WO2000042184A1 - Zlrr3: proteine humaine repetee riche en leucine - Google Patents

Zlrr3: proteine humaine repetee riche en leucine Download PDF

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Publication number
WO2000042184A1
WO2000042184A1 PCT/US2000/000742 US0000742W WO0042184A1 WO 2000042184 A1 WO2000042184 A1 WO 2000042184A1 US 0000742 W US0000742 W US 0000742W WO 0042184 A1 WO0042184 A1 WO 0042184A1
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zlrr3
nucleic acid
amino acid
seq
antibody
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PCT/US2000/000742
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English (en)
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Christopher S. Piddington
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Zymogenetics, Inc.
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Priority to AU25034/00A priority Critical patent/AU2503400A/en
Priority to CA002360577A priority patent/CA2360577A1/fr
Publication of WO2000042184A1 publication Critical patent/WO2000042184A1/fr

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • ZLRR3 A Human Leucine-Rich Repeat Protein
  • the present invention relates generally to a new polypeptide having diagnostic and therapeutic uses.
  • the present invention relates to a novel polypeptide, designated "Zlrr3,” and to nucleic acid molecules encoding Zlrr3.
  • LRR Leucine-rich repeats
  • proteins which appear to be involved in various aspects of protein-protein interaction, such as cell-to-cell communication and signal transduction (for a review, see Kobe and Deisenhofer, TIBS 79:415 (1994); Kobe and Deisenhofer, Curr. Opin. Struct. Biol. 5:409 (1995); Kajava, J. Mol. Biol. 277:5 9 (1998)).
  • Proteins that contain an LRR motif include hormone receptors, enzyme subunits, cell adhesion proteins, and ribosome-binding proteins.
  • a subfamily of the LRR superfamily referred to as the Small Leucine- Rich Proteoglycan family, illustrates the critical functions fulfilled by proteins containing an LRR motif.
  • Members of this subfamily are believed to play essential biological roles during inflammation and cancer invasion, a regulatory role in collagen fibril formation, suppression of the malignant phenotype of cancer cells, and an inhibition of the growth of certain normal cells (see, for example, Iozzo, Annu. Rev. Biochem. 67:609 (1998)).
  • the present invention provides a novel polypeptide, designated "Zlrr3.”
  • the present invention also provides variant Zlrr3 polypeptides and Zlrr3 fusion proteins, as well as nucleic acid molecules encoding such polypeptides and proteins.
  • the present invention provides nucleic acid molecules that encode a new human polypeptide that is a member of the leucine-rich repeat superfamily.
  • An illustrative nucleic acid molecule containing a sequence that encodes the polypeptide designated as "Zlrr3" has the nucleotide sequence of SEQ ID NO:l.
  • the encoded polypeptide has the following amino acid sequence: MGDTWAQLPW PGPPHPAMLL ISLLLAAGLM HSDAGTSCPV LCTCRNQVVD CSSQRLFSVP PDLPMDTRNL SLAHNRITAV PPGYLTCYME LQVLDLHNNS LMELPRGLFL HAKRLAHLDL SYNNFSHVPA DMFQEAHGLV HIDLSHNPWL RRVHPQAFQG LMQLRDLDLS YGGLAFLSLE ALEGLPGLVT LQIGGNPWVC GCTMEPLLKW LRNRIQRCTA DSQLAECRGP PEVEGAPLFS LTEESFKACH LTLTLDDYLF lAFVGFVVSI ASVATNFLLG ITANCCHRWS KASEEEEI (SEQ ID NO:2).
  • the ZlrrS gene described herein encodes a polypeptide of 298 amino acids, as shown in SEQ ID NO:2.
  • the Zlrr3 protein has a signal sequence, N- and C-terminal flanking cysteine-rich regions, six leucine-rich repeat regions (with the typical 24 amino acid periodicity), and a transmembrane domain.
  • Table 1 identifies the locations of these structural features and the corresponding nucleotide sequences that encode the polypeptide domains.
  • the leucine-rich motif (LxxLxLxxNxL, where "x” is any amino acid) is present in proteins that are involved in specific protein-protein interaction or cell adhesion (see, for example, Schneider and Schweiger, Oncogene (5:1807 (1991), and Kobe and Deisenhofer, Trends Biochem. Sci. 79:415 (1994)).
  • the largest subfamily of proteins that contain a leucine-rich domain are extracellular proteins having the following motif: LxxLxxLxLxxNxLxxLPxxOFxx, where "x" is any amino acid and "O” is a non-polar residue (Kajava, J. Mol. Biol. 277:519 (1998)).
  • This motif is present in Zlrr3 at amino acid residues 88 to 111, with the exception that a tyrosine residue is substituted for the first leucine residue of the consensus sequence.
  • cysteine-rich domains flank the leucine-rich repeat region.
  • Kobe and Deisenhofer, TIBS 79:415 (1994) have identified the N-terminal cysteine-rich motif as "CP[about 2x]CxC[about 6x]C,” while the C-terminal-like cysteine-rich motif is represented by "PxxCxC[about 20x]C[about 20x]C.”
  • These motifs reside at amino acid residues 38 to 51 and at amino acid residues 197-249 of SEQ ID NO:2.
  • cysteine-rich domains in a leucine-rich repeat protein indicates that the protein is extracellular, and that it may function as an adhesive protein or receptor (Kobe and Deisenhofer, 77ES 79:415 (1994); Kajava, J. Mol. Biol. 277:519 (1998)).
  • the present invention provides isolated polypeptides having an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to the amino acid sequence of SEQ ID NO:2, wherein such isolated polypeptides can specifically bind with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
  • polypeptide is a polypeptide that comprises the amino acid sequence of SEQ ID NO:2.
  • Additional exemplary polypeptides include the following: (a) polypeptides comprising a leucine-rich region having the following amino acid residue motif: "Y-x(2)-L-x(2)-L- x-L-x(2)-N-x-L-x(2)-L-P-x(2)-L-F-L,” wherein "x" is any amino acid, and wherein the values in parentheses indicate the number of occurrences of "x,” (b) polypeptides having a leucine-rich region comprising amino acid residues 88 to 110 of SEQ ID NO:2, (c) polypeptides comprising a leucine-rich region with the recited motif and at least one cysteine-rich region that resides in an N-terminal or C-terminal position relative to the leucine-rich region, wherein the N-terminal cysteine-rich region has the motif of "C-P-x(2)-C-
  • the present invention also provides isolated polypeptides comprising at least 15, or at least 30, contiguous amino acid residues of an amino acid sequence of SEQ ID NO:2 selected from the group consisting of: (a) amino acid residues amino acid residues 29 to 254, (b) amino acid residues 255 to 284, (c) amino acid residues 285 to 298, and (d) amino acid residues 70 to 249.
  • Illustrative polypeptides include polypeptides that either comprise, or consist of, amino acid residues (a) to (d).
  • the present invention further provides antibodies and antibody fragments that specifically bind with such polypeptides.
  • Exemplary antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
  • Illustrative antibody fragments include F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, and minimal recognition units.
  • the present invention further includes compositions comprising a carrier and a peptide, polypeptide, or antibody described herein.
  • the present invention also provides isolated nucleic acid molecules that encode a Zlrr3 polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:3, (b) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2, and (c) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule having the nucleotide sequence of nucleotides 123-932 of SEQ ID NO:l, or the complement of nucleotides 123-932 of SEQ ID NO:l .
  • Illustrative nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO:2 is due to a conservative amino acid substitution.
  • the present invention further contemplates isolated nucleic acid molecules that comprise a nucleotide sequence of nucleotides 123 to 932 of SEQ ID NO:l.
  • the present invention also includes vectors and expression vectors comprising such nucleic acid molecules.
  • Such expression vectors may comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.
  • the present invention further includes recombinant host cells comprising these vectors and expression vectors.
  • Illustrative host cells include bacterial, yeast, fungal, insect, mammalian, and plant cells.
  • Recombinant host cells comprising such expression vectors can be used to produce Zlrr3 polypeptides by culturing such recombinant host cells that comprise the expression vector and that produce the Zlrr3 protein, and, optionally, isolating the Zlrr3 protein from the cultured recombinant host cells.
  • the present invention also contemplates methods for detecting the presence of Zlrr3 RNA in a biological sample, comprising the steps of (a) contacting a Zlrr3 nucleic acid probe under hybridizing conditions with either (i) test RNA molecules isolated from the biological sample, or (ii) nucleic acid molecules synthesized from the isolated RNA molecules, wherein the probe has a nucleotide sequence comprising a portion of the nucleotide sequence of nucleotides 123 to 932 of SEQ ID NO:l, or its complement, and (b) detecting the formation of hybrids of the nucleic acid probe and either the test RNA molecules or the synthesized nucleic acid molecules, wherein the presence of the hybrids indicates the presence of ZlrrS RNA in the biological sample.
  • the biological sample may be a human biological sample, such as a biopsy or autopsy specimen.
  • the present invention further provides methods for detecting the presence of Zlrr3 polypeptide in a biological sample, comprising the steps of: (a) contacting the biological sample with an antibody or an antibody fragment that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, wherein the contacting is performed under conditions that allow the binding of the antibody or antibody fragment to the biological sample, and (b) detecting any of the bound antibody or bound antibody fragment.
  • an antibody or antibody fragment may further comprise a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold.
