CA2384324A1 - Proteins associated with cell differentiation - Google Patents

Abstract

The invention provides human proteins involved in cell differentiation (CDIF F) and polynucleotides which identify and encode CDIFF. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, o r preventing disorders associated with expression of CDIFF.

Description

PROTEINS ASSOCIATED WITH CELL DIFFERENTIATION
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteins involved in cell differentiation and to the use of these sequences in the diagnosis, treatment, and prevention of cell .
proliferative, developmental, and neurological disorders.
BACKGROUND OF THE INVENTION
Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function, despite the fact that each cell is like the others in its hereditary endowment.
Cell differentiation is the process by which cells come to differ in their structure and physiological function. The cells of a multicellular organism all arise from mitotic divisions of a single-celled zygote. The zygote is totipotent, meaning that it has the ability to give rise to every type of cell in the adult body. During development the cellular descendants of the zygote lose their totipotency and become determined. Once its prospective fate is achieved, a cell is said to have differentiated. All descendants of this cell will be of the same type.
Human growth and development requires the spatial and temporal regulation of cell differentiation, along with cell proliferation and regulated cell death. These processes coordinately control reproduction, aging, embryogenesis, morphogenesis, organogenesis, and tissue repair and maintenance. The processes involved in cell differentiation are also relevant to disease states such as cancer, in which case the factors regulating normal cell differentiation have been altered, allowing the cancerous cells to proliferate in an anaplastic, or undifferentiated state.
The mechanisms of differentiation involve cell-specific regulation of transcription and translation, so that different genes are selectively expressed at different times in different cells.
Genetic experiments using the fruit fly Drosophila melano ag ster have identified regulated cascades of transcription factors which control pattern formation during development and differentiation. These include the homeotic genes, which encode transcription factors containing homeobox motifs. The products of homeotic genes determine how the insect's imaginal discs develop from masses of undifferentiated cells to specific segments containing complex organs. Many genes found to be involved in cell differentiation and development in Drosophila have homologs in mammals. For example, human homologs have recently been found for the Drosophila ash2 gene.
The ash2 gene product is a transcriptional regulator of homeotic selector genes and is implicated in early development and formation of various disc patterns in the fruit fly (Ikegawa, S. (1999) Cytogenet.
Cell Genet. 84:167-172). The ariadne-2 protein, a retinoic-acid inducible protein with a RING finger transcription factor motif, also has a human homolog (GenBank Entry g5453556, Homo Sapiens ariadne-2 (D. melano ag ster) homology.
There is evidence in some cases that the human genes have equivalent developmental roles as their Drosophila homologs. The human homolog of the Drosophila eyes absent gene (eya) underlies branchio-oto-renal syndrome, a developmental disorder affecting the ears and kidneys (Abdelhak, S.
et al. ( 1997) Nat. Genet. 15:157-164). The Drosophila slit gene encodes a secreted leucine-rich repeat containing protein expressed by the midline glial cells and required for normal neural development. Two mammalian homologs, SLIT1 and SLTT 2, have recently been identified in both humans and mice. In mice both genes are expressed during CNS development in the floor plate (the vertebrate equivalent of midline glial cells), roof plate and developing motor neurons, suggesting a conservation of protein function between Drosophila and mammals (Holmes, G. P.
( 1998) Mech.
Dev. 79:57-72).
At the cellular level, growth and development are governed by the cell's decision to enter into or exit from the cell cycle and by the cell's commitment to a terminally differentiated state. The schlafen family of genes, a novel family of at least 7 members, are involved in maintenance of T cell quiescence. These genes are differentially regulated during thymocyte maturation and are preferentially expressed in lymphoid tissues. Expression of schlafen genes in fibroblasts or thyoma cells either retards or ablates cell growth, indicating that the schlafen proteins probably participate in regulation of the cell cycle (Schwarz, D. A. (1998) Immunity 9:657-668).
Differential gene expression within cells is triggered in response to extracellular signals and other environmental cues. Such signals include growth factors and other mitogens such as retinoic acid; cell-cell and cell-matrix contacts; and environmental factors such as nutritional signals, toxic substances, and heat shock. Candidate genes that may play a role in differentiation can be identified by altered expression patterns upon induction of cell differentiation in vitro. For example, the REX
genes display reduced expression during retinoic acid induced differentiation of murine teratocarcinoma cells (Faria et al. (1998) Mol. Cell Endocrinol. 143:155-166).
The murine embryonal carcinoma cell line P19 responds to retinoic acid by differentiating into neuronal cell types. The shyc gene was isolated from differentiating P19 cells and found to be predominantly expressed in the developing and embryonic nervous system, as well as the olfactory pathway of the adult mouse brain (Koster, F. et al. (1998) Neurosci. Lett. 252:69-71). Similarly, the Bdml gene is upregulated during differentiation of P19 cells to neuronal cells by retinoic acid, and was widely expressed in the olfactory bulb, cerebral cortex, hippocampus, cerebellum, thalamus, and medulla oblongata (Yamauchi, Y. et al. ( 1999) Brain Res. Mol. Brain Res. 68:149-58). These proteins therefore appear to play a role in the differentiation and later function of neuronal cells.
The final step in cell differentiation results in a specialization that is characterized by the production of particular proteins, such as contractile proteins in muscle cells, serum proteins in liver cells and globins in red blood cell precursors. The expression of these specialized proteins depends at least in part on cell-specific transcription factors. For example, the homobox-containing transcription factor PAX-6 is essential for early eye determination, specification of ocular tissues, and normal eye development in vertebrates. PAX-6 is also involved in regulating the expression of crystalline, the dominant structural proteins of the eye lens. Defects in crystallin proteins can cause formation of cataracts, the most common cause of visual impairment world-wide (Francis, P.
J. et al. (1999) Trends Genet. 15:191-196).
In the case of epidermal differentiation, the induction of differentiation-specific genes occurs either together with or following growth arrest and is believed to be linked to the molecular events that control irreversible growth arrest. Irreversible growth arrest is an early event which occurs when cells transit from the basal to the innermost suprabasal layer of the skin and begin expressing squamous-specific genes. These genes include those involved in the formation of the cross-linked envelope, such as transglutaminase I and III, involucrin, loricin, and small proline-rich repeat (SPRR) proteins. The SPRR proteins are 8-10 kDa in molecular mass, rich in proline, glutamine, and cysteine, and contain similar repeating sequence elements. The SPRR proteins may be structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins.
(Jetten, A. M. and Harvat, B. L. (1997) J. Dermatol. 24:711-725; PRINTS Entry Small proline-rich protein signature.) The discovery of new proteins involved in cell differentiation and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, developmental, and neurological disorders.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, proteins involved in cell differentiation, referred to collectively as "CDIFF" and individually as "CDIFF-1," "CDIFF-2,"
"CDIFF-3," "CDIFF-4," "CDIFF-5," "CDIFF-6," "CDIFF-7," "CDIFF-8," "CDIFF-9," "CDIFF-10," "CDIFF-11,"
"CDIFF-12," "CDIFF-13," "CDIFF-14," "CDIFF-15," "CDIFF-16," "CDIFF-17," "CDIFF-18,"
"CDIFF-19," "CDIFF-20," "CDIFF-21," "CDIFF-22," "CDIFF-23," "CDIFF-24," "CDIFF-25,"
"CDIFF-26," "CDIFF-27," and "CDIFF-28." In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:I-28. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-28.
The invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ )D NO:1-28. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:29-56.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: I-28, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:I-28. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: I-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28.
The invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:29-56, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID
N0:29-56, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:29-56, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID
N0:29-56, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:29-56, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ 1D
N0:29-56, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional CDIFF, comprising administering to a patient in need of such treatment the composition.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-28. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional CDIFF, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ 117 NO:1-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-28. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional CDIFF, comprising administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that modulates the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-28. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID N0:29-56, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID
N0:29-56, ii) a naturally occurnng polynucleotide sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:29-56, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID N0:29-56, ii) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:29-56, iii) a polynucleotide sequence complementary to i}, iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above;
c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs), clone identification numbers (clone )Ds), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding CDIFF.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of CDIFF.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis; diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding CDIFF were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
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 machines, materials and methods 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 same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors 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 invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"CDIFF" refers to the amino acid sequences of substantially purified CDIFF
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of CDIFF. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of CDIFF either by directly interacting with CDIFF or by acting on components of the biological pathway in which CDIFF
participates.
An "allelic variant" is an alternative form of the gene encoding CDIFF.
Allelic variants 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. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants 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 CDIFF include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as CDIFF or a polypeptide with at least one functional characteristic of CDIFF. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding CDIFF, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding CDIFF. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent CDIFF. 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 CDIFF is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of CDIFF. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of CDIFF either by directly interacting with CDIFF or by acting on components of the biological pathway in which CDIFF participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind CDIFF polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized 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" refers to that region 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 (particular regions or three-dimensional structures on the protein). An antigenic 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" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "irrimunogenic"
refers to the capability of the natural, recombinant, or synthetic CDIFF, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding CDIFF or fragments of CDIFF 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. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" 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" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
A "fragment" is a unique portion of CDIFF or the polynucleotide encoding CDIFF
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID N0:29-56 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:29-56, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:29-56 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:29-56 from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:29-56 and the region of SEQ ID N0:29-56 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO:1-28 is encoded by a fragment of SEQ ID N0:29-56. A
fragment of SEQ ID NO:1-28 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-28. For example, a fragment of SEQ ID NO:1-28 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-28.
The precise length of a fragment of SEQ ID NO:1-28 and the region of SEQ ID NO:1-28 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full-length" polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A
"full-length" polynucleotide sequence encodes a "full-length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Cap: 2 penalties Gap x drop-off:' S0 Expect: l0 Word Size: 1l Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (Apr-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Open Gap: 1I and Extension Gap: 1 penalties Gap x drop-off:' S0 Expect: 10 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 lrg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 pg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., 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" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of CDIFF
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of CDIFF which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of CDIFF. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CDIFF.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an CDIFF may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of CDIFF.
"Probe" refers to nucleic acid sequences encoding CDIFF, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerise enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerise chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laborat~ Manual, 2"° ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. ( 1990) PCR
Protocols, A Guide to Methods and Annlications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding CDIFF, or fragments thereof, or CDIFF 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 substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising 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.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least SEQ ID N0:29-56 free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or 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 chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants, and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook, J. et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic"
variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing.
The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human proteins involved in cell differentiation (CDIFF), the polynucleotides encoding CDIFF, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, developmental, and neurological disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding CDIFF. Columns I and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each CDIFF were identified, and column 4 shows the cDNA
libraries from which these clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. In some cases, GenBank sequence identifiers are also shown in column S.
The Incyte clones and GenBank cDNA sequences, where indicated, in column 5 were used to assemble the consensus nucleotide sequence of each CDIF'F and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 shows homologous sequences as identified by BLAST analysis along with relevant citations, all of which are expressly incorporated by reference herein in their entirety; and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding CDIFF. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID
N0:29-56 and to distinguish between SEQ ID N0:29-56 and related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 lists tissue categories which express CDIFF as a fraction of total tissues expressing CDIFF.
Column 4 lists diseases, disorders, or conditions associated with those tissues expressing CDIFF as a fraction of total tissues expressing CDIFF. Column 5 lists the vectors used to subclone each cDNA
library.
The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding CDIFF were isolated. Column 1 references the nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
SEQ ID N0:32 maps to chromosome 1 within the interval from 152.2 to 157.4 centiMorgans, to chromosome 3 within the interval from 157.4 to 158.0 centiMorgans, and to the X chromosome within the interval from 104.9 to 150.3 centiMorgans. The interval on chromosome 1 from 152.2 to 157.4 centiMorgans also contains genes associated with leukemia, hypothyroidism, and adrenal hyperplasia. The interval on the X chromosome from 104.9 to 150.3 centiMorgans also contains genes associated with X-linked lissencephaly, leiomyomatosis with Alport syndrome, lymphoproliferative syndrome, Bruton agammaglobulinemia, and diffuse angiokeratoma. SEQ ID
N0:37 maps to chromosome 11 within the interval from 19.6 to 23.2 centiMorgans. SEQ ID N0:39 maps to chromosome 16 within the interval from 109.1 to 130.8 centiMorgans, and to chromosome 22 within the interval from 45.5 to 58.1 centiMorgans. The interval on chromosome 16 from 109.1 to 130.8 centiMorgans also contains a gene associated with gastric cancer susceptibility. SEQ ID
N0:45 maps to chromosome 7 within the interval from 105.2 to 109.0 centiMorgans, to chromosome 17 within the interval from 65.0 to 90.2 centiMorgans, and to chromosome 20 within the interval from 50.2 to 54.9 centiMorgans. The interval on chromosome 7 from 105.2 to 109.0 centiMorgans also contains a gene associated with osteogenesis imperfecta. The interval on chromosome 17 from 65.0 to 90.2 centiMorgans also contains genes associated with breast cancer, hepatic leukemia, myeloperoxidase deficiency, muscular dystrophy, periodic paralysis, and placental growth. SEQ ID
N0:54 maps to chromosome 12 within the interval from 21.3 to 36.1 centiMorgans. SEQ ID N0:55 maps to chromosome 1 within the interval from 22.9 to 39.9 centiMorgans and to chromosome 3 within the interval from 30.9 to 43.0 centiMorgans.
The invention also encompasses CDIFF variants. A preferred CDIFF variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the CDIFF amino acid sequence, and which contains at least one functional or structural characteristic of CDIFF.

The invention also encompasses polynucleotides which encode CDIFF. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:29-56, which encodes CDIFF. The polynucleotide sequences of SEQ ID N0:29-56, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding CDIFF. In particular, such a variant polynucleotide sequence will have at least about 80%, or alternatively at least about 90%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding CDIFF. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID
N0:29-56 which has at least about 80%, or alternatively at least about 90%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:29-56. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CDIFF.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding CDIFF, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring 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 CDIFF, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode CDIFF and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring CDIFF under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CDIFF or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding CDIFF 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 CDIFF
and CDIFF 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 well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding CDIFF 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
N0:29-56 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (PE
Biosystems, Foster City CA), thermostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerises and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI
CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biolo~v and Biotechnology, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding CDIFF may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence 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 PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, 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 template at temperatures of about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), 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 sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode CDIFF may be cloned in recombinant DNA molecules that direct expression of CDIFF, 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 used to express CDIFF.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter CDIFF-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of CDIFF, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding CDIF'F 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) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.) Alternatively, CDIFF itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (PE Biosystems). Additionally, the amino acid sequence of CDIFF, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high 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, supra, pp. 28-53.) In order to express a biologically active CDIFF, the nucleotide sequences encoding CDIFF or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding CDIFF. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CDIFF. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding CDIFF and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. 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 host cell system used. (See, e.g., Scharf, D. et al. ( 1994) Results Probl. Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding CDIFF and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. ( 1989) Molecular Cloning Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular BioloQV, 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 CDIFF. 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 viral expression vectors (e.g., baculovirus);
plant cell systems transformed with viral 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. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-31 I; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
( 1984) Science 224:838-843; Winter, J. et al. ( 1991 ) Results Probl. Cell Differ. 17:85-105; The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) 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. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding CDIFF. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding CDIFF can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding CDIFF into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of CDIFF are needed, e.g. for the production of antibodies, vectors which direct high level expression of CDIFF may be used.
For example, vectors containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of CDIFF. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia nastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, supra; Bitter, su ra; and Scorer, supra.) Plant systems may also be used for expression of CDIFF. Transcription of sequences encoding CDIFF may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV 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, su ra;~Broglie, su ra; and Winter, supra.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding CDIFF
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses CDIFF in host cells. (See, e.g., Logan, J. and T. Shenk ( 1984) Proc.
Natl. Acad. Sci. USA 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. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from 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. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of CDIFF in cell lines is preferred. For example, sequences encoding CDIFF can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated 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 and adenine phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; 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; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate f3-glucuronide, or luciferase and its substrate luciferin may be used. 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. (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 CDIFF is inserted within a marker gene sequence, transformed cells containing sequences encoding CDIFF can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding CDIFF under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding CDIF'F
and that express CDIF'F 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, PCR
amplification, 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.
Immunological methods for detecting and measuring the expression of CDIFF
using either specific polyclonal or monoclonal antibodies 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 CDIFF is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) 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 CDIFF
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding CDIFF, or any fragments thereof, may be cloned into a vector for the production of an 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 Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. 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 CDIFF 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 retained 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 CDIFF may be designed to contain signal sequences which direct secretion of CDIF'F through a prokaryotic or eukaryotic cell membrane.
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 which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
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, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding CDIFF may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric CDIFF protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of CDIFF
activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the CDIFF encoding sequence and the heterologous protein sequence, so that CDIFF may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, su ra, ch. 10).
A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled CDIFF may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
CDIFF of the present invention or fragments thereof may be used to screen for compounds that specifically bind to CDIFF. At least one and up to a plurality of test compounds may be screened for specific binding to CDIFF. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of CDIFF, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunoloay 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which CDIFF
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express CDIFF, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosonhila, or E. coli. Cells expressing CDIFF or cell membrane fractions which contain CDIFF
are then contacted with a test compound and binding, stimulation, or inhibition of activity of either CDIFF or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with CDIFF, either in solution or affixed to a solid support, and detecting the binding of CDIFF to the compound.
Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
CDIFF of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of CDIFF. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for CDIFF activity, wherein CDIFF is combined with at least one test compound, and the activity of CDIFF in the presence of a test compound is compared with the activity of CDIFF in the absence of the test compound. A change in the activity of CDIFF in the presence of the test compound is indicative of a compound that modulates the activity of CDIFF. Alternatively, a test compound is combined with an in vitro or cell-free system comprising CDIFF under conditions suitable for CDIFF
activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of CDIFF may do so indirectly and need not come in direct contact with the test compound.
At least one and up to a plurality of test compounds may be screened.

In another embodiment, polynucleotides encoding CDIFF or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding CDIFF may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
( 1998) Science 282:1145-1147).
Polynucleotides encoding CDIFF can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding CDIFF is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress CDIFF, e.g., by secreting CDIFF
in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of CDIFF and proteins involved in cell differentiation. In addition, the expression of CDIFF is closely associated with cell proliferative disorders (including cancer) as well as reproductive and nervous tissue disorders. Therefore, CDIFF appears to play a role in cell proliferative, developmental, and neurological disorders. In the treatment of disorders associated with increased CDIFF expression or activity, it is desirable to decrease the expression or activity of CDIFF. In the treatment of disorders associated with decreased CDIFF
expression or activity, it is desirable to increase the expression or activity of CDIFF.
Therefore, in one embodiment, CDIFF or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CDIFF. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, inflammatory disorders, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), 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, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; and a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis;
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis; mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD);
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia.
In another embodiment, a vector capable of expressing CDIFF or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CDIFF including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified CDIFF in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CDIFF
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CDIFF
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CDIFF including, but not limited to, those listed above.
In a further embodiment, an antagonist of CDIFF may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CDIFF.
Examples of such disorders include, but are not limited to, those cell proliferative, developmental, and neurological disorders described above. In one aspect, an antibody which specifically binds CDIFF may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express CDIFF.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding CDIFF may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CDIFF including, but not limited to, those described 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 CDIFF may be produced using methods which are generally known in the art. In particular, purified CDIFF may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind CDIFF.
Antibodies to CDIFF 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 generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with CDIFF or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological 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 Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to CDIFF have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist 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. Short stretches of CDIFF 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 CDIFF 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. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. 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 specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (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 CDIFF-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for CDIFF 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 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 CDIFF and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CDIFF epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for CDIFF.
Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of CDIFF-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple CDIFF epitopes, represents the average affinity, or avidity, of the antibodies for CDIFF. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular CDIFF epitope, represents a true measure of affinity. High-affinity antibody preparations with K~ ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in which the CDIFF-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of CDIFF, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of CDIFF-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, sera, and Coligan et al., s_ upra.) In another embodiment of the invention, the polynucleotides encoding CDIFF, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding CDIFF. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CDIFF. (See, e.g., Agrawal, S., ed. ( 1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial vital envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res.
25( 14):2730-2736.) In another embodiment of the invention, polynucleotides encoding CDIFF may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia ( 1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in CDIFF expression or regulation causes disease, the expression of CDIFF from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in CDIFF are treated by constructing mammalian expression vectors encoding CDIFF
and introducing these vectors by mechanical means into CDIFF-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson ( 1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of CDIFF include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). CDIFF may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding CDIFF from a normal individual.
Cornmercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to CDIFF expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding CDIFF under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. ( 1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding CDIFF to cells which have one or more genetic abnormalities with respect to the expression of CDIFF. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. ( 1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding CDIFF to target cells which have one or more genetic abnormalities with respect to the expression of CDIFF. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing CDIFF to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. ( 1999) Exp. Eye Res.169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol.
73:519-532 and Xu, H. et al.
(1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding CDIFF to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for CDIFF into the alphavirus genome in place of the capsid-coding region results in the production of a large number of CDIFF-coding RNAs and the synthesis of high levels of CDIFF in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of CDIFF into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. 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. Can, Molecular and Immunologic Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding CDIFF.
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 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.
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 in vivo transcription of DNA
sequences encoding CDIFF. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA 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.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding CDIFF.
Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased CDIFF expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding CDIFF may be therapeutically useful, and in the treament of disorders associated with decreased CDIFF expression or activity, a compound which specifically promotes expression of the polynucleotide encoding CDIFF may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding CDIFF is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding CDIFF are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding CDIFF. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Amdt, G.M. et al.
(2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691 ).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo 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) Nat.
Biotechnol. 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of CDIFF, antibodies to CDIFF, and mimetics, agonists, antagonists, or inhibitors of CDIFF.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
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.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising CDIFF or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, CDIFF or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, 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 CDIFF or fragments thereof, antibodies of CDIFF, and agonists, antagonists or inhibitors of CDIFF, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDSO (the dose therapeutically effective in 50% of the population) or LDSo (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDSO
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 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. I ~g to 100,000 fig, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind CDIFF may be used for the diagnosis of disorders characterized by expression of CDIFF, or in assays to monitor patients being treated with CDIFF or agonists, antagonists, or inhibitors of CDIFF.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CDIFF include methods which utilize the antibody and a label to detect CDIFF 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 CDIF'F, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of CDIFF expression. Normal or standard values for CDIFF expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to CDIFF under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of CDIF'F 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 CDIFF 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 quantify gene expression in biopsied tissues in which expression of CDIFF
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of CDIFF, and to monitor regulation of CDIFF levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CDIFF or closely related molecules may be used to identify nucleic acid sequences which encode CDIFF. 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 will determine whether the probe identifies only naturally occurring sequences encoding CDIFF, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the CDIFF encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:29-56 or from genomic sequences including promoters, enhancers, and introns of the CDIFF
gene.
Means for producing specific hybridization probes for DNAs encoding CDIFF
include the cloning of polynucleotide sequences encoding CDIFF or CDIFF derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 3'-P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding CDIFF may be used for the diagnosis of disorders associated with expression of CDIFF. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, inflammatory disorders, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), 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 Syndenharri s chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; and a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis;
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis; mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD);
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia. The polynucleotide sequences encoding CDIFF may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR
technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered CDIFF expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding CDIFF may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding CDIFF 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 signal is quantified 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 CDIFF 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 monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of CDIFF, 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 CDIFF, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
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 an abnormal amount of transcript (either under- or overexpressed) 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.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding CDIFF
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 CDIFF, or a fragment of a polynucleotide complementary to the polynucleotide encoding CDIFF, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding CDIFF may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited.or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding CDIFF are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of CDIFF include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. ( I 993) J. Immunol. Methods 159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described in Seilhamer, J.J. et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, antibodies specific for CDIFF, or CDIFF or fragments thereof may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent Number 5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurnng environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson (2000) Toxicol. Lett. 112-I 13:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families.
Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for CDIFF
to quantify the levels of CDIFF expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem.
270:103-11 l; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer ( 1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A
difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
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.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding CDIFF
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134;
and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP). (See, e.g., Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding CDIFF on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11 q22-23, 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 instant 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, CDIFF, 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 CDIFF and the agent being tested may be measured.
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. The test compounds are reacted with CDIFF, or fragments thereof, and washed. Bound CDIFF is then detected by methods well known in the art.
Purified CDIFF 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 neutralizing antibodies capable of binding CDIFF specifically compete with a test compound for binding CDIFF.
In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with CDIFF.
In additional embodiments, the nucleotide sequences which encode CDIFF may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above and below, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
(Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, su ra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), peDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI

or 377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VI.
The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software (DNASTAR). Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector, linker, and polyA
sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM is a probabilistic approach which analyzes consensus primary structures of gene families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:29-56. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression 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; Ausubel, 1995, supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). 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 similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity 5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding CDIFF occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
V. Chromosomal Mapping of CDIFF Encoding Polynucleotides The sequences which were used to assemble SEQ ID N0:29-56 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:29-56 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, or human chromosomes.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:32 maps to chromosome 1 within the interval from 152.2 to 157.4 centiMorgans, to chromosome 3 within the interval from 157.4 to 158.0 centiMorgans, and to the X chromosome within the interval from 104.9 to 150.3 centiMorgans. The interval on chromosome 1 from 152.2 to 157.4 centiMorgans also contains genes associated with leukemia, hypothyroidism, and adrenal hyperplasia. The interval on the X chromosome from 104.9 to 150.3 centiMorgans also contains genes associated with X-linked lissencephaly, leiomyomatosis with Alport syndrome, lymphoproliferative syndrome, Breton agammaglobulinemia, and diffuse angiokeratoma. SEQ ID N0:37 maps to chromosome 11 within the interval from 19.6 to 23.2 centiMorgans. SEQ ID N0:39 maps to chromosome 16 within the interval from 109.1 to 130.8 centiMorgans, and to chromosome 22 within the interval from 45.5 to 58.1 centiMorgans. The interval on chromosome 16 from 109.1 to 130.8 centiMorgans also contains a gene associated with gastric cancer susceptibility. SEQ ID N0:45 maps to chromosome 7 within the interval from 105.2 to 109.0 centiMorgans, to chromosome 17 within the interval from 65.0 to 90.2 centiMorgans, and to chromosome 20 within the interval from 50.2 to 54.9 centiMorgans. The interval on chromosome 7 from 105.2 to 109.0 centiMorgans also contains a gene associated with osteogenesis imperfecta. The interval on chromosome 17 from 65.0 to 90.2 centiMorgans also contains genes associated with breast cancer, hepatic leukemia, myeloperoxidase deficiency, muscular dystrophy, periodic paralysis, and placental growth. SEQ ID N0:54 maps to chromosome 12 within the interval from 21.3 to 36.1 centiMorgans. SEQ ID NO:55 maps to chromosome 1 within the interval from 22.9 to 39.9 centiMorgans and to chromosome 3 within the interval from 30.9 to 43.0 centiMorgans.
More than one map location is reported for SEQ ID N0:32, SEQ ID N0:39, SEQ ID
N0:45, and SEQ ID NO:55, indicating that sequences having different map locations were assembled into a single cluster. This situation occurs, for example, when sequences having strong similarity, but not complete identity, are assembled into a single cluster.
VI. Extension of CDIFF Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:29-56 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5'extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR

was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)ZS04, and (3-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p1 of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ~cl to 10 ~1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise (Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide ( 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM

BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:29-56 are used to obtain 5' regulatory sequences using the procedure above, along with oligonucleotides designed for such extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:29-56 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 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of [y-3zP~ adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
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 following endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% 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 are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
VIII. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing, See, e.g., Baldeschweiler, supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. ( 1995) Science 270:467-470; Shalom D. et al. ( 1996) Genome Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/pl oligo-(dT) primer (2lmer), 1X
first strand buffer, 0.03 units/pl RNase inhibitor, 500 NM dATP, 500 liM dGTP, 500 NM dTTP, 40 _ liM dCTP, 40 I.dVI dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of O.SM sodium hydroxide and incubated for 20 minutes at 85 °C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 p1 SX SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 pg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a I10°C oven.
Array elements are applied to the coated glass substrate using a procedure described in US
Patent No. 5,807,522, incorporated herein by reference. 1 p1 of the array element DNA, at an average concentration of 100 ng/pl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 °C followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 pg each of Cy3 and Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65 °C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 p1 of SX SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60 °C. The arrays are washed for 10 min at 45 °C in a first wash buffer ( 1X SSC, 0.1 % SDS), three times for 10 minutes each at 45 °C
in a second wash buffer (0.1 X
SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
IX. Complementary Polynucleotides Sequences complementary to the CDIFF-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring CDIFF. 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 oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CDIFF. 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 CDIF'F-encoding transcript.
X. Expression of CDIFF
Expression and purification of CDIFF is achieved using bacterial or virus-based expression systems. For expression of CDIFF in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21 (DE3).
Antibiotic resistant bacteria express CDIFF upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of CDIFF in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autogranhica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CDIFF by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Svodoptera fru~iperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems, CDIFF is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from CDIFF at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified CDIFF obtained by these methods can be used directly in the assays shown in Examples XI and XV.
XI. Demonstration of CDIFF Activity CDIFF activity is demonstrated by measuring the induction of terminal differentiation or cell cycle progression when CDIFF is expressed at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include pCMV SPORTTM
(Life Technologies, Gaithersburg, MD) and pCRTM 3.1 (Invitrogen, Carlsbad, CA), both of which contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 ~g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo Alto, CA), CD64, or a CD64-GFP fusion protein. Flow cytometry detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell cycle progression or terminal differentiation. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; up or down-regulation of DNA
synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V
protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. ( 1994) Flow C try, Oxford, New York, NY.
XII. Functional Assays CDIFF function is assessed by expressing the sequences encoding CDIFF at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ~g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of CDIFF on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding CDIFF and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art.
Expression of mRNA encoding CDIFF and other genes of interest can be analyzed by northern analysis or microarray techniques.
XIII. Production of CDIFF Specific Antibodies CDIFF substantially purified using polyacrylamide gel electrophoresis (PAGE;
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.
Alternatively, the CDIFF amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-CDIFF activity by, for example, binding the peptide or CDIFF to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIV. Purification of Naturally Occurring CDIFF Using Specific Antibodies Naturally occurring or recombinant CDIFF is substantially purified by immunoaffinity chromatography using antibodies specific for CDIFF. An immunoaffinity column is constructed by covalently coupling anti-CDIFF antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing CDIFF are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of CDIFF (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/CDIFF 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 CDIFF is collected.
XV. Identification of Molecules Which Interact with CDIFF
CD1FF, or biologically active fragments thereof, are labeled with 'z5I Bolton-Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled CDIFF, washed, and any wells with labeled CDIFF complex are assayed. Data obtained using different concentrations of CDIFF are used to calculate values for the number, affinity, and association of CDIFF with the candidate molecules.
CDIFF may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101 ).
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 certain 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.

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g7 SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
TANG, Y. Tom HILLMAN, Jennifer L.
YUE, Henry REDDY, Roopa LAL, Preeti SHAH, Purvi AZIMZAI, Yalda BAUGHN, Mariah R.
LU, Dyung Aina M.
BANDMAN, Olga SHIH, Leo L.
PATTERSON, Chandra <120> PROTEINS ASSOCIATED WITH CELL DIFFERENTIATION
<130> PF-0741 PCT
<140> To Be Assigned <141> Herewith <150> 60/154,140; 60/169,155 <151> 1999-09-15; 1999-12-06 <160> 56 <170> PERL Program <210> 1 <211> 367 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1681724CD1 <400> 1 Met Ala Thr Pro Asn Asn Leu Thr Pro Thr Asn Cys Ser Trp Trp Pro Ile Ser Ala Leu Glu Ser Asp Ala Ala Lys Pro Ala Glu Ala Pro Asp Ala Pro Glu Ala Ala Ser Pro Ala His Trp Pro Arg Glu Ser Leu Val Leu Tyr His Trp Thr Gln Ser Phe Ser Ser Gln Lys Val Arg Leu Val Ile Ala Glu Lys Gly Leu Val Cys Glu Glu Arg Asp Val Ser Leu Pro Gln Ser Glu His Lys Glu Pro Trp Phe Met Arg Leu Asn Leu Gly Glu Glu Val Pro Val Ile Ile His Arg Asp Asn Ile Ile Ser Asp Tyr Asp Gln Ile Ile Asp Tyr Val Glu Arg Thr Phe Thr Gly Glu His Val Val Ala Leu Met Pro Glu Val Gly Ser Leu Gln His Ala Arg Val Leu Gln Tyr Arg Glu Leu Leu Asp Ala Leu Pro Met Asp Ala Tyr Thr His Gly Cys Ile Leu His Pro Glu Leu Thr Thr Asp Ser Met Ile Pro Lys Tyr Ala Thr Ala Glu Ile Arg Arg His Leu Ala Asn Ala Thr Thr Asp Leu Met Lys Leu Asp His Glu Glu Glu Pro Gln Leu Ser Glu Pro Tyr Leu Ser Lys Gln Lys Lys Leu Met Ala Lys Ile Leu Glu His Asp Asp Val Ser Tyr Leu Lys Lys Ile Leu Gly Glu Leu Ala Met Val Leu Asp Gln Ile Glu Ala Glu Leu Glu Lys Arg Lys Leu Glu Asn Glu Gly Gln Lys Cys Glu Leu Trp Leu Cys Gly Cys Ala Phe Thr Leu Ala Asp Val Leu Leu Gly Ala Thr Leu His Arg Leu Lys Phe Leu Gly Leu Ser Lys Lys Tyr Trp Glu Asp Gly Ser Arg Pro Asn Leu Gln Ser Phe Phe Glu Arg Val Gln Arg Arg Phe Ala Phe Arg Lys Val Leu Gly Asp Ile His Thr Thr Leu Leu Ser Ala Val Ile Pro Asn Ala Phe Arg Leu Val Lys Arg Lys Pro Pro Ser Phe Phe Gly Ala Ser Phe Leu Met Gly Ser Leu Gly Gly Met Gly Tyr Phe Ala Tyr Trp Tyr Leu Lys Lys Lys Tyr Ile <210> 2 <211> 102 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1718047CD1 <400> 2 Met Ala Leu Leu Lys Ala Asn Lys Asp Leu Ile Ser Ala Gly Leu Lys Glu Phe Ser Val Leu Leu Asn Gln Gln Val Phe Asn Asp Pro Leu Val Ser Glu Glu Asp Met Val Thr Val Val Glu Asp Trp Met Asn Phe Tyr Ile Asn Tyr Tyr Arg Gln Gln Val Thr Gly Glu Pro Gln Glu Arg Asp Lys Ala Leu Gln Glu Leu Arg Gln Glu Leu Asn Thr Leu Ala Asn Pro Phe Leu Ala Lys Tyr Arg Asp Phe Leu Lys Ser His Glu Leu Pro Ser His Pro Pro Pro Ser Ser <210> 3 <211> 205 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1980323CD1 <400> 3 Met Ala Glu Pro Leu Gln Pro Asp Pro Gly Ala Ala Glu Asp Ala Ala Ala Gln Ala Val Glu Thr Pro Gly Trp Lys Ala Pro Glu Asp Ala Gly Pro Gln Pro Gly Ser Tyr Glu Ile Arg His Tyr Gly Pro Ala Lys Trp Val Ser Thr Ser Val Glu Ser Met Asp Trp Asp Ser Ala Ile Gln Thr Gly Phe Thr Lys Leu Asn Ser Tyr Ile Gln Gly Lys Asn Glu Lys Glu Met Lys Ile Lys Met Thr Ala Pro Val Thr Ser Tyr Val Glu Pro Gly Ser Gly Pro Phe Ser Glu Ser Thr Ile Thr Ile Ser Leu Tyr Ile Pro Ser Glu Gln Gln Phe Asp Pro Pro Arg Pro Leu Glu Ser Asp Val Phe Ile Glu Asp Arg Ala Glu Met Thr Val Phe Val Arg Ser Phe Asp Gly Phe Ser Ser Ala Gln Lys Asn Gln Glu Gln Leu Leu Thr Leu Ala Ser Ile Leu Arg Glu Asp Gly Lys Val Phe Asp Glu Lys Val Tyr Tyr Thr Ala Gly Tyr Asn Ser Pro Val Lys Leu Leu Asn Arg Asn Asn Glu Val Trp Leu Ile Gln Lys Asn Glu Pro Thr Lys Glu Asn Glu <210> 4 <211> 120 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1990956CD1 <400> 4 Met Glu Ser Lys Glu Glu Leu Ala Ala Asn Asn Leu Asn Gly Glu Asn Ala Gln Gln Glu Asn Glu Gly Gly Glu Gln Ala Pro Thr Gln Asn Glu Glu Glu Ser Arg His Leu Gly Gly Gly Glu Gly Gln Lys Pro Gly Gly Asn Ile Arg Arg Gly Arg Val Arg Arg Leu Val Pro Asn Phe Arg Trp Ala Ile Pro Asn Arg His.Ile Glu His Asn Glu Ala Arg Asp Asp Val Glu Arg Phe Val Gly Gln Met Met Glu Ile Lys Arg Lys Thr Arg Glu Gln Gln Met Arg His Tyr Met Arg Phe Gln Thr Pro Glu Pro Asp Asn His Tyr Asp Phe Cys Leu Ile Pro <210> 5 <211> 108 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2009069CD1 <400> 5 Met Ala Lys Val Thr Ser Glu Pro Gln Lys Pro Asn Glu Asp Val Asp Glu His Thr Pro Ser Thr Ser Ser Thr Lys Gly Arg Lys Lys Gly Lys Thr Pro Arg Gln Arg Arg Ser Arg Ser Gly Val Lys Gly Leu Lys Thr Thr Arg Lys Ala Lys Arg Pro Leu Arg Gly Ser Ser Ser Gln Lys Ala Gly Glu Thr Asn Thr Pro Ala Gly Lys Pro Lys Lys Ala Arg Gly Pro Ile Leu Arg Gly Arg Tyr His Arg Leu Lys Glu Lys Met Lys Lys Glu Glu Ala Asp Lys Glu Gln Ser Glu Thr Ser Val Leu <210> 6 <211> 308 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2009435CD1 <400> 6 Met Ala Lys Met Glu Leu Ser Lys Ala Phe Ser Gly Gln Arg Thr Leu Leu Ser Ala Ile Leu Ser Met Leu Ser Leu Ser Phe Ser Thr Thr Ser Leu Leu Ser Asn Tyr Trp Phe Val Gly Thr Gln Lys Val Pro Lys Pro Leu Cys Glu Lys Gly Leu Ala Ala Lys Cys Phe Asp Met Pro Val Ser Leu Asp Gly Asp Thr Asn Thr Ser Thr Gln Glu Val Val Gln Tyr Asn Trp Glu Thr Gly Asp Asp Arg Phe Ser Phe Arg Ser Phe Arg Ser Gly Met Trp Leu Ser Cys Glu Glu Thr Val Glu Glu Pro Ala Leu Leu His Pro Gln Ser Trp Lys Gln Phe Arg Ala Leu Arg Ser Ser Gly Thr Ala Ala Ala Lys Gly Glu Arg Cys Arg Ser Phe Ile Glu Leu Thr Pro Pro Ala Lys Arg Gly Glu Lys Gly Leu Leu Glu Phe Ala Thr Leu Gln Gly Pro Cys His Pro Thr Leu Arg Phe Gly Gly Lys Arg Leu Met Glu Lys Ala Ser Leu Pro Ser Pro Pro Leu Gly Leu Cys Gly Lys Asn Pro Met Val Ile Pro Gly Asn Ala Asp His Leu His Arg Thr Ser Ile His Gln Leu Pro Pro Ala Thr Asn Arg Leu Ala Thr His Trp Glu Pro Cys Leu Trp Ala Gln Thr Glu Arg Leu Cys Cys Cys Phe Leu Cys Pro Val Arg Ser Pro Gly Asp Gly Gly Pro His Asp Val Phe Thr Ser Leu Pro Ser Asp Cys Gln Leu Gly Ser Arg Arg Leu Glu Thr Thr Cys Leu Glu Leu Trp Leu Gly Leu Leu His Gly Leu Ala Leu Leu His Leu Leu His Gly Val Gly Cys His His Leu Gln His Val His Gln Asp Gly Ala Gly Val Gln Val Gln Ala <210> 7 <211> 116 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2027937CD1 <400> 7 Met Ser Phe Ser Glu Gln Gln Cys Lys Gln Pro Cys Val Pro Pro Pro Cys Leu Pro Lys Thr Gln Glu Gln Cys Gln Ala Lys Ala Glu Glu Val Cys Leu Pro Thr Cys Gln His Pro Cys Gln Asp Lys Cys Leu Val Gln Ala Gln Glu Val Cys Leu Ser Gln Cys Gln Glu Ser Ser Gln Glu Lys Cys Pro Gln Gln Gly Gln Glu Pro Tyr Leu Pro Pro Cys Gln Asp Gln Cys Pro Pro Gln Cys Ala Glu Pro Cys Gln Glu Leu Phe Gln Thr Lys Cys Val Glu Val Cys Pro Gln Lys Val Gln Glu Lys Cys Ser Ser Pro Gly Lys Gly Lys <210> 8 <211> 1253 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2722347CD1 <400> 8 Met Thr Thr His Val Thr Leu Glu Asp Ala Leu Ser Asn Val Asp Leu Leu Glu Glu Leu Pro Leu Pro Asp Gln Gln Pro Cys Ile Glu Pro Pro Pro Ser Ser Ile Met Tyr Gln Ala Asn Phe Asp Thr Asn Phe Glu Asp Arg Asn Ala Phe Val Thr Gly Ile Ala Arg Tyr Ile Glu Gln Ala Thr Val His Ser Ser Met Asn Glu Met Leu Glu Glu Gly His Glu Tyr Ala Val Met Leu Tyr Thr Trp Arg Ser Cys Ser Arg Ala Ile Pro Gln Val Lys Cys Asn Glu Gln Pro Asn Arg Val Glu Ile Tyr Glu Lys Thr Val Glu Val Leu Glu Pro Glu Val Thr Lys Leu Met Lys Phe Met Tyr Phe Gln Arg Lys Ala Ile Glu Arg Phe Cys Ser Glu Val Lys Arg Leu Cys His Ala Glu Arg Arg Lys Asp Phe Val Ser Glu Ala Tyr Leu Leu Thr Leu Gly Lys Phe Ile Asn Met Phe Ala Val Leu Asp Glu Leu Lys Asn Met Lys Cys Ser Val Lys Asn Asp His Ser Ala Tyr Lys Arg Ala Ala Gln Phe Leu Arg Lys Met Ala Asp Pro Gln Ser Ile Gln Glu Ser Gln Asn Leu Ser Met Phe Leu Ala Asn His Asn Arg Ile Thr Gln Cys Leu His Gln Gln Leu Glu Val Ile Pro Gly Tyr Glu Glu Leu Leu Ala Asp Ile Val Asn Ile Cys Val Asp Tyr Tyr Glu Asn Lys Met Tyr Leu Thr Pro Ser Glu Lys His Met Leu Leu Lys Val Met Gly Phe Gly Leu Tyr Leu Met Asp Gly Asn Val Ser Asn Ile Tyr Lys Leu Asp Ala Lys Lys Arg Ile Asn Leu Ser Lys Ile Asp Lys Phe Phe Lys Gln Leu Gln Val Val Pro Leu Phe Gly Asp Met Gln Ile Glu Leu Ala Arg Tyr Ile Lys Thr Ser Ala His Tyr Glu Glu Asn Lys Ser Lys Trp Thr Cys Thr Gln Ser Ser Ile Ser Pro Gln Tyr Asn Ile Cys Glu Gln Met Val Gln Ile Arg Asp Asp His Ile Arg Phe Ile Ser Glu Leu Ala Arg Tyr Ser Asn Ser Glu Val Val Thr Gly Ser Gly Leu Asp Ser Gln Lys Ser Asp Glu Glu Tyr Arg Glu Leu Phe Asp Leu Ala Leu Arg Gly Leu Gln Leu Leu Ser Lys Trp Ser Ala His Val Met Glu Val Tyr Ser Trp Lys Leu Val His Pro Thr Asp Lys Phe Cys Asn Lys Asp Cys Pro Gly Thr Ala Glu Glu Tyr Glu Arg Ala Thr Arg Tyr Asn Tyr Thr Ser Glu Glu Lys Phe Ala Phe Val Glu Val Ile Ala Met Ile Lys Gly Leu Gln Val Leu Met Gly Arg Met Glu Ser Val Phe Asn Gln Ala Ile Arg Asn Thr Ile Tyr Ala Ala Leu Gln Asp Phe Ala Gln Val Thr Leu Arg Glu Pro Leu Arg Gln Ala Val Arg Lys Lys Lys Asn Val Leu Ile Ser Val Leu Gln Ala Ile Arg Lys Thr Ile Cys Asp Trp Glu Gly Gly Arg Glu Pro Pro Asn Asp Pro Cys Leu Arg Gly Glu Lys Asp Pro Lys Gly Gly Phe Asp Ile Lys Val Pro Arg Arg Ala Val Gly Pro Ser Ser Thr Gln Leu Tyr Met Val Arg Thr Met Leu Glu Ser Leu Ile Ala Asp Lys Ser Gly Ser Lys Lys Thr Leu Arg Ser Ser Leu Asp Gly Pro Ile Val Leu Ala Ile Glu Asp Phe His Lys Gln Ser Phe Phe Phe Thr His Leu Leu Asn Ile Ser Glu Ala Leu Gln Gln Cys Cys Asp Leu Ser Gln Leu Trp Phe Arg Glu Phe Phe Leu Glu Leu Thr Met Gly Arg Arg Ile Gln Phe Pro Ile Glu Met Ser Met Pro Trp Ile Leu Thr Asp His Ile Leu Glu Thr Lys Glu Pro Ser Met Met Glu Tyr Val Leu Tyr Pro Leu Asp Leu Tyr Asn Asp Ser Ala Tyr Tyr Ala Leu Thr Lys Phe Lys Lys Gln Phe Leu Tyr Asp Glu Ile Glu Ala Glu Val Asn Leu Cys Phe Asp Gln Phe Val Tyr Lys Leu Ala Asp Gln Ile Phe Ala Tyr Tyr Lys Ala Met Ala Gly Ser Val Leu Leu Asp Lys Arg Phe Arg Ala Glu Cys Lys Asn Tyr Gly Val Ile Ile Pro Tyr Pro Pro Ser Asn Arg Tyr Glu Thr Leu Leu Lys Gln Arg His Val Gln Leu Leu Gly Arg Ser Ile Asp Leu Asn Arg Leu Ile Thr Gln Arg Ile Ser Ala Ala Met Tyr Lys Ser Leu Asp Gln Ala Ile Ser Arg Phe Glu Ser Glu Asp Leu Thr Ser Ile Val Glu Leu Glu Trp Leu Leu Glu Ile Asn Arg Leu Thr His Arg Leu Leu Cys Lys His Met Thr Leu Asp Ser Phe Asp Ala Met Phe Arg Glu Ala Asn His Asn Val Ser Ala Pro Tyr Gly Arg Ile Thr Leu His Val Phe Trp Glu Leu Asn Phe Asp Phe Leu Pro Asn Tyr Cys Tyr Asn Gly Ser Thr Asn Arg Phe Val Arg Thr Ala Ile Pro Phe Thr Gln Glu Pro Gln Arg Asp Lys Pro Ala Asn Val Gln Pro Tyr Tyr Leu Tyr Gly Ser Lys Pro Leu Asn Ile Ala Tyr Ser His Ile Tyr Ser Ser Tyr Arg Asn Phe Val Gly Pro Pro His Phe Lys Thr Ile Cys Arg Leu Leu Gly Tyr Gln Gly Ile Ala Val Val Met Glu Glu Leu Leu Lys Ile Val Lys Ser Leu Leu Gln Gly Thr Ile Leu Gln Tyr Val Lys Thr Leu Ile Glu Val Met Pro Lys Ile Cys Arg Leu Pro Arg His Glu Tyr Gly Ser Pro Gly Ile Leu Glu Phe Phe His His Gln Leu Lys Asp Ile Ile Glu Tyr Ala Glu Leu Lys Thr Asp Val Phe Gln Ser Leu Arg Glu Val Gly Asn Ala Ile Leu Phe Cys Leu Leu Ile Glu Gln Ala Leu Ser Gln Glu Glu Val Cys Asp Leu Leu His Ala Ala Pro Phe Gln Asn Ile Leu Pro Arg Val Tyr Ile Lys Glu Gly Glu Arg Leu Glu Val Arg Met Lys Arg Leu Glu Ala Lys Tyr Ala Pro Leu His Leu Val Pro Leu Ile Glu Arg Leu Gly Thr Pro Gln Gln Ile Ala Ile Ala Arg Glu Gly Asp Leu Leu Thr Lys Glu Arg Leu Cys Cys Gly Leu Ser Met Phe Glu Val Ile Leu Thr Arg Ile Arg Ser Tyr Leu Gln Asp Pro Ile Trp Arg Gly Pro Pro Pro Thr Asn Gly Val Met His Val Asp Glu Cys Val Glu Phe His Arg Leu Trp Ser Ala Met Gln Phe Val Tyr Cys Ile Pro Val Gly Thr Asn Glu Phe Thr Ala Glu Gln Cys Phe Gly Asp Gly Leu Asn Trp Ala Gly Cys Ser Ile Ile Val Leu Leu Gly Gln Gln Arg Arg Phe Asp Leu Phe Asp Phe Cys Tyr His Leu Leu Lys Val Gln Arg Gln Asp Gly Lys Asp Glu Ile Ile Lys Asn Val Pro Leu Lys Lys Met Ala Asp Arg Ile Arg Lys Tyr Gln Ile Leu Asn Asn Glu Val Phe Ala Ile Leu Asn Lys Tyr Met Lys Ser Val Glu Thr Asp Ser Ser Thr Val Glu His Val Arg Cys Phe Gln Pro Pro Ile His Gln Ser Leu Ala Thr Thr Cys <210> 9 <211> 98 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2759876CD1 <400> 9 Met Ser Val Asp Met Asn Ser Gln Gly Ser Asp Ser Asn Glu Glu Asp Tyr Asp Pro Asn Cys Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Pro Gly Asp Ile Glu Asp Tyr Tyr Val Gly Val Ala Ser Asp Val Glu Gln Gln Gly Ala Asp Ala Phe Asp Pro Glu Glu Tyr Gln Phe Thr Cys Leu Thr Tyr Lys Glu Ser Glu Gly Ala Leu Asn Glu His Met Thr Ser Leu Ala Ser Val Leu Lys Val Ser Ser Val Val Asn Ser Ser Val Ile Pro Pro Ser <210> 10 <211> 524 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2763735CD1 <400> 10 Met Glu Glu Glu Gln Asp Leu Pro Glu Gln Pro Val Lys Lys Ala Lys Met Gln Glu Ser Gly Glu Gln Thr Ile Ser Gln Val Ser Asn Pro Asp Val Ser Asp Gln Lys Pro Glu Thr Ser Ser Leu Ala Ser Asn Leu Pro Met Ser Glu Glu Ile Met Thr Cys Thr Asp Tyr Ile Pro Arg Ser Ser Asn Asp Tyr Thr Ser Gln Met Tyr Ser Ala Lys Pro Tyr Ala His Ile Leu Ser Val Pro Val Ser Glu Thr Ala Tyr Pro Gly Gln Thr Gln Tyr Gln Thr Leu Gln Gln Thr Gln Pro Tyr Ala Val Tyr Pro Gln Ala Thr Gln Thr Tyr Gly Leu Pro Pro Phe Ala Ser Ser Thr Asn Ala Ser Leu Ile Ser Thr Ser Ser Thr Ile Ala Asn Ile Pro Ala Ala Ala Val Ala Ser Ile Ser Asn Gln Asp Tyr Pro Thr Tyr Thr Ile Leu Gly Gln Asn Gln Tyr Gln Ala Cys Tyr Pro Ser Ser Ser Phe Gly Val Thr Gly Gln Thr Asn Ser Asp Ala Glu Ser Thr Thr Leu Ala Ala Thr Thr Tyr Gln Ser Glu Lys Pro Ser Val Met Ala Pro Ala Pro Ala Ala Gln Arg Leu Ser Ser Gly Asp Pro Ser Thr Ser Pro Ser Leu Ser Gln Thr Thr Pro Ser Lys Asp Thr Asp Asp Gln Ser Arg Lys Asn Met Thr Ser Lys Asn Arg Gly Lys Arg Lys Ala Asp Ala Thr Ser Ser Gln Asp Ser Glu Leu Glu Arg Val Phe Leu Trp Asp Leu Asp Glu Thr Ile Ile Ile Phe His Ser Leu Leu Thr Gly Ser Tyr Ala Gln Lys Tyr Gly Lys Asp Pro Thr Val Val Ile Gly Ser Gly Leu Thr Met Glu Glu Met Ile Phe Glu Val Ala Asp Thr His Leu Phe Phe Asn Asp Leu Glu Glu Cys Asp Gln Val His Val Glu Asp Val Ala Ser Asp Asp Asn Gly Gln Asp Leu Ser Asn Tyr Ser Phe Ser Thr Asp Gly Phe Ser Gly Ser Gly Gly Ser Gly Ser His Gly Ser Ser Val Gly Val Gln Gly Gly Val Asp Trp Met Arg Lys Leu Ala Phe Arg Tyr Arg Lys Val Arg Glu Ile Tyr Asp Lys His Lys Ser Asn Val Gly Gly Leu Leu Ser Pro Gln Arg Lys Glu Ala Leu Gln Arg Leu Arg Ala Glu Ile Glu Val Leu Thr Asp Ser Trp Leu Gly Thr Ala Leu Lys Ser Leu Leu Leu Ile Gln Ser Arg Lys Asn Cys Val Asn Val Leu Ile Thr Thr Thr Gln Leu Val Pro Ala Leu Ala Lys Val Leu Leu Tyr Gly Leu Gly Glu Ile Phe Pro Ile Glu Asn Ile Tyr Ser Ala Thr Lys Ile Gly Lys Glu Ser Cys Phe Glu Arg Ile Val Ser Arg Phe Gly Lys Lys Val Thr Tyr Val Val Ile Gly Asp Gly Arg Asp Ala Ala Lys Gln His Asn Met Pro Phe Trp Arg Ile Thr Asn His Gly Asp Leu Val Ser Leu His Gln Ala Leu Glu Leu Asp Phe Leu <210> 11 <211> 628 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2848676CD1 <400> 11 Met Ala Ala Ala Gly Ala Gly Pro Gly Gln Glu Ala Gly Ala Gly Pro Gly Pro Gly Ala Val Ala Asn Ala Thr Gly Ala Glu Glu Gly Glu Met Lys Pro Val Ala Ala Gly Ala Ala Ala Pro Pro Gly Glu Gly Ile Ser Ala Ala Pro Thr Val Glu Pro Ser Ser Gly Glu Ala Glu Gly Gly Glu Ala Asn Leu Val Asp Val Ser Gly Gly Leu Glu Thr Glu Ser Ser Asn Gly Lys Asp Thr Leu Glu Gly Ala Gly Asp Thr Ser Glu Val Met Asp Thr Gln Ala Gly Ser Val Asp Glu Glu Asn Gly Arg Gln Leu Gly Glu Val Glu Leu Gln Cys Gly Ile Cys Thr Lys Trp Phe Thr Ala Asp Thr Phe Gly Ile Asp Thr Ser Ser Cys Leu Pro Phe Met Thr Asn Tyr Ser Phe His Cys Asn Val Cys His His Ser Gly Asn Thr Tyr Phe Leu Arg Lys Gln Ala Asn Leu Lys Glu Met Cys Leu Ser Ala Leu Ala Asn Leu Thr Trp Gln Ser Arg Thr Gln Asp Glu His Pro Lys Thr Met Phe Ser Lys Asp Lys Asp Ile Ile Pro Phe Ile Asp Lys Tyr Trp Glu Cys Met Thr Thr Arg Gln Arg Pro Gly Lys Met Thr Trp Pro Asn Asn Ile Val Lys Thr Met Ser Lys Glu Arg Asp Val Phe Leu Val Lys Glu His Pro Asp Pro Gly Ser Lys Asp Pro Glu Glu Asp Tyr Pro Lys Phe Gly Leu Leu Asp Gln Asp Leu Ser Asn Ile Gly Pro Ala Tyr Asp Asn Gln Lys Gln Ser Ser Ala Val Ser Thr Ser Gly Asn Leu Asn Gly Gly Ile Ala Ala Gly Ser Ser Gly Lys Gly Arg Gly Ala Lys Arg Lys Gln Gln Asp Gly Gly Thr Thr Gly Thr Thr Lys Lys Ala Arg Ser Asp Pro Leu Phe Ser Ala Gln Arg Leu Pro Pro His Gly Tyr Pro Leu Glu His Pro Phe Asn Lys Asp Gly Tyr Arg Tyr Ile Leu Ala Glu Pro Asp Pro His Ala Pro Asp Pro Glu Lys Leu Glu Leu Asp Cys Trp Ala Gly Lys Pro Ile Pro Gly Asp Leu Tyr Arg Ala Cys Leu Tyr Glu Arg Val Leu Leu Ala Leu His Asp Arg Ala Pro Gln Leu Lys Ile Ser Asp Asp Arg Leu Thr Val Val Gly Glu Lys Ala Lys Gln His Asn Met Pro Phe Trp Arg Ile Thr Asn H

