Abstract:
This invention provides an isolated receptor having the amino acid sequence of FIG.  1  (SEQ ID NO:2) or substantially the same amino acid sequence as the amino acid sequence shown in FIG.  1  (SEQ ID NO:2) or an amino acid sequence functionally similar to that sequence, and DNA sequences encoding such a receptor.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to cellular nuclear receptors. 
     2. Brief Description of the Art 
     A large family of nuclear receptors has been identified which confer cells with responsiveness to molecules such as retinoic acid, vitamin D3, steroid hormones and thyroid hormones. Extensive studies have shown that the members of this superfamily of nuclear receptors activate and/or repress gene transcription through direct binding to discrete cis-acting elements termed “hormone response elements” (HRE). It has been shown that these HRE&#39;s comprise repeats of consensus palindromic hexanucleotide DNA motifs. The specificity of the HRE&#39;s is determined by the orientation of, and spacing between, halfsites (i.e. half a palindromic sequence)(Umenesono K., et al, 1991  Cell  65, 1255-1266). 
     Specific DNA binding is mediated by a distinct DNA binding domain, containing two zinc fingers, which is conserved among all thus discovered nuclear receptors. Three amino acids at the C-terminal base of the first zinc finger (known as the “P-box”) are important for the recognition of the half site nucleotide sequence. Members of the nuclear receptor superfamily have been classified into different groups on the basis of the amino acid sequence within the P box. 
     Molecules thought to be nuclear receptors, as they are structurally related to characterized receptors, but for which no ligand has been identified are termed “orphan receptors”. Many such orphan receptors have been identified (see for example Evans R. M, (1988)  Science  240,889-895 and O&#39;Malley, B. (1990)  Mol. Endocrinol.  4 363-369). 
     BRIEF SUMMARY OF THE INVENTION 
     According to one aspect of the invention there is provided a novel nuclear receptor, hereinafter termed “OR- 1 ”, having the amino acid sequence of FIG. 1 (SEQ ID NO:2) or substantially the same amino acid sequence as the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or an amino acid sequence functionally similar to that sequence. 
     An amino acid sequence which is more than about 90%, preferably more than 95%, identical with the sequence shown in FIG. 1 (SEQ ID NO:2) is substantially the same amino acid sequence for the purposes of the present application. 
     According to another aspect of the invention there is provided a DNA sequence encoding a nuclear receptor according to the first aspect of the invention. Preferably, the DNA sequence is that given in FIG. 2 (SEQ ID NO:1) or is a DNA sequence encoding a protein or polypeptide having the functionality of OR- 1 . 
     The nuclear receptor of the invention has a similar P-box configuration to the retinoic acid receptor (RAR), the vitamin D receptor (VDR), and the thyroid hormone receptor (TR) and can be placed in the same subfamily as those receptors. 
     Preferably, the receptor heterodimerizes with RXR to form a complex. 
     Preferably, the receptor interacts with RXR and binds to a DNA sequence comprising at least one repeat of the DNA sequence -AGGTCA- (SEQ ID NO:10). Preferably the sequence is AGTCAGGTCACTCGAGGTCAGTCA (SEQ ID NO:11). 
     Preferably, the receptor modulates 9-cis retinoic acid signalling. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The nuclear receptor of the invention, OR- 1 , and its method of production will now be described, by way of example only, with reference to the accompanying drawings FIGS. 1-5, in which: 
     FIG. 1 shows the amino acid sequence of a nuclear receptor of the invention (SEQ ID NO:2); 
     FIGS. 2A and 2B show the DNA sequence of a nuclear receptor of the invention (SEQ ID NO:1); 
