EP1051488A2 - Menschliche wachstumsfaktor-homologe - Google Patents

Menschliche wachstumsfaktor-homologe

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Publication number
EP1051488A2
EP1051488A2 EP99902197A EP99902197A EP1051488A2 EP 1051488 A2 EP1051488 A2 EP 1051488A2 EP 99902197 A EP99902197 A EP 99902197A EP 99902197 A EP99902197 A EP 99902197A EP 1051488 A2 EP1051488 A2 EP 1051488A2
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EP
European Patent Office
Prior art keywords
seq
hgfh
fragment
polynucleotide
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP99902197A
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English (en)
French (fr)
Inventor
Jennifer L. Hillman
Preeti Lal
Neil C. Corley
Mariah Baughn
Karl J. Guegler
Y. Tom Tang
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Incyte Corp
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Incyte Pharmaceuticals Inc
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Application filed by Incyte Pharmaceuticals Inc filed Critical Incyte Pharmaceuticals Inc
Publication of EP1051488A2 publication Critical patent/EP1051488A2/de
Withdrawn legal-status Critical Current

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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
    • C07K14/475—Growth factors; Growth regulators
    • 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

Definitions

  • This invention relates to nucleic acid and amino acid sequences of human growth factor homologs, and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and developmental disorders.
  • Intercellular communication is essential for the development and survival of multicellular organisms. Communication is achieved through the secretion of proteins by signaling cells and the intemalization of these proteins by their target cells.
  • Growth factors are an example of secreted proteins that mediate communication between signaling and target cells. Inside the signaling cell, growth factors are synthesized and transported through the secretory pathway. Entry into the secretory pathway is mediated by the signal peptide sequence, a protein sorting motif at the N-terminus of most secreted proteins. Within the secretory pathway, the signal sequence is proteolytically removed from its cognate growth factor. Most growth factors also undergo further post-translational modifications within the secretory pathway. These modifications can include glycosylation, phosphorylation, and intramolecular disulfide bond formation.
  • growth factors Following their secretion into the extracellular space, some growth factors oligomerize or associate with extracellular matrix components.
  • the secreted growth factors bind to specific receptors on the surfaces of their target cells, and the bound receptors trigger second messenger signal transduction pathways. These signal transduction pathways elicit specific cellular responses in the target cells. These responses can include the modulation of gene expression and the stimulation or inhibition of cell division, cell differentiation, and cell motility.
  • growth factors are local mediators that act on cells in the immediate environment. Such local activity is maintained by physical proximity of the signaling cell to its target cell, sequestration of the growth factor by extracellular matrix components, intemalization and degradation of the growth factor by the target cell, and exclusion of the growth factor from circulation.
  • Growth factors fall into three broad and overlapping classes. The first and broadest class includes the large polypeptide growth factors, which are wide-ranging in their effects. These factors include epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor- ⁇ (TGF- ⁇ ), insulin-like growth factor (IGF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF), each defining a family of numerous related factors.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • IGF insulin-like growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • the large polypeptide growth factors generally act as mitogens on diverse cell types to stimulate wound healing, bone synthesis and remodeling, extracellular matrix synthesis, and proliferation of epithelial, epidermal, and connective tissues. Some members of the TGF- ⁇ , EGF, and FGF families also function as inductive signals in the differentiation of embryonic tissue. However, some of the large polypeptide growth factors carry out specific functions on a restricted set of target tissues. For example, mouse growth/differentiation factor 9 (GDF-9) is a TGF- ⁇ family member that is expressed solely in the ovary. (McPherron, A. C. and Lee, S.-J. (1993) J. Biol. Chem. 268:3444-3449.) NGF functions specifically as a neurotrophic factor, promoting neuronal growth and differentiation.
  • GDF-9 mouse growth/differentiation factor 9
  • the second class of growth factors includes the hematopoietic growth factors, which have a narrow target specificity. These factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors. These factors include the colony-stimulating factors (e.g., G-CSF, M-CSF, GM-CSF, and CSF1-3), erythropoietin, and the cytokines. Cytokines are specialized hematopoietic factors secreted by cells of the immune system in response to external insults, such as tissue damage and viral or microbial infection.
  • hematopoietic growth factors which have a narrow target specificity. These factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, bas
  • Cytokines function in tissue repair, inflammation, and modulation of the immune response. Cytokines include the interleukins, IL1-IL13; the interferons, IFN- ⁇ , - ⁇ , and -y; and the tumor necrosis factors, TNF- ⁇ and - ⁇ .
  • the third class of growth factors includes the small peptide factors, which primarily function as hormones in the regulation of highly specialized processes other than cellular proliferation. These factors, which are typically less than 20 amino acids in length, are generated by the proteolytic processing of larger precursor proteins.
  • Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Overexpression of the large polypeptide growth factors promotes the proliferation and transformation of cells in culture. Moreover, tumor cells and normal cells show differential expression of certain growth factors. For example, northern analysis shows the expression of human hepatoma-derived growth factor messenger RNA at various levels in hepatoma and other transformed cell lines and in normal tissues. (Nakamura, H. et al. (1994) J. Biol. Chem. 269:25143-25149.) Inappropriate expression of large polypeptide growth factors by tumor cells in vivo may contribute to vascularization and metastasis of tumors.
  • FGF and PDGF family members are themselves homologous to oncoproteins, whereas receptors for some members of the EGF, NGF, and FGF families are encoded by proto-oncogenes. Growth factors also affect the transcriptional regulation of both proto-oncogenes and oncosuppressor genes. (Pimentel, supra.)
  • Twisted Gastrulation is a secreted protein similar to human connective tissue growth factor, a PDGF-related protein induced by TGF- ⁇ activity.
