EP1025128A1 - Dna molecules encoding imidazoline receptive polypeptides and polypeptides encoded thereby - Google Patents

Abstract

A genomic DNA encoding a human imidazoline receptor is described. cDNAs encoding the receptor and fragments thereof are also provided. An amino acid sequence predicted to be 120,000 MW for nearly the entire protein is identified, as well as a middle fragment believed to contain the imidazoline binding site of the receptor. The protein is highly unique in its sequence and may represent the first in a novel family of receptor proteins. Methods of cloning the cDNA and expressing the imidazoline receptor in a host cell are described. Methods of preparing antibodies against the transfected protein are also described. Also, a screening method for identifying additional subtypes of this receptor are identified. Also, screening methods for identifying drugs that interact with the imidazoline receptor are described.

Description

DNA MOLECULES ENCODING IMIDAZOLINE RECEPTIVE POLYPEPTIDES AND POLYPEPTIDES ENCODED THEREBY

BACKGROUND OF THE INVENTION

1. Field of the Invention The present invention is directed to DNA molecules encoding imidazoline receptive polypeptides, preferably encoding human imidazoline receptive polypeptides, that can be used as an imidazoline receptor (abbreviated IR) . In addition, transcript (s) and protein sequences are predicted from the DNA clones. The invention is also directed to a genomic DNA clone designated as JEP-1A. The cDNA clones according to the invention comprise cDNA homologous to portion (s) of this genomic clone; including 5A-1 cDNA, cloned by the inventors that established the open-reading frame for translation of mRNA from the gene, and established the immunoreactive properties of its polypeptide sequence in an expression systems. Also, the invention relates to cDNA clone EST04033, which is another clone identified to contain cDNA sequences from the JEP-1A gene, and of which the 5A-1 is a part, that encodes an active fragment of the IR polypeptide in transfection assays, and the protein sequences thereof. The invention also relates to methods for producing such genomic and cDNA clones, methods for expressing the IR protein and fragments, and uses thereof. 2. Description of Related Art It is believed that brainstem imidazoline receptors possess binding site(s) for therapeutically relevant imidazoline compounds, such as clonidine and idazoxan. These drugs represent the first generation of ligands discovered for the binding site(s) of imidazoline receptors. However, clonidine and idazoxan were developed based on their high affinity for α₂-adrenergic receptors. Second generation ligands, such as moxonidine, possess somewhat improved selectivity for IR over α₂-adrenergic receptors, but more selective compounds for IR are needed.

An imidazoline receptor clone is of particular interest because of its potential utility in identifying novel pharmaceutical agents having greater potency and/or more selectivity than currently available ligands have for imidazoline receptors. Recent technological advances permit pharmaceutical companies to use combinatorial chemistry techniques to rapidly screen a cloned receptor for ligands (drugs) binding thereto. Thus, a cloned imidazoline receptor would be of significant value to a drug discovery program. Until now, the molecular nature of imidazoline receptors remains unknown. For instance, no amino acid sequence data for a novel IR, e.g., by N-terminal sequencing, has been reported. Three different techniques have been described in the literature by three different laboratories to visualize imidazoline-selective binding proteins (imidazoline receptor candidates) using gel electrophoresis. Some important consistencies have emerged from these results despite the diversity of the techniques employed. On the other hand, multiple protein bands have been identified, which suggests heterogeneity amongst imidazoline receptors. These reports are discussed below.

Some of the abbreviations used hereinbelow, have the following meanings: ₂AR Alpha-2 adrenoceptor

BAC Bovine adrenal chromaffin

ECL Enhanced chemiluminescence (protein detection procedure) EST Expressed Sequence Tag (a one-pass cDNA documentation without identification)

I-site Any imidazoline-receptive binding site (e.g., encoded on IR)

IR] Imidazoline receptor subtype, IR-Ab Imidazoline receptor antibody

I₂Site Imidazoline binding subtype₂ kDa Kilodaltons (molecular size)

MAO monoa ine oxidase

MW molecular weight NRL European abbreviation for RVLM (see below)

PC-12 Phaeochromocytoma-12 cells

¹²⁵PIC [¹²⁵I]p-iodoclonidine

PKC Protein Kinase C

RVLM Rostral Ventrolateral Medulla in brainstem SDS sodium dodecyl sulfate gel electrophoresis

Reis et al. [Wang et al., Mol. Pharm. , 42: 792-801

(1992); Wang et al., Mol. Pharm. , 43: 509-515 (1993)] were the first to characterize an imidazoline-selective binding protein and to demonstrate it as having MW = 70 kDa. This was accomplished using bovine cells (BAC) , which lack an ₂AR

[Powis & Baker, Mol. Pharm.. 29:134-141 (1986)]. The 70 kDa imidazoline-selective protein in those studies had high affinities for both idazoxan and p-aminoclonidine affinity chromatography columns and was eluted by another imidazoline compound (phentolamine) . Unfortunately, those investigators failed to isolate sufficient 70 kDa protein to determine its other biochemical properties. To date, no one has reported the complete purification of an imidazoline receptor protein. Likewise, no amino acid sequences have been reported for IR. Their 70 kDa protein was used by Reis and co-workers to raise "I-site binding antiseru ", designated herein as Reis antiserum. The term "I-site" refers to the imidazoline binding site, presumably defined within the imidazoline receptor protein. Reis antiserum was prepared by injecting the purified protein into rabbits [Wang et al, 1992]. The first immunization was done subcutaneously with the protein antigen (10 μg) emulsified in an equal volume of complete Freund's adjuvant, and the next three booster shots were given at 15-day intervals with incomplete Freund's adjuvant. The polyclonal antiserum has been mostly characterized by immunoblotting, but radioimmunoassays (RIA) and/or conjugated assay procedures, i.e., ELISA assays, are also conceivable [see "Radioimmunoassay of Gut Regulatory Peptides: Methods in Laboratory Medicine," Vol. 2, chapters 1 and 2, Praeger Scientific Press, 1982].

The present inventors and others [Escriba et al., Neurosci. Lett. 178: 81-84 (1994)] have characterized the Reis antiserum in several respects. For instance, the present inventors have discovered that human platelet immunoreactivity with Reis antiserum is mainly confined to a single protein band of MW α 33 kDa, although a trace band at ~ 85 kDa was also observed. The « 33 and « 85 kDa bands were enriched in plasma membrane fractions as expected for an imidazoline receptor. Furthermore, the intensity of the « 33 kDa band was found to be positively correlated with non-adrenergic ^{I 5}PIC Bmax values at platelet IR, sites in samples from the same subjects, with an almost one-to-one slope factor. In addition, the nonadrenergic ^I25PIC binding sites on platelets were discovered by the present inventors to have the same rank order of affinities as IR, binding sites in brainstem [Piletz and Sletten, J. Pharm. & Exper. Therap.. 267: 1493-1502

(1993)]. The platelet » 33 kDa band may also be a product of a larger protein, since in human megakaryoblastoma cells, which are capable of forming platelets in tissue cultures, an « 85 kDa immunoreactive band was found to predominate. Immunoreactivity with Reis antiserum does not appear to be directed against human ₂AR and/or MAO A/B. This is a significant point because α₂AR and MAO A/B have previously been cloned and also bind to imidazolines. The present inventors have obtained selective antibodies and recombinant preparations for ₂AR and MAO A/B, and these proteins do not correspond to the « 33, 70, or 85 kDa putative IR, bands. Thus, there is substantial evidence that, at least in human platelets, the Reis antiserum is IR, selective.

Another antiserum was raised by Drs. Dontenwill and Bousquet in France [Greney et al., Europ. J. Pharmacol., 265: R1-R2 (1994); Greney et al., Neurochem. Int.. 25: 183-191 (1994); Bennai et al., Annals NY Acad. Sci., 763:140-148 (1995) ] against polyclonal antibodies for idazoxan (designated Dontenwill antiserum) . This anti-idiotypic antiserum inhibits ³H-clonidine but not H-rauwolscine (α₂-selective) binding sites in the brainstem, suggesting it also interacts with IR, [Bennai et al., 1995]. As shown in Fig. 1, human RVLM (same as NRL) membrane fractions displayed bands of « 41 and 44 kDa, as detected by the present inventors using this anti-idiotypic antiserum.

The present inventors have found that the bands of MW ~ 41 and 44 kDa detected by Dontenwill antiserum may be derived from an ~ 85 kDa precursor protein, similar to that occurring in platelet precursor cells. An 85 kDa im unoreactive protein is obtained in fresh rat brain membranes only when a cocktail of 11 protease inhibitors is used. Also, as shown in Fig. 1, it is found that Reis antiserum detects the ~ 41 and 44 kDa bands in human brain when fewer protease inhibitors are used. Additionally, the Dontenwill antiserum weakly detects a platelet « 33 kDa band. Thus, the present inventors have hypothesized that the « 41 and 44 kDa immunoreactive proteins may be alternative breakdown products of an ~ 85 kDa protein, as opposed to the platelet « 33 kDa breakdown product.

In summary, the main conclusion from the above results is that, despite vastly different origins, the Reis and Dontenwill antisera both detect identical bands in human platelets, RVLM, and hippocampus. Using yet another technique, a photoaffinity imidazoline ligand, ¹⁵AZIPI, has also been developed to preferentially label I₂-imidazoline binding sites [Lanier et al., J. Biol. Chem.. 268: 16047-16051 (1993)]. The ¹⁵AZIPI photoaffinity ligand was used to visualize ~ 55 kDa and ~ 61 kDa binding proteins from rat liver and brain. It is believed that the « 61 kDa protein is probably MAO, in agreement with other findings [Tesson et al., J. Biol. Chem. , 270: 9856-9861 (1995) ] showing that MAO proteins bind certain imidazoline compounds. The different molecular weights between these bands and those detected immunologically by the present inventors is one of many pieces of evidence that distinguishes IR_j from I₂ sites. To the inventors' knowledge and as described herein, we are first to clone the gene, cDNAs and fragments thereof encoding a protein with the immunological and ligand binding properties expected of an IR. On this basis, we are first to identify the nucleotide sequences of DNA molecules encoding an imidazoline receptor and active fragments thereof, and the first to determine the amino acid sequence of an imidazoline receptor and active fragments thereof. The polypeptides described herein are clearly distinct from α₂AR or MAO A/B proteins.

SUMMARY OF THE INVENTION

The present invention involves various cDNA clones (ie., 5A-1 and EST04033) and a genomic clone (JEP-1A) which are directed to an isolated polypeptide (s) that is receptive to (bind to) imidazoline compound (s) , and can be used to identify other compounds of interest. Currently available imidazoline compounds in this context are p-iodoclonidine and moxonidine. Initially, the inventors detected a polypeptide expressed by their cDNA clone (5A-1 isolated from a human hippocampus cDNA library) that immunoreacted with Reis antiserum and/or Dontenwill antiserum. The DNA sequence of the 5A-1 clone is encapsulated within a portion of the other clones (EST04033 and JEP-1A genomic clone) . In one aspect of the invention, a polypeptide includes a 651 amino acid sequence as shown in SEQ ID No. 5. This polypeptide is predicted from non-plasmid cDNA in EST04033; a clone which the inventors showed possesses sequences inclusive of 5A-1. Furthermore, transfection of EST04033 into COS cells yielded imidazoline receptivity by radioligand binding assays (described in detail later) . Other imidazoline receptive proteins homologous to this polypeptide are also contemplated. Such polypeptide (s) generally have a molecular weight of about 50 to 80 kDa. More particularly, one can have a molecular weight of about 70 kDa.

In another aspect of this invention, a polypeptide includes a 390 amino acid sequence as shown in SEQ ID No. 6. This represents the polypeptide predicted from the non-plasmid DNA of the original 5A-1 clone. Such a polypeptide generally has a molecular weight of about 35 to 50 kDa. More particularly, it can have a molecular weight of about 43 kDa.

DNA molecules encoding aforementioned imidazoline- receptive polypeptide (s) are also contemplated. Such a DNA molecule, e.g., a cDNA derived from mRNA, can contain a nucleotide sequence encoding the 651 amino acid sequence shown in SEQ ID No. 5. Thus, a DNA molecule containing the 1954 base pairs (b.p.) (1954 b.p. encodes 651 amino acids) nucleotide sequence shown in SEQ ID No. 2 is contemplated. This represents the coding sequence for the polypeptide predicted by EST04033 transfections . In another embodiment, a DNA molecule includes the longer nucleotide sequence shown in SEQ ID No. 3. This represents the cDNA predicted to have been translated + not predicted to have been translated in transfections experiments of EST04033.

In another embodiment of the invention, a DNA molecule contains a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 6. In another aspect, it can include the

1171 b.p. nucleic acid sequence shown in SEQ ID No. 4. The 1171 b.p. nucleic acid sequence shown in SEQ ID No. 4 is the 5A-1 non-plasmid DNA. The nucleic acid sequence of the genomic clone encoding the imidazoline receptor is further shown in SEQ ID No. 21. The nucleic acid and amino acid sequence of the predicted transcript (ie., cDNA) can be predicted from the description hereinbelow. The polypeptide encoded by the genomic DNA is shown in SEQ ID No. 22.

Sequence similarity with the sequences indicated in SEQ ID protocols of the attached Sequence Listing is defined in connection with the present invention as a very close structural relationship of the relevant sequences with the sequences indicated in the respective SEQ ID protocols. To determine the sequence similarity, in each case the structurally mutually corresponding sections of the sequence of the SEQ ID protocol and of the sequence to be compared therewith are superimposed in such a way that the structural correspondence between the sequences is a maximum, account being taken of differences caused by deletion or insertion of individual sequence members (DNA-codon or amino acid respectively) , and being compensated by appropriate shifts in sections of the sequences. The sequence similarity in % results from the number of sequence members which now correspond to one another in the sequences ("homologous positions") relative to the total number of members contained in the sequences of the SEQ ID protocols. Differences in the sequences may be caused by variation, insertion or deletion of sequence members. Additionally in DNA sequences, different DNA-codons encoding for the same amino acid are considered identical in the context of the present invention. For amino acid sequences, conservative amino acid substitutions encoded by their corresponding DNA-codons, as well as naturally occurring homologs of the sequences, are considered within the context of sequence similarity.

DNA molecules of substantial homology (> 75 %) are an implicit aspect of this sort of invention. As will be discussed later, the inventors have already identified two possible splice variants in the amino acid coding sequence. In addition, artificially mutated receptor cDNA molecules can be routinely constructed by methods such as site-directed polymerase chain reaction-mediated mutagenesis [Nelson and Long, Anal. Biochem. 180: 147-151 (1989)]. It is commonly appreciated that highly homologous mutants frequently mimic their natural receptor. A study by Kjelsberg et al. [J. Biol. Chem. 267: 1430-1433 (1992)] showed that all 20 amino acid substitutions produce an active receptor at a single site in the α_lb-adrenergic receptor. RNA molecules of > 75 % complementarity to an instant DNA molecule, e.g., an mRNA molecule (sense) or a complementary cRNA molecule (antisense) , are a further aspect of the invention.

A further aspect of the invention is for a recombinant vector, as well as a host cell transfected with the recombinant vector, wherein the recombinant vector contains at least one of the nucleotide sequences shown in SEQ ID Nos. 1- 4, or sequences predicted by the genomic clone, or nucleotide sequences > 75 % homologous thereto.

A method of producing an imidazoline receptor protein is another aspect of the invention. Such a method entails transfecting a host cell with an aforementioned vector, and culturing the transfected host cell in a culture medium to generate the imidazoline receptor.

A method for producing homologous imidazoline receptor proteins, and the proteins produced thereby, are also considered an aspect of this invention.

A significant further aspect of the invention is a method of screening for a ligand that binds to an imidazoline receptor. Such a method can comprise culturing an above- mentioned transfected cell in a culture medium to express imidazoline receptor proteins, followed by contacting the proteins with a labelled ligand for the imidazoline receptor under conditions effective to bind the labelled ligand thereto. The imidazoline receptor proteins can then be contacted with a candidate ligand, and any displacement of the labelled ligand from the proteins can be detected.

Displacement of labelled ligand signifies that the candidate ligand is a ligand for the imidazoline receptor. These steps could be performed on intact host cells, or on proteins isolated from the cell membranes of the host cells.

The invention will now be described in more detail with reference to specific examples.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 depicts a comparison of Reis antiserum (lane 1, 1:2000 dilution) and Dontenwill antiserum (lane 2, 1:5000 dilution) immunoreactivities for human NRL (same as RVLM) and hippocampus, as discussed in Example 1.

Fig. 2 depicts a comparison of Reis antiserum (1:15,000 dilution) and Dontenwill antiserum (1:20,000 dilution) immunoreactivities for plaques isolated from the human hippocampal cDNA library used in cloning as discussed in Example 2. The plaques contain the initial clone, designated herein as 5A-1, in a third stage of purification. Fig. 3 depicts the restriction map of the EST04033 cDNA clone.

Fig. 4 depicts a competitive binding assay between ¹⁵I- labelled p-iodoclonidine (PIC) and various ligands for the imidazoline receptor on membranes expressed in COS cells transfected with the EST04033 cDNA clone, as discussed in Example 4.

Fig. 5 depicts the prediction of introns and exons of the genomic clone (as analyzed by the GENESCAN program and verified by the available CDNAS) . Fig. 6 depicts the distribution of MRNA homologous to our CDNA in human adult tissues (bar graph) and the two species of MRNA (6 and 9.5 kb) . DETAILED DESCRIPTION OF THE INVENTION The present invention is concerned with multiple aspects of an imidazoline receptor protein, and DNA molecules encoding the same, and fragments thereof, which have now been discovered.

First, a polypeptide having imidazoline binding activity has been identified, which contains the putative active site for binding, as discussed hereinafter. Although polypeptide (s) described herein has a binding affinity for an imidazoline compound, it may also have an enzymatic activity, such as do catalytic antibodies and ribozymes. In fact, one such domain within our protein predicts a cytochrome p450 activity (described later) .

Exemplary "binding" polypeptides are those containing either of the amino acid sequences shown in SEQ ID Nos. 5 or 6 (with the amino acid sequence predicted by EST04033 given in SEQ ID No. 5) . Functionally equivalent polypeptides are also contemplated, such as those having a high degree of homology with such aforementioned polypeptides, particularly when they contain the Glu-Asp-rich region described hereinafter which we believe may define an active imidazoline binding site.

A polypeptide of the invention can be formed by direct chemical synthesis on a solid support using the carbodiimide method [R. Merrifield, JACS, 85: 2143 (1963)]. Alternatively, and preferably, an instant polypeptide can be produced by a recombinant DNA technique as described herein and elsewhere [e.g., U.S. Patent No. 4,740,470 (issued to Cohen and Boyer) , the disclosure of which is incorporated herein by reference] , followed by culturing transformants in a nutrient broth.

Second, a DNA molecule of the present invention encodes aforementioned polypeptide. Thus, any of the degenerate set of codons encoding an instant polypeptide is contemplated. A particularly preferred coding sequence is the 1954 b.p. sequence set forth in SEQ ID No. 2, which has now been discovered to be a nucleotide sequence that encodes a polypeptide capable of binding imidazoline compound (s) . In another embodiment, a DNA molecule includes the 3318 b.p. nucleotide sequence shown in SEQ ID No. 3. This latter sequence is the entire EST04033 insert. It includes the nucleotide sequence of SEQ ID No. 2 which was predicted to have been translated into protein in the transfection experiments. In another embodiment of the invention, a DNA molecule contains a nucleic acid sequence encoding the amino acid sequence (390 residues) shown in SEQ ID No. 6. This amino acid sequence corresponds to that derived from direct sequencing of the 5A-1 clone and represents a fragment of the native protein. The 5A-1 DNA molecule is defined by the 1171 b.p. nucleic acid sequence shown in SEQ ID No. 4.

A DNA molecule of the present invention can be synthesized according to the phosphotriester method [Matteucci et al., JACS, 103: 3185 (1988)]. This method is particularly suitable when it is desired to effect site-directed mutagenesis of an instant DNA sequence, whereby a desired nucleotide substitution can be readily made. Another method for making an instant DNA molecule is by simply growing cells transformed with plasmids containing the DNA sequence, lysing the cells, and isolating the plasmid DNA molecules. Preferably, an isolated DNA molecule of the invention is made by employing the polymerase chain reaction (PCR) [e.g., U.S. Patent No. 4,683,202 (issued to Mullis)] using synthetic primers that anneal to the desired DNA sequence, whereby DNA molecules containing the desired nucleotide sequence are amplified. Also, a combination of the above methods can be employed, such as one in which synthetic DNA is ligated to CDNA to produce a quasi-synthetic gene [e.g., U.S. Patent No. 4,601,980 (issued to Goeddel et al.)].

