GB2265376A - Human steroid hormone receptor (nuci) - Google Patents
Human steroid hormone receptor (nuci) Download PDFInfo
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- GB2265376A GB2265376A GB9306043A GB9306043A GB2265376A GB 2265376 A GB2265376 A GB 2265376A GB 9306043 A GB9306043 A GB 9306043A GB 9306043 A GB9306043 A GB 9306043A GB 2265376 A GB2265376 A GB 2265376A
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- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
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Abstract
A member of the steroid hormone receptor superfamily (hereinafter identified as NUCI) is disclosed which has been prepared by cDNA cloning from a human osteosarcoma SAOS-2/BiO cell library. Also disclosed is the complete sequence of human NUCI complementary DNA; expression systems, including a COS stable expression system; and an assay using the COS expression system. NUCI can be used in an assay to identify and evaluate chemical entities that bind to this receptor.
Description
TITLE OF THE INVENTION
HUMAN STEROID HORMONE RECEPTOR NUCI
SUMMARY OF THE INVENTION
The present invention relates generally to ligand-responsive regulatory proteins and genes encoding them. In particular, a novel recombinant human steroid hormone receptor (hereinafter identified as NUCI) is disclosed which has been prepared by polymerase chain reaction techniques.
Also disclosed is the complete sequence of human NUCI complementary DNA; expression systems, including a
COS stable expression system; and an assay using the Cos expression system. In addition, the invention relates to a method for identifying functional ligands of the NUCI receptor.
BACKGROUND OF THE INVENTION
Retinoids, steroid and thyroid hormones and possibly other molecules produce their biological effects by binding to proteins of the steroid receptor superfamily. These receptors interact with specific DNA.sequences and modulate gene expression (for reviews see JM Berg, Cell 57:1065-1068 (19890 ;
RM Evans, Science 240:899-895 (1988); M Beato, Cell 56:335-344 (1989)). Sequence analysis and functional studies of these receptors revealed two important regions which exhibit a high degree of amino acid residue conservation.The highest level of similarity among the receptors is found in a region which contains nine cystein residues that bind zinc atoms to form two "zinc fingers," which interact with the cognate steroid response elements of DNA (J Miller, et al., EMBO J 4:1609-1614 (1985) ; RM Evans, Cell 52:1-3 (1988)). The second region, which is less conserved, is the ligand binding domain responsible for the interaction with the hormone (J Carlstedt-Duke, et al., Proc Natl Acad
Sci USA 79:4260-4264 (1982). J. Caristedt-Duke, et al., Proc Natl Acad sci USA 84:4437-4440 (1987)).
Recent studies have attributed additional functions to other domains of these receptors, such as protein protein interaction that participates in transcriptional regulation (R Scule, et al., Cell 62:1217-1226 (1990); HF Tang, Cell 62:1205-1215 (1990) ; JM Holloway et al., Proc Natl Acad Sci USA 87:8160-8164 (1990)). The amino acid conservation in the DNA binding domain has led to the identification of new members of the steroid receptor superfamily.
For example, hER1 and hER2 have been cloned by low stringency hybridization of cDNA libraries with a DNA probe coding for the DNA binding domain of the estrogen receptor (V Giguere, et al., Nature 331:91-94 (1988)). Similar approaches have led to the discovery of the retinoic acid receptors and the peroxisome proliferator activator receptor (PPAR)(i Issemann, et al., Nature 347:645-650 (1990) ; DJ
Mangelsdorf, et al., Nature 345:224-229 (1990)).
Recently, three novel members of the Xenopus nuclear hormone receptor superfamily have been disclosed (C Dreyer, Cell 68:879-887 (1992)), In addition, U.S.
Patent No. 4,981,784 to Evans, . discloses the identification of a retinoic acid receptor and the use of chimeric constructs to produce hybrid receptors for the identification qf novel ligands.
The above references, however, neither disclose nor suggest the instant invention.
DETAILED DESCRIPTION OF THE INVENTION
One embodiment of the invention concerns human steroid hormone receptor NUCI, said receptor being free of other human receptor proteins.
In one class this embodiment concerns human steroid hormone receptor NUCI, said receptor being free of other human proteins.
Within this class, this embodiment concerns human steroid hormone receptor NUCI from human cells such as osteosarcoma, said receptor being free of other human proteins.
In a second class* this embodiment concerns a protein comprising the following 441 amino acid sequence (SEQ ID NO: depicted from the.amino to the carboxy terminus:
Met Glu Glu Pro Gln Glu Glu Ala Pro Glu Val Arg Golu Glu Glu Glu 1 5 10 15
Lys Glu Glu Val Ala Glu Ala Glu Gly Ala Pro Glu Leu Asn Gly Gly
20 25 30
Pro Gln His Ala Leu Pro Ser Ser Ser Tyr Thr Asp Leu Ser Arg Ser
35 40 45
Ser Ser Pro Pro Ser Leu Leu Asp Gin Leu Gin Met Gly Cys Asp Gly
50 55 60
Ala Ser Cys Gly Ser Leu Asn Met Glu Cys Arg Val Cys Gly Asp lays 65 70 75 80
Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly
85 90 95
Phe Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Glu 100 105 110 Arg Ser Cye Lys Ile Gln Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys
115 120 125
Arg Phe Gin Lys Cys teu Ala Leu Gly Met Ser His Asn Ala Ile Arg
130 135 140
Phe Gly Arg Met Pro Glu Ala Glu lays Arg Lys Leu Val Ala Gly Leu 145 150 155 160
Thr Ala Asn Glu Gly Ser Gln Tyr Asn Pro Gln Val Ala Asp Leu Lys
165 170 175
Ala Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met
180 185 190
Thr Lys Lys Lys Als Arg Ser Ile Leu Thr Gly Lys Ala Ser His Thr
195 200 205
Ala Pro Phe Val lie His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys
210 215 220
Gly Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu 225 230 235 240
Ile Ser Val His Val Phe Tyr Arg Cys Gln Cys Thr Thr Val Glu Thr
245 250 255
Val Arg Glu Leu Thr Glu Phe Ala Lys Ser Ile Pro Ser Phe Ser Ser
260 265 270
Leu Phe Leu Asn Asp Gln Vat Thr Leu Leu Lys Tyr Gly Val His Gin 275 280 285 Ala Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Lue
290 295 300
Val Ala Asn Gly Ser Gly Phe Val Thr Arg Glu Phe Leu Arg Ser Leu
305 310 315 320 Arg lays Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val
325 330 335 lays Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile
340 345 350
Ala Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro
355 360 365
Arg Val Glu Ala Ile Gln Asp Thr Ile Leu Arg Ala Leu Glu Phe His
370 375 380
Leu Gin Ala Asn His Pro Asp Ala Gln Tyr ten Phe Pro Lys Leu Leu
385 390 395 400
Gln Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met
405 410 415
Met Gln Arg Ile Lys Lys Thr Glu Thr Glu Thr Ser Leu His Pro Leu
420 425 430
Leu Cln Glu Ile Tyr Lys Asp Met Tyr
435 440 the protein being free of other human receptor proteins.
Within the second class, this embodiment concerns a protein consisting of the foregoing 441 amino acid sequence (SEQ ID No : 5 :).
A second embodiment concerns a DNA sequence encoding human steroid hormone receptor NUCI complementary DNA, said DNA, said sequence being free of other human DNA sequences.
As will be appreciated by those of skill in the art, there is a substantial amount af redundancy in the set of codons which translate specific amino acids. Accordingly, the invention also includes alternative base sequences wherein a codon (or codons) are replaced with another codon, such that the amino acid sequence translated by the DNA sequence remains unchanged. For purposes of this specification, a sequence bearing one or more such replaced codons will be defined as a degenerate variation. Also included are mutations (exchange of individual amino acids) which one of skill in the art would expect to have no effect on functionality, such as valine for leucine, arginine fox lysine and asparagine for glutamine.
One class of the second embodiment of the invention concerns the following nucleotide sequence (SEQ ID NO:I:) of complementary DNA depicted from the 5 to the 3' terminus:
GAATTCTGCG GAGCCTGCGG GACCGCGGCG GGTTGGCCCG TAGGCAGCCG GGACAGTGTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CACACAGTGG CTTCTGCTCA CCAACAGATG 120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG 180
ATACCCCTGG AAAACTGAAG CCCOTGGAGC AATGATCTQT ACAGGACTGC TTQAAGGCTG 240
ATGGGAACCA CCCTGTAGAG GTCCATCTGC GTTCAGACCC AGACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTGTGGCG CQGAGGGGAG ATCAGCOATG GAGCAGCCAC AGGAGGAAGC 360 CCCTCAGGTC CGGGAAGAGG AGGAGAAAGA GGAAGTGGCA GAGGCACAAG GAGCCCCAGA 420
GCTCAATGGG GGACCACAGC ATGCACTTCC TTCCAGCAGC TACACAGACC TCTCCCGGAG 480
CTCCTCGCCA CCCTCACTGC TGGACCAACT GCAGATGGGC TGTGACGGGG CCTCATGCGG 540
CAGCCTCAAC ATGGAGTGCC GGGTGTGCGG GGACAAGGCA TCGGGCTTCC ACTACGGTGT 600
TCATGCATGT GAGGGGTGCA AGGGCTTCTT CCGTCGTACG ATCCGCATGA AGCTGGAGTA 660
CGAGAAGTGT GAGCGCAGCT GCAAGATTCA GAAGAAGAAC CGCAACAAGT GCCAGTACTG 720
CCGCTTCCAG AAGTGCCTGG CACTGGGCAT GTCACACAAC GCTATCCGTT TTGGTGGGAT 780
GCCGGAGGCT GAGAAGAGGA AGCTGGTGGC AGGGCTGACT GCAAACGAGG GGAGCCAGTA 840
CAACCCACAG GTGGCCGACC TGAAGGCCTT CTCCAAGCAC ATCTACAATG CCTACCTGAA 900
AAACTTCAAC ATGACCAAAA AGAAGGCCCG CAGCATCCTC ACCGGCAAAG CCAGCCACAC 960
GGCGCCCTTT GTGATCCACG ACATCGAGAC ATTGTGGCAG GCAGAGAAGG GGCTGGTGTG 1020
GAAGCAGTTG GTGAATGGCC TGCCTCCCTA CAAGGAGATC AGCGTGCACG TCTTCTACCG 1080
CTGCCAGTGC ACCACAGTGG AGACCGTGCG GGAGCTCACT GAGTTCGCCA AGAGCATCCC 1140
CAGCTTCAGC AGCCTCTTCC TCAACGACCA GGTTACCCTT CTCAAGTATG GCGTGCACGA 1200
GGCCATCTTC GCCATGCTGG CCTCTATCGT CAACAAGGAC GGGCTGCTGG TAGCCAACGG 1260
CAGTGGCTTT GTCACCCGTG AGTTCCTGCG CAGCCTCCGC AAACCCTTCA GTGATATCAT 1320
TGAGCCTAAG TTTGAATTTG CTGTCAAGTT CAACGCCCTG GAACTTGATG ACAGTGACCT 1380
GGCCCTATTC ATTGCGGCCA TCATTCTGTG TGGAGACCGG CCAGGCCTCA TGAACGTTCC 1440
ACGGGTGGAG GCTATCCAGG ACACCATCCT GCGTGCCCTC GAATTCCACC TGCAGGCCAA 1500
CCACCCTGAT GCCCAGTACC TCTTCCCCAA GCTGCTGCAG AAGATGGCTG ACCTGCGGCA 1560
ACTGGTCACC GAGCACGCCC AGATGATGCA GCGGATCAAG AAGACCGAAA CCGAGACCTC 1620
GCTGCACCCT CTGCTCCAGG AGATCTACAA GGACATGTAC TAACGGCGGC ACCCAGGCCT 1680
CCCTGCAGAC TCCAATGGGG CCAGCACTGG AGGGGCCCAC CCACATGACT TTTCCATTGA 1740
CCAGCTCTCT TCCTGTCTTT GTTGTCTCCC TCTTTCTCAG TTCCTCTTTC TTTTCTAATT 1800
CCTGTTGCTG TGTTTCTTCC TTTCTGTAGG TTTCTCTCTT CCCTTCTCCC TTCTCCCTTG 1860
CCCTCCCTTT CTCTCTCCTA TCCCCACGTC TGTCCTCCTT TCTTATTCTG TGAGATGTTT 1920
TGTATTATTT CACCAGCAGG ATAGAACAGG ACCTCTGCTT TTGCACACCT TTTCCCCAGG 1980
AGCAGAAGAG AGTGGGCCTG CCCTCTGCCC CATCATTGCA CCTGCAGGCT TAGGTCCTCA 2040
CTTCTGTCTC CTGTCTTCAG AGCAAAAGAC TTGAGCCATC CAAAGAAACA CTAAGCTCTC 2100
TGGGCCTGGG TTCCAGGGAA GGCTAAGCAT GGCCTGGACT GACTGCAGCC CCCTATAGTC 2160
ATGGGGTCCC TGCTGCAAAG GACAGTGGCA GACCCCGGCA GTAGAGCCGA GATGCCTCCC 2220
CAAGACTGTC ATTGCCCCTG CGATGGTGAG GCCACCCACT GACCCAATGA TCCTCTCCAG 2280
CAGCACACCT CAGCCCCACT GACACCCAGT GTCCTTCCAT CTTCACACTG GTTTGCCAGG 2340
CCAATGTTGC TGATGGCCCC TCCAGCACAC ACACATAAGC ACTGAAATCA CTTTACCTGC 2400
AGGCACCATG CACCTCCCTT CCCTCCCTGA GGCAGGTGAG AACCCAGAGA GAGGGGCCTG 2460
CAGGTGAGCA GGCAGGGCTG GGCCAGGTCT CCGGGGAGGC AGGGGTCCTG CAGGTCCTGG 2520
TGGGTCAGCC CAGCACCTCG CCCAGTGGGA GCTTCCCGGG ATAAACTGAG CCTGTTCATT 2580
CTGATGTCCA TTTGTGGCAA TAGCTCTACT GCCCTCCCCT TCCCCTTTAC TCAGCCCAGC 2640
TGGCCACCTA GAAGTCTCCC TGCACAGCCT CTAGTGTCCG GGGACCTTGT GGGACCAGTC 2700
CCACACCGCT GGTCCCTGCC CTCCCCTGCT CCCAGGTTGA GGTGCGCTCA CCTCAGAGCA 2760
GGGCCAAAGC ACAGCTGGGC ATGCCATGTC TGAGCGGCGC AGAGCCCTCC AGGCCTGCAG 2820
GGGCAAGGGG CTGGCTGGAG TCTCAGAGCA CAGAGGTAGG AGAACTGGGG TTCAAGCCCA 2880
GGCTTCCTGG GTCCTGCCTG GTCCTCCCTC CCAAGGAGCC ATTCTATGTG ACTCTGGGTG 2940
GAAGTGCCCA GCCCCTGCCT GACGGGATCA CTCTCTGCTG GCAGGATTCT TCCCGCTCCC 3000
CACCTACCCA GCTGATGGGG GTTGGGGTGC TTCTTTCAGC CAAGGCTATG AAGGGACAGC 3060
TGCTGGGACC CACCTCCCCC CTTCCCGGGC CACATGCCGC GTCCCTGCCC CCACCCGGGT 3120
CTGGTGCTGA GGATACAGCT CTTCTCAGTG TCTGAACAAT CTCCAAAATT GAAATGTATA 3180
TTTTTGCTAG GAGCCCCAGC TTCCTGTGTT TTTAATATAA ATAGTGTACA CAGACTGACG 3240
AAACTTTAAA TAAATGGGAA TTAAATATTT AAAAAAAAAA GCGGCCGCGA ATTC 3294 or a degenerate variation thereof.