Gly Tyr Ser Met Val Arg Ala Ser His Gly Val Arg Lys Gly Ala Trp Tyr Phe Glu Ile Thr Val Asp Glu Met Pro Pro Asp Thr Ala Ala Arg Leu Gly Trp Ser Gln Pro Leu Gly Asn Leu Gln Ala Pro Leu Gly Tyr Asp Lys Phe Ser Tyr Ser Trp Arg Ser Lys Lys Gly Thr Lys Phe His Gln Ser Ile Gly Lys His Tyr Ser Ser Gly Tyr Gly Gln Gly Asp Val Leu Gly Phe Tyr Ile Asn Leu Pro Glu Asp Thr Glu Thr Ala Lys Ser Leu Pro Asp Thr Tyr Lys Asp Lys Ala Leu Ile Lys Phe Lys Ser Tyr Leu Tyr Phe Glu Glu Lys Asp Phe Val Asp Lys Ala Glu Lys Ser Leu Lys Gln Thr Pro His Ser Glu Ile Ile Phe Tyr Lys Asn Gly Val Asn Gln Gly Val Ala Tyr Lys Asp Ile Phe Glu Gly Val Tyr Phe Pro Ala Ile Ser Leu Tyr Lys Ser Cys Thr Val Ser Ile Asn Phe Gly Pro Cys Phe Lys Tyr Pro Pro Lys Asp Leu Thr Tyr Arg Pro Met Ser Asp Met Gly Trp Gly Ala Val Val Glu His Thr Leu Ala Asp Val Leu Tyr His Val Glu Thr Glu Val Asp Gly Arg Arg Ser Pro Pro Trp Glu Pro <210> 12 <211> 259 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2956153CD1 <400> 12 Met Asn Leu Val Asp Leu Trp Leu Thr Arg Ser Leu Ser Met Cys Leu Leu Leu Gln Ser Phe Val Leu Met Ile Leu Cys Phe His Ser Ala Ser Met Cys Pro Lys Gly Cys Leu Cys Ser Ser Ser Gly Gly Leu Asn Val Thr Cys Ser Asn Ala Asn Leu Lys Glu Ile Pro Arg Asp Leu Pro Pro Glu Thr Val Leu Leu Tyr Leu Asp Ser Asn Gln Ile Thr Ser Ile Pro Asn Glu Ile Phe Lys Asp Leu His Gln Leu Arg Val Leu Asn Leu Ser Lys Asn Gly Ile Glu Phe Ile Asp Glu His Ala Phe Lys Gly Val Ala Glu Thr Leu Gln Thr Leu Asp Leu Ser Asp Asn Arg Ile Gln Ser Val His Lys Asn Ala Phe Asn Asn Leu Lys Ala Arg Ala Arg Ile Ala Asn Asn Pro Trp His Cys Asp Cys Thr Leu Gln Gln Val Leu Arg Ser Met Ala Ser Asn His Glu Thr Ala His Asn Val Ile Cys Lys Thr Ser Val Leu Asp Glu His Ala Gly Arg Pro Phe Leu Asn Ala Ala Asn Asp Ala Asp Leu Cys Asn Leu Pro Lys Lys Thr Thr Asp Tyr Ala Met Leu Val Thr Met Phe Gly Trp Phe Thr Met Val Ile Ser Tyr Val Val Tyr Tyr Val Arg Gln Asn Gln Glu Asp Ala Arg Arg His Leu Glu Tyr Leu Lys Ser Leu Pro Ser Arg Gln Lys Lys Ala Asp Glu Pro Asp Asp Ile Ser Thr Val Val <210> 13 <211> 380 <212> PRT
<213> Homo sapiens <220>
<221> misC_feature <223> Incyte ID No: 3333139CD1 <400> 13 Met Ala Ala Pro Trp Trp Arg Ala Ala Leu Cys Glu Cys Arg Arg Trp Arg Gly Phe Ser Thr Ser Ala Val Leu Gly Arg Arg Thr Pro Pro Leu Gly Pro Met Pro Asn Ser Asp Ile Asp Leu Ser Asn Leu Glu Arg Leu Glu Lys Tyr Arg Ser Phe Asp Arg Tyr Arg Arg Arg Ala Glu Gln Glu Ala Gln Ala Pro His Trp Trp Arg Thr Tyr Arg Glu Tyr Phe Gly Glu Lys Thr Asp Pro Lys Glu Lys Ile Asp Ile Gly Leu Pro Pro Pro Lys Val Ser Arg Thr Gln Gln Leu Leu Glu Arg Lys Gln Ala Ile Gln Glu Leu Arg Ala Asn Val Glu Glu Glu Arg Ala Ala Arg Leu Arg Thr Ala Ser Val Pro Leu Asp Ala Val Arg Ala Glu Trp Glu Arg Thr Cys Gly Pro Tyr His Lys Gln Arg Leu Ala Glu Tyr Tyr Gly Leu Tyr Arg Asp Leu Phe His Gly Ala Thr Phe Val Pro Arg Val Pro Leu His Val Ala Tyr Ala Val Gly Glu Asp Asp Leu Met Pro Val Tyr Cys Gly Asn Glu Val Thr Pro Thr Glu Ala Ala Gln Ala Pro Glu Val Thr Tyr Glu Ala Glu Glu Gly Ser Leu Trp Thr Leu Leu Leu Thr Ser Leu Asp Gly His Leu Leu Glu Pro Asp Ala Glu Tyr Leu His Trp Leu Leu Thr Asn Ile Pro Gly Asn Arg Val Ala Glu Gly Gln Val Thr Cys Pro Tyr Leu Pro Pro Phe Pro Ala Arg Gly Ser Gly Ile His Arg Leu Ala Phe Leu Leu Phe Lys Gln Asp Gln Pro Ile Asp Phe Ser Glu Asp Ala Arg Pro Ser Pro Cys Tyr Gln Leu Ala Gln Arg Thr Phe Arg Thr Phe Asp Phe Tyr Lys Lys His Gln Glu Thr Met Thr Pro Ala Gly Leu Ser Phe Phe Gln Cys Arg Trp Asp Asp Ser Val Thr Tyr Ile Phe His Gln Leu Leu Asp Met Arg Glu Pro Val Phe Glu Phe Val Arg Pro Pro Pro Tyr His Pro Lys Gln Lys Arg Phe Pro His Arg Gln Pro Leu Arg Tyr Leu Asp Arg Tyr Arg Asp Ser His Glu Pro Thr Tyr Gly Ile Tyr <210> 14 <211> 130 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3432292CD1 <400> 14 Met Ser Cys Gln Gln Asn Gln Gln Gln Cys Gln Pro Pro Pro Lys Cys Pro Pro Lys Cys Pro Pro Lys Cys Pro Pro Lys Cys Arg Pro Gln Cys Pro Ala Pro Cys Pro Pro Pro Val Ser Ser Cys Cys Gly Pro Ser Ser Gly Gly Cys Cys Gly Ser Ser Ser Gly Gly Cys Cys Ser Ser Gly Gly Gly Gly Cys Cys Leu Ser His His Arg Pro Arg Leu Phe His Arg His Arg His Gln Ser Pro Asp Cys Cys Glu Ser Glu Leu Leu Gly Ala Leu Ala Ala Ser Thr Ala Leu Gly Thr Ala Ala Asp Gln Thr Ser Asn Ile Thr Glu Gln Ala Phe Met Glu Lys Thr Cys Lys Arg Gly Thr Cys Pro Gln Glu <210> 15 <211> 761 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3478571CD1 <400> 15 Met Ser Leu Arg Ile Asp Val Asp Thr Asn Phe Pro Glu Cys Val Val Asp Ala Gly Lys Val Thr Leu Gly Thr Gln Gln Arg Gln Glu Met Asp Pro Arg Leu Arg Glu Lys Gln Asn Glu Ile Ile Leu Arg Ala Val Cys Ala Leu Leu Asn Ser Gly Gly Gly Ile Ile Lys Ala Glu Ile Glu Asn Lys Gly Tyr Asn Tyr Glu Arg His Gly Val Gly Leu Asp Val Pro Pro Ile Phe Arg Ser His Leu Asp Lys Met Gln Lys Glu Asn His Phe Leu Ile Phe Val Lys Ser Trp Asn Thr Glu Ala Gly Val Pro Leu Ala Thr Leu Cys Ser Asn Leu Tyr His Arg Glu Arg Thr Ser Thr Asp Val Met Asp Ser Gln Glu Ala Leu Ala Phe Leu Lys Cys Arg Thr Gln Thr Pro Thr Asn Ile Asn Val Ser Asn Ser Leu Gly Pro Gln Ala Ala Gln Gly Ser Val Gln Tyr Glu Gly Asn Ile Asn Val Ser Ala Ala Ala Leu Phe Asp Arg Lys Arg Leu Gln Tyr Leu Glu Lys Leu Asn Leu Pro Glu Ser Thr His Val Glu Phe Val Met Phe Ser Thr Asp Val Ser His Cys Val Lys Asp Arg Leu Pro Lys Cys Val Ser Ala Phe Ala Asn Thr Glu Gly Gly Tyr Val Phe Phe Gly Val His Asp Glu Thr Cys Gln Val Ile Gly Cys Glu Lys Glu Lys Ile Asp Leu Thr Ser Leu Arg Ala Ser Ile Asp Gly Cys Ile Lys Lys Leu Pro Val His His Phe Cys Thr Gln Arg Pro Glu Ile Lys Tyr Val Leu Asn Phe Leu Glu Val His Asp Lys Gly Ala Leu Arg Gly Tyr Val Cys Ala Ile Lys Val Glu Lys Phe Cys Cys Ala Val Phe Ala Lys Val Pro Ser Ser Trp Gln Val Lys Asp Asn Arg Val Arg Gln Leu Pro Thr Arg Glu Trp Thr Ala Trp Met Met Glu Ala Asp Pro Asp Leu Ser Arg Cys Pro Glu Met Val Leu Gln Leu Ser Leu Ser Ser Ala Thr Pro Arg Ser Lys Pro Val Cys Ile His Lys Asn Ser Glu Cys Leu Lys Glu Gln Gln Lys Arg Tyr Phe Pro Val Phe Ser Asp Arg Val Val Tyr Thr Pro Glu Ser Leu Tyr Lys Glu Leu Phe Ser Gln His Lys Gly Leu Arg Asp Leu Ile Asn Thr Glu Met Arg Pro Phe Ser Gln Gly Ile Leu Ile Phe Ser Gln Ser Trp Ala Val Asp Leu Gly Leu Gln Glu Lys Gln Gly Val Ile Cys Asp Ala Leu Leu Ile Ser Gln Asn Asn Thr Pro Ile Leu Tyr Thr Ile Phe Ser Lys Trp Asp Ala Gly Cys Lys Gly Tyr Ser Met Ile Val Ala Tyr Ser Leu Lys Gln Lys Leu Val Asn Lys Gly Gly Tyr Thr Gly Arg Leu Cys Ile Thr Pro Leu Val Cys Val Leu Asn Ser Asp Arg Lys Ala Gln Ser Val Tyr Ser Ser Tyr Leu Gln Ile Tyr Pro Glu Ser Tyr Asn Phe Met Thr Pro Gln His Met Glu Ala Leu Leu Gln Ser Leu Val Ile Val Leu Leu Gly Phe Lys Ser Phe Leu Ser Glu Glu Leu Gly Ser Glu Val Leu Asn Leu Leu Thr Asn Lys Gln Tyr Glu Leu Leu Ser Lys Asn Leu Arg Lys Thr Arg Glu Leu Phe Val His Gly Leu Pro Gly Ser Gly Lys Thr Ile Leu Ala Leu Arg Ile Met Glu Lys Ile Arg Asn Val Phe His Cys Glu Pro Ala Asn Ile Leu Tyr Ile Cys Glu Asn Gln Pro Leu Lys Lys Leu Val Ser Phe Ser Lys Lys Asn Ile Cys Gln Pro Val Thr Arg Lys Thr Phe Met Lys Asn Asn Phe Glu His Ile Gln His Ile Ile Ile Asp Asp Ala Gln Asn Phe Arg Thr Glu Asp Gly Asp Trp Tyr Gly Lys Ala Lys Phe Ile Thr Gln Thr Ala Arg Asp Gly Pro Gly Val Leu Trp Ile Phe Leu Asp Tyr Phe Gln Thr Tyr His Leu Ser Cys Ser Gly Leu Pro Pro Pro Ser Asp Gln Tyr Pro Arg Glu Glu Ile Asn Arg Val Val Arg Asn Ala Gly Pro Ile Ala Asn Tyr Leu Gln Gln Val Met Gln Glu Ala Arg Gln Asn Pro Pro Pro Asn Leu Pro Pro Gly Ser Leu Val Met Leu Tyr Glu Pro Lys Trp Ala Gln Gly Cys Pro Arg Gln Leu Arg Asp Tyr <210> 16 <211> 197 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3495166CD1 <400> 16 Met Ser Ser Ala Pro Ala Ser Gly Pro Ala Pro Ala Ser Leu Thr Leu Trp Asp Glu Glu Asp Phe Gln Gly Arg Arg Cys Arg Leu Leu Ser Asp Cys Ala Asn Val Cys Glu Arg Gly Gly Leu Pro Arg Val Arg Ser Val Lys Val Glu Asn Gly Val Trp Val Ala Phe Glu Tyr Pro Asp Phe Gln Gly Gln Gln Phe Ile Leu Glu Lys Gly Asp Tyr Pro Arg Trp Ser Ala Trp Ser Gly Ser Ser Ser His Asn Ser Asn Gln Leu Leu Ser Phe Arg Pro Val Leu Cys Ala Asn His Asn Asp Ser Arg Val Thr Leu Phe Glu Gly Asp Asn Phe Gln Gly Cys Lys Phe Asp Leu Val Asp Asp Tyr Pro Ser Leu Pro Ser Met Gly Trp Ala Ser Lys Asp Val Gly Ser Leu Lys Val Ser Ser Gly Ala Trp Val Ala Tyr Gln Tyr Pro Gly Tyr Arg Gly Tyr Gln Tyr Val Leu Glu Arg Asp Arg His Ser Gly Glu Phe Cys Thr Tyr Gly Glu Leu Gly Thr Gln Ala His Thr Gly Gln Leu Gln Ser Ile Arg Arg Val Gln His <210> 17 <211> 339 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3554748CD1 <400> 17 Met Pro Glu Cys Trp Asp Gly Glu His Asp Ile Glu Thr Pro Tyr Gly Leu Leu His Val Val Ile Arg Gly Ser Pro Lys Gly Asn Arg Pro Ala Ile Leu Thr Tyr His Asp Val Gly Leu Asn His Lys Leu Cys Phe Asn Thr Phe Phe Asn Phe Glu Asp Met Gln Glu Ile Thr Lys His Phe Val Val Cys His Val Asp Ala Pro Gly Gln Gln Val Gly Ala Ser Gln Phe Pro Gln Gly Tyr Gln Phe Pro Ser Met Glu Gln Leu Ala Ala Met Leu Pro Ser Val Val Gln His Phe Gly Phe Lys Tyr Val Ile Gly Ile Gly Val Gly Ala Gly Ala Tyr Val Leu Ala Lys Phe Ala Leu Ile Phe Pro Asp Leu Val Glu Gly Leu Val Leu Val Asn Ile Asp Pro Asn Gly Lys Gly Trp Ile Asp Trp Ala Ala Thr Lys Leu Ser Gly Leu Thr Ser Thr Leu Pro Asp Thr Val Leu Ser His Leu Phe Ser Gln Glu Glu Leu Val Asn Asn Thr Glu Leu Val Gln Ser Tyr Arg Gln Gln Ile Gly Asn Val Val Asn Gln Ala Asn Leu Gln Leu Phe Trp Asn Met Tyr Asn Ser Arg Arg Asp Leu Asp Ile Asn Arg Pro Gly Thr Val Pro Asn Ala Lys Thr Leu Arg Cys Pro Val Met Leu Val Val Gly Asp Asn Ala Pro Ala Glu Asp Gly Val Val Glu Cys Asn Ser Lys Leu Asp Pro Thr Thr Thr Thr Phe Leu Lys Met Ala Asp Ser Gly Gly Leu Pro Gln Val Thr Gln Pro Gly Lys Leu Thr Glu Ala Phe Lys Tyr Phe Leu Gln Gly Met Gly Tyr Met Pro Ser Ala Ser Met Thr Arg Leu Ala Arg Ser Arg Thr Ala Ser Leu Thr Ser Ala Ser Ser Val Asp Gly Ser Arg Pro Gln Ala Cys Thr His Ser Glu Ser Ser Glu Gly Leu Gly Gln Val Asn His Thr Met Glu Val Ser Cys <210> 18 <211> 109 <212> PRT
<213> Homo sapiens <220>

