EP1294887A1 - Polypeptide zins4 homologue de l'insuline - Google Patents

Polypeptide zins4 homologue de l'insuline

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Publication number
EP1294887A1
EP1294887A1 EP01909021A EP01909021A EP1294887A1 EP 1294887 A1 EP1294887 A1 EP 1294887A1 EP 01909021 A EP01909021 A EP 01909021A EP 01909021 A EP01909021 A EP 01909021A EP 1294887 A1 EP1294887 A1 EP 1294887A1
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EP
European Patent Office
Prior art keywords
seq
polypeptide
amino acid
residue
zins4
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01909021A
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German (de)
English (en)
Inventor
James L. Holloway
Si Lok
Stephen R. Jaspers
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Zymogenetics Inc
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Zymogenetics Inc
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Publication of EP1294887A1 publication Critical patent/EP1294887A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/04—Drugs for genital or sexual disorders; Contraceptives for inducing labour or abortion; Uterotonics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/64—Relaxins

Definitions

  • hormones and polypeptide growth factors are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form organs, and to repair and regenerate damaged tissue.
  • hormones and growth factors include the steroid hormones (e.g., estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO), insulin and calcitonin. Hormones and growth factors influence cellular metabolism by binding to receptors. Receptors may be integral membrane proteins, that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble molecules, such as certain transcription factors.
  • Insulin belongs to a group of protein/polypeptide hormones. Insulin increases the rate of synthesis of glycogen, fatty acids, and proteins and stimulates glycolysis. It also promotes the transport of glucose, some other sugars, and amino acids into muscle and fat cells.
  • the mature form of insulin consists of a 30 amino acid residue B chain, that is at the N-terminus of the propeptide form, and a 21 amino acid residue A chain, that is C-terminal.
  • Proinsulin also contains a connecting peptide, C- peptide, between the B chain and A chain that is cleaved out to form mature insulin. The B chain and A chain are covalently joined by two disulfide bonds.
  • the B-chain, C- peptide, A-chain motif is found in several other proteins including, relaxin (U.S. Patent No. 4,835,251), insulin-like growth factors (IGF) I and ⁇ (Bang and Hall, In “Insulinlike Growth Factors", P. N. Schofield (eds.), 151-177, Oxford Univ. Press, Oxford), Leydig factor (Bullesbach et al., J. Biol. Chem. 270:16011-15, 1995), Leydig insulinlike peptide (LEY I-L, Burkhardt et al., Hum. Genet.
  • IGF I and IGF II have D and E domains that are cleaved post-franslationally. Cysteines that are involved in disulfide bonds are conserved in all the members of the family and play a role in the tertiary structure ofthe molecules.
  • the invention provides an isolated polypeptide comprising amino acid residues 26-52 of SEQ ID NO:2.
  • the polypeptide further comprises amino acid residues 119-142 of SEQ ID NO:2.
  • the said polypeptide further comprises amino acid residues 55-114 of SEQ ID NO:2.
  • the polypeptide comprises the amino acid residues 26-142 of SEQ ID NO:2.
  • polypeptide comprises the amino acid residues 1-142 of SEQ ID NO:2.
  • the invention also provides an isolated polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2, wherein said polypeptide specifically binds with an antibody to which a polypeptide having the amino acid sequence of SEQ ID NO:2 specifically binds.
  • isolated polypeptide comprises the sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • any difference between said amino acid sequence and said corresponding amino acid sequence of SEQ ID NO:2 is due to one or more conservative amino acid substitutions.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2. Additionally, the invention provides an isolated polypeptide selected from the group consisting of: a) a polypeptide consisting of the sequence of amino acid residues from residue 26 to residue 52 of SEQ ID NO:2; b) a polypeptide consisting of the sequence of amino acid residues from residue 26 to residue 53 of SEQ ID NO:2; c) a polypeptide consisting of the sequence of amino acid residues from residue 26 to residue 54 of SEQ ID NO:2; d) a polypeptide consisting of the sequence of amino acid residues from residue 55 to residue 114 of SEQ ID NO:2; e) a polypeptide consisting of the sequence of amino acid residues from residue 55 to residue 115 of SEQ ID NO:2; f) a polypeptide consisting of the sequence of amino acid residues from residue 55 to residue 116 of SEQ ID NO:2; g) a polypeptide consisting of the sequence of amino acid residues from residue 55 to residue
  • the invention also provides an isolated protein having: a B chain comprising amino acid residue 26 to amino acid residue 52 of SEQ ID NO:2; and an A chain comprising amino acid residue 119 to amino acid residue 142 of SEQ ID NO:2; wherein the B chain and A chain are joined by inter- and infra-chain disulfide bonds.
  • polypeptide as described above, further comprising an affinity tag or binding domain.
  • the invention provides an isolated polynucleotide molecule that encodes a polypeptide as described above.
  • the invention also provides an isolated polynucleotide molecule of SEQ ID NO:l.
  • the invention provides an isolated polynucleotide selected from the group consisting of: a) a polynucleotide molecule consisting of nucleotides 74-156 of SEQ ID NO:l; b) a polynucleotide consisting of nucleotides 74-159 of SEQ ID NO:l; c) a polynucleotide consisting of nucleotides 74-162 of SEQ ID NO:l; d) a polynucleotide consisting of nucleotides 163-342 of SEQ ID NO:l; e) a polynucleotide consisting of nucleotides 163-345 of SEQ ID NO: 1; f) a polynucleotide consisting of nucleot
  • the invention provides cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule that encodes a polypeptide according to claim 1; and a transcription terminator, wherein said cultured cell expresses said polypeptide encoded by said polynucleotide segment.
  • the cell further comprises a second expression vector comprising the following operably linked elements: a franscriptional promoter; a DNA sequence encoding a prohormone convertase; and a ttanscriptional terminator.
  • the prohormone convertase is selected from the group consisting of prohormone convertase 1/3, prohormone convertase 2, prohormone convertase 4, PACE, PACE4, furin, and kex2.
  • the invention also provides a method of producing a protein comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a polynucleotide molecule that encodes a polypeptide as described above; and a transcription terminator; whereby said cell expresses said polypeptide encoded by said polynucleotide segment; and recovering said expressed protein.
  • the invention also provides a fusion protein, comprising the polypeptide as described above.
  • the invention provides an antibody or antibody fragment that specifically binds to a polypeptide as described above.
  • 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.
  • the antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.
  • the invention also provides an anti-idiotype antibody that specifically binds to an antibody as described above.
  • the invention also provides a polypeptide as described above, in combination with a pharmaceutically acceptable vehicle.
  • the invention further provides a method for producing, from a genomic DNA source, a contiguous polypeptide encoding polynucleotide sequence free from introns comprising the steps of: a) amplifying ex ⁇ n segments using exon specific primer pairs having a type II-S restriction endonuclease site near the primer 5' terminus; b) digest the amplified exon segments with the type II-S restriction endonuclease to produce cohesive overhangs; and c) ligate digested exon segments.
  • the genomic DNA source is purified genomic DNA, a genomic clone, a BAC, a PAC, genomic library, or chromosomal DNA clone.
  • 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.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al., ⁇ MBO J. 4:1015, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc.
  • 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.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • amino-terminal N-terminal
  • carboxyl-terminal C- terminal
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/hgand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 ⁇ M" x .
  • polynucleotide molecule denotes 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'.
  • sequence denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide.
  • representative contigs to the polynucleotide sequence 5'-ATGGCTTAGCTT-3' are 5'- T AGCTTgagtct-3 ' and 3 ' -gtcgacT ACCGA-5 ' .
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 promoter 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 316:114-1 , 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • 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 DNA, and may be isolated from natural sources, synthesized in vitro, 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-sttanded.
  • 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”.
  • promoter is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA 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 "receptor” denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
  • Membrane-bound receptors are characterized by a multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal fransduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • 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.
  • 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
  • the present invention is based in part upon the discovery of a novel DNA sequence that encodes a novel polypeptide having homology to the relaxin family.
  • the polypeptide has been designated zins4.
  • zins4 polypeptide of SEQ JD NO:2 shares 28% identity over a 117 amino acid residue overlap with spiny dogfish relaxin (RELX_SQUAC, Bullesbach et al., Eur. J. Biochem.