A second class of the second embodiment of the invention concerns the following nucleotide sequence (SEQ ID NO:2:) of complementary DNA depicted from the 5' to the 3' terminus:
ATG GAGCAGCCAC AGGAGGAAGC 360
CCCTGAGGTC CGGGAAGAGG AGGAGAAAGA GGAAGTGGCA GAGGCAGAAG GAGCCCCAGA 420
GCTCAATGGG GGACCACAGC ATGCACTTCC TTCCAGCAGC TACACAGACC TCTCCCGGAG 480
CTCCTCGCCA CCCTCACTGC TGGACCAACT GCAGATGGGC TGTGACGGGG CCTCATGCGG 540
CAGCCTCAAC ATGGAGTGCC GGGTGTGCGG GGACAAGGCA TCGGGCTTCC ACTACGGTGT 600
TCATGCATGT GAGGGGTGCA AGGGCTTCTT CCGTCGTACG ATCCGCATGA AGCTGGAGTA 660
CGAGAAGTGT GAGCGCAGCT GCAAGATTCA GAAGAAGAAC CCCAACAAGT GCCAGTACTG 720
CCGCTTCCAG AAGTGCCTGG GACTGGGCAT GTCACACAAC GCTATCCGTT TTGGTCGGAT 780
GCCGGAGGCT GAGAAGAGGA AGCTGGTGGC AGGGCTGACT GCAAACGAGG GGAGCCAGTA 840
CAACCCACAG GTGGCCGACC TGAAGGCCTT CTCCAAGCAC ATCTACAATG CCTACCTGAA 900
AAACTTCAAC ATGACCAAAA AGAAGGCCCG CAGCATCCTC ACCGGCAAAG CCAGCCACAC 960
GGCGCCCTTT GTGATCCACG ACATCGAGAC ATTGTGGCAG GCAGAGAAGG GGCTGGTGTG 1020
GAAGCAGTTG GTGAATGGCC TGCCTCCCTA CAAGGAGATC AGCGTGCACG TCTTCTACCG 1080
CTGCCAGTGC ACCACAGTGG AGACCGTGCG GGAGCTCACT GAGTTCGCCA AGAGCATCCC 1140
CAGCTTCAGC AGCCTCTTCC TCAACGACCA GGTTACCCTT CTCAAGTATG GCGTGCACGA 1200
GGCCATCTTC GCCATGCTGG CCTCTATCGT CAACAAGGAC GGGCTGCTGG TAGCCAACGG 1260
CAGTGGCTTT GTCACCCGTG AGTTCCTGCG CAGCCTCCGC AAACCCTTCA GTGATATCAT 1320
TGAGCCTAAG TTTGAATTTG CTGTCAAGTT CAACGCCCTG GAACTTGATG ACAGTGACCT 1380
GGCCCTATTC ATTGCGGCCA TCATTCTGTG TGGAGACCGG CCAGGCCTCA TGAACGTTCC 1440
ACGGGTGGAG GCTATCCAGG ACACCATCCT GCGTGCCCTC GAATTCCACC TGCAGGCCAA 1500
CCACCCTGAT GCCCAGTACC TCTTCCCCAA GCTGCTGCAG AAGATGGCTG ACCTGCGGCA 1560
ACTGGTCACC GAGCACGCCC AGATGATGCA GCGGATCAAG AAGACCGAAA CCGAGACCTC 1620
GCTGCACCCT CTGCTCCAGG AGATCTACAA GGACATGTA 1659 or a degenerate variation thereof.
Also within this second class of the second embodiment of the invention is the foregoing DNA sequence (SEQ ID NO:2:) further comprising the following nucleotide sequence (SEQ tD NO:3:) of complementary DNA depicted from the 5' to the 32 terminus:
GAATTCTGCG GAGCCTGCGG GACGGCGGCG GGTTGGCCCG TAGGCAGCCG GGACAGTGTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CACACAGTGG CTTCTGCTCA CCAACAGATG 120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG 180
ATACCCCTGG AAAACTGAAG CCCGTGGAGC AATGATCTCT ACAGGACTGC TTCAAGGCTG 240
ATGGGAACCA CCCTGTAGAG GTCCATCTGC GTTCAGACCC AGACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTGTGGCG CCGAGGGGAG ATCAGCC 337 or a degenerate variation thereof.
Further within this second class of the second embodiment of the invention is the foregoing
DNA sequence (SEQ ID NO:2:) further comprising the following nucleotide sequence (SEQ ID NO:4:) of complementary DNA depicted from the 5' to the 3' terminus::
C TAACGGCGGC ACCCAGGCCT 1680
CCCTGCAGAC TCCAATGGGG CCACCACTGG AGGGGCCCAC CCACATGACT TTTOCATTGA 1740
CCAGCTCTCT TCCTGTCTTT GTTGTCTCCC TCTTTCTGAG TTCCTCTTTC TTTTCTAATT 1800
CCTGTTGCTC TGTTTCTTCC TTTCTGTAGG TTTCTCTCTT CCCTTCTCCC TTCTCCCTTG 1860
CCCTCCCTTT CTCTCTCCTA TCCCCACGTC TGTCCTCCTT TCTTATTCTG TGAGATGTTT 1920
TCTATTATTT CACCAGCAGC ATAGAACAGG ACCTCTGCTT TTGCACACCT TTTCCCCAGG 1980
AGCAGAAGAG AGTGGGCCTG CCCTCTGCCC CATCATTGCA CCTGCAGGCT TAGGTCCTCA 2040
CTTCTGTCTC CTGTCTTCAG AGCAAAAGAC TTGAGCCATC CAAAGAAACA CTAAGCTCTC 2100
TGGGCCTGGG TTCCAGGGAA GGCTAAGCAT GGCCTGGACT GACTGCAGCC CCCTATAGTC 2160
ATGGGGTCCC TGCTGCAAAG GACAGTGGCA GACCCCGGCA GTAGAGCCGA GATGCCTCCC 2220
CAAGACTGTC ATTGCCCCTC COATCGTGAG GCCACCCACT GACCCAATGA TCCTCTCCAG 2280
CAGCACACCT CAGCCCCACT GACACCCAGT GTCCTTCCAT CTTCACACTG GTTTGCCAGG 2340 CCAATGTTGC TGATGGCCCC TCCAGCACAC ACACATAAGC ACTGAAATCA CTTTACCTGC 2400
AGGCACCATG CACCTCCCTT CCCTCCCTGA GGCAGGTGAG AACCCAGAGA GAGGGGCCTG 2460
CAGGTGAGCA GGCAGGGCTG GGCCAGGTCT CCGGGGAGGC AGGGGTCCTG CAGGTCCTGG 2520
TGGGTCAGCC CAGCACCTCG CCCAGTGGGA GCTTCCCGGG ATAAACTGAG CCTGTTCATT 2580
CTGATGTCCA TTTGTCCCAA TAGCTCTACT GCCCTCCCCT TCCCCTTTAC TCAGCCCAGC 2640
TGGCCACCTA GAAGTCTCCC TGCACAGCCT CTAGTGTCCG GGGACCTTGT GGGACCAGTC 2700 CCACACCGCT GGTCCCTGCG CTCCCCTGCT CCCAGGTTGA GGTGCGCTCA CCTCAGAGCA 2760
GGGCCAAAGC ACAGCTGGGC ATGCCATGTC TGAGCGGCGC AGAGCCCTCC AGGCCTGCAG 2820
GGGCAAGGGG CTGGCTGGAG TCTCAGAGCA CAGAGGTAGG AGAACTGGGO TTCAAGCCCA 2880
GGCTTCCTGG GTCCTGCCTG GTCCTCCCTC CCAAGGAGCC ATTCTATGTG ACTCTGGGTG 2940
GAAGTGCCCA GCCCCTGCCT GACGGGATCA CTCTCTGCTG GCAGGATTCT TCCCGCTCCC 3000
CACCTACCCA GCTGATGGGG GTTGGGGTGC TTCTTTCAGC CAAGGCTATG AAGGGACAGC 3060
TGCTGGGACC CACCTCCCCC CTTCCCCGGC CACATGCCGC GTCCCTGCCC CCACCCGGGT 3120
CTGGTGCTGA GGATACAGCT CTTCTCAGTG TCTGAACAAT CTCCAAAATT GAAATGTATA 3180
TTTTTGCTAG GAGCCCCAGC TTCCTGTGTT TTTAATATAA ATAGTGTACA CAGACTGACG 3240 AAACTTTMA TAAATGGGAA TTAAATATTT AAAAAAAAAA GCGGCCGCGA ATTC 3294 or a degenerate variation thereof.
A third embodiment of this invention concerns systems for expressing all or part of the human steroid hormone receptor NUCf.
One class of this third embodiment of the invention comprises:
An expression construct, such as a plasmid which comprises:
a) an expression vector, such as
PJ3NUCI, and
b) a base sequence encoding human steroid
hormone receptor NUCI protein.
Within this class of the third embodiment, the steroid hormone receptor NUCI comprises the nucleotide sequence (SEQ ID NO:1:) of complementary
DNA as shown above.
A second class of this third embodiment of the invention concerns a system for the transientexpression of human steroid hormone receptor NUCI in a suitable host cell, such as a monkey kidney cell line (COS), the system comprised of a vector which expresses human steroid hormone receptor NUCI cDNA.
It is understood, and is readily apparent to those skilled in the art that a wide variety of commonly used cell lines are suitable for use in the present invention. Suitable cell lines derived from various species include, but are not limited to, cell lines of human, bovine, porcine, monkey, and rodent origin, or from yeast and bacterial strains.
A fourth embodiment of the invention concerns a method of using any of the above eukaryote or prokaryote expression systems for determining the binding affinity of a test sample for steroid hormone receptor NUCI.
hollowing the isolation of a DNA sequence encoding human steroid hormone receptor NUCI cDNA, a chimeric gene can be created by substituting the
DNA-binding domain region in the DNA sequence encoding NUCt cDNA with a DNA-binding domain region taken from a DNA sequence coding for another steroid hormone receptor protein, e.g., glucocorticoid receptor protein, thyroid receptor protein, mineralocorticoid receptor protein or retinoic acid receptor protein. Next, a suitable xeceptor-defifient host cell is transfected with: (1) the chimeric receptor gene, which is preferably carried on an expression plasmid, and (2) a reporter gene, such as the CAT gene or the firefly luciferase gene, which is also preferably carried on a plasmid.
In any cases the reporter gene is functionally linked to an operative hormone response element (ERE) (either wild-type or engineered) wherein the hormone response element is capable of being activated by the
DNA-binding domain used to make the chimeric receptor gene. (For example, if the chimeric receptor gene contains the DNA-binding domain region from NUCI receptor coding DNA, then the .HRE should be a wild-type, an engineered, or a synthetic GRE, i.e., one that can be activated by the operative portion of the DNA-binding region of a NUCI receptor protein.)
