JP2002034583A - Retinoic acid receptor epsilon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To disclose an amino acid sequence of retinoic acid receptor epsilon (RARε), and a nucleotide sequence encoding the RARε. SOLUTION: The RARε and a DNA (RNA) encoding such a polypeptide are provided. The method for producing the peptide by a recombinant technique, and the method for utilizing the peptide for therapy, e.g. tissue regeneration, and stimulation of immune or hematopoietic systems are also provided. The method for identifying a ligand stimulating the RARε is also provided.

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 also the use of such polynucleotides and polypeptides. Related to the manufacture of. Above all,
The polypeptide of the present invention is the retinoic acid receptor epsilon (RARε). The invention also relates to inhibiting the action of such polypeptides.

[0002] Retinoids (vitamin A derivatives) are responsible for the normal growth, vision, maintenance, and reproduction of many tissues.
ction) and overall survival (Wolbach, S. et al.
B., J. Exp. Med. , 42: 753-777 (192)
5)). In addition, offspring of Vitamin A deficient (VAD) female parents show many developmental defects, indicating that retinoids are also important during embryogenesis (Wilson, J.G. et al., Am.J.
Anat. , 92: 189-217 (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 striking effects of local administration of retinoids on vertebrate embryonic development and rim regeneration in amphibians are described by R
A is actually a morphogen (giving location information during outbreak)
And also contributes significantly to the notion that important roles can also be played during organogenesis (Tabin, CJ, C
ell , 66: 199-217 (1991)).

[0003] Retinoids are also important for the normal growth, appearance, maintenance, reproduction and overall survival of many tissues.
(Wolbach, SB and Howe, PR , J. Exp . Me
d. , 42: 753-777 (1925)).

[0004] 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 vitamin D3 receptors ( Evans, RM, Science , 240: 88.
9-895 (1988)).

[0005] 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 region encodes the 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 repressing receptor transcriptional activity (Hollenberg et al., Cell , 55: 899-906).
(1988)).

The RA receptor (RAR) family (RAR
α, β and γ and their isoforms) are activated by both all-trans and non-cis RA. RARδ has also been cloned (Mech, Dev., 40: 99-112 (19
93)). Within certain species, three RAR-type DNs
The A-binding region and the ligand-binding region domain are very similar, whereas the C-terminal and middle 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 Differs in the N-terminal A region of
(Leid, M. et al., Trends Biochem. Sci. , 17: 427.
-433 (1992)).

[0007] RARγ mutant mice exhibit impaired growth, early lethality, and male sterility due to squamous metaplasia of the seminal vesicles and prostate (Lohnes,
D. et al., Cell 73: 643-658 (1993)).

[0008] RARγ transcripts are found in pre-cartilaginous compacts, regardless of their embryo origin, and are restricted to cartilage,
And differentiation of squamous keratinized epithelium follow (Dolle,
P. et al., Nature , 342: 702-705 (198).
9)). These observations suggest a role for RARγ in morphogenesis and chondrogenesis (Dolle et al., Develo
pment , 100: 1133-1151 (1990)).

DNA sequences of the invention and such DNs
The polypeptide encoded by the A sequence belongs to the retinoic acid receptor family and is 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 polypeptide of the present invention
It is of human origin.

According to another aspect of the present invention, there is provided a polynucleotide (DNA or RNA) encoding such a polypeptide.

[0011] According to yet a further aspect of the present invention, there is provided a method for producing such a polypeptide by recombinant techniques.

In accordance with yet a further aspect of the present invention, such polypeptides, or polynucleotides encoding such polypeptides, are identified for therapeutic purposes, eg, identifying their novel hormones, DNA binding sites, gene targets and tissue specificity. And provides therapeutic targets for promoting development, differentiation, tissue regeneration, reproduction, and stimulation of the immune and hematopoietic system.

[0013] According to yet a further aspect of the present invention, there are provided antibodies against such polypeptides.

[0014] These and other aspects of the invention include:
It will be apparent to those skilled in the art from the teachings herein.

