WO1999029722A1 - DNA MOLECULES ENCODING VERTEBRATE NUCLEAR RECEPTOR PROTEIN, nNR4 - Google Patents

DNA MOLECULES ENCODING VERTEBRATE NUCLEAR RECEPTOR PROTEIN, nNR4 Download PDF

Info

Publication number
WO1999029722A1
WO1999029722A1 PCT/US1998/026446 US9826446W WO9929722A1 WO 1999029722 A1 WO1999029722 A1 WO 1999029722A1 US 9826446 W US9826446 W US 9826446W WO 9929722 A1 WO9929722 A1 WO 9929722A1
Authority
WO
WIPO (PCT)
Prior art keywords
nnr4
protein
expression vector
host cell
recombinant
Prior art date
Application number
PCT/US1998/026446
Other languages
French (fr)
Inventor
Fang Chen
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to EP98963116A priority Critical patent/EP1037912A1/en
Priority to JP2000524313A priority patent/JP2001525196A/en
Priority to CA002315273A priority patent/CA2315273A1/en
Publication of WO1999029722A1 publication Critical patent/WO1999029722A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates in part to isolated nucleic acid molecules (polynucleotide) which encode a mouse nuclear receptor protein, referred to throughout as nNR4.
  • the present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding nNR4, substantially purified forms of associated nNR4 protein, mutant proteins, and methods associated with identifying compounds which modulate nNR4 activity.
  • the nuclear receptor superfamily which includes steroid hormone receptors, are small chemical ligand-inducible transcription factors which have been shown to play roles in controlling development, differentiation and physiological function. Isolation of cDNA clones encoding nuclear receptors reveal several characteristics. First, the NH2-terminal regions, which vary in length between receptors, is hypervariable with low homology between family members. There are three internal regions of conservation, referred to as domain I, II and III. Region I is a cysteine-rich region which is referred to as the DNA binding domain (DBD). Regions II and III are within the COOH-terminal region of the protein and is also referred to as the ligand binding domain (LBD). For a review, see Power et al. (1992, Trends in Pharmaceutical Sciences 13: 318-323).
  • a nucleic acid molecule expressing a nuclear receptor protein will be useful in screening for compounds acting as a modulator of cell differentiation, cell development and physiological function.
  • the present invention addresses and meets these needs by disclosing isolated nucleic acid molecules which express a nuclear receptor protein which will have a role in cell differentiation and development.
  • the present invention relates to isolated nucleic acid molecules (polynucleotides) which encode novel nuclear receptor proteins which are herein designated as members of the nuclear receptor superfamily.
  • the isolated polynucleotides of the present invention encode vertebrate members of this nuclear receptor superfamily, and preferably an isolated polynucleotide encoding a murine nuclear receptor protein, such as a nuclear receptor protein exemplified and referred to throughout this specification as nNR4.
  • the nuclear receptor proteins encoded by the isolated polynucleotides of the present invention are involved in the regulation of in vivo cell proliferation and/or cell development.
  • nNR4 nuclear receptor proteins encoded by isolated polynucleotides of the present invention are involved in the regulation of gene expression, expressed in substantial quantity in the liver and show structural similarity to another member of the nuclear receptor superfamily, the vitamin D receptor.
  • the nuclear receptor proteins encoded by the isolated polynucleotides of the present invention are involved in the regulation of gene expression.
  • nNR4 is mainly expressed in mouse liver at high level, and the most related nuclear receptor with known function is vitamin D receptor, it is likely that nNR4 plays important roles in metabolism and the endogenous ligand regulating nNR4 may very likely to be a metabolic intermediate.
  • the present invention also relates to isolated nucleic acid fragments which encode mRNA expressing a biologically active novel vertebrate nuclear receptor which belongs to the nuclear receptor superfamily.
  • a preferred embodiment relates to isolated nucleic acid fragments of SEQ ID NO: 1 which encode mRNA expressing a biologically functional derivative of nNR4. Any such nucleic acid fragment will encode either a protein or protein fragment comprising at least an intracellular DNA-binding domain and/or ligand binding domain, domains conserved throughout the nuclear receptor family domain which exist in nNR4 (SEQ ID NO: 2).
  • any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy- terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNR4.
  • the isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
  • DNA deoxyribonucleic acid molecule
  • cDNA complementary DNA
  • synthetic DNA such as a synthesized, single stranded polynucleotide.
  • the isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • recombinant vectors and recombinant hosts both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • a preferred aspect of the present invention is disclosed in
  • Figure 1A-B and SEQ ID NO: 1 an isolated cDNA encoding a novel nuclear trans-acting receptor protein, nNR4.
  • nNR4 novel nuclear trans-acting receptor protein
  • the present invention also relates to biologically functional derivatives of nNR4 as set forth as SEQ ID NO: 2, including but not limited to nNR4 mutants and biologically active fragments such as amino acid substitutions, deletions, additions, amino terminal truncations and carboxy- terminal truncations, such that these fragments provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and which would be useful for screening for agonists and/or antagonists of nNR4 function.
  • the present invention also relates to polyclonal and monoclonal antibodies raised in response to either form of nNR4 disclosed herein, or a biologically functional derivative thereof. It will be especially preferable to raise antibodies against epitopes within the NH2-terminal domain of nNR4, which show the least homology to other known proteins belonging to the nuclear receptor superfamily.
  • the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of nNR4.
  • the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of nNR4.
  • the present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type nNR4 activity.
  • a preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase GST-nNR4 fusion constructs. These fusion constructs include, but are not limited to, all or a portion of the ligand-binding domain of nNR4, respectively, as an in-frame fusion at the carboxy terminus of the GST gene.
  • the disclosure of SEQ ID Nos: 1-2 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein.
  • Soluble recombinant GST-nuclear receptor fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac- N-Blue DNA from Invitrogen or pAcG2T from Pharmingen). It is an object of the present invention to provide an isolated polynucleotide as set forth in SEQ ID NO: 1.
  • a nuclear receptor protein such as nNR4, nuclear receptor protein fragments of full length proteins such as nNR4, and mutants which are derivatives of SEQ ID NO: 2.
  • Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for nNR4 function.
  • Another object of this invention is tissue typing using probes or antibodies of this invention.
  • polynucleotide probes are used to identify tissues expressing nNR4 mRNA.
  • probes or antibodies can be used to identify a type of tissue based on nNR4 expression or display of nNR4 receptors.
  • DBD refers to DNA binding domain.
  • LBD refers to ligand binding domain
  • mamalian host refers to any mammal, including a human being.
  • Figure 1A-B show the nucleotide sequence (SEQ ID NO: 1) which comprises the open reading frame encoding the nuclear receptor protein, nNR4.
  • Figure 2A-B show the coding strand of the isolated cDNA molecule (SEQ ID NO: 1) which encodes nNR4, and the amino acid sequence (SEQ ID NO: 2) of nNR4. The region in bold is the DNA binding domain.
  • Figure 3 shows the amino acid sequence (SEQ ID NO: 2) of nNR4. The region in bold is the DNA binding domain.
  • the present invention relates to isolated nucleic acid and protein forms which represent vertebrate nuclear receptors. These expressed proteins are novel nuclear receptors which are useful in the identification of downstream target genes and ligands regulating their activity.
  • the nuclear receptor proteins encoded by the isolated polynucleotides of the present invention are involved in the regulation of in vivo cell proliferation and/or cell development.
  • the nuclear receptor superfamily is composed of a group of structurally related receptors which are regulated by chemically distinct ligands.
  • the common structure for a nuclear receptor is a highly conserved DNA binding domain (DBD) located in the center of the peptide and the ligand-binding domain (LBD) at the COOH-terminus.
  • DBD DNA binding domain
  • LBD ligand-binding domain
  • the DBDs share at least 50% to 60% amino acid sequence identity even among the most distant members in vertebrates.
  • the superfamily has been expanded within the past decade to contain approximately 25 subfamilies.
  • EST clones corresponding to GenBank Ass. #'s AA106163 and AA396982 were obtained from Genome Systems Inc. (St. Louis, Missouri, http://www. genomesysytems.com).
  • the cDNA clone AA106163 is also identified as Image Consortium ID No. 521858 and dbEST ID No. 743956 while the cDNA clone AA396982 is also identified as Image Consortium ID No.
  • PCR nested primer analysis was utilized to scan a mouse brain cDNA library.
  • a PCR fragment of with expected size (approximately 350 bps) was amplified from mouse brain cDNA and the DNA fragment was purified using Qiagen gel extraction kit and subjected to automated sequencing. The sequence information showed a complete DBD region.
  • the full length cDNA clone for nNR4 was obtained via PCR using a 5'-end primer and a 3'-end primer on mouse brain cDNA, mouse embryo cDNA (Clontech, Palo Alto, CA, USA) and mouse testis cDNA (Clontech, Palo Alto, CA, USA).
  • a 1.2 kb DNA fragment from mouse embryo cDNA was cloned into the pCRII vector (Invitrogen, San Diego, CA) and multiple clones were sequenced to identify the clone coding wild type protein sequence.
  • the present invention also relates to isolated nucleic acid fragments of nNR4 (SEQ ID NO: 1) which encode mRNA expressing a biologically active novel nuclear receptor. Any such nucleic acid fragment will encode either a protein or protein fragment comprising at least an intracellular DNA-binding domain and/or ligand binding domain, domains conserved throughout the nuclear receptor family domain which exist in nNR4 (SEQ ID NO: 2).
  • any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these biologically functional derivatives encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for nNR4 function.
  • the isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
  • DNA deoxyribonucleic acid molecule
  • cDNA complementary DNA
  • synthetic DNA such as a synthesized, single stranded polynucleotide.
  • the isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • FIG. 1A-B A preferred aspect of the present invention is disclosed in Figure 1A-B and SEQ ID NO: 1, a cDNA encoding a novel nuclear trans- acting receptor protein, nNR4, disclosed as follows:
  • the present invention also relates to a substantially purified form of the novel nuclear trans-acting receptor protein, nNR4, which is shown in Figures 2A-B and Figure 3 and as set forth in SEQ ID NO: 2, disclosed as follows:
  • the present invention also relates to biologically functional derivatives and/or mutants of nNR4 as set forth as SEQ ID NO: 2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNR4 function.
  • the present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type nNR4 activity.
  • a preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase GST-nNR4 and/or GST-nNR4 fusion constructs.
  • These fusion constructs include, but are not limited to, all or a portion of the ligand-binding domain of nNR4, respectively, as an in-frame fusion at the carboxy terminus of the GST gene.
  • the disclosure of SEQ ID NOS: 1-2 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein.
  • Soluble recombinant GST-nuclear receptor fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen).
  • Spodoptera frugiperda Sf21 insect cells
  • baculovirus expression vector e.g., Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen.
  • the isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
  • DNA deoxyribonucleic acid molecule
  • cDNA complementary DNA
  • synthetic DNA such as a synthesized, single stranded polynucleotide.
  • the isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
  • this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below:
  • the present invention discloses codon redundancy which may result in differing DNA molecules expressing an identical protein.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
  • DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide.
  • Methods of altering the DNA sequences include but are not limited to site directed mutagenesis.
  • altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
  • nucleic acid, protein, or respective fragment thereof in question has been substantially removed from its in vivo environment so that it may be manipulated by the skilled artisan, such as but not limited to nucleotide sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in pure quantities so as to afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the nucleic acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or substantially purified form.
  • a nucleic acid is considered substantially purified when it is purified away from environmental contaminants.
  • a nucleic acid sequence isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • the present invention also relates to methods of expressing nNR4 and biological equivalents disclosed herein, assays employing these recombinantly expressed gene products, cells expressing these gene products, and agonistic and/or antagonistic compounds identified through the use of assays utilizing these recombinant forms, including, but not limited to, one or more modulators of the nNR4 either through direct contact with the LBD or through direct or indirect contact with a ligand which either interacts with the DBD or with the wild-type transcription complex which nNR4 interacts in trans, thereby modulating cell differentiation or cell development.
  • a "biologically functional derivative" of a wild-type nNR4 possesses a biological activity that is related to the biological activity of the wild type nNR4.
  • the term “functional derivative” is intended to include “fragments,” “mutants,” “variants,” “degenerate variants,” “analogs” and “homologues” of the wild type nNR4 protein.
  • fragment is meant to refer to any polypeptide subset of wild-type nNR4, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations.
  • mutant is meant to refer a subset of a biologically active fragment that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the nNR4 or nNR4 functional derivative.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof. A molecule is "substantially similar" to a wild-type nNR4-like protein if both molecules have substantially similar structures or if both molecules possess similar biological activity.
  • analog refers to a molecule substantially similar in function to either the full-length nNR4 protein or to a biologically functional derivative thereof.
  • nNR4 any of a variety of procedures may be used to clone nNR4. These methods include, but are not limited to, (1) a RACE PCR cloning technique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998- 9002). 5' and/or 3' RACE may be performed to generate a full-length cDNA sequence. This strategy involves using gene-specific oligonucleotide primers for PCR amplification of nNR4 cDNA.
  • These gene-specific primers are designed through identification of an expressed sequence tag (EST) nucleotide sequence which has been identified by searching any number of publicly available nucleic acid and protein databases; (2) direct functional expression of the nNR4 cDNA following the construction of a nNR4-containing cDNA library in an appropriate expression vector system; (3) screening a nNR4- containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino acid sequence of the nNR4 protein; (4) screening a nNR4-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the nNR4 protein.
  • EST expressed sequence tag
  • This partial cDNA is obtained by the specific PCR amplification of nNR4 DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other kinases which are related to the nNR4 protein; (5) screening a nNR4-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the nNR4 protein.
  • This strategy may also involve using gene-specific oligonucleotide primers for PCR amplification of nNR4 cDNA identified as an EST as described above; or (6) designing 5' and 3' gene specific oligonucleotides using SEQ ID NO: 1 as a template so that either the full-length cDNA may be generated by known PCR techniques, or a portion of the coding region may be generated by these same known PCR techniques to generate and isolate a portion of the coding region to use as a probe to screen one of numerous types of cDNA and/or genomic libraries in order to isolate a full-length version of the nucleotide sequence encoding nNR4.
  • libraries as well as libraries constructed from other cell types-or species types, may be useful for isolating a nNR4-encoding DNA or a nNR4 homologue.
  • Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than cells or tissue such as human cells, rodent cells or any other such vertebrate host which may contain a homologue of nNR4.
  • a nNR4 gene and homologues may be isolated by oligonucleotide- or polynucleotide-based hybridization screening of a vertebrate genomic library, including but not limited to, a human genomic library and a rodent genomic library.
  • cDNA libraries may be prepared from cells or cell lines which have nNR4 activity.
  • the selection of cells or cell lines for use in preparing a cDNA library to isolate a cDNA encoding nNR4 may be done by first measuring cell-associated nNR4 activity using any known assay available for such a purpose.
  • cDNA libraries can be performed by standard techniques well known in the art.
  • Well known cDNA library construction techniques can be found for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
  • Complementary DNA libraries may also be obtained from numerous commercial sources, including but not limited to Clontech Laboratories, Inc. and Stratagene. It is also readily apparent to those skilled in the art that DNA encoding nNR4 may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Sambrook, et al., supra.
  • the amino add sequence or DNA sequence of nNR4 or a homologous protein may be necessary.
  • the nNR4 protein or a homologous protein may be purified and partial amino add sequence determined by automated sequenators. It is not necessary to determine the entire amino add sequence, but the linear sequence of two regions of 6 to 8 amino adds can be determined for the PCR amplification of a partial nNR4 DNA fragment. Once suitable amino add sequences have been identified, the DNA sequences capable of encoding them are synthesized.
  • the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the nNR4 sequence but others in the set will be capable of hybridizing to nNR4 DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still suffidently hybridize to the nNR4 DNA to permit identification and isolation of nNR4 encoding DNA.
  • the nucleotide sequence of a region of an expressed sequence may be identified by searching one or more available genomic databases.
  • Gene-specific primers may be used to perform PCR amplification of a cDNA of interest from either a cDNA library or a population of cDNAs.
  • the appropriate nucleotide sequence for use in a PCR-based method may be obtained from SEQ ID NO: 1, either for the purpose of isolating overlapping 5' and 3' RACE products for generation of a full-length sequence coding for nNR4, or to isolate a portion of the nucleotide sequence coding for nNR4 for use as a probe to screen one or more cDNA- or genomic-based libraries to isolate a full-length sequence encoding nNR4 or nNR4-like proteins.
  • the nNR4 full-length cDNA of the present invention were generated by confirmation of DNA sequence analysis of 5' and 3' ESTs from mouse and PCR-based cloning of a missing portion of the coding region.
  • a full length cDNA clone for nNR4 was obtained via PCR using a 5'-end primer and a 3 '-end primer on mouse brain cDNA, mouse embryo cDNA and mouse testis cDNA.
  • a 1.2 kb DNA fragment from mouse embryo cDNA was cloned into plasmid pCRII (Invitrogen, San Diego, CA) and multiple clones were sequenced, which resulted in the identification of the mouse nNR4 clone.
  • the mouse nNR4 cDNA encodes a peptide of 357 amino adds, which has the distinctive DBD and LBD structures. It is most related to human orphan nuclear hormone receptor (hONR) and shares 75% a.a. sequence identity in the overlapping peptide region (Baes et al.,1994,
  • nNR4 does not seem to be a mouse homolog of hONR as the two receptors have 10 different amino acids in the most conserved DBD region while homologs usually have almost identical DBD. Thus, nNR4 most likely represents a subtype of this orphan subfamily. nNR4 is highly expressed in liver but not in other tissue as detected through northern analysis.
  • a variety of mammalian expression vectors may be used to express recombinant nNR4 in mammalian cells.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host.
  • Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria- animal cells.
  • An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • mammalian expression vectors which may be suitable for recombinant nNR4 expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565
  • bacterial expression vectors may be used to express recombinant nNR4 in bacterial cells.
  • Commercially available bacterial expression vectors which may be suitable for recombinant nNR4 expression include, but are not limited to pQE (Qiagen), pETlla (Novagen), lambda gtll (Invitrogen), and pKK223-3 (Pharmada).
  • fungal cell expression vectors may be used to express recombinant nNR4 in fungal cells.
  • Commercially available fungal cell expression vectors which may be suitable for recombinant nNR4 expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
  • insect cell expression vectors may be used to express recombinant receptor in insect cells.
  • Commercially available insect cell expression vectors which may be suitable for recombinant expression of nNR4 include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
  • An expression vector containing DNA encoding a nNR4- like protein may be used for expression of nNR4 in a recombinant host cell.
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of , bovine, pordne, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines.
  • L cells L-M(TK") ATCC CCL 1.3
