JPH10501986A - Retinoic acid receptor epsilon - Google Patents

Abstract

  (57) [Summary] Disclosed are RARε polynucleotides, and DNA (RNA) encoding such polypeptides. Also provided are methods of producing such polypeptides by recombinant techniques, and methods of using such polypeptides for therapeutic purposes, eg, tissue regeneration, and stimulation of the immune and hematopoietic systems. Also disclosed is a method of identifying a ligand that stimulates RARε.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, and , The production of such polynucleotides and polypeptides. In particular, the polypeptide of the invention is the retinoic acid receptor epsilon (RARε). The invention also relates to inhibiting the action of such polypeptides. Retinoids (vitamin A derivatives) are important for the normal growth, vision, maintenance, reproduction and overall survival of many tissues (Wolbach, SB , J. Exp. Med. , 42: 753-777 (1925)). In addition, offspring of Vitamin A deficient (VAD) female parents show many developmental defects, indicating that retinoids are also important during embryogenesis (Wilson, GG et al., Am . J. Anat ., 92: 189-21. 7 (1953)). Retinoic acid (RA) administration can prevent and / or reverse the effects of vitamin A deficiency in fetuses and young and adult animals (Wilson, JG et al., Am. J. Anat. , 92 189-217 (1953)). The dramatic teratogenic effects of maternal RA administration on mammalian embryos and the impressive effects of topical administration of retinoids on vertebrate embryonic development and rim regeneration in amphibians indicate that RA is actually a morphogen (giving positional information during development) And contributes significantly to the notion that important roles may also play a role during organogenesis (Tabin, CJ, Cell , 66: 199-217 (1991)). Retinoids are also important for the normal growth, appearance, maintenance, reproduction and overall survival of many tissues (Wolbach, SB and Howe, PR , J. Exp. Med. , 42). : 753-777 (1925)). The action of the RA signal is thought to be mediated by two families of receptors that belong to the superfamily of ligand-inducible transcriptional regulators, including steroid thyroid hormones and the vitamin D3 receptor (Evans, R M., Science , 240: 889-895 (1988)). The retinoic acid receptor gene belongs to a superfamily of genes known as the steroid hormone receptor family. All genes in this family can be divided into distinct regions or domains, which are sometimes referred to as regions A / B, C, D, E, and F. The C region encodes a DNA binding domain, the E region encodes a ligand binding domain, and the F region encodes a carboxy terminal domain. The D region is thought to function as a "hinge". The function of the A / B (or N-terminal) region has not been fully elucidated, but may be involved in enhancing and suppressing receptor transcriptional activity (Hollenberg et al., Cell , 55: 899-906 (1988) )). The RA receptor (RAR) family (RARα, β and γ and their isoforms) is activated by both all-trans and non-cis RAs. RARδ has also been cloned (Mech, Dev., 40: 99-112 (1993)). Within certain species, the DNA-binding and ligand-binding domain domains of the three RAR types are very similar, while the C-terminal and intermediate regions show little or no similarity. The amino acid sequences of the three RAR types are also significantly different in their B regions, and their major isoforms (α1 and α2, β1 to β4, γ1 and γ2, and δ1 and δ2) further indicate that (Leid, M. et al., Trends Biochem. Sci. , 17: 427-433 (1992)). RARγ mutant mice exhibit growth impairment, early lethality, and male sterility due to seminal vesicle and squamous metaplasia of the prostate (Lohnes, D. et al., Cell , 73: 643). -658 (1993)). RARγ transcripts are found in prochondral compacts, regardless of their embryo origin, followed by restriction to cartilage and differentiation of squamous keratinized epithelium (Dolle, P. et al., Nature , 342: 702-705 (1989)). These observations suggest a role for RARγ in morphogenesis and chondrogenesis (Dolle et al., Developmen t, 100: 1133-1151 ( 1990)). The DNA sequences of the present invention and the polypeptides encoded by such DNA sequences belong to the retinoic acid receptor family and are most homologous to retinoic acid receptor gamma. Both the modulator and the DNA binding domain share the highest homology with the corresponding part of the Drosophila ecdysone receptor. During development, the ecdysone receptor plays an important role. According to one aspect of the present invention, there is provided a novel mature polypeptide that is RARε, as well as fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin. According to another aspect of the present invention, there is provided a polynucleotide (DNA or RNA) encoding such a polypeptide. According to yet a further aspect of the invention there is provided a method for producing such a polypeptide by recombinant techniques. According to yet a further aspect of the invention, such polypeptides, or polynucleotides encoding such polypeptides, are useful for therapeutic purposes, e.g., to identify their novel hormones, DNA binding sites, gene targets and tissue specificity. Methods of use are provided, which provide therapeutic targets for promoting development, differentiation, tissue regeneration, reproduction, and stimulation of the immune and hematopoietic systems. According to yet a further