WO2000024757A9 - Human retinoid-like orphan receptor gamma - Google Patents
Human retinoid-like orphan receptor gammaInfo
- Publication number
- WO2000024757A9 WO2000024757A9 PCT/US1999/024309 US9924309W WO0024757A9 WO 2000024757 A9 WO2000024757 A9 WO 2000024757A9 US 9924309 W US9924309 W US 9924309W WO 0024757 A9 WO0024757 A9 WO 0024757A9
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- WO
- WIPO (PCT)
- Prior art keywords
- polypeptide
- hrorγ
- nucleic acid
- seq
- antibody
- Prior art date
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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)
Definitions
- the invention provides, for example, isolated nucleic acid and amino acid sequences of hROR ⁇ , antibodies to hROR ⁇ , transgenic animals, methods of identifying ligands for hROR ⁇ , and methods of screening for modulators of hROR ⁇ .
- Nuclear receptors encode ligand-activated transcriptional regulators that play important roles in embryonic development, cell proliferation, and differentiation (see, e.g., Kastner et al, Cell 83:859-869 (1995)). These diverse nuclear receptors are all members of a gene superfamily, which share a common structure, including a conserved DNA-binding domain that contains two zinc finger motifs (see, e.g., Beato et al, Cell 83:851-857 (1995)).
- the nuclear receptors bind as monomers, homo-, or heterodimers to specific response elements in nucleic acids, typically characterized by a single half site motif, PuGGTCA, preceded by an AT-rich sequence or direct palindromic or inverted palindromic repeats of the core motif spaced by one or more nucleotides (see, e.g., Giguere, Endo. Rev. 15:61-795)).
- the superfamily includes receptors for steroid hormones, thyroid hormone, retinoids, and vitamin D, as well as a large number of orphan receptors, for which no ligand has been identified (see, e.g., Evans et al, Science 240:889-895 (1988)).
- nuclear receptors typically have two major domains: a DNA binding domain and a ligand binding domain.
- the DNA binding domain appears to be ligand independent, while the binding of ligand to the ligand binding domain is typically required for the transcriptional activation or repression activity of the receptor.
- the ROR ⁇ nuclear receptor is a member of the ROR/RZR subfamily, and both mouse and human versions of the gene encoding this receptor have been cloned (Hirose et al, Biochem. Biophys. Res. Comm. 205:1976-1983 (1994); Medvedev et al, Gene 181:199-206 (1996)).
- Each member of the ROR/RZR subfamily appears to exhibit a different tissue distribution; for example, ROR ⁇ is preferentially expressed in thymus, T cell lymphomas, and skeletal muscle (see, e.g., Hirose et al, supra).
- Members of this subfamily are involved in regulation of the immune system and adipocyte differentiation (Ortiz et al, Mol Endo. 9: 1679-1691 ; Austin et al, Cell Growth & Diff. 9:267-276
- ROR ⁇ appears to play a role in controlling gene expression in tissues of the immune system such as the thymus, and in skeletal muscle, no biological ligand has yet been identified.
- the open reading frame of human ROR ⁇ has been reported to encode a protein of 560 amino acids with a predicted molecular weight of 63 kDa (Id.). This amino acid sequence is set forth herein as SEQ ID NO:2.
- SEQ ID NO:2 The genomic structure and chromosomal location of hROR ⁇ has also been identified (Medvedev et al, Genomics 46:93-102 (1997)). Identification of the ligand for hROR ⁇ would be useful for modulating hROR ⁇ -mediated transcriptional regulation.
- the present invention provides the surprising discovery of a sequence of hROR ⁇ that is substantially different than SEQ ID NO:2, the previously hypothesized amino acid sequence of hROR ⁇ .
- the new amino acid sequence for hROR ⁇ set forth as SEQ ID NO:4, is truncated in the C-terminal region relative to SEQ ID NO:2 (a nucleic acid encoding the hROR ⁇ of SEQ ID NO:2 is set forth in SEQ ID NO: 1 ; a nucleic acid encoding the hROR ⁇ of SEQ ID NO:4 is set forth in SEQ ID NO:3).
- the end of the C-terminal domain comprises the amino acid subsequence LSK, which is not found in SEQ ID NO:2.
- the present invention provides new polypeptides which include a subsequence having structural similarity to the C-terminal domain of SEQ ID NO:4.
- One convenient method of determining whether an hROR ⁇ polypeptide has a C-terminal domain structurally similar SEQ ID NO:4 is to determine whether an antibody specific to the polypeptide of SEQ ID NO:4 specifically binds to the hROR ⁇ polypeptide.
- Such an antibody is specific to the polypeptide of SEQ ID NO:4 when the antibody binds to the polypeptide of SEQ ID NO:4 with an affinity at least 100 fold higher than to the polypeptide of SEQ ID NO:2.
- the antibody should also preferentially bind SEQ ID NO:4 over known hROR ⁇ homologs, such as the murine ROR.
- the hROR ⁇ polypeptide comprises the C-terminal domain of the polypeptide of SEQ ID NO:4, the hROR ⁇ polypeptide is essentially similar to the polypeptide of SEQ ID NO:4.
- a test polypeptide with a truncation in the N- terminal region relative to the polypeptide of SEQ ID NO:4 which has the same C- terminal sequence as the polypeptide of SEQ ID NO:4 is in the same family as the polypeptide of SEQ ID NO:4.
- the test polypeptide can be tested for other biological activities similar to the polypeptide of SEQ ID NO:4, such as binding to the hROR ⁇ ligand melatonin.
- the hROR ⁇ polypeptide is optionally chimeric, comprising, e.g., heterologous amino acid subsequences such as DNA binding domains from proteins other than hROR ⁇ .
- the hROR ⁇ polypeptide can also include chemically linked moieties which confer specific properties such as nucleic acids (e.g., to target the C-terminal region to specific nucleic acids), carbohydrates, and the like.
- the hROR ⁇ polypeptide can be free in solution, bound to a solid support, in a cell, in a biological mixture or the like.
- Nucleic acids encoding the hROR ⁇ polypeptide are also provided.
- non-lambda expression vectors including a recombinant expression cassette encoding the hROR ⁇ polypeptides above are provided.
- An example nucleic acid sequence which the vector can encode is set forth at SEQ ID NO:3.
- hROR ⁇ nucleic acids can be used in various assays, and can be free in solution, bound to a solid support, in a cell, in a biological mixture or the like. Animals comprising the recombinant expression cassette are also provided.
- Methods of identifying a ligand that binds to the C-terminal domain of the hROR ⁇ polypeptide are also provided, both in vitro and in vivo.
- the hROR ⁇ polypeptide is contacted with a first ligand and ligand binding is monitored to determine whether the ligand binds to the polypeptide.
- assay formats is appropriate, e.g., where hROR ⁇ is optionally bound to a solid support; expressed in a cell (naturally or recombinantly); or used in assays with peptide sensors as depicted in Figures 1 and 2, etc.
- a peptide sensor which binds to the nuclear receptor only after a conformational change has occurred upon ligand binding.
- Libraries of ligands can be screened.
- the effects of potential modulators on ligand binding can also be determined by contacting the hROR ⁇ polypeptide with the potential modulator of ligand binding, before, during, or after incubating the ligand and hROR ⁇ polypeptide. Essentially any compound can be tested for such modulatory activity.
- Kits embodying any of the compositions or methods above e.g., comprising an hROR ⁇ polypeptide or nucleic acid and further comprising one or more of a container, instructions for practicing a method herein, buffers, ligands, antibodies to hROR ⁇ , or the like, are provided.
- Figure 1 provides a schematic diagram of a nuclear receptor- ligand binding assay, in which a peptide sensor detects binding of the ligand to the nuclear receptor via conformational changes.
- the peptide sensor is coated on a plate, and then binds to the receptor-ligand complex.
- Figure 2 provides a schematic diagram of a nuclear receptor-ligand binding assay, in which differences in rotation rates between bound and unbound receptor are measured using fluorescence polarization and a peptide sensor, which binds to the receptor-ligand complex.