  • L cells L-M ATCC CCL 1.2
  • Saos-2 ATCC HTB-85
  • 293 ATCC CRL 1573
  • Raji ATCC CCL 86
  • CV-1 ATCC CCL 70
  • COS-1 ATCC CRL 1650
  • COS-7 ATCC CRL 1651
  • CHO-K1 ATCC CCL 61
  • 3T3 ATCC CCL 92
  • NIH/3T3 ATCC CRL 1658
  • HeLa ATCC CCL 2
  • C127I ATCC CRL 1616
  • BS-C-1 ATCC CCL 26
  • MRC-5 ATCC CCL 171
  • CPAE ATCC CCL 209
  • the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
  • the expression vector-containing cells are individually analyzed to determine whether they produce nNR4 protein. Identification of nNR4 expressing cells may be done by several means, including but not limited to immunological reactivity with anti-nNR4 antibodies, labeled ligand binding and the presence of host cell-associated nNR4 activity.
  • nNR4 cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant nNR4.
  • an expression vector such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28
  • Techniques for such manipulations can be found described in Sambrook, et al., supra , are discussed at length in the Example section and are well known and easily available to the artisan of ordinary skill in the art.
  • Expression of nNR4 DNA may also be performed using in vitro produced synthetic mRNA.
  • Synthetic mRNA can be effidently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
  • nNR4 cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full- length open reading frame for nNR4 as well as various constructs containing portions of the cDNA encoding only spedfic domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a nNR4 cDNA. The expression levels and activity of nNR4 can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells.
  • this nNR4 cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.
  • expression vectors including recombinant viruses
  • the present invention also relates to polyclonal and monoclonal antibodies raised in response to either the human form of nNR4 disclosed herein, or a biologically functional derivative thereof. It will be espedally preferable to raise antibodies against epitopes within the NH2-te ⁇ ninal domain of nNR4, which show the least homology to other known proteins belonging to the human nuclear receptor superfamily.
  • Recombinant nNR4 protein can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies spedfic for full-length nNR4 protein, or polypeptide fragments of nNR4 protein. Additionally, polyclonal or monoclonal antibodies may be raised against a synthetic peptide (usually from about 9 to about 25 amino adds in length) from a portion of the protein as disclosed in SEQ ID NO: 2. Monosperific antibodies to nNR4 are purified from mammalian antisera containing antibodies reactive against nNR4 or are prepared as monoclonal antibodies reactive with nNR4 using the technique of Kohler and Milstein (1975, Nature 256: 495-497).
  • Monosperific antibody as used herein is defined as a single antibody spedes or multiple antibody species with homogenous binding characteristics for nNR4.
  • Homogenous binding as used herein refers to the ability of the antibody species to bind to a spedfic antigen or epitope, such as those assodated with nNR4, as described above.
  • nNR4-spedfic antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of nNR4 protein or a synthetic peptide generated from a portion of nNR4 with or without an immune adjuvant.
  • Preimmune serum is collected prior to the first immunization.
  • Each animal receives between about 0.1 mg and about 1000 mg of nNR4 protein associated with an acceptable immune adjuvant.
  • acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA.
  • the initial immunization consists of nNR4 protein or peptide fragment thereof in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both.
  • SC subcutaneously
  • IP intraperitoneally
  • Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization.
  • nNR4 in Freund's incomplete adjuvant
  • Booster injections are given at about three week intervals until maximal titers are obtained.
  • the animals are bled, the serum collected, and aliquots are stored at about -20°C.
  • Monoclonal antibodies (mAb) reactive with nNR4 are prepared by immunizing inbred mice, preferably Balb/c, with nNR4 protein.
  • the mice are immunized by the IP or SC route with about 1 mg to about 100 mg, preferably about 10 mg, of nNR4 protein in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred.
  • the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to about 100 mg of nNR4 in a buffer solution such as phosphate buffered saline by the intravenous (IV) route.
  • IV intravenous
  • Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NSl/Ag 4-1, MPC-11, S-194 and Sp 2/0, with Sp 2/0 being preferred.
  • the antibody produdng cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%.
  • Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using nNR4 as the antigen. The culture fluids are also tested in the Ouchterlony predpitation assay to determine the isotype of the mAb.
  • SPIRA solid phase immunoradioassay
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press.
  • Monoclonal antibodies are produced in vivo by injection of pristine primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 106 to about 6 x 106 hybridoma cells about 4 days after priming. Asdtes fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
  • In vitro production of anti-nNR4 mAb is carried out by growing the hybridoma in DMEM containing about 2% fetal calf serum to obtain suffirient quantities of the spedfic mAb.
  • the mAb are purified by techniques known in the art.
  • Antibody titers of asdtes or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, predpitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of nNR4 in body fluids or tissue and cell extracts.
  • Mouse nNR4 antibody affinity columns are made, for example, by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HCl (pH 8.0). The column is washed with water followed by 0.23 M glydne HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • the column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supematants or cell extracts containing full-length nNR4 or nNR4 protein fragments are slowly passed through the column.
  • the column is then washed with phosphate buffered saline until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6).
  • the purified nNR4 protein is then dialyzed against phosphate buffered saline.
  • nNR4-spedfic affinity beads or nNR4-spedfic antibodies are used to isolate 35S-methionine labeled or unlabelled nNR4. Labeled nNR4 protein is analyzed by SDS-PAGE. Unlabelled nNR4 protein is detected by Western blotting, ELISA or RIA assays employing either nNR4 protein spedfic antibodies and/or antiphosphotyrosine antibodies.
  • nNR4 protein may be recovered to provide nNR4 protein in active form.
  • nNR4 protein purification procedures are available and suitable for use.
  • Recombinant nNR4 protein may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
  • the present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding a nNR4 protein.
  • Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
  • Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding nNR4, or the function of nNR4.
  • Compounds that modulate the expression of DNA or RNA encoding nNR4 or the biological function thereof may be detected by a variety of assays.
  • the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
  • the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
  • Kits containing nNR4, antibodies to nNR4, or modified nNR4 may be prepared by known methods for such uses.
  • the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of nNR4.
  • the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of nNR4.
  • Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
  • the carrier would further comprise reagents such as recombinant nNR4 or anti-nNR4 antibodies suitable for detecting nNR4.
  • the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
  • Pharmaceutically useful compositions comprising modulators of nNR4 may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, modified nNR4, or either nNR4 agonsits or antagonists.
  • compositions of the invention are administered to an individual in amounts suffident to treat or diagnose disorders.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
  • chemical derivative describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxidty of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • the active agents can be administered concurrently, or they each can be administered at separately staggered times.
  • the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, spedes, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed.
  • a physirian or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxidty requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
  • AA396982 were obtained from Genome Systems Inc. (St. Louis, Missouri, http://www. genomesysytems.com).
  • the cDNA clone AA106163 is also identified as Image
  • TTCTGTCTTC AAACAGAGAA TTTCTTCTGT GGGCCTCTTT GCTACAAGAT GGAGGACGCA GTCATGGGTT CCAGTACGAG TTTTTGGAGT CGATCCTCCA CTTCCATAA AACCTGAAAG GACTGCATCT CCAGGAGCCT GAGTATGTGC TCATGGCTGC CACGGCCCTC TTCTCCCCTG ACAGACCCGG AGTTACCCAA AGAGAAGAGA TAGATCAGCT ACAAGAGGAG ATGGCGCTGA TTCTGAACAA CCACATTATG GAACAACAGT CTCGGCTCCA AAGTCGGTTT CTGTATGCAA AGCTGATGGG CCTGCTGGCT GACCTCCGGA GTATAAACAA TGCATACTCC TATGAACTTC AGCGCTTGGA GGAACTGTCT GCTATGACGC CGCTGCTCGG GGAGATTTGC AGTTGAGGCC CAGGCTTGCA TCAGATGGCT GACCTCCGGA GTATAAACAA TGCATACTCC TATGAACT
  • PCR primers R6R2 (5'-TCTGGACCAGTTCTTTCTG-3'; SEQ ID NO: 5) and R6F2 (5'-CCATGGCCAGTGAAGAAGAA-3'; SEQ ID NO: 6) flanking the missing exon were designed to scan mouse brain cDNA and a mouse brain cDNA library which was distributed in 96- well plate.
  • a PCR fragment of with expected size (approximately 350 bps) was amplified from mouse brain cDNA. The DNA fragment was purified using Qiagen gel extraction kit (QIAGEN, Santa Clarita, CA) and submitted for automated sequencing with primers R6R2 and R6F3
  • the sequence information showed a complete DBD region.
  • the full length cDNA clone for nNR4 was obtained via PCR using the very 5'-end primer R6F5 (5'-ATTCCTGGCAGTTCCTGAGA-3'; SEQ ID NO: 8) and the very 3'-end primer R6R4 9 (5'-TCCAGGCCAGTGTATCCCTG-3'; SEQ ID NO: 9) on mouse (Mus musculus) brain cDNA, mouse (Mus musculus) embryo cDNA (Clontech, Palo Alto, CA, USA) and mouse (Mus musculus) testis cDNA (Clontech, Palo Alto, CA, USA).
  • a 1.2 kb DNA fragment from mouse embryo cDNA was cloned into the pCRII vector (Invitrogen, San Diego, CA) and multiple clones were sequenced to identify the clone coding wild type protein sequence. Sequence assembly and analysis were performed with SEQUENCHERTM 3.0 (Gene Codes Corporation, Ann Arbor, MI). Ambiguities and/or discrepancies between automated base calling in sequencing reads were visually examined and edited to the correct base call.
  • nNR4 The final nucleotide sequence encoding nNR4 is shown as set forth in Figure 1A-B and as set forth as SEQ ID NO: 1 and the open reading frame for nNR4 results in a 357 amino add mouse nuclear receptor protein as shown in Figure 2A-B, Figure 3 and as also set forth in SEQ ID NO: 2.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • High Energy & Nuclear Physics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention discloses the isolation and characterization of cDNA molecules encoding a novel member to the human nuclear receptor superfamily, designated nNR4. Also within the scope of the disclosure are recombinant vectors, recombinant host cells, methods of screening for modulators of nNR4 activity, and production of antibodies against nNR4, or epitopes thereof.

