EP0815219A1 - Human b-cell translocation genes-2 and 3 - Google Patents

Human b-cell translocation genes-2 and 3

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Publication number
EP0815219A1
EP0815219A1 EP95913736A EP95913736A EP0815219A1 EP 0815219 A1 EP0815219 A1 EP 0815219A1 EP 95913736 A EP95913736 A EP 95913736A EP 95913736 A EP95913736 A EP 95913736A EP 0815219 A1 EP0815219 A1 EP 0815219A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
btg
polynucleotide
seq
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95913736A
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German (de)
French (fr)
Other versions
EP0815219A4 (en
Inventor
Charles A. Kunsch
Arvind Chopra
Craig A. Rosen
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Publication date
Application filed by Human Genome Sciences Inc filed Critical Human Genome Sciences Inc
Priority to EP02021846A priority Critical patent/EP1284292A3/en
Publication of EP0815219A1 publication Critical patent/EP0815219A1/en
Publication of EP0815219A4 publication Critical patent/EP0815219A4/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides.
  • the polypeptides of the present invention have been put tively identified as human B- cell translocation genes-2 and 3, hereinafter referred to as "BTG-2 and BTG-3".
  • the invention also relates to inhibiting the action of such polypeptides.
  • the polynucleotides and polypeptides of the present invention are thought to be members of a family of anti- proliferative genes.
  • BTG-1 is a member of this group and has been cloned and expressed. (Rovault, J.P., et al., The EMBO Journal, 11(4) :1663-1670 (1992). BTG-1 was shown to negatively regulate N1H3T3 cell proliferation when over- or inappropriately expressed.
  • BTG stands for B-cell translocation gene, and the BTG-1 gene has been shown to be involved in a chromosomal translocation [t(8 12) (q24,-22)] in B-cell chronic lymphocytic leukemia.
  • the BTG-1 open reading frame is 60% homologous to PC3, an immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analyses indicate that BTG-l and PC3 are not cognate genes but are thought to be members of this new family of anti-prolifera ion genes.
  • the BTG-l gene is preferentially expressed in quiescent cells during the early sub-phases of G, in a serum-dependent manner and it is then down-regulated to reach a minimum level as the cells enter the S phase. This suggests a functional link between BTG-l and the cell cycle process.
  • BTG-l is expressed in tissues (lymphoid, liver, placenta) containing non- dividing cells likely to re-enter the cell cycle upon different stimuli, whereas the expression of BTG-l is barely detectable in fully differentiated tissues such as brain and muscle.
  • the BTG-l gene was shown to be highly conserved in evolution and a similar 1.8 Kb transcript can be detected in murine and chicken tissue by using a human BTG-l DNA probe (Rimokh, R. et al . , Genes Chr ⁇ m. Cancer. 3:24-36 (1991)).
  • the BTG-2 and BTG-3 genes and gene products have been putatively identified as members of this family as a result of amino acid sequence homology to BTG-l.
  • the term "gene” or "cistron” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
  • novel mature polypeptides which have been putatively identified as BTG-2 and BTG-3, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments and derivatives thereof.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the gene sequences of the present invention.
  • a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences which encode for the polypeptides of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
  • antagonists to such polypeptides which may be used to inhibit the action of such polypeptides, for example, in the treatment of diseases related to chromosomal translocation, for example, lymphocytic leukemia.
  • diagnostic assays for detecting diseases related to the under-expression of the polypeptides of the present invention and mutations in the nucleic acid sequences encoding such polypeptides are provided.
  • Figure 1 is an illustration of the cDNA and corresponding deduced amino acid sequence the putative BTG-2. One-letter abbreviations for amino acids are used. Sequencing was performed, for both BTG-2 and BTG-3, using a 373 Automated DNA sequencer (Applied Biosystems, Inc.) . Sequencing accuracy is predicted to be greater than 97% accurate.
  • Figure 2 shows the cDNA and corresponding deduced amino acid sequence of the putative BTG-3.
  • Figure 3 is an amino acid sequence alignment between BTG-l, BTG-2 and BTG-3 proteins, wherein m represents mouse and h represents human.
  • Figure 4 is a photograph of a gel after in vitro translation and electrophoresis of BTG-2 and BTG-3.
  • Lane l Low molecular weight markers
  • Lane 2 High molecular weight markers
  • Lane 3 Blank
  • Lane 4 T3
  • Lane 5 BTG-3
  • Lane 6 BTG-2.
  • nucleic acids which encode for the mature polypeptides having the deduced amino acid sequences of Figures 1 and 2 (SEQ ID No. 2 and 4) or for the mature polypeptides encoded by the cDNAs of the clone( ⁇ ) deposited as ATCC Deposit No. 97025 deposited January 18, 1995 for BTG-2 and ATCC No. 97010 deposited on January 5, 1995 for BTG-3.
  • a polynucleotide encoding the BTG-2 polypeptide of the present invention may be obtained from liver, lymphoid tissue and placenta.
  • the polynucleotide of this invention was discovered in a cDNA library derived from a human endometrial tumor. It is structurally related to BTG-l. It contains an open reading frame encoding a protein of 345 amino acid residues of which approximately the first 25 amino acids residues are the putative leader sequence such that the mature protein comprises 320 amino acids.
  • the protein exhibits the highest degree of homology to BTG-l with 49 % identity and 72 % similarity over a 91 amino acid stretch.
  • a polynucleotide encoding a BTG-3 polypeptide of the present invention may be obtained from synovial sarcoma, cerebellum, embryonic tissues and placenta.
  • the polynucleotide of this invention was discovered in a cDNA library derived from human synovial carcinoma. It is structurally related to the BTG family. It contains an open reading frame encoding a protein of 345 amino acid residues of which approximately the first 25 amino acids residues are the putative leader sequence such that the mature protein comprises 320 amino acids.
  • the protein exhibits the highest degree of homology to BTG-l with 48 % identity and 74 % similarity over an 85 amino acid stretch.
  • BTG-2 and BTG-3 are 83 % identical and 87 % similar to each other.
  • BTG-2 is approximately 143 amino acids longer at the carboxy terminus as compared to BTG-l, while BTG-3 is approximately 162 amino acids longer at the carboxy terminus.
  • the polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figures 1 and 2 (SEQ ID No. l and 3) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptides as the DNA of Figures l and 2 (SEQ ID No. l and 3) or the deposited cDNA.
  • polynucleotides which encodes for the mature polypeptide of Figures l and 2 (SEQ ID No. 2 and 4) or for the mature polypeptides encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequences of Figures 1 and 2 (SEQ ID No. 2 and 4) or the polypeptides encoded by the cDNAs of the deposited clones.
  • the variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.
  • the present invention includes polynucleotides encoding the same mature polypeptides as shown in Figures 1 and 2 (SEQ ID No. 2 and 4) or the same mature polypeptides encoded by the cDNAs of the deposited clones as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of Figures 1 and 2 (SEQ ID No. 2 and 4) or the polypeptides encoded by the cDNAs of the deposited clones.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in Figures 1 and 2 (SEQ ID No. 1 and 3) or of the coding sequence of the deposited clones.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptides.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention.
  • the marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptides encoded by the cDNAs of Figures l and 2 (SEQ ID No. 1 and 3) or the deposited cDNAs.
  • the present invention further relates to polypeptides which have the deduced amino acid sequences of Figures 1 and 2 (SEQ ID No. 2 and 4) or which have the amino a id sequences encoded by the deposited cDNAs, as well as fragments, analogs and derivatives of such polypeptides.
  • fragment when referring to the polypeptides of Figures 1 and 2 (SEQ ID No. 2 and 4) or that encoded by the deposited cDNAs, means polypeptides which retain essentially the same biological function or activity as such polypeptides.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptides of Figures 1 and 2 (SEQ ID No. 2 and 4) or that encoded by the deposited cDNAs may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptides are fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus,- yeast plasmids,- vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site( ⁇ ) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter for example, LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cells such as E. coli. Streptomyces. Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, HEK, COS or Bowes melanoma
  • adenoviruses plant cells, etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • regulatory sequences including, for example, a promoter, operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen) , pBS, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)) .
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are ci ⁇ -acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancer ⁇ .
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPi gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , or-factor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3-phosphoglycerate kinase
  • the heterologous structural sequence is assembled in appropriate phase with translation, initiation and termination sequences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus sub ilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although other ⁇ may al ⁇ o be employed a ⁇ a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured 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 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 know to those skilled in the art.
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary riboso e binding sites, polyadenylation site, splice donor and acceptor ⁇ ite ⁇ , transcriptional termination sequence ⁇ , and 5' flanking nontran ⁇ cribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the polypeptide ⁇ of the present invention can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, pho ⁇ phocellulo ⁇ e chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configura ion of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • Fragments of the full length genes of the present invention may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type generally have at least 50 bases, although they may have a greater number of bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns.
  • a ⁇ an example of a screen comprises isolating the coding region of the genes by using the known DNA sequence to ⁇ ynthesize an oligonucleotide probe. Labeled oligonucleotides having a ⁇ equence complementary to that of the gene ⁇ of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the BTG-2 and BTG-3 polypeptides have an anti- proliferative ability and may be employed to treat diseases or pathological conditions associated with aberrant cellular proliferation.
  • the polypeptides may be employed as a tumor/growth suppression regulator. They may also be employed to inhibit cancer cell proliferation.
  • BTG-2 and BTG-3 may also be employed to prevent uncontrolled wound healing which would otherwise cause scarring. Restenosis, which is re-occlusion of arterial wall ⁇ after balloon angioplasty, may also be treated with BTG-2 and BTG-3 since arteries re-occlude through cell proliferation. Similarly angiogenesis of tumors may be inhibited.
  • the BTG-2 and BTG-3 genes and gene products may also be employed for modulation of cellular growth. Due to their anti-proliterative effect they could be selectively administered or possibly inhibited when it is desirable to have certain cells proliferate. An example would be a disorder related to the underproduction of certain cells, where proliferation and differentiation of these cells would help to treat the disorder.
  • polynucleotides and polypeptides encoded by such polynucleotides may also be utilized for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors and for designing therapeutics and diagnostic ⁇ for the treatment of human disease.
  • the nucleic acid sequences of the present invention may be employed as part of a diagnostic a ⁇ say for detecting susceptibility to disease ⁇ associated with aberrant cellular proliferation. Since, the polypeptides of the present invention are anti-proliferative genes, a disruption in the transcription of the genes and corresponding lack of production of the gene product will likely be involved in aberrant cellular proliferation associated with a malignant phenotype.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, including but not limited to blood, urine, saliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analy ⁇ i ⁇ .
  • RNA or cDNA may al ⁇ o be u ⁇ ed for the ⁇ ame purpose.
  • PCR primers complementary to the nucleic acid encoding the polypeptides of the present invention can be used to identify and analyze mutations.