  • An exemplary biological sample is a human biological sample, such as a biopsy or autopsy specimen.
  • kits for performing these detection methods may comprise a container that comprises a nucleic acid molecule, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucleotide sequence of nucleotides 123 to 932 of SEQ ID NO:l, (b) a nucleic acid molecule comprising the complement of nucleotides 123 to 932 of the nucleotide sequence of SEQ ID NO:l, (c) a nucleic acid molecule that is a fragment of (a) consisting of at least eight nucleotides, and (d) a nucleic acid molecule that is a fragment of (b) consisting of at least eight nucleotides.
  • kit may also comprise a second container that comprises one or more reagents capable of indicating the presence of the nucleic acid molecule.
  • a kit for detection of Zlrr3 protein may comprise a container that comprises an antibody, or an antibody fragment, that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
  • the present invention also contemplates anti-idiotype antibodies, or anti- idiotype antibody fragments, that specifically bind an antibody or antibody fragment that specifically binds a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
  • the present invention provides fusion proteins that comprise a Zsig67 polypeptide moiety, and an immunoglobulin moiety, such as an immunoglobulin heavy chain constant region.
  • nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • nucleic acid molecule also includes so- called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • nucleic acid molecule refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a nucleic acid molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule. Contiguous sequences are said to "overlap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid 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).
  • structural gene refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • an "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • a nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
  • nucleic acid molecule construct is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • Linear DNA denotes non-circular DNA molecules having free 5' and 3' ends.
  • Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
  • cDNA complementary DNA
  • cDNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA to refer to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • the term “cDNA” also refers to a clone of a cDNA molecule synthesized from an RNA template.
  • a “promoter” is a nucleotide sequence that directs the transcription of a structural gene.
  • a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al. , Mol. Endocrinol. 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol.
  • CREs cyclic AMP response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O'Reilly et al., J. Biol. Chem. 2(57:19938 (1992)), AP2 (Ye et al., J. Biol. Chem. 269:2572% (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem.
  • a promoter contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
  • a “regulatory element” is a nucleotide sequence that modulates the activity of a core promoter.
  • a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific,” “tissue-specific,” or “organelle-specific” manner.
  • An “enhancer” is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
  • Heterologous DNA refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell.
  • DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA).
  • a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.
  • a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter.
  • a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.
  • 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.”
  • 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.
  • a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
  • An "integrated genetic element” is a segment of DNA that has been incorporated into a chromosome of a host cell after that element is introduced into the cell through human manipulation.
  • integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its progeny.
  • a "cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, that has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • an “expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked to” the promoter.
  • a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
  • a “recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
  • a recombinant host is a cell that produces Zlrr3 from an expression vector.
  • Zlrr3 can be produced by a cell that is a "natural source" of Zlrr3, and that lacks an expression vector.
  • fusion protein is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • a fusion protein can comprise at least part of a Zlrr3 polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of Zlrr3 using affinity chromatography.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • 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).
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the 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, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often 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.
  • secretory signal sequence denotes a nucleotide sequence that encodes a peptide (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.
  • secretory peptide a nucleotide sequence that encodes a 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.
  • secretory signal sequence denotes a nucleotide sequence that encodes a peptide (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.
  • isolated polypeptide is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • SDS sodium dodecyl sulfate
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • expression refers to the biosynthesis of a gene product.
  • expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • the term "splice variant” is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence.
  • the term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
  • immunomodulator includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.
  • 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/ligand 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 less than 10 9 M '1 .
  • an "anti-idiotype antibody” is an antibody that binds with the variable region domain of an immunoglobulin.
  • an anti-idiotype antibody binds with the variable region of an anti-Zlrr3 antibody, and thus, an anti-idiotype antibody mimics an epitope of Zlrr3.
  • an “antibody fragment” is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-Zlrr3 monoclonal antibody fragment binds with an epitope of Zlrr3.
  • antibody fragment also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • scFv proteins peptide linker
  • a “chimeric antibody” is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
  • Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • a “detectable label” is a molecule or atom which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis. Examples of detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
  • 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, EMBO J. 4: 075 (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.
  • naked antibody is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
  • antibody component includes both an entire antibody and an antibody fragment.
  • an “immunoconjugate” is a conjugate of an antibody component with a therapeutic agent or a detectable label.
  • antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a therapeutic agent.
  • therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein") and toxins ("antibody-toxin fusion protein”).
  • a “target polypeptide” or a “target peptide” is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen.
  • T cells recognize peptide epitopes presented by a major histocompatibility complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.
  • antigenic peptide is a peptide which will bind a major histocompatibility complex molecule to form an MHC -peptide complex which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell.
  • antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell which binds or expresses the antigen.
  • the antigenic peptide can be bound in the context of a class I or class II major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.
  • RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA.
  • a nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA.
  • the RNA transcript is termed an "anti- sense RNA” and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene.”
  • Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
  • an "anti-sense oligonucleotide specific for Zlrr3" or an “Zlrr3 anti-sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the Zlrr3 gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Zlrr3 gene.
  • a "ribozyme” is a nucleic acid molecule that contains a catalytic center.
  • the term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
  • a nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene.”
  • an “external guide sequence” is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P.
  • a nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene.”
  • variant Zlrr3 gene refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO:2. Such variants include naturally-occurring polymorphisms of Zlrr3 genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO:2. Additional variant forms of Zlrr3 genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences /42184
  • a variant Zlrr3 gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, or its complement, under stringent conditions.
  • variant Zlrr3 genes can be identified by sequence comparison. Two amino acid sequences have "100% amino acid sequence identity” if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence. Similarly, two nucleotide sequences have "100% nucleotide sequence identity” if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin).
  • a variant gene or polypeptide encoded by a variant gene may be characterized by the ability to bind specifically to an anti-Zlrr3 antibody.
  • 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.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counte ⁇ art 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.
  • the present invention includes functional fragments of Zlrr3 genes.
  • a "functional fragment" of a Zlrr3 gene refers to a nucleic acid molecule that encodes a portion of a Zlrr3 polypeptide which specifically binds with an anti-Zlrr3 antibody.
  • Nucleic acid molecules encoding a human Zlrr3 gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon SEQ ID NO: 1. These techniques are standard and well-established.
  • a nucleic acid molecule that encodes a human Zlrr3 gene can be isolated from a human cDNA library.
  • the first step would be to prepare the cDNA library by isolating RNA from tissue, such as thymus tissue, using methods well-known to those of skill in the art.
  • tissue isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants.
  • total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells, extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and separating RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et ⁇ l (eds.), Short Protocols in Molecular Biology, 3 r d Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)”]; Wu et al, Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)”]).
  • total RNA can be isolated from tissue by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation (see, for example, Chirgwin et al, Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages 33-41).
  • poly(A) + RNA In order to construct a cDNA library, poly(A) + RNA must be isolated from a total RNA preparation. Poly(A) + RNA can be isolated from total RNA using the standard technique of oligo(dT)-cellulose chromatography (see, for example, Aviv and Leder, Proc. Nat 'I Acad. Sci.
  • Double-stranded cDNA molecules are synthesized from poly(A) + RNA using techniques well-known to those in the art. (see, for example, Wu (1997) at pages 41-46).
  • kits can be used to synthesize double- stranded cDNA molecules.
  • such kits are available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Co ⁇ oration (Madison, WI) and STRATAGENE (La Jolla, CA).
  • Various cloning vectors are appropriate for the construction of a cDNA library.
  • a cDNA library can be prepared in a vector derived from bacteriophage, such as a ⁇ gtlO vector. See, for example, Huynh et al., "Constructing and Screening cDNA Libraries in ⁇ gtlO and ⁇ gtl l," in DNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985); Wu (1997) at pages 47-52.
  • double-stranded cDNA molecules can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Co ⁇ .) or other commercially available vectors.
  • Suitable cloning vectors also can be obtained from the American Type Culture Collection (Manassas, VA).
  • the cDNA library is inserted into a prokaryotic host, using standard techniques.
  • a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained, for example, from Life Technologies, Inc. (Gaithersburg, MD).
  • a human genomic library can be prepared by means well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).
  • Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl, digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient.
  • DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases.
  • Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307- 327).
  • Nucleic acid molecules that encode a human Zlrr3 gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the human Zlrr3 gene, as described herein.
  • PCR polymerase chain reaction
  • General methods for screening libraries with PCR are provided by, for example, Yu et al. , "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).
  • human genomic libraries can be obtained from commercial sources such as Research Genetics (Huntsville, AL) and the American Type Culture Collection (Manassas, NA).
  • a library containing cD ⁇ A or genomic clones can be screened with one or more polynucleotide probes based upon SEQ ID ⁇ O:l, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).
  • Anti-Zlrr3 antibodies produced as described below, can also be used to isolate DNA sequences that encode human Zlrr3 genes from cDNA libraries.
  • the antibodies can be used to screen ⁇ gtl l expression libraries, or the antibodies can be used for immunoscreening following hybrid selection and translation (see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al., "Screening ⁇ expression libraries with antibody and protein probes," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University Press 1995)).
  • a Zlrr3 gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).
  • Established techniques using the polymerase chain reaction provide the ability to synthesize DNA molecules at least two kilobases in length (Adang et al., Plant Molec. Biol.