aspect of the present invention, there are provided antibodies against such polypeptides. These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein. The following drawings illustrate embodiments of the invention and are not intended to limit the scope of the invention, which is encompassed by the claims. FIG. 1 shows the predicted mature RARε polypeptide polynucleotide sequence and the corresponding deduced amino acid sequence. Use the standard one-letter abbreviations for amino acids. FIG. 2 illustrates the high homology between RARε and RARγ in the DNA and ligand binding regions. FIG. 3 shows the results of Northern blot analysis of RARε in adult tissues. FIG. 4 shows the results of electrophoresis of RARε on a gel after transcription / translation in vitro. FIG. 5 illustrates the baculovirus transfer plasmid pA2 used for the expression of RARε. According to one aspect of the present invention, the mature polypeptide encoding the mature polypeptide having the deduced amino acid sequence of FIG. 1 or the cDNA of the clone deposited as ATCC Deposit No. 75754 on Mar. 18, 1994. The present invention provides an isolated nucleic acid (polynucleotide). Polynucleotides encoding the polypeptides of the present invention can be obtained from testis, spleen and thymus. The polynucleotides of the present invention have been found in a cDNA library obtained from adult lung. It is structurally related to the retinoic acid receptor family. It contains an open reading frame encoding a protein of about 433 amino acid residues. The protein exhibits the highest degree of homology to retinoic acid receptor gamma with greater than 70 amino acids in length of the modulator region with 48% identity and 58% similarity. Similarly, the DNA binding and ligand binding regions are also very homologous to retinoic acid receptor gamma (FIG. 5). The polynucleotides of the present invention can be in the form of RNA or in the form of DNA, including cDNA, genomic DNA, and synthetic DNA. The DNA can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (anti-sense) strand. The coding sequence encoding the mature polypeptide may be the same as the coding sequence shown in FIG. 1 or the coding sequence of the deposited clone, or as a result of duplication or degeneracy of the genetic code, the DNA of FIG. Alternatively, it may be a different coding sequence encoding the same mature polypeptide as the deposited cDNA. The polynucleotide encoding the mature polypeptide of FIG. 1 or the mature polypeptide encoded by the deposited cDNA may include: the coding sequence of the mature polypeptide only; the coding sequence of the mature polypeptide; And additional coding sequences, such as leader or secretory sequences; the coding sequence of the mature polypeptide (and optionally additional coding sequences); and the introns or non-coding sequences 5 'and / or 3' of the coding sequence of the mature polypeptide. Non-coding array such as. Thus, the term "polynucleotide encoding a polypeptide" includes polynucleotides that include only the coding sequence of the polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences. The present invention further relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of FIG. 1 or the cDNA of the deposited clone. A variant of a polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide. Accordingly, the present invention includes polynucleotides encoding the same mature polypeptide as shown in FIG. 1 or the same mature polypeptide encoded by the cDNA of the deposited clone, as well as mutations of such polynucleotides. These variants encode fragments, derivatives or analogs of the polypeptide of FIG. 1 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition and insertion variants. As indicated above, the polynucleotide is a naturally occurring variant of the coding sequence shown in FIG. It may have a coding sequence that is a naturally occurring allelic variant of the coding sequence of an allelic variant or a deposited clone. As is known in the art, an allelic variant is another form of a polynucleotide sequence that can have one or more nucleotide substitutions, deletions or additions, which comprises the encoded polynucleotide. It does not substantially alter the function of the peptide. The polynucleotide of the invention may also have the coding sequence fused in frame to a marker sequence that allows for purification of the polypeptide of the invention. In the case of a cellular host, the marker sequence may be a hexa-histidine tag provided by the pQE-9 (Qiagen) vector to provide for purification of the mature polypeptide fused to the marker, or For example, when using a mammalian host, for example, COS-7 cells, the marker sequence may be a hemagglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al., Cell, 37: 767 (1984)). The invention further relates to polynucleotides that hybridize to the above sequences when there is at least 50%, and preferably 70%, identity between the sequences. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides. As used herein, the term "stringent conditions" means that hybridization will only occur if there is at least 95%, and preferably at least 97%, identity between the sequences. I do. In a preferred embodiment, the polynucleotide that hybridizes to the above-described polynucleotide is a polypeptide that possesses substantially the same biological function or activity as the cDNA of FIG. 1 or the mature polypeptide encoded by the deposited cDNA. Code. The deposit referred to herein will be kept under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. These deposits are provided solely as a convenience to those of skill in the art and are subject to 35 USC. It is not an admission that it is required under §112. The sequence of the polynucleotide contained in the deposited material, and also the amino acid sequence of the polypeptide encoded thereby, form part of the present specification and may include any of the sequences herein. Whenever it conflicts with the description, it is checked. A license may be required to make, use, or sell the deposited material, and no such license is granted herein. The invention further relates to RARε polypeptides having the deduced amino acid sequence of FIG. 1 or having the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptides. When referring to the polypeptide of FIG. 1 or the polypeptide encoded by the deposited cDNA, the terms "fragment,""derivative," and "analog" refer to substantially the same biological function as such a polypeptide. Or a polypeptide that retains activity. Thus, analogs include protein that can be activated by cleavage of the protein portion to produce an active mature polypeptide. The polypeptide of the present invention may be a recombinant, natural or synthetic polypeptide, preferably a recombinant polypeptide. Fragments, derivatives or analogs of the polypeptide of FIG. 1 or of the polypeptide encoded by the deposited cDNA may comprise (i) one or more amino acid residues of the same or non-homologous amino acid residues (preferably, Amino acid residue), and such substituted amino acid residue may or may not be encoded by the genetic code; (ii) one or Those in which further amino acid residues contain substituents, or (iii) the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (eg, polyethylene glycol). Or (iv) a leader or secretory sequence, or a sequence used to purify the mature polypeptide, or a protein sequence. Tsu and as may be those additional amino acids are fused to the mature polypeptide. Such fragments, derivatives and analogs are deemed to be within the skill of the art in light of 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 substance has been removed from its original environment (eg, the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide in a living animal has not been isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting materials in the natural system has been isolated. ing. Such polynucleotides can be part of a vector, and / or such polynucleotides or polypeptides can be part of a composition, and such vectors or compositions are not part of their natural environment. And still isolated. The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention, and recombinant production of the polypeptides of the present invention. A host cell is genetically engineered (transduced, transformed, or transfected) with a vector of the invention, which can be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, virus particle, phage, and the like. The engineered host cells can be cultured in a conventional nutrient medium that has been modified to activate the promoter, select for transformants, or amplify the RARε gene. Culture conditions, such as temperature, pH, etc., are those previously used in the host cell selected for expression and will be apparent to those skilled in the art. The polynucleotides of the present invention can be used to produce peptides by recombinant techniques. Thus, for example, the polynucleotide can be included in any one of various expression vectors for expressing the polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, eg, derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; Viral DNA such as viruses, fowlpox virus, and pseudorabies. However, any other vector can be used as long as it is replicable and viable in the host. The appropriate DNA sequence can be inserted into the vector by various methods. Generally, the DNA sequence is inserted into an appropriate restriction endonuclease site by methods known in the art. Such and other methods are deemed to be within the purview of those skilled in the art. The DNA sequence of the expression vector is operably linked to an appropriate expression control sequence (promoter) to cause mRNA synthesis. As representative examples of such promoters include the following:. LTR or SV40 promoter, E. coli lac or the trp, the phage lambda P _L promoter, and expression of genes in prokaryotic or eukaryotic cells or their viruses Other promoters that are known to control. 