Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/66—Thymopoietins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• proteins which share regions of sequence homology and localization to the nucleus. These proteins include the thymopoietins,
• Emerin is a nuclear membrane protein responsible for the X-linked recessive disorder Emery-Dreifuss muscular dystrophy. Mouse, rat and human emerin sequences have been reported (Bione et al .
• mice, rat and human emerin share 73-95% nucleotide and amino acid identity. All share some structural homology with the thymopoietins and LAP2 , in particular within portions of the conserved N-terminal region and the hydrophobic putative transmembrane domain of thymopoietin.
• emerin is ubiquitous expressed and it is predicted that emerin has the same inner nuclear membrane organization as do thymopoietin and LAP2 (Manual et al . , ibid. ) .
• Antisera raised against emerin peptides localized expression of the protein to the nuclear membranes of normal skeletal and cardiac muscle cells, but found it to be absent in those cells of patients with muscular dystrophy. It is unclear how a deficiency of a nuclear protein results in the disease (Nagano et al . , Nat . Genet . 12.:254-9, 1996 and Small et al . , ibid.).
• the present invention provides associated polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
• the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 876 of SEQ ID NO : 2. Within one embodiment the sequence of amino acid residues is at least 90% identical. Within another embodiment any differences between said polypeptide and residues 1 through 876 of SEQ ID NO : 2 are due to conservative amino acid substitutions.
• the polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.
• the polypeptide is covalently linked to a moiety selected from the group consisting of affinity tags, radionucleotides, enzymes and fluorophores .
• the moiety is an affinity tag selected from the group consisting of polyhistidine, FLAG, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region.
• polypeptide comprising the amino acid sequence of SEQ ID NO : 2.
• the invention provides a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion consisting of a polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 876 of SEQ ID NO: 2; and said second portion comprising another polypeptide .
• the invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a polypeptide as described above, in combination with a pharmaceutically acceptable vehicle.
• an antibody or antibody fragment that specifically binds to a polypeptide as described above.
• the antibody is selected from the group consisting of: a) polyclonal antibody; b) murine monoclonal antibody; c) humanized antibody derived from b) ; and d) human monoclonal antibody.
• the antibody fragment is selected from the group consisting of F(ab'), F(ab), Fab', Fab, Fv, scFv, and minimal recognition unit.
• an anti-idiotype antibody that specifically binds to the antibody described above.
• binding protein that specifically binds to an epitope of a polypeptide as described above.
• an isolated polynucleotide selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 876 of SEQ ID NO : 2 ; b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO : 5 ; c) a polynucleotide that remains hybridized following stringent wash conditions to a polynucleotide consisting of the nucleotide sequence of SEQ ID NO : 1 , or the complement of SEQ ID NO:l.
• the sequence of amino acid residues is at least 90% identical.
• any difference between the amino acid sequence encoded by the polynucleotide and the corresponding amino acid sequence of SEQ ID NO : 2 is due to a conservative amino acid substitution.
• the polynucleotide comprises nucleotide 127 to nucleotide 2754 of SEQ ID NO : 1.
• the polynucleotide is DNA.
• the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment consisting of a polynucleotide as described above; and a transcriptional terminator.
• a transcription promoter a DNA segment consisting of a polynucleotide as described above; and a transcriptional terminator.
• sequence of amino acid residues is at least 90% identical.
• any difference between the amino acid sequence encoded by the polynucleotide and the corresponding amino acid sequence of SEQ ID NO : 2 is due to a conservative amino acid substitution.
• the DNA segment encodes a polypeptide covalently linked to an affinity tag selected from the group consisting of polyhistidine, Glu-Glu, glutathione S transferase and an immunoglobulin heavy chain constant region.
• the expression vector further comprises a secretory signal sequence operably linked to said DNA segment.
• the invention provide a method of producing a ZTMPO-1 polypeptide comprising: culturing a cell into which has been introduced an expression vector as described above, whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.
• Also provided by the invention is a method for detecting a genetic abnormality in a patient, comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NO : 1 or the complement of SEQ ID N0:1, under conditions wherein said polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing said first reaction product to a control reaction product, wherein a difference between said first reaction product and said control reaction product is indicative of a genetic abnormality in the patient.
• the figure shows a multiple amino acid sequence alignment for ZTMPO-1 (SEQ ID NO:2), human emerin (EMD_HU) Bione et al . , Nat . Genet . 8 . :323-27, 1994 (SEQ ID NO:3), human thymopoietin ⁇ (PIR_A5) Harris et al . , Proc . Natl . Acad. Sci. USA 91: 6283-7, 1994 (SEQ ID NO : 4 ) , human thymopoietin ⁇ (PIR_B5) Harris et al . , ibid. (SEQ ID NO:30) and human thymopoietin ⁇ (PIR_C5) Harris et al . , ibid. (SEQ ID NO:31) .
• affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
• affinity tag any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
• Affinity tags include a poly-histidine tract, protein A
• allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
• allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene .
• amino-terminal 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.
• polynucleotide molecule is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence.
• sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3 ' . 7
• contig denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or alone a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5 ' - ATGGCTTAGCTT-3 ' are 5 ' -TAGCTTgagtct-3 ' and 3'- gtcgacTACCGA-5 ' .
• degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .
• Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
• expression vector is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
• additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
• Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
• isolated when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
• isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
• Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985) .
• an "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
• the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
• the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
• operably linked when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
• ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
• polynucleotide is a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
• Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt”), or kilobases ("kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded.
• 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” . "Probes and/or primers" as used herein can be
• RNA or DNA can be either cDNA or genomic DNA.
• Polynucleotide probes and primers are single or double- stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used.
• PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt . Short polynucleotides can be used when a small region of the gene is targeted for analysis.
• a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersha Corp., Arlington Heights, IL, using techniques that are well known in the art. Examples of ZTMPO-1 probes and primers include, but are not limited to, the sequences disclosed herein as SEQ ID NOs:6-29. 10
• promoter is used herein for its art- recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.
• a “protein” is a macromolecule comprising one or more polypeptide chains.
• a protein may also comprise non- peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
• receptor denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
• a bioactive molecule i.e., a ligand
• 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. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell.
• Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
• receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, 11
• IL-3 receptor GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor
• secretory signal sequence denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
• the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
• splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence.
• splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
• the present invention is based in part upon the discovery of a novel protein having regions of homology to members of the thymopoietin-emerin family of nuclear membrane proteins.
• This protein has been designated "ZTMPO-1".
• the human ZTMPO-1 nucleotide sequence is represented in SEQ ID NO : 1 and the deduced amino acid sequence in SEQ ID NO: 2.
• the ZTMPO-1 proteins and polypeptides encoded by polynucleotides of the present invention were initially identified by querying an EST (Expressed Sequence Tag) database for sequences homologous to conserved motifs within the thymopoietin family. 12
• ZTMPO-1 as represented in SEQ ID NO : 1 is a 2,754 bp polynucleotide which has an open reading frame encoding an 876 amino acid residue protein. Sequence analysis of the deduced amino acid sequence as represented in SEQ ID NO : 2 does not indicate the presence of a secretion signal sequence or transmembrane domain. There is a putative ankyrin-like region, amino acid residues 333-385 of SEQ ID NO: 2, having an ankyrin repeat (residues 347-379 of SEQ ID NO: 2) which may indicate that ZTMPO-1 is retained in the plasma membrane. Ankyrin repeats have been described as a
• Ankyrin repeats have been proposed as a generalized protein binding motif, one function of ankyrin repeats is to serve as adaptors, associating with the spectrin-based cytoplasmic skeleton and membrane proteins .
• Ankyrin is used as a membrane attachment site in neurons and may provide a transport mechanism through secretory vesicles.
• ZTMPO-1 At the C-terminal end of ZTMPO-1 is a calcium binding protein-like region having two potential calcium binding sites (residues 678-692 and residues 719-731 of SEQ ID NO: 2) similar to that seen in the sea urchin calcium binding protein LPSl-beta (Xiang et al . , J. Biol. Chem. 16 . : 10524-33, 1991).
• the ZTMPO-1 polynucleotide of SEQ ID NO : 1 encodes an 876 amino acid residue protein which is much larger than other members of the thymopoietin/emerin family.
• Human thymopoietin ⁇ is a 693 amino acid residue protein
• human emerin is a 254 amino acid residue protein
• rat LAP2 is a 452 amino acid residue protein. 13
• the amino acid sequence of ZTMPO-1 does not contain the 42 amino acid thymopoietin peptide originally identified by Goldstein (Nature 247 : 11-14 ,
• ZTMPO-1 shares 50% amino acid identity with the corresponding regions of the mouse and human thymopoietins and 30% with human emerin.
• the region defined by amino acid residues 30-44 of SEQ ID NO : 2 is highly conserved between the proteins, see Figure.
• ZTMPO-1 also shares discrete regions of homology with rat lamina associated protein 2, LAP2 , (Furukawa et al . , ibid. , Genbank Accession No. U18314) . These regions correspond to many of the same regions with which ZTMPO-1 shares identity with the thymopoietins. ZTMPO-1 and rat LAP2 share 70% amino acid identity over the region corresponding to amino acid residues 13 to 44 of SEQ ID NO : 2. ZTMPO-1 also shares a limited degree of homology to regions of the yeast transcription factor IIF alpha subunit over the region corresponding to amino acid residues 86 to 160 and amino acid residues 205 to 260 of SEQ ID NO:2.
• ZTMPO-2 shares 27% amino acid identity with Trypanosoma brucei ribonuclease HI (Hesslein and Campbell, Mol. Biochem. Parasitol . 16:121-6, 1997, Genbank Accession No. U74470) over the region corresponding to amino acid residues 156 to 203 of SEQ ID NO: 2. This homology, along with that shared with LAP2 , as well as the possible ankyrin repeat, suggests the 14
• ZTMPO-1 possesses chromatin or DNA binding properties.
• a 3.2 and a 5 kb transcript corresponding to ZTMPO-1 were ubiquitously expressed with the highest level being in testis tissue. Similar ubiquitous expression patterns were also reported for the thymopoietins and emerin (Harris et al . , ibid, and Small et al . , ibid. ) .
• Thymopoietin maps to chromosome 12q22 (Harris et al . , ibid. ) .
• nucleotide sequences encoding regions of conserved amino acid residues between ZTMPO-1 and nuclear proteins such as the thymopoietins, LAP2 and emerin, for example, the region between nucleotides 163 and 258 of SEQ ID N0:1, in particular the region between nucleotides 214 15
• RT-PCR reverse transcription- polymerase chain reaction
• highly degenerate primers designed from the ZTMPO-1 sequences are useful for this purpose.
• SEQ ID NO : 5 is a degenerate DNA sequence that encompasses all DNAs that encode the ZTMPO-1 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO : 5 also provides all RNA sequences encoding SEQ ID NO : 2 by substituting U
• ZTMPO-1 polypeptide- encoding polynucleotides comprising nucleotide 1 to nucleotide 2628 of SEQ ID NO : 5 and their RNA equivalents are contemplated by the present invention.
• Table 1 sets forth the one-letter codes used within SEQ ID NO : 5 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. "Nucleotide
• Complement indicates the code for the complementary nucleotide (s) .
• the code Y denotes either C
• degenerate codons used in SEQ ID NO : 5 encompassing all possible codons for a given amino acid, are set forth in Table 2.
• Gln Z SAR Any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
• the degenerate codon for serine can, in some circumstances, encode arginine (AGR)
• the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) .
• 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. Introduction of preferential codon sequences into recombinant DNA can, for example, 19
• the degenerate codon sequence disclosed in SEQ ID NO : 5 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 also provides polypeptide fragments or peptides comprising an epitope-bearing portion of an ZTMPO-1 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. Acad. Sci . USA 11:3998, 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 preferably 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 20
• epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr . Opin. Immunol. 1:268, 1993, and Cortese et al . , Curr. Opin. Biotechnol . 1_ : 616 , 1996) . Standard methods for identifying epitopes and producing antibodies from small peptides that comprise an epitope are described, for example, by Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992), Price,
• Potential antigenic sites in ZTMPO-1 can be identified using the Jameson-Wolf method (Jameson and Wolf, CABIPS 4_:181, 1988), as implemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR; Madison, WI) .
