Patent Publication Number: US-6037131-A

Title: Melatonin 1a receptor gene regulatory regions an uses thereof

Description:
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made at least in part with funds from the Federal government (NIH grant R37 HD 14427), and the government therefore has rights in the invention. 
    
    
     CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a division of Ser. No. 08/895,701, filed Jul. 17, 1997, now U.S. Pat. No. 5,889,177; claims priority from provisional application 60/022,185, filed on Jul. 18, 1996. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to nucleic acids encoding transcriptional regulatory sequences of a high-affinity melatonin receptor gene. 
     The high-affinity melatonin receptor is a membrane protein that is coupled to guanine nucleotide binding proteins (G proteins). G proteins, in turn, communicate ligand-activated receptor signals to the appropriate intracellular effector system(s). The hormone, melatonin, inhibits adenylyl cyclase causing a decrease in intracellular cyclic AMP (cAMP) concentration. 
     Melatonin, the principal hormone of the vertebrate pineal gland, elicits potent neurobiological effects. Melatonin influences circadian rhythm and mediates the effects of photoperiod on reproductive function in seasonally breeding mammals. In humans, melatonin administration has been shown to alleviate the symptoms of jet lag after air travel across several time zones. The hormone also has potent sedative effects in humans and may be a useful hypnotic agent. 
     Melatonin exerts its photoperiodic and circadian effects through pharmacologically specific, high-affinity receptors (Dubocovich, M. L. and Takahashi, J., Proc. Natl. Acad Sci. USA (1987) 84:3916-3920; Vanecek, J., J. Neurochem. (1988) 51:1436-1440; Reppert et al., (1988) supra). In seasonally breeding mammals, pineal melatonin secretion regulates seasonal responses to changes in day length (Bartness, T. J. and Goldman, B. D., Experientia (1989) 45:939-945; Karsch et al., Recent Prog. Homm. Res. (1984) 40:185-232). The only site containing melatonin 1a receptors in all photoperiodic species examined to date (Weaver, et al., Suprachiasmatic nucleus: the mind&#39;s clock. Klein, D. C., Moore, R. Y, and Reppert, S. M., eds. New York: Oxford University Press; (1991) pp. 289-308) is the pars tuberalis (PT), a portion of the pituitary gland. In contrast to other species, in humans melatonin 1a receptors are not consistently present in the PT. 
     High-affinity melatonin 1a (Mel 1a ) receptors are located in discrete regions of the vertebrate central nervous system of several mammalian species, including humans. Binding studies using the ligand 2-[ 125  I]-iodomelatonin ( 125  I-melatonin or [ 125  I]MEL) have identified high-affinity melatonin 1a receptors (K d  &lt;2×10 -10  M) in sites such as the suprachiasmatic nuclei (SCN), the site of a biological clock that regulates numerous circadian rhythms (Reppert et al., Science (1988) 242:78-81). To date, high-affinity melatonin receptors have not been identified in central nervous system tissues other than brain. 
     Receptor affinity is sensitive to guanine nucleotides and activation of the receptors consistently leads to the inhibition of adenylyl cyclase through a pertussis toxin-sensitive mechanism (Rivkees, S. A. et al., Proc. Natl. Acad. Sci. USA (1989) 86:3883-3886; Carlson, L. L. et al., Endocrinology (1989) 125:2670-2676; Morgan, P. J. et al., Neuroendocrinology (1989) 50:358-362; Morgan, P. J. et al., J. Neuroendocrinol. (1990) 2:773-776; Laitinen, J. T. and Saavedra, J. M., Endocrinology (1990) 126:2110-2115). High-affinity melatonin receptors thus appear to belong to the superfamily of G protein-coupled receptors. 
     SUMMARY OF THE INVENTION 
     In general, the invention features substantially pure DNA (cDNA or genomic DNA) encoding the 3&#39; and 5&#39; transcriptional regulatory regions and coding region of a high-affinity melatonin 1a receptor in brain. In preferred embodiments, the invention includes nucleotide sequences for 5&#39; and 3&#39; transcriptional regulatory regions of a melatonin 1a receptor gene. Such sequences of the invention include a promoter as well as upstream and downstream enhancer elements and/or cis- and trans-acting factor binding sites functional in the transcriptional regulation of the melatonin 1a receptor gene. Sequences functional in the transcription of the melatonin 1a receptor gene may be located more than 1.5 kilobase pairs from the start or termination of the receptor coding sequence. In other preferred embodiments of the invention, the transcriptional regulatory sequences make up a portion of SEQ ID NO:1. The nucleotide sequence of the coding regions and the amino acid sequence of the melatonin 1a receptor is also included in SEQ ID NO:1 and are disclosed in applicant&#39;s U.S. application Ser. No. 08/261,857 filed Jun. 17, 1994, Ser. No. 08/319,887 filed Oct. 7, 1994, and Ser. No. 08/446,103 filed Jun. 6, 1995, which applications are incorporated herein by reference in their entirety. 
     In various preferred embodiments, the transcriptional regulatory regions are derived from a vertebrate animal, such as a mouse, human, or sheep. 
     In an aspect of the invention, a nucleic acid construct is provided which includes a melatonin 1a promoter operably linked to a gene of interest, such as a reporter gene. Preferably, a melatonin 1a promoter has a nucleotide sequence substantially identical to a portion of SEQ ID NO:1 that contains transcriptional regulatory sequences of a melatonin 1a receptor gene. 
     In an embodiment of the invention, the melatonin 1a transcriptional regulatory region of the construct hybridizes under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:1. 
     In another embodiment, the construct of the invention includes a melatonin 1a regulatory region having approximately 95% homology to transcriptional regulatory sequences of SEQ ID NO:1. 
     In other related aspects, the invention features vectors which contain such isolated DNA and which are preferably capable of directing expression of a gene of interest encoded by the DNA in a vector-containing cell; and cells containing such vectors (preferably eukaryotic cells, e.g., RT2-2 cells (obtained from C. Mahle, Bristol-Myers Squibb, New Jersey) or CHO cells (ATCC; Cat. No. CCL 61 or COS-7 cells (ATCC; Cat. No. CRL 1651). Preferably, such cells are stably transfected with such isolated DNA. 
     In another aspect, the invention features a method of screening candidate compounds for their ability to alter transcription from a functional melatonin 1a receptor gene promoter. The method involves: a) providing a cell comprising a recombinant mouse melatonin 1a receptor gene promoter operably linked to a reporter gene; b) contacting the cell with a candidate compound; and c) monitoring the expression of the reporter gene in the presence of the candidate compound. 
     In preferred embodiments, the method includes a cell comprising a recombinant melatonin 1a receptor gene promoter, which promoter has a nucleotide sequence substantially identical to transcriptional regulatory sequences of SEQ ID NO:1. 
     In another preferred embodiment, the promoter is capable of hybridizing under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence complementary to SEQ ID NO:1, and preferably the portion of SEQ ID NO:1 corresponding to part or all of the 5&#39; untranslated region and/or the 3&#39; untranslated region. 
     In yet another preferred embodiment, the promoter includes the nucleotide sequence from approximately nucleotide -1142 to nucleotide +26 of SEQ ID NO:1 (FIG. 2), which sequence contains genetic elements functional in the transcription and translation of operably attached genetic sequences. An embodiment of the invention further includes sequences in the region 3&#39; of the coding sequence, preferably from nucleotide +1165 (immediately 3&#39; of the stop codon) to nucleotide +1606 (3&#39; of the poly(A) signal) (FIG. 2). Intronic sequences (FIG. 2) containing functional genetic elements of the melatonin 1a receptor gene are a further embodiment of the instant invention. Such functional genetic elements include, but are not limited to, enhancers, response elements, cis and/or trans acting factor binding sites, and the like. 