The following drawings illustrate embodiments of the present invention and are not intended to limit the scope of the invention, which is encompassed by the claims. 1 to 4 show the polynucleotide sequence of the putative mature RARε polypeptide and the corresponding putative amino acid sequence. Use the standard one-letter abbreviations for amino acids. FIGS. 5 and 6 show D
RARε and R at the NA and Ligand Binding Regions
Explains the high homology with ARγ. FIG. 7 shows the results of Northern blot analysis of RARε in adult tissues. FIG. 8 shows that after transcription / translation in vitro,
The result of electrophoresis of RARε on a gel is shown. FIG.
The baculovirus transfer plasmid pA2 used for the expression of RARε will be described.

According to one aspect of the present invention, a mature polypeptide having the deduced amino acid sequence of FIGS.
Provided is an isolated nucleic acid (polynucleotide) encoding a mature polypeptide encoded by the cDNA of a clone deposited on March 18, 2008 under ATCC Deposit No. 75754.

The polynucleotide encoding the polypeptide 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 responds to retinoic acid receptor gamma
Over 70 amino acids in length of the modulator region shows the highest degree of homology with 48% identity and 58% similarity. Similarly, the DNA binding and ligand binding regions are also very homologous to retinoic acid receptor gamma (FIG. 9).

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, wherein the DNA comprises cD
NA, 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 FIGS. 1-4 or the coding sequence of the deposited clone, or as a result of the duplication or degeneracy of the genetic code, of FIG. It may be a different coding sequence that encodes the same mature polypeptide as the DNA or deposited cDNA.

Polynucleotides encoding the mature polypeptide of FIGS. 1-4 or the mature polypeptide encoded by the deposited cDNA may include: the coding sequence of the mature polypeptide only; the mature polypeptide And additional coding sequences, such as a leader or secretory sequence; the coding sequence of the mature polypeptide (and optionally, the additional coding sequence); and introns or non-coding sequences 5 'of the coding sequence of the mature polypeptide and And / or non-coding sequences such as 3 '.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide that includes only the coding sequence of the polypeptide, as well as a polynucleotide that includes additional coding and / or non-coding sequences. I do. The present invention further encodes polypeptides having the deduced amino acid sequence of FIGS. 1-4 or fragments, analogs and derivatives of the polypeptide encoded by the cDNA of the deposited clone.
It relates to variants of the above polynucleotides. A variant of a polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention provides for the cD of the same mature polypeptide or deposited clone as shown in FIGS.
Included are polynucleotides encoding the same mature polypeptide encoded by NA, as well as variants of such polynucleotides, which are variants of the polypeptides of FIGS. 1-4 or the deposited clone. cD
A fragment of the polypeptide encoded by NA,
Encode the derivative or analog. Such nucleotide variants include deletion variants, substitution variants, and addition and insertion variants.

As indicated above, the polynucleotide may be a naturally occurring allelic variant of the coding sequence shown in FIGS. 1-4 or a naturally occurring allelic variant of the coding sequence of the deposited clone. May have an array. 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,
It does not substantially alter the function of the encoded polypeptide.

The polynucleotide of the present invention may also have the coding sequence fused in frame to a marker sequence that allows for purification of the polypeptide of the present invention. In the case of a cellular host, the marker sequence may include pQE-9 (Qiagen) to provide for purification of the mature polypeptide fused to the marker.
Hex-histidine tag (ta
g) 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, 3;
7: 767 (1984)).

The present invention further provides that at least 50
%, And preferably 70% identity, to polynucleotides that hybridize to the above sequences. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the above-described polynucleotides. As used herein, the term "stringent conditions" refers to at least 95%
It also preferably means that hybridization will only occur if there is at least 97% identity. In a preferred embodiment, the polynucleotide that hybridizes to the above polynucleotide is the polynucleotide of FIGS.
Encodes a polypeptide that substantially retains the same biological function or activity as the cDNA or the mature polypeptide encoded by the deposited cDNA.

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 merely as a convenience to those skilled in the art, and
It is not an admission that it is required under U.S.C. §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 present invention further provides a RARε polypeptide having the deduced amino acid sequence of FIGS. 1-4 or having the amino acid sequence encoded by the deposited cDNA, and also fragments of such a polypeptide;
For analogs and derivatives.

The polypeptides of FIGS. 1-4 or deposited
When referring to a polypeptide encoded by a cDNA,
The terms "fragment,""derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as such a polypeptide. Thus, analogs include protein that can be activated by cleavage of the protein portion to produce an active mature polypeptide.

[0028] The polypeptide of the present invention may be a recombinant, natural or synthetic polypeptide, preferably a recombinant polypeptide.

