CA2317812A1 - Human growth factor homologs - Google Patents

Abstract

The invention provides human growth factor homologs (HGFH) and polynucleotides which identify and encode HGFH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of HGFH.

Description

HUMAN GROWTH FACTOR HOMOLOGS
TECHNICAL FIELD
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.
BACKGROUND OF THE INVENTION
Intercellular communication is essential for the development and survival of l0 multicellular organisms. Communication is achieved through the secretion of proteins by signaling cells and the internalization 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 fiuther post-translational modifications within the secretory pathway. These modifications can include glycosylation, phosphorylation, and intramolecular disulfide bond formation.
Following 2o 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.
Most 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, 3o internalization 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-ø (TGF-ø), insulin-like growth factor (IGF}, nerve s growth factor (NGF), and platelet-derived growth factor (PDGF), each defining a family of numerous related factors. 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-ø, EGF, and FGF families also function as inductive ~o 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-ø 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 ~ s growth and differentiation.
Large polypeptide growth factors and the signal transduction pathways they trigger are often conserved among eukaryotes ranging from nematodes, fruit flies, yeast, and mammals. Prokaryotes also produce "growth factors" required for morphogenetic and life cycle transitions such as sporulation. (Albertini, A. M. et al. (1987) J.
Bacteriol.
20 169:1480-1484.) Recently, a yeast pmtein similar to bacterial sporulation outgrowth factor B has been identified. (Johnston, M. et al. (1994) Science 2b5:2077-2082.}
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, 2s 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. Cytokines function in tissue repair, 3o inflammation, and modulation of the immune response. Cytokines include the interleukins, IL 1-IL 13; the interferons, IFN-a, -ø, and -y; and the tumor necrosis factors, TNF-a and -ø.