A further aspect of the invention is for a vector, e.g., a plasmid, that contains at least one of the nucleotide sequences shown in SEQ ID Nos. 1-4 or those predicted by the genomic clone in SEQ ID No. 21. Whenever the reading frame of the vector is appropriately selected, the vector encodes an IR polypeptide of the invention. Hence, as well as full-length protein, fragments of the native IR protein are contemplated; as well as fusion proteins that incorporate an amino acid sequence as described herein. Also, a vector containing a nucleotide sequence having a high degree of homology with any of SEQ ID Nos. 1-4 or 21 is contemplated within the invention, particularly when it encodes a protein having imidazoline binding activity. A recombinant vector of the invention can be formed by ligating an afore-mentioned DNA molecule to a preselected expression plasmid, e.g., with T4 DNA ligase. Preferably, the plasmid and DNA molecule are provided with cohesive (overlapping) terminii, with the plasmid and DNA molecule operatively linked (i.e., in the correct reading frame).

Another aspect of the invention is a host cell transfected with a vector of the invention. Relatedly, a protein expressed by a host cell transfected with such a vector is contemplated, which protein may be bound to the cell membrane. Such a protein can be identical with an aforementioned polypeptide, or it can be a fragment thereof, such as when the polypeptide has been partially digested by a protease in the cell. Also, the expressed protein can differ from an aforementioned polypeptide, as whenever it has been subjected to one or more post-translational modifications. For the protein to be useful within the context of the present invention, it should exhibit imidazoline binding capacity. A method of producing an imidazoline receptor protein is another aspect of the invention, which entails transfecting a host cell with an aforementioned vector, and culturing the transfected host cell in a culture medium to generate the imidazoline receptor. The receptor molecule can undergo any post-translational modification (s) , including proteolytic decomposition, whereby its structure is altered from the basic amino acid residue sequence encoded by the vector. A suitable transfection method is electroporation, and the like.

With respect to transfecting a host cell with a vector of the invention, it is contemplated that a vector encoding an instant polypeptide can be transfected directly in animals. For instance, embryonic stem cells can be transfected, and the cells can be manipulated in embryos to produce transgenic animals. Methods for performing such an operation have been previously described [Bond et al., Nature , 374:272-276 (1995) ] . These methods for expressing an instant CDNA molecule in either tissue culture cells or in animals can be especially useful for drug discovery.

Possibly the most significant aspect of the present invention is in its potential for affording a method of screening for a ligand (drug) that binds to an imidazoline receptor. Such a method comprises culturing an above- mentioned host cell in a culture medium to express an instant imidazoline receptive polypeptide, then contacting the polypeptides with a labelled ligand, e.g., radiolabelled p- iodoclonidine, for the imidazoline receptor under conditions effective to bind the labelled ligand thereto. The polypeptides are further contacted with a candidate ligand, and any displacement of the labelled ligand from the polypeptides is detected. Displacement signifies that the candidate ligand actually binds to the imidazoline receptor. These steps could be performed on intact host cells, or on proteins isolated from the cell membranes of the host cells. Typically, a suitable drug screening protocol involves preparing cells (or possibly tissues from transgenic animals) that express an instant imidazoline receptive polypeptide. In this process, categories of chemical structure are systematically screened for binding affinity or activation of the receptor molecule encoded by the transfected CDNA. This process is currently referred to as combinatorial chemistry. With respect to the imidazoline receptor, a number of commercially available radioligands, e.g., ¹²⁵PIC, can be used for competitive drug binding affinity screening.

An alternative approach is to screen for drugs that elicit or block a second messenger effect known to be coupled to activation of the imidazoline receptor, e.g., moxonidine- stimulated arachidonic acid release. Even with a weak binding affinity or activation by one category of chemicals, systematic variations of that chemical structure can be studied and a preferred compound (drug) can be deduced as being a good pharmaceutical candidate. Identification of this compound would lead to animal testing and upwards to human trials. However, the initial rationale for drug discovery becomes vastly improved with an instant cloned imidazoline receptor. Along these lines, a drug screening method is contemplated in which a host cell of the invention is cultured in a culture medium to express an instant imidazoline receptive polypeptide. Intact cells are then exposed to an identified agent (ie., agonist, inverse agonist, or antagonist) under conditions effective to elicit a second messenger or other detectable responses upon interacting with the receptor molecule. The imidazoline receptive polypeptides are then contacted with one or more candidate chemical compounds (drugs) , and any modification in a second messenger response is detected. Compounds that mimic an identified agonist would be agonist candidates, and those producing the opposite response would be inverse agonist candidates. Those compounds that block the effects of a known agonist would be antagonist candidates for an in vivo imidazoline receptor. For meaningful results, the contacting step with a candidate compound is preferably conducted at a plurality of candidate compound concentrations. A method of probing for another gene encoding an imidazoline receptor or homologous protein is further contemplated. Such a method comprises providing a radiolabelled DNA molecule identical or complementary to one of the above-described CDNA molecules (probe) . The probe is then placed in contact with genetic material suspected of containing a gene encoding an imidazoline receptor or encoding a homologous protein, under stringent hybridization conditions (e.g., a high stringency wash condition is 0.1 x SSC, 0.5% SDS at 65 °C) , and identifying any portion of the genetic material that hybridizes to the DNA molecule.

Still further, a method of selectively producing antibodies, (e.g., monoclonal antibodies, immunoreactive with an instant imidazoline-receptive protein) comprises injecting a mammal with an aforementioned polypeptide, and isolating the antibodies produced by the mammal. This aspect is discussed in more detail in an example presented hereinafter.

The present inventors began their search for a human imidazoline receptor CDNA by screening a λgtll phage human hippocampus CDNA expression library. Their research had indicated that both of the known antisera (Reis and

Dontenwill) that are directed against human imidazoline receptors were immunoreactive with identical bands on SDS gels of membranes prepared from the human hippocampus (an in other tissues) . By contrast, other brain regions either were commercially unavailable as cDNA expression libraries or yielded inconsistencies between the two antisera. Therefore, it was felt that a human hippocampal cDNA library held the best opportunity for obtaining a CDNA for an imidazoline receptor. Immunoexpression screening was chosen over other cloning strategies because of its sensitivity when coupled with the ECL detection system used by the present inventors, as discussed hereinbelow. A number of unique discoveries led to identifying the first 5A-1 clone as an imidazoline receptor CDNA. These included discoveries that led to the choice of a hippocampal CDNA library and adapting ECL to the antisera. Once the initial clone (5A-1) was identified and sequenced, a more complete clone (EST04033) was purchased without restriction from ATCC Inc. (Catalogue # 82815; American Type Culture Collection, Rockville, MD) . EST 04033 was the only EST clone available at the time of the discovery of 5A-1, that contained a segment of complete homology (the origination of EST04033 is discussed later on) . The binding affinities of the expressed protein after transfection in COS cells were determined by radioligand binding procedures developed in the inventor's laboratory [Piletz and Sletten, 1993, ibid].

To identify an instant CDNA clone encoding an imidazoline receptor it was preferable to employ both of the known antibodies to imidazoline receptors. These antibodies were obtained by contacting Dr. D. Reis (Cornell University Medical Center, New York City) , and Drs. M. Dontenwill and P. Bousquet (Laboratoire de Pharmacologie Cardiovascular et Renale, CNRS, Strasbourg, France) . These antisera were obtained free of charge and without confidentiality or restrictions on their use. The former antiserum (Reis antiserum) was derived from a published imidazoline receptor protein [Wang et al., (1992, 1993) , the disclosures of which are incorporated herein by reference] . The method for raising the latter antiserum (Dontenwill antiserum) has also been published [Bennai et al., (1995) , the disclosure of which is also incorporated herein by reference] . The latter antiserum was developed using an anti- idiotypic approach that identified the pharmacologically correct (clonidine and idazoxan selective) binding site structure.

Example 1. Selectivity of the Antisera. The obtained Reis antiserum had been prepared against a purified imidazoline binding protein isolated from BAC cells, which protein runs in denaturing-SDS gels at 70 Kda [Wang et al. , 1992, 1993]. The Dontenwill antiserum is anti-idiotypic, and thus is believed to detect the molecular configuration of an imidazoline binding site domain in any species. Prior to being used for screening plaques, both antisera were cleaned by stripping out possible antibacterial antibodies.

Both antisera have been tested to ensure that they are in fact selective for a human imidazoline receptor. In particular, we found that both of these antisera detected identical bands in human platelets and hippocampus, and in brainstem RVLM (NRL) by Western blotting (see Fig. 1) . In these studies, in order to increase sensitivity over previously published detection methods, an ECL (Enhanced Chemiluminescence) system was employed. The linearity of response of the ECL system was demonstrated with a standard curve. ECL detection was demonstrated to be very quantifiable and about ten times more sensitive than other screening methods previously used with these antisera. Western blots with antiserum dilutions of 1:3000 revealed immunoreactivity with as little as 1 ng of protein from a human hippocampal homogenate by dot blot analysis.

For the studies depicted in Fig. 1, human hippocampal homogenate (30μg) and NRL membrane proteins (lOμg) were electrophoresed through a 12.5% SDS-polyacrylamide gel, electrotransfered to nitrocellulose and sequentially incubated with (1) the Reis antibody (1:2000 dilution) and (2) the

Dontenwill antibody (1:5000 dilution). Immunoreactive bands were visualized with an Enhanced Chemiluminescence (ECL) detection kit (Amersham) using anti-rabbit Ig-HRP conjugated antibody at a dilution of 1:3000 and the ECL detection reagents. Following detection with the antibody, blots were stripped and reprocessed omitting the primary antibody to check for complete removal of this antibody. In panels A and B, lane 1 shows the immunoreactive bands observed with the Reis antibody and lane 2 shows the bands detected with the Dontenwill antibody. Protein molecular weight standards are indicated to the left of each panel (in Kda) .

Despite the diverse origins of the Reis and Dontenwill antisera, both of these antisera detected a similar 85 Kda protein in human brain and other tissues. But, a 33 Kda band was found in human platelets. Although the 33 Kda band is of smaller size than that reported for other tissues [Wang et al., 1993; Escriba et al., 1994; Greney et al., 1994], the fact that both antisera detected it, suggests that both the 85 Kda and 33 Kda bands may be imidazoline binding polypeptides. The 85 and 33 Kda bands were enriched in plasma membrane fractions, as is known to be the case for IR, binding, but not I₂ binding [Piletz and Sletten, 1993]. A significant positive correlation was established for the 85 Kda band detected by the Dontenwill antiserum with IR, Bmax values across nine rat tissues (r² = 0.8736). A similar positive correlation was established amongst platelet samples from 15 healthy platelet donors between radioligand IR, Bmax values (but not I₂ or α₂AR Bmax values) , and the 33 Kda band (presumed IR, immunoreactivity) on Western blots. This correlation exhibited a slope factor close to unity (results not shown) . These correlations strongly suggested that an IR, binding protein might be revealed in an imidazoline receptor- antibody Western blotting assay. Furthermore, the Reis antiserum failed to detect authentic α₂AR, MAO A, or MAO B bands on gels, i.e., it was not immunoreactive with MAO at MW = 61 Kda, or ₂AR at MW = 64 Kda. Additionally, no immunoreactive bands were observed using preimmune antiserum. Thus, after extensively characterizing these antisera with human and rat materials, it was concluded that these antisera are indeed selective for human imidazoline receptor protein. Example 2. Cloning of cDNA For An Imidazoline Receptor

A commercially available human hippocampal cDNA λgtll expression library (Clontech Inc. , Palo Alto, CA) was screened for immunoreactivity sequentially using both the anti- idiotypic Dontenwill antiserum and the Reis antiserum.

Standard techniques to induce protein and transference to a nitrocellulose overlay were employed. [See, for instance, Sambrook et al., 1989, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press]. After washing and blocking with 5% milk, the Dontenwill antiserum was added to the overlay at 1:20,000 dilution in Tris-buffered saline, 0.05% Tween20, and 5% milk. The Reis antiserum was employed similarly, but at 1:15,000 dilution. These high dilutions of primary antiserum were chosen to avoid false positives. The secondary antibody was added, and positive plaques were identified using ECL. Representative results are shown in Fig. 2.

Positive plaques were pulled and rescreened until tertiary screenings yielded only positive plaques. Four separate positive plaques were identified from more than

300,000 primary plaques in our library. Recombinant λgtll DNA purified from each of the four plaques was subsequently subcloned into E_;_ coli pBluescript vector (Stratagene, La Jolla, CA) . Sequencing of these four cDNA inserts in pBluescript demonstrated that they were identical, suggesting that only one cDNA had actually been identified four times. Thus, the screening had been verified as being highly reproducible and the frequency of occurrence was as expected for a single copy gene, i.e., one in 75,000 transcripts. As shown in Fig. 2, the protein produced by the first positive clone, designated 5A-1, tested positive with both the Reis antiserum and the Dontenwill antiserum. Clone 5A-1 has been deposited under the Budapest Treaty with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD, USA, 20852, on August 28, 1997 and has been assigned deposit accession no. ATCC 209217. Tertiary-screened plaques of 5A-1 were all immuno-positive with either of the two known anti-imidazoline receptor antisera, but not with either preimmune antisera. These results suggested that clone 5A-1 encoded a fusion peptide similar to or identical with one of the predominant bands detected in human Western blots by both the Dontenwill and Reis antisera. Sequencing of the first four clones was performed by contracting with ACGT Company (Chicago, IL) after subcloning them into pBluescript vector SK (Stratagene) . Both manual and automatic sequencing strategies were employed which are outlined as follows: Manual Sequencing

1. DNA sequencing was performed using T7 DNA polymerase and the dideoxy nucleotide termination reaction.

2. The primer walking method [Sambrook et al., ibid. ] was used in both directions. 3. (³⁵S)dATP was used for labelling.

4. The reactions were analyzed on 6% polyacrylamide wedge or non-wedge gels containing 8 M urea, with samples being loaded in the order of A C G T. 5. DNA sequences were analyzed by MacVector Version 5.0. and by various Internet-available programs, i.e., the BLAST program.

Automatic Sequencing 1. DNA sequencing was performed by the fluorescent dye terminator labelling method using AmpliTaq DNA polymerase (Applied Biosystems Inc. , Prizm DNA Sequencing Kit, Perkin- Elmer Corp. , Foster City, CA) .

2. The primer walking method was used. The primers actually used were a subset of those shown in SEQ ID Nos. 7- 20.

3. Sequencing reactions were analyzed on an Applied Biosystems, Inc. (Foster City, CA) sequence analyzer.

These results demonstrated that the initial clone (5A-1) contained a 1171 base pair insert (see SEQ ID No. 4) . The entire 5A-1 cDNA was found to exist as extended open reading frame for translation into protein. Consequently, it was determined that the 5A-1 cDNA must be a fragment of a larger mRNA.

cDNA Sequence Homologies

Using programs and databases available on the Internet (retrieved from NCBI Blast E-mail Server address blast@ncbi.nlm.nih.gov), it was determined that the 5A-1 clone encodes a previously undefined unique molecule. The BLASTP program [1.4.8MP, 20-June-1995 (build 11/13/95)] was used to compare all of the possible frames of amino acid sequences encoded by 5A-1 versus all known amino acid sequences available within multiple international databases [Altschul et al., J. Mol. Biol.. 215: 403-410 (1990)]. Only one protein, from Micrococcus luteus , possessed a marginally significant homology (p=0.04) (41%) over a short stretch of 75 of the 390 amino acids encoded by 5A-1. Otherwise, there were not any amino acid homologies (i.e., with p < 0.05) for any known proteins. Therefore, the protein encoded by 5A-1 is not significantly related to MAO A or B, α₂AR, or any other known eukaryotic protein in the literature. In contrast to the amino acid search on BLASTP, two nearly homologous EST cDNA sequences of undefined nature covering 155 and 250 b.p. of the 5A-1 clone were reported to exist using BLASTN (reached from the same Internet server on 11/13/95) . BLASTN is a program used to compare known DNA sequences from international databases, regardless of whether they encode a polypeptide. Neither of the two EST cDNA sequences having high homology to 5A-1, to our knowledge have been reported anywhere else except on the Internet. Both were derived as Expressed Sequence Tags (ESTs) in random attempts to sequence the human cDNA repertoire [as described in Adams et al., Science, 252: 1651-1656 (1991)]. As far as can be determined, the people who generated these ESTs lack any knowledge of what protein (s) they encode. One cDNA, designated HSA09H122, contained 250 b.p. with 7 unknown/ incorrect base pairs (97% homology) versus 5A-1 over the same region. HSA09H122 was generated in France (Genethon, B.P. 60, 91002 Evry Cedex France) from a human lymphoblast cDNA library. The other EST, designated EST04033, contained 155 b.p. with 12 unknown/ incorrect base pairs (92% homology) versus 5A-1 over the same region. EST04033 was generated at the Institute for Genomic Research (Gaithersburg, MD) from a human fetal brain cDNA clone (HFBDP28) . Thus, both of these ESTs are short DNA sequences and contain a number of errors

(typical of single-stranded sequencing procedures as used when randomly screening ESTs) .

Based on the BLASTN search, the owner of HSA09H122 was contacted in an effort to obtain that clone. The current owner of the clone appears to be Dr. Charles Auffret (Paul Brousse Hospital, Genetique, B.P. 8, 94801 Villejuif Cedex, France) . Dr. Auffret indicated by telephone that his clone came from a lot of clones believed to be contaminated with yeast DNA, and he did not trust it for release. Contamination with yeast DNA of that clone was later confirmed to have been reported within an Internet database. Thus, HSA09H122 was not reliable.

The other partial clone (EST04033) was purchased from American Type Culture Collection in Rockville, MD (ATCC Catalog no. 82815) . A telephone call to the Institute for

Genomic Research revealed that it had been deposited at ATCC under [insert terms]. As far as can be determined, the present inventors were the first to completely sequence EST04033. The complete size of EST04033 was 3389 b.p. (SEQ ID No. 1) , with a 3,318 b.p. nonplasmid insert (see SEQ ID No. 3). Within this sequence of EST04033 the remaining 783 base pairs of the coding sequence presumed for a 70 kDa imidazoline receptor were predicted at the 5' side of 5A-1 (i.e., 783 coding nucleotides unique to EST04033 + 1171 coding nucleotides of 5A-1 = 1954 predicted total coding nucleotides; assuming b.p.# 1397-1400 in SEQ. No. 1 encodes the initiating methionine) . The entire 1954 b.p. coding region for an « 70 kDa protein is shown in SEQ ID No. 2. The nucleotide sequence of EST04033 was determined in the same manner as described previously for the 5A-1 clone. The nucleotide sequence of the entire clone is shown in SEQ ID No. 1. In this sequence, an identical overlap was observed for the sequence obtained previously for the 5A-1 clone and the sequence obtained for EST04033. The 5A- 1 overlap began at EST04033 b.p. 2,181 (SEQ. No.l) and continued to the end of the molecule (b.p. 3,351).

Conclusions About Our cDNA Clones cDNA of the present invention encode a protein that is immunoreactive with both of the known selective antisera for an imidazoline receptor, i.e., Reis antiserum and Dontenwill antiserum. Thus, an instant cDNA molecule produces a protein immunologically related to a purified imidazoline receptor and has the antigenic specificity expected for an imidazoline binding site. These antisera have been documented in the scientific literature as being selective for an "imidazoline receptor" , which provides strong evidence that such an imidazoline receptor has indeed been cloned.

As mentioned, our instant cDNA sequence contains open reading frame distinct from any previously described proteins. Therefore, the encoded protein is novel, and it is unrelated to α₂-adrenoceptors or monoamine oxidases. Small hydrophobic domains in the predicted amino acid sequence suggest that the protein is probably membrane bound, as expected for an imidazoline receptor.

