WO1996019566A1 - E2 binding proteins - Google Patents
E2 binding proteins Download PDFInfo
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- WO1996019566A1 WO1996019566A1 PCT/US1995/016806 US9516806W WO9619566A1 WO 1996019566 A1 WO1996019566 A1 WO 1996019566A1 US 9516806 W US9516806 W US 9516806W WO 9619566 A1 WO9619566 A1 WO 9619566A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
Definitions
- Papillomaviruses have been linked to widespread, serious human diseases, especially carcinomas of the genital and oral mucosa.
- Tens of millions of women suffer from human papilloma virus (HPV) infection of the genital tract. Significant number of these women eventually develop cancer of the cervix. It has been estimated that perhaps twenty percent (20%) of all cancer deaths in women worldwide are from cancers which are associated with HPV. As many as 90% of all cervical cancer maybe linked to HPV.
- HPV human papilloma virus
- Papillomaviruses also induce benign, dysplastic and malignant hyperproliferations of skin and mucosal epithelium (see, for example, Mansur and Androphy, (1993) Biochim Biophys Acta 1 155:323-345; Pfister (1984) Rev. Physiol. Biochem. Pharmacol. 99: 1 11-181; and Broker et al. (1986) Cancer Cells 4:17-36, for reviews of the molecular, cellular, and clinical aspects of the papillomaviruses).
- HPV's are a heterogeneous group of DNA tumor viruses associated with hyperplastic (warts, condylomata), pre-malignant and malignant lesions (carcinomas) of squamous epithelium. Almost 70 HPV types have been identified, and different papillomavirus types are known to cause distinct diseases, Pfister, (1987) Adv. Cancer Res.. 48:1 13-147, Syrjanen, (1984) Obstet. Gynecol. Survey 39:252-265. For example, HPV types 1 and 2 cause common warts, and types 6 and 1 1 cause warts of the external genitalia, anus and cervix. HPV's can be isolated from the majority of cervical cancers.
- HPV-16 is present in about 50 percent of all cervical cancers. These HPV's are referred to as “high risk” HPV's. While HPV 6 and 1 1 are common isolates for cervical papillomas. these infections rarely progress to invasive cancer, and therefore these HPV's are referred to as "low risk” HPV's.
- the biological life cycle of the papillomaviruses appears to differ from most other viral pathogens. These viruses are believed to infect the basal or germ cells of the epithelium. Rather than proceeding to a lytic infection in which viral replication kills the cell, viral DNA transcription and replication are maintained at very low levels until higher strata of the epithelium are achieved. There, presumably in response to differentiation-specific signals, viral transcription accelerates, DNA synthesis begins and virions assemble. It is within this terminally differentiating cellular environment that the virus must recruit the panoply of cellular factors necessary for its reproduction.
- the product of the E2 open reading frame plays an important role in the complex transcriptional pattern of the HPV's.
- the E2 transcriptional activation protein (“the E2 protein”) is a trans-acting factor that activates transcription through specific binding to cis- acting E2 enhancer sequences in viral DNA (Androphy et al., (1987) Nature, 324:70-73), and has been shown to induce promoter expression in a classical enhancer mechanism (Spalholz et al., (1985) Cell 42:183-91).
- the E2 gene product exerts trans-regulatory effects in the upstream regulatory region ("URR") of the viral genome and disruption of E2 is thought to alter regulation of expression of E6 and E7 genes.
- UTR upstream regulatory region
- the upstream regulatory region is found immediately 5' to the early genes of bovine papilloma viruses (BPV's) and other papillomaviruses.
- the URR contains cis-acting regulatory signals, including an origin of DNA replication and several promoters that function in early transcription.
- the URR also contains enhancer elements that activate transcription from the URR promoters and heterologous promoters (Sousa et al., (1990) Biochemica et Biophysica Acta 1032: 19-37).
- the E2 enhancer elements are conditional, in that they stimulate transcription only when activated by a protein encoded by the E2 open reading frame.
- E2 protein As with other transcription factors, the functions of E2 protein appear to be localized to discrete modular domains (Giri et al., (1988) EMBO J, 7:2923-29).
- Gene products from the E2 gene include the full-length transcriptional activator E2 protein and at least two truncated versions of the E2 protein BPV1 that function as transcriptional repressors. Transcriptional activation and repression of viral genes by E2 gene products constitute critical regulatory circuits in papillomavirus gene expression and DNA replication.
- transcriptional regulation by the E2 protein depends on its direct binding to the nucleotide sequence 5'ACC(G)NNNN(C)GGT3' (SEQ ID NO:9) (Androphy et al., sw/jr ⁇ ; Dartmann et al., (1986) Virology, 151 :124-30; Hirochika et al., (1987) J. Virol, 61 :2599-606 ; P. Hawley-Nelson et al., (1988) EMBO J., 7:525-31 ; McBride et al., (1988) EMBO J., 7:533-39).
- N represents any nucleotide
- X is any nucleotide, but is usually G
- Y represents any nucleotide, but is usually C.
- E2 binding sites appear to be positioned in close proximity to the viral promoters, with multiple E2 binding sites present throughout the papillomavirus genome (R. Li et al., (1989) Genes Dew, 3: 510-26), in the URR's of all papillomaviruses, as well as in other sites near promoters throughout the viral genome.
- E2 binding sites may function as an element in viral DNA replication, as well as a classical transcriptional enhancer element.
- the present invention relates, in part, to the discovery in eukaryotic cells, particularly human cells, of novel protein-protein interactions between the papillomavirus regulatory protein E2 and certain cellular proteins, referred to hereinafter as "E2-binding proteins" or "E2-BP", some of which, e.g., E2-BP 42A , E2-BP 23 , E2-BP 24 , and E2-BP 2 " 7 are themselves novel proteins.
- the invention features, a E2-BP 42A polypeptide, preferably a substantially pure preparation of an E2-BP 42A polypeptide, or a recombinant E2-BP 42A polypeptide.
- the polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein: the polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence in SEQ ID NO:5; the polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:5; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:5; the E2-BP 42A polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring E2-binding protein.
- the E2-BP 2A polypeptide is an agonist or antagonist of the regulation of E2 function, e.g. of E2-mediated transcription of PV oncogenes.
- an exemplary E2-BP which is antagonistic of E2 transcriptional activation is the portion of the 42 A clone initially isolated, 357 amino acid fragment, in the E2-mediated two hybrid assay. (When expressed without the VP16 domain (i.e. not as a VP16/42A fusion protein), the 42A polypeptide was able to inhibit transactivation of the reporter construct by the wild-type E2 protein.)
- the E2-BP 42A polypeptide is encoded by the nucleic acid in SEQ ID NO: 1 , or by a nucleic acid having at least 60%, 70%, 80%, 90% or 95% homology with the nucleic acid of SEQ ID NO: 1.
- the subject E2-BP 42A polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NO:5.
- the differences are such that: the E2-BP polypeptide exhibits an E2-BP biological activity, e.g., the E2-BP 4 A polypeptide retains a biological activity of a naturally occurring E2-BP 4 A , e.g., the E2-BP 42A of SEQ ID NO:5.
- the E2-binding protein is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to E2-BP 2A .
- the second polypeptide portion can be any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain.
- the fusion protein can be used in a two-hybrid assay.
- the E2-BP polypeptide encodes amino acid residues 40- 259 of SEQ ID NO:5.
- the E2-binding polypeptide has antagonistic activity, and is capable of: suppressing tumor growth, e.g. in a tumor cell in which endogenous E2-BP is misexpressed; suppressing growth of papillomavirus-infected cells, e.g. HPV-infected cells; inhibiting growth of a papillomavirus-infected cell, e.g., an HPV-infected cell, e.g.. a low-risk or a high-risk HPV-infected cell, e.g.. and HPV- 16.
- a bovine papillomavirus e.g., a bovine papillomavirus (BPV); inhibiting infection of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31 , or 33, e.g. a bovine papillomavirus (BPV); inhibiting transformation of a cell by a papillomavirus, e.g.. an HPV, e.g.. a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g.
- a bovine papillomavirus e.g. an HPV, e.g.. a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g.
- a papillomavirus e.g.. an HPV, e.g.. a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g. a bovine papillomavirus; inhibiting the growth of, or diminishing the size of a wart.
- HPV high-risk HPV
- HPV-16, -18, -31, or 33 e.g. a bovine papillomavirus
- the E2-BP polypeptide is a fragment of E2-BP 42A which inhibits the E2 transcriptional activation.
- the invention includes an immunogen which includes an E2-BP A polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said E2-BP 42A polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
- the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:5.
- the present invention also includes an antibody preparation specifically reactive with an epitope of the E2-BP 2A immunogen or generally of an E2-BP 42A polypeptide.
- the E2-BP polypeptide differs in amino acid sequence from the amino acid sequence of residues 130-262 of SEQ ID NO:5.
- the E2-BP polypeptide is other than the amino acid sequence 130-262 of SEQ ID NO:5, e.g., it differs from residues 130-262 of SEQ ID NO:5 by at least one amino acid residue, e.g., the E2-BP polypeptide is at least one amino acid residue shorter, one amino acid residue longer, differs in sequence at least at one position, has a different N terminus, or has a different C terminus, as compared with residues 130-262 of SEQ ID NO:5.
- the E2-BP polypeptide is encoded by a nucleic acid which hybridizes to a nucleic acid corresponding to a sequence encoding at least 4 consecutive amino acids, more preferably at least 10 consecutive amino acid residues, and even more preferably at least 20 amino acid residues between residues 1 and 129, or between residues 263 and 357, of SEQ ID NO:5 .
- the E2-BP polypeptide includes at least 1, 2, 3 or 5, and preferably at least 10, 20, or 50 amino acid residues from the region of SEQ ID NO:5 which encodes amino acid residues 1-129, or between residues 263-357.
- the E2-BP polypeptide differs in amino acid sequence from the amino acid sequence of residues 1-134 of SEQ ID NO: 19.
- the E2-BP polypeptide is other than the amino acid sequence 1-134 of SEQ ID NO:19, e.g., it differs from residues 1-134 of SEQ ID NO:19 by at least one amino acid residue, e.g., the E2-BP polypeptide is at least one amino acid residue shorter, one amino acid residue longer, differs in sequence at least at one position, has a different N terminus, or has a different C terminus, as compared with residues 1-134 of SEQ ID NO:19.
- the E2-BP polypeptide differs in amino acid sequence from the amino acid sequence encoded by vector EST 01427 described in WO 93/00353 and deposited with the American Type Culture Collection under the designation EST 01427 and subsequently assigned ATCC deposit number 75915.
- the E2-BP polypeptide is other than the amino acid sequence encoded by vector EST 01427, e.g., it differs from the amino acid sequence encoded by vector EST 01427 by at least one amino acid residue, e.g., the E2-BP polypeptide is at least one amino acid residue shorter, one amino acid residue longer, differs in sequence at least at one position, has a different N terminus, or has a different C terminus, as compared with the amino acid sequence encoded by vector EST 01427.
- the invention features, a E2-BP SD*23 polypeptide, preferably a substantially pure preparation of an E2-BP SD_23 polypeptide, or a recombinant E2-BP SD " 23 polypeptide.
- the polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein;
- the polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence in SEQ ID NO:7;
- the polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:7;
- the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length;
- the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:7;
- the E2-BP SD " 23 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring E
- the E2-BP SD23 polypeptide is encoded by the nucleic acid in SEQ ID NO:3, or by a nucleic acid having at least 60%, 70%, 80%, 90% or 95% homology with the nucleic acid of SEQ ID NO:3.
- a subject E2-BP 23 polypeptide differs in amino acid sequence at 1 , 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NO: 7. The differences, however, are such that: the E2-BP polypeptide exhibits an E2-BP biological activity, e.g., the E2-BP 23 polypeptide retains a biological activity of a naturally occurring E2-BP 23 . e.g.. the E2-BP 23 of SEQ ID NO:7.
- the E2-binding protein is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to E2-BP 23 .
- the second polypeptide portion can be any of glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain.
- the fusion protein can be used in a two-hybrid assay.
- the E2-binding polypeptide has antagonistic activity , and is capable of: suppressing tumor growth, e.g., in a tumor cell in which endogenous E2-BP is misexpressed; suppressing growth of papillomavirus-infected cells, e.g., HPV-infected cells; inhibiting growth of a papillomavirus-infected cell, e.g., an HPV-infected cell, e.g., a low-risk or a high-risk HPV-infected cell, e.g.. and HPV- 16. -18, -31 infected cell, e.g..
- a bovine papillomavirus inhibiting infection of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16. -18. -31 , or 33, e.g..
- a papillomavirus e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16. -18. -31 , or 33, e.g..
- a bovine papillomavirus inhibiting transformation of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g., a human cell, by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g., a bovine papillomavirus; inhibiting the growth of, or diminishing the size of a wart.
- a papillomavirus e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g., a bovine papillomavirus
- the invention includes an immunogen which includes an E2-BP23 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said E2-BP23 polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
- the immunogen includes an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:7.
- the invention also includes an antibody preparation specifically reactive with an epitope of the E2-BP SD " 23 immunogen or generally of an E2-BP SD " 23 polypeptide.
- the invention features, a E2-BP SD - 24 polypeptide, preferably a substantially pure preparation of an E2-BP SD_24 polypeptide, or a recombinant E2-BP SD " 24 polypeptide.
- the polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein;
- the polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence in SEQ ID NO: 8;
- the polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:8;
- the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length;
- the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:8;
- the E2-BP SD " 24 polypeptide is either, an antagonist or an antagonist, of a biological activity of a naturally occurring E
- the E2-BP SD24 polypeptide is encoded by the nucleic acid in SEQ ID NO:4, or by a nucleic acid having at least 60%, 70%, 80%, 90% or 95% homology with the nucleic acid of SEQ ID NO:4.
- the subject E2-BP SD " 24 polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NO:8.
- the differences are such that: the E2-BP polypeptide exhibits an E2-BP biological activity, e.g.. the E2-BP SD*24 polypeptide retains a biological activity of a naturally occurring E2-BP SD " 24 . e.g., the E2-BP S * 24 of SEQ ID NO:8.
- the subject E2-binding protein is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to the E2-BP SD " 24 .
- the second polypeptide portion can be, e.g., any of: glutathione-S-transferase; a DNA domain; or a polymerase activating domain.
- the fusion protein is functional in a two-hybrid assay.
- the invention includes an immunogen which includes an E2-BP SD " 24 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the E2-BP SD " 24 polypeptide, e.g., a humoral response; an antibody response; or a cellular response.
- the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:8.
- the invention also includes an antibody preparation specifically reactive with an epitope of the E2-BP SD*24 immunogen or generally of an E2-BP SD " 24 polypeptide.
- the E2-binding polypeptide has antagonistic activity, and is capable of: suppressing tumor growth, e.g., in a tumor cell in which endogenous E2-BP is misexpressed; suppressing growth of papillomavirus-infected cells, e.g., HPV-infected cells; inhibiting growth of a papillomavirus-infected cell, e.g., an HPV-infected cell, e.g., a low-risk or a high-risk HPV-infected cell, e.g., and HPV-16, -18, -31 infected cell, e.g., a bovine papillomavirus (BPV); inhibiting infection of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g., a
- the invention feature, a E2-BP SD2 " 7 polypeptide, preferably a substantially pure preparation of an E2-BP SD2_7 polypeptide, or a recombinant E2-BP SD2 " 7 polypeptide.
- the polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein;
- the polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence in SEQ ID NOS:6 or 21 ;
- the polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NOS:6 or 21 ;
- the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length;
- the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NOS:6 or 21 ;
- the E2-BP SD2 " 7 polypeptide is either, an antagonist
- the E2-BP SD2 " 7 polypeptide is an agonist or antagonist of the regulation of E2 function, e.g. of E2 -mediated transcription of PV oncogenes.
- the E2-BP SD2 " 7 polypeptide is encoded by the nucleic acid in SEQ ID NOS:2 or 20, or by a nucleic acid having at least 60%. 70%, 80%, 90% or 95% homology with the nucleic acid of SEQ ID NOS:2 or 20.
- the subject E2-BP SD2 " 7 polypeptide differs in amino acid sequence at 1 , 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NOS:6 or 21.
- the differences are such that: the E2-BP polypeptide exhibits an E2-BP biological activity, e.g., the E2-BP SD2 ' 7 polypeptide retains a biological activity of a naturally occurring E2-BP SD2 " 7 , e.g.. the E2-BP SD2 ' 7 of SEQ ID NOS:2 or 20.
- the subject E2-binding protein is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g.. a second polypeptide portion having an amino acid sequence unrelated to the E2-BP SD2 " 7 .
- the second polypeptide portion can be e.g., any of: glutathione-S-transferase; a DNA domain; or a polymerase activating domain.
- the fusion protein is functional in a two-hybrid assay.
- the invention includes an immunogen which includes an E2-BP SD2 " 7 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the E2-BP SD2 " 7 polypeptide, e.g., a humoral response; an antibody response; or a cellular response.
- the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NOS:6 or 21.
- the invention also includes an antibody preparation specifically reactive with an epitope of the E2-BP SD2 " 7 immunogen or generally of an E2-BP SD2 " 7 polypeptide.
- the E2-binding polypeptide has antagonistic activity, and is capable of: suppressing tumor growth, e.g., in a tumor cell in which endogenous E2-BP is misexpressed; suppressing growth of papillomavirus-infected cells, e.g., HPV-infected cells; inhibiting growth of a papillomavirus-infected cell, e.g., an HPV-infected cell, e.g., a low-risk or a high-risk HPV-infected cell, e.g., and HPV-16, -18, -31 infected cell, e.g., a bovine papillomavirus (BPV); inhibiting infection of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g., a
- a papillomavirus e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g., a bovine papillomavirus; inhibiting the growth of, or diminishing the size of a wart.
- HPV high-risk HPV
- HPV-16, -18, -31, or 33 e.g., a bovine papillomavirus
- Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an E2-BP 42A polypeptide.
- the encoded polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein; the encoded polypeptide has an amino acid sequence at least 60%. 80%. 90% or 95% homologous to the amino acid sequence in SEQ ID NO:5; the encoded polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:5; the encoded polypeptide is at least 5, 10, 20. 50. 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20.
- the encoded E2-BP 2A polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring E2-BP.
- the encoded E2-BP 42A polypeptide is an agonist or antagonist of the regulation of E2 function, e.g. of E2-mediated transcription of PV oncogenes.
- an exemplary E2-BP which is antagonistic of E2 transcriptional activation is the portion of the 42A clone initially isolated, 357 amino acid residues long, in the E2-mediated two hybrid assay. (When expressed without the VP16 domain (i.e. not as a VP16/42A fusion protein), the 42A polypeptide was able to inhibit transactivation of the reporter construct by the wild-type E2 protein.)