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. Some of these factors include bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin II, vasoactive intestinal peptide, bradykinin, and related peptides.
(Pimentel, E. ( 1994) Handbook of CTrowth Factors, CRC Press, Ann Arbor, MI; McKay, I. and Leigh, L, eds.
(1993) Growth Factors A Practical_ A roach, Oxford University Press, New York, NY;
and Habenicht, A., ed. (1990) C'Irnwth Factorc Differentiation Fac or and C3 okinec, 1 o Springer-Verlag, New York, NY.) Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression inin 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 'm vivo may contribute to vascularization and metastasis of tumors. Furthermore, some of the large 2o polypeptide growth factors are both structurally and functionally related to oncoproteins, the cancer-correlated products of oncogenes. Certain 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, 2s In addition, some of the large polypeptide growth factors play essential roles in embryonic development. In the fruit fly Drosophila mela_~ogaster, for example, Twisted Gastrulation (TSG) is a secreted protein similar to human connective tissue growth factor, a PDGF-related protein induced by TGF-p activity. TSG protein is required for the 3o 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. 1 I 1:359-371.) The discovery of new human growth factor homologs and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cell proliferative and developmental disorders.
SUMMARY OF THE INVENTION
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." In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3, SEQ ID NO:S, SEQ ID N0:7, a fragment of SEQ ID NO:1, is a fragment of SEQ ID N0:3, a fragment of SEQ ID NO:S, and a fragment of SEQ
ID
N0:7.
The invention further provides a substantially purified variant having at least 90%
amino acid identity to the amino acid sequences of SEQ ID NO:1, SEQ ID N0:3, SEQ ID
NO:S, or SEQ ID N0:7, or to a fragment of any of these sequences. The invention also 2o provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
N0:3, SEQ ID NO:S, SEQ ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID
N0:3, a fragment of SEQ ID NO:S, and a fragment of SEQ ID N0:7. The invention also includes an isolated and purified polynucleotide variant having at least 90%
polynucleotide 25 sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:3, SEQ ID
NO:S, SEQ ID N0:7, a fragment of SEQ ID NO:l, a fragment of SEQ ID N0:3, a fragment of SEQ ID NO:S, and a fragment of SEQ ID N0:7.
Additionally, the invention provides an isolated and purified polynucIeotide which 30 hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:I, SEQ ID N0:3, SEQ ID NO:S, SEQ ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID N0:3, a fragment of SEQ ID NO:S, and a fragment of SEQ ID N0:7, 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:1, SEQ ID N0:3, SEQ ID N0:5, SEQ ID
N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID N0:3, a fragment of SEQ ID
N0:5, and a fragment of SEQ ID N0:7.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:2, SEQ
ID
N0:4, SEQ ID N0:6, SEQ ID N0:8, a fragment of SEQ ID N0:2, a fragment of SEQ
ID
to N0:4, a fragment of SEQ ID N0:6, and a fragment of SEQ ID N0:8. 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 N0:2, SEQ
ID
N0:4, SEQ ID N0:6, SEQ ID N0:8, a fragment of SEQ ID N0:2, a fragment of SEQ
ID
~ 5 N0:4, a fragment of SEQ ID N0:6, and a fragment of SEQ ID N0:8, 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 N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8, a fragment of SEQ ID
N0:2, a fragment of SEQ ID N0:4, a fragment of SEQ ID N0:6, and a fragment of SEQ
20 ID N0:8. The invention also provides a poIynucleotide 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
N0:2, nucleotides 124-156 of SEQ ID N0:4, nucleotides 30-62 of SEQ ID N0:6, and nucleotides 147-182 of SEQ ID N0:8.
25 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:1, SEQ ID N0:3, SEQ ID NO:S, SEQ
ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID N0:3, a fragment of SEQ ID
NO:S, and a fragment of SEQ ID N0:7. In another aspect, the expression vector is 3o contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID N0:3, SEQ ID NO:S, SEQ ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID N0:3, a fragment of SEQ ID NO:S, or a fragment of SEQ ID N0:7, 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 comprising a substantially purified polypeptide having the amino acid sequence of SEQ ID
NO:1, SEQ
ID N0:3, SEQ ID NO:S, SEQ ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ
ID N0:3, a fragment of SEQ ID NO:S, or a fragment of SEQ ID N0:7 in conjunction with l0 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:1, SEQ ID N0:3, SEQ ID NO:S, SEQ
ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID N0:3, a fragment of SEQ ID
NO:S, or a fragment of SEQ ID N0:7, 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 2o 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:1, SEQ ID N0:3, SEQ
ID N0:5, SEQ ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID N0:3, a fragment of SEQ ID NO:S, or a fragment of SEQ ID N0:7 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: l, SEQ ID N0:3, SEQ ID NO:S, SEQ ID N0:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID N0:3, a fragment of SEQ ID NO:S, or a fragment of SEQ ID
N0:7 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization 3o complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
Figures lA, 1B, and 1C show the amino acid sequence alignment between HGFH-1 (862403; SEQ ID NO:1) and mouse growth and differentiation factor 9 (GI
567206;
SEQ ID N0:9).
Figures 2A and 2B show the amino acid sequence alignment between HGFH-2 (2676869; SEQ ID N0:3) and human hepatoma-derived growth factor (GI 598956;
SEQ
ID NO:10).
io Figures 3A, 3B, 3C, and 3D show the amino acid sequence alignment between HGFH-3 (1568019; SEQ ID NO:S) and yeast Yhr074wp (GI 500832; SEQ ID NO:11).
Figures 4A and 4B show the amino acid sequence alignment between HGFH-4 (3577857; SEQ ID N0:7) and fruit fly Twisted Gastrulation (GI 529900; SEQ ID
N0:12).
The alignments were produced using the multisequence alignment program of DNASTARTM software (DNASTAR Inc, Madison WI).
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the 3o same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the s invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"HGFH," as used herein, refers to the amino acid sequences of substantially purified HGFH obtained from any species, particularly a mammalian species, including to bovine, ovine, porcine, marine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist," as used herein, 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 15 the effect of HGFH.
An "allele" or an "allelic sequence," as these terms are used herein, is 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 20 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, as described herein, include 25 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 3o 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 s 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. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and gIutamine; serine and threonine; and phenylalanine and tyrosine.
The terms "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. In this context, "fragments", "immunogenic fragments", or "antigenic 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. Where "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," as used herein, relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain 2o reaction (PCR) technologies well known in the art. (See, e.g., Dieffenbach, C. W. and G.S. Dveksler (1995) PCR Primer_ a T.aboratorv h~a- ual, Cold Spring Harbor Press, Plainview, NY, pp.l-5.) The term "antagonist," as it is used herein, 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.
As used herein, 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 3o 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) can be derived from the translation of RNA, or synthesized WO 99/389'76 PCT/US99/00654 chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant," as used herein, refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (given regions or three-dimensional structures on the protein).
An antigenic l0 determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense," as used herein, 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 15 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.
20 As used herein, the term "biologically active," refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
Likewise, "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.
25 The terms "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 3o complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the e~ciency and strength of the hybridization between the nucleic acid strands. This is of particular io importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
A "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.
to In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
The phrase "consensus sequence," as used herein, 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 GELVIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence .
2o As used herein, 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.
The term "derivative," as used herein, refers to the chemical modification of HGFH, of a polynucleotide sequence encoding HGFH, or of a polynucleotide sequence 3o 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 m 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 ~ at least one biological or immunological function of the polypeptide from which it was derived.
The term "homology," as used herein, refers to a degree of complementarity.
There may be partial homology or complete homology. 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 to 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. This is not to say that conditions of 15 reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% homology or identity). In the absence of non-specific binding, the 2o substantially homologous sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" or "% identity" refer 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 25 (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 CIustal 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 30 between two amino acid sequences, e.g., sequence A and sequence B, 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.
"Human artificial chromosomes" (HACs), as described herein, 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.) The term "humanized antibody," as used herein, 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," as the term is used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
As used herein, the term "hybridization complex" as used herein, refers to a complex formed between two nucleic acid sequences by virtue of the formation of 2o hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., Cot 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).
The words "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.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be 3o characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
The term "microarray," as used herein, 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.
The term "modulate," as it appears herein, 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.
The phrases "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 to represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material: In this context, "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.
is The terms "operabIy associated" or "operably linked," as used herein, refer 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 operabIy associated or operably linked nucleic acid sequences can be contiguous and in reading frame, certain genetic elements, e.g., repressor genes, are not 2o contiguously linked to the encoded polypeptide but still bind to operator sequences that contml expression of the polypeptide.
The term "oligonucleotide," as used herein, 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 2s a hybridization assay or microarray. As used herein, the term "oligonucleotide" is substantially equivalent to the terms "amplimers," "primers," "oligomers," and "probes,"
as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA), as used herein, refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in 30 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 pegyIated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. {1993) Anticancer Drug Des. 8:53-63.) The term "sample," as used herein, 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.
As used herein, 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 1o 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.
As used herein, the term "stringent conditions" refers to conditions which permit hybridization between polynucleotide sequences and the claimed polynucleotide sequences. Suitably 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. In particular, stringency 2o can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
For example, 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. In particular, hybridization could occur under high stringency conditions at 42°C in 50%
formamide, SX 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 3o purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art.
The term "substantially purified," as used herein, refers to nucleic acid or amino is WO 99/389'I6 PCT/US99/00654 acid sequences that are removed fi~om 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.
A "substitution," as used herein, refers to the replacement of one or more amino s acids or nucleotides by different amino acids or nucleotides, respectively.
"Transformation," as defined herein, 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 to 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. The term "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 1s chromosome, and refers to cells which transiently express the inserted DNA
or RNA for limited periods of time.
A "variant" of HGFH, as used herein, 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., 2o 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 2s well known in the art, for example, DNASTAR software.
THE INVENTION
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 3o diagnosis, treatment, or prevention of cell proliferative and developmental disorders.
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
N0:2, 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.
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:l. HGFH-1 is 446 amino acids in length and has five potential N-glycosylation sites at N,ob, NzzB, N247~ Nzso~ ~d N33o; one potential cAMP- and cGMP-dependent protein kinase phosphorylation site at Sz~; three potential casein kinase II phosphorylation sites at SzB, T~48, and 5,93; six potential protein kinase C
1o phosphoryIation sites at S76, S,os, T"3, S,7g, Sz~e, and T~4; a potential signal peptide sequence from M, to Pzo; and a TGF-(3 family signature from I363 to C37g. The region of HGFH-1 from C345 to 8446 also shows similarity to a TGF-a protein domain. As shown in Figures lA, 1B, and 1C, HGFH-1 has chemical and structural homology with mouse growth/differentiation factor 9 (GDF-9), a member of the TGF-a family (GI
567206; SEQ
ID N0:9). In particular, HGFH-1 and mouse GDF-9 share 66% identity overall and 95%
identity in their C-terminal regions from A3~ to R,~6 of HGFH-1. Within this region, cysteines important for intramolecular disulfide bond formation in GDF-9 are conserved in HGFH-1 at C~,,s, C374, C378, C4", C~a3, and Cps. S4os of GDF-9, which distinguishes GDF-9 from other TGF-li-like proteins that contain a cysteine at this position, is conserved in 2o HGFH-1 at S4,o. In addition, the potential N-glycosylation sites at NzzB, Nz~o, and N330, the potential phosphorylation sites at SzB, S76, T"3, T~48, S,7g, and T4,~; and the potential signal peptide sequence of HGFH-1 are conserved in GDF-9. The fragment of SEQ ID N0:2 from about nucleotide 535 to nucleotide 570 is useful for designing oligonucleotides or to be used directly as a hybridization prnbe. 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 N0:4, was derived from the following overlapping and/or extended nucleic acid sequences:
Incyte 3o Clones 2676869 (KIDNNOT19), 488482 (HNT2AGT01), 2571655 (HIPOAZTO1), 2150727 (BRAINOT09), 899636 (BRSTTUT03), 2311937 (NGANNOT01), 2613941 (THYRNOT09), 776810 (COLNNOTOS), 968484 (BRSTNOT05), 1292885 m (PGANNOT03), and 2288692 (BRAINONO1 ).
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID N0:3. HGFH-2 is 203 amino acids in length and has three potential N-glycosylation sites at N,76, N,87, and N,9,; two potential cAMP- and cGMP-dependent protein kinase phosphorylation sites at 5,49 and 5,56; eight potential casein kinase II phosphorylation sites at S,ob, S,oB, T"o, 5,2,, 5,22, S,6z, S,7a, and T,92; and six potential protein kinase C phosphorylation sites at 575, S,44, T,s,, 5,52, S,ss, and 5,59. As shown in Figures 2A and 2B, HGFH-2 has chemical and structural homology with human hepatoma-derived growth factor (HDGF) (GI 598956; SEQ ID NO:10). In particular, HGFH-2 and HDGF share 49% identity. In addition, the potential phosphorylation sites at S,ob, 5,22, S,s2, and 5,78 of HGFH-2 are conserved in HDGF. The fragment of SEQ ID
N0:4 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 ~ 5 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 (UTRSNOTOS) using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID N0:6, was derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones 1568019 (UTRSNOTOS), 2504437 (CONUTUTO1), 136194 (SYNORABO1), 1849286 (LUNGFET03) and shotgun sequences SAEA02891, SAEA03437, and SAFC02217.
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO:S. HGFH-3 is 706 amino acids in length and has four potential N-glycosylation sites at N,98, N2s,, N422, and N677; one potential cAMP- and cGMP-dependent protein kinase phosphorylation site at T6; sixteen potential casein kinase II phosphorylation sites at T,Si, T2", 5262 'S308~ 5327 5382 T401~ 5423 '5474 TS27~ 5532 '~543~ 5584 T586~ Ts~4~ ~d 5663 ~d six potential protein kinase C phosphorylation sites at T~°, S28o, S2s6, T462, 5474, and T62g. As shown in Figures 3A, 3B, 3C, and 3D, HGFH-3 has chemical 3o and structural homology with yeast Yhr074wp (GI 500832; SEQ ID NO:11 ), an open reading frame with similarity to prokaryotic sporuladon outgrowth factor B. In particular, HGFH-3 and Yhr074wp share 57% identity. In addition, the potential N-glycosylation is sites at N,98, Nzs,, and N4z2, and the potential phosphorylation sites at T22o, S262~ Szao~ S3z7 '5423 T462~ s474~ T527~ 5532 '~543~ T594~ T628~ ~d Ss~3 of HGFH-3 are conserved In Yhr074wp.
The fragment of SEQ ID N0:6 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 1o in Incyte Clone 3577857 from the bronchial tissue cDNA library BRONNOTO1 using a computer search for amino acid sequence alignments. A consensus sequence, SEQ
ID
N0:8, was derived from the following overlapping and/or extended nucleic acid sequences: Incyte Clones 3577857 (BRONNOTO1), 2799484 (NPOLNOTO1), 1685583 (PROSNOT15), 1210438 (BRSTNOT02), 3601074 (DRGTNOTO1), 3279304 (STOMFET02), and shotgun sequences SAFC00098, SAYA00353, and SAFC00552.
In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID N0:7. HGFH-4 is 223 amino acids in length and has three potential N-glycosylation sites at Ns2, NB,, and N,47; six potential casein kinase II
phosphorylation sites at 59,, T9z, T,oB, 5,28, S,3s, and 5,6,; three potential protein kinase C
2o phosphorylation sites at TgB, S,6,, and T2,s ; and a potential signal peptide sequence from M, to S2s. As shown in Figures 4A and 4B, HGFH-4 has chemical and structural homology with fruit fly Twisted Gastrulation (TSG) (GI 529900; SEQ ID N0:12).
In particular, HGFH-4 and TSG share 29% amino acid identity. In addition, 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 phosphoryIation sites at T88 and T,oB of HGFH-4 are conserved in TSG. The fragment of SEQ ID N0:8 from about nucleotide I47 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 3o 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. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID N0:2, which encodes an HGFH-1. In another embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ
ID
N0:4, which encodes an HGFH-2. In a further embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID N0:6, which encodes an 1 o HGFH-3. In still another embodiment, the invention encompasses a polynucleotide sequence comprising the sequence of SEQ ID N0:8, which encodes an HGFH-4.
The invention also encompasses a variant of a polynucleotide sequence encoding HGFH. In particular, such 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 N0:2 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 N0:2. The invention further encompasses a polynucleotide variant of SEQ ID N0:4 having at least about 80%, more preferably at least about 90%, 2o and most preferably at least about 95% polynucleotide sequence identity to SEQ ID N0:4.
The invention further encompasses a polynucleotide variant of SEQ ID N0:6 having at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID N0:6. The invention further encompasses a polynucleotide variant of SEQ ID N0:8 having at least about 80%, more 2s preferably at least about 90%, and most preferably at least about 95%
polynucleotide sequence identity to SEQ ID N0:8. 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.
It will be appreciated by those skilled in the art that as a result of the degeneracy of 30 the genetic code, a multitude of polynucleotide sequences encoding HGFH, some bearing minimal homology to the polynucleotide sequences of any known and naturally occurnng gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HGFH, and all such variations are to be considered as being specifically disclosed.
Although 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 to the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding HGFH and its derivatives without altering the encoded amino acid sequences include the production of 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. After production, 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.
2o Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HGFH or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:8, a fragment of SEQ ID
N0:2, a fragment of SEQ ID N0:4, a fragment of SEQ ID N0:6 or a fragment of SEQ ID
.N0:8, under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L.
Berger (1987) Methods Enzymol. 152:399-407; and Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Methods for DNA sequencing are well known and generally available in the art 3o 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 polymerise I, Sequenase~ (US
Biochemical Corp., Cleveland, OH), Taq polymerise (Perkin Elmer), thermostable WO 99!38976 PCT/US99/00654 polymerise (Amersham, Chicago, IL), or combinations of polymerises and proofreading exonucleases such as those found in the ELONGASE Amplification System (GIBCOBRL, Gaithersburg, MD). Preferably, the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV}, Pettier 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. For example, one method which to may be employed, restriction-site PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) In particular, 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
polymerise and sequenced using reverse transcriptase.
Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
2o 16:8186.) The primers may be designed using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, 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.
Another method which may be used is capture PCR, which involves PCR
amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. {See, e.g., Lagerstrom, M. et al. ( 1991 } PCR
Methods 3o Applic. 1:111-119.) In this method, 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) Nucleic Acids Res. 19:3055-3060.) Additionally, one may use PCR, nested primers, and 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.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, 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 to 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. In particular, 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.
OutputJlight 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 2o pieces of DNA which might be present in limited amounts in a particular sample.
In another embodiment of the invention, 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.
As will be understood by those of skill in the art, it may be advantageous to produce HGFH-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can 3o 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, andlor expression of the gene product. DNA shuffling by random s fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, 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.
In another embodiment of the invention, natural, modified, or recombinant nucleic ~o acid sequences encoding HGFH may be ligated to a heterologous sequence to encode a fusion protein. 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 ~ s sequence, so that HGFH may be cleaved and purified away from the heterologous moiety.
In another embodiment, sequences encoding HGFH may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al.
(1980) Nucl. Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl.
Acids Res.
Symp. Ser. 225-232.) Alternatively, the protein itself may be produced using chemical 2o methods to synthesize the amino acid sequence of HGFH, or a fragment thereof. For example, 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 2s performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T.
(1983) Proteinc_ . tn,~cfi.~r c and M~~~~,,tar n,..,neriies, WH Freeman and Co., New York, NY.) Additionally, the amino acid sequence of HGFH, or any part thereof, may be altered 3o during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
In order to express a biologically active HGFH, 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.
Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HGFH and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and inin vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning A Laboraton- A~~1~ Cold Spring Harbor Press, Plainview, NY, ch. 4, 8, and 16-17; and Ausubel, F.M. et al. (1995, and periodic i0 supplements) Current Protocoh in Molec lar Biology, John Wiley & Sons, New York, NY, ch. 9, 13, and 16.) 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 virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
2o The invention is not limited by the host cell employed.
The "control elements" or "regulatory sequences" 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. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters, e.g., hybrid lacZ promoter of the Bluescript~ phagemid (Stratagene, La Jolla, CA) or pSportlTM
plasmid (GIBCO/BRL), may be used. The baculovirus polyhedrin promoter may be used 3o 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. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding HGFH, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
In bacterial systems, 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), l0 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 !3-galactosidase so that a hybrid protein is produced, and pIN vectors. (See, e.g., Van Heeke, G.
and S.M.
Schuster (1989) J. Biol. Chem. 264:5503-5509.) pGEX vectors (Pharmacia Biotech, Uppsala, Sweden) may also be used to express foreign polypeptides as fusion proteins 15 with glutathione S-transferase (GST). In general, such 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.
2o In the yeast Saccha_mmvces 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, ; and Grant et al. (1987) Methods Enzymol. 153:516-544.) In cases where plant expression vectors are used, the expression of sequences encoding HGFH may be driven by any of a number of promoters. For example, viral 25 promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV. (Takamatsu, N. ( 1987) EMBO
J. 6:307-311.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO
J.