Example 3. Cloning of a Human Gene A pre-made genomic library of human placental DNA was purchased from Stratagene (La Jolla, CA) to screen for an IR gene by hybridization. The genomic library was constructed in Stratagene's vector λ FIX® II (catalog # 946206), and it was grown in XLl-Blue MRA (P2) host bacteria. It was titered to yield approximately 50,000 plaques per 137 mm plate. Lifts from six such plates were screened in duplicate by hybridization. The DNA probe used for screening was a 1.85 kb EcoRI fragment from EST 04033 cDNA (uniquely related to our sequences based on the BLASTN) . After the restriction digestion of EST 04033 DNA, the 1.85 kb fragment was extracted from an agarose electrophoresis gel, cleaned according to the GENECLEAN® III kit manual (BIO 101, Inc., P.O. Box 2284, La Jolla, CA) , and radiolabeled with [α-³²P] d-CTP according to Stratagene's Prime-It® II Random Primer Labeling Kit manual. Plaques were lifted onto 137 mm Duralon-UV™ membranes

(Stratagene's catalog #420102), denatured, and cross-linked with Stratgene's UV-Stratalinker™ 1800. Hybridization was conducted under high stringency conditions: prehybridization = 6 X SSC, 1 % SDS, 50 % formamide, and 100 lμg/ml of sheared, denatured salmon sperm DNA at 42°C for 2 hrs; hybridization = 6 X SSC, 1 % SDS, 50 % formamide, and 100 μg/ml of sheared, denatured salmon sperm DNA at 45°C overnight; wash = 2 washes of 1 X SSC, 0.1 % SDS at 65°C and 3 washes of 0.2 X SSC, 0.2 % SDS at 65°C. This hybridization procedure is essentially described in Stratagene's vector λ FIX® II instruction manual. Positive plaques were localized by developing Kodak BioMax films. Two positive genomic clones of identical size were retained through three rounds of screening.

One of the positive genomic clones (designated JEP 1-A) was selected for complete characterization. It was found to contain an « 17 kb insert. Large-scale preparations of this genomic clone DNA were performed using the λ QUICK! SPIN kit (BIO101, La Jolla, CA) . To verify that we had cloned a gene corresponding to 5A-1 and EST04033 cDNA, some restriction site positions in the genomic clone were determined using the FLASH Nonradioactive Gene Mapping Kit (Stratagene) and compared to Southern blots of human DNA. The location of genomic sequences highly related to (or identical to) those of our cDNA clones was determined by high stringency hybridization (as above) with the following ²P-labeled probe: a 1110 bp Apal-EcoRI fragment from the cDNA clone 5A-1. This fragment was chosen as the probe because it lacks the GAG repeat (encoding glutamic acids) , which might have complicated matters if it were found to be repeated elsewhere in the genome. With genomic clone JEPl-A, we detected a 14.1 kb .EcoRI fragment and a 7.7 kb SacI fragment that hybridized with this probe. Southern blots containing i?coRI- or Sacl-digested human genomic DNA (from human blood) with the 1110 bp Apal-EcoRI cDNA probe also resulted in the detection of a 14.1 kb EcoRI fragment and a 7.7 kb SacI fragment. No additional restriction fragments of human genomic DNA appeared to hybridize with this probe under lower stringency conditions. These results strongly suggested that this gene (JEP-1A) encodes transcript (s) giving rise to the 5A-1 and EST04033 cDNA clones. Clone JEP-1A has been deposited under the

Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD, USA, 20852, on August 28, 1997 and has been assigned deposit accession no. ATCC 209216. Genomic DNA sequencing was done by contract with Cadus Pharmaceutical Corporation (Tarrytown, NY) . The original lambda JEPl-A clone was subcloned into pZero (Invitrogen) as a convenient vector. The initial fragments for sequencing were derived from Sac I and Xba I restriction enzymes. The short Sac I fragments of 1.5, 3.0 and 3.5 kb were further digested with Hind III, Pst I, and Kpn I yielding 15 subclones of varying length. The procedure consisted of sequencing all these subclones and parent clones with vector forward and reverse primers. Subsequently, this initial round of sequencing was supplemented with primer walking using custom oligonucleotides. The Sac I fragments were joined together by primer walking using the 2 Xba I fragments of 3 and 10 Kb. Then, the largest Sac I fragment (8 kb) and the 10 kb Xba I fragment were used as templates for a transposon sequencing method. The method used was the Primer Island Transposition Kit (Perkin-Elmer Corp. , Norwalk, CT; Applied Biosystems) (ABI) . The kit consists of a synthetic transposon (Tyl) containing forward and reverse primers and the integrase enzyme which inserts the transposon randomly into the target plasmid DNA. Transposon insertion is an alternative to subcloning or primer walking when sequencing a large region of DNA (Devine and Boeke, Nucleic Acids Res. 22: 3765-3772 (1994); Devine et al., Genome Res., in press, (1997); Kimmel et al., In Genome Analysis, a Laboratory Manual, Cold Spring Harbor Press, NY, NY, in press (1997). A total of over 250 individual sequencing reactions were performed. Sequencing was done on ABI model 373 and 377 automated sequencers using ABI dye-terminator sequencing kits. Primers were designed using Gene Runner software (Hastings Software, Hastings On Hudson, NY) . Oligonucleotides were purchased from Gibco-BRL (Gaithersburg, MD) . Sequence assembly was performed using Sequencer Software (Gene Codes Corp. , Ann Arbor, MI) from 4- fold redundancy of sequences.

The entire sequence of our JEP-1A genomic clone is shown in SEQ. 21. The computer program, GENSCAN 1.0, was able to identify splice sites of known topology. As expected, this gene contained a number of introns. See Table 1 hereinbelow. Only one continuous open reading frame was identified within our genomic clone. This open reading frame was interrupted by a number of introns (which is typical of eukaryotic transcripts) as shown in Fig. 5. The predicted polypeptide is encoded by the genomic DNA beginning at b.p. # 971 of SEQ ID No. 21. The predicted amino acid sequence of the polypeptide encoded thereby is shown in SEQ ID No. 22. As can be seen, the entire 5A-1 DNA and polypeptide sequence was encapsulated within this predicted genomic transcript. Therefore, there is no question that this is the gene encoding 5A-1 and EST04033 cDNA. In addition, JEP-1A has more nearly defined the full- length transcript (by at least 102 more coding nucleotides than the cDNAs alone) .

TABLE 1

Position of Predicted Introns and Exons

GENSCAN 1.0 Date run: 26-Aug-97 Time: 12:35:39

Sequence gs_seqfile : 15202 bp : 58.36% C+G : Isochore 4 (57.00 - 100.00 C+G%) Parameter matrix: Humanlso.smat Predicted genes/exons: Gn.Ex Type S .Begin ...End .Len Fr Ph I/Ac Do/T CodRg P.. Tscr..

1.01 Intr + 971 1084 114 1 0 69 98 200 0. .836 20.91

1.02 Intr + 4096 4177 82 0 1 37 53 81 0. .358 -0.13

1.03 Intr + 5732 5856 125 0 2 117 95 84 0. .953 13.48

1.04 Intr + 6997 7046 50 0 2 95 116 44 0. .998 6.52

1.05 Intr + 8416 9825 1410 1 0 96 94 2914 0. .970 283.09

1.06 Intr + 10489 10897 409 1 1 15 59 318 o. .517 17.19

1.07 Intr + 11293 11449 157 0 1 57 61 236 o. .998 18.57

1.08 Intr + 11923 12051 129 2 0 84 63 224 o. .997 21.34

1.09 Intr + 12570 12731 162 1 0 95 80 229 o. .996 23.94

1.10 Term + 13090 13700 611 2 2 59 41 1012 o. .942 89.44

1.11 PlyA + 14257 14262 6 1.05

A BLASTN analysis of the entire genomic sequence (on 08/26/97) demonstrated again that this gene has not been previously defined in the literature.

As with the cDNA clones, some EST sequences of identity were found (listed below and later) . Of particular interest was a variance in the first intron splice site predicted by the computer. Upstream of that site (ie., upstream of amino acids PEKKGGE = amino acids predicted after first splice site) we have identified two types of transcripts. Genomic clone JEP-1A predicted 34 amino acids upstream of that sequence before entering another intron upstream. In an identical manner, three ESTs (H61282, AA428790 and AA428250) overlapped that entire region in our clones and they contained the identical nucleotides for those 34 amino acids, plus an additional 22 more amino acids further upstream. By comparison, however, our EST04033 varied from all of these ESTs upstream of that site. This means, the first 1,532 nucleotides of EST04033 (thought to encode translation of amino acids 1-56 of EST04033 beginning at b.p. 1,398 in SEQ. 1) are completely at variance with the other ESTs down to that splice site, but from there on they are identical. This provides strong evidence that this site can generate two alternatively spliced transcripts which can produce at least one functional protein (ie., the transfections with EST04033). For the reader's information, this splice site is upstream of b.p. # 1,565 in SEQ.l, b.p. # 168 in SEQ.2, b.p. # 1,532 in

SEQ.3, amino acid # 57 in SEQ.5, and b.p. # 971 in the genomic SEQ.21.

Genomic Sequence Analysis

Of interest is a unique glutamic- and aspartic acid-rich region within our predicted protein. This region of the IR protein delineates a highly unique span of 59 amino acids, 36 of which are Glu or Asp residues (61%) . This region was largely discovered within clone 5A-1 and it is present within all discovered and predicted transcripts from the gene (EST04033 included) . This sequence lies between two potential transmembrane loops (hydrophobic domains) . The identification of this unique Glu/Asp-rich domain within our clones is consistent with an expected negatively charged pocket capable of binding clonidine and agmatine, both of which are highly positively charged ligands. Also, since the Dontenwill antiserum was specifically developed against an idazoxan/clonidine binding site, and its immunoreactivity is directed against the clone 5A-l/λgtll fusion protein, this suggests that clone 5A-1 might encode an imidazoline binding site. Furthermore, this glu/asp-rich sequence is located within the longest stretch of homology that the clone has with any known protein, i.e., the ryanodine receptor (as determined by on BLASTN) . Specifically, we have discovered four regions of homology between the imidazoline receptor and the ryanodine receptor, which are all Glu/Asp-rich. The total nucleic acid homology is 67% with the ryanodine receptor DNA over the stretches encompassing this region. However, this is not sufficient to indicate that the imidazoline receptor is a subtype of the ryanodine receptor, because this homologous stretch is still a minor portion of the overall transcript (s) identified in the gene. Instead, this significant homology may reflect a commonality in function between this region of the IR and the ryanodine receptor.

The Glu/Asp-rich region within the ryanodine receptor has also been reported to define a calcium and ruthenium red dye binding domain that modulates the ryanodine receptor/Ca⁺⁺ release channel located within the sarcoplasmic reticulum. The only other charged amino acids within the Glu/Asp-rich region of our clones are two arginines (the ryanodine receptor has uncharged amino acids at the corresponding positions) . Based on this identification of Arg residues within the Glu/Asp-rich region of the predicted imidazoline binding site, the assistance of Dr. Paul Ernsberger (Case Western Reserve University, Cleveland, Ohio) was enlisted. Dr. Ernsberger performed phenylglyoxal attack of arginine on native PC-12 membranes. Dr. Ernsberger was able to demonstrate that this treatment completely eliminated imidazoline binding sites in these membranes. This provides some indirect evidence that the native imidazoline binding site also contains an Arg residue. On the other hand, attempts to chemically modify cysteine and tyrosine residues, which are not located near the Glu/Asp-rich region did not affect PC-12 membrane binding of imidazolines.

As a further test of the sequence, it was determined whether native IR binding sites in PC-12 cells would be sensitive to ruthenium red. From the structure of the cloned sequence, it was reasoned that native IR should bind ruthenium red. Accordingly, a competition of ruthenium red with ^I25PIC at native IR sites on PC-12 membranes was studied. In these studies it was observed that ruthenium red competed for ^I25PIC binding to the same extent as did the potent imidazoline compound, moxonidine, i.e., 100% competition. Furthermore, the IC₅₀ for competition of ruthenium red at IR was slightly more robust than reported for ruthenium red on the activation of calcium-dependent cyclic nucleotide phosphodiesterase - the previous most potent effect of ruthenium red on any biological site - indicating possible pharmacological importance. It is also noteworthy that calcium failed to compete for ¹²⁵PIC binding at PC-12 IR sites (as did a calcium substitute, lanthanum) . We and others have previously reported that a number of other cations robustly interfere with IR binding [Ernsberger et al., Annals NY Acad. Sci.. 763: 22-42 (1995); Ernsberger et al., Annals NY Acad. Sci., 763: 163-168 (1995)]. Attempts were also made to directly stain the proteins in SDS gels with ruthenium red [Chen and MacLennan, J. Biol. Chem., 269: 22698-22704 (1994)]. It was found that ruthenium red stains the same platelet (33 kDa) and brain (85 kDa) bands that Reis antiserum detects. (Remember, the same 33 kDa band was verified to directly correlate with ¹²⁵PIC Bmax values for IR.) Thus, these results linked the attributes predicted from the cloned sequence to a native IR binding site.

Two other facets of the predicted polypeptide from JEP-1A suggest that we have identified most of the functional sequences. First, our predicted protein is comparable in regard to both the order and size of three regions of importance to the function of the interleukin-2R receptor (IL-2R/3) . Specifically, IL-2R/5 possesses the following regions over a span of 286 amino acids: ser-rich region, followed by glu/asp-rich region, followed by proline-rich region. Likewise, our predicted protein has the same three regions, in the same order, over a span of about 625 amino acids. This suggests that our protein might function similarly as cytokine receptors. Secondly, our predicted protein possesses a cytochrome p450 heme-iron ligand signature sequence [Nelson et al., Pharmacogenetics 6: 1-42 (1996)]. This suggests that our protein might also function as do cytochrome p450s in oxidative, peroxidative and reductive metabolism of endogenous compounds.

Some additional findings about the amino acid sequence of our instant IR polypeptide are: (1) The glu/asp-rich region also bears similarity to an amino acid sequence within a

GTPase activator protein. (2) There appear to be four small hydrophobic domains indicative of transmembrane domain receptors. (3) A number of potential protein kinase C (PKC) phosphorylation sites appear near to the carboxy side of the protein, and we have previously found that treatment of membranes with PKC leads to an enhancement of native IR binding. Thus, these observations are all consistent with other observations expected for native IR.

RNA Studies Northern blotting has also been performed on polyA⁺ mRNA from human tissues in order to ascertain the regional expression of the mRNA corresponding to our cDNA. The same 1110 b.p. Apal-EcoRI fragment from cDNA clone 5A-1 used in Southern blots was used for these studies. This probe region was not found within any other known sequences on the BLASTN database. The results revealed a 6 kb mRNA band, which predominated over a much fainter 9.5 kb mRNA in most regions (Fig. 6) . Some exceptions to this pattern were in lymph nodes and cerebellum (Fig. 6), where the 9.5 kb band was equally or more intense. Although the 6 kb band is weakly detectable in some non-CNS tissues, it is enriched in brain. An enrichment of the 6 kb mRNA is observed in brainstem, although not exclusively. The regional distribution of the mRNA is somewhat in keeping with the reported distribution of IR binding sites, when extrapolated across species (Fig. 6). Thus, the rank order of Bmax values for IR in rat brain has been reported to be frontal cortex > hippocampus > medulla oblongata > cerebellum [Kamisaki et al., Brain Res. , 514: 15- 21 (1990)]. Therefore, with the exception of human cerebellum, which showed two mRNA bands, the distribution of the mRNA for our the present cloned cDNA is consistent with it belonging to IR.

[It should be noted that while IR binding sites are commonly considered to be low in cerebral cortex compared to brainstem, this is in fact a misinterpretation of the literature based only on comparisons to the alpha-2 adrenoceptor ' s Bmax, rather than on absolute values. Thus, IR Bmax values have actually been reported to be slightly higher in the cortex than the brainstem, but they only "appear" to be low in the cortex in comparison to the abundance of alpha-2 binding sites in cortex. Therefore, the distribution of the IR mRNA is reasonably in keeping with the actual Bmax values for radioligand binding to the receptor [Kamisaki et al., (1990)].

A final point to emphasize about the Northern blots is that they clearly demonstrate two high-stringency transcripts (Fig. 6) . This is in keeping with the alternatively spliced EST cDNAs mentioned earlier. Thus, we suggest this may be the basis for both the 6 and 9.5 kb transcripts.

Summary of Genomic Sequence Results The JEP-lA clone clearly contains most of the gene. Within it we have identified at least 3,776 nucleotides for transcript (s) (encoding 1,065 amino acids plus 587 b.p. of untranslated region down to the polyT⁺ tail) . This has been lengthened by at least 66 coding nucleotides upstream (22 amino acids) in comparison to overlapping ESTs. In addition to this, we are quite confident of the splice site for the two observed mRNA sizes. Most of the functional sequences are predicted to be encoded within our genomic clone. A summary of the evidence that a gene encoding an imidazoline receptor protein has been cloned is summarized in Table 2 hereinbelow.

TABLE 2 Comparison of Protein Predicted From Our Clones with Properties of Native IR, and I₂ Sites

Example 4. Transient Transfection Studies

COS-7 cells were transfected with a vector containing EST04033 cDNA, which was predicted based on sequence analysis to contain the glu/asp rich region thought to be important for ligand binding to the imidazoline receptor protein. The EST04033 cDNA was subcloned into pSVK3 (Pharmacia LKB Biotechnology, Piscataway, NJ) using standard techniques [Sambrook, supra] , and transfected via the DEAE-dextran technique as previously described [Choudhary et al., Mol. Pharmacol. , 42: 627-633 (1992); Choudhary et al., Mol. Pharmacol. , 43: 557-561 (1993); Kohen et al., J.Neurochem. , 66: 47-56 (1996)]. A restriction map of the EST04033 cDNA is shown in Fig. 3. The restriction enzymes Sal I and Xba I were used for subcloning into pSVK3. Briefly stated, COS-7 cells were seeded at 3 x 10⁶ cells/100 mm plate, grown overnight and exposed to 2 ml of DEAE-dextran/plasmid mixture. After a 10-15 min. exposure, 20 ml of complete medium (10% fetal calf serum; 5 μg/ml streptomycin; 100 units/ml penicillin, high glucose, Dulbeccos' modified Eagle's medium) containing 80 μM chloroquine was added and the incubation continued for 2.5 hr. at 37 °C in a 5% C0₂ incubator. The mixture was then aspirated and 10 ml of complete medium containing 10% dimethyl sulfoxide was added with shaking for 150 seconds. Following aspiration, 15 ml of complete medium with dialyzed serum was added and the incubation continued for an additional 65 hours. After this time period, the cells from 6 plates were harvested and membranes were prepared as previously described [Ernsberger et al., Annals NY Acad. Sci., 763: 22-42 (1995), the disclosure of which is incorporated herein by reference] . Parent, untransfected COS-7 cells were prepared as a negative control. Some membranes were treated with and without PKC for 2 hrs prior to analysis, since previous studies had indicated that receptor phosphorylation could be beneficial to detect IR binding.

Transfected samples were also analyzed by Western blots. The protocol used for Western blot assay of transfected cells is as follows. Cell membranes were prepared in a special cocktail of protease inhibitors (1 mM EDTA, 0.1 mM EGTA, 1 mM phenylmethyl-sufonylfluoride, 10 M e-aminocaproic acid, 0.1 mM benzamide, 0.1 mM benzamide-HCl , 0.1 mM phenanthroline, 10 μg/ml pepstatin A, 5 mM iodoacetamide, 10 μg/ml antipain, 10 μg/ml trypsin-chymotrypsin inhibitor, 10 μg/ml leupeptin, and 1.67 μg/ml calpain inhibitor) in 0.25 M sucrose, 1 mM MgCl₂, 5 mM Tris, pH 7.4. Fifteen μg of total protein were denatured and separated by SDS gel electrophoresis. Gels were equilibrated and electrotransferred to nitrocellulose membranes. Blots were then blocked with 10% milk in Tris- buffered saline with 0.1% Tween-20 (TBST) during 60 min. of gentle rocking. Afterwards, blots were incubated in anti- imidazoline receptor antiserum (1:3000 dil.) for 2 hours. Following the primary antibody, blots were washed and incubated with horseradish peroxidase-conjugated anti-rabbit goat IgG (1:3000 dil.) for 1 hr. Blots were extensively washed and incubated for 1 min. in a 1:1 mix of Amersham ECL detection solution. The blots were wrapped in cling-film (SARAN WRAP) and exposed to Hyperfil -ECL (Amersham) for 2 minutes. Quantitation was based on densitometry using a standard curve of known amounts of protein containing BAC membranes or platelet membranes run in each gel. One nM [¹²⁵I ]p-iodoclonidine was employed in the radioligand binding competition assays, since at this low concentration this radioligand is selective for the IR site much more than for I₂ binding sites. The critical processes of membrane preparation of tissue culture cells and the radioligand binding assays of IR and I₂ have been reviewed by Piletz and colleagues [Ernsberger et al., Annals NY Acad Sci.. 763: 510-519 (1995)]. Total binding (n = 12 per experiment) was determined in the absence of added competitive ligands and nonspecific binding was determined in the presence of 10^"4 M moxonidine (n = 6 per experiment) . Log normal competition curves were generated against unlabeled moxonidine, p- iodoclonidine, and (-) epinephrine. Each concentration of the competitors was determined in triplicate and the experiment was repeated thrice. The protocol to fully characterize radioligand binding in the transfected cells entails the following. First, the presence of IR and/or I₂ binding sites are scanned over a range of protein concentrations using a single concentration of [¹²⁵I]-p-iodoclonidine (l.OnM) and H-idazoxan (8nM) , respectively. Then, rate of association binding experiments (under a 10 μM mask of NE to remove α₂AR interference) are performed to determine if the kinetic parameters are similar to those reported for native imidazoline receptors [Ernsberger et al. Annals NY Acad. Sci.. 763: 163-168 (1995)]. Then, full Scatchard plots of [¹²⁵I]-p-iodoclonidine (2-20 nM if like IR) and Η-idazoxan (5-60 n if like I₂) binding are conducted under a 10 μM mask of NE. Total NE (10 μM) -displaceable binding is ascertained as a control to rule out α₂-adrenergic binding. The Bmax and K_D parameters for the transfected cells are ascertained by computer modeling using the LIGAND program [McPherson, G., J.Pharmacol .Meth. 14: 213-228 (1985) ] using 20 μM moxonidine to define IR nonspecific binding, or 20 μM cirazoline to define I₂ nonspecific binding. The results of the transient transfection experiments of the imidazoline receptor vector into COS-7 cells are shown in Fig. 4. Competition binding experiments were performed using membrane preparations from these cells and ¹²⁵PIC was used to radiolabel IR sites. A mask of 10 μM norepinephrine was used to rule out any possible α₂AR binding in each assay even though parent COS-7 cells lacked any α₂AR sites. Moxonidine and p-iodoclondine (PIC) were the compounds tested for their affinity to the membranes of transfected cells. As can be seen, the affinities of these compounds in competition with ^I25PIC were well within the high affinity (nM) range.