- the nucleic acid is that of SEQ ID NO: 1 ; the nucleic acid is at least 60%, 70%, 80%, 90% or 95% homologous with the nucleic acid sequence of SEQ ID NO:l .
- the encoded polypeptide has antagonistic activity, and is preferably capable of: suppressing tumor growth, e.g., in a tumor cell in which endogenous E2-BP is misexpressed; suppressing growth of papillomavirus-infected cells, e.g., HPV- infected cells; inhibiting growth of a papillomavirus-infected cell, e.g., an HPV-infected cell, e.g., a low-risk or a high-risk HPV infected cell, e.g., and HPV- 16, -18, -31, or -33 infected cell, e.g., a bovine papillomavirus (BPV)-infected cell; inhibiting infection of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or
- a bovine papillomavirus inhibiting transformation of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or -33, e.g., a bovine papillomavirus; or inhibiting immortalization of a cell, e.g., a human cell, by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV- 16, -18, -31, or -33, e.g., a bovine papillomavirus; inhibiting the growth of, or diminishing the size of a wart.
- a papillomavirus e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16,
- the encoded E2-BP 2A polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NO:5.
- the differences are such that: the E2-BP encoded polypeptide exhibits an E2-BP biological activity, e.g., the encoded E2-BP 2A polypeptide retains a biological activity of a naturally occurring E2-BP 42A , e.g., the E2-BP 42A of SEQ ID NO:5.
- the encoded polypeptide is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to the E2-BP 2A .
- the second polypeptide portion can be glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain.
- the fusion protein can be used in a two-hybrid assay.
- the subject E2-BP 2A nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the E2-BP 42A gene sequence, e.g., to render the E2-BP 2A gene sequence suitable for use as an expression vector.
- a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the E2-BP 42A gene sequence, e.g., to render the E2-BP 2A gene sequence suitable for use as an expression vector.
- the nucleic acid which encodes an E2-BP 2A polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO: 1 ; more preferably to at least 20 consecutive nucleotides of SEQ ID NO:l ; more preferably to at least 40 consecutive nucleotides of SEQ ID NO: 1.
- the E2-BP encoding nucleic acid sequence encodes amino acid residues 40-259 of SEQ ID NO:5.
- the nucleic acid encodes a peptide which differs by at least one amino acid residue from the region of 130-262 of SEQ ID NO:5.
- the nucleic acid differs by at least one nucleotide from the nucleotide sequence which encodes amino acids 130-262 of SEQ ID NO:5.
- the E2-BP encoding nucleic acid is other than a nucleic acid sequence which encodes the amino acid sequence 130-262 of SEQ ID NO:5, e.g., it differs from a sequence which encodes residues 130-262 of SEQ ID NO:5 by at least one nucleotide, e.g., the E2-BP encoding nucleic acid is at least one nucleotide shorter, one nucleotide longer, differs in sequence at least one position, has a different 5' terminus, or has a different 3' terminus, as compared with a sequence which encodes residues 130-262 of SEQ ID NO:5.
- the E2-BP encoding nucleic acid hybridizes to a nucleic acid corresponding to a sequence encoding at least 4 consecutive amino acids, more preferably at least 10 consecutive amino acid residues, and even more preferably at least 20 amino acid residues between residues 1 and 129, or between residues 263 and 357, of SEQ ID NO:5.
- the E2-BP encoding nucleic acid sequence includes at least 1, 2, 3 or 5, and preferably at least 10, 20, 50, or 100 nucleotides from the region of SEQ ID NO:5 which encodes amino acid residues 1-129, or between residues 263-357, of SEQ ID NO:5.
- the nucleic acid encodes a peptide which differs by at least one amino acid residue from the region of 1 -134 of SEQ ID NO: 19.
- the nucleic acid differs by at least one nucleotide from the nucleotide sequence which encodes amino acids 1-134 of SEQ ID NO:19.
- the E2-BP encoding nucleic acid is other than a nucleic acid sequence which encodes the amino acid sequence 1-134 of SEQ ID NO: 19. e.g.. it differs from a sequence which encodes residues 1 -134 of SEQ ID NO: 1 by at least one nucleotide. e.g.. the E2-BP encoding nucleic acid is at least one nucleotide shorter, one nucleotide longer, differs in sequence at least one position, has a different 5' terminus, or has a different 3' terminus, as compared with a sequence which encodes residues 1-134 of SEQ) ID NO: 19.
- the nucleic acid encodes a peptide which differs by at least one amino acid residue from the amino acid sequence encoded by vector EST 01427 described in WO 93/00353 and deposited with the American Type Culture Collection under the designation ESR 01427 and subsequently assigned ATCC deposit number 75915.
- the nucleic acid differs by at least one nucleotide from the nucleotide sequence in vector EST 01427 described in WO 93/00353 and deposited with the American Type Culture Collection under the designation EST 01427 and subsequently assigned ATCC deposit number 75915.
- the E2-BP encoding nucleic acid is other than a nucleic acid sequence which encodes the amino acid sequence encoded by vector EST 01427, e.g., it differs from the sequence encoded by vector EST 01427 by at least one nucleotide, e.g., the E2-BP encoding nucleic acid is at least one nucleotide shorter, one nucleotide longer, differs in sequence at least one position, has a different 5' terminus, or has a different 3' terminus, as compared with a sequence encoded by vector EST 01427.
- the encoded polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein; the encoded polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence in SEQ ID NO:7, the encoded polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:7; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO: 7; the encoded E2-BP SD " 23 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring E2-BP.
- the encoded E2-BP SD " 23 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring E2-BP.
- the nucleic acid is that of SEQ ID NO:3; the nucleic acid is at least 60%, 70%, 80%, 90% or 95% homologous with the nucleic acid sequence of SEQ ID NO:3.
- the encoded polypeptide has antagonistic activity, and is preferably capable of: suppressing tumor growth, e.g., in a tumor cell in which endogenous E2-BP is misexpressed; suppressing growth of papillomavirus-infected cells, e.g.. HPV- infected cells; inhibiting growth of a papillomavirus-infected cell, e.g., an HPV-infected cell, e.g..
- a low-risk or a high-risk HPV infected cell e.g., and HPV- 16, -18, -31, or -33 infected cell, e.g., a bovine papillomavirus (BPV)-infected cell; inhibiting infection of a cell by a papillomavirus.
- a papillomavirus e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV- 16, -18, -31 , or -33, e.g.
- a bovine papillomavirus inhibiting transformation of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV- 16, -18, -31 , or -33, e.g., a bovine papillomavirus; or inhibiting immortalization of a cell, e.g., a human cell, by a papillomavirus, e.g., an HPV, e.g...
- a papillomavirus e.g., an HPV, e.g.
- HPV high-risk HPV
- HPV-16, -18, -31, or -33 e.g., a bovine papillomavirus
- the encoded E2-BP SD " 23 polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NO:7.
- the differences are such that: the encoded E2-BP polypeptide exhibits an E2-BP biological activity, e.g., the encoded E2-BP SL%23 polypeptide retains a biological activity of a naturally occurring E2-BPSD-23, e .g., the E2-BP SD " 23 of SEQ ID NO:7.
- the encoded polypeptide is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to the E2-BP SL 23 .
- the second polypeptide portion can be glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain.
- the fusion protein can be used in a two-hybrid assay.
- the subject E2-BP SD " 23 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the E2-BP S " 23 gene sequence, e.g., to render the E2-BP SD " 23 gene sequence suitable for use as an expression vector.
- the nucleic acid which encodes an E2-BP polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:3; more preferably to at least 20 consecutive nucleotides of SEQ ID NO:3; more preferably to at least 40 consecutive nucleotides of SEQ ID NO:3.
- Another aspect of the present invention provides a substantially pure nucleic acid having a nucleotide sequence which encodes an E2-BP SD " 24 polypeptide.
- the encoded polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein; the encoded polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence in SEQ ID NO:8; the encoded polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:8; the encoded polypeptide is at least 5.
- the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:8;
- the encoded E2-BP SD " 24 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring E2-BP.
- the encoded E2-BP SD " 24 polypeptide is an agonist or antagonist of the regulation of E2 function, e.g. of E2-mediated expression of PV oncogenes.
- the nucleic acid is that of SEQ ID NO:4; the nucleic acid is at least 60%, 70%, 80%. 90% or 95% homologous with the nucleic acid sequence of SEQ ID NO:4.
- the encoded polypeptide has antagonistic activity, and is preferably capable of: suppressing tumor growth, e.g.. in a tumor cell in which endogenous E2-BP is misexpressed; suppressing growth of papillomavirus-infected cells, e.g., HPV- infected cells; inhibiting growth of a papillomavirus-infected cell, e.g.. an HPV-infected cell, e.g..
- a low-risk or a high-risk HPV infected cell e.g., and HPV- 16. -18. -31, or -33 infected cell, e.g., a bovine papillomavirus (BPV)-infected cell; inhibiting infection of a cell by a papillomavirus, e.g., an HPV. e.g., a high-risk HPV, e.g.. and HPV-16, -18, -31, or -33, e.g., a bovine papillomavirus (BPV); inhibiting transformation of a cell by a papillomavirus, e.g., an HPV. e.g..
- a high-risk HPV e.g., and HPV-16. -18. -31. or -33.
- a bovine papillomavirus or inhibiting immortalization of a cell, e.g.. a human cell, by a papillomavirus, e.g.. an HPV, e.g., a high-risk HPV, e.g., and HPV- 16, -18, -31, or -33, e.g., a bovine papillomavirus; inhibiting the growth of, or diminishing the size of a wart.
- the encoded E2-BP SD " 24 polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NO:8. The differences, however, are such that: the encoded E2-PB polypeptide exhibits an E2-BP biological activity, e.g., the encoded E2-BP SD_24 polypeptide retains a biological activity of a naturally occurring E2-BP SD " 24 , e.g., the E2-BP SD " 24 of SEQ ID NO:4.
- the encoded polypeptide is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to the E2-BP SD " 24 .
- the second polypeptide portion can be glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain.
- the fusion protein can be used in a two-hybrid assay.
- the subject E2-BP SD " 24 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the E2-BP SD " 24 gene sequence, e.g., to render the E2-BP SD*24 gene sequence suitable for use as an expression vector.
- a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence
- operably linked to the E2-BP SD " 24 gene sequence e.g., to render the E2-BP SD*24 gene sequence suitable for use as an expression vector.
- the nucleic acid which encodes an E2-BP polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:4; more preferably to at least 20 consecutive nucleotides of SEQ ID NO:4; more preferably to at least 40 consecutive nucleotides of SEQ ID NO:4.
- the encoded polypeptide has biological activity, e.g., it specifically binds a papillomavirus E2 protein; the encoded polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence in SEQ ID NOS:6 or 21 ; the encoded polypeptide has an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NOS:6 or 21; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide comprises at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NOS:6 or 21 ; the encoded E2-BP SD2 " 7 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally
- the nucleic acid is that of SEQ ID NOS:2 or 20; the nucleic acid is at least 60%, 70%, 80%, 90% or 95% homologous with the nucleic acid sequence of SEQ ID NOS:2 or 20.
- the encoded polypeptide has antagonistic activity, and is preferably capable of: suppressing tumor growth, e.g..
- papillomavirus-infected cells e.g., HPV- infected cells
- inhibiting growth of a papillomavirus-infected cell e.g., an HPV-infected cell, e.g., a low-risk or a high-risk HPV infected cell, e.g., and HPV-16, -18, -31, or -33 infected cell, e.g., a bovine papillomavirus (BPV)-infected cell
- BPV bovine papillomavirus
- the encoded E2-BP SD2 " 7 polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from the sequence in SEQ ID NOS:6 or 21. The differences, however, are such that: the encoded E2-BP polypeptide exhibits an E2-BP biological activity, e.g., the encoded E2-BP SD2 " 7 polypeptide retains a biological activity of a naturally occurring E2-BP SD2 " 7 , e.g., the E2-BP SD2 " 7 of SEQ ID NOS:6 or 21.
- the encoded polypeptide is a recombinant fusion protein having a first E2-BP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to the E2-BP SD2 " 7 .
- the second polypeptide portion can be glutathione-S-transferase; a DNA binding domain; or a polymerase activating domain.
- the fusion protein can be used in a two-hybrid assay.
- the subject E2-BP SD2 " 7 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the E2-BP SD2 " 7 gene sequence, e.g.. to render the E2-BP SD2 " 7 gene sequence suitable for use as an expression vector.
- a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence
- the nucleic acid which encodes an E2-BP polypeptide of the invention hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NOS:2 or 20; more preferably to at least 20 consecutive nucleotides of SEQ ID NOS:2 or 20; more preferably to at least 40 consecutive nucleotides of SEQ ID NOS.2 or 20.
- the invention also provides a probe or primer which includes a substantially purified oligonucleotide.
- the oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of one of SEQ ID NOS:l-4 and 20. or naturally occurring mutants thereof.
- the probe or primer further includes a label group attached thereto.
- the label group can be. e.g.. a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor.
- Probes of the invention can be used as a part of a diagnostic test kit for identifying transformed cells, such as for measuring in a sample of cells isolated from a patient, a level of a nucleic acid encoding one of the subject E2-binding proteins; e.g. measuring the E2-BP mRNA level in a cell; e.g. determining whether the genomic E2-BP gene has been mutated or deleted.
- the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
- the invention features a purified preparation of cells, e.g., cells having an E2-BP transgene, or cells which misexpress an E2-BP.
- the cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells, having an E2-BP transgene, e.g., a cell which includes (and preferably express) a heterologous form of one of the E2-BP genes described herein, e.g.
- a gene derived from humans in the case of a non-human cell
- a gene which misexpress an endogenous E2-BP gene e.g., a cell in which expression of one or more of the subject E2-BP's is disrupted.
- Such a transgenic cell can serve as a model for studying cellular disorders which are related to mutated or mis-expressed E2-BP alleles or for use in drug screening.
- the invention includes a method of evaluating the effect of the expression or misexpression of an E2-BP on any of: a parameter related to the viral life cycle, e.g., transcription, translation, infectivity; a parameter related to transformation, e.g., cell growth; a parameter related to immortalization, e.g., cell growth; a parameter related to unwanted cell proliferation, e.g., the formation of warts or tumors.
- a parameter related to the viral life cycle e.g., transcription, translation, infectivity
- a parameter related to transformation e.g., cell growth
- a parameter related to immortalization e.g., cell growth
- unwanted cell proliferation e.g., the formation of warts or tumors.
- the method includes: providing a transgenic cell having an E2-BP transgene; contacting the cell with a PV, e.g., an HPV; and evaluating the effect of the transgene on the parameter (e.g., by comparing the value of the parameter for a transgenic cell with the value for a control, e.g., a wild type cell).
- a PV e.g., an HPV
- evaluating the effect of the transgene on the parameter e.g., by comparing the value of the parameter for a transgenic cell with the value for a control, e.g., a wild type cell.
- invention features, a transgenic non-human animal, e.g., a rodent, e.g.. a mouse or a rat, a rabbit, or a pig, having an E2-BP transgene, e.g., an animal which includes (and preferably express) a heterologous form of one of the E2-BP genes described herein, e.g.. a gene derived from humans, or a gene which misexpress an endogenous E2-BP gene, e.g., an animal in which expression of one or more of the subject E2-BP's is disrupted.
- an E2-BP transgene e.g., an animal which includes (and preferably express) a heterologous form of one of the E2-BP genes described herein, e.g.. a gene derived from humans, or a gene which misexpress an endogenous E2-BP gene, e.g., an animal in which expression of one or more of the subject E2-BP's is disrupted.
- Such a transgenic animal can serve as a model for studying cellular disorders which are related to mutated or mis-expressed E2-BP alleles or for use in drug screening.
- the invention includes a method of evaluating the effect of the expression or misexpression of an E2-BP on any of: a parameter related to the viral life cycle, e.g., transcription, translation, infectivity; a parameter related to transformation, e.g.. cell growth; a parameter related to immortalization, e.g., cell growth; a parameter related to unwanted cell proliferation, e.g., the formation of warts or tumors.
- the method includes: providing a transgenic animal having an E2-BP transgene; contacting the animal with a PV, e.g., an HPV; and evaluating the effect of the transgene on the parameter (e.g., by comparing the value of the parameter for a transgenic animal with the value for a control, e.g.. a wild type animal).
- the invention features a method for evaluating a test compound, e.g., for the ability to modulate an interaction, e.g., to inhibit an interaction of an E2-BP polypeptide with a papillomavirus E2 protein.
- the method includes the steps of (i) combining a viral E2 protein (or preferably a purified preparation thereof), an E2-BP(or preferably a purified preparation thereof), e.g., an E2-BP of the invention (e.g., a protein expressed from one of the clones selected from the group 42A, SD-23, SD-24 or SD2-7), and a test compound, e.g., under conditions wherein in the absence of the test compound the E2 protein and the E2 -binding protein are able to interact, e.g., to form a complex; and (ii) detecting the interaction, e.g., detecting the formation (or dissolution) of a complex which includes the E2 protein and the E2-binding protein.
- a viral E2 protein or preferably a purified preparation thereof
- an E2-BP of the invention e.g., a protein expressed from one of the clones selected from the group 42A, SD-23, SD-24 or SD2-7
- a test compound
- a change, e.g., a decrease or increase, in the formation of the complex in the presence of a test compound (relative to what is seen in the absence of the test compound) is indicative of a modulation, e.g., an inhibition or promotion, of the interaction between the E2 protein and the E2-binding protein.
- the E2 protein is an HPV E2 protein, e.g. from a high-risk HPV, e.g. from HPV- 16, - 18, -31 or -33 ;
- the E2 protein is a BPV E2 protein;
- the E2 protein and the E2- binding protein are combined in a cell-free system and contacted with the test compound; i.e.
- the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the E2-binding protein are simultaneously expressed in a cell, and the cell is contacted with the test compound, e.g. the E2 -binding protein comprise an interaction trap assay (e.g., a two-hybrid assay).
- the E2 -binding protein comprise an interaction trap assay (e.g., a two-hybrid assay).
- the invention features a two-phase method (having an in vitro and an in vivo phase) for evaluating a test compound, e.g., for the ability to modulate, e.g., to inhibit or promote, an interaction of an E2-BP polypeptide with a papillomavirus E2 protein.
- the method includes: (i) combining, in vitro, a viral E2 protein (or preferably a purified preparation thereof), an E2-BP (or preferably a purified preparation thereof), e.g., an E2-BP of the invention (e.g., a protein expressed from one of the clones selected from the group 42 A. SD-23.