3:1671-1680; Brogue, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) 30 Results Probl. Cell Differ. 17:85-105.) These constructs 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.

wo ~r~sr~s rcr~s~roo6sa 1ri McGraw Hlll Yea_rhnnlt of ~CienrP an TeC nnlnvv (1992) McGraw Hill, New York, NY; pp. 191-196.) An insect system may also be used to express HGFH. For example, in one such system, Autog~nha califoriica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frug~ rda cells or in Trichonlusia larvae.
The sequences encoding HGFH may be cloned into a non-essential region of the virus, 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 virus lacking coat protein. The recombinant viruses may then be 1o used to infect, for example, S. fruginerda cells or Trichoplusia larvae in which HGFH may be expressed. (See, e.g., Engelhard, E.K. et al. (1994) Proc. Nat. Acad. Sci.
91:3224-3227.) In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding HGFH may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E 1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing HGFH in infected host cells. (See, e.g., Logan, J. and T. Shenk ( 1984) Proc.
Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
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 constructed 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 3o 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 WO 99138976 PCf/US99100654 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.) In addition, 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. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing to 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.
For long term, high yield production of recombinant proteins, stable expression is preferred. For example, 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.
2o 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 stabIy transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus 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. For example, dhfr confers resistance to methotrexate; npt confers resistance to the aminoglycosides neomycin and G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Muny, supra.) Additional selectable genes have been described, e.g., trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Recently, the use of visible markers has gained popularity with such markers as anthocyanins,13 glucuronidase and its substrate GUS, luciferase and its substrate luciferin. Green fluorescent proteins (GFP) (Clontech, Palo Alto, CA) are also used (See, e.g., Chalfie, M. et al. (1994) Science l0 263:802-805.) These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C.A. et al. (1995) Methods Mol. Biol.
55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
For example, if the sequence encoding HGFH is inserted within a marker gene sequence, transfonmed cells containing sequences encoding HGFH can be identified by the absence of marker gene function. Alternatively, 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 2o tandem gene as well.
Alternatively, 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, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
The presence of 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. Nucleic acid amplification based assays 3o involve the use of oligonucleotides or oligomers based on the sequences encoding HGFH
to detect transformants containing DNA or RNA encoding HGFH.
A variety of protocols for detecting and measuring the expression of HGFH, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art.
Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A
two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HGFH is preferred, but a competitive binding assay may be employed. These and other assays are well described in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods a LaboratQ annul, ApS Press, St Paul, MN, Section IV; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. 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. Alternatively, the sequences encoding HGFH, or any fragments thereof, may be cloned into a vector for the production of an ~5 mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Pharmacia & Upjohn (Kalamazoo, MI), Promega (Madison, WI), and U.S. Biochemical Corp. (Cleveland, OH).
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. As will be understood by those of skill in the art, 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 constructions 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). The inclusion of cleavable linker sequences, such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA), between the purification domain and the HGFH
encoding sequence may be used to facilitate purification. 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) (See, to e.g., Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281.) 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.) Fragments of HGFH may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, T.E.
(1984) Protein: Structures and Molecular Properties, pp. 55-60, W.H. Freeman and Co., New York, NY.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments of HGFH
may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
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.
Chemical and structural homology exists between HGFH-3 and Yhr074wp from yeast (GI 500832). In addition, HGFH-3 is expressed in cancerous tissues.
Therefore, 3o HGFH-3 appears to play a role in cell proliferative and developmental disorders.
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, appears to play a role in cell proliferative and developmental disorders.
Therefore, in one embodiment, an antagonist of HGFH may be administered to a subject to treat or prevent a cell proliferative disorder. Such 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, 1 o ovary, pancreas, parathyroid, penis; prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibody which specifically binds HGFH
may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HGFH.
In an additional embodiment, 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.
In a further embodiment, HGFH or a fragment or derivative thereof may be administered to a subject to treat or prevent a developmental disorder. Such a disorder can include, but is not limited to, renal tubular acidosis, anemia, Cushing's syndrome, 2o 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 Syndenham's chorea and cerebral palsy, spinal bifida, and congenital glaucoma, cataract, or sensorineural hearing loss.
In another embodiment, 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.
In a still another embodiment, a pharmaceutical composition comprising a 3o 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.

In still another embodiment, 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.
In other embodiments, 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. In particular, 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.
2o For the production of antibodies, 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. Depending on the host species, various adjuvants may be used to increase imlnunological response. Such 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. Among adjuvants used in humans, BCG
(bacilli Calmette-Guerin) and Conrnebacteriurn ~,m are especially preferable.
It is preferred that 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. (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.
to Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specifcity and biological activity, can be used.
(See, e.g., Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M.S. et al.
Is (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of 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.
20 (See, e.g., Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.) Antibodies may also be produced by inducing inin 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.) 25 Antibody fragments which contain specific binding sites for HGFH may also be generated. For example, 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. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab 3o fragments with the desired specificity. (See, e.g., Huse, W.D. et al.
(1989) Science 246:1275-1281.) Various 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.) In another embodiment of the invention, the polynucleotides encoding HGFH, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, ~ o 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. In particular, cells may be transformed with sequences complementary to polynucleotides encoding HGFH.
Thus, complementary molecules or fragments may be used to modulate HGFH activity, or to achieve regulation of gene function. Such technology is now well known in the art, and 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 retroviruses, adenoviruses, or herpes or vaccinia viruses, 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 2o to those skilled in the art can be used to construct vectors which will express nucleic acid sequences complementary to the polynucleotides of the gene encoding HGFH.
{See, e.g., Sambrook, ; and Ausubel, .) 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 constructs 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.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the WO 9913$976 PCT/US99/00654 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. Similarly, 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. and B.I.
Carr, Molecular and IrrL unologic AApproaches, Futura Publishing Co., Mt.
Kisco, NY, pp.
163-177.) A complementary sequence or antisense molecule may also be designed to 1o 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. For example, engineered hammerhead motif ribozyme ~ s molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HGFH.
Specific 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 2o 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.
2s Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and inin vivo transcription of DNA sequences encoding HGFH. Such DNA
3o sequences may be incorporated into a wide variety of vectors with suitable RNA
polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs 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. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use 'm vivo, in vitro, and ex vivo. For iv therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. 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 I 5:462-466.) 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 2o 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, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, 3o infra-arterial, intramedullary, mtrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, 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 R mineton's Pha_rma~P"tic~t CrjencPC (M~ck Publishing Co., Easton, PA).
Pharmaceutical 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, syrups, slurries, suspensions, and the like, for 1 o ingestion by the patient.
Pharmaceutical preparations 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. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as 1 s sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, 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. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, 20 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, polyvinylpyrroIidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may 25 be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include 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 3o binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations 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 s suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, 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 1o polymers may also be used for delivery. Optionally, 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.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the 15 art.
The pharmaceutical 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 lyaphilizing processes.
2o 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. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 25 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.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of HGFH, such labeling would include amount, frequency, and method of 30 administration.
Pharmaceutical 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.
For any compound, the therapeutically effective dose can be estimated initially either in cell cultwe 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 l0 inhibitors of HGFH, which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedwes 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 cultwe 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, drug 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 ~cg to 100,000 ,ug, up to a total 3o 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.
DIAGNOSTICS
In another embodiment, 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 to 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.
A variety of protocols for measuring HGFH, including ELISAs, RIAs, and FRCS, are known in the art and provide a basis for diagnosing altered or abnormal levels of HGFH expression. Normal or standard values for HGFH expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HGFH under conditions suitable for complex formation The 2o amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of HGFH expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, 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 3o regulation of HGFH levels during therapeutic intervention.
In one aspect, 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 ~o may be derived from the sequence of SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, SEQ
ID N0:8, 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 1 s 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 32P or 355, or by enzymatic labels, such as alkaline phosphatase 2o 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. Examples of such a disorder include, but are not limited to, a cell proliferative disorder, such as arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, 25 paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary . thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myelorna, 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, 30 penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; and a developmental disorder such as 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 Syndenham's chorea and cerebral palsy, spinal bifida, and congenital glaucoma, cataract, or sensorineural hearing loss. 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.
1o In a particular aspect, 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. After a suitable incubation period, the sample is washed and the ~5 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. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to 2o monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of HGFH, 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 25 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 3o values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, 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.
With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
to Additional diagnostic uses for 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 l5 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.
Methods which may also be used to quantitate the expression of HGFH include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and 2o interpolating results from standard curves. (See, e.g., Melby, P.C. et a1.
(1993) J.
Immunol. Methods 159:235-244; and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
2s In further embodiments, 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 3o 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.