     In preferred embodiments of the screening methods, by operable attachment of the recombinant high-affinity melatonin receptor gene promoter to a gene of interest, the gene of interest is stably expressed by a mammalian cell which does not normally express the gene of interest. The gene of interest may be any gene expressible in a cell and whose transcription is detected after introduction into a cell. Preferred genes of interest are reporter genes which include, but are not limited to, the coding sequence for β-galactosidase, antibiotic resistance genes, SV40 T antigen, chloramphenicol acetyltransferase (CAT), human growth hormone (hGH), and the like. 
     By &#34;transcriptional regulatory regions&#34; or &#34;transcriptional regulatory sequences&#34; or &#34;promoter&#34; is meant nucleic acid sequences upstream and downstream of the coding region of a melatonin 1a receptor gene, which sequences function in the initiation of transcription of the coding region into mRNA and encode sequences functional in translation of the mRNA into the amino acid sequence of the high-affinity melatonin receptor polypeptide. The transcriptional regulatory sequences are the minimal sequences sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5&#39; or 3&#39; regions of the native gene. 
     By &#34;reporter gene&#34; is meant a gene whose expression may be assayed; such genes include, without limitation, luciferase, β-galactosidase, and chloramphenicol transacetylase (CAT). 
     By &#34;operably linked&#34; is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). 
     By &#34;positioned for expression&#34; is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a recombinant protein or a RNA molecule). 
     By &#34;high-affinity melatonin receptor polypeptide&#34; is meant all or part of a vertebrate cell surface protein which specifically binds melatonin and signals the appropriate melatonin-mediated cascade of biological events (e.g., a decrease in intracellular cAMP) concentration. The polypeptide is characterized as having the ligand binding properties (including the agonist and antagonist binding properties) and tissue distribution described herein. 
     By a &#34;polypeptide&#34; is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation). 
     By a &#34;substantially identical&#34; nucleic acid sequence is meant a nucleic acid sequence which differs by less than 10%, preferably less than 5% of nucleic acids of the transcriptional regulatory regions of the melatonin 1a receptor gene. Preferably, a substantially identical nucleic acid sequence of the transcriptional regulatory regions are functional initiating transcription at substantially the same frequency as the regulatory regions of SEQ ID NO:1, preferably at a frequency of at least 50%, more preferably 80% of the frequency initiated by the regulatory regions of SEQ ID NO:1. The nucleic acid sequence of SEQ ID NO:1 or a substantially identical sequence may be isolated from a vertebrate animal or may be chemically synthesized by standard techniques. 
     By &#34;derived from&#34; is meant encoded by the genome of that organism and present on the surface of a subset of that organism&#39;s cells. 
     By &#34;isolated DNA&#34; is meant a DNA that is not immediately contiguous with (i.e., covalently linked to) both of the coding sequences with which it is immediately contiguous (i.e., one at the 5&#39; end and one at the 3&#39; end) in the naturally-occurring genome of the organism from which the DNA of the invention is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. 
     By &#34;transformed cell&#34; is meant a cell into which (or into an ancestor of which) has been introduced, by means of genetic engineering, a DNA molecule encoding the transcriptional regulatory regions of a high-affinity melatonin receptor (or a fragment or analog, thereof). Such a DNA molecule is &#34;positioned for expression&#34; meaning that the DNA molecule is positioned adjacent to a DNA sequence and directs transcription and translation of the sequence (i.e., facilitates the production of genetic transcript and/or protein of interest, or fragment or analog, thereof). 
     A &#34;transgenic animal&#34; as used herein denotes an animal (such as a non-human mammal) bearing in some or all of its nucleated cells one or more genes derived from a different species (exogenous); if the cells bearing the exogenous gene include cells of the animalls germline, the gene may be transmissible to the animal&#39;s offspring. As used herein, genes derived from a different species of animal are exogenous genes. Preferably the exogenous genes include nucleotide sequences which effect expression of the gene in its endogenous tissue distribution. 
     The melatonin 1a receptor transcriptional regulatory regions of the invention control the expression of melatonin 1a, a receptor likely to be involved in the function of vertebrate circadian rhythm. Such transcriptional regulatory regions are therefore useful to develop therapeutics to treat such conditions as jet lag, facilitate reentrainment of some endogenous melatonin rhythms, synchronize the disturbed sleep-wake cycle of blind people, alleviate sleep disorders in shift workers, facilitate the emergence of a diurnal sleep-wake pattern in neonates, regulate ovarian cyclicity in human females, control the initiation and timing of puberty in humans, and alter the mating cycle in seasonally breeding animals, such as sheep. Preferred therapeutics include 1) transcriptional activators, e.g., compounds which increase transcription of an operably attached gene of interest through interaction with the high affinity melatonin receptor gene promoter of the invention; and 2) transcriptional inactivators, e.g., compounds which block high-affinity melatonin receptor gene promoter function by interfering with the transcriptional activator:promoter interaction. 
     Because the receptor gene promoter component may now be produced by recombinant techniques and because candidate compounds may be screened using transformed, cultured cells, the instant invention provides a simple and rapid approach to the identification of useful therapeutics. Such an approach was previously difficult because of the localization of the receptor and its genetic regulatory regions to a few discrete regions in the central nervous system of most mammals. Isolation of the high-affinity melatonin receptor transcriptional regulatory regions allows expression of a gene of interest in a cell type which does not normally express the gene of interest, providing a system for assaying a melatonin 1a receptor transcriptional activator or inhibitor. 
     Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. 
     DETAILED DESCRIPTION 
     The drawings will first briefly be described. 
    
    
     DRAWINGS 
     FIG. 1 is a schematic representation of the structure of the mouse Mel 1a  melatonin receptor gene. The genomic map shows the receptor gene with exons depicted as bars. Translated regions of the exons are black bars, while the 5&#39; and 3&#39; untranslated regions are open bars. Below the gene, the receptor cDNA is shown with the coding region depicted as a wide bar; transmembrane domains I to VII are indicated. The thin lines depict the untranslated regions. Dashed lines between the gene and cDNA show the portions of the gene encoding the cDNA. 
     FIGS. 2A and 2B are digrams is a diagram of the nucleotide sequence of the mouse Mel 1a  receptor gene. Nucleotide sequence is numbered from the major transcription start site which defines the start of the first exon. In the coding region, consensus sites for N-linked glycosylation (box) and protein kinase C phosphorylation (oval) are indicated and the transmembrane regions are underlined. In the 3&#39; untranslated region, an ATTTA sequence (bold print) and consensus sequence for a polyadenylation signal (underlined) are highlighted. The nucleotide and amino acid numbers are to the right of each line. The sequence has been deposited in GenBank under accession number U52222. 
     FIGS. 3A and 3B are diagrams of strategies for determining the transcription start sites of the mouse Mel 1a  receptor gene. A. 5&#39;-RACE analysis was used to clone upstream sequences. After reverse transcription, PCR amplification was performed using a primer in the amino terminus and a 5&#39; anchor primer. The vertical lines depict the location of 15 clones sequenced. The star (*) denotes 6 clones that ended 103 nucleotides upstream of the translation start codon;  +   denotes 2 clones that ended 133 nucleotides upstream of the translation start codon. B. RNase protection analysis was used to further define the transcription start site. The cRNA probe (335 nucleotides in length) and protected fragment (186 base pairs in length) are shown. 
     FIG. 4 is a bar graph showing that the 5&#39; flanking region of the mouse Mel 1a  receptor gene functions as a promoter in RT2-2 cells. RT2-2 cells were transfected with a pGL2-Basic luciferase reporter vector containing 1.1 kb of the 5&#39; flanking region or the pGL2 vector without the insert. Results are mean ± SEM of four independent experiments. 
    
    
     EXAMPLES 
     There now follows a description of the cloning and characterization of the high-affinity melatonin 1a receptor transcriptional regulatory regions from mouse. Transformed cells containing and expressing DNA operably attached to the transcriptional regions of mouse melatonin 1a receptor are also described. These examples are provided for the purpose of illustrating the invention, and should not be construed as limiting. 