- the present invention provides, for the first time, the correct nucleotide sequence for human retinoid-like orphan receptor gamma ("hROR ⁇ ”), which encodes an approximately 60 kDa protein with a C-terminal domain that differs significantly from previously reported hROR ⁇ protein sequences.
- hROR ⁇ receptors are ligand-activated transcription factors that have both DNA-binding and ligand binding domains.
- hROR ⁇ is a member of the nuclear receptor superfamily and the ROR/RZR subfamily, the members of which share a structure consisting of a DNA-binding domain that contains two zinc fingers, as well as a C-terminal domain that is involved in ligand recognition (see, e.g., Medvedev et al.
- Nuclear transcription factors typically bind as monomers, homo- or heterodimers to response elements in DNA, and regulate transcription in response to extracellular signals.
- the C-terminal domain of hROR ⁇ is involved in ligand binding and in homo/heterodimer formation, and is thus an important domain for hROR ⁇ regulation.
- hROR ⁇ nucleic acids provide specific probes for tissues such as skeletal muscle and thymus, as nucleic acids encoding hROR ⁇ are highly expressed in immune tissue such as the thymus, T cell lymphomas, and in skeletal muscle. Furthermore, the nucleic acids and the proteins they encode can be used to identify ligands that bind to hROR ⁇ , and to investigate transcriptional regulation in allergic, and inflammatory reactions, as well as in adipocyte differentiation.
- the invention also provides methods of screening for ligands of hROR ⁇ and methods of screening for modulators, e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists, of hROR ⁇ activity.
- modulators e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists
- Such modulators of hROR ⁇ activity are useful for transcriptional regulation in allergic and inflammatory reactions, as well as in adipocyte differentiation.
- These methods of screening can also be used to identify ligands of hROR ⁇ activity.
- the invention provides assays for hROR ⁇ ligands and modulators, where hROR ⁇ acts as an direct or indirect reporter molecule for the effect of modulators and ligands on transcriptional activation.
- hROR ⁇ can be used in ligand binding and modulator assays, e.g., to measure changes in reporter gene transcription (luciferase, CAT, ⁇ -galactosidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)); signal transduction; phosphorylation and dephosphorylation; voltage, membrane potential and conductance changes; ion flux assays; receptor-ligand interactions; changes in second messenger concentrations (e.g., cAMP, IP3, Ca ), in vitro, in vivo, and ex vivo.
- reporter gene transcription luciferase, CAT, ⁇ -galactosidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)
- signal transduction phosphorylation and dephosphorylation
- voltage, membrane potential and conductance changes ion flux assays
- receptor-ligand interactions changes in
- the C-terminal domain (containing the ligand binding domain) of hROR ⁇ is fused to a reporter such as the Gal4 DNA binding domain.
- This construct is co-transfected into a cell with a second construct having a promoter with a reporter-responsive (e.g., Gal4-responsive) element operably linked to a reporter gene such as luciferase.
- a reporter-responsive element operably linked to a reporter gene such as luciferase.
- Ligand binding to hROR ⁇ activates the Gal4 DNA-binding domain and promotes transcription of the reporter gene.
- This system can also be used to test for modulators of ROR ⁇ ligand binding (see, e.g., Medvedev et al, 1996, supra).
- the invention also provides for methods of detecting hROR ⁇ nucleic acid and protein expression, allowing investigation of transcriptional regulation.
- hROR ⁇ s also provide useful nucleic acid probes for paternity and forensic investigations.
- hROR ⁇ s are useful nucleic acid probes identifying immune tissue such as the thymus, as well as skeletal muscle tissue.
- hROR ⁇ s can also be used to generate monoclonal and polyclonal antibodies useful for identifying, e.g., thymus and skeletal muscle tissue.
- hROR ⁇ can be identified using techniques such as reverse transcription and amplification of mRNA, isolation of total RNA or poly A+ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, SI digestion, probing DNA microchip arrays, western blots, and the like.
- hROR ⁇ functionally represents a nuclear receptor involved in transcriptional activation in response to ligand binding.
- the nucleotide sequence of hROR ⁇ (see, e.g., SEQ ID NO:3) encodes a polypeptide of approximately 518 amino acids with a predicted molecular weight of approximately 60 kDa (see, e.g., SEQ ID NO:4).
- Related hROR ⁇ alleles and polymorphic variants are homologous to the C-terminal domain of SEQ ID NO:4; in particular, at the C-terminal end of the polypeptide they comprise the amino acid subsequence LSK or a conservatively modified variant thereof.
- hROR ⁇ is highly expressed in immune tissue such as the thymus, as well as several other tissues such as skeletal muscle.
- the present invention also provides polymorphic variants of the hROR ⁇ depicted in SEQ ID NO:4: variant #1, in which an isoleucine residue or a valine residue is substituted for a leucine acid residue at amino acid position 516; variant #2, in which a threonine residue is substituted for a serine residue at amino acid position 517; and variant #3, in which an arginine or a histidine residue is substituted for a lysine residue at amino acid position 518, or any combination of these substitutions. Additional polymorphic variants comprise conservatively modified variations at any amino acid position in SEQ ID NO:4.
- the C-terminal domain of the hROR ⁇ nucleotide and amino acid sequence may be used to identify polymorphic variants and alleles of hROR ⁇ . This identification can be made in vitro, e.g., under stringent hybridization conditions or PCR (using primers comprising the amino acid subsequence at positions 516-518 of SEQ ID NO:4) and sequencing, or by using the sequence information in a computer system for comparison with other nucleotide sequences. Sequence comparison can be performed using any of the sequence comparison algorithms discussed below. Antibodies that bind specifically to the C-terminal domain of hROR ⁇ or a conserved region thereof but not to the sequence of SEQ ID NO:2 can also be used to identify alleles and polymorphic variants.
- hROR ⁇ nucleotide and amino acid sequence information may also be used to construct models of nuclear receptors and their ligands in a computer system. These models are subsequently used to identify ligands and compounds that can activate or inhibit hROR ⁇ . Such compounds that modulate the activity of hROR ⁇ or that bind to hROR ⁇ can be used to investigate the role of hROR ⁇ in transcriptional regulation.
- the identification of the correct amino acid and nucleotide sequence of hROR ⁇ thus provides, for the first time, a means for assaying for ligands, inhibitors, and activators of hROR ⁇ mediated transcriptional regulation.
- Methods of detecting hROR ⁇ nucleic acids and expression of hROR ⁇ are also useful for identifying thymus and skeletal muscle and for studying regulation of transcriptional activation.
- hROR ⁇ refers to a polypeptide that comprises a C-terminal domain that includes, at the end the amino acid subsequence, the sequence LSK, or a conservatively modified variation thereof.
- the term hROR ⁇ therefore refers to polymo ⁇ hic variants and alleles having conservatively modified variations of the amino acid or nucleotide sequence of SEQ ID NO:3 or 4.
- C-terminal domain refers to the C-terminal or "second half of a polypeptide, i.e., the part that is encoded by the 3' half of the gene.
- C-terminal domain of SEQ ID NO:4 refers to the C-terminal half of the polypeptide encoded by SEQ ID NO:4, or a conservatively modified variation thereof, comprising at the end the subsequence LSK or a conservatively modified variation thereof.
- N-terminal domain refers to the N-terminal or "first half of a polypeptide, i.e., the part that is encoded by the 5' end of the gene. A polypeptide that is "truncated" at the N-terminal domain is lacking a subsequence of the N-terminal domain.
- hROR ⁇ activity refers to the ability of a polypeptide to bind ligand via an hROR ⁇ ligand binding domain and regulate transcription (“ligand-binding activity"). hROR ⁇ activity can also refer to the ability of a polypeptide to bind DNA via a DNA- binding domain ("DNA-binding activity").
- the hROR ⁇ ligand binding domain can be fused to a heterologous DNA binding moiety or domain.
- DNA-binding moiety refers to a compound that has the ability to selectively associate with a nucleic acid.
