WO2000020583A1 - Ribonucleoprotein homolog zrnp1, having also homology to the gnrh receptor - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zrnp1, a novel ribonucleoprotein. The polypeptides, and polynucleotides encoding them may be used for mediating mRNA processing and for diagnosis and treatment of diseases associated with connective tissues. The present invention also includes antibodies to the zrnp1 polypeptides.

Description

Description RIBONUCLEOPROTEIN HOMOLOG ZRNP1, HAVING ALSO HOMOLOGY TO THE GNRH RECEPTOR

BACKGROUND OF THE INVENTION

When nascent RNA emerges from RNA polymerase II it is associated with numerous "mRNA precursors", which include a group of nuclear proteins collectively known as heterogeneous ribonucleoprotein particles or proteins

(hnRNPs) which are formed by association of these proteins with heterogeneous nuclear RNA (hnRNA) and are responsible for RNA processing. A second group are known as the small nuclear ribonucleoproteins (snRNPs) which also form RNA- protein clusters and are active in RNA processing. In particular are the uridine-rich, small nuclear RNP complexes, UsnRNPs (for a review see Luhrmann et al . , Biochim. Biophys. Acta 1087:265-92, 1990; Dreyfuss et al . , Annu. Rev. Biochem. 61:289-321, 1993 and Transcription, Termination, RNA Processing, and Posttranslational Control , Molecular Cell Biology 3^rd Edition, Lodish et al . Eds., Scientific American Books, New York, 1995). The proteins which make up the snRNPs and hnRNPs associate with mRNA with great affinity, but differing specificity.

The RNP proteins comprise an N-terminal conserved "RNA-binding domain" and a charged C-terminal region which is responsible for the unique action and interactions of each of the proteins. The RNA-binding domain contains about 90 amino acid residues made up of four β strands and two α helices. Located within the RNA- binding domain are "RNA-binding motifs" which have been characterized within the RNA-binding domains of various RNP proteins. Examples of RNA-binding motifs include a highly conserved octapeptide (RNP1) sequence near the C- terminal end of the RNA-binding domain and a second region (RNP2) of hydrophobic amino acid residues found near the N-terminal end of the domain, see Query et al . (Cell 57:89-101, 1989) and Bandziulis et al . (Genes & Development 3:431-7, 1989) which provide multiple sequence alignments of ribonucleoproteins from various species, illustrating the conserved nature of the RNP1 and RNP2 regions . The structure of the RNA-binding domain has been determined to include four β strands and two α helices,

Other RNA-binding motifs include an "RGG box" , a 26 -amino acid residue sequence containing five Arg-Gly-Gly (RGG) repeats interspersed with aromatic amino acids, found in various RNA-binding proteins. The "KH motif" is found in the hnRNP K protein and other RNA binding proteins. It is about 45 amino acids in length and generally contains an RGG box between the KH repeats. A "double stranded RNA-binding motif" found in proteins involved in post-transcriptional gene regulation by RNA- binding proteins . Binding proteins having this motif do not bind double stranded DNA (Burd and Dreyfuss, Nature 265:615-21, 1994) . The heterogeneous nuclear ribonucleoprotein complex, hnRNP, contains a group of proteins, Al, A2 , Bl, B2, Cl, C2 and C3 , collectively termed "core proteins" which are involved in mRNA processing. Al is the most abundant of these proteins (Dreyfuss et al . , ibid, and Monroe and Dong, Proc. Natl. Acad. Sci. USA 89:895-99, 1992) . Proteolysis of the native Al protein yields an N- terminal single stranded DNA binding protein, UP1 (Williams et al . , Proc. Natl. Acad. Sci. USA 82:5666-70, 1985, Morandi et al . , EMBO J. 5:2267-73, 1996 and Kumar et al., J. Biol. Chem. 261:11266-73, 1986)

The small nuclear RNP complexes, snRNP, contain all of the major small nuclear RNAs (UI, U2 , U3 , U4 , U5 and U6) and the common core proteins, B/B', D (Dl, D2 , D3) , E, F and G. In addition, each UsnRNP complex also contains a collection of proteins unique to each snRNP group. UI RNA is the most abundant of the small nuclear RNAs . The UlsnRNP complex has been found to be involved in pre-mRNA splicing. UlsnRNP contains the unique proteins 70 kD, A and C.

Autoantibodies to UlsnRNP proteins and RNA have been found in sera of patients with autoimmune diseases. Systemic lupus erythematosus patients develop antibodies to four cellular ribonucleoproteins, nRNP, Sm, Ro/SSA and La/SSb and with U1RNA. Antibodies against the snRNP 70kD protein are found most often in patients with mixed connective tissue disease (MTCD) , less frequently in systemic lupus erythematosus (SLE) or other rheumatic diseases (Maddison et al . , Clin. Exp. Immunol. 62 : 337-45 , 1985; Hoet and Venrooij , Mol. Biol. Rep. 16:199-205, 1992; Lundberg et al . , Brit. J. Rheum. 31:811-17, 1992; Hoet et al., Nuc. Acid Res. 21:5130-6, 1993; Zhang and Reichlin, Clin. Immuno . Immunopath. 74:70-6, 1995 and

Vlachoyiannopoulos et al . , Brit. J. Rheum. 35:534-41, 1996) .

A need exists to identify other ribonucleoproteins to more completely understand their role in mRNA processing and the association of anti-RNP antibodies with autoimmune disease. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.

SUMMARY OF THE INVENTION

Within one aspect of the invention provides an isolated polynucleotide molecule that encodes a polypeptide, wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 34 to amino acid residue 518, and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2; b) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO: 7, from amino acid residue 1 to amino acid residue 125, and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 7; or c) a polynucleotide molecule having the sequence of SEQ ID NO:l, SEQ ID NO : 6 or SEQ ID NO : 8. Within one embodiment any difference between the amino acid sequence encoded by the polynucleotide molecule and the corresponding amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 is due to a conservative amino acid substitution. Within another embodiment amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=blosum62, with other parameters set as default . Within yet another embodiment the polynucleotide hybridizes under stringent conditions to a polynucleotide molecule having the nucleotide sequence selected from the group consisting of: a) SEQ ID NO:l; b) SEQ ID NO: 6; c) the complement of SEQ ID NO:l; or d) the complement of SEQ ID NO: 6. Within a further embodiment the polypeptide further comprises an affinity tag or binding domain. Within still another embodiment the polynucleotide molecule comprises nucleotides 144-518 of SEQ ID NO:l.

The invention also provides an isolated polynucleotide molecule selected from the group consisting of: a) nucleotides 219-449 of SEQ ID NO:l; b) nucleotides 462-518 of SEQ ID NO : 1 ; c) nucleotides 92-109 of SEQ ID NO: 6; and d) nucleotides 335-391 of SEQ ID NO: 6.

Within another aspect of the invention is provided an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule as described above; and a transcription terminator. Within one embodiment the expression vector further comprises a secretory signal sequence operably linked to the DNA segment. Within another embodiment the polynucleotide encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag.

The invention also provides a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule as described above; and a transcription terminator, wherein the cultured cell expresses the polypeptide encoded by the polynucleotide segment.

The invention further provides a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule as described above; and a transcription terminator; whereby the cell expresses the polypeptide encoded by the polynucleotide segment; and recovering the expressed polypeptide. Within one embodiment the expression vector further comprises a secretory signal sequence operably linked to the polynucleotide segment, the cultured cell secretes the polypeptide into a culture medium, and the polypeptide is recovered from the culture medium.

Within another aspect the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 85% identical to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO : 2 , from amino acid residue 34 to amino acid residue 518, b) a polypeptide comprising the amino acid sequence as shown in SEQ ID N0:2, from amino acid residue 1 to amino acid residue 125 of SEQ ID NO : 7 ; wherein the isolated polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7. Within one embodiment the polypeptide comprises a sequence of amino acid residues that is at least 90% identical to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ ID N0:2, from amino acid residue 34 to amino acid residue 518, b) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 125 of SEQ ID NO : 7 ; wherein the isolated polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7. Within another embodiment the polypeptide comprises a sequence of amino acid residues that is at least 95% identical to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 34 to amino acid residue 518, b) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 125 of SEQ ID NO: 7; wherein the isolated polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7. Within still another embodiment any difference between the amino acid sequence encoded by the polynucleotide molecule and the corresponding amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 is due to a conservative amino acid substitution. Within still another embodiment the amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=blosum62 , with other parameters set as default. Still another embodiment provides a polypeptide further comprising an affinity tag or binding domain.