Description

TITLE OF THE INVENTION DNA MOLECULES ENCODING VERTEBRATE NUCLEAR RECEPTOR PROTEIN, nNR4
CROSS-REFERENCE TO RELATED APPLICATIONS Provisional application 60/068,144 filed December 12, 1997.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not applicable.
REFERENCE TO MICROFICHE APPENDK Not applicable.
FIELD OF THE INVENTION
The present invention relates in part to isolated nucleic acid molecules (polynucleotide) which encode a mouse nuclear receptor protein, referred to throughout as nNR4. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding nNR4, substantially purified forms of associated nNR4 protein, mutant proteins, and methods associated with identifying compounds which modulate nNR4 activity.
BACKGROUND OF THE INVENTION
The nuclear receptor superfamily, which includes steroid hormone receptors, are small chemical ligand-inducible transcription factors which have been shown to play roles in controlling development, differentiation and physiological function. Isolation of cDNA clones encoding nuclear receptors reveal several characteristics. First, the NH2-terminal regions, which vary in length between receptors, is hypervariable with low homology between family members. There are three internal regions of conservation, referred to as domain I, II and III. Region I is a cysteine-rich region which is referred to as the DNA binding domain (DBD). Regions II and III are within the COOH-terminal region of the protein and is also referred to as the ligand binding domain (LBD). For a review, see Power et al. (1992, Trends in Pharmaceutical Sciences 13: 318-323).
The lipophilic hormones that activate steroid receptors are known to be associated with human diseases. Therefore, the respective nuclear receptors have been identified as possible targets for therapeutic intervention. For a review of the mechanism of action of various steroid hormone receptors, see Tsai and O'Malley (1994, Annu. Rev. Biochem. 63: 451-486).
Recent work with non-steroid nuclear receptors has also shown the potential as drug targets for therapeutic intervention. This work reports that peroxisome proliferator activated receptor g (PPARg), identified by a conserved DBD region, promotes adipocyte differentiation upon activation and that thiazolidinediones, a class of antidiabetic drugs, function through PPARg (Tontonoz et al., 1994, Cell 79: 1147-1156; Lehmann et al., 1995, J. Biol. Chem. 270(22): 12953-12956; Teboul et al., 1995, J. Biol. Chem. 270(47): 28183-28187). This indicates that PPARg plays a role in glucose homeostasis and lipid metabolism.
Mangelsdorf et al. (1995, Cell 83: 835-839) provide a review of known members of the nuclear receptor superfamily.
Baes et al. (1994, Mol. Cell. Biol. 14(3): 1544-1552) disclose a cDNA clone which encodes a new member of the nuclear receptor superfamily, referred herein as hONR. The authors present data showing that the gene is expressed mainly in human liver and suggests that this nuclear receptor may play a role in regulating expression of retinoid- responsive genes.
It would be advantageous to identify additional genes which are members of the nuclear receptor superfamily, especially vertebrate members from such species as mouse, rat and human. A nucleic acid molecule expressing a nuclear receptor protein will be useful in screening for compounds acting as a modulator of cell differentiation, cell development and physiological function. The present invention addresses and meets these needs by disclosing isolated nucleic acid molecules which express a nuclear receptor protein which will have a role in cell differentiation and development.
SUMMARY OF THE INVENTION The present invention relates to isolated nucleic acid molecules (polynucleotides) which encode novel nuclear receptor proteins which are herein designated as members of the nuclear receptor superfamily. The isolated polynucleotides of the present invention encode vertebrate members of this nuclear receptor superfamily, and preferably an isolated polynucleotide encoding a murine nuclear receptor protein, such as a nuclear receptor protein exemplified and referred to throughout this specification as nNR4. The nuclear receptor proteins encoded by the isolated polynucleotides of the present invention are involved in the regulation of in vivo cell proliferation and/or cell development. Additionally, the nNR4 nuclear receptor proteins encoded by isolated polynucleotides of the present invention are involved in the regulation of gene expression, expressed in substantial quantity in the liver and show structural similarity to another member of the nuclear receptor superfamily, the vitamin D receptor. The nuclear receptor proteins encoded by the isolated polynucleotides of the present invention are involved in the regulation of gene expression. As nNR4 is mainly expressed in mouse liver at high level, and the most related nuclear receptor with known function is vitamin D receptor, it is likely that nNR4 plays important roles in metabolism and the endogenous ligand regulating nNR4 may very likely to be a metabolic intermediate.
The present invention also relates to isolated nucleic acid fragments which encode mRNA expressing a biologically active novel vertebrate nuclear receptor which belongs to the nuclear receptor superfamily. A preferred embodiment relates to isolated nucleic acid fragments of SEQ ID NO: 1 which encode mRNA expressing a biologically functional derivative of nNR4. Any such nucleic acid fragment will encode either a protein or protein fragment comprising at least an intracellular DNA-binding domain and/or ligand binding domain, domains conserved throughout the nuclear receptor family domain which exist in nNR4 (SEQ ID NO: 2). Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy- terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNR4.
The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification. A preferred aspect of the present invention is disclosed in
Figure 1A-B and SEQ ID NO: 1, an isolated cDNA encoding a novel nuclear trans-acting receptor protein, nNR4.
Another preferred aspect of the present invention relates to a substantially purified form of the novel nuclear trans-acting receptor protein, nNR4, which is disclosed in Figures 2A-B and Figure 3 and as set forth in SEQ ID NO: 2.
The present invention also relates to biologically functional derivatives of nNR4 as set forth as SEQ ID NO: 2, including but not limited to nNR4 mutants and biologically active fragments such as amino acid substitutions, deletions, additions, amino terminal truncations and carboxy- terminal truncations, such that these fragments provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and which would be useful for screening for agonists and/or antagonists of nNR4 function.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either form of nNR4 disclosed herein, or a biologically functional derivative thereof. It will be especially preferable to raise antibodies against epitopes within the NH2-terminal domain of nNR4, which show the least homology to other known proteins belonging to the nuclear receptor superfamily. To this end, the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of nNR4. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of nNR4.
The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type nNR4 activity. A preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase GST-nNR4 fusion constructs. These fusion constructs include, but are not limited to, all or a portion of the ligand-binding domain of nNR4, respectively, as an in-frame fusion at the carboxy terminus of the GST gene. The disclosure of SEQ ID Nos: 1-2 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein. Soluble recombinant GST-nuclear receptor fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac- N-Blue DNA from Invitrogen or pAcG2T from Pharmingen). It is an object of the present invention to provide an isolated polynucleotide as set forth in SEQ ID NO: 1.
It is an object of the present invention to provide an isolated nucleic acid molecule which encodes a novel form of a nuclear receptor protein such as nNR4, nuclear receptor protein fragments of full length proteins such as nNR4, and mutants which are derivatives of SEQ ID NO: 2. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for nNR4 function.
Another object of this invention is tissue typing using probes or antibodies of this invention. In a particular embodiment, polynucleotide probes are used to identify tissues expressing nNR4 mRNA. In another embodiment, probes or antibodies can be used to identify a type of tissue based on nNR4 expression or display of nNR4 receptors.
It is a further object of the present invention to provide the nuclear receptor proteins or protein fragments encoded by the nucleic acid molecules referred to in the preceding paragraph.
It is a further object of the present invention to provide recombinant vectors and recombinant host cells which comprise a nucleic acid sequence encoding nNR4 or a biologically functional derivative thereof.
It is an object of the present invention to provide a substantially purified form of nNR4, as set forth in SEQ ID NO: 2.
It is an object of the present invention to provide for biologically functional derivatives of nNR4, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these fragment and/or mutants provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
It is also an object of the present invention to provide for nNR4-based in-frame fusion constructions, methods of expressing these fusion constructions and biological equivalents disclosed herein, related assays, recombinant cells expressing these constructs and agonistic and/or antagonistic compounds identified through the use DNA molecules encoding nuclear receptor proteins such as nNR4. As used herein, "DBD" refers to DNA binding domain.