     FIG. 3 gives a comparison between the primary amino acid sequences of the nuclear receptor of the invention and those of other members of the nuclear receptor superfamily; 
     FIG. 4 Localization of OR- 1  mRNA—producing cells in rat tissues with in situ hybridization; 
     FIG. 5A gives the DNA sequences of seven potential HRE&#39;s DR- 0 -DR- 6 ; 
     FIG. 5B illustrates the interaction between OR- 1  or the retinoid X receptor (RXR) and the potential HRE&#39;s, DR- 2  and DR 4 ; and 
     FIG. 6 illustrates experiments showing that OR- 1  confers 9-cis retinoic acid-responsiveness of RXR on a DR 4 -containing promoter. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Cloning and Expression of OR- 1   
     Rat OR- 1  was cloned from a cDNA library from Sprague Dawley rat liver in the commercially-available λZAP vector (Stratagene, USA) using the techniques described in Göttlicher, M. et al (1992)  Proc. Natl. Acad. Sci.  USA 89, 4653-4657. Foetal and adult rat tissues were excised after decapitation and frozen on dry ice. Cryostat sections were hybridized to 48-mer oligonucleotides complementary to OR- 1  mRNA positions 100-151 and 850-900 as described in Dagerlind, Å et al (1992)  Histochemistry  98 34-49. 
     Several unrelated oligonucleotides were also used as controls. The addition of 100 fold of the respective nonlabelled control oligonucleotide abolished all labelling observed with the OR- 1  probes. 
     Plasmids 
     OR- 1  cDNA was subcloned as an Eco RI fragment in pGEM-3Z (Promega) to produce the plasmid pROR- 1 -Sp6, or in the multiple cloning site of pCMV5 (described in Andersson, S. et al 1989  J. Biol. Chem.,  264, 8222-8229) to produce the plasmid pCMV-OR- 1 . The reporter construct pDR 4 -AF contains a SphI-XhoI fragment of the cDNA for a secreted form of human PAP (placental alkaline phosphatase) described in (Berger, J. et al. 1988  Gene  66,1-10) under the control of a DR 4 -TK-containing promoter, pRRXR-T7 and pCMV-RXR described previously in Gearing, K. L. et al 1993  Proc. Natl. Acad. Sci. USA  90, 1440-1444. 
     DNA Binding Studies 
     Gel shifts were performed using in vitro-translated OR- 1  and RXR with the commercially-available TNT™-coupled reticulocyte lysate system (Promega, Madison USA). Proteins were incubated on ice for 15 min with 4 μg of poly (dI-dC) and with unlabelled competitor DNA where indicated in a solution comprising 100 mM KCl; 10 mM Hepes, pH 7.6; 1 mM dithiothreitol; 1 mM EDTA; 10% (wt./vol) glycerol, before addition of 0.5 ng of a  32 P-end labelled oligonucleotide probe. The reaction mixtures were incubated for a further 10 min at 22° C. before electrophoresis at 200V and 4° C. in pre-run 4% polyacryliamide/0.25 TBE (0.089 m tris-borate pH 8.3, 0.025 EDTA) gels. 
     The following oligonucleotides and their complements were used as probes: 
     DR 0  AGCTTCAGGTCAAGGTCAGGTTCA (SEQ ID NO:3) 
     DR 1  AGCTTCAGGTCACAGGTCAGTTCA (SEQ ID NO:4) 
     DR 2  AGCTTAGGTCACCAGGTCAGTTCA (SEQ ID NO:5) 
     DR 3  AGTCCAGGTCACTCAGGTCAGTCA (SEQ ID NO:6) 
     DR 4  AGTCAGGTCACTCGAGGTCAGTCA (SEQ ID NO:7) 
     DR 5  AGTCAGGTCACTCGTAGGTCAGTCA (SEQ ID NO:8) 
     DR 6  AGTCAGGTCACTCGTTAGGTCAGTCA (SEQ ID NO:9) 
     Cells and Transfection 
     Embryonal carcinoma P19 EC cells were cultured in Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% foetal calf serum, nonessential amino acids, penicillin (100 units/ml) and streptomycin (100 mg/ml). Chinese Hamster Ovary (CHO) cells were cultured in Ham&#39;s F-12 medium supplemented with 10% foetal calf serum, penicillin (100 units/ml) and streptomycin (100 mg/ml). Cells were plated in duplicate in 35 mm Petri dishes and transfected at 30% confluency, using lipofectin reagent (Bethesada Research Laboratories, USA) according to the recommendations of the supplier. After 12 hours the medium was changed and supplemented or not supplemented as the case may be with 100 nM 9-cis retinoic acid (a gift of Hoffman-LaRoche) as indicated, and incubated for an additional 36 h. Cell culture supernatants were then heated to 65° C. for 30 min. PAP activity was determined as the increase in A 405  at 30° C. in a 1 ml reaction mixture containing 0.75 ml of supernatant, 200 nM Tris (pH 8.8.), 275 mM NaCl, 0.5 mM MgCl 2 , and 5 mM p-nitrophenylphosphate. Transfections were repeated 6 times with different plasmid preparations and data from a representative experiment is presented here. 