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the present invention or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresi ⁇ . DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985) ) .
  • Sequence changes at specific locations may also be revealed by nuclease protection a ⁇ says, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
  • nuclease protection a ⁇ says such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphism ⁇ (RFLP) ) and Southern blotting of genomic DNA.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphism ⁇ (RFLP)
  • the present invention also relates to a diagnostic assay for detecting altered levels of the proteins of the present invention in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of a disease or susceptibility to a disease, for example, abnormal cellular proliferation and differentiation.
  • Assays used to detect levels of these proteins in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassay ⁇ , competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich" assay.
  • An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to the antigens to the polypeptide ⁇ of the present invention, preferably a monoclonal antibody.
  • a reporter antibody is prepared against the monoclonal antibody.
  • a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradi ⁇ h peroxida ⁇ e enzyme.
  • a sample is removed from a ho ⁇ t and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample.
  • any free protein binding sites on the dish are then covered by incubating with a non-specific protein, for example, bovine ⁇ erum albumen.
  • a non-specific protein for example, bovine ⁇ erum albumen.
  • the monoclonal antibody specific to the polypeptides of the present invention is incubated in the dish during which time the monoclonal antibodies attach to any proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradi ⁇ h peroxida ⁇ e i ⁇ now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the proteins of the present invention. Unattached reporter antibody is then washed out.
  • Peroxida ⁇ e ⁇ ub ⁇ trate ⁇ are then added to the di ⁇ h and the amount of color developed in a given time period is a mea ⁇ urement of the amount of protein pre ⁇ ent in a given volume of patient sample when compared against a standard curve.
  • a competition assay may be employed wherein antibodies specific to the polypeptides of the present invention are attached to a solid support. Labeled polypeptides and a sample derived from the host are then passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of the polypeptides of the present invention in the sample.
  • a “sandwich” assay is similar to an ELISA assay.
  • the polypeptides of the present invention are passed over a solid support and bind to antibodies attached to a solid support.
  • a second antibody is then bound to the polypeptide ⁇ .
  • a third antibody which i ⁇ labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.
  • This invention provides a method for identification of the receptors for the polypeptides of the present invention.
  • the gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al. , Current Protocols in Immun. , 1(2), Chapter 5, (1991)) .
  • expression cloning is employed wherein polyadenylated RNA i ⁇ prepared from a cell responsive to the polypeptides, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slide ⁇ are exposed to the labeled polypeptides.
  • the polypeptide ⁇ can be labeled by a variety of mean ⁇ including iodination or inclu ⁇ ion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and ⁇ ub-pool ⁇ are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
  • the labeled polypeptide ⁇ can be photo-affinity linked with cell membrane or extract preparation ⁇ that express the receptor molecule.
  • Cross-linked material is resolved by PAGE analysis and exposed to X-ray film.
  • the labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing.
  • the amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
  • the present invention further provides a method of identifying agonist and antagonist compounds to the gene and gene products of the present invention.
  • An example of such an assay comprises contacting a mammalian cell or membrane preparation expres ⁇ ing the receptor ⁇ of the polypeptide ⁇ with labeled polypeptide ⁇ , eg. by radioactivity, in the presence of a compound to be screened. The ability of the compound to block and enhance the interaction of the polypeptides of the present invention with its receptor is then measured, for example, by liquid scintillation chromatography.
  • This invention provides a method of screening drugs to identify those which enhance or inhibit interaction of the polypeptide ⁇ with their receptors.
  • a mammalian cell or membrane preparation expressing the receptor would be incubated with labeled polypeptides in the presence of the drug. The ability of the drug to enhance or block this interaction could then be measured.
  • second messenger sy ⁇ tem ⁇ include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
  • BTG-2 or BTG-3 polypeptides include an antibody, or in some cases, an oligopeptide, which binds to the polypeptide.
  • a potential antagonist may be a closely related protein which binds to the receptor sites, however, they are inactive forms of the polypeptide and thereby prevent their action since receptor sites are occupied.
  • Anti ⁇ en ⁇ e technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide sequence which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • the anti ⁇ ense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptides (Antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • the oligonucleotides described above can al ⁇ o be delivered to cells such that the antisen ⁇ e RNA or DNA may be expressed in vivo to inhibit production of the polypeptides of the present invention.
  • Potential antagonists also include a small molecule which binds to and occupies the active site of the polypeptides thereby making them inaccessible to substrate such that normal biological activity is prevented.
  • small molecules include but are not limited to small peptides or peptide-like molecules.
  • the antagonists may be employed to treat leukemia, which result ⁇ from oncogene activation in hemopoietic cell ⁇ due to a chromosomal translocation.
  • the polypeptides of the present invention may have a direct or indirect function in the activation of a cellular oncogene resulting in leukemia.
  • the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
  • the invention al ⁇ o provide ⁇ a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form pre ⁇ cribed by a governmental agency regulating the manufacture, u ⁇ e or ⁇ ale of pharmaceutical ⁇ or biological product ⁇ , which notice reflect ⁇ approval by the agency of manufacture, u ⁇ e or ⁇ ale for human administration.
  • the polypeptide ⁇ and agoni ⁇ t ⁇ or antagoni ⁇ t ⁇ of the present invention may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical composition ⁇ may be admini ⁇ tered in a convenient manner ⁇ uch a ⁇ by the topical, intravenous, intramuscular or subcutaneous routes.
  • the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ⁇ g/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage i ⁇ from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
  • polypeptide ⁇ of the present invention and agonists and antagonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
  • cell ⁇ from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cell ⁇ may be engineered by procedure ⁇ known in the art by u ⁇ e of a retroviral particle containing RNA encoding a polypeptide of the pre ⁇ ent invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expres ⁇ ion of the polypeptide in vivo.
  • the ⁇ e and other method ⁇ for admini ⁇ tering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teaching ⁇ of the pre ⁇ ent invention.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be u ⁇ ed to engineer cells in vivo after combination with a ⁇ uitable delivery vehicle.
  • the sequence ⁇ of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosome ⁇ by preparing PCR primer ⁇ (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of ⁇ omatic cell hybrids containing individual human chromo ⁇ ome ⁇ . Only tho ⁇ e hybrid ⁇ containing the human gene corre ⁇ ponding to the primer will yield an amplified fragment.
  • mapping of ⁇ omatic cell hybrid ⁇ i ⁇ a rapid procedure for a ⁇ signing a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragment ⁇ from specific chromosome ⁇ or pools of large genomic clones in an analogous manner.
  • Other mapping strategie ⁇ that can ⁇ imilarly be u ⁇ ed to map to it ⁇ chromo ⁇ ome include in si tu hybridization, prescreening with labeled flow-sorted chromosome ⁇ and pre ⁇ election by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromo ⁇ omal location in one ⁇ tep.
  • This technique can be u ⁇ ed with cDNA a ⁇ ⁇ hort as 500 or 600 bases,- however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for ⁇ imple detection. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • Verma et al. Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
  • a cDNA preci ⁇ ely localized to a chromo ⁇ omal region associated with the disea ⁇ e could be one of between 50 and 500 potential cau ⁇ ative genes. (This as ⁇ umes l megabase mapping resolution and one gene per 20 kb) .
  • the polypeptide ⁇ , their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also include ⁇ chimeric, ⁇ ingle chain, and humanized antibodies, a ⁇ well a ⁇ Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Lis ⁇ , Inc., pp. 77-96).
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes u ⁇ ed herein are commercially available and their reaction condition ⁇ , cofactors and other requirements were used as would be known to the ordinarily skilled arti ⁇ an.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are dige ⁇ ted with 20 to 250 unit ⁇ of enzyme in a larger volume.
  • buffer ⁇ and ⁇ ub ⁇ trate amount ⁇ for particular re ⁇ triction enzyme ⁇ are ⁇ pecified by the manufacturer.
  • Incubation time ⁇ of about l hour at 37'C are ordinarily u ⁇ ed, but may vary in accordance with the supplier's instructions.
  • Oligonucleotides refer ⁇ to either a ⁇ ingle ⁇ tranded polydeoxynucleotide or two complementary polydeoxynucleotide ⁇ trand ⁇ which may be chemically ⁇ ynthe ⁇ ized. Such ⁇ ynthetic oligonucleotide ⁇ have no 5' pho ⁇ phate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A ⁇ ynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double ⁇ tranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146) . Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • the DNA sequence encoding the full length BTG-2 protein, ATCC # 97025, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the gene:
  • the 5' primer has the sequence 5' CAGTGGATCCGCCAC CATGCAGCTTGAAATCCAAGTAGCAC 3' (SEQ ID No. 5) and contain ⁇ a Bam HI restriction enzyme site (in bold) followed by 6 nucleotides re ⁇ embling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol . Biol., 196:947-950 (1987) and just behind the first 26 nucleotides of the BTG-2 gene (the initiation codon for translation "ATG" is underlined) .
  • the 3' primer has the sequence 5' CAGTGGTACCATACATTT TCTTTTTTTTAGTTAGCCAT 3' (SEQ ID No. 6) and contains the cleavage site for the restriction endonuclea ⁇ e A ⁇ p718 and 25 nucleotide ⁇ complementary to the 3' non-tran ⁇ lated sequence of the BTG-2 gene.
  • the amplified sequences are isolated from a 1% agaro ⁇ e gel u ⁇ ing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, a.) .
  • the fragment i ⁇ then digested with the endonucleases Bam HI and Asp7l8 and purified again on a 1% agarose gel. This fragment is designated F2.
  • the vector pRGl (modification of pVL941 vector, discussed below) is used for the expression of the BTG-2 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
  • This expression vector contains the ⁇ trong polyhedrin promoter of the Autographa californica nuclear polyhidro ⁇ i ⁇ viru ⁇ (AcMNPV) followed by the recognition sites for the re ⁇ triction endonucleases.
  • the polyadenylation site of the simian virus (SV)40 is u ⁇ ed for efficient polyadenylation.
  • the beta- galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA.
  • Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31- 39) .
  • the plasmid is digested with the restriction enzymes Bam HI and Asp7l8 and dephosphorylated using calf intestinal phosphata ⁇ e by procedure ⁇ known in the art.
  • the DNA i ⁇ then i ⁇ olated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated V2.
  • Fragment F2 and the dephosphorylated pla ⁇ mid V2 are ligated with T4 DNA ligase.
  • E.coli HB101 cells are then transformed and bacteria identified that contained the pla ⁇ mid (pBac BTG-2) with the BTG-2 gene using the respective re ⁇ triction enzyme ⁇ .
  • the ⁇ eguence of the cloned fragment i ⁇ confirmed by DNA sequencing. 5 ⁇ g of the plasmid pBac BTG-2 is co-transfected with
  • the plate is rocked back and forth to mix the newly added solution.
  • the plate is then incubated for 5 hours at 27°C.
  • the transfection solution i ⁇ removed from the plate and l ml of Grace' ⁇ insect medium supplemented with 10% fetal calf serum i ⁇ added.
  • the plate i ⁇ put back into an incubator and cultivation continued at 27°C for four day ⁇ .
  • plaque as ⁇ ay After four day ⁇ the supernatant is collected and a plaque as ⁇ ay performed ⁇ imilar as described by Summers and Smith ( ⁇ upra) .
