WO2000059934A2 - Proteine specifique des testicules - Google Patents

Proteine specifique des testicules Download PDF

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Publication number
WO2000059934A2
WO2000059934A2 PCT/US2000/008836 US0008836W WO0059934A2 WO 2000059934 A2 WO2000059934 A2 WO 2000059934A2 US 0008836 W US0008836 W US 0008836W WO 0059934 A2 WO0059934 A2 WO 0059934A2
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zaiphalό
amino acid
nucleic acid
sequence
seq
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PCT/US2000/008836
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WO2000059934A3 (fr
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Darrell C. Conklin
Andrew L. Feldhaus
Susan D. Holderman
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Zymogenetics, Inc.
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Priority to AU41917/00A priority Critical patent/AU4191700A/en
Publication of WO2000059934A2 publication Critical patent/WO2000059934A2/fr
Publication of WO2000059934A3 publication Critical patent/WO2000059934A3/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

Definitions

  • the present invention relates generally to a new testis specific protein having diagnostic and therapeutic uses.
  • the present invention relates to a novel testis-specific protein, and to nucleic acid molecules encoding this protein.
  • Cancer is a complex disease, often having a mixed etiology. Although environmental factors appear to play a dominant role in the development of certain cancers, it is also clear that genetic factors determine cancer susceptibility (for a review, see Cornelisse and Devilee, Patient Education and Counseling 32:9 (1997), Brandt- Rauf and Pincus, Pharmacol. Ther. 77:135 (1998), and Jameson, "Oncogenes and Tumor Suppressor Genes," in Principles of Molecular Medicine, Jameson (ed.), pages 73- 82 (Humana Press, Inc. 1998)).
  • Genetic changes can affect many aspects of cellular function, such as an increased rate of cellular proliferation, resistance to apoptosis, altered tissue invasiveness, production of growth and angiogenic factors, and the ability to avoid immune defenses.
  • genetic alterations of oncogenes and tumor suppressor genes can be identified in a variety of human tumor tissues.
  • Tissue-specific markers can be used to enhance diagnosis, staging, and treatment of cancer (see, for example, Dean and Moul, Urol. Clin. North Am. 25:365 (1998)). Moreover, proteins that are preferentially expressed by one tissue can be used to detect metastases of tumors derived from that tissue. A need therefore exists for the identification of new tissue-specific marker genes.
  • the present invention provides a novel testis-specific protein, designated
  • Zaiphal ⁇ The present invention also provides Zaiphal ⁇ polypeptides and Zaiphal ⁇ fusion proteins, nucleic acid molecules encoding such polypeptides and proteins, and methods for using these polypeptides, proteins, and nucleic acid molecules.
  • a nucleic acid molecule containing a sequence that encodes the human Zaiphal ⁇ gene has the nucieotide sequence of SEQ ID NO: 1.
  • the encoded polypeptide has the following amino acid sequence: MAKGGEALPQ GSPAPVQDPH LIKVTVKTPK DKEDFSVTDT CTIQQLKEEI SQRFKAHPDQ LVLIFAGKIL KDPDSLAQCG VRDGLTVHLV IKRQHRAMGN ECPAASVPTQ GPSPGSLPQP SSIYPADGPP AFSLGLLTGL SRLGLAYRGF PDQPSSLMRQ HVSVPEFVTQ LIDDPFIPGL LSNTGLVRQL VLDNPHMQQL IQHNPEIGHI LNNPEIMRQT LEFLRNPAMM QEMIRSQDRV LSNLESIPGG YNVLCTMYTD IMDPMLNAVQ EQFGGNPFAT ATTDNATTTT SQPSRMENCD PLPNPWTSTH GGSGSRQGRQ DGD
  • the Zaiphal ⁇ gene has been mapped to human chromosome 11 at 1 lp.15. This locus is associated with various diseases, as described below. Northern analyses indicate that the Zaiphal ⁇ gene is strongly expressed in human testis, while there is little or no expression in other human tissues, including heart, lung, skeletal muscle, kidney, spleen, brain, placenta, liver, pancreas, thymus, prostate, ovary, small intestine, colon, peripheral blood lymphocytes, stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, and bone marrow.
  • Zaiphal ⁇ gene in amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe, hippocampus, medulla oblongata, occipital lobe, putamen, substantia nigra, temporal lobe, thalamus, subthalamic nucleus, aorta, bladder, uterus, pituitary gland, thyroid gland, salivary gland, mammary gland, appendix, fetal brain, fetal liver, fetal kidney, fetal heart, fetal spleen, fetal thymus, and fetal lung.
  • Zaiphal ⁇ RNA was present in murine testis, while there appeared to be little or no expression in the following murine tissues: heart, brain, spleen, lung, liver, skeletal muscle, kidney, eye, thyroid, pancreas, smooth muscle, ovary, submaxillary gland, uterus, and epididymis.
  • Ubiquitin is a 76 amino acid protein that exists in cells free or covalently conjugated to other proteins. Conjugation occurs between the C-terminus of ubiquitin and the lysine side chains of the target protein.
  • the number of ubiquitin molecules conjugated to a target protein is regulated by multiple conjugating and deconjugating enzymes (see, for example, Ciechanover and Schwartz, Proc. Nat'l Acad. Sci. 95:2121 (1998); D'Andrea and Pellman, Crit. Rev. Biochem. Mol. Biol. 33:331 (1998)). Many of the known functions of ubiquitin conjugation involve the targeted degradation of the modified protein.
  • the ubiquitin protein is highly conserved in a wide variety of organisms from which it has been cloned (Pfeifer et al., J. Cell. Sci. 106:545 (1993)). This high degree of conservation has allowed the cloning of several families of ubiquitin-like proteins.
  • One class of ubiquitin-like proteins consists solely of ubiquitin-like domains and is generally capable of conjugation to other proteins. These ubiquitin-like proteins appear to function as post-translational protein modifiers in a similar manner to ubiquitin (Rao-Naik et al., J. Biol. Chem. 275:34976 (1998); Whitby et al., J. Biol. Chem. 275:34983 (1998)).
  • a second class of ubiquitin-like proteins consists of very large proteins with limited similarities to a particular domain within ubiquitin (UBQ domain).
  • UBQ domain For example, the Parkin gene encodes a 465 amino acid protein that contains a UBQ domain near the amino terminus. Although the function of the parkin protein is unknown, mutations within the Parkin gene have been shown to result in juvenile parkinsonism (Kitada et al., Nature 392:605 (1998)).
  • the ubiquitin-associated domain is a 45 amino acid region predicted to have an a-helical structure and is present in a variety of proteins of the ubiquination pathway such as ubiquitin-carrier proteins, ubiquitin-protein ligases and ubiquitin carboxy-terminal hydrolases (Hofmann and Bucher, Trends Biochem. Sci. 27:172 (1996)).
  • the UBA domain is also found in several large proteins that possess UBQ domains.
  • One such protein that contains both UBQ and UBA domains is the yeast Dsk2 protein. Dsk2 is involved in spindle pole body duplication and thus, important for cell cycle progression (Biggins et al, J. Cell. Biol. 755:1331 (1986)).
  • PLIC-1 and PLIC-2 Two murine genes, PLIC-1 and PLIC-2, also contain UBQ and UBA domains and function to regulate the association between vimentin and integrin-associated protein at the plasma membrane (Wu et al, Molec. Cell 4:619 (1999)). Thus, although these proteins contain UBQ and UBA domains, they function outside of the ubiquitination pathway.
  • Zaiphal ⁇ contains a UBQ domain (Lys 23 to Asp 74 ).
  • a 93 amino acid region, designated as the "NP domain,” is present in Zaiphal ⁇ from Arg 188 to Thr 280 .
  • Zaiphal ⁇ also includes a UBA domain from Thr 605 to Ser 655 .
  • a novel motif resides within the Zaiphal ⁇ UBA domain from amino acid residue 619 to amino acid residue 648, which has the following sequence: QLEQL[R,S,N][S,A]MGF[L,I]NREANLQALIATGGD[V,I][D,N]AA, wherein acceptable amino acids for a given position are indicated within square brackets.
  • cyclin Al is expressed only in testes and cyclin A2 is expressed in a wide variety of cell types, both cyclins are expressed in different cell types within the spermatogenetic lineage. Cyclin Al expression is restricted to stages IX to XII (Ravnik and Wolgemuth, Dev. Biol. 207:408 (1999)) and is critical for the first meiotic division in spermatocytes (Kiu et al, Nature Genetics 20:311 (1998)).
  • Cyclin A2 is expressed during earlier stages of spermatogenesis and is absent from meiotic cells (Ravnik and Wolgemuth, Dev. Biol. 207:408 (1999)).
  • the Xenopus protein XDRPl interacts specifically with cyclin Al and
  • the present invention provides isolated polypeptides comprising an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to an amino acid sequence selected from the group consisting of amino acid residues 1 to 22 of SEQ ID NO:2, amino acid residues 75 to 187 of SEQ ID NO:2, amino acid residues 281 to 604 of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:2.
  • Certain of these 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.
  • An illustrative polypeptide is a polypeptide that comprises the amino acid sequence of SEQ ID NO:2.