The invention also provides an isolated polypeptide comprising amino acid residues 59-135 of SEQ ID NO: 2.

The invention further provides an isolated polypeptide according to claim 20, wherein the polypeptide comprises four β strands corresponding to amino acid residues 59-64, 83-89, 98-105 and 130-135 of SEQ ID N0:2 and two α helices corresponding to amino acid residues 70- 77 and 109-117 of SEQ ID NO : 2.

Also provided by the invention is an isolated polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 7; c) amino acid residues 59-135 of SEQ ID NO:2; and d) amino acid residues 140-158 of SEQ ID NO:2.

Within another aspect of the invention is an antibody or antibody fragment that specifically binds to a polypeptide as described above. Within one embodiment the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody. Within one embodiment the antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit. Within another embodiment is provided an anti-idiotype antibody that specifically binds to the antibody as described above.

The invention also provides a polypeptide as described above, in combination with a pharmaceutically acceptable vehicle.

These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an alignment of the amino acid sequence of zrnpl (SEQ ID NO: 2) with the sequence of the crystal structure of human heterogeneous nuclear ribonucleoprotein Al (1UP1) (Xu et al . , Structure 5_:559- 70, 1997). Conserved amino acid residues (:) and conserved substitutions (.) are indicated. Figure 2 shows a multiple sequence alignment of the amino acid residues between and including the RNP-2 (marked by a series of "2"s) and RNP-CS motifs (marked by a series of "l"s) for human zrnpl (amino acid residue 59 (Leu) to amino acid residue 104 (Tyr) of SEQ ID NO: 2 and corresponding amino acid residues 282-328 of human UI small nuclear ribonucleoprotein 70dK (snRNP70) (Theissen et al., EMBO J . 5_:3209-17, 1986) and corresponding amino acid sequence 106-152 of human heterogeneous nuclear ribonucleoprotein Al (1UP1) (Riva et al . , EMBO J. 5:2267- 73, 1986) . Conserved amino acid residues "*" and conservative substitutions " . " between the three sequences are indicated.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al . , EMBO J . 4:1075, 1985; Nilsson et al . , Methods Enzymol . 198 :3 , 1991), glutathione S transferase

(Smith and Johnson, Gene £7:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag™ peptide (Hopp et al . , Biotechnology 6:1204-10, 1988), phage T7 gene 10 (glO) protein (Rosenberg et al . , Gene 5_6:125-35, 1987), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al . , Protein Expression and Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ) . The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.

The terms "amino-terminal" and " carboxyl- terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl- terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3' .

The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) . Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .

The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985) .

An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

The term "operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.

The term "ortholog" denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

A "polynucleotide" is a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DΝA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs" . It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.

A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides" .

The term "promoter" is used herein for its art- recognized meaning to denote a portion of a gene containing DΝA sequences that provide for the binding of RΝA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non- peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene. Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to +10%.

The present invention is based in part upon the discovery of a novel DNA sequence that encodes a polypeptide having homology to the family of human ribonucleoproteins. The zrnpl polynucleotide sequence is disclosed in SEQ ID NO:l and contains an open reading frame encoding an 158 amino acid residue polypeptide (SEQ ID NO: 2) . The mature polypeptide ranges from amino acid 1 (Met) or 34 (Met) to amino acid 158 (Arg) of SEQ ID NO: 2, nucleotides 45 to 518 or 144 'to 518 of SEQ ID NO:l. Within the mature polypeptide is a putative RNA binding domain from amino acid residues 59 (Leu) to amino acid residue 135 (Val) of SEQ ID NO : 2 , nucleotides 219 to 449 or SEQ ID NO : 1. The RNA binding domain is made up of four beta strands (B) and two alpha helixes (A) . The structure is predicted to be a two-layer alpha/beta sandwich comprising the two amphipathic helices packed against a four-stranded beta sheet in the order, BABBAB, with the topology UDUDUD, wherein U denotes up and D denotes down. The beta sheet formed by the four beta strands serves as a general RNA-binding platform.

Beta strand 1 comprises amino acid residues 59 (Leu) to amino acid residue 64 (Val) of SEQ ID NO: 2, nucleotides 219 to 236 of SEQ ID N0:1; alpha helix 1 comprises amino acid residue 70 (Glu) to amino acid residue 77 (Phe) of SEQ ID NO: 2, nucleotides 252 to 275 of SEQ ID NO:l; beta strand 2 comprises amino acid residue 83 (lie) to amino acid residue 89 (Asn) of SEQ ID NO: 2, nucleotides 291 to 311 of SEQ ID NO;l; beta strand 3 comprises amino acid residue 98 (Lys) to amino acid residue 105 (Try) of SEQ ID NO: 2, nucleotides 336 to 359 of SEQ ID NO : 1 ; alpha helix 2 comprises amino acid residue 109 (Lys) to amino acid residue 117 (Gly) of SEQ ID NO: 2, nucleotides 369 to 395 of SEQ ID NO:l; and beta strand 4 comprises amino acid residue 130 (Val) to amino acid residue 135 (Val) of SEQ ID NO: 2, nucleotides 432 to 449 Of SEQ ID NO:l. The C-terminal region of zrnpl, from amino acid residue 140 (Lys) to amino acid residue 158 (Arg) of SEQ ID NO: 2, nucleotides 462-518 of SEQ ID NO:l, is highly basic and may interact with repetitive structures in RNA and modify the overall strength of the RNA binding.

Within the RNA binding domain of zrnpl are two motifs conserved among the ribonucleoproteins. A hexapeptide "RNP-2 motif" occurs from amino acid residue 59 (Leu) to amino acid residue 64 (Val) of SEQ ID NO: 2, nucleotides 336 to 359 of SEQ ID NO:l, and corresponds to beta strand 1 of the RNA binding domain. The second, an octapeptide "RNP-CS motif, sometimes known as a "RNP consensus octamer or RNP-1" occurs from amino acid residue 98 (Lys) to amino acid residue 105 (Tyr) of SEQ ID NO: 2, nucleotides 219 to 236 of SEQ ID NO:l, and corresponds to beta strand 3 of the RNA binding domain. These motifs have been observed among RNA binding proteins, see Query et al . , ibid, and Bandziulis et al . , ibid. , features multiple alignments highlighting the two conserved motifs and other conserved features within the RNA binding domains of various ribonucleoproteins.

Each motif contains one conserved aromatic amino acid residue, amino acid residue 60 (Phe) and amino acid residue 100 (Tyr) of SEQ ID NO: 2, that participates in RNA binding. The aromatic side chains of these residues base- stack with the single-strand bases of bound RNA (Xu et al . , ibid. ) . Figure 2 shows a multiple sequence alignment of the amino acid sequences, including the RNP-2 and RNP- CS motifs, for human zrnpl amino acid residue 59 (Leu) to amino acid residue 104 (Tyr) of SEQ ID NO: 2 and corresponding amino acid residues 282-328 of human UI small nuclear ribonucleoprotein 70dK (snRNP70kD) (Theissen et al . , ibid.) and corresponding amino acid sequence 106- 152 of human heterogeneous nuclear ribonucleoprotein Al (1UP1) (Riva et al . , ibid.).

Zrnpl shares 55% identity at the amino acid level with Caenorhabdi tis elegans ribonucleoprotein Snf5 homolog R07E5.3 (Genbank Accession No. S43599) over the region of 140 amino acid residues from 390 through 529 of UlsnRNP. A partial nucleotide sequence, (PSN-Q57436, WIPO Publication, WO94/03599) described as a UlsnRNP 70kDa- like protein shares identity with the human zrnpl sequence from nucleotide 219 to 437 of SEQ ID NO:l.

Interestingly, a portion of the 3 ' untranslated region of zrnpl (from about nucleotide 1130 to nucleotide 2800 of SEQ ID NO:l) was found to be an antisense transcript of the complementary polynucleotide sequence encoding a region of the C-terminus of the Type II human gonadotropin releasing hormone receptor (WIPO Publication WO 97/47743) . Similarly the gene for snRNP SM D3 was found in the reverse orientation within one of the introns of the Drosophila antizyme gene (Ivanov et al . , Mol. Cell. Biol. 18:1553-61, 1998).