Next, the transfected host cell is challenged with a test sample which contains one or more ligand(s) which can potentially bind with the ligand-binding domain region of the chimeric protein coded for by the chimeric gene.To determine the extent that ligands can functionally complex with the chimeric receptor protein, induction of the reporter gene is monitored by monitoring changes in the protein levels of the protein coded for by the reporter gene. (For example, if luciferase is the reporter gene, the production of luciferase is indicative of receptor-regulated gene transcription.) Finally, when a ligand(s) is found that can induce transcription of the reporter gene,
It is concluded that this ligand(s) can bind to the receptor protein coded for by the initial sample DNA sequence. This conclusion can be further verified by testing the binding properties of the receptor protein, coded for by the initial sample DNA sequences, vis-a-vis the ligand{s) that induce expression of the reporter gene.
The fourth embodiment further concerns a method for determining the affinity of a test sample for activation of a steroid hormone receptor NUCI, the method cotprDsing: (a) constructing a chimeric gene by substituting
portions of a DNA-binding domain region of a DNA
sequence encoding human steroid hormone receptor NUCT cDNA with operative portions of a DRA-binding domain region from a known
ligand-responsive receptor protein; (b) introducing into a suitable receptor-deficient
host cell::
(i) the chimeric gene from step (a), and
(ii) a reporter gene functionally linked to an
operative hormone response element
wherein the hormone response element is
capable of being activated by the
DNA-binding domain region of the receptor
protein encoded by the chimeric gene of
step (a); (c) challenging the transfected host cell from step
(b) with the test sample to be evaluated for
ligand-binding activity with the chimeric
receptor protein encoded by the chimeric gene of
step (a); (d) assaying induction of the reporter gene by
monitoring changes in the protein levels of the
protein coded for by the reporter gene.
One class of this embodiment concerns a method of using a monkey kidney cell line (COS) as the suitable receptor-deficient host cell. In addition tbe COS host cell line may be transfected with a plasmid, the plasmid comprising:
(a) an expression vector, such as
PJ3NUCI, and
(b) the base sequence encoding human steroid
hormone receptor NUCI protein.
The aforementioned fourth embodiment is further useful for identifying compounds which may be peroxisome proliferators and, hence, are potentially hepatocarcinogens. This embodiment is also useful in identifying ligands for new hormone systems which regulate bodily function.
In overview, the present invention describes methods to isolate the human steroid hormone receptor
NUCI complementary DRA (cDNA) without prior knowledge of its protein sequence or gene sequence. Polymerase chain reaction (PCR) technique was utilized for the isolation of human steroid hormone receptor NUCI cDNA.
The complete sequence of the human steroid hormone receptor NUCI cDNA was determined, and its encoded protein sequence was deduced. Among other things, such Sequence information is useful in the process of developing novel steroid hormone antagonists.
An expression system was used to express the cloned human steroid hormone receptor NUCI cDNA. The
COS (a monkey kidney cell line) expression system can be used to measure the ligand binding properties of human steroid hormone receptor NUCI.
Assay protocols use the heterologously expressed human steroid hormone receptor NUCI for determination of the activation of steroid hormone receptor NUCI by antagonists.
The present invent ion generally relates to a new member of the steroid hormone receptor superfamily. The amino.9cid sequence deduced from the DNA sequence (Bases'338 to~l659) shows the characteristic features of both the DNA and the ligand binding domains of this family of receptors.
Sequence analysis predicted a protein of 451 amino acids which includes the conserved amino acrid residues characteristic of the DNA and ligand-binding domains of nuclear receptors. The putative DNA binding domain of NUCI contains the conserved nine cystein.residues, which are found in all the members of this receptor superfamily. Interestingly, two additional cystein residues (cys62 and cys67) are located in proximity to the 9 conserved Cyetein residues. The presence of additional cystein residues in this region could lead to the formation of alternative structures for the DNA binding domain.
Sequence comparison to the different receptors of this superfamily indicates that NUCI is closely related to the peroxisome proliferator activator receptor (I Issemann, et al., Nature 347:645-650 (1990)) with which it shares an overall homology of 62%, in particular, homology of 86% of the amino acids In the DNA binding domain and 72% of the amino acids in the ligand-binding domain.
Hybridization experiments revealed that the distribution of YUCS receptor is different from that of the PPAR. Northern blot analysis showed that in mature rats the receptor is highly expressed in heart. kidney and lungs as a transcript of approximately 3500 nucleotides. In human cells the size of the mRNA is approximately 4000 nucleotides.
NUCT is highly expressed in heart, kidney, lung.
spleen and ovaries and has very low expression in liver. In contrast, PPAR was reported to show highest expression in liver. This suggests that although the two receptors seem to be related. they may have regulatory roles in different target tissues.
Various peroxisome proliferators have been identified as being hepatocarcinogens. Because NUCI has some homology with PPAR, it is useful for identification of compounds which may have activity as peroxisome proliferators and accordingly allow screening of compounds for potential hepatocarcinogenicity.
Consistent with the sequence similarity, the chimeric NUCI receptor was activated by peroxisome proliferator W -14643 at a concentration of 10
Lower concentrations did not activate the chimeric
NUCI receptor. The lower sensitivity of NUCI receptor to the peroxisome proliferator, in spite of the similarity in the ligand binding domain, could be attributed either to differences in key amino acids in the ligand binding domain or possibly to some unknown technical aspects of the assay.Since W-14643 and other peroxisome proliferators are similar synthetic molecules, it is not known if they are the only ligands for these receptors or merely structurally related to the '!real" physiological ligand(s). The structure of NUCI and its related receptor PPAR suggest that they belong to a subgroup of the steroid receptor superfamily.
As used herein, "steroid hormone receptor superfamily" refers to the class of related receptors comprised of giucocorticoid, mineralocorticoid.
progesterone, estrogen, estrogen-related, vitamin
D3, thyroid, v-erb-A, retinoic acid and E75 (Drosophilia) receptors. As used herein steroid hormone receptor" refers to members within the steroid hormone receptor superfamily.
As used herein, "ligand" means an inducer, such as a hormone or growth substance. Inside a cell the ligand binds to a receptor protein, thereby creating a ligand-receptor complex, which in turn can bind to an appropriate hormone response element.
Single ligands may have multiple receptors.
As used herein, "expression construct" refers to a plasmid or vector comprising a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein.
and (3) appropriate transcription initiation and termination sequences. "Recombinant expression system" means a combination of. an expression construct and a suitable host microorganism.
The following examples are given for the purpose of illustrating the present invention and shall not be construed as being limitations on the scope or spirit of the instant invention EXAflPLl PCR Amplification
To use the polymerase chain reaction (PCR) method, degenerate oligonucleotides were synthesized according to the amino acid sequence of two conserved segments shared by members of the nuclear receptor superfamily (RM Evans, Sciene 240:899-895 (1988)).
The 5'end primers, ES11 and ES 12, were designed according to a segment of the DNA binding domain.
The primer at the 3' end, ES 15, was prepared according to a conserved amino acid sequence in the ligand binding domain of the retinoid receptor subfamily and the vitamin D receptor. Since this conserved region contains two nonconserved amino acid residues, inosine nucleotides were used as part of this primer. Human cDNA prepared from mRNA of osteosarcoma cells SAOS-2/310, amplified with the primers ES11 and ES15, yielded multiple DNA fragments with various sizes after the first round of amplification. A portion of the reaction was subjected to a second round of amplification using the nested primer ESI2 and the same 3 end primer
ES15.
A random primed cDNA library was prepared from 2 mg total RNA isolated from the Osteosarcoma
SAOS-2/B10 cells by the Moloney reverse transcriptase enzyme RTH according to the manufacturer recommendations (Bethesda Research Laboratoies).
The cDNA reaction (25 ml) was diluted into 300 ml water and heat denatured at 95 C for 5 minutes and quickly chilled on ice. The cDNA (2.5 ml) and the first primer pair, ES11 and ES15 (0.5 mM each) were employed in the amplification reaction with the amplitaq kit and the DNA thermal cycler (Perkin-Elmer-Cetus).
Primer ESlt has the following sequence (SEQ
ID NO:6:):
CGAATTCTGT GAGGGCTGGA ARGSC 25 wherein: R represents A or G; and
S represents C or G; and Primer E515 has the following sequence (SBQ ID
NO:7:): GGAATTCRAA NCCNGGNANN NNYTTNOCRA A 31 wherein: N (at the 11, 14 & 26 positions) represents
A or C or G or T; N (at the 17. 19, 20, 21 & BR<
.22 positions) represent inosine; R
represents A or G; S represents C or G; and
Y represents C or T.
The following amplification cycles were conducted: denaturation at 94 C. 1.S minutes; annealing at 650C, 3 minutes; extension at 72 C, 5 minutes for 3 cycles; denaturing at 94 C, 1 minute; annealing at 600C, 3 minutes; extension at 72 C, 5 minutes for 15 cycles; and denaturing at 940C, 1 minute; annealing at 57 C, 3 minutes; extension at 720C, 5 minutes for 20 cycles.
After completion of the first round of amplification, 5 ml of the reaction was added to an amplification reaction buffer containing a second set of primers: a partially nested oligomer ES12 and the same 3' end primer ES15 (O.S mX each).
Primer ES 12 has the following sequence
(SEQ ID NO:8:):
CGAATTCTGT GAGGGCTGCA ARGSCTTCTT C 31 wherein: R represents A or G; and
S represents Cor G.
The second round of amplification was performed with the same program used for the first amplification cycles. The amplification products were separated on 5% polyacrylamide gel and stained by ethidium bromide. The DNA products were isolated from the gel, phosphorylated by T4 polynucleotide kinase and cloned into PUC 18 vector by blunt end ligation. Clones were identified by digestion of mini-prep plasmid DNA with PvuII enzyme. The DNA insert was analyzed by double-stranded DNA sequencing by the dideoxy termination method using sequenase enzyme kit (United States Biohemicals).
This amplification produced two major DNA fragments of 270 bp and 320, respectively,
EXAMPLE 2
Cloning and Sequencing of cDNA
A human oligo-dT cDNA library was constructed from osteosarcoma SAOS-2/BlO cells in ggtll using the Lambda Librarian cloning kit (Invitrogen Corp.). Several positive clones were identified by plaque screening with the [32P] labeled DNA 'probe of the cloned amplified product (NUCI). The hybridization conditions were as described by A Schmidt, et al., J Biol Chem 259:7411-7415 (1984). The cDNA inserts were cloned into EcoRI site of the cloning vector PUCKS. The complete DNA sequence of both strands was determined by the dideoxy sequencing method using a series of oligonucleotides synthesized as the DNA sequence data became available.
The fragments from PCR amplification were cloned into plasmids and sequenced. The amino acid residues predicted by the DNA sequences, indicated that both DNA fragments may code for genuine and novel receptors belonging to the steroid hormone superfamily. To obtain the complete cDNA clone the amplified cDNA fragment of 320 bp NUCI was used for the screening of a human osteosarcoma SAOS-2/B10 cells cDNA library. All the highly positive clones were identical and matched the sequence for the amplified NUCI DNA fragment.
EXAMPLE 3 Northern Blot analysis
RNA from various tissues or the listed cell lines were prepared by using guanidine thiocyanate or by the guanidine hydrochloride method (GGA Nemeth, et
al., Anal Biochem 183:301-304 (1989) ; JM Chirgwin, et al., Biochemistry, 18:5294-5299 (1979)), RNA samples were analyzed by formaldehyde agarose gel electrophoresis as described by (KM Rosen, et
Focus 12:23-24 (1990)). The RNA was transferred by blotting to N-Hybond (Amersham Corp.). and hybridized with 32P-labeled eDNA of NUCI as deacribed by (A Schmidt, et al., J Biol Chem 259 : 7411-7415 (1984) ; KM Rosen, et al., Focus 12:23-24 (1990)).
Analysis of the deduced amino acid sequence revealed a long open reading frame, which starts at the putative initiation methionine codon, at nucleotide 338 and codes for a protein of 441 amino acid residues. The putative protein contains a cystein-rich region at the amino terminus, which mostprobably represents the conserved DNA-binding domain of this receptor. The carboxyl terminus of the protein.contains conserved amino acid residues, which may be part of a ligand binding domain (JM
Berg, et al., Cell 57:1065-1068 ; RM Evans Science 240:899-895 et al., M Beato, Cell S6;335-344 (1989)).The DNA binding region containS eleven cystein residues, compared to nine residues in other steroid receptors, suggesting the potential formation of an alternative Zn±dependent "finger" structure(s). As in the peroxisome proliferator activator receptor (I Issemann et L., Nature 347:645-650) (1990)), the conserved loop structure of the second Zn+ "finger" (cys to cys 115) contains only three amino acid residues, compared to five in other members of the receptor family.
Comparison of the amino acid sequence of
NUCI with that of other members of the nuclear receptor family revealed best overall similarity (627.) to the peroxisome proliferator activator receptor (I Issemann, Nature 347:645-650 (1990)).