- SD-24 or SD2-7) a test compound, e.g., under conditions wherein in the absence of the test compound the E2 protein and the E2-binding protein are able to interact, e.g.. form a complex; (ii) detecting an interaction, e.g., the formation (or dissolution) of a complex which includes the E2 protein and the E2-binding protein (a change, e.g.. a decrease, in the interaction, e.g..
- test compound relative to what is seen in the absence of the test compound being indicative of a modulation, e.g., an inhibition, of the interaction between the E2 protein and the E2-binding protein
- a modulation e.g., an inhibition, of the interaction between the E2 protein and the E2-binding protein
- determining if the test compound modulates the interaction in vitro and if so e.g., administering the test compound to a cell or animal; and evaluating the in vivo effect of the compound on an interaction, e.g., inhibition, of an E2-BP polypeptide with a papillomavirus E2 protein, e.g., by the effect on cell growth or by the effect on the expression of a PV gene or a reporter gene.
- the E2 protein is an HPV E2 protein, e.g. from a high-risk HPV, e.g. from HPV-16, -18, -31 or -33; the E2 protein is a BPV E2 protein.
- the invention features a method of evaluating the ability of a compound to modulate any of: the viral life cycle, e.g., transcription, translation, infectivity; the ability of a PV to effect transformation of a cell; the ability of a PV to effect immortalization of a cell; or unwanted cell proliferation, e.g., the formation of warts or tumors.
- the viral life cycle e.g., transcription, translation, infectivity
- the ability of a PV to effect transformation of a cell e.g., the ability of a PV to effect immortalization of a cell
- unwanted cell proliferation e.g., the formation of warts or tumors.
- the method includes the steps of (i) combining a viral E2 protein (or preferably a purified preparation thereof), an E2-BP (or preferably a purified preparation thereof), e.g., an E2-BP of the invention (e.g., a protein expressed from one of the clones selected from the group 42A, SD-23, SD-24 or SD2-7), and a test compound, e.g., under conditions wherein in the absence of the test compound, the E2 protein and the E2-binding protein are able to interact; and (ii) detecting the interaction, e.g., the formation (or dissolution) of a complex which includes the E2 protein and the E2-binding protein.
- a viral E2 protein or preferably a purified preparation thereof
- an E2-BP of the invention e.g., a protein expressed from one of the clones selected from the group 42A, SD-23, SD-24 or SD2-7
- a test compound e.g., under conditions wherein in the absence of the
- a change, e.g., a decrease, in the formation of the complex in the presence of a test compound is indicative of the ability to modulate one of: the viral life cycle, e.g., transcription, translation, infectivity; the ability of a PV to effect transformation of a cell; the ability of a PV to effect immortalization of a cell; or unwanted cell proliferation, e.g., the formation of warts or tumors.
- the E2 protein is an HPV E2 protein, e.g. from a high-risk HPV, e.g.
- the E2 protein is a BPV E2 protein
- the E2 protein and the E2-binding protein are combined in a cell-free system and contacted with the test compound; i.e. the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture
- the E2-binding protein are simultaneously expressed in a cell, and the cell is contacted with the test compound, e.g. the E2-binding protein comprise an interaction trap assay (e.g., a two hybrid assay).
- Contacting cells with agents that alter the formation of one or more E2-BP/E2 complexes can inhibit pathological progression of papillomavirus infection, such as preventing or reversing the formation of warts, e.g. Plantar warts (verruca plantaris), common warts (verruca plana), Butcher's common warts, flat warts, genital warts (condyloma acuminatum), or epidermodysplasia verruciformis; as well as treating papillomavirus cells which have become, or are at risk of becoming, transformed and/or immortalized, e.g. cancerous, e.g. a laryngeal papilloma, a focal epithelial, a cervical carcinoma.
- warts e.g. Plantar warts (verruca plantaris), common warts (verruca plana), Butcher's common warts, flat warts, genital warts
- the invention features a two-phase method (having an in vitro and an in vivo phase) of evaluating the ability of a compound to modulate on any of: the viral life cycle, e.g., transcription, translation, infectivity; the ability of a PV to effect transformation of a cell; the ability of a PV to effect immortalization of a cell; or unwanted cell proliferation, e.g., the formation of warts or tumors.
- the method includes (i) combining, in vitro, a viral E2 protein (or preferably a purified preparation thereof), an E2-BP (or preferably a purified preparation thereof), e.g., an E2-BP of the invention (e.g.
- the E2 protein is an enzyme that catalyzes the oxidation of a protein to form a complex that includes the E2 protein and the E2-binding protein and if the compound has an effect on formation or dissolution; (iii) administering the test compound to a cell or animal; and (iv) evaluating the in vivo effect of the compound on any of: the viral life cycle, e.g., transcription, translation, infectivity; the ability of a PV to effect transformation of a cell; the ability of a PV to effect immortalization of a cell; or unwanted cell proliferation, e.g., the formation of warts or tumors, e.g., by the effect on cell growth or by the effect on the expression of a PV gene.
- the E2 protein is an enzyme that cata protein that has been modified by the formation of warts or tumors.
- HPV e.g. from HPV-16, -18, -31 or -33; the E2 protein is a BPV E2 protein.
- the invention features a method of evaluating the ability of an E2-BP peptide to modulate propagation of a virus including: (i) providing a cell; (ii) administering an E2-BP to the cell; and (ii) contacting the cell with a PV; and (iii) determining the effect of the E2-BP on a a parameter related to the propagation of a virus, e.g., viral replication, transcription, translation, transformation, or immortalization.
- a parameter related to the propagation of a virus e.g., viral replication, transcription, translation, transformation, or immortalization.
- the E2-BP is: E2-BP 42A , E2-BP SE) - 23 , E2-BP SD - 24 or E2-BP SD2 -7.
- the invention features a method of evaluating the ability of a compound to interact with, e.g., to bind, an E2-BP, e.g., E2-BP 42A , E2-BP SD " 23 , E2-BP SD " 24 or BPSD2-7 ⁇ ne method includes: providing the E2-BP (preferably in purified form); contacting the E2-BP with the compound; and determining whether the compound can interact with. e.g. bind to the E2-BP. The method can be used to identify compounds which interrupt the interaction of E2 with an E2-BP.
- E2-BP e.g., E2-BP 42A , E2-BP SD " 23 , E2-BP SD " 24 or BPSD2-7
- ne method includes: providing the E2-BP (preferably in purified form); contacting the E2-BP with the compound; and determining whether the compound can interact with. e.g. bind to the E2-BP.
- the method can be used to identify compounds which interrupt the interaction of E2 with an E2-BP
- the ability of the compound to interact with the E2-BP can be evaluated in a variety of ways, e.g., by determining the ability of the compound to bind to immobilized E2-BP, or by determining the ability of the compound to prevent or disrupt binding of a ligand. e.g.. and anti E2-BP antibody or E2, to the E2-BP.
- a ligand e.g.. and anti E2-BP antibody or E2
- E2-BP proteins are thought to interact with cellular and/or viral components, thus compounds which interact with E2-BP's maybe useful for studying or modulating, e.g.. inhibiting or promoting, viral related processes, e.g., compounds maybe useful for studying or modulating unwanted cell proliferation; compounds maybe useful for studying or treating the formation of warts or tumors, or for studying the effect on cell growth, or the effect on the expression of a PV gene.
- the invention features, a method of determining the presence of. or evaluating the stage of an E2-BP related disorder in a subject mammal, e.g., a primate, e.g., a human.
- the method includes: evaluating expression of an E2-BP in the subject, e.g.. by obtaining a sample from the subject, e.g., a blood sample, and evaluating that sample for the expression of an E2-BP, e.g.. an E2-BP 42A . an E2-BP S °- 23 . an E2-BP S1 24 or an BP SD2" 7 .
- Non-wild type expression of E2-BP is indicative of an E2-BP related disorder.
- Another aspect of the present invention provides a method of determining if a subject mammal, e.g., a primate, e.g., a human, is at risk for a disorder characterized by unwanted cell proliferation.
- the method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of: (i) an alteration affecting the integrity of a gene encoding an E2 -binding protein, e.g., a protein represented by one of SEQ ID NOS:5-8 and 21, or a homolog thereof; or (ii) the mis-expression of a gene encoding a protein represented by one of SEQ ID NOS:5-8 and 21 or a homolog thereof.
- detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from an E2-BP gene, an addition of one or more nucleotides to an E2-BP gene, a substitution of one or more nucleotides of an E2-BP gene, a gross chromosomal rearrangement of an E2-BP gene, a gross alteration in the level of a messenger RNA transcript of an E2-BP gene, aberrant modification of an E2-BP gene, such as of the methylation pattern of the genomic DNA, the presence of a non-wild type splicing pattern of a messenger RNA transcript of an E2-BP gene, a non-wild type level of an E2- binding protein, and inappropriate post-translational modification of an E2 -binding protein.
- the assay can detect point mutational events in one or more of an E2-BP coding sequences or regulatory regions, such as in the promoter of an E2-BP gene.
- the subject method can detect chromosomal abnormalities, e.g., abnormalities characterized by extra or missing individual chromosomes. extra or missing portions of a chromosome (segmental duplications or deletions), breaks, rings and chromosomal rearrangements.
- the diagnostic assay can be used to detect rearrangement, amplification and/or deletion of an E2-BP gene, or a portion thereof.
- detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence of one of SEQ ID NOS: l -4 and 20. or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the E2-BP gene: ( ii ) exposing the probe/primer to nucleic acid of the tissue; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion: e.g.
- detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the E2-BP gene and, optionally, of the flanking nucleic acid sequences; e.g. wherein detecting the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR); e.g. wherein detecting said lesion comprises utilizing the probe/primer in a ligation chain reaction (LCR).
- the level of said protein is detected in an immunoassay using an antibody which is specifically immunoreactive with. e.g.. a protein represented by one of SEQ ID NOS:5-8 and 21.
- the invention also provides a method for treating a subject mammal, e.g.. a primate, e.g., a human, having unwanted cell growth characterized by a loss of wild-type function of one or more of the subject E2-binding proteins.
- the method includes: administering a therapeutically effective amount of an agent able to inhibit the interaction of the E2-binding protein with other cellular or viral proteins.
- the method comprises administering a nucleic acid construct encoding a polypeptides represented in one of SEQ ID NOS.5-8 and 21, under conditions wherein the construct is incorporated by cells deficient in that E2-binding protein, and under conditions wherein the recombinant gene is expressed, e.g., by gene therapy techniques.
- the therapeutic agent can be an antibody, such as a single chain antibody, or an antisense construct. Such agents can be delivered in formulations amenable to cellular transport, or in the form of a gene therapy construct.
- the invention features, a mutant E2 polypeptide, preferably a substantially pure preparation of a mutant E2 polypeptide, or a recombinant mutant E2 polypeptide, which can bind E2 responsive elements in the PV genome, but which is defective in the activation of the transcription of genes which are under transcriptional control of E2.
- the mutant E2 results in a reduction of at least 25%, more preferably 50%, and even more preferably 75% or 90% of the activation of a PV ordinarily under the control of an E2 polypeptide.
- the mutation is a point mutation; the mutation is in the first 10, 20, 50, 100, 150, or 250 amino acids of a naturally occurring E2; the E2 mutant has an amino acid change, preferably a non conservative amino acid change, at a residue between amino acid residues 15 and 208 of E2, inclusive; the mutation is at any of the following amino acid residues: Q 15 H, D 24 A, K 25 E, E 39 G, A 46 E, F 87 S, W 92 R, W 99 C, P 106 S, N 127 Y, Y 138 H, W 145 R, or R 208 G; the mutant E2 polypeptide is other than a truncation mutant, e.g., other than a truncation in which the first 10, 20, 50, or 100 N-terminal amino acids of a naturally occurring E2 are deleted; the mutant E2 polypeptide is encoded by nucleic acid which can hybridize under stringent conditions to nucleic acid which encodes first 10, 20, 50, or 100 amino acids of a
- the mutant E2 polypeptide is a deletion mutant, wherein, preferably less than 10 nucleotides, more preferably less than 5 nucleotides, even more preferably less than 3 nucleotides of a naturally occurring E2 are deleted.
- the polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence of a naturally occurring E2 polypeptide; the polypeptide is at least 5. 10, 20, 50. 100. or 150 amino acids in length; the polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50. 100.
- the mutant E2 polypeptide disrupts the biological activity of a naturally occurring E2 polypeptide.
- the mutant E2 polypeptide can exert an antiviral effect by interfering with E2-mediated transcription of PV oncogenes.
- the mutant E2 polypeptide is encoded by the nucleic acid of a wild type E2. or by a nucleic acid having at least 60%. 70%, 80%. 90% or 95% homology with the nucleic acid of the wild type E2.
- the mutant E2 polypeptide competes with wild type E2 for binding to E2 responsive elements in the PV genome, and is capable of: suppressing tumor growth, e.g., in a tumor cell in which endogenous E2 is expressed; suppressing growth of papillomavirus-infected cells, e.g., HPV-infected cells; inhibiting growth of a papillomavirus- infected cell, e.g., an HPV-infected cell, e.g..
- a high-risk HPV-infected cell e.g., and HPV- 16, -18, -31 infected cell, e.g., a bovine papillomavirus (BPV); inhibiting infection of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or 33, e.g., a bovine papillomavirus (BPV); inhibiting transformation of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV- 16, -18, -31, or 33, e.g., a human cell, by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g.,
- the invention includes an immunogen which includes a mutant E2 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said mutant E2 polypeptide, e.g., a humoral response, an antibody response, or a cellular response.
- the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by mutant E2 polypeptide.
- the present invention also includes an antibody preparation specifically reactive with an epitope of a mutant E2 immunogen, or generally of a mutant E2 polypeptide. In a preferred embodiment the antibody does not cross react with a wild type E2 polypeptide or with the E2 truncation polypeptide referred to herein.
- the invention features, a substantially pure nucleic acid , or a recombinant nucleic acid, which encodes a mutant E2 polypeptide which can bind E2 responsive elements in the PV genome, but which is defective in the activation of the transcription of genes which are under transcriptional control of E2.
- the mutant E2 results in a reduction of at least 25%. more preferably 50%. and even more preferably 75% or 90% of the activation of a PV ordinarily under the control of an E2 polypeptide.
- the mutation is a point mutation; the mutation is in the first 10.
- the E2 mutant has an amino acid change, preferably a non conservative amino acid change, at a residue between amino acid residues 15 and 208 of E2, inclusive; the mutation is at any of the following amino acid residues: Q 15 H, D 24 A, K 25 E, E 39 G, A 46 E, F 87 S, W 92 R. W
- the mutant E2 encoded polypeptide is other than a truncation mutant, e.g., other than a truncation in which the first 10, 20. 50. or
- the mutant E2 encoded polypeptide is encoded by nucleic acid which can hybridize under stringent conditions to nucleic acid which encodes first 10. 20, 50, or 100 amino acids of a naturally occurring E2.
- the mutant E2 encoded polypeptide is a deletion mutant, wherein, preferably less than 10 nucleotides. more preferably less than 5 nucleotides. even more preferably less than 3 nucleotides of a naturally occurring E2 are deleted.
- the encoded polypeptide has an amino acid sequence at least 60%, 80%, 90% or 95% homologous to the amino acid sequence of a naturally occurring E2 polypeptide; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded polypeptide includes at least 5, preferably at least 10, more preferably at least 20, more preferably at least 50, 100, or 150 contiguous amino acids from sequence of the naturally occurring E2 polypeptide; the mutant E2 encoded polypeptide disrupts the biological activity of a naturally occurring E2 polypeptide.
- the mutant E2 polypeptide can exert an antiviral effect by interfering with E2-mediated transcription of PV oncogenes.
- the nucleic acid is that of naturally occurring E2; the nucleic acid is at least 60%, 70%, 80%, 90% or 95% homologous with the nucleic acid sequence of naturally occurring E2.
- the encoded polypeptide competes with wild type E2 for binding to E2 responsive elements in the PV genome, and is preferably capable of: suppressing tumor growth, e.g., in a tumor cell in which endogenous E2 is expressed; suppressing growth of papillomavirus-infected cells, e.g., HPV-infected cells; inhibiting growth of a papillomavirus-infected cell, e.g., an HPV-infected cell, e.g., a high-risk HPV infected cell, e.g., and HPV-16, -18, -31, or -33 infected cell, e.g., a bovine papillomavirus (BPV)-infected cell; inhibiting infection of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18,
- a bovine papillomavirus e.g., a bovine papillomavirus (BPV); inhibiting transformation of a cell by a papillomavirus, e.g., an HPV, e.g., a high-risk HPV, e.g., and HPV-16, -18, -31, or -33, e.g., a bovine papillomavirus; or inhibiting immortalization of a cell, e.g., a human cell, by a papillomavirus, e.g., an HPV, e.g., a high- risk HPV, e.g., and HPV-16, -18, -31, or -33, e.g., a bovine papillomavirus; inhibiting the growth of, or diminishing the size of a wart.
- a papillomavirus e.g., an HPV, e.g
- the subject mutant E2 nucleic acid will include a transcriptional regulatory sequence, e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the mutant E2 gene sequence, e.g.. to render the mutant E2 gene sequence suitable for use as an expression vector.
- a transcriptional regulatory sequence e.g., at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the mutant E2 gene sequence, e.g.. to render the mutant E2 gene sequence suitable for use as an expression vector.
- the invention also provides a method for treating a subject mammal, e.g., a primate, e.g., a human, having a disorder characterized by the presence of PV, e.g., a disorder characterized by unwanted cell proliferation: or by the interaction of an E2 protein with the PV genome.
- the method includes: administering a therapeutically effective amount of the mutant E2 polypeptide, or a fragment thereof, to a subject mammal, e.g., a primate, e.g.. a human.
- the method comprises administering a nucleic acid construct encoding a mutant E2 polypeptide. under conditions wherein the construct is incorporated by cells in which PV genes are under transcriptional control of a wild type E2. and under conditions wherein the recombinant gene is expressed, e.g. by gene therapy techniques.
- FIG. 1 is the general map of the pEJA plasmid used to construct the VP16/cDNA fusion gene for expression in a two hybrid assay.
- Figure 2 is a schematic diagram illustrating the interaction of E2 and E2 variants with 42A as defined by in vivo and in vitro binding assays.
- Figure 3 is a schematic diagram of the ⁇ -galactosidase reporters pLG-178. pL72 and pLg-312.
- Figure 4 is a schematic diagram illustrating the interaction of 42A deletions with
- Figure 5 is a schematic diagram illustrating E2, 42a transcriptional activation data in S. cervisiae.