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 vector may be used to express a transformed host cell, such as dihydrofolate reductase or neomycin resistance in eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli. It is preferred to include one or more selectable marker genes to confer type characteristics. Using a suitable DNA sequence, as described above, as well as a vector containing a suitable promoter or control sequence, to transform a suitable host to enable that host to express the protein. Can be. As representative examples of appropriate hosts, include the following: E.coli, Streptomyc es, bacterial cells such as Salmonella typhimurium; insect cells such as Drosophila and Sf9;; fungal cells such as yeast CHO, COS or Animal cells such as Bowes melanoma; plant cells and the like. The selection of a suitable host will be within the skill of the art in light of the teachings herein. In particular, the present invention also includes a recombinant construct comprising one or more of the sequences broadly described above. The construct comprises a vector, such as a plasmid or viral vector, into which the sequences of the invention have been inserted in forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises control sequences, including, for example, a promoter operably linked to the sequence. Many suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are examples. For bacteria: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3 pKK233-3, pDR54, pRIT5 (Pharmacia), pRS and pGEM. For eukaryotes: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used as long as they are replicable and viable in the host. The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector having a selectable marker. Two suitable vectors are PKK232-8 and PCM7. The Particular named bacterial promoters include lac I, lacZ, T3, T7 , gpt, lambda P _R, P _L and rp. 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 aspect, the present invention relates to a host cell comprising the above-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, 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 host cells can be by calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, L., Basic Methods in Molecular Biology (1986)). The construct in a host cell can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptide of the present invention can be produced synthetically using a conventional peptide synthesizer. 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 used to produce such proteins using RNA obtained from the DNA constructs of the present invention. Cloning and expression vectors suitable for use in prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, New York, (1989), the disclosure of which is hereby incorporated by reference. , Constitute a part of this specification. Transcription of a DNA encoding a polypeptide of the invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers at bp 100-270. Typically, recombinant expression vectors include an origin of replication and a selectable marker that allows for transformation of the host cell, such as the E. coli ampicillin resistance gene and the S. cerevisiae TRP1 gene, and the downstream structural sequences. A promoter derived from a highly expressed gene to drive transcription would be included. Such promoters can be obtained from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in an appropriate phase, with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. . Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that confers desired properties, such as stabilization or purification of the expressed recombinant product. Expression vectors useful for use in bacteria are constructed by inserting a structural DNA sequence encoding the desired protein, along with appropriate translation initiation and termination signals, into an operable reading phase having a functional promoter. You. The vector includes one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification in the host. Will be. Prokaryotic hosts suitable for transformation include E. coli , Bacillus subtilis , Salmonella typhimurium , and various species within the genus Pseudomonas, Streptomyces, and Staphylococcus, but others may also be selected substances. Can be used as As a representative, but non-limiting example, a vector useful for use in bacteria is a selectable vector obtained from a commercially available plasmid comprising the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). It may comprise a marker and a bacterial origin of replication. Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "bone nuclei" portions are combined with an appropriate promoter and the structural sequence to be expressed. After transformation of a suitable host strain and growth of the host strain to a suitable cell density, the selected promoter is induced by a suitable method (e.g., temperature shift or chemical induction) to further promote the cells. Incubate for a period. The cells are generally collected by centrifugation, disrupted by physical or chemical methods, and the resulting crude extract is retained for further purification. Microbial cells used for protein expression can be disrupted by any conventional method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents, and such methods are known to those of skill in the art. It is well known. Various mammalian cell culture systems can also be used to express the recombinant protein. Examples of mammalian expression systems include the COS-7 system of monkey kidney fibroblasts, as described by Gluzman, Cell, 23: 175 (1981), and other capable of expressing compatible vectors. Cell lines such as C127, 3T3, CHO, HeLa and BHK cell lines are included. Mammalian expression vectors contain an origin of replication, a suitable promoter and enhancer, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and non-transcribed sequences flanking the 5 '. Will also comprise. The DNA sequence obtained from SV40 splicing, and the polyadenylation site can be used to provide the required non-transcribed genetic elements. RARε polypeptides include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. The method allows for recovery and purification from recombinant cell culture. It is preferred