  • the cysteine motif is highly conserved in the B and A chains, where the B chain motif can be represented as LCGX ⁇ 10 ⁇ C, where X ⁇ ⁇ is the number of amino acid residues. X is any amino acid residue except cysteine.
  • the A chain motif is CCX ⁇ 3 ⁇ CX ⁇ 8 ⁇ C (SEQ ID NO:4), where X ⁇ ⁇ is the number of any amino acid residues. X is any amino acid residue except cysteine.
  • the B chain also contains a conserved motif RXXXR (SEQ JD NO:5). X represents any amino acid residue except cysteine.
  • the two arginine residues are necessary for relaxin binding to cells and tissues. Ii either arginine is changed synthetically, there is no competition for relaxin binding.
  • the DNA sequence encoding the zins4 polypeptide revealed that the predicted a ino acid sequence contained the B chain-C peptide-A chain motif found in the relaxins and insulin.
  • Preprorelaxin and preproinsulin both have a signal sequence, followed by the B chain, C peptide, and then the A chain.
  • the mature relaxins and insulin have the signal peptide and C peptide removed, with the B and A chains joined by both inter- and infra-chain disulfide bonds (James et al., Nature 267:544-546, 1977). Sequence analysis indicates that the human polypeptide sequence (SEQ ID NO: 2) is structurally equivalent to other members of the family.
  • Processing of the protein involves cleavage at the C-terminus of the signal peptide, and, based on predicted structural homology with other mature members of the insulin family, a cleavage at the C-terminus of the-B chain and at the N-terminus of the A chain, resulting in removal of the C-peptide.
  • Analysis of the zins4 polypeptide of SEQ ID NO:2 with other known members of the insulin family suggests a signal peptide cleavage site in the region of amino acid residue 25 (Ala) of SEQ ID NO: 2.
  • Cleavage at the C-terminus of the B chain is predicted to be at the C-terminal of amino acid residue 53 (Arg) or residue 54 (Arg) followed by cleavage of the Arg residues by carboxypeptidase to leave amino acid residue 52 (Trp) as the C-terminal amino acid residue.
  • Cleavage sites resulting in the N-terminus of the A chain are suggested in the region of amino acid residue 115 (Arg) to 118 (Arg).
  • Cleavage is predicted to be after the C-terminus of amino acid residue 118 (Arg) leaving amino acid residue 119 (Asp) as the N-terminal amino acid residue of the A chain.
  • the C-terminal amino acid is residue 142 (Cys).
  • the cleavage site at the junction of the C-peptide and A chain is highly conserved, occurring after Arg-X-X-Arg (wherein X is any amino acid residue), Arg- Arg or Lys-Arg; however, the cleavage sites at the junction of the signal sequence and B chain, and at the junction of the B chain and C-peptide, do not maintain a similarly high degree of conservation within the insulin family.
  • the amino acid sequence of mature zins4 includes a B chain having the sequence of SEQ ID NO:2 from amino acid residue 26 (Arg) to amino acid residue 52 (Trp), and an A chain having the sequence of SEQ JD NO:2 from amino acid residue 119 (Asp) to amino acid residue 142 (Cys).
  • the enzymology of proinsulin conversion suggests that prohormone convertase 1/3 (PCl/3), or prohormone convertase 2 (PC2), cleave zins4 primarily at the B chain-C-peptide junction.
  • the B chain contains a conserved motif CCX ⁇ 3 ⁇ CX ⁇ 8 ⁇ C (amino acid residues 128-142 of SEQ ID NO:2), where X ⁇ ⁇ is the number of any amino acid residues, and X is any amino acid residue except cysteine.
  • the C peptide comprises amino acid residue 55 (Ser) to amino acid residue 118 (Arg) of SEQ ID NO:2
  • zins4 designates nucleotide sequence or related matters and zins4 designates polypeptide sequences.
  • the present invention also provides polynucleotide molecules, including
  • SEQ JD NO:6 is a degenerate DNA sequence that encompasses all DNAs that encode the zins4 polypeptide of SEQ JD NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ JD NO: 6 also provides all RNA sequences encoding polypeptides of SEQ ID NO:2 by substituting U for T.
  • zins4 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 429 of SEQ ID NO:l and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ JD NO: 6 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
  • degenerate codons used in SEQ JD NO:6, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • 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.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY).
  • WSN can, in some circumstances, encode arginine
  • MGN degenerate codon for arginine
  • AGY serine
  • 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 sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
  • 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).
  • 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.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein franslation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ JD NO: 6 serves 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 isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic sfrength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake, MN) and Primer Premier 4.0 (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 identify suitable probe sequences. Typically, hybridization of longer polynucleotide sequences, >50 base pairs, is performed at temperatures of about 20-25°C below the calculated T m . For smaller probes, ⁇ 50 base pairs, 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.
  • Suitable stringent hybridization conditions are equivalent to about a 5 h to overnight incubation at about 42°C in a solution comprising: about 40-50% formamide, up to about 6X SSC, about 5X Denhardt's solution, zero up to about 10% dextran sulfate, and about 10-20 ⁇ g/ml denatured commercially-available carrier DNA.
  • stringent conditions include temperatures of 20-70°C and a hybridization buffer containing up to 6x SSC and 0-50% formamide; hybridization is then followed by washing filters in up to about 2X SSC.
  • a suitable wash stringency is equivalent to 0.1X SSC to 2X SSC, 0.1% SDS, at 55°C to 65°C.
  • Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
  • the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes.
  • Stringent hybridization and wash conditions depend on the length of the probe, reflected in the Tm, hybridization and wash solutions used, and are routinely determined empirically by one of skill in the art.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • Methods for preparing DNA and RNA are well known in the art and are discussed herein.
  • Zins4 is representative of an emerging class of genes that were first discovered through interrogation of genomic databases. The absence of information pointing to a source of mRNA template makes it difficult to employ conventional means to isolate a full-length cDNA for these genes. Additionally, many of these genes are not represented in current EST databases suggesting that they are either expressed at very low levels or by limited cell-types or at restricted times.
  • Isolation of a cDNA encoding the full-length polypeptide is a necessary first step for producing recombinant protein. While it is possible for some eukaryotic expression system to produce recombinant proteins encoded by introduced genomic sequences, many genes contain multiple introns whose collective length renders the expression unit too large to be efficiently inserted in most plasmid-based expression vectors. Another challenge posed by genomic sequence is the possible presence of repeated elements within introns. These elements may promote plasmid-instability while the expression vector is being propagated through the bacterial host. The presence of intronic sequences within the expression cassette leaves open the possibility for the recipient mammalian host cell to use cryptic splice donor and acceptor sites within the inttons.
  • Such alternative splice sites may be natural to the gene or may be artifactural to the expression host cell.
  • the use of these cryptic splice sites by the host cell could lead to the production of a different recombinant polypeptide than intended.
  • the splicing mechanisms of mammalian, yeast or insect cells may also be sufficiently different from one another to preclude accurate or efficient splicing of some mRNAs transcribed by heterologous genes.
  • the lack of mRNA splicing in bacterial host cells necessitates the removal of all intronic sequence within the expression unit for the production of recombinant protein in such systems.
  • PCR polymerase chain reaction
  • the resulting PCR product derived from these primers would incorporate the restriction endonuclease site at its terminus, which upon digestion with the corresponding type II restriction enzyme, would produce suitable cohesive overhangs to promote efficient ligation in the presence of DNA ligase.
  • This method cannot be employed to ligate exon segments to produce a contiguous polypeptide coding sequence. Exon segments ligated together in this manner would contain a foreign resttiction endonuclease recognition sequence between ligated segments thereby introducing one or more added amino acid residues at each ligation junction.
  • blunt-end ligation reaction would enable the ligation of PCR generated exon segments without the need to incorporate restriction endonuclease sites to the primers and thereby the ligated products would be free of foreign sequences.
  • This approach has severe limitations. Since the blunt-end ligation reaction does not make use of defined cohesive ends, the number of combinations and permutations of incorrect ligation products increases exponentially with the number of exons to be ligated, rendering this method impractical for general use.
  • the type IJ-S restriction enzymes cleave double-sttanded DNA and generate complementary cohesive ends at a defined distance and orientation outside an asymmetric DNA recognition site.
