WO1995032284A1 - Facteur agissant en interaction avec des proteines nucleaires - Google Patents

Facteur agissant en interaction avec des proteines nucleaires Download PDF

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Publication number
WO1995032284A1
WO1995032284A1 PCT/EP1995/001834 EP9501834W WO9532284A1 WO 1995032284 A1 WO1995032284 A1 WO 1995032284A1 EP 9501834 W EP9501834 W EP 9501834W WO 9532284 A1 WO9532284 A1 WO 9532284A1
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obf
nucleic acid
protein
dna
oct
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PCT/EP1995/001834
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English (en)
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WO1995032284A9 (fr
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Patrick Matthias
Michel Strubin
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Ciba-Geigy Ag
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Priority to EP95922452A priority Critical patent/EP0763109A1/fr
Priority to JP7530029A priority patent/JPH10500311A/ja
Priority to AU27345/95A priority patent/AU2734595A/en
Publication of WO1995032284A1 publication Critical patent/WO1995032284A1/fr
Publication of WO1995032284A9 publication Critical patent/WO1995032284A9/fr
Priority to FI964606A priority patent/FI964606A0/fi
Priority to NO964981A priority patent/NO964981L/no

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity

Definitions

  • the present invention provides nucleic acids and transcription factor proteins encoded thereby.
  • host cells containing or expressing such nucleic acids are provided.
  • immunoglobulin (Ig) genes are controlled by promoter and enhancer elements which are B cell-specific in their activity (Staudt and Lenardo, 1991).
  • the octamer motif with the consensus sequence -ATGCAAAT-, or its inverse complement -ATTTGCAT-, is one of the most conspicuous regulatory elements of Ig genes, as it is present in every Ig promoter as well as in most of the Ig enhancers, intronic or 3' (Staudt and Lenardo, 1991).
  • a single octamer motif of the above sequence is able to confer B cell-specific expression to a linked reporter gene: insertion of the octamer site into a non-related minimal promoter renders it largely B cell-specific (Dreyfus et al., 1987; Wirth et al., 1987). Multimerisation of the octamer motif creates a potent B cell- specific enhancer (Gerster et al., 1987). Furthermore, the octamer motif is also a functionally important element of the promoter or enhancer of many non lymphoid-specific genes such as the histone H2B or U small nuclear (sn) RNA genes (LaBella et al., 1988).
  • Oct-1 an ubiquitous protein of about 750 amino acids.
  • NF-A1 NF-A1 , OBP100, NF III or OTF-1.
  • Oct-2 a protein of about 479 amino acids which exists in several different isoforms due to alternative splicing.
  • Oct-2 which is also known as OTF-2 or NF-A2
  • OTF-2 is expressed mostly, if not exclusively, in B cells (M ⁇ ller et al., 1988; Staudt et al., 1988).
  • Oct proteins all belong to the POU family of homeodomain proteins and are highly homologous in their DNA binding domain but quite divergent outside of it (Herr et al., 1988). So far, the two best characterised Oct proteins are Oct-1 and Oct-2.
  • Ig promoters or other artificial octamer-dependent promoters are highly active in B cells, which contain Oct-1 and Oct-2, and weakly active in other cells such as fibroblasts, which contain only the Oct-1 protein. These results suggested that the B cell-spec if ic activity of the octamer motif is largely due to the presence of the Oct-2 protein in B cells. I nis early model was further supported by the demonstration that overexpression of Oct-2 in non-B cells could efficiently activate octamer-containing promoters (M ⁇ ller et al., 1988).
  • HeLa extracts (LeBowitz et al., 1988; Johnson et al., 1990; Pierani et al., 1990; Luo et al., 1992).
  • a protein fraction has been recently isolated from a B cell nuclear extract which, in conjunction with purified Oct-1 or Oct-2, specifically stimulates transcription from an Ig promoter. This fraction, which could not be isolated from HeLa nuclear extracts, has been designated OCA-B (Oct coactivator from B cells) (Luo et al., 1992).
  • Oct-2-deficient mice have recently been generated by gene targeting in embryonic stem (ES) cells; in these mice, which totally lack the Oct-2 protein, Ig genes are rearranged and transcribed efficiently and B cell development appears normal until the stage of the surface IgM-bearing virgin B cell, suggesting that Oct-2 is dispensable in the first, antigen- independent phase of B cell differentiation (Corcoran et al., 1993). At later stages, the B cells from these animals show an impaired capacity to synthesize high levels of immuno- globulin. This finding therefore directly demonstrates a role for Oct-2 in Ig expression, if only at a late B cell stage.
  • ES embryonic stem
  • the present invention provides nucleic acids encoding a protein specifically interacting with the POU proteins Oct-1 or Oct-2. Such proteins are denominated OBF-1 (Oct binding factor 1).
  • a nucleic acid encoding a B-lymphocyte specific activator of octamer site-mediated gene transcription, which interacts with the POU domain of Oct-1 and Oct-2 in order to activate gene transcription.
  • Such activators are referred to herein as OBF-1 and the present invention therefore relates to isolated nucleic acid (DNA, RNA) coding for OBF-1.
  • these nucleic acids are also useful as probes, thus readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding OBF-1.
  • the nucleic acid may be unlabelled or labelled with a detectable moiety.
  • nucleic acid according to the invention is useful e.g.
  • the invention provides nucleic acid sequence that is complementary to, or hybridizes under stringent conditions to, a nucleic acid sequence encoding OBF-1.
  • the invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) OBF-1.
  • the nucleic acid is DNA and further comprises a replicable vector comprising the nucleic acid encoding OBF-1 operably linked to control sequences recognized by a host transformed by the vector.
  • the invention provides host cells transformed with such vector and a method of using a nucleic acid encoding OBF-1 to effect the production of OBF-1, comprising expressing OBF-1 nucleic acid in a culture of the transformed host cells and, if desired, recovering OBF-1 from the host cell culture.
  • the present invention relates to isolated OBF-1 proteins encoded by the above-described nucleic acids.
  • isolated is intended to refer to a molecule of the invention in an enriched or, preferably, pure form obtainable from a natural source or by means of genetic engineering.
  • the isolated DNAs, RNAs and proteins of the invention may be useful in ways that the DNAs, RNAs and proteins as they naturally occur are not, such as identification of com ⁇ pounds selectively modulating the activity of Oct-1 or Oct-2.
  • Isolated OBF-1 nucleic acid includes nucleic acid that is free from at least one contaminant nucleic acid with which it is ordinarily associated in the natural source of OBF-1 nucleic acid. Isolated nucleic acid thus is present in other than in the form or setting in which it is found in nature.
  • isolated OBF-1 encoding nucleic acid includes OBF-1 nucleic acid in ordinarily OBF-1 -expressing cells where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature.
  • isolated nucleic acids e.g. DNAs or RNAs, encoding OBF-1 , particularly mammalian OBF-1, e.g. murine or human OBF-1 , or fragments thereof.
  • the invention provides a DNA molecule encoding OBF-1 , or a fragment thereof.
  • a DNA comprises a coding single stranded DNA, a double stranded DNA of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself.
  • Exemplary nucleic acids encoding OBF-1 are represented in SEQ ID NOs. 1 and 3.
  • a cDNA encoding human OBF-1 is obtainable from plasmid pRS314/UNVPl6/clone 9 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-381 4 Braunschweig, under accession number 9200 on May 9, 1994.
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • Preferred sequences encoding OBF-1 are those having substantially the same nucleotide sequence as the coding sequences in SEQ ID NOs. 1 and 3, with the nucleic acids having the same sequence as the coding sequence in SEQ ID NOs. 1 and 3 being most preferred.
  • nucleotide sequences which are substantially the same share at least about 90 % identity. However, in the case of splice variants having e.g. an additional exon sequence homology may be lower.