     Example 1 
     Molecular Cloning of Transcriptional Regulatory Regions from Mouse Melatonin 1a Receptor 
     A 466 bp fragment of the mouse Mel 1a  melatonin receptor gene was cloned from genomic DNA by PCR. Genomic DNA or first strand cDNA underwent 30-35 cycles of amplification by PCR using two oligonucleotide primers (200 nM final concentration). Each reaction cycle consisted of incubation at 94° C. for 45 sec, 45° C. for 2 min (for degenerate primers) or 60° C. for 2 min (for specific primers), and 72° C. for 2 min, with AmpliTaq DNA Polymerase (Perkin-Elmer Cetus, Foster City Calif.). Amplified DNA was separated on an agarose gel and paper-eluted as previously described (Reppert, S. M. et al. (1994) Neuron 13:1177-1185). The DNA was then subcloned into PCRII using a TA cloning kit (Invitrogen, San Diego Calif.), or digested with restriction enzymes and subcloned into pBluescriptII SK+ or KS+ (Stratagene, La Jolla Calif.). 
     PCR was performed using degenerate oligonucleotide primers based on amino acid residues in the third and seventh transmembrane domains conserved among the previously cloned mammalian Mel 1a  receptors (see, for example, U.S. application Ser. No. 08/466,103, incorporated herein by reference in its entirety). Polymerase chain reaction (PCR) of mouse genomic DNA yielded a 466 bp fragment that was 94% identical at the amino acid level to the rat and Djungarian hamster Mel 1a  receptor cDNAs. 
     The PCR-generated fragment of the mouse Mel 1a  receptor was used to probe a mouse genomic library. A BALB/c adult mouse liver EMBL3 SP6/T7 genomic library (Clontech, Palo Alto Calif.) was plated (12 plates; 100,000 pfu/plate) and transferred to Colony Plaque Screen filters (New England Nuclear, Boston Mass.). The filters were screened under conditions of either high or reduced stringency as previously described (Reppert, S. M. et al. (1994) supra). Probes were cDNA fragments labeled with [alpha- 32  P]dCTP (3000 Ci/mmol) by the method of random priming (Pharmacia, Piscataway N.J.). Phage that hybridized to probe were plaque-purified. 
     Ten positively hybridizing clones were isolated and plaque-purified. In the sheep and human Mel 1a  receptor genes, there is an intron in the region coding the first intracellular loop. Thus, the genomic library was reprobed with a 160 bp fragment of the coding region of the sheep Mel 1a  receptor immediately upstream from the GN motif that resides in the first cytoplasmic loop (Reppert, S. M. et al. (1994) Neuron 13:1177-1185). The sheep fragment hybridized to a separate population of ten clones. These clones did not overlap with the clones found using the mouse fragment, suggesting the presence of a large intron. Both sets of phage clones were mapped by restriction endonucleases, and PCR and Southern analyses. Selected genomic fragments were subcloned and sequenced by standard techniques. 
     Comparison of the genomic sequence with the coding and untranslated regions of the cDNA showed that the mouse Mel 1a  receptor gene is composed of two exons. Exon 1 encodes the entire 5&#39; untranslated region and the coding region through the first cytoplasmic loop (see FIG. 1). Exon 2 encodes the rest of the coding region and the entire 3&#39;-untranslated region. Based on restriction endonuclease mapping of genomic clones, the intron length is greater than 13 kb. The intron has conserved 5&#39; GT-AG 3&#39; sequence for splicing. Previous studies have shown that the mouse Mel 1a  receptor gene (Mtnr1a) is a single copy gene and resides on chromosome 8 (Slaugenhaupt, S. A. et al. (1994) Genomics 27:355-357). 
     Example 2 
     Expression of Melatonin 1a Receytor Gene in Mammalian Cells 
     Based on studies in rats and hamsters, Mel 1a  melatonin receptor mRNA is expressed at low levels with limited distribution in brain (Reppert, S. M. et al. (1994) Neuron 13:1177-1185). A vertebrate cell line expressing the Mel 1a  receptor was sought for analysis of the 5&#39;- and 3&#39;-untranslated regions of the receptor DNA. RT2-2 cells are a cell line of immortalized retinal amacrine cells derived from SV40 T antigen-induced tumors in transgenic mice (Hammang, J. P. et al. (1990) Neuron 4:775-782; RT2-2 cells were provided by C. Mahle, Bristol-Myers Squibb, New Jersey). RT2-2 cells bind melatonin with high affinity (K d  =50 to 100 pM) and modest capacity (B max  20 to 40 fmol/mg protein) suggesting that these cells express a high-affinity melatonin receptor (Mahle, C. D. et al. (1994) Abstract, Program of the 24th Annual Meeting of The Society for Neuroscience, Miami, Fla., p. 535). 
     RNA extraction from RT2-2 cells was performed as follows. RT2-2 cells were grown as a monolayer at 37° C. in 5% CO 2  in OPTI-MEM I (GIBCO/BRL, Grand Island N.Y.) with 10% heat-inactivated fetal calf serum. Total RNA was extracted from RT2-2 cells using an acid-phenol method (Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem 162:156-159). Poly(A) +   RNA was prepared as previously described (Reppert, S. M. et al. (1991) Mol. Endocrinol. 5:1037-1048). Abundant mRNA for the Mel 1a  receptor was identified in RT2-2 cells by RNase protection analysis using the PCR-generated receptor fragment. 
     Sequence analysis of two sets of clones from the mouse genomic library delineated the apparent 5&#39; and 3&#39; ends of the coding region. RT-PCR of MRNA from RT2-2 cells using specific primers flanking the 5&#39; and 3&#39; ends of the coding region amplified the expected cDNA with the intron spliced out at the predicted splice sites. The PCR-generated cDNA was subcloned into pcDNA3 for sequence analysis and in situ hybridization. 
     The mouse Mel 1a  receptor encodes a protein of 353 amino acids and has structural features characteristic of the melatonin receptor family (see applicant&#39;s related U.S. application Ser. No. 08/466,103). These include an NRY motif just downstream from the third transmembrane domain, a C(C/Y)ICH motif immediately downstream from NRY and an NAXXY motif in transmembrane 7. Overall, the coding region of the mouse receptor is 83% and 85% identical at the amino acid level with the sheep and human Mel 1a  receptors, respectively; the sheep and human receptors are the only other members of this subtype in mammals in which the full length coding region has been cloned, sequenced, and expressed (U.S. application Ser. No. 08/466,103; and Reppert, S. M. et al. (1994) Neuron, supra). The mouse Mel 1a  receptor has 2 consensus sites for N-linked glycosylation in the amino terminus. The second cytoplasmic loop contains two consensus sites for protein kinase C phosphorylation that may be involved in receptor regulation. The carboxyl tail also has several serine residues that might serve as phosphorylation sites. 
     Example 3 
     Analysis of the Mouse Melatonin 1a Receptor Gene 5&#39; Untranslated Region 
     Transcription start sites for the Mel 1a  receptor gene were examined using 5&#39;-RACE. 5&#39;-RACE was performed using the 5&#39;-AmpliFINDER RACE Kit (Clontech). First strand cDNA was generated by reverse transcription of poly(A) +   RNA from RT2-2 cells using a primer corresponding to a nucleotide sequence in transmembrane 1 of the mouse Mel 1a  melatonin receptor. The AmpliFINDER anchor was ligated to the cDNA ends using T4 RNA ligase. The cDNA was then amplified using an anchor primer (complementary to the AmpliFINDER anchor) and a primer nested immediately upstream of the primer used for reverse transcription. The amplified cDNA was separated on an agarose gel. Specific bands were paper eluted, subcloned into pCRII, and sequenced. 