  • This property of he type JJ-S endonucleases was exploited to design sets of exon-specific PCR primers. Digestion of the resulting amplified exon products produces cohesive overhangs consisting entirely of exon derived sequences. To achieve the ligation of exon segments in the correct order and orientation, exon-primer-pairs were designed such that adjacent exon segments have unique but complementary cohesive overhangs.
  • This method can be used to convert zins4 genomic DNA into a contiguous polypeptide coding sequence (cPCS).
  • the human zcytor-1 gene (Sprecher et al., Biochem. Biophys. Res. Comm. 245:82-90, 1998; Baumgartner et al., U.S. Patent Number 5,792,850) is located 2 kb from the zins4 locus.
  • Zcytor-1 gene primers were used to isolate a BAC clone containing the gene encoding human zins4.
  • a "Down to the Well" human BAC H DNA library (Genome Systems fric, St.
  • Zins4 contains two exons.
  • the zins4 exon 1 DNA segment is isolated using sense oligonucleotide primer (SEQ JD NO:9); and antisense oligonucleotide primer (SEQ JD NO: 10), respectively.
  • the zins4 exon 2 DNA segment is isolated using sense oligonucleotide primer (SEQ JD NO: 11) and antisense oligonucleotide primer (SEQ JD NO: 12), respectively.
  • a PCR reaction mix is prepared as follows: to a final volume of 100 ⁇ l is added 10 ⁇ l 10X native PFU DNA polymerase buffer (Stratagene, La Jolla, CA); 1 ⁇ l 20 mM dNTP mix; 30 pmoles each of sense and antisense primers; 10 ng of 218M2 BAC clone DNA template; and 2 units of native PFU DNA polymerase (Stratagene).
  • DNA amplification is carried out as follows: 1 cycle (95°C for 1 minute), followed by 3 cycle of (95 ° C for 10 seconds; 45°C for 10 seconds; 72°C for 2 minutes) followed by 17 cycles of (95 ° C for 10 seconds; 60°C for 10 seconds; 72°C for 2 minutes), followed by a further 5 minute extension at 72 ° C.
  • the amplified zins4 exon products are extracted with phenol and chloroform and are precipitated with ethanol.
  • the PCR products are digested with the type-JJS restriction endonuclease, Eco311 (MBI Fermentas, Amherst, NY) according to manufacturer's instruction.
  • the digested PCR product is purified using a Gel Extraction Kit (Qiagen, Chatsworth, CA) according to manufacturer's instructions.
  • Ligation of zins4 exons into EcoR-1 and Xba-1 digested mammalian expression vector pSAM-8 is carried out in a 5 ⁇ l ligation reaction consisted of: 1 ⁇ l vector (40 ng); 0.5 ⁇ l 10X Promega ligase buffer (Promega Life Science, Madison, WI),
  • ligation reaction is carried at 6°C for 2 hrs; 8°C for 2 hrs; 10°C for 2 hrs; 12.5°C for 2 hrs; 15°C for 2 hrs; followed by an overnight incubation at 10°C overnight.
  • One ⁇ l of the ligation mixture is used to ttansform MAX efficiency DH10B competent cells (Life Technologies, Rockville, MD). Transformants are selected by plating onto Ampicillin LB agar plates. The resulting clone carrying the zins4 cPCS is confirmed by DNA sequence analysis.
  • RNA may also be isolated from a tissue or cell that produces large amounts of zins4 RNA. Such tissues and cells are identified by using the sequence information provide herein in combination with Northern blotting (Thomas, Proc. Natl.
  • Total RNA can be prepared using guanidinium isothiocyanate extraction followed by isolation by centtifugation 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 59:1408-12, 1972).
  • Complementary DNA is prepared from poly(A) + RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zins4 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • a full-length clone encoding zins4 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 zins4, receptor fragments, or other specific binding partners.
  • the polynucleotides of the present invention can also be synthesized using DNA synthesis machines.
  • each complementary strand is made separately, for example via the phosphoramidite method known in the art.
  • the production of short polynucleotides 60 to 80 bp is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • special strategies are usually employed for producing longer polynucleotides (longer than about 300 bp).
  • synthetic DNAs double-stranded are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length.
  • One method for building a synthetic DNA involves producing a set of overlapping, complementary oligonucleotides. Each internal section of the DNA has complementary 3' and 5' terminal extensions designed to base pair precisely with an adjacent section. After the DNA is assembled, the process is completed by ligating the nicks along the backbones of the two strands.
  • synthetic DNAs can be designed with terminal sequences that facilitate insertion into a resttiction endonuclease site of a cloning vector.
  • Alternative ways to prepare a full-length DNA are also known in the art. See 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 87:633-1, 1990.
  • the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are zins4 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human zins4 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zins4 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 zins4-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.
  • 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 zins4 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.
  • SEQ JD NO:l represents a single allele of human zins4 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 sequence shown in SEQ JD NO:l, 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 NO:2.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zins4 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 zins4 polypeptides that are substantially similar to the polypeptides of SEQ JD NO:2 and their orthologs.
  • the term "substantially similar” is used herein to denote polypeptides having 70%, preferably 75%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its 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.
  • 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 zins4.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 55:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990.
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution mattix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
  • the BLOSUM62 table (Table 3) is an amino acid substitution mattix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins
  • 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.
  • conservative amino acid substitution preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • 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 zins4 polypeptides or substantially homologous zins4 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 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.
  • the present invention thus includes polypeptides of from about 14 to about 500 amino acid residues that comprise a sequence that is at least 80%, preferably at least 90%, and more preferably 95% or more identical to the corresponding region of SEQ ID NO:2.
  • Aromatic phenylalanine tryptophan tyrosine
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trarcs-3-methylproline, 2,4-methanoproline, cz's-4-hychoxyproline, trans-4- hydroxyproline, N-methylglycine, Z/o-threonine, methylthreonine, hydroxy- ethylcysteine, hydroxyethylhomocysteine, nittoglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-di- methylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • 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. 33:1410-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro 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 zins4 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 ⁇ 5:4498-502, 1991).
  • site-directed mutagenesis or alanine-scanning mutagenesis
  • 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. 271:4699-108, 1996.
  • Sites of ligand-receptor or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, 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. 309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related polypeptide sequences or proteins. Determination of amino acid residues that are within regions or domains that are critical to maintaining structural integrity can be determined.
  • Methods for analyzing sequence structure include, but are not Umited to, alignment of multiple sequences with high amino acid or nucleotide identity and computer analysis using available software (e.g., the Insight U ® viewer and homology modeling tools; MSI, San Diego, CA), secondary structure propensities, binary patterns, complementary packing and buried polar interactions (Barton, Current Opin. Struct. Biol. 5:312-316, 1995 and Cordes et al., Current Opin. Struct. Biol. 5:3-10, 1996).
  • available software e.g., the Insight U ® viewer and homology modeling tools; MSI, San Diego, CA
  • secondary structure propensities binary patterns, complementary packing and buried polar interactions
  • Amino acid sequence changes are made in zins4 polypeptides so as to minimize disruption of higher order structure essential to biological activity.
  • changes in amino acid residues will be made so as not to disrupt the structures and other components of the molecule where changes in conformation abate some critical function, for example, binding of the molecule to its binding partners.
  • the effects of amino acid sequence changes can be predicted by, for example, computer modeling as disclosed herein or determined by analysis of crystal structure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268, 1995).
  • CD Crohn's disease
  • hydrophobic residues selected from the group consisting of Val, Leu and JJe or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.
  • residues tolerant of substitution could include these hydrophobic residues as shown in SEQ JD NO: 2. Cysteine residues in the cysteine motifs of SEQ JD NO: 2, described herein, will be relatively intolerant of substitution.
  • the identities of essential amino acids can also be inferred from analysis of sequence similarity between insulin/relaxin family members and zins4. Using methods such as "FASTA" analysis described previously, regions of high similarity are identified within a family of proteins and used to analyze amino acid sequence for conserved regions.
  • An alternative approach to identifying a variant zins4 polynucleotide on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant zins4 polynucleotide can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ JD NO: 1, as discussed above.
  • the present invention also includes functional fragments of zins4 polypeptides and nucleic acid molecules encoding such functional fragments.