<221> misc_feature <223> Incyte ID No: 3555629CD1 <400> 18 Met Glu Arg Gln Gln Gln Gln Gln Gln Gln Leu Arg Asn Leu Arg Asp Phe Leu Leu Val Tyr Asn Arg Met Thr Glu Leu Cys Phe Gln Arg Cys Val Pro Ser Leu His His Arg Ala Leu Asp Ala Glu Glu Glu Ala Cys Val Pro Ser Cys Ala Gly Lys Leu Ile His Ser Asn His Arg Leu Met Ala Ala Tyr Val Gln Leu Met Pro Ala Leu Val Gln Arg Arg Ile Ala Asp Tyr Glu Ala Ala Ser Ala Val Pro Gly Val Ala Ala Glu Gln Pro Gly Val Ser Pro Ser Gly Ser Ser Asp Unk Unk Unk Unk <210> 19 <211> 131 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 639636CD1 <400> 19 Met Thr Lys Lys Lys Val Ser Gln Lys Lys Gln Arg Gly Arg Pro Ser Ser Gln Pro Arg Arg Asn Ile Val Gly Cys Arg Ile Ser His Gly Trp Lys Glu Gly Asp Glu Pro Ile Thr Gln Trp Lys Gly Thr Val Leu Asp Gln Leu Leu Asp Asp Tyr Lys Glu Gly Asp Leu Arg Ile Met Pro Glu Ser Ser Glu Ser Pro Pro Thr Glu Arg Glu Pro 65 70 ' 75 Gly Gly Val Val Asp Gly Leu Ile Gly Lys His Val Glu Tyr Thr Lys Glu Asp Gly Ser Lys Arg Ile Gly Met Val Ile His Gln Val Glu Ala Lys Pro Ser Val Tyr Phe Ile Lys Phe Asp Asp Asp Phe His Ile Tyr Val Tyr Asp Leu Val Lys Lys Ser <210> 20 <211> 194 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 902218CD1 <400> 20 Met Gly Ala Asn Gln Leu Val Val Leu Asn Val Tyr Asp Met Tyr Trp Met Asn Glu Tyr Thr Ser Ser Ile Gly Ile Gly Val Phe His Ser Gly Ile Glu Val Tyr Gly Arg Glu Phe Ala Tyr Gly Gly His Pro Tyr Pro Phe Ser Gly Ile Phe Glu Ile Ser Pro Gly Asn Ala Ser Glu Leu Gly Glu Thr Phe Lys Phe Lys Glu Ala Val Val Leu Gly Ser Thr Asp Phe Leu Glu Asp Asp Ile Glu Lys Ile Val Glu Glu Leu Gly Lys Glu Tyr Lys Gly Asn Ala Tyr His Leu Met His Lys Asn Cys Asn His Phe Ser Ser Ala Leu Ser Glu Ile Leu Cys Gly Lys Glu Ile Pro Arg Trp Ile Asn Arg Leu Ala Tyr Phe Ser Ser Cys Ile Pro Phe Leu Gln Ser Cys Leu Pro Lys Glu Trp Leu Thr Pro Ala Ala Leu Gln Ser Ser Val Ser Gln Glu Leu Gln Asp Glu Leu Glu Glu Ala Glu Asp Ala Ala Ala Ser Ala Ser Val Ala Ser Thr Ala Ala Gly Ser Arg Pro Gly Arg His Thr Lys Leu <210> 21 <211> 184 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1360522CD1 <400> 21 Met Ala Thr Ala Leu Ala Leu Arg Ser Leu Tyr Arg Ala Arg Pro Ser Leu Arg Cys Pro Pro Val Glu Leu Pro Trp Ala Pro Arg Arg Gly His Arg Leu Ser Pro Ala Asp Asp Glu Leu Tyr Gln Arg Thr Arg Ile Ser Leu Leu Gln Arg Glu Ala Ala Gln Ala Met Tyr Ile Asp Ser Tyr Asn Ser Arg Gly Phe Met Ile Asn Gly Asn Arg Val Leu Gly Pro Cys Ala Leu Leu Pro His Ser Val Val Gln Trp Asn Val Gly Ser His Gln Asp Ile Thr Glu Asp Ser Phe Ser Leu Phe Trp Leu Leu Glu Pro Arg Ile Glu Ile Val Val Val Gly Thr Gly Asp Arg Thr Glu Arg Leu Gln Ser Gln Val Leu Gln Ala Met Arg Gln Arg Gly Ile Ala Val Glu Val Gln Asp Thr Pro Asn Ala Cys Ala Thr Phe Asn Phe Leu Cys His Glu Gly Arg Val Thr Gly Ala Ala Leu Ile Pro Pro Pro Gly Gly Thr Ser Leu Thr Ser Leu Gly Gln Ala Ala Gln <210> 22 <211> 528 <212> PRT
<213> Homo Sapiens <220>