  • an agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques.
  • a detailed description of a "plaque as ⁇ ay” can al ⁇ o be found in the u ⁇ er' ⁇ guide for in ⁇ ect cell culture and baculovirology di ⁇ tributed by Life Technologies Inc., Gaithersburg, page 9- 10.
  • the viruses are added to the cells and blue stained plaque ⁇ are picked with the tip of an Eppendorf pipette.
  • the agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
  • the agar i ⁇ removed by a brief centrifugation and the ⁇ upernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
  • the supernatants of these culture dishes are harvested and then stored at 4°C.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
  • the cells are infected with the recombinant baculovirus V-BTG-2 at a multiplicity of infection (MOD of 2.
  • MOD multiplicity of infection
  • the medium is removed -and replaced with SF900 II medium minus methionine and cy ⁇ teine (Life Technologies Inc., Gaithersburg).
  • the cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins vi ⁇ ualized by SDS-PAGE and autoradiography.
  • the DNA sequence encoding the full length BTG-3 protein, ATCC # 97010, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the gene:
  • the 5' primer has the ⁇ equence 5' CAGTGGATCCGCCACCATGCAG CTAGATCAAAGTGGCCC 3' (SEQ ID No. 7) and contain ⁇ a Bam HI restriction enzyme site (in bold) followed by 6 nucleotides resembling an efficient ⁇ ignal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol. Biol., 196:947-950 (1987) and just behind the first 228 nucleotide ⁇ of the BTG-3 gene (the initiation codon for tran ⁇ lation "ATG" i ⁇ underlined) .
  • the 3' primer ha ⁇ the ⁇ equence 5' CAGTGGTACCACGGGCCA GGTAGATGGTCAGTTGGCCAGCAC 3' (SEQ ID No. 8) and contain ⁇ the cleavage ⁇ ite for the re ⁇ triction endonuclea ⁇ e Asp718 and 25 nucleotides complementary to the 3' non-translated ⁇ equence of the BTG-3 gene.
  • the amplified ⁇ equence ⁇ are i ⁇ olated from a 1% agaro ⁇ e gel u ⁇ ing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.). The fragment i ⁇ then digested with the endonucleases Bam HI and A ⁇ p7l8 and purified again on a 1% agarose gel. This fragment is designated F2.
  • the vector pRGl (modification of pVL941 vector, discussed below) is used for the expression of the BTG-3 protein using the baculovirus expression sy ⁇ tem (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of method ⁇ for baculoviru ⁇ vector ⁇ and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
  • Thi ⁇ expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosi ⁇ viru ⁇ (AcMNPV) followed by the recognition ⁇ ite ⁇ for the re ⁇ triction endonucleases Bam HI and A ⁇ p7l8.
  • SV simian viru ⁇
  • the polyhedrin ⁇ equence ⁇ are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA.
  • baculovirus vector ⁇ could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
  • the plasmid is dige ⁇ ted with the re ⁇ pective restriction enzymes and dephosphorylated using calf intestinal phosphata ⁇ e by procedure ⁇ known in the art.
  • the DNA i ⁇ then i ⁇ olated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) .
  • This vector DNA is designated V2.
  • Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase.
  • E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid (pBac BTG-3) with the BTG-3 gene using the enzymes Bam HI and A ⁇ p718.
  • the sequence of the cloned fragment i ⁇ confirmed by DNA sequencing.
  • plasmid pBac BTG-3 5 ⁇ g of the plasmid pBac BTG-3 is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl.
  • the plate is rocked back and forth to mix the newly added solution.
  • the plate is then incubated for 5 hours at 27°C.
  • the transfection ⁇ olution i ⁇ removed from the plate and 1 ml of Grace' ⁇ in ⁇ ect medium supplemented with 10% fetal calf serum is added.
  • the plate is put back into an incubator and cultivation continued at 27°C for four days.
  • plaque assay After four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra) . As a modification an agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A detailed de ⁇ cription of a "plaque assay” can also be found in the user' ⁇ guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaither ⁇ burg, page 9- 10) .
  • the viruses are added to the cells and blue stained plaque ⁇ are picked with the tip of an Eppendorf pipette.
  • the agar containing the recombinant viru ⁇ e ⁇ i ⁇ then re ⁇ u ⁇ pended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
  • the agar is removed by a brief centrifugation and the ⁇ upernatant containing the recombinant baculoviru ⁇ i ⁇ used to infect Sf9 cells seeded in 35 mm dishes.
  • the supernatants of these culture dishes are harvested and then ⁇ tored at 4°C.
  • Sf9 cell ⁇ are grown in Grace's medium ⁇ upplemented with 10% heat-inactivated FBS.
  • the cell ⁇ are infected with the recombinant baculoviru ⁇ V-BTG-3 at a multiplicity of infection (MOD of 2.
  • MOD multiplicity of infection
  • the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 3S S cysteine (Amersham) are added.
  • the cell ⁇ are further incubated for 16 hours before they are harvested by centrifugation and the labelled protein ⁇ vi ⁇ ualized by SDS-PAGE and autoradiography.
  • a DNA fragment encoding the entire BTG-2 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously de ⁇ cribed (I.
  • the plasmid construction ⁇ trategy i ⁇ de ⁇ cribed a ⁇ follow ⁇ The DNA sequence encoding BTG-2, ATCC # 97025, i ⁇ constructed by PCR using two primers: the 5' primer 5' GGCCC AAGCTTGCCGCC ⁇ TGCAGCTTGAAATCCAAGTAG 3' (SEQ ID No. 9) contains a Hind III site followed by 23 nucleotides of BTG-2 coding sequence starting from the initiation codon; the 3' sequence 5' ATCGTCTAC ⁇ TTAGTTAGCCATAACAGGCrcGAATTGC SGTTAGAATACro TTATTTAAGCTAAAATTCAAGCCATCTA 3' (SEQ ID No.
  • the PCR product contains complementary sequences to Xbal site, translation stop codon, HA tag and the last 68 nucleotides of the BTG-2 coding sequence (not including the stop codon) . Therefore, the PCR product contains a Hind III site, BTG-2 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are dige ⁇ ted with Hind III and Xbal restriction enzyme and ligated. The ligation mixture is transformed into E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected.
  • Plasmid DNA is isolated from transformants and examined by re ⁇ triction analy ⁇ is for the presence of the correct fragment.
  • COS cell ⁇ are tran ⁇ fected with the expre ⁇ ion vector by DEAE-DEXTRAN method (J. Sambrook, E. Frit ⁇ ch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Pres ⁇ , (1989)) .
  • the expre ⁇ ion of the BTG-2 HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodie ⁇ : A Laboratory Manual, Cold Spring Harbor Laboratory Pre ⁇ , (1988) ) .
  • Cell ⁇ are labelled for 8 hours with 35 S-cysteine two day ⁇ post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tri ⁇ , pH 7.5) . (Wil ⁇ on, I. et al., Id. 37:767 (1984)). Both cell ly ⁇ ate and culture media are precipitated with a HA ⁇ pecific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.
  • RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tri ⁇ , pH 7.5
  • BTG-3 HA The expression of plasmid, BTG-3 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire BTG-3 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previou ⁇ ly de ⁇ cribed (I. Wilson, H.
  • HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the DNA sequence encoding BTG-3, ATCC # 97010 is constructed by PCR using two primers: the 5' primer 5' GGCCC AAGCTTGCCGCCATGCAGCTAGAGATCAAAGTGGC 3' (SEQ ID No. 11) contains a Hind III site followed by 23 nucleotides of BTG-3 coding sequence ⁇ tarting from the initiation codon; the 3' ⁇ equence 5' ATCGTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGGTTG TAGCTGAGGCCTTCCACAAAGGGTGTCTTCTCCAGG 3' (SEQ ID No.
  • the PCR product contains a Hind III site, BTG-3 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Hind III and Xbal restriction enzymes and ligated. The ligation mixture is transformed into E.
  • coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniati ⁇ , Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) .
  • the expression of the BTG- HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) ) .
  • Cells are labelled for 8 hours with 35 S-cysteine two day ⁇ po ⁇ t tran ⁇ fection. Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA ⁇ pecific monoclonal antibody. Protein ⁇ precipitated are analyzed on 15% SDS-PAGE gel ⁇ .
  • RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5
  • the DNA ⁇ equence encoding BTG-2, ATCC # 97025 is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end ⁇ equence ⁇ of the processed BTG-2 gene (minus the signal peptide sequence) and the vector sequences 3' to the BTG-2 gene. Additional nucleotides corresponding to Ncol and Bgl II re ⁇ triction enzyme ⁇ ites were added to the 5' and 3' end sequence ⁇ re ⁇ pectively.
  • the 5' oligonucleotide primer has the sequence ATCGCCATGGGACAGCTT GAAATCCAAGTAGCACTA 3' (SEQ ID No.
  • the restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expres ⁇ ion vector pQE-60 (Qiagen, Inc. Chatsworth, CA) .
  • pQE- 60 encode ⁇ antibiotic re ⁇ i ⁇ tance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator
  • pQE-60 is then digested with Nco I and Bgl II.
  • the amplified sequences are ligated into pQE- 60 and are inserted in frame with the sequence encoding for the histidine tag and the RBS.
  • the ligation mixture i ⁇ then u ⁇ ed to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Pre ⁇ ,
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expres ⁇ e ⁇ the lad repre ⁇ or and al ⁇ o confers kanamycin resi ⁇ tance (Kan r ) . Tran ⁇ formants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysi ⁇ . Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ l) and Kan (25 ug/ml) . The 0/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG I ⁇ opropyl-B-D-thiogalacto pyrano ⁇ ide
  • IPTG induces by inactivating the lad repre ⁇ or, clearing the P/O leading to increa ⁇ ed gene expre ⁇ ion.
  • Cell ⁇ are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.
  • BTG-2 ⁇ olubilized BTG-2 i ⁇ purified from thi ⁇ ⁇ olution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)) .
  • BTG-2 (95% pure) is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.
  • the 5' oligonucleotide primer ha ⁇ the ⁇ equence 5' ATCGCCATGGGACAGCTAGAGATCAAAGTGGCCCTG 3' (SEQ ID No.
  • the 15 contain ⁇ an Ncol re ⁇ triction enzyme site followed by 24 nucleotides of BTG-3 coding ⁇ equence ⁇ tarting from the presumed terminal amino acid of the processed protein.
  • the 3' sequence 5' ATCGAGATCTGTTGGCCAGCACCACGGGCTGG 3' contains complementary sequences to a Bgl II restriction site and the last 21 nucleotides of BTG-3 coding sequence.
  • the restriction enzyme sites correspond to the re ⁇ triction enzyme ⁇ ite ⁇ on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, CA) .
  • pQE-60 encodes antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6-His tag and restriction enzyme sites.
  • pQE-60 is then digested with Nco I and Bgl II.
  • the amplified sequences are ligated into pQE- 60 and are inserted in frame with the sequence encoding for the histidine tag and the RBS.
  • the ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J.
  • M15/rep4 contain ⁇ multiple copies of the plasmid pREP4, which expresses the lad repressor and al ⁇ o confer ⁇ kanamycin resistance (Kan r ) .
  • Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is i ⁇ olated and confirmed by restriction analysis. Clones containing the desired construct ⁇ are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the O/N culture is u ⁇ ed to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cell ⁇ are grown to an optical den ⁇ ity 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG I ⁇ opropyl-B-D-thiogalacto pyrano ⁇ ide
  • IPTG induces by inactivating the lad repres ⁇ or, clearing the P/O leading to increased gene expres ⁇ ion.
  • Cell ⁇ are grown an extra 3 to 4 hour ⁇ . Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.
  • BTG-3 i ⁇ purified from this solution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (16084)).
  • BTG-3 (95 % pure) is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM ⁇ odium pho ⁇ phate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) .
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • GTGACAGCCC CCTTAACCTC AGTCCTCTCC AGTACAGTAA TGCCTTTGAT GTGTTTGCAG 960 CTATGGAGG CCTCAATGAG AAGTCTTTTG TAGATGGCTT GAATTTTAGC TTAAATAACA 1020 TGCAGTATTC TAACC ⁇ GCAA TTCCAGCCTG TTATGGCTAA CTAAAAAAAA GAAAATGTAT 1080 CGTACAAGTT AAAATGCAC ⁇ GGCCAAGGGGGGATTTTTTT TTTCACCTCC TTGAGAAT ⁇ T 1140 TTTTTTAA GCTTATAGTA AGGATACATT CAAGCTTGGT TAAAAAAATAATAATAAAAC 1200 ATGCATCATT TTTCATTTGC CAACCAAGCA CAAAGTTATT TTATGCTGCC TGTATATTTT 1260 AAAGTATACT CTCAGATATG CCCTCTTACA GTATTTTAAG ATATTAGCAA AGGACATGGC 1320 TTGATTTTTT TTTATAAAAA TTGGCACTAA
  • GGAATTCGGC ACGAGCAACC CTCAACGACG AAAAGGACTT CGGTCCCCTG GCCCGGCGAC 60 GCCCGGRAAG GAAAGGAGAG CGACCTCCGC CCCGCGCTCA GGCCACCCTG GAGGGAGAAG 120 CCGCCCCGCG GCGGGTAGAG CGCCCCGCCG CCNCGYHAGN ACCCGAAGCC GCCTGGAGCC 180 CAAGGCTGTA CACGTGCCCT GTGCTGAGGC TCTGCCTAGG AAAGGACCAT GCAGCTAGAG 240 ATCAAAGTGG CCCTGAACTT CATCCATCTC CTACTTGTAC AACAAGCTGC CCCGGCGCCC 300 GGGCAGAACC TG TTGGGGA GGAGCTAGAG CGGCTTTTGA AAAGGAAATA TGAAGGCCAC 360 TGGTACCCTG AGAAGCCACT GAAAGGCTCT GGCTTCCGCT GTGTTCACAT TGGGGAGATG 420 GTGGACCCCG TGGTGGAGCT GGCCGC
  • MOLECULE TYPE PROTEIN
  • xi SEQUENCE DESCRIPTION: SEQ ID N0:4:
  • Leu Asn Phe lie Thr Ala Asn Pro Ala Pro Gin Ser Gin Leu Ser