  • a "functional" zins4 or fragment thereof defined herein is characterized by its proliferative or differentiating activity, by its ability to induce or inhibit specialized cell functions, or by its ability to bind specifically to an anti-zins4 antibody or zins4 receptor (either soluble or immobilized).
  • zins4 is characterized by several cleavage sites that generate a number of bioactive zins4 peptides.
  • the present invention further provides fusion proteins encompassing: (a) polypeptide molecules comprising one or more of the of the zins4 peptides described above; and (b) functional fragments comprising one or more of these peptides.
  • the other polypeptide portion of the fusion protein may be contributed by another peptide hormone, such as insulin, glucagon, POMC, growth hormone, neuropeptide hormones, and the like, or by a non-native and/or an unrelated secretory signal peptide that facilitates secretion of the fusion protein.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a zins4 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ JD NO:l or fragments thereof can be digested with Bal31 nuclease to obtain a series of nested deletions. These DNA fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for zins4 activity, or for the ability to bind anti-zins4 antibodies or zins4 receptor.
  • oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired zins4 fragment.
  • particular fragments of a zins4 polynucleotide can be synthesized using the polymerase chain reaction.
  • variants of the disclosed zins4 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 91:10141-51, 1994 and WJPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro 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., secreted and detected by antibodies, binding assays, or measured by a signal fransduction type assay
  • 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.
  • polypeptides that are substantially similar to SEQ JD NO:2 or allelic variants thereof and retain the properties of the wild-type protein. For example, using the methods described above, one could identify a receptor binding domain on zins4; an extracellular ligand-binding domain of a receptor for zins4; other functional or structural domains; affinity tags; or other domains important for protein- protein interactions or signal ttansduction.
  • polypeptides may also include additional polypeptide segments as generally disclosed above.
  • any zins4 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.
  • Zins4 chimeric molecules are also provided by the invention. Zins4 in combination with other members of the insulin/relaxin family can be use to form zwitterhormons, proteins having two unrelated biological functions (Bullesbach et al., Biochem. 35:9154-60, 1996). Zins4 can be combined with insulin, relaxins, insulin-like growth factors, Leydig factors, Leydig insulin-like peptides, early placenta insulin-like factors, INSL5, INSL6, bombyxins, and their analogs, among others.
  • the zins4 polypeptides of the present invention 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.
  • a DNA sequence encoding a zins4 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.
  • cells transfected with expression vectors containing DNA sequences encoding zins4 are co-ttansfected with expression vectors encoding a suitable prohormone convertase, for example furin, PCl/3, PC2, PACE, PACE4, or PC4.
  • suitable prohormone convertase for example furin, PCl/3, PC2, PACE, PACE4, or PC4.
  • 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 zins4, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo.
  • the secretory signal sequence is operably linked to the zins4 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).
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from zins4 (amino acid residues 1-25 of SEQ ID NO:2) is operably linked to a DNA sequence encoding another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non- secreted protein. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.
  • 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 14:125, 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.
  • 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. 36:59-12, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
  • 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
  • Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61
  • Suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Manassas, VA.
  • 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 "fransfectants”. 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 fransfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neornycin-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 fransfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for ⁇ ells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methottexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetylttansferase
  • drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetylttansferase
  • 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.
  • eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:41-58, 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 WJPO publication WO 94/06463.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV).
  • 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, C. D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995.
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, et al., J Virol 57:4566-79, 1993). This system is sold in the Bac-to-BacTM kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, pFasfBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zins4 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.”
  • the pFastBaclTM ttansfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case zins4.
  • pFastBaclTM can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Peer, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins.
  • the baculovirus basic protein promoter also known as Peer, p6.9 or MP promoter
  • Peer, p6.9 or MP promoter which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins and Possee, J. Gen. Virol. 71:911-6, 1990; Bonning, et al., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-9, 1995. In such ttansfer vector constructs, a short or long version of the basic protein promoter can be used.
  • ttansfer vectors can be constructed which replace the native zins4 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invittogen, Carlsbad, CA), or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native zins4 secretory signal sequence.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed zins4 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). The tag can then be removed after purification.
  • a transfer vector containing zins4 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 zins4 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 FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435). Commercially available serum-free media are used to grow and maintain the cells.
  • Suitable media are Sf900 IJTM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x IO 5 cells to a density of 1-2 x IO 6 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.
  • MOI multiplicity of infection
  • 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.
  • Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Surnino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications 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.
  • the promoter and terminator in the plasmid be that of a E. methanolica gene, such as a E. methanolica alcohol utilization gene (AUG1 or AUG2).
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • 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.
  • host cells 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. Electtoporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to ttansform E.
  • methanolica cells by electtoporation 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 (t) 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.).
  • the polypeptide When expressing a zins4 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.
  • 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.
  • 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.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nufrients and other components required for the growth of the chosen host cells.
  • 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.
  • E. 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 Y ⁇ PD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI), 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
  • polypeptides of the present invention 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.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Expressed recombinant zins4 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 can 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.
  • Phenyl-Sepharose FF Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like
  • 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 rtolyacrylamide 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.
  • 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.
  • the polypeptides of the present invention can be isolated by exploitation of their structural and biological properties.
  • immobilized metal ion adsorption (MAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-1, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • MAC immobilized metal ion adsorption
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., HIS tag, Glu-Glu, FLAG, maltose- binding protein, an immunoglobulin domain
  • Such fusions include polypeptides comprising affinity tags which further comprise a proteolytic cleavage site between the zins4 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites, factor Xa cleavage sites, endoprotease (Arg-Cys or Lys-Cys) sites.
  • the C peptide can be tagged or replaced.
  • zins4 polypeptides can be purified using prohormone cleavage to isolate the mature protein.
  • the unprocessed polypeptides are first exposed to a prohormone convertase, such as PCl/3, PC3, PC 4, PACE, PACE4, furin, kex-2, to cleave RR or RXXR sites, followed by cleavage of remaining R residues by a carboxypeptidase.
  • a prohormone convertase such as PCl/3, PC3, PC 4, PACE, PACE4, furin, kex-2
  • polypeptide fusions, or hybrid zins4 proteins are constructed using regions or domains of zins4 in combination with those of paralogs, orthologs, or heterologous proteins (Sambrook et al., ibid., Altschul et al., ibid., Picard Cur. Opin. Biology 5:511-5, 1994, and references therein).
  • Fusion polypeptides 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 one or more components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein.
  • a domain(s) conferring a biological function may be swapped between zins4 of the present invention with ' the functionally equivalent domain(s) from another family member.
  • Such domains include, but are not limited to the secretory signal sequence, the A chain, the B chain and the C peptide, described herein.
  • 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 family proteins or to a heterologous protein, depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
  • Standard molecular biological and cloning techniques can be used to swap the equivalent domains between the zins4 polypeptide and those polypeptides to which they are fused.
  • a DNA segment that encodes a domain of interest e.g., a zins4 N-terminal polypeptide, the A chain, the B chain, or the C peptide, or motif described herein, is operably linked in frame to at least one other DNA segment encoding an additional polypeptide and inserted into an appropriate expression vector, as described herein.
  • DNA constructs are made such that the several DNA segments that encode the corresponding regions of a polypeptide are operably linked in frame to make a single construct that encodes the entire fusion protein, or a functional portion thereof.
  • a DNA construct would encode from N-terminus to C- terminus a fusion protein comprising a signal polypeptide followed by a mature polypeptide; or a DNA construct would encode from N-terminus to C-terminus a fusion protein comprising a signal polypeptide followed by B chain, C peptide and A chain, or as interchanged with equivalent regions from another protein.
  • fusion proteins can be expressed, isolated, and assayed for activity as described herein.
  • zins4 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • zins4 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.
  • Polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. Methods for synthesizing polypeptides are well known in the art. See, for example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Kaiser et al., Anal. Biochem. 34:595, 1970. After the entire synthesis of the desired peptide on a solid support, the peptide-resin is washed with a reagent which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Such methods are well established in the art.
  • Nucleic acid molecules disclosed herein can be used to detect the expression of a zins4 gene in a biological sample.