• the Jameson-Wolf method predicts potential antigenic determinants by combining six major subroutines for protein structural prediction. Briefly, the Hopp-Woods method (Hopp et al . , Proc. Nat. Acad. Sci. USA 71:3824, 1981) , is first used to identify amino acid sequences representing areas of greatest local hydrophilicity (parameter: seven residues averaged) .
• secondary structure predictions are applied to the data using the methods of Chou-Fasman, Chou, 21
• a surface contour value designated as the "antigenic index.”
• a peak broadening function is 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.
• variant ZTMPO-1 encodes a polypeptide that is characterized by its glycoprotein synthesis or cell-cell interaction activity, or by the ability to bind specifically to an anti-ZTMPO-1 antibody. More specifically, variant ZTMPO-1 genes encode polypeptides which exhibit at least 50%, and preferably, greater than 70, 80, or 90%, of the activity of polypeptide encoded by the human ZTMPO-1 gene described herein.
• 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 : 1 , SEQ ID NO : 2 or SEQ ID NO : 5. Suitable forms of computer-readable media include 22
• magnetic media and optically-readable 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.
• RAM random access memory
• DLT digital linear tape
• 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) .
• the isolated polynucleotides can hybridize under stringent conditions to polynucleotides having the nucleotide sequence of SEQ ID NO:l or to nucleic acid molecules having a nucleotide sequence complementary to SEQ ID NO:l.
• 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.
• DNA, RNA-RNA and DNA-RNA can hybridize if the nucleotide sequences have some degree of complementarity. Hybrids can tolerate mismatched base pairs in the double helix, but the stability of the hybrid is influenced by the degree of mismatch. The T m of the mismatched hybrid decreases by 1°C for every 1-1.5% base pair mismatch. Varying the stringency of the hybridization conditions allows control over the degree of mismatch that will be present in the hybrid. The degree of stringency increases as the hybridization temperature increases and the ionic strength of the hybridization buffer decreases. Stringent hybridization conditions encompass temperatures of about 5-25°C below the T m of the hybrid and a hybridization buffer having up to 1 M Na + . Higher degrees of stringency at lower temperatures can be achieved with the addition of formamide which reduces the T m of the hybrid about 1°C for 23
• each 1% formamide in the buffer solution includes temperatures of 20-70°C and a hybridization buffer containing up to 6xSSC and 0-50% formamide.
• a higher degree of stringency can be achieved at temperatures of from 40-70°C with a hybridization buffer having up to 4xSSC and from 0-50% formamide.
• Highly stringent conditions typically encompass temperatures of 42-70°C with a hybridization buffer having up to lxSSC and 0-50% formamide.
• Different degrees of stringency can be used during hybridization and washing to achieve maximum specific binding to the target sequence.
• the washes following hybridization are performed at increasing degrees of stringency to remove non-hybridized polynucleotide probes from hybridized complexes .
• the above conditions are meant to serve as a guide and it is well within the abilities of one skilled in the art to adapt these conditions for use with a particular polypeptide hybrid.
• the T m for a specific target sequence is the temperature (under defined conditions) at which 50% of the target sequence will hybridize to a perfectly matched probe sequence.
• Those conditions which influence the T m include, the size and base pair content of the polynucleotide probe, the ionic strength of the hybridization solution, and the presence of destabilizing agents in the hybridization solution.
• Numerous equations for calculating T m are known in the art, and are specific for DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences of varying length (see, for example, Sambrook et al . , Molecular Cloning: A Laboratory
• OLIGO 6.0 LSR; Long Lake, MN
• Primer Premier 4.0 Premier Biosoft International; Palo Alto, CA
• sites on the Internet are available tools for analyzing a given sequence and calculating T m based on user defined criteria.
• Such programs can also analyze a given sequence under defined conditions and identify suitable probe sequences.
• hybridization of longer polynucleotide sequences, >50 base pairs is performed at temperatures of about 20-25°C below the calculated T m .
• hybridization is typically carried out at the T ra or 5-10°C below. This allows for the maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
• the length of the polynucleotide sequence influences the rate and stability of hybrid formation. Smaller probe sequences, ⁇ 50 base pairs, reach equilibrium with complementary sequences rapidly, but may form less stable hybrids. Incubation times of anywhere from minutes to hours can be used to achieve hybrid formation. Longer probe sequences come to equilibrium more slowly, but form more stable complexes even at lower temperatures . Incubations are typically allowed to proceed overnight or longer. Generally, incubations are carried out for a period equal to three times the calculated Cot time. Cot time, the time it takes for the polynucleotide sequences to reassociate, can be calculated for a particular sequence by methods known in the art .
• the base pair composition of polynucleotide sequence will effect the thermal stability of the hybrid complex, thereby influencing the choice of hybridization temperature and the ionic strength of the hybridization buffer.
• A-T pairs are less stable than G-C pairs in aqueous solutions containing sodium chloride. Therefore, the higher the G-C content, the more stable the hybrid. Even distribution of G and C residues within the sequence also contribute positively to hybrid stability.
• the base pair composition can be manipulated to 25
• T m alters the T m of a given sequence.
• 5- methyldeoxycytidine can be substituted for deoxycytidine and 5-bromodeoxuridine can be substituted for thymidine to increase the T m _ whereas 7-deazz-2 ' -deoxyguanosine can be substituted for guanosine to reduce dependence on T m .
• Hybridization buffers generally contain blocking agents such as Denhardt ' s solution (Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO) , heparin or SDS, and a Na + source, such as SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M NaCl , 10 mM NaH 2 P0 4 , 1 mM EDTA, pH 7.7) .
• SSC Denhardt ' s solution
• BLOTTO denatured salmon sperm DNA
• tRNA milk powders
• BLOTTO milk powders
• heparin or SDS heparin or SDS
• Na + source such as SSC (lx SSC: 0.15 M sodium chloride, 15 mM sodium citrate) or SSPE (lx SSPE: 1.8 M NaCl , 10 mM NaH 2
• hybridization buffers typically contain from between 10 mM - 1 M Na + .
• destabilizing or denaturing agents such as formamide, tetralkylammonium salts, guanidinium cations or thiocyanate cations to the hybridization solution will alter the T m of a hybrid.