(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 W095I251116; Shalon, D. et al. (1995) PCT application W095/35505; Heller, R.A, et al.
{1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No.
5,605,562.) In another embodiment of the invention, 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 constructions, e.g., human ~ o artificial chromosomes (HACs), yeast artificial chromosomes (PACs);
bacterial artificial chromosomes (BACs), bacterial P 1 constructions, or single chromosome cDNA
libraries.
(See, e.g., Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet.
7:149-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical ~ 5 chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al.
(1995) in Meyers, R.A. (ed.) Molecular Blolog,IOtP~hn~i~~v, VCH Publishers New York, NY, pp. 965-968.) 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 2o 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, such as linkage analysis using established chromosomal markers, may be used 25 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 3o techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., AT to l 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. {1988) Nature 336:577-580.) 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.
In another embodiment of the invention, HGFH, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug 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.
1o Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application W084/03564.) In this method, large numbers of different small 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 ~5 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 drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which 2o neutralizing antibodies capable of binding HGFH specifically compete with a test compound for binding HGFH. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HGFH.
In additional embodiments, the nucleotide sequences which encode HGFH may be used in any molecular biology techniques that have yet to be developed, provided the new 25 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 examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.
EXAMPLES
I. cDNA Library Construction 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 Instruments, Westbury, N~. 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.
1o UTRSNOTOS
The UTRSNOTOS cDNA library was constructed 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 CsCI 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. KIDNNOT19 AND BRONNOTO1 The KIDNNOT19 cDNA library was constructed from nontumorous kidney tissue 2o removed from a 65-year-old Caucasian male during an exploratory laparotomy and .
nephroureterectomy. The BRONNOTO1 cDNA library was constructed 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. For each library, the frozen tissue was homogenized and lysed in TRIzoI reagent ( 1 g tissue/l 0 ml TRIzoI; Catalog #
10296-028, GibcoBRL, 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 3o with DNase. The RNA was reprecipitated using sodium acetate and ethanol.
For BRONNOTO1, RNA was extracted with acid phenol prior to this final precipitation step.
BRAITUT03, UTRSNOTOS, KIDNNOT19, and BRONNOTO1 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 construct each cDNA library according to the recommended protocols in the Superscript Plasmid System (Catalog #18248-013, GibcoBRL, Gaithersburg, MD).
The cDNAs were fractionated on a Sepharose CL4B column (Catalog #2751 O5, Pharmacia, Piscataway, NJ). For BRAITUT03, those cDNAs exceeding 400 by were Iigated into the pSport I plasmid {GibcoBRL). For UTRSNOTOS, KIDNNOT19, AND BRONNOTO1, those cDNAs exceeding 400 by were ligated into the pINCY I plasmid (Incyte), a derivative of pSPORT I. All plasmids were subsequently transformed into DHSaTM
1o competent cells (Catalog #18258-012, GibcoBRL).
II. Isolation and Sequencing of cDNA Clones (Alt cDNA Libraries) 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 ~5 except for the following changes: 1) the bacteria were cultured in 1 ml of sterile Ternfic Broth (Catalog #22711, GibcoBRL) 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.
2o The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.
Biol.
94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with Pettier Thermal Cyclers (PTC200 from MJ Research, Watertown, MA) and Applied Biosystems 377 DNA Sequencing Systems, and the reading frames were determined.
25 III. Homology Searching of cDNA Clones and Their Deduced Proteins The 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 identif ed and annotated sequences;
were searched for regions of homology using BLAST (Basic Local Alignment Search 3o Tool). (See, e.g., Altschul, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. MoI. 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 structure 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).
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 10'25 for nucleotides and 10'g 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.
IV. Northern Analysis 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. , 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. In addition, 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:
% seauence i~lPnr;r3r~l° maximum BL A ST score 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.
The results of northern analysis are reported as a list of libraries in which the transcript encoding HGFH occurs. Abundance and percent abundance are also reported.
Abundance directly reflects the number of times a particular transcript is represented in a cDNA library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library.
V. Extension of HGFH Encoding Polynucleotides The 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. For each nucleic acid sequence, 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, Plymouth, MN), or another appropriate 2o 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 structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries (G~BCOBRL) were used to extend the sequence.
If more than one extension is necessary or desired, additional sets of primers are designed to further extend the known region.
High fidelity amplification was obtained by following the instructions for the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. PCR
was performed using the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, MA), 3o beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, with the following parameters:
Step 1 94 ° C for 1 min (initial denaturation) WO 9913897b PCTNS99/00654 Step 2 65 C for 1 min Step 3 68 C for 6 min Step 4 94 C for 15 sec Step 5 65 C for 1 min Step 6 68 C for 7 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 1 min Step 10 68 C for 7:15 min to Step 11 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72 C for 8 min Step 13 4 C (and holding) A 5 ul to 10 ~cl aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6% to 0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQuickTM (QIAGEN Inc., Chatsworth, CA), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 ~cl of ligation 2o buffer, l,ul T4-DNA ligase ( 15 units) and l,ul T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16 ° C.
Competent E.E. coli cells (in 40 ,ul of appropriate media) were transformed with 3 ~1 of ligation mixture and cultured in 80 ~l of SOC medium. (See, e.g., Sambrook, sub, Appendix A, p. 2.) After incubation for one hour at 37° C, the E.E.
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 ,ul 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 ~cl of each overnight culture was transferred into a non-sterile 96-well plate and, after 3o dilution 1:10 with water, 5 ~cl from each sample was transferred into a PCR
array.
For PCR amplification, 18 ~cl of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerise, 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 1 94 ° C for 60 sec Step 2 94 ° C for 20 sec Step 3 55 ° C for 30 sec WO 99/38976 PCTIUS99l00654 Step 72 C for 90 sec Step Repeat steps 2 through 4 for an additional 29 cycles Step 72 C for 180 sec Step 4 C (and holding) Aliquots of the PCR reactions were run on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs, and appropriate clones were selected, ligated into plasmid, and sequenced.
In like manner, the nucleotide sequences of SEQ ID N0:2, SEQ ID N0:4, SEQ
1o ID N0:6, and SEQ ID N0:8 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:2, SEQ ID N0:4, SEQ ID N0:6, 1s and SEQ ID N0:8 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 s0 pmol of each oligomer, 250 ,uCi of 20 ~y 32P~ 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 10' 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 2s following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II
(DuPont NEN, Boston, MA).
The DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham, NH).
Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots 3o are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM
film (Kodak, Rochester, N~ is exposed to the blots to film for several hours, hybridization patterns are compared visually.

VII. Microarrays A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, sub.) 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. After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns io 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.
In another alternative, full-length cDNAs or Expressed Sequence Tags (ESTs) comprise the elements of the microarray. Full-length cDNAs or ESTs corresponding to is 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., U.V. cross-linking followed, by thermal and chemical and subsequent drying. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470; and Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent 2o 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. In one 25 embodiment, 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 3o cDNA sequences. {See, e.g., Bairoch, A. et al. (1997) Nucleic Acids Res.
25:217-221; and Attwood, T. K. et al. (1997) J. Chem. Inf. Comput. Sci. 37:417-424.) PROSITE
may be used to identify functional or structural domains that cannot be detected using conserved WO 99138976 PCTNS9910065a 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.
In another embodiment, Hidden Markov models (HMMs) may be used to find shared motifs, specifically consensus sequences. (See, e.g., Pearson, W.R. and D.J.
Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and Smith, T.F. and M.S.
Waterman ( 1981 ) J. Mol. Biol. 147:195-197.) 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 structure. (See, e.g.;
Krogh, A. et al.
l0 (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.
VIII. Complementary Polynucleotides Sequences complementary to the HGFH-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HGFH. Although use of 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 2~ oligonucleotides are designed using Oligo 4.06 software and the coding sequence of HGFH. To inhibit transcription, 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.
IX, Expression of HGFH
Expression of HGFH 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., Q-galactosidase upstream of the cloning site, operably associated with the cDNA of interest. (See, e.g.,Sambrook, supra, pp. 404-433;
and Rosenberg, M. et al. (1983) Methods Enzymol. 101:123-138.) Induction of an isolated, transformed bacterial strain with isopropyl beta-D-WO 99138976 PCT/US99/E)0654 thiogalactopyranoside (IPTG) using standard methods produces a fusion protein which consists of the first 8 residues of 13-galactosidase, about 5 to 15 residues of linker, and the full length protein. The signal residues direct the secretion of HGFH into bacterial growth media which can be used directly in the following assay for activity.
X. Demonstration of HGFH Activity The prototypical assay for growth factor activity measures the stimulation of DNA
synthesis in Swiss mouse 3T3 cells. (McICay and Leigh, .) Initiation of DNA
synthesis indicates the cells' entry into the mitotic cycle and their commitment to undergo 1o 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 inin 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 15 added to quiescent 3T3 cultured cells 'in the presence of [3H]thymidine, a radioactive DNA
precursor. HGFH for this assay can be obtained by recombinant means or from biochemical preparations. Incorporation of [3H]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 2o 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 [3H]thymidine into acid-precipitable DNA .
25 XI. Production of HGFH Specific Antibodies HGFH substantially purified using PAGE electrophoresis (see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, 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 3o 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. , ch. 1 I .) Typically, the oligopeptides are 15 residues in length, and are synthesized using an Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry and coupled to ICLH (Sigma, St. Louis, MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel et al.
supra.) Rabbits are immunized with the oligopeptide-ICLH 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.
XII. Purification of Naturally Occurring HGFH Using Specific Antibodies Naturally occurnng or recombinant HGFH is substantially purified by immunoaffinity chromatography using antibodies specific for HGFH. An immunoaffnity column is constructed 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 instructions.
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 disrupt 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.
XIII. Identification of Molecules Which Interact with HGFH
HGFH, or biologically active fragments thereof, are labeled with '25I
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.
3o Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

