to a reporter gene.
• Transgenic animals may be made through homologous recombination, where the TBC-1 locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome.
• Vectors for stable integration include for example plasmids, retroviruses and other animal viruses, and YACs. Of interest are transgenic mammals e.g. cows, pigs, goats, horses, and particularly rodents such as rats and mice. Transgenic animals allow to study both efficacy and toxicity of the candidate drug.
• compositions according to the present invention comprise advantageously an oligonucleotide fragment of the nucleic sequence of TBC-1 as an antisense tool that inhibits the expression of the co ⁇ esponding TBC-1 gene.
• Prefe ⁇ ed methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al. (1995).
• the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5 'end of the TBC-1 mRNA.
• a combination of different antisense polynucleotides complementary to different parts of the desired targetted gene are used.
• Prefe ⁇ ed antisense polynucleotides according to the present invention are complementary to a sequence of the mRNAs of TBC-1 that contains the translation initiation codon ATG.
• the antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. They comprise a nucleotide sequence complementary to the targeted sequence of the PTCA-1 genomic DNA, the sequence of which can be determined using one of the detection methods of the present invention.
• the targeted DNA or RNA sequence preferably comprises at least one of the biallelic markers according to the present invention.
• the antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an mtracellular duplex having sufficient stability to inhibit the expression of the TBC-1 mRNA m the duplex.
• Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al., (1986) and Izant and Wemtraub, (1984), the disclosures of which are inco ⁇ orated herein by reference.
• antisense molecules are obtained by reversing the o ⁇ entation of the TBC-1 coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed m the cell.
• the antisense molecules may be transcnbed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript.
• Another approach involves transcription of TBC-1 antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter a suitable expression vector.
• an alternative to the antisense technology that is used according to the present invention consists m using ⁇ bozymes that will bind to a target sequence via their complementary polynucleotide tail and that will cleave the co ⁇ esponding RNA by hydrolyzmg its target site (namely « hammerhead ⁇ bozymes »).
• the simplified cycle of a hammerhead ribozyme consists of (1) sequence specific binding to the target RNA via complementary antisense sequences; (2) site-specific hydrolysis of the cleavable motif of the target strand; and (3) release of cleavage products, which gives nse to another catalytic cycle.
• a prefe ⁇ ed delivery system for antisense ⁇ bozyme is achieved by covalently linking these antisense nbozymes to hpophihc groups or to use liposomes as a convenient vector.
• Prefe ⁇ ed antisense nbozymes according to the present invention are prepared as described by Sczakiel et al. (1995), the specific preparation procedures being refe ⁇ ed to in said article being herein inco ⁇ orated by reference.
• EXAMPLE 1 Analysis of the first mRNA encoding a TBC-1 polypeptide synthesized by the cells.
• TBC-1 cDNA was obtained as follows : 4 ⁇ l of ethanol suspension containing 1 mg of human prostate total RNA (Clontech laboratories, Inc., Palo Alto, USA; Catalogue N. 64038-1) was centrifuged, and the resulting pellet was air dried for 30 minutes at room temperature.
• First strand cDNA synthesis was performed using the AdvantageTM RT-for- PCR kit (Clontech laboratories Inc., catalogue N. K1402-1).
• 1 ⁇ l of 20 mM solution of a specific oligo dT primer was added to 12.5 ⁇ l of RNA solution in water, heated at 74°C for 2.5 min and rapidly quenched in an ice bath.
• 10 ⁇ l of 5 x RT buffer 50 mM Tris-HCl, pH 8.3, 75 mM KCI, 3 mM MgCl 2
• 1.25 ⁇ l of human recombinant placental RNA inhibitor were mixed with 1 ml of MMLV reverse transcriptase (200 units).
• 6.5 ⁇ l of this solution were added to RNA-primer mix and incubated at 42°C for one hour.
• 80 ⁇ l of water were added and the solution was incubated at 94°C for 5 minutes.
• 5 ⁇ l of the resulting solution were used in a Long Range PCR reaction with hot start, in 50 ⁇ l final volume, using 2 units of rtTHXL, 20 pmol/ ⁇ l of each of 5'- TGACCACCATGCCCATGCT-3' (271-289 in SEQ ID No 3) and 5'- GCATTTATTCACGTCCACGCC-3' (3929-3949 in SEQ ID No 3) primers with 35 cycles of elongation for 6 minutes at 67°C in fhermocycler.
• the amplification products co ⁇ esponding to both cDNA strands were partially sequenced in order to ensure the specificity of the amplification reaction.
• Donors were unrelated and healthy. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 individuals was extracted and tested for the detection of the biallelic markers.
• the pellet of white cells was lysed overnight at 42°C with 3.7 ml of lysis solution composed of: - 3 ml TE 10-2 (Tns-HCl 10 mM, EDTA 2 mM) / NaCl 0.4 M
• the pellet was dried at 37°C, and resuspended 1 ml TE 10-1 or 1 ml water.
• the pool was constituted by mixing equivalent quantities of DNA from each individual.
• Example 3 Detection of the biallelic markers: amplification of genomic DNA by PCR
• Each pair of first pnmers was designed using the sequence information of the TBC-1 gene disclosed herein and the OSP software (Hillier & Green, 1991). This first pair of pnmers was about 20 nucleotides in length and had the sequences disclosed in Table 1 in the columns labeled PU and RP. Table 1
• the primers contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing.
• Primers PU contain the following additional PU 5' sequence : TGTAAAACGACGGCCAGT (SEQ ID No 6); primers RP contain the following RP 5' sequence : CAGGAAACAGCTATGACC (SEQ ED No 7).
• DNA amplification was performed on a Genius II thermocycler. After heating at 95°C for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 95°C, 54°C for 1 min, and 30 sec at 72°C. For final elongation, 10 min at 72°C ended the amplification.
• the quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).
• Detection of the biallelic markers sequencing of amplified genomic DNA and identification of polymorphisms.
• the sequencing of the amplified DNA obtained in example 3 was carried out on ABI 377 sequencers.
• the sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol.
• the products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel image analysis [ABI Prism DNA Sequencing Analysis software (2.1.2 version)].
• sequence data were further evaluated to detect the presence of biallelic markers among the pooled amplified fragments.
• the polymo ⁇ hism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occu ⁇ ing at the same position as described previously.
• BM refers to "biallelic marker”. Alll and all2 refer respectively to a lele 1 and allele 2 of the biallelic marker.
• the biallelic markers identified in example 4 were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was ca ⁇ ied out for each individual DNA sample described in Example 2.
• the prefe ⁇ ed primers used in microsequencing were about 19 nucleotides in length and hybridized just upstream of the considered polymo ⁇ hic base. According to the invention, the primers used in microsequencing are detailed in Table 4.
• microsequencing reaction was performed as follows :
• the microsequencing reaction mixture was prepared by adding, in a 20 ⁇ l final volume: 10 pmol microsequencing oligonucleotide, 1 U Thermosequenase (Amersham E79000G), 1.25 ⁇ l Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mM MgCl 2 ), and the two appropriate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to the nucleotides at the polymo ⁇ hic site of each biallelic marker tested, following the manufacturer's recommendations.
• the software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous.
• the software identifies significant peaks (according to shape and height criteria). Among the significant peaks, peaks co ⁇ esponding to the targeted site are identified based on their position. When two significant peaks are detected for the same position, each sample is categorized classification as homozygous or heterozygous type based on the height ratio.

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