  • probe molecules include double-sttanded nucleic acid molecules comprising the nucleotide sequences of SEQ JD NO:l, or fragments thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequences of SEQ JD NO:l, or a fragment thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • suitable probes include nucleic acid molecules that bind with a portion of a zins4 A chain, B chain or C peptide, or the sequences of SEQ JD Nos: 3, 4, or 5.
  • 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 zins4 RNA species. After separating unbound probe from hybridized molecules, the amount of hybrids is detected.
  • RNA detection includes 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 32 P or 35 S.
  • zins4 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).
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates.
  • Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
  • Zins4 oligonucleotide probes are also useful for in vivo diagnosis.
  • 18 F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al., Nature Medicine 4:461, 1998).
  • PCR polymerase chain reaction
  • 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.
  • PCR primers can be designed to amplify a sequence encoding a particular zins4 domain or motif, such as the A chain, B chain, C peptide, or the sequences of SEQ ID Nos:3, 4, or 5.
  • a particular zins4 domain or motif such as the A chain, B chain, C peptide, or the sequences of SEQ ID Nos:3, 4, or 5.
  • RT-PCR reverse ttanscriptase-PCR
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with zins4 primers (see, for example, Wu et al.
  • RNA is isolated from biological sample using, for example, the guanidinium-thiocyanate cell lysis procedure described herein.
  • 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 zins4 anti-sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • Zins4 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.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be ttansferred to a membrane, hybridized with a detectably-labeled zins4 probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid ttiphosphates to provide chemiluminescence detection, and the C-TRAK colorimettic assay.
  • a fluorogenic probe consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers.
  • 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.
  • CPT cycling probe technology
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross-hybridization
  • LCR ligase chain reaction
  • Zins4 probes and primers can also be used to detect and to localize zins4 gene expression in tissue samples.
  • Methods for such in situ hybridization are well- known to those of skill in the art (see, for example, Choo (ed.), Jn Situ Hybridization Protocols, Humana Press, Inc., 1994; Wu et al. (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Situ 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 Situ Hybridization (RISH),” in Methods in Gene Biotechnology, CRC Press, Inc., pages 279-289, 1997).
  • RISH Radioactive In Situ Hybridization
  • Polypeptides associated with the ovaries are useful for modulating steroidogenesis, both in vivo and in vitro, and modulating aspects of the ovarian cycle such as oocyte maturation, ovarian cell-cell interactions, follicular development and rupture, luteal function, promoting uterine implantation of fertilized oocytes as well as modulating activities associated the pregnancy, such as gestation and labor, including the force and rate of contractions.
  • Imbalances in reproductive polypeptides can be used to assess the existence of diseases, as well as determine whether normal hormonal balance has been restored after administration of a therapeutic agent. Determination of esttadiol, progesterone, LH, and FSH, for example, from serum is known by one of skill in the art.
  • Such assays can be used to monitor hormone levels after administration of zins4 in vivo, or in a transgenic mouse model where the zins4 gene is expressed or the murine ortholog is deleted.
  • Zins4 polypeptides can be used alone or in combination with other reproductive proteins such as relaxin, as a therapeutic application for pregnancy support.
  • Proteins of the present invention may also be used in applications for enhancing fertilization during assisted reproduction in humans and in animals.
  • assisted reproduction methods are known in the art and include artificial insemination, in vitro fertilization, embryo ttansfer, and gamete inttafallopian ttansfer. Such methods are useful for assisting those who may have physiological or metabolic disorders that prevent or impede natural conception.
  • animal breeding programs e.g., for livestock, racehorses, domestic and wild animals, and could be used as methods for the creation of transgenic animals.
  • Proteins of the present invention may also be useful in therapies for treating reproductive disorders. Disorders such as luteal phase deficiency would benefit from such therapy (Soules, "Luteal phase deficiency: A subtle abnormality of ovulation” in, Infertility: Evaluation and Treatment. Keye et al., eds., Philadelphia, WB Saunders, 1995). Administration of reproductive hormones, such as gonadotropin- releasing hormone, is shown to stimulate reproductive behavior (Riskin and Moss, Res. Bull. ⁇ :481-5, 1983; Kadar et al., Physiol. Behav. 51:601-5, 1992 and Silver et al., J. Neruoendocrin.
  • reproductive hormones such as gonadotropin- releasing hormone
  • polypeptides which may modulate or enhance reproductive activity can find application in developing treatments for these conditions.
  • Proteins associated with the ovaries may modulate hormones, hormone receptors, growth factors, or cell-cell interactions, of the reproductive cascade or have an involvement in oocyte or ovarian development.
  • the polypeptides, nucleic acid and/or antibodies of the present invention may be used in treatment of disorders associated with gonadal development, pregnancy, pubertal changes, menopause, ovarian cancer, fertility, ovarian function, polycystic ovarian syndrome and other reproductive functions.
  • the molecules of the present invention may be used to modulate or to treat or prevent development of pathological conditions in ovary.
  • certain syndromes or diseases may be amenable to such diagnosis, treatment or prevention.
  • natural functions, such as ovulation may be suppressed or controlled for use in birth control by molecules of the present invention.
  • Diagnostic methods of the present invention involve the detection of zins4 polypeptides in the serum or tissue biopsy of a patient undergoing analysis of reproductive function or evaluation for possible ovarian cancer.
  • polypeptides can be detected using immunoassay techniques and antibodies, described herein, that are capable of recognizing polypeptide epitopes.
  • Such methods include using probes or primers derived, for example, from the nucleotide sequences disclosed herein to detect zins4 expression in a patient sample, such as a blood, saliva, sweat, tissue sample, or the like.
  • probes can be hybridized to tumor tissues and the hybridized complex detected by in situ hybridization.
  • Zins4 sequences can also be detected by PCR amplification using cDNA generated by reverse ttanslation of sample mRNA as a template (PCR Primer A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Press, 1995). When compared with a normal control, both increases or decreases of zins4 expression in a patient sample, relative to that of a control, can be monitored and used as an indicator or diagnostic for disease.
  • the polypeptides of the present invention may modulate contractility in certain tissues.
  • assays known in the art can be applied to tissue samples, such as aortic rings, vas deferens, ileum, uterine and other contractile tissue samples, as well as to organ systems, such as atria, and can be used to determine whether zins4 polypeptide, its agonists or antagonists, enhance or depress conttactility.
  • Molecules of the present invention are hence useful for treating dysfunction associated with contractile tissues or can be used to suppress or enhance contractility in vivo.
  • molecules of the present invention have utility in treating cardiovascular disease, infertility, in vitro fertilization, birth control, treating impotence or other male reproductive dysfunction, as well as inducing birth.
  • zins4 polypeptides, antagonists and agonists of the present invention can be measured in a tensiometer that measures conttactility and relaxation in tissues. See, Dainty et al., J. Pharmacol. 100:161, 1990; Rhee et al., Neurotox. 16: 179, 1995; Anderson, Endocrinol. 114:364-8, 1984; and Downing and Sherwood, Endocrinol. 775:1206-14, 1985. For example, measuring vasodilatation of aortic rings is well known in the art.
  • aortic rings are taken from 4 month old Sprague Dawley rats and placed in a buffer solution, such as modified Krebs solution (118.5 mM NaCl, 4.6 mM KC1, 1.2 mM MgSO 4 .7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 .2H 2 O, 24.8 mM NaHCO 3 and 10 mM glucose).
  • a buffer solution such as modified Krebs solution (118.5 mM NaCl, 4.6 mM KC1, 1.2 mM MgSO 4 .7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 .2H 2 O, 24.8 mM NaHCO 3 and 10 mM glucose).
  • modified Krebs solution 118.5 mM NaCl, 4.6 mM KC1, 1.2 mM MgSO 4 .7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl
  • the rings are then attached to an isometric force transducer (Radnoti Inc., Monrovia, CA) and the data recorded with a Ponemah physiology platform (Gould Instrument systems, Inc., Valley View, OH) and placed in an oxygenated (95% O , 5% CO 2 ) tissue bath containing the buffer solution.
  • the tissues are adjusted to 1 gram resting tension and allowed to stabilize for about one hour before testing.
  • the integrity of the rings can be tested with norepinepherin (Sigma Co., St. Louis, MO) and Carbachol, a muscarinic acetylcholine agonist (Sigma Co.). After integrity is checked, the rings are washed three times with fresh buffer and allowed to rest for about one hour.
