WO2000024757A1 - Human retinoid-like orphan receptor gamma - Google Patents

Abstract

The invention provides isolated nucleic acid and amino acid sequences of hRORη, antibodies to hRORη, transgenic animals, methods of identifying ligands for hRORη, and methods of screening for modulators of hRORη.

Description

HUMAN RETINOID-LIKE ORPHAN RECEPTOR GAMMA

CROSS-REFERENCES TO RELATED APPLICATIONS This application claims priority to USSN 09/178,358, filed October 23, 1998, herein incorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH AND

DEVELOPMENT

Not applicable

FIELD OF THE INVENTION The invention provides, for example, isolated nucleic acid and amino acid sequences of hRORγ, antibodies to hRORγ, transgenic animals, methods of identifying ligands for hRORγ, and methods of screening for modulators of hRORγ.

BACKGROUND OF THE INVENTION 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 fmger 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)). Typically, nuclear receptors 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 RORγ 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, RORγ 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

(1998)). These subfamily receptors have also been reported to recognize retinoic acid and thyroid hormone, as well as melatonin (Ortiz et al, supra; Carlberg & Wiesenberg, J. Pineal Res. 18:171-178 (1995)).

Although RORγ 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 RORγ 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. The genomic structure and chromosomal location of hRORγ has also been identified (Medvedev et al, Genomics 46:93-102 (1997)). Identification of the ligand for hRORγ would be useful for modulating hRORγ -mediated transcriptional regulation.

SUMMARY OF THE INVENTION The present invention provides the surprising discovery of a sequence of hRORγ that is substantially different than SEQ ID NO:2, the previously hypothesized amino acid sequence of hRORγ. In particular, the new amino acid sequence for hRORγ, 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 hRORγ of SEQ ID NO:2 is set forth in SEQ ID NO:l; a nucleic acid encoding the hRORγ of SEQ ID NO:4 is set forth in SEQ ID NO:3). In addition, as shown in SEQ ID NO:4, the end of the C-terminal domain comprises the amino acid subsequence LSK, which is not found in SEQ ID NO:2.

This discovery of a new C-terminal domain for hRORγ provides new hRORγ polypeptides, nucleic acids, assays for hRORγ ligand binding (mediated by the C- terminal of the new hRORγ polypeptide domain) and other features which will be apparent upon complete review of this disclosure.

As noted, 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 hRORγ 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 hRORγ 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. Similarly, the antibody should also preferentially bind SEQ ID NO:4 over known hRORγ homologs, such as the murine ROR.

If the hRORγ polypeptide comprises the C-terminal domain of the polypeptide of SEQ ID NO:4, the hRORγ polypeptide is essentially similar to the polypeptide of SEQ ID NO:4. For example, 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. In addition to antibody binding, the test polypeptide can be tested for other biological activities similar to the polypeptide of SEQ ID NO:4, such as binding to the hRORγ ligand melatonin. The hRORγ polypeptide is optionally chimer , comprising, e.g., heterologous amino acid subsequences such as DNA binding domains from proteins other than hRORγ. The hRORγ 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 hRORγ 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 hRORγ polypeptide are also provided. In particular, non-lambda expression vectors including a recombinant expression cassette encoding the hRORγ polypeptides above are provided. An example nucleic acid sequence which the vector can encode is set forth at SEQ ID NO:3. As with the polypeptide above, hRORγ 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 hRORγ polypeptide are also provided, both in vitro and in vivo. In the methods, the hRORγ polypeptide is contacted with a first ligand and ligand binding is monitored to determine whether the ligand binds to the polypeptide. A variety of assay formats is appropriate, e.g., where hRORγ 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. In one embodiment, a peptide sensor is used, 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 hRORγ polypeptide with the potential modulator of ligand binding, before, during, or after incubating the ligand and hRORγ polypeptide. Essentially any compound can be tested for such modulatory activity.

Kits embodying any of the compositions or methods above, e.g., comprising an hRORγ polypeptide or nucleic acid and further comprising one or more of a container, instructions for practicing a method herein, buffers, ligands, antibodies to hRORγ, or the like, are provided.

BRIEF DESCRIPTION OF THE DRAWINGS 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. In this assay, 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.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides, for the first time, the correct nucleotide sequence for human retinoid-like orphan receptor gamma ("hRORγ"), which encodes an approximately 60 kDa protein with a C-terminal domain that differs significantly from previously reported hRORγ protein sequences. hRORγ receptors are ligand-activated transcription factors that have both DNA-binding and ligand binding domains. hRORγ 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. Gene 181 :199-206 (1996); Medvedev et al, Genomics 46:93-102 (1997); Hirose et al, Bioch. BiophysRes. Comm. 205:1976-1983 (1994); Ortiz et al, Mol. Endo. 9:1679-1691 (1995)). 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 hRORγ is involved in ligand binding and in homo/heterodimer formation, and is thus an important domain for hRORγ regulation. hRORγ nucleic acids provide specific probes for tissues such as skeletal muscle and thymus, as nucleic acids encoding hRORγ 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 hRORγ, 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 hRORγ and methods of screening for modulators, e.g., activators, inhibitors, stimulators, enhancers, agonists, and antagonists, of hRORγ activity. Such modulators of hRORγ 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 hRORγ activity. Thus, the invention provides assays for hRORγ ligands and modulators, where hRORγ acts as an direct or indirect reporter molecule for the effect of modulators and ligands on transcriptional activation. hRORγ can be used in ligand binding and modulator assays, e.g., to measure changes in reporter gene transcription (luciferase, CAT, β-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, Ca²⁺), in vitro, in vivo, and ex vivo. In one embodiment, the C-terminal domain (containing the ligand binding domain) of hRORγ 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. Ligand binding to hRORγ activates the Gal4 DNA-binding domain and promotes transcription of the reporter gene. This system can also be used to test for modulators of RORγ ligand binding (see, e.g., Medvedev et al, 1996, supra).

The invention also provides for methods of detecting hRORγ nucleic acid and protein expression, allowing investigation of transcriptional regulation. hRORγs also provide useful nucleic acid probes for paternity and forensic investigations. hRORγs are useful nucleic acid probes identifying immune tissue such as the thymus, as well as skeletal muscle tissue. hRORγs can also be used to generate monoclonal and polyclonal antibodies useful for identifying, e.g., thymus and skeletal muscle tissue. hRORγ 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.

As described above, hRORγ functionally represents a nuclear receptor involved in transcriptional activation in response to ligand binding. Structurally, the nucleotide sequence of hRORγ (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 hRORγ 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. hRORγ 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 hRORγ 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 hRORγ nucleotide and amino acid sequence may be used to identify polymoφhic variants and alleles of hRORγ. 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 hRORγ or a conserved region thereof but not to the sequence of SEQ ID NO:2 can also be used to identify alleles and polymoφhic variants. The polymoφhic variants and alleles are expected to retain the ability to bind a ligand and activate transcription. hRORγ 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 hRORγ. Such compounds that modulate the activity of hRORγ or that bind to hRORγ can be used to investigate the role of hRORγ in transcriptional regulation.

The identification of the correct amino acid and nucleotide sequence of hRORγ thus provides, for the first time, a means for assaying for ligands, inhibitors, and activators of hRORγ mediated transcriptional regulation. Methods of detecting hRORγ nucleic acids and expression of hRORγ are also useful for identifying thymus and skeletal muscle and for studying regulation of transcriptional activation.

II. Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

"hRORγ" 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 hRORγ therefore refers to polymoφhic 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. The "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.

"hRORγ activity" refers to the ability of a polypeptide to bind ligand via an hRORγ ligand binding domain and regulate transcription ("ligand-binding activity"). hRORγ activity can also refer to the ability of a polypeptide to bind DNA via a DNA- binding domain ("DNA-binding activity"). Optionally, the hRORγ ligand binding domain can be fused to a heterologous DNA binding moiety or domain.

The term "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," as used herein, is a sample of biological tissue or fluid that contains hRORγ or nucleic acid encoding hRORγ 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 puφoses. 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 hRORγ, and cellular extracts from such cells.

The phrase "modulates the activity" in the context of assays for screening compounds that modulate hRORγ includes the determination of any parameter that is indirectly or directly under the influence of hRORγ activity. The phrase "determining whether a ligand binds the polypeptide" also includes the determination of any parameter that is indirectly or directly under the influence of hRORγ activity. Such parameters include, e.g., measuring changes in reporter gene transcription (luciferase, CAT, β- 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, Ca²⁺), in vitro, in vivo, and ex vivo. 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 hRORγ expression; tissue culture cell hRORγ expression; transcriptional activation of hRORγ; 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.

"Ligands," and "modulators" of hRORγ refer to binding, inhibitory or activating molecules for hRORγ activity identified using in vitro and in vivo assays, e.g., ligands, agonists, antagonists, and their homologs and mimetics. 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 hRORγ activity. Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate hRORγ activity. Ligands bind to hRORγ at twice background and can function as either activators or inhibitors. Activators and inhibitors of hRORγ activity can also modulate ligand-hRORγ interactions (i.e., "modulators of ligand binding"). Modulators include genetically modified versions of hRORγ, 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 hRORγ in cells, cell extracts or providing hRORγ in vitro reactions, applying putative modulator compounds, including putative ligands, and then determining the functional effects on hRORγ activity, as described above.

Samples or assays comprising hRORγ 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 hRORγ activity value of 100%. Modulation/inhibition of hRORγ activity is achieved when the hRORγ activity value relative to the control is about 90%, preferably 50%, more preferably 25%. Modulation/activation of hRORγ activity is achieved when the hRORγ activity value relative to the control is 110%, more preferably 150%, more preferable 200% higher.

The terms "isolated" "purified" or "biologically pure" refer 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 hRORγ nucleic acid is separated from open reading frames that flank the hRORγ gene and encode proteins other than hRORγ. The term "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). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer 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. The terms "polypeptide," "peptide" and "protein" include glycoproteins, as well as non-glycoproteins. The term "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.).

"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular 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. Specifically, 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 al, J. Biol. Chem. 260:2605- 2608 (1985); Rossolini et al, Mol. Cell Probes 8:91-98 (1994)). Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon in an amino acid herein, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such 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. One of skill will recognize that 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) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

As to 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 polymoφhic variants and alleles of the invention.

The following groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G); 2) Serine (S), Threonine (T);

3) Aspartic acid (D), Glutamic acid (E);

4) Asparagine (N), Glutamine (Q);

5) Cysteine (C), Methionine (M);

6) Arginine (R), Lysine (K), Histidine (H); 7) Isoleucine (I), Leucine (L), Valine (V); and

8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, Proteins (1984) for a discussion of amino acid properties).

A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes

(e.g., as commonly used in an ELISA), biotin, digoxigenin, or 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 incoφorating 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.

The term "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. Thus, for example, 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.

The term "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. For instance, 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. Similarly, 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. As used herein, 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. As used herein, 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., hRORγ, 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. Typically, 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.

The terms "identical" or percent "identity," in the context of two or more 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. For example, 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. Preferably, 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.

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. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, 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).. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a 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. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al, supra).

One example of a useful algorithm is PILEUP. 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 & Shaφ, 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. Using 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).

Another example algorithm that is suitable for determining percent sequence identity (i.e., substantial similarity or identity) is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These 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 BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, 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 'I. Acad. Sci. USA

90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a 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.

An indication that two 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. Thus, 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 puφoses of the present application, SEQ ID NOS:2 and 4, and SEQ ID NOS:l and 3 are not considered substantially identical.

The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).

The phrase "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. 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. For selective or specific hybridization, 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. Alternatively, 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. Thus, for example, 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). While various 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. Thus, the term 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-hRORγ" 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.

The term "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. Thus, under designated immunoassay conditions, 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. For example, polyclonal antibodies raised to hRORγ from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with hRORγ and not with other proteins, except for polymoφhic variants and alleles of hRORγ. This selection may be achieved by suotracting out antibodies that cross-react with hRORγ molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratoiy Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

The phrase "selectively associates with" refers to the ability of a nucleic acid to "selectively hybridize" with another as defined above, or the ability of an antibody to "selectively (or specifically) bind" to a protein, as defined above. By "host cell" is meant 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.

III. Assays for ligands and modulators of hRORγ

A. Assays for hRORy activity hRORγ and its alleles and polymoφhic variants are nuclear receptors that regulate transcription. Assays for hRORγ activity can be used to test for ligands, inhibitors, and activators of hRORγ, which can then be used to modulate hRORγ- mediated transcription. For example, hRORγ is involved in transcriptional regulation of allergic and inflammatory reactions, as well as in adipocyte differentiation. The activity of hRORγ 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 Ca²⁺); 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.

Preferably, the hRORγ of the assay will be selected from a polypeptide having a sequence of the C-terminal domain of SΕQ ID NO:4 or conservatively modified variant thereof. In one embodiment, amino acids 96-518 of SΕQ ID NO:4 are used as the hRORγ of the assay, either alone or as part of a fusion protein. In one embodiment, amino acids 96-518 of the C-terminal domain of hRORγ are fused to a GST domain.

Modulators of hRORγ activity are tested using biologically active hRORγ 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. hRORγ activity can also be examined in vitro with soluble or solid state reactions. Samples or assays that are treated with a potential hRORγ 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 hRORγ activity value of 100. Inhibition of hRORγ is achieved when the hRORγ activity value relative to the control is about 90%, preferably 50%, more preferably 25%. Activation of hRORγ is achieved when the hRORγ activity value relative to the control is 110%, more preferably 150%, more preferably 200%> higher. Generally, 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 hRORγ activity can be used to assess the influence of a test compound on the polypeptides of this invention. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca²⁺, IP3 or cAMP. Preferred assays for hRORγ activity can be performed in vitro or in vivo.

One example of a preferred in vitro assay format is described in USSN 09/052,995, herein incoφorated by reference in its entirety. In this assay, hRORγ activity is detected by examining transcription of a reporter RNA. Transcription of the reporter RNA is regulated either directly or indirectly by hRORγ. For example, hRORγ can regulate transcription when an hRORγ responsive element is present in the promoter operably linked to the reporter gene. Alternatively, the hRORγ 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 hRORγ 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 hRORγ is detected. In another variation of this in vitro assay, high throughput in vitro expression assays are provided. In such assays, 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.

Another example of a preferred assay format useful for monitoring hRORγ activity is performed in vivo. The general format is described in detail in USSN

09/052,841, herein incoφorated by reference in its entirety. In this assay, hRORγ 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 hRORγ, as described above. The in vivo reaction is first contacted with test ligands or modulators of hRORγ 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.