  • Exemplary nucleic acids can alternatively be characterized as those nucleotide sequences which encode an OBF-1 protein and hybridize to the DNA sequences set forth in SEQ ID NOs. 1 and 3, or a selected portion (fragment) of said DNA sequence.
  • selected fragments useful for hybridisation are those employed in the Examples, e.g. the cDNA used for the isolation of the mouse homologue , i.e. the 2 kb Sfi 1 cDNA insert present in plasmid pR314/UNVP16/clone 9.
  • Preferred are such sequences encoding OBF-1 which hybridize under high-stringency conditions to the above-mentioned 2 kb Sfi 1 cDNA insert.
  • Stringency of hybridization refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T m ) of the hybrid which decreases approximately 1 to 1.5°C with every 1 % decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of higher stringency, followed by washes of varying stringency.
  • high stringency refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 1 M Na + at 65-68 °C.
  • High stringency conditions can be provided, for example, by hybridization in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulfate), 0.1 Na + pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor.
  • high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2- 0.1 x SSC, 0.1 % SDS.
  • Moderate stringency refers to conditions equivalent to hybridization in the above described solution but at about 60-62°C. In that case the final wash is performed at the hybridization temperature in 1x SSC, 0.1 % SDS.
  • Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridization temperature in 2x SSC, 0.1 % SDS.
  • nucleic acids of the invention are obtainable according to methods well known in the art.
  • a DNA of the invention is obtain ⁇ able by chemical synthesis, using polymerase chain reaction (PCR) or by screening a genomic library or a suitable cDNA library prepared from a source believed to possess OBF- 1 and to express it at a detectable level.
  • PCR polymerase chain reaction
  • Chemical methods for synthesis of a nucleic acid of interest include triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods as well as oligonucleotide synthesis on solid supports. These methods may be used if the entire nucleic acid sequence of the nucleic acid is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.
  • cDNA expression libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it.
  • suitable means include monoclonal or polyclonal antibodies that recognize and specifically bind to OBF-1 ; oligonucleotides of about 20 to 80 bases in length that encode known or suspected OBF-1 cDNA from the same or different species; and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridizing gene.
  • Appropriate probes for screening genomic DNA libraries include, but are not limited to oligonucleotides, cDNAs or fragments thereof that encode the same or hybridizing DNA; and/or homologous genomic DNAs or fragments thereof.
  • a nucleic acid encoding OBF-1 may be isolated by screening suitable cDNA or genomic libraries under suitable hybridization conditions with a probe, i.e. a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth in SEQ ID NOs. 1 and 3.
  • a probe i.e. a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth in SEQ ID NOs. 1 and 3.
  • Suitable libraries are commercially available or can be prepared e.g. from cell lines, tissue samples, and the like.
  • a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous bases set forth in SEQ ID NOs. 1 and 3.
  • the nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimized.
  • the nucleotide sequences are usually based on conserved or highly homologous nucleotide sequences or regions of OBF-1.
  • the nucleic acids used as probes may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is not known.
  • nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridization.
  • suitable label means is a radiolabel.
  • the preferred method of labelling a DNA fragment is by incorporating ⁇ 32 P- dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art.
  • Oligonucleotides are usually end-labelled with ⁇ 32 P-labelled ATP and polynucleotide kinase.
  • other methods e.g. non-radioactive
  • positive clones are identified by detecting a hybridization signal; the identified clones are characterized by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding a complete OBF-1 (i.e., if they include translation initiation and termination codons). If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones.
  • the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNAs and deduced amino acid sequences provided herein.
  • genetic screening may be carried out using the nucleotide sequences of the invention as hybridization probes. Also, based on the nucleic acid sequences provided herein antisense-type therapeutic agents may be designed.
  • nucleic acid of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof.
  • Such mutants can be used e.g. to produce an OBF-1 mutein (mutant protein) that has an amino acid sequence differing from the OBF-1 sequences as found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.
  • the cDNA or genomic DNA encoding native or mutant OBF-1 can be incorporated into vectors for further manipulation.
  • vector refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplifi ⁇ cation of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
  • Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
  • the origin of repli ⁇ cation from the plasmid pBR322 is suitable for most Gram-negative bacteria
  • the 2 ⁇ plasmid origin is suitable for yeast
  • various viral origins e.g. SV 40, polyoma, adenovirus
  • the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.
  • Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression.
  • a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome.
  • DNA may also be replicated by insertion into the host genome.
  • the recovery of genomic DNA encoding OBF-1 is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise OBF-1 DNA.
  • DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.
  • an expression and cloning vector may contain a selection gene also referred to as selectable marker.
  • This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.
  • any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene.
  • Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3. LEU2. LYS2. TRP1. or HIS3 gene.
  • E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript (TM) vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
  • E. coli plasmids such as pBR322, Bluescript (TM) vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up OBF-1 nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase or genes conferring resistance to G418 or hygromycin.
  • the mammalian cell transformants are placed under selection pressure which only those transformants which have taken up and are expressing the marker are uniquely adapted to survive.
  • selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes OBF-1.
  • Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the chromosomes of recombinant cells. Increased quantities of desired protein are usually synthesized from thus amplified DNA.
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to OBF-1 nucleic acid.
  • Such a promoter may be inducible or constitutive.
  • the promoters are operably linked to DNA encoding OBF-1 by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native OBF-1 promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of OBF-1 DNA.
  • Promoters suitable for use with prokaryotic hosts include, for example, the ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding OBF-1 , using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding OBF-1.
  • the OBF-1 gene according to the invention preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.
  • the peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.
  • Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene.
  • hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene
  • a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene PH05-GAP hybrid promoter
  • a suitable constitutive PHQ5 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide -173 and ending at nucleotide -9 of the PH05 gene.
  • OBF-1 gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated with OBF-1 sequence, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or
  • Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5' or 3' to OBF-1 DNA, but is preferably located at a site 5' from the promoter.
  • a eukaryotic expression vector encoding OBF-1 may comprise a locus control region (LCR).
  • LCRs are capable of directing high-level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the OBF-1 gene is to be expressed in the context of a permanently- transfected eukaryotic cell line in which chromosomal integration of the vector has occurred, in vectors designed for gene therapy applications or in transgenic animals.
  • Suitable eukaryotic host cells for expression of OBF-1 include yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding OBF-1.
  • An expression vector includes any vector capable of expressing OBF-1 nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • DNAs encoding OBF-1 may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias et al., 1989).
  • a CMV enhancer-based vector such as pEVRF (Matthias et al., 1989).
  • Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesizes high levels of OBF-1.
  • transient expression systems are useful e.g. for identifying OBF-1 muteins, to identify potential phosphorylation sites, or to characterize functional domains of the protein.
  • Plasmids according to the invention employs convent onal ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and relieved in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing OBF-1 expression and function are known to those skilled in the art.
  • Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridization, using an appropriately labelled probe based on a sequence provided herein. Suitable methods include those described in detail in the Examples. Those skilled in the art will readily envisage how these methods may be modified, if desired.
  • cells containing the above-described nucleic acids i.e., DNA or mRNA.
  • Such host cells such as prokaryote, yeast and higher eukaryote cells may be used for replicating DNA and pro ⁇ ducing OBF-1.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram- positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5a and HB101 , or Bacilli.
  • Further hosts suitable for OBF-1 encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae.
  • Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells.
  • vertebrate cells in recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • useful mammalian host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or293T cells.
  • the host cells referred to in this disclosure comprise cells in ]n vitro culture as well as cells that are within a host animal.
  • DNA may be stably incorporated into cells or may be transiently expressed using methods known in the art.
  • Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.