The following IC₅₀ values and Hill slopes were obtained in this study: moxonidine, IC₅₀ = 45.1 nM (Hill slope = 0.35 ± 0.04); p-iodoclonidine without PKC pretreatment of the membranes, IC₅₀ = 2.3 nM (Hill slope = 0.42 ± 0.06); p-iodoclonidine with PKC pretreatment of the membranes, IC₅₀ = 19.0 nM (Hill slope = 0.48 ± 0.08). Shallow Hill slopes for [¹²⁵I]p-iodoclonidine have been reported before in studies of the interaction of moxonidine and p-iodoclonidine with the human platelet IR, binding site [Piletz and Sletten, (1993)]. Epinephrine failed to displace any of the [¹²⁵I]p-iodoclonidine binding in the transfected cells, as expected since this is a nonadrenergic imidazoline receptor. Furthermore, in untransfected cells less than 5% of the amount of displaceable binding was observed as for the transfected cells - and this "noise" in the parent cells all appeared to be low affinity (data not shown) . These results thus demonstrate the high affinities of two imidazoline compounds, p-iodoclonidine and moxonidine, for the portion of our cloned receptor encoded within EST04033. PKC pretreatment of the membranes had no effect in the transfected COS cells.

It was also observed that the level of the expressed protein, as measured by Western blotting of the transfected cells, was consistent with the level of IR binding that was detected. In other words, a protein band was uniquely detected in the transfected cells, and it was of a density consistent with the amount of radioligand binding. Hence, the present results are in keeping with those expected for an imidazoline receptor. In summary, these data provide direct evidence that the EST04033 clone encodes an imidazoline binding site having high affinities for moxonidine and p-iodoclonidine, which is expected for an IR protein.

Example 5. Stable Transfection Methods.

Stable transfections can be obtained by subcloning the imidazoline receptor cDNA into a suitable expression vector, e.g., pRc/CMV (Invitrogen, San Diego, CA) , which can then be used to transform host cells, e.g. CHO and HEK-293 cells, using the Lipofectin reagent (Gibco/BRL, Gaithersburg, MD) according to the manufacturer's instructions. These two host cell lines can be used to increase the permanence of expression of an instant clone. The inventors have previously ascertained that parent CHO cells lack both alpha₂-adrenoceptor and IR binding sites [Piletz et al., J. Pharm. & Exper. Ther.. 272: 581-587 (1995)], making them useful for these studies. Twenty-four hours after transfection, cells are split into culture dishes and grown in the presence of 600 μg/ml G418-supplemented complete medium (Gibco/BRL) . The medium is changed every 3 days and clones surviving in G418 are isolated and expanded for further investigation .

Example 6. Direct Cloning of More Complete Gene and Other Homologous Human IR.

Direct probing of other human genomic and cDNA libraries can be performed by preparing labelled cDNA probes from different subcloned regions of our clone. Commercially available human DNA libraries can be used. Besides the cDNA and genomic libraries we have already screened, another genomic library is EMBL (Clontech) , which integrates genomic fragments up to 22 kbp long. It is reasonable to expect that introns may exist within other human IR genes so that only by obtaining overlapping clones can the full-length genes be sequenced. A probe encompassing the 5' end of an instant cDNA is generally useful to obtain the gene promoter region. Clontech's Human PromoterFinder DNA Walking procedure provides a method for "walking" upstream or downstream from cloned sequences such as cDNAs into adjacent genomic DNA.

Example 7. Methods for Preparing Antibodies to Imidazoline Receptive Proteins.

An instant imidazoline receptive polypeptide can also be used to prepare antibodies immunoreactive therewith. Thus, synthetic peptides (based on deduced amino acid sequences from the DNA) can be generated and used as immunogens . Additionally, transfected cell lines or other manipulations of the DNA sequence of an instant imidazoline receptor can provide a source of purified imidazoline receptor peptides in sufficient quantities for immunization, which can lead to a source of selective antibodies having potential commercial value.

In addition, various kits for assaying imidazoline receptors can be developed that include either such antibodies or the purified imidazoline receptor protein. A purification protocol has already been published for the bovine imidazoline receptor in BAC cells [Wang et al, 1992] and an immunization protocol has also been published [Wang et al., 1993]. These same protocols can be utilized with little if any modification to afford purified human IR protein from transfected cells and to yield selective antibodies thereto. In order to obtain antibodies to a subject peptide, the peptide may be linked to a suitable soluble carrier to which antibodies are unlikely to be encountered in human serum. Illustrative carriers include bovine serum albumin, keyhole limpet hemocyanin, and the like. The conjugated peptide is injected into a mouse, or other suitable animal, where an immune response is elicited. Monoclonal antibodies can be obtained from hybridomas formed by fusing spleen cells harvested from the animal and myeloma cells [see, e.g., Kohler and Milstein, Nature, 256: 495-497 (1975)]. Once an antibody is prepared (either polyclonal or monoclonal) , procedures are well established in the literature, using other proteins, to develop either RIA or ELISA assays [see, e.g., "Radioimmunoassay of Gut Regulatory Peptides; Methods in Laboratory Medicine," Vol. 2, chapters 1 and 2, Praeger Scientific Press, 1982]. In the case of RIA, the purified protein can also be radiolabelled and used as a radioactive antigen tracer.

Currently available methods to assay imidazoline receptors are unsuitable for routine clinical use, and therefore the development of an assay kit in this manner could have significant market appeal. Suitable assay techniques can employ polyclonal or monoclonal antibodies, as has been previously described [U.S. Patent No. 4,376,110 (issued to David et al.), the disclosure of which is incorporated herein by reference] . Summary

In summary, we have identified unique DNA sequences that have properties expected of a gene and the cDNA transcript (s) of an imidazoline receptor. Prior to our first cloning the cDNA, only two sequences of EST cDNA were identified within public databases having similar nature. But, these were both partial and imprecise sequences - not identified at all with respect to any encoded protein. Indeed, one of them (HSA09H122) was reported to be contaminated. In our hands, the other EST 04033 clone was correctly sequenced for the first time (in its entirety = 3318 bp) . Prior to this, even the size of EST 04033 was unknown. The present inventors also demonstrated that an imidazoline receptive site can be expressed in cells transfected with the EST 04033 cDNA clone, and this site has the proper potencies of an IR. We have deduced most of the complete cDNA encoding this protein.

The present invention has been described with reference to specific examples for purposes of clarity and explanation. Certain obvious modifications of the invention readily apparent to one skilled in the art can be practiced within the scope of the appended claims.

SEQUENCE LISTING

GENERAL INFORMATION:

APPLICANT: John E. Piletz

Tina R. Ivanov TITLE OF INVENTION: DNA MOLECULES ENCODING IMIDAZOLINE RECEPTIVE POLYPEPTIDES AND POLYPEPTIDES ENCODED THEREBY NUMBER OF SEQUENCES: 22 CORRESPONDENCE ADDRESS:

ADDRESSEE: WENDEROTH, LIND & PONACK STREET: 805 Fifteenth St. N.W., Suite 700 CITY: Washington STATE: District of Columbia COUNTRY: U.S.A. ZIP: 20005 COMPUTER READABLE FORM:

MEDIUM TYPE: 3.50" 1.44 Mb diskette COMPUTER: IBM PC compatible OPERATING SYSTEM: MS-DOS SOFTWARE: WordPerfect 5.1+ CURRENT APPLICATION DATA:

APPLICATION NUMBER: PCT/US97/15695 FILING DATE: September 3, 1997 CLASSIFICATION: PRIOR APPLICATION DATA:

APPLICATION NO.: USSN 60/012,600 FILING DATE: March 1, 1996 PRIOR APPLICATION DATA:

APPLICATION NO.: USSN 08/650,766 FILING DATE: May 20, 1996 ATTORNEY/AGENT INFORMATION:

NAME: Warren Cheek REGISTRATION NUMBER: 33,367 REFERENCE/DOCKET NUMBER: WMC-1342/clone TELECOMMUNICATION INFORMATION:

TELEPHONE: (202) 371-8850 TELEFAX: (202) 371-8856 INFORMATION FOR SEQ ID NO: 1 SEQUENCE CHARACTERISTICS:

LENGTH: 3389 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear ORIGINAL SOURCE:

ORGANISM: Homo sapiens IMMEDIATE SOURCE:

LIBRARY: cDNA CLONE: EST04033 (HFBDP28) FEATURE :

NAME/KEY: predicted translation product when transfected

LOCATION: 1398 ... 3389 SEQUENCE DESCRIPTION: SEQ ID NO: 1

GCTCTAGAAC TAGTGGATCC CCCGGGCTGC AGGAATTCCA GTTTAATACT AACCCTAATG 60

TGTGACTGCG GTTTACAAAG AGCTCTGTAT CACCTGGGAT AGCTTTCAGT AGCAATTCAC 120

TACAACTGGT CCTAAAAAAT AATAACAATA ATAATAATAA TTAGAGAATT AAAACCCAAC 180

AGCATGTTGA ATGGTTAAAA TCACGTAAGA ACTGAAATTT GGGGTGGGGG TGTCCTCAAC 240

AGCTGAGCTT GTCCTAGCAG TGAAAATGCT CGCCTCCAAG CAGGGCTCAG AAAGGTCTGG 300

AGCCCTCCAG GCAGAGGGCT GAGCTCAGGG GGCTCTTGGA GGACACTCAC CCCATGGTCC 360

ATGGGATGCT TCTGGCTTCC TTAAAAACAG TTGGGCATCC GCATTGTATA AGTAGGTGGA 420

GACCCTAGTG TGGTTCTTTT GAAGGATATG GGAAGGGAGG ATGACGAACT AGAGAAGTGG 480

GAGGGGACCA AAATCACTGA GGTCCCAGAA TATCATAGAT TTGGGTATAG GATTGGGGTC 540

ACTAAGAATT GAGCACCAGG AATTCCAGCT TCTTCCCATT AAAGAAACTG GGACTGGTTT 600

TGCCTTGGAG GCCTATGTAG TGTTTTCTGC CCCTGTCCCA TACCAAGTCT CATTGATATT 660

TCTGCAGAAT ATCAGATGAA AATCTATTTC TAAAGACCAT TGGGAGAATG GGTGGTGGAG 720

AAGGAGTTGG AGTGGGGTTG GGGGGCAGTT AAAAATGAAT AAAAATCTCT CAGCTACAGA 780

ACCCAAACAT CACTTCCCTC CGCATTCACA GCATTTCCCA GCAGTCCCCA GATGGTTGTT 840 TCCGTGGGGA CACAGCAGCT GCCTCATTTC CCTTCAGGCC CCATGGGCTG CTGGTCAACC 900

TCAGGATCTA CTAAAGATGA CGCAAATGCC GACTGAACAA TCTGAAACCC AAAGGACTCG 960

AGGAGAGACA TGTTCTGCTG AGGAGAGAAA GGTGAGCCAA GGGCAGGGCC CAGGTCCCCC 1020

AGGGGGCCCC CGAGAGCCCG GACATGCACC TTCTGGATGT GTTTGTTCAA GTAGGACTTA 1080

GAGCGGAAGA AGCTCCCACA TTCAGGGCAT GGGTACTTCT TCTCCCCATC AGACTCCATT 1140

TTGTTTTTGG GGACTGCCAT GTCGCAGGAG AAAGAGCCAT TGGCACTCTG CTTCTCTGGC 1200

GTCTTCAGGT CGCTGGCATC TGAGAGGTCA CCATAGGAGT CAGAGCTCTC AATCGGATCC 1260

TGATGTGAGC ATTTCTGGCC TTCTCGGTTA CAGATACTGC AGAAGTTGCT GGGCCCCTCG 1320

CTGTGCTTCT TCAGGTGGTC TGCCATGTAT GCTGCCCGCA AGTACTTCCC ACACACCTGG 1380

CAGGGCACCT TGTCTTC ATG ACA GGC CAG GTG GGA GCG CAG ACG GTC TCG 1430

Met Thr Gly Gin Val Gly Ala Gin Thr Val Ser 1 5 10

GGT GGC AAA AGA AGC ATT GCA GGT CTG ACA CTT GTG AGG CCG CTC AGA 1478 Gly Gly Lys Arg Ser lie Ala Gly Leu Thr Leu Val Arg Pro Leu Arg 15 20 25

AGT GTG CAC CTG CTT GAT ATG TCC GTT CAA GTG ATC AGG CCT GGA GAA 1526 Ser Val His Leu Leu Asp Met Ser Val Gin Val lie Arg Pro Gly Glu 30 35 40

GCC TTT CCC ACA GCT CTG GCA GAT GTA AGG CGG AAT TCC CCA GAG AAG 1574 Ala Phe Pro Thr Ala Leu Ala Asp Val Arg Trp Asn Ser Pro Glu Lys 45 50 55

AAG GGT GGT GAA GAC TCC CGG CTC TCA GCT GCC CCC TGC ATC AGA CCC 1622 Lys Gly Gly Glu Asp Ser Trp Leu Ser Ala Ala Pro Cys lie Arg Pro 60 65 70 75

AGC AGC TCC CCT CCC ACT GTG GCT CCC GCA TCT GCC TCC CTG CCC CAG 1670 Ser Ser Ser Pro Pro Thr Val Ala Pro Ala Ser Ala Ser Leu Pro Gin 80 85 90

CCC ATC CTC TCT AAC CAA GGA ATC ATG TTC GTT CAG GAG GAG GCC CTG 1718 Pro lie Leu Ser Asn Gin Gly lie Met Phe Val Gin Glu Glu Ala Leu 95 100 105

GCC AGC AGC CTC TCG TCC ACT GAC AGT CTG ACT CCC GAG CAC CAG CCC 1766 Ala Ser Ser Leu Ser Ser Thr Asp Ser Leu Thr Pro Glu His Gin Pro 110 115 120

ATT GCC CAG GGA TGT TCT GAT TCC TTG GAG TCC ATC CCT GCG GGA CAG 1814 lie Ala Gin Gly Cys Ser Asp Ser Leu Glu Ser lie Pro Ala Gly Gin 125 130 135

GCA GCT TCC GAT GAT TTA AGG GAC GTG CCA GGA GCT GTT GGT GGT GCA 1862 Ala Ala Ser Asp Asp Leu Arg Asp Val Pro Gly Ala Val Gly Gly Ala 140 145 150 155

AGC CCA GAA CAT GCC GAG CCG GAG GTC CAG GTG GTG CCG GGG TCT GGC 1910 Ser Pro Glu His Ala Glu Pro Glu Val Gin Val Val Pro Gly Ser Gly 160 165 170

CAG ATC ATC TTC CTG CCC TTC ACC TGC ATT GGC TAC ACG GCC ACC AAT 1958 Gin lie lie Phe Leu Pro Phe Thr Cys lie Gly Tyr Thr Ala Thr Asn 175 180 185

CAG GAC TTC ATC CAG CGC CTG AGC ACA CTG ATC CGG CAG GCC ATC GAG 2006 Gin Asp Phe lie Gin Arg Leu Ser Thr Leu lie Trp Gin Ala lie Glu 190 195 200

CGG CAG CTG CCT GCC TGG ATC GAG GCT GCC AAC CAG CGG GAG GAG GGC 2054 Trp Gin Leu Pro Ala Trp lie Glu Ala Ala Asn Gin Trp Glu Glu Gly 205 210 215

CAG GGT GAA CAG GGC GAG GAG GAG GAT GAG GAG GAG GAA GAA GAG GAG 2102 Gin Gly Glu Gin Gly Glu Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu 220 225 230 235 GAC GTG GCT GAG AAC CGC TAC TTT GAA ATG GGG CCC CCA GAC GTG GAG 2150 Asp Val Ala Glu Asn Arg Tyr Phe Glu Met Gly Pro Pro Asp Val Glu 240 245 250

GAG GAG GAG GGA GGA GGC CAG GGG GAG GAA GAG GAG GAG GAA GAG GAG 2198 Glu Glu Glu Gly Gly Gly Gin Gly Glu Glu Glu Glu Glu Glu Glu Glu 255 260 265

GAT GAA GAG GCC GAG GAG GAG CGC CTG GCT CTG GAA TGG GCC CTG GGC 2246 Asp Glu Glu Ala Glu Glu Glu Arg Leu Ala Leu Glu Trp Ala Leu Gly 270 275 280

GCG GAC GAG GAC TTC CTG CTG GAG CAC ATC CGC ATC CTC AAG GTG CTG 2294 Ala Asp Glu Asp Phe Leu Leu Glu His lie Arg lie Leu Lys Val Leu 285 290 295

TGG TGC TTC CTG ATC CAT GTG CAG GGC AGT ATC CGC CAG TTC GCC GCC 2342 Trp Cys Phe Leu lie His Val Gin Gly Ser lie Arg Gin Phe Ala Ala 300 305 310 315

TGC CTT GTG CTC ACC GAC TTC GGC ATC GCA GTC TTC GAG ATC CCG CAC 2390 Cys Leu Val Leu Thr Asp Phe Gly lie Ala Val Phe Glu lie Pro His 320 325 330

CAG GAG TCT CGG GGC AGC AGC CAG CAC ATC CTC TCC TCC CTG CGC TTT 2438 Gin Glu Ser Trp Gly Ser Ser Gin His lie Leu Ser Ser Leu Arg Phe 335 340 345

GTC TTT TGC TTC CCG CAT GGC GAC CTC ACC GAG TTT GGC TTC CTC ATG 2486 Val Phe Cys Phe Pro His Gly Asp Leu Thr Glu Phe Gly Phe Leu Met 350 355 360

CCG GAG CTG TGT CTG GTG CTC AAG GTA CGG CAC AGT GAG AAC ACG CTC 2534 Pro Glu Leu Cys Leu Val Leu Lys Val Arg His Ser Glu Asn Thr Leu 365 370 375

TTC ATT ATC TCG GAC GCC GCC AAC CTG CAC GAG TTC CAC GCG GAC CTG 2582 Phe lie lie Ser Asp Ala Ala Asn Leu His Glu Phe His Ala Asp Leu 380 385 390 395

CGC TCA TGC TTT GCA CCC CAG CAC ATG GCC ATG CTG TGT AGC CCC ATC 2630 Arg Ser Cys Phe Ala Pro Gin His Met Ala Met Leu Cys Ser Pro lie 400 405 410

CTC TAC GGC AGC CAC ACC AGC CTG CAG GAG TTC CTG CGC CAG CTG CTC 2678 Leu Tyr Gly Ser His Thr Ser Leu Gin Glu Phe Leu Arg Gin Leu Leu 415 420 425

ACC TTC TAC AAG GTG GCT GGC GGC TGC CAG GAG CGC AGC CAG GGC TGC 2726 Thr Phe Tyr Lys Val Ala Gly Gly Cys Gin Glu Arg Ser Gin Gly Cys 430 435 440

TTC CCC GTC TAC CTG GTC TAC AGT GAC AAG CGC ATG GTG CAG ACG GCC 2774 Phe Pro Val Tyr Leu Val Tyr Ser Asp Lys Arg Met Val Gin Thr Ala 445 450 455