  • Additional exemplary polypeptides include polypeptides that comprise an amino acid motif having the sequence QLEQL[R,S,N][S,A]MGF[L,I]NREANL QALIATGGDfV,I][D,N]AA, wherein the sequence is further defined by at least one condition selected from the group consisting of: (a) the sixth residue of the motif is R, (b) the twenty-seventh residue is V, (c) the twenty-eighth residue is D, and (d) the seven residue is S when the eleventh residue is L.
  • polypeptides include that comprise an amino acid sequence comprising at least 15, at least 20, at least 30, or at least 40 contiguous amino acid residues of the following regions of SEQ ID NO:2: amino acid residues 1 to 22, amino acid residues 75 to 187, and amino acid residues 281 to 604.
  • the present invention also includes polypeptides consisting of amino acid residues of the following regions of SEQ ID NO:2: amino acid residues 1 to 22, amino acid residues 75 to 187, and amino acid residues 281 to 604.
  • 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 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 further includes compositions comprising a carrier and a peptide, polypeptide, antibody, or anti-idiotype antibody described herein.
  • the present invention also provides isolated nucleic acid molecules that encode a Zaiphal ⁇ polypeptide, wherein the nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule comprising the nucieotide 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 consisting of the nucieotide sequence of SEQ ID NO: 1 , or the complement of SEQ ID NO: 1.
  • 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 nucieotide sequence of nucleotides 86 to 2050 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, avian, insect, mammalian, and plant cells.
  • Recombinant host cells comprising such expression vectors can be used to prepare Zaiphal ⁇ polypeptides by culturing such recombinant host cells that comprise the expression vector and that produce the Zaiphal ⁇ protein, and, optionally, isolating the Zaiphal ⁇ protein from the cultured recombinant host cells.
  • the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and at least one of such an expression vector or recombinant virus comprising such expression vectors.
  • the present invention also contemplates methods for detecting the presence of Zaiphal ⁇ RNA in a biological sample, comprising the steps of (a) contacting a Zaiphal ⁇ 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 nucieotide sequence comprising a portion of the nucieotide sequence 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 Zaiphal ⁇ RNA in the biological sample.
  • the present invention further provides methods for detecting the presence of Zaiphal ⁇ 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 can further comprise a detectable label selected from the group consisting of radioisotope, fluorescent label, chemiluminescent label, enzyme label, bioluminescent label, and colloidal gold.
  • the present invention also provides kits for performing these detection methods.
  • a kit for detection of Zaiphal ⁇ gene expression can 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 nucieotide sequence of nucleotides 86 to 2050 of SEQ ID NO:l, (b) a nucleic acid molecule comprising the complement of nucleotides 86 to 2050 of the nucieotide 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.
  • kits can 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 Zaiphal ⁇ protein can 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 further provides variant Zaiphal ⁇ polypeptides, wherein the amino acid sequence of the variant is characterized by at least one amino acid substitution within SEQ ID NO:2 selected from the group consisting of: (a) an aspartate for glutamate 613 , (b) an arginine for histidine 615 , (c) an isoleucine for valine 645 , (d) an isoleucine for valine 649 , and (e) an arginine for lysine 651 .
  • SEQ ID NO:2 selected from the group consisting of: (a) an aspartate for glutamate 613 , (b) an arginine for histidine 615 , (c) an isoleucine for valine 645 , (d) an isoleucine for valine 649 , and (e) an arginine for lysine 651 .
  • variant Zaiphal ⁇ polypeptides have an amino acid sequence that shares an identity with the amino acid sequence of SEQ ID NO:2 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO:2 is due to one or more conservative amino acid substitutions.
  • the present invention also includes isolated polypeptides consisting of an amino acid sequence of amino acid residues 1-655 of SEQ ID NO:2.
  • the present invention further provides fusion proteins a Zaiphal ⁇ polypeptide and an immunoglobulin moiety.
  • the immunoglobulin moiety may be an immunoglobulin heavy chain constant region, such as a human F c fragment.
  • the present invention further includes isolated nucleic acid molecules that encode such fusion proteins.
  • 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 nucieotide sequence and reverse orientation as compared to a reference nucieotide sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • sequence 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 nucieotide 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 nucieotide 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 nucieotide 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.
  • DSEs differentiation-specific elements
  • CREs cyclic AMP response elements
  • SREs serum response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O'Reilly et al, J. Biol. Chem. 257:19938 (1992)), AP2 (Ye et al, J. Biol. Chem. 269:2512% (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr. 5:253 (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. J.
  • a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter.
  • Repressible promoters are also known.
  • a “core promoter” contains essential nucieotide 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 nucieotide sequence that modulates the activity of a core promoter.
  • a regulatory element may contain a nucieotide 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.
  • the Zaiphal ⁇ regulatory element preferentially induces gene expression in testis, as opposed to heart, lung, skeletal muscle, kidney, spleen, brain, placenta, liver, pancreas, thymus, prostate, ovary, small intestine, colon, peripheral blood lymphocytes, stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, and bone marrow.
  • Heterologous DNA refers to a DNA molecule, or a population of
  • 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).
  • endogenous DNA DNA derived from the host cell species
  • exogenous DNA DNA derived from the host cell species
  • 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.
  • a "polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides.”
  • 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.
  • 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 nucieotide 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 Zaiphal ⁇ from an expression vector.
  • Zaiphal ⁇ can be produced by a cell that is a "natural source" of
  • “Integrative transformants” are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
  • a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucieotide sequences of at least two genes.
  • a fusion protein can comprise at least part of a Zaiphal ⁇ polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of Zaiphal ⁇ 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 nucieotide 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 nucieotide 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 nucieotide 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
  • 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.
  • splice variant is used herein to denote alternative forms of
  • RNA transcribed from a gene 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.
  • the term "immunomodulator” includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.
  • the term "complement/anti-complement pair” denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) 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. Where subsequent dissociation of the complement/anti-complement pair is desirable, 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-Zalphal ⁇ antibody, and thus, an anti-idiotype antibody mimics an epitope of Zaiphal ⁇ .
  • 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-Zalphal ⁇ monoclonal antibody fragment binds with an epitope of Zaiphal ⁇ .
  • 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 "therapeutic agent” is a molecule or atom, which is conjugated to an antibody moiety to produce a conjugate, which is useful for therapy.
  • therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
  • detectable label is a molecule or atom, which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis.
  • 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. 4:1015 (1985); Nilsson et al, Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al, Proc.
  • 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”).
  • tumor associated antigen is a protein normally not expressed, or expressed at lower levels, by a normal counterpart cell.
  • tumor associated antigens include alpha-fetoprotein, carcinoembryonic antigen, and Her-2/neu. Many other illustrations of tumor associated antigens are known to those of skill in the art. See, for example, Urban et al, Ann. Rev. Immunol. 10:6 ⁇ 1 (1992).
  • 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 Zaiphal ⁇ ” or a “Zaiphal ⁇ anti-sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the Zaiphal ⁇ gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the Zaiphal ⁇ gene.
  • a “ribozyme” is a nucleic acid molecule that contains a catalytic center.
  • 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 Zaiphal ⁇ 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 Zaiphal ⁇ 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 Zaiphal ⁇ genes are nucleic acid molecules that contain insertions or deletions of the nucieotide sequences described herein. A variant Zaiphal ⁇ gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucieotide sequence of SEQ ID NO:l, or its complement, under stringent conditions.
  • variant Zaiphal ⁇ 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 nucieotide sequences have "100% nucieotide sequence identity” if the nucieotide residues of the two nucieotide 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 can be functionally characterized the ability to bind specifically to an anti- Zalphal ⁇ 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 counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Parenters are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • a "functional fragment" of a Zaiphal ⁇ gene refers to a nucleic acid molecule that encodes a portion of a Zaiphal ⁇ polypeptide, which specifically binds with an anti-Zalphal ⁇ antibody.
  • a functional fragment of a Zaiphal ⁇ gene comprises a portion of the nucieotide sequence of SEQ ID NO:l, and encodes a polypeptide that binds with a Zalphal ⁇ -specific antibody.
  • Nucleic acid molecules encoding a human Z ⁇ lph ⁇ l ⁇ gene can be obtained by screening a human cDNA or genomic library using polynucleotide probes based upon SEQ ID NO:l. These techniques are standard and well-established. As an illustration, a nucleic acid molecule that encodes a Zaiphal ⁇ gene can be isolated from a cDNA library. In this case, the first step would be to prepare the cDNA library by isolating RNA from a tissue, such testicular tissue, using methods well- known to those of skill in the art.
  • RNA 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 al.
  • total RNA can be isolated from testicular 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'l Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at pages 4-11 to 4- 12).
  • 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). Moreover, commercially available kits can be used to synthesize double- stranded cDNA molecules. For example, such kits are available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, Wl) 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 Corp.) or other commercially available vectors.
  • Suitable cloning vectors also can be obtained from the American Type Culture Collection
  • 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
  • a human genomic library can be prepared by means well-known in the art
  • 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 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-
  • human genomic libraries can be obtained from commercial sources such as Research Genetics (Huntsville, AL) and the American Type Culture
  • a library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon S ⁇ Q ID NO:l, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).