  • the cells should be transfected with a sufficient amount of OBF-1 -encoding nucleic acid to form OBF-1.
  • the precise amounts of DNA encoding OBF-1 may be empirically determined and optimized for a particular cell and assay.
  • Host cells are transfected or, preferably, transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognized when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.
  • Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby OBF-1 encoded by the DNA is expressed.
  • the composition of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also commercially available.
  • DNA provided herein may be expressed in any suitable host cell, e.g. those referred to above, preferred for expression of DNA encoding functional OBF-1 are eukaryotic expression systems such as baculovirus-based systems and, particularly, mammalian expression systems, including commercially available systems and other systems known to those of skill in the art, which express the Oct-1 protein and/or the Oct-2 protein (either endogenously or recombinantly).
  • eukaryotic expression systems such as baculovirus-based systems and, particularly, mammalian expression systems, including commercially available systems and other systems known to those of skill in the art, which express the Oct-1 protein and/or the Oct-2 protein (either endogenously or recombinantly).
  • OBF-1 encoding DNA is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express OBF-1.
  • the resulting cell lines can then be produced in quantity for reproducible qualitative and/or quantitative analysis of the effect(s) of potential drugs affecting OBF-1 function.
  • OBF-1 expressing cells may be employed for the identification of compounds, particularly small molecules capable of penetrating the nucleus, which compounds enhance the specific interaction between OBF-1 and Oct factors (agonists), thereby increasing the potency of OBF-1.
  • antagonizing molecules interfering with the Oct / OBF-1 interaction are useful.
  • OBF-1 antisense OBF-1 RNA.
  • host cells expressing OBF-1 are useful for drug screening and it is a further object of the present invention to provide a method for identifying compounds which modulate the activity of OBF-1 , said method comprising exposing cells containing heterologous DNA encoding OBF-1 , wherein said cells produce functional OBF-1 , to at least one compound or signal whose ability to modulate the activity of said OBF-1 is sought to be determined, and thereafter monitoring said cells for changes caused by said modulation.
  • Such an assay enables the identification of agonists, antagonists and allosteric modulators of OBF-1.
  • Cell-based screening assays can be designed e.g. by constructing cell lines in which the expression of a reporter protein, i.e. an easily assayable protein, such as ⁇ galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on OBF-1.
  • a reporter protein i.e. an easily assayable protein, such as ⁇ galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase
  • CAT chloramphenicol acetyltransferase
  • CAT chloramphenicol acetyltransferase
  • luciferase e.g. an easily assayable protein, such as ⁇ galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase
  • CAT chloramphenicol acetyltransfera
  • OBF-1 modulates the activity of these transcription factors and thus is capable of contributing directly to the regulation of Ig genes expression.
  • OBF-1 may be useful for boosting Ig production, e.g. for boosting monoclonal antibody production. This can be achieved, for example, by overexpressing in B cells OBF-1 itself or even more potent OBF-1 based hybrid proteins such as, for example, a VP16-OBF-1 chimera.
  • molecules interfering with Oct factor/ OBF-1 expression may be useful.
  • the invention accordingly provides an expression system which is regulatable directly or indirectly by OBF-1 , comprising a host cell which is transfectable with one or more vectors which encode a desired protein.
  • Oct proteins which are necessary for OBF-1 activity, may be provided exogenously, by transfected recombinant transcription unit(s) or may be endogenous to the host cell.
  • OBF-1 may be endogenous to the host cell, but preferably it is provided by means of a recombinant transcription unit expressing an OBF-1 gene. It has been found that OBF-1 is expressed mostly in cells of lymphoid origin.
  • the present invention also provides a method to exogenously affect OBF-1 dependent processes occurring in such cells.
  • Recombinant OBF-1 producing host cells e.g.
  • mammalian cells can be contacted with a test compound, and the modulating effect(s) thereof can then be evaluated by comparing the OBF-1 -mediated response in the presence and absence of test compound, or relating the OBF-1 -mediated response of test cells, or control cells (i.e., cells that do not express OBF-1), to the presence of the compound.
  • a compound or signal that modulates the activity of OBF-1 refers to a compound that alters the activity of OBF-1 in such a way that the activity of OBF-1 is different in the presence of the compound or signal (as compared to the absence of said compound or signal).
  • the invention also provides a transgenic non-human mammal which has been modified to modulate the expression of endogenous OBF-1.
  • the transgenic non-human mammal is a transgenic mouse.
  • a transgenic mouse may be designed in which OBF-1 production is greatly reduced or eliminated
  • the transgenic mouse of the invention may express elevated levels of OBF-1 , or may be subject to regulation of OBF-1 expression in a developmentally or tissue-specific manner, or via control by exogenous agents. Study of such an animal provides insights into the importance of OBF-1 in vivo.
  • the invention provides a transcription unit encoding OBF-1 for use in a method of treatment of a condition involving aberrant Ig gene expression by gene therapy techniques.
  • the transcription unit provided according to the present aspect of the invention comprises regulatable control regions which include a promoter, together with one or more enhancers and/or LCRs.
  • the transcription unit may be delivered to the subject by any suitable means, including viral vectors, especially retroviral vectors, adeno- and adeno associated viral vectors, non-viral delivery systems, including liposomal and antibody targeted delivery systems, and direct uptake of naked DNA.
  • the target tissue is advantageously a lymphoid tissue and preferably the transcription unit is delivered to haematopoietic stem cells.
  • the haematopoietic stem cells are removed from a patient, transfected ex vivo and subsequently returned to the patient.
  • the cells may be targeted in vivo, for example using antibody targeting approaches.
  • proteins encoded by an above-described nucleic acid comprising a B-lymphocyte specific activator of octamer site-mediated gene transcription, which interacts with the POU domain of Oct-1 and Oct-2 in order to activate gene transcription.
  • proteins are designated OBF-1 (Oct binding factor 1 ). Being a transcription factor, OBF-1 is capable of influencing transcription.
  • This biological activity can be shown in a suitable assay such as a transactivation assay, e.g. the assay as described in the Examples.
  • the protein of the invention is provided in isolated form.
  • isolated OBF-1 means OBF-1 which has been identified and is free of one or more components of its natural environment.
  • Isolated OBF-1 includes OBF-1 in a recombinant cell culture.
  • OBF-1 includes the amino acid sequences of human and murine OBF-1 set forth in SEQ ID NOs. 2 and 4, respectively, as well as peptides comprising all or part of said sequences and additional sequences, polypeptide or peptide fragments of said sequences and the OBF-1 protein producible from the plasmid pRS314/UNVP16/clone 9.
  • the definition of OBF-1 includes functional or immunogenic equivalents of OBF-1.
  • “functional equivalent” means a protein displaying the in vivo effector function that is directly or indirectly performed by OBF-1 (whether in its native or denatured conformation), or by any subsequence thereof. Effector functions include receptor binding and activation, induction of differentiation, DNA regulatory functions and the like.
  • a principal known effector function of OBF-1 is its ability to interact with Oct-1 and Oct-2.
  • Immunogenic equivalent means a protein or peptide having the antigenic functions of OBF-1. Antigenic functions includes possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured OBF-1 polypeptide or fragment thereof.
  • OBF-1 as provided by the present invention includes splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants (muteins), glycosylation variants and other covalent derivatives of OBF-1 which retain the physiological and/or physical properties of OBF-1. Exemplary derivatives include molecules wherein the protein of the invention is covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid.
  • Such a moiety may be a detectable moiety such as an enzyme or a radioisotope.
  • a detectable moiety such as an enzyme or a radioisotope.
  • Naturally occurring variants of OBF-1 found with a particular species, preferably a mammal Such a variant may be encoded by a related gene of the same gene family, by an allelic variant of a particular gene, or represent an alternative splicing variant of the OBF-1 gene.