     Results 
     The OR- 1  clone spans 1940 bp including a 55 bp long poly-A tail and contains an open reading frame starting with an ATG corresponding to a protein of 446 amino acids with a predicted molecular weight of 50 kD. The complete amino acid and nucleotide sequences of OR- 1  are given in FIGS. 1 and 2A and  2 B (SEQ ID NO:2 and SEQ ID NO:1) respectively. OR- 1  shows no striking homology to known members of the nuclear receptors superfamily: the highest homologies represent less than 10% in the N-terminal domain, about 50% in the DNA binding domain, and between 20-30% in the putative ligand binding domain as shown in FIG.  3 . The amino-terminal domain of OR- 1  (underlined in FIG. 1 (SEQ ID NO:2)) is 77 amino acids long and to a large extent comprises a so-called “PEST” sequence, meaning that it is an amino acid sequence rich in proline, glutamic acid, serine, threonine, and aspartic acid residues. The DNA binding domain consists of 68 amino acids including the nine invariable cysteines characteristic of the members of the nuclear receptor superfamily, as well as other amino acids that are found to be conserved in all members of this protein family. 
     Genomic Cloning 
     A rat genomic fragment has been isolated, that spans the DNA binding domain or OR 1  and all the exons downstream of it. Most nuclear receptors for which the genomic structure has been determined have the two zinc “fingers” of the DNA binding domain encoded on separate exons. We have shown that the whole DNA-binding domain is encoded by one exon in OR 1 . We have furthermore shown that this is also the case with RLD- 1  ( Mol. Endocrinol.  infra), a closely related receptor “knock-out” mice of OR 1  and RLD- 1 . 
     Tissue Distribution of OR- 1   
     To analyse the tissue distribution of OR- 1  transcripts, in situ hybridizations were performed on foetal and adult rat tissues. Labelling for OR- 1  was found in several tissues of both foetal and adult rats. As discussed, below, prominent expression was observed in liver, lung, thymus, brown fat, salivary gland, thyroid gland, pituitary gland and retina whereas moderate levels were seen in developing cerebrum and cerebellum, in perichondrium around developing bones, heart and skin. Low levels of OR- 1  mRNA was present in skeletal muscle as shown in FIG.  4 . In adult rats, strong labelling was found in lymph node, prostate, adrenal cortex and the intermediate lobe of the pituitary gland. Moderate levels were seen in liver, testis, salivary gland, thyroid and parathyroid gland, adrenal medulla, anterior pituitary and kidney. In the brain, a moderate signal was observed in neurons in the granular cell layer of the cerebellum and hippocampus. 
     1) Immune System 
     Prominent expression of OR- 1  mRNA was seen in the cortex of the thymus with lower levels in the medulla. In dipped sections grains were seen over most of the thymocytes in the cortex. Significant expression was also seen in the lymph nodes, whereas low levels were observed in spleen. Some cells in the bone marrow expressed OR- 1  mRNA. 
     2) Endocrine System 