     Primer 5&#39;-GTACACAGACAGGATGACCAGCAG-3&#39; (SEQ ID NO:3) corresponding to +248 to +271 in transmembrane 1 was used for reverse transcription, while primer 5&#39;-CAGCAGGTTGCCCAGAATGTCCACCAC-3&#39; (SEQ ID NO:4), which nested just upstream at +227 to +253, was used for PCR amplification. Sequence analysis of the 5&#39;-RACE clones indicated the presence of multiple potential start sites (FIG. 3A). In the 5&#39;-untranslated region, the cDNA sequence matched the genomic sequence, indicating that introns were not present. Of 15 clones sequenced, 6 ended 103 bp upstream of the translation start codon suggesting that this was the major transcription start site. The two longest cDNAs extended 133 bp upstream of the translation start codon (FIG. 3A). 
     To verify the 5&#39;-RACE results, RNase protection analysis was performed using a 335 bp fragment generated by PCR using primers derived from the mel 1a  genomic sequence (FIG. 3B). One primer (5&#39;-TTCAAGCTTAGCCAGGACGGTCGTGGTCTCCCT-3&#39; (SEQ ID NO:5)) corresponded to +166 to +189 in the amino terminus. The other primer (5&#39;-TCAGAATTCGAGTCCAAGTTGCTGGGCAGTGGA-3&#39; (SEQ ID NO:6)) was 125 to 149 nucleotides upstream of the putative transcription start site suggested by 5&#39;-RACE analysis. The resulting fragment was subcloned and linearized to generate a CRNA probe. The probe was made with  32  p-UTP (up to 6000 Ci/mmol) using the MAXIscript system (Ambion, Austin Tex.). Ribonuclease protection assays were run using the RPAII system (Ambion), as previously described (Reppert, S. M. et al. (1994) Neuron 13:1167-1176). Total RNA or poly(A) +   RNA was mixed with approximately 10 5  cpm of each probe in a 20 ml volume of hybridization buffer. Hybridization mixtures were heated to 95° C. for 3 min and then incubated at 45° C. overnight. Unhybridized RNA was digested by a RNase A/RNase T1 mixture for 1 hr at 37° C. Samples were boiled and separated on 8M urea/5% acrylamide gels. For all RNase protection analyses, control samples were run using yeast RNA, sense probes, and omission of RNase. 
     RNase protection analysis results confirmed that the predominant transcription start site was 103 bp upstream of the translation start codon, with minor sites to 133 bp upstream. Sequencing of the cloned 5&#39; upstream region was performed by standard techniques. 
     Sequence analysis of 1.1 kb of the 5&#39; flanking region revealed that it did not contain typical TATA or CAAT boxes in the region of the transcription start sites (FIG. 2). However, there was an AT rich region at -27 to -34 nucleotides. No consensus binding sequences for cAMP regulatory elements were identified in the sequenced region. 
     To establish that this region functioned as a promoter, a 1.1 kb fragment of the 5&#39; flanking region, corresponding to the region -1132 to +26 relative to the transcription start site (see FIG. 2), was subcloned into the pGL2-Basic luciferase reporter vector (Promega, Madison Wis.) by standard techniques. The insert-containing vector or vector without insert was transfected into RT2-2 cells (2 μg cDNA/35 mm well) using a modified diethylaminoethyl dextran method (Cullen, B. R. (1987) Methods Enzymol. 152:684-704). Two days after transfection, cell lysates were harvested with Reporter Lysis Buffer (Promega). Luciferase activity in lysates was measured by a Luminoskan luminometer (Labsystems, Helsinki) (dewet, J. R. et al. (1987) Mol. Cell. Biol. 7:725-737). For each cDNA construct, triplicate 35 mm wells were assayed in duplicate. Consistent transfection efficiency was verified by cotransfection with a pCMV-β-galactosidase vector in preliminary studies (Rosenthal, N. (1987) Methods Enzymol. 152:704-720). As a positive control, parallel plates were transfected with pCMV-luciferase in each experiment (Treacy, M. N. et al. (1991) Nature 350:577-584). 
     Luciferase activity in RT2-2 cells transfected with the 5&#39; flanking/pGL2 construct was 160±3.4 (mean±SEM), while luciferase activity was 1.4±0.4 in RT2-2 cells transfected with the pGL2 vector lacking insert (FIG. 4). Thus, the 1.1 kb flanking region of the mouse receptor gene caused a mean 114-fold induction of luciferase activity over control (vector alone) values. This showed that the 5&#39; flanking region immediately upstream of the transcription start site from approximately nucleotide -1132 to nucleotide +62 relative to the transcription start site (FIG. 2) acts as a functional promoter in RT2-2 cells. 
     A TATA box is not present in the mouse receptor gene promoter to position RNA polymerase II for the initiation of transcription. Lack of a TATA sequence is common among genes possessing several transcription start sites, as is the case for the mouse Mel 1a  receptor gene. Indeed, many G-protein coupled receptor genes are likewise missing a TATA box in their promoters, including genes for luteinizing hormone (Tsai-Morris, C. H. et al. (1991) J. Biol. Chem. 266:11355-11359), follicle-stimulating hormone (Heckert, L. L. et al. (1992) Molecular Endocrinology 6:70-80), thyrotropin (Ikuyama, S. et al. (1992) Molecular Endocrinology 6:793-804), gonadotropin-releasing hormone (Albarracin, C. T. et al. (1994) Endocrinology 135:2300-2306), and oxytocin (Rozen, F. (1995) Proc. Natl. Acad. Sci. USA 92:200-204) receptors; multiple start sites are found in the genes for all of these receptors. In the mouse Mel 1a  receptor gene promoter, an AT-rich region which may be involved in the initiation of transcription (Hahn, S. et al. (1989) Proc. Natl. Acad. Sci. USA 86:5718-5722; Singer, V. L. et al. (1990) Genes and Development 4:636-645; and Wobbe, C. R. and Struhl, K. (1990) Molecular and Cellular Biology 10:3859-3867) is located 27 to 34 nt upstream of the major cap site. GC-rich regions, which often surround a TATA box, surround this AT-rich region. The GC content of the first 200 bases upstream of the major transcription start site is 65%, similar to that reported for other G protein-coupled receptor genes (Ikuyama, S. et al. (1992) supra). However, there are no canonical GC or CAAT boxes present in the mouse mel 1a  receptor gene promoter. 
     A functional promoter was shown to be present in the 1.1 kb 5&#39; flanking region of the mouse Mel 1a  receptor gene. The 114-fold increase in luciferase activity in RT2-2 cells transfected with vector containing the Mel 1a  receptor gene promoter compared to cells transfected with a promoterless reporter vector showed that the promoter was functional in RT2-2 cells. 
     The sequence of the 5&#39; flanking region of the mouse Mel 1a  receptor gene was analyzed for elements involved in regulation of transcription. The GCG (Genetics Computer Group, Madison Wis.) Wisconsin Package MAP command was utilized with the data file tfsites.dat, defined by the logical name genmoredata. 
     Example 4 
     Analysis of the 3&#39; Untranslated Region of Mouse Melatonin 1a Receptor Gene 
     The length of the 3&#39; untranslated region was determined by 3&#39;-RACE using two rounds of PCR amplification. 3&#39;-RACE was performed using the 3&#39;-RACE System (GIBCO/BRL, Bethesda Md.). First strand cDNA was generated using an Adapter Primer which is an oligo(dT) primer engineered to contain three restriction endonuclease sites at the 5&#39; end. The cDNA was then amplified by PCR using an Anchor Primer complementary to the specific 5&#39; sequence of the Adapter Primer and a primer corresponding to nucleotide sequence in transmembrane 6 of the mouse mel 1a  receptor. A second amplification step used the Anchor Primer and a primer nested in transmembrane 7 downstream of the one used in the first amplification. The cDNA of the second amplification was separated on an agarose gel. A single band was paper eluted, subcloned into pCRII, and sequenced. 