  • OBF-1 muteins may be produced from a DNA encoding OBF-1 which has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids.
  • substitutional, deletional or insertional variants of OBF-1 are prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of OBF-1.
  • a protein of the invention is obtainable from a natural source, e.g. from nuclear extracts of lymphoid cells, by chemical synthesis or by recombinant techniques. Due to its capability of competing with the endogenous OBF-1 counterpart for an endogenous ligand, a fragment displaying a selective physiological characteristic of OBF-1 , e.g. a fragment interacting with Oct-1 or Oct-2, is envisaged as a therapeutic agent.
  • the invention accordingly provides OBF-1 for use in medicine.
  • the invention provides a method for preparing a protein of the invention characterized in that suitable host cells producing said protein are multiplied in vitro or jn vivo.
  • the host cells are transformed (transfected) with a vector comprising an expression cassette comprising a promoter and a DNA sequence coding for OBF-1 which DNA is controlled by said promoter.
  • the protein of the invention may be recovered. Recovery comprises e.g. isolating the protein from the culture broth or from the host cells.
  • Preferred is a method for preparation of a functionally active protein. Any method known in the art for purification of proteins from recombinant cell culture may be used, including chemical solubilisation of proteins produced as inclusion bodies.
  • the protein is produced in soluble form and advantageously it is secreted by the host microorganism.
  • OBF-1 may also be derivatized in vitro, e.g. to prepare immobilized OBF-1 and labelled OBF-1 , e.g. for affinity purification of OBF-1 antibodies.
  • the proteins of the invention are useful e.g. as immunogens, in drug screening assays, as reagents for immunoassays and in purification methods, such as affinity purification of a binding ligand, and as therapeutics.
  • antibodies specifically recognizing and binding to OBF-1 may be generated against the OBF-1 having the amino acid sequences set forth in SEQ ID Nos. 2 or 4.
  • OBF-1 or OBF-1 fragments are fused (by recombinant expression or an in vitro peptidyl bond) to an immunogenic polypeptide and this fusion polypeptide, in turn, is used to raise antibodies against an OBF-1 epitope.
  • Anti-OBF-1 antibodies are recovered from the serum of immunized animals. Alternatively, monoclonal antibodies are prepared from cells in vitro or from in vivo immunized animals in conventional manner. Preferred antibodies identified by routine screening inhibit the interaction of OBF-1 with Oct-1 or Oct-2.
  • the antibodies of the invention are useful for studying OBF-1 tissue localization, screening of an expression library to identify nucleic acids encoding OBF-1 or the structure of functional domains, as well as in diagnostic applications, for the purification of OBF-1 , and the like.
  • the invention particularly relates to the specific embodiments as described in the Examples which serve to illustrate the present invention but should not be construed as a limitation thereof.
  • a cDNA library is then prepared in a yeast expression vector such that the cDNA (derived from the appropriate cell line or tissue) is fused randomly to a transcription activation domain active in yeast (this is hybrid protein #2, thus the name two- hybrid). If a particular cDNA encodes a protein (or protein domain) interacting with the target protein (and if the cDNA is in-frame with the expression vector-derived activation domain) an elevated transcription from the reporter gene can be measured. The corresponding cDNA can then be rescued from the yeast cells, amplified in E. coli and characterised further.
  • a plasmid is constructed to express an essentially intact Oct-1 protein from a centromeric yeast vector, using the promoter and termination sequences of the yeast TATA binding protein gene.
  • a nuclear localisation sequence (NLS) derived from the SV40 T-antigen protein is engineered at the N terminus of Oct-1.
  • the his 3 gene which codes for the enzyme imidazole glycerol phosphate dehydratase (IGP), an enzyme required for the biosynthesis of the amino acid histidine, is used as a reporter gene.
  • IGP imidazole glycerol phosphate dehydratase
  • his 3 gene expression is required for growth (e. g. Struhl, 1983).
  • 3- aminotriazole (3-AT) 3- aminotriazole
  • 3-AT 3- aminotriazole
  • a his 3 reporter is made which contains, in the his 3 promoter upstream of the TATA box, six copies of an octamer site derived from the Ig heavy chain intron enhancer (Ylp55- AT/H36).
  • This reporter construct is reintroduced in yeast at the original his 3 locus, to generate the strain called Y:AT.H36.
  • the expression vector coding for Oct-1 is then introduced, generating the screening strain Y:AT.H36/OCT1.
  • An inducible expression vector which contains the transcription activation domain from VP16, a very potent transcription activator from Herpes Simplex Virus, under the control of a hybrid Gal1-10/his 3 promoter (pRS314/UASQ-NLS.VP16(Sfi1)).
  • This vector allows the expression, upon galactose induction, of fusion proteins between the VP16 activation domain and any cDNA that is inserted downstream of it.
  • a cDNA library with mRNA extracted from the human B cell line Namalwa (ATCC CRL-1432) is constructed. Plasmid DNA is then prepared from the cDNA library and used to transform the yeast strain containing the his 3 reporter and expressing Oct-1. This allows the isolation of a cDNA coding for OBF-1 , a novel protein.
  • YPD 1 % (w/v) Bacto-yeast extract (Difco), 2 % (w/v) Bacto-peptone, 2 % (w.v)glucose or galactose (for galactose, a quality containing less than 0.01 % glucose should be used;
  • YPAD as YPD with the addition of 0.01 volume of 0.25 % adenine.
  • Synthetic medium lacking histidine and containing AT 0.17 % (w/v) yeast nitrogen base without amino acids and A1T1SO4 (Difco), 0.5 % (w/v) ammonium sulfate, 2 % (w/v) glucose or galactose, 0.01 volume of each: 0.5 % (w/v) tryptophan, 0.25 % (w/v) uracil, 1 % (w/v) leucine, 0.25 % (w/v) adenine, 1 % (w/v) lysine.
  • 3-Aminotriazole (AT) is added at the appropriate concentration.
  • FOA 5-fluoroorotic acid
  • CAA medium is made with CAA medium supplemented with 0.1% (w/v) FOA, 0.01 volume of 0.5 % (w/v) tryptophan and 0.02 volume of 0.25 % (w/v) uracil.
  • Solid medium for plates contains, in addition to the above, 2 % agar.
  • the Oct1 protein is expressed constitutively from a single copy plasmid using the native transcriptional initiation and termination signals of the yeast TATA binding protein (TBP) gene.
  • TBP yeast TATA binding protein
  • the parental plasmid p2DN-1 from which these regulatory sequences are derived carries a 2.3 kb Pstl-BamH1 genomic fragment containing the entire yeast TBP gene and extending 1 kb upstream and 500bp downstream of the coding region (Cormack et al, 1991).
  • This gene differs from wild type by the presence of an EcoR1 site introduced upstream of the naturally occurring ATG initiator codon (TTTTTTGAATTCAT ATG; Cormack et al., 1991).
  • the promoter and 5'-untranslated region of the TBP gene is first subcloned as a ca.
  • pRS316 is a centromeric plasmid carrying the URA 3 selectable marker.
  • Nde1 site is introduced at the first ATG of the Oct1 sequence described by Sturm et al. (1988) by polymerase chain reaction (PCR) using pBS-Oct1+ (Sturm et al., 1988) as template and the following forward and reverse oligonucleotide primers: forward 5 ' -GGG CAT ATG AAC AAT CCG TCA GAA ACC-3 ' reverse 5 ' -GAG TAG TAA CTG TTG CTG GGC AGG-3 1 .
  • the resulting 375 bp PCR fragment is then directly ligated into the pCRII vector from a T/A cloning kit (Invitrogen) and the DNA sequence of the resulting clone called pCRII/Oct1 (Nde)5' is confirmed.
  • pCRII/Oct1 Nde5' is confirmed.