As used herein, "LBD" refers to ligand binding domain.
As used herein, the term "mammalian host" refers to any mammal, including a human being.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A-B show the nucleotide sequence (SEQ ID NO: 1) which comprises the open reading frame encoding the nuclear receptor protein, nNR4.
Figure 2A-B show the coding strand of the isolated cDNA molecule (SEQ ID NO: 1) which encodes nNR4, and the amino acid sequence (SEQ ID NO: 2) of nNR4. The region in bold is the DNA binding domain.
Figure 3 shows the amino acid sequence (SEQ ID NO: 2) of nNR4. The region in bold is the DNA binding domain.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to isolated nucleic acid and protein forms which represent vertebrate nuclear receptors. These expressed proteins are novel nuclear receptors which are useful in the identification of downstream target genes and ligands regulating their activity. The nuclear receptor proteins encoded by the isolated polynucleotides of the present invention are involved in the regulation of in vivo cell proliferation and/or cell development. The nuclear receptor superfamily is composed of a group of structurally related receptors which are regulated by chemically distinct ligands. The common structure for a nuclear receptor is a highly conserved DNA binding domain (DBD) located in the center of the peptide and the ligand-binding domain (LBD) at the COOH-terminus. Eight out of the nine non-variant cysteines form two type II zinc fingers which distinguish nuclear receptors from other DNA-binding proteins. The DBDs share at least 50% to 60% amino acid sequence identity even among the most distant members in vertebrates. The superfamily has been expanded within the past decade to contain approximately 25 subfamilies. EST clones corresponding to GenBank Ass. #'s AA106163 and AA396982 were obtained from Genome Systems Inc. (St. Louis, Missouri, http://www. genomesysytems.com). The cDNA clone AA106163 is also identified as Image Consortium ID No. 521858 and dbEST ID No. 743956 while the cDNA clone AA396982 is also identified as Image Consortium ID No. 693202 and dbEST ID No. 1038315. Data base searches may also be conducted through the National Center for Biotechnology Information homepage at http://www.ncbi.nlm.nih.gov/dbEST/index.html. Plasmid DNA prepared from AA106163 [which is subcloned in pBSSK(-)] was sequenced using M13 forward and reverse primers, while plasmid DNA for containing AA396982 [which is subcloned in pT7T3D-Pac (Pharmacia) vector] was sequenced using T3 and T7 primers. It was determined by assembling the two sequences that an exon coding the second finger of the DBD was missing. PCR nested primer analysis was utilized to scan a mouse brain cDNA library. A PCR fragment of with expected size (approximately 350 bps) was amplified from mouse brain cDNA and the DNA fragment was purified using Qiagen gel extraction kit and subjected to automated sequencing. The sequence information showed a complete DBD region. The full length cDNA clone for nNR4 was obtained via PCR using a 5'-end primer and a 3'-end primer on mouse brain cDNA, mouse embryo cDNA (Clontech, Palo Alto, CA, USA) and mouse testis cDNA (Clontech, Palo Alto, CA, USA). A 1.2 kb DNA fragment from mouse embryo cDNA was cloned into the pCRII vector (Invitrogen, San Diego, CA) and multiple clones were sequenced to identify the clone coding wild type protein sequence.
The present invention also relates to isolated nucleic acid fragments of nNR4 (SEQ ID NO: 1) which encode mRNA expressing a biologically active novel nuclear receptor. Any such nucleic acid fragment will encode either a protein or protein fragment comprising at least an intracellular DNA-binding domain and/or ligand binding domain, domains conserved throughout the nuclear receptor family domain which exist in nNR4 (SEQ ID NO: 2). Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these biologically functional derivatives encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for nNR4 function.
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
A preferred aspect of the present invention is disclosed in Figure 1A-B and SEQ ID NO: 1, a cDNA encoding a novel nuclear trans- acting receptor protein, nNR4, disclosed as follows:
GGGCCCGGGT GTTTTCCAGG CACTGAGGAC CGCAGTCCCT AATTCCTGGC AGTTCCTGAG ATCTCAAGGA AAGCAGGGTC AGCGAGGAGG CCTGGGGAGA GGAGGCATCC TACACCCGAT CTTGTGGCCT GCTGCCTAAG GGAAACAGGA GACCATGACA GCTATGCTAA CACTAGAAAC CATGGCCAGT GAAGAAGAAT ATGGGCCGAG GAACTGTGTG GTGTGTGGAG ACCGGGCCAC AGGCTATCAT TTCCACGCCC TGACTTGTGA GGGCTGCAAG GGCTTCTTCA GACGAACAGT CAGCAAAACC ATTGGTCCCA TCTGTCCGTT TGCTGGAAGG TGTGAGGTCA GCAAGGCCCA GAGACGCCAC TGTCCAGCCT GCAGGTTGCA GAAGTGTCTA AATGTTGGCA TGAGGAAAGA CATGATACTG TCAGCAGAAG CCCTGGCATT GCGGCGAGCC AGACAGGC C AGCGGCGGGC AGAGAAAGCA TCTTTGCAAC TGAATCAGCA GCAGAAAGAA CTGGTCCAGA TCCTCCTCGG GGCCCACACT CGCCATGTGG GCCCCATGTT TGACCAGTTT GTGCAGTTCA AGCCTCCAGC CTATCTGTTC ATGCATCACC GGCCTTTCCA GCCTCGGGGC CCCGTGTTGC CTCTGCTCAC ACACTTTGCA GATATCAACA CGTTTATGGT GCAACAGATC ATCAAGTTCA CCAAGGATCT GCCGCTCTTC CGGTCCCTAA CCATGGAGGA CCAGATCTCC CTTCTCAAGG GAGCGGCTGT GGAAATATTG CATATCTCAC TCAACACTAC GTTCTGTCTT CAAACAGAGA ATTTCTTCTG TGGGCCTCTT TGCTACAAGA TGGAGGACGC AGTCCATGGG TTCCAGTACG AGTTTTTGGA GTCGATCCTC CACTTCCATA AAAACCTGAA AGGACTGCAT CTCCAGGAGC CTGAGTATGT GCTCATGGCT GCCACGGCCC TCTTCTCCCC TGACAGACCC GGAGTTACCC AAAGAGAAGA GATAGATCAG CTACAAGAGG AGATGGCGCT GATTCTGAAC AACCACATTA TGGAACAACA GTCTCGGCTC CAAAGTCGGT TTCTGTATGC AAAGCTGATG GGCCTGCTGG CTGACCTCCG GAGTATAAAC AATGCATACT CCTATGAACT TCAGCGCTTG GAGGAACTGT CTGCTATGAC GCCGCTGCTC GGGGAGATTT GCAGTTGAGG CCCAGGCTTG CATCCTTTCC CCAGACCCCC AGGGATACAC TGGCCTGGAA (SEQ ID NO: 1) .
The present invention also relates to a substantially purified form of the novel nuclear trans-acting receptor protein, nNR4, which is shown in Figures 2A-B and Figure 3 and as set forth in SEQ ID NO: 2, disclosed as follows:
MTAMLTLETM ASEEEYGPRN CWCGDRATG YHFHALTCEG CKGFFRRTVS KTIGPICPFA GRCEVSKAQR RHCPACRLQK CLNVGMRKDM I SAEALALR RARQAQRRAE KASLQLNQQQ KELVQILLGA HTRHVGPMFD QFVQFKPPAY LFMHHRPFQP RGPVLPLLTH FADINTFMVQ QIIKFTKDLP LFRSLTMEDQ ISLLKGAAVE ILHISLNTTF CLQTENFFCG PLCYKMEDAV HGFQYEFLES ILHFHKNLKG HLQEPEYVL MAATALFSPD RPGVTQREEI DQLQEEMALI LNNHIMEQQS RLQSRFLYAK LMG LADLRS INNAYSYELQ RLEELSAMTP LLGEICS (SEQ ID NO: 2).
The present invention also relates to biologically functional derivatives and/or mutants of nNR4 as set forth as SEQ ID NO: 2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists of nNR4 function. The present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type nNR4 activity. A preferred aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase GST-nNR4 and/or GST-nNR4 fusion constructs. These fusion constructs include, but are not limited to, all or a portion of the ligand-binding domain of nNR4, respectively, as an in-frame fusion at the carboxy terminus of the GST gene. The disclosure of SEQ ID NOS: 1-2 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST-nuclear receptor fusion protein. Soluble recombinant GST-nuclear receptor fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen).
The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below: A=Ala=Alanine: codons GCA, GCC, GCG, GCU C=Cys=Cysteine: codons UGC, UGU D=Asp=Aspartic acid: codons GAC, GAU E=Glu=Glutamic acid: codons GAA, GAG F=Phe=Phenylalanine: codons UUC, UUU G=Gly=Glycine: codons GGA, GGC, GGG, GGU H=His =Histidine: codons CAC, CAU I=Ile =Isoleucine: codons AUA, AUC, AUU K=Lys=Lysine: codons AAA, AAG
L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU M=Met=Methionine: codon AUG N=Asp=Asparagine: codons AAC, AAU P=Pro=Proline: codons CCA, CCC, CCG, CCU Q=Gln=Glutamine: codons CAA, CAG
R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T=Thr=Threonine: codons ACA, ACC, ACG, ACU V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Therefore, the present invention discloses codon redundancy which may result in differing DNA molecules expressing an identical protein. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis.
Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
As used herein, "purified" and "isolated" are utilized interchangeably to stand for the proposition that the nucleic acid, protein, or respective fragment thereof in question has been substantially removed from its in vivo environment so that it may be manipulated by the skilled artisan, such as but not limited to nucleotide sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in pure quantities so as to afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the nucleic acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or substantially purified form. A nucleic acid is considered substantially purified when it is purified away from environmental contaminants. Thus, a nucleic acid sequence isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
The present invention also relates to methods of expressing nNR4 and biological equivalents disclosed herein, assays employing these recombinantly expressed gene products, cells expressing these gene products, and agonistic and/or antagonistic compounds identified through the use of assays utilizing these recombinant forms, including, but not limited to, one or more modulators of the nNR4 either through direct contact with the LBD or through direct or indirect contact with a ligand which either interacts with the DBD or with the wild-type transcription complex which nNR4 interacts in trans, thereby modulating cell differentiation or cell development.