WO 99/38976 PCT/US99/OOb54 SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS. INC.
HILLMAN, Jennifer L.
LAL, Preeti CORLEY, Neil C.
BAUGHN, Mariah GUEGLER, Karl J.
TANG. Y. Tom <120> HDMAN GROWTH FACTOR HOMOLOGS
<130> PF-0472 PCT
<140> To Be Assigned <141> Herewith <150> 09/015.412 <151> 1998-O1-29 <160> 12 <170> PERL PROGRAM
<210> 1 <211> 446 <212> PRT
<213> Homo sapiens <220> -<223> 862403 <400> 1 Met Ala Arg Pro Asn Lys Phe Leu Leu Trp Phe Cys Cys Phe Ala Trp Leu Cys Phe Pro Ile Ser Leu Gly Ser Gln Ala Ser Gly Gly Glu Ala Gln Ile Ala Ala Ser Ala Glu Leu Glu Ser Gly Ala Met Pro Trp Ser Leu Leu Gln His Ile Asp Glu Arg Aap Arg Ala Gly Leu Leu Pro Ala Leu Phe Lys Val Leu Ser Val Gly Arg Gly Gly Ser Pro Arg Leu Gln Pro Asp Ser Arg Ala Leu His Tyr Met Lys Lys Leu Tyr Lys Thr Tyr Ala Thr Lys Glu Gly Ile Pro Lys Ser Asn Arg Ser His Leu Tyr Asn Thr Val Arg Leu Phe Thr Pro Cys Thr Arg His Lys Gln Ala Pro Gly Asp Gln Val Thr Gly Ile Leu Pro Ser Val Glu Leu Leu Phe Asn Leu Asp Arg Ile Thr Thr Val Glu His Leu Leu Lys Ser Val Leu Leu Tyr Pro Leu Leu Ser Lys Cys Val Cys Asn Leu Met Ile Lys Glu Pro Lys Ser Ser Ser Arg WO 991389'16 PCT/US99/00654 Thr Leu Gly Arg Ala Pro Tyr Ser Phe Thr Phe Asn Ser Gln Phe Glu Phe Gly Lys Lys His Lys Trp Ile Gln Ile Asp Val Thr Ser Leu Leu Gln Pro Leu Val Ala Ser Asn Lys Arg Ser Ile His Met Ser Ile Asn Phe Thr Cys Met Lys Asp Gln Leu Glu His Pro Ser Ala Gln Asn Gly Leu Phe Asn Met Thr Leu Val Ser Pro Ser Leu Ile Leu Tyr Leu Asn Asp Thr Ser Ala Gln Ala Tyr His Ser Trp Tyr Ser Leu His Tyr Lys Arg Arg Pro Ser Gln Gly Pro Asp Gln Glu Arg Ser Leu Ser Ala Tyr Pro Val Gly Glu Glu Ala Ala Glu Asp Gly Arg Ser Ser His His Arg His Arg Arg Gly Gln Glu Thr Val Ser Ser Glu Leu Lys Lys Pro Leu Gly Pro Ala Ser Phe Asn Leu Ser Glu Tyr Phe Arg Gln Phe Leu Leu Pro Gln Asn Glu Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser Gln Leu Lys Trp Asp Asn Trp Ile Val Ala Pro His Arg Tyr Asn Pro Arg Tyr Cys Lys Gly Asp Cys Pro Arg Ala val Gly His Arg Tyr Gly Ser Pro Val His Thr Met Val Gln Asn Ile Ile Tyr Glu Lys Leu Asp Ser Ser Val Pro Arg Pro Ser Cys Val Pro Ala Lys Tyr Ser Pro Leu Ser Val Leu Thr Ile Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu Tyr Glu Asp Met Ile Ala Thr Lys Cys Thr Cys Arg <210> 2 <211> 2592 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 135, 2396, (2402}...(2412), (2417)...(2422), <221> unsure <222> (2429}...(2432), 2434, 2436, 2441, 2442, 2444 <221> unsure <222>
<223> a or g or c or t, unknown, or other <220> -<223> 862403 WO 99!38976 PCTNS99/00654 <400> z ggggtagata cgcgtccggt gggtcataaa caggaatttg cttgtcgggt tcgtactcag 60 gaacgaacat cttaggcttc aatttctcaa aaaatgagat attttattta agatcattcc 120 tcaatttatc ttagnacttt cagctacata gttagtccgg tagtgtaaca gtaacgggtt 180 ttacgtaaat gccaagggaa aagagaggaa agctaaataa ccaagtaaat ctgtatctaa 240 ttaacaaatt ggctaaataa ggtgttatca gctgcttgat atagagctga taaaatcttc 300 agctaggcat acttgaggcc tgattacaga agtgaccgta gtccacccac acacctgaaa 360 tttatttaag aagaccaagc taggctcttc ctggccttta ggaagaggac tggcatggag 420 aaatatgttc ctcactagtt ctcccaagcc atggcacgtc ccaacaaatt cctcctttgg 480 ttttgctgct ttgcctggct gtgttttcct attagccttg gttctcaggc ttctggggga 540 gaagctcaga ttgctgctag tgctgagttg gaatctgggg ctatgccttg gtccttgctg 600 cagcatatag atgagagaga cagagctggc ctccttcccg cgcttttcaa agttctatct 660 gttgggcgag gtgggtcacc taggctgcag ccagactcca gagctttgca ctacatgaag 720 aagctctata agacatatgc taccaaggaa gggattccta aatccaatag aagtcacctc 780 tacaacactg ttcggctctt caccccctgt acccggcaca agcaggctcc tggagaccag 840 gtaacaggaa tccttccatc agtggaactg ctatttaacc tggatcgcat tactaccgtt 900 gaacacttac tcaagtcagt cttgctgtac cctctgctgt ccaaatgtgt gtgcaatcta 960 atgataaagg agccaaagtc ttctagcagg actctcggca gagctccata ctcatttacc 1020 tttaactcac agtttgaatt tggaaagaaa cacaaatgga ttcagattga tgtgaccagc 1080 ctccttcaac ctttagtggc ctccaacaag agaagtattc acatgtctat aaattttact 1140 tgcatgaaag accagctgga gcatccttca gcacagaatg gtttgtttaa catgactctg 1200 gtgtccccct cactgatctt atatttgaat gacacaagtg ctcaggctta tcacagctgg 1260 tattcccttc actataaaag gaggccttcc cagggtcctg accaggagag aagtctgtct 1320 gcctatcctg tgggagaaga ggctgctgag gatgggagat cttcccatca ccgtcaccgc 1380 agaggtcagg aaactgtcag ttctgaattg aagaagccct tgggcccagc ttccttcaat 1440 ctgagtgaat acttcagaca atttcttctt ccccaaaatg agtgtgagct ccatgacttt 1500 agacttagct ttagtcagct gaagtgggac aactggattg tggctccgca caggtacaac 1560 cctcgatact gtaaagggga ctgtccaagg gcagttggac atcggtatgg ctctccagtt 1620 cacaccatgg tacagaacat catctatgag aagctggact cctcagtgcc aagaccgtca 1680 tgtgtacctg ccaaatacag ccccttgagt gttttgacca ttgagcccga tggctcaatt 1740 gcctataaag agtacgaaga tatgatagct acaaagtgca cctgtcgtta acaaatggtc 1800 ctcttaaaac cttgagccta tttggcaaag taactactgt gtgcctatgt gtgccttcaa 1860 gagaaagctt catatattaa gtctctaaat gtagcatatg ttatataagg aggagcctgt 1920 gtaggttagt accttctatg gcatctatca ggataaaggg ataacatcaa ttgttgctac 1980 agagcctttt ttttatttcc aaatttaaat gaaatataat tattgtggag aactttacat 2040 ttttttcctt gagtgatttt ttttcttttc ataggagtct tattcttgat agggaaaaaa 2100 ccttaattag catcaatcct ggatggactt gcagctataa ataggcaatt cagattgctg 2160 tagtcttaat agaagaataa atttctgtca atggcaaaaa aaaaaaaaaa aaaaaaaaaa 2220 aaaagggggg gcccctcaaa gggccccaag ttttgggagc cgggggttgg ggggtaaaac 2280 ccccttgaag gggcccccaa atttcatttc cggggggggg tttttaaaag ggggggtggg 2340 ggaaaccccg ggggttcccc attttaggcc gcttcagggc ccccccccaa aggggnacgg 2400 gnnnnnnnnn nnaattnnnn nntcctttnn nntncncctc nncncccttg aagttgcatt 2460 tggttcttag tggccggatt tagagattca ggacttttcc tgtttggcca ggttgatgct 2520 attgttgtgt ccttgtggga tttccaccag gtttgtgctg ttgtgggcgt gagtgtcccc 2580 ggttgtcgcg gt 2592 <210> 3 <211> 203 <212> PRT
<213> Homo sapiens <220> -<223> 2676869 <400> 3 Met Ala Arg Pro Arg Pro Arg Glu Tyr Lys Ala Gly Asp Leu Val Phe Ala Lys Met Lys Gly Tyr Pro His Trp Pro Ala Arg Ile Asp Glu Leu Pro Glu Gly Ala Val Lys Pro Pro Ala Asn Lys Tyr Pro Ile Phe Phe Phe Gly Thr His Glu Thr Ala Phe Leu Gly Pro Lys Asp Leu Phe Pro Tyr Lys Glu Tyr Lys Asp Lys Phe Gly Lys Ser Asn Lys Arg Lys Gly Phe Asn Glu Gly Leu Trp Glu Ile Glu Asn Asn Pro Gly Val Lys Phe Thr Gly Tyr Gln Ala Ile Gln Gln Gln Ser Ser Ser Glu Thr Glu Gly Glu Gly Gly Asn Thr Ala Asp Ala 110 lI5 120 Ser Ser Glu Glu Glu Gly Asp Arg Val Glu Glu Asp Gly Lys Gly Lys Arg Lys Asn Glu Lys Ala Gly Ser Lys Arg Lys Lys Ser Tyr Thr Ser Lys Lys Ser Ser Lys Gln Ser Arg Lys Ser Pro Gly Asp Glu Asp Asp Lys Asp Cys Lys Glu Glu Glu Asn Lys Ser Ser Ser Glu Gly Gly Asp Ala Gly Asn Asp Thr Arg Asn Thr Thr Ser Asp Leu Gln Lys Thr Ser Glu Gly Thr <210> 4 <211> 3805 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 3788 <223> a or g or c or t, unknown, or other <220> -<223> 2676869 <400> 4 gcgaccgctc gtccgccggc ttgaggcccg cggggaggcg cgcaattcgt cggcccgcgg 60 gggggcggcc atcccggcag tcttcgcggc gaccaaggac taccaggaag gggagcggct 120 gggatggcgc gtccgcggcc ccgcgagtac aaagcgggcg acctggtctt cgccaagatg 180 aagggctacc cgcactggcc ggcccggatt gatgaactcc cagagggcgc tgtgaagcct 240 ccagcaaaca agtatcctat cttctttttt ggcacccatg aaactgcatt tctaggtccc 300 aaagaccttt ttccatataa ggagtacaaa gacaagtttg gaaagtcaaa caaacggaaa 360 ggatttaacg aaggattgtg ggaaatagaa aataacccag gagtaaagtt tactggctac 420 caggcaattc agcaacagag ctcttcagaa actgagggag aaggtggaaa tactgcagat 480 gcaagcagtg aggaagaagg tgatagagta gaagaagatg gaaaaggcaa aagaaagaat 540 gaaaaagcag gctcaaaacg gaaaaagtca tatacttcaa agaaatcctc taaacagtcc 600 cggaaatctc caggagatga agatgacaaa gactgcaaag aagaggaaaa caaaagcagc 660 tctgagggtg gagatgcggg caacgacaca agaaacacaa cttcagactt gcagaaaacc 720 agtgaaggga cctaactacc ataatgaatg ctgcatatta agagaaacca caagaaggtt 780 atatgtttgg ttgtctaata ttcttggatt tgatatgaac caacacatag tccttgttgt 840 cattgacaga accccagttt gtatgtacat tattcatatt cctctctgtt gtgtttcggg 900 gggaaaagac attttagcct tttttaaaag ttactgattt aatttcatgt tatttggttg 960 catgaagttg cccttaacca ctaaggatta tcaagatttt tgcgcagact tatacatgtc 1020 taggatcctt ttatcaaggc agttatgatc atcgttttcc tgccttgacc ccaccatcat 1080 caaacactca gttaaatata aattaacatt ttttagatga ccactcaaca taatgcttaa 1140 gaatggaatt tcctctctgt gacagaaccc aggaattaat tcctaaatac ataacgttgg 1200 tatattgaag acgaaattaa aattgtcctt cagttttgag gccatgtgta aagtttaacc 1260 atattgtaaa atatctattc cgtattagaa atagctagtt gacagcttat acttctcaaa 1320 attcatattg ttatgtacac aaactaagtt tctatatgtg aagttagtga gtctttttgt 1380 gttactccaa aataaaggca atgatttatt tttttcccag tgccaataca attttgagct 1440 aagcactcaa ggtggatact ttacatttta aagctggaat cagcaacagc cctatgggaa 1500 accagacaaa gcattgactt ttaaatgtag acttttaaaa taaactgttt tcttttggaa 1560 ctacaattag aatagttaat attcatcctt aaaccattat tatgtgtaca ttattgttgc 1620 tattgtgata atagagaatt ttatttattt ttatgccagc ttatattgtg agaacacatt 1680 tagtcagttt gggttttatc aatcctgtta atgcttgtcc ttggaacatc tttcgcgtat 1740 tcacggtttg tagttgaaaa gtttactgta