The highest similarity is in the putative DNA binding domain, where 86% of the 66 amino acid residues are identical. The ligand binding domain of NUCI also exhibits close similarity with the corresponding region of the peroxisome proliferator activator receptor. These domains share 71% of the amino acid residues. Outside these two regions, the similarity between NUCI and the peroxisome proliferator activator receptor is lower. While 57% of the amino acid residues in the region between the DNA and the ligand binding domains are identical, in the N-turminal portion only 27% are the same. Although the similarity between NUCI and the peroxisome proliferator activator receptor it substantial, it is much lower than the similarity among members of the retinoic acid receptor family (RM Evans, Science 240:899-895 (1988). NUCI showed the next best similarity with members of this subfamily, the retinoic acid/thyroid roceptors including RARa, RXRa, thyroid hormone receptor or vitamin D receptor (DJ mangelsdorf, Nature 345:224-229 (1990).
M Petkovich, et al., Nature 330: 444-450 (1987) ;
V Giuere, et al., Nature 330 : 624-629 (1987) ; C Weinberger, ., Nature 324: 642-646 (1985); AR Baker, et al., Proc. Natal. Acad. Sci, USA 85;3294-3298 (1988)). The DNA binding domains are 58-627. identical and the ligand binding domains exhibit a similarity in the range of 29-31%.
Regarding the PCR procedure, it is of interest that sequence analysis of the amplified DNA products revealed that the primer ES12 was incorporated at both the 5' and the 3' ends. At the 3' end the primer only partially matched the actual cDNA sequence. Thus, the amplification was the product of a single primer. Surprisingly, the nucleotide at the 3' end of primer ES12 did not match at all the cDNA sequence. It is not clear whether primer ES12 was incorporated into the DNA fragments at the the 3' end at the beginning of the amplification process or only during late amplification cycles.
Expression of NUCI mRN Northern analysis with a NUCt cDNA probe revealed that NUCI receptor mRNA is expressed as a 4.0 Kb transcript in the human osteosarcoma
SAOS-2/B10 cells. In mice and rats the transcript for NUCI is about 500 nucleotides shorter than that observed in human cells and baboon tissues. In mature rats we found high levels of NUCI expression in heart, lung and kidney. About five-fold lower levels were found in skin and bone RNA prepared from tibia. High expression levels were observed also in spleen and ovaries.
EXAMPLE 4
Ligand Screeing Assay
The hybrid receptor GR-NUCI was prepared by inserting a shot site into the NUCI DNA sequence by the polymerase chain reaction method (I Issemann, et. al., Nature 347:645-650 (1990)). In NUCI receptor, introducing the Xho T site resulted in a substitution of residues ser139 and his 140 to leu139 and glu140. The DNA binding domain of the mouse glucocorticoid receptor, which has a kho I site at the amino acid residues leu495 and glu496 was obtained. The chimeric receptor pJ3GR/NUCl was prepared by ligation of the DNA coding. for amino terminal of the glucocorticoid receptor to the DNA coding for the ligand binding region-of NUCI at the
Xho I site.The cDNA molecules of the human NUCI receptor (pJ3NUCI) and the native mouse glucocorticoid receptor (pSV2wrec) cloned by Ringold (M Danielsen, et al., EMBO J 5:2513-2525 (1985)) were expressed under the control of SV40 base expression vectors (R. White, et al., Mol Endo 1:735-745 (1987)). The reporter gene was pJA358 plasmid in which the expression of the firefly luciferase cDNA was controlled by a modified MMTV promoter. This promoter is regulated by the dimer form of the GRE hormone response element.
Transient transfection assays of COS cells were based on described protocols (t Issemann, et al., Nature 347:645-650 (1990); DJ Nangelsdorf, et al., Nature 345:224-229 (1990) ; CM Gorman, et al., flol Cell Biol 2:2044-1051 (1982)). Cells were plated (l.5xlO 5 in 1 ml) into 12 well dishes in phenols red-free medium supplemented with activated charcoal treated fetal calf' serum. The next day 0.1 ml of DNA (a mixture of S mg receptor DNA and 5 mg reporter plasmid), as a calcium phosphate precipitate, was added to each well of cells.
Ligands were added to the cells 30 minutes after transfection. The next day (18 hours), the cells were washed and fresh ligands were added. Twenty-four hours later cell extracts were prepared and assayed for the luciferase enzyme activity according to the instructions supplied in the luciferase assay system (Promega,). The samples were read in the Auto
Clinilumat, Berthold. Each transfection was performed in triplicates and each sample was read three times.
The values varied by less than 5%.
Receptor activation
To search for a putative ligand for the NUCI receptor, the fact that the ligand binding domains of the various -recept,ors of the steroid superfamily can be interchanged to form chimeric receptors was employed. These hybrid receptors are capable of exhibiting ligand-dependent transcription activation of a heterologous responsive DNA sequence (I
Issemann, et al., Nature 347:645-650 (1990);
S Greene, et al., Nature 325:75-78 (1987) ;
NJG Wbster, et al., Cell 54:199-207 (1988)).
A chimeric receptor for NUCI (pJ3GR/NUCI) was prepared. The amino acid terminal, which included the DNA binding domain of the mouse glucocorticoid receptor (mGR) (M Danielsen, EMBO J 5:2513-2525 1986),.was fused to the ligand binding domain of NUCI in the manner described for PPAR (I issemann, et al., Nature 347:645-650 (1990)). The plasmid pJA358 that contains the luciferase gene under the control of the modified MNTV promoter was used as the reporter of the transcription unit.The screening assay was performed by transient tranfection of COS cells as previously described (I Issemann, al., Nat 347:645-650 (1990); DJ Mangelsdorf et al.,
Nature 345:224-229 (1990); CM Gorman, et
Mol Cell Biol 2:20440-1051 (1982)). Based on the similarity of NUCI receptor to the PPAR, we tested if the Wy-14643 molecule could activate the NUCI hybrid receptor. We found that this compound, at a concentration of 100 mM, stimulates luciferase levels about four-fold. No-increase of luciferase activity was observed when the WY-14643 was added to either cells expressing the unaltered mGR or NUCI receptors. The low activation level may be attributed to the fact that the chimeric receptor was constitutively activated in the absence of exogenous ligands. We observed a five-fold higher level of luciferase enzyme in cells transfected with the hybrid receptor than in cells expressing the NUCI receptor itself or the native mGR. Similar observations were found in experiments where the ligand binding domain of NUCI was fused to the estrogen receptor or RXR. No transcription activation mediated by the hybrid receptor was found with any of the other ligands tested,- including 1,25-vitamin D3, 24,25-(OH2) vitamin D3, tyroxine, estrogen, vitamin E, retinoic acid, dexamethasone, progesterone, androgen and other putative commercially available ligands at concentrations of 1-10 mM.
While the foregoing specifications teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the casual variations, adaptations, modifications, deletions, or additions of procedures and protocols described herein, as come within the scope of the following claims and it equivalents.
SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPLICANT: Schmidt, A.
Rodan, G.A.
Rutledge, s.J.
Vogel, R. L.
(ii) TITLE OF INVENTION : HUMAN STEROID HORMONE
RECEPTOR NUCI
(iii) UMBER OF SEQUENCES 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co., Inc.
(B) STREET: P.O. Box 2000
(C) CITY: Rahway
(D) STATE: New Jersey
(E) COUNTRY : US
(F) ZIP: 07065-Q907 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE : PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA :
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION :
(viii) ATTORNE?/AGENT INFORMATION:
(A) NAME: Thies, J.Eric
(B) REGISTRATION NUMBER: p-35,382 (C) REFERENCE/DOCKET NUMBER: 18574
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE': (908)594-3904 (B) TELEFAX : (908)594-4720
(C) TELEX : 138825 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTIOS :
(A) LENGTH : 3294 base pains
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D).TOPOLOGY : linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAATTCTGCG GAGCCTGCGG GACGGCGGCG GGTTGGCCCG TAGGCAGCCG GGACAGTGTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CAC4CAGTGG CTTCTGCTCA CCAACAGATG .120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG 180
ATACCCCTGG AAAACTGAAG CCCGTGGAGC AATGATCTCT ACAGGACTGC TTCAAGGCTG 240
ATGGGAACCA CCCTGTAGAG GTCCATCTGC GTTCAGACCC AGACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTGTGGCG CCGAGGGGAG ATCAGCCATG GAGCAGCCAC AGGAGGAAGC 360
CCCTGAGGTC CGGGAAGAGG AGGAGAAAGA GGAAGTGGCA GAGGCAGAAG GAGCCCCAGA 420
GCTCAATGGG GGACCACAGC ATGCACTTCC TTCCAGCAGC TACACAGACC TCTCCCGGAG 480
CTCCTCGCCA CCCTCACTGC TGGACCAACT GCAGATGGGC TGTGACGGGG CCTCATGCGG 540
CAGCCTCAAC ATGGAGTGCC GGGTGTGCGG GGACAAGGCA TCGGGCTTCC ACTACGGTGT 600
TCATGCATGT GAGGGGTGCA AGGGCTTCTT CCGTCGTACG ATCCGCATGA AGCTGGAGTA 660
CGAGAAGTGT GAGCGCAGCT GCAAGATTCA GAAGAAGAAC CGCAACAAGT GCCAGTACTG 720
CCGCTTCCAG AAGTGCCTGG CACTGGGCAT GTCACACAAC GCTATCCGTT TTGGTCGGAT 780
GCCGGAGGCT GAGAAGAGGA AGCTGGTGGC AGGGCTGACT GCAAACGAGG GGAGCCAGTA 840
CAACCCACAG GTGGCCGACC TGAAGGCCTT CTCCAAGCAC ATCTACAATG CCTACCTGAA 900
AAACTTCAAC ATGACCAAAA AGAAGGCCCG CAGCATCCTC ACCGGCAAAG CCAGCCACAC 960