  • Nucleic acid molecules that encode a human Zaiphal ⁇ gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucieotide sequences that are based upon the nucieotide sequences of the PCR
  • Zaiphal ⁇ gene as described herein.
  • General methods for screening libraries with PCR are provided by, for example, Yu et al. , "Use of the Polymerase Chain Reaction to
  • Anti-Zalphal ⁇ antibodies produced as described below, can also be used to isolate DNA sequences that encode human Zaiphal ⁇ genes from cDNA libraries.
  • the antibodies can be used to screen ⁇ gtl 1 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 Zaiphal ⁇ gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucieotide 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. 27:1131 (1993), Bambot et al, PCR Methods and Applications 2:266 (1993), Dillon et al., "Use of the Polymerase Chain Reaction for the Rapid Construction of Synthetic Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 263-268, (Humana Press, Inc. 1993), and Holowachuk et al, PCR Methods Appl 4:299 (1995)).
  • 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%.
  • Zaiphal ⁇ polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of a Zaiphal ⁇ gene.
  • this gene region is expected to provide for preferential expression in testicular tissue.
  • Promoter elements from a Zaiphal ⁇ gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, testicular tissue of transgenic animals or patients treated with gene therapy.
  • the identification of genomic fragments containing a Zaiphal ⁇ 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 Zaiphal ⁇ proteins by "gene activation," as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous Zaiphal ⁇ gene in a cell is altered by introducing into the Zaiphal ⁇ 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 Zaiphal ⁇ 5' non-coding sequence that permits homologous recombination of the construct with the endogenous Zaiphal ⁇ locus, whereby the sequences within the construct become operably linked with the endogenous Zaiphal ⁇ coding sequence.
  • an endogenous Zaiphal ⁇ 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 nucieotide sequence that encompasses all nucleic acid molecules that encode the Zaiphal ⁇ polypeptide of SEQ ID NO:2.
  • 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 Zaiphal ⁇ polypeptide-encoding nucleic acid molecules comprising nucieotide 86 to nucieotide 2050 of SEQ ID NO:l, and their RNA equivalents.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO:3 to denote degenerate nucieotide positions. "Resolutions” are the nucleotides denoted by a code letter. “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C. Table 1
  • degenerate codons used in SEQ ID NO:3, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • 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 2).
  • the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein 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 counterparts 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 Zaiphal ⁇ polypeptides from other mammalian species, including mouse, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zaiphal ⁇ can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a Zaiphal ⁇ cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zaiphal ⁇ 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 Zalphal ⁇ -encoding cDNA can 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 Zaiphal ⁇ sequences disclosed herein.
  • a 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 Zaiphal ⁇ polypeptide.
  • SEQ ID NO:l represents a single allele of human Zaiphal ⁇ , 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 nucieotide 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 Zaiphal ⁇ 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 nucieotide sequences disclosed herein.
  • such nucleic acid molecules can hybridize under stringent conditions to nucleic acid molecules comprising the nucieotide sequence of SEQ ID NO:l, or a sequence complementary thereto, under "stringent conditions.”
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • a nucleic acid molecule encoding a variant Zaiphal ⁇ polypeptide can be hybridized with a nucleic acid molecule having the nucieotide 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 lOOx
  • 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 Zaiphal ⁇ polypeptide remain hybridized with a nucleic acid molecule having the nucieotide sequence of SEQ ID NO:l (or its complement) following stringent washing conditions, 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 Zaiphal ⁇ polypeptide remain hybridized with a nucleic acid molecule having the nucieotide sequence of SEQ ID NO:l (or its complement) following highly stringent washing conditions, 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 Zaiphal ⁇ 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 at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the sequences shown in SEQ ID NO:2, or their orthologs.
  • Zaiphal ⁇ 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 Zaiphal ⁇ variants include nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucieotide sequence of SEQ ID NO:l (or its complement) following stringent washing conditions, 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 at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • Zaiphal ⁇ variants can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule having the nucieotide sequence of SEQ ID NO:l (or its complement) following highly stringent washing conditions, 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 at least 70%, at least 80%, at least 90%, at least 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.
  • 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, SIAMJ. 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 four to six.
  • 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 Zaiphal ⁇ amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in a Zaiphal ⁇ amino acid sequence, a sulfur- containing amino acid is substituted for a sulfur-containing amino acid in a Zaiphal ⁇ amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy- containing amino acid in a Zaiphal ⁇ amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in a Zaiphal ⁇ amino acid sequence, a basic amino acid is substituted for a basic amino acid in a Zaiphal ⁇ amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino
  • 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.
  • variant Zaiphal ⁇ polypeptides that have an amino acid sequence that differs from SEQ ID NO:2 can be obtained by substituting aspartate for glutamate 613 , arginine for histidine 615 , isoleucine for valine 645 , isoleucine for valine 649 , or arginine for lysine 651 . Additional variants can be obtained by producing polypeptides having two or more of these amino acid substitutions.
  • 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. Nat'l Acad. Sci. USA 5P:10915 (1992)).
  • 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.
  • conservative amino acid substitution preferably refers to a substitution represented by a BLOSUM62 value of greater than -1.
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Zaiphal ⁇ are characterized by having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the corresponding amino acid sequence (i.e., SEQ ID NO:2), wherein the variation in amino acid sequence is due to one or more conservative amino acid substitutions.
  • Zaiphal ⁇ 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 Zaiphal ⁇ polypeptide can be identified by the ability to specifically bind anti-Zalphal ⁇ 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- s-S-methylproline, 2,4-methanoproline, c/.s-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ //o-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 incorporated into the protein in place of its natural counterpart. See, Koide et al, Biochem. 55:7470 (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)).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zaiphal ⁇ 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: ⁇ 08 ⁇ (1989), Bass et al, Proc. Nat'l 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)).
  • Sequence analysis can identify motifs that reside within Zaiphal ⁇ polypeptides.
  • sequence analysis revealed that the following novel motif resides in the carboxy terminus of Zaiphal ⁇ from amino acid residue 619 to amino acid residue 648: QLEQL[R,S,N][S,A]MGF[L,I]NREANLQALIA TGGD[V,I][D,N]AA, wherein acceptable amino acids for a given position are indicated within square brackets.
  • This motif is also found in the carboxy terminus of a protein (amino acid residues 369 to 398), expressed in human fetal brain, which contains a potential nuclear targeting signal (Ueki et al, Nature Biotechnology 75:1338 (1998); GenBank Accession No.
  • Zaiphal ⁇ can be distinguished from these polypeptides when the motif is further defined by at least one of the following conditions: (1) the sixth residue of the motif is R, (2) the twenty- seventh residue is V, (3) the twenty-eighth residue is D, or (4) the seven residue is S and the eleventh residue is L.
  • the present invention includes polypeptides that comprise an amino acid motif having the sequence QLEQL[R,S,N][S,A]MGF[L,I]NREANLQALIATGGD[V,I][D,N]AA, wherein the sequence is further defined by at least one of the following conditions: the sixth residue of the motif is R, the twenty-seventh residue is V, the twenty-eighth residue is D, or the seven residue is S when the eleventh residue is L.
  • Zaiphal ⁇ 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 photoaffmity 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, Zaiphal ⁇ labeled with biotin or FITC can be used for expression cloning of Zaiphal ⁇ receptors.
  • variants of the disclosed Zaiphal ⁇ nucieotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA 91:10741 (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-Zalphal ⁇ 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 Zaiphal ⁇ 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 Zaiphal ⁇ polypeptide.
  • DNA molecules having the nucieotide sequence of SEQ ID NO:l can be digested with Bal31 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-Zalphal ⁇ antibodies.
  • oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of a Zaiphal ⁇ gene can be synthesized using the polymerase chain reaction.
  • Zaiphal ⁇ gene that has amino acid changes, compared with the amino acid sequence of SEQ ID NO:2.
  • a variant Zaiphal ⁇ gene can be identified on the basis of structure by determining the level of identity with nucieotide and amino acid sequence of SEQ ID NO: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 Zaiphal ⁇ gene can hybridize to a nucleic acid molecule having the nucieotide sequence of SEQ ID NO:l, as discussed above.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zaiphal ⁇ 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. Nat'l 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 Zaiphal ⁇ 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). 5. Production ofZalpha16 Fusion Proteins and Conjugates
  • Fusion proteins of Zaiphal ⁇ can be used to express Zaiphal ⁇ in a recombinant host, and to isolate expressed Zaiphal ⁇ . As described below, particular Zaiphal ⁇ fusion proteins also have uses in diagnosis and therapy.
  • One type of fusion protein comprises a peptide that guides a Zaiphal ⁇ polypeptide from a recombinant host cell.
  • a secretory signal sequence also known as a signal peptide, a leader sequence, prepro sequence or pre sequence
  • Zaiphal ⁇ expression vector is provided in the Zaiphal ⁇ expression vector.
  • secretory signal sequence may be derived from Zaiphal ⁇
  • 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 Zalphal ⁇ -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 nucieotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucieotide sequence of interest (see, e.g., Welch et ⁇ l, U.S. Patent No. 5,037,743; Holland et ⁇ l, 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 MF ⁇ l gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene). See, for example, Romanos et ⁇ l. , "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).
  • Zaiphal ⁇ can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide.