aaaaaatcaa aaacaaaaaa atgtattgtt 1800 tttacagaat aaatttattg gaatgtgtac tgggagtaag atttgaggtt gtaagcaaac 1860 taagttagtg taatttggct tcatatatgt aacgtgaggt attaatgtaa ttcatatatt 1920 aaagcaaaaa ttgttcacag caagctgaca atagaatcaa gtgcaggtga gggtttttct 1980 ttttctttct tttttttttt ttaaacactc tctgggtttt agattatttg aaaacactgt 2040 aggggatgat ggggagatga ctacatgatt tgcttacact tacatattta ccagcccagt 2100 ggaccctaga ttttgtaatt tgatattatc catgcattga tttgtatccc attttctttt 2160 ggttttcttg atgagagcta ataaaaacat gagaaaatgt gagcagacct aaaagcctag 2220 ggccgttaat aatgatgatg gtgatgatgg cagctaaggg tatgaaagca ccagtccatt 2280 tatcattcca gggcacagtg aggaggtggc tgtttgtatt tttggtgtgt ttctttcatt 2340 tagtgaatac tgattttctc tagtatactc aacatacaat cccaaacatg aaaagttcat 2400 aaaaagatag tagggatgtc gatctaatac ttcttcctta aaaggtcctt tttaattttg 2460 aatatttgaa tgtatttttt aattaatgaa atttaattca tcaaaagacc tcttcattgc 2520 tcacactgaa acataagggg aatgtagatg gcattttgaa ttggaatata caactcacaa 2580 attttcttta aaagcgtttt atgtgtatat atgaaatgtt tcaaatttaa taagatttgt 2640 ctttataaga ggccaaaaat aattttatta aaaatataca cagaaataca atagaaatat 2700 ttccagccac caataaaaca aatgttcatt aaggatagag ataaagttac tgatccctat 2760 ttagctgttt aaattcacac aaaaaaattt agtaaaagta attttcttga aacagcagca 2820 aataataaac tttccttggg aaaacatttt tcaaacaaca tttaaaaata ttctgatgat 2880 ttgcctttta tattgagttt aataatgaag catattcatc ttatttgcaa aagttactcc 2940 tccagtctgc attcactgtt ttgaattttt taatatgcac aggccatatt ctcttttgaa 3000 tagtttcatt tcatgctcac tactgttggc actaaattgt aattttttac acttataatt 3060 attttatatg tcaaacaata ttgagatgga tgatcaagtt tattcacaaa tgattttgaa 3120 tatatttctt taaacaaact gtttctcaag aatttccatt ttccttcaaa aaaaatttac 3180 acaataccat atagctcaca catacagtgc agagcaagaa tgtacatatt cttaagtact 3240 cacgtatgag aagagagtga aatgggacaa aatatcagta ccagtataac aaaagttagt 3300 aatttagaaa ggaggaacat gaagtaatat ttttattcca ctttttcttc tgcctttgtt 3360 tttatgaaga actctggttt gtagatacaa cgataggcca agagctgagg aagaactcca 3420 tatgctatgt ttaatgctaa aaaaaggatt tttgcttctt cagggactct gtagacataa 3480 gcagttctag catgaagaga tgcaccaatg tgagaaaact gagcctgagc cagacctcca 3540 gcatgtatca atgtgatgtc aggcatccag gaacatccag gaaccactaa gccatacagt 3600 gcagtcacaa agtaaggaac agaatagaac atatatgcca gcatctgaat tttaggataa 3660 gcagcaggat cctttagata gggctcttga aattgcgtat ataatcggca gagctcagat 3720 gggcaatcca aagcaatcaa acctctgaac aggcaaaatc cagttgcccg gacgcgtggg 3780 cccacgcntt ccgcccacgc gtctg 3805 <210> 5 <211> 706 <212> PRT
$~ 15 <213> Homo sapiens <220> -<223> 1568019 <400> 5 Met Gly Arg Lys Val Thr Val Ala Thr Cys Ala Leu Asn Gln Trp Ala Leu Aap Phe Glu Gly Asn Leu Gln Arg Ile Leu Lys Ser Ile Glu Ile Ala Lys Asn Arg Gly Ala Arg Tyr Arg Leu Gly Pro Glu Leu Glu Ile Cys Gly.Tyr Gly Cys Trp Asp His Tyr Tyr Glu Ser Asp Thr Leu Leu His Ser Phe Gln Val Leu Ala Ala Leu Leu Glu Ser Pro Val Thr Gln Asp Ile Ile Cys Asp Val Gly Met Pro Val Met His Arg Asn Val Arg Tyr Asn Cys Arg Val Ile Phe Leu Asn Arg Lys Ile Leu Leu Ile Arg Pro Lys Met Ala Leu Ala Asn Glu Gly Asn Tyr Arg Glu Leu Arg Trp Phe Thr Pro Trp Ser Arg Ser Arg His Thr Glu Glu Tyr Phe Leu Pro Arg Met Ile Gln Asp Leu Thr Lys Gln Glu Thr Val Pro Phe Gly Asp Ala Val Leu Val Thr Trp Asp Thr Cys Ile Gly Ser Glu Ile Cys Glu Glu Leu Trp Thr Pro His Ser Pro His Ile Asp Met Gly Leu Asp Gly Val Glu Ile Ile Thr Asn Ala Ser Gly Ser His His Val Leu Arg Lys Ala Asn Thr Arg Val Asp Leu Val Thr Met Val Thr Ser Lys Asn Gly Gly Ile Tyr Leu Leu Ala Asn Gln Lys Gly Cys Asp Gly Asp Arg Leu Tyr Tyr Asp Gly Cys Ala Met Ile Ala Met Asn Gly Ser Val Phe Ala Gln Gly Ser Gln Phe Ser Leu Asp Asp Val Glu Val Leu Thr Ala Thr Leu Asp Leu Glu Asp Val Arg Ser Tyr Arg Ala Glu Ile Ser Ser Arg Asn Leu Ala Ala Ser Arg Ala Ser Pro Tyr Pro Arg Val Lys Val Asp Phe Ala Leu Ser Cys His Glu Asp Leu Leu Ala Pro Ile Ser Glu Pro Ile Glu Trp Lys Tyr His Ser Pro Glu Glu Glu Ile Ser Leu Gly Pro Ala Cys Trp Leu Trp Asp Phe Leu Arg Arg Ser Gln Gln Ala Gly Phe Leu Leu Pro Leu Ser Gly Gly Val Asp Ser Ala Ala Thr Ala Cys Leu Ile Tyr Ser Met Cys Cys Gln Va1 Cys Glu Ala Val Arg Ser Gly Asn Glu Glu Val Leu Ala Asp Val Arg Thr Ile Val Asn Gln Ile Ser Tyr Thr Pro Gln Asp Pro Arg Asp Leu Cys Gly Arg Ile Leu Thr Thr Cys Tyr Met Ala Ser Lys Asn Ser Ser Gln Glu Thr Cys Thr Arg Ala Arg Glu Leu Ala Gln Gln ile Gly Ser His His Ile Ser Leu Asn Ile Asp Pro Ala Val Lys Ala Val Met Gly Ile.Phe Ser Leu Val Thr Gly Lys Ser Pro Leu Phe Ala Ala His Gly Gly Ser Ser Arg Glu Asn Leu AIa Leu Gln Asn Val Gln Ala Arg Ile Arg Met Val Leu Ala Tyr Leu Phe Ala Gln Leu Ser Leu Trp Ser Arg Gly Val His Gly Gly Leu Leu Val Leu Gly Ser Ala Asn Val Asp Glu Ser Leu Leu Gly Tyr Leu Thr Lys Tyr Asp Cys Ser Ser Ala Asp Ile Asn Pro Ile Gly Gly Ile Ser Lys Thr Asp Leu Arg Ala Phe Val Gln Phe Cys Ile Gln Arg Phe Gln Leu Pro Ala Leu Gln Ser Ile Leu Leu Ala Pro Ala Thr Ala Glu Leu Glu Pro Leu Ala Asp Gly Gln Val Ser Gln Thr Asp Glu Glu Asp Met Gly Met Thr Tyr Ala Glu Leu Ser Val Tyr Gly Lys Leu Arg Lys Val Ala Lys Met Gly Pro Tyr Ser Met Phe Cys Lys Leu Leu Gly Met Trp Arg His Ile Cys Thr Pro Arg Gln Val Ala Asp Lys Val Lys Arg Phe Phe Ser Lys Tyr Ser Met Asn Arg His Lys Met Thr Thr Leu Thr Pro Ala Tyr His Ala Glu Asn Tyr Ser Pro Glu Asp Asn Arg Phe Asp Leu Arg Pro Phe Leu Tyr Asn Thr Ser Trp Pro Trp Gln Phe Arg Cys Ile Glu Asn Gln Val Leu Gln Leu Glu Arg Ala Glu Pro Gln Ser Leu Asp Gly Val Asp <210> 6 <211> 2762 <212> DNA
<213> Homo sapiens <220> -<223> 1568019 <400> 6 ggcctcctgc ccaagcgact gcggccagga tgggccggaa ggtgaccgtg gccacctgcg 60 cactcaacca gtgggccctg gacttcgagg gcaatttgca aagaatttta aagagtattg 120 aaattgccaa aaacagagga gcaagataca ggcttggacc agagctggaa atatgcggct 180 acggatgttg ggatcattat tacgagtcgg acaccctctt gcactcgttt caagtcctag 240 cggcccttct ggagtctccc gtcactcagg acatcatctg cgacgtgggg atgcctgtaa 300 tgcaccgaaa cgtccgctac aactgcagag tgatattcct caacaggaag atcctgctca 360 tcagacccaa gatggccttg gccaatgaag gcaactaccg cgagctgcgc tggttcaccc 420 cgtggtcgag gagtcggcac acagaggagt actttctgcc tcggatgata caggacctga 480 caaagcagga aaccgtaccc ttcggagatg cggtgctggt gacatgggac acctgcattg 540 gaagtgagat ctgtgaggag ctctggacac cccacagccc gcacatcgac atgggcctgg 600 atggcgtgga gatcatcacc aacgcctcgg gcagccacca cgtgctgcgc aaagccaaca 660 ccagggtgga tctcgtgact atggtcacca gcaagaacgg tgggatttac ttgctggcca 720 accagaaggg ttgcgacggg gaccgcctgt actacgacgg ctgtgccatg atcgccatga 780 acggaagcgt ctttgctcaa ggatcccagt tttctctgga tgacgtggaa gtcctgacgg 840 ccacgctgga tctggaggac gtccggagct acagggcgga gatttcatct cgaaacctgg 900 cggccagcag ggcgagcccc taccccagag tgaaggtgga ctttgccctc tcgtgccacg 960 aggacttgct ggcacccatc tctgagccca tcgagtggaa ataccacagc cctgaggagg 1020 agataagcct tggacctgcc tgctggctct gggatttttt aagacgaagt caacaggcag 1080 ggtttttgct gcccttgagt ggcggggtgg acagcgcagc caccgcctgc ctcatctact 1140 ccatgtgctg ccaggtctgc gaggccgtga ggagtggaaa tgaggaagtg ctggctgatg 1200 tccgcaccat cgtgaaccag atcagctaca ccccccagga tccccgagac ctctgtggac 1260 gcatactgac cacctgctac atggccagca agaactcctc ccaggagacg tgcacccggg 1320 ccagagagtt ggcccagcag attggaagcc accacatcag tctcaacatc gatccagccg 1380 tgaaggccgt catgggcatc ttcagcctgg tgacggggaa gagccctctg tttgcagctc 1440 atggaggaag cagcagggaa aacctggcgc tgcaaaatgt gcaggctcga atacggatgg 1500 tcctcgccta tctgtttgct cagttgagcc tctggtctcg gggtgtccac ggtgggctcc 1560 tcgtgctggg atccgccaac gtggatgaga gtctcctggg ctacctgacc aagtacgact 1620 gctccagtgc ggacatcaac cccataggcg ggatcagcaa gacggacctc agggccttcg 1680 tccagttctg catccagcgc ttccagcttc ctgccctgca gagcatcctg ttggcgccgg 1740 ccaccgcaga gctggagccc ttggctgatg gacaggtgtc ccagaccgac gaggaagata 1800 tggggatgac atatgcggag ctctcggtct atgggaaact caggaaggtg gccaagatgg 1860 ggccctacag catgttctgc aaactcctcg gcatgtggag acacatctgc accccgagac 1920 aggtcgctga caaagtgaag cggtttttct ccaagtactc catgaacaga cacaagatga 1980 ccacgctcac acccgcgtac cacgccgaga actacagccc tgaggacaac aggtttgatc 2040 tgcgaccatt tctgtacaac acaagctggc cttggcagtt tcggtgcata gaaaatcagg 2100 tgctacagct cgagagggca gagccacagt ccctggacgg cgtggactga ggccggttcc 2160 ttcctggagg cctcctgtcc tcggggaccc cagcacctca tcatcagcat tgctggagcc 2220 aagggtagga gccctacact aggagcccag gatgggacgg cgcatcagcc gagagggagg 2280 gaacttttca gtcaaattcc tcaaaaagag gctggaataa agcctgggct taaaaagagg 2340 ctggaaaaaa aaaaaagaaa gagaaggaaa aaatagtatc cattagagct atgggtatac 2400 catcacatcg atagtaaaaa gagagtcaaa agtcacacca ggacacaact agaacaaaaa 2460 aaggggggcg cccccacttt gtaggccctc taaccggggt aatttttggg gcgggtacct 2520 ctgggggaac caattttccc caaagggcgg cgtttttaag cctaagggga acaaaggggc 2580 aaaatattcc cccgggggga aattttttcc ccgcccaaat ttccccacaa tttaaggggg 2640 gggggggcta aaagtttaac acccgagggg ggccccatgg ggggggggaa cccccccttt 2700 tattgggtgg ggcacacgtg ccccgttttc cccggggggg gacccttttg ttaccactat 2760 at <210> 7 <211> 223 <212> PRT
<213> Homo sapiens <220> -<223> 3577857 8/1$