     Significant expression of OR- 1  was seen in the anterior and intermediate lobes of the pituitary. In dipped sections grains could be seen over most of the cells in the intermediate lobe and over the majority of the cells in the anterior lobe. The posterior lobe appeared virtually nonlabelled. Prominent expression of OR- 1  was detected in the parathyroid glands where most of the cells expressed OR- 1  mRNA. In the thyroid gland moderate expression was observed and OR- 1  mRNA was heterogenously distributed in different cell types. Most of the parafollicular cells expressed OR- 1 , whereas only part of the follicular cells were labelled. High expression in the adrenal gland was observed in all layers of the cortex, whereas lower levels were seen in the medulla. Expression of OR- 1  was slightly higher in the zona glomerulosa than in the rest of the cortex. In the adrenal medulla the labelling was heterogenous and part of the chromaffin cells and ganglion cells expressed OR- 1 . In pineal gland some cells contained OR- 1  mRNA. 
     3) Reproductive System 
     OR- 1  could be detected both in male and female genital organs. In the testis OR- 1  mRNA was present in all cross-sections of the seminiferous tubules. The labelling localizes to the basal compartment of the seminiferous epithelium and grains could be seen mainly over primary spermatocytes, whereas spermatogonia and germ cells at later developmental stages were non-labelled. The Sertoli cells and Leydig cells did not express OR- 1  mRNA. A strong signal for OR- 1  was evident in the epithelium of the prostate gland and also in the epididymis, whereas low levels were seen in the epithelium of the vesicula seminalis. In the ovary oocytes at different stages of development expressed OR- 1  mRNA while other cells appeared non-labelled. In the uterus the epithelium was strongly labelled and lower levels of OR- 1  mRNA were seen in the myometrium. 
     4) Urinary System 
     Moderate expression of OR- 1  could be detected in the outer medulla of the kidney, whereas in the cortex and inner medulla the labelling was very low or nondetectable. In dipped sections grains were seen over different parts of the loop of Henle. The glomeruli, proximal and distal convoluted tubules and collecting tubules did not express OR- 1  at detectable levels. The transitional epithelium of the renal pelvis expressed OR- 1 . 
     5) Digestive System 
     In salivary glands the secretory acini and the ducts expressed moderate levels of OR- 1  mRNA. In the liver OR- 1  mRNA was evenly distributed throughout the liver and most, if not all, hepatocytes were labelled. In the intestinal tract OR- 1  was expressed in the epithelium of stomach and small and large intestine. 
     6) Nervous System 
     Significant expression of OR- 1  was seen in the sympathetic and sensory ganglia. In superior cervical ganglion most of the sympatetic neurons expressed OR- 1  at high level and also the satellite cells were labelled. In dorsal root ganglion the labelling was heterogenous and varied between individual neurons. The Schwan cells of peripheral nerves expressed OR- 1  whereas oligodendrocytes in optic nerve were nonlabelled. In the retina the bipolar cells expressed OR- 1 . In the central nervous system OR- 1  mRNA was seen in several areas including hippocampus and cerebellum. 
     7) Respiratory System 
     Moderate expression of OR- 1  was seen in the bronchial epithelium and in the alveoli. 
     8) Other Tissues 
     Low or non-detectable levels of OR- 1  were seen in sketal muscle and heart. Also in white adipose tissue OR- 1  expression was below the detection limit. In skin a clear signal was observed in keratinocytes in the basal part of the epidermis. A strong signal was seen in perichondrium around the cartilage in trachea. Low expression of OR- 1  could be seen in intra and extraorbital lacrimal glands. 