GCC GGG GAC TAC TCA GGC AAC ATC GAG TGG GCC AGC TGC ACA CTC TGT 2822 Ala Gly Asp Tyr Ser Gly Asn lie Glu Trp Ala Ser Cys Thr Leu Cys 460 465 470 475

TCA GCC GTG CGG CGC TCC TGC TGC GCG CCC TCT GAG GCC GTC AAG TCC 2870 Ser Ala Val Arg Arg Ser Cys Cys Ala Pro Ser Glu Ala Val Lys Ser 480 485 490

GCC GCC ATC CCC TAC TGG CTG TTG CTC ACG CCC CAG CAC CTC AAC GTC 2918 Ala Ala lie Pro Tyr Trp Leu Leu Leu Thr Pro Gin His Leu Asn Val 495 500 505 ATC AAG GCC GAC TTC AAC CCC ATG CCC AAC CGT GGC ACC CAC AAC TGT 2966 lie Lys Ala Asp Phe Asn Pro Met Pro Asn Arg Gly Thr His Asn Cys 510 515 520

CGC AAC CGC AAC AGC TTC AAG CTC AGC CGT GTG CCG CTC TCC ACC GTG 3014 Arg Asn Arg Asn Ser PHe Lys Leu Ser Arg Val Pro Leu Ser Thr Val 525 530 535

CTG CTG GAC CCC ACA CGC AGC TGT ACC CAG CCT CGG GGC GCC TTT GCT 3062 Leu Leu Asp Pro Thr Arg Ser Cys Thr Gin Pro Arg Gly Ala Phe Ala 540 545 550 555

GAT GGC CAC GTG CTA GAG CTG CTC GTG GGG TAC CGC TTT GTC ACT GCC 3110 Asp Gly His Val Leu Glu Leu Leu Val Gly Tyr Arg Phe Val Thr Ala 560 565 570

ATC TTC GTG CTG CCC CAC GAG AAG TTC CAC TTC CTG CGC GTC TAC AAC 3158 lie Phe Val Leu Pro His Glu Lys Phe His Phe Leu Arg Val Tyr Asn 575 580 585

CAG CTG CGG GCC TCG CTG CAG GAC CTG AAG ACT GTG GTC ATC GCC AAG 3206 Gin Leu Arg Ala Ser Leu Gin Asp Leu Lys Thr Val Val lie Ala Lys 590 595 600

ACC CCC GGG ACG GGA GGC AGC CCC CAG GGC TCC TTT GCG GAT GGC CAG 3254 Thr Pro Gly Thr Gly Gly Ser Pro Gin Gly Ser Phe Ala Asp Gly Gin 605 610 615

CCT GCC GAG CGC AGG GCC AGC AAT GAC CAG CGT CCC CAG GAG GTC CCA 3302 Pro Ala Glu Arg Arg Ala Ser Asn Asp Gin Arg Pro Gin Glu Val Pro 620 625 630 635

GCA GAG GCT CTG GCC CCG GCC CCA GTG GAA GTC CCA GCT CCA GCC CCG 3350 Ala Glu Ala Leu Ala Pro Ala Pro Val Glu Val Pro Ala Pro Ala Pro 640 645 650

GAA TTC GAT ATC AAG CTT ATC GAT ACC GTC GAC CTG CAG 3389 Glu Phe Asp lie Lys Leu He Asp Thr Val Asp Leu Gin 655 660 664

INFORMATION FOR SEQ ID NO: 2

SEQUENCE CHARACTERISTICS:

LENGTH: 1954 base pairs

TYPE: nucleic acid

STRANDEDNESS: double

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 2

ATGACAGGCC AGGTGGGAGC GCAGACGGTC TCGGGTGGCA AAAGAAGCAT TGCAGGTCTG 60

ACACTTGTGA GGCCGCTCAG AAGTGTGCAC CTGCTTGATA TGTCCGTTCA AGTGATCAGG 120

CCTGGAGAAG CCTTTCCCAC AGCTCTGGCA GATGTAAGGC GGAATTCCCC AGAGAAGAAG 180

GGTGGTGAAG ACTCCCGGCT CTCAGCTGCC CCCTGCATCA GACCCAGCAG CTCCCCTCCC 240

ACTGTGGCTC CCGCATCTGC CTCCCTGCCC CAGCCCATCC TCTCTAACCA AGGAATCATG 300

TTCGTTCAGG AGGAGGCCCT GGCCAGCAGC CTCTCGTCCA CTGACAGTCT GACTCCCGAG 360

CACCAGCCCA TTGCCCAGGG ATGTTCTGAT TCCTTGGAGT CCATCCCTGC GGGACAGGCA 420

GCTTCCGATG ATTTAAGGGA CGTGCCAGGA GCTGTTGGTG GTGCAAGCCC AGAACATGCC 480

GAGCCGGAGG TCCAGGTGGT GCCGGGGTCT GGCCAGATCA TCTTCCTGCC CTTCACCTGC 540

ATTGGCTACA CGGCCACCAA TCAGGACTTC ATCCAGCGCC TGAGCACACT GATCCGGCAG 600

GCCATCGAGC GGCAGCTGCC TGCCTGGATC GAGGCTGCCA ACCAGCGGGA GGAGGGCCAG 660

GGTGAACAGG GCGAGGAGGA GGATGAGGAG GAGGAAGAAG AGGAGGACGT GGCTGAGAAC 720

CGCTACTTTG AAATGGGGCC CCCAGACGTG GAGGAGGAGG AGGGAGGAGG CCAGGGGGAG 780

GAAGAGGAGG AGGAAGAGGA GGATGAAGAG GCCGAGGAGG AGCGCCTGGC TCTGGAATGG 840

GCCCTGGGCG CGGACGAGGA CTTCCTGCTG GAGCACATCC GCATCCTCAA GGTGCTGTGG 900

TGCTTCCTGA TCCATGTGCA GGGCAGTATC CGCCAGTTCG CCGCCTGCCT TGTGCTCACC 960

GACTTCGGCA TCGCAGTCTT CGAGATCCCG CACCAGGAGT CTCGGGGCAG CAGCCAGCAC 1020

ATCCTCTCCT CCCTGCGCTT TGTCTTTTGC TTCCCGCATG GCGACCTCAC CGAGTTTGGC 1080

TTCCTCATGC CGGAGCTGTG TCTGGTGCTC AAGGTACGGC ACAGTGAGAA CACGCTCTTC 1140

ATTATCTCGG ACGCCGCCAA CCTGCACGAG TTCCACGCGG ACCTGCGCTC ATGCTTTGCA 1200 CCCCAGCACA TGGCCATGCT GTGTAGCCCC ATCCTCTACG GCAGCCACAC CAGCCTGCAG 1260

GAGTTCCTGC GCCAGCTGCT CACCTTCTAC AAGGTGGCTG GCGGCTGCCA GGAGCGCAGC 1320

CAGGGCTGCT TCCCCGTCTA CCTGGTCTAC AGTGACAAGC GCATGGTGCA GACGGCCGCC 1380

GGGGACTACT CAGGCAACAT CGAGTGGGCC AGCTGCACAC TCTGTTCAGC CGTGCGGCGC 1440

TCCTGCTGCG CGCCCTCTGA GGCCGTCAAG TCCGCCGCCA TCCCCTACTG GCTGTTGCTC 1500

ACGCCCCAGC ACCTCAACGT CATCAAGGCC GACTTCAACC CCATGCCCAA CCGTGGCACC 1560

CACAACTGTC GCAACCGCAA CAGCTTCAAG CTCAGCCGTG TGCCGCTCTC CACCGTGCTG 1620

CTGGACCCCA CACGCAGCTG TACCCAGCCT CGGGGCGCCT TTGCTGATGG CCACGTGCTA 1680

GAGCTGCTCG TGGGGTACCG CTTTGTCACT GCCATCTTCG TGCTGCCCCA CGAGAAGTTC 1740

CACTTCCTGC GCGTCTACAA CCAGCTGCGG GCCTCGCTGC AGGACCTGAA GACTGTGGTC 1800

ATCGCCAAGA CCCCCGGGAC GGGAGGCAGC CCCCAGGGCT CCTTTGCGGA TGGCCAGCCT 1860

GCCGAGCGCA GGGCCAGCAA TGACCAGCGT CCCCAGGAGG TCCCAGCAGA GGCTCTGGCC 1920

CCGGCCCCAG TGGAAGTCCC AGCTCCAGCC CCGG 1954

INFORMATION FOR SEQ ID NO: 3

SEQUENCE CHARACTERISTICS:

LENGTH: 3318 base pairs

TYPE: nucleic acid

STRANDEDNESS: double

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 3

AATTCCAGTT TAATACTAAC CCTAATGTGT GACTGCGGTT TACAAAGAGC TCTGTATCAC 60

CTGGGATAGC TTTCAGTAGC AATTCACTAC AACTGGTCCT AAAAAATAAT AACAATAATA 120

ATAATAATTA GAGAATTAAA ACCCAACAGC ATGTTGAATG GTTAAAATCA CGTAAGAACT 180

GAAATTTGGG GTGGGGGTGT CCTCAACAGC TGAGCTTGTC CTAGCAGTGA AAATGCTCGC 240

CTCCAAGCAG GGCTCAGAAA GGTCTGGAGC CCTCCAGGCA GAGGGCTGAG CTCAGGGGGC 300

TCTTGGAGGA CACTCACCCC ATGGTCCATG GGATGCTTCT GGCTTCCTTA AAAACAGTTG 360

GGCATCCGCA TTGTATAAGT AGGTGGAGAC CCTAGTGTGG TTCTTTTGAA GGATATGGGA 420

AGGGAGGATG ACGAACTAGA GAAGTGGGAG GGGACCAAAA TCACTGAGGT CCCAGAATAT 480

CATAGATTTG GGTATAGGAT TGGGGTCACT AAGAATTGAG CACCAGGAAT TCCAGCTTCT 540

TCCCATTAAA GAAACTGGGA CTGGTTTTGC CTTGGAGGCC TATGTAGTGT TTTCTGCCCC 600

TGTCCCATAC CAAGTCTCAT TGATATTTCT GCAGAATATC AGATGAAAAT CTATTTCTAA 660 AGACCATTGG GAGAATGGGT GGTGGAGAAG GAGTTGGAGT GGGGTTGGGG GGCAGTTAAA 720 AATGAATAAA AATCTCTCAG CTACAGAACC CAAACATCAC TTCCCTCCGC ATTCACAGCA 780 TTTCCCAGCA GTCCCCAGAT GGTTGTTTCC GTGGGGACAC AGCAGCTGCC TCATTTCCCT 840 TCAGGCCCCA TGGGCTGCTG GTCAACCTCA GGATCTACTA AAGATGACGC AAATGCCGAC 900 TGAACAATCT GAAACCCAAA GGACTCGAGG AGAGACATGT TCTGCTGAGG AGAGAAAGGT 960

GAGCCAAGGG CAGGGCCCAG GTCCCCCAGG GGGCCCCCGA GAGCCCGGAC ATGCACCTTC 1020

TGGATGTGTT TGTTCAAGTA GGACTTAGAG CGGAAGAAGC TCCCACATTC AGGGCATGGG 1080

TACTTCTTCT CCCCATCAGA CTCCATTTTG TTTTTGGGGA CTGCCATGTC GCAGGAGAAA 1140

GAGCCATTGG CACTCTGCTT CTCTGGCGTC TTCAGGTCGC TGGCATCTGA GAGGTCACCA 1200

TAGGAGTCAG AGCTCTCAAT CGGATCCTGA TGTGAGCATT TCTGGCCTTC TCGGTTACAG 1260

ATACTGCAGA AGTTGCTGGG CCCCTCGCTG TGCTTCTTCA GGTGGTCTGC CATGTATGCT 1320

GCCCGCAAGT ACTTCCCACA CACCTGGCAG GGCACCTTGT CTTCATGACA GGCCAGGTGG 1380

GAGCGCAGAC GGTCTCGGGT GGCAAAAGAA GCATTGCAGG TCTGACACTT GTGAGGCCGC 1440

TCAGAAGTGT GCACCTGCTT GATATGTCCG TTCAAGTGAT CAGGCCTGGA GAAGCCTTTC 1500

CCACAGCTCT GGCAGATGTA AGGCGGAATT CCCCAGAGAA GAAGGGTGGT GAAGACTCCC 1560

GGCTCTCAGC TGCCCCCTGC ATCAGACCCA GCAGCTCCCC TCCCACTGTG GCTCCCGCAT 1620

CTGCCTCCCT GCCCCAGCCC ATCCTCTCTA ACCAAGGAAT CATGTTCGTT CAGGAGGAGG 1680

CCCTGGCCAG CAGCCTCTCG TCCACTGACA GTCTGACTCC CGAGCACCAG CCCATTGCCC 1740

AGGGATGTTC TGATTCCTTG GAGTCCATCC CTGCGGGACA GGCAGCTTCC GATGATTTAA 1800

GGGACGTGCC AGGAGCTGTT GGTGGTGCAA GCCCAGAACA TGCCGAGCCG GAGGTCCAGG 1860

TGGTGCCGGG GTCTGGCCAG ATCATCTTCC TGCCCTTCAC CTGCATTGGC TACACGGCCA 1920

CCAATCAGGA CTTCATCCAG CGCCTGAGCA CACTGATCCG GCAGGCCATC GAGCGGCAGC 1980

TGCCTGCCTG GATCGAGGCT GCCAACCAGC GGGAGGAGGG CCAGGGTGAA CAGGGCGAGG 2040

AGGAGGATGA GGAGGAGGAA GAAGAGGAGG ACGTGGCTGA GAACCGCTAC TTTGAAATGG 2100

GGCCCCCAGA CGTGGAGGAG GAGGAGGGAG GAGGCCAGGG GGAGGAAGAG GAGGAGGAAG 2160

AGGAGGATGA AGAGGCCGAG GAGGAGCGCC TGGCTCTGGA ATGGGCCCTG GGCGCGGACG 2220

AGGACTTCCT GCTGGAGCAC ATCCGCATCC TCAAGGTGCT GTGGTGCTTC CTGATCCATG 2280

TGCAGGGCAG TATCCGCCAG TTCGCCGCCT GCCTTGTGCT CACCGACTTC GGCATCGCAG 2340

TCTTCGAGAT CCCGCACCAG GAGTCTCGGG GCAGCAGCCA GCACATCCTC TCCTCCCTGC 2400

GCTTTGTCTT TTGCTTCCCG CATGGCGACC TCACCGAGTT TGGCTTCCTC ATGCCGGAGC 2460

TGTGTCTGGT GCTCAAGGTA CGGCACAGTG AGAACACGCT CTTCATTATC TCGGACGCCG 2520

CCAACCTGCA CGAGTTCCAC GCGGACCTGC GCTCATGCTT TGCACCCCAG CACATGGCCA 2580

TGCTGTGTAG CCCCATCCTC TACGGCAGCC ACACCAGCCT GCAGGAGTTC CTGCGCCAGC 2640

TGCTCACCTT CTACAAGGTG GCTGGCGGCT GCCAGGAGCG CAGCCAGGGC TGCTTCCCCG 2700 TCTACCTGGT CTACAGTGAC AAGCGCATGG TGCAGACGGC CGCCGGGGAC TACTCAGGCA 2760

ACATCGAGTG GGCCAGCTGC ACACTCTGTT CAGCCGTGCG GCGCTCCTGC TGCGCGCCCT 2820

CTGAGGCCGT CAAGTCCGCC GCCATCCCCT ACTGGCTGTT GCTCACGCCC CAGCACCTCA 2880

ACGTCATCAA GGCCGACTTC AACCCCATGC CCAACCGTGG CACCCACAAC TGTCGCAACC 2940

GCAACAGCTT CAAGCTCAGC CGTGTGCCGC TCTCCACCGT GCTGCTGGAC CCCACACGCA 3000

GCTGTACCCA GCCTCGGGGC GCCTTTGCTG ATGGCCACGT GCTAGAGCTG CTCGTGGGGT 3060

ACCGCTTTGT CACTGCCATC TTCGTGCTGC CCCACGAGAA GTTCCACTTC CTGCGCGTCT 3120

ACAACCAGCT GCGGGCCTCG CTGCAGGACC TGAAGACTGT GGTCATCGCC AAGACCCCCG 3180

GGACGGGAGG CAGCCCCCAG GGCTCCTTTG CGGATGGCCA GCCTGCCGAG CGCAGGGCCA 3240

GCAATGACCA GCGTCCCCAG GAGGTCCCAG CAGAGGCTCT GGCCCCGGCC CCAGTGGAAG 3300

TCCCAGCTCC AGCCCCGG 3318

INFORMATION FOR SEQ ID NO: 4

SEQUENCE CHARACTERISTICS:

LENGTH: 1171 base pairs

TYPE: nucleic acid

STRANDEDNESS: double

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 4

GAGGAGGAGG AAGAGGAGGA TGAAGAGGCC GAGGAGGAGC GCCTGGCTCT GGAATGGGCC 60

CTGGGCGCGG ACGAGGACTT CCTGCTGGAG CACATCCGCA TCCTCAAGGT GCTGTGGTGC 120

TTCCTGATCC ATGTGCAGGG CAGTATCCGC CAGTTCGCCG CCTGCCTTGT GCTCACCGAC 180

TTCGGCATCG CAGTCTTCGA GATCCCGCAC CAGGAGTCTC GGGGCAGCAG CCAGCACATC 240

CTCTCCTCCC TGCGCTTTGT CTTTTGCTTC CCGCATGGCG ACCTCACCGA GTTTGGCTTC 300

CTCATGCCGG AGCTGTGTCT GGTGCTCAAG GTACGGCACA GTGAGAACAC GCTCTTCATT 360

ATCTCGGACG CCGCCAACCT GCACGAGTTC CACGCGGACC TGCGCTCATG CTTTGCACCC 420

CAGCACATGG CCATGCTGTG TAGCCCCATC CTCTACGGCA GCCACACCAG CCTGCAGGAG 480

TTCCTGCGCC AGCTGCTCAC CTTCTACAAG GTGGCTGGCG GCTGCCAGGA GCGCAGCCAG 540

GGCTGCTTCC CCGTCTACCT GGTCTACAGT GACAAGCGCA TGGTGCAGAC GGCCGCCGGG 600

GACTACTCAG GCAACATCGA GTGGGCCAGC TGCACACTCT GTTCAGCCGT GCGGCGCTCC 660

TGCTGCGCGC CCTCTGAGGC CGTCAAGTCC GCCGCCATCC CCTACTGGCT GTTGCTCACG 720

CCCCAGCACC TCAACGTCAT CAAGGCCGAC TTCAACCCCA TGCCCAACCG TGGCACCCAC 780 AACTGTCGCA ACCGCAACAG CTTCAAGCTC AGCCGTGTGC CGCTCTCCAC CGTGCTGCTG 840 GACCCCACAC GCAGCTGTAC CCAGCCTCGG GGCGCCTTTG CTGATGGCCA CGTGCTAGAG 900 CTGCTCGTGG GGTACCGCTT TGTCACTGCC ATCTTCGTGC TGCCCCACGA GAAGTTCCAC 960

TTCCTGCGCG TCTACAACCA GCTGCGGGCC TCGCTGCAGG ACCTGAAGAC TGTGGTCATC 1020

GCCAAGACCC CCGGGACGGG AGGCAGCCCC CAGGGCTCCT TTGCGGATGG CCAGCCTGCC 1080

GAGCGCAGGG CCAGCAATGA CCAGCGTCCC CAGGAGGTCC CAGCAGAGGC TCTGGCCCCG 1140

GCCCCAGTGG AAGTCCCAGC TCCAGCCCCG G 1171

INFORMATION FOR SEQ ID NO: 5

SEQUENCE CHARACTERISTICS:

LENGTH: 651 amino acids

TYPE: polypeptide

STRANDEDNESS: single

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 5

Met Thr Gly Gin Val Gly Ala Gin Thr Val Ser

1 5 10

Gly Gly Lys Arg Ser He Ala Gly Leu Thr Leu Val Arg Pro Leu Arg

15 20 25

Ser Val His Leu Leu Asp Met Ser Val Gin Val He Arg Pro Gly Glu

30 35 40

Ala Phe Pro Thr Ala Leu Ala Asp Val Arg Trp Asn Ser Pro Glu Lys

45 50 55

Lys Gly Gly Glu Asp Ser Trp Leu Ser Ala Ala Pro Cys He Arg Pro 60 65 70 75

Ser Ser Ser Pro Pro Thr Val Ala Pro Ala Ser Ala Ser Leu Pro Gin

80 85 90

Pro He Leu Ser Asn Gin Gly He Met Phe Val Gin Glu Glu Ala Leu

95 100 105

Ala Ser Ser Leu Ser Ser Thr Asp Ser Leu Thr Pro Glu His Gin Pro

110 115 120

He Ala Gin Gly Cys Ser Asp Ser Leu Glu Ser He Pro Ala Gly Gin 125 130 135

Ala Ala Ser Asp Asp Leu Arg Asp Val Pro Gly Ala Val Gly Gly Ala 140 145 150 155

Ser Pro Glu His Ala Glu Pro Glu Val Gin Val Val Pro Gly Ser Gly

160 165 170

Gin He He Phe Leu Pro Phe Thr Cys He Gly Tyr Thr Ala Thr Asn

175 180 185

Gin Asp Phe He Gin Arg Leu Ser Thr Leu He Trp Gin Ala He Glu

190 195 200

Trp Gin Leu Pro Ala Trp He Glu Ala Ala Asn Gin Trp Glu Glu Gly

205 210 215

Gin Gly Glu Gin Gly Glu Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu 220 225 230 235