<400> 7 Met Lys Leu His Tyr Val Ala Val Leu Thr Leu Ala Ile Leu Met Phe Leu Thr Trp Leu Pro Glu Ser Leu Ser Cys Asn Lys Ala Leu Cys Ala Ser Asp Val Ser Lys Cys Leu Ile Gln Glu Leu Cys Gln Cys Arg Pro Gly Glu Gly Asn Cys Ser Cys Cys Lys Glu Cys Met Leu Cys Leu Gly Ala Leu Trp Asp Glu Cys Cys Asp Cys Val Gly Met Cys Asn Pro Arg Asn Tyr Ser Asp Thr Pro Pro Thr Ser Lys Ser Thr Val Glu Glu Leu His Glu Pro Ile Pro Ser Leu Phe Arg Ala Leu Thr Glu Gly Asp Thr Gln Leu Asn Trp Aan Ile Val Ser Phe Pro Val Ala Glu Glu Leu Ser His His Glu Asn Leu Val Ser Phe Leu Glu Thr Val Asn Gln Pro His His Gln Asn Val Ser Val Pro Ser Asn Asn Val His Ala Pro Tyr Ser Ser Asp Lys Glu His Met Cys Thr Val Val Tyr Phe Asp Asp Cys Met Ser Ile His Gln Cys Lys Ile Ser Cys Glu Ser Met Gly Ala Ser Lys Tyr Arg Trp Phe His Asn Ala Cys Cys Glu Cys Ile Gly Pro Glu Cys Ile Asp Tyr Gly Ser Lys Thr Val Lys. Cys Met Aen Cys Met Phe <210> 8 <211> 2204 <212> DNA
<213> Homo sapiens <220> -<223> 3577857 <400> 8 gcgggaggcg cggcctggcc tcgcactcaa agccgccgca gcgcgccccg ggctcggccg 60 acccggcggg gatctagggg tgggcgactt cgcgggaccg tggcgcatgt ttcctgggag 120 ttactgatca tcttctttga agaaacatga agttacacta tgttgctgtg cttactctag 180 ccatcctgat gttcctgaca tggcttccag aatcactgag ctgtaacaaa gcactctgtg 240 ctagtgatgt gagcaaatgc ctcattcagg agctctgcca gtgccggccg ggagaaggca 300 attgctcctg ctgtaaggag tgcatgctgt gtcttggggc cctttgggac gagtgctgtg 360 actgtgttgg tatgtgtaat cctcgaaatt atagtgacac acctccaact tcaaagagca 420 cagtggagga gctgcatgaa ccgatccctt ctctcttccg ggcactcaca gaaggagata 480 ctcagttgaa ttggaacatc gtttctttcc ctgttgcaga agaactttca catcatgaga 540 atctggtttc atttttagaa actgtgaacc agccacacca ccagaatgtg tctgtcccca 600 gcaataatgt tcacgcgcct tattccagtg acaaagaaca catgtgtact gtggtttatt 660 ttgatgactg catgtccata catcagtgta aaatatcctg tgagtccatg ggagcatcca 720 aatatcgctg gtttcataat gcctgctgcg agtgcattgg tccagaatgt attgactatg 780 gtagtaaaac tgtcaaatgt atgaactgca tgttttaaag aagacaaatg caaaccaaag 840 caacttagta aaataatagg tataaaaagt tattctgtaa gtctgttggt tgtatcttgt 900 atcagaatcc cagtaagtta agttgtaaag actttggaat aagtttcttt taaaaatatg 960 acatagccag tgatgtgttt aattatataa ctgttcttac tgattttatt gccccctagc 1020 aataagccct ttcctttgaa tacatgtaca actttggtca tatgagaagc aggtgcgcag 1080 agaattcctt gaaagatctg aggtttttaa cataaagtct gatgtggttt tcctctagca 1140 ttccaaaagg tttttgcttt gaaagtgtta gcagaagcat gttgatgtga attatgattt 1200 cttcatgtgc tactgttagc acactgagtt tttatagttg cacatcattc ctcattgtgc 1260 cttgttttat ccattttata aatagagtag atatttgata taccactctg ataactcata 1320 taaaaatatc atcataaaaa gcttaatttc atccctttta tgttggtttt aaaaggtaaa 1380 tgcttaccat attttataat tgagaactct tacatagtag aatccattct ataatacatg 1440 tgttgacaaa gctttagaga aagtttccta ttctcttcca tttcccctgc ccaaagtgct 1500 gacataggca gtgatgaaga atctttacca agattttcag ggtgtaccta tgaaattgct 1560 ttaaatgcac tgctggtgta aataattagc aagcaaaagc gtttctgtga cttcaggtac 1620 cagcttaaag agcactaggg atggggaacg aatgccaaat cagactccac ctagagcacc 1680 aggaaacagc ttgtaccctg gtagggaaat ggtgttgctg aaaggggagg ctgagccagt 1740 gcgagactga acttgtgcag ccttagccaa gacaaagcag tgtttttcag cagacggctg 1800 atgggacagg aattgaagaa gagaattgac tcgtatgaac aggacagggt gaaaatgctg 1860 ggaattataa tgggaaacaa aactatctat gttcatattt tgtaatattt catttgttaa 1920 gtttatatct ggatataatg ttctttttaa acaagtataa tcatatcgtc ggaggttaag 1980 attatgaaat tttagaatct ctattcaaga tgatgttcac tccaaataca ctacagaatt 2040 tagtcaacat tttatataat gtttcaataa atgtttcttt caataaagat actatgtgcc 2100 cttcatagta ataaaatctc aatcttaaag catgaatcta aaacataaat atttatatta 2160 cagactcaat tttacaaaat tactaattgc tctagtgcat attt 2204 <210> 9 <211> 441 <212> PRT
<213> Mus musculus <220> -<223> 8567206 <400> 9 Met Ala Leu Pro Ser Aan Phe Leu Leu Gly Val Cys Cys Phe Ala Trp Leu Cys Phe Leu Ser Ser Leu Ser Ser Gln Ala Ser Thr Glu Glu Ser Gln Ser Gly Ala Ser Glu Asn Val Glu Ser Glu Ala Asp Pro Trp Ser Leu Leu Leu Pro Val Asp Gly Thr Asp Arg Ser Gly Leu Leu Pro Pro Leu Phe Lys Val Leu Ser Asp Arg Arg Gly Glu Thr Pro Lys Leu Gln Pro Asp Ser Arg Ala Leu Tyr Tyr Met Lys Lys Leu Tyr Lys Thr Tyr Ala Thr Lys Glu Gly Val Pro Lys Pro Ser Arg Ser His Leu Tyr Asn Thr Val Arg Leu Phe Ser Pro Cys Ala Gin Gln Glu Gln Ala Pro Ser Asn Gln Val Thr Gly Pro Leu Pro Met Val Asp Leu Leu Phe Asn Leu Asp Arg Val Thr Ala Met Glu His Leu Leu Lys Ser Val Leu Leu Tyr Thr Leu Asn Asn Ser WO 99!38976 PCT/US99/00654 Ala Ser Ser Ser Ser Thr Val Thr Cys Met Cys Asp Leu Val Val Lys Glu Ala Met Ser Ser Gly Arg Ala Pro Pro Arg Ala Pro Tyr Ser Phe Thr Leu Lys Lys His Arg Trp Ile Glu Ile Asp Val Thr Ser Leu Leu Gln Pro Leu Val Thr Ser Ser Glu Arg Ser Ile His Leu Ser Val Asn Phe Thr Cys Thr Lys Asp Gln Val Pro Glu Asp Gly Val Phe Ser Met Pro Leu Ser Val Pro Pro Ser Leu Ile Leu Tyr Leu Asn Asp Thr Ser Thr Gln Ala Tyr His Ser Trp Gln Ser Leu Gln Ser Thr Trp Arg Pro Leu Gln His Pro Gly Gln Ala Gly Val Ala Ala Arg Pro Val Lys Glu Glu Ala Thr Glu Val Glu Arg Ser Pro Arg Arg Arg Arg Gly Gln Lys Ala Ile Arg Ser Glu Ala Lys Gly Pro Leu Leu Thr Ala Ser Phe Asn Leu Ser Glu Tyr Phe Lys Gln Phe Leu Phe Pro Gln Asn Glu Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser Gln Leu Lys Trp Asp Asn Trp Ile Val Ala Pro His Arg Tyr Asn Pro Arg Tyr Cys Lys Gly Asp Cys Pro Arg Ala Val Arg His Arg Tyr Gly Ser Pro Val His Thr Met Val Gln Asn Ile Ile Tyr Glu Lys Leu Asp Pro Ser Val Pro Arg Pro Ser Cys Val Pro Gly Lys Tyr Ser Pro Leu Ser Val Leu Thr Ile Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu Tyr Glu Asp Met Ile Ala Thr Arg Cys Thr Cys Arg <210> 10 <211> 240 <212> PRT
<2I3> Homo sapiens <220> -<223> 8598956 <400> 10 Met Ser Arg Ser Asn Arg Gln Lys Glu Tyr Lys Cys Gly Asp Leu Val Phe Ala Lye Met Lys Gly Tyr Pro His Trp Pro Ala Arg Ile Asp Glu Met Pro Glu Ala Ala Val Lys Ser Thr Ala Asn Lys Tyr Gln Val Phe Phe Phe Gly Thr His Glu Thr Ala Phe Leu Gly Pro Lys Asp Leu Phe Pro Tyr Glu Glu Ser Lys Glu Lys Phe Gly Lys 65 7p 75 Pro Asn Lys Arg Lys Gly Phe Ser Glu Gly Leu Trp Glu Ile Glu Asn Asn Pro Thr Val Lys Ala Ser Gly Tyr Gln Ser Ser Gln Lys Lye Ser Cys Val Glu Glu Pro Glu Pro Glu Pro Glu Ala Ala Glu Gly Asp Gly Asp Lys Lys Gly Asn Ala Glu Gly Ser Ser Asp Glu Glu Gly Lys Leu Val Ile Asp Glu Pro Ala Lys Glu Lys Asn Glu Lys Gly Ala Leu Lys Arg Arg Ala Gly Asp Leu Leu Glu Asp Ser Pro Lys Arg Pro Lys Glu Ala Glu Asn Pro Glu Gly Glu Glu Lys Glu Ala Ala Thr Leu Glu Val Glu Arg Pro Leu Pro Met Glu Val Glu Lys Asn Ser Thr Pro Ser Glu Pro Gly Ser Gly Arg Gly Pro Pro Gln Glu Glu Glu Glu Glu Glu Asp Glu Glu Glu Glu Ala Thr Lys Glu Asp Ala Glu Ala Pro Gly Ile Arg Asp His Glu Ser Leu <210> 11 <211> 714 <212> PRT
c213> Saccharomyces cerivisiae <220> -<223> 8500832 <400> 11 Met Ser His Leu Ile Thr Leu Ala Thr Cys Asn Leu Asn Gln Trp Ala Leu Asp Phe Glu Gly Asn Arg Asp Arg Ile Leu Gln Ser Ile Lys 21e Ala Lys Glu Arg Gly Ala Arg Leu Arg Val Gly Pro Glu 35 . 40 45 Leu Glu Ile Thr Gly Tyr Gly Cys Leu Asp His Phe Leu Glu Asn Asp Val Cys Leu His Ser Trp Glu Met Tyr Ala Gln Ile Ile Lys Asn Lys Glu Thr His Gly Leu Ile Leu Asp Ile Gly Met Pro Val Leu His Lys Asn Val Arg Tyr Asn Cys Arg Leu Leu Ser Leu Asp Gly Glu Ile Leu Phe Ile Arg Pro Lys Ile Trp Leu Ala Asn Asp Gly Asn Tyr Arg Glu Met Arg Phe Phe Thr Pro Trp Met Lys Pro Gly Val Val Glu Asp Phe Ile Leu Pro Pro Glu Ile Gln Lys Val Thr Gly Gln Arg Leu Val Pro Phe Gly Asp Ala Val Ile Asn Ser Leu Asp Thr Cys Ile Gly Thr Glu Thr Cys Glu Glu Leu Phe Thr Pro Gln Ser Pro His Ile Ala Met Ser Leu Asp Gly Val Glu Ile Met Thr Asn Ser Ser Gly Ser His His Glu Leu Arg Lys Leu Asn Lys Arg Leu Asp Leu Ile Leu Asn Ala Thr Lys Arg Cys Gly Gly Val Tyr Leu Tyr Ala Asn Gln Arg Gly Cys Asp Gly Asp Arg Leu Tyr Tyr Asp Gly Cys Ala Leu Ile Ala Ile Asn Gly Thr Ile Val Ala Gln Gly Ser Gln Phe Ser Leu Asp Asp Val Glu Val Val Thr Ala Thr Val Asp Leu Glu Glu Val Arg Ser Tyr Arg Ala Ala Val Met Ser Arg Gly Leu Gln Ala Ser Leu Ala Glu Ile Lys Phe Lys Arg Ile Asp Ile Pro Val Glu Leu Ala Leu Met Thr Ser Arg Phe Asp Pro Thr Val Cys Pro Thr Lys Val Arg Glu Pro Phe Tyr His Ser Pro Glu Glu Glu Ile Ala Leu Gly Pro Ala Cys Trp Met Trp Asp Tyr Leu Arg Arg Cys Asn Gly Thr Gly Phe Phe Leu Pro Leu Ser Gly Gly Ile Asp Ser Cys Ala Thr Ala Met Ile Val His Ser Met Cys Arg Leu Val Thr Asp Ala Ala Gln Asn Gly Asn Glu Gln Val Ile Lys Asp Val Arg Lys Ile Thr Arg Ser Gly Asp Asp Trp Ile Pro Asp Ser Pro Gln Asp Leu Ala Ser Lys Ile Phe His Ser Cys Phe Met Gly Thr Glu Asn Ser Ser Lys Glu Thr Arg Asn Arg Ala Lys Asp Leu Ser Asn Ala Ile Gly Ser Tyr His Val Asp Leu Lys Met Asp Ser Leu Val Ser Ser Val Val Ser Leu Phe Glu Val Ala Thr Gly Lys Lys Pro Ile Tyr Lys Ile Phe Gly Gly Ser Gln Ile Glu Asn Leu Ala Leu Gln Asn Ile Gln Ala Arg Leu Arg Met Val Leu Ser Tyr Leu Phe Ala Gln Leu Leu Pro Trp Val Arg Gly Ile Pro Asn Ser Gly Gly Leu Leu Val Leu Gly Ser Ala Asn Val Asp Glu Cys Leu Arg Gly Tyr Leu Thr Lys Tyr Asp Cys Ser Ser Ala Asp Ile Asn Pro Ile Gly Gly Ile Ser Lys Thr Asp Leu Lys Arg Phe Ile Ala Tyr Ala Ser Lys Gln Tyr Asn Met Pro Ile Leu WO 99/389'76 PCT/US99/00654 sso s65 570 Asn Asp Phe Leu Asn Ala Thr Pro Thr Ala Glu Leu Glu Pro Met Thr Lys Asp Tyr VaI Gln Ser Asp Glu Ile Asp Met Gly Met Thr Tyr Glu Glu Leu Gly Val Phe Gly Tyr Leu Arg Lys Val Glu Lys Cys Gly Pro Tyr Ser Met Phe Leu Lys Leu Leu His Gln Trp Ser Pro Lys Leu Thr Pro Arg Gln Ile Ser Glu Lys Val Lys Arg Phe Phe Phe Phe Tyr Ala Ile Asn Arg His Lys Gln Thr Val Leu Thr Pro Ser Tyr His Ala Glu Gln Tyr Ser Pro Glu Asp Asn Arg Phe Asp Leu Arg Pro Phe Leu Ile Asn Pro Arg Phe Pro Trp Ala Ser Arg Lys Ile Asp Glu Val Val Glu Gln Cys Glu Ala His Lys Gly Ser Thr Leu Asp Ile Met Ser Ile Asp <210> 12 <211> 249 <212> PRT
<213> Drosophila melanogaster <220> -<223> 8529900 <400> 12 Met Gln Leu Leu Cys Tyr Phe Val Ile Leu Phe Val Gly Ile Ala Pro Trp Ser Ser Leu Ala Aan Asp Asp Gly Cys Asn Glu Val Val Cys Gly Ser Val Val Ser Lys Cys Leu Ile Thr Gln Ser Cys Gln Cys Lys Leu Asn Asp Cya His Cys Cys Lys Asp Cys Leu Asn Cys Leu Gly Glu Leu Tyr Ile Glu Cys Cys Gly Cys Leu Asp Met Cys Pro Lys His Lys Asp Val Leu Pro Ser Leu Thr Pro Arg Ser Glu Ile Gly Asp Ile Glu Gly Val Pro Glu Leu Phe Asp Thr Leu Thr Ala Glu Asp Asp Glu Gly Trp Ser Thr Ile Arg Phe Ser Met Arg Ala Gly Phe Lys Gln Arg Val Gln Gly Gly Ala Ser Gly Asp Ala Gly Asn Gly Asn Gly Asn Gly Asn Ala Gly Ser Ala Gly Val Thr Leu Cys Thr Val Ile Tyr Val Asn Ser Cys Ile Arg Ala Asn Lys Cys Arg Gln Gln Cys Glu Ser Met Gly Ala Ser Ser Tyr Arg Trp Phe His Asp Gly Cys Cys Glu Cys Val Gly Glu Asn Cys Leu Asn Tyr Gly Ile Asn Glu Ser Arg Cys Arg Gly Cys Pro Glu Asp Gln 200 205 . 210 Asp Gln Leu Leu Thr Ala Asp Thr Val Pro Ala Glu Ala Glu Gln Asp Leu Glu Arg Phe Phe Gly Asn Glu Glu Ile Glu Asp Glu Trp 230 235_ 240 Gly Tyr Gly Glu Glu Asp Glu Phe Ser ISIS