  • a complete Oct-1 cDNA with the Nde1 site at the ATG is then reconstructed by ligating the three DNA fragments described below:
  • the accepting vector is Bluescript KS- (Stratagene) which is cleaved with Pstl (compatible with Nsi1 ) and Hind3.
  • the final Oct-1 expression vector is then constructed by ligating four DNA fragments as follows:
  • oligonucleotide which has 5' EcoR1 and 3' Nde1 compatible single-stranded extensions and provides an ATG as well as a nuclear localisation sequence (NLS) derived from SV40:
  • the accepting vector is the previously described pRS316 derivative pRS316.TBP5' containing the 5' regulatory region and promoter of the yeast TBP gene.
  • This plasmid is cleaved with EcoR1 and BamH1 (site found in the pRS316 polylinker).
  • the resulting final plasmid is confirmed by DNA sequencing and is called pRS316/TBP5'3'- OCT1.
  • a his 3 allele with six octamer sites 73bp upstream of the TATA box is constructed from Ylp55-Sc3760, an integrative plasmid carrying the URA 3 selectable marker and containing a 6.1 kbp segment of yeast chromosomal DNA with the entire pet56-his 3-ded1 gene region (Harbury and Struhl, 1989).
  • a ca. 300bp Xhol fragment containing 6 copies of an octamer motif (called here 6x octa fragment) derived from the mouse immunoglobulin heavy chain intron enhancer (positions 518-564; numbering as in Ephrussi et al., 1985) is obtained from p6W+, a pUC derivative (described in Gerster et al., 1987).
  • the 6x octa Xhol fragment is first inserted into the Xhol site of BluescriptKS- and results in clone Blue6W. In Blue6W, the former Hinfl site of the IgH enhancer DNA fragment (position ca. 560) is next to the Kpn1 site of the Bluescript polylinker.
  • the final reporter construct is then made by ligating together three fragments:
  • the accepting vector is the large (ca. 8kbp) EcoRI -Kpn1 fragment from Ylp55-Sc3760.
  • These various steps effectively replace the native EcoRI -Kpn1 fragment from Ylp55- Sc3760 by the chimeric 6x octa-his 3 promoter fragment, and generate the final reporter plasmid called Ylp55-AT/H36.
  • the nucleotide sequence of the Yip55-AT/H36 promoter region, presented from the EcoRI site 5' of the 6x octa fragment to the 4th nucleotide past the end of the his 3 TATA box, is as presented below.
  • the borders of the 6 times repeated IgH enhancer fragment, the oct site and the his 3 TATA box are highlighted in bold.
  • the screening of the library expressing hybrid proteins between the VP16 activation domain and random cDNA-encoded polypeptides requires a yeast strain containing an integrated copy of the AT/H36 his3 allele and expressing constitutively OCT1.
  • yeast strain KY320 Choen and Struhl, 1988
  • the resulting Ura + integrants are then grown on nonselective YPD medium before being streaked on 5-fluoroorotic acid (5-FOA) plates.
  • This step selects against the URA 3 gene and hence for the loss of the plasmid sequence as a result of homologous recombination events between the parental and the new copies of the his 3 gene.
  • the segregants that retain the AT/H36 his 3 allele are identified by their ability to grow in medium lacking histidine.
  • the final Y:AT.H36/OCT1 strain is generated by introducing pRS316/TBP5'3'-OCT1 into the segregant with the desired His + phenotype using the lithium-acetate method (Becker and Guarente, 1991) and selecting for growth on plates lacking uracil.
  • the library of hybrid proteins between the VP16 acidic activation domain and random cDNA fragments is expressed from a centromeric plasmid under control of the tightly regulated gal-his 3 hybrid promoter.
  • This expression vector is constructed as follows. The 365 bp Gall -10 UASQ element fused to the his 3 promoter is derived from plasmid
  • the PCR reaction is carried out using as template DNA a derivative of plasmid 3801 in which the unique EcoRI restriction site located between the UASQ and the his 3 TATA box is deleted by filling the recessed 3' termini resulting from EcoRI digestion with the Klenow fragment of DNA polymerase I.
  • the PCR product is digested with Sail and EcoRI and inserted into pUC19 for DNA sequencing.
  • the resulting clone is called pUC19.Gal.his 3.
  • the VP16 acidic activation domain (amino acids 413 to 490) is amplified by PCR from plasmid pMSVP16 D1 D3 (Triezenberg et al., 1988), using the following forward and reverse oligonucleotide primers: forward 5 ' -CCC GAATTC ACCATGGCCCCCCCGACCGATGTC-3 ' reverse 5 ' -CCG CATATG CCCACCGTACTCGTCAATTC-3 ' .
  • This step introduces a 5' EcoRI site flanking an ATG initiator codon fused in frame to Ala-
  • PCR product is cloned between the EcoRI and Sma1 sites of pUC19 to generate pUC19 VP16. The DNA sequence of the clone is confirmed.
  • a nuclear localisation sequence is subsequently fused in frame to the amino terminus of the VP16 activation domain by ligating three fragments as follows:
  • Hind3-Nde1 (Klenow filled-in) ca. 1kbp fragment including the 5' flanking region of yeast TBP coding region and sequences encoding the NLS prepared from pRS316/TBP5'3'- OCT1
  • the accepting vector is pRS314 (Sikorski and Hieter, 1989) cleaved with Hind3 at the polylinker site (partial digest) and BamH1.
  • the gal-his 3 hybrid promoter prepared from pUC19.Gal.his 3 is then introduced into this intermediate construct as a Sail -EcoRI fragment between the Xhol (compatible with Sail) and EcoRI sites, hence replacing the TBP promoter region upstream of the NLS.VP16 coding sequence.
  • the unique EcoRI site present in this plasmid (located 5' of the NLS.VP16 coding sequence) is deleted by filling the EcoRI recessed 3' termini with T4 DNA polymerase.
  • the resulting plasmid is called pRS314. UASf3-NLS.VP16.
  • the final expression vector is prepared by inserting, at the 3' end of the VP16 activation domain (Nde1 site) in pRS314/UASQ-NLS.VP16, a cDNA cloning cassette comprising a 800 bp long stuffer fragment derived from the chloramphenicol acetyltransferase (CAT) gene flanked by non-palindromic Sfi1 sites and the 3' termination signals of the yeast TBP gene. This is done as follows:
  • Two double-stranded phosphorylated adaptors having non-palindromic Sfi1 extensions are synthesized; these adaptors are kinased and annealed and have the following sequence:
  • dsAdaptorl provides a Nde1 5' overhang and contains an internal EcoRI site; dsAdaptor * 2 introduces stop codons in each translational reading frame (CGGCCGCTAACTGACTAGGTAC) and provides a 3' Kpn1 overhang.
  • An equimolar mixture of these two adaptors is first ligated for 3 hrs to ca. 1 ⁇ g of the purified 800 bp CAT Sfi1 fragment prepared from plasmid EBO-Sfi (Steimle et al., 1993).
  • CAT fragment is a mixture of CAT fragments having a copy of dsAdaptorl at each end, or a copy of dsAdaptor2 at each end, or dsAdaptorl at one end and dsAdaptor2 at the other end.
  • the accepting vector is pRS314.
  • UASQ-NLS.VP16 cleaved with Nde1 and BamH1. Restriction analysis and DNA sequencing allows to identify the final clone having the following structure: Gal-his3 promoter/NLS/VP16 activation domain/ Nde1 -EcoRI -Sfi1 -CAT- Sfi1 -stop3x-Kpn1/3' TBP termination signals-BamH1.
  • This expression vector is called pRS314/ UAS G -NLS.VP16(Sfi1 ).