to have a low concentration (about 0.15-5 mM) of calcium ions during the purification (Price et al., J. Biol. Chem., 244: 917 (1969)). If desired, a protein regeneration step can be used to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HP LC) can be used for the final purification step. The polypeptides of the present invention can be naturally purified products, or products of chemical synthesis, or from prokaryotic or eukaryotic hosts, such as bacteria, yeast, higher plants, insects and mammals in culture. It can be produced by recombinant technology (by cells). Depending on the host used in the recombinant production method, the polypeptide of the present invention may be glycosylated or may not be glucosylated. The polypeptides of the present invention may also include a first methionine amino acid residue. The RARε hormone receptor can be used to identify its novel hormones, DNA binding sites, gene targets and tissue specificity. RARε polypeptides can also be used as therapeutic targets for tissue regeneration, reproduction, stimulation of the immune and hematopoietic system, and for the treatment of male sterility. This is particularly important since the RAR mutant mice exhibited the aforementioned abnormalities. Thus, RARε receptors can be used to treat these disorders. RARε polypeptides can also be used to screen for putative hormone molecules, such as steroid agonists, that can modulate the hormonal response mediated by this receptor. To assay these and other ligands for RARε, the gene encoding the RARε binding site was cloned into a pRS eukaryotic expression vector (Giguere et al., Cell , 46: 645 (1986)) to produce pRShRARε. I do. The plasmid is then introduced into monkey kidney CV-1 cells by calcium phosphate transfection together with the reporter plasmid DELTA MTV-TRE sub p-CAT. TRE means thyroid receptor response factor, and TRE sub p is a TRE engineered to maximize the palindromic nature of this factor. As a control, pRSerbAsup-1 (which does not encode any protein, which is a negative control) is also tested. Transfected cells were incubated for 36 hours in the presence or absence of 100 nM retinoic acid or other potential ligand, and the induced CAT activity was analyzed by chromatography. The results will indicate that the ligand binds to RARε. The present invention further provides a method of screening a drug to identify a drug (agonist) that enhances the interaction of the ligand with RARε. As an example, a reporter plasmid and pRShRARε are introduced into monkey cells, as discussed above. The transfected cells are then incubated with labeled retinoic acid in the presence of the drug. The ability of the drug to enhance this interaction could then be measured. The reporter gene can be a CAT or luciferase gene, whose activity with respect to ligand and receptor interaction is possible or can be measured (Promega Technical Bulletin 126, July 1993). Transfection systems are useful as biological cell cultures for competitive binding assays between known or candidate drugs and ligands that bind to the receptor and are labeled by radioactivity, spectroscopy or other reagents . The transfection system may be used to generate natural or synthetic compounds that have the potential for the development of drugs that can be further modified or used directly as therapeutic compounds to activate or inhibit the natural function of human RARε. Construct a "drug discovery system" useful for identification. The transfection system is also useful for measuring the affinity and potency of a known drug at a human RARε site. Agonists and drugs that are polypeptides can be used in accordance with the present invention due to the in vivo expression of such polypeptides, often referred to as "gene therapy." Thus, for example, cells from a patient are engineered ex vivo with a polynucleotide (DNA or RNA) encoding a polypeptide, and the engineered cells are then provided to a patient to be treated with the peptide. Such methods are well-known in the art. For example, cells can be engineered by methods known in the art by use of retroviral particles comprising RNA encoding a polypeptide of the invention. Similarly, cells can be engineered in vivo for expression of the polypeptide in vivo, for example, by methods known in the art. As is known in the art, producing cells for producing retroviral particles comprising RNA encoding a polypeptide of the invention, in order to treat the cells in vivo and express the polypeptide in vivo. It can be administered to a patient. These and other methods for administering a polypeptide of the present invention by such methods will be apparent to those skilled in the art from the teachings of the present invention. For example, an expression vehicle for manipulating cells may be other than a retrovirus, for example, an adenovirus that can be used to manipulate cells in vivo after being combined with a suitable delivery vehicle. The RARε polypeptides of the present invention are also useful for identifying other molecules with similar biological activity. An example of a screen in this regard is to isolate the coding region of the RARε gene by synthesizing an oligonucleotide probe using a known DNA sequence. A probe is hybridized to any member of the library using a labeled oligonucleotide having a sequence complementary to the sequence of the gene of the invention to screen a library of human cDNA, genomic DNA or