• formamide is used at a concentration of up to 50% to allow incubations to be carried out at more convenient and lower temperatures. Formamide also acts to reduce non-specific background when using RNA probes.
• a polynucleotide encoding a variant ZTMPO-1 polypeptide can be hybridized with a polynucleotide having the nucleotide sequence of SEQ ID NO:l (or its complement) at 42 °C overnight in a solution comprising 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate) , 50 mM sodium phosphate
• the hybridization mixture can be incubated at a 26
• premixed hybridization solutions are available ( e . g. ,
• 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 ZTMPO-1 polypeptide hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2x SSC with 0.1% SDS at 65°C.
• wash stringency is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2x SSC 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 0.1x-0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50-65°C.
• SDS sodium dodecyl sulfate
• polynucleotides encoding a variant ZTMPO-1 polypeptide hybridize with a polynucleotide having the nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1x-0.2x SSC with 0.1% SDS at 50-65°C, including 0. lx SSC with 0.1% SDS at 50°C, or 0.2x SSC with 0.1% SDS at 65°C.
• the present invention also contemplates ZTMPO-1 variant polypeptides 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:
• ZTMPO-1 variants include nucleic acid molecules (1) that 27
• nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 (or its complement) under stringent washing conditions, in which the wash stringency is equivalent to 0.5x-2x SSC with 0.1% SDS at 50-65°C, and (2) that encode a polypeptide having 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.
• ZTMPO-1 variants can be characterized as nucleic acid molecules (1) that hybridize with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to 0.1x-0.2x SSC with 0.1% SDS at 50-65°C, and (2) that encode a polypeptide having 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.
• RNA is isolated from a tissue or cell that produces large amounts of ZTMPO-1 RNA. Such tissues and cells are identified by Northern blotting (Thomas, Proc. Natl . Acad. Sci. USA T7:5201, 1980), an exemplary source being human testis tissue.
• Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
• Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder
• cDNA Complementary DNA
• poly (A) + RNA using known methods.
• genomic DNA can be isolated.
• Polynucleotides encoding ZTMPO-1 polypeptides are then identified and isolated by, for example, hybridization or PCR.
• the polynucleotides of the present invention can also be synthesized using techniques widely known in the art. See, for example, Glick and Pasternak, Molecular 28
• the present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs) . These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species.
• ZTMPO-1 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides.
• Orthologs of human ZTMPO-1 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
• a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses ZTMPO-1 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 ZTMPO-1-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences.
• a cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human ZTMPO-1 sequence disclosed herein.
• the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to ZTMPO-1 polypeptide. Similar techniques can also be applied to the isolation of genomic clones .
• sequence disclosed in SEQ ID NO : 1 represents a single allele of human ZTMPO-1 and that allelic variation and 29
• Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.
• Allelic variants of the DNA sequence shown in SEQ ID NO : 2 including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO : 2.
• cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ZTMPO-1 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
• Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art .
• the present invention also provides isolated ZTMPO-1 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO : 2 and their orthologs.
• substantially homologous is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO: 2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 2 or its orthologs) . Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9, 1992.
• the present invention further includes nucleic acid molecules that encode such polypeptides. Methods for determining percent identity are described below.
• the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant ZTMPO-1.
• the FASTA algorithm is described by Pearson and Lipman, Proc . Nat. Acad. Sci. USA 15:2444, 1988, and by Pearson, Meth. Enzvmol . 183 :63, 1990.
• the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
• 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.
• Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
• Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zsig37 polypeptide and the affinity tag.
• Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites.
• the present invention includes nucleic acid molecules that encode a polypeptide having one or more "conservative amino acid substitutions," compared with the amino acid sequence of SEQ ID NO : 2.
• Conservative amino acid substitutions can be based upon the chemical properties of the amino acids. 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 ZTMPO-1 amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in a ZTMPO-1 amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur- containing amino acid in a ZTMPO-1 amino acid sequence, a hydroxy-containing amino acid is substituted for a hydroxy-containing amino acid in a ZTMPO-1 amino acid 34
• an acidic amino acid is substituted for an acidic amino acid in a ZTMPO-1 amino acid sequence
• a basic amino acid is substituted for a basic amino acid in a ZTMPO-1 amino acid sequence
• a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxylic amino acid in a ZTMPO-1 amino acid sequence.
• 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.
• conservative amino acid substitutions are provided in Table 4.
• Aromatic phenylalanine tryptophan tyrosine
• the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Natl. Acad. Sci . USA 19:10915, 1992) . Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above) , the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to this system, preferred 3 6
• 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) .
• Conservative amino acid changes in a ZTMPO-1 gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO : 1.
• 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
• variants to promote proliferation and cardiac functions as will as other properties of the wild-type protein can be determined using a standard methods, such as the assays described herein.
• a variant ZTMPO-1 polypeptide can be identified by the ability to specifically bind anti-ZTMPO-1 antibodies.
• the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
• Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methyl - glycine, allo-threonine, methylthreonine, hydroxy- ethylcysteine, hydroxyethylhomocysteine, nitro-glutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3- dimethylproline, tert-leucine, norvaline, 2-azaphenyl- alanine, 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-natural amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 11:7470-6, 1994.
• Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions
• 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 ZTMPO-1 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 38
• Variants of the disclosed ZTMPO-1 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370 :389-91 , 1994 and Stemmer, Proc. Natl. Acad. Sci. USA ⁇ : 10747-51, 1994. 39
• variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations.
• This technique can be modified by using a family of parent DNAs, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
• Mutagenesis methods as disclosed herein can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
• Preferred assays in this regard include cell proliferation assays and biosensor-based ligand-binding assays, which are described below.
• Mutagenized DNA molecules that encode active polypeptides 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.
• polypeptide fragments or variants of SEQ ID NO: 2 can be identified and/or prepare a variety of polypeptide fragments or variants of SEQ ID NO: 2 or that retain the receptor binding properties of the wild-type ZTMPO-1 protein.
• polypeptides may also include additional polypeptide segments as generally disclosed herein.
• any ZTMPO-1 polypeptide including variants and fusion proteins
• one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above. 40
• a fusion protein consists essentially of a first portion and a second portion joined by a peptide bond.