Claims (21)

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, 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.

2. A substantially purified variant having at least 90% amino acid identity to the amino acid sequence of claim 1.

3. An isolated and purified polynucleotide encoding the HGFH of claim 1.

4. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 3.

5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3.

6. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide sequence of claim 3.

7. 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.

8. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 7.

9. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 7.

10. A fragment of the polynucleotide of claim 7 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-I82 of SEQ ID NO:8.

11. An expression vector containing at least a fragment of the polynucleotide of claim 3.

12. A host cell containing the expression vector of claim 11.

13. A method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1, 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, or a fragment of SEQ ID
NO:7, the method comprising the steps of:
a) culturing the host cell of claim 12 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.

14. A pharmaceutical composition comprising the polypeptide of claim 1 in conjunction with a suitable pharmaceutical carrier.

15. A purified antibody which specifically binds to the polypeptide of claim 1.

16. A purified agonist of the polypeptide of claim 1.

17. A purified antagonist of the polypeptide of claim 1.

18. 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 the antagonist of claim 17.

19. 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 the pharmaceutical composition of claim 14.

20. A method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1, 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, or a fragment of SEQ ID NO:7 in a biological sample, the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 to at least one of the nucleic acids in the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of the polynucleotide encoding the polypeptide in the biological sample.

21. The method of claim 20 wherein the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to hybridization.