  • TSG protein is required for the specification of certain dorsal cell fates in the fruit fly embryo. This specification contributes to the establishment of the dorsoventral axis of the embryo. The dorsoventral axis, in turn, provides the framework for the entire adult body plan. Disruption of TSG activity is lethal with the manifestation of developmental defects commencing at the time of gastrulation. (Mason, E. D. et al. (1994) Genes Dev. 8:1489-1501; and Zusman, S. B. and Weischaus, E. F. (1985) Dev. Biol. 111 :359-371.)
  • the invention features substantially purified polypeptides, human growth factor homologs, referred to collectively as "HGFH” and individually as “HGFH-1,” “HGFH-2,” “HGFH-3,” and “HGFH-4.”
  • HGFH human growth factor homologs
  • the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO.T, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, and a fragment of SEQ ID NO:7.
  • the invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO: 7, or to a fragment of any of these sequences.
  • the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, and a fragment of SEQ ID NO:7.
  • the invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, and a fragment of SEQ ID NO:7.
  • the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, and a fragment of SEQ ID NO:7, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO: 1 , a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, and a fragment of SEQ ID NO:7.
  • the invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:6, and a fragment of SEQ ID NO:8.
  • the invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:6, and a fragment of SEQ ID NO:8, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:6, and a fragment of SEQ ID NO: 8.
  • the invention also provides a polynucleotide fragment useful for designing oligonucleotides or to be used as a hybridization probe comprising a polynucleotide sequence selected from the group consisting of nucleotides 535-570 of SEQ ID NO:2, nucleotides 124-156 of SEQ ID NO:4, nucleotides 30-62 of SEQ ID NO:6, and nucleotides 147-182 of SEQ ID NO: 8.
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO: 1 , a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, and a fragment of SEQ ID NO:7.
  • the expression vector is contained within a host cell.
  • the invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO.T, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, or a fragment of SEQ ID NO: 7, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, or a fragment of SEQ ID NO:7 in conjunction with a suitable pharmaceutical carrier.
  • the invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, or a fragment of SEQ ID NO:7, as well as a purified agonist and a purified antagonist to the polypeptide.
  • the invention also provides a method for treating or preventing a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of HGFH.
  • the invention also provides a method for treating or preventing a developmental disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified HGFH.
  • the invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO.T , a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, or a fragment of SEQ ID NO:7 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising SEQ ID NO.T, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:5, or a fragment of SEQ ID NO:7 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein
  • Figures 1 A, IB, and IC show the amino acid sequence alignment between HGFH- 1 (862403; SEQ ID NOT) and mouse growth and differentiation factor 9 (GI 567206; SEQ ID NO:9).
  • Figures 2A and 2B show the amino acid sequence alignment between HGFH-2 (2676869; SEQ ID NO:3) and human hepatoma-derived growth factor (GI 598956; SEQ ID NO: 10).
  • Figures 3A, 3B, 3C, and 3D show the amino acid sequence alignment between HGFH-2 and HGFH-2 (2676869; SEQ ID NO:3) and human hepatoma-derived growth factor (GI 598956; SEQ ID NO: 10).
  • Figures 3A, 3B, 3C, and 3D show the amino acid sequence alignment between
  • HGFH-3 (1568019; SEQ ID NO:5) and yeast Yhr074wp (GI 500832; SEQ ID NOT 1).
  • Figures 4A and 4B show the amino acid sequence alignment between HGFH-4 (3577857; SEQ ID NO:7) and fruit fly Twisted Gastrulation (GI 529900; SEQ ID NO: 12). The alignments were produced using the multisequence alignment program of DNASTARTM software (DNASTAR Inc, Madison WI).
  • HGFH refers to the amino acid sequences of substantially purified HGFH obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which, when bound to HGFH, increases or prolongs the duration of the effect of HGFH.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of HGFH.
  • alleles are an alternative form of the gene encoding HGFH. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding HGFH include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same HGFH or a polypeptide with at least one functional characteristic of HGFH. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HGFH, and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HGFH.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HGFH. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of HGFH is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
  • amino acid or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
  • fragments or “anti genie fragments” refer to fragments of HGFH which are preferably about 5 to about 15 amino acids in length and which retain some biological activity or immunological activity of HGFH.
  • amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art. (See, e.g., Dieffenbach, C.W. and G.S. Dveksler (1995) PCR Primer, a Laboratory Manual. Cold Spring Harbor Press, Plainview, NY, pp.1-5.)
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which, when bound to HGFH, decreases the amount or the duration of the effect of the biological or immunological activity of HGFH.
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of HGFH.
  • the term "antibody” refers to intact molecules as well as to fragments thereof, such as Fa, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
  • Antibodies that bind HGFH polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • antigenic determinant refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition containing a nucleic acid sequence which is complementary to a specific nucleic acid sequence.
  • the term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
  • Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant, or synthetic HGFH, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementary or “complementarity,” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base pairing. For example, the sequence “A-G-T” binds to the complementary sequence "T-C-A.” Complementarity between two single-stranded molecules may be “partial,” such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence,” as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation, an aqueous solution, or a sterile composition.
  • Compositions comprising polynucleotide sequences encoding HGFH or fragments of HGFH may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GEL VIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence .
  • the term "correlates with expression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding HGFH, by northern analysis is indicative of the presence of nucleic acids encoding HGFH in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding HGFH.
  • a “deletion,” as the term is used herein, refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to the chemical modification of HGFH, of a polynucleotide sequence encoding HGFH, or of a polynucleotide sequence complementary to a polynucleotide sequence encoding HGFH. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains a at least one biological or immunological function of the polypeptide from which it was derived.
