DNAENCODING A HUMANIMIDAZOLINERECEPTOR

REFERENCE TO RELATED APPLICATION The present application is related to provisional application Serial No. 60/12,600, filed March 1, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to a cDNA clone encoding a human imidazoline receptive protein, designated as an imidazoline receptor subtype-1 (abbreviated IR,) , and fragments thereof.

Also, the invention relates to an IR, polypeptide encoded by the cDNA, as well as fragments containing the receptor binding site(s) . The invention also relates to methods for producing such a cDNA clone, methods for expressing the 1R ₁ protein, 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 for the binding site(s) of imidazoline receptors. However, clonidine and idazoxan are also known to possess high affinity for α ₂ -adrenergic receptors. Second generation ligands, such as moxonidine, possess somewhat improved selectivity for IR, over α ₂ -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 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: α ₂ AR Alpha-2 adrenoceptor BAC Bovine adrenal chromaffin

ECL Enhanced chemiluminescence (protein detection procedure) EST Expressed Sequence Tag

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

IR, Imidazoline receptor subtype,

IR-Ab Imidazoline receptor antibody I ₂ Site Imidazoline binding subtype ₂ kDa Kilodaltons (molecular size)

MAO monoamine oxidase

MW molecular weight

NRL European abbreviation for RVLM (see below) PC-12 Phaeochromocytoma-12 cells

^I25 PIC [ ¹²⁵ I]p-iodoclonidine

PKC Protein Kinase C

RVLM Rostral Ventrolateral Medulla in brainste

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 demonstrate partial purification of an imidazoline-selective binding protein and to characterize it as having MW = 70 kDa. This was accomplished using bovine cells (BAC) , which lack an α ₂ AR [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. To date, no one has reported the complete purification of this imidazoline receptor protein. Likewise, no amino acid sequences have been reported for IR,.

The partially purified 70 kDa protein was used by Reis and co-workers to raise "I-site binding antiserum", 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 = 33 kDa, although a trace band at 85 kDa was also observed. This 33 kDa band was enriched in plasma membrane fractions es expected for an imidazoline receptor. Furthermore, the intensity of this band was found to be positively correlated with non-adrenergic ¹²⁵ 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 ^,25 PIC 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. & Exoer. 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 α ₂ AR and/or MAO A/B. This is significant because α ₂ AR and MAO A/B have previously been cloned and also bind to imidazolines. The present inventors have obtained selective antibodies and recombinant preparations for α ₂ AR 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 ³ H-clonidine but not ³ H-rauwolscine (α ₂ -selective) binding sites in the brainstem, suggesting it 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 a d 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 immunoreactive 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 the 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 plόtelets, RVLM, and hippocampus.

Using yet another technique, a photoaffinity imidazoline ligand, ¹²⁵ AZIPI, has also been developed to preferentially label I ₂ -imidazoline binding sites [Lanier et al., J.Biol.Chem.. 268: 16047-16051 (1993)]. The ¹²⁵ AZIPI 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 studied by the present inventors is one of many pieces of evidence that distinguishes IR, from I ₂ sites. To the inventors' knowledge and as described herein, we are first to clone a cDNA encoding a protein with the immunological

and ligand binding properties expected of an IR,. We are first to identify the nucleotide sequence of a DNA molecule encoding an imidazoline receptor, and first to determine the amino acid sequence of an imidazoline receptor. The polypeptides described herein are clearly distinct from α ₂ AR or MAO A/B proteins.

SUMMARY OF THE INVENTION The present invention is for an isolated polypeptide that is receptive to (binds to) an imidazoline compound. Exemplary imidazoline compounds in this context are p-iodoclonidine and moxonidine. Typically, such a polypeptide immunoreacts with Reis antiserum and/or Dontenwill antiserum.

In one aspect of the invention, a polypeptide includes a 559 amino acid sequence as shown in SEQ ID No. 6. Other imidazoline receptive proteins homologous to this polypeptide are also contemplated. Such a protein generally has a molecular weight of about 60 to 80 kDa. More particularly, it can have a molecular weight of about 70 kDa.

In another aspect of the invention, a polypeptide includes a 298 amino acid sequence as shown in SEQ ID No. 7. Such a polypeptide generally has a molecular weight of about 35 to 45 kDa. More particularly, it can have a molecular weight of about 37 kDa.

A DNA molecule encoding an aforementioned imidazoline- receptive polypeptide is also contemplated. Such a DNA molecule, e.g., a cDNA derived from mRNA, can contain a nucleotide sequence

encoding the amino acid sequence shown in SEQ ID No. 6. Thus, a DNA molecule containing the 1677 base pair (b.p.) (1677/3 = 559) nucleotide sequence shown in SEQ ID No. 2 is contemplated. In another embodiment, a DNA molecule includes the longer nucleotide sequence shown in SEQ ID No. 3.

In another embodiment of the invention, a DNA molecule contains a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 7. Such a DNA molecule can include the 894 b.p. nucleic acid sequence shown in SEQ ID No. 4. In another aspect, it can include the 1170 b.p. nucleic acid sequence shown in SEQ ID No. 5.

RNA molecules complementary to an instant DNA molecule, e.g. , an mRNA molecule (sense) or a complementary cRNA molecule (antisense) , is 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. 2-5, or a nucleotide sequence 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 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 host 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 can 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 IR, cDNA clone.

Fig. 4 depicts a competitive binding assay between ¹²⁵ I- labelled p-iodoclonidine (PIC) and various ligands for the imidazoline receptor on membranes expressed in COS cells transfected with the IR, cDNA clone, as discussed in Example 3.

DETAILED DESCRIPTION OF THE INVENTION The present invention concerns multiple aspects of the imidazoline receptor protein, and DNA molecule encoding the same, which have now been discovered. First, a polypeptide having imidazoline binding activity has been identified, as well as fragments thereof which contain the putative active site for binding, as discussed hereinafter. Although a polypeptide described herein has a binding affinity for an imidazoline compound, it may also have an enzymatic activity, such as do catalytic antibodies and ribozymes.