  • Glutathione S-transferease fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • a Zaiphal ⁇ 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), e-mye 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. 329:2X5 (1996), Morganti et al, Biotechnol Appl Biochem. 23:61 (1996), and Zheng et al, Gene 755:55 (1997). Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO).
  • fusion protein comprises a Zaiphal ⁇ polypeptide and an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two or three constant region domains and a hinge region but lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • F c fragment an immunoglobulin heavy chain constant region
  • Chang et al, U.S. Patent No. 5,723,125 describe 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.
  • An example of 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 Zaiphal ⁇ fusion protein that comprises a Zaiphal ⁇ moiety and a human Fc fragment, wherein the C-terminus of the Zaiphal ⁇ 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 Zaiphal ⁇ moiety can be a Zaiphal ⁇ molecule or a fragment thereof.
  • a Zaiphal ⁇ fusion protein comprises an IgG sequence, a Zaiphal ⁇ moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that is covalently joined to the aminoterminal of the Zaiphal ⁇ 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 Zaiphal ⁇ moiety displays a Zaiphal ⁇ activity, as described herein, such as the ability to bind with a Zaiphal ⁇ receptor.
  • Fusion proteins comprising a Zaiphal ⁇ moiety and an Fc moiety can be used, for example, as an in vitro assay tool.
  • the presence of an Zaiphal ⁇ receptor in a biological sample can be detected using a Zalphal ⁇ -immunoglobulin fusion protein, in which the Zaiphal ⁇ 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 Zaiphal ⁇ to its receptor. 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.
  • 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 16-19 to 16- 25.
  • the present invention also contemplates chemically modified Zaiphal ⁇ compositions, in which a Zaiphal ⁇ polypeptide is linked with a polymer.
  • the polymer is water soluble so that the Zaiphal ⁇ conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • 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 Zaiphal ⁇ conjugates.
  • Zaiphal ⁇ 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, bt-.-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 Zaiphal ⁇ conjugate can also comprise a
  • Zaiphal ⁇ conjugate comprises a Zaiphal ⁇ moiety and a polyalkyl oxide moiety attached to the N-terminus of the Zaiphal ⁇ moiety.
  • PEG is one suitable polyalkyl oxide.
  • Zaiphal ⁇ can be modified with PEG, a process known as "PEGylation.” PEGylation of Zaiphal ⁇ 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. 27:290 (1994), and Francis et al, Int J Hematol 55:1 (1998)).
  • PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule.
  • Zaiphal ⁇ 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 Zaiphal ⁇ polypeptide.
  • An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide.
  • acylation includes the following types of linkages between Zaiphal ⁇ and a water soluble polymer: amide, carbamate, urethane, and the like.
  • Methods for preparing PEGylated Zaiphal ⁇ by acylation will typically comprise the steps of (a) reacting a Zaiphal ⁇ polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to Zaiphal ⁇ , 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:Zalphal ⁇ , the greater the percentage of polyPEGylated Zaiphal ⁇ product.
  • the product of PEGylation by acylation is typically a polyPEGylated
  • Zaiphal ⁇ product wherein the lysine ⁇ -amino groups are PEGylated via an acyl linking group.
  • An example of a connecting linkage is an amide.
  • the resulting Zaiphal ⁇ 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 Zaiphal ⁇ 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 Zaiphal ⁇ in the presence of a reducing agent.
  • PEG groups are preferably attached to the polypeptide via a -CH 2 -NH 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.
  • a water-soluble polymer that contains a reactive group such as an aldehyde
  • the present invention provides a substantially homogenous preparation of Zaiphal ⁇ monopolymer conjugates.
  • Reductive alkylation to produce a substantially homogenous population of monopolymer Zaiphal ⁇ conjugate molecule can comprise the steps of: (a) reacting a Zaiphal ⁇ 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 Zaiphal ⁇ , and (b) obtaining the reaction product(s).
  • the reducing agent used for reductive alkylation should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation.
  • Preferred 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 Zaiphal ⁇ .
  • 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 :Zalpha 16 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 the higher the molecular weight of the polymer, the fewer number of polymer molecules that may be attached to the protein. For PEGylation reactions, the typical molecular weight 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 Zaiphal ⁇ will generally be in the range of 1:1 to 100:1. Typically, the molar ratio of water-soluble polymer to Zaiphal ⁇ will be 1:1 to 20:1 for polyPEGylation, and 1 :1 to 5:1 for monoPEGylation.
  • 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 nucieotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell.
  • a Zaiphal ⁇ expression vector may comprise a Z ⁇ lph ⁇ l ⁇ gene and a secretory sequence derived from any secreted gene.
  • Zaiphal ⁇ 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 ⁇ l., 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 Zaiphal ⁇ gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al, Mol Cell. Biol. 70:4529 (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 are 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.
  • Zaiphal ⁇ polypeptides can also be produced by cultured mammalian cells using a viral delivery system.
  • viruses for this purpose include adenovirus, herpesvirus, 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)).
  • Zaiphal ⁇ can 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 Zaiphal ⁇ genes into insect cells.
  • Suitable expression vectors are based upon the Autographa californica 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 (ie-1) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter.
  • hsp Drosophila heat shock protein
  • ie-1 Autographa californica nuclear polyhedrosis virus immediate-early gene promoter
  • baculovirus plO promoter the Drosophila metallothionein promoter.
  • a second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, et al, J. Virol. 57:4566 (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 Zaiphal ⁇ 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 Zaiphal ⁇ polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al, Proc. Nat'l Acad. Sci. 52:7952 (1985)).
  • a transfer vector containing a Zaiphal ⁇ 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 Pcor, p ⁇ .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. 270:1543 (1995).
  • a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed, which replace the native Zaiphal ⁇ secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from ⁇ cdysteroid Glucosyltransferase ( ⁇ GT), honey bee Melittin (Invitrogen Corporation; Carlsbad, CA), or baculovirus gp67 (PharMingen: San Diego, CA) can be used in constructs to replace the native Zaiphal ⁇ secretory signal sequence.
  • the recombinant virus or bacmid is used to transfect host cells.
  • suitable insect host cells include cell lines derived from IPLB-5/ 21, a Spodoptera frugiperda pupal ovarian cell line, such as Sfl (ATCC CRL 1711), SJ21A ⁇ , and S21 (Invitrogen Corporation; 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).
  • Sfl ATCC CRL 1711
  • SJ21A ⁇ S21
  • S21 Invitrogen Corporation
  • 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
  • Fungal cells including yeast cells, can also be used to express the genes described herein.
  • yeast species of particular interest in this regard include
  • yeast Suitable promoters for expression in yeast include promoters from GALl (galactose), PGK (phosphogly cerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase),
  • HIS4 histidinol dehydrogenase
  • yeast cloning vectors include YIp-based vectors, such as
  • YIp5 YRp vectors, such as YRp 17, YEp vectors such as YEp 13 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,
  • 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).
  • Saccharomyces cerevisiae is the EOE7 vector system disclosed by Kawasaki et al.
  • 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 752:3459 (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 are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a E. methanolica gene, such as a E. methanolica alcohol utilization gene (AUG1 or AUG2).
  • 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 E. 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.
  • E. 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.
  • AUG2 methanol utilization genes
  • host cells deficient in vacuolar protease genes PEP4 and PRB1 are suitable. 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 70:268 (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).
  • Zaiphal ⁇ genes can be expressed in prokaryotic host cells.
  • Suitable promoters that can be used to express Zaiphal ⁇ polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp ⁇ 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 transferase 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. ( 1995J .
  • Illustrative prokaryotic hosts include E. coli and Bacillus subtilus. Suitable strains of E. coli include BL21(D ⁇ 3), 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, Ml 120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)).
  • the polypeptide When expressing a Zaiphal ⁇ 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, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Standard methods for introducing expression vectors into bacterial, yeast, insect, and plant cells are provided, for example, by Ausubel (1995).
  • General methods for expressing and recovering foreign protein produced by a mammalian cell system are provided by, for example, Etcheverry, "Expression of Engineered Proteins in Mammalian Cell Culture,” in Protein Engineering: Principles and Practice, Cleland et al (eds.), pages 163 (Wiley-Liss, Inc. 1996).
  • Standard techniques for recovering protein produced by a bacterial system is provided by, for example, Grisshammer et al. , "Purification of over-produced proteins from E. coli cells," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
  • 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. Within certain embodiments of the invention, the polypeptides comprise 20, 30, 40, 50, 75, or more contiguous residues of SEQ ID NO:2.
  • the present invention includes at least 15, at least 20, at least 30, or at least 40 contiguous amino acid residues of the following regions of SEQ ID NO:2: amino acid residues 1 to 22, amino acid residues 75 to 187, and amino acid residues 281 to 604. Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
  • 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. Examples of carriers include water, buffer solution, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.
  • 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.
  • a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Fractionation and/or conventional purification methods can be used to obtain preparations of Zaiphal ⁇ purified from natural sources (e.g., testicular tissue), and recombinant Zaiphal ⁇ polypeptides and fusion Zaiphal ⁇ polypeptides purified from recombinant host cells.
  • natural sources e.g., testicular tissue
  • recombinant Zaiphal ⁇ polypeptides and fusion Zaiphal ⁇ polypeptides purified from recombinant host cells e.g., testicular tissue
  • ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl- Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica- based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996).