- a DNA-binding moiety therefore includes polypeptides, peptides, proteins, nucleic acids, e.g., oligonucleotides, lectins, glycoproteins, carbohydrates, and small organic molecules.
- Biological sample is a sample of biological tissue or fluid that contains hROR ⁇ or nucleic acid encoding hROR ⁇ protein. Such samples include, but are not limited to, tissue isolated from humans, mice, and rats, in particular, thymus and skeletal muscle. Biological samples may also include sections of tissues such as frozen sections taken for histological pu ⁇ oses.
- a biological sample is typically obtained from a eukaryotic organism, such as insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mouse, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
- Preferred samples include skeletal muscle tissue, thymus tissue, T cells, T cell lymphomas, cultured cells that recombinantly express hROR ⁇ , and cellular extracts from such cells.
- modulates the activity in the context of assays for screening compounds that modulate hROR ⁇ includes the determination of any parameter that is indirectly or directly under the influence of hROR ⁇ activity.
- determining whether a ligand binds the polypeptide also includes the determination of any parameter that is indirectly or directly under the influence of hROR ⁇ activity.
- Such parameters include, e.g., measuring changes in reporter gene transcription (luciferase, CAT, ⁇ - galactosidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)); signal transduction; phosphorylation and dephosphorylation; voltage, membrane potential and conductance changes; ion flux assays; receptor-ligand interactions; changes in second messenger concentrations (e.g., cAMP, LP3, Ca 2+ ), in vitro, in vivo, and ex vivo.
- reporter gene transcription luciferase, CAT, ⁇ - galactosidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)
- signal transduction phosphorylation and dephosphorylation
- voltage, membrane potential and conductance changes ion flux assays
- receptor-ligand interactions changes in second messenger concentrations (e.g., cAMP,
- Such functional effects can be measured by any means known to those skilled in the art, e.g., patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte hROR ⁇ expression; tissue culture cell hROR ⁇ expression; transcriptional activation of hROR ⁇ ; ligand binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, identification of reporter gene expression (CAT, luciferase, - gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, and the like.
- patch clamping voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte hROR ⁇ expression; tissue culture cell hROR ⁇ expression; transcriptional activation
- Binding compounds are those that have an association constant higher than an unrelated negative control compounds, usually with a KD of 10 "6 or better.
- Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, antagonize, or down regulate hROR ⁇ activity.
- Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate hROR ⁇ activity.
- Ligands bind to hROR ⁇ at twice background and can function as either activators or inhibitors. Activators and inhibitors of hROR ⁇ activity can also modulate ligand-hROR ⁇ interactions (i.e., "modulators of ligand binding"). Modulators include genetically modified versions of hROR ⁇ , e.g., with altered activity, as well as naturally occurring, genetically modified, and synthetic ligands, modulators. Modulators and ligands are typically peptides, proteins, polypeptides, oligonucleotides, small chemical or organic molecules, and the like.
- Such assays for modulators and ligands include, e.g., expressing hROR ⁇ in cells, cell extracts or providing hROR ⁇ in vitro reactions, applying putative modulator compounds, including putative ligands, and then determining the functional effects on hROR ⁇ activity, as described above.
- Samples or assays comprising hROR ⁇ that are treated with a potential ligand or modulator are compared to control samples without the ligand or modulator to examine the extent of inhibition.
- Control samples (untreated with the test compound) are assigned a relative hROR ⁇ activity value of 100%.
- Modulation/inhibition of hROR ⁇ activity is achieved when the hROR ⁇ activity value relative to the control is about 90%, preferably 50%, more preferably 25%.
- Modulation/activation of hROR ⁇ activity is achieved when the hROR ⁇ activity value relative to the control is 110%, more preferably 150%, more preferable 200% higher.
- isolated refers to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated hROR ⁇ nucleic acid is separated from open reading frames that flank the hROR ⁇ gene and encode proteins other than hROR ⁇ .
- purified denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
- Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
- the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral- methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
- PNAs peptide-nucleic acids
- nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
- polypeptide refers to a polymer of amino acid residues.
- the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
- Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
- polypeptide refers to glycoproteins, as well as non-glycoproteins.
- amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, carboxyglutamate, and O-phosphoserine.
- Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group., e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
- Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes (A, T, G, C, U, etc.).
- Constantly modified variants applies to both amino acid and nucleic acid sequences.
- conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
- degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ⁇ /., J. Biol. Chem.
- nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
- Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
- each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
- TGG which is ordinarily the only codon for tryptophan
- amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymo ⁇ hic variants and alleles of the invention.
- label or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
- useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes
- biotin e.g., as commonly used in an ELISA
- digoxigenin e.g., as commonly used in an ELISA
- haptens and proteins for which antisera or monoclonal antibodies are available e.g., the polypeptide of SEQ ID NO:2 can be made detectable, e.g., by inco ⁇ orating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).
- a "labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
- recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
- recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
- heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
- the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
- a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). See, e.g., Ausubel, supra, for an introduction to recombinant techniques.
- a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
- a promoter typically includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
- a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoters often have an element that is responsive to transactivation by a DNA-binding moiety such as a polypeptide, e.g., hROR ⁇ , Gal4, the lac repressor and the like.
- a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
- An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
- the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
- An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
- the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
- the expression vector includes an "expression cassette," which comprises a nucleic acid to be transcribed operably linked to a promoter.
- a "non- lambda expression vector” refers to an expression vector that does not replicate or transduce a cell in a lambda bacteriophage-dependent manner, e.g., does not require lambda packaging elements for transduction, or does not replicate using lambda proteins.
- nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
- This definition also refers to the complement of a test sequence, which has a designated percent sequence or subsequence complementarity when the test sequence has a designated or substantial identity to a reference sequence.
- a designated amino acid percent identity of 86% refers to sequences or subsequences that have at least about 86% amino acid identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
- the percent identity exists over a region of the sequence that is at least about 25 amino acids in length, more preferably over a region that is 50 amino acids in length.
- sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
- a conservative substitution is given a score between zero and 1.
- the scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
- PC/GENE Intelligents, Mountain View, California, USA.
- sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
- sequence comparison algorithm test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
- the sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated or default program parameters.
- a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 25 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- Methods of alignment of sequences for comparison are well-known in the art.
- Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J.
- PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sha ⁇ , CABIOS 5:151-153 (1989). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
- This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
- Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
- the final alignment is achieved by a series of progressive, pairwise alignments.
- the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
- PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
- PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12:387-395 (1984).
- HSPs high scoring sequence pairs
- initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
- the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues, always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
- the BLASTP program uses as default parameters a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl Acad. Sci. USA 89:10915 (1989)).
- the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat 7. Acad. Sci. USA
- nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
- nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
- a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
- Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. For the pu ⁇ oses of the present application, SEQ ID NOS:2 and 4, and SEQ ID NOS:l and 3 are not considered substantially identical.
- stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
- T m thermal melting point
- the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
- Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
- Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
- a positive signal is at least two times background, preferably 10 time background hybridization (see, e.g., the conditions described in Sambrook et al, supra).
- Exemplary "highly stringent" hybridization conditions include hybridization in a buffer comprising 50% formamide, 5x SSC, and 1% SDS at 42°C, or hybridization in a buffer comprising 5x SSC and 1% SDS at 65°C, both with a wash of 0.2x SSC and 0.1% SDS at 65°C.
- Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
- Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
- a further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers or a pool of degenerate primers that encode a conserved amino acid sequence, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.
- another indication that the sequences are substantially identical is if the same set of PCR primers can be used to amplify both sequences.
- Antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
- the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
- Light chains are classified as either kappa or lambda.
- Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
- An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
- Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
- the N- terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
- Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
- pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to V H -CH1 by a disulfide bond.
- the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
- the Fab' monomer is essentially an Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
- antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
- antibody as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).
- a “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
- an “anti-hROR ⁇ ” antibody is an antibody or antibody fragment that specifically binds to the C-terminal domain of SEQ ID NO:4 or a conservatively modified variation thereof but does not specifically bind to the C-terminal domain of SEQ ID NO:2.