  • homoology refers to a degree of complementarity.
  • the word "identity” may substitute for the word "homology.”
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially homologous.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency.
  • a substantially homologous sequence or hybridization probe will compete for and inhibit the binding of a completely homologous sequence to the target sequence under conditions of reduced stringency.
  • Percent identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MegAlign program (Lasergene software package, DNASTAR, Inc., Madison WI). The MegAlign program can create alignments between two or more sequences according to different methods, e.g., the Clustal Method. (Higgins, D.G. and P. M. Sharp (1988) Gene 73:237-244.) The Clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
  • the percentage similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be calculated by the Clustal Method, or by other methods known in the art, such as the Jotun Hein Method. (See, e.g., Hein, J. (1990) Methods in Enzymology 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance. (See, e.g., Harrington, J.J. et al. (1997) Nat Genet. 15:345-355.)
  • humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion or “addition,” as used herein refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • microarray refers to an array of distinct polynucleotides or oligonucleotides arrayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • modulate refers to a change in the activity of HGFH. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HGFH.
  • nucleic acid or “nucleic acid sequence,” as used herein, refer to an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA- like or RNA-like material.
  • fragments refers to those nucleic acid sequences which are greater than about 60 nucleotides in length, and most preferably are at least about 100 nucleotides, at least about 1000 nucleotides, or at least about 10,000 nucleotides in length.
  • operably associated refers to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the encoded polypeptide but still bind to operator sequences that control expression of the polypeptide.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
  • oligonucleotide is substantially equivalent to the terms “amplimers,” “primers,” “oligomers,” and “probes,” as these terms are commonly defined in the art.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentially bind complementary single stranded DNA and RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)
  • sample is used in its broadest sense.
  • a biological sample suspected of containing nucleic acids encoding HGFH, or fragments thereof, or HGFH itself may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solid support; a tissue; a tissue print; etc.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • stringent conditions refers to conditions which permit hybridization between polynucleotide sequences and the claimed polynucleotide sequences.
  • stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • hybridization under high stringency conditions could occur in about 50% formamide at about 37°C to 42°C.
  • Hybridization could occur under reduced stringency conditions in about 35% to 25% formamide at about 30°C to 35°C.
  • hybridization could occur under high stringency conditions at 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and 200 ⁇ g/ml sheared and denatured salmon sperm DNA.
  • Hybridization could occur under reduced stringency conditions as described above, but in 35% formamide at a reduced temperature of 35°C.
  • the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • Transformation describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, and refers to cells which transiently express the inserted DNA or RNA for limited periods of time.
  • a “variant" of HGFH refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have
  • nonconservative changes e.g., replacement of glycine with tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.
  • the invention is based on the discovery of new human growth factor homologs (HGFH), the polynucleotides encoding HGFH, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative and developmental disorders.
  • HGFH human growth factor homologs
  • Nucleic acids encoding the HGFH-1 of the present invention were first identified in Incyte Clone 862403 from the brain tumor cDNA library (BRAITUT03) using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:2 was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 862403 and 860725 (BRAITUT03) and shotgun sequences SAAB00148, SAAC00081, SAAB00364, SAAB00198, and SZZZ00645.
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • HGFH-1 is 446 amino acids in length and has five potential N-glycosylation sites at N 106 , N 228 , N 247 , N 260 , and N 330 ; one potential cAMP- and cGMP-dependent protein kinase phosphorylation site at S 280 ; three potential casein kinase II phosphorylation sites at S 28 , T ]48 , and S 193 ; six potential protein kinase C phosphorylation sites at S 76 , S 105 , T n3 , S I7g , S 2I8 , and T 444 ; a potential signal peptide sequence from M, to P 20 ; and a TGF- ⁇ family signature from I 363 to C 37g .
  • HGFH-1 has chemical and structural homology with mouse growth/differentiation factor 9 (GDF-9), a member of the TGF- ⁇ family (GI 567206; SEQ ID NO:9).
  • GDF-9 mouse growth/differentiation factor 9
  • HGFH-1 and mouse GDF-9 share 66% identity overall and 95% identity in their C-terminal regions from A 327 to R 446 of HGFH-1.
  • cysteines important for intramolecular disulfide bond formation in GDF-9 are conserved in HGFH-1 at C 345 , C 374 , C 378 , C 411 , C 443 , and C 445 .
  • S 405 of GDF-9 which distinguishes GDF-9 from other TGF- ⁇ -like proteins that contain a cysteine at this position, is conserved in HGFH-1 at S 4I0 .
  • the potential N-glycosylation sites at N 228 , N 260 , and N 330 ; the potential phosphorylation sites at S 28 , S 76 , T I 13 , T 148 , S 178 , and T 444 ; and the potential signal peptide sequence of HGFH-1 are conserved in GDF-9.
  • the fragment of SEQ ID NO:2 from about nucleotide 535 to nucleotide 570 is useful for designing oligonucleotides or to be used directly as a hybridization probe.
  • Northern analysis shows the expression of this sequence in a cDNA library derived from tumorous brain tissue.
  • Nucleic acids encoding the HGFH-2 of the present invention were first identified in Incyte Clone 2676869 from the kidney cDNA library (KIDNNOT19) using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:4 was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 2676869 (KIDN OT19), 488482 (HNT2AGT01), 2571655 (HIPOAZT01), 2150727 (BRAINOT09), 899636 (BRSTTUT03), 2311937 (NGANNOT01), 2613941 (THYRNOT09), 776810 (COLNNOT05), 968484 (BRSTNOT05), 1292885 (PGANNOT03), and 2288692 (BRAINON01).
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:3.