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Abstract

Human B-cell translocation genes-2 and 3 and polypeptides encoded by such genes and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for the treatment of aberrant cellular proliferation. Also disclosed are diagnostic assays for detecting susceptibility to diseases associated with mutations in the nucleic acid sequences encoding said polypeptides and for detecting altered levels of such polypeptides.

Description

HUMAN B-CELL TRANSLOCATION GENES-2 AND 3
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptides of the present invention have been put tively identified as human B- cell translocation genes-2 and 3, hereinafter referred to as "BTG-2 and BTG-3". The invention also relates to inhibiting the action of such polypeptides.
In normal tissues, ho eostasis is maintained through negative and positive growth controls which effect the proliferation and differentiation related to cellular genetic programs. An alteration of this subtle balance can result in developmental abnormalities or in neoplasia. Proto- oncogenes, genes that promote cell division, were the first growth-inducing elements to be identified and more than sixty of them have been described so far (Bishop, J.M., Cell, 64:235:248 (1991)) . The genes that negatively regulate cell proliferation are crucial to counteract the growth-inducing elements and are likely to have the same importance as proto- oncogenes in controlling cell division (Marshall, C. . , Cell. 64:313-326 (1991), especially since the loss of their function has been reported to be associated with irregular
-l- cellular differentiation and proliferation or with alteration of embryonic development (Weinberg, R.A. , Science. 254:1138- 1146 (1991) ) .
The polynucleotides and polypeptides of the present invention are thought to be members of a family of anti- proliferative genes. BTG-1 is a member of this group and has been cloned and expressed. (Rovault, J.P., et al., The EMBO Journal, 11(4) :1663-1670 (1992). BTG-1 was shown to negatively regulate N1H3T3 cell proliferation when over- or inappropriately expressed. BTG stands for B-cell translocation gene, and the BTG-1 gene has been shown to be involved in a chromosomal translocation [t(8 12) (q24,-22)] in B-cell chronic lymphocytic leukemia.
The BTG-1 open reading frame is 60% homologous to PC3, an immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analyses indicate that BTG-l and PC3 are not cognate genes but are thought to be members of this new family of anti-prolifera ion genes. The BTG-l gene is preferentially expressed in quiescent cells during the early sub-phases of G, in a serum-dependent manner and it is then down-regulated to reach a minimum level as the cells enter the S phase. This suggests a functional link between BTG-l and the cell cycle process. BTG-l is expressed in tissues (lymphoid, liver, placenta) containing non- dividing cells likely to re-enter the cell cycle upon different stimuli, whereas the expression of BTG-l is barely detectable in fully differentiated tissues such as brain and muscle.
The BTG-l gene was shown to be highly conserved in evolution and a similar 1.8 Kb transcript can be detected in murine and chicken tissue by using a human BTG-l DNA probe (Rimokh, R. et al . , Genes Chrσm. Cancer. 3:24-36 (1991)).
The BTG-2 and BTG-3 genes and gene products have been putatively identified as members of this family as a result of amino acid sequence homology to BTG-l. The term "gene" or "cistron" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
In accordance with one aspect of the present invention, there are provided novel mature polypeptides which have been putatively identified as BTG-2 and BTG-3, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments and derivatives thereof.
In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the gene sequences of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques, comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing nucleic acid sequences which encode for the polypeptides of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, to treat disease states characterized by aberrant cellular proliferation, and to modulate cellular growth. In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of diseases related to chromosomal translocation, for example, lymphocytic leukemia.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases related to the under-expression of the polypeptides of the present invention and mutations in the nucleic acid sequences encoding such polypeptides.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vi tro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1 is an illustration of the cDNA and corresponding deduced amino acid sequence the putative BTG-2. One-letter abbreviations for amino acids are used. Sequencing was performed, for both BTG-2 and BTG-3, using a 373 Automated DNA sequencer (Applied Biosystems, Inc.) . Sequencing accuracy is predicted to be greater than 97% accurate.
Figure 2 shows the cDNA and corresponding deduced amino acid sequence of the putative BTG-3. Figure 3 is an amino acid sequence alignment between BTG-l, BTG-2 and BTG-3 proteins, wherein m represents mouse and h represents human.
Figure 4 is a photograph of a gel after in vitro translation and electrophoresis of BTG-2 and BTG-3. Lane l: Low molecular weight markers; Lane 2: High molecular weight markers; Lane 3: Blank; Lane 4: T3; Lane 5: BTG-3; Lane 6: BTG-2.
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequences of Figures 1 and 2 (SEQ ID No. 2 and 4) or for the mature polypeptides encoded by the cDNAs of the clone(ε) deposited as ATCC Deposit No. 97025 deposited January 18, 1995 for BTG-2 and ATCC No. 97010 deposited on January 5, 1995 for BTG-3.
A polynucleotide encoding the BTG-2 polypeptide of the present invention may be obtained from liver, lymphoid tissue and placenta. The polynucleotide of this invention was discovered in a cDNA library derived from a human endometrial tumor. It is structurally related to BTG-l. It contains an open reading frame encoding a protein of 345 amino acid residues of which approximately the first 25 amino acids residues are the putative leader sequence such that the mature protein comprises 320 amino acids. The protein exhibits the highest degree of homology to BTG-l with 49 % identity and 72 % similarity over a 91 amino acid stretch.
A polynucleotide encoding a BTG-3 polypeptide of the present invention may be obtained from synovial sarcoma, cerebellum, embryonic tissues and placenta. The polynucleotide of this invention was discovered in a cDNA library derived from human synovial carcinoma. It is structurally related to the BTG family. It contains an open reading frame encoding a protein of 345 amino acid residues of which approximately the first 25 amino acids residues are the putative leader sequence such that the mature protein comprises 320 amino acids. The protein exhibits the highest degree of homology to BTG-l with 48 % identity and 74 % similarity over an 85 amino acid stretch. Over the same stretch of 85 amino acids, BTG-2 and BTG-3 are 83 % identical and 87 % similar to each other. In addition, BTG-2 is approximately 143 amino acids longer at the carboxy terminus as compared to BTG-l, while BTG-3 is approximately 162 amino acids longer at the carboxy terminus.
The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figures 1 and 2 (SEQ ID No. l and 3) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptides as the DNA of Figures l and 2 (SEQ ID No. l and 3) or the deposited cDNA.
The polynucleotides which encodes for the mature polypeptide of Figures l and 2 (SEQ ID No. 2 and 4) or for the mature polypeptides encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequences of Figures 1 and 2 (SEQ ID No. 2 and 4) or the polypeptides encoded by the cDNAs of the deposited clones. The variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.
Thus, the present invention includes polynucleotides encoding the same mature polypeptides as shown in Figures 1 and 2 (SEQ ID No. 2 and 4) or the same mature polypeptides encoded by the cDNAs of the deposited clones as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of Figures 1 and 2 (SEQ ID No. 2 and 4) or the polypeptides encoded by the cDNAs of the deposited clones. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequences shown in Figures 1 and 2 (SEQ ID No. 1 and 3) or of the coding sequence of the deposited clones. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptides.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptides encoded by the cDNAs of Figures l and 2 (SEQ ID No. 1 and 3) or the deposited cDNAs.
The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
The present invention further relates to polypeptides which have the deduced amino acid sequences of Figures 1 and 2 (SEQ ID No. 2 and 4) or which have the amino a id sequences encoded by the deposited cDNAs, as well as fragments, analogs and derivatives of such polypeptides. The terms "fragment," "derivative" and "analog" when referring to the polypeptides of Figures 1 and 2 (SEQ ID No. 2 and 4) or that encoded by the deposited cDNAs, means polypeptides which retain essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptides of Figures 1 and 2 (SEQ ID No. 2 and 4) or that encoded by the deposited cDNAs may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptides are fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus,- yeast plasmids,- vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host. The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(ε) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomyces. Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, HEK, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) , pBS, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)) .
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are ciε-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancerε.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPi gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , or-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation, initiation and termination sequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus sub ilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although otherε may alεo be employed aε a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell_density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured 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 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 know to those skilled in the art.
Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary riboso e binding sites, polyadenylation site, splice donor and acceptor εiteε, transcriptional termination sequenceε, and 5' flanking nontranεcribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The polypeptideε of the present invention can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phoεphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configura ion of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
Fragments of the full length genes of the present invention may be used as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type generally have at least 50 bases, although they may have a greater number of bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. Aε an example of a screen comprises isolating the coding region of the genes by using the known DNA sequence to εynthesize an oligonucleotide probe. Labeled oligonucleotides having a εequence complementary to that of the geneε of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The BTG-2 and BTG-3 polypeptides have an anti- proliferative ability and may be employed to treat diseases or pathological conditions associated with aberrant cellular proliferation. The polypeptides may be employed as a tumor/growth suppression regulator. They may also be employed to inhibit cancer cell proliferation.
BTG-2 and BTG-3 may also be employed to prevent uncontrolled wound healing which would otherwise cause scarring. Restenosis, which is re-occlusion of arterial wallε after balloon angioplasty, may also be treated with BTG-2 and BTG-3 since arteries re-occlude through cell proliferation. Similarly angiogenesis of tumors may be inhibited.
The BTG-2 and BTG-3 genes and gene products may also be employed for modulation of cellular growth. Due to their anti-proliterative effect they could be selectively administered or possibly inhibited when it is desirable to have certain cells proliferate. An example would be a disorder related to the underproduction of certain cells, where proliferation and differentiation of these cells would help to treat the disorder.
The polynucleotides and polypeptides encoded by such polynucleotides may also be utilized for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors and for designing therapeutics and diagnosticε for the treatment of human disease.