- immunoassay is an assay that uses an antibody to specifically bind an antigen.
- the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
- the phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
- the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
- Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
- polyclonal antibodies raised to hROR ⁇ from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with hROR ⁇ and not with other proteins, except for polymo ⁇ hic variants and alleles of hROR ⁇ . This selection may be achieved by subtracting out antibodies that cross-react with hROR ⁇ molecules from other species.
- a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
- solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
- a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
- host cell refers to a cell that contains an expression vector and supports the replication or expression of the expression vector.
- Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells in vivo.
- Assays for hRORy activity hROR ⁇ and its alleles and polymo ⁇ hic variants are nuclear receptors that regulate transcription. Assays for hROR ⁇ activity can be used to test for ligands, inhibitors, and activators of hROR ⁇ , which can then be used to modulate hROR ⁇ - mediated transcription. For example, hROR ⁇ is involved in transcriptional regulation of allergic and inflammatory reactions, as well as in adipocyte differentiation.
- hROR ⁇ polypeptides can be assessed using a variety of in vitro and in vivo assays, e.g., measuring transcriptional activation of a reporter gene; current; membrane potential; ion flux, e.g., sodium, potassium, or calcium; intracellular ion concentration; second messengers levels (e.g., cAMP, IP3, or Ca 2+ ); transcription levels; phosphorylation and dephosphorylation of a reporter protein; receptor activation; neurotransmitter, cytokine, and hormone production levels; using e.g., voltage-sensitive dyes, radioactive tracers, patch-clamp electrophysiology, immunoassays, hybridization assays, and the like.
- in vitro and in vivo assays e.g., measuring transcriptional activation of a reporter gene; current; membrane potential; ion flux, e.g., sodium, potassium, or calcium; intracellular ion concentration; second messengers levels (e.g., cAMP, IP3,
- the hROR ⁇ of the assay will be selected from a polypeptide having a sequence of the C-terminal domain of S ⁇ Q ID NO:4 or conservatively modified variant thereof.
- amino acids 96-518 of S ⁇ Q ID NO:4 are used as the hROR ⁇ of the assay, either alone or as part of a fusion protein.
- amino acids 96-518 of the C-terminal domain of hROR ⁇ are fused to a GST domain.
- Modulators of hROR ⁇ activity are tested using biologically active hROR ⁇ and fragments thereof, either recombinant or naturally occurring.
- the protein can be isolated, expressed in a cell, or expressed in a membrane derived from a cell. Modulation is tested using one of the in vitro or in vivo assays described herein. Membranes or whole cells can be used.
- hROR ⁇ activity can also be examined in vitro with soluble or solid state reactions. Samples or assays that are treated with a potential hROR ⁇ inhibitor or activator are compared to control samples without the test compound, to examine the extent of modulation or ligand binding. Control samples (untreated with activators or inhibitors) are assigned a relative hROR ⁇ activity value of 100.
- Inhibition of hROR ⁇ is achieved when the hROR ⁇ activity value relative to the control is about 90%, preferably 50%, more preferably 25%. Activation of hROR ⁇ is achieved when the hROR ⁇ activity value relative to the control is 110%, more preferably 150%, more preferably 200% higher.
- the compounds to be tested are present in the range from 1 pM to 100 mM. The effects of the test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects hROR ⁇ activity can be used to assess the influence of a test compound on the polypeptides of this invention.
- hROR ⁇ activity can be performed in vitro or in vivo.
- hROR ⁇ activity is detected by examining transcription of a reporter RNA. Transcription of the reporter RNA is regulated either directly or indirectly by hROR ⁇ .
- hROR ⁇ can regulate transcription when an hROR ⁇ responsive element is present in the promoter operably linked to the reporter gene.
- the hROR ⁇ ligand binding domain can be linked to a heterologous DNA binding domain from another transcription factor such as Gal4, or to a DNA-binding moiety such as an oligonucleotide or a lac repressor. The heterologous DNA-binding domain or the DNA-binding moiety recognizes a sequence in the promoter operably linked to the reporter nucleic acid.
- the in vitro reaction mixture is first contacted with test ligands or modulators of hROR ⁇ activity.
- the selected reporter RNA which has been transcribed in the sample, is then contacted with oligonucleotides that are complementary to the selected RNA.
- Single-stranded RNA in the sample is cleaved, typically with RNases such as RNase A.
- the sample is then incubated with a recognition agent that binds to RNA duplexes.
- the recognition agent is detected either directly or indirectly, indicating the level of expression of the selected RNA. In this manner, a reporter RNA whose transcription is regulated by hROR ⁇ is detected.
- high throughput in vitro expression assays are provided.
- the transcription mixtures contain an expression cassette with a promoter operably linked to a DNA encoding a selected G-less or A-less RNA.
- the single-stranded RNA in the sample is cleaved at guanine residues, typically by RNase Tl, or at adenine residues, typically by RNase U2.
- the sample is then incubated with a recognition reagent that captures the RNA.
- the selected RNA is then detected either directly or indirectly, indicating the level of expression of the selected RNA.
- hROR ⁇ activity is detected by examining transcription of a reporter RNA in a cell. Transcription of the reporter RNA is regulated either directly or indirectly by hROR ⁇ , as described above.
- the in vivo reaction is first contacted with test ligands or modulators of hROR ⁇ activity.
- the selected reporter RNA which has been transcribed in the cell (from a transduced expression cassette or an endogenous gene), is contacted with an oligonucleotide which has a region complementary to the selected RNA.
- the oligonucleotide enters the cell (typically following permeabilization of the cell membrane, e.g., with a mild detergent or alcohol or a mild chaotropic agent), and forms an RNA duplex in the cell. Single stranded RNA in the cell is cleaved to reduce background in subsequent assays steps.
- the RNA duplex is then detected by contacting the RNA duplex with a recognition reagent such as an antibody which binds RNA homo- or hetero-duplexes.
- the recognition reagent is directly or indirectly detectable. This method is broadly applicable to the detection of RNA levels which result from transcription and processing of the RNA.
- hROR ⁇ activity is assessed using a dual construct system.
- This system can be used either in vitro or in vivo.
- a first expression cassette is made encodes a polypeptide comprising the C-terminal, ligand binding domain of hROR ⁇ fused to the DNA binding domain of Gal4.
- construct is encoded by an expression vector encoding a fusion protein having, from N- to C-terminus, Flag-Gal4- hROR ⁇ amino acids 96-518.
- a second expression cassette comprises a promoter with a Gal4 responsive element operably linked to a reporter gene such as luciferase.
- the second construct is an expression vector with four copies of the Gal4 responsive element in front of a gene encoding luciferase.
- a control plasmid is used as a control for transfection efficiency, e.g., a plasmid encoding ⁇ -gal.
- a plasmid encoding ⁇ -gal When the hROR ⁇ is contacted with a ligand, it stimulates transactivation of the luciferase reporter through the Gal4 DNA binding domain. Expression of the reporter gene is used to detect hROR ⁇ activity.
- This system thus can be used to screen for hROR ⁇ ligands as well as modulators of hROR ⁇ activity (see, e.g., Medvedev et al, Gene 181:199-206 (1996)).
- the plasmids are transfected into human embryonic kidney 293 cells via calcium phosphate transfection. after 24-48 hours, the cells are then treated with a chemical ligand library, and lucifera
- a peptide sensor is used to detect the hROR ⁇ -ligand complex (see, e.g., USSN 08/975,614, filed November 21, 1997, and USSN 09/163,713, filed September 30, 1998).
- the peptide sensor is derived from a co-activator protein, which contains short signature motifs that bind to the nuclear receptor only upon conformational changes that occur when the nuclear receptor binds its ligand (see Figure 1). Peptides that contain these short signature motifs retain the ability to bind the nuclear receptor-ligand complex.