     The expression of OR- 1  thus appears to be ubiquitous, suggesting that this receptor might have a house keeping function and/or mediate many effects by regulating the transcriptor of various genes. The tissue distribution or OR- 1  is different from the tissue distribution of RLD- 1  ( Mol Endocrinol  9, 72-85, 1995) suggesting that these two isoforms might have different functions. OR- 1  is particularly well expressed in tissues involved in the immue system. It has been described that 9-cis retinoic acid plays a role in thymocyte development, being a potent negative regulator of activation-induced T-cell apoptosis. Since OR- 1  dimerizes with RXR and is expressed at a high level in the thymus during the fetal stages, it may play a role in regulating T-cells development. OR- 1  is also well expressed in peripheral endocrine glands, in male and female genital organs and in the nervous system. The tissue distribution of OR- 1  is thus different from that of RXRα which has been described to be noticeably abundant in visceral tissues such as liver, kidney, lung, brain, heart, intestine and testis. We previously suggested that OR- 1  could act as a helper of RXRα in mediating the effects of 9-cis retinoic acid. Nevertheless we do not know whether OR- 1  could also act as a monomer, as a homodimer or as a heterodimer with another protein than RXRα. For example, it is possible that OR- 1  modulates the actions of RXRβ that shows a diffuse and probably ubiouitous expression, and of RXRγ which has a more specific distribution. 
     OR- 1  Interacts with RXR on a DR4 Motif in Vitro 
     A set of potential HRE&#39;s, DR 0 -DR 6 , having the DNA sequences described above predicted by the  3 - 4 - 5  rule (Umensono et al supra) was synthesized and assayed in gel shift experiments using in vitro translated OR- 1  alone or in combination with RXR also translated in vitro. In vitro translation of OR- 1  produced a protein of the predicted size of 50 kD. In the gel shift assays, OR- 1  was unable to bind to any of the potential HRE&#39;s but OR- 1  combined with RXR, recognized the potential HRE DR 4  which is usually described as the thyroid hormone response element (TRE)(Umensono et al supra). 
     FIG. 5B shows that although OR- 1  or RXR alone was not able to bind to DR 4 , together these proteins were able to form a specific complex with this DNA element. The appearance of this complex depends on the presence of RXR and is inhibited by a 10-fold excess of the specific DNA target element, but not by a 100-fold excess of an unrelated DNA element—see FIG. 5B, lane 7) 
     OR- 1  Confers 9-Cis Retinoic Acid Responsiveness or RXR on a DR 4 -Containing Promoter 
     Since OR- 1  and RXR formed a specific complex on the DR 4  sequence in vitro, coexpression of OR- 1  in embryonal carcinoma (EC) cells that express endogenous RXR was tested to determine whether it could affect the activity of a reporter gene under the control of a DR 4 -containing promoter. RXR has been shown to be an auxiliary receptor for several classes of hormone receptors, controlling the ligand responses of receptors that form heterodimers with RXR (Yu, V. C. et al 1991  Cell  67, 251-1266 and Bugge, T. H. et al 1992  EMBO J.  11, 1409-1418). In addition, it has been shown that 9-cis retinoic acid leads to effective RXR homodimer formation and that these homodimers bind and activate several retinoic acid response elements (“RARE&#39;s”), but not natural thyroid hormone response elements (Zhang, X. K. et al 1992  Nature  (London)358, 587-591). As previously described by others (Hallenbeck, P. L. et al 1993,  J. Biol Chem. 268, 3825-3828) our transfection studies showed no induction by 9-cis retinoic acid of RXR on a reporter containing DR 4  (FIG.  5 ). Expression of OR- 1  allowed activation of RXR by 9-cis retinoic acid on a DR 4 -containing promoter. In CHO cells that do not express endogenous RXR at as high a level as EC cells, cotransfection of RXR together with OR- 1  is necessary to obtain induction by 9-cis retinoic acid. Thus acting as a helper of RXR, OR- 1  appears to confer 9-cis retinoic acid signalling on DR 4 -containing promoters. 
     