The nucleic acid sequences of the present invention may be employed as part of a diagnostic aεsay for detecting susceptibility to diseaseε associated with aberrant cellular proliferation. Since, the polypeptides of the present invention are anti-proliferative genes, a disruption in the transcription of the genes and corresponding lack of production of the gene product will likely be involved in aberrant cellular proliferation associated with a malignant phenotype.
Individuals carrying mutations in the genes may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, including but not limited to blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analyεiε. RNA or cDNA may alεo be uεed for the εame purpose. As an example, PCR primers complementary to the nucleic acid encoding the polypeptides of the present invention can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the present invention or alternatively, radiolabeled antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresiε. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985) ) .
Sequence changes at specific locations may also be revealed by nuclease protection aεsays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphismε (RFLP) ) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can alεo be detected by in situ analysis. The present invention also relates to a diagnostic assay for detecting altered levels of the proteins of the present invention in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of a disease or susceptibility to a disease, for example, abnormal cellular proliferation and differentiation. Assays used to detect levels of these proteins in a sample derived from a host are well-known to those of skill in the art and include radioimmunoassayε, competitive-binding assays, Western Blot analysis, ELISA assays and "sandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to the antigens to the polypeptideε of the present invention, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horseradiεh peroxidaεe enzyme. A sample is removed from a hoεt and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein, for example, bovine εerum albumen. Next, the monoclonal antibody specific to the polypeptides of the present invention is incubated in the dish during which time the monoclonal antibodies attach to any proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradiεh peroxidaεe iε now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to the proteins of the present invention. Unattached reporter antibody is then washed out. Peroxidaεe εubεtrateε are then added to the diεh and the amount of color developed in a given time period is a meaεurement of the amount of protein preεent in a given volume of patient sample when compared against a standard curve.
A competition assay may be employed wherein antibodies specific to the polypeptides of the present invention are attached to a solid support. Labeled polypeptides and a sample derived from the host are then passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to a quantity of the polypeptides of the present invention in the sample.
A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay the polypeptides of the present invention are passed over a solid support and bind to antibodies attached to a solid support. A second antibody is then bound to the polypeptideε. A third antibody which iε labeled and specific to the second antibody is then passed over the solid support and binds to the second antibody and an amount can then be quantified.
This invention provides a method for identification of the receptors for the polypeptides of the present invention. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al. , Current Protocols in Immun. , 1(2), Chapter 5, (1991)) . Preferably, expression cloning is employed wherein polyadenylated RNA iε prepared from a cell responsive to the polypeptides, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slideε are exposed to the labeled polypeptides. The polypeptideε can be labeled by a variety of meanε including iodination or incluεion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and εub-poolε are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
Aε an alternative approach for receptor identification, the labeled polypeptideε can be photo-affinity linked with cell membrane or extract preparationε that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
The present invention further provides a method of identifying agonist and antagonist compounds to the gene and gene products of the present invention. An example of such an assay comprises contacting a mammalian cell or membrane preparation expresεing the receptorε of the polypeptideε with labeled polypeptideε, eg. by radioactivity, in the presence of a compound to be screened. The ability of the compound to block and enhance the interaction of the polypeptides of the present invention with its receptor is then measured, for example, by liquid scintillation chromatography.
This invention provides a method of screening drugs to identify those which enhance or inhibit interaction of the polypeptideε with their receptors. As an example, a mammalian cell or membrane preparation expressing the receptor would be incubated with labeled polypeptides in the presence of the drug. The ability of the drug to enhance or block this interaction could then be measured.
Alternatively, the response of a known second raesεenger system following interaction of the polypeptides and their receptors would be measured and compared in the presence or absence of the drug. Such second messenger syεtemε include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
Potential antagonists to BTG-2 or BTG-3 polypeptides include an antibody, or in some cases, an oligopeptide, which binds to the polypeptide. Alternatively, a potential antagonist may be a closely related protein which binds to the receptor sites, however, they are inactive forms of the polypeptide and thereby prevent their action since receptor sites are occupied.
Another potential antagonist is an antisenεe construct prepared using antisense technology. Antiεenεe technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide iε deεigned to be complementary to a region of the gene involved in tranεcription (triple helix - see Lee et al., Nucl. Acidε Reε., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991) ) , thereby preventing tranεcription and the production of BTG-2 and/or BTG-3. The antiεense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptides (Antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can alεo be delivered to cells such that the antisenεe RNA or DNA may be expressed in vivo to inhibit production of the polypeptides of the present invention.
Potential antagonists also include a small molecule which binds to and occupies the active site of the polypeptides thereby making them inaccessible to substrate such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules.
The antagonists may be employed to treat leukemia, which resultε from oncogene activation in hemopoietic cellε due to a chromosomal translocation. The polypeptides of the present invention may have a direct or indirect function in the activation of a cellular oncogene resulting in leukemia.
The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
The polypeptides and agonists or antagonists of the present invention may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The invention alεo provideε a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, uεe or εale of pharmaceuticalε or biological productε, which notice reflectε approval by the agency of manufacture, uεe or εale for human administration. In addition, the polypeptideε and agoniεtε or antagoniεtε of the present invention may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositionε may be adminiεtered in a convenient manner εuch aε by the topical, intravenous, intramuscular or subcutaneous routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excess of about 8 mg/Kg body weight per day. In most cases, the dosage iε from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
The polypeptideε of the present invention, and agonists and antagonists which are polypeptides, may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which iε often referred to aε "gene therapy."
Thus, for example, cellε from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methodε are well-known in the art. For example, cellε may be engineered by procedureε known in the art by uεe of a retroviral particle containing RNA encoding a polypeptide of the preεent invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expresεion of the polypeptide in vivo. Theεe and other methodε for adminiεtering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachingε of the preεent invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be uεed to engineer cells in vivo after combination with a εuitable delivery vehicle. The sequenceε of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of εomatic cell hybrids containing individual human chromoεomeε. Only thoεe hybridε containing the human gene correεponding to the primer will yield an amplified fragment.
PCR mapping of εomatic cell hybridε iε a rapid procedure for aεsigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragmentε from specific chromosomeε or pools of large genomic clones in an analogous manner. Other mapping strategieε that can εimilarly be uεed to map to itε chromoεome include in si tu hybridization, prescreening with labeled flow-sorted chromosomeε and preεelection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromoεomal location in one εtep. This technique can be uεed with cDNA aε εhort as 500 or 600 bases,- however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for εimple detection. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al. , Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkinε University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes) .
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals . If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current reεolution of phyεical mapping and genetic mapping techniqueε, a cDNA preciεely localized to a chromoεomal region associated with the diseaεe could be one of between 50 and 500 potential cauεative genes. (This asεumes l megabase mapping resolution and one gene per 20 kb) .
The polypeptideε, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includeε chimeric, εingle chain, and humanized antibodies, aε well aε Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Lisε, Inc., pp. 77-96).
Techniques described for the production of single chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce εingle chain antibodieε to immunogenic polypeptide productε of this invention. Alεo, tranεgenic mice may be uεed to expreεs humanized antibodies to immunogenic polypeptide products of this invention.
The present invention will be further described with reference to the following exampleε; however, it iε to be underεtood that the preεent invention is not limited to εuch exampleε. All partε or amounts, unleεε otherwiεe εpecified, are by weight.
In order to facilitate underεtanding of the following exampleε certain frequently occurring methods and/or terms will be described. "Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes uεed herein are commercially available and their reaction conditionε, cofactors and other requirements were used as would be known to the ordinarily skilled artiεan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digeεted with 20 to 250 unitε of enzyme in a larger volume. Appropriate bufferε and εubεtrate amountε for particular reεtriction enzymeε are εpecified by the manufacturer. Incubation timeε of about l hour at 37'C are ordinarily uεed, but may vary in accordance with the supplier's instructions. After digeεtion the reaction iε electrophoresed directly on a polyacrylamide gel to isolate the deεired fragment.
Size εeparation of the cleaved fragments is performed uεing 8 percent polyacrylamide gel deεcribed by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).
"Oligonucleotides" referε to either a εingle εtranded polydeoxynucleotide or two complementary polydeoxynucleotide εtrandε which may be chemically εyntheεized. Such εynthetic oligonucleotideε have no 5' phoεphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A εynthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double εtranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146) . Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973) .
Example 1 Cloning and expression of BTG-2 using the baculovirus expression system
The DNA sequence encoding the full length BTG-2 protein, ATCC # 97025, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the gene:
The 5' primer has the sequence 5' CAGTGGATCCGCCAC CATGCAGCTTGAAATCCAAGTAGCAC 3' (SEQ ID No. 5) and containε a Bam HI restriction enzyme site (in bold) followed by 6 nucleotides reεembling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol . Biol., 196:947-950 (1987) and just behind the first 26 nucleotides of the BTG-2 gene (the initiation codon for translation "ATG" is underlined) .