- the papillomaviruses are infectious agents that can cause benign epithelial tumors, or warts, in their natural hosts. Infection with specific human papillomaviruses (HPV) is also associated with human epithelial malignancies, including malignancies of the uterine cervix, genitalia, skin and less frequently, other sites.
- HPV human papillomaviruses
- the analysis of the temporal expression of mRNAs and the nucleotide sequence of human and animal papillomavirus (PV) genomes has revealed overall structural similarities in their genetic organization. There are at least seven open reading frames (ORFs) in all PVs, and differential RNA splicing provides the capability to produce several additional proteins.
- E2 for "early”
- L the genes that are required for viral replication
- E2 open reading frame plays a key role in the regulation of the viral cycle, affecting both transcription and replication.
- E2 proteins can activate or repress viral promoters apparently by several distinct mechanisms, though binding to conserved DNA motifs in the viral genome is a common requirement.
- the E2 protein appears to provide both positive and negative feedback loops for viral gene expression, and appears to be part of a necessary mechanism for achieving long-term persistence of infection.
- E2 proteins of various papillomaviruses Although amino acid sequence homology among E2 proteins of various papillomaviruses is low (typically about 35 percent), the E2 proteins share conserved motifs that constitute unique structural domains having distinct functions.
- the C- terminal domain of the E2 protein is responsible for recognition of E2 binding sites on viral DNA.
- the N-terminal domain of the E2 protein is responsible for transcriptional activation following binding of homodimers of the E2 protein to viral DNA.
- the large size of the N-terminal transactivation domain has led us to predict that this domain defines multiple interactions with other cellular proteins, e.g. transcriptional factors, the interplay with which is a prerequisite for physiological responses, persistence, and cellular specificity of papillomavirus infections.
- a two hybrid assay (an example of a two hybrid assay can be found in U.S. Patent Serial No: 5,283,173) was used to identify human cellular proteins which interact with the viral E2 protein and which are candidate proteins participation in PV infectivity and/or transformation.
- the two hybrid system used to screen cDNA libraries for E2 interactors includes an E2 responsive ⁇ -galactosidase ⁇ lac Z) reporter construct and an expression construct encoding a transactivation defective E2 protein.
- the transactivation defective E2 mutants used were derived by screening a library of mutants containing point mutations in the N-terminal portion of the protein. The E2 mutants isolated retained DNA binding activity despite an inability to activate transcription. Activation of the E2 responsive reporter occurred only when a protein containing a functional transcriptional activation domain interacted with the defective E2 protein as a complex bound to the E2- responsive elements in the reporter gene.
- a yeast strain expressing the transcriptionally defective E2 and harboring the E2-responsive reporter construct was transformed with a library of yeast shuttle vector plasmid in which randomly primed HeLa cell cDNA was inserted C-terminal to the strong VP 16 transcription activation domain. Interaction of VP16/cD A fusion proteins with the defective E2 protein recruits the VP 16 transcriptional activation domain to the E2 binding sites and activates expression of the lacZ gene. This assay was used to isolate a number of human genes which encode proteins that specifically interact with E2.
- Embodiments of the invention derive, in part, from the discovery that in addition to other viral proteins such as El, the papillomavirus protein E2 can also associate with several cellular proteins (hereinafter termed "cellular E2- binding proteins" or "E2-BPs"), which association appears to be important to the pathogenesis of papillomavirus infection and papillomavirus-mediated disease states.
- E2-BPs cellular E2- binding proteins
- association of an E2-binding protein with E2 could be important for regulating expression of the viral oncogenes, e.g. E5, E6 or E7, or for providing a mechanism to ensure specificity of infection based on cell-type (e.g. epithelial) and stage of differentiation of infected tissue.
- E2-binding proteins may also function in the regulation of E2- mediated replication of the viral genome. Consequently, the interaction of E2 with one or more E2 -binding proteins may be significant in the modulation of cellular homeostasis of infection and/or neoplastic transformation.
- embodiments of the invention provide diagnostic and therapeutic assays and reagents for detecting and treating papillomavirus- infected cells.
- E2-BP's of the invention can be used as the basis of assays to identify agents which alter, e.g. decrease, the ability of a particular E2-binding protein to bind a papillomavirus E2 protein and thereby, through modulation, e.g., inhibition, of E2-BP/E2 complexes, modulate, e.g., inhibit papillomavirus infection, transformation and/or immortalization.
- agents can be of use therapeutically to prevent E2-BP/E2 complexes in cells infected by, for example, human papillomaviruses, e.g.
- agents can be identified which inhibit formation of E2-BP E2 complexes in other PVs, such as bovine papillomaviruses (BPVs), e.g. BPV-1 , or cottontail rabbit papillomaviruses (CRPVs).
- BPVs bovine papillomaviruses
- CPPVs cottontail rabbit papillomaviruses
- Contacting cells with agents that alter the formation of one or more E2-BP/E2 complexes can inhibit pathological progression of papillomavirus infection, such as preventing or reversing the formation of warts, e.g. Plantar warts (verruca plantaris). common warts (verruca plana), Butcher's common warts. flat warts, genital warts (condyloma acuminatum). or epidermodysplasia verruciformis; as well as treating papillomavirus cells which have become, or are at risk of becoming, transformed and/or immortalized, e.g. cancerous, e.g. a laryngeal papilloma, a focal epithelial, a cervical carcinoma.
- warts e.g. Plantar warts (verruca plantaris). common warts (verruca plana), Butcher's common warts. flat warts, genital warts
- nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
- the term "gene” or “recombinant gene” refers to a nucleic acid including an open reading frame encoding an E2-binding polypeptide, including both exon and (optionally) intron sequences.
- Exemplary recombinant genes encoding the subject E2- binding proteins are represented by any one of SEQ ID NOS: 1 -4 and 20.
- recombinant genes encoding each of the subject E2-binding proteins can be isolated from ATCC deposit No:75915, as described below.
- the term “intron” refers to a DNA sequence present in a given E2-BP gene which is not translated into protein and is generally found between exons.
- the term “transfection” means the introduction of a nucleic acid, e.g. , an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
- "Transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of the E2-binding protein of the present invention or where anti-sense expression occurs from the transferred gene, the expression of a naturally- occurring form of the E2-binding protein is disrupted.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
- Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked.
- Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
- expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
- plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
- vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
- Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
- transcription of a recombinant E2-BP gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of the E2 -binding protein.
- tissue-specific promoter means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as cells of a epithelial lineage, e.g. cervical squamous cells.
- gene constructs can be used as a part of gene therapy to deliver, for example, an E2-BP antagonist in order to modulate levels of E2/E2-BP complexes comprising one of the subject E2-binding proteins in papillomavirus-mediated disorders, e.g.
- papillomas or to direct expression of an antisense construct of one of the subject E2 -binding proteins in only epithelial tissue.
- the term also covers so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
- a purified preparation of cells refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cell, it consists of a preparation of at least 10% or more preferably 50% of the subject cells.
- a "transgenic animal” is any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
- the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
- transgenic animal In the typical transgenic animals described herein, the transgene causes cells to express a recombinant form of one of the subject E2-BP proteins, e.g. either agonistic or antagonistic forms.
- transgenic animals in which the recombinant E2-BP gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below.
- transgenic animal also includes those recombinant animals in which gene disruption of one or more E2-BP genes is caused by human intervention, including both recombination and antisense techniques.
- non-human animals include vertebrates such as rodents, non- human primates, sheep, dog, cow. chickens, amphibians, reptiles, etc.
- Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
- transgenic amphibians such as members of the Xenopus genus
- transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
- chimeric animal is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal.
- tissue-specific chimeric animal indicates that one of the recombinant E2-BP genes is present and/or expressed or disrupted in some tissues but not others.
- transgene means a nucleic acid sequence (encoding, e.g., one or more E2-BP's), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
- a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
- Genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity.
- the term "DNA sequence encoding an E2-binding protein" may thus refer to one or more genes within a particular individual.
- certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
- Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- the term "antibody” in its various grammatical forms is art-recognized and includes immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen.
- the simplest naturally occurring antibody comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
- the light chains exist in two distinct forms called kappa (K) and lambda ( ⁇ ).
- K kappa
- ⁇ lambda
- Each chain has a constant region (C) and a variable region (V).
- Each chain is organized into a series of domains.
- the light chains have two domains, corresponding to the C region and the other to the V region.
- the heavy chains have four domains, one corresponding to the V region and three domains (1 ,2 and 3) in the C region.
- the naturally occurring antibody has two arms (each arm being an Fab region), each of which comprises a V L and a V H region associated with each other. It is this pair of V regions (V L and V H ) that differ from one antibody to another (owing to amino acid sequence variations).
- the variable domains for each of the heavy and light chains have the same general structure, including four framework regions (FRs). whose sequences are relatively conserved, connected by three hypervariable or complementarity determining regions (CDRs).
- variable region of each chain can typically be represented by the general formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
- the CDRs for a particular variable region are held in close proximity to one and other by the framework regions, and with the CDRs from the other chain and which together are responsible for recognizing the antigen and providing an antigen binding site (ABS).
- ABS antigen binding site
- binding fragments encompassed within the term antibody include (i) the Fab fragment consisting of the NL, V H , CL and CHI domains; (ii) the Fd fragment consisting of the V H and C ⁇ j domains; (iii) the Fv fragment consisting of the VL and V ⁇ domains of a single arm of an antibody, (iv) the dAb fragment (Ward et al., (1989) Nature 341 :544-546 ) which consists of a V H domain; (v) isolated CDR regions; and (vi) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
- a “chimeric protein” or “fusion protein” is a fusion of an acid sequence encoding one of the subject E2-binding proteins with an amino acid sequence defining a domain foreign to and not substantially homologous with any domain of the subject E2-BP.
- a chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
- E2-binding protein refers to nucleic acid sequences which have arisen naturally in an organism, including naturally occurring mutants.
- the term also refers to nucleic acid sequences which, while derived from a naturally occurring E2-BP, have been altered by mutagenesis. as for example, combinatorial mutagenesis described below, yet still encode polypeptides which have at least one activity of an E2-binding protein.
- Such evolutionarily derived E2-binding proteins preferred by the present invention are at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in either SEQ ID ⁇ O:5. SEQ ID NO:6.
- SEQ ID NO:7 SEQ ID NO:8 or SEQ ID NO:21.
- Polypeptides having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence shown in any of SEQ ID NOS:5-8 and 21 are also within the scope of the invention.
- nucleic acid As described herein, one aspect of the invention features an isolated (or recombinant) nucleic acid which includes a nucleotide sequence encoding one of the subject E2-binding proteins, and/or equivalents of such nucleic acids.
- nucleic acid as used herein can include fragments and equivalents.
- equivalent refers to nucleotide sequences encoding functionally equivalent E2-binding proteins or functionally equivalent peptides which, for example, retain the ability to bind to E2.
- Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions.
- allelic variants will, therefore, include sequences that differ from the nucleotide sequence of E2-binding proteins shown in any of SEQ ID NOS: 1-4 and 20 due to the degeneracy of the genetic code. Equivalents will also include nucleotide sequences that hybridize under stringent conditions (i.e., equivalent to about 20-27°C below the melting temperature (T m ) of the DNA duplex formed in about IM salt) to the nucleotide sequence of E2 -binding proteins represented in SEQ ID NOS: 1-4 and 20, or to the nucleotide sequence of an E2 -binding protein from the pRS306-E2BP library (ATCC accession No:75915).
- stringent conditions i.e., equivalent to about 20-27°C below the melting temperature (T m ) of the DNA duplex formed in about IM salt
- equivalents will further include nucleic acid sequences derived from and evolutionarily related to, a nucleotide sequences shown in any of SEQ ID NOS: 1-4 and 20.
- isolated or substantially pure as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.
- An isolated or substantially pure nucleic acid protein preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks that sequence in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence.
- isolated or substantially pure as used herein also refers to a nucleic acid that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
- isolated or substantially pure nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
- the nucleic acid is a cDNA encoding a peptide having at least one activity of a subject E2-binding protein.
- the nucleic acid is a cDNA molecule comprising at least a portion of the nucleotide sequence represented in one of SEQ ID NOS: 1 -4 and 20. A preferred portion of these cDNA molecules includes the coding region of the gene.
- E2-binding proteins which function in a limited capacity as one of either an E2-BP agonists or an E2-BP antagonist, in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein.
- specific biological effects can be elicited by treatment with a homolog of limited function, and with fewer side effects relative to treatment with agonists or antagonists which are directed to all of an E2-binding proteins biological activities.
- E2-BP homologs can be generated, as described below, which interfere with the function of the viral E2 protein but which do not substantially interfere with the normal cellular function of the wild-type E2-binding protein.
- homologs of the subject E2-binding proteins can be generated by mutagenesis. such as by discrete point mutation(s). or by truncation of a nucleic acid encoding a naturally occurring form of the protein. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the E2-BP from which it was derived.
- antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to E2.
- a protein has E2-BP biological activity if it has one or more of the following properties: the ability to modulate gene expression, e.g., expression of a PV gene, or replication, e.g., of a PV genome; the ability to modulate the efficacy of papillomavirus infection, e.g., human papillomaviruses, e.g., infection by HPV-l ⁇ , HPV-18, HPV-31 or HPV-33; the ability to affect the efficacy of cell transformation, e.g., PV-mediated transformation, e.g., PV-mediated transformation, e.g., high risk HPV -mediated transformation; the ability to affect the efficacy of cellular immortalization, e.g., PV- mediated transformation, e.g., HPV-mediated transformation, e.g., high risk HPV-mediated immortalization; or the ability to bind a PV E2 protein, e.g., an HPV E2 protein, e.g.,
- Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
- the recombinant E2-BP genes can include, in addition to nucleotides encoding the amino acid sequences shown in SEQ ID NOS: 1-4 and 20, additional nucleotide sequences which encode amino acids at the C-terminus and N-terminus of each protein.
- a recombinant E2-BP gene can include nucleotide sequences of a PCR fragment generated by amplifying one of the coding sequences for one of the E2-BP clones of ATCC deposit No:75915 using sets of primers derived from Table 1 below.
- Another aspect of the invention provides a nucleic acid which hybridizes under high or low stringency conditions to a nucleic acid which encodes a peptide having all or a portion of an amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6. SEQ ID NO:7. SEQ ID NO:8 or SEQ ID NO:21.
- Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1- 6.3.6.
- the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
- the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
- Nucleic acids having a sequence that differs from the nucleotide sequence shown any of SEQ ID NOS: 1 -4 and 20 due to degeneracy in the genetic code are also within the scope of the invention.
- Such nucleic acids encode functionally equivalent peptides (i.e., a peptide having a biological activity of an E2-binding protein) but differ in sequence from the sequence shown in said sequence listings due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in "silent" mutations which do not affect the amino acid sequence of the E2-binding protein.
- DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject E2-binding proteins will exist among vertebrates.
- these variations in one or more nucleotides (up to e.g., about 3-5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of an E2-binding protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention.
- a fragment of the nucleic acid encoding the active portion of an E2 -binding protein refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of an E2-binding protein but which nevertheless encodes a peptide having an E2- BP biological activity, e.g. the fragment retains the ability to bind to E2 .
- Nucleic acid fragments within the scope of the present invention include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species for use in screening protocols to detect E2-BP homologs, as well as those capable of hybridizing with nucleic acids from human specimens for use in detecting the presence of a nucleic acid encoding one of the subject E2-BPs, including alternate isoforms, e.g. mRNA splicing variants. Nucleic acids within the scope of the invention may also contain linker sequences, modified restriction endonuclease sites and other sequences useful for molecular cloning, expression or purification of recombinant forms of the subject E2-binding proteins.
- a nucleic acid encoding a peptide having an activity of an E2-binding protein may be obtained from mRNA present in eukaryotic cells. It should also be possible to obtain nucleic acids encoding E2-binding proteins of the present invention from genomic DNA obtained from both adults and embryos.
- a gene encoding an E2-binding protein can be cloned from either a cDNA or a genomic library in accordance with protocols herein described, as well as those known to persons skilled in the art.
- a cDNA encoding one of the subject E2-binding proteins can be obtained by isolating total mRNA from a cell, e.g. a mammalian cell, e.g.
- the nucleic acid of the invention can be DNA or RNA.
- a preferred nucleic acid is a cDNA represented by the sequence shown in SEQ ID NO: 1.
- Another nucleic acid is a cDNA represented by the sequence shown in SEQ ID NOS: 2 and 20.
- nucleic acids include cDNA molecules represented by the sequences shown in one of SEQ ID NOS:3-4.
- a preferred nucleic acid is a cDNA derived from the pRS306-E2BP library (ATCC deposit No:75915).
- This invention also provides expression vectors containing a nucleic acid encoding a peptide having an activity of an E2-binding protein, operably linked to at least one transcriptional regulatory sequence.
- Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
- Regulatory sequences are art-recognized and are selected to direct expression of the peptide having an activity of an E2 -binding protein.
- transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
- any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the E2-binding proteins of this invention.
- useful expression control sequences include, for example, the early and late promoters of S V40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the tip system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase.
- the major operator and promoter regions of phage lambda the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast alpha -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
- the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
- the expression vector includes a recombinant gene encoding a peptide having an activity of a subject E2-binding protein, or alternatively, encoding a peptide which is an antagonistic form of the subject E2-binding protein.
- Such expression vectors can be used to transfect cells and thereby produce proteins or peptides. including fusion proteins or peptides, encoded by nucleic acids as described herein.
- the gene constructs of the invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of one of the E2-binding proteins, or alternatively, an antisense construct or an antibody (e.g., sFv) against one of the subject E2-binding proteins.
- another aspect of the invention features expression vectors for in vivo transfection and expression of an E2-binding protein, an antisense nucleic acid to an E2-BP gene sequence, and/or an antibody selective for an E2-BP gene product, in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of one of the subject E2-binding proteins in a cell.
- such constructs can be used to inhibit PV-infection by interfering with the biological function of E2.
- a "therapeutic gene” refers to a nucleic acid which may provide, e.g., a coding sequence for an E2-binding protein or an antibody against an E2-binding protein, or the nucleic acid may be provide, upon transcription in a targeted cell, an E2-BP antisense sequence.
- the therapeutic gene constructs may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the gene to cells in vivo.
- Approaches include insertion of the subject therapeutic gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
- Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO- ⁇ . precipitation carried out in vivo.
- transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g., locally or systemically.
- the particular gene construct provided for in vivo transduction of E2-BP expression may also be useful for in vitro transduction of cells, such as for use in the diagnostic assays described above.
- a preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing the therapeutic gene, e.g.. a cDNA, encoding an E2-binding protein. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
- Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
- a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
- the development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
- recombinant retro virus can be constructed in which part of the retroviral coding sequence ⁇ gag, pol, env) has been replaced by nucleic acid encoding one of the subject receptors rendering the retrovirus replication defective.