  • HGFH-2 is 203 amino acids in length and has three potential N-glycosylation sites at N 176 , N 187 , and N 191 ; two potential cAMP- and cGMP-dependent protein kinase phosphorylation sites at S 149 and S 156 ; eight potential casein kinase II phosphorylation sites at S 106 , S 108 , T 110 , S 12) , S 122 , S, 62 , S 178 , and T 192 ; and six potential protein kinase C phosphorylation sites at S 75 , S 144 , T, 51 , S 152 , S ]55 , and S 159 .
  • HGFH-2 has chemical and structural homology with human hepatoma-derived growth factor (HDGF) (GI 598956; SEQ ID NO: 10).
  • HDGF human hepatoma-derived growth factor
  • HGFH-2 and HDGF share 49% identity.
  • the potential phosphorylation sites at S ⁇ o 6> S 122 , S 152 , and S 178 of HGFH-2 are conserved in HDGF.
  • the fragment of SEQ ID NO:4 from about nucleotide 124 to nucleotide 156 is useful for designing oligonucleotides or to be used directly as a hybridization probe.
  • Northern analysis shows the expression of this sequence in various libraries, at least 44% of which are associated with immortalized or cancerous tissue. Of particular note is the expression of HGFH-2 in neural tissue.
  • Nucleic acids encoding the HGFH-3 of the present invention were first identified in Incyte Clone 1568019 from the uterus cDNA library (UTRSNOT05) using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:6, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 1568019 (UTRSNOT05), 2504437 (CONUTUT01), 136194 (SYNORAB01), 1849286 (LUNGFET03) and shotgun sequences SAEA02891, SAEA03437, and SAFC02217.
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:5.
  • HGFH-3 is 706 amino acids in length and has four potential N-glycosylation sites at N 198 , N 25I , N 422 , and N 677 ; one potential cAMP- and cGMP-dependent protein kinase phosphorylation site at T 6 ; sixteen potential casein kinase 11 phosphorylation sites at T 151 , T 2U , S 62 , S 308 , S 327 , S 382 , T 401 , S 423 , S 474 , T 527 , S 532 , S 543 , S 584 , T 586> T 594 , and S 663 ; and six potential protein kinase C phosphorylation sites at T 220 , S 280 , S 286> T 462 , S 474 , and T 628 .
  • HGFH-3 has chemical and structural homology with yeast Yhr074wp (GI 500832; SEQ ID NO: 11 ), an open reading frame with similarity to prokaryotic sporulation outgrowth factor B.
  • yeast Yhr074wp GI 500832; SEQ ID NO: 11
  • HGFH-3 and Yhr074wp share 57% identity.
  • the potential N-glycosylation sites at N 198 , N 251 , and N 422 , and the potential phosphorylation sites at T 220 , S 262 , S 280 , S 327 , S 423 , T 462 , S 474 , T 527 , S 532 , S 543 , T 594 , T 628 , and S 663 of HGFH-3 are conserved in Yhr074wp.
  • the fragment of SEQ ID NO: 6 from about nucleotide 30 to nucleotide 62 is useful for designing oligonucleotides or to be used directly as a hybridization probe.
  • Northern analysis shows the expression of this sequence in various cDNA libraries, at least 39% of which are associated with cancerous or immortalized tissue and at least 31% with the immune response. Of particular note is the expression of HGFH-3 in reproductive and gastrointestinal tissues.
  • Nucleic acids encoding the HGFH-4 of the present invention were first identified in Incyte Clone 3577857 from the bronchial tissue cDNA library BRON OT01 using a computer search for amino acid sequence alignments.
  • a consensus sequence, SEQ ID NO:8, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 3577857 (BRONNOT01), 2799484 (NPOLNOT01), 1685583 (PROSNOT15), 1210438 (BRSTNOT02), 3601074 (DRGTNOT01), 3279304 (STOMFET02), and shotgun sequences SAFC00098, SAYA00353, and SAFC00552.
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:7.
  • HGFH-4 is 223 amino acids in length and has three potential N-glycosylation sites at N 52 , N g consult and N 147 ; six potential casein kinase II phosphorylation sites at S 91 , T 92 , T 10g , S I28 , S ]35 , and S 161 ; three potential protein kinase C phosphorylation sites at T 88 , S 161 , and T 2I5 ; and a potential signal peptide sequence from M, to S 25 .
  • HGFH-4 has chemical and structural homology with fruit fly Twisted Gastrulation (TSG) (GI 529900; SEQ ID NO: 12).
  • TSG fruit fly Twisted Gastrulation
  • HGFH-4 and TSG share 29% amino acid identity.
  • all 24 cysteine residues in HGFH-4 are conserved in TSG.
  • the potential signal peptide sequence, acidic isoelectric point, and high cysteine content (-10%) of TSG are conserved in HGFH-4.
  • the potential phosphorylation sites at T g8 and T 108 of HGFH-4 are conserved in TSG.
  • the fragment of SEQ ID NO: 8 from about nucleotide 147 to nucleotide 182 is useful for designing oligonucleotides or to be used directly as a hybridization probe.
  • Northern analysis shows the expression of HGFH-4 in various libraries, at least 45% of which are associated with cancerous or immortalized tissue and at least 24% with immune response. Of particular note is the expression of HGFH-4 in reproductive and neural tissues.
  • the invention also encompasses HGFH variants.
  • a preferred HGFH variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the HGFH amino acid sequence, and which contains at least one functional or structural characteristic of HGFH.
  • the invention also encompasses polynucleotides which encode HGFH.
  • the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO:2, which encodes an HGFH-1.
  • the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO:4, which encodes an HGFH-2.
  • the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO: 6, which encodes an HGFH-3.
  • the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID NO: 8, which encodes an HGFH-4.
  • the invention also encompasses a variant of a polynucleotide sequence encoding HGFH.