     The first round of amplification used primer 5&#39;-ACTCAACCTCATAGGTCTTATTGT-3&#39; (SEQ ID NO:7) corresponding to +871 to +894 in transmembrane 6, while the second round used primer 5&#39; CCAGAGTGGCTGTTCGTGGCTAGT 3&#39; (SEQ ID NO:8) nested downstream at +929 to +952 in transmembrane 7 (FIG. 2). Sequence analysis of 3&#39;-RACE clones consistently revealed a 650 bp cDNA fragment which included 444 bp of the 3&#39; untranslated region ending with a poly(A) tail. The fragment contained an ATTTA sequence that may be involved in regulating mRNA stability (Sachs, A. B. (1993) Cell 74:413-421) and the polyadenylation consensus sequence ATTAAA 18 bp before the start of the poly(A) tail (FIG. 2). The 3&#39; untranslated sequence matched the sequence found in genomic DNA downstream of the coding region, indicating that introns are not present in the 3&#39; untranslated region. 
     The 3&#39; untranslated regions of genes are believed to be involved in mRNA stability, and may contain sequences that regulate gene activity (Jackson, R. J. and Standart, N. (1990) Cell 62:15-24). Thus, it is noteworthy that the 3&#39; untranslated region of the mouse Mel 1a  receptor gene contains an AU-rich element (ATTTA). This element can induce mRNA instability (Sachs, A. B. (1993) supra), suggesting that the mouse Mel 1a  receptor transcript might be unstable and short-lived. Deletion or alteration of the AU-rich element to alter Mel 1a  receptor transcript stability is within the scope of the invention for increasing expression of Mel 1a . 
     Example 5 
     Tissue Distribution of Mouse Melatonin 1a Receytor Gene Expression 
     Northern blot analysis of RNA from RT2-2 cells confirmed the RNase protection analysis result that the Mel 1a  receptor was expressed moderately in these cells. Hybridization of the blot with the PCR-generated Mel 1a  probe from exon 2 revealed two transcripts: an intense hybridization signal at approximately 1.8 kb and a much weaker signal at approximately 7 kb relative to transcript size standards. No hybridization signal was detected in whole mouse brain. 
     In rats and hamsters, the Mel 1a  melatonin receptor transcript has been identified in PT and SCN (Reppert, S. M. et al. (1994) Neuron, supra). The distribution of mel 1a  receptor mRNA was examined in the mouse SCN and pituitary by in situ hybridization using a cRNA probe complementary to the entire coding region.  35  S-labeled antisense cRNA probes were generated by HindIII digestion of pCDNA3 containing the entire coding region of the mouse receptor, followed by in vitro transcription with SP6 RNA polymerase (Ambion) in the presence of [ 35  S]alpha-thio-UTP (New England Nuclear; 1100-1200 ci/mmol) as previously described (Reppert, S. M. (1991) Mol. Endocrinol. 5:1037-1048). As a control, sense CRNA probes were generated from each plasmid and processed along with the antisense probes. In addition to the distribution of Mel 1a  receptor mRNA in mouse SCN and pituitary, the distribution of high-affinity melatonin binding sites was examined on adjacent sections by in vitro autoradiography using  125  I-Mel. 
     The highest level of receptor MRNA was found in mouse PT. The SCN of adult mice also displayed a moderate hybridization signal. Intense  125  I-Mel binding was found in both PT and SCN. In situ hybridization analysis of the SCN in newborn mice showed an intense hybridization signal in SCN and a diffuse signal in lateral hypothalamus. Examination of emulsion autoradiograms from these animals showed that the hybridization signal was more intense in the ventral SCN. Within this region, only scattered cells displayed silver grains. There was no obvious day-night difference in hybridization intensity in the adult SCN. 
     Example 6 
     Expression of an Exogenous Gene from a Vertebrate Melatonin 1a Receptor Gene Promoter 
     An expression cassette, genetic construct, or expression vector containing a gene of interest (such as a reporter gene) operably linked to the transcriptional regulatory regions of a vertebrate Mel 1a  receptor gene promoter (such as from a mouse) according to the invention may be expressed by transformation of a suitable host cell. 
     Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant expression construct. The precise host cell used is not critical to the invention provided the host cell expresses DNA operably linked to the mouse Mel 1a  receptor gene promoter. A host cell useful for the expression from the mouse Mel 1a  receptor gene promoter is the RT2-2 cell (Bristol-Myers Squibb, New Jersey). The method of transfection and the choice of expression vehicle will depend on the host system selected. Transformation and mammalian cell transfection methods are described, e.g., in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, (1989)); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (Pouwels, P. H. et al., (1985), Supp. 1987). 
     One particularly preferred expression system is the RT2-2 cell transfected with a pGL2-Basic luciferase vector containing the 1.1 kb 5&#39; flanking sequence from -1132 to +26 bp upstream of the transcription start site of the mouse Mel 1a  receptor gene as described above. DNA encoding the mouse Mel 1a  receptor gene promoter was inserted into the pGL2-Basic luciferase vector in an orientation designed to allow expression of luciferase. The expression construct may be used in a screening method of the invention. 
     Alternatively, the mouse Mel 1a  receptor gene promoter:reporter gene construct is expressed by a stably-transfected mammalian cell line. 
     A number of vectors suitable for stable transfection of mammalian cells are available to the public, e.g., see Pouwels et al. (supra); methods for constructing such cell lines are also publicly available, e.g., in Ausubel et al. (supra). In one example, cDNA encoding the Mel 1a  promoter:reporter gene construct is cloned into an expression vector which includes a promoterless second reporter gene. Such a construct allows integration of the plasmid and, therefore, the Mel 1a  promoter:reporter gene construct into the host cell chromosome to be selected for, such as by inclusion of a substrate for the second reporter gene (as described in Ausubel et al., supra). This dominant selection can be accomplished in most cell types. 
     Expression of a Mel 1a  promoter:reporter gene (e.g., produced by any of the expression systems described herein) may be assayed by immunological procedures, such as Western blot or immunoprecipitation analysis of recombinant cell extracts, or by immunofluorescence of intact recombinant cells (using, e.g, the methods described in Ausubel et al., supra); by light emission assays (using, e.g., luciferase as described herein); by colorimetric assay (using, e.g., β-galactosidase expression), for example. 
     Example 7 
     Screening For Melatonin 1a Receptor Gene Promoter Activators and Inhibitors 
     As discussed above, one aspect of the invention features screening for compounds that activate transcription from the mouse Mel 1a  receptor gene transcriptional regulatory sequences, thereby inducing an increase in the amount of Mel 1a  receptor expressed cells of the SCN and PT, and in turn increasing sensitivity to circulating melatonin. An element of the screen is recombinant Mel 1a  receptor gene promoter:reporter gene transfected into a cell allowing expression of the Mel 1a  receptor gene promoter (such as RT2-2 cells). Preferably, the Mel 1a  receptor gene promoter is from a vertebrate animal such as a mouse. Other elements of the screen include contacting the transfected cell with a candidate compound and monitoring an increase in reporter gene activity as indicative of increased expression from the Mel 1a  receptor gene promoter. 
     A screen for a compound capable of inhibiting Mel 1a  receptor gene promoter function is screened in the same manner as described for transcription activators except that a decrease in reporter gene activity is monitored. 
     Preferably, such screening assays are carried out using a cell line (such as RT2-2) stably transfected with the Mel 1a  receptor gene promoter:reporter gene construct. Most preferably, the untransfected cell line expresses substantially no reporter gene. 
     Transcriptional activators may be expected to be a useful therapeutic agent for circadian rhythm disorders such as jet lag, day/night cycle disorders in humans or mating cycle alterations in animals such as sheep by increasing the number of melatonin receptors on the surface of cells in the SCN or PT and thereby increasing the sensitivity of the animal to circulating melatonin. 