In a particularly preferred method for screening ligands and modulators of hRORγ, hRORγ activity is assessed using a dual construct system. This system can be used either in vitro or in vivo. In this system, a first expression cassette is made encodes a polypeptide comprising the C-terminal, ligand binding domain of hRORγ fused to the DNA binding domain of Gal4. In one embodiment, construct is encoded by an expression vector encoding a fusion protein having, from N- to C-terminus, Flag-Gal4- hRORγ 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. In one embodiment, the second construct is an expression vector with four copies of the Gal4 responsive element in front of a gene encoding luciferase. Typically, a control plasmid is used as a control for transfection efficiency, e.g., a plasmid encoding β-gal. When the hRORγ 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 hRORγ activity. This system thus can be used to screen for hRORγ ligands as well as modulators of hRORγ activity (see, e.g., Medvedev et al, Gene 181 :199-206 (1996)). In one embodiment, 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 luciferase activity is determined.

In yet another method for screening ligands and modulators of hRORγ activity, a peptide sensor is used to detect the hRORγ-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.

In one embodiment, (an "ELISA" type assay) a plate is coated with the peptide sensor, and then the hRORγ protein and potential ligands are added to each well in the plate. The plate is then subjected to an ELISA assay, u.ing an antibody linked to an enzyme. When a ligand binds to hRORγ it induces a conformational change in the nuclear receptor, allowing the peptide sensor to bind to the nuclear receptor (see Figure 1). In a preferred embodiment of the ELISA assay, the hRORγ is a fusion protein, with the N-terminal domain derived from GST and the C-terminal domain comprising amino acids 96-518 of hRORγ. For this embodiment, the antibody used in the ELISA is typically an anti-GST antibody.

In another embodiment, a fluorescence polarization detection scheme is used to detect binding of the peptide sensor to the hRORγ-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 hRORγ-ligand complexes (due to the altered conformation of the receptor), the peptides have a lower rotational rate, and polarization is preserved. R. Computer assisted drug design

Yet another assay for compounds that modulate hRORγ activity involves computer assisted drug design, in which a computer system is used to generate a three- dimensional structure of hRORγ 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 hRORγ and modulators of ligand binding and hRORγ 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 hRORγ 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 (or a nucleotide sequence encoding 10 amino acids) 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. 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. In modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.

Once the structure has been generated, 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 hRORγ protein to identify ligands that bind to hRORγ. 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.

C. Ligands and modulators The compounds tested as modulators and ligands of hRORγ can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and peptides. A preferred ligand is melatonin, or a melatonin analogue. Alternatively, modulators can be genetically altered versions of hRORγor its ligands. Essentially 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. For example, 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.

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such 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)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al, Science 261 :1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)), 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. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; moφholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

As noted, 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. For each of the assay formats described, "no modulator" control reactions which do not include a modulator provide a background level of expression from a given coding DNA.

In some assays it will be desirable to have controls to ensure that the components of the assays are working properly. For example, a known inhibitor of transcription such as α-amanitin (a strong inhibitor of the pol II transcription complex) can be added, and the resulting decrease in transcription similarly detected. It will be appreciated that modulators can also be combined with ligands to find modulators which inhibit transcriptional activation or transcriptional repression.

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, 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. Thus, 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, microfluidic approaches to reagent manipulation have been developed, e.g., by Caliper Technologies (Palo Alto, CA).

IN. Isolation of the gene encoding hRORγ

A. General Recombinant DNA Methods hRORγ 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)). In addition, essentially any nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others. Similarly, peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio.Synthesis, Inc., and many others.

For 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 DΝA sequences. For 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.

R. Cloning methods for the isolation of nucleotide sequences encoding hRORy

In general, the nucleic acid sequences encoding hRORγ 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. For example, hRORγ 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 hRORγ 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 hRORγ 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 hRORγ (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 hRORγ.

Nucleic acids encoding hRORγ 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. hRORγ polymoφhic variants and alleles that are substantially identical to hRORγ can be isolated using hRORγ nucleic acid probes, and oligonucleotides under stringent hybridization conditions, by screening libraries. Alternatively, expression libraries can be used to clone hRORγ and hRORγ polymoφhic variants and alleles, by detecting expressed homologs immunologically with antisera or purified antibodies made against hRORγ, which also recognize and selectively bind to the hRORγ homolog. To make a cDNA library, one should choose a source that is rich in hRORγ 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).

For a genomic library, 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).

An alternative method of isolating hRORγ 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 hRORγ directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. Degenerate oligonucleotides can be designed to amplify hRORγ homologs using the sequences provided herein. Restriction endonuclease sites can be incoφorated 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 hRORγ encoding mRNA in physiological samples, for nucleic acid sequencing, or for other puφoses. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.

These techniques can also be used to make hRORγ chimeras, such as constructs encoding the C-terminal ligand binding domain of hRORγ fused to the DNA binding domain of a heterologous protein such as Gal4 or the lac repressor. In addition, constructs encoding N-terminal deletions of hRORγ 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 hRORγ 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 hRORγ 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 hRORγ nucleic acid. The specific subsequence is then ligated into an expression vector.

The nucleic acid encoding hRORγ 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.

C. Expression in prokaryotes and eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, such as those cDNAs encoding hRORγ, one typically subclones hRORγ into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. 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 hRORγ protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al, Gene 22:229-235 (1983)). 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., hRORγ responsive elements, Gal4 responsive elements, lac repressor responsive elements, and the like. In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the hRORγ encoding nucleic acid in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding hRORγ and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The nucleic acid sequence encoding hRORγ may typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell. Such 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. In addition to a promoter sequence, 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-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/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. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a hRORγ 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 hRORγ protein, which are then purified using standard techniques (see, e.g., Colley et αl, 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 hRORγ.

After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of hRORγ, which is recovered from the culture using standard techniques identified below. D. Chimeric hRORy proteins

The C-terminal domain of hRORγ has at least two major regulatory functions: it mediates ligand binding and homo-or heterodimer formation. Consequently, both the full length hRORγ protein and the C-terminal domain of hRORγ 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 hRORγ 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. kinases, acetylases and deacetylases); DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases) and their associated factors and modifiers; 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) and factors that associate with or modify them. While a hRORγ 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 hRORγ domain. To identify such nucleic acid binding domains, one can perform 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. Association of a portion of the protein with the nucleic acid will result in a retardation of the electrophoretic mobility of the nucleic acid. 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. 2:46-9 (1995) and Roeder, Methods Enzymol. 273: 165-71 (1996). Databases dedicated to transcription factors are known. See, e.g., Science 269:630. Intracellular receptor transcription factors are described in, for example, Rosen et al, J. Med. Chem. 38:4855-74 (1995). The C/EBP family of transcription factors are reviewed in Wedel et al, Immunobiology 193:171-85 (1995). Coactivators and co-repressors that mediate transcription regulation by nuclear hormone receptors are reviewed in, for example, Meier, Eur. J. Endocrinol 134(2):158-9 (1996) and Kaiser et al, Trends Biochem. Sci. 21:342-5 (1996). GATA transcription factors, which are involved in regulation of hematopoiesis, are described in, for example, Simon, Nat. Genet. 11 :9-l 1 (1995); Weiss et al, Exp. Hematol. 23:99-107. TATA box binding protein (TBP) and its associated TAF polypeptides (which include TAF30, TAF55, TAF80, TAF110, TAF150, and TAF250) are described in Goodrich & Tjian, Curr. Opin. Cell Biol. 6:403-9 (1994) and Hurley, Curr. Opin. Struct. Biol. 6:69-75 (1996). The STAT family of transcription factors are reviewed in, for example,

Barahmand-pour et α/., Curr. Top. Microbiol Immunol. 211 :121-8 (1996). Transcription factors involved in disease are reviewed in Aso et al, J. Clin. Invest. 97:1561-9 (1996).