  • a variant polynucleotide sequence will have at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding HGFH.
  • a particular aspect of the invention encompasses a variant of SEQ ID NO:2 which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:2.
  • the invention further encompasses a polynucleotide variant of SEQ ID NO:4 having at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:4.
  • the invention further encompasses a polynucleotide variant of SEQ ID NO:6 having at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:6.
  • the invention further encompasses a polynucleotide variant of SEQ ID NO: 8 having at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID NO:8. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HGFH.
  • nucleotide sequences which encode HGFH and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HGFH under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HGFH or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode HGFH and HGFH derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding HGFH or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:6 or a fragment of SEQ ID NO:8, under various conditions of stringency.
  • SEQ ID NO:2 SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:6 or a fragment of SEQ ID NO:8, under various conditions of stringency.
  • Methods for DNA sequencing are well known and generally available in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp., Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE Amplification System (GlBCO/BRL, Gaithersburg, MD).
  • the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • the nucleic acid sequences encoding HGFH may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • one method which may be employed, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus.
  • genomic DNA is first amplified in the presence of a primer complementary to a linker sequence within the vector and a primer specific to the region predicted to encode the gene.
  • the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region.
  • the primers may be designed using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, MN) or another appropriate program to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to 72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations may be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
  • PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size-selected to include larger cDNAs it is preferable to use libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable in that they will include more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GenotyperTM and Sequence NavigatorTM, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode HGFH may be used in recombinant DNA molecules to direct expression of HGFH, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced, and these sequences may be used to clone and express HGFH.
  • HGFH-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • the nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HGFH-encoding sequences for a variety of reasons including, but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • natural, modified, or recombinant nucleic acid sequences encoding HGFH may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence For example, to screen peptide libraries for inhibitors of HGFH activity, it may be useful to encode a chimeric HGFH protein that can be recognized by a commercially available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the HGFH encoding sequence and the heterologous protein sequence, so that HGFH may be cleaved and purified away from the heterologous moiety.
  • sequences encoding HGFH may be synthesized, in whole or in part, using chemical methods well known in the art.
  • the protein itself may be produced using chemical methods to synthesize the amino acid sequence of HGFH, or a fragment thereof.
  • peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431 A Peptide Synthesizer (Perkin Elmer).
  • the newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography.
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing.
  • amino acid sequence of HGFH, or any part thereof may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • the nucleotide sequences encoding HGFH or derivatives thereof may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding HGFH. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic vims (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors e.g., insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • plant cell systems transformed with virus expression vectors e.g.
  • control elements are those non-translated regions, e.g., enhancers, promoters, and 5' and 3' untranslated regions, of the vector and polynucleotide sequences encoding HGFH which interact with host cellular proteins to carry out transcription and translation.
  • Such elements may vary in their strength and specificity.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters e.g., hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, La Jolla, CA) or pSportlTM plasmid (GlBCO/BRL
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable.
  • vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • a number of expression vectors may be selected depending upon the use intended for HGFH. For example, when large quantities of HGFH are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, multifunctional E.
  • coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding HGFH may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced, and pIN vectors.
  • Bluescript® Stratagene
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel, supra: and Grant et al.
  • HGFH a gene that encodes HGFH
  • promoters such as the 35 S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
  • constmcts can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews. (See, e.g., Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, NY; pp. 191-196.)
  • An insect system may also be used to express HGFH.
  • HGFH Autographa califomica nuclear polyhedrosis vims (AcNPV) is used as a vector to express foreign genes in Spodoptera fmgiperda cells or in Trichoplusia larvae.
  • the sequences encoding HGFH may be cloned into a non-essential region of the vims, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of sequences encoding HGFH will render the polyhedrin gene inactive and produce recombinant vims lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding HGFH may be ligated into an adenovims transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable vims which is capable of expressing HGFH in infected host cells.
  • transcription enhancers such as the Rous sarcoma vims (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma vims
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • HACs of about 6 kb to 10 Mb are constmcted and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HGFH. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding HGFH and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding, and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, MD) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • cell lines capable of stably expressing HGFH can be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex vims thymidine kinase genes and adenine phosphoribosyltransferase genes, which can be employed in tk or apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11 :223-232; and Lowy, I. et al. (1980) Cell 22:817-823) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • npt confers resistance to the aminoglycosides neomycin and G-418
  • als ox pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB, which allows cells to utilize indole in place of tryptophan, or hisD,
  • sequence encoding HGFH is inserted within a marker gene sequence
  • transformed cells containing sequences encoding HGFH can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding HGFH under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the
  • host cells which contain the nucleic acid sequence encoding HGFH and express HGFH may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane,
  • polynucleotide sequences encoding HGFH can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding HGFH.
  • oligonucleotides or oligomers based on the sequences encoding HGFH to detect transformants containing DNA or RNA encoding HGFH.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HGFH include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding HGFH, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • RNA polymerase such as T7, T3, or SP6
  • Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding HGFH may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode HGFH may be designed to contain signal sequences which direct secretion of HGFH through a prokaryotic or eukaryotic cell membrane. Other constmctions may be used to join sequences encoding HGFH to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp., Seattle, WA.
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA)
  • One such expression vector provides for expression of a fusion protein containing HGFH and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
  • the histidine residues facilitate purification on immobilized metal ion affinity chromatography.
  • IMAC immobilized metal ion affinity chromatography
  • the enterokinase cleavage site provides a means for purifying HGFH from the fusion protein. (See, e.g., Kroll, D.J. et al. (1993) DNA Cell Biol. 12:441-453.)
  • HGFH fragments of HGFH may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques.
  • Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • Various fragments of HGFH may be synthesized separately and then combined to produce the full length molecule.
  • HGFH-1 Chemical and structural homology exists between HGFH-1 and GDF-9 from mouse (GI 567206). In addition, HGFH-1 is expressed in cancerous tissues. Therefore, HGFH-1 appears to play a role in cell proliferative and developmental disorders. Chemical and structural homology exists between HGFH-2 and HDGF from human (GI 598956). In addition, HGFH-2 is expressed in cancerous tissues. Therefore, HGFH-2 appears to play a role in cell proliferative and developmental disorders.
  • HGFH-3 Chemical and structural homology exists between HGFH-3 and Yhr074wp from yeast (GI 500832). In addition, HGFH-3 is expressed in cancerous tissues. Therefore, HGFH-3 appears to play a role in cell proliferative and developmental disorders.
  • HGFH-4 Chemical and structural homology exists between HGFH-4 and TSG from fruit fly (GI 529900). In addition, HGFH-4 is expressed in cancerous tissues. Therefore, HGFH-4 appears to play a role in cell proliferative and developmental disorders.
  • an antagonist of HGFH may be administered to a subject to treat or prevent a cell proliferative disorder.
  • a disorder may include, but is not limited to, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,
  • a vector expressing the complement of the polynucleotide encoding HGFH may be administered to a subject to treat or prevent a cell proliferative disorder including, but not limited to, those described above.
  • HGFH or a fragment or derivative thereof may be administered to a subject to treat or prevent a developmental disorder.
  • a developmental disorder can include, but is not limited to, renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome, Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenhan ⁇ s chorea and cerebral palsy, spinal bifida, and congenital glaucoma, cataract, or sensorineural hearing loss.
  • a vector capable of expressing HGFH or a fragment or derivative thereof may be administered to a subject to treat or prevent a developmental disorder including, but not limited to, those described above.
  • a pharmaceutical composition comprising a substantially purified HGFH in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a developmental disorder including, but not limited to, those provided above.
  • an agonist which modulates the activity of HGFH may be administered to a subject to treat or prevent a developmental disorder including, but not limited to, those listed above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of HGFH may be produced using methods which are generally known in the art.
  • purified HGFH may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HGFH.
  • Antibodies to HGFH may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • Neutralizing antibodies i.e., those which inhibit dimer formation are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HGFH or with any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to HGFH have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HGFH amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to HGFH may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • the hybridoma technique the human B-cell hybridoma technique
  • EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 :31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • single chain antibodies may be adapted, using methods known in the art, to produce HGFH-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for HGFH may also be generated.
  • such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constmcted to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between HGFH and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HGFH epitopes is preferred, but a competitive binding assay may also be employed. (Maddox, supra.)
  • the polynucleotides encoding HGFH, or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding HGFH may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding HGFH.
  • complementary molecules or fragments may be used to modulate HGFH activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HGFH.
  • Expression vectors derived from retrovimses, adenovimses, or herpes or vaccinia vimses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to constmct vectors which will express nucleic acid sequences complementary to the polynucleotides of the gene encoding HGFH. (See, e.g., Sambrook, supra: and Ausubel, supra.)
  • Genes encoding HGFH can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding HGFH. Such constmcts may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding HGFH.
  • Oligonucleotides derived from the transcription initiation site e.g., between about positions -10 and +10 from the start site, are preferred.
  • inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HGFH.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HGFH. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constmcts that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-466.)
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of HGFH, antibodies to HGFH, and mimetics, agonists, antagonists, or inhibitors of HGFH.
  • the compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, dmgs, or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, symps, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example HGFH or fragments thereof, antibodies of HGFH, and agonists, antagonists or inhibitors of HGFH, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED50 (the dose therapeutically effective in 50%) of the population) or LD50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED50/LD50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, dmg combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • antibodies which specifically bind HGFH may be used for the diagnosis of disorders characterized by expression of HGFH, or in assays to monitor patients being treated with HGFH or agonists, antagonists, or inhibitors of HGFH.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for HGFH include methods which utilize the antibody and a label to detect HGFH in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • HGFH human-specific kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinas, and kinase-specific kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinas
  • the polynucleotides encoding HGFH may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HGFH may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of HGFH, and to monitor regulation of HGFH levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HGFH or closely related molecules may be used to identify nucleic acid sequences which encode HGFH.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding HGFH, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the HGFH encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, or from genomic sequences including promoters, enhancers, and introns of the HGFH gene.
  • Means for producing specific hybridization probes for DNAs encoding HGFH include the cloning of polynucleotide sequences encoding HGFH or HGFH derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding HGFH may be used for the diagnosis of a disorder associated with expression of HGFH.
  • a disorder associated with expression of HGFH include, but are not limited to, a cell proliferative disorder, such as arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pan
  • the polynucleotide sequences encoding HGFH may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered HGFH expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding HGFH may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding HGFH may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
  • the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HGFH in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding HGFH, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • oligonucleotides designed from the sequences encoding HGFH may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro.
  • Oligomers will preferably contain a fragment of a polynucleotide encoding HGFH, or a fragment of a polynucleotide complementary to the polynucleotide encoding HGFH, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
  • HGFH HGFH
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.
  • nucleic acid sequences encoding HGFH may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constmctions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constmctions, or single chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constmctions or single chromosome cDNA libraries.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding HGFH on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been cmdely localized by genetic linkage to a particular genomic region, e.g., AT to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • HGFH its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of dmg screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between HGFH and the agent being tested may be measured.
  • Another technique for dmg screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al.
  • test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with HGFH, or fragments thereof, and washed. Bound HGFH is then detected by methods well known in the art.