The 3' primer has the sequence 5' CAGTGGTACCATACATTT TCTTTTTTTTAGTTAGCCAT 3' (SEQ ID No. 6) and contains the cleavage site for the restriction endonucleaεe Aεp718 and 25 nucleotideε complementary to the 3' non-tranεlated sequence of the BTG-2 gene. The amplified sequences are isolated from a 1% agaroεe gel uεing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, a.) . The fragment iε then digested with the endonucleases Bam HI and Asp7l8 and purified again on a 1% agarose gel. This fragment is designated F2.
The vector pRGl (modification of pVL941 vector, discussed below) is used for the expression of the BTG-2 protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) . This expression vector contains the εtrong polyhedrin promoter of the Autographa californica nuclear polyhidroεiε viruε (AcMNPV) followed by the recognition sites for the reεtriction endonucleases. The polyadenylation site of the simian virus (SV)40 is uεed for efficient polyadenylation. For an eaεy εelection of recombinant viruses the beta- galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31- 39) .
The plasmid is digested with the restriction enzymes Bam HI and Asp7l8 and dephosphorylated using calf intestinal phosphataεe by procedureε known in the art. The DNA iε then iεolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plaεmid V2 are ligated with T4 DNA ligase. E.coli HB101 cells are then transformed and bacteria identified that contained the plaεmid (pBac BTG-2) with the BTG-2 gene using the respective reεtriction enzymeε. The εeguence of the cloned fragment iε confirmed by DNA sequencing. 5 μg of the plasmid pBac BTG-2 is co-transfected with
1.0 μg of a commercially available linearized baculovirus
("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)). lμg of BaculoGold™ viruε DNA and 5 μg of the plasmid pBac BTG-2 are mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologieε Inc., Gaitherεburg, MD) . Afterwardε 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to the Sf9 insect cellε (ATCC CRL 1711) seeded in a 35 mm tiεsue culture plate with 1ml Grace' medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hourε the transfection solution iε removed from the plate and l ml of Grace'ε insect medium supplemented with 10% fetal calf serum iε added. The plate iε put back into an incubator and cultivation continued at 27°C for four dayε.
After four dayε the supernatant is collected and a plaque asεay performed εimilar as described by Summers and Smith (εupra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A detailed description of a "plaque asεay" can alεo be found in the uεer'ε guide for inεect cell culture and baculovirology diεtributed by Life Technologies Inc., Gaithersburg, page 9- 10) .
Four days after the serial dilution the viruses are added to the cells and blue stained plaqueε are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar iε removed by a brief centrifugation and the εupernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then stored at 4°C.
Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-BTG-2 at a multiplicity of infection (MOD of 2. Six hours later the medium is removed -and replaced with SF900 II medium minus methionine and cyεteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 μCi of 35S-methionine and 5 μCi 35S cyεteine (Amersham) are added. The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins viεualized by SDS-PAGE and autoradiography.
Example 2 Cloning and expreεεion of BTG-3 uεing the baculovirus expression system
The DNA sequence encoding the full length BTG-3 protein, ATCC # 97010, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end sequences of the gene:
The 5' primer has the εequence 5' CAGTGGATCCGCCACCATGCAG CTAGAGATCAAAGTGGCCC 3' (SEQ ID No. 7) and containε a Bam HI restriction enzyme site (in bold) followed by 6 nucleotides resembling an efficient εignal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol. Biol., 196:947-950 (1987) and just behind the first 228 nucleotideε of the BTG-3 gene (the initiation codon for tranεlation "ATG" iε underlined) .
The 3' primer haε the εequence 5' CAGTGGTACCACGGGCCA GGTAGATGGTCAGTTGGCCAGCAC 3' (SEQ ID No. 8) and containε the cleavage εite for the reεtriction endonucleaεe Asp718 and 25 nucleotides complementary to the 3' non-translated εequence of the BTG-3 gene. The amplified εequenceε are iεolated from a 1% agaroεe gel uεing a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.). The fragment iε then digested with the endonucleases Bam HI and Aεp7l8 and purified again on a 1% agarose gel. This fragment is designated F2.
The vector pRGl (modification of pVL941 vector, discussed below) is used for the expression of the BTG-3 protein using the baculovirus expression syεtem (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methodε for baculoviruε vectorε and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) . Thiε expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosiε viruε (AcMNPV) followed by the recognition εiteε for the reεtriction endonucleases Bam HI and Aεp7l8. The polyadenylation site of the simian viruε (SV)40 iε uεed for efficient polyadenylation. For an eaεy εelection of recombinant viruses the beta-galactoεidase gene from E.coli iε inεerted in the εame orientation aε the polyhedrin promoter followed by the polyadenylation εignal of the polyhedrin gene. The polyhedrin εequenceε are flanked at both sides by viral sequences for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectorε could be used in place of pRGl such as pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39).
The plasmid is digeεted with the reεpective restriction enzymes and dephosphorylated using calf intestinal phosphataεe by procedureε known in the art. The DNA iε then iεolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This vector DNA is designated V2.
Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid (pBac BTG-3) with the BTG-3 gene using the enzymes Bam HI and Aεp718. The sequence of the cloned fragment iε confirmed by DNA sequencing.
5 μg of the plasmid pBac BTG-3 is co-transfected with 1.0 μg of a commercially available linearized baculovirus ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)). lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac BTG-3 are mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologieε Inc., Gaithersburg, MD) . Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minuteε at room temperature. Then the transfection mixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1ml Grace' medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection εolution iε removed from the plate and 1 ml of Grace'ε inεect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation continued at 27°C for four days.
After four days the supernatant is collected and a plaque assay performed similar as described by Summers and Smith (supra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allows an easy isolation of blue stained plaques. (A detailed deεcription of a "plaque assay" can also be found in the user'ε guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaitherεburg, page 9- 10) .
Four dayε after the serial dilution the viruses are added to the cells and blue stained plaqueε are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruεeε iε then reεuεpended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the εupernatant containing the recombinant baculoviruε iε used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then εtored at 4°C.
Sf9 cellε are grown in Grace's medium εupplemented with 10% heat-inactivated FBS. The cellε are infected with the recombinant baculoviruε V-BTG-3 at a multiplicity of infection (MOD of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 μCi of 35S-methionine and 5 μCi 3SS cysteine (Amersham) are added. The cellε are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteinε viεualized by SDS-PAGE and autoradiography.
Example 3 Expression of Recombinant BTG-2 in COS cells
The expresεion of plaεmid, BTG-2 HA iε derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire BTG-2 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously deεcribed (I. Wilεon, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767) . The fusion of HA tag to the target protein allowε eaεy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction εtrategy iε deεcribed aε followε: The DNA sequence encoding BTG-2, ATCC # 97025, iε constructed by PCR using two primers: the 5' primer 5' GGCCC AAGCTTGCCGCCΛTGCAGCTTGAAATCCAAGTAG 3' (SEQ ID No. 9) contains a Hind III site followed by 23 nucleotides of BTG-2 coding sequence starting from the initiation codon; the 3' sequence 5' ATCGTCTAC^TTAGTTAGCCATAACAGGCrcGAATTGC SGTTAGAATACro TTATTTAAGCTAAAATTCAAGCCATCTA 3' (SEQ ID No. 10) contains complementary sequences to Xbal site, translation stop codon, HA tag and the last 68 nucleotides of the BTG-2 coding sequence (not including the stop codon) . Therefore, the PCR product contains a Hind III site, BTG-2 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digeεted with Hind III and Xbal restriction enzyme and ligated. The ligation mixture is transformed into E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by reεtriction analyεis for the presence of the correct fragment. For expresεion of the recombinant BTG-2, COS cellε are tranεfected with the expreεεion vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritεch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Presε, (1989)) . The expreεεion of the BTG-2 HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodieε: A Laboratory Manual, Cold Spring Harbor Laboratory Preεε, (1988) ) . Cellε are labelled for 8 hours with 35S-cysteine two dayε post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Triε, pH 7.5) . (Wilεon, I. et al., Id. 37:767 (1984)). Both cell lyεate and culture media are precipitated with a HA εpecific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels.
Example 4 Expression of Recombinant BTG-3 in COS cellε
The expression of plasmid, BTG-3 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire BTG-3 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previouεly deεcribed (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767) . The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plaεmid conεtruction strategy iε described aε follows:
The DNA sequence encoding BTG-3, ATCC # 97010, is constructed by PCR using two primers: the 5' primer 5' GGCCC AAGCTTGCCGCCATGCAGCTAGAGATCAAAGTGGC 3' (SEQ ID No. 11) contains a Hind III site followed by 23 nucleotides of BTG-3 coding sequence εtarting from the initiation codon; the 3' εequence 5' ATCGTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGGTTG TAGCTGAGGCCTTCCACAAAGGGTGTCTTCTCCAGG 3' (SEQ ID No. 12) containε complementary εequences to an Xbal site, translation stop codon, HA tag and the last 68 nucleotideε of the BTG-3 coding εequence (not including the εtop codon) . Therefore, the PCR product contains a Hind III site, BTG-3 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Hind III and Xbal restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant BTG-3, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) . The expression of the BTG- HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) ) . Cells are labelled for 8 hours with 35S-cysteine two dayε poεt tranεfection. Culture media is then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA εpecific monoclonal antibody. Proteinε precipitated are analyzed on 15% SDS-PAGE gelε.
Example 5 Bacterial Expreεεion and Purification of BTG-2
The DNA εequence encoding BTG-2, ATCC # 97025, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' end εequenceε of the processed BTG-2 gene (minus the signal peptide sequence) and the vector sequences 3' to the BTG-2 gene. Additional nucleotides corresponding to Ncol and Bgl II reεtriction enzyme εites were added to the 5' and 3' end sequenceε reεpectively. The 5' oligonucleotide primer has the sequence ATCGCCATGGGACAGCTT GAAATCCAAGTAGCACTA 3' (SEQ ID No. 13) contains a Nco I restriction enzyme site followed by 24 nucleotides of BTG-2 coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5' ATC GAGATCTTTAGTTAGCCATAACΑGGCTGGAAT 3' (SEQ ID No. 14) contains complementary sequences to a Bgl II restriction site and the last 21 nucleotides of BTG-2. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expresεion vector pQE-60 (Qiagen, Inc. Chatsworth, CA) . pQE- 60 encodeε antibiotic reεiεtance (Ampr) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator
(P/O) , a ribosome binding εite (RBS) , a 6-Hiε tag and restriction enzyme siteε. pQE-60 is then digested with Nco I and Bgl II. The amplified sequences are ligated into pQE- 60 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture iε then uεed to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε,