• the first portion consists of a polypeptide comprising a sequence of amino acid residues that is at least 80% identical in amino acid sequence to residues 1 through 876 of SEQ ID NO : 2 and the second portion is any other polypetide.
• the other polypeptide may be alternative or additional domains from other members of the thymopoietin or emerin family, a signal peptide to facilitate secretion of the fusion protein, affinity tags, Ig domains or the like.
• the ZTMPO-1 polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
• Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al .
• a DNA sequence encoding a ZTMPO-1 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
• the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of 41
• the exogenous DNA may be provided by integration into the host cell genome.
• Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
• a secretory signal sequence (also known as a leader sequence, signal sequence, prepro sequence or pre sequence) is provided in the expression vector.
• the secretory signal sequence may be derived from another secreted protein (e.g., t-PA) or synthesized e novo .
• the secretory signal sequence is operably linked to the ZTMPO-1 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
• Secretory signal sequences are commonly positioned 5 ' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al . , U.S. Patent No. 5,037,743; Holland et al . , U.S. Patent No. 5,143,830).
• Cultured mammalian cells are suitable hosts within the present invention.
• Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol. 3_6: 59-72, 1977) and
• Chinese hamster ovary e.g. CHO-K1; ATCC No. CCL 61 cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus . See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
• Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
• a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.
• Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
• a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
• Other drug resistance genes e.g. hygromycin resistance, 43
• puromycin acetyltransferase can also be used.
• Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
• Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by
• Insect cells can be infected with recombinant baculovirus vectors, which are commonly derived from Autographa calif ornica multiple nuclear polyhedrosis virus (AcMNPV) .
• AcMNPV Autographa calif ornica multiple nuclear polyhedrosis virus
• DNA encoding the polypeptide of interest is inserted into the viral genome in place of the polyhedrin gene coding sequence by homologous recombination in cells infected with intact, wild-type AcMNPV and transfected with a transfer vector comprising the cloned gene operably linked to polyhedrin gene promoter, terminator, and flanking sequences.
• the resulting recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general,
• Fungal cells including yeast cells, can also be used within the present invention.
• Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pas tor is , and Pichia methanolica . Methods for transforming S . cerevisiae cells with 44
• exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. 4,845,075.
• Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
• a preferred vector system for use in Saccharomyces cerevisiae is the POTl vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
• Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosaccharomyces pombe ,
• Kl uyveromyces la ctis , Kluyveromyces fragil is , Us tilago maydis , Pichia pastoris , Pichia methanol ica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol . 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al . , U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S.
• Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 9717450 and W09717451.
• DNA molecules for use in transforming P . methanolica will commonly be 45
• the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P . methanolica alcohol utilization gene (AUG1 or AUG2) .
• Other useful promoters include those of the dihydroxyacetone synthase (DHAS) , formate dehydrogenase (FMD) , and catalase (CAT) genes.
• DHAS dihydroxyacetone synthase
• FMD formate dehydrogenase
• CAT catalase
• a preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine .
• P. methanolica ADE2 gene which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine .
• AIRC phosphoribosyl-5-aminoimidazole carboxylase
• Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant ( ⁇ ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
• Prokaryotic host cells including strains of the bacteria Escherichia col i , Bacill us and other genera are also useful as host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) . When expressing 46
• the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
• the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
• the denatured polypeptide can then be refolded and dimerized by diluting the denaturant , such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
• the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
• Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells .
• suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
• the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
• P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors .
• a preferred culture medium for P. methanolica is YEPD (2% D-glucose, 47
• polypeptides of the present invention it is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
• a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
• Expressed recombinant ZTMPO-1 polypeptides can be purified using fractionation and/or conventional purification methods and media.
• Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
• Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography .
• Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
• Exemplary chromatographic media include those media derivatized with phenyl , butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA) , Octyl -Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
• Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino 48
• coupling chemistries include cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries.
• solid media are well known and widely used in the art, and are available from commercial suppliers.
• Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography : Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
• the polypeptides of the present invention can be isolated by exploitation of their binding 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. 1:1-7, 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
• ZTMPO-1 polypeptides may be prepared as monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; and may or may not include an initial methionine amino acid residue.
• 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. 41:161-89, 1994; and Douglas and Curiel, Science & Medicine 4 . : 44 -53) .
• adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts may be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
• the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary) .
• the host cell the human 293 cell line is exemplary.
• adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will 50
• the heterologous protein express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
• the adenovirus system can also be used for protein production in vi tro .
• the cells can produce proteins for extended periods of time.
• BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest.
• the cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
• adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al . , Cvtotechnol . 11:145-55, 1994).
• ZTMPO-1 polypeptides, agonists and antagonists have potential in both in vitro and in vivo applications.
• ZTMPO-1 is expressed ubiquitously, many of those tissues are characterized by a high rate of cellular 51
• ZTMPO-1 polypeptides would find use as regulators of cellular proliferation and/or differentiation.
• Proliferation and differentiation can be measured using cultured cells or in vivo by administering molecules of the present invention to the appropriate animal model. Suitable cultured cells, include but are not limited to, testicular, muscle, lymphatic and tumor cell lines which are all readily available to one skilled in the art from such sources as American Type Culture Collection, Rockville, MD.
• proliferation can be measured using cultured cardiac cells or in vivo by administering molecules of the present invention to the appropriate animal model.
• proliferative effects are seen as an increase in cell number, and may include inhibition of apoptosis as well as stimulation of mitogenesis.
• Cultured cells for use in these assays include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, and human umbilical vein endothelial cells from primary cultures. Suitable established cell lines include: NIH 3T3 fibroblasts (ATCC No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC No. CRL-1446), Shionogi mammary carcinoma cells (Tanaka et al . , Proc. Natl. Acad. Sci. 89 : 8928-32 , 1992), and LNCap . FGC adenocarcinoma cells (ATCC No. CRL- 1740).
• Cultured testicular cells include dolphin DBl.Tes cells (ATCC No. CRL- 6258) ; mouse GC-1 spg cells (ATCC No. CRL-2053); TM3 cells (ATCC No. CRL-1714); TM4 cells (ATCC No. CRL-1715) ; and pig ST cells (ATCC No. CRL-1746) .
• Mouse skeletal muscle ATCC No. CRL-2174
• human muscle ATCC No. CRL-7522
• Raji (Burkitt's human lymphoma, ATCC No. CCL86)
• Ramos Burkitt's lymphoma cell line, ATCC No.