The sequences provided herein were used to search a murine EST database for homologous sequences. A polynucleotide encoding a full length murine zrnpl ortholog is disclosed in SEQ ID NO: 6 and the deducted amino acid sequence is disclosed in SEQ ID NO: 7 and was obtained by an assembly algorithm restricted to mouse ESTs . Human and mouse zrnpl share 100% identity at the amino acid level and 85% nucleotide identity over a 519 bp overlap. The mouse zrnpl polypeptide sequence begins at Met 34 of the human sequence as disclosed in SEQ ID NO: 2. It is likely that this Met is the start Met for the human sequence as well, based on structural analysis of the human sequence and lack of additional 5 ' sequence for the mouse sequence .

Northern blot analysis, as described in the Example below, resulted in five transcript sizes, 7, 5.5, 3.2, 1.5 and 0.9 kb, seen in all tissues tested. The strongest signal was seen in the 0.9 kd transcript, both the 5.5 and 3.2 kb transcripts were of moderate intensity and the 7 and 1.5 kb transcripts were weak.

Chromosomal localization of the zrnpl gene was done using radiation hybrids as described in more detail below. The results showed that zrnpl maps 1.01 cR_3000 from the framework marker WI-6253 on the chromosome 14 WICGR radiation hybrid map. Proximal and distal framework markers were WI-6253 and WI-5815, respectively. The use of surrounding markers positions zrnpl in the 14q22.2- 14q23.1 region on the integrated LDB chromosome 14 map. The present invention further provides polynucleotide molecules, including DNA and RNA molecules, encoding zrnpl proteins. The polynucleotides of the present invention include the sense strand; the anti-sense strand; and the DNA as double-stranded, having both the sense and anti-sense strand annealed together by their respective hydrogen bonds . Representative DNA sequences encoding zrnpl proteins are set forth in SEQ ID NOs : 1 and 6. DNA sequences encoding other zrnpl proteins can be readily generated by those of ordinary skill in the art based on the genetic code. Counterpart RNA sequences can be generated by substitution of U for T.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO: 5 is a degenerate DNA sequence that encompasses all DNAs that encode the zrnpl polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 5 also provides all RNA sequences encoding SEQ ID NO: 2 by substituting U for T. Thus, zrnpl polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 474 of SEQ ID NO: 5 or nucleotide 1 to nucleotide 375 of SEQ ID NO: 8 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NOs : 5 and 8 to denote degenerate nucleotide positions. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide (s) . For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C. TABLE 1

Nucleotide Resolution Complement Resolution

A A T T C C G G G G C C T T A A R A|G Y |T Y C|T R |G M A|C K |T K G|T M |c S C|G S |G W A|T w |T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T

AICIGIT AICIGIT

The degenerate codons used in SEQ ID NOs : 5 and 8, encompassing all possible codons for a given amino acid, are set forth in Table 2.

TABLE 2

One

Amino Letter Codons Degenerate

Acid Code Codon

Cys C TGC TGT TGY

Ser S AGC AGT TCA TCC TCG TCT SN

Thr T ACA ACC ACG ACT ACN

Pro P CCA CCC CCG CCT CCN

Ala A GCA GCC GCG GCT GCN

Gly G GGA GGC GGG GGT GGN

Asn N AAC AAT AAY

Asp D GAC GAT GAY

Glu E GAA GAG GAR

Gin Q CAA CAG CAR

His H CAC CAT CAY

Arg R AGA AGG CGA CGC CGG CGT MGN

Lys K AAA AAG AAR

Met M ATG ATG

He I ATA ATC Aπ ATH

Leu L CTA CTC CTG cπ πA πG YTN

Val V GTA GTC GTG Gπ GTN

Phe F πc πr πY

Tyr Y TAC TAT TAY

Trp W TGG TGG

Ter TAA TAG TGA TRR

Asn |Asp B RAY

Glu|Gln Z SAR

Any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate codon for serine (WSN) can, in some circumstances, encode arginine (AGR) , and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) . A similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of SEQ ID NOs : 2 and 7. Variant sequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." In general, see, Grantham, et al., Nuc . Acids Res . _.8:1893-912, 1980; Haas, et al . Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al . , Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18_.:199-209, 1982; Holm, Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158_.: 573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) . For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art . Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequences disclosed in SEQ ID NOs : 5 and 8 serve as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein. The highly conserved amino acids in the RNA- binding domain of zrnpl can be used as a tool to identify new family members. For instance, reverse transcription- polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved RNA-binding domain from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the zrnpl sequences are useful for this purpose. Preferably, the region encompassed by amino acid residues 59-135 of SEQ ID NO : 2 or amino acid residues 1-125 of SEQ ID NO: 7 would be useful.

The present invention also contemplates degenerate probes based upon the polynucleotides described above . Probes corresponding to complements of the polynucleotides set forth above are also encompassed. Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NOs : 1 or 6, other polynucleotide probes, primers, fragments and sequences recited herein or sequences complementary thereto. Polynucleotide hybridization is well known in the art and widely used for many applications, see for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; Ausubel et al . , eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987; Berger and Kimmel, eds., Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, 1987 and Wetmur, Crit . Rev. Biochem. Mol. Biol . 26:227-59, 1990. Polynucleotide hybridization exploits the ability of single stranded complementary sequences to form a double helix hybrid. Such hybrids include DNA-DNA, RNA-RNA and DNA-RNA. Hybridization will occur between sequences which contain some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch. The T_m of the mismatched hybrid decreases by 1°C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. Stringent hybridization conditions encompass temperatures of about

5-25°C below the thermal melting point (T_m) of the hybrid and a hybridization buffer having up to 1 M Na⁺. Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the

T_m of the hybrid about 1°C for each 1% formamide in the buffer solution. Generally, such stringent conditions encompass temperatures of 20-70°C and a hybridization buffer containing up to 6X SSC and 0-50% formamide. A higher degree of stringency can be achieved at temperatures of from 40-70°C with a hybridization buffer having up to 4X SSC and from 0-50% formamide. Highly stringent conditions typically encompass temperatures of

42-70°C with a hybridization buffer having up to IX SSC and 0-50% formamide. Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence. Typically, the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes . The above conditions are meant to serve as a guide and it is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid. The T_m for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence. Those conditions which influence the T_m include, the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution. Numerous equations for calculating T_m are known in the art, see for example (Sambrook et al . , ibid. ; Ausubel et al . , ibid. ; Berger and Kimmel, ibid, and Wetmur, ibid. ) and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length. Sequence analysis software such as Oligo 4.0 (publicly available shareware) and Primer Premier (PREMIER Biosoft International, Palo Alto, CA) as well as sites on the Internet, are available tools for analyzing a given sequence and calculating T_m based on user defined criteria. Such programs can also analyze a given sequence under defined conditions and suggest suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 bp, is done at temperatures of about 20-

25°C below the calculated T_m. For smaller probes, <50 bp, hybridization is typically carried out at the T_m or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids. The length of the polynucleotide sequence influences the rate and stability of hybrid formation. Smaller probe sequences, <50 bp, come to equilibrium with complementary sequences rapidly, but may form less stable hybrids. Incubation times of anywhere from minutes to hours can be used to achieve hybrid formation. Longer probe sequences come to equilibrium more slowly, but form more stable complexes even at lower temperatures . Incubations are allowed to proceed overnight or longer. Generally, incubations are carried out for a period equal to three times the calculated Cot time. Cot time, the time it takes for the polynucleotide sequences to reassociate, can be calculated for a particular sequence by methods known in the art .

The base pair composition of polynucleotide sequence will effect the thermal stability of the hybrid complex, thereby influencing the choice of hybridization temperature and the ionic strength of the hybridization buffer. A-T pairs are less stable than G-C pairs in aqueous solutions containing NaCl. Therefore, the higher the G-C content, the more stable the hybrid. Even distribution of G and C residues within the sequence also contribute positively to hybrid stability. Base pair composition can be manipulated to alter the T_m of a given sequence, for example, 5-methyldeoxycytidine can be substituted for deoxycytidine and 5-bromodeoxuridine can be substituted for thymidine to increase the T_m. 7-deazo- 2 ' -deoxyguanosine can be substituted for guanosine to reduce dependence on T_m .