Kinases, phosphatases, and other proteins that modify polypeptides involved in gene regulation are also useful for making hRORγ 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).

As described, 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 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, Eur. J. Biochem. 211 :7-18 (1993) and Crepieux et al, Crit. Rev. Oncog. 5:615-38 (1994). Myc oncogenes are reviewed in, for example, Ryan et al, Biochem. J. 314:713- 21 (1996). The jun and fos transcription factors are described in, for example, The Fos and Jun Families of Transcription Factors, Angel & Herrlich, eds. Boca Raton, FL, CRC Press, 1994. The max oncogene is reviewed in Hurlin et al, Cold Spring Harb. Symp. Quant. Biol. 59:109-16. The myb gene family is reviewed in Kanei-Ishii et al, Curr. Top. Microbiol Immunol. 21 1 :89-98 (1996). The mos family is reviewed in Yew et al, Curr. Opin. Genet. Dev. 3:19-25 (1993).

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).

Invasiveness promoters and suppressors are reviewed in, for example, Mareel et al, Mol. Biol Rep. 19:45-67 (1994).

Chimeric hRORγ 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); Sancaτ, Ann. Rev. Genet. 29:69-105 (1995); Lehmann, Genet. Eng. 17:1-19 (1995); and Wood, Ann. Rev. Biochem. 65:135-67 (1996). DNA reaπangement enzymes and their associated factors and modifiers (see, e.g., Gangloff et αl, Experientiα 50:261-9 (1994); Sadowski, FASEB J. 7:760-7 (1993)), cell cycle proteins and their associated factors and modifiers are also useful as domains for addition to hRORγ. For example, proteins involved in DNA replication can be used to construct chimeric hRORγs. DNA replication proteins are described in Kearsey et αl, Curr. Opin. Genet. Dev. 6:208-14 (1996) and Donovan et αl., 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).

Preferred embodiments of hRORγ chimeras include fusions between Gal4 and the hRORγ C-terminal domain, and fusions between GST (glutathione-S-transferase) and the RORγ C-terminal domain.

Similarly, chimeric polypeptides can be derived from DNA modifying enzymes (e.g., methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases) and their associated factors and modifiers. 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), such as histone deacylase (Wolffe, Science 272:371-2 (1996)) are also useful as domains for addition to the hRORγ C-terminal domain herein.

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 hRORγ C-terminal domains. For a review of protein-RNA interactions, see, Draper, Ann. Rev. Biochem. 64:593-620 (1995) and Burd et al, Science 29:615-21 (1994). 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 incoφorated 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. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

In addition to recombinant production of hRORγ proteins, chemical methods can be used to produce molecules with hRORγ C-terminal regions attached to non-peptide components. For example, nucleic acid-protein constructs have been made in which an 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).

V. Purification of hRORγ

Either naturally occurring or recombinant hRORγ can be purified for use in functional assays. Preferably, recombinant hRORγ is purified. Naturally occurring hRORγ is purified, e.g., from mammalian tissue such as thymus or skeletal muscle tissue, and any other source of a hRORγ homolog. Recombinant hRORγ is purified from any suitable expression system, e.g., by expressing hRORγ in E. coli and then purifying the recombinant protein via affinity purification, e.g., by using antibodies that recognize a specific epitope on the protein or on part of the fusion protein, or by using glutathione affinity gel, which binds to GST. In some embodiments, the recombinant hRORγ is a fusion protein, e.g., with GST or Gal4 at the N-terminus. hRORγ 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).

A number of procedures can be employed when recombinant hRORγ is being purified. For example, proteins having established molecular adhesion properties can be reversible fused to hRORγ. With the appropriate ligand, hRORγ 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, hRORγ could be purified using immunoaffinity columns. A. Purification of hRORy from 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.

Proteins expressed in bacteria may form insoluble aggregates ("inclusion bodies"). Several protocols are suitable for purification of hRORγ inclusion bodies. For example, 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 MgCl₂, 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).

If necessary, 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, for example SDS (sodium dodecyl sulfate), 70% formic acid, are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of immunogenicity and/or activity. Although 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. hRORγ is separated from other bacterial proteins by standard separation techniques, e.g., with Ni- NTA agarose resin.

Alternatively, it is possible to purify hRORγ from bacteria periplasm. After lysis of the bacteria, when hRORγ is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art. To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ 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.

R. Standard Protein Separation Techniques For Purifying hRORy Solubility Fractionation

Often as an initial step, particularly if the protein mixture is complex, 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.

Size Differential Filtration The molecular weight of hRORγ 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). As a first step, 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 Chromatography hRORγ can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands. In addition, 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).

VI. Immunological Detection of hRORγ

In addition to the detection of hRORγ genes and gene expression using nucleic acid hybridization technology, one can also use immunoassays to detect hRORγ or to measure hRORγ activity, e.g., to identify hRORγ ligands and modulators of hRORγ activity. Immunoassays can be used to qualitatively or quantitatively analyze hRORy. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).

A. Antibodies to hRORy

Methods of producing polyclonal and monoclonal antibodies that react specifically with hRORγ are known to those of skill in the art (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). 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)). In addition, as noted above, 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 hRORγ comprising immunogens may be used to produce antibodies specifically reactive with hRORγ. For example, recombinant hRORγ 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. Alternatively, 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.

Methods of production of polyclonal antibodies are known to those of skill in the art. To improve reproducibility, an inbred strain of mice (e.g., BALB/C mice) can be immunized to make the antibody; however, standard animals (mice, rabbits, etc.) used to make antibodies are immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol (see Harlow & Lane, supra). The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to hRORγ. When appropriately high titers of antibody to the immunogen are obtained, 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. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof 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 10⁴ or greater are selected and tested for their cross reactivity against non-hRORγ proteins or even other related proteins, e.g., from other organisms, using a competitive binding immunoassay. The 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 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better.

Once hRORγ specific antibodies are available, hRORγ can be detected by a variety of immunoassay methods. For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991). Moreover, 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.

B. Immunological binding assays hRORγ 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). For a review of the general immunoassays, see also 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 (or immunoassays) typically use an antibody that specifically binds to a protein or antigen of choice (in this case the hRORγ or antigenic subsequence thereof). The antibody (e.g., anti-hRORγ) 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. Thus, the labeling agent may be a labeled hRORγ polypeptide or a labeled anti-hRORγ antibody. Alternatively, the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/hRORγ 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. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al, J. Immunol. 111 :1401-1406 (1973); Akerstrom et al, J. Immunol.

135:2589-2542 (1985)). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.

Throughout the assays, 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 hRORγ in samples may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of antigen is directly measured. In one preferred "sandwich" assay, for example, the anti- hRORγ antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture hRORγ present in the test sample. hRORγ is thus immobilized is then bound by a labeling agent, such as a second hRORγ antibody bearing a label. Alternatively, 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.