The polypeptides of FIGS. 1-4 or deposited
A fragment, derivative or analog of the polypeptide encoded by the cDNA may comprise (i) one or more amino acid residues having been replaced by homologous or non-homologous amino acid residues (preferably homologous amino acid residues); Also, such substituted amino acid residues may or may not be those encoded by the genetic code, and (ii) one or more amino acid residues may have a substituent. Included, 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) leader or secretion A sequence, or a sequence used to purify a mature polypeptide, or a protein sequence,
Additional amino acids may be 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 or transformed or transfected) with a vector of the invention, which may 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 cell activates the promoter, selects for the transformant,
Alternatively, the cells can be cultured in a conventional nutrient medium modified so as to be suitable for amplifying the RARε gene. Temperature, p
Culture conditions such as H are those previously used in the host cell selected for expression and will be apparent to those skilled in the art.

The polynucleotide of the present invention can be used to produce a peptide 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 such as SV40.
Bacterial plasmid; phage DNA; baculovirus; yeast plasmid; plasmid and phage DN
A vector obtained from the combination with A; viral DNA such as vaccinia, adenovirus, 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:
It is believed to be within the skill of the art.

The DNA sequence of the expression vector is operably linked to an appropriate expression control sequence (promoter), and
Let the composition take place. Representative examples of such promoters include: LTR or SV40 promoter, E. coli lac or trp , phage lambda PL promoter, and expression of genes in prokaryotic or eukaryotic cells or their viruses. Other promoters 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, expression vectors may be used to select for transformed host cells, 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 phenotypic characteristics.

A suitable DNA sequence, as described above, as well as a vector containing a suitable promoter or control sequence, can be used to transform a suitable host to enable that host to express the protein. Can be Representative of suitable hosts include the following: E. coli , Streptomyces , Salmonella ty
bacterial cells such as phimurium ; fungal cells such as yeast; insect cells such as Drosophila and Sf9 ; animal cells such as CHO, COS or Bowes melanoma; 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 regulatory 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 given as examples. For bacteria: pQE70, pQE
60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript S
K, pbsks, pNH8A, pNH16a, pNH18A, pN
H46A (Stratagene); ptrc99a, pKK223-
3, pKK233-3, pDR540, pRIT5 (Pharm
acia), pRS and pGEM. For eukaryotes: pWLNE
O, pSV2CAT, pOG44, pXT1, pSG (Stra
tagene), pSVK3, pBPV, pMSG, pSVL (Pha
rmacia). 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 another vector having a selection marker. Two suitable vectors are PKK232-8 and PCM7. Individually named bacterial promoters include lacI, lacZ,
T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early
early), HSV thymidine kinase, early and late S
V40, LTR 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., Di
bner, M., Battey, L., Basic Methods in Molecula
r 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.

The mature protein can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of a suitable promoter. Such a protein,
Cell-free translation systems can also be used for production with RNA obtained from the DNA constructs of the present invention. Cloning and expression vectors suitable for use in prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular.
Cloning: A Laboratory Manual, Second Edition, Cold S
Spring Harbor, New York, (1989), the disclosure of which is hereby incorporated by reference.
Transcription of a DNA encoding a polypeptide of the invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers act on a promoter to increase its transcription, usually about 10-30.
0 bp is a cis-acting element of DNA. For example, bp
SV40 on the late side of the replication origin, 100-270
Includes enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers on the late side of the replication origin, and adenovirus enhancers.

Typically, the recombinant expression vector contains an origin of replication and a selectable marker that allows transformation of the host cell, such as the ampicillin resistance gene of E. coli and the S. cerevisiae TRP1 gene, and the downstream gene. A promoter derived from a highly expressed gene to drive transcription of the structural sequence would be included. Such promoters include, inter alia, 3
-It can be obtained from operons encoding glycolytic enzymes such as phosphoglycerate kinase (PGK), alpha factor, acid phosphatase or heat shock proteins. 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 include structural DNA sequences encoding the desired protein.
It is constructed by insertion into an operable reading phase with a functional promoter, with appropriate translation initiation and termination signals. 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 typ.
himurium , genus Pseudomonas, genus Streptomyces,
And various species within the genus Staphylococcus, but others can also be used as selection agents.