Asp Val Ala Glu Asn Arg Tyr Phe Glu Met Gly Pro Pro Asp Val Glu

240 245 250

Glu Glu Glu Gly Gly Gly Gin Gly Glu Glu Glu Glu Glu Glu Glu Glu

255 260 265

Asp Glu Glu Ala Glu Glu Glu Arg Leu Ala Leu Glu Trp Ala Leu Gly

270 275 280

Ala Asp Glu Asp Phe Leu Leu Glu His He Arg He Leu Lys Val Leu

285 290 295

Trp Cys Phe Leu He His Val Gin Gly Ser He Arg Gin Phe Ala Ala 300 305 310 315

Cys Leu Val Leu Thr Asp Phe Gly He Ala Val Phe Glu He Pro His

320 325 330

Gin Glu Ser Trp Gly Ser Ser Gin His He Leu Ser Ser Leu Arg Phe

335 340 345

Val Phe Cys Phe Pro His Gly Asp Leu Thr Glu Phe Gly Phe Leu Met

350 355 360

Pro Glu Leu Cys Leu Val Leu Lys Val Arg His Ser Glu Asn Thr Leu

365 370 375

Phe He He Ser Asp Ala Ala Asn Leu His Glu Phe His Ala Asp Leu 380 385 390 395

Arg Ser Cys Phe Ala Pro Gin His Met Ala Met Leu Cys Ser Pro He 400 405 410

Leu Tyr Gly Ser His Thr Ser Leu Gin Glu Phe Leu Arg Gin Leu Leu

415 420 425

Thr Phe Tyr Lys Val Ala Gly Gly Cys Gin Glu Arg Ser Gin Gly Cys

430 435 440

Phe Pro Val Tyr Leu Val Tyr Ser Asp Lys Arg Met Val Gin Thr Ala

445 450 455

Ala Gly Asp Tyr Ser Gly Asn He Glu Trp Ala Ser Cys Thr Leu Cys 460 465 470 475

Ser Ala Val Arg Arg Ser Cys Cys Ala Pro Ser Glu Ala Val Lys Ser

480 485 490

Ala Ala He Pro Tyr Trp Leu Leu Leu Thr Pro Gin His Leu Asn Val

495 500 505

He Lys Ala Asp Phe Asn Pro Met Pro Asn Arg Gly Thr His Asn Cys

510 515 520

Arg Asn Arg Asn Ser Phe Lys Leu Ser Arg Val Pro Leu Ser Thr Val

525 530 535

Leu Leu Asp Pro Thr Arg Ser Cys Thr Gin Pro Arg Gly Ala Phe Ala 540 545 550 555

Asp Gly His Val Leu Glu Leu Leu Val Gly Tyr Arg Phe Val Thr Ala

560 565 570

He Phe Val Leu Pro His Glu Lys Phe His Phe Leu Arg Val Tyr Asn

575 580 585

Gin Leu Arg Ala Ser Leu Gin Asp Leu Lys Thr Val Val He Ala Lys

590 595 600

Thr Pro Gly Thr Gly Gly Ser Pro Gin Gly Ser Phe Ala Asp Gly Gin

605 610 615

Pro Ala Glu Arg Arg Ala Ser Asn Asp Gin Arg Pro Gin Glu Val Pro 620 625 630 635

Ala Glu Ala Leu Ala Pro Ala Pro Val Glu Val Pro Ala Pro Ala Pro 640 645 650

INFORMATION FOR SEQ ID NO: 6

SEQUENCE CHARACTERISTICS: LENGTH: 390 amino acids TYPE: polypeptide STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 6

Glu Glu Glu Glu Glu Glu

1 5

Asp Glu Glu Ala Glu Glu Glu Arg Leu Ala Leu Glu Trp Ala Leu Gly

10 15 20

Ala Asp Glu Asp Phe Leu Leu Glu His He Arg He Leu Lys Val Leu

25 30 35

Trp Cys Phe Leu He His Val Gin Gly Ser He Arg Gin Phe Ala Ala

40 45 50

Cys Leu Val Leu Thr Asp Phe Gly He Ala Val Phe Glu He Pro His 55 60 65 70

Gin Glu Ser Trp Gly Ser Ser Gin His He Leu Ser Ser Leu Arg Phe

75 80 85

Val Phe Cys Phe Pro His Gly Asp Leu Thr Glu Phe Gly Phe Leu Met

90 95 100

Pro Glu Leu Cys Leu Val Leu Lys Val Arg His Ser Glu Asn Thr Leu

105 110 115

Phe He He Ser Asp Ala Ala Asn Leu His Glu Phe His Ala Asp Leu

120 125 130

Arg Ser Cys Phe Ala Pro Gin His Met Ala Met Leu Cys Ser Pro He 135 140 145 150

Leu Tyr Gly Ser His Thr Ser Leu Gin Glu Phe Leu Arg Gin Leu Leu

155 160 165

Thr Phe Tyr Lys Val Ala Gly Gly Cys Gin Glu Arg Ser Gin Gly Cys

170 175 180

Phe Pro Val Tyr Leu Val Tyr Ser Asp Lys Arg Met Val Gin Thr Ala

185 190 195

Ala Gly Asp Tyr Ser Gly Asn He Glu Trp Ala Ser Cys Thr Leu Cys 200 205 210 Ser Ala Val Arg Arg Ser Cys Cys Ala Pro Ser Glu Ala Val Lys Ser 215 220 225 230

Ala Ala He Pro Tyr Trp Leu Leu Leu Thr Pro Gin His Leu Asn Val

235 240 245

He Lys Ala Asp Phe Asn Pro Met Pro Asn Arg Gly Thr His Asn Cys

250 255 260

Arg Asn Arg Asn Ser Phe Lys Leu Ser Arg Val Pro Leu Ser Thr Val

265 270 275

Leu Leu Asp Pro Thr Arg Ser Cys Thr Gin Pro Arg Gly Ala Phe Ala

280 285 290

Asp Gly His Val Leu Glu Leu Leu Val Gly Tyr Arg Phe Val Thr Ala 295 300 305 310

He Phe Val Leu Pro His Glu Lys Phe His Phe Leu Arg Val Tyr Asn

315 320 325

Gin Leu Arg Ala Ser Leu Gin Asp Leu Lys Thr Val Val He Ala Lys

330 335 340

Thr Pro Gly Thr Gly Gly Ser Pro Gin Gly Ser Phe Ala Asp Gly Gin

345 350 355

Pro Ala Glu Arg Arg Ala Ser Asn Asp Gin Arg Pro Gin Glu Val Pro

360 365 370

Ala Glu Ala Leu Ala Pro Ala Pro Val Glu Val Pro Ala Pro Ala Pro 375 380 385 390

INFORMATION FOR SEQ ID NO: 7

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 7

CTTGAGGATG CGGATGTGCT 20 INFORMATION FOR SEQ ID NO: 8

SEQUENCE CHARACTERISTICS:

LENGTH: 18 base pairs

TYPE: nucleic acid

STRANDEDNESS: single

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 8

CCATGGGGTG AGTGTCCT 18

INFORMATION FOR SEQ ID NO: 9

SEQUENCE CHARACTERISTICS:

LENGTH: 18 base pairs

TYPE: nucleic acid

STRANDEDNESS: single

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 9

AGGACACTCA CCCCATGG 18

INFORMATION FOR SEQ ID NO: 10

SEQUENCE CHARACTERISTICS :

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 10

GTATGGGACA GGGGCAGAAA 20

INFORMATION FOR SEQ ID NO: 11

SEQUENCE CHARACTERISTICS :

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 11

TTTCTAAAGA CCATTGGGAG 20

INFORMATION FOR SEQ ID NO: 12

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 12

CCATTTTAAA GTAGCGGTTC 20

INFORMATION FOR SEQ ID NO: 13

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs

TYPE: nucleic acid

STRANDEDNESS: single

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 13

AGGAGAGAAA GGTGAGCCAA 20

INFORMATION FOR SEQ ID NO: 14

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 14 GTAGATCCTG AGGTTGACCA 20 INFORMATION FOR SEQ ID NO: 15

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs

TYPE: nucleic acid

STRANDEDNESS: single

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 15

TGTGAGCATT TCTGGCCTTC 20

INFORMATION FOR SEQ ID NO: 16

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 16

TGAAGACGCC AGAGAAGCAG 20

INFORMATION FOR SEQ ID NO: 17

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 17

GCCTCACAAG TGTCAGACCT 20

INFORMATION FOR SEQ ID NO: 18

SEQUENCE CHARACTERISTICS:

LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 18

AGAAGGGTGG TGAAGACT 18

INFORMATION FOR SEQ ID NO: 19

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 19

CTTGGTTAGA GAGGATGGGC 20

INFORMATION FOR SEQ ID NO: 20

SEQUENCE CHARACTERISTICS:

LENGTH: 20 base pairs

TYPE: nucleic acid

STRANDEDNESS: single

TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 20

GCCCATCCTC TCTAACCAAG 20

INFORMATION FOR SEQ ID NO: 21

SEQUENCE CHARACTERISTICS:

LENGTH: 15202 nucleic acids TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY : 1inear FEATURE : LOCATION :

IDENTIFICATION METHOD:

OTHER INFORMATION: /note="N is unknown or other" SEQUENCE DESCRIPTION: SEQ ID NO: 21

GATCCGAGCT CAATTAACCC TCACTAAAGG GAGTCGACTC GATCCTTAAA ATATTCATAT 60

CTCCTGGACA ACCTGTGGCC ATAGTGCCTG ACTGTAAACC CAAAGGGTTT GCCTTTGCCA 120

GTGTAGCCCA GCCTGGTGTC TGCTGCCCCT CGCGGTGTCT GTGCACCTGC CACGATGCTG 180

ACCAGACACC CTTAACCAGG TTCACCCATC GCCTGGGCCT GGAGCAGTCC CCCTGATGCT 240

CTGATTGGTC CTTGGACCTT CTGTTCTCCC AAAATCCCAG GTCAGAAAAT ACCTGGAAGT 300

CTATTTGTGT CCCACCTCCC TCTTTGTGGC CGCAAGTGCC CCTTCCTCCA CACAGTCACA 360

AGACCATGAG ATGCCATCTC CTCCCCTCCT GGGCTGCAGA CTTTGGGAAG CTCCCAGGCC 420

ACAGAGGTGT CAGCTCCTGT CCAGGCCCTT GGGACCTTCC CTCATTCAAC CACCCTACCC 480

AACCCCCCAC TGCCTGCCAG CCACCACTCC CTCCCACATT TGCAGGCGGG GGCCCTGCCC 540

TCTCCTGCCG CTGGTTCCCC TACCCAGGAG GCTCTCCCAT CGCTCTTTTG AGAGTCTGCC 600

TCCCACCTCT AACTGGGGGC TTAGTTCAAG TTGCCCCCTT ACCCTAGTCC CAGCTGCCCA 660

AGAGCTTGCT GCCTCCTGTT CTTGGTGAGG GACTCCAGAG ACAGATGTGA GACCTCCCTG 720

GACCCCTCCA AGGCATTCCC AGGTCACTTC CATGAGTAGT GAAGAACCGC CTCTGAGCAG 780

GCTGAGCCTC CCTCAGCCTA TGGTGTCCTC ACGTGGCTTG GCCCACAGCA GGTGCTCACG 840

CCTCCTCCTC AGCAGAGCCT ACCATCCTCC TGCCATGCTC ACCAGTCCCC ATGCTGATAG 900

CCATCACCAG TCCCCATGCT GATAGCCATC ACCAGTCCCC ATGCTGATAG CCACTTTCTG 960

GATGCTCTAG GTCTGTCTGG ATGACACAGT GACCACAGAG AAGGAGCTGG ACACTGTGGA 1020

AGTGCTGAAA GCAATTCAGA AAGCCAAGGA GGTCAAGTCC AAACTGAGCA ACCCAGAGAA 1080

GAAGGTGGGT TTGTGTGGCA GGTGGGAGGG CAGTGGTGCA GAGCCAGCCG GGATAGGAGC 1140

CAGTTCGGGG GGCTTGGGCC ATGGGACTGC TCAGGGCTGC CGAGTCCCAG CTGCGCCCCT 1200

CCCTGGCTGC ATGACCTCGG GCAAGTCGCG GCCTCTCTGT TCTCTGTGGG GTGGGGACAG 1260

TGGTAGTTCC TGCTCTAAGG ATATGATGAG ACCATCTTTA CCACCCAGTT GGTGGGAACC 1320

GTTGCGCTCC CTCCTCACAC CCCTGGCCTT GGGGAGCTCT GTGCTTCCTC TTCTCTCCCG 1380

GGCTGACTCA AGCACTCGTC CTCAGGGTGG TGAAGACTCC CGGCTCTCAG CTGCCCCCTG 1440

CATCAGACCC AGCAGCTCCC CTCCCACTGT GGCTCCCGCA TCTGCCTCCC TGCCCCAGCC 1500

CATCCTCTCT AACCAAGGTA ATCGTGTATG TATCTTGCTT CTAGTGGAGC CACACAGCCC 1560

TGCCTGGGCC CCCTGGCTGG GCTGGGGTTG GGGGAGAGGT GCCAGCACCT GCTTCCAACA 1620

GGGTCAGACA CAGGGAGGGC AGTGCCTTCT GCAGGCTGGT CCTCGCGGGG GGACACATGG 1680

CAGGGGTGCC TGGCCTGATG CCAGCTGTTG CTTGCTTGGT GAGGACTCCC AATTGCTCTG 1740 ATGCCCACAT CCAGCTCCTC TAGGAGACCG CAGGGTGTCT GACAGGCCCT GAGGCTGCCC 1800

TCTGAACAGG CTCGGGGCTG TTGGCTCATG GGACCCATTC CCTCACCGGC AGCACAAGCA 1860

GGTTGGCTCC TGGTTACAGG AAGCCGGGCT TGTGACTTTA CTGTCTGGAG CCCGAATCCC 1920

TGTGCAGGGA AAAGCTTGCT TTTATCACTG CCTCATCTCT GTGGGGTGAC CCAGCCCCAG 1980

AACACCATGT TTGTGGGGCC AAGATGGGCC ATCTCTGTCC CTGTGGACCC ATGGAAGACC 2040

AGGCCCATTC GTCTGCCCAC TATCTTAGCG TTTTCAAAGG GCTTTCACCT CTGAACCCAG 2100

GCATCCTCGG AGATGAGTGA GTGAAGCAGG TCTCATGAGC GTGTCTGCTG GCCCGGCCCC 2160

CACGGAAGAG GGGAGGGTGT GCCGTCCCGA GTGGAGCCGA GGCTCGGGAC ACGCAGGAAA 2220

GGACGCCGCC TGCCCGGGCT CCTGGAGACG CAGAACTTGG TGTGAGGTCT TGGGAAAACA 2280

GTTCAACCCG ATGTTTTAAG AGCCAGAAAA ACATTCCCAC CCCTTGACCT GGTAACCCCA 2340

CTGGTGGGGA TTTTCTCTTA GAGGGATAAG ATACCGGGAA GGGGAGGTGA AATGCTCACC 2400

ACTGCCAAAA CACGGGCTGC AACTGCAACA TCGGAGGATG AGAGGGAGAG TCGGCTGTGG 2460

TGCAGAATGC TCAGCAGCCC TCCCAGCAGG GACAGGAAGA CTGGGCAGGA AGAGGGGAGA 2520

AGCATTCAAG TTAAGGCAAA AGGCCCAACG CAGAGCAGCA CACTGAGGTC ACACCTGTGA 2580

GATGTGGAAG AGAATTCCTG AGCGTGGAGC GATGGGGTTA GGTGCCAGGA TGATTGCCCA 2640

TTTTGCTTCT GTCAGACTCT TGACTAAGGA TTTCTGGTTG CATTTTATTA CATAAAAGCC 2700

AGGGAGGTTA TATCACGGTG AGAAAGCTTC CCTGACGCCG CCTCCTGTAG CGCAGCCAAG 2760

CGAGCCTGTG GAGGTACCAT ATGACTGTAG GCCTCTGGGG ACAGGGAGCT GCATCTGCTT 2820

CTCAAGGCCA GGGACACAGC CATTTCTGCC AGCATCTGTT GATCAGTGAG TGAGTGAGTG 2880

GGCAGGTAGA GCAGGAGCCA GTGAAGAGCA GGCCCTGGAT GGGTGGGGAT GCACCATGTC 2940

CCCAGGCTGC AGCTGCAGGC AGCCCCCCAC ATTGTCGGAG AAGCCTCTGC ACCAGCTCAG 3000

CCCCCTCCTC ACTCCCCTTG TGCCCTGGGG ACACTCTGCA GAGGGGCACT CTGCAGTCTG 3060

TCCCCGCCAT CGCTGGACTT CTGGACATGG CCTCCAGATT TGCACCTCTT AAATAAATCT 3120

GCAGTGGATG TCTTTGTGTG CACCTCTCTT TCCTTTTGGT GAGAAACAGC AAAGATCGGA 3180

CCCCTAAGGA CTCTCCTGAT GTCTCCGCTC TATCCGCTGA GTGCCCTTTC TGACCACTTG 3240

TTTGTACAGG CCACGGTCCA GGACGGGAGC AGATAGACTG TCCCTGTCCC TGTCCACATT 3300

TCCTTGGTCC AAACAGGGCT TGTGGGAGGT AGTGGCAAAA GGTGTTGGTC TTTTTCTCAC 3360

TGATTTGGAG GCCTCCCCGT GTGTTTTTTC AGCCGCGTGT TCCTGGGTCT TGCCTGGATG 3420

GACAGGGTTT TTTAGCGCGT GGGAGCAGCT TTGCTGACCA TGCCTGTTGC TTCCAGCCTG 3480

ATTCCCGAGA AGGGAGCGTG CTTGCGAAGG AACTGGCACT CGGGCCTGCC TGAAGGGGGC 3540

GCTGTCCAGA CACACCCAGC CTCCCGTCGT GGCAGGCGCT GTCGGAGCCA TGGATGATTG 3600

TGACCAATAG GGGTGGTCGC CAGAGTTGAT TGTCCAGCCA GGCCCAGGGG CTGAGAGGAG 3660

GCTGTGTGGA GAGGTGGTTA GGAGCCAGGG CTCGGTCAGC TGAGTTCGCA TGCCAGCTTC 3720

CTAGCTGTGG GACCTCAAGC AACTTGTAGC CCCTCTGAAG CTGTTTTCTC AACTGTGAAG 3780 TGGACGCACC CTACTTCATT GATTCTAAGA GGCACGCATT TCCACCTTGT GACTTCTCTG 3840