Competitive assay formats

In competitive assays, the amount of hRORγ present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) hRORy displaced (competed away) from an anti-hRORγ antibody by the unknown hRORy present in a sample. In one competitive assay, a known amount of hRORγ is added to a sample and the sample is then contacted with an antibody that specifically binds to hRORγ. The amount of exogenous hRORγ bound to the antibody is inversely proportional to the concentration of hRORγ present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of hRORγ bound to the antibody may be determined either by measuring the amount of hRORγ present in a hRORγ/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein. The amount of hRORγ may be detected by providing a labeled hRORγ molecule. A hapten inhibition assay is another preferred competitive assay. In this assay the known hRORγ, is immobilized on a solid substrate. A known amount of anti- hRORγ antibody is added to the sample, and the sample is then contacted with the immobilized hRORγ. The amount of anti-hRORγ antibody bound to the known immobilized hRORγ is inversely proportional to the amount of hRORγ present in the sample. Again, 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.

Cross-reactivity determinations Immunoassays in the competitive binding format can also be used for crossreactivity determinations. For example, a protein at least partially encoded by SEQ ID NO:4 can be immobilized to a solid support. Proteins (e.g., hRORy 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 hRORγ 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 (SEQ ID NO:2) are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsoφtion 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 polymoφhic variant of hRORγ, to the immunogen protein (i.e., hRORγ of SEQ ID NO:4). In order to make this comparison, 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 hRORγ immunogen.

Other assay formats Western blot (immunoblot) analysis is used to detect and quantify the presence of hRORγ in .he sample. 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 hRORγ. The anti-hRORγ antibodies specifically bind to the hRORγ 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-hRORγ antibodies.

Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al, Amer. Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of non-specific binding One of skill in the art will appreciate that it is often desirable to minimize non-specific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of non-specific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this technique involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered milk being most preferred.

Labels 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. Thus, 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., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), 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.).

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. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. 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. The ligands and their targets can be used in any suitable combination with antibodies that recognize hRORγ, or secondary antibodies that recognize anti-hRORγ. 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. For a review of various labeling or signal producing systems that may be used, see U.S. Patent No. 4,391,904. Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it 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. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally 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.

Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.

NIL Transgenic mice

Transgenic mice expressing hRORγ can be made by simple insertion of hRORγ into the mouse genome or by homologous recombination, in a pluripotent cell line that is capable of differentiating into germ cell tissue. A DΝA construct that contains hRORγ is introduced into the nuclei of embryonic stem cells. In a portion of the cells, the introduced DΝA recombines with the endogenous copy of the mouse gene, replacing it with the human copy. Alternatively, cells can be selected that express both the endogenous and human genes. Alternatively, knock-out mice can be made, in which the endogenous RORγ 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. For example, a transgenic mouse that is heterozygous or homozygous for integrated transgenes that have functionally disrupted the endogenous hRORγ gene can be used as a sensitive in vivo screening assay for hRORγ ligands and modulators of hRORγ 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).

VIII. Kits hRORγ 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 hRORγ ligands and modulators of hRORγ activity. hRORγ specific reagents that specifically hybridize to hRORγ nucleic acid, such as hRORγ probes and primers, and hRORγ specific reagents that specifically bind to the hRORγ protein, e.g., hRORγ antibodies are typically used to examine transcriptional regulation.

Nucleic acid assays for the presence of hγ 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, SI analysis, amplification techniques such as PCR and LCR, and in situ hybridization. In 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 moφhology for subsequent inteφretation and analysis. The following articles provide an overview of the art of zw situ hybridization: Singer et al, Biotechniques 4:230-250 (1986); Haase et al, Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach (Hames et al, eds. 1987). In addition, hRORγ 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 hRORγ) and a negative control.

The present invention also provides for kits for screening for modulators of hRORγ. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: hRORγ, reaction tubes, and instructions for testing hRORγ 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. For example, the kit can be tailored for in vitro or in vivo assays for measuring the activity of hRORγ.

All publications and patent applications cited in this specification are herein incoφorated by reference as if each individual publication or patent application were specifically and individually indicated to be incoφorated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