• assays measuring proliferation include chemosensitivity to neutral red dye (Cavanaugh et al . , Investigational New Drugs 1:347-54, 1990), incorporation of radiolabelled nucleotides (Cook et al . , Analytical Biochem. 179 :l-7, 1989), incorporation of 5-bromo-2'- deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al . , J. Immunol. Methods 12:169-79, 1985), and use of tetrazolium salts (Mosmann, J. Immunol. Methods £5:55-63, 1983; Alley et al . , Cancer Res .
• Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 1:281-4, 1991; Francis, Differentiation £2:63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-71, 1989) .
• Bioassays and ELISAs are available to measure cellular response to ZTMPO-1, in particular are those which measure changes in cytokine production as a measure of cellular response (see for example, Current Protocols in Immunology ed. John E. Coligan et al . , NIH, 1996) .
• In vivo assays are available for evaluating cardiac neogenesis or hyperplasia include treating neonatal and mature rats with the molecules of the present invention.
• the animals' cardiac function is measured as heart rate, blood pressure, and cardiac output to determine left ventricular function.
• Post-mortem methods for assessing cardiac decline or improvement include: increased or decreased cardiac weight, nuclei/cytoplasmic volume, and staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al . , Circulation Res. 53
• Cardiac defects related to conduction have been reported in patients having a deleted emerin gene (Emery, J. Med. Genet. 2£:637-41, 1989).
• the resulting cardiac conduction defect is life threatening in these patients.
• Defects in the intrinsic conduction system can cause irregularities in the heart rhythm, such as arrhythmia and fibrillation.
• Tissue distribution and sequence similarities between emerin and ZTMPO-1 suggest that ZTMPO-1 may be involved in re-polarization of cardiac cell membranes. Localization of emerin to the desmosomes and fasciae adherentes suggests that association with the connection between epithelial cells accounts for the cardiac conduction defect when the gene is absent.
• ZTMPO-1 polypeptides and antagonists may influence cell- cell communication, either independently, or in conjunction with other proteins, such as emerin, and may regulate messages between cell membranes.
• agonists or antagonists of the present invention such ZTMPO-1 polypeptides, agonists or antagonists are evaluated with respect to their ability to modulate cardiac conductance according to procedures known in the art. If desired, ZTMPO-1 polypeptide performance in this regard can be compared to emerin and may be evaluated in combination with emerin to identify synergistic effects.
• a resulting increase or decrease is measured by assessing voltage-dependent conductance, sodium or calcium ion flux in an appropriate assay system known in the art.
• Changes in the voltage conductance or in indicator substrates reflect the activities of ZTMPO-1 polypeptides on enhancing or inhibition cardiac conductance relative to a control not subjected to treatment.
• An electrocardiograph is used to monitor the electrical currents generated and transmitted through the heart. Changes in the electrocardiogram (ECG) 54
• tracing (wave pattern and/or timing) would indicate an alteration in the heart's conduction system. Therefore a return to a normal ECG pattern following ZTMPO-1 administration would indicate a re-establishment of a regular heart rhythm.
• the invention also provides isolated and purified ZTMPO-1 polynucleotide probes or primers.
• polynucleotide probes can be RNA or DNA.
• DNA can be either cDNA or genomic DNA.
• Polynucleotide probes are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least 16 nucleotides, more often from 17 nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion, domain or even the entire ZTMPO-1 gene or cDNA.
• Probes and primers are generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) can be used. PCR primers are at least 5 nucleotides in length, preferably 15 or more nucleotides, more preferably 20-30 nucleotides. Short polynucleotides can be used when a small region of the gene is targeted for analysis. For gross analysis of genes, a polynucleotide probe may comprise an entire exon or more.
• Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
• Preferred regions from which to construct probes include regions of homology with other thymopoietins and emerin as described herein, the 55
• ankyrin-like region the calcium binding protein-like region, the signal sequence, and the like.
• Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1991.
• ZTMPO-1 polypeptides may be used within diagnostic systems to detect the presence of ZTMPO-1.
• the information derived from such detection methods would provide insight into the significance of ZTMPO-1 polypeptides in various diseases, and as a would serve as diagnostic tools for diseases for which altered levels of ZTMPO-1 are significant.
• Altered levels of ZTMPO-1 receptor polypeptides may be indicative of pathological conditions including cancer, cardiac and autoimmune disorders and infectious diseases.
• RNA isolated from a biological sample
• RNA isolated from a biological sample
• RNA detection include northern analysis and dot/slot blot hybridization (see, for example, Ausubel ibid, and Wu et al . (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997)) .
• Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S .
• ZTMPO-1 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 56
• nonradioactive moieties include biotin, fluorescein, and digoxigenin.
• ZTMPO-1 oligonucleotide probes are also useful for in vivo diagnosis.
• 18 F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al . , Nature Medicine 4:467, 1998).
• PCR polymerase chain reaction
• PCR primers can be designed to amplify a sequence encoding a particular ZTMPO-1 domain or region of homology as described herein.
• RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with ZTMPO-1 primers (see, for example, Wu et al . (eds.),
• RNA is isolated from biological sample using, for example, the guanidinium- thiocyanate cell lysis procedure described above.
• a solid-phase technique can be used to isolate mRNA from a cell lysate.
• Oligo-dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences.
• ZTMPO-1 sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically at least 5 bases in length.
• PCR amplification products can be detected using a variety of approaches.
• PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
• fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled ZTMPO-1 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.
• a fluorogenic probe consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers.
• the reporter dye is separated from the quencher dye and a sequence-specific signal is generated and increases as amplification increases.
• the fluorescence intensity can be continuously monitored and quantified during the PCR reaction.
• CPT cycling probe technology
• NASBA nucleic acid sequence-based amplification
• CATCH cooperative amplification of templates by cross- hybridization
• LCR ligase chain reaction
• ZTMPO-1 probes and primers can also be used to detect and to localize ZTMPO-1 gene expression in tissue samples.