- the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology. Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
- retroviruses examples include pLJ, pZIP, pWE and pEM which are known to those skilled in the art.
- suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am.
- Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
- retroviral vectors as a gene delivery system for the therapeutic genes, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses. and therefore of stable introduction of the recombinant gene, is that the target cells must be dividing. With certain exceptions, such as lymphatic cancers, this requirement will not be a hindrance to use of retroviral vectors.
- gene therapy constructs of the present invention are intended to be delivered to papillomavirus-transformed cells, such limitation on infection can be beneficial in that the tissue (e.g., nontransformed cells) surrounding the target cells do not likely undergo as extensive cell division and is therefore somewhat refractory to infection with retroviral vectors.
- retroviral-based vectors it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications W093/25234 and WO94/06920).
- strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Man et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al. (1983) Virology 163:251-254); or coupling cell surface receptor ligands to the viral env proteins (Neda et al.
- Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g., single-chain antibody/e/iv fusion proteins).
- a protein or other variety e.g., lactose to convert the env protein to an asialoglycoprotein
- fusion proteins e.g., single-chain antibody/e/iv fusion proteins
- retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the therapeutic gene from the retroviral vector.
- adenovirus- derived vectors The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.
- Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are known to those skilled in the art.
- adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral
- DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal. thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
- the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra: Haj-Ahmand and Graham (1986) J. Virol. 57:267).
- adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80 % of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, E.J. Murray. Ed. (Humana. Clifton, NJ, 1991) vol. 7. pp. 109-127).
- Expression of the inserted therapeutic gene can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
- MLP major late promoter
- Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
- AAV adeno-associated virus
- AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
- a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell.
- non-viral methods can also be employed to cause expression of the therapeutic gene in the tissue of an animal.
- Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
- non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject therapeutic gene by the targeted cell.
- Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
- a gene encoding one of the subject E2-binding proteins can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551 ; PCT publication WO91/06309; Japanese patent application 1047381 ; and European patent publication EP-A- 43075).
- lipofection of papilloma-virus infected epithelial cells can be carried out using liposomes tagged with monoclonal antibodies against, for example, squamous cells.
- the gene delivery systems for the therapeutic gene can be introduced into a patient by methods, known to those skilled in the art.
- a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell- type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
- initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
- the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al.
- an antagonistic form of one of the subject E2-binding proteins such as the fragment of the 42 A clone described in the examples below, can be delivered in a gene therapy construct to PV-infected squamous cells by electroporation using techniques described, for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115.
- a single-chain antibody or E2-BP antisense construct can be provided to the PV-infected cells.
- the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
- the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
- antisense therapy refers to administration or in situ generation of oligonucleotides or their derivatives which specifically hybridizes (e.g. binds) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one of an E2-binding proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation.
- the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
- antisense refers to the range of techniques generally employed in the art. and includes any therapy which relies on specific binding to oligonucleotide sequences.
- an antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes an E2- binding protein.
- the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and or genomic sequences of an E2-BP gene.
- Such oligonucleotide probes are preferably modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and or endonucleases, and is therefore stable in vivo.
- nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659- 2668. Accordingly, the modified oligomers of the invention are useful in therapeutic, diagnostic, and research contexts. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general.
- the oligomers of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences. Meade Publishing Co., Easton, PA.
- systemic administration injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous for injection
- the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
- the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included in the invention.
- the compounds can be administered orally, or by transmucosal or transdermal means.
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- penetrants are known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, and detergents.
- Transmucosal administration may be through nasal sprays or using suppositories.
- the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
- the oligomers of the invention are formulated into ointments, salves, gels, or creams as known in the art.
- the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail herein.
- the antisense constructs of the present invention by antagonizing the normal biological activity of E2-BP, can be used in the manipulation of tissue, both in vivo and in ex vivo tissue cultures.
- the invention includes transgenic animals which include cells (of that animal) which contain an E2-BP transgene and which preferably (though optionally) express (or misexpress) an endogenous or exogenous E2-binding protein in one or more cells in the animal.
- the E2- BP transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs.
- the expression of the transgene is restricted to specific subsets of cells, or tissues utilizing, for example, cis-acting sequences that control expression in the desired pattern.
- such mosaic expression of the subject E2-BP polypeptide can provide a means to assess the effects of.
- Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns.
- Temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
- target sequence refers to a nucleotide sequence that is genetically recombined by a recombinase.
- the target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity.
- Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of the subject E2-BP polypeptide.
- excision of a target sequence which interferes with the expression of a recombinant E2-BP gene can be designed to activate expression of that gene.
- This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the E2-BP gene from the promoter element or an internal stop codon.
- the transgene can be made wherein the coding sequence of the gene is flanked recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element.
- inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.
- cre/loxP recombinase system of bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991 ) Science 251 : 1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences.
- loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
- the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
- genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
- Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage- specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
- the activation expression of the recombinant E2-binding protein can be regulated via control of recombinase expression.
- crelloxP recombinase system to regulate expression of a recombinant E2- binding protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject E2-BP protein.
- Animals containing both the Cre recombinase and a recombinant E2-BP gene can be provided through the construction of "double" transgenic animals.
- a convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., an E2-BP gene and recombinase gene.
- BP transgene in a recombinase-mediated expressible format particularly derives from the likelihood that the subject protein will be deleterious upon expression in the transgenic animal.
- a founder population in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues.
- an antagonistic E2-BP transgene is silent will allow the study of progeny from that founder in which disruption of E2-BP mediated induction in a particular tissue or at developmental stages would result in, for example, a lethal phenotype.
- conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the transgene.
- Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
- expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner. By this method, the E2-BP transgene could remain silent into adulthood until "turned on" by the introduction of the trans-activator.
- the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
- Embryonic target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonic target cell.
- the zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of l-2pl of DNA solution.
- the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic non-human animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
- Retroviral infection can also be used to introduce transgene into a non-human animal.
- the developing non-human embryo can be cultured in vitro to the blastocyst stage.
- the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
- Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida ⁇ Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.
- the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
- transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
- ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. ( 1981 ) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258: Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction.
- Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- Jaenisch, R. 1988) Science 240: 1468-1474.
- the invention includes recombinant E2-binding polypeptides which are encoded by genes derived from eukaryotic organisms, e.g. mammals, e.g., primates, e.g. humans.
- recombinant protein refers to a protein of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding the subject E2 -binding protein is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
- derived from is meant to include within the meaning of "recombinant protein” those proteins having an amino acid sequence of a native E2-BP of the present invention, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring E2-binding protein of a organism.
- Recombinant proteins preferred by the present invention are at least 60% homologous, more preferably 70% homologous and most preferably 80% homologous with an amino acid sequence shown in one of SEQ ID NOS:5-8 and 21.
- Polypeptides having an activity of the subject E2-binding proteins i.e. either agonistic or antagonistic
- having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% homology with a sequence of either in SEQ ID NO:5-8 and 21 are also within the scope of the invention.
- the invention includes recombinant forms of the subject E2 -binding proteins which are encoded by genes derived from a organism and which have amino acid sequences evolutionarily related to an E2-binding protein of either SEQ ID NO:5-8 and 21.
- Such recombinant E2 -binding proteins preferably are capable of functioning in one of either role of an agonist or antagonist of at least one biological activity of the present E2-BPs.
- the E2-BP produced by expression of the nucleic acids of the invention can be obtained from cell culture using conventional techniques, e.g., by inserting the nucleic acid into a cell, culturing the cell, and obtaining the protein from the culture, e.g., from the cultured cell or from the medium in which the cell grows.
- the invention also includes methods of producing the subject E2-binding proteins. For example, a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject E2-binding protein can be cultured under appropriate conditions to allow expression of the peptide to occur. The peptide may be secreted and isolated from a mixture of cells and medium containing the recombinant E2-BP.
- the peptide may be retained cytoplasmically and the cells harvested, lysed and the protein isolated.
- a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
- the recombinant E2-BP peptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion- exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide.
- the recombinant E2-binding protein is a fusion protein containing a domain which facilitates its purification, such as an E2-BP/GST fusion protein.
- the invention includes a host cell transfected to express a recombinant form of at least one of the subject E2-binding proteins.
- the host cell may be any prokaryotic or eukaryotic cell.
- a nucleotide sequence derived from the cloning of the E2-binding proteins of the present invention, encoding all or a selected portion of a protein can be used to produce a recombinant form of an E2-BP via microbial or eukaryotic cellular processes.
- hosts either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells) are standard procedures used in producing other well-known proteins, e.g. insulin, interferons, human growth hormone, IL-1, IL-2, and the like. Similar procedures, or modifications thereof, can be employed to prepare recombinant E2-binding proteins, or portions thereof, by microbial means or tissue-
- the recombinant E2-binding gene can be produced by ligating nucleic acid encoding a subject E2-binding protein, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both.
- Expression vectors for production of recombinant forms of the subject E2-binding proteins include plasmids and other vectors.
- suitable vectors for the expression of an E2-BP include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
- YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83. incorporated by reference herein).
- These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
- drug resistance markers such as ampicillin can be used.
- an E2-binding protein is produced recombinantly utilizing an expression vector generated by sub-cloning a gene encoding the protein from pRS306-E2BP library (ATCC accession No:75915) using, for example, primers based on SEQ ID NO: 1-4 and 20 and or primers based on the flanking plasmid sequence (e.g. the primers represented by SEQ ID NOS: 13- 15 ).
- the preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
- the pcDNAl/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
- viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
- BBV-1 bovine papillomavirus
- pHEBo Epstein-Barr virus
- pHEBo Epstein-Barr virus
- pHEBo Epstein-Barr virus
- baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
- pVL-derived vectors such as pVL1392, pVL1393 and pVL941
- pAcUW-derived vectors such as pAcUWl
- pBlueBac-derived vectors such as the ⁇ -gal containing pBlueBac III
- MAP methionine aminopeptidase
- removal of an N-terminal methionine can be achieved either in vivo by expressing E2-BP-derived polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purified MAP (e.g., procedure of Miller et al., supra).
- MAP e.g., E. coli or CM89 or S. cerevisiae
- purified MAP e.g., procedure of Miller et al., supra.
- the coding sequences for polypeptides of the invention can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide. This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of an E2-binding protein.
- the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the E2-BP polypeptide, either in the monomeric form or in the form of a viral particle.
- the nucleic acid sequences corresponding to the portion of a subject E2-binding protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising a portion of the protein E2-BP as part of the virion.
- the Multiple Antigen Peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of a subject E2-binding protein is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992) J. fmmunol. 148:914).
- Antigenic determinants of the subject E2-binding proteins can also be expressed and presented by bacterial cells.
- fusion proteins can also facilitate the expression of proteins, such as any one of the E2-binding protein of the present invention.
- an E2 -binding protein of the present invention can be generated as a glutathione-S-transferase (GST- fusion protein).
- GST fusion proteins can enable easy purification of the E2 -binding protein, such as by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausabel et al. (N.Y.: John Wiley & Sons, 1991)).
- a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the E2-binding protein, can allow purification of the poly(His)- expressed E2-BP-fusion protein by affinity chromatography using a Ni + metal resin.
- the purification leader sequence can then be subsequently removed by treatment with enterokinase (e.g., see Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht et al. PNAS 88:8972).
- fusion genes are known to those skilled in the art. For example, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger- ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
- the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
- PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
- the invention also includes isolated E2-binding polypeptides which are isolated from, or otherwise substantially free of other cellular or viral proteins, especially papillomavirus proteins, normally associated with the E2-binding protein.
- the term "substantially pure or purified preparations” are defined as E2-BP preparations which are substantially free of at least one polypeptide, polysaccaride, or lipid with which the E2-BP protein naturally occurs with in the cell.
- Preparations which are "substantially free of other cellular or viral proteins" also referred to herein as "contaminating proteins” have less than 20% (by dry weight) contaminating protein, and preferably having less than 5% contaminating protein.
- Functional forms of the subject E2- binding proteins can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
- the subject E2-binding proteins can be isolated by affinity purification using, for example. E2-derivatized matrices. Isolated peptidyl portions of the subject E2-binding proteins can also be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
- fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
- an E2-binding protein of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.
- the fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of an E2-binding protein activity, such as by microinjection assays.
- E2-binding proteins can be modified, e.g., to enhance therapeutic or prophylactic efficacy, or stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo).
- modified peptides when designed to retain at least one activity of the naturally-occurring form of the protein, are considered functional equivalents of the E2- binding protein described in more detail herein.
- modified peptide can be produced, for instance, by amino acid substitution, deletion, or addition.
- Phenylalanine. tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
- the invention includes a method of generating sets of combinatorial mutants of the subject E2-binding proteins, as well as truncation and fragmentation mutants, and is especially useful for identifying potential variant sequences which are functional in binding to a PV E2 protein, especially an E2 protein of a high risk HPV.
- One purpose for screening such combinatorial libraries is, for example, to isolate novel E2-BP homologs which function as one of either an agonist or antagonist of the biological activities of the wild-type ("authentic") protein, or alternatively, possess novel activities all together.
- E2- BP homologs can be engineered by the present method to provide proteins which bind E2 and which block E2-mediated gene transcription in order to inhibit papillomavirus infection, transformation and/or immortalization.
- Such proteins when expressed from recombinant DNA constructs, can be used in gene therapy protocols. Mutagenesis can give rise to E2-BP homologs which have intracellular half-lives significantly different than the corresponding wild-type protein. For example, the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular process which result in destruction of, or otherwise inactivation of, an E2-binding protein.
- E2-BP homologs, and the genes which encode them can be utilized to alter the envelope of expression for the particular recombinant E2 binding protein by modulating the half-life of the recombinant protein.
- a short half-life can give rise to more transient biological effects associated with a particular recombinant E2-BP and, when part of an inducible expression system, can allow tighter control of recombinant protein levels within the cell.
- proteins, and particularly their recombinant nucleic acid constructs can be used in gene therapy protocols.
- the amino acid sequences for a population of E2-BP homologs or other related proteins are aligned, preferably to promote the highest homology possible.
- a population of variants can include, for example, E2- BP homologs from one or more species, or E2-BP homologs from the same species but which differ due to mutation.
- Amino acids which appear at each position of the aligned sequences are selected to create a degenerate set of combinatorial sequences.
- the combinatorial E2-BP library is produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential E2-BP sequences.
- a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential E2-BP sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display) containing the set of E2-BP sequences therein.
- the library of potential E2-BP homologs can be generated from a degenerate oligonucleotide sequence.
- Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes then be ligated into an appropriate gene for expression.
- the purpose of a degenerate set of genes is to provide, in one mixture, all of the sequences encoding the desired set of potential E2-BP sequences.
- the synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA. Proc 3rd Cleveland Sympos.
- Techniques for screening gene products of combinatorial libraries made by point mutations, and for screening cDNA libraries for gene products having a preselected property are known in the art. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of E2-BP homologs.
- the most widely used techniques for screening large gene libraries typically includes cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
- Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate E2-BP sequences created by combinatorial mutagenesis techniques.
- the candidate E2-BP gene products are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an E2 protein, such HPV-16 E2, via this gene product is detected in a "panning assay".
- the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991 ) Bio/Technology 9: 1370-1371 ; and Goward et al. (1992) TIBS 18: 136-140).
- E2 fluorescently labeled E2 can be used to score for potentially functional E2-BP homologs.
- Cells can be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, separated by a fluorescence-activated cell sorter.
- the gene library is expressed as a fusion protein on the surface of a viral particle.
- foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
- coli filamentous phages Ml 3, fd., and fl are most often used in phage display libraries, as either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBOJ 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al (1992) PNAS 89:4457-4461).
- the recombinant phage antibody system (RPAS, Pharmacia Catalog number 27-9400-01) can be easily modified for use in expressing and screening E2-BP combinatorial libraries.
- the pCANTAB 5 phagemid of the RPAS kit contains the gene which encodes the phage gill coat protein.
- the E2-BP combinatorial gene library can be cloned into the phagemid adjacent to the gill signal sequence such that it will be expressed as a gill fusion protein. After ligation, the phagemid is used to transform competent E. coli TGI cells. Transformed cells are subsequently infected with M13KO7 helper phage to rescue the phagemid and its candidate E2-BP gene insert.
- the resulting recombinant phage contain phagemid DNA encoding a specific candidate E2-BP, and display one or more copies of the corresponding fusion coat protein.
- Those phage-di splayed candidate E2-BPs which are capable of binding a E2 are selected or enriched by panning with E2.
- the phage library can be panned on glutathione immobilized E2-GST fusion proteins, and unbound phage washed away from the cells.
- the bound phage is then isolated, and if the recombinant phage express at least one copy of the wild type gill coat protein, they will retain their ability to infect E. coli.
- successive rounds of reinfection of E. coli, and panning will greatly enrich for E2-BP homologs, which can retain an ability to bind E2 which can subsequently be screened for further biological activities in order to differentiate agonists and antagonists.
- E2-BP homologs can be generated and screened using, for example, alanine scanning mutagenesis and the like (Ruf et al. ( 1994) Biochemistry 33:1565-1572: Wang et al. (1994) J. Biol. Chem. 269:3095- 3099; Balint et al. (1993) Gene 137:109-1 18; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601 ; Nagashima et al. (1993) J Biol.
- the invention also provides for reduction of the E2-binding domains of the subject E2-binding proteins to generate mimetics, e.g., peptide or non-peptide agents, which are able to disrupt binding of an E2-BP of the present invention with a papillomavirus E2 protein.
- mimetics e.g., peptide or non-peptide agents
- mutagenic techniques are particularly useful to map the determinants of the E2- BP which participate in protein-protein interactions involved in. for example, binding of the subject E2-binding protein to a PV E2 protein.
- E2 -binding protein which are involved in molecular recognition of E2 can be determined and used to generate E2-BP-derived peptidomimetics which competitively inhibit binding of the E2-BP with E2 (see, for example, "Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein" European patent applications EP-412,762 A and EP- B31,080A).
- peptidomimetic compounds e.g., diazepine or isoquinoline derivatives
- non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R.
- the invention also includes antibodies specifically reactive with one of the subject E2-binding proteins.
- immunogens derived from the present activity E2-binding proteins based on the cDNA sequences
- anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies. A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
- a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., E2-binding protein or an antigenic fragment which is capable of eliciting an antibody response).
- an immunogenic portion of the subject E2-binding proteins can be administered in the presence of adjuvant.
- the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
- Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
- the subject antibodies are immunospecific for antigenic determinants of the E2-binding proteins of the present invention, e.g. antigenic determinants of a protein represented by one of SEQ ID NOS:5-8 and 21 or a closely related human or non-human mammalian homolog (e.g.