  • Zaiphal ⁇ isolation and purification can be devised by those of skill in the art.
  • anti-Zalphal ⁇ antibodies obtained as described below, can be used to isolate large quantities of protein by immunoaffinity purification.
  • methods for binding ligands, such as Zaiphal ⁇ , 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 adsorption (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. 5:1 (1985)). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • IMAC immobilized metal ion adsorption
  • Zaiphal ⁇ 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.
  • Zalpha16 Analogs and the Zalpha16 Receptor 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.
  • Zaiphal ⁇ analogs are variants having an amino acid sequence that is a mutation of the amino acid sequence disclosed herein.
  • Another general class of Zaiphal ⁇ 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 705:420 (1996)). Since the variable domains of anti- idiotype Zaiphal ⁇ antibodies mimic Zaiphal ⁇ , these domains can provide either Zaiphal ⁇ agonist or antagonist activity.
  • Lim and Langer J Interferon Res. 75:295 (1993), describe anti-idiotypic interferon- ⁇ antibodies that have the properties of either interferon- ⁇ agonists or antagonists.
  • Zaiphal ⁇ antagonists are useful as research reagents for characterizing sites of interaction between Zaiphal ⁇ and its receptor.
  • pharmaceutical compositions comprising Zaiphal ⁇ antagonists can be used to inhibit Zaiphal ⁇ activity.
  • the activity of Zaiphal ⁇ 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 Corp. (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.
  • microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells.
  • the microphysiometer directly measures cellular responses to various stimuli, including Zaiphal ⁇ , its agonists, or antagonists.
  • the microphysiometer is used to measure responses of a Zaiphal ⁇ responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to Zaiphal ⁇ polypeptide.
  • Zaiphal ⁇ responsive eukaryotic cells comprise cells into which a receptor for Zaiphal ⁇ has been transfected to create a cell that is responsive to Zaiphal ⁇ , or cells that are naturally responsive to Zaiphal ⁇ .
  • Zaiphal ⁇ modulated cellular responses are measured by a change (e.g., an increase or decrease in extracellular acidification) in the response of cells exposed to Zaiphal ⁇ , compared with control cells that have not been exposed to Zaiphal ⁇ .
  • a microphysiometer can be used to identify cells, tissues, or cell lines, which respond to a Zaiphal ⁇ stimulated pathway, and which express a functional Zaiphal ⁇ receptor.
  • cells that express a functional Zaiphal ⁇ receptor can be identified by (a) providing test cells, (b) incubating a first portion of the test cells in the absence of Zaiphal ⁇ , (c) incubating a second portion of the test cells in the presence of Zaiphal ⁇ , 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 Zaiphal ⁇ receptor.
  • a change e.g., an increase or decrease in extracellular acidification rate, as measured by a microphysiometer
  • An additional negative control may be included in which a portion of the test cells is incubated with Zaiphal ⁇ and an anti-Zalphal ⁇ antibody to inhibit the binding of Zaiphal ⁇ with its cognate receptor.
  • the microphysiometer also provides one means to identify Zaiphal ⁇ agonists.
  • agonists of Zaiphal ⁇ can be identified by a method, comprising the steps of (a) providing cells responsive to Zaiphal ⁇ , (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 Zaiphal ⁇ 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 Zalphal 6 and in the absence of a test compound can be used as a positive control for the Zaiphal ⁇ responsive cells, and as a control to compare the agonist activity of a test compound with that of Zaiphal ⁇ .
  • An additional control may be included in which a portion of the cells is incubated with a test compound (or Zaiphal ⁇ ) and an anti- Zalphal ⁇ antibody to inhibit the binding of the test compound (or Zaiphal ⁇ ) with the Zaiphal ⁇ receptor.
  • the microphysiometer also provides a means to identify Zaiphal ⁇ antagonists.
  • Zaiphal ⁇ antagonists can be identified by a method, comprising the steps of (a) providing cells responsive to Zaiphal ⁇ , (b) incubating a first portion of the cells in the presence of Zaiphal ⁇ and in the absence of a test compound, (c) incubating a second portion of the cells in the presence of both Zaiphal ⁇ 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 Zaiphal ⁇ antagonist.
  • An illustrative change in cellular response is a measurable change extracellular acidification rate, as measured by a microphysiometer.
  • Zaiphal ⁇ , its analogs, and anti-iodiotype Zaiphal ⁇ antibodies can be used to identify and to isolate Zaiphal ⁇ 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 Zaiphal ⁇ polypeptides can also be used to identify or to localize Zaiphal ⁇ receptors in a biological sample (see, for example, Deutscher (ed.), Methods in Enzymol, vol.
  • Antibodies to Zaiphal ⁇ can be obtained, for example, using the product of a Zaiphal ⁇ expression vector or Zaiphal ⁇ isolated from a natural source as an antigen. Particularly useful anti-Zalphal ⁇ antibodies "bind specifically" with Zaiphal ⁇ . Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to Zaiphal ⁇ with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to Zaiphal ⁇ .
  • antibodies specifically bind if they bind to a Zaiphal ⁇ polypeptide, peptide or epitope with a binding affinity (K ) of 10 6 M “1 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.
  • K 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 Ac ⁇ d Sci. 51:660 (1949)).
  • antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zaiphal ⁇ , but not known polypeptides using a standard Western blot analysis.
  • Suitable antibodies of the present invention include antibodies that bind the C-terminus motif of Zaiphal ⁇ (i.e., at amino acid residues 619 to 648), as well as antibodies that bind a region of Zaiphal ⁇ other than amino acid residues 619 to 648.
  • Other suitable antibodies of the present invention bind with one of the following regions of SEQ ID NO:2: amino acid residues 1 to 22, amino acid residues 75 to 187, and amino acid residues 281 to 604.
  • Anti-Zalphal ⁇ antibodies can be produced using antigenic Zaiphal ⁇ epitope-bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, 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 Zaiphal ⁇ .
  • 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.
  • Zaiphal ⁇ potential antigenic sites in Zaiphal ⁇ were identified using the Jameson- Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), as implemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison, Wl). Default parameters were used in this analysis.
  • a surface contour value designated as the "antigenic index.”
  • a peak broadening function was applied to the antigenic index, which broadens major surface peaks by adding 20, 40, 60, or 80% of the respective peak value to account for additional free energy derived from the mobility of surface regions relative to interior regions. This calculation was not applied, however, to any major peak that resides in a helical region, since helical regions tend to be less flexible.
  • amino acids 1 to 131 amino acids 1 to 131
  • amino acids 149 to 158 amino acids 292 to 303
  • amino acids 315 to 332 amino acids 315 to 332
  • amino acids 369 to 393 amino acids 417 to 436
  • amino acids 560 to 577 amino acids 560 to 577.
  • the present invention contemplates the use of any one of antigenic peptides 1 to 7 to generate antibodies to Zaiphal ⁇ .
  • the present invention also contemplates polypeptides comprising at least one of antigenic peptides 1 to 7.
  • Polyclonal antibodies to recombinant Zaiphal ⁇ protein or to Zaiphal ⁇ isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al, "Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al, "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995).
  • the immunogenicity of a Zaiphal ⁇ 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 Zaiphal ⁇ 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
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • polyclonal antibodies are typically raised in animals such as horses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, or sheep
  • an anti- Zaiphal ⁇ 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
  • monoclonal anti-Zalphal ⁇ 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 Zaiphal ⁇ 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-Zalphal ⁇ 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 555:856 (1994), and Taylor et al, Int. Immun. 5:579 (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- Zalphal ⁇ antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • 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.
  • 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.
  • 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. 12:431 (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 Zaiphal ⁇ polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zaiphal ⁇ protein or peptide).
  • Genes encoding polypeptides having potential Zaiphal ⁇ polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucieotide 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 Zaiphal ⁇ sequences disclosed herein to identify proteins, which bind to Zaiphal ⁇ .
  • CDR peptides (“minimal recognition units") can be obtained by constmcting 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-Zalphal ⁇ antibody may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 55:3833 (1989).
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-Zalphal ⁇ 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-Zalphal ⁇ 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 Z ⁇ lph ⁇ l ⁇ gene in a biological sample.
  • Certain probe molecules include double- stranded nucleic acid molecules comprising the nucieotide sequence of SEQ ID NO:l, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucieotide sequence of SEQ ID NO:l, or a portion thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • the term "portion" refers to at least eight nucleotides to at least 20 or more nucleotides.
  • Certain probes bind with regions of the Z ⁇ lph ⁇ l ⁇ gene that have a low sequence similarity to comparable regions in other proteins.
  • suitable probes can comprise a portion of the following regions of SEQ ID NO:l: nucleotides 86 to 151, nucleotides 308 to 646, nucleotides 926 to 1897, and the like. Those of skill in the art can determine additional suitable regions of the nucieotide sequence disclosed herein. In a basic assay, a single-stranded probe molecule is incubated with
  • RNA isolated from a biological sample, under conditions of temperature and ionic strength that promote base pairing between the probe and target Zaiphal ⁇ RNA species.
  • 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 al. (eds.), "Analysis of Gene Expression at the RNA
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S.
  • Zaiphal ⁇ 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.