• Methods for such in si tu hybridization are well- known to those of skill in the art (see, for example, Choo (ed.), In Situ Hybridization Protocols, Humana Press, Inc., 1994; Wu et al . (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Si tu Hybridization (RISH),” in Methods in Gene Biotechnology, CRC Press, Inc., pages 259-278, 1997 and Wu et al . (eds.), “Localization of DNA or Abundance of mRNA by Fluorescence In Si tu Hybridization (RISH),” in Methods in Gene Biotechnology, CRC Press, Inc., pages 279-289, 1997).
• RISH Radioactive In Si tu Hybridization
• the invention also provides antagonists or inhibitors of ZTMPO-1 activity.
• Such antagonists would include anti-ZTMPO-1 antibodies, soluble ZTMPO-1 receptors, as well as other peptidic and non-peptidic agents (including ribozymes) .
• Such antagonists would have use as research reagents for characterizing sites of ligand-receptor interaction.
• Antagonists would also find use in modulating cellular proliferation and differentiation such as in tumor growth and development. 59
• ZTMPO-1 antagonists would be useful for inhibiting spermatogenesis and sperm activation.
• Such ZTMPO-1 antagonists can be used for contraception in humans and animals, and in particular, domestic and zoological animals and livestock, where they would act to prevent fertilization of an egg.
• ZTMPO-1 antagonists could be used, for instance, in place of surgical forms of contraception (such as spaying and neutering) , and would allow for the possibility of future breeding of treated animals if desired.
• ZTMPO-1 antagonists could also be used to mediate immune response, for instance by boosting the humoral response in individuals at risk for an infectious disease or as a supplement to vaccination.
• ZTMPO-1 can be used to identify inhibitors
• test compounds are transfected into cells or possibly added to the assays disclosed herein to identify compounds that inhibit the activity of ZTMPO-1.
• samples can be tested for inhibition of ZTMPO-1 activity within a variety of assays designed to measure receptor binding or the stimulation/inhibition of ZTMPO-1-dependent cellular responses.
• ZTMPO- 1-responsive cell lines can be transfected with a reporter gene construct that is responsive to a ZTMPO-1-stimulated cellular pathway. Reporter gene constructs of this type are known in the art, and will generally comprise a ZTMPO- 1-DNA response element operably linked to a gene encoding an assayable protein, such as luciferase.
• compounds or other samples can be tested for direct blocking of ZTMPO-1 binding to receptor using ZTMPO-1 tagged with a detectable label (e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like) .
• a detectable label e.g., 125 I, biotin, horseradish peroxidase, FITC, and the like
• Receptors used within binding assays may be cellular receptors or isolated, immobilized receptors.
• ZTMPO-1 polypeptides can also be used to prepare antibodies that specifically bind to ZTMPO-1 epitopes, peptides or polypeptides.
• the ZTMPO-1 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
• Suitable antigens would be the ZTMPO-1 polypeptide encoded by SEQ ID NO : 2 from amino acid number 1 to amino acid number 876, or contiguous 9 to 25 amino acid residue fragments thereof.
• Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al .
• polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a ZTMPO-1 polypeptide or a fragment thereof.
• the immunogenicity of a ZTMPO-1 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
• Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZTMPO-1 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
• the polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like" , such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
• a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
• antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 anc ⁇ Fab proteolytic fragments.
• Non-human antibodies may be humanized by grafting non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody) .
• humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper 62
• Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
• These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
• Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , US Patent NO. 5,223,409; Ladner et al . , US Patent NO. 4,946,778; Ladner et al . , US Patent NO. 5,403,484 and
• Random peptide display libraries can be screened using the ZTMPO-1 sequences disclosed herein to identify proteins which bind to ZTMPO-1. These "binding proteins" which interact with ZTMPO-1 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; 63
• binding proteins can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as ZTMPO-1 "antagonists" to block ZTMPO-1 binding and signal transduction in vi tro and in vivo . These anti-ZTMPO-1 binding proteins would be useful for inhibiting binding.
• Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules.
• antibodies herein specifically bind if they bind to a ZTMPO-1 polypeptide, peptide or epitope with a binding affinity
• binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann .
• antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect ZTMPO-1 but not known related polypeptides using a standard Western blot analysis
• antibodies may be 64
• screened against known related polypeptides, such as non-human ZTMPO-1, and ZTMPO-1 mutant polypeptides, to isolate a population that specifically binds to the inventive polypeptides.
• antibodies raised to ZTMPO-1 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to ZTMPO-1 will flow through the matrix under the proper buffer conditions .
• Screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al . (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995).
• assays known to those skilled in the art can be utilized to detect antibodies and binding proteins which specifically bind to ZTMPO-1 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant ZTMPO-1 protein or polypeptide. 65
• Antibodies to ZTMPO-1 may be used for tagging cells that express ZTMPO-1; for isolating ZTMPO-1 by affinity purification; for diagnostic assays for determining circulating levels of ZTMPO-1 polypeptides; for detecting or quantitating soluble ZTMPO-1 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZTMPO-1 binding in vi tro and in vivo .
• Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates .
• Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
• antibodies to ZTMPO-1 or fragments thereof may be used in vi tro to detect denatured ZTMPO-1 or fragments thereof in assays, for example, Western Blots or other assays known in the art.
• Antibodies or polypeptides herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
• polypeptides or antibodies of the present invention may be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance) .
• ZTMPO-1 polypeptides or anti-ZTMPO-1 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having 66
• Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
• Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like) , as well as therapeutic radionuclides, such as iodine- 131, rhenium- 188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance) .
• Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
• cytotoxic drugs such as adriamycin.
• the detectable or cytotoxic molecule may be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
• biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
• Molecules of the present invention can be used to identify and isolate receptors involved in ZTMPO-1 binding.
• proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques, Hermanson et al . , eds., Academic Press, San Diego, CA, 1992, pp.195-202).
• Proteins and peptides can also be radiolabeled (Methods in Enzymol . , vol. 182, "Guide to Protein Purification", M. Deutscher, ed. , Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled (Brunner et al . , Ann. Rev. Biochem.£ . :483-514, 1993 and Fedan et al . , Biochem. Pharmacol. 11:1167-80, 1984) and specific cell-surface proteins can be identified. 67
• the molecules of the present invention will be useful regulators in multiple cellular organisms.
• the molecules of the present invention may be used to modulate cellular proliferation and differentiation, for example spermatogenesis.