- the anti-E2-BP antibodies do not substantially cross react (i.e. react specifically) with a protein which is: e.g., less than 90 percent homologous to one of SEQ ID NOS:5-8 and 21; e.g. less than 95 percent homologous with one of SEQ ID NOS: 5-8 and 21; e.g. less than 98-99 percent homologous with one of SEQ ID NOS:5-8 an 21.
- the antibody has a binding affinity for a non-homologous protein (e.g., E2) which is less than 10 percent, more preferably less than 5 percent, and even more preferably less than 1 percent, of the binding affinity for a protein of SEQ ID NOS:5-8 and 21.
- E2 non-homologous protein
- anti-E2-BP antisera can be obtained and, if desired, polyclonal anti-E2-BP antibodies isolated from the serum.
- antibody producing cells lymphocytes
- myeloma cells can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
- Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an E2-binding protein of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
- antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the subject E2-binding protein.
- Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
- the antibody of the present invention is further intended to include bispecific and chimeric molecules having an anti-E2-BP portion.
- Both monoclonal and polyclonal antibodies (Ab) directed against E2-BP or E2-BP variants, and antibody fragments such as Fab ' and F(ab ' )2, or the single chain antibody sFv, can be used to block the action of E2-BP and allow the study of the role of a particular E2 binding protein of the present invention in papillomavirus infection, transformation and/or immortalization, as well as the normal cellular function of the E2-binding protein, e.g. by microinjection of anti-E2-BP antibodies of the present invention.
- Such antibodies, particularly fragments such as the sFv. can also be used therapeutically.
- Antibodies which specifically bind E2-BP epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of each of the subject E2-binding proteins.
- Anti-E2-BP antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate E2-BP levels in tissue or bodily fluid as part of a clinical testing procedure. For instance. such measurements as the level of E2-BP/E2 complexes can be useful in predictive valuations of the onset or progression of HPV infection.
- the ability to monitor E2- BP levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.
- the level of an E2-binding protein can be measured in cells found in bodily fluid, such as in samples of cerebral spinal fluid, or can be measured in tissue, such as produced by biopsy.
- Diagnostic assays using anti-E2-BP antibodies can include, for example, immunoassays designed to aid in early diagnosis of a neoplastic or hyperplastic disorder, e.g., the presence of cancerous cells in the sample, e.g., PV-infected cells, e.g., PV-transformed cells, e.g., PV-immortalized cells, e.g., to detect cells in which a lesion of the E2-BP gene has occurred.
- anti-E2-BP antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as ⁇ gtl 1, ⁇ gtl 8-23, ⁇ ZAP, and ⁇ ORF8.
- Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
- ⁇ gtl 1 will produce fusion proteins whose amino termini consist of ⁇ -galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
- Antigenic epitopes of a subject E2-BP can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-E2-BP antibodies.
- E2-BP Nucleic Acids can be detected and cloned from other animals, and alternate isoforms (including splicing variants) can be detected and cloned from human sources.
- the nucleotide sequence determined from the cloning of the subject E2-binding proteins from a human cell line will further allow for the generation of probes designed for use in identifying E2-BP homologs in other human cell types, as well as E2-BP homologs from other animals.
- the present invention also provides a probe/primer including a substantially purified oligonucleotide, wherein the oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or anti-sense sequence of one of SEQ ID NOS: 1 -4 and 20, or naturally occurring mutants thereof.
- the probe/primer further comprises a label group attached thereto and able to be detected, e.g. the label group is selected from the group consisting of radioisotopes. fluorescent compounds, enzymes, and enzyme co-factors.
- the label group is selected from the group consisting of radioisotopes. fluorescent compounds, enzymes, and enzyme co-factors.
- Such probes can also be used as a part of a diagnostic test kit for identifying transformed cells, such as for measuring a level of an E2-BP nucleic acid in a sample of cells from a patient; e.g. measuring an E2-BP mRNA level; e.g. determining whether a genomic E2-BP gene has been mutated or deleted.
- Nucleotide probes can be generated from the cloned sequence of the subject E2- binding proteins which allow for histological screening of intact tissue and tissue samples for the presence of an E2-BP mRNA. Similar to the diagnostic uses of anti-E2-BP antibodies. the use of probes directed to E2-BP mRN As, or to genomic E2-BP sequences, can be used f both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, neoplastic or hyperplastic disorders (e.g. unwanted cell growth). Used in conjunction with anti-E2-BP antibody immunoassays, the nucleotide probes can help facilitate the determination of the molecular basis for a developmental disorder which may involve some abnormality associated with expression (or lack thereof) of an E2-binding protein.
- variation in E2-BP synthesis can be differentiated from a mutation in the E2-BP coding sequence.
- Diagnostic Screens The invention further provides methods for determining if a subject is at risk for a disorder characterized by aberrant cell proliferation, differentiation and/or death (e.g., apoptosis).
- the subject method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gen encoding an E2-binding protein, or (ii) the mis-expression of the E2-BP gene.
- such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an E2-BP gene, (ii) an addition of one or more nucleotides to an E2-BP gene, (iii) a substitution of one or more nucleotides of an E2-BP gene, (iv) a gross chromosomal rearrangement of an E2-BP gene, (v) a gross alteration in th level of a messenger RNA transcript of an E2-BP gene, (vii) aberrant modification of an E2- BP gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a no wild type splicing pattern of a messenger RNA transcript of an E2-BP gene, (viii) a non-wil type level of an .
- E2-binding protein and (ix) inappropriate post-translational modification of an E2-binding protein.
- point mutational events may occur not only in coding sequences, but also in regulatory regions, such as in the promoter of the gene, leading to los or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the E2-BP gene product.
- Chromosomal abnormalities can also be of several types, including extra or missing individual chromosomes, extra or missing portions of a chromosome (segmental duplications or deletions), breaks, rings and chromosomal rearrangements. Chromosomal rearrangements include translocations. dicentrics.
- the present invention provides a large number of assay techniques for detecting lesions in an E2-BP gene, and importantly, provides the ability to discern between different molecular causes underlying E2-BP-dependent aberrant cell proliferation, differentiation and/or death.
- tissue In order to detect the alteration of the wild-type E2-BP gene in a tissue, it is helpful t isolate the tissue free from surrounding normal tissues.
- Means for enriching a tissue preparation for particular cells are known in the art.
- the tissue may be isolated from paraffin or cryostat sections.
- Cells of certain phenotype may also be separated from larger samples by, for example, flow cytometry. These as well as other techniques for separating cells are well known in the art.
- a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an E2-BP gene, such as represented by any of SEQ ID Nos: 1-13, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject E2-BP genes or naturally occurring mutants thereof.
- the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
- Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
- detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 :1077-1080; and Nakazawa et al. (1944) PNAS 91 :360-364), the later of which can be particularly useful for detecting point mutations in the E2-BP gene.
- PCR polymerase chain reaction
- LCR ligation chain reaction
- the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to an E2-BP gene under conditions such that hybridization and amplification of the E2-BP gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
- nucleic acid e.g., genomic, mRNA or both
- Detection of mismatches with probes of the present invention provides yet another techniques for discovering mutations resulting from, for example, deletions, insertions, inversions, substitutions or frameshift mutations.
- Mismatch detection can be used to detect point mutations in the gene or its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of samples.
- An example of a mismatch cleavage technique is the RNase protection method, which is described in detail in Winter et al. (1985) PNAS 82:7575, 1985; and Meyers et al. (1985) Science 230:1242.
- the method involves the use of a labeled riboprobe which is complementary to the wild-type E2-BP gene coding sequence.
- the riboprobe and either mRNA or DNA isolated from a cell sample are hybridized and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A. it cleaves at the site of the mismatch.
- RNase A cleaves at the site of the mismatch.
- RNA product which is smaller than the full-length duplex RNA for the riboprobe and the mRNA or DNA.
- the riboprobe need not b the full length of the E2-BP mRNA or gene but can be a segment of either.
- DNA probes can be used to detect mismatches, through enzymati or chemical cleavage (see, e.g., Cotton et al. (1988) PNAS 85:4397).
- mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexe relative to matched duplexes (see, e.g., Cariello,(1988) Human Genetics 42:726).
- the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see above) before hybridization.
- DNA sequences of the E2-BP gene which have been amplified by use of polymerase chain reaction may also be screened using allele-specific probes.
- These probes are nucleic acid oligomers, each of which contains a region of the E2-BP gene, e.g., which may harbor known mutation or which may be deleted or amplified in certain disorders. For example, on oligomer may be about 30-60 nucleotides in length, corresponding to a portion of the E2-BP gene sequence.
- Hybridization of allele-specific probes with amplified E2-BP sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the sample tissue as in the allele-specific probe. Alteration of E2-BP mRNA expression can be detected by any technique known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type E2-BP gone.
- chromosome banding techniques are widely used to identify numerical and/or structural chromosome aberrations.
- Current procedures for genetic screening and biological dosimetry can involve the analysis of karyotypes.
- a karyotype is a collection of indices which characterize the state of an organism's chromosomal complement. It includes such things as total chromosome number, copy number of individual chromosome types, and chromosomal morphology, e.g., as measured by length, centromeric index, connectedness, or the like.
- Chromosomal abnormalities can be detected by examination of karyotypes.
- metaphase chromosomes can be stained by a number of cytological techniques to reveal a longitudinal segmentation into entities generally referred to as bands.
- the banding pattern o each chromosome within an organism is unique, permitting unambiguous chromosome identification regardless of morphological similarity, Latt, "Optical Studies ofMetaphase Chromosome Organization, " in Annual Review of Biophysics and Bioengineering. Vol. 5, pgs. 1-37 (1976).
- cloned probes can be used to assess the amount of a given E2-BP gene sequence in a chromosome by Southern blotting. This method is effective even if the genome is heavily rearranged so as to eliminate useful karyotype information.
- Comparative genomic hybridization offers yet another approach to identify the presence and localization of amplified/deleted E2-BP gene sequences. See, for example, Kallioniemi et al. (1992) Science 258:818. CGH, like Southern blotting, reveals amplifications and deletions irrespective of genome rearrangement. Additionally, CGH provides a more quantitative estimate of copy number than Southern blotting, and also provides information of the localization of the amplified or deleted sequence in the normal chromosome.
- FISH fluorescence in situ hybridization
- This procedure basically involves the steps of preparing interphase or metaphase spreads from cells; hybridizing labelled probes to the interphase or metaphase spreads; and visualizing the labeled probes to determine the order and distance between the hybridization sites.
- the probes can be labelled with biotin-dUTP or digoxigenin-dUTP. Using probes with mixed labels allows visualization of space, order and distance between hybridization sites.
- the method is practiced according to U.S. Patent no. 5,472,842, and comprises (i) providing a mapped library of labeled probes specific for a chromosome, which library includes probe(s) for one or more of the subject E2-BP genes; (ii) contacting a chromosome sample from a patient with the probes under conditions in which the probes bind selectively with target polynucleotide sequences in the sample to form hybridization complexes; (iii) detecting the hybridization complexes; and (iv) determining the copy number of each complex.
- the mapped libraries of probes consist of a set of probes which, when hybridized to a normal chromosome, are distributed relatively uniformly across the region of interest, e.g., flanking the E2-BP gene of interest.
- the region typically is one chromosome or a part of one chromosome.
- the library of probes can encompass the entire genome.
- a reference probe which binds selectively to a sequence within the centromere of the preselected chromosome can be used as a control.
- Each probe in the library is hybridized to the normal chromosome in a metaphase spread in situ. The physical location of the probe on the chromosome is determined by visualization of a marker.
- Probe locations are typically expressed as the average fractional length from the p telomere (FLpter). Hybridization spots may be counted by regular fluorescence microscopy to give t copy number as a function of FLpter. The copy number relative to normal cells is then indicative of various chromosome abnormalities such as amplifications, deletions and the like.
- a "chromosome sample” as used herein refers to a tissue or cell sample prepared fo hybridization methods as described herein.
- the sample is prepared such that individual chromosomes remain substantially intact and typically comprises metaphase spreads or interphase nuclei prepared according to standard techniques. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification.
- restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. Such a method is particularly useful for screening among kindre persons of an affected individual for the presence of a mutation found in that individual.
- RFLP restriction fragment length polymorphism
- Single stranded conformation polymorphism (SSCP) analysis can also be used to detect bas change variants of an allele (c.f, Orita et al., (1989) PNAS 86:2766-2770; and Orita et al., (1989) Genomics 5:874-879).
- Other techniques for detecting insertions and deletions as are known in the art can be used.
- Still another embodiment of the method for measuring the gene amplification or deletion is by pulsed field gel electrophoresis.
- This procedure is basically that described by Schwartz et al. (1982) Cold Spring Harbor Sy p., Quant. Biol. 47:189-195.
- the procedure basically comprises running a standard electrophoresis gel (agarose, polyacrylamide or othe gel known to those skilled in the art) under pulsing conditions.
- a standard electrophoresis gel agarose, polyacrylamide or othe gel known to those skilled in the art
- the strength of the field as well as the direction of the field is pulsed and rotated in order to separate megabase DNA molecules.
- Current commercial systems are computer controlled and select the strength, direction and time of pulse depending on the molecular weight of DNA to be separated.
- Bind(s) substantially refers to complementary hybridization between an oligonucleotide and a target sequence and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
- Hybridizing refers the binding of two single stranded nucleic acids via complementary base pairing.
- polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 90% sequence identity, more preferably at least 95%, compared to a reference sequence using the methods described below using standard parameters.
- Alteration of wild-type E2-BP genes can also be detected by screening for alteration in the cellular levels of an E2-binding protein.
- antibodies immunoreactive with an E2-binding protein can be used to screen a cell or tissue sample by standard immunoassay techniques. For instance, the cells of a biopsy sample can be lysed, and the level of an E2- binding protein present in the cell can be quantitated by standard immunoassay techniques. Lack of cognate antigen would indicate an E2-BP mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant E2-BP gene product.
- Such immunological assays can be done in any convenient format known in the art. These include Western blots, irnmunohistochemical assays and ELISA assays. Any means for detecting an altered E2 -binding protein can be used to detect alteration of wild-type E2-BP genes.
- aberrant methylation patterns of an E2-BP gene can be detected by digesting genomic DNA from a patient sample with one or more restriction endonucleases that are sensitive to methylation and for which recognition sites exist in the E2-BP gene (including in the flanking and intronic sequences). See, for example, Buiting et al. (1994) Human Mol Genet 3:893-895. Digested DNA is separated by gel electrophoresis, and hybridized with probes derived from, for example, genomic or cDNA sequences. The methylation status of the E2-BP gene can be determined by comparison of the restriction pattern generated from the sample DNA with that for a standard of known methylation. Other E2-BP Screens
- anti-sense techniques e.g. microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to an E2-BP mRNA or gene sequence
- HPV- mediated events infection, transformation and/or immortalization
- normal cellular function of each of the novel E2-BPs e.g. in regulation of transcription and/or replication, by inhibiting endogenous production of a particular E2-binding protein.
- Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
- the invention includes assays which can be used to screen for drugs which are either agonists or antagonists of the normal cellular function of the subject E2-binding proteins, or of their role in papillomavirus infection.
- the assay evaluates the ability of a compound to modulate binding between an E2-binding protein and an E2 protein, e.g., E2 from a high risk HPV.
- E2 protein e.g., E2 from a high risk HPV.
- the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity with oth proteins or change in enzymatic properties of the molecular target.
- the compound of interest is contacted with an isolated and purified E2-binding protein.
- the mixture of the compound and E2-binding protein is then added to a composition containing a papillomavirus E2 protein but which do not contain E2-BP.
- Detection and quantification of E2 E2-BP complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the E2 protein and the E2-binding protein.
- the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentration of the test compound.
- a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified E2-BP is added to a composition containing the E2 protein, and the formation of E2 E2-BP complex is quantitated in the absence of the test compound.
- Complex formation between the E2-binding protein and an E2 may be detected by a variety of techniques.
- modulation of the formation of complexes can be quantitated using, for example, defectably labeled proteins (e.g. radiolabeled, fluorescently labeled, or enzymatically labeled, by immunoassay, or by chromatographic detection.
- defectably labeled proteins e.g. radiolabeled, fluorescently labeled, or enzymatically labeled, by immunoassay, or by chromatographic detection.
- a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
- glutathione-S-transferase E2 (GST E2) fusion proteins described below, can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the E2-binding protein, e.g.
- an 35 S-labeled E2-binding protein and the test compound and incubated under conditions conducive to complex formation, e.g., at 4°C in a buffer of 2mM Tris-HCl (pH 8 lnM EDTA, 0.5% Nonidet P-40, and lOOmM NaCl .
- the beads are washed to remove any unbound E2-BP, and the matrix bead-bound radiolabel determined directly (e.g. beads placed in scintilant), or in the supemtantant after the E2/E2-BP complex are dissociated.
- the complexes can dissociated from the bead, separated by SDS-PAGE gel, and the level of E2-BP found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
- Other techniques for immobilizing proteins on matrices are also available for use in the subject assay.
- the E2 protein can be immobilized utilizing conjugation of biotin and streptavidin.
- biotinylated E2 can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
- antibodies reactive with E2 can be derivatized to the wells of the plate, and E2 tapped in the wells by antibody conjugation.
- preparations of an E2 -binding protein and a test compound are incubated in the E2-presenting wells of the plate, and the amount of E2/E2-BP complex trapped in the well can be quantitated.
- Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the E2-binding protein, or which are reactive with the E2 protein and compete for binding with the E2-BP; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the E2-binding protein.
- the enzyme can be chemically conjugated or provided as a fusion protein with the E2-binding protein.
- the E2-binding protein can be chemically cross-linked with alkaline phosphatase, and the amount of E2-BP trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. paranitrophenyl phosphate.
- a fusion protein comprising the E2-BP and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
- the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the E2-BP or E2 sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources).
- the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
- Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991 ) JR/o/ Chem 266:21 150-21 157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system
- the present invention further contemplates cell- based and whole animal assays for identifying agents which affect the biological function of a PV E2 protein or an E2-binding protein. While these assay systems can certainly be used as primary screens for initial identification of compounds which alter the binding of the two proteins, the in vivo assays are more typically used as "secondary" screens for further accessing compounds identified in the primary screen (e.g., the "hits"). For example, secondary screens can be used to evaluate the ability of the agent to actually modulate the function of E2 in vivo, as well as to assess the cytotoxicity. bioavailability, and other pharmacokinetic parameters of the test compound.
- the subject E2 -binding proteins can be used to generate an interaction trap assay, as described in the examples below (see also, U.S. Patent No.: 5,283,317; PCT publication WO94/10300; Zervo et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8: 1693-1696), which can be used to detect agents which disrupt or enhance binding of the E2-BP to an E2 protein.