  • the nucleic acid molecules of the present invention can also be synthesized with "gene machines” using protocols such as the phosphoramidite method. If chemically-synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes 60 to 80 base pairs is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 base pairs), however, special strategies may be required, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%.
  • Zlrr3 cDNA or Zlrr3 genomic fragment can be determined using standard methods.
  • Zlrr3 polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a Zlrr3 gene.
  • Promoter elements from a Zlrr3 gene can be used to direct the expression of heterologous genes in, for example, thymus tissue of transgenic animals or patients undergoing gene therapy.
  • the identification of genomic fragments containing a Zlrr3 promoter or regulatory element can be achieved using well-established techniques, such as deletion analysis (see, generally, Ausubel (1995)).
  • Cloning of 5' flanking sequences also facilitates production of Zlrr3 proteins by "gene activation," according to the general methods disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous Zlrr3 gene in a cell is altered by introducing into the Zlrr3 locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is a Zlrr3 5' non-coding sequence that permits homologous recombination of the construct with the endogenous Zlrr3 locus, whereby the sequences within the construct become operably linked with the endogenous Zlrr3 coding sequence.
  • an endogenous Zlrr3 promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • SEQ ID NO:3 is a degenerate nucleotide sequence that encompasses all nucleic acid molecules that encode the Zlrr3 polypeptides of SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:3 also provides all RNA sequences encoding SEQ ID NO:2, by substituting U for T.
  • the present invention contemplates Zlrr3 polypeptide-encoding nucleic acid molecules comprising nucleotide 39 to nucleotide 932 of SEQ ID NO:l, and their RNA equivalents.
  • Table 2 sets forth the one-letter codes used within SEQ ID NO: 3 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.
  • degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 3.
  • degenerate codon representative of all possible codons encoding an amino acid.
  • WSN can, in some circumstances, encode arginine
  • MGN can, in some circumstances, encode 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 3).
  • 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 translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 3 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 present invention further provides variant polypeptides and nucleic acid molecules that represent counte ⁇ arts 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 Zlrr3 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zlrr3 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 Zlrr3 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 Zlrr3 -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 with primers designed from the representative human Zlrr3 sequences disclosed herein.
  • 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 Zlrr3 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence disclosed in
  • SEQ ID NO:l represents a single allele of human Zlrr3, 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 nucleotide sequence shown in SEQ ID 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.
  • cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the Zlrr3 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 isolated nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules comprising nucleotide sequences disclosed herein.
  • such nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:l, or a sequence complementary thereto.
  • 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 strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • a nucleic acid molecule encoding a variant Zlrr3 polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) at 42°C overnight in a solution comprising 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • 5xSSC lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution lOO
  • hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide.
  • a higher temperature such as about 65°C
  • premixed hybridization solutions are available (e.g., EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed according to the manufacturer's instructions.
  • nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions.
  • Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C.
  • nucleic acid molecules encoding a variant Zlrr3 polypeptide remain hybridized, following stringent washing conditions, with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement), in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • SSPE for SSC in the wash solution.
  • Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65°C.
  • SDS sodium dodecyl sulfate
  • nucleic acid molecules encoding a variant Zlrr3 polypeptide remain hybridized, following highly stringent washing conditions, with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement), in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 0.1% SDS at 50°C, or 0.2xSSC with 0.1% SDS at 65°C.
  • the present invention also provides isolated Zlrr3 polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO:2, or their orthologs.
  • substantially similar sequence identity is used herein to denote polypeptides having 70%, 80%, 90%, 95% or greater than 95% sequence identity to the sequences shown in SEQ ID NO:2, or their orthologs.
  • the present invention also contemplates Zlrr3 variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ID NO:2, and a hybridization assay, as described above.
  • Such Zlrr3 variants include nucleic acid molecules (1) that remain hybridized, following stringent washing conditions, with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement), in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, and (2) that encode a polypeptide having 70%, 80%, 90%, 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • Zlrr3 variants can be characterized as nucleic acid molecules (1) that remain hybridized, following highly stringent washing conditions, with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement), in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, and (2) that encode a polypeptide having 70%, 80%, 90%, 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA £9:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 4 (amino acids are indicated by the standard one- letter codes). The percent identity is then calculated as: ([Total number of identical matches]/ [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
  • 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 Zlrr3 variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat 'I 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 matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman- Wunsch- Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl Math. 26:787 (1974)), which allows for amino acid insertions and deletions.
  • 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 described above.
  • the present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared with the amino acid sequence of SEQ ID NO:2. That is, variants can be obtained that contain one or more amino acid substitutions of SEQ ID NO:2, in which an alkyl amino acid is substituted for an alkyl amino acid in a Zlrr3 amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in a Zlrr3 amino acid sequence, a sulfur- containing amino acid is substituted for a sulfur-containing amino acid in a Zlrr3 amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy- containing amino acid in a Zlrr3 amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a Zlrr3 amino acid sequence, a basic amino acid is substituted for a basic amino acid in a Zlrr3 amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic
  • a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Natl Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • certain conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while other conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Zlrr3 are characterized by having greater than 96%, at least 97%, at least 98%, or at least 99% sequence identity to the corresponding amino acid sequence (e.g., SEQ ID NO:2), wherein the variation in amino acid sequence is due to one or more conservative amino acid substitutions.
  • amino acid sequence e.g., SEQ ID NO:2
  • Conservative amino acid changes in a Zlrr3 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO:l.
  • Such "conservative amino acid” variants can be obtained, for example, by oligonucleotide- directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).
  • a variant Zlrr3 polypeptide can be identified by the ability to specifically bind anti-Zl ⁇ antibodies.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tr ⁇ , -3-methylproline, 2,4-methanoproline, ew-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ // ⁇ -threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine.
  • non-naturally occurring amino acid is inco ⁇ orated into the protein in place of its natural counte ⁇ art. See, Koide et al, Biochem. 33:7470 (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 (1993)).
  • 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 Zlrr3 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 244:108 (1989), Bass et al, Proc. Natl Acad. Sci. USA 55:4498 (1991), Coombs and Corey, "Site-Directed Mutagenesis and Protein Engineering," in Proteins: Analysis and Design, Angeletti (ed.), pages 259-311 (Academic Press, Inc. 1998)).
  • Zlrr3 receptor binding domains 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 (1992), Smith et al, J. Mol. Biol. 224:899 (1992), and Wlodaver et al, FEBS Lett. 309:59 (1992). Moreover, Zlrr3 labeled with biotin or FITC can be used for expression cloning of Zlrr3 receptors.
  • variants of the disclosed Zlrr3 nucleotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Natl Acad. Sci. USA 97:10747 (1994), and international publication No. 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.
  • 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 biologically active polypeptides, or polypeptides that bind with anti-Zlrr3 antibodies, can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the present invention also includes "functional fragments" of Zlrr3 polypeptides and nucleic acid molecules encoding such functional fragments.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes a Zlrr3 polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ID NO:l can be digested with Bal3l nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for the ability to bind anti-Zlrr3 antibodies.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of a Zlrr3 gene can be synthesized using the polymerase chain reaction.
  • Methods for identifying functional domains are well-known to those of skill in the art. For example, studies on the truncation at either or both termini of interferons have been summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995).
  • standard techniques for functional analysis of proteins are described by, for example, Treuter et al, Molec. Gen. Genet.
  • the present invention also contemplates functional fragments of a Zlrr3 gene that has amino acid changes, compared with the amino acid sequence of SEQ ID NO:2.
  • a variant Zlrr3 gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequences of SEQ ID NOs:l and 2, as discussed above.
  • An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant Zlrr3 gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 , as discussed above.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zlrr3 polypeptide described herein.
  • Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen.
  • Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al, Proc. Natl Acad. Sci. USA 57:3998 (1983)).
  • polypeptide fragments or peptides may comprise an
  • antigenic epitope which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al, Science 219:660 (1983)). Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.
  • Antigenic epitope-bearing peptides and polypeptides can contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID NO:2.
  • Such epitope-bearing peptides and polypeptides can be produced by fragmenting a Zlrr3 polypeptide, or by chemical peptide synthesis, as described herein.
  • epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al, Curr. Opin. Biotechnol. 7:616 (1996)).
  • the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3. Suitable forms of computer-readable media include magnetic media and optically-readable media.
  • magnétique media examples include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIP disk.
  • Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
  • compact discs e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable
  • DVD digital versatile/video discs
  • Fusion proteins of Zlrr3 can be used to express Zlrr3 in a recombinant host, and to isolate expressed Zlrr3. As described below, particular Zlrr3 fusion proteins also have uses in diagnosis and therapy.
  • fusion protein comprises a peptide that guides a Zlrr3 polypeptide from a recombinant host cell.
  • a secretory signal sequence also known as a signal peptide, a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence may be derived from Zlrr3
  • a suitable signal sequence may also be derived from another secreted protein or synthesized de novo.
  • the secretory signal sequence is operably linked to a Zlrr3- encoding sequence such that 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 nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide 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).
  • yeast signal sequence is preferred for expression in yeast cells.
  • suitable yeast signal sequences are those derived from yeast mating phermone ⁇ -factor (encoded by the MFal gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene). See, for example, Romanos et al. , "Expression of Cloned Genes in Yeast," in DNA Cloning 2: A Practical Approach, 2 nd Edition, Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).