  • zins4 polypeptide sample is then added to 1, 2 or 3 of the 4 baths, without flushing, and tension on the rings recorded and compared to the control rings containing buffer only. Enhancement or relaxation of conttactility by zins4 polypeptides, their agonists and antagonists is directly measured by this method, and it can be applied to other contractile tissues such as uterus, prostate, and testis. Zins4 could be useful as modulator of blood pressure, muscle tension or and osmotic balance.
  • blood pressure modification is important in situations such as heart attack, stroke, traumatic shock, surgery, and any number of bleeding complications.
  • zins4 may modulate conttactility in the organ systems and tissues that it effects.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure cell contractility and discussed below. Such assays are well known in the art, and described herein.
  • Zins4 may also have CNS functions as well as cardiovascular functions. Assays and models to test for such zins4 activity are well known in the art and described herein. For example, see Feng et al., Ada. Physiol. Scand. 166:285-91, 1999; (pithed rat heart failure model to assess vascular sympathetic nerve activity); Horackova, et al., Cell Tissue Res. 297:409-21, 1999 (guinea pig atria model); McLean et al., Neuroscience 92:1377-87, 1999 (CNS response to hypotensive challenge to assess neuron response or activation within cardiovascular control); Potter, et al; Regul. Pept.
  • the activity of molecules of the present invention can be measured using a variety of assays that measure cell differentiation and proliferation as well as assays that measure cell contractility and cardiovascular function. Such assays are well known in the art.
  • the molecules of the present invention are useful as components of defined cell culture media, as described herein, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Molecules of the present invention are particularly useful in specifically promoting the growth, development, differentiation, and/or maturation of ovarian cells in culture, and may also prove useful in the study of the ovarian cycle, reproductive function, ovarian cell-cell interactions, and fertilization.
  • molecules of the present invention may...be useful for promoting the growth and/or development of myocytes in culture, such as cardiac myocytes or myoblasts; skeletal myocytes or myoblasts and smooth muscle cells; chrondrocytes; endothelial cells; adipocytes and osteoblasts in vitro.
  • myocytes in culture such as cardiac myocytes or myoblasts; skeletal myocytes or myoblasts and smooth muscle cells; chrondrocytes; endothelial cells; adipocytes and osteoblasts in vitro.
  • molecules of the present invention are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture, and may also prove useful in the study of cardiac myocyte hyperplasia and regeneration.
  • Proteins of the present invention are useful for example, in treating reproductive, prostate, heart, kidney and other disorders, and can be measured in vitro using cultured cells or in vivo by administering molecules of the present invention to the appropriate animal model.
  • An in vivo approach for assaying proteins of the present invention involves viral delivery systems.
  • Exemplary viruses for this purpose include adenovirus, herpesvirus, retroviruses, vaccinia virus, and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for review, see Becker et al., Meth. Cell Biol.
  • adenovirus can accommodate relatively large DNA inserts; (ii) can be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) can be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-ttansfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).
  • the host cell the human 293 cell line is exemplary.
  • adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells.
  • adenoviral vectors containing various deletions of viral genes can be used in an attempt to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El deleted, and in addition contain deletions of E2A or E4 (Lusky, et al., J. Virol. 72:2022-32, 1998; Raper, et al., Human Gene Therapy 9:671-9, 1998).
  • deletion of E2b is reported to reduce immune responses (Amalfitano, et al., J. Virol. 72:926-33, 1998).
  • deletion of the entire adenovirus genome very large inserts of heterologous DNA can be accommodated.
  • Generation of so called "gutless" adenoviruses where all viral genes are deleted are particularly advantageous for insertion of large inserts of heterologous DNA.
  • Yeh and Perricaudet. FASEB J. 11:615-23, 1997.
  • the adenovirus system can also be used for protein production in vitro.
  • the cells 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.
  • 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. 75:145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant, lysate, or membrane fractions depending on the disposition of the expressed protein in the cell. Within the infected 293 cell production protocol, non-secreted proteins may also be effectively obtained.
  • the activity of zins4 polypeptide can be measured by a silicon-based biosensor microphysiometer which measures the exfracellular acidification rate or proton excretion associated with receptor binding and subsequent physiologic cellular responses.
  • An exemplary device is the CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • CytosensorTM Microphysiometer manufactured by Molecular Devices, Sunnyvale, CA.
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method. See, for example, McConnell et al., Science 257:1906-12, 1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli et al., J. Immunol.
  • the microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including zins4 polypeptide, its agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zins4-responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zins4 polypeptide.
  • Zins4- responsive eukaryotic cells comprise cells into which a receptor for zins4 has been transfected creating a cell that is responsive to zins4; or cells naturally responsive to zins4 such as cells derived from prostate, testis, uterine tissue, or the like. Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zins4 polypeptide, relative to a control not exposed to zins4, are a direct measurement of zins4-modulated cellular responses. Moreover, such zins4-modulated responses can be assayed under a variety of stimuli.
  • a method of identifying agonists of zins4 polypeptide comprising providing cells responsive to a zins4 polypeptide, culturing a first portion of the cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test compound, and detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • culturing a third portion of the cells in the presence of zins4 polypeptide and the absence of a test compound can be used as a positive control for the zins4-responsive cells, and as a control to compare the agonist activity of a test compound with that of the zins4 polypeptide.
  • a method of identifying antagonists of zins4 polypeptide comprising providing cells responsive to a zins4 polypeptide, culturing a first portion of the cells in the presence of zins4 and the absence of a test compound, culturing a second portion of the cells in the presence of zins4 and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells.
  • the change in cellular response is shown as a measurable change extracellular acidification rate.
  • Antagonists and agonists, for zins4 polypeptide can be rapidly identified using this method.
  • zins4 can be used to identify cells, tissues, or cell lines which respond to a zins4-stimulated pathway.
  • the microphysiometer, described above can be used to rapidly identify ligand-responsive cells, such as cells responsive to zins4 of the present invention.
  • Cells can be cultured in the presence or absence of zins4 polypeptide. Those cells which elicit a measurable change in extracellular acidification in the presence of zins4 are responsive to zins4.
  • Such cell lines can be used to identify antagonists and agonists of zins4 polypeptide as described above.
  • agonists including the natural ligand substrate/cofactor/etc.
  • antagonists have enormous potential in both in vitro and in vivo applications.
  • zins4 polypeptide and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with cytokines and hormones to replace serum that is commonly used in cell culture. Agonists are thus useful in specifically promoting the growth and/or development . of mammalian cells in vitro, particularly of those derived from reproductive tissues. As such, zins4 polypeptides or agonists are added to tissue culture media for these cell types.
  • Zins4 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to assays disclosed herein to identify compounds that inhibit the activity of zins4. In addition to those assays disclosed herein, samples can be tested for inhibition of zins4 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of zins4-dependent cellular responses. For example, zins4-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zins4-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a zins4-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase.
  • DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE) insulin response element (IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990) and serum response elements (SRE) (Shaw et al. Cell 56: 563-72, 1989).
  • Cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec. Endocrinol. 4 (8): 1087-94; 1990.
  • Hormone response elements are reviewed in Beato, Cell 55:335-44; 1989.
  • Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zins4 on the target cells as evidenced by a decrease in zins4 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zins4 binding to cell-surface receptors, as well as compounds that block processes in the cellular pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of zins4 binding to receptor using zins4 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like).
  • a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like.
  • Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
  • a zins4 ligand-binding polypeptide can also be used for purification of ligand.
  • the polypeptide is immobilized on a solid support, such as agarose beads, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross- linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting medium will generally be configured in the form of a column, and fluids containing • Jigand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
  • the ligand is then eluted using changes in salt concentration, chaottopic agents (guanidine HCl), or pH to disrupt ligand-receptor binding.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complement anti-complement pair
  • a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ)
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 745:229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-63, 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dexttan fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell. Jf a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimettic assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).
  • Zins4 polypeptides can also be used to prepare antibodies that bind to zins4 epitopes, peptides or polypeptides. The zins4 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
  • antigenic, epitope-bearing polypeptides contain a sequence of at least 6, preferably at least 9, and more preferably at least 15 to about 30 contiguous amino acid residues of a zins4 polypeptide (e.g., SEQ ID NO:2). Polypeptides comprising a larger portion of a zins4 polypeptide, i.e., from 10 to 30 residues up to the entire length of the amino acid sequence are included.