  • RNA is isolated from the human B lymphoid cell line Namalwa (ATCC CRL 1432) using an RNA Extraction Kit from Pharmacia (product # 27-9270-01 ) and following exactly the manufacturers instructions. A total of 3 x 10*-- cells are used and ca. 4.5 mg of total RNA is obtained. The RNA is diluted in sterile 10 mM Tris pH 7.5, 1 mM EDTA (TE) to a con ⁇ centration of 2 mg/ml and an aliquot (0.8 ml, equivalent to ca.
  • TE sterile 10 mM Tris pH 7.5, 1 mM EDTA
  • cDNA is synthesized using a Superscript Choice System from Life Technologies (product # 530-8090SA). To 5 ⁇ g of polyA + RNA, 2 ⁇ l of oligodT (tube A1 ) and 1 ⁇ l of random hexamer (tube A2) are added; after 10 min at 70°C, the mixture is chilled on ice. To that tube, 0.5 ⁇ l RNAsin (Promega), 4 ⁇ l 5x first strand buffer (tube A3), 2 ⁇ l 0.1 M DTT, 1 ⁇ l dNTPs (tube A5) and 1 ⁇ l ⁇ - 32 P-dATP (Amersham, diluted 1 :5; 0.666 pmole) are added.
  • the first strand synthesis reaction is started by adding 5 ⁇ l Superscript reverse transcriptase (from the kit) and incubating the reaction for 60 min at 42°C. The reaction is then transferred on ice. To the tube the following are added sequentially: 93 ⁇ l H2O, 30 ⁇ l 5x second strand buffer (tube B1 ), 3 ⁇ l dNTPs (tube A5), 1 ⁇ l E. coli ligase (tube B2), 4 ⁇ l E. coli DNA polymerase (tube B3), 1 ⁇ l RNAse H (tube B4).
  • reaction is incubated for 2 hours at 16°C, 2 ⁇ l T4 DNA polymerase (tube B5) are added and the reaction is incubated for another 10 min at 16°C and then finally quenched on ice. 10 ⁇ l 0.5 M EDTA and 16 ⁇ l 3M NaAc are added, the reaction is extracted with phenol: chloroform (1 :1 ), and the nucleic acids in the supernatant are precipitated by addition of 425 ⁇ l 100 % EtOH.
  • the sample is centrifuged in a microfuge, washed with EtOH 80 %, resuspended in 75 ⁇ l TE and passed over a Sepharose 4CLB column to get rid of the small cDNAs (SizeSep column from Pharmacia; product # 27-5105-01) following exactly the manufacturers instructions.
  • the eluate is divided in three equal aliquots (of ca. 20 ⁇ l each) and each aliquot is used for a separate ligation reaction to double-stranded Sfi1 adaptors, in each translational reading frame.
  • the adaptors have previously been kinased and annealed and have the following sequence:
  • Each ligation reaction contains 20 ⁇ l cDNA and 190 pmoles kinased and annealed adaptor in a final volume of 30 ⁇ l. After 15 hrs at 16°C the 3 reactions are pooled and precipitated with NH4AC and EtOH. The cDNA is collected by centrifugation, washed with 80% EtOH and resuspended in 100 ⁇ l TEN (10 mM Tris pH 7.5; 1 mM EDTA; 25 mM NaCl). The cDNA is then size-fractionated by passing over a Sephacryl column (provided in the cDNA synthesis kit), following exactly the manufacturers instructions. The different cDNA fractions are EtOH precipitated individually, and each cDNA pellet is finally resuspended in 10 ⁇ l TE.
  • the reaction is then precipitated with NH4AC and EtOH, the DNA is collected by centrifugation and resuspended in 200 ⁇ l TE.
  • the cut vector is deposited on 2 sucrose gradients prepared (as described by Kieffer, 1991 ) in SW41 centrifuge tubes. The gradients are centrifuged for 16 hrs at 30 000 rpm in a SW41 rotor. The lower band (vector) is collected, the EtBr is removed by 1 -Butanol extraction, the sample is diluted with 1 vol. H2O and the DNA is precipitated with isopropanol after adjusting the NaCl concentration to 0.2 M (final) and adding 12mg linear polyacrylamide as carrier.
  • the vector DNA is collected by centrifugation and resuspended in TE at a concentration of ca. 50 ng/ ⁇ . cDNA ligation and E. coli transformation
  • Ligation reactions are set up with 50 ng vector (prepared as above) and varying amounts of size-fractionated, Sfi1 adaptors-ligated cDNA in 20 ⁇ l reactions containing 50 mM Tris pH 7.6, 10 mM MgCI , 1 mM ATP, 5% (w/v) PEG 8000, 1 mM DTT and 20 u T4 DNA ligase (N. E. Biolabs). After ligation for 12 hrs at 16°C, the DNA is phenol and phenol-CHC.3 extracted and then EtOH precipitated after addition of NaAc. (0.3 M final concentration) and 1.5mg yeast RNA as carrier.
  • the DNA is collected by centrifugation, the pellet is washed extensively with 80% EtOH and then resuspended in 4 ⁇ l TE. 1 ⁇ l of each ligation is then used for electroporation of ElectroMax DH10B electrocompetent bacteria (Life Technologies product # 530-8290 SA) following exactly the manufacturers instructions. On the basis of the number of transformants obtained the optimal cDNA:vector ratio is determined and additional ligation reactions are set up and subsequently processed as described.
  • the products corresponding to several electroporations as described above are pooled (corresponding to a total number of ca. 8 x 10 6 individual transformants) and plated onto LB/agar plates containing 100 ⁇ g/ml ampicillin at a density of 50 000 colony forming units (cfus)/132 mm plate. After overnight growth at 37°C, the colonies are washed from the plates with LB medium and pooled. An aliquot corresponding to ca. half of the sample is frozen away for future reamplification, and the rest is used for plasmid DNA preparation using a Magic Maxiprep Kit from Promega (product # A7270). The resulting DNA is then used for the yeast screening.
  • the library of fusion proteins is introduced into the yeast screening strain Y:AT.H36/OCT1 according to Schiestl and Gietz (1989), with the following modifications: an overnight culture, grown to 1x10 7 cells/ml in glucose minimal medium lacking uracil to maintain selection for the plasmid expressing OCT1 , is diluted to 2x10 6 cells/ml in fresh YPAD medium and regrown to 1x10 7 cells/ml. Cells from a 50ml culture are resuspended in 500 ⁇ l TE/LiAc buffer and directly mixed with 20 ⁇ g cDNA library plasmid DNA and 500 ⁇ g human polyA- RNA.
  • the cell suspension is dispensed equally in five eppendorf tubes. The subsequent steps are done exactly according to the published protocol. After heat shock, the cells are pooled and incubated for 1 hour at 30°C in 500 ml of YPAD with agitation. The complexity of the library (2x10 6 independent double transformants) is estimated by plating an aliquot of the culture on glucose minimal plates lacking uracil and tryptophan.
  • Transformants are recovered by centrifugation, inoculated into 500ml of glucose minimal medium lacking uracil and tryptophan to select for double transformants, and incubated for 16 hr at 30°C, at which time the culture consists of approximately 25% Trp +// Ura + cells.
  • An aliquot of the culture (7x10 8 cells representing 1.7x10 8 transformants) is harvested by centrifugation, resuspended in 50ml YP medium supplemented with galactose, and incubated for 5 hr at 30°C with constant agitation. This step, during which the cells do not divide, is required to induce the expression of the hybrid protein library.
  • the cells After centrifugation, the cells are resuspended in 5ml TE/LiAc buffer, and the transformants plated on galactose synthetic medium lacking histidine and containing 10mM AT; approximately 2x10 7 cells (5x10*-- double transformants) are plated on each of 20 plates.
  • mouse homologue of OBF-1 is also isolated from a cDNA library prepared from the mouse B cell line S194.