  • Zlrr3 can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide.
  • Glutathione S-transferase fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • a Zlrr3 fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resin column, while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose.
  • Established techniques for expressing a heterologous polypeptide as a fusion protein in a bacterial cell are described, for example, by Williams et al. , "Expression of Foreign Proteins in E. coli Using Plasmid Vectors and Purification of Specific Polyclonal Antibodies," in DNA Cloning 2: A Practical Approach, 2 nd Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995).
  • the PINPOINT Xa protein purification system provides a method for isolating a fusion protein comprising a polypeptide that becomes biotinylated during expression with a resin that comprises avidin.
  • Peptide tags that are useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaryotic cells include polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies). See, for example, Luo et al, Arch. Biochem. Biophys. 329:2 5 (1996), Morganti et al, Biotechnol. Appl Biochem.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1 to 28 of SEQ ID NO:2 is operably linked to 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.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor.
  • Such fusions may be used in a transgenic animal or in a cultured recombinant host to direct peptides through the secretory pathway.
  • exemplary polypeptides include pharmaceutically active molecules such as Factor Vila, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, and IL-15), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferons- ⁇ , - ⁇ , - ⁇ , - ⁇ , and - ⁇ ), the stem cell growth factor designated "SI factor,” erythropoietin, and thrombopoietin.
  • interleukins e.g., interleukin-1 (IL
  • the Zlrr3 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.
  • Fusion proteins comprising a Zlrr3 secretory signal sequence can be constructed using standard techniques.
  • Another form of fusion protein comprises a Zlrr3 polypeptide and an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two constant region domains and a hinge region but lacks the variable region.
  • an F c fragment typically a fusion protein comprising a human interferon and a human immunoglobulin Fc fragment.
  • the C- terminal of the interferon is linked to the N-terminal of the Fc fragment by a peptide linker moiety.
  • a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert.
  • An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO:4).
  • a preferred Fc moiety is a human ⁇ 4 chain, which is stable in solution and has little or no complement activating activity.
  • the present invention contemplates a Zlrr3 fusion protein that comprises a Zlrr3 moiety and a human Fc fragment, wherein the C-terminus of the Zlrr3 moiety is attached to the N-terminus of the Fc fragment via a peptide linker, such as a peptide consisting of the amino acid sequence of SEQ ID NO:4.
  • the Zlrr3 moiety can be a Zlrr3 molecule or a fragment thereof.
  • An illustrative Zlrr3 fragment is a polypeptide comprising amino acid residues 29 to 254 of SEQ ID NO:2.
  • a Zlrr3 fusion protein comprises an IgG sequence, a Zlrr3 moiety covalently joined to the amino terminal end of the IgG sequence, and a signal peptide that is covalently joined to the amino terminal of the Zlrr3 moiety, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH 2 domain, and a CH 3 domain. Accordingly, the IgG sequence lacks a CH, domain.
  • the Zlrr3 moiety displays a Zlrr3 activity, as described herein, such as the ability to bind with a Zlrr3 receptor.
  • Fusion proteins comprising a Zlrr3 moiety and an Fc moiety can be used, for example, as an in vitro assay tool.
  • the presence of a Zlrr3 receptor in a biological sample can be detected using a Zlrr3 -antibody fusion protein, in which the Zlrr3 moiety is used to target the cognate receptor, and a macromolecule, such as Protein A or anti-Fc antibody, is used to detect the bound fusion protein- receptor complex.
  • fusion proteins can be used to identify agonists and antagonists that interfere with the binding of Zlrr3 to its receptor.
  • antibody-Zlrr3 fusion proteins comprising antibody variable domains, are useful as therapeutic proteins, in which the antibody moiety binds with a target antigen, such as a tumor associated antigen.
  • hybrid Zlrr3 proteins can be constructed using regions or domains of the inventive protein in combination with those of related leucine-rich repeat proteins, or heterologous proteins (see, for example, Picard, Cur. Opin. Biology 5:511 (1994)).
  • domains include, but are not limited to, the secretory signal sequence, extracellular domain, transmembrane domain, and intracellular domain.
  • These hybrids may be characterized by altered reaction kinetics, altered binding, limited or expanded substrate specificity, or altered tissue and cellular localization of a polypeptide.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. General methods for enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) at pages 16-
  • polypeptides of the present invention can be produced in recombinant host cells following conventional techniques.
  • a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the expression vector.
  • Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
  • expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell.
  • a Zlrr3 expression vector may comprise a Zlrr3 gene and a secretory sequence derived from a Zlrr3 gene or another secreted gene.
  • Zlrr3 proteins of the present invention may be expressed in mammalian cells.
  • suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin et al, Som. Cell. Molec. Genet.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Molec. Appl Genet. 7:273 (1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 early promoter (Benoist et al, Nature 290:304 (1981)), the Rous sarcoma virus promoter (Gorman et al, Proc. Nat'l Acad. Sci.
  • a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter, can be used to control Zlrr3 gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al, Mol Cell. Biol. 10:4529 (1990), and Kaufman et al, Nucl Acids Res. 79:4485 (1991)).
  • An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like.
  • the transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).
  • one suitable selectable marker is a gene that provides resistance to the antibiotic neomycin.
  • selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a suitable amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • 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.
  • Zlrr3 polypeptides can also be produced by cultured mammalian cells using a viral delivery system.
  • exemplary viruses for this pu ⁇ ose include adenovirus, he ⁇ esvirus, 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 a review, see Becker et al, Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)).
  • Advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters.
  • Adenovirus vector-infected human 293 cells ATCC Nos. CRL- 1573, 45504, 45505
  • Adenovirus vector-infected human 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 (1994)).
  • Zlrr3 genes may also be expressed in other higher eukaryotic cells, such as avian, fungal, insect, yeast, or plant cells.
  • the baculovirus system provides an efficient means to introduce cloned Zlrr3 genes into insect cells.
  • Suitable expression vectors are based upon the Autographa calif ornica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, Autographa californica nuclear polyhedrosis virus immediate-early gene promoter (z ' e-7) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter.
  • hsp Drosophila heat shock protein
  • z ' e-7 the delayed early 39K promoter
  • baculovirus plO promoter and the
  • a second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, et al, J. Virol. 67:4566 (1993)).
  • This system which utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Zlrr3 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 77:971 (1990), Bonning, et al, J. Gen.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Zlrr3 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al, Proc. Natl Acad. Sci. 82:7952 (1985)).
  • a transfer vector containing a Zlrr3 gene 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 then isolated using common techniques.
  • the illustrative PFASTBAC vector can be modified to a considerable degree.
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Vcor, 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, for example, Hill-Perkins and Possee, J. Gen. Virol. 77:971 (1990), Bonning, et al, J. Gen. Virol 75:1551 (1994), and Chazenbalk and Rapoport, J. Biol. Chem. 270: 543 (1995).
  • a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native Zlrr3 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from ⁇ cdysteroid Glucosyltransferase ( ⁇ GT), honey bee Melittin (Invitrogen Co ⁇ oration; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native Zlrr3 secretory signal sequence.
  • the recombinant virus or bacmid is used to transfect host cells.
  • suitable insect host cells include cell lines derived from IPLB-S 21, a Spodoptera frugiperda pupal ovarian cell line, such as S ⁇ (ATCC CRL 1711), S ⁇ lAE, and Sf2l (Invitrogen Co ⁇ oration; San Diego, CA), as well as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).
  • S ⁇ ATCC CRL 1711
  • S ⁇ lAE S ⁇ lAE
  • Sf2l Invitrogen Co ⁇ oration
  • Drosophila Schneider-2 cells Drosophila Schneider-2 cells
  • HIGH FIVEO cell line Invitrogen
  • Commercially available serum-free media can be used to grow and to maintain the cells.
  • Suitable media are Sf900 IITM (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 typically grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 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
  • yeast cells can also be used to express the genes described herein.
  • yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
  • GAL1 galactose
  • PGK phosphoglycerate kinase
  • ADH alcohol dehydrogenase
  • AOX1 alcohol oxidase
  • HIS4 histidinol dehydrogenase
  • yeast cloning vectors have been designed and are readily available. These vectors include Yip-based vectors, such as YIp5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCp 19. 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 suitable vector system for use in Saccharomyces cerevisiae is the POT1 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.
  • Additional suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al, U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446, 5,063,154, 5,139,936, and 4,661,454.
  • Transformation systems for other yeasts including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol 132:3459 (1986), and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349.
  • methanolica will commonly be prepared as double-stranded, circular plasmids, which can be linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUGl or AUG2).
  • Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes.
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a suitable 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), and which allows ade2 host cells to grow in the absence of adenine.
  • a suitable 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), and which allows ade2 host cells to grow in the absence of adenine.
  • methanol utilization genes AUG2
  • host cells deficient in vacuolar protease genes PEP4 and PRB1 are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P.
  • P. methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells.
  • Methods for introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch et al, Science 227:1229 (1985), Klein et al, Biotechnology 10:268 (1992), and Miki et al, "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press, 1993).
  • Zlrr3 genes can be expressed in prokaryotic host cells.
  • Suitable promoters that can be used to express Zlrr3 polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
  • subtilis subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl trans- ferase gene.
  • Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol 7:277 (1987), Watson et al, Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995).
  • Suitable prokaryotic hosts include E. coli and Bacillus subtilus.
  • Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)).
  • Suitable strains of Bacillus subtilus include BR151, YB886, Mil 19, MI 120, and B 170 (see, for example, Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)).
  • the polypeptide When expressing a Zlrr3 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the denaturant such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione
  • 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.
  • polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well-known to those of skill in the art (see, for example, Merrifield, J. Am. Chem. Soc. 55:2149 (1963), Stewart et al, "Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot.
  • Peptides and polypeptides of the present invention comprise at least six, at least nine, or at least 15 contiguous amino acid residues of SEQ ID NO:2.
  • polypeptides can comprise at least six, at least nine, or at least 15 contiguous amino acid residues of any of the following amino acid sequences of SEQ ID NO:2: amino acid residues amino acid residues 29 to 254, amino acid residues 255 to 284, amino acid residues 285 to 298, and amino acid residues 70 to 249.
  • the polypeptides comprise 20, 30, 40, 50, or more contiguous residues of these amino acid sequences. Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
  • Polypeptides of the present invention can be purified to at least about 80% purity, to at least about 90% purity, to at least about 95% purity, or even greater than 95% purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • the polypeptides of the present invention may also be purified to a pharmaceutically pure state, which is greater than 99.9% pure.
  • Certain preparations contain purified polypeptides that are substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Fractionation and/or conventional purification methods can be used to obtain preparations of Zlrr3 purified from natural sources (e.g., thymus tissue), and recombinant Zlrr3 polypeptides and fusion Zlrr3 polypeptides purified from recombinant host cells.
  • natural sources e.g., thymus tissue
  • recombinant Zlrr3 polypeptides and fusion Zlrr3 polypeptides purified from recombinant host cells Numerous methods for purifying proteins are known in the art. In general, ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like.
  • chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomery ville, PA), Octyl- Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl-Sepharose FF Pharmacia
  • Toyopearl butyl 650 Toso Haas, Montgomery ville, PA
  • Octyl- Sepharose Pharmacia
  • polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
  • Zlrr3 isolation and purification can be devised by those of skill in the art.
  • anti-Zlrr3 antibodies obtained as described below, can be used to isolate large quantities of protein by immunoaffinity purification.
  • methods for binding ligands, such as Zlrr3, to receptor polypeptides bound to support media are well known in the art.
  • the polypeptides of the present invention can also be isolated by exploitation of particular properties.
  • immobilized metal ion adso ⁇ tion (IMAC) 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: (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.
  • IMAC immobilized metal ion adso ⁇ tion
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • Zlrr3 polypeptides or fragments thereof may also be prepared through chemical synthesis, as described below. Zlrr3 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.
  • Zlrr3 contains a transmembrane domain, there is a potential thrombin cleavage site (amino acid residues 228-229 of SEQ ID NO:2) located between the N-terminus and the transmembrane region. This observation indicates that, in vivo, thrombin can liberate the extracellular domain of Zlrr3. This is similar to the thrombin cleavage of human platelet glycoprotein V, another leucine-rich protein (Lanza et ⁇ l, J. Biol. Chem. 268:20801 (1993)). Accordingly, the present invention contemplates the use of polypeptides comprising the Zlrr3 extracellular domain as a ligand for a Zlrr3 receptor.
  • Zlrr3 polypeptide or fragments of the polypeptide can be used as a ligand.
  • Illustrative Zlrr3 fragments include polypeptides comprising amino acid residues 29-254 of SEQ ID NO:2 and polypeptides comprising amino acid residues 29-228 of SEQ ID NO:2.
  • Zlrr3 analogs are variants having an amino acid sequence that is a mutation of the amino acid sequence disclosed herein.
  • Another general class of Zlrr3 analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below.
  • recombinant antibodies comprising anti- idiotype variable domains can be used as analogs (see, for example, Monfardini et ⁇ l, Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domains of anti- idiotype Zlrr3 antibodies mimic Zlrr3, these domains can provide either Zlrr3 agonist or antagonist activity.
  • Lim and Langer, J. Interferon Res. 13:295 (1993) describe anti-idiotypic interferon- ⁇ antibodies that have the properties of either interferon- ⁇ agonists or antagonists.
  • Zlrr3 and its analogs can be used to identify and to isolate Zlrr3 receptors.
  • proteins and peptides of the present invention can be immobilized on a column and used to bind receptor proteins from membrane preparations that are run over the column (Hermanson et al. (eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)).
  • Radiolabeled or affinity labeled Zlrr3 polypeptides can also be used to identify or to localize Zlrr3 receptors in a biological sample (see, for example, Lieber (ed.), Methods in Enzymol, vol. 182, pages 721-37 (Academic Press 1990); Brunner et al, Ann. Rev. Biochem.
  • the activity of Zlrr3 can be measured by a silicon- based biosensor microphysiometer which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent cellular responses.
  • An exemplary device is the CYTOSENSOR Microphysiometer manufactured by Molecular Devices Co ⁇ . (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 (1992), Pitchford et al, Meth. Enzymol. 228:84 (1997), Arimilli et al, J. Immunol. Meth.
  • the microphysiometer can be used for assaying adherent or non-adherent eukaryotic cells. Since energy metabolism is coupled with the use of cellular ATP, any event which alters cellular ATP levels, such as receptor activation and the initiation of signal transduction, will cause a change in cellular acid section. By measuring extracellular acidification changes in cell media over time, therefore, the microphysiometer directly measures cellular responses to various stimuli, including Zlrr3, its agonists, or antagonists.
  • the microphysiometer is used to measure responses of a Zlrr3 -responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to Zlrr3 polypeptide.
  • Zlrr3 responsive eukaryotic cells comprise cells into which a receptor for Zlrr3 has been transfected to create a cell that is responsive to Zlrr3, or cells that are naturally responsive to Zlrr3.
  • Zlrr3 modulated cellular responses are measured by a change (e.g., an increase or decrease in extracellular acidification) in the response of cells exposed to Zlrr3, compared with control cells that have not been exposed to Zlrr3.
  • a microphysiometer can be used to identify cells, tissues, or cell lines which respond to a Zlrr3 stimulated pathway, and which express a functional Zlrr3 receptor.
  • cells that express a functional Zlrr3 receptor can be identified by (a) providing test cells, (b) incubating a first portion of the test cells in the absence of Zlrr3, (c) incubating a second portion of the test cells in the presence of Zlrr3, and (d) detecting a change (e.g., an increase or decrease in extracellular acidification rate, as measured by a microphysiometer) in a cellular response of the second portion of the test cells, as compared to the first portion of the test cells, wherein such a change in cellular response indicates that the test cells express a functional Zlrr3 receptor.
  • An additional negative control may be included in which a portion of the test cells is incubated with Zlrr3 and an anti-Zlrr3 antibody to inhibit the binding of Zh ⁇ 3 with its
  • the microphysiometer also provides one means to identify Zlrr3 agonists.
  • agonists of Zlrr3 can be identified by a method, comprising the steps of (a) providing cells responsive to Zlrr3, (b) incubating a first portion of the cells in the absence of a test compound, (c) incubating a second portion of the cells in the presence of a test compound, and (d) 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, wherein such a change in cellular response indicates that the test compound is a Zlrr3 agonist.
  • An illustrative change in cellular response is a measurable change in extracellular acidification rate, as measured by a microphysiometer.
  • incubating a third portion of the cells in the presence of Zlrr3 and in the absence of a test compound can be used as a positive control for the Zlrr3 responsive cells, and as a control to compare the agonist activity of a test compound with that of Zlrr3.
  • An additional control may be included in which a portion of the cells is incubated with a test compound (or Zlrr3) and an anti-Zlrr3 antibody to inhibit the binding of the test compound (or Zlrr3) with the Zlrr3 receptor.
  • a Zlrr3 variant gene product that lacks biological activity may be a Zlrr3 antagonist.
  • These biologically-inactive Zlrr3 variants can be initially identified on the basis of hybridization analysis, sequence identity determination, or by the ability to specifically bind anti-Zlrr3 antibody.
  • a Zlrr3 antagonist can be further characterized by its ability to inhibit the biological response induced by Zlrr3 or by a Zlrr3 agonist. This inhibitory effect may result, for example, from the competitive or non-competitive binding of the antagonist to the Zlrr3 receptor.
  • Zlrr3 antagonists can be identified by a method, comprising the steps of (a) providing cells responsive to Zlrr3, (b) incubating a first portion of the cells in the presence of Zlrr3 and in the absence of a test compound, (c) incubating a second portion of the cells in the presence of both Zlrr3 and the test compound, and (d) comparing the cellular responses of the first and second cell portions, wherein a decreased response by the second portion, compared with the response of the first portion, indicates that the test compound is a Zlrr3 antagonist.
  • Zlrr3 its agonists and antagonists are valuable in both in vivo and in vitro uses.
  • Zlrr3 and its agonists may be used to supplement serum-free media, while Zlrr3 antagonists are useful as research reagents for characterizing sites of interaction between Zlrr3 and its receptor.
  • pharmaceutical compositions comprising Zlrr3 antagonists can be used to inhibit Zlrr3 activity.
  • the present invention also contemplates chemically modified Zlrr3 compositions, in which a Zlrr3 polypeptide is linked with a polymer.