  • Antigens or immunogenic epitopes can also include attached tags, adjuvants and carriers, as described herein.
  • Suitable antigens include the zins4 polypeptide encoded by SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 142 (Cys), or a contiguous 9 to 142 amino acid fragment thereof. Still other suitable antigens include, but are not limited to, polypeptides from amino acid residue 25-52, 26-52, 26- 53, 26-54, 55-114, 55-115, 55-116, 55-117, 55-118, 26-114, 55-142, 1-25, 1-52, 26-54, 1-118, 119-142, and 26-142 of SEQ JD NO:2. Other suitable antigens include the signal sequence, the A chain, the B chain and the C peptide, as disclosed herein.
  • peptides to use as antigens are hydrophilic peptides such as those predicted by one of skill in the art from a hydrophobicity plot.
  • Antibodies from an immune response generated by inoculation of an animal with these antigens can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology. Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982.
  • polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zins4 polypeptide or a fragment thereof.
  • the immunogenicity of a zins4 polypeptide may 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 zins4 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. Jf 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 (BS A) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BS A) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • 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.
  • 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 fhiol reducing agent to produce 3.5S Fab' monovalent fragments.
  • the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
  • 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. Biophvs. 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.
  • Fv fragments comprise an association of V H and V L chains.
  • variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as gluteraldehyde (see, for example, Sandhu, Grit. Rev. Biotech. 12:437, 1992).
  • the Fv fragments may comprise V H and V chains which are connected by a peptide linker.
  • scFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli.
  • 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, ibid.
  • a scFV can be obtained by exposing lymphocytes to zins4 polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zins4 protein or peptide).
  • Genes encoding polypeptides having potential zins4 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.
  • 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.
  • 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.
  • Random peptide display libraries can be screened using the zins4 sequences disclosed herein to identify proteins which bind to zins4.
  • 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.
  • Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zins4 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zins4 protein or peptide).
  • Genes encoding polypeptides having potential zins4 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.
  • 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.
  • 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., US Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and Ladner et al., US Patent NO.
  • Random peptide display libraries can be screened using the zins4 sequences disclosed herein to identify proteins which bind to zins4. These "binding polypeptides" which interact with zins4 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding polypeptides can also be used in analytical methods such as for screening expression libraries and neuttalizing activity, e.g., for blocking interaction between ligand and receptor, or viral binding to a receptor.
  • the binding polypeptides can also be used for diagnostic assays for determining circulating levels of zins4 polypeptides; for detecting or quantitating soluble zins4 polypeptides as marker of underlying pathology or disease.
  • These binding polypeptides can also act as zins4 "antagonists" to block zins4 binding and signal ttansduction in vitro and in vivo. These anti-zins4 binding polypeptides would be useful for inhibiting zins4 activity or protein-binding.
  • Antibodies are considered to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules.
  • a threshold level of binding is determined if anti-zins4 antibodies herein bind to a zins4 polypeptide, peptide or epitope with an affinity at least 10-fold greater than the binding affinity to control (non-zins4) polypeptide. It is preferred that the antibodies exhibit a binding affinity (K a ) of 10 M " or greater, preferably 10 7 M- " 1 or greater, more preferably 108 M- " 1 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, G., Ann.
  • anti-zins4 antibodies do not significantly cross-react with related polypeptide molecules is shown, for example, by the antibody detecting zins4 polypeptide but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.).
  • known related polypeptides are those disclosed in the prior art, such as known orthologs, and paralogs, and similar known members of. a protein family, Screening can also be done using non-human zins4, and zins4 mutant polypeptides.
  • antibodies can be "screened against" known related polypeptides, to isolate a population that specifically binds to the zins4 polypeptides.
  • antibodies raised to zins4 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zins4 will flow through the matrix under the proper buffer conditions.
  • Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to known closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol.
  • Specifically binding anti-zins4 antibodies can be detected by a number of methods in the art, and disclosed below.
  • assays known to those skilled in the art can be utilized to detect antibodies which bind to zins4 proteins or polypeptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zins4 protein or polypeptide.
  • Antibodies to zins4 may be used for tagging cells that express zins4; for isolating zins4 by affinity purification; for diagnostic assays for determining circulating levels of zins4 polypeptides; for detecting or quantitating soluble zins4 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block zins4 activity in vitro and in vivo.
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drags, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • antibodies to zins4 or fragments thereof may be used in vitro to detect denatured zins4 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
  • Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance).
  • zins4 polypeptides or anti-zins4 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti- complementary molecule.
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium- 188 or ytttium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance).
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin stteptavidin is an exemplary complementary/ anticomplementary pair.
  • polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
  • a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
  • the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule.
  • Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.
  • bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, inttaarterially or intraductally, or may be introduced locally at the intended site of action.
  • Molecules of the present invention can be used to identify and isolate receptors that bind zins4 polypeptide.
  • proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques. Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202).
  • Proteins and peptides can also be radiolabeled (Methods in Enzymol.. vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 52:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-80, 1984) and specific cell-surface proteins can be identified.
  • the polypeptides, antagonists, agonists, nucleic acid and/or antibodies of the present invention may be used in tteatment of disorders associated with gonadal development, pregnancy, pubertal changes, menopause, ovarian cancer, fertility, ovarian function, polycystic ovarian syndrome, uterine cancer, endometriosis, libido, mylagia and neuralgia associated with reproductive phenomena, male sexual dysfunction, impotency, prostate cancer, testicular cancer, and dysfunction.
  • the molecules of the present invention may used to modulate or to treat or prevent development of pathological conditions in such diverse tissue as ovary, heart, testis, and kidney. In particular, certain syndromes or diseases may be amenable to such diagnosis, tteatment or prevention.
  • natural functions, such as embryo implantation or spermatogenesis may be suppressed or controlled for use in birth control by molecules of the present invention.
  • Polynucleotides and polypeptides of the present invention will be useful as educational tools in laboratory practicum kits for courses related to genetics and molecular biology, protein chemistry, and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequences, molecules of zins4 can be used as standards or as "unknowns" for testing purposes.
  • zins4 polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constructs for bacterial, viral, or mammalian expression, including fusion constructs, wherein zins4 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of zins4 polynucleotides in tissues (i.e., by northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization.
  • Zins4 polypeptides can be used as an aid to teach preparation of antibodies; identifying proteins by western blotting; protein purification; determining the weight of produced zins4 polypeptides as a ratio to total protein produced; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein in vitro and in vivo.
  • Zins4 polypeptides can also be used to teach analytical skills such as mass specfrometry, circular dichroism to determine conformation, especially of the four alpha helices, x-ray crystallography to determine the three-dimensional structure in atomic detail, nuclear magnetic resonance specfroscopy to reveal the structure of proteins in solution.
  • a kit containing the zins4 can be given to the student to analyze. Since the amino acid sequence would be known by the instructor, the protein can be given to the student as a test to determine the skills or develop the skills of the student, the instructor would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of zins4 would be unique unto itself.
  • the antibodies which bind specifically to zins4 can be used as a teaching aid to instruct students how to prepare affinity chromatography columns to purify zins4, cloning and sequencing the polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies.
  • the zins4 gene, polypeptide, or antibody would then be packaged by reagent companies and sold to educational institutions so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum. Such educational kits containing the zins4 gene, polypeptide, or antibody are considered within the scope of the present invention.
  • Polynucleotides encoding zins4 polypeptides are useful within gene therapy or gene ttansfer applications where it is desired to increase or inhibit zins4 activity. If a mammal has a mutated or absent zins4 gene, the zins4 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zins4 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like.
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Sttatford-Perricaudet et al., J. Clin. Invest.
  • HSV1 herpes simplex virus 1
  • a zins4 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Ternin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 52:1120, 1988; Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No.
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo ttansfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non- peptide molecules can be coupled to liposomes chemically.
  • DNA vectors for gene therapy or gene ttansfer can be introduced into the desired host cells by methods known in the art, e.g., ttansfection, electtoporation, microinjection, transduction, cell fusion, DEAE dexttan, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al., /. Biol. Chem. 257:963-7, 1992; Wu et al., J. Biol. Chem. 253:14621-4, 1988.