- an "ELISA” type assay a plate is coated with the peptide sensor, and then the hROR ⁇ protein and potential ligands are added to each well in the plate. The plate is then subjected to an ELISA assay, using an antibody linked to an enzyme. When a ligand binds to hROR ⁇ it induces a conformational change in the nuclear receptor, allowing the peptide sensor to bind to the nuclear receptor (see Figure 1).
- the hROR ⁇ is a fusion protein, with the N-terminal domain derived from GST and the C-terminal domain comprising amino acids 96-518 of hROR ⁇ .
- the antibody used in the ELISA is typically an anti-GST antibody.
- a fluorescence polarization detection scheme is used to detect binding of the peptide sensor to the hROR ⁇ -ligand complex (see Figure 2).
- This detection scheme is based on the differences in rotation rates between unbound and bound states of fluorescently-labeled peptides. When the peptide is free in solution and has high rotational rates, the polarized light will be depolarized. When the peptides are bound to hROR ⁇ -ligand complexes (due to the altered conformation of the receptor), the peptides have a lower rotational rate, and polarization is preserved.
- Yet another assay for compounds that modulate hROR ⁇ activity involves computer assisted drug design, in which a computer system is used to generate a three- dimensional structure of hROR ⁇ based on the structural information encoded by the amino acid sequence.
- the input amino acid sequence interacts directly and actively with a preestablished algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the protein.
- the models of the protein structure are then examined to identify regions of the structure that have the ability to bind, e.g., ligands. These regions are then used to identify ligands that bind to hROR ⁇ and modulators of ligand binding and hROR ⁇ activity.
- the three-dimensional structural model of the protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a hROR ⁇ polypeptide into the computer system.
- the amino acid sequence of the polypeptide is selected from the group consisting of SEQ ID NO:4 and conservatively modified versions thereof.
- the amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein.
- At least 10 residues of the amino acid sequence are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM.
- electronic storage media e.g., magnetic diskettes, tapes, cartridges, and chips
- optical media e.g., CD ROM
- the three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
- the amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the protein of interest.
- the software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
- the tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure.
- the user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure.
- the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
- potential ligand binding regions are identified by the computer system.
- Three-dimensional structures for potential ligands are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above.
- the three-dimensional structure of the potential ligand is then compared to that of the hROR ⁇ protein to identify ligands that bind to hROR ⁇ . Binding affinity between the protein and ligands is determined using energy terms to determine which ligands have an enhanced probability of binding to the protein.
- the compounds tested as modulators and ligands of hROR ⁇ can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid.
- test compounds will be small chemical molecules and peptides.
- a preferred ligand is melatonin, or a melatonin analogue.
- modulators can be genetically altered versions of hROR ⁇ or its ligands.
- any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
- the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like. In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds).
- Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity.
- the compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
- a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
- a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
- combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)).
- chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No.
- nucleic acid libraries see Ausubel, Berger and Sambrook, all supra
- peptide nucleic acid libraries see, e.g., U.S. Patent 5,539,083
- antibody libraries see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287)
- carbohydrate libraries see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853
- small organic molecule libraries see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S.
- the invention provides solid phase based in vitro assays in a high throughput format.
- Control reactions that measure the expression level of the selected RNA in a reaction that does not include a transcription modulator are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in a preferred embodiment, the methods of the invention include such a control reaction.
- "no modulator" control reactions which do not include a modulator provide a background level of expression from a given coding DNA.
- ⁇ -amanitin a strong inhibitor of the pol II transcription complex
- modulators can also be combined with ligands to find modulators which inhibit transcriptional activation or transcriptional repression.
- each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
- a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds is possible using the integrated systems of the invention. More recently, micro fluidic approaches to reagent manipulation have been developed, e.g., by Caliper Technologies (Palo Alto, CA).
- polypeptides and nucleic acids are used in the assays described above. Such polypeptides and nucleic acids can be made using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994)).
- nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company ([email protected]), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others.
- peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic ([email protected]), HTI Bio-products, inc. (http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio. Synthesis, Inc., and many others.
- nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.
- kb kilobases
- bp base pairs
- proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
- Oligonucleotides can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al, Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
- the sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al, Gene 16:21-26 (1981). Again, as noted above, companies such as Operon Technologies, Inc. provide an inexpensive commercial source for essentially any oligonucleotide.
- nucleic acid sequences encoding hROR ⁇ and related nucleic acid sequence homologs are cloned from cDNA and genomic DNA libraries by hybridization with a probe, or isolated using amplification techniques with oligonucleotide primers.
- hROR ⁇ sequences are typically isolated from mammalian nucleic acid (genomic or cDNA) libraries by hybridizing with a nucleic acid probe, the sequence of which can be derived from SEQ ID NO:3.
- a suitable tissue from which hROR ⁇ RNA and cDNA can be isolated is skeletal muscle tissue or thymus tissue.
- Amplification techniques using primers can also be used to amplify and isolate hROR ⁇ from DNA or RNA.
- Degenerate primers comprising the amino acid subsequence "LSK" at the C-terminal end of the molecule, or conservatively modified variations thereof, can also be used to amplify a sequence of hROR ⁇ (see, e.g., Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995)). These primers can be used, e.g., to amplify either the full length sequence or a probe of one to several hundred nucleotides, which is then used to screen a mammalian library for full-length hROR ⁇ .
- Nucleic acids encoding hROR ⁇ can also be isolated from expression libraries using antibodies as probes. Such polyclonal or monoclonal antibodies can be raised using the sequence of SEQ ID NO:4 and are subtracted for cross-reactivity with antibodies that recognize the C-terminal domain of SEQ ID NO:2.
- hROR ⁇ polymo ⁇ hic variants and alleles that are substantially identical to hROR ⁇ can be isolated using hROR ⁇ nucleic acid probes, and oligonucleotides under stringent hybridization conditions, by screening libraries.
- expression libraries can be used to clone hROR ⁇ and hROR ⁇ polymo ⁇ hic variants and alleles, by detecting expressed homologs immunologically with antisera or purified antibodies made against hROR ⁇ , which also recognize and selectively bind to the hROR ⁇ homolog.
- a source that is rich in hROR ⁇ mRNA, e.g., skeletal muscle tissue or thymus tissue.
- the mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning.
- Methods for making and screening cDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook et al, supra; Ausubel et al, supra).
- the DNA is extracted from the tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in non-lambda expression vectors. These vectors are packaged in vitro. Recombinant phage are analyzed by plaque hybridization as described in Benton & Davis, Science 196:180-182 (1977). Colony hybridization is carried out as generally described in Grunstein et al, Proc. Natl Acad. Sci. USA., 72:3961-3965 (1975).
- hROR ⁇ nucleic acid and its homologs combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al, eds, 1990)).
- Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify nucleic acid sequences of hROR ⁇ directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
- Degenerate oligonucleotides can be designed to amplify hROR ⁇ homologs using the sequences provided herein. Restriction endonuclease sites can be inco ⁇ orated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of hROR ⁇ encoding mRNA in physiological samples, for nucleic acid sequencing, or for other pu ⁇ oses. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
- hROR ⁇ chimeras such as constructs encoding the C-terminal ligand binding domain of hROR ⁇ fused to the DNA binding domain of a heterologous protein such as Gal4 or the lac repressor.
- constructs encoding N-terminal deletions of hROR ⁇ can be constructed. These constructs can then be optionally linked to DNA binding moieties such as oligonucleotides, using methodology known to those of skill in the art.
- Gene expression of hROR ⁇ can also be analyzed by techniques known in the art, e.g., reverse transcription and PCR amplification of mRNA, isolation of total RNA or poly A+ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, probing DNA microchip arrays, and the like. All of these techniques are standard in the art.
- Synthetic oligonucleotides can be used to construct recombinant hROR ⁇ genes for use as probes or for expression of protein. This method is performed using a series of overlapping oligonucleotides usually 40-120 bp in length, representing both the sense and non-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned. Alternatively, amplification techniques can be used with precise primers to amplify a specific subsequence of the hROR ⁇ nucleic acid. The specific subsequence is then ligated into an expression vector.