  • Purified HGFH can also be coated directly onto plates for use in the aforementioned dmg screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • nucleotide sequences which encode HGFH may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • the BRAITUT03 cDNA library was constructed from brain tumor tissue removed from the left frontal lobe of a 17-year-old Caucasian female during excision of a cerebral meningeal lesion.
  • the frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a Brinkmann Homogenizer Polytron-PT 3000 (Brinkmann Instmments, Westbury, NY).
  • the lysate was extracted once with acid phenol, and RNA was isolated according to Stratagene's protocol (Stratagene, La Jolla, CA). The RNA was extracted twice with acid phenol, precipitated with sodium acetate and ethanol, resuspended in RNase-free water, and treated with DNase.
  • UTRSNOT05 The RNA was extracted twice with acid phenol, precipitated with sodium acetate and ethanol, resuspended in RNase-free water, and treated with DNase.
  • the UTRSNOT05 cDNA library was constmcted from nontumorous uterine tissue excised from a 45-year-old Caucasian female during a total abdominal hysterectomy and total colectomy.
  • the frozen tissue was homogenized and lysed in guanidinium isothiocyanate solution using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments).
  • the lysate was centrifuged over a CsCl cushion to isolate RNA.
  • the RNA was extracted with acid phenol, precipitated with sodium acetate and ethanol, resuspended in RNase-free water, and treated with DNase.
  • the RNA was re-extracted and reprecipitated as described above.
  • the KIDNNOT19 cDNA library was constmcted from nontumorous kidney tissue removed from a 65-year-old Caucasian male during an exploratory laparotomy and nephroureterectomy.
  • the BRONNOT01 cDNA library was constmcted from bronchial tissue removed from a 15 -year-old Caucasian male with a history of alcohol, tobacco, and marijuana use who died an accidental death.
  • the frozen tissue was homogenized and lysed in TRIzol reagent (1 g tissue/10 ml TRIzol; Catalog #10296-028, Gibco/BRL, Gaithersburg, MD), a monoplastic solution of phenol and guanidinium isothiocyanate, using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments). After a brief incubation on ice, chloroform was added (1 :5 v/v), and the mixture was centrifuged. The upper aqueous layer was removed to a fresh tube, and the RNA was precipitated with isopropanol, resuspended in RNase-free water, and treated with DNase. The RNA was reprecipitated using sodium acetate and ethanol. For
  • RNA was extracted with acid phenol prior to this final precipitation step.
  • BRAITUT03, UTRSNOT05, KIDNNOT19, and BRONNOT01 From each of the four RNA preparations described above, Poly(A+) RNA was isolated using the Qiagen Oligotex kit (QIAGEN Inc, Chatsworth, CA). Poly(A+) RNA was used to constmct each cDNA library according to the recommended protocols in the Superscript Plasmid System (Catalog #18248-013, Gibco/BRL, Gaithersburg, MD). The cDNAs were fractionated on a Sepharose CL4B column (Catalog #275105, Pharmacia, Piscataway, NJ).
  • BRAITUT03 those cDNAs exceeding 400 bp were ligated into the pSport I plasmid (Gibco/BRL).
  • pSport I plasmid For UTRSNOT05, KIDNNOT19, AND BRONNOT01, those cDNAs exceeding 400 bp were ligated into the pINCY 1 plasmid (Incyte), a derivative of pSPORT I. All plasmids were subsequently transformed into DH5 ⁇ TM competent cells (Catalog #18258-012, Gibco/BRL).
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 Plasmid Kit (Catalog #26173; QIAGEN Inc). The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #22711, Gibco/BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and then lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellets were resuspended in 0.1 ml of distilled water. The plasmid DNA samples were stored at 4°C The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol. Biol.
  • nucleotide sequences and/or amino acid sequences of the Sequence Listing were used to query sequences in the GenBank, SwissProt, BLOCKS, and Pima II databases. These databases, which contain previously identified and annotated sequences, were searched for regions of homology using BLAST (Basic Local Alignment Search Tool). (See, e.g., Altschul, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol. Biol. 215:403-410.)
  • BLAST produced alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST was especially useful in determining exact matches or in identifying homologs which may be of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Other algorithms could have been used when dealing with primary sequence patterns and secondary stmcture gap penalties. (See, e.g., Smith, T. et al. (1992) Protein Engineering 5:35-51.) The sequences disclosed in this application have lengths of at least 49 nucleotides and have no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
  • BLAST The BLAST approach searched for matches between a query sequence and a database sequence. BLAST evaluated the statistical significance of any matches found, and reported only those matches that satisfy the user-selected threshold of significance. In this application, threshold was set at IO "25 for nucleotides and IO "8 for peptides.
  • Incyte nucleotide sequences were searched against the GenBank databases for primate (pri), rodent (rod), and other mammalian sequences (mam), and deduced amino acid sequences from the same clones were then searched against GenBank functional protein databases, mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp), for homology.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; and Ausubel, F.M. et al. supra, ch. 4 and 16.)
  • Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQTM database (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • the basis of the search is the product score, which is defined as:
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • nucleic acid sequences of Incyte Clones 862403, 2676869, 1568019, and 3577857 were used to design oligonucleotide primers for extending partial nucleotide sequences to full length.
  • one primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide.
  • Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new unknown nucleotide sequence for the region of interest.
  • the initial primers were designed from the cDNA using OLIGO 4.06 (National Biosciences, Madison, MN), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin stmctures and primer-primer dimerizations was avoided.