As a representative, but non-limiting example, a vector useful for bacterial use is obtained from a commercially available plasmid comprising the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). It may comprise a selectable marker and a bacterial origin of replication. Such commercially available vectors include, for example, pKK2
23-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madis)
on, 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 (eg, temperature shift or chemical induction). The cells are cultured for an additional 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.

[0049] A variety of mammalian cell culture systems can also be used to express the recombinant protein.
Examples of mammalian expression systems include Gluzman, Cell, 23: 1.
75 (1981), COS-7 line of monkey kidney fibroblasts, and other cell lines capable of expressing compatible vectors, such as C12
7, 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.

The RARε polypeptide can be purified by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Can be recovered from the recombinant cell culture and purified. It is preferred to have a low concentration (about 0.15-5 mM) of calcium ions during purification (Price et al., J. Biol. Chem., 244: 91).
7 (1969)). If desired, a protein regeneration step can be used to complete the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

The polypeptides of the present invention may be naturally purified products, or products of chemical synthesis, or may be derived from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects in culture). (And by mammalian cells). Depending on the host used in the recombinant production method, the polypeptide of the present invention may be glycosylated or not 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. Therefore, RA
Rε receptors can be used to treat these disorders.

The RARε polypeptide 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)).
Produces RShRARε. The plasmid was then transferred to the reporter plasmid DELTA MTV-TRE sub
It is introduced into monkey kidney CV-1 cells by calcium phosphate transfection together with p-CAT. TR
E means thyroid receptor response factor and TRE sub
p is the TRE engineered to maximize the palindromic nature of this factor. As a control, pRSerbAsup-
1 (which does not encode any protein, is a negative control) is also tested. Transfected cells
The induced CAT activity was analyzed by chromatography after incubation for 36 hours in the presence or absence of 100 nM retinoic acid or other potential ligand. The results will indicate that the ligand binds to RARε.

The present invention further provides a method for screening a drug to identify a drug (agonist) that enhances the interaction of a ligand with RARε. As an example, as discussed above, the reporter plasmid and p
RShRARε is introduced into monkey cells. 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 (Pr
omega Technical Bulletin No. 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 with radioactive, spectroscopic, 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 also comprises human RA
It is also useful to determine the affinity and potency of a known drug at the Rε 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,
By using retroviral particles comprising RNA encoding a polypeptide of the present invention, cells can be manipulated by methods known in the art.

Similarly, cells can be engineered in vivo for expression of the polypeptide in vivo, eg, 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 that have similar biological activities. 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 labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention was converted to human cD
Used to screen libraries of NA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The availability of RARε allows its use in diagnostic assays to determine the 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 useful 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. D according to the invention
Mapping NAs to chromosomes is an important first step in relating their sequences to genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. cDNA
Using computer analysis of genomic DNA
The rapid selection of primers that do not span more than one exon complicates 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 give the amplified fragment.

[0061] 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 that chromosome include in s
It includes itu hybridization, prescreening using labeled flow-sorted chromosomes, and preselection by hybridization to construct a chromosome-specific cDNA library.

Fluorescence in situ hybridization of cDNA clones to metaphase chromosomal spreads (FIS
Using H), the exact chromosome location can be provided in one step. This technique can be used with cDNAs as short as 500 or 600 bases;
Clones larger than 00 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, 2,0
00bp is better, 4000 is better,
More than 4,000 is probably not necessary to get good results at a reasonable time rate. A review of this technique includes Verma
Et al., Human Chromosomes: a Manual of Basic Tech
niques, Pergamon Press, New York (1988)
See

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
For example, V. McKusick, Mendelian Inheritance in
Man (Johns Hopkins University Welch Medical)
(Available online via 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 physical and genetic mapping analyses, 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).

[0066] Comparisons between affected and unaffected individuals are usually visible from chromosomal spreads or can be detected using PCR based on their cDNA sequences, deletions or deletions. First look for structural changes in the chromosome, such as translocation. Finally, complete sequencing of genes from several individuals is required to confirm the presence of the mutation and to distinguish the mutation from polymorphism.

