EP0769023A1 - Retinoic acid receptor epsilon - Google Patents

Abstract

Disclosed is an RARε polynucleotide and DNA (RNA) encoding such polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and utilizing such polypeptide for therapeutic purposes, for example, tissue regeneration and stimulation of the immune and hematopoietic system. Also disclosed are methods of identifying ligands which stimulate the RARε.

Description

RETINOIC ACID RECEPTOR EPSILON

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Retinoic Acid Receptor Epsilon (RARe) . The invention also relates to inhibiting the action of such polypeptides.

Retinoids (Vitamin A derivatives) are crucial for normal growth, vision, maintenance of numerous tissues, reproduction and overall survival (Wolbach, S.B., J. Exp. Med.. 42:753-777 (1925)) . In addition, offspring of Vitamin A deficient (VAD) dams exhibit a number of developmental defects, indicating that retinoids are also important during embryogenesis (Wilson, J.G., et al., Am. J. Anat.. 92:189-217 (1953)). The effects of Vitamin A deficiency in fetuses and young and adult animals can be prevented and/or reversed by retinoic acid (RA) administration (Wilson, J.G., et al., Am. . Anat. , 92:189-217 (1953)). The dramatic teratogenic effects of maternal RA administration on mammalian embryos and the spectacular effects of topical administration of retinoids on embryonic development of vertebrates and limb-regeneration in amphibians have markedly contributed to the belief that RA could, in fact, be a morphogen (conferring positional information during development) and may also play a critical role during organogenesis (Tabin, C.J., Cell, 66:199-217 (1991) ) .

Retinoids are also crucial for normal growth, vision, maintenance of numerous tissues, reproduction and overall survival (Wolbach, S.B. and Howe, P.R., J. Exp. Med. , 42:753- 777 (1925)) .

It is thought that the effects of the RA signal are mediated through two families of receptors that belong to the superfamily of ligand-inducible transcriptional regulatory factors, which includes steroid-thyroid hormone and vitamin D3 receptors (Evans, R.M., Science. 240:889-895 (1988)).

The retinoic acid receptor genes belong to the superfamily of genes known as the steroid hormone receptor family. All genes in this family can be divided into discrete regions or domains that are sometimes referred to as regions A/B, C, D, E, and F. The C region encodes the DNA- binding domain, the E region encodes the ligand-binding domain and the F region encodes the carboxy-terminus domain. The D region is believed to function as a "hinge". The function of the A/B (or N-terminus) region is not entirely clear but it may be involved with enhancement and repression of receptor transcription activity. (Hollenberg et al., Cell, 55:899-906 (1988)).

The RA receptor (RAR) family (RARcϋ, β and and their isoforms) are activated by both all-trans and non-cis RA. RARδ has also been cloned (Mech, Dev. , 40:99-112 (1993)). Within a given species, the DNA-binding region and the ligand-binding region domains of the three RAR types are highly similar, whereas the C-terminal region and the middle region exhibit no or little similarity. The amino acid sequences of the three RAR types are also notably different in their B regions, and their main isoforms (αl and α_2, βl to β4, 1 and γ2 and δl and δ2) further differ in their N- terminal A regions (Leid, M. et al., Trends Biochem. Sci., 17:427-433 (1992) ) .

RAR7 mutant mice exhibit growth deficiency, early lethality, and male sterility due to squamous metaplasia of the seminal vesicles and prostate (Lohnes, D. et al., Cell, 73:643-658 (1993)).

RAR7 transcripts are found in precartilaginous condensations, with subsequent restriction to cartilage and differentiating squamous keratinizing epithelia, regardless of their embryonic origin (Dolle, P. et al., Nature, 342:702- 705 (1989) ) . These observations suggest a role for RARγ in morphogenesis and chondrogenesis (Dolle et al., Development, 100:1133-1151 (1990)) .

The DNA sequence and polypeptide encoded by such DNA sequence of the present invention belongs to the retinoic acid receptor family and is most homologous to retinoic acid receptor gamma. Both modulator and the DNA binding domains share the highest homology with the corresponding parts of the ecdysone receptor of Drosophila. The ecdysone receptor plays an important role during development.