Exemplary polypeptides are those containing either of the amino acid sequences shown in SEQ ID Nos. 6 or 7 (with the full length (559 residue) amino acid sequence given in SEQ ID No. 6) . Functionally equivalent polypeptides are also contemplated, such as those having a high degree of homology with an aforementioned polypeptide, particularly when they contain the Glu-Asp-rich region described hereinafter which is believed to 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 (isεαed 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 an aforementioned polypeptide. Thus, any of the degenerate set of codons encoding an instant polypeptide is contemplated. A particularly preferred coding sequence is the 1677 b.p. sequence set forth in SEQ ID No. 2, which has now been discovered to be the native nucleotide sequence that encodes the 70 kDa human IR, protein of the invention. In another embodiment, a DNA molecule includes the 3317 b.p. nucleotide sequence shown in SEQ ID No. 3. This latter sequence includes the nucleotide sequence of SEQ ID No. 2, as well as additional nonplasmid, noncoding cDNA at the 5' and 3' ends of the protein-coding sequence.

In another embodiment of the invention, a DNA molecule contains a nucleic acid sequence encoding the amino acid sequence (298 residues) shown in SEQ ID No. 7. This amino acid sequence corresponds to that derived from direct sequencing of the 5A-1 clone described hereinafter, and represents a C-terminal fragment of the native protein. Preferably, a DNA molecule includes the native 894 b.p. nucleic acid sequence shown in SEQ ID No. 4, which was derived from the 5A-1 clone. A DNA molecule can also include the 1170 b.p. nucleic acid sequence shown in SEQ ID No. 5, which includes non-plasmid cDNA at its 3' (noncoding) end.

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. 2-5. Whenever the reading frame of the vector is appropriately selected, the vector encodes an IR, polypeptide of the invention. Hence, a fragment of the native IR, protein is 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. 2-5 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 still should retain imidazoline binding activity.

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, 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 can be performed on intact host cells, or on proteins

iso _l ated 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. This process is currently referred to as combinatorial chemistry. In this process, categories of chemical structure are systematically screened for binding affinity or activation of the receptor molecule encoded by the transfected cDNA. With respect to the imidazoline receptor, a number of commercially available radioligands, e.g. , ¹²⁵ PIC, 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., oxonidine- 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 is 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 (agonist,

inverse agonist, or antagonist) under conditions effective to elicit a second messenger or other detectable response 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 a gene encoding an imidazoline receptor or homologous protein is further contemplated. Such a method comprises providing a DNA molecule identical or complementary to an above-described cDNA molecule, contacting the DNA molecule with genetic material suspected of containing a gene encoding an imidazoline receptor, or 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 preliminary research had indicated that both of the known antisera (Reis and Dontenwill) that are directed against human imidazoline receptors were immunoreactive with identical bands in the human hippocampus. 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.

Once an initial clone (5A-1) was identified, a more full- length clone was obtainable after DNA sequence analysis. The binding affinities of the expressed protein after transfection in COS cells were determined by radioligand binding procedures developed in the inventors' laboratory [Piletz and Sletten, 1993, ibid], A number of unique discoveries, i.e., brain immunoreactivity regionalization studies, and adapting ECL to these antisera, led to our identification of an imidazoline

receptor cDNA described herein.

To identify an instant cDNA clone encoding an imidazoline receptor it was necessary 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 incorporated herein by reference] . The latter antiserum was derived 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.

Both of these 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 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% 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 Reis and Dontenwill antisera, both of these antisera detected a 33 kDa band in human platelets. Although this 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 an imidazoline binding peptide. The 33 kDa band was enriched in platelet plasma membrane fractions, as is known to be the case for IR, binding, but not I ₂ binding [Piletz and Sletten, 1993]. The present investigators also found that human cortex imidazoline receptor - antibody immunoreactivity is enriched in a plasma membrane fraction (pelleted at 100,000 x g) , but not in a mitochondrial fraction (pelleted at 20,000 x g) , which suggests that the brain receptor also exists within plasma membranes. A significant positive correlation was observed within samples from 15 healthy platelet donors between IR, Bmax values (but not I ₂ or ₂ AR Bmax values) , with the IR, immunoreactivity on Western blots. This correlation exhibited a slope factor close to unity (results not shown) . This correlation strongly suggested that an IR, binding protein could be revealed in an imidazoline receptor - antibody Western blotting assay. Furthermore, the Reis antiserum failed to detect authentic α ₂ AR, MAO A or MAO B bands on gels, i.e., it was not immunoreactive

with MAO at MW = 61 kDa, or ctjAR at MW = 64 kDa. Additionally, no immunoreactive bands were observed using preimmune antiserum. Thus, after extensively characterizing these antisera with human materials, it was concluded that these antisera are indeed selective for a human imidazoline receptor protein.

Example 2. Cloning of the 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. 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 Ej_ 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. 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 four clones was performed by 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. ( ^3S S)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. 8-21.

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 1170 base pair insert (see SEQ ID No. 5) . Only one extended open reading frame for translation into protein was found possible. Consequently, it was determined that the 5A-1 cDNA derived from mRNA encoding 298 amino acids, which ended at a carboxy terminal cysteine (the TAG termination codon was at base pairs 895-898 in the 5A-1 sequence) . The 894 b.p. nucleotide sequence of the coding region of the 5A-1 clone is shown in SEQ ID No. 4. Thus, clone 5A-1 defined approximately 53% of the 70,000 MW protein predicted.

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 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 Microcoσcus luteus, possessed a marginally significant homology (p=0.04) (41%) over a short stretch of 75 of the 298 amino acids encoded by 5A-1. Otherwise, there were no significant 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, α ₂ AR, or any other known eukaryotic protein in the literature. In contrast to the amino acid search on BLASTP, two partially homologous cDNA sequences covering 155 and 250 b.p. of the 5A-1 clone were discovered to exist using BLASTN (reached from the same Internet server) . BLASTN is a program used to compare known DNA sequences from international databases, regardless of whether they encode a polypeptide. Neither of the two cDNA sequences having high homology to 5A-1 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 discoverers of these ESTs lack any knowledge of the protein 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 choose to release it. Contamination with yeast DNA was later confirmed to have been reported within an Internet database. Thus, HSA09H122 was not deemed reliable.