  • Zaiphal ⁇ 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
  • PCR polymerase chain reaction
  • Certain PCR primers are designed to amplify a portion of the Zaiphal ⁇ gene that has a low sequence similarity to a comparable region in other proteins.
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with Zaiphal ⁇ 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 gunadinium-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 Zaiphal ⁇ anti-sense oligomers.
  • Oligo- dT primers offer the advantage that various mRNA nucieotide sequences are amplified that can provide control target sequences.
  • Zaiphal ⁇ 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 Zaiphal ⁇ 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
  • Zaiphal ⁇ probes and primers can also be used to detect and to localize Zaiphal ⁇ 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.
  • Zaiphal ⁇ nucieotide sequences are used to detect the metastasis of testicular tumors to other tissues.
  • Suitable test samples include blood, urine, saliva, tissue biopsy, and autopsy material.
  • nucleic acid molecules comprising Zaiphal ⁇ nucieotide sequences can also be used to determine whether a subject's chromosomes contain a mutation in the Zaiphal ⁇ gene, which resides at chromosome l lpl5.
  • Detectable chromosomal aberrations at the Zaiphal ⁇ gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • alterations in the l ip 15 region are associated with Beckwith-Wiedemann syndrome, arthrogryposis multiplex congenita, Jansky- Bielschowsky disease, and insulin-dependent diabetes mellitus (see, for example, Koufos et al, Am. J. Hum. Genet. 44:711 (1989); Davies et al, Nature 577:130 (1994); Krakowiak et al, Am. J. Hwm. Genet. 60:426 (1997); Sharp et al, Hum. Molec. Genet. 6:591 (1997)).
  • Zaiphal ⁇ locus Aberrations associated with the Zaiphal ⁇ locus can be detected using nucleic acid molecules of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 705:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • molecular genetic techniques such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluor
  • RNA is isolated from a biological sample, and used to synthesize cDNA. PCR is then used to amplify the Zaiphal ⁇ 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 9.11.1 - 9.11.18 (John Wiley & Sons 1998).
  • kits for performing a diagnostic assay for Zaiphal ⁇ gene expression or to detect mutations in the Zaiphal ⁇ gene comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucieotide sequence of SEQ ID NO:l, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucieotide 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 Zaiphal ⁇ probe or primer.
  • the kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zaiphal ⁇ 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 Zaiphal ⁇ probes and primers are used to detect Zaiphal ⁇ gene expression.
  • written instructions may state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes Zaiphal ⁇ , or a nucleic acid molecule having a nucieotide sequence that is complementary to a Zalphal 6-encoding nucieotide 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-Zalphal ⁇ antibodies to screen biological samples in vitro for the presence of Zaiphal ⁇ .
  • anti-Zalphal ⁇ antibodies are used in liquid phase.
  • the presence of Zaiphal ⁇ in a biological sample can be tested by mixing the biological sample with a trace amount of labeled Zaiphal ⁇ and an anti-Zalphal ⁇ antibody under conditions that promote binding between Zaiphal ⁇ and its antibody.
  • Complexes of Zaiphal ⁇ and anti- Zaiphal ⁇ 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 Zaiphal ⁇ in the biological sample will be inversely proportional to the amount of labeled Zaiphal ⁇ bound to the antibody and directly related to the amount of free labeled Zalphal 6.
  • in vitro assays can be performed in which anti- Zaiphal ⁇ 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.
  • polymer such as aminodextran
  • anti-Zalphal ⁇ antibodies can be used to detect Zaiphal ⁇ in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of Zaiphal ⁇ and to determine the distribution of Zaiphal ⁇ in the examined tissue.
  • General immunochemistry techniques are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application,” in Mammalian Development: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular Biology, Vol 10: Immunochemical Protocols (The Humana Press, Inc. 1992)).
  • Immunochemical detection can be performed by contacting a biological sample with an anti-Zalphal ⁇ 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-Zalphal ⁇ antibody.
  • the anti-Zalphal ⁇ 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-Zalphal ⁇ antibody can be conjugated with a detectable label to form an anti-Zalphal ⁇ immunoconjugate.
  • 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 purpose of the present invention are 3 H, 125 1, 131 1, 35 S and 14 C.
  • Anti-Zalphal ⁇ 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, rhoda- mine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • anti-Zalphal ⁇ 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.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • a bioluminescent compound can be used to label anti-Zalphal ⁇ 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-Zalphal ⁇ immunoconjugates can be detectably labeled by linking an anti-Zalphal ⁇ 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 ⁇ - galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • the convenience and versatility of immunochemical detection can be enhanced by using anti-Zalphal ⁇ 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). Methods for performing immunoassays are well-established.
  • biotin- or FITC-labeled Zaiphal ⁇ can be used to identify cells that bind Zaiphal ⁇ . Such can binding can be detected, for example, using flow cytometry.
  • anti-Zalphal ⁇ antibodies are used to detect the metastasis of testicular tumors to other tissues.
  • Illustrative test samples include blood, urine, saliva, tissue biopsy, and autopsy material.
  • kits for performing an immunological diagnostic assay for Zaiphal ⁇ gene expression comprise at least one container comprising an anti-Zalphal ⁇ antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of Zaiphal ⁇ antibody or antibody fragments.
  • 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 Zaiphal ⁇ antibodies or antibody fragments are used to detect Zaiphal ⁇ protein.
  • written instructions may state that the enclosed antibody or antibody fragment can be used to detect Zaiphal ⁇ .
  • 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 includes the use of proteins, polypeptides, and peptides having Zaiphal ⁇ activity (such as Zaiphal ⁇ polypeptides, Zaiphal ⁇ analogs, and Zaiphal ⁇ fusion proteins) to a subject who lacks an adequate amount of this polypeptide.
  • Zaiphal ⁇ antagonists e.g., anti-idiotype antibodies
  • the dosage of administered Zaiphal ⁇ 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 Zaiphal ⁇ , 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 Zaiphal ⁇ 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.
  • Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous.
  • Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid- based systems (see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).
  • the feasibility of an intranasal delivery is exemplified by such a mode of insulin administration (see, for example, Hinchclif e and Ilium, Adv. Drug Deliv. Rev. 55:199 (1999)).
  • Dry or liquid particles comprising Zaiphal ⁇ can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH 75:343 (1998); Patton et al, Adv. Drug Deliv. Rev. 35:235 (1999)).
  • This approach is illustrated by the AERX diabetes management system, which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs.
  • proteins as large as 48,000 kDa have been delivered across skin at therapeutic concentrations with the aid of low-frequency ultrasound, which illustrates the feasibility of trascutaneous administration (Mitragotri et al, Science 269:850 (1995)).
  • Transdermal delivery using electroporation provides another means to administer a molecule having Zaiphal ⁇ activity (Potts et al, Pharm. Biotechnol 10:213 (1997)).
  • a pharmaceutical composition comprising a protein, polypeptide, or peptide having Zaiphal ⁇ 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 Zaiphal ⁇ 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 Zaiphal ⁇ 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.
  • a pharmaceutical composition comprising Zaiphal ⁇ (or Zaiphal ⁇ analog or fusion protein) can be furnished in liquid form, in an aerosol, or in solid form. Liquid forms, are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al, Pharm.
  • Liposomes provide one means to deliver therapeutic polypeptides to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation, or intranasal administration.
  • Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol Infect. Dis.
  • Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
  • a variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy et al, Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al, American J. Hosp. Pharm. 46:1576 (1989)). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.
  • Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent.
  • an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof et al, Ann. N Y. Acad. Sci. 446:368 (1985)).
  • small liposomes (0.1 to 1.0 ⁇ m) are typically taken up by cells of the reticuloendothelial system, located principally in the liver and spleen, whereas liposomes larger than 3.0 ⁇ m are deposited in the lung. This preferential uptake of smaller liposomes by the cells of the reticuloendothelial system has been used to deliver chemotherapeutic agents to macrophages and to tumors of the liver.
  • the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means (Claassen et al, Biochim. Biophys. Acta 502:428 (1984)).
  • incorporation of glycolipid- or polyethelene glycol- derivatized phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Allen et al, Biochim. Biophys. Acta 1068:133 (1991); Allen et al, Biochim. Biophys. Acta 1150:9 (1993)).
  • Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes.
  • liposomes prepared with a high content of a nonionic surfactant, have been used to target the liver (Hayakawa et al, Japanese Patent 04- 244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)).
  • These formulations were prepared by mixing soybean phospatidylcholine, ⁇ -tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water.
  • DPPC dipalmitoylphosphatidylcholine
  • SG soybean-derived sterylglucoside mixture
  • Cho cholesterol
  • various targeting ligands can be bound to the surface of the liposome, such as antibodies, antibody fragments, carbohydrates, vitamins, and transport proteins.
  • liposomes can be modified with branched type galactosyllipid derivatives to target asialoglycoprotein (galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst.
  • Polyaconitylated human serum albumin liposomes provide another approach for targeting liposomes to liver cells (Kamps et al, Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).
  • Geho, et al. U.S. Patent No. 4,603,044 describe a hepatocyte-directed liposome vesicle delivery system, which has specificity for hepatobiliary receptors associated with the specialized metabolic cells of the liver.