  • Illustrative Zlrr3 polypeptides include polypeptides comprising amino acid residues 29 to 254 of SEQ ID NO:2.
  • the polymer is water soluble so that the Zlrr3 conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation, In this way, the degree of polymerization can be controlled.
  • a reactive aldehyde is polyethylene glycol propionaldehyde, or mono-(Cl-ClO) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al, U.S. Patent No. 5,252,714).
  • the polymer may be branched or unbranched.
  • a mixture of polymers can be used to produce Zlrr3 conjugates.
  • Zlrr3 conjugates used for therapy can comprise pharmaceutically acceptable water-soluble polymer moieties.
  • Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(Cl-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, t ⁇ -succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers.
  • Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000.
  • a Zlrr3 conjugate can also comprise a
  • Zlrr3 conjugate comprises a Zlrr3 moiety and a poly alkyl oxide moiety attached to the N-terminus of the Zlrr3 moiety.
  • PEG is one suitable polyalkyl oxide.
  • Zlrr3 can be modified with PEG, a process known as "PEGylation.”
  • PEGylation of Zlrr3 can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316, Delgado et al, Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet.
  • PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule.
  • Zlrr3 conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, Karasiewicz et al, U.S. Patent No. 5,382,657).
  • PEGylation by acylation typically requires reacting an active ester derivative of PEG with a Zlrr3 polypeptide.
  • An example of an activated PEG ester is PEG esterified to N-hydroxy succinimide.
  • acylation includes the following types of linkages between Zlrr3 and a water soluble polymer: amide, carbamate, urethane, and the like.
  • Methods for preparing PEGylated Zlrr3 by acylation will typically comprise the steps of (a) reacting a Zlrr3 polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to Zlrr3, and (b) obtaining the reaction product(s).
  • PEG such as a reactive ester of an aldehyde derivative of PEG
  • the optimal reaction conditions for acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG:Zlrr3, the greater the percentage of polyPEGylated Zlrr3 product.
  • the product of PEGylation by acylation is typically a polyPEGylated Zlrr3 product, wherein the lysine ⁇ -amino groups are PEGylated via an acyl linking group.
  • An example of a connecting linkage is an amide.
  • the resulting Zlrr3 will be at least 95% mono-, di-, or tri-pegylated, although some species with higher degrees of PEGylation may be formed depending upon the reaction conditions.
  • PEGylated species can be separated from unconjugated Zlrr3 polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, and the like.
  • PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with Zlrr3 in the presence of a reducing agent.
  • PEG groups can be attached to the polypeptide via a -CH 2 - ⁇ H group.
  • Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization.
  • the reaction is performed at a pH that allows one to take advantage of the pKa differences between the ⁇ -amino groups of the lysine residues and the ⁇ -amino group of the N-terminal residue of the protein.
  • Reductive alkylation to produce a substantially homogenous population of monopolymer Zlrr3 conjugate molecule can comprise the steps of: (a) reacting a Zlrr3 polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the ⁇ -amino group at the amino terminus of the Zlrr3, and (b) obtaining the reaction product(s).
  • the reducing agent used for reductive alkylation should be stable in aqueous solution and able to reduce only the Schiff base formed in the initial process of reductive alkylation.
  • Illustrative reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, and pyridine borane.
  • the reductive alkylation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the N-terminus of Zlrr3.
  • Such reaction conditions generally provide for pKa differences between the lysine amino groups and the ⁇ -amino group at the N-terminus.
  • the pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal ⁇ -group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polymer:Zlrr3 need not be as large because more reactive groups are available. Typically, the pH will fall within the range of 3 to 9, or 3 to 6.
  • the molecular weight of the water-soluble polymer is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25 kDa.
  • the molar ratio of water-soluble polymer to Zlrr3 will generally be in the range of 1 :1 to 100:1. Typically, the molar ratio of water-soluble polymer to Zlrr3 will be 1:1 to 20: 1 for polyPEGylation, and 1 : 1 to 5:1 for monoPEGylation.
  • compositions comprising a peptide or polypeptide described herein.
  • Such compositions can further comprise a carrier.
  • the carrier can be a conventional organic or inorganic carrier.
  • carriers include water, buffer solution, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.
  • Antibodies to Zlrr3 can be obtained, for example, using the product of a Zlrr3 expression vector or Zlrr3 isolated from a natural source as an antigen. Particularly useful anti-Zlrr3 antibodies "bind specifically" with Zlrr3. Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to Zlrr3 with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides known in the art, such as human decorin.
  • antibodies specifically bind if they bind to a Zlrr3 polypeptide, peptide or epitope with a binding affinity (Ka) of 10 6 M “ ' or greater, preferably 10 7 M '1 or greater, more preferably 10 8 M “ 1 or greater, and most preferably 10 9 M “1 or greater.
  • Ka binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 57:660 (1949)).
  • Anti-Zlrr3 antibodies can be produced using antigenic Zlrr3 epitope- bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least six, or between 15 to about 30 amino acids contained within SEQ ID NO:2.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with Zlrr3. It is desirable that the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.
  • antigenic peptide 1 amino acids 33 to 39 of SEQ ID NO:2
  • antigenic peptide 2 amino acids 33 to 38 of SEQ ID NO:2
  • antigenic peptide 3 amino acids 33 to 38 of SEQ ID NO:2
  • amino acids 61 to 70 amino acids 61 to 70 ("antigenic peptide 4"), amino acids 61 to 66 ("antigenic peptide 5"), amino acids 62 to 67 (“antigenic peptide 6"), amino acids 63 to 68 (“antigenic peptide 7"), amino acids 64 to 69 ("antigenic peptide 8"), amino acids 65 to 70 (“antigenic peptide 9"), amino acids 217 to 233 (“antigenic peptide 10"), amino acids 217 to 222 (“antigenic peptide 11"), amino acids 218 to 223 (“antigenic peptide 12”), amino acids 219 to 224 (“antigenic peptide 13”), amino acids 220 to 225 (“antigenic peptide 14"), amino acids 221 to 226 (“antigenic peptide 15”), amino acids 222 to 227 (“antigenic peptide 16”), amino acids 223 to 228 (“antigenic peptide 17”), amino acids 224 to 229
  • the present invention contemplates the use of any one of antigenic peptides 1 to 25 to generate antibodies to Zlrr3.
  • the present invention also contemplates polypeptides comprising at least one of antigenic peptides 1 to 25.
  • Polyclonal antibodies to recombinant Zlrr3 protein or to Zlrr3 isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al., "Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al, "Expression of foreign proteins in E.
  • the immunogenicity of a Zlrr3 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • 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 Zlrr3 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.
  • polypeptide portion is "hapten-like,” such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • an anti- Zlrr3 antibody of the present invention may also be derived from a subhuman primate antibody.
  • General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al, international patent publication No. WO 91/11465, and in Losman et al, Int. J. Cancer 46:310 (1990).
  • monoclonal anti-Zlrr3 antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al, Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1- 2.6.7 (John Wiley & Sons 1991) ["Coligan”], Picksley et al, "Production of monoclonal antibodies against proteins expressed in E. coli " in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising a Zlrr3 gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B- lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • an anti-Zlrr3 antibody of the present invention may be derived from a human monoclonal antibody.
  • Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al, Nature Genet. 7:13 (1994), Lonberg et al, Nature 368:856 (1994), and Taylor et al, Int. Immun. 6:579 (1994).
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al, "Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)). For particular uses, it may be desirable to prepare fragments of anti-
  • 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 thiol 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. Biophys. 89:230 (I960), Porter, Biochem. J. 75:119 (1959), Edelman et al., in Methods in Enzymology Vol. 7, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of V H and V L chains.
  • This association can be noncovalent, as described by Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659 (1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 12:437 (1992)).
  • the Fv fragments may comprise V H and V L chains which are connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • a scFV can be obtained by exposing lymphocytes to Zlrr3 polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zlrr3 protein or peptide).
  • Genes encoding polypeptides having potential Zlrr3 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 Zlrr3 sequences disclosed herein to identify proteins which bind to Zlrr3.
  • 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.
  • an anti-Zlrr3 antibody may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counte ⁇ arts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 56:3833 (1989).
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zlrr3 antibodies or antibody fragments, using standard techniques. See, for example, Green et al. , "Production of Polyclonal Antisera,” in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7.
  • monoclonal anti- idiotype antibodies can be prepared using anti-Zlrr3 antibodies or antibody fragments as immunogens with the techniques, described above.
  • humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques.
  • Nucleic acid molecules can be used to detect the expression of a Zlrr3 gene in a biological sample.
  • probe molecules include double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:l, or a fragment thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:l, or a fragment thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • portion refers to at least eight nucleotides to at least 20 or more nucleotides.
  • RNA isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target Zlrr3 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 (1995) at pages 4-1 to 4-27, and Wu et ⁇ l. (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.
  • Zlrr3 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.
  • Zlrr3 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 4:467 (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. 1998), and Meltzer (ed.), PCT?
  • PCR primers can be designed to amplify a portion of the Zlrr3 gene that has a low sequence similarity to a comparable region in other genes.
  • One variation of PCR for diagnostic assays is reverse transcriptase-
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with Zlrr3 primers (see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)). PCR is then performed and the products are analyzed using standard techniques.