In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is RARe, as well as fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.

In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, to identify its novel hormone, DNA binding sites, gene targets and tissue specificity, which provide therapeutic targets for stimulating development, differentiation, tissue regeneration, reproduction and stimulation of immune and hematopoietic systems.

In accordance with yet a further aspect of the present invention, there is provided an antibody against such polypeptides.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure 1 depicts the polynucleotide sequence and the corresponding deduced amino acid sequence of the putative mature RARe polypeptide. The standard one letter abbreviation for amino acids is used.

Figure 2 illustrates the high homology between RARe and RAR7 at the DNA binding region and ligand binding region.

Figure 3 depicts the results of a Northern blot analysis of the RARe in human adult tissues.

Figure 4 shows the results of electrophoresing RARe on a gel after in vi tro transcription/translation.

Figure 5 illustrates the baculovirus transfer plasmid pA2 used for expression of RARe .

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75754 on March 18, 1994.

A polynucleotide encoding a polypeptide of the present invention may be obtained from testes, spleen and thymus. The polynucleotide of this invention was discovered in a cDNA library derived from human adult lung. It is structurally related to the retinoic acid receptor family. It contains an open reading frame encoding a protein of approximately 433 amino acid residues. The protein exhibits the highest degree of homology to retinoic acid receptor gamma with 48% identity and 58% similarity over a 70 amino acid stretch of the modulator region. Similarly, the DNA binding region and ligand binding region are also highly homologous to retinoid acid receptor gamma (Figure 5) .

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure l or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa- histidine tag supplied by a pQE-9 (Qiagen) vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984) ) . The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The present invention further relates to a RARe polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the RARe genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence (s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or _______ the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomvces, Salmonella t phimurium; fungal cells, such as yeast; insect cells such as Drosophila and Sf_9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs

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SUBSTITUTE SHEET (RULE 25) comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) , pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia), pRS and pGEM. Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P_R/ P_L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986) ) .

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers .

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al . , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989) , the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ctf-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplas ic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The RARe polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion present during purification. (Price et al., J. Biol. Chem. , 244:917 (1969)). Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedure^'s, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The RARe hormone receptor may be used for the identification of its novel hormones, DNA binding sites, gene targets and tissue specificity. The RARe polypeptide may also be employed as a therapeutic target for tissue regeneration, reproduction, stimulation of the immune and hematopoietic system, and for the treatment of male sterility. This is particularly important since RAR mutant mice exhibited the previously stated abnormalities. Accordingly, the RARe receptor may be used to treat these disorders.

The RARe polypeptide may also be used for screening of putative hormone molecules, such as steroid agonists that can modulate the hormone response mediated through this receptor. To assay for these and other ligands for the RARe, the gene encoding for RARe binding sites is cloned in the pRS eukaryotic expression vector (Giguere et al., Cell, 46:645 (1986)) producing pRShRARe . The plasmid is then introduced into monkey kidney CV-1 cells via calcium- phosphate transfection together with a reporter plasmid DELTA MTV-TRE sub p-CAT. TRE means thyroid receptor response element and TRE sub p is a TRE that has been engineered to maximize the palindorminicity of this element. As a control, pRSerbA sup-1 (encodes no protein, stands as a negative control) , is also examined. The transfected cells are incubated in the presence or absence of 100 nM retinoic acid, or other potential ligands, for 36 hours, and the induced CAT activities were analyzed by chromatography. The results will indicate which ligands bind to RARe .

This invention further provides a method of screening drugs to identify those which enhance (agonists) interaction of ligands to the RARe. As an example, a reporter plasmid and pRShRARe are introduced into monkey cells as discussed above. The transfected cells are then incubated with labeled retinoic acid in the presence of drug. The ability of drug to enhance this interaction could then be measured. The reporter gene can be a CAT or luciferase gene whose activities with respect to the interaction of ligand and receptor are capable or being measured (Promega Technical Bulletin No. 126, July, 1993) . Transfection systems are useful as living cell cultures for competitive binding assays between known or candidate drugs and ligands which bind to the receptor and which are labeled by radioactive, spectroscopic or other reagents. A transfection system constitutes a "drug discovery system" useful for the identification of natural or synthetic compounds with potential for drug development that can be further modified or used directly as therapeutic compounds to activate or inhibit the natural functions of the human RARe. The transfection system is also useful for determining the affinity and efficacy of known drugs at the human RARe sites.