SEQUENCE LISTING

Human RORγ nucleotide sequence (encoding 62.5 kDa)— SEQ ID NO:l

ATG GAC AGG GCC CCA CAG AGA CAG CAC CGA GCC TCA CGG GAG CTG CTG GCT GCA

AAG AAG ACC CAC ACC TCA CAA ATT GAA GTG ATC CCT TGC AAA ATC TGT GGG GAC

AAG TCG TCT GGG ATC CAC TAC GGG GTT ATC ACC TGT GAG GGG TGC AAG GGC TTC

TTC CGC CGG AGC CAG CGC TGT AAC GCG GCC TAC TCC TGC ACC CGT CAG CAG AAC

TGC CCC ATC GAC CGC ACC AGC CGA AAC CGA TGC CAG CAC TGC CGC CTG CAG AAA

TGC CTG GCG CTG GGG ATG TCC CGA GAT GCT GTC AAG TTC GGC CGC ATG TCC AAG

AAG CAG AGG GAC AGC CTG CAT GCA GAA GTG CAG AAA CAG CTG CAG CAG CGG CAA

CAG CAG CAA CAG GAA CCA GTG GTC AAG ACC CCT CCA GCA GGG GCC CAA GGA GCA

GAT ACC CTC ACC TAC ACC TTG GGG CTC CCA GAC GGG CAG CTG CCC CTG GGC TCC

TCG CCT GAC CTG CCT GAG GCT TCT GCC TGT CCC CCT GGC CTC CTG AAA GCC TCA

GGC TCT GGG CCC TCA TAT TCC AAC AAC TTG GCC AAG GCA GGG CTC AAT GGG GCC

TCA TGC CAC CTT GAA TAC AGC CCT GAG CGG GGC AAG GCT GAG GGC AGA GAG AGC

TTC TAT AGC ACA GGC AGC CAG CTG ACC CCT GAC CGA TGT GGA CTT CGT TTT GAG

GAA CAC AGG CAT CCT GGG CTT GGG GAA CTG GGA CAG GGC CCA GAC AGC TAC GGC

AGC CCC AGT TTC CGC AGC ACA CCG GAG GCA CCC TAT GCC TCC CTG ACA GAG ATA

GAG CAC CTG GTG CAG AGC GTC TGC AAG TCC TAC AGG GAG ACA TGC CAG CTG CGG

CTG GAG GAC CTG CTG CGG CAG CGC TCC AAC ATC TTC TCC CGG GAG GAA GTG ACT

GGC TAC CAG AGG AAG TCC ATG TGG GAG ATG TGG GAA CGG TGT GCC CAC CAC CTC

ACC GAG GCC ATT CAG TAC GTG GTG GAG TTC GCC AAG AGG CTC TCA GGC TTT ATG

GAG CTC TGC CAG AAT GAC CAG ATT GTG CTT CTC AAA GCA GGA GCA ATG GAA GTG

GTG CTG GTT AGG ATG TGC CGG GCC TAC AAT GCT GAC AAC CGC ACG GTC TTT TTT

GAA GGC AAA TAC GGT GGC ATG GAG CTG TTC CGA GCC TTG GGC TGC AGC GAG CTC

ATC AGC TCC ATC TTT GAC TTC TCC CAC TCC CTA AGT GCC TTG CAC TTT TCC GAG

GAT GAG ATT GCC CTC TAC ACA GCC CTT GTT CTC ATC AAT GCC CAT CGG CCA GGG

CTC CAA GAG AAA AGG AAA GTA GAA CAG CTG CAG TAC AAT CTG GAG CTG GCC TTT

CAT CAT CAT CTC TGC AAG ACT CAT CGC CAA AGC ATC CTG GCA AAG CTG CCA CCC

AAG GGG AAG CTT CGG AGC CTG TGT AGC CAG CAT GTG GAA AGG CTG CAG ATC TTC

CAG CAC CTC CAC CCC ATC GTG GTC CAA GCC GCT TTC CCT CCA CTC TAC AAG GAG

CTC TTC AGC ACT GAA ACC GAG TCA CCT GTG GGC TGT CCA AGT GAC CTG GAA GAG

GGA CTC CTT GCC TCT CCC TAT GGC CTG CTG GCC ACC TCC CTG GAC CCC GTT CCA

CCC TCA CCC TTT TCC TTT CCC ATG AAC CCT GGA GGG TGG TCC CCA CCA GCT CTT TGG AAG TGA Human RORγ amino acid sequence (62.5 kDa)— SEQ ID O:2

AT CAC TAC 130

TC AAC GCG GCC TAC TCC TGC ACC CCT CAG CAG AAC TGC CCC ATC GAC CGC ACC AGC CGA 240

" U "-i « -- -^, -. 3 _. 7 _{3 R} AAC CGA TGC CAG CAC TGC CGC t—^- r^r I TV- r~τ~~ r- ~ , .-

GTC AAG TTC GGC CGC ATG TCC .AAG AAG CAG AGG GAC ACC CC CAT GCA GAA GTG CAG AAA 350 ^{s Λ S (}-! ^R D -> - H A H V Q :< C^G CTG CAG CAG CGG CAA CAG CAG CAA C^G GAA CCA GTG GTC A AAAGG A ACCCC C C~CT* C CCCAA GC~A. r GG~

R G

Q Q Ξ ? V V 420 ^:< T ? ? AA σG

GCC CAA GGA GCA GAT ACC CTC ACC TAC ACC TTG GCG CTC CCA GAC GGG CAG CTG CCC CTG

480

CCC TCC TCG CCT GAC CTG CCT GAG GCT TCT GCC TGT CCC CCT GGC C.

CTG AAA GCC TCA G S S ? D L P Ξ A S A C ? ? σ -- 540 !-^■ A S

GGC TCT GGG CCC TCA TAT TCC AAC AAC GCC AAG GCA GGG CTC AAT GGG GCC TCA TGC G S G P S Y S N N S00 A X A G L N G A S c

CAC GAA TAC AGC CCT GAG CGG GGC AAG GCT GAG GGC AGA GAG =-3C _T-

TAT AGC ACA S50 ^Ξ - ^S ? Ξ R G X A E G R -. s -^' Y s T

GGC AGC CAG CTG ACC CCT GAC CGA TGT GGA CTT CGT TTT GAG GAA CAC ACC CAT CCT GGG 720

- - ^•- R --. ? G

CTT GGG GAA CTG GGA CAG GGC CCA GAC AGC TAC GGC .AGC CCC AGT ^ CGC AGT -r. r~ -ran

L G S L G Q G ? D 3 Y _{G 3 ?} «. ,~ ^- J∞ ^ACA =~- 780

GAG GCA CCC TAT GCC TCC CTG ACA GAG ATA GAG CAC CTG GTG CAG AGC GTC TGC AAG TCC 840 ^c- ^{A ? Y} A S I- E -: Ξ H V Q _{S v c κ: Ξ}

O 00/24757

CA -f*j--?G i-.u ACLΛ. TGC GAG IX CG * CTC *-v*A-. *--«*.>«-, CCG CTC C -J G G GC TCC AA C V^r"_{' '}i'"* * <3 fl fl

Y R Ξ T C Q -- R L Ξ D -. R Q R 5 . ^{" "} _z ^""~ f^"

TCC CGG GAG GAA GTG ACT CCC -AC CAG AGG AAG TCC ATG TGG GAG ATG TGG CAA C_GG TG~ 500

S R Ξ Ξ V T G Y ^Q R X S v Ξ M W Ξ R c

GCC CAC CAC CTC ACC GAG GCC ATT CAG TAC GTG GTG GAG TTC GCC AAG AGG CTC TCA _CCC 1020 A H H T Ξ A I Q Y V V E F A X R r,^" 5 ^' _G

TTT ATG GAG CTC TGC CAG AAT GAC CAG ATT GTG CTT CTC AAA GCA GGA GCA ATG GAA _GT_G 1080 F M -- -- C ^Q N D ^Q I V L -- K A G A M S ^"

GTG CTG GTT AGG ATG TGC CGG GCC TAC AAT GCT GAC AAC CGC ACG GTC TTT TTT GAA GGC 1140

V I- V R M C R A Y N A D M R T V F F Ξ G

AAA T.AC GGT GGC ATG GAG CTG TTC CGA GCC TTG GGC TGC AGC GAG CTC ATC GC TCC ATC 1200 r G G M -- -- F A -. G C S -- -. I S S Z

TTT GAC TTC TCC CAC TCC CTA AGT GCC TTG CAC TTT TCC GAG GAT GAG ATT GCC CTC TAC 12S0 F D F S H S -. S A -- H F S Ξ D Ξ I Λ -. Y

ACA GCC CTT GTT CTC ATC AAT GCC CAT CGG CCA GGG CTC CAA GAG AAA AGG AAA GTA GAA 1320 T A V ϊ N A H R 2 G Q Ξ X R X V S

CAG CTG CAG TAC AAT CTG GAG CTG GCC TTT CAT CAT CAT CTC TGC AA.G ACT CAT CGC CAA 1380 Q -- Q V N L 2 L A ? H r- K L C :< T K S Q

AGC ATC CTG GCA AAG CTG CCA CCC AAG GGG AAG CTT CGG AGC CTG TGT AGC CAG CAT GTG 1440 s r -- A K L ? ? :< G :< -- S -- c s Q H v

GAA AGG CTC CAG ATC TTC CAG CAC CTC CAC CCC ATC GTG GTC CAA GCC GCT TTC CCT CCA 1S00 £ R Q X F Q K K ? I V V Q A A F ? ?