- the nucleic acid encoding hROR ⁇ is typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
- These intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors.
- a cloned gene or nucleic acid such as those cDNAs encoding hROR ⁇
- Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al.
- Bacterial expression systems for expressing the hROR ⁇ protein are available in, e.g., E.
- Kits for such expression systems are commercially available.
- Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
- the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
- the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
- the promoter typically also includes elements that are responsive to transactivation, e.g., hROR ⁇ responsive elements, Gal4 responsive elements, lac repressor responsive elements, and the like.
- the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the hROR ⁇ encoding nucleic acid in host cells.
- a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding hROR ⁇ and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
- the nucleic acid sequence encoding hROR ⁇ may typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell.
- signal peptides would include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens.
- Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
- the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
- the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc. Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Ban * virus.
- SV40 vectors e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Ban * virus.
- exemplary eukaryotic vectors include pMSG, pAN009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
- Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
- high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a hROR ⁇ encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
- the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
- the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
- the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
- Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of hROR ⁇ protein, which are then purified using standard techniques (see, e.g., Colley et al, J. Biol. Chem. 264:17619- 17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101 :347-362 (Wu et al, eds, 1983).
- Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing hROR ⁇ .
- the transfected cells are cultured under conditions favoring expression of hROR ⁇ , which is recovered from the culture using standard techniques identified below.
- the C-terminal domain of hROR ⁇ has at least two major regulatory functions: it mediates ligand binding and homo-or heterodimer formation. Consequently, both the full length hROR ⁇ protein and the C-terminal domain of hROR ⁇ are suitable for ligand screening. Both the full length protein and the C-terminal domain are also suitable for examining regulation of hetero- and homodimer formation.
- Common domains for addition to the C-terminal domain of hROR ⁇ include transcription factors (activators), silencers, nuclear receptors, general transcription machinery and modifiers of these factors, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members etc.), tumor promoters, metastasis and invasiveness promoters or suppressors and their associated factors and modifiers; tumor suppressors (e.g., p53, WT1, MDM2, Rb family) and their associated factors and modifiers; DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and their associated factors and modifiers, cell cycle proteins and their associated factors and modifiers; chromatin associated proteins and their modifiers (e.g.
- DNA modifying enzymes e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases
- DNA modifying enzymes e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases
- RNA modifying enzymes and their associated factors and modifiers RNA binding factors (directly or indirectly) and their associated factors and modifiers, factors that control chromatin, DNA, RNA and RNP (ribonuclear protein) structure, movement and localization and their associated factors and modifiers
- factors derived from microbes e.g., prokaryotes, eukaryotes and virus
- hROR ⁇ protein can include a subsequence derived from a complete protein
- portions of proteins that are capable of binding to nucleic acids, directly or indirectly, are also useful as domains for addition to the C-terminal hROR ⁇ domain.
- assays such as an electrophoretic mobility shift assay (EMSA) (Scott et al, J. Biol. Chem. 269:19848- 19858 (1994)), in which a nucleic acid sequence of interest is allowed to associate with various fragments of a molecule that is capable of binding to the nucleic acid sequence.
- ESA electrophoretic mobility shift assay
- nucleic acid binding moieties Another method by which one can identify nucleic acid binding moieties is DNase I footprinting.
- Common polypeptides from which one can obtain a protein domain include polypeptides that are involved in transcription, for either or both regulated and basal transcription. Such polypeptides include transcription factors and coactivators, silencers, nuclear receptors, general transcription machinery and modifiers of these factors. See, e.g., Goodrich et al, Cell 84:825-30 (1996) for a review of proteins and nucleic acid elements involved in transcription. Transcription factors in general are reviewed in Barnes & Adcock, Clin. Exp. Allergy 25 Suppl.
- TATA box binding protein TBP
- TAF TAF box binding protein
- TAF28, TAF55, TAF80, TAF110, TAF150, and TAF250 TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250
- TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250 TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250
- STAT family of transcription factors are reviewed in, for example,
- Kinases, phosphatases, and other proteins that modify polypeptides involved in gene regulation are also useful for making hROR ⁇ C-terminal chimeras. Such modifiers are often involved in switching on or off transcription mediated by, for example, hormones.
- Kinases involved in transcription regulation are reviewed in Davis, Mol Reprod. Dev. 42:459-67 (1995), Jackson et al, Adv. Second Messenger Phosphoprotein Res. 28:279-86 (1993), and Boulikas, Crit. Rev. Eukaryot. Gene Expr. 5:1-77 (1995), while phosphatases are reviewed in, for example, Schonthal & Semin, Cancer Biol 6:239-48 (1995).
- Nuclear tyrosine kinases are described in Wang, Trends Biochem. Sci. 19:373-6 (1994).
- useful domains can also be obtained from the gene products of oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.) and tumor suppressors (e.g., p53, WT1, MDM2, Rb family, and the like) and their associated factors and modifiers.
- oncogenes e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.
- tumor suppressors e.g., p53, WT1, MDM2, Rb family, and the like
- Oncogenes are described in, for example, Cooper, Oncogenes, 2nd ed., The Jones and Bartlett Series in Biology, Boston, MA, Jones and Bartlett Publishers, 1995.
- the ets transcription factors are reviewed in Waslylk et al
- Tumor promoters, metastasis and invasiveness promoters or suppressors and their associated factors and modifiers are also suitable. Proteins involved in carcinogenesis, including tumor suppressors and activators, are reviewed in Schmandt et al, Clin. Chem. 39(11 Pt 2):2375-85 (1993) and Kelley et al, Adv. Intern. Med. 39:93- 122 (1994). Tumor suppressors are reviewed in Hinds et al, Curr. Opin. Genet. Dev. 4:135-41 (1994). The p53 tumor suppressor in particular is described in Hainaut, Curr. Opin. Oncol. 7:76-82 (1995) and Cox & Lane, Bioessays, 17:501-8 (1995), while the Rb family is reviewed in Sidle et al, Crit. Rev. Biochem. Mol Biol. 31 :237-71 (1996).
- Chimeric hROR ⁇ proteins can include a polypeptide subsequence that is obtained from DNA repair enzymes and their associated factors and modifiers.
- DNA repair systems are reviewed in, for example, Vos, Curr. Opin. Cell Biol. 4:385-95 (1992); S ca ⁇ , Ann. Rev. Genet. 29:69-105 (1995); Lehmann, Genet. Eng. 17:1-19 (1995); and Wood, Ann. Rev. Biochem. 65:135-67 (1996).
- DNA rearrangement enzymes and their associated factors and modifiers see, e.g., Gangloff et al, Experientia 50:261-9 (1994); Sadowski, FASEB J.
- cell cycle proteins and their associated factors and modifiers are also useful as domains for addition to hROR ⁇ .
- proteins involved in DNA replication can be used to construct chimeric hROR ⁇ s.
- DNA replication proteins are described in Kearsey et al, Curr. Opin. Genet. Dev. 6:208-14 (1996) and Donovan et al, Curr. Opin. Genet. Dev. 6:203-7 (1996).
- Cell cycle proteins are also described in Stein et al, Int. J. Obes. Relat. Metab. Disord. 20 Suppl 3:S84-90 (1996).
- hROR ⁇ chimeras include fusions between Gal4 and the hROR ⁇ C-terminal domain, and fusions between GST (glutathione-S-transferase) and the ROR ⁇ C-terminal domain.
- chimeric polypeptides can be derived from DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases) and their associated factors and modifiers.
- DNA modifying enzymes e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases
- Helicases are reviewed in Matson et al, Bioessays, 16:13-22 (1994), and methyltransferases are described in Cheng, Curr. Opin. Struct. Biol. 5:4-10 (1995).
- Chromatin associated proteins and their modifiers e.g., kinases, acetylases and deacetylases
- histone deacylase Wolffe, Science 272:371-2 (1996)
- RNA modifying enzymes and their associated factors and modifiers, RNA binding factors (directly or indirectly) and their associated factors and modifiers can also be added to hROR ⁇ C-terminal domains.