AAACTGAGGT GCGTCTTTCA GTCAGTGGCG TCTCATAGTC GCTGTCAGCC AGCTGGTATT 3900

CGAGATGGAG TCGTGGAAAA CCCGTGGACA CCTTCCGCTA GGACCAAGAT GGCGCCACCT 3960

GCCGCATCTT AGATTTGATG AAATGTGGTA AATAACGAGA GGCATGCATG AGCGAATGCT 4020

GGGGAGGCGC TTGGCACTAC CCAGAGCTCC ACAGAGGTGG TCGATGAGGG CTGCCCTTTC 4080

CCACATCCTT AGTAGGGGGT TCAAGATGAC CCAGACTGTG CCCCTGGGGA GCTTGGAGCC 4140

ATGCGGGAGG ATGAGCCATG TGCTGGAGGA GAACAGGGTA GGATGGTGTG GGGCTTTTGT 4200

AGACTGTCTA GAGCAGAGAA GGTCTGCAGT GGAGGTGGTG TCTGAGGTGA ATCTCGAAGG 4260

TGAATAGGAG TTGAACGTTA GCAGGCAGAG GGTGGATTGC AGGAGAGCAG CGGCCTGGGC 4320

AGGTGCCCAG CGTGGCCCAT CAGGGTGCTT CATGCATGGC TGTGTGCTTG CCATCCTTCC 4380

TGCCTGCCTA CCCCCTGCTG CTTCGCTTCA TGGGGGCGTT TGAGCTTGGG CCCACCTGCC 4440

TGCCTCGCTT GTGGGCAGAG GACCCAGGCT GTGTGAGTTG TCCTGTCCCG GGGAGCAGCT 4500

GAGCTTGTCC GGGTTCCTCG ACCTGTGGGG CTTCAGAGGA CTTCGGGTCA TTTCAATGGG 4560

CTGTGGCGAT GCTGGCTGTG GAGGTAGCCT AGGGCTCCTG TAGCCTTCAG TGAGACTGGC 4620

GGCCCGATGC CCAGTGTTCA CCCTGCTGGC GGCAGTCAGG AACATGTTCA CAAAGCTTTA 4680

CTTCAAGTGG TCTAGAGGTG ATCTGAGGTG GAGTAACAGG TCCAGATAGG CTACGTTCAT 4740

AAAACAGCTT CAGCGGGGTT TAGGAACACT GTGCATTTAC GGGACGCAGT GGGTCAGAGT 4800

GCTGCTGTCC GTGGGAGGTG GCCCCAGGGC AGGTCAGTGG GCACGTCCTG TGGTAAGTGG 4860

GACTGTGGAT GTGGGCTCAG GCTGGACTCA GCAGCCCTGC TGGATACCAA GGCCTGCAAG 4920

GGCTGGCCCC CTGGTGAATT GTCCCGTGCC CTGTGTATCT ATGAGTCCTG CAGAGATGAC 4980

AAATCAGGGG ACGGGGTCAT GTCTAGTCAC CGTCTGGGAA AATGCTCCAG GAGTGAACAC 5040

ATTTCAGGCT CTTGATGGAT GTACCTCCAA ACTCTTCTCT GGATGGGTGG GCCAGCTTGC 5100

ATGCCTGTGC CGGCCTCTGC CCAGCGAGGT CAGGGCCAGG CCACACAGTC AGTCTGACTT 5160

TGGCAGAAGT TGAGAGGCAA CACTTGTCTC TTGTTTCAGC TTGCCTTTCT TTGTGTACTT 5220

CTGAGAGCGA GCATTCTTTT CATGTTCTAT CCGCTGGCCG TTCTTCTGCG GAATGTCTGT 5280

TCACGTCCTT TGCAGTCTGT TAATGAGGTT TCCAACCTTC CCTCATTTTT GTAATCTGTA 5340

AGAACTTTTT CCAGACTAGC GATATAAATC CTTGTCAAAT ATTGCAAACA CTTTTCTCAT 5400

TTCATCTGGT TTTAATCTAT CCTGGTTTTT AAAAAATGTG TCTGTGGAAG TTTAATTTTT 5460

ATGTAGTCAC ATCTCAGTTT TTTTCCATTG CATTTATTCT CAGAATGCTT CTCCCTGCCC 5520

TGAGATTAGA TAAGCAGTCA TTTGTTCTTT CTTGAGTTAT TTTGAGATTT CAGTTTTAAC 5580

ATTTTCTTCT ATAATCCATG TGGCTGGGTT TTGGGATCTG GCTAACCCCC GCCATGCCAG 5640

TAGCCTGAGG GGCCCAGCCC CACTTGTTGA ACAGCCGCTC TCCCCGCCCC ACCCACCCTG 5700

CCTGCCTGCC CACCCGCCCT GGTCTCTCCA GGAATCATGT TCGTTCAGGA GGAGGCCCTG 5760

GCCAGCAGCC TCTCGTCCAC TGACAGTCTG ACTCCCGAGC ACCAGCCCAT TGCCCAGGGA 5820 TGTTCTGATT CCTTGGAGTC CATCCCTGCG GGACAGGTAA TGCCCTCTTC CCGCTTCTGG 5880

GGACCATACA TCTGTGGGTG GACTCTTCTG CTTGGGGTTG TGTGCAGTAG GAAGTGGCCT 5940

AGCTGGAGCT GAGGCAGATG CTTCCAGGGT TTGGCGTCCT CTGCTTTGCG CCACGGTCTT 6000

TCTCTTGGAC CTGTCTCTGG TTGAGTGTCT TCCTGACAAA CACAGTGGTT AAGGGTTTAT 6060

TTTCAGCCTC CCTCCTTCCC TTCCCCACCC ACCTTGGTTG ATGGGAACAG GCAGTTCTCT 6120

GTCACTGGGC CCAGGGCACG AGGGGGGCAG GTGGAGAGGG TGGCCCTTGA CCCTGTGAGC 6180

AGGCTTCCCT GGGGAAGGCA TTTCAAAAGA CCCTCGTGCA GGGGCTTGTT TGGGTTTCTT 6240

CTCTGTTTCC TGGCACCCCT GGAGCCACTC GGCGCCTTTC CGCATGTCAC CCTGGTGGTC 6300

TGGGAAACAG TCTCACTCTG GCGCCTCCTC TGTGGTTGTT ACTGAGAGTT CTGGGGCCCC 6360

TTCCTTTGTC CTGAGGAAAG ACAGGAGGAA AGCAAGGGTG CTTGCTGTGT GCTTCGCAAA 6420

TGTGCTTGGT GCCTGGGCCT CCCTCCAGCC CCATCTCTGC AGCAGCACAA GGTTATGGCC 6480

TTGTGACACT GGGACAGTTT GCAGAGTCCT TGTCTGTCCT CAGTACTCCA CAGTATTCTG 6540

CCATCACCCT TTCCAGGGTC ACACAGCAAG AGATTCCCAA GCCCTAGGTA TTCCCCAGTG 6600

CACAGAGACC ATTGGGAGGG ACTTGCCAGG GCTGTGTCCA CTGCTGGCCA GTTAGGGTCG 6660

GACCAAATTT GTAGACTGTC TACCTGGACC CTTGCGTGGC ACAAGGAGCA GTCAGATGCT 6720

GGATCCCTGG AGAGTGGCGA GAGGCTCTGG CCTTAGGTTG CGAGTGGGAA TCCCAGCCCT 6780

GCTGTGTGCT GGTGGGATAA CCAAGTGGGT CTCTGCCCTT GGGTCCCAGA GTGGGCCCCA 6840

GGGTCCCAGA GTGGGCTCCA GGGTACAGCG TGGGGATGGG GAGCCTCCTC AGGGCGGTGA 6900

TGGAGGGCAG AATGCCCAGC TCAGGGTCTG GCAACCAGTA AATGGCTGGG GCTGGCTGCA 6960

GTAGGTGGGG ACTGACTGTG TTTCTTTCTC CATCAGGCAG CTTCCGATGA TTTAAGGGAC 7020

GTGCCAGGAG CTGTTGGTGG TGCAAGGTAA GGAAGAGGTT GGAAAGGGAC CTGGGCCTGG 7080

CCACACAGCC TTATGCACAC ACACTGCTGT GGGCCAGGGG TGGCCAGTCA GGTTTTTTTA 7140

AAAATCCGTT CACAGAAGGC CTATAGAACT ATTTCTTCCT CTAAAGAGAC ACAGATGAGA 7200

TGGACTTTTC AATCTGTTTC CAAATTCTAA TACCTAAACT CTGCTCAGCA CATGTTGCCC 7260

TACACCAGGG GTTGGCAAAT CAAGGCCTGT GTGTGGCCCA CAGCCTGGGA GCTAAGAATG 7320

ACAGTTACAT TCTTTTTTCT TTTTTTGAGA CTGAGTCTCG CTCTGTCGCC CAGGCTGGAG 7380

TGCAGTGGCG TGTTCTTGGC TCACTGCAAC CCCCGCCTCC CAGATTAATG CAATTTTCCT 7440

GTCTCAGCCT CAGCCTTCTG AGTAGCCCGG ACCACAGGCG CACGCCACCA CGCCCAACTA 7500

ATTTTTTATA TTTTTAGTAG AGACAGAGAT TCACCATGTG GCCTAGCTGG TCTCGAACTC 7560

CTGAACTCCA GTGATCCACC AACCTCGGCT TCCTAAAGTA CTGGAATTAC AGGCATGAGC 7620

CACCGCGCCT GGCTAGAATA ACAGTTACTT TTTTTTTCTT TGAGACTGAG TCTTGCTTTG 7680

TCACCCAGGC TGGAGTGCAG TGGCACGATC TCAGCTCGCT GCAACCTCCG CCTCCCGGGT 7740

TCAAGCGATT CTTCTGCCTC AGCCACCCAA GGTGCCCGCC ACCACACCTG GCTAATTTTT 7800

CTGTTTTTAG TAGGGACAGG ATTTCGCCAT GTTGGACAGT TACATTCTTA AAGGGCTGCT 7860 GAAGATCGTA TGGACATGGT AGCCCATAAA TCCCAAAATG TGTACTCTGA CCCTTTACAG 7920

AAGCTTACTA ACTCCCACTC TACATGTGAG GGCTGCGGTG GCCAAGAAGA GCTGGAATTT 7980

AAGTGTGAAG GTCCTAAGAC CTGCCCCAGC CCACTTCCCT GCCCCGGAGG CCACCAGGGG 8040

TGACAAGTAG ATTCATGCCC TGGAGTGTTC CTTCTCTCCG GGGCTTATGG CAGCAACTGA 8100

ATGACTTAGA AGTCCATGGG AGTGCTTTCT GTTGTGGGAA CTCGTGTGGT CTGGGCATAG 8160

CTGTGCCAGG CACCTATGGT CCAAGCCCCT AGAAGCATAG ACTCTGACCA AACTGGCGAC 8220

CCAGCCTTCC AGCAGGCAGC ACTGGCTCCC ACCAGGGCCC TCATCCTGGG AACTGACTTG 8280

GCCATGTGGG AGGCTTGGGA GACCCATGGG TTGGTTTCTC AGGGTCAGGG TGTAGCAGTG 8340

GGCTCCAGAT GTGGCAGGTG GGAGGTGGGA GGGGCCCCTC CCAGCATGCC ACTGACCTGG 8400

CCTCTCCCTG CACAGCCCAG AACATGCCGA GCCGGAGGTC CAGGTGGTGC CGGGGTCTGG 8460

CCAGATCATC TTCCTGCCCT TCACCTGCAT TGGCTACACG GCCACCAATC AGGACTTCAT 8520

CCAGCGCCTG AGCACACTGA TCCGGCAGGC CATCGAGCGG CAGCTGCCTG CCTGGATCGA 8580

GGCTGCCAAC CAGCGGGAGG AGGGCCAGGG TGAACAGGGC GAGGAGGAGG ATGAGGAGGA 8640

GGAAGAAGAG GAGGACGTGG CTGAGAACCG CTACTTTGAA ATGGGGCCCC CAGACGTGGA 8700

GGAGGAGGAG GGAGGAGGCC AGGGGGAGGA AGAGGAGGAG GAAGAGGAGG ATGAAGAGGC 8760

CGAGGAGGAG CGCCTGGCTC TGGAATGGGC CCTGGGCGCG GACGAGGACT TCCTGCTGGA 8820

GCACATCCGC ATCCTCAAGG TGCTGTGGTG CTTCCTGATC CATGTGCAGG GCAGTATCCG 8880

CCAGTTCGCC GCCTGCCTTG TGCTCACCGA CTTCGGCATC GCAGTCTTCG AGATCCCGCA 8940

CCAGGAGTCT CGGGGCAGCA GCCAGCACAT CCTCTCCTCC CTGCGCTTTG TCTTTTGCTT 9000

CCCGCATGGC GACCTCACCG AGTTTGGCTT CCTCATGCCG GAGCTGTGTC TGGTGCTCAA 9060

GGTACGGCAC AGTGAGAACA CGCTCTTCAT TATCTCGGAC GCCGCCAACC TGCACGAGTT 9120

CCACGCGGAC CTGCGCTCAT GCTTTGCACC CCAGCACATG GCCATGCTGT GTAGCCCCAT 9180

CCTCTACGGC AGCCACACCA GCCTGCAGGA GTTCCTGCGC CAGCTGCTCA CCTTCTACAA 9240

GGTGGCTGGC GGCTGCCAGG AGCGCAGCCA GGGCTGCTTC CCCGTCTACC TGGTCTACAG 9300

TGACAAGCGC ATGGTGCAGA CGGCCGCCGG GGACTACTCA GGCAACATCG AGTGGGCCAG 9360

CTGCACACTC TGTTCAGCCG TGCGGCGCTC CTGCTGCGCG CCCTCTGAGG CCGTCAAGTC 9420

CGCCGCCATC CCCTACTGGC TGTTGCTCAC GCCCCAGCAC CTCAACGTCA TCAAGGCCGA 9480

CTTCAACCCC ATGCCCAACC GTGGCACCCA CAACTGTCGC AACCGCAACA GCTTCAAGCT 9540

CAGCCGTGTG CCGCTCTCCA CCGTGCTGCT GGACCCCACA CGCAGCTGTA CCCAGCCTCG 9600

GGGCGCCTTT GCTGATGGCC ACGTGCTAGA GCTGCTCGTG GGGTACCGCT TTGTCACTGC 9660

CATCTTCGTG CTGCCCCACG AGAAGTTCCA CTTCCTGCGC GTCTACAACC AGCTGCGGGC 9720

CTCGCTGCAG GACCTGAAGA CTGTGGTCAT CGCCAAGACC CCCGGGACGG GAGGCAGCCC 9780

CCAGGGCTCC TTTGCGGATG GCCAGCCTGC CGAGCGCAGG GCCAGGTGAG ATCAAGCACA 9840

GCTCTCAGGG GCCCCGGGGG^4'CACGGGTCTG GCATGTGTGT GATCTCAGCA TCTGCGGCTA 9900 GTGTGGGCTG GGAGTTGCTG CGAGAGCTGG GCCCCCTCCC CCCTGCCCCT CGCCCCCCCC 9960 GGGCCTCCCT CTACATCACC ACCCCAGGTT TGGTGCCAGG CTGCTCCTTA TCTCAGTGCT 10020 GTAGAAGAAG CCCAGGAAAG CTGTCCTCTC ACAAAATGGG TTGGCCCAGC CTCTTGCCAC 10080 CCATGAAGGG CAGGCCAAGG GGGCTGCCCC ACCTTTGCCT GCCCAGTGGG AGAGCAACAG 10140 GCTGCAGCAC ACCGAGGCCA GGAGAGCTGT CACCCTGGCT GCTGTGCTCC TCTGGGCCCA 10200 AGCATGGCCT CTGGGCACTA CCTCCTCCAG GGTCACAGTC CCACGGATGG CTCTGTGGGC 10260 CAGGATCTGC CTTAGGCTTC ACCCACCTCA ACATCTTGCT GTGTTGTTCA GGCTGGTCTC 10320 AAACTTTGGG CTCAAACAAT CCTCCGCCTC AGCCTCCCAA AGTGCTGGGA TTACAGACAT 10380 GAGCCACCGT GCCCGGCCGT GCTGTTCTGT TCTCCAATAG AGAAGCTGGT GGAAGTCCCC 10440 AGTAACCCAG AGGTGATGTG TGATGCACAC AGTCTCCTCA CTCTGAAGCT GCACATGCGA 10500 TGTGAATCTT CATTTGGGGT CCGCTGTTAA TATGGTGTTT TTCGGGGGAT ACAGCAATGA 10560 CCAGCGTCCC CAGGAGGTCC CAGCAGAGGC TCTGGCCCCG GCCCCAGTGG AAGTCCCAGC 10620 TCCAGCCCCT GCAGCAGCCT CAGCCTCAGG CCCAGCGAAG ACTCCGGCCC CAGCAGAGGC 10680 CTCAACTTCA GCTTTGGTCC CAGAGGAGAC GCCAGTGGAA GCTCCAGCCC CACCCCCAGC 10740 CGAGGCCCCT GCCCAGTACC CGAGTGAGCA CCTCATCCAG GCCACCTCGG AGGAGAATCA 10800 GATCCCCTCG CACTTGCCTG CCTGCCCGTC GCTCCGGCAC GTCGCCAGCC TGCGGGGCAG 10860 CGCCATCATC GAGCTCTTCC ACAGCAGCAT TGCTGAGGTA GCGGCCCGGG TGTGGGTGCC 10920 AGCTATGGCA CGGCCAGTCC TGAGGGCGAG GCCAAGCTTG GCTTCAGGTC AGCCTCAGGT 10980 CCCTGGACTT CCCTGATGTC GGAGTCCTCA GCTGAGCTGC TCACAGCTTT GAGGACCTGG 11040 GCAGTGAGGT CCTGAGTTGC CCTCCCTGGC CATTTGTGCT GTGTCACCAC CTCCTGTGCC HIOO ACTTCCAGCC CCAGGTAGAC CTCCCACCAA CAGCCATCTC CCACCCCTCT CTTCCTCTCT 11160 GCCTTGAAGC ATACGGATTC ATTGGTGAGC CAAGAGGGGC TTCCCATGTC TCCTTGTGGA 11220 AGCTGTGGGC ATGTCCCTGG TATGTGCAGG TTGCTAGGGT GGTGGAGCTG ACAGGAGGCC 11280 CCCCGTCTTC AGGTTGAAAA CGAGGAGCTG AGGCACCTCA TGTGGTCCTC GGTGGTGTTC 11340 TACCAGACCC CAGGGCTGGA GGTGACTGCC TGCGTGCTGC TCTCCACCAA GGCTGTGTAC 11400 TTTGTGCTCC ACGACGGCCT CCGCCGCTAC TTCTCAGAGC CACTGCAGGG TAGGCACAGG 11460 GCCTGCTGGG GCTCAGGAGC TTGGAGTGTG TGGTTGGGGC AGGCCTGGGG GGTCATTCTC 11520 TGGAGCCAGC TGTGTGGCTT CAGGCAGCAG TCAGCGACTT GGCTGCAGTG GGCTGAGAGT 11580 TCCTTGTCTG AGGAAGGGAG CTGTCATGAG GGAGGGGTCC ATGGCCAGAT GTGAACGCAG 11640 AATGCACTGA GCCAGGGCCT GGTGACTGCT TGGGAACAGC CTGTGATGAG AAGGGGTTAG 11700 GCAGCCTTTG CCCCTGGGGC TGCACAGGAA GCCCTAGCCA GCGACCTGGT GACTCCCCTG 11760 AGCTGGAAGA GGCTCAGACT CCAGAGGGCA TTGCCTATGG GGCTTTGCAC GGGTGGAAGC 11820 CAGGCCAGCC AAGAGGACCT GTTCCTGCTG GATGTGCTGC ACACCTAGGA ACCTTGTGCT 11880 TGCCTGCCAC CGCCTCCCTC TGTCCCTTTC TCCATCACAC AGATTTCTGG CATCAGAAAA 11940 ACACCGACTA CAACAACAGC CCTTTCCACA TCTCCCAGTG CTTCGTGCTA AAGCTTAGTG 12000 ACCTGCAGTC AGTCAATGTG GGGCTTTTCG ACCAGCATTT CCGGCTGACG GGTGGGTGAC 12060 CCTCTGTGCT TTGTCCTATT TCGGGTGAAG GCCAGCATCA CCAGTGGGCT TCCACCTTCC 12120 GTACGTGGGT GGGTTATCAT AGACAGTTAT CTCTGTGCTC AAGAGCCACT TCTTACCCGG 12180 GGTGGGAGGA AGCAGCTTCA GGAACTGCTG AGAGAGCAGA ACTCACGCTC CAGGGCTCAG 12240 AGCAGGAGGT AGGGTGTGCG GCAAGCGCTG GCCCGGACAG AAGCAGAGTG GGCCCTGGTC 12300 TCGGGCAGGA TGTTTCTGAC TCACATTTCC TGAGGAGAGA AAGCTAAGCT CTTTGCCTAA 12360 TGTCTCTGTC TCCCCTTCCA GAAAAATGCC TCAGCTCTTC CGGCCTGAAG GAATGGCCTC 12420 CTCCCGGGCC CCATGATTCT TTCCTGTGTG GGCCCTCCTG GCCCTGGCCT CTGGGCTGAG 12480 GCTTGCTAGG GACTCGGGGT GGCTCTAAGG GGCAGGGATA GGGCTGGGGA GCGCCGGCCT 12540 GTGGCCCTGA CCAGCCCCTT CTCGTGCAGG TTCCACCCCG ATGCAGGTGG TCACGTGCTT 12600 GACGCGGGAC AGCTACCTGA CGCACTGCTT CCTCCAGCAC CTCATGGTCG TGCTGTCCTC 12660 TCTGGAACGC ACGCCCTCGC CGGAGCCTGT TGACAAGGAC TTCTACTCCG AGTTTGGGAA 12720 CAAGACCACA GGTACCCCTG TCTAGCTCAG GCTGCAGACA GGCTGCCTGG ACAGACGTCA 12780 TGGGCCCCAG GGTGGCTCTC TGTGCCCCAG AACCCTCTCT GCCTCTATGT CTCTCTTTTC 12840 TCACTTAGCT GGCCAGGGTT TTATGTGGGG CTTTTCGATG GCAGAGTCTC CACTCCAGCA 12900 GTCCCTCAAC CATCTGGCAG ACACATCTCC AGTGCCTGCT TTGGGCTCCT GGCCTGTGGG 12960 CCCCACACTT GGAGCATCCT CTCCTGCCTG TCTCATGCCG GGGTCTCTCG GTTGGCTTGG 13020 GGCCCTTGGT GCTCCCAGCC CCACCAGGGG CCGGTTCCAG GCTATAGCCC AGGTGGCATC 13080 TCTCTGCAGG GAAGATGGAG AACTACGAGC TGATCCACTC TAGTCGCGTC AAGTTTACCT 13140 ACCCCAGTGA GGAGGAGATT GGGGACCTGA CGTTCACTGT GGCCCAAAAG ATGGCTGAGC 13200 CAGAGAAGGC CCCAGCCCTC AGCATCCTGC TGTACGTGCA GGCCTTCCAG GTGGGCATGC 13260 CACCCCCTGG GTGCTGCAGG GGCCCCCTGC GCCCCAAGAC ACTCCTGCTC ACCAGCTCCG 13320 AGATCTTCCT CCTGGATGAG GACTGTGTCC ACTACCCACT GCCCGAGTTT GCCAAAGAGC 13380 CGCCGCAGAG AGACAGGTAC CGGCTGGACG ATGGCCGCCG CGTCCGGGAC CTGGACCGAG 13440 TGCTCATGGG CTACCAGACC TACCCGCAGG CCCTCACCCT CGTCTTCGAT GACGTGCAAG 13500 GTCATGACCT CATGGGCAGT GTCACCCTGG ACCACTTTGG GGAGGTGCCA GGTGGCCCGG 13560 CTAGAGCCAG CCAGGGCCGT GAAGTCCAGT GGCAGGTGTT TGTCCCCAGT GCTGAGAGCA 13620 GAGAGAAGCT CATCTCGCTG TTGGCTCGCC AGTGGGAGGC CCTGTGTGGC CGTGAGCTGC 13680 CTGTCGAGCT CACCGGCTAG CCCAGGCCAC AGCCAGCCTG TCGTGTCCAG CCTGACGCCT 13740 ACTGGGGCAG GGCAGCAGGC TTTTGTGTTC TCTAAAAATG TTTTATCCTC CCTTTGGTAC 13800 CTTAATTTGA CTGTCCTCGC AGAGAATGTG AACATGTGTG TGTGTTGTGT TAATTCTTTC 13860 TCATGTTGGG AGTGAGAATG CCGGGCCCCT CAGGGCTGTC GGTGTGCTGT CAGCCTCCCA 13920 CAGGTGGTAC AGCCGTGCAC ACCAGTGTCG TGTCTGCTGT TGTGGGACCG TTGTTAACAC 13980 GTGACACTGT GGGTCTGACT TTCTCTTCTA CACGTCCTTT CCTGAAGTGT CGAGTCCAGT 14040 CCTTTGTTGC TGTTGCTGTT GCTGTTGCTG TTGCTGTTGG CATCTTGCTG CTAATCCTGA 14100 GGCTGGTAGC AGAATGCACA TTGGAAGCTC CCACCCCATA TTGTTCTTCA AAGTGGAGGT 14160 CTCCCCTGAT CCAGACAAGT GGGAGAGCCC GTGGGGGCAG GGGACCTGGA GCTGCCAGCA 14220 CCAAGCGTGA TTCCTGCTGC CTGTATTCTC TATTCCAATA AAGCAGAGTT TGACACCGTC 14280 TGCATCTTCT AAACCAAGGG TCACTGGGAT CGAGTCGACG GCCCTATAGT GAGTCGTATT 14340 AGAGCTCGCG GCCGCGAGCT CTAGATGCAT GCTCGAGCGG CCGCCAGTGT GATGGATATC 14400 TGCAGAATTC CAGCACACTG GCGGCCGTTA CTAGTGGATC CGAGCTCCAC AGAGGTGGTC 14460 GATGAGGGCT GCCCTTTCCC ACATCCTTAG TAGGGGGTTC AAGATGACCC AGACTGTGCC 14520 CCTGGGGAGC TTGGAGCCAT GCGGGAGGAT GAGCCATGTG CTGGAGGAGA ACAGGGTAGG 14580 ATGGTGTGGG GCTTTTGTAG ACTGTCTAGA AGCAAAGAAG GTCTGCAGTG GAGGTGGTGT 14640 CTGAGGTGAA TCTCGAAGGT GAATAGGAGT TGAACGTTAG CAGGCAGAGG GTGGATTGCA 14700 GGAGAGCAGC GGCCTGGGCA GGTGCCCAGC GTGGCCCATC AGGGTGCTTC ATGCATGGCT 14760 GTGTGCTTGC CATCCTTCCT GCCTGCCTAC CCCCTGCTGC TTCGCTTCAT GGGGGCGTTT 14820 GAGCTTGGGC CCACCTGCCT GCCTCGCTTG TGGGCAGAGG ACCCAAGCTG TGTGAGTTGT 14880 CCTGTCCCGG GGAGCAGCTG AACTGGTCCG GGGTCTCGAA CTGTGGGGCT CAAAAGGACT 14940 CCGGGGTCAT TTCACTGGGG CTGTGCCGAT TCCTGGGGGC TGTTNGGAAN GTAAAGGCCT 15000 AAAGGGGCTC CCTGGTTANG GCCCTCAANT TTAANAACCT GGGGCCGGGG CCCGGAATTG 15060 CCCCCAANTT TGTTTCAACN CCCCTTGGCC TTNGGCNGGG GCAAATTTCC ANGGGGAACC 15120 AATGGNTTTC CCCCAAAAAN GGGGCCNTTT TAACCCNTTT CCAAANTTTG GGNCCTAAAA 15180 AAGGGTGGAN TTCCTGAANG GG 15202