  • Step 1 94° C for 1 min (initial denaturation)
  • Step 2 65° C for l min
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 5 65° C for l min
  • Step 7 Repeat steps 4 through 6 for an additional 15 cycles
  • Step 8 94° C for 15 sec
  • Step 9 65° C for l min
  • Step 11 Repeat steps 8 through 10 for an additional 12 cycles
  • coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra. Appendix A, p. 1) containing 2x Carb. The following day, several colonies were randomly picked from each plate and cultured in 150 ⁇ l of liquid LB/2x Carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 ⁇ l of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1 :10 with water, 5 ⁇ l from each sample was transferred into a PCR array.
  • LB Luria Bertani
  • PCR amplification For PCR amplification, 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:
  • Step 2 94° C for 20 sec
  • Step 3 55° C for 30 sec
  • Step 4 72° C for 90 sec
  • Step 5 Repeat steps 2 through 4 for an additional 29 cycles
  • nucleotide sequences of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
  • Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments.
  • Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham, Chicago, IL), and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA).
  • the labeled oligonucleotides are substantially purified using a Sephadex G-25 superfine resin column (Pharmacia & Upjohn, Kalamazoo, MI).
  • An aliquot containing IO 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II (DuPont NEN, Boston, MA).
  • a chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate.
  • An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using or thermal, UV, mechanical, or chemical bonding procedures, or a vacuum system.
  • a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
  • nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
  • full-length cDNAs or Expressed Sequence Tags comprise the elements of the microarray.
  • Full-length cDNAs or ESTs corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevent to the present invention are arranged on an appropriate substrate, e.g., a glass slide.
  • the cDNA is fixed to the slide using, e.g., UV. cross-linking followed, by thermal and chemical and subsequent drying.
  • Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
  • Probe sequences for microarrays may be selected by screening a large number of clones from a variety of cDNA libraries in order to find sequences with conserved protein motifs common to genes coding for signal sequence containing polypeptides.
  • sequences identified from cDNA libraries are analyzed to identify those gene sequences with conserved protein motifs using an appropriate analysis program, e.g., the Block 2 Bioanalysis Program (Incyte, Palo Alto, CA).
  • This motif analysis program based on sequence information contained in the Swiss-Prot Database and PROSITE, is a method of determining the function of uncharacterized proteins translated from genomic or cDNA sequences. (See, e.g., Bairoch, A. et al. (1997) Nucleic Acids Res.
  • PROSITE may be used to identify functional or structural domains that cannot be detected using conserved motifs due to extreme sequence divergence. The method is based on weight matrices. Motifs identified by this method are then calibrated against the SWISS-PROT database in order to obtain a measure of the chance distribution of the matches.
  • HMMs Hidden Markov models
  • HMMs may be used to find shared motifs, specifically consensus sequences.
  • HMMs were initially developed to examine speech recognition patterns, but are now being used in a biological context to analyze protein and nucleic acid sequences as well as to model protein stmcture.
  • Krogh A. et al. (1994) J. Mol. Biol. 235:1501-1531; and Collin, M. et al. (1993) Protein Sci. 2:305-314.
  • HMMs have a formal probabilistic basis and use position-specific scores for amino acids or nucleotides. The algorithm continues to incorporate information from newly identified sequences to increase its motif analysis capabilities.
  • Sequences complementary to the HGFH-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HGFH.
  • oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate oligonucleotides are designed using Oligo 4.06 software and the coding sequence of HGFH.
  • a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the HGFH-encoding transcript.
  • HGFH HGFH expression is accomplished by subcloning the cDNA into an appropriate vector and transforming the vector into host cells.
  • This vector contains an appropriate promoter, e.g., ⁇ -galactosidase upstream of the cloning site, operably associated with the cDNA of interest.
  • an appropriate promoter e.g., ⁇ -galactosidase upstream of the cloning site, operably associated with the cDNA of interest.
  • IPTG isopropyl beta-D- thiogalactopyranoside
  • the prototypical assay for growth factor activity measures the stimulation of DNA synthesis in Swiss mouse 3T3 cells.
  • Initiation of DNA synthesis indicates the cells' entry into the mitotic cycle and their commitment to undergo later division.
  • 3T3 cells are competent to respond to most growth factors, not only those that are mitogenic, but also those that are involved in embryonic induction. This competency is possible because the in vivo specificity demonstrated by some growth factors is not necessarily inherent but is determined by the responding tissue. Therefore, this assay is generally applicable to HGFH. In this assay, varying amounts of HGFH are added to quiescent 3T3 cultured cells in the presence of [ 3 H]thymidine, a radioactive DNA precursor.
  • HGFH for this assay can be obtained by recombinant means or from biochemical preparations. Incorporation of [ 3 H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold HGFH concentration range is indicative of growth factor activity. One unit of activity per milliliter is defined as the concentration of HGFH producing a 50% response level, where 100% represents maximal incorporation of [ 3 H]thymidine into acid-precipitable DNA .
  • HGFH substantially purified using PAGE electrophoresis is used to immunize rabbits and to produce antibodies using standard protocols.
  • the HGFH amino acid sequence is analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel et al. supra, ch. 11.)
  • the oligopeptides are 15 residues in length, and are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to KLH (Sigma, St. Louis, MO) by reaction with N-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • MBS N-maleimidobenzoyl-N- hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinant HGFH is substantially purified by immunoaffinity chromatography using antibodies specific for HGFH.
  • An immunoaffinity column is constmcted by covalently coupling anti-HGFH antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instmctions.
  • HGFH Media containing HGFH are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HGFH (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that dismpt antibody/HGFH binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HGFH is collected.
  • dismpt antibody/HGFH binding e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion
  • HGFH HGFH, or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton et al. (1973) Biochem. J. 133:529.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HGFH, washed, and any wells with labeled HGFH complex are assayed. Data obtained using different concentrations of HGFH are used to calculate values for the number, affinity, and association of HGFH with the candidate molecules.

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