BACKGROUND OF THE INVENTION The voltage activated calcium channels ("VSCCs") of vertebrates have been shown to be involved in a variety of different physiological processes including muscle contraction, insulin release from the pancreas, and neurotransmitter release in the nervous system (Greenberg D. Annals of Neurology, 1997; 42 : 275-82; Catterall W. A., Trends in Neurosciences, 1993 ; 16: 500-506; Catterall W., Epstein P. N., Diabetologia, 35 (Suppl 2: S23-33) 1992; Birnbaumer L., et al., Neuron., 1994: 13; Rorsman P., et al., Diabete.

Metab., 1994 ; 20: 138-145).

VSCCs are most highly expressed in excitable tissues including brain, skeletal muscle, and heart. They are multiprotein complexes composed of a

central al pore-forming subunit variably associated with beta, gamma, and/or an a25 subunit. Nine different functional classes of VSCCs have been described, based on biophysical and pharmacological studies. These functional classes are mainly determined by the al subunit composition. The beta, gamma, and a28 subunits modulate channel function, affecting the kinetics of activation and inactivation, voltage-dependence, peak amplitude, and ligand binding. Walker N., De Waard M., Trends in Neurosciences, 1998 ; 21 (4): 148-154.

A number of compounds useful in treating various diseases in animals, including humans, are thought to exert their beneficial effects by modulating functions of voltage-dependent calcium channels. Many of these compounds bind to calcium channels and alter cellular calcium flux in response to a depolarizing signal. However, a lack of understanding of the structure of channel subunits and the genes that code for them has hampered scientists both in discerning the pharmacology of compounds that interact with calcium channels and in the ability to rationally design compounds that will interact with calcium channels to have desired therapeutic effects. The lack of understanding is due in part to the fact that it has not been possible to obtain the large amounts of highly purified channel subunits that are required to understand, at the molecular level, the nature of the subunits and their interactions with one another, with the cell membranes across which the channels allow calcium ions to pass, with calcium and other ions, and with low molecular weight compounds that affect channel function.

Further, the lack of information on genes that code for calcium channel subunits has prevented the understanding of the molecular properties of the mature calcium channel subunits and their precursor proteins (i. e., the mature subunits with signal peptides appended to the amino-terminus) and the regulation of expression of calcium channel subunits. An understanding of these properties, and of how expression of calcium channel subunits genes is regulated, may provide the basis for designing therapeutic agents which have beneficial effects through affecting calcium channel function or concentration. Furthermore, the availability of sequences of genes coding for calcium channel subunits would make possible the diagnosis of defects, which might underlie a number of diseases, in genes coding for such subunits.

Expression experiments in Xenopus oocytes have demonstrated that in order to produce fully functional calcium channels, the al and a28 subunits must both be expressed. Absence of the a28 subunit results in a nonfunctional channel, even though the al subunit, through which ions flow, is fully expressed. Indeed, not only the ion flux through these channels, but the pharmacological properties of the al are different in the absence of the a28 subunit. The a2S subunit, therefore, is a critical component of VSCCs and one that must be studied if one is to better characterize VSCC function.

A detailed understanding of VSCC operation is beginning to reveal some mechanisms for interceding in the progression of diseases associated with abnormal VSCC functions. US Patent No. 5,618,720, which issued April 8,1997, references al and a28 subunits and the polynucleotide sequences that encode the subunits. The publication, however, does not disclose any additional a28 subunits and in light of the importance of the a28 subunit, it can be understood that the identification and characterization of new a28 subunits and the genes encoding these subunits would advance molecular genetic and pharmacological studies to understand the relations between the structure and the function of VSCCs.

Also, a further understanding of the biochemical mechanisms behind these subunits and their effect on mammals may lead to new opportunities for treating and diagnosing diseases related to abnormal (high or low) VSCC operation.

Stated another way, a better understanding of the molecular mechanisms of VSCC operation will allow improved design of therapeutic drugs that treat diseases related to abnormal VSCC expression, and specifically abnormal a28 expression.

The cDNAs, oligonucleotides, peptides, antibodies for the a28 proteins, which are the subject of this invention, provide a plurality of tools for studying VSCC operations in various cells and tissues and for diagnosing and selecting inhibitors or drugs with the potential to intervene in various disorders or diseases in which altered a28 expression is implicated. Such disease states affected include epilepsy and other seizure-related syndromes, migraine, ataxia and other vestibular defects (for review, Terwindt, GM et. Al., Eur J Hum Genet 1998 Jul- Aug; 6 (4): 297-307), chronic pain (Backonja M, JAMA 1998 Dec 2; 280 (21): 1831-6), mood, sleep interference (Rowbotham M, JAMA 1998 Dec

2 ; 280 (21): 1837-42), anxiety (Singh et al., Psychopharmocology 1996 Sep.

127 (1) : 1-9), ALS (Mazzini L et. Al., J Neurol Sci 1998 Oct, 160 Suppl 1 : S57- 63), multiple sclerosis (Metz L, Semin Neurol 1998; 18 (3): 389-95), mania (Erfurth A, et al., J Psychiatr Res 1998 Sep-Oct ; 32 (5): 261-4), tremor (Evidente VG, et al., Mov Disord 1998 Sep ; 13 (5) : 829-31), parkinsonism (Olson WL, et al., Am J Med 1997 Jan; 102 (1) : 60-6) substance abuse/addiction syndromes (Watson, WP et al., Neuropharmacology 1997 Oct; 36 (10) : 1369-75), depression, and cancer, since at least one a28 gene is located in a region of the genome which is thought to harbor an important tumor suppressor gene (Kok K., et al., Adv Cancer Res 1997 ; 71: 27- 92).

The a28 gene is also thought to play a part in proliferative diseases other than cancer, such as inflammation. Treatment with compounds which bind to a28 lead to changes in the signal transduction mechanism of certain proteins.

This includes altered levels of MEK (eg, MEK1 and MEK2) which activates the MAP kinase. Inhibitors of MEK appear to mimic the analgesic activities associated with the binding of gabapentin to a28. Activation of MAP kinase by mitogens appears to be essential for proliferation, and constitutive activation of this kinase is sufficient to induce cellular transformation.

SUMMARY OF THE INVENTION While the al subunit is known to be coded for by 9 genes, the beta subunit by 4 genes, and the gamma subunit by 2 genes, previously only two human a28 genes were known :"axa (cDNA Accession No. M76559.1 and protein Accession No. P54289.1) and a28-B (cDNA SEQ ID NO 1 and protein SEQ ID NO 2). The a28-A gene codes for at least five different splice variants which show tissue-specific expression (Angelotti T., Hoffman F., FEBS, 1996 ; 397 : 331- 337). Translation of the a28-A gene produces a polypeptide which is post- translationally cleaved into the a2 and the 8 subunits. A2 and 8 are then joined by disulfide bonds (De Jongh K., JBC, 1990 ; 265 (25): 14738-14741; Jay S., JBC, 1991; 266 (5): 3287-3293). A2 is thought to be completely extracellular and is heavily glycosylated, while 8 probably forms a single transmembrane domain with five intracellular amino acids at its c-terminus (Brickley K., FEBS,

1995 ; 364: 129-133). This transmembrane domain anchors the protein to the membrane. A28-B is related to a28-A and is available in the public database, GENBANK The inventors, however, have discovered the existence of two new human a28 genes, hereinafter referred to as"a28-C", and"a28-D"genes (gene names CACNA2C and CACNA2D). The present invention, therefore, relates to the isolation of polynucleotide sequences which identify and encode novel a28- related proteins (preferably a28-C and (x28-D proteins) that are expressed in various cells and tissues, both the polynucleotide sequences for the full length genes and any splice variants and their encoded proteins. The polynucleotide sequences are identified in SEQ ID NOS 3-4 and the amino acid sequences of the a28 proteins encoded by the three novel genes are set forth in SEQ ID NOS 5-6.

The invention also concerns a purified or isolated nucleic acid comprising at least 20 consecutive nucleotides of the nucleotide sequences SEQ ID NOS 3-4, or a nucleotide sequence complementary thereto.

A25-C protein of SEQ ID NO 5 is 28% identical and 48% similar at the protein level to a2§-A protein. A28-C protein is 28% identical and 47% similar to a28-B. A28-C gene of SEQ ID NO 3 contains a mapped marker (known as an STS) within its nucleotide sequence which has been mapped to human chromosome 3p21. 1. This region of the human genome is thought to harbor an important tumor suppressor gene, thus a28-C gene is a candidate tumor suppressor gene (Kersemaekers AM, et al., Br J Cancer 1998 ; 77 (2); 192-200).

A28-D protein of SEQ ID NO 6 is 28% identical and 47% similar at the protein level to a28-A protein. A28-D protein is 28% identical and 46% similar to a28-B protein. A28-D gene of SEQ ID 4 maps to a previously published cosmid contig on human chromosome 12pl3. 3.

The unique full length polynucleotides of the present invention were initally discovered by mining the genbank database for sequences with homology to a25, by utilizing known nucleotide sequences and various methods known in the art, including tools provided by Compugen Systems Ltd. See Sequence Analysis Primer by Michael Gribskkov, John Devereux, Oxford University Press, 1994. After identification of expressed sequenced tags (ESTs) and full-length

sequences related to a28-A, cloning methods were used to obtain, in hand, full- length sequences for a28-C and a2b-D, see Examples 1, 2 and 3. In short, an arrayed human, kidney cDNA library obtained from Origene, was screened by PCR, using oligonucleotide primers derived from the database sequences. Clones identified from the library screen were sequenced by standard methods for verification. A summary of the sequencing information is provided in Example 3.

Analysis of the cloned sequences for a28-B, a28-C, and a28-D led to the identification of a conserved domain and of a number of splice variants. The conserved domain is known as a vonWillebrand factor A3 domain (Huizinga, EG, et. al., Structure 1997, Sept 15; 5 (9): 1147-56). This domain has been described in a large number of proteins and is thought to mediate cell adhesion. Interesting splice-variants of a2â-C and a2â-D were also identified. These variants result in a c-terminal truncation of the respective protein sequences. Truncation of the c- terminus may lead to the production of a soluble, secreted a25-C or a28-D protein with new functions beyond that previously described for a28.

The a28 proteins are of interest because they play an important role in many disease states. In one example, a28-A has been shown to be a high-affinity binding target of the anti-convulsant drug gabapentin (NEURONTIN) (Gee N., JBC 1996 ; 271: 5768-5776). This property of the a28-A protein has the potential to have profound physiological effects. Thus, by regulating the levels or activities of a28-C and/or a28-D protein, or by modulating their function, desirable physiological effects may be obtained. Such effects may be used to treat a variety of diseases involving abnormal expression of a28 or the abnormal expression of VSCCs (i. e., disease states include, but are not limited to epilepsy, chronic pain, anxiety, diabetes, ALS, mania, cancer, tremor, parkinsonism, migraine, ataxia, mood, sleep interference, depression, multiple sclerosis, inflammation).

The rationale for the therapeutic use of a25-C and/or a28-D proteins to design or discover treatment for these diseases is based upon the fact that gabapentin has been successfully used for treating epilepsy, chronic pain, and ALS, and has implications for use in the treatment of mania, tremor, parkinsonism, migraine, ataxia, mood, inflammation, sleep interference, and/or multiple sclerosis). Gabapentin is known to bind to a28-A with high affinity and

this binding is thought to represent the mechanism of action of gabapentin.

Therefore, gabapentin and/or other compounds which bind to a25-C and/or a28- D proteins may have similar, or related, therapeutic effects to the effects seen with gabapentin. Also, compounds which are known to have therapeutic effects on calcium channels are regulated in their affinity by the presence of a28. Thus, pharmacological or genetic approaches to alleviating this deficiency will have a major impact on the diseases described above.

One aspect of the invention is to provide purified a28-C and/or a28-D proteins. The purified proteins may be obtained from either recombinant cells or naturally occurring cells. The purified a28-C and/or a28-D proteins of the invention may be mammalian in origin. Primate, including human-derived a28-C and/or a28-D proteins, are examples of the various proteins specifically provided for. The invention also provides allelic variants and biologically active derivatives of naturally occurring a28-C and/or a28-D proteins.

Another aspect of the invention is to provide polynucleotides encoding the a28-C and/or a28-D proteins of the invention and to provide polynucleotides complementary to polynucleotide coding strand. The polynucleotides of the invention may be used to provide for the recombinant expression of a28-C and/or a28-D proteins. The polynucleotides of the invention may also be used for genetic therapy purposes so as to 1) treat diseases which may result from alterations of a28-C and/or a28-D genes or from alterations of cellular pathways involving a28-C and/or a28-D, 2) test for presence of a disease, or susceptibility to a disease, due to alterations or deletions in a28-C and/or a2b-D, 3) analyze or alter the subcellular localization of the a28-C and/or a28-D polypeptide, 4) clone or isolate discrete classes of RNA similar to a28-C and/or a28-D genes, 5) express discrete classes of RNA in order to alter the levels of a28-C and/or a28-D genes.

The invention also relates to oligonucleotide molecules useful as probes or primers, wherein said oligonucleotide molecules hybridize specifically with any nucleotide sequence comprising or related to the a28-C and/or a28-D genes, particularly the sequences of SEQ ID NOS 3-4. These oligonucleotides are useful

either as primers for use in various processes such as DNA amplification and microsequencing or as probes for DNA recognition in hybridization analyses.

A nucleic acid probe or primer according to the invention comprises at least 8 consecutive nucleotides of a polynucleotide of SEQ ID NOS 3-4, preferably from 8 to 200 consecutive nucleotides, more particularly from 10,15, 20 or 30 to 100 consecutive nucleotides, more preferably from 10 to 90 nucleotides, and most preferably from 20 to 80 consecutive nucleotides of a polynucleotide of SEQ ID NOS 3 or 4. Preferred probes or primers of the invention comprise the oligonucleotides selected from the group consisting of the oligonucleotides set forth in the examples below.

The invention also concerns a method for the amplification of a region of the a28-C and/or a28-D genes. The method comprises the step of : contacting a test sample suspected of containing the desired a28-C and/or a28-D sequence or portion thereof with amplification reaction reagents, comprising a pair of amplification primers such as those described above, the primers being located on either side of the a28-C and/or a28-D nucleotide region to be amplified. The method may further comprise the step of detecting the amplification product. For example, the amplification product may be detected using a detection probe that can hybridize with an internal region of the amplified sequences. Alternatively, the amplification product may be detected with any of the primers used for the amplification reaction themselves, optionally in a labeled form.

The invention also concerns diagnostic kits for detecting the presence of at least one copy of a a28-C and/or a28-D DNA in a test sample, said kits containing a primer, a pair of primers or a probe according to the invention.

In a first embodiment, the kit comprises primers such as those described above, preferably forward and reverse primers which are used to amplify the a28- C and/or a28-D gene or a fragment thereof.

In a second embodiment, the kit comprises a hybridization DNA probe, that is or eventually becomes immobilized on a solid support, which is capable of hybridizing with the a28-C and/or a28-D gene or a fragment thereof. The

techniques for immobilizing a nucleotide primer or probe on a solid support are well-known to the skilled person.

The kits of the present invention can also comprise optional elements including appropriate amplification reagents such as DNA polymerases when the kit comprises primers, reagents useful in hybridization reactions and reagents useful to reveal the presence of a hybridization reaction between a labeled hybridization probe and the a28-C and/or a26-D gene.

Another aspect of the invention is to provide antibodies capable of binding to a28-C and/or a25-D proteins of the invention. The antibodies may be polyclonal or monoclonal. The invention also provides methods of using the subject antibodies to detect and measure expression of a28-C and/or a25-D proteins either in vitro or in vivo, or for detecting proteins that interact with a28-C and/or a28-D proteins, or molecules that regulate any of the activities of a28-C and/or a28-D proteins.

Another aspect of the invention is to provide assays for the detection of proteins that interact with a28-C or a28-D using genetic approaches. A preferred embodiment involves the use of yeast two-hybrid approaches for this screening.

(Bartel and Fields, The Yeast Two-Hybrid System, Oxford University Press, 1997) Another aspect of the invention is to provide assays for the detection or screening of therapeutic compounds that interfere with, or mimic in any way, the interaction between a28-C and/or a28-D proteins and ligands that bind to a28-C and/or a28-D proteins.

In a first embodiment, such a method for the screening of a candidate substance comprises the following steps: a) providing a polypeptide comprising the amino acid sequence of SEQ ID NO 5 and/or 6, or a peptide fragment or a variant thereof ; b) obtaining a candidate substance ; c) bringing into contact said polypeptide with said candidate substance; and d) detecting the complexes formed between said polypeptide and said candidate substance.

In one embodiment of the screening method defined above, the complexes formed between the polypeptide and the candidate substance are further incubated in the presence of a polyclonal or a monoclonal antibody that specifically binds to the a28-C and/or a25-D protein of the invention or to the peptide fragment or variant thereof.

The candidate substance or molecule to be assayed for interacting with the a28-C and/or a28-D polypeptide may be of diverse nature, including, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides.

In another embodiment of the present screening method, increasing concentrations of a substance competing for binding to the a28-C and/or a28-D protein with the considered candidate substance is added, simultaneously or prior to the addition of the candidate substance or molecule, when performing step c) of said method. By this technique, the detection and optionally the quantification of the complexes formed between the a28-C and/or a28-D protein or the peptide fragment or variant thereof and the candidate substance or molecule to be screened allows the one skilled in the art to determine the affinity value of said substance or molecule for said a28-C and/or a28-D protein or the peptide fragment or variant thereof.

The invention also pertains to kits useful for performing the hereinbefore described screening method. Preferably, such kits comprise a a28-C and/or a28-D protein having the amino acid sequence of SEQ ID NO 5 and/or 6 or a peptide fragment or a variant thereof, and optionally means useful to detect the complex formed between the a28-C and/or a28-D protein or its peptide fragment or variant and the candidate substance. In a preferred embodiment the detection means consist in monoclonal or polyclonal antibodies directed against the a28-C and/or a28-D protein or a peptide fragment or a variant thereof.

The assays of the invention therefore comprise the step of measuring the effect of a compound of interest on binding between a28-C and/or a28-D protein and the ligands that bind to a28-C and/or a28-D proteins. Binding may be measured in a variety of ways, including the use of labeled a28-C and/or a28-D protein or labeled ligands. These ligands may include, but are not limited to,

neutral alpha-amino acids, which have been shown to bind to a28-A, or therapeutic compounds such as gabapentin or related analogues.

Another aspect of the invention is to provide assays for the discovery of proteins that interact directly or indirectly with a25-C and/or a25-D proteins. The assays of the invention comprise a method for detecting such interactions in cells, or in biochemical assays. These interactions may be detected in a variety of ways, including the use of the cDNA encoding a28-C and/or a25-D proteins, or a28-C and/or a28-D proteins themselves, or fragments or modifications thereof. The assays may also comprise a method for detecting the interaction between a28 subunits and other subunits of the calcium channel, such as al subunits. These assays may involve measuring the interaction between the proteins directly, or assaying the activity of a fully assembled calcium channel.

Before the present sequences, polypeptides, methods for making and using the invention are described, it is to be understood that the invention is not to be limited only to the particular sequences, polypeptides and methods described. The sequences, polypeptides and methodologies may vary, and the terminology used herein is for the purpose of describing particular embodiments. The foregoing is not intended and should not be construed as limiting the invention in any way since the scope of protection will ultimately depend upon the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All U. S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto.

BRIEF DESCRIPTION OF THE INVENTION Figure 1: Fine-mapping of a28-B to mouse chromosome 9 Figure 2: Human a25-B tissue distribution Figure 3: [3H] gabapentin binding activity by human a28-B in transiently transfected COS7 Figure 4: Human a28-C tissue distribution

DETAILED DESCRIPTION OF THE INVENTION Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning : A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol.

185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA),"Guide to Protein Purification"in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols : A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells : A Manual of Basic Technique, 2d Ed. (R. I. Freshney. 1987. Liss, Inc. New York, NY), and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N. J.) Sequence Analysis Primer (Gribskov, et al., 1994, Oxford University Press).

In one aspect, the present invention provides novel isolated and purified polynucleotides, hereinafter referred to as alpha-2-delta-C and alpha-2-delta-D ("a28-C","a28-D") genes, encoding a26-C and a25-D proteins, wherein the polynucleotide sequences are substantially similar to those shown in SEQ ID NOS 3-4 and the polypeptide sequences are substantially similar to those shown in SEQ ID NOS 5-6. The terms"a26-C"and"a28-D"are used broadly herein. Unless noted otherwise, the terms"a26-C"and"a25-D"include any natural mammalian- derived form ofa25-C and a28-D and the like. It is preferred that the terms a26- C and a28-D include all mammals, including but not limited to primates and humans.

The polynucleotides provided for may encode complete a25-C and/or a28-D proteins or portions thereof. The polynucleotides of the invention may be produced by a variety of methods including in chemical synthesis using well known solid phase synthesis technique, by cloning or combinations thereof. The polynucleotide of the invention may be derived from cDNA or genomic libraries.

Persons of ordinary skill in the art are familiar with the degeneracy of the genetic code and may readily design polynucleotides that encode a28-C and/or a28-D proteins that have either partial or polynucleotide sequence homology to naturally

occurring polynucleotide sequences encoding a26-C and/or a25-D proteins. The polynucleotides of the invention may be single stranded or double stranded.

Polynucleotide complementary to polynucleotides encoding a25-C and/or a25-D proteins are also provided.

Polynucleotides encoding an a25-C or a28-D protein can be obtained from cDNA libraries prepared from tissue believed to possess a25-C and/or a25- D protein or mRNA and to express it at a detectable level. For example, a cDNA library can be constructed by obtaining polyadenylated mRNA from a cell line known to express a25-C and/or a25-D protein, and using the mRNA as a template to synthesize double stranded cDNA.

Libraries, either cDNA or genomic, are screened with probes designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, suitable probes include monoclonal and polyclonal antibodies that recognize and specifically bind to an a28-C or a25-D protein. For cDNA libraries, suitable probes include carefully selected oligonucleotide probes (usually of about 20-80 bases in length) that encode known or suspected portions of an a25-C or a25-D protein from the same or different species, and/or complementary or homologous cDNAs or fragments thereof that encode the same or a similar gene, and/or homologous genomic DNAs or fragments thereof.

Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in Chapters 10-12 of Sambrook et al., Molecular Cloning : A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989).

A preferred method of practicing this invention is to use carefully selected oligonucleotide sequences to screen cDNA libraries from various tissues. The oligonucleotide sequences selected as probes should be sufficient in length and sufficiently unambiguous that false positives are minimized. The actual nucleotide sequence (s) is/are usually designed based on regions of an a25 protein that have the least codon redundance. The oligonucleotides may be degenerate at one or more positions. The use of degenerate oligonucleotides is of particular importance where a library is screened from a species in which preferential codon usage is not known.

The oligonucleotide must be labeled such that it can be detected upon hybridization to DNA in the library being screened. The preferred method of labeling is to use ATP (e. g., T32P) and polynucleotide kinase to radiolabel the 5' end of the oligonucleotide. However, other methods may be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labeling. cDNAs encoding a28 proteins can also be identified and isolated by other known techniques of recombinant DNA technology, such as by direct expression cloning or by using the polymerase chain reaction (PCR) as described in U. S.

Patent No. 4,683,195, in section 14 of Sambrook, et al., Molecular Cloning : A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, New York, 1989, or in Chapter 15 of Current Protocols in Molecular Biology. Ausubel et al. eds., Green Publishing Associates and Wiley-Interscience 1991. This method requires the use of oligonucleotide probes that will hybridize to DNA encoding an a28-C and/or a28-D protein.

As defined herein,"substantially similar"includes identical sequences, as well as deletions, substitutions or additions to a DNA, RNA or protein sequence that maintain any biologically active portion thereof of the protein product and possess any of the conserved motifs. This includes, but is not limited to, any splice variants of a28-C and/or a28-D which are found to exist. Preferably, the DNA sequences according to the invention consist essentially of the DNA sequence of SEQ ID NOS 3-4. These novel purified and isolated DNA sequences can be used to direct expression of the a25-C and/or a28-D protein and for mutational analysis of a28-C and/or a28-D protein function.

Mutated sequences according to the invention can be identified in a routine manner by those skilled in the art using the teachings provided herein, and techniques well known in the art.

In a preferred embodiment, the present invention comprises a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NOS 3-4 under high stringency hybridization conditions. As used herein, the term"high stringency hybridization conditions"refers to hybridization on a filter support at 65°C in a low salt hybridization buffer to the probe of interest at 2 x 108 cpm/llg for between about 8 hours to 24 hours, followed by washing in 1% SDS, 20 mM

phosphate buffer and 1 mM EDTA at 65°C, for between about 30 minutes to 4 hours. In a preferred embodiment, the low salt hybridization buffer comprises between, 0.5-10% SDS, and 0. 05M and 0.5 M sodium phosphate. In a most preferred embodiment, the low salt hybridization buffer comprises, 7% SDS, and 0.125M sodium phosphate.

As known in the art, numerous equivalent conditions may be employed to comprise either low or high stingency conditions. Factors such as the length and nature (DNA, RNA, base composition) of the sequence, nature of the target (DNA, RNA, base composition, presence in solution or immobilization, etc.), and the concentration of the salts and other components (e. g., the presence or absence of formamide, dextran sulfate and/or polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency different from, but equivalent to, the above listed conditions.

The term"stringent conditions", as used herein, is the"stringency"which occurs within a range from about Tm-5°C (5°C below the melting temperature (Tm) of the probe) to about 20°C to 25°C below Tm. As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences.

The polynucleotides of the invention have a variety of uses, some of which have been indicated or will be addressed in greater detail, infra. The particular uses for a given polynucleotide depend, in part, on the specific polynucleotide embodiment of interest. The polynucleotides of the invention may be used as hybridization probes to recover a28-C and/or a28-D proteins from genetic libraries. The polynucleotides of the invention may also be used as primers for the amplification of a20-C and/or a25-D protein encoding polynucleotides or a portion thereof through the polymerase chain reaction (PCR) and other similar amplification procedures. The polynucleotides of the invention may also be used as probes and amplification primers to detect mutations in a28-C and/or a28-D protein encoding genes that have been correlated with diseases, particularly diseases related to an altered function for a28-A protein. Including, but not limited to, those diseases stated above.

The invention also provides a variety of polynucleotide expression vectors, comprising a25-C and/or a28-D, or a sequence substantially similar to it subcloned into an extra-chromosomal vector. This aspect of the invention allows for in vitro expression of the a25-C and/or a25-D gene, thus permitting an analysis of a25-C and/or a28-D gene regulation and a28-C and/or a28-D protein structure and function. As used herein, the term"extra-chromosomal vector" includes, but is not limited to, plasmids, bacteriophages, cosmids, retroviruses and artificial chromosomes. In a preferred embodiment, the extra-chromosomal vector comprises an expression vector that allows for a26-C and/or a25-D protein production when the recombinant DNA molecule is inserted into a host cell. Such vectors are well known in the art and include, but are not limited to, those with the T3 or T7 polymerase promoters, the SV40 promoter, the CMV promoter, or any promoter that either can direct gene expression, or that one wishes to test for the ability to direct gene expression.

In a preferred embodiment, the subject expression vectors comprise a polynucleotide sequence encoding an a25-C and/or a26-D protein in functional combination with one or more promoter sequences so as to provide for the expression of the a25-C and/or a25-D protein (or an anti-sense copy of the sequence suitable for inhibition of expression of an endogenous gene). The vectors may comprise additional polynucleotide sequences for gene expression, regulation, or the convenient manipulation of the vector, such additional sequences include terminators, reporters, enhancers, selective markers, packaging sites, and the like. Detailed description of polynucleotide expression vectors and their use can be found in, among other places Gene Expression Technologv Methods in Enzymology Volume 185 Goeddel ed, Academic Press Inc., San Diego, CA (1991), Protein Expression in Animal Cells Roth ea., Academic Press, San Diego, CA (1994).

The polynucleotide expression vectors of the invention have a variety of uses. Such uses include the genetic engineering of host cells to express a25-C and/or a25-D proteins. In a further aspect, the present invention provides recombinant host cells that are stably transfected with a recombinant DNA molecule comprising a25-C and/or a26-D subcloned into an extra-chromosomal

vector. The host cells of the present invention may be of any type, including, but not limited to, bacterial, yeast, mammalian cells, and Xenopus oocytes.

Transfection of host cells with recombinant DNA molecules is well known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989) and, as used herein, includes, but is not limited to calcium phosphate transfection, dextran sulfate transfection, electroporation, lipofection and viral infection. This aspect of the invention allows for in vitro and in vivo expression of a25-C and/or a28-D and its gene product, thus enabling high-level expression of a28-C and/or a25-D protein. In a further aspect of the invention the RNA molecules containing a28-C or a28-D can be injected into Xenopus oocytes along with other calcium channel subunit clones and calcium flux across the oocyte membrane can be measured using standard electrophysiological techniques.

In another aspect of the invention transgenic animals can be constructed by injection of the nucloetide sequence for a28-C or a28-D cloned in suitable expression vectors into germ cells.

Other uses of the polynucleotide expression vectors, discussed in greater detail, infra, include, their use for genetic therapy for diseases and conditions in which it may be desirable use to express a28-C and/or a28-D proteins at levels greater than naturally occurring expression levels. Alternatively, it may be desirable to use the subject vectors for anti-sense expression to reduce the naturally occurring levels of a28-C and/or a28-D protein.

A28-C and a28-D share amino acid homology to a28-A, thus it is very likely that they share some structural and functional characteristics with a28-A.

A28-A is known to interact with other subunits of voltage-sensitive calcium channels, such as al and beta. When calcium channels are expressed in oocytes, a functional channel is only produced when an a28 subunit is present. Therefore, a28 is required for calcium channel function. In addition, a28-A has been shown to bind to gabapentin, a drug used to treat epilepsy, chronic pain, ALS, and potentially other neurological diseases. The mechanism of action of gabapentin is thought to be through its interaction with a28. Given the homology between the a28 proteins, it is likely that a28-C and a28-D also share these functions.

The polynucleotide sequences of SEQ ID NOS 3-4 were mapped to human chromosomes using the nucleotide sequences for the cDNA from library sources (See Examples 2-3) to generate probes. The sequences were mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridization to chromosomal spreads, and PCR-based mapping by amplifying DNA from standard radiation hybrid cell lines. (Verma et al (1988) Human Chromosomes : A Manual of Basic Techniques, Pergamon Press, NYC. A28-C of SEQ ID NO 3 maps to human chromosome 3p21. 1. A25-D of SEQ ID NO 4 maps to a previously published cosmid contig on human chromosome 12pl3. 3.

In another aspect, the present invention provides a substantially purified recombinant protein comprising a polypeptide substantially similar to the a26-C and/or a28-D polypeptides shown in SEQ ID NOS 5-6. Furthermore, this aspect of the invention enables the use of a28 protein in several in vitro assays described below. As used herein, the term"substantially similar"includes deletions, substitutions and additions to the sequences of SEQ ID NOS 5-6 introduced by any in vitro means, or any genetic alterations naturally seen in vivo. As used herein, the term"substantially purified"means that the protein should be free from detectable contaminating protein, but the oc26-C and/or a25-D protein may be co-purified with an interacting protein, or as an oligomer. In a most preferred embodiment, the protein sequence according to the invention comprises an amino acid sequence of SEQ ID NOS 5-6. Mutated sequences according to the invention can be identified in a routine manner by those skilled in the art using the teachings provided herein and techniques well known in the art. This aspect of the invention provides a novel purified protein that can be used for in vitro assays, and as a component of a pharmaceutical composition.

A25-C and/or a2S-D proteins may be used to discover molecules that interfere with its activities. For example, molecules that prevent the binding of a25-C and/or a28-D to ligands such as neutral alpha-amino acids (for example (L)-leucine), or to other molecules such as other subunits of the voltage-sensitive calcium channels.. Additionally, a26-C and/or a28-D proteins may be used to

find other proteins with which it directly interacts, and potentially representing additional important regulators of VSCC transport.

The a28-C and/or a28-D proteins of the present invention have a putative biological activity of modulating the cellular flux of calcium, potentially including both intracellular and extracellular calcium stores. The a25-C and/or a28-D protein of the invention may be isolated from a variety of mammalian animal species. Preferred mammalian species for isolation are primates and humans. The invention also contemplates allelic variants of a28-C and/or a28-D protein. A28- C and/or a28-D proteins may be prepared from a variety of mammalian tissues.

Preferably, a28-C and/or a28-D proteins are obtained from recombinant host cells genetically engineered to express significant quantities of a28-C and/or a28-D proteins. A28-C and/or a28-D proteins may be isolated from non-recombinant or recombinant cells in a variety of ways well known to a person of ordinary skill in the art.

The terms"a28-C protein"and"a28-D protein"as used herein refers not only to proteins having the amino acid residue sequence of naturally occurring a28-C and/or a28-D proteins, but also refers to functional derivatives and variants of naturally occurring a28-C and/or a28-D protein. A"functional derivative"of a native polypeptide is a compound having a qualitative biological activity in common with the native a28-C and/or a28-D protein. Thus, a functional derivative of a native a28-C and/or a28-D protein is a compound that has a qualitative biological activity in common with a native a28-C and/or a28-D protein, e. g., binding to other calcium channel subunits and modulating the flux of calcium in cells, or binding to neutral alpha-amino acids and other cognate ligands."Functional derivatives"include, but are not limited to, fragments of native polypeptides from any animal species (including human), and derivatives of native (human and non-human) polypeptides and their fragments, provided that they have a biological activity in common with a respective native polypeptide.

"Fragments"comprise regions within the sequence of a mature native polypeptide. The term"derivative"is used to define amino acid sequence and glycosylation variants, and covalent modifications of a native polypeptide, whereas the term"variant"refers to amino acid sequence and glycosylation

variants within this definition. Preferably, the functional derivatives are polypeptides which have at least about 70% amino acid sequence similarity, more preferably about 80% amino acid sequence similarity, even more preferably at least 90% amino acid sequence similarity, most preferably at least about 99% amino acid sequence similarity with the sequence of a corresponding native polypeptide. Most preferably, the functional derivatives of a native a28-C and/or a28-D protein retain or mimic the region or regions within the native polypeptide sequence that directly participate in ligand binding. The phrase"functional derivative"specifically includes peptides and small organic molecules having a qualitative biological activity in common with a native a28-C and/or a28-D protein.

"Identity"or"homology"with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are similar to residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Neither N-or C-terminal extensions nor insertions, nor alternatively-spliced variants, shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art.

Amino acid sequence variants of native a28-C and/or a28-D proteins and a28-C and/or a28-D protein fragments are prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant a28-C and/or a28-D protein encoding DNA, or by in vitro synthesis of the desired polypeptide. There are two principal variables in the construction of amino acid sequence variants: the location of the mutation site and the nature of the mutation.

With the exception of naturally-occurring alleles, which do not require the manipulation of the DNA sequence encoding the a28-C and/or a28-D protein, the amino acid sequence variants of a28-C and/or a28-D protein are preferably constructed by mutating the DNA, either to arrive at an allele or an amino acid sequence variant that does not occur in nature.

Alternatively or in addition, amino acid alterations can be made at sites that differ in a28-C and/or a28-D proteins from various species, or in highly conserved regions, depending on the goal to be achieved.

Sites at such locations will typically be modified in series, e. g. by (1) substituting first with conservative choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue or residues, or (3) inserting residues of the same or different class adjacent to the located site, or combinations of options 1-3.

One helpful technique is called"alanine scanning"Cunningham and Wells, Science 244, 1081-1085 (1989). Here, a residue or group of target resides is identified and substituted by alanine or polyalanine. Those domains demonstrating functional sensitivity to the alanine substitutions are then refined by introducing further or other substituents at or for the sites of alanine substitution.

After identifying the desired mutation (s), the gene encoding an a28-C and/or a28-D protein variant can, for example, be obtained by chemical synthesis.

More preferably, DNA encoding an a28-C and/or a28-D protein amino acid sequence variant is prepared by site-directed mutagenesis of DNA that encodes an earlier prepared variant or a nonvariant version of the a28-C and/or a28-D protein. Site-directed (site-specific) mutagenesis allows the production of a28-C and/or a28-D protein variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the techniques of site-specific mutagenesis are well known in the art, as exemplified by publications such as, Edelman et al., DNA 2: 183 (1983). As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. This and other phage vectors are commercially available and their use is well known to those skilled in the art.

A versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site-specific mutations in DNA fragments using M13-derived vectors was published by Zoller, M. J. and Smith, M., Nucleic Acids Res. 10,6487-6500 [1982]). Also, plasmid vectors that contain a single-stranded phage origin of replication, Veira et al., Meth. Enzymol. 153 : 3 (1987)] may be employed to obtain single-stranded DNA. Alternatively, nucleotide substitutions are introduced by synthesizing the appropriate DNA fragment in vitro, and amplifying it by PCR procedures known in the art.

In general, site-specific mutagenesis may be performed by obtaining either a double-stranded or a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., Proc. Natl. Acad. Sci. USA 75,5765 (1978). This primer is then annealed with the single-stranded protein sequence-containing vector, and subjected to DNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desires mutation. This heteroduplex vector is then used to transform appropriate host cells such as HB101 cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. Thereafter, the mutated region may be removed and placed in an appropriate expression vector for protein production.

The PCR technique may also be used in creating amino acid sequence variants of an a28-C and/or a26-D protein. When small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template. For introduction of a mutation into a plasmid DNA, one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a stretch of sequence of the

opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 500-5000 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primes can be easily sequenced. PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.

Further details of the foregoing and similar mutagenesis techniques are found in general textbooks, such as, for example, Sambrook et al., Molecular Cloning: H Laboratorv Manual 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor (1989), and Current Protocols in Molecular Biologv, Ausubel et al. eds., John Wiley and Sons (1995).

Naturally-occurring amino acids are divided into groups based on common side chain properties : (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophobic: cys, ser. tier; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, erg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic : trp, tyr, pine.

Conservative substitutions involve exchanging a member within one group for another member within the same group, whereas non-conservative substitutions will entail exchanging a member of one of these classes for another.

Variants obtained by non-conservative substitutions are expected to result in significant changes in the biological properties/function of the obtained variant, and may result in a25-C and/or a25-D protein variants which block a25-C and/or a28-D protein biological activities, i. e., modulation of calcium flux, or binding to neutral, alpha-amino acids. Amino acid positions that are conserved among

various species are generally substituted in a relatively conservative manner if the goal is to retain biological function.

Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues, and typically are contiguous.

Deletions may be introduced into regions not directly involved in ligand binding.

Amino acid insertions include amino-and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions (i. e. insertions within the a28-C and/or a28-D protein amino acid sequence) may range generally from about 1 to 10 residues, more preferably 1 to 5 residues, more preferably 1 to 3 residues. Examples of terminal insertions include the a26-C and/or a28-D proteins with an N-terminal methionyl residue, a naturally-occuring N-terminal signal sequence, an artifact of direct expression in bacterial recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the a28-C and/or a28-D protein to facilitate the secretion of the mature a28-C and/or a28-D protein from recombinant host cells. Such signal sequences will generally be obtained from, and thus homologous to, the intended host cell species. Suitable sequences include STII or Ipp for E. coli. alpha factor for yeast, and viral signals such as herpes gD for mammalian cells. Other insertional variants of the native a28-C and/or a28-D protein molecules include the fusion of the N-or C-terminus of an a25-C and/or a28-D protein to immunogenic polypeptides, e. g. bacterial polypeptides such as betalactamase or an enzyme encoded by the E. cold trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as immunoglobulin regions (preferably immunoglobulin constant regions), albumin, or ferritin, as described in PCT published application WO 89/02922.

Since it is often difficult to predict in advance the characteristics of a variant a28-C and/or a28-D protein, it will be appreciated that screening will be needed to select the optimum variant. For this purpose biochemical screening assays, such as those described herein below, will be readily available.

In a further aspect, the present invention provides antibodies and methods

for detecting antibodies that selectively bind polypeptides with an amino acid sequence substantially similar to the amino acid sequence of SEQ ID NOS 5-6.

As discussed in greater detail, infra, the antibody of the present invention can be a polyclonal or a monoclonal antibody, prepared by using all or part of the sequence of SEQ ID NOS 5-6, or modified portions thereof, to elicit an immune response in a host animal according to standard techniques (Harlow and Lane (1988), eds.

Antibody: A Laboratory Manual, Cold Spring Harbor Press). In a preferred embodiment, the entire polypeptide sequence of SEQ ID NOS 5-6 is used to elicit the production of polyclonal antibodies in a host animal.

The method of detecting a25-C and/or a25-D antibodies comprises contacting cells with an antibody that recognizes a26-C and/or a28-D protein and incubating the cells in a manner that allows for detection of the a25-C and/or a28-D protein-antibody complex. Standard conditions for antibody detection of antigen can be used to accomplish this aspect of the invention (Harlow and Lane, 1988). This aspect of the invention permits the detection of a25-C and/or a25-D protein both in vitro and in vivo.

The subject invention provides methods for the treatment of a variety of diseases characterized by undesirably abnormal cellular levels of a25-C and/or a25-D. Diseases may be treated through either in vivo or in vitro genetic therapy.

Protocols for genetic therapy through the use of viral vectors can be found, among other places, in Viral Vector Gene Therapy and Neuroscience Applications, Kaplit and Lowry, Academic Press, San Diego (1995). Gene therapy applications typically involve identifying target host cells or tissues in need of the therapy, designing vector constructs capable of expressing a desired gene product in the identified cells, and delivering the constructs to the cells in a manner that results in efficient transduction of the target cells. The cells or tissues targeted by gene therapy are typically those that are affected by the disease that the vector construct is designed to treat. For example, in the case of cancer, the targeted tissues are malignant tumors.

The genetic therapy methods of the present invention comprise the step of introducing a vector for the expression of a25-C and/or a25-D protein (or inhibitory anti-sense RNA) into a patient cell. The patient cell may be either in

the patient, i. e., in vivo genetic therapy, or external to the patient and subsequently reintroduced into the patient, i. e., in vitro genetic therapy. Diseases that may be treated by the subject genetic therapy methods include, but are not limited to epilepsy, chronic pain, ALS, mania, cancer, anxiety, diabetes, tremor, parkinsonism, migraine, ataxia, mood, sleep interference, multiple sclerosis and inflammation).

In a preferred aspect of the invention, a method is provided for protecting mammalian cells from abnormal levels of a28-C and/or a28-D in cells, comprising introducing into mammalian cells an expression vector comprising a DNA sequence substantially similar to the DNA sequence shown in SEQ ID NOS 3 or 4, that is operatively linked to a DNA sequence that promotes the expression of the DNA sequence and incubating the cells under conditions wherein the DNA sequence of SEQ ID NOS 3 or 4 will be expressed at high levels in the mammalian cells. Suitable expression vectors are as described above. In a preferred embodiment, the coding region of the human a25-C and/or a26-D gene r » CCjNn vactor under the transcriptional control ofthe --ive a25-C and/or a26-D MONumUtr 2 tion, a method is provided and/or a2b-D in VSCCs, .. ils an expression vector Nombredepages......... yA Nombre : antially similar to the DNA eratively linked to a DNA .......... DNA sequence. The cells BD/AB DNA sequence of SEQ ID DE 3 zmalian cells. ....... 3... uence consists essentially of ment, the expression vector DR \ < ... a25-D cDNA is operatively ...... s (CMV) promoter to allow SUR D antisense cDNA in a host Controle par............. Scaimc par. ... Nbre de copies Article 20

cell. In a preferred embodiment, the a25-C and/or a25-D adenoviral expression vector is introduced into cells by injection into a mammal.

Another aspect of the invention is to provide assays useful for determining if a compound of interest can bind to a25-C and/or a25-D proteins. This binding may interfere with, or mimic, the binding of ligands to the VSCCs, or this binding may affect the function of a28-C and/or a26-D in modulating calcium flux. The assay comprises the steps of measuring the binding of a compound of interest to an a25-C and/or a25-D protein. Either the a25-C and/or the a25-D protein or the compound of interest to be assayed may be labeled with a detectable label, e. g., a radioactive or fluorescent label, so as to provide for the detection of complex formation between the compound of interest and the a25-C and/or a26-D protein.

In another embodiment of the subject assays, the assays involve measuring the interference, i. e., competitive binding, of a compound of interest with the binding interaction between an a25-C and/or a25-D protein and a ligand already known to bind to a26-A protein. For example, the effect of increasing quantities of a compound of interest on the formation of complexes between radioactivity labeled ligand and an a25-C and/or a25-D protein may be measured by quantifying the formation of labeled ligand-a20-C and/or a25-D protein complex formation. In another embodiment of the subject assays, the assays involve measuring the alteration, i. e., non-competitive inhibition, of a compound of interest with the activity of a25-C and/or a25-D protein (compounds which bind to a different region of a26 and inhibit a25 activity, but don't prevent binding of ligands such as gabapentin).

Polyclonal antibodies to a25-C and/or a25-D proteins generally are raised in animals by multiple subcutaneous (se) or intraperitoneal (ip) injections of an a25 protein and an adjuvant. It may be useful to conjugate the a26-C and/or cl26- D protein or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, e. g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues),

N-hydroxysuccinimide (through lysine resides), glutaraldehyde, succinic anhydride, SOC12, or Rl~N=C=NR, where R and Rl are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 fig of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for anti-a28-C and/or o25-D protein antibody titer.

Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same a28-C and/or a25-D protein, but also may be conjugated to a different protein and/or through a different cross-linking reagent.

Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i. e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier"monoclonal"indicates the character of the antibody as not being a mixture of discrete antibodies. For example, the anti-a25- C and/or a28-D protein monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256: 495 (1975), or may be made by recombinant DNA methods [Cabilly, et al, U. S. Pat.

No. 4,816,567].

Antibodies can also be generated using phage display. In this approach libraries of peptides of random sequence are generated in antibody genes cloned into phage. These phage libraries are screened for antibodies by screening against the immobilized protein. (Hoogenboom-HR, Trends-Biotechnol. 1997 Feb; 15 (2): 62-70) In the hybridoma method, a mouse or other appropriate host animal, such a hamster is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro.

Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Coding, Monoclonal Antibodies : Principles and Practice, pp. 59-103 (academic Press, 1986)].

The nanti-arc and/or a28-D protein specific antibodies of the invention have a number of uses. The antibodies may be used to purify a25-C and/or a28- D proteins from either recombinant or non-recombinant cells. The subject antibodies may be used to detect and/or quantify the presence of a26-C and/or a28-D proteins in tissue samples, e. g., from blood, skin, and the like.

Quantitation of a26-C and/or a28-D proteins may be used diagnostically for those diseases and physiological or genetic conditions that have been correlated with particular levels of a26-C and/or a28-D protein expression levels.

In a further aspect, the present invention provides a diagnostic assay for detecting cells containing a28-C and/or a28-D deletions, comprising isolating total genomic DNA from the cell and subjecting the genomic DNA to PCR amplification using primers derived from the DNA sequence of SEQ ID NOS 3 or 4.

This aspect of the invention enables the detection of a26-C and/or a28-D deletions in any type of cell, and can be used in genetic testing or as a laboratory tool. The PCR primers can be chosen in any manner that allows the amplification of an a28-C and/or a28-D gene fragment large enough to be detected by gel electrophoresis. Detection can be by any method, including, but not limited to ethidium bromide staining of agarose or polyacrylamide gels, autoradiographic detection of radio-labeled a28-C and/or a28-D gene fragments, Southern blot hybridization, and DNA sequence analysis. In a preferred embodiment, detection is accomplished by polyacrylamide gel electrophoresis, followed by DNA sequence analysis to verify the identity of the deletions. PCR conditions are routinely determined based on the length and base-content of the primers selected according to techniques well known in the art (Sambrook et al., 1989).

An additional aspect of the present invention provides a diagnostic assay for detecting cells containing a28-C and/or a28-D deletions, comprising isolating total cell RNA and subjecting the RNA to reverse transcription-PCR amplification using primers derived from the DNA sequence of SEQ ID NOS 3 or 4. This

aspect of the invention enables the detection of a26-C and/or a26-D deletions in any type of cell, and can be used in genetic testing or as a laboratory tool.

Reverse transcription is routinely accomplished via standards techniques (Ausubel et al., in Current Protocols in Molecular Biology, ed. John Wiley and Sons, Inc., 1994) and PCR is accomplished as described above.

The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended hereto.

Examples Example 1 The sequence for human a28-A, c-DNA Accession No. M76559.1, was used to perform standard BLASTP searches against the Genbank non-redundant protein database and TBLASTN searches against the expressed sequence tag database (dbEST). Four full-length RNA sequences were identified (c-DNA Accession Nos. AF040709.1, AF042792.1, AF042793. 1, and AB011130. 1) which were highly homologous to a25-A. The DNA sequence of a28-B is provided by SEQ ID NO 1 and the amino acid sequence of a26-B is provided by SEQ ID NO 2. Using standard alignment tools, these four sequences were found to represent 4 different variants of the same gene. This gene was named a28-B.

Further searches of the sequence databases, and analysis of proprietary clustered sequences generated using Compugen software, led to the identification of additional sequences related to a2O-B. This includes human ESTs (Accession Nos. T80372.1, AA360556.1, AI563965. 1, N53512.1, a mouse EST (Accession No. AA000341.1), and a sequence from C. elegans (Accession No. CAA90091. 1).

Since the initial identification of a28-B, additional related sequences have been deposited into the Genbank database. These correspond to Accession Nos. ( human: AI027237. 1, AI026646. 1, AA994701.1, AA887514.1, AI275868. 1, AI675521. 1, AA906993.1, AA301068.1, AI884536. 1, AI862563. 1, AI191453. 1, AI241832. 1, AA534927.1, AA329137.1, AI586961. 1, AA394008.1,

AW007700.1, R38827.1, AA255807.1, H11152. 1, R60736.1, T16903.1, AA435601. AI094263. 1; mouse: AA008996.1; rat: AI105056. 1, AI502878. 1).

A26-B is 53 % identical and 69% similar at the amino acid level to a28-A.

The a28-B mRNA is 5482 bp long, and codes for a protein of 1145 amino acids.

The three splice-variants of a28-B which were identified differ only in the 5' untranslated region, and do not alter the amino acid sequence. A28-B aligns to genomic sequence from a previously published cosmid contig on human chromosome 3p21. 3. This DNA contig covers more than 600kb of sequence. The Accession Nos. for these genomic sequences are Z84493. 1, Z84494.1, Z75743.1, Z75742.1, and Z84492.1. Analysis of the DNA sequences flanking a28-B led to the identification of genes flanking a28-B on human chromosome 3p21. 3 which had been mapped in both human and mouse. These flanking genes include CIS, HyaLl, GNAI-2, and GNAT-1. In mouse, all of the flanking genes were localized to mouse chromosome 9, Analysis of mapping data stored in the Mouse Genome Database, by Jackson Laboratory, led to the identification of three mouse neurological phenotypes that had been genetically mapped to the same mouse chromosome 9,60cM region. These phenotypes include epilepsyl, ducky and tippy. Epilepsyl and ducky both have spike-wave activity consistent with epilepsy. This tentatively maps a28-B to the chromosome 9,60cM region in mouse, and identifies a28-B as a candidate gene for the mouse mutants ducky, tippy, and Ell (for overview of mapping data see Figure 1).

Northern and RT-PCR analysis of RNA expression levels of human a28-B were performed to analyze the expression pattern of a28-B. For Northern analysis, multiple tissue Northern blots and brain blots were obtained from Clontech. Non-isotopic DNA probes for a28-B were generated by PCR using SEQ ID NOS 7-8 and SEQ ID NO 1 as a template. Hybridization and washing conditions were in accordance with the manufacturer's instructions (Boehringer Mannheim). A28-B was found to have highest expression in lung, and was also detected in brain, heart, skeletal muscle, and at lower levels in all tissues tested (Figure 2). For the Northern blot surveying different areas of human brain, a28-B had the highest level of expression in the cerebral cortex, but was detected in all

areas of the human brain (Figure 2). RT-PCR expression analysis of human a28- B was also performed. RT-PCR analysis, using a cDNA tissue panel obtained from Clontech and SEQ ID NOS 7-8 for PCR-based gene amplication (cycles: <BR> <BR> <BR> <BR> 1X at 94 C 1', 35X at 94 C 0. 5', 55 C 1', 72 C 2' ; IX at 72 C 10'), produced an expression pattern for a28-B consistent with results from Northern analysis.

Overall, the expression pattern of a26-B is consistent with a proposed role of a28-B in epilepsy.

In order to determine if a28-B has functional properties comparable to a28-A, the ability of a28-B to bind to amino acids and gabapentin was measured.

For this analysis, COS-7 cells were transiently transfected with the full-length porcine a28-A, and human a28-B genes each in the vector pcDNA3 (Invitrogen) (pcDNA3.1 for a28-B) by the lipofectamine mediated transfection method. The cells were transfected and membranes harvested by the generic methods outlined below. The KD for binding of [3H] gabapentin to a28-B, as compared to a28-A, can be found in Table 1. Additional binding studies were performed using techniques similar to those outlined below. Alterations to the protocol are listed below under the subheading"Alternative Method for Measuring [3H] Gabapentin binding". The data for these binding studies are in figure 3. Overall, the binding and Western data demonstrated that the porcine a28-A and human a28-B full-length gene-products expressed transiently in the COS-7 system bind [3H] gabapentin with high affinity.

Table 1 : Saturation binding data for a28-B Porcine oc28-A Human ct26-B (n=2) (n=2) KD,23. KD,21.2 nM KD, 87.2 nM Mean = 22.2 nM Mean = 59. 9 nM

Transient Transfection method (150mm plate) 1 : Seed 3.9 x 106 COS-7 cells/150mm plate in 42ml DMEM + 10% FBS + 5u/ml Penicillin/5ßg/ml Streptomycin on 150mm plate. Grow O/N.

2: Steup Tube A-30ug DNA in 300ul TE + 1. Optimem (5u/ml Penicillin/ 5 gel Streptomycin) Tube B-150ul Lipofectamine + 1. Optimem (5u/ml Penicillin/ 5ug/ml Streptomycin) 3: At time=0 mix tubes A and B and leave at RT for 45 minutes.

4: Wash cells with 30ml Optimem (5u/ml Penicillin/5u. g/ml Streptomycin) twice then add 16. Optimem (5u/ml Penicillin/5, ug/ml Streptomycin) to the plates. At t=45 minutes add A/B mix to plates.

5 : At t=6 hours add 21ml of Optimem (5u/ml Penicillin/5Rg/ml Streptomycin).

6: At t=24 hours replace medium with 42ml Optimem (5u/ml Penicillin/ 5, ug/ml Streptomycin) 7: At t=48 hours rinse the cells twice with 20ml of PBS then harvest.

Membrane preparation (perform at 4°C) 1 Harvest cells into a 2ml eppendorf in 1.5ml 1mM EDTA/1mM EGTA/0. 1mM PMSF (added immediately prior to use from a 1000x stock)/20% Glycerol/lOmM HEPES pH7.4 @ 4°C using a cell scraper.

2 Mix cells end-over-end for 30 minutes at 4°C then centrifuge at 20,000 x g for 5 minutes.

3 Resuspend pellet in 1. 5ml ImM EDTA/lmM EGTA/20% Glycerol/lOmM HEPES pH7.4 @ 4°C then immediately re-centrifuge at 20, 000 x g for 5 minutes.

4 Resuspend pellet to-Img/ml (protein concentration as determined by the Bradford protein assay) in 1mM EDTA/lmM EGTA/20% Glycerol/lOmM HEPES pH7.4 @ 4°C For total [H3] binding, cells were sonicated for 30-40 seconds, centrifuged for 10'at 750-lOOOXg, and the supernatent was centrifuged for 30'at 50, 000Xg.

The resulting pellet was resuspended in 5mM f3HlGabapentin saturation binding assay methodotosv and data analysis Assays were carried out at 21°C in a final volume of 250ß1 in 96-well deep- well plates. Duplicate wells were set up for both'total'and'non-specific' binding. Specific binding was defined as that remaining after subtraction of the'non-specific binding'values from the'total'binding values. Assay components were added in the following order (all reagents were diluted in 10mM HEPES (pH 7.4 at 21°C)) : Total binding 2001l1 10mM HEPES pH 7. 4 Non-specific binding 175µl 10mM HEPES pH 7.4 and 25µl 100µM (S+)-3-isobutyl GABA 25p1 Appropriate COS membrane sample 251 [Hjgabapentin The reaction was incubated at 21°C for 45 minutes then filtered through GF/B filters soaked in 50 mM Tris-Cl pH 7.4 @4°C (wash buffer), filters were washed three times with wash buffer.

The filters were then counted in a scintillation counter.

Saturation experiments were performed with 12 duplicate data points ('Total'and'Non-Specific'binding determined in duplicate for each concentration of [3H] gabapentin tested) and a [3H] gabapentin concentration range from to 400nM. Data was analyzed using KEL-RADLIG for Windows.

Alternative Method for Measuring f3Hl Gabapentin binding The method described above was followed with the following exceptions: 1) COS7 transfection : 20ug of a2O-A or a2O-B plasmid DNA were incubated with 30ul of lipofectamine. The mixture was overlaid onto the cells in 1. 5ml of serum-free medium and incubated for 5 hours. Then FBS was added to the dishes to bring the final concentration to 10%. The medium was changed the next morning. Forty-eight hours after transfection the cells were harvested for membrane preparation.

2) Membrane preparation: Cells were washed twice with cold PBS and then scraped offthe tissue culture plates in cold 5mM of Tris/5mM EDTA (pH7. 4) containing PMSF (0. leupeptin (0.02mM), and pepstatin (0.02mM). The cells were incubated on ice for 30 minutes and then sonicated for 30-40 seconds. The homogenate was centrifuged for 10 minutes at 750-1000xg, and then the supernatent was centrifuged for 30 minutes at 50, 000xg. The resulting pellet was resuspended in the same buffer as described above.

3) Binding Assays: The radioligand binding assay was done using 0.05 mg of membrane protein incubated in the presence of 20 nM of [3H] gabapentin. The membranes were incubated in 10 mM Hepes (pH 7.4) for 40-50 minutes at room temperature, and then filtered onto pre- wetted GF/C membranes and quickly washed five times with 3ml of ice cold 50mM tris buffer pH7.4. The filters were dried and counted in a liquid scintillation counter. To determine background binding, 10 uM of isobutyl GABA was used and the resulting counts subtracted from the total counts of each sample.

Detection of a26-A and a26-B expression with anti-oc2 polyclonal antibodies Using affinity purified anti-a2 polyclonal antibodies (antigen derived from porcine a26-A ; See Brown and Gee (1998) JBC 273 25458-25465 for pAb

generation details) the expression of the porcine a28-A and human azS-B proteins (over and above control levels-COS cells transfected with the parent pcDNA3 vector) was confirmed. N. B. Cross reaction of the pAb's with a28-B was not unexpected given the-50% amino acid sequence identity. Furthermore, and with reference to Example 2, expression ouf 028- C was not detected using this antibody (sequence identity with OC25-A -30%).

Example 2 The sequence for human a2o-A, Accession No. M76559. 1, was used to perform standard BLASTP searches against the Genbank non-redundant protein database and TBLASTN searches against the expressed sequence tag database (dbEST). EST sequences were identified (Accession Nos. AA815447.

AA190607.1, AI223142. 1, AA188635.1, R43629.1, R20288.1, AA459684.1, AA662058.1, Z44942.1, Z40693.1, AI051759. 1) corresponding to a new gene, with similarity to (X28-A, named a2o-C. Additional searches of the sequence databases led to the identification of other sequences related to a28-C. This includes a mouse EST (Accession No. AU022914.1, AI843362. 1), and an STS (Accession No. G36524.1) which maps to human chromosome 3p21. 1. Since the initial identification of azb-B, additional related sequences have been deposited into the Genbank database. These correspond to Accession Nos. (human ESTs : AA459804.1, AI696320. 1, AI051759. 1, AI696214. 1; human genomic sequence: AC010180.1; mouse EST : AA445859.1, mouse RNA : AJ010949. 1).

In order to clone a full-length a2o-C, a PCR-based cDNA library screen was carried out by Origene using primers (SEQ ID NOS 9-10) based on sequence derived from EST clone accession number AA190607.1 which were designed to amplify a 273 bp fragment. A positive clone was identified in a kidney library.

After sequencing, this clone was identified as a novel 3'splice variant (SEQ ID NO 43). The protein sequence, which can be derived from SEQ ID NO 43, of this novel splice variant is a truncated, potentially secreted soluble form of a28-C.

PCR was performed, using primers (SEQ ID NOS 9 and 11) and a human adult

brain library from LTI as a template, and the resulting fragment of 248 bp was cloned in pBS and sequence verified. A SacI-NcoI fragment from the kidney clone, a NcoI-KpnI fragment from the PCR center clone, and a KpnI-NotI fragment from a clone obtained from the IMAGE consortium (corresponding to Accession No. R43629. 1) were ligated together, using methods standard to the art, to create a full-length clone. Each individual clone, and the full-length clone (SEQ ID NO 3), were sequence verified. A number of other EST clones from the IMAGE consortium were also obtained and sequenced. One of these clones (corresponding to Accession No. AI051759. 1) contained a two novel splice- variants which result in a truncated, potentially soluble CC25-C (SEQ ID NO 44).

Full-length a28-C is 28 % identical and 48% similar at the amino acid level to a28-A. The 028-0 mRNA sequence (SEQ ID NO 3) is 3770 bp long, and codes for a protein of 1085 amino acids (SEQ ID NO 5). In addition, three splice variants of a26-C were identified. Two of the variants contain deletions of internal exons. The third variant contains a novel 3'end. Two of these splice- variants produce a truncated protein which is devoid of the membrane anchoring delta subunit. These variants may represent a secreted alpha2 protein which could have additional functions beyond regulation of voltage-sensitive calcium channels.

In order to identify sequences for a28-C from other species, human and mouse specific primers (SEQ ID NOS 9-10 and 12-13, respectively) were used to amplify a28-C RT-PCR products. RNA from human brain was purchased from Invitrogen, Carlsbad, CA (catalog #D6030-15). RNA from rat and mouse brain was isolated using standard in-house protocols. First-strand cDNA synthesis was completed using Superscript Choice System (LTI, Bethesda, MD, catalog &num 18090-019). Ethanol precipitated cDNA was added to PCR mix containing 1X PCR buffer, 0.2mM dNTP, l Opmol/well forward primer, l Opmol/well reverse primer, and 0.5 units Platinum TAQ High Fidelity (LTI, Bethesda, MD).

Products were amplified at 95 °C for 5 minutes, followed by 35 cycles of 95 °C for 1 minute, 58° C for 1 min, 68° C for 2 minutes, and final extension at 72 °C for 10 minutes. PCR products were assayed on 1% agarose (TAE) gels at 100 volts for 45 minutes. Gels were visualized under UV and photographed. Products

were purified using Millipore Ultrafree-MC PCR purification filter units (catolog # UFC3LTKOO) prior to DNA sequence analyses. Using this approach, three sets of primers (SEQ ID NO 36,37; SEQ ID NO 12,13, SEQ ID NO 38, 39) where used for PCR amplification of rat a28-C. Three partial rat sequences for a28-C were identified (SEQ ID NOS 40, SEQ ID NO 14, SEQ ID NO 41).

RT-PCR analysis of RNA expression levels were performed to analyze the expression pattern of a28-C. cDNA Expression Panels were purchased from OriGene Technologies, Inc. (Rockville, Maryland). Human (catalog # HSC-101) and Mouse (catalog # MSCB-101) cDNAs from 24 tissue sources were pre- arrayed in a 96-well PCR format. PCR mix containing 1X PCR buffer, 0. dNTP, lOpmol/well forward primer, lOpmol/well reverse primer, and 0.5 units Platinum TAQ (LTI, Bethesda, MD) was added to each well. Products were amplified at 95 °C for 5 minutes, followed by 35 cycles of 95 °C for 1 minute, 58° C for 1 min, 68° C for 2 minutes, and final extension at 72 °C for 10 minutes.

PCR products were assayed on 1% agarose (TAE) gels at 100 volts for 45 minutes. Gels were visualized under UV and photographed. The primers used for this amplification from the human template correspond to SEQ ID NOS 9-10, and from the mouse template correspond to SEQ ID NOS 12-13. By RT-PCR, a28-C was found to be expressed in a wide variety of tissues (Table 2). The highest levels of a28-C were detected in human brain, and also in human testis and kidney. In addition to RT-PCR, the cDNA sequence for this gene has been detected in a human, adult brain library and also libraries from: infant brain, hNT neural cell line, testis, total fetus, alveolar rhabdomyosarcoma, adenocarcinoma, and a pooled germ cell library.

Northern blot analysis was performed using a28-C as a probe. Human total RNA was obtained from Invitrogen (Carlsbad, CA) (brain. total RNA (Cat &num D6030-01), kidney total RNA (Cat &num D6070-01), testis total RNA (Cat &num D6121- 01), liver total RNA (Cat # D6080-015)) or Ambion Inc (Austin, TX) (placenta total RNA Cat#7950, heart total RNA Cat &num 7966), lung total RNA (Cat #7968)) RNA was electrophoresed in formaldehyde agarose gels then transferred to charged nylon membranes (Ambion Inc. (Austin TX) Cat #10104. The EST clone (SEQ ID NO 47) was digested with BamHI and used as template in an RNA synthesis

reaction that yielded 32P labeled probe. The nylon membranes containing the RNA were prehybridized for 2 hours in ExpressHyb hybridization solution (Clontech Inc. (Palo Alto, CA) (Cat # 8015-1). After the prehybridization 4X106 cpm of RNA probe labeled with 32P wre added to the solution and the hybridization was performed in the same solution for 2 hours. After hybridization the nylon filter was washed for 1 hour with 4 changes of 2X SSC, 0. at room temperature. The nylon filter was transferred to a solution of 0. 2X SSC, 0.5% SDS at 68°C and washed with 4 changes of solution. The nylon filters were then exposed to phosphoroimager screens Molecular Dynamics (Sunnyvale, CA) and read on a Storm phosphorimager. Molecular Dynamics (Sunnyvale, CA).

Results from Northern analysis (Figure 4) indicate that a25-C has highest levels of expression in human brain, kidney, and testis.

Since 0. 28-0 has sequence homology to a26-A, and CC25-A functions as a subunit of VSCCs, experiments were undertaken to determine if a25-C can replace (X25-A and produce functional VSCCs. Xenopus oocytes were isolated using standard techniques and injected with cRNA for alB, Pic and a28- C subunits of voltage-gated Ca2+ channels. Four days to 1 week following injection of cRNA, Ca2+ channel currents were measured using two-electrode voltage clamp with 5 mM Ba2+ as the charge carrier. Test pulses to +10 mV from a holding membrane potential of-80 mV were applied to evoke Ca2+ channel currents. Peak inward currents evoked during the test pulse were measured. The amplitude of inward currents is proportional to the expression level of voltage- gated Ca2+ channels.

Expression of 0. 13, Pic without α2# subunits produced currents with an average amplitude of 105 13 nA (n=20). Co-injection of α2#o with als and Pic subunits produced a significant increase in current amplitude to 213 12 nA (n=20, p < 0.01 compared to no a28 subunits). These data suggest that 0. 280 has an effect on Ca2+ channels similar to α2#A, enhancing the level of channel expression. However, U-25,, did not produce as large of an effect on channel expression as a2bA"producing a 2-fold increase in current compared to a 20-fold increase observed with the co-injection of a28A. Overall, these initial functional

studies indicate that a26-C can replace a26-A in voltage-sensitive calcium channels after co-injection into Xenopus oocytes with the al and beta subunits.

Table 2: RT-PCR EXPRESSION PROFILE FOR ALPHA2-DELTA C Tissue Human a2d-C Mouse a2d-C Brain +++ + Heart ++++ Kidney ++ ++ Liver Colon + not assayed Lung + ++ Small Intestine ++ + Muscle ++++ ++ Stomach ++ Testis +++ ++ Placenta ++ not assayed Salivary Gland ++ not assayed Thyroid Gland ++ not assayed Adrenal Gland _ Pancreas ++ not assayed Ovary ++ Uterus ++ Prostrate ++ ++ Skin ++ PBL-not assayed Bone Marrow-not assayed Fetal Brain ++ not assayed Fetal Liver ++ not assayed Example 3 The sequence for human a28-A, Accession No. M76559.1, was used to perform BLASTP searches against the Genbank non-redundant protein database

and TBLASTN searches against the expressed sequence tag database (dbEST).

EST sequences were identified (Accession No. T70594.1, T96901.1, AA766033.1, AI160471. 1, AA719773.1, AI003601. 1, AA442451.1, AA521470.1, AA770076.1, AA001411.1, AA001473.1, W22650.1, H86016.1) corresponding to a new gene, with similarity to a26-A, named a26-D. Additional searches of the sequence databases led to the identification of other sequences related to a25-D.

This includes genomic sequence derived from human chromosome 12pl3. 3 (Accession No. AC005342.1, AC005343.1). Since the initial identification of a26-D, additional related sequences have been deposited into the Genbank database. These sequences correspond to Accession Nos. (human ESTs : T96900.1, AI457823. 1, AI377638. 1, and AI433691. 1).

To isolate a full-length a26-D clone, a PCR-based cDNA library screen was carried out by Origene using primers (SEQ ID NOS 18-19) based on sequence derived from EST clone Accession No. AA001473.1 which were designed to amplify a 372 bp fragment. A positive clone was identified in a placental library, and was confirmed using a nested internal primer (SEQ ID NO 20). This clone was fully sequenced. The sequence extended 350 bp 5'of the sequence obtained from the EST sequences, but did not include the 5'end.

To obtain the 5'end, two approaches were undertaken. One approach utilized 5'RACE (Rapid Amplification of cDNA Ends). For 5'RACE, placenta poly A+ RNA from Clontech was used to construct a RACE-ready cDNA library using a Marathon cDNA Amplification kit purchased from Clontech. The 5'-end sequence of a28-D was obtained by 5'RACE PCR using first set of primers: Marathon cDNA adapter primer 1 (SEQ ID NO 45) and gene specific primer I (SEQ ID NO 21). The PCR product was re-amplified using a set of nested primers: adapter primer 2 (SEQ ID NO 46) and gene specific primer II (SEQ ID NO 22). A resulting 1 kb PCR product was cloned into a TA vector (Invitrogen) and sequenced. Sequence analysis revealed that it contains the 5'sequence of a25-D.

A second method undertaken to identify the 5'end of a28-D was a PCR- based library screen performed by Edge, using the 5'most sequence known for a25-D. Nine clones were PCR amplified by the methods indicated above, for

verification using primers with SEQ ID NOS 48 and 49. These nine positive clones were then sequenced for verification by standard methods. All nine clones were identical to each other, and all were short of the 5'end by approximately 500 bp. However, these clones contained novel splice-variants of a26-D, with insertions of novel nucleotide sequences (SEQ ID NO 16).

The full-length sequence of a26-D is 28 % identical and 47% similar at the amino acid level to ct26-A. The a26-D mRNA is 5,073 bp long (SEQ ID NO 4), and codes for a protein of 1120 amino acids (SEQ ID NO 6). In addition, two splice variants of a2O-D were identified. One of the variants contains a 72 bp deletion of an internal exon (SEQ ID NO 15). The amino acid sequence of this variant can be found in SEQ ID NO 17. The second variant contains two novel insertions, one of 338 bp and one of 305 bp (SEQ ID NO 16). These insertions appear to result in a truncated protein (SEQ ID NO 42), comparable to the truncated protein sequence identified for a26-C in Example 2.

RT-PCR analysis of RNA expression levels of human a28-D were performed in order to analyze the tissue distribution ofa25-D. cDNA Expression Panels were purchased from OriGene Technologies, Inc. (Rockville, Maryland).

Human (catalog # HSC-101) and Mouse (catalog # MSCB-101) cDNAs from 24 tissue sources were pre-arrayed in a 96-well PCR format. PCR mix containing 1X PCR buffer, 0. 2mM dNTP, lOpmol/well forward primer, lOpmol/well reverse primer, and 0.5 units Platinum TAQ (LTI, Bethesda, MD) was added to each well.

Products were amplified at 95 °C for 5 minutes, followed by 35 cycles of 95 °C for 1 minute, 58° C for 1 min, 68° C for 2 minutes, and final extension at 72 °C for 10 minutes. PCR products were assayed on 1% agarose (TAE) gels at 100 volts for 45 minutes. Gels were visualized under W and photographed. In the case of the a28-D human panels two separate sets of primers were used to distinguish splice variants and wild type species (SEQ ID NOS 18 & 20, SEQ ID NOS 23 & 19, respectively).

Analysis of the results from RT-PCR of () C25-D (see Table 3) indicate that a28-D is expressed in a wide variety of tissues, with highest levels in placenta, adrenal gland and pancreas, but also detected in all tissues other than colon. Of note, a28-D was detected in human brain, consistent with a potential role in

neurological disease. In addition, based on the tissue distribution of EST sequences, the cDNA sequence for ad has been detected in human libraries from: adult brain, retina, fetal liver/spleen, fetal heart, pineal gland, and testis.

Table 3. RT-PCR EXPRESSION PROFILE FOR ALPHA2-DELTA D Tissue Human α2#-D ** Human α2#-D <BR> <BR> <BR> <BR> <BR> <BR> Brain +++ +++<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> Hart...<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <P>Kidney +++* Liver ++ Colon Lung ++ Small Intestine +* Muscle _ Stmach ++ - Testis +++ - Placenta ++++* Salivary Gland ++++ Thyroid Gland +++ ++++ Adrenal Gland ++++ +++ Pancreas ++++* ++ Ovary ++* ++ Uterus +* ++ Prostrate ++* + Skin + -

Pal--<BR> <BR> <BR> <BR> <BR> <BR> BoneMarrow l l l Fetal Brain +++ Fetal Liver ++ *2 products: wt and splice variant ** Primers d2+dhD-2 detects splice region Example 4. Knockout of a28-B In order to create a mouse knockout of a26-B, Genome Systems (Catalog: BAC 4922 Mouse ES 129Svj PCR based Library Screen) performed a PCR- based screen of a mouse BAC library using primers SEQ ID NOS 25-26, which were predicted to amplify an 650 bp cDNA or genomic fragment. One positive BAC clone (Genome Systems DNA control number: BAC-22401) from this screen was received. The same primers were used to generate a human DNA probe. This probe was used on a Southern blot to identify a-10kb HindIII mouse genomic fragment from the BAC, which was subcloned into the HindII site of plasmid vector pRS416 (Stratagene). Two separate subclones were sequenced by standard techniques, using the T3 and T7 primers and SEQ ID NOS (25-32).

Two 500 bp regions of sequence from the 5'and 3'ends of the 10kb genomic fragment (SEQ ID NOS 33 and 34, respectively), plus a 1. 8kb sequence contig (SEQ ID NO 35) were identified. This genomic sequence can be used to identify the intron/exon structure of a portion of mouse a26-B gene, and may contain regulatory elements important for a28-B gene expression.

Example 5. Identification of amino acids encoded by a28 gene The amino acid sequences of a26-C and a28-D, indicated in SEQ ID NOS 5 and 6, were determined by translating the nucleotide sequences described in SEQ ID NOS 3 and 4, and aligning the amino acid sequences of a26-A, a26-B, a28-C, and a25-D. The correct open reading frame for each amino acid sequence was determined based on homology of the amino acid sequences to other a26-A

homologs. At the amino acid level, a28-C is 28% identical and 48% similar to a25-A and is 28% identical and 47% similar to a25-B, and a2§-D is 28% identical and 47% similar to a2§-A and is 28% identical and 46% similar to a26- B. Although a25-C and a28-D are related to a28-A, they are distinctly new and different genes.

Example 6. Proposed method of detecting the a28-C and a28-D proteins by using an a28-C and a28-D antibody Antibodies could be developed which specifically detect epitopes unique to a28-C and a28-D, or which detect all a28 proteins. These antibodies could be developed by either synthesizing a peptide which is identical to a28-C and/or a26-D, or by bacterially-expressing a fusion protein containing either a28-C or a28-D, and then injecting these peptides into a research animal in order to stimulate an immunogenic response. Antibodies generated in such a manner could be used to detect levels of a28-C and/or a28-D protein in cells. This could be done by immunocytochemistry, where whole cells are fixed and then the antibody is used on the whole cells to detect expression of α2#-C or a28-D, and to detect the subcellular localization of a28-C or a28-D. Or, cells may be lysed and protein extracts generated and analyzed for α2#-C and/or a28-D expression.

Example 7. Isolation of RNA for cDNA Library In order to isolate a26-C or a28-D from cells, RNA could be isolated by lysing cells from any tissue of interest using standard methods known in the field.

After isolation, RNA is reverse-transcribed into cDNA using the enzyme reverse transcriptase and a poly (T) primer or a mix of random primers. A mix opf cDNA is produced, representing a large number of the genes which are expressed in the beginning cell population at a particular point in time. Once the cDNA pool has been created, it can be restricted and then ligated into a cloning vector using methods standard in the field. This results in a cDNA library.

Example 8. cDNA Cloning Procedure A28-C or a28-D could be cloned from a cDNA library, created as above, by using primers specific for a28-C or a28-D nucleotide sequences in a polymerase chain reaction, with the cDNA used as a template. Alternatively, a25-C or a28-D sequences could be used as a probe in order to screen the cDNA library by hybridization. Using either technique, single clones are ulimately isolated from the library and sequenced using standard techniques. By sequencing multiple clones from a library, one could look for the existence of alternatively- spliced variants of a28-C or a28-D, or for the existence of single nucleotide polymorphisms, or for mutations/alterations in a28-C or a28-D.

Example 9. Screening cDNA Library with Antibody A cDNA library could also be screened by using an antibody to a28-C or a28- D. The cDNA library is cloned into a vector which allows induction of protein expression of the cloned inserts. The complete cDNA library is induced to express a protein representing the cloned insert, then single clones which contain an insert that codes for a28-C or a28-D are identified if they hybridize to an antibody generated against a28-C or a28-D. Positive clones are isolated, and then sequenced using standard methods.

It is to be understood that the invention is not to be limited to the exact details of operation, or to the exact compounds, compositions, methods, procedures or embodiments shown and described, as obvious modifications and equivalents will be apparent to one skilled in the art, and the invention is therefore to be limited only by the full scope of the appended claims.