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 .alpha..sub.2 -adrenergic receptors. Second generation ligands, such as moxonidine, possess somewhat improved selectivity for IR over .alpha..sub.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 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:
______________________________________.alpha..sub.2 AR Alpha-2 adrenoceptorBAC Bovine adrenal chromaffinECL 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.sub.1 Imidazoline receptor subtype.sub.1IR-Ab Imidazoline receptor antibodyI.sub.2 Site Imidazoline binding subtype.sub.2kDa Kilodaltons (molecular size)MAO monoamine oxidaseMW molecular weightNRL European abbreviation for RVLM (see below)PC-12 Phaeochromocytoma-12 cells.sup.125 PIC [.sup.125 I]p-iodoclonidinePKC Protein Kinase CRVLM Rostral Ventrolateral Medulla in brainstemSDS 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 .alpha..sub.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 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 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 .mu.g) 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.apprxeq.33 kDa, although a trace band at .apprxeq.85 kDa was also observed. The .apprxeq.33 and .apprxeq.85 kDa bands were enriched in plasma membrane fractions as expected for an imidazoline receptor. Furthermore, the intensity of the .apprxeq.33 kDa band was found to be positively correlated with non-adrenergic .sup.125 PIC Bmax values at platelet IR.sub.1 sites in samples from the same subjects, with an almost one-to-one slope factor. In addition, the nonadrenergic .sup.125 PIC binding sites on platelets were discovered by the present inventors to have the same rank order of affinities as IR.sub.1 binding sites in brainstem [Piletz and Sletten, J.Pharm. & Exper. Therap., 267: 1493-1502 (1993)]. The platelet .apprxeq.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 .apprxeq.85 kDa immunoreactive band was found to predominate.
Immunoreactivity with Reis antiserum does not appear to be directed against human .alpha..sub.2 AR and/or MAO A/B. This is a significant point because .alpha..sub.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 .alpha..sub.2 AR and MAO A/B, and these proteins do not correspond to the .apprxeq.33, 70, or 85 kDa putative IR.sub.1 bands. Thus, there is substantial evidence that, at least in human platelets, the Reis antiserum is IR.sub.1 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 .sup.3 H-clonidine but not .sup.3 H-rauwolscine (.alpha..sub.2 -selective) binding sites in the brainstem, suggesting it also interacts with IR.sub.1 [Bennai et al., 1995]. As shown in FIG. 1, human RVLM (same as NRL) membrane fractions displayed bands of .apprxeq.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.apprxeq.41 and 44 kDa detected by Dontenwill antiserum may be derived from an .apprxeq.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 .apprxeq.41 and 44 kDa bands in human brain when fewer protease inhibitors are used. Additionally, the Dontenwill antiserum weakly detects a platelet .apprxeq.33 kDa band. Thus, the present inventors have hypothesized that the .apprxeq.41 and 44 kDa immunoreactive proteins may be alternative breakdown products of an .apprxeq.85 kDa protein, as opposed to the platelet .apprxeq.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, .sup.125 AZIPI, has also been developed to preferentially label I.sub.2 -imidazoline binding sites [Lanier et al., J.Biol.Chem., 268: 16047-16051 (1993)]. The .sup.125 AZIPI photoaffinity ligand was used to visualize .apprxeq.55 kDa and .apprxeq.61 kDa binding proteins from rat liver and brain. It is believed that the .apprxeq.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.sub.1 from I.sub.2 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 .alpha..sub.2 AR 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 11710 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 (.gtoreq.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 .alpha..sub.1b -adrenergic receptor. RNA molecules of .gtoreq.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 .gtoreq.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
FIGS. 1A-B depicts a comparison of Reis antiserum (lane 1, 1:2000 dilution) and Dontenwill antiserum (lane 2, 1:5000 dilution) immunoreactivities for human NRL (FIG. 1B) (same as RVLM) and hippocampus (FIG. 1A), as discussed in Example 1.
FIGS. 2A-B depicts a comparison of Reis antiserum (FIG. 2A) (1:15,000 dilution) and Dontenwill antiserum (FIG. 2B) (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 .sup.125 I-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).
FIGS. 6A-B depicts the distribution of MRNA homologous to our CDNA in human adult tissues, FIG. 6A (bar graph) and the two species of MRNA (6 and 9.5 kb), FIG. 6B.

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. Pat. 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. Pat. 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. Pat. 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., .sup.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., 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.times. SSC, 0.5% SDS at 65.degree. 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 .lambda.gt11 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 (30 .mu.g) and NRL membrane proteins (10 .mu.g) 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.sub.1 binding, but not I.sub.2 binding [Piletz and Sletten, 1993].
A significant positive correlation was established for the 85 Kda band detected by the Dontenwill antiserum with IR.sub.1 Bmax values across nine rat tissues (r.sup.2 =0.8736). A similar positive correlation was established amongst platelet samples from 15 healthy platelet donors between radioligand IR.sub.1 Bmax values (but not I.sub.2 or .alpha..sub.2 AR Bmax values), and the 33 Kda band (presumed IR.sub.1 immunoreactivity) on Western blots. This correlation exhibited a slope factor close to unity (results not shown). These correlations strongly suggested that an IR.sub.1 binding protein might be revealed in an imidazoline receptor-antibody Western blotting assay. Furthermore, the Reis antiserum failed to detect authentic .alpha..sub.2 AR, MAO A, or MAO B bands on gels, i.e., it was not immunoreactive with MAO at MW=61 Kda, or .alpha..sub.2 AR 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 .lambda.gt11 expression library (Clontech Inc., Palo Alto, Calif.) 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 .lambda.gt11 DNA purified from each of the four plaques was subsequently subcloned into E. coli pBluescript vector (Stratagene, La Jolla, Calif.). 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), 10801 University Boulevard, Manassas Va. 20110-2209, on Aug. 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, Ill.) 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. (.sup.35 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, Calif.).
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, Calif.) 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, Jun. 20, 1995 (build Nov. 13, 1995)] 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.ltoreq.0.05) for any known proteins. Therefore, the protein encoded by 5A-1 is not significantly related to MAO A or B, .alpha..sub.2 AR, 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 Nov. 13, 1995). 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 .apprxeq.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.1) 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 .alpha..sub.2 -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, Calif.) to screen for an IR gene by hybridization. The genomic library was constructed in Stratagene's vector .lambda. FIX.RTM. II (catalog # 946206), and it was grown in XL1-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 EcoR1 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.RTM. III kit manual (BIO 101, Inc., P.O. Box 2284, La Jolla, Calif.), and radiolabeled with (.alpha.-.sup.32 p]d-CTP according to Stratagene's Prime-It.RTM. II Random Primer Labeling Kit manual. Plaques were lifted onto 137 mm Duralon-UV.TM. membranes (Stratagene's catalog #420102), denatured, and cross-linked with Stratgene's UV-Stratalinker.TM. 1800. Hybridization was conducted under high stringency conditions: prehybridization=6.times. SSC, 1% SDS, 50% formamide, and 100 1 .mu.g/ml of sheared, denatured salmon sperm DNA at 42.degree. C. for 2 hrs; hybridization=6.times. SSC, 1% SDS, 50% formamide, and 100 .mu.g/ml of sheared, denatured salmon sperm DNA at 45.degree. C. overnight; wash=2 washes of 1.times. SSC, 0.1% SDS at 65.degree. C. and 3 washes of 0.2.times. SSC, 0.2% SDS at 65.degree. C. This hybridization procedure is essentially described in Stratagene's vector .lambda. FIX.RTM. 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 .apprxeq.17 kb insert. Large-scale preparations of this genomic clone DNA were performed using the .lambda. QUICK! SPIN kit (BIO101, La Jolla, Calif.). 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 .sup.32 P-labeled probe: a 1110 bp ApaI-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 JEP1-A, we detected a 14.1 kb EcoRI fragment and a 7.7 kb SacI fragment that hybridized with this probe. Southern blots containing EcoRI- or SacI-digested human genomic DNA (from human blood) with the 1110 bp ApaI-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), 10801 University Boulevard, Manassas Va. 20110-2209, on Aug. 28, 1997 and has been assigned deposit accession no. ATCC 209216.
Genomic DNA sequencing was done by contract with Cadus Pharmaceutical Corporation (Tarrytown, N.Y.). The original lambda JEP1-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, Conn.; Applied Biosystems) (ABI). The kit consists of a synthetic transposon (Ty1) 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, N.Y., 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, N.Y.). Oligonucleotides were purchased from Gibco-BRL (Gaithersburg, Md.). Sequence assembly was performed using Sequencer Software (Gene Codes Corp., Ann Arbor, Mich.) 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 ExonsGENSCAN 1.0 Date run: 26-Aug-97 Time: 12:35:39Sequence gs.sub.-- seqfile: 15202 bp: 58.36% C + G: Isochore 4(57.00-100.00 C + G%)Parameter matrix: HumanIso.smatPredicted 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 20.911.02 Intr + 4096 4177 82 0 1 37 53 81 0.358 -0.131.03 Intr + 5732 5856 125 0 2 117 95 84 0.953 13.481.04 Intr + 6997 7046 50 0 2 95 116 44 0.998 6.521.05 Intr + 8416 9825 1410 1 0 96 94 2914 0.970 283.091.06 Intr + 10489 10897 409 1 1 15 59 318 0.517 17.191.07 Intr + 11293 11449 157 0 1 57 61 236 0.998 18.571.08 Intr + 11923 12051 129 2 0 84 63 224 0.997 21.341.09 Intr + 12570 12731 162 1 0 95 80 229 0.996 23.941.10 Term + 13090 13700 611 2 2 59 41 1012 0.942 89.441.11 PlyA + 14257 14262 6 1.05__________________________________________________________________________
A BLASTN analysis of the entire genomic sequence (on Aug. 26, 1997) 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.1, 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-1/.lambda.gt11 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.sup.++ 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 .sup.125 PIC at native IR sites on PC-12 membranes was studied. In these studies it was observed that ruthenium red competed for .sup.125 PIC binding to the same extent as did the potent imidazoline compound, moxonidine, i.e., 100% competition. Furthermore, the IC.sub.50 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 .sup.125 PIC 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 .sup.125 PIC 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.beta. receptor (IL-2R.beta.). Specifically, IL-2R.beta. 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.sup.+ mRNA from human tissues in order to ascertain the regional expression of the mRNA corresponding to our cDNA. The same 1110 b.p. ApaI-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-1A 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.sup.+ 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 withProperties of Native IR.sub.1 and I.sub.2 SitesImidazoline Receptor-like Clone Authentic IR.sub.1 Authentic I.sub.2__________________________________________________________________________Original phage fusion Dontenwill-Ab activity Dontenwill & Reis Absprotein (from 5A-1) is (a) inhibits RVLM IR.sub.1 both inhibit brain I.sub.2immunoreactive with binding (.sup.3 H-Clonidine), sites (.sup.3 H-IDX).Dontenwill and Reis & (b) correlates withantibodies 85 kDa Western band. Reis-Ab activity correlates w platelet IR.sub.1 Bmax (.sup.125 PIC binding)Segment homologous to a Weak to moderate Not sensitive to GTPGTPase-activator prot'n sensitivity to GTPPredicts .gtoreq. 120,000 MW 85,000 MW 59-61,000 MWprotein immunoreactivity photoaffinityPredicts 1-4 Enriched in plasma Enriched inhydrophobic domains membranes mitochondriaEncodes Glu/Asp-rich .cndot. Binds (+)-charged .cndot. Binds (+)-charged(negatively charged) imidazolines imidazolinesdomain consistent with .cndot. Sensitive to .cndot. Not sensitive toCa.sup.++ and ruthenium red divalent cations divalent cationsbinding .cndot. Sensitive to .cndot. Unknown ruthenium red sensitivity for Ruthen. redArginine is only .cndot. Arg attack Unknownpositively charged elminatesamino acid near Glu/Asp bindingdomain .cndot. Cys & Tyr attack w/o effect on bindingEncodes PKC sites PKC treatment enhances Unknown bindingHuman mRNA Rat IR.sub.1 Bmax (.sup.125 PIC): Rat I.sub.2 Bmax (.sup.3 H-IDX):Distribution; F. Cortex > F. Cortex > hippocampus > Medulla > F. Cortexhippocampus > medulla medullaTransfected COS-7 cells High affinity for Low affinity forexpressed high affinity moxonidine and PIC moxonidine and PICfor moxonidine &p-iodoclonidine (PIC)__________________________________________________________________________
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, N.J.) 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.times.10.sup.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 .mu.g/ml streptomycin; 100 units/ml penicillin, high glucose, Dulbeccos' modified Eagle's medium) containing 80 .mu.M chloroquine was added and the incubation continued for 2.5 hr. at 37.degree. C. in a 5% CO.sub.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.
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 mM .epsilon.-aminocaproic acid, 0.1 mM benzamide, 0.1 mM benzamide-HCl, 0.1 mM phenanthroline, 10 .mu.g/ml pepstatin A, 5 mM iodoacetamide, 10 .mu.g/ml antipain, 10 .mu.g/ml trypsin-chymotrypsin inhibitor, 10 .mu.g/ml leupeptin, and 1.67 .mu.g/ml calpain inhibitor) in 0.25 M sucrose, 1 mM MgCl.sub.2, 5 mM Tris, pH 7.4. Fifteen .mu.g 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 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 [.sup.125 I]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 I.sub.2 binding sites. The critical processes of membrane preparation of tissue culture cells and the radioligand binding assays of IR and I.sub.2 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.sup.-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 I.sub.2 binding sites are scanned over a range of protein concentrations using a single concentration of [.sup.125 I]-p-iodoclonidine (1.0 nM) and .sup.3 H-idazoxan (8 nM), respectively. Then, rate of association binding experiments (under a 10 .mu.M mask of NE to remove .alpha..sub.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 [.sup.125 I]-p-iodoclonidine (2-20 nM if like IR) and .sup.3 H-idazoxan (5-60 nM if like I.sub.2) binding are conducted under a 10 .mu.M mask of NE. Total NE (10 .mu.M)-displaceable binding is ascertained as a control to rule out .alpha..sub.2 -adrenergic binding. The Bmax and K.sub.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 .mu.M moxonidine to define IR nonspecific binding, or 20 .mu.M cirazoline to define I.sub.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 .sup.125 PIC was used to radiolabel IR sites. A mask of 10 .mu.M norepinephrine was used to rule out any possible .alpha..sub.2 AR binding in each assay even though parent COS-7 cells lacked any .alpha..sub.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 .sup.125 -PIC were well within the high affinity (nM) range.
The following IC.sub.50 values and Hill slopes were obtained in this study: moxonidine, IC.sub.50 =45.1 nM (Hill slope=0.35.+-.0.04); p-iodoclonidine without PKC pretreatment of the membranes, IC.sub.50 =2.3 nM (Hill slope=0.42.+-.0.06); p-iodoclonidine with PKC pretreatment of the membranes, IC.sub.50 =19.0 nM (Hill slope=0.48.+-.0.08). Shallow Hill slopes for [.sup.125 I]p-iodoclonidine have been reported before in studies of the interaction of moxonidine and p-iodoclonidine with the human platelet IR.sub.1 binding site [Piletz and Sletten, (1993)]. Epinephrine failed to displace any of the [.sup.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 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, Calif.), 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.sub.2 -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 .mu.g/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. Pat. 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- <160> NUMBER OF SEQ ID NOS: 22- <210> SEQ ID NO 1<211> LENGTH: 3385<212> TYPE: DNA<213> ORGANISM: Homo sapiens<220> FEATURE:<221> NAME/KEY: CDS<222> LOCATION: (1398)..(3383)- <400> SEQUENCE: 1- gctctagaac tagtggatcc cccgggctgc aggaattcca gtttaatact aa - #ccctaatg 60- tgtgactgcg gtttacaaag agctctgtat cacctgggat agctttcagt ag - #caattcac 120- tacaactggt cctaaaaaat aataacaata ataataataa ttagagaatt aa - #aacccaac 180- agcatgttga atggttaaaa tcacgtaaga actgaaattt ggggtggggg tg - #tcctcaac 240- agctgagctt gtcctagcag tgaaaatgct cgcctccaag cagggctcag aa - #aggtctgg 300- agccctccag gcagagggct gagctcaggg ggctcttgga ggacactcac cc - #catggtcc 360- atgggatgct tctggcttcc ttaaaaacag ttgggcatcc gcattgtata ag - #taggtgga 420- gaccctagtg tggttctttt gaaggatatg ggaagggagg atgacgaact ag - #agaagtgg 480- gaggggacca aaatcactga ggtcccagaa tatcatagat ttgggtatag ga - #ttggggtc 540- actaagaatt gagcaccagg aattccagct tcttcccatt aaagaaactg gg - #actggttt 600- tgccttggag gcctatgtag tgttttctgc ccctgtccca taccaagtct ca - #ttgatatt 660- tctgcagaat atcagatgaa aatctatttc taaagaccat tgggagaatg gg - #tggtggag 720- aaggagttgg agtggggttg gggggcagtt aaaaatgaat aaaaatctct ca - #gctacaga 780- acccaaacat cacttccctc cgcattcaca gcatttccca gcagtcccca ga - #tggttgtt 840- tccgtgggga cacagcagct gcctcatttc ccttcaggcc ccatgggctg ct - #ggtcaacc 900- tcaggatcta ctaaagatga cgcaaatgcc gactgaacaa tctgaaaccc aa - #aggactcg 960- aggagagaca tgttctgctg aggagagaaa ggtgagccaa gggcagggcc ca - #ggtccccc1020- agggggcccc cgagagcccg gacatgcacc ttctggatgt gtttgttcaa gt - #aggactta1080- gagcggaaga agctcccaca ttcagggcat gggtacttct tctccccatc ag - #actccatt1140- ttgtttttgg ggactgccat gtcgcaggag aaagagccat tggcactctg ct - #tctctggc1200- gtcttcaggt cgctggcatc tgagaggtca ccataggagt cagagctctc aa - #tcggatcc1260- tgatgtgagc atttctggcc ttctcggtta cagatactgc agaagttgct gg - #gcccctcg1320- ctgtgcttct tcaggtggtc tgccatgtat gctgcccgca agtacttccc ac - #acacctgg1380#cag acg gtc tcg 1430ca ggc cag gtg gga gcg#Sert Thr Gly Gln Val Gly Ala Gln Thr Val#10- ggt ggc aaa aga agc att gca ggt ctg aca ct - #t gtg agg ccg ctc aga1478Gly Gly Lys Arg Ser Ile Ala Gly Leu Thr Le - #u Val Arg Pro Leu Arg# 25- agt gtg cac ctg ctt gat atg tcc gtt caa gt - #g atc agg cct gga gaa1526Ser Val His Leu Leu Asp Met Ser Val Gln Va - #l Ile Arg Pro Gly Glu# 40- gcc ttt ccc aca gct ctg gca gat gta agg cg - #g aat tcc cca gag aag1574Ala Phe Pro Thr Ala Leu Ala Asp Val Arg Ar - #g Asn Ser Pro Glu Lys# 55- aag ggt ggt gaa gac tcc cgg ctc tca gct gc - #c ccc tgc atc aga ccc1622Lys Gly Gly Glu Asp Ser Arg Leu Ser Ala Al - #a Pro Cys Ile Arg Pro# 75- agc agc tcc cct ccc act gtg gct ccc gca tc - #t gcc tcc ctg ccc cag1670Ser Ser Ser Pro Pro Thr Val Ala Pro Ala Se - #r Ala Ser Leu Pro Gln# 90- ccc atc ctc tct aac caa gga atc atg ttc gt - #t cag gag gag gcc ctg1718Pro Ile Leu Ser Asn Gln Gly Ile Met Phe Va - #l Gln Glu Glu Ala Leu# 105- gcc agc agc ctc tcg tcc act gac agt ctg ac - #t ccc gag cac cag ccc1766Ala Ser Ser Leu Ser Ser Thr Asp Ser Leu Th - #r Pro Glu His Gln Pro# 120- att gcc cag gga tgt tct gat tcc ttg gag tc - #c atc cct gcg gga cag1814Ile Ala Gln Gly Cys Ser Asp Ser Leu Glu Se - #r Ile Pro Ala Gly Gln# 135- gca gct tcc gat gat tta agg gac gtg cca gg - #a gct gtt ggt ggt gca1862Ala Ala Ser Asp Asp Leu Arg Asp Val Pro Gl - #y Ala Val Gly Gly Ala140 1 - #45 1 - #50 1 -#55- agc cca gaa cat gcc gag ccg gag gtc cag gt - #g gtg ccg ggg tct ggc1910Ser Pro Glu His Ala Glu Pro Glu Val Gln Va - #l Val Pro Gly Ser Gly# 170- cag atc atc ttc ctg ccc ttc acc tgc att gg - #c tac acg gcc acc aat1958Gln Ile Ile Phe Leu Pro Phe Thr Cys Ile Gl - #y Tyr Thr Ala Thr Asn# 185- cag gac ttc atc cag cgc ctg agc aca ctg at - #c cgg cag gcc atc gag2006Gln Asp Phe Ile Gln Arg Leu Ser Thr Leu Il - #e Arg Gln Ala Ile Glu# 200- cgg cag ctg cct gcc tgg atc gag gct gcc aa - #c cag cgg gag gag ggc2054Arg Gln Leu Pro Ala Trp Ile Glu Ala Ala As - #n Gln Arg Glu Glu Gly# 215- cag ggt gaa cag ggc gag gag gag gat gag ga - #g gag gaa gaa gag gag2102Gln Gly Glu Gln Gly Glu Glu Glu Asp Glu Gl - #u Glu Glu Glu Glu Glu220 2 - #25 2 - #30 2 -#35- gac gtg gct gag aac cgc tac ttt gaa atg gg - #g ccc cca gac gtg gag2150Asp Val Ala Glu Asn Arg Tyr Phe Glu Met Gl - #y Pro Pro Asp Val Glu# 250- gag gag gag gga gga ggc cag ggg gag gaa ga - #g gag gag gaa gag gag2198Glu Glu Glu Gly Gly Gly Gln Gly Glu Glu Gl - #u Glu Glu Glu Glu Glu# 265- gat gaa gag gcc gag gag gag cgc ctg gct ct - #g gaa tgg gcc ctg ggc2246Asp Glu Glu Ala Glu Glu Glu Arg Leu Ala Le - #u Glu Trp Ala Leu Gly# 280- gcg gac gag gac ttc ctg ctg gag cac atc cg - #c atc ctc aag gtg ctg2294Ala Asp Glu Asp Phe Leu Leu Glu His Ile Ar - #g Ile Leu Lys Val Leu# 295- tgg tgc ttc ctg atc cat gtg cag ggc agt at - #c cgc cag ttc gcc gcc2342Trp Cys Phe Leu Ile His Val Gln Gly Ser Il - #e Arg Gln Phe Ala Ala300 3 - #05 3 - #10 3 -#15- tgc ctt gtg ctc acc gac ttc ggc atc gca gt - #c ttc gag atc ccg cac2390Cys Leu Val Leu Thr Asp Phe Gly Ile Ala Va - #l Phe Glu Ile Pro His# 330- cag gag tct cgg ggc agc agc cag cac atc ct - #c tcc tcc ctg cgc ttt2438Gln Glu Ser Arg Gly Ser Ser Gln His Ile Le - #u Ser Ser Leu Arg Phe# 345- gtc ttt tgc ttc ccg cat ggc gac ctc acc ga - #g ttt ggc ttc ctc atg2486Val Phe Cys Phe Pro His Gly Asp Leu Thr Gl - #u Phe Gly Phe Leu Met# 360- ccg gag ctg tgt ctg gtg ctc aag gta cgg ca - #c agt gag aac acg ctc2534Pro Glu Leu Cys Leu Val Leu Lys Val Arg Hi - #s Ser Glu Asn Thr Leu# 375- ttc att atc tcg gac gcc gcc aac ctg cac ga - #g ttc cac gcg gac ctg2582Phe Ile Ile Ser Asp Ala Ala Asn Leu His Gl - #u Phe His Ala Asp Leu380 3 - #85 3 - #90 3 -#95- cgc tca tgc ttt gca ccc cag cac atg gcc at - #g ctg tgt agc ccc atc2630Arg Ser Cys Phe Ala Pro Gln His Met Ala Me - #t Leu Cys Ser Pro Ile# 410- ctc tac ggc agc cac acc agc ctg cag gag tt - #c ctg cgc cag ctg ctc2678Leu Tyr Gly Ser His Thr Ser Leu Gln Glu Ph - #e Leu Arg Gln Leu Leu# 425- acc ttc tac aag gtg gct ggc ggc tgc cag ga - #g cgc agc cag ggc tgc2726Thr Phe Tyr Lys Val Ala Gly Gly Cys Gln Gl - #u Arg Ser Gln Gly Cys# 440- ttc ccc gtc tac ctg gtc tac agt gac aag cg - #c atg gtg cag acg gcc2774Phe Pro Val Tyr Leu Val Tyr Ser Asp Lys Ar - #g Met Val Gln Thr Ala# 455- gcc ggg gac tac tca ggc aac atc gag tgg gc - #c agc tgc aca ctc tgt2822Ala Gly Asp Tyr Ser Gly Asn Ile Glu Trp Al - #a Ser Cys Thr Leu Cys460 4 - #65 4 - #70 4 -#75- tca gcc gtg cgg cgc tcc tgc tgc gcg ccc tc - #t gag gcc gtc aag tcc2870Ser Ala Val Arg Arg Ser Cys Cys Ala Pro Se - #r Glu Ala Val Lys Ser# 490- gcc gcc atc ccc tac tgg ctg ttg ctc acg cc - #c cag cac ctc aac gtc2918Ala Ala Ile Pro Tyr Trp Leu Leu Leu Thr Pr - #o Gln His Leu Asn Val# 505- atc aag gcc gac ttc aac ccc atg ccc aac cg - #t ggc acc cac aac tgt2966Ile Lys Ala Asp Phe Asn Pro Met Pro Asn Ar - #g Gly Thr His Asn Cys# 520- cgc aac cgc aac agc ttc aag ctc agc cgt gt - #g ccg ctc tcc acc gtg3014Arg Asn Arg Asn Ser Phe Lys Leu Ser Arg Va - #l Pro Leu Ser Thr Val# 535- ctg ctg gac ccc aca cgc agc tgt acc cag cc - #t cgg ggc gcc ttt gct3062Leu Leu Asp Pro Thr Arg Ser Cys Thr Gln Pr - #o Arg Gly Ala Phe Ala540 5 - #45 5 - #50 5 -#55- gat ggc cac gtg cta gag ctg ctc gtg ggg ta - #c cgc ttt gtc act gcc3110Asp Gly His Val Leu Glu Leu Leu Val Gly Ty - #r Arg Phe Val Thr Ala# 570- atc ttc gtg ctg ccc cac gag aag ttc cac tt - #c ctg cgc gtc tac aac3158Ile Phe Val Leu Pro His Glu Lys Phe His Ph - #e Leu Arg Val Tyr Asn# 585- cag ctg cgg gcc tcg ctg cag gac ctg aag ac - #t gtg gtc atc gcc aag3206Gln Leu Arg Ala Ser Leu Gln Asp Leu Lys Th - #r Val Val Ile Ala Lys# 600- acc ccc ggg acg gga ggc agc ccc cag ggc tc - #c ttt gcg gat ggc cag3254Thr Pro Gly Thr Gly Gly Ser Pro Gln Gly Se - #r Phe Ala Asp Gly Gln# 615- cct gcc gag cgc agg gcc agc aat gac cag cg - #t ccc cag gag gtc cca3302Pro Ala Glu Arg Arg Ala Ser Asn Asp Gln Ar - #g Pro Gln Glu Val Pro620 6 - #25 6 - #30 6 -#35- gca gag gct ctg gcc ccg gcc cca gtg gaa gt - #c cca gct cca gcc ccg3350Ala Glu Ala Leu Ala Pro Ala Pro Val Glu Va - #l Pro Ala Pro Ala Pro# 650# 3385at atc aag ctt atc gat acc gtc ga - #c ctGlu Phe Asp Ile Lys Leu Ile Asp Thr Val As - #p# 660- <210> SEQ ID NO 2<211> LENGTH: 1954<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 2- atgacaggcc aggtgggagc gcagacggtc tcgggtggca aaagaagcat tg - #caggtctg 60- acacttgtga ggccgctcag aagtgtgcac ctgcttgata tgtccgttca ag - #tgatcagg 120- cctggagaag cctttcccac agctctggca gatgtaaggc ggaattcccc ag - #agaagaag 180- ggtggtgaag actcccggct ctcagctgcc ccctgcatca gacccagcag ct - #cccctccc 240- actgtggctc ccgcatctgc ctccctgccc cagcccatcc tctctaacca ag - #gaatcatg 300- ttcgttcagg aggaggccct ggccagcagc ctctcgtcca ctgacagtct ga - #ctcccgag 360- caccagccca ttgcccaggg atgttctgat tccttggagt ccatccctgc gg - #gacaggca 420- gcttccgatg atttaaggga cgtgccagga gctgttggtg gtgcaagccc ag - #aacatgcc 480- gagccggagg tccaggtggt gccggggtct ggccagatca tcttcctgcc ct - #tcacctgc 540- attggctaca cggccaccaa tcaggacttc atccagcgcc tgagcacact ga - #tccggcag 600- gccatcgagc ggcagctgcc tgcctggatc gaggctgcca accagcggga gg - #agggccag 660- ggtgaacagg gcgaggagga ggatgaggag gaggaagaag aggaggacgt gg - #ctgagaac 720- cgctactttg aaatggggcc cccagacgtg gaggaggagg agggaggagg cc - #agggggag 780- gaagaggagg aggaagagga ggatgaagag gccgaggagg agcgcctggc tc - #tggaatgg 840- gccctgggcg cggacgagga cttcctgctg gagcacatcc gcatcctcaa gg - #tgctgtgg 900- tgcttcctga tccatgtgca gggcagtatc cgccagttcg ccgcctgcct tg - #tgctcacc 960- gacttcggca tcgcagtctt cgagatcccg caccaggagt ctcggggcag ca - #gccagcac1020- atcctctcct ccctgcgctt tgtcttttgc ttcccgcatg gcgacctcac cg - #agtttggc1080- ttcctcatgc cggagctgtg tctggtgctc aaggtacggc acagtgagaa ca - #cgctcttc1140- attatctcgg acgccgccaa cctgcacgag ttccacgcgg acctgcgctc at - #gctttgca1200- ccccagcaca tggccatgct gtgtagcccc atcctctacg gcagccacac ca - #gcctgcag1260- gagttcctgc gccagctgct caccttctac aaggtggctg gcggctgcca gg - #agcgcagc1320- cagggctgct tccccgtcta cctggtctac agtgacaagc gcatggtgca ga - #cggccgcc1380- ggggactact caggcaacat cgagtgggcc agctgcacac tctgttcagc cg - #tgcggcgc1440- tcctgctgcg cgccctctga ggccgtcaag tccgccgcca tcccctactg gc - #tgttgctc1500- acgccccagc acctcaacgt catcaaggcc gacttcaacc ccatgcccaa cc - #gtggcacc1560- cacaactgtc gcaaccgcaa cagcttcaag ctcagccgtg tgccgctctc ca - #ccgtgctg1620- ctggacccca cacgcagctg tacccagcct cggggcgcct ttgctgatgg cc - #acgtgcta1680- gagctgctcg tggggtaccg ctttgtcact gccatcttcg tgctgcccca cg - #agaagttc1740- cacttcctgc gcgtctacaa ccagctgcgg gcctcgctgc aggacctgaa ga - #ctgtggtc1800- atcgccaaga cccccgggac gggaggcagc ccccagggct cctttgcgga tg - #gccagcct1860- gccgagcgca gggccagcaa tgaccagcgt ccccaggagg tcccagcaga gg - #ctctggcc1920# 1954 tccc agctccagcc ccgg- <210> SEQ ID NO 3<211> LENGTH: 3318<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 3- aattccagtt taatactaac cctaatgtgt gactgcggtt tacaaagagc tc - #tgtatcac 60- ctgggatagc tttcagtagc aattcactac aactggtcct aaaaaataat aa - #caataata 120- ataataatta gagaattaaa acccaacagc atgttgaatg gttaaaatca cg - #taagaact 180- gaaatttggg gtgggggtgt cctcaacagc tgagcttgtc ctagcagtga aa - #atgctcgc 240- ctccaagcag ggctcagaaa ggtctggagc cctccaggca gagggctgag ct - #cagggggc 300- tcttggagga cactcacccc atggtccatg ggatgcttct ggcttcctta aa - #aacagttg 360- ggcatccgca ttgtataagt aggtggagac cctagtgtgg ttcttttgaa gg - #atatggga 420- agggaggatg acgaactaga gaagtgggag gggaccaaaa tcactgaggt cc - #cagaatat 480- catagatttg ggtataggat tggggtcact aagaattgag caccaggaat tc - #cagcttct 540- tcccattaaa gaaactggga ctggttttgc cttggaggcc tatgtagtgt tt - #tctgcccc 600- tgtcccatac caagtctcat tgatatttct gcagaatatc agatgaaaat ct - #atttctaa 660- agaccattgg gagaatgggt ggtggagaag gagttggagt ggggttgggg gg - #cagttaaa 720- aatgaataaa aatctctcag ctacagaacc caaacatcac ttccctccgc at - #tcacagca 780- tttcccagca gtccccagat ggttgtttcc gtggggacac agcagctgcc tc - #atttccct 840- tcaggcccca tgggctgctg gtcaacctca ggatctacta aagatgacgc aa - #atgccgac 900- tgaacaatct gaaacccaaa ggactcgagg agagacatgt tctgctgagg ag - #agaaaggt 960- gagccaaggg cagggcccag gtcccccagg gggcccccga gagcccggac at - #gcaccttc1020- tggatgtgtt tgttcaagta ggacttagag cggaagaagc tcccacattc ag - #ggcatggg1080- tacttcttct ccccatcaga ctccattttg tttttgggga ctgccatgtc gc - #aggagaaa1140- gagccattgg cactctgctt ctctggcgtc ttcaggtcgc tggcatctga ga - #ggtcacca1200- taggagtcag agctctcaat cggatcctga tgtgagcatt tctggccttc tc - #ggttacag1260- atactgcaga agttgctggg cccctcgctg tgcttcttca ggtggtctgc ca - #tgtatgct1320- gcccgcaagt acttcccaca cacctggcag ggcaccttgt cttcatgaca gg - #ccaggtgg1380- gagcgcagac ggtctcgggt ggcaaaagaa gcattgcagg tctgacactt gt - #gaggccgc1440- tcagaagtgt gcacctgctt gatatgtccg ttcaagtgat caggcctgga ga - #agcctttc1500- ccacagctct ggcagatgta aggcggaatt ccccagagaa gaagggtggt ga - #agactccc1560- ggctctcagc tgccccctgc atcagaccca gcagctcccc tcccactgtg gc - #tcccgcat1620- ctgcctccct gccccagccc atcctctcta accaaggaat catgttcgtt ca - #ggaggagg1680- ccctggccag cagcctctcg tccactgaca gtctgactcc cgagcaccag cc - #cattgccc1740- agggatgttc tgattccttg gagtccatcc ctgcgggaca ggcagcttcc ga - #tgatttaa1800- gggacgtgcc aggagctgtt ggtggtgcaa gcccagaaca tgccgagccg ga - #ggtccagg1860- tggtgccggg gtctggccag atcatcttcc tgcccttcac ctgcattggc ta - #cacggcca1920- ccaatcagga cttcatccag cgcctgagca cactgatccg gcaggccatc ga - #gcggcagc1980- tgcctgcctg gatcgaggct gccaaccagc gggaggaggg ccagggtgaa ca - #gggcgagg2040- aggaggatga ggaggaggaa gaagaggagg acgtggctga gaaccgctac tt - #tgaaatgg2100- ggcccccaga cgtggaggag gaggagggag gaggccaggg ggaggaagag ga - #ggaggaag2160- aggaggatga agaggccgag gaggagcgcc tggctctgga atgggccctg gg - #cgcggacg2220- aggacttcct gctggagcac atccgcatcc tcaaggtgct gtggtgcttc ct - #gatccatg2280- tgcagggcag tatccgccag ttcgccgcct gccttgtgct caccgacttc gg - #catcgcag2340- tcttcgagat cccgcaccag gagtctcggg gcagcagcca gcacatcctc tc - #ctccctgc2400- gctttgtctt ttgcttcccg catggcgacc tcaccgagtt tggcttcctc at - #gccggagc2460- tgtgtctggt gctcaaggta cggcacagtg agaacacgct cttcattatc tc - #ggacgccg2520- ccaacctgca cgagttccac gcggacctgc gctcatgctt tgcaccccag ca - #catggcca2580- tgctgtgtag ccccatcctc tacggcagcc acaccagcct gcaggagttc ct - #gcgccagc2640- tgctcacctt ctacaaggtg gctggcggct gccaggagcg cagccagggc tg - #cttccccg2700- tctacctggt ctacagtgac aagcgcatgg tgcagacggc cgccggggac ta - #ctcaggca2760- acatcgagtg ggccagctgc acactctgtt cagccgtgcg gcgctcctgc tg - #cgcgccct2820- ctgaggccgt caagtccgcc gccatcccct actggctgtt gctcacgccc ca - #gcacctca2880- acgtcatcaa ggccgacttc aaccccatgc ccaaccgtgg cacccacaac tg - #tcgcaacc2940- gcaacagctt caagctcagc cgtgtgccgc tctccaccgt gctgctggac cc - #cacacgca3000- gctgtaccca gcctcggggc gcctttgctg atggccacgt gctagagctg ct - #cgtggggt3060- accgctttgt cactgccatc ttcgtgctgc cccacgagaa gttccacttc ct - #gcgcgtct3120- acaaccagct gcgggcctcg ctgcaggacc tgaagactgt ggtcatcgcc aa - #gacccccg3180- ggacgggagg cagcccccag ggctcctttg cggatggcca gcctgccgag cg - #cagggcca3240- gcaatgacca gcgtccccag gaggtcccag cagaggctct ggccccggcc cc - #agtggaag3300#3318 gg- <210> SEQ ID NO 4<211> LENGTH: 1171<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 4- gaggaggagg aagaggagga tgaagaggcc gaggaggagc gcctggctct gg - #aatgggcc 60- ctgggcgcgg acgaggactt cctgctggag cacatccgca tcctcaaggt gc - #tgtggtgc 120- ttcctgatcc atgtgcaggg cagtatccgc cagttcgccg cctgccttgt gc - #tcaccgac 180- ttcggcatcg cagtcttcga gatcccgcac caggagtctc ggggcagcag cc - #agcacatc 240- ctctcctccc tgcgctttgt cttttgcttc ccgcatggcg acctcaccga gt - #ttggcttc 300- ctcatgccgg agctgtgtct ggtgctcaag gtacggcaca gtgagaacac gc - #tcttcatt 360- atctcggacg ccgccaacct gcacgagttc cacgcggacc tgcgctcatg ct - #ttgcaccc 420- cagcacatgg ccatgctgtg tagccccatc ctctacggca gccacaccag cc - #tgcaggag 480- ttcctgcgcc agctgctcac cttctacaag gtggctggcg gctgccagga gc - #gcagccag 540- ggctgcttcc ccgtctacct ggtctacagt gacaagcgca tggtgcagac gg - #ccgccggg 600- gactactcag gcaacatcga gtgggccagc tgcacactct gttcagccgt gc - #ggcgctcc 660- tgctgcgcgc cctctgaggc cgtcaagtcc gccgccatcc cctactggct gt - #tgctcacg 720- ccccagcacc tcaacgtcat caaggccgac ttcaacccca tgcccaaccg tg - #gcacccac 780- aactgtcgca accgcaacag cttcaagctc agccgtgtgc cgctctccac cg - #tgctgctg 840- gaccccacac gcagctgtac ccagcctcgg ggcgcctttg ctgatggcca cg - #tgctagag 900- ctgctcgtgg ggtaccgctt tgtcactgcc atcttcgtgc tgccccacga ga - #agttccac 960- ttcctgcgcg tctacaacca gctgcgggcc tcgctgcagg acctgaagac tg - #tggtcatc1020- gccaagaccc ccgggacggg aggcagcccc cagggctcct ttgcggatgg cc - #agcctgcc1080- gagcgcaggg ccagcaatga ccagcgtccc caggaggtcc cagcagaggc tc - #tggccccg1140# 1171 cagc tccagccccg g- <210> SEQ ID NO 5<211> LENGTH: 651<212> TYPE: PRT<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 5- Met Thr Gly Gln Val Gly Ala Gln Thr Val Se - #r Gly Gly Lys Arg Ser# 15- Ile Ala Gly Leu Thr Leu Val Arg Pro Leu Ar - #g Ser Val His Leu Leu# 30- Asp Met Ser Val Gln Val Ile Arg Pro Gly Gl - #u Ala Phe Pro Thr Ala# 45- Leu Ala Asp Val Arg Arg Asn Ser Pro Glu Ly - #s Lys Gly Gly Glu Asp# 60- Ser Arg Leu Ser Ala Ala Pro Cys Ile Arg Pr - #o Ser Ser Ser Pro Pro# 80- Thr Val Ala Pro Ala Ser Ala Ser Leu Pro Gl - #n Pro Ile Leu Ser Asn# 95- Gln Gly Ile Met Phe Val Gln Glu Glu Ala Le - #u Ala Ser Ser Leu Ser# 110- Ser Thr Asp Ser Leu Thr Pro Glu His Gln Pr - #o Ile Ala Gln Gly Cys# 125- Ser Asp Ser Leu Glu Ser Ile Pro Ala Gly Gl - #n Ala Ala Ser Asp Asp# 140- Leu Arg Asp Val Pro Gly Ala Val Gly Gly Al - #a Ser Pro Glu His Ala145 1 - #50 1 - #55 1 -#60- Glu Pro Glu Val Gln Val Val Pro Gly Ser Gl - #y Gln Ile Ile Phe Leu# 175- Pro Phe Thr Cys Ile Gly Tyr Thr Ala Thr As - #n Gln Asp Phe Ile Gln# 190- Arg Leu Ser Thr Leu Ile Arg Gln Ala Ile Gl - #u Arg Gln Leu Pro Ala# 205- Trp Ile Glu Ala Ala Asn Gln Arg Glu Glu Gl - #y Gln Gly Glu Gln Gly# 220- Glu Glu Glu Asp Glu Glu Glu Glu Glu Glu Gl - #u Asp Val Ala Glu Asn225 2 - #30 2 - #35 2 -#40- Arg Tyr Phe Glu Met Gly Pro Pro Asp Val Gl - #u Glu Glu Glu Gly Gly# 255- Gly Gln Gly Glu Glu Glu Glu Glu Glu Glu Gl - #u Asp Glu Glu Ala Glu# 270- Glu Glu Arg Leu Ala Leu Glu Trp Ala Leu Gl - #y Ala Asp Glu Asp Phe# 285- Leu Leu Glu His Ile Arg Ile Leu Lys Val Le - #u Trp Cys Phe Leu Ile# 300- His Val Gln Gly Ser Ile Arg Gln Phe Ala Al - #a Cys Leu Val Leu Thr305 3 - #10 3 - #15 3 -#20- Asp Phe Gly Ile Ala Val Phe Glu Ile Pro Hi - #s Gln Glu Ser Arg Gly# 335- Ser Ser Gln His Ile Leu Ser Ser Leu Arg Ph - #e Val Phe Cys Phe Pro# 350- His Gly Asp Leu Thr Glu Phe Gly Phe Leu Me - #t Pro Glu Leu Cys Leu# 365- Val Leu Lys Val Arg His Ser Glu Asn Thr Le - #u Phe Ile Ile Ser Asp# 380- Ala Ala Asn Leu His Glu Phe His Ala Asp Le - #u Arg Ser Cys Phe Ala385 3 - #90 3 - #95 4 -#00- Pro Gln His Met Ala Met Leu Cys Ser Pro Il - #e Leu Tyr Gly Ser His# 415- Thr Ser Leu Gln Glu Phe Leu Arg Gln Leu Le - #u Thr Phe Tyr Lys Val# 430- Ala Gly Gly Cys Gln Glu Arg Ser Gln Gly Cy - #s Phe Pro Val Tyr Leu# 445- Val Tyr Ser Asp Lys Arg Met Val Gln Thr Al - #a Ala Gly Asp Tyr Ser# 460- Gly Asn Ile Glu Trp Ala Ser Cys Thr Leu Cy - #s Ser Ala Val Arg Arg465 4 - #70 4 - #75 4 -#80- Ser Cys Cys Ala Pro Ser Glu Ala Val Lys Se - #r Ala Ala Ile Pro Tyr# 495- Trp Leu Leu Leu Thr Pro Gln His Leu Asn Va - #l Ile Lys Ala Asp Phe# 510- Asn Pro Met Pro Asn Arg Gly Thr His Asn Cy - #s Arg Asn Arg Asn Ser# 525- Phe Lys Leu Ser Arg Val Pro Leu Ser Thr Va - #l Leu Leu Asp Pro Thr# 540- Arg Ser Cys Thr Gln Pro Arg Gly Ala Phe Al - #a Asp Gly His Val Leu545 5 - #50 5 - #55 5 -#60- Glu Leu Leu Val Gly Tyr Arg Phe Val Thr Al - #a Ile Phe Val Leu Pro# 575- His Glu Lys Phe His Phe Leu Arg Val Tyr As - #n Gln Leu Arg Ala Ser# 590- Leu Gln Asp Leu Lys Thr Val Val Ile Ala Ly - #s Thr Pro Gly Thr Gly# 605- Gly Ser Pro Gln Gly Ser Phe Ala Asp Gly Gl - #n Pro Ala Glu Arg Arg# 620- Ala Ser Asn Asp Gln Arg Pro Gln Glu Val Pr - #o Ala Glu Ala Leu Ala625 6 - #30 6 - #35 6 -#40- Pro Ala Pro Val Glu Val Pro Ala Pro Ala Pr - #o# 650- <210> SEQ ID NO 6<211> LENGTH: 390<212> TYPE: PRT<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 6- Glu Glu Glu Glu Glu Glu Asp Glu Glu Ala Gl - #u Glu Glu Arg Leu Ala# 15- Leu Glu Trp Ala Leu Gly Ala Asp Glu Asp Ph - #e Leu Leu Glu His Ile# 30- Arg Ile Leu Lys Val Leu Trp Cys Phe Leu Il - #e His Val Gln Gly Ser# 45- Ile Arg Gln Phe Ala Ala Cys Leu Val Leu Th - #r Asp Phe Gly Ile Ala# 60- Val Phe Glu Ile Pro His Gln Glu Ser Arg Gl - #y Ser Ser Gln His Ile# 80- Leu Ser Ser Leu Arg Phe Val Phe Cys Phe Pr - #o His Gly Asp Leu Thr# 95- Glu Phe Gly Phe Leu Met Pro Glu Leu Cys Le - #u Val Leu Lys Val Arg# 110- His Ser Glu Asn Thr Leu Phe Ile Ile Ser As - #p Ala Ala Asn Leu His# 125- Glu Phe His Ala Asp Leu Arg Ser Cys Phe Al - #a Pro Gln His Met Ala# 140- Met Leu Cys Ser Pro Ile Leu Tyr Gly Ser Hi - #s Thr Ser Leu Gln Glu145 1 - #50 1 - #55 1 -#60- Phe Leu Arg Gln Leu Leu Thr Phe Tyr Lys Va - #l Ala Gly Gly Cys Gln# 175- Glu Arg Ser Gln Gly Cys Phe Pro Val Tyr Le - #u Val Tyr Ser Asp Lys# 190- Arg Met Val Gln Thr Ala Ala Gly Asp Tyr Se - #r Gly Asn Ile Glu Trp# 205- Ala Ser Cys Thr Leu Cys Ser Ala Val Arg Ar - #g Ser Cys Cys Ala Pro# 220- Ser Glu Ala Val Lys Ser Ala Ala Ile Pro Ty - #r Trp Leu Leu Leu Thr225 2 - #30 2 - #35 2 -#40- Pro Gln His Leu Asn Val Ile Lys Ala Asp Ph - #e Asn Pro Met Pro Asn# 255- Arg Gly Thr His Asn Cys Arg Asn Arg Asn Se - #r Phe Lys Leu Ser Arg# 270- Val Pro Leu Ser Thr Val Leu Leu Asp Pro Th - #r Arg Ser Cys Thr Gln# 285- Pro Arg Gly Ala Phe Ala Asp Gly His Val Le - #u Glu Leu Leu Val Gly# 300- Tyr Arg Phe Val Thr Ala Ile Phe Val Leu Pr - #o His Glu Lys Phe His305 3 - #10 3 - #15 3 -#20- Phe Leu Arg Val Tyr Asn Gln Leu Arg Ala Se - #r Leu Gln Asp Leu Lys# 335- Thr Val Val Ile Ala Lys Thr Pro Gly Thr Gl - #y Gly Ser Pro Gln Gly# 350- Ser Phe Ala Asp Gly Gln Pro Ala Glu Arg Ar - #g Ala Ser Asn Asp Gln# 365- Arg Pro Gln Glu Val Pro Ala Glu Ala Leu Al - #a Pro Ala Pro Val Glu# 380- Val Pro Ala Pro Ala Pro385 3 - #90- <210> SEQ ID NO 7<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 7# 20 tgct- <210> SEQ ID NO 8<211> LENGTH: 18<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 8# 18 ct- <210> SEQ ID NO 9<211> LENGTH: 18<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 9# 18 gg- <210> SEQ ID NO 10<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 10# 20 gaaa- <210> SEQ ID NO 11<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 11# 20 ggag- <210> SEQ ID NO 12<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 12# 20 gttc- <210> SEQ ID NO 13<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 13# 20 ccaa- <210> SEQ ID NO 14<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 14# 20 acca- <210> SEQ ID NO 15<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 15# 20 cttc- <210> SEQ ID NO 16<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 16# 20 gcag- <210> SEQ ID NO 17<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 17# 20 acct- <210> SEQ ID NO 18<211> LENGTH: 18<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 18# 18 ct- <210> SEQ ID NO 19<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 19# 20 gggc- <210> SEQ ID NO 20<211> LENGTH: 20<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 20# 20 caag- <210> SEQ ID NO 21<211> LENGTH: 15202<212> TYPE: DNA<213> ORGANISM: Homo sapiens- <400> SEQUENCE: 21- gatccgagct caattaaccc tcactaaagg gagtcgactc gatccttaaa at - #attcatat 60- ctcctggaca acctgtggcc atagtgcctg actgtaaacc caaagggttt gc - #ctttgcca 120- gtgtagccca gcctggtgtc tgctgcccct cgcggtgtct gtgcacctgc ca - #cgatgctg 180- accagacacc cttaaccagg ttcacccatc gcctgggcct ggagcagtcc cc - #ctgatgct 240- ctgattggtc cttggacctt ctgttctccc aaaatcccag gtcagaaaat ac - #ctggaagt 300- ctatttgtgt cccacctccc tctttgtggc cgcaagtgcc ccttcctcca ca - #cagtcaca 360- agaccatgag atgccatctc ctcccctcct gggctgcaga ctttgggaag ct - #cccaggcc 420- acagaggtgt cagctcctgt ccaggccctt gggaccttcc ctcattcaac ca - #ccctaccc 480- aaccccccac tgcctgccag ccaccactcc ctcccacatt tgcaggcggg gg - #ccctgccc 540- tctcctgccg ctggttcccc tacccaggag gctctcccat cgctcttttg ag - #agtctgcc 600- tcccacctct aactgggggc ttagttcaag ttgccccctt accctagtcc ca - #gctgccca 660- agagcttgct gcctcctgtt cttggtgagg gactccagag acagatgtga ga - #cctccctg 720- gacccctcca aggcattccc aggtcacttc catgagtagt gaagaaccgc ct - #ctgagcag 780- gctgagcctc cctcagccta tggtgtcctc acgtggcttg gcccacagca gg - #tgctcacg 840- cctcctcctc agcagagcct accatcctcc tgccatgctc accagtcccc at - #gctgatag 900- ccatcaccag tccccatgct gatagccatc accagtcccc atgctgatag cc - #actttctg 960- gatgctctag gtctgtctgg atgacacagt gaccacagag aaggagctgg ac - #actgtgga1020- agtgctgaaa gcaattcaga aagccaagga ggtcaagtcc aaactgagca ac - #ccagagaa1080- gaaggtgggt ttgtgtggca ggtgggaggg cagtggtgca gagccagccg gg - #ataggagc1140- cagttcgggg ggcttgggcc atgggactgc tcagggctgc cgagtcccag ct - #gcgcccct1200- ccctggctgc atgacctcgg gcaagtcgcg gcctctctgt tctctgtggg gt - #ggggacag1260- tggtagttcc tgctctaagg atatgatgag accatcttta ccacccagtt gg - #tgggaacc1320- gttgcgctcc ctcctcacac ccctggcctt ggggagctct gtgcttcctc tt - #ctctcccg1380- ggctgactca agcactcgtc ctcagggtgg tgaagactcc cggctctcag ct - #gccccctg1440- catcagaccc agcagctccc ctcccactgt ggctcccgca tctgcctccc tg - #ccccagcc1500- catcctctct aaccaaggta atcgtgtatg tatcttgctt ctagtggagc ca - #cacagccc1560- tgcctgggcc ccctggctgg gctggggttg ggggagaggt gccagcacct gc - #ttccaaca1620- gggtcagaca cagggagggc agtgccttct gcaggctggt cctcgcgggg gg - #acacatgg1680- caggggtgcc tggcctgatg ccagctgttg cttgcttggt gaggactccc aa - #ttgctctg1740- atgcccacat ccagctcctc taggagaccg cagggtgtct gacaggccct ga - #ggctgccc1800- tctgaacagg ctcggggctg ttggctcatg ggacccattc cctcaccggc ag - #cacaagca1860- ggttggctcc tggttacagg aagccgggct tgtgacttta ctgtctggag cc - #cgaatccc1920- tgtgcaggga aaagcttgct tttatcactg cctcatctct gtggggtgac cc - #agccccag1980- aacaccatgt ttgtggggcc aagatgggcc atctctgtcc ctgtggaccc at - #ggaagacc2040- aggcccattc gtctgcccac tatcttagcg ttttcaaagg gctttcacct ct - #gaacccag2100- gcatcctcgg agatgagtga gtgaagcagg tctcatgagc gtgtctgctg gc - #ccggcccc2160- cacggaagag gggagggtgt gccgtcccga gtggagccga ggctcgggac ac - #gcaggaaa2220- ggacgccgcc tgcccgggct cctggagacg cagaacttgg tgtgaggtct tg - #ggaaaaca2280- gttcaacccg atgttttaag agccagaaaa acattcccac cccttgacct gg - #taacccca2340- ctggtgggga ttttctctta gagggataag ataccgggaa ggggaggtga aa - #tgctcacc2400- actgccaaaa cacgggctgc aactgcaaca tcggaggatg agagggagag tc - #ggctgtgg2460- tgcagaatgc tcagcagccc tcccagcagg gacaggaaga ctgggcagga ag - #aggggaga2520- agcattcaag ttaaggcaaa aggcccaacg cagagcagca cactgaggtc ac - #acctgtga2580- gatgtggaag agaattcctg agcgtggagc gatggggtta ggtgccagga tg - #attgccca2640- ttttgcttct gtcagactct tgactaagga tttctggttg cattttatta ca - #taaaagcc2700- agggaggtta tatcacggtg agaaagcttc cctgacgccg cctcctgtag cg - #cagccaag2760- cgagcctgtg gaggtaccat atgactgtag gcctctgggg acagggagct gc - #atctgctt2820- ctcaaggcca gggacacagc catttctgcc agcatctgtt gatcagtgag tg - #agtgagtg2880- ggcaggtaga gcaggagcca gtgaagagca ggccctggat gggtggggat gc - #accatgtc2940- cccaggctgc agctgcaggc agccccccac attgtcggag aagcctctgc ac - #cagctcag3000- ccccctcctc actccccttg tgccctgggg acactctgca gaggggcact ct - #gcagtctg3060- tccccgccat cgctggactt ctggacatgg cctccagatt tgcacctctt aa - #ataaatct3120- gcagtggatg tctttgtgtg cacctctctt tccttttggt gagaaacagc aa - #agatcgga3180- cccctaagga ctctcctgat gtctccgctc tatccgctga gtgccctttc tg - #accacttg3240- tttgtacagg ccacggtcca ggacgggagc agatagactg tccctgtccc tg - #tccacatt3300- tccttggtcc aaacagggct tgtgggaggt agtggcaaaa ggtgttggtc tt - #tttctcac3360- tgatttggag gcctccccgt gtgttttttc agccgcgtgt tcctgggtct tg - #cctggatg3420- gacagggttt tttagcgcgt gggagcagct ttgctgacca tgcctgttgc tt - #ccagcctg3480- attcccgaga agggagcgtg cttgcgaagg aactggcact cgggcctgcc tg - #aagggggc3540- gctgtccaga cacacccagc ctcccgtcgt ggcaggcgct gtcggagcca tg - #gatgattg3600- tgaccaatag gggtggtcgc cagagttgat tgtccagcca ggcccagggg ct - #gagaggag3660- gctgtgtgga gaggtggtta ggagccaggg ctcggtcagc tgagttcgca tg - #ccagcttc3720- ctagctgtgg gacctcaagc aacttgtagc ccctctgaag ctgttttctc aa - #ctgtgaag3780- tggacgcacc ctacttcatt gattctaaga ggcacgcatt tccaccttgt ga - #cttctctg3840- aaactgaggt gcgtctttca gtcagtggcg tctcatagtc gctgtcagcc ag - #ctggtatt3900- cgagatggag tcgtggaaaa cccgtggaca ccttccgcta ggaccaagat gg - #cgccacct3960- gccgcatctt agatttgatg aaatgtggta aataacgaga ggcatgcatg ag - #cgaatgct4020- ggggaggcgc ttggcactac ccagagctcc acagaggtgg tcgatgaggg ct - #gccctttc4080- ccacatcctt agtagggggt tcaacatgac ccagactgtg cccctgggga gc - #ttggagcc4140- atgcgggagg atgagccatg tgctggagga gaacagggta ggatggtgtg gg - #gcttttgt4200- agactgtcta gagcagagaa ggtctgcagt ggaggtggtg tctgaggtga at - #ctcgaagg4260- tgaataggag ttgaacgtta gcaggcagag ggtggattgc aggagagcag cg - #gcctgggc4320- aggtgcccag cgtggcccat cagggtgctt catgcatggc tgtgtgcttg cc - #atccttcc4380- tgcctgccta ccccctgctg cttcgcttca tgggggcgtt tgagcttggg cc - #cacctgcc4440- tgcctcgctt gtgggcagag gacccaggct gtgtgagttg tcctgtcccg gg - #gagcagct4500- gagcttgtcc gggttcctcg acctgtgggg cttcagagga cttcgggtca tt - #tcaatggg4560- ctgtggcgat gctggctgtg gaggtagcct agggctcctg tagccttcag tg - #agactggc4620- ggcccgatgc ccagtgttca ccctgctggc ggcagtcagg aacatgttca ca - #aagcttta4680- cttcaagtgg tctagaggtg atctgaggtg gagtaacagg tccagatagg ct - #acgttcat4740- aaaacagctt cagcggggtt taggaacact gtgcatttac gggacgcagt gg - #gtcagagt4800- gctgctgtcc gtgggaggtg gccccagggc aggtcagtgg gcacgtcctg tg - #gtaagtgg4860- gactgtggat gtgggctcag gctggactca gcagccctgc tggataccaa gg - #cctgcaag4920- ggctggcccc ctggtgaatt gtcccgtgcc ctgtgtatct atgagtcctg ca - #gagatgac4980- aaatcagggg acggggtcat gtctagtcac cgtctgggaa aatgctccag ga - #gtgaacac5040- atttcaggct cttgatggat gtacctccaa actcttctct ggatgggtgg gc - #cagcttgc5100- atgcctgtgc cggcctctgc ccagcgaggt cagggccagg ccacacagtc ag - #tctgactt5160- tggcagaagt tgagaggcaa cacttgtctc ttgtttcagc ttgcctttct tt - #gtgtactt5220- ctgagagcga gcattctttt catgttctat ccgctggccg ttcttctgcg ga - #atgtctgt5280- tcacgtcctt tgcagtctgt taatgaggtt tccaaccttc cctcattttt gt - #aatctgta5340- agaacttttt ccagactagc gatataaatc cttgtcaaat attgcaaaca ct - #tttctcat5400- ttcatctggt tttaatctat cctggttttt aaaaaatgtg tctgtggaag tt - #taattttt5460- atgtagtcac atctcagttt ttttccattg catttattct cagaatgctt ct - #ccctgccc5520- tgagattaga taagcagtca tttgttcttt cttgagttat tttgagattt ca - #gttttaac5580- attttcttct ataatccatg tggctgggtt ttgggatctg gctaaccccc gc - #catgccag5640- tagcctgagg ggcccagccc cacttgttga acagccgctc tccccgcccc ac - #ccaccctg5700- cctgcctgcc cacccgccct ggtctctcca ggaatcatgt tcgttcagga gg - #aggccctg5760- gccagcagcc tctcgtccac tgacagtctg actcccgagc accagcccat tg - #cccaggga5820- tgttctgatt ccttggagtc catccctgcg ggacaggtaa tgccctcttc cc - #gcttctgg5880- ggaccataca tctgtgggtg gactcttctg cttggggttg tgtgcagtag ga - #agtggcct5940- agctggagct gaggcagatg cttccagggt ttggcgtcct ctgctttgcg cc - #acggtctt6000- tctcttggac ctgtctctgg ttgagtgtct tcctgacaaa cacagtggtt aa - #gggtttat6060- tttcagcctc cctccttccc ttccccaccc accttggttg atgggaacag gc - #agttctct6120- gtcactgggc ccagggcacg aggggggcag gtggagaggg tggcccttga cc - #ctgtgagc6180- aggcttccct ggggaaggca tttcaaaaga ccctcgtgca ggggcttgtt tg - #ggtttctt6240- ctctgtttcc tggcacccct ggagccactc ggcgcctttc cgcatgtcac cc - #tggtggtc6300- tgggaaacag tctcactctg gcgcctcctc tgtggttgtt actgagagtt ct - #ggggcccc6360- ttcctttgtc ctgaggaaag acaggaggaa agcaagggtg cttgctgtgt gc - #ttcgcaaa6420- tgtgcttggt gcctgggcct ccctccagcc ccatctctgc agcagcacaa gg - #ttatggcc6480- ttgtgacact gggacagttt gcagagtcct tgtctgtcct cagtactcca ca - #gtattctg6540- ccatcaccct ttccagggtc acacagcaag agattcccaa gccctaggta tt - #ccccagtg6600- cacagagacc attgggaggg acttgccagg gctgtgtcca ctgctggcca gt - #tagggtcg6660- gaccaaattt gtagactgtc tacctggacc cttgcgtggc acaaggagca gt - #cagatgct6720- ggatccctgg agagtggcga gaggctctgg ccttaggttg cgagtgggaa tc - #ccagccct6780- gctgtgtgct ggtgggataa ccaagtgggt ctctgccctt gggtcccaga gt - #gggcccca6840- gggtcccaga gtgggctcca gggtacagcg tggggatggg gagcctcctc ag - #ggcggtga6900- tggagggcag aatgcccagc tcagggtctg gcaaccagta aatggctggg gc - #tggctgca6960- gtaggtgggg actgactgtg tttctttctc catcaggcag cttccgatga tt - #taagggac7020- gtgccaggag ctgttggtgg tgcaaggtaa ggaagaggtt ggaaagggac ct - #gggcctgg7080- ccacacagcc ttatgcacac acactgctgt gggccagggg tggccagtca gg - #ttttttta7140- aaaatccgtt cacagaaggc ctatagaact atttcttcct ctaaagagac ac - #agatgaga7200- tggacttttc aatctgtttc caaattctaa tacctaaact ctgctcagca ca - #tgttgccc7260- tacaccaggg gttggcaaat caaggcctgt gtgtggccca cagcctggga gc - #taagaatg7320- acagttacat tcttttttct ttttttgaga ctgagtctcg ctctgtcgcc ca - #ggctggag7380- tgcagtggcg tgttcttggc tcactgcaac ccccgcctcc cagattaatg ca - #attttcct7440- gtctcagcct cagccttctg agtagcccgg accacaggcg cacgccacca cg - #cccaacta7500- attttttata tttttagtag agacagagat tcaccatgtg gcctagctgg tc - #tcgaactc7560- ctgaactcca gtgatccacc aacctcggct tcctaaagta ctggaattac ag - #gcatgagc7620- caccgcgcct ggctagaata acagttactt tttttttctt tgagactgag tc - #ttgctttg7680- tcacccaggc tggagtgcag tggcacgatc tcagctcgct gcaacctccg cc - #tcccgggt7740- tcaagcgatt cttctgcctc agccacccaa ggtgcccgcc accacacctg gc - #taattttt7800- ctgtttttag tagggacagg atttcgccat gttggacagt tacattctta aa - #gggctgct7860- gaagatcgta tggacatggt agcccataaa tcccaaaatg tgtactctga cc - #ctttacag7920- aagcttacta actcccactc tacatgtgag ggctgcggtg gccaagaaga gc - #tggaattt7980- aagtgtgaag gtcctaagac ctgccccagc ccacttccct gccccggagg cc - #accagggg8040- tgacaagtag attcatgccc tggagtgttc cttctctccg gggcttatgg ca - #gcaactga8100- atgacttaga agtccatggg agtgctttct gttgtgggaa ctcgtgtggt ct - #gggcatag8160- ctgtgccagg cacctatggt ccaagcccct agaagcatag actctgacca aa - #ctggcgac8220- ccagccttcc agcaggcagc actggctccc accagggccc tcatcctggg aa - #ctgacttg8280- gccatgtggg aggcttggga gacccatggg ttggtttctc agggtcaggg tg - #tagcagtg8340- ggctccagat gtggcaggtg ggaggtggga ggggcccctc ccagcatgcc ac - #tgacctgg8400- cctctccctg cacagcccag aacatgccga gccggaggtc caggtggtgc cg - #gggtctgg8460- ccagatcatc ttcctgccct tcacctgcat tggctacacg gccaccaatc ag - #gacttcat8520- ccagcgcctg agcacactga tccggcaggc catcgagcgg cagctgcctg cc - #tggatcga8580- ggctgccaac cagcgggagg agggccaggg tgaacagggc gaggaggagg at - #gaggagga8640- ggaagaagag gaggacgtgg ctgagaaccg ctactttgaa atggggcccc ca - #gacgtgga8700- ggaggaggag ggaggaggcc agggggagga agaggaggag gaagaggagg at - #gaagaggc8760- cgaggaggag cgcctggctc tggaatgggc cctgggcgcg gacgaggact tc - #ctgctgga8820- gcacatccgc atcctcaagg tgctgtggtg cttcctgatc catgtgcagg gc - #agtatccg8880- ccagttcgcc gcctgccttg tgctcaccga cttcggcatc gcagtcttcg ag - #atcccgca8940- ccaggagtct cggggcagca gccagcacat cctctcctcc ctgcgctttg tc - #ttttgctt9000- cccgcatggc gacctcaccg agtttggctt cctcatgccg gagctgtgtc tg - #gtgctcaa9060- ggtacggcac agtgagaaca cgctcttcat tatctcggac gccgccaacc tg - #cacgagtt9120- ccacgcggac ctgcgctcat gctttgcacc ccagcacatg gccatgctgt gt - #agccccat9180- cctctacggc agccacacca gcctgcagga gttcctgcgc cagctgctca cc - #ttctacaa9240- ggtggctggc ggctgccagg agcgcagcca gggctgcttc cccgtctacc tg - #gtctacag9300- tgacaagcgc atggtgcaga cggccgccgg ggactactca ggcaacatcg ag - #tgggccag9360- ctgcacactc tgttcagccg tgcggcgctc ctgctgcgcg ccctctgagg cc - #gtcaagtc9420- cgccgccatc ccctactggc tgttgctcac gccccagcac ctcaacgtca tc - #aaggccga9480- cttcaacccc atgcccaacc gtggcaccca caactgtcgc aaccgcaaca gc - #ttcaagct9540- cagccgtgtg ccgctctcca ccgtgctgct ggaccccaca cgcagctgta cc - #cagcctcg9600- gggcgccttt gctgatggcc acgtgctaga gctgctcgtg gggtaccgct tt - #gtcactgc9660- catcttcgtg ctgccccacg agaagttcca cttcctgcgc gtctacaacc ag - #ctgcgggc9720- ctcgctgcag gacctgaaga ctgtggtcat cgccaagacc cccgggacgg ga - #ggcagccc9780- ccagggctcc tttgcggatg gccagcctgc cgagcgcagg gccaggtgag at - #caagcaca9840- gctctcaggg gccccggggg cacgggtctg gcatgtgtgt gatctcagca tc - #tgcggcta9900- gtgtgggctg ggagttgctg cgagagctgg gccccctccc ccctgcccct cg - #cccccccc9960- gggcctccct ctacatcacc accccaggtt tggtgccagg ctgctcctta tc - #tcagtgct10020- gtagaagaag cccaggaaag ctgtcctctc acaaaatggg ttggcccagc ct - #cttgccac10080- ccatgaaggg caggccaagg gggctgcccc acctttgcct gcccagtggg ag - #agcaacag10140- gctgcagcac accgaggcca ggagagctgt caccctggct gctgtgctcc tc - #tgggccca10200- agcatggcct ctgggcacta cctcctccag ggtcacagtc ccacggatgg ct - #ctgtgggc10260- caggatctgc cttaggcttc acccacctca acatcttgct gtgttgttca gg - #ctggtctc10320- aaactttggg ctcaaacaat cctccgcctc agcctcccaa agtgctggga tt - #acagacat10380- gagccaccgt gcccggccgt gctgttctgt tctccaatag agaagctggt gg - #aagtcccc10440- agtaacccag aggtgatgtg tgatgcacac agtctcctca ctctgaagct gc - #acatgcga10500- tgtgaatctt catttggggt ccgctgttaa tatggtgttt ttcgggggat ac - #agcaatga10560- ccagcgtccc caggaggtcc cagcagaggc tctggccccg gccccagtgg aa - #gtcccagc10620- tccagcccct gcagcagcct cagcctcagg cccagcgaag actccggccc ca - #gcagaggc10680- ctcaacttca gctttggtcc cagaggagac gccagtggaa gctccagccc ca - #cccccagc10740- cgaggcccct gcccagtacc cgagtgagca cctcatccag gccacctcgg ag - #gagaatca10800- gatcccctcg cacttgcctg cctgcccgtc gctccggcac gtcgccagcc tg - #cggggcag10860- cgccatcatc gagctcttcc acagcagcat tgctgaggta gcggcccggg tg - #tgggtgcc10920- agctatggca cggccagtcc tgagggcgag gccaagcttg gcttcaggtc ag - #cctcaggt10980- ccctggactt ccctgatgtc ggagtcctca gctgagctgc tcacagcttt ga - #ggacctgg11040- gcagtgaggt cctgagttgc cctccctggc catttgtgct gtgtcaccac ct - #cctgtgcc11100- acttccagcc ccaggtagac ctcccaccaa cagccatctc ccacccctct ct - #tcctctct11160- gccttgaagc atacggattc attggtgagc caagaggggc ttcccatgtc tc - #cttgtgga11220- agctgtgggc atgtccctgg tatgtgcagg ttgctagggt ggtggagctg ac - #aggaggcc11280- ccccgtcttc aggttgaaaa cgaggagctg aggcacctca tgtggtcctc gg - #tggtgttc11340- taccagaccc cagggctgga ggtgactgcc tgcgtgctgc tctccaccaa gg - #ctgtgtac11400- tttgtgctcc acgacggcct ccgccgctac ttctcagagc cactgcaggg ta - #ggcacagg11460- gcctgctggg gctcaggagc ttggagtgtg tggttggggc aggcctgggg gg - #tcattctc11520- tggagccagc tgtgtggctt caggcagcag tcagcgactt ggctgcagtg gg - #ctgagagt11580- tccttgtctg aggaagggag ctgtcatgag ggaggggtcc atggccagat gt - #gaacgcag11640- aatgcactga gccagggcct ggtgactgct tgggaacagc ctgtgatgag aa - #ggggttag11700- gcagcctttg cccctggggc tgcacaggaa gccctagcca gcgacctggt ga - #ctcccctg11760- agctggaaga ggctcagact ccagagggca ttgcctatgg ggctttgcac gg - #gtggaagc11820- caggccagcc aagaggacct gttcctgctg gatgtgctgc acacctagga ac - #cttgtgct11880- tgcctgccac cgcctccctc tgtccctttc tccatcacac agatttctgg ca - #tcagaaaa11940- acaccgacta caacaacagc cctttccaca tctcccagtg cttcgtgcta aa - #gcttagtg12000- acctgcagtc agtcaatgtg gggcttttcg accagcattt ccggctgacg cg - #tgggtgac12060- cctctgtgct ttgtcctatt tcgggtgaag gccagcatca ccagtgggct tc - #caccttcc12120- gtacgtgggt gggttatcat agacagttat ctctgtgctc aagagccact tc - #ttacccgg12180- ggtgggagga agcagcttca ggaactgctg agagagcaga actcacgctc ca - #gggctcag12240- agcaggaggt agggtgtgcg gcaagcgctg gcccggacag aagcagagtg gg - #ccctggtc12300- tcgggcagga tgtttctgac tcacatttcc tgaggagaga aagctaagct ct - #ttgcctaa12360- tgtctctgtc tccccttcca gaaaaatgcc tcagctcttc cggcctgaag ga - #atggcctc12420- ctcccgggcc ccatgattct ttcctgtgtg ggccctcctg gccctggcct ct - #gggctgag12480- gcttgctagg gactcggggt ggctctaagg ggcagggata gggctgggga gc - #gccggcct12540- gtggccctga ccagcccctt ctcgtgcagg ttccaccccg atgcaggtgg tc - #acgtgctt12600- gacgcgggac agctacctga cgcactgctt cctccagcac ctcatggtcg tg - #ctgtcctc12660- tctggaacgc acgccctcgc cggagcctgt tgacaaggac ttctactccg ag - #tttgggaa12720- caagaccaca ggtacccctg tctagctcag gctgcagaca ggctgcctgg ac - #agacgtca12780- tgggccccag ggtggctctc tgtgccccag aaccctctct gcctctatgt ct - #ctcttttc12840- tcacttagct ggccagggtt ttatgtgggg cttttcgatg gcagagtctc ca - #ctccagca12900- gtccctcaac catctggcag acacatctcc agtgcctgct ttgggctcct gg - #cctgtggg12960- ccccacactt ggagcatcct ctcctgcctg tctcatgccg gggtctctcg gt - #tggcttgg13020- ggcccttggt gctcccagcc ccaccagggg ccggttccag gctatagccc ag - #gtggcatc13080- tctctgcagg gaagatggag aactacgagc tgatccactc tagtcgcgtc aa - #gtttacct13140- accccagtga ggaggagatt ggggacctga cgttcactgt ggcccaaaag at - #ggctgagc13200- cagagaaggc cccagccctc agcatcctgc tgtacgtgca ggccttccag gt - #gggcatgc13260- caccccctgg gtgctgcagg ggccccctgc gccccaagac actcctgctc ac - #cagctccg13320- agatcttcct cctggatgag gactgtgtcc actacccact gcccgagttt gc - #caaagagc13380- cgccgcagag agacaggtac cggctggacg atggccgccg cgtccgggac ct - #ggaccgag13440- tgctcatggg ctaccagacc tacccgcagg ccctcaccct cgtcttcgat ga - #cgtgcaag13500- gtcatgacct catgggcagt gtcaccctgg accactttgg ggaggtgcca gg - #tggcccgg13560- ctagagccag ccagggccgt gaagtccagt ggcaggtgtt tgtccccagt gc - #tgagagca13620- gagagaagct catctcgctg ttggctcgcc agtgggaggc cctgtgtggc cg - #tgagctgc13680- ctgtcgagct caccggctag cccaggccac agccagcctg tcgtgtccag cc - #tgacgcct13740- actggggcag ggcagcaggc ttttgtgttc tctaaaaatg ttttatcctc cc - #tttggtac13800- cttaatttga ctgtcctcgc agagaatgtg aacatgtgtg tgtgttgtgt ta - #attctttc13860- tcatgttggg agtgagaatg ccgggcccct cagggctgtc ggtgtgctgt ca - #gcctccca13920- caggtggtac agccgtgcac accagtgtcg tgtctgctgt tgtgggaccg tt - #gttaacac13980- gtgacactgt gggtctgact ttctcttcta cacgtccttt cctgaagtgt cg - #agtccagt14040- cctttgttgc tgttgctgtt gctgttgctg ttgctgttgg catcttgctg ct - #aatcctga14100- ggctggtagc agaatgcaca ttggaagctc ccaccccata ttgttcttca aa - #gtggaggt14160- ctcccctgat ccagacaagt gggagagccc gtgggggcag gggacctgga gc - #tgccagca14220- ccaagcgtga ttcctgctgc ctgtattctc tattccaata aagcagagtt tg - #acaccgtc14280- tgcatcttct aaaccaaggg tcactgggat cgagtcgacg gccctatagt ga - #gtcgtatt14340- agagctcgcg gccgcgagct ctagatgcat gctcgagcgg ccgccagtgt ga - #tggatatc14400- tgcagaattc cagcacactg gcggccgtta ctagtggatc cgagctccac ag - #aggtggtc14460- gatgagggct gccctttccc acatccttag tagggggttc aagatgaccc ag - #actgtgcc14520- cctggggagc ttggagccat gcgggaggat gagccatgtg ctggaggaga ac - #agggtagg14580- atggtgtggg gcttttgtag actgtctaga agcaaagaag gtctgcagtg ga - #ggtggtgt14640- ctgaggtgaa tctcgaaggt gaataggagt tgaacgttag caggcagagg gt - #ggattgca14700- ggagagcagc ggcctgggca ggtgcccagc gtggcccatc agggtgcttc at - #gcatggct14760- gtgtgcttgc catccttcct gcctgcctac cccctgctgc ttcgcttcat gg - #gggcgttt14820- gagcttgggc ccacctgcct gcctcgcttg tgggcagagg acccaagctg tg - #tgagttgt14880- cctgtcccgg ggagcagctg aactggtccg gggtctcgaa ctgtggggct ca - #aaaggact14940- ccggggtcat ttcactgggg ctgtgccgat tcctgggggc tgttnggaan gt - #aaaggcct15000- aaaggggctc cctggttang gccctcaant ttaanaacct ggggccgggg cc - #cggaattg15060- cccccaantt tgtttcaacn ccccttggcc ttnggcnggg gcaaatttcc an - #ggggaacc15120- aatggntttc ccccaaaaan ggggccnttt taacccnttt ccaaantttg gg - #ncctaaaa15180# 15202ang gg- <210> SEQ ID NO 22- <211> LENGTH: 1070- <212> TYPE: PRT- <213> ORGANISM: Homo sapiens- <400> SEQUENCE: 22- Val Cys Leu Asp Asp Thr Val Thr Thr Glu Ly - #s Glu Leu Asp Thr Val# 15- Glu Val Leu Lys Ala Ile Gln Lys Ala Lys Gl - #u Val Lys Ser Lys Leu# 30- Ser Asn Pro Glu Lys Lys Gly Gly Glu Asp Se - #r Arg Leu Ser Ala Ala# 45- Pro Cys Ile Arg Pro Ser Ser Ser Pro Pro Th - #r Val Ala Pro Ala Ser# 60- Ala Ser Leu Pro Gln Pro Ile Leu Ser Asn Gl - #n Gly Ile Met Phe Val# 80- Gln Glu Glu Ala Leu Ala Ser Ser Leu Ser Se - #r Thr Asp Ser Leu Thr# 95- Pro Glu His Gln Pro Ile Ala Gln Gly Cys Se - #r Asp Ser Leu Glu Ser# 110- Ile Pro Ala Gly Gln Ala Ala Ser Asp Asp Le - #u Arg Asp Val Pro Gly# 125- Ala Val Gly Gly Ala Ser Pro Glu His Ala Gl - #u Pro Glu Val Gln Val# 140- Val Pro Gly Ser Gly Gln Ile Ile Phe Leu Pr - #o Phe Thr Cys Ile Gly145 1 - #50 1 - #55 1 -#60- Tyr Thr Ala Thr Asn Gln Asp Phe Ile Gln Ar - #g Leu Ser Thr Leu Ile# 175- Arg Gln Ala Ile Glu Arg Gln Leu Pro Ala Tr - #p Ile Glu Ala Ala Asn# 190- Gln Arg Glu Glu Gly Gln Gly Glu Gln Gly Gl - #u Glu Glu Asp Glu Glu# 205- Glu Glu Glu Glu Glu Asp Val Ala Glu Asn Ar - #g Tyr Phe Glu Met Gly# 220- Pro Pro Asp Val Glu Glu Glu Glu Gly Gly Gl - #y Gln Gly Glu Glu Glu225 2 - #30 2 - #35 2 -#40- Glu Glu Glu Glu Glu Asp Glu Glu Ala Glu Gl - #u Glu Arg Leu Ala Leu# 255- Glu Trp Ala Leu Gly Ala Asp Glu Asp Phe Le - #u Leu Glu His Ile Arg# 270- Ile Leu Lys Val Leu Trp Cys Phe Leu Ile Hi - #s Val Gln Gly Ser Ile# 285- Arg Gln Phe Ala Ala Cys Leu Val Leu Thr As - #p Phe Gly Ile Ala Val# 300- Phe Glu Ile Pro His Gln Glu Ser Arg Gly Se - #r Ser Gln His Ile Leu305 3 - #10 3 - #15 3 -#20- Ser Ser Leu Arg Phe Val Phe Cys Phe Pro Hi - #s Gly Asp Leu Thr Glu# 335- Phe Gly Phe Leu Met Pro Glu Leu Cys Leu Va - #l Leu Lys Val Arg His# 350- Ser Glu Asn Thr Leu Phe Ile Ile Ser Asp Al - #a Ala Asn Leu His Glu# 365- Phe His Ala Asp Leu Arg Ser Cys Phe Ala Pr - #o Gln His Met Ala Met# 380- Leu Cys Ser Pro Ile Leu Tyr Gly Ser His Th - #r Ser Leu Gln Glu Phe385 3 - #90 3 - #95 4 -#00- Leu Arg Gln Leu Leu Thr Phe Tyr Lys Val Al - #a Gly Gly Cys Gln Glu# 415- Arg Ser Gln Gly Cys Phe Pro Val Tyr Leu Va - #l Tyr Ser Asp Lys Arg# 430- Met Val Gln Thr Ala Ala Gly Asp Tyr Ser Gl - #y Asn Ile Glu Trp Ala# 445- Ser Cys Thr Leu Cys Ser Ala Val Arg Arg Se - #r Cys Cys Ala Pro Ser# 460- Glu Ala Val Lys Ser Ala Ala Ile Pro Tyr Tr - #p Leu Leu Leu Thr Pro465 4 - #70 4 - #75 4 -#80- Gln His Leu Asn Val Ile Lys Ala Asp Phe As - #n Pro Met Pro Asn Arg# 495- Gly Thr His Asn Cys Arg Asn Arg Asn Ser Ph - #e Lys Leu Ser Arg Val# 510- Pro Leu Ser Thr Val Leu Leu Asp Pro Thr Ar - #g Ser Cys Thr Gln Pro# 525- Arg Gly Ala Phe Ala Asp Gly His Val Leu Gl - #u Leu Leu Val Gly Tyr# 540- Arg Phe Val Thr Ala Ile Phe Val Leu Pro Hi - #s Glu Lys Phe His Phe545 5 - #50 5 - #55 5 -#60- Leu Arg Val Tyr Asn Gln Leu Arg Ala Ser Le - #u Gln Asp Leu Lys Thr# 575- Val Val Ile Ala Lys Thr Pro Gly Thr Gly Gl - #y Ser Pro Gln Gly Ser# 590- Phe Ala Asp Gly Gln Pro Ala Glu Arg Arg Al - #a Ser Asn Asp Gln Arg# 605- Pro Gln Glu Val Pro Ala Glu Ala Leu Ala Pr - #o Ala Pro Val Glu Val# 620- Pro Ala Pro Ala Pro Ala Ala Ala Ser Ala Se - #r Gly Pro Ala Lys Thr625 6 - #30 6 - #35 6 -#40- Pro Ala Pro Ala Glu Ala Ser Thr Ser Ala Le - #u Val Pro Glu Glu Thr# 655- Pro Val Glu Ala Pro Ala Pro Pro Pro Ala Gl - #u Ala Pro Ala Gln Tyr# 670- Pro Ser Glu His Leu Ile Gln Ala Thr Ser Gl - #u Glu Asn Gln Ile Pro# 685- Ser His Leu Pro Ala Cys Pro Ser Leu Arg Hi - #s Val Ala Ser Leu Arg# 700- Gly Ser Ala Ile Ile Glu Leu Phe His Ser Se - #r Ile Ala Glu Val Glu705 7 - #10 7 - #15 7 -#20- Asn Glu Glu Leu Arg His Leu Met Trp Ser Se - #r Val Val Phe Tyr Gln# 735- Thr Pro Gly Leu Glu Val Thr Ala Cys Val Le - #u Leu Ser Thr Lys Ala# 750- Val Tyr Phe Val Leu His Asp Gly Leu Arg Ar - #g Tyr Phe Ser Glu Pro# 765- Leu Gln Asp Phe Trp His Gln Lys Asn Thr As - #p Tyr Asn Asn Ser Pro# 780- Phe His Ile Ser Gln Cys Phe Val Leu Lys Le - #u Ser Asp Leu Gln Ser785 7 - #90 7 - #95 8 -#00- Val Asn Val Gly Leu Phe Asp Gln His Phe Ar - #g Leu Thr Gly Ser Thr# 815- Pro Met Gln Val Val Thr Cys Leu Thr Arg As - #p Ser Tyr Leu Thr His# 830- Cys Phe Leu Gln His Leu Met Val Val Leu Se - #r Ser Leu Glu Arg Thr# 845- Pro Ser Pro Glu Pro Val Asp Lys Asp Phe Ty - #r Ser Glu Phe Gly Asn# 860- Lys Thr Thr Gly Lys Met Glu Asn Tyr Glu Le - #u Ile His Ser Ser Arg865 8 - #70 8 - #75 8 -#80- Val Lys Phe Thr Tyr Pro Ser Glu Glu Glu Il - #e Gly Asp Leu Thr Phe# 895- Thr Val Ala Gln Lys Met Ala Glu Pro Glu Ly - #s Ala Pro Ala Leu er# 910- Ile Leu Leu Tyr Val Gln Ala Phe Gln Val Gl - #y Met Pro Pro Pro ly# 925- Cys Cys Arg Gly Pro Leu Arg Pro Lys Thr Le - #u Leu Leu Thr Ser er# 940- Glu Ile Phe Leu Leu Asp Glu Asp Cys Val Hi - #s Tyr Pro Leu Pro lu945 9 - #50 9 - #55 6 -#0- Phe Ala Lys Glu Pro Pro Gln Arg Asp Arg Ty - #r Arg Leu Asp Asp ly# 975- Arg Arg Val Arg Asp Leu Asp Arg Val Leu Me - #t Gly Tyr Gln Thr yr# 990- Pro Gln Ala Leu Thr Leu Val Phe Asp Asp Va - #l Gln Gly His Asp eu# 10050- Met Gly Ser Val Thr Leu Asp His Phe Gly Gl - #u Val Pro Gly Gly ro# 10205- Ala Arg Ala Ser Gln Gly Arg Glu Val Gln Tr - #p Gln Val Phe Val ro# 14035 0- Ser Ala Glu Ser Arg Glu Lys Leu Ile Ser Le - #u Leu Ala Arg Gln rp# 10550- Glu Ala Leu Cys Gly Arg Glu Leu Pro Val Gl - #u Leu Thr Gly# 10705__________________________________________________________________________

DNA molecules encoding imidazoline receptive polypeptides and polypeptides encoded thereby

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

REFERENCE TO RELATED APPLICATION

Non-Patent Literature Citations (15)

Continuation in Parts (1)

Entry
P. Ernsberger et al. "Role of Imidazole Receptors in the Vasodepressor Response to Clonidine Analogs in the Rostral Ventrolateral Medulla.sup.1 ", The Journal of Pharmacology and Experimental Therapeutics vol. 253, No. 1, pp. 408-418, (1990).
J. Piletz et al., "Nonadrenergic Imidazoline Binding Sites on Human Platelets", The Journal of Pharmacology and Experimental Therapeutics, vol. 267, No. 3, pp. 1493-1502, (1993).
B. Lanier et al., "Structural and Ligand Recognition Properties of Imidazoline Binding Proteins in Tissues of Rat and Rabbit", The American Society for Pharmacology and Experimental Therapeutics, vol. 48, pp. 703-710, (1995).
F. Bennai et al., "Antiidiotypic Antibodies as Tools to Study Imidazoline Receptors", Annals New York Academy of Sciences, vol. 763, pp. 140-148, (1995).
H. Wang et al., "Isolation and Characterization of Imidazoline Receptor Protein from Bovine Adrenal Chromaffin Cells", The American Society for Pharmacology and Experimental Therapeutics, vol. 42, pp. 792-801, (1992).
H. Wang et al., "Production and Characterization of Antibodies Specific for the Imidazoline Receptor Protein", The American Society for Pharmacology and Experimental Therapeutics, vol. 43, pp. 509-515, (1993).
P. Ernsberger et al., "I.sup.1 -Imidazoline Receptors Definition, Characterization, Distribution, and Transmembrane Signaling.sup.a ", Annals New York Academy of Sciences, vol. 763, pp. 22-42, (1995).
Fourth IBRO World Congress of Neuroscience.
J. Piletz et al., "Imidazoline Receptors in Depression", American College of Neuropsychopharmacology, 34th Annual Meeting, p. 119, (1995).
A. Parini et al., "The Elusive Family of Imidazoline Binding Sites", TiPS, vol. 17, pp. 13-16, (1996).
J. Piletz et al., "Desipramine Lowers Tritiated Para-Aminoclonidine Binding in Platelets of Depressed Patients", Arch Gen Psychiatry, vol. 48, pp. 813-820, (1991).
J. Piletz et al., "Psychopharmacology of Imidazoline and .alpha..sub.2 -Adrenergic Receptors: Implications for Depression", Critical Reviews in Neurobiology, vol. 9, No. 1, pp. 29-66, (1994).
Lin, et al., Science 190:61-63 (1975).
George et al., Macromolecular sequencing and synthesis, Alan Riss , pp. 127-149, 1988.
EST04033, in : Adams et al., Nature Genet.4:256-67, 1993.