Ionic concentration of the hybridization buffer also effects the stability of the hybrid. Hybridization buffers generally contain blocking agents such as Denhardt ' s solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO) , heparin or SDS, and a Na⁺ source, such as SSC (IX SSC: 0.15 M NaCl, 15 mM sodium citrate) or SSPE (IX SSPE: 1.8 M NaCl, 10 mM NaH₂P0₄, 1 mM EDTA, pH 7.7) . By decreasing the ionic concentration of the buffer, the stability of the hybrid is increased. Typically, hybridization buffers contain from between 10 mM-1 M Na⁺ . Premixed hybridization solutions are also available from commercial sources such as Clontech Laboratories (Palo Alto, CA) and Promega Corporation (Madison, WI) for use according to manufacturer's instruction. Addition of destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the T_m of a hybrid. Typically, formamide is used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures. Formamide also acts to reduce non-specific background when using RNA probes.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zrnpl RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980). Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al . , Biochemistry 18 : 52-94 ,

1979) . Poly (A) ⁺ RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA .69:1408-12, 1972) . Complementary DNA (cDNA) is prepared from poly (A) ⁺ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zrnpl polypeptides are then identified and isolated by, for example, hybridization or PCR.

A full-length clone encoding zrnpl can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to zrnpl, receptor fragments, or other specific binding partners . The polynucleotides of the present invention can also be synthesized using automated equipment. The current method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp) , however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. Gene synthesis methods are well known in the art. See, for example, Glick and

Pasternak, Molecular Biotechnology, Principles &

Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994; Itakura et al . , Annu. Rev. Biochem. 53 : 323- 56, 1984; and Climie et al . , Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

Zrnpl polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5 ' non- coding regions of a zrnpl gene. In view of the tissue- specific expression observed for zrnpl by Northern blotting, this gene region is expected to provide for brain, ovary and testis-specific expression. Promoter elements from a zrnpl gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5' flanking sequences also facilitates production of zrnpl proteins by "gene activation" as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous zrnpl gene in a cell is altered by introducing into the zrnpl locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is a zrnpl 5' non-coding sequence that permits homologous recombination of the construct with the endogenous zrnpl locus, whereby the sequences within the construct become operably linked with the endogenous zrnpl coding sequence. In this way, an endogenous zrnpl promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression. The zrnpl polynucleotide sequences disclosed herein can be used to isolate polynucleotides encoding other zrnpl proteins. Such other proteins include alternatively spliced cDNAs (including cDNAs encoding secreted zrnpl proteins) and counterpart polynucleotides from other species (orthologs) . These orthologous polynucleotides can be used, inter alia, to prepare the respective orthologous proteins. Other species of interest include, but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zrnpl polynucleotides and proteins from other mammalian species, including human and other primate, porcine, ovine, bovine, canine, feline, and equine polynucleotides and proteins. The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs) . Orthologs of human zrnpl can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zrnpl as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A zrnpl- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zrnpl sequence disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zrnpl polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Use can also be made of sequence information available for many species which can be found in electronic databases . Zrnpl sequences provided herein can be used as probes to screen such databases for orthologous zrnpl sequences. Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOs : 1 and 6 represent a single allele of human zrnpl and a single allele of murine zrnpl and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequences shown in SEQ ID NOs : 1 and 6, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NOs : 2 and 7. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zrnpl polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art . The present invention also provides isolated zrnpl polypeptides that are substantially homologous to the polypeptides of SEQ ID NOs : 2 or 7 and their orthologs. The term "substantially homologous" is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NOs : 2 or 7 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NOs: 2 or 7 or their orthologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48 : 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89_.: 10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid. ) as shown in Table 3 (amino acids are indicated by the standard one-letter codes) . The percent identity is then calculated as:

Total number of identical matches x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]

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H CN Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .

Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zrnpl. The FASTA algorithm is described by Pearson and Lipman, Proc . Nat. Acad. Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol . 183:63, 1990.

Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2) , without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff" value (calculated by a predetermined formula based upon the length of the sequence and the ktup value) , then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48=444, 1970; Sellers, SIAM J. Appl . Math. 26_.:787, 1974), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol . 183 : 63 , 1990.

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from four to six.

The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1992) . Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above) , the language "conservative amino acid substitution" preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3) .

Variant zrnpl polypeptides or substantially homologous zrnpl polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl -terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zrnpl polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 4

Conservative amino acid substitutions

Basic : arginine lysine histidine

Acidic : glutamic acid aspartic acid

Polar : glutamine asparagine

Hydrophobic leucine isoleucine valine

Aromatic : phenylalanine tryptophan tyrosine

Small glycine alanine serine threonine methionine

The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3 -methylproline, 2 , 4-methanoproline, ci -4-hydroxyproline, trans-4-hydroxyproline, N-methyl - glycine, allo-threonine, methylthreonine, hydroxyethyl- cysteine, hydroxyethylhomocysteine, nitroglutamine, homo- glutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3 , 3 -dimethyl- proline, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4-fluoro- phenylalanine . Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs . Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell- free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al . , J. Am. Chem. Soc . 113 : 2722 , 1991; Ellman et al . , Methods Enzvmol . 202 :301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al . , Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al . , J. Biol . Chem. 271:19991-8, 1996) . Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) . The non- naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 33_.: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification.

Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions

(Wynn and Richards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for zrnpl amino acid residues . Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244 : 1081-5, 1989; Bass et al . , Proc. Natl. Acad. Sci. USA 88 = 4498-502, 1991) . In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al . , J. Biol. Chem. 271:4699-708, 1996. Sites of RNA-binding can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography (see for example, Xu et al . , ibid.), electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al . , Science 255:306- 12, 1992; Smith et al . , J. Mol. Biol. 224:899-904, 1992; Wlodaver et al . , FEBS Lett. 3_09: 59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related ribonucleoproteins, such as snRNP UI 70 kd protein.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86 = 2152-6, 1989) . Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 3_0 = 10832-7, 1991; Ladner et al . , U.S. Patent

No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al . , Gene 46=145, 1986; Ner et al . , DNA 7:127, 1988).

Variants of the disclosed zrnpl DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370 : 389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., those retaining RNA-binding capacity) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NOs: 2 or 7 or that retain the RNA-binding properties of the wild-type zrnpl protein.

For any zrnpl polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.

The zrnpl polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al . , Molecular Cloning: A Laboratory Manual , 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al . , eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a zrnpl polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. To direct a zrnpl polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of zrnpl, or may be derived from another secreted protein (e.g., t-PA) or synthesized e novo . The secretory signal sequence is operably linked to the zrnpl DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell . Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al . , U.S. Patent No. 5,037,743; Holland et al . , U.S. Patent No. 5,143,830). Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 14_.: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al . , EMBO J . 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al . , ibid.), and liposome-mediated transfection (Hawley-Nelson et al . , Focus 15:73, 1993; Ciccarone et al . , Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996) . The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al . , U.S. Patent No. 4,713,339; Hagen et al . , U.S. Patent No. 4,784,950; Palmiter et al . , U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol. 36_.= 59-72, 1977) and

Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus . See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants" . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate . Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci . fBangalorej 11:47-58, 1987.

Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al . , U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) . See, King and Possee, The Baculovirus Expression System: A Laboratory Guide,

London, Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and, Richardson Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995. A second method of making recombinant zrnpl baculovirus utilizes a transposon-based system described by Luckow (Luckow et al . , J. Virol. β_7:4566-79, 1993). This system, which utilizes transfer vectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville, MD) . This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zrnpl polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid. " See, Hill- Perkins and Possee, J. Gen. Virol. 7JL.971-6, 1990; Bonning et al., J. Gen. Virol. 75_: 1551-6, 1994; and, Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-9, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zrnpl polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al . , Proc. Natl. Acad. Sci. 8_2: 7952-4, 1985) . Using a technique known in the art, a transfer vector containing zrpnl is transformed into E . coli , and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus . The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zrnpl is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921™

(Expression Systems) for the Sf9 cells; and Ex-cellO405™

(JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 10⁵ cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. Procedures used are generally described in available laboratory manuals (King and Possee, ibid. ; O'Reilly et al . , ibid. ; Richardson, ibid. ) . Subsequent purification of the zrnpl polypeptide from the supernatant can be achieved using methods described herein. Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica .

Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POTl vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosaccharomyces pombe,

Kluyveromyces lactis , Kluyveromyces frag His , Ustilago maydis , Pichia pastoris , Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol . 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acre-Tioniu-n chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S.

Patent No. 4,486,533.

For example, the use of Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808, Raymond,

U.S. Patent No. 5,736,383, Raymond et al . , Yeast 14:11-23, 1998, and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2) . Other useful promoters include those of the dihydroxyacetone synthase (DHAS) , formate dehydrogenase (FMD) , and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine . For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes {AUG1 and AUG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes {PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about

3.75 kV/cm, and a time constant (τ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid.). When expressing a zrnpl polypeptide in bacteria such as E . coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.

The adenovirus system can also be used for protein production in vi tro . By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovirus vector infected 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al . , Cytotechnol . 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant . Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose,

2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1%

Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine) .

It is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. Expressed recombinant zrnpl polypeptides (or chimeric zrnpl polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography : Principles & Methods, Pharmacia LKB

Biotechnology, Uppsala, Sweden, 1988.

Moreover, using methods described in the art, polypeptide fusions, or hybrid zrnpl proteins, are constructed using regions or domains of the inventive zrpnl in combination with those of other human ribonucleoproteins (e.g. UlsnRNP 70kD protein, hnRNP Al protein), or heterologous proteins (Sambrook et al . , ibid. , Altschul et al . , ibid. , Picard, Cur . Opin . Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.

Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain (s) conferring a biological function may be swapped between zrnpl of the present invention with the functionally equivalent domain (s) from another family member, such as snRNP UI 70kD protein or hnRNP Al protein. Such domains include, but are not limited to, the RNA- binding domain, conserved motifs [RNP1, RNP2] , or charged C-terminal regions. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known ribonucleoprotein proteins (e.g. snRNP UI 70kD or hnRNP Al protein) , depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein. Zrnpl polypeptides or fragments thereof may also be prepared through chemical synthesis, zrnpl polypeptides may be monomers or multimers; glycosylated or non- glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue. See for example, Merrifield, J . Am . Chem . Soc . 85:2149, 1963; Stewart et al . , "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co., Rockford, IL, 1984); Bayer and Rapp, Chem. Pept . Prot . 3:3, 1986; and Atherton et al . , Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989.

The activity of molecules of the present invention can be measured using a variety of assays that measure the interaction of RNA-binding proteins with RNA. Specific assays include, but are not limited to bioassays measuring RNA splicing, annealing, binding and UV crosslinking. Such assays are well known in the art, see for example, Mayeda et al . , EMBO J. 13_.: 5483-95, 1994. If desired, zrnpl polypeptide performance in this regard can be compared to other ribonucleoproteins, such as UlsnRNP

70 kD, A or C proteins or hnRNP Al protein, and the like.

Alternative RNA splicing can be measured using the methods of Reed and Maniatis, Cell 46_. = 681-90, 1986;

Krainer et al . , Cell 62 : 35-42, 1990 and Mayeda et al . , Mol. Cell. Biol. 13:2993-3001, 1993. RNA annealing can be measured using assays described by Munroe and Dung, Proc.

Natl. Acad. Sci. USA 89 = 895-99, 1992 and Mayeda et al . , ibid. UV crosslinking can be measured using the methods of Mayeda et al . , ibid, and Meyeda and Ohshima, Mol. Cell Biol. 8:4484-91, 1988.

Antagonists and agonists are also useful as research reagents for characterizing sites of RNA-binding. Inhibitors of zrnpl activity (zrnpl antagonists) include anti- zrnpl antibodies, as well as other peptidic and non- peptidic agents. Zrnpl can be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zrnpl.

As described above, the disclosed polypeptides can be used to construct zrnpl variants and functional fragments of zrnpl. Such variants and extracellular domain fragments are considered to be zrnpl agonists. Another type of zrnpl agonist is provided by anti-idiotype antibodies, and fragments thereof, which mimic the RNA- binding domain of zrnpl, for example. Zrnpl agonists can also be constructed using combinatorial libraries.

Methods for constructing and screening phage display and other combinatorial libraries are provided, for example, by Kay et al . , Phage Display of Peptides and Proteins

(Academic Press 1996), Verdine, U.S. Patent No. 5,783,384, Kay, et. al . , U.S. Patent No. 5,747,334, and Kauffman et al . , U.S. Patent No. 5,723,323.

Antibodies that associate with the zrnpl RNA- binding domain may be used to mask the zrnpl polypeptide from its corresponding RNA sequence, thus altering mRNA processing. Another approach to negate the effects of zrnpl expression is to inhibit the zrnpl synthesis. For example, cells can be transfected with an expression vector comprising a nucleotide sequence that encodes zrnpl anti-sense RNA. Suitable sequences for anti-sense molecules can be derived from the nucleotide sequences of zrnpl disclosed herein.

The zrnpl polypeptides disclosed herein can be used to determine the sequence specificity of the zrnpl

RNA-binding domain. Such methods are known in the art, see for example Tsai et al . , Nucleic Acids Research 19:4931-6, 1991. Such methods can be used to make phylogeneic comparisons with other RNA-binding proteins, predict conserved recognition elements, and to test the effects on RNA binding of altering amino acid residues in the zrnpl RNA-binding domain.

Nucleic acid molecules disclosed herein can be used to detect the expression of a zrnpl gene in a biological sample. Such probe molecules include double- stranded nucleic acid molecules comprising the nucleotide sequences of SEQ ID NOs : 1 or 6 , or fragments thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequences of SEQ ID NOs : 1 or 6, or a fragment thereof. Probe molecules may be DNA, RNA, oligonucleotides, and the like.

As an illustration, suitable probes include nucleic acid molecules that bind with a portion of a zrnpl domain or motif, such as the zrnpl RNA-binding domain (nucleotides 219 to about 135 of SEQ ID NO:l or nucleotides 92 to about 322 of SEQ ID N0:6), zrnpl structural features such as beta strands (located at about nucleotides 219-236, 291-311, 336-359 and 432-449 of SEQ ID N0:1 or nucleotides 92-100, 164-184, 109-132, and 305- 322 of SEQ ID NO:6), alpha helixes (located at about nucleotides 252-275 and 369-395 of SEQ ID NO:l or nucleotides 113-148 and 242-268 of SEQ ID N0:6), zrnpl motifs such as RNP-1 and RNP-2 (located at about nucleotides 219-236 and 336-359 of SEQ ID NO:l or nucleotides 109-132 and 92-100 of SEQ ID NO: 6) .

In a basic assay, a single-stranded probe molecule is incubated with RNA, isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target zrnpl RNA species . After separating unbound probe from hybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northern analysis and dot/slot blot hybridization (see, for example, Ausubel ibid, and Wu et al . (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectably labeled with radioisotopes such as ³²P or ³⁵S . Alternatively, zrnpl RNA can be detected with a nonradioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana Press, Inc., 1993). Typically, nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates. Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.

Zrnpl oligonucleotide probes are also useful for in vivo diagnosis. As an illustration, ¹⁸F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al . , Nature Medicine 4:467, 1998).

Numerous diagnostic procedures take advantage of the polymerase chain reaction (PCR) to increase sensitivity of detection methods. Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana

Press, Inc. 1991), White (ed.), PCR Protocols .- Current

Methods and Applications (Humana Press, Inc. 1993), Cotter

(ed.), Molecular Diagnosis of Cancer (Humana Press, Inc.

1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.), Clinical

Applications of PCR (Humana Press, Inc. 1998), and Meltzer

(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).

PCR primers can be designed to amplify a sequence encoding a particular zrnpl domain or motif, such as the zrnpl RNA- binding domain (encoded by about nucleotide 219 to 449 of

SEQ ID N0:1 or nucleotides 92 to 322 of SEQ ID NO: 6) .