INFORMATION FOR SEQ ID NO: 22

SEQUENCE CHARACTERISTICS:

LENGTH: 1070 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear SEQUENCE DESCRIPTION: SEQ ID NO: 22

Val Cys Leu Asp Asp Thr Val Thr Thr Glu Lys Glu Leu Asp Thr Val 1 5 10 15

Glu Val Leu Lys Ala He Gin Lys Ala Lys Glu Val Lys Ser Lys Leu 20 25 30 Ser Asn Pro Glu Lys Lys Gly Gly Glu Asp Ser Arg Leu Ser Ala Ala

35 40 45

Pro Cys He Arg Pro Ser Ser Ser Pro Pro Thr Val Ala Pro Ala Ser

50 55 60

Ala Ser Leu Pro Gin Pro He Leu Ser Asn Gin Gly He Met Phe Val 65 70 75 80

Gin Glu Glu Ala Leu Ala Ser Ser Leu Ser Ser Thr Asp Ser Leu Thr

85 90 95

Pro Glu His Gin Pro He Ala Gin Gly Cys Ser Asp Ser Leu Glu Ser

100 105 110

He Pro Ala Gly Gin Ala Ala Ser Asp Asp Leu Arg Asp Val Pro Gly

115 120 125

Ala Val Gly Gly Ala Ser Pro Glu His Ala Glu Pro Glu Val Gin Val

130 135 140

Val Pro Gly Ser Gly Gin He He Phe Leu Pro Phe Thr Cys He Gly 145 150 155 160

Tyr Thr Ala Thr Asn Gin Asp Phe He Gin Arg Leu Ser Thr Leu He

165 170 175

Arg Gin Ala He Glu Arg Gin Leu Pro Ala Trp He Glu Ala Ala Asn

180 185 190

Gin Arg Glu Glu Gly Gin Gly Glu Gin Gly Glu Glu Glu Asp Glu Glu

195 200 205

Glu Glu Glu Glu Glu Asp Val Ala Glu Asn Arg Tyr Phe Glu Met Gly

210 215 220

Pro Pro Asp Val Glu Glu Glu Glu Gly Gly Gly Gin Gly Glu Glu Glu 225 230 235 240

Glu Glu Glu Glu Glu Asp Glu Glu Ala Glu Glu Glu Arg Leu Ala Leu

245 250 255

Glu Trp Ala Leu Gly Ala Asp Glu Asp Phe Leu Leu Glu His He Arg

260 265 270

He Leu Lys Val Leu Trp Cys Phe Leu He His Val Gin Gly Ser He

275 280 285

Arg Gin Phe Ala Ala Cys Leu Val Leu Thr Asp Phe Gly He Ala Val 290 295 300 Phe Glu He Pro His Gin Glu Ser Arg Gly Ser Ser Gin His He Leu 305 310 315 320

Ser Ser Leu Arg Phe Val Phe Cys Phe Pro His Gly Asp Leu Thr Glu

325 330 335

Phe Gly Phe Leu Met Pro Glu Leu Cys Leu Val Leu Lys Val Arg His

340 345 350

Ser Glu Asn Thr Leu Phe He He Ser Asp Ala Ala Asn Leu His Glu

355 360 365

Phe His Ala Asp Leu Arg Ser Cys Phe Ala Pro Gin His Met Ala Met

370 375 380

Leu Cys Ser Pro He Leu Tyr Gly Ser His Thr Ser Leu Gin Glu Phe 385 390 395 400

Leu Arg Gin Leu Leu Thr Phe Tyr Lys Val Ala Gly Gly Cys Gin Glu

405 410 415

Arg Ser Gin Gly Cys Phe Pro Val Tyr Leu Val Tyr Ser Asp Lys Arg

420 425 430

Met Val Gin Thr Ala Ala Gly Asp Tyr Ser Gly Asn He Glu Trp Ala

435 440 445

Ser Cys Thr Leu Cys Ser Ala Val Arg Arg Ser Cys Cys Ala Pro Ser

450 455 460

Glu Ala Val Lys Ser Ala Ala He Pro Tyr Trp Leu Leu Leu Thr Pro 465 470 475 480

Gin His Leu Asn Val He Lys Ala Asp Phe Asn Pro Met Pro Asn Arg

485 490 495

Gly Thr His Asn Cys Arg Asn Arg Asn Ser Phe Lys Leu Ser Arg Val

500 505 510

Pro Leu Ser Thr Val Leu Leu Asp Pro Thr Arg Ser Cys Thr Gin Pro

515 520 525

Arg Gly Ala Phe Ala Asp Gly His Val Leu Glu Leu Leu Val Gly Tyr

530 535 540

Arg Phe Val Thr Ala He Phe Val Leu Pro His Glu Lys Phe His Phe 545 550 555 560

Leu Arg Val Tyr Asn Gin Leu Arg Ala Ser Leu Gin Asp Leu Lys Thr 565 570 575 Val Val He Ala Lys Thr Pro Gly Thr Gly Gly Ser Pro Gin Gly Ser

580 585 590

Phe Ala Asp Gly Gin Pro Ala Glu Arg Arg Ala Ser Asn Asp Gin Arg

595 600 605

Pro Gin Glu Val Pro Ala Glu Ala Leu Ala Pro Ala Pro Val Glu Val

610 615 620

Pro Ala Pro Ala Pro Ala Ala Ala Ser Ala Ser Gly Pro Ala Lys Thr 625 630 635 640

Pro Ala Pro Ala Glu Ala Ser Thr Ser Ala Leu Val Pro Glu Glu Thr

645 650 655

Pro Val Glu Ala Pro Ala Pro Pro Pro Ala Glu Ala Pro Ala Gin Tyr

660 665 670

Pro Ser Glu His Leu He Gin Ala Thr Ser Glu Glu Asn Gin He Pro

675 680 685

Ser His Leu Pro Ala Cys Pro Ser Leu Arg His Val Ala Ser Leu Arg

690 695 700

Gly Ser Ala He He Glu Leu Phe His Ser Ser He Ala Glu Val Glu 705 710 715 720

Asn Glu Glu Leu Arg His Leu Met Trp Ser Ser Val Val Phe Tyr Gin

725 730 735

Thr Pro Gly Leu Glu Val Thr Ala Cys Val Leu Leu Ser Thr Lys Ala

740 745 750

Val Tyr Phe Val Leu His Asp Gly Leu Arg Arg Tyr Phe Ser Glu Pro

755 760 765

Leu Gin Asp Phe Trp His Gin Lys Asn Thr Asp Tyr Asn Asn Ser Pro

770 775 780

Phe His He Ser Gin Cys Phe Val Leu Lys Leu Ser Asp Leu Gin Ser 785 790 795 800

Val Asn Val Gly Leu Phe Asp Gin His Phe Arg Leu Thr Gly Ser Thr

805 810 815

Pro Met Gin Val Val Thr Cys Leu Thr Arg Asp Ser Tyr Leu Thr His

820 825 830

Cys Phe Leu Gin His Leu Met Val Val Leu Ser Ser Leu Glu Arg Thr 835 840 845 Pro Ser Pro Glu Pro Val Asp Lys Asp Phe Tyr Ser Glu Phe Gly Asn

850 855 860

Lys Thr Thr Gly Lys Met Glu Asn Tyr Glu Leu He His Ser Ser Arg 865 870 875 880

Val Lys Phe Thr Tyr Pro Ser Glu Glu Glu He Gly Asp Leu Thr Phe

885 890 895

Thr Val Ala Gin Lys Met Ala Glu Pro Glu Lys Ala Pro Ala Leu Ser

900 905 910

He Leu Leu Tyr Val Gin Ala Phe Gin Val Gly Met Pro Pro Pro Gly

915 920 925

Cys Cys Arg Gly Pro Leu Arg Pro Lys Thr Leu Leu Leu Thr Ser Ser

930 935 940

Glu He Phe Leu Leu Asp Glu Asp Cys Val His Tyr Pro Leu Pro Glu 945 950 955 960

Phe Ala Lys Glu Pro Pro Gin Arg Asp Arg Tyr Arg Leu Asp Asp Gly

965 970 975

Arg Arg Val Arg Asp Leu Asp Arg Val Leu Met Gly Tyr Gin Thr Tyr

980 985 990

Pro Gin Ala Leu Thr Leu Val Phe Asp Asp Val Gin Gly His Asp Leu

995 1000 1005

Met Gly Ser Val Thr Leu Asp His Phe Gly Glu Val Pro Gly Gly Pro

1010 1015 1020

Ala Arg Ala Ser Gin Gly Arg Glu Val Gin Trp Gin Val Phe Val Pro 1025 1030 1035 1040

Ser Ala Glu Ser Arg Glu Lys Leu He Ser Leu Leu Ala Arg Gin Trp

1045 1050 1055

Glu Ala Leu Cys Gly Arg Glu Leu Pro Val Glu Leu Thr Gly 1060 1065 1070

Claims

WHAT IS CLAIMED IS :CLAIMS

1. A DNA molecule encoding for a polypeptide including an amino acid sequence which is receptive to imidazoline compounds, said DNA molecule containing a DNA sequence with at least 75% sequence similarity with the DNA sequence shown in SEQ ID No. 4.

2. A DNA molecule according to claim 1, containing a DNA sequence with at least 75% sequence similarity with the DNA sequence shown in SEQ ID No. 2.

3. A DNA molecule according to claim 2, containing a DNA sequence with at least 75% sequence similarity with the DNA sequence of SEQ ID No. 3.

4. A DNA molecule according to claim 3, containing a DNA sequence with at least 75% sequence similarity with the DNA sequence of SEQ ID No. 1.

5. A DNA molecule according to any one of claims 1 to 4 , containing a DNA sequence with at least 80% sequence similarity with the sequence of said SEQ ID No.

6. A DNA molecule according to any one of claims 1 to 4 , containing a DNA sequence with at least 85% sequence similarity with the sequence of said SEQ ID No.

7. A DNA molecule according to any one of claims 1 to 4 , containing a DNA sequence with at least 90% sequence similarity with the sequence of said SEQ ID No.

8. A DNA molecule according to any one of claims 1 to 4 , containing a DNA sequence with at least 95% sequence similarity with the sequence of said SEQ ID No.

9. A DNA molecule according to claim 1, which is deposited with the ATCC under deposit accession no. ATCC 209217.

10. A genomic DNA molecule encoding for a polypeptide including an amino acid sequence which is receptive to imidazoline compounds, and wherein exon portions of said genomic DNA molecule include the DNA sequence as defined in claim 1.

11. A genomic DNA molecule according to claim 10, which is deposited with the ATCC under deposit accession no. ATCC 209216.

12. A 1110 bp Apal-EcoRI restriction fragment of the DNA molecule according to claim 1.

13. A 1.85 kb EcoRI restriction fragment of the DNA molecule according to claim 4.

14. A vector containing a DNA sequence as defined in any one of claims 1-13.

15. A host cell transfected with a vector as defined in claim 14.

16. An isolated polypeptide including a site which is receptive to imidazoline compounds, said polypeptide containing an amino acid sequence with at least 80% sequence similarity with the amino acid sequence shown in SEQ ID No. 6.

17. A polypeptide as defined in claim 16, having a molecular weight of about 35 to 45 kDa.

18. A polypeptide as defined in claim 17, having a molecular weight of about 37 kDa.

19. An isolated polypeptide including a site which is receptive to imidazoline compounds, said polypeptide containing an amino acid sequence with at least 80% sequence similarity with the amino acid sequence shown in SEQ ID No. 5.

20. A polypeptide as defined in claim 19, having a molecular weight of about 60 to 85 kDa.

21. A polypeptide as defined in claim 20, having a molecular weight of about 70 kDa.

22. A fragment of the amino acid sequence shown in SEQ ID No. 5 or 6, which fragment is receptive to imidazoline compounds.

23. A polypeptide according to any one of claims 16 to 22, which is immunoreactive with at least one of Reis antiserum and Dontenwill antiserum.

24. A polypeptide according to any one of claims 16 to 23, which is a human polypeptide.

25. A method of producing an isolated polypeptide including an amino acid sequence which is receptive to imidazoline compounds, said method comprising: transfecting a host cell with a vector as defined in claim 14 ; and culturing the transfected host cell in a culture medium to express the polypeptide.

26. An isolated polypeptide including an amino acid sequence which is receptive to imidazoline compounds, which polypeptide is expressed by the method of claim 25.

27. A method of screening for a ligand of an imidazoline receptor, which method comprises: culturing a host cell as defined in claim 15 in a culture medium to express a polypeptide including an amino acid sequence which is receptive to imidazoline compounds; contacting said polypeptide with a labelled ligand for the imidazoline receptor under conditions effective to bind the labelled ligand thereto; contacting said polypeptide with a candidate ligand; and detecting any displacement of the labelled ligand from said polypeptide, wherein displacement signifies that the candidate ligand is a ligand for the imidazoline receptor.

28. The method of claim 27, wherein said contacting steps are performed in an intact cultured host cell.

29. The method of claim 27, further comprising isolating the cell membrane of said cultured host cell prior to performing said contacting steps.

30. The method of claim 27, wherein said contacting of said imidazoline receptive polypeptide with said candidate ligand is conducted at a plurality of candidate ligand concentrations.

31. The method of claim 27, wherein the labelled ligand is radiolabelled .

32. A method of obtaining a DNA material encoding a polypeptide which is receptive to imidazoline compounds, said method comprising: providing a labelled DNA probe by labelling a DNA molecule identical or complementary to a DNA molecule as defined in any one of claims 1 to 9 or a restriction fragment thereof; contacting said DNA probe with genetic material suspected of encoding said imidazoline receptive polypeptide; hybridizing said DNA probe and said genetic material under stringent hybridization conditions; identifying any portion of the genetic material which hybridizes to said DNA probe; and isolating said identified material.

33. A method according to claim 32, wherein the genetic material is derived from a library selected from the group consisting of RNA library, cDNA library and genomic DNA library.

34. A method according to claim 33, wherein said library is a human library.

35. A method according to claim 32, wherein the labelled DNA probe is provided by labelling a restriction fragment according to claim 12 or 13.

36. A method of raising antibodies immunoreactive with a polypeptide which is receptive to an imidazoline compound, which method comprises: injecting an animal with a polypeptide as defined in any one of claims 16 to 24 and 26; and isolating antibodies produced by the animal.