<221> misc_feature <223> Incyte ID No: 1400678CD1 <400> 22 Met Ala Ser Met Arg Glu Ser Asp Thr Gly Leu Trp Leu His Asn Lys Leu Gly Ala Thr Asp Glu Leu Trp Ala Pro Pro Ser Ile Ala Ser Leu Leu Thr Ala Ala Val Ile Asp Asn Ile Arg Leu Cys Phe His Gly Leu Ser Ser Ala Val Lys Leu Lys Leu Leu Leu Gly Thr Leu His Leu Pro Arg Arg Thr Val Asp Glu Met Lys Gly Ala Leu Met Glu Ile Ile Gln Leu Ala Ser Leu Asp Ser Asp Pro Trp Val Leu Met Val Ala Asp Ile Leu Lys Ser Phe Pro Asp Thr Gly Ser Leu Asn Leu Glu Leu Glu Glu Gln Asn Pro Asn Val Gln Asp Ile Leu Gly Glu Leu Arg Glu Lys Val Gly Glu Cys Glu Ala Ser Ala Met Leu Pro Leu Glu Cys Gln Tyr Leu Asn Lys Asn Ala Leu Thr Thr Leu Ala Gly Pro Leu Thr Pro Pro Val Lys His Phe Gln Leu Lys Arg Lys Pro Lys Ser Ala Thr Leu Arg Ala Glu Leu Leu Gln Lys Ser Thr Glu Thr Ala Gln Gln Leu Lys Arg Ser Ala Gly Val Pro Phe His Ala Lys Gly Arg Gly Leu Leu Arg Lys Met Asp Thr Thr Thr Pro Leu Lys Gly Ile Pro Lys Gln Ala Pro Phe Arg Ser Pro Thr Ala Pro Ser Val Phe Ser Pro Thr Gly Asn Arg Thr Pro Ile Pro Pro Ser Arg Thr Leu Leu Arg Lys Glu Arg Gly Val Lys Leu Leu Asp Ile Ser Glu Leu Asp Met Val Gly Ala Gly Arg Glu Ala Lys Arg Arg Arg Lys Thr Leu Asp Ala Glu Val Val Glu Lys Pro Ala Lys Glu Glu Thr Val Val Glu Asn Ala Thr Pro Asp Tyr Ala Ala Gly Leu Val Ser Thr Gln Lys Leu Gly Ser Leu Asn Asn Glu Pro Ala Leu Pro Ser Thr Ser Tyr Leu Pro Ser Thr Pro Ser Val Val Pro Ala Ser Ser Tyr Ile Pro Ser Ser Glu Thr Pro Pro Ala Pro Ser Ser Arg Glu Ala Ser Arg Pro Pro Glu Glu Pro Ser Ala Pro Ser Pro Thr Leu Pro Ala Gln Phe Lys Gln Arg Ala Pro Met Tyr Asn Ser Gly Leu Ser Pro Ala Thr Pro Thr Pro Ala Ala Pro Thr Ser Pro Leu Thr Pro Thr Thr Pro Pro Ala Val Ala Pro Thr Thr Gln Thr Pro Pro Val Ala Met Val Ala Pro Gln Thr Gln Ala Pro Ala Gln Gln Gln Pro Lys Lys Asn Leu Ser Leu Thr Arg Glu Gln Met Phe Ala Ala Gln Glu Met Phe Lys Thr Ala Asn Lys Val Thr Arg Pro Glu Lys Ala Leu Ile Leu Gly Phe Met Ala Gly Ser Arg Glu Asn Pro Cys Gln Glu Gln Gly Asp Val Ile Gln Ile Lys Leu Ser Glu His Thr Glu Asp Leu Pro Lys Ala Asp Gly Gln Gly Ser Thr Thr Met Leu Val Asp Thr Val Phe Glu Met Asn Tyr Ala Thr Gly Gln Trp Thr Arg Phe Lys Lys Tyr Lys Pro Met Thr Asn Val Ser <210> 23 <211> 298 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1435556CD1 <400> 23 Met Thr Thr Ile Tyr Asp Leu Lys Lys Gln Lys Asp Lys Leu Leu Lys Phe Tyr Ala Glu Ser Asp Glu Gln Ile Leu Met Lys Asn Arg Lys Thr Leu~His Lys Ala Lys Asn Glu Asp Leu Asp Arg Val Leu Lys Glu Trp Ile Arg Gln Arg Arg Ser Glu His Met Pro Leu Asn Gly Met Leu Ile Met Lys Gln Ala Lys Ile Tyr His Asn Glu Leu Lys Ile Glu Gly Asn Cys Glu Tyr Ser Thr Gly Trp Leu Gln Lys Phe Lys Lys Arg His Gly Ile Lys Phe Leu Lys Thr Cys Gly Asn Lys Ala Ser Ala Gly His Glu Ala Thr Glu Lys Phe Thr Gly Asn Phe Ser Asn Asp Asp Glu Gln Asp Gly Asn Phe Glu Gly Phe Ser Met Ser Ser Glu Lys Lys Ile Met Ser Asp Leu Leu Thr Tyr Thr Lys Asn Ile His Pro Glu Thr Val Ser Lys Leu Glu Glu Glu Asp Ile Lys Asp Val Phe Asn Ser Asn Asn Glu Ala Pro Val Val His Ser Leu Ser Asn Gly Glu Val Thr Lys Met Val Leu Asn Gln Asp Asp His Asp Asp Asn Asp Asn Glu Asp Asp Val Asn Thr Ala Glu Lys Val Pro Ile Asp Asp Met Val Lys Met Cys Asp Gly Leu Ile Lys Gly Leu Glu Gln His Ala Phe Ile Thr Glu Gln Glu Ile Met Ser Val Tyr Lys Ile Lys Glu Arg Leu Leu Arg Gln Lys Ala Ser Leu Met Arg Gln Met Thr Leu Lys Glu Thr Phe Lys Lys Ala Ile Gln Arg Asn Ala Ser Ser Ser Leu Gln Asp Pro Leu Leu Gly Pro Ser Thr Ala Ser Asp Ala Ser Ser His Leu Lys Ile Lys <210> 24 <211> 630 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1546633CD1 <400> 24 Met Pro Gln Gln Gln His Lys Val Ser Pro Ala Ser Glu Ser Pro Phe Ser Glu Glu Glu Ser Arg Glu Phe Asn Pro Ser Ser Ser Gly Arg Ser Ala Arg Thr Val Ser Ser Asn Ser Phe Cys Ser Asp Asp Thr Gly Cys Pro Ser Ser Gln Ser Val Ser Pro Val Lys Thr Pro Ser Asp Ala Gly Asn Ser Pro Ile Gly Phe Cys Pro Gly Ser Asp Glu Gly Phe Thr Arg Lys Lys Cys Thr Ile Gly Met Val Gly Glu Gly Ser Ile Gln Ser Ser Arg Tyr Lys Lys Glu Ser Lys Ser Gly Leu Val Lys Pro Gly Ser Glu Ala Asp Phe Ser Ser Ser Ser Ser Thr Gly Ser Ile Ser Ala Pro Glu Val His Met Ser Thr Ala Gly Ser Lys Arg Ser Ser Ser Ser Arg Asn Arg Gly Pro His Gly Arg Ser Asn Gly Ala Ser Ser His Lys Pro Gly Ser.Ser Pro Ser Ser Pro Arg Glu Lys Asp Leu Leu Ser Met Leu Cys Arg Asn Gln Leu Ser Pro Val Asn Ile His Pro Ser Tyr Ala Pro Ser Ser Pro Ser Ser Ser Asn Ser Gly Ser Tyr Lys Gly Ser Asp Cys Ser Pro Ile Met Arg Arg Ser Gly Arg Tyr Met Ser Cys Gly Glu Asn His Gly Val Arg Pro Pro Asn Pro Glu Gln Tyr Leu Thr Pro Leu Gln Gln Lys Glu Val Thr Val Arg His Leu Lys Ile Lys Leu Lys Glu Ser Glu Arg Arg Leu His Glu Arg Glu Ser Glu Ile Val Glu Leu Lys Ser Gln Leu Ala Arg Met Arg Glu Asp Trp Ile Glu Glu Glu Cys His Arg Val Glu Ala Gln Leu Ala Leu Lys Glu Ala Arg Lys Glu Ile Lys Gln Leu Lys Gln Val Ile Glu Thr Met Arg Ser Ser Leu Ala Asp Lys Asp Lys Gly Ile Gln Lys Tyr Phe Val Asp Ile Asn Ile Gln Asn Lys Lys Leu Glu Ser Leu Leu Gln Ser Met Glu Met Ala His Se-r Gly Ser Leu Arg Asp Glu Leu Cys Leu Asp Phe Pro Cys Asp Ser Pro Glu Lys Ser Leu Thr Leu Asn Pro Pro Leu Asp Thr Met Ala Asp Gly Leu Ser Leu Glu Glu Gln Val Thr Gly Glu Gly Ala Asp Arg Glu Leu Leu Val Gly Asp Ser Ile Ala Asn Ser Thr Asp Leu Phe Asp Glu Ile Val Thr Ala Thr Thr Thr Glu Ser Gly Asp Leu Glu Leu Val His Ser Thr Pro Gly Ala Asn Val Leu Glu Leu Leu Pro Ile Val Met Gly Gln Glu Glu Gly Ser Val Val Val Glu Arg Ala Val Gln Thr Asp Val Val Pro Tyr Ser Pro Ala Ile Ser Glu Leu Ile Gln Ser Val Leu Gln Lys Leu Gln Asp Pro Cys Pro Ser Ser Leu Ala Ser Pro Asp Glu Ser Glu Pro Asp Ser Met Glu Ser Phe Pro Glu Ser Leu Ser Ala Leu Val Val Asp Leu Thr Pro Arg Asn Pro Asn Ser Ala Ile Leu Leu Ser Pro Val Glu Thr Pro Tyr Ala Asn Val Asp Ala Glu Val His Ala Asn Arg Leu Met Arg Glu Leu Asp Phe Ala Ala Cys Val Glu Glu Arg Leu Asp Gly Val Ile Pro Leu Ala Arg Gly Gly Val Val Arg Gln Tyr Trp Ser Ser Ser Phe Leu Val Asp Leu Leu Ala Val Ala Ala Pro Val Val Pro Thr Val Leu Trp Ala Phe Ser Thr Gln Arg Gly Gly Thr Asp Pro Val Tyr Asn Ile Gly Ala Leu Leu Arg Gly Cys Cys Val Val Ala Leu His Ser Leu Arg Arg Thr Ala Phe Arg Ile Lys Thr <210> 25 <211> 339 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1794031CD1 <400> 25 Met Asp Glu Asp Leu Ser Ala Ser Gln Asp His Ser Gln Ala Val Thr Leu Ile Gln Glu Lys Met Thr Leu Phe Lys Ser Leu Met Asp Arg Phe Glu His His Ser Asn Ile Leu Leu Thr Phe Glu Asn Lys Asp Glu Asn His Leu Pro Leu Val Pro Pro Asn Lys Leu Glu Glu Met Lys Arg Arg Ile Asn Asn Ile Leu Glu Lys Lys Phe Ile Leu Leu Leu Glu Phe His Tyr Tyr Lys Cys Leu Val Leu Gly Leu Val Asp Glu Val Lys Ser Lys Leu Asp Ile Trp Asn Ile Lys Tyr Gly Ser Arg Glu Ser Val Glu Leu Leu Leu Glu Asp Trp His Lys Phe Ile Glu Glu Lys Glu Phe Leu Ala Arg Leu Asp Thr Ser Phe Gln 125 ' 130 135 Lys Cys Gly Glu Ile Tyr Lys Asn Leu Ala Gly Glu Cys Gln Asn Ile Asn Lys Gln Tyr Met Met Val Lys Ser Asp Val Cys Met Tyr Arg Lys Asn Ile Tyr Asn Val Lys Ser Thr Leu Gln Lys Val Leu Ala Cys Trp Ala Thr Tyr Val Glu Asn Leu Arg Leu Leu Arg Ala Cys Phe Glu Glu Thr Lys Lys Glu Glu Ile Lys Glu Val Pro Phe Glu Thr Leu Ala Gln Trp Asn Leu Glu His Ala Thr Leu Asn Glu Ala Gly Asn Phe Leu Val Glu Val Ser Asn Asp Val Val Gly Ser Ser Ile Ser Lys Glu Leu Arg Arg Leu Asn Lys Arg Trp Arg Lys Leu Val Ser Lys Thr Gln Leu Glu Met Asn Leu Pro Leu Met Ile Lys Lys Gln Asp Gln Pro Thr Phe Asp Asn Ser Gly Asn Ile Leu Ser Lys Glu Glu Lys Ala Thr Val Glu Phe Ser Thr Asp Met Ser Val Glu Leu Pro Glu Asn Tyr Asn Gln Asn Ile Lys Ala Gly Glu Lys His Glu Lys Glu Asn Glu Glu Phe Thr Gly Gln Leu Lys Val Ala Lys Asp Val Glu Lys Leu Ile Gly <210> 26 <211> 189 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2060563CD1 <400> 26 Met Leu Gly Met Ile Lys Asn Ser Leu Phe Gly Ser Val Glu Thr Trp Pro Trp Gln Val Leu Ser Lys Gly Asp Lys Glu Glu Val Ala Tyr Glu Glu Arg Ala Cys Glu Gly Gly Lys Phe Ala Thr Val Glu Val Thr Asp Lys Pro Val Asp Glu Ala Leu Arg Glu Ala Met Pro Lys Val Ala Lys Tyr Ala Gly Gly Thr Asn Asp Lys Gly Ile Gly Met Gly Met Thr Val Pro Ile Ser Phe Ala Val Phe Pro Asn Glu Asp Gly Ser Leu Gln Lys Lys Leu Lys Val Trp Phe Arg Ile Pro Asn Gln Phe Gln Ser Asp Pro Pro Ala Pro Ser Asp Lys Ser Val Lys Ile Glu Glu Arg Glu Gly Ile Thr Val Tyr Ser Met Gln Phe Gly Gly Tyr Ala Lys Glu Ala Asp Tyr Val Ala Gln Ala Thr Arg Leu Arg Ala Ala Leu Glu Gly Thr Ala Thr Tyr Arg Gly Asp Ile Tyr Phe Cys Thr Gly Tyr Asp Pro Pro Met Lys Pro Tyr Gly Arg Arg Asn Glu Ile Trp Leu Leu Lys Thr <210> 27 <211> 530 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2573955CD1 <400> 27 Met Leu Leu Trp Pro Leu Leu Leu Leu Leu Leu Leu Leu Pro Thr Leu Ala Leu Leu Arg Gln Gln Arg Ser Gln Asp Ala Arg Leu Ser Trp Leu Ala Gly Leu Gln His Arg Val Ala Trp Gly Ala Leu Val Trp Ala Ala Thr Trp Gln Arg Arg Arg Leu Glu Gln Ser Thr Leu His Val His Gln Ser Gln Gln Gln Ala Leu Arg Trp Cys Leu Gln Gly Ala Gln Arg Pro His Cys Ser Leu Arg Arg Ser Thr Asp Ile Ser Thr Phe Arg Asn His Leu Pro Leu Thr Lys Ala Ser Gln Thr Gln Gln Glu Asp Ser Gly Glu Gln Pro Leu Ala Pro Thr Ser Asn Gln Asp Leu Gly Glu Ala Ser Leu Gln Ala Thr Leu Leu Gly Leu Ala Ala Leu Asn Lys Ala Tyr Pro Glu Val Leu Ala Gln Gly Arg Thr Ala Arg Val Thr Leu Thr Ser Pro Trp Pro Arg Pro Leu Pro Trp Pro Gly Asn Thr Leu Gly Gln Val Gly Thr Pro Gly Thr Lys Asp Pro Arg Ala Leu Leu Leu Asp Ala Leu Arg Ser Pro Gly Leu Arg Ala Leu Glu Ala Gly Thr Ala Val Glu Leu Leu Asp Val Phe Leu Gly Leu Glu Thr Asp Gly Glu Glu Leu Ala Gly Ala Ile Ala Ala Gly Asn Pro Gly Ala Pro Leu Arg Glu Arg Ala Ala Glu Leu Arg Glu Ala Leu Glu Gln Gly Pro Arg Gly Leu Ala Leu Arg Leu Trp Pro Lys Leu Gln Val Val Val Thr Leu Asp Ala Gly Gly Gln Ala Glu Ala Val Ala Ala Leu Gly Ala Leu Trp Cys Gln Gly Leu Ala Phe Phe Ser Pro Ala Tyr Ala Ala Ser Gly Gly Val Leu Gly Leu Asn Leu Gln Pro Glu Gln Pro His Gly Leu Tyr Leu Leu Pro Pro Gly Ala Pro Phe Ile Glu Leu Leu Pro Val Lys Glu Gly Thr Gln Glu Glu Ala Ala Ser Thr Leu Leu Leu Ala Glu Ala Gln Gln Gly Lys Glu Tyr Glu Leu Val Leu Thr Asp Arg Ala Ser Leu Thr Arg Cys Arg Leu Gly Asp Val Val Arg Val Val Gly Ala Tyr Asn Gln Cys Pro Val Val Arg Phe Ile Cys Arg Leu Asp Gln Thr Leu Ser Val Arg Gly Glu Asp Ile Gly Glu Asp Leu Phe Ser Glu Ala Leu Gly Arg Ala Val Gly Gln Trp Ala Gly Ala Lys Leu Leu Asp His Gly Cys Val Glu Ser Ser Ile Leu Asp Ser Ser Ala Gly Ser Ala Pro His Tyr Glu Val Phe Val Ala Leu Arg Gly Leu Arg Asn Leu Ser Glu Glu Asn Arg Asp Lys Leu Asp His Cys Leu Gln Glu Ala Ser Pro Arg Tyr Lys Ser Leu Arg Phe Trp Gly Ser Val Gly Pro Ala Arg Val His Leu Val Gly Gln Gly Ala Phe Arg Ala Leu Arg Ala Ala Leu Ala Ala Cys Pro Ser Ser Pro Phe Pro Pro Ala Met Pro Arg Val Leu Arg His Arg His Leu Ala Gln Cys Leu Gln Glu Arg Val Val Ser <210> 28 <211> 356 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3404792CD1 <400> 28 Met Ala Gly Leu Gly Ser Asp Pro Trp Trp Lys Lys Thr Leu Tyr Leu Thr Gly Gly Ala Leu Leu Ala Ala Ala Ala Tyr Leu Leu His Glu Leu Leu Val Ile Arg Lys Gln Gln Glu Ile Asp Ser Lys Asp Ala Ile Ile Leu His Gln Phe Ala Arg Pro Asn Asn Gly Val Pro Ser Leu Ser Pro Phe Cys Leu Lys Met Glu Thr Tyr Leu Arg Met Ala Asp Leu Pro Tyr Gln Asn Tyr Phe Gly Gly Lys Leu Ser Ala Gln Gly Lys Met Pro Trp Ile Glu Tyr Asn His Glu Lys Val Ser Gly Thr Glu Phe Ile Ile Asp Phe Leu Glu Glu Lys Leu Gly Val Asn Leu Asn Lys Asn Leu Gly Pro His Glu Arg Ala Ile Ser Arg Ala Val Thr Lys Met Val Glu Glu His Phe Tyr Trp Thr Leu Ala Tyr Cys Gln Trp Val Asp Asn Leu Asn Glu Thr Arg Lys Met Leu Ser Leu Ser Gly Gly Gly Pro Phe Ser Asn Leu Leu Arg Trp Val Val Cys His Ile Thr Lys Gly Ile Val Lys Arg Glu Met His Gly His Gly Ile Gly Arg Phe Ser Glu Glu Glu Ile Tyr Met Leu Met Glu Lys Asp Met Arg Ser Leu Ala Gly Leu Leu Gly Asp Lys Lys Tyr Ile Met Gly Pro Lys Leu Ser Thr Leu Asp Ala Thr Val Phe Gly His Leu Ala Gln Ala Met Trp Thr Leu Pro Gly Thr Arg Pro Glu Arg Leu Ile Lys Gly Glu Leu Ile Asn Leu Ala Met Tyr Cys Glu Arg Ile Arg Arg Lys Phe Trp Pro Glu Trp His His Asp Asp Asp Asn Thr Ile Tyr Glu Ser Glu Glu Ser Ser Glu Gly Ser Lys Thr His Thr Pro Leu Leu Asp Phe Ser Phe Tyr Ser Arg Thr Glu Thr Phe Glu Asp Glu Gly Ala Glu Asn Ser Phe Ser Arg Thr Pro Asp Thr Asp Phe Thr Gly His Ser Leu Phe Asp Ser Asp Val Asp Met Asp Asp Tyr Thr Asp His Glu Gln Cys Lys <210> 29 <211> 1364 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1681724CB1 <400> 29 gagagagccg ccgcgccgga gcctccttct ttcctgcctc tgattccggg ctgtcatggc 60 gacccccaac aatctgaccc ccaccaactg cagctggtgg cccatctccg cgctggagag 120 cgatgcggcc aagccagcgg aggcccccga cgctcccgag gcggccagcc ccgcccattg 180 gcccagggag agcctggttc tgtaccactg gacccagtcc ttcagctcgc agaaggtgcg 240 gctggtgatc gccgagaagg gcctggtgtg cgaggagcgg gacgtgagcc tgccacagag 300 cgagcacaag gagccctggt tcatgcggct caacctgggc gaggaggtgc ccgtcatcat 360 ccaccgcgac aacatcatca gtgactatga ccagatcatt gactatgtgg agcgcacctt 420 cacaggagag cacgtggtgg ccctgatgcc cgaggtgggc agcctgcagc acgcacgggt 480 gctgcagtac cgggagctgc tggacgcact gcccatggat gcctacacgc atggctgcat 540 cctgcatccc gagctcacca ccgactccat gatccccaag tacgccacgg ccgagatccg 600 cagacattta gccaatgcca ccacggacct catgaaactg gaccatgaag aggagcccca 660 gctctccgag ccctaccttt ctaaacaaaa gaagctcatg gccaagatct tggagcatga 720 tgatgtgagc_tacctgaaga agatcctcgg ggaactggcc atggtgctgg accagattga 780 ggcggagctg gagaagagga agctggagaa cgaggggcag aaatgcgagc tgtggctctg 840 tggctgtgcc ttcaccctcg ctgatgtcct cctgggagcc accctgcacc gcctcaagtt 900 cctgggactg tccaagaaat actgggaaga tggcagccgg cccaacctgc agtccttctt 960 tgagagggtc cagagacgct ttgccttccg gaaagtcctg ggtgacatcc acaccaccct 1020 gctgtcggcc gtcatcccca atgctttccg gctggtcaag aggaaacccc catccttctt 1080 cggggcgtcc ttcctcatgg gctccctggg tgggatgggc tactttgcct actggtacct 1140 caagaaaaaa tacatctagg gccaggcctg gggcttggtg tctgactgtc ggtgtctctg 1200 tgctgtgtga ttccccgtga gctctcagta actcactgtc tcatgaacac ttggacagcc 1260 ctccccgccc ttcgttctga gtaataatac cgtcagtgtg aaaacattcc gtagtttaga 1320 agtagacgtt gccaatgctg tgactcaagg ccagggttca atta 1364 <210> 30 <211> 505 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1718047CB1 <400> 30 cggctcgagg agagattcac gcaccctcaa gagtgtgggt gagacatata cagcctgtta 60 gacctgaagg cagatggctc ttcttaaggc caataaggat ctcatttccg caggattgaa 120 ggagttcagc gttctgctga atcagcaggt cttcaatgat cctctcgtct ctgaagaaga 180 catggtgact gtggtggagg actggatgaa cttctacatc aactattaca ggcagcaggt 240 gacaggggag ccccaagagc gagacaaggc tctgcaggag cttcggcaag agctgaacac 300 tctggccaac cctttcctgg ccaagtacag ggacttcctg aagtctcatg agctcccgag 360 tcacccaccg ccctcctcct agctcaggga cccagccccc cctctctgag aaactctgac 420 cttcatgtcc ttaggctgtg ctcctgccac tctaccctga cacctcaata aagaccagtg 480 ctggttttgt tggaaaaaaa aaaaa 505 <210> 31 <211> 926 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1980323CB1 <400> 31 gtccggggtc gtgagccggc cccgccttgg tggcggcgcc ccctcgcggt ccagaggcag 60 acgcatcggg tgggctcggg tctccagccc ggccgggagg agggaccggg tctgcggagc 120 ggggactcgg ggcctcggcg gggcgcgcac acgcaggcgg ggcggcccgg ggtgcggggc 180 ctctgcgcgg ctgaccaggc tcccagagcg tcacgccgcc catggccgag ccgctccagc 240 cagaccccgg ggcggccgag gacgcggcgg cccaagctgt ggagacgccg ggctggaagg 300 ccccggagga cgccggcccc cagcccggaa gttatgagat ccgacactat ggaccagcca 360 agtgggtcag cacgtccgtg gagtctatgg actgggattc agccatccag acgggcttta 420 cgaaactgaa cagctacatt caaggcaaaa acgagaaaga gatgaaaata aagatgacag 480 ctccagtgac aagctacgtg gagcctggtt caggtccttt tagtgagtct accattacca 540 tttccctgta tattccctct gaacagcaat ttgatccacc caggccttta gagtcagatg 600 tcttcattga agatagagcc gaaatgactg tgtttgtacg gtctttcgat ggattttcta 660 gtgcccaaaa gaatcaagaa caacttttga cattagcaag cattttaagg gaagatggaa 720 aagttttcga tgagaaggtt tactacactg caggctacaa cagtcctgtc aaattgctta 780 atagaaataa tgaagtgtgg ttgattcaaa aaaatgaacc caccaaagaa aacgaatgag 840 aaaaatgaaa ggaagttctg ctgtcagagg caaaacatct gtttatcata gacatcaaca 900 tgacctataa gtaaaaaaaa aaaaaa 926 <210> 32 <211> 1364 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1990956CB1 <400> 32 gacaacagcc ccacgtgacc ggccaacact gagtgttgtc tcgctctggc gtcagagccg 60 tcgtggctcg ttccattctc ggcggtggta cctgctcccg gtggccctga ggacgtgtgg 120 gccaggggcg gccccgaaat taggaagcgg agggggagca gtctgcaggt ctgcggggct 180 aagtgtcgcg gcggcgcacc tcgcgtcaag aatccggagg aggagactgc aaggataggc 240 ccaggagtaa tggagtccaa agaggaacta gcggcaaaca atctcaacgg ggaaaatgcc 300 caacaagaaa acgaaggagg ggagcaggcc cccacgcaga atgaagaaga atcccgccat 360 ttgggagggg gtgaaggcca gaagcctgga ggaaatatca ggcgggggcg agttaggcga 420 cttgtcccta attttcgatg ggccatacct aataggcata ttgagcacaa tgaagcgaga 480 gatgatgtag aaaggtttgt agggcagatg atggaaatca agagaaagac tagggaacag 540 cagatgaggc actatatgcg cttccaaact cctgaacctg acaaccatta tgacttttgc 600 ctcatacctt gaatcctaaa agttttcgct gaggttaatg tgaacactgc tttacaagct 660 tgtatttttg tgatttactt tttctgtaag ccttttggtg tttacactta ccagtttcta 720 atggaaatta gaattctaat tgaatattgt tttgtctcag cctaaaagtt acggtcagca 780 tggcaattca cctattttag gaaaaatact cttttcataa tatgaaatgc ataaagcagt 840 tcaaaaagca gtctgtattc catcatcttc ctttttcatt ccagtcctta tttttgtaag 900 tattactttt cctcctccgg ctacctggac tcaaaatctc agttgtcttt gacagttttt 960 ttcttgtccc tgaccaaaaa agaatgatca tacccagaat tcaatgtttg atattttaag 1020 aatgtatgtt ctagtgtttt tcagagtgag tctaccatct gtataaaaac accttggggg 1080 caggcagggg catttaaaaa tgtaggacct atcgtccaga ctcacagagt ggggctccag 1140 aatctccatt tttaacaaac tctcttaagt aattctgatg tgtaccaaaa tcagtgccat 1200 tggtgtgtgt gtacgtaact atatacatat gtgtgtgtgt gtatatatat aatgtgtcat 1260 aaccgtaaac aataaacaat atcaagataa atctgacttt gatgggcaag taattaaaaa 1320 agaaaagtat gagaccttaa aaaaaaaaaa aaaaaaaaaa aaaa 1364 <210> 33 <211> 464 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2009069CB1 <400> 33 gagcaggaat tcggcacgag gaattcagta tggcaaaggt gaccagtgag ccacagaagc 60 ctaatgaaga tgtggacgaa cacaccccat caacctcaag taccaaaggg aggaagaagg 120 ggaagacacc ccgtcaacga aggtccagaa gcggcgttaa gggcctaaag accaccagga 180 aggcgaaaag accccttcga gggagctcga gccaaaaagc cggtgaaact aacacccctg 240 caggaaaacc taagaaagct agaggaccaa tactgcgtgg tcgttatcac cggctgaaag 300 aaaaaatgaa gaaagaagag gccgacaaag agcaaagcga gacctcagtt ctgtgatgtc 360 tctagaggtc cgccactgaa aagtcatcaa tcatacagtc agtgaattct acaccaacag 420 gttaaaacca tgaaaataaa atcaacctga atcgaaaaaa aaaa 464 <210> 34 <211> 1549 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2009435CB1 <400> 34 ttccctgggt cgacccacgc gtccgggaag acagtttgca ttcttgcaac attaaaccaa 60 agggacttgg agtgcagatg gcatccttcg gttcttccag acaagctgca agacgctgac 120 catggccaag atggagctct cgaaggcctt ctctggccag cggacactcc tatctgccat 180 cctcagcatg ctatcactca gcttctccac aacatccctg ctcagcaact actggtttgt 240 gggcacacag aaggtgccca agcccctgtg cgagaaaggt ctggcagcca agtgctttga 300 catgccagtg tccctggatg gagataccaa cacatccacc caggaggtgg tacaatacaa 360 ctgggagact ggggatgacc ggttctcctt ccggagcttc cggagtggca tgtggctatc 420 ctgtgaggaa actgtggaag aaccagcact gctccatccc cagtcctgga aacaatttag 480 agcccttcgg tccagtggta cagcggcagc aaaaggggag aggtgccgaa gtttcattga 540 acttacacca ccagccaaga gaggtgagaa aggactactg gaatttgcca cgttgcaagg 600 cccatgtcac cccactctcc gatttggagg gaagcggttg atggagaagg cttccctccc 660 ctcccctccc ttggggcttt gtggcaaaaa tcctatggtt atccctggga acgcagatca 720 cctacatcgg acttcaattc atcagcttcc tcctgctact aacagacttg ctactcactg 780 ggaaccctgc ctgtgggctc aaactgagcg cctttgctgc tgtttcctct gtcctgtcag 840 gtctcctggg gatggtggcc cacatgatgt attcacaagt cttccaagcg actgtcaact 900 tgggtccaga agactggaga ccacatgttt ggaattatgg ctgggccttc tacatggcct 960 ggctctcctt cacctgctgc atggcgtcgg ctgtcaccac cttcaacacg tacaccagga 1020 tggtgctgga gttcaagtgc aagcatagta agagcttcaa ggaaaacccg aactgcctac 1080 cacatcacca tcagtgtttc cctcggcggc tgtcaagtgc agcccccacc gtgggtcctt 1140 tgaccagcta ccaccagtat cataatcagc ccatccactc tgtctctgag ggagtcgact 1200 tctactccga gctgcggaac aagggatttc aaagaggggc cagccaggag ctgaaagaag 1260 cagttaggtc atctgtagag gaagagcagt gttaggagtt aagcgggttt ggggagtagg 1320 cttgagccct accttacacg tctgctgatt atcaacatgt gcttaagcca aaaagctctg 1380 gagctatttc cagattaaat agtttttcta aaactttctg tccttcttta ctgggggcct 1440 gtcagcatca ctgatgaata tttcttgcca cagaggtttt tcttgttttt cccggattcc 1500 tttggatgtg gatcaacttt aaaatatcct gggtgactcc ggttcacca 1549 <210> 35 <211> 1205 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 2027937CB1 <400> 35 ctttgttgag tcttgttgaa gatcaggctc tgcaaatcac cctaggatgt ccttcagtga 60 gcagcagtgc aagcagccat gtgtgccccc tccatgcctc ccaaagaccc aggagcagtg 120 ccaagcaaag gctgaggagg tgtgcctccc cacatgccag cacccctgcc aagataagtg 180 tctagtgcag gcccaggagg tatgtctttc tcagtgccag gaatcaagtc aagaaaaatg 240 cccacagcaa ggccaagagc catacctacc tccatgccaa gaccagtgtc cacctcagtg 300 tgcagagcca tgccaggagc tattccagac aaaatgtgtg gaggtttgcc cacagaaagt 360 tcaggagaag tgctcatccc ctggcaaggg aaagtagctg ctcatatgtc atctgggttc 420 aagaagatgg ccagcagatg aaaccctgac cccagcccac gctctggtga ccttcttctg 480 tgggtacctc tgtgtgcaat gtaccttctt gcctcctggc ttccttagca ttccaggact 540 tggtctgtgt ctctgaagac agttctttct gtatttcatc accctctgtg aataagcatt 600 gttctcagca gtctgatgga aggtctcaaa tgtaggaatg gtgtggttgt cagggaagac 660 cacagaagcc tagcacagct tccttggtgc aaaaattcac ccagctctgg gtgtgtaaca 720 gccaaggata ccttcattca tctttcagag tttcaggctc tcaaataagc ctcaaaacaa 780 actgaattct gatggacttt ccacttatca ccaccaccac cacctccacc accaccacca 840 ccacaggtgt tgagacaaag cggcttgcgg tgtttcaaaa atcaaaaatt tggtgatttt 900 gtctctgtac actaaaaaga acagcaaatt attgaacttt gaaatgttgg ttgtgctttt 960 ttaaaatgaa ccgaaagtat gtaaacgatt gaatccaaac aaccagaagg gaaagataag 1020 atggatgttt ggtgcccttg tcaatctctg tgcttcatag ctggtctaat gtgggccctt 1080 agttctcacc atgtacactc tttagagatg tgatttgtgt ctgtgtgatg caggctggtc 1140 tgttctccag cttccttgcc ttcttctccc tggtgaaggt aaaatacata ataaagctga 1200 tcctg 1205 <210> 36 <211> 4061 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2722347CB1 <400> 36 ccggcgcggg gccctgcggt agcctcaggc Ccctcccctg gacccgccgc agagccagtg 60 cagaatacag aaactgcagc catgaccacg cacgtcaccc tggaagatgc cctgtccaac 120 gtggacctgc ttgaagagct tcccctcccc gaccagcagc catgcatcga gcctccacct 180 tcctccatca tgtaccaggc taactttgac acaaactttg aggacaggaa tgcatttgtc 240 acgggcattg caaggtacat tgagcaggct acagtccact ccagcatgaa tgagatgctg 300 gaggaaggac atgagtatgc ggtcatgctg tacacctggc gcagctgttc ccgggccatt 360 ccccaggtga aatgcaacga gcagcccaac cgagtagaga tctatgagaa gacagtagag 420 gtgctggagc cggaggtcac caagctcatg aagttcatgt attttcagcg caaggccatc 480 gagcggttct gcagcgaggt gaagcggctg tgccatgccg agcgcaggaa ggactttgtc 540 tctgaggcct acctcctgac ccttggcaag ttcatcaaca tgtttgctgt cctggatgag 600 ctaaagaaca tgaagtgcag cgtcaagaat gaccactctg cctacaagag ggcagcacag 660 ttcctgcgga agatggcaga tccccagtct atccaggagt cgcagaacct ttccatgttc 720 ctggccaacc acaacaggat cacccagtgt ctccaccagc aacttgaagt gatcccaggc 780 tatgaggagc tgctggctga cattgtcaac atctgtgtgg attactacga gaacaagatg.840 tacctgactc ccagtgagaa acatatgctc ctcaaggtga tgggctttgg cctctaccta 900 atggatggaa atgtcagtaa catttacaaa ctggatgcca agaagagaat taatcttagc 960 aaaattgata aattctttaa gcagctgcag gtggtgcccc ttttcggcga catgcagata 1020 gagctggcca gatacattaa gaccagtgct cactatgaag agaacaagtc caagtggacg 1080 tgcacccaga gcagcatcag cccccagtac aatatctgcg agcagatggt tcagatccgg 1140 gatgaccaca tccgcttcat ctccgagctc gctcgctaca gcaacagtga ggtggtgacg 1200 ggctcagggc tggacagcca gaagtcagac gaggagtatc gcgagctctt cgacctagcc 1260 ctgcggggtc tgcagcttct atccaagtgg agcgcccacg tcatggaggt gtactcttgg 1320 aagctggttc atcccacaga caagttctgc aacaaggact gtcctggcac cgcggaggaa 1380 tatgagagag ccacacgcta caattacacc agtgaggaaa aatttgcctt cgttgaggtg 1440 atcgccatga tcaaaggcct gcaggtgctc atgggcagga tggagagcgt cttcaaccag 1500 gccatcagga acaccatcta cgcggcattg caggacttcg cccaggtgac gctgcgtgag 1560 cccctgcggc aggcggtacg gaagaagaag aatgtcctca tcagcgtcct acaggcaatt 1620 cgaaagacca tctgtgactg ggagggaggg cgagagcccc ctaatgaccc atgcttgaga 1680 