  • RNA is isolated from biological sample using, for example, the guanidinium-thiocyanate cell lysis procedure described above.
  • 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 Zlrr3 anti-sense oligomers.
  • Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
  • Zlrr3 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 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 transferred to a membrane, hybridized with a detectably-labeled Zlrr3 probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • CPT cycling probe technology
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross-hybridization
  • LCR ligase chain reaction
  • Zlrr3 probes and primers can also be used to detect and to localize Zlrr3 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.), In 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, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.
  • the Zlrr3 gene resides in chromosome l lql l, a region that is associated with diseases and disorders, such as angioneurotic edema. Moreover, mutations in genes encoding leucine-rich repeat proteins have been associated with diseases, including Bernard-Soulier syndrome and polycystic kidney disease (see, for example, European Polycystic Kidney Disease Consortium, Cell 77:81 (1994); Suzuki et al, Br. J. Haematol 99:794 (1997)). Thus, Zlrr3 nucleotide sequences can be used in linkage-based testing for various diseases, and to determine whether a subject's chromosomes contain a mutation in the Zlrr3 gene.
  • Detectable chromosomal aberrations at the Zlrr3 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Of particular interest are genetic alterations that inactivate the Zlrr3 gene.
  • Aberrations associated with the Zlrr3 locus can be detected using nucleic acid molecules of the present invention by employing molecular genetic techniques, such as restriction fragment length polymo ⁇ hism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymo ⁇ hism 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.
  • molecular genetic techniques such as restriction fragment length polymo ⁇ hism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymo ⁇ hism detection, RNase cleavage methods, denaturing gradient gel electrophoresis
  • RNA is isolated from a biological sample, and used to synthesize cDNA. PCR is then used to amplify the Zlrr3 target sequence and to introduce an RNA polymerase promoter, a translation initiation sequence, and an in- frame ATG triplet. PCR products are transcribed using an RNA polymerase, and the transcripts are translated in vitro with a T7-coupled reticulocyte lysate system.
  • the translation products are then fractionated by SDS-PAGE to determine the lengths of the translation products.
  • the protein truncation test is described, for example, by Dracopoli et al. (eds.), Current Protocols in Human Genetics, pages 9.1 1.1 - 9.11.18 (John Wiley & Sons 1998).
  • the present invention also contemplates kits for performing a diagnostic assay for Zlrr3 gene expression or to examine the Zlrr3 locus.
  • kits comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:l, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NO:l, or a portion thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • Kits may comprise nucleic acid primers for performing PCR.
  • kits can contain all the necessary elements to perform a nucleic acid diagnostic assay described above.
  • a kit will comprise at least one container comprising a Zlrr3 probe or primer.
  • the kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zlrr3 sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like.
  • a kit may also comprise a means for conveying to the user that the Zlrr3 probes and primers are used to detect Zlrr3 gene expression.
  • written instructions may state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes Zlrr3, >or a nucleic acid molecule having a nucleotide sequence that is complementary to a Zlrr3 -encoding nucleotide sequence.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • the present invention contemplates the use of anti-Zlrr3 antibodies to screen biological samples in vitro for the presence of Zlrr3.
  • anti-Zlrr3 antibodies are used in liquid phase.
  • the presence of Zlrr3 in a biological sample can be tested by mixing the biological sample with a trace amount of labeled Zlrr3 and an anti-Zlrr3 antibody under conditions that promote binding between Zlrr3 and its antibody.
  • Complexes of Zlrr3 and anti-Zlrr3 in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein which binds with the antibody, such as an Fc antibody or Staphylococcus protein A.
  • the concentration of Zlrr3 in the biological sample will be inversely proportional to the amount of labeled Zlrr3 bound to the antibody and directly related to the amount of free labeled Zlrr3.
  • in vitro assays can be performed in which anti-Zlrr3 antibody is bound to a solid-phase carrier.
  • antibody can be attached to a polymer, such as aminodextran, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube.
  • an insoluble support such as a polymer-coated bead, a plate or a tube.
  • anti-Zlrr3 antibodies can be used to detect Zlrr3 in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of Zlrr3 and to determine the distribution of Zlrr3 in the examined tissue.
  • Immunochemical detection can be performed by contacting a biological sample with an anti-Zlrr3 antibody, and then contacting the biological sample with a detectably labeled molecule which binds to the antibody.
  • the detectably labeled molecule can comprise an antibody moiety that binds to anti-Zlrr3 antibody.
  • the anti-Zh ⁇ 3 antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin). Numerous variations of this basic technique are well-known to those of skill in the art.
  • an anti-Zlrr3 antibody can be conjugated with a detectable label to form an anti-Zlrr3 immunoconjugate.
  • Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
  • the detectable label can be a radioisotope that is detected by autoradiography.
  • Isotopes that are particularly useful for the pu ⁇ ose of the present invention are ⁇ , 1 5 1, 13 T, 35 S and 14 C.
  • Anti-Zlrr3 immunoconjugates can also be labeled with a fluorescent compound.
  • the presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence.
  • Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • anti-Zlrr3 immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemi- luminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • Bioluminescent compound can be used to label anti-Zlrr3 immunoconjugates of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-Zlrr3 immunoconjugates can be detectably labeled by linking an anti-Zlrr3 antibody component to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galac- tosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • the convenience and versatility of immunochemical detection can be enhanced by using anti-Zlrr3 antibodies that have been conjugated with avidin, streptavidin, and biotin (see, for example, Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al, "Immunochemical Applications of Avidin-Biotin Technology,” in Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).
  • kits for performing an immunological diagnostic assay for Zlrr3 gene expression comprise at least one container comprising an anti-Zlrr3 antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zlrr3 antibody or antibody fragments. Examples of such indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like.
  • a kit may also comprise a means for conveying to the user that Zlrr3 antibodies or antibody fragments are used to detect Zlrr3 protein. For example, written instructions may state that the enclosed antibody or antibody fragment can be used to detect Zlrr3. The written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • Zlrr3 may be used to treat various pathological conditions, such as abnormal cell proliferation. Therefore, the present invention includes the use of proteins, polypeptides, and peptides having Zlrr3 activity (such as Zlrr3 polypeptides, Zlrr3 variants, and Zlrr3 fusion proteins) to treat conditions characterized by abnormal cell proliferation.
  • Zlrr3 polypeptides can be used to treat wounds, to promote wound healing, to inhibit scar formation, to inhibit fibrosis in general, and to inhibit invasion of glial cells or neurite outgrowth.
  • the dosage of administered polypeptide, protein or peptide will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of a molecule having Zlrr3 activity which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.
  • Administration of a molecule having Zlrr3 activity to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
  • the administration may be by continuous infusion or by single or multiple boluses.
  • a pharmaceutical composition comprising a protein, polypeptide, or peptide having Zlrr3 activity can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known to those in the art. See, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995).
  • molecules having Zlrr3 activity and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount.
  • a combination of a protein, polypeptide, or peptide having Zlrr3 activity and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • an agent having Zlrr3 activity when used to treat a tumor, then that agent is physiologically significant if it inhibits tumor growth, as indicated, for example, by a decrease in the number of tumor cells, decreased metastasis, a decrease in the size of a solid tumor, or increased necrosis of a tumor.
  • a pharmaceutical composition comprising molecules having Zlrr3 activity can be furnished in liquid form, or in solid form.
  • Liquid forms, including liposome-encapsulated formulations, are illustrated by injectable solutions and oral suspensions.
  • Exemplary solid forms include capsules, tablets, and controlled-release forms, such as a miniosmotic pump or an implant.
  • Other dosage forms can be devised by those skilled in the art, as shown, for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5 th Edition (Lea & Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19 th Edition (Mack Publishing Company 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).
  • Zlrr3 pharmaceutical compositions may be supplied as a kit comprising a container that comprises Zlrr3.
  • Zlrr3 can be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • Such a kit may further comprise written information on indications and usage of the pharmaceutical composition.
  • such information may include a statement that the Zlrr3 composition is contraindicated in patients with known hypersensitivity to Zlrr3.
  • the present invention includes the use of Zlrr3 nucleotide sequences to provide Zlrr3 to a subject in need of such treatment.
  • a therapeutic expression vector can be provided that inhibits Zlrr3 gene expression, such as an anti- sense molecule, a ribozyme, or an external guide sequence molecule.
  • an expression vector is constructed in which a nucleotide sequence encoding a Zlrr3 gene is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription.
  • a Zlrr3 gene can be delivered using recombinant viral vectors, including for example, adeno viral vectors (e.g., Kass-Eisler et ⁇ l., Proc. Nat 7 Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci. USA 97:215 (1994), Li et al, Hum. Gene Ther.
  • adeno viral vectors e.g., Kass-Eisler et ⁇ l., Proc. Nat 7 Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci. USA 97:215 (1994), Li et al, Hum. Gene Ther.
  • retroviruses e.g., Baba et al, J. Neurosurg 79:729 (1993), Ram et al, Cancer Res. 53:83 (1993), Takamiya et al, J. Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 55:962 (1993), Vile and Hart, Cancer Res. 55:3860 (1993), and Anderson et al, U.S. Patent No. 5,399,346).
  • the viral vector itself, or a viral particle which contains the viral vector may be utilized in the methods and compositions described below.
  • adenovirus a double-stranded DNA virus
  • the adenovirus system offers several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii) the ability to be grown to high-titer, (iii) the ability to infect a broad range of mammalian cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters.