As used herein, a "biologically functional derivative" of a wild-type nNR4 possesses a biological activity that is related to the biological activity of the wild type nNR4. The term "functional derivative" is intended to include "fragments," "mutants," "variants," "degenerate variants," "analogs" and "homologues" of the wild type nNR4 protein. The term "fragment" is meant to refer to any polypeptide subset of wild-type nNR4, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations. The term "mutant" is meant to refer a subset of a biologically active fragment that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the nNR4 or nNR4 functional derivative. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof. A molecule is "substantially similar" to a wild-type nNR4-like protein if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical. The term "analog" refers to a molecule substantially similar in function to either the full-length nNR4 protein or to a biologically functional derivative thereof.
Any of a variety of procedures may be used to clone nNR4. These methods include, but are not limited to, (1) a RACE PCR cloning technique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998- 9002). 5' and/or 3' RACE may be performed to generate a full-length cDNA sequence. This strategy involves using gene-specific oligonucleotide primers for PCR amplification of nNR4 cDNA. These gene-specific primers are designed through identification of an expressed sequence tag (EST) nucleotide sequence which has been identified by searching any number of publicly available nucleic acid and protein databases; (2) direct functional expression of the nNR4 cDNA following the construction of a nNR4-containing cDNA library in an appropriate expression vector system; (3) screening a nNR4- containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino acid sequence of the nNR4 protein; (4) screening a nNR4-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the nNR4 protein. This partial cDNA is obtained by the specific PCR amplification of nNR4 DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other kinases which are related to the nNR4 protein; (5) screening a nNR4-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the nNR4 protein. This strategy may also involve using gene-specific oligonucleotide primers for PCR amplification of nNR4 cDNA identified as an EST as described above; or (6) designing 5' and 3' gene specific oligonucleotides using SEQ ID NO: 1 as a template so that either the full-length cDNA may be generated by known PCR techniques, or a portion of the coding region may be generated by these same known PCR techniques to generate and isolate a portion of the coding region to use as a probe to screen one of numerous types of cDNA and/or genomic libraries in order to isolate a full-length version of the nucleotide sequence encoding nNR4. It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cell types-or species types, may be useful for isolating a nNR4-encoding DNA or a nNR4 homologue. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than cells or tissue such as human cells, rodent cells or any other such vertebrate host which may contain a homologue of nNR4. Additionally a nNR4 gene and homologues may be isolated by oligonucleotide- or polynucleotide-based hybridization screening of a vertebrate genomic library, including but not limited to, a human genomic library and a rodent genomic library.
It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have nNR4 activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate a cDNA encoding nNR4 may be done by first measuring cell-associated nNR4 activity using any known assay available for such a purpose.
Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Complementary DNA libraries may also be obtained from numerous commercial sources, including but not limited to Clontech Laboratories, Inc. and Stratagene. It is also readily apparent to those skilled in the art that DNA encoding nNR4 may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Sambrook, et al., supra. In order to clone the nNR4 gene or a homologue thereof by one of the preferred methods, the amino add sequence or DNA sequence of nNR4 or a homologous protein may be necessary. To accomplish this, the nNR4 protein or a homologous protein may be purified and partial amino add sequence determined by automated sequenators. It is not necessary to determine the entire amino add sequence, but the linear sequence of two regions of 6 to 8 amino adds can be determined for the PCR amplification of a partial nNR4 DNA fragment. Once suitable amino add sequences have been identified, the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino add, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the nNR4 sequence but others in the set will be capable of hybridizing to nNR4 DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still suffidently hybridize to the nNR4 DNA to permit identification and isolation of nNR4 encoding DNA. Alternatively, the nucleotide sequence of a region of an expressed sequence may be identified by searching one or more available genomic databases. Gene-specific primers may be used to perform PCR amplification of a cDNA of interest from either a cDNA library or a population of cDNAs. The appropriate nucleotide sequence for use in a PCR-based method may be obtained from SEQ ID NO: 1, either for the purpose of isolating overlapping 5' and 3' RACE products for generation of a full-length sequence coding for nNR4, or to isolate a portion of the nucleotide sequence coding for nNR4 for use as a probe to screen one or more cDNA- or genomic-based libraries to isolate a full-length sequence encoding nNR4 or nNR4-like proteins.
In an exemplified method, the nNR4 full-length cDNA of the present invention were generated by confirmation of DNA sequence analysis of 5' and 3' ESTs from mouse and PCR-based cloning of a missing portion of the coding region. A full length cDNA clone for nNR4 was obtained via PCR using a 5'-end primer and a 3 '-end primer on mouse brain cDNA, mouse embryo cDNA and mouse testis cDNA. A 1.2 kb DNA fragment from mouse embryo cDNA was cloned into plasmid pCRII (Invitrogen, San Diego, CA) and multiple clones were sequenced, which resulted in the identification of the mouse nNR4 clone. The mouse nNR4 cDNA encodes a peptide of 357 amino adds, which has the distinctive DBD and LBD structures. It is most related to human orphan nuclear hormone receptor (hONR) and shares 75% a.a. sequence identity in the overlapping peptide region (Baes et al.,1994,
Mol. Cell. Biol. 14(3):1544-1552). The mouse nuclear receptor nNR4 does not seem to be a mouse homolog of hONR as the two receptors have 10 different amino acids in the most conserved DBD region while homologs usually have almost identical DBD. Thus, nNR4 most likely represents a subtype of this orphan subfamily. nNR4 is highly expressed in liver but not in other tissue as detected through northern analysis.
A variety of mammalian expression vectors may be used to express recombinant nNR4 in mammalian cells. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria- animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
Commercially available mammalian expression vectors which may be suitable for recombinant nNR4 expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
A variety of bacterial expression vectors may be used to express recombinant nNR4 in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant nNR4 expression include, but are not limited to pQE (Qiagen), pETlla (Novagen), lambda gtll (Invitrogen), and pKK223-3 (Pharmada).
A variety of fungal cell expression vectors may be used to express recombinant nNR4 in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant nNR4 expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
A variety of insect cell expression vectors may be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of nNR4 include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
An expression vector containing DNA encoding a nNR4- like protein may be used for expression of nNR4 in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of , bovine, pordne, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commerdally available, include but are not limited to, L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. The expression vector-containing cells are individually analyzed to determine whether they produce nNR4 protein. Identification of nNR4 expressing cells may be done by several means, including but not limited to immunological reactivity with anti-nNR4 antibodies, labeled ligand binding and the presence of host cell-associated nNR4 activity. The cloned nNR4 cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant nNR4. Techniques for such manipulations can be found described in Sambrook, et al., supra , are discussed at length in the Example section and are well known and easily available to the artisan of ordinary skill in the art. Expression of nNR4 DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be effidently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
To determine the nNR4 cDNA sequence(s) that yields optimal levels of nNR4, cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full- length open reading frame for nNR4 as well as various constructs containing portions of the cDNA encoding only spedfic domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a nNR4 cDNA. The expression levels and activity of nNR4 can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the nNR4 cDNA cassette yielding optimal expression in transient assays, this nNR4 cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.