       
         
           
             11 
           
           
             
               1934 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             1
CAAGTGCTGT GGAGGAGCAA TCACCGGTGC GGACACAGAG CTCCCGCCTC CCACAGCCAT     60
TTCCAGGGTA ACGAAGTAGG AGACCCCCTC CTGCGACCCC CTCACGATCG CCGGTGCAGT    120
CATGAGCCCC GCCTCCCCCT GGTGCACGGA GAGGGGCGGG GCCTGGAACG AGGCTGCTTC    180
GTGACCCACT ATGTCTTCCC CCACAAGTTC TCTGGACACT CCCTTGCCTG GGAATGGTTC    240
TCCCCAGCCC AGTACCTCCT CCACTTCACC CACTATTAAG GAGGAGGGAC AGGAGACTGA    300
TCCACCTCCA GGCTCTGAAG GGTCCAGCTC TGCCTACATC GTGGTCATCT TAGAGCCAGA    360
GGATGAACCT GAGCGCAAGC GGAAGAAGGG TCCGGCCCCG AAGATGCTGG GCCATGAGCT    420
GTGCCGCGTG TGCGGGGACA AGGCCTCGGG CTTCCACTAC AATGTGCTCA GTTGTGAAGG    480
CTGCAAAGGC TTCTTCCGGC GTAGCGTGGT CCATGGTGGG GCCGGGCGCT ATGCCTGTCG    540
GGGCAGCGGA ACCTGCCAGA TGGATGCCTT CATGCGGCGC AAGTGCCAGC TCTGCAGACT    600
GCGCAAGTGC AAGGAGGCTG GCATGCGGGA GCAGTGCGTG CTTTCTGAGG AGCAGATTCG    660
GAAGAAAAAG ATTCAGAAGC AGCAACAGCA GCAGCCACCG CCCCCGACTG AGCCAGCATC    720
CGGTAGCTCA GCCCGGCCTG CAGCCTCCCC TGGCACTTCG GAAGCAAGTA GCCAGGGCTC    780
CGGGGAAGGA GAGGGCATCC AGCTGACAGC GGCTCAGGAG CTGATGATCC AACAGTTAGT    840
TGCCGCGCAG CTGCAGTGCA ACAAGCGATC TTTCTCCGAC CAGCCTAAAG TCACGCCCTG    900
GCCCTTGGGT GCAGACCCTC AGTCCCGAGA CGCTCGTCAG CAACGCTTTG CCCACTTCAC    960
TGAGCTAGCC ATCATCTCAG TCCAGGAGAT CGTGGACTTC GCCAAGCAGG TGCCAGGGTT   1020
CCTGCAGCTG GGCCGGGAGG ACCAGATCGC CCTCCTGAAG GCATCCACCA TCGAGATCAT   1080
GTTGCTAGAG ACAGCCAGAC GCTACAACCA CGAGACAGAG TGCATCACGT TCCTGAAGGA   1140
CTTCACCTAC AGCAAGGACG ACTTCCACCG TGCAGGCTTG CAGGTGGAGT TCATCAATCC   1200
CATCTTTGAG TTCTCTCGGG CTATGCGTCG GCTGGGCCTA GACGATGCAG AGTATGCCTT   1260
GCTCATTGCC ATCAACATCT TCTCAGCGGA CCGGCCTAAT GTGCAGGAGC CCAGCCGTGT   1320
GGAGGCTCTG CAGCAGCCCT ATGTGGAGGC CCTCCTCTCC TACACGAGGA TCAAGCGGCC   1380
GCAGGACCAG CTGCGCTTCC CACGAATGCT CATGAAGCTG GTGAGCCTGC GCACCCTCAG   1440
CTCCGTGCAC TCGGAGCAGG TTTTCGCATT GCGTCTCCAG GACAAGAAGC TGCCGCCTTT   1500
GCTGTCCGAG ATCTGGGATG TGCATGAGTA GGGGCCGCCA CAAGTGCCCC AGCCTTGGTG   1560
GTGTCTACTT GCAGATGGAC GCTTCCTTTG CCTTTCCTGG GGTGGGAGGA CACTGTCACA   1620
GCCCAGTCCC CTGGGCTCGG GCTGAGCGAG TGGCAGTTGG CACTAGAAGG TCCCACCCCA   1680
CCCGCTGAGT CTTCCAGGAG TGGTGAGGGT CACAGGCCCT AGCCTCTGAT CTTTACCAGC   1740
TGCCCTTCCT CCCGAGCTTA CACCTCAGCC TACCACACCA TGCACCTTGA GTGGAGAGAG   1800
GTTAGGGCAG GTGGCTCCCC ACAGTTGGGA GACCACAGGC CCCCTCTTCT GCCCCTTTTA   1860
TTTAATAAAA AAATAAAATA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA   1920
AAAAAAAAAA AAAA                                                     1934 
           
           
             
               446 amino acids 
               amino acid 
               single 
               linear 
             