The agonists and drugs which are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy." Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For

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SUBSTITUTE SHEET (RULE 25) example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

The RARe polypeptides of the present invention are also useful for identifying other molecules which have similar biological activity. An example of a screen for this is isolating the coding region of the RARe gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

Availability of the RARe allows its use in diagnostic assays to determine levels of retinoic acid present in various tissues and body fluids. The determination of these levels allows the diagnosis and treatment of diseases related to low levels of retinoic acid.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in si tu hybridization (FISH) of a cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes) .

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .

Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.

The present invention further provides a method for diagnosing a predisposition to a disorder associated with the underexpression of the RARe polypeptide, which comprises obtaining DNA of subjects suffering from the disorder, performing a restriction digest of the DNA with a panel of

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SUBSTITUTE SHEET (RULE 25) restriction enzymes, electrophoretically separating the resulting DNA fragments on a sizing gel, contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding an RARe polypeptide and labelled with a detectable marker, detecting labelled bands which have hybridized to the DNA encoding an RARe polypeptide with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder, preparing DNA obtained for diagnosis by the above steps and comparing the unique band pattern specific to the DNA of subjects suffering from the disorder and the DNA obtained from the diagnosis step to diagnose predisposition to the disorder.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For

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SUBSTITUTE SHEET (RULE 25) analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37"C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980) .

"Oligonucleotideε" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146) . Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973) .

Example 1 Expression of RARe by in vi tro transcription and translation The in vi tro transcription and translation of RARe was carried out using the TNΓ Coupled Reticulocyte Lysate System (Promega, 2800 Woods Hollow Road, Madison, WI 53771-5399) . The cDNA encoding for RARe, ATCC # 75754, was cloned directionally EcoRI to Xhol with the EcoRI site defining the 5' end of the gene and the Xhol site defining the 3' end of the gene. The gene was inserted in the T3 direction. T3 defines a bacteriophage RNA polymerase which recognizes a specific promoter, and transcribes the DNA into a mRNA. A rabbit reticulocyte lysate is supplemented with T3 RNA polymerase and directs the expression of proteins with a T3 promoter utilizing the T3 RNA polymerase to transcribe the message, and the reticulocyte lysate to translate the nascent RNA. Iμg of circular (or linear) plasmid containing the RARe DNA was added directly to TNT™ lysate and incubated in a 50 μl reaction volume at 30°C for 1 hour with T3 RNA polymerase and [³⁵S] -Methionine. After incubation, the translation product was separated by 10% SDS-PAGE gel electrophoresis. The gel was fixed in 10% acetic acid, 15% methanol for 30 minutes followed by drying on a Bio-Rad gel dryer for one hour. Autoradiography was carried out with Kodak XAR film. The film was exposed at -80°C with intensifying screen. A prominent translation product is visible at 49 kd (Figure 4)

Example 2 Expression and Purification of RARe in baculovirus-insect cell system

Sf9 insect cells are maintained in culture using Grace's Insect medium (Gibco) supplemented with 10% FBS at 27°C. The baculovirus transfer plasmid pA2 was constructed by Gentz et al (unpublished results) (Figure 5) . The entire human RARe cDNA including the BamHl site at 5' end and Asp 718 site at 3' end were amplified using PCR. The PCR oligonucleotide primers used for the amplification of the human RARe are:

5' -GCGCGGATCCACCΑTGTCCTTGTGGC_TGGGG-3 ' 5' -GCGCGGTACCTCATTCGTGCACATCCCAGAT-3 ' . The amplified fragment containing human RARe cDNA, and plasmid pA2 were digested by BamHI and Asp 718 and purified by Geneclean kit. The amplified fragment containing human RARe cDNA was inserted into vector pA2 to generate the recombinant transfer vector pA2RARe . The accuracy of the construct was verified using restriction endonuclease, PCR and DNA sequencing. Transfer of the human RARe cDNA from and pA2RAR to an AcNPV genome can be achieved by co-transfection using Lipofectin (Gibco) . AcNPV is a wild-type virus abailable from Pharmagene (San Diego, CA) . Techniques for manipulation of baculovirus and insect cell culture such as cultivation, co-transfection, virus infection, isolation, screening and purification of putative recombinant plaques and virus titer determination was described by O'Reilly D.R. et al [Baculovirus expression vectors: A Laboratory Manual (1992) , W. H. Freeman and Company] . The plaques will be screened for recombinant baculovirus by using the blue-white selection. The visually screened plaques are to be analyzed and confirmed by PCR screening. Once the recombinant virus is obtained, Sf9 cells (2X10⁶ cells/ml) can be infected with recombinant RAR virus (10⁸ plaque-forming units/ml) for 2 hr at room temperature. After transfection, the viral inoculum is replaced with fresh medium. The supernatant and cells are harvested after 60 - 72 hr. The extra cellular and intracellular fractions are then analyzed for the expression of RARe by SDS-PAGE. The receptor can be partially purified by sequential anion-exchange, gel-filtration and DNA affinity chromatography.

Example 3 Expression pattern of RARe in human tissue Northern blot analysis was carried out to examine the levels of expression of RARe in human tissues. Total cellular RNA samples were isolated with RNAzol™ B system

(Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033) . About 10μg of total RNA isolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press,

(1989) ) . The labeling reaction was done according to the Stratagene Prime-It kit with 50ng DNA fragment. The labeled DNA was purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303) . The filter was then hybridized with radioactive labeled full length RARe gene at 1,000,000 cpm/ml in 0.5 M NaP0₄, pH 7.4 and 7% SDS overnight at 65^°C. After wash twice at room temperature and twice at 60°C with 0.5 x SSC, 0.1% SDS, the filter was then exposed at -70°C overnight with an intensifying screen. The message RNA for RARe is abundant in testes, placenta, spleen, thymus and lung.

(Figure 3) .

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: CAO, ET AL.

(ii) TITLE OF INVENTION: Retinoic Acid Receptor Epsilon

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Concurrently

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-125

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1649 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AGTCCAGGTC CTGCTTGTGC TCAGCTCCAG CTCACTGGCT GGCCACCGAA ACTTCTGGAC 60