- the interaction trap assay described for the isolation of clone 42 A, as well as the LexA based assay described in the examples, are each forms of the interaction trap assay suitable for screening test compounds.
- Saccharomyces cerevisiae YPB2 cells are transformed simultaneously with a plasmid encoding a G AL4 ad -E fusion and with a plasmid encoding the GAL4 db domain fused to subject E2-BP.
- the strain is transformed such that the GAL4-responsive promoter drives expression of a phenotypic marker.
- the ability to grow in the absence of histidine can depend on the expression of HIS3 gene.
- the HIS3 gene When the HIS3 gene is placed under the control of a GAL4 responsive promoter, relief of this auxotrophic phenotype indicates that a functional GAL4 activator has been reconstituted through the interaction of E2 and the E2-BP. Thus, agent able to inhibit E2-BP interaction with E2 will result in yeast cells unable to grow in the absence of histidine.
- the phenotypic marker e.g. instead of the HIS3 gene
- the invention features an animal model for developmental diseases, which has an E2-BP allele which is misexpressed.
- a mouse can be bred which has an E2-BP allele deleted, or in which all or part of one or more E2-BP exons are deleted. Such a mouse model can then be used to study disorders arising from misexpressed E2-BP genes.
- the E2 mutants defective in transactivation are also useful in disrupting the biological activity of the wild- type E2 protein (Androphy et al., (1987) Nature, 324:70-73), and therefore can be used to alter the regulation of the papillomavirus viral cycle.
- each of the gene constructs described above, particularly those useful for gene therapy can also be used to deliver a gene encoding one of the subject E2 transactivation-defective mutants to a papillomavirus infected cell.
- these E2 mutants can advantageously exe their anti-viral effect by interfering with transcriptional activation of viral genes by native E2 proteins in cells infected with papillomavirus.
- the E2 transactivation repressors of the present invention are characterized by an ability to bind to E2 responsive elements in the PV genome, but an inability to cause activation of transcription of PV genes ordinarily under the transcriptional control of E2. Accordingly, the mutant E2 proteins form complexes consisting of homodimers and heterodimers (e.g. includes a wild- type E2) which compete with homodimers of wild-type E2 for binding to E2 responsive elements in the PV genome.
- This vector contains a BamHl/Kpnl fragment encoding the first 286 amino acids of E2, and was used here because, unlike YEplacl 12GE2, the Accl and Bgll sites in E2 are unique in this vector. Sequences encoding amino acids 1 to 286 for both mutant libraries in M13mpl9E2-286 were transferred to Yeplacl 12GE2 as BamHl/Kpnl fragments placing the mutations in the context of full length E2. The two YEplacl 12GE2 mutant libraries spanning E2 amino acids 1 to 71 and 72 to 133 were used to isolate transactive defective mutants by screening in Saccharomyces cerevesiae.
- a misincorporation library of sequences encoding amino acids 134 to 215 was made using primers EA 37 (5' CCAGGGTGGTAGAGGTG 3')(SEQ ID NO: 16) and EA 107 (5' CCTTCGCTAGCGACCCAGACTC 3')(SEQ ID NO: 17).
- the PCR product was cleaved with Accl and Nhel, and after gel purification was used to construct a library in M13mpl 9E2-286 N .
- This vector is identical to M13mpl9E2-286, except that silent changes at amino acids 215 and 216 have been made creating an Nhel cleavage site.
- This library was transfeffed to the context of full length E2 as was described for the others.
- COS-7 cells were electroporated at .25KV and 960 ⁇ FD. Electroporations into COS-7 cell lines introduced 20 ⁇ g of reporter plasmid, 20 ⁇ g of effector plasmid and 360 ⁇ g of salmon sperm DNA. These conditions and input DNA concentrations were determined to be optimal for COS-7 cell electroporations and are known in the art. Each COS-7 electroporation was plated and fed with fresh media at 24 hours post electroporation. Cell media was assayed for human growth hormone (hGH) concentration at 3 days post-electroporation.
- hGH human growth hormone
- hGH was connected to the E2 responsive promoter in the same manner as in the ⁇ -GAL assay described herein.
- Whole cell extracts were prepared from electroporated COS-7 cells by lysing directly in denaturing buffer. To confirm protein levels, western blots of all mutants reported were performed using the E2 antibody II-2 (EA).
- R 208 G indicates Argenine at position 208 that has been substituted with Glycine at th position.
- YES absolutely conserved across PV's
- Transcriptionally defective E2 point mutants are those that exhibit ⁇ 50% activity of the wild ty ⁇ e E2.
- Another aspect of the present invention concerns a novel in vivo method for the isolation of genes encoding proteins which physically interact with a "bait" protein, e.g. a protein suspected or known to be a molecular target for therapeutic intervention or of diagnostic value.
- the method relies on detecting the reconstitution of a functional transcriptional activator mediated by protein-protein interactions with the bait protein.
- the method makes use of chimeric genes which express hybrid proteins.
- the first hybrid comprises the DNA-binding domain of a papillomavirus E2 protein fused to the bait protein.
- the second hybrid protein contains a transcriptional activation domain fused to a "fish" protein, e.g. a test protein derived from a cDNA library.
- the fish and bait proteins are able to interact, they bring into close proximity the DNA-binding and transcriptional activator domains. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to an E2 transcriptional regulatory site (e.g. an E2 binding site), and expression of the marker gene can be detected and used to score for the interaction of the bait protein with another protein.
- an E2 transcriptional regulatory site e.g. an E2 binding site
- a multiplicity of proteins can be simultaneously tested to determine whether any interact with the bait protein.
- a DNA fragment encoding the E2 DNA-binding domain can be fused to a DNA fragment encoding the bait protein in order to provide one hybrid.
- This hybrid is introduced into the cells carrying the marker gene and the transfected cells caused to express the E2/bait fusion protein.
- a library of plasmids can be constructed which may include, for example, total mammalian complementary DNA (cDNA) fused to the DNA sequence encoding the activation domain. This library is introduced into the cells carrying the first hybrid.
- the method comprises providing a host cell, preferably a yeast cell, more preferably Saccharomyces cerevisiae or Schizosaccharomyces pombe, containing gene constructs encoding each of the chimeric "bait" and "fish” proteins, as well as an E2- responsive reporter gene which, when expressed, causes a phenotypic change in the host cell.
- each of the chimeric proteins is induced (although constitutive expression of one or both of the proteins is also acceptable), and the host cells are isolated or otherwise selectively enriched based on the phenotypic characteristics conferred by the expression of the reporter gene in cells in which the bait and fish chimeras are capable of binding to one and other.
- the reporter gene can provide a protein which confers a drug resistant phenotype (e.g., resistance to chloroamphenicol or neomycin), an enzyme which provides for colorimetric or luminescence detection (e.g., GAL4 or luciferase).
- a gene product which rescues an auxotrophic phenotype e.g., LEU2. HIS3, Ura3 or LYS2. or any cell surface antigen for which antibodies are available (e.g., for panning).
- the method of the present invention may be practiced using a kit for detecting interaction between a first test protein and a second test protein.
- the kit includes a container, two vectors, and a host cell.
- the first vector contains a promoter and may include a transcription termination signal functionally associated with the first chimeric gene in order to direct the transcription of the first gene.
- the first gene includes a DNA sequence that encodes an E2 DNA-binding domain and a unique restriction site(s) for inserting a DNA sequence encoding a first test protein (e.g.. the "bait" protein) or protein fragment in such a manner that the first test protein is expressed as part of a hybrid protein with the E2 DNA-binding domain.
- the first vector also includes a means for replicating itself in the host cell and in bacteria. Also included on the first vector is a first marker gene, the expression of which in the host cell permits selection of cells containing the first marker gene from cells that do not contain the first marker gene.
- the first vector is a plasmid.
- the kit also includes a second vector which contains a second gene.
- the second gene also includes a promoter and a transcription termination signal to direct transcription.
- the second gene also includes a DNA sequence that encodes a transcriptional activation domain and a unique restriction site(s) to insert a DNA sequence encoding the second test protein (e.g., the fish proteins) or protein fragment into the vector, in such a manner that the second test protein is capable of being expressed as part of a hybrid protein with the transcriptional activation domain.
- the transcriptional activation domain of the second hybrid protein is derived from transcriptional activators having separate DNA-binding and transcriptional activation domains.
- transcriptional activators are known to be found in the yeast GAL4 protein, and are also known to be found in the yeast GCN4 and ADR1 proteins.
- the viral VP16 protein is known to have a strong transcriptional activation domain.
- Many other proteins involved in transcription also have separable binding and transcriptional activation domains, the latter of which make them useful for the present invention.
- the second hybrid protein may be encoded on a library of plasmids that contain genomic, cDNA or synthetically generated DNA sequences fused to the DNA sequence encoding the transcriptional activation domain.
- the second vector further includes a means for replicating itself in the host cell and in bacteria.
- the second vector also includes a second marker gene, the expression of which in the host cell permits selection of cells containing the second marker gene from cells that do not contain the second marker gene.
- the kit includes a host cell, preferably a yeast strain of Saccharomyces cerevisiae or Schizosaccharomyces pombe.
- the host cell contains the detectable gene having a binding site for the E2 DNA-binding domain of the first hybrid protein, e.g., is under the transcriptional control of at least one E2-responsive element.
- the E2 binding sites are positioned so that the detectable gene expresses a detectable protein when the gene is activated by the transcriptional activation domain encoded by the second vector. Activation of the gene is possible when the transcriptional activation domain is in sufficient proximity to the detectable gene.
- the host cell by itself, should be incapable of expressing a protein having a function of the first marker gene, the second marker gene, the DNA-binding domain of the E2 protein, or the transcriptional activation domain.
- the interaction of the bait protein and fish protein in the host cell causes a measurably greater expression of the detectable gene than when the E2 DNA-binding domain and the transcriptional activation domain are present but fail to interact.
- the detectable gene may encode an enzyme or other product that can be readily measured. Such measurable activity may include the ability of the cell to grow only when the marker gene is transcribed, or the presence of detectable enzyme activity only when the marker gene is transcribed.
- Various other markers are well known within the skill of workers in the art.
- the cells containing the two hybrid proteins are incubated in an appropriate medium and the culture is monitored for the measurable activity.
- a positive test for this activity is an indication that the bait protein and the fish protein have interacted. Such interaction brings their respective DNA-binding and transcriptional activation domains into sufficiently close proximity to cause transcription of the marker gene.
- VP16/cDNA fusion proteins that can interact with the E2 protein, or that can bind the reporter promoter directly, would recruit the VP16 activation domain to the E2 binding sites and activate expression of the lacZ gene, and these yeast cells would subsequently stain blue on x-gal plates.
- this assay used autonomously replicating yeast plasmids with selectable markers to maintain an E2 responsive ⁇ -galactosidase reporter (pL72, 2 micron, LEU2) and a BPV-1 E2 expression construct ( URA3).
- E2 responsive ⁇ -galactosidase reporter pL72, 2 micron, LEU2
- BPV-1 E2 expression construct URA3
- E2 responsive ⁇ -galactosidase reporter pL72, 2 micron, LEU2
- URA3 BPV-1 E2 expression construct
- the reporter construct contains the E. coli ⁇ -galactosidase (lacZ) gene under control of a minimal yeast CYC 1 promoter to which E2 binding sites (E2RE) were added (Androphy et al. (1987) Nature 325:70-73).
- the lacZ gene is transcribed when E2 binds the E2RE in the promoter and activates transcription, and can be detected as blue color formation in colonies grown on yeast minimal medium (YMM) containing galactose to induce synthesis of E2 and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (XGAL) which is the substrate for lacZ.
- YMM yeast minimal medium
- XGAL 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
- BGWl -7a MATa leu2,2- leu2,-l I;his4-his519,adel-adel00,ura3-ura52) previously described (Guarente and Mason, (1983) Cell 32:1279-1286).
- the TRP 1 gene of BGWl -7a was inactivated by one step gene replacement (Alani al. (1987) Genetics 1 16:541-545).
- the inactivation vector, pNKY1009 was cleaved with EcoRI, and, following phenol: chloroform extraction and ethanol precipitation, was transformed into BGWl -7a using the lithium acetate method (Ito et al. (1983) J Bacteriol
- DBY1 was transformed with YEplacl 12 which contains the TRP1 gene and the 2 micron replicon (Gietz and Sugino (1988) Gene 74: 527-534).
- YEplac 112 transformants of DBY 1 to grow on YMM glucose minus tryptophan, but not those receiving YEplacl 81 (LEU2, 2 micron) DNA confirmed tha the TRP1 gene of DBY1 was inactivated.
- DBY 1 allows the simultaneous presence of the three extrachromosomal replicons required for screening the cDNA library: VP16-HeLa cD A library, URA3; pL72 LacZ reporter, LEU 2; and YEplacl 12GE2, TRP1.
- YEplacl 12GE2 construct contains the same pGAL4-BPVl E2 cassette as does pKpE2. Success of the two hybrid screen requires an E2 molecule with is capable of site specific DNA binding, but unable to activate transcription when bound to the E2RE in the promoter of pL72. As wild type E2 transactivates the pL72 reporter it was necessary to construct mutants of E2 which have these properties.
- a carboxy terminal 85 amino acid (325 to 410) of E2 is capable of site specific DNA binding, and when fused to the LexA DNA binding domain, the amino terminal 300 amino acids of E2 are able to activate transcription of a reporter containing LexA DNA binding sites in yeast.
- the transcriptional activation domain of E2 is less well characterized, but is thought to comprise some or all of amino acids 1 to 161. For this reason we have used region specific mutagenesis to isolate transactivation defective mutants in the amino terminal 161 amino acids of E2.
- a library of mutations affecting amino acids 1 to 150 of E2 was prepared by performing the polymerase chain reaction on YEplacl 12GE2 using primers: EA-59 (5'-, CTCGGATCCCATGGAGACAGCATG-3')(SEQ ID NO: 10) and EA-18 (5'-GCTG CCAGCCGTCCTC-3'XSEQ ID NO:l l) under conditions favoring misincorporation at adenine residues (Leung et al. ( 1989) Method Cell Mol Biol 1 :11-19).
- the PCR product was cleaved with Ace I and Bgl I, and the 186 nt. fragment spanning E2 amino acids 69 to 133 was resolved by electrophoresis in a 1% Tris-acetate-EDTA agarose minigel, cut out of the gel and purified using a Quiex Kit (Quiagen Corp.).
- the Bgl I/Acc I fragment was first inserted into M13mpl9E2-286, which is a bacteriophage M13 vector into which the Bam HI/Kpn I fragment encoding amino acids 1 to 300 from YEplacl 12GE2 was cloned.
- M13mpl9E2-286 is a bacteriophage M13 vector into which the Bam HI/Kpn I fragment encoding amino acids 1 to 300 from YEplacl 12GE2 was cloned.
- the Ace I site in the Ml 3 multiple cloning site was destroyed by Ace I digestion, Klenow treatment, ligation and transformation prior to insertion of the E2 fragment.
- the purified fragment was ligated to M13mpl9E2-286 which had been cleaved with Ace I and Bgl I and purified as described above.
- the ligation reaction was reintroduced into E. coli MV1190 by electroporation (Dower et al. (1988) Nucleic Acids Res 16:6127- 6145).
- Virus extracted from the top agar of the transformation plates was pooled and used to infect MV1 190 from which double stranded replicative form DNA (RF DNA) was prepared using a Quiagen Tip 100 column (Quiagen Corp.).
- the mutant RF DNA was cleaved with Bam HI and Kpn I, and the E2 1 to 286 amino acid fragment was gel purified, and ligated to the YEplacl 12GE2 fragment containing E2 amino acids 287 to 410.
- the ligation reaction was introduced into E. coli DH5 ⁇ by electroporation, generating library of approximately 30,000 transformants. Colonies were scraped off transformation plates with a glass spreader and combined in a volume of 50 ml of terrific broth (TB) with 50 ⁇ g/ml ampicillin. After 2 hr. of incubation at 37°C with shaking, YEplacl 12GE2 DNA was prepared from the culture with a Quiagen Tip 100 kit.
- the YEplacl 12GE2 mutant library was transformed into DBY1 containing pBsy72, an E2 responsive LacZ reporter identical to pL72, except that it contains an intact URA3 gene (Morrissey and Androphy (1989) J Virol 63:4422-4425). Transformants were selected on YMM glucose minus tryptophan and uracil, and replica plated to the same medium and YMM GAL XGAL minus tryptophan and uracil. After 48 hr. at 30°C colonies were scored for color formation, and white and light blue colonies were picked off the glucose replica plate into 5 ml. of YMM glucose minus tryptophan and uracil. The next day 2.5 ml.
- E2 The presence of full length E2 was determined by probing Western blots first with an rabbit anti-BPVl-E2 polyclonal serum, and second with a sheep anti-rabbit alkaline phosphatase conjugate (Sigma) (Prakash et al. (1992) Genes Dev 6:105- 116). After visualization of E2 bands with a BIORAD alkaline phosphatase kit, mutant extracts were scored for the presence of full length BPV1 E2.
- Putative mutant E2 plasmids were then recovered from isolates which scored positive for the presence of full length E2. The remaining 2.5 ml. of the original culture was pelleted and DNA was extracted, as described by Hoffman and Winston ((1987) Gene 57: 262-272), except that after phenol/chloroform extraction DNA was precipitated, washed twice with 70% ethanol, and resuspended in 50 ⁇ l of TE (50 mM Tris pH8.0, lOmM EDTA). Five microliters of each DNA sample was used to transform E. coli DH5 ⁇ as described by Hanahan 1983. Transformation reactions were plated on LB with 50 ⁇ g/ml ampicillin and 0.4% XGAL. On this medium the reporter construct (pBsy72) forms blue colonies, while transformants containing YEplac 112GE2 are white.
- Putative YEplacl 12GE2 transformants were picked in to 2.5 ml of TB (50 ⁇ g/ml ampicillin) and incubated overnight at 37°C with shaking. Plasmid DNA was isolated using the alkaline lysis method, and the identity of plasmid was confirmed by restriction endonuclease digestion (Birnboim and Doly (1979) Nucleic Acids Res 7:1513). Isolates were also reintroduced into DBYl with pBsy72 and tested on GAL/XGAL plates for activation of the reporter. Any putative mutants which activated transcription of the reporter to wild type levels when retested were discarded.
- the transactivation potential of defective mutants was further characterized by quantitative ⁇ -galactosidase assay.
- Colonies used for the GAL/XGAL plate assay were also inoculated into YMM 2% raffinose (minus tryptophan and uracil) and incubated 12 hr. at 30°C with shaking. Cells were pelleted at 1000 x g for 5 minutes at room temperature, washed with sterile distilled water, and resuspended in YMM 4% High purity galactose (Sigma) minus tryptophan and uracil and incubated as above. Afte 12 hr.