  • the ca. 2 kbp Sfi cDNA insert present in human clone pRS314/UNVP16/clone 9, DSM accession number 9200) is used.
  • the hybridisation is performed at 67°C for 16 hours in a solution containing 6 x SSC (20 x SSC is: 3 M NaCl, 0.3 M trisodium citrate), 5 x Denhardt's (100 x Denhardt's is 2 % (w/v) bovine serum albumin, 2 % (w/v) Ficoll 400, 2 % (w/v) polyvinylpyrollidone), 0.5 % SDS (sodium dodecyl sulfate) and 0.1 mg/ml denatured salmon sperm DNA.
  • 6 x SSC is: 3 M NaCl, 0.3 M trisodium citrate
  • 5 x Denhardt's 100 x Denhardt's is 2 % (w/v) bovine serum albumin, 2 % (w/v) Ficoll 400, 2 % (w/v) polyvinylpyrollidone
  • 0.5 % SDS sodium dodecyl sulfate
  • the filters are then washed as follows: 2 x 5 min at room temperature in 2 x SSC, 0.1 % SDS, 3 x 30 min at 60°C in 2 x SSC, 0.1 % SDS; 2 x 30 min at 60°C in 1 x SSC, 0.1 % SDS.
  • Several clones are isolated and confirmed by secondary and tertiary screenings under the same conditions.
  • the nucleotide sequence of the mouse OBF-1 cDNA (SEQ ID No. 3) is determined after progressive deletions are generated from either end of the cDNA subcloned in Bluescript IIKS+.
  • the OBF-1 cDNA can also be isolated from another species (e.g. rat) by using a PCR- based strategy.
  • Degenerate primers because of the genetic code degeneracy
  • it is then possible to attempt the amplification of the corresponding DNA fragment from the species of interest by using cDNA derived from B cells (or any other OBF-1 expressing cells) from that species (an already prepared cDNA library is also suitable for that purpose).
  • the amplified DNA fragment can then be subcloned in a standard vector and its nucleotide sequence determined. Once the correct fragment is obtained (on the basis of the sequence similarity with the presented mouse or human OBF- 1 sequence), it can then be used to rescreen cDNA libraries from the species of interest in order to isolate a complete OBF-1 clone from that species.
  • OBF-1 256 amino acid OBF-1 proteins
  • SEQ ID Nos. 2 and 4 which do not contain any known protein motif (such as a leucine zipper, a homeodomain, etc.).
  • OBF-1 does not show any obvious feature.
  • Known transcription factors, such as CTF-1 have been shown to be rich in proline residues and to contain proline-rich activation domains (Mermod et al., 1989). Thus it is possible that some of the proline residues of OBF- 1 might serve a similar function.
  • RNAs from various sources either organ (polyA + RNAs) or cell lines (total RNAs) shows that OBF-1 expression is highly restricted.
  • a major RNA species is detected ca. 3.0 to 3.2 kb in size.
  • Analysis with a radiolabelled probe derived from hOBF-1 shows strong expression in spleen and peripheral blood leukocytes, weak expression in thymus and small intestine and no detectable expression in prostate, testis, ovary and colon (polyA + RNAs).
  • RNA derived from various human cell lines shows strong expression in Namalwa and BJA-B (B cell lines), weak expression in Molt3 and Hut78 (T cell lines) and in HepG2 (hepatocytes), and no detectable expression in the following cells: K562 (myeloid leukemia), U937 (monocyte/macrophage), 293T (fibroblast), HeLa (cervix carcinoma, epithelial), MCF-7 (mammary carcinoma).
  • analysis of total RNA from several mouse B cell lines with the mouse OBF-1 probe gives the following pattern: intermediate to high expression in J558L, MPC11 and S194 B cell lines and weak expression in 70Z/3, 40E-1 , 18-81 and 220- 8 pre-B cell lines. ln conclusion the expression of the OBF-1 gene is highly cell-specific, being expressed mostly in cells of lymphoid origin. In addition the gene appears to be developmentally regulated, as several pre-B cell lines show significantly lower levels of expression than the mature B cell lines tested.
  • the yeast assay genetically identifies an interaction between Oct-1 and OBF-1.
  • the hOBF-1 cDNA was recloned in pEVRF, a CMV enhancer-based expression vector suitable for expression of cDNAs in mammalian cells (Matthias et al., 1989) giving rise to plasmid pEV-OBF.
  • the pEV-OBF-1 construct was made by ligating together the three DNA fragments indicated below: a Sma I to Sfi I fragment from pEVRFO (Matthias et al., 1989); this fragment contains the ampicillin resistance gene, prokaryotic origin of replication and CMV eukaryotic promoter/enhancer sequences as well as an ATG translation initiation codon in an optimised context; an Eco Rl (filled in) to Hind III OBF-1 cDNA fragment from plasmid pRS314/UNVP16/clone 9; this fragment is derived from the complete OBF-1 cDNA clone (the Eco Rl site is derived from the vector and the Hind III site is within the 3' untranslated region of the OBF-1 cDNA) and includes the 5' leader sequences present in the sequence shown as Seq ID No: 1 ; and a Hind III to Sfi I fragment from plasmid p3S/2-457 (M ⁇ ller-lmmerg
  • the pEV-OBF-1 plasmid leads to the expression, in eukaryotic cells, of the OBF-1 protein with translation starting at the ATG derived from the pEVRF vector.
  • the pEV-OBF-1 plasmid was then transiently transfected, alone or in combination with an Oct-2 expression vector (OEV1+ , M ⁇ ller et al, 1988), into 293T cells, a highly transfectable human fibroblastic cell line.
  • nuclear extracts were prepared from the transfected cells and used in an electrophoretic mobility shift assay (EMSA, also called gel retardation or gel shift assay; Rezvin, 1989) done with a labelled DNA probe containing an octamer site derived from the intron heavy chain enhancer (similar to a monomer of the oct site present in plasmid Ylp55-AT/H36).
  • a transactivated mammalian expression system was designed.
  • an expression plasmid encoding a desired polypeptide in this case a reporter polypeptide, is cotransfected with a second plasmid directing expression of OBF-1 (pEV-OBF) or with an empty expression vector as a control, and the resulting activity from the reporter is measured after 2 to 3 days.
  • pEV-OBF second plasmid directing expression of OBF-1
  • the expression plasmid used contains a promoter with an octamer motif (derived from the intron heavy chain enhancer) controlling transcription of the luciferase gene (this reporter is based on the pGL2-enhancer plasmid from Promega and the promoter is identical to the promoter present in the OCTA(1 ) plasmid described by M ⁇ ller et al., 1988).
  • This reporter is based on the pGL2-enhancer plasmid from Promega and the promoter is identical to the promoter present in the OCTA(1 ) plasmid described by M ⁇ ller et al., 1988).
  • the result obtained shows that OBF-1 activates transcription from this plasmid ca. 10 fold (through the endogenous Oct-1 protein). Additional transactivation experiments done in HeLa cells and with other reporter plasmids confirm the initial results. As expected, activation by OBF-1 is dependent of the integrity of the oct site present in the promoter of the reporter plasm
  • OBF-1 is a strong transcription activator, perhaps defining a novel class of such proteins. It is perhaps the first cell-specific coactivator to be isolated.
  • nlasmid pRS314/UNVP16/clone9 was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1 b, D- 38124 Braunschweig, under accession no. 9200.