AGGAAACTGC ACCATCCTCT TCTCCCAGCA AGGGGGCTCC AGAGAACTGC CCACCCAGGA 120

AGTCTGGTGG CCTGGGGATT TGGACAGTGC CTTGGTAATG ACCAGGGCTC CAGGAAGAGA 180

TGTCCTTGTG GCTGGGGGCC CCTGTGCCTG ACATTCCTCC TGACTCTGCG GTGGAGCTGT 240

GGAAGCCAGG CGCACAGGAT GCAGGCAGCC AGGCCCAGGG AGGCAGCAGC TGCATCCTCA 300

GAGAGGAAGC CAGGATGCCC CACTCTGCTG GGGGTACTGC AGGGGTGGGG CTGGAGGCTG 360

CAGAGCCCAC AGCCCTGCTC ACCAGGGCAG AGCCCCCTTC AGAACCCACA GAGATCCGTC 420

CACCAAAGCG GAAAAAGGGG CCAGCCCCCA AAATGCTGGG GAACGAGCTA TGCAGCGTGT 480

GTGGGGACAA GGCCTCGGGC TTCCACTACA ATGTTCTGAG CTGCGAGGGC TGCAAGGAAT 540

TCTTCCGCCG CAGCGTCATC AAGGGAGCGC ACTACATCTG CCACAGTGGC GGCCACTGCC 600

CATGGAACAC CTACATGCGT CGCAAGTGCC AGGAGTGTGT CCTGTCAGAA GAACAGATCC 660

GCCTGAAGAA ACTGAAGCGG CAAGAGGAGG AACAGGTTCA TGCCACATCC TTGCCCCCCA 720

GGGCTTCCTC ACCCCCCCAA ATCCTGCCCC AGCTCAACCC GGAACAACTG GGCATGATCG 780

AGAAGCTCGT CCCTGCCCAG CAACAGTGTA ACCGGCGCTC CTTTTCTGAC CGGCTTCGAG 840

TCACGCCTTG GCCCATGGCA CCAGATCCCC ATAGCCGGGA GGCCCGTCAG CAGCGCTTTG 900

CCCACTTCAC TGAGCTGGCC ATCGTCTCTG TGCAGGAGAT AGTTGACTTT GCTAAACAGC 960 TACCCGGCTT CCTGCAGCTC AGCCGGGAGG AC AGATTGC CCTGCTGAAG ACCTCTGCGA 1020

TCGAGGTGAT GCTTGTGGAG ACATCTCGGA GGTACAACCC TGGGAGTGAG AGTATCACCT 1080

TCCTCAAGGA TTTCAGTTAT AACCGGGAAG ACTTTGCCAA AGCAGGGCTG CAAGTGGAAT 1140

TCATCAACCC CATCTTCGAG TTCTCCAGGG CCATGAATGA GCTGCAACTC AATGATCCCG 1200

AGTTTGCCTT GCTCATTGCT ATCAGCATCT TCTCTGCAGA CCGGCCCAAC GTGCAGGACC 1260

AGCTCCAGGT AGAGAGGCTG CAGCACACAT ATGTGGAAGC CCTGCATGCC TACGTCTCCA 1320

TCCACCATCC CCATGACCGA CTGATGTTCC CACGGATGCT AATGAAACTG GTGAGCCTCC 1380

GGACCCTGAG CAGCGTCCAC TCAGAGCAAG TGTTTGCACT GCGTCTGCAG GACAAAAAGC 1440

TCCCACCGCT GCTCTCTGAG ATCTGGGATG TGCACGAATG ACTGTTCTGT CCCCATATTT 1500

TCTGTTTTCT TGGCCGGATG GCTGAGGCCT GGTGGCTGCC TCCTAGAAGT GGAACAGACT 1560

GAGAAGGGCA AACATTCCTG GGAGCTGGGA AAGGAGATCC TCCCGTGGCA TTAAAAGAGA 1620

GTCAAAGGGT AAAAAAAAAA AAAAAAAAA 1649

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 433 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Leu Trp Leu Gly Ala Pro Val Pro Asp lie Pro Pro Asp