CTC T-AC AAG GAG CTC TTC AGC ACT GAA ACC GAG TCA CCT GTG GGC TGT CCA ACT GAC CTG 1550 Y K. Ξ -. F S T E T Ξ S P V G C P S -. --

GAA GAG GGA CTC CTT GCC TCT CCC TAT GGC CTG CTG GCC ACC TCC CTG GAC CCC GTT CCA 1520 Ξ Ξ G -. -- A S P Y G I- A T S -- -. P V P

CCC TCA CCC TTT TCC TTT CCC ATG AAC CCT GGA GGG TGG TCC CCA CCA GCT CTT TGG AAG 1S80 P S P F 'S P M N P G G S P P A W K

TGA 1683 Human RORγ nucleotide sequence (encoding 60 kDa)— SEQ ID NO:3

ATG GAC AGG GCC CCA CAG AGA CAG CAC CGA GCC TCA CGG GAG CTG CTG GCT GCA

AAG AAG ACC CAC ACC TCA CAA ATT GAA GTG ATC CCT TGC AAA ATC TGT GGG GAC

AAG TCG TCT GGG ATC CAC TAC GGG GTT ATC ACC TGT GAG GGG TGC AAG GGC TTC

TTC CGC CGG AGC CAG CGC TGT AAC GCG GCC TAC TCC TGC ACC CGT CAG CAG AAC

TGC CCC ATC GAC CGC ACC AGC CGA AAC CGA TGC CAG CAC TGC CGC CTG CAG AAA

TGC CTG GCG CTG GGG ATG TCC CGA GAT GCT GTC AAG TTC GGC CGC ATG TCC AAG

AAG CAG AGG GAC AGC CTG CAT GCA GAA GTG CAG AAA CAG CTG CAG CAG CGG CAA

CAG CAG CAA CAG GAA CCA GTG GTC AAG ACC CCT CCA GCA GGG GCC CAA GGA GCA

GAT ACC CTC ACC TAC ACC TTG GGG CTC CCA GAC GGG CAG CTG CCC CTG GGC TCC

TCG CCT GAC CTG CCT GAG GCT TCT GCC TGT CCC CCT GGC CTC CTG AAA GCC TCA

GGC TCT GGG CCC TCA TAT TCC AAC AAC TTG GCC AAG GCA GGG CTC AAT GGG GCC

TCA TGC CAC CTT GAA. TAC AGC CCT GAG CGG GGC AAG GCT GAG GGC AGA GAG AGC

TTC TAT AGC ACA GG 2 AGC CAG CTG ACC CCT GAC CGA TGT GGA CTT CGT TTT GAG

GAA CAC AGG CAT CCT GGG CTT GGG GAA CTG GGA CAG GGC CCA GAC AGC TAC GGC

AGC CCC AGT TTC CG-: AGC ACA CCG GAG GCA CCC TAT GCC TCC CTG ACA GAG ATA

GAG CAC CTG GTG CAG AGC GTC TGC AAG TCC TAC AGG GAG ACA TGC CAG CTG CGG

CTG GAG GAC CTG CTG CGG CAG CGC TCC AAC ATC TTC TCC CGG GAG GAA GTG ACT

GGC TAC CAG AGG AAG TCC ATG TGG GAG ATG TGG GAA CGG TGT GCC CAC CAC CTC

ACC GAG GCC ATT CAG TAC GTG GTG GAG TTC GCC AAG AGG CTC TCA GGC TTT ATG

GAG CTC TGC CAG AAT GAC CAG ATT GTG CTT CTC AAA GCA GGA GCA ATG GAA GTG

GTG CTG GTT AGG ATG TGC CGG GCC TAC AAT GCT GAC AAC CGC ACG GTC TTT TTT

GAA GGC AAA TAC GGT GGC ATG GAG CTG TTC CGA GCC TTG GGC TGC AGC GAG CTC

ATC AGC TCC ATC TTT GAC TTC TCC CAC TCC CTA AGT GCC TTG CAC TTT TCC GAG

GAT GAG ATT GCC CTC TAC ACA GCC CTT GTT CTC ATC AAT GCC CAT CGG CCA GGG

CTC CAA GAG AAA AGG AAA GTA GAA CAG CTG CAG TAC AAT CTG GAG CTG GCC TTT

CAT CAT CAT CTC TGC AAG ACT CAT CGC CAA AGC ATC CTG GCA AAG CTG CCA CCC

AAG GGG AAG CTT CGG AGC CTG TGT AGC CAG CAT GTG GAA AGG CTG CAG ATC TTC

CAG CAC CTC CAC CCC ATC GTG GTC CAA GCC GCT TTC CCT CCA CTC TAC AAG GAG

CTC TTC AGC ACT GAA ACC GAG TCA CCT GTG GGG CTG TCC AAG TGA Human RORγ amino acid sequence (60 kDa)— SEQ ID NO:4

ATG GAC A----J G*-.-. CCA CAG AGA CAG CAC CGA GCC TCA CGC GAG A AAG 1 A P O R 0 K R A , S R A

AC" CAC ACC TCA CAA ATT GAA GTG ATC CCT TGC AAA ATC TGT GGG GAC AAG TCG TCT GGG 120 ^ " _{H T} ^' s _Q I H '- -: ? C X C G D X S S G — CAC TAC GGG GTT ATC ACC TGT GAG GGG TGC AAG GGC TTC TTC CGC CGG AGC CAG CGC 130

-" _{K G V I T C Ξ G} C K G F F R R S Q R

TC-" AAC GCG GCC TAC TCC TGC ACC CGT CAG CAG AAC TGC CCC ATC GAC CGC ACC AGC CG? 240 C^' N A A Y S C T Q Q N C P I D S R iiC CGA TTGC CAG CAC TGC CGC CTG CAG AAA TGC CTG GCG CTG GGG ATG TCC CGA GAT GCT 300 ii^"" C Q H C R 1 Q X C ^« A G M S R D A

GT" AAG TC GGC CGC ATG TCC AAG AAG CAG .AGG GAC AGC CTG CAT GCA GAA GTG CAG AAA 350 _V"~ X T G R S X X Q 3. D 3 L H A E V Q K

CAG CTG CAG CAG CGG CAA CAG CAG CAA CAG GAA CCA GTG GTC AAG ACC CCT CCA GCA GGG 420

_Q L _{Q Q} R _{Q Q Q}. _{Q Q} Ξ ? v v τ ? ? λ G r~- CAA GGA GCA GAT ACC CTC ACC TAC ACC TTG GGG CTC CCA GAC GGG CAG CTG CCC CTG 430

A Q G A D T T Y T G P D G Q L P

GGC TCC TCG CCT GAC CTG CCT GAG GCT TCT GCC TGT CCC CCT GGC CTC CTG AAA GCC TCA 540 _{G S} S P D -- P Ξ A S A C P P G -. A S

__ QJ3JJ _ccc T^ _TAT T C AAC AAC TTG GCC AAG GCA GGG CTC AAT GGG GCC TCA TGC 500 G S G ? S Y S M N L A X A G L N G A S C

CAC CT~ CAA T-AC.AGC CCT GAG CGG GGC AAG GCT GAG GGC AGA GAG AGC TTC TAT .AGC ACA SSO „ L ^* E Y S ? E R G X A Ξ G R Ξ S F Y S T

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 E E H H ? G

C _T" GGG GAA CTG GGA CAG GGC CCA GAC AGC TAC GGC AGC CCC .AGT TTC CGC .AGC ACA CCG 730 L G E L G Q G P D S G S P S F R S T ?