The polypeptides, fragments or other derivatives thereof, or analogs thereof, or cells expressing them can be used as immunogens to produce antibodies thereto. These antibodies are
For example, it can be a polyclonal or monoclonal antibody. 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. Can be. The antibody so obtained will then bind to the polypeptide itself. In this way, even a sequence encoding only a fragment of a polypeptide,
It can be used to produce antibodies that bind the whole 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, 25
6: 495-497), trioma technology, human B
Cell hybridoma technology (Kozbor et al., 1983, Immun
ology Today 4:72), and EBV-hybridoma technology (Cole) for producing human monoclonal antibodies.
Et al., 1985, Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).

The techniques described for the production of single-chain antibodies (US Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to the immunogenic polypeptide products of the invention.

The present 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 NA, performing restriction digestion of DNA with a group of restriction enzymes,
The resulting DNA fragments were separated by electrophoresis on a sizing gel, and the resulting gel was
A labeled band capable of specifically hybridizing to the DNA encoding the RARε polypeptide and contacting with a nucleic acid probe labeled with a detectable marker to hybridize to the DNA encoding the RARε polypeptide. Is detected with a detectable marker to create a unique band pattern specific to the DNA of the subject suffering from the disorder, the DNA obtained in the previous step is prepared for diagnosis, and the disorder is identified. Diagnosing a predisposition to the disorder by comparing the unique band pattern specific to the DNA of the affected subject and the DNA obtained from the diagnostic process.

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

To facilitate understanding of the following embodiment,
Some frequently occurring methods and / or terms are 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, DN
Digest 5-50 μg of A with 20-250 units of enzyme.
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. The size separation of the cleaved fragments is
Goeddel, D et al., Nucleic Acids Res., 8: 4057.
Performed using 8% polyacrylamide gel as described by (1980).

An “oligonucleotide” is a single-stranded polydeoxynucleotide that can be chemically synthesized.
Or two complementary polynucleotide strands. 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 are
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 was performed with 10 units of T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amount of the DNA fragment to be ligated.
This can be accomplished using known buffers and conditions. Unless otherwise stated, transformations were performed by Graham, F. and Van der Eb, A., Virology,
52: 456-457 (1973).

[0077]

EXAMPLES Example 1 Generation of RARε by In Vitro Transcription and Translation
Current TNT Coupled Reticulocyte Lysate System (Pro
mega, 2800 Woods Hollow Road, Madison, W
I53771-5399) was used for in vitro transcription and translation of RARε. The cDNA encoding RARε, ATCC No. 75754, is
It was cloned in the 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 T3R is used to transcribe the message.
Protein expression is driven by the T3 promoter using NA polymerase and reticulocyte lysate to translate nascent RNA. 1 μg of circular (or linear) plasmid containing RARε DNA was
Add directly to the lysate and add T3R in a reaction volume of 50 μl.
With NA polymerase and [35S] -methionine
Incubated at 30 ° C. for 1 hour. After incubation, the translation products were separated by 10% SDS-PAGE gel electrophoresis. 10% acetic acid, 15%
After fixing in methanol for 30 minutes, it was 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 main translation products are
It can be seen at 49 Kd (FIG. 8).

Example 2 RARε in Baculovirus-Insect Cell System
Expression and purification of Grace's insect medium supplemented with 10% FBS (Grace's
(Insect medium) (Gibco).
9 Maintain insect cells at 27 ° C. The baculovirus transfer plasmid pA2 was constructed by Gentz et al. (Unpublished results) (FIG. 9). 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:

(Equation 1) It is. The amplified fragment containing the human RARε cDNA, and the plasmid pA2 were transformed 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 and the recombinant transfer vector pA2 was inserted.
A2RARε was generated. Restriction endonuclease, P
CR 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 available from Pharmagene
(San Diego, CA). Techniques for the manipulation of baculovirus and insect cell cultures, such as cultivation of putative recombinant plaques, co-transfection, virus infection, isolation, screening and purification, 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 were P
It must be analyzed and confirmed by CR screening. Once the recombinant virus was obtained, Sf9 cells (2 × 1
06 cells / ml) can be infected with the recombinant RAR virus (108 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. Then, SDS
-PAGE of extracellular and intracellular fractions by RARε
Is analyzed for expression of 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
The expression level of Rε was examined. The total cell RNA sample was
NAzol ™ B system (Biotecx Laboratories, In
c. 6023 South Loop East, Houston, TX7
7033). Approximately 10 μg of total RNA isolated from each designated human tissue was separated on a 1% agarose gel and blotted onto nylon filters (Samb
rook, Fritsch, and Maniatis, Molecular Cloni
ng, Cold Spring Harbor Press, (1989)).
Labeling reactions were performed with 50 ng of DNA fragments using the Stratagene Prime-It kit. Labeled DNA with S
elect-G-50 column (5 Prime-3 Prime, Inc. 5
603 Arapahoe Road, Boulder, CO 80303)
Was purified. The filter was then filtered at 1,000,000 c in 0.5M NaPO4 (pH 7.4) and 7% SDS.
Hybridized overnight at 65 ° C. with pm / ml of the full-length radiolabeled RARε gene. 0.5 × SS
C, twice with 0.1% SDS at room temperature and 60 ° C.
After washing twice, the filter was placed on an intensifying screen at -70 ° C
And exposed overnight. RARε message RNA is abundant in testis, placenta, spleen, thymus and lung (FIG. 7). 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.