5 10 15

Ser Ala Val Glu Leu Trp Lys Pro Gly Ala Gin Asp Ala Gly Ser

20 25 30

Gin Ala Gin Gly Gly Ser Ser Cys lie Leu Arg Glu Glu Ala Arg

35 40 45

Met Pro His Ser Ala Gly Gly Thr Ala Gly Val Gly Leu Glu Ala

50 55 60

Ala Glu Pro Thr Ala Leu Leu Thr Arg Ala Glu Pro Pro Ser Glu

65 70 75

Pro Thr Glu lie Arg Pro Pro Lys Arg Lys Lys Gly Pro Ala Pro

80 85 90 Lys Met Leu Gly Asn Glu Leu Cys Ser Val Cys Gly Asp Lys Ala

95 100 105

Ser Gly Phe His Tyr Asn Val Leu Ser Cys Glu Gly Cys Lys Glu

110 115 120

Phe Phe Arg Arg Ser Val lie Lys Gly Ala His Tyr lie Cys His

125 130 135

Ser Gly Gly His Cys Pro Trp Asn Thr Tyr Met Arg Arg Lys Cys

140 145 150

Gin Glu Cys Val Leu Ser Glu Glu Gin lie Arg Leu Lys Lys Leu

155 160 165

Lys Arg Gin Glu Glu Glu Gin Val His Ala Thr Ser Leu Pro Pro

170 175 180

Arg Ala Ser Ser Pro Pro Gin lie Leu Pro Gin Leu Asn Pro Glu

185 190 195

Gin Leu Gly Met lie Glu Lys Leu Val Pro Ala Gin Gin Gin Cys

200 205 210

Asn Arg Arg Ser Phe Ser Asp Arg Leu Arg Val Thr Pro Trp Pro

215 220 225

Met Ala Pro Asp Pro His Ser Arg Glu Ala Arg Gin Gin Arg Phe

230 235 240

Ala His Phe Thr Glu Leu Ala He Val Ser Val Gin Glu He Val

245 250 255

Asp Phe Ala Lys Gin Leu Pro Gly Phe Leu Gin Leu Ser Arg Glu

260 265 270

Asp Gin He Ala Leu Leu Lys Thr Ser Ala He Glu Val Met Leu

275 280 285

Val Glu Thr Ser Arg Arg Tyr Asn Pro Gly Ser Glu Ser He Thr

290 295 300

Phe Leu Lys Asp Phe Ser Tyr Asn Arg Glu Asp Phe Ala Lys Ala

305 310 315

Gly Leu Gin Val Glu Phe He Asn Pro He Phe Glu Phe Ser Arg

320 325 330

Ala Met Asn Glu Leu Gin Leu Asn Asp Pro Glu Phe Ala Leu Leu

335 340 345 He Ala He Ser He Phe Ser Ala Asp Arg Pro Asn Val Gin Asp

350 355 360

Gin Leu Gin Val Glu Arg Leu Gin His Thr Tyr Val Glu Ala Leu

365 370 375

His Ala Tyr Val Ser He His His Pro His Asp Arg Leu Met Phe

380 385 390

Pro Arg Met Leu Met Lys Leu Val Ser Leu Arg Thr Leu Ser Ser

395 400 405

Val His Ser Glu Gin Val Phe Ala Leu Arg Leu Gin Asp Lys Lys

410 415 420

Leu Pro Pro Leu Leu Ser Glu He Trp Asp Val His Glu

425 430

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide encoding for RARe , said polynucleotide selected from the groups consisting of

(a) a polynucleotide encoding for the RARe polypeptide having the deduced amino acid sequence of Figure 1 or a fragment, analog or derivative of said polypeptide;

(b) a polynucleotide encoding for the RARe polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75754 or a fragment, analog or derivative of said polypeptide.

2. The polynucleotide of Claim l wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.

4. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

5. The polynucleotide of Claim 2 wherein said polynucleotide encodes for RARe having the deduced amino acid sequence of Figure 1.

6. The polynucleotide of Claim 2 wherein said polynucleotide encodes for the RARe polypeptide encoded by the CDNA of ATCC Deposit No. 75754.

7. The polynucleotide of Claim 1 having the coding sequence for RARe as shown in Figure 1.

8. The polynucleotide of Claim 2 having the coding sequence for RARe deposited as ATCC Deposit No. 75754.

9. A vector containing the DNA of Claim 2.

10. A host cell genetically engineered with the vector of Claim 9.

11. A process for producing a polypeptide comprising: expressing from the host cell of Claim 10 the polypeptide encoded by said DNA.

12. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 9.

13. An isolated DNA hybrid!zable to the DNA of Claim 2 and encoding a polypeptide having RARe activity.

14. A polypeptide selected from the group consisting of (i) a RARe polypeptide having the deduced amino acid sequence of Figure l and fragments, analogs and derivatives thereof and (ii) a RARe polypeptide encoded by the cDNA of ATCC Deposit No. 75754 and fragments, analogs and derivatives of said polypeptide.

15. The polypeptide of Claim 14 wherein the polypeptide is RARe having the deduced amino acid sequence of Figure 1.

16. An antibody against the polypeptide of claim 14.

17. An agonist for the polypeptide of claim 14.

18. A pharmaceutical composition comprising the polypeptide of Claim 14 and a pharmaceutically acceptable carrier.

19. A method for the treatment of a patient having need of stimulation of RARe receptors comprising: administering to the patient a therapeutically effective amount of the agonist of claim 17.

20. The method of Claim 19 wherein said therapeutically effective amount of the agonist is administered by providing to the patient DNA encoding said agonist and expressing said agonist in vivo.

21. A method of identifying compounds which interact with the RARe comprising: introducing an RARe gene and a reporter gene into a eukaryotic cell; contacting the cell with a compound; and determining whether the compound interacts with the

RARe .