• certain proliferative disorders such as cancers may be amenable to such diagnosis, treatment or prevention.
• ZTMPO-1 would be useful in modulating the cell cycle such as during differentiation or in rapidly proliferating cells such as in tumor tissues.
• ZTMPO-1 would find application in a diverse array of tissues as testis, skeletal muscle, thyroid and adrenal gland for example.
• Polynucleotides encoding ZTMPO-1 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit ZTMPO-1 activity. If a mammal has a mutated or absent ZTMPO-1 gene, the ZTMPO-1 gene can be introduced into the cells of the mammal . In one embodiment, a gene encoding a ZTMPO-1 polypeptide is introduced in vivo in a viral vector.
• viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV) , papillomavirus , Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV), and the like.
• Defective viruses which entirely or almost entirely lack viral genes, are preferred.
• a defective virus is not infective after introduction into a cell .
• Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1
• HSV1 vector Keratt et al . , Molec. Cell. Neurosci. 2:320-30, 1991
• an attenuated adenovirus vector such as the vector described by Stratford-Perricaudet et al . , J. Clin. Invest. 10:626-30, 1992
• a defective adeno- associated virus vector (Samulski et al . , J. Virol. £1:3096-101, 1987; Samulski et al . , J. Virol. £1:3822-8, 1989) .
• a ZTMPO-1 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . Cell H:153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol.£:1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al .
• the vector can be introduced 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. Natl. Acad.
• lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
• Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
• Lipids may be chemically coupled to other molecules for the purpose of targeting.
• Targeted peptides e.g., hormones or neurotransmitters
• proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.
• DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use 69
• the present invention also provides reagents for use in diagnostic applications.
• the ZTMPO-1 gene, a probe comprising ZTMPO-1 DNA or RNA, or a subsequence thereof can be used to determine if the ZTMPO- 1 gene is present on chromosome 12 or if a mutation has occurred.
• Detectable chromosomal aberrations at the ZTMPO-1 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level .
• these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
• Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA.
• the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO : 1 , the complement of SEQ ID N0:1, or an RNA equivalent thereof. Suitable assay 70
• RFLP restriction fragment length polymorphism
• STR short tandem repeat
• PCR techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5- 16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid.; Marian,
• Ribonuclease protection assays comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase . Hybridized regions of the RNA are protected from digestion.
• PCR assays a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient.
• Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991) .
• SSCP single strand conformational polymorphism
• the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods.
• Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours.
• compositions will include a ZTMPO-1 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
• a pharmaceutically acceptable vehicle such as saline, buffered saline, 5% dextrose in water or the like.
• Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.
• Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, PA, 19th ed. , 1995. Determination of dose is within the level of ordinary skill in the art.
• the proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years.
• Evaluation of therapeutic effect of ZTMPO-1 for cardiac applications can be done by looking for changes in ECG. Decreases in creatine kinase levels and a decrease in weakness would serve as indicators for changes in muscle wasting associated with muscular dystrophy.
• the invention is further illustrated by the following non-limiting examples.
• Novel ZTMPO-1 encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST database. To identify the corresponding cDNA, two clones from which an identified EST was derived that were considered likely to contain the entire human ZTMPO-1 sequence were used for sequencing. Using a QIAwe11 8 plasmid kit (Qiagen, Inc., Chatsworth, CA) according to manufacturer's instructions, a 5 ml overnight culture in LB + 50 ⁇ g/ml ampicillin was prepared. The templates were sequenced on an Applied 72
• Oligonucleotides ZC15976 (SEQ ID N0:11), ZC15485 (SEQ ID N0:12), ZC15526 (SEQ ID N0:13), Z15620 (SEQ ID N0:15) and ZC15823 (SEQ ID NO: 16) were used to complete the sequence from the clones.
• Sequencing reactions were carried out in a Hybaid OmniGene Temperature Cycling System (National Labnet Co., Woodbridge, NY). SequencherTM 3.0 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI) was used for data analysis. The sequences from the two clones overlapped by 740 bp and contained the 3 ' end of the gene and the poly A tail . A third clone prepared as described above was sequenced resulting in the remaining 5' sequence. Oligonucleotides ZC447 (SEQ ID NO:14), ZC976 (SEQ ID NO:10), ZC16162 (SEQ ID NO: 17), ZC16038 (SEQ ID
• ZC16163 (SEQ ID NO:21), ZC16165 (SEQ ID NO:22) and ZC16037
• SEQ ID NO: 23 were used in sequencing. Differences between the original EST sequences and the final sequence of ZTMPO-1 were detected. The lack of identity arose from ambiguity in the original EST sequences.
• RACE reaction was carried out at 94°C, for 2 minutes, followed by 25 cycles at 94°C for 15 seconds, 61°C for 20 73
• PCR products from the first round reaction were diluted 1/100 and used as templates for a second round of PCR using oligonucleotides AP2 (SEQ ID NO: 32, supplied with the Marathon Kit or synthesized) and ZC15526 (SEQ ID NO: 13) as primers.
• the PCR derived DNA fragments were resolved by gel electrophoresis , excised and ligated into the expression vector was the vector pCR2.1 (TA Cloning Kit, Invitrogen Inc., San Diego, CA) according to manufacturer's instructions.
• the sequence of the inserts was confirmed by sequence analysis using oligos ZC694 (SEQ ID NO:9) and ZC695 (SEQ ID NO:26) as primers, as described above and confirmed that the Met
• amino acid residue 1 of SEQ ID NO : 2 was indeed the start methionine.
• the resulting 2,754 bp polynucleotide (SEQ ID NO:l) had an open reading frame encoding an 876 amino acid residue protein sequence (SEQ ID NO: 2) and was designated ZTMPO-1.
• the probe was purified using a NUCTRAP push column (Stratagene) .
• EXPRESSHYB (Clontech) solution was 74
• ZTMPO-1 was mapped to chromosome 12 using the commercially available GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) .
• the GeneBridge 4 Radiation Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) .
• a publicly available WWW server http://www-genome.wi.mit.edu/cgi-bin/contig/ rhmapper.pl) allows mapping relative to the Whitehead
• the PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 95°C, 35 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 62°C and 1.5 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72°C.
• the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD) .

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