The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the human form of nNR4 disclosed herein, or a biologically functional derivative thereof. It will be espedally preferable to raise antibodies against epitopes within the NH2-teπninal domain of nNR4, which show the least homology to other known proteins belonging to the human nuclear receptor superfamily.
Recombinant nNR4 protein can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies spedfic for full-length nNR4 protein, or polypeptide fragments of nNR4 protein. Additionally, polyclonal or monoclonal antibodies may be raised against a synthetic peptide (usually from about 9 to about 25 amino adds in length) from a portion of the protein as disclosed in SEQ ID NO: 2. Monosperific antibodies to nNR4 are purified from mammalian antisera containing antibodies reactive against nNR4 or are prepared as monoclonal antibodies reactive with nNR4 using the technique of Kohler and Milstein (1975, Nature 256: 495-497). Monosperific antibody as used herein is defined as a single antibody spedes or multiple antibody species with homogenous binding characteristics for nNR4. Homogenous binding as used herein refers to the ability of the antibody species to bind to a spedfic antigen or epitope, such as those assodated with nNR4, as described above. nNR4-spedfic antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of nNR4 protein or a synthetic peptide generated from a portion of nNR4 with or without an immune adjuvant.
Preimmune serum is collected prior to the first immunization. Each animal receives between about 0.1 mg and about 1000 mg of nNR4 protein associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The initial immunization consists of nNR4 protein or peptide fragment thereof in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of nNR4 in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20°C.
Monoclonal antibodies (mAb) reactive with nNR4 are prepared by immunizing inbred mice, preferably Balb/c, with nNR4 protein. The mice are immunized by the IP or SC route with about 1 mg to about 100 mg, preferably about 10 mg, of nNR4 protein in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to about 100 mg of nNR4 in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NSl/Ag 4-1, MPC-11, S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody produdng cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using nNR4 as the antigen. The culture fluids are also tested in the Ouchterlony predpitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press.
Monoclonal antibodies are produced in vivo by injection of pristine primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 106 to about 6 x 106 hybridoma cells about 4 days after priming. Asdtes fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
In vitro production of anti-nNR4 mAb is carried out by growing the hybridoma in DMEM containing about 2% fetal calf serum to obtain suffirient quantities of the spedfic mAb. The mAb are purified by techniques known in the art.
Antibody titers of asdtes or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, predpitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of nNR4 in body fluids or tissue and cell extracts.
It is readily apparent to those skilled in the art that the above described methods for producing monosperific antibodies may be utilized to produce antibodies spedfic for nNR4 peptide fragments, or full-length nNR4.
Mouse nNR4 antibody affinity columns are made, for example, by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HCl (pH 8.0). The column is washed with water followed by 0.23 M glydne HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supematants or cell extracts containing full-length nNR4 or nNR4 protein fragments are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). The purified nNR4 protein is then dialyzed against phosphate buffered saline.
Levels of nNR4 in host cells is quantified by a variety of techniques including, but not limited to, immunoaffinity and/or ligand affinity techniques. nNR4-spedfic affinity beads or nNR4-spedfic antibodies are used to isolate 35S-methionine labeled or unlabelled nNR4. Labeled nNR4 protein is analyzed by SDS-PAGE. Unlabelled nNR4 protein is detected by Western blotting, ELISA or RIA assays employing either nNR4 protein spedfic antibodies and/or antiphosphotyrosine antibodies.
Following expression of nNR4 in a host cell, nNR4 protein may be recovered to provide nNR4 protein in active form. Several nNR4 protein purification procedures are available and suitable for use. Recombinant nNR4 protein may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding a nNR4 protein. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding nNR4, or the function of nNR4. Compounds that modulate the expression of DNA or RNA encoding nNR4 or the biological function thereof may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Kits containing nNR4, antibodies to nNR4, or modified nNR4 may be prepared by known methods for such uses. The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of nNR4. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of nNR4. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant nNR4 or anti-nNR4 antibodies suitable for detecting nNR4. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like. Pharmaceutically useful compositions comprising modulators of nNR4 may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, modified nNR4, or either nNR4 agonsits or antagonists.
Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts suffident to treat or diagnose disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
The term "chemical derivative" describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxidty of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co- administration or sequential administration of other agents may be desirable. The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, spedes, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed. A physirian or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxidty requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
The following examples are provided to illustrate the present invention without, however, limiting the same hereto.
EXAMPLE 1:
Isolation and Characterization of a DNA Molecule Encoding nNR4
EST clones corresponding to GenBank Ass. #AA106163 and
AA396982 were obtained from Genome Systems Inc. (St. Louis, Missouri, http://www. genomesysytems.com).
The cDNA clone AA106163 is also identified as Image
Consortium ID No. 521858 and dbEST ID No. 743956, and is as follows:
GTTTTCCAGG CACTGAGGAC CGCAGTCCCT AATTCCTGGC AGTTCCTGAG
ATCTCAAGGA AAGCAGGGTC AGCGAGGAGG CCTGGGGAGA GGAGGCATCC TACACCCGAT CTTGTGGCCT GCTGCCTAAG GGAAACAGGA GACCATGACA
GCTATGCTAA CACTAGAAAC CATGGCCAGT GAAGAAGAAT ATGGGCCGAG GAACTGTGTG GTGTGTGGAG ACCGGGCCAC AGGCTATCAT TTCCACGCCC TGACTTGTGA GGGCTGCAAG GGCTTCTTCA GTGATACTGT CAGCAGAAGC CCTGGCATTG CGGCGACCAG ACAGGCACAG CGGCGGGCAG AGAAAGCATC TTTGCAACTG AATCAGCAGC AGAAAGAACT GGTCCAGA ( SEQ ID NO : 3 ) . The cDNA clone AA396982 is also identified as Image
Consortium ID No.693202 and dbEST ID No.1038315, and is as follows: TTCTGTCTTC AAACAGAGAA TTTCTTCTGT GGGCCTCTTT GCTACAAGAT GGAGGACGCA GTCATGGGTT CCAGTACGAG TTTTTGGAGT CGATCCTCCA CTTCCATAA AACCTGAAAG GACTGCATCT CCAGGAGCCT GAGTATGTGC TCATGGCTGC CACGGCCCTC TTCTCCCCTG ACAGACCCGG AGTTACCCAA AGAGAAGAGA TAGATCAGCT ACAAGAGGAG ATGGCGCTGA TTCTGAACAA CCACATTATG GAACAACAGT CTCGGCTCCA AAGTCGGTTT CTGTATGCAA AGCTGATGGG CCTGCTGGCT GACCTCCGGA GTATAAACAA TGCATACTCC TATGAACTTC AGCGCTTGGA GGAACTGTCT GCTATGACGC CGCTGCTCGG GGAGATTTGC AGTTGAGGCC CAGGCTTGCA TCCTTTCCCC AGACCCCCAG GGATACACTG GCCTGGAA (SEQ ID NO : 4) .
Plasmid DNA prepared from AA106163 [which is subcloned in pBSSK(-)] was sequenced using M13 forward and reverse primers, while plasmid DNA for containing AA396982 [which is subcloned in pT7T3D- Pac (Pharmacia) vector] was sequenced using T3 and T7 primers. It was determined by assembling the two sequences that an exon coding the second finger of the DBD was missing. PCR primers R6R2 (5'-TCTGGACCAGTTCTTTCTG-3'; SEQ ID NO: 5) and R6F2 (5'-CCATGGCCAGTGAAGAAGAA-3'; SEQ ID NO: 6) flanking the missing exon were designed to scan mouse brain cDNA and a mouse brain cDNA library which was distributed in 96- well plate. A PCR fragment of with expected size (approximately 350 bps) was amplified from mouse brain cDNA. The DNA fragment was purified using Qiagen gel extraction kit (QIAGEN, Santa Clarita, CA) and submitted for automated sequencing with primers R6R2 and R6F3
(5'-GTGAAGAAGAATATGGGCC-3'; SEQ ID NO: 7). The sequence information showed a complete DBD region. The full length cDNA clone for nNR4 was obtained via PCR using the very 5'-end primer R6F5 (5'-ATTCCTGGCAGTTCCTGAGA-3'; SEQ ID NO: 8) and the very 3'-end primer R6R4 9 (5'-TCCAGGCCAGTGTATCCCTG-3'; SEQ ID NO: 9) on mouse (Mus musculus) brain cDNA, mouse (Mus musculus) embryo cDNA (Clontech, Palo Alto, CA, USA) and mouse (Mus musculus) testis cDNA (Clontech, Palo Alto, CA, USA). A 1.2 kb DNA fragment from mouse embryo cDNA was cloned into the pCRII vector (Invitrogen, San Diego, CA) and multiple clones were sequenced to identify the clone coding wild type protein sequence. Sequence assembly and analysis were performed with SEQUENCHER™ 3.0 (Gene Codes Corporation, Ann Arbor, MI). Ambiguities and/or discrepancies between automated base calling in sequencing reads were visually examined and edited to the correct base call. The final nucleotide sequence encoding nNR4 is shown as set forth in Figure 1A-B and as set forth as SEQ ID NO: 1 and the open reading frame for nNR4 results in a 357 amino add mouse nuclear receptor protein as shown in Figure 2A-B, Figure 3 and as also set forth in SEQ ID NO: 2.