One variation of PCR for diagnostic assays is reverse transcriptase-PCR (RT-PCR) . In the RT-PCR technique, RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with zrnpl primers (see, for example, Wu et al . (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR, " in Methods in Gene Biotechnology, CRC Press, Inc., pages 15- 28, 1997) . PCR is then performed and the products are analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, for example, the guanidinium- thiocyanate cell lysis procedure described above. Alternatively, a solid-phase technique can be used to isolate mRNA from a cell lysate . A reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or zrnpl anti- sense oligomers. Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences . Zrnpl sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically at least 5 bases in length.

PCR amplification products can be detected using a variety of approaches. For example, PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining. Alternatively, fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled zrnpl probe, and examined by autoradiography. Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach is real time quantitative PCR (Perkin-Elmer Cetus, Norwalk, Ct . ) . A fluorogenic probe, consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers. Using the 5' endonuclease activity of Taq DNA polymerase, the reporter dye is separated from the quencher dye and a sequence-specific signal is generated and increases as amplification increases. The fluorescence intensity can be continuously monitored and quantified during the PCR reaction.

Another approach for detection of zrnpl expression is cycling probe technology (CPT) , in which a single-stranded DNA target binds with an excess of DNA- RNA-DNA chimeric probe to form a complex, the RNA portion is cleaved with RNase H, and the presence of cleaved chimeric probe is detected (see, for example, Beggs et al., J. Clin. Microbiol . 34:2985, 1996 and Bekkaoui et al . , Biotechniques 20_:240, 1996). Alternative methods for detection of zrnpl sequences can utilize approaches such as nucleic acid sequence-based amplification (NASBA) , cooperative amplification of templates by cross- hybridization (CATCH) , and the ligase chain reaction (LCR) (see, for example, Marshall et al . , U.S. Patent No. 5,686,272 (1997), Dyer et al . , J. Virol. Methods 60:161, 1996; Ehricht et al . , Eur. J. Biochem. 243:358, 1997 and Chadwick et al . , J. Virol. Methods 70:59, 1998). Other standard methods are known to those of skill in the art.

Zrnpl probes and primers can also be used to detect and to localize zrnpl gene expression in tissue samples. Methods for such in si tu hybridization are well- known to those of skill in the art (see, for example, Choo (ed.), In Situ Hybridization Protocols, Humana Press, Inc., 1994; Wu et al. (eds.), "Analysis of Cellular DNA or

Abundance of mRNA by Radioactive In Si tu Hybridization

(RISH) , " in Methods in Gene Biotechnology, CRC Press, Inc., pages 259-278, 1997 and Wu et al . (eds.), "Localization of

DNA or Abundance of mRNA by Fluorescence In Si tu

Hybridization (RISH) , " in Methods in Gene Biotechnology, CRC Press, Inc., pages 279-289, 1997).

Various additional diagnostic approaches are well-known to those of skill in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics Humana Press, Inc., 1991; Coleman and Tsongalis, Molecular Diagnostics , Humana Press, Inc., 1996 and Elles, Molecular Diagnosis of Genetic Diseases, Humana Press, Inc., 1996) . Probes and primers generated from the sequences disclosed herein can be used to map the zrnpl gene to a particular chromosome. Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990) . Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) . These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence- tagged sites (STSs) , and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a gene's position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.

Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS) , GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm. nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences. The present invention also contemplates use of such chromosomal localization for diagnostic applications. Briefly, the zrpnl gene, a probe comprising zrpnl DNA or RNA or a subsequence thereof, can be used to determine if the zrnpl gene is present on human chromosome 14 or if a mutation has occurred. Detectable chromosomal aberrations at the zrnpl gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest

108 = 255-65, 1995) .

In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID N0:1 or SEQ ID NO : 6 , the complement of SEQ ID NO:l or SEQ ID NO: 6, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ;

Ausubel et. al . , ibid. ; Marian, Chest 108 :255-65, 1995). Ribonuclease protection assays (see, e.g., Ausubel et al . , ibid. , ch. 4) comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA are protected from digestion. Within PCR assays, a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991) .

The invention also provides anti-zrnpl antibodies. Antibodies to zrnpl can be obtained, for example, using as an antigen the product of a zrnpl expression vector, or zrnpl isolated from a natural source. Particularly useful anti-zrnpl antibodies "bind specifically" with zrnpl. Antibodies are considered to be specifically binding if the antibodies bind to a zrnpl polypeptide, peptide or epitope with a binding affinity

(K_a) of 10 6 M-1 or greater, preferably 107 M-1 or greater,

8 -1 more preferably 10 M or greater, and most preferably 10 9 M-1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann.

NY Acad. Sci. 5_1:660, 1949) . Suitable antibodies include antibodies that bind with zrnpl in particular domains, such as the zrnpl RNA-binding domain (amino acid residues

59 to about 135 of SEQ ID NO : 2 or amino acid residues 26 to about 102 of SEQ ID NO:7), the RNP-1 motif (located at about amino acid residues 98 to 105 of SEQ ID NO: 2 or amino acid residues 65 to 72 of SEQ ID NO:7), or the RNP-2 motif (located at about amino acid residues 59 to 64 of SEQ ID NO: 2 or amino acid residues 26 to 31 of SEQ ID NO : 7) . Anti- zrnpl antibodies can be produced using antigenic zrnpl epitope-bearing peptides and polypeptides. Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within SEQ ID NO : 2 or SEQ ID NO : 7. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with zrnpl. It is desirable that the amino acid sequence of the epitope- bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided) . Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.

Polyclonal antibodies to recombinant zrnpl protein or to zrnpl isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al . , "Production of Polyclonal Antisera, " in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al . , "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al . (eds.), page 15 (Oxford University Press 1995) . The immunogenicity of a zrnpl polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zrnpl or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like, " such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, hamsters, guinea pigs, goats, or sheep, an anti-zrnpl antibody of the present invention may also be derived from a subhuman primate antibody. General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al . , international patent publication No. WO 91/11465, and in Losman et al . , Int. J. Cancer 46_.: 310, 1990. Antibodies can also be raised in transgenic animals such as transgenic sheep, cows, goats or pigs, and can also be expressed in yeast and fungi in modified forms as will as in mammalian and insect cells.

Alternatively, monoclonal anti-zrnpl antibodies can be generated. Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al . , Nature 256 : 495 (1975), Coligan et al . (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991), Picksley et al . , "Production of monoclonal antibodies against proteins expressed in E. coli , " in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al . (eds.), page 93 (Oxford University Press 1995) ) .

Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a zrnpl gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures. In addition, an anti-zrnpl antibody of the present invention may be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al . , Nature Genet . 7:13, 1994, Lonberg et al . , Nature 368:856, 1994, and Taylor et al . , Int . Immun . 6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well- established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size- exclusion chromatography, and ion-exchange chromatography

(see, for example, Coligan at pages 2.7.1-2.7.12 and pages

2.9.1-2.9.3; Baines et al . , "Purification of

Immunoglobulin G (IgG)," in Methods in Molecular Biology,

Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). For particular uses, it may be desirable to prepare fragments of anti-zrnpl antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. As an illustration, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')₂. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments. Optionally, the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages. As an alternative, an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al . , Arch Biochem. Biophys. 89:230, 1960, Porter, Biochem. J. 73_.: 119, 1959, Edelman et al . , in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and by Coligan, ibid.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of V_H and V_L chains. This association can be noncovalent, as described by Inbar et al . , Proc . Nat ' 1 Acad. Sci. USA 69:2659, 1972. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as gluteraldehyde (see, for example, Sandhu, Crit . Rev. Biotech. 12:437, 1992) .