  • target cells are prelabeled with biotinylated antibodies specific for a ligand expressed by the target cell (Harasym et al, Adv. Drug Deliv. Rev. 52:99 (1998)). After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered.
  • targeting antibodies are directly attached to liposomes (Harasym et al, Adv. Drug Deliv. Rev. 52:99 (1998)).
  • Polypeptides having Zaiphal ⁇ activity can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al, Infect. Immun. 57:1099 (1981), Anderson et al, Cancer Res.
  • liposomes may contain a variety of components.
  • liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allen et al, Biochim. Biophys. Acta 1150:9 (1993)).
  • Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins.
  • Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer (Gombotz and Pettit, Bioconjugate Chem.
  • the present invention also contemplates chemically modified polypeptides having Zaiphal ⁇ activity and Zaiphal ⁇ antagonists, in which a polypeptide is linked with a polymer, as discussed above.
  • 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).
  • compositions may be supplied as a kit comprising a container that comprises a molecule having Zaiphal ⁇ activity or a Zaiphal ⁇ antagonist.
  • Therapeutic polypeptides 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.
  • a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a therapeutic polypeptide.
  • Such a kit may further comprise written information on indications and usage of the pharmaceutical composition.
  • such information may include a statement that the Zaiphal ⁇ composition is contraindicated in patients with known hypersensitivity to Zaiphal ⁇ .
  • the present invention includes the use of Zaiphal ⁇ nucieotide sequences to provide Zaiphal ⁇ to a subject in need of such treatment.
  • a therapeutic expression vector can be provided that inhibits Z ⁇ lph ⁇ l ⁇ gene expression, such as an anti-sense molecule, a ribozyme, or an external guide sequence molecule. Inhibition of Z ⁇ lph ⁇ l ⁇ gene expression can be used to repress spermatogenesis.
  • an expression vector is constmcted in which a nucieotide sequence encoding a Z ⁇ lph ⁇ l ⁇ gene is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription.
  • a Zaiphal ⁇ gene can be delivered using recombinant viral vectors, including for example, adenoviral vectors (e.g., Kass-Eisler et al, Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci.
  • herpes viral vectors e.g., U.S. Patent Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688
  • parvovirus vectors Kelpes viral vectors
  • pox vims vectors Ozaki et al, Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali and Paoletti, Proc. Nat'l Acad. Sci.
  • pox vimses such as canary pox vims or vaccinia vims (Fisher-Hoch et al, Proc. Nat'l Acad. Sci. USA 86:317 (1989), and Flexner etal, Ann. NY. Acad. Sci. 569:86 (1989)), and retrovimses (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. 53:962 (1993), Vile and Hart, Cancer Res.
  • pox vimses such as canary pox vims or vaccinia vims (Fisher-Hoch et al, Proc. Nat'l Acad. Sci. USA 86:317 (1989), and Flexner etal, Ann. NY. Acad. Sci. 569:86 (1989)
  • adenovims a double-stranded DNA vims
  • a viral particle which contains the viral vector
  • adenovims 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.
  • adeno vimses can be administered by intravenous injection, because the vimses are stable in the bloodstream.
  • inserts are incorporated 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 vims will not replicate unless the El gene is provided by the host cell.
  • adenovims When intravenously administered to intact animals, adenovims 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. Secreted proteins will enter the circulation from tissue that expresses the heterologous gene (e.g., the highly vascularized liver).
  • adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector.
  • Such adenovimses 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)). By deleting the entire adenovims genome, very large inserts of heterologous DNA can be accommodated.
  • High titer stocks of recombinant vimses 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 Zaiphal ⁇ 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. Nat'l Acad. Sci. USA 85:8027 (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 purpose 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. For example, 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 Zaiphal ⁇ anti-sense RNA that inhibits the expression of Zaiphal ⁇ .
  • Suitable sequences for anti-sense molecules can be derived from the nucieotide sequences of Zaiphal ⁇ disclosed herein.
  • an expression vector can be constmcted in which a regulatory element is operably linked to a nucieotide 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.
  • ribozymes include nucieotide sequences that bind with Zaiphal ⁇ mRNA.
  • expression vectors can be constmcted in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of mRNA molecules that encode a Zaiphal ⁇ gene.
  • an external guide sequence can be constmcted 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 nucieotide sequence complementary to Zaiphal ⁇ mRNA, and a 3'-NCCA nucieotide 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 nucieotide located at the 5 '-side of the base-paired region.
  • the dosage of a composition comprising a therapeutic vector having a Zaiphal ⁇ nucieotide acid sequence, such as a recombinant vims will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history.
  • 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.
  • Horton et al Proc. Nat'l Acad. Sci. USA 95:1553 (1999) demonstrated that intramuscular injection of plasmid DNA encoding interferon- ⁇ produces potent antitumor effects on primary and metastatic tumors in a murine model.
  • 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 vimses 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 vims 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 vims) 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.
  • the therapy is preferably somatic cell gene therapy. That is, the preferred treatment of a human with a therapeutic gene expression vector or a recombinant vims 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).
  • nucleic acid molecules and proteins of the present invention can be used as nutritional sources or supplements. Such uses include the use as a protein or amino acid supplement, the use as a carbon source, the use as a nitrogen source, or the use as a carbohydrate source.
  • the nucleic acid molecules or proteins of the present invention can be added to the feed of an organism, or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions, or capsules.
  • Exemplary nutritional supplements for human consumption include CytoVol (EAS, Inc.), which contains ribonucleic acid, and Precision Protein (EAS, Inc.), which contains proteins and protein fragments.
  • the nucleic acid molecules or proteins can be added to the culture medium.
  • Transgenic mice can be engineered to over-express the Z ⁇ lph ⁇ l ⁇ gene in all tissues or under the control of a tissue-specific or tissue-preferred regulatory element. These over-producers of Zaiphal ⁇ 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 Zaiphal ⁇ . Transgenic mice that over-express Zaiphal ⁇ also provide model bioreactors for production of Zaiphal ⁇ in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses a Zaiphal ⁇ 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 , 5% O 2 ,. and 90% N 2 at 37°C.
  • the eg egos are then stored in a 37°C/5% CO 2 incubator until microinjection.
  • 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 overnight in a 37°C/5% CO 2 incubator.
  • 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 serrefine (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 serrefine 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 instmctions. Genomic DNA is analyzed by PCR using primers designed to amplify a Zaiphal ⁇ 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 bom 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 Zaiphal ⁇ mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
  • transgenic mice that over-express Zaiphal ⁇
  • Such transgenic mice provide useful models for diseases associated with a lack of Zaiphal ⁇ .
  • Zaiphal ⁇ gene expression can be inhibited using anti-sense genes, ribozyme genes, or external guide sequence genes.
  • inhibitory sequences are targeted to Zaiphal ⁇ mRNA.
  • An alternative approach to producing transgenic mice that have little or no Zaiphal ⁇ gene expression is to generate mice having at least one normal Zaiphal ⁇ allele replaced by a nonfunctional Zaiphal ⁇ gene.
  • One method of designing a nonfunctional Zaiphal ⁇ gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes Zaiphal ⁇ . 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 ofCytokines 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)).
  • 5' RACE was performed to obtain 5' sequence from an expressed sequence tag.
  • the 5' RACE was performed as follows: 5 ⁇ l of 1/50 diluted human testis cDNA library pools of 1000, 20 pmoles each of oligonucleotide primers ZC6,583 (5' GTC CAA CGA CTA TAA AGA GGG 3'; SEQ ID NO:7) and ZC 19,529 (5' TTT GGG AGA GGA TCC TCA GAC ATA AG 3'; SEQ ID NO:8), 10 mM dNTP's, 5 ⁇ l lOx Pwo buffer and 1 U of Pwo Polymerase (Roche Molecular Biochemicals; Indianapolis, IN) were combined in 50 ⁇ l reactions.
  • the reactions were run as follows: 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 2 minutes, 72°C for 2 minutes. The reaction was stopped with a 7 minute incubation at 72°C followed by a 4°C hold indefinitely. Five microliters each of 1/20 diluted first PCR product were used as template for a nested PCR.
  • oligonucleotide primers ZC 13,006 (5* GGC TGT CCT CTA AGC GTC AC 3*; SEQ ID NO:9) and ZC 19,530 (5' TAG GCA AGG CAT GAA CCA GAG TAG G 3'; SEQ ID NO:10), lOmM dNTP's, 5 ⁇ l lOx Pwo buffer and 1 U of Pwo Polymerase were combined in 50 ⁇ l reactions. The reactions were run as follows: 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 2 minutes, and 72°C for 2 minutes.
  • the reaction was stopped with a 7 minute incubation at 72°C followed by a 4°C hold indefinitely.
  • the PCR products were fractionated on 2% agarose gels. Obvious bands were obtained in library pools A2-4, F2-3, F2-4 and D10-6, purified with QIAprep Spin Miniprep Kit (QIAGEN Inc.; Valencia, CA) and sequenced. The results indicated that the fragment amplified from the D10-6 pool did extend the original Zaiphal ⁇ expressed sequence tag 5 '-ward, but additional upstream sequence was needed to find the initiating Met and upstream STOP codon.