             
               protein 
             
             
               not provided 
             
             2
Met Ser Ser Pro Thr Ser Ser Leu Asp Thr Pro Leu Pro Gly Asn Gly
 1               5                  10                  15
Ser Pro Gln Pro Ser Thr Ser Ser Thr Ser Pro Thr Ile Lys Glu Glu
            20                  25                  30
Gly Gln Glu Thr Asp Pro Pro Pro Gly Ser Glu Gly Ser Ser Ser Ala
        35                  40                  45
Tyr Ile Val Val Ile Leu Glu Pro Glu Asp Glu Pro Glu Arg Lys Arg
    50                  55                  60
Lys Lys Gly Pro Ala Pro Lys Met Leu Gly His Glu Leu Cys Arg Val
65                  70                  75                  80
Cys Gly Asp Lys Ala Ser Gly Phe His Tyr Asn Val Leu Ser Cys Glu
                85                  90                  95
Gly Cys Lys Gly Phe Phe Arg Arg Ser Val Val His Gly Gly Ala Gly
            100                 105                 110
Arg Tyr Ala Cys Arg Gly Ser Gly Thr Cys Gln Met Asp Ala Phe Met
        115                 120                 125
Arg Arg Lys Cys Gln Leu Cys Arg Leu Arg Lys Cys Lys Glu Ala Gly
    130                 135                 140
Met Arg Glu Gln Cys Val Leu Ser Glu Glu Gln Ile Arg Lys Lys Lys
145                 150                 155                 160
Ile Gln Lys Gln Gln Gln Gln Gln Pro Pro Pro Pro Thr Glu Pro Ala
                165                 170                 175
Ser Gly Ser Ser Ala Arg Pro Ala Ala Ser Pro Gly Thr Ser Glu Ala
            180                 185                 190
Ser Ser Gln Gly Ser Gly Glu Gly Glu Gly Ile Gln Leu Thr Ala Ala
        195                 200                 205
Gln Glu Leu Met Ile Gln Gln Leu Val Ala Ala Gln Leu Gln Cys Asn
    210                 215                 220
Lys Arg Ser Phe Ser Asp Gln Pro Lys Val Thr Pro Trp Pro Leu Gly
225                 230                 235                 240
Ala Asp Pro Gln Ser Arg Asp Ala Arg Gln Gln Arg Phe Ala His Phe
                245                 250                 255
Thr Glu Leu Ala Ile Ile Ser Val Gln Glu Ile Val Asp Phe Ala Lys
            260                 265                 270
Gln Val Pro Gly Phe Leu Gln Leu Gly Arg Glu Asp Gln Ile Ala Leu
        275                 280                 285
Leu Lys Ala Ser Thr Ile Glu Ile Met Leu Leu Glu Thr Ala Arg Arg
    290                 295                 300
Tyr Asn His Glu Thr Glu Cys Ile Thr Phe Leu Lys Asp Phe Thr Tyr
305                 310                 315                 320
Ser Lys Asp Asp Phe His Arg Ala Gly Leu Gln Val Glu Phe Ile Asn
                325                 330                 335
Pro Ile Phe Glu Phe Ser Arg Ala Met Arg Arg Leu Gly Leu Asp Asp
            340                 345                 350
Ala Glu Tyr Ala Leu Leu Ile Ala Ile Asn Ile Phe Ser Ala Asp Arg
        355                 360                 365
Pro Asn Val Gln Glu Pro Ser Arg Val Glu Ala Leu Gln Gln Pro Tyr
    370                 375                 380
Val Glu Ala Leu Leu Ser Tyr Thr Arg Ile Lys Arg Pro Gln Asp Gln
385                 390                 395                 400
Leu Arg Phe Pro Arg Met Leu Met Lys Leu Val Ser Leu Arg Thr Leu
                405                 410                 415
Ser Ser Val His Ser Glu Gln Val Phe Ala Leu Arg Leu Gln Asp Lys
            420                 425                 430
Lys Leu Pro Pro Leu Leu Ser Glu Ile Trp Asp Val His Glu
        435                 440                 445 
           
           
             
               24 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             3
AGCTTCAGGT CAAGGTCAGG TTCA                                            24 
           
           
             
               24 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             4
AGCTTCAGGT CACAGGTCAG TTCA                                            24 
           
           
             
               24 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             5
AGCTTAGGTC ACCAGGTCAG TTCA                                            24 
           
           
             
               24 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             6
AGTCCAGGTC ACTCAGGTCA GTCA                                            24 
           
           
             
               24 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             7
AGTCAGGTCA CTCGAGGTCA GTCA                                            24 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA to scRNA 
             
             
               not provided 
             
             8
AGTCAGGTCA CTCGTAGGTC AGTCA                                           25 
           
           
             
               26 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             9
AGTCAGGTCA CTCGTTAGGT CAGTCA                                          26 
           
           
             
               6 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             10
AGGTCA                                                                 6 
           
           
             
               24 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             11
AGTCAGGTCA CTCGAGGTCA GTCA                                            24