The other partial clone (EST04033) was purchased from the American Type Culture Collection (ATCC catalog no. 82815) (Rockville, MD) . A telephone call to the Institute for Genomic Research revealed that it had been deposited at ATCC recently.

As far as can be determined, the present inventors were the first

to sequence the full length insert of EST04033. The full length of EST04033 sequenced was 3387 b.p. (SEQ ID No. 1), with a 3,317 b.p. nonplasmid insert (see SEQ ID No. 3) . Within this sequence of EST04033 the remaining 783 base pairs of the coding sequence predicted for a 70 kDa imidazoline receptor were obtained (i.e., 783 b.p. in EST04033 + 894 b.p. of 5A-1 = 1677 total coding nucleotides) . The entire 1677 b.p. coding region for the 70 kDa protein is shown in SEQ ID No. 2. It is important to note that all of the 155 b.p. reported for EST04033 on the Internet were located at the 3' end outside of the coding region. Thus, the present inventors are first to sequence any of the coding region of an instant imidazoline receptor.

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 and continued to the end of the molecule (b.p. 3,350). The nucleotide sequence identified contains an open reading frame for a 70 kDa protein, which is the same size as the protein originally isolated as the putative bovine imidazoline receptor purified by Reis and coworkerε [Wang et al., 1992] from which the Reis antiserum was subsequently derived [Wang et al., 1993]. Of further interest is a unique glutamic- and aspartic acid-rich coding region within the clone. This region of the IR,

cDNA encodes a highly unique span of 59 amino acids, 36 of which are Glu or Asp residues (61%) . This region is largely contained within clone 5A-1 and is just upstream from apparent transmembrane loops and an ultimate polar carboxy terminus tail. Since the Dontenwill antiserum is specifically directed 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 encodes the binding site of the imidazoline receptor. The identification of this unique Glu/Asp-rich domain within the 5A-1 clone is consistent with an expected negatively charged pocket capable of binding clonidine and agmatine, both of which are highly positively charged ligands. Furthermore, this stretch is located within the longest overall region of homology that the sequence has for any known protein, i.e., the ryanodine receptor (as determined by running EST04033 on BLAST) . Specifically, we have discovered four regions of homology between the imidazoline receptor and the ryanodine receptor, which are all Glu/Asp-rich. In this region of the clone the total nucleic acid homology is 67% with the ryanodine receptor DNA sequence. However, this is not sufficient to indicate that the imidazoline receptor is a subtype of the ryanodine receptor, because the homologous sequence is still a minor portion of the 3317 bp sequence identified in the cloned cDNA. 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, which is significantly homologous to clone 5A-1, has been reported to define a calcium and ruthenium red dye binding domain that modulates the ryanodine receptor/Ca ⁺⁺ release channel located within sarcoplasmic reticulum. The only other charged amino acids within the Glu/Asp-rich region are two arginines (the ryanodine receptor has uncharged amino acids at the corresponding positions) .

Based on the 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, who 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 provided indirect evidence that the native imidazoline binding site also contains an Arg residue. 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. 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. Inasmuch as the cloned sequence had suggested a similarity with the ryanodine receptor in terms of ruthenium red binding, it was reasoned that native IR, should bind ruthenium red. Accordingly, a competition of ruthenium red with ^,25 PIC at PC-12 IR, sites was studied. In these studies it was

observed that ruthenium red competed for ¹²⁵ PIC binding equally well as did the potent imidazoline compound, moxonidine, i.e., 100% competition. Furthermore, the IC _$0 for competition of ruthenium red at IR, was more robust than has been previously reported for ruthenium red on the activation of calcium-dependent cyclic nucleotide phosphodiesterase - indicating that it might have pharmacological importance. It is also noteworthy that calcium failed to compete for ¹²⁵ PIC binding at PC-12 IR, sites (as did a calcium substitute, lanthanum) . We 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, and it was found that ruthenium red stains the same platelet (33 kDa) and brain (85 kDa) bands that Reis antiserum detects. That 33 kDa band was verified to directly correlate with ¹⁵ PIC Bmax values for IR,. Thus, these results strongly link the attributes predicted from the cloned sequence to a native IR, binding site. Some additional findings about the amino acid sequence of an instant IR, polypeptide are: (1) it bears a similarity to an amino acid sequence within a GTPase activator protein; (2) it contains at least seven small hydrophobic domains indicative of seven transmembrane domain receptors; and (3) three potential protein kinase C (PKC) phosphorylation sites are near to the carboxy terminus, 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 the observations previously expected for IR,.

Northern blotting has also been performed on polyA ⁺ mRNA from 22 human tissues in order to ascertain the regional expression of the mRNA corresponding to our cDNA. For these studies, a carboxy terminal coding sequence of clone 5A-1 (minus the Glu/Asp-rich region) was used as the labelled probe. This region was not found on 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. The two exceptions to this pattern were in lymph nodes and cerebellum, where the 9.5 kb band was equally or more intense. In either case, the size of these mRNAs could easily encode a 70 kDa protein. Although the 6 kb band is weakly detectable in some non-CNS tissues, it is strikingly enriched in brain. An enrichment of the 6 kb mRNA is observed in brainstem, although not exclusively. Importantly, the regional distribution of the mRNA is generally in keeping with the known distribution of IR, binding sites, when extrapolated across species. 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 the present cloned cDNA is consistent with it belonging to IR,.

It should be noted that while IR, binding sites are widely 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 "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 very much in keeping with the actual Bmax values for radioligand binding to the receptor [Kamisaki et al., (1990)].

Conclusion

A DNA molecule of the present invention expresses 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.

An instant cDNA sequence contains an open reading frame for a 70 kDa protein distinct from any previously described proteins having affinity for imidazoline compounds, i.e., the protein is

not an α ₂ -adrenoceptor or monoamine oxidase. Also, at least seven small hydrophobic domains in the amino acid sequence corresponding to the DNA sequence have been identified, which suggests that the protein is probably membrane bound, as is expected for an imidazoline receptor. A short sequence homology was also observed with a domain in the human ryanodine receptor that encodes the ruthenium red binding site in the ryanodine receptor.

Further evidence that an IR, cDNA has been cloned is apparent from the fact that native IR, binding sites in PC-12 cells were found to be inhibited by ruthenium red dye. Moreover, ruthenium red was found to stain the protein band previously identified by us as the IR, protein in human platelets [Chen and MacLennan, J. Biol. Chem.. 269: 22698-22704 (1994)]. Thus, based solely on our sequence we were able to predict that native imidazoline receptors would possess ruthenium red binding capacity, a finding that further establishes that an imidazoline receptor has indeed been cloned. A summary of this evidence that a cDNA encoding an imidazoline receptor (probably IR,) protein has been cloned is summarized in the Table hereinbelow.

TABLE Comparison of Properties of cDNA Clone with Properties of IR, and I ₂ Sites

Imidazoline Receptor¬ Authentic IR, Authentic I ₂ like Clone λ phage fusion protein Dontenwill Ab Dontenwill & Reis Abs is immunoreactive with inhibits RVLM IR, both inhibit brain I- Dontenwill and Reis binding site (*H- site ( ³ H-IDX) . antibodies CLON) Reis Ab immunoreactivity correlates with platelet IR, Bmax ( ^12S PIC).

No G-Protein-binding Weak-1o-moderate Not sensitive to GTP consensus sequence; but senβitivity to GTP similar to a GTPase activator protein

Predicts 70,000 MW 85,000 MW 59-61,000 MW protein immunoreactivity photoaffinity

Predicts 7 hydrophobic Enriched in plasma Enriched in domains membranes mitochondria

Encodes Glu/Asp-rich • Binds (+)-charged • Binds (+)-charged (negatively charged) imidazolines imidazolines domain consistent with • Sensitive to • Not sensitive to Ca ⁺⁺ and ruthenium red divalent cations divalent cations binding • Sensitive to • Unknown sensitivity ruthenium red for Ru red

Arginine is only • Arg attack Unknown positively charged eliminates binding amino acid near Glu/Asp • Cys & Tyr attack w/o domain effect

Encodes PKC sites PKC treatment enhances Unknown binding

Human mRNA Rat IR, Bmax ( ^l25 PIC): Rat I- Bmax ( ³ H-IDX) :

Distribution; F.Cortex F.Cortex > hippocampus Medulla > F. Cortex > hippocampus > medulla > medulla

Transfected COS-7 cells High affinity for Low affinity for have high affinity for moxonidine and PIC moxonidine and PIC moxonidine & p-iodoclonidine (PIC)

Example 3. Transfection Studies

Transient transfection studies using the above-described full length EST04033 cDNA have been performed. COS-7 cells were transfected with a vector containing the EST04033 cDNA, which was predicted based on sequence analysis to contain the entire coding region of the imidazoline receptor protein, lacking only some 5' and 3' untranslated sequences. 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 10 ⁶ 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% C0 ₂ 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. The protocol used for Western blot assay of transfected cells is as follows. Cell membranes are prepared in a special cocktail of protease inhibitors (1 mM EDTA, 0.1 mM EGTA, 1 mM phenyImethy1-sufonylfluoride, 10 mM 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 MgCl ₂ , 5 mM Tris, pH 7.4. Fifteen μg of total protein are denatured and separated by SDS gel electrophoresis. Gels are equilibrated and electrotransferred to nitrocellulose membranes. Blots are then blocked with 10% milk in Tris-buffered saline with 0.1% Tween-20 (TBST) during 60 min. of gentle rocking. Afterwards, blots are incubated in anti-imidazoline receptor antiserum (1:3000 dil.) for 2 hours. Following the primary antibody, blots are washed and incubated with horseradish peroxidase-conjugated anti-rabbit goat IgG (1:3000 dil.) for 1 hr. Blots are extensively washed

and incubated for 1 min. in a 1:1 mix of Amersham ECL detection solution. The blots are wrapped in cling-film (SARAN WRAP) and exposed to Hyperfilm-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 [ ⁿ⁵ I]p-iodoclonidine was employed as radioligand in the competition assays, since at this low concentration this radioligand is selective for the IR, site much more than for I ₂ binding sites. The critical processes of membrane preparation of tissue culture cells and the radioligand binding assays of IR, and I ₂ have recently 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" 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 I ₂ binding sites are scanned over a range of protein concentrations using a single concentration of [ ¹²⁵ I]-p- iodoclonidine (l.OnM) and ³ H-idazoxan (8nM) , respectively. Then, rate of association binding experiments (under a 10 μM mask of NE

to remove α ₂ AR 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 [ ¹²⁵ I]-p- iodoclonidine (2-20 nM if like IR,) and ³ H-idazoxan (5-60 nM if like I ₂ ) binding are conducted under a 10 μM mask of NE. Total NE (10 μM)-displaceable binding is ascertained as a control to rule out α ₂ -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 I ₂ 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 ¹²⁵ PIC was used to radiolabel I- sites. A mask of 10 μM norepinephrine was used to rule out any possible α ₂ AR binding in each assay even though parent COS-7 cells lacked any ₂ AR 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 ^{I 5} PIC were well within the high affinity (nM) range.

The following IC _J0 values and Hill slopes were obtained in this study: moxonidine, IC _S0 = 45.1 nM (Hill slope = 0.35 ±

0.04); p-iodoclonidine without PKC pretreatment, IC ₅₀ = 2.3 nM

(Hill slope = 0.42 ± 0.06); p-iodoclonidine with PKC pretreatment, IC ₅₀ = 19.0 nM (Hill slope = 0.48 ± 0.08). Shallow Hill slopes for [ ¹²⁵ I]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 [ ¹²⁵ I]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 our cloned receptor. PKC pretreatment had no effect: in the transfected COS cells.

It was also observed that the level of the expressed protein, as measured by immunoblotting of the transfected cells, was consistent with the level of IR, binding that was detected. Hence, the present results are in keeping with those expected for the native 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 the IR, protein.

Example 4. 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 alpha ₂ -adrenoceptor and IR, binding sites [Piletz et al., . Pharm♦& Exper. Ther. , 272: 581-587 (1995)], making them useful fcr these studies. Twenty-four hours after transfection, cells are split into culture dishes and grown in the presence of 600 μg/ml G 18-supplemented complete medium (Gibco/BRL) . The medium is changed every 3 days and clones surviving in G 18 are isolated and expanded for further investigation.

Example 5. Direct Cloning of Human Genomic IR,.

Direct cloning from a human genomic library can be done by preparing labelled cDNA probes from different subcloned regions of our full-length cDNA and using the probes to screen a commercially available human brain genomic library. One genomic library is EMBL (Clontech) , which integrates genomic fragments up to 22 kbp long. It is reasonable to expect that introns may exist within the human gene so that only by obtaining overlapping clones can the full gene 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 6. Methods for Preparing Antibodies to Imidazoline Receptive Proteins.

An instant imidazoline receptive polypeptide can also be used to prepare antibodies immunoreactive therewith. Thus, transfected cell lines or other manipulations of the DNA sequence of an instant imidazoline receptor can provide a source of purified imidazoline receptor protein in sufficient quantities for immunization, which can lead to a source of selective antibodies having 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 and 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 for imidazoline receptive protein [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 would 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 o _f which is incorporated herein by reference] .

summary

In summary, we have identified a unique cDNA sequence and have shown that it has the properties expected of an imidazoline receptor. Although two partial sequences of the entire sequence were found in public databases on the Internet, these are partial

sequences (155-250 bp) and were not identified at all with respect to any encoded protein. Moreover, neither of these sequences reliably defines any portion of the coding region for an instant IR, protein. The EST04033 clone, which was reported to contain the 155 bp sequence, was sequenced for the first time by the present inventors in its entirety (3316 bp) by the inventors. Prior to this, even the size of EST04033 was unknown. The present inventors also demonstrated that an imidazoline receptive site can be expressed in cells transfected with the entire cDNA clone, and this site has the proper potencies for an IR,. Thus, a DNA sequence with an open reading frame for a 70 kDa human IR, protein has been identified from our clones and the amino acid sequence of the protein has been deduced. The authenticity of the expressed protein as that defining an IR, protein has been demonstrated in a variety of ways.

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

(1) GENERAL INFORMATION:

(i) APPLICANT: John E. Piletz Tina R. Ivanov (ii) TITLE OF INVENTION: DNA Sequence Encoding a Human Imidazoline Receptor and Method for Cloning the Same (iii) NUMBER OF SEQUENCES: 21 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: WENDEROTH, LIND & PONACK

(B) STREET: 805 Fifteenth St. N.W. , Suite 700

(C) CITY: Washington

(D) STATE: District of Columbia

(E) COUNTRY: U.S.A.

(F) ZIP: 20005 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.50" 1.44 Mb diskette

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: Wordperfect 5.1+ (Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: new

(B) FILING DATE:

(C) CLASSIFICATION: (Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NO. : 60/012,600

(B) FILING DATE: March 1, 1996 (Viii) ATTORNE /AGENT INFORMATION:

(A) NAME: Warren Cheek

(B) REGISTRATION NUMBER: 33,367

(C) REFERENCE/DOCKET NUMBER: WMC-1342/clone (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202) 371-8850

(B) TELEFAX: (202) 371-8856

(2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3387 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE:

(A) LIBRARY : cDNA

(B) CLONE : ESTO4033 (HFBDP28 ) ( ix) FEATURE :

(A) NAME/KEY : open reading frame

(B) LOCATION : 1398 . . . 3074

(xi) 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 AAG-3AGTTGG 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 He Ala Gin Gly Cys Ser Asp Ser Leu Glu Ser He 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 He He Phe Leu Pro Phe Thr Cys He 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 He Gin Arg Leu Ser Thr Leu He Trp Gin Ala He 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 He 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 He Arg He 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 He His Val Gin Gly Ser He 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 He Ala Val Phe Glu He 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 He Leu Ser Ser Leu Arg Phe 335 340 345

GTC TTT TGC TTC CGC ATG GCG ACC TCA CCG AGT TTG GCT TCC TCA TGC 2486 Val Phe Cys Phe Arg Met Ala Thr Ser Pro Ser Leu Ala Ser Ser Cys 350 355 360

CGG AGC TGT GTC TGG TGC TCA AGG TAC GGC ACA GTG AGA ACA CGC TCT 2534 Trp Ser Cys Val Trp Cys Ser Arg Tyr Gly Thr Val Arg Thr Arg Ser 365 370 375

TCA TTA TCT CGG ACG CCG CCA ACC TGC ACG AGT TCC ACG CGG ACC TGC 2582 Ser Leu Ser Trp Thr Pro Pro Thr Cys Thr Ser Ser Thr Trp Thr Cys 380 385 390 395

GCT CAT GCT TTG CAC CCC AGC ACA TGG CCA TGC TGT GTA GCC CCA TCC 2630 Ala His Ala Leu His Pro Ser Thr Trp Pro Cys Cys Val Ala Pro Ser

400 405 410

TCT ACG GCA GCC ACA CCA GCC TGC AGG AGT TCC TGC GCC AGC TGC TCA 2678 Ser Thr Ala Ala Thr Pro Ala Cys Arg Ser Ser Cys Ala Ser Cys Ser 415 420 425

CCT TCT ACA AGG TGG CTG GCG GCT GCC AGG AGC GCA GCC AGG GCT GCT 2726

Pro Ser Thr Arg Trp Leu Ala Ala Ala Arg Ser Ala Ala Arg Ala Ala 430 435 440

TCC CCG TCT ACC TGG TCT ACA GTG ACA AGC GCA TGG TGC AGA CGG CCG 2774 Ser Pro Ser Thr Trp Ser Thr Val Thr Ser Ala Trp Cys Arg Trp Pro

445 450 455

CCG GGG ACT ACT CAG GCA ACA TCG AGT GGG CCA GCT GCA CAC TCT GTT 2822 Pro Gly Thr Thr Gin Ala Thr Ser Ser Gly Pro Ala Ala His Ser Val 460 465 470 475

CAG CCG TGC GGC GCT CCT GCT GCG CGC CCT CTG AGG CCG TCA AGT CCG 2870 Gin Pro Cys Gly Ala Pro Ala Ala Arg Pro Leu Arg Pro Ser Ser Pro

480 485 490

CCG CCA TCC CCT ACT GGC TGT TGC TCA CGC CCC AGC ACC TCA ACG TCA 2918 Pro Pro Ser Pro Thr Gly Cys Cys Ser Arg Pro Ser Thr Ser Thr Ser 495 500 505

TCA AGG CCG ACT TCA ACC CCA TGC CCA ACC GTG GCA CCC ACA ACT GTC 2966 Ser Arg Pro Thr Ser Thr Pro Cys Pro Thr Val Ala Pro Thr Thr Val 510 515 520

GCA ACC GCA ACA GCT TCA AGC TCA GCC GTG TGC CGC TCT CCA CCG TGC 3014 Ala Thr Ala Thr Ala Ser Ser Ser Ala Val Cys Arg Ser Pro Pro Cys 525 530 535

TGC TGG ACC CCA CAC GCA GCT GTA CCC AGC CTC GGG GCG CCT TTG CTG 3062 Cys Trp Thr Pro His Ala Ala Val Pro Ser Leu Gly Ala Pro Leu Leu 540 545 550 555

ATG GCC ACG TGC TAGAGC TGCTCGTGGG GTACCGCTTT GTCACTGCCA TCTTCGTGCT 3120 Met Ala Thr Cys 559

GCCCCACGAG AAGTTCCACT TCCTGCGCGT CTACAACCAG CTGCGGGCCT CGCTGCAGGA 3180

CCTGAAGACT GTGGTCATCG CCAAGACCCC CGGGACGGGA GGCAGCCCCC AGGGCTCCTT 3240

TGCGGATGGC CAGCCTGCCG AGCGCAGGGC CAGCAATGAC CAGCGTCCCC AGGAGGTCCC 3300

AGCAGAGGCT CTGGCCCCGG CCCCAGTGGA AGTCCCAGCT CCAGCCCCGG AATTCGATAT 3360

CAAGCTTATC GATACCGTCG ACCTCGA 3387

( 2 ) INFORMATION FOR SEQ ID NO : 2

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 1677 base pairs

(B) TYPE : nucleic acid

( C) STRANDEDNESS : double

( D) TOPOLOGY : linear

( ix) 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 TTCCGCATGG CGACCTCACC GAGTTTGGCT 1080

TCCTCATGCC GGAGCTGTGT CTGGTGCTCA AGGTACGGCA CAGTGAGAAC ACGCTCTTCA 1140

TTATCTCGGA CGCCGCCAAC CTGCACGAGT TCCACGCGGA CCTGCGCTCA TGCTTTGCAC 1200

CCCAGCACAT GGCCATGCTG TGTAGCCCCA TCCTCTACGG CAGCCACACC AGCCTGCAGG 1260

AGTTCCTGCG CCAGCTGCTC ACCTTCTACA AGGTGGCTGG CGGCTGCCAG GAGCGCAGCC 1320

AGGGCTGCTT CCCCGTCTAC CTGGTCTACA GTGACAAGCG CATGGTGCAG ACGGCCGCCG 1380

GGGACTACTC AGGCAACATC GAGTGGGCCA GCTGCACACT CTGTTCAGCC GTGCGGCGCT 1440

CCTGCTGCGC GCCCTCTGAG GCCGTCAAGT CCGCCGCCAT CCCCTACTGG CTGTTGCTCA 1500

CGCCCCAGCA CCTCAACGTC ATCAAGGCCG ACTTCAACCC CATGCCCAAC CGTGGCACCC 1560

ACAACTGTCG CAACCGCAAC AGCTTCAAGC TCAGCCGTGT GCCGCTCTCC ACCGTGCTGC 1620

TGGACCCCAC ACGCAGCTGT ACCCAGCCTC GGGGCGCCTT TGCTGATGGC CACGTGC 1677

(3) INFORMATION FOR SEQ ID NO: 3

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3317 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 3

AATTCCAGTT TAATACTAAC CCTAATGTGT GACTGCGGTT TACAAAG GC 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 TTGCTTCCGC ATGGCGACCT CACCGAGTTT GGCTTCCTCA TGCCGGAGCT 2460

GTGTCTGGTG CTCAAGGTAC GGCACAGTGA GAACACGCTC TTCATTATCT CGGACGCCGC 2520

CAACCTGCAC GAGTTCCACG CGGACCTGCG CTCATGCTTT GCACCCCAGC ACATGGCCAT 2580

GCTGTGTAGC CCCATCCTCT ACGGCAGCCA CACCAGCCTG CAGGAGTTCC TGCGCCAGCT 2640

GCTCACCTTC TACAAGGTGG CTGGCGGCTG CCAGGAGCGC AGCCAGGGCT GCTTCCCCGT 2700

CTACCTGGTC TACAGTGACA AGCGCATGGT GCAGACGGCC GCCGGGGACT ACTCAGGCAA 2760

CATCGAGTGG GCCAGCTGCA CACTCTGTTC AGCCGTGCGG CGCTCCTGCT GCGCGCCCTC 2820

TGAGGCCGTC AAGTCCGCCG CCATCCCCTA CTGGCTGTTG CTCACGCCCC AGCACCTCAA 2880

CGTCATCAAG GCCGACTTCA ACCCCATGCC CAACCGTGGC ACCCACAACT GTCGCAACCG 2940

CAACAGCTTC AAGCTCAGCC GTGTGCCGCT CTCCACCGTG CTGCTGGACC CCACACGCAG 3000

CTGTACCCAG CCTCGGGGCG CCTTTGCTGA TGGCCACGTG CTAGAGCTGC TCGTGGGGTA 3060

CCGCTTTGTC ACTGCCATCT TCGTGCTGCC CCACGAGAAG TTCCACTTCC TGCGCGTCTA 3120

CAACCAGCTG CGGGCCTCGC TGCAGGACCT GAAGACTGTG GTCATCGCCA AGACCCCCGG 3180

GACGGGAGGC AGCCCCCAGG GCTCCTTTGC GGATGGCCAG CCTGCCGAGC GCAGGGCCAG 3240

CAATGACCAG CGTCCCCAGG AGGTCCCAGC AGAGGCTCTG GCCCCGGCCC CAGTGGAAGT 3300

CCCAGCTCCA GCCCCGG 3317

(4) INFORMATION FOR SEQ ID NO: 4

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 894 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) 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 CGCATGGCGA CCTCACCGAG TTTGGCTTCC 300

TCATGCCGGA GCTGTGTCTG GTGCTCAAGG TACGGCACAG TGAGAACACG CTCTTCATTA 360

TCTCGGACGC CGCCAACCTG CACGAGTTCC ACGCGGACCT GCGCTCATGC TTTGCACCCC 420

AGCACATGGC CATGCTGTGT AGCCCCATCC TCTACGGCAG CCACACCAGC CTGCAGGAGT 480

TCCTGCGCCA GCTGCTCACC TTCTACAAGG TGGCTGGCGG CTGCCAGGAG CGCAGCCAGG 540

GCTGCTTCCC CGTCTACCTG GTCTACAGTG ACAAGCGCAT GGTGCAGACG GCCGCCGGGG 600

ACTACTCAGG CAACATCGAG TGGGCCAGCT GCACACTCTG TTCAGCCGTG CGGCGCTCCT 660

GCTGCGCGCC CTCTGAGGCC GTCAAGTCCG CCGCCATCCC CTACTGGCTG TTGCTCACGC 720

CCCAGCACCT CAACGTCATC AAGGCCGACT TCAACCCCAT GCCCAACCGT GGCACCCACA 780

ACTGTCGCAA CCGCAACAGC TTCAAGCTCA GCCGTGTGCC GCTCTCCACC GTGCTGCTGG 840

ACCCCACACG CAGCTGTACC CAGCCTCGGG GCGCCTTTGC TGATGGCCAC GTGC 894

(5) INFORMATION FOR SEQ ID NO: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1170 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 5

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 CGCATGGCGA CCTCACCGAG TTTGGCTTCC 300

TCATGCCGGA GCTGTGTCTG GTGCTCAAGG TACGGCACAG TGAGAACACG CTCTTCATTA 360

TCTCGGACGC CGCCAACCTG CACGAGTTCC ACGCGGACCT GCGCTCATGC TTTGCACCCC 420

AGCACATGGC CATGCTGTGT AGCCCCATCC TCTACGGCAG CCACACCAGC CTGCAGGAGT 480

TCCTGCGCCA GCTGCTCACC TTCTACAAGG TGGCTGGCGG CTGCCAGGAG CGCAGCCAGG 540

GCTGCTTCCC CGTCTACCTG GTCTACAGTG ACAAGCGCAT GGTGCAGACG GCCGCCGGGG 600

ACTACTCAGG CAACATCGAG TGGGCCAGCT GCACACTCTG TTCAGCCGTG CGGCGCTCCT 660

GCTGCGCGCC CTCTGAGGCC GTCAAGTCCG CCGCCATCCC CTACTGGCTG TTGCTCACGC 720

CCCAGCACCT CAACGTCATC AAGGCCGACT TCAACCCCAT GCCCAACCGT GGCACCCACA 780

ACTGTCGCAA CCGCAACAGC TTCAAGCTCA GCCGTGTGCC GCTCTCCACC GTGCTGCTGG 840

ACCCCACACG CAGCTGTACC CAGCCTCGGG GCGCCTTTGC TGATGGCCAC GTGCTAGAGC 900

TGCTCGTGGG GTACCGCTTT GTCACTGCCA TCTTCGTGCT GCCCCACGAG AAGTTCCACT 960 rCCTGCGCGT CTACAACCAG CTGCGGGCCT CGCTGCAGGA CCTGAAGACT GTGGTCATCG 1020

CCAAGACCCC CGGGACGGGA GGCAGCCCCC AGGGCTCCTT TGCGGATGGC CAGCCTGCCG 1080

:.GCGCAGGGC CAGCAATGAC CAGCGTCCCC AGGAGGTCCC AGCAGAGGCT CTGGCCCCGG 1140

CCCCAGTGGA AGTCCCAGCT CCAGCCCCGG 1170

(6) INFORMATION FOR SEQ ID NO: 6

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 559 amino acids

(B) TYPE: polypeptide

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 6

Met Thr Gly Gin Val Gly Ala Gin Thr Val Ser Gly Gly Lys Arg

1 5 10 15

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

20 25 30

Leu Leu Asp Met Ser Val Gin Val He Arg Pro Gly Glu Ala Phe

35 40 45

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

50 55 60

Gly Gly Glu Asp Ser Trp Leu Ser Ala Ala Pro Cys He Arg Pro

65 70 75

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

80 85 90

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

95 100 105

Ala Leu Ala Ser Ser Leu Ser Ser Thr Asp Ser Leu Thr Pro Glu

110 115 120

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

125 130 135

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

140 145 150

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

155 160 165

Val Val Pro Gly Ser Gly Gin He He Phe Leu Pro Phe Thr Cys

170 175 180

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

185 190 195

Thr Leu He Trp Gin Ala He Glu Trp Gin Leu Pro Ala Trp He

200 205 210

Glu Ala Ala Asn Gin Trp Glu Glu Gly Gin Gly Glu Gin Gly Glu

215 220 225

Glu Glu Asp Glu Glu Glu Glu Glu Glu Glu Asp Val Ala Glu Asn

230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315

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

320 325 330

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

335 340 345

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

350 355 360

Ser Ser Cys Trp Ser Cys Val Trp Cys Ser Arg Tyr Gly Thr Val

365 370 375

Arg Thr Arg Ser Ser Leu Ser Trp Thr Pro Pro Thr Cys Thr Ser

380 385 390

Ser Thr Trp Thr Cys Ala His Ala Leu His Pro Ser Thr Trp Pro

395 400 405

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

410 415 420

Ser Ser Cys Ala Ser Cys Ser Pro Ser Thr Arg Trp Leu Ala Ala

425 430 435

Ala Arg Ser Ala Ala Arg Ala Ala Ser Pro Ser Thr Trp Ser Thr

440 445 450

Val Thr Ser Ala Trp Cys Arg Trp Pro Pro Gly Thr Thr Gin Ala

455 460 465

Thr Ser Ser Gly Pro Ala Ala His Ser Val Gin Pro Cys Gly Ala

470 475 480

Pro Ala Ala Arg Pro Leu Arg Pro Ser Ser Pro Pro Pro Ser Pro

485 490 495

Thr Gly Cys Cys Ser Arg Pro Ser Thr Ser Thr Ser Ser Arg Pro

500 505 510

Thr Ser Thr Pro Cys Pro Thr Val Ala Pro Thr Thr Val Ala Thr

515 520 525

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

530 535 540

Trp Thr Pro His Ala Ala Val Pro Ser Leu Gly Ala Pro Leu Leu

545 550 555

Met Ala Thr Cys 559

(7) INFORMATION FOR SEQ ID NO: 7

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 298 amino acids

(B) TYPE: polypeptide

(C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 7

Glu Glu Glu Glu Glu Glu Asp Glu Glu Ala Glu Glu Glu Arg Leu 1 5 10 15

Ala Leu Glu Trp Ala Leu Gly Ala Asp Glu Asp Phe Leu Leu Glu

20 25 30

His He Arg He Leu Lys Val Leu Trp Cys Phe Leu He His Val

35 40 45

Gin Gly Ser He Arg Gin Phe Ala Ala Cys Leu Val Leu Thr Asp

50 55 60

Phe Gly He Ala Val Phe Glu He Pro His Gin Glu Ser Trp Gly

65 70 75

Ser Ser Gin His He Leu Ser Ser Leu Arg Phe Val Phe Cys Phe

80 85 90

Arg Met Ala Thr Ser Pro Ser Leu Ala Ser Ser Cys Trp Ser Cys

95 100 105

Val Trp Cys Ser Arg Tyr Gly Thr Val Arg Thr Arg Ser Ser Leu

110 115 120

Ser Trp Thr Pro Pro Thr Cys Thr Ser Ser Thr Trp Thr Cys Ala

125 130 135

His Ala Leu His Pro Ser Thr Trp Pro Cys Cys Val Ala Pro Ser

140 145 150

Ser Thr Ala Ala Thr Pro Ala Cys Arg Ser Ser Cys Ala Ser Cys

155 160 165

Ser Pro Ser Thr Arg Trp Leu Ala Ala Ala Arg Ser Ala Ala Arg

170 175 180

Ala Ala Ser Pro Ser Thr Trp Ser Thr Val Thr Ser Ala Trp Cys

185 190 195

Arg Trp Pro Pro Gly Thr Thr Gin Ala Thr Ser Ser Gly Pro Ala

200 205 210

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

215 220 225

Arg Pro Ser Ser Pro Pro Pro Ser Pro Thr Gly Cys Cys Ser Arg

230 235 240

Pro Ser Thr Ser Thr Ser Ser Arg Pro Thr Ser Thr Pro Cys Pro

245 250 255

Thr Val Ala Pro Thr Thr Val Ala Thr Ala Thr Ala Ser Ser Ser

260 265 270

Ala Val Cys Arg Ser Pro Pro Cys Cys Trp Thr Pro His Ala Ala

275 280 285

Val Pro Ser Leu Gly Ala Pro Leu Leu Met Ala Thr Cys

290 295 298

(8) INFORMATION FOR SEQ ID NO: 8

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 8

CTTGAGGATG CGGATGTGCT 20

(9) INFORMATION FOR SEQ ID NO: 9

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO:

CCATGGGGTG AGTGTCCT 18

(10) INFORMATION FOR SEQ ID NO: 10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 10

AGGACACTCA CCCCATGG 18

(11) INFORMATION FOR SEQ ID NO: 11 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 11

GTATGGGACA GGGGCAGAAA 20

(12) INFORMATION FOR SEQ ID NO: 12 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 12

TTTCTAAAGA CCATTGGGAG 20

(13) INFORMATION FOR SEQ ID NO: 13 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 13

CCATTTTAAA GTAGCGGTTC 20

(14) INFORMATION FOR SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 14

AGGAGAGAAA GGTGAGCCAA 20

(15) INFORMATION FOR SEQ ID NO: 15 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 15

GTAGATCCTG AGGTTGACCA 20

(16) INFORMATION FOR SEQ ID NO: 16 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 16

TGTGAGCATT TCTGGCCTTC 20

(17) INFORMATION FOR SEQ ID NO: 17 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 17

TGAAGACGCC AGAGAAGCAG 20

(18) INFORMATION FOR SEQ ID NO: 18

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 18

GCCTCACAAG TGTCAGACCT 20

(19) INFORMATION FOR SEQ ID NO: 19 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 19

AGAAGGGTGG TGAAGACT 18

(20) INFORMATION FOR SEQ ID NO: 20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 20

CTTGGTTAGA GAGGATGGGC 20

(21) INFORMATION FOR SEQ ID NO: 21 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) SEQUENCE DESCRIPTION: SEQ ID NO: 21

GCCCATCCTC TCTAACCAAG 20