GAG GCA CCC _T-. GCC TCC CTG ACA GAG ATA GAG CAC CTG GTG CAG AGC GTC TGC AAG TCC 840 - _{A ?} Y A S -- T E I -- H V Q S V C K S

TAC AGG GAG ACA TGC CAG CTG CGG CTG GAG GAC CTG CTG CGG CAG CGC TCC AAC ATC TTC 900 _V P. E T C Q -. R -. E D -. -. R Q R S N -: --

^C CGG GAG GAA GTG ACT GGC TAC CAG AGG AAG TCC ATG TGG GAG ATG TGG GAA CGG TGT 950 S R E S V T G Y Q R X S M W E W Ξ R C

GCC CAC CAC CTC ACC GAG GCC ATT CAG T-AC GTG GTG GAG TTC GCC AAG AGG CTC TCA GGC 1020 A H H T E A T Q Y V V Ξ F A K R S G

TTT ATG GAG CTC TGC CAG AAT GAC CAG ATT GTG CTT CTC AAA GCA GGA GCA ATG GAA GTG 1080 _f M E -. C Q N D Q -- V -. -. X A G A M -- V

CT . CTG GTT AGG ATG TGC CGG GCC T-AC AAT GCT GAC AAC CGC ACG GTC TTT TTT GAA GGC 1140 V L V R M C R A Y N A D N R T V - - -- G

-G GGC TCC AGC GAG CTC ATC AGC TCC ATC UGO

AAA T.AC GGT ATG GAG CTG TTC

;< Y G GA.C TTC TCC CAC TCC CT .AGT GCC TTG CAC TTT TCC GAG GAC GAG ATT GCC CTC TAC 1250 1" „ _• =; -r ς S A L H - => - ^J "

ACA GCC CTT GTT CTC ATC AAT G C CAT CGG CCA GGG CTC CAA GAG AAA AGC AAA GTA GAA 1320

C_AG CTG CAG TAC .AAT CTG GAG CTG GCC TTT CAT CAT CAT CTC TGC AAG ACT CAT CGC CAA 1330 _{Q L} Q Y N L E A F .ι Λ r. C X - rt R U

AGC ATC CIG GCA AAG CTG CCA CCC .AAG GGG AAG CTT CGG .AGC CTG TGT .AGC CAG CAT CTG 1440

5 . L _" " ^* ____ _ _. : _ ^ „_ r^" r CAC CCC ATC GTG GTC CAA GCC GCT TTC CCT CCA

GAA AGG CTG CAG ATC TTC ^ CAC CTC CAC C ^C ^ ^ _Q ^ _{λ ? ? ?}

£ R Q I F Q a - ^Λ -

„„ ,__ _., , _f- GAG " -A. CCT GTG GGG CTG TCC AAG TGA CTC TAC AAG GAG CTC TTC AGC ACT GAA ACC GAG TCA C^C ^ ^ ^ _g ^ _ r_i X Ξ ^{L F S}

Claims

WHAT IS CLAIMED IS:

1. An isolated polypeptide which:

(i) specifically binds to an antibody, which antibody specifically binds to the C-terminal domain of SEQ ID NO:4, which antibody does not specifically bind to the C-terminal domain of SEQ ID NO:2; or

(ii) comprises the C-terminal domain of SEQ ID NO:4.

2. The isolated polypeptide of claim 1 , wherein the polypeptide has the amino acid sequence of SEQ ID NO:4.

3. The isolated polypeptide of claim 1, wherein the polypeptide is truncated at its N-terminal domain as compared to the N-terminal domain of SEQ ID NO:4.

4. The isolated polypeptide of claim 3, wherein the polypeptide comprises a heterologous amino acid subsequence.

5. The isolated polypeptide of claim 1, wherein the polypeptide comprises a heterologous amino acid subsequence.

6. The isolated polypeptide of claim 1, wherein the polypeptide further comprises an DNA-binding moiety.

7. The isolated polypeptide of claim 6, wherein the DNA-binding moiety is selected from the group consisting of a nucleic acid and a polypeptide.

8. The isolated polypeptide of claim 1 , wherein the polypeptide binds melatonin with the same specificity as SEQ ID NO:4, or with a higher specificity than SEQ ID NO:4.

9. The isolated polypeptide of claim 1, wherein the polypeptide is bound to a solid support.

10. A recombinant nucleic acid comprising an expression cassette, which expression cassette comprises a nucleic acid subsequence which encodes the isolated polypeptide of claim 1, which isolated nucleic acid is a non-lambda expression vector.

11. The nucleic acid of claim 10, wherein the nucleic acid subsequence encodes a polypeptide which has the amino acid sequence of SEQ ID NO:4.

12. The nucleic acid of claim 10, wherein the nucleic acid subsequence has the nucleotide sequence of SEQ ID NO:3.

13. The nucleic acid of claim 10, wherein the nucleic acid subsequence encodes a polypeptide .hat is truncated at its N-terminal domain as compared to the N- terminal domain of SEQ ID NO:4.

14. The isolated polypeptide of claim 13, wherein the polypeptide comprises a heterologous amino acid subsequence.

15. The isolated polypeptide of claim 10, wherein the polypeptide comprises a heterologous amino acid subsequence.

16. The isolated polypeptide of claim 10, wherein the polypeptide further comprises an DNA-binding moiety.

17. The isolated polypeptide of claim 16, wherein the DNA-binding moiety is selected from the group consisting of a nucleic acid and a polypeptide.

18. The nucleic acid of claim 10, wherein the nucleic acid subsequence encodes a polypeptide that binds to melatonin with the same specificity as SEQ ID NO:4.

19. The nucleic acid of claim 10, wherein the nucleic acid is bound to a solid support.

20. A transgenic animal comprising the recombinant expression cassette of claim 10.

21. An antibody which specifically binds to the polypeptide of claim 1 , which antibody does not specifically bind to the polypeptide of SEQ ID NO:2.

22. The antibody of claim 21 , wherein the antibody specifically binds to the C-terminal domain of the polypeptide.

23. The antibody of claim 21 , wherein the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO:4.

24. The antibody of claim 21, wherein the antibody specifically binds to the C-terminal domain of a polypeptide having the amino acid sequence of SEQ ID NO:4.

25. A method of identifying a ligand that binds to the C-terminal domain of the polypeptide of claim 1, the method comprising:

(i) contacting the polypeptide with a first ligand; and, (ii) determining whether the ligand binds to the polypeptide.

26. The method of claim 25, wherein the polypeptide is bound to a solid support.

27. The method of claim 25, wherein the polypeptide is expressed in a cell.

28. The method of claim 25, wherein the polypeptide is recombinant.

29. The method of claim 28, wherein the polypeptide comprises a heterologous amino acid subsequence.

30. The method of claim 25, wherein the polypeptide further comprises a DNA binding moiety.

31. The method of claim 30, wherein the DNA-binding moiety is selected from the group consisting of a nucleic acid and a polypeptide.

32. The method of claim 25, further comprising the step of contacting the polypeptide with a second ligand.

33. The method of claim 25, wherein the method comprises screening a library of ligands.

34. The method of claim 25, further comprising the step of contacting the polypeptide with a modulator of ligand binding.

35. A method of identifying modulators of the polypeptide of claim 1, the method comprising:

(i) contacting the polypeptide with a compound; and, (ii) determining whether the compound modulates the activity of the polypeptide.

36. The method of claim 35, wherein the compound modulates DNA binding activity of the polypeptide.

37. The method of claim 35, wherein the compound modulates ligand binding activity of the polypeptide.

38. The method of claim 35, wherein the polypeptide is bound to a solid support.

39. The method of claim 35, wherein the polypeptide is expressed in a cell.

40. The method of claim 35, wherein the polypeptide is recombinant.

41. The method of claim 40, wherein the polypeptide comprises a heterologous amino acid sequence.

42. The method of claim 35, wherein the polypeptide further comprises a DNA binding moiety.

43. The method of claim 42, wherein the DNA-binding moiety is selected from the group consisting of a nucleic acid and a polypeptide.