  • New primers ZC20,491 (5' TGT GGG GTA TCT CAG GAA AGC TGG 3*; SEQ ID NO:l 1) and ZC20,530 (5' CCC CGA GAC AAG ATC AGG CAA GTT T 3'; SEQ ID NO: 12) were designed according to the new 5' sequence information on the clone.
  • 5' RACE was performed on the library pools named above (A2-3, F2-3, F2-4, F2-9 and D10-6).
  • PCR products Five microliters each of 1/20 diluted PCR product were used as template for a nested PCR. Twenty pmoles of each oligonucleotide primers ZC13,006 and ZC20.491, 10 mM dNTP's, 5 ⁇ l lOx Advantage Klentaq buffer and 1 U of Advantage Klentaq Polymerase were combined in 50 ⁇ l reactions. The reactions were n as follows: 94°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 2 minutes, and 72°C for 2 minutes. The reaction was stopped with a 7 minute incubation at 72°C followed by a 4°C hold indefinitely. The PCR products were fractionated on 1% agarose gels.
  • Filter lifts were performed on the two library pools that yielded 5' nested RACE products (A2-4 and F2-9).
  • One microliter of human testis pools of 1000 A2-4 and F2-9 were mixed with 20 ⁇ l DH10B (Gibco BRL) electrocompetent cells. Cells were electroporated at 2.0KV, 400 ohms, 25 ⁇ F.
  • the cell/DNA mixture was outgrown in 1 ml of SOC for 1 hour at 37°C.
  • the 1 ml of transformation mixture was mixed with 1 ml of 50% glycerol and flash frozen on dry ice. The mixture was then thawed, diluted, and plated for titer on LB Amp plates. Only pool F2-9 was used for subsequent analysis.
  • the frozen glycerol stock was thawed and plated on LB Amp maxiplates. Filter lifts were then made from these transformation plates. The filter lifts were then probed with radiolabeled Zaiphal ⁇ .
  • the probe was made by digesting the original expressed sequence tag with Ev_.II and gel purifying a 316 base pair band that corresponded to Zaiphal ⁇ . Fifty nanograms of probe was radiolabeled using the R ⁇ DIPRIM ⁇ II labeling kit (AM ⁇ RSHAM PHARMACIA BIOTECH, Inc.; Piscataway, NJ) according to the manufacturer's protocol.
  • the probe was purified using a NUCTRAP push column (STRATAGENE; La Jolla, CA). EXPRESSHYB (CLONTECH) solution was used for the prehybridization and hybridization solutions for the northern blots. Hybridization took place overnight at 65°C. Following hybridization, the lifts were washed 4 times, 12 minutes each, at 25°C in 2xSSC, 0.1% SDS. The blots were then washed 2 times, 30 minutes each, at 50°C in O.lxSSC, 0.1% SDS. A final wash was conducted for 30 minutes at 55°C in O.lxSSC, 0.1%SDS. Lifts were exposed to Kodak BioMax film. Five clones were selected for sequencing. The results indicated pool F2-9 clone #2-1 to be full-length Zaiphal ⁇ cDNA with a Met and upstream STOP codon confirmed.
  • RNA Master Blot (dot blot), a Mouse Multiple Tissue Northern Blot, and a Mouse RNA Master Blot (dot blot) (CLONTECH Laboratories, Inc.; Palo Alto, CA).
  • the hybridization probe was generated as described for Example 1. Fifty nanograms of probe were radiolabeled as described in Example 1. EXPRESSHYB (CLONTECH) solution was used for the prehybridization and hybridization solutions for the northern blots. Hybridization took place overnight at 65°C. Following hybridization, lifts were washed 4 times, 12 minutes each, at 25°C in 2xSSC, 0.1% SDS.
  • the blots were then washed 2 times, 30 minutes each, at 50°C in O.lxSSC, 0.1% SDS. A final wash was conducted for 1 hour at 53°C in O.lxSSC, 0.1%SDS for the human sample northerns.
  • the blots were exposed to Kodak BioMax film. A band at 3.0 kilobases was clearly visible in testis only on the northern blots (both human and mouse).
  • the RNA Master Blot (human and mouse) also showed a positive signal from testis only.
  • Southern analysis was performed using a commercially prepared Interspecies Zoo-Blot from CLONTECH Laboratories, Inc.
  • the Southern blot contained EcoRI-digested DNA.
  • the hybridization probe was generated as described for Example 1. Fifty nanograms of probe were radiolabeled as described in Example 1. EXPRESSHYB (CLONTECH) solution was used for the prehybridization and hybridization solutions for the Southern blots. Hybridization took place overnight at 65°C. Following hybridizaion, lifts were washed 4 times, 12 minutes each, at 25°C in 2xSSC, 0.1% SDS. The blots were then washed 2 times, 30 minutes each, at 50°C in O.lxSSC, 0.1% SDS.
  • the blots were exposed to Kodak BioMax film.
  • the human and monkey DNA samples contained a hybridizing fragment at approximately 3.8 kilobases.
  • the dog and cow DNA samples contained a hybridizing fragment at approximately 4.0 kilobases; the dog sample also contained a 3.8 kilobase band (similar to the human and monkey samples).
  • the rabbit DNA sample contained a hybridizing fragment at approximately 3.2 kilobases.
  • the rat DNA sample contained a hybridizing fragment at approximately 9.4 kilobases.
  • the mouse DNA sample contained a hybridizing fragment at approximately 20 kilobases.
  • Zaiphal ⁇ was mapped to chromosome 11 using the commercially available version of the "Stanford G3 Radiation Hybrid Mapping Panel” (Research Genetics, Inc.; Huntsville, AL).
  • the "Stanford G3 RH Panel” contains DNA molecules from each of 83 radiation hybrid clones of the whole human genome, plus two control DNA molecules (the RM donor and the A3 recipient).
  • a WWW server http://shgc- www.stanford.edu) allows chromosomal localization of markers.
  • Each of the 85 PCR reactions consisted of 2 ⁇ l lOx KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc.; Palo Alto, CA), 1.6 ⁇ l dNTPs mix (2.5 mM each; PERKIN- ELMER; Foster City, CA), 1 ⁇ l sense primer, ZC22,152 (5' CAG CCA AGC TCC ATT TAC 3'; SEQ ID NO:5), 1 ⁇ l antisense primer, ZC22,153 (5' TCA GGG AAG CCA CGA TAG 3'; SEQ ID NO:6), 2 ⁇ l "RediLoad” (Research Genetics, Inc.; Huntsville, AL), 0.4 ⁇ l 50x Advantage KlenTaq Polymerase Mix (CLONTECH Laboratories, Inc.), 25 ng of DNA from an individual hybrid clone or control and ddH 2 O for a total volume of 20 ⁇ l.
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 94°C, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 64°C, and a 1 minute and 15 seconds extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
  • the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies; Gaithersburg, MD).

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Abstract

L'invention porte sur des marqueurs spécifiques d'un tissu pouvant être utilisé pour améliorer le diagnostic, la détermination du stade et le traitement du cancer, ainsi que pour détecter des métastases de tumeurs dérivées de ce tissu. La présente invention porte sur un nouveau gène humain, appelé « Zalpha16 », qui est de préférence exprimé dans un tissu de testicule.
PCT/US2000/008836 1999-04-07 2000-04-03 Proteine specifique des testicules WO2000059934A2 (fr)

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Citations (1)

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WO1998033916A2 (fr) * 1997-01-31 1998-08-06 Genetics Institute, Inc. Proteines secretees et polynucleotides codant celles-ci

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Publication number Priority date Publication date Assignee Title
WO1998033916A2 (fr) * 1997-01-31 1998-08-06 Genetics Institute, Inc. Proteines secretees et polynucleotides codant celles-ci

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CONKLIN DARRELL ET AL: "Molecular cloning, chromosome mapping and characterization of UBQLN3 a testis-specific gene that contains an ubiquitin-like domain." GENE (AMSTERDAM), vol. 249, no. 1-2, 16 May 2000 (2000-05-16), pages 91-98, XP002152071 ISSN: 0378-1119 *
DATABASE EMBL [Online] Accesion number AA725499, 8 January 1998 (1998-01-08) STRAUSBERG R.: " ai18e05.s1 Soares_testis_NHT Homo sapiens cDNA clone 1343168 3', mRNA sequence" XP002152073 *
DATABASE EMBL [Online] Accession number AA628294, 28 October 1997 (1997-10-28) HILLIER L. ET AL.: "af99d06.s1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:1055915 3', mRNA sequence" XP002152074 *
DATABASE EMBL [Online] Accession number HSB9806, 17 July 1996 (1996-07-17) EVANS G.A. ET AL.: "Genomic Sequence Sampled Map of Chromosome 11" XP002152075 *
KITADA TOHRU ET AL: "Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism." NATURE (LONDON), vol. 392, no. 6676, 9 April 1998 (1998-04-09), pages 605-608, XP002152069 ISSN: 0028-0836 cited in the application *
RAO-NAIK CHETANA ET AL: "The Rub family of ubiquitin-like proteins: Crystal structure of arabidopsis Rub1 and expression of multiple Rubs in arabidopsis." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 52, 25 December 1998 (1998-12-25), pages 34976-34982, XP002152070 ISSN: 0021-9258 cited in the application *

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