[0080]

[Sequence list]

(1) General information: (i) Patent applicant: Cao et al. (Ii) Title of the invention: Retinoic acid receptor epsilon (iii) Number of sequences: 2 (iv) Contact information: (A) Address: Carrera, Baan , Vane, Gilfiran, Sec, Stuart and Olstein (B) Street: Becker Farm Road No. 6 (C) City: Roseland (D) State: New Jersey (E) Country: United States (F) ZIP: 07068 (V) Computer reading / reading ceremony: (A) Medium type: 3.5 inch diskette (B) Computer: IBM PS / 2 (C) Operating system: MS-DOS (D) Software: Word Perfect 5.1 (vi ) Data of this application: (A) Application number: (B) Filing date: Same date (C) Classification: (vii) Technical data: (A) Application number: (B) Filing date: (viii) Patent attorney / agent information: (A) Name: Ferraro, Gregory di (B) Registration number: 36,134 (C) See / Reference number: 325800-125 (ix) Telephone contact information: (A) Telephone number: 201-994-1700 (B) Fax number: 201-994-1744 (2) Information of sequence number 1: (i) Characteristics (A) Length: 1,649 base pairs (B) Type: Nucleic acid (C) Number of strands: Single strand (D) Topology: Linear (ii) Type of molecule: cDNA (xi) Sequence description : SEQ ID NO: 1:

(Equation 2) (2) Information of SEQ ID NO: 2: (i) Sequence characteristics: (A) Length: 433 amino acids (B) Type: amino acids (C) Number of chains: (D) Topology: linear (ii) Sequence Type: protein (xi) Sequence description: SEQ ID NO: 2

(Equation 3)

(Equation 4)

[Brief description of the drawings]

FIG. 1 shows the polynucleotide sequence of the predicted mature RARε polypeptide and the corresponding deduced amino acid sequence.

FIG. 2 shows the polynucleotide sequence of the deduced mature RARε polypeptide and the corresponding deduced amino acid sequence (continuation of FIG. 1).

FIG. 3 shows the polynucleotide sequence of the deduced mature RARε polypeptide and the corresponding deduced amino acid sequence (continuation of FIG. 2).

FIG. 4 shows the polynucleotide sequence of the predicted mature RARε polypeptide and the corresponding deduced amino acid sequence (continuation of FIG. 3).

FIG. 5 illustrates the high homology between RARε and RARγ in the DNA and ligand binding regions.

FIG. 6 illustrates the high homology between RARε and RARγ in the DNA and ligand binding regions (continuation of FIG. 5).

FIG. 7 shows the results of Northern blot analysis of RARε in adult tissues.

FIG. 8. After transcription / translation in vitro, RA
The results of electrophoresis of Rε on a gel are shown.

FIG. 9 illustrates the baculovirus transfer plasmid pA2 used for expression of RARε.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Liang Zhao Hong Kong University of Hong Kong, Department of Microbiology, Queen Mary Hospital (72) Inventor Chian Nee United States 20853 Maryland No. 5502, Manonfield Road, Rockville, NC (72) Inventor Robert Di Fleischman No. 470, Chiffry Square Road, Gay Saarsburg, Maryland, United States 20878 F-Term (reference) 4B024 AA01 AA11 BA63 CA04 CA09 CA20 DA02 EA02 EA04 GA13 4H045 AA10 BA10 CA40 DA50 EA20 EA50 FA74

Claims (1)

[Claims] 1. Any invention described or claimed herein.