(1989) . M15/rep4 contains multiple copies of the plasmid pREP4, which expresεeε the lad repreεεor and alεo confers kanamycin resiεtance (Kanr) . Tranεformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysiε. Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ l) and Kan (25 ug/ml) . The 0/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG ("Iεopropyl-B-D-thiogalacto pyranoεide") iε then added to a final concentration of l mM. IPTG induces by inactivating the lad repreεεor, clearing the P/O leading to increaεed gene expreεεion. Cellε are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized BTG-2 iε purified from thiε εolution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)) . BTG-2 (95% pure) is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.
Example 6 Bacterial Expression and Purification of BTG-3
The DNA sequence encoding BTG-3, ATCC # 97010 iε initially amplified uεing PCR oligonucleotide primerε corresponding to the 5' and 3' end sequenceε of the procesεed BTG-3 gene. Additional nucleotideε correεponding to Ncol and Bgl II reεtriction enzyme sites are added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer haε the εequence 5' ATCGCCATGGGACAGCTAGAGATCAAAGTGGCCCTG 3' (SEQ ID No. 15) containε an Ncol reεtriction enzyme site followed by 24 nucleotides of BTG-3 coding εequence εtarting from the presumed terminal amino acid of the processed protein. The 3' sequence 5' ATCGAGATCTGTTGGCCAGCACCACGGGCTGG 3' (SEQ ID No. 16) contains complementary sequences to a Bgl II restriction site and the last 21 nucleotides of BTG-3 coding sequence. The restriction enzyme sites correspond to the reεtriction enzyme εiteε on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, CA) . pQE-60 encodes antibiotic resistance (Ampr) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6-His tag and restriction enzyme sites. pQE-60 is then digested with Nco I and Bgl II. The amplified sequences are ligated into pQE- 60 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Presε, (160860) . M15/rep4 containε multiple copies of the plasmid pREP4, which expresses the lad repressor and alεo conferε kanamycin resistance (Kanr) . Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is iεolated and confirmed by restriction analysis. Clones containing the desired constructε are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/N culture is uεed to inoculate a large culture at a ratio of 1:100 to 1:250. The cellε are grown to an optical denεity 600 (O.D.600) of between 0.4 and 0.6. IPTG ("Iεopropyl-B-D-thiogalacto pyranoεide") iε then added to a final concentration of l mM. IPTG induces by inactivating the lad represεor, clearing the P/O leading to increased gene expresεion. Cellε are grown an extra 3 to 4 hourε. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, solubilized BTG-3 iε purified from this solution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (16084)). BTG-3 (95 % pure) is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM εodium phoεphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in thiε εolution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT: KUNSCH, ET AL.
(ii) TITLE OF INVENTION: Human B-Cell Tranεlocation
Genes-2 and 3
(iii) NUMBER OF SEQUENCES: 16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART _ OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07068
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: Submitted herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER:
(B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134
(C) REFERENCE/DOCKET NUMBER: 325800-262
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1843 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE : CDNA
(xi) SEQUENCE DESCRIPTION : SEQ ID NO: l :
GGCACGAGAT TTTGTGGCGT AGAGCTATGC AGCTTGAAAT CCAAGTAGCA CTAAATTTTA 60
TTATTTCGTA TTTGTACAAT AAGCTTCCCA GGAGACGTGT CAACATTTTT GGTGAAGAAC 120
TTGAAAGAC TCTTAAGAAG AAATATGAAG GGCACTGGTA TCCTGAAAAG CCATACAAAG 180
GATCGGGGTT TAGATGTATA CACATAGGGG AGAAAGTGGA CCCAGTGATT GAACAAGCAT 240
CCAAAGAGAG TGGTTTGGAC ATTGATGATG TTCGTGGCAA TCTGCCACAG GATCTTAGTG 300
TTTGGATCGA CCCATTTGAG GTTTCTTACC AAATTGGTGA AAAGGGACCA GTGAAGGTGC 360
TTTACGTGGA TGATAATAAT GAAAATGGAT GTGAGTTGGA TAAGGAGATC AAAAACAGCT 420
TTAACCCAGA GGCCCAGGTT TTTATGCCCA TAAGTGACCC AGCCTCATCA GTGTCCAGCT 480
CTCCATCGCC TCCTTTTGGT CACTCTGCTG CTGTAAGCCC TACCTTCATG CCCCGGTCCA 540
CTCAGACTTT AACCTTTACC ACTGCCACTT TTGGCTGCCA CCAAGTTCGG CTCTACCAAA 600
ATGAAGATAG TGGCCGTAGC AACAAGGTTG CACGTACTTC TCCCATCAAC CTCGGCTTGA 660
ATGTGAATGA CCTCTTGAAG CAGAAAGCCA TCTCTTCCTC AATGCACTCT CTGTATGGGC 720
TTGGCTTGGG TAGCCAGCAG CAGCCACAGC AACAGCAGCA GCCAGCCCAG CCGCCACCGC 780
CACCACCACC ACCACAGGAG GAACAANAGC AGAAAACCTC TGCTCTTTCT CCTAATGCCA 840
AGGAATTTAT TTTTCCTAAT ATGCAGGGTC AAGGTAGTAG TACCAATGGA ΛΓGTTCCCAG 900
GTGACAGCCC CCTTAACCTC AGTCCTCTCC AGTACAGTAA TGCCTTTGAT GTGTTTGCAG 960 CTATGGAGG CCTCAATGAG AAGTCTTTTG TAGATGGCTT GAATTTTAGC TTAAATAACA 1020 TGCAGTATTC TAACCΆGCAA TTCCAGCCTG TTATGGCTAA CTAAAAAAAA GAAAATGTAT 1080 CGTACAAGTT AAAATGCACσ GGCCAAGGGG GGATTTTTTT TTTCACCTCC TTGAGAATΓT 1140 TTTTTTTTAA GCTTATAGTA AGGATACATT CAAGCTTGGT TAAAAAAATA ATAATAAAAC 1200 ATGCATCATT TTTCATTTGC CAACCAAGCA CAAAGTTATT TTATGCTGCC TGTATATTTT 1260 AAAGTATACT CTCAGATATG CCCTCTTACA GTATTTTAAG ATATTAGCAA AGGACATGGC 1320 TTGATTTTTT TTTATAAAAA TTGGCACTAA TAAGTGGGTT TATTGGTCTT TTCTAATTGT 1380 ATAATTTAAT TTAGTACCAA AGTTTGTAAA ATATCAGAGG ATATATATAT ATTGTATCCT 1440 ACGACATGGT ATTGCATTTA TATCTTTTTA CTACAGTGAT CTGTGACAGC AGCAGCCTCA 1500 TGTTGTATTT TTTTTACTGA AATTGTAAAA TATCCATCTT AAAGACATCA ACTATTCTAA 1560 AAATTGTGTA CAGGATATTC CTTTAGTGGT GGAATTAAAA TGTGCGAATA CTTGCTTTCT 1620 CCAAAAAAAT GTATTTTCTG TTAAAAGTTT AAAGATTTTT GCTATATATT ATGGAAGGAA 1680 AATGTAATCG TAAATATTAA TTTTGTACCT ATATTGTGCA ATACTTGAAA AAAACGGTAT 1740 AAAAGTATTT TGAGTCAGTG TCTTACATGT TAAGAGGGAC TGAAATAGTT TATATTAAGT 1800 TTGTATTAAA ATTCTTTAAA ATTAAAAAAA AAAAAAAAAA AAA 1843
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 345 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Gin Leu Glu lie Gin Val Ala Leu Asn Phe lie lie Ser Tyr -25 -20 -15
Leu Tyr Aεn Lys Leu Pro Arg Arg Arg Val Asn lie Phe Gly Glu -10 -5 1 5
Glu Leu Glu Arg Leu Leu Lys Lys Lys Tyr Glu Gly His Trp Tyr
10 15 20
Pro Glu Lys Pro Tyr Lys Gly Ser Gly Phe Arg Cys lie His lie
25 30 35
Gly Glu Lys Val Asp Pro Val lie Glu Gin Ala Ser Lys Glu Ser
40 45 50
Gly Leu Asp lie Asp Asp Val Arg Gly Asn Lys Pro Gin Asp Lys
55 60 65 Ser Val Trp lie Asp Pro Phe Glu Val Ser Tyr Gin lie Gly Glu
70 75 80
Lys Gly Pro Val Lys Val Leu Tyr Val Aεp Asp Asn Asn Glu Asn
85 90 95
Gly Cys Glu Leu Asp Lys Glu lie Lys Asn Ser Phe Asn Pro Glu
100 105 110
Ala Gin Val Phe Met Pro lie Ser Asp Pro Ala Ser Ser Val Ser
115 120 125
Ser Ser Pro Ser Pro Pro Phe Gly His Ser Ala Ala Val Ser Pro
130 135 140
Thr Phe Met Pro Arg Ser Thr Gin Thr Leu Thr Phe Thr Thr Ala
145 150 155
Thr Phe Gly Cys His Gin Val Arg Leu Tyr Gin Asn Glu Asp Ser
160 165 170
Gly Arg Ser Asn Lys Val Ala Arg Thr Ser Pro lie Aεn Leu Gly
175 180 185
Leu Asn Val Asn Asp Leu Leu Lys Gin Lys Ala lie Ser Ser Ser
190 195 200
Met His Ser Leu Tyr Gly Leu Gly Leu Gly Ser Gin Gin Gin Pro
205 210 215
Gin Gin Gin Gin Gin Pro Ala Gin Pro Pro Pro Pro Pro Pro Pro
220 225 230
Pro Gin Glu Glu Gin Gin Gin Lyε Thr Ser Ala Leu Ser Pro Aεn
235 240 245
Ala Lys Glu Phe lie Phe Pro Asn Met Gin Gly Gin Gly Ser Ser
250 255 260
Thr Asn Gly Met Phe Pro Gly Asp Ser Pro Leu Asn Leu Ser Pro
265 270 275
Leu Gin Tyr Ser Asn Ala Phe Asp Val Phe Ala Ala Tyr Gly Gly
280 285 290
Leu Asn Glu Lys Ser Phe Val Asp Gly Leu Asn Phe Ser Leu Asn
295 300 305
Asn Met Gin Tyr Ser Asn Gin Gin Phe Gin Pro Val Met Ala Asn
310 315 320 (2 ) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH : 1352 BASE PAIRS
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS : SINGLE
(D) TOPOLOGY : LINEAR
(ii) MOLECULE TYPE : CDNA
(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 3 :
GGAATTCGGC ACGAGCAACC CTCAACGACG AAAAGGACTT CGGTCCCCTG GCCCGGCGAC 60 GCCCGGRAAG GAAAGGAGAG CGACCTCCGC CCCGCGCTCA GGCCACCCTG GAGGGAGAAG 120 CCGCCCCGCG GCGGGTAGAG CGCCCCGCCG CCNCGYHAGN ACCCGAAGCC GCCTGGAGCC 180 CAAGGCTGTA CACGTGCCCT GTGCTGAGGC TCTGCCTAGG AAAGGACCAT GCAGCTAGAG 240 ATCAAAGTGG CCCTGAACTT CATCCATCTC CTACTTGTAC AACAAGCTGC CCCGGCGCCC 300 GGGCAGAACC TG TTGGGGA GGAGCTAGAG CGGCTTTTGA AAAGGAAATA TGAAGGCCAC 360 TGGTACCCTG AGAAGCCACT GAAAGGCTCT GGCTTCCGCT GTGTTCACAT TGGGGAGATG 420 GTGGACCCCG TGGTGGAGCT GGCCGCCAAG CGGAGTGGCC TGGCGGTGGA AGATGTGCGG 480 GCCAATGTGC CTGAGGAGCT GAGTGTCTGG ATTGATCCCT TTGAGGTGTC CTACCAGATT 540 GGTGAGAAGG GAGCTGTGAA AGTGCTGTAC CTGGATGACA GTGAGGGTTG CGGTGCCCCA 600 GAGCTGGACA AGGAGATCAA GAGCAGCTTC AACCCTGACG CCCAGGTGTT CGTGCCCATT 660 GGCAGCCAGG ACAGCTCCCT GTCCAACTCC CCATCGCCAT CCTTTGGCCA GTCACCCAGC 720 CCTACCTTCA TTCCCCGCTC CGCTCAGCCC ATCACCTTCA CCACCGCCTC CTTCGCTGCC 780 ACCAAATTTG GCTCCACTAA GATGAAGAAG GGGGGCGGGG CAGCAAGTGG TGGGGGTGTA 840 GCCAGCAGTG GGGCGGGTGG CCAGCAGCCA CCACAGCAGC CTCGCATGGC CCGCTCACCC 900 ACCAACAGCC TGCTGAAGCA CAAGAGCCTC TCTCTGTCTA TGCATTCACT GAACTTCATC 960 ACGGCCAACC CGGCCCCTCA GTCCCAGCTC TCACCCAATG CCAAGGAGTT CGTGTACAAC 1020 GGTGGTGGCT CACCCAGCCT CTTCTTTGAT GCGGCCGATG GCCAGGGCAG CGGCACCCCA 1080 GGCCCGTTTG GAGGCAGTGG GGCTGGCACC TGCAACAGCA GCAGCTTTGA CATGGCCCAG 1140 GTATTTGGAG GTGGTGCCAA CAGCCTCTTC CTGGAGAAGA CACCCTTTGT GGAAGGCCTC 1200 AGCTACAACC TGAACACCAT GCAGTATCCC AGCCAGCAGT TCCAGCCCGT GGTGCTGGCC 1260 AACTGACCAT CTACCTGGCC CGTGGGGGCA GGAGCACCCA AGACCACAGA AAAGAGAAAG 1320 GAAAGGCCAA AAAAAAAAAA AAAAAACTCG AG 1352 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 345 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Gin Leu Glu lie Lys Val Ala Leu Asn Phe lie His Leu Leu -25 -20 -15
Leu Val Gin Gin Ala Ala Pro Ala Pro Gly Gin Asn Leu Phe Gly -10 -5 1 5
Glu Glu Leu Glu Arg Leu Leu Lys Arg Lys Tyr Glu Gly His Trp
10 15 20
Tyr Pro Glu Lyε Pro Leu Lyε Gly Ser Gly Phe Arg Cys Val His
25 30 35 lie Gly Glu Met Val Asp Pro Val Val Glu Leu Ala Ala Lyε Arg
40 45 50
Ser Gly Leu Ala Val Glu Aεp Val Arg Ala Aεn Val Pro Glu Glu
55 60 65
Leu Ser Val Trp lie Aεp Pro Phe Glu Val Ser Tyr Gin lie Gly
70 75 80
Glu Lyε Gly Ala Val Lys Val Leu Tyr Leu Asp Aεp Ser Glu Gly
85 90 95
Cyε Gly Ala Pro Glu Leu Asp Lys Glu lie Lys Ser Ser Phe Asn
100 105 110
Pro Asp Ala Gin Val Phe Val Pro lie Gly Ser Gin Asp Ser Ser
115 120 125
Leu Ser Asn Ser Pro Ser Pro Ser Phe Gly Gin Ser Pro Ser Pro
130 135 140
Thr Phe lie Pro Arg Ser Ala Gin Pro lie Thr Phe Thr Thr Ala
145 150 155 Ser Phe Ala Ala Thr Lys Phe Gly Ser Thr Lys Met Lys Lys Gly
160 165 170
Gly Gly Ala Ala Ser Gly Gly Gly Val Ala Ser Ser Gly Ala Gly
175 180 185
Gly Gin Gin Pro Pro Gin Gin Pro Arg Met Ala Arg Ser Pro Thr
190 195 200
Asn Ser Leu Leu Lys His Lys Ser Leu Ser Leu Ser Met His Ser
205 210 215
Leu Asn Phe lie Thr Ala Asn Pro Ala Pro Gin Ser Gin Leu Ser
220 225 230
Pro Asn Ala Lys Glu Phe Val Tyr Asn Gly Gly Gly Ser Pro Ser
235 240 245
Leu Phe Phe Asp Ala Ala Asp Gly Gin Gly Ser Gly Thr Pro Gly
250 255 260
Pro Phe Gly Gly Ser Gly Ala Gly Thr Cys Asn Ser Ser Ser Phe
265 270 275
Asp Met Ala Gin Val Phe Gly Gly Gly Ala Asn Ser Leu Phe Leu
280 285 290
Glu Lys Thr Pro Phe Val Glu Gly Leu Ser Tyr Asn Leu Aεn Thr
295 300 305
Met Gin Tyr Pro Ser Gin Gin Phe Gin Pro Val Val Leu Ala Aεn
310 315 320
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 41 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CAGTGGATCC GCCACCATGC AGCTTGAAAT CCAAGTAGCA C 41
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 38 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CAGTGGTACC ATACATTTTC TTTTTTTTAG TTAGCCAT 38
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 41 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CAGTGGATCC GCCACCATGC AGCTAGAGAT CAAAGTGGCC C 41
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 42 BASE PAIRS
(B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CAGTGGTACC ACGGGCCAGG TAGATGGTCA GTTGGCCAGC AC 42
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 39 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GGCCCAAGCT TGCCGCCATG CAGCTTGAAA TCCAAGTAG 39
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 84 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ATCGTCTAGA TTAGTTAGCC ATAACAGGCT GGAATTGCTG GTTAGAATAC TGCATGTTAT 60 TTAAGCTAAA ATTCAAGCCA TCTA 84
(2 ) INFORMATION FOR SEQ ID NO : 11 :
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH : 40 BASE PAIRS
(B) TYPE : NUCLEIC ACID
(C) STRANDEDNESS : SINGLE
(D) TOPOLOGY : LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GGCCCAAGCT TGCCGCCATG CAGCTAGAGA TCAAAGTGGC 40
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 81 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii ) MOLECULE TYPE : Oligonucleotide
(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 12 :
ATCGTCTAGA TCAAGCGTAG TCTGGGACGT CGTATGGGTA GGTTGTAGCT GAGGCCTTCC 60 ACAAAGGGTG TCTTCTCCAG G 81
(2 ) INFORMATION FOR SEQ ID NO : 13 :
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH : 36 BASE PAIRS (B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
ATCGCCATGG GACAGCTTGA AATCCAAGTA GCACTA 36
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 35 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATCGAGATCT TTAGTTAGCC ATAACAGGCT GGAAT 35
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 36 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: ATCGCCATGG GACAGCTAGA GATCAAAGTG GCCCTG 36
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 32 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ATCGAGATCT GTTGGCCAGC ACCACGGGCT GG 32