     Transcriptional inhibitors may be expected to be a useful therapeutic agent for disorders treatable by decreasing the number of melatonin receptors on the surface of SCN or PT cells thereby decreasing the sensitivity of the animal to melatonin. Such disorders include, but are not limited to, controlling reproductive cycles in mammals, such as controlling the initiation of timing of puberty in humans. 
     Example 8 
     Preparation of a Transgenic Animal Containing a Recombinant Melatonin 1a Regulatory Seguence:Reporter Gene Cassette 
     There are several means by which transgenic animals can be made. A transgenic animal (such as a mammal) may be constructed by one of several techniques, including targeted insertion of an exogenous melatonin receptor gene into the endogenous gene of the animal, or other methods well known to those skilled in the art. 
     A transgenic mammal whose germ cells and somatic cells contain an exogenous reporter gene under the control of a melatonin receptor gene transcriptional regulatory region is produced by methods known in the art. See, for example, U.S. Pat. No. 4,736,866 describing production of a transgenic mammal, herein incorporated by reference in its entirety. Generally, the DNA sequence encoding an exogenous melatonin 1a receptor gene promoter operably linked to a reporter gene of interest is introduced into the animal, or an ancestor of the animal, at an embryonic stage (preferably the one-cell, or fertilized oocyte, stage, and generally not later than about the 8-cell stage). There are several methods known to the art of introducing a foreign gene into an animal embryo to achieve stable expression of the foreign gene. One method is to transfect the embryo with the gene as it occurs naturally, and select transgenic animals in which the foreign gene has integrated into the genome at a locus which results in its expression. Other methods involve modifying the foreign gene or its control sequences prior to introduction into the embryo. 
     Tissues of transgenic mammals are analyzed for the presence of exogenous reporter gene product, either by directly analyzing mRNA, or by assaying the tissue for exogenous product. 
     Example 9 
     Using the Transgenic Mammal to Determine Melatonin Receptor Gene Promoter Activation or Inhibition 
     The animals described above can be used to determine whether candidate compounds are melatonin promoter activators or inhibitors in vivo. 
     One aspect of the invention features screening for compounds that agonize or antagonize melatonin activity in vivo. The elements of the screen are a Mel 1a  promoter:reporter gene transgenic mammal and a potential melatonin 1a receptor gene promoter activator or inhibitor in a suitable formulation for administration to the mammal. Detection of a change in the phenotype of interest (e.g., sleep/wake cycle or reproductive cycle) relative to a control transgenic mammal to which no promoter activator or inhibitor has been administered indicates a potentially useful candidate compound. 
     Therapy 
     Particularly suitable therapeutics for the treatment of circadian rhythm disorders in humans as well as for regulating changes in the reproductive cycle of seasonally breeding animals are the transcriptional activators and inhibitors described above formulated in an appropriate buffer such as physiological saline. 
     The therapeutic preparation is administered in accordance with the condition to be treated. ordinarily, it will be administered intravenously, at a dosage, of a duration, and with the appropriate timing to elicit the desired response. Appropriate timing refers to the time in the natural circadian rhythm at which administration of therapeutic preparation elicits the desired response. Alternatively, it may be convenient to administer the therapeutic orally, nasally, or topically, e.g., as a liquid or a spray. Again, the dosages are as described above. Treatment may be repeated as necessary for alleviation of disease symptoms. 
     Melatonin 1a receptor gene promoter transcriptional activators can be used to reentrain the endogenous melatonin rhythm of humans; alleviate jet lag symptoms in humans; phase shift the sleep/wake cycle of some blind people, reinforce entrainment of endogenous melatonin rhythm using low intensity light/dark cycle; control ovulation in humans; and alter reproductive cycles in seasonally breeding animals. 
     Melatonin 1a receptor gene promoter transcriptional inhibitors may be useful in controlling reproductive cycles in mammals, such as controlling the initiation or timing of puberty in humans. 
     The methods of the invention may be used to screen therapeutic receptor gene promoter transcriptional activators or inhibitors for their effectiveness in altering expression of a gene of interest in vitro or in vivo by the assays described above. Where a non-human mammal is treated or where a therapeutic for a non-human animal is screened, a melatonin 1a receptor gene regulatory region is preferably specific for that species. 
     Other Embodiments 
     Melatonin 1a receptor transcriptional regulatory regions according to the invention include any melatonin 1a receptor gene promoters (e.g., regulatory regions as described herein). Such promoters may be derived from any source, but are preferably derived from a vertebrate animal, e.g., a mouse, a human, a sheep, or a frog. These promoters are used, e.g., to screen for transcriptional inhibitors which can be used to alter production of melatonin 1a receptor and thereby alter sensitivity to melatonin in cell lines or in whole transgenic animals. 
     Specific receptor gene promoter regions of interest include sequences upstream of the transcription start site of the melatonin 1a receptor gene, within the intron, or downstream of the Mel 1a  coding sequence, which sequences function in a cell to express a gene of interest operably linked to the receptor gene promoter region of the invention. Nucleotide substitutions, deletions, or insertions located at positions of the nucleotide sequence which do not destroy the promoter&#39;s ability to initiate transcription or destroy the ability of the resultant mRNA to be translated are within the scope of the present invention. 
     
         __________________________________________________________________________
#             SEQUENCE LISTING                                            
  - -  - - (1) GENERAL INFORMATION:                                       
  - -    (iii) NUMBER OF SEQUENCES: 8                                     
  - -  - - (2) INFORMATION FOR SEQ ID NO:1:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 2772 base - #pairs                                 
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (ix) FEATURE:                                                   
           (A) NAME/KEY: Coding Se - #quence                              
           (B) LOCATION: 1270...2328                                      
           (D) OTHER INFORMATION: - #intron coding sequence between       
                positions - #1464 and 1465                                
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                         
  - - CGGAGGATGA CCTTGAACCT CTGATCCTTT GCCTTCCCTC CTGGGTGCTG GG -         
#GTTACGTG     60                                                          
   - - ACTGAGGTGC CACATCCAGT TTATACAGCA CTAGAAATGG AGTCTAAGAT TT -        
#TGCAAATG    120                                                          
   - - CTGCACAGGA GCCCTAACGA CAGAGCCACA CGCTCAGGCC CCTCAATTCT GC -        
#ATTGCATT    180                                                          
   - - TCTTCTTGAA ATTATTGATG AACACAACCA TTTTACTTAA TATGATTTGT TG -        
#AGACAGGA    240                                                          
   - - TTTTATACAG CAACACTGAT TGACCACACA GCTCAAGATG CCCCCAAACT GG -        
#TGTTGATT    300                                                          
   - - CTCCTGCCTC AGCCTCCAGA GCTACGACAC ATTGTTTAAT TTTAATACAG AT -        
#TTTAATAT    360                                                          
   - - TGTCATGTCA TGCTTTTCTG GTATTCATCT TCTTAAAATG TATTTTCTTC AT -        
#TTTTCTTC    420                                                          
   - - ACTCTTTCAA AGGGACTTTG GAAATGCTTA GGAATTGGAC AGCCATAAAA TA -        
#TATGGAGA    480                                                          
   - - GCATGAAAAT TTAAGTGTTC AATATGAGAA CATCATATGT TTTGTGTAAG TC -        