ggggagaagg accccaaagg tggatttgat atcaaggtgc cccggcgtgc tgtggggcca 1740 tccagcacac agctgtacat ggtgcggacc atgcttgaat cactcattgc agacaaaagc 1800 ggctccaaga agaccctgag gagcagcctg gatggaccca ttgtcctcgc catagaggac 1860 tttcacaaac agtccttctt cttcacacat ctgctcaaca tcagtgaagc cctgcagcag 1920 tgttgtgacc tctcccagct ctggttccga gaattcttcc tggagttaac catgggccga 1980 cgaatccagt tccccatcga gatgtccatg ccctggattc taacggacca tatcctggaa 2040 accaaagaac cttccatgat ggagtatgtc ctctaccctc tggatctgta caacgacagc 2100 gcctactatg ctctgaccaa gtttaaaaag cagttcctgt acgatgagat agaagctgag 2160 gtgaacctgt gttttgatca gtttgtctac aagctggcag accagatctt tgcttactac 2220 aaagccatgg ctggcagtgt cctgttggat aaacgttttc gagctgagtg taagaattat 2280 ggcgtcatca ttccgtatcc accgtccaat cgctatgaaa cactgctgaa gcagagacac 2340 gtccagctgt tgggtagatc aattgacttg aacagactca ttacccagcg catctctgcc 2400 gccatgtata aatccttgga ccaagctatc agccgctttg agagtgagga cctgacctcc 2460 attgtggagc tggagtggct gctggagatt aaccggctca cgcatcggct gctctgtaag 2520 catatgacgc tggacagctt cgatgccatg ttccgagagg ccaatcacaa tgtgtccgcc 2580 ccctatggcc gtatcaccct gcatgtcttc tgggaactga actttgactt tctccccaac 2640 tactgctaca atgggtccac taaccgtttt gtgcggactg ccattccttt Cacccaagaa 2700 ccacaacgag acaaacctgc caacgtccag ccttattacc tctatggatc caagcctctc 2760 aacattgcct acagccacat ctacagctcc tacaggaatt tcgtggggcc acctcatttc 2820 aagactatct gcagactcct gggttatcag ggcatcgctg tggtcatgga ggaactgcta 2880 aagattgtga agagcttgct ccaaggaacc attctccagt atgtgaaaac actgatagag 2940 gtgatgccca agatatgccg cttgccccga catgagtatg gctccccagg gatcctggag 3000 ttcttccacc accagctgaa ggacatcatt gagtacgcag agctcaaaac agacgtgttc 3060 cagagcctga gggaagtggg caatgccatc ctcttctgcc tcctcataga gcaagctctg 3120 tctcaggagg aggtctgcga tttgctccat gccgcaccct tccaaaacat cttgcctaga 3180 gtctacatca aagaggggga gcgcctggag gtccggatga aacgtctgga agccaagtat 3240 gccccgctcc acctggtccc tctgatcgag cggctgggga cccctcagca aatcgccatt 3300 gctcgcgagg gtgacctcct gaccaaggag cggctgtgct gtggcctgtc catgttcgag 3360 gtcatcctga cccgcattcg gagctacctg caggacccca tctggcgggg cccaccgccc 3420 accaatggcg tcatgcacgt cgatgagtgt gtggagttcc accggctgtg gagcgccatg 3480 cagttcgtgt actgcatccc tgtgggaacc aacgagttca cagctgagca gtgtttcggc 3540 gatggcttga actgggctgg ttgctccatc attgtcctgc tgggccagca gcgtcgcttt 3600 gacctgttcg acttctgtta ccacctgcta aaagtgcaga ggcaggacgg gaaggatgaa 3660 atcattaaga atgtgcccct gaagaagatg gccgaccgga tcaggaagta tcagatcttg 3720 aacaatgagg tttttgccat cctgaacaaa tacatgaagt ccgtggagac agacagttcc 3780 actgtggagc atgtgcgctg cttccagcca cccatccacc agtccttggc caccacttgc 3840 taagcagaag atcctgcaga cccttatctg gaggaggaag agaagcagga gagagaaagc 3900 cacagccagc ctgccatagg atccaactgg acaacgtgtg ggatggacct ggaaacaagc 3960 acctccccaa acacatcacc actccctagg gcggggcctg tgcatgctct cccatgacat 4020 ctccatgctg gtttctccat agcataaatg aaaaaaaaaa a 4061 <210> 37 <211> 773 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2759876CB1 <400> 37 gcagttctcc tgcctcagcc tcctgagtag ctgggattac agacaaaaat actaatgcat 60 ttgagaaagc ggtagttttg gttggagggg gaaaaatcaa ctgctttcct gatctgcaac 120 ttggctggat gctaagatgt cagtggacat gaatagccag gggtctgaca gcaatgaaga 180 ggactatgac ccaaattgtg aggaagagga agaagaagaa gaagacgacc ctggggacat 240 agaggactat tacgtgggag tagccagcga tgtggagcag cagggggctg atgcctttga 300 tcccgaggag taccagttca cttgcttgac ctacaaggaa tctgagggtg ccctcaatga 360 gcacatgacc agcttagctt ctgtcctaaa ggtgagcagt gttgtaaact ccagtgtaat 420 cccccccagt taaatctaag gatgagctgt tgttaattaa tgtctcctcc agactaattg 480 aatggccttg tagcttgaaa ccaaatttaa gtgtatgtca acatcatttg gacattttgt 540 tacatttact ttgttttcta atatatgcat gtctaaggtc ctcagattcc caaattagta 600 agaattagaa agtaggggtg ggacagattc aagaaagatg taccatacca gaaatgtgta 660 gtagcagaat tgcacaactt tgcacgtttc aggaagtgag ttttcagaat tttatgaggt 720 tgatacaata agcatcagaa tcaccatctt ctgtacgact gtgagtgact gat 773 <210> 38 <211> 2116 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2763735CB1 <400> 38 ctcgattggt tctactgtgg gtctggactg atctccatgt cctgttgtgg ggcttttaca 60 gcctttggat tgtgaaaact gctgagagag acttgcaatc cagtcacata agtataataa 120 agaaatattg gtcctcatgg aagaagagca agatttacca gagcaaccag tgaaaaaagc 180 caagatgcag gaatcaggag agcaaactat aagtcaagta agcaatccag atgtcagtga 240 tcagaagcct gaaacatcaa gccttgcttc aaaccttccc atgtcagagg aaattatgac 300 atgcaccgat tacatccctc gctcatccaa tgattatacc tcacaaatgt attctgcaaa 360 accttatgca catattctct cagttcctgt ttcggaaact gcttaccctg gacagactca 420 ataccagaca ctacagcaga ctcaacccta tgctgtctac cctcaggcaa cccaaacgta 480 tggactacct ccttttgctt caagcacaaa tgccagcctg atatctactt cttctacaat 540 tgccaatatt ccagcagcag cagtagccag catctcaaac caggattatc ccacctatac 600 tattcttggt cagaatcagt accaggcctg ctaccccagc tccagctttg gagtcacagg 660 tcagactaac agtgatgcag agagcaccac attagcagca accacatacc agtcggagaa 720 gcctagtgtc atggcgcctg cacctgcagc acagagactt tcctctggag acccttctac 780 aagtccatct ttgtcccaga ctacaccaag taaagatact gatgatcagt ccaggaaaaa 840 catgactagc aagaaccggg gcaagaggaa agctgatgcc acttcttccc aagacagtga 900 attagaacgg gtatttctgt gggacttgga tgaaaccatc atcatcttcc actcacttct 960 tactggatcc tatgcccaga aatatggaaa ggacccaaca gtagtgattg gctcaggttt 1020 aacaatggaa gaaatgattt ttgaagtggc tgatactcat ctatttttca atgacttaga 1080 ggagtgtgac caggtacatg tggaagatgt ggcttctgat gacaatggcc aagacttgag 1140 caactacagt ttctcaacag atggtttcag tggctcagga ggtagtggca gccatggttc 1200 atctgtgggt gttcagggag gtgtggactg gatgaggaaa ctagctttcc gctaccggaa 1260 agtgagagaa atctatgata agcataaaag caacgtgggt ggtctcctca gtccccagag 1320 gaaggaagca ctgcagagat taagagcaga aattgaagtt ttaacagatt cctggttagg 1380 aactgcatta aagtccttac ttctcatcca gtccagaaag aattgtgtga atgttctgat 1440 cactaccacc cagctggttc cagccctggc caaggttctc ctatatggac taggagaaat 1500 atttcctatt gagaacatct atagtgctac caaaattggt aaggagagct gctttgagag 1560 aattgtgtca aggtttggaa agaaagtcac atatgtagtg attggagatg gacgagatgc 1620 agccaaacag cacaacatgc ctttctggag gatcacaaac catggagacc tagtatccct 1680 tcaccaggct ttagagcttg attttctcta agaactggaa tgaggagcct tccccttgag 1740 ctccttttca ctcctgaagg gagctggaga ctggaaccaa ctgagaactt tctctgtctg 1800 tctctctctg tgtctctgtc tctatctctc tctctttctc tctttctctc tctccctctc 1860 tccctccctc ccttcctctc tccctccctc tctgcctctc tctctctctc tctctctgtc 1920 tttatccatg gaatgctggc gagaacacaa tcagaaccaa cagctgcaat ttttgctaag 1980 agtgagctgc agccccgtgt tcatctccat acagaagcag ggacagttgg atagagagaa 2040 caatggactc actgcagcta cagtgctttt aatttttctg tgttttgttt ttgttttttt 2100 gttttcaaaa aaaaaa 2116 <210> 39 <211> 2556 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2848676CB1 <400> 39 cgagaagtcc aggggtggcc gtgatggcgg cggcaggagc aggacctggc caggaagcgg 60 gtgccgggcc tggcccagga gcggtcgcaa atgcaacagg ggcagaagag ggggagatga 120 agccggtggc agcgggagca gccgctcctc ctggagaggg gatctctgct gctccgacag 180 ttgagcccag ttccggggag gctgaaggcg gggaggcaaa cttggtcgat gtaagcggtg 240 gcttggagac agaatcatct aatggaaaag atacactaga aggtgctggg gatacatcag 300 aggtgatgga tactcaggcg ggctccgtgg atgaagagaa tggccgacag ttgggtgagg 360 tagagctgca atgtgggatt tgtacaaaat ggttcacggc tgacacattt ggcatagata 420 cctcatcctg tctacctttc atgaccaact acagttttca ttgcaacgtc tgccatcaca 480 gtgggaatac ctatttcctc cggaagcaag caaacttgaa ggaaatgtgc cttagtgctt 540 tggccaacct gacatggcag tcccgaacac aggatgaaca tccgaagaca atgttctcca 600 aagataagga tattatacca tttattgata aatactggga gtgcatgaca accagacaga 660 gacctgggaa aatgacttgg ccaaataaca ttgttaaaac aatgagtaaa gaaagagatg 720 tattcttggt aaaggaacac ccagatccag gcagtaaaga tccagaagaa gattacccca 780 aatttggact tttggatcag gaccttagta acattggtcc tgcttatgac aaccaaaaac 840 agagcagtgc tgtgtctact agtgggaatt taaatggggg aattgcagca ggaagcagcg 900 gaaaaggacg aggagccaag cgcaaacagc aggatggagg gaccacaggg accaccaaga 960 aggcccggag tgaccctttg ttttctgctc agcgccttcc ccctcatggc tacccattgg 1020 aacacccgtt taacaaagat ggctatcggt atattctagc tgagcctgat ccgcacgccc 1080 ctgaccccga gaagctggaa cttgactgct gggcaggaaa acctattcct ggagacctct 1140 acagagcctg cttgtatgaa cgggttttgt tagccctaca tgatcgagct ccccagttaa 1200 agatctcaga tgaccggctg actgtggttg gagagaaggg ctactctatg gtgagggcct 1260 ctcatggagt acggaagggt gcctggtatt ttgaaatcac tgtggatgag atgccaccag 1320 ataccgctgc cagactgggt tggtcccagc ccctaggaaa ccttcaagct cctttaggtt 1380 atgataaatt tagctattct tggcggagca aaaagggaac caagttccac cagtccattg 1440 gcaaacacta ctcttctggc tatggacagg gagacgtcct gggattttat attaatcttc 1500 ctgaagacac agagacagcc aagtcattgc cagacacata caaagataag gctttgataa 1560 aattcaagag ttatttgtat tttgaggaaa aagactttgt ggataaagca gagaagagcc 1620 tgaagcagac tccccatagt gagataatat tttataaaaa tggtgtcaat caaggtgtgg 1680 cttacaaaga tatttttgag ggggtttact tcccagccat ctcactgtac aagagctgca 1740 cggtttccat taactttgga ccatgcttca agtatcctcc gaaggatctc acttaccgcc 1800 ctatgagtga catgggctgg ggcgccgtgg tagagcacac cctggctgac gtcttgtatc 1860 acgtggagac agaagtggat gggaggcgca gtcccccatg ggaaccctga ccaggtccct 1920 cttttctgtc aaggactttc tgggaataat actgggggtt ttgtttttgt ttttgaactg 1980 tctcaaatgt tctcccaaag atgctaaaaa cacagcctct ccttttagca agttaaaagg 2040 ctgggtagga ctgcgggaga ctgcctgcct ttcaccattt tctccccact tccagtgact 2100 gctcttattt tgtgtaccat aagccaacaa ccgctgactc caggattgca taagccccct 2160 gtgaaatcgg tgctgtactg cataccctgc cagctgtgac ttgttatcct actatatttt 2220 ctaaggagtg aataatattg tccgagtaac taacttattt aaaagacatt tccttctgtg 2280 ggcattgact gtatcccacc tgttttccaa ggaaatggta acctgtttct gagaacacct 2340 gaaatcaatg gctatacatt ccaaaccaat ctaaacgcta tttccttttg gtgtgggttt 2400 ggttttgttc attttgaaat acacttttga acactgagat ccgtaaaact actagatctc 2460 tggaagtgta attgtgaaag aaacttgctt gcagctttaa caaaatgaga aacttcccaa 2520 ataaaacttg ttttgaagtt taaaaaaaaa aaaaaa 2556 <210> 40 <211> 1394 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2956153CB1 <400> 40 gcccagacga attcccttgc ggccgctgca gccctggcag gcgcacaccc ctaagcatac 60 gcacccaacg cctcctccct gagccacgag gatggagcag ccaccccggc ccgggactgg 120 cgcaaggtgc ccaagcaagg aaagaaataa tgaagagaca catgtgttag ctgcagcctt 180 ttgaaacacg caagaaggaa atcaatagtg tggacagggc tggaaccttt accacgcttg 240 ttggagtaga tgaggaatgg gctcgtgatt atgctgacat tccagcatga atctggtaga 300 cctgtggtta acccgttccc tctccatgtg tctcctccta caaagttttg ttcttatgat 360 actgtgcttt cattctgcca gtatgtgtcc caagggctgt ctttgttctt cctctggggg 420 tttaaatgtc acctgtagca atgcaaatct caaggaaata cctagagatc ttcctcctga 480 aacagtctta ctgtatctgg actccaatca gatcacatct attcccaatg aaatttttaa 540 ggacctccat caactgagag ttctcaacct gtccaaaaat ggcattgagt ttatcgatga 600 gcatgccttc aaaggtgtag ctgaaacctt gcagactctg gacttgtccg acaatcggat 660 tcaaagtgtg cacaaaaatg ccttcaataa cctgaaggcc agggccagaa ttgccaacaa 720 cccctggcac tgcgactgta ctctacagca agttctgagg agcatggcgt ccaatcatga 780 gacagcccac aacgtgatct gtaaaacgtc cgtgttggat gaacatgctg gcagaccatt 840 cctcaatgct gccaacgacg ctgacctttg taacctccct aaaaaaacta ccgattatgc 900 catgctggtc accatgtttg gctggttcac tatggtgatc tcatatgtgg tatattatgt 960 gaggcaaaat caggaggatg cccggagaca cctcgaatac ttgaaatccc tgccaagcag 1020 gcagaagaaa gcagatgaac ctgatgatat tagcactgtg gtatagtgtc caaactgact 1080 gtcattgaga aagaaagaaa gtagtttgcg attgcagtag aaataagtgg tttacttctc 1140 ccatccattg taaacatttg aaactttgta tttcagtttc ttttgaatta tgccactgct 1200 gaacttttaa caaacactac aacataaata atttgagttt aggtgatcca ccccttaatt 1260 gtacccccga tggtatattt ctgagtaagc tactatctga acattagtta gatccatctc 1320 actatttaat aatgaaattt atttttttaa tttaaaagca aataaaagct taactttgaa 1380 ccatgaaaaa aaaa 1394 <210> 41 <211> 1376 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3333139CB1 <400> 41 gtcgcaggcc ggagggaaga tggcggcgcc ctggtggcga gccgcgctgt gcgagtgtcg 60 gagatggcgg ggcttcagca cctcggccgt cctgggccgc cggacacccc cgctggggcc 120 gatgcccaac agtgacatcg acttgagcaa cctggagcgg ctggagaagt accggagctt 180 cgaccgctac cggcgccggg cagagcagga ggcgcaggcc ccgcactggt ggcggaccta 240 ccgagagtat ttcggggaga agacagatcc caaagagaag attgatattg ggctgcctcc 300 acccaaagtc tcccggaccc aacagctact ggaacggaaa caggccatcc aggagcttcg 360 ggccaatgtg gaagaggagc gggctgcccg cctccgcaca gccagtgtcc cgctggatgc 420 cgtgcgggcc gagtgggaga ggacctgtgg cccctaccac aagcagcgtc tggctgagta 480 ttacggcctc taccgagacc tgttccacgg tgccaccttt gtgccccgag tccccctgca 540 cgtggcctac gctgtgggtg aggatgacct gatgcctgtg tactgtggca atgaggtgac 600 tccaaccgag gctgcccaag cgccagaggt gacctatgag gcagaagagg gctccttgtg 660 gacgttgcta ctcactagct tggatgggca cctgctggag ccagatgctg agtacctcca 720 ctggctgcta accaacatcc cgggtaaccg ggtggctgaa ggacaggtga cgtgtcccta 780 cctccccccc ttccctgccc gaggctccgg catccaccgt cttgccttcc tgctcttcaa 840 gcaggaccag ccgattgact tctctgagga cgcacgcccc tcaccctgct atcagctggc 900 ccagcggacc ttccgcactt ttgatttcta caagaaacac caagaaacca tgactccagc 960 cggcttgtcc ttcttccagt gccgctggga tgactccgtc acctacatct tccaccagct 1020 tctggacatg cgggagccgg tgtttgagtt cgtgcggccg cccccttacc accccaagca 1080 gaagcgcttc ccccaccggc agcccctgcg ctacctggac cggtacaggg acagtcatga 1140 gcccacctat ggcatctact aaggagccag agtgtgcgca tttcagagca tgggattgat 1200 cggcagcaag agtaaagaca cagctccaga ggcccacact gtggggtctg ggccctgcct 1260 taggcagccc ccctctttgg ccccctcccg tcaggcccag ggcttggagt gaaagtgact 1320 ctcaggtggt ggggtgggga atgtgaataa acatgatttc ttgccgggaa aaaaaa 1376 <210> 42 <211> 526 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3432292CB1 <400> 42 caggacgtgt ctgtgctcct gtgtgtgacc agggttgaaa aagtcgcact gagatgtcct 60 gccagcaaaa ccagcagcag tgccagcccc ctcccaagtg ccccccaaaa tgcccaccca 120 agtgtcctcc aaagtgccga cctcagtgcc cagccccatg cccacctcca gtctcttcct 180 gctgtggtcc cagctctggg ggctgctgcg gctccagctc tgggggctgc tgcagctctg 240 ggggtggcgg ctgctgcctg agccaccaca ggccccgtct cttccaccgg caccggcacc 300 agagccccga ttgttgtgag tctgaacttc tgggggctct ggctgctagc acagctctgg 360 ggactgctgc tgaccaaacc tcgaacatca cagagcaagc ctttatggag aaaacttgca 420 aacgaggaac ctgtccccaa gagtgatagc ttcttcctga ccccttgttg tctccttatc 480 ccctgggggc tcgacaacac ctttgttgag agttgttttg gctctc 526 <210> 43 <211> 2431 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3478571CB1 <400> 43 gcctgctccc ggaggagacg cgctgccgag gagaacccag cgggagaaca tttcaggata 60 ggaataggcc aagtgctgag aagatgagtc ttaggattga tgtggataca aactttcctg 120 agtgtgttgt agatgcagga aaagtcaccc ttgggactca gcagaggcag gagatggacc 180 ctcgcctgcg ggagaaacag aatgaaatca tcctgcgagc agtatgtgct ctgctgaatt 240 ctggtggggg cataatcaag gctgagattg agaacaaagg ctacaattat gaacgtcatg 300 gagtaggatt ggatgtgcct ccaattttca gaagccattt agataagatg cagaaggaaa 360 accacttttt gatttttgtg aaatcatgga acacagaggc tggtgtgcca cttgctacct 420 tatgctccaa tttgtaccac agagagagaa catccaccga tgtcatggat tctcaggaag 480 ctctggcatt cctcaaatgc aggactcaga ctccaacgaa tattaatgtt tccaattcat 540 taggtccaca ggcagctcag ggtagtgtac aatatgaagg taacataaat gtgtcagctg 600 ctgctttatt tgatagaaag cggcttcagt atctggaaaa actcaacctt cctgagtcca 660 cacatgttga atttgtaatg ttctcgacag acgtgtcaca ctgtgttaaa gacagacttc 720 cgaagtgtgt ttctgcattt gcaaatactg aaggaggata tgtatttttt ggtgtgcatg 780 atgagacttg tcaagtgatt ggatgtgaaa aagagaaaat agaccttacg agcttgaggg 840 cttctattga tggctgtatt aagaagctac ctgtccatca tttctgcaca cagaggcctg 900 agataaaata tgtccttaac ttccttgaag tgcatgataa gggggccctc cgtggatatg 960 tctgtgcaat caaggtggag aaattctgct gtgcggtgtt tgccaaagtg cctagttcct 1020 ggcaggtgaa ggacaaccgt gtgagacaat tgcccacaag agaatggact gcttggatga 1080 tggaagctga cccagacctt tccaggtgtc ctgagatggt tctccagttg agtttgtcat 1140 ctgccacgcc ccgcagcaag cctgtgtgca ttcataagaa ttcggaatgt ctgaaagagc 1200 agcagaaacg ctactttcca gtattttcag acagagtggt atatactcca gaaagcctct 1260 acaaggaact cttctcacaa cataaaggac tcagagactt aataaataca gaaatgcgcc 1320 ctttctctca aggaatattg attttttctc aaagctgggc tgtggattta ggtctgcaag 1380 agaagcaggg agtcatctgt gatgctcttc taatttccca gaacaacacc cctattctct 1440 acaccatctt cagcaagtgg gatgcggggt gcaagggcta ttctatgata gttgcctatt 1500 ctttgaagca gaagctggtg aacaaaggcg gctacactgg gaggttatgc atcaccccct 1560 tggtctgtgt gctgaattct gatagaaaag cacagagcgt ttacagttcg tatttacaaa 1620 tttaccctga atcctataac ttcatgaccc cccagcacat ggaagccctg ttacagtccc 1680 tcgtgatagt cttgcttggg ttcaaatcct tcttaagtga agagctgggc tctgaggttt 1740 tgaacctact gacaaataaa cagtatgagt tgctttcaaa gaaccttcgc aagaccagag 1800 agttgtttgt tcatggctta cctggatcag ggaagactat cttggctctt aggatcatgg 1860 agaagatcag gaatgtgttt cactgtgaac cggctaacat tctctacatc tgtgaaaacc 1920 agcccctgaa gaagttggtg agtttcagca agaaaaacat ctgccagcca gtgacccgga 1980 aaaccttcat gaaaaacaac tttgaacaca tccagcacat tatcattgat gacgctcaga 2040 atttccgtac tgaagatggg gactggtatg ggaaagcaaa gttcatcact cagacagcaa 2100 gggatggccc aggagttctc tggatctttc tggactactt tcagacctat cacttgagtt 2160 gcagtggcct cccccctccc tcagaccagt atccaagaga agagatcaac agagtggtcc 2220 gcaatgcagg tccaatagct aattacctac aacaagtaat gcaggaagcc cgacaaaatc 2280 ctccacctaa cctcccccct gggtccctgg tgatgctcta tgaacctaaa tgggctcaag 2340 ggtgtcccag gcaacttaga gattattgaa gacttgaact tggaggagat actgatctat 2400 gtagcgaata aatgccgttt tctcttgcgg g 2431 <210> 44 <211> 714 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3495166CB1 <400> 44 gcgcaccgtc gcaccggtcc atttccctcg cgtgccgcgc tcacccaccc gcggcatgag 60 cagcgccccc gcgtcgggcc cggcgcccgc cagcctcacg ctctgggacg aggaggactt 120 ccagggccgt cgctgtcggc tgctaagcga ctgtgcgaac gtctgcgagc gcggaggcct 180 gcccagggtg cgctcggtca aggtggaaaa cggcgtttgg gtggcctttg agtaccccga 240 cttccaggga cagcagttca ttctggagaa gggagactat cctcgctgga gcgcctggag 300 tggcagcagc agccacaaca gcaaccagct gctgtccttc cggccagtgc tctgcgcgaa 360 ccacaatgac agccgtgtga cactgtttga gggggacaac ttccaaggct gcaagtttga 420 cctcgttgat gactacccat ccctgccctc catgggctgg gccagcaagg atgtgggttc 480 cctcaaagtc agctccggag cgtgggtggc ctaccagtac ccaggctacc gaggctacca 540 gtatgtgttg gagcgggacc ggcacagcgg agagttctgt acttacggtg agctcggcac 600 acaggcccac actgggcagc tgcagtccat ccggagagtc cagcactagg ctccacggcc 660 ccagacacct tccctgagga cactcaataa aggttcctga atcttcctgc caaa 714 <210> 45 <211> 3154 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3554748CB1 <400> 45 accatctccc actcgagctg cccccgccct ctggacccga gtgactcagg cctttgtttg 60 tccttcctgg tagaggcggg ttccctccct cggcaagatg ccggagtgct gggatgggga 120 acatgacatc gagacaccct acggccttct gcatgtagtg atccggggct cccccaaggg 180 gaaccgccca gccatcctca cctaccatga tgtgggcctc aaccacaaac tatgcttcaa 240 caccttcttc aacttcgagg acatgcagga gatcaccaag cactttgtgg tgtgtcacgt 300 ggatgcccct ggacaacagg tgggggcgtc gcagtttcct caggggtacc agttcccctc 360 catggagcag ctggctgcca tgctccccag cgtggtgcag catttcgggt tcaagtatgt 420 gattggcatc ggagtgggcg ccggagccta tgtgctggcc aagtttgcac tcatcttccc 480 cgacctggtg gaggggctgg tgctggtgaa catcgacccc aatggcaaag gctggataga 540 ctgggctgcc accaagctct ccggcctaac tagcacttta cccgacacgg tgctctccca 600 cctcttcagc caggaggagc tggtgaacaa cacagagttg gtgcagagct accggcagca 660 gattgggaac gtggtgaacc aggccaacct gcagctcttc tggaacatgt acaacagccg 720 cagagacctg gacattaacc ggcctggaac ggtgcccaat gccaagacgc tccgctgccc 780 cgtgatgctg gtggttgggg ataatgcacc cgctgaggac ggggtggtgg agtgcaactc 840 caaactggac ccgaccacta cgaccttcct gaagatggca gactctggag ggctgcccca 900 ggtcacacag ccagggaagc tgactgaagc cttcaaatac ttcctgcaag gcatgggcta 960 catgccctca gccagcatga cccgcctggc acgctcccgc actgcatccc tcaccagtgc 1020 cagctcggtg gatggcagcc gcccacaggc ctgcacccac tcagagagca gcgaggggct 1080 gggccaggtc aaccacacca tggaggtgtc ctgttgaagc ccttgatccc gctgacgacg 1140 cccacgtcga ggccccaccg ccatccttgc gccggctcat gttcccttta gtttattttt 1200 gtgagggcaa aggggaggaa atggggttct gtttgaaaaa aatgagggga tcttagatgc 1260 tgcagcagaa cagtctccag gtgttttaag gggctcagtc ctcctcatcc catctcactc 1320 tccgtggtaa cttagccaac ttgacccctc tcatcccact cccggcggcc caggcacaga 1380 agggcagggc catagggagg gagattcgct acggatccag gccattcctg ggtgagccct 1440 tgggcaggca tgtttggaga tgagagaggc ttcgagaggg tgggtgctgg gccacagggg 1500 tgcggggcca gctcaggcac tggcgtggga gccctgggag accccttccc ccaccctcca 1560 ccaagcacac ctgtttctgt ctcatagcac atgtgacaat catctggaca acagccacaa 1620 gggggcgctc ggaccaggca gccactttcc tggtgctctc tgggcccagc tggtgctgta 1680 gggccacgca ggcaggggcg tcaaggggtt tctctgccca aggaagacag aacatggaga 1740 accgtcaggg caggaacccc acagactgtc ccttccagcc cacactctgc cacctcctgg 1800 ccctgtccca attctgagcc aaggcctccc cgaggcagaa gttgcctggt cctctgtccc 1860 cacagtgacc tgactggggg tgagggagaa ggaggagaga gcccatgtgt ggtgtgtgtg 1920 cccctgagaa cttcgtggtg actgcctttg ggagcccgca ggtggccaga ggcaggggta 1980 gctgagttcc tggagacccc ttttttgccc ccaggttccc cagagggcaa cgccatcagt 2040 agcagtgtgg tgtttcaggc agagctctgg ccaggctgtg ccagtgtgtc ccggacgcat 2100 cactaaggaa gagagagttt atttagtcaa ctggcccaag gcagcgaggc ttctacagtc 2160 ccacacccca tagccgcctg ggctggggct tactgggggc tgaaggttct ggacatgaac 2220 aagggtcagg tagaagagaa aggcttcccc tacaccccag cctcctgctg tcccctgaag 2280 cccaggactg cgttgtatgc tttccatcca ctcaccttac cccatagcat cttgcggccc 2340 agaaaccaga gccatttgtc tcagacccta aatcaataat cacaaacccc aaaacgggag 2400 agagcagtga aaacatgcag ggctgtggac gggggaaggg ttgtggcggg tgttctgagg 2460 ctgagaggac acctatatgc gtatttcctc tacacacatc accccccttc tataatctta 2520 agccatgact agcctggtgg cgtgttagtt tctgcccagt tctaccccct catgtgcttc 2580 ttctgaatac tgaatgtgac tgtttgaaag ctggtagaat tcatccctct tactgtagat 2640 aacactgcaa atcttggaat tttgtttttt gctgtttcca gatgtatcta taaatatcta 2700 tacattatat gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtacatcggg tcctcccatg 2760 tgtggtgttc ttctggaggt tgtctctttg gtcaaggtga acttttaatg tttattattt 2820 tcttctccgc acaaagtaaa gagcctaatt ttgtgtattc tggtggctgc tgtcatgaga 2880 tgataaaatg taaaacaaaa ctctagtcaa cgtagaaaga gttaactgtg ctgaaaaact 2940 aataaagaac ctaagaagaa ttccagtgtg gtgatgccat gcccatcatg ggaggctttt 3000 ggagaaacag aatgtttggg caggggctgc tggtgctgct tgggttttgg gttgagggtg 3060 ctaggagagg atggtctcca cccatctttc tatttccagt acacgtcaca ttattttacc 3120 ggtgagatga gaatgtcaca aacattaaaa gcct 3154 <210> 46 <211> 2204 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3555629CB1 <400> 46 agggcatgcg ccggtggcgt gatggagcgg cagcagcagc agcaacagca actgcgaaac 60 ctgcgtgact tcctgttggt ctacaatcgg atgacagaac tctgcttcca gcgctgtgtg 120 cccagcttgc accaccgagc tctggacgct gaggaggagg cctgtgtgcc cagctgtgct 180 gggaagctga tccattccaa ccaccgcctc atggccgctt acgtgcagct catgcctgcc 240 ctggtacagc gccgcatcgc agactacgag gctgcctcgg ctgtgccagg cgttgctgct 300 gaacagcctg gggtctctcc atcaggcagc tagccatacc caaccccagg aaggaaggcc 360 ttggatggac cctcagattg aaggacccgg tggaccttgg ggttggtgaa tcctaaacag 420 agagaattcg aggttgcctg aaagctgggt gtccttgctc cttttcctgg agccaatata 480 cccagttttt actcagtttg atttatattc tgggcaagga agctttgcct actttattgg 540 cacaatccgt tgttctgtcg tttagtgcat atctgctggc ttcagccctg gcagctgaga 600 aattgttttc tatatgtaga aggaaaacct gagcatttgc aggcatctgg ttaaagcagg 660 gtctgtgtgt acaattttaa aacgggtaat atgtcatgct cttagttcat cttcacaaca 720 aaactatgag taagcggtat tagcctcact taacagatga ggaagcaaga ttccagaaag 780 taccagaagg tcattttata caacaggaga ttggttcctg cccagatgac agaaaatggg 840 agctctgtct agttgtcctt aagtctgact gacttcagtg gctcataacc gtgagccaag 900 tatttgttgg ttcataactg ttgttttgtg aactatgtct tacatgtcta gagttctgct 960 ggatctaggg aaaggaggag ctatcgaagt acaacggatc aaaaaaccac agggcttttg 1020 ggcactgcct ccttgggaag ttagtggcca cagaagagag atgaaacctg taagaagtct 1080 ggagtctttt ggaacttcag ccatttcccc aggttgttac tttcttagta tgtacagtct 1140 tctcaggatg agcagtaaaa cctttgaaca aaggtctgtg tggttgtctt cacgggcaat 1200 caggaaggga gagagctggg gaccatattc tgcaatgcag ccaaatccga ggaagagaaa 1260 ctgaagggag aagtagatgg caatggttat gataaaaagg gataaaacta aatcttcggg 1320 acttctttaa tgctacgtta atgtttcact gctcgtctag aaactcctaa atccagcttt 1380 ctatcatctg ccccacattg gtcccattga gtacattctg tgatttctaa ttccagcctc 1440 tccattcttt tctcattatt gcctcccccg ccccccaact ttgtgtaatt tacttctgta 1500 ttcagcagcc tggatagcat atcattccat caccccattt tcttgccacc attggccatc 1560 tttttgtatc attccactta ttctgtcttt tccattcctt cattcaaact gctagagaaa 1620 aacagttgtg taatgaatgc cactaaatat tcaaggcctc caacctcagc caagtcctca 1680 caccaacacg cagtcacacc aacacacacc tttatgggtt