Claims

WHAT IS CLAIMED:
l.A purified DNA molecule encoding a nNR4 protein wherein said protein comprises the amino acid sequence as follows: MTAMLTLETM ASEEEYGPRN CWCGDRATG YHFHALTCEG CKGFFRRTVS KTIGPICPFA GRCEVSKAQR RHCPACRLQK CLNVGMRKDM ILSAEALALR RARQAQRRAE KASLQLNQQQ KELVQILLGA HTRHVGPMFD QFVQFKPPAY LFMHHRPFQP RGPVLPLLTH FADINTFMVQ QIIKFTKDLP LFRSLTMEDQ ISLLKGAAVE ILHISLNTTF CLQTENFFCG PLCYKMEDAV HGFQYEFLES ILHFHKNLKG LHLQEPEYVL MAATALFSPD RPGVTQREEI DQLQEEMALI LNNHIMEQQS RLQSRFLYAK LMGLLADLRS INNAYSYELQ RLEELSAMTP LLGEICS , as set forth in three-letter abbreviation in SEQ ID NO : 2 .
2. An expression vector for expressing a nNR4 protein in a recombinant host cell wherein said expression vector comprises a DNA molecule of claim 1.
3. A host cell which expresses a recombinant nNR4 protein wherein said host cell contains the expression vector of claim 2.
4. A process for expressing a nNR4 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 2 into a suitable host cell; and,
(b) culturing the host cells of step (a) under conditions which allow expression of said the nNR4 protein from said expression vector.
5. A purified DNA molecule encoding a nNR4 protein wherein said protein consists of the amino acid sequence as follows: MTAMLTLETM ASEEEYGPRN CWCGDRATG YHFHALTCEG CKGFFRRTVS KTIGPICPFA GRCEVSKAQR RHCPACRLQK CLNVGMRKDM ILSAEALALR RARQAQRRAE KASLQLNQQQ KELVQILLGA HTRHVGPMFD QFVQFKPPAY LFMHHRPFQP RGPVLPLLTH FADINTFMVQ QIIKFTKDLP LFRSLTMEDQ ISLLKGAAVE ILHISLNTTF CLQTENFFCG PLCYKMEDAV HGFQYEFLES ILHFHKNLKG LHLQEPEYVL MAATALFSPD RPGVTQREEI DQLQEEMALI LNNHIMEQQS RLQSRFLYAK LMGLLADLRS INNAYSYELQ RLEELSAMTP LLGEICS , as set forth in three-letter abbreviation in SEQ ID NO : 2 .
6. An expression vector for expressing a nNR4 protein in a recombin╬╡mt host cell wherein said expression vector comprises a DNA molecule of claim 5.
7. A host cell which expresses a recombinant nNR4 protein wherein said host cell contains the expression vector of claim 6.
8. A process for expressing a nNR4 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 6 into a suitable host cell; and,
(b) culturmg the host cells of step (a) under conditions which allow expression of said the nNR4 protein from said expression vector.
9. A purified DNA molecule encoding a nNR4 protein wherein said DNA molecule comprises the nucleotide sequence as set forth in SEQ ID NO: 1, as follows:
GGGCCCGGGT GTTTTCCAGG CACTGAGGAC CGCAGTCCCT AATTCCTGGC AGTTCCTGAG ATCTCAAGGA AAGCAGGGTC AGCGAGGAGG CCTGGGGAGA GGAGGCATCC TACACCCGAT CTTGTGGCCT GCTGCCTAAG GGAAACAGGA GACCATGACA GCTATGCTAA CACTAGAAAC CATGGCCAGT GAAGAAGAAT ATGGGCCGAG GAACTGTGTG GTGTGTGGAG ACCGGGCCAC AGGCTATCAT TTCCACGCCC TGACTTGTGA GGGCTGCAAG GGCTTCTTCA GACGAACAGT CAGCAAAACC ATTGGTCCCA TCTGTCCGTT TGCTGGAAGG TGTGAGGTCA GCAAGGCCCA GAGACGCCAC TGTCCAGCCT GCAGGTTGCA GAAGTGTCTA AATGTTGGCA TGAGGAAAGA CATGATACTG TCAGCAGAAG CCCTGGCATT GCGGCGAGCC AGACAGGCAC AGCGGCGGGC AGAGAAAGCA TCTTTGCAAC TGAATCAGCA GCAGAAAGAA CTGGTCCAGA TCCTCCTCGG GGCCCACACT CGCCATGTGG GCCCCATGTT TGACCAGTTT GTGCAGTTCA AGCCTCCAGC CTATCTGTTC ATGCATCACC GGCCTTTCCA GCCTCGGGGC CCCGTGTTGC CTCTGCTCAC ACACTTTGCA GATATCAACA CGTTTATGGT GCAACAGATC ATCAAGTTCA CCAAGGATCT GCCGCTCTTC CGGTCCCTAA CCATGGAGGA CCAGATCTCC CTTCTCAAGG GAGCGGCTGT GGAAATATTG CATATCTCAC TCAACACTAC GTTCTGTCTT CAAACAGAGA ATTTCTTCTG TGGGCCTCTT TGCTACAAGA TGGAGGACGC AGTCCATGGG TTCCAGTACG AGTTTTTGGA GTCGATCCTC CACTTCCATA AAAACCTGAA AGGACTGCAT CTCCAGGAGC CTGAGTATGT GCTCATGGCT GCCACGGCCC TCTTCTCCCC TGACAGACCC GGAGTTACCC AAAGAGAAGA GATAGATCAG CTACAAGAGG AGATGGCGCT GATTCTGAAC AACCACATTA TGGAACAACA GTCTCGGCTC CAAAGTCGGT TTCTGTATGC AAAGCTGATG GGCCTGCTGG CTGACCTCCG GAGTATAAAC AATGCATACT CCTATGAACT TCAGCGCTTG GAGGAACTGT CTGCTATGAC GCCGCTGCTC GGGGAGATTT GCAGTTGAGG CCCAGGCTTG CATCCTTTCC CCAGACCCCC AGGGATACAC TGGCCTGGAA (SEQ ID NO: 1) .
10. A DNA molecule of claim 9 which consists of nucleotide 155 to about nucleotide 1218 ofSEQ ID NO: 1.
11. An expression vector for expressing a nNR4 protein wherein said expression vector comprises a DNA molecule of claim 9.
12. An expression vector for expressing a nNR4 protein wherein said expression vector comprises a DNA molecule of claim 11.
13. A host cell which expresses a recombinant nNR4 protein wherein said host cell contains the expression vector of claim 11.
14. A host cell which expresses a recombinant nNR4 protein wherein said host cell contains the expression vector of claim 12.
15. A process for expressing a nNR4 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 11 into a suitable host cell; and,
(b) culturing the host cells of step (a) under conditions which allow expression of said the nNR4 protein from said expression vector.
16. A purified DNA molecule encoding a nNR4 protein wherein said DNA molecule consists of the nucleotide sequence as set forth in SEQ ID NO: 1, as follows:
GGGCCCGGGT GTTTTCCAGG CACTGAGGAC CGCAGTCCCT AATTCCTGGC AGTTCCTGAG ATCTCAAGGA AAGCAGGGTC AGCGAGGAGG CCTGGGGAGA GGAGGCATCC TACACCCGAT CTTGTGGCCT GCTGCCTAAG GGAAACAGGA GACCATGACA GCTATGCTAA CACTAGAAAC CATGGCCAGT GAAGAAGAAT ATGGGCCGAG GAACTGTGTG GTGTGTGGAG ACCGGGCCAC AGGCTATCAT TTCCACGCCC TGACTTGTGA GGGCTGCAAG GGCTTCTTCA GACGAACAGT CAGCAAAACC ATTGGTCCCA TCTGTCCGTT TGCTGGAAGG TGTGAGGTCA GCAAGGCCCA GAGACGCCAC TGTCCAGCCT GCAGGTTGCA GAAGTGTCTA AATGTTGGCA TGAGGAAAGA CATGATACTG TCAGCAGAAG CCCTGGCATT GCGGCGAGCC AGACAGGCAC AGCGGCGGGC AGAGAAAGCA TCTTTGCAAC TGAATCAGCA GCAGAAAGAA CTGGTCCAGA TCCTCCTCGG GGCCCACACT CGCCATGTGG GCCCCATGTT TGACCAGTTT GTGCAGTTCA AGCCTCCAGC CTATCTGTTC ATGCATCACC GGCCTTTCCA GCCTCGGGGC CCCGTGTTGC CTCTGCTCAC ACACTTTGCA GATATCAACA CGTTTATGGT GCAACAGATC ATCAAGTTCA CCAAGGATCT GCCGCTCTTC CGGTCCCTAA CCATGGAGGA CCAGATCTCC CTTCTCAAGG GAGCGGCTGT GGAAATATTG CATATCTCAC TCAACACTAC GTTCTGTCTT CAAACAGAGA ATTTCTTCTG TGGGCCTCTT TGCTACAAGA TGGAGGACGC AGTCCATGGG TTCCAGTACG AGTTTTTGGA GTCGATCCTC CACTTCCATA AAAACCTGAA AGGACTGCAT CTCCAGGAGC CTGAGTATGT GCTCATGGCT GCCACGGCCC TCTTCTCCCC TGACAGACCC GGAGTTACCC AAAGAGAAGA GATAGATCAG CTACAAGAGG AGATGGCGCT GATTCTGAAC AACCACATTA TGGAACAACA GTCTCGGCTC CAAAGTCGGT TTCTGTATGC AAAGCTGATG GGCCTGCTGG CTGACCTCCG GAGTATAAAC AATGCATACT CCTATGAACT TCAGCGCTTG GAGGAACTGT CTGCTATGAC GCCGCTGCTC GGGGAGATTT GCAGTTGAGG CCCAGGCTTG CATCCTTTCC CCAGACCCCC AGGGATACAC TGGCCTGGAA (SEQ ID NO : 1) .
17. A DNA molecule of claim 16 which consists of nucleotide 155 to about nucleotide 1218 of SEQ ID NO: 1.
18. An expression vector for expressing a nNR4 protein wherein said expression vector comprises a DNA molecule of claim 16.
19. An expression vector for expressing a nNR4 protein wherein said expression vector comprises a DNA molecule of claim 17.
20. A host cell which expresses a recombinant nNR4 protein wherein said host cell contains the expression vector of claim 18.
21. A host cell which expresses a recombinant nNR4 protein wherein said host cell contains the expression vector of claim 19.
22. A process for expressing a nNR4 protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 18 into a suitable host cell; and,
(b) culturing the host cells of step (a) under conditions which allow expression of said the nNR4 protein from said expression vector.
23. A purified mouse nNR4 protein which comprises the amino acid sequence as set forth in SEQ ID NO: 2.
24. The purified mouse nNR4 protein of claim 23 which consists of the amino acid sequence as set forth in SEQ ID NO: 2.
PCT/US1998/026446 1997-12-12 1998-12-11 DNA MOLECULES ENCODING VERTEBRATE NUCLEAR RECEPTOR PROTEIN, nNR4 WO1999029722A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP98963116A EP1037912A1 (en) 1997-12-12 1998-12-11 DNA MOLECULES ENCODING VERTEBRATE NUCLEAR RECEPTOR PROTEIN, nNR4
JP2000524313A JP2001525196A (en) 1997-12-12 1998-12-11 DNA molecule encoding vertebrate nuclear receptor protein nNR4
CA002315273A CA2315273A1 (en) 1997-12-12 1998-12-11 Dna molecules encoding vertebrate nuclear receptor protein, nnr4

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6814497P 1997-12-12 1997-12-12
US60/068,144 1997-12-12

Publications (1)

Publication Number Publication Date
WO1999029722A1 true WO1999029722A1 (en) 1999-06-17

Family

ID=22080682

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/026446 WO1999029722A1 (en) 1997-12-12 1998-12-11 DNA MOLECULES ENCODING VERTEBRATE NUCLEAR RECEPTOR PROTEIN, nNR4

Country Status (4)

Country Link
EP (1) EP1037912A1 (en)
JP (1) JP2001525196A (en)
CA (1) CA2315273A1 (en)
WO (1) WO1999029722A1 (en)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BERTILSSON G, ET AL.: "IDENTIFICATION OF A HUMAN NUCLEAR RECEPTOR DEFINES A NEW SIGNALING PATHWAY FOR CYP3A INDUCTION", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 95, no. 21, 1 October 1998 (1998-10-01), US, pages 12208 - 12213, XP002920310, ISSN: 0027-8424, DOI: 10.1073/pnas.95.21.12208 *

Also Published As

Publication number Publication date
JP2001525196A (en) 2001-12-11
EP1037912A1 (en) 2000-09-27
CA2315273A1 (en) 1999-06-17

Similar Documents

Publication Publication Date Title
US20060160140A1 (en) Human nuclear receptor protein nNR7-1
EP1017811A1 (en) G-protein coupled glycoprotein hormone receptor hg38
US6054295A (en) DNA molecules encoding human nuclear receptor proteins
US7029865B2 (en) DNA molecules encoding the melanocortin 4 receptor protein from rhesus monkey
EP1140968B1 (en) Dna molecules encoding splice variants of the human melanocortin 1 receptor protein
EP1007540A1 (en) Dna molecules encoding human nuclear receptor proteins
CA2314434A1 (en) Dna molecules encoding human nuclear receptor protein, nnr5
EP1037912A1 (en) DNA MOLECULES ENCODING VERTEBRATE NUCLEAR RECEPTOR PROTEIN, nNR4
US6645738B1 (en) DNA molecules encoding the melanocortin 5 receptor protein from rhesus monkey
US7060463B2 (en) DNA molecules encoding Macaca mulatta androgen receptor
US20030119100A1 (en) DNA molecules encoding human nuclear receptor proteins
US7001742B1 (en) Human uncoupling protein 3
EP1133515A1 (en) Dna molecules encoding hg51, a g-protein-coupled receptor
EP1129104A1 (en) Dna molecules encoding the melanocortin 3 receptor protein from rhesus monkey

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 09581380

Country of ref document: US

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 524313

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1998963116

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2315273

Country of ref document: CA

Ref country code: CA

Ref document number: 2315273

Kind code of ref document: A

Format of ref document f/p: F

WWP Wipo information: published in national office

Ref document number: 1998963116

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1998963116

Country of ref document: EP