- RNA binding factors directly or indirectly
- RNA binding factors can also be added to hROR ⁇ C-terminal domains.
- RNP domains are reviewed in Nagai et al, Trends Biochem. Sci. 20:235-40 (1995).
- Factors that control chromatin, DNA, RNA and RNP (ribonuclear protein) structure, movement and localization and their associated factors and modifiers; factors derived from microbes (e.g., prokaryotes, eukaryotes and virus) and factors that associate with or modify them can also be used to obtain chimeric proteins.
- Linker domains between polypeptide domains can be included. Such domains are typically polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. In some embodiments, proline residues are inco ⁇ orated into the linker to prevent the formation of significant secondary structural elements by the linker. Preferred linkers are often flexible amino acid subsequences which are synthesized as part of a recombinant fusion protein. In one embodiment, the flexible linker is an amino acid subsequence comprising a proline such as Gly(x)-Pro-Gly(x) where x is a number between about 3 and about 100. In other embodiments, a chemical linker is used to connect synthetically or recombinantly produced domain sequences.
- Such flexible linkers are known to persons of skill in the art.
- poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
- oligonucleotide is chemically linked to a polypeptide to confer specific DNA binding activity upon the protein.
- An example of an oligonucleotide being chemically linked to a protein (micrococcal endonuclease) by chemical coupling is found in Corey et al, Biochemistry 28:8277-8286 (1989).
- Either naturally occurring or recombinant hROR ⁇ can be purified for use in functional assays.
- recombinant hROR ⁇ is purified.
- Naturally occurring hROR ⁇ is purified, e.g., from mammalian tissue such as thymus or skeletal muscle tissue, and any other source of a hROR ⁇ homo log.
- Recombinant hROR ⁇ is purified from any suitable expression system, e.g., by expressing hROR ⁇ in E.
- the recombinant hROR ⁇ is a fusion protein, e.g., with GST or Gal4 at the N-terminus.
- hROR ⁇ may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al, supra; and Sambrook et al, supra).
- hROR ⁇ proteins having established molecular adhesion properties can be reversible fused to hROR ⁇ . With the appropriate ligand, hROR ⁇ can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, hROR ⁇ could be purified using immunoaffmity columns.
- A. Purification ofhRORyfrom recombinant bacteria Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is a one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein.
- inclusion bodies Proteins expressed in bacteria may form insoluble aggregates ("inclusion bodies").
- purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF.
- the cell suspension can be lysed using 2-3 passages through a French press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et al, supra; Ausubel et al, supra).
- the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter. Proteins that formed the inclusion bodies may be renatured by dilution or dialysis with a compatible buffer.
- suitable solvents include, but are not limited to urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M).
- Some solvents which are capable of solubilizing aggregate- forming proteins are inappropriate for use in this procedure due to the possibility of i ⁇ eversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
- SDS sodium dodecyl sulfate
- 70% formic acid are inappropriate for use in this procedure due to the possibility of i ⁇ eversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity.
- guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of immunologically and/or biologically active protein.
- Other suitable buffers are known to those skilled in the art.
- hROR ⁇ is separated from other bacterial proteins by standard separation techniques, e.g., with Ni- NT A agarose resin.
- the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art.
- the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose.
- the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO 4 and kept in an ice bath for approximately 10 minutes.
- the cell suspension is centrifuged and the supernatant decanted and saved.
- the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
- an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
- the preferred salt is ammonium sulfate.
- Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
- a typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins.
- the precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest.
- the precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration.
- Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
- the molecular weight of hROR ⁇ can be used to isolated it from proteins of greater and lesser size using ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes).
- membranes of different pore size for example, Amicon or Millipore membranes.
- the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cutoff than the molecular weight of the protein of interest.
- the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
- the recombinant protein will pass through the membrane into the filtrate.
- the filtrate can then be chromatographed as described below.
- Column Chromatographv hROR ⁇ can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands.
- antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
- immunoassays In addition to the detection of hROR ⁇ genes and gene expression using nucleic acid hybridization technology, one can also use immunoassays to detect hROR ⁇ or to measure hROR ⁇ activity, e.g., to identify hROR ⁇ ligands and modulators of hROR ⁇ activity. Immunoassays can be used to qualitatively or quantitatively analyze hROR ⁇ . A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
- Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science 246:1275-1281 (1989); Ward et al, Nature 341 :544-546 (1989)).
- many companies such as BMA Biomedicals, Ltd., HTI Bio-products, and the like, provide the commercial service of making an antibody to essentially any peptide.
- a number of hROR ⁇ comprising immunogens may be used to produce antibodies specifically reactive with hROR ⁇ .
- Recombinant hROR ⁇ or an antigenic fragment thereof is isolated as described herein.
- Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above.
- Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
- a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
- Naturally occurring protein may also be used either in pure or impure form.
- the product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
- mice e.g., BALB/C mice
- standard animals such as Freund's adjuvant, and a standard immunization protocol
- the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to hROR ⁇ .
- blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see Harlow & Lane, supra).
- Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
- DNA sequences which encode a monoclonal antibody or a binding fragment thereof may be isolated by screening a DNA library from human B cells according to the general protocol outlined by Huse et al, Science 246:1275-1281 (1989). Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Typically, polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non-hROR ⁇ proteins or even other related proteins, e.g., from other organisms, using a competitive binding immunoassay.
- polypeptides of SEQ ID NO:2 and SEQ ID NO:4 can be distinguished in this manner.
- Specific polyclonal antisera and monoclonal antibodies will usually bind with a KD of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
- hROR ⁇ can be detected by a variety of immunoassay methods.
- immunoassay methods see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991).
- the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.
- Immunological binding assays hROR ⁇ can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241 ; 4,376,110; 4,517,288; and 4,837,168).
- U.S. Patents 4,366,241 ; 4,376,110; 4,517,288; and 4,837,168 See, e.g., U.S. Patents 4,366,241 ; 4,376,110; 4,517,288; and 4,837,168.
- Methods in Cell Biology Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).
- Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case the hROR ⁇ or antigenic subsequence thereof).
- the antibody e.g., anti -hROR ⁇
- the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
- Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen.
- the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
- the labeling agent may be a labeled hROR ⁇ polypeptide or a labeled anti-hROR ⁇ antibody.
- the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/hROR ⁇ complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
- Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent.
- the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin.
- detectable moieties are well known to those skilled in the art.
- incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.
- Non-Competitive Assay Formats Immunoassays for detecting hROR ⁇ in samples may be either competitive or noncompetitive.
- Noncompetitive immunoassays are assays in which the amount of antigen is directly measured.
- the anti- hROR ⁇ antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture hROR ⁇ present in the test sample. hROR ⁇ is thus immobilized is then bound by a labeling agent, such as a second hROR ⁇ antibody bearing a label.
- the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
- the second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.
- the amount of hROR ⁇ present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) hROR ⁇ displaced (competed away) from an anti-hROR ⁇ antibody by the unknown hROR ⁇ present in a sample.
- a known amount of hROR ⁇ is added to a sample and the sample is then contacted with an antibody that specifically binds to hROR ⁇ .
- the amount of exogenous hROR ⁇ bound to the antibody is inversely proportional to the concentration of hROR ⁇ present in the sample.
- the antibody is immobilized on a solid substrate.
- the amount of hROR ⁇ bound to the antibody may be determined either by measuring the amount of hROR ⁇ present in a hROR ⁇ /antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein.
- the amount of hROR ⁇ may be detected by providing a labeled hROR ⁇ molecule.
- a hapten inhibition assay is another preferred competitive assay. In this assay the known hROR ⁇ , is immobilized on a solid substrate. A known amount of anti- hROR ⁇ antibody is added to the sample, and the sample is then contacted with the immobilized hROR ⁇ .
- the amount of anti-hROR ⁇ antibody bound to the known immobilized hROR ⁇ is inversely proportional to the amount of hROR ⁇ present in the sample.