#TCCTTCCC    540                                                          
   - - CATGTTGAAG AAAGTTTTGG GGTTTTGTTC CATTCTGTAC AGAGCTGGCT AA -        
#TGCACTTC    600                                                          
   - - CCAGAAATCT TACACTGTGG TTCTACGTCT GCTTCTATTA TCTCAAGTTT CT -        
#GTTTTCAC    660                                                          
   - - TGATAGTTTC AAAAGAACAT ATACACCTGT CATCTGCAAA TATTTACATT TT -        
#GTTTCGTT    720                                                          
   - - TAATTGCCAG AAACACCCAG AACAGACATT AAATCGTGCT ATGCCAGACG GA -        
#AAGGGTGT    780                                                          
   - - GATTTAATAT CATTATTACT TTATTTTTAC ACATCATTGA CTATTAAAGT TG -        
#ATGAGTTA    840                                                          
   - - GATTCTATTT TCATCTTCAT TTTCCGGATA AGACATCCAG GGCATCTAGT CC -        
#TGGTTAGG    900                                                          
   - - ATTCAAGTTC GTGAATCGAG GCCTTCCAGG GTGCAAGTTT CCCTCCACTT GA -        
#TGCCTCCA    960                                                          
   - - CGTGTCTCAC CGAGTCTCGC CACACGGGGG CGCAACGTGC ACGCACTGTG GG -        
#ACCTCCGA   1020                                                          
   - - GTCCAAGTTG CTGGGCAGTG GACAGCAGGT GTCAGCAGGC GGCAGTGGCC AA -        
#GTGCAGAG   1080                                                          
   - - AGGGTGTCCT ACCACCGGGA GGGGGCTGGA GTGGGCAGGA CAGCCGCGAA GC -        
#AATCATAA   1140                                                          
   - - GGATGCAAAG TAGACGCGGG AGGGCCATAA AAAGTGGCGG AGAGGGCTCG AG -        
#CAGAGCTG   1200                                                          
   - - AGCAGTTGAG GGCTCCGGGG CGACAGGACA ATGGCCCTGG CTGTGCTGCG GT -        
#GAGGCACC   1260                                                          
   - - CAGGGGACC  ATG AAG GGC AAT GTC AGC GAG CTG - #CTC AAT GCC ACT CAG  
CAG  1311                                                                 
             Met Lys Gly Asn - #Val Ser Glu Leu Leu Asn Ala Thr Gln Gln   
              1     - #          5        - #          10                 
  - - GCT CCA GGC GGC GGG GAG GGA GGG AGA CCA CG - #A CCG TCC TGG CTG GCC 
    1359                                                                  
 Ala Pro Gly Gly Gly Glu Gly Gly Arg Pro Ar - #g Pro Ser Trp Leu Ala      
  15                 - # 20                 - # 25                 - # 30 
  - - TCT ACA CTG GCC TTC ATC CTC ATC TTT ACC AT - #C GTG GTG GAC ATT CTG 
    1407                                                                  
 Ser Thr Leu Ala Phe Ile Leu Ile Phe Thr Il - #e Val Val Asp Ile Leu      
                  35 - #                 40 - #                 45        
  - - GGC AAC CTG CTG GTC ATC CTG TCT GTG TAC CG - #C AAC AAG AAG CTC AGG 
    1455                                                                  
 Gly Asn Leu Leu Val Ile Leu Ser Val Tyr Ar - #g Asn Lys Lys Leu Arg      
              50     - #             55     - #             60            
  - - AAC TCA GGG AAT ATA TTT GTG GTG AGT TTA GC - #T GTG GCA GAC CTC GTG 
    1503                                                                  
 Asn Ser Gly Asn Ile Phe Val Val Ser Leu Al - #a Val Ala Asp Leu Val      
          65         - #         70         - #         75                
  - - GTG GCT GTT TAC CCT TAT CCC TTG GTG CTG AC - #A TCT ATC CTT AAC AAC 
    1551                                                                  
 Val Ala Val Tyr Pro Tyr Pro Leu Val Leu Th - #r Ser Ile Leu Asn Asn      
      80             - #     85             - #     90                    
  - - GGA TGG AAT CTG GGA TAT CTA CAC TGT CAA GT - #C AGC GCA TTT CTA ATG 
    1599                                                                  
 Gly Trp Asn Leu Gly Tyr Leu His Cys Gln Va - #l Ser Ala Phe Leu Met      
  95                 - #100                 - #105                 - #110 
  - - GGC TTG AGT GTC ATC GGC TCG ATA TTC AAC AT - #C ACG GGG ATC GCT ATG 
    1647                                                                  
 Gly Leu Ser Val Ile Gly Ser Ile Phe Asn Il - #e Thr Gly Ile Ala Met      
                 115  - #               120  - #               125        
  - - AAC CGT TAC TGC TAC ATT TGC CAC AGC CTC AA - #G TAC GAC AAA ATA TAC 
    1695                                                                  
 Asn Arg Tyr Cys Tyr Ile Cys His Ser Leu Ly - #s Tyr Asp Lys Ile Tyr      
             130      - #           135      - #           140            
  - - AGT AAC AAG AAC TCG CTC TGC TAC GTG TTC CT - #G ATA TGG ATG CTG ACA 
    1743                                                                  
 Ser Asn Lys Asn Ser Leu Cys Tyr Val Phe Le - #u Ile Trp Met Leu Thr      
         145          - #       150          - #       155                
  - - CTC ATC GCC ATC ATG CCC AAC CTG CAA ACC GG - #A ACA CTC CAG TAC GAT 
    1791                                                                  
 Leu Ile Ala Ile Met Pro Asn Leu Gln Thr Gl - #y Thr Leu Gln Tyr Asp      
     160              - #   165              - #   170                    
  - - CCC CGG ATC TAC TCC TGT ACC TTC ACC CAG TC - #T GTC AGC TCA GCG TAC 
    1839                                                                  
 Pro Arg Ile Tyr Ser Cys Thr Phe Thr Gln Se - #r Val Ser Ser Ala Tyr      
 175                 1 - #80                 1 - #85                 1 -  
#90                                                                       
   - - ACG ATA GCA GTG GTG GTT TTC CAT TTC ATC GT - #G CCT ATG ATT ATT    
GTC     1887                                                              
  Thr Ile Ala Val Val Val Phe His Phe Ile Va - #l Pro Met Ile Ile Val     
                 195  - #               200  - #               205        
  - - ATC TTC TGC TAC TTA AGG ATA TGG GTC CTG GT - #C CTT CAG GTC AGA CGG 
    1935                                                                  
 Ile Phe Cys Tyr Leu Arg Ile Trp Val Leu Va - #l Leu Gln Val Arg Arg      
             210      - #           215      - #           220            
  - - AGG GTG AAA CCC GAC AAC AAG CCC AAA CTG AA - #G CCC CAG GAC TTC AGG 
    1983                                                                  
 Arg Val Lys Pro Asp Asn Lys Pro Lys Leu Ly - #s Pro Gln Asp Phe Arg      
         225          - #       230          - #       235                
  - - AAC TTT GTC ACC ATG TTC GTA GTT TTT GTA CT - #T TTT GCC ATT TGT TGG 
    2031                                                                  
 Asn Phe Val Thr Met Phe Val Val Phe Val Le - #u Phe Ala Ile Cys Trp      
     240              - #   245              - #   250                    
  - - GCC CCA CTC AAC CTC ATA GGT CTT ATT GTG GC - #C TCA GAC CCT GCC ACC 
    2079                                                                  
 Ala Pro Leu Asn Leu Ile Gly Leu Ile Val Al - #a Ser Asp Pro Ala Thr      
 255                 2 - #60                 2 - #65                 2 -  
#70                                                                       
   - - ATG GTC CCC AGG ATC CCA GAG TGG CTG TTC GT - #G GCT AGT TAC TAC    
CTG     2127                                                              
  Met Val Pro Arg Ile Pro Glu Trp Leu Phe Va - #l Ala Ser Tyr Tyr Leu     
                 275  - #               280  - #               285        
  - - GCG TAC TTC AAC AGC TGC CTC AAC GCA ATT AT - #A TAC GGA CTA CTG AAT 
    2175                                                                  
 Ala Tyr Phe Asn Ser Cys Leu Asn Ala Ile Il - #e Tyr Gly Leu Leu Asn      
             290      - #           295      - #           300            
  - - CAG AAT TTC AGA AAG GAA TAC AAA AAG ATT AT - #T GTC TCG TTG TGC ACA 
    2223                                                                  
 Gln Asn Phe Arg Lys Glu Tyr Lys Lys Ile Il - #e Val Ser Leu Cys Thr      
         305          - #       310          - #       315                
  - - GCC AAG ATG TTC TTT GTG GAG AGT TCA AAT GA - #A GAA GCA GAT AAG ATT 
    2271                                                                  
 Ala Lys Met Phe Phe Val Glu Ser Ser Asn Gl - #u Glu Ala Asp Lys Ile      
     320              - #   325              - #   330                    
  - - AAA TGT AAG CCC TCT CCA CTA ATA CCC AAT AA - #T AAC TTA ATA AAG GTG 
    2319                                                                  
 Lys Cys Lys Pro Ser Pro Leu Ile Pro Asn As - #n Asn Leu Ile Lys Val      