GGCGCCCTTT GTGATCCACG ACATCGAGAC ATTGTGGCAG GCAGAGAAGG GGCTGGTGTG 1020
GAAGCAGTTG GTGAATGGCC TGCCTCCCTA CAAGGAGATC AGCGTGCACG TCTTCTACCG 1080
CTGCCAGTGC ACCACAGTGG AGACCGTGCG GGAGCTCACT GAGTTCGCCA AGAGCATCCC 1140
CAGCTTCAGC AGCCTCTTCC TCAACGACCA GGTTACCCTT CTCAAGTATC CCGTGCACGA 1200
GGCCATCTTC GCCATGCTGG CCTCTATCGT CAACAAGGAC GGGCTGCTGG TAGCCAACGG 1260
CAGTGGCTTT GTCACCCGTG AGTTCCTGCG CAGCCTCCGC AAACCCTTCA GTGATATCAT 1320
TGAGCCTAAG TTTGAATTTG CTGTCAAGTT CAACGCCCTG GAACTTGATG ACAGTGACCT 1380
GGCCCTATTC ATTGCGGCCA TCATTCTGTG TGGAGACCGG CCAGGCCTCA TGAACGTTCC 1440
ACGGGTGCAG GCTATCCAGG ACACCATCCT GCGTGCCCTC GAATTCCACC TGCAGGCCAA 1500
CCACCCTGAT GCCCAGTACC TCTTCCCCAA GCTGCTGCAG AAGATGGCTG ACCTGCGGCA 1560
ACTGGTCACC GAGCACGCCC AGATGATGCA GCGGATCAAG AAGACCGAAA CCGAGACCTC 1620
GCTGCACCCT CTGCTCCAGG AGATCTACAA GGACATGTAC TAACGGCGGC ACCCAGGCCT 1680
CCCTGCAGAC TCCAATGGGG CCAGCACTGG AGGGGCCCAC CCACATGACT TTTCCATTGA 1740
CCAGCTCTCT TCCTGTCTTT GTTGTCTCCC TCTTTCTCAG TTCCTCTTTC TTTTCTAATT 1800
CCTGTTGCTC TGTTTCTTCC TTTCTGTAGG TTTCTCTCTT CCCTTCTCCC TTCTCCCTTG 1860
CCCTCCCTTT CTCTCTCCTA TCCCCACGTC TGTCCTCCTT TCTTATTCTG TGAGATGTTT 1920
TGTATTATTT CACCAGCAGC ATAGAACAGG ACCTCTGCTT TTGCACACCT TTTCCCCAGG 1980
AGCAGAAGAG AGTGGGCCTG CCCTCTGCCC CATCATTGCA CCTGCAGGCT TAGGTCCTCA 2040
CTTCTGTCTC CTGTCTTCAG AGCAAAAGAC TTGAGCCATC CAAAGAAACA CTAAGCTCTC 2100
TGGGCCTGGG TTCCAGGGAA GGCTAAGCAT GGCCTGGACT GACTGCAGCC CCCTATAGTC 2160
ATGGGGTCCC TGCTGCAAAG GACAGTGGCA GACCCCGGCA GTAGAGCCGA GATGCCTCCC 2220
CAAGACTGTC ATTGCCCCTC CGATCGTGAG GCCACCCACT GACCCAATGA TCCTCTCCAG 2280
CAGCACACCT CAGCCCCACT GACACCCAGT GTCCTTCCAT CTTCACACTG GTTTGCCAGG 2340
CCAATGTTGC TGATGGCCCC TCCAGCACAC ACACATAAGC ACTGAAATCA CTTTACCTGC 2400
AGGCACCATG CACCTCCCTT CCCTCCCTGA GGCAGGTGAG AACCCAGAGA GAGGGGCCTG 2460
CAGGTGAGCA GGCAGGGCTG GGCCAGGTCT CCGGGGAGGC AGGGGTCCTG CAGGTCCTGG 2520
TGGGTCAGCC CAGCACCTCG CCCAGTGGGA GCTTCCCGGG ATAAACTGAG CCTGTTCATT 2580
CTGATGTCCA TTTGTCCCAA TAGCTCTACT GCCCTCCCCT TCCCCTTTAC TCAGCCCAGC 2640
TGGCCACCTA GAAGTCTCCC TGCACAGCCT CTAGTGTCCG GGGACCTTGT GGGACCAGTC 2700
CCACACCGCT GGTCCCTGCC CTCCCCTGCT CCCAGGTTGA GGTGCGCTCA CCTCAGAGCA 2760
GGGCCAAAGC ACAGCTGGGC ATGCCATGTC TGAGCGGCGC AGAGCCCTCC AGGCCTGCAG 2820
GGGCAAGGGG CTGGCTGGAG TCTCAGAGCA CAGAGGTAGG AGAACTGGGG TTCAAGCCCA 2880
GGCTTCCTGG GTCCTGCCTG GTCCTCCCTC CCAAGGAGCC ATTCTATGTG ACTCTGGGTG 2940
GAAGTGCCCA GCCCCTGCCT GACGGGATCA CTCTCTGCTG GCAGGATTCT TCCCGCTCCC 3000
CACCTACCCA GCTGATGGGG GTTGGGGTGC TTCTTTCAGC CAAGGCTATG AAGGGACAGC 3060
TGCTGGGACC CACCTCCCCC CTTCCCCGGC CACATGCCGC GTCCCTGCCC CCACCCGGGT 3120
CTGGTGCTGA GGATACAGCT CTTCTCAGTG TCTGAACAAT CTCCAAAATT GAAATGTATA 3180
TTTTTGCTAG GAGCCCCAGC TTCCTGTGTT TTTAATATAA ATAGTGTACA CAGACTGACG 3240
AAACTTTAAA TAAATGGGAA TTAAATATTT AAAAAAAAAA GCGGCCGCGA ATTC 3294 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 1322 base pairs
(B) T?PE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:SEQ In NO:2 :
ATGGAGCAGC CACAGGAGGA AGCCCCTGAG GTCCGGGAAG AGGAGGAGAA AGAGGAAGTG 60
GCAGAGGCAG AAGGAGCCCC AGAGCTCAAT GGGGGACCAC AGCATGCACT TCCTTCCAGC 120 AGCTACACAG ACCTCTCCCG GAGCTCCTCG CCACCCTCAC TGCTGGACCA ACTGCAGATG 180
GGCTGTGACG GGGCCTCATG CGGCAGCCTC AACATGGAGT GCCGGGTGTG CGGGGACAAC 240
GCATCGGGCT TCCACTACGG TGTTCATGCA TGTGAGGGGT GCAAGGGCTT CTTCCGTCGT 300
ACGATCCGCA TGAAGCTGGA GTACGAGAAG TGTGAGCGCA GCTGCAAGAT TCAGAAGAAG 360
AACCGCAACA AGTGCCAGTA CTGCCGCTTC CAGAAGTGCC TGGCACTGGG CATGTCACAC 420
AACGCTATCC GTTTTGGTCG GATGCCGGAG GCTGAGAAGA GGAAGCTGGT GGCAGGGCTG 480
ACTGCAAACG AGGGGAGCCA GTACAACCCA CAGGTGGCCG ACCTGAAGGC CTTCTCCAAG 540
CACATCTACA ATGCCTACCT GAAAAACTTC AACATGACCA AAAAGAAGGC CCGCAGCATC 600
CTCACCGGCA AAGCCAGCCA CACGGCGCCC TTTGTGATCC ACGACATCGA GACATTGTGG 660
CAGGCAGAGA AGGGGCTGGT GTGGAAGCAG TTGGTGAATG GCCTGCCTCC CTACAAGGAG 720
ATCAGCGTGC ACGTCTTCTA CCGCTGCCAG TGCACCACAG TGGAGACCGT GCGGGAGCTC 780
ACTGAGTTCG CCAAGAGCAT CCCCAGCTTC AGCAGCCTCT TCCTCAACGA CCAGGTTACC 840
CTTCTCAAGT ATGGGGTGCA CGAGGCCATC TTCGCCATGC TGGCCTCTAT CGTCAACAAG 900
GACGGGCTGC TGGTAGCCAA CGGCAGTGGC TTTGTCACCC GTGAGTTCCT GCGCAGCCTC 960
CGCAAACCCT TCAGTGATAT CATTGAGCCT AAGTTTGAAT TTGCTGTCAA GTTCAACGCC 1020
CTGGAACTTG ATGACAGTGA CCTGGCCCTA TTCATTGCGG CCATCATTCT GTGTGGAGAC 1080
CGGCCAGGCC TCATGAACGT TCCACGGGTG GAGGCTATCC AGGACACCAT CCTGCGTGCC 1140
CTCGAATTCC ACCTGCAGGC CAACCACCCT GATGCCCAGT ACCTCTTCCC CAAGCTGCTG 1200 CAGAAGATGG CTGACCTGCG GCAACTGGTC AOCGAGCACG CCCAGATGAT GCAGCGGATC 1260
AAGAAGACCG AAACCGAGAC CTCGCTGCAC CCTCTGCTCC AGGAGATCTA CAAGGACATG 1320
TA 1322 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CKAtACTERISTICS: (A) LENGTH : 337 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY : linear
(ii) MOLECULE TYPE : cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAATTCTGCG GAGCCTGCGG GACGGCGGCG GGTTGGCCCG TAGGCAGCCG GGACAGTGTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CACACAGTGG CTTCTGCTCA CCAACAGATG 120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG 180
ATACCCCTGG AAAACTGAAG CCCGTGGAGC AATGATCTCT ACAGGACTGC TTCAAGGCTG 240
ATGGGAACCA CCCTGTAGAC OTCOATCTGC GTTCAGACCC AGACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTOTCCCC CCGAGGGGAG ATCAGCC 337 (2) INFORMATION FOR SEQ ID NO:4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1635 base pairs
(B) TYPE? nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION SEQ ID NO:4: CTAACGGCGG CACCCAGGCC TCCCTGCAGA CTCCAATGGG GCCAGCACTG GAGGGGCCCA 60
CCCACATGAC TTTTCCATTG ACCAGCTCTC TTCCTGTCTT TGTTGTCTCC CTCTTTCTCA 120
GTTCCTCTTT CTTTTCTAAT TCCTGTTGCT CTGTTTCTTC CTTTCTGTAG GTTTCTCTCT 180
TCCCTTCTCC CTTCTCCCTT GCCCTCCCTT TCTCTCTCCT ATCCCCACGT CTOTCCTCCT 240
TTCTTATTCT GTGAGATGTT TTGTATTATT TCACCAGCAG CATAGAACAG GACCTCTGCT 300
TTTGCACACC TTTTCCCCAG GAGCAGAAGA GAGTGGGCCT GCCCTCTGCC CCATCATTGC 360
ACCTGCAGGC TTAGGTCCTC ACTTCTGTCT CCTGTCTTCA GAGCAAAAGA CTTGAGCCAT 420
CCAAAGAAAC ACTAAGCTCT CTGGGCCTGG GTTCCAGGGA AGGCTAAGCA TGGCCTGGAC 480
TGACTGCAGC CCCCTATAGT CATGGGGTCC CTGCTGCAAA GGACAGTGGC AGACCCCGGC 540 AGTAGAGCCG AGATGCCTCC CCAAGACTGT CATTGCCCCT CCGATCGTGA GGCCACCCAC 600
TGACCCAATG ATCCTCTCCA GCAGCACACC TCAGCCCCAC TGACACCCAG TGTCCTTCCA 660
TCTTCACAGT GGTTTGCCAG GCCAATGTTG CTGATGGCCC CTCCAGCACA CACACATAAG 720
CACTGAAATC ACTTTACCTG CAGGCACCAT GCACCTCCCT TCCCTCCCTG AGGCAGGTGA 780
GAACCCAGAG AGAGGGGCCT GCAGGTGAGC AGGCAGGGCT GGGCCAGGTC TCCGGGGAGG 840
CAGGGGTCCT GCAGGTCCTG GTGGGTCAGC CCAGCACCTC GCCCAGTGGG AGCTTCCCGG 900
GATAAACTGA GCCTGTTCAT TCTGATGTCC ATTTGTCCCA ATAGCTCTAC TGCCCTCCCC 960
TTCCCCTTTA CTCAGCCCAG CTGGCCACCT AGAACTCTCC CTGCACAGCC TCTAGTGTCC 1020
GGGGACCTTG TGGGACCAGT CCCACACGGC TGGTCCCTGC CCTCCCCTGC TCCCAGGTTG 1080
AGGTGCGCTC ACCTCAGAGC AGGGCCAAAG CACAGCTGGG CATGCCATGT CTGAGCGGCG 1140
CAGAGCCCTC CAGGCCTGCA GGGGCAAGGG GCTGGCTGGA GTCTCAGAGC ACAGAGGTAG 1200
GAGAACTGGG GTTCAAGCCC AGGCTTCCTG GGTCCTGCCT GGTCCTCCCT CCCAAGGAGC 1260
CATTCTATGT GACTCTGGGT GGAAGTGCCC AGCCCCTGCC TGACGGGATC ACTCTCTGCT 1320
GGCAGGATTC TTCCCGCTCC CCACCTACCC AGCTGATGGG GGTTGGGGTG CTTCTTTCAG 1380 CCAAGGCTAT GAAGGGACAG CTGCTGGGAC CCACCTCCCC CCTTCCCCGG CCACATGCCG 1440
CGTCCCTGCC CGCACCCGGG TCTGGTGCTG AGGATACAGC TCTTCTCAGT GTCTGAACAA 1500
TCTCCAAAAT TGAAATGTAT ATTTTTGCTA GGAGCCCCAG CTTCCTGTGT TTTTAATATA 1560
AATAGTGTAC ACAGACTGAC GAAACTTTAA ATAAATGGGA ATTAAATATT TAAAAAAAAA 1620
AGCGGCCGCG AATTC 1635 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 441 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION :SEQ ID NO : 5 :
Met Glu Gln Pro Gln Glu Glu Ala Pro Glu Val Arg Glu Glu Glu Glu 1 5 10 15
Lys Glu Glu Val Ala Glu Ala Glu Gly Ala Pro Glu Leu Asn Gly Gly
20 25 30
Pro Gln His Ala Leu Pro Ser Ser Ser Tyr Thr Asp Leu Ser Arg Ser
35 40 45
Ser Ser Pro Pro Ser Leu Leu Asp Gln Leu Gln Met Gly Cys Asp Gly
50 55 60
Ala Ser Cys Gly Ser Leu Asn Met Glu Cys Arg Val Cys Cly Asp Lys 65 70 75 80
Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly
85 90 95
Phe Phe Arg Arg Thr Ile Arg Met Lys Leu Glu Tyr Glu Lys Cys Glu
'100 105 110
Arg Ser Cys Lys Ile Gin Lys Lys Asn Arg Asn Lys Cys Gin Tyr Cys
115 120 125
Arg Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg
130 135 140
Phe Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Leu 145 150 155 160
Thr Ala Asn Glu Gly Ser Gln Tyr Asn Pro Gln Val Ala Asp Leu Lys
165 170 175
Ala Phe Ser Lys His Ile Tyr Asn Ala Tyr Leu Lys Asn Phe Asn Met
180 185 190
Thr Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ala Ser His Thr
195 200 205
Ala Pro Phe Val Ile His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys
210 215 220
Gly Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu 225 ' 230 235 240
lie Ser Val His Val Phe Tyr Arg Cys Gln Cys Thr Thr Val Glu Thr
245 250 255
Val Arg Glu Leu Thr Glu Phe Ala Lys Ser Ile Pro Ser Phe Ser Ser
260 265 270
Leu Phe Leu Asn Asp Gin Val Thr Leu Leu Lys Tyr Gly Val His Glu
275 280 285
Ala Ile Phe Ala Met Leu Ala Ser Ile Val Asn Lys Asp Gly Leu Leu
290 295 300
Val Ala Asn Gly Ser Gly Phe Val Thr Arg Glu Phe Leu Arg Ser Leu
305 310 315 320
Arg Lys Pro Phe Ser Asp lie lie Glu Pro Lys Phe Glu Phe Ala Val
325 330 335
Lys Phe Asn Ala Leu Glu Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile
340 345 350
Ala Ala Ile Ile Leu CYs Gly Asp Arg Pro Gly Leo Met Asn Val Pro 355 360 365
Arg Val Glu Ala Ile Gln Asp Thr lie Leu Arg Ala Leu Glu Phe His
370 375 380
Leu Gln Ala Asn His Pro Asp Ala Gin Tyr Leu Phe Pro Lys Len Leu
385 390 395 400
Gin Lys Met Ala Asp Leu Arg Gln Leu Val Thr Glu His Ala Gln Met
405 410 415
Met Gln Arg Ile Lys Lys Thr glu Thr Glu Thr Ser Leu His Pro Leu
420 425 430
Leu Gln Glu Ile Tyr Lys Asp Met Tyr
435 440 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY : linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ XD NO:6:.