- the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
- Immunoassays in the competitive binding format can also be used for crossreactivity determinations.
- a protein at least partially encoded by SEQ ID NO:4 can be immobilized to a solid support.
- Proteins e.g., hROR ⁇ having the sequence of SEQ ID NO:2 are added to the assay that compete for binding of the antisera to the immobilized antigen.
- the ability of the added protein to compete for binding of the antisera to the immobilized protein is compared to the ability of hROR ⁇ encoded by SEQ ID NO:4 to compete with itself.
- the percent crossreactivity for the above proteins is calculated, using standard calculations.
- Those antisera with less than 10% crossreactivity with the added protein are selected and pooled.
- the cross-reacting antibodies are optionally removed from the pooled antisera by immunoabso ⁇ tion with the added protein corresponding to SEQ ID NO:2.
- the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps an allele or polymo ⁇ hic variant of hROR ⁇ , to the immunogen protein (i.e., hROR ⁇ of SEQ ID NO:4).
- the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the protein encoded by SEQ ID NO:4 that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to a hROR ⁇ immunogen.
- the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind hROR ⁇ .
- a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
- the anti-hROR ⁇ antibodies specifically bind to the hROR ⁇ on the solid support.
- These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-hROR ⁇ antibodies.
- LOA liposome immunoassays
- the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
- the detectable group can be any material having a detectable physical or chemical property.
- Such detectable labels have been well- developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
- a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
- magnetic beads e.g., DYNABEADSTM
- fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
- radiolabels e.g., 3 H, 125 1, 35 S, 14 C, or 32 P
- enzymes e.g., horse radish per
- the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
- Non-radioactive labels are often attached by indirect means.
- a ligand molecule e.g., biotin
- the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- the ligands and their targets can be used in any suitable combination with antibodies that recognize hROR ⁇ , or secondary antibodies that recognize anti-hROR ⁇ .
- the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
- Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases.
- Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
- Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
- means for detection include a scintillation counter or photographic film as in autoradiography.
- the label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence.
- the fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
- enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
- simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
- agglutination assays can be used to detect the presence of the target antibodies.
- antigen-coated particles are agglutinated by samples comprising the target antibodies.
- none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
- Transgenic mice expressing hROR ⁇ can be made by simple insertion of hROR ⁇ into the mouse genome or by homologous recombination, in a pluripotent cell line that is capable of differentiating into germ cell tissue.
- a DNA construct that contains hROR ⁇ is introduced into the nuclei of embryonic stem cells. In a portion of the cells, the introduced DNA recombines with the endogenous copy of the mouse gene, replacing it with the human copy.
- cells can be selected that express both the endogenous and human genes.
- knock-out mice can be made, in which the endogenous ROR ⁇ gene is replaced by a marker gene such as neo.
- Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re- implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al, Science 244:1288 (1989)). Cells and animals that have one or more functionally disrupted endogenous genes or that express an exogenous gene have various commercial applications.
- a transgenic mouse that is heterozygous or homozygous for integrated transgenes that have functionally disrupted the endogenous hROR ⁇ gene can be used as a sensitive in vivo screening assay for hROR ⁇ ligands and modulators of hROR ⁇ activity.
- Chimeric targeted mice can be derived according to Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
- Kits hROR ⁇ and its homologs are a useful tool for identifying thymus and skeletal muscle tissue, for forensics and paternity determinations, and for examining transcriptional regulation by assaying for hROR ⁇ ligands and modulators of hROR ⁇ activity.
- hROR ⁇ specific reagents that specifically hybridize to hROR ⁇ nucleic acid such as hROR ⁇ probes and primers
- hROR ⁇ specific reagents that specifically bind to the hROR ⁇ protein e.g., hROR ⁇ antibodies are typically used to examine transcriptional regulation.
- Nucleic acid assays for the presence of h ⁇ DNA and RNA in a sample include numerous techniques are known to those skilled in the art, such as Southern analysis, northern analysis, dot blots, RNase protection, S 1 analysis, amplification techniques such as PCR and LCR, and in situ hybridization.
- in situ hybridization for example, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular mo ⁇ hology for subsequent inte ⁇ retation and analysis.
- hROR ⁇ protein can be detected with the various immunoassay techniques described above.
- the test sample is typically compared to both a positive control (e.g., a sample expressing recombinant hROR ⁇ ) and a negative control.
- kits for screening for modulators of hROR ⁇ can be prepared from readily available materials and reagents.
- such kits can comprise any one or more of the following materials: hROR ⁇ , reaction tubes, and instructions for testing hROR ⁇ activity.
- a wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user.
- the kit can be tailored for in vitro or in vivo assays for measuring the activity of hROR ⁇ .
- GTC AAG TTC GGC CGC ATG TCC AAG AAG CAG AGG GAC .AGC CT CAT GCA GAA GTG CAG .AAA 350
- GAG GCA CCC TAT GCC TCC CTG ACA GAG ATA GAG CAC CTG GTG CAG AGC GTC - ⁇ C AAG -*-- 840
- GGC 1020 A a K T ⁇ A Z Q Y V V E F A X R l 3 G
- CAG C*G CAG "AC AAT CTG GAG CTG GCC TTT CAT CAT CAT CTC TGC AAG ACT CAT CGC CAA 1330
- ATC GAC AGG GCC CCA CAG AGA CAG CAC CGA GCC TCA CGG GAG CTG CTG GCT GCA AAG AAG SO lr " n -J A ? Q R Q K R A , S R ⁇ ___.
- GGC AGC CAG CTG ACC CCT GAC CGA TGT GGA CTT CGT TTT GAG GAA CAC AGG CAT CCT GGG 720 G S Q L T ? D R C G R F ⁇ ⁇ K R H ? G
- TAC AGG GAG ACA TGC CAG CTG CGG CTG GAG GAC CTG CTG CGG CAG CGC TCC AAC ATC TTC 900 ⁇ a ⁇ T C Q L R L E D L L R Q R S N .. -- ⁇ G CGG GAG GAA GTG ACT GGC TAC CAG AGG -AAG TCC ATG TGG GAG ATG TGG GAA CGG TGT 960 S R S ⁇ V T G Y Q K S M E M E R C
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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AU12103/00A AU1210300A (en) | 1998-10-23 | 1999-10-18 | Human retinoid-like orphan receptor gamma |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US17835898A | 1998-10-23 | 1998-10-23 | |
US09/178,358 | 1998-10-23 |
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WO2000024757A1 WO2000024757A1 (en) | 2000-05-04 |
WO2000024757A9 true WO2000024757A9 (en) | 2000-10-19 |
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PCT/US1999/024309 WO2000024757A1 (en) | 1998-10-23 | 1999-10-18 | Human retinoid-like orphan receptor gamma |
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AU (1) | AU1210300A (en) |
WO (1) | WO2000024757A1 (en) |
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US20040265809A1 (en) * | 2001-05-07 | 2004-12-30 | Dino Moras | Polypeptides derived from retinoic acid-related orphan receptor(ror) and their applications |
WO2004024879A2 (en) | 2002-09-16 | 2004-03-25 | Exelixis, Inc. | RORs AS MODIFIERS OF THE p21 PATHWAY AND METHODS OF USE |
AU2003292144A1 (en) * | 2002-11-27 | 2004-06-18 | Develogen Aktiengesellschaft Fur Entwicklungsbiologische Forschung | Proteins involved in the regulation of energy homeostasis |
US8389739B1 (en) | 2006-10-05 | 2013-03-05 | Orphagen Pharmaceuticals | Modulators of retinoid-related orphan receptor gamma |
EP2181710A1 (en) * | 2008-10-28 | 2010-05-05 | Phenex Pharmaceuticals AG | Ligands for modulation of orphan receptor-gamma (NR1F3) activity |
-
1999
- 1999-10-18 AU AU12103/00A patent/AU1210300A/en not_active Abandoned
- 1999-10-18 WO PCT/US1999/024309 patent/WO2000024757A1/en active Application Filing
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