mRNA. To decide. The availability of RARε allows its use in diagnostic assays to measure levels of retinoic acid present in various tissues and body fluids. Measurement of these levels allows diagnosis and treatment of diseases associated with low levels of retinoic acid. The sequences of the present invention are also valuable for chromosome identification. The sequence can be specifically targeted to a particular location on an individual human chromosome and hybridized. Moreover, there is currently a need to identify specific sites on the chromosome. Chromosome marking reagents based on actual sequence data (repetitive polymorphisms) are currently scarcely available for marking chromosomal locations. The mapping of DNA to chromosomes according to the present invention is an important first step in relating those sequences to genes associated with disease. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. The amplification process is complicated by the rapid selection of primers that do not span one or more exons in genomic DNA using computer analysis of the cDNA. 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 give the amplified fragment. PCR mapping of somatic cell hybrids is a rapid method for assigning specific DNA to specific chromosomes. Utilizing the present invention with the same oligonucleotide primers, sublocalization can be achieved in a similar manner using panels of fragments from a pool of specific chromosomes or large genomic clones. Other mapping methods that can also be used to map to the chromosome include in situ hybridization, prescreening using labeled flow-sorted chromosomes, and live chromosome-specific cDNAs. Preselection by hybridization to construct a rally is included. Fluorescent in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNAs as short as 500 or 600 bases; however, clones larger than 2,000 bp are more likely to bind to unique chromosomal locations with sufficient signal intensity for easy detection. FISH requires the use of clones from which ESTs are obtained, and the longer the better. For example, 2,000 bp is better, 4,000 is better, and 4,000 or more is probably not necessary to get good results at a reasonable time rate. 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 location of the sequence on the chromosome can be associated with genetic map data. Such data is found, for example, in V. McKusick, Mendelian Inheritance in Man (available online via the Johns Hopkins University Welch Medical Library). The relationship between the gene and the disease mapped to the same chromosomal region is then confirmed by binding 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 the mutation is found in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be disease-causing. At present, from the analysis of physical and genetic mapping, a cDNA that is correctly localized to a chromosomal region associated with a disease could be one of 50-500 possible causative genes. (This assumes a 1 megabase mapping analysis and one gene per 20 kb). Comparisons between affected and unaffected individuals typically include deletions or translocations that can be viewed from chromosomal spreads or detected using PCR based on their cDNA sequences. First, look for structural changes in the chromosome. Finally, complete sequencing of genes from several individuals is required to confirm the presence of the mutation and to distinguish the mutation from polymorphism. Antibodies to them can be produced using the polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them as an immunogen. 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 products of a Fab expression library. Various methods known in the art can be used for the production of such antibodies and fragments. Antibodies raised against a polypeptide corresponding to a sequence of the present invention can be obtained by injecting the polypeptide directly into an animal, or by administering the polypeptide to an animal, preferably a non-human animal. The antibody so obtained will then bind to the polypeptide itself. In this way, even sequences encoding only fragments of the polypeptide can be used to produce antibodies that bind the entire naturally occurring polypeptide. Such an antibody can then be used to isolate the polypeptide from tissues expressing the polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include hybridoma technology (Kohler and Milstein, 1975, Nature, 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today 4:72), and humans. EBV-hybridoma technology for producing monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) is included. The techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) may be adapted for producing single-chain antibodies to the immunogenic polypeptide products of the invention. The invention further provides a method of diagnosing a predisposition to a disorder associated with an under-expression of a RARε polypeptide, the method comprising obtaining DNA from a subject suffering from the disorder, and restricting the restriction digestion of the DNA by a group of restriction agents. Performed with an enzyme, the resulting DNA fragments are separated by electrophoresis on a sizing gel, and the resulting gel can be specifically hybridized to DNA encoding the RARε polypeptide and detected. Contacting with a nucleic acid probe labeled with a possible marker, detecting a labeled band that hybridizes to the