Claims

WHAT IS CLAIMED IS:
1. An iεolated polynucleotide selected from the group consisting of:
(a) a polynucleotide encoding the polypeptide having the deduced amino acid sequence of SEQ ID No. 2 or a fragment, analog or derivative of said polypeptide,-
(b) a polynucleotide encoding the polypeptide having the deduced amino acid sequence of SEQ ID No. 4 or a fragment, analog or derivative of said polypeptide,-
(c) a polynucleotide encoding the polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Depoεit No. 97025 or a fragment, analog or derivative of said polypeptide; and
(d) a polynucleotide encoding the polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Depoεit No. 97010 or a fragment, analog or derivative of εaid polypeptide.
2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.
3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA. . The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.
5. The polynucleotide of Claim 2 wherein said polynucleotide encodes a polypeptide having the deduced amino acid sequence of SEQ ID No. 2.
6. The polynucleotide of Claim 2 wherein εaid polynucleotide encodes a polypeptide having the deduced amino acid sequence of SEQ ID No. 4.
7. The polynucleotide of Claim 2 wherein εaid polynucleotide encodeε the polypeptide encoded by the cDNA of ATCC Depoεit No. 97025.
8. The polynucleotide of Claim 2 wherein εaid polynucleotide encodeε the polypeptide encoded by the cDNA of ATCC Deposit No. 97010. 9. The polynucleotide of Claim 1 having the coding sequence as shown in SEQ ID No. 1.
10. The polynucleotide of Claim 1 having the coding sequence as shown in SEQ ID No. 3.
11. The polynucleotide of Claim 2 having the coding sequence deposited as ATCC Deposit No. 97025.
12. The polynucleotide of Claim 2 having the coding sequence deposited as ATCC Deposit No. 97010.
13. A BTG-2 polypeptide fragment comprising the amino acid sequence as set forth in SEQ ID No. 2 from residue 1 to residue 320.
14. A BTG-3 polypeptide fragment comprising the amino acid sequence as set forth in SEQ ID No. 4 from residue l to residue 320.
15. A vector containing the DNA of Claim 2.
16. A host cell genetically engineered with the vector of Claim 15.
17. A proceεs for producing a polypeptide comprising: expressing from the host cell of Claim 16 the polypeptide encoded by said DNA.
18. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 15.
19. A polypeptide selected from the group consisting of (i) a polypeptide having the deduced amino acid sequence of SEQ ID No. 2 and fragments, analogs and derivativeε thereof (ii) a polypeptide encoded by the cDNA of ATCC Depoεit No. 97025 and fragmentε, analogε and derivativeε of εaid polypeptide (iii) a polypeptide having the deduced amino acid εequence of SEQ ID No. 4 and fragmentε, analogs and derivatives thereof; and (iv) a polypeptide encoded by the cDNA of ATCC Deposit No. 97010 and fragments, analogs and derivatives of said polypeptide. 20. The polypeptide of Claim 19 wherein the polypeptide is BTG-2 having the deduced amino acid sequence of SEQ ID No. 2.
21. The polypeptide of Claim 19 wherein the polypeptide is BTG-3 having the deduced amino acid sequence of SEQ ID No. 4.
22. An isolated polynucleotide encoding BTG-2 comprising the sequence aε set forth in SEQ ID No. l from nucleotide 27 to nucleotide 1061.
23. An isolated polynucleotide encoding BTG-3 compriεing the sequence as set forth in SEQ ID No. 3 from nucleotide 229 to nucleotide 1263.
24. An antibody against the polypeptide of claim 17.
25. A compound effective aε an agoniεt to the polypeptide of claim 19.
26. A compound effective aε an antagonist against the polypeptide of claim 19.
27. A method for the treatment of a patient having need of BTG-2 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 19.
28. A method for the treatment of a patient having need of BTG-3 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 19.
29. A method for the treatment of a patient having need to inhibit BTG-2 comprising-, administering to the patient a therapeutically effective amount of the compound of Claim 26.
30. A method for the treatment of a patient having need to inhibit BTG-3 comprising: administering to the patient a therapeutically effective amount of the compound of Claim 26.
31. The method of Claim 27 wherein εaid therapeutically effective amount of the polypeptide iε administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.
32. The method of Claim 28 wherein said therapeutically effective amount of the polypeptide iε administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.
33. A process for identifying compounds active aε agonists or antagonists to BTG-2 and BTG-3 comprising: combining a compound to be screened, labelled BTG-2 or BTG-3 and a reaction mixture containing cells which express a receptor for BTG-2 or BTG-3; and determining the extent of binding of BTG-2 or BTG-3 to the receptors.
34. A process for diagnosing a disease or a susceptibility to a disease related to an altered level of expression of the polypeptide of claim 19 comprising-. determining a mutation in the nucleic acid sequence encoding εaid polypeptide.
35. A diagnostic process comprising: analyzing for the presence of the polypeptide of claim 19 in a sample derived from a host.
EP95913736A 1995-03-17 1995-03-17 Human b-cell translocation genes-2 and 3 Withdrawn EP0815219A4 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02021846A EP1284292A3 (en) 1995-03-17 1995-03-17 Human B-cell Translocation Gene-3

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1995/003323 WO1996029401A1 (en) 1995-03-17 1995-03-17 Human b-cell translocation genes-2 and 3

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP02021846A Division EP1284292A3 (en) 1995-03-17 1995-03-17 Human B-cell Translocation Gene-3

Publications (2)

Publication Number Publication Date
EP0815219A1 true EP0815219A1 (en) 1998-01-07
EP0815219A4 EP0815219A4 (en) 2001-09-19

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP95913736A Withdrawn EP0815219A4 (en) 1995-03-17 1995-03-17 Human b-cell translocation genes-2 and 3

Country Status (4)

Country Link
EP (1) EP0815219A4 (en)
JP (1) JPH11502119A (en)
AU (1) AU2101195A (en)
WO (1) WO1996029401A1 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013469A (en) 1995-06-02 2000-01-11 Human Genome Sciences, Inc. Human B-cell translocation genes-2 and 3
AU7364596A (en) * 1996-09-18 1998-04-14 Human Genome Sciences, Inc. Human b-cell translocation genes-2 and 3
US6525185B1 (en) 1998-05-07 2003-02-25 Affymetrix, Inc. Polymorphisms associated with hypertension
CN1160119C (en) * 2001-02-27 2004-08-04 中国科学院上海生物化学研究所 Functions of Tob gene in central nervous system of mammol and its application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No further relevant documents disclosed *
See also references of WO9629401A1 *

Also Published As

Publication number Publication date
AU2101195A (en) 1996-10-08
JPH11502119A (en) 1999-02-23
EP0815219A4 (en) 2001-09-19
WO1996029401A1 (en) 1996-09-26

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