CGAATTCTGT GAGGGCTGGA ARGSC 25 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 31 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CGAATTCTGT GAGGGCTGCA ARGSCTTCTT C 31 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GGAATTCRAA NCCNGGNANN NNYTTNGCRA A 31
Claims (20)
1. A human steroid receptor NUCI, the receptor being substantially free of other human receptor proteins.
2. The human steroid receptor NUCI of Claim 1, the receptor being free of other human proteins.
3. . The human steroid receptor NUCI of Claim 2, the receptor being a recombinantly produced receptor from human cells.
4. A protein corresponding to the amino acid sequence of human steroid receptor NUCT, the protein comprising 441 amino acids.
5. The protein of Claim 4 comprising the amino acid sequence (SEQ ID NO:5:) which is:
Met Glu Gln Pro Gln Glu Glu Ala Pro Glu Val Arg Glu Glu Glu Glu
1 5 10 15
Lys Glu Glu Val Ala Gin Ala Glu Gly Ala Pro Glu Leu Asn Gly Gly
20 25 30
Pro Gln His Ala Leu Pro Ser Ser Ser Tyr Thr Asp Leu Ser Arg Ser
35 40 45
Ser Ser Pro Pro Ser Leu Leu Asp Gin Leu Gln Met Gly Cys Asp Gly
SO 55 60
Ala Ser Cys Gly Ser Leu Asn Met Glu Cys Arg Vel Cys Gly Asp Lys
65 70 75 80
Ala Ser Gly Phe His Tyr Gly Val His Ala Cys Glu Gly Cys Lys Gly
85 90 95
Phe Phe Arg Arg Thr Ile ARg Met Lys Leu Glu Tyr Glu Lys Cys Glu
100 105 110
Arg Ser Cys Lys Ile Gin Lys Lys Asn Arg Asn Lys Cys Gln Tyr Cys
115 120 125
Arg Phe Gln Lys Cys Leu Ala Leu Gly Met Ser His Asn Ala Ile Arg
130 135 140
Phe Gly Arg Met Pro Glu Ala Glu Lys Arg Lys Leu Val Ala Gly Len 145 150 155 160 Thr Ala Asn Glu Gly Ser Gin Tyr Asn Pro Gln Val Ala Asp Leu Lys 165 170 175
Ala Phe Ser Lys His Ile 5r Asn Ala Tyr Leu Lys Asn Phe Asn Met
180 185 190
Thr Lys Lys Lys Ala Arg Ser Ile Leu Thr Gly Lys Ala Ser His Thr
195 200 205
Ala Pro Phe Val lie His Asp Ile Glu Thr Leu Trp Gln Ala Glu Lys
210 ' 215 220
Gly Leu Val Trp Lys Gln Leu Val Asn Gly Leu Pro Pro Tyr Lys Glu 225 230 235 240
Ile Ser Val His Val Phe Tyr Arg Cys Cin Cys Thr Thr Val Glu Thr
245 250 255
Val Arg Glu Leu Thr Glu Phe Ala tys Ser Ile Pro Ser Phe Ser Ser
260 265 270
Leu Phe Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly Val His Glu
275 280 285
Ala lie Pbe Ala Met Leu Ala Ser lie Val Asn Lys Asp Gly Leu Leu
290 295 300 Val Ala Asn Gly Ser Gly Phe Val Thr Arg Glu Phe Leu Arg Ser Leu 305 310 315 320
Arg Lys Pro Phe Ser Asp Ile Ile Glu Pro Lys Phe Glu Phe Ala Val
325 330 335
Lys Phe Asn Ala Leu Gin Leu Asp Asp Ser Asp Leu Ala Leu Phe Ile
340 345 350
Ala Ala Ile Ile Leu Cys Gly Asp Arg Pro Gly Leu Met Asn Val Pro
355 360 365
Arg Val Glu Ala lie Gln Asp Thr Ile Leu Arg Ala Leu Gin Phe His
370 375 380
Leu Gin Ala Asn His Pro Asp Ala Gin Tyr Leu Phe Pro Lys Leu Leu
385 390 395 400
Gln Lys Met Ala Asp Leu Arg Gin Leu Vai Thr Glu His Ala Gln Met
405 410 415
Met Gin Arg Ile Lys Lys Thr Glu Thr Glu Thr Ser Leu His Pro Leu
420 425 430
Leu Gln Glu Ile Tyr Lys Asp Met Tyr
435 440
6.A DNA sequence encoding human steroid receptor NUCT, the sequence being free of other human DNA sequences.
7. The DNA sequence of Claim 6 comprising the sequence (SEQ ID NO:1:) which is:
GAATTCTGCG GAGCCTGCGG GCGGCGGCC GGTTGGCCCG TAGGCAGCCG GGACAGTGTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CACACAGTGG CTTCTGCTCA CCAACAGATG 120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG 180
ATACCCCTGG AAAACTGAAG CCCGTGGAGC AATATCTCT ACAGGACTGC TTCAAGGCTG 240
ATGGGAACCA CCCTGTAGAG GTCCATCTGC GTTCAGACCC AGACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTGTGGCG CCGAGGGGAG ATCAGCCATG GAGCAGCCAC AGGAGGAAGC 360
CCCTGAGGTC CGGGAAGAGG AGGAGAAAGA GGAAGTGGCA GAGGCAGAAG GACCCCCAGA 420
GCTCAATGGG GGACCACAGC ATGCACTTCC TTCCAGCAGC TACACAGACC TCTCCCGGAG 480
CTCCTCGCCA CCCTCACTGC TGGACCAACT GCAGATGGGC TGTGACGGGG CCTCATGOGG 540
CAGCCTCAAC ATGGAGTGCC GGGTGTGCGG GGACAAGGCA TCGGGCTTCC ACTACGGTGT 600
TCATGCATGT GAGGGGTGCA AGGGCTTCTT CCGTCGTACG ATCCGCATGA AGCTGGAGTA 660
CGAGAAGTGT GAGCGCAGCT GCAAGATTCA GAAGAAGAAC CGCAACAAGT GCCAGTACTG 720
CCGCTTCCAG AAGTGCCTGG CACTGGGCAT GTCACACAAC GCTATCCGTT TTGGTCGGAT 780
GCCGGAGGCT GAGAAGAGGA AGCTGGTGGC AGGGCTGACT GCAAACCAGG GGAGCCAGTA 840
CAACCCACAG GTGGCCGACC TGAAGGCCTT CTCCAAGCAC ATCTACAATG CCTACCTGAA 900
AAACTTCAAC ATGACCAAAA AGAAGGCCCG CAGCATCCTC ACCGGCAAAG CCAGCCACAC 960
GGCGCCCTTT GTGATCCACG ACATCGAGAC ATTGTGGCAG GCAGAGAAGG GGCTGGTGTG 1020
GAAGCAGTTG GTGAATGGCC TGCCTCCCTA CAAGGAGATC AGCGTGCACG TCTTCTACCG 1080
CTGCCAGTGC ACCACAGTGG AGACCGTGCG GGAGCTCACT GAGTTCGCCA AGAGCATCCC 1140
CAGCTTCAGC AGCCTCTTCC TCAACGACCA GGTTACCCTT CTCAAGTATG GCGTGCACGA 1200
GGCCATCTTC GCCATGCTGG CCTCTATCGT CAACAAGGAC GGGCTGCTGG TAGCCAACGG 1260
CAGTGGCTTT GTCACCCGTG AGTTCCTGCG CAGCCTCCGC AAACCCTTCA GTGATATCAT 1320
TGAGCCTAAG TTTGAATTTG CTGTCAAGTT CAACGCCCTG GAACTTGATG ACAGTGACCT 1380
GGCCCTATTC ATTGCGGCCA TCATTCTGTG TGGAGACCGG CCAGGCCTCA TGAACGTTCC 1440
ACGGGTGGAG GCTATCCAGG ACACCATCCT GCGTGCCCTC GAATTCCACC TGCAGGCCAA 1500
CCACCCTGAT GCCCAGTACC TCTTCCCCAA GCTGCTGCAG AAGATGGCTG ACCTGCGGCA 1560
ACTGGTCACC GAGCACGCCC AGATGATGCA GCGGATCAAG AAGACCGAAA CCGAGACCTC 1620
GCTGCACCCT CTGCTCCAGG AGATCTACAA GGACATGTAC TAACGGCGGC ACCCAGGCCT 1680
CCCTGCAGAC TCCAATGGGG CCAGCACTGG AGGGGCCCAC CCACATGACT TTTCCATTGA 1740
CCAGCTCTCT TCCTGTCTTT GTTGTCTCCC TCTTTCTCAG TTCCTCTTTC TTTTCTAATT 1800
CCTGTTGCTC TGTTTCTTCC TTTCTGTAGG TTTCTCTCTT CCCTTCTCCC TTCTCCCTTG 1860
CCCTCCCTTT CTCTCTCCTA TCCCOACGTC TGTOOTCCTT TCTTATTCTG TGAGATGTTI 1920
TGTATTATTT CACCAGCAGC ATAGAACAGG ACCTCTGCTT TTGCACACCT TTTCCCCAGG 1980
AGCAGAAGAG AGTOGOCCTG CCCTCTGCCC CATCATTGCA CCTGCAGGCT TAGGTCCTCA 2040
CTTCTGTCTC CTGTCTTCAG AGCAAAAGAC TTGAGCCATC CAAAGAAACA CTAAGCTCTC 2100
TGGGCCTGGG TTCCAGGGAA GGCTAAGCAT GGCCTGGACT GACTGCAGCC CCCTATAGTC 2160
ATGGGGTCCC TGCTGCAAAG GACAGTGGCA GACCCCGGCA GTAGAGCCGA GATGCCTCCC 2220
CAAGACTGTC ATTGCCCCTC CGATCGTGAG GCCACCCACT GACCCAATGA TCCTCTCCAG 2280
CAGCACACCT CACOCOCACT CACACCCAGT GTCCTTCCAT CTTCACACTG GTTTGCCAGG 2340
CCAATGTTGC TGATGGCCCC TCCAGCACAC ACACATAAGC ACTGAAATCA CRTTACCTGC 2400
AGGCACCATG CACCTCCCTT CCCTCCCTGA GGCAGGTGAG AACCCAGAGA GAGGGGCCTG 2460
CAGGTGAGCA GGCAGGGCTG GGCCAGGTCT CCGGGGAGGC AGGGGTCCTG GAGGTCCTGG 2520
TGGGTCAGCC CAGCACCTCC CCCAGTGGGA GCTTCCCGGG ATAAACTGAG CCTGTTCATT 2580
CTGATGTCCA TTTGTCOCAA TAGCTCTACT GCCCTCCCCT TCCCCTTTAC TCACCCCACC 2640
TGGCCACCTA GAAGTCTCCC TGCACAGCCT CTAGTGTCCC GGGACCTTGT GGGACCAGTC 2700
CCACACCGCT GGTCCCTGCC CTCCCCTGCT CCCAGGTTGA GGTGCGCTCA CCTCAGAGCA 2760
GGGCCAAAGC ACAGCTGGGC ATGCCATGTC TGAGCGGCGC AGAGCCCTCC AGGGCTGCAG 2820
GGGCAAGGGG CTGGCTGGAG TCTCAGAGCA CAGAGGTAGG AGAACTGGGG TTCAAGCCCA 2880
GGCTTCCTGG GTCCTGCCTG GTCCTCCCTC CCAAGGAGCC ATTCTATGTG ACTCTGGGTG 2940
GAAGTGCCCA GCCCCTGCCT GACGGGATCA CTCTCTGCTG GCAGGATTCT TCCCGCTCCC 3000
CACCTACCCA GCTGATGGGG GTTGGGGTGC TTCTTTCAGC CAAGGCTATG AAGGGACAGC 3060
TGCTGGGACC CACCTCCCCC CTTCCCCGGC CACATGCCGC GTCCCTGCCC CCACCCGGGT 3120
CTGGTGCTGA GGATACAGCT CTTCTCAGTG TCTGAACAAT CTCCAAAATT GAAATGTATA 3180
TTTTTGCTAG GAGCCCCAGC TTCCTGTGTT TTTAATATAA ATAGTGTACA CAGACTGACG 3240
AAACTTTAAA TAAATGGGAA TTAAATATTT AAAAAAAAAA GCGGCCGCGA ATTC 3294 or a degenerate variation thereof.