  • Antisense methodology can be used to inhibit zins4 gene transcription, such as to inhibit cell proliferation in vivo.
  • Polynucleotides that are complementary to a segment of a zins4-encoding polynucleotide e.g., a polynucleotide as set froth in SEQ
  • ID NO:l are designed to bind to z s4-encoding mRNA and to inhibit translation of such mRNA.
  • antisense polynucleotides are used to inhibit expression of zins4 polypeptide-encoding genes in cell culture or in a subject.
  • the present invention also provides reagents which will find use in diagnostic applications.
  • the zins4 gene a probe comprising zins4 DNA or RNA or a subsequence thereof can be used to confirm that the zins4 gene is present on human chromosome 19 and can be used as a marker to determine if a mutation has occurred.
  • Zins4 is located in the 19pl3.11 region of chromosome 19. Detectable chromosomal aberrations at the zins4 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 resttiction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, 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.).
  • molecular genetic techniques such as resttiction fragment length polymorphism (RFLP) analysis, fluorescence in situ hybridization methods, 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.).
  • Such probes include, but are not limited to, polynucleotides 74-156, 74-159, 74-162, 163-342, 163-345, 163-348, 163-351, 163-354, 355-426, 1-73, 1-162, 1-342, 74-342, 74-345, 74-348, 74-351, and 74-354.
  • 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.
  • the diagnostic methods used in genetic linkage analysis, to detect a genetic abnormality or aberration in a patient are known in the art.
  • Most diagnostic methods comprise the steps of (a) obtaining a genetic sample from a potentially diseased patient, diseased patient or potential non-diseased carrier of a recessive disease allele; (b) producing a first reaction product by incubating the genetic sample with a zins4 polynucleotide probe wherein the polynucleotide will hybridize to complementary polynucleotide sequence, such as in RFLP analysis or by incubating the genetic sample with sense and antisense primers in a PCR reaction under appropriate PCR reaction conditions; (iii) Visualizing the first reaction product by gel electrophoresis and/or other known method such as visualizing the first reaction product with a zins4 polynucleotide probe wherein the polynucleotide will hybridize to the complementary polynucleotide sequence of the first reaction; and (iv) comparing the visualized first reaction product to a second control reaction product of a genetic sample from wild type patient.
  • a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the diseased or potentially diseased patient, or the presence of a heterozygous recessive carrier phenotype for a non-diseased patient, or the presence of a genetic defect in a tumor from a diseased patient, or the presence of a genetic abnormality in a fetus or pre- implantation embryo.
  • a difference in restriction fragment pattern, length of PCR products, length of repetitive sequences at the zins4 genetic locus, and the like are indicative of a genetic abnormality, genetic aberration, or allelic difference in comparison to the normal wild type control. Controls can be from unaffected family members, or unrelated individuals, depending on the test and availability of samples.
  • Genetic samples for use within the present invention include genomic DNA, mRNA, and cDNA isolated form any tissue or other biological sample from a patient, such as but not limited to, blood, saliva, semen, embryonic cells, amniotic fluid, and the like.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ JD NO:l, the complement of SEQ JD NO:l, or an RNA equivalent thereof.
  • Such methods of showing genetic linkage analysis to human disease phenotypes are well known in the art. For reference to PCR based methods in diagnostics see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
  • Aberrations associated with the zins4 locus can be detected using nucleic acid molecules of the present invention by employing standard methods for direct mutation analysis, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • standard methods for direct mutation analysis such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-as
  • Sequence tagged sites 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.
  • 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.
  • polynucleotide primers generating a zins4 sequence mapping to human chromosome 19pl3.11 can be used to generate and STS.
  • mice engineered to express the zins4 gene referred to as "transgenic mice,” and mice that exhibit a complete absence of zins4 gene function, referred to as “knockout mice,” may also be generated (Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature 355:740-42, 1993; Capecchi., Science 244: 1288-92, 1989; Palmiter et al. Annu Rev Genet. 20: 465-99, 1986).
  • transgenic mice that over-express zins4 either ubiquitously or under a tissue-specific or tissue-restricted promoter can be used to ask whether over-expression causes a phenotype.
  • over-expression of a wild-type zins4 polypeptide, polypeptide fragment or a mutant thereof may alter normal cellular processes, resulting in a phenotype that identifies a tissue in which zins4 expression is functionally relevant and may indicate a therapeutic target for the zins4, its agonists or antagonists.
  • a preferred fransgenic mouse to engineer is one that over-expresses the zins4 mature polypeptide (residue 1 (Met) to residue 142 (Cys) of SEQ JD NO:2).
  • Transgenic mice engineered to over- expresses zins4 A chain, B chain or C peptide can also be used.
  • knockout zins4 mice can be used to determine where zins4 is absolutely required in vivo.
  • the phenotype of knockout mice is predictive of the in vivo effects of that a zins4 antagonist, such as those described herein, may have.
  • the human zins4 cDNA can be used to isolate murine zins4 mRNA, cDNA and genomic DNA, which are subsequently used to generate knockout mice. Relaxin knock out mice have been generated (Zhao et al., Ednocrin. 140:445-52, 1999).
  • Transgenic mice engineered to over-expresses human polypeptides or mouse polypeptides corresponding to the human A chain, B chain or C peptide, can also be used. These mice may be employed to study the zins4 gene and the protein encoded thereby in an in vivo system, and can be used as in vivo models for corresponding human diseases. Moreover, transgenic mice expression of zins4 antisense polynucleotides or ribozymes directed against zins4, described herein, can be used analogously to transgenic mice described above.
  • a pharmaceutically effective amount of a zins4 polypeptide of the present invention can be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal, topical, ttansdermal 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, ttansdermal 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 zins4 polypeptide or antagonist.
  • Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use.
  • 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.
  • a "pharmaceutically effective amount" of a zins4 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.
  • an effective amount of a zins4 polypeptide is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • Effective amounts of the zins4 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.
  • 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.
  • 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.

Abstract

L'invention concerne des molécules polynucléotidiques et polypeptidiques destinées au polypeptide zins4, un nouveau polypeptide présentant une homologie avec les relaxines. On peut, par exemple, utiliser les polynucléotides codant pour le polypeptide zins4 afin d'identifier une région du génome associée à des états pathologiques humains. L'invention concerne également des procédés permettant de produire cette protéine, ses utilisateurs et ses anticorps.
EP01909021A 2000-03-10 2001-02-09 Polypeptide zins4 homologue de l'insuline Withdrawn EP1294887A1 (fr)

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KR20020093053A (ko) * 2000-04-21 2002-12-12 다케다 야쿠힌 고교 가부시키가이샤 신규 인슐린/ｉｇｆ/릴랙신 패밀리 폴리펩티드 및 그의ｄｎａ
AUPR814401A0 (en) 2001-10-08 2001-11-01 Howard Florey Institute Of Experimental Physiology And Medicine Human 3 relaxin
US20050026194A1 (en) * 2003-06-20 2005-02-03 Tularik Inc. Gene amplification and overexpression in cancer
CA2555469A1 (fr) 2004-02-09 2005-08-18 Eisai R & D Management Co., Ltd. Procede de test de selection
US7893197B2 (en) 2004-08-25 2011-02-22 Janssen Pharmaceutica N.V. Relaxin-3 chimeric polypeptides and their preparation and use
WO2006026355A2 (fr) * 2004-08-25 2006-03-09 Janssen Pharmaceutica N.V. Polypeptides chimeres de relaxine-3 et leur preparation et leur utilisation
WO2008094437A2 (fr) 2007-01-30 2008-08-07 Janssen Pharmaceutica N.V. Peptide chimère antagoniste du gpcr1 35 ou du gpcr1 42

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AU3972797A (en) * 1996-08-02 1998-02-25 Zymogenetics Inc. Testis-specific insulin homolog polypeptides
WO1998016635A1 (fr) * 1996-10-15 1998-04-23 Zymogenetics, Inc. Homologues de l'insuline
WO1998027210A1 (fr) * 1996-12-16 1998-06-25 Zymogenetics, Inc. Compositions et methodes pour stimuler la regeneration des cellules des ilots pancreatiques

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