DNA encoding the RAR ε polypeptide with a detectable marker, and detecting the presence of the subject suffering from the disorder. Create a unique band pattern specific to DNA, prepare the DNA obtained in the previous step for diagnosis, That in comparison subjects DNA and their own band pattern specific to the obtained DNA from the diagnostic process consists of diagnosing the predisposition to said disorders. The invention will be further described with reference to the following examples; however, it should be understood that the invention is not limited to such examples. All parts or amounts are by weight unless otherwise indicated. To facilitate understanding of the following examples, some frequently occurring methods and / or terms will be described. "Plasmids" are indicated 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 a non-limiting basis, or can be constructed from plasmids available by published methods. In addition, plasmids equivalent to the described plasmids are known in the art and will be apparent to those skilled in the art. "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 one of skill in the art. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, generally 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 usually utilized, but can be varied according to the supplier's instructions. After digestion, the reaction is directly electrophoresed on a polyacrylamide gel to isolate the desired fragment. Size separation of the cleaved fragments is performed using an 8% polyacrylamide gel as described by Goeddel, D et al., Nucleic Acids Res., 8: 4057 (1980). "Oligonucleotide" refers to a single-stranded polydeoxynucleotide, or two complementary polynucleotide strands, that can be chemically synthesized. Since such a synthetic oligonucleotide does not have a 5 'phosphate, it will not ligate to another oligonucleotide without adding a phosphate with ATP in the presence of a kinase. Synthetic oligonucleotides will ligate to fragments that have not been dephosphorylated. "Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments (Maniatis, T. et al., Ibid., P. 146). Unless otherwise stated, ligation can be accomplished using known buffers and conditions with approximately 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amount of the DNA fragment to be ligated. Unless otherwise stated, transformations were performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52: 456-457 (1973). Example 1 Expression of RARε by In Vitro Transcription and Translation In vitro transcription and translation of RARε was performed using the TNT Coupled Reticulocyte Lysate System (Promega, 2800 Woods Hollow Road, Madison, WI 53775-1399). The cDNA encoding RARε, ATCC No. 75754, was cloned in the EcoRI to XhoI direction. An EcoRI site defines the 5 'end of the gene, and an XhoI site defines the 3' end of the gene. The gene was inserted in the direction of T3. T3 defines a bacteriophage RNA polymerase that recognizes a specific promoter and transcribes DNA into mRNA. Rabbit reticulocyte lysate is supplemented with T3 RNA polymerase and protein expression is driven by the T3 promoter using T3 RNA polymerase to transcribe messages and reticulocyte lysate to translate nascent RNA. 1 μg of circular (or linear) plasmid containing RARε DNA was added directly to TNT ^™ lysate and incubated with T3 RNA polymerase and [ ³⁵ S] -methionine for 1 hour at 30 ° C in a 50 μl reaction volume. After incubation, the translation products were separated by 10% SDS-PAGE gel electrophoresis. The gel was fixed in 10% acetic acid and 15% methanol for 30 minutes, and then dried for 1 hour in a Bio-Rad gel dryer. Autoradiography was performed using Kodak XAR film. The film was exposed to an intensifying screen at -80C. The major translation product can be found at 49 Kd (FIG. 4). Example 2 Expression and Purification of RARε in a Baculovirus-Insect Cell System Sf9 insect cells are maintained at 27 ° C. in culture in Grace's Insect medium (Gibco) supplemented with 10% FBS. . The baculovirus transfer plasmid pA2 was constructed by Gentz et al. (Unpublished results) (FIG. 5). PCR was used to amplify a complete human RARε cDNA containing a BamHI site at the 5 ′ end and an Asp 718 site at the 3 ′ end. The PCR oligonucleotide primers used for the amplification of human RARε were: It is. The amplified fragment containing human RARε cDNA and plasmid pA2 were digested with BamHI and Asp 718 and purified with the Geneclean kit. The amplified fragment containing the human RARε cDNA was inserted into the vector pA2 to generate the recombinant transfer vector pA2RARε. Restriction endonuclease, PCR and DNA sequencing were used to confirm the accuracy of the construct. Transfer of human RARε cDNA from pA2RAR to the AcNPV genome can be accomplished by co-transfection with lipofectin (Gibco). AcNPV is a wild-type virus available from Pharmagene (San Diego, CA). Techniques for the manipulation of baculovirus and insect cell cultures, such as culturing, co-transfection, viral infection, isolation, screening and purification of putative recombinant plaques, and virus titer measurement, are described in O'Reilly DR. [Baculovirus expression vector: A Laboratory Manual (1992), WH Freeman and Company]. Plaques will be screened for recombinant baculovirus by utilizing blue-white selection. Plaques screened visually must be confirmed by analysis by PCR screening. Once the recombinant virus