8. The DNA sequence of Claim 6 comprising the sequence (SEQ ID NO:2:) which is:
ATGGAGCAGC CACAGGAGGA AGCCCCTGAG GTCCGGGAAG AGGAGGAGAA AGAGGAAGTG 60
GCAGAGGCAG AAGGAGCCCC AGAGCTCAAT GGGGGACCAC AGCATGCACT TCCTTCCAGC 120
AGCTACACAG ACCTCTCCCG GAGCTCCTCG CCACCCTCAC TGCTGGACCA ACTGCAGATG 180
GGCTGTGACG GGGCCTCATG CGGCAGCCTC AACATGGAGT GCCGGGTGTG CGGGGACAAG 240
GCATCGGGCT TCCACTACGG TGTTCATGCA TCTGAGGGGT GCÁGGGCTT CTTCCGTCGT 300
ACGATCCGCA TGAAGCTGGA GTACGAGAAG TGTGAGCGCA GCTGCAAGAT TCAGAAGAAG 360
AACCGCAACA AGTGCCAGTA CTGCCGCTTC CAGAAGTGCC TGGCACTGGG CATGTCACAC 420
AACGCTATCC GTTTTGGTCG GATGCCGGAG GCTGAGAAGA GGAAGCTGGT GGCAGGGCTG 480
ACTGCAAACG AGGGGAGCCA GTACAACCCA CAGGTGGCCC ACCTGAAGGC CTTCTCCAAG 540
CACATCTACA ATGCCTACCT GAAAAACTTC AACATGACCA AAAAGAAGGC CCGCAGCATC 600
CTCACCGGCA AAGCCAGCCA CACGGCGCCC TTTGTGATCC ACGACATCGA GACATTGTGG 660
CAGGCAGAGA AGGGGCTGGT GTGGAAGCAG TTGGTGAATG GCCTGCCTCC CTACAACGAG 720
ATCAGCGTGC ACGTCTTCTA CCGCTGCCAG TGCACCACAG TGGAGACCGT GCGGGAGCTC 780
ACTGAGTTCG CCAAGAGCAT CCCCAGCTTC AGCAGCCTCT TCCTCAACGA CCAGGTTACC 840 CTTCTCAAGT ATGGCGTGCA CGAGGCCATC TTCGCCATGC TGGCCTCTAT CGTCAACAAG 900
GACGGGCTGC TGGTAGCCAA CGGCAGTGGC TTTGTCACCC GTGAGTTCCT GCGCAGCCTC 960
CGCAAACCCT TCAGTGATAT CATTGAGCCT AAGTTTGAAT TTGCTGTCAA GTTCAACCCC 1020
CTGGAACTTG ATGACAGTGA CCTGGCCCTA TTCATTGCGG CCATCATTCT GTGTGGAGAC 1080
CGGCCAGGCC TCATGAACGT TCCACGGGTG GAGGCTATCC AGGACACCAT CCTGCGTGCC 1140
CTCGAATTCC ACCTGCAGGC CAACCACCCT GATGCCCAGT ACCTCTTCCC CAAGCTGCTG 1200
CAGAAGATGG CTGACCTGCG GCAACTGGTC ACCGAGCACG CCCAGATGAT GCAGCGGATC 1260 AAGAAGACCG AAACCGAGAC CTCGCTGCAC CCTCTGCTCC AGGAGATCTA CAAGGACATG 1320
TA 1322 or a degenerate variation thereof.
9. The DNA sequence of Claim 8 further comprising the sequence (SEQ ID NO:3:) which is; GAATTCTGCG GAGCCTGCGG GACGGCGGCG GGTTGGCCCG TAGGCAGCCG GGACAGTGTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CACACAGTGG CTTCTGCTCA CCAACAGATG 120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG 180
ATACCCCTGG AAAACTGAAG CCCGTGGAGC AATGATCTCT ACAGGACTGC TTCAAGGCTG 240
ATGGGAACCA CCCTGTAGAG GTCCATCTGC GTTCAGACCC AGACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTGTGGCG CCGAGGGGAG ATCAGCC 337 or a degenerate variation thereof.
10. The DNA sequence of Claim 9 further comprising the sequence (SEQ ID NO:4:) which is:
CTAACGGCGG CACCCAGGCC TCCCTGCAGA CTCCAATGGG GCCAGCACTG GAGGGGCCCA 60
CCCACATGAC TTTTCCATTG ACCAGCTCTC TTCCTGTCTT TGTTGTCTCC CTCTTTCTCA 120
GTTCCTCTTT CTTTTCTAAT TCCTGTTGCT CTGTTTCTTC CTTTCTGTAG GTTTCTCTCT 180
TCCCTTCTCC CTTCTCCCTT GCCCTCCCTT TCTCTCTCCT ATCCCCACGT CTGTCCTCCT 240
TTCTTATTCT GTGAGATGTT TTGTATTATT TCACCAGCAG CATAGAACAG GACCTCTGCT 300 TTTGCACACC TTTTCCCCAG GAGCAGAAGA GAGTGGGCCT GCCCTCTGCC CCATCATTGC 360
ACCTGGAGGC TTAGGTCCTC ACTTCTGTCT CCTGTGTTCA GAGCAAAAGA CTTGAGCCAT 420
CCAAAGAAAC ACTAAGCTCT CTGGGCCTGG GTTCCAGGGA AGGCTAAGCA TGGCCTGGAC 480
TGACTGCAGC CCCCTATAGT CATGGGGTCC CTGCTGCAAA GGACAGTGGC AGACCCCGGC 540
AGTAGAGCCG AGATGCCTCC CCAAGACTGT CATTGCCCCT CCGATCGTGA GGCCACCCAC 600
TGACCCAATG ATCCTCTCCA GCAGCACACC TCAGCCCCAC TGACACCCAG TGTCCTTCCA 660
TCTTCACACT GGTTTGCCAG GCCAATGTTG CTGATGGCCC CTCCAGCACA CACACATAAG 720
CACTGAAATC ACTTTACCTG CAGGCACCAT GCACCTCCCT TCCCTCCCTG AGGCAGGTGA 780
GAACCCAGAG AGAGGGGCCT GCAGGTGAGC AGGCAGGGCT GGGCCAGGTC TCCGGGGAGG 840
CAGGGGTCCT GCAGGTCCTG GTGGGTCAGC CCAGCACCTC GCCCAGTGGG AGCTTCCCGG 900
GATAAACTGA GCCTGTTCAT TCTGATGTCC ATTTGTCCCA ATAGCTCTAC TGCCCTCCCC 960
TTCCCCTTTA CTCAGCCCAG CTGGCCACCT AGAAGTCTCC CTGCACAGCC TCTAGTGTCC 1020
GGGGACCTTG TGGGACCAGT CCCACACCGC TGGTCCCTGC CCTCCCCTGC TCCCAGGTTG 1080
AGGTGCGCTC ACCTCAGAGC AGGGCCAAAG CACACCTGGG CATGCCATGT CTGAGCGGCG 1140
CAGAGCCCTC CAGGCCTGCA GGGGCAAGGG GCTGGCTGGA GTCTCAGAGC ACAGAGGTAG 1200
GAGAACTGGG GTTCAAGCCC AGGCTTCCTG GGTCCTGCCT GGTCCTCCCT CCCAAGGAGC 1260
CATTCTATGT GACTCTGGGT GGAAGTGCCC AGCCCCTGCC TGACGGGATC ACTCTCTGCT 1320
GGCAGGATTC TTCCCGCTCC CCACCTACCC AGCTGATGGG GGTTGGGGTG CTTCTTTCAG 1380
CCAAGGCTAT GAAGGGACAG CTGCTGGGAC CCACCTCCCC CCTTCCCCGG CCACATGCCG 1440
CGTCCCTGCC CCCACCCGGG TCTGGTGCTG AGGATACAGC TCTTCTCAGT GTCTGAACAA 1500
TCTCCAAAAT TGAAATGTAT ATTTTTGCTA GGAGCCCCAG CTTCCTGTGT TTTTAATATA 1560
AATAGTGTAC ACAGACTGAC GAAACTTTAA ATAAATGGGA ATTAAATATT TAAAAAAAAA 1620
AGCGGCCGCG AATTC 1635 or a degenerate variation thereof.
11. An expression construct which comprises:
(a) a mammalian cell vector, and
(b) a base sequence encoding human
steroid receptor NUCI protein.
12. The expression construct of Claim 11 which comprises:
(a) vector PUC18, and
(b) a base sequence encoding human steroid
receptor NUCI protein.
13. The expression construct of Claim 11 wherein the base sequence comprises the sequence (SEQ
ID NO:1:) which is:
GAATTCTGCC GAGCCTGCGG GACGGCGGCG GGTTGGCCCG TAGGCACCCG GGACAGTCTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CACACAGTGG CTTCTGCTGA CCAACAGATG 120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG 180
ATACCCCTGG AAAACTGAAG CCCGTGGACC AATGATCTCT ACAGGACTGC TTCAAGGCTG 240 ATGOCAACCA CCCTGTAGAG GTCCATCTGC GTTCAGACCC ACACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTGTGGGG CCGAGGGGAG ATGAGCCATG GAGCAGCCAC AGGAGGAAGC 360
CCCTGAGGTC CGGGAAGAGG AGGAGAAAGA GGAAGTCCCA GAGGCAGAAG GAGCCCCAGA 420
GCTCAATGGG GGACCACAGC ATGCACTTCC TTCCAGGAGC TACACAGACC TCTCCCGGAG 480
CTCCTCGCCA CCCTCACTGC TGGACCAACT GCAGATGGGC TGTGACGGGG CCTCATGCGG 540
CAGCCTCAAC ATGGAGTGCC GGGTGTGCGG GGACAAGGCA TCGGGCTTCC ACTACGGTGT 600
TCATGCATGT GAGGGGTGCA AGGGCTTCTT CCGTCGTACG ATCCGCATGA AGCTGGAGTA 660
CGAGAAGTGT GAGCGCAGCT GCAAGATTCA GAAGAAGAAC CGCAACAAGT GCCAGTACTG 720
CCGCTTCCAG AAGTGCCTGG CACTGGGCAT GTCACACAAC GCTATCCGTT TTGGTCGGAT 780
GCCGGAGGCT GAGAAGAGGA AGCTGGTGGC AGGGCTGACT GCAAACGAGG GGAGCCAGTA 840
CAACCCACAG GTGGCCGACC TGAAGGCCTT CTCCAAGCAC ATCTACAATG CCTACCTGAA 900
AAACTTCAAC ATGACCAAAA AGAAGGCCCG CAGCATCCTC ACCGGCAAAG CCAGCCACAC 960
GGCGCCCTTT GTGATCCACG ACATCGAGAC ATTGTGGCAG GCAGAGAAGG GGCTGGTGTG 1020
GAAGCAGTTG GTGAATGGCC TGCCTCCCTA CAAGGAGATC AGCGTGCACG TCTTCTACCG tO8O CTGCCAGTGC ACCACAGTGG AGACCGTGCG GGAGCTCACT GAGTTCGCCA AGAGCATCCC 1140
CAGCTTCAGC AGCCTCTTCC TCAACGACCA GGTTACCCTT CTCAAGTATG GCGTGCACGA 1200
GGCCATCTTC GCCATGCTGG CCTCTATCGT CAACAAGGAC GGGCTGCTGG TAGCCAACGG 1260
CAGTGGCTTT CTCACCCGTG AGTTCCTGCG CAGCCTCCGC AAACCCTTCA GTGATATCAT 1320
TGAGCCTAAG TTTGAATTTG CTGTCAAGTT CAACGCCCTG GAACTTGATG ACAGTGACCT 1380
GGCCCTATTC ATTGCGGCCA TCATTCTGTG TGGAGACCGG CCAGGCCTCA TGAACGTTCC 1440
ACGGGTGGAG GCTATCCAGG ACACCATCCT GCGTGCCCTC GAATTCCACC TGCAGGCCAA 1500
CCACCCTGAT GCCCAGTACC TCTTCCCCAA GCTGCTGCAG AAGATGGCTG ACCTGCGGCA 1560
ACTGGTCACC GAGCACGCCC AGATGATGCA GCGGATCAAG AAGACCCAAA CCGAGACCTC 1620
GCTGCACCCT CTGCTCCAGG AGATCTACAA GGACATGTAC TAACGGCGGC ACCCAGGCCT 1680
CCCTGCAGAC TCCAATGGGG CCAGCACTGG AGGGGCCCAC CCACATGACT TTTCCATTGA 1740
CCAGCTCTCT TCCTGTCTTT GTTGTCTCCC TCTTTCTCAG TTCCTCTTTC TTTTCTAATT 1800
CCTGTTGCTC TGTTTCTTCC TTTGTGTAGG TTTCTCTCTT CCCTTCTCCC TTCTCCCTTG 1860
CCCTCCCTTT CTCTCTCCTA TCCCCACGTC TGTCCTCCTT TCTTATTCTG TGAGATGTTT 1920
TGTATTATTT CACCAGCAGC ATAGAACAGG ACCTCTGCTT TTGCACACCT TTTCCCCAGG 1980
AGCAGAAGAG AGTGGGCCTG CCCTCTGCCC CATCATTGCA CCTGCAGGCT TAGGTCCTCA 2040
CTTCTGTCTC CTGTCTTCAG AGCAAAAGAC TTGAGCCATC CAAAGAAACA CTAAGCTCTC 2100
TGGGCCTGGG TTCCAGGGAA GGCTAAGCAT GGCCTGGACT GACTGCAGCC CCCTATAGTC 2160
ATGGGGTCCC TGCTGCAAAG GACAGTGGCA GACCCCGGCA GTAGAGCCGA GATGCCTCCC 2220
CAAGACTGTC ATTGCCCCTC OCATCGTGAG GOCACOCACT GACCCAATGA TCCTCTCOAG 2280
CAGCACACCT CAGCCCCACT GACACCCAGT GTCCTTCCAT CTTCACACTG GTTTGCCAGG 2340
CCAATGTTGC TGATGGCCCC TCCAGCACAC ACACATAAGC ACTGAAATCA CTTTACCTGC 2400
AGGCACCATG CACCTCCCTT CCCTCCCTGA GGCAGGTGAG AACCCAGAGA GAGGGGCCTG 2460
CAGGTGAGCA GGCAGGGCTG GGCCAGGTCT CCGGGGAGGC AGGGGTCCTG CAGGTCCTGG 2520
TGGGTCAGCC CAGCACCTCG CCCAGTGGGA GCTTCCCGGG ATAAACTGAG CCTGTTCATT 2580
CTGATGTCCA TTTGTCCCAA TAGCTCTACT GCCCTCCCCT TCCCCTTTAC TCAGGCCAGC 2640
TGGCCACCTA GAAGTCTCCC TGCACAGCCT CTAGTGTCCG GGGACCTTGT GGGACCAGTC 2700
CCACACCGCT OGTCCCTGCC CTCCCCTGCT CCCAGGTTGA GGTGCGCTCA CCTCAGAGCA 2760
GGGCCAAAGC ACAGCTGGGC ATGCCATGTC TGAGCGGCGC AGAGCCCTCC AGGCCTGCAG 2820
GGGCAAGGGG CTGGCTGGAG TCTCAGAGCA CAGAGGTAGG AGAACTGGGG TTCAAGCCCA 2880
GGCTTCCTGG CTCCTGCCTG GTCCTCCCTC CCAAGGAGCC ATTCTATGTG ACTCTGGGTG 2940
GAAGTGCCCA GCCCCTGCCT GACGGGATCA CTCTCTGCTG GCAGGATTCT TCCCGCTCCC 3000
CACCTACCCA GCTGATGGGG GTTGGGGTGC TTCTTTCAGC CAAGGCTATG AAGGGACAGC 3060
TGCTGGGACC CACCTCCCCC CTTCCCCGGC CACATGCCGC GTCCCTGCCC CCACCCGGGT 3120
CTGGTGCTGA GGATACAGCT CTTCTCAGTG TCTGAACAAT CTCCAAAATT GAAATGTATA 3180
TTTTTGCTAG GAGCCCCAGC TTCCTGTGTT TTTAATATAA ATAGTGTACA CAGACTGACG 3240
AAACTTTAAA TAAATGGGAA TTAAATATTT AAAAAAAAAA GCGGCCGCGA ATTC 3294 or a degenerate variation thereof.