The Fv fragments may comprise V_H and V_L chains which are connected by a peptide linker. These single- chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V_H and V_L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli . The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are described, for example, by Whitlow et al . , Methods : A Companion to Methods in Enzymology 2:97, 1991, also see, Bird et al . , Science 242 :423 , 1988, Ladner et al., U.S. Patent No. 4,946,778, Pack et al . , Bio/Technology 11:1271, 1993, and Sandhu, supra . As an illustration, a scFV can be obtained by exposing lymphocytes to zrnpl polypeptide in vi tro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zrnpl protein or peptide) . Genes encoding polypeptides having potential zrnpl polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli . Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , U.S. Patent No. 5,223,409, Ladner et al . , U.S. Patent No. 4,946,778, Ladner et al . , U.S. Patent No. 5,403,484, Ladner et al . , U.S. Patent No. 5,571,698, and Kay et al . , Phage Display of Peptides and Proteins (Academic Press, Inc. 1996)) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA) , Invitrogen Inc. (San Diego, CA) , New England Biolabs, Inc. (Beverly, MA), and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) . Random peptide display libraries can be screened using the zrnpl sequences disclosed herein to identify proteins which bind to zrnpl . Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR) . CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody- producing cells (see, for example, Larrick et al . , Methods : A Companion to Methods in Enzymology 2_.:106, 1991) , Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies, " in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al . (eds.), page 166 (Cambridge University Press 1995), and Ward et al .. "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al . , (eds.), page 137 (Wiley-Liss, Inc. 1995) ) . Alternatively, an anti-zrnpl antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al . , Proc. Nat ' 1 Acad. Sci. USA 8_.6: 3833, 1989. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986, Carter et al . , Proc . Nat ' 1 Acad. Sci. USA 89:4285, 1992, Sandhu, Crit . Rev. Biotech. 12:437, 1992, Singer et al . , J . Immun . 150_.:2844, 1993, Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic Antibodies," in Protein Engineering: Principles and Practice, Cleland et al . (eds.), pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al . , U.S. Patent No. 5,693,762 (1997) .

Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-zrnpl antibodies or antibody fragments, using standard techniques. See, for example, Green et al . , "Production of Polyclonal Antisera, " in Methods In Molecular Biology: Immunochemical Protocols , Manson (ed.), pages 1-12 (Humana Press 1992) . Also, see Coligan, ibid. at pages 2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can be prepared using anti-zrnpl antibodies or antibody fragments as immunogens with the techniques, described above. As another alternative, humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques. Methods for producing anti-idiotype antibodies are described, for example, by Irie, U.S. Patent No. 5,208,146, Greene, et . al., U.S. Patent No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol. 77:1875, 1996. Antibodies directed toward intracellular and nuclear antigens from UsnRNP associated proteins and RNAs have been found in sera of patients suffering from a variety of connective tissues diseases (Maddison et al . , ibid. and Hoet et al . , ibid. ) . Anti-Sm antibodies, directed to the common proteins B/B', Dl, D2 , D3 , E, F or G, are highly specific for SLE and are considered a diagnostic marker of the disease (Tan, Adv . Immunol . 44=93-120, 1989) . Ro/SSA and La/SSb are associated with and specific for sera from patients suffering from SLE and its variants, photosensitive dermatitis, as well as in

Sjόgren's syndrome. Anti-nRNP antibodies are associated with mixed connective tissue disease, having features of

SLE, systemic sclerosis, rheumatoid arthritis and polymyositis . Methods for detecting the presence of antibodies directed towards intracellular and nuclear RNA-binding proteins are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) , see Seelig et al . , J. Immunol. Meth. 143:11-24, 1991; Maddison et al . , ibid. and Vlachoyiannopoulos et al . , ibid. ; immunoblot analysis (Lundberg et al . , ibid.), counterimmunoelectrophoresis (Kurata and Tan, Arth. Rheum. 19:574-645, 1996). Such methods could be used in diagnostic methods to monitor and quantify levels of anti- RNA-binding protein antibodies.

Polypeptides of the present invention may be used within diagnostic systems to detect the presence of anti-zrnpl antibodies in a biological sample. Biological samples include cells, cell components or cell products, including, but not limited to, cell culture supernatants, cell lysates, cleared cell lysates, cell extracts, tissue extracts, blood plasma, serum, and fractions thereof, from a patient. Polypeptides or other agents that specifically bind to zrnpl antibodies may be used to detect the presence of such circulating antibodies. More specifically, the present invention contemplates methods for detecting zrnpl antibodies comprising: exposing a biological sample possibly containing anti-zrpnl antibodies to an polypeptide attached to a solid support, wherein the antibody binds to a first epitope of a zrnpl polypeptide; washing the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zrpnl polypeptide-antibody complex, wherein the second antibody is associated with a detectable label; and detecting the detectable label. Serum or biopsy anti-zrnpl antibody concentration (relative to normal serum or tissue concentration) may be indicative of dysfunction related to altered levels of anti-zrnpl antibodies. Such indications include autoimmune diseases, in particular diseases associated with connective tissues. The zrnpl polynucleotides, polypeptides, agonists, antagonists and antibodies of the present invention can used to diagnose, treat or prevent such diseases.

Animal models, such as the SWR x SJC mouse, which spontaneously develop anti-nuclear antibodies (Vidal et al. J. Exp. Med. 179:1429-35, 1994) are available.

Antisense methodology can be used to inhibit zrnpl gene transcription, such as to inhibit cell proliferation in vivo . Polynucleotides that are complementary to a segment of a zrnpl-encoding polynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO:l or SEQ ID NO : 6 ) are designed to bind to zrnpl- encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zrnpl polypeptide-encoding genes in cell culture or in a subject.

Transgenic mice, engineered to express the zrnpl gene, and mice that exhibit a complete absence of zrnpl gene function, referred to as "knockout mice" (Snouwaert et al., Science 257:1083 , 1992), may also be generated (Lowell et al . , Nature 366_.: 740-42 , 1993). These mice may be employed to study the zrnpl gene and the protein encoded thereby in an in vivo system.

Pharmaceutically effective amounts of zrnpl polypeptides or zrnpl antagonists of the present invention can be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal, topical, transdermal administration or the like, according to conventional methods. Formulations may further include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets, for example. Slow or extended-release delivery systems, including any of a number of biopolymers (biological-based systems) , systems employing liposomes, and polymeric delivery systems, can also be utilized with the compositions described herein to provide a continuous or long-term source of the zrnpl polypeptide or antagonist. Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use. The term "pharmaceutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient. One skilled in the art may formulate the compounds of the present invention in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington: The Science and Practice of Pharmacy, Gennaro, ed. , Mack Publishing Co., Easton PA, 19th ed. , 1995.

As used herein a "pharmaceutically effective amount" of a zrnpl polypeptide, agonists or antagonist is an amount sufficient to induce a desired biological result. The result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an effective amount of a zrnpl polypeptide is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer. For example, such an effective amount of a zrnpl polypeptide or antagonist would decrease the amount of circulating anti-zrnpl antibodies in patients suffering from autoimmune diseases. Such an amount would include that sufficient to alter zrnpl-mediated mRNA processing or to inhibit zrnpl polypeptide synthesis. Effective amounts of the zrnpl polypeptides can vary widely depending on the disease or symptom to be treated. The amount of the polypeptide to be administered and its concentration in the formulations, depends upon the vehicle selected, route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and the stability of the compound in the formulation. Thus, the clinician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients. Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health of the patient, and other factors evident to those skilled in the art. Typically a dose will be in the range of 0.1-100 mg/kg of subject. Doses for specific compounds may be determined from in vitro or ex vivo studies in combination with studies on experimental animals. Concentrations of compounds found to be effective in vitro or ex vivo provide guidance for animal studies, wherein doses are calculated to provide similar concentrations at the site of action.

The invention is further illustrated by the following non-limiting example.

EXAMPLES EXAMPLE 1

Tissue Distribution

Northern analysis was performed using human brain-specific Northern blots and human fetal tissue- specific Northern blots from Clontech. An approximately 415 bp DNA fragment was generated by PCR amplification using oligonucleotide primers ZC10,071 (SEQ ID NO: 9) and ZC10,063 (SEQ ID NO.10). This 415 bp fragment is from the 3' untranslated region of zrnpl. The 415 bp DNA fragment was electrophoresed on a 1% agarose gel, the fragment was electroeluted, and then radioactively labeled using a random priming MEGAPRIME DNA labeling system (Amersham, Arlington Heights, IL) according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA) . EXPRESSHYB (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 68°C, and the blots were then washed in 2X SSC and 0.05% SDS at RT, followed by a wash in 0. IX SSC and 0.1% SDS at 50°C. Three transcript sizes were detected. Two transcript sizes were observed in all areas of the brain tested, one at approximately 3.2 kb and one at 5.5 kb. Signal intensity was highest for the 5.5 kb transcript. A third transcript size of 7.1 kb was present at varying intensity, with highest expression in cerebral cortex, medulla, spinal cord and corpus callosum. On the fetal blot, the highest level of expression of all three transcripts was in fetal kidney.