- YEplacl 12GE2 (87S,131H) was chosen for screening of the HeLa cDNA library.
- Screening HeLa Cell cDNA Library The VP16-cDNA library and YElpacl 12GE2 (87S. 131H) were co-transformed into DBYl(pL72) and plated on YMM glucose minus tryptophan, leucine, and uracil. After 72 hr.
- the cDNA plasmid was then recovered from colonies which were positive when retested.
- the vector used to construct the cDNA library (pRS306) has a low copy yeast replicon, but the YEplacl 12GE2 and pL72 vectors have high copy replicons making it necessary to cure the yeast of the high copy plasmid prior to isolation of cDNA plasmid. This was done by growing the yeast several generations while selecting only for the low copy replicon. The colonies were inoculated into 2.5 ml. of YMM glucose minus uracil medium, and incubated overnight at 30°C with shaking. The overnight was diluted 1 to 50 in the same medium and incubated over night.
- Total DNA was prepared from these isolates and transformed into E. coli as described previously. Plasmid DNA was prepared by the alkaline lysis method and digested with restriction endonucleases to confirm the identity of the plasmid. The plasmid DNA was also transformed into DBYl (pL72, YElpacl 12GE2 87S, 131H) and replica plated to GAL/XGAL plates to determine if the plasmid recovered was responsible for the color formation in the original screen.
- the activation of the reporter plasmid was also quantitated, essentially as was described for the E2 mutants, except that YMM minus tryptophan. leucine, and uracil was used for growth and galactose induction. From the original colonies isolated in the screen, only colonies which retested positive after repeat isolation of the cDNA plasmid were retained.
- the cDNA plasmids that specifically interacted with E2 were subjected to DNA sequence analysis using a primer initiating within the VP16 coding sequence. This provides information on the reading frame at the fusion point with the cDNA. In general using this primer we have determined about 200-300 nucleotides of DNA sequence. Provided in Table 1 below is a guide to the sequence listings for each of these clones.
- each of these clones as a library of pEJA plasmids (designated "pRS306-E2BP") containing the 4 different novel clones isolated in the E2 interaction trap has been ma ⁇ e with the American Type Culture Collection (Rockville, MD) on October 19, 1994, under the terms of the Budapest Treaty. ATCC Accession number 75915 has been assigned to the deposit.
- pRS306-E2BP pEJA plasmids
- each individual clone of the ATCC deposit can be isolated using primers based on the nucleotide sequences provided by SEQ ID Nos. 1 -4, or a combination of such primers and th primers based on flanking plasmid sequences.
- Isolated clones can be subcloned into expression vectors in order to produce a recombinant protein, or can be used to generate anti-sense constructs, or can be used to generate oligonucleotide probes.
- oligonucleotide probes have been generated using the coding sequences for each of the clones of the subject ATCC deposit, and used in Southern hybridization and in situ hybridization assays to detect the pattern and abundance of expression of each of the E2-binding proteins.
- each member of the ATCC deposit is a plasmid encoding a fusion protein identified from an interaction trap assay
- the clone can be utilized directly from the deposit in a similar ITS employed as, for example, a drug screening assay, or alternatively, a mutagenesis assay for mapping E2 binding epitopes.
- a full length cDNA for the SD2-7 clone was obtained and sequenced.
- SD24 SEQ ID NO:4 SEQ ID NO:8 E2-BPSD-24 The 42 A clone represented an insert of about 1000 bases, encoding an open reading frame of 357 amino acid residues. Internally within the reading frame is an exact copy of a brain expressed sequence tag (EST) previously described in Adams et al. ((1993) Nat Genet 4:256-267, although the homology ends at the 3' end of the EST sequence and the 42A clone continues on for several hundred additional nucleotides.
- EST brain expressed sequence tag
- Northern blot analyses under high stringency conditions with the 42A sequence reveals that it hybridizes to a transcript of about 2,000 nucleotides in size that is expressed in both cultured human keratinocytes and HeLa cells.
- a fusion of GST and the VP16 transactivation domain from the original 42A isolate was made in pGEX2T for use as a negative control.
- the GST fusion plasmids were transformed into E. coli BL21(DE3) pLysS and synthesis of recombinant protein was induced by addition of ImM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) (Studier et al. (1990) Meth Enzymol 185:60-89). Fusion proteins were extracted from frozen cell pellets, bound to glutathione beads, and visualized by Coomassie staining after SDS/PAGE.
- IPTG ImM isopropyl- ⁇ -D-thiogalactopyranoside
- Binding reactions contained about 5 ⁇ g of E2 fusion protein (as determined by BCA protein assay, Pierce Scientific) and 100,000 cpm of 35 S-labeled 42 A protein in a total volume of 250 ⁇ l of NETN (20 mM tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonident P-40) with protease inhibitors. Reactions were incubated for 3 to 5 hr. at 4°C with shaking, and the beads were pelleted. The pellets were washed five times with 1 ml of ice cold NETN, and an additional three times with NETN with 500 mM NaCl. After the last wash, samples were resuspended in sample buffer and resolved by SDS/PAGE using a 12% gels. Proteins were visualized by autoradiography.
- 1 12G 2 ⁇ replicon with the GAL 1-10 UAS, TRP 1.
- 1 12G-E2 87L, 131 H (used in the screen that isolated the 42A cDNA) and 1 12G-E2 15H are TAD mutants expressed at wt levels in S. cerevisiae.
- 1 12G-E2 1 13-410 begins with amino acid 1 13 of BPV E2 (i.e. deletion of AA 1 - 1 13)
- the two hybrid system used to isolate the 42A open reading frame (ORF) employed a mutation of BPV- 1 E2 with a very low level of intrinsic transactivation potential.
- ORF open reading frame
- transcriptional activation (transactivation) of the reporter is stimulated by the VPl 6-42 A fusion as compared with a randomly chosen ORF fused to VPl 6.
- WT wild type
- transactivation transcriptional activation
- the VPl 6 moiety presumably stimulates initiation of transcription of the reporter independently of the transactivation domain of E2.
- Construction of the pL72 reporter originally used involved deletion of nucleotides -250 to -178 of pLG -250 (a chimeric yeast pCYCl/LacZ reporter) and insertion of 4 copies of E2 response elements (E2RE) at position -178 (Figure 3) (Morissey et al. (1989) S «/?r ⁇ ,Guarente and Mason (1983), Supra).
- reporter pLG -178 which is otherwise identical to pL72, is not activated by WT E2 (line 1). Furthermore, VPl 642 A does not effect this reporter either in the presence or absence of E2 (column 2).
- Table 2 shows that the effects of VP1642A on transactivation of pL72 by various E2 constructs (Table 2) requires E2 bound to the promoter. Consistent with this observation E2 340R, a mutant defective for binding to E2RE. fails stimulate transcription of pL72 plus or minus VP1642A (data not shown).
- VP1642A could be acting in a promoter specific manner, but only when transcription is activated through an upstream DNA binding element (ie. E2RE or UASc) In this case it would be expected that VP1642A has no effect of pLG -178, but response elements for other transcriptional activators (such as HAP) could substitute for those of E2.
- the pLG -250 reporter lacks E2RE and contains additional native pCYCl sequence with an UASc element ( Figure 3). Characterizing the effect of VP1642A on this reporter should indicate whether it is able to affect another transcriptional activator (yeast HAP activator) as it does E2.
- the transcription of pLG -312 is activated by endogenous yeast HAP (Table 3 line 3).
- the level activation is not increased in the presence of VP1642A or by two other VI 6 fusions to previously unidentified ORFS from the VP16-cDNA library. These results further confirm the VPl 642 A acts in an E2 rather than a promoter specific manner in the original two hybrid assay.
- VPl 642 A affects pGAL4 (promoter used to drive expression of E2 in yeast) enhancing the steady state level of E2 present by increasing the activity of pGAL4, possibly acting through an upstream response element.
- pGAL4 promoter used to drive expression of E2 in yeast
- Increasing the protein level of WT E2 or a mutant with very low intrinsic activation potential could mimic physical interaction in the two hybrid assay, resulting in a false positive.
- VP1642A When the expression of WT E2 was driven with pADH, a constitutive yeast promoter unrelated to the galactose inducible GAL4 promoter, VP1642A enhanced the level of transactivation of the E2 dependent pL72 reporter by E2 (data not shown). Thus, the effect of VP1642A is not dependent of the promoter used to drive E2 expression. We consider it unlikely that VPl 642 A would similarly affect two unrelated yeast promoters.
- LexA Fusion Two Hybrid Assav: LexA Fusion
- LexA-42A construct (YEplacl 8 lGLexA42A) and YEplacl 12G were introduced into DBYl containing LexA/LacZ reporter pSH18-34 (Gyuris et al., supra).
- LexA -42 A was induced on GAL/XGAL plates, colonies remained white after extended incubation, suggesting the 42A fusion bound LexA operators in pSH 18-34, but was unable to activate transcription of the LacZ gene (Table 4, column 1).
- YEplacl 12GE2 340R is a mutant of E2 which is unable to bind E2 elements, and is defective for transactivation when assayed with the E2 dependent reporter pL72 (Prakash et al. Supra). ND, not done.
- the transactivation defective mutant of E2 (YEplacl 12GE2, 87L, 131H) used to isolate 42A in the original two hybrid screen was unable to activate the LexA reporter in the presence of LexA42A.
- This mutant E2 protein presumably associates with the 42A moiety of the LEexA42A fusion, but is unable to activate transcription due to the presence of mutations in its transactivation domain.
- Interaction of Deletions of 42A with BPV-1 E2 in S. Cervisiae Truncations of the 42 A ORF were translationally fused to the VP 16 transactivation domain (TAD) in the SD10 vector.
- This hybrid protein consists of the VPl 6 transcriptional activation domain fused to the C-terminal 125 amino acids of E2, which comprises the E2 DNA binding domain.
- the region of E2 required for interaction with 42A lies between amino acids 134 and 162, which are absent in the VP16E2-125 protein.
- overexpression of the 42A ORF saturates the 42A association domain on E2 molecules, preventing association of E2 with the endogenous 42 A protein which is required for transcriptional activation by E2 in mammalian cells. This assumption is not unreasonable, because the 42A ORF isolated in the two hybrid screen is only a portion of a larger molecule.
- Antisera specific for 42A was generated in a rabbit using recombinant 42A protein made in Escherichia coli.
- a bacterial expression construct (pET8c42/ 10) was made by transferring the 42A ORF to pET8c. creating an amino terminal translational fusion to the first 10 amino acids of the bacteriophage T7 gene 10 present in this vector.(Studier et al. (1990) Meth. Enzymol.
- the gene 10-42A fusion protein ( 10/42A) could be detected in extracts of Escherichia coli BL21 pLysS, pET8c42/10 resolved using SDS- PAGE by Coomasie blue staining one hour after induction of synthesis with isopropyl- ⁇ -D- thiogalactopyranoside (IPTG).
- IPTG isopropyl- ⁇ -D- thiogalactopyranoside
- Cell pellets from 500 ml of culture were resuspended in 20 ml of NETN ( 20 mM tris pH 8.0, lOOmM NaCl, ImM EDTA. 0.5% Nonidet P-40) with protease inhibitors (Phenylmethylsulfonylfluoride. leupeptin. pepstatin). and sonicated for 10 sec. at a setting of 7. After sonication, triton X-100 was added to 1%, and the extract was centrifuged for 30 min. at 12,000 x G for 30 min at 4°C.
- NETN 20 mM tris pH 8.0, lOOmM NaCl, ImM EDTA. 0.5% Nonidet P-40
- protease inhibitors Phenylmethylsulfonylfluoride. leupeptin. pepstatin
- the pellet was suspended in the same volume of NETN with protease inhibitors and centrifuged as before, except that the time was reduced to 10 minutes.
- the pellet was extracted twice with 20 ml. of 1.5% n-octylglucosid, ImM EDTA, 15 mM NaCl, 0.25M KPO 4 , pH 8.0. After each extraction the pellet was recovered by centrifugation as before for 15 min.
- the 10/42A in the pellet was then solubilized in 8 M urea, and subjected to ion exchange chromatography using SP Sepharose Fast Flow (Pharmacia), which was monitored by Coomasie blue staining of fractions resolved by SDS/PAGE.
- the 10/42A 8 M urea fraction was adjusted to 50 mM Hepes pH 7.1 , and loaded on a column equilibrated with 8 M urea, 50 mM Hepes pH 7.1. After the column was washed with loading buffer, protein on the column was eluted by increasing the NaCl concentration in 100 mM steps. Most of 10/42A was eluted from the column at the 200 mM NaCl step, and this fraction was used for immunization of a rabbit. For the initial injection and first boost two weeks afterward, approximately 2 mg of the 10/42 A 200 mM eluate was emulsified with Hunters Titer Max (Sigma) after the urea concentration was reduced to 1 M.
- a third injection was done after a additional two week interval using eluate emulsified with Freunds incomplete adjuvant (Sigma) with out reducing the urea concentration.
- the presence of antibodies specific for the 10/42 A protein was monitored by probing western blots of IPTG induced Escherichia coli BL21 pLysS, pET8c42/10 with sera from the immunized rabbit. Detection of specific interaction was done as described previously with a alkaline phosphatase assay. Two weeks after the third injection serum from the immunized rabbit was found to specifically detect 10/42A at dilutions of up to 1 :5000 in this assay.
- This serum was found to immunoprecipitate 35 S-labeled 42A protein synthesized in rabbit reticulocyte lysate, but not an unrelated protein (TATA binding protein, from Saccharomyces cerevisiae) also synthesized in reticulocute lysate.
- TATA binding protein from Saccharomyces cerevisiae
- Gly Phe Thr Leu Arg lie Tyr Cys Tyr Cys Cys Cys Cys Cys Phe 1 5 10 15 TTA ATG ATA AGC TAC TTC TGC AAT TTT AAC GTT GTA GAA AAG ATG CTA 95 Leu Met lie Ser Tyr Phe Cys Asn Phe Asn Val Val Glu Lys Met Leu 20 25 30
- Pro Ser Gly Pro Pro lie lie Pro Pro Pro Pro Pro lie Cys Pro Asp 115 120 125
- MOLECULE TYPE cDNA
- cDNA SEQUENCE DESCRIPTION: SEQ ID NO:12: TCGTCACCAA AGCGAG 16 (2) INFORMATION FOR SEQ ID NO: 13:
- AAA CAA ATG TTT GGA CCC CAA GTG CTT ACG ACC CGG CAC TAC GTG GGC 190
- ATA GAA CAG AAT GCT CAA GAG AAT GAA AAT GAA AGC CAA GTT TCA ACA 534 lie Glu Gin Asn Ala Gin Glu Asn Glu Asn Glu Ser Gin Val Ser Thr 155 160 165 GAT GAA AGT GAG AAC TCC AGG TCT CCT GGA AAT AAA TCA GAT AAC ATC 582 Asp Glu Ser Glu Asn Ser Arg Ser Pro Gly Asn Lys Ser Asp Asn lie 170 175 180
- Lys Cys Ser Ala lie Trp Ser Glu Asp Gly Cys lie Tyr Pro Ala Thr 100 105 110 lie Ala Ser lie Asp Phe Lys Arg Glu Thr Cys Val Val Val Tyr Thr 115 120 125
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US6110663A (en) * | 1998-11-12 | 2000-08-29 | The Penn State Research Foundation | Methods for detecting, titering, and determining susceptibility to papillomavirus |
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---|---|---|---|---|
US5464936A (en) * | 1990-12-21 | 1995-11-07 | Cetus Oncology Corporation | Compositions for identification of papillomavirus replication inhibitors |
EP0711833B1 (en) * | 1994-10-19 | 2005-08-03 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Survival motor neuron (SMN) gene: a gene for spinal muscular atrophy |
EP0708178A1 (en) * | 1994-10-19 | 1996-04-24 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Survival motor neuron (SMN) gene: a gene for spinal muscular atrophy |
-
1994
- 1994-12-22 US US08/361,806 patent/US5792833A/en not_active Expired - Fee Related
-
1995
- 1995-12-22 CA CA002208211A patent/CA2208211A1/en not_active Abandoned
- 1995-12-22 WO PCT/US1995/016806 patent/WO1996019566A1/en not_active Application Discontinuation
- 1995-12-22 JP JP8520020A patent/JPH10511546A/en active Pending
- 1995-12-22 AU AU46070/96A patent/AU711651B2/en not_active Ceased
- 1995-12-22 EP EP95944210A patent/EP0796321A4/en not_active Withdrawn
-
1996
- 1996-03-06 US US08/612,986 patent/US5770384A/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5219990A (en) * | 1991-01-28 | 1993-06-15 | Biogen, Inc. | Papillomavirus e2 trans-activation repressors |
Non-Patent Citations (2)
Title |
---|
JOURNAL OF VIROLOGY, Volume 68, Number 10, issued October 1994, USHIKAI et al., "Trans Activation by the Full-Length E2 Proteins of Human Papillomavirus Type 16 and Bovine Papillomavirus Type 1 in Vitro and in Vivo: Cooperation With Activation Domains of Cellular Transcription Factors", pages 6655-6666. * |
See also references of EP0796321A4 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110294226A1 (en) * | 1994-10-19 | 2011-12-01 | Judith Melki | Survival motor Neurons (SMN) gene: a gene for spinal muscular atrophy |
US8394932B2 (en) * | 1994-10-19 | 2013-03-12 | Institut National De La Sante Et De La Recherche Medicale | Survival motor neurons (SMN) gene: a gene for spinal muscular atrophy |
US8962269B2 (en) | 1994-10-19 | 2015-02-24 | Institut National De La Sante Et De La Recherche Medicale | Spinal muscular atrophy diagnostic methods |
US6818412B2 (en) | 1996-08-23 | 2004-11-16 | Human Genome Sciences, Inc. | Human growth factors |
US7060801B2 (en) | 1996-08-23 | 2006-06-13 | Human Genome Sciences, Inc. | Antibodies to human growth factor huXAG-3 and methods of use |
US7611846B2 (en) | 1996-08-23 | 2009-11-03 | Human Genome Sciences, Inc. | Diagnostic methods involving human growth factor huXAG-1 |
Also Published As
Publication number | Publication date |
---|---|
AU4607096A (en) | 1996-07-10 |
JPH10511546A (en) | 1998-11-10 |
EP0796321A1 (en) | 1997-09-24 |
EP0796321A4 (en) | 1999-01-27 |
US5770384A (en) | 1998-06-23 |
US5792833A (en) | 1998-08-11 |
AU711651B2 (en) | 1999-10-21 |
CA2208211A1 (en) | 1996-06-27 |
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