14. COS cells transfected with the expression construct of Claim Ii.
15. COS cells transfected with the expression construct of Claim 12.
16. COS cells transfected with the expression construct of Claim 13.
17. A method for determining the affinity of a test sample for a steroid hormone receptor NUCI, the method comprising: (a) constructing a chimeric gene by substituting
portions of a DNA-binding domain region of a DNA
sequence encoding human steroid hormone receptor
NUCI cDNA with operative portions of a
DNA-binding domain region from a known
ligand-responsive receptor protein; (b) introducing into a suitable host cell::
(i) the chimeric gene from step (a), and
(ii) a reporter gene functionally linked to an
operative hormone response element
wherein the hormone response element is
capable of being activated by the
DNA-binding domain region of the receptor
protein encoded by the chimeric gene of
step (a); (c) challenging the transfected host cell from step
(b) with the test sample to be evaluated for ligand-binding-activity with the chimeric
receptor protein encoded by the chimeric gene of
step (a); (d) assaying induction of the reporter gene by
monitoring changes in the protein levels of the
protein coded for by the reported gene.
18. The method of Claim 17 wherein the suitablehost cell of step (b) is a COS cell.
19. The method of Claim 17 wherein the reporter gene of step (b)(ii) is a firefly luciferase gene.
20. The method of Claim 17 wherein the DNA sequence encoding human steroid hormone receptor NUCI comprises the sequence (SEQ ID NO:I:) which is:
GAATTCTGCG GAGCCTGCGG GACGGCGGCG GGTTGGCCCG TAGGCAGCCG GGACACTGTT 60
GTACAGTGTT TTGGGCATGC ACGTGATACT CACACAGTGG CTTCTGCTCA CCAACAGATG 120
AAGACAGATG CACCAACGAG GGTCTGGAAT GGTCTGGAGT GGTCTGGAAA GCAGGGTCAG ISO
ATACCCCTGG AAAACTGAAG CCCGTGGAGC AATGATCTCT ACAGGACTGC TTCAAGGCTG 240
ATGGGAACCA CCCTGTAGAG GTCCATCTGC GTTCAGACCC AGACGATGCC AGAGCTATGA 300
CTGGGCCTGC AGGTGTGGCG CCGAGGGGAG ATCAGCCATG GAGCAGCCAC AGGAGGAAGC 360
CCCTGAGGTC CGGGAAGAGG AGGAGAAAGA GGAAGTGGCA GAGGCAGAAG GAGCCCCAGA 420
GCTCAATGGG GGACCACAGC ATGCACTTCC TTCCAGCAGC TACACAGACC TCTCCCGGAG 480
CTCCTCGCCA CCCTCACTGC TGGACCAACT GCAGATGGGC TGTGACGGGG CCTCATGCGG 540
CAGCCTCAAC ATGGAGTGCC GGGTGTGCGG GGACAAGGCA TCGGGCTTCC ACTACGGTGT 600
TCATGGATGT GAGGGGTGCA AGGGGTTCTT CCGTCGTACG ATCCGCATGA AGCTGGAGTA 660
CGAGAAGTGT GAGCGCAGCT GCAAGATTCA GAAGAAGAAC CGCAACAAGT GCCAGTACTG 720
CCGCTTCCAG AAGTGCCTGG CACTGGGCAT GTCACACAAC GCTATCCGTT TTGGTCGGAT 780
GCCGGAGGCT GAGAAGAGGA AGCTGCTCGC AGGGCTGACT GCAAACGAGG GGAGCCAGTA 840
CAACCCACAG GTGGCCGACC TGAAGGCCTT CTCCAAGCAC ATCTACAATG CCTACCTGAA 900
AAACTTCAAC ATGACCAAAA AGAAGGCCCG CAGCATCCTC ACCGGCAAAG CCAGCCACAC 960
GGCGCCCTTT GTGATCCACG ACATCGAGAC ATTGTGGCAG GCAGAGAAGG GGCTGGTGTG 1020
GAAGCAGTTG GTGAATGGCC TGCCTCCCTA CAAGGAGATC AGCGTGCACG TCTTCTACCG 1080 CTGCCACTCC ACCACAGTGG AGACCGTGCG CCAOCTCACT GAGTTCGCCA AGAGCATCCC 1140
CAGCTTCAGC AGCCTCTTCC TCAACGACCA GGTTACCCTT CTCAAGTATG GCGTGCACGA 1200
GGCCATCTTC GCCATGCTGG CCTCTATCGT CAACAAGGAC GGGCTGCTGG TAGCCAACGG 1260
CAGTGGCTTT GTCACCCGTG AGTTCCTGCG CAGCCTCCGC AAACCCTTCA GTGATATCAT 1320
TGAGCCTAAG TTTGAATTTG CTGTCAAGTT CAACGCCCTG GAACTTGATG ACAGTGACCT 1380
GCCCCTATTC ATTGCGGCCA TCATTCTGTG TGGAGACCGG CCAGGCCTCA TGAACGTTCC 1440
ACGGGTGGAG GCTATCCAGG ACACCATCCT GCGTGCCCTC GAATTCCACC TGCAGGCCAA 1500
CCACCCTGAT GCCCAGTACC TCTTCCCCAA GCTGCTGCAG AAGATGGCTG ACCTGGGGCA 1560
ACTGGTCACC GAGCACGCCC AGATGATGCA GCGGATCAAG AAGACCGAAA CCGAGACCTC 1620
GCTGCACCCT CTGCTCCAGG AGATCTACAA GGACATGTAC TAACGGCGGC ACCCAGGCCT 1680
CCCTCCAGAC TCCAATGGGG CCAGCACTGG AGGGGCCCAC CCACATGACT TTTCCATTGA 1740
CCACCTCTCT TCCTGTCTTT GTTGTCTCCC TCTTTCTCAG TTCCTCTTTC TTTTCTAATT 1800
CCTGTTGCTC TGTTTCTTCC TTTCTGTAGG TTTGTCTCTT CCCTTCTCCC TTCTCCCTTG 1860
CCCTCCCTTT CTCTCTCCTA TCCCCACGTC TGTCCTCCTT TCTTATTCTG TGAGATGTTT 1920
TGTATTATTT CACCAGCAGC ATAGAACAGG ACCTCTGCTT TTGCACACCT TTTCCCCAGG 1980
AGCAGAAGAG AGTGGGCCTG CCCTCTGCCC CATCATTGCA CCTGCAGGCT TAGGTCCTCA 2040
CTTCTGTCTC CTGTCTTCAG AGCAAAAGAC TTGAGCCATC CAAAGAAACA CTAAGCTCTC 2100
TGGGCCTGGG TTCCAGGGAA GGCTAAGCAT GGCCTGGACT GACTGCAGCC CCCTATAGTC 2160
ATGGGGTCCC TGCTGCAAAG GACAGTGGCA GACCCCGGCA GTAGAGCCGA GATGCCTCCC 2220
CAAGACTGTC ATTGCCCCTC CGATCGTGAG GCCACCCACT GACCCAATGA TCCTCTCCAG 2280
CAGCACACCT CAGOCOCACT GACACCCAGT GTCCTTCCAT CTTCACACTG CTTTGCCAGG 2340
CCAATGTTGC TGATGGCCCC TCCAGCACAC ACACATAAGC ACTGAAATCA CTTTACCTGC 2400
AGGCACCATG CACCTCCCTT CCCTCCCTGA GGCACGTGAG AACCCAGAGA GAGGGGCCTG 2460
CAGGTGAGCA GGCAGGGCTG GGCCAGGTCT CCGGGGAGGC AGGGGTCCTG CAGGTCCTGG 2520
TGGGTCAGCC CAGCACCTCG CCCAGTGGGA GCTTCCCGGG ATAAACTGAG CCTGTTCATT 2580
CTGATGTCCA TTTGTCCCAA TAGCTCTACT GCCCTCCCCT TCCCCTTTAC TCAGCCCAGC 2640
TGGGCACCTA GAAGTCTCCC TGCACAGCCT CTAGTGTCCG GGGACCTTGT GGGACCAGTC 2700
CCACACCGGT GGTCCCTGCC CTCCCCTGCT CCCAGGTTGA GGTGCGCTCA CCTCAGAGGA 2760
GGGCCAAAGC ACAGCTGGGC ATGCCATGTC TGAGCGGCGC AGAGCCCTCC AGGCCTGCAG 2820 tGGCAAGGGG CTGGCTGGAG TCTCAGACCA CAGAGCTAGG AGAACTGGGG TTCAACCCCA 2880
GGCTTCCTGG GTCCTGCCTG GTCCTCCCTC CCAAGGAGCC ATTCTATGTG ACTCTGGGTG 2940
GAAGTGCCCA GCCCCTGCCT GACGGGATCA CTCTCTGCTG GCAGGATTCT TCCCCCTCCC 3000
CACCTACCCA GCTGATGGGG GTTGGGGTGC TTCTTTCAGC CAAGGCTATG AAGGGAGAGC 3060
TGCTGGGACC CACCTCCCCC CTTCCCCGGC CACATGCCGC GTCCCTGCCC CCACCCGGGT 3120
CTGGTGCTGA GGATACAGCT CTTCTCAGTG TCTGAACAAT CTCCAAAATT GAAATGTATA 3180
TTTTTGCTAG GAGCCCCAGC TTCCTGTGTT TTTAATATAA ATAGTGTACA CAGACTGACG 3240
AAACTTTAAA TAAATGGGAA TTAAATATTT AAAAAAAAAA GCGGCCGCGA ATTC 3294 or a degenerate variation thereof.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US85705592A | 1992-03-24 | 1992-03-24 |
Publications (2)
Publication Number | Publication Date |
---|---|
GB9306043D0 GB9306043D0 (en) | 1993-05-12 |
GB2265376A true GB2265376A (en) | 1993-09-29 |
Family
ID=25325082
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB9306043A Withdrawn GB2265376A (en) | 1992-03-24 | 1993-03-23 | Human steroid hormone receptor (nuci) |
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Country | Link |
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GB (1) | GB2265376A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2292885A (en) * | 1994-09-08 | 1996-03-13 | Merck & Co Inc | Method of treating hyperlipidemia |
-
1993
- 1993-03-23 GB GB9306043A patent/GB2265376A/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
Chemical Abstracts 92:509970 & Mol.Endocrinol.,6(10), (1992)pages 1634-41 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2292885A (en) * | 1994-09-08 | 1996-03-13 | Merck & Co Inc | Method of treating hyperlipidemia |
Also Published As
Publication number | Publication date |
---|---|
GB9306043D0 (en) | 1993-05-12 |
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