is obtained, Sf9 cells (2 × 10 ⁶ cells / ml) can be infected with the recombinant RAR virus (10 ⁸ plaque forming units / ml) for 2 hours at room temperature. After transfection, the virus inoculum is replaced with fresh medium. Supernatants and cells are collected after 60-72 hours. The extracellular and intracellular fractions are then analyzed for RARε expression by SDS-PAGE. The receptor can be partially purified by continuous anion exchange, gel filtration and DNA affinity chromatography. Example 3 Expression pattern of RARε in human tissues Northern blot analysis was performed to examine the expression level of RARε in human tissues. Total cellular RNA samples were isolated on the RNAzol ^™ B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033). Approximately 10 μg of total RNA isolated from each designated human tissue was separated on a 1% agarose gel and blotted on nylon filters (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, ( 1989)). Labeling reactions were performed with 50 ng of DNA fragments using the Stratagene Prime-It kit. The labeled DNA was purified on a Select-G-50 column (5 Prime-3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filters were then hybridized with 1,000,000 cpm / ml of the full-length radiolabeled RARε gene in 0.5 M NaPO ₄ (pH 7.4) and 7% SDS at 65 ° C. overnight. Soy. After washing twice with 0.5 × SSC, 0.1% SDS at room temperature and twice at 60 ° C., the filter was exposed to an intensifying screen at −70 ° C. overnight. RARε message RNA is abundant in testis, placenta, spleen, thymus and lung (FIG. 3). Since many modifications and variations of the present invention are possible in light of the above teachings, the present invention can be practiced otherwise than as specifically described within the scope of the following claims.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI C07H 21/04 7823-4B C12Q 1/68 Z C07K 14/705 0276-2J G01N 33/566 16/28 9358- 4B C12P 21/08 C12N 5/10 9735-4B C12N 5/00 A C12Q 1/68 9051-4C A61K 37/02 ACV G01N 33/566 9051-4C ABD // C12P 21/08 9051-4C ABY (72) Inventor Knee, Chian Mannonfield Road, Rockville, MD 20853, United States No. 5502 (72) Inventor Freishman, Robert Di United States 20878 Chiefly Square Road, Gaithersburg, Maryland No. 470

Claims (1)

[Claims]   1. An isolated polynucleotide encoding RARε, comprising: (A) a RARε polypeptide having the deduced amino acid sequence of FIG. 1, or the polypeptide Polynucleotides encoding peptide fragments, analogs or derivatives Do; (B) encoded by the cDNA contained in ATCC Deposit No. 75754 A RARε polypeptide having an amino acid sequence, or a fragment of the polypeptide; A polynucleotide encoding an antigen, analog or derivative; A polynucleotide selected from the group consisting of:   2. The polynucleotide according to claim 1, wherein the polynucleotide is DNA.   3. The polynucleotide according to claim 1, wherein the polynucleotide is RNA.   4. The polynucleotide according to claim 1, wherein the polynucleotide is genomic DNA. Chid.   5. The polynucleotide encodes RARε having the deduced amino acid sequence of FIG. The polynucleotide according to claim 2, which is loaded.   6. The polynucleotide is derived from the cDNA of ATCC Deposit No. 75754. 3. The polynucleic acid of claim 2, which encodes the encoded RARε polypeptide. Leotide.   7. 2. The polynucleic acid according to claim 1, which has the coding sequence of RARε shown in FIG. Leotide.   8. RARε coding sequence deposited under ATCC Deposit No. 75754 The polynucleotide according to claim 2, having the formula:   9. A vector comprising the DNA according to claim 2.   10. A host cell genetically engineered with the vector of claim 9.   11. A method for producing a polypeptide, said method being encoded by said DNA Expressing the polypeptide from the host cell of claim 10. Way.   12. A method for producing a cell capable of expressing a polypeptide. A method comprising genetically engineering a cell with the vector of claim 9. Law.   13. It is possible to hybridize to the DNA according to claim 2, An isolated DNA encoding a polypeptide having ARε activity.   14． (I) a RARε polypeptide having the deduced amino acid sequence of FIG. 1; Fragments, analogs and derivatives thereof; and (Ii) RARε encoded by the cDNA of ATCC Deposit No. 75754 A polypeptide, and fragments, analogs and derivatives of the polypeptide; A polypeptide selected from the group consisting of:   15. The polypeptide is RARε having the deduced amino acid sequence of FIG. 1, The polypeptide according to claim 14.   16. An antibody against the polypeptide of claim 14.   17． An agonist of the polypeptide of claim 14.   18. 15. A polypeptide comprising the polypeptide of claim 14 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:   19. A method for treating a patient in need of RARε receptor stimulation, comprising: Administering a therapeutically effective amount of the agonist of claim 17 to the patient. How to be.   20. A DNA encoding the agonist is given to a patient, and the agonist is injected into the patient. Administer a therapeutically effective amount of the agonist by expressing in vivo 20. The method of claim 19.   21. A method for identifying a compound that interacts with RARε, comprising: Introducing the RARε gene and the receptor gene into a eukaryotic cell; Contacting the cell with a compound; and Determine whether the compound interacts with RARε; A method consisting of: