WO1999021983A1 - Human atrophin-1 related gene - Google Patents

Abstract

Human atrophin-1 related gene polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing human atrophin-1 related gene polypeptides and polynucleotides in the design of protocols for the treatment of Charcot-Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Corneal dystrophy, Dentatorubral pallidoluysian atrophy and atxia, among others, and diagnostic assays for such conditions.

Description

HUMAN ATROPHIN-1 RELATED GENE

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the atrophin family, hereinafter referred to as human atrophin-l related gene. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Atrophin- 1 is the gene responsible for dentatorubral pallidoluysian atrophy (DRPLA) and Haw River sydrome. As one EST (M78755) was 64% identical to atrophin-l, it is proposed that a novel gene family of atrophin exists. Atrophin- 1 related gene was cloned in rat by Khan. This indicates that the atrophin family has an established, proven history as therapeutic targets . Clearly there is a need for identification and characterization of further members of the atrophin family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, Charcot-Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral pallidoluysian atrophy and atxia.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to human atrophin-l related gene polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such human atrophin-l related gene polypeptides and polynucleotides. Such uses include the treatment of Charcot-Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral pallidoluysian atrophy and atxia, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with human atrophin-l related gene imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate human atrophin- 1 related gene activity or levels .

DESCRIPTION OF THE INVENTION Definitions The following definitions are provided to facilitate understanding of certain terms used frequently herein.

"Human atrophin-l related gene" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allelic variant thereof. "Human atrophin-l related gene activity or human atrophin-l related gene polypeptide activity" or "biological activity of the human atrophin-l related gene or human atrophin-l related gene polypeptide" refers to the metabolic or physiologic function of said human atrophin-l related gene including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said human atrophin-l related gene.

"Human atrophin-l related gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:l or allelic variants thereof and/or their complements.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND

MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al, "Protein Synthesis: Posttranslational Modifications and Aging", Ann NYAcad Sci (1992) 663:48-62.

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Identity" is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SUM J Applied Math (1988) 48: 1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H, and Lipton, D., SIAM J Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al, Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al, JMolec Biol (1990) 215:403). As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Polypeptides of the Invention

In one aspect, the present invention relates to human atrophin-l related gene polypeptides (or human atrophin-l related gene proteins). The human atrophin-l related gene polypeptides include the polypeptide of SEQ ID NO:2; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO:2 over its entire length, and still more preferably at least 90-91% identity, and even still more preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with at least 97-99% are highly preferred. Also included within human atrophin-l related gene polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO:2 over its entire length, and still more preferably at least 90-91% identity, and still more preferably at least 95% identity to SEQ ID NO:2. Furthermore, those with at least 97-99% are highly preferred. Preferably human atrophin-l related gene polypeptide exhibit at least one biological activity of human atrophin-l related gene.

The human atrophin-l related gene polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Fragments of the human atrophin-l related gene polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned human atrophin-l related gene polypeptides. As with human atrophin-l related gene polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of human atrophin-l related gene polypeptide. In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of human atrophin-l related gene polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate human atrophin-l related gene activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human. Preferably, all of these polypeptide fragments retain the biological activity of the human atrophin-l related gene, including antigenic activity. Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions - i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.

The human atrophin-l related gene polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

Polynucleotides of the Invention Another aspect of the invention relates to human atrophin- 1 related gene polynucleotides. human atrophin-l related gene polynucleotides include isolated polynucleotides which encode the human atrophin-l related gene polypeptides and fragments, and polynucleotides closely related thereto. More specifically, human atrophin-l related gene polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a human atrophin-l related gene polypeptide of SEQ ID NO: 2, and polynucleotide having the particular sequence of SEQ ID NO:l. human atrophin-l related gene polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the human atrophin-l related gene polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to of SEQ ID NO: 1 over its entire length. In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred. Also included under human atrophin-l related gene polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO:l to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such human atrophin-l related gene polynucleotides.

Human atrophin-l related gene of the invention is structurally related to other proteins of the atrophin family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1) encoding human atrophin-l related gene. The cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 565 to 3600) encoding a polypeptide of 1012 amino acids of SEQ ID NO:2. The amino acid sequence of Table 2 (SEQ ID NO:2) has about 49 3% identity (using FASTA) in 858 aπnno acid residues with human atrophin-l protein (S. Nagafuchi e al. Nat. Genet. 8:177-182, 1994). Furthermore, human atrophin-l related protein is about 90% identical to rat atrophin-l related protein over 1012 amino acid residues (F.A. Khan et al. Neurobiol Dis 3(2).121-128, 1996). The nucleotide sequence of Table 1 (SEQ ID NO.1) has about 57.0% identity (using FASTA) in 1307 nucleotide residues with human atrophin-l gene (R. L. Margohs et al. Brain Res. Mol. Brain Res. 36(2).219-226, 1 96). Furthermore, human atrophin- 1 related gene is 83 4% identical to rat atrophin- 1 related gene over 2843 nucleotide residues (F.A. Khan et al. Neurobiol Dis. 3(2):121-128, 1996). Thus, human atrophin-l related gene polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art

Table 1'

GAGCCGGAGCCCAGTGACAGGCGACGGCCTCTTGCCGCCCCGGACCCCGGGCGCTTACCTGTGGCTGCTGGGGTCGGCCG

CCCGGGACCAGGCTGGGAGCCCCGAGAAACGCTTCGTTAAGGGACTCAGGCAGTACGGGAAGAACTTCTTCAGAATTAGA

AAGGAGCTGCTTCCCAATAAGGAAACAGGGGAGCTGATCACCTTCTATTACTATTGGAAGAAGACCCCCGAAGCAGCCAG

CTCCCGAGCCCATCGTAGGCACCGCAGGCAGGCCGTGTTCAGGAGGATTAAGACTCGCACCGCGTCCACACCCGTCAACA

CACCCTCCAGACCCCCGTCCAGTGAATTCTTGGACCTAAGTTCAGCCAGTGAAGATGACTTCGACAGTGAGGACAGTGAG

CAGGAGTGAAGGGGTACGCCTGCCGCCACTGCTTCACCACCACCTCCAAAGATTGGCACCACGGAGGCCGGGAGAACATC

CTGCTTTGCACCGACTGTCGCATCCACTTCAAGAAATACGGTGAGCTCCCGCCCATTGAGAAGCCCGTGGACCCGCCACC

GTTTATGTTCAAACCCGTCAAGGAAGAGGATGATGGGCTCAGTGGGAAGCATAGCATGAGGACACGGCGGAGTCGGGGCT

CGATGTCGACACTACGCAGTGGTGGAAGAAGCAGCCAGCCAGCCCTGGATGGTCGCACCTCACCCATCAATGAAGACATC

CGCTCCAGCGGCCGGAACTCCCCCAGCGCTGCCAGTACCTCCAGCAATGACAGTAAAGCAGAGACAGTGAAGAAGTCGGC

CAAGAAGGTGAAGGAGGAAGCTTCTTCCCTTCTTAAGAGTAACAAACGCCAGCGGGAGAAGGTGGCCTCTGATACGGAGG

AGGCTGACAGGACCAGCTCCAAGAAGACAAAAACGCAGGAGATCAGCAGGCCCAACTCGCCATCTGAAGGTGAGGGAGAG

AGTTCAGACAGTCGCAGCGTCAACGATGAGGGTAGCAGTGACCCCAAAGACATCGACCAGGACAATCGCAGCACGTCCCC

GAGCATCCCCAGCCCCCAGGACAATGAGAGTGACTCGGACTCGTCAGCCCAGCAGCAGATGCTGCAGGCCCAGCCCCCAG

CCTTGCAGGCTCCCACTGGGGTCACCCCAGCTCCCTCCTCAGCTCCTCCAGGGACCCCTCAGCTGCCCACGCCAGGGCCC

ACGCCCTCTGCCACTGCAGTTCCCCCACAGGGCTCCCCCACGGCCTCCCAGGCCCCTAACCAGCCACAGGCTCCCACAGC

GCCTGTTCCCCACACCCACATCCAACAGGCACCGGCCTTGCACCCCCAGCGGCCGCCCTCACCGCATCCCCCGCCGCATC

CCTCGCCACATCCCCCACTGCAGCCTCTGACTGGGTCGGCGGGCCAGCCTTCTGCACCCTCTCATGCCCAGCCCCCACTG

CACGGTCAGGGCCCACCCGGCCCTCACAGCCTGCAGGCTGGGCCCCTGCTGCAGCACCCAGGCCCCCCACAGCCCTTTGG

CCTCCCTCCCCAGGCCTCCCAAGGCCAGGCCCCTCTGGGGACCTCCCCAGCAGCAGCGTACCCTCACACCTCCCTGCAGC

TGCCAGCCTCTCAGTCAGCGCTGCAGTCCCAACAGCCTCCACGGGAGCAGCCCCTGCCACCAGCGCCCTTGGCCATGCCC

CACATCAAGCCCCCGCCTACCACTCCCATCCCCCAGCTGCCGGCGCCACAGGCCCACAAGCACCCTCCCCACCTCTCGGG

GCCCTCACCCTTCTCCATGAATGCCAACCTGCCTCCCCCTCCAGCCCTGAAGCCCCTGAGCTCCCTGTCCACACATCACC

CCCCGTCGGCTCACCCCCCACCCCTGCAACTCATGCCTCAGAGCCAGCCATTGCCCTCCTCGCCCGCCCAGCCCCCCGGG

CTGACCCAGAGCCAGAACCTGCCCCCGCCCCCTGCCTCCCACCCCCCTACAGGCCTCCACCAGGTGGCCCCCCAACCCCC

GTTTGCTCAGCACCCCTTTGTCCCTGGAGGCCCTCCTCCCATCACCCCTCCGACCTGCCCCTCCACCTCTACCCCACCGG

CGGGACCTGGCACCTCGGCCCAGCCACCCTGCTCTGGTGCGGCGGCTTCAGGAGGCAGCATAGCGGGGGGGTCGTCCTGC

CCACTCCCCACCGTCCAGATCAAGGAGGAGGCTCTGGACGACGCTGAGGAGCCTGAGAGCCCCCCTCCCCCACCAAGGAG

CCCGTCCCCGGAGCCCACTGTGGTGGACACCCCCAGTCACGCCAGCCAGTCAGCTAGGTTCTACAAACACCTGGACCGGG

GCTACAACTCGTGTGCCCGGACAGACCTGTACTTCATGCCTCTGGCCGGGTCCAAGCTGGCCAAGAAGAGGGAGGAGGCC

ATTGAGAAGGCCAAGCGCGAGGCTGAGCAGAAAGCCCGAGAGGAGCGAGAGCGGGAGAAGGAGAAGGAGAAGGAGCGGGA GCGGGAGCGAGAGCGGGAGCGCGAGGCAGAGCGGGCGGCTAAGGCGTCCAGCTCAGCGCATGAAGGTCGCCTCAGTGACC

CACAGCTCAGTGGTCCTGGCCACATGCGGCCATCCTTCGAGCCACCACCAACCACCATTGCTGCTGTGCCCCCCTACATC

GGGCCCGACACACCTGCCCTTCGGACTCTGAGCGAGTACGCCCGGCCCCACGTCATGTCGCCCACCAACCGCAACCACCC

CTTCTACATGCCCCTTAACCCCACGGACCCCCTGCTGGCCTACCACATGCCTGGCCTCTACAACGTCGACCCCACCATCC

GCGAGCGGGAGCTCCGGGAGCGGGAGATCCGAGAGCGGGAGATCCGAGAGCGGGAGCTGCGGGAGAGGATGAAGCCGGGC

TTCGAGGTGAAGCCCCCAGAGCTGGACCCCCTGCACCCAGCCGCCAACCCCATGGAGCACTTTGCCCGGCACAGCGCCCT

CACCATCCCCCCGACCGCCGGGCCCCACCCTTTTGCTTCTTTCCACCCGGGCCTGAACCCCTTGGAGAGGGAGAGACTGG

CCCTGGCGGGCCCCCAGCTGCGGCCCGAGATGAGCTACCCTGACAGACTGGCAGCCGAGCGTATCCACGCAGAGCGCATG

GCATCGCTGACCAGCGATCCCCTGGCCCGACTGCAGATGTTCAACGTGACTCCGCACCATCACCAGCACTCTCACATTCA

CTCCCACCTCCACCTCCACCAGCAGGACCCCCTCCACCAAGGTTCAGCAGGCCCCGTTCACCCGCTGGTCGACCCCCTGA

CTGCCGGTCCCCACCTGGCTCGCTTCCCCTACCCGCCTGGCACTCTCCCCAACCCTCTGCTTGGACAGCCCCCACACGAG

CACGAGATGCTTCGCCACCCAGTTTTCGGCACCCCCTACCCCCGTGACCTGCCTGGGGCCATCCCACCCCCCATGTCAGC

AGCCCACCAGCTGCAGGCCATGCATGCCCAGTCGGCCGAGCTGCAGAGACTGGCCATGGAGCAGCAGTGGCTGCATGGAC

ACCCCCACATGCATGGTGGCCACCTACCAAGTCAGGAAGATTATTACAGTCGACTGAAGAAAGAAGGTGACAAGCAGTTA

TAAGTTATTTATTTGTTAACGCTGGCTGTGGAAACCCCAGTTCTTGGGGGGAGAAACAGGACTTTTTACATAAAATAGGA

GCTGCAAAAGCAAAAAGAATATCTTCTAAAGATTCTTTATATATTTAAAAACCCACAACTAAAAATGTATCCACATAGTA

GTGTTCGTTTGTCGAGAGGATTTCCTGAGACTGGTTTGGATCTCCCTGCATGACAGTCCCCCAGAAACTTAGTGAGTCCT

GGACTGGACTGAACATCCAGAAAGCTTCCCTGCAATCTTGGGGTTTGGCTTTAGTTTTCTTTTCCTTGATTTCTCAGTAG

GTGCTAGAATCCAGTTCACACCCTTCACTGTGCGTGCAGACACACTGACACACTCCGCCACGAGTGCTCCAGAGCCCACG

AGGCTTGCAGATCGGGGGCATAGGAATTTGGAATCCAAGAGCTATAATTTTTAAAAAAAAAAAAATCTTTTATTTTAATA

CATTGTAGGAAATCTTCATAATTGGAGAAAGTTCTGCAGCATGGCTTTTTACGTCTGTAAATAAATAATTTTAGAACAGC

CTTTTTTTCTTCCATAAACTACTATTGTGATCTATTTTTTCCAGCCATGTCACTAATTGTGAATTCCTACCAACTATTGA

CAGAATACAGAGTTGATTTTTTAATAAAAAGTTATATATAATTATCCCTTTAATTAAAGGGAGCAAAGGGGCGTTCCACA

TGGACAGAGGCTTGGACCGAGGCCTGGTCACAGCAGCGAGCATCCAGGGTTTGCAGGGACGATGTTACAGACTCTGTTTT

CTGCCTGGCGTTTCACTTGTGTCTGCTCCTAGCCTGTGCTCTGCCAGCAGCACAGACATCTGCTCCATCAGACCTCTTCC

ATTTTGCACAGGGAGTGCAGGAGGTGAATGTTCACTTTCTGTTCTCCAGTGTCACTGTTCTGTTTCCACGGGATGGAAAG

CGCATGGGCCTGTGTCCATTGTAGATTTCCTTCTAGAT

A nucleotide sequence of a human atrophin-l related gene (SEQ ID NO: 1).

Table 2^b

MFKPVKEEDDGLSGKHSMRTRRSRGSMSTLRSGGRSSQPALDGRTSPINEDIRS5GRNSPSAASTSSNDSKAETVKKSAK

KVKEEASSLLKSNKRQREKVASDTEEADRTSSKKTKTQEISRPNΞPSEGEGESSDSRSV DEGSSDPKDIDQDNRSTSPS

IPSPQDNESDSDSSAQQQMLQAQPPALQAPTGVTPAPSSAPPGTPQ PTPGPTPSATAVPPQGSPTASQAPNQPQAPTAP

VPHTHIQQAPALHPQRPPSPHPPPHPSPHPPLQPLTGSAGQPSAPSHAQPPLHGQGPPGPHSLQAGPLLQHPGPPQPFGL

PPQASQGQAPLGTSPAAAYPHTS QLPASQSAQSQQPPREQPLPPAP AMPHIKPPPTTPIPQLPAPQAHKHPPHLSGP

SPFSMNAN PPPPALKPLSSLSTHHPPSAHPPPLQLMPQSQPLPSSPAQPPGLTQSQNLPPPPASHPPTGLHQVAPQPPF

AQHPFVPGGPPPITPPTCPSTSTPPAGPGTSAQPPCSGAAASGGSIAGGSSCPLPTVQIKEEA DDAEEPESPPPPPRSP

SPEPTVVDTPSHASQSARFYKH DRGYNSCARTDLYFMPIAGSK AKϊCREEAIEKAKREAEQKAREEREREKEKEKERER

EREREREAERAAKASSSAHEGR SDPQ SGPGHMRPSFEPPPTTIAAVPPYIGPDTPA RTLSEYARPHVMSPTNRNHPF

YMPLNPTDPL AYHMPGLYNVDPTIRERELREREIREREIRERE RERMKPGFEVKPPELDPLHPAA PMEHFARHSALT

IPPTAGPHPFASFHPGLNPLERERLALAGPQLRPEMSYPDRLAAERIHAERMASLTSDPLARLQMFNVTPHHHQHSHIHS

HLHLHQQDP HQGSAGPVHPLVDPLTAGPHLARFPYPPGTLPNPLLGQPPHEHE LRHPVFGTPYPRDLPGAIPPPMSAA

HQ QAMHAQSAELQRLAMEQQWHGHPHMHGGH PSQEDYYSRLKKEGDKQL ^b An amino acid sequence of a human atrophin-l related gene (SEQ ID NO: 2).

One polynucleotide of the present invention encoding human atrophin-l related gene may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human heart and testis using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252:1651-1656; Adams, M.D. et al, Nature, (1992) 555:632-634; Adams, M.D., et al, Nature (1995) 377 Supp:3-174). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.

The nucleotide sequence encoding human atrophin-l related gene polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 565 to 3600 of SEQ ID NO: 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. When the polynucleotides of the invention are used for the recombinant production of human atrophin-l related gene polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatlAcadSci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding human atrophin-l related gene variants comprise the amino acid sequence human atrophin-l related gene polypeptide of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination. The present invention further relates to polynucleotides that hybridize to the herein above- described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences.

Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding human atrophin-l related gene polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the human atrophin- 1 related gene Such hybndization techniques are known to those of skill in the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent The probes generally will compπse at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly prefeπed probes will range between 30 and 50 nucleotides

In one embodiment, to obtain a polynucleotide encoding human atrophin- 1 related gene polypeptide, mcludmg homologs and orthologs from species other than human, comprises the steps of screening an appropπate hbrary under stingent hybndization conditions with a labeled probe having the SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence Thus in another aspect, human atrophin- 1 related gene polynucleotides of the present invention further include a nucleotide sequence compnsmg a nucleotide sequence that hybπdize under stringent condition to a nucleotide sequence having SEQ ED NO 1 or a fragment thereof Also included with human atrophin- 1 related gene polypeptides are polypeptide compnsmg ammo acid sequence encoded by nucleotide sequence obtained by the above hybndization condition Such hybndization techniques are well known to those of skill m the art Stringent hybndization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising 50% formamide, 5xSSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA. followed by washing the filters in 0 lx SSC at about 65°C

The polynucleotides and polypeptides of the present mvention may be employed as research reagents and mateπals for discovery of treatments and diagnostics to animal and human disease

Vectors, Host Cells, Expression

The present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engmeered with vectors of the mvention and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs deπved from the DNA constructs of the present mvention

For recombinant production, host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention Introduction of polynucleotides into host cells can be effected by methods descπbed m many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE- extran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells. A great variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If the human atrophin-l related gene polypeptide is to be expressed for use in screening assays, generally, it is prefeπed that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If human atrophin-l related gene polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

Human atrophin-l related gene polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of human atrophin-l related gene polynucleotides for use as diagnostic reagents. Detection of a mutated form of human atrophin-l related gene gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of human atrophin-l related gene. Individuals carrying mutations in the human atrophin-l related gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled human atrophin-l related gene nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al, Science (1985) 230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method. See Cotton etal., ProcNatlAcadSci USA (1985) 85: 4397-4401. In another embodiment, an array of ohgonucleotides probes comprising human atrophin-l related gene nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al. Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining a susceptibility to Charcot- Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Corneal dystrophy, Dentatorubral pallidoluysian atrophy and atxia, through detection of mutation in the human atrophin- 1 related gene by the methods descπbed

In addition, Charcot-Maπe-Tooth disease, Schwartz-Jampel syndrome, Crystalline Corneal dystrophy, Dentatorubral pallidoluysian atrophy and atxia , can be diagnosed by methods compnsmg determimng from a sample denved from a subject an abnormally decreased or mcreased level of human atrophin- 1 related gene polypeptide or human atrophin- 1 related gene mRNA Decreased or mcreased expression can be measured at the RNA level usmg any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection,

Northern blotting and other hybndization methods Assay techniques that can be used to determine levels of a protein, such as an human atrophin- 1 related gene polypeptide, m a sample deπved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays

Thus in another aspect, the present mvention relates to a diagonostic kit for a disease or suspectabi ty to a disease, particularly Charcot-Mane-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral pallidoluysian atrophy and atxia, which compπses

(a) a human atrophin- 1 related gene polynucleotide, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,

(b) a nucleotide sequence complementary to that of (a),

(c) a human atrophin- 1 related gene polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a human atrophm-1 related gene polypeptide, preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component

Chromosome Assays

The nucleotide sequences of the present mvention are also valuable for chromosome identification The sequence is specifically targeted to and can hybπdize with a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present mvention is an important first step m coπelating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be coπelated with genetic map data Such data are found, for example, in V McKusick, Mende an Inheπtance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. The human atrophin-l related gene was mapped to chromosome lp36 where Charcot- Marie-Tooth disease, Schwartz-Jampel syndrome, and Crystalline Comeal dystrophy were localized.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the human atrophin-l related gene polypeptides. The term "immunospecific" means that the antibodies have substantial! greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.

Antibodies generated against the human atrophin-l related gene polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV- hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R Liss, Ine, 1985).

Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Antibodies against human atrophin-l related gene polypeptides may also be employed to treat Charcot-Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral pallidoluysian atrophy and atxia, among others.

Vaccines Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with human atrophin-l related gene polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from Charcot-Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral palhdoluysian atrophy and atxia, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering human atrophin-l related gene polypeptide via a vector directing expression of human atrophin-l related gene polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases. Further aspect of the invention relates to an immunological/vaccine formulation

(composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a human atrophin-l related gene polypeptide wherein the composition comprises a human atrophin-l related gene polypeptide or human atrophin-l related gene. The vaccine formulation may further comprise a suitable carrier. Since human atrophin-l related gene polypeptide may be broken down in the stomach, it is preferably administered parenterally

(including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit- dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Screening Assays

The human atrophin-l related gene polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the human atrophin-l related gene polypeptide of the present invention. Thus, polypeptides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These agonists or antagonists may be natural or modified substrates, hgands, receptors, enzymes, etc, as the case may be, of the polypeptide of the present invention; or may be structural or functional mimetics of the polypeptide of the present invention. See Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991). Human atrophin-l related gene polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate human atrophin-l related gene polypeptide on the one hand and which can inhibit the function of human atrophin-l related gene polypeptide on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as Charcot-Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral pallidoluysian atrophy and atxia. Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as Charcot- Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral palhdoluysian atrophy and atxia .

In general, such screening procedures may involve using appropriate cells which express the human atrophin-l related gene polypeptide or respond to human atrophin-l related gene polypeptide of the present invention. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells which express the human atrophin-l related gene polypeptide (or cell membrane containing the expressed polypeptide) or respond to human atrophin-l related gene polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for human atrophin-l related gene activity.

The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the human atrophin-l related gene polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the human atrophin-l related gene polypeptide, using detection systems appropriate to the cells bearing the human atrophin-l related gene polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing a human atrophin-l related gene polypeptide to form a mixture, measuring human atrophin-l related gene activity in the mixture, and comparing the human atrophin-l related gene activity of the mixture to a standard. The human atrophm-1 related gene cDNA, protem and antibodies to the protein may also be used to configure assays for detectmg the effect of added compounds on the production of human atrophm-1 related gene mRNA and protein m cells For example, an ELISA may be constructed for measuring secreted or cell associated levels of human atrophm-1 related gene protem usmg monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents which may inhibit or enhance the production of human atrophm-1 related gene (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues

The human atrophm-1 related gene protem may be used to identify membrane bound or soluble receptors, if any, through standard receptor bmdmg techniques known in the art These include, but are not limited to, ligand bmdmg and crosslinkmg assays m which the human atrophm-1 related gene is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotmylated), or fused to a peptide sequence suitable for detection or punfication, and mcubated with a source of the putative receptor (cells, cell membranes, cell supematants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy In addition to bemg used for punfication and clonmg of the receptor, these bmdmg assays can be used to identify agomsts and antagomsts of human atrophm-1 related gene which compete with the bmdmg of human atrophm-1 related gene to its receptors, if any Standard methods for conductmg screening assays are well understood in the art

Examples of potential human atrophm-1 related gene polypeptide antagomsts mclude antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, etc , as the case may be, of the human atrophm-1 related gene polypeptide, e g , a fragment of the hgands. substrates, receptors, enzymes, etc , or small molecules which bind to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented Thus m another aspect, the present invention relates to a screening kit for identifying agomsts, antagonists, hgands, receptors, substrates, enzymes, etc for human atrophm-1 related gene polypeptides, or compounds which decrease or enhance the production of human atrophm-1 related gene polypeptides, which compnses (a) a human atrophm-1 related gene polypeptide, preferably that of SEQ ID NO 2, (b) a recombmant cell expressing a human atrophm-1 related gene polypeptide, preferably that of SEQ ID NO 2,

(c) a cell membrane expressmg a human atrophm-1 related gene polypeptide, preferably that of SEQ ID NO 2, or (d) antibody to a human atrophin-l related gene polypeptide, preferably that of SEQ ID NO: 2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as, Charcot-Marie-Tooth disease, Schwartz-Jampel syndrome, Crystalline Comeal dystrophy, Dentatorubral pallidoluysian atrophy and atxia , related to both an excess of and insufficient amounts of human atrophin-l related gene polypeptide activity. If the activity of human atrophin-l related gene polypeptide is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the human atrophin-l related gene polypeptide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc, or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of human atrophin-l related gene polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous human atrophin-l related gene polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the human atrophin-l related gene polypeptide. In still another approach, expression of the gene encoding endogenous human atrophin-l related gene polypeptide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988). Alternatively, ohgonucleotides which form triple helices with the gene can be supplied. See, for example, Lee et al, Nucleic Acids Res (1979) 6:3073; Cooney et al, Science (1988) 241:456; Dervan et al, Science (1991) 251: 1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of human atrophin-l related gene and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates human atrophin-l related gene polypeptide, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of human atrophin-l related gene by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Pubhshers Ltd (1996). Another approach is to administer a therapeutic amount of human atrophin-l related gene polypeptides in combination with a suitable pharmaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of human atrophin-l related gene polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Such carriers include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

Prefeπed forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often refeπed to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: XIA, JIA-HUI LIU, CHUN-YU RUAN, QUIN-GUO WANG, DE-AN

DENG, HAN-XIANG

(ii) TITLE OF THE INVENTION: Human Atrophin-l Related Gene

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: RATNER & PRESTIA (B) STREET: P.O. BOX 980

(C) CITY: VALLEY FORGE

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19482

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: TO BE ASSIGNED

(B) FILING DATE: (C) CLASSIFICATION: UNKNOWN

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: PRESTIA, PAUL F (B) REGISTRATION NUMBER: 23,031

(C) REFERENCE/DOCKET NUMBER: GP-70296 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-407-0700

(B) TELEFAX: 610-407-0701

(C) TELEX: 846169

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4598 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAGCCGGAGC CCAGTGACAG GCGACGGCCT CTTGCCGCCC CGGACCCCGG GCGCTTACCT 60

GTGGCTGCTG GGGTCGGCCG CCCGGGACCA GGCTGGGAGC CCCGAGAAAC GCTTCGTTAA 120 GGGACTCAGG CAGTACGGGA AGAACTTCTT CAGAATTAGA AAGGAGCTGC TTCCCAATAA 180

GGAAACAGGG GAGCTGATCA CCTTCTATTA CTATTGGAAG AAGACCCCCG AAGCAGCCAG 240

CTCCCGAGCC CATCGTAGGC ACCGCAGGCA GGCCGTGTTC AGGAGGATTA AGACTCGCAC 300

CGCGTCCACA CCCGTCAACA CACCCTCCAG ACCCCCGTCC AGTGAATTCT TGGACCTAAG 360

TTCAGCCAGT GAAGATGACT TCGACAGTGA GGACAGTGAG CAGGAGTGAA GGGGTACGCC 420 TGCCGCCACT GCTTCACCAC CACCTCCAAA GATTGGCACC ACGGAGGCCG GGAGAACATC 480

CTGCTTTGCA CCGACTGTCG CATCCACTTC AAGAAATACG GTGAGCTCCC GCCCATTGAG 540

AAGCCCGTGG ACCCGCCACC GTTTATGTTC AAACCCGTCA AGGAAGAGGA TGATGGGCTC 600

AGTGGGAAGC ATAGCATGAG GACACGGCGG AGTCGGGGCT CGATGTCGAC ACTACGCAGT 660

GGTGGAAGAA GCAGCCAGCC AGCCCTGGAT GGTCGCACCT CACCCATCAA TGAAGACATC 720 CGCTCCAGCG GCCGGAACTC CCCCAGCGCT GCCAGTACCT CCAGCAATGA CAGTAAAGCA 780

GAGACAGTGA AGAAGTCGGC CAAGAAGGTG AAGGAGGAAG CTTCTTCCCT TCTTAAGAGT 840

AACAAACGCC AGCGGGAGAA GGTGGCCTCT GATACGGAGG AGGCTGACAG GACCAGCTCC 900

AAGAAGACAA AAACGCAGGA GATCAGCAGG CCCAACTCGC CATCTGAAGG TGAGGGAGAG 960

AGTTCAGACA GTCGCAGCGT CAACGATGAG GGTAGCAGTG ACCCCAAAGA CATCGACCAG 1020 GACAATCGCA GCACGTCCCC GAGCATCCCC AGCCCCCAGG ACAATGAGAG TGACTCGGAC 1080

TCGTCAGCCC AGCAGCAGAT GCTGCAGGCC CAGCCCCCAG CCTTGCAGGC TCCCACTGGG 1140

GTCACCCCAG CTCCCTCCTC AGCTCCTCCA GGGACCCCTC AGCTGCCCAC GCCAGGGCCC 1200

ACGCCCTCTG CCACTGCAGT TCCCCCACAG GGCTCCCCCA CGGCCTCCCA GGCCCCTAAC 1260

CAGCCACAGG CTCCCACAGC GCCTGTTCCC CACACCCACA TCCAACAGGC ACCGGCCTTG 1320 CACCCCCAGC GGCCGCCCTC ACCGCATCCC CCGCCGCATC CCTCGCCACA TCCCCCACTG 1380

CAGCCTCTGA CTGGGTCGGC GGGCCAGCCT TCTGCACCCT CTCATGCCCA GCCCCCACTG 1440

CACGGTCAGG GCCCACCCGG CCCTCACAGC CTGCAGGCTG GGCCCCTGCT GCAGCACCCA 1500

GGCCCCCCAC AGCCCTTTGG CCTCCCTCCC CAGGCCTCCC AAGGCCAGGC CCCTCTGGGG 1560

ACCTCCCCAG CAGCAGCGTA CCCTCACACC TCCCTGCAGC TGCCAGCCTC TCAGTCAGCG 1620 CTGCAGTCCC AACAGCCTCC ACGGGAGCAG CCCCTGCCAC CAGCGCCCTT GGCCATGCCC 1680

CACATCAAGC CCCCGCCTAC CACTCCCATC CCCCAGCTGC CGGCGCCACA GGCCCACAAG 1740

CACCCTCCCC ACCTCTCGGG GCCCTCACCC TTCTCCATGA ATGCCAACCT GCCTCCCCCT 1800 CCAGCCCTGA AGCCCCTGAG CTCCCTGTCC ACACATCACC CCCCGTCGGC TCACCCCCCA 1860

CCCCTGCAAC TCATGCCTCA GAGCCAGCCA TTGCCCTCCT CGCCCGCCCA GCCCCCCGGG 1920

CTGACCCAGA GCCAGAACCT GCCCCCGCCC CCTGCCTCCC ACCCCCCTAC AGGCCTCCAC 1980

CAGGTGGCCC CCCAACCCCC GTTTGCTCAG CACCCCTTTG TCCCTGGAGG CCCTCCTCCC 2040 ATCACCCCTC CGACCTGCCC CTCCACCTCT ACCCCACCGG CGGGACCTGG CACCTCGGCC 2100

CAGCCACCCT GCTCTGGTGC GGCGGCTTCA GGAGGCAGCA TAGCGGGGGG GTCGTCCTGC 2160

CCACTCCCCA CCGTCCAGAT CAAGGAGGAG GCTCTGGACG ACGCTGAGGA GCCTGAGAGC 2220

CCCCCTCCCC CACCAAGGAG CCCGTCCCCG GAGCCCACTG TGGTGGACAC CCCCAGTCAC 2280

GCCAGCCAGT CAGCTAGGTT CTACAAACAC CTGGACCGGG GCTACAACTC GTGTGCCCGG 2340 ACAGACCTGT ACTTCATGCC TCTGGCCGGG TCCAAGCTGG CCAAGAAGAG GGAGGAGGCC 2400

ATTGAGAAGG CCAAGCGCGA GGCTGAGCAG AAAGCCCGAG AGGAGCGAGA GCGGGAGAAG 2460

GAGAAGGAGA AGGAGCGGGA GCGGGAGCGA GAGCGGGAGC GCGAGGCAGA GCGGGCGGCT 2520

AAGGCGTCCA GCTCAGCGCA TGAAGGTCGC CTCAGTGACC CACAGCTCAG TGGTCCTGGC 2580

CACATGCGGC CATCCTTCGA GCCACCACCA ACCACCATTG CTGCTGTGCC CCCCTACATC 2640 GGGCCCGACA CACCTGCCCT TCGGACTCTG AGCGAGTACG CCCGGCCCCA CGTCATGTCG 2700

CCCACCAACC GCAACCACCC CTTCTACATG CCCCTTAACC CCACGGACCC CCTGCTGGCC 2760

TACCACATGC CTGGCCTCTA CAACGTCGAC CCCACCATCC GCGAGCGGGA GCTCCGGGAG 2820

CGGGAGATCC GAGAGCGGGA GATCCGAGAG CGGGAGCTGC GGGAGAGGAT GAAGCCGGGC 2880

TTCGAGGTGA AGCCCCCAGA GCTGGACCCC CTGCACCCAG CCGCCAACCC CATGGAGCAC 2940 TTTGCCCGGC ACAGCGCCCT CACCATCCCC CCGACCGCCG GGCCCCACCC TTTTGCTTCT 3000

TTCCACCCGG GCCTGAACCC CTTGGAGAGG GAGAGACTGG CCCTGGCGGG CCCCCAGCTG 3060

CGGCCCGAGA TGAGCTACCC TGACAGACTG GCAGCCGAGC GTATCCACGC AGAGCGCATG 3120

GCATCGCTGA CCAGCGATCC CCTGGCCCGA CTGCAGATGT TCAACGTGAC TCCGCACCAT 3180

CACCAGCACT CTCACATTCA CTCCCACCTC CACCTCCACC AGCAGGACCC CCTCCACCAA 3240 GGTTCAGCAG GCCCCGTTCA CCCGCTGGTC GACCCCCTGA CTGCCGGTCC CCACCTGGCT 3300

CGCTTCCCCT ACCCGCCTGG CACTCTCCCC AACCCTCTGC TTGGACAGCC CCCACACGAG 3360

CACGAGATGC TTCGCCACCC AGTTTTCGGC ACCCCCTACC CCCGTGACCT GCCTGGGGCC 3420

ATCCCACCCC CCATGTCAGC AGCCCACCAG CTGCAGGCCA TGCATGCCCA GTCGGCCGAG 3480

CTGCAGAGAC TGGCCATGGA GCAGCAGTGG CTGCATGGAC ACCCCCACAT GCATGGTGGC 3540 CACCTACCAA GTCAGGAAGA TTATTACAGT CGACTGAAGA AAGAAGGTGA CAAGCAGTTA 3600

TAAGTTATTT ATTTGTTAAC GCTGGCTGTG GAAACCCCAG TTCTTGGGGG GAGAAACAGG 3660

ACTTTTTACA TAAAATAGGA GCTGCAAAAG CAAAAAGAAT ATCTTCTAAA GATTCTTTAT 3720

ATATTTAAAA ACCCACAACT AAAAATGTAT CCACATAGTA GTGTTCGTTT GTCGAGAGGA 3780

TTTCCTGAGA CTGGTTTGGA TCTCCCTGCA TGACAGTCCC CCAGAAACTT AGTGAGTCCT 3840 GGACTGGACT GAACATCCAG AAAGCTTCCC TGCAATCTTG GGGTTTGGCT TTAGTTTTCT 3900

TTTCCTTGAT TTCTCAGTAG GTGCTAGAAT CCAGTTCACA CCCTTCACTG TGCGTGCAGA 3960

CACACTGACA CACTCCGCCA CGAGTGCTCC AGAGCCCACG AGGCTTGCAG ATCGGGGGCA 4020

TAGGAATTTG GAATCCAAGA GCTATAATTT TTAAAAAAAA AAAAATCTTT TATTTTAATA 4080

CATTGTAGGA AATCTTCATA ATTGGAGAAA GTTCTGCAGC ATGGCTTTTT ACGTCTGTAA 4140 ATAAATAATT TTAGAACAGC CTTTTTTTCT TCCATAAACT ACTATTGTGA TCTATTTTTT 4200

CCAGCCATGT CACTAATTGT GAATTCCTAC CAACTATTGA CAGAATACAG AGTTGATTTT 4260

TTAATAAAAA GTTATATATA ATTATCCCTT TAATTAAAGG GAGCAAAGGG GCGTTCCACA 4320

TGGACAGAGG CTTGGACCGA GGCCTGGTCA CAGCAGCGAG CATCCAGGGT TTGCAGGGAC 4380

GATGTTACAG ACTCTGTTTT CTGCCTGGCG TTTCACTTGT GTCTGCTCCT AGCCTGTGCT 4440 CTGCCAGCAG CACAGACATC TGCTCCATCA GACCTCTTCC ATTTTGCACA GGGAGTGCAG 4500

GAGGTGAATG TTCACTTTCT GTTCTCCAGT GTCACTGTTC TGTTTCCACG GGATGGAAAG 4560

CGCATGGGCC TGTGTCCATT GTAGATTTCC TTCTAGAT 4598 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1012 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2

Met Phe Lys Pro Val Lys Glu Glu Asp Asp Gly Leu Ser Gly Lys His 1 5 10 15 Ser Met Arg Thr Arg Arg Ser Arg Gly Ser Met Ser Thr Leu Arg Ser 20 25 30

Gly Gly Arg Ser Ser Gin Pro Ala Leu Asp Gly Arg Thr Ser Pro lie

35 40 45

Asn Glu Asp lie Arg Ser Ser Gly Arg Asn Ser Pro Ser Ala Ala Ser 50 55 60

Thr Ser Ser Asn Asp Ser Lys Ala Glu Thr Val Lys Lys Ser Ala Lys 65 70 75 80

Lys Val Lys Glu Glu Ala Ser Ser Leu Leu Lys Ser Asn Lys Arg Gin 85 90 95 Arg Glu Lys Val Ala Ser Asp Thr Glu Glu Ala Asp Arg Thr Ser Ser 100 105 110

Lys Lys Thr Lys Thr Gin Glu lie Ser Arg Pro Asn Ser Pro Ser Glu

115 120 125

Gly Glu Gly Glu Ser Ser Asp Ser Arg Ser Val Asn Asp Glu Gly Ser 130 135 140

Ser Asp Pro Lys Asp lie Asp Gin Asp Asn Arg Ser Thr Ser Pro Ser

145 150 155 160 lie Pro Ser Pro Gin Asp Asn Glu Ser Asp Ser Asp Ser Ser Ala Gin

165 170 175 Gin Gin Met Leu Gin Ala Gin Pro Pro Ala Leu Gin Ala Pro Thr Gly

180 185 190

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

195 200 205

Thr Pro Gly Pro Thr Pro Ser Ala Thr Ala Val Pro Pro Gin Gly Ser 210 215 220

Pro Thr Ala Ser Gin Ala Pro Asn Gin Pro Gin Ala Pro Thr Ala Pro

225 230 235 240

Val Pro His Thr His lie Gin Gin Ala Pro Ala Leu His Pro Gin Arg

245 250 255 Pro Pro Ser Pro His Pro Pro Pro His Pro Ser Pro His Pro Pro Leu

260 265 270

Gin Pro Leu Thr Gly Ser Ala Gly Gin Pro Ser Ala Pro Ser His Ala 275 280 285

Gin Pro Pro Leu His Gly Gin Gly Pro Pro Gly Pro His Ser Leu Gin

290 295 300

Ala Gly Pro Leu Leu Gin His Pro Gly Pro Pro Gin Pro Phe Gly Leu 305 310 315 320

Pro Pro Gin Ala Ser Gin Gly Gin Ala Pro Leu Gly Thr Ser Pro Ala

325 330 335

Ala Ala Tyr Pro His Thr Ser Leu Gin Leu Pro Ala Ser Gin Ser Ala 340 345 350 Leu Gin Ser Gin Gin Pro Pro Arg Glu Gin Pro Leu Pro Pro Ala Pro 355 360 365

Leu Ala Met Pro His lie Lys Pro Pro Pro Thr Thr Pro lie Pro Gin

370 375 380

Leu Pro Ala Pro Gin Ala His Lys His Pro Pro His Leu Ser Gly Pro 385 390 395 400

Ser Pro Phe Ser Met Asn Ala Asn Leu Pro Pro Pro Pro Ala Leu Lys

405 410 415

Pro Leu Ser Ser Leu Ser Thr His His Pro Pro Ser Ala His Pro Pro 420 425 430 Pro Leu Gin Leu Met Pro Gin Ser Gin Pro Leu Pro Ser Ser Pro Ala 435 440 445

Gin Pro Pro Gly Leu Thr Gin Ser Gin Asn Leu Pro Pro Pro Pro Ala

450 455 460

Ser His Pro Pro Thr Gly Leu His Gin Val Ala Pro Gin Pro Pro Phe 465 470 475 480

Ala Gin His Pro Phe Val Pro Gly Gly Pro Pro Pro lie Thr Pro Pro

485 490 495

Thr Cys Pro Ser Thr Ser Thr Pro Pro Ala Gly Pro Gly Thr Ser Ala 500 505 510 Gin Pro Pro Cys Ser Gly Ala Ala Ala Ser Gly Gly Ser lie Ala Gly 515 520 525

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

530 535 540

Asp Asp Ala Glu Glu Pro Glu Ser Pro Pro Pro Pro Pro Arg Ser Pro 545 550 555 560

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

565 570 575

Ala Arg Phe Tyr Lys His Leu Asp Arg Gly Tyr Asn Ser Cys Ala Arg 580 585 590 Thr Asp Leu Tyr Phe Met Pro Leu Ala Gly Ser Lys Leu Ala Lys Lys 595 600 605

Arg Glu Glu Ala lie Glu Lys Ala Lys Arg Glu Ala Glu Gin Lys Ala

610 615 620

Arg Glu Glu Arg Glu Arg Glu Lys Glu Lys Glu Lys Glu Arg Glu Arg 625 630 635 640

Glu Arg Glu Arg Glu Arg Glu Ala Glu Arg Ala Ala Lys Ala Ser Ser 645 650 655 Ser Ala His Glu Gly Arg Leu Ser Asp Pro Gin Leu Ser Gly Pro Gly

660 665 670

His Met Arg Pro Ser Phe Glu Pro Pro Pro Thr Thr lie Ala Ala Val 675 680 685 Pro Pro Tyr lie Gly Pro Asp Thr Pro Ala Leu Arg Thr Leu Ser Glu 690 695 700

Tyr Ala Arg Pro His Val Met Ser Pro Thr Asn Arg Asn His Pro Phe 705 710 715 720

Tyr Met Pro Leu Asn Pro Thr Asp Pro Leu Leu Ala Tyr His Met Pro 725 730 735

Gly Leu Tyr Asn Val Asp Pro Thr lie Arg Glu Arg Glu Leu Arg Glu

740 745 750

Arg Glu lie Arg Glu Arg Glu lie Arg Glu Arg Glu Leu Arg Glu Arg 755 760 765 Met Lys Pro Gly Phe Glu Val Lys Pro Pro Glu Leu Asp Pro Leu His 770 775 780

Pro Ala Ala Asn Pro Met Glu His Phe Ala Arg His Ser Ala Leu Thr 785 790 795 800 lie Pro Pro Thr Ala Gly Pro His Pro Phe Ala Ser Phe His Pro Gly 805 810 815

Leu Asn Pro Leu Glu Arg Glu Arg Leu Ala Leu Ala Gly Pro Gin Leu

820 825 830

Arg Pro Glu Met Ser Tyr Pro Asp Arg Leu Ala Ala Glu Arg lie His 835 840 845 Ala Glu Arg Met Ala Ser Leu Thr Ser Asp Pro Leu Ala Arg Leu Gin 850 855 860

Met Phe Asn Val Thr Pro His His His Gin His Ser His lie His Ser 865 870 875 880

His Leu His Leu His Gin Gin Asp Pro Leu His Gin Gly Ser Ala Gly 885 890 895

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

900 905 910

Arg Phe Pro Tyr Pro Pro Gly Thr Leu Pro Asn Pro Leu Leu Gly Gin 915 920 925 Pro Pro His Glu His Glu Met Leu Arg His Pro Val Phe Gly Thr Pro 930 935 940

Tyr Pro Arg Asp Leu Pro Gly Ala lie Pro Pro Pro Met Ser Ala Ala 945 950 955 960

His Gin Leu Gin Ala Met His Ala Gin Ser Ala Glu Leu Gin Arg Leu 965 970 975

Ala Met Glu Gin Gin Trp Leu His Gly His Pro His Met His Gly Gly

980 985 990

His Leu Pro Ser Gin Glu Asp Tyr Tyr Ser Arg Leu Lys Lys Glu Gly 995 1000 1005 Asp Lys Gin Leu 1010

Claims

What is claimed is:

1. An isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the human atrophin-l related gene polypeptide of SEQ ID NO:2; or a nucleotide sequence complementary to said isolated polynucleotide.

2. The polynucleotide of claim 1 wherein said polynucleotide comprises the nucleotide sequence contained in SEQ ID NO: 1 encoding the human atrophin-l related gene polypeptide of SEQ ID N02.

3. The polynucleotide of claim 1 wherein said polynucleotide comprises a nucleotide sequence that is at least 80% identical to that of SEQ ID NO: 1 over its entire length.

4. The polynucleotide of claim 3 which is polynucleotide of SEQ ID NO: 1.

5. The polynucleotide of claim 1 which is DNA or RNA.

6. A DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing a human atrophin-l related gene polypeptide comprising an amino acid sequence, which has at least 91% identity with the polypeptide of SEQ ID NO:2 when said expression system is present in a compatible host cell.

7. A host cell comprising the expression system of claim 6.

8. A process for producing a human atrophin-l related gene polypeptide comprising culturing a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.

9. A process for producing a cell which produces a human atrophin-l related gene polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a human atrophin- 1 related gene polypeptide.

10. A human atrophin-l related gene polypeptide comprising an amino acid sequence which is at least 91% identical to the amino acid sequence of SEQ ID NO:2 over its entire length.

11. The polypeptide of claim 10 which comprises the amino acid sequence of SEQ ID NO.2.

12. An antibody immunospecific for the human atrophin-l related gene polypeptide of claim 10.

13. A method for the treatment of a subject in need of enhanced activity or expression of human atrophin-l related gene polypeptide of claim 10 comprising:

(a) administering to the subject a therapeutically effective amount of an agonist to said polypeptide; and/or

(b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the human atrophin-l related gene polypeptide of SEQ ID NO:2 over its entire length; or a nucleotide sequence complementary to said nucleotide sequence in a form so as to effect production of said polypeptide activity in vivo.

14. A method for the treatment of a subject having need to inhibit activity or expression of human atrophin-l related gene polypeptide of claim 10 comprising:

(a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or (b) administering to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said polypeptide; and/or

(c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate , or receptor.

15. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of human atrophin-l related gene polypeptide of claim 10 in a subject comprising: (a) determining the presence or absence of a mutation in the nucleotide sequence encoding said human atrophin-l related gene polypeptide in the genome of said subject; and/or

(b) analyzing for the presence or amount of the human atrophin-l related gene polypeptide expression in a sample derived from said subject.

16. A method for identifying compounds which inhibit (antagonize) or agonize the human atrophin-l related gene polypeptide of claim 10 which comprises:

(a) contacting a candidate compound with cells which express the human atrophin-l related gene polypeptide (or cell membrane expressing human atrophin-l related gene polypeptide) or respond to human atrophin- 1 related gene polypeptide; and

(b) observing the binding, or stimulation or inhibition of a functional response; or comparing the ability of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for human atrophin-l related gene polypeptide activity.

17. An agonist identified by the method of claim 16.

18. An antagonist identified by the method of claim 16.

19. A recombinant host cell produced by a method of Claim 9 or a membrane thereof expressing a human atrophin-l related gene polypeptide.