 335                 3 - #40                 3 - #45                 3 -  
#50                                                                       
   - - GAC TCT GTT TAAAAAGCCA GTGGTGCTAG CAGATTATCC ACACTGGTT - #G        
GGGTCTTCC   2377                                                          
  Asp Ser Val                                                             
   - - TGCTCTCCTT GTTTGCTTTC TTTTGTCTAG AAATCAGTCT ATCCAACTTG AA -        
#GCTCTTCA   2437                                                          
   - - GGGTTGCCTC CATAGTGTTG GAAAGGATCT CCTGTCTGCC CCATAATCAG AT -        
#TGCTAGTA   2497                                                          
   - - TCAAGGGGAA TGCTGAACAG GCACACCATA GTTTAAATGG ACAACTTGTA TC -        
#AGCAGAGG   2557                                                          
   - - AGGTCGTGGT GCAGACTCTC TCGTCTCTGG GGCAACCAGG TCTTGGGGGT TG -        
#CCCACATT   2617                                                          
   - - TAGGATTACA ATATACAGCA ACAGACCAAA CCTGAACAAA ATGTGGAAGG AA -        
#CTCAAGAC   2677                                                          
   - - AAGAGGGACC ATGGGGACCT TCCTTTTATT GTAAGCGAGT GATACAGAGT GT -        
#TTATTCTT   2737                                                          
   - - ACCTATGGCT GAATTAAAAT AGTCAAAAAA CTTAA       - #                   
- #     2772                                                              
   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                              
   - -      (i) SEQUENCE CHARACTERISTICS:                                 
            (A) LENGTH: 353 amino - #acids                                
            (B) TYPE: amino acid                                          
            (D) TOPOLOGY: linear                                          
   - -     (ii) MOLECULE TYPE: protein                                    
   - -      (v) FRAGMENT TYPE: internal                                   
   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                        
   - - Met Lys Gly Asn Val Ser Glu Leu Leu Asn Al - #a Thr Gln Gln Ala    
Pro                                                                       
    1               5 - #                 10 - #                 15       
  - - Gly Gly Gly Glu Gly Gly Arg Pro Arg Pro Se - #r Trp Leu Ala Ser Thr 
              20     - #             25     - #             30            
  - - Leu Ala Phe Ile Leu Ile Phe Thr Ile Val Va - #l Asp Ile Leu Gly Asn 
          35         - #         40         - #         45                
  - - Leu Leu Val Ile Leu Ser Val Tyr Arg Asn Ly - #s Lys Leu Arg Asn Ser 
      50             - #     55             - #     60                    
  - - Gly Asn Ile Phe Val Val Ser Leu Ala Val Al - #a Asp Leu Val Val Ala 
  65                 - # 70                 - # 75                 - # 80 
  - - Val Tyr Pro Tyr Pro Leu Val Leu Thr Ser Il - #e Leu Asn Asn Gly Trp 
                  85 - #                 90 - #                 95        
  - - Asn Leu Gly Tyr Leu His Cys Gln Val Ser Al - #a Phe Leu Met Gly Leu 
             100      - #           105      - #           110            
  - - Ser Val Ile Gly Ser Ile Phe Asn Ile Thr Gl - #y Ile Ala Met Asn Arg 
         115          - #       120          - #       125                
  - - Tyr Cys Tyr Ile Cys His Ser Leu Lys Tyr As - #p Lys Ile Tyr Ser Asn 
     130              - #   135              - #   140                    
  - - Lys Asn Ser Leu Cys Tyr Val Phe Leu Ile Tr - #p Met Leu Thr Leu Ile 
 145                 1 - #50                 1 - #55                 1 -  
#60                                                                       
   - - Ala Ile Met Pro Asn Leu Gln Thr Gly Thr Le - #u Gln Tyr Asp Pro    
Arg                                                                       
                  165  - #               170  - #               175       
  - - Ile Tyr Ser Cys Thr Phe Thr Gln Ser Val Se - #r Ser Ala Tyr Thr Ile 
             180      - #           185      - #           190            
  - - Ala Val Val Val Phe His Phe Ile Val Pro Me - #t Ile Ile Val Ile Phe 
         195          - #       200          - #       205                
  - - Cys Tyr Leu Arg Ile Trp Val Leu Val Leu Gl - #n Val Arg Arg Arg Val 
     210              - #   215              - #   220                    
  - - Lys Pro Asp Asn Lys Pro Lys Leu Lys Pro Gl - #n Asp Phe Arg Asn Phe 
 225                 2 - #30                 2 - #35                 2 -  
#40                                                                       
   - - Val Thr Met Phe Val Val Phe Val Leu Phe Al - #a Ile Cys Trp Ala    
Pro                                                                       
                  245  - #               250  - #               255       
  - - Leu Asn Leu Ile Gly Leu Ile Val Ala Ser As - #p Pro Ala Thr Met Val 
             260      - #           265      - #           270            
  - - Pro Arg Ile Pro Glu Trp Leu Phe Val Ala Se - #r Tyr Tyr Leu Ala Tyr 
         275          - #       280          - #       285                
  - - Phe Asn Ser Cys Leu Asn Ala Ile Ile Tyr Gl - #y Leu Leu Asn Gln Asn 
     290              - #   295              - #   300                    
  - - Phe Arg Lys Glu Tyr Lys Lys Ile Ile Val Se - #r Leu Cys Thr Ala Lys 
 305                 3 - #10                 3 - #15                 3 -  
#20                                                                       
   - - Met Phe Phe Val Glu Ser Ser Asn Glu Glu Al - #a Asp Lys Ile Lys    
Cys                                                                       
                  325  - #               330  - #               335       
  - - Lys Pro Ser Pro Leu Ile Pro Asn Asn Asn Le - #u Ile Lys Val Asp Ser 
             340      - #           345      - #           350            
  - - Val                                                                 
  - -  - - (2) INFORMATION FOR SEQ ID NO:3:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 24 base - #pairs                                   
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                         
  - - GTACACAGAC AGGATGACCA GCAG          - #                  - #        
        24                                                                
  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 27 base - #pairs                                   
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                         
  - - CAGCAGGTTG CCCAGAATGT CCACCAC          - #                  - #     
        27                                                                
  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 33 base - #pairs                                   
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                         
  - - TTCAAGCTTA GCCAGGACGG TCGTGGTCTC CCT       - #                  - # 
        33                                                                
  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 33 base - #pairs                                   
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                         
  - - TCAGAATTCG AGTCCAAGTT GCTGGGCAGT GGA       - #                  - # 
        33                                                                
  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 24 base - #pairs                                   
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                         
  - - ACTCAACCTC ATAGGTCTTA TTGT          - #                  - #        
        24                                                                
  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                               
  - -      (i) SEQUENCE CHARACTERISTICS:                                  
           (A) LENGTH: 24 base - #pairs                                   
           (B) TYPE: nucleic acid                                         
           (C) STRANDEDNESS: single                                       
           (D) TOPOLOGY: linear                                           
  - -     (ii) MOLECULE TYPE: cDNA                                        
  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                         
  - - CCAGAGTGGC TGTTCGTGGC TAGT          - #                  - #        
        24                                                                
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