cctggtccat ttccttctct 1740 aattaccatg gcagttattt tacacctcta ctgctgtcct taatcccata ccccaccctc 1800 atcaggtgac cctgtttcct tttttagaga aattgaagct cttagacatt ggtttccaca 1860 gtaataattt taaaacttct tacaactacc tacaaagcag atgttctatc ctatctacag 1920 agcagaaaat tgaaattctc aagtagcaga cccggtatta aagtgcagat ctgactttaa 1980 agtccatgtt cattttacac agcaggctgc ctcttaagat agtatttatt gagcacacac 2040 tttgtgtagg ttctgatttt ggtagatgtc atgctttata ttaatcttta caacaactat 2100 aagtaagagg tattaacctc acttaacaga tgaggaagca agaatccaga atatgccaga 2160 aggcacattc tgcagatttc gtgcaaacat ttatacacag cttc 2204 <210> 47 <211> 863 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 639636CB1 <400> 47 cggctcgagg gctgaggggc cgccaccgcc agcagggagg cggagggcta gcgagccaag 60 cggtggggac gccgctgcct tcctctttgc ctgtctcgcc gccctcctag acacgctcct 120 catctacgga tcctaccctc cccgctcttg caagcattca ctcggccggt cgcctgctga 180 ccctccttcg ccacaggctc gtagcggagg cagcagcgag gcatgaagac ccccaacgca 240 caggaagccg aagggcaaca aaccagggca gctgcaggac gggccactgg gtctgcaaac 300 atgacaaaga aaaaagtctc ccaaaagaag cagagaggcc gaccttcatc ccagccccgc 360 aggaacatcg tgggctgcag aatttctcat ggatggaagg aaggagatga gcccatcacg 420 cagtggaaag gaaccgttct ggatcagctt ctagatgatt ataaggaagg tgacctccgc 480 atcatgccag aatccagtga gtctcctcca acagagaggg agccaggagg agttgtagat 540 ggcctaatag gtaagcatgt ggaatatacc aaagaagatg gctccaaaag gatcggcatg 600 gtcattcacc aagtggaagc caaaccctct gtgtatttca tcaagtttga tgatgatttc 660 catatctatg tctacgattt ggtgaaaaag tcctaactgt tagggtaaaa tttggcacat 720 gtgtggaaac aaatgtataa tttgtagaca tgcaaaaaat gttgcctttc agtgtattga 780 aagcttatgg aatccctgat aactaaacat ctttgccagc attaactgtt gttttgctct 840 aaaaaataca aacttaatga aat 863 <210> 48 <211> 3860 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 902218CB1 <400> 48 taagcttgcg gccgcgggga ggaggatggg ggctaaccag ttagtggtgc tcaacgtgta 60 cgacatgtat tggatgaacg aatatacctc atccattgga attggagttt ttcattcagg 120 aattgaagtc tatggcagag aatttgctta tggtggccat ccttacccct tttctggaat 180 atttgaaatt tccccaggaa atgcttctga actaggagaa acatttaaat ttaaagaagc 240 tgttgtttta gggagcacgg acttcctaga agatgatata gaaaaaattg tagaagaact 300 gggaaaagaa tacaaaggca atgcttatca tttaatgcat aaaaactgca atcatttttc 360 ttcagcttta tcagagattc tttgtgggaa agagattcct cgctggatca atcgacttgc 420 ctacttcagc tcctgtatac cctttctaca gagttgcctc ccgaaggagt ggctcacgcc 480 cgcagccctg cagtctagtg tcagccaaga actccaggat gaactggagg aagcagagga 540 tgctgccgca tccgcttccg tggcaagcac tgcagcaggc tccagacccg ggcgccacac 600 taaactataa atgtctccaa agtcacacat tcagaactgt ctctggcagt cgaatatcac 660 tagagaaaag taaacagaga agcatccttt agatattttg tatgcaaaga tggctctccc 720 ccaaatccca gtttttcagc tcaggattat atttgtaatc aaaaaaaaaa aatcacttgg 780 cgcaggaggg agaactttgt aagaagctgc cctctgtttt ttttatccac tcgtaaatct 840 ggatttattt cttctgtttt atacaagctc tgttaagtta tgtttacagt atcttgtatc 900 gctgtttaca aatcttgcat ggactcctgc cacagtgaaa gaagaaaatc ttcatgtctt 960 caaaaattag gcaggtaatc tttgtatact tctgacaatt gccagatcta tggcataaat 1020 aggcacacaa aaaggtactt aaacagttat agtcaccatc acctgcttca gaatggtctt 1080 ttagatttgt gtttgttttg ttaaagttgt tggcaccagg atgcagagaa tcagactggc 1140 ctgaggtgaa ggagcacaca gccctgaggg cttggaaccc tgggtccagt tcctcttcac 1200 acccccttcc actctgagta gcacatctcc ccaggtgccc atggaacacc tgctttcatc 1260 ccaaatatcc gtccacctag gcggggtggt atgttcttac gtctctctga ctttgatgcc 1320 actcattcta tagtttagct ggttttcgtt caagatattc ttggtagtaa ctgacaagta 1380 tgttgcacat gtattggggg aggcgcttca tttttatttt aatacacatg tatttcctcc 1440 ttgcacagga ttttgatggt gtgggaatat cctaagtggt agccttccaa gtagcagtga 1500 gttgacattc agctgctttt aactattcag gctacctttt atactaaacc ttgaaaacta 1560 gaatctaatg tctaccccaa aaaagtagtt ctttgatatt ttatactttt tatgtaccat 1620 gtcagaaaga gtatgttggc gtttgtcatg ggactcattt cacataatag aatgcctagt 1680 ctcattgacc aatcgttaaa aaatcatatt tgtgtgtctt aagattcata tttatatgtt 1740 ctctcaaatg tatgtctctt acgtaacata ctctaagaat gaaactgtca ccacaggtaa 1800 atccttgtta gcaaggaatc tgtctgctcc agtctactcc tagtttgatc cttggatgta 1860 agaaccaagt cattacatgt caaattcaat ttttctgcct taagaatgaa tgtccttcat 1920 aaaatattgg atgcagtgta acactatcca aggcagtgac ttcagcttta tatacatata 1980 aaatatagtt agttttaaaa ttattgacat ttatttaaac ttttagattg gattgtttgc 2040 tattgctctg tgtgaggata cataatcttt cagtaaactg tatttttaac ttttccataa 2100 gctgattttg gttcatttta tcaacgtaag cacaccctgt tcatagggaa aataaacctt 2160 gggttataag cattagcctg aggacaatga agccacttaa cctaatttat gctttcgact 2220 gttctgtttc cagagaggaa agcctttaca aattactctc agttctttag gggcagaagg 2280 cttgtttcaa gaggtttgac agaagaaagg aatatatgaa cttaatgaga tgtcgacttg 2340 gttcaggtct aaaaatgagg gcaaaacact aaggctctag cagtgacttg ttcactaaaa 2400 agagagagtc ctgtccccag acggttagta caaagccttg gatacagttt gcttgtaata 2460 tttttaataa tgtgaggagt acagtgtttt ctaattcatt caagtatata tgatttaaac 2520 ctgggctact gacacacaca cagtagccat tagttagact cttcttagtg aatatcagga 2580 acatcccatc tgtgcttaac cagaatccag caagtcagca cacaagtgat tttattgtta 2640 ttttgttgta tttacttgca tttgttgtat ttactttcat ctgcagcatt tggagtttaa 2700 aaataatgta aagggttcta gtagaaatag tgtcctaagg ccaattacct accatactaa 2760 caatcagcag ataaaattct ggacgtgaga ttccttataa tctaattata cctgaggttg 2820 agcaagaaat gtcttccttt agaaaatctc attcaagtca ggttcttctc tacagttcaa 2880 aattgagaat ggatttaatt aactagcatt tagccagctt tttcttgccc ttggagaaaa 2940 agaatcattc tcaacctgat aatctgttaa gaaaaatccc atatgaacaa tctggtcatt 3000 aacatacata tgatacggag tctctttgtt gtcaccaagt gaacatactt ctcatggtgg 3060 gttggacagt aatacatgtt acagggtcag aagcttctgg tttctgctgt ttgctttaaa 3120 tacccttggg gttttttttt aaacccttac aaggggagca tcagctttgg aaagtgtgac 3180 tctgtaggag tgtagaaggc agtggtgtat gatcttagcc tcgtcctgat gcctgaatcc 3240 agccagctgt tgctctgacc cacagcaata gagcaagtta cccatcacca gcatttgtac 3300 agagcaggga attctggttt tagtccattg gtagcattgt gtgtatgagg agattcaaca 3360 ccacagacag ctgcaggact cgatatccat ggcttctttc catcacaaaa cgggtagaaa 3420 cacattcact gcttcagggt tctaatctgt gtgtctcctt atgactccat ttctgtaagc 3480 tactctgtaa ctttgatata tgctgtattt tctttcttta aaagatttag atgttttttc 3540 agcaagctag ccatacaacc attgtatctc tttctcttca gtatggttta gagcccagat 3600 cagttagtag gctttcgttg tcttctcttt caatacatgt acatctttac tgtttgaaaa 3660 gtgttacagc tgtcaaagaa tcttcatgga cctgaagata atttcttgtg aagttgaatg 3720 caagtgtact gtcattcata gtgtttatat caaaatacca ggaatcttca cttttgctac 3780 cttgatatag cattgggcta tcatgttaca acattgaaat acattgattt attaaaaaat 3840 acttttataa gaaaaaaaaa 3860 <210> 49 <211> 726 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1360522CB1 <400> 49 cccggggact aacggcgccg gtgacgactt cgccgcgcgt tggtcagcca tggccaccgc 60 tctcgcgcta cgtagcttgt accgagcgcg accctcgctg cgctgtccgc ccgttgagct 120 tccctgggcc ccgcggcgag ggcatcggct ctcgccggcg gatgacgagc tgtatcagcg 180 gacgcgcatc tctctgctgc aacgcgaggc cgctcaggca atgtacatcg acagctacaa 240 cagccgcggc ttcatgataa acggaaaccg cgtgctcggc ccctgcgctc tgctcccgca 300 ctcggtggtg cagtggaacg tgggatccca ccaggacatc accgaagaca gcttttccct 360 cttctggttg ctggagcccc ggatagagat cgtggtggtg gggactggag accggaccga 420 gaggctgcag tcccaggtgc ttcaagccat gaggcagcgg ggcattgctg tggaagtgca 480 ggacacgccc aatgcctgtg ccaccttcaa cttcctgtgt catgaaggcc gagtaactgg 540 agctgctctc atccctccac caggagggac ttcacttaca tctttgggcc aagctgctca 600 atgaaccgcc aggaactgac ctgctgactg cactctgcca ggcttcccaa tgctttcact 660 cttatctacc ctttggcact tatcttgctt atcaacataa taatttatac acttctaaaa 720 aaaaaa 726 <210> 50 <211> 2196 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1400678CB1 <400> 50 gatggcgtcc atgcgggaga gcgacacggg cctgtggctg cacaacaagc tgggggccac 60 ggacgagctg tgggcgccgc ccagcatcgc gtccctgctc acggccgcgg tcatcgacaa 120 catccgtctc tgcttccatg gcctctcgtc ggcagtgaag ctcaagttgc tactcgggac 180 gctgcacctc ccgcgccgca cggtggacga gatgaagggc gccctaatgg agatcatcca 240 gctcgccagc ctcgactcgg acccctgggt gctcatggtc gccgacatct tgaagtcctt 300 tccggacaca ggctcgctta acctggagct ggaggagcag aatcccaacg ttcaggatat 360 tttgggagaa cttagagaaa aggtgggtga gtgtgaagcg tctgccatgc tgccactgga 420 gtgccagtac ttgaacaaaa acgccctgac gaccctcgcg ggacccctca ctcccccggt 480 gaagcatttt cagttaaagc ggaaacccaa gagcgccacg ctgcgggcgg agctgctgca 540 gaagtccacg gagaccgccc agcagttgaa gcggagcgcc ggggtgccct tccacgccaa 600 gggccggggg ctgctgcgga agatggacac caccacccca ctcaaaggca tcccgaagca 660 ggcgcccttc agaagcccca cggcgcccag cgtcttcagc cccacaggga accggacccc 720 catcccgcct tccaggacgc tgctgcggaa ggaacgaggt gtgaagctgc tggacatctc 780 tgagctggat atggttggcg ctggccgaga ggcgaagcgg agaaggaaga ctctcgatgc 840 ggaggtggtg gagaagccgg ccaaggagga aacggtggtg gagaacgcca ccccggacta 900 cgcagccggc ctggtgtcca cgcagaaact tgggtccctg aacaatgagc ctgcgctgcc 960 ctccacgagc taccttccct ccacgcccag cgtggttccc gcctcctcct acatccccag 1020 ctccgagacg cccccagccc catcttcccg ggaagccagc cgcccaccag aggagcccag 1080 cgccccgagc cccacgttgc cagcgcagtt caagcagcgg gcgcccatgt acaacagcgg 1140 cctgagccct gccacaccca cgcctgcggc gcccacctcg cctctgacac ccaccacacc 1200 tccggctgtc gcccctacca ctcagacacc cccggttgcc atggtggccc cgcagaccca 1260 ggcccctgct cagcagcagc ctaagaagaa cctgtccctc acgagagagc agatgttcgc 1320 tgcccaggag atgttcaaga cggccaacaa agtcacgcgg cccgagaagg ccctcatcct 1380 gggcttcatg gccggctccc gagagaaccc gtgccaggag cagggggacg tgatccagat 1440 caagctgagc gagcacacgg aggacctgcc caaggcggac ggccagggta gcacaaccat 1500 gctggtggac acagtgtttg agatgaacta tgccacgggc cagtggacgc gcttcaagaa 1560 gtacaagccc atgaccaatg tgtcctagaa ccacctgcct cacagctggc cgtcacttgt 1620 gggggtccac gggacgatgg ctttgccagc ttaaagtaac cggatggcgg acacctggcc 1680 cccgaggtcc cccggccgcc gccctgctgc tgacccagcc tgttttaagt tctggatgca 1740 tttctctggg gtatttgggg cttattttta aaattttaat atgggttctt ttttgtgtga 1800 tttaagacac tttttggact caacgttaca tttttgaatg tagtaagtaa attaaccaaa 1860 aaagttacaa cttcctaatt ttagtgacag ctctgcctgt tagactctta ctttttaaaa 1920 tcttttctat tttccctcgc tggggcagtg ccctcctacc cccagggttg aggggaccaa 1980 ggtggcacgg tggtactggg ggtgcggcag ggacacccga ccacaccaga gcgtgggaga 2040 cggtgggcct tgtcccctgc ctgtgcctgc ctgggagttt tgtattcatc ttttgtatag 2100 ttgtggacat ttaagacagt ctttgggtac ctattttcat tgtaaaacta tctgaaccat 2160 taaagtcgag cttttctaaa gaaaaaaaaa aaaaaa 2196 <210> 51 <211> 1495 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1435556CB1 <400> 51 gagcagaaat tcggcacgag gaaaaatctg aaatctgaaa tgctccaaaa tcctaaactt 60 tttgagtgct gacattatgc cacaaatgga aaatttcata cctgacctta tgtgagttgc 120 agtcaaaaca caggtgcaca acacccagtt catgcaacat ccccaatggg aaaaaagacc 180 cccccagctc tcttctgctg cagtttttct gctcacacct ggattcccca tgcattccca 240 caaaaagtaa ttaaatggca tgcgtgcagg ctggacacgc caacaacagg tttcccacaa 300 tgccccacat gggcgaagac ctgtgtgcat tactcattgc atttttttgc ttattctctg 360 ctgtgtggta taaatatatt gttgaaaatg tcaaaaagac ctaaagatac ccctgtgaat 420 atcagtgata agaaaaagag gaagcattta tgtttatcta tagcacagaa agtcaagttg 480 ttggagaaac tggacagtgg tgtaagtgtg aaacatctta cagaagagta tggtgttgga 540 atgaccacca tatatgacct gaagaaacag aaggataaac tgttgaagtt ttatgctgaa 600 agtgatgagc agatattaat gaaaaataga aaaacacttc ataaagctaa aaatgaagat 660 cttgatcgtg tattgaaaga gtggatccgt cagcgtcgca gtgaacacat gccacttaat 720 ggtatgctga tcatgaaaca agcaaagata tatcacaatg aactaaaaat tgaggggaac 780 tgtgaatatt caacaggctg gttgcagaaa tttaagaaaa gacatggcat taaattttta 840 aagacttgtg gcaataaagc atctgctggt catgaagcaa cagagaagtt tactggcaat 900 ttcagtaatg atgatgaaca agatggtaac tttgaaggat tcagtatgtc aagtgagaaa 960 aaaataatgt ctgacctcct tacatataca aaaaatatac atccagagac tgtcagtaag 1020 ctggaagaag aggatatcaa agatgttttt aacagtaata atgaggctcc agttgttcat 1080 tcattgtcca atggtgaagt aacaaaaatg gttctgaatc aagatgatca tgatgataat 1140 gataatgaag atgatgttaa cactgcagaa aaagtgccta tagacgacat ggtaaaaatg 1200 tgtgatgggc ttattaaagg actagagcag catgcattca taacagagca agaaatcatg 1260 tcagtttata aaatcaaaga gagacttcta agacaaaaag catcattaat gaggcagatg 1320 actctgaaag aaacatttaa aaaagccatc cagaggaatg cttcttcctc tctacaggac 1380 ccacttcttg gtccctcaac tgcttctgat gcttcttctc acctaaaaat aaaataaaat 1440 acagtgtaca gtaacctttt agtcaaaaca gcatcatact tggaaactga aagcc 1495 <210> 52 <211> 2794 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1546633CB1 <220>
<221> unsure <222> 2705, 2709, 2711, 2713-2714, 2717, 2719, 2731, 2735-2736, 2745, 2747, 2750, 2757, 2763-2764, 2766, 2770, 2782, 2785, 2787 <223> a, t, c, g, or other <400> 52 ccgcgagagc aaggagcaca gagtgcagca tcatgacaag gagatttctc gaagccgaat 60 tccccggttg attcttcggc cccatatgcc ccaacaacag cacaaagtgt ccccagcctc 120 tgagtctcct ttctctgagg aagagagcag agagttcaac cccagcagct ctgggcgctc 180 agcgaggacc gttagcagca acagcttctg ctcagatgac acaggctgtc ctagcagcca 240 gtcagtgtct cctgtgaaga caccctcaga tgctggaaac agccccattg gcttttgccc 300 tggaagtgat gaaggcttca ccagaaagaa atgcacgatt ggaatggttg gtgaaggaag 360 cattcagtcc tctcgatata agaaggaatc aaagtcaggc cttgtgaaac caggtagtga 420 agctgatttt agctcctcga gcagcacagg cagcatttcc gctcctgagg tccatatgtc 480 gactgcggga agcaagcggt cttcttcttc acgcaatcga ggtcctcatg ggcggagtaa 540 tggagcttcg tcacacaagc ctggcagcag cccatcatcc ccgcgggaaa aggaccttct 600 gtccatgctg tgcaggaatc agctgagccc tgtcaatatc catcccagtt atgcaccttc 660 ttccccaagc agtagcaact caggctccta caaaggaagc gactgtagcc ccatcatgag 720 gcgttctgga aggtacatgt cttgcggtga aaatcatggt gtcagacccc caaacccaga 780 gcagtatttg actccactgc agcagaaaga ggtgacagtg agacacctca aaatcaagct 840 gaaggaatct gagcgccgac tccatgaaag ggaaagtgaa atcgtggagc ttaagtccca 900 gctggcccgc atgcgagagg actggattga ggaggagtgt caccgggtag aggcccagtt 960 ggcactcaaa gaagccagga aagagattaa acagctcaaa caggtcatcg aaaccatgcg 1020 gagcagcttg gctgataaag ataaaggcat tcagaaatat tttgtggaca taaacatcca 1080 aaacaagaag ctggagtctc tccttcagag catggagatg gcacacagtg gctctctgag 1140 ggacgaactg tgcctagact ttccatgtga ttccccagag aagagcttaa ccctcaaccc 1200 ccctcttgac acaatggcag atgggttatc tctggaagag caggtcacgg gggaaggggc 1260 tgacagggag ctactggtag gagatagcat agccaacagc acagatttgt tcgatgagat 1320 agtgacagcc accaccacag aatctggtga cctggagctt gtgcattcca cccctggggc 1380 taacgtcctg gagctgctgc ccatagtcat gggtcaggag gagggcagtg tggtggtgga 1440 gcgagccgtt cagaccgacg tggtgcccta cagcccagcc atctcagagc tcattcagag 1500 tgtgctgcag aagctccagg acccctgtcc ctcgagcttg gcgtcccctg atgagtctga 1560 accagactcg atggagagct tcccagagtc cctctctgcc ttagtggttg atttaactcc 1620 aagaaatcca aactcagcca tccttttgtc tcccgtggag accccctacg ccaatgtgga 1680 tgcagaagtt catgcaaacc gcctcatgag agagctggat tttgcagcct gcgtggaaga 1740 gaggttggat ggtgtcatcc cactggctcg cgggggcgtc gtgaggcagt actggagcag 1800 cagcttcctg gtggatctcc tggctgtggc tgcccccgtg gtccccacgg ttctgtgggc 1860 attcagtact cagagagggg gaacggatcc tgtgtataac atcggggcct tgctcagggg 1920 ctgttgcgtg gttgccctgc attcgctccg ccgcaccgcc ttccgtatca aaacctaaat 1980 agaagttgtt gttaccgtgt gccaatgtgt cccatgtggg ttgtgccagg tagagaaaca 2040 ggaagtcaat catctgtgac agtctctatt ctgtcgtttt gctccttggt atttgatttg 2100 cactatattt agttgaagcc tgttcactgt ttaaaaccgg aggtatcttc aaaggcatgg 2160 agacctggtt ccagtaaatg tcccaccagt ggggtataga aagcatgctc atgaccctgc 2220 cgtgtcgtct gaggtacccg ttcttatcct agtggttcag gaagagaaaa cgcagtttgc 2280 actttcaaga cagcttctct aaggctggca tgttatctcc ttgctttgct ttttgccgtt 2340 ttaaaatgtg taattgttcc agcattccaa tggtcttgtg catagcaggg gactgtaacc 2400 aaaaataaac atgtatttgt gtaattggtt tgaagaagtc ttgaatagct ctttactgtc 2460 ttacttgggg ttgataagat ttgagtgttt gcaatttttt actaaatgta gctccaaagt 2520 cttaaatggc ttgtttgttc ttaaactgtt aattgatgaa actgtgcata agtttacaat 2580 gtactaactt attttgctta ttatatatag tgttttattg gaaattgtaa ccacacactt 2640 cagcatgatg aaaataaaga ttagtgtttc catttaaata aatgttttat cctccccata 2700 aaatnaagna ngnnttnang ggggaggttt ncaannttgg gttgnanaan atttagngcc 2760 ttnngntgtn tcgggggttt gngcnangca aaaa 2794 <210> 53 <211> 1516 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1794031CB1 <400> 53 acgggatctg gatgagctgg acaaggatca tttacagttg agagaagcct gggatggcct 60 cgatcaccag attaatgcat ggaaaataaa gctaaattat gccttgcccc cacccctcca 120 tcaaactgaa gcttggctcc aggaggtaga agagcttatg gatgaagatt tgtcagcctc 180 ccaggatcac tctcaagccg tgactctgat acaagagaaa atgactttat tcaagagcct 240 gatggataga tttgagcatc attcgaacat tctccttacc tttgaaaata aggatgaaaa 300 tcacttgcca ttggtaccac ctaacaaatt ggaggaaatg aaaagacgaa tcaacaacat 360 tttggagaaa aaatttattc tacttctaga atttcattac tacaagtgct tagttcttgg 420 tttggtagat gaagtgaaat caaaattgga tatttggaac attaaatatg ggagcagaga 480 atctgtggaa ttattgctgg aagactggca taaatttatt gaagaaaaag aattcctagc 540 tcgacttgat acttcttttc aaaaatgtgg agaaatttat aagaatttgg ctggagaatg 600 tcagaatatt aataaacagt atatgatggt gaaatctgat gtttgtatgt atagaaaaaa 660 tatatataat gtgaagtcca ctctacaaaa agtgctggca tgttgggcta cttatgtgga 720 aaaccttcgc ttactaaggg cttgctttga ggagacaaag aaggaagaaa ttaaagaggt 780 accctttgag acactagccc agtggaatct agaacacgct actttaaatg aagcaggaaa 840 tttcttagtc gaagtcagca atgatgtggt tggatcatct atttctaaag aactgagaag 900 gctgaataaa agatggagaa agttggtttc aaaaactcaa cttgaaatga acctgccact 960 gatgataaaa aaacaggatc agcccacttt tgacaattct ggaaatattc tatctaaaga 1020 agagaaagca actgttgagt tttcaacaga tatgtcagta gaacttcctg aaaattataa 1080 tcaaaatata aaggctggag agaaacatga aaaagaaaat gaagaattca cagggcaact 1140 aaaagtggct aaagatgttg aaaaactcat tggataagtg gaaatctggg aggcagaagc 1200 caaatctgtt ttggatcaag atgatgtgga cacctcaatg gaagaatctt tgaaggtatg 1260 tgtgtaaaag tattaagagg gtactttcat ggttgtgcat ttatgtttta agttaaataa 1320 gaagttttaa agtaagtagt aataagccta cagttttaat tttctttgtt gggagtttta 1380 aaaatgaatg gattttatcc ctggatcatt tgctgttatt ttgcttgaca gcagaggata 1440 gattaggaga ccactgataa tacctatgaa tgttaagctc ttggacttat tttcttagct 1500 ataacatggg ggttaa 1516 <210> 54 <211> 1146 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2060563CB1 <400> 54 tgccgccctg ccaccctgcc gccctgccgc cctgccgccc tgccgccctg ccgccggtgg 60 tcgctgcccg tggtgctccg tcgcccccgc cacctcacgt cctcccgtgc gtcgggagcg 120 tctcggctac aacatgttgg gcatgatcaa gaactcgctg ttcggaagcg tagagacgtg 180 gccttggcag gtcctaagca aaggggacaa ggaagaagtt gcctatgaag aaagggcctg 240 tgaaggcggc aaatttgcca cagtagaagt gacagataag cctgtggatg aggctctacg 300 ggaagcaatg cccaaggtcg caaagtatgc ggggggcacc aatgacaagg gaattgggat 360 ggggatgaca gtccctattt cctttgctgt gttccccaat gaagatggct ctctgcagaa 420 gaaattaaaa gtctggttcc ggattccaaa ccaatttcaa agcgacccac cagctcccag 480 tgacaaaagc gttaagattg aggaacggga aggcatcact gtctattcca tgcagtttgg 540 tggttatgcc aaggaagcag actacgtagc acaagccacc cgtctgcgtg ctgccctgga 600 gggcacagcc acctaccggg gggacatcta cttctgcacg ggttatgacc ctcccatgaa 660 gccctacgga cggcgcaatg agatctggct gttgaagaca tgagtgaccc actgaaccaa 720 gaacttactg gaagtgtgcc tctgtgtctc cttcctcggg ggtaaggagg ggacagtgct 780 tcccaagttc cagctgcaag tccaacttaa ccaactttcc ttcaaagtca gttactgcca 840 attttctgaa aaaagcatgt tccatatact aagtctcttt tctcacagta ggaaataata 900 cagccaagat atgcagcatc cttctcattg atgtagaaaa ttctgcgata gaccagaaaa 960 atcctggcag cttttctcca ggcatctggg tcactaaaaa ctgattttct aaaattattg 1020 gatttgtatt ttgttattaa gggggaaaat gtgatttgtg cctgatcttt catctgtgat 1080 tcttataaga gctttgtctt cagaaaaact aaaaataaaa ggcattgact taaaaaaaaa 1140 aaaaaa 1146 <210> 55 <211> 2761 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2573955CB1 <400> 55 cggctcgagg ctgagaccca gagtcaccca ggggtctccg tcacgtgcca ggagtaggca 60 gaagtgggct gtgacagatc aggaaacaga gctcagtgca gcccactaaa ttgctcaggg 120 ccctacagct aacaagcggc agaggcagga tctgcactca ggagctgctt ggagatgctg 180 ctgtggccac tgctgctgct gctgctgctg ctgccaacat tggccctgct caggcagcag 240 cggtcccagg atgccaggct gtcctggctt gctggcctcc agcaccgagt ggcatggggg 300 gccctggtct gggcagccac ctggcagcgg cggaggctgg agcagagcac gctccatgtg 360 caccagagcc agcagcaggc cctgaggtgg tgtctacagg gagcccagcg cccccactgt 420 tccctcagaa ggagcacgga cataagcacc ttccggaatc atctccctct gaccaaggcc 480 agccagaccc agcaggaaga cagtggagag cagccactgg ccccgacctc aaaccaggac 540 cttggggagg cctctctgca ggccaccttg ctgggtctgg cagccctaaa caaggcctac 600 ccagaagtgc tggctcaggg acgcactgcc cgtgtgacgc ttacatcccc ttggccccga 660 cccctgcctt ggcctgggaa taccctgggc caggtgggca cccctggaac caaggaccct 720 agggccctgc tgctggacgc actgaggtcc ccagggctga gggcactgga ggctgggacg 780 gctgtcgaac ttctggatgt tttcttgggc ctggagactg atggtgaaga gctagctggg 840 gcgatagctg ccgggaaccc tggagcgcct ctccgtgaac gggcagctga gctccgggag 900 gccctagagc aggggccacg gggactggcc cttcggctct ggccaaagct gcaggtggtg 960 gtgactctgg atgcaggagg ccaggccgag gctgtggctg ccctcggggc cttgtggtgc 1020 caaggactag ccttcttctc tcctgcttat gctgcctcgg gaggggtgct gggcctaaac 1080 ctacagccag agcagcccca tgggctctac cttctgcccc ctggggcccc ctttatcgag 1140 ctgctcccag tcaaggaagg cacccaggag gaagctgcct ccaccctcct tttggccgag 1200 gcccagcagg gcaaggagta tgagctggtg ctgacggacc gcgccagcct caccaggtgc 1260 cgcctgggtg atgtggtgcg agtggttggt gcctacaatc agtgtccagt cgtcaggttc 1320 atctgcaggc tggaccagac cctgagtgtg cgaggggaag atattggtga agacctgttc 1380 tctgaggccc tgggccgggc agtggggcag tgggcggggg ccaagctgct ggaccatggc 1440 tgtgtggaga gcagcattct ggattcctct gcgggctctg ctccccacta cgaggtgttt 1500 gtggcgctga gggggctgag gaatctgtca gaggaaaatc gagacaagct ggaccactgc 1560 cttcaggaag cctctccccg ctacaagtcc ctgcggttct ggggcagcgt gggccctgcc 1620 agagtccacc tggtggggca gggagccttc cgagcactcc gggcagccct cgctgcctgc 1680 ccctcctccc ccttcccccc tgcgatgccc cgggtccttc ggcacaggca cctggcccag 1740 tgtctgcagg agagggtggt gtcctgagtc aagtcctgcc ccaccgccca gctcccccca 1800 gaggccacct cgcccctccc tctgggacct ctccggatgg ggagtccttg gccagggtct 1860 ctgactctgt gtcacctgac atttgcccat gagagccgct gggccttaga gaggccttgg 1920 cccagctgac cggttctgaa gtatgggcct ccggggttag cagatgccag cagtgcctgc 1980 ccgtgtcccc atgtcccggc atgaaggaca ctgctagaga gttaccatgc acaccgatgg 2040 tttcctgtat cacagcccaa agaggttctc tggtggccac agctgtgtgc tcagtcagtg 2100 cactgggcaa gctagaagtg ttggggggtt aatgtcccca ggagcagcaa ccctgagtca 2160 ataaggagca ggacctcagc ttcattgtcc ttgagcagga caattctgaa gtgtattcta 2220 cataaactct cagaggatgc ccagcaggat ggagtcccag ttgcccgcag cagtaaccca 2280 ctcattcatg tacttcctgc gggggctctc ccttccctct cttccccact cccccgcctt 2340 gggcttcctg ggatggctcc caaataaacc tcttgcaccc agaaaaaaaa aaaaaaaaaa 2400 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaagg 2460 gggggccgct ctaggggttc caggtttagg tacgggtgca tgggaggtca tagctcttct 2520 aaggtgtccc ctaatttcat ttcacgggcg gtggttttaa aaggtcgtga ctgggaaaac 2580 cctggggtta cccaatttaa tcgctttgag gaaattcccc ttttggcaag ttggggtaat 2640 agcgaagggg cccgcacggt tggcctttcc aaaaatttgg gccctctgat tggcgattgg 2700 gacgcgcctg taggggggtt ttaagccggc ggttttggtg gttacgccgg tgacccgtta 2760 c 2761 <210> 56 <211> 1164 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3404792CB1 <400> 56 atcatggcag ggctgggctc cgatccctgg tggaagaaaa ccctttactt gaccggggga 60 gctttgctgg ccgcagctgc gtatctgctc cacgaactcc tggtcattag gaaacagcaa 120 gagattgact ctaaagatgc tattattttg catcagtttg caagacctaa caatggtgtt 180 ccaagtttat ctcctttctg tttaaagatg gaaacttatt taaggatggc tgacttaccg 240 tatcagaact attttggtgg aaaactctct gctcaaggga aaatgccttg gattgaatat 300 aatcatgaaa aagtttctgg cacagaattc ataattgact ttctggaaga gaagcttgga 360 gtgaatttaa acaaaaacct tggccctcat gaaagagcca tctccagagc ggtgaccaag 420 atggtggagg agcacttcta ctggacgtta gcttattgcc agtgggtgga caatctcaat 480 gagacccgga agatgctctc tcttagtggt ggtggtccct tcagcaacct gctgaggtgg 540 gttgtgtgcc acataacgaa aggaattgtg aaacgcgaga tgcacggcca cggcattggc 600 cgcttctccg aggaagagat ttacatgctg atggagaagg acatgcggtc tttagcaggg 660 cttttgggtg ataagaagta catcatgggg cccaagcttt ccactcttga cgccactgtc 720 tttggacact tggcacaggc aatgtggacc ttaccaggga caagacccga acggctgatc 780 aaaggtgagc tgatcaacct tgccatgtac tgtgagagga taaggaggaa attttggcca 840 gagtggcacc acgatgatga caataccatc tatgagtctg aggagagcag cgaaggcagc 900 aaaacccaca ccccgctgct ggattttagc ttttactcaa ggacagagac ctttgaagat 960 gagggagcag aaaacagttt ttccagaacc ccagacacag attttactgg acactcactc 1020 tttgattcgg atgtggacat ggatgactat acagaccacg aacagtgcaa gtgacgtcca 1080 gcctcactga ccctcttcct tgggacctgc cactccctgg gtcggtccat tttcccaggt 1140 agcaatccat ccgagctggg agga 1164