  • adenoviruses can be administered by intravenous injection, because the viruses are stable in the bloodstream.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are inco ⁇ orated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell.
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. Although an adenoviral delivery system with an El gene deletion cannot replicate in the host cells, the host's tissue will express and process an encoded heterologous protein. Host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence.
  • adenoviral vectors containing various deletions of viral genes can be used 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 (1998); Raper et al, Human Gene Therapy 9:671 (1998)).
  • the deletion of E2b has also been reported to reduce immune responses (Amalfitano et al, J. Virol. 72:926 (1998)).
  • High titer stocks of recombinant viruses capable of expressing a therapeutic gene can be obtained from infected mammalian cells using standard methods.
  • recombinant HSV can be prepared in Vero cells, as described by Brandt et al, J. Gen. Virol. 72:2043 (1991), Herold et al, J. Gen. Virol. 75:1211 (1994), Visalli and Brandt, Virology 755:419 (1991), Grau et al, Invest. Ophthalmol Vis. Sci. 30:2474 (1989), Brandt et al, J. Virol. Meth. 36:209 (1992), and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).
  • an expression vector comprising a Zlrr3 gene can be introduced into a subject's cells by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al, Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al, Proc. Natl Acad. Sci. USA 55:8027 (1988)).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Liposomes can be used to direct transfection to particular cell types, which is 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 pu ⁇ ose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
  • Electroporation is another alternative mode of administration of a Zlrr3 nucleic acid molecules.
  • Aihara and Miyazaki Nature Biotechnology 16:867 (1998), have demonstrated the use of in vivo electroporation for gene transfer into muscle.
  • a therapeutic gene may encode a Zlrr3 anti-sense RNA that inhibits the expression of Zlrr3.
  • Suitable sequences for Zlrr3 anti-sense molecules can be derived from the nucleotide sequences of Zlrr3 disclosed herein.
  • an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
  • Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example, Draper and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337).
  • ribozymes include nucleotide sequences that bind with Zlrr3 mRNA.
  • expression vectors can be constructed in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of mRNA molecules that encode a Zlrr3 gene.
  • an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see, for example, Altaian et al, U.S. Patent No. 5,168,053, Yuan et al, Science 263:1269 (1994), Pace et al, international publication No. WO 96/18733, George et al, international publication No.
  • the external guide sequence comprises a ten to fifteen nucleotide sequence complementary to Zlrr3 mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine.
  • the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5 '-side of the base-paired region.
  • a composition comprising a therapeutic vector having a Zlrr3 nucleotide acid sequence, such as a recombinant virus
  • Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into a cavity that contains a tumor.
  • a composition comprising viral vectors, non-viral vectors, or a combination of viral and non- viral vectors of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby vectors or viruses are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition such as phosphate-buffered saline is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient subject.
  • suitable carriers are well-known to those in the art (see, for example, Remington 's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman 's the Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)).
  • a therapeutic gene expression vector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount.
  • a combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject.
  • that agent is physiologically significant if it inhibits tumor growth, as indicated, for example, by a decrease in the number of tumor cells, decreased metastasis, a decrease in the size of a solid tumor, or increased necrosis of a tumor.
  • the therapy is preferably somatic cell gene therapy.
  • the preferred treatment of a human with a therapeutic gene expression vector or a recombinant virus does not entail introducing into cells a nucleic acid molecule that can form part of a human germ line and be passed onto successive generations (i.e., human germ line gene therapy).
  • Transgenic mice can be engineered to over-express the Zlrr3 gene in all tissues or under the control of a tissue-specific or tissue-preferred regulatory element. These over-producers of Zlrr3 can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serve as models for human disease caused by excess Zlrr3. Transgenic mice that over-express Zlrr3 also provide model bioreactors for production of Zlrr3, such as soluble Zlrr3, in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses a Zlrr3 gene can begin with adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl, 2-4 months, (Taconic Farms)).
  • the donors are acclimated for one week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, MO) I. P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.
  • Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection, Copulation is confirmed by the presence of a vaginal plug the morning following mating.
  • Fertilized eggs are collected under a surgical scope.
  • the oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma).
  • Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (described, for example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated with 5% CO 2 , 5% O 2 , and 90% N 2 at 37°C.
  • the eggs are then stored in a 37°C/5% CO 2 incubator until microinjection.
  • plasmid DNA containing a Zlrr3 encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinjection.
  • the Zlrr3 encoding sequences can encode a polypeptide comprising amino acid resides 29 to 254 of SEQ ID NO:2. Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO -equilibrated mineral oil.
  • the DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei. Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue-culture dish with pre-gassed W640 medium for storage overnig toht in a 37°C/5% CO 2 incubator. The following day, two-cell embryos are transferred into pseudopregnant recipients.
  • the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
  • Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope.
  • a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
  • the reproductive organs are exteriorized onto a small surgical drape.
  • the fat pad is stretched out over the surgical drape, and a baby serrefme (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
  • a baby serrefme Robot, Rockville, MD
  • the pipette is transferred into the nick in the oviduct, and the embryos are blown in, allowing the first air bubble to escape the pipette.
  • the fat pad is gently pushed into the peritoneum, and the reproductive organs allowed to slide in.
  • the peritoneal wall is closed with one suture and the skin closed with a wound clip.
  • the mice recuperate on a 37°C slide warmer for a minimum of four hours.
  • the recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning.
  • the weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
  • Genomic DNA is prepared from the tail snips using, for example, a QIAGEN DNEASY kit following the manufacturer's instructions. Genomic DNA is analyzed by PCR using primers designed to amplify a Zlrr3 gene or a selectable marker gene that was introduced in the same plasmid. After animals are confirmed to be transgenic, they are back-crossed into an inbred strain by placing a transgenic female with a wild-type male, or a transgenic male with one or two wild-type female(s). As pups are born and weaned, the sexes are separated, and their tails snipped for genotyping.
  • a partial hepatectomy is performed.
  • a surgical prep is made of the upper abdomen directly below the zyphoid process.
  • a small 1.5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized.
  • a tie is made around the lower lobe securing it outside the body cavity.
  • An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed proximal to the first tie.
  • a distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish.
  • the excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice.
  • the surgical site is closed with suture and wound clips, and the animal's cage placed on a 37°C heating pad for 24 hours post operatively.
  • the animal is checked daily post operatively and the wound clips removed 7-10 days after surgery.
  • the expression level of Zlrr3 mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
  • Such transgenic mice provide useful models for diseases associated with a lack of Zlrr3.
  • Zlrr3 gene expression can be inhibited using anti-sense genes, ribozyme genes, or external guide sequence genes.
  • anti-sense genes ribozyme genes, or external guide sequence genes.
  • inhibitory sequences are targeted to Zlrr3 mRNA.
  • Methods for producing transgenic mice that have abnormally low expression of a particular gene are known to those in the art (see, for example, Wu et al. , “Gene Underexpression in Cultured Cells and Animals by Antisense DNA and RNA Strategies," in Methods in Gene Biotechnology, pages 205-224 (CRC Press 1997)).
  • An alternative approach to producing transgenic mice that have little or no Zlrr3 gene expression is to generate mice having at least one normal Zlrr3 allele replaced by a nonfunctional Zlrr3 gene.
  • One method of designing a nonfunctional Zlrr3 gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes Zlrr3. Standard methods for producing these so-called “knockout mice” are known to those skilled in the art (see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice," in Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et al. , “New Strategies for Gene Knockout,” in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)).

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Abstract

Des motifs riches en leucine apparaissent dans un nombre important de protéines remplissant des fonctions biologiques critiques, telles que l'adhésion cellulaire et la transduction de signaux. Zlrr3 est un nouveau membre de la superfamille humaine répétée riche en leucine, pouvant éventuellement jouer un rôle dans la différenciation et le développement neuraux.
PCT/US2000/000742 1999-01-13 2000-01-12 Zlrr3: proteine humaine repetee riche en leucine WO2000042184A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072961A2 (fr) * 2000-03-24 2001-10-04 Smithkline Beecham Corporation Nouveaux composes
EP3225633A1 (fr) * 2004-05-21 2017-10-04 The UAB Research Foundation Recepteurs de lymphocytes variables, polypeptides et acides nucleiques associes, et leurs utilisations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010518A1 (fr) * 1990-12-07 1992-06-25 Yale University Proteine slit purifiee et ses elements de sequence

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010518A1 (fr) * 1990-12-07 1992-06-25 Yale University Proteine slit purifiee et ses elements de sequence

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KAJAVA A V: "Structural diversity of leucine-rich repeat proteins.", JOURNAL OF MOLECULAR BIOLOGY APRIL 3, 1998, vol. 277, no. 3, 3 April 1998 (1998-04-03), pages 519 - 527, XP000907339, ISSN: 0022-2836 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072961A2 (fr) * 2000-03-24 2001-10-04 Smithkline Beecham Corporation Nouveaux composes
WO2001072961A3 (fr) * 2000-03-24 2002-06-20 Smithkline Beecham Corp Nouveaux composes
EP3225633A1 (fr) * 2004-05-21 2017-10-04 The UAB Research Foundation Recepteurs de lymphocytes variables, polypeptides et acides nucleiques associes, et leurs utilisations

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