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • GTC ACG ACA AGA AGC TCC GCC ACG CCC GCA GTG GGG CCC CCG CTG GAG 594 Val Thr Thr Arg Ser Ser Ala Thr Pro Ala Val Gly Pro Pro Leu Glu 155 160 165
  • Pro Pro Leu lie Thr Asn Val Thr Thr Arg Ser Ser Ala Thr Pro Ala 145 150 155 160
  • AGG GCC ATC AGC TCC CTG ACC ATT GAC AAG CTG CTT CTG GAG GAA GAG 897 Arg Ala lie Ser Ser Leu Thr lie Asp Lys Leu Leu Leu Glu Glu 225 230 235 240
  • Pro Pro Leu lie Thr Asn Val Thr Pro Arg Ser Thr Ala Thr Pro Ala 145 150 155 160
  • Arg Ala lie Ser Ser Leu Thr lie Asp Lys Leu Leu Leu Glu Glu Glu 225 230 235 240

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Abstract

Cette invention se rapporte à un acide nucléique codant un activateur de la transcription génétique véhiculée par site octamère, activateur qui est spécifique des lymphocytes B et qui agit en interaction avec le domaine POU des protéines nucléaires Oct-1 et Oct-2, afin d'activer la transcription génétique, ainsi qu'à la protéine codée par un tel acide nucléique. Un autre aspect de cette invention se rapporte à des cellules hôtes contenant ou exprimant de tels acides nucléiques, ainsi qu'aux procédés d'utilisation de ces acides nucléiques et aux cellules transformées à l'aide de ces procédés.
PCT/EP1995/001834 1994-05-24 1995-05-15 Facteur agissant en interaction avec des proteines nucleaires WO1995032284A1 (fr)

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EP95922452A EP0763109A1 (fr) 1994-05-24 1995-05-15 Facteur agissant en interaction avec des proteines nucleaires
JP7530029A JPH10500311A (ja) 1994-05-24 1995-05-15 核タンパク質と相互作用する因子
AU27345/95A AU2734595A (en) 1994-05-24 1995-05-15 Factor interacting with nuclear proteins
FI964606A FI964606A0 (fi) 1994-05-24 1996-11-18 Tumaproteiinien kanssa vuorovaikutuksessa oleva faktori
NO964981A NO964981L (no) 1994-05-24 1996-11-22 Faktor som reagerer med nukleære proteiner

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EP94810299.1 1994-05-24
EP94810299 1994-05-24

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013502A2 (fr) * 1996-09-27 1998-04-02 Icos Corporation Procedes d'identification de composes pour la rupture des interactions entre proteines
WO2000078951A1 (fr) * 1999-06-18 2000-12-28 Ariad Gene Therapeutics, Inc. Facteurs de transcription oca-b chimeriques
WO2001092286A1 (fr) * 2000-05-09 2001-12-06 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine 48 de liaison oct, et polynucleotide codant ce polypeptide
WO2003018836A2 (fr) * 2001-08-22 2003-03-06 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Modele de maladie auto-immune et procedes permettant d'identifier des agents actifs contre une maladie auto-immune
EP1589030A1 (fr) * 2004-04-14 2005-10-26 Friedrich-Alexander-Universität Erlangen-Nürnberg Lymphocytes spécifiques à l' antigène Bob-1 et leurs utilisations
CN100340674C (zh) * 2005-04-28 2007-10-03 中国人民解放军总医院 耳聋相关基因突变及其检测方法
EP1905845A2 (fr) * 2006-09-21 2008-04-02 FUJIFILM Corporation Procédé de détection de plusieurs myélomes et son procédé d'inhibition

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* Cited by examiner, † Cited by third party
Title
C. FLETCHER ET AL.: "Purification and characterization of OTF-1, a transcription factor regulating cell cycle expression of a human histone H2b gene", CELL, vol. 51, no. 5, 4 December 1987 (1987-12-04), CELL PRESS, CAMBRIDGE, MA,US;, pages 773 - 781 *
C. SCHEIDEREIT ET AL.: "Identification and purification of a human lymphoid-specific octamer-binding protein (OTF-2) that activates transcription of an immunglobulin promoter in vitro", CELL, vol. 51, no. 5, 4 December 1987 (1987-12-04), CELL PRESS, CAMBRIDGE, MA,US;, pages 783 - 793 *
D.G. JOHNSON ET AL.: "The ubiquitous octamer-binding-protein(s) is sufficient for transcription of immunglobulin genes", MOL. CELL. BIOL., vol. 10, no. 3, AM. SOC. MICROBIOL., WASHINGTON, D.C. US;, pages 982 - 990 *
L.M. CORCORAN ET AL.: "Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival", GENES & DEVELOPMENT, vol. 7, no. 4, CSH LABORATORY PRESS, CSH, N.Y., US;, pages 570 - 582 *
M. GSTAIGER ET AL.: "A B-cell coactivator of octamer-binding transcription factor", NATURE, vol. 373, 26 January 1995 (1995-01-26), MACMILLAN JOURNALS LTD., LONDON,UK, pages 360 - 362 *
STRUBIN, MICHEL ET AL: "OBF -1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins", CELL (CAMBRIDGE, MASS.) (1995), 80(3), 497-506 CODEN: CELLB5;ISSN: 0092-8674, 10 February 1995 (1995-02-10) *
Y. LUO ET AL.: "A novel B cell-derived coactivator potentiates the activation of immunglobulin promoters by octamer-binding transcription factors", CELL, vol. 71, no. 2, 16 October 1992 (1992-10-16), CELL PRESS, CAMBRIDGE, MA,US;, pages 231 - 241 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013502A2 (fr) * 1996-09-27 1998-04-02 Icos Corporation Procedes d'identification de composes pour la rupture des interactions entre proteines
WO1998013502A3 (fr) * 1996-09-27 1998-07-16 Icos Corp Procedes d'identification de composes pour la rupture des interactions entre proteines
WO2000078951A1 (fr) * 1999-06-18 2000-12-28 Ariad Gene Therapeutics, Inc. Facteurs de transcription oca-b chimeriques
WO2001092286A1 (fr) * 2000-05-09 2001-12-06 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine 48 de liaison oct, et polynucleotide codant ce polypeptide
WO2003018836A2 (fr) * 2001-08-22 2003-03-06 Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research Modele de maladie auto-immune et procedes permettant d'identifier des agents actifs contre une maladie auto-immune
WO2003018836A3 (fr) * 2001-08-22 2003-10-30 Novartis Forschungsstiftung Modele de maladie auto-immune et procedes permettant d'identifier des agents actifs contre une maladie auto-immune
EP1589030A1 (fr) * 2004-04-14 2005-10-26 Friedrich-Alexander-Universität Erlangen-Nürnberg Lymphocytes spécifiques à l' antigène Bob-1 et leurs utilisations
WO2005100390A2 (fr) * 2004-04-14 2005-10-27 Friedrich-Alexander Universität Erlanger/Nürnberg Lymphocytes t specifiques de bob-1 et leurs procedes d'utilisation
WO2005100390A3 (fr) * 2004-04-14 2006-04-27 Univ Friedrich Alexander Er Lymphocytes t specifiques de bob-1 et leurs procedes d'utilisation
JP2008509878A (ja) * 2004-04-14 2008-04-03 フリードリッヒ−アレクサンダー ウニベルジテート アーランガー／ニュールンベルク Ｂｏｂ−１特異的ｔ細胞及び使用法
CN100340674C (zh) * 2005-04-28 2007-10-03 中国人民解放军总医院 耳聋相关基因突变及其检测方法
EP1905845A2 (fr) * 2006-09-21 2008-04-02 FUJIFILM Corporation Procédé de détection de plusieurs myélomes et son procédé d'inhibition
EP1905845A3 (fr) * 2006-09-21 2008-09-17 FUJIFILM Corporation Procédé de détection de plusieurs myélomes et son procédé d'inhibition

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AU2734595A (en) 1995-12-18
FI964606A0 (fi) 1996-11-18
JPH10500311A (ja) 1998-01-13
NO964981L (no) 1996-11-22
CA2188423A1 (fr) 1995-11-30
EP0763109A1 (fr) 1997-03-19

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