Claims (35)

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
a) an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID
NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ
ID NO:28, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID
NO:28.

2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 selected from the group consisting of SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID
NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID
NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID
NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID
NO:56.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method for producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. An isolated antibody which specifically binds to a polypeptide of claim 1.

11. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
a) a polynucleotide sequence selected from the group consisting of SEQ ID
NO:29, SEQ ID
NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID
NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:37, SEQ ID
NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID

NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52, SEQ ID
NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).

12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 11.

13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising:
a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.

15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising:
a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

16. A composition comprising an effective amount of a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

17. A composition of claim 16, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:13, SEQ
ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID
NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID

NO:27, and SEQ ID NO:28.

18. A method for treating a disease or condition associated with decreased expression of functional CDIFF, comprising administering to a patient in need of such treatment the composition of claim 16.

19. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

20. A composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.

21. A method for treating a disease or condition associated with decreased expression of functional CDIFF, comprising administering to a patient in need of such treatment a composition of claim 20.

22. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

23. A composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.

24. A method for treating a disease or condition associated with overexpression of functional CDIFF, comprising administering to a patient in need of such treatment a composition of claim 23.

25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of:
a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

26. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

27. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.

28. A method for assessing toxicity of a test compound, said method comprising:
a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 11 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 11 or fragment thereof;
c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

29. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:40 and SEQ ID NO:50.

30. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 29.

31. A cell transformed with a recombinant polynucleotide of claim 30.

32. A transgenic organism comprising a recombinant polynucleotide of claim 30.

33. A method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:22, the method comprising:
a) culturing the cell of claim 31 under conditions suitable for expression of the polypeptide, and b) recovering the polypeptide so expressed.

34. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 29, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.

35. A method for assessing toxicity of a test compound, said method comprising:
a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 29 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 29 or fragment thereof;
c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.