The same probe was used to probe human multiple tissue Northern blots (MTN, MTN II and MTN III, Clontech) using the same hybridization and washing conditions described above. The same three transcript sizes, 3.2, 5.5 and 7.1 kb, were seen in varying intensity in all tissues, with the highest expression of the 3.2 and 5.5 kb transcripts in skeletal muscle, ovary, thyroid and spinal cord. The 7.1 transcript was most intense in spinal cord, brain, ovary, skeletal muscle and testis. A more stringent wash at 65°C did not alter the binding patterns described above .

A second Northern was performed using a hybridization probe from the 5 ' end of the coding region of zrnpl cDNA. An approximately 74 bp DNA fragment was generated by PCR amplification using oligonucleotide primers ZC17,615 (SEQ ID NO: 11) and ZC17,295 (SEQ ID NO: 12) . The 74 bp DNA fragment was electrophoresed on a 1.5% agarose gel, the fragment recovered using a QIAGEN QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's instructions, and then radioactively labeled using a MULTIPRIME DNA labeling system (Amersham, Arlington Heights, IL) according to the manufacturer's specifications. The labeled probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA) . EXPRESSHYB (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. The 5' specific zrnpl probe was used to probe human multiple tissue Northern blots (MTN, MTN II and MTN III, Clontech) . Hybridization took place overnight at 65°C, and the blots were then washed in 2X SSC and 0.1% SDS at RT, followed by a wash in 0. IX SSC and 0.1% SDS at 50°C. Five transcript sizes were detected in all tissue samples represented on the Clontech blots. The three largest transcripts coincided with the transcript sizes seen above at 7.1 kb, 5.5 kb, and 3.2 kb. In addition, two additional transcripts sizes were observed at 1.5 kb and 0.9 kb .

EXAMPLE 2

Chromosomal Localization of Zrnpl

Zrnpl was mapped to chromosome 14 using the commercially available "GeneBridge 4 Radiation Hybrid Panel" (Research Genetics, Inc., Huntsville, AL) . The GeneBridge 4 Radiation Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) . A publicly available WWW server (http://www-genome.wi.mit.edu/cgi- bin/contig/rhmapper .pi) allows mapping relative to the Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome (the "WICGR" radiation hybrid map) which was constructed with the GeneBridge 4 Radiation Hybrid Panel .

For the mapping of zrnpl with the GeneBridge 4 RH Panel, 20 μl reactions were set up in a 96 -well microtiter plate (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2 μl 10X PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA) , 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, CA) , 1 μl sense primer, ZC 20,192 (SEQ ID NO:13), 1 μl antisense primer, ZC 20,193 (SEQ ID NO:14), 2 μl . ediLoad (Research Genetics, Inc.), 0.4 μl 50X Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH₂0 for a total volume of 20 μl . The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows : an initial 1 cycle 4 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 64°C and 1 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD) .

The results showed that zrnpl maps 1.01 cR_3000 from the framework marker WI-6253 on the chromosome 14 WICGR radiation hybrid map. Proximal and distal framework markers were WI-6253 and WI-5815, respectively. The use of surrounding markers positions zrnpl in the 14q22.2- 14q23.1 region on the integrated LDB chromosome 14 map (The Genetic Location Database, University of Southhampton, WWW server: http: //cedar .genetics . soton. ac.uk/public_html/).

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

What is claimed is :

1. An isolated polynucleotide molecule that encodes a polypeptide, wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 34 to amino acid residue 518, and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2; b) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 85% identical to the amino acid sequence as shown in SEQ ID NO: 7, from amino acid residue 1 to amino acid residue 125, and specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 7 ; or c) a polynucleotide molecule having the sequence of SEQ ID NO:l, SEQ ID NO : 6 or SEQ ID NO : 8.

2. An isolated polynucleotide molecule according to claim 1, wherein any difference between said amino acid sequence encoded by the polynucleotide molecule and said corresponding amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7 is due to a conservative amino acid substitution.

3. An isolated polynucleotide of claim 1, wherein said amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=blosum62, with other parameters set as default .

4. An isolated polynucleotide of claim 1, wherein said polynucleotide hybridizes under stringent conditions to a polynucleotide molecule having the nucleotide sequence selected from the group consisting of: a) SEQ ID NO:l; b) SEQ ID NO: 6; c) the complement of SEQ ID N0:1; or d) the complement of SEQ ID NO: 6.

4. An isolated polynucleotide molecule of claim 1, wherein said polypeptide further comprises an affinity tag or binding domain.

6. An isolated polynucleotide molecule of claim 1, comprising nucleotides 144-518 of SEQ ID NO:l.

7. An isolated polynucleotide molecule selected from the group consisting of: a) nucleotides 219-449 of SEQ ID NO:l; b) nucleotides 462-518 of SEQ ID NO:l; c) nucleotides 92-109 of SEQ ID NO: 6; and d) nucleotides 335-391 of SEQ ID NO: 6.

8. An expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule according to claim 1; and a transcription terminator.

9. An expression vector according to claim 8 further comprising a secretory signal sequence operably linked to said DNA segment .

10. An expression vector according to claim 8, wherein said polynucleotide encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag.

11. A cultured cell into which has been introduced an expression vector comprising the following operably linked elements : a transcription promoter; a polynucleotide molecule according to claim 1; and a transcription terminator, wherein said cultured cell expresses said polypeptide encoded by said polynucleotide segment .

12. A method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements : a transcription promoter; a polynucleotide molecule according to claim 1; and a transcription terminator; whereby said cell expresses said polypeptide encoded by said polynucleotide segment; and recovering said expressed polypeptide.

13. A method according to claim 12, wherein said expression vector further comprises a secretory signal sequence operably linked to said polynucleotide segment, said cultured cell secretes said polypeptide into a culture medium, and said polypeptide is recovered from said culture medium.

14. An isolated polypeptide comprising a sequence of amino acid residues that is at least 85% identical to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 34 to amino acid residue 518, b) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 125 of SEQ ID NO: 7; wherein said isolated polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7.

15. An isolated polypeptide according to claim 14 wherein said polypeptide comprises a sequence of amino acid residues that is at least 90% identical to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 34 to amino acid residue 518, b) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 125 of SEQ ID NO: 7; wherein said isolated polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO : 2 or SEQ ID NO: 7.

16. An isolated polypeptide according to claim 15, wherein said polypeptide comprises a sequence of amino acid residues that is at least 95% identical to a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 34 to amino acid residue 518, b) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO: 2, from amino acid residue 1 to amino acid residue 125 of SEQ ID NO: 7; wherein said isolated polypeptide specifically binds with an antibody that specifically binds with a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7.

17. An isolated polynucleotide molecule according to claim 14, wherein any difference between said amino acid sequence encoded by the polynucleotide molecule and said corresponding amino acid sequence of SEQ ID NO : 2 or SEQ ID NO : 7 is due to a conservative amino acid substitution.

18. An isolated polynucleotide of claim 14, wherein the amino acid percent identity is determined using a FASTA program with ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=blosum62 , with other parameters set as default.

19. An isolated polypeptide according to claim 14, further comprising an affinity tag or binding domain.

20. An isolated polypeptide comprising amino acid residues 59-135 of SEQ ID NO:2.

21. An isolated polypeptide according to claim 20, wherein said polypeptide comprises four β strands corresponding to amino acid residues 59-64, 83-89, 98-105 and 130-135 of SEQ ID NO : 2 and two α helices corresponding to amino acid residues 70-77 and 109-117 of SEQ ID NO : 2.

22. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) a polypeptide comprising the amino acid sequence of SEQ ID NO: 7; c) amino acid residues 59-135 of SEQ ID NO: 2; and d) amino acid residues 140-158 of SEQ ID NO:2.

23. An antibody or antibody fragment that specifically binds to a polypeptide according to claim 14.

24. An antibody according to claim 23, wherein said antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody.

25. An antibody fragment according to claim 23, wherein said antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.

26. An anti-idiotype antibody that specifically binds to said antibody of claim 14.

27. A polypeptide